The Interactive Fly

Zygotically transcribed genes

Circadian behavior and sleep

  • Proteins involved in photoperiod response and regulation of sleep

  • Proteins involved in photoperiod response

    Proteins involved in regulation of sleep

    Circadian regulation of gene expression systems in the Drosophila head

    Mechanisms composing Drosophila's clock are conserved within the animal kingdom. To learn how such clocks influence behavioral and physiological rhythms, the complement of circadian transcripts in adult Drosophila heads was determined. High-density oligonucleotide arrays were used to collect data in the form of three 12-point time course experiments spanning a total of 6 days. Analyses of 24 hr Fourier components of the expression patterns revealed significant oscillations for ~400 transcripts. Based on secondary filters and experimental verifications, a subset of 158 genes showed particularly robust cycling and many oscillatory phases. Circadian expression is associated with genes involved in diverse biological processes, including learning and memory/synapse function, vision, olfaction, locomotion, detoxification, and areas of metabolism. Data collected from three different clock mutants (per0, tim01, and ClkJrk), are consistent with both known and novel regulatory mechanisms controlling circadian transcription (Claridge-Chang, 2001).

    A genome-wide expression analysis was performed aimed at identifying all transcripts from the fruit fly head that exhibit circadian oscillations in their expression. By taking time points every 4 hr, a data set was obtained that has a high enough sampling rate to reliably extract 24 hr Fourier components. Time course experiments spanning a day of entrainment followed by a day of free-running were performed to take advantage of both the self-sustaining property of circadian patterns and the improved amplitude and synchrony of circadian patterns found during entrainment. 36 RNA isolates from wild-type adult fruit fly heads, representing three 2 day time courses, were analyzed on high-density oligonucleotide arrays. Each array contained 14,010 probe sets (each composed of 14 pairs of oligonucleotide features) including ~13,600 genes annotated from complete sequence determination of the Drosophila genome. To identify different regulatory patterns underlying circadian transcript oscillations, four-point time course data was colleced from three strains of mutant flies with defects in clock genes (per0, tim01, and ClkJrk) during a single day of entrainment. Because all previously known clock-controlled genes cease to oscillate in these mutants but exhibit changes in their average absolute expression levels, the analysis of the mutant data was focused on changes in absolute expression levels rather than on evaluations of periodicity (Claridge-Chang, 2001).

    To organize the 158 statistically significant circadian transcripts in a way that was informed by the data, hierarchical clustering was performed. Both the log ratio wild-type data (normalized per experiment) and the log ratios for each of the three clock mutants (normalized to the entire data set) were included to achieve clusters that have both a more or less uniform phase and a uniform pattern of responses to defects in the circadian clock. One of the most interesting clusters generated by this organization is the per cluster. This cluster contains genes that have an expression peak around ZT16 and a tendency to be reduced in expression in the ClkJrk mutant. Strikingly, all genes previously known to show this pattern of oscillation (per, tim, vri) are found in this cluster. In fact, the tim gene, which has multiple representations on the oligonucleotide arrays, has two independent representations in this cluster. Together with the novel oscillator CG5798, per, tim, and vri form a subcluster (average phase ZT14) that shows upregulation in both the per0 and tim01 mutants. The fact that per, tim, and vri all function in the central circadian clock raises the possibility that several other genes from this cluster, including the ubiquitin thiolesterase gene CG5798 and the gene coding for the channel modulator Slowpoke binding protein (Slob) may function in the circadian clock or directly downstream of it (Claridge-Chang, 2001).

    The genes in a second cluster (Clock cluster are primarily grouped together based on their peak phase (average phase ZT2). By virtue of the mutant expression data, several subclusters within this phase group can be identified. The known circadian genes Clock and takeout (to) are part of this cluster. Clk is found in a clustered pair with the leucyl aminopeptidase gene CG9285. In terms of chromosomal organization, to, CG11891, and CG10513 map closely together on chromosome 3R. Two additional circadian genes in this chromosomal region (CG11852, CG1055). Interestingly, the Clk cluster contains three pairs of homologous genes with very similar expression patterns: the UDP-glycosyl transferase genes Ugt35a and Ugt35b, the enteropeptidase genes CG9645 and CG9649, and the long-chain fatty acid transporter genes CG6178 and CG11407. In the first two cases, the homologous genes are also directly adjacent to each other on the chromosome. An overview of the map positions of all circadian genes in this study is available as supplemental information online ( Apart from Ugt35a and Ugt35b, several other genes with a predicted function in detoxification are members of the Clk cluster (CG17524, CG8993, CG3174, Cyp6a21). It may also be noteworthy that the genes for three oxidoreductases found in this group [Photoreceptor dehydrogenase (Pdh), CG15093, CG12116] have almost identical phases (ZT3) (Claridge-Chang, 2001).

    All genes of the apterous (ap) cluster are defined by both the oscillatory phase of their expression pattern (average phase ZT17) and by a distinct expression profile in the three clock mutants. Although the 6 hr sampling interval in the mutant data makes it difficult to reliably detect oscillations, it seems that the majority of the genes in this cluster shows some degree of periodicity in the three mutant light-dark regime (LD) time courses. Although it cannot be ruled out that there are circadian oscillations independent from the known clock genes, the hypothesis that there may be a light-driven response underlying the observed mutant expression pattern is favored. The genes in this group may, therefore, be regulated not only by the circadian clock, but also by a direct light-dependent mechanism. It should be mentioned that evidence of gene expression patterns that are purely light-driven in wild-type flies was sought, but little indication was found of such regulation. Instead, genes with both a strong light-driven oscillation and a weak circadian component were encountered. apterous (ap) encodes a LIM-homeobox transcription factor, which is known to act both in neural development and in neuropeptide expression. The ap cluster includes the genes for the transcription factor moira, the synaptic regulator syndapin, two septins (Sep1 and CG9699), and two ATP binding cassette (ABC) transporters (CG6162, CG9990). In terms of chromosomal organization, CG6166, the gene adjacent to CG6162 on chromosome 3R is homologous to CG9990 and coregulated with CG6162 and CG9990 (Claridge-Chang, 2001).

    The founding member of the fourth cluster, ebony (e), encodes β-alanyl-dopamine synthase and has roles in both cuticle tanning and regulating circadian locomotor behavior. Among the other cluster members are six genes that function in protein cleavage (CG9377, Ser99Da, SP1029, CG7828, CG11531, BcDNA:GH02435), three transcription factor genes (CG15632, CG17257, CG6755), as well as two genes each that act in signal transduction (prune, loco), the cytoskeleton (TpnC47D, Chd64), and lipid metabolism (ATPCL, CG1583) (Claridge-Chang, 2001).

    The per0, tim01, and ClkJrk mutations affect genes that are essential for maintaining circadian rhythms and result in both molecular and behavioral arrhythmicity. In addition to abrogating the oscillations of the per, tim, vri, to, and Clk transcripts, these mutations also affect their absolute levels of expression. per0 and tim01 flies have intermediate or somewhat elevated levels of per, tim, vri, and to transcript, and decreased levels of Clk transcript whereas ClkJrk mutants have the opposite effect. Based on these observations, genome-wide expression data was gathered from per0, tim01, and ClkJrk mutant flies in three separate four-point time course experiments. A rank-sum Wilcoxon test was employed to determine if any of the interrogated transcripts were significantly up- or down-regulated in any of the mutants when compared to the total wild-type expression data set (Claridge-Chang, 2001).

    Out of the 14010 probe sets on the arrays, 4865 showed up- or down-regulation in one or more of the three mutants with a p value lower than 0.05; 2544 were significantly changed in the tim01 flies; 1810 were significantly different in per0, and 2181 in ClkJrk. It is unclear what proportion of these changes depends on the actual mutations themselves, because (1) the three mutant fly strains have different genetic backgrounds and (2) data was collected for only one population of each mutant strain. Although there are known examples of noncircadian genes whose expression is affected by clock mutations, it was decided that it would be more informative to consider effects of the clock mutations only with respect to the subset of 158 strong oscillators. Among this set, 72 genes were found with one or more significant expression changes in the three clock mutant strains (Claridge-Chang, 2001).

    Included in the regulated set are tim (twice independently), vri, to, and Clk, and their patterns agree with previously published observations. The hierarchical clustergram shows four basic patterns of regulation: (type I) increased in per0 and tim01 but decreased in ClkJrk (e.g., vri, CG5798); (type II) decreased in per0 and tim01 but increased in ClkJrk (e.g., Clk, CG15447); (type III) decreased in all three mutants (e.g., Ugt36Bc/CG17932, ea), and (type IV) increased in all three mutants (e.g., Pdh, CG11891). Type I and II match the two known modes of regulation for circadian genes. The behavioral and molecular phenotypes of the per0 and tim01 mutations are almost identical. It may, therefore, be relevant that no circadian genes are found that are significantly upregulated in per0 and significantly downregulated in tim01 or vice versa. Apart from genes that were a priori predicted to have expression patterns of type I (vri, tim, to) or II (Clk), novel genes were found for each of these two expression patterns. The average phases for the type I and type II subclusters are, respectively, ZT12 and ZT7, but there is large variation in phase among the members of each of these subclusters. to is in the type I subcluster and has a phase peak at ZT2, whereas CG15447 is in the type II subcluster and peaks at ZT10. This phenomenon of phase differences among transcripts with a similar response to clock defects has been described previously for type I regulation in the case of to. Here, a similar phenomenon was detected for genes with Clk-like type II regulation. Type III and IV predict a novel and unexpected response to the circadian mutants (Claridge-Chang, 2001).

    The promoter sequences of the set of 158 genes was tested for the presence of known and candidate circadian enhancer motifs. The results suggest that in fact this set is enriched in such elements. For example, the frequency of E boxes in the set of oscillators (42 hits in total) is significantly higher than the frequency in random selections of genes. The significance of this result does, however, depend on the inclusion of well-studied genes (per, tim, vri). Some novel oscillators with remarkable frequencies of 'circadian transcription elements' are loco (1 PDP1 site; 3 W boxes; 8 CRE elements; 3 TER elements); trpl (1 E box; 1 PDP1 site; 9 W boxes; 6 CRE elements; 12 TER elements); Rh5 (5 W boxes; 5 CRE elements; 6 TER elements), and Slob (1 E box; 1 PERR element; 2 PDP1 sites; 6 W boxes; 15 TER elements). It is noteworthy that many robustly cycling genes have no known circadian transcription elements in their promoters or first introns (Claridge-Chang, 2001).

    The set of 158 circadian genes were organized according to annotated or predicted molecular function. Several of these functional classes may provide insights into pathways influencing rhythmic behavior (Claridge-Chang, 2001).

    Synaptic Transmission and Plasticity
    An emerging theory of the function of sleep postulates that it is required for neural plasticity, synaptic maintenance, and remodelling. Behaviorally defined sleep has been identified in the fly, with behavioral recordings in LD indicating increased rest during the dark phase. The assay for circadian expression uncovered a number of genes known to be involved in synaptic function and synaptic plasticity (Claridge-Chang, 2001).

    Three oscillating trancripts encode synaptic vesicle endocytosis factors: ß-adaptin (Bap), AP-1gamma, and syndapin. The first two are adaptors between the budding membrane and clathrin lattice, while syndapin is thought to connect vesicle endocytosis to actin. Clock modulation of the synaptic vesicle pool is consistent with the idea of modulated synaptic function, although it is unclear what effect raising or lowering endocytotic factors would have on synaptic function (Claridge-Chang, 2001).

    The Slob transcript peaks at ZT15 and is downregulated in the ClkJrk mutant, suggesting that CLK acts as an activator of Slob. There is an E box 5.4 kb upstream of the Slob transcriptional start site raising the possibility that CLK acts directly on the Slob promoter (Claridge-Chang, 2001).

    If the cycling transcript can be shown to produce oscillating Slob protein, this could indicate a potent mechanism for rhythmic control of synaptic function, including synaptic plasticity: Slob protein binds the calcium-dependent potassium channel Slowpoke (Slo) and has been shown to increase Slo activity and voltage sensitivity. Slob is in turn bound by a second channel regulator, Leonardo. Hypomorphic mutations of leonardo produce defects in learning, and electrophysiological analyses of the larval neuromuscular junction (NMJ) in these mutants show presynaptic function and plasticity is greatly impaired in these animals. In contrast to Slob, Leonardo is a strong inhibitor of Slo, but requires Slob for this interaction. All three proteins colocalize to the presynaptic bouton at larval NMJs. Thus, Slob may contribute to a switching mechanism that ultimately places Slo channel activity under circadian control (Claridge-Chang, 2001).

    Slo channels are widely expressed in the adult fly head, including the eye, lamina, medulla, central brain, and mushroom bodies, but it is not known which subset of these areas contain oscillating Slob expression. In situ hybridization was performed with larval brains to localize Slob RNA expression. Prominent staining was observed in a restricted region consistent with placement of the developing mushroom body. The staining also corresponds well with that region of the larval brain receiving PDF-rich projections of the circadian pacemaker cells, the lateral neurons. In future studies, it will be important to determine whether presence of the innervating LNs is required for cycling Slob expression (Claridge-Chang, 2001).

    leonardo was initially implicated in presynaptic function by the effect of mutations on learning. Mutations of latheo also cause learning defects, and this protein is also found at larval NMJs. Lowered latheo function has been associated with elevated synaptic transmission and reduced synaptic plasticity. latheo shows cycling expression with peak accumulation at ZT12-15. Rhythmicity was detected in the expression of dunce and Calpain-B genes involved in learning and synaptic long-term potentiation, respectively (Claridge-Chang, 2001).

    Amine Neurotransmitter-Related Functions
    Two serotonin receptor transcripts, 5-HT2 and 5-HT1A, were found to oscillate with phases of ZT15 and ZT18, respectively. Serotonin is known to be involved in a variety of neuronal processes in animals, including synaptic plasticity, clock entrainment, and mating behavior. The 104 serotonergic neurons in the adult CNS have been mapped, but no studies have been done of either 5-HT receptor localization or receptor mutant phenotypes. Neither of these receptors are orthologs of the mammalian 5-HT receptor implicated in photic clock entrainment; this would be represented by theDrosophila 5-HT7 gene. 5-HT1A belongs in a class of receptors that respond to agonists by decreasing cellular cAMP, while 5-HT2 is homologous to mammalian receptors whose main mode of action involves activation of phospholipase C, a function involved in synaptic plasticity (Claridge-Chang, 2001).

    The ebony transcript was found to oscillate, showing a peak of expression around ZT5. ebony oscillation connects with a body of earlier evidence linking ebony to circadian activity rhythms. ebony hypomorphs show severe defects in circadian rhythm including arrhythmicity/aberrant periodicity in the free-running condition, as well as abnormal activity patterns in LD conditions. Ebony is a putative ß-alanyl dopamine synthetase, and hypomorphs show elevated levels of dopamine. Dopamine has been implicated in control of motor behavior, since it induces reflexive locomotion in decapitated flies, and this response is under circadian control. The results suggest that oscillations of ebony contribute to the assembly of rhythmic locomotor behavior. Other evidence points to a role in clock resetting. In addition to impaired entrainment in LD, ebony flies show an abnormal ERG, and ebony is strongly expressed in the lamina and the medulla optic neuropile, a region associated with vision rather than motor control (Claridge-Chang, 2001).

    The Drosophila eye is both a likely target of clock control and partly responsible for photic input to the central pacemaker. Several genes found to oscillate by microarray assay are components of visual processes (Claridge-Chang, 2001).

    Photoreceptor cells contain peripheral clocks, suggesting that visual function may be regulated by the clock. In vertebrates, the synthesis of various visual components is known to be under circadian control. In Drosophila, electroretinogram (ERG) measurements of visual sensitivity reveal a 4-fold cycle in sensitivity, with a minimum at ZT4 and a broad peak around lights off (ZT12). This suggests that some of the fly visual components are clock controlled. However, a previous study of five major phototransduction components found no cycling of either mRNA or protein. In this genome-wide assay, the trpl transcript was found to oscillate with peak expression at ZT11. TRPL is one of two ion channels in the visual transduction pathway, along with TRP, a paralog. TRPL and TRP open in response to a G protein-coupled phosphoinositide cascade that is initiated by the isomerization of rhodopsin by light. Their opening produces the light-sensitive conductance in the photoreceptors. Amorphic mutants of each channel show visual defects, while the double null genotype results in a blind fly. A cycling trpl transcript could contribute to the visual sensitivity cycle: (1) the two phenomena are in the same phase with both sensitivity and trpl expression peaking around lights-off; (2) it is estimated that TRPL contributes about half of the wild-type conductance, allowing for a substantial range of modulation by reducing TRPL function. Another possible role for oscillating TRPL function is connected with circadian entrainment. In addition to being blind, trp/trpl double null mutants show reduced circadian behavioral resetting and less TIM degradation in response to light pulses. It is noted that the established entrainment factor CRY is known to cycle and that interactions of CRY and TIM are essential for light-dependent TIM degradation. The oscillating, clock-related protein VIVID has also been shown to regulate photo-entrainment in Neurospora (Claridge-Chang, 2001).

    The microarray experiments show that two opsin genes are under circadian control: Rh5 and Rh4. The Rh5 mRNA rhythm peaks at ~ZT 21, while the Rh4 array data show a circadian pattern with a peak 4 hr later, at ZT1. Rh5 is a blue-absorbing rhodopsin expressed in a subset of R8 cells at the base of the retina, while Rh4 is a UV-absorbing pigment expressed in apical R7 cells. Rh5 is never expressed in an R8 cell underlying an Rh4-expressing R7 cell, so in this way all ommatidia would contain one cycling rhodopsin. In terms of regulating sensory receptiveness to light, it is unclear why these two opsins should be targets for clock control. The major blue rhodopsin Rh1 does not cycle so it seems unlikely that an oscillation in these two minor pigments would produce overall tuning of the sensitivity of the fly visual system (Claridge-Chang, 2001).

    NinaA is a rhodopsin chaperone and is required to move Rh1 from the endoplasmic reticulum (ER) to the rhabdomeric membrane. ninaA mutants display aberrant accumulation of Rh1 protein in the ER. ninaA mRNA shows cycling expression in fly heads by both array and Northern blot, with peak expression around ZT2. It is tempting to hypothesize that early morning ninaA upregulation would have the effect of releasing a reservoir of Rh1 from the ER, making it available for use in visual transduction. However, a previous assay of NinaA levels in an LD regime revealed no oscillation in protein levels. If true, this would represent a surprising example of a robustly oscillating mRNA producing a constitutive cognate protein. Finally, although its function is still unknown, Photoreceptor dehydrogenase is a robustly oscillating transcript with an extremely high level of expression in the screening pigment cells of the eye (Claridge-Chang, 2001).

    Rhythmic Proteases and Accessory Factors
    Fifteen of the identified oscillatory genes are implicated in protein cleavage. CG7828 and BcDNA:GH02435 (peak phase ZT6 and ZT10, respectively) mediate ubiquitination of protein substrates, thus targeting them for degradation. Conversely, CG5798 and CG7288 cycle with a peak at respectively, ZT14 and ZT16, and each produces a ubiquitin specific protease (Ubp; cleaves ubiquitin from ubiquitin-protein conjugates) that may act to prevent the degradation of specific protein targets. Two metalloprotease genes, two aminopeptidase genes, and five serine peptidases show circadian oscillation. Four of the five serine peptidase genes cycle with a peak phase between ZT4-7. This profusion of oscillating proteases suggests that circadian proteolysis may represent a broad mechanism of clock control, both of clock components themselves, as well as output factors (Claridge-Chang, 2001).

    While circadian transcriptional mechanisms are relatively well understood, less is known about posttranslational mechanisms of circadian regulation. Proteases are known to be involved in circadian control of the degradation of some central clock components, and the clock proteins PER, TIM, CLK, CRY, and VRI are all known to undergo daily cycles of protein accumulation. Degradation of TIM is responsible for photic resetting of the Drosophila clock. This is thought to be mediated by interaction with CRY, followed by ubiquitination and proteasome-dependent loss of TIM. Nothing is known about the factors mediating TIM degradation in the dark, yet patterns of CG5798 expression may be of interest in this regard as this gene encodes a cycling Ubp whose peak expression (ZT14) immediately precedes an interval of rapid TIM accumulation in pacemaker cells (Claridge-Chang, 2001).

    A likely clock-related target of one or more proteases is the neuropeptide PDF, whose regulation may be crucial to linking the clock to behavior. While pdf RNA is expressed constitutively, the peptide accumulates rhythmically under indirect control of the clock gene vri. This mechanism has not been further explored, but a clear possibility is that a PDF propeptide is cleaved rhythmically, allowing cyclical release of active PDF. Of the 15 cycling proteases suggested by this study, CG4723 may be of special interest due to its inclusion in a class of proteases known to cleave neuropeptides. The phase of CG4723 expression, ZT4, also coincides with that of PDF immunoreactivity in the Drosophila head (Claridge-Chang, 2001).

    Detoxification and Oxidative Stress
    One hypothesis of sleep characterizes it as a cellular detoxification and repair process. Twelve genes were found that have a predicted role in detoxification. Three additional redox enzymes may also participate in this process. Toxins are initially modified into reactive species by reductases/dehydrogenases and cytochrome P450 molecules. Then glutathione-S-transferases (GSTs) and UDP-glycosyl transferases add polar groups to the substrates to render them hydrophilic for elimination by secretion. Four results are noteworthy: (1) following this pathway, the genes for three circadian dehydrogenases: Pdh, CG10593, and CG12116, were found.; (2) both morning and night cytochrome P450 genes (Cyp6a21 and Cyp305a1) were found to peak early in the day (ZT0 and ZT5), whereas Cyp18a1 and Cyp4d21 peak at approximately the same time late at night (ZT18 and ZT19); (3) while CG17524 is the only GST gene found in the core set, it was noticed that two other members of the GST type III gene cluster on chromosome 2R show 24 hr periodicity (CG17523; CG17527) and (4) UDP-glycosyl transferase genes are represented in the circadian set by Ugt35a, Ugt35b, and Ugt36Bc. Of these, Ugt35b encodes an antennal specific transcript with a potential role in olfaction, whereas Ugt35a is expressed more uniformly (Claridge-Chang, 2001).

    CG13848 is an alpha-tocopherol transfer protein (alpha-TTP) that is strongly expressed in certain basal cells of the eye, showing peak expression around dawn. Humans with mutations in the homologous alpha-TTP show neurodegenerative ataxia that is associated with a deficiency in alpha-tocopherol (vitamin E) incorporation into lipoprotein particles. Vitamin E acts as an antioxidant, and it is thought that this activity allows it to protect neurons from damage by free radicals. Also from human studies, it is known that photoreceptor cells are particularly susceptible to oxidative damage due to high levels of polyunsaturated fatty acids in the photoreceptor membrane, and their exposure to visible light. Thus, it is proposed that the dawn phase of CG13848 represents an upregulation of alpha-TTP for increased daytime transfer of photoprotective vitamin E into the photoreceptor membrane. Also in the circadian set, Catalase (encoded by Cat) and a thioredoxin (encoded by CG8993) are both involved in neutralizing reactive oxygen species (Claridge-Chang, 2001).

    Different aspects of metabolism are represented among the selected set of oscillating transcripts: lipid metabolism (five genes), amino acid metabolism (three), carbohydrate metabolism (three), and glycoprotein biosynthesis (two). Intriguingly, Zw encodes glucose-6-phosphate 1-dehydrogenase of the pentose-phosphate pathway (PPP), while CG10611 encodes fructose-bisphosphatase in gluconeogenesis. The two pathways have antagonizing roles in glucose metabolism; both genes are key control points in their respective pathway and are maximally expressed in opposite phases. Zw transcripts are at their zenith just before dusk (ZT11), whereas CG10611 transcripts peak at dawn (ZT0). Thus, the antiphase oscillation of these two genes may produce daily alternation between glucose anabolism and catabolism. Zw and CG10611 transcript levels also respond in opposite fashion to clock defects: Zw levels are significantly decreased in per0 and tim01 mutants, whereas, CG10611 is significantly upregulated in tim01 (Claridge-Chang, 2001).

    In more direct relation to the clock itself, Zw is the first committed step in the PPP and generally thought to control flux through this pathway. The PPP is the major pathway of NAD (or NADP) conversion to NAD(P)H. It was recently shown that NAD(P)H can bind homologs of CLK and CYC, promoting their dimerization and DNA binding. Maximal Zw expression at ZT11 -- and therefore presumably NAD(P)H production via the PPP -- is coincident with maximal per and tim transcription by CLK/CYC. This information is consistent with Zw participating in a NAD(P)H-mediated autoregulatory loop of the clockworks (Claridge-Chang, 2001).

    Nucleic Acid Metabolism
    A subset of 15 genes involved in nucleic acid metabolism were found. This includes five genes encoding specific RNA polymerase II transcription factors. Four of these encode parts of the circadian clock itself (per, tim, vri, and Clk), whereas the fifth one, apterous (ap), generates a homeobox transcription factor with a role in neurogenesis and the expression of neuropeptides. Taf30alpha2 is a subunit of the general transcription factor TFIID, while moira (mor) is part of the SWI-SNF chromatin-remodeling complex. One splicing factor gene (DebB) and one DNA repair gene (CG4049) are also found among this set of oscillating transcripts (Claridge-Chang, 2001).

    Circadian genes with a role in the cytoskeleton include those encoding two actin binding proteins (Chd64, CG11605), a troponin C (TpnC47D), and two septins (Sep-1 and CG9699). Chd64 (phase peak ZT4) and TpnC47D (phase peak ZT7) function specifically in muscle contraction. Another Drosophila Troponin C, TpnC73F, is found to peak at ZT6 (Claridge-Chang, 2001).

    In conclusion, a set of 158 genes expressed with a robust circadian rhythm in the adult Drosophila head was found by microarray screening. These encompass a wide variety of molecular functions, and expression patterns represented essentially all circadian phases. A larger set of genes was identified (393 entries; 293 entries after secondary filters), and the statistical approach again indicated significant circadian rhythmicity for these, but they were characterized by somewhat less robust oscillations than those of the smaller set. Independent verifications indicated substantial enrichment for cycling gene expression in this larger set and beyond. 532 genes passed secondary filters for 24 hr autocorrelation, noise, and the range-to-noise measure. From the frequency of Northern-verified oscillations detected in this larger pool of candidate genes, it is believed that the total complement of circadian genes would include ~400-500 in the adult head (Claridge-Chang, 2001).

    There are important factors that might lead to an underestimation of the total complement of circadian genes. The approach that was used would favor genes that are homogeneously expressed in the head. If the same gene is expressed with varied phases in different head tissues, this will lessen the robustness of the apparent oscillation and phase. Similarly, if only a restricted portion of the head generates the cycling gene pattern, but constitutive expression is found elsewhere in the head, amplitude of the signal will be diminished. Differences of this sort might be expected in cases where a cycling gene product produces a limited physiological effect. Regulation of this type might be expected in the antennae, where, for example, electrophysiological responses to odorants vary with a circadian rhythm. It should also be stressed that only the fully differentiated adult head has been sampled. If transient patterns of circadian expression occur during development, these would go unnoticed in the experiment. Given the plethora of tissues housing autonomous circadian clocks, an expanded list of rhythmic genes would probably be derived from any related sampling of the body (e.g., wings, legs, excretory, and digestive tissues), especially as this tissue autonomy may reflect a requirement for tissue-specific pathways of circadian control that lie downstream from a largely uniform clock mechanism (Claridge-Chang, 2001).

    How will the many patterns of cycling gene expression be further explored? Molecular tools that reveal the importance of oscillating gene activity have already been applied to a study of several clock genes in Drosophila. In these studies, oscillating patterns of a target gene's expression have been replaced with constitutive activity. Central questions related to vri, per, and tim function have each been explored in this manner. The present study allows an expansion of such work to address the molecular connections between individual behaviors and circadian clocks (Claridge-Chang, 2001).

    Control of daily transcript oscillations in Drosophila by light and the circadian clock

    The transcriptional circuits of circadian clocks control physiological and behavioral rhythms. Light may affect such overt rhythms in two ways: (1) by entraining the clock circuits and (2) via clock-independent molecular pathways. In this study the relationship between autonomous transcript oscillations and light-driven transcript responses were examined. Transcript profiles of wild-type and arrhythmic mutant Drosophila were recorded both in the presence of an environmental photocycle and in constant darkness. Systematic autonomous oscillations in the 12- to 48-h period range were detectable only in wild-type flies and occurred preferentially at the circadian period length. However, an extensive program of light-driven expression was confirmed in arrhythmic mutant flies. Many light-responsive transcripts are preferentially expressed in the compound eyes and the phospholipase C component of phototransduction, NORPA (no receptor potential), is required for their light-dependent regulation. Although there is evidence for the existence of multiple molecular clock circuits in cyanobacteria, protists, plants, and fungi, Drosophila appears to possess only one such system. The sustained photic expression responses identified here are partially coupled to the circadian clock and may reflect a mechanism for flies to modulate functions such as visual sensitivity and synaptic transmission in response to seasonal changes in photoperiod (Wijnen, 2006).

    In recent years, five different sets of circadian transcripts have been proposed for the Drosophila head. Unfortunately, the overlap between these transcript sets is very poor (seven transcripts), and it falsely excludes numerous confirmed circadian transcript oscillations. These recent genome-wide surveys for rhythmic transcription have defined groups of circadian transcripts based on empirical ranking and filtering approaches, often using necessarily arbitrary cut-offs. To complement these studies a method was developed for examining periodic expression at the systems level, allowing pursuit of a number of new investigations. This new strategy enabled description of the programs of circadian and light-driven transcription in the adult fly head. Because this method emphasizes uniformity in period length and peak phase while tolerating inter-experimental variability in amplitude, it is particularly successful at measuring oscillatory trends across different independent experiments. Integrative analysis of all available microarray time-series data allowed detection and ranking of oscillatory transcript profiles with improved resolution and revealed a circadian expression program that is much more substantial than the apparent consensus (or lack thereof) between different published studies indicates. Some of the best described and strongest circadian oscillations (per, Clk, Pdp1, cry, and to) were missed in one or more of the previously published studies, but all of these rank high in the current integrative analysis. Although there are relatively few genes (~50) that show the same level of circadian regulation as the oscillating components in the core clock circuits (per, tim, Clk, cry, vri, and Pdp1), the results provide evidence for a substantially broader circadian expression program downstream of the core oscillator. This suggests that the circadian clock is responsible for both the purely circadian expression patterns of a limited set of genes and the partial circadian regulation of a much greater group (Wijnen, 2006).

    Whereas many of the genes composing the Drosophila clock are expressed with a circadian rhythm in wild-type flies, all known clock gene oscillations cease if just one of them is lost by mutation. It was reasoned that all of the circadian oscillations in gene expression that were identified in this study should stop in tim01 mutants if these were truly devoid of a circadian clock. Alternatively, rhythmicity could theoretically persist in a subset of the genes if their expression depended on a parallel, novel circadian clock. The distribution analyses allowed addressing of these two alternative possibilities. No alternative systems of oscillatory expression are detectable for the 12-48-h range of period lengths. In the absence of tim-dependent clock circuits, no circadian patterns of gene expression were detected. This latter result, from microarray and Northern analyses, is in agreement with earlier observations, with limited sampling of individual circadian transcripts. Moreover, the absence of detectable molecular circadian rhythms fits well with the abolition of circadian eclosion and locomotor rhythms in tim01 flies. Thus, Drosophila appears to possess only one, tim-dependent, circadian clock. This observation contrasts with results from cyanobacteria, protists, fungi, and plants that suggest the presence of multiple oscillators, sometimes even in the same cell. Although there is no compelling evidence supporting the existence of alternative circadian clocks in Drosophila that are not entrainable to light or independent from transcriptional rhythms, this study does not disprove these possibilities. The results complement and extend previous microarray and differential display analyses using different arrhythmic mutants (per0 or Clkjrk) in which few or apparently no daily transcript oscillations persisted in the mutant context (Wijnen, 2006).

    Comparative analysis of data collected from wild-type and arrhythmic mutant flies in the presence or absence of an environmental photocycle allowed identification of a program of light-driven regulation. The tim01 mutant flies used for these experiments do not just have a defective circadian clock, but because TIM degradation is a major mechanism of clock re-setting, they have also lost the main photic input pathway that entrains the clock circuits to light. In a wild-type context, light can directly entrain clock-bearing tissues in a cell-autonomous manner by activating the circadian photoreceptor CRY, or it can entrain the pacemaker neurons in the brain via phototransduction in the visual organs. TIM is the target for CRY's effect on the clock circuits, and it may also play a role in mediating entrainment via the visual organs. In spite of their defective clock circuits and circadian entrainment pathways, tim01 mutant flies retain an extensive set of daily transcript oscillations in the presence of an environmental photocycle. By comparing the light-driven expression signature that was found for tim01 with the microarray analysis for per0 LD and with confirmatory northern analyses, it was established that many light-driven transcripts show the same expression profiles in per0 and tim01 arrhythmic mutants. Moreover, the light-driven expression response found in a combined per0 and tim01 LD microarray dataset is comparable in size to the clock-dependent circadian expression program (Wijnen, 2006).

    Light-regulated genes fall into two classes, a clock-independent class, and a group of genes that are also clock-controlled. That there are clock-independent patterns of light-regulated gene expression suggests that coordinate clock- and light-control can be disadvantageous in some circumstances. For example, although the clock carries phase information about the photocycle, it may not be able to carry information about day length and sunlight intensity, and some photoprotective functions might be better linked to acute light activation so that they are delivered only when needed. Such a case might be made for ultraviolet-induced melanogenesis in human skin. In contrast, it is suspected that many genes controlled by light and the clock contribute to processes that require both daily anticipation of changes in light and light responsiveness (Wijnen, 2006).

    A survey of published expression studies for the selection of light-regulated genes indicates that many of them are prominently expressed in the adult compound eyes (trpl, CdsA, Pkc53E, dlg1, Slob, CG17352, CG5798, CG7077, CdsA, dlg1, Slob, and trpl). Indeed, comparative transcript profiling studies of wild-type and eya2 mutant flies predict expression in the adult compound eyes for 22 of the 27 light-dependent transcripts (Wijnen, 2006).

    Two of the confirmed light-regulated transcripts (trpl and CdsA) encode known regulators of phototransduction. Daily oscillations in the transcript levels have been observed for trpl, which encodes a light-activated calcium channel. Although some effects on light-activated conductance have been observed in a trpl null mutant, the major light-dependent cation channel in Drosophila appears to be encoded by its homolog trp (transient receptor potential). Instead, the TRPL protein may have a specific function in phototransduction during extended illuminations and for adaptation of the light response to dim background light. The effect of TRPL on long-term adaptation is thought to be mediated via light-dependent subcellular translocation of TRPL protein, resulting in a preferred localization at the photoreceptor membranes in the dark and in the cell-bodies in the light. Experiments in the blowfly Calliphora vicina indicate that this translocation does not require regulation at the transcript level, but it is possible that the daily evening peaks of the trpl transcript in Drosophila facilitate efficient accumulation of TRPL protein at the rhabdomeres around dusk. Daily fluctuations are also exhibited by the transcript for CdsA (CDP diglyceride synthetase). The CDSA protein is localized to photoreceptor neurons and catalyzes the synthesis of CDP-diacyl glycerol from phosphatidic acid and CTP071. This enzymatic function helps generate the signaling compound phosphatidyl inositol 4,5-bisphosphate, which is consumed during phototransduction by the phospholipase C NORPA. Studies of CdsA loss-of-function and gain-of-function mutants indicate that by controlling availability of phosphatidyl inositol 4,5-bisphosphate, CDSA expression levels affect the gain of the phototransduction response. Periodic variation of CdsA expression under influence of the environmental photocycle could, therefore, be hypothesized to promote daily variations in visual sensitivity (Wijnen, 2006).

    Two other light-driven transcripts, dlg1 and Slob, are associated with the regulation of synaptic transmission. The dlg1 (discs large 1) gene has roles in control of cell growth and differentiation as well as synaptic function. DLG1 spatial expression pattern includes synaptic sites in the adult brain and the outer membrane of photoreceptors, where it localizes Sh (Shaker) potassium channels (Wijnen, 2006).

    Slob is negatively regulated by light in a clock-independent manner in addition to being one of the most robustly oscillating circadian transcripts in the adult head. The clock-dependent and light-dependent fluctuations that were uncovered for the Slob transcript are reflected in the SLOB protein levels observed in photoreceptor cells and whole heads. A number of findings point to a possible role for SLOB in mediating overt behavioral rhythms. SLOB protein is thought to bind the SLO and EAG potassium channels, and can directly enhance SLO activity, as well as mediate the inhibitory effect of 14-3-3ζ on SLO. slo mutants have altered potassium channel currents and reported defects in flight, male courtship, and circadian locomotor behavior, whereas mutations of eag display hyperactivity, and affect potassium currents and courtship behavior (Wijnen, 2006).

    As mentioned above, circadian rhythms in adult Drosophila can be entrained to a LD cycle via either opsin-mediated photoreception in the light-sensing organs (compound eyes, ocelli, and eyelets) or cell-autonomous activation of the circadian blue-light photoreceptor CRY. Interestingly, the contribution of visual photo-transduction to circadian photo-entrainment is apparently restricted to a few pacemaker neurons in the brain, a situation reminiscent of photo-entrainment of the clock circuits in the mammalian brain via the retina and the retino-hypothalamic tract. In contrast, Drosophila CRY contributes to photo-entrainment in many more clock-bearing tissues, including the visual organs. CRY mediates the light-dependent degradation of TIM, which in turn affects CLK/CYC transcriptional activity in a manner that depends on the phase of the circadian cycle (Wijnen, 2006).

    The light-driven transcript responses identified in this study resemble circadian responses in amplitude and duration in the context of a photocycle, and are found for a number of genes with a verified circadian expression profile. It was, therefore, asked whether these light-driven transcript responses depend on the same light sensors as the circadian system. For the most part light-driven regulation was found not to require CRY. Given TIM's status as a target for CRY-mediated light responses, it is perhaps not surprising that light-driven expression responses that do not require TIM function also persist in the absence of CRY. There is one interesting exception to this rule: The light-mediated repression of the Slob transcript apparently requires CRY, but not TIM. If this observation indeed represents a previously unappreciated function for CRY, it may share this role with the phospholipase C enzyme NORPA, since norpA mutants similarly affect the Slob transcript (Wijnen, 2006).

    In contrast with CRY, it was found that NORPA phototransduction mediates many if not all of the other clock-independent light responses identified in this study. Based on the overlapping expression of both NORPA and its target transcripts in the adult compound eyes and NORPA's well-documented role in phototransduction, the simplest interpretation of these observations would be that light-driven expression responses are mediated by visual phototransduction. Nevertheless, NORPA is known to be expressed outside of the visual organs, and it has been reported to affect functions unrelated to phototransduction, such as olfaction and temperature-controlled clock gene oscillations. Additionally, norpA loss-of-function mutants show a number of defects in circadian locomotor behavior. Their activity profiles reveal an advanced evening activity peak under LD conditions and a shortened intrinsic period length under DD conditions, and they are slow to adjust their behavior to shifting cycles of light and dark. One possible interpretation of these observations is that NORPA plays a role in seasonal photoperiodic control of locomotor behavior. The norpA mutant phenotype partially mimics the effect of a shortened photoperiod, which also leads to advanced evening activity peaks and shortened period lengths. Recent studies provide further evidence connecting norpA to seasonal control of daily locomotor activity patterns. norpA mutants show abnormally high levels of splicing in the 3' untranslated region of per mRNA. Increased splicing of per transcripts at this site has been shown to contribute to the advanced accumulation of PER protein and the advanced timing of evening locomotor activity that is observed for shorter photoperiods and lower temperatures. Thus, NORPA's effect on splicing of per may be an important determinant of the 'short day' locomotor behavior phenotype of norpA mutants. The sustained photic expression responses that are identified here may reflect yet another mechanism for flies to translate a seasonal environmental signal (photoperiod) into a set of molecular signals. Photoperiodic control of transcripts associated with functions in visual sensitivity (trpl and CdsA) and synaptic transmission (Slob and dlg1) may be relevant to adaptive responses in the visual system and the brain. NORPA's involvement in both regulating per splicing and mediating photoresponses at the transcript level raises questions as to if and how these two molecular functions are connected. One possibility is that both reflect NORPA-dependent selective regulation of mRNA stability that takes place in the compound eyes (and perhaps also the brain). Whether or not NORPA's function in circadian locomotor behavior involves some of the light-dependent expression responses that have been identified could be examined by targeted misexpression studies. The subset of transcripts that have been independently confirmed to exhibit both NORPA-dependent light responses and strong clock-dependent circadian regulation might be particularly relevant to these experiments (Wijnen, 2006).

    This paper has reported a new strategy for analyzing oscillatory patterns in microarrray data that allowed answer general questions about oscillatory gene systems in the fly head. By applying this strategy to 17 d of data, it was conclusively demonstrated that there are more than a hundred circadian transcript oscillations in the fly head. Additionally, in a search for rhythmic gene activity over a wide range of periods (from 12 to 48 h), it was established that 24-h periodicity constitutes the only broad program of transcriptional oscillation. It was further found that the tim-dependent clock is the sole transcriptional circadian clock in Drosophila. Thus, the fly appears to differ from cyanobacteria, protists, plants, and fungi, which are thought to possess multiple circadian clocks. Lastly, a novel, light-regulated system of gene regulation was found in Drosophila that is largely dependent on norpA-mediated phototransduction. This system regulates about the same number of genes as the clock, including a number of circadian genes. This study defines three types of transcripts that oscillate in wild-type flies: those from purely clock-regulated genes, those that are purely photocycle-regulated, and those expressed by genes that respond to both inputs (Wijnen, 2006).

    The HisCl1 histamine receptor acts in photoreceptors to synchronize Drosophila behavioral rhythms with light-dark cycles

    In Drosophila, the clock that controls rest-activity rhythms synchronizes with light-dark cycles through either the blue-light sensitive cryptochrome (Cry) located in most clock neurons, or rhodopsin-expressing histaminergic photoreceptors. This study shows that, in the absence of Cry, each of the two histamine receptors Ort and HisCl1 contribute to entrain the clock whereas no entrainment occurs in the absence of the two receptors. In contrast to Ort, HisCl1 does not restore entrainment when expressed in the optic lobe interneurons. Indeed, HisCl1 is expressed in wild-type photoreceptors and entrainment is strongly impaired in flies with photoreceptors mutant for HisCl1. Rescuing HisCl1 expression in the Rh6-expressing photoreceptors restores entrainment but it does not in other photoreceptors, which send histaminergic inputs to Rh6-expressing photoreceptors. These results thus show that Rh6-expressing neurons contribute to circadian entrainment as both photoreceptors and interneurons, recalling the dual function of melanopsin-expressing ganglion cells in the mammalian retina (Alejevski, 2019).

    The Drosophila sleep–wake rhythms are controlled by a brain circadian clock that includes about 150 clock neurons. Light synchronizes the clock neuronal network through cell-autonomous and non-cell-autonomous light input pathways. Cry is a blue-light sensitive photoreceptor protein that is expressed in most clock neurons. In the absence of Cry, flies do not phase-shift their behavioral rhythms in response to a short light pulse but still synchronize to light–dark (LD) cycles. Only flies devoid of both Cry and rhodopsin-expressing photoreceptors fail to entrain to LD cycles. Six different rhodopsins (Rhs) have been characterized in the Drosophila photoreceptive structures, which include the compound eye, the Hofbauer-Buchner (H-B) eyelet, and ocelli. The compound eye strongly contributes to circadian photoreception, whereas a modest contribution appears to be brought by the H-B eyelet and the ocelli. A circadian function has been recently associated with the yet poorly characterized rhodopsin 7, although its exact contribution and localization in the brain and/or the eye remains controversial. In addition to entrainment, the visual system controls other features of the clock neuron network by conveying light information to either promote or inhibit the behavioral output of specific clock neuron subsets (Alejevski, 2019).

    The compound eye includes about 800-unit eyes (ommatidia), each of which contains eight photoreceptors. The six Rh1-expressing outer photoreceptors (R1–6) are involved in motion detection and project to the lamina neuropile of the optic lobe. The two inner photoreceptors (R7–8) are important for color detection and project to the medulla. They express four different rhodopsins and thus define two types of ommatidia: 'pale' (p) ommatidia (30%) include a Rh3-expressing R7 and a Rh5-expressing R8, whereas 'yellow' (y) ommatidia (70%) include a Rh4-expressing R7 and a Rh6-expressing R8. Each extra-retinal H-B eyelet contains four Rh6-expressing photoreceptors that project to the accessory medulla, in the vicinity of key pacemaker neurons, the ventral lateral neurons (LNvs) that produce the pigment-dispersing factor (PDF) neuropeptide9,20–24. Each of the three ocelli contains about 80 photoreceptors that express Rh225. The Drosophila rhodopsins cover a wide range of wavelengths from 300 nm to 600 nm18,19, with only Rh1 and Rh6 being sensitive to red light (Alejevski, 2019).

    Rhodopsin-dependent circadian entrainment involves two downstream signaling pathways, the canonical one that relies on the phospholipase C encoded by the no receptor potential A gene (norpA)2 or an unknown pathway that does not contribute in very low light levels. All but Rh2- and Rh5- expressing photoreceptors support synchronization in very low light, and at least Rh1, Rh5, and Rh6 can signal through the NorpA-independent pathway. Photoreceptors of the compound eye are histaminergic but the H-B eyelet expresses both histamine and acetylcholine. Although the two neurotransmitters might contribute to circadian entrainment, flies devoid of Cry and histidine decarboxylase do not synchronize their rest–activity rhythms with LD cycles. This suggests that besides Cry, there is no histamine-independent pathway to entrain the clock (Alejevski, 2019).

    Two genes encoding histamine-gated chloride channels, ora transientless (ort) and Histamine-gated chloride channel subunit 1 (HisCl1), have been identified in Drosophila. The ort-null mutants are visually blind and their electroretinograms have no ON and OFF transients. In contrast, HisCl1 mutants show increased OFF transients, whereas slower responses were observed in the postsynaptic laminar monopolar cells. Based on transcriptional reporters, ort expression in the optic lobes was observed in neurons of both the lamina and medulla/lobula neuropils. Based on reporter gene expression, HisCl1 was localized in glial cells of the lamina. However, recent work reported expression in photoreceptors, in particular in the R7 and R8 inner photoreceptor subtypes. Indeed, Ort and HisCl1 support color opponency between the two subtypes of 'inner' photoreceptors, the ultraviolet (UV)-sensitive R7 and non-UV-sensitive R8, with HisCl1 and Ort mediating direct and indirect inhibition, respectively. The histaminergic pathways that are involved in circadian entrainment are unknown and are the subject of the present study. The results show that both Ort and HisCl1 define two different pathways for circadian entrainment. Whereas Ort contributes through its expression in the interneurons of the optic lobe, HisCl1 mostly contributes through its expression in the Rh6-expressing retinal photoreceptors. The work thus reveals that Rh6-expressing neurons contribute to light-mediated entrainment as both photoreceptors and interneurons (Alejevski, 2019).

    This work reveals that the Cryptochrome-independent entrainment of rest–activity rhythms relies on distinct pathways that are contributed by the two histamine receptors Ort and HisCl1. Whereas Ort mediates circadian entrainment through the optic lobe interneurons that are involved in visual functions, HisCl1 defines a new photoreceptive pathway through Rh6-expressing photoreceptors. Although both receptors mediate synchronization with a shifted LD cycle, it seems likely that the two pathways will show differences in specific light conditions. It was not possible to rescue Ort function with HisCl1 expression in the ort-expressing cells, whereas the Ort could replace HisCl1 in Rh6 photoreceptors. It is possible that HisCl1 has a lower affinity for histamine with Rh6 cells receiving more neurotransmitter than optic lobe interneurons. Alternatively, interneurons could sufficiently differ from photoreceptors for their physiology or specific receptor-interacting protein content, preventing HisCl1 from working efficiently. HisCl1 downregulation in Rh6 cells slows down synchronization and flies with HisCl1134 mutant eyes synchronize very poorly with advanced LD cycles, and fail to synchronize with delays. It cannot be excluded that non-photoreceptor cells contribute to HisCl1-dependent entrainment but other pathways appear to have a modest contribution if any (Alejevski, 2019).

    HisCl1 is expressed in the H-B eyelet, which could thus contribute to this synchronization pathway. However, the cell-killing experiments indicate that H-B eyelet is not required for HisCl1-mediated synchronization through Rh6 cells. In the recently described color opponency mechanism, retinal R7 cells inhibit R8 and vice versa through HisCl1 expression in the photoreceptors (Schnaitmann, 2018). It is supposed that HisCl1-dependent clock synchronization is also mediated by the hyperpolarization of Rh6-expressing cells. How this hyperpolarization interacts with the light-induced depolarization in Rh6 photoreceptors to result in a synchronization message to the clock neurons remains to be understood. Since only Rh6-expressing R8 and not the other inner photoreceptors contribute to this circadian photoreception pathway, Rh6 cells might have specific connections with downstream interneurons. Such specificity has been described for color vision where each of the four inner photoreceptor subtypes connects to a different type of TmY interneuron in the Medulla. This study shows that HisCl1 expression in Rh6 cells supports synchronization with red light, in the absence of Rh1, indicating that an intra-Rh6-photoreceptor circuit is sufficient. This indicates that Rh6-expressing R8 photoreceptors play a dual photoreceptor/interneuron role in this pathway (Model for the retinal input pathways to the brain clock). Whether the same individual cells have the two roles is unknown, although the HisCl1-dependent color opponency mechanism suggests that it could be the case. It is also unclear whether all Rh6-expressing R8 photoreceptors or only a fraction of them contribute to circadian synchronization. The results imply that, in addition to histaminergic neurotransmission, Rh6-expressing photoreceptors can talk to downstream interneurons through histamine-independent neurotransmission. A recent transcriptomics study indeed revealed the expression of cholinergic markers in R7 and R8 cells, supporting cholinergic transmission in the inner photoreceptors, in addition to histaminergic transmission (Alejevski, 2019).

    The data indicate that histaminergic inputs from both outer and inner photoreceptors converge to Rh6 cells to contribute to circadian entrainment. It is possible that some of these inputs rely on Rh7, which seems to be expressed in Rh6-expressing photoreceptors, according to transcriptional reporter data. Putative connections between photoreceptors have been described in Drosophila and other insects. How R1–6 photoreceptors might be connected to Rh6-expressing R8 cells remains difficult to understand, but a few putative contacts between presynaptic outer cells and postsynaptic inner cells have been observed in Musca. The intra-retinal functional connectivity that this study reports in Drosophila is reminiscent to the circuit logic of circadian entrainment in the mammalian retina, where intrinsically photoreceptive retinal ganglion cells express the melanopsin photopigment in addition to receiving inputs from rods and cones. Interestingly, melanopsin appears to share light-sensing properties with the rhabdomeric photoreceptors of invertebrates. It has been shown that the mammalian circadian clock can synchronize with day–night cycles by tracking light color changes in addition to light intensity changes. It will be interesting to investigate the possible contribution of the dual function of Rh6-expressing photoreceptors to integrating different color cues into the retinal information that is sent to the clock (Alejevski, 2019).

    Drosophila CLOCK target gene characterization: implications for circadian tissue-specific gene expression

    Clock (Clk) is a master transcriptional regulator of the circadian clock in Drosophila. To identify Clk direct target genes and address circadian transcriptional regulation in Drosophila, chromatin immunoprecipitation (ChIP) tiling array assays (ChIP-chip) were performed with a number of circadian proteins. Clk binding cycles on at least 800 sites with maximal binding in the early night. The Clk partner protein Cycle (Cyc) is on most of these sites. The Clk/Cyc heterodimer is joined 4-6 h later by the transcriptional repressor Period (Per), indicating that the majority of Clk targets are regulated similarly to core circadian genes. About 30% of target genes also show cycling RNA polymerase II (Pol II) binding. Many of these generate cycling RNAs despite not being documented in prior RNA cycling studies. This is due in part to different RNA isoforms and to fly head tissue heterogeneity. Clk has specific targets in different tissues, implying that important Clk partner proteins and/or mechanisms contribute to gene-specific and tissue-specific regulation (Abruzzi, 2011).

    Previous circadian models in Drosophila suggested a transcriptional cascade in which Clk directly controls a limited number of genes, including core clock genes, which then drive the oscillating expression of many different output genes. The results of this study indicate that Clk directly regulates not only the five core clock genes (i.e., pdp1, vri, tim, per, and cwo), but also many output genes, including ~60 additional transcription factors. Some of these are tissue-specific; e.g., lim1 and crp. In addition, Clk direct target gene regulation may impact timekeeping in yet unforeseen ways. For example, Clk, Per, and Cyc bind to three of the four known circadian kinases; i.e., dbt, nmo, and sgg. Although none of these mRNAs have been previously reported to cycle, both dbt and sgg have cycling Pol II, and dbt shows weak oscillations via qRT-PCR. nmo expression is enriched in circadian neurons and has been shown to cycle in l-LNvs. The data, taken together, indicate that this simple ChIP-chip strategy has uncovered important relationships and suggest that the transcriptional regulation of some of these new target genes warrants further investigation (Abruzzi, 2011).

    Most of the 1500 Clk direct target genes are also bound by two other circadian transcription factors: Cyc and Per. Because a previous study showed that there is a progressive, ordered recruitment of Clk, Pol II, and Per on per and tim (Menet, 2010), the same basic mechanism is conserved on most Clk direct targets. Clk binding increases in late morning and gives rise to an increase in Pol II signal where detectable (ZT6-ZT10). Clk binding is maximal in the early night (ZT14), and both Clk binding and Pol II occupancy decrease rapidly after the repressor Per is bound to chromatin 4-6 h later, at ZT18. Interestingly, Per binds to nearly all Clk direct targets at the identical Clk/Cyc locations, suggesting Per recruitment via protein-protein interactions (Abruzzi, 2011).

    The identical binding sites for Clk, Cyc, and Per suggest that binding is not background binding or 'sterile' binding with no functional consequence. This is because three components of the circadian transcription machinery are present with proper temporal regulation. Pol II cycling on ~30% of cycling Clk targets further supports this interpretation. The Pol II signal is maximal from mid- to late morning (ZT6-ZT10), which slightly anticipates the maximal transcription times of core circadian genes like per and tim. Most Pol II signals are promoter-proximal and may reflect poised Pol II complexes often found on genes that respond quickly to environmental stimuli (Abruzzi, 2011).

    To address RNA cycling, ten direct target genes with Pol II cycling were examined. Eight of these genes show oscillating mRNA with >1.5-fold amplitude, suggesting that oscillating Pol II indeed reflects cycling transcription. Because this assay may underestimate cycling transcription due to tissue heterogeneity (i.e., masking by noncycling gene expression elsewhere in the head), ~30% is a minimal estimate of Clk direct targets with cyclical mRNA (Abruzzi, 2011).

    Interestingly, previous microarray studies did not detect many of these genes. One possibility is that the alternative start sites that characterize 55% of Clk direct targets are not detectable on microarrays; e.g., moe and mnt. However, several mRNAs that cycle robustly by qRT-PCR are not isoform-specific and are also not consistently identified in microarray studies. A second possibility is that the relatively low cycling amplitude of many target genes -- twofold or less, compared with the much greater amplitudes of core clock genes such as tim, per, and pdp1, assayed in parallel -- may be more difficult to detect on microarrays (Abruzzi, 2011).

    Low-amplitude cycling may result from relatively stable mRNA, which will dampen the amplitude of cycling transcription. Alternatively, or in addition, low-amplitude cycling may reflect cycling in one head location and noncycling elsewhere within the head, which will dampen head RNA cycling amplitude. This is likely for many eye-specific Clk targets, which appear expressed elsewhere in the head via a Clk-independent mechanism (Abruzzi, 2011).

    A third and arguably more interesting explanation for low-amplitude cycling is that Clk binds on promoters with other transcription factors within single tissues. These could include chromatin modifiers and would function together with Clk in a gene- and tissue-specific fashion. For example, a gene could be constitutively expressed at a basal level by one transcription factor, with temporal Clk binding causing a modest boost to transcription. For example, gol is a Clk target exclusively in the eye, and gol mRNA cycles with a fourfold amplitude. Rather than cycling from 'OFF' (no or very low mRNA levels) to 'ON,' however, gol mRNA levels are quite high even at the trough or lowest time points. This suggests that gol cycles from a substantial basal level in the late night and daytime to an even higher level of expression in the evening and early night. Since mRNA levels decrease by >10-fold in GMR-hid flies, trough transcription levels are not likely from other tissues. Therefore, Clk probably acts on gol and other targets not as an 'ON/OFF switch,' but rather in concert with other factors to boost a basal level of gene expression at a particular time of day and cause low-amplitude cycling within a single tissue (Abruzzi, 2011).

    The large number of Clk target genes in fly heads is explained in part by tissue-specific Clk binding. Transcription assays that measure the cycling of mRNA and Pol II binding in one head tissue can be masked by noncycling expression in another. The ChIP assays, in contrast, are not plagued with the same problem. They can identify a gene bound by the cycling circadian transcription machinery even if the same gene is constitutively expressed elsewhere in the head. Surprisingly 44% of Clk direct targets were no longer detected when eyes were ablated with GMR-hid. Because many of these mRNAs are not particularly eye-enriched, it is inferred that their genes are constitutively expressed under the control of other transcription factors elsewhere in the head (Abruzzi, 2011).

    The large number of target genes is also explained by the efficiency and sensitivity of the ChIP assay. It is inferred that it can detect Clk binding from a relatively low number of cells within the fly head. Lim1 is one example and is expressed predominantly in a subset of circadian neurons (l-LNvs; enriched more than four times relative to head). Preliminary cell-specific Clk ChIP-chip experiments from LNvs confirm that lim1 is an enriched Clk direct target in these cells, suggesting that this is the source of a large fraction of the binding signal in the head ChIP-chip experiments. Experiments are under way to more clearly define circadian neuron-specific Clk-binding patterns (Abruzzi, 2011).

    This tissue specificity also suggests the existence of factors and/or chromatin modifications that help regulate Clk-mediated gene expression. They could enable Clk binding to specific genes in one tissue or inhibit binding in another tissue. These tissue-specific factors are strongly indicated by the pdp1 and lk6 Clk-binding patterns, which change so strikingly and specifically in GMR-hid. Although not unprecedented, tissue-specific factors that enable or inhibit specific DNA-binding locations are intriguing and warrant further investigation and identification (Abruzzi, 2011).

    Temporally and spatially partitioned neuropeptide release from individual clock neurons

    Neuropeptides control rhythmic behaviors, but the timing and location of their release within circuits is unknown. Imaging in the brain shows that synaptic neuropeptide release by Drosophila clock neurons is diurnal, peaking at times of day that were not anticipated by prior electrical and Ca(2+) data. Furthermore, hours before peak synaptic neuropeptide release, neuropeptide release occurs at the soma, a neuronal compartment that has not been implicated in peptidergic transmission. The timing disparity between release at the soma and terminals results from independent and compartmentalized mechanisms for daily rhythmic release: consistent with conventional electrical activity-triggered synaptic transmission, terminals require Ca(2+) influx, while somatic neuropeptide release is triggered by the biochemical signal IP(3). Upon disrupting the somatic mechanism, the rhythm of terminal release and locomotor activity period are unaffected, but the number of flies with rhythmic behavior and sleep-wake balance are reduced. These results support the conclusion that somatic neuropeptide release controls specific features of clock neuron-dependent behaviors. Thus, compartment-specific mechanisms within individual clock neurons produce temporally and spatially partitioned neuropeptide release to expand the peptidergic connectome underlying daily rhythmic behaviors (Klose, 2021).

    FAP imaging revealed synaptic neuropeptide release from LNv clock neurons that does not conform to predictions from previously used indirect methods. Earlier neuropeptide-content measurements could not resolve whether somatic changes were due to release or traffic and did not detect l-LNv rhythmic neuropeptide release, likely because it is relatively modest and/or obscured by DCV capture that replenishes synaptic neuropeptide stores. Furthermore, Ca2+ measured at the soma was not reflective of release at terminals likely because of somatic IP3 signaling. Thus, presynaptic Ca2+ may be more predictive of release by LNv termini. Finally, somatic electrical recording does not take into account regulation by presynaptic inputs. Thus, direct live imaging of neuropeptide release is essential for monitoring peptidergic transmission in the brain (Klose, 2021).

    Indeed, this approach demonstrates that central clock neurons release neuropeptide from terminals and the soma, with each compartment operating with different mechanisms and timing. Release from LNv clock neuron terminals is conventional (i.e., mediated by extracellular Ca2+ influx); because cell specific genetic Ca2+-channel inhibition was not used, the contributions of Ca2+ channels in LNv neurons and their presynaptic inputs was not determined. In contrast, somatic neuropeptide release is triggered by IP3 signaling that operates in the absence of action potential-induced Ca2+ influx. This shows that the two compartments use different mechanisms. It also raises the possibility that release by the two compartments differ in cell autonomy. Most importantly, different release mechanisms allow for multiphasic temporal control of neuropeptide release from separate compartments of the same neuron, each of which releases onto different parts of the clock circuit, thereby providing separate output avenues to independently influence different parameters of behavior (Klose, 2021).

    Circadian deep sequencing reveals stress-response genes that adopt robust rhythmic expression during aging

    Disruption of the circadian clock, which directs rhythmic expression of numerous output genes, accelerates aging. To enquire how the circadian system protects aging organisms, compare circadian transcriptomes in heads of young and old Drosophila melanogaster were compared. The core clock and most output genes remained robustly rhythmic in old flies, while others lost rhythmicity with age, resulting in constitutive over- or under-expression. Unexpectedly, a subset of genes was identified that adopted increased or de novo rhythmicity during aging, enriched for stress-response functions. These genes, termed late-life cyclers, were also rhythmically induced in young flies by constant exposure to exogenous oxidative stress, and this upregulation is CLOCK-dependent. Age-onset rhythmicity was identified in several putative primary piRNA transcripts overlapping antisense transposons. These results suggest that, as organisms age, the circadian system shifts greater regulatory priority to the mitigation of accumulating cellular stress (Kuintzle, 2017).

    Short-term maintenance on a high-sucrose diet alleviates aging-induced sleep fragmentation in Drosophila

    Sleep is a fundamental behavior in an animal's life influenced by many internal and external factors, such as aging and diet. Critically, poor sleep quality places people at risk of serious medical conditions. Because aging impairs quality of sleep, measures to improve sleep quality for elderly people are needed. Given that diet can influence many aspects of sleep, this study investigated whether a high-sucrose diet (HSD) affected aging-induced sleep fragmentation using the fruit fly, Drosophila melanogaster. Drosophila is a valuable model for studying sleep due to its genetic tractability and many similarities with mammalian sleep. Total sleep duration, sleep bout numbers (SBN), and average sleep bout length (ABL) were compared between young and old flies on a normal sucrose diet (NSD) or HSD. On the NSD, old flies slept slightly more and showed increased SBN and reduced ABL, indicating increased sleep fragmentation. Short-term maintenance of flies in HSD (up to 8 days), but not long-term maintenance (up to 35 days), suppressed aging-induced sleep fragmentation. This study provides meaningful strategies for preventing the deterioration of sleep quality in the elderly (Lee, 2021).

    Dietary restriction and the transcription factor Clock delay eye aging to extend lifespan in Drosophila Melanogaster

    Many vital processes in the eye are under circadian regulation, and circadian dysfunction has emerged as a potential driver of eye aging. Dietary restriction is one of the most robust lifespan-extending therapies and amplifies circadian rhythms with age. This study demonstrates that dietary restriction extends lifespan in Drosophila melanogaster by promoting circadian homeostatic processes that protect the visual system from age- and light-associated damage. Altering the positive limb core molecular clock transcription factor, CLOCK, or CLOCK-output genes, accelerates visual senescence, induces a systemic immune response, and shortens lifespan. Flies subjected to dietary restriction are protected from the lifespan-shortening effects of photoreceptor activation. Inversely, photoreceptor inactivation, achieved via mutating rhodopsin or housing flies in constant darkness, primarily extends the lifespan of flies reared on a high-nutrient diet. These findings establish the eye as a diet-sensitive modulator of lifespan and indicates that vision is an antagonistically pleiotropic process that contributes to organismal aging (Hodge, 2022).

    Interneurons of fan-shaped body promote arousal in Drosophila

    Progressive declines in circadian rhythms are one of the most common hallmarks of aging observed across most lifeforms. Quantifying the strength, or amplitude, of circadian rhythms is an accurate metric for predicting chronological age. Many cellular processes involved in aging (e.g., metabolism, cellular proliferation, DNA repair mechanisms, etc.) display robust cyclic activities. Both genetic and environmental disruptions to circadian rhythms are associated with accelerated aging and reduced longevity. These observations suggest that circadian rhythms may not merely be a biomarker of aging; rather, declines in circadian rhythms might play a causal role. The observation that DR and DR-memetics, such as calorie restriction and time-restricted feeding, improve biological rhythms suggests that clocks may play a fundamental role in mediating their lifespan-extending benefits (Kato, 2022).

    This study identified circadian processes that are selectively amplified by DR. The findings demonstrate that DR amplifies circadian homeostatic processes in the eye, some of which are required for DR to delay visual senescence and improve longevity in Drosophila. Disrupting CLK function within photoreceptors accelerates visual declines and shortens lifespan, while overexpressing wild-type CLK protects against age-associated declines in vision and rescues AL-dependent declines in photoreceptor function. These data also demonstrate that photoreceptor stress has deleterious effects on organismal health; overstimulation of the photoreceptors induced a systemic immune response and reduced longevity (Kato, 2022).

    Among the more interesting and unexpecting findings of this study is the observation that the Drosophila eye influences systemic immune responses, as elevated AMP expression was observed in the bodies of flies overexpressing CLK-Δ pan-neuronally and in flies with forced photoreceptor degeneration (ATPα-RNAi). It is possible that GAL4 misexpression may promote inflammatory responses in the fly bodies, although this study found a reduction in systemic inflammation in the rhodopsin-null lines suggesting that this phenomenon can originate at the photoreceptor. These systemic immune responses correlated with lifespan changes (increased body AMP expression is associated with declines in longevity and vice versa), similar to what is observed with chronic inflammation or “inflammaging” in other models. However, it cannot be concluded whether neuronal or eye-mediated increases in systemic inflammation are causal to aging in other tissues. Furthermore, the mechanisms by which the Drosophila eye, and, more specifically, the photoreceptor influence systemic immune responses are unclear. It is speculated that photoreceptor degeneration may disrupt the retinal-blood barrier such that damage signals from the eye propagate through to the hemolymph and activate AMP expression in distal tissues. Future studies are aimed at elucidating this mechanism, and its effect on longevity (Kato, 2022).

    The findings of this study establish the eye as a diet-sensitive regulator of lifespan. DR's neuroprotective role in the photoreceptors appears to be mediated via the transcription factor CLK, which promotes the rhythmic oscillation of genes involved in the suppression of phototoxic cell stress. Given that CLK transcriptionally regulates circadian and non-circadian transcripts, future investigations may determine whether the time-of-day regulation of these genes by CLK is germane to promoting eye health with age. These studies may also examine whether the DR-mediated benefits on visual senescence and photoreceptor viability are mediated solely by CLK as a transcription factor (as demonstrated here) or whether circadian clock function (rhythmic output) is required. The findings also support the notion that age-related declines in the visual system impose a high cost on an organism's physiology. Perhaps this provides an alternative hypothesis for why several cave-dwelling animals, whose visual systems have undergone regressive evolution (e.g., cave-dwelling fish and naked-mole rats), are especially long-lived. Failing to develop a visual system may act as a pro-survival mechanism allowing organisms to avoid the damage and inflammation triggered by age-related retinal degeneration. Ultimately, developing a visual system, which is critical for reproduction and survival, may be detrimental to an organism later in life. Thus, vision may be an example of an antagonistically pleiotropic mechanism that shapes lifespan (Kato, 2022).

    A new promoter element associated with daily time keeping in Drosophila

    Circadian clocks are autonomous daily timekeeping mechanisms that allow organisms to adapt to environmental rhythms as well as temporally organize biological functions. Clock-controlled timekeeping involves extensive regulation of rhythmic gene expression. To date, relatively few clock-associated promoter elements have been identified and characterized. In an unbiased search of core clock gene promoters from 12 species of Drosophila, a 29-bp consensus sequence was discovered that has been designated as the Clock-Associated Transcriptional Activation Cassette or 'CATAC'. To experimentally address the spatiotemporal expression information associated with this element, constructs were generated with four separate native CATAC elements upstream of a basal promoter driving expression of either the yeast Gal4 or firefly luciferase reporter genes. Reporter assays showed that presence of wild-type, but not mutated CATAC elements, imparted increased expression levels as well as rhythmic regulation. Part of the CATAC consensus sequence resembles the E-box binding site for the core circadian transcription factor CLOCK/CYCLE (CLK/CYC), and CATAC-mediated expression rhythms are lost in the presence of null mutations in either cyc or the gene encoding the CLK/CYC inhibitor, period (per). Nevertheless, the results indicate that CATAC's enhancer function persists in the absence of CLK/CYC. Thus, CATAC represents a novel cis-regulatory element encoding clock-controlled regulation (Sharp, 2017).

    Drosophila melanogaster rhodopsin Rh7 is a UV-to-visible light sensor with an extraordinarily broad absorption spectrum

    The genome of Drosophila melanogaster contains seven rhodopsin genes. Rh1-6 proteins are known to have respective absorption spectra and function as visual pigments in ocelli and compound eyes. In contrast, Rh7 protein was recently revealed to function as a circadian photoreceptor in the brain. However, its molecular properties have not been characterized yet. This study successfully prepared a recombinant protein of Drosophila Rh7 in mammalian cultured cells. Drosophila Rh7 bound both 11-cis-retinal and 11-cis-3-hydroxyretinal to form photo-pigments which can absorb UV light. Irradiation with UV light caused formation of a visible-light absorbing metarhodopsin that activated Gq-type of G protein. This state could be photoconverted back to the original state and, thus Rh7 is a Gq-coupled bistable pigment. Interestingly, Rh7 (lambda max = 350 nm) exhibited an unusual broad spectrum with a longer wavelength tail reaching 500 nm, whose shape is like a composite of spectra of two pigments. In contrast, replacement of lysine at position 90 with glutamic acid caused the formation of a normal-shaped absorption spectrum with maximum at 450 nm. Therefore, Rh7 is a unique photo-sensor that can cover a wide wavelength region by a single pigment to contribute to non-visual photoreception (Sakai, 2017).

    Circadian modulation of light-evoked avoidance/attraction behavior in Drosophila

    Many insects show strong behavioral responses to short wavelength light. Drosophila melanogaster exhibit Cryptochrome- and Hyperkinetic-dependent blue and ultraviolet (UV) light avoidance responses that vary by time-of-day, suggesting that these key sensory behaviors are circadian regulated. This study shows mutant flies lacking core clock genes exhibit defects in both time-of-day responses and valence of UV light avoidance/attraction behavior. Non-genetic environmental disruption of the circadian clock by constant UV light exposure leads to complete loss of rhythmic UV light avoidance/attraction behavior. Flies with ablated or electrically silenced circadian lateral ventral neurons have attenuated avoidance response to UV light. It is concluded that circadian clock proteins and the circadian lateral ventral neurons of the adult brain regulate both the timing and the valence of UV light avoidance/attraction. These results provide mechanistic support for Pittendrigh's "escape from light" hypothesis regarding the co-evolution of phototransduction and circadian systems (Baik, 2018).

    Haeme oxygenase protects against UV light DNA damages in the retina in clock-dependent manner
    This study has shown that in the retina of Drosophila, the expression of the ho gene, encoding haeme oxygenase (HO), is regulated by light but only at the beginning of the day. This timing must be set by the circadian clock as light pulses applied at other time points during the day do not increase the ho mRNA level. Moreover, light-induced activation of HO does not depend on the canonical phototransduction pathway but instead involves cryptochrome and is enhanced by ultraviolet (UV) light. Interestingly, the level of DNA damage in the retina after UV exposure was inversely related to the circadian oscillation of the ho mRNA level during the night, being the highest when the HO level was low and reversed during the day. Accordingly, induction of HO by hemin was associated with low DNA damage, while inhibition of HO activity by SnPPIX aggravated the damage. These data suggest that HO acts in the retina to decrease oxidative DNA damage in photoreceptors caused by UV-rich light in the morning (Damulewicz, 2017).

    AMPK signaling linked to the schizophrenia-associated 1q21.1 deletion is required for neuronal and sleep maintenance

    The human 1q21.1 deletion of ten genes is associated with increased risk of schizophrenia. This deletion involves the beta-subunit of the AMP-activated protein kinase (AMPK) complex, a key energy sensor in the cell. Although neurons have a high demand for energy and low capacity to store nutrients, the role of AMPK in neuronal physiology is poorly defined. This study shows that AMPK is important in the nervous system for maintaining neuronal integrity and for stress survival and longevity in Drosophila. To understand the impact of this signaling system on behavior and its potential contribution to the 1q21.1 deletion syndrome, this study focused on sleep, an important role of which is proposed to be the reestablishment of neuronal energy levels that are diminished during energy-demanding wakefulness. Sleep disturbances are one of the most common problems affecting individuals with psychiatric disorders. This study shows that AMPK is required for maintenance of proper sleep architecture and for sleep recovery following sleep deprivation. Neuronal AMPKbeta loss specifically leads to sleep fragmentation and causes dysregulation of genes believed to play a role in sleep homeostasis. These data also suggest that AMPKbeta loss may contribute to the increased risk of developing mental disorders and sleep disturbances associated with the human 1q21.1 deletion (Nagy, 2018).

    Loss of Prune in circadian cells decreases the amplitude of the circadian locomotor rhythm in Drosophila

    The circadian system, which has a period of about 24 h, is import for organismal health and fitness. The molecular circadian clock consists of feedback loops involving both transcription and translation, and proper function of the circadian system also requires communication among intracellular organelles. As important hubs for signaling in the cell, mitochondria integrate a variety of signals. Mitochondrial dysfunction and disruption of circadian rhythms are observed in neurodegenerative diseases and during aging. However, how mitochondrial dysfunction influences circadian rhythm is largely unknown. This study reports that Drosophila prune (pn), which localizes to the mitochondrial matrix, most likely affects the function of certain clock neurons. Deletion of pn in flies caused decreased expression of mitochondrial transcription factor TFAM and reductions in levels of mitochondrial DNA, which resulted in mitochondrial dysfunction. Loss of pn decreased the amplitude of circadian rhythms. In addition, depletion of mtDNA by overexpression of a mitochondrially targeted restriction enzyme mitoXhoI also decreased the robustness of circadian rhythms. This work demonstrates that pn is important for mitochondrial function thus involved in the regulation of circadian rhythms (Chen. 2019).

    Bidirectional regulation of sleep and synapse pruning after neural injury

    Following acute neural injury, severed axons undergo programmed Wallerian degeneration over several following days. While sleep has been linked with synaptic reorganization under other conditions, the role of sleep in responses to neural injuries remains poorly understood. To study the relationship between sleep and neural injury responses, Drosophila melanogaster was examined following the removal of antennae or other sensory tissues. Daytime sleep is elevated after antennal or wing injury, but sleep returns to baseline levels within 24 h after injury. Similar increases in sleep are not observed when olfactory receptor neurons are silenced or when other sensory organs are severed, suggesting that increased sleep after injury is not attributed to sensory deprivation, nociception, or generalized inflammatory responses. Neuroprotective disruptions of the E3 ubiquitin ligase highwire and c-Jun N-terminal kinase basket in olfactory receptor neurons weaken the sleep-promoting effects of antennal injury, suggesting that post-injury sleep may be influenced by the clearance of damaged neurons. Finally, pre-synaptic active zones were shown to be preferentially removed from severed axons within hours after injury, and depriving recently injured flies of sleep slows the removal of both active zones and damaged axons. These data support a bidirectional interaction between sleep and synapse pruning after antennal injury: locally increasing the need to clear neural debris is associated with increased sleep, which is required for efficient active zone removal after injury (Singh, 2020).

    Regulation of circadian behavioural output via clock-responsive miR-276b

    Growing evidence indicates that microRNAs play numerous important roles. However, the roles of some microRNAs involved in regulation of circadian rhythm and sleep are still not well understood. This study shows that the miR-276b is essential for maintaining both sleep and circadian rhythm by targeting tim, NPFR and DopR1 genes, with miR-276b deleted mutant flies sleeping more, and vice versa in miR-276b overexpressing flies. Through analysing its promoter, mir-276b was found to be responsive to CLOCK and regulates circadian rhythm through the negative feedback loop of the CLK/CYC-TIM/PER. Furthermore, miR-276b is broadly expressed in the clock neurons and the central complexes such as the mushroom body and the fan-shape body of Drosophila brain, in which up-regulation of miR-276b in tim, npfr1 and DopR1 expressing tissues significantly causes sleep decreases. This study clarifies that the mir-276b is very important for participating in regulation of circadian rhythm and sleep (Zhang, 2020).

    Glial control of sphingolipid levels sculpts diurnal remodeling in a circadian circuit
    Structural plasticity in the brain often necessitates dramatic remodeling of neuronal processes, with attendant reorganization of the cytoskeleton and membranes. Although cytoskeletal restructuring has been studied extensively, how lipids might orchestrate structural plasticity remains unclear. This study shows that specific glial cells in Drosophila produce glucocerebrosidase (GBA) to locally catabolize sphingolipids. Sphingolipid accumulation drives lysosomal dysfunction, causing gba1b mutants to harbor protein aggregates that cycle across circadian time and are regulated by neural activity, the circadian clock, and sleep. Although the vast majority of membrane lipids are stable across the day, a specific subset that is highly enriched in sphingolipids cycles daily in a gba1b-dependent fashion. Remarkably, both sphingolipid biosynthesis and degradation are required for the diurnal remodeling of circadian clock neurites, which grow and shrink across the day. Thus, dynamic sphingolipid regulation by glia enables diurnal circuit remodeling and proper circadian behavior (Vaughen, 2022).

    The data demonstrate that glia produce Gba1b to non-autonomously control brainsph ingolipids, protein degradation, and neurite remodeling in a circadian circuit. This study identified two specific glial subtypes, ensheathing glia (EG) and perineural glia (PNG), as critical sources of gba1b required for lysosomal function, proteostasis, circadian behaviors and neurite remodeling. While previous genetic studies in vertebrate models did not determine whether GBA was required in glia or neurons, lysosomal GBA expression in mice and humans is ~5-fold higher in glia and microglia compared to neurons, and gba mice harbor aggregates in astrocytes. Given these expression patterns and the striking similarities in brain phenotypes seen across GBA mutants in flies, fish, and mammals, there appears to be an evolutionarily ancient role for glia in regulating sphingolipid metabolism in the brain (Vaughen, 2022).

    Previous work identified the cytoskeletal effector Rho1 and the transcription factor Mef2 as important for sLNv neurite remodeling. The current work demonstrates that these changes must be coordinated with membrane remodeling, particularly sphingolipid degradation and biosynthesis, both of which are required for membrane retraction and growth. In control animals, sLNv neurites are enlarged at dawn, retract across the day to become stunted at dusk, and then re-extend during the night. This cycle coincides with a diurnal sphingolipid cycle in which specific GlcCer and Cer species are elevated during the retraction phase, and reduced during the growth phase. In gba1bΔ mutants, the cycle of sLNv growth and retraction is blocked such that sLNv neurites remain in a stunted state across the day. In parallel, the sphingolipid cycle is also substantially blocked, and many sphingolipids are present at highly elevated levels. Conversely, in animals in which sphingolipid biosynthesis is globally reduced (Pdf>laceRNAi), the cycle of sLNv remodeling is also blocked but sLNv neurites remain aberrantly extended throughout the day. Notably, Lace is bound by the transcription factor Clock and undergoes circadian changes in expression, increasing before dusk and peaking at midnight, the same stage at which RNAi perturbation would be expected to be strongest (given the Pdf promoter. Finally, changing the timing of gba1b expression in sLNv neurites preserved the diurnal cycle of neurite growth and retraction, but inverted its phase such that sLNv neurites were reduced at dawn and enlarged at night. Taken together, these results demonstrate that carefully timed cycles of sphingolipid degradation and biosynthesis are instructive for the diurnal pattern of neurite remodeling. Lipidomics analysis demonstrates that the levels of Cer and GlcCer 14:1/18:1 and 14:1/20:1 species, as well as CerPE14:1/18:0 and CerPe 14:2/18:0, are elevated during the phase of sLNv neurite retraction, suggesting that these specific species might play an important regulatory role. Although sphingolipids are substantially less abundant than phospholipids (with Cer and GlcCer species representing <0.5% of neural membranes), their unique effects on membrane biophysics makes them well-poised to strongly affect membrane remodeling and structural plasticity. Finally, given that GlcT depletion (which blocks GlcCer but not Cer formation) did not increase sLNv volume compared to lace manipulations (which blocks GlcCer, Cer, and CerPE), a model is favored whereby Cer species drive membrane retraction. This hypothesis would be consistent with recent work revealing that diurnal changes in Cer species trigger retraction of microglia processes (Vaughen, 2022).

    There is also strong evidence for sphingolipids regulating the cytoskeleton. For example, blocking de novo sphingolipid synthesis in fibroblasts acutely reduced cell area and lamellipodia formation, and altered sphingolipid profiles were associated with shortened neurites and upregulated RhoA following acid ceramidase knockdown in neuroblastoma lines. Moreover, GBA2 (non-lysosomal) knockout mice have cytoskeletal defects, shorter neurites, and dysregulate Rho GTPase localization. Additionally, activity within larval LNv neurites triggers lipid-mediated structural alterations critical for circuit function dependent on Ceramide Synthase. Taken with this work, sphingolipids and their regulatory enzymes may function coordinately or even upstream to Rho1 and attendant cytoskeletal changes in sLNv remodeling, with more precise spatiotemporal control facilitated by glial degradation of remodeled neurite membranes (Vaughen, 2022).

    While sLNv neurites are a dramatic example of membrane remodeling, many neurons grow and shrink across circadian cycles and varied environmental conditions. Membrane turnover is likely important during structural plasticity at synapses, and indeed gba1b flies have memory defects. As EG and PNG are broadly distributed throughout the brain, glia-mediated sphingolipid degradation may be central to membrane remodeling in many neural circuits. Interestingly, EG engulf neuronal membranes to clear damage in a sleep- dependent fashion, a cellular response potentially co-opted from a role in the daily remodeling of neurite membranes. Daily neurite remodeling is also a feature of the mouse suprachiasmatic nucleus. Moreover, microglia, which express GBA in mice and humans, locally prune neurites during development and injury, regulate sleep, and are enriched for sphingolipid catabolizing genes. Taken together, this suggests GBA could control many forms of structural plasticity across species. Protein aggregation is dynamically controlled by circadian state and neural activity Our characterization of gba1b mutant animals revealed a surprising circadian cycle of protein aggregate accumulation and removal. This study observed both activity-dependent and direct circadian control of aggregate burden. Protein aggregates have been described in wide-ranging disease models from flies to mice and are a prominent feature of neurodegeneration in humans, including cells derived from GBA mutant mice and patients. Given the observations of this study, it may prove critical to characterize aggregate accumulation (and lipid abundance) with respect to circadian time and neural activity. Indeed, circadian phagocytosis of amyloid-beta, a component of Alzheimer's aggregates, was recently observed in cultured macrophages to be driven by circadian biosynthesis of heparan sulfate proteoglycans (Vaughen, 2022).

    These data provide a direct mechanistic connection between the enzymatic activity of gba1b in glia to circadian behavior through sLNv neurite remodeling, as well as broader effects on sleep behavior likely mediated by other circuits. Notably, gba1bΔ mutant animals display deficits in activity and sleep prior to overt accumulation of aberrant protein aggregates. Similarly, many Parkinson's patients have sleep defects prior to development of clinical characteristics typically associated with neuropathological aggregate accumulation. Many genes that impinge on lysosomal function and sphingolipid degradation are linked to Parkinson's disease, and glia are key regulators of Parkinson's and other neurodegenerative diseases. Moreover, precise control of lipid species is central to many neurodegenerative models and is often modulated by neural activity. Recently, long-chain saturated lipids were found to mediate neurotoxicity by reactive astrocytes, and this study also observed increased nighttime long-chain saturated Cer/GlcCer species in gba1bΔ mutants, which coincides with circadian aggregate burden. Based on these observations, mutations in lipid-regulating genes could impair glial remodeling of sleep circuits, and specific lipid species may diurnally drive cyclic aggregates. Sleep has been proposed to drive clearance of aggregates from the brain. Moreover, cell-type specific functions for sphingolipids are known in glia, and regional lipid heterogeneity pervades the human brain\. Unraveling the complicated mechanisms of cell-type specific lipid synthesis and degradation may provide crucial insights into the connections between sleep, circadian rhythms, neurodegenerative diseases, and neuronal membrane dynamics (Vaughen, 2022).

    These studies revealed that the brain is sensitive to the level of expression of gba1b when doing rescue experiments, likely reflecting the fact that gba1b itself is under tight transcriptional control and that very low levels of expression are sufficient to rescue most gba1b phenotypes (consistent with scRNA-seq data). A key gap in the field is the absence of tools that afford both cell-type specificity and quantitative control of expression across physiologically relevant levels alongside precise temporal actuation. As such tools develop, further exploring the relative balance of sphingolipid biosynthesis and degradation in shaping neurite growth and retraction would be fascinating. In addition, while analyzing sLNv neurites at specific timepoints has provided key insights, being able to capture the dynamics of lipid turnover in a live imaging preparation would deepen this understanding. This requires the application of fluorescent probes for specific lipids that can be engaged cell-type specifically, combined with a chronic imaging preparation that can span both sleep and wake cycles. Finally, while this study has quantitatively measured changes in membrane lipid (Vaughen, 2022).

    Identification of Genes Contributing to a Long Circadian Period in Drosophila Melanogaster

    The endogenous circadian period of animals and humans is typically very close to 24 h. Individuals with much longer circadian periods have been observed, however, and in the case of humans, these deviations have health implications. Previously, a line of Drosophila was observed with a very long average period of 31.3 h for locomotor activity behavior. Preliminary mapping indicated that the long period did not map to known canonical clock genes but instead mapped to multiple chromosomes. Using RNA-Seq, the whole transcriptome of fly heads from this line was surveyed across time and compared with a wild-type control. A three-way generalized linear model revealed that approximately two-thirds of the genes were expressed differentially among the two genotypes, while only one quarter of the genes varied across time. Using these results, algorithms were applied to search for genes that oscillated over 24 h, identifying genes not previously known to cycle. 166 differentially expressed genes were identified that overlapped with a previous Genome-wide Association Study (GWAS) of circadian behavior, strongly implicating them in the long-period phenotype. Mutations were tested in 45 of these genes for their effect on the circadian period. Mutations in Alk, alph, CG10089, CG42540, CG6034, Kairos (CG6123), CG8768, klg, Lar, sick, and tinc had significant effects on the circadian period, with seven of these mutations increasing the circadian period of locomotor activity behavior. Genetic rescue of mutant Kairos restored the circadian period to wild-type levels, suggesting it has a critical role in determining period length in constant darkness (Kumar, 2020).

    Clk post-transcriptional control denoises circadian transcription both temporally and spatially

    The transcription factor CLOCK (CLK) is essential for the development and maintenance of circadian rhythms in Drosophila. However, little is known about how CLK levels are controlled. This study shows that Clk mRNA is strongly regulated post-transcriptionally through its 3' UTR. Flies expressing Clk transgenes without normal 3' UTR exhibit variable CLK-driven transcription and circadian behaviour as well as ectopic expression of CLK-target genes in the brain. In these flies, the number of the key circadian neurons differs stochastically between individuals and within the two hemispheres of the same brain. Moreover, flies carrying Clk transgenes with deletions in the binding sites for the miRNA bantam have stochastic number of pacemaker neurons, suggesting that this miRNA mediates the deterministic expression of CLK. Overall these results demonstrate a key role of Clk post-transcriptional control in stabilizing circadian transcription, which is essential for proper development and maintenance of circadian rhythms in Drosophila (Lerner, 2021).

    DPP acutely defines the connectivity of central pacemaker neurons in Drosophila

    Rhythmic rest-activity cycles are controlled by an endogenous clock. In Drosophila, this clock resides in ∼150 neurons organized in clusters whose hierarchy changes in response to environmental conditions. The concerted activity of the circadian network is necessary for the adaptive responses to synchronizing environmental stimuli. Thus far work was devoted to unravel the logic of the coordination of different clusters focusing on neurotransmitters and neuropeptides. This study further explored communication in the adult male brain through ligands belonging to the Bone Morphogenetic Protein (BMP) pathway. This study shows that the Lateral Neurons ventral (LNvs) express the small morphogen Decapentaplegic (DPP). DPP expression in the large LNvs triggered a period lengthening phenotype, while its downregulation caused reduced rhythmicity and affected anticipation at dawn and dusk, underscoring DPP per se conveys time-of-day relevant information. Surprisingly, its expression in the large LNvs impaired circadian remodeling of the small LNv axonal terminals, likely through local modulation of the guanine nucleotide exchange factor (GEF) Trio. These findings open the provocative possibility that the BMP pathway is recruited to strengthen/reduce the connectivity among specific clusters along the day and thus modulate their contribution to the circadian network (Polcownuk, 2021).

    The Neuropeptide PDF Is Crucial for Delaying the Phase of Drosophila's Evening Neurons Under Long Zeitgeber Periods

    Full comprehension of circadian clocks function requires precise understanding of their entrainment to the environment. The phase of entrained clock is plastic, which depends on different factors such as the period of endogenous oscillator, the period of the zeitgeber cycle (T), and the proportion of light and darkness (day length). This study investigated the importance of the neuropeptide Pigment-Dispersing Factor (PDF) for entrainment by systematically studying locomotor activity rhythms of Pdf mutants and wild-type flies under different T-cycles (T22 to T32) and different day lengths (8, 12, and 16 hour [h]). Furthermore, this study analysed Period protein oscillations in selected groups of clock neurons in both genotypes under T24 and T32 at a day length of 16 h. As expected, it was found that the phase of Drosophila's evening activity and evening neurons advanced with increasing T in all the day lengths. This advance was much larger in Pdf mutants (~7 h) than in wild-type flies causing (1) pronounced desynchrony between morning and evening neurons and (2) evening activity to move in the morning instead of the evening. Most interestingly, it was found that the lights-off transition determines the phase of evening neurons in both genotypes and that PDF appears necessary to delay the evening neurons by ~3 h to their wild-type phase (, 2021). Thus, in T32, PDF first delays the molecular cycling in the evening neurons, and then, as shown in previous studies, delays their neuronal firing rhythms to produce a total delay of ~7 h necessary for a wild-type evening activity phase. It is concluded that PDF is crucial for appropriate phasing of Drosophila activity rhythm (Vaze, 2021).

    Light triggers a network switch between circadian morning and evening oscillators controlling behaviour during daily temperature cycles

    Proper timing of rhythmic locomotor behavior is the consequence of integrating environmental conditions and internal time dictated by the circadian clock. Rhythmic environmental input like daily light and temperature changes (called Zeitgeber) reset the molecular clock and entrain it to the environmental time zone the organism lives in. This study attempted to entrain the clock with a Zeitgeber different from the environmental input used for phasing the behavior. 150 clock neurons in the Drosophila melanogaster brain control different aspects of the daily activity rhythms and are organized in various clusters, During regular 12 hr light: 12 hr dark cycles at constant mild temperature (LD 25°C, LD being the Zeitgeber), so called morning oscillator (MO) neurons control the increase of locomotor activity just before lights-on, while evening oscillator (EO) neurons regulate the activity increase at the end of the day, a few hours before lights-off. This study, using 12 h: 12 h 25°C:16°C temperature cycles as Zeitgeber, attempted to look at the impact of light on phasing locomotor behavior. While in constant light and 25°C:16°C temperature cycles (LLTC), flies show an unimodal locomotor activity peak in the evening, during the same temperature cycle, but in the absence of light (DDTC), the phase of the activity peak is shifted to the morning. This study shows that the EO is necessary for synchronized behavior in LLTC but not for entraining the molecular clock of the other clock neuronal groups, while the MO controls synchronized morning activity in DDTC. Interestingly, the data suggest that the influence of the EO on the synchronization increases depending on the length of the photoperiod (constant light vs 12h of light). Hence, these results show that effects of different environmental cues on clock entrainment and activity phase can be separated, allowing to decipher their integration by the circadian clock (Lorber, 2022).

    The daily pattern of locomotor behavior is highly plastic and not only depends on the time of day but also on the current environmental condition. How the brain clock integrates Zeitgeber information and various environmental inputs to time locomotor activity is not clear. To address this important question, a specific daily temperature oscillation (25°C-16&frhC) was used as Zeitgeber and light input as the environmental variable. By genetically probing clock- and neuronal function of different parts of the circadian neuronal network in different environmental conditions, it was revealed that the ambient light status determines circadian network balance (Lorber, 2022).

    An important question in chronobiology that so far remains unanswered is the mechanism of entrainment by light and temperature when these two environmental parameters are actually not so reliable considering their substantial daily variation. Notably, substantial weather-inflicted variations of temperature and light intensity can occur during the day and these must not lead to circadian clock resetting. Nonetheless, animals behave quite differently depending on the current environmental status regardless of their clock entrainment status. For example, fruit flies lose their two anticipatory activities present in LD 25°C at different constant temperatures. At warm temperature (≥30°C), flies only anticipate the light-on transition while at colder temperatures (≤20°C) they only anticipate the lights-off. On the other hand, when temperature cycles are used as Zeitgeber in constant darkness, the absolute temperatures determine the phase of the activity peak, with TCs of 25°C:16°C and 29°C:20°C inducing activity in the first half or the second half of the thermophase, respectively. In constant light however, both temperature cycles result in an activity peak during the second half of the thermophase (Lorber, 2022).

    This study demonstrates that the EO that drives the evening peak in LD25°C is necessary to drive the evening peak in LLTC25°C-16°C, while the MO that drives the morning peak in LD25°C controls the phase in DDTC25°C-16°C. According to this model the ambient environmental condition modulates the clock network balance. Hence, the environmental condition must be taken into account in order to understand the role of each oscillator. Previously, trying to understand how temperature cycles synchronize flies, a similar approach has been performed, although in the absence of light and different absolute temperatures, but with the same amplitude of temperature oscillations (29°C-20°C). Consistent with these findings, ablating both the MO and EO using a cry-gal4 line lead to largely desynchronized behavior. As mentioned above, in DDTC 29°C:20°C conditions flies exhibit an afternoon activity peak, and ablating the PDF neurons does not affect this synchronization. In contrast, the data show that a functional clock in the PDF neurons is important for synchronized morning activity in DDTC 25°C:16°C. Following the model, it is postulated therefore that the DN1p clock neurons also contribute to the behavioral evening activity during warm temperature cycles in constant darkness. The DN1p can drive evening activity during DDTC 29°C:20°C and during low intensity LD cycles when the temperature is constant, while at high light intensity LD cycles they support morning activity. Furthermore, the DN1p receive warm input via the TrpA1 expressing AC neurons. Interestingly however, TrpA1 is not required for temperature entrainment as such, but shapes behavior under warm conditions. Notably, while the loss of TrpA1 function has no effect on the behavioral phase in DDTC 25°C:16°C, TrpA1 mutant flies show an advanced increase of activity and a reduced siesta in DDTC 29°C:20°C compared to controls. Hence, taken together these data re-enforce the model where, depending on the environmental condition, the clock network can change its balance to set behavioral activity phase, bypassing a change of clock entrainment (Lorber, 2022).

    The molecular clock in brain clock neurons is entrained by light and temperature. However, these two environmental inputs vary on a day to day basis (e.g., cloudy versus sunny days), and animals change their activity at different times of day in response to the ambient environmental condition. How does the circadian system distinguish between an input that entrains the circadian clock (Zeitgeber) and one that after integration by the clock system, sets the daily activity phase? This study demonstrates that the EO determines the behavioral evening peak (Φ) in the presence of light and 25°C, while the circadian clock is entrained with a 25°C:16°C temperature cycle. In contrast, the MO determines the morning peak in constant darkness and 25°C, during the same temperature cycle. A model is presented explaining how the environment and the circadian clock shape locomotor activity (see Environmental cues act as Zeitgeber and set behavioral activity Phase). On one hand, the on/up and off/down environmental changes that happen once a day entrain the molecular clocks in the system. On the other hand, different environmental input such as light (quality and intensity) and temperature (different levels) are perceived by different oscillators in different manners. Depending on the time of day (the molecular clock status of the system), this will lead to a modification of the network balance and a dominancy of one or several oscillators (blue clocks vs grey ones), resulting in a behavioral activity phase (Φ) according to the internal timing and the current condition. Therefore, to biologically demonstrate this model, a Zeitgeber (in this study TC 25°C-16°C) was fixed, and how light (here presence/absence) modifies the balance was tested. From this basis, it is possible to apply more subtle modifications such as the intensity or the quality of light to change the phase of the behavior and use this to understand the principles underlying neuronal network switches (Lorber, 2022).

    Furthermore, the data suggest that the length of the light period (here 12 h vs 24 h) influences the involvement of the EO. Ablating the PDF neurons not only advances the evening peak in LD but it also drastically reduces the synchronization of the flies (S6B and S6C Fig), while they remain strongly synchronized in LLTC. Furthermore, while ablating the EO, even with incomplete penetrance, drastically affected the presence of the synchronized evening peak in LLTC, it only slightly reduced synchronization in LD. Hence, although the clock of the EO can be entrained independently to light , the data indicate that the PDF neurons have a strong influence on the robustness of EO-output in LD25°C but not in LLTC. This suggests that the extent of dark periods increases the influence of the PDF neurons during the day. The evening peak occurs under the same environmental condition (lights-on and 25°C), however with a slight advance in LLTC compared to LD. Therefore, it is proposed that the past experience during the night/cryophase determines the influence of one group toward the other. In summary, these data confirm that the network status varies with the environmental condition, and therefore it is crucial to consider the specific environmental conditions when deciphering the different contributions of circadian network components in regulating behavioural activity. This simple environmental protocol provides a template to further test and extend this model. For example, using temperature cycles as Zeitgeber, it is now possible to modify light quality and intensity to test how the clock network responds to these light variations while the clock is stably entrained (Lorber, 2022).

    Synchronization of clock protein oscillations in LPN neurons is preferentially sensitive to temperature. However, it has been observed that these neurons are not necessary for rhythmic behavior under TC conditions in both LL and constant darkness. This study confirms the non-requirement of clock function within the LPN for rhythmic locomotor behavior in LLTC, suggesting that LPN sensitivity for molecular synchronization to TC serves another function unrelated to entrainment. Indeed, a recent study shows that the LPN are activated above 27° and they are important for increased siesta sleep at 30°C. CRY is expressed in about 1/3rd of the clock network in the brain and plays an important role in light entrainment, consistent with the idea that CRY+ neurons are more sensitive to light and CRY- cells are more sensitive to temperature. Notably, isolated brains can still be entrained to LD cycles, and in the absence of visual input all clock neurons are entrained to LD, indicating that the CRY+ neurons synchronize the remaining CRY- neurons non-cell autonomously. However, this study demonstrates an essential role of the CRY+ neurons in controlling rhythmic behavior in LLTC. Nevertheless, even in the absence of all CRY+ neurons, flies exhibit weak synchronized behavior with an evening phase during temperature cycles (in LL and DD), suggesting that CRY- clock neurons (subsets of DN1p and LNd, DN2, and DN3) also play a role in temperature synchronization. Nevertheless, because they are not able to instruct the CRY+ neurons during TC , their role in temperature entrainment is not as prominent as that of CRY+ neurons in LD. Moreover, ablation of PDF+ or all three groups of LN (LNv, LNd, LPN) still allows for weak behavioral evening synchronization to 30°C: 25°C TC in LL. Furthermore, the fact that in the absence of a clock in the Mai179+ cells, after ablation of all CRY+ cells, or in the absence of all LN, molecular oscillations in other clock neurons can be synchronized, supports the idea that multiple and independent pathways contribute to temperature entrainment (Lorber, 2022).

    Protein phosphatase 4 controls circadian clock dynamics by modulating CLOCK/BMAL1 activity

    In all organisms with circadian clocks, post-translational modifications of clock proteins control the dynamics of circadian rhythms, with phosphorylation playing a dominant role. All major clock proteins are highly phosphorylated, and many kinases have been described to be responsible. In contrast, it is largely unclear whether and to what extent their counterparts, the phosphatases, play an equally crucial role. To investigate this, a systematic RNAi screen was performed in human cells and protein phosphatase 4 (PPP4) was identified with its regulatory subunit PPP4R2 as critical components of the circadian system in both mammals and Drosophila. Genetic depletion of PPP4 (Pp4-19C in Drosophila) shortens the circadian period, whereas overexpression lengthens it. PPP4 inhibits CLOCK/BMAL1 transactivation activity by binding to BMAL1 (Cycle in Drosophila) and counteracting its phosphorylation. This leads to increased CLOCK/BMAL1 DNA occupancy and decreased transcriptional activity, which counteracts the "kamikaze" properties of CLOCK/BMAL1. Through this mechanism, PPP4 contributes to the critical delay of negative feedback by retarding PER/CRY/CK1Δ-mediated inhibition of CLOCK/BMAL1 (Klemz, 2021).

    Sleep deprivation results in diverse patterns of synaptic scaling across the Drosophila mushroom bodies

    Sleep is essential for a variety of plastic processes, including learning and memory. However, the consequences of insufficient sleep on circuit connectivity remain poorly understood. To better appreciate the effects of sleep loss on synaptic connectivity across a memory-encoding circuit, changes were examined in the distribution of synaptic markers in the Drosophila mushroom body (MB). Protein-trap tags for active zone components indicate that recent sleep time is inversely correlated with Bruchpilot (BRP) abundance in the MB lobes; sleep loss elevates BRP while sleep induction reduces BRP across the MB. Overnight sleep deprivation also elevated levels of dSyd-1 and Cacophony, but not other pre-synaptic proteins. Cell-type-specific genetic reporters show that MB-intrinsic Kenyon cells (KCs) exhibit increased pre-synaptic BRP throughout the axonal lobes after sleep deprivation; similar increases were not detected in projections from large interneurons or dopaminergic neurons that innervate the MB. These results indicate that pre-synaptic plasticity in KCs is responsible for elevated levels of BRP in the MB lobes of sleep-deprived flies. Because KCs provide synaptic inputs to several classes of post-synaptic partners, a fluorescent reporter for synaptic contacts was used to test whether each class of KC output connections is scaled uniformly by sleep loss. The KC output synapses that were observed in this study can be divided into three classes: KCs to MB interneurons; KCs to dopaminergic neurons, and KCs to MB output neurons. No single class showed uniform scaling across each constituent member, indicating that different rules may govern plasticity during sleep loss across cell types (Weiss, 2021).

    This study used genetic reporters to quantify the effects of sleep loss on pre-synaptic active zone markers and putative synaptic contacts in the Drosophila MB lobes. Abundance of Brp, dSyd-1, and Cacophony broadly increase across all MB lobes after overnight sleep deprivation and that acutely increasing sleep for 6 h is sufficient to reduce Brp levels across the α, 'β, γ, and 'β' lobes. KCs strongly contribute to the increase in Brp across each MB lobe following sleep loss, while pre-synapses of other MB cell types are less sensitive to sleep disruption. Because release of Drosophila neuromodulators likely occurs through a combination of classical neurotransmission and extrasynaptic release, these studies do not rule out the possibility that BRP-independent secretion of dopaminergic dense core vesicles might be altered in the MB lobes by sleep loss. The elevated levels of Brp present in KCs of sleep-deprived flies return to control levels within 48 h of ab libitum recovery sleep. While associative learning can recover within only a few hours after sleep deprivation, these studies indicate that some synaptic consequences of prolonged waking may persist for at least 24 h of recovery. These findings parallel those from humans and rodents, suggesting that some measures of cognition and neurophysiology recover rapidly after acute sleep loss while others last much longer, even for several days in some cases. The tractability of Drosophila may provide opportunities for future studies to investigate the processes that mediate recovery from sleep loss and to test whether similar trends in plasticity occur in other neuropil regions across the brain (Weiss, 2021).

    Interestingly, sleep deprivation does not seem to increase other active zone components: Rim and Syt1 only show localized changes in some MB lobes, and the primarily post-synaptic marker Dlg shows no significant changes across the MB after sleep loss. Additionally, it was found that the abundance of vesicular proteins Rab3 and nSyb decreases across all MB lobes following overnight sleep deprivation. The varying responses between pre-synaptic components may indicate that sleep deprivation may alter the abundance of some active zone constituents along differing time courses or that active zone release machinery may be regulated differently from synaptic vesicle pools. The varied responses of each synaptic reporter that was observed suggests that Brp, dSyd-1, and Cac levels may underlie the consequences of sleep loss on MB functioning, but the precise physiological consequences of these changes on KC neurotransmitter release are unclear. Previous work finds that increasing BRP gene copy number drives changes in other active zone proteins that recapitulate protein levels observed in short sleeping mutants and also increases sleep in a dose-dependent manner (Weiss, 2021).

    It is tempting to speculate that increases in Brp with sleep loss may drive concomitant increases in some core active zone scaffolding components and compensatory decreases in some proteins regulating synaptic vesicle release. Experiments at the Drosophila larval NMJ indicate that elevated Brp levels increase the rate of spontaneous release and enhance facilitation with pairs of stimuli, while other markers of synapse strength, including the amplitudes of evoked and spontaneous junction potentials, remained unchanged (Weiss, 2021).

    It is unclear whether acute changes in Brp with sleep loss induce the same physiological changes at MB-output synapses, and additional studies will be required to understand how plastic mechanisms that contribute to memory formation might be altered by the pre-synaptic changes described above. Recent work finds that pan-neuronal knockdown of dSyd-1 can reduce sleep and dampen homeostatic rebound, even in flies with elevated BRP (Weiss, 2021).

    Consistent with the idea that dSyd-1 levels may influence sleep pressure, decreased dSyd-1MI05387-GFSTF abundance was found in previously sleep-deprived flies after 48 h of recovery. While the MB contains several different cell types, pre-synapses in the axons of KCs appear to be uniquely plastic during sleep loss. Use of an activity-dependent fluorescent GRASP reporter of synaptic contacts observed that sleep loss altered synaptic contacts between KCs and distinct post-synaptic partners in different ways (Weiss, 2021).

    Among these changes, it was found that GRASP fluorescence reporting contacts from KCs to PPL1 DANs is strongly decreased after sleep loss, indicating a weakening of the KC > PPL1 DAN contacts. Interestingly, these connections may be vital for recurrent activation within MB compartments during learning and could contribute to prediction error signals (Weiss, 2021).

    While further studies will be required to examine the contribution of these particular connections to learning deficits after sleep loss, human subjects have been reported to exhibit impaired error prediction and affective evaluation in learning tasks following sleep loss (Weiss, 2021).

    Because reduced GRASP signal was observed in KC > PPL1 DAN connections, which mediate aversive reinforcement, and not in KC > PAM DAN connections, which influence appetitive reinforcement, it is also possible that sleep loss may not equally degrade the encoding of reinforcement signals across all valences or modalities. Recent findings also suggest that not all forms of memory require sleep for consolidation; appetitive olfactory memories can be consolidated without sleep when flies are deprived of food, and sleep-dependent and independent memory traces in these conditions are stored in separate MB zones (Weiss, 2021).

    The KC > MBON connections that contribute to sleep-dependent memory (KC > γ2α'1) also show an overall decrease in GRASP signal with sleep loss, while those that are vital for sleep-independent memory (MBON-γ1pedc) show no GRASP change after sleep deprivation. These compartment-specific variations in the effects of sleep on both memory and synaptic distribution further indicate that local MB zones may follow distinct plasticity rules under physiological stressors, including sleep loss (Weiss, 2021).

    Additionally, GRASP signal from KCs to APL is significantly elevated following sleep loss, suggesting a strengthening of KC > APL connections. KCs and APL form a negative feedback circuit, where KCs activate APL and APL inhibits KCs: this feedback inhibition maintains sparseness of odor coding and odor specificity of memories (Weiss, 2021).

    It is possible that KCs compensate for increased synaptic abundance accumulated during sleep loss by recruiting inhibition from APL. While further experimentation is needed to examine the role of these connections in the regulation of net synaptic strength during sleep loss, sleep deprivation results in increased cortical excitability in humans and rodents, and hyperexcitability is often counteracted by increased synaptic inhibition (Weiss, 2021).

    Conversely, sleep loss reduces connectivity between KCs and DPM, a second large interneuron that may facilitate recurrent activity in the MB lobes. The current results also indicate that KC > MBON synaptic contacts exhibit a variety of changes in response to sleep deprivation. The specific KC > MBON connections that show significantly elevated or reduced GRASP signal in this study are not clearly assorted based on valence encoding, contribution to specific associative memory assays, or influence on sleep/wake regulation (Weiss, 2021).

    Activity in several MB cell types, including α'/'β' KCs, MBON-γ5'β'2, MBON-γ2 α'1, DPM, and PAM DANs regulate sleep. The observation that KC > MBON-γ5'β'2a labeling is reduced with sleep loss complements previous observations of reduced electrical activity in MBON-γ5'β'2 following sleep deprivation (Weiss, 2021).

    Other sleep-promoting MB neurons, however, such as DPM, do not show an overall increase in BRP abundance, suggesting either that other changes in excitability, synaptic drive, or post-synaptic adaptations might drive homeostatic sleep regulation in these cells or that distinct subsets of connections within the populations that were labelled in this study might be sleep regulatory. The compartment-to-compartment variance in KC > MBON responses to sleep loss also parallels previous findings that plasticity rules can vary between MBONs during heterosynaptic plasticity (Weiss, 2021).

    While GRASP results suggest diverse changes in putative synaptic contacts with sleep loss, the functional effects of these changes require further study. It is important to note that a significant portion of MB synapses are composed of connections between either pairs or groups of KCs. The genetic strategies that were used in this study have prevented reliable visualization and quantification of these connections. As a result, the effect of sleep loss on KC > KC synapses has not been examined in this study but may comprise a portion of the increase in KC pre-synaptic abundance that was observed in this study. While these studies identify synaptic classes that exhibit altered GRASP labeling across sleep loss, future studies using super resolution imaging and/or physiology could examine the structural and molecular changes that underlie this plasticity. Connections between neurons in the MB may be also influenced by non-neuronal cell types, including astrocytes. Astrocytic contact with KCs can be reduced by sleep loss and astrocytic calcium levels correlate with sleep pressure, which suggests that astrocytic processes could be positioned to mediate sleep-dependent plasticity in the MB (Weiss, 2021).

    The broad conservation of release machinery across active zones within and between cell types has simplified examination of pre-synaptic plasticity during sleep loss. Assays of both Hebbian and homeostatic plasticity have also identified a variety of post-synaptic adaptations. Interestingly, post-synaptic densities isolated from rodent cortex show significant reorganization of post-synaptic GluR5 receptors. This depends upon the activity of Homer and sleep-dependent phosphorylation of CaMKII and GluR1, that contribute to consolidation of visual cortex plasticity (Weiss, 2021).

    Because MBONs exhibit post-synaptic plasticity during other contexts, including the formation of associative memories, sleep deprivation may also alter post-synaptic organization of MBONs or other cell types in the MB. Although the distribution of Dlg is not significantly changed by sleep loss, the rich variety of post-synaptic receptors for acetylcholine, dopamine, GABA, and other signals in the MB requires development of additional reporters to examine these post-synaptic consequences of insufficient sleep in MB neurons. Additionally, while the data outline changes in pre-synaptic protein abundance and pre-synaptic KC contacts that result from sleep loss, the possibility that these synaptic changes may be accompanied by homeostatic compensation in neuronal excitability or firing patterns remains to be tested. Because sleep-deprived flies can recover the capacity to learn after only a brief nap, homeostatic adjustments in post-synaptic strength and/or excitability may permit MBs to compensate for pre-synaptic changes that appear to persist for at least 24 h after sleep deprivation. Further, recovery sleep or pharmacological sleep enhancement may not simply reverse the effects of sleep loss, and it is unclear how particular subsets of synaptic proteins or connections may be selected for removal during times of elevated sleep (Weiss, 2021).

    The consequences of sleep loss on synaptic connectivity are not clearly understood, but previous work has found net changes in synaptic abundance or size across brain regions. This study characterized a diverse array of synaptic responses to sleep loss among different cell types within the same circuit. These findings may suggest that distinct cell types and connections within the MB are governed by heterogeneous plasticity rules during sleep disruption. While previous studies have characterized the synaptic effects of sleep history on individual cell types within plastic circuits, the data provide a more comprehensive understanding of the consequences of sleep loss on MB circuits. While this project outlines the local effects of sleep loss on MB connectivity, it is unclear whether specific neural subsets also drive BRP increases within other neuropil compartments of sleep-deprived brains (Weiss, 2021).

    This study found an overall increase in the abundance of reporters for some, but not all, pre-synaptic proteins. These pre-synaptic changes are not distributed equally across all cell types: they are most pronounced in MB-intrinsic KCs. Further, output connections from KCs to different classes of synaptic partners show varying patterns of plasticity in MB sub-circuits that contribute to encoding odor valence, comprise recurrent feedback loops, or relay reinforcement signals. The results indicate that sleep loss may degrade MB-dependent memory by altering several different classes of synapses, but future studies will be required to test the specific roles of changes at individual synapse types and the mechanisms by which prolonged waking reorganizes MB connectivity (Weiss, 2021).

    Compartment specific regulation of sleep by mushroom body requires GABA and dopaminergic signaling

    Sleep is a fundamental behavioral state important for survival and is universal in animals with sufficiently complex nervous systems. Biogenic amines like dopamine, serotonin and norepinephrine have been shown to be critical for sleep regulation across species but the precise circuit mechanisms underlying how amines control persistence of sleep, arousal and wakefulness remain unclear. The fruit fly, Drosophila melanogaster, provides a powerful model system for the study of sleep and circuit mechanisms underlying state transitions and persistence of states to meet the organisms motivational and cognitive needs. In Drosophila, two neuropils in the central brain, the mushroom body (MB) and the central complex (CX) have been shown to influence sleep homeostasis and receive aminergic neuromodulator input critical to sleep-wake switch. Dopamine neurons (DANs) are prevalent neuromodulator inputs to the MB but the mechanisms by which they interact with and regulate sleep- and wake-promoting neurons within MB are unknown. This study investigated the role of subsets of PAM-DANs that signal wakefulness and project to wake-promoting compartments of the MB. This study found that PAM-DANs are GABA responsive and require GABA(A)-Rdl receptor in regulating sleep. In mapping the pathways downstream of PAM neurons innervating γ5 and β'2 MB compartments it was found that wakefulness is regulated by both DopR1 and DopR2 receptors in downstream Kenyon cells (KCs) and mushroom body output neurons (MBONs). Taken together, a dopamine modulated sleep microcircuit has been identified within the mushroom body that has previously been shown to convey information about positive and negative valence critical for memory formation. These studies will pave the way for understanding how flies balance sleep, wakefulness and arousal (Driscoll, 2021).

    Protocerebral Bridge Neurons That Regulate Sleep in Drosophila melanogaster

    The central complex is one of the major brain regions that control sleep in Drosophila. However, the circuitry details of sleep regulation have not been elucidated yet. This study shows a novel sleep-regulating neuronal circuit in the protocerebral bridge (PB) of the central complex. Activation of the PB interneurons labeled by the R59E08-Gal4 and the PB columnar neurons with R52B10-Gal4 promoted sleep and wakefulness, respectively. A targeted GFP reconstitution across synaptic partners (t-GRASP) analysis demonstrated synaptic contact between these two groups of sleep-regulating PB neurons. Furthermore, it was found that activation of a pair of dopaminergic (DA) neurons projecting to the PB (T1 DA neurons) decreased sleep. The wake-promoting T1 DA neurons and the sleep-promoting PB interneurons formed close associations. Dopamine 2-like receptor (Dop2R) knockdown in the sleep-promoting PB interneurons increased sleep. These results indicated that the neuronal circuit in the PB, regulated by dopamine signaling, mediates sleep-wakefulness (Tomita, 2021).

    Hugin (+) neurons provide a link between sleep homeostat and circadian clock neurons

    Sleep is controlled by homeostatic mechanisms, which drive sleep after wakefulness, and a circadian clock, which confers the 24-h rhythm of sleep. These processes interact with each other to control the timing of sleep in a daily cycle as well as following sleep deprivation. However, the mechanisms by which they interact are poorly understood. These studies show that hugin (+) neurons, previously identified as neurons that function downstream of the clock to regulate rhythms of locomotor activity, are also targets of the sleep homeostat. Sleep deprivation decreases activity of hugin (+) neurons, likely to suppress circadian-driven activity during recovery sleep, and ablation of hugin (+) neurons promotes sleep increases generated by activation of the homeostatic sleep locus, the dorsal fan-shaped body (dFB). Also, mutations in peptides produced by the hugin (+) locus increase recovery sleep following deprivation. Transsynaptic mapping reveals that hugin (+) neurons feed back onto central clock neurons, which also show decreased activity upon sleep loss, in a Hugin peptide-dependent fashion. It is proposed that hugin (+) neurons integrate circadian and sleep signals to modulate circadian circuitry and regulate the timing of sleep (Schwarz, 2021).

    Circuit analysis reveals a neural pathway for light avoidance in Drosophila larvae

    Understanding how neural circuits underlie behaviour is challenging even in the connectome era because it requires a combination of anatomical and functional analyses. This is exemplified in the circuit underlying the light avoidance behaviour displayed by Drosophila melanogaster larvae. While this behaviour is robust and the nervous system relatively simple, the circuit is only partially delineated with some contradictions among studies. This study devised trans-Tango MkII, an offshoot of the transsynaptic circuit tracing tool trans-Tango, and implement it in anatomical tracing together with functional analysis. Neuronal inhibition was used to test necessity of particular neuronal types in light avoidance and selective neuronal activation to examine sufficiency in rescuing light avoidance deficiencies exhibited by photoreceptor mutants. These studies reveal a four-order circuit for light avoidance connecting the light-detecting photoreceptors with a pair of neuroendocrine cells via two types of clock neurons. This approach can be readily expanded to studying other circuits (Sorkac, 2022).

    The study revealed a circuit consisting of four orders of neurons that connect the Rh5 photoreceptors to PTTH neurons via the 5th-LaN and DN2s. While this circuit mediates the response to bright light, the observation that DN1s are necessary for photophobic response only to low light intensity indicates the existence of an additional pathway for dim light. It is noteworthy that a third, independent system has been reported in which a gustatory receptor mediates photophobic response to high-intensity light in class IV multidendritic neurons. This study has no information as to at which level these pathways might meet, if at all, upstream of the motor neurons (Sorkac, 2022).

    The results clarify several earlier studies regarding the role of Pdf-LaNs in light avoidance. In the current experiments, Pdf-LaNs are dispensable for light avoidance, yet their activation is attractive. A potential explanation is that Pdf-LaNs may modulate larval photophobia via inhibition, especially since adult Pdf-LaNs are glycinergic. In addition, the results contradict a previous study reporting that Pdf-LaNs are presynaptic to PTTH neurons. This study relied on a version of GRASP that is in fact not synaptic. Thus, the proposed connection could have been the result of a non-synaptic reconstitution of GFP due to proximity especially since reconstituted GFP was not observed using a synaptic version of GRASP. This result, however, does not rule out a Pdf-LaN-mediated inhibition of the light avoidance circuit from the Rh5 photoreceptors to PTTH neurons. It is conceivable that, alongside their roles in alternative circuits that mediate this behaviour, Pdf-LaNs play inhibitory roles in this circuit as well. Indeed, ablating Pdf-LaNs increases the activity in PTTH neurons as revealed by the GCaMP signal (Sorkac, 2022).

    Our analysis of the robust light avoidance response in larvae exemplifies the importance of employing a comprehensive approach combining circuit tracing together with neuronal inhibition and activation to test necessity and sufficiency. Circuit epistasis analysis was made possible by trans-Tango MkII, a new version of trans-Tango that allows researchers to trace and manipulate neural circuits in Drosophila larvae. The combination of a robust and user-friendly genetic tool such as trans-Tango MkII with careful functional analysis constitutes a powerful approach that can be readily expanded to studying other circuits and behaviours (Sorkac, 2022).

    A thermometer circuit for hot temperature adjusts Drosophila behavior to persistent heat

    Small poikilotherms such as the fruit fly Drosophila depend on absolute temperature measurements to identify external conditions that are above (hot) or below (cold) their preferred range and to react accordingly. Hot and cold temperatures have a different impact on fly activity and sleep, but the circuits and mechanisms that adjust behavior to specific thermal conditions are not well understood. This study use patch-clamp electrophysiology to show that internal thermosensory neurons located within the fly head capsule (the AC neurons(1)) function as a thermometer active in the hot range. ACs exhibit sustained firing rates that scale with absolute temperature-but only for temperatures above the fly's preferred ~25°C (i.e., "hot" temperature). ACs were identified in the fly brain connectome and demonstrate that they target a single class of circadian neurons, the LPNs.(2) LPNs receive excitatory drive from ACs and respond robustly to hot stimuli, but their responses do not exclusively rely on ACs. Instead, LPNs receive independent drive from thermosensory neurons of the fly antenna via a new class of second-order projection neurons (TPN-IV). Finally, silencing LPNs blocks the restructuring of daytime "siesta" sleep, which normally occurs in response to persistent heat. Previous work described a distinct thermometer circuit for cold temperature.(3) Together, the results demonstrate that the fly nervous system separately encodes and relays absolute hot and cold temperature information, show how patterns of sleep and activity can be adapted to specific temperature conditions, and illustrate how persistent drive from sensory pathways can impact behavior on extended temporal scales (Alpert, 2022).

    Interneurons of fan-shaped body promote arousal in Drosophila

    Sleep is required to maintain physiological functions and is widely conserved across species. To understand the sleep-regulatory mechanisms, sleep-regulating genes and neuronal circuits are studied in various animal species. In the sleep-regulatory neuronal circuits in Drosophila melanogaster, the dorsal fan-shaped body (dFB) is a major sleep-promoting region. However, other sleep-regulating neuronal circuits were not well identified. It was recently found that arousal-promoting T1 dopamine neurons, interneurons of protocerebral bridge (PB) neurons, and PB neurons innervating the ventral part of the FB form a sleep-regulatory circuit, which was named "the PB-FB pathway". In the exploration of other sleep-regulatory circuits, it was found that activation of FB interneurons, also known as pontine neurons, promoted arousal. FB interneurons had possible connections with the PB-FB pathway and dFB neurons. Ca2+ imaging revealed that FB interneurons received excitatory signals from the PB-FB pathway. The possible role of FB interneurons to regulate dFB neurons was demonstrated. These results suggested the role of FB interneurons in sleep regulation (Kato, 2022).

    This study reports a novel sleep-regulatory pathway that promotes arousal. This study first focused on FB interneurons and found that cholinergic FB interneurons promoted arousal. The arousal-promoting effect of FB interneurons was confirmed by using more specific drivers. These drivers label FB interneurons which receive input from P-FN neurons (that project from the protocerebral bridge to the ventral FB and the NO) and send output to dFB neurons. There should be other FB interneurons that do not have a connection with P-FN neurons or dFB neurons. It means that FB interneurons labeled by two split drivers are only a part of FB interneurons. Therefore we considered that the weaker effects were due to the smaller number of neurons labeled by split-Gal4 lines than NP2320, not the effect of neurons other than FB interneurons. No aclear sleep rebound was observed after neuronal activation. In the previous study, R52B10-Gal4 was used which is reported to drive sleep rebound in the female fly. A clear sleep rebound was observed in female flies but not in male flies. These results indicated that there is a sex difference in the regulation of sleep rebound, at least, in R52B10 neurons. The current study used only male flies and this could be one of the reasons why no clear sleep rebound was observed. It was next asked about the relationship between FB interneurons and known sleep-regulatory circuits. GRASP and Ca2+ experiments were performed, and FB interneurons and R52B10 neurons which label the output neurons of the PB-FB pathway were shown to be anatomically and functionally connected . Although the possibility of other pathways downstream to R52B10 cannot be excluded, this study demonstrated clearly that FB interneurons are one of the downstream to R52B10. Further study will show the impact size of the connection between them in sleep regulation. To investigate the postsynaptic partners of FB interneurons, a GRASP experiment was conducted. FB interneurons and dFB neurons labeled by R23E10 were found to form close associations. Besides, according to the connectome paper and connectome dataset, vDeltaB, C, D, and hDeltaC receive input from P-FN neurons and send output to FB tangential neurons which arborize in layers 6 and 7. This information also supported the idea that R52B10 neurons including P-FN neurons, FB interneurons like vDeltaB, C, D, and hDeltaC neurons, and dFB neurons that are consisted of FB tangential neurons which arborize in layers 6 and 7 form a neuronal circuit. Future study will clarify their functional connection and the role of this circuit in sleep regulation. Furthermore, a previous study showed that neurons that project to the ventral FB (vFB neurons) promote sleep and mediate consolidation of long-term memory. Since axon terminals and dendrites of FB interneurons arborize in both the dorsal and ventral FB, there would be interactions between dFB and vFB neurons via FB interneurons. Further research will clarify the functional relationship between these neurons (Kato, 2022).

    According to previous reports, FB interneurons regulate optomotor behavior and express tachykinin, a neuropeptide that regulates aggression. Additionally, T1 dopamine neurons, which are upstream of R52B10 neurons, regulate aggression as well. Besides, P2 neurons, which include FB interneurons, regulate chronic isolation evoked sleep loss. Moreover, courtship-regulator P1 neurons activate T1 neurons and modulate sleep/courtship balance based on the nutritional status. Taking all the information mentioned above into account, it is considerd that arousal signals related to aggression, courtship, nutrition, and vision converge into the PB-FB pathway including FB interneurons to regulate arousal. Further studies should clarify the role of these arousal signals on the PB-FB pathway and FB interneurons in sleep regulation (Kato, 2022).

    In conclusion, the results provided possible sleep-regulatory neurons that may connect with the PB-FB pathway and dFB neurons. It is hypothesized that arousal signals are sent from the PB-FB pathway to FB interneurons, inhibit dFB neurons via inhibitory signals, and regulate sleep (Kato, 2022).

    Compartment specific regulation of sleep by mushroom body requires GABA and dopaminergic signaling
    Sleep is a fundamental behavioral state important for survival and is universal in animals with sufficiently complex nervous systems. As a highly conserved neurobehavioral state, sleep has been described in species ranging from jellyfish to humans. Biogenic amines like dopamine, serotonin and norepinephrine have been shown to be critical for sleep regulation across species but the precise circuit mechanisms underlying how amines control persistence of sleep, arousal and wakefulness remain unclear. The fruit fly, Drosophila melanogaster, provides a powerful model system for the study of sleep and circuit mechanisms underlying state transitions and persistence of states to meet the organisms motivational and cognitive needs. In Drosophila, two neuropils in the central brain, the mushroom body (MB) and the central complex (CX) have been shown to influence sleep homeostasis and receive aminergic neuromodulator input critical to sleep-wake switch. Dopamine neurons (DANs) are prevalent neuromodulator inputs to the MB but the mechanisms by which they interact with and regulate sleep- and wake-promoting neurons within MB are unknown. This study investigated the role of subsets of PAM-DANs that signal wakefulness and project to wake-promoting compartments of the MB. PAM-DANs were found to be GABA responsive and required GABA(A)-Rdl receptor in regulating sleep. In mapping the pathways downstream of PAM (protocerebral anterior medial) neurons innervating γ5 and β'2 MB compartments it was found that wakefulness is regulated by both DopR1 and DopR2 receptors in downstream Kenyon cells (KCs) and mushroom body output neurons (MBONs). Taken together, this study has identified and characterized a dopamine modulated sleep microcircuit within the mushroom body that has previously been shown to convey information about positive and negative valence critical for memory formation. These studies will pave way for understanding how flies balance sleep, wakefulness and arousal (Driscoll, 2021).

    The mushroom body lobes are tiled by discrete anatomic compartments defined by the axons of a specific subset of DANs and the dendrites of one or two mushroom body output neurons (MBONs). This anatomical arrangement positions DANs to strategically convey positive and negative reinforced information by changing the synaptic weight of KC-MBONs in producing aversive and appetitive responses (Driscoll, 2021).

    While, the most in-depth analysis of these synapses and distinct DAN-KC-MBON connectivity and behavioral output comes from studies of olfactory conditioning, there is evidence that these synapses play a critical role in innate behaviors like feeding and sleep. Although, role of DA on sleep has been extensively investigated in Drosophila, the commonly used TH-Gal4 driver line labels most dopamine neuron clusters, but is absent from the several PAM clusters that projects to MB (Driscoll, 2021).

    This study specifically probed PAM subsets that project to γ5, γ4, and β'2 MB compartments. This study focused on this subset because KCs and MBONs downstream of these PAM neurons can be neuroanatomically resolved and have been shown to be required for wakefulness. Further, KCs and MBONs that form the γ5, γ4, and β'2 synaptic compartments alter their spontaneous neural activity in response to sleep need (induced by mechanical sleep-deprivation). The ability to use cell-specific split-GAL4 tools provides opportunity to resolve the precise circuit mechanisms by which PAM neurons regulate wakefulness (Driscoll, 2021).

    GABA signaling also modulates sleep and wake microcircuits within MB. The key source of GABA in the MB is anterior paired lateral neurons, APL and dorsal paired medial neurons (DPM), which are electrically coupled and increase sleep by GABAergic inhibition of wake-promoting KCs. In the context of associative learning, there is strong evidence for interactions between KCs, APL, DPM and DANs but it is not clear if GABA and dopamine signaling represent opposing inputs to the KCs and MBONs in the regulation of sleep. This study found that the excitability of PAM DANs involved in wakefulness is blocked by sleep-promoting GABA signaling and mediated by ionotropic receptor subtype GABAA-Rdl (Driscoll, 2021).

    A recent study showed that GABA inhibitory input to the presynaptic terminals of the PAM neurons regulates appetitive memory and that this interaction is mediated by GABA-B3 receptors that are clustered in PAM boutons localized to PAM-γ5 and -α1 compartments. These data are consistent with the findings that PAM-γ5 are GABA responsive and that multiple receptors are critical to this interaction. Since, no role was found for GABA-B3 in PAM mediated sleep regulation, it is likely that PAM γ5, γ4, and β'2 express multiple GABA receptors which are differentially recruited in sleep and learning. How and what regulates the expression of these receptors in PAM subsets presents a potential mechanism of presynaptic gating to MB core circuits. Transcriptomic analysis of PAM neurons reveals extremely high levels of Rdl expression followed by GABA-B3. Among the PAM subsets mean TPM or transcripts per million of Rdl receptor in PAM γ5, γ4, and β'2 are much higher as compared to other PAM subsets (Driscoll, 2021).

    Simple connection query search of the recently released hemibrain data85 reveals there is significant bidirectional connectivity between APL, DPM, and PAM neurons. Further, a recent study showed that APL neurons express the inhibitory D2R receptor55. APL mediated GABAergic inhibition of the PAM neurons was recently shown to control the intensity and specificity of olfactory appetitive memory but previous results show that blocking GABA release from APL neurons only modestly affects sleep phenotypes (Driscoll, 2021).

    While, the role of APL in GABA signaling to PAM γ5, γ4, and β'2 cannot be completely ruled out, other inputs to wake-regulating PAM DANs could also be GABAergic and critical for promoting sleep. A recent study using EM dataset of a Full Adult Female Fly Brain (FAFB) mapped the inputs and outputs of the PAMγ5 DANs and identified that this cell type is highly heterogenous and in addition to recurrent feedback from MBON01 γ5β'2a, it receives extensive input from other MBONs, sub-esophageal output neurons (SEZONs) and lateral horn output neurons86. The EM data also reveals that octopaminergic neurons synapse onto PAM γ5, γ4, and β'2 DANs. Whether, these inputs play a role in wakefulness is unknown but suggests that the PAMγ5 could serve as a key link between sensory inputs, wake-promoting octopamine signal and core sleep regulating circuitry within the MB. Each of these inputs could modulate PAM-DAN activity and dopamine release in regulating wakefulness via the MB (Driscoll, 2021).

    In addition to probing the release and activity of these PAM-DANs the dopamine receptors and their location within the MB in signaling wakefulness were also explored. To this end validated RNAi lines were expressed in subsets of KCs and MBONs; DopR1 and DopR2 were found to be critical in mediating the wakefulness signal via KCs and γ5β'2 MBONs. Knocking down the receptor consistently increased total sleep and bout length. Furthermore, specific manipulations of DopR receptors within the MB did not directly alter locomotor activity as observed by manipulation of these receptors in CX. Although, loss-of-function mutations of D1 dopamine receptor DopR are shown to enhance repetitive air puff startle-induced arousal and increase sleep. Expression and restoration of DopR in the mutant background specifically in the central complex rescues the startle response, while, the sleep phenotype is rescued via a broad MB driver. The current data extends these findings by showing that the DopR receptors regulate sleep via the MB γ5 and β'2 compartment. Although, targeted RNAi experiments show that DopR's are required for sleep regulation by KCs and MBONs, the lack of a sleep phenotype in DopR2 mutant could be a result of global loss of receptor in the mutant as opposed to targeted loss of receptor function within MB. Dopamine signals wakefulness by activation of wake-promoting neurons of MB via DopR1 and DopR2 and within. the central complex, neurons of dFB are inhibited by dopamine via DopR2. Hence, DopR2 has opposing effects within MB and CX (Driscoll, 2021).

    In vitro characterization indicates that DopR's signal through distinct G-proteins, with DopR1 via Gαs to stimulate cAMP production and DopR2 coupling to Gαq via increased calcium. These receptors are thought to have differential sensitivity to dopamine and could be potentially recruited by varying DA release or DAN activity. In the context of sleep regulation, this work reveals that both DopR1 and DopR2 induce wakefulness via the γ5 β'2 MB compartment but not γ4 compartment. Although, chronic activation of PAM γ4 induces wakefulness, the glutamatergic MBON γ4 < γ1,2 projects to multiple compartments and could potentially activate or inhibit MBONs and PAMs projecting to γ1 and γ2 compartment. The interaction between compartments is not well understood in the context of sleep and wake regulation and requires further investigation to better understand the role of DopR2 in regulating the γ4 compartment. The neuroanatomical specificity obtained from split-Gal4 lines combined with EM data has paved way for more detailed analysis of the role of dopamine signaling to MB in the context of sleep and other behaviors (Driscoll, 2021).

    The sleep-regulating PAM DANs and associated KCs and MBONs identified in this study are also involved in mediating satiety, novelty, caffeine induced arousal, punishment and reward associated experiences suggesting that the activity of these neurons is tuned to several wake and arousal associated behaviors. This is further supported by the EM connectome data showing that MB receives extensive gustatory, auditory and visual input in addition to olfactory input (Driscoll, 2021).

    Current models of sleep regulation rely on two main processes, the circadian clock and the sleep homeostat and don't completely account for multiple external and internal factors that influence wakefulness. The ability to sleep, however, is influenced by motivational or cognitive stimuli. It is therefore envisioned that sleep, wakefulness and arousal within MB are not located in distinct circuits, but rather mediated by distinct processes within a common circuit (Driscoll, 2021).

    Circadian autophagy drives iTRF-mediated longevity

    Time-restricted feeding (TRF) has recently gained interest as a potential anti-ageing treatment for organisms from Drosophila to humans. TRF restricts food intake to specific hours of the day. Because TRF controls the timing of feeding, rather than nutrient or caloric content, TRF has been hypothesized to depend on circadian-regulated functions; the underlying molecular mechanisms of its effects remain unclear. To exploit the genetic tools and well-characterized ageing markers of Drosophila, this study developed an intermittent TRF (iTRF) dietary regimen that robustly extended fly lifespan and delayed the onset of ageing markers in the muscles and gut. iTRF enhanced circadian-regulated transcription, and iTRF-mediated lifespan extension required both circadian regulation and autophagy, a conserved longevity pathway. Night-specific induction of autophagy was both necessary and sufficient to extend lifespan on an ad libitum diet and also prevented further iTRF-mediated lifespan extension. By contrast, day-specific induction of autophagy did not extend lifespan. Thus, these results identify circadian-regulated autophagy as a critical contributor to iTRF-mediated health benefits in Drosophila. Because both circadian regulation and autophagy are highly conserved processes in human ageing, this work highlights the possibility that behavioural or pharmaceutical interventions that stimulate circadian-regulated autophagy might provide people with similar health benefits, such as delayed ageing and lifespan extension (Ulgherait, 2021).

    A sleep-inducing gene, nemuri, links sleep and immune function in Drosophila

    Sleep remains a major mystery of biology. In particular, little is known about the mechanisms that account for the drive to sleep. In an unbiased screen of more than 12,000 Drosophila lines, identified a single gene, nemuri (CG31813), that induces sleep. The NEMURI protein is an antimicrobial peptide that can be secreted ectopically to drive prolonged sleep (with resistance to arousal) and to promote survival after infection. Loss of nemuri increased arousability during daily sleep and attenuated the acute increase in sleep induced by sleep deprivation or bacterial infection. Conditions that increase sleep drive induced expression of nemuri in a small number of fly brain neurons and targeted it to the sleep-promoting, dorsal fan-shaped body. It is proposed that NEMURI is a bona fide sleep homeostasis factor that is particularly important under conditions of high sleep need; because these conditions include sickness, these findings provide a link between sleep and immune function (Toda, 2019).

    Neurocalcin regulates nighttime sleep and arousal in Drosophila

    Sleep-like states in diverse organisms can be separated into distinct stages, each with a characteristic arousal threshold. However, the molecular pathways underlying different sleep stages remain unclear. The fruit fly, Drosophila melanogaster, exhibits consolidated sleep during both day and night, with night sleep associated with higher arousal thresholds compared to day sleep. This study identified a role for the neuronal calcium sensor protein Neurocalcin (NCA) in promoting sleep during the night but not the day by suppressing nocturnal arousal and hyperactivity. Both circadian and light-sensing pathways define the temporal window in which NCA promotes sleep. Furthermore, NCA promotes sleep by suppressing synaptic release from a dispersed wake-promoting neural network and the mushroom bodies, a sleep-regulatory center, are a module within this network. These results advance the understanding of how sleep stages are genetically defined (ChenK, 2019).

    Conserved properties of Drosophila Insomniac link sleep regulation and synaptic function

    Sleep is an ancient animal behavior that is regulated similarly in species ranging from flies to humans. Various genes that regulate sleep have been identified in invertebrates, but whether the functions of these genes are conserved in mammals remains poorly explored. Drosophila insomniac (inc) mutants exhibit severely shortened and fragmented sleep. Inc protein physically associates with the Cullin-3 (Cul3) ubiquitin ligase, and neuronal depletion of Inc or Cul3 strongly curtails sleep, suggesting that Inc is a Cul3 adaptor that directs the ubiquitination of neuronal substrates that impact sleep. Three proteins similar to Inc exist in vertebrates-KCTD2, KCTD5, and KCTD17-but are uncharacterized within the nervous system and their functional conservation with Inc has not been addressed. This study shows that Inc and its mouse orthologs exhibit striking biochemical and functional interchangeability within Cul3 complexes. Remarkably, KCTD2 and KCTD5 restore sleep to inc mutants, indicating that they can substitute for Inc in vivo and engage its neuronal targets relevant to sleep. Inc and its orthologs localize similarly within fly and mammalian neurons and can traffic to synapses, suggesting that their substrates may include synaptic proteins. Consistent with such a mechanism, inc mutants exhibit defects in synaptic structure and physiology, indicating that Inc is essential for both sleep and synaptic function. These findings reveal that molecular functions of Inc are conserved through ~600 million years of evolution and support the hypothesis that Inc and its orthologs participate in an evolutionarily conserved ubiquitination pathway that links synaptic function and sleep regulation (Li, 2017).

    The presence of sleep states in diverse animals has been suggested to reflect a common purpose for sleep and the conservation of underlying regulatory mechanisms. This study has shown that attributes of the Insomniac protein likely to underlie its impact on sleep in Drosophila-its ability to function as a multimeric Cul3 adaptor and engage neuronal targets that impact sleep-are functionally conserved in its mammalian orthologs. This comparative analysis of Inc family members in vertebrate and invertebrate neurons furthermore reveals that these proteins can traffic to synapses and that Inc itself is essential for normal synaptic structure and excitability. These findings support the hypothesis that Inc family proteins serve as Cul3 adaptors and direct the ubiquitination of conserved neuronal substrates that impact sleep and synaptic function (Li, 2017).

    The ability of KCTD2 and KCTD5 to substitute for Inc in the context of sleep is both surprising and notable given the complexity of sleep-wake behavior and the likely functions of these proteins as Cul3 adaptors. Adaptors are multivalent proteins that self-associate, bind Cul3, and recruit substrates, and these interactions are further regulated by additional post-translational mechanisms. The findings indicate that KCTD2 and KCTD5 readily substitute for Inc within oligomeric Inc-Cul3 complexes, and strongly suggest that these proteins recapitulate other aspects of Inc function in vivo including the ability to engage neuronal targets that impact sleep. The simplest explanation for why KCTD2 and KCTD5 have retained the apparent ability to engage Inc targets despite the evolutionary divergence of Drosophila and mammals is that orthologs of Inc targets are themselves conserved in mammals. This inference draws support from manipulations of Drosophila Roadkill/HIB and its mammalian ortholog SPOP, Cul3 adaptors of the MATH-BTB family that regulate the conserved Hedgehog signaling pathway. While the ability of SPOP to substitute for HIB has not been assessed by rescue at an organismal level, clonal analysis in Drosophila indicates that ectopically expressed mouse SPOP can degrade the endogenous HIB substrate Cubitus Interruptus (Ci), and conversely, that HIB can degrade mammalian Gli proteins that are the conserved orthologs of Ci and substrates of SPOP. By analogy, Inc targets that impact sleep are likely to have orthologs in vertebrates that are recruited by KCTD2 and KCTD5 to Cul3 complexes. While the manipulations do not resolve whether KCTD17 can substitute for Inc in vivo, the ability of KCTD17 to assemble with fly Inc and Cul3 suggests that functional divergence among mouse Inc orthologs may arise outside of the BTB domain, and in particular may reflect properties of their C-termini including the ability to recruit substrates (Li, 2017).

    The finding that Inc can transit to synapses and is required for normal synaptic function is intriguing in light of hypotheses that invoke synaptic homeostasis as a key function of sleep. While ubiquitin-dependent mechanisms contribute to synaptic function and plasticity and sleep is known to influence synaptic remodeling in both vertebrates and invertebrates, molecular links between ubiquitination, synapses, and sleep remain poorly explored. Other studies in flies have indicated that regulation of RNA metabolism may similarly couple synaptic function and the control of sleep. Alterations in the activity of the Fragile X mental retardation protein (FMR), a regulator of mRNA translation, cause defects in the elaboration of neuronal projections and the formation of synapses as well as changes in sleep duration and consolidation. Loss of Adar, a deaminase that edits RNA, leads to increased sleep through altered glutamatergic synaptic function. Like Inc, these proteins are conserved in mammals, suggesting that further studies in flies may provide insights into diverse mechanisms by which sleep influences synaptic function and conversely, how changes in synapses may impact the regulation of sleep (Li, 2017).

    These findings at a model synapse suggest that the impact of Inc on synaptic function may be intimately linked to its influence on sleep but do not yet resolve important aspects of such a mechanism. The synaptic phenotypes of inc mutants-increased synaptic growth, decreased evoked neurotransmitter release, and modest effects on spontaneous neurotransmission-are qualitatively distinct from those of other short sleeping mutants. Shaker (Sh) and Hyperkinetic (Hk) mutations decrease sleep in adults but increase both excitability and synaptic growth at the NMJ, suggesting that synaptic functions of Inc may affect sleep by a mechanism different than broad neuronal hyperexcitability. While a parsimonious model is that Inc directs the ubiquitination of a target critical for synaptic transmission both at the larval NMJ and in neuronal populations that promote sleep, this hypothesis awaits the elucidation of Inc targets, definition of the temporal requirements of Inc activity, and further mapping of the neuronal populations through which Inc impacts sleep. Finally, determining the localization of endogenous Inc within neurons is essential to distinguish possible presynaptic and postsynaptic functions of Inc and whether Inc engages local synaptic proteins or extrasynaptic targets that ultimately influence synaptic function (Li, 2017).

    A clear implication of these findings is that neuronal targets and synaptic functions of Inc may be conserved in other animals. While the impact of Inc orthologs on sleep in vertebrates is as yet unknown, findings from C. elegans support the notion that conserved molecular functions of Inc and Cul3 may underlie similar behavioral outputs in diverse organisms. INSO-1/C52B11.2, the only C. elegans ortholog of Inc, interacts with Cul3, and RNAi against Cul3 and INSO-1 reduces the duration of lethargus, a quiescent sleep-like state, suggesting that effects of Cul3- and Inc-dependent ubiquitination on sleep may be evolutionarily conserved. The functions of Inc orthologs and Cul3 in the mammalian nervous system await additional characterization, but emerging data suggest functions relevant to neuronal physiology and disease. Human mutations at the KCTD2/ATP5H locus are associated with Alzheimer's disease, and mutations of KCTD17 with myoclonic dystonia. Cul3 lesions have been associated in several studies with autism spectrum disorders and comorbid sleep disturbances. More generally, autism spectrum disorders are commonly associated with sleep deficits and are thought to arise in many cases from altered synaptic function, but molecular links to sleep remain fragmentary. Studies of Inc family members and their conserved functions in neurons are likely to broaden understanding of how ubiquitination pathways may link synaptic function to the regulation of sleep and other behaviors (Li, 2017).

    Dissecting the Genetic Basis of Variation in Drosophila Sleep Using a Multiparental QTL Mapping Resource

    There is considerable variation in sleep duration, timing and quality in human populations, and sleep dysregulation has been implicated as a risk factor for a range of health problems. Human sleep traits are known to be regulated by genetic factors, but also by an array of environmental and social factors. These uncontrolled, non-genetic effects complicate powerful identification of the loci contributing to sleep directly in humans. The model system, Drosophila melanogaster, exhibits a behavior that shows the hallmarks of mammalian sleep, and this study used a multitiered approach, encompassing high-resolution QTL mapping, expression QTL data, and functional validation with RNAi to investigate the genetic basis of sleep under highly controlled environmental conditions. A battery of sleep phenotypes was measured in >750 genotypes derived from a multiparental mapping panel and identified several, modest-effect QTL contributing to natural variation for sleep. Merging sleep QTL data with a large head transcriptome eQTL mapping dataset from the same population allowed refining the list of plausible candidate causative sleep loci. This set includes genes with previously characterized effects on sleep and circadian rhythms, in addition to novel candidates. Finally, adult, nervous system-specific RNAi was used on the Dopa decarboxylase, dyschronic, and timeless genes, finding significant effects on sleep phenotypes for all three. The genes resolved in this study are strong candidates to harbor causative, regulatory variation contributing to sleep (Smith, 2020).

    The genetic basis of diurnal preference in Drosophila melanogaster

    Most animals restrict their activity to a specific part of the day, being diurnal, nocturnal or crepuscular. The genetic basis underlying diurnal preference is largely unknown. Under laboratory conditions, Drosophila melanogaster is crepuscular, showing a bi-modal activity profile. However, a survey of strains derived from wild populations indicated that high variability among individuals exists, including flies that are nocturnal. Using a highly diverse population, an artificial selection experiment was performed, selecting flies with extreme diurnal or nocturnal preference. After 10 generations, highly diurnal and nocturnal strains were obtained. Whole-genome expression analysis was used to identify differentially expressed genes in diurnal, nocturnal and crepuscular (control) flies. Other than one circadian clock gene (pdp1), most differentially expressed genes were associated with either clock output (pdf, to) or input (Rh3, Rh2, msn). This finding was congruent with behavioural experiments indicating that both light masking and the circadian pacemaker are involved in driving nocturnality. This study demonstrates that genetic variation segregating in wild populations contributes to substantial variation in diurnal preference. Candidate genes associated with diurnality/nocturnality, while data emerging from expression analysis and behavioural experiments suggest that both clock and clock-independent pathways are involved in shaping diurnal preference. The diurnal and nocturnal selection strains provide us with a unique opportunity to understand the genetic architecture of diurnal preference (Pegoraro, 2020).

    Regulatory mechanism of daily sleep by miR-276a

    MiRNAs have attracted more attention in recent years as regulators of sleep and circadian rhythms after their roles in circadian rhythm and sleep were discovered. This study explored the roles of the miR-276a on daily sleep in Drosophila melanogaster, and found a regulatory cycle for the miR-276a pathway, in which miR-276a, regulated by the core CLOCK/CYCLE (CLK/CYC) transcription factor upstream, regulates sleep via suppressing targets TIM and NPFR1. (a) Loss of miR-276a function makes the flies sleep more during both daytime and nighttime, while flies with gain of miR-276a function sleep less; (b) MiR-276a is widely expressed in the mushroom body (MB), the pars intercerebralis (PI) and some clock neurons lateral dorsal neurons (LNds), in which tim neurons is important for sleep regulation; (c) MiR-276a promoter is identified to locate in the 8th fragment (aFrag8) of the pre-miR-276a, and this fragment is directly activated and regulated by CLK/CYC; (4) MiR-276a is rhythmically oscillating in heads of the wild-type w(1118), but this oscillation disappears in the loss of function mutant clk(jrk) ; (5) The neuropeptide F receptor 1 (npfr1) was found to be a downstream target of miR-276a. These results clarify that the miR-276a is a very important factor for sleep regulation (Zhang, 2021).

    Integrative Role of 14-3-3epsilon in Sleep Regulation

    Sleep is a crucial factor for health and survival in all animals. This study found by proteomic analysis that some cancer related proteins were impacted by the circadian clock. The 14-3-3ε protein, expression of which is activated by the circadian transcription factor Clock, regulates adult sleep of Drosophila independent of circadian rhythm. Detailed analysis of the sleep regulatory mechanism shows that 14-3-3ε directly targets the Ultrabithorax (Ubx) gene to activate transcription of the pigment dispersing factor (PDF). The dopamine receptor (Dop1R1) and the octopamine receptor (Oamb), are also involved in the 14-3-3ε pathway, which in 14-3-3ε mutant flies causes increases in the dopR1 and OAMB, while downregulation of the DopR1 and Oamb can restore the sleep phenotype caused by the 14-3-3ε mutation. In conclusion, 14-3-3ε is necessary for sleep regulation in Drosophila (Wei, 2021).

    The E3 ubiquitin ligase adaptor Tango10 links the core circadian clock to neuropeptide and behavioral rhythms

    Circadian transcriptional timekeepers in pacemaker neurons drive profound daily rhythms in sleep and wake. This study revealed a molecular pathway that links core transcriptional oscillators to neuronal and behavioral rhythms. Using two independent genetic screens, mutants of Transport and Golgi organization 10 (Tango10) were identified with poor behavioral rhythmicity. Tango10 expression in pacemaker neurons expressing the neuropeptide PIGMENT-DISPERSING FACTOR (PDF) is required for robust rhythms. Loss of Tango10 results in elevated PDF accumulation in nerve terminals even in mutants lacking a functional core clock. TANGO10 protein itself is rhythmically expressed in PDF terminals. Mass spectrometry of TANGO10 complexes reveals interactions with the E3 ubiquitin ligase CULLIN 3 (CUL3). CUL3 depletion phenocopies Tango10 mutant effects on PDF even in the absence of the core clock gene timeless. Patch clamp electrophysiology in Tango10 mutant neurons demonstrates elevated spontaneous firing potentially due to reduced voltage-gated Shaker-like potassium currents. It is proposed that Tango10/Cul3 transduces molecular oscillations from the core clock to neuropeptide release important for behavioral rhythms (Lee, 2021).

    The microtubule associated protein tau suppresses the axonal distribution of PDF neuropeptide and mitochondria in circadian clock neurons

    Disrupted circadian rhythms is a prominent feature of multiple neurodegenerative diseases. Yet mechanisms linking Tau (see Drosophila Tau) to rhythmic behavior remain unclear. This study found that expression of a phosphomimetic human Tau mutant (TauE14) in Drosophila circadian pacemaker neurons disrupts free-running rhythmicity. While cell number and oscillations of the core clock protein PERIOD are unaffected in the small LNv (sLNv) neurons important for free running rhythms, a near complete loss of the major LNv neuropeptide pigment dispersing factor (PDF) in the dorsal axonal projections of the sLNvs. This was accompanied by a ~ 50% reduction in the area of the dorsal terminals and a modest decrease in cell body PDF levels. Expression of wild-type Tau also reduced axonal PDF levels but to a lesser extent than TauE14. TauE14 also induces a complete loss of mitochondria from these sLNv projections. However, mitochondria were increased in sLNv cell bodies in TauE14 flies. These results suggest that TauE14 disrupts axonal transport of neuropeptides and mitochondria in circadian pacemaker neurons, providing a mechanism by which Tau can disrupt circadian behavior prior to cell loss (Zhang, 2021).

    Mitochondrial LETM1 drives ionic and molecular clock rhythms in circadian pacemaker neurons

    The mechanisms that generate robust ionic oscillation in circadian pacemaker neurons are under investigation. This study demonstrates critical functions of the mitochondrial cation antiporter leucine zipper-EF-hand-containing transmembrane protein 1 (LETM1), which exchanges K(+)/H(+) in Drosophila and Ca(2+)/H(+) in mammals, in circadian pacemaker neurons. Letm1 knockdown in Drosophila pacemaker neurons reduced circadian cytosolic H(+) rhythms and prolonged nuclear PERIOD/TIMELESS expression rhythms and locomotor activity rhythms. In rat pacemaker neurons in the hypothalamic suprachiasmatic nucleus (SCN), circadian rhythms in cytosolic Ca(2+) and Bmal1 transcription were dampened by Letm1 knockdown. Mitochondrial Ca(2+) uptake peaks late during the day were also observed in rat SCN neurons following photolytic elevation of cytosolic Ca(2+). Since cation transport by LETM1 is coupled to mitochondrial energy synthesis, it is proposed that LETM1 integrates metabolic, ionic, and molecular clock rhythms in the central clock system in both invertebrates and vertebrates (Morioka, 2022).

    The Statin Target HMG-Coenzyme a Reductase (Hmgcr) Regulates Sleep Homeostasis in Drosophila

    Statins, HMG Coenzyme A Reductase (HMGCR) inhibitors, are a first-line therapy, used to reduce hypercholesterolemia and the risk for cardiovascular events. While sleep disturbances are recognized as a side-effect of statin treatment, the impact of statins on sleep is under debate. Using Drosophila, this study discovered a novel role for Hmgcr in sleep modulation. Loss of pan-neuronal Hmgcr expression affects fly sleep behavior, causing a decrease in sleep latency and an increase in sleep episode duration. The pars intercerebralis (PI), equivalent to the mammalian hypothalamus, was identified as the region within the fly brain requiring Hmgcr activity for proper sleep maintenance. Lack of Hmgcr expression in the PI insulin-producing cells recapitulates the sleep effects of pan-neuronal Hmgcr knockdown. Conversely, loss of Hmgcr in a different PI subpopulation, the corticotropin releasing factor (CRF) homologue-expressing neurons (DH44 neurons), increases sleep latency and decreases sleep duration. Interestingly, loss of Hmgcr in the PI does not affect circadian rhythm, suggesting that Hmgcr regulates sleep by pathways distinct from the circadian clock. Taken together, these findings suggest that Hmgcr activity in the PI is essential for proper sleep homeostasis in flies (Alsehli, 2022).

    A screen for sleep and starvation resistance identifies a wake-promoting role for the auxiliary channel unc79

    The regulation of sleep and metabolism are highly interconnected, and dysregulation of sleep is linked to metabolic diseases that include obesity, diabetes, and heart disease. Furthermore, both acute and long-term changes in diet potently impact sleep duration and quality. To identify novel factors that modulate interactions between sleep and metabolic state, a genetic screen was performed for their roles in regulating sleep duration, starvation resistance, and starvation-dependent modulation of sleep. This screen identified a number of genes with potential roles in regulating sleep, metabolism, or both processes. One such gene encodes the auxiliary ion channel UNC79, which was implicated in both the regulation of sleep and starvation resistance. Genetic knockdown or mutation of unc79 results in flies with increased sleep duration, as well as increased starvation resistance. Previous findings have shown that unc79 is required in pacemaker for 24-hours circadian rhythms. This study found that unc79 functions in the mushroom body, but not pacemaker neurons, to regulate sleep duration and starvation resistance. Together, these findings reveal spatially localized separable functions of unc79 in the regulation of circadian behavior, sleep, and metabolic function (Murakami, 2021).

    The opposing chloride cotransporters KCC and NKCC control locomotor activity in constant light and during long days

    Cation chloride cotransporters (CCCs) regulate intracellular chloride ion concentration ([Cl(-)](i)) within neurons, which can reverse the direction of the neuronal response to the neurotransmitter GABA. Na(+) K(+) Cl(-) (NKCC) and K(+) Cl(-) (KCC) cotransporters transport Cl(-) into or out of the cell, respectively. When NKCC activity dominates, the resulting high [Cl(-)](i) can lead to an excitatory and depolarizing response of the neuron upon GABA(A) receptor opening, while KCC dominance has the opposite effect. This inhibitory-to-excitatory GABA switch has been linked to seasonal adaption of circadian clock function to changing day length, and its dysregulation is associated with neurodevelopmental disorders such as epilepsy. In Drosophila melanogaster, constant light normally disrupts circadian clock function and leads to arrhythmic behavior. This study demonstrates a function for CCCs in regulating Drosophila locomotor activity and GABA responses in circadian clock neurons because alteration of CCC expression in circadian clock neurons elicits rhythmic behavior in constant light. The same effects were observed after downregulation of the Wnk and Fray kinases, which modulate CCC activity in a [Cl(-)](i)-dependent manner. Patch-clamp recordings from the large LNv clock neurons show that downregulation of KCC results in a more positive GABA reversal potential, while KCC overexpression has the opposite effect. Finally, KCC and NKCC downregulation reduces or increases morning behavioral activity during long photoperiods, respectively. In summary, these results support a model in which the regulation of [Cl(-)](i) by a KCC/NKCC/Wnk/Fray feedback loop determines the response of clock neurons to GABA, which is important for adjusting behavioral activity to constant light and long-day conditions (Eick, 2022).

    Wolfram syndrome 1 regulates sleep in dopamine receptor neurons by modulating calcium homeostasis

    Sleep disruptions are quite common in psychological disorders, but the underlying mechanism remains obscure. Wolfram syndrome 1 (WS1) is an autosomal recessive disease mainly characterized by diabetes insipidus/mellitus, neurodegeneration and psychological disorders. It is caused by loss-of function mutations of the WOLFRAM SYNDROME 1 (WFS1) gene, which encodes an endoplasmic reticulum (ER)-resident transmembrane protein. Heterozygous mutation carriers do not develop WS1 but exhibit 26-fold higher risk of having psychological disorders. Since WS1 patients display sleep abnormalities, this study aimed to explore the role of WFS1 in sleep regulation so as to help elucidate the cause of sleep disruptions in psychological disorders. It was found in Drosophila that knocking down wfs1 in all neurons and wfs1 mutation lead to reduced sleep and dampened circadian rhythm. These phenotypes are mainly caused by lack of wfs1 in dopamine 2-like receptor (Dop2R) neurons which act to promote wake. Consistently, the influence of wfs1 on sleep is blocked or partially rescued by inhibiting or knocking down the rate-limiting enzyme of dopamine synthesis, suggesting that wfs1 modulates sleep via dopaminergic signaling. Knocking down wfs1 alters the excitability of Dop2R neurons, while genetic interactions reveal that lack of wfs1 reduces sleep via perturbation of ER-mediated calcium homeostasis. Taken together, a role is proposed for wfs1 in modulating the activities of Dop2R neurons by impinging on intracellular calcium homeostasis, and this in turn influences sleep. These findings provide a potential mechanistic insight for pathogenesis of diseases associated with WFS1 mutations (Hao, 2023).

    Sleep disruptions are common in individuals with psychiatric disorders, and sleep disturbances are risk factors for future onset of depression. However, the mechanism underlying sleep disruptions in psychiatric disorders are largely unclear. Wolfram Syndrome 1 (WS1) is an autosomal recessive neurodegenerative disease characterized by diabetes insipidus, diabetes mellitus, optic atrophy, deafness and psychiatric abnormalities such as severe depression, psychosis and aggression. It is caused by homozygous (and compound heterozygous) mutation of the WOLFRAM SYNDROME 1 (WFS1) gene, which encodes wolframin, an endoplasmic reticulum (ER) resident protein highly expressed in the heart, brain, and pancreas. On the other hand, heterozygous mutation of WFS1 does not lead to WS1 but increase the risk of depression by 26 fold. A study in mice further confirmed that WFS1 mutation is causative for depression. Consistent with the comorbidity of psychiatric conditions and sleep abnormalities, WS1 patients also experience increased sleep problems compared to individuals with type I diabetes and healthy controls. It has been proposed that sleep symptoms can be used as a biomarker of the disease, especially during relatively early stages, but the mechanisms underlying these sleep disturbances are unclear. Considering that heterozygous WFS1 mutation is present in up to 1% of the population and may be a significant cause of psychiatric disorder in the general population, it was decided to investigate the role of wolframin in sleep regulation so as to probe the mechanism underlying sleep disruptions in psychiatric disorders (Hao, 2023).

    Although the wolframin protein does not possess distinct functional domains, a number of ex vivo studies in cultured cells demonstrated a role for it in regulating cellular responses to ER stress and calcium homeostasis, as well as ER-mitochondria cross-talk. Mice that lack Wfs1 in pancreatic β cells develop glucose intolerance and insulin deficiency due to enhanced ER stress and apoptosis. Knocking out Wfs1 in layer 2/3 pyramidal neurons of the medial prefrontal cortex in mice results in increased depression-like behaviors in response to acute restraint stress. This is accompanied by hyperactivation of the hypothalamic-pituitary-adrenal axis and altered accumulation of growth and neurotrophic factors, possibly due to defective ER function. A more recent study in Drosophila found that knocking down wfs1 in the nervous system does not increase ER stress, but enhances the susceptibility to oxidative stress-, endotoxicity- and tauopathy-induced behavioral deficits and neurodegeneration (Sakakibara, 2018). Overall, the physiological function of wolframin in vivo, especially in the brain, remains elusive for the most part. This study identified a role for wolframin in regulating sleep and circadian rhythm in flies. Wfs1 deficiency in the dopamine 2-like receptor (Dop2R) neurons leads to reduced sleep, while inhibiting dopamine synthesis blocks the effect of wfs1 on sleep, implying that wfs1 influences sleep via dopaminergic signaling. It was further found that these Dop2R neurons function to promote wakefulness. Depletion of wfs1 alters neural activity, which leads to increased wakefulness and reduced sleep. Consistent with this, it was found that knocking down the ER calcium channel Ryanodine receptor (RyR) or 1,4,5-trisphosphate receptor (Itpr) rescues while knocking down the sarco(endoplasmic)reticulum ATPase SERCA synergistically enhances the short-sleep phenotype caused by wfs1 deficiency, indicating that wolframin regulates sleep by modulating calcium homeostasis. Taken together, these findings provide a potential mechanism for the sleep disruptions associated with WFS1 mutation, and deepen understanding of the functional role of wolframin in the brain (Hao, 2023).

    Sleep problems have been reported in WS1 patients. Their scores on Pediatric Sleep Questionnaire are more than 3 times higher than healthy controls and doubled compared to individuals with type I diabetes, indicating that the sleep issues are not merely due to metabolic disruptions. Indeed, this study suggests that the sleep problems in human patients are of neural origin, specifically in the wake-promoting Dop2R neurons. Given that the rebound sleep is not significantly altered in wfs1 depleted flies, it is believed that lack of wfs1 does not shorten sleep duration by impairing the sleep homeostasis system. Instead, wfs1 deficiency leads to excessive wakefulness which in turn results in decreased sleep. Considering that heterozygous WFS1 mutation is present in up to 1% of the population, it would be interesting to examine whether these heterozygous mutations contribute to sleep disruptions in the general population (Hao, 2023).

    In mouse, chick, quail and turtle, Wfs1 has been shown to be expressed in brain regions where dopamine receptor Drd1 is expressed. D1-like dopamine receptor binding is increased while striatal dopamine release is decreased in Wfs1-/- mice. The current results also implicate a role for wolframin in dopamine receptor neurons and that lack of wfs1 impacts dopaminergic signaling, as the effects of wfs1 deficiency on both sleep and mushroom body (MB) calcium concentration is blocked by the tyrosine hydroxylase inhibitor AMPT. Both Dop2RGAL4 and GoαGAL4 exhibit prominent expression in the MB, and to be more specific, in the α and β lobes of MB. Previous studies have shown that dopaminergic neurons innervate wake promoting MB neurons, and this study found Dop2R and Goα+ cells to be wake-promoting as well. Therefore, it is suspected that wolframin functions in MB Dop2R/Goα+ neurons to influence sleep. Taken together, these findings suggest an evolutionarily conserved role of wolframin within the dopaminergic system. As this system is also important for sleep/wake regulation in mammals, it is reasonable to suspect that wolframin functions in mammals to modulate sleep by influencing the dopaminergic tone as well (Hao, 2023).

    MB neural activity appears to be enhanced in wfs1 deficient flies based on the results obtained using CaLexA and spH reporters. This elevated activity is consistent with behavioral data, as activation of Dop2R/Goα+ cells reduces sleep, similar to the effects of wfs1 deficiency. Moreover, silencing Dop2R neurons rescues the short-sleep phenotype of wfs1 mutants, while over-expressing wfs1 restores the decreased sleep induced by activation of Dop2R neurons. These findings suggest that wolframin functions to suppress the excitability of MB Dop2R neurons, which in turn reduces wakefulness and promotes sleep. Comparable cellular changes have been observed in SERCA mutant flies. Electric stimulation leads to an initial increase followed by prolonged decrease of calcium concentration in mutant motor nerve terminal compared to the control, while action potential firing is increased in the mutants. This series of results underpin the importance of ER calcium homeostasis in determining membrane excitability and thus neural function (Hao, 2023).

    GCaMP6 monitoring reveals that wfs1 deficiency selectively reduces fluorescence signal in the MB both under baseline condition and after dopamine treatment, which should reflect a reduction of cytosolic calcium level that is usually associated with decreased excitability. Previous studies have shown that lack of wolframin leads to increased basal calcium level in neural progenitor cells derived from induced pluripotent stem (iPS) cells of WS1 patients and primary rat cortical neurons, but after stimulation the rise of calcium concentration is smaller in Wfs1 deficient neurons, resulting in reduced calcium level compared to controls. Similarly, evoked calcium increase is also diminished in fibroblasts of WS1 patients and MIN6 insulinoma cells with WFS1 knocked down. Notably, wolframin has been shown to bind to calmodulin (CaM) in rat brain, and is capable of binding with calcium/CaM complex in vitro and in transfected cells. This may undermine the validity of using GCaMP to monitor calcium level in wfs1 deficient animals and cells, and could potentially account for the contradictory data acquired using CaLexA vs GCaMP (Hao, 2023).

    It is intriguing that in this study wfs1 deficiency appears to selectively impair the function of Dop2R/Goα+ neurons. It has been shown that in the rodent brain Wfs1 is enriched in layer II/III of the cerebral cortex, CA1 field of the hippocampus, central extended amygdala, striatum, and various sensory and motor nuclei in the brainstem. Wfs1 expression starts to appear during late embryonic development in dorsal striatum and amygdala, and the expression quickly expands to other regions of the brain at birth. It is suspect that in flies wfs1 may be enriched in Dop2R/Goα+ cells during a critical developmental period, and that sufficient level of wolframin is required for their maturation and normal functioning in adults. Another possibility is that these cells are particularly susceptible to calcium dyshomeostasis induced by loss of wfs1. Indeed, this is believed to be an important cause of selective dopaminergic neuron loss in Parkinson's Disease, as dopaminergic neurons are unique in their autonomic excitability and selective dependence on calcium channel rather than sodium channel for action potential generation. It is reasoned that Dop2R/Goα+ neurons may also be more sensitive to abnormal intracellular calcium concentration, making them particularly vulnerable to wfs1 deficiency. The pathogenic mechanism underlying the neurodegeneration of WS1 is quite complex, possibly involving brain-wide neurodegenerative processes and neurodevelopmental dis-regulations. The findings of this study provide some evidence supporting a role for altered dopaminergic system during development. Obviously, much more needs to be done to test these hypotheses (Hao, 2023).

    The precise role of wolframin in ER calcium handling is not yet clear. It has been shown in human embryonic kidney (HEK) 293 cells that knocking down WFS1 reduces while over-expressing WFS1 increases ER calcium level. The authors concluded that wolframin upregulates ER calcium concentration by increasing the rate of calcium uptake. Consistently, this study found by genetic interaction that knocking down RyR or Itpr (which act to reduce ER calcium level and thus knocking down either one will increase ER calcium level) rescues the short-sleep phenotype caused by wfs1 mutation, while knocking down SERCA (which acts to increase ER calcium level and thus knocking down this gene will reduce ER calcium level) synergistically enhances the short-sleep phenotype. Based on the results of these genetic interactions, it is proposed that lack of wfs1 increases cytosolic calcium while decreasing ER calcium, leading to hyperexcitability of Dop2R neurons and thus reduced sleep. Knocking down RyR or Itpr decreases cytosolic calcium and increases ER calcium, counteracting the influences of wfs1 deficiency and thus rescuing the short-sleep phenotype. On the other hand, knocking down SERCA further increases cytosolic calcium and decreases ER calcium, rendering an enhancement of the short-sleep phenotype. In line with this, study conducted in neural progenitor cells derived from iPS cells of WS1 patients demonstrated that pharmacological inhibition of RyR can prevent cell death caused by WFS1 mutation. In addition, inhibiting the function of IP3R may mitigate ER stress in wolframin deficient cells. One caveat is that SERCA protein level is increased in primary islets isolated from Wfs1 conditional knock-out mice, as well as in MIN6 cells and neuroblastoma cell line with WFS1 knocked down. It is reasoned that this may be a compensatory increase to make up for the reduced ER calcium level due to wolframin deficiency. It is acknowledged that the hypothesis proposed in in the papert is not supported by GCaMP data, which indicates decreased cytosolic calcium level in Dop2R neurons of wfs1 deficient flies. It is suspected that since the sleep phenotype associated with lack of wfs1 is of developmental origin, it is possible there is an initial increase of cytosolic calcium during critical developmental period in wfs1 deficient flies and this influences the function of Dop2R neurons in adults. Clearly, further characterizations need to be done to fully elucidate this issue, and preferably another calcium indicator independent of the GCaMP system should be employed (Hao, 2023).

    In conclusion, this study identified a role for wolframin in the wake-promoting Dop2R neurons. wfs1 depletion in these cells lead to impaired calcium homeostasis and altered neural activity, which in turn leads to enhanced wakefulness and reduced sleep. This study may provide some insights for the mechanisms underlying the sleep disruptions in individuals with WFS1 mutation, as well as for the pathogenesis of WS1 (Hao, 2023).

    Circadian programming of the ellipsoid body sleep homeostat in Drosophila

    Homeostatic and circadian processes collaborate to appropriately time and consolidate sleep and wake. To understand how these processes are integrated, brief sleep deprivation was scheduled at different times of day in Drosophila, and elevated morning rebound was compared to evening. These effects depend on discrete morning and evening clock neurons, independent of their roles in circadian locomotor activity. In the R5 ellipsoid body sleep homeostat, this study identified elevated morning expression of activity dependent and presynaptic gene expression as well as the presynaptic protein BRUCHPILOT consistent with regulation by clock circuits. These neurons also display elevated calcium levels in response to sleep loss in the morning, but not the evening consistent with the observed time-dependent sleep rebound. These studies reveal the circuit and molecular mechanisms by which discrete circadian clock neurons program a homeostatic sleep center (Andreani, 2022).

    This study describes the neural circuit and molecular mechanisms by which discrete populations of the circadian clock network program the R5 sleep homeostat to control the homeostatic response to sleep loss. A novel protocol was developed to administer brief duration sleep deprivation (SD) and robustly measure homeostatic rebound sleep. Using this strategy, it was demonstrated that homeostatic rebound is significantly higher in the morning than in the evening. Distinct subsets of the circadian clock network and their downstream neural targets were identified that mediate the enhancement and suppression of morning and evening rebound respectively. Using unbiased transcriptomics, very little gene expression significantly altered was observed in response to 2.5 hr sleep deprivation. On the other hand, this study did identify elevated expression of activity-dependent and presynaptic genes in the morning independent of sleep deprivation. Consistent with this finding, elevated levels of the presynaptic protein BRP were observed, that is absent in the absence of Clk. These baseline changes are accompanied by an elevated calcium response to sleep deprivation in the morning mirroring the enhanced behavioral rebound in the morning. Taken together, these data support the model of a circadian regulated homeostat that turns the homeostat up late at night to sustain sleep and down late in the day to sustain wake (Andreani, 2022).

    These studies suggest that homeostatic drive in the R5 neurons is stored post-transcriptionally. As part of these studies, a novel protocol using minimal amounts of SD was developed; this protocol could be useful for minimizing mechanical stress effects and isolating underlying molecular processes crucial for sleep homeostasis. Six to 24 hr of SD in Drosophila is commonly used despite the potential stressful or even lethal effects. This study demonstrated that shorter 2.5 hr deprivations not only induce a robust rebound sleep response, but also the percent of sleep lost recovered at ZT0 is close to 100% versus 14-35% seen in 12 hr SD protocols. Using this shorter SD, this study now found that many effects observed in R5 neurons with 12 hr SD (e.g. increased BRP and upregulation of nmdar subunits) are no longer observed with shorter SD, even though the necessity of R5 neurons for rebound is retained after 2.5 hr SD. Previously, translating ribosome affinity purification (TRAP) was used to show upregulation of nmdar subunits following 12 hr SD. FACS and TRAP are distinct methodologies for targeted collection of RNA for sequencing and can yield unique gene lists. One possibility is that upregulation of nmdar subunits is occurring locally in neuronal processes, which are often lost during FACS, and/or is at the level of translation initiation or elongation. Nonetheless, in agreement with previous work, this study observed SD-induced increases in calcium correlated with behavioral rebound in the morning, suggesting that this process is a core feature of the cellular homeostatic response (Andreani, 2022).

    Using genetically targeted 'loss-of-function' manipulations, this study has defined small subsets of circadian clock neurons and downstream circuits that are necessary for intact clock modulation of sleep homeostasis. The use of intersectional approaches enabled highly resolved targeting not possible with traditional lesioning experiments in the SCN. Collectively these studies defined a potential Glu+ DN1p-TuBu-R4m circuit important for enhancing morning rebound as well as a discrete group of LNds important for suppressing evening rebound. Importantly, most of these effects on sleep rebound are evident in the absence of substantial changes in baseline activity, despite other studies indicating their necessity for normal circadian behavior. Of note, the proposed roles of the DN1p and LNd clock neurons are sleep and wake promotion consistent with the findings after sleep deprivation. It is hypothesized that by using chronic silencing methods, baseline effects may not be evident due to compensatory changes but that these effects are only revealed when the system is challenged by sleep deprivation. Similar genetic strategies in mammals may be useful in uncovering which SCN neurons are driving circadian regulation of sleep homeostasis given the comparable suppression of sleep rebound in the evening in humans. Nonetheless, the finding of sleep homeostasis phenotypes in the absence of significant baseline effects suggests that a major role of these clock neuron subsets may be to manage homeostatic responses (Andreani, 2022).

    These studies suggest that circadian and homeostatic processes do not compete for influence on a downstream neural target but that the circadian clock programs the homeostat itself. Using an unbiased transcriptomic approach, this study discovered time-dependent expression of activity dependent and presynaptic genes, consistent with previous data that the R5 neurons exhibit time-dependent activity. This study observed significant upregulation of several genes involved in synaptic transmission (Syx1a, Rim, nSyb, unc-104, Srpk79D, para, CG5890) evincing a permissive active state for R5 neurons in the morning. This is accompanied by elevated levels of the key presynaptic protein BRP in the morning compared to evening. It is notable that elevated BRP in the morning is the opposite of what would be expected based on a sleep-dependent reduction in BRP proposed by the synaptic homeostasis hypothesis, suggesting a sleep-wake independent mechanism. Previous studies have shown that modulation of BRP levels in the R5 are important for its sleep function, suggesting that changes in BRP levels impact R5 function. It is hypothesized that these baseline transcriptomic changes underlie the differential R5 sensitivity to sleep deprivation is evident as calcium increases in the morning and not the evening. Indeed, trancriptomic and proteomic studies of the mouse forebrain across time and after sleep deprivation are consistent with the model that the circadian clock programs the transcriptome while homeostatic process function post-trranscriptionally, paralleling what was found for R5. It will be of great interest to understand the circuit and molecular mechanisms by which circadian clocks regulate the R5 neuronal calcium and synaptic properties and whether similar circuit architectures underlie daily mammalian sleep-wake (Andreani, 2022).

    Dissecting the Genetic Basis of Variation in Drosophila Sleep Using a Multiparental QTL Mapping Resource

    There is considerable variation in sleep duration, timing and quality in human populations, and sleep dysregulation has been implicated as a risk factor for a range of health problems. Human sleep traits are known to be regulated by genetic factors, but also by an array of environmental and social factors. These uncontrolled, non-genetic effects complicate powerful identification of the loci contributing to sleep directly in humans. The model system, Drosophila melanogaster, exhibits a behavior that shows the hallmarks of mammalian sleep, and this study used a multitiered approach, encompassing high-resolution QTL mapping, expression QTL data, and functional validation with RNAi to investigate the genetic basis of sleep under highly controlled environmental conditions. A battery of sleep phenotypes was measured in >750 genotypes derived from a multiparental mapping panel and identified several, modest-effect QTL contributing to natural variation for sleep. Merging sleep QTL data with a large head transcriptome eQTL mapping dataset from the same population allowed refining the list of plausible candidate causative sleep loci. This set includes genes with previously characterized effects on sleep and circadian rhythms, in addition to novel candidates. Finally, adult, nervous system-specific RNAi was used on the Dopa decarboxylase, dyschronic, and timeless genes, finding significant effects on sleep phenotypes for all three. The genes resolved in this study are strong candidates to harbor causative, regulatory variation contributing to sleep (Smith, 2020).

    Rhythmic behavior is controlled by the SRm160 splicing factor in Drosophila melanogaster
    While many transcription factors underlying circadian oscillations are known, the splicing factors that modulate these rhythms remain largely unexplored. A genome-wide assessment of the alterations of gene expression in a null mutant of the alternative splicing regulator SR-related matrix protein of 160 kD (SRm160) revealed the extent to which alternative splicing impacts on behavior-related genes. SRm160 affects gene expression in pacemaker neurons of the Drosophila brain to ensure proper oscillations of the molecular clock. A reduced level of SRm160 in adult pacemaker neurons impairs circadian rhythms in locomotor behavior, and this phenotype is caused, at least in part, by a marked reduction in period (per) levels. Moreover, rhythmic accumulation of the neuropeptide Pigment-dispersing factor (PDF) in the dorsal projections of these neurons is abolished after SRm160 depletion. The lack of rhythmicity in SRm160 downregulated flies is reversed by a fully spliced per construct, but not by an extra copy of the endogenous locus, showing that SRm160 positively regulates per levels in a splicing-dependent manner. These findings highlight the significant effect of alternative splicing on the nervous system and particularly on brain function in an in vivo model (Beckwith, 2017).

    Mapping quantitative trait loci underlying circadian light sensitivity in Drosophila

    Despite the significant advance in understanding of the molecular basis of light entrainment of the circadian clock in Drosophila, the underlying genetic architecture is still largely unknown. The aim of this study was to identify loci associated with variation in circadian photosensitivity, which are important for the evolution of this trait. Complementary approaches were used that combined quantitative trait loci (QTL) mapping, complementation testing, and transcriptome profiling to dissect this variation. A major QTL was identified on chromosome 2, which was subsequently fine mapped using deficiency complementation mapping into 2 smaller regions spanning 139 genes, some of which are known to be involved in functions that have been previously implicated in light entrainment. Two genes implicated with the clock and located within that interval, timeless and cycle, failed to complement the QTL, indicating that alleles of these genes contribute to the variation in light response. Specifically, the timeless s/ls polymorphism that has been previously shown to constitute a latitudinal cline in Europe is also segregating in the recombinant inbred lines and is contributing to the phenotypic variation in light sensitivity. This study also profiled gene expression in 2 recombinant inbred strains that differ significantly in their photosensitivity and a total of 368 transcripts were identified that showed differential expression (false discovery rate < 0.1). Of 131 transcripts that showed a significant recombinant inbred line by treatment interaction (i.e., putative expression QTL), 4 are located within QTL2 (Adewoye, 2017).

    LAT1-like transporters regulate dopaminergic transmission and sleep in Drosophila

    Amino-acid transporters are involved in functions reportedly linked to the sleep/wake cycle: neurotransmitter synthesis and recycling, the regulation of synaptic strength, protein synthesis and energy metabolism. In addition, the existence of bidirectional relationships between extracellular content, transport systems and sleep/wake states is receiving emerging support. Nevertheless, the connection between amino-acid transport and sleep/wake regulation remains elusive. To address this question, this study used Drosophila melanogaster and investigated the role of LAT1 (Large neutral Amino-acid Transporter 1) transporters. This study shows that the two Drosophila LAT1-like transporters: JhI-21 and minidiscs (Mnd) are required in dopaminergic neurons for sleep/wake regulation. Down-regulating either gene in dopaminergic neurons resulted in higher daily sleep and longer sleep bout duration during the night, suggesting a defect in dopaminergic transmission. Since LAT1 transporters can mediate in mammals the uptake of L-DOPA, a precursor of dopamine, amino-acid transport efficiency was assessed by L-DOPA feeding. Downregulation of JhI-21, but not Mnd, reduced the sensitivity to L-DOPA as measured by sleep loss. JhI-21 downregulation also attenuated the sleep loss induced by continuous activation of dopaminergic neurons. Since LAT1 transporters are known to regulate TOR (Target Of Rapamycin) signaling, the role of this amino-acid sensing pathway in dopaminergic neurons was investigated. Consistently, it is reported that TOR activity in dopaminergic neurons modulates sleep/wake states. Altogether, this study provides evidence that LAT1 mediated amino-acid transport in dopaminergic neurons, is playing a significant role in sleep/wake regulation, and is providing several entry points to elucidate the role of nutrients such as amino-acids in sleep/wake regulation (Aboudhiaf, 2018).

    Emerging evidence suggests bidirectional relationships between extracellular space content and vigilance states, emphasizing the so far little explored sleep-regulatory role of the transmembrane transport of ions and small molecules. Sleep and wakefulness have a pervasive impact on brain cellular activities linked to neurotransmission, neuronal plasticity, neurotransmitter synthesis, nutrient supply, and waste elimination, relying on the efficient and precise coordination of transport systems. Investigating how transporters and underlying molecular and cellular mechanisms are involved in these mutual interactions requires the targeting of individual genes in specific cell types. This strategy is highly amenable to the Drosophila model. Among the large array of cellular transporters present in the genome and conserved between insects and mammals, the well-characterized large neutral amino acid transporters are particularly relevant given their role in neurotransmitter synthesis and recycling, in the regulation of synaptic strength, in protein synthesis and energy metabolism. In addition, the de novo synthesis of brain monoamines associated with wakefulness and neuromodulation, such as serotonin, dopamine, and noradrenaline, is dependent on large neutral essential amino acids provided by the blood (Aboudhiaf, 2018).

    The SLC7A5 (or large neutral amino-acid transporter, light chain or LAT1) and SLC7A8 (LAT2) amino acid transporters are present in most cell types and appear to play a prominent role in the Na+-independent transport of large branched and aromatic neutral amino acids. These transporters belong to the heterodimeric amino acid transporters (HAT) family and require co-expression of the CD98hc /4F2hc (SLC3A2) heavy chain, to which they can be covalently linked by a di-sulfur bridge. The heavy subunit does not appear to confer transport-specific properties, nor to be confined to HAT transporter function. In the mammalian brain, LAT1 is highly expressed in the cells of the blood-brain-barrier and is thought to play a critical role in providing the central nervous system with essential amino acids such as phenylalanine, tyrosine, leucine, and tryptophan, which are nutrients and precursors for monoamine synthesis. The uptake of leucine through LAT1 is a major activator signal for target of rapamycin complex 1 (TORC1), a cellular pathway dependent on the TOR kinase that controls protein synthesis, brain excitability, and plasticity. Reciprocally, inhibition of mTOR by rapamycin has been shown to significantly reduce the activity and the mRNA expression of LAT1. Despite a few pieces of evidence, it remains to be investigated whether LAT1, LAT2, or other SLC7A transporters are also localized in neurons, and whether their function is linked to sleep and wake. In Drosophila, the HAT family of transporters is represented by one heavy chain, CD98hc, and five light chains: juvenile hormone inducible-21 (JHI-21), minidiscs (MND), genderblind (GB), CG9413 protein, and CG1607 protein. The specific activity of these transporters cannot be easily predicted from their sequences and requires functional testing. JHI-21 and MND can transport leucine, are inhibited by BCH (2-aminobicyclo[2.2.1]heptane-2-carboxylic acid), and, at least for JHI-21, require CD98hc to become functional, thus classifying them as LAT1-LAT2 homologs. Both genes are expressed at high levels in the central nervous system and have been shown to play important neurophysiological functions. JHI-21 and GB regulate glutamatergic synaptic strength, primarily through the regulation of extracellular glutamate levels, and are, respectively, required in motor neurons and in glial cells. In a recent report, it has also been shown that MND is required to activate brain insulin-producing neurons in response to circulating leucine. Interestingly those same insulin-producing neurons are connected to wakefulness promoting circuits. This study used the molecular-genetic tools of Drosophila to investigate the potential impact of JhI-21 and Mnd downregulation in neuronal subsets on sleep/wake regulation (Aboudhiaf, 2018).

    Evidence is provided that the LAT-1 like transporters JhI-21 and Mnd are required in adult fly dopaminergic neurons to achieve adequate sleep/wake regulation. The results demonstrate that a downregulation of these transporters in dopaminergic neurons results in a decrease in wakefulness, under baseline conditions but also in conditions that increase dopaminergic transmission. This implies that the activity of dopaminergic neurons and/or their ability to release neurotransmitter requires JhI-21 and Mnd. These two amino acid transporters are the closest drosophila LAT1 homologs based on sequence and functional data, suggesting that some amino acid availability plays a critical role in dopaminergic neuronal function. Supporting this hypothesis, this study finds that downregulating the TOR pathway in dopaminergic neurons results in a decrease in wakefulness (Aboudhiaf, 2018).

    As in mammals, dopaminergic transmission plays a major role in Drosophila wakefulness and has been suggested to constitute a core ancestral regulator of arousal and sleep entry across invertebrates. Drosophila and mammals share homologs for genes playing a central role in dopamine synthesis, reuptake, and signaling. This includes two D1-like receptors and one D2-like dopamine receptor. Among those, the D1-like receptor Dop1R1 (dDA1) plays a prominent role in sleep/wake regulation. Dop1R1 mutant flies display high daily sleep, longer sleep bout duration, and normal waking activity, a phenotype that closely resembles the one obtained in this studu by downregulating JhI-21 or Mnd in dopaminergic neurons. The effect of Dop1R1 on sleep/wake depends on its expression in the dorsal fan-shaped body (dFB), a key sleep-wake regulatory structure, and on the release of dopamine by a very limited set of dopaminergic neurons projecting to the dFB and located in the PPL1 and PPM3 cluster. Thus, it is possible that Mnd and JhI-21 are required in those specific neurons to achieve normal sleep-wake regulation. Expressing Mnd and JhI-21 UAS-RNAi constructs in PPL1 and PPM3 neurons failed to produce an abnormal sleep/wake phenotype. This lack of effect may be attributable to the limited efficiency of the UAS-RNAi constructs. Alternatively, the inhibition of JhI-21 and Mnd may affect multiple dopamine dependent microcircuits throughout the brain and thus cannot be easily replicated by a more specific manipulation (Aboudhiaf, 2018).

    What function could JHI-21 and MND fulfill in dopaminergic neurons? At the glutamatergic neuromuscular junction, JHI-21 appears to regulate the clustering of post-synaptic glutamate receptors. JHI-21 does not appear to trigger directly the release of glutamate in this context, but possibly mediates the entry of amino acids such as leucine to activate molecular pathways controlling glutamatergic physiology. Although abnormal sleep/wake regulation was not observed when JhI-21 was inhibited in glutamatergic neurons, the results indicate that JhI-21 could play a role in dopaminergic physiology similar to the one hypothesized for the neuromuscular junction. The synthesis of brain monoamines depends on the supply of essential amino acids that are provided by food intake, thus requiring efficient cellular transport systems. Although dopaminergic neuron function is impaired when JhI-21 expression is downregulated, no evidence was found of reduced brain dopamine levels under baseline conditions. Dopamine levels were increased in the mutant flies fed with the dopamine precursor L-DOPA, an amino acid that has been shown to be transported by LAT1, further suggesting that JhI-21 expression is not critical for dopamine synthesis. In this experiment, dopamine synthesis could also take place ectopically in dopa-decarboxylase (ddc) expressing serotoninergic neurons, in which the UAS-JhI-21-RNAi construct was not expressed. Dopamine content was still increased, although moderately, in flies in which the UAS-JhI-21-RNAi construct was targeted to both dopaminergic and serotoninergic neurons using the ddc-Gal4 driver. The lack of impact of JhI21 downregulation on L-DOPA-induced increase in dopamine synthesis could be due to functional redundancy between JhI-21 and Mnd, or could be a consequence of the partial inhibition provided by the UAS-RNAi constructs. In contrast to those findings on tissue content, this study clearly observed that JhI-21 downregulation in dopaminergic neurons reduced the sensitivity to L-DOPA as measured with sleep loss, and significantly attenuated the sleep loss phenotype of the TH-GaL4 > UAS-TrpA1 hyperdopaminergic condition. This raises the possibility that JhI-21 mediates the entry of amino acids critical for the physiology of dopaminergic neurons, such as leucine that could activate TOR-dependent processes influencing protein synthesis and energy metabolism, but is not directly involved in dopamine synthesis through the transport of precursors such as tyrosine or phenylalanine. The phenotype observed in flies where both Rheb and JhI-21 are manipulated indicates that JhI-21 could be required for TOR pathway activation. This hypothetical model is conceivable since in mammals enriched intracellular leucine levels via LAT1 transport activate mTORC1. Alternatively, TOR signaling could modulate JHI-21 function and affect LAT1-like-dependent transport mechanisms required for neuronal activity. Elucidating the relationships between TOR signaling and LAT1-like transporters, and understanding how they play a critical role in dopaminergic neurons function will require further work (Aboudhiaf, 2018).

    This study showed that the MND and JHI-21 transporters are broadly expressed in the brain. However, targeting the JhI-21 and Mnd UAS-RNAi constructs to most nondopaminergic cell types, including the wake-promoting octopaminergic neurons, failed to affect sleep-wake regulation. This lack of effect may be attributable to the efficiency of genetic tools or alternatively to the more stringent requirement for these transporters in dopaminergic neurons. Further studies are warranted to further evaluate this question. Interestingly, accumulating pieces of evidence support the existence of a dynamic regulation for JhI-21 and Mnd: JhI-21 is modulated during larval development in close correlation with behavioral changes and in adult after long-term memory conditioning, whereas Mnd is differentially expressed after sleep deprivation. The expression of LAT1 transporters in mammalian dopaminergic neurons has not been investigated yet; however, a report using pharmacological methods suggested that such transporters could modulate neuronal activity. The fact that sleep deprivation induces changes in TOR signaling in the brain opens the possibility that LAT1 could be modulated by sleep/wake in mammals. Of note, sleep deprivation could induce changes in LAT1 expression at the blood-brain barrier (Aboudhiaf, 2018).

    In conclusion, this study reveals the role of LAT1-like transporters in the function of dopaminergic neurons, adding one more element to the array of cellular and molecular events affecting sleep/wake regulation. Since these transporters are known to be dynamically regulated by physiological cellular states, they provide an entry point to elucidate the role of nutrient in sleep/wake regulation (Aboudhiaf, 2018).

    Daily regulation of phototransduction, circadian clock, DNA repair, and immune gene expression by Heme Oxygenase in the retina of Drosophila

    The daily expression of genes and the changes in gene expression after silencing the heme oxygenase (ho) gene were examined in the retina of Drosophila using microarray and SybrGreen qPCR (quantitative polymerase chain reaction) methods. The HO decrease in the morning upregulated 83 genes and downregulated 57 genes. At night, 80 genes were upregulated and 22 were downregulated. The top 20 genes downregulated after ho silencing in the morning modulate phototransduction, immune responses, autophagy, phagocytosis, apoptosis, the carbon monoxide (CO) response, the oxidative stress/UV response, and translation. In turn, the genes that upregulated at night were involved in translation-the response to oxidative stress, DNA damage, and phototransduction. Among the top 20 genes downregulated at night were genes involved in phototransduction, immune responses, and autophagy. For some genes, a low level of HO had an opposite effect in the morning compared to those at night. Silencing ho also changed the expression of circadian clock genes, while the HO decrease during the night enhanced the expression of immune system genes. The results showed that the cyclic expression of HO is important for controlling several processes in the retina, including neuroprotection and those involved in the innate immune system (Damulewicz, 2018).

    The Clock:Cycle complex is a major transcriptional regulator of Drosophila photoreceptors that protects the eye from retinal degeneration and oxidative stress

    The aging eye experiences physiological changes that include decreased visual function and increased risk of retinal degeneration. Although there are transcriptomic signatures in the aging retina that correlate with these physiological changes, the gene regulatory mechanisms that contribute to cellular homeostasis during aging remain to be determined. This study integrated ATAC-seq and RNA-seq data to identify 57 transcription factors that showed differential activity in aging Drosophila photoreceptors. These 57 age-regulated transcription factors include two circadian regulators, Clock and Cycle, that showed sustained increased activity during aging. When the Clock:Cycle complex was disrupted by expressing a dominant negative version of Clock (ClkDN) in adult photoreceptors, changes were observed in expression of 15-20% of genes including key components of the phototransduction machinery and many eye-specific transcription factors. Using ATAC-seq, expression of ClkDN in photoreceptors was shown to lead to changes in activity of 37 transcription factors and causes a progressive decrease in global levels of chromatin accessibility in photoreceptors. Supporting a key role for Clock-dependent transcription in the eye, expression of ClkDN in photoreceptors also induced light-dependent retinal degeneration and increased oxidative stress, independent of light exposure. Together, these data suggests that the circadian regulators Clock and Cycle act as neuroprotective factors in the aging eye by directing gene regulatory networks that maintain expression of the phototransduction machinery and counteract oxidative stress (Jauregui-Lozano, 2022).

    Nitric oxide mediates neuro-glial interaction that shapes Drosophila circadian behavior

    Drosophila circadian behavior relies on the network of heterogeneous groups of clock neurons. Short- and long-range signaling within the pacemaker circuit coordinates molecular and neural rhythms of clock neurons to generate coherent behavioral output. The neurochemistry of circadian behavior is complex and remains incompletely understood. This study demonstrates that the gaseous messenger nitric oxide (NO) is a signaling molecule linking circadian pacemaker to rhythmic locomotor activity. mutants lacking nitric oxide synthase (NOS) have behavioral arrhythmia in constant darkness, although molecular clocks in the main pacemaker neurons are unaffected. Behavioral phenotypes of mutants are due in part to the malformation of neurites of the main pacemaker neurons, s-LNvs. Using cell-type selective and stage-specific gain- and loss-of-function of NOS, this study also demonstrated that NO secreted from diverse cellular clusters affect behavioral rhythms. Furthermore, perineurial glia, one of the two glial subtypes that form the blood-brain barrier, as the major source of NO that regulates circadian locomotor output. These results reveal for the first time the critical role of NO signaling in the Drosophila circadian system and highlight the importance of neuro-glial interaction in the neural circuit output (Kozlov, 2020).

    It is rather surprising that the lack of NOS enzyme is not lethal as NO is part of various developmental processes. NOSΔ mutants are nonetheless strongly arrhythmic in DD and have reduced morning anticipation in LD. The data suggest that both congenital impairments and lack of NO signaling in adulthood contribute to the behavioral phenotype of the mutants. Axonal terminals of the master pacemaker, s-LNvs, in NOS mutants are profoundly disordered, suggesting the wrong or absent synaptic connections with the downstream partners. However, molecular rhythms in the pacemaker neurons are unaffected. During adulthood, NOS activity in the perineurial glia is required for producing free-running locomotor rhythms but not for maintaining PDF rhythms and structure of the s-LNvs. This finding indicates that NO produced in the perineurial glia is necessary for proper the functioning of circadian locomotor output circuits. Taken together, these results demonstrate that NO signaling is essential for establishing and controlling circadian output circuit (Kozlov, 2020).

    The functional isoform dNOS1 shows a circadian variation of its RNA levels throughout the day, which suggest that levels of NO could cycle at least in LD. However, dNOS is likely to be regulated by its truncated isoforms in a stage- and cell-type-specific manner, which lays an additional complexity to the regulation of NO production and probably leads to the heterogeneous and context-specific variations of NO. Hyperproduction of NO modulates molecular clockwork, albeit modestly, and is generally detrimental to locomotor rhythms. Therefore, the level and potentially the rhythms of NO production should be tightly controlled in wild-type flies (Kozlov, 2020).

    In an NOS RNAi mini screen, two optic lobe-specific drivers, GMR79D04 and GMR85B12, reduced locomotor rhythmicity in DD. This phenotype was observed when NOS was downregulated constitutively in these cells but not when knockdown was restricted to adulthood. These results reinforce the idea that NO is necessary for a proper establishment of neuronal circuits. A low rhythmicity phenotype caused by NOS knockdown with the pan-neuronal driver GMR57C10-GAL4 is congruent with the above findings. Intriguingly, however, in addition to the low rhythmicity, GMR57C10 > NOS RNAi in adulthood resulted in an extended period. What might be the neuronal subsets that produce NO and regulate period length of locomotor activity? A recent study by Aso (2019), NO was shown to act as a co-transmitter in a subset of dopaminergic neurons, specifically in some of the PAMs, PPL1s and PPL2abs. It is thus possible that dopamine signaling modulated by NO is involved in the control of the locomotor activity period. It is also noteworthy that NO-mediated signaling has a profound neuromodulatory effect on spinal motor networks and regulates frequency and amplitude of motor activity in various vertebrate species. NO-mediated regulation of circadian locomotor output in flies might involve a similar mechanism (Kozlov, 2020).

    DAR4-M staining showed an enrichment of NO in glial cells, including the surface glia. Targeting glial cells leads to the strongest and most persistent phenotype in locomotor activity both for gain- and loss-of-function of NOS. Among glial subpopulations, the perineurial glia appears to be the major site of NOS activity that regulates locomotor rhythms. The importance of glia in circadian rhythms have been recognized, especially those containing the molecular clocks and exert reciprocal communication with the pacemaker neural circuit. It has been shown that the perineurial glial cells harbor molecular clocks, which drive daily rhythms in the blood-brain barrier permeability but are not required for locomotor activity rhythms. This study is the first to identify NO as a signaling molecule produced in glia and mediates part of the role of glia, independently of their molecular clocks, in Drosophila circadian rhythms (Kozlov, 2020).

    It has been shown that in mammals NO mediates light-induced phase-shifts through the cGMP pathway. It is an interesting parallel to note that forced production of NO in the s-LNvs caused phase shift rather than amplitude dampening. Since high levels of NO inhibit E75/UNF dimerization and E75/UNF normally enhances per transcription, it is speculated that NO-induced phase-shift may be partly mediated by the inhibition of E75/UNF (Hormone receptor 51) heterodimerization. It will be interesting to test this hypothesis in future studies. Mammalian clocks contain E75 homologs REV-ERB α/β, which repress Bmal1 transcription. Analogous to the notion in flies, NO is thought to decrease REV-ERB α/β activity. Consistently, in vitro studies in mammalian cell culture showed that excessive presence of NO increases the production of Bmal1 mRNA. These findings altogether point out that NO is an evolutionarily conserved regulator of circadian rhythms (Kozlov, 2020).

    In line with recent studies, this research expands the view on the factors that participate in neuronal and molecular mechanisms of circadian rhythmicity. The finding that gaseous messenger NO contributes to the various aspects of circadian rhythmicity emphasizes that non-cell-autonomous, systemic regulation is integral to the circadian circuit operation. These results set a foundation for future studies addressing the mechanism by which NO signaling modulates the state of the pacemaker circuit and its output (Kozlov, 2020).

    Mesencephalic Astrocyte-Derived Neurotrophic Factor Regulates Morphology of Pigment-Dispersing Factor-Positive Clock Neurons and Circadian Neuronal Plasticity in Drosophila melanogaster

    Mesencephalic Astrocyte-derived Neurotrophic Factor (MANF) is one of a few neurotrophic factors described in Drosophila melanogaster (DmMANF) but its function is still poorly characterized. The present study found that DmMANF is expressed in different clusters of clock neurons. In particular, the PDF-positive large (l-LNv) and small (s-LNv) ventral lateral neurons, the CRYPTOCHROME-positive dorsal lateral neurons (LNd), the group 1 dorsal neurons posterior (DN1p) and different tim-positive cells in the fly's visual system. Importantly, DmMANF expression in the ventral lateral neurons is not controlled by the clock nor it affects its molecular mechanism. However, silencing DmMANF expression in clock neurons affects the rhythm of locomotor activity in light:dark and constant darkness conditions. Such phenotypes correlate with abnormal morphology of the dorsal projections of the s-LNv and with reduced arborizations of the l-LNv in the medulla of the optic lobe. Additionally, it was shown that DmMANF is important for normal morphology of the L2 interneurons in the visual system and for the circadian rhythm in the topology of their dendritic tree. These results indicate that DmMANF is important not only for the development of neurites but also for maintaining circadian plasticity of neurons (Krzeptowski, 2021).

    Astrocytic GABA transporter controls sleep by modulating GABAergic signaling in Drosophila circadian neurons

    A precise balance between sleep and wakefulness is essential to sustain a good quality of life and optimal brain function. GABA is known to play a key and conserved role in sleep control, and GABAergic tone should, therefore, be tightly controlled in sleep circuits. This study examined the role of the astrocytic GABA transporter (Gat) in sleep regulation using Drosophila melanogaster. A hypomorphic Gat mutation (Gat33-1) increased sleep amount, decreased sleep latency, and increased sleep consolidation at night. Interestingly, sleep defects were suppressed when Gat33-1 was combined with a mutation disrupting wide-awake (wake), a gene that regulates the cell-surface levels of the GABA(A) receptor Resistance to dieldrin (Rdl) in the wake-promoting large ventral lateral neurons (l-LNvs). Moreover, RNAi knockdown of Rdl and its modulators dnlg4 and wake in these circadian neurons also suppressed Gat33-1 sleep phenotypes. Brain immunohistochemistry showed that GAT-expressing astrocytes were located near RDL-positive l-LNv cell bodies and dendritic processes. It is concluded that astrocytic GAT decreases GABAergic tone and RDL activation in arousal-promoting LNvs, thus determining proper sleep amount and quality in Drosophila (Chaturvedi, 2022).

    Intrinsic disorder is an essential characteristic of components in the conserved circadian circuit

    The circadian circuit, a roughly 24 h molecular feedback loop, or clock, is conserved from bacteria to animals and allows for enhanced organismal survival by facilitating the anticipation of the day/night cycle. Recent research has demonstrated that proteins comprising the circadian clock network display a significant amount of intrinsic disorder. This work focussed on the extent of intrinsic disorder in the circadian clock and its potential mechanistic role in circadian timing. The conservation of disorder was highlighted by quantifying the extent of computationally-predicted protein disorder in the core clock of the key eukaryotic circadian model organisms Drosophila melanogaster, Neurospora crassa, and Mus musculus. Previously published work, as well as feature novel experimental evidence, was examined, demonstrating that the core negative arm circadian period drivers FREQUENCY (Neurospora crassa) and PERIOD-2 (PER2) (Mus musculus), possess biochemical characteristics of intrinsically disordered proteins. Finally, the potential contributions are discussed of the inherent biophysical principals of intrinsically disordered proteins that may explain the vital mechanistic roles they play in the clock to drive their broad evolutionary conservation in circadian timekeeping. It is concluded that the pervasive conservation of disorder amongst the clock in the crown eukaryotes suggests that disorder is essential for optimal circadian timing from fungi to animals, providing vital homeostatic cellular maintenance and coordinating organismal physiology across phylogenetic kingdoms (Pelham, 2020).

    AKH-FOXO pathway regulates starvation-induced sleep loss through remodeling of the small ventral lateral neuron dorsal projections

    AKH-FOXO pathway regulates starvation-induced sleep loss through remodeling of the small ventral lateral neuron dorsal projections

    Starvation caused by adverse feeding stresses or food shortages has been reported to result in sleep loss in animals. However, how the starvation signal interacts with the central nervous system is still unknown. In this study, the adipokinetic hormone (AKH)-Fork head Box-O (FOXO) pathway is shown to respond to energy change and adjust the sleep of Drosophila through remodeling of the s-LNv (small ventral lateral neurons) dorsal projections. The results show that starvation prevents flies from going to sleep after the first light-dark transition. The LNvs are required for starvation-induced sleep loss through extension of the pigment dispersing factor (PDF)-containing s-LNv dorsal projections. Further studies reveal that loss of AKH or AKHR (akh receptor) function blocks starvation-induced extension of s-LNv dorsal projections and rescues sleep suppression during food deprivation. FOXO, which has been reported to regulate synapse plasticity of neurons, acts as starvation response factor downstream of AKH, and down regulation of FOXO level considerably alleviates the influence of starvation on s-LNv dorsal projections and sleep. Taking together, these results outline the transduction pathways between starvation signal and sleep, and reveal a novel functional site for sleep regulation (He, 2020).

    Manipulations of the olfactory circuit highlight the role of sensory stimulation in regulating sleep amount

    While wake duration is a major sleep driver, an important question is if wake quality also contributes to controlling sleep. In particular, this study sought to determine whether changes in sensory stimulation affect sleep in Drosophila. As Drosophila rely heavily on their sense of smell, this study focused on manipulating olfactory input and the olfactory sensory pathway. Sensory deprivation was first performed by removing antennae or applying glue to antennae. Sleep was then measured in response to neural activation, via TRPA1, or inhibition, via KIR2.1, of subpopulations of neurons in the olfactory pathway. Genetically restricting manipulations to adult animals prevented developmental effects. This study found that olfactory deprivation reduces sleep, largely independently of mushroom bodies that integrate olfactory signals for memory consolidation and have previously been implicated in sleep. However, specific neurons in the lateral horn, the other third order target of olfactory input, affect sleep. Also, activation of inhibitory second order projection neurons increases sleep. No single neuronal population in the olfactory processing pathway was found to bidirectionally regulate sleep, and reduced sleep in response to olfactory deprivation may be masked by temperature changes. These findings demonstrate that Drosophila sleep is sensitive to sensory stimulation, and identify novel sleep-regulating neurons in the olfactory circuit. Scaling of signals across the circuit may explain the lack of bidirectional effects when neuronal activity is manipulated. It is proposed that olfactory inputs act through specific circuit components to modulate sleep in flies (Hsu, 2020).

    Slowpoke functions in circadian output cells to regulate rest:activity rhythms

    The circadian system produces ~24-hr oscillations in behavioral and physiological processes to ensure that they occur at optimal times of day and in the correct temporal order. At its core, the circadian system is composed of dedicated central clock neurons that keep time through a cell-autonomous molecular clock. To produce rhythmic behaviors, time-of-day information generated by clock neurons must be transmitted across output pathways to regulate the downstream neuronal populations that control the relevant behaviors. An understanding of the manner through which the circadian system enacts behavioral rhythms therefore requires the identification of the cells and molecules that make up the output pathways. To that end, the Drosophila pars intercerebralis (PI) was recently characterized as a major circadian output center that lies downstream of central clock neurons in a circuit controlling rest:activity rhythms. Single-cell RNA sequencing (scRNAseq) was performed to identify potential circadian output genes expressed by PI cells, and cell-specific RNA interference (RNAi) was used to knock down expression of ~40 of these candidate genes selectively within subsets of PI cells. Knockdown of the slowpoke (slo) potassium channel in PI cells reliably decreases circadian rest:activity rhythm strength. Interestingly, slo mutants have previously been shown to have aberrant rest:activity rhythms, in part due to a necessary function of slo within central clock cells. However, rescue of slo in all clock cells does not fully reestablish behavioral rhythms, indicating that expression in non-clock neurons is also necessary. These results demonstrate that slo exerts its effects in multiple components of the circadian circuit, including PI output cells in addition to clock neurons, and it is hypothesized that it does so by contributing to the generation of daily neuronal activity rhythms that allow for the propagation of circadian information throughout output circuits (Ruiz, 2021).

    Light Stimuli and Circadian Clock Affect Neural Development in Drosophila melanogaster

    Endogenous clocks enable organisms to adapt cellular processes, physiology, and behavior to daily variation in environmental conditions. Metabolic processes in cyanobacteria to humans are under the influence of the circadian clock, and dysregulation of the circadian clock causes metabolic disorders. In mouse and Drosophila, the circadian clock influences translation of factors involved in ribosome biogenesis and synchronizes protein synthesis. Notably, nutrition signals are mediated by the insulin receptor/target of rapamycin (InR/TOR) pathways to regulate cellular metabolism and growth. However, the role of the circadian clock in Drosophila brain development and the potential impact of clock impairment on neural circuit formation and function is less understood. This study demonstrates that changes in light stimuli or disruption of the molecular circadian clock cause a defect in neural stem cell growth and proliferation. Moreover, this study shows that disturbed cell growth and proliferation are accompanied by reduced nucleolar size indicative of impaired ribosomal biogenesis. Further, this study defines that light and clock independently affect the InR/TOR growth regulatory pathway due to the effect on regulators of protein biosynthesis. Altogether, these data suggest that alterations in InR/TOR signaling induced by changes in light conditions or disruption of the molecular clock have an impact on growth and proliferation properties of neural stem cells in the developing Drosophila brain (Dapergola, 2021).

    Circadian structural plasticity drives remodeling of E cell output

    The Drosophila circadian network dictates the temporal organization of locomotor activity; under light-dark (LD) conditions, flies display a robust bimodal pattern. PDF-positive small ventral lateral neurons (sLNv) have been linked to the generation of the morning activity peak (the "M cells"), whereas the CRY-positive dorsal lateral neurons (LNds) and the PDF-negative sLNv are necessary for the evening activity peak (the "E cells"). While each group directly controls locomotor output pathways, an interplay between them along with a third dorsal cluster (the DN1ps) is necessary for the correct timing of each peak and for adjusting behavior to changes in the environment. M cells set the phase of roughly half of the circadian neurons (including the E cells) through PDF. This study shows the existence of synaptic input provided by the evening oscillator onto the M cells. Both structural and functional approaches revealed that E-to-M cell connectivity changes across the day, with higher excitatory input taking place before the day-to-night transition. Two different neurotransmitters, acetylcholine and glutamate, released by E cells were identified that are relevant for robust circadian output. Indeed, this study shows that acetylcholine is responsible for the excitatory input from E cells to M cells, which show preferential responsiveness to acetylcholine during the evening. These findings provide evidence of an excitatory feedback between circadian clusters and unveil an important plastic remodeling of the E cells' synaptic connections (Duhart, 2020a).

    In addition to timed neuronal activation and neuropeptide release, circadian remodeling of neuronal connectivity provides a mechanism to timely restrict circadian neuronal input/output. To explore mechanisms modulating E cell communication, this study sought for changes in presynaptic sites of the E cells by means of expressing an RFP-tagged version of Bruchpilot (BRP), a component of presynaptic active zones. Although the complexity of the E cell arborization pattern precluded a description based on membrane labeling, quantification of BRP particle numbers have proven to uncover circadian synaptic remodeling. The MB122B-splitGal4 and the Mai179Gal4;pdfGal80 drivers were employed to direct expression to E cells. As previously described, E cells showed an extensive arborization of putative presynaptic zones, marked by dense BRPRFP staining, in close proximity to M cell somas, axons, and dorsal terminals; particularly, in the dorsal region, a significantly lower number of active zones were detected at the beginning of the day (ZT2) and higher numbers toward the end of the light phase (at ZT10, 'late-day'). This time-of-day difference was confirmed through a second E cell driver (Mai179Gal4;pdfGal80). Interestingly, the timing of E cell maximum synaptic density coincides with the highest level of neuronal activity, similar to what has previously been described for M cells, suggesting this might be a general mechanism coupling circadian control of structural synaptic output to cellular excitability. E cell presynaptic changes were not detected near the cell bodies or the main axonal tract of the M cells but were paralleled by changes in M cell postsynaptic markers at the dorsal protocerebrum (Gorostiza, 2014), suggesting that these two groups are reciprocally remodeling their terminals in this region (Duhart, 2020a).

    To test the possibility that E cells could provide input to M cells, its targets were trans-synaptically tagging with trans-Tango. Out of the two available drivers, the MB122B-splitGal4 was selected because it shows minimal expression beyond this circadian subset, thus providing a more precise tool to map E cell postsynaptic contacts. This approach uncovered a restricted number of yet unidentified neurons (likely including the short neuropeptide F (sNPF)-positive dorsal lateral neurons [LNds], which are postsynaptic to the ion transport peptide (ITP)-positive LNd and 5th small ventral lateral neurons [sLNv]) whose somas and profuse arborizations are located within the dorsolateral region; more importantly, trans-synaptic labeling revealed that pigment-dispersing factor (PDF)+ M cells are postsynaptic to E cells (Duhart, 2020a).

    Trans-Tango experiments do not allow any temporal resolution. To gain more insight on the sites and plastic changes of E-to-M connectivity, GFP reconstitution across synaptic partners (GRASP) was employed to direct the expression of a presynaptically tagged GFP fragment to the E cells and describe their degree of contact across the day. Using both the strong Mai179Gal4;pdfGal80 and the specific MB122B-splitGal4 E cell drivers, evidence was found of E-to-M synaptic contacts in the M cell dorsal terminals and axonal tracts. Indeed, GRASP intensity at the dorsal region (but not in the axonal tracts) was higher at dusk compared to dawn, a further indication of strengthened E-to-M connectivity at the dorsal protocerebrum during late day. Of note, GRASP signal was also high toward the end of the night, as it had originally been reported employing a non-targeted split GFP, suggesting that the connection between E and M cells might also be relevant for the night-day transition (Duhart, 2020a).

    E cells have the ability to affect the morning activity component, but a functional assessment for E-to-M cell communication has not been reported yet. To test this possibility, E cells were depolarized by means of an ATP perfusion that activates the ectopically expressed P2X2 channel. An excitatory response was uncovered in M cells, revealed by an increase in intracellular calcium levels , indicating a functional excitatory feedback from E to M cells (Duhart, 2020a).

    The observation of structural changes in the E cell terminals suggests that the functional E-to-M communication might be subject to daily regulation. Accordingly, it was found that forced depolarization of the E cells led to M cell responses predominantly during the second half of the light period; specifically, at ZT8-10, responses are statistically different from those obtained in the remaining intervals, with the exception of those triggered at ZT6-8. This coincides with the window of maximum E cell presynaptic contacts at the dorsal terminals. Single trace analysis enabled comparison between the individual responses triggered at different times, which highlighted not only the variability among the responses observed at any given time point but also the differential kinetics of M cell responses, which appear to differ at ZT22-0 (faster) compared to ZT8-10 (slower, albeit no statistical analysis of this property was performed (Duhart, 2020a).

    Taken together, these findings confirm the E-to-M cell excitatory communication and show daily changes in the functional synaptic connectivity between key components of the Drosophila circadian network (Duhart, 2020a).

    Within the circadian network it has been shown that fast neurotransmission from M cells promotes network synchronization; likewise, DN1ps contribute to shape the activity profile under light-dark (LD) cycles through fast neurotransmission. It was hypothesized that fast neurotransmission from E cells would be part of the mechanisms underlying proper circadian outputs and the identity of the putative neurotransmitter(s) were sought by means of expressing specific RNAis to downregulate genes involved in their synthesis, release, or reuptake. Acetylcholine (ACh) and glutamate (Glut) were identified as strong candidates to play a role in circadian timekeeping. To avoid any compensatory or deleterious effect throughout development, RNAi expression was restricted to adulthood by means of the Target system. E cell downregulation of the vesicular acetylcholine transporter (vAChT) or the choline acetyltransferase (ChAT; that catalyzes the biosynthesis of the neurotransmitter acetylcholine) affected the consolidation of the endogenous locomotor activity rhythms, without inducing changes in the clock's speed, confirming the importance of ACh released by the E cells for network synchronization. Interestingly, the evening anticipatory activity was reduced by vAChT and ChAT downregulation in E cells, a behavioral signature of E cell activity under LD cycles (Duhart, 2020a).

    Fast neurotransmitter co-release has been described both in vertebrates and invertebrates, and peptidergic/fast neurotransmitter co-transmission is present within the Drosophila circadian network, which led to examining the role of Glut as a second E cell neurotransmitter. Adult-restricted expression of the vesicular glutamate transporter (vGluT) RNAi impaired behavioral rhythmic output, triggering a reduced consolidation of the activity patterns as well as a small change in the circadian period of endogenous rhythms without significantly affecting the LD locomotor patterns. These results confirmed the role of glutamatergic neurotransmission released by E cells, at least under free running conditions. Reduction in rhythm robustness was previously reported by silencing E cells with the inward-rectifying potassium channel Kir2.1, which coincides with the behavioral phenotype uncovered upon cholinergic and glutamatergic downregulation in E cells (Duhart, 2020a).

    In addition to classical neurotransmitters, the PDF neuropeptide is crucial for proper coupling among circadian clusters and for modulating the activity of locomotor centers. Interestingly, constant PDF levels are found in flies with defective consolidation of activity rhythms. Thus, it was reasoned that the phenotype associated to prevent neurotransmitter release from E cells could possibly involve alterations in PDF cycling as an output of the network. Along those lines, it was found that impairing cholinergic neurotransmission in E cells abolished PDF oscillations at the sLNv dorsal terminals, with similar levels to those found at night in controls. Likewise, impairing glutamatergic neurotransmission particularly affected PDF levels in the morning, albeit some residual cycling was still observed, suggesting that a balanced neurotransmission from E cells is necessary for proper neuropeptidergic output of the M cells. The idea is favored that reduced amplitude of PDF cycling is the result of an altered balance of sLNv activity, which depends not only on clock-dependent control of ion channel expression but also on direct/indirect input from the circadian network [tested at least for the large LNvs (lLNvs)] (Duhart, 2020a).

    Since E cell activation led to M cell depolarization, and ACh from E cells impacted on circadian timekeeping, it was hypothesized that the M cells would be one of the cholinergic targets of the E cells. Bath application of the nicotine antagonist curare prevented M cell excitatory responses triggered by acute depolarization of E cells between ZT6-10. As previously reported, its effects were difficult to wash; however, about 30% of the M cells partially recovered after washing the antagonist (i.e., the fluorescence after washout was at least two times higher than with curare). Taking into account that the M cell response to E cell activation showed an unequivocal time-of-day dependency, and that PDF-positive cells have previously been shown to respond to bath application of Ach, whether M cells exhibit a differential response to ACh was tested at times where E-to-M connectivity differed significantly. Bath application of ACh to brains expressing GCaMP6m in PDF cells led to strong calcium responses in both the lLNv (also PDF-positive cells but not formally part of the M oscillator) and M cells during the evening. However, although ACh application induced equivalent responses in the lLNv during the morning, M cells' activation was highly reduced at this time point, underscoring that the sensitivity of these cells to cholinergic inputs is time-of-day dependent. Cholinergic inputs lead to both Ca2+ and cAMP increases in M cells through the nicotinic ACh receptor (nAChR). Therefore, changes in the levels of the nAChR itself or in the intracellular pathways activated by ACh throughout the day might also contribute to the control of E to M cell cholinergic circuit (Duhart, 2020a).

    In addition to the well-studied role of neuropeptides, classical neurotransmitters contribute to the coordination of the multi-oscillator circadian network. Both E cells and DN1ps respond to an inhibitory glycine tone likely released by M cells. Additionally, an inhibitory feedback from the glutamatergic DN1ps to the M and E cells further refines the temporal organization of the activity pattern. The notion of reciprocal regulation between oscillator pairs prompted an investigate of whether E cells feedback onto the M oscillator. The results point toward an excitatory cholinergic feedback from E to M cells during the late day (i.e., around ZT8-10). In M cells, intracellular calcium levels show a strong time-of-day dependence, with a peak shortly before lights-on, declining over the light phase, and rising after lights-off. Thus, increased excitability of E cells and the higher synaptic excitatory input to M cells could provide activating signals which, integrated with other inputs and self-autonomous mechanisms, would ultimately lead to the change in intracellular calcium. Loss of excitatory feedback would then prevent the increase of PDF levels at the dorsal terminals of M cells (through recruitment of PDF-filled dense-core vesicles) and thus affect other timed signaling outputs, leading to network desynchronization and hence, deconsolidated behavioral rhythms. Within the circadian network, E, M, and DN1p clusters express mGluRA RNA, and glutamatergic input from DN1ps impacts both M and E cells. Thus, glutamate released from E cells could impact in any of these clusters, in a time-of-day controlled manner, providing an inhibitory output from E cells into the network. Specifically, the highest connectivity detected through GRASP opens the possibility that glutamatergic input from E to M cells reaches its maximum toward the end of the night, consistent with the inhibitory responses observed during that window, which could directly or indirectly imbalance the active recruitment of PDF dense-core vesicles to the terminals, hence PDF release in the morning. Alternatively, loss of glutamatergic inhibitory input onto glutamatergic DN1ps might result in a persisting increase of the activity of this cluster, which in turn could increase their inhibition onto M cells and also lead to impaired PDF release, a change in the pace of the molecular clock, and hence, deconsolidation of rest-activity patterns (Duhart, 2020a).

    This work uncovers that the E cells communicate through fast neurotransmitters with a tight time-dependent control and a versatile output nature. Daily axonal remodeling of neuronal terminals-along with clock-controlled changes in the postsynaptic response to neurotransmitters-leads to plastic functional connectivity and provides novel building blocks to the flexible circadian network. This, in turn, enables fine-tuning reciprocal connections between oscillator pairs, further expanding its ability to adapt to environmental cycles. Thus, rhythmic behavior represents an emerging property of the plastic communication among clock neurons (Duhart, 2020a).

    A deep sleep stage in Drosophila with a functional role in waste clearance

    Sleep is a highly conserved state, suggesting that sleep's benefits outweigh the increased vulnerability it brings. Yet, little is known about how sleep fulfills its functions. This study used video tracking in tethered flies to identify a discrete deep sleep stage in Drosophila, termed proboscis extension sleep, that is defined by repeated stereotyped proboscis extensions and retractions. Proboscis extension sleep is accompanied by highly elevated arousal thresholds and decreased brain activity, indicative of a deep sleep state. Preventing proboscis extensions increases injury-related mortality and reduces waste clearance. Sleep deprivation reduces waste clearance and during subsequent rebound sleep, sleep, proboscis extensions, and waste clearance are increased. Together, these results provide evidence of a discrete deep sleep stage that is linked to a specific function and suggest that waste clearance is a core and ancient function of deep sleep (van Alphen, 2021).

    Quantitative investigation reveals distinct phases in Drosophila sleep

    The fruit fly, Drosophila melanogaster, has been used as a model organism for the molecular and genetic dissection of sleeping behaviors. However, most previous studies were based on qualitative or semi-quantitative characterizations. This study quantified sleep in flies. An assay was set up to continuously track the activity of flies using infrared camera, which monitored the movement of tens of flies simultaneously with high spatial and temporal resolution. Accurate statistics were obtained regarding the rest and sleep patterns of single flies. Analysis of the data has revealed a general pattern of rest and sleep: the rest statistics obeyed a power law distribution and the sleep statistics obeyed an exponential distribution. Thus, a resting fly would start to move again with a probability that decreased with the time it has rested, whereas a sleeping fly would wake up with a probability independent of how long it had slept. Resting transits to sleeping at time scales of minutes. This method allows quantitative investigations of resting and sleeping behaviors and these results provide insights for mechanisms of falling into and waking up from sleep (Xu, 2021).

    Circadian autophagy drives iTRF-mediated longevity

    Time-restricted feeding (TRF) has recently gained interest as a potential anti-ageing treatment for organisms from Drosophila to humans. TRF restricts food intake to specific hours of the day. Because TRF controls the timing of feeding, rather than nutrient or caloric content, TRF has been hypothesized to depend on circadian-regulated functions; the underlying molecular mechanisms of its effects remain unclear. To exploit the genetic tools and well-characterized ageing markers of Drosophila, this study developed an intermittent TRF (iTRF) dietary regimen that robustly extended fly lifespan and delayed the onset of ageing markers in the muscles and gut. iTRF enhanced circadian-regulated transcription, and iTRF-mediated lifespan extension required both circadian regulation and autophagy, a conserved longevity pathway. Night-specific induction of autophagy was both necessary and sufficient to extend lifespan on an ad libitum diet and also prevented further iTRF-mediated lifespan extension. By contrast, day-specific induction of autophagy did not extend lifespan. Thus, these results identify circadian-regulated autophagy as a critical contributor to iTRF-mediated health benefits in Drosophila. Because both circadian regulation and autophagy are highly conserved processes in human ageing, this work highlights the possibility that behavioural or pharmaceutical interventions that stimulate circadian-regulated autophagy might provide people with similar health benefits, such as delayed ageing and lifespan extension (Ulgherait, 2021).

    Sleep correlates with behavioral decision making critical for reproductive output in Drosophila melanogaster

    Balance between sleep, wakefulness and arousal is important for survival of organisms and species as a whole. While, the benefits of sleep both in terms of quantity and quality is widely recognized across species, sleep has a cost for organismal survival and reproduction. here the study focuses on how sleep duration, sleep depth and sleep pressure affect the ability of animals to engage in courtship and egg-laying behaviors critical for reproductive success. Using isogenic lines from the Drosophila Genetic Reference Panel with variable sleep phenotypes this study investigated the relationship between sleep and reproductive behaviors, courtship and oviposition. This study found that three out of five lines with decreased sleep and increased arousal phenotypes, showed increased courtship and decreased latency to court as compared to normal and long sleeping lines. However, the male courtship phenotype is dependent on context and genotype as some but not all long sleeping-low courting lines elevate their courtship in the presence of short sleeping-high courting flies. Sleep phenotypes were less variable and minimally susceptible to social experience. In addition to male courtship, Oviposition was found to be less sensitive to sleep length. Taken together this extensive behavioral analysis shows complex bidirectional interactions between genotype and environment and add to the growing evidence linking sleep duration and sleep-wake switch parameters to behavioral decision making critical to reproductive output (Buchert, 2021).

    Hsp40 overexpression in pacemaker neurons delays circadian dysfunction in a Drosophila model of Huntington's disease

    Circadian disturbances are early features of neurodegenerative diseases, including Huntington's disease (HD). Emerging evidence suggests that circadian decline feeds into neurodegenerative symptoms, exacerbating them. Therefore, it was asked whether known neurotoxic modifiers can suppress circadian dysfunction. A screen was performed of neurotoxicity-modifier genes to suppress circadian behavioural arrhythmicity in a Drosophila circadian HD model. The molecular chaperones Hsp40 and HSP70 emerged as significant suppressors in the circadian context, with Hsp40 being the more potent mitigator. Upon Hsp40 overexpression in the Drosophila circadian ventrolateral neurons (LNv), the behavioural rescue was associated with neuronal rescue of loss of circadian proteins from small LNv soma. Specifically, there was a restoration of the molecular clock protein Period and its oscillations in young flies and a long-lasting rescue of the output neuropeptide Pigment dispersing factor. Significantly, there was a reduction in the expanded Huntingtin inclusion load, concomitant with the appearance of a spot-like Huntingtin form. Thus, this study provided evidence implicating the neuroprotective chaperone Hsp40 in circadian rehabilitation. The involvement of molecular chaperones in circadian maintenance has broader therapeutic implications for neurodegenerative diseases (Prakash, 2022).

    DBT affects sleep in both circadian and non-circadian neurons

    Sleep is a very important behavior observed in almost all animals. Importantly, sleep is subject to both circadian and homeostatic regulation. The circadian rhythm determines the daily alternation of the sleep-wake cycle, while homeostasis mediates the rise and dissipation of sleep pressure during the wake and sleep period. As an important kinase, Dbt plays a central role in both circadian rhythms and development. This study investigated the sleep patterns of several ethyl methanesulfonate-induced dbt mutants and discusses the possible reasons why different sleep phenotypes were shown in these mutants. In order to reduce DBT in all neurons in which it is expressed, CRISPR-Cas9 was used to produce flies that expressed GAL4 in frame with the dbt gene at its endogenous locus, and knock-down of DBT with this construct produced elevated sleep during the day and reduced sleep at night. Loss of sleep at night is mediated by dbt loss during the sleep/wake cycle in the adult, while the increased sleep during the day is produced by reductions in dbt during development and not by reductions in the adult. Additionally, using targeted RNA interference, this study uncovered the contribution of dbt on sleep in different subsets of neurons in which dbt is normally expressed. Reduction of dbt in circadian neurons produced less sleep at night, while lower expression of dbt in noncircadian neurons produced increased sleep during the day. Importantly, independently of the types of neurons where dbt affects sleep, this study demonstrated that the PER protein is involved in DBT mediated sleep regulation (Wang, 2022).

    Expression of a constitutively active insulin receptor in Drosulfakinin (Dsk) neurons regulates metabolism and sleep in Drosophila

    The ability of organisms to sense their nutritional environment and adjust their behavior accordingly is critical for survival. Insulin-like peptides (ilps) play major roles in controlling behavior and metabolism; however, the tissues and cells that insulin acts on to regulate these processes are not fully understood. In the fruit fly, Drosophila melanogaster, insulin signaling has been shown to function in the fat body to regulate lipid storage, but whether ilps act on the fly brain to regulate nutrient storage is not known. This study manipulated insulin signaling in defined populations of neurons in Drosophila and measure glycogen and triglyceride storage. Expressing a constitutively active form of the insulin receptor (dInR) in the insulin-producing cells had no effect on glycogen or triglyceride levels. However, activating insulin signaling in the Drosulfakinin (Dsk)-producing neurons led to triglyceride accumulation and increased food consumption. The expression of ilp2, ilp3 and ilp5 was increased in flies with activated insulin signaling in the Dsk neurons, which along with the feeding phenotype, may cause the triglyceride storage phenotypes observed in these flies. In addition, expressing a constitutively active dInR in Dsk neurons resulted in decreased sleep in the fed state and less starvation-induced sleep suppression suggesting a role for insulin signaling in regulating nutrient-responsive behaviors. Together, these data support a role for insulin signaling in the Dsk-producing neurons for regulating behavior and maintaining metabolic homeostasis (Palermo, 2022).

    Regulation of feeding and energy homeostasis by clock-mediated Gart in Drosophila

    Feeding behavior is essential for growth and survival of animals; however, relatively little is known about its intrinsic mechanisms. This study demonstrates that Gart is expressed in the glia, fat body, and gut and positively regulates feeding behavior via cooperation and coordination. Gart in the gut is crucial for maintaining endogenous feeding rhythms and food intake, while Gart in the glia and fat body regulates energy homeostasis between synthesis and metabolism. These roles of Gart further impact Drosophila lifespan. Importantly, Gart expression is directly regulated by the CLOCK/CYCLE heterodimer via canonical E-box, in which the CLOCKs (CLKs) in the glia, fat body, and gut positively regulate Gart of peripheral tissues, while the core CLK in brain negatively controls Gart of peripheral tissues. This study provides insight into the complex and subtle regulatory mechanisms of feeding and lifespan extension in animals (He, 2023).

    Feeding is a necessary behavior for animals to grow and survive, with a characteristic of taking food regularly. The quality and quantity of feeding directly impact the normal growth and development of animals. Time-restricted feeding or fasting is beneficial for preventing obesity, alleviating inflammation, and attenuating cardiac diseases and even has antitumor effects. Metabolic syndrome has become a global health problem. Shift work and meal irregularity disrupt circadian rhythms, with an increased risk of developing metabolic syndrome. The maintenance of the daily feeding rhythm is very important in metabolic homeostasis.Irregular feeding perturbs circadian metabolic rhythms and results in adverse metabolic consequences and chronic diseases (He, 2023).

    Most behaviors in animals are synchronized to a ~24 h (circadian) rhythm induced by circadian clocks in both the central nervous system and peripheral tissues. Circadian rhythmic behaviors, such as feeding and locomotion, are involved in complex connections and specific output pathways under the control of the circadian clock. Although the core clock feedback loop has been well established in recent decades, the crucial genes responsible for rhythmic feeding regulation as well as for the interrelation between the core clocks and feeding are still unclear (He, 2023).

    To increase the understanding of how the circadian clock regulates feeding and metabolism, this study sought to identify the output genes in the circadian feeding and metabolism control network, in which the model animal Drosophila provides special advantages to explore the mechanistic underpinnings and the complex integration of these primitive responses. Previous studies confirmed that one of juvenile hormone receptors, methoprene tolerance (Met), is important for the control of neurite development and sleep behavior in Drosophila. Many genes related to metabolic regulation have attracted attention in the transcriptome data from Met27, a Met-deficient fly line, in which this study focused on the target genes regulated by CLOCK/CYCLE (CLK/CYC). As a basic Helix-Loop-Helix-Per-ARNT-Sim (bHLH-PAS) transcription factor with a canonical binding site “CACGTG," the CLK/CYC heterodimer is a crucial core oscillator that regulates circadian rhythms (He, 2023).

    The Gart trifunctional enzyme, a homologous gene of adenosine-3 in mammals, is a trifunctional polypeptide with the activities of phosphoribosylglycinamide formyltransferase, phosphoribosylglycinamide synthetase, and phosphoribosylaminoimidazole synthetase (Tiong, 1990). Gart in astrocytes of vertebrates plays a role in the lipopolysaccharide-induced release of proinflammatory factors (Zhang, 2014), and Gart expressed in the liver and heart is required for de novo purine synthesis. However, there is no information yet for Gart's functions in feeding rhythm. In this study, Gart was identified as a candidate that was controlled by the core clock gene CLK/CYC heterodimer and was found to be related to feeding behavior in Drosophila. Thus this study focused Gart studies on the role of feeding rhythms and further regulatory mechanisms. This study provides a critical foundation for understanding the complex feeding mechanism. (He, 2023).

    In animals, hundreds of genes exhibit daily oscillation under clock regulation, and some of them are involved in metabolic functions. This study identified a CLK/CYC-binding gene, Gart, which is involved in feeding rhythms and energy metabolism independent of locomotor rhythms. Previous research reported that blocking CLK in different tissues yields different phenotypes. This study found that MET, like CYC, can combine with CLK to regulate the transcription of Gart. Knocking down Gart in different tissues exhibits different phenotypes, and Gart in different tissues can rescue the phenotype caused by CLK deletion; thus, the phenomenon caused by CLK deletion is due to the change in Gart (He, 2023).

    CLK regulates the feeding rhythms of Drosophila, and its loss can cause disorders of feeding rhythms and abnormal energy storage. Tim01, Cry01, and Per01 mutants have significantly lower levels of truactkglycerides (TAGs). The maintenance of energy homeostasis is achieved by a dynamic balance of energy intake (feeding), storage, and expenditure (metabolic rate), which are crucial factors for longevity and resistance to adverse environments in Drosophila. Additionally, studies have shown that mutations of Timeless and per shorten the adult lifespan of Drosophila. This study further reveals that peripheral CLKs control the oscillation of Gart among different peripheral tissues; however, core CLKs in the brain can negatively regulate Gart expression in peripheral tissues, indicating that a complex and refined network regulatory system exists between CLK and Gart in the brain and in different peripheral tissues to coordinate feeding behavior and energy homeostasis in Drosophila and that it further affects sensitivity to starvation and longevity. These novel findings enrich the network of regulatory mechanisms by the clocks-Gart pathway on feeding, energy homeostasis, and longevity (He, 2023).

    Glial cells have vital functions in neuronal development, activity, plasticity, and recovery from injury. This study reveals that flies lacking Gart in glial cells display a significant decline in the viability of Drosophila under starvation, caused by a decrease in energy storage that puts flies under a state of energy deficit. This discovery extends the functions of glial cells in feeding, energy storage, and starvation resistance controlled by Gart (He, 2023).

    The fat body is the primary energy tissue for the storage of fuel molecules, such as TAG and glycogen, which play an important role in the regulation of metabolic homeostasis and provide the most energy during starvation. Indeed, functional defects of the fat body increase starvation sensitivity in Drosophila. In this study, flies lacking Gart in the fat body led to decreased energy storage, which reduces the survival rate and the survival time under starvation conditions. However, flies lacking gut Gart still maintain normal energy storage, which is not sensitive to food shortage or starvation. In addition, this study found that although high temperature can stimulate the food intake of Drosophila, which is consistent with previous reports, it does not affect the feeding rhythm (He, 2023).

    This study reveals that Gart in the glia and the fat body collectively regulate the homeostasis of energy intake, storage, and expenditure, thereby influencing the viability of flies under starvation stress. Although Gart in the gut strongly influences feeding behavior, it does not play similar functions as the glia and the fat body in adversity resistance. This occurs possibly because the gut has vital roles in digestion and absorption, while the fat body has crucial functions in energy metabolism. In addition, Gart in the glia and the fat body has biased roles in the synthesis of glycogen and TAG, despite having similar functions in energy storage. The biased role of the glia and the fat body may be coordinated to provide effective energy homeostasis. These findings provide new insight into how specific circadian coordination of various tissues modulates adversity resistance and aging (He, 2023).

    Such robust findings in Drosophila suggest that a decrease in lifespan and an increase in sensitivity to starvation in Drosophila is a faithful readout of disordered feeding rhythms and abnormal metabolism. Gart effects on metabolism in both glia cells and the fat body indicate the intricacy of the circadian network and energy homeostasis. It is crucial for animals to have a well-organized network to coordinate and ensure that these various tissue regions are in a normal state (He, 2023).

    This study has demonstrated that CLK regulates feeding, energy homeostasis, and longevity via Gart. Even though attempts were made to explore more comprehensively how Gart coordinates and regulates the physiological functions in different tissues of D. melanogaster, there are still some limitations. For instance, it is still unclear that how Gart achieves functional differentiation in different tissues, as well as whether Gart regulates lifespan through autophagy and/or bacterial content or not, which are two critical factors related to lifespan. These future studies are of great significance for understanding the relationship between feeding and longevity regulated by Gart (He, 2023).

    Chloride oscillation in pacemaker neurons regulates circadian rhythms through a chloride-sensing WNK kinase signaling cascade

    Central pacemaker neurons regulate circadian rhythms and undergo diurnal variation in electrical activity in mammals and flies. Whether and how intracellular chloride regulates circadian rhythms remains controversial. This study demonstrates a signaling role for intracellular chloride in the Drosophila small ventral lateral (sLN(v)) pacemaker neurons. In control flies, intracellular chloride increases in sLN(v)s over the course of the morning. Chloride transport through sodium-potassium-2-chloride (NKCC) and potassium-chloride (KCC) cotransporters is a major determinant of intracellular chloride concentrations. Drosophila melanogaster with loss-of-function mutations in the NKCC encoded by Ncc69 have abnormally low intracellular chloride 6 h after lights on, loss of morning anticipation, and a prolonged circadian period. Loss of kcc, which is expected to increase intracellular chloride, suppresses the long-period phenotype of Ncc69 mutant flies. Activation of a chloride-inhibited kinase cascade, consisting of WNK (with no lysine [K]) kinase and its downstream substrate, Fray, is necessary and sufficient to prolong period length. Fray activation of an inwardly rectifying potassium channel, Irk1, is also required for the long-period phenotype. These results indicate that the NKCC-dependent rise in intracellular chloride in Drosophila sLN(v) pacemakers restrains WNK-Fray signaling and overactivation of an inwardly rectifying potassium channel to maintain normal circadian period length (Schellinger, 2022).

    Intracellular chloride oscillates in central pacemaker neurons of the mammalian SCN, but the functional significance of this oscillation has remained unclear. This study demonstrates an NKCC-dependent increase in intracellular chloride in Drosophila sLNv pacemaker neurons over the course of the morning, which constrains activity of the chloride-sensitive WNK kinase; its downstream substrate, Fray; and an inwardly rectifying potassium channel, Irk1, to maintain normal circadian periodicity. Loss of the Ncc69 NKCC in LNv pacemaker neurons results in loss of morning anticipation and lengthening of the circadian period in free-running conditions. These observations are consistent with studies in the SCN implicating NKCC in the determination of intracellular chloride in mammalian pacemaker neurons and connects intracellular chloride to behavioral circadian phenotypes (Schellinger, 2022).

    Diurnal variations in intracellular chloride have been proposed to influence the effect of GABAergic neurotransmission on clock neurons in mammals, but a recent study challenged this idea. Existing data indicate a minor role for sLNv ligand-gated chloride channels in the regulation of circadian period. Rather, this study demonstrated a signaling role for chloride in sLNvs via inhibition of WNK-Fray signaling. As chloride-sensitive kinases, WNKs are poised to interpret changes in intracellular chloride and initiate downstream signal transduction cascades. This has been studied in transepithelial ion transport in Drosophila and mammalian renal epithelia, as well as in the clearance of apoptotic corpses (Schellinger, 2022).

    These findings further extend this concept to circadian pacemaker neurons. KCC and another cation-chloride cotransporter, encoded by the NKCC gene, have been linked to the electrophysiological response to GABA in lLNvs, which express GABAA receptor chloride channels. The transport activity of the NKCC-encoded transporter differs from the Ncc69 NKCC. Expression of Ncc69 and NKCC may also differ. Consistent with this idea, broad clock neuron knockout of NKCC has no effect on morning anticipation or period length but leads to abnormal rhythmicity in constant light, as does NKCC overexpression or loss or gain of kcc. Thus, intracellular chloride likely affects clock neurons both by affecting the driving force for chloride through neurotransmitter-gated chloride channels and via inhibition of WNK-Fray signaling. Interestingly, knocking down WNK and Fray broadly in clock neurons phenocopies loss of kcc and NKCC in the same neurons, suggesting that WNK and Fray act in their usual regulatory roles upstream of the transporters in this case, and further highlighting distinct actions of this pathway in different subpopulations of Drosophila clock neurons (Schellinger, 2022).

    Pacemaker neurons in flies and mammals undergo cell-autonomous, molecular clock-controlled circadian variation in electrical activity, and altering the excitability of the LNv pacemakers disrupts circadian rhythms. Two voltage-gated potassium channels have been implicated in LNv neuron electrical oscillations, and a sodium leak current and potassium channels contribute to the day/night cycling of resting membrane potential in Drosophila dorsal clock neurons. Because inwardly rectifying potassium channels play an important role in determining cellular membrane potential, which is thought to be a determinant of circadian variation in electrical activity, chloride regulation of Irk1 activity could also contribute to the diurnal variation in sLNv excitability. Specifically, Irk1 activation at ZT6 due to low intracellular chloride and activation of the WNK-Fray pathway in Ncc69 mutants may disrupt the usual circadian pattern of membrane depolarization and hyperpolarization in sLNvs, thereby altering period length (Schellinger, 2022).

    Could intracellular chloride play a signaling role in SCN pacemaker neurons? NKCC1, KCCs, and WNK3 are expressed in the rat SCN. The repertoire of ion channels regulating pacemaker neuron excitability is complex and incompletely understood, but large-conductance Ca2+-activated potassium channels have been implicated and are regulated by mammalian WNKs. Whether oscillating intracellular chloride observed in SCN neurons regulates these or other ion channels modulating pacemaker neuron electrical properties will be of interest for future investigation (Schellinger, 2022).

    Insulin signaling in clock neurons regulates sleep in Drosophila

    Sleeprelates to numerous biological functions, including metabolism. Both dietary conditions and genes related to metabolism are known to affect sleep behavior. Insulin signaling is well conserved across species including the fruit fly and relates to both metabolism and sleep. However, the neural mechanism of sleep regulation by insulin signaling is poorly understood. Here, we report that insulin signaling in specific neurons regulates sleep in Drosophila melanogaster. This study analyzed the sleep behavior of flies with the mutation in insulin-like ligands expressed in the brain and found that three insulin-like ligands participate in sleep regulation with some redundancy. Twenty-one Gal4 drivers were used to express a dominant-negative form of the insulin receptor (InR DN) in various neurons including circadian clock neurons, which express the clock gene, and the pars intercerebralis (PI). Inhibition of insulin signaling in the anterior dorsal neuron group 1 (DN1a) decreased sleep. Additionally, the same manipulation in PI also decreased sleep. Pan-neuronal induced expression of InR DN also decrease4ple, it was demonstrated that H3K27ac and H3K9ac represent universal active marks in promoters, rather than worm-specific marks as previously reported. Second, acting regions of the studied active marks that are common across species (and across a wide range of tissues at different developmental stages) were found to extend beyond the previously defined regions. Thus, it appears that the active histone codes analyzed have a universality that has previously been underappreciated. These results suggested that these universal codes, including those previously considered species-specific, could have an ancient origin, and are important in regulating animal gene expression abundance (Yamaguchi, 2022).

    LKB1 is physiologically required for sleep from Drosophila melanogaster to the Mus musculus

    Liver Kinase B1 (LKB1) is known as a master kinase for 14 kinases related to the adenosine monophosphate-activated protein kinase. Two of them salt inducible kinase 3 and adenosine monophosphate-activated protein kinase α have previously been implicated in sleep regulation. This study generated loss-of-function mutants for Lkb1 in both Drosophila and mice. Sleep, but not circadian rhythms, was reduced in Lkb1-mutant flies and in flies with neuronal deletion of Lkb1. Genetic interactions between Lkb1 and threonine to alanine mutation at residue 184 of adenosine monophosphate-activated protein kinase in Drosophila sleep or those between Lkb1 and Threonine to Glutamic Acid mutation at residue 196 of salt inducible kinase 3 in Drosophila viability have been observed. Sleep was reduced in mice after virally mediated reduction of Lkb1 in the brain. Electroencephalography analysis showed that nonrapid eye movement sleep and sleep need were both reduced in Lkb1-mutant mice. These results indicate that liver kinase B1 plays a physiological role in sleep regulation conserved from flies to mice (Liu, 2022).

    Optogenetic activation of SIFamide (SIFa) neurons induces a complex sleep-promoting effect in the fruit fly Drosophila melanogaster

    Sleep is a universal and extremely complicated function. Sleep is regulated by two systems-sleep homeostasis and circadian rhythms. In a wide range of species, neuropeptides have been found to play a crucial role in the communication and synchronization between different components of both systems. In the fruit fly Drosophila melanogaster, SIFamide (SIFa) is a neuropeptide that has been reported to be expressed in 4 neurons in the pars intercerebralis (PI) area of the brain. Previous work has shown that transgenic ablation of SIFa neurons, mutation of SIFa itself, or knockdown of SIFa receptors reduces sleep, suggesting that SIFa is sleep-promoting. However, those were all constitutive manipulations that could have affected development or resulted in compensation, so the role of SIFa signaling in sleep regulation during adulthood remains unclear. This study, examined the sleep-promoting effect of SIFa through an optogenetic approach, which allowed for neuronal activation with high temporal resolution, while leaving development unaffected. Activation of the red-light sensor Chrimson in SIFa neurons was found to promote sleep in flies in a sexually dimorphic manner, where the magnitude of the sleep effect was greater in females than in males. Because neuropeptidergic neurons often also release other transmitters, RNA interference was used to knock down SIFa while also optogenetically activating SIFa neurons. SIFa knockdown only partially reduced the magnitude of the sleep effect, suggesting that release of other transmitters may contribute to the sleep induction when SIFa neurons are activated. Video-based analysis showed that activation of SIFa neurons for as brief a period as 1 second was able to decrease walking behavior for minutes after the stimulus. Future studies should aim to identify the transmitters that are utilized by SIFa neurons and characterize their upstream activators and downstream targets. It would also be of interest to determine how acute optogenetic activation of SIFa neurons alters other behaviors that have been linked to SIFa, such as mating and feeding (Huang, 2021).

    The HisCl1 histamine receptor acts in photoreceptors to synchronize Drosophila behavioral rhythms with light-dark cycles

    In Drosophila, the clock that controls rest-activity rhythms synchronizes with light-dark cycles through either the blue-light sensitive cryptochrome (Cry) located in most clock neurons, or rhodopsin-expressing histaminergic photoreceptors. This study shows that, in the absence of Cry, each of the two histamine receptors Ort and HisCl1 contribute to entrain the clock whereas no entrainment occurs in the absence of the two receptors. In contrast to Ort, HisCl1 does not restore entrainment when expressed in the optic lobe interneurons. Indeed, HisCl1 is expressed in wild-type photoreceptors and entrainment is strongly impaired in flies with photoreceptors mutant for HisCl1. Rescuing HisCl1 expression in the Rh6-expressing photoreceptors restores entrainment but it does not in other photoreceptors, which send histaminergic inputs to Rh6-expressing photoreceptors. These results thus show that Rh6-expressing neurons contribute to circadian entrainment as both photoreceptors and interneurons, recalling the dual function of melanopsin-expressing ganglion cells in the mammalian retina (Alejevski, 2019).

    The Drosophila sleep–wake rhythms are controlled by a brain circadian clock that includes about 150 clock neurons. Light synchronizes the clock neuronal network through cell-autonomous and non-cell-autonomous light input pathways. Cry is a blue-light sensitive photoreceptor protein that is expressed in most clock neurons. In the absence of Cry, flies do not phase-shift their behavioral rhythms in response to a short light pulse but still synchronize to light–dark (LD) cycles. Only flies devoid of both Cry and rhodopsin-expressing photoreceptors fail to entrain to LD cycles. Six different rhodopsins (Rhs) have been characterized in the Drosophila photoreceptive structures, which include the compound eye, the Hofbauer-Buchner (H-B) eyelet, and ocelli. The compound eye strongly contributes to circadian photoreception, whereas a modest contribution appears to be brought by the H-B eyelet and the ocelli. A circadian function has been recently associated with the yet poorly characterized rhodopsin 7, although its exact contribution and localization in the brain and/or the eye remains controversial. In addition to entrainment, the visual system controls other features of the clock neuron network by conveying light information to either promote or inhibit the behavioral output of specific clock neuron subsets (Alejevski, 2019).

    The compound eye includes about 800-unit eyes (ommatidia), each of which contains eight photoreceptors. The six Rh1-expressing outer photoreceptors (R1–6) are involved in motion detection and project to the lamina neuropile of the optic lobe. The two inner photoreceptors (R7–8) are important for color detection and project to the medulla. They express four different rhodopsins and thus define two types of ommatidia: 'pale' (p) ommatidia (30%) include a Rh3-expressing R7 and a Rh5-expressing R8, whereas 'yellow' (y) ommatidia (70%) include a Rh4-expressing R7 and a Rh6-expressing R8. Each extra-retinal H-B eyelet contains four Rh6-expressing photoreceptors that project to the accessory medulla, in the vicinity of key pacemaker neurons, the ventral lateral neurons (LNvs) that produce the pigment-dispersing factor (PDF) neuropeptide9,20–24. Each of the three ocelli contains about 80 photoreceptors that express Rh225. The Drosophila rhodopsins cover a wide range of wavelengths from 300 nm to 600 nm18,19, with only Rh1 and Rh6 being sensitive to red light (Alejevski, 2019).

    Rhodopsin-dependent circadian entrainment involves two downstream signaling pathways, the canonical one that relies on the phospholipase C encoded by the no receptor potential A gene (norpA)2 or an unknown pathway that does not contribute in very low light levels. All but Rh2- and Rh5- expressing photoreceptors support synchronization in very low light, and at least Rh1, Rh5, and Rh6 can signal through the NorpA-independent pathway. Photoreceptors of the compound eye are histaminergic but the H-B eyelet expresses both histamine and acetylcholine. Although the two neurotransmitters might contribute to circadian entrainment, flies devoid of Cry and histidine decarboxylase do not synchronize their rest–activity rhythms with LD cycles. This suggests that besides Cry, there is no histamine-independent pathway to entrain the clock (Alejevski, 2019).

    Two genes encoding histamine-gated chloride channels, ora transientless (ort) and Histamine-gated chloride channel subunit 1 (HisCl1), have been identified in Drosophila. The ort-null mutants are visually blind and their electroretinograms have no ON and OFF transients. In contrast, HisCl1 mutants show increased OFF transients, whereas slower responses were observed in the postsynaptic laminar monopolar cells. Based on transcriptional reporters, ort expression in the optic lobes was observed in neurons of both the lamina and medulla/lobula neuropils. Based on reporter gene expression, HisCl1 was localized in glial cells of the lamina. However, recent work reported expression in photoreceptors, in particular in the R7 and R8 inner photoreceptor subtypes. Indeed, Ort and HisCl1 support color opponency between the two subtypes of 'inner' photoreceptors, the ultraviolet (UV)-sensitive R7 and non-UV-sensitive R8, with HisCl1 and Ort mediating direct and indirect inhibition, respectively. The histaminergic pathways that are involved in circadian entrainment are unknown and are the subject of the present study. The results show that both Ort and HisCl1 define two different pathways for circadian entrainment. Whereas Ort contributes through its expression in the interneurons of the optic lobe, HisCl1 mostly contributes through its expression in the Rh6-expressing retinal photoreceptors. The work thus reveals that Rh6-expressing neurons contribute to light-mediated entrainment as both photoreceptors and interneurons (Alejevski, 2019).

    This work reveals that the Cryptochrome-independent entrainment of rest–activity rhythms relies on distinct pathways that are contributed by the two histamine receptors Ort and HisCl1. Whereas Ort mediates circadian entrainment through the optic lobe interneurons that are involved in visual functions, HisCl1 defines a new photoreceptive pathway through Rh6-expressing photoreceptors. Although both receptors mediate synchronization with a shifted LD cycle, it seems likely that the two pathways will show differences in specific light conditions. It was not possible to rescue Ort function with HisCl1 expression in the ort-expressing cells, whereas the Ort could replace HisCl1 in Rh6 photoreceptors. It is possible that HisCl1 has a lower affinity for histamine with Rh6 cells receiving more neurotransmitter than optic lobe interneurons. Alternatively, interneurons could sufficiently differ from photoreceptors for their physiology or specific receptor-interacting protein content, preventing HisCl1 from working efficiently. HisCl1 downregulation in Rh6 cells slows down synchronization and flies with HisCl1134 mutant eyes synchronize very poorly with advanced LD cycles, and fail to synchronize with delays. It cannot be excluded that non-photoreceptor cells contribute to HisCl1-dependent entrainment but other pathways appear to have a modest contribution if any (Alejevski, 2019).

    HisCl1 is expressed in the H-B eyelet, which could thus contribute to this synchronization pathway. However, the cell-killing experiments indicate that H-B eyelet is not required for HisCl1-mediated synchronization through Rh6 cells. In the recently described color opponency mechanism, retinal R7 cells inhibit R8 and vice versa through HisCl1 expression in the photoreceptors (Schnaitmann, 2018). It is supposed that HisCl1-dependent clock synchronization is also mediated by the hyperpolarization of Rh6-expressing cells. How this hyperpolarization interacts with the light-induced depolarization in Rh6 photoreceptors to result in a synchronization message to the clock neurons remains to be understood. Since only Rh6-expressing R8 and not the other inner photoreceptors contribute to this circadian photoreception pathway, Rh6 cells might have specific connections with downstream interneurons. Such specificity has been described for color vision where each of the four inner photoreceptor subtypes connects to a different type of TmY interneuron in the Medulla. This study shows that HisCl1 expression in Rh6 cells supports synchronization with red light, in the absence of Rh1, indicating that an intra-Rh6-photoreceptor circuit is sufficient. This indicates that Rh6-expressing R8 photoreceptors play a dual photoreceptor/interneuron role in this pathway (Model for the retinal input pathways to the brain clock). Whether the same individual cells have the two roles is unknown, although the HisCl1-dependent color opponency mechanism suggests that it could be the case. It is also unclear whether all Rh6-expressing R8 photoreceptors or only a fraction of them contribute to circadian synchronization. The results imply that, in addition to histaminergic neurotransmission, Rh6-expressing photoreceptors can talk to downstream interneurons through histamine-independent neurotransmission. A recent transcriptomics study indeed revealed the expression of cholinergic markers in R7 and R8 cells, supporting cholinergic transmission in the inner photoreceptors, in addition to histaminergic transmission (Alejevski, 2019).

    The data indicate that histaminergic inputs from both outer and inner photoreceptors converge to Rh6 cells to contribute to circadian entrainment. It is possible that some of these inputs rely on Rh7, which seems to be expressed in Rh6-expressing photoreceptors, according to transcriptional reporter data. Putative connections between photoreceptors have been described in Drosophila and other insects. How R1–6 photoreceptors might be connected to Rh6-expressing R8 cells remains difficult to understand, but a few putative contacts between presynaptic outer cells and postsynaptic inner cells have been observed in Musca. The intra-retinal functional connectivity that this study reports in Drosophila is reminiscent to the circuit logic of circadian entrainment in the mammalian retina, where intrinsically photoreceptive retinal ganglion cells express the melanopsin photopigment in addition to receiving inputs from rods and cones. Interestingly, melanopsin appears to share light-sensing properties with the rhabdomeric photoreceptors of invertebrates. It has been shown that the mammalian circadian clock can synchronize with day–night cycles by tracking light color changes in addition to light intensity changes. It will be interesting to investigate the possible contribution of the dual function of Rh6-expressing photoreceptors to integrating different color cues into the retinal information that is sent to the clock (Alejevski, 2019).

    Electrical hyperexcitation of lateral ventral pacemaker neurons desynchronizes downstream circadian oscillators in the fly circadian circuit and induces multiple behavioral periods

    Coupling of autonomous cellular oscillators is an essential aspect of circadian clock function but little is known about its circuit requirements. Functional ablation of the pigment-dispersing factor-expressing lateral ventral subset (LNV) of Drosophila clock neurons abolishes circadian rhythms of locomotor activity. The hypothesis that LNVs synchronize oscillations in downstream clock neurons was tested by rendering the LNVs hyperexcitable via transgenic expression of a low activation threshold voltage-gated sodium channel. When the LNVs are made hyperexcitable, free-running behavioral rhythms decompose into multiple independent superimposed oscillations and the clock protein oscillations in the dorsal neuron 1 and 2 subgroups of clock neurons are phase-shifted. Thus, regulated electrical activity of the LNVs synchronize multiple oscillators in the fly circadian pacemaker circuit (Nitabach, 2006).

    Understanding the mechanisms for synchronizing multiple independent neural oscillators in circadian circuits is a key issue in circadian biology. This study provides evidence that the excitability state of the LNV subset of clock neurons plays a critical role in coordinating multiple oscillators in the fly brain. When the LNVs are made electrically hyperexcitable by genetically targeted expression of a voltage-gated sodium channel cloned from a halophilic bacterium, NaChBac, transgenic flies exhibit complex free-running behavioral rhythms with multiple periods along with desynchronization of clock protein cycling throughout the pacemaker circuit and disrupted cycling of PDF levels in the dorsomedial terminal projections of the small LNVs (sLNVs) (Nitabach, 2006).

    Anti-PDF immunofluorescence was observed in the dorsomedial terminals of the sLNVs in control flies. However, anti-PDF immunofluorescence in the dorsomedial terminals of the sLNVs of experimental flies expressing NaChBac in the LNVs is maintained at constitutively higher levels. This result is unexpected if PDF release at nerve terminals is the only cellular function influenced by alterations in cellular electrical excitability. Although there remains a formal possibility that NaChBac expression does not cause increased electrical excitability in pacemaker neurons, this is considered highly unlikely because of the robust and opposite effects of NaChBac expression compared with open-rectifier potassium-channel expression on behavior, reciprocal rescue of behavior by coexpression, clock oscillation, and direct electrophysiological recordings of muscle and photoreceptor neurons expressing NaChBac. Furthermore, hyperpolarization of LNv membrane potential after the targeted expression of open-rectifier potassium channels to these cells causes accumulations of PDF in the cell bodies of the LNVs, providing further evidence that membrane potential regulates the rates of synthesis and/or trafficking of PDF as well as release. These results together suggest that regulated electrical excitability of the sLNV plasma membrane underlies cycling PDF levels in the dorsomedial terminals, and that rendering the sLNVs hyperexcitable through NaChBac expression disrupts one or more of the cellular processes (synthesis, trafficking, or release) that determine PDF accumulation in the dorsomedial terminals. It remains unclear whether changes in neuronal membrane excitability directly influences PDF accumulation or whether this is caused by indirect effects via the molecular clock, because PDF accumulation appears to be restricted to pacemaker neurons (Nitabach, 2006).

    The behavioral and circuit alterations caused by NaChBac expression in the LNVs may be attributable in part to an altered pattern of PDF release or a yet-unidentified neurotransmitter released by the LNVs, or to complex circuit properties of the pacemaker circuit. Regulated membrane electrical excitability of other neuropeptide-secreting neurons of the insect nervous system is known to be essential for appropriate control of the temporal patterns of peptide release. PDF may act as an intrinsic coupling signal within the circadian clock circuit that synchronizes multiple oscillators that otherwise free-run independently. This interpretation is consistent with a synchronizing role for PDF proposed on the basis of gradual phase dispersal within the sLNV subgroup of Pdf01-null mutant flies in constant darkness. In addition, the results are consistent with the idea that temporally regulated PDF release by the LNVs synchronizes the circuit, and are inconsistent with the hypothesis that PDF plays a purely permissive role (Nitabach, 2006).

    Recent electrophysiological evidence in another insect suggests a mechanism for PDF- and GABA-mediated synchronization of multiple oscillators of pacemaker circuits (Schneider, 2005). Extracellular multiunit recordings of the candidate circadian neurons in excised preparations of the cockroach accessory medulla exhibit ultradian oscillatory action potential firing that is synchronized by local application of pressure ejected PDF and GABA through glass micropipettes or bath applied GABA (Schneider, 2005). Similarly, circadian neurons in the fly may fire in PDF-regulated assemblies. Although there is as yet insufficient electrophysiological evidence to allow direct comparison of the results in Drosophila with this recent finding in the cockroach, this raises the interesting possibility that NaChBac expression in the Drosophila LNVs may result in desynchronized firing of pacemaker neurons throughout the circuit, starting with the LNVs themselves. This would be consistent with the biophysical property of NaChBac of low-threshold voltage activation. Interestingly, similar mechanisms for oscillator coupling at the circuit level may also be important in mammals. GABA also modulates phase coupling between the ventral and dorsal oscillators in brain slices prepared from the rat SCN (Nitabach, 2006).

    The behavioral results confirm that the Drosophila circadian control circuit contains multiple clocks capable of oscillating independently and capable of independently controlling the pattern, but not the amount, of locomotor activity. They further indicate that properly regulated electrical excitability of the LNVs (and perhaps of particular importance, the LNVs) is required to synchronize these multiple clocks throughout the pacemaker neuronal circuit. The synchronization of multiple oscillators appears to be necessary to generate coherent single-period behavioral rhythms (Nitabach, 2006).

    The reciprocal suppression by NaChBac of the arrhythmicity induced by Kir2.1, and by Kir2.1 of the complex rhythmicity induced by NaChBac, strongly supports the interpretation that NaChBac and Kir2.1 have opposite effects on the electrical excitability of the LNVs, with Kir2.1 decreasing excitability and NaChBac increasing excitability. When expressed individually in the LNVs, K+ channels and Na+ channels have opposite behavioral effects: hyperpolarizing K+-channel expression results in arrhythmic behavior, whereas depolarizing Na+-channel expression results in hyper-rhythmic behavior. The coexpression of these two channel types together results in functional reciprocal compensation, yielding nearly normal behavior (Nitabach, 2006).

    In a previous studies, LNV membrane potential was manipulated to be hypoexcitable through the targeted expression of modified open-rectifier or inward-rectifier potassium channels (Nitabach, 2002). This caused behavioral arrhythmicity and cell autonomous dampening of the free-running molecular clock in LNV neurons in constant darkness, along with no discernable changes in the cycling of the molecular clock in downstream pacemaker neuronal subgroups at circadian day 2. Those results are consistent with the findings that desynchrony of downstream cell groups does not become apparent in pdf01-null mutant flies until 2 d in constant darkness. In the present study, LNV hyperexcitability induces trans-synaptic changes in the free-running temporal pattern of clock protein accumulation in the dorsal neuron subgroups DN1 and DN2. Thus, the DN neuronal groups appear to be functionally downstream of the LNV neurons in the pacemaker circuit. In negative control flies, the DN1s oscillate in phase with the sLNVs and LNDs, maintaining synchrony on both days 2 and 5 after release into constant darkness from a diurnal 12 h light/dark entraining regime, whereas the DN2s gradually advance from synchrony in 12 h light/dark to a 12 h phase difference by circadian day 5. The DN2s of control flies exhibit peak PDP1 accumulation at CT14 on day 2 in constant darkness and at CT10-CT14 on day 5 in constant darkness. This gradual shift of DN2 PDP1 oscillation from synchrony with the other cell groups in LD to a 12 h phase advance after 5 d in constant darkness is consistent with observations of DN2 PER cycling. In pdf>NaChBac1 flies expressing NaChBac in the LNVs, the DN1s exhibit a PDP1 molecular peak 8 h earlier than control flies on day 2 in constant darkness, and by circadian day 5 this peak has significantly damped and an additional significant peak has appeared at CT22. The DN2s of pdf>NaChBac1 flies exhibit a peak of PDP1 accumulation at CT14 on day 2 in constant darkness, in phase with control flies; by day 5 in constant darkness they peak at CT6, 4–8 h earlier than in controls. This phase shift suggests that the DN2 molecular oscillator of pdf>NaChBac1 flies is running faster than that of control flies. These differences in the temporal pattern of PDP1 accumulation in the DN1s and DN2s induced by NaChBac expression in the LNVs indicate that properly regulated electrical activity is required for normal patterns of molecular oscillation in these dorsal cell groups (Nitabach, 2006).

    The DN2s may be capable of independently driving behavioral outputs, and are possibly the cellular substrate for the ~22 h short-period component of the complex behavioral rhythmicity exhibited by flies expressing NaChBac in the LNVs. The cellular substrates for the ~25.5 h long-period component are likely to reside in other cells within the circuit. In control pdf>TM3 flies, robust free-running PER oscillation is observed in the sLNV,LND, and DN1 neurons after 5 d in constant darkness, with trough levels of PER in the second half of subjective day. The differences in the spatiotemporal pattern of PER accumulation induced by NaCh-Bac expression in the LNVs confirm, as indicated by the effects on PDP1 accumulation, that hyperexcitation of electrical activity in the LNVs causes desynchronization of the coupling and phase of molecular oscillation in dorsal clock neurons (Nitabach, 2006).

    Multiple oscillators are distributed throughout the pacemaker circuit in Drosophila. The present study confirms and extends evidence for multiple oscillators in the pacemaker circuit in Drosophila. The independent oscillators driving the multiple period components of the behavioral rhythms that were observed do not appear to correspond directly to the 'morning' and 'evening' oscillators, which have been localized to the LNVs and LNDs, respectively. The current results emphasize that the activity of the LNVs controls the synchronization of independent oscillators throughout the pacemaker circuit. The normal pattern of DN1 and DN2 clock oscillation requires properly regulated electrical excitability of the LNVs. Further, the results suggest that the DN2s, and at least some other cell groups, possess independent output pathways to the downstream locomotor circuitry (Nitabach, 2006).

    This study introduces a novel method for inducing electrical hyperexcitability in neurons of interest by the expression of the low-threshold voltage-gated sodium channel NaChBac. This method is likely to be useful for the analysis of other neural circuits. In another study (Luan, 2006), the utility of the NaChBac channel for enhancing excitability in other neurons has also been demonstrated. Targeted expression of ion channel subunits in vivo provides a powerful means for precisely perturbing neuronal membrane excitability to probe the role of activity on neuronal development and function. Initial methods to exogenously regulate electrical excitability in neurons in vivo have used potassium channel expression to electrically silence neurons. Exogenous manipulation of electrical excitability within specific Drosophila neurons can be combined with finer parsing of neural circuits using GAL80 and other genetic approaches (Nitabach, 2006).

    This study has shown that aberrations of electrical excitability in Drosophila neurons, either hyperexcitability induced by NaChBac or hypoexcitability induced by Kir2.1, can be rescued by coexpression of an ion channel with an opposite effect on excitability. This provides reason to believe that such an approach to neurological disorders of aberrant electrical activity such as epilepsy might indeed be feasible (Nitabach, 2006).

    Functional analysis of circadian pacemaker neurons in Drosophila melanogaster

    The molecular mechanisms of circadian rhythms are well known, but how multiple clocks within one organism generate a structured rhythmic output remains a mystery. Many animals show bimodal activity rhythms with morning (M) and evening (E) activity bouts. One long-standing model assumes that two mutually coupled oscillators underlie these bouts and show different sensitivities to light. Three groups of lateral neurons (LN) and three groups of dorsal neurons govern behavioral rhythmicity of Drosophila. Recent data suggest that two groups of the LN (the ventral subset of the small LN cells and the dorsal subset of LN cells) are plausible candidates for the M and E oscillator, respectively. Evidence is provided that these neuronal groups respond differently to light and can be completely desynchronized from one another by constant light, leading to two activity components that free-run with different periods. As expected, a long-period component starts from the E activity bout. However, a short-period component originates not exclusively from the morning peak but more prominently from the evening peak. This reveals an interesting deviation from the original Pittendrigh and Daan (1976) model and suggests that a subgroup of the ventral subset of the small LN acts as 'main' oscillator controlling M and E activity bouts in Drosophila (Rieger, 2006).

    Daily biological rhythms are governed by inherent timekeeping mechanisms, called circadian clocks. Such clocks reside in discrete sites of the brain and consist of multiple autonomous single-cell oscillators. Within each neuron, interacting transcriptional and translational molecular feedback loops as well as ionic signaling pathways constitute the oscillatory mechanism of the clock. It is not understood how individual pacemaker neurons interact to drive behavioral rhythmicity. The long-standing model of Pittendrigh and Daan (1976) assumes that the clock consists of two groups of oscillators with different responsiveness to light, one governing the morning (M) and the other the evening (E) activity of the animal. Typical M and E activity bouts are present in animals ranging from insects to mammals and suggest that the two-oscillatory model is generally valid. It has been shown that M and E bouts could be eliminated or reinstated by manipulating different circadian pacemaker neurons in Drosophila. This work has suggested that the ventral (LNv) and dorsal (LNd) subsets of the lateral neurons are the neuronal substrates for the M and E oscillators. It is not known whether these two oscillators respond differently to light (Rieger, 2006).

    The particular power of the two-oscillator model is that it explains observed adaptations to seasonal changes in day length. The model predicts that the M oscillator will shorten and the E oscillator will lengthen its period when exposed to extended constant light (LL). As a consequence, the M activity occurs earlier and the E activity occurs later in long summer days, helping day-active animals avoid the midday heat. The model also predicts that the M oscillator will free-run with short period and the E oscillator with long period when animals are placed in constant light. However, such internal desynchronization between oscillators does not occur, because high-intensity constant light usually results in arrhythmicity. In Drosophila, the clock protein Timeless (TIM) is permanently degraded during light-induced interaction with Cryptochrome (CRY), leading finally to the arrest of the clock. Without functional CRY, this does not happen. Indeed, internal desynchronization into two free-running components (one with a short period and the other with a long period) has been described for cryb mutants under constant-light conditions. The present study aims to analyze the molecular state of all clock gene-expressing neurons during behavioral rhythm dissociation to test the Pittendrigh–Daan model and refine the neuronal substrates of the E and M oscillators (Rieger, 2006).

    This study supports the notion that the activity rhythm of Drosophila is controlled by at least two sets of neuronal oscillators. Furthermore, the definition of these neuronal substrates of both oscillators were refined more precisely than previously. As proposed by Pittendrigh and Daan (1976), the two oscillators show different responses to light: one is accelerated and the other decelerated by constant light. However, a deviation from the original model was observed. In contrast to previous observations, the current results suggest that the PDF-positive s-LNv cells control not only the M but also the E activity bout. Therefore, the discussion should perhaps not focus of a 'morning' oscillator but rather of an M–E or 'main' oscillator (to keep the 'M'), for the following reasons. The PDF-positive s-LNv cells are essential for maintaining activity rhythms after several days under constant conditions, and electrical silencing of the LNv cells severely impairs free-running rhythms. In the present study, the PDF-positive s-LNv cells appear to dominate the rhythms in those flies that did couple E and M components after the first crossing-over on day 11 in LL, because such flies free-ran with short period (Rieger, 2006).

    The hypothesis that the PDF-positive LNv cells control not only the M activity but also partly the E activity can also explain other findings. The E activity bout is always the most prominent peak, which persists under constant-dark conditions, whereas the M activity bout is much reduced under such conditions and may even disappear. Thus, mainly the E component constitutes the free-running rhythm, and it seems implausible that the neurons responsible for rhythmicity under these conditions should have no impact on the E component. Indeed, it has been found that the s-LNv show the most robust cycling after extended time under constant conditions. Furthermore, another study emphasizes the importance of the s-LNv cells for the timing of activity peaks under constant conditions (Rieger, 2006 and references therein).

    Despite their dominance, the PDF-positive s-LNv cells depend on functional LNd and DN cells to provoke a normal E activity bout under light-dark conditions. Flies with the clock gene PER present only in PDF-positive LNv cells have a prominent M activity bout but lack the E activity bout. It is unclear whether this is attributable to the E activity fusing with the M activity or whether the E activity is suppressed, but these findings show that the output from the PDF cells requires PER in the LNd and DN cells to manifest wild-type activity patterns (Rieger, 2006).

    It was found that the PDF-negative 5th s-LNv cell cycles in-phase with the LNd cells under LL and thus may contribute to the E component. Notably, the PDF-negative 5th s-LNv cell shows high-amplitude cycling. Although this is not proof of the involvement of this cell, it suggests that it is an important circadian pacemaker neuron. Little is known about this cell because it could not be distinguished from the other lateral neurons in the former studies in which single-labeled clock protein staining was performed, but the PDF-negative 5th s-LNv cell is the only clock cell beside the PDF-positive s-LNv cells that appears to work from the first larval instar onward. Thus, it might have the same strong impact on the activity rhythm that has been revealed for the PDF-positive s-LNv cells. More work is necessary to reveal the role of the PDF-negative s-LNv cell in more detail (Rieger, 2006).

    Additional studies are also necessary to fully reveal the function of the DN cells. The current results suggest that the DN1 and the DN3 cells may contain different subclusters. Indeed, the DN1 cells develop at different times and appear to have distinct projection patterns. It is very likely that some DN1 cells contribute to the M oscillator whereas others supply the E oscillator. Again, there are data that support this hypothesis: if the lateral neurons (s-LNv, l-LNv, and LNd) are absent as a result of mutation or genetic ablation but the dorsal neurons (DN1, DN2, and DN3) are left intact, morning and evening activity bouts are still present under LD conditions, although with reduced amplitude and changed phase. The DN2 cells might play a special role for bimodal activity patterns because, in wild-type flies, they cycle 12 h out-of-phase with the s-LNv and LNd cells under DD conditions. The present study indicates that this is not the case in cryb flies under LL conditions, because the DN2 cells were in-phase with all other neurons on the first day in LL. The same applies for wild-type flies under LD conditions. It has been shown that the DN2 are indeed pacemaker neurons that cycle independently of the s-LNv cells. However, despite their autonomous function, the DN2 cells did not visibly contribute to the activity patterns of the flies under constant darkness. This suggests a minor role of the DN2 cells in the control of the activity rhythm, but the possiblity cannot be exclude that the DN2, together with the other DN groups, may contribute to morning and evening activity bouts under certain conditions (Rieger, 2006).

    The blue-light photopigment cryptochrome is regarded as the main photoreceptor of the fruit flies' circadian clock. This study shows that the compound eyes are responsible for period shortening and period lengthening of the molecular oscillations in different subsets of pacemaker neurons (the M and E oscillators) under LL. Their special role may lie in the adaptation of the clock to seasonal changes. This is in line with previous findings showing that the compound eyes are necessary for the adequate timing of M and E activity bouts in long summer days and short winter days. Cryptochrome, conversely, appears to lengthen the period in all clock neurons as can be deduced from the periods of the wild-type flies that showed internal desynchronization under 'moonlight LL.' In such flies, the periods of both components were clearly longer than those of internally desynchronized cryb flies (Rieger, 2006).

    The internal desynchronization of activity into long- and short-period components described in this study is reminiscent of previous results for Drosophila mutants with primarily reduced optic lobes or ectopic expression of PDF. Both of these fly strains have ectopic PDF-containing nerve fibers in the dorsal brain that might lead to elevated and/or nonrhythmic secretion of PDF in this brain area and may disturb normal communication between the pacemaker cells. It is unknown whether such a perturbed communication results in internal desynchronization between the s-LNv and the 5th s-LNv and extra LNd as observed in the present study. Dual-oscillator systems have been also described for mammals, but in no case they could be traced to the level of single neurons. Like the circadian pacemaker center of flies, the mammalian pacemaker center, the suprachiasmatic nucleus (SCN), contains a heterogeneous neuronal population. A recent study has shown that internal desynchronization of motor activity into short and long periods similar to the one shown in this study can be provoked in rats by special light schedules. As in Drosophila, both components reflect the separate activities of two oscillators in anatomically defined subdivisions of the SCN. Furthermore, there is some evidence to suggest that the SCN is composed of two oscillating M and E components. These results underline the universality of dual-oscillator systems (Rieger, 2006).

    Other studies strongly implicate the PDF-expressing LNv and the LNd cells as the respective neuronal loci for the morning and evening activity bouts. Despite the near 12 h phase difference between the morning and evening locomotor peaks under LD, no obvious molecular phase differences between these pacemakers have been observed that would explain them. Work in mammals suggests that the relationship between molecular phase and locomotion is complex. For example, nocturnal and diurnal rodents show the same phases of PER oscillations. Furthermore, different rat strains that displayed unimodal or multimodal activity patterns, respectively, all exhibited the same unimodal rhythm in melatonin synthesis. Individual Nile grass rats changed their activity patterns from unimodal–diurnal to bimodal–nocturnal after introducing a running wheel. Despite this dramatic effect on the activity patterns, the wheel had little effect on the circadian pacemaker, and the spatial and temporal patterns of c-Fos expression in the SCN remained similar. All of these data indicate that the relationship between molecular and behavioral phase is not straightforward. Clearly, a multitude of phase relationships between the molecular rhythm and behavior are possible. Brain regions outside the pacemaker center may be responsible for these different phases as was shown recently for mammals. It appears that the same is true within the circadian system of the fly. The present data show that, during the internally synchronized state, the trough in PER level of all neurons correlates with the main activity bout (the E peak). No second trough appears to correlate with the M peak. However, a second small peak can be seen at closer inspection of the PDF immunoreactivity in the terminals of the s-LNv. This suggests that the unimodal rhythm in clock protein cycling might be converted into a bimodal output already within the neurons (Rieger, 2006).

    During the state of behavioral desynchronization under LL conditions, an internal desynchronization was observed simultaneously in PER oscillations among subsets of pacemaker neurons. One interpretation of these data is that constant light causes internal desynchronization between these pacemaker neurons that then in turn drive the behavioral outputs. However, it must be acknowledged that this is only a correlation, and, although the hypothesis is favored that the split molecular rhythms are driving the split locomotor rhythms, it is possible that they are merely tracking or entraining to a split rhythm driven by other pacemakers. For example, the split rhythms might be driven by subsets of dorsal neurons. The hypothesis is preferred that the split behavioral rhythms were driven by the desynchronized PDF-positive LNv and the 5th s-LNv/extra LNd cells for two reasons. First, accumulating evidence points to the lateral neurons (LNv and LNd cells) as major pacemaker cells, whereas the dorsal neurons (the DN1, DN2, and DN3 cells) are not sufficient for locomotor rhythms under constant darkness. Second, in rodents, a similar behavioral desynchronization was correlated with a dissociation of clock gene expression between ventrolateral and dorsomedial subdivisions of the SCN. The established role of this brain center as the circadian clock has led to the uncontroversial conclusion that the split molecular oscillations drive the split behavioral oscillations. It is suggested that the same phenomenon is occurring in main (i.e., small LNv cells) and evening (i.e., 5th s-LNv and extra LNd cells) neuronal oscillators in Drosophila (Rieger, 2006).

    The Drosophila circadian network is a seasonal timer

    Work in Drosophila has defined two populations of circadian brain neurons, morning cells (M-cells) and evening cells (E-cells), both of which keep circadian time and regulate morning and evening activity, respectively. It has long been speculated that a multiple oscillator circadian network in animals underlies the behavioral and physiological pattern variability caused by seasonal fluctuations of photoperiod. This study manipulated separately the circadian photoentrainment pathway within E- and M-cells and shows that E-cells process light information and function as master clocks in the presence of light. M-cells in contrast need darkness to cycle autonomously and dominate the network. The results indicate that the network switches control between these two centers as a function of photoperiod. Together with the different entraining properties of the two clock centers, the results suggest that the functional organization of the network underlies the behavioral adjustment to variations in daylength and season (Stoleru, 2007).

    Two populations of circadian brain neurons, morning cells (M-cells) and evening cells (E-cells), have been connected to morning and evening locomotor activity, respectively (Grima, 2004; Stoleru, 2004). Interactions between the two oscillator populations were studied by selectively overexpressing sgg to speed up the clock in only one cell population or the other (Stoleru, 2005). This study has found that sgg overexpression gives rise to LL rhythmicity, which led to a search for the cellular substrates of entrainment. The rhythmicity is predominantly due to sgg overexpression in E-cells, which suggested that this subset of the clock network is particularly important in the light and that Sgg affects the biochemical pathway through which light impacts clock molecules and adjusts phase to the correct time of day. Indeed, strong evidence is presented that Sgg modulates Cry function, which affects in turn the core clock proteins Per and Tim. The separate manipulation of the Sgg/Cry pathway within E- and M-cells also reveals that the E-clocks drive the behavioral rhythm in light, with prominent Per oscillations of nuclear localization. This light dependence of E-cells contrasts with M-cells, which need darkness to cycle autonomously and dominate the activity output pathway. This distinction suggests a simple dual-oscillator model for how the clock adjusts to photoperiod changes, and support for this seasonal model was obtained by examining E- and M-cell cooperation under different photoperiods (Stoleru, 2007).

    The free-running pacemaker and entrainment are two important and increasingly understood aspects of circadian rhythms. In contrast, little information exists about seasonal adjustment, namely, how a constant ~24-hr timekeeper accommodates dramatically different photoperiods. This study shows that the previously defined dual oscillator system in Drosophila, M-cells and E-cells, creates different rhythmic patterns by alternating master clock roles. This understanding emerged from restricting Sgg overexpression to E-cells, which allowed the E-oscillator to function and render flies rhythmic in LL. Sgg probably modulates Cry activity and, when overexpressed, provides sufficient Per and Tim to allow E-oscillator function under constant illumination conditions. The E-clocks therefore manifest free-running properties and function as the master pacemakers in LL, analogous to a previous finding that the M-oscillator is the master in DD (Stoleru, 2005). Nonetheless, these constant conditions, and even the perfect standard LD cycles commonly used in the laboratory, are poor approximations of the changing LD environments found in nature. Circadian oscillators and their entrainment mechanisms have adapted to the dramatic seasonal changes in photoperiod. The previous strategy of using oscillators with different speeds, combined with different photoperiods, has led to a model of alternating control between the M-oscillator and E-oscillator (Stoleru, 2007).

    Sgg appears to attenuate, rather than inactivate, Cry activity in E-cells. This is because the LL period of timSgg/PdfGAL80 (~23.5 hr) is longer than the intrinsic period of Sgg-expressing E-clocks in DD (~21 hr) (Stoleru, 2005). A longer period in light is compatible with attenuated light perception under high light intensity conditions (1600 lx, which renders wild-type flies completely arrhythmic) and the application of Aschoff's rule to insects [Aschoff, 1979; One of the earliest observations in the study of circadian rhythms was that continuous light (LL) lengthens circadian period in most nocturnal animal species. 'Aschoff's Rule' posits that there is a positive log-linear relationship between the LL intensity and period]. As there is also a prominent effect on Cry stability, Sgg may be the regulator previously predicted to bind to the Cry C terminus (Busza, 2004; Dissel, 2004). Although Cry is favored as the major circadian substrate of Sgg, there may be others, e.g., the serotonin receptor. Biochemical support for GSK3 involvement in mammalian rhythms has recently been obtained (Yin, 2006). Since GSK3 is a proposed therapeutic target of lithium, the relationship between Sgg and Cry reported in this study recalls the intriguing relationship between mood disorders, light sensitivity, and circadian rhythms (Stoleru, 2007).

    The cryb genotype markedly affects DD period in some of the rhythmic genotypes described in this study. Although Cry is probably unnecessary for M-cell rhythmicity, this could reflect some redundancy or assay insensitivity. Moreover, the DD period of cryb is slightly shorter than that of wild-type (23.7 versus 24.4), suggesting that 'dark Cry' makes some contribution to pacemaker function in M-cells as well as E-cells. For these reasons, it is suggested that Drosophila Cry is closer to the central pacemaker than previously believed, and therefore closer to the level of importance of its mammalian paralogs in influencing free-running pacemaker activity. Unlike mammalian Cry, however, Drosophila Cry still appears to function predominantly at a posttranslational level. Indeed, the effects of cryb on Sgg overexpression in DD suggest that the proposed effect of Sgg on Tim stability is really an effect of Sgg on Cry followed by an altered Cry-Tim interaction. It is noted that there is a recent proposal (Collins, 2006) that Drosophila Cry, like mammalian Cry, also functions as a transcription factor in peripheral clocks (Stoleru, 2007).

    The importance of E-cells in LL rhythmicity is underscored by the staining results of timSgg/PdfGAL80 brains. Only some E-cells and DN2s manifest robust cycling. It has been suspected that E-cells are important in light because they can rescue the output of arrhythmic M-cells in LD, but not in DD (Stoleru, 2004). Indeed, all of these observations make it attractive to view E-cells as autonomous pacemakers. There is, however, evidence that M-cells may not be completely dispensable. Moreover, a synchronizing or stabilization function is compatible with previous observations under different conditions (Stoleru, 2007).

    In the timSgg/PdfGAL80 genotype, only Per nuclear localization changes were detectable near the end of LL cycle. The nature of the assay makes it hard to conclude that there were no differences in total Per staining intensity, i.e., no oscillations in Per levels, so the unique nature of the Per nuclear localization cycling is a tentative conclusion. The same caveat applies to the absence of Tim oscillations and nuclear staining, i.e., negative results cannot exclude low-amplitude oscillations; it is noted, however, that Tim cytoplasmic sequestration has been previously observed in cryb flies after several days in LL. Furthermore, the circadian nuclear accumulation of Tim has been shown to respond differently than that of Per to changes in photoperiod. Nonetheless, Tim could be shuttling with a predominant steady-state cytoplasmic localization, nuclear Tim could be rapidly degraded to create a low nuclear pool, or both (Stoleru, 2007).

    The importance of E-cells in entrainment is strongly supported by the potent effect of restricted Cry rescue of cryb: E-cell rescue is much more impressive than M-cell rescue. Moreover, the differences between the two rescued phase response curves (PRCs) are striking; E-cell rescue is virtually complete, whereas the M-cell rescue is notably deficient in the delay zone. In addition, flies with Sgg overexpression in E-cells show altered PRCs, whereas flies with Sgg overexpression in M-cells respond normally to light. The results are strikingly different in darkness, as M-cell-restricted expression causes the typical short period determined by Sgg overexpression, whereas E-cell overexpression has no systemic effect (Stoleru, 2007).

    The PRC delay zone is the region impacted most strongly by E-cell Sgg overexpression, indicating that the lights-off early night region is most important to E-cell function and light entrainment. Exposure to light in this interval should mimic long days (summer), which, it is speculated, will delay phase by many hours so that “evening” output of the following day will coincide with the objective evening of the environment. Even the short nights of summer are probably enough time for E-clocks to accumulate sufficient Tim and Per, shuttle them into the nucleus, and reconstitute the rhythmic substrate observed in the Sgg-overexpressing brains in LL. In contrast, M-cells need darkness to cycle robustly. They will become the master clocks and drive the system whenever lights fail to turn on more than 12 hr past lights-off, i.e., during the long nights of winter that mimic the beginning of a DD cycle. Since the intrinsic pacemaker program of M-cells in darkness relies on the changing nature of clock proteins during the night, it is hypothesized that the activity phases under long nights (winter) are locked to lights-off. This suggestion is supported by preliminary data and previous observations showing that per transcription remains locked to lights-off under different entrainment regimes. M-cells are also capable of fully entraining the system in the PRC interval that determines a phase advance (late night). This is consistent with their predicted role in generating an advanced evening output, coincident with the early evenings typical of winter. Otherwise put, long summer days should underlie light primacy as well as long and prominent evening delay zones; both suggest E-cell dominance. Night primacy and M-cells should dominate under winter conditions. This concept endows E- and M-cells with the properties originally envisioned by the Pittendrigh and Daan (1976) dual-oscillator model of entrainment (Stoleru, 2007).

    Moonlight shifts the endogenous clock of Drosophila melanogaster

    The ability to be synchronized by light-dark cycles is a fundamental property of circadian clocks. Although there are indications that circadian clocks are extremely light-sensitive and that they can be set by the low irradiances that occur at dawn and dusk, this has not been shown on the cellular level. This study demonstrates that a subset of Drosophila's pacemaker neurons responds to nocturnal dim light. At a nighttime illumination comparable to quarter-moonlight intensity, the flies increase activity levels and shift their typical morning and evening activity peaks into the night. In parallel, clock protein levels are reduced, and clock protein rhythms shift in opposed direction in subsets of the previously identified morning and evening pacemaker cells. No effect was observed on the peripheral clock in the eye. These results demonstrate that the neurons driving rhythmic behavior are extremely light-sensitive and capable of shifting activity in response to the very low light intensities that regularly occur in nature. This sensitivity may be instrumental in adaptation to different photoperiods. This adaptation depends on retinal input but is independent of cryptochrome (Bachleitner, 2007).

    Nocturnal light provoked an advance of the morning (M) activity and a delay of the evening (E) activity into the night. Simultaneously, the midday trough broadened and the midnight trough diminished, making the flies nocturnal in a cycle of 12 h:12 h. In other words, they switched their temporal niche. Upon transfer to constant conditions, they reverted, with activity in continuous moonlight (MM) always starting from the preceding light phase. A similar switch was observed in night-active white-fronted lemurs (Eulemur fulvus albifrons). These animals switched from night-active to day-active after reduction of the nocturnal illumination below a certain threshold; but on release into constant conditions, free-running activity always started from the preceding dark phase. This switching was thought to be caused by direct effects of light on activity (masking effects) that do not interfere with the circadian clock. In other words, the animals have strong preferences for certain light conditions, and they accordingly avoid being active under both total darkness, because it precludes visual orientation, and in high irradiances, because it may damage their sensitive eyes. Studies on mice (Mus musculus) yield similar results, with nocturnal animals becoming diurnal after mutations, genetic manipulations, or brain lesions that interfere with photoreceptor input to the circadian clock. One possible explanation is that mice with impaired photoreception simply prefer higher irradiances than wild-type mice (Bachleitner, 2007).

    This study demonstrates that temporal niche switching may be caused by a change in the phasing of the endogenous clock as induced by dim light levels. Masking effects may still be involved, because the masking that is typically seen in conventional light-dark (LD) is lost under light-'moonlight' (LM) conditions. Instead, the flies' activity appears to be promoted by dim light. Despite a contribution of masking effects, this study shows that Drosophila's circadian pacemaker neurons are highly light-sensitive and respond to nocturnal light levels comparable to moonlight. The peripheral oscillators in the eye fail to do so. This finding is extremely interesting with respect to photoreceptor sensitivity. Whereas photoreceptors for visual image detection should quickly adapt to alterations in light intensity to ensure optimal vision, photoreceptors for the circadian clock should not adapt, at least not in the lower ranges. Otherwise, it would be impossible to measure increasing and decreasing irradiances during dawn and dusk. The observed clock protein oscillations in the photoreceptor cells of the compound eyes might regulate light sensitivity of the circadian system. If true, the clock protein levels and oscillations should not be altered by dim light, and the high sensitivity of the clock during early dawn and late dusk should be preserved. Indeed, no alterations were observed in PER and TIM protein levels under LM conditions. Consistent with this finding, it was found that the compound eyes, not the photoreceptor cryptochrome, mediate the responses to moonlight. This finding is in line with previous observations showing that the compound eyes and, thus, rhodopsins are necessary for adaptation of activity times to long and short days. Interestingly, the insect rhodopsins are closely related to the mammalian melanopsin, which is critically involved in mouse circadian photoreception (Bachleitner, 2007).

    But then, what role remains for cryptochrome? Cryptochrome may contribute to the phase delay of the E peak under LM conditions, because cryb mutants show a less dramatic delay of this peak than wild-type flies. However, the E peak was not at all delayed in clieya mutants, suggesting that a phase-delaying effect of cryptochrome is either dependent on the compound eyes or is negligible. Cryptochrome is a blue-light photoreceptor and, thus, most appropriate to detect intensity changes in blue light. Furthermore, the proportion of blue light increases during dawn and decreases during dusk. Thus, cryptochrome appears particularly suited to distinguish dawn or dusk light transitions from moonlight, which does not change its spectrum over time (Bachleitner, 2007).

    These results are interpreted as evidence for a differential action of dim light on the pace of M and E oscillators as was originally proposed in the dual-oscillator model for rodents and recently verified for D. melanogaster. Because the M cells phase advance and a subset of the E cells phase delay their clock in response to dim light, these cells are optimally suited to adapt activity rhythms to seasonal changes in day length. In addition, the proposed role of the LNd cells as E cells is not supported by this study. This finding is in line with earlier work demonstrating that the LNd represent a heterogenous group of cells and that, putatively, only one LNd cell behaves as an E oscillator. It is quite possible that this LNd cell also phase delayed in the present study; but without a specific marker, it was not possible to distinguish it from the other cells. In summary, these results are consistent with the involvement of the PDF-positive s-LNv cells and the PDF-negative fifth s-LNv cells on behavioral rhythmicity. The relevance of the two-oscillator model under natural conditions is also shown (Bachleitner, 2007).

    Not all animals may be as light-sensitive as are fruit flies, but recent studies showed that even species such as hamsters and humans are more sensitive than supposed. The synchronization of Syrian and Siberian hamsters to different photoperiods was facilitated under dim night illumination (<0.005 lux) as compared with DD. Furthermore, the incidence of bimodal activity patterns and the interval between both components increased under LM conditions. In humans, dawn simulations at low light intensities were found to phase advance the circadian melatonin and the activity rhythm. Together with the results presented here, these studies suggest that clock function in many species is conspicuously altered by nocturnal illumination as experienced under dim moonlight. This finding might go back to the ability of primordial marine animals to synchronize their reproduction to the lunar cycle, an ability that is apparently lost in humans and other terrestrial animals. Presumably, many terrestrial organisms do not use their light sensitivity for moonlight detection, but for timing their clock to the increasing and decreasing irradiances during dusk and dawn. Further studies are necessary to reveal whether these animals hide at night from the moonlight so as not to confound their clocks, whether they switch to nocturnal activity (or become sleepless) during the full moon, or whether they use cryptochrome to distinguish moonlight from dawn and dusk (Bachleitner, 2007).

    Large ventral lateral neurons modulate arousal and sleep in Drosophila

    Large ventral lateral clock neurons (lLNvs) exhibit higher daytime-light-driven spontaneous action-potential firing rates in Drosophila, coinciding with wakefulness and locomotor-activity behavior. To determine whether the lLNvs are involved in arousal and sleep/wake behavior, the effects of altered electrical excitation of the LNvs were examined. LNv-hyperexcited flies reverse the normal day-night firing pattern, showing higher lLNv firing rates at night and pigment-dispersing-factor-mediated enhancement of nocturnal locomotor-activity behavior and reduced quantity and quality of sleep. lLNv hyperexcitation impairs sensory arousal, as shown by physiological and behavioral assays. lLNv-hyperexcited flies lacking sLNvs exhibit robust hyperexcitation-induced increases in nocturnal behavior, suggesting that the sLNvs are not essential for mediation of arousal. It is concluded that light-activated lLNvs modulate behavioral arousal and sleep in Drosophila (Sheeba, 2008b).

    The small and large ventral lateral clock neurons (henceforth sLNvs and lLNvs) were among the first cells identified as crucial for normal light entrainment of circadian behavior. Several studies suggest that the sLNvs are responsible for sustained circadian locomotor activity in constant darkness, whereas the lLNvs are less characterized. Recently, it was shown by using whole-cell patch clamp electrophysiology that lLNvs acutely increase their firing rate in response to light in a cryptochrome-dependent fashion (Sheeba, 2008a). Because light is a well-known sensory cue for arousal, as well as circadian entrainment, this study tested whether altered electrical activity of the lLNvs influences locomotor-activity behavior, sleep, and arousal (Sheeba, 2008b).

    These studies show that alteration of the balance of day-night neuronal firing by hyperexcitation of lLNvs in D. melanogaster directs behavioral-activity preference toward increased nocturnality and modulates the quantity and quality of nocturnal sleep. Furthermore, other peptidergic neurons encompassed within the c929 expression pattern modulate both day and night wakefulness and sleep. These results, in combination with an earlier detailed electrophysiological analysis (Sheeba, 2008a), suggest that lLNvs constitute a light-activated arousal circuit. Additional support for this is shown by decreased behavioral responsiveness to day onset in PDF-lacking flies and flies with electrically altered LNvs. It has been shown previously that plasticity in temporal day versus night behavioral preference in mammals and other animals can result from changes in environmental sensory time cues or manipulations that alter sensory-input pathways to circadian clocks. In Drosophila, mutants with partial or complete loss of photoreceptors sometimes show greater activity at night. Mutations in a widely expressed putative cation channel DMα1U (narrow abdomen [na]) also results in a switch from diurnal to nocturnal activity (Nash, 2002). Rescue of diurnal activity was achieved by expression of wild-type channel in parts of the circuit that included lLNvs (Sheeba, 2008b and references therein).

    In the case of NaChBac-induced hyperexcitation of lLNvs, in which NaChBac, a voltage-gated sodium channel, was expressed in LNvs) the normal pattern of light-driven activity during the day is reversed to a novel pattern of firing rate that favors higher activity in the night. The lLNvs are not likely to be 'nocturnal' neurons. Rather, they appear to drive locomotor activity according to their relative day versus night pattern of excitation. Considering that the wild-type electrophysiological firing properties of lLNvs are so dramatically different from NaChBac-evoked firing and sustained hyperexcitation, it is reasonable to wonder how NaChBac expression in LNvs, and specifically a lLNv subset, yields such a coherent pattern of behavioral activity. The results above show that lLNvs modulate arousal and sleep and that altering the relative pattern of day versus night excitability is sufficient to evoke a temporal change in behavioral output. On the basis of these observations, it is hypothesized that the precise firing pattern or timing of lLNv electrical activity is probably not important, thereby making NaChBac expression an appropriate tool to study these neurons and probably other modulatory circuits for which general changes in the gain of activity rather than the precise pattern of activity dictate functional output (Sheeba, 2008b).

    The results reveal that the PDF-expressing peptidergic lLNvs modulate arousal and wakeful behavior as well as sleep stability. Considering these functional studies, lLNvs appear to act as an arousal circuit that is physiologically activated by light and borders with, but is distinct from, the circadian pacemaker and downstream sleep circuits. A number of other recently described modulatory systems in Drosophila influence behavioral locomotion and sleep, including aminergic and GABAergic neurons. As noted, many of the overall features of morning and evening peaks in locomotor activity are retained when lLNv and other peptidergic neuronal subsets are hyperexcited. Considering the naturalistic implications, temporal-niche switching has been observed in a few animals, including the social ant species Camponotus compressus. Among the worker class of these ants, some individuals are diurnal and others nocturnal, and these plastic behavioral differences are associated with differences in their underlying free-running circadian period. On the basis of the current results, it is possible that activity changes in a relatively small number of arousal neurons could influence both short-term temporal-niche switching or long-term evolutionary commitment to a given temporal niche (Sheeba, 2008b).

    A role for blind DN2 clock neurons in temperature entrainment of the Drosophila larval brain

    Circadian clocks synchronize to the solar day by sensing the diurnal changes in light and temperature. In adult Drosophila, the brain clock that controls rest-activity rhythms relies on neurons showing Period oscillations. Nine of these neurons are present in each larval brain hemisphere. They can receive light inputs through Cryptochrome (CRY) and the visual system, but temperature input pathways are unknown. This study investigated how the larval clock network responds to light and temperature. Focus was placed on the CRY-negative dorsal neurons (DN2s), in which light-dark (LD) cycles set molecular oscillations almost in antiphase to all other clock neurons. The phasing of the DN2s in LD depends on the pigment-dispersing factor (PDF) neuropeptide in four lateral neurons (LNs), and on the PDF receptor in the DN2s. In the absence of PDF signaling, these cells appear blind, but still synchronize to temperature cycles. Period oscillations in the DN2s were stronger in thermocycles than in LD, but with a very similar phase. Conversely, the oscillations of LNs were weaker in thermocycles than in LD, and were phase-shifted in synchrony with the DN2s, whereas the phase of the three other clock neurons was advanced by a few hours. In the absence of any other functional clock neurons, the PDF-positive LNs were entrained by LD cycles but not by temperature cycles. These results show that the larval clock neurons respond very differently to light and temperature, and strongly suggest that the CRY-negative DN2s play a prominent role in the temperature entrainment of the network (Picot, 2009).

    Although the absence of PDF severely affects Drosophila activity rhythms in DD, the exact function of the neuropeptide in the adult clock neuronal network remains unclear. In LD, PDF is required to produce a morning activity peak and to properly phase the evening peak, but not to entrain the brain clock. The behavioral phenotypes of PDF receptor mutants resemble that of the pdf01 mutant. PDFR is expressed in all clock neurons except the large ventral lateral neurons (l-LNvs), supporting a role of PDF in maintaining phase coherence within the adult clock network in DD. The loss of PER oscillations in the DN2s of pdf01 larvae demonstrates a clear and novel role of PDF in transmitting not only phase information but also a synchronizing signal without which the receiving neurons are not entrained in LD (Picot, 2009).

    The current results show that the CRY-less DN2s are 'blind' neurons that perceive light indirectly. The PDF receptor rescue experiments strongly suggest that PDF acts on its receptor on the larval DN2s themselves, which are located in the vicinity of the LN axon terminals. Furthermore, DN2s possess a wide and dense neuritic network that borders on the axons of the LNs over a large fraction of their length. However, it cannot be ruled out that expression of the receptor in the (PDF-negative) fifth LN is involved in synchronizing the DN2s downstream, through PDF-independent mechanisms (Picot, 2009).

    The PDF-negative fifth LN is also a CRY-negative clock neuron, but it cycles in phase with the CRY-positive neurons of the larval brain. The visual input to the PDF-expressing LNs appears sufficient to phase them normally even in cryb mutants. It could thus be expected to entrain the CRY-less fifth LN in phase with the other larval LNs, as observed, in contrast to the CRY-less DN2s. A direct input from the visual system to the fifth LN is also consistent with its PDF-independent entrainment by LD cycles. Similarly, light entrainment of the larval DN1s in cryb mutants is consistent with their suggested connection to the visual system. Thus, the CRY-less DN2s would be the only larval clock neurons devoid of such a connection (Picot, 2009).

    Adult eclosion rhythms depend on the PDF-expressing LNs and appear to require the PDF-dependent clock that resides in the prothoracic gland. Since the larval DN2s project in the pars intercerebralis, a region of the brain that sends projections to the prothoracic gland, they could play a role in this physiologically important clock function. These results raise the possibility that the damped PER oscillations in the DN2s of the pdf01 mutants participate to their eclosion phenotype (Picot, 2009).

    The DN2s are the only larval clock neurons that are phased identically by light and temperature, but their temperature entrainment appears independent of any LN-derived signal. PER oscillations in the DN2s have a larger amplitude in HC cycles than in LD cycles, also suggesting a prominent role of temperature in their entrainment. Conversely, the molecular oscillations of the PDF-positive LNs have a larger amplitude in LD compared with HC cycles. In the latter, the molecular oscillations of the PDF-expressing LNs seem to follow those in the DN2s, with a large phase change compared with LD conditions. The DN1s and the PDF-negative fifth LN, in contrast, share another phase that is slightly advanced. Interestingly, behavioral and transcriptome data in adult flies indicate that HC cycles result in a general phase advance relative to LD cycles. Cooperative synchronization of the clock by light and temperature likely requires temperature changes to act earlier than light changes since changes in temperature always lag behind changes in solar illumination in nature. The very different relative phasing of the larval clock neurons in HC versus LD cycles suggests different ecological constraints on this life stage, spent mostly burrowed in food, in which light may be a weaker Zeitgeber, and in which the lag between temperature and light changes may be quite different (Picot, 2009).

    When a functional clock is absent from the DN2s (and the fifth LN), the larval PDF-expressing LNs are unable to entrain to thermocycles, whereas they autonomously entrain to LD cycles. It remains possible that autonomous temperature entrainment of the larval LNs (but not the DN2s) requires per transcriptional regulation, which the GAL4-UAS system is lacking. But the results demonstrate the existence of a control exerted on the LN clock by CRY-negative clock cells when temperature is the synchronizing cue. Although a role of the fifth LN cannot be ruled out, the absence of autonomous photoperception by the DN2s nicely fits with a role in temperature entrainment. The high cycling amplitude of the DN2s in thermocycles and the locking of the phase of the LNs on that of the DN2s in these conditions strongly support their role in the temperature entrainment of the LNs (Picot, 2009).

    Additional studies should investigate whether the DN2s communicate with the LNs via fibers that appear to run along the dorsal projection of the LNs. Alternatively, the dense dendritic-like network of the DN2s could ensure reciprocal exchanges between them and the LNs. A model is thus proposed whereby, in the larval brain, the DN2s and the four PDF-positive LNs form a distinct subnetwork, with the LNs entraining the DN2s in LD, whereas the opposite is true in HC). What becomes of their hierarchy in constant conditions, after entrainment stops? Their relative phases appear to change little at least during the first 2 d after entrainment, whether they have been set in antiphase by LD entrainment, or in phase by HC entrainment. This suggests that, whatever the entraining regimen, the LNs and the DN2s run autonomously in constant conditions. However, it cannot be excluded that one of the two groups still dominates but requires more time after the end of entrainment to shift the phase of the other (Picot, 2009).

    The rhythmic behavior of the adult flies that emerge from the temperature-entrained larvae is almost in antiphase compared with the one of flies entrained by light during the larval stage. This strongly suggests that the phase of the adult rhythms is set by the antiphasic oscillations of the larval PDF-positive LNs, consistent with these cells being the only neurons in which molecular cycling persists throughout metamorphosis. It is thus believed that the large phase shift of adult activity can be accounted for simply by the large phase shift of molecular oscillations in the PDF-expressing LNs (Picot, 2009).

    It is often assumed that temperature affects the molecular clock directly and identically in all clock cells, as opposed to light, which requires dedicated input pathways. However, in the adult, thermocycles phase the brain clock differently from all peripheral clocks, as judged from whole-tissue oscillations of a luciferase reporter enzyme (Glaser, 2005). Recent data suggest that subsets of clock neurons in the Drosophila adult brain may indeed be dedicated to temperature entrainment. In experiments combining LD and HC entrainment, all DN groups, as well as the less studied lateral posterior neurons (LPNs), seem to preferentially follow thermocycles, whereas the other LNs preferentially follow LD cycles (Miyasako, 2007). Although adult PDF+ LNs are able to entrain to thermocycles in the absence of any other functional clock, they do not seem to be required for (and actually slowed down) the temperature entrainment of activity rhythms, whereas the PDF-negative LPNs appear to play a prominent role in such conditions (Picot, 2009).

    The current results indicate that a similar specialization toward light or temperature entrainment exists in the larval brain. The DN2s, which appear to be the most temperature-responsive clock neurons, are by themselves completely blind. Conversely, the four PDF-positive LNs, which may be the most light-sensitive clock neurons (with both CRY and the visual system as inputs), appear almost temperature blind, and depend on the DN2s for temperature entrainment. PER-negative DN2s do not allow PER oscillations in the larval LNs, suggesting that entrainment of the latter in HC cycles depends on clock function in the former. The hierarchy of clock neurons thus appears very different during entrainment of the clock network by one or the other Zeitgeber (Picot, 2009).

    Roles of dopamine in circadian rhythmicity and extreme light sensitivity of circadian entrainment

    Light has profound behavioral effects on almost all animals, and nocturnal animals show sensitivity to extremely low light levels. Crepuscular, i.e., dawn/dusk-active animals such as Drosophila melanogaster are thought to show far less sensitivity to light. This study reports that Drosophila respond to extremely low levels of monochromatic blue light. Light levels three to four orders of magnitude lower than previously believed impact circadian entrainment and the light-induced stimulation of locomotion known as positive behavioral masking. GAL4;UAS-mediated rescue of tyrosine hydroxylase (DTH) mutant (ple) flies was used to study the roles of dopamine in these processes. Evidence is presented for two roles of dopamine in circadian behaviors. First, rescue with either a wild-type DTH or a DTH mutant lacking neural expression leads to weak circadian rhythmicity, indicating a role for strictly regulated DTH and dopamine in robust circadian rhythmicity. Second, the DTH rescue strain deficient in neural dopamine selectively shows a defect in circadian entrainment to low light, whereas another response to light, positive masking, has normal light sensitivity. These findings imply separable pathways from light input to the behavioral outputs of masking versus circadian entrainment, with only the latter dependent on dopamine (Hirsch, 2010).

    Sensitivity to extremely low levels of light is most commonly found in nocturnal animals. These animals, such as nocturnal geckos or insects such as nocturnal hawkmoths, can not only sense extremely low levels of light but can also discern colors at light intensities well below those to which diurnal animals are sensitive. Humans and diurnal vertebrates lose color vision at light intensities comparable to dim moonlight at irradiances of 3-10 nW/cm2. In contrast, nocturnal hawkmoths and geckos can discern colors even at intensities of ~0.01-0.3 nW/cm2 and normally function in starlight, ~0.001 nW/cm2. Extreme light sensitivity in nocturnal insects commonly involves adaptations to their compound eyes to allow summation of photons from many individual ommatidia. These visual system adaptations are not seen in diurnal insects such as the fruit fly Drosophila melanogaster. Accordingly, current data accord Drosophila with rather modest light sensitivity. For light-dependent entrainment of circadian rhythmicity, ~40 nW/cm2 blue light was thought to be required, although subsequent studies show entrainment by 1-5 nW/cm2 white light. Wild-type flies are now thought to entrain at ~0.04 nW/cm2 blue light (C. Helfrich-Forster, personal communication to Hirsch, 2010). An intensity of ~0.5 nW/cm2 white light is reported to cause positive behavioral masking, the largely circadian clock-independent stimulation of locomotion. For comparison, this study found that a dark-adapted human observer loses the ability to perceive the diffuse planar blue light sources used in the present study at intensities of ~0.01-0.03 nW/cm2. This intensity is difficult to compare to published human perception studies, which commonly use short duration flashes of focal light (Hirsch, 2010).

    This study found unexpectedly strong light sensitivity for Drosophila melanogaster, with behavioral masking and circadian entrainment at intensities as low as 0.001 nW/cm2 and at least two roles for dopamine in circadian rhythmicity. First, DTH rescue flies showed poor behavioral rhythmicity in constant dark conditions, independent of whether dopamine levels were rescued in the nervous system. Second, it was found that neuronal DTH rescue flies lacking neuronal dopamine showed reduced light sensitivity for circadian entrainment, whereas light sensitivity of behavioral masking was unaffected. Dopamine has several roles in Drosophila neural function, from modulation of locomotor behaviors and arousal states to learning and memory, but a role for dopamine in insect light-dependent circadian behavioral entrainment is novel (Hirsch, 2010).

    The two circadian phenotypes likely represent separate roles for dopamine, presumably in different regions of the nervous system, because reduced amplitude of rhythmicity, as seen in DTH rescue lines, is normally associated with higher rather than lower efficacy of reentrainment. The dopaminergic system in Drosophila is highly rhythmic, as evidenced by rhythmicity in responsiveness to dopamine agonists and by the rhythmic transcription of the tyrosine hydroxylase gene ple, which encodes the rate-limiting enzyme in dopamine biosynthesis. The rhythmicity of the ple transcript may explain the poor rhythmicity in ple rescue animals. These animals have near-normal levels of brain dopamine in an apparently normal cellular pattern, but the inclusion of the GAL4 transcription factor into the regulatory cascade will almost certainly interfere with normal temporal cycling of the DTH transcript. Note that significant diurnal variation in levels of brain dopamine in brain extracts have not been detected, but this does not preclude diurnal variation in dopamine neuron subsets (Hirsch, 2010).

    Low-light circadian entrainment is disrupted in the brain dopamine-deficient DTHgFS±ple flies. The simplest mechanism for the disruption of low-light circadian entrainment would be due to alterations in the photoreceptive pathway, which could be via cryptochrome (CRY) or visual photoreceptors. There is some support for dopaminergic involvement in the CRY pathway, because Sathyanarayanan (2008) identified ple in a screen for genes that, when targeted by RNA interference, have a strong inhibitory effect on light-dependent degradation of CRY and timeless (TIM) in cultured cells. This could indicate a positive role for dopamine in light-dependent degradation of these molecules, providing a potential mechanism for the reduced light sensitivity for circadian entrainment that was observed in the absence of dopamine (Hirsch, 2010).

    Alternatively, it is known that visual photoreceptors are involved in dim-light entrainment because genetic loss of all photoreceptive visual organs results in at least a three-order-of-magnitude reduction in blue light sensitivity for circadian entrainment. Analogous studies in mice show an ~60-fold reduction in dim-light sensitivity for entrainment in animals lacking both rods and cones (Hirsch, 2010).

    A role for dopamine in fly visual function has some support in that cyclic AMP (cAMP) can slow the response to light in a preparation of isolated Drosophila photoreceptors (Chyb, 1999), and this effect can be mimicked by application of octopamine or dopamine, an effect interpreted as enhanced adaptation to dark. Dopamine signaling, via cAMP second-messenger pathways, is not currently considered part of the main insect visual transduction pathway. However, dopamine involvement could have been missed if it has an exclusive role in a neural pathway selectively required for circadian entrainment by dim light (Hirsch, 2010).

    There is strong support of a role for dopamine functioning in the vertebrate retina, which makes visual involvement of dopamine in the fly all the more likely. The vertebrate retina contains autonomous circadian oscillators that are thought to allow the retina to prepare for the large difference in light intensity between day and night. Central to this rhythmicity are opposing and rhythmic roles for melatonin and dopamine, with release of each modulator inhibiting synthesis and/or release of the other. The best defined role for dopamine in the vertebrate circadian oscillator is in entraining fetal rodents prior to light exposure, a capacity lost in adults. This role of dopamine could be related to the roles that have been uncovered in adult Drosophila (Hirsch, 2010).

    The selective effect of neural dopamine on low-light entrainment versus low-light masking behavior implies separable pathways involved in modulating these behaviors, a novel finding because previous studies have only identified circadian components with parallel effects on masking (Mazzoni, 2005). The best defined synaptic connections from eye to circadian neurons are the projections from the Drosophila eyelet, a remnant of the larval photoreceptive Bolwig's organ. This photoreceptive organ makes projections that terminate in close apposition to neurites from the small and large ventral lateral neurons, neurons key to circadian rhythmicity. Connections from the main visual photoreceptors to these circadian neurons must be indirect because the rod-like outer photoreceptor ommatidia terminate in the optic lamina, and the cone-like central ommatidia terminate in the optic medulla. Nonetheless, dopamine could be acting as a neuromodulator in any of these pathways to increase sensitivity to a light-dependent signal. The genetic tools available in Drosophila should prove useful to precisely identify these pathways (Hirsch, 2010).

    Deep conservation of genes required for both Drosophila melanogaster and Caenorhabditis elegans sleep includes a role for dopaminergic signaling

    Cross-species conservation of sleep-like behaviors predicts the presence of conserved molecular mechanisms underlying sleep. However, limited experimental evidence of conservation exists. This prediction is tested directly in this study. During lethargus, Caenorhabditis elegans spontaneously sleep in short bouts that are interspersed with bouts of spontaneous locomotion. Twenty-six genes required for Drosophila melanogaster sleep were identified. Twenty orthologous C. elegans genes were selected based on similarity. Their effect on C. elegans sleep and arousal during the last larval lethargus was assessed. The 20 most similar genes altered both the quantity of sleep and arousal thresholds. In 18 cases, the direction of change was concordant with Drosophila studies published previously. Additionally, a conserved genetic pathway was delineated by which dopamine regulates sleep and arousal. In C. elegans neurons, G-alpha S, adenylyl cyclase, and protein kinase A act downstream of D1 dopamine receptors to regulate these behaviors. Finally, a quantitative analysis of genes examined herein revealed that C. elegans arousal thresholds were directly correlated with amount of sleep during lethargus. However, bout duration varies little and was not correlated with arousal thresholds. The comprehensive analysis presented in this study suggests that conserved genes and pathways are required for sleep in invertebrates and, likely, across the entire animal kingdom. The genetic pathway delineated in this study implicates G-alpha S and previously known genes downstream of dopamine signaling in sleep. Quantitative analysis of various components of quiescence suggests that interdependent or identical cellular and molecular mechanisms are likely to regulate both arousal and sleep entry (Singh, 2014).

    Epigenetic regulator Stuxnet modulates octopamine effect on sleep through a Stuxnet-Polycomb-Octbeta2R cascade

    Sleep homeostasis is crucial for sleep regulation. The role of epigenetic regulation in sleep homeostasis is unestablished. Previous studies showed that octopamine is important for sleep homeostasis. However, the regulatory mechanism of octopamine reception in sleep is unknown. This study identified an epigenetic regulatory cascade (Stuxnet-Polycomb-Octβ2R) that modulates the octopamine receptor in Drosophila. stuxnet positively regulates Octβ2R through repression of Polycomb in the ellipsoid body of the adult fly brain and Octβ2R is one of the major receptors mediating octopamine function in sleep homeostasis. In response to octopamine, Octβ2R transcription is inhibited as a result of stuxnet downregulation. This feedback through the Stuxnet-Polycomb-Octβ2R cascade is crucial for sleep homeostasis regulation. This study demonstrates a Stuxnet-Polycomb-Octβ2R-mediated epigenetic regulatory mechanism for octopamine reception, thus providing an example of epigenetic regulation of sleep homeostasis (Zhao, 2021).

    Drosophila has been used as a model system to study mechanisms of sleep regulation. The first studies on sleep in Drosophila revealed that they periodically enter a quiescence state that meets a set of criteria for sleep. Drosophila sleep is monitored normally by a Drosophila activity monitoring system (DAMS) and is defined as immobility for 5 min or longer which is a sleep bout. Drosophila sleep mainly happens at night, while a period of siesta is in the mid-day. For example, total sleep time is around 380 min (male) and 250 min (female) during the day time, and 480 min (male) and 490 min (female) during the night time in w1118 (Zhao, 2021).

    In Drosophila, central complex structures, especially the ellipsoid body (EB) and fan-shaped body (FSB), are important for sleep homeostasis regulation. Activation of dorsal FSB neurons is sufficient to induce sleep. The dorsal FSB also integrates some sleep inhibiting signals. Both dorsal FSB and EB ring 2 are important in sleep homeostasis. Recently, the helicon cells were found to connect the dorsal FSB and EB Ring 2, indicating that these EB and FSB are connected (Zhao, 2021).

    Multiple studies indicate that the epigenetic mechanisms are involved in circadian regulation. However, a direct link between epigenetic regulation and sleep homeostasis is not yet established (Zhao, 2021).

    Octopamine (OA) in Drosophila is a counterpart of vertebrate noradrenaline. Previous studies in Drosophila showed that OA is a wake-promoting neurotransmitter and plays an important role in regulating both sleep amount and sleep homeostasis. The mutants of the OA synthesis pathway show an increased total sleep. Activation of OA signaling inhibits sleep homeostasis, while in OA synthesis pathway mutants, an enhanced sleep homeostasis is observed. Study of the neural circuit responsible for the sleep/wake effect of OA showed that octopaminergic ASM neuronsproject to the pars intercerebralis (PI), where OAMB (one of the OA receptors)-expressing insulin-like peptide (ILP)-secreting neurons act as downstream mediators of OA signaling. However, the effects of manipulating ASM neurons or ILP-secreting neurons are much weaker than those observed by manipulating all OA secreting neurons. Moreover, the effect of octopamine is not completely suppressed in the OAMB286 mutant, arguing that another receptor or circuit may participate in this process (Zhao, 2021).

    Eight OA receptors are identified to date: OAMB, Octβ1R, Octβ2R, Octβ3R, TAR1, TAR2, TAR3, and Octα2R. Although the expression pattern of OA is identified, the endogenous expression profile of these receptors is lacking. A previous study demonstrated that the mushroom body-expressed OAMB mediates the sleep:wake effect of OA. Recently, Octβ2R was shown to be important for the OA effect on endurance exercise adaptation. How the versatility of OA function is mediated by the diverse array of its receptors needs further study. Moreover, the upstream regulatory mechanisms of OA receptors are still unknown (Zhao, 2021).

    A previous study showed that Stuxnet (Stx) is important in mediating Polycomb (Pc) protein degradation in the proteasome (Du, 2016). Stx, which is an ubiquitin like protein, mediates Polycomb (Pc) protein degradation through binding to the proteasome with a UBL domain at its N terminus and to Polycomb through a Pc-binding domain. stx level changes result in a series of homeotic transformation phenotypes. Pc is an epigenetic regulator functioning in Polycomb Group (PcG) Complexes. Although it is reported that PcG component E(Z) is involved in circadian regulation, the role of stx in adult physiological process is unknown (Zhao, 2021).

    This study identified the role of the epigenetic regulator stx in sleep regulation. stx positively regulates Octβ2R through regulation of Polycomb in the EB of the adult fly brain. Further study demonstrated that the Stuxnet-Polycomb-Octβ2R cascade plays an important role in sleep regulation. In order to elucidate the role of this Stuxnet-Polycomb-Octβ2R cascade in sleep regulation, the role of various Octβ receptors was systematically identified in sleep regulation. Octβ2R was found to be one of the receptors that mediates OA function in sleep homeostasis. More interestingly, it was found that stx was OA-responsive depending on the Octβ1R. Based on these data, it is proposed that the Stuxnet-Polycomb-Octβ2R cascade provides a feedback mechanism for OA signals to the EB to regulate sleep homeostasis and sleep amount (Zhao, 2021).

    This study highlights the importance of epigenetic regulation on sleep. Although epigenetic regulation was intensively studied in adult pathological processes such as cancer, epigenetic factors have been far less studied in other physiological processes such as sleep. This study provides an example of the maintenance role of PcG complex in sleep regulation. Although the core PcG complex component Pc is ubiquitously expressed, its regulator stx is tissue specifically distributed, and this distribution may keep appropriate activity of Pc as well as the PcG complex in a tissue-specific manner. The factors regulating the tissue specificity of stx expression need to be further investigated (Zhao, 2021).

    A previous study found that mutation of Octβ2R does not have an obvious sleep phenotype. The current data were compared with the published Octβ2Rf05679 mutant data. Although Octβ2Rf05679 mutant was shown not significantly affected total sleep, this study found that the Octβ2Rf05679 has mild effect on sleep. Other Octβ2R mutants were tested, and it was found that the male flies from these mutations indeed have sleep phenotype (Zhao, 2021).

    Published studies have shown that the sleep phenotype of octopamine pathway mutants is different between video-based method and DAM-based method. For example, based on DAM data, the TβH mutant resulted in increased sleep per day, while the same mutant showed decreased sleep based on video data. This study used the video-based method to repeat the TβH mutant phenotype. The results showed that compared with the control flies, the TβH mutant got significantly less sleep. This result is consistent with the previously published data. Through close observation of TβH mutant and control flies, this study found that this mutant has much more frequent grooming behavior than the controls. The TβH mutant and control flies were video recorded for 10 min between ZT3.5 and ZT4.5. The results showed a statistically significant increase of the total number of grooming case. The difference between video-based method and DAM-based method is that these grooming behaviors can be detected in video-based methods, but not in DAM-based methods. Multiple studies have established a positive correlation between octopamine treatment and grooming behavior. Theoretically, TβH allele should result in a decrease in octopamine synthesis. The opposite phenotype may be caused by increased tyramine in TβH mutant or by other feedback regulation. The alleles for Octβ2R receptor used in this study show a similar grooming behavior as the control flies. The previously published octβ2R knockout allele should be a stronger one. The difference of sleep phenotypes between video-based and DAM-based methods may be due to the grooming behavior induced by the massive decrease of octopamine detection. Or other unrelated effects caused by the compensation effect previously reported. One hypothesis is that the significant change of grooming behavior probably masks the sleep behavior. The relationship between grooming and sleep needs to be further clarified. The detection of the sleep phenotype without significant changes in grooming phenotype may be a better strategy to get reliable sleep phenotype. If the increase of grooming in TβH mutant is a side effect caused by the increased tyramine, the identification of the phenotypes of octopamine treatment or collective phenotype of octopamine receptors may be more reliable ways to draw conclusions on the function of octopamine. Furthermore, whether grooming is epistatic to sleep is a problem worthy of further study (Zhao, 2021).

    Two aspects of sleep homeostasis need to be further studied. First, this study found that Octβ2R and stx colocalize in a subset of EB neurons. In a previously study, EB R2 neurons were found to be responsible for sleep homeostasis regulation. The relationship of these two groups of EB neurons needs further study. Second, the OA-treated Octβ2R mutant has more sleep recovery than the control. This indicates that OA induces more sleep recovery in the condition of Octβ2R downregulation. It seems that in this condition OA induces certain pathways to counteract its role in sleep homeostasis. One possibility is that Octβ2R negatively regulates Octβ3R which results in increased sleep pressure in the absence of Octβ2R. Further studies are needed to clarify the mechanism (Zhao, 2021).

    The results suggest the stx-Pc-Octβ2R regulatory cascade serves as a buffering step for OA function in sleep homeostasis. Two-way regulation of OA on stx leads to reverse changes of stx-the more OA, the less stx and vice versa. Through the function of stx-Pc-Octβ2R regulatory cascade, the Octβ2R transcription is changed accordingly. Variation of Octβ2R transcription could buffer the OA response. As a result, the unfavorable effect of OA causing dramatic decrease of sleep amount and homeostasis could be compensated by its receptor (Zhao, 2021).

    Propagation of homeostatic sleep signals by segregated synaptic microcircuits of the Drosophila mushroom body

    The Drosophila mushroom body (MB) is a key associative memory center that has also been implicated in the control of sleep. However, the identity of MB neurons underlying homeostatic sleep regulation, as well as the types of sleep signals generated by specific classes of MB neurons, has remained poorly understood. Two MB output neuron (MBON) classes whose axons convey sleep control signals from the MB to converge in the same downstream target region: a cholinergic sleep-promoting MBON class and a glutamatergic wake-promoting MBON class have been previously identified. This study deploys a combination of neurogenetic, behavioral, and physiological approaches to identify and mechanistically dissect sleep-controlling circuits of the MB. The existence of two segregated excitatory synaptic microcircuits that propagate homeostatic sleep information from different populations of intrinsic MB "Kenyon cells" (KCs) to specific sleep-regulating MBONs was revealed: sleep-promoting KCs increase sleep by preferentially activating the cholinergic MBONs, while wake-promoting KCs decrease sleep by preferentially activating the glutamatergic MBONs. Importantly, activity of the sleep-promoting MB microcircuit is increased by sleep deprivation and is necessary for homeostatic rebound sleep (i.e., the increased sleep that occurs after, and in compensation for, sleep lost during deprivation). These findings reveal for the first time specific functional connections between subsets of KCs and particular MBONs and establish the identity of synaptic microcircuits underlying transmission of homeostatic sleep signals in the MB (Sitaraman, 2015a).

    This study has used a combination of sophisticated cell-specific genetic manipulations with behavioral sleep analysis and optical electrophysiology to provide an unprecedented level of detailed understanding of the propagation of homeostatic sleep signals through microcircuits of the Drosophila MB. Specifically, two parallel segregated compartment-specific microcircuits were identified that regulate sleep: a wake-promoting microcircuit that originates in α'/β' and γm KCs and converges onto MBON-γ5β'2a/β'2mp/β'2mp_bilateral and a sleep-promoting microcircuit that originates in γd KCs and converges onto MBON-γ2α'1. Importantly, it was shown not only that exogenous activation of these microcircuits is sufficient to regulate sleep, but also that physiological manipulation of sleep need by sleep deprivation alters their endogenous neural activity, and propagation of these neural signals to downstream targets outside the MB is essential for the generation of homeostatic rebound sleep (Sitaraman, 2015a).

    Previous studies using broadly expressed traditional GAL4 drivers have implicated the MB in the control of sleep, but due to lack of appropriate cell-specific drivers, were unable to resolve specific sleep-regulating MB cell types, although very recent studies have specifically implicated α'/β' KCs and MB-MV1/PPL1 dopaminergic MB neurons in regulating sleep. Moreover, previous studies have not established a role for the MB in the generation and/or propagation of homeostatic sleep signals necessary for rebound following sleep deprivation. While a mutation of the amnesiac gene, which is expressed in a pair of neurons innervating the MB lobes, was shown to impair homeostatic sleep rebound, rebound was not found to be strongly affected by very broad synaptic inactivation of the MB KCs that comprise the lobes. This report has established a comprehensive catalog of the KCs and MBONs that control sleep, making use of a novel library of split-GAL4 lines targeting each of the cell types of the MB. Combining sophisticated behavioral genetic and optical electrophysiology approaches has allowed determination of the roles of specific MB cell types in encoding homeostatic sleep signals under physiological conditions. Then specific synaptic microcircuits were identified linking sleep-controlling KCs to sleep-controlling MBONs, revealing the synaptic mechanisms underlying the propagation of homeostatic sleep signals through the MB associative network (Sitaraman, 2015a).

    Based on these results, a detailed mechanistic model is proposed for homeostatic control of sleep by excitatory microcircuits in the Drosophila MB. Wake-promoting MBON-γ5β'2a/β'2mp/β'2mp_bilateral and sleep-promoting MBON-γ2α'1 each receive anatomical inputs from both wake-promoting γm and α'/β' KCs and sleep-promoting γd KCs. However, functional segregation of sleep control information into separate microcircuits is enforced by greater synaptic weights between γm and α'/β' KCs and MBON-γ5β'2a/β'2mp/β'2mp_bilateral and between γd KCs and MBON-γ2α'1. Anatomical studies indicate that the axons of MBON-γ5β'2a/β'2mp/β'2mp_bilateral and MBON-γ2α'1 exit the MB and terminate convergently in the superior medial protocerebrum (SMP) and crepine (CRE) neuropils. Intriguingly, dendrites of some central complex (CX) neurons-a brain region involved in locomotor control and implicated in sleep and sleep homeostasis-arborize in SMP and CRE. It is thus speculated that segregated homeostatic sleep-promoting and wake-promoting signals are generated in the KC-to-MBON microcircuits of the MB and propagate to the CX, where they are integrated to ultimately control sleep. Future studies are needed to further refine the understanding of the neurochemistry and physiology of the MB sleep control microcircuits, explore the mechanisms by which sleep deprivation alters microcircuit activity, and elucidate the connections between the MB and its downstream sleep control targets. In light of the relationship between sleep, learning, and synaptic homeostasis and the recent discovery that sleep-regulating MBON-γ5β'2a/β'2mp/β'2mp_bilateral and MBON-γ2α'1 are also important for some forms of associative learning, it will be of great interest to determine how activity of the sleep-controlling MB synaptic microcircuits influences memory formation and consolidation (Sitaraman, 2015a).

    A Paradoxical Kind of Sleep in Drosophila melanogaster

    The dynamic nature of sleep in many animals suggests distinct stages that serve different functions. Genetic sleep induction methods in animal models provide a powerful way to disambiguate these stages and functions, although behavioral methods alone are insufficient to accurately identify what kind of sleep is being engaged. In Drosophila, activation of the dorsal fan-shaped body (dFB) promotes sleep, but it remains unclear what kind of sleep this is, how the rest of the fly brain is behaving, or if any specific sleep functions are being achieved. This study developed a method to record calcium activity from thousands of neurons across a volume of the fly brain during spontaneous sleep and compared this to dFB-induced sleep. Spontaneous sleep was found to typically transition from an active "wake-like" stage to a less active stage. In contrast, optogenetic activation of the dFB promotes sustained wake-like levels of neural activity even though flies become unresponsive to mechanical stimuli. When flies were probed with salient visual stimuli, it was found that the activity of visually responsive neurons in the central brain was blocked by transient dFB activation, confirming an acute disconnect from the external environment. Prolonged optogenetic dFB activation nevertheless achieved a key sleep function by correcting visual attention defects brought on by sleep deprivation. These results suggest that dFB activation promotes a distinct form of sleep in Drosophila, where brain activity appears similar to wakefulness, but responsiveness to external sensory stimuli is profoundly suppressed (Tainton-Heap, 2020).

    Control of sleep by dopaminergic inputs to the Drosophila mushroom body

    The Drosophila mushroom body (MB) is an associative learning network that is important for the control of sleep. Particular intrinsic MB Kenyon cell (KC) classes have been identified that regulate sleep through synaptic activation of particular MB output neurons (MBONs) whose axons convey sleep control signals out of the MB to downstream target regions. Specifically, it was found that sleep-promoting KCs increase sleep by preferentially activating cholinergic sleep-promoting MBONs, while wake-promoting KCs decrease sleep by preferentially activating glutamatergic wake-promoting MBONs. By using a combination of genetic and physiological approaches to identify wake-promoting dopaminergic neurons (DANs) that innervate the MB, it was shown that they activate wake-promoting MBONs. These studies reveal a dopaminergic sleep control mechanism that likely operates by modulation of KC-MBON microcircuits (Sitaraman, 2015b).

    This study used a combination of sophisticated cell-specific genetic manipulations with behavioral sleep analysis and optical electrophysiology to identify compartment-specific wake-promoting MB DANs that activate wake-promoting microcircuits. Previous studies have implicated DANs innervating the central complex (CX) - a brain region involved in locomotor control - in regulating sleep, and other non-dopamingeric CX neurons have been implicated in homeostatic control of sleep. In addition, it has recently been shown that manipulations of dopamine signaling in the MB alter sleep, although the precise DANs involved remains unclear. This study has now identified specific wake-promoting MB DANs and shown that they innervate lobe compartments also innervated by wake-promoting KCs and MBONs. Importantly, this study has also shown that dopamine secretion by DANs innervating a particular MB lobe compartment acts through D1 subtype receptors to activate the wake-promoting microcircuit specific to that compartment to a much greater extent than it activates the sleep-promoting microcircuit residing in different compartments. This provides direct physiological evidence for compartment-specific dopamine signaling in the regulation of sleep by the MB, and is consistent with a previous study in the context of learning and memory (Boto, 2014). Future studies are required to determine additional cellular and molecular details of how dopamine signals modulate sleep-regulating microcircuits (Sitaraman, 2015b).

    On the basis of recently published studies of MB control of sleep and the results presented in this study, a unified mechanistic model is proposed for homeostatic control of sleep by excitatory microcircuits in the Drosophila MB. Wake-promoting MBON-γ5β'2a/β'2mp/β'2mp_bilateral and sleep-promoting γ2α'1 each receive anatomical inputs from both wake-promoting γm and α'/β' KCs KCs and sleep-promoting γd KCs. However, segregation of sleep control information into separate microcircuits is enforced by greater synaptic weights between γ and γm and α'/β' KCs and MBON-γ5β'2a/β'2mp/β'2mp_bilateral, and between γm and α/β' KCs and MBON-γ5β'2a/β'2mp/β'2mp_bilateral, and between γd KCs and MBON-γ2α'1 (Sitaraman, 2015a). Thus it is hypothesize that compartment-specific dopamine signals from MB DANs could potentially determine these differences in synaptic weight. Future studies will test this hypothesis (Sitaraman, 2015b).

    Interestingly, other fly behaviors have recently been found to be regulated by sleep-controlling compartment-specific MB microcircuits. For example, the integration of food odor to suppress innate avoidance of CO2 is mediated by MBON-γ5β'2a/β'2mp/β'2mp_bilateral and PAM DANs that innervate the β'2 compartment (Lewis, 2015). Optogenetic activation experiments reveal that wake-promoting γ5β'2a/β'2mp/β'2mp_bilateral mediates innate avoidance, while MBON-γ2α'1 mediates attraction. However, thermogenetic inactivation studies reveal that both MBON-γ5β'2a/β'2mp/β'2mp_bilateral and MBON-γ2α'1 are important for various forms of associative memory formation. These diverse waking behaviors that involve the activity of sleep-regulating neurons raises the interesting question whether such roles are independent, or causally linked, which future studies can address (Sitaraman, 2015b).

    Importantly, this study has provided for the first time a cellular and molecular mechanism for for dopaminergic control of sleep through modulation of an associative network. While dopaminergic projections to cerebral cortex are known to be important for regulating sleep and arousal in mammals, underlying cellular and molecular mechanisms remain poorly understood, although D2 subtype dopamine receptors have been implicated in the control of REM sleep. Because of the possible evolutionary relationship between the MB and vertebrate forebrain associative networks (such as mammalian cerebral cortex), these studies thus provide a framework for the design of analogous experiments in genetically tractable vertebrate model systems such as zebrafish and mice (Sitaraman, 2015b)

    PDF cells are a GABA-responsive wake-promoting component of the Drosophila sleep circuit

    Daily sleep cycles in humans are driven by a complex circuit within which GABAergic sleep-promoting neurons oppose arousal. Drosophila sleep has recently been shown to be controlled by GABA, which acts on unknown cells expressing the Rdl GABAA receptor. This study has identified the relevant Rdl-containing cells as PDF-expressing small and large ventral lateral neurons (LNvs) of the circadian clock. LNv activity regulates total sleep as well as the rate of sleep onset; both large and small LNvs are part of the sleep circuit. Flies mutant for pdf or its receptor are hypersomnolent, and PDF acts on the LNvs themselves to control sleep. These features of the Drosophila sleep circuit, GABAergic control of onset and maintenance as well as peptidergic control of arousal, support the idea that features of sleep-circuit architecture as well as the mechanisms governing the behavioral transitions between sleep and wake are conserved between mammals and insects (Parisky, 2008).

    Using a variety of mutants and novel genetic strategies to manipulate chronic and acute circuit activity, this study has shown that a small set of circadian clock cells in Drosophila has a critical role in the GABAergic initiation and maintenance of sleep. New genetic tools (dnATPase, ShawRNAi), were developed that allow an increase in the chronic response of neurons to their endogenous inputs. This adds greatly to the arsenal of activity-manipulating tools, most of which suppress firing or neurotransmitter release. Bidirectional manipulation of activity provides much more information about circuit function and dynamics. The utility was demonstrated of a new tool for acute activity manipulation (dTrpA1), which can be used on small numbers of neurons deep within the fly brain. The data suggest a model in which the pdf-GAL4-positive large LNvs (l-LNvs) translate light inputs (and perhaps other arousal signals) into wakefulness. The release of PDF from these cells is required, and l-LNv PDF signals to the smaller s-LNvs. The data demonstrating somnolence after downregulation of PDFR in LNvs indicates that s-LNvs participate in sleep control, although experiments in which they have been ablated suggest that they are not be the only sleep-relevant l-LNv targets. PDF signaling to PDFR-expressing neurons outside the clock that directly control activity is likely to be important. GABA may modulate the ability of LNvs to suppress sleep by acting on either or both s- and l-LNvs (Parisky, 2008).

    In mammals, the role of the circadian clock in sleep is not completely understood. It is nonetheless clear that there are genetic (e.g., familial advance sleep phase syndrome) and environmental (e.g., jet-lag, shift work) conditions that disrupt sleep despite primarily affecting the circadian rhythms. The clock has been shown to regulate both when an animal sleeps and how much sleep occurs. The current consensus view is that the mammalian clock is primarily wake-promoting, acting along with the homeostatic sleep drive to shape sleep over the day and night (Parisky, 2008).

    The data indicate that in flies PDF and the circadian LNvs more generally regulate both the maintenance of sleep as well as the ability of flies to respond to the wake-promoting effects of light. Although these effects recall the role of the mammalian SCN in sleep regulation, there are few prior links between the Drosophila circadian clock and the regulation of fly sleep. The almost complete elimination of the difference in total sleep between subjective day and subjective night in the pdf01 background adds substantially to this connection, i.e., light regulation of sleep appears to be substantially circadian clock-mediated Therefore, the contribution of the circadian machinery and fly brain clock circuitry to the control of sleep will probably parallel the important role of the mammalian circadian clock and the SCN in sleep regulation (Parisky, 2008).

    PDF neurons have been recently shown to be light-responsive, like some neurons of the mammalian SCN. The l-LNvs also act as the dawn photoreceptor for the clock, sending a reset signal each morning to the rest of the clock. There is also good evidence that fly cryptochrome responds directly to light in addition to influencing circadian timekeeping, and a cry mutant substantially decreases the PDF neuron acute light response. Therefore, some of the waking effects described in this study probably reflect a role of PDF cells on acute processes involving light stimulation. Indeed, the phenotypes of flies without PDF or with decreased LNv neuronal excitability resemble some of the acute effects of the loss of orexin/hypocretin in narcoleptic mice. PDF neurons are also regulated by GABAergic inputs, analogous to those from the basal forebrain that regulate orexin/hypocretin neurons (Parisky, 2008).

    Despite these similarities, there are also important organizational differences between systems. Most notable is the wide distribution of sleep circuitry in mammals. There are for example many targets of sleep-promoting GABAergic neurons, and the role of the circadian clock may be largely modulatory. The sleep circuitry of flies is almost certainly more circumscribed and simpler. Indeed, the surprisingly large effects of manipulating Rdl in the 16 LNvs argue that they are a principal target of sleep promoting GABAergic neurons and constitute part of the 'core' sleep circuitry. The fact that activation of a subset of these cells, the l-LNvs, has an effect on sleep homeostasis, further suggests that these cells sit at the heart of the sleep circuit. The fly sleep circuitry may therefore have condensed mammalian stimulatory systems (e.g., histaminergic, cholinergic and adrenergic, as well as orexin) into a simpler and more compact region, which may even largely coincide with the sixteen PDF cells of the circadian circuit (Parisky, 2008).

    A limited number of other fly brain regions have been proposed to contribute to fly sleep. Manipulations of a broad set of peptidergic (PHM+) cells indicate that peptidergic neurons other than PDF neurons are wake promoting. An attractive hypothesis is that some these other peptidergic cells reside in the pars intercerebralis, a group of neurohumoral cells shown to an important sleep output center. The targets of these cells may even overlap with the targets of LNvs, e.g. the ellipsoid bodies. The PDFR is a class II G-protein coupled receptor and is fairly promiscuous: PDF is the highest affinity ligand, but this receptor is also activated by DH31 and PACAP-38. Since peptidergic modulation may occur by 'volume' transmission instead of by direct synaptic contact, both LNv peptides and peptides from the pars could together affect this motor center to regulate sleep and activity. The role of the pars may be to inform the sleep generation machinery about nutritional and metabolic state, i.e., animals undergoing starvation exhibit hyperlocomotor activity that is believed to be evolutionarily useful as a method for finding food, and alteration of this pars-generated locomotor program affects sleep. The role of l-LNvs is clearly different from that of other PHM+ neurons, and their unique involvement in homeostatic sleep suggests they are central to sleep control (Parisky, 2008).

    The only other brain region that has been implicated in Drosophila sleep regulation is the paired structure known as the mushroom bodies. These studies showed that GAL4-driven manipulation of signaling or of neurotransmitter release in this neuropil had complex effects on sleep, not inconsistent with a modulatory role for this sensory integration center. The exact mechanism of these effects is not clear, however, especially since all of the mushroom body GAL4 lines that were examined in this study also express in multiple subsets of clock cells (Parisky, 2008).

    The small circuit this study describes presents a tractable model system for understanding the circuit-level control of sleep, the relationship between homeostatic and circadian control as well as the dynamics of sleep-wake transitions; the latter are critical to an understanding of episodic and age-related insomnia (Parisky, 2008).

    The clock input to the first optic neuropil of Drosophila melanogaster expressing neuronal circadian plasticity

    In the first optic neuropil (lamina) of the fly's visual system, two interneurons, L1 and L2 monopolar cells, and epithelial glial cells show circadian rhythms in morphological plasticity. These rhythms depend on clock gene period (per) and cryptochrome (cry) expression. This study found that rhythms in the lamina of Drosophila may be regulated by circadian clock neurons in the brain since the lamina is invaded by one neurite extending from ventral lateral neurons; the so-called pacemaker neurons. These neurons and the projection to the lamina were visualized by green fluorescent protein (GFP). GFP reporter gene expression was driven by the cry promotor in cry-GAL4/UAS-GFP transgenic lines. It was observed that the neuron projecting to the lamina forms arborizations of varicose fibers in the distal lamina. These varicose fibers do not form synaptic contacts with the lamina cells and are immunoreactive to the antisera raised against a specific region of Schistocerca gregaria ion transport peptide (ITP). ITP released in a paracrine way in the lamina cortex, may regulate the swelling and shrinking rhythms of the lamina monopolar cells and the glia by controlling the transport of ions and fluids across cell membranes at particular times of the day (Damulewicz, 2011).

    This study showed a single projection from the pacemaker cells in the brain to the lamina, in which several structural circadian rhythms have been detected. Moreover, this input probably originates from the 5th small LNv. Since the 5th s-LNv does not express PDF, this cell is different from the other LNvs. The possibility that this process originates from other clock cells, for example from the LNds, and extends to the aMe first, and next to the lamina cannot be excluded. A CRY-positive LNd, which is immunoreactive to ITP, could invade the lamina by passing the aMe first. This neuron, however, is also immunoreactive to sNPF, but the projection detected in the lamina is immunoreactive to ITP only. It indicates that this projection originates from the 5th s-LNv, which is immunoreactive to ITP but not to sNPF. This study examined GFP expression driven by cry-GAL4 in thin, 20 microm cryostat sections and thick 100 microm vibratom sections of the Drosophila brain. In most earlier studies on clock neurons and their projections, whole-mount preparations of the Drosophila brain were used, or the lamina was cut-off during preparation. Such procedures from previous studies meant that the very fine projection from the brain to the lamina could not be observed. This study detected the projection by using 20 microm sections and collecting confocal optical sections at a 1 microm interval (Damulewicz, 2011).

    In several previous studies, it has been suggested that CRY is present in different types of clock neurons. These results have been obtained using various methods; cry-GAL4 driven GFP expression, cry mRNA in situ hybridization, immunolocalization and cry deletion mutants. Using cry-GAL4 line and 20 microm sections of the D. melanogaster brain, it was found that CRY is located in all s-LNvs, l-LNvs, LNds, DN1s and DN3s but is absent in DN2s and LPNs. These results only partly confirm the results of earlier studies. It has been shown that LNvs but only some DN1, and three or four from the six LNd are CRY - positive, while DN2, DN3 and LPNs are CRY-negative. One study did d not detect CRY in DN2s and DN3s, and in about half of the LNds and DN1, but cry promoter dependent reporter genes and cry mRNA can be detected in these neurons. In this study, all of LNds showed GFP fluorescence in the cry-GAL4 strain, but only 3-4 cells were found to be CRY-immunopositive using antibodies. In turn, using the in situ hybridization method, cry mRNA was not detected in those cells. Since the pattern of cry-GAL4 driven GFP expression depends on the transgene insertion site and whether the first intron of the transgene has been inserted, spatial and circadian regulation of cry was examined. A series of cry-GAL4 transgenes containing different portions of cry upstream and intron 1 sequences was examined. The first intron was shown to drive expression in eyes and antennae, and upstream sequences induce cry expression in brain clock neurons and in peripheral oscillators; in eyes and antennae. In addition, upstream sequences also induce expression of cry, in other non-clock cells in the optic lobe (Damulewicz, 2011).

    The results obtained using various methods suggest that in the case of CRY, translation and cry transcription may be specifically regulated. CRY-positive labeling in the 4th LNd was observed in flies kept for 5 days in constant darkness. Flies kept longer in this condition brought on weak staining in one of the DN2 neurons. Thus, the level of CRY in this neuron may be very low, and the CRY level may only be detected after it has accumulated for several days in DD. It is possible, that in some of the LNds, DN1 and DN3 cry expression is very low and protein is undetectable by the immunohistochemistry method, or that cry mRNA is unstable and CRY protein is not synthesized. Among six LNds, three neurons, that show a strong signal of GFP in the brain cryostat sections used in this study, may correspond to CRY-positive cells detected in the studies of other authors. In turn, three LNds with weak GFP in these preparations may correspond to CRY immunonegative cells. These cells had about a 50% lower GFP level than the rest of the LNds at all time points, except at ZT4 when their GFP fluorescence was lower by 20% (Damulewicz, 2011).

    Beside neurons, clock genes have also been detected in glial cells. A subpopulation of glial cells in the brain of Drosophila have rhythmic expression of per gene, and they are necessary for maintaining circadian locomotor activity. However, the presence of CRY in glia was not detected in this study. In the optic lobes, GFP driven by cry-GAL4 was observed in many non-clock cells in which the localization pattern was very similar to the distribution of glial cells. But these non-clock cells were not labeled with the antibody against REPO protein, a specific marker for glial cells. The REPO protein is required for glia development and differentiation and has been detected in all types of glia in the adult brain of Drosophila. The analysis of cry-GAL4 driven GFP and REPO immunolabeling showed no co-localization between CRY and REPO. However, in the close vicinity of GFP-positive cells, REPO-positive glial cells were observed. A similar result was obtained using the antibody against the Drosophila vesicular monoamine transporter (DVMAT), which enabled labeling the fenestrated glia in the optic lobe. These results suggest that CRY is present in non-clock neurons in the optic lobe, but not in glial cells (Damulewicz, 2011).

    In addition to localization of cry-GAL4 driven GFP in cell bodies of neurons, GFP processes were also detected invading three neuropils in the optic lobe. In the medulla, a dense network of processes originate from DN3s and their terminals seem to form synaptic contacts with not-yet identified target cells. The regular network of processes was also detected in the lobula but their origin is unknown. The most interesting finding is the projection of CRY-positive processes to the lamina. Although the lamina showed robust circadian remodeling of neuron morphology, a circadian input had not been previously detected. In the lamina, per is probably expressed in the epithelial glial cells, however, maintaining the lamina structural rhythms also requires per expression in the retina photoreceptors and in the LNs (Damulewicz, 2011).

    Beside PER, CRY is also important for circadian rhythms in the lamina. In an earlier study, it was shown that the circadian rhythm in morphological plasticity of L2 dendritic trees, is not present in per01 mutant while its phase depends on CRY. In cryb mutant, the pattern of daily changes in size of the L2 dendritic tree was different than in wild-type Canton-S flies. In males and females of Canton-S wild-type flies, the largest L2 dendritic tree was found at the beginning of the day. This daily pattern of the structural changes of L2 dendrite resembles the pattern of cry mRNA cycling in Drosophila heads and bodies, and in the 5th s-LNv detected in this study. Although the L2 dendritic tree is the largest at the beginning of the day in the distal lamina, its axon, as well as the axon of L1 monopolar cell, swell at the beginning of both day and night. These changes have been detected in the proximal lamina. Moreover, the α-subunit of the Na+/K+-ATPase and subunits of the V-ATPase also show diurnal changes in abundance in the lamina. Such an occurrence indicates that circadian rhythms in cell structural plasticity are correlated with rhythmic changes in the level of proteins involved in the transport of ions. The rhythm in the α-subunit of the Na+/K+-ATPase level is bimodal with two peaks; in the morning and in the evening. This pattern is changed in the cry0 mutant. It indicates that CRY is not only important for the maintenance of the daily pattern of morphological changes of the L2 dendritic tree but CRY also helps to maintain cycling of the Na+/K+-ATPase in the epithelial glial cells in the lamina (Damulewicz, 2011).

    It is uncertain whether there is regulation of lamina rhythms by the brain pacemaker because connections between the pacemaker neurons in the accessory medulla and the lamina have not been observed. It was found, however, that rhythms in axon plasticity of neurons in the lamina are circadian, have two peaks (morning and evening) and are synchronized with locomotor activity. The present results now show, that thin neurite extends from the aMe and arborizes in the distal lamina. In the aMe, the s-LNvs are regarded as the main pacemaker cells maintaining circadian rhythms. The l-LNvs are involved in behavioral arousal and sleep. For these reasons, the LNvs are good candidates as oscillators controlling lamina rhythms. Moreover, all LNvs except the 5th s-LNv, express PDF which may synchronize central oscillators with each other and with peripheral ones. In the housefly, large PDF-immunoreactive neurons, similar to Drosophila's l-LNvs, have terminals in the lamina which show circadian structural changes. Moreover, these neurons cyclically release PDF that affects circadian plasticity in the lamina. In Drosophila, release of PDF from PDF-immunoreactive processes in the medulla, where these processes form a dense network of varicose processes, is also possible. These processes, however, do not extend to the lamina. In the present study, PDF immunolabeling of the newly described Drosophila's CRY-positive terminals in the lamina was negative. This does not exclude PDF action in the lamina, particularly when PDF receptors have been detected in non-neuronal cells between the lamina and the retina. PDF may diffuse in the lamina after release from terminals in the distal medulla (Damulewicz, 2011).

    Ion transport peptide (ITP) and short neuropeptide F (sNPF) have been detected in the LNvs. Among the five s-LNvs, ITP was found in the 5th s-LNv, while sNPF was observed in four other s-LNvs which also express PDF. In the present study, ITP-immunoreactive fibers were detected, using the Schgr-ITP antisera, in the distal lamina, co-localized with cry-GAL4 driven GFP. The co-localization with ITP suggests that the projection into the lamina may originate from the 5th s-LNv. Little is known about the function of the 5th s-LNv. It has been suggested, that this neuron, together with LNds and some DN1s, drive the evening peak of D. melanogaster bimodal activity. The finding indicates a possible new function of the 5th s-LNv in regulating circadian structural rhythms in the lamina, since this neuron is immunoreactive to ITP. Like other peptides in the optic lobe, ITP seems to be released from varicose terminals in a paracrine way. This conclusion was reached because no synaptic contacts between ITP-immunoreactive processes and cells in the lamina were detected. This peptide probably diffuses in the distal lamina and may facilitate chloride and/or other ion-dependent swelling and shrinking of the L1 and L2 axons. At least two ion pumps; the V-ATPase and Na+/K+-ATPase, show robust cyclical activity in the epithelial glial cells. The epithelial glial cells swell and shrink in anti-phase to the L1 and L2 interneurons. Preliminary results showed that in a transgenic line carrying RNAi to block ITP expression, the pattern of rhythmic changes in the level of the α-subunit of the Na+/K+-ATPase in the lamina glial cells of Drosophila is different than the pattern in wild-type flies. Thus, not only CRY but also ITP is important for maintaining rhythmic activity changes of the Na+/K+-ATPase (Damulewicz, 2011).

    The function of ITP in the nervous system is unknown. In the lamina ITP may play a similar regulatory role as in hindgut of insects, transporting ions and fluids across cell membranes (Damulewicz, 2011).

    Since the L1 and L2 monopolar cells swell in the morning and in the evening, ITP released from the 5th s-LNv may drive the evening peak of this rhythm. This is thought to be so, because the 5th s-LNv and LNd are regarded as the lateral neurons' evening oscillator. In turn, PDF may drive the morning peak because PDF is thought to control the morning peak of locomotor activity, in a LD 12:12 regime. However, PDF's role in promoting locomotor activity in the evening has also been shown. The role of ITP as a neurotransmitter of circadian information to the lamina and as a possible regulator of rhythmic swelling and shrinking of the L1 and L2 monopolar cells, requires more experimentation and will be the subject of the next study (Damulewicz, 2011).

    Sleep and synaptic homeostasis: structural evidence in Drosophila

    The functions of sleep remain elusive, but a strong link exists between sleep need and neuronal plasticity. This study tested the hypothesis that plastic processes during wake lead to a net increase in synaptic strength and sleep is necessary for synaptic renormalization. In three Drosophila neuronal circuits it was found that synapse size or number increases after a few hours of wake and decreases only if flies are allowed to sleep. A richer wake experience resulted in both larger synaptic growth and greater sleep need. Finally, it was demonstrated that the gene Fmr1 (fragile X mental retardation 1) plays an important role in sleep-dependent synaptic renormalization (Bushey, 2011).

    Sleep is present in every species that has been carefully studied, including Drosophila, but its functions remain elusive. Increasing evidence points to a link between sleep need and neuronal plasticity. A recent hypothesis suggests that a consequence of staying awake is a progressive increase in synaptic strength, as the awake brain learns and adapts to an ever-changing environment mostly through synaptic potentiation. However, such increase would soon become unsustainable, because stronger synapses consume more energy, occupy more space, require more supplies, and cannot be further potentiated, saturating the ability to learn. Thus, according to the synaptic homeostasis hypothesis, sleep may serve an essential function by promoting a homeostatic reduction in synaptic strength down to sustainable levels. Also, the hypothesis predicts that the more one learns and adapts (i.e., the more intense is the wake experience), the more one needs to sleep. Findings in rodents are consistent with this hypothesis. For instance, molecular and electrophysiological markers of synaptic strength are higher after wake and lower after sleep. Moreover, presynaptic terminals of hypocretin neurons in zebrafish larvae undergo both circadian and sleep-wake-dependent structural changes, the latter consistent with sleep-dependent down-regulation. Finally, in the fly brain, overall levels of synaptic proteins increase after wake and decrease after sleep (Gilestro, 2009), and synaptic structural changes have been described after very long sleep deprivation (Donlea, 2009). These results suggest that a role for sleep in synaptic homeostasis may hold in phylogenetically distant species and may thus be of general importance (Bushey, 2011).

    The evidence in support of the synaptic homeostasis hypothesis is mainly correlative, and thus it is important to seek direct proof that sleep is necessary for synaptic renormalization and to do so at the level of individual synapses. Moreover, the synaptic homeostasis hypothesis predicts that behavioral paradigms that enhance wake-related plasticity in specific neural circuits should increase synaptic strength in those circuits as well as sleep need, but this prediction has never been tested. Finally, the cellular mechanisms that underlie synaptic and sleep changes remain unexplored. This study exploited the power of Drosophila genetics, combined with confocal microscopy and behavioral analysis, to address these questions (Bushey, 2011).

    Changes in synaptic strength are often associated with changes in synaptic structure, including synapse number and size, although the link between structural and functional plasticity is complex. In mammals, the diameter and length of synaptic spines correlate with the size of the postsynaptic density and with the magnitude of electric signals transmitted to the dendritic shaft. Moreover, the induction of synaptic potentiation leads to growth of synapses and spines, whereas synaptic depression causes synapses and spines to retract or shrink. Similarly, in Drosophila, synaptic morphology at the neuromuscular junction changes depending on experience, and these changes correlate with synaptic strength. Previous in vivo experiments in mammals and flies measured overall changes in electrophysiological and molecular markers of synaptic strength, without cellular resolution, and without direct evidence for morphological changes in synaptic terminals. Three specific cell populations in the fly brain were selected, and it was asked whether sleep and wake affect synaptic density and size (Bushey, 2011).

    The first cell group studied included the small ventral lateral neurons (LNvs), a subset of circadian oscillator neurons that are part of the wake promoting system and express the neuropeptide pigment dispersing factor (PDF). To visualize changes in presynaptic morphology, a fusion protein between synaptotagmin and enhanced GFP (syt-eGFP) was expressed, whose protein product colocalizes with native synaptic vesicles. PDF expression was also measured, because the latter is another marker of presynaptic boutons in small LNvs. First, adult females (7 days old) collected either during the light period were tested after 7 hours of mainly (>75%) spontaneous wake or during the dark period after 7 hours of mostly sleep (>80%) or sleep deprivation (>90%). Syt-eGFP and PDF staining were both higher in the presynaptic region of sleep-deprived and spontaneously awake flies relative to sleeping flies, whereas no differences were found in the axonal processes extending from the cell bodies to the presynaptic region, suggesting that the changes are independent of circadian time and specific to the presynaptic terminal. Males were then tested because they have less consolidated wake during the day than females. Flies were only tested at night, after sleep or sleep deprivation. Sleep-deprived 3- and 7-day-old males consistently showed higher presynaptic syt-eGFP and PDF staining than sleeping flies. In contrast, 1-day-old flies showed low syt-eGFP and PDF staining after both sleep and sleep deprivation. The lack of PDF staining in very young flies suggests that these neurons are still inactive soon after eclosure. Moreover, because PDF promotes arousal, low PDF staining is consistent with flies being predominantly asleep after eclosure, even if mechanical stimulation was used to try to keep them awake, consistent with high sleep need and elevated arousal threshold in newborn mammals. Syt-eGFP staining did not change in newly eclosed flies, whose PDF levels were very low. Syt-eGFP and PDF expression were also measured in Per01 flies carrying a null mutation of the clock gene Period. Because Per01 mutants have no spontaneous consolidated sleep, flies were collected immediately after 7 hours of sleep deprivation or after 5 additional hours of either recovery sleep or sleep deprivation. Overall, syt-eGFP and PDF staining in presynaptic terminals was reduced in Per01 mutants relative to wild-type (WT) flies but was still high after both 7 and 12 hours of sleep deprivation and low after recovery sleep (Bushey, 2011).

    The second cell group analyzed included γ neurons of the mushroom bodies, because they can be targeted by mosaic analysis with a repressible cell marker (MARCM) to visualize single cells, show a relatively simple morphology, and undergo activity-dependent pruning. Moreover, the mushroom bodies are involved in sleep regulation, and mutations altering cyclic adenosine monophosphate-dependent protein kinase signaling or Fmr1 (fragile X mental retardation 1) expression in these brain regions affect both sleep need and experience-dependent structural plasticity . Flies were collected at night after 7 hours of sleep or sleep deprivation, and dissected brains were immunostained for GFP-tagged CD8 to visualize neuronal membranes. It was found that the axonal tips were larger after sleep deprivation than after sleep, consistent with an increase in volume of presynaptic terminals. To confirm this result, fly stocks were generated with γ MARCM clones expressing syt-eGFP, and flies were collected after 7 hours of mostly spontaneous wake, or during the dark period after 7 hours of mostly sleep or sleep deprivation. As expected, syt-eGFP tended to accumulate in puncta along lightly stained processes, in contrast to the diffuse CD8-GFP staining. Syt-eGFP puncta were larger in sleep deprived and spontaneously awake flies relative to sleeping flies (Bushey, 2011).

    Next, whether postsynaptic morphological changes also occur as a function of sleep and wake was tested. To do so, focus was placed on the first giant tangential neuron of the lobula plate vertical system (VS). This cell (VS1) is unambiguously recognizable, and its stereotyped dendritic tree shows small actin-enriched protrusions morphologically and functionally similar to mammalian dendritic spines. Flies were compared that were spontaneously awake during the day or that slept or were sleep deprived during the first 7 hours of the night. Single VS1 spines were visualized using an antibody against actin-GFP and counted in one easily identifiable branch. The total number of spines was similar in spontaneously awake and sleeping flies but increased after sleep deprivation relative to both conditions, mainly because of an increase in stubby spines (which were the majority of scored spines). The number of mushroom spines did not change. The increase in spine number after sleep loss was associated with increased branching and lengthening of the dendritic tree, whereas spine density (number of spines divided by branch length) was similar in all conditions. Because sleep-deprived female flies had been mostly awake during the previous light period, this suggests that these postsynaptic changes may need sustained periods of wake. Another possibility, not mutually exclusive, is that changes in VS1 spines require a wake condition richer than that experienced by flies spontaneously awake alone inside small glass tubes. Indeed, sleep-deprived flies were kept awake using vibratory stimuli, resulting in the flies often falling from the top to the bottom of the tubes. Because visually driven responses in VS neurons are stronger during flight than during nonflight, it is possible that these cells were activated by the fall (Bushey, 2011).

    To test whether a rich wake experience that engages the VS circuit is sufficient to affect VS1 synaptic morphology, up to 100 flies were housed inside a large lighted chamber ('fly mall') for an entire light period (12 hours). In the mall, flies could fly ad libitum, explore, and interact with each other. Flies were collected immediately after the mall experience and compared with flies that, as usual, had remained awake during the day in single tubes. The enriched experience in the mall had profound morphological effects on the VS1 dendritic tree: Total branch length increased because of the addition of more branches with spines (mainly stubby), resulting in an overall increase in spine number (Bushey, 2011).

    Once experience-dependent synaptic changes have occurred, are they stable? If not, is sleep necessary to bring synaptic morphology back to pre-enrichment levels? To answer these questions, two other groups of flies were moved back to single tubes after 12 hours of mall experience; one group was allowed to sleep for 7 hours, whereas the other was kept awake as before using mechanical stimuli. In flies that were sleep-deprived after enrichment, branch length, branch points, and spine number were at levels similar to those seen in flies collected immediately after enrichment. In contrast, in flies that were allowed to sleep after the mall experience, all morphological parameters reverted to the levels observed in awake flies kept in single tubes. Moreover, spine density was negatively correlated with the amount of sleep during the last 7 hours, as well as with the maximal duration of sleep bouts. In another experiment, flies were housed in the mall for 12 hours during the day and then moved back to single tubes to record their sleep. During the 24 hours after the enrichment, flies slept more, both during the day and at. Finally, in the last experiment, flies were housed in the mall for 12 hours during the day, moved back to single tubes and sleep deprived all night (12 hours), and then either collected immediately, allowed to sleep for 6 hours, or kept awake for 6 more hours. Consistent with the previous experiments, decreases in all morphological parameters were seen only in flies that could sleep, and spine density was negatively correlated with the amount of sleep during the last 6 hours, as well as with mean and maximal duration of sleep bouts (Bushey, 2011).

    Previous experiments suggest that Fmr1 could mediate at least some of the effects of sleep/wake on synapses. Fmr1 protein product (FMRP) is present in dendritic spines, and loss of FMRP in flies is associated with overgrown dendritic trees, larger synaptic boutons, and defects in developmental and activity-dependent pruning. Notably, Fmr1 overexpression results in the opposite phenotype, with dendritic and axonal underbranching and loss of synapse differentiation. Moreover, Fmr1 expression is reduced by sensory deprivation in flies and increased by sensory stimulation and enrichment in mammals (Bushey, 2011).

    It was recently shown that FMRP levels increase in the adult fly brain during wake relative to sleep, independent of time of day or light, suggesting that waking experience is sufficient to affect Fmr1 expression even after the end of development. It has also been shown that Fmr1 overexpression in either the whole brain or in the mushroom bodies is associated with an ~30% decrease in sleep duration, and it is hypothesized that this reduced need for sleep occurs because chronically high Fmr1 levels may allow synaptic pruning to occur at all times, independent of sleep. If so, Fmr1 overexpressing (OE) flies should fail to show increased spine density after prolonged wake. Thus Fmr1 was overexpressed specifically in the vertical and horizontal system of the lobula plate. OE flies were collected at night after 7 hours of either sleep or sleep deprivation and were compared to corresponding sleeping and sleep-deprived WT controls. As expected, Fmr1 expression was concentrated in granules along the VS1 dendritic tree, and overall Fmr1 levels were higher in sleeping and sleep-deprived OE flies than in their corresponding controls, due to larger Fmr1 granules in OE flies. Crucially, in contrast to WT controls, OE flies showed no increase in either spine number, branch length, or branch points after sleep deprivation relative to sleep; all these parameters were similar between the two experimental groups, and their levels were close to those observed in WT flies after sleep. Finally, OE flies slept less than their WT controls during baseline and showed a reduced sleep rebound after 12 hours of sleep deprivation at night. Thus, it seems that Fmr1 overexpression was sufficient to completely abolish the wake-dependent increase in VS1 spine number, whereas the effects on sleep were small. The latter result is not surprising, because sleep need presumably results from the overall amount of synaptic plasticity occurring during wake in many brain areas, whereas Fmr1 overexpression was restricted to a few VS neurons (Bushey, 2011).

    Sleep is perhaps the only major behavior still in search of a function. The results of this study support the hypothesis that plastic processes during wake lead to a net increase in synaptic strength in many brain circuits and that sleep is required for synaptic renormalization. A wake-related increase in synapse number and strength, if unopposed, would lead to a progressive increase in energy expenditure and saturation of learning. A sleep-dependent synaptic homeostasis may explain why sleep is required to maintain cognitive performance. How sleep would bring about a net decrease in synaptic strength remains unknown, but in mammals, potential mechanisms favoring synaptic depression during non-rapid eye movement sleep may require the repeated sequences of depolarization/synchronous firing and hyperpolarization/silence at ~1Hz observed in corticothalamic cells, as well as the low levels of neuromodulators such as noradrenaline and of plasticity-related molecules such as brain-derived neurotrophic factor. To what extent such mechanisms may also apply to flies remains to be determined (Bushey, 2011).

    CRY-dependent plasticity of tetrad presynaptic sites in the visual system of Drosophila at the morning peak of activity and sleep

    Tetrad synapses are formed between the retina photoreceptor terminals and postsynaptic cells in the first optic neuropil (lamina) of Drosophila. They are remodelled in the course of the day and show distinct functional changes during activity and sleep. These changes result from fast degradation of the presynaptic scaffolding protein Bruchpilot (BRP) by Cryptochrome (CRY) in the morning and depend on BRP-170, one of two BRP isoforms. This process also affects the number of synaptic vesicles, both clear and dense-core, delivered to the presynaptic elements. In cry01 mutants lacking CRY and in brpΔ170, the number of synaptic vesicles is lower in the morning peak of activity than during night-sleep while in wild-type flies the number of synaptic vesicles is similar at these two time points. CRY may also set phase of the circadian rhythm in plasticity of synapses. The process of synapse remodelling stimulates the formation of clear synaptic vesicles in the morning. They carry histamine, a neurotransmitter in tetrad synapses and seem to be formed from glial capitate projections inside the photoreceptor terminals. In turn dense-core vesicles probably carry synaptic proteins building the tetrad presynaptic element (Damulewicz, 2020).

    The results confirmed earlier studies that the presynaptic element (T-bar) of tetrad synapses is remodelled during the day and night and this rhythm is regulated by light and circadian clock (Gorska-Andrzejak, 2013; Woznicka, 2015). In the present study, it was found additionally that cyclic changes occur in the T-bar ultrastructure and its volume. Ultrastructural changes in T-bars and synaptic vesicles were possible because of using high resolution electron microscope tomography (EMT). In the present study it was also found that the number of synaptic vesicles cycles during the day, but this rhythm is masked by light. This result indicates that in the case of tetrad synapses, which are sites of fast neurotransmission between photoreceptors and the first order interneurons, intense neurotransmission occurs during the morning peak of locomotor activity, when more synaptic vesicles are attached to the T-bar platform than during sleep (ZT16) and are transported from capitate projections located next to the T-bars. At ZT16, tetrad synapses are ready for neurotransmission, since the total number of synaptic vesicles near tetrad synapses is similar at ZT1 and ZT16, but it is in a standby mode with lower frequency of synapses and vesicles, which are not attached to the T-bar platform and are not delivered from glial cells, respectively. However, transmission can be activated and is efficient because synapses during sleep have larger volume, and synaptic vesicles can be transported to the T-bar platform, if necessary, in response to an unexpected light pulse. Cyclic remodelling of tetrad presynaptic sites depends on BRP, which must be delivered to T-bars and degraded after light exposure after binding to CRY. This fast remodelling of synapses affects the number of vesicles transported to the presynaptic element (Damulewicz, 2020).

    The present study was carried out only in light/dark (LD 12:12) conditions because in earlier studies it was found that the rhythm of the changes in BRP abundance in tetrad synapses of D. melanogaster is circadian. The rhythm is maintained in constant darkness (DD) and abolished in the per01 clock null mutant. On the basis of these results, it is assumed that rhythmic changes during the 24 h cycle reported is this paper are also circadian; however, in DD, the morning peak in BRP is not present because it depends on light (Damulewicz, 2020).

    Electron microscope tomography (EMT) used in this study showed that synaptic vesicles are attached to the platform of the T-bar with filamentous proteins that are reduced in brpΔ170 and brpΔ190 mutants, which have fewer synaptic vesicles compared with wild-type flies. Two types of vesicles, clear and dense-core were detected. Dense-core synaptic vesicles were less numerous than clear ones. It is known that synaptic vesicles of tetrad synapses contain histamine, while the content of dense-core vesicles is unknown. It is possible that they carry presynaptic proteins to the T-bar. The comparison of ultrastructure of tetrad synapses in the morning peak of activity (ZT1) and during sleep (ZT16) indicates that more vesicles are attached to the T-bar platform and to capitate projections at ZT1, but this pattern is not present in brpΔ170, brpΔ190 and cry01 . This result confirmed an earlier study that both high motor activity in the morning and light exposure increase activity of the visual system. In the morning, there is intense transport of synaptic vesicles to T-bars and delivery of histamine in clear vesicles from the epithelial glial cells through capitate projections. The evidence for a role of capitate projections in neurotransmitter recycling has already been reported and now this study showed, using EMT, that vesicles are produced from capitate projections and directly delivered to T-bars. This intense transport is damaged in all mutants studied, suggesting that both BRP isoforms and CRY are needed for this process. In addition, in the brpΔ190 mutant lacking BRP-190, the T-bar structure is less dense than in the other strains studied. In a previous study, it was found that there are approximately 50% fewer synapses in brpΔ190 than in Canton S and brpΔ170. Another study reported that BRP isoforms are important for the formation of T-bars in neuromuscular junctions, and in brp mutants T-bars are smaller than in controls. T-bar height was reduced in brpΔ190, whereas the widths of pedestal and platform were reduced in both mutants. They also decrease transmission since the active zone was smaller in both mutants and the number of synaptic vesicles was reduced. These ultrastructural changes are correlated with cell physiology since the amplitude of evoked excitatory junctional current was decreased in both mutants with a stronger effect in brpΔ190 (Damulewicz, 2020).

    The obtained reconstructions of tetrad T-bars from TEM serial sections of the lamina showed that although there were fewer synapses during the night (ZT16), the volume of the T-bar was larger at that time than in the morning (ZT1), while the total number of synaptic vesicles was similar. In contrast, a day/night difference (ZT1 vs. ZT16) in the number of vesicles was observed in brpΔ170 and cry01 . This suggests that the CRY protein and BRP-170 are responsible for an increase in the number of synaptic vesicles during the morning peak of activity. Since CRY co-localizes with BRP and is involved in BRP degradation in the morning, CRY is probably also involved in the degradation of other proteins of synaptic vesicle organization in the morning since the number of vesicles in cry01 was low in the morning but high at night (ZT16). It seems that in cry01, synaptic vesicles are not delivered to the photoreceptor terminals from capitate projections and tethered to the cytomatrix in the morning. It is also interesting that the daily rhythm in the number of vesicles is not maintained in BRP mutants, which confirms an earlier study, and the BRP N-terminus, which lacks brpΔ190, is necessary to maintain daily remodelling of the T-bar structure. Although both isoforms participate in building the cytomatrix their functions seem to be different in the course of the day. It is also possible that CRY is not only responsible for degradation of synaptic proteins but also as a protein, what is known, affecting the clock. In another cell types, in clock neurons l-LNvs, CRY, except interaction with TIM, is responsible for blue light response and firing of the l-LNvs. In the lamina it was found that in cry01 mutant the daily rhythm in synapse number and their remodelling was delayed in phase and the day/night difference in Canton S increased when peaks in the number of synapses were shift to ZT4 and ZT16. In result the difference between ZT1 and ZT16 was increased in cry01 (Damulewicz, 2020).

    BRP is also responsible for rapid remodelling of the presynaptic active zone (AZ), and as reported in Drosophila NMJ, presynaptic homeostatic potentiation increases the number of BRP molecules and other AZ proteins, Unc13A and RBP, within minutes (Damulewicz, 2020).

    When synaptic vesicles were counted at two different distances from the T-bar, to 200 nm and above 200 nm, there were differences between clear vesicles containing histamine and dense-core ones located in both areas. More clear vesicles were located near the T-bar and fewer above 200 nm. In the case of dense-core vesicles, their number was similar in both areas. This difference was not so striking in mutants in the case of vesicles located next to the platform, but in brpΔ170 and cry01, there were more dense-core vesicles at ZT16 in the distance above 200 nm than closer to the T-bar. This result indicates that BRP-170 and CRY are important for the distribution of clear synaptic vesicles next to the T-bar as well as dense-core vesicles located above 200 nm from the presynaptic element. It is possible that dense-core vesicles contain T-bar proteins, probably BRP. When transport along the actin cytoskeleton is disrupted, the number of tetrad synapses decreases, and rapid AZ remodelling also fails (Damulewicz, 2020).

    The above mentioned ultrastructural changes depend on the level of the presynaptic scaffolding protein BRP, which changes in abundance during the day and night. These changes are controlled by the daily expression of CRY, which seems to have many functions in photoreceptors in addition to being the circadian clock photoreceptor. In an earlier study, it was found that CRY interacts with BRP but only during light exposure and leads to the degradation of BRP during the day/light phase of the 24 h cycle. This seems to be responsible for the decrease in BRP level in the middle of the day after its peak at the beginning of the day. The lack of CRY in cry01 mutants changes the pattern of the tetrad presynaptic profile frequency during the day and the size of the T-bar. However, the rhythm is not completely abolished, which indicates that other proteins are also involved in the daily remodelling of tetrad synapses. Since CRY plays several functions in photoreceptors, changes in the number of tetrad synapse and T-bar size in cry01 may result from different processes and lack of interactions of CRY with TIM and BRP. CRY is a component of the molecular clock and interacts with TIM, and this may affect daily changes in the number and size of T-bars. In addition, light-activated CRY binds BRP and targets it to degradation. Previous work showed that flies with constitutively active CRY have low BRP level. In turn, cry01 mutants show changes in the pattern of BRP expression, with higher BRP level during the day (ZT4), at the time when wild-type flies have minimum of BRP expression. The pattern of BRP expression is similar to the pattern of daily changes in tetrad synapse number, so it is possible that the number of synapses or T-bar size is directly dependent on the amount of BRP which oscillates during the day. However, CRY in the retina photoreceptors binds also actin and is involved in the organization of phototransduction cascade of proteins in rhabdomeres38. This may be also involved in the regulation of T-bar structure. The differences in T-bar size of cry01 are shown at time when in Canton S CRY is active (during the day) or its level increases (ZT16). At the beginning of the night the level of CRY is very low, so there was no effect on T-bar structure and no difference between CS and cry01 was observed (Damulewicz, 2020).

    The epithelial glial cells are important for many processes during phototransduction and in recycling neurotransmitters and other compounds during the night. Glia take up histamine and metabolize it to carcinin, which is next delivered to the photoreceptor terminals, and capitate projections are involved in this process. Activity of glial cells is also controlled by the circadian clock. During the night, glial cells seem to be more active than neurons to recycle neurotransmitters, and many proteins, including proteins of ion pumps, are found at higher concentrations at that time. The high number of synaptic vesicles near the tetrad T-bar during the morning peak of activity in Drosophila seems to depend on capitate projections invaginating from the epithelial glia to the photoreceptor terminals in the lamina of Drosophila (Damulewicz, 2020).

    Although the presynaptic cytomatrix can be rapidly remodelled with transmission strength, it is also affected by motor and visual system activity, external factors, such as light in the case of the visual system, and the circadian clock, showing plasticity and correlation to changes in behaviour during the day/night cycle. As was shown in the present study synaptic plasticity and synapse remodelling during the day is a complex process which involves presynaptic proteins of the T-bar as well as two types of synaptic vesicles, clear and dense-core, and glial cells. It was also found that fast degradation of proteins involved in transmission is as important as pre- and postsynaptic protein synthesis (Damulewicz, 2020).

    Synchronized bilateral synaptic inputs to Drosophila melanogaster neuropeptidergic rest/arousal neurons

    Neuropeptide PDF (pigment-dispersing factor)-secreting large ventrolateral neurons (lLNvs) in the Drosophila brain regulate daily patterns of rest and arousal. These bilateral wake-promoting neurons are light responsive and integrate information from the circadian system, sleep circuits, and light environment. To begin to dissect the synaptic circuitry of the circadian neural network, simultaneous dual whole-cell patch-clamp recordings of pairs of lLNvs were performed. Both ipsilateral and contralateral pairs of lLNvs exhibit synchronous rhythmic membrane activity with a periodicity of ~5-10 s. This rhythmic lLNv activity is blocked by TTX, voltage-gated sodium blocker, or α-bungarotoxin, nicotinic acetylcholine receptor antagonist, indicating that action potential-dependent cholinergic synaptic connections are required for rhythmic lLNv activity. Since injecting current into one neuron of the pair had no effect on the membrane activity of the other neuron of the pair, this suggests that the synchrony is attributable to bilateral inputs and not coupling between the pairs of lLNvs. To further elucidate the nature of these synaptic inputs to lLNvs, a variety of neurotransmitter receptors were blocked or activated, and effects on network activity and ionic conductances were measured. These measurements indicate the lLNvs possess excitatory nicotinic ACh receptors, inhibitory ionotropic GABAA receptors, and inhibitory ionotropic GluCl (glutamate-gated chloride) receptors. It was demonstrated that cholinergic input, but not GABAergic input, is required for synchronous membrane activity, whereas GABA can modulate firing patterns. It is concluded that neuropeptidergic lLNvs that control rest and arousal receive synchronous synaptic inputs mediated by ACh (McCarthy, 2011).

    Previously studies have shown that neural circuits responsible for generating circadian rhythms and also those neural networks controlling rest and arousal exhibit synchronous membrane activity both in mammals and in insects. Furthermore, neuropeptides, VIP and PDF, in mammals and flies, respectively, and the classical neurotransmitter, GABA, play critical roles in this synchrony. Furthermore, this study found that stereotyped neurons that were positive for a well studied driver exhibited varying degrees of synchrony with lLNv membrane activity. This is consistent with a model in which certain neurons receive some of the same inputs as lLNvs and some unique inputs. Similar to the observations in Drosophila, neurons in some mammalian brain regions outside the suprachiasmatic nucleus (SCN) exhibit synchronized membrane activities with SCN neurons. The data do not, however, preclude the possibility that the synchrony that was observe is attributable to widespread epileptiform or other widespread synchronous brain activity that is not specific to lLNvs. This explanation is not favored because lLNv pairs exhibiting varying degrees of synchrony were observed and also lLNv pairs in which one cell is burst firing while the other is tonically firing, as shown in the paired recording pharmacology experiments. Even if it is the case that the synchronized activity that was seen is attributable to some sort of epileptiform or other widespread synchronous activity, the nature of and mechanisms underlying this activity are still informative, as it provides insight into the connectivity of the network (McCarthy, 2011).

    To characterize the nature of synaptic inputs to lLNvs, a combination of agonists and antagonists against neurotransmitter receptors were used in both current-clamp and voltage-clamp mode. Current-clamp mode was used to monitor modulation of membrane activity in the lLNvs in the context of the functional neural network, whereas voltage-clamp mode was used to determine the presence of underlying receptors in the lLNvs themselves. lLNvs were found to receive excitatory cholinergic input through nAChR. Treatment of brains with cholinergic receptor agonists, acetylcholine and nicotine, enhances membrane activity, depolarizing the neurons and increasing action potential firing rate, whereas treatment with ACh receptor antagonists, curare and a-BuTX, inhibits membrane activity. Voltage-clamp recordings in the presence of TTX revealed that ACh- and nicotine-induced currents occur in lLNvs themselves, and these currents reverse near the equilibrium potential for nonselective monovalent cation channels, as expected for currents through nicotinic acetylcholine receptors. In insects, acetylcholine is the primary excitatory neurotransmitter in the CNS, and nAChRs are widely expressed in the Drosophila brain. These receptors are known to mediate fast synaptic transmission in Kenyon cells in the adult mushroom body. A previous study has also shown that dissociated PDF-positive neurons from the larval Drosophila brain, which are developmental precursors of the LNvs, express nAChRs and exhibit both ACh-induced and nicotine-induced increases in intracellular calcium that are dependent on both external sodium and calcium concentrations. The current findings confirm that lLNvs in the adult circadian neural network possess nAChRs and that these receptors mediate excitatory synaptic input and synchrony of rhythmic firing (McCarthy, 2011).

    GABA is a major neurotransmitter in the Drosophila CNS, mediating fast inhibitory synaptic transmission through the GABAA receptor. This receptor has been shown to be expressed in LNvs and has been shown genetically to play a major role in the regulation of arousal and sleep by lLNvs specifically (Parisky, 2008; Shang, 2008; Chung, 2009). GABA-induced decreases in intracellular calcium and Cl- currents have been recorded in dissociated PDF neurons from the larval and adult fly brain, respectively, but previous studies have not analyzed effects of GABA on lLNv membrane activity in the context of the intact circadian rest/arousal control network. This study demonstrated that GABA inhibits the membrane activity of lLNvs, whereas the ionotropic GABAR antagonist, picrotoxin, is excitatory. In contrast, another study has shown no rescue of the inhibitory effect of GABA by picrotoxin on PDF+ LNv precursors but does show alleviation of GABA-induced inhibitory responses in these neurons by metabotropic GABABR antagonists. Although the current studies do not exclude a role for GABABRs in lLNvs, the discrepancy of the effect of picrotoxin could be attributable to changes in different GABAR subtype expression at different developmental stages (McCarthy, 2011).

    Through an extensive series of voltage-clamp experiments it was determined that GABA induces currents in lLNvs that reverse at the equilibrium potential of Cl-, indicating that these currents are mediated by GABAARs. These data demonstrate that lLNvs express the GABAAR and that lLNvs receive GABAergic inhibitory synaptic input. These data from paired recordings in lLNvs show that GABAergic synaptic input, in conjunction with PDF signaling, plays a critical role in modulating the membrane activity of lLNvs but is not required for the robust synchrony of firing in these neurons, as application of picrotoxin does not abolish synchronous firing. Conversely, in the cockroach, picrotoxin leads to desynchrony within circadian neural networks. In this system, PDF also serves to synchronize these neural populations by inhibiting GABAergic interneurons. This mechanism does not seem to be conserved in Drosophila, but additional experiments are needed to elucidate the effect of PDF on the synchronous electrical activity of the circadian neural circuit (McCarthy, 2011).

    Glutamate and its excitatory ionotropic receptors, homologs of the AMPA, kainate, and NMDA receptors in mammals, have been shown to mediate fast excitatory neurotransmission at the neuromuscular junction (NMJ) in Drosophila. Interestingly, the current data demonstrate that treatment of lLNvs with glutamate led to an inhibition of membrane activity, which is opposite to the effect seen at the NMJ. Through voltage-clamp experiments, it was shown that this glutamate-induced current in lLNvs reverses near the equilibrium potential of Cl-. Furthermore, when the Cl- concentration of the external solution was altered and the reversal potential of the current was measured, the experimental value was well predicted by the calculated equilibrium potential for each specific Cl- concentration. These data together indicate that lLNvs possess a glutamate-gated Cl- channel. Members of the GluCl family have been cloned from bothDrosophila and C. elegans but have not been found in vertebrate species. Their functional roles in neural circuits in Drosophila remain enigmatic. The current studies indicate these channels are present in lLNvs, which also express metabotropic glutamate receptors. Their role in synchronous membrane activity between lLNvs remains to be elucidated (McCarthy, 2011).

    Given the variety of the synaptic inputs to lLNvs described in this study, the ability of lLNvs to autonomously detect light through the blue light-activated photopigment CRY, and the convergence of the arousal and circadian circuits on lLNvs, these neurons are clearly in a position to integrate complicated signals from all these systems. The data also show that the rhythmic oscillation in membrane activity seen in these neurons is most likely not attributable to intrinsic pacemaking, but instead arises from synchronized synaptic inputs, both excitatory and inhibitory. It remains to be determined where these cholinergic, GABAergic, and glutamatergic synaptic inputs converging on lLNvs originate. Previous studies have demonstrated that the Hofbauer-Büchner adult eyelets, which are derived developmentally from Bolwig's organ in the larvae, send axon bundles to the dendritic region on LNvs. These cholinergic neurons may provide excitatory input to lLNvs via nAChRs. However, it is not considered likely that Hofbauer-Büchner cholinergic inputs to the lLNvs contribute to rhythmic activity in the whole-brain explant. As far as anatomical characterization of the inhibitory inputs into lLNvs, varicosities in the accessory medulla, which abut lLNv dendrites, express glutamic acid decarboxylase, a marker for GABAergic neurons; however, it is not known where cell bodies reside from which these processes originate. In addition, it has been previously shown that other circadian clock neurons are glutamatergic. The axon terminals of these neurons are in close proximity to the dendritic arbors of the lLNvs in the larval optic center and in the accessory medulla of the adult fly. These data, in combination with the current findings, suggest that GluCl within lLNvs may mediate inhibitory synaptic inputs from other clock neurons in the circadian circuit (McCarthy, 2011).

    Through the use of whole-cell patch-clamp electrophysiology techniques, this study has demonstrated synchronous membrane activity of lLNvs of the circadian rest/arousal neural network of Drosophila arising from bilateral synchronized synaptic inputs. This synchronous membrane activity is mediated by cholinergic inputs to the lLNvs themselves. However, GABAergic inputs modulate membrane activity of these neurons but are not required for synchrony. The role of glutamatergic signaling in synchronous membrane activity between lLNv pairs remains to be revealed, as agents to pharmacologically inhibit GluCl are not currently available. Building on these findings, future studies are required to elucidate the overlapping neural circuitry of the circadian, rest/arousal, and light input systems, and will discern how these systems are integrated and finely coordinated to generate a robust and complex pattern of behavior (McCarthy, 2011).

    Two different forms of arousal in Drosophila are oppositely regulated by the dopamine D1 receptor ortholog DopR via distinct neural circuits

    Arousal is fundamental to many behaviors, but whether it is unitary or whether there are different types of behavior-specific arousal has not been clear. In Drosophila, dopamine promotes sleep-wake arousal. However, there is conflicting evidence regarding its influence on environmentally stimulated arousal. This study shows that loss-of-function mutations in the D1 dopamine receptor DopR enhance repetitive startle-induced arousal while decreasing sleep-wake arousal (i.e., increasing sleep). These two types of arousal are also inversely influenced by cocaine, whose effects in each case are opposite to, and abrogated by, the DopR mutation. Selective restoration of DopR function in the central complex rescues the enhanced stimulated arousal but not the increased sleep phenotype of DopR mutants. These data provide evidence for at least two different forms of arousal, which are independently regulated by dopamine in opposite directions, via distinct neural circuits (Lebestky, 2009).

    'Arousal', a state characterized by increased activity, sensitivity to sensory stimuli, and certain patterns of brain activity, accompanies many different behaviors, including circadian rhythms, escape, aggression, courtship, and emotional responses in higher vertebrates. A key unanswered question is whether arousal is a unidimensional, generalized state. Biogenic amines, such as dopamine (DA), norepinephrine (NE), serotonin (5-HT), and histamine, as well as cholinergic systems, have all been implicated in arousal in numerous behavioral settings. However, it is not clear whether these different neuromodulators act on a common 'generalized arousal' pathway or rather control distinct arousal pathways or circuits that independently regulate different behaviors. Resolving this issue requires identifying the receptors and circuits on which these neuromodulators act, in different behavioral settings of arousal (Lebestky, 2009).

    Most studies of arousal in Drosophila have focused on locomotor activity reflecting sleep-wake transitions, a form of 'endogenously generated' arousal. Several lines of evidence point to a role for DA in enhancing this form of arousal in Drosophila. Drug-feeding experiments, as well as genetic silencing of dopaminergic neurons, have indicated that DA promotes waking during the subjective night phase of the circadian cycle. Similar conclusions were drawn from studying mutations in the Drosophila DA transporter (dDAT). Consistent with these data, overexpression of the vesicular monoamine transporter (dVMAT-A), promoted hyperactivity in this species, as did activation of DA neurons in quiescent flies (Lebestky, 2009).

    Evidence regarding the nature of DA effects on 'exogenously generated' or environmentally stimulated arousal, such as that elicited by startle, is less consistent. Classical genetic studies and quantitative trait locus (QTL) analyses have suggested that differences in DA levels may underlie genetic variation in startle-induced locomotor activity (see Carbone, 2006 and Jordan, 2006). Fmn (dDAT; Dopamine transporter) mutants displayed hyperactivity in response to mechanical shocks, implying a positive-acting role for DA in controlling environmentally induced arousal (Kume, 2005). In contrast, other data imply a negative-acting role for DA in controlling stimulated arousal. Mutants in Tyr-1, which exhibit a reduction in dopamine levels, show an increase in stimulated but not spontaneous levels of locomotor activity. Genetic inhibition of tyrosine hydroxylase-expressing neurons caused hyperactivity in response to mechanical startle (Friggi-Grelin, 2003). Finally, transient activation of DA neurons in hyperactive flies inhibited locomotion (Lima, 2005). Whether these differing results reflect differences in behavioral assays, the involvement of different types of DA receptors, or an 'inverted U'-like dosage sensitivity to DA (Birman, 2005), is unclear (Lebestky, 2009).

    This investigation has developed a novel behavioral paradigm for environmentally stimulated arousal, using repetitive mechanical startle as a stimulus, and a screen was carried out for mutations that potentiate this response. One such mutation is a hypomorphic allele of the D1 receptor ortholog, DopR. This same mutation caused decreased spontaneous activity during the night phase of the circadian cycle, due to increased rest bout duration. In both assays, cocaine influenced behavior in the opposite direction as the DopR mutation, and the effect of cocaine was abolished in DopR mutant flies, supporting the idea that DA inversely regulates these two forms of arousal. Genetic rescue experiments, using Gal4 drivers with restricted CNS expression, indicate that these independent and opposite influences of DopR are exerted in different neural circuits. These data suggest the existence of different types of arousal states mediated by distinct neural circuits in Drosophila, which can be oppositely regulated by DA acting via the same receptor subtype (Lebestky, 2009).

    Previous studies of arousal in Drosophila have focused on sleep-wake transitions, a form of 'endogenous' arousal. This study has introduced and characterized a quantitative behavioral assay for repetitive startle-induced hyperactivity, which displays properties consistent with an environmentally triggered ('exogenous') arousal state. A screen was conducted for mutations affecting this behavior, the phenotype of one such mutation (DopR) was analyzed, and the neural substrates of its action was mapped by cell-specific genetic rescue experiments. The results reveal that DopR independently regulates Repetitive Startle-induced Hyperactivity (ReSH) and sleep in opposite directions by acting on distinct neural substrates. Negative regulation of the ReSH response requires DopR function in the ellipsoid body (EB) of the central complex (CC), while positive regulation of waking reflects a function in other populations of neurons, including PDF-expressing circadian pacemaker cells. Both of these functions, moreover, are independent of the function of DopR in learning and memory, which is required in the mushroom body. These data suggest that ReSH behavior and sleep-wake transitions reflect distinct forms of arousal that are genetically, anatomically, and behaviorally separable. This conclusion is consistent with earlier suggestions, based on classical genetic studies, that spontaneous and environmentally stimulated locomotor activity reflect 'distinct behavioral systems' in Drosophila (Lebestky, 2009).

    Several lines of evidence suggest that ReSH behavior represents a form of environmentally stimulated arousal. First, hyperactivity is an evolutionarily conserved expression of increased arousal. Although not all arousal is necessarily expressed as hyperactivity, electrophysiological studies indicate that mechanical startle, the type of stimulus used in this study, evokes increases in 20-30 Hz and 80-90 Hz brain activity, which have been suggested to reflect a neural correlate of arousal in flies (Nitz, 2002; van Swinderen, 2004). Second, ReSH does not immediately dissipate following termination of the stimulus, as would be expected for a simple reflexive stimulus-response behavior, but rather persists for an extended period of time, suggesting that it reflects a change in internal state. Third, this state, like arousal, is scalable: more puffs, or more intense puffs, produce a stronger and/or longer-lasting state of hyperactivity. Fourth, this state exhibits sensitization: even after overt locomotor activity has recovered to prepuff levels, flies remain hypersensitive to a single puff for several minutes. Fifth, this sensitization state generalizes to a startle stimulus of at least one other sensory modality (olfactory). In Aplysia, sensitization of the gill/siphon withdrawal reflex has been likened to behavioral arousal. Taken together, these features strongly suggest that ReSH represents an example of environmentally stimulated ('exogenous') arousal in Drosophila (Lebestky, 2009).

    DopR mutant flies exhibited longer rest periods during their subjective night phase, suggesting that DopR normally promotes sleep-wake transitions. These data are consistent with earlier studies indicating that DA promotes arousal by inhibiting sleep (Andretic, 2005, Kume, 2005; Wu, 2008). In contrast, prior evidence regarding the role of DA in startle-induced arousal is conflicting. Some studies have suggested that DA negatively regulates locomotor reactivity to environmental stimuli, consistent with the current observations, while others have suggested that it positively regulates this response. Even within the same study, light-stimulated activation of TH+ neurons produced opposite effects on locomotion, depending on the prestimulus level of locomotor activity (Lima, 2005; Lebestky, 2009 and references therein).

    This study has found that DA and DopR negatively regulate environmentally stimulated arousal: the DopR mutation enhanced the ReSH response, while cocaine suppressed it. Furthermore, the effect of cocaine in the ReSH assay was eliminated in the DopR mutant but could be rescued by Gal4-driven DopR expression, confirming that the effect of the drug is mediated by DA. Taken together, these results reconcile apparently conflicting data on the role of DA in 'arousal' in Drosophila by identifying two different forms of arousal -- repetitive startle-induced arousal and sleep-wake arousal -- that are regulated by DA in an inverse manner (Lebestky, 2009).

    The finding that DopR negatively regulates one form of environmentally stimulated arousal leaves open the question of whether this is true for all types of exogenous arousing stimuli. The 'sign' of the influence of DA on exogenously generated arousal states may vary depending on the type or strength of the stimulus used, the initial state of the system prior to exposure to the arousing stimulus (Birman, 2005; Lima and Miesenbock, 2005), or the precise neural circuitry that is engaged. Future studies using arousing stimuli of different sensory modalities or associated with different behaviors should shed light on this question (Lebestky, 2009).

    Several lines of evidence suggest that endogenous DopR likely acts in the ellipsoid body (EB) of the central complex (CC) to regulate repetitive startle-induced arousal. First, multiple Gal4 lines that drive expression in the EB rescued the ReSH phenotype of DopR mutants. Second, endogenous DopR is expressed in EB neurons, including those in which the rescuing Gal4 drivers are expressed. Third, the domain of DopR expression in the EB overlaps the varicosities of TH+ fibers. In an independent study of dopaminergic inputs required for regulating EtOH-stimulated hyperactivity TH+ neurons were identified that are a likely source of these projections to the EB. Fourth, rescue of the ReSH phenotype is associated with re-expression of DopR in EB neurons. Finally, rescue is observed using conditional DopR expression in adults. Taken together, these data argue that rescue of the ReSH phenotype by the Gal4 lines tested reflects their common expression in the EB and that this is a normal site of DopR action in adult flies (Lebestky, 2009).

    A requirement for DopR in the EB in regulating ReSH behavior is consistent with the fact that the CC is involved in the control of walking activity. However, the mushroom body has also been implicated in the control of locomotor behavior, and DopR is strongly expressed in this structure as well. Rescue data argue against the MB and in favor of the CC as a neural substrate for the ReSH phenotype of DopR mutants. Unexpectedly, the nocturnal hypoactivity phenotype of DopR mutants was not rescued by restoration of DopR expression to the CC. Thus, not all locomotor activity phenotypes of the DopR mutant necessarily reflect a function for the gene in the CC (Lebestky, 2009).

    Interestingly, Gal4 line c547 expresses in R2/R4m neurons of the EB, while lines 189y and c761 express in R3 neurons, yet both rescued the ReSH phenotype of DopR mutants. Similar results have been obtained in experiments to rescue the deficit in ethanol-induced behavior exhibited by the DopR mutant. Double-labeling experiments suggest that endogenous DopR is expressed in all of these EB neuronal subpopulations. Perhaps the receptor functions in parallel or in series in R4m and R3 neurons, so that restoration of DopR expression in either population can rescue the ReSH phenotype. Whether these DopR-expressing EB subpopulations are synaptically interconnected is an interesting question for future investigation (Lebestky, 2009).

    Despite its power as a system for studying neural development, function, and behavior, Drosophila has not been extensively used in affective neuroscience, in part due to uncertainty about whether this insect exhibits emotion-like states or behaviors. Increased arousal is a key component of many emotional or affective behaviors. The data presented in this study indicate that Drosophila can express a persistent arousal state in response to repetitive stress. ReSH behavior exhibits several features that distinguish it from simple, reflexive stimulus-response behaviors: scalability, persistence following stimulus termination, and sensitization. In addition, the observation that mechanical trauma promotes release from Drosophila of an odorant that repels other flies suggests that the arousal state underlying ReSH behavior may have a negative 'affective valence' as well. These considerations, taken together with the fact that ReSH is influenced by genetic and pharmacologic manipulations of DA, a biogenic amine implicated in emotional behavior in humans, support the idea that the ReSH response may represent a primitive 'emotion-like' behavior in Drosophila (Lebestky, 2009).

    The phenotype of DopR flies is reminiscent of attention-deficit hyperactivity disorder (ADHD), an affective disorder linked to dopamine, whose symptoms include hyper-reactivity to environmental stimuli. If humans, like flies, have distinct circuits for different forms of arousal, then the current data suggest that ADHD may specifically involve dopaminergic dysfunction in those circuits mediating environmentally stimulated, rather than endogenous (sleep-wake), arousal. Given that DA negatively regulates environmentally stimulated arousal circuits in Drosophila, such a view would be consistent with the fact that treatment with drugs that increase synaptic levels of DA, such as methylphenidate (ritalin), can ameliorate symptoms of ADHD (Lebestky, 2009).

    In further support of this suggestion, in mammals, dopamine D1 receptors in the prefrontal cortex (PFC) have been proposed to negatively regulate activity, while D1 receptors in the nucleus accumbens are thought to promote sleep-wake transitions. Numerous studies have linked dopaminergic dysfunction in the PFC to ADHD. While most research has focused on the role of the PFC in attention and cognition, rather than in environmentally stimulated arousal per se, dysfunction of PFC circuits mediating phasic DA release has been invoked to explain behavioral hypersensitivity to environmental stimuli in ADHD (Sikstrom, 2007). This view of ADHD as a disorder of circuits mediating environmentally stimulated arousal suggests that further study of such circuits in humans and in vertebrate animal models, as well as in Drosophila, may improve understanding of this disorder and ultimately lead to improved therapeutics (Lebestky, 2009).

    Identification of a neural circuit that underlies the effects of octopamine on sleep:wake behavior

    An understanding of sleep requires the identification of distinct cellular circuits that mediate the action of specific sleep:wake-regulating molecules, but such analysis has been very limited. This study identifies a circuit that underlies the wake-promoting effects of octopamine in Drosophila. Using MARCM, the ASM cells in the medial protocerebrum were identified as the wake-promoting octopaminergic cells. Octopamine signaling was then blocked in random areas of the fly brain, and the postsynaptic effect was mapped to insulin-secreting neurons of the pars intercerebralis (PI). These PI neurons show altered potassium channel function as well as an increase in cAMP in response to octopamine, and genetic manipulation of their electrical excitability alters sleep:wake behavior. Effects of octopamine on sleep:wake are mediated by the cAMP-dependent isoform of the OAMB receptor. These studies define the cellular and molecular basis of octopamine action and suggest that the PI is a sleep:wake-regulating neuroendocrine structure like the mammalian hypothalamus (Crocker, 2010).

    Drosophila pacemaker neurons require G protein signaling and GABAergic inputs to generate twenty-four hour behavioral rhythms

    Intercellular signaling is important for accurate circadian rhythms. In Drosophila, the small ventral lateral neurons (s-LNvs) are the dominant pacemaker neurons and set the pace of most other clock neurons in constant darkness. This study shows that two distinct G protein signaling pathways are required in LNvs for 24 hr rhythms. Reducing signaling in LNvs via the G alpha subunit Gs, which signals via cAMP, or via the G alpha subunit Go, which signals via Phospholipase 21c, lengthens the period of behavioral rhythms. In contrast, constitutive Gs or Go signaling makes most flies arrhythmic. Using dissociated LNvs in culture, it was found that Go and the metabotropic GABA(B)-R3 receptor are required for the inhibitory effects of GABA on LNvs and that reduced GABA(B)-R3 expression in vivo lengthens period. Although no clock neurons produce GABA, hyperexciting GABAergic neurons disrupts behavioral rhythms and s-LNv molecular clocks. Therefore, s-LNvs require GABAergic inputs for 24 hr rhythms (Dahdal, 2010).

    The long-periods observed with reduced Gs signaling are consistent with four other manipulations of cAMP levels or PKA activity that alter fly circadian behavior. First, long-period rhythms with dnc over-expression complement the short periods of dnc hypomorphs and suggest that the latter are due to loss of dnc from LNvs. dnc mutants also increase phase shifts to light in the early evening. However, this study found no difference in phase delays or advances between Pdf > dnc and control flies, suggesting that altered light-responses of dnc hypomorphs are due to dnc acting in other clock neurons. The period-altering effects seen when manipulating cAMP levels are also consistent with finding stat expressing the cAMP-binding domain of mammalian Epac1 in LNvs lengthens period. This Epac1 domain likely reduces free cAMP levels in LNvs, although presumably not as potently as UAS-dnc. Third, mutations in PKA catalytic or regulatory subunits that affect the whole fly disrupt circadian behavior. Fourth, over-expressing a PKA catalytic subunit in LNvs rescues the period-altering effect of a UAS-shibire transgene that alters vesicle recycling, although the PKA catalytic subunit had no effect by itself. The long periods observed with reduced Gs signaling in LNvs also parallel mammalian studies in which pharmacologically reducing Adenylate cyclase activity lengthened period in SCN explants and mice (Dahdal, 2010).

    G-proteins typically transduce extracellular signals. What signals could activate Gs in s-LNvs? PDF is one possibility since PDFR induces cAMP signaling in response to PDF in vitro, indicating that it likely couples to Gs. PDF could signal in an autocrine manner since PDFR is present in LNvs. However, the long-periods observed with reduced Gs signaling differ from the short-period and arrhythmic phenotypes of Pdf and pdfr mutants. The likeliest explanation for these differences is that the altered behavior of Pdf and pdfr mutants results from effects of PDF signaling over the entire circadian circuit, whereas the current manipulations specifically targeted LNvs. Indeed, LNvs are not responsible for the short-period rhythms in Pdf01 null mutant flies. Other possible explanations for the differences between the long-period rhythms with decreased Gs signaling in LNvs and the short-period rhythms of Pdf and pdfr mutants are that additional GPCRs couple to Gs in s-LNvs and influence molecular clock speed and that the current manipulations decrease rather than abolish reception of PDF. In summary, the data shows that Gs signaling via cAMP in s-LNvs modulates period length (Dahdal, 2010).

    Go signaling via PLC21C constitutes a novel pathway that regulates the s-LNv molecular clock. This study found that Go and the metabotropic GABAB-R3 receptor are required for the inhibitory effects of GABA on larval LNvs, which develop into adult s-LNvs. The same genetic manipulations that block GABA inhibition of LNvs in culture (expression of Ptx or GABAB-R3-RNAi) lengthened the period of adult locomotor rhythms. Furthermore, the molecular clock in s-LNvs is disrupted when a subset of GABAergic neurons are hyper-excited. Since the LNvs do not produce GABA themselves, s-LNvs require GABAergic inputs to generate 24hr rhythms. Thus s-LNvs are less autonomous for determining period length in DD than previously anticipated (Dahdal, 2010).

    Activation of G-proteins can have both short- and long-term effects on a cell. With Go signaling blocked by Ptx, short-term effects on LNv responses were detected in response to excitatory ACh and longer-term effects on the molecular clock. The latter are presumably explained by PLC activation since the behavioral phenotypes of Pdf > GoGTP flies were rescued by reducing Plc21C expression (Dahdal, 2010).

    Since s-LNv clocks were unchanged even when the speed of all non-LNv clock neurons were genetically manipulated, it is surprising to find s-LNv clocks altered by signaling from GABAergic non-clock neurons. Why would LNvs need inputs from non-clock neurons to generate 24hr rhythms? One possibility is that LNvs receive multiple inputs which either accelerate or slow down the pace of their molecular clock but overall balance each other to achieve 24hr rhythms in DD. Since reducing signaling by Gs and Go lengthens period, these pathways normally accelerate the molecular clock. According to this model, there are unidentified inputs to LNvs which delay the clock. Identifying additional receptors in LNvs would allow this idea to be tested (Dahdal, 2010).

    Previous work showed that GABAergic neurons project to LNvs and that GABAA receptors in l-LNvs regulate sleep. The current data show that constitutive activation of Go signaling dramatically alters behavioral rhythms, suggesting that LNvs normally receive rhythmic GABAergic inputs. But how can s-LNvs integrate temporal information from non clock-containing GABAergic neurons? s-LNvs could respond rhythmically to a constant GABAergic tone by controlling GABAB-R3 activity. Indeed, a recent study found that GABAB-R3 RNA levels in s-LNvs are much higher at ZT12 than at ZT0 (Kula-Eversole, 2010). Strikingly, this rhythm in GABAB-R3 expression is in antiphase to LNv neuronal activity. Thus regulated perception of inhibitory GABAergic inputs could at least partly underlie rhythmic LNv excitability. GABAergic inputs could also help synchronize LNvs as in the cockroach circadian system. Thus GABA's short-term effects on LNv excitability, likely mediated by Gβ/γ, and GABA's longer-term effects on the molecular clock via Go may both contribute to robust rhythms (Dahdal, 2010).

    This work adds to the growing network view of circadian rhythms in Drosophila where LNvs integrate information to set period for the rest of the clock network in DD. The period-altering effects of decreased G-protein signaling in LNvs point to a less hierarchical and more distributed network than previously envisioned. Since the data strongly suggests that GABA inputs are novel regulators of 24hr rhythms, the GABAergic neurons that fine-tune the s-LNv clock should be considered part of the circadian network (Dahdal, 2010).

    GABAB receptors play an essential role in maintaining sleep during the second half of the night in Drosophila melanogaster

    GABAergic signalling is important for normal sleep in humans and flies. This study has advance the current understanding of GABAergic modulation of daily sleep patterns by focusing on the role of slow metabotropic GABAB receptors in Drosophila. It was asked whether GABAB-R2 receptors are regulatory elements in sleep regulation in addition to the already identified fast ionotropic Rdl GABAA receptors. By immunocytochemical and reporter-based techniques it was shown that the pigment dispersing factor (PDF)-positive ventrolateral clock neurons (LNv) express GABAB-R2 receptors. Downregulation of GABAB-R2 receptors in the large PDF neurons (l-LNv) by RNAi reduced sleep maintenance in the second half of the night, whereas sleep latency at the beginning of the night that was previously shown to depend on ionotropic Rdl GABAA receptors remained unaltered. The results confirm the role of the l-LNv neurons as an important part of the sleep circuit in D. melanogaster and also identify the GABAB-R2 receptors as the thus far missing component in GABA-signalling that is essential for sleep maintenance. Despite the significant effects on sleep, no changes were observed changes in circadian behaviour in flies with downregulated GABAB-R2 receptors, indicating that the regulation of sleep maintenance via l-LNv neurons is independent of their function in the circadian clock circuit (Gmeiner, 2013).

    The fruit fly has become a well-accepted model for sleep research. As in mammals, it has been shown that the sleep-like state of Drosophila is associated with reduced sensory responsiveness and reduced brain activity, and is subject to both circadian and homeostatic regulation. Similarly to in humans, monaminergic neurons (specifically dopaminergic and octopaminergic neurons) enhance arousal in fruit flies, whereas GABAergic neurons promote sleep (Agosto, 2008). As in humans, GABA advances sleep onset (reduces sleep latency) and prolongs total sleep (increases sleep maintenance). Brain regions possibly implicated in the regulation of sleep in D. melanogaster are the pars intercerebralis, the mushroom bodies and a subgroup of the pigment dispersing factor (PDF)-positive neurons called the l-LNv neurons. The l-LNv belong to the circadian clock neurons, indicating that in flies, as in mammals, the sleep circuit is intimately linked to the circadian clock and that the mechanisms employed to govern sleep in the brain are evolutionarily ancient (Gmeiner, 2013).

    The l-LNv are conspicuous clock neurons with wide arborisations in the optic lobe, fibres in the accessory medulla -- the insect clock centre -- and connections between the brain hemispheres. Thus, the l-LNv neurons are anatomically well suited to modulate the activity of many neurons. In addition, their arborisations overlap with those of monaminergic neurons. Several studies show that they indeed receive dopaminergic, octopaminergic and GABAergic input and that they control the flies' arousal and sleep. Furthermore, the l-LNv are directly light sensitive and promote arousal and activity in response to light, especially in the morning (Gmeiner, 2013).

    A part of the sleep-promoting effect of GABA on the l-LNv has been shown to be mediated via the fast ionotropic GABAA receptor Rdl (Resistance to dieldrin) (Agosto, 2008). Rdl Cl- channels are expressed in the l-LNv (Agosto, 2008) and, similar to mammalian GABAA receptors, they mediate fast inhibitory neurotransmission. As expected, GABA application reduced the action potential firing rate in the l-LNv, whereas application of picrotoxin, a GABAA receptor antagonist, increased it (McCarthy, 2011). Furthermore, an Rdl receptor mutant with prolonged channel opening and consequently increased channel current significantly decreased sleep latency of the flies after lights-off, whereas the downregulation of the Rdl receptor via RNAi increased it (Agosto, 2008; Gmeiner, 2013 and references therein).

    Nevertheless, the manipulation of the Rdl receptor had no effect on sleep maintenance. Because the latter is significantly reduced after silencing the GABAergic neurons (Parisky, 2008), other GABA receptors must be responsible for maintaining sleep. Suitable candidates are slow metabotropic GABAB receptors that are often co-localised with ionotropic GABAA receptors (Enell, 2007). In Drosophila, like in mammals, the metabotropic GABAB receptors are G-protein-coupled seven-transmembrane proteins composed of two subunits, GABAB-R1 and GABAB-R2 (Kaupmann, 1998; Mezler, 2001). The GABAB-R1 is the ligand binding unit and GABAB-R2 is required for translocation to the cell membrane and for stronger coupling to the G-protein (Kaupmann, 1998; Galvez, 2001). This study shows that the l-LNv do express metabotropic GABAB-R2 receptors and that these receptors are relevant for sleep maintenance but not for sleep latency. Thus, metabotropic and ionotropic GABA receptors are cooperating in sleep regulation (Gmeiner, 2013).

    This study shows that metabotropic GABAB-R2 receptors are expressed on the PDF-positive clock neurons (LNv neurons), and that their downregulation in the l-LNv by RNAi results in: (1) a higher activity level throughout the day and night and (2) reduced sleep maintenance in the second half of the night. Neither sleep onset nor circadian rhythm parameters were affected by the downregulation. It is concluded that GABA signalling via metabotropic receptors on the l-LNv is essential for sustaining sleep throughout the night and for keeping activity at moderate levels throughout the 24-h day (preventing flies from hyperactivity). A major caveat of RNAi is off-target effects, particularly when Gal4 drivers are expressed in large numbers of non-target neurons. Though GABAB-R2 was downregulated in only eight neurons per brain hemisphere and the behavioural effects of the knockdown experiments were carefully correlated with observation and measures of GABAB-R2 immunostaining in the s-LNv and l-LNv, it is still possible that some effects were due to off-target knockdown of other membrane proteins. Nevertheless, given the fact that no such effects have been reported in the previous paper that used the same GABAB-R2 RNAi line (Root, 2008), it is thought unlikely that the behavioural effects described in this study were due to off-target knockdown of other genes (Gmeiner, 2013).

    The results are in line with a former study describing the location of GABAB receptors in D. melanogaster (Hamasaka, 2005). The ionotropic GABAA receptor Rdl has also been identified on the l-LNv neurons and has been shown to regulate sleep, but its downregulation delayed only sleep onset and did not perturb sleep maintenance (Parisky, 2008). In contrast, silencing GABAergic signalling influenced sleep onset and sleep maintenance, indicating that GABA works through the fast Rdl receptor, and also implying a longer-lasting signalling pathway. GABAB receptors are perfect candidates in mediating slow but longer-lasting effects of GABA. Often, GABAA and GABAB receptors cooperate in mediating such fast and slow effects. For example, in the olfactory system, GABAA receptors mediate the primary modulatory responses to odours whereas GABAB receptors are responsible for long-lasting effects (Wilson, 2005; Gmeiner, 2013 and references therein).

    In D. melanogaster, GABAB receptors consist of the two subunits GABAB-R1 and GABAB-R2, and only the two units together can efficiently activate the metabotropic GABA signalling cascade (Galvez, 2001; Mezler, 2001). In the current experiments, only GABAB-R2 was downregulated, but this manipulation should also have decreased the amount of functional GABAB-R1/GABAB-R2 heterodimers and, therefore, reduced GABAB signalling in general. Taking into account that sleep maintenance in the second half of the night was already significantly impaired by an ~46% reduction in detectable GABAB-R2 immunostaining intensity in the l-LNv clock neurons, it can be assumed that GABAB receptors account for an even larger portion of the sleep maintenance than detected in these experiments. Thus GABAB receptors play a crucial role in mediating GABAergic signals to the l-LNv neurons, which are needed to sustain sleep throughout the night. This is mainly due to the maintenance of extended sleep bout durations in the second half of the night. When signalling by the GABAB receptor is reduced, sleep bouts during this interval are significantly shortened, leading to less total sleep (Gmeiner, 2013).

    Most importantly, this study confirmed the l-LNv as important components in regulation of sleep and arousal (Agosto, 2008; Parisky, 2008; Kula-Eversole, 2010; Shang, 2011). In contrast, the s-LNv seem to be not involved in sleep-€“arousal regulation but are rather important for maintaining circadian rhythmicity under DD (reviewed by Helfrich-Förster, 2007). One caveat in clearly distinguishing the function of s-LNv and l-LNv is the fact that both cell clusters express the neuropeptide PDF and, as a consequence, Pdf-GAL4 drives expression in both subsets of clock neurons. Though no significant GABAB-R2 knock-down in the s-LNv is seen, it cannot be completely excluded that GABAB-R2 was slightly downregulated in these clock neurons and that this knock-down contributes to the observed alterations in sleep. To restrict the knock-down to the s-LNv the R6-GAL4 line was used that is expressed in the s-LNv but not in the l-LNv. Neither a reduction in GABAB-R2 staining intensity in the s-LNv nor any effects on sleep in the second half of the night was seen. The lack of any visible GABAB-R2 downregulation in the s-LNv with R6-GAL4 is in agreement with observations of Shafer and Taghert (Shafer, 2009), who could completely downregulate PDF in the s-LNv using Pdf-GAL4 but not using R6-GAL4. Thus, R6-GAL4 is a weaker driver than Pdf-GAL4 and is obviously not able to influence GABAB-R2 in the s-LNv. Nevertheless, in the current experiments the R6-Gal4-driven GABAB-R2 RNAi led to flies that had slightly higher diurnal activity levels and less diurnal rest than the control flies. This suggests that GABAB-R2 was downregulated somewhere else. When checking the R6-GAL4 expression more carefully it was found that R6-GAL4 was not restricted to the brain, but was also present in many cells of the thoracic and especially the abdominal ganglia. Given the broad expression of GABAB-R2, a putative knock-down in the ventral nervous system is likely to affect locomotor activity (Gmeiner, 2013).

    The results on the l-LNv certainly do not exclude a role of GABA in the circadian clock controlling activity rhythms under DD conditions (here represented by the s-LNv). In mammals, GABA is the most abundant neurotransmitter in the circadian clock centre in the brain -- the suprachiasmatic nucleus. GABA interacts with GABAA and GABAB receptors, producing primarily but not exclusively inhibitory responses through membrane hyperpolarisation. GABA signalling is important for maintaining behavioural circadian rhythmicity, it affects the amplitude of molecular oscillations and might contribute to synchronisation of clock cells within the suprachiasmatic nucleus. The same seems to be true for fruit flies. The s-LNv neurons of adults alter cAMP levels upon GABA application on isolated brains in vitro (Lelito, 2012). Hyperexcitation of GABAergic neurons disrupts the molecular rhythms in the s-LNv and renders the flies arrhythmic (Dahdal, 2010). Thus, GABA signalling affects the circadian clock in the s-LNv. Flies with downregulated GABAB-R2 receptors were found to have slightly longer free-running periods than the control flies, but this turned out to be only significant in comparison with Control 2 and not to Control 1. Dahdal (2010) found similar small effects on period after downregulating GABAB-R2 receptors, but a significant period lengthening after downregulating GABAB-R3 receptors. This indicates that GABA signals via GABAB-R3 receptors to the s-LNv and was confirmed in vitro in the larval Drosophila brain by Ca2+ imaging (Dahdal, 2010). Nevertheless, the study of Dahdal does not rule out that GABA signals via GABAB-R3 plus GABAB-R2 receptors on the adult s-LNv. This study found a rather strong expression of GABAB-R2 receptors in these clock neurons, and were not able to downregulate it significantly by RNAi, although dicer2 was used as amplification. Dahdal did not use dicer2, and they also did not measure the effectiveness of the downregulation of GABAB-R2 by RNAi immunocytochemically directly in the s-LNv. Thus, the exact GABAB receptors that mediate GABA responses in the adult s-LNv need still to be determined (Gmeiner, 2013).

    In summary, it is concluded that the l-LNv subgroup of the PDF-positive clock neurons is a principal target of sleep-promoting and activity-repressing GABAergic neurons and sits at the heart of the sleep circuit in D. melanogaster. Thus, the sleep circuitry of flies is clearly more circumscribed and simpler than that of mammals. Mammals have many targets of sleep-promoting GABAergic neurons, and the circadian clock seems to have a mainly modulatory and less direct influence on sleep (Mistlberger, 2005). The fly sleep circuitry may therefore have condensed the mammalian arousal and sleep stimulating systems (e.g., monaminergic, cholinergic, peptidergic and GABAergic systems) into a simpler and more compact region, which seems to largely coincide with the eight PDF-positive l-LNv cells of the circadian circuit (Gmeiner, 2013).

    Sleep-promoting effects of threonine link amino acid metabolism in Drosophila neuron to GABAergic control of sleep drive

    Emerging evidence indicates the role of amino acid metabolism in sleep regulation. This study demonstrates sleep-promoting effects of dietary threonine (SPET) in Drosophila. Dietary threonine markedly increased daily sleep amount and decreased the latency to sleep onset in a dose-dependent manner. High levels of synaptic GABA or pharmacological activation of metabotropic GABA receptors (GABAB-R) suppressed SPET. By contrast, synaptic blockade of GABAergic neurons or transgenic depletion of GABAB-R in the ellipsoid body R2 neurons enhanced sleep drive non-additively with SPET. Dietary threonine reduced GABA levels, weakened metabotropic GABA responses in R2 neurons, and ameliorated memory deficits in plasticity mutants. Moreover, genetic elevation of neuronal threonine levels was sufficient for facilitating sleep onset. Taken together, these data define threonine as a physiologically relevant, sleep-promoting molecule that may intimately link neuronal metabolism of amino acids to GABAergic control of sleep drive via the neuronal substrate of sleep homeostasis (Ki, 2019).

    The circadian clock and sleep homeostasis are two key regulators that shape daily sleep behaviors in animals. In stark contrast to the homeostatic nature of sleep, the internal machinery of sleep is vulnerable to external (e.g., environmental change) or internal conditions (e.g., genetic mutation) that lead to adaptive changes in sleep behaviors. Sleep behavior is conserved among mammals, insects, and even lower eukaryotes. Since the identification of the voltage-gated potassium channel Shaker as a sleep-regulatory gene in Drosophila, fruit flies have been one of the most advantageous genetic models to dissect molecular and neural components that are important for sleep homeostasis and plasticity (Ki, 2019).

    To date, a number of sleep-regulatory genes and neurotransmitters have been identified in animal models as well as in humans. For instance, the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) is known to have a sleep-promoting role that is conserved in invertebrates and vertebrates. Hypomorphic mutations in mitochondrial GABA-transaminase (GABA-T) elevate GABA levels and lengthen baseline sleep in flies (Chen, 2015). The long sleep phenotype in GABA-T mutants accompanies higher sleep consolidation and shorter latency to sleep onset, consistent with the observations that pharmacological enhancement of GABAergic transmission facilitates sleep in flies and mammals, including humans. In addition, resistance to dieldrin (Rdl), a Drosophila homolog of the ionotropic GABA receptor, suppresses wake-promoting circadian pacemaker neurons in adult flies to exert sleep-promoting effects. Similarly, 4,5,6,7-tetrahydroisoxazolo[5,4 c]pyridin-3-ol (THIP), an agonist of the ionotropic GABA receptor, promotes sleep in insects and mammals (Ki, 2019).

    Many sleep medications modulate GABAergic transmission. A prominent side effect of anti-epileptic drugs relevant to GABA is causing drowsiness. Conversely, glycine supplements improve sleep quality in a way distinct from traditional hypnotic drugs, minimizing deleterious cognitive problems or addiction. In fact, glycine or D-serine acts as a co-agonist of N-methyl-D-aspartate receptors (NMDARs) and promotes sleep through the sub-type of ionotropic glutamate receptors. Emerging evidence further supports the roles of amino acid transporters and metabolic enzymes in sleep regulation. In particular, it has been demonstrated that starvation induces the expression of metabolic enzymes for serine biosynthesis in Drosophila brains, and elevates free serine levels to suppress sleep via cholinergic signaling (Sonn, 2018). These observations prompted a hypothesis that other amino acids may also display neuro-modulatory effects on sleep behaviors (Ki, 2019).

    The molecular and neural machinery of sleep regulation intimately interacts with external (e.g., light, temperature) and internal sleep cues (e.g., sleep pressure, metabolic state) to adjust the sleep architecture in animals. Using a Drosophila genetic model, this study has investigated whether dietary amino acids could affect sleep behaviors, through this investigation SPET was discovered. Previous studies have demonstrated that the wake-promoting circadian pacemaker neurons are crucial for timing sleep onset after lights-off in LD cycles. In addition, WAKE-dependent silencing of clock neurons and its collaborative function with RDL have been suggested as a key mechanism in the circadian control of sleep onset. However, the current evidence indicates that SPET facilitates sleep onset in a manner independent of circadian clocks. It was further elucidated that SPET operates likely via the down-regulation of metabotropic GABA transmission in R2 EB neurons, a neural locus for generating homeostatic sleep drive (Ki, 2019).

    Both food availability and nutritional quality substantially affect sleep behaviors in Drosophila. Sucrose contents in food and their gustatory perception dominate over dietary protein to affect daily sleep. Starvation promotes arousal in a manner dependent on the circadian clock genes Clock and cycle as well as neuropeptide F (NPF), which is a fly ortholog of mammalian neuropeptide Y. On the other hand, protein is one of the nutrients that contribute to the postprandial sleep drive in Drosophila and this observation is possibly relevant to SPET. While Leucokinin (Lk) and Lk receptor (Lkr) play important roles in dietary protein-induced postprandial sleepand in starvation-induced arousal, comparable SPET was observed between hypomorphic mutants of Lk or Lkr and their heterozygous controls. Therefore, SPET and its neural basis reveal a sleep-regulatory mechanism distinct from those involved in sleep plasticity relevant to food intake (Ki, 2019).

    What will be the molecular basis of SPET? Given the general implication of GABA in sleep promotion, a simple model will be that a molecular sensor expressed in a subset of GABAergic neurons (i.e., LN) directly responds to an increase in threonine levels, activates GABA transmission, and thereby induces sleep. Several lines of evidence, however, favored the other model that dietary threonine actually down-regulates metabotropic GABA transmission in R2 EB neurons, de-represses the neural locus for generating homeostatic sleep drive, and thereby enhances sleep drive. The latter model does not necessarily conflict with sleep-promoting effects of genetic or pharmacological conditions that generally elevate GABA levels or enhance GABAergic transmission since those effects will be the net outcome of activated GABA transmission via various sub-types of GABA receptors expressed in either wake- or sleep-promoting neurons and their (Ki, 2019).

    The structural homology among threonine, GABA, and their metabolic derivatives (e.g., alpha-ketobutyrate and gamma-hydroxybutyrate) led to the hypothesis that these relevant chemicals may act as competitive substrates in enzymatic reactions for their overlapping metabolism. Consequently, dietary threonine may limit the total flux of GABA-glutamate-glutamine cycle possibly through substrate competition, decreases the size of available GABA pool, and thereby down-scales GABA transmission for SPET. This accounts for why genetic or pharmacological elevation of GABA levels rather suppresses SPET. Threonine, GABA, and their derivatives may also act as competitive ligands for metabotropic GABA receptors, explaining weak GABA responses in R2 EB neurons of threonine-fed flies. Biochemical and neural evidence supportive of this hypothesis is quite abundant. It has been previously shown that alpha-ketobutyrate, GABA, and the ketone body beta-hydroxybutyrate act as competitive substrates in common enzymatic reactions. Moreover, functional interactions of beta-hydroxybutyrate or gamma-hydroxybutyrate with GABAergic signaling have been well documented. Finally, threonine and GABA derivatives have anti-convulsive effects, which further support their common structural and functional relevance to GABAergic signaling (Ki, 2019).

    The removal of the amino group is the initial step for amino acid metabolism, and various transaminases mediate its transfer between amino acids and alpha-keto acids. On the other hand, a group of amino acids (i.e., glutamate, glycine, serine, and threonine) has their own deaminases that can selectively remove the amino group. The presence of these specific deaminases is indicative of active mechanisms that individually fine-tune the baseline levels of these amino acids in metabolism, and possibly in the context of other physiological processes as well. This idea is further supported by the conserved roles of glutamate, glycine, and serine as neurotransmitters or neuromodulators important for brain function, including sleep regulation. In fact, serine, glycine, and threonine constitute a common metabolic pathway, and threonine may contribute indirectly to glycine- or serine-dependent activation of sleep-promoting NMDAR. Nonetheless, this study found that sleep-modulatory effects of dietary glycine were distinct from SPET and thus, it is speculated that threonine may act as an independent neuromodulator, similar to other amino acids with their dedicated deaminases (Ki, 2019).

    While several lines of the data support that threonine is likely to be an endogenous sleep driver in fed conditions, it wa recently demonstrated that starvation induces serine biosynthesis in the brain and neuronal serine subsequently suppresses sleep via cholinergic signaling (Sonn, 2018). These two pieces of relevant works establish a compelling model that the metabolic pathway of serine-glycine-threonine functions as a key sleep-regulatory module in response to metabolic sleep cues (e.g., food ingredients and dietary stress). It is further hypothesized that the adaptive control of sleep behaviors by select amino acids and their conserved metabolic pathway suggests an ancestral nature of their sleep regulation. Future studies should address if the serine-glycine-threonine metabolic pathway constitutes the sleep homeostat that can sense and respond to different types of sleep needs. In addition, it will be interesting to determine if this metabolic regulation of sleep is conserved among other animals, including humans (Ki, 2019).

    Increased food intake after starvation enhances sleep in Drosophila melanogaster

    Feeding and sleep are highly conserved, interconnected behaviors essential for survival. Starvation has been shown to potently suppress sleep across species; however, whether satiety promotes sleep is still unclear. This study used the fruit fly, Drosophila melanogaster, as a model organism to address the interaction between feeding and sleep. The sleep of flies that had been starved for 24 h was monitored, and sleep amount was found to increase in the first 4 h after flies were given food. Increased sleep after starvation was due to an increase in sleep bout number and average sleep bout length. Mutants of translin or adipokinetic hormone, which fail to suppress sleep during starvation, still exhibited a sleep increase after starvation, suggesting that sleep increase after starvation is not a consequence of sleep loss during starvation. It was also found that feeding activity and food consumption were higher in the first 10-30 min after starvation. Restricting food consumption in starved flies to 30 min was sufficient to increase sleep for 1 h. Although flies ingested a comparable amount of food at differing sucrose concentrations, sleep increase after starvation on a lower sucrose concentration was undetectable. Taken together, these results suggest that increased food intake after starvation enhances sleep and reveals a novel relationship between feeding and sleep (Regalado, 2017).

    Sleep deprivation negatively impacts reproductive output in Drosophila melanogaster

    Most animals sleep or exhibit a sleep-like state, yet the adaptive significance of this phenomenon remains unclear. Although reproductive deficits are associated with lifestyle induced sleep deficiencies, how sleep loss affects reproductive physiology is poorly understood, even in model organisms. This study aimed to bridge this mechanistic gap by impairing sleep in female fruit flies and testing its effect on egg output. Sleep deprivation by feeding caffeine or by mechanical perturbation was shown to result in decreased egg output. Transient activation of wake-promoting dopaminergic neurons decreases egg output in addition to sleep levels, thus demonstrating a direct negative impact of sleep deficit on reproductive output. Similarly, loss-of-function mutation in dopamine transporter fumin (fmn) leads to both significant sleep loss and lowered fecundity. This demonstration of a direct relationship between sleep and reproductive fitness indicates a strong driving force for the evolution of sleep (Potdar, 2018a).

    Characterization of sleep-related neurochemicals in the different developmental stages and insomnia models of Drosophila melanogaster

    Neurotransmitters play an important role in regulating the physiological activity of the animal, especially in emotion and sleep. While nucleotides are involved in almost all cellular processes. However, the characteristics of sleep-related neurochemicals under different life cycles and environment remain poorly understood. A rapid and sensitive analytical method was established with LC-MS/MS to determine eight endogenous neurochemicals in Drosophila melanogaster and the levels of neurochemicals in the different developmental stages of Drosophila melanogaster were evaluated. The results indicated that there were significant discrepancies among different stages, especially from pupal stage to adult. The levels of these compounds in caffeine-induced insomnia model of Drosophila melanogaster were investigated. Compared with normal group the eight endogenous metabolites did not fluctuate significantly in insomnia Drosophila melanogaster, which may be due to the mechanism of caffeine-induced insomnia through other pathways, such as adenosine. The results provide a reference for decoding of neurochemicals involved in the development of the full cycle of mammalian life and exploration of insomnia even other mental diseases induced by exogenous substances in the future (Sun, 2022).

    Wakefulness is promoted during day time by PDFR signalling to dopaminergic neurons in Drosophila melanogaster

    Circadian clocks modulate timing of sleep/wake cycles in animals; however, the underlying mechanisms remain poorly understood. In Drosophila melanogaster, large ventral lateral neurons (l-LNv) are known to promote wakefulness through the action of the neuropeptide pigment dispersing factor (PDF), but the downstream targets of PDF signalling remain elusive. In a screen using downregulation or overexpression (OEX) of the gene encoding PDF receptor (pdfr), this study found that a subset of dopaminergic neurons responds to PDF to promote wakefulness during the day. Moreover, this study found that small LNv (sLNv) and dopaminergic neurons form synaptic contacts, and PDFR signalling inhibited dopaminergic neurons specifically during day time. It is proposed that these dopaminergic neurons that respond to PDFR signalling are sleep-promoting and that during the day when PDF levels are high, they are inhibited, thereby promoting wakefulness. Thus, this study has identified a novel circadian clock pathway that mediates wake promotion specifically during day time (Potdar, 2018b).

    Daily cycles in several environmental factors synchronize endogenous circadian clocks which drive rhythmic sleep/wake patterns in many organisms. Homeostatic mechanisms modulate the amount and depth of sleep, and also allow animals to recover from any sleep deprivation they may have incurred. Together, these processes control the timing and occurrence of sleep and wake states, thereby modulating sleep/wake cycles. Since the discovery that sleep behavior of Drosophila melanogaster is similar to mammalian sleep in several aspects, many pathways and neuronal circuits involving sleep homeostat and circadian clocks have been uncovered. Genes such as minisleep (mns) and hyperkinetic (hk) encoding subunits of Shaker potassium channel function in the sleep homeostat. More recently, central complex structures such as dorsal fan-shaped body (FB) and the ellipsoid body (EB) have been shown to function as effector and modulator of the sleep homeostat, respectively. Meanwhile, mutations in core circadian clock genes such as Clock (clk) and Cycle (cyc) have been shown to cause impaired timing of sleep as they tend to become nocturnal. The circadian neuropeptide pigment dispersing factor (PDF) and its receptor (PDFR) are involved in relaying wake-promoting signals from the circadian pacemaker ventral lateral neurons (LNvs) in response to light input as well as dopamine. While it has been suggested that the EB may be the downstream target of this wake-promoting PDF/PDFR signaling, the evidence in favor of the same is limited (Potdar, 2018b).

    In the recent past, in the quest to uncover output pathways of the circadian clocks that help in timing of sleep/wake cycles, a few dedicated circuits have been mapped. Most notably, timing of sleep onset at the beginning of night is a function of increased inhibition of wake-promoting large LNv (l-LNv) by GABA. In contrast, sleep is suppressed at the end of night by the action of PDF on the PDFR+ dorsal neuron 1 (DN1) group of the circadian network that in turn secretes the wake-promoting neuropeptide diuretic hormone 31 (DH31). Furthermore, yet another group showed that DN1s through glutamate modulate day-time siesta and night-time sleep by inhibiting the morning (small LNv; s-LNv) and evening (dorsal lateral neurons; LNds) activity controlling circadian neurons. Yet, none of the studies so far have shed light on how circadian neurons may induce wakefulness during the day (Potdar, 2018b).

    This question was addressed by screening for putative downstream targets of PDFR signaling by altering the levels of pdfr expression in several subsets of neurons - namely, circadian neurons that are known to express pdfr, subsets of mushroom body (MB) neurons that are sleep- or wake-promoting, wake-promoting pars intercerebralis (PI), sleep homeostat EB, and sleep-promoting FB neurons as well as aminergic neuronal groups, most of which are reported to be wake-promoting. Strikingly, this study found that a subset of dopaminergic neurons responds to changes in pdfr expression by changing the levels of day-time sleep, increasing pdfr levels decreases day-time sleep and vice versa. Moreover, PDF+ and dopaminergic neurons were found to form synaptic contacts with one another, along with the possibility of the former inhibiting the latter. Thus, these results uncover a dedicated pathway involving signaling from the PDF+ neurons perhaps to the PPM3 dopaminergic neurons in the regulation of wakefulness during the day (Potdar, 2018b).

    Dopamine is primarily involved in promoting wakefulness and is known to act on l-LNv as well as inhibit sleep-promoting dFB to carry out its wake-promoting function. This study has revealed that certain dopamine neurons are in fact sleep-promoting and through the inhibitory action of PDFR signaling, wakefulness gets promoted specifically during the day. additional experiments that use optogenetic techniques can shed more light on whether these dopaminergic neurons promote sleep directly, or indirectly by preventing wakefulness either through a gating mechanism or by a permissive role. Interestingly, a previous study has found that dopamine acts on l-LNv to promote wakefulness and this study found that PDFR signaling acts on dopamine neurons, suggesting a feed-forward pathway for wake promotion, where dopamine acting on l-LNv promotes the inhibition of sleep-promoting dopaminergic neurons by PDFR signaling. The identity of dopamine neurons acting on l-LNv and those responding to PDFR signaling may differ which can be uncovered with additional experiments (Potdar, 2018b).

    The role of s-LNv in modulating sleep and wake has been explored in some detail in the recent years. s-LNv have also been shown to promote sleep via short NPF (sNPF) as well as myoinhibitory peptide (MiP) by inhibiting the wake-promoting l-LNv. This study shows that PDF+ s-LNv make synaptic contacts with dopaminergic neurons and that PDFR signaling inhibits the downstream dopaminergic neurons to promote wakefulness during the day. Moreover, this study has shown a secondary role for s-LNv in modulating wake-promoting effects of l-LNv. Yet, how this wake-promoting signal which originates in the l-LNv gets relayed to the s-LNv is not understood. Furthermore, from the screen it is clear that this function is not mediated via PDFR signaling among the LNv, as downregulating and overexpressing pdfr in s-LNv (Clk 9M GAL4 and Pdf GAL4) do not result in any sleep defects. Thus, l-LNv to s-LNv wake-promoting signal is independent of PDF while s-LNv to dopamine wake-promoting signal requires PDFR signaling (Potdar, 2018b).

    PDFR being a class B1 GPCR utilizes cAMP as its second messenger, although there is evidence for Ca2+ also acting as the second messenger. For most of the functions of PDF including stabilizing core clock proteins such as TIMELESS and PERIOD in different target neurons such as DN1s and s-LNv, cAMP is the major secondary messenger. Moreover, it is thought that different actions of PDF of slowing and speeding up of morning and evening clock neurons is also mediated by different components of cAMP signaling mechanism. However, this study shows that for the function of regulating wake levels during the day time, PDFR signaling changes levels of intracellular Ca2+ in dopamine neurons with negligible role for cAMP signaling, suggesting a mechanism by which a neuropeptide that has diverse effects on its downstream targets can modulate different functions independently. This study therefore identified a unique subset of downstream targets for PDFR signaling among the dopamine neurons that promote wakefulness depending on time of day (Potdar, 2018b).

    Interestingly, in this screen it is noted that there are several driver lines which there are significant changes in day-time sleep but with only one type of manipulation of pdfr levels (Clk 4.1M, 30y, 104y, 121y GAL4). This may be due to ineffective downregulation of pdfr achieved through the Pdfr RNAi line with these particular drivers. Given that PDF is a neuropeptide which can have long-range non-synaptic effects, even misexpressing it (104y and 121y GAL4) in different substrates has resulted in altered day-time sleep levels. Because DH31 can also respond to PDFR, it is possible that these effects could be mediated by DH31 binding to misexpressed PDFR. However, this may not be the case as downregulating DH31-receptor in these regions does not cause changes in sleep levels. Thus, it can be concluded that in regions previously not known to express pdfr, misexpression of pdfr can cause sleep level deficits suggesting that PDF can act in regions which are not direct targets yet may lie in the vicinity of LNv projections (Potdar, 2018b).

    The role of PDF/PDFR signaling is well-known in synchronizing the free-running molecular rhythms in neurons across the circadian network. PDFR signaling in the 'evening' neurons (LNd and 5th s-LNv) is important for appropriate phasing of the evening bout of activity in light/dark cycles. While the role of PDF as a wake-signal has been known, this study demonstrates that a subset of dopaminergic neurons is downstream of the PDF/PDFR signaling. While the PDFR expression is not conclusive, it is shown that perhaps one PPM3 neuron per hemisphere may express the PDFR. Additional experiments that more directly test the functional connectivity between dopaminergic neurons and PDF+ neurons, as well as responsiveness of dopaminergic neurons to PDF may result in a clearer picture. Downregulating pdfr in these neurons results in increase of day-time sleep, which is a phenocopy of the sleep behavior of loss-of-function pdfr whole-body mutants. On the other hand, overexpressing pdfr in these neurons leads to decrease of day-time sleep specifically. It was further shown that PDF and dopaminergic neurons make synaptic contacts with each other at the site of the axonal projection of s-LNv. Moreover, the effect of PDFR signaling on the PPM3 neurons appears to be inhibitory, suggesting that the PDFR+ PPM3 neurons promote sleep. Taken together, it is concluded that wake-promoting LNv make synaptic connections with sleep-promoting dopaminergic neurons and promote wakefulness specifically during the day time through inhibitory PDFR signaling (Potdar, 2018b).

    Regulation of PDF receptor signaling controlling daily locomotor rhythms in Drosophila

    Each day and in conjunction with ambient daylight conditions, neuropeptide PDF regulates the phase and amplitude of locomotor activity rhythms in Drosophila through its receptor, PDFR, a Family B G protein-coupled receptor (GPCR). The in vivo process was studied by which PDFR signaling turns off, by converting as many as half of the 28 potential sites of phosphorylation in its C terminal tail to a non-phosphorylatable residue (alanine). Many such sites are conserved evolutionarily, and their conversion creates a specific behavioral syndrome opposite to loss-of-function phenotypes previously described for pdfr. That syndrome includes increases in the amplitudes of both Morning and Evening behavioral peaks, as well as multi-hour delays of the Evening phase. The precise behavioral effects were dependent on day-length, and most effects mapped to conversion of only a few, specific serine residues near the very end of the protein and specific to its A isoform. Behavioral phase delays of the Evening activity under entraining conditions predicted the phase of activity cycles under constant darkness. The behavioral phenotypes produced by the most severe PDFR variant were ligand-dependent in vivo, and not a consequence of changes to their pharmacological properties, nor of changes in their surface expression, as measured in vitro. The mechanisms underlying termination of PDFR signaling are complex, subject to regulation that is modified by season, and central to a better understanding of the peptidergic modulation of behavior (Li, 2022).

    In Drosophila, neuropeptide PDF signaling helps pattern the output of the fly circadian pacemaker network that controls rhythmic daily locomotor activity. Its functions have been compared to those of neuropeptide VIP in regulating mammalian circadian physiology. Historically PDF was first isolated as an active principle (Pigment Dispersing Hormone) that mediates light-dependent dispersion of pigment granules in diverse chromatophores of crustacea. In the insect circadian system, PDF acts for a specified period within each 24 hr cycle, and works in conjunction with environmental light to set phase and amplitude for locomotor activity rhythms that normally occur around dawn and dusk. Each day, the precise times of dusk and of dawn change, which alters the time interval between them. These facts require that the time of PDF signaling, the point when it starts and the point when it stops, must also be adjusted each day to appropriately follow and reflect these daily variations in the light: dark transitions. PDF signaling starts following its release by specific pacemaker neurons, whose period of activity in vivo tracks the dawn in a variety of photoperiodic conditions. A comparable understanding of how PDF receptor signaling normally stops is lacking: this work addresses that mechanism (Li, 2022).

    The PDF receptor (PDFR) is a member of the Family B (secretin receptor-like) GPCR group: it is Gs-coupled and its activation elevates cAMP levels in vivo. It regulates different adenylate cyclases (AC) in diverse target pacemakers (Duval, 2012; Duval, 2013), which, through PKA activation, ultimately regulate the pace of the molecular clock through regulation of Timeless (Seluzicki, 2014). PDFR autoreceptor signaling promotes dramatic, daily morphological changes in the axonal terminals of sLNv pacemakers. In addition to its effects on the pace of the molecular pacemaker, PDFR activation also regulates calcium dynamics in subsets of pacemaker neuron groups to help dictate their group-specific, daily phases of activation (in the sLNv, in the 5th sLNv and in subsets of LNd, and DN3 groups). Such target cell-specific delays of PER-dependent neuronal activity illustrate the basis by which the circadian network produces a daily series of staggered phasic, neuronal outputs. Finally, PDF/PDFR signaling is long-lasting: its depression of basal calcium levels in target neurons persists without abatement over many hours. These observations raise fundamental questions regarding the mechanism and the time course by which PDFR signaling diminishes in anticipation of the next day's cycle of signaling (Li, 2022).

    The canonical model of GPCR phosphorylation and homologous desensitization features G protein-coupled receptor kinases (GRKs) which associate with activated GPCRs and phosphorylate cytosolic segments, thereby recruiting βarrestin via its β-arrestins uncouple the receptors from G proteins. or enhance receptor endocytosis; they can also serve as signal transducers by recruiting distinct signaling molecules. A second major regulatory mechanism to reduce GPCR signaling is heterologous desensitization, whereby second-messenger-dependent kinases (PKA or PKC) phosphorylate GPCRs. Thus, experiments were to modify evolutionarily-conserved residues in the C terminal tail of PDFR that could conceivably serve as substrates for phosphorylation and subsequent signal termination. The working hypothesis was that, by their actions in vivo, such modified PDF receptors would reveal extended lifetimes of activation (Li, 2022).

    Little is known about the mechanisms that underlie normal termination of PDFR signaling. In the small LNv, pdfr levels are higher late in the day than in early morning. Sensitivity to PDF in vivo peaks in the early day and is regulated by the PER-dependent clock, via post-transcriptional mechanisms: the EC50 for PDF responses in identified neurons varies systematically 5-10 fold: as a consequence of RalA action, as a function of time of day, and as a function of seasonality. PDFR signaling is long-lasting: it persists for many hours in vivo, for as long as free peptide ligand is available in the bath. In addition, β-arrestin2-GFP is not efficiently recruited to activated PDFR when the receptor is functionally-expressed in hEK-293T cells. In contrast, each of 13 other Drosophila neuropeptide GPCRs (including two Family B GPCRs, CG8422 and CG17415) efficiently recruit β-arrestin2-GFP, when they are activated by their cognate ligands in that cellular environment (Li, 2022).

    This study reports (1) that PDFR is normally phosphorylated in vivo at conserved C terminal residues; (2) that loss of conserved PDFR phosphorylatable sites leads to a behavioral syndrome opposite to loss-of-function pdf and pdfr phenotypes, with effects on both the amplitude and phase of the daily locomotor peaks. This 'gain of function' approach reveals a multi-hour range of potential phases for both the Morning and Evening activity peaks, within which neuropeptide PDF:PDFR signaling normally specifies rhythmic behavior, according to season. In addition, using a structure-function approach, this work identifies specific PDFR sequence elements that are major points at which the duration of receptor activity is regulated, and through which behavior is modulated in season-specific fashion (Li, 2022).

    A gain-of-function approach was used to measure neuropeptide GPCR function in vivo. In doing so, attempts were made to learn about the maximal consequences of PDFR GPCR signaling and also learn something about the phosphorylation events that normally control the duration of PDFR's signaling lifetime. Expression of a PDF receptor variant, containing numerous Ala-substitutions of conserved phosphorylatable residues in the C terminal domain (1-7A), fundamentally altered rhythmic locomotor behavior in Drosophila. The changes included increases in the amplitudes of the Morning and/or Evening activity peaks, and delays in the Phase of the Evening peak. Such results are generally consistent with a prediction whereby non-phosphorylatable PDFR variants-those with a potential to increase the duration of PDFR signaling-would produce behavioral actions opposite to those seen in loss-of-function pdf, or pdfr mutant stocks. Accordingly, it is speculated that phosphorylation of the PDFR at these conserved residues normally defines the duration of its active signaling state each day. The longer duration that follows the substitutions of Ala may allow receptor signal strength to also increase by accrual: such an effect could explain the increased amplitude of the Morning and Evening peaks, as seen with expression of some of the variants. In this context, it is acknowledged that modification of GPCRs by phosphorylation does not exclusively lead to signal termination. GRK2-dependent phosphorylation of the smoothened (smo) GPCR follows reception of the Hh signal and helps mediate its signal: phosphorylation leads to Smo activation in both Drosophila and mammalian systems. Thus modification of putative phosphorylation sites on GPCRs (to preclude phosphorylation) will not exclusively promote extended signaling. Therefore the interpretations must correspondingly consider outcomes without a priori assumption of mechanisms (Li, 2022).

    PDFR belongs to the Secretin Receptor Family (Family B) of neuropeptide receptors: there is no clear consensus regarding mechanisms of desensitization and internalization for this receptor family. For VPAC2 receptors, phosphorylation and internalization is mediated exclusively by GRK2. Likewise, Drosophila orthologues of Cortocotrophin Releasing Factor receptors (CG8422, DH44-R1) and Calcitonin receptors (CG17415, DH31-R1) are internalized in hEK cells following recruitment of β-arrestin2. In contrast, PDFR, which is also related to the mammalian Calcitonin receptor, is not internalized following exposure to PDF. VPAC2 receptors are also regulated by heterologous receptor signaling: M3 cholinergic receptors via PKC signaling can block VPAC2 phosphorylation, desensitization and internalization. Furthermore, secretin receptors and VPAC1 receptors undergo phosphorylation by GRKs and β-arrestin2-dependent desensitization, but these are not sufficient to facilitate or mediate internalization. Finally, GLP2-Receptor associates with β-arrestin2 via its distal C terminal sequences, but that receptor domain is required neither for GLP2-R desensitization nor its internalization. Thus kinases other than GRKs and effectors other than β-arrestin2 may regulate internalization of diverse Family B receptors (Li, 2022).

    Following activation of rhodopsin in the mammalian retina, visual arrestin is recruited with a time constant of < 80 ms; for many Drosophila neuropeptide receptors, β-arrestin2 is recruited within a minute of exposure to ligand. The PDFR GPCR signals over a time base of many hours: could phosphorylation/de-phosphorylation also regulate its activity? The mammalian blue-light sensitive GPCR melanopsin (OPN4) mediates intrinsic light sensitivity in certain classes of retinal ganglion cells (RGCs): melanopsin signaling is distinguished by long latencies, graded responses and sustained RGC depolarization that can outlast the duration of the light stimulus. Melanopsin-expressing RGCs produce exceptionally long time-course integration because that GPCR behaves differently from other opsins in two fundamental ways. Firstly, it undergoes photoequilibration between signaling and silent states: a property that maintains the availability of pigment molecules for activation, termed 'tristability'. Secondly, phosphorylation and β-arrestin recruitment do contribute to the kinetics of melanopsin signaling, but over a long time-course. It has been proposed that the distal portion of the melanopsin C terminal tail creates a steric blockade covering a cluster of specific serine residues situated in more proximal regions. The time required for relief of that blockade dictates the time course of GPCR phosphorylation and therefore delays subsequent desensitization (Li, 2022).

    The 1-7A PDFR variant modified the greatest number of phosphorylatable residues in the series that was tested, and typically produced the strongest behavioral effects. Notably, the 6A variant-which modified only two specific Ser residues-produced effects that were nearly identical to those exhibited by flies expressing the 1-7A form. This suggests that much PDFR post-translational modification, that which is capable of restricting its signaling time-course, may be directed to specific phosphorylation sites near the extreme C terminal end. There are two broadly divergent hypotheses to describe the mechanisms by which GPCR phosphorylation promotes desensitization. The first proposes that modification of specific residues have the greatest significance for downstream desensitizing mechanisms: such a mechanism can explain the effects seen with the highly limited 6A PDFR variant. The second hypothesis invokes triggering of a termination processes by the aggregate negative charge accumulated with bulk phosphorylation, regardless of where it might occur along a GPCR's intracellular sequences. For some GPCRs (e.g., OPN4), the evidence supports both models. The current data concerning PDFR desensitization also support both viewpoints and a model is presented (see A model predicting the effects of phosphorylating different sites on the PDFR CT on downstream signaling.). In particular CL6 and CL7 are considered to be the specific residues especially critical in terminating the time course of PDFR signaling. Notably, converting just the single pair of CL6 AAs, or the single pair of CL7 AAs, is enough to generate hours-long delays in the peaks of locomotor rhythms under Light:Dark conditions. It is noted that the splicing event that distinguishes the PDFR-A and PDFR-D isoforms occurs just prior to the position of sequences encoding CL6 and 7, such that the D form lacks these two highly conserved domains ( Other clusters (like CL2-3) also appear to have potential for 'specific' contributions to PDFR regulation. Together, these results speak to the potency of the inferred specific termination mechanisms for PDFR GPCR signaling. In contrast, the evidence for the bulk phosphorylation hypothesis comes from comparison of the 1-4A, versus the 1-5A, 1-6A and 1-7A variants. This series inactivates increasing numbers of phosphorylatable residues, and with it increasingly delayed evening phases in short day conditions are observed. The tandem mass-spectroscopy results, indicating endogenous phosphorylation of certain PDFR residues demonstrates such post-translational modifications can occur. However, it is cautioned that these phospho-peptide measurements derive from whole head extracts and may not provide a complete or relevant accounting of those sites that are modified in critical E pacemaker neurons (Li, 2022).

    The results also point to what is proposed as 'context-dependent' effects of modifying specific GPCR residues-ones that may produce opposing behavioral effects, depending on the phosphorylation status of neighboring sites. In particular, opposing results were observed with Ala-variants of the CL5 site. In comparing results from the series 1-4A, 1-5A, 1-6A and 1-7A, it was found that under Short Day conditions, the 1-4A had only mild effects on the evening peak phase compared to over-expressing WT PDFR. Whereas 1-5A, 1-6A and 1-7A all produced significant delays. The difference between the 1-$A and 1-5A variants is a change in a single S residue at CL5 (S633A): Those observations suggest modification of the CL5 residue normally promotes desensitization of PDFR and termination of PDFR signaling. However, the PDFR variant 5-7A was constructed with the expectation that it would be as effective as 1-5A, 1-6A or 1-7A in delaying the evening phase. Instead it proved only weakly effective. This same mis-match of expected behavioral effects for 5-7A was also seen in other photoperiodic conditions. Likewise, Langlet (2005) reported that effects on mutating Serine residues in the terminus of VPAC1 did not produce additive effects. It is proposed that phosphorylation of CL5 will have either positive or negative consequences on PDFR desensitization depending on which other neighboring residues are also modified. Thus it is speculated that CL5 takes on outsize importance in determining desensitization rates for PDFR and so may itself be subject to exceptional regulation. Such complex interactions between phosphorylation sites is reminiscent of interactions documented between diverse phosphorylation sites in the circadian clock protein PERIOD. Better resolution of these two paradoxical mechanisms [(1) specific phosphorylation versus bulk negative charge, and (2) context-dependent effects of CL5 site phosphorylation] awaits precise molecular definition of where and when PDFR is modified in key pacemaker neurons in vivo, and by which post-translational modifications (Li, 2022).

    The actions of PDFR variants that were described, following substitutions of Ala for various Ser/Thr or Tyr residues, are not easily explained by a hypothesis invoking neomorphic or constitutive GPCR properties. The evidence for this conclusion is three-fold. First, the effects on both amplitude and phase of activity peaks by these variants are not of a random assortment: rather they are all strictly opposite to that of loss-of-function models for pdf and pdfr. Second, the actions of PDFR variants display strong dependence on wild type pdf gene function: this strongly argues that the actions of PDFR variants reflect responses to normal, endogenous PDF signaling. Third, even the strongest actions of PDFR variants on the phases and amplitudes of locomotor activity (that of the 1-7A variant) reflected PDFR sequence variation, and not the properties of the GFP C-terminal fusion. Together these observations support the hypothesis that phosphorylation of some or all of the C terminal residues that were studied are normally modified to attenuate the strength and duration of PDFR signaling during the 24-hr day (Li, 2022).

    PDF neuropeptide actions were discovered in the context of physiological adaptions to daylight in crustacea. PDF expression was discovered within a defined subset of the insect Drosophila circadian pacemaker network. Pdf has been shown to make a fundamental contribution by setting the normal behavioral phase of Evening activity in Drosophila during light:dark entrainment and promoting normal rhythmicity during constant darkness. PDF signaling coordinates with that driven by light: It works in parallel to direct photosensitivity along with the CRY blue light photoreceptor. Flies doubly-mutant for cry and pdf display pronounced deficits of locomotor rhythmicity, and can be as severe as measured in clock-deficient flies. PDF and environmental light also work coordinately at the level of neuronal activity patterns: in the case of the evening pacemakers (LNd and the 5th s-LNv), both light and PDF signaling promote a delay in their PER-dependent activation period, and together help align it to a phase just prior to dusk. Based on the close association of PDF signaling and photoperiodic signaling, it is not surprising that the duration of the photoperiod strongly influenced the effects of PDFR sequence variants on the Morning versus the Evening activity peaks. Delayed Evening activity peak produced by PDFR gain-of-function variants under Short Day conditions has been reported: that delay appears strongly inhibited by light durations > 8 hr. It remains unclear at what molecular level light exerts such an inhibitory effect, however, a similarity is noted of these observations with effects recently reported from loss-of-function states for the phosphatase PRL-1. Like the most active PDFR variants this study described (e.g., 6A, 7A, 1-5A 1-6A and 1-7A), PRL-1 mutants produce a 3-4 h delay in the phase of the evening activity peak, but only under winter-like (short day) conditions, and not under equinox or summer-like (long day) conditions. It has been shown that TIM phosphorylation is affected by PRL-1 activity and suggest the seasonal action of PRL-1 to advance the evening locomotor activity phase is mediated by modification of TIM levels within the transcription-translation feedback loop. The extensive similarity of these two sets of behavioral phenotypes reveals either serial or parallel pathways to effect comparable outcomes on locomotor activity. If serial, then according to the simplest model, PRL1 acts downstream of PDFR, and the combined results to-date suggest PRL-1 would be inhibited by PDFR activation. Further genetic and biochemical experiments are needed to evaluate if and how these pathways converge (Li, 2022).

    Using an in vitro assay for cyclic AMP generation, this study found that modifying phosphorylation properties of PDFR does not affect the strength of signaling. That conclusion suggests that in vivo other GPCR features are normally affected to regulate the extent/duration of PDFR signaling according to season. This speculation corresponds to findings described with other Family B GPCRs like VPAC1: Mutation of all the Ser and Thr residues of the C terminal tail, and of Ser250 to Ala, led to a receptor with binding properties and adenylate cyclase activity similar to the wild type receptor; however that variant receptor was neither phosphorylated nor internalized. It is proposed that the PDFR variants this study has described do not modify locomotor behavior by virtue of greater second messenger signaling. Rather they do so by generating cAMP (and perhaps other second messengers) over time periods longer than that normal sustained by a WT receptor over a portion of each 24-hr cycle (Li, 2022).

    Is PDFR modulation of diurnal phase independent of its modulation of circadian period? A conventional interpretation of the phenotypes that have been described is that PDFR signaling affects the pace of the molecular clock (re-setting the period) and consequently affects behavioral phase indirectly, which is downstream of the clock. However, PDFR variants reliably delayed the Evening activity phase under winter-like conditions, but these variants did not significantly lengthen circadian period. All effects of the 1-7A variant on circadian period were derived from the GFP fusion. Therefore, an alternative hypothesis cannot be ruled out: that the duration of PDF>PDFR signaling to target pacemakers in the Drosophila brain is modulated each day to directly delay Evening pacemaker neuronal activity independent of its effects on entrainment of the molecular clock. In fact, the pdfr han mutant flies exhibited quasi-normal tau values in this report, although they more traditionally exhibit shortened ones. Evening locomotor activity derives in large part from the activity of the primary Evening oscillators, termed E cells, the LNd and the 5th small LNv. However, numerous network interactions are also known to provide critical contributions to the accuracy, precision and adaptability of that timing system. The Evening oscillator group itself is known to be heterogeneous and to constitute at least three separate, functionally distinct oscillators, only some of which express PDFR-A and respond to PDF. It has been reported that the phase of Period protein accumulation in E neurons is phase-locked to the previous lights-off transition and also sensitive to PDF signaling. It was concluded that the peak of PER accumulation is key to determining the phase of the evening activity peak, but also note that the correlation between the Period-clock timing cue and the Evening activity peak is not perfect. It has been suggested the Evening behavioral phase is better described by also factoring in the delay in E cell neuronal activity driven by PDF neuromodulation. It is speculated that it is the combination of two separate PDFR signaling effects that is essential for proper rhythmic behavioral outcomes across seasons. If valid, the hypothesis predicts a bifurcation of signaling pathways downstream of PDFR, to independently regulate diurnal phase and also circadian period within E pacemaker neurons (Li, 2022).

    A sleep state in Drosophila larvae required for neural stem cell proliferation

    Sleep during development is involved in refining brain circuitry, but a role for sleep in the earliest periods of nervous system elaboration, when neurons are first being born, has not been explored. This study has identified a sleep state in Drosophila larvae that coincides with a major wave of neurogenesis. Mechanisms controlling larval sleep are partially distinct from adult sleep: octopamine, the Drosophila analog of mammalian norepinephrine, is the major arousal neuromodulator in larvae, but dopamine is not required. Using real-time behavioral monitoring in a closed-loop sleep deprivation system, sleep loss in larvae was found to impair cell division of neural progenitors. This work establishes a system uniquely suited for studying sleep during nascent periods, and demonstrates that sleep in early life regulates neural stem cell proliferation (Szuperak, 2018).

    Serine metabolism in the brain regulates starvation-induced sleep suppression in Drosophila melanogaster

    Sleep and metabolism are physiologically and behaviorally intertwined; however, the molecular basis for their interaction remains poorly understood. This study identified a serine metabolic pathway as a key mediator for starvation-induced sleep suppression. Transcriptome analyses revealed that enzymes involved in serine biosynthesis were induced upon starvation in Drosophila melanogaster brains. Genetic mutants of astray (aay), a fly homolog of the rate-limiting phosphoserine phosphatase in serine biosynthesis, displayed reduced starvation-induced sleep suppression. In contrast, a hypomorphic mutation in a serine/threonine-metabolizing enzyme, serine/threonine dehydratase (stdh), exaggerated starvation-induced sleep suppression. Analyses of double mutants indicated that aay and stdh act on the same genetic pathway to titrate serine levels in the head as well as to adjust starvation-induced sleep behaviors. RNA interference-mediated depletion of aay expression in neurons, using cholinergic Gal4 drivers, phenocopied aay mutants, while a nicotinic acetylcholine receptor antagonist selectively rescued the exaggerated starvation-induced sleep suppression in stdh mutants. Taken together, these data demonstrate that neural serine metabolism controls sleep during starvation, possibly via cholinergic signaling. It is proposed that animals have evolved a sleep-regulatory mechanism that reprograms amino acid metabolism for adaptive sleep behaviors in response to metabolic needs (Sonn, 2018).

    High-salt diet causes sleep fragmentation in young Drosophila through circadian rhythm and dopaminergic Systems

    Salt (sodium chloride) is an essential dietary requirement, but excessive consumption has long-term adverse consequences. A high-salt diet (HSD) increases the risk of chronic diseases such as cardiovascular conditions and diabetes and is also associated with poor sleep quality. Little is known, however, about the neural circuit mechanisms that mediate HSD-induced sleep changes. This study sought to identify the effects of HSD on the sleep and related neural circuit mechanisms of Drosophila. Strikingly, it was found that HSD causes young Drosophila to exhibit a fragmented sleep phenotype similar to that of normal aging individuals. Importantly, it was further shown that HSD slightly impairs circadian rhythms and that the HSD-induced sleep changes are dependent on the circadian rhythm system. In addition, it was demonstrated that HSD-induced sleep changes are dopaminergic-system dependent. Together, these results provide insight into how elevated salt in the diet can affect sleep quality (Xie, 2019).

    Balance of activity between LNvs and glutamatergic dorsal clock neurons promotes robust circadian rhythms in Drosophila

    Circadian rhythms offer an excellent opportunity to dissect the neural circuits underlying innate behavior because the genes and neurons involved are relatively well understood. This study sought to understand how Drosophila clock neurons interact in the simple circuit that generates circadian rhythms in larval light avoidance. Genetics was used to manipulate two groups of clock neurons, increasing or reducing excitability, stopping their molecular clocks, and blocking neurotransmitter release and reception. The results revealed that lateral neurons (LNvs) promote and dorsal clock neurons (DN1s) inhibit light avoidance, these neurons probably signal at different times of day, and both signals are required for rhythmic behavior. Similar principles apply in the more complex adult circadian circuit that generates locomotor rhythms. Thus, the changing balance in activity between clock neurons with opposing behavioral effects generates robust circadian behavior and probably helps organisms transition between discrete behavioral states, such as sleep and wakefulness (Collins, 2012).

    This study identified some of the network logic that helps generate a simple rhythmic behavior through precise genetic manipulations of the larval circadian circuit and extended these findings to the more complex adult circadian network. Previous studies have shown that intercellular signaling in clock neuron networks promotes molecular clock synchrony and can strengthen genetically weak molecular clocks. This study increases the importance of circadian neural networks by finding that non-LNv clock neurons are as important as the 'master' pacemaker LNv clock neurons for rhythmic behavior both in larvae and adult flies. However, LNvs can still be considered pacemakers in DD because most manipulations to non-LNv clock neurons do not affect period length (Collins, 2012).

    Non-LNv signals appear to gate pacemaker neuron activity. Why is this necessary when LNvs have their own intrinsic excitability rhythms? It is proposed that the interaction of two oscillators with opposite signs helps reduce the time when LNvs signal. Without signaling from non-LNvs, adult locomotor activity rhythms are weak and activity is distributed throughout the day and night as in tim-Gal4; Pdf-Gal80 > dORKΔC flies. In contrast, in tim-Gal4; Pdf-Gal80 > NaChBac flies, the timing of locomotor activity is narrowed. Thus, the gating of LNv activity by non-LNvs may help turn gradual changes in the excitability of each neuronal group into thresholds that promote a switch in overall output and allow flies to abruptly transition from inactivity to activity (Collins, 2012).

    This gating system can only function if LNvs and non-LNvs have differently phased neuronal activity. However, most Drosophila clock neurons have similarly phased molecular clocks. It is proposed that molecular clocks in different clock neurons regulate divergent sets of output genes to generate distinct phases of neuronal excitability. This would be analogous to the mammalian circadian system, in which molecular clocks in different tissues drive tissue-specific outputs. In summary, this genetic dissection of a circadian neural circuit reveals an unexpected and essential role for inhibitory signals from non-LNvs (E cells) in shaping activity profiles at dawn and a mechanism for how clock neurons couple together to promote robust rhythms (Collins, 2012).

    Dorsal clock neurons in Drosophila sculpt locomotor outputs but are dispensable for circadian activity rhythms

    The circadian system is comprised three components: a network of core clock cells in the brain that keeps time, input pathways that entrain clock cells to the environment, and output pathways that use this information to ensure appropriate timing of physiological and behavioral processes throughout the day. Core clock cells can be divided into molecularly distinct populations that likely make unique functional contributions. This study clarifies the role of the dorsal neuron 1 (DN1) population of clock neurons in the transmission of circadian information by the Drosophila core clock network. Using an intersectional genetic approach that allowed selective and comprehensive targeting of DN1 cells, this study shows the suppressing DN1 neuronal activity alters the magnitude of daily activity and sleep without affecting overt rhythmicity. This suggests that DN1 cells are dispensable for both the generation of circadian information and the propagation of this information across output circuits (Nettnin, 2021).

    Dorsal clock networks drive temperature preference rhythms in Drosophila

    Animals display a body body temperature rhythm (BTR). Little is known about the mechanisms by which a rhythmic pattern of BTR is regulated and how body temperature is set at different times of the day. As small ectotherms, Drosophila exhibit a daily temperature preference rhythm (TPR), which generates BTR. This study demonstrates dorsal clock networks that play essential roles in TPR. Dorsal neurons 2 (DN2s) are the main clock for TPR. It was found that DN2s and posterior DN1s (DN1ps) contact and the extent of contacts increases during the day and that the silencing of DN2s or DN1ps leads to a lower temperature preference. The data suggest that temporal control of the microcircuit from DN2s to DN1ps contributes to TPR regulation. This study also identified anterior DN1s (DN1as) as another important clock for TPR. Thus, this study shows that the DN networks predominantly control TPR and determine both a rhythmic pattern and preferred temperatures (Chen, 2022).

    Dorsal-lateral clock neurons modulate consolidation and maintenance of long-term memory in Drosophila

    A newly formed memory is initially unstable. However, if it is consolidated into the brain, the consolidated memory is stored as stable long-term memory (LTM). Despite the recent progress, the molecular and cellular mechanisms of LTM have not yet been fully elucidated. The fruitfly Drosophila melanogaster, for which various genetic tools are available, has been used to clarify the molecular mechanisms of LTM. Using the Drosophila courtship-conditioning assay as a memory paradigm, it was previously identified that the circadian clock gene period (per) plays a vital role in consolidating LTM, suggesting that per-expressing clock neurons are critically involved in LTM. However, it is still incompletely understood which clock neurons are essential for LTM. This study shows that dorsal-lateral clock neurons (LNds) play a crucial role in LTM. Using an LNd-specific split-GAL4 line, it wa confirmed that disruption of synaptic transmission in LNds impaired LTM maintenance. On the other hand, induction of per RNAi or the dominant-negative transgene of Per in LNds impaired LTM consolidation. These results reveal that transmitter release and Per function in LNds are involved in courtship memory processing (Suzuki, 2022).

    The lateral posterior clock neurons of Drosophila melanogaster express three neuropeptides and have multiple connections within the circadian clock network and beyond

    Drosophila's lateral posterior neurons (LPNs) belong to a small group of circadian clock neurons that is so far not characterized in detail. Thanks to a new highly specific split-Gal4 line, this study describes LPNs' morphology in fine detail, their synaptic connections, daily bimodal expression of neuropeptides, and a putative role of this cluster in controlling daily activity and sleep patterns is proposed. The three LPNs were found to be heterogeneous. Two of the neurons with similar morphology arborize in the superior medial and lateral protocerebrum and most likely promote sleep. One unique, possibly wakefulness-promoting, neuron with wider arborizations extends from the superior lateral protocerebrum toward the anterior optic tubercle. Both LPN types exhibit manifold connections with the other circadian clock neurons, especially with those that control the flies' morning and evening activity (M- and E-neurons, respectively). In addition, they form synaptic connections with neurons of the mushroom bodies, the fan-shaped body, and with many additional still unidentified neurons. Both LPN types rhythmically express three neuropeptides, Allostatin A, Allostatin C, and Diuretic Hormone 31 with maxima in the morning and the evening. The three LPN neuropeptides may, furthermore, signal to the insect hormonal center in the pars intercerebralis and contribute to rhythmic modulation of metabolism, feeding, and reproduction. These findings are discussed in the light of anatomical details gained by the recently published hemibrain of a single female fly on the electron microscopic level and of previous functional studies concerning the LPN (Reinhard, 2021).

    Identification of a circadian output circuit for rest:activity rhythms in Drosophila

    Though much is known about the cellular and molecular components of the circadian clock, output pathways that couple clock cells to overt behaviors have not been identified. A screen was conducted for circadian-relevant neurons in the Drosophila brain, and this study reports that cells of the pars intercerebralis (PI), a functional homolog of the mammalian hypothalamus, comprise an important component of the circadian output pathway for rest:activity rhythms. GFP reconstitution across synaptic partners (GRASP) analysis demonstrates that PI cells are connected to the clock through a polysynaptic circuit extending from pacemaker cells to PI neurons. Molecular profiling of relevant PI cells identified the corticotropin-releasing factor (CRF) homolog, DH44, as a circadian output molecule that is specifically expressed by PI neurons and is required for normal rest:activity rhythms. Notably, selective activation or ablation of just six DH44+ PI cells causes arrhythmicity. These findings delineate a circuit through which clock cells can modulate locomotor rhythms (Cavanaugh, 2014).

    Given its location near the axonal projections of several groups of clock neurons and its function in metabolic, locomotor, and sleep processes, the PI has been proposed as a possible component of the output pathway in Drosophila, but direct evidence supporting a contribution to behavioral or physiological rhythms has been lacking. This study used a combined genetic, anatomical, and molecular approach to unequivocally identify specific subsets of PI cells as comprising part of the circadian output circuit for rest:activity rhythms. Ectopic activation of PI neurons is sufficient to induce behavioral arrythmicity, and similarly, ablation of small subsets of PI neurons results in loss of rest:activity rhythms. This latter result is consistent with previous studies showing that surgical destruction of the PI in both crickets and cockroaches results in loss of locomotor rhythms. It was further shown that manipulations of the PI that result in behavioral arrhythmicity do not affect the underlying molecular clock in s-LNvs, thus demonstrating that the PI exerts its effects downstream of clock neurons (Cavanaugh, 2014).

    Importantly, this study has uncovered a segregation of different behavioral and physiological outputs by specific neurons of the PI. Thus, kurs58-GAL4+ PI neurons function to modulate locomotor behavior, whereas insulin-like peptide-producing PI cells, which constitute a nonoverlapping subset, influence metabolic processes. It will be of interest to determine whether Dilp2+ cells are also modulated by the clock, because such a result would suggest that the PI is a common relay for multiple circadian output circuits that couple to unique physiological functions, each subserved by discrete subpopulations of PI neurons. Furthermore, within kurs58-GAL4+ cells, there appear to be at least two subsets of neurons that contribute to rest:activity cycles. Interestingly, ablation of the SIFa-GAL4+ subset results in reduced rhythmicity, accompanied by decreases in sleep, whereas ablation of the DH44VT-GAL4+ subset also results in reduced rhythmicity, but in this case, the effect on sleep, if any, is an increase. Thus, it is possible that these two molecularly distinct populations control behavioral rhythms through opposing effects on locomotion and/or sleep, and thus, that the contribution of a particular subset predominates depending on time of day (Cavanaugh, 2014).

    In conjunction with behavioral studies, GRASP analysis was used to trace neuronal connections emanating from the clock network. It was found that s-LNvs, which function as master pacemakers, make limited connections within the clock cell network and do not appear to directly access output cells of the PI. Instead, PI output cells receive time-of-day information through inputs from DN1 clock cells, as demonstrated by the fact that presynaptic components of DN1 cells adjoin dendrites of PI neurons, in the same brain region where GRASP analysis reveals cellular contacts between these two cell groups. Several studies corroborate a function of DN1 neurons downstream of s-LNvs to mediate rest:activity rhythms. Dorsal neurons are responsive to bath application of PDF, and restoration of the PDF receptor selectively in these neurons of pdfr mutant flies is sufficient to rescue multiple aspects of circadian locomotor rhythms. Furthermore, speeding up the molecular clock in s-LNvs causes concomitant acceleration of molecular cycling in several groups of dorsal neurons, including DN1s. These experiments, along with the current study, argue that DN1 neurons serve an important output function within the clock network and likely reside downstream of s-LNvs in the output circuit for rest:activity rhythms. The data are therefore consistent with a very simple circadian output circuit, in which time-of-day information from the clock network, which is generated by master pacemaker cells (s-LNvs and possibly LNds), passes through dorsal clock neurons (including DN1s) before accessing downstream output neurons of the PI, which then integrate these signals to modulate locomotor rhythms. Whether the PI also lies downstream of other groups of dorsal clock neurons, in addition to DN1s, or whether all time-of-day signals received by the PI pass through DN1 cells remains to be determined (Cavanaugh, 2014).

    Within the brain, projections from the PI primarily terminate in the dorsal tritocerebrum; however, more diffuse termination patterns throughout the central brain and optic lobes have been observed for SIFa+ PI neurons. The PI also accesses neurohemal organs via the esophageal canal, as well as directly releasing peptides into the hemolymph. Thus, signals released from the PI could either act within neuronal tissue or systemically via release of peptide neurotransmitters and other hormones. The latter possibility is consistent with studies that showed that transplantation of pers brains into the abdomen of per mutant flies rescued locomotor rhythms, demonstrating that release of a secreted factor underlies brain control of rest:activity rhythms in flies. Similarly, abdominal transplantation of PI cells is sufficient to alter sexually dimorphic locomotor patterns, indicating that the PI can modulate locomotor behavior in a neuroendocrine manner (Cavanaugh, 2014).

    Through single-cell transcriptome analysis, the CRF-like peptide, DH44, was identified as a candidate molecule through which PI neurons might influence locomotor behavior. Consistent with this possibility, RNAi-mediated knockdown, or genetic antagonism, of DH44 resulted in altered locomotor behavior and weakened rest:activity rhythms. In addition, selective activation or destruction of DH44+ PI neurons also substantially weakened rest:activity rhythms. In flies, DH44 acts as a diuretic hormone, which stimulates fluid secretion from Malpighian tubules through a cyclic AMP (cAMP) pathway. Its role as a stress molecule is less clear, but DH44 receptor has also been localized to corazonin+ cells of the lateral protocerebrum, which may be involved in the stress response of the fly. Notably, manipulations of neuronal excitability in corazonin+ cells alter stress-induced locomotor activity. In mammals, stress hormones, such as glucocorticoids, show diurnal cycles of secretion and serve as entrainment signals for peripheral clocks. Thus, stress hormones may play a conserved role in circadian regulation of behavioral and physiological processes (Cavanaugh, 2014).

    Light-induced structural and functional plasticity in Drosophila larval visual system

    How to build and maintain a reliable yet flexible circuit is a fundamental question in neurobiology. The nervous system has the capacity for undergoing modifications to adapt to the changing environment while maintaining its stability through compensatory mechanisms, such as synaptic homeostasis. This study describes findings in the Drosophila larval visual system, where the variation of sensory inputs induces substantial structural plasticity in dendritic arbors of the postsynaptic neuron and concomitant changes to its physiological output. Furthermore, a genetic analysis has identified the cyclic adenosine monophosphate (cAMP) pathway and a previously uncharacterized cell surface molecule as critical components in regulating experience-dependent modification of the postsynaptic dendrite morphology in Drosophila (Yuan, 2011).

    Proper functions of neuronal circuits rely on their fidelity, as well as plasticity, in responding to experience or changing environment, including the Hebbian form of plasticity, such as long-term potentiation, and the homeostatic plasticity important for stabilizing the circuit. Activity-dependent modification of neuronal circuits helps to establish and refine the nervous system and provides the cellular correlate for cognitive functions, such as learning and memory. Multiple studies have examined synaptic strength regulation by neuronal activity, whereas to what extent and how the dendritic morphology may be modified by neuronal activity remain open questions (Yuan, 2011).

    Drosophila melanogaster has facilitated genetic studies of nervous system development and remodeling. Notwithstanding the relatively stereotyped circuitry, flies exhibit experience-induced alterations in neuronal structures and behaviors such as learning and memory). In a study of experience-dependent modifications of the Drosophila larval CNS, it has been found that different light exposures dramatically altered dendritic arbors of ventral lateral neurons [LN(v)s], which are postsynaptic to the photoreceptors. Unlike the visual activity-induced dendrite growth in Xenopus optic tectum, extending the light exposure of Drosophila larvae reduced the LN(v)s' dendrite length and functional output, a homeostatic plasticity for compensatory adaptation to alterations in sensory inputs. It was further shown that the cyclic adenosine monophosphate (cAMP) pathway and an immunoglobulin domain-containing cell surface protein, CG3624, are critical for this sensory experience-induced structural plasticity in Drosophila CNS (Yuan, 2011).

    In Drosophila larvae, Bolwig's organ (BO) senses light, and its likely postsynaptic targets are LN(v)s. As the major circadian pacemaker, LN(v)s are important for the entrainment to environmental light-dark cycles and larval light avoidance behavior. In the larval brain, Bolwig's nerve (BN), the axonal projection from BO, terminates in an area overlapping the dendritic field of LN(v)s. Using the FRT-FLP system [in which DNA sequences flanked by flippase recognition targets (FRT) are snipped out by flippase (FLP)] along with three-dimensional (3D) tracing, the dendritic arbor of individual LN(v) neurons were labeled and analyzed. Then potential synaptic connections were demonstrated between BN and LN(v)s using the GRASP [green fluorescent protein (GFP) reconstitution across synaptic partners] technique to drive expression of one-half of the split GFP in the BN by means of Gal4/UAS and expression of the other half of the split GFP in LN(v)s via LexA/LexAop. The proximity of putative synaptic connections between BN and LN(v)s' dendrites reconstituted GFP fluorescence for photoreceptors expressing either rhodopsin 5 (Rh5) or rhodopsin 6 (Rh6) in BO, which suggested that both groups of photoreceptors may have synaptic connections with LN(v)s (Yuan, 2011).

    To test whether LN(v)s can be activated by BN inputs through light stimulation, calcium imaging was performed using GCaMP3 transgenic flies with the larval brain-eye preparation, which included BO, BN, developing eye disks, the larval brain, and ventral nerve cord. Because BO senses blue and green light, the confocal laser at 488 nm (blue) and 543 nm (green) were used to stimulate these larval photoreceptors. LN(v)s' axon terminals displayed a relatively stable baseline of GCaMP3 fluorescence and, upon light stimulation, yielded large calcium responses, which increased with the greater intensity and longer duration of the light pulses (Yuan, 2011).

    Recent studies suggest that Cryptochrome (CRY) in adult large LN(v)s senses light and elicits neuronal firing. In larvae, however, severing BN abolished light-induced calcium responses in LN(v)s. The loss-of-function mutation of NorpA (no-receptor-potential A), encoding a phospholipase C crucial for phototransduction, also eliminated these calcium responses, which indicated that light-elicited responses in LN(v)s are generated via phototransduction in larval photoreceptors rather than as a direct response to light by LN(v)s (Yuan, 2011).

    In animals with BO genetically ablated, the dendritic field of LN(v) is absent. To test whether BO is required for LN(v)s' dendrite maintenance, the expression of cell death genes rpr and hid was induced in BO after synapse formation, and the LN(v) dendrite length was also found to be greatly reduced. Whereas physical contacts with BN or growth-promoting factors released from presynaptic axons could be important for LN(v)s' dendrite maintenance, it is also possible that synaptic activity from BN promotes LN(v) dendrite growth, as suggested by previous studies. To explore the latter scenario, newly hatched larvae were provided with different visual experiences through various light regimes-including the standard 12 hours of light and 12 hours of dark daily cycle (LD); constant darkness (DD) for sensory deprivation; constant light (LL) for enhanced light input; 16-hour light and 8-hour dark cycle, mimicking a long day; and 8-hour light and 16-hour dark cycle, mimicking a short day. The dendrite morphology of LN(v)s of late third instar larvae was examined. Whereas different light exposure had no detectable effects on larval developmental timing, increasing light exposure reduced the total dendrite length of individual LN(v) neurons, with the longest dendrite in constant darkness and the shortest dendrite length in constant light condition. Thus, not only is the LN(v) dendrite dependent on the presence of presynaptic nerve fibers, its length is modified by the sensory experience in a compensatory fashion, whereby an increase in sensory inputs causes a reduction in the dendrite length and vice versa (Yuan, 2011).

    Whereas adult LN(v)s alter their axon terminal structures in a circadian cycle-controlled fashion, no difference was found in dendrite morphology of LN(v)s from larvae collected at four different time points around the clock, which indicated that circadian regulation is not involved in the light-induced modification of LN(v) dendrites. Under regular light-dark conditions, LN(v) dendrites expanded as the larval brain size increased from the second to the third instar stage. However, the dendrite length of the LL group increased at a significantly slower rate than the DD group. It thus appears that light exposure retards the growth of LN(v) dendrites throughout the larval development (Yuan, 2011).

    To test the contribution of different light-sensing pathways, loss-of-function mutations of Cry (cry01) or NorpA (norpA36) and of double mutants lacking both Rh5 and Rh6 (rh52;rh61) were examined. Although wild-type and cry01 larvae displayed differences in their dendrite length when exposed to constant darkness versus constant light, such light-induced changes were absent in the rh52;rh61 double mutant and the norpA36 mutant. Thus, similar to the calcium response to light, light-induced modification of LN(v) dendritic structure requires visual transduction mediated by rhodopsin and NorpA in BO but not Cry function in LN(v)s (Yuan, 2011).

    To manipulate the level of synaptic activity, the BO excitability was weither increased by expressing the heat-activated Drosophila transient-receptor-potential A1 (dTrpA) channel, or transmitter release from BN was reduced through a temperature-sensitive form of the dominant-negative dynamin, Shibirets (Shits). These manipulations eliminated light-induced modification of LN(v) dendrites at 29°C. Reducing BO activity by means of Shits caused dendrite expansion, as if the animal detected no light, whereas increasing BO activity by means of the dTrpA channel resulted in reduction of LN(v) dendrites, a process reminiscent of constant light exposure (Yuan, 2011).

    Whether intrinsic LN(v) neuronal activity drives modification of its dendrite morphology was further tested by expression of either the sodium channel NaChBac to increase excitability or the potassium channel Kir2.1 to reduce excitability. LN(v)s expressing Kir2.1 showed reduced or no calcium responses upon light stimulation. In contrast, LN(v)s expressing NaChBac displayed numerous peaks in GCaMP3 signals in the presence or absence of light stimulation, indicative of elevated spontaneous activities. Upon examining LN(v) dendrites, it was found that neuronal excitability of the LN(v) was inversely proportional to its dendrite length (Yuan, 2011).

    These results obtained using genetic approaches agreed with findings in experiments with different environmental light conditions. They suggested that LN(v)'s dendritic structures are modified according to its neuronal activity, which varies with light-induced synaptic inputs (Yuan, 2011).

    To test whether synaptic contacts of BN on LN(v)s are modified by light, synapses formed by BN with EGFP (enhanced green fluorescent protein)-tagged Cacophony (Cac-EGFP) were marked, because Cacophony is a calcium channel localized at presynaptic terminals and its distribution correlates with the number of synapses. Close association was found of Cac-EGFP-expressing structures with LN(v)s' dendritic arbors. Compared with regular light-dark conditions, constant darkness increased, whereas constant light reduced, the total intensity of Cac-EGFP, which suggested that light modified not only dendritic arbors of LN(v)s but also the number of synaptic contacts impinging on LN(v) dendrites (Yuan, 2011).

    Next, using calcium imaging, whether there are light-induced functional modifications of LN(v)s was examined. Increased light exposure caused LN(v)s to be less responsive. Conversely, sensory deprivation in constant darkness increased LN(v)s' sensitivity to light. Thus, in contrast to stable synaptic responses observed in synaptic homeostasis, light-induced responses of central neurons postsynaptic to photoreceptors in the Drosophila larval visual circuit have a dynamic range, modifiable by sensory experiences and positively correlated to the dendrite length (Yuan, 2011).

    In dunce1, a loss-of-function mutant of the fly homolog of 3'5'-cyclic nucleotide phosphodiesterase, the LN(v)s' dendrite length was comparable among LD, LL, and DD groups. Reducing dunce gene expression specifically in LN(v)s through RNA interference (dncIR) resulted in a similar indifference of LN(v)s' dendrite size to the light exposure, which implicated a cell-autonomous action of dunce in LN(v) neurons (Yuan, 2011).

    To explore the possibility that the elevated cAMP level caused by the dunce mutation interfered with dendrite plasticity, tests were performed for the involvement of downstream components of the cAMP pathway, including the catalytic subunit of protein kinase A (PKAmc), which up-regulates cAMP signaling, and a dominant-negative form of the cAMP response element-binding protein (CREBdn), which inhibits cAMP-induced transcription activation. Expression of either transgene specifically in LN(v)s obliterated their ability to adjust dendrite length under different light-dark conditions. Calcium imaging further revealed that the expression of PKAmc or CREBdn eliminated changes of LN(v)s' light responses produced by different light-dark conditions. Thus, the cAMP pathway regulates both structural and functional plasticity of LN(v)s (Yuan, 2011).

    The screen for mutants with defective LN(v) dendritic plasticity also identified babos-1, a mutant with a P-element insertion near the transcriptional start site of CG3624, a previously uncharacterized immunoglobulin domain-containing cell surface protein. The LN(v) dendrite length of babos-1 mutant larvae was comparable to controls in LD and LL but has no compensatory increase in DD. Similar phenotypes were found in larvae expressing an RNAi transgene targeting CG3624 in LN(v)s. Moreover, flies carrying a hypomorphic allele of CG3624, CG3624[KG05061], also showed defective light-induced dendritic plasticity, which was fully rescued by expressing the UAS-CG3624 transgene specifically in LN(v)s. Thus, the function of this immunoglobulin domain-containing protein in LN(v)s is important for the dendrite expansion in constant darkness (Yuan, 2011).

    Bioinformatic analyses suggest that CG3624 is a cell surface protein containing an N-terminal signal peptide, extracellular immunoglobulin domains followed by a transmembrane helix, and a short C-terminal cytoplasmic tail. CG3624 is widely expressed in the nervous system throughout development. Its specific requirement for the adjustment of LN(v)s' dendrite length in constant darkness suggests that elevation or reduction of sensory inputs likely invokes separate mechanisms for compensatory modifications of central neuronal dendrites (Yuan, 2011).

    A functioning nervous system must have the capacity for adaptive modifications while maintaining circuit stability. This study of the Drosophila larval visual circuit reveals large-scale, bidirectional structural adaptations in dendritic arbors invoked by different sensory exposure. Whereas the circuit remains functional with modified outputs, this type of homeostatic compensation may modify larval light sensitivity according to its exposure during development and could facilitate adaption of fly larvae toward altered light conditions, such as seasonal changes. The observations also suggest shared molecular machinery between homeostasis and the Hebbian plasticity with respect to the cAMP pathway and indicate the feasibility of genetic studies of experience-dependent neuronal plasticity in Drosophila (Yuan, 2011).

    Selection for reproduction under short photoperiods changes diapause-associated traits and induces widespread genomic divergence

    The incidence of reproductive diapause is a critical aspect of life history in overwintering insects from temperate regions. Much has been learned about the timing, physiology and genetics of diapause in a range of insects, but how the multiple changes involved in this and other photoperiodically regulated traits are interrelated is not well understood. This study performed quasinatural selection on reproduction under short photoperiods in a northern fly species, Drosophila montana, to trace the effects of photoperiodic selection on traits regulated by the photoperiodic timer and / or by a circadian clock system. Selection changed several traits associated with reproductive diapause, including the critical day length for diapause (CDL), the frequency of diapausing females under photoperiods that deviate from daily 24 h cycles and cold tolerance, towards the phenotypes typical of lower latitudes. However, selection had no effect on the period of free-running locomotor activity rhythm regulated by the circadian clock in fly brain. At a genomic level, selection induced extensive divergence between the selection and control line replicates in 16 gene clusters involved in signal transduction, membrane properties, immunologlobulins and development. These changes resembled ones detected between latitudinally divergent D. montana populations in the wild and involved SNP divergence associated with several genes linked with diapause induction. Overall, this study shows that photoperiodic selection for reproduction under short photoperiods affects diapause-associated traits without disrupting the central clock network generating circadian rhythms in fly locomotor activity (Kauranen, 2019).

    Plasticity in Photoperiodism: Drosophila montana Females Have a Life-Long Ability to Switch From Reproduction to Diapause

    Photoperiodic reproductive diapause is an essential part of female life cycle in several insect species living on high latitudes, where overwintering in reproductive stage involves high risks for survival and progeny production. The sensitive period (SP), during which photoperiodic cues can trigger the switch from direct development to diapause, can last from a few hours or days after emergence to the entire life span of females. Moreover, in some species, sexually mature females can enter post-reproductive diapause as a response to decreasing day length and/or temperature. The duration of SP for diapause induction and the females' ability to enter post-reproductive diapause at short day lengths in Drosophila montana strains was assessed from different latitudes in Europe, North America, and Japan. This study shows that the females of this species have a life-long SP and that they retain an ability to switch between reproduction and diapause as a response to back-and-forth changes in day length for at least 3 months. D. montana strains from different latitudes showed high variation in females' ability to enter post-reproductive diapause; females of the southern strains generally requiring longer time and/or lower temperature to enter this stage than those of the northern strains. Moreover, the proportion of females that switched to post-reproductive diapause in 3 weeks in short day conditions at 16 °C showed positive correlation with the critical day length (CDL) for diapause induction and the latitudinal and continental origin of the strains. Life-long SP increases females' flexibility to respond to short-term changes in environmental conditions and enables reproducing females to switch to post-reproductive diapause when the days get shorter and colder toward the autumn. This ability can play a major role in species phenology and should be taken into account in theoretical and empirical studies on insect adaptation to seasonal variation (Lankinen, 2022).

    Selection for timing of eclosion results in co-evolution of temperature responsiveness in Drosophila melanogaster

    Circadian rhythms in adult eclosion of Drosophila are postulated to be regulated by a pair of coupled oscillators: one is the master clock that is light sensitive and temperature compensated and the other that is a slave oscillator whose period is temperature sensitive and whose phase is reflected in the overt behavior. Within this framework, it was reasoned that in populations of Drosophila melanogaster that have been artificially selected for highly divergent phases of eclosion rhythm, there may be changes in this network of the master-slave oscillator system, via changes in the temperature-sensitive oscillator and/or the coupling of the light- and temperature-sensitive oscillators. Light/dark cycles were used in conjunction with different constant ambient temperatures and two different amplitudes of temperature cycles in an overall cool or warm temperature and analyzed phases, gate width, and normalized amplitude of the rhythms in each of these conditions. The populations selected for eclosion in the morning (early flies) do not vary their phases with change in temperature regimes, whereas the populations selected for eclosion in the evening (late flies) show phase lability of up to ~5 h. These results imply a genetic correlation between timing of behavior and temperature sensitivity of the circadian clock (Abhilash, 2019).

    Phenotypic coupling of sleep and starvation resistance evolves in D. melanogaster

    One hypothesis for the function of sleep is that it serves as a mechanism to conserve energy. Recent studies have suggested that increased sleep can be an adaptive mechanism to improve survival under food deprivation in Drosophila melanogaster. To test the generality of this hypothesis, Sleep and its plastic response to starvation was compared in a temperate and tropical population of Drosophila melanogaster. Flies from the temperate population were found to be more starvation resistant, and it was hypothesized that they would engage in behaviors that are considered to conserve energy, including increased sleep and reduced movement. Surprisingly, temperate flies slept less and moved more when they were awake compared to tropical flies, both under fed and starved conditions, therefore sleep did not correlate with population-level differences in starvation resistance. In contrast, total sleep and percent change in sleep when starved were strongly positively correlated with starvation resistance within the tropical population, but not within the temperate population. Thus, unexpectedly complex relationships between starvation and sleep were observed that vary both within and across populations. These observations falsify the simple hypothesis of a straightforward relationship between sleep and energy conservation. The hypothesis that starvation is correlated with metabolic phenotypes was tested by investigating stored lipid and carbohydrate levels; stored metabolites were found to partially contributed towards variation starvation resistance. These findings demonstrate that the function of sleep under starvation can rapidly evolve on short timescales and raise new questions about the physiological correlates of sleep and the extent to which variation in sleep is shaped by natural selection (Sarikaya, 2020).

    Screening of sleep assisting drug candidates with a Drosophila model

    Lately, Drosophila has been favored as a model in sleep and circadian rhythm research due to its conserved mechanism and easily manageable operation. These studies have revealed the sophisticated parameters in whole-day sleep profiles of Drosophila, drawing connections between Drosophila sleep and human sleep. This study tested several sleep deprivation protocols (mechanical shakes and light interruptions) on Drosophila and delineated their influences on Drosophila sleep. A daytime light-deprivation protocol (DD) was applied mimicking jet-lag to screen drugs that alleviate sleep deprivation. Characteristically, classical sleep-aid compounds exhibited different forms of influence: phenobarbital and pentobarbital modified total sleep time, while melatonin only shortened the latency to sleep. Such results construct the basis for further research on sleep benefits in other treatments in Drosophila. Seven herb extracts were screened, and very diverse results were found regarding their effect on sleep regulation. For instance, Panax notoginseng and Withania somnifera extracts displayed potent influence on total sleep time, while Melissa officinalis increased the number of sleep episodes. By comparing these treatments, it was possible to rank drug potency in different aspects of sleep regulation. Notably, the presence of sleep difficulties in a Drosophila Alzheimer's disease (AD) model was confirmed with an overexpression of human Aβ, and clear differences were recognized between the portfolios of drug screening effects in AD flies and in the control group. Overall, potential drug candidates and receipts for sleep problems can be identified separately for normal and AD Drosophila populations, outlining Drosophila's potential in drug screening tests in other populations if combined with the use of other genetic disease tools (Wang, 2020).

    Chronic social isolation signals starvation and reduces sleep in Drosophila

    Social isolation and loneliness have potent effects on public health. Research in social psychology suggests that compromised sleep quality is a key factor that links persistent loneliness to adverse health conditions. Although experimental manipulations have been widely applied to studying the control of sleep and wakefulness in animal models, how normal sleep is perturbed by social isolation is unknown. This study reports that chronic, but not acute, social isolation reduces sleep in Drosophila. Quantitative behavioural analysis and transcriptome profiling were used to differentiate between brain states associated with acute and chronic social isolation. Although the flies had uninterrupted access to food, chronic social isolation altered the expression of metabolic genes and induced a brain state that signals starvation. Chronically isolated animals exhibit sleep loss accompanied by overconsumption of food. This resonates with anecdotal findings of loneliness-associated hyperphagia in humans. Chronic social isolation reduces sleep and promotes feeding through neural activities in the peptidergic fan-shaped body columnar neurons of the fly. Artificial activation of these neurons causes misperception of acute social isolation as chronic social isolation and thereby results in sleep loss and increased feeding. These results present a mechanistic link between chronic social isolation, metabolism, and sleep, addressing a long-standing call for animal models focused on loneliness (Li, 2021).

    Fruit flies are social animals, and exhibit dynamic social network structures and collective behaviours, that contribute to environmental sensing, foraging, feeding, fighting, mating, oviposition, circadian time setting and even the existence of 'culture'. These important aspects of social interactions imply that insects can provide suitable models for studying how the objective absence of social relationships is perceived and represented in the brain (Li, 2021).

    Social experience affects sleep need in Drosophila. This study revisited this finding by exploring how social isolation affects sleep in flies that have prior social experience. Sleep behaviour was tested after maintaining 1, 2, 5, 25 or 100 male flies in a food vial for 7 days. Group-housed flies, regardless of group size (2, 5, 25 or 100), exhibited similar sleep profiles. By contrast, flies housed in isolation displayed a significant loss of sleep, mainly distributed during the daytime (Li, 2021).

    The duration of social isolation was manipulated: flies were either isolated or housed in a group of 25 flies for 1, 3, 5, or 7 days, before sleep was measured in a Drosophila activity monitor (DAM). Sleep profiles, which display the proportion of time spent sleeping in consecutive 30-min segments over 24 h, showed that chronic social isolation (5 or 7 days) changed sleep architecture primarily during the daytime and especially during an interval of several hours following dawn (lights on). Although short durations of social isolation (1 or 3 days) did not induce sleep loss, chronic social isolation (5 or 7 days) significantly reduced daily total sleep, daytime sleep and sleep between Zeitgeber time (ZT) 0 and ZT4 (corresponding to the first 4 h after lights-on in a light-dark (LD) cycle) (Li, 2021).

    To assess how social isolation alters daytime sleep, all daytime sleep bouts were pooled from all animals tested for a given condition and their distributions were plotted as cumulative relative fractions for bout lengths. Acute social isolation (1 day) produced sleep bout distributions that were indistinguishable from those of 1-day group-housed flies. Flies that were socially isolated for 3 days showed slightly different sleep bout distributions from their group-housed counterparts. However, there was no deficit in total daily sleep, daytime sleep or ZT0-4 sleep in these flies. Flies that were isolated for longer periods (5 or 7 days) had sleep distributions that were significantly different from those of their group-housed counterparts. Cumulative relative frequency curves of daytime sleep bouts from chronically isolated flies climbed faster than those of their group-housed counterparts as shorter sleep bouts accumulated (5 or 7 days). Over seven days of isolation, daily total sleep, daytime sleep and ZT0-4 sleep all decreased progressively (Li, 2021).

    Age-matched flies were used to rule out the possibility that chronic social isolation induced sleep loss because the flies were older. Chronic social isolation (7 days) induced sleep loss consistently in various isogenic strains, in aged (4-week-old) wild-type flies, and in sleep inbred panel (SIP) strains with different baseline levels of sleep (Li, 2021).

    RNA sequencing (RNA-seq) libraries were prepared for three conditions: socially enriched flies (group treated, Grp), chronically isolated flies (isolated for 7 days, Iso_7D) and acutely isolated flies (isolated for 1 day, Iso_1D). Raster plots demonstrate sleep bouts of individual animals during a 24-h period measured immediately after group enrichment or social isolation. Daytime sleep was reduced and much more fragmented in chronically isolated flies than in group-housed or acutely isolated flies. Fly heads were collected between ZT0.5 and ZT2, a window of time within ZT0-4 that immediately preceded significant loss of daytime sleep in chronically isolated flies. Using differential gene expression analyses, intersectional and clustering strategies, candidate genes were identified for the sleep loss induced by chronic social isolation. These 214 candidate genes showed differences in expression in chronically isolated flies compared with both acutely isolated and group-housed flies and underwent unidirectional changes during the process of chronic social isolation. Gene ontology enrichment analysis suggested that these 214 genes are enriched for biological pathways associated with oxidation-reduction processes, one-carbon metabolic processes and carbohydrate metabolic processes. The rest of the gene ontology of biological pathways showed a strong preference for metabolic functions, such as fatty acid, pyruvate, glucose and amino acid metabolic processes. Consistent with the sleep loss phenotype, sleep was also among the most overrepresented gene ontologies for biological pathways (Li, 2021).

    Among the top 20 genes in this list, two genes stood out: Limostatin (Lst, CG8317), expression of which increased 1.67-fold after chronic isolation, and Drosulfakinin (Dsk), expression of which decreased 2.03-fold after chronic isolation. Limostatin is a decretin hormone that is induced by starvation and suppresses insulin release. Drosulfakinin, a satiety-inducing cholecystokinin-like peptide, is secreted when the animal is fed. As a signal of satiety, drosulfakinin is depleted under starvation conditions. A third gene, target of brain insulin (tobi), also showed significantly increased expression (1.76-fold) during chronic social isolation. tobi encodes an α-glucosidase that is regulated by Drosophila insulin and glucagon analogues. In addition, 7 of these top 20 genes and 32 of the total 214 candidate genes were previously identified as being regulated in Drosophila heads after 24 h of starvation. Thus, from a transcriptomic perspective, the brain of a fly maintained in chronic social isolation closely resembles the brain of a starving fly, despite continuous access to food. It was reasoned that such a 'starvation brain state' might broadly affect gene expression associated with metabolic processes. Massive changes in mitochondrial functions and oxidation-reduction processes could be direct consequences of starvation and/or elevated feeding (Li, 2021).

    The activity recording capillary feeder (ARC) assay, a video recording capillary feeder (CAFE) assay that monitors sleep and feeding behaviours simultaneously and continuously in individual Drosophila, was used. ARC assays validated the isolation-induced sleep loss phenotype previously observed with DAM assays: daily total sleep, daytime sleep and ZT0-4 sleep were reduced significantly after 7 days of social isolation. In addition, nighttime sleep was also reduced, probably owing to higher sensitivity in detecting movements using the positional tracking method, or differences in chamber shape and food source between the ARC and DAM systems. As predicted from the gene expression profiling results, increased feeding was observed in socially isolated animals compared to their group-treated counterparts. Flies isolated for 7 days showed significant increases in total food consumption, daytime food consumption, nighttime food consumption and ZT0-4 food consumption in comparison to flies that were group-housed for 7 days. Thus, chronic social isolation induces a starvation state in Drosophila at the levels of both gene expression in the brain and behaviour (Li, 2021).

    The altered feeding pattern produced by chronic social isolation is not merely a consequence of sleep loss, because several classic sleep mutants all exhibited normal feeding behaviour. In addition, acutely isolated flies did not show a strong increase in food consumption (Li, 2021).

    The candidate gene Lst is normally induced by nutrient restriction in endocrine neurons in the corpora cardiaca. However, the RNA profiling experiment suggested that there could be a previously unknown brain source for LST production. A resource of high-resolution transcriptomes of 100 GAL4 driver lines suggested that cells labelled by the driver line NPF-GAL4 (NPF, neuropeptide F [the fly homologue of neuropeptide Y)] are likely to express LST. Using a monoclonal antibody against LST, co-localized LST immunoreactivity and NPF-GAL4-driven GFP signals were detected. Among six known neuronal clusters that express NPF, LST immunoreactivity appeared to be co-localized with NPF-GAL4-driven GFP signals at the dorsal stratum of the fan-shaped body (dorsal fan-shaped body, dFB) and in a cluster of small cell bodies in the dorsal brain. Neurons without known function that comprise this cluster of NPF cells were previously named P2. A recent study used a split-GAL4 driver, SS0020-split-GAL4 (abbreviated as P2-GAL4 below), to strongly label the majority of P2 neurons that showed positive immunoreactivity for LST and NPF22 (Li, 2021).

    Notably, the projections of the P2 neurons overlapped with the axonal projections of the dFB neurons labelled by R23E10-GAL4, which suggests that P2 neurons might signal to sleep-promoting dFB neurons. At the cell body level, P2 neurons differ from the R23E10-GAL4 labelled cells. A MultiColor FlpOut (MCFO) approach was used to stochastically decorate individual neurons labelled by P2-GAL4, and it was found that they are fan-shaped body columnar neurons. The hemibrain connectome allowed determination that P2 neurons include, as a dominant constituent, the hDeltaK cell type—a columnar cell class, where each neuron has a stereotypical dendritic input in the ellipsoid body (EB) in addition to the FB innervation. hDeltaK cells exhibit extensive synaptic connections with a known subset of R23E10-GAL4-labelled sleep-promoting dFB neurons27 . On the basis of the above connectome data and existing evidence that NPF/NPY is involved in animal metabolism and stress responses, focus was placed on P2 neurons (Li, 2021).

    To test whether P2 neurons contribute to sleep loss induced by chronic social isolation, these neurons were chronically silenced by expressing the inward-rectifying potassium channel Kir2.1 under the control of P2-GAL4. In flies carrying both P2-GAL4 and UAS-Kir2.1, chronic isolation no longer induced an altered sleep profile when compared to their group-housed counterparts. The cumulative relative frequency curve of daytime sleep bouts for socially isolated animals no longer climbed faster than that of group-reared animals. Raster plots of sleep bouts in individual flies showed little difference between chronically isolated and group-housed flies. No difference was found between isolated and group-housed animals for daily total sleep, daytime sleep, or ZT0-4 sleep. By contrast, in heterozygous parental control animals carrying either the P2-GAL4 or the UAS-Kir2.1 transgene, chronic social isolation robustly induced sleep loss. Temporally silencing P2 neurons using UAS-shibirets1 during group enrichment or social isolation did not block social isolation-induced sleep loss. Although isolated flies carrying both P2-GAL4 and UAS-Kir2.1 still showed some overconsumption of food, they no longer showed excessive food consumption for ZT0-4, and the total increase in daytime food consumption was much smaller than in parental controls (Li, 2021).

    Using [Ca2+] imaging, it was found that the activity of P2 neurons was correlated with locomotor activity in both group-housed and isolated flies. One might expect that P2 neurons would be tonically more active in isolated flies than in group-housed flies, but this effect in baseline [Ca2+] levels could not be detected. Alternatively, it can be hypothesize that locomotion drives more P2 neuron total activity during 7 days of isolation than during 1 day of isolation (Li, 2021).

    Therefore whether boosting activity in P2 neurons during acute social isolation (1 day) is sufficient to promote behavioural changes that resemble the effects of chronic social isolation (7 days) was measured. To activate P2 neurons, a Drosophila warmth-gated cation channel, UAS-dTRPA1, was expressed with P2-GAL4. The P2-GAL4-labelled neurons were activated by treating the flies at 28 °C during acute social isolation or group enrichment (1 day). Control experiments, using flies of the same genotype, were conducted by treating the flies at 22 °C during acute social isolation or group enrichment (1 day). Following these treatments, all flies were subsequently maintained at 22 °C for measurement of sleep or feeding behaviour. In animals carrying both P2-GAL4 and UAS-dTRPA1, there were significant interactions between temperature treatment and isolation status for total, daytime, and ZT0-4 sleep and food consumption: activation of P2 neurons during acute social isolation promoted significant sleep loss and excessive feeding, whereas activation of P2 neurons in group housing did not alter either sleep or feeding behaviour. In control experiments, no evidence was found for interactions between temperature treatment and isolation status in the heterozygous parental flies (Li, 2021).

    Notably, P2 neurons are connected to the dFB neurons that are known to regulate sleep homeostasis and couple energy metabolism to sleep. Artificial activation of P2 neurons can produce a behavioural state that resembles the effects of chronic isolation after social isolation for a single day; however, activation of P2 neurons failed to produce these behaviours in the complete absence of social isolation. This indicates that both activity in P2 neurons and a status of being socially isolated are required to induce reduced sleep and increased feeding. Social isolation might be sensed by P2 neurons or elsewhere in the brain, but in either case appears to cause the activity of P2 neurons to be interpreted differently and thereby to generate novel behaviours. Downregulation of a secreted cytokine in a non-neural tissue, the fat body, suppresses sleep and promotes feeding in Drosophila. It would be interesting to determine whether these behavioural responses also depend on P2 neuronal activity (Li, 2021).

    Modifications of feeding circuits appear to be crucial for the evolution of complex social behaviours. For example, in C. elegans, a single-residue difference in the neuropeptide Y receptors of naturally occurring strains determines whether the strains exhibit solitary or social feeding behaviour. As antibodies to neuropeptide F, the fly homologue of neuropeptide Y, label P2 neurons, future work may ascertain whether Drosophila's P2 neurons influence social patterns of feeding behaviour as well as mediating feeding and sleep responses to social isolation (Li, 2021).

    In humans, social isolation promotes new emotional states that intensify with the passage of time. Sleep loss in Drosophila is a faithful readout of the duration of social isolation, and this allowed identification of specific patterns of gene and behavioural states that emerge as social isolation becomes chronic. This unexpected association between social isolation, sleep and metabolism in an insect model is reminiscent of the connection observed by social psychologists between loneliness, sleep difficulties and hyperphagia. Such robust findings in Drosophila suggest that studies of animal models might identify conserved brain states, genes, and neural circuits that are associated with social isolation (Li, 2021).

    Identification of Ultrastructural Signatures of Sleep and Wake in the Fly Brain

    The cellular consequences of sleep loss are poorly characterized. In the pyramidal neurons of mouse frontal cortex this study found that mitochondria and secondary lysosomes occupy a larger proportion of the cytoplasm after chronic sleep restriction compared to sleep, consistent with increased cellular burden due to extended wake. For each morphological parameter the within-animal variance was high, suggesting that the effects of sleep and sleep loss vary greatly among neurons. However, the analysis was based on 4-5 mice/group and a single section/cell. this study applied serial block-face scanning electron microscopy to identify signatures of sleep and sleep loss in the Drosophila brain. Stacks of images were acquired and used to obtain full 3D reconstructions of the cytoplasm and nucleus of 263 Kenyon cells from adult flies collected after a night of sleep (S) or after 11 hours (SD11) or 35 hours (SD35) of sleep deprivation (9 flies/group). Relative to S flies, SD35 flies showed increased density of dark clusters of chromatin and of Golgi apparata and a trend increase in the percent of cell volume occupied by mitochondria, consistent with increased need for energy and protein supply during extended wake. Logistic regression models could assign each neuron to the correct experimental group with good accuracy, but in each cell nuclear and cytoplasmic changes were poorly correlated, and within-fly variance was substantial in all experimental groups. Together, these results support the presence of ultrastructural signatures of sleep and sleep loss but underscore the complexity of their effects at the single-cell level (Flores, 2022).

    An extra-clock ultradian brain oscillator sustains circadian timekeeping

    The master circadian clock generates 24-hour rhythms to orchestrate daily behavior, even running freely under constant conditions. Traditionally, the master clock is considered self-sufficient in sustaining free-running timekeeping via its cell-autonomous molecular clocks and interneuronal communications within the circadian neural network. This study found a set of bona fide ultradian oscillators in the Drosophila brain that support free-running timekeeping, despite being located outside the master clock circuit and lacking clock gene expression. These extra-clock electrical oscillators (xCEOs) generate cell-autonomous ultradian bursts, pacing widespread burst firing and promoting rhythmic resting membrane potentials in clock neurons via parallel monosynaptic connections. Silencing xCEOs disrupts daily electrical rhythms in clock neurons and impairs cycling of neuropeptide pigment dispersing factor, leading to the loss of free-running locomotor rhythms. Together, it is concluded that the master clock is not self-sufficient to sustain free-running behavior rhythms but requires additional endogenous inputs to the clock from the extra-clock ultradian brain oscillators (Tang, 2022).

    Real time, in vivo measurement of neuronal and peripheral clocks in Drosophila melanogaster

    Circadian clocks are highly conserved transcriptional regulators that control ~24 hr oscillations in gene expression, physiological function, and behavior. Circadian clocks exist in almost every tissue and are thought to control tissue-specific gene expression and function, synchronized by the brain clock. Many disease states are associated with loss of circadian regulation. How and when circadian clocks fail during pathogenesis remains largely unknown because it is currently difficult to monitor tissue-specific clock function in intact organisms. This study developed a method to directly measure the transcriptional oscillation of distinct neuronal and peripheral clocks in live, intact Drosophila; the method was termed Locally Activatable BioLuminescence, or LABL. Using this method, it was observed that specific neuronal and peripheral clocks exhibit distinct transcriptional properties. Loss of the receptor for PDF, a circadian neurotransmitter critical for the function of the brain clock, disrupts circadian locomotor activity but not all tissue-specific circadian clocks. While peripheral clocks in non-neuronal tissues were less stable after the loss of PDF signaling, they continued to oscillate. It was also demonstrated that distinct clocks exhibit differences in their loss of oscillatory amplitude or their change in period, depending on their anatomical location, mutation, or fly age. These results demonstrate that LABL is an effective tool that allows rapid, affordable, and direct real-time monitoring of individual clocks in vivo (Johnstone, 2022).

    The Drosophila circadian phase response curve to light: Conservation across seasonally relevant photoperiods and anchorage to sunset

    Photic history, including the relative duration of day versus night in a 24-hour cycle, is known to influence subsequent circadian responses to light in mammals. Whether such modulation is present in Drosophila is currently unknown. This study constructed the first high-resolution Drosophila seasonal atlas for light-induced circadian phase-resetting. Testing the light responses of over 4,000 Drosophila at 120 timepoints across 5 seasonally-relevant rectangular photoperiods (i.e., LD 8:16, 10:14, 12:12, 14:10, and 16:8; 24 hourly intervals surveyed in each), it was determined that many aspects of the fly circadian PRC waveform are conserved with increasing daylength. Surprisingly though, irrespective of LD schedule, the start of the PRCs always remained anchored to the timing of subjective sunset, creating a differential overlap of the advance zone with the morning hours after subjective sunrise that was maximized under summer photoperiods and minimized under winter photoperiods. These data suggest that there may be differences in flies versus mammals as to how the photoperiod modulates the waveform and amplitude of the circadian PRC to light. On the other hand, they support the possibility that the lights-off transition determines the phase-positioning of photic PRCs across seasons and across species. More work is necessary to test this claim and whether it might factor into the timing of seasonal light responses in humans (Dollish, 2022).

    Alterations in lifespan and sleep/wake duration under selective monochromes of visible light in Drosophila melanogaster

    Rapid technology development, exposure to gadgets, and artificial lights (with different monochromes) have disturbed lifestyles and the circadian clock, which otherwise confers better regulation of behavioral patterns and sleep/wake cycles in most organisms including Drosophila melanogaster. This study assayed the effect of LD12:12 hr (light: dark) monochromatic lights (violet, blue, green, yellow, orange, and red) on the lifespan, activity, and sleep of the D. melanogaster. A shortened lifespan was observed under 12h of violet, blue, green, and yellow lights, while significantly reduced activity levels under the light phase of blue and green light as compared to their dark phase is observed. Significant increase in the evening anticipation index of flies under blue and green light alongside increased and decreased sleep depth during the day and night respectively suggests the light avoidance, while there is no effect of colored light on the waking time, daily active time, and sleep time. Thus, this study shows short and long-term exposure to certain colored lights in terms of reduced lifespan and locomotor activity, which cause qualitative as well as quantitative changes in the sleep of flies; probably as a sign of aversion towards a specific light (Krittika, 2022).

    Alignment between glioblastoma internal clock and environmental cues ameliorates survival in Drosophila

    Virtually every single living organism on Earth shows a circadian (i.e. "approximately a day") internal rhythm that is coordinated with planet rotation (i.e. 24 hours). External cues synchronize the central clock of the organism. Consequences of biological rhythm disruptions have been extensively studied on cancer. Still, mechanisms underlying these alterations, and how they favor tumor development remain largely unknown. This study shows that glioblastoma-induced neurodegeneration also causes circadian alterations in Drosophila. Preventing neurodegeneration in all neurons by genetic means reestablishes normal biological rhythms. Interestingly, in early stages of tumor development, the central pacemaker lengthens its period, whereas in later stages this is severely disrupted. The re-adjustment of the external light:dark period to longer glioblastoma-induced internal rhythms delays glioblastoma progression and ameliorates associated deleterious effects, even after the tumor onset (Jarabo, 2022).

    Sleep Deprivation Does Not Influence Photic Resetting of Circadian Activity Rhythms in Drosophila Clocks

    Previous investigations in humans and rodent animal models have assessed the interplay of sleep in the circadian system's phase responses to nighttime light exposure. The resulting data have been mixed, but generally support a modulatory role for sleep in circadian photic resetting (not an absolute requirement). Drosophila have been historically used to provide important insights in the sleep and circadian sciences. However, no experiments to date have evaluated how immediate sleep need or recent sleep history affects their pacemaker's phase readjustments to light. We did so in the current study by (1) forcing separate groups of animals to stay awake for 1 or 4 h after they were shown a broadspectrum pulse (15 min during the first half of the night, 950 lux), or (2) placing them on a restricted sleep schedule for a week before light presentation without any subsequent sleep disruption. Forced sleep restriction, whether acute or chronic, did not alter the size of light-induced phase shifts. These data are consistent with observations made in other diurnal animals and raise the possibility, more broadly, that phototherapies applied during sleep-such as may be necessary during the winter months-may still be efficacious in individuals experiencing sleep-continuity problems such as insomnia (Negelspach, 2022).

    Analysis of sleep in individual Drosophila melanogaster reveals a self-regulatory role for cuticular hydrocarbons pheromones

    It is well established that pheromones are used by insects to transmit information between individuals. However, research has revealed that individual insects can be both the sender and the receiver of some pheromonal signals. It is therefore interesting to consider whether the pheromonal state of an individual insect can exert an effect on itself. This study monitored the sleep activity of single flies exhibiting a mutation that leads to pheromonal deficiency and found that cuticular hydrocarbons (CHs) exerted self-regulatory effects on the amount of sleep experienced by these flies. To identify the physiological significance of this mechanism, the amounts of sleep were compared in individual young flies and individual old flies (flies are known to sleep less as they get older), and this data was compared with young and old flies exhibiting mutations that lead to CH reception defects. The differences in the amount of sleep experienced by young and old mutant flies were significantly lower than those of the control flies. These data show that hydrocarbon signals produced by the cuticle in Drosophila can be self-perceived and regulate the amount of sleep acquired in a maturation-dependent manner (Wu, 2022).

    Sleep Induction by Mechanosensory Stimulation in Drosophila

    People tend to fall asleep when gently rocked or vibrated. Experimental studies have shown that rocking promotes sleep in humans and mice. However, the mechanisms underlying the phenomenon are not well understood. A habituation model proposes that habituation, a form of non-associative learning, mediates sleep induction by monotonous stimulation. This study showed that gentle vibration promotes sleep in Drosophila in part through habituation. Vibration-induced sleep (VIS) leads to increased homeostatic sleep credit and reduced arousability, and can be suppressed by heightened arousal or reduced GABA signaling. Multiple mechanosensory organs mediate VIS, and the magnitude of VIS depends on vibration frequency and genetic background. Sleep induction improves over successive blocks of vibration. Furthermore, training with continuous vibration does not generalize to intermittent vibration, demonstrating stimulus specificity, a characteristic of habituation. These findings suggest that habituation plays a significant role in sleep induction by vibration (Ozturk-Colak, 2020).

    Sleep contributes to preference for novel food odours in Drosophila melanogaster

    The importance of sleep in maintaining cognitive functions such as learning and memory has been reported in both vertebrates and invertebrates. Previous studies demonstrated that sleep deprivation impaired the olfactory memory retention of fruit flies as described in the classical conditioning paradigm. This study shows that sleep deprivation leads to a preference for the odours of the rearing environment in Drosophila melanogaster. Flies whose sleep had been disturbed with periodic rotation stimuli during night-time preferred apple cider vinegar (ACV) to broth, while this preference was lower in flies without sleep deprivation and those rotated during daytime. Experiments using single odours showed an increase in responses to ACV due to sleep deprivation. These results suggest that sleep functions in food odour preference. Flies grown on medium supplemented with ACV showed greater preference for ACV, and those grown with broth supplementation showed a greater preference for broth under sleep-deprived conditions. These results suggest that flies with night-time sleep deprivation become attached to the environment on which they have developed, and that sleep contributes to preference for novel food odours. This study offers an approach to investigating the interaction between sleep and neural disorders concerning cognitive deficits towards novel stimuli (Tanizawa, 20021).

    Daily increasing or decreasing photoperiod affects stress resistance and life history traits in four Drosophila species

    Photoperiod is one of the most reliable seasonal cues that organisms can use to prepare for upcoming environmental changes. Evidence suggests that exposure to different photoperiod can activate plastic responses in stress resistance traits, while there is limited evidence on the plastic response induced by daily progressive cumulative changes in photoperiod. This study assayed the effect of within generation daily uni-directional and cumulative changes in photoperiod on stress resistance and life history traits in four Drosophila species. It was predicted that daily increasing photoperiod, mimicking upcoming summer conditions, should lead to an increase in heat resistance and establish trade-offs with other fitness related traits. On the other hand, it was predicted that daily decreasing photoperiod should reflect upcoming winter conditions leading to an increase in cold resistance. It was found that within genreation changes in photoperiod had a significant effect on life history and stress resistance traits in the four Drosophila species. The observed response was different across species, with D. melanogaster showing five out of six studied traits affected, while in D. mercatorum only one trait was significantly affected. The exposure to changing photoperiod led to an increased upper thermal resistance in D. melanogaster and D. mercatorum and a decreased lower thermal resistance in D. melanogaster and D. simulans, as well as a decreased starvation and desiccation resistance in D. virilis. The developmental time was shorter when flies were exposed to the two photoperiod regimes compared to constant day length control in D. melanogaster and D. simulans. A limited effect was observed on egg-to-adult-viability and desiccation resistance. The results of this study show that daily change in photoperiod induced a plastic response in different traits of drosophilids, suggesting that this environmental parameter needs to be carefully considered in evolutionary studies (Manenti, 2021).

    Simulated mobile communication frequencies (3.5 GHz) emitted by a signal generator affects the sleep of Drosophila melanogaster

    With the rapid development of science and technology, 5G technology will be widely used, and biosafety concerns about the effects of 5G radiofrequency radiation on health have been raised. Drosophila melanogaster was selected as the model organism for this study, in which a 3.5 GHz radiofrequency radiation (RF-EMR) environment was simulated at intensities of 0.1 W/m(2), 1 W/m(2), and 10 W/m(2). The activity of parent male and offspring (F1) male flies was measured using a Drosophila activity monitoring system under short-term and long-term 3.5 GHz RF-EMR exposure. Core genes associated with heat stress, the circadian clock and neurotransmitters were detected by QRT-PCR technology, and the contents of GABA and glutamate were detected by UPLC-MS. The results show that short-term RF-EMR exposure increased the activity level and reduced the sleep duration while long-term RF-EMR exposure reduced the activity level and increased the sleep duration of F1 male flies. Under long-term RF-EMR, the expression of heat stress response-related hsp22, hsp26 and hsp70 genes was increased, the expression of circadian clock-related per, cyc, clk, cry, and tim genes was altered, the content of GABA and glutamate was reduced, and the expression levels of synthesis, transport and receptor genes were altered. In conclusion, long-term RF-EMR exposure enhances the heat stress response of offspring flies and then affects the expression of circadian clock and neurotransmitter genes, which leads to decreased activity, prolonged sleep duration, and improved sleep quality (Wang, 2021).

    Disrupting flight increases sleep and identifies a novel sleep-promoting pathway in Drosophila

    Sleep is plastic and is influenced by ecological factors and environmental changes. The mechanisms underlying sleep plasticity are not well understood. This study shows that manipulations that impair flight in Drosophila increase sleep as a form of sleep plasticity. Flight was disrupted by blocking the wing-expansion program, genetically disrupting flight, and by mechanical wing perturbations. A new sleep regulatory circuit is defined starting with specific wing sensory neurons, their target projection neurons in the ventral nerve cord, and the neurons they connect to in the central brain. In addition, a critical neuropeptide (Burs) and its receptor (Rickets) were identified that link wing expansion and sleep. Disrupting flight activates these sleep-promoting projection neurons, as indicated by increased cytosolic calcium levels, and stably increases the number of synapses in their axonal projections. These data reveal an unexpected role for flight in regulating sleep and provide new insight into how sensory processing controls sleep need (Melnattur, 2020).

    Adult-specific electrical silencing of pacemaker neurons uncouples molecular clock from circadian outputs

    Circadian rhythms regulate physiology and behavior through transcriptional feedback loops of clock genes running within specific pacemaker cells. In Drosophila, molecular oscillations in the small ventral lateral neurons (sLNvs) command rhythmic behavior under free-running conditions releasing the neuropeptide Pigment Dispersing Factor (PDF) in a circadian fashion. Electrical activity in the sLNvs is also required for behavioral rhythmicity. Yet, how temporal information is transduced into behavior remains unclear. This study developed a new tool for temporal control of gene expression to obtain adult-restricted electrical silencing of the PDF circuit, which led to reversible behavioral arrhythmicity. Remarkably, Period (Per) oscillations during the silenced phase remained unaltered, indicating that arrhythmicity is a direct consequence of the silenced activity. Accordingly, circadian axonal remodeling and PDF accumulation were severely affected during the silenced phase. It is concluded that although electrical activity of the sLNvs is not a clock component, it coordinates circuit outputs leading to rhythmic behavior (Depetris-Chauvin, 2011).

    Work from many laboratories has shaped the current view of the molecular clockworks. Although the relative contribution of specific molecular mechanisms is still a matter of debate, it is clear that a transcriptional and translational negative feedback loop is key to give rise to and sustain molecular oscillations. Years ago it was proposed that circadian oscillations arise from interactions between ion transport systems across the cell membrane and the resulting ion concentration gradients. In fact, in support of such possibility, electrical silencing of a key pacemaker circuit in Drosophila stopped the free-running clock both in the larval and adult brains, leading to the proposition that active ionic conductances are an essential component of this cellular mechanism. One potential caveat of those experiments is that they rely on the long-term expression of ion channels from early circuit development, which could not only trigger compensatory mechanisms to avoid net changes in excitability but also trigger cell death (Depetris-Chauvin, 2011).

    To more precisely examine the connection between the membrane and the molecular clock, expression of an inward rectifier K+ channel (KIR) was restricted to adult stages. Such genetic manipulation rendered the flies as behaviorally arrhythmic as those expressing the channel from early circuit development and prevented action potential firing to a similar extent. Interestingly, however, no effects were observed in the pace of the molecular oscillations after several days under free-running conditions (i.e., on DD4, and even in DD9), which, along with the reversibility observed once kir 2.1 expression was turned off in several affected outputs (free-running locomotor behavior, PDF immunoreactivity), strongly support the notion of an unaltered molecular clock during the silenced phase. In favor of an alternative interpretation of the original observations, a rundown in the molecular oscillations - and even no oscillations whatsoever - was noticed after prolonged KIR expression, opening the possibility that long-term changes on intrinsic properties of the neurons, likely through the alteration of second messenger cascades, as it has been shown in a different but also extreme condition, ultimately impinge upon cell viability and thus indirectly result in abnormal clock function. In fact, adult-restricted silencing of the PDF circuit triggered morphological changes in second order processes, giving rise to a less complex arborization pattern; it follows that a more severe treatment, such as long-term KIR expression, could result in stronger structural phenotypes indicative of defective cell physiology (Depetris-Chauvin, 2011).

    In addition, constantly low PDF levels could potentially account for the progressive run-down in molecular oscillations. Along this line it has been shown that, in the absence of PDF, the sLNvs eventually desynchronize, becoming evident by DD6. Because acute electrical silencing of PDF neurons clamps the neuropeptide to trough levels that are insufficient to sustain synchronicity in dorsal oscillators, affecting excitability for longer terms could eventually result in reduced amplitude oscillations and internal desynchronization in central pacemakers. In the mammalian SCN, evidence from different laboratories has lent support to the notion that membrane excitability or, more precisely, a certain degree of depolarization and activation of Ca2+ and cAMP second messenger cascades, may be required for sustained molecular oscillations. These observations underscore that intercellular communication is important to reinforce high amplitude molecular oscillations through synchronization of independent cellular oscillators, as opposed to being an essential component within the mechanism responsible for the generation of the molecular oscillations. Interestingly, it has been reported that, in a subset of SCN neurons, molecular oscillations of a circadian reporter still take place even in the absence of synaptic connectivity, highlighting the autonomy of the molecular oscillator (Depetris-Chauvin, 2011).

    Adult-restricted silencing of the PDF circuit impairs locomotor behavior to a similar extent compared to constitutively silencing them, demonstrating that regardless of the overall levels of KIR achieved through the inducible system, short-term expression effectively prevents communication with other neuronal targets. Such a scenario offers the possibility to identify the direct consequences of reducing the excitability of the PDF circuit in a defined temporal window. Surprisingly, despite kir expression being limited to the adult brain, it correlated with axonal arbors of reduced complexity throughout the day in the dorsal protocerebrum, even though the circadian remodeling phenomenon continued to take place. The latter lends further support to the notion that no effect on the pace of the molecular oscillator became evident during the acutely silenced phase (Depetris-Chauvin, 2011).

    In addition, adult-restricted silencing correlated with noncycling PDF levels. PDF is transported along the axonal tract in large dense core vesicles (DCV), which apparently are released outside of the chemical synapse. Although no precise information is available on PDF, it is expected for neuropeptides to be released after high frequency stimulation, suggesting that during the silenced phase, the DCV would accumulate in the axonal terminals. It has been proposed that the trough of PDF accumulation at dusk might represent the depletion of the PDF readily releasable pool, and it correlates with the time of day when the sLNvs are most hyperpolarized. Interestingly, despite no release expected to occur while KIR is expressed, PDF intensity at the axonal terminals stayed at trough levels throughout the day, underscoring that reduced excitability affected additional steps such as peptide synthesis, processing, or transport. In favor of this possibility, hyperexcitation of the PDF circuit correlates with constantly high (daytime) PDF levels at the dorsal protocerebrum. Moreover, once kir expression was turned off, PDF levels resume to cycle, indicating a direct modulatory effect of membrane excitability on this specific output. In line with a defective output from the sLNvs, desynchronization of dorsal oscillators (i.e., the DN1s) became evident as early as in DD4. Gaining more insight into the mechanisms of communication within the circadian network, as well as those connecting the cell membrane with the molecular clock, will provide a better understanding on how these components interact to sustain temporal and spatial order to shape rhythmic overt behavior (Depetris-Chauvin, 2011).

    Taken together, these results confirm that in Drosophila, altering membrane excitability mainly affects the output of pacemaker cells and thus intercellular communication, as is the case in the eye of the mollusk Bulla and the rodent SCN, highlighting the degree of conservation in the mechanisms underlying the biological clock in distant organisms (Depetris-Chauvin, 2011).

    Circadian rhythm of temperature preference and its neural control in Drosophila

    A daily body temperature rhythm (BTR) is critical for the maintenance of homeostasis in mammals. Whereas mammals use internal energy to regulate body temperature, ectotherms typically regulate body temperature behaviorally. Some ectotherms maintain homeostasis via a daily temperature preference rhythm (TPR), but the underlying mechanisms are largely unknown. This study shows that Drosophila exhibit a daily circadian clock-dependent TPR that resembles mammalian BTR. Pacemaker neurons critical for locomotor activity are not necessary for TPR; instead, the dorsal neuron 2 s (DN2s), whose function was previously unknown, is sufficient. This indicates that TPR, like BTR, is controlled independently from locomotor activity. Therefore, the mechanisms controlling temperature fluctuations in fly TPR and mammalian BTR may share parallel features. Taken together, these results reveal the existence of a novel DN2-based circadian neural circuit that specifically regulates TPR; thus, understanding the mechanisms of TPR will shed new light on the function and neural control of circadian rhythms (Kaneko, 2012).

    Drosophila exhibit a daily TPR-low in the morning, high in the evening-that follows a similar pattern as in humans. This study is not only the first demonstration of fly TPR, but also the first systematic analysis of the molecular and neural mechanisms underlying TPR. TPR is controlled by the DN2s, which might explain why temperature preference remains rhythmic in LL. The DN2s do not express CRYPTOCHROME (CRY), a blue-light sensor crucial for circadian photoreception. Arrhythmicity in LL is caused by constant activation of CRY and thus constant degradation of Tim. Therefore, CRY-negative DN2 neurons may maintain residual rhythms in LL for a longer period of time than CRY-positive circadian neurons. To explore this possibility, immunostaining of brains were performed with Tim-antibody, and the staining levels of DN2 cells were analyzed in LL 4 days. Although Tim signal was found to be weakly rhythmic in DN2 neurons, these oscillations were not statistically significant. Further studies will thus be needed to verify that DN2 neurons maintain residual rhythm in LL. Because locomotor activity is controlled by CRY-positive circadian neurons and rapidly becomes arrhythmic in LL, the maintenance of TPR rhythms in LL also supports the conclusion that locomotor activity and TPR are controlled by independent circadian neural pathways (Kaneko, 2012).

    The data reveal striking parallel features between fly TPR and mammalian BTR, although the modes of heat production are not the same. Flies exhibit robust temperature increases during the daytime, which is the same phenomenon as diurnal mammalian BTR. Furthermore, ablation studies in rats show that BTR is controlled by SCN neurons that target a different subset of subparaventricular zone neurons than those that control locomotor activity. Thus, both fly TPR and mammalian BTR exhibit circadian clock dependent temperature fluctuations, independently regulated from locomotor activity. Taken together, these data raises the possibility that mammalian BTR and fly TPR are evolutionally conserved, which may be because temperature fluctuation in an animal's body is fundamental for maintenance of its homeostasis (Kaneko, 2012).

    Why do flies exhibit TPR? Flies probably exhibit TPR primarily to maintain homeostasis, like mammals. Mammalian BTR has been shown to have a clear interaction with sleep, and it has been reported that mechanisms controlling fly sleep are analogous to those controlling mammalian sleep. Therefore, fly TPR may also have a relationship with sleep. Intriguingly, it was observed that PER expression limited to pdf neurons can generate a weak TPR with an abnormal phase in per01 mutants, suggesting that pdf neurons may have a role in the TPR circuits. pdf-positive (sLNv and lLNv) neurons regulate sleep and sLNvs are known to project near the DN2s. Therefore, pdf neurons may be able to modulate DN2 activity even when these neurons are arrhythmic and may represent the neural basis for an interaction between TPR and sleep (Kaneko, 2012).

    Additionally, TPR may provide feedback to circadian pacemakers. Ambient temperature fluctuations can entrain not only peripheral clocks in mammals and flies but also circadian pacemaker neurons in the fly brain, which contribute to morning and evening locomotor activity. Because TPR can generate temperature fluctuations in the fly body, the output of TPR may thus reinforce or refine circadian rhythm entrainment. For circadian locomotor behavior, the DN2s could actually participate in the reinforcement, because in the larval brain the DN2s help the sLNvs entraining to temperature cycles. Therefore, by further exploring this newly discovered circadian rhythm, Drosophila TPR might not only help understanding the mechanisms underlying body temperature control in animals but also contribute to a greater understanding of circadian rhythm's mammalian CSLs plasticity (Kaneko, 2012).

    Circadian clock neurons constantly monitor environmental temperature to set sleep timing

    Circadian clocks organize biological processes to occur at optimized times of day and thereby contribute to overall fitness. While the regular daily changes of environmental light and temperature synchronize circadian clocks, extreme external conditions can bypass the temporal constraints dictated by the clock. Despite advanced knowledge about how the daily light-dark changes synchronize the clock, relatively little is known with regard to how the daily temperature changes influence daily timing and how temperature and light signals are integrated. In Drosophila, a network of approximately 150 brain clock neurons exhibit 24-hr oscillations of clock gene expression to regulate daily activity and sleep. This study shows that a temperature input pathway from peripheral sensory organs, which depends on the gene nocte, targets specific subsets of these clock neurons to synchronize molecular and behavioral rhythms to temperature cycles. Strikingly, while nocte1 mutant flies synchronize normally to light-dark cycles at constant temperatures, the combined presence of light-dark and temperature cycles inhibits synchronization. nocte1 flies exhibit altered siesta sleep, suggesting that the sleep-regulating clock neurons are an important target for nocte-dependent temperature input, which dominates a parallel light input into these cells. In conclusion, this study reveals a nocte-dependent temperature input pathway to central clock neurons and shows that this pathway and its target neurons are important for the integration of sensory light and temperature information in order to temporally regulate activity and sleep during daily light and temperature cycles (Chen, 2018).

    This study reveals a nocte-dependent and presumably chordotonal organ (Cho)-dependent temperature input to LNd and DN subsets of the Drosophila clock circuit. While it has previously been shown that nocte and Cho contribute to temperature entrainment of the circadian clock, it was not known which of the clock neurons are targeted by this peripheral sensory input pathway. The neuronal targets that have been identified in this study are largely consistent with previous studies showing that most of the clock neurons not expressing CRY are more responsive to fluctuating temperature signals compared to CRY+ cells. The lack of normal synchronized TIM expression in the DN1-DN3 and LNds of nocte1 flies shows that nocte-dependent temperature input serves as the main thermal entrainment signal for these cells. Interestingly, loss of the variant glutamate receptor IR25a, which impairs entrainment to temperature cycles (TCs), also strongly affected the DN1s and LNds, highlighting the importance of these neuronal groups for temperature entrainment. In fact, blocking synaptic transmission from the DN1 via expression of tetanus toxin interfered with behavioral synchronization to TCs. The low-amplitude oscillations of TIM in the LNv groups are either caused by alternative temperature inputs into these cells or result from the initial exposure to LD cycles before the flies were transferred to TCs (Chen, 2018).

    In addition to uncovering the neuronal targets of the NOCTE-Cho temperature pathway, this study revealed that its function is also important during more natural environmental situations, i.e., the combined presence of in-phase or conflicting TCs and LD cycles. This is very surprising, considering that nocte mutants synchronize normally to LD cycles at constant temperatures. During combined LD and TC conditions, the DN1-DN3 and LNd groups were most severely affected as evidenced by the lack (DN2) or severe amplitude reduction of synchronized TIM and PER-LUC oscillations. In contrast to the situation in DD and TC, all three exclusively CRY-positive LNv groups now exhibit synchronized TIM oscillations, which can be explained by CRY-mediated light resetting within these neurons. Only about 50% of the DN1s and LNds express CRY, so that the non-significant, low-amplitude TIM oscillations within these groups could potentially be explained by light synchronization within the CRY-positive neurons. However, this idea is not favored, because in the absence of LD cycles, imilar non-significant TIM oscillations were observed within these neuronal groups. Moreover, the CRY-negative DN3s exhibit the same low-amplitude oscillations in the presence of combined LD and TCs. Based on the observation that low-amplitude TIM oscillations are abolished in non-entrained and increased in weakly entrained nocte1 flies, it is argued that the lack of full penetrance contributes to the shallow TIM oscillations observed when both nocte1 phenotypic classes are averaged (Chen, 2018).

    Over the last few years, the DN1s have been shown to be a versatile subset of the clock neuronal circuit. They receive and integrate multiple environmental sensory inputs to regulate accurate timing of locomotor activity and sleep. Avery recent study elegantly shows that the DN1s are activated by very brief (<20 s) temperature reductions from 23°C to 16°C in a nocte- and Cho-dependent manner. Because nocte1 mutants or reducing nocte function in ChO impairs temperature entrainment, these rapid DN1 temperature responses most likely also contribute to the detection of long-term temperature fluctuations relevant for daily temperature entrainment (Chen, 2018).

    DN1s support morning activity at warmer (25°C) and evening activity at colder (20°C) temperatures, while at low light levels, they also support evening activity at 25°C, presumably revealing a light and temperature integration function of the DN1s. This study shows that the DN1s are among the clock neurons that are strongly affected by the lack of nocte function, both during 16°C:25°C TC and combined TCs and LD cycles, supporting a role for this neuronal group in light and temperature integration. The strong nocte1 effects on the CRY-positive LNvs during combined LD and TCs raise the possibility that they are coupled to the temperature-input-receiving LNd and DN groups. This would also explain the 'dominant-negative' effects of nocte1 on synchronization during combined LD and TCs. It is proposed that, in the presence of TCs, nocte prevents inappropriate signaling from the DN and LNd groups to the LNvs, thereby contributing to the correct integration of light and temperature (Chen, 2018).

    With regard to sleep regulation, temperature-dependent functions of the DN1 are more complex. In general, warmer temperatures increase daytime sleep and decrease sleep at night. The daytime increase in sleep depends on a functional clock and PER expression in the DN1s, showing that a subset of this heterogeneous neuronal group can have sleep-promoting functions. This DN1 subset presumably represses the activity-promoting LNvs and LNds via glutamate (Chen, 2018).

    This study shows that nocte1 flies exhibit significantly reduced siesta sleep (ZT3-ZT9) during the warm phase of combined TC and LD cycles-a condition disrupting molecular synchronization in the DN1. Although this study did not directly address a role for nocte in regulating DN1 function in sleep, these results are in line with a sleep-promoting function for the DN1s below 30°C. In the absence of light cues (TCs in DD), wild-type flies gradually initiate a period of inactivity and sleep during the warm phase starting after ZT6 and reaching peak inactivity and sleep levels toward the end of the warm phase at ZT10. During this interval (ZT6-ZT10) nocte1 flies show the exact opposite behavior and drastically decrease their sleep levels. This points again to a sleep-supporting role for the DN1s, in line with the results obtained for combined LD and TC synchronization and results by Yadlapalli (2018) showing that the DN1s promote sleep during ramped TCs. It is proposed that, in nocte1 flies, DN1 activity is altered during TCs, resulting in impaired behavioral and molecular synchronization, as well as in a reduced ability to repress activity promoting clock neurons (Chen, 2018).

    Drosophila temperature preference rhythms: An innovative model to understand body temperature rhythms

    Human body temperature increases during wakefulness and decreases during sleep. The body temperature rhythm (BTR) is a robust output of the circadian clock and is fundamental for maintaining homeostasis, such as generating metabolic energy and sleep, as well as entraining peripheral clocks in mammals. However, the mechanisms that regulate BTR are largely unknown. Drosophila are ectotherms, and their body temperatures are close to ambient temperature; therefore, flies select a preferred environmental temperature to set their body temperature. This study identified a novel circadian output, the temperature preference rhythm (TPR), in which the preferred temperature in flies increases during the day and decreases at night. TPR, thereby, produces a daily BTR. Fly TPR shares many features with mammalian BTR. Diuretic hormone 31 receptor (DH31R) was found to mediates Drosophila TPR, and the closest mouse homolog of DH31R, calcitonin receptor (Calcr), is essential for mice BTR. Importantly, both TPR and BTR are regulated in a distinct manner from locomotor activity rhythms, and neither DH31R nor Calcr regulates locomotor activity rhythms. These findings suggest that DH31R/Calcr is an ancient and specific mediator of BTR. Thus, understanding fly TPR will provide fundamental insights into the molecular and neural mechanisms that control BTR in mammals (Goda, 2019).

    A Robust and Self-Sustained Peripheral Circadian Oscillator Reveals Differences in Temperature Compensation Properties with Central Brain Clocks

    Circadian clocks are characterized by three properties: they run in constant conditions with a period of ∼24 h, synchronize to the environmental cycles of light and temperature, and are temperature compensated, meaning they do not speed up with temperature. Central brain clocks regulate daily activity rhythms, whereas peripheral clocks are dispersed throughout the body of insects and vertebrates. Using a set of luciferase reporter genes, this study shows that Drosophila peripheral clocks are self-sustained but over-compensated, i.e., they slow down with increasing temperature. In contrast, central clock neurons in the fly brain, both in intact flies and in cultured brains, show accurate temperature compensation. Although this suggests that neural network properties contribute to temperature compensation, the circadian neuropeptide Pigment Dispersing Factor (PDF) is not required for temperature-compensated oscillations in brain clock neurons. These findings reveal a fundamental difference between central and peripheral clocks, which likely also applies for vertebrate clocks (Versteven, 2020).

    Drosophila PSI controls circadian period and the phase of circadian behavior under temperature cycle via tim splicing

    The Drosophila circadian pacemaker consists of transcriptional feedback loops subjected to post-transcriptional and post-translational regulation. While post-translational regulatory mechanisms have been studied in detail, much less is known about circadian post-transcriptional control. Thus, this study targeted 364 RNA binding and RNA associated proteins with RNA interference. Among the 43 hits that were identified was the alternative splicing regulator P-element somatic inhibitor (PSI). PSI regulates the thermosensitive alternative splicing of timeless (tim), promoting splicing events favored at warm temperature over those increased at cold temperature. Psi downregulation shortens the period of circadian rhythms and advances the phase of circadian behavior under temperature cycle. Interestingly, both phenotypes were suppressed in flies that could produce TIM proteins only from a transgene that cannot form the thermosensitive splicing isoforms. Therefore, it is concluded that PSI regulates the period of Drosophila circadian rhythms and circadian behavior phase during temperature cycling through its modulation of the tim splicing pattern (Foley, 2019).

    Increasing evidence indicates that post-transcriptional mechanisms controlling gene expression are also critical for the proper function of circadian clocks in many organisms. In Drosophila, the post-transcriptional regulation of per mRNA has been best studied. per mRNA stability changes as a function of time. In addition, per contains an intron in its 3'UTR (dmpi8) that is alternatively spliced depending on temperature and lighting conditions. On cold days, the spliced variant is favored, causing an advance in the accumulation of per transcript levels as well as an advance of the evening activity peak. This behavioral shift means that the fly is more active during the day when the temperature would be most tolerable in their natural environment. The temperature sensitivity of dmpi8 is due to the presence of weak non-canonical splice sites. However, the efficiency of the underlying baseline splicing is affected by four single nucleotide polymorphisms (SNPs) in the per 3'UTR that vary in natural populations and form two distinct haplotypes. Also, while this splicing is temperature-sensitive in two Drosophila species that followed human migration, two species that remained in Africa lack temperature sensitivity of dmpi8 splicing. Furthermore, it has been recently demonstrated that the the trans-acting splicing factor B52 enhances dmpi8 splicing efficiency, and this effect is stronger with one of the two haplotypes. per is also regulated post-transcriptionally by the TWENTYFOUR-ATAXIN2 translational activation complex. This complex works by binding to per mRNA as well as the cap-binding complex and poly-A binding protein. This may enable more efficient translation by promoting circularization of the transcript. Interestingly, this mechanism appears to be required only in the circadian pacemaker neurons. Non-canonical translation initiation has also been implicated in the control of PER translation. Regulation of PER protein translation has also been studied in mammals, with RBM4 being a critical regulator of mPER1 expression. In flies however, the homolog of RBM4, LARK, regulates the translation of DBT, a PER kinase. miRNAs have emerged as important critical regulators of circadian rhythms in Drosophila and mammals, affecting the circadian pacemaker itself, as well as input and output pathways controlling rhythmic behavioral and physiological processes (Foley, 2019).

    RNA-associated proteins (RAPs) include proteins that either bind directly or indirectly to RNAs. They mediate post-transcriptional regulation at every level. Many of these regulated events - including alternative splicing, splicing efficiency, mRNA stability, and translation - have been shown to function in molecular clocks. Thus, to obtain a broad view of the Drosophila circadian RAP landscape and its mechanism of action, an RNAi screen was performed targeting 364 of these proteins. This led to the discovery of a role for the splicing factor P-element somatic inhibitor (PSI) in regulating the pace of the molecular clock through alternative splicing of tim (Foley, 2019).

    The results identify a novel post-transcriptional regulator of the circadian clock: PSI. PSI is required for the proper pace of both brain and body clock, and for proper phase-relationship with ambient temperature cycles. When Psi is downregulated, the circadian pacemaker speeds up and behavior phase under temperature cycles is advanced by 3 hr, and these phenotypes appear to be predominantly caused by an abnormal tim splicing pattern. Indeed, the circadian period and behavior phase of flies that can only produce functional TIM protein from a transgene missing most introns is insensitive to Psi downregulation. It is noted however that cwo's splicing pattern is also affected by Psi downregulation, and sgg splicing pattern was not studied, although it might also be controlled by PSI. It is therefore not possible to exclude a small contribution of non-tim splicing events to PSI downregulation phenotypes, or that in specific tissues these other splicing events play a greater role than in the brain (Foley, 2019).

    Interestingly, Psi downregulation results in an increase in intron inclusion events that are favored under cold conditions (tim-sc and tim-cold), while an intron inclusion event favored under warm conditions is decreased (tim-M). However, the ability of tim splicing to respond to temperature changes is not abolished when Psi is downregulated. This could imply that an as yet unknown factor specifically promotes or represses tim splicing events in a temperature-dependent manner. Another possibility is that the strength of splice sites or tim's pre-mRNA structure impacts splicing efficiency in a temperature-dependent manner. For example, suboptimal per splicing signals explain the lower efficiency of per's most 3' splicing event at warm temperature (Foley, 2019).

    How would the patterns of tim splicing affect the pace of the circadian clock, or advance the phase of circadian behavior under temperature cycles? In all splicing events that were studied, intron retention results in a truncated TIM protein. It is therefore possible that the balance of full length and truncated TIM proteins, which may function as endogenous dominant-negatives, determines circadian period. For example, truncated TIM might be less efficient at protecting PER from degradation, thus accelerating the pacemaker, or affecting its phase. Consistent with this idea, overexpression of the shorter cold-favored tim isoform (tim-sc) shortens period. Strikingly, Psi downregulation increases this isoform's levels and also results in a short phenotype. It has been proposed that production of tim-M transcripts (called tim-tiny in their study) delays the rate of TIM accumulation. Such a mechanism could also contribute to the short period observed when Psi is downregulated, since this reduces tim-M levels, which may accelerate TIM accumulation. Another interesting question is how PSI differentially affects specific splice isoforms of tim. One possibility is that the execution of a specific tim splicing event negatively influences the probability of the occurrence of other splicing events. For example, PSI could downregulate tim-sc and tim-cold by enhancing splicing and removal of the introns whose retention is necessary for production of these isoforms. This could indirectly reduce splicing of the intron that is retained in the warm tim-M isoform and result in tim-M upregulation. Conversely, PSI could directly promote tim-M intron retention and indirectly downregulate production of tim-sc and tim-cold (Foley, 2019).

    Other splicing factors have been shown to be involved in the control of circadian rhythms in Drosophila. SRm160 contributes to the amplitude of circadian rhythms by promoting per expression, while B52/SMp55 and PRMT5 regulate per's most 3' splicing, which is temperature sensitive. Loss of PRMT5 results in essentially arrhythmic behavior, but this is unlikely to be explained by its effect on per's thermosensitive splicing. B52/SMp55 knockdown flies show a reduced siesta, which is controlled by the same per splicing. With the identification of Psi, this study has uncover a key regulator of tim alternative splicing pattern and shows that this pattern determines circadian period length, while per alternative splicing regulates the timing and amplitude of the daytime siesta. Interestingly, a recent study identified PRP4 kinase and other members of tri-snRNP complexes as regulators of circadian rhythms. Downregulation of prp4 caused excessive retention of the tim-M intron. PSI and PRP4 might thus have complementary functions in tim mRNA splicing regulation, working together to maintain the proper balance of tim isoform expression (Foley, 2019).

    An unexpected finding is the role played by both PDF neurons and other circadian neurons in the short period phenotype observed with circadian locomotor rhythms when Psi was knocked-down. Indeed, it is quite clear from multiple studies that under constant darkness, the PDF-positive sLNvs dictate the pace of circadian behavior. Why, in the case of Psi downregulation, do PDF negative neurons also play a role in period determination? The explanation might be that PSI alters the hierarchy between circadian neurons, promoting the role of PDF negative neurons. This could be achieved by weakening PDF/PDFR signaling, for example (Foley, 2019).

    While this study focused on PSI, several other interesting candidates were identified in the screen. The presence of a large number of splicing factors is noted. This adds to the emerging notion that alternative splicing plays a critical role in the control of circadian rhythms. Several per splicing regulators have been mentioned that can impact circadian behavior. In addition, a recent study demonstrated that specific classes of circadian neurons express specific alternative splicing variants, and rhythmic alternative splicing is widespread in these neurons. Interestingly, in this study, the splicing regulator barc, which was identified in the screen and which has been shown to causes intron retention in specific mRNAs, was found to be rhythmically expressed in LNds. Moreover, in mammals, alternative splicing appears to be very sensitive to temperature, and could explain how body temperature rhythms synchronize peripheral clocks. Another intriguing candidate is cg42458, which was found to be enriched in circadian neurons (LNvs and Dorsal Neurons 1). In addition to emphasizing the role of splicing, the screen suggests that regulation of polyA tail length is important for circadian rhythmicity, since several members of the CCR4-NOT complex and deadenylation-dependent decapping enzymes were identified. Future work will be required to determine whether these factors directly target mRNAs encoding for core clock components, or whether their effect on circadian period is indirect. Interestingly, the POP2 deadenylase, which is part of the CCR4-NOT complex, was recently shown to regulate tim mRNA levels post-transcriptionally. It should be noted that while the screen targeted 364 proteins binding or associated with RNA, it did not include all of them. For example, LSM12, which was recently shown to be a part of the ATXN2/TYF complex, was not included in the screen because it had not been annotated as a potential RAP when the screen was initiated (Foley, 2019).

    In summary, this work provides an important resource for identifying RNA associated proteins regulating circadian rhythms in Drosophila. It identifies PSI is an important regulator of circadian period and circadian phase in response to thermal cycles, and points at additional candidates and processes that determine the periodicity of circadian rhythms (Foley, 2019).

    The Summer Is Coming: nocte and timeless Genes Are Influenced by Temperature Cycles and May Affect Aedes aegypti Locomotor Activity

    Mosquitoes exhibit activity rhythms, crucial for the transmission of pathogens, under the control of a circadian clock. Aedes aegypti is one of the world's leading vectors. For decades, several studies have linked the rise in ambient temperature with the increase in their activity. This study identified candidate genes whose expression is influenced by temperature cycles and may affect Aedes locomotor activity. timeless completely lost its rhythmic expression in light/dark, with out-of-phase temperature cycles, and by RNAi mediated knockdown of nocte, an important gene for Drosophila circadian synchronization by temperature cycles. Thus, timeless and nocte are important genes for synchronization by temperature cycles in Aedes aegypti. To reinforce these findings, the gradual temperature fluctuations that were as close as possible to daily temperature variations in Brazil were simulated in the laboratory. It was observed that the activity and the expression of the molecular circadian clock of Ae. aegypti differs significantly from that of mosquitoes subjected to constant or rectangular abrupt changes in temperature. It is suggested that for understanding the circadian behavior of Aedes with possible implications for intervention strategies, the seminatural paradigm needs to replace the traditional laboratory study (Teles-de-Freitas, 2020).

    Na(+)/Ca(2+) exchanger mediates cold Ca(2+) signaling conserved for temperature-compensated circadian rhythms

    Circadian rhythms are based on biochemical oscillations generated by clock genes/proteins, which independently evolved in animals, fungi, plants, and cyanobacteria. Temperature compensation of the oscillation speed is a common feature of the circadian clocks, but the evolutionary-conserved mechanism has been unclear. This study shows that Na(+)/Ca(2+) exchanger (NCX) mediates cold-responsive Ca(2+) signaling important for the temperature-compensated oscillation in mammalian cells. In response to temperature decrease, NCX elevates intracellular Ca(2+), which activates Ca(2+)/calmodulin-dependent protein kinase II and accelerates transcriptional oscillations of clock genes. The cold-responsive Ca(2+) signaling is conserved among mice, Drosophila, and Arabidopsis. The mammalian cellular rhythms and Drosophila behavioral rhythms were severely attenuated by NCX inhibition, indicating essential roles of NCX in both temperature compensation and autonomous oscillation. NCX also contributes to the temperature-compensated transcriptional rhythms in cyanobacterial clock. These results suggest that NCX-mediated Ca(2+) signaling is a common mechanism underlying temperature-compensated circadian rhythms both in eukaryotes and prokaryotes (Kon, 2021).

    Circadian TTFLs are an elaborate system that drives a wide range of overt rhythms with various phase angles and amplitudes. The oscillation speed of the TTFLs is temperature compensated, although many of the biochemical reactions in TTFLs are slowed down by decreasing temperature. This study demonstrates that the temperature compensation of the TTFL in mammalian cells was compromised when Ca2+-dependent phosphorylation signaling was inhibited. An important role was found for NCX-CaMKII activity as the state variable of the circadian oscillator. This present study and a series of preceding works demonstrate that the Ca2+ oscillator plays essential roles in the circadian oscillation mechanism. Functional studies clearly demonstrated essential roles of NCX-dependent Ca2+ signaling in the three important properties of the circadian clock, i.e., cell-autonomous oscillation, temperature compensation, and entrainment. The circadian Ca2+ oscillation is observed in mice lacking Bmal1 or Cry1/Cry2, implicating that the Ca2+ oscillator is an upstream regulator of the TTFL in mammals (Kon, 2021).

    The effects of NCX2 and NCX3 deficiencies on the regulation of mouse behavioral rhythms suggest involvement of Na+/Ca2+ exchanging activity in the Ca2+ dynamics of the SCN. Previous studies showed that L-type Ca2+ channel (LTCC) and voltage-gated Na+ channel (VGSC) are required for high-amplitude Ca2+ rhythms in the SCN. Because NCX activities are regulated by local concentrations of Na+/Ca2+ and the membrane potential, cooperative actions of LTCC, VGSC, and NCX seem to play important roles in generation mechanism of the robust Ca2+ oscillations in the SCN (Kon, 2021).

    It should be emphasized that the role of Ca2+/calmodulin-dependent protein kinases is conserved among clockworks in insects, fungi, and plants, suggesting that the Ca2+ oscillator might be a core timekeeping mechanism in their common ancestor (see Involvement of ancient Ca2+ signaling for temperature-compensated circadian rhythms). After divergence of each lineage, a subset of clock genes should have independently evolved in association with the Ca2+ oscillator. It is noteworthy that NCX is also required for temperature compensation of PTO-based cyanobacterial clock. Because intracellular Ca2+ in cyanobacteria is elevated in response to temperature decrease, YrbG-mediated Ca2+ signaling may regulate the PTO in vivo. Conservation of NCX among eukaryotes, eubacteria, and archaea suggests that NCX-dependent temperature signaling is essential for adaptation of a wide variety of organisms to environment. Further studies on NCX-regulated Ca2+ flux will provide evolutionary insights into the origin of the circadian clocks (Kon, 2021).

    Daily temperature cycles prolong lifespan and have sex-specific effects on peripheral clock gene expression in Drosophila melanogaster

    Circadian rhythms optimize health by coordinating the timing of physiological processes to match predictable daily environmental challenges. The circadian rhythm of body temperature is thought to be an important modulator of molecular clocks in peripheral tissues, but how daily temperature cycles impact physiological function is unclear. This study examined the effect of constant (25°C, T(CON)) and cycling (28°C/22°C during light/dark, T(CYC)) temperature paradigms on lifespan of Drosophila melanogaster, and the expression of clock genes, Heat shock protein 83 (Hsp83), Frost (Fst), and Senescence-associated protein 30 (smp-30). Male and female Drosophila housed at T(CYC) had longer median lifespans than those housed at T(CON) T(CYC) induced robust Hsp83 rhythms and rescued the age-related decrease in smp-30 expression that was observed in flies at T(CON), potentially indicating an increased capacity to cope with age-related cellular stress. Ageing under T(CON) led to a decrease in the amplitude of expression of all clock genes in the bodies of male flies, except for cyc, which was non-rhythmic, and for per and cry in female flies. Strikingly, housing under T(CYC) conditions rescued the age-related decrease in amplitude of all clock genes, and generated rhythmicity in cyc expression, in the male flies, but not the female flies. The results suggest that ambient temperature rhythms modulate Drosophila lifespan, and that the amplitude of clock gene expression in peripheral body clocks may be a potential link between temperature rhythms and longevity in male Drosophila Longevity due to T(CYC) appeared predominantly independent of clock gene amplitude in female Drosophila (Goh, 2021).

    A subset of DN1p neurons integrates thermosensory inputs to promote wakefulness via CNMa signaling

    Sleep is an essential and evolutionarily conserved behavior that is modulated by many environmental factors. Ambient temperature shifting usually occurs during climatic or seasonal change or travel from high-latitude area to low-latitude area that affects animal physiology. Increasing ambient temperature modulates sleep in both humans and Drosophila. Although several thermosensory molecules and neurons have been identified, the neural mechanisms that integrate temperature sensation into the sleep neural circuit remain poorly understood. This study reveals that prolonged increasing of ambient temperature induces a reversible sleep reduction and impaired sleep consolidation in Drosophila via activating the internal thermosensory anterior cells (ACs). ACs form synaptic contacts with a subset of posterior dorsal neuron 1 (DN1p) neurons and release acetylcholine to promote wakefulness. Furthermore, this study identified that this subset of DN1ps promotes wakefulness by releasing CNMamide (CNMa) neuropeptides to inhibit the Dh44-positive pars intercerebralis (PI) neurons through CNMa receptors. This study demonstrates that the AC-DN1p-PI neural circuit is responsible for integrating thermosensory inputs into the sleep neural circuit. Moreover, the CNMa signaling pathway was identified as a newly recognized wakefulness-promoting DN1 pathway (Jin, 2021).

    Sleep is an essential and evolutionarily conserved behavior across species and it is critical for synaptic plasticity, learning and memory, neuronal development, and metabolite clearance in both vertebrates and invertebrates. Sleep is a reversible status characterized by quiescence and an increased arousal threshold which is modulated by many environmental factors. Among these, temperature shifting is a common and essential factor that is associated with climatic or seasonal change or travel from high-latitude area to low-latitude area to affect the physiology of all animals. In humans, increased ambient temperature influences sleep patterns and correlates with reductions in rapid eye movement sleep. In Drosophila, numerous studies have shown that increasing ambient temperature modifies sleep architecture. However, how thermosensory input integrates into the sleep neural circuit remains unclear (Jin, 2021).

    Drosophila melanogaster is sensitive to temperature, and several thermosensory molecules with different temperature sensitivities have been identified, including TRPA1, Gr28b, Pyrexia, and Painless. Moreover, internal and external thermosensory neurons have also been identified in the brain and in the antenna. These thermosensory pathways enable Drosophila to adapt behavioral responses to attractive or noxious temperatures. Drosophila also exhibits sleep-like behavior and have been used to dissect the neural mechanism that regulates sleep as well as the neurobiological function of sleep. Dorsal neuron 1 (DN1) neurons include several subsets with distinct anatomical projection patterns that play distinct roles in waking and sleep behaviors. Recent studies have shown that DN1 neurons receive environmental temperature inputs. Thus, DN1 neurons might be a critical site to integrate various thermosensory inputs into the sleep neural circuit (Jin, 2021).

    This study shows that thermal-sensing anterior cell (AC) neurons monitor ambient temperature shifting and release acetylcholine (ACh) to promote wakefulness. This study demonstrates that the AC-DN1p-pars intercerebralis (PI) neural circuit integrates thermosensory inputs to promote wakefulness. Moreover, this study identified the CNMamide (CNMa) signaling as a newly recognized wakefulness-promoting signaling (Jin, 2021).

    Sleep is a reversible status characterized by quiescence and an increased arousal threshold. Thus, animals exhibit reduced alertness to risks during sleep. The increasing of ambient temperature usually occurs during climatic or seasonal change, but the excessively high temperature is fatal to animals. Increasing ambient temperature modifies sleep architecture, prolongs the latency for sleep onset, and reduces nighttime sleep. Warmth-induced wakefulness ensures that Drosophila increases their alertness to avoid fatal harm during ambient temperature shifting. It has been shown that increasing ambient temperature prolonged daytime sleep onset and increased total day sleep time and characterized the mechanism of warmth-induced morning wakefulness. This study characterized the effects of increased ambient temperature on nighttime sleep reduction and identified the circuit and molecular mechanisms of warmth-induced nighttime sleep reduction (Jin, 2021).

    Internal thermosensory AC neurons, expressing TRPA1 channels, have been shown to set preferred temperature before dawn and to regulate prolonged morning wakefulness. Moreover, loss of TRPA1 function affects siesta behavior, but not synchronization to temperature cycles. In addition, increasing ambient temperature with different protocols may induce different effects on the flies. Consistent with a the previous report, this study showed that both the strong loss-of-function mutant and depletion of TRPA1 in ACs exhibited the suppression in warmth-induced wakefulness (Jin, 2021).

    Although TrpA1ins-GAL4 is able to label TRPA1-expressing AC neurons, it also labels some other neurons that project to the same brain region as AC neurons. This study identified the NP5130-GAL4 driver that labels AC neurons and a pair of neurons in SOG. Previous GRASP analysis has shown that AC neurons form synaptic connections with DN1ps by using TrpA1ins-GAL4 and Clk4.1M-lexA lines. However, blocking the synaptic transmission of Clk4.1M+ DN1ps failed to suppress warmth-induced wakefulness. GRASP analysis narrowed down the neurons that synaptically contact with AC neurons by using NP5130-GAL4 and R18H11-lexA lines. Supportive to this connectivity, calcium-imaging studies and genetic and behavioral analysis further support that ACs make synaptic contact with a subset of DN1ps and promote wakefulness via ACh signaling. Consistent with the previous studies, this study reveals that R18H11+, but not Clk4.1M+ or R18H11+/Clk4.1M−, DN1ps promote warmth-induced wakefulness. These results suggest that R18H11+/Clk4.1M+ DN1ps are essential for warmth-induced wakefulness. The potential roles of R18H11−/Clk4.1M+ DN1 neurons in warmth-induced wakefulness need to be further investigated (Jin, 2021).

    DN1 neurons have been shown to integrate circadian, light, and temperature inputs to influence locomotor rhythms. Consistent with previous reports, consecutive calcium imaging reveals that acute heating rapidly inhibits CNMa+ DN1ps, whereas prolonged heating slowly increases Ca2+ level in CNMa+ DN1ps. These observations suggest that DN1ps receive multiple thermosensory inputs through different pathways. This study further reveals that CNMa signaling promotes warmth-induced wakefulness and demonstrates that DN1ps promote wakefulness via releasing the CNMa neuropeptide to inhibit Dh44+ PI neurons. Moreover, this study reveals that CNMa is involved in daytime sleep regulation and DN1 neurons also receive light inputs to influence locomotor rhythms. The mechanism of CNMa that regulates daytime sleep needs to be further investigated (Jin, 2021).

    To sum up, the AC-DN1p-PI neural circuit integrates thermosensory inputs to promote wakefulness. These newly identified circuits initiate a further road map to understand integrative mechanisms at the circuit level (Jin, 2021).

    SRP54 mediates circadian rhythm-related, temperature-dependent gene expression in Drosophila

    Recent studies have shown that alternative splicing (AS) plays an important role in regulating circadian rhythm. However, it is not clear whether clock neuron-specific AS is circadian rhythm dependent and what genetic and environmental factors mediate the circadian control of AS. By genome-wide RNA sequencing, SRP54 was identified is one of the Clock (Clk) dependent alternative splicing factors. Genetic interaction between Clock and SRP54 alleles showed that the enhancement of the circadian phenotype increased with temperature, being strongest at 29 °C and weakest at 18 °C. The alternative splicing and differential gene expression profile of Clock and SRP54 overlapped with the circadian-related gene profiles identified in various genome-wide studies, indicating that SRP54 is involved in circadian rhythm. By analyzing of the RNA-seq results at different temperatures, it was found that the roles of Clock and SRP54 are temperature dependent. Multiple novel temperature-dependent transcripts not documented in current databases were also found (Li, 2022).

    PERIOD Phosphoclusters Control Temperature Compensation of the Drosophila Circadian Clock

    Ambient temperature varies constantly. However, the period of circadian pacemakers is remarkably stable over a wide-range of ecologically- and physiologically-relevant temperatures, even though the kinetics of most biochemical reactions accelerates as temperature rises. This thermal buffering phenomenon, called temperature compensation, is a critical feature of circadian rhythms, but how it is achieved remains elusive. This study uncovered the important role played by the Drosophila PERIOD (PER) phosphodegron in temperature compensation. This phosphorylation hotspot is crucial for PER proteasomal degradation and is the functional homolog of mammalian PER2 S478 phosphodegron, which also impacts temperature compensation. Using CRISPR-Cas9, a series of mutations was introduced that altered three Serines of the PER phosphodegron. While all three Serine to Alanine substitutions lengthened period at all temperatures tested, temperature compensation was differentially affected. S44A and S45A substitutions caused undercompensation, while S47A resulted in overcompensation. These results thus reveal unexpected functional heterogeneity of phosphodegron residues in thermal compensation. Furthermore, mutations impairing phosphorylation of the pers phosphocluster showed undercompensation, consistent with its inhibitory role on S47 phosphorylation. It was observed that S47A substitution caused increased accumulation of hyper-phosphorylated PER at warmer temperatures. This finding was corroborated by cell culture assays in which S47A slowed down phosphorylation-dependent PER degradation at high temperatures, causing PER degradation to be excessively temperature-compensated. Thus, these results point to a novel role of the PER phosphodegron in temperature compensation through temperature-dependent modulation of the abundance of hyper-phosphorylated PER. This work reveals interesting mechanistic convergences and differences between mammalian and Drosophila temperature compensation of the circadian clock (Joshi, 2022).

    Natural populations of Drosophila melanogaster reveal features of an uncharacterized circadian property: the lower temperature limit of rhythmicity

    Most cyclic biological processes are under control of a circadian molecular timing system that synchronizes these phenomena to the 24-h day. One generic property of circadian-controlled processes is that they operate within a specific temperature range, below which the manifestation of rhythm ceases. Little is known about the evolutionary relevance of the lower temperature limit of rhythmicity or about the mechanism underlying the loss of overt circadian behavior below this lower limit, especially in one model organism of chronobiology, Drosophila melanogaster. Natural populations of Drosophila are evolving under divergent selection pressures and so provide a source of diversity necessary to address these issues. Using lines derived from African populations, this study found that there is natural variation in the expression of rhythmic behavior under low-temperature conditions. Evidence was found that this variability is evolutionarily relevant at extremely low temperature (12 degrees C) because high-altitude populations exhibit selection for locally adapted genomes that contribute to rhythmic behavior. Lines resistant to 15 degrees C show an additional layer of diversity in their response to temperature extremes because some lines are resistant to low temperature (15 degrees C) only, whereas others are cross-resistant to high and low temperature (15 degrees C and 30 degrees C). Genetic analysis of one cold-resistant circadian line at 15 degrees C reveals that the phenotype maps to the X-chromosome but not to the core clock genes, per and sgg. Analysis of the central clock cells of this line reveals that maintenance of rhythm is associated with robust clock function, which is compromised in a standard laboratory strain. These data indicate that the cold-resistant circadian phenotype is clock based. This study highlights the importance of using natural populations to inform us of the basic features of circadian traits, especially those that might be under temperature-based selection (Maguire, 2014).

    Drosophila Ionotropic Receptor 25a mediates circadian clock resetting by temperature

    Circadian clocks are endogenous timers adjusting behaviour and physiology with the solar day. Synchronized circadian clocks improve fitness and are crucial for physical and mental well-being. Visual and non-visual photoreceptors are responsible for synchronizing circadian clocks to light, but clock-resetting is also achieved by alternating day and night temperatures with only 2-4oC difference. This temperature sensitivity is remarkable considering that the circadian clock period (~24 h) is largely independent of surrounding ambient temperatures. This study shows that Drosophila Ionotropic Receptor 25a (IR25a) is required for behavioural synchronization to low-amplitude temperature cycles. This channel is expressed in sensory neurons of internal stretch receptors previously implicated in temperature synchronization of the circadian clock. IR25a is required for temperature-synchronized clock protein oscillations in subsets of central clock neurons. Extracellular leg nerve recordings reveal temperature- and IR25a-dependent sensory responses, and IR25a misexpression confers temperature-dependent firing of heterologous neurons. It is proposed that IR25a is part of an input pathway to the circadian clock that detects small temperature differences. This pathway operates in the absence of known 'hot' and 'cold' sensors in the Drosophila antenna, revealing the existence of novel periphery-to-brain temperature signalling channels (Chen, 2015).

    In Drosophila, daily activity rhythms are controlled by a network of ~150 clock neurons expressing the clock genes period (per) and timeless (tim). These encode repressor proteins that negatively feedback on their own promoters resulting in 24 h oscillations of clock molecules. Temperature cycles (TC) synchronize molecular clocks present in peripheral appendages in a tissue-autonomous manner, whereas synchronization of clock neurons in the brain mainly depends on peripheral temperature receptors located in the chordotonal organs (ChO) and the ChO-expressed gene nocte (Chen, 2015).

    To discover novel factors involved in temperature entrainment, this study identified NOCTE-interacting proteins by co-immunoprecipitation and mass-spectrometry. Focus was placed on IR25a, a member of a divergent subfamily of ionotropic glutamate receptors, and the interaction was verified by co-immunoprecipitation after overexpressing IR25a and NOCTE in all clock cells using tim-gal4 . IR25a is expressed in different populations of sensory neurons, including those in the antenna and labellum. In the olfactory system IR25a acts as a co-receptor with different odour-sensing IRs (Chen, 2015).

    To investigate if IR25a is co-expressed with nocte in ChO, IR25a expression was analyzed in femur and antennal ChO using an IR25a-gal4 line (Chen, 2015).

    IR25a-gal4-driven mCD8-GFP labelled subsets of ChO neurons in the femur, overlapped substantially with nompC-QF driven QUAS-Tomato signals (using the QF binary transcriptional activation system). nompC-QF is expressed in larval ChO18 and in the adult femur ChO. Comparison of IR25a-driven mCD8-GFP and nuclear DsRed signals with those of other ChO neuron drivers (F-gal4 and nocte-gal4 suggests that IR25a is expressed in a subset of femur ChO neurons and Johnston's Organ (JO) neurons. To determine if IR25a-gal4 ChO signals reflect endogenous IR25a expression, the presence of IR25a mRNA was confirmed in the femur and leg and the co-localization of anti-IR25a immunofluorescence signals in femur ChO neurons. IR25a was detected in ChO neuron cell bodies and ciliated dendrites, as was an mCherry-IR25a fusion protein expressed in these cells (Chen, 2015).

    As nocte mutants do not synchronize to temperature cycles in constant light (LL), IR25a-/- mutants were analyzed under these conditions. Unlike nocte, the IR25a-/- flies synchronized well to this regime and similar results were obtained at warmer temperature cycles. To test whether IR25a is specifically required for synchronization to small temperature intervals, IR25a-/- flies were subjected to various temperature cycles with an amplitude of only 2oC. Surprisingly, and in contrast to wild-type, IR25a-/- mutants did not synchronize to any of the shallow temperature cycles in LL or constant darkness (DD). In LL, wild-type and IR25 rescue flies showed a clear activity peak in the second part of the warm period before and after the 6 h shift of the temperature cycle. By contrast, IR25a-/- mutants were constantly active throughout the temperature cycle, apart from a short period of reduced activity at the beginning of the warm phase of TC1. In DD, control flies slowly advanced (or delayed) their evening activity peak during phase-advanced (or delayed) temperature cycles. The phase of this activity peak was maintained in the subsequent free-running conditions (DD, constant 25°C) indicating stable re-entrainment of the circadian clock. By contrast, IR25a mutants did not shift their evening peak during the temperature cycle, keeping their original phase throughout the experiment (Chen, 2015).

    To quantify entrainment in LL, the 'entrainment index' (EI) was determined, whereas for most DD experiments the phase difference of the main activity peak was calculated upon release into constant conditions between IR25a mutants and controls. In all 2oC amplitude temperature cycles tested the entrainment index of IR25a-/- flies was significantly lower, and phase calculation indicated no phase shift or a significantly reduced phase shift compared to controls. The same non-synchronization phenotype was observed in IR25a-/Df(IR25a) flies, and temperature synchronization was fully restored in IR25a-/- rescue flies. IR25a-/- mutants synchronize to light and have normal free-running and temperature compensated periods. These results suggest that IR25a enables the circadian clock to sense subtle temperature changes across the entire physiological range, rather than mediating synchronization to a specific range. Increasing the temperature cycle amplitude to 4oC consistently restored temperature entrainment in IR25a-/- flies (Chen, 2015).

    Temperature receptors located in fly antennae and arista are not required for temperature-synchronized behaviour. As expected, it was found that antennal IR25a function is not required for temperature entrainment. To reveal the importance of IR25a expression in ChO neurons, tissue-specific IR25a RNA interference (RNAi) was performed using validated transgenes. IR25a RNAi in all or subsets of ChO neurons. By contrast, IR25a RNAi in multidendritic, TRPA1-expressing or clock neurons did not impair temperature entrainment. These findings are consistent with the absence of IR25a expression in clock neurons and the brain and show that IR25a functions in ChO neurons for temperature entrainment to 25°C:27°C temperature cycles in LL (Chen, 2015).

    To identify the neural substrates underlying the lack of behavioural synchronization,clock protein levels was quantified in wild-type, IR25a-/-, and IR25a-/- rescue flies exposed to a shallow temperature cycle in LL. Although TIM expression was robustly rhythmic and synchronized in all clock neuronal groups in controls, TIM was barely detectable in the Dorsal Neuron 1 (DN1) and DN2 of IR25a-/- flies. Moreover, in the small and large ventral lateral neurons (s-LNv and l-LNv), TIM expression exhibited an additional peak during the warm phase. In the DN3, TIM declined earlier compared to controls and there was no effect on the dorsal lateral neurons (LNd). In temperature cycles and DD, TIM levels in DN1 were also blunted but oscillations in the DN2 and DN3 were similar to controls. In contrast to LL, TIM did not oscillate in any of the LN groups and was at constantly low levels. The alterations of TIM expression are temperature specific, as normal oscillations were observed in LD cycles at 25°C. An increase of the temperature cycle amplitude to 4°C also restored normal TIM expression in IR25a-/- flies, in agreement with the behavioural rescue. In summary, in low-amplitude temperature cycles, IR25a is required for normally synchronized TIM oscillations in DN1-3 and LNv in LL and in DN1 and LN clock neurons in DD (Chen, 2015).

    Tests were performed to see if the clock neurons affected by the lack of IR25a are indeed involved in regulating behavioural synchronization to shallow temperature cycles by blocking synaptic transmission using tetanus-toxin (TNT). Indeed, TNT-expression in DN1 and DN2 blocked synchronization in LL, whereas in DD only DN1 blockage interfered with temperature entrainment. Consistent with the differential effect on TIM oscillations in LL and DD these results strongly suggest that IR25a is required for the synchronized output of the DN1 (LL and DD) and DN2 (LL) to control temperature-entrained behaviour (Chen, 2015).

    Next, it was asked if ChO might directly sense temperature in an IR25a-dependent manner. Leg nerve activity was measured in restrained preparations and ChO units were identified in the compound signal. In both wild-type and -/- flies, spontaneous leg movement changed as a function of temperature along with motor and sensory activity. Additionally, presumed ChO activity of wild-type flies also increased during periods without movement. This temperature-induced but movement-independent, ChO activity was absent in -/- flies, showing that temperature is sensed in the legs in an IR25a-dependent manner. To test if IR25a contributes directly to temperature-sensing, this channel was expressed in the physiologically well-characterized, IR25a-negative, l-LNv. As a positive control, the temperature-sensitive Drosophila TRPA1 channel was also expressed in the l-LNv. Isolated brains were exposed to a temperature ramp, and spike frequency of individual l-LNv was recorded. Control l-LNv did not show a significant temperature-dependent change in neural activity. As expected, the firing rate of TRPA1 expressing neurons drastically increased linearly with temperature, as did other cellular parameters. IR25a expression resulted in a linear and reversible temperature-dependent increase in action potential firing frequency, whereas other cellular parameters showed no difference. Increasing the temperature by only 2-3°C also lead to a reversible increase in firing frequency in IR25a expressing l-LNv. By contrast, expression of the related, but olfactory-specific co-receptor IR8a (which is not required for temperature entrainment) did not confer temperature-sensitivity. These observations suggest that IR25a is at least part of a thermosensory receptor required for temperature entrainment (Chen, 2015).

    These data indicating that IR25a contributes to temperature sensing within ChO extend the roles of IR's beyond chemoreception, reminiscent of the requirement for the 'gustatory receptor' Gr28b in warmth-avoidance. Although this study shows that IR25a-expressing leg neurons are capable of sensing temperature and mediating temperature entrainment, it is possible that this receptor has a similar role elsewhere in the peripheral nervous system. IR25a responds to small temperature changes and it is proposed that the fly continuously integrates temperature signals received from multiple ChO across the whole body for synchronization of the clock. This potential reliance on weakly responding temperature receptors might explain why the Drosophila circadian clock is insensitive to brief temperature pulses, which could help maintain synchronized clock function in natural conditions of rapid and large temperature fluctuations (Chen, 2015).

    Harbison, S. T., Serrano Negron, Y. L., Hansen, N. F. and Lobell, A. S. (2017). Selection for long and short sleep duration in Drosophila melanogaster reveals the complex genetic network underlying natural variation in sleep. PLoS Genet 13(12): e1007098. PubMed ID: 29240764

    Selection for long and short sleep duration in Drosophila melanogaster reveals the complex genetic network underlying natural variation in sleep

    Why do some individuals need more sleep than others? Forward mutagenesis screens in flies using engineered mutations have established a clear genetic component to sleep duration, revealing mutants that convey very long or short sleep. Whether such extreme long or short sleep could exist in natural populations was unknown. This study applied artificial selection for high and low night sleep duration to an outbred population of Drosophila melanogaster for 13 generations. Night sleep duration diverged by 9.97 hours in the long and short sleeper populations, and 24-hour sleep was reduced to 3.3 hours in the short sleepers. Whole genome sequence data revealed several hundred thousand changes in allele frequencies at polymorphic loci across the genome. Combining the data from long and short sleeper populations across generations in a logistic regression implicated 126 polymorphisms in 80 candidate genes, and three of these genes and a larger genomic region were confirmed with mutant and chromosomal deficiency tests, respectively. Many of these genes could be connected in a single network based on previously known physical and genetic interactions. Candidate genes have known roles in several classic, highly conserved developmental and signaling pathways-EGFR, Wnt, Hippo, and MAPK. The involvement of highly pleiotropic pathway genes suggests that sleep duration in natural populations can be influenced by a wide variety of biological processes, which may be why the purpose of sleep has been so elusive (Harbison, 2017).

    Unexpected features of Drosophila circadian behavioural rhythms under natural conditions

    Circadian clocks have evolved to synchronize physiology, metabolism and behaviour to the 24-h geophysical cycles of the Earth. Drosophila melanogaster's rhythmic locomotor behaviour provides the main phenotype for the identification of higher eukaryotic clock genes. Under laboratory light-dark cycles, flies show enhanced activity before lights on and off signals, and these anticipatory responses have defined the neuronal sites of the corresponding morning (M) and evening (E) oscillators. However, the natural environment provides much richer cycling environmental stimuli than the laboratory, so this study sought to examine fly locomotor rhythms in the wild. Several key laboratory-based assumptions about circadian behaviour are not supported by natural observations. These include the anticipation of light transitions, the midday 'siesta', the fly's crepuscular activity, its nocturnal behaviour under moonlight, and the dominance of light stimuli over temperature. Also, a third major locomotor component in addition to M and E, which was termed 'A' (afternoon). Furthermore, it was shown that these natural rhythm phenotypes can be observed in the laboratory by using realistic temperature and light cycle simulations. The results suggest that a comprehensive re-examination of circadian behaviour and its molecular readouts under simulated natural conditions will provide a more authentic interpretation of the adaptive significance of this important rhythmic phenotype. Such studies should also help to clarify the underlying molecular and neuroanatomical substrates of the clock under natural protocols (Vanin, 2012).

    Identification of a dopamine pathway that regulates sleep and arousal in Drosophila

    Sleep is required to maintain physiological functions, including memory, and is regulated by monoamines across species. Enhancement of dopamine signals by a mutation in the dopamine transporter (DAT) decreases sleep, but the underlying dopamine circuit responsible for this remains unknown. This study found that the D1 dopamine receptor (DA1, also known as DopR) in the dorsal fan-shaped body (dFSB) mediates the arousal effect of dopamine in Drosophila. The short sleep phenotype of the DAT mutant was completely rescued by an additional mutation in the DA1 gene, but expression of wild-type DA1 in the dFSB restored the short sleep phenotype. Anatomical and physiological connections were found between dopamine neurons and the dFSB neuron. Finally, mosaic analysis with a repressive marker found that a single dopamine neuron projecting to the FSB activates arousal. These results suggest that a local dopamine pathway regulates sleep (Ueno, 2012).

    Neurons in the dFSB are involved in dopaminergic sleep regulation in Drosophila an the PPM3-FSB dopamine pathway, which is distinct from that required for memory formation, regulates arousal. A previous study found that the rescue of DA1 mutants outside of the mushroom body using a pan-neuronal GAL4 driver, elav-GAL4, coupled with the mushroom body suppressor MB-GAL80 in DA1dumb1 mutants can recover methamphetamine sensitivity. This suggests that dopamine regulates arousal outside of the mushroom body. Previous findings have shown that DA1 in the PDF neurons (lateral ventral neurons) regulates sleep-wake arousal and that DA1 in the ellipsoid body regulates stress-induced arousal (Ueno, 2012).

    A previous study identified PDF neurons that mediate the buffering effects of light on dopamine-induced arousal. However, the current ablation experiments showed that dopamine can elicit strong arousal effects without PDF neurons. The previous report used heterozygous DA1 mutant flies in a wild-type background for most experiments. However, this study used homozygous (null) DA1 mutants, as we found that the heterozygous DA1 mutants crossed with DATfmn showed an almost equivalent short sleep phenotype. Thus, one possible explanation for the difference between the previous study and this one is that heterozygous expression of DA1 in the FSB is sufficient to elicit the arousal effects of dopamine, which is also regulated partly through PDF neurons. Given that DA1 expression in the dFSB neurons alone is sufficient for the majority of the wake-promoting effects of dopamine, the arousal regulating dopamine pathway appears to converge at the FSB (Ueno, 2012).

    Activation of the dopamine neurons required for aversive memory formation had little effect on sleep. On the other hand, TrpA1 stimulation in a single FSB-projecting PPM3 dopamine neuron was able to reduce sleep. As the magnitude of the decrease in sleep by the activation of the single PPM3 neuron was smaller than that of most dopamine neurons with TH-GAL4, it is possible that other dopamine neurons, which were not labeled in MARCM screening, also affect arousal. For example, in the PPL1 cluster, at least five types of projections to the mushroom body and FSB-projecting neurons have been described (Ueno, 2012).

    However, in a MARCM experiment, many of the labeled neurons in the PPL1 cluster were seen to project to the alpha lobe of the mushroom body; it is possible that the contribution of other PPL1 neurons were not fully determine. Alternatively, combinatorial activation of the FSB-projecting PPM3 neurons and other dopamine neurons may have a synergistic effect. Further MARCM experiments using various clone induction protocols may help to formulate a more comprehensive characterization of single dopamine neurons. In addition, it is also possible that dopamine neurons that are not labeled by TH-GAL4 also have an arousal effect. It was noticed that TH-GAL4–induced GFP expression and tyrosine hydroxylase staining do not overlap completely, and some clusters, such as the PAM cluster, are covered only partially by TH-Gal4 (Ueno, 2012).

    The association between sleep and memory in Drosophila has recently been described in various reports. Other short sleep flies, hyperkinetic and calcineurin knockdown, also suffer from impaired memory. Sleep deprivation has been shown to impair aversive olfactory memory and learned phototaxis suppression. Conversely, memory formation increased sleep duration in normal flies, and this has not been observed in learning-deficient mutants. In the Drosophila brain, the expression of synaptic component proteins decreased during sleep and increased during waking, suggesting that sleep is required for the maintenance of synapses. In contrast, the current data imply a functional dissociation between sleep and memory circuits by different dopamine neurons. These results will provide clues to uncover a possible physiological relationship between them, for example, by the activation of only one to examine the causal relationship (Ueno, 2012).

    The FSB has been reported to have a role in visual memory processing, but its involvement in other behaviors is not yet fully understood. A recent report showed that activation of dFSB neurons induces sleep. This suggests that the dopamine signal regulates sleep via control of the neural properties of the dFSB neurons through DA1. In mammals, dopamine signaling via the D1-type dopamine receptor is thought to increase firing probability. However, dopamine signaling via the D1-like receptor DA1 in flies results in the inhibition of neural activity. Although the previous report suggested that the inhibition is independent of protein kinase A, activation of adenylyl cyclase with Gs* in dFSB decreased sleep levels. Taken together, these findings indicate that DA1 activation in the dFSB inhibits neural activity and results in the promotion of wakefulness. It has been reported that sleep induction through the activation of the dFSB neurons promotes memory consolidation after courtship conditioning. However, this study found that DA1 expression in the FSB itself had little effect on aversive olfactory memory. The contradiction between these findings might be a result of the difference in the memory tasks. It is also possible that functional interaction between sleep and memory is implemented downstream of the FSB. Further studies are required to elucidate the causal relationship between sleep and memory (Ueno, 2012).

    Short neuropeptide f is a sleep-promoting inhibitory modulator

    To advance the understanding of sleep regulation, a screen was performed in Drosophila for sleep-promoting cells, and neurons expressing neuropeptide Y-like short neuropeptide F (sNPF) were identifed. Sleep induction by sNPF meets all relevant criteria. Rebound sleep following sleep deprivation is reduced by activation of sNPF neurons, and flies experience negative sleep rebound upon cessation of sNPF neuronal stimulation, indicating that sNPF provides an important signal to the sleep homeostat. Only a subset of sNPF-expressing neurons, which includes the small ventrolateral clock neurons, is sleep promoting. Their release of sNPF increases sleep consolidation in part by suppressing the activity of wake-promoting large ventrolateral clock neurons, and suppression of neuronal firing may be the general response to sNPF receptor activation. sNPF acutely increases sleep without altering feeding behavior, which it affects only on a much longer time scale. The profound effect of sNPF on sleep indicates that it is an important sleep-promoting molecule (Shang, 2013).

    This study presents several independent lines of evidence indicating that sNPF acutely increases sleep and alters sleep homeostasis. This is because release of animals from sNPF neuron activation after several days of hypersomnolence resulted in a transient decrease in sleep or negative sleep rebound. Moreover, activation of sNPF neurons during mechanical sleep deprivation blunted the rebound sleep following the deprivation. This suggests that sNPF might alter the internal perception of sleep state during the deprivation despite an apparently behaviorally awake state. It also suggests that sNPF might directly modulate the sleep homeostat (Shang, 2013).

    The most potent in vivo manipulations of sNPF function, mutation of the sNPF gene and strong activation of sNPF neurons with dTRPA1, affect daytime as well as nighttime sleep levels. These manipulations also strongly alter sleep bout duration, a measure of consolidation, in the opposite direction to the sleep duration effects. More limited manipulations of sNPF signaling (cell-specific downregulation of sNPF levels or of sNPF signaling) indicate that sNPF is most important for promoting sleep at night. It also affects the structure of daytime sleep, a function of sNPF circuitry normally suppressed during the day by wake-promoting GABAergic neurons, acting via GABAA receptors. Suppression of excitability with Kir2.1 likely mimics this daytime GABAergic function. These results in aggregate suggest that sNPF action differs depending on the time of day, a result that supports the idea that daytime and nighttime sleep may be regulated by different circuitries (Shang, 2013).

    The role of sNPF in promoting more consolidated sleep is consistent with a general antiarousal function. As in mammals, Drosophila arousal can be measured electrophysiologically, but the most straightforward measure of arousal state is behavioral, and sleep fragmentation is indicative of a less stable, more easily aroused state. The main neurochemical previously implicated in fly arousal is DA, and l-LNvs play a prominent role in the arousal circuitry (Shang, 2013).

    Although the imaging assays indicated that sNPF alone did not lead to significant cAMP changes in the l-LNvs, it mildly suppressed the activation effect of DA on the l-LNvs. As one subset of clock neurons in the sleep circuit releases sNPF and promotes sleep at night and an adjacent subset responds to sNPF and suppresses nighttime sleep, sNPF may be used by the s-LNv-to-l-LNv pathway to coordinate the timing of sleep with other circadian behaviors. Indeed, sNPF mRNA is a potent cycling mRNA in s-LNvs (Kula-Eversole, 2010). Importantly, the electrophysiological assays in larval central neurons suggest that inhibition of neuronal firing may be a general feature of sNPF function and relevant to other sleep centers in addition to the clock neurons (Shang, 2013).

    sNPF and other sleep-relevant neuromodulators like DA are likely to act at multiple sites in the brain given the major state change effected by the sleep/wake transition. This expectation also reflects the modest effects of sNPFR manipulation within l-LNvs on total sleep time. Moreover, fan-shaped body neurons have recently been shown to be important for DA-mediated arousal (Liu, 2012; Ueno, 2012). The ability of these neuromodulators to act on many circuits may allow for more flexible integration of sleep with other behaviors and with other external and internal factors (Shang, 2013)

    An important influence on sleep is metabolic state. Indeed, sNPF facilitated the OA-to-DILP circuit, which may reflect its role in sleep/wake, feeding and/or metabolic regulation. However, the wake-promoting effect of activating the DILP pathway is context-dependent, occurring only in LD. Moreover, acute activation of octopaminergic neurons by dTRPA1 only mildly affects sleep and also in a condition-dependent manner, and feeding animals with octopamine only significantly suppresses total sleep after 2-€“3 days of exposure. Although long-term activation of octopaminergic neurons leads to long-lasting increases in food dwelling, these effects contrast sharply with the rapid and condition-independent effects seen with acute increases in dopamine signaling (Shang, 2011). Dopaminergic neurons have also been shown to be a critical part of NPF-regulated changes in satiety and response to food, and activation of these neurons indeed led to an immediate onset of food dwelling, which reversed rapidly upon dTRPA1 inactivation. As expected, tracker analysis shows that these food-dwelling flies also sleep very little, indicating that dopamine affects both sleep and feeding rapidly. These effects contrast with the slow effects on food dwelling by sNPF neuronal activation (Shang, 2013)

    The simplest interpretation of this slow food-dwelling response is that it is secondary to a more primary effect of sNPF on sleep. Indeed, a slow buildup in hunger or even starvation as a consequence of too much sleep is a simple explanation consistent with most if not all of these data. Behavioral effects as a secondary consequence of some other more direct effect is also an interpretation of many of the sleep effects of activation of peptidergic neurons seen in this study, in which only sNPF robustly increased sleep, i.e., under both LD and DD conditions. It is therefore suggested that a necessary condition for serious consideration of a molecule as behavior-relevant is a rapid response, which is also relatively condition independent. Dopamine as a wake-promoting molecule and now sNPF as a sleep-promoting molecule meet these criteria (Shang, 2013).

    Identification of Redeye, a new sleep-regulating protein whose expression is modulated by sleep amount

    This study reports a new protein involved in the homeostatic regulation of sleep in Drosophila. A forward genetic screen was conducted of chemically mutagenized flies to identify short-sleeping mutants, and one, redeye (rye; nicotinic Acetylcholine Receptor α4), was found that shows a severe reduction of sleep length. Cloning of rye reveals that it encodes a nicotinic acetylcholine receptor alpha subunit required for Drosophila sleep. Levels of RYE oscillate in light-dark cycles and peak at times of daily sleep. Cycling of RYE is independent of a functional circadian clock, but rather depends upon the sleep homeostat, as protein levels are up-regulated in short-sleeping mutants and also in wild type animals following sleep deprivation. It is proposed that the homeostatic drive to sleep increases levels of RYE, which responds to this drive by promoting sleep (Shi, 2014).

    The molecular mechanism of sleep homeostasis is a mystery and a subject of intense research in the sleep field. In addition to the investigation of mechanisms underlying sleep drive, considerable effort is being put into identifying biomarkers of sleep need. Based on what is known about the so-called sleep homeostat, which consists of increasing sleep pressure during wakefulness and dissipation of such pressure following sleep, it is suggested that a component or direct output of the homeostat should satisfy three criteria: (1) the gene product should regulate the sleep:wake cycle (i.e., genetic alleles of this gene should have some sleep phenotype); (2) expression levels or activity of the gene product should go up during wakefulness or during sleep deprivation and (3) expression levels or activity should decrease after sleep. The function of RYE and the molecular kinetics of the RYE protein largely satisfy these criteria. However, while RYE builds up during sleep deprivation, it does not accumulate gradually over the wake period in a daily cycle. Rather, it displays a marked increase close to the time of sleep onset, suggesting that it is not a central component of the homeostat, but responds to an upstream homeostatic signal, perhaps when that signal reaches a certain threshold. The fact that over-expression of RYE does not promote sleep also supports the idea that it is not the sleep-inducing homeostatic signal. Nevertheless, RYE is not simply a sleep output gene or sleep biomarker. It is required for implementation of signals from the homeostat and it functions to maintain sleep. Thus, it is proposed that rye is a sleep-regulating gene immediately downstream of the homeostat (Shi, 2014).

    It is suggested that RYE represents a molecular correlate of delta power, a characteristic of an electroencephalogram (EEG) that reflects sleep drive. Recently, a few other molecules were reported to change with sleep drive, but the effects were at the level of the mRNA, the magnitude of the increase was less than we report here for RYE and loss of the molecules did not affect baseline sleep duration. In addition, only one is expressed cyclically, indicating that others reflect sleep drive only under pathological conditions of sleep deprivation. RYE levels oscillate robustly in a daily cycle, although the phase is not as coherent as seen for circadian clock proteins. The timing of the peak varies within a temporal range, such that there is almost always a daytime peak and a night-time peak but not necessarily at the exact same time. It is suggested that RYE cycles under control of the sleep homeostat, which may not time behavior as precisely as the circadian clock, perhaps because sleep can be influenced by many factors. The variability in RYE cycling is particularly pronounced in short-sleeping mutants and in the ClkJrk circadian clock mutant, suggesting that the clock does influence RYE expression although it is not required for its cycling in an LD cycle. Interestingly, RYE cycles exclusively at the level of the protein, indicating translational or post-translational mechanisms. It is worth noting that a recently identified sleep regulator, Insomniac, is a component of specific protein degradation pathways in the cell. Although this study indicates that RYE cycling does not require Insomniac, it is possible that it is regulated by other protein turn-over machinery. Thus, translational/posttranslational regulation appears to be part of the mechanism of sleep homeostasis (Shi, 2014).

    This study shows that RYE not only reflects sleep drive, but is also required for sleep maintenance. Given that RYE is induced by sleep deprivation and it promotes sleep, one might expect over-expression of the protein to increase sleep. However, transgenic expression of rye in a wild type background does not increase sleep, suggesting that while rye is necessary, it is not sufficient for sleep onset. The possibility cannot be excluded that RYE functions together with other signals as part of the sleep-inducing homeostatic drive. On the other hand, it is also possible that transgenic expression does not produce adequate amounts of RYE protein in relevant cells. This might be the case if RYE is tightly regulated at the level of protein stability. For the moment, though, the parsimonious explanation noted above is preferred, that RYE is not part of the homeostat, but immediately downstream of it (Shi, 2014).

    Acetylcholine signaling has been long proposed as an arousal factor, as the nAChR complex is a cation channel that normally promotes neuronal activity and ACh is released during wakefulness in mammals. In contrast, this study indicates that at least one nAChR subunit (RYE) promotes sleep in the fly. There are more than 10 paralogs of nAChR subunits in the fly genome. One possibility is that RYE is expressed specifically in sleep promoting neurons, while other subunits of AChRs are in wake-promoting cells. An increase in ACh during wakefulness may contribute to the accumulation of sleep drive and to the increase in RYE. Alternatively, sleep drive may increase RYE independently of ACh, but in either scenario, RYE then promotes sleep. The precise site of RYE action is currently not known. rye-gal4 driven GFP is expressed widely in the brain, but it is not certain that endogenous RYE is as widespread, as an antibody was not effective in immunohistochemistry experiments, and GAL4 drivers are often quite promiscuous (Shi, 2014).

    Sleepless (SSS) was previously identified as a sleep promoting factor, essential for maintaining baseline sleep and for homeostatic rebound. An interaction between rye and sss is therefore not surprising. What is surprising is that overexpression of SSS promotes wakefulness in ryeT227M heterozygotes. SSS is a GPI-anchored protein that functions as a neuronal modulator. Previous studies indicate that SSS promotes activity of the voltage-gated potassium channel, Shaker. This study reports that SSS acts like a brake on nAChR (RYE) activity, as does Lynx-1, a SSS-like molecule in mammals. Although the data shown for Drosophila receptors used only the RYE α subunit, it is likely that SSS also inhibits activity of other Drosophila nAChR receptors. As both sssP1 (a null mutation) and sssP2 (a hypomorphic allele) are short-sleeping mutants, it is proposed that the overall effect of SSS is to promote sleep. The reduced sleep in sss mutants probably results from an increase of neuronal excitability, through inactivation of potassium channels (Shaker), or from hyperactivity of nAChR channels in wake-promoting neurons. Thus, typically the sleep-inhibiting effect of SSS, mediated through RYE, is masked by these other more dominant influences. However, in a sensitized background (i.e., rye/+), this effect is evident. RYE promotes sleep, and so loss of RYE results in a decrease in sleep, which is further impacted by SSS overexpression (Shi, 2014).

    It is noted that there are some caveats to these data. For instance, the ryeT227M allele could confer a neomorphic function that accounts for the interaction with sss. Likewise, the effects in oocytes could be non-physiological, not necessarily reflecting what happens in the fly brain. However, given that interactions were observed in these two very different types of assays, and both assays indicate repression of nAchR function by SSS, which is the effect predicted from the role of the mammalian SSS-like protein, Lynx1, it is believed SSS does indeed regulate nAchRs such as RYE. Interactions between SSS and nicotinic acetylcholine receptors are also confirmed by other recent unpublished studies (Shi, 2014).

    It is interesting that genes identified through independent genetic screens in Drosophila are turning out to interact with one another. SSS and Shaker were isolated independently as sleep-regulating genes, and subsequently shown to interact, and now it turns out that RYE interacts with SSS. Given that each of these genes represents a relatively infrequent hit in an unbiased screen, the interactions suggest that genetic approaches are converging upon specific sleep-regulating pathways. Interestingly, a recent Genome-wide association study for sleep-altering loci in humans identified significant effects of SNPs in an nAchR subunit as well as in a regulatory subunit of Shaker, suggesting that these mechanisms are also conserved across species (Shi, 2014).

    Wide awake mediates the circadian timing of sleep onset

    How the circadian clock regulates the timing of sleep is poorly understood. This study identifies a Drosophila mutant, wide awake (wake), that exhibits a marked delay in sleep onset at dusk. Loss of Wake in a set of arousal-promoting clock neurons, the large ventrolateral neurons (l-LNvs), impairs sleep onset. Wake levels cycle, peaking near dusk, and the expression of Wake in l-LNvs is Clock dependent. Strikingly, Clock and cycle mutants also exhibit a profound delay in sleep onset, which can be rescued by restoring Wake expression in LNvs. Wake interacts with the GABAA receptor Resistant to Dieldrin (Rdl), upregulating its levels and promoting its localization to the plasma membrane. In wake mutant l-LNvs, GABA sensitivity is decreased and excitability is increased at dusk. It is proposed that Wake acts as a clock output molecule specifically for sleep, inhibiting LNvs at dusk to promote the transition from wake to sleep (Liu, 2014).

    The molecular pathways by which the circadian clock modulates the timing of sleep are unknown. This study identified a molecule, Wide Awake, that promotes sleep and is required for circadian timing of sleep onset. The data argue for a direct role for the circadian oscillator in regulating sleep and support a model whereby Wake acts as a molecular intermediary between the circadian clock and sleep. In this model, Wake transmits timing information from the circadian clock to inhibit arousal circuits at dusk, thus facilitating the transition from wake to sleep. wake is transcriptionally upregulated by Clk activity, specifically in LNv clock neurons. Wake levels in l-LNvs rise during the day and peak at the early night, near the wake/sleep transition. This increase in Wake levels upregulates Rdl in l-LNvs, enhancing their sensitivity to GABA signaling and serving to inhibit the l-LNv arousal circuit. In this manner, cycling of Wake promotes cycling of the excitability of l-LNv cells. In wake mutants, l-LNvs lose this circadian electrical cycling; the higher firing rate of these cells at dusk leads to increased release of Pdf, which would act on Pdfr on downstream neurons to inhibit sleep onset. The identity of the GABAergic neurons signaling to the l-LNvs is currently unknown, but if they serve to convey information about sleep pressure from homeostatic circuits, the l-LNvs could serve as a site of integration for homeostatic and circadian sleep regulatory signals (Liu, 2014).

    Although Wake is expressed in clock neurons and its levels vary throughout the day, Wake itself is not a core clock molecule, since period length and activity rhythm strength are intact in wake mutants in constant darkness. The effects of Wake on sleep latency are not attributable to alterations in core clock function. In addition, because locomotor rhythm strength is intact in wake mutants, Wake is not a clock output molecule for locomotor rhythms. Rather, Wake is the first clock output molecule shown to specifically regulate sleep timing (Liu, 2014).

    Previous studies have demonstrated that Rdl in LNvs regulates sleep in Drosophila. This work further implicates Rdl as a key factor in the circadian modulation of sleep. In mammals, the localization and function of GABAA receptors are regulated by a variety of cytosolic accessory proteins, some of which are associated with the plasma membrane and cytoskeletal elements. The data suggest that Wake acts as an accessory protein for Rdl, upregulating its levels and promoting its targeting to the plasma membrane. Rdl is broadly expressed throughout the adult Drosophila brain, whereas Wake appears more spatially restricted. It is likely that Rdl is regulated by Wake in specific cells (e.g., Wake+ cells), while in other cells that express Rdl but not Wake, other factors are involved. Together, these data suggest a model in which increased GABA sensitivity is required in specific arousal circuits to facilitate rapid and complete switching between sleep/wake states at the appropriate circadian time (Liu, 2014).

    Intriguingly, the data, as well as data from the Allen Brain Atlas, suggest that the putative mouse homolog of Wake (ANKFN1) is enriched in the mouse SCN, the master circadian pacemaker in mammals. Specifically, ANKFN1 is expressed in the 'core' region of the SCN, which is analogous to the large LNvs in flies, in that it receives light input and its molecular oscillator does not cycle or cycles weakly in DD. These observations support a potential conservation of Wake function in regulating clock-dependent timing of sleep onset, which will be evaluated by ongoing genetic analysis in mice. The pronounced difficulty of wake flies to fall asleep at lights off is reminiscent of sleep-onset insomnia in humans. Moreover, the most widely used medications for the treatment of insomnia are GABA agonists. Thus, the identification of a molecule that mediates circadian timing of sleep onset by promoting GABA signaling may lead to a deeper understanding of mechanisms underlying insomnia and its potential therapies (Liu, 2014).

    Clock-generated temporal codes determine synaptic plasticity to control sleep

    Neurons use two main schemes to encode information: rate coding (frequency of firing) and temporal coding (timing or pattern of firing). While the importance of rate coding is well established, it remains controversial whether temporal codes alone are sufficient for controlling behavior. Moreover, the molecular mechanisms underlying the generation of specific temporal codes are enigmatic. This study shows in Drosophila clock neurons that distinct temporal spike patterns, dissociated from changes in firing rate, encode time-dependent arousal and regulate sleep. From a large-scale genetic screen, this study identified the molecular pathways mediating the circadian-dependent changes in ionic flux and spike morphology that rhythmically modulate spike timing. Remarkably, the daytime spiking pattern alone is sufficient to drive plasticity in downstream arousal neurons, leading to increased firing of these cells. These findings demonstrate a causal role for temporal coding in behavior and define a form of synaptic plasticity triggered solely by temporal spike patterns (Tabuchi, 2018).

    Understanding how the brain represents and processes information is a fundamental goal of neuroscience research. For the first half of the 20th century, the dominant neural coding model postulated that simple action potential (spike) counts in the relevant time window encode information about the environment or internal states (i.e., the rate-coding model). However, it has long been recognized that neural coding schemes using temporal codes (timing and/or pattern of spiking) would be computationally more powerful than traditional rate codes. In contrast to rate coding, which comprises a singular mechanism, temporal coding encompasses a diverse repertoire of coding schemes in individual or groups of neurons, ranging from latency to first spike to synchronization of oscillatory activity between spatially segregated neuronal populations (Tabuchi, 2018).

    A wide variety of temporal codes have now been observed to correlate with specific external stimuli in different settings, including sensory systems, hippocampal place cells, and neocortical circuits. However, the assessment of whether and how temporal codes embody neurobiologically relevant information is complicated by multiple factors, such as the concomitant presence of changes in firing rate, integration of spatial with temporal information, and the requirement for multiple interacting brain regions in the regulation of behavior. The rigorous demonstration of a causal role for temporal codes in representing biological information requires the fulfillment of three criteria. First, the temporal code should exist under native conditions or be elicited by naturalistic stimuli. Second, as articulated in the 'reader-actuator' model, a meaningful neural code should trigger a distinct response in the downstream neural circuit. Third, the temporal code should have physiological significance and be utilized by the brain to inform behavioral choices (Tabuchi, 2018).

    While many studies have identified temporal codes occurring in response to naturally occurring sensory stimuli, relatively few studies have shown that induction of specific temporal codes alter the firing of target neural circuits or affect behavior. For instance, in olfactory sensory neurons in mammals, varying the timing of firing relative to other neurons or the sniff cycle impacts firing of downstream neurons. However, other studies have found that temporal coding in olfactory, visual, and somatosensory systems had no effect on the activity of target neurons or behavioral readouts. Thus, the functional relevance for temporal coding alone to represent information about the environment and internal states remains controversial. Moreover, the molecular mechanisms that underlie the generation of different temporal codes within a neural circuit are largely unknown (Tabuchi, 2018).

    This study, in the clock neuron network in Drosophila, demonstrates the presence of naturally occurring temporal spiking patterns associated with daytime versus nighttime; the cycling of these patterns was found to depend on the core clock and wide awake (wake), a recently identified clock output gene required for circadian regulation of sleep. Using optogenetic approaches in vivo, this study shows that these distinct patterns of clock neuron firing, in the absence of changes in firing rate, serve as a temporal code to signify time-dependent arousal and directly impact sleep behavior. From a large-scale forward genetic screen, this study identified the molecular mechanisms underlying the generation of these clock-dependent temporal codes. Electrophysiological and computational analyses was used to delineate the biophysical processes that rhythmically shape spike morphology and tune the firing patterns of these clock neurons. Remarkably, the temporal spiking pattern alone was found to drive neural plastic changes, which mediate the transformation of temporal codes in clock neurons to increased firing of a downstream arousal circuit. Together, these data demonstrate a causal role for temporal coding in behavior that is mediated by a distinct form of synaptic plasticity specifically triggered by the pattern of neural spiking (Tabuchi, 2018).

    The molecular mechanisms underlying the generation of different temporal codes are largely unknown. This study shows that the circadian clock drives distinct temporal spiking patterns, as defined by the second-order temporal structure of interspike intervals, by adjusting ionic flux in clock neurons in a time-dependent manner. These changes are mediated by the clock output molecule WAKE, which controls the membrane targeting of SLOB and a Na+/K+ ATPase β subunit. This dynamic regulation of ionic flux leads to cycling of specific aspects of spike waveforms, which in turn induces the temporal spiking patterns seen during the day versus the night (Tabuchi, 2018).

    From a broader perspective, this work addresses a central issue in neuroscience: the functional importance of temporal codes in encoding information and impacting behavior. One challenge in demonstrating a causal role of temporal coding is identifying systems with a defined neural circuit where changes in the pattern or timing of spiking occur naturally, lead to measurable effects in target neurons, and regulate a specific behavior. An additional confounding factor is that information can be coded in a multiplexed manner with concurrent spatial, temporal, and rate-related features. This study shows that the Drosophila clock network fulfills these criteria and finds that time is encoded unidimensionally by the spiking patterns of these neurons in the absence of changes in firing rate or network timing (due to synchronization of neural firing within a cluster). Moreover, using computational, in vivo optogenetic, and electrophysiological approaches in these clock neurons, it was demonstrated that this temporal coding has functional consequences on the firing of a target arousal circuit and on sleep behavior. While the findings suggest that the irregular second-order spiking pattern is critical for this process, it is also possible that the temporal code consists of brief periods of faster spiking that are repeated over a >40-s time frame (Tabuchi, 2018).

    Previous work has demonstrated that WAKE is critical for clock-dependent regulation of sleep onset at dusk and that it upregulates and properly targets GABAA receptor to mediate this process by markedly suppressing the firing rate of clock neurons (Liu, 2014). Why would multiple neural coding mechanisms (rate coding changes at dusk and temporal coding at mid-day and mid-night) evolve to underlie circadian clock regulation of sleep at different times? One possibility relates to the dynamics of sleep onset versus sleep quality. Transitions between sleep and wake are major changes in brain state occurring on a relatively short timescale and hence may require dramatic changes in firing rate (i.e., rate coding) that are energetically costly. In contrast, maintenance of sleep quality occurs over hours; thus, it may be more energetically favorable for the relevant neurons to alter the pattern, instead of the rate, of their firing. Because of these potential energy savings, it is speculated that the use of temporal spiking patterns to encode information could be a broadly used mechanism for representing persistent internal states, such as hunger or emotion (Tabuchi, 2018).

    Finally, this study demonstrate that changes in the pattern of spike firing in the DN1ps, independent of changes in firing rate, triggers NMDA-receptor-dependent postsynaptic plasticity in the Dilp2+ PI neurons. Importantly, these data suggest a specific mechanism for inducing synaptic plasticity distinct from previously described processes that are dependent on changes in rate coding (e.g., long-term potentiation) or relative timing of individual spike events (e.g., STDP). These data represent one of the first examples of synaptic plasticity being induced specifically by the intrinsic temporal pattern of spiking, expanding the repertoire by which neural codes can generate plasticity. Together, these findings suggest that temporal patterns of spike firing are a crucial mechanism for driving neural plastic changes that mediate how internal states modulate behavior (Tabuchi, 2018).

    Circadian pacemaker neurons change synaptic contacts across the day

    Daily cycles of rest and activity are a common example of circadian control of physiology. In Drosophila, rhythmic locomotor cycles rely on the activity of 150-200 neurons grouped in seven clusters. Work from many laboratories points to the small ventral lateral neurons (sLNvs) as essential for circadian control of locomotor rhythmicity. sLNv neurons undergo circadian remodeling of their axonal projections, opening the possibility for a circadian control of connectivity of these relevant circadian pacemakers. This study shows that circadian plasticity of the sLNv axonal projections has further implications than mere structural changes. First, it was found that the degree of daily structural plasticity exceeds that originally described, underscoring that changes in the degree of fasciculation as well as extension or pruning of axonal terminals could be involved. Interestingly, the quantity of active zones changes along the day, lending support to the attractive hypothesis that new synapses are formed while others are dismantled between late night and the following morning. More remarkably, taking full advantage of the GFP reconstitution across synaptic partners (GRASP) technique, this study showed that, in addition to new synapses being added or removed, sLNv neurons contact different synaptic partners at different times along the day. These results lead to a proposal that the circadian network, and in particular the sLNv neurons, orchestrates some of the physiological and behavioral differences between day and night by changing the path through which information travels (Gorostaza, 2014).

    Circadian remodeling of the small ventral lateral neuron (sLNv) dorsal terminals was first described at the peak and trough levels of pigment-dispersing factor (PDF) immunoreactivity, that is at zeitgeber time 2 (ZT2) and ZT14 (2 hr after lights ON and lights OFF, respectively), as well as their counterparts under constant darkness (DD) (circadian time 2 [CT2] and CT14). For a more precise examination of the extent of structural remodeling, a time course was carried out. An inducible GAL4 version termed GeneSwitch restricted to PDF neurons (pdf-GS) combined with a membrane-tethered version of GFP (mCD8GFP) was used as control. As expected from the original observations, a significant reduction in complexity of the axonal arbor-measured as total axonal crosses-could be seen between CT2 and CT14 and between CT18 and CT22, which remained unchanged at nighttime. However, toward the end of the subjective night (CT22), the primary processes appeared to be shorter. To more precisely describe this additional form of plasticity, the length of the maximum projection was measure from the lateral horn toward the midbrain. This analysis revealed that toward the end of the subjective night (CT22), PDF projections are significantly shorter than at the beginning of the day (CT2). These observations imply that mechanisms other than the proposed changes in the degree of fasciculation are recruited during circadian plasticity. To get a deeper insight into the nature of the phenomena, the changes were monitored in brain explants kept in culture for 48 hr after dissection. Transgenic pdf-GAL4; UAS-mCD8RFP flies (referred to as pdf>RFP) were dissected under safe red light, and brains were maintained under DD. Imaging of individual brains at two different time points highlighted three types of changes experienced by axonal terminals: (1) changes in the degree of fasciculation/defasciculation, more common in primary branches, (2) the addition/retraction of new processes, mostly affecting those of secondary or tertiary order, and (3) positional changes of minor terminals, thus confirming and extending previous observations. Altogether, these results indicate that a rather complex remodeling process takes place on daily basis in the axonal terminals of PDF neurons (Gorostaza, 2014).

    The level of structural remodeling occurring at the dorsal terminals suggested that synapses themselves could undergo changes in a time-dependent fashion. The presynaptic protein Synaptotagmin (SYT) was examined at different times across the day as an indicator of vesicle accumulation. A GFP-tagged version of SYT was expressed in PDF neurons (pdf >sytGFP), and both the number and area span by SYT+ puncta (most likely describing the accumulation of several dense core vesicles) were analyzed separately at the sLNv dorsal terminals. No statistical differences were observed in the number of SYT+ puncta (although there is a tendency for higher numbers in the early morning), perhaps as a result of the nature of the signal, which is too diffuse for precise identification of individual spots. On the other hand, SYT+ puncta were larger and, as a result, the area covered by SYT+ immunoreactivity was significantly different at CT2 compared to CT14, but not between CT22 and CT2, perhaps reflecting that vesicles started to accumulate at the end of the day in preparation for the most dramatic membrane change taking place between CT22 and the beginning of the following morning (Gorostaza, 2014).

    The observation that a more complex structure correlated with a larger area covered by presynaptic vesicles reinforced the notion that indeed the number of synapses could be changing throughout the day and prompted analysis of Bruchpilot (BRP), a well-established indicator of active zones. Expressing a tagged version of BRP in PDF neurons, the number of BRP+ puncta was quantitated as a proxy for active zones at times when the most dramatic changes in structure had been detected (i.e., CT2, CT14, and CT22). Interestingly, the number of active zones was significantly larger at CT2 than at CT14 or CT22; in fact, no statistical differences were observed between the last two time points, underscoring that axonal remodeling can occur (i.e., pruning of major projections taking place toward the end of the night) without significantly affecting overall connectivity. Thus, circadian structural plasticity is accompanied by changes in the number of synapses. Not only are more vesicles recruited toward CT2, but also a higher number of active zones are being established (Gorostaza, 2014).

    Circadian changes in the abundance of the presynaptic active zone BRP have also been shown in the first optic neuropil of the fly brain, although BRP abundance in the lamina increases in the early night under DD conditions, in contrast to the oscillations in BRP levels observed at the dorsal protocerebrum that peak in the early subjective day just described. In addition, rhythmic changes in the number of synapses have also been described in the terminals of adult motor neurons in Drosophila examined through transmission electron microscopy, as well as BRP+ light confocal microscopy, underscoring the validity of the approach employed herein. Interestingly, in different brain areas, the level of presynaptic markers (such as BRPRFP or SYTGFP) also changes in response to the sleep/wake 'state,' being high when the animals are awake and lower during sleep; this observation led to the proposal that sleep could be involved in maintaining synaptic homeostasis altered during the awaking state. This trend coincides with observation of higher levels during the subjective morning and lower levels at the beginning of the subjective night; however, no changes were detected through the night, suggesting that, at least in clock neurons, there is a circadian rather than a homeostatic control of synaptic activity. Given that clock outputs are predominantly regulated at the transcriptional level and that there is circadian regulation of MEF2, a transcription factor that turns on a program involved in structural remodeling, this correlation opens the provocative possibility that the circadian clock is controlling the ability of assembling novel synapses in particularly plastic neurons, which might become recruited and/or stabilized, or otherwise pruned (disassembled), toward the end of the day (Gorostaza, 2014).

    Adult-specific electrical silencing of PDF neurons reduces the complexity of dorsal arborizations, although a certain degree of circadian remodeling of the axonal terminals still takes place. To examine whether electrical alterations could affect circadian changes in the number of active zones, either Kir2.1 or NaChBac was expressed (to hyperpolarize or depolarize PDF neurons, respectively). To avoid any undesired developmental defects, pdf-GS was used to drive expression of the channels only during adulthood. Interestingly, Kir2.1 expression abrogated circadian changes in the number of active zones. In fact, PDF neurons displayed a reduced number of active zones compared to controls at CT2 and remained at similar levels throughout the day, indistinguishable from nighttime controls. On the other hand, when neurons were depolarized through NaChBac expression, the number of active zones did not change along the day and was maintained at daytime levels even at CT14 and CT22 (Gorostaza, 2014).

    It has recently been shown that MEF2, a transcription factor involved in activity-dependent neuronal plasticity and morphology in mammals, is circadianly regulated and mediates some of the remodeling of PDF dorsal terminals through the regulation of Fasciclin2. In contrast, adult-specific silencing (and depolarization) of PDF neurons abolishes cycling in the number of BRP+ active zones, despite the fact that it does not completely obliterate the remodeling of the axonal terminals, suggesting that some of the mechanisms underlying structural plasticity are clearly activity independent and are most likely the result of additional clock-controlled output pathways still to be identified (Gorostaza, 2014).

    Since structural remodeling of PDF neurons results in the formation and disappearance of new synapses on daily basis, it was anticipated that not only the number but also the postsynaptic partners of these contacts could concomitantly be changing. To shed light on this possibility, GFP reconstitution across synaptic partners (GRASP), which labels contacts between adjacent membranes, was used. In brief, two complementary fragments of GFP tethered to the membrane are expressed in different cells. If those cells are in contact, GFP is reconstituted and becomes fluorescent. GRASP has previously been employed to monitor synapses in adult flies. Given the complex arborization at the dorsal protocerebrum, it was asked whether specific subsets of circadian neurons projecting toward that area could be contacting across the day. Perhaps not surprisingly, an extensive reconstituted GFP signal could be observed between the sLNv dorsal projections and those of the posterior dorsal neuron 1 cells (DN1ps, lighted up by the dClk4.1-GAL4 line, suggesting contacts along the entire area, which are detectable across all time points analyzed (ZT2, ZT14, and ZT22). Consistent with these observations, extensive physical contact between the sLNv projections and those of the DN1p neurons has just been reported at the dorsal protocerebrum with no clear indication of the time of day examined. Next the study examined whether a subset of dorsal LNs (LNds), projecting toward both the accessory medulla and the dorsal protocerebrum (through the combined expression of Mai179-GAL4; pdf-GAL80), could also contact the profuse dorsal arborization of sLNv neurons; this genetic combination enables expression of split-GFP in a restricted number of circadian cells (which are part of the evening oscillator, i.e., up to four LNds, including at least a CRYPTOCHROME-positive one, and the fifth sLNv), as well as others located within the pars intercerebralis (PI), a neurosecretory structure recently identified as part of the output pathway relevant in the control of locomotor behavior. In contrast to the extensive connections between DN1p and sLNv clusters, only very discreet reconstituted puncta were detected. Quite strikingly, the degree of connectivity appeared to change across the day, reaching a maximum (when almost every brain exhibited reconstituted signal) at ZT22. However, due to the nature of the signal, no quantitation of its intensity was attempted. Although a more detailed analysis is required to define the identity (i.e., whether it is one or several LNds, the fifth sLNv, or both groups that directly contact the sLNvs), this finding highlights a potentially direct contact between the neuronal substrates of the morning and evening oscillators. In sum, through GRASP analysis, this study has begun to map the connectivity within the circadian network; commensurate with a hierarchical role, the sLNvs appear to differentially contact specific subsets in a distinctive fashion (Gorostaza, 2014).

    To address the possibility that PDF neurons could be contacting noncircadian targets at different times across the day, an enhancer trap screen was carried out employing a subset of GAL4 enhancers selected on the basis of their expression pattern in the adult brain, i.e., known to drive expression in the dorsal protocerebrum, and an additional requirement imposed was that none of the selected GAL4 lines could direct expression to the sLNv neurons to avoid internal GFP reconstitution. Reconstitution of the GFP signal at the sLNv dorsal terminals by recognition through specific antibodies was assessed at three different time points for each independent GAL4 line (ZT2, ZT14, and ZT22). Some of the GAL4 lines showed reconstituted GFP signal at every time point analyzed, suggesting that those neuronal projections are indeed in close contact across the day and might represent stable synaptic contacts. No GFP signal was detected in the negative parental controls. Despite the fact that several GAL4 drivers directed expression to the proximity of the PDF dorsal terminals, some of the selected lines did not result in reconstituted GFP signal (Gorostaza, 2014).

    Quite remarkably, a proportion of the GAL4 lines showed GFP+ signal only at a specific time point. One such example is line 3-86, where reconstitution was detected in most of the brains analyzed at ZT2, but not at nighttime. Being able to identify putative postsynaptic contacts to the sLNvs in the early morning is consistent with the observation of a higher number of BRP+ active zones in the early day. This enhancer trap spans different neuropils, such as the mushroom body (MB) lobes and lateral horn, and directs expression to particularly high levels in the PI, a structure that has recently been implicated in the rhythmic control of locomotor activity. In fact, some yet unidentified somas in the PI appear to arborize profusely near the PDF dorsal terminals, underscoring a potential link between the two neuronal groups. These direct contacts are unlikely to be the ones reported by Mai179-GAL4; pdf-GAL80 since those connect to the sLNv neurons preferentially at night. Interestingly, a subset of neurons in the PI is relevant in mediating the arousal promoting signal from octopamine; in addition, sleep promoting signals are also derived from a different subset of neurons in the PI, opening the attractive possibility that both centers could be under circadian modulation (Gorostaza, 2014).

    GRASP analysis also uncovered a different neuronal cluster (4-59) that contacts PDF neurons preferentially during the early night (ZT14), which is in itself striking, since this time point corresponds to that with fewer arborizations and an overall decrease in the number of synapses. This enhancer trap is expressed in the MBs, subesophagic ganglion, antennal lobes, and accessory medulla. Among those structures, the MBs are important for higher-order sensory integration and learning in insects. Interestingly, circadian modulation of short-term memory and memory retrieval after sleep deprivation has been reported; short-term memory was found to peak around ZT15-ZT17, coinciding with the window of GFP reconstitution, thus providing a functional connection to the synaptic plasticity observed. To corroborate whether there is a direct contact between the two neuronal clusters, the extensively used GAL4 driver OK107, which is expressed in the α'/β'and the γ lobes of the MBs and to a lower extent in the PI, was employed for GRASP analysis. Surprisingly, reconstituted GFP signal could be observed at every time point analyzed, suggesting that MB lobes contact PDF neurons throughout the day but that specific clusters (for example those highlighted by the 4-59 line) establish plastic, time-of-day-dependent physical contact with PDF neurons (Gorostaza, 2014).

    It was next asked whether these prospective postsynaptic targets of PDF neurons could play a role in the output pathway controlling rhythmic locomotor activity. To address this possibility, the impact of adult-specific alteration of excitability of distinct neuronal groups was examined through expression of TRPA1. Interestingly, adult-specific depolarization of specific neuronal populations triggered a clear deconsolidation of the rhythmic pattern of activity, which resulted in less-rhythmic flies accompanied by a significant decrease in the strength of the underlying rhythm. These results lend support to the notion that the underlying neuronal clusters are relevant in the control of rest/activity cycles (Gorostaza, 2014).

    Over the years, it has become increasingly clear that the circadian clock modulates structural properties of different cells. In fact, a number of years ago, it was reported that the projections of a subset of core pacemaker fly PDF+ and mammalian VIP+ neurons undergo structural remodeling on daily basis. The work presented in this study lends support to the original hypothesis that circadian plasticity represents a means of encoding time-of-day information. By changing their connectivity, PDF neurons could drive time-specific physiological processes. As new synapses assemble while others are dismantled, the information flux changes, allowing PDF neurons to promote or inhibit different processes at the same time. This type of plasticity adds a new level to the complex information encoded in neural circuits, where PDF neurons could not only modulate the strength in the connectivity between different partners, but also define which neuronal groups could be part of the circadian network along the day. Although further analysis of the underlying process is ensured, evidence so far supports the claim that structural plasticity is an important circadian output (Gorostaza, 2014).

    Genetic rescue of functional senescence in synaptic and behavioral plasticity

    Aging has been linked with decreased neural plasticity and memory formation in humans and in laboratory model species such as the fruit fly, Drosophila melanogaster. This study examined plastic responses following social experience in Drosophila as a high-throughput method to identify interventions that prevent these impairments. Young (5-day old) or aged (20-day old) adult female Drosophila were housed in socially enriched or isolated environments, then assayed for changes in sleep and for structural markers of synaptic terminal growth in the ventral lateral neurons (LNVs) of the circadian clock. When young flies are housed in a socially enriched environment, they exhibit synaptic elaboration within a component of the circadian circuitry, the LNVs, which is followed by increased sleep. Aged flies, however, no longer exhibit either of these plastic changes. Because of the tight correlation between neural plasticity and ensuing increases in sleep, sleep after enrichment was used as a high-throughput marker for neural plasticity to identify interventions that prolong youthful plasticity in aged flies. To validate this strategy, three independent genetic manipulations were used that delay age-related losses in plasticity: (1) elevation of dopaminergic signaling, (2) over-expression of the serum response factor transcription factor blistered (bs) in the LNVs, and (3) reduction of the Imd immune signaling pathway. These findings provide proof-of-principle evidence that measuring changes in sleep in flies after social enrichment may provide a highly scalable assay for the study of age-related deficits in synaptic plasticity. These studies demonstrate that Drosophila provides a promising model for the study of age-related loss of neural plasticity and begin to identify genes that might be manipulated to delay the onset of functional senescence (Donlea, 2014a).

    The MAP kinase p38 is part of Drosophila melanogaster's circadian clock

    All organisms have to adapt to acute as well as to regularly occurring changes in the environment. To deal with these major challenges organisms evolved two fundamental mechanisms: the p38 mitogen-activated protein kinase (MAPK) pathway, a major stress pathway for signaling stressful events, and circadian clocks to prepare for the daily environmental changes. Both systems respond sensitively to light. Recent studies in vertebrates and fungi indicate that p38 is involved in light-signaling to the circadian clock providing an interesting link between stress-induced and regularly rhythmic adaptations of animals to the environment, but the molecular and cellular mechanisms remained largely unknown. This study demonstrates by immunocytochemical means that p38 is expressed in Drosophila melanogaster's clock neurons and that it is activated in a clock-dependent manner. Surprisingly, it was found that p38 is most active under darkness and, besides its circadian activation, additionally gets inactivated by light. Moreover, locomotor activity recordings revealed that p38 is essential for a wild-type timing of evening activity and for maintaining approximately 24 h behavioral rhythms under constant darkness: flies with reduced p38 activity in clock neurons, delayed evening activity and lengthened the period of their free-running rhythms. Furthermore, nuclear translocation of the clock protein Period was significantly delayed on the expression of a dominant-negative form of p38b in Drosophila's most important clock neurons. Western Blots revealed that p38 affects the phosphorylation degree of Period, what is likely the reason for its effects on nuclear entry of Period. In vitro kinase assays confirmed the Western Blot results and point to p38 as a potential 'clock kinase' phosphorylating Period. Taken together, these findings indicate that the p38 MAP Kinase is an integral component of the core circadian clock of Drosophila in addition to playing a role in stress-input pathways (Dusik, 2014 - Open access: 25144774).

    Class IIa histone deacetylases are conserved regulators of circadian function

    Class IIa histone deacetylases (HDACs) regulate the activity of many transcription factors to influence liver gluconeogenesis and the development of specialized cells including muscle, neurons and lymphocytes. This study describes a conserved role for class IIa HDACs in sustaining robust circadian behavioral rhythms in Drosophila and cellular rhythms in mammalian cells. In mouse fibroblasts, over-expression of HDAC5 severely disrupts transcriptional rhythms of core clock genes. HDAC5 over-expression decreases BMAL1 acetylation on Lys537 and pharmacological inhibition of Class IIa HDACs increases BMAL1 acetylation. Furthermore, cyclical nucleocytoplasmic shuttling of HDAC5 was observed in mouse fibroblasts that is characteristically circadian. Mutation of the Drosophila homolog HDAC4 impairs locomotor activity rhythms of flies and decreases period mRNA levels. RNAi-mediated knockdown of HDAC4 in Drosophila clock cells also dampens circadian function. Given that the localization of Class IIa HDACs is signal-regulated and influenced by Ca2+ and cAMP signals, these findings offer a mechanism by which extracellular stimuli that generate these signals can feed into the molecular clock machinery (Fogg, 2014).

    Functional conservation of MBD proteins: MeCP2 and Drosophila MBD proteins alter sleep
    Proteins containing a methyl-CpG-binding domain (MBD) bind 5-hydroxymethylcytosine and convert the methylation pattern information into appropriate functional cellular states. Recent evidence indicates the genome of Drosophila melanogaster is methylated and two MBD proteins, dMBD2/3 and dMBD-R2, are present. Are Drosophila MBD proteins required for neuronal function, and as MBD-containing proteins have diverged and evolved, does the MBD domain retain the molecular properties required for conserved cellular function across species? To address these questions, the human MBD-containing protein, hMeCP2, was expressed in distinct amine neurons and functional changes were quantified in sleep circuitry output using a high throughput assay in Drosophila. hMeCP2 expression resulted in phase-specific sleep loss and sleep fragmentation with the hMeCP2-mediated sleep deficits requiring an intact MBD-domain. Reducing endogenous dMBD2/3 and dMBD-R2 levels also generated sleep fragmentation, with an increase in sleep occurring upon dMBD-R2 reduction. To examine if hMeCP2 and dMBD-R2 are targeting common neuronal functions, dMBD-R2 levels were reduced in combination with hMeCP2 expression and a complete rescue of sleep deficits was observed. Furthermore, chromosomal binding experiments indicate MBD-R2 and MeCP2 associate on shared genomic loci. These results provide the first demonstration that Drosophila MBD-containing family members are required for neuronal function and suggest the MBD domain retains considerable functional conservation at the whole organism level across species (Gupta, 2016).

    The ROP vesicle release factor is required in adult Drosophila glia for normal circadian behavior

    It is known that endocytosis and/or vesicle recycling mechanisms are essential in adult Drosophila glial cells for the neuronal control of circadian locomotor activity. The goal of this study was to identify specific glial vesicle trafficking, recycling, or release factors that are required for rhythmic behavior. From a glia-specific, RNAi-based genetic screen, eight glial factors were identified and were found to be required for normally robust circadian rhythms in either a light-dark cycle or in constant dark conditions. In particular, it was shown that conditional knockdown of the ROP vesicle release factor in adult glial cells results in arrhythmic behavior. Immunostaining for ROP reveals reduced protein in glial cell processes and an accumulation of the Par Domain Protein 1ε (PDP1ε) clock output protein in the small lateral clock neurons. These results suggest that glia modulate rhythmic circadian behavior by secretion of factors that act on clock neurons to regulate a clock output factor (Ng, 2015).

    The transcription factor Cabut coordinates energy metabolism and the circadian clock in response to sugar sensing

    Nutrient sensing pathways adjust metabolism and physiological functions in response to food intake. For example, sugar feeding promotes lipogenesis by activating glycolytic and lipogenic genes through the Mondo/ChREBP-Mlx transcription factor complex. Concomitantly, other metabolic routes are inhibited, but the mechanisms of transcriptional repression upon sugar sensing have remained elusive. This study characterizes cabut (cbtDrosophila. cbt was rapidly induced upon sugar feeding through direct regulation by Mondo-Mlx. CBT repressed several metabolic targets in response to sugar feeding, including both isoforms of phosphoenolpyruvate carboxykinase (pepck). Deregulation of pepck1 (CG17725) in mlx mutants underlay imbalance of glycerol and glucose metabolism as well as developmental lethality. Furthermore, cbt provided a regulatory link between nutrient sensing and the circadian clock. Specifically, a subset of genes regulated by the circadian clock were also targets of CBT. Moreover, perturbation of CBT levels led to deregulation of the circadian transcriptome and circadian behavioral patterns (Bartok, 2015).

    This study establishes the Drosophila klf10 ortholog, cbt, as a repressive effector of the sugar sensing transcriptional network. Specifically, (1) cbt expression is activated by dietary sugars in mlx-dependent manner; (2) cbt is a direct target of Mlx; (3) many key metabolic genes are rapidly repressed by CBT upon sugar feeding; (4) CBT binds to the proximity of pepck genes; (5) pepck1 is dispensable for viability, but essential for glucose and glycerol homeostasis; (6) deregulation of pepck1 underlies lethality of mlx mutants, and (7) CBT modulates the circadian system by controlling the cycling of a subset of circadian output genes. Based on these findings, a model is proposed in which CBT serves as a repressive downstream effector of the Mondo-Mlx-mediated sugar sensing, which contributes to diet-induced physiological readjustment, including flux of central carbon metabolism and cycling of metabolic circadian clock targets (Bartok, 2015).

    By uncovering the CBT-mediated repression of pepck isoforms downstream of Mondo-Mlx, This study provides a mechanistic explanation to the regulation of cataplerosis in response to intracellular sugar sensing. Drosophila Mondo-Mlx is known to drive activation of glycolysis, for example, by promoting the expression of phosphofructokinase 2. Placing the rate-limiting enzymes of gluconeogenesis downstream, the same sensor mechanism that activates glycolysis provides an elegant mechanism to adjust the direction of flux of glucose metabolism in response to sugar input. Such simple network topology provides a robust safeguard against loss of energy in futile cycles caused by simultaneous high activity of glycolysis and gluconeogenesis (Bartok, 2015).

    Mondo-Mlx also activates the expression of lipogenic gene expression (e.g., FAS and ACC) in order to promote conversion of excess sugars into triglycerides. In addition to fatty acid moieties, which are built by FAS and ACC, triglyceride biosynthesis requires glycerol-3-phosphate. Substrate-labeling studies in mammals have shown that a significant portion of glycerol in triglycerides is in fact derived from the PEPCK-dependent glyceroneogenesis pathway. This is supported by the current findings showing significantly lower circulating glycerol levels in well-fed pepck1-mutant larvae. The impact of glyceroneogenesis on triglyceride homeostasis is also likely reflected in the reduced triglyceride levels in pepck1 mutant flies. Similarly, mammalian studies have shown that elevated expression of PEPCK-C in adipose tissue increases fat mass, whereas reduced PEPCK-C expression leads to lower fat content. Moreover, in humans, adipose tissue expression of PEPCK-C positively correlates with adiposity and plasma triacylglycerol levels. Control of pepck through CBT places both branches of triglyceride biosynthesis under Mondo-Mlx. Inhibition of PEPCK-mediated cataplerosis upon high sugar intake allows maximal conversion of excess glucose-6-phosphate into the glycerol moieties of triglycerides through the glycolytic route of glycerol-3-phosphate synthesis. Simultaneous impairment of de novo lipogenesis and failure to suppress glyceroneogenesis likely leads to the breakage of glycerol homeostasis and massive accumulation of circulating glycerol, as observed in mlx mutants. Interestingly, a recent study showed that fasting serum levels of glycerol predicted development of hyperglycemia and type 2 diabetes. It will be interesting to learn whether this diagnostic marker is associated with deregulation of pepck isoforms and the activity of ChREBP/MondoA-Mlx and Klf10-dependent transcriptional network (Bartok, 2015).

    According to the current view, Mondo/ChREBP and Mlx act mainly in nutrient sensing and metabolic regulation. In contrast, CBT and its human ortholog Klf10 are multifunctional transcription factors. In Drosophila, cbt was originally identified as a developmental regulator with an essential function in dorsal closure early in development. Moreover, cbt is a direct transcriptional target of the circadian transcription factor CLK, and this study establishes it is deeply involved in the control of the circadian transcriptional network. While CBT overexpression leads to strong behavioral abnormalities, they are not accompanied by noticeable changes in the oscillation of the core clock components in the fly heads. This suggests that it reflects a specific effect on circadian output. If the behavioral patterns were due to an effect in the general health of the animal, deregulation of core clock components would be expected. Despite the null effect of cbt overexpression in the expression of core clock genes, this study observed that cbt modulates the expression of an important subset of CLK and circadian-controlled genes, most of which are involved in metabolic functions. Strong effects of cbt downregulation were observed in circadian oscillation of metabolic genes, establishing CBT as a new regulator of the circadian transcriptome. Interestingly, downregulation of CBT in circadian cells decreases the amplitude of oscillation of a large number of circadian-controlled genes, providing a direct link between food intake, circadian gene expression, and behavior. Given the established link between feeding time, metabolism, and the circadian system in Drosophila, it will be interesting to further analyze the importance of CBT in this coordination (Bartok, 2015).

    Although the functional analysis in this study largely focused on the metabolic role of pepck regulation by Mondo-Mlx-CBT network, microarray and RNA-seq analyses revealed other interesting CBT transcriptional targets including bmm. This gene is an ortholog of the human adipocyte triglyceride lipase gene, and it is an essential regulator of triglyceride stores in Drosophila. bmm expression is positively regulated by the Forkhead transcription factor FoxO, which is activated during starvation through inhibition of insulin-like signaling. The sugar-dependent repression of bmm expression by CBT is in perfect agreement with the lipogenic role of Mondo-Mlx (Bartok, 2015).

    It has been proposed that CBT mammalian ortholog Klf10 acts as a negative feedback regulator for ChREBP-activated genes, including lipogenic genes FAS and ACC. This conclusion was based on suppression of ChREBP-activated transcription upon Klf10 overexpression in primary liver cells. This model was tested by analyzing the expression of Mondo-Mlx targets FAS and ACC, but no effect was observed by cbtRNAi. In contrast, genome-wide expression analysis of CBT loss-of-function flies revealed that the CBT-dependent branch of the sugar sensing transcriptional network mediates rapid repression of gene expression. It is interesting to note that while most metabolic targets of CBT are rapidly and persistently downregulated, cbt expression is rapidly attenuated during prolonged sugar feeding. This is likely due to the negative autoregulation demonstrated earlier and supported by ChIP data. The finding that most of the identified CBT-dependent mRNAs are stably repressed for many hours after cbt levels have significantly attenuated suggests that CBT-mediated repression might involve regulation at the chromatin level. This is in agreement with the possible involvement of Sin3A in CBT-mediated repression. Through such persistent regulatory marks, sugar feeding may have a long-lasting influence on metabolic homeostasis, which is a topic that certainly deserves to be more thoroughly analyzed in the future (Bartok, 2015).

    In sum, this work provides a mechanistic explanation for the transcriptional repression upon Mondo-Mlx-mediated intracellular sugar sensing through the transcription factor CBT. The CBT-mediated repressive branch of the sugar sensing network is involved in securing the mutually exclusive activity of glycolysis and gluconeogenesis and coordination of fatty acid and glycerol biosynthesis with respect to dietary sugar intake. This study also establishes a mechanism for nutrient input into the circadian gene expression. As intracellular sugar-sensing and circadian regulation are highly conserved processes, the insight achieved in this study in the genetically tractable Drosophila model should provide a new conceptual framework for forthcoming studies in human subjects and mammalian model systems (Bartok, 2015).

    The metabolites NADP(+) and NADPH are the targets of the circadian protein Nocturnin (Curled)

    Nocturnin (NOCT) is a rhythmically expressed protein that regulates metabolism under the control of circadian clock. It has been proposed that NOCT deadenylates and regulates metabolic enzyme mRNAs. However, in contrast to other deadenylases, purified NOCT lacks the deadenylase activity. To identify the substrate of NOCT, a mass spectrometry screen was conducted, and NOCT was found to specifically and directly convert the dinucleotide NADP(+) into NAD(+) and NADPH into NADH. Further, it was demonstrated that the Drosophila NOCT ortholog, Curled, has the same enzymatic activity. The 2.7 A crystal structure of the human NOCT*NADPH complex, revealed that NOCT recognizes the chemically unique ribose-phosphate backbone of the metabolite, placing the 2'-terminal phosphate productively for removal. Evidence is provided for NOCT targeting to mitochondria and it is proposed that NADP(H) regulation, which takes place at least in part in mitochondria, establishes the molecular link between circadian clock and metabolism (Estrella, 2019).

    Ade2 functions in the Drosophila fat body to promote sleep

    Metabolic state is a potent modulator of sleep and circadian behavior and animals acutely modulate their sleep in accordance with internal energy stores and food availability. Growing evidence suggests the fat body is a critical regulator of complex behaviors, but little is known about the genes that function within the fat body to regulate sleep. To identify molecular factors functioning in non-neuronal tissues to regulate sleep, an RNAi screen selectively knocking down genes in the fat body. Knockdown was performed of Phosphoribosylformylglycinamidine synthase/Pfas(Ade2), a highly conserved gene involved the biosynthesis of purines, sleep regulation and energy stores. Flies heterozygous for multiple Ade2 mutations are also short sleepers and this effect is partially rescued by restoring Ade2 to the Drosophila fat body. Targeted knockdown of Ade2 in the fat body does not alter arousal threshold or the homeostatic response to sleep deprivation, suggesting a specific role in modulating baseline sleep duration. Together, these findings suggest Ade2 functions within the fat body to promote both sleep and energy storage, providing a functional link between these processes (Yurgel, 2018).

    The lysine demethylase dKDM2 is non-essential for viability, but regulates circadian rhythms in Drosophila

    Post-translational modification of histones, such as histone methylation controlled by specific methyltransferases and demethylases, play critical roles in modulating chromatin dynamics and transcription in eukaryotes. Misregulation of histone methylation can lead to aberrant gene expression, thereby contributing to abnormal development and diseases such as cancer. As such, the mammalian lysine-specific demethylase 2 (KDM2) homologs, KDM2A and KDM2B, are either oncogenic or tumor suppressive depending on specific pathological contexts. However, the role of KDM2 proteins during development remains poorly understood. Unlike vertebrates, Drosophila has only one KDM2 homolog (dKDM2), but its functions in vivo remain elusive due to the complexities of the existing mutant alleles. To address this problem, two dKdm2 null alleles were generated using the CRISPR/Cas9 technique. These dKdm2 homozygous mutants are fully viable and fertile, with no developmental defects observed under laboratory conditions. However, the dKdm2 null mutant adults display defects in circadian rhythms. Most of the dKdm2 mutants become arrhythmic under constant darkness, while the circadian period of the rhythmic mutant flies is approximately 1 h shorter than the control. Interestingly, lengthened circadian periods are observed when dKdm2 is overexpressed in circadian pacemaker neurons. Taken together, these results demonstrate that dKdm2 is not essential for viability; instead, dKDM2 protein plays important roles in regulating circadian rhythms in Drosophila. Further analyses of the molecular mechanisms of dKDM2 and its orthologs in vertebrates regarding the regulation of circadian rhythms will advance understanding of the epigenetic regulations of circadian clocks (Zheng, 2018).

    Identification and functional analysis of early gene expression induced by circadian light-resetting in Drosophila

    The environmental light-dark cycle is the dominant cue that maintains 24-h biological rhythms. In Drosophila, light entrainment is mediated by the photosensitive protein Cryptochrome. This study analyzed light-induced global transcriptional changes in the fly's head by using microarrays. Flies were subjected to a 30-min light pulse during the early night (3 h after lights-off). 200 genes were identified whose transcripts are significantly altered in response to the light pulse. Analysis suggests the involvement of at least six biological processes in light-induced delay phase shifts of rhythmic activities, include signalling, ion channel transport, receptor activity, synaptic organisation, signal transduction, and chromatin remodelling. Using RNAi, the expression of 22 genes was downregulated in the clock neurons, leading to significant effects on circadian output. For example, while continuous light normally causes arrhythmicity in wild-type flies, the knockdown of Kr-h1, Nipped-A, Thor, nrv1, Nf1, CG11155 (ionotropic glutamate receptor), and Fmr1 results in flies that are rhythmic, suggesting a disruption in the light input pathway to the clock. These analyses provides a first insight into the early responsive genes that are activated by light and their contribution to light resetting of the Drosophila clock (Adewoye, 2015).

    A conserved bicycle model for circadian clock control of membrane excitability

    Circadian clocks regulate membrane excitability in master pacemaker neurons to control daily rhythms of sleep and wake. This study found that two distinctly timed electrical drives collaborate to impose rhythmicity on Drosophila clock neurons. In the morning, a voltage-independent sodium conductance via the NA/NALCN ion channel Narrow abdomen depolarizes these neurons. This current is driven by the rhythmic expression of NCA localization factor-1 (CG10420), linking the molecular clock to ion channel function. In the evening, basal potassium currents peak to silence clock neurons. Remarkably, daily antiphase cycles of sodium and potassium currents also drive mouse clock neuron rhythms. Thus, this study reveals an evolutionarily ancient strategy for the neural mechanisms that govern daily sleep and wake (Flourakis, 2015).

    Circadian clock properties of fruit flies Drosophila melanogaster exhibiting early and late emergence chronotypes

    The role of circadian clock in timing daily behaviors is widely acknowledged, and while empirical evidence suggests that clock period is correlated with the preferred phase of a rhythmic behavior (chronotype), other clock properties have also been hypothesized to underlie chronotype variation. This study reports that fruit fly Drosophila melanogaster populations exhibiting evening emergence chronotype (late) are characterized by higher incidence of behavioral arrhythmicity in constant dim light, wider range of entrainment, reduced rates of re-entrainment to simulated jet-lag and higher amplitude of both entrained and free-running rhythms as compared to those exhibiting morning emergence chronotype (early). These results thus highlight the role of circadian clock properties such as zeitgeber sensitivity, amplitude and coupling in driving chronotype variation (Nikhil, 2015).

    Life-history traits of Drosophila melanogaster populations exhibiting early and late eclosion chronotypes

    The hypothesis that circadian clocks confer adaptive advantage to organisms has been proposed based on its ubiquity across almost all levels of complexity and organization of life-forms. This thought has received considerable attention, and studies employing diverse strategies have attempted to investigate it. However, only a handful of them have examined how selection for circadian clock controlled rhythmic behaviors influences life-history traits which are known to influence Darwinian fitness. The 'early' and 'late' chronotypes are amongst the most widely studied circadian phenotypes; however, life-history traits associated with these chronotypes, and their consequences on Darwinian fitness remain largely unexplored, primarily due to the lack of a suitable model system. Several life-history traits of Drosophila melanogaster populations were studied that were subjected to laboratory selection for morning (early) and evening (late) emergence. This paper reports that the late eclosion chronotypes evolved longer pre-adult duration as compared to the early eclosion chronotypes both under light/dark (LD) and constant dark (DD) conditions, and these differences appear to be mediated by both clock dependent and independent mechanisms. Furthermore, longer pre-adult duration in the late chronotypes does not lead to higher body-mass at pupariation or eclosion, but the late females were significantly more fecund and lived significantly shorter as compared to the early females. It is concluded that coevolution of multiple life-history traits in response to selection on timing of eclosion highlights correlations of the genetic architecture governing timing of eclosion with that of fitness components which suggests that timing ecologically relevant behaviors at specific time of the day might confer adaptive advantage (Nikhil, 2016).

    Responses of activity rhythms to temperature cues evolve in Drosophila populations selected for divergent timing of eclosion

    Even though the rhythm in adult emergence and rhythm in locomotor activity are two different rhythmic phenomena that occur at distinct life-stages of the fly life cycle, previous studies have hinted at similarities in certain aspects of the organisation of the circadian clock driving these two rhythms. For instance, the period gene plays an important regulatory role in both rhythms. Previous work showed that selection on timing of adult emergence behaviour in populations of Drosophila melanogaster leads to the co-evolution of temperature sensitivity of circadian clocks driving eclosion. In this study, it was asked if temperature sensitivity of the locomotor activity rhythm has evolved in the selected populations with divergent timing of adult emergence rhythm, with the goal of understanding the extent of similarity (or lack of it) in circadian organisation between the two rhythms. In response to simulated jetlag with temperature cycles, late chronotypes (populations selected for predominant emergence during dusk) indeed re-entrain faster than early chronotypes (populations selected for predominant emergence during dawn) to 6-h phase-delays, thereby indicating enhanced sensitivity of the activity/rest clock to temperature cues in these stocks (entrainment is the synchronisation of internal rhythms to cyclic environmental time-cues). Additionally, it was found that late chronotypes show higher plasticity of phases across regimes, day-to-day stability in phases and amplitude of entrainment, all indicative of enhanced temperature sensitive activity/rest rhythms. These results highlight remarkably similar organisation principles between emergence and activity/rest rhythms (Abhilash, 2020).

    Mechanisms of photic entrainment of activity/rest rhythms in populations of Drosophila selected for divergent timing of eclosion

    It is a common notion that phases-of-entrainment of circadian rhythms are adaptive, in that they enable organisms to time their behavior to specific times of the day to enhance their fitness. Previous studies have shown that selection for morning and evening phasing of adult emergence in Drosophila melanogaster populations leads to divergent coevolution of free-running periods of both adult emergence and activity/rest rhythms, such that early (morning) and late (evening) adult emergence chronotypes have shorter and longer circadian periods, respectively. However, there is little evidence to support the notion that phases-of-entrainment in these fly stocks is indeed driven by non-parametric mechanisms. Extending from a previous hypothesis based on anecdotal evidence for parametric mechanisms being in play, this study explored the extent of non-parametric and parametric effects of light on circadian clocks of early and late chronotypes. Predictions of the non-parametric model of entrainment were systematically tested, the Circadian Integrated Response Characteristic (CIRC) of the stocks , the effect of light pulses on amplitude of the behavior and the effect of duration of light pulse on phase-shifts of the clock were assessed were sketched. In addition to the differences in clock period, divergent CIRCs contribute to entrainment of the activity/rest rhythm. The differences in CIRC could be explained by differential transient amplitude responses and duration responses of the clock's phase between the early and late chronotypes. This study thus highlights the role of amplitude responses and phase-shifts due to long durations of light in entrainment of circadian rhythms of D. melanogaster (Abhilash, 2020).

    Functional contributions of strong and weak cellular oscillators to synchrony and light-shifted phase dynamics

    Light is the primary signal that calibrates circadian neural circuits and thus coordinates daily physiological and behavioral rhythms with solar entrainment cues. Drosophila and mammalian circadian circuits consist of diverse populations of cellular oscillators that exhibit a wide range of dynamic light responses, periods, phases, and degrees of synchrony. How heterogeneous circadian circuits can generate robust physiological rhythms while remaining flexible enough to respond to synchronizing stimuli has long remained enigmatic. Cryptochrome is a short-wavelength photoreceptor that is endogenously expressed in approximately half of Drosophila circadian neurons. This study applied analysis of real-time bioluminescence experimental data to show detailed dynamic ensemble representations of whole circadian circuit light entrainment at single neuron resolution. Organotypic whole-brain explants were either maintained in constant darkness (DD) for 6 days or exposed to a phase-advancing light pulse on the second day. Stronger circadian oscillators were found to support robust overall circuit rhythmicity in DD, whereas weaker oscillators can be pushed toward transient desynchrony and damped amplitude to facilitate a new state of phase-shifted network synchrony. Additionally, mathematical modeling was used to examine how a network composed of distinct oscillator types can give rise to complex dynamic signatures in DD conditions and in response to simulated light pulses. Simulations suggest that complementary coupling mechanisms and a combination of strong and weak oscillators may enable a robust yet flexible circadian network that promotes both synchrony and entrainment. A more complete understanding of how the properties of oscillators and their signaling mechanisms facilitate their distinct roles in light entrainment may allow direction and augmentation of the circadian system to speed recovery from jet lag, shift work, and seasonal affective disorder (Roberts, 2016).

    Evolution of robust circadian clocks in Drosophila melanogaster populations reared in constant dark for over 330 generations

    Robustness, the ability to maintain stable biological phenotypes across environments, is thought to be of adaptive value. Previous studies have reported higher intrinsic activity levels and power of rhythm in Drosophila populations (stocks) reared in constant darkness (DD stocks) as compared to those reared in constant light (LL stocks) and 12:12-h light-dark cycles (LD stocks) for over 19 years (approximately 330 generations). The current study intended to examine whether the enhanced levels of activity observed in DD stocks persist under various environments such as photoperiods, ambient temperatures, non-24-h light-dark (LD) cycles, and semi-natural conditions (SN). DD stocks largely retain their phenotype of enhanced activity levels across most of the above-mentioned environments suggesting the evolution of robust circadian clocks in DD stocks. Furthermore, the change in peak activity levels upon entrainment was not significantly different across the three stocks for any of the examined environmental conditions. This suggests that the enhancement of activity levels in DD stocks is not due to differential sensitivity to environment. Thus, these results suggest that rearing in constant darkness (DD) leads to evolution of robust circadian clocks suggesting a possible adaptive value of possessing such rhythms under constant dark environments (Shindey, 2016).

    Interspecific studies of circadian genes period and timeless in Drosophila

    The level of rescue of clock function in genetically arrhythmic Drosophila melanogaster hosts using interspecific clock gene transformation was used to study the putative intermolecular coevolution between interacting clock proteins. Among them PER and TIM are the two important negative regulators of the circadian clock feedback loop. This study transformed either the D. pseudoobscura per or tim transgenes into the corresponding arrhythmic D. melanogaster mutant (per01 or tim01) and observed >50% rhythmicity but the period of activity rhythm was either longer (D. pseudoobscura-per) or shorter than 24h (D. pseudoobscura-tim) compared to controls. By introducing both transgenes simultaneously into double mutants, it was observed that the period of the activity rhythm was rescued by the pair of hemizygous transgenes (~24h). These flies also showed a more optimal level of temperature compensation for the period. Under LD 12:12 these flies have a D. pseudoobscura like activity profile with the absence of morning anticipation as well as a very prominent earlier evening peak of activity rhythm. These observation are consistent with the view that TIM and PER form a heterospecific coevolved module at least for the circadian period of activity rhythms. However the strength of rhythmicity was reduced by having both transgenes present, so while evidence for a coevolution between PER and TIM is observed for some characters it is not for others (Noreen, 2018).

    Chvalova, D. and Dolezel, D. (2022). Loss of Timeless Underlies an Evolutionary Transition within the Circadian Clock

    Most organisms possess time-keeping devices called circadian clocks. At the molecular level, circadian clocks consist of transcription-translation feedback loops (TTFLs). Although some components of the negative TTFL are conserved across the animals, important differences exist between typical models, such as mouse and the fruit fly. In Drosophila, the key components are PERIOD (PER) and TIMELESS (TIM-d) proteins, whereas the mammalian clock relies on PER and CRYPTOCHROME (CRY-m). Importantly, how the clock has maintained functionality during evolutionary transitions between different states remains elusive. Therefore, this study systematically described the circadian clock gene setup in major bilaterian lineages and identified marked lineage-specific differences in their clock constitution. Then a thorough functional analysis was performed of the linden bug Pyrrhocoris apterus, an insect species comprising features characteristic of both the Drosophila and the mammalian clocks. Unexpectedly, the knockout of timeless-d, a gene essential for the clock ticking in Drosophila, did not compromise rhythmicity in P. apterus, it only accelerated its pace. Furthermore, silencing timeless-m, the ancestral timeless type ubiquitously present across animals, resulted in a mild gradual loss of rhythmicity, supporting its possible participation in the linden bug clock, which is consistent with timeless-m role suggested by research on mammalian models. The dispensability of timeless-d in P. apterus allows drawing a scenario in which the clock has remained functional at each step of transition from an ancestral state to the TIM-d-independent PER + CRY-m system operating in extant vertebrates, including humans (Kotwica-Rolinska, 2022).

    Adaptation of Drosophila melanogaster to Long Photoperiods of High-Latitude Summers Is Facilitated by the ls-Timeless Allele

    Circadian clocks help animals to be active at the optimal time of the day whereby for most species the daily light-dark cycle is the most important zeitgeber for their circadian clock. In this respect, long arctic summer days are particularly challenging as light is present almost 24 h per day, and continuous light makes the circadian clocks of many animals arrhythmic. This is especially true for the fruit fly, Drosophila melanogaster, which possesses a very light-sensitive clock. The blue-light photoreceptor Cryptochrome (CRY) and the clock protein Timeless (TIM) are the light-sensitive components of the circadian clock and are responsible for constant light-induced arrhythmicity even at very low light intensities. Nevertheless, D. melanogaster was able to spread from its tropical origin and invade northern latitudes. This study tested whether a natural polymorphism at the timeless (tim) locus, s-tim and ls-tim, helped adaptation to very long photoperiods. The recently evolved natural allele, ls-tim, encodes a longer, less light sensitive form of TIM (L-TIM) in addition to the shorter (S-TIM) form, the only form encoded by the ancient s-tim allele. ls-tim has evolved in southeastern Italy and slowly spread to higher latitudes. L-TIM is known to interact less efficiently with CRY as compared with S-TIM. The locomotor activity patterns of ~40 wild s-tim and ls-tim isofemale lines caught at different latitudes was measured under simulated high-latitude summer light conditions (continuous light or long photoperiods with 20-h daily light). It was found that the ls-tim lines were significantly more rhythmic under continuous light than the s-tim lines. Importantly, the ls-tim lines can delay their evening activity under long photoperiods, a behavioral adaptation that appears to be optimal under high-latitude conditions. These observations suggest that the functional gain associated with ls-tim may drive the northern spread of this allele by directional selection (Deppisch, 2022).

    CRTC potentiates light-independent timeless transcription to sustain circadian rhythms in Drosophila

    Light is one of the strongest environmental time cues for entraining endogenous circadian rhythms. Emerging evidence indicates that CREB-regulated transcription co-activator 1 (CRTC1) is a key player in this pathway, stimulating light-induced Period1 (Per1) transcription in mammalian clocks. This study demonstrates a light-independent role of Drosophila CRTC in sustaining circadian behaviors. Genomic deletion of the crtc locus causes long but poor locomotor rhythms in constant darkness. Overexpression or RNA interference-mediated depletion of CRTC in circadian pacemaker neurons similarly impairs the free-running behavioral rhythms, implying that Drosophila clocks are sensitive to the dosage of CRTC. The crtc null mutation delays the overall phase of circadian gene expression yet it remarkably dampens light-independent oscillations of TIMELESS (TIM) proteins in the clock neurons. In fact, CRTC overexpression enhances CLOCK/CYCLE (CLK/CYC)-activated transcription from tim but not per promoter in clock-less S2 cells whereas CRTC depletion suppresses it. Consistently, TIM overexpression partially but significantly rescues the behavioral rhythms in crtc mutants. Taken together, these data suggest that CRTC is a novel co-activator for the CLK/CYC-activated tim transcription to coordinate molecular rhythms with circadian behaviors over a 24-hour time-scale. The study proposes that CRTC-dependent clock mechanisms have co-evolved with selective clock genes among different species (Kim, 2016).

    CREB-dependent transcription has long been implicated in different aspects of circadian gene expression. In mammalian clocks, light exposure triggers intracellular signaling pathways that activate CREB-dependent Per1 transcription, thereby adjusting the circadian phase of master circadian pacemaker neurons in the suprachiasmatic nucleus (SCN). The phase-resetting process involves the specific CREB coactivator CRTC1 and its negative regulator SIK1, constituting a negative feedback in the photic entrainment via a CREB pathway (Sakamoto, 2013; Jagannath, 2013). This report demonstrates a novel role of Drosophila CRTC that serves to coordinate circadian gene expression with 24-hour locomotor rhythms even in the absence of light. CRTC may regulate several clock-relevant genes, including those clock output genes that might be involved in the rhythmic arborizations and PDF cycling of the circadian pacemaker neurons. However, tim transcription was identified as one of the primary targets of Drosophila CRTC to sustain circadian rhythms in the free-running conditions, thus defining its light-independent clock function (Kim, 2016).

    CREB could employ another transcriptional coactivator CBP (CREB-binding protein) to activate CRE-dependent transcription. In fact, CBP is a rather general coactivator recruited to gene promoters by other DNA-binding transcription factors. Previous studies have shown that Drosophila CBP associates with CLK, titrating its transcriptional activity. Mammalian CBP and the closely related coactivator p300 also form a complex with CLOCK-BMAL1, a homolog of the Drosophila CLK-CYC heterodimer, to stimulate their transcriptional activity. One possible explanation for CRTC-activated tim transcription is that Drosophila CRTC may analogously target the CLK-CYC heterodimer to stimulate CLK-CYC-tivate CRE-dependent transcription. Under these circumstances, a circadian role of light-sensitive TIM might have degenerated, while-induced clock genes. Moreover, CRTC associates with the bZIP domain in CREB protein, whereas CBP/p300 binds CREB through the phosphorylated KID domain, indicating that they might not necessarily target the same transcription factors apart from CREB. Finally, a protein complex of CLK and CRTC could not be detected in Drosophila S2 cells. Thus, it is likely that CRTC and CBP/p300 play unique roles in circadian transcription through their interactions with different DNA-binding transcription factors (Kim, 2016).

    If CRTC augments CLK-CYC-dependent tim transcription indirectly, then why do crtc effects require CLK? A recent study suggested that mammalian CLOCK-BMAL1 may regulate the rhythmic access of other DNA-binding transcription factors to their target promoters in the context of chromatin, acting as a pioneer-like transcription factor. Given the structural and functional homology between Drosophila CLK-CYC and mammalian CLOCK-BMAL1, the presence of CLK-CYC in the tim promoter might allow the recruitment of additional transcription factors (e.g., CREB) and their co-activators including CRTC for maximal tim transcription. The transcriptional context of tim promoter might thus define its sensitivity to crtc effects among other clock promoters. In addition, the differential assembly of transcription factors on the tim promoter could explain tissue-specific effects of crtc on TIM oscillations (i.e., circadian pacemaker neurons versus peripheral clock tissues). Interestingly, chromatin immunoprecipitation with V5-tagged CLK protein revealed that CLK-CYC heterodimers associate with both tim and Sik2 gene promoters in fly heads. In LD cycles, however, their rhythmic binding to the Sik2 promoter is phase-delayed by ~4.5 hours compared with that to the tim promoter. These modes of transcriptional regulation may gate crtc effects on tim transcription in a clock-dependent manner, particularly in the increasing phase of tim transcription (Kim, 2016).

    Transcription from CREB-responsive reporter genes shows daily oscillations, both in Drosophila and mammals, implicating this transcriptional strategy in the evolution of molecular clocks. In fact, cAMP signaling and CRE-dependent transcription constitute the integral components of core molecular clocks, serving to regulate daily rhythmic transcription of circadian clock genes. For instance, reciprocal regulation of dCREB2 and per at the transcription level has been reported to sustain free-running circadian rhythms in Drosophila. During fasting in mammals, a transcriptional program for hepatic gluconeogenesis is induced by CREB phosphorylation and CRTC2 dephosphorylation. Fasting-activated CREB-CRTC2 then stimulates Bmal1 expression61, whereas CLOCK-BMAL1-induced CRY rhythmically gates CREB activity in this process by modulating G protein-coupled receptor activity and inhibiting cAMP-induced CREB phosphorylation62. This molecular feedback circuit thus mutually links mammalian clocks and energy metabolism in terms of CREB-dependent transcription (Kim, 2016).

    On the basis of these observations, a model is proposed for the evolution of CRTC-dependent clocks to explain the distinctive circadian roles of CRTC homologs (see A model for the evolution of CRTC-dependent clocks). CRTC is a transcriptional effector that integrates various cellular signals (Altarejos, 2011). It was reasoned that ancestral clocks may have employed CREB-CRTC-mediated transcription to sense extracellular time cues cell-autonomously and integrate this timing information directly into the earliest transcription-translation feedback loop (TTFL). This strategy would have generated simple but efficient molecular clocks to tune free-running molecular rhythms in direct response to environmental zeitgebers, such as light and the availability of nutrients. A circadian role of CRTC then has differentially evolved along with a selective set of clock targets. In poikilothermic Drosophila, light is accessible directly to circadian pacemaker neurons in the adult fly brain. Therefore, TIM degradation by the blue-light photoreceptor CRY plays a major role in the light entrainment of Drosophila clocks, although the photic induction of CLK/CYC-dependent tim transcription has been reported specifically at lower temperatures. Accordingly, Drosophila CRTC retained a constitutive co-activator function from the ancestral TTFL to support CLK/CYC-activated tim transcription and sustain free-running circadian behaviors. In homeothermic mammals, light input to the SCN is indirectly mediated by neurotransmitter release from presynaptic termini of the retinohypothalamic tract (RHT). Intracellular signaling relays in the SCN converge on the dephosphorylation and nuclear translocation of CRTC1 to activate CRE-dependent transcription. Under these circumstances, a circadian role of light-sensitive TIM might have degenerated, while per took over a role in the light-entrainment pathway by retaining CREB-CRTC1-dependent transcriptional regulation from the primitive TTFL. Consequently, mammalian clocks have lost a homolog of the Drosophila-like cry gene family, but instead evolved CRY homologs of the vertebrate-like cry gene family with transcriptional repressor activities in CLOCK-BMAL1-dependent transcription (Kim, 2016).

    Regulation of metabolism and stress responses by neuronal CREB-CRTC-SIK pathways has been well documented in Drosophila. Given the demonstration of a circadian role of CRTC in the pacemaker neurons, it is possible that CRTC might sense metabolic cues in the context of circadian neural circuits to entrain molecular clocks cell-autonomously. Alternatively, but not exclusively, CRTC could participate in the regulation of clock-relevant metabolism as clock outputs from pacemaker neurons. These hypotheses remain to be validated in future studies (Kim, 2016).

    Understanding the role of chromatin remodeling in the regulation of circadian transcription in Drosophila

    Circadian clocks enable organisms to anticipate daily changes in the environment and coordinate temporal rhythms in physiology and behavior with the 24-hour day-night cycle. The robust cycling of circadian gene expression is critical for proper timekeeping, and is regulated by transcription factor binding, RNA polymerase II (RNAPII) recruitment and elongation, and post-transcriptional mechanisms. Recently, it has become clear that dynamic alterations in chromatin landscape at the level of histone posttranslational modification and nucleosome density facilitate rhythms in transcription factor recruitment and RNAPII activity, and are essential for progression through activating and repressive phases of circadian transcription. This study discusses the characterization of the Brahma (Brm) chromatin-remodeling protein in Drosophila in the context of circadian clock regulation. By dissecting its catalytic vs. non-catalytic activities, a model is proposed in which the non-catalytic activity of Brm functions to recruit repressive factors to limit the transcriptional output of Clock (Clk) during the active phase of circadian transcription, while the primary function of the ATP-dependent catalytic activity is to tune and prevent over-recruitment of negative regulators by increasing nucleosome density. Finally, ongoing efforts and investigative directions towards a deeper mechanistic understanding of transcriptional regulation of circadian gene expression at the chromatin level are described (Kwok, 2016).

    Phosphatase of regenerating liver-1 selectively times circadian behavior in darkness via function in PDF neurons and dephosphorylation of TIMELESS

    The timing of behavior under natural light-dark conditions is a function of circadian clocks and photic input pathways, but a mechanistic understanding of how these pathways collaborate in animals is lacking. This study demonstrates in Drosophila that the Phosphatase of Regenerating Liver-1 (PRL-1) sets period length and behavioral phase gated by photic signals. PRL-1 knockdown in PDF clock neurons dramatically lengthens circadian period. PRL-1 mutants exhibit allele-specific interactions with the light and clock-regulated gene timeless (tim). Moreover, this study shows that PRL-1 promotes TIM accumulation and dephosphorylation. Interestingly, the PRL-1 mutant period lengthening is suppressed in constant light, and PRL-1 mutants display a delayed phase under short, but not long, photoperiod conditions. Thus, these studies reveal that PRL-1-dependent dephosphorylation of TIM is a core mechanism of the clock that sets period length and phase in darkness, enabling the behavioral adjustment to change day-night cycles (Kula-Eversole, 2020).

    This study has identified a novel circadian clock gene PRL-1 that mediates the behavioral response to changes in photoperiod. Circadian regulation of this phosphatase provides a mechanism for mediating rhythmic phosphorylation, a widely conserved feature of circadian clocks. Reduction or loss-of-function in PRL-1 can induce potent effects on circadian period length even larger than those caused by the reduction of canonical clock genes. These effects are widely conserved and are evident even in human cells. PRL-1 selectively dephosphorylates and suppresses the accumulation of the light-sensitive clock component TIM. PRL-1 affects behavioral phase in short but not long photoperiods, revealing a novel mechanism for appropriate timing across ecologically relevant photoperiods (Kula-Eversole, 2020).

    The data suggest PRL-1 plays an especially important role in setting period length comparable to or exceeding those of the canonical core clock components. RNAi knockdown of PRL-1 with two independent RNAi lines results in among the strongest RNAi-induced period-altering phenotypes seen in Drosophila with period lengths exceeding 28 h. Interestingly, the period effects observed through PRL-1 knockdown in the PDF-positive LNv (pdfGAL4) are larger than those observed with a broader circadian driver (timGAL4) or in the putative null allele of PRL-1, suggesting that PRL-1 may have distinct and even antagonistic period setting roles in different clock neurons. The other clock genes with RNAi effects comparable to PRL-1 are the regulatory beta subunit of the protein kinase CK2 and the E3 ubiquitin ligase circadian trip, further highlighting the role of post-translation modifications in determining period length (Kula-Eversole, 2020).

    Several lines of evidence support the model that TIM is an in vivo target of PRL-1. PRL-1 mutants exhibit non-additive effects with a tim allele, timS1, suggesting PRL-1 and tim function within the same pathway. Non-additive genetic interactions with specific alleles can reflect functional, even direct biochemical, interactions such as between per and tim78 and between per and Dbt. Simple additive effects on period length with timUL flies probably reflect the fact that TIMUL is stable and exhibits prolonged nuclear localization; therefore, stabilizing effects of timUL are not counteracted by the destabilizing effects of PRL-1 mutation during the rising phase of TIM accumulation. Loss of PRL-1 results in substantially reduced and delayed nuclear accumulation of TIM during the first day of DD. These effects are much larger than those simply predicted based on the lengthened period and much larger than effects on PER, suggesting a selective effect on TIM in vivo. Using PRL-1 overexpression and knockdown in clock-less Drosophila S2 cells, it was found that PRL-1 dephosphorylates TIM but not PER. Given that PRL-1 mRNA expression peaks around dusk, it is proposed that PRL-1 accumulation during the early night results in TIM dephosphorylation, stabilization, and nuclear localization (Kula-Eversole, 2020).

    Strikingly, period lengthening is evident even after siRNA knockdown of all three highly conserved human orthologs of PRL-1, PTP4a-1, -2, and -3 in human U2OS cells, suggesting a widely conserved role in period determination. Like fly PRL-1, mammalian orthologs of PRL-1 also undergo rhythmic expression in many tissues. PTPs may also function via TIM. Mammalian TIM interacts with CRY1 as well as PER1/2, is involved in CLOCK-BMAL1 repression of CLOCK-BMAL1 activation, and sets period length. Mutation of human TIMELESS also results in familial advanced sleep phase syndrome perhaps via altered light responses. Thus, PTP4 action may similarly function to regulate TIM in mammals (Kula-Eversole, 2020).

    The connection between PRL-1 and photosensitive TIM suggested that PRL-1 may function to mediate clock responses to light. PRL-1 mutants exhibit a light-dependent and CRY-independent period phenotype. To examine rhythms in LL, PRL-1 mutant period length was examined in a cry mutant background in which flies exhibit free-running rhythms. These flies retain their long periods in DD. On the other hand, in LL they exhibit period lengths comparable to their PRL-1+ controls. Thus, light can act independently of CRY photoreception to compensate for the PRL-1 mutant period lengthening. It is hypothesized that signaling through the visual system could accomplish this (Kula-Eversole, 2020).

    The finding of LL-dependent period phenotypes led to an examination of the role of PRL-1 in mediating behavioral adaptations to seasonal changes in photoperiod. PRL-1 mutants display delayed morning and evening activity phases under short (6:18) photoperiods. In contrast, they have no alteration of evening activity phase under long (18:6) photoperiods. These photoperiods approximate those that would be experienced by wild Drosophila melanogaster at more extreme latitudes. It is hypothesized that PRL-1 defines a molecular pathway through which the clock adjusts behavioral phase in response to different photoperiods (Kula-Eversole, 2020).

    The phenomenon of photoperiod-dependent network hierarchy suggests that M and E cells differ in their core clock properties, responses to light, and/or their network connectivity. It is hypothesized that PRL-1 represents such a specialization of M cell clocks that enables the appropriate behavioral adjustments to different photoperiods. PRL-1 exhibits robust PDF neuron specific cycling, and PRL-1 knockdown selectively in PDF neurons substantially lengthens period. The finding of period phenotypes in DD and not LL is also consistent with M cell specific function. The findings of photoperiod-dependent effects in PRL-1 mutants could reflect enhanced coupling between LNv and CRY-negative DN1p in short photoperiods, consistent with prior findings of light intensity-dependent sLNv-DN1p coupling. Under this model, selective PRL-1-dependent changes in the M cell phase are propagated to E cells in a photoperiod-dependent manner as observed for other M cell-specific changes. Of note, other core clock components have been identified with differential functions between M and E cells, including those involved in TIM phosphorylation, suggesting a wider role for specialized core clock components in M and E cells. It will be of interest to determine if M or E cell-specific mechanisms also function to control behavioral adaptations to different photoperiods and, if so, how they work with respect to PRL-1 (Kula-Eversole, 2020).

    A neural network underlying circadian entrainment and photoperiodic adjustment of sleep and activity in Drosophila

    Sensitivity of the circadian clock to light/dark cycles ensures that biological rhythms maintain optimal phase relationships with the external day. In animals, the circadian clock neuron network (CCNN) driving sleep/activity rhythms receives light input from multiple photoreceptors, but how these photoreceptors modulate CCNN components is not well understood. This study shows that the Hofbauer-Buchner eyelets, located between the retina and the medulla in the fly optic lobes, differentially modulate two classes of ventral lateral neurons (LNvs) within the Drosophila CCNN. The eyelets antagonize Cryptochrome (CRY)- and compound-eye-based photoreception in the large LNvs while synergizing CRY-mediated photoreception in the small LNvs. Furthermore, it was shown that the large LNvs interact with subsets of 'evening cells' to adjust the timing of the evening peak of activity in a day length-dependent manner. This work identifies a peptidergic connection between the large LNvs and a group of evening cells that is critical for the seasonal adjustment of circadian rhythms (Schlichting, 2016).

    Circadian clocks create an endogenous sense of time that is used to produce daily rhythms in physiology and behavior. A defining characteristic of a circadian clock is a modest deviation of its endogenous period from the 24.0 h period of daily environmental change. For example, the average human clock has an endogenous period of 24 h and 11 min. Thus, to maintain a consistent phase relationship with the environment, the human clock must be sped up by 11 min every day. A sensitivity of the circadian clock to environmental time cues (zeitgebers) ensures that circadian clocks are adjusted daily to match the period of environmental change. This process, called entrainment, is fundamental to the proper daily timing of behavior and physiology. For most organisms, daily light/dark (LD) cycles are the most salient zeitgeber (Schlichting, 2016).

    Although most tissues express molecular circadian clocks in animals, the clock is required in small islands of neural tissue for the presence of sleep/activity rhythms and many other daily rhythms in physiology. Within these islands, a circadian clock neuron network (CCNN) functions as the master circadian clock. Subsets of neurons within the CCNN receive resetting signals from photoreceptors, and physiological connections between these neurons and their clock neuron targets ensure light entrainment of the CCNN as a whole (Schlichting, 2016).

    In both mammals and insects, the CCNN receives light input from multiple photoreceptor types. In Drosophila, the CCNN is entrained by photoreceptors in the compound eye, the ocelli, the Hofbauer-Buchner (HB) eyelets, and by subsets of clock neurons that express the blue light photoreceptor Cryptochrome (CRY). Understanding how multiple light input pathways modulate the CCNN to ensure entrainment to the environmental LD cycle is critical for understanding of the circadian system and its dysfunction when exposed to the unnatural light regimens accompanying much of modern life (Schlichting, 2016).

    This study investigates the physiological basis and circadian role of a long-suspected circadian light input pathway in Drosophila: the HB eyelets. These simple accessory eyes contain four photoreceptors located at the posterior edges of the compound eyes and project directly to the accessory medullae (AMe), neuropils that support circadian timekeeping in insects. In Drosophila, the AMe contain projections from ventral lateral neurons (LNvs), important components of the CCNN that express the neuropeptide pigment dispersing factor (PDF), an output required for robust circadian rhythms in locomotor activity. The axon terminals of the HB eyelets terminate near PDF-positive LNv projections and analysis of visual system and cry mutants reveals a role for the HB eyelet in the entrainment of locomotor rhythms to LD cycles, but how the eyelets influence the CCNN to support light entrainment is not well understood (Schlichting, 2016).

    This study presents evidence that this circadian light input pathway excites the small LNvs (s-LNvs) and acts to phase-dependently advance free-running rhythms in sleep/activity while inhibiting the large LNvs (l-LNvs). This work reveals that input from external photoreceptors differentially affects specific centers within the fly CCNN. Furthermore, it was shown that, under long summer-like days, the l-LNvs act to modulate subsets of so-called evening cells to delay the onset of evening activity. These results reveal a neural network underlying the photoperiodic adjustment of sleep and activity (Schlichting, 2016).

    The experiments described in this study lead to two unexpected findings regarding the network properties of circadian entrainment in Drosophila. First, the l-LNvs govern the phase of evening peak of activity through PdfR-dependent effects on evening cells that bypass the s-LNvs. Although previous work has implicated the l-LNvs in the control of evening peak phase, the current results are the first to provide evidence that there is a direct connection between the l-LNvs and evening cells within the AMe and that this connection mediates the photoperiodic adjustment of sleep and activity in the fly. Second, the HB eyelets light input pathways, long implicated in circadian entrainment, have opposing effects on the l-LNvs and s-LNvs, inhibiting the former and exciting the latter. These results reveal not only a differential effect of a light input pathway on specific nodes of the CCNN but also establish that light from extraretinal photoreceptors can have synergistic or antagonistic effects on CRY- and compound eye-mediated light responses, depending on the clock neuron target in question (Schlichting, 2016).

    Both the l-LNvs and s-LNvs express the blue light circadian photoreceptor CRY, the expression of which renders neurons directly excitable by light entering the brain through the cuticle. How such CRY-mediated light input interacts with input from external photoreceptors is not well understood, although it is known that each system alone is sufficient for the entrainment of locomotor rhythms. Genetic evidence suggests that the HB eyelets have relatively weak effects on circadian entrainment: flies with functional eyelets that lack compound eyes, ocelli, and CRY entrain relatively poorly to LD cycles relative to flies with functional eyes or CRY. The small phase responses of locomotor rhythms to HB eyelet excitation further supports a relatively weak effect of the eyelet on free-running locomotor rhythms (Schlichting, 2016).

    The LNvs are critical nodes in the CCNN and are closely associated with input pathways linking the central brain to external photoreceptors. Work on the LNvs has provided evidence for a division of labor among the l-LNvs and s-LNvs: the l-LNvs are wake-promoting neurons that acutely govern arousal and sleep independently of the s-LNvs, whereas the s-LNvs act as key coordinators of the CCNN to support robust circadian timekeeping. Anatomical and genetic evidence has long supported the notion that the dorsal projections of the s-LNvs represent the key connection between the LNvs and the remaining components of the CCNN. However, a smaller body of work has suggested that the l-LNvs also contribute to the entrainment of sleep/activity rhythms under LD cycles. The Pdf knockdown and PdfR rescue experiments under long day conditions indicate that, as the day grows longer, the l-LNvs play a greater role in the timing of the evening peak. Moreover, the effects of PDF released from the l-LNvs are mediated not by the PDF receptive s-LNvs but rather by the fifth s-LNvs and a subset of the LNds, the NPF and ITP coexpressing LNds in particular (with some influence of the other PDF-receptor positive LNds). These same neurons were recently identified as evening cells that are physiologically responsive to PDF but relatively weakly coupled to LNv clocks under conditions of constant darkness. The results suggest that the l-LNvs differentially modulate the NPF/ITP-positive evening oscillators as a function of day length, producing stronger PDF-dependent delays under long day conditions through increased release of PDF from the l-LNvs, thereby delaying the evening activity peak. Thus, the l-LNvs mediate their effects on the evening peak of activity through their action on the NPF/ITP-positive subset of evening oscillators. The proposed PDF release from the l-LNvs under long days requires their activation via CRY and/or the compound eyes via ACh release from lamina L2 interneurons. It is hypothesized that the inhibitory influence of the HB eyelets ceases under long days allowing the compound eyes and CRY to maximally excite the l-LNvs. Indeed, previous work has established that the compound eyes are especially important for adapting fly evening activity to long days. Furthermore, several studies have suggested that the compound eyes signal to the l-LNvs leading to enhanced PDF release and a slowing-down of the evening oscillators. A recent paper measuring Ca2+ rhythms in the different clock neurons in vivo supports this view (Liang, 2016): Ca2+ rhythms in the l-LNvs peak in the middle of the day, unlike the s-LNvs, which display Ca2+ peaks in the late night/early morning. It is suggested that this phasing is produced by the inhibition of l-LNvs by the eyelets in the morning, followed by the excitation of the l-LNvs by the compound eyes and CRY. Interestingly, the only other clock neuron classes to display Ca2+ increases during the day are the LNds and fifth-sLNv, which phase lag the l-LNvs by ~2.5 h and display peak Ca2+ levels in the late afternoon, a time that coincides with the evening peak of activity (Liang, 2016). It is proposed that the relative coordination of Ca2+ rhythms between the l-LNvs and the LNds/fifth-sLNv is produced by the connection this study has identified between these neurons and the action of the eyelet and visual system on the l-LNvs (Schlichting, 2016).

    Recent work has revealed that evening activity is promoted directly by the evening oscillator neurons and that the mid-day siesta is produced by the daily inhibition of evening oscillators by a group of dorsal clock neurons (Guo, 2016). It is proposed that the connections described in this study govern the timing of the evening peak of activity through the PDF-dependent modulation of the molecular clocks within the evening oscillator neurons, although PDF modulation likely results in the excitation of target neurons, which would promote evening activity. The results reveal new and unexpected network properties underlying the entrainment of the circadian clock neuron network to LD cycles. Excitatory effects of light on the LNvs are differentially modulated by the HB eyelets via cholinergic excitation of the s-LNvs and histaminergic inhibition of the l-LNvs. The work further reveals PDF-dependent modulatory connections in the AMe between the l-LNvs and the s-LNvs and, most surprisingly, between the l-LNvs and a small subset of evening oscillators. This work indicates that the latter connection is critical for the adjustment of evening activity phase during long, summer-like days. This network model of entrainment reveals not only how CRY and external photoreceptors interact within specific nodes of the CCNN, but also how photoreception is likely to drive changes in CCNN output in the face of changing day length (Schlichting, 2016).

    Quasimodo mediates daily and acute light effects on Drosophila clock neuron excitability

    The light-input factor Quasimodo (Qsm) regulates rhythmic electrical excitability of clock neurons, presumably via an Na+, K+, Cl- cotransporter (NKCC) and the Shaw K+ channel (dKV3.1). Because of light-dependent degradation of the clock protein Timeless (Tim), constant illumination (LL) leads to a breakdown of molecular and behavioral rhythms. Both overexpression (OX) and knockdown (RNAi) of qsm, NKCC, or Shaw led to robust LL rhythmicity. Whole-cell recordings of the large ventral lateral neurons (l-LNv) showed that altering Qsm levels reduced the daily variation in neuronal activity: qsmOX led to a constitutive less active, night-like state, and qsmRNAi led to a more active, day-like state. Qsm also affected daily changes in K+ currents and the GABA reversal potential, suggesting a role in modifying membrane currents and GABA responses in a daily fashion, potentially modulating light arousal and input to the clock. When directly challenged with blue light, wild-type l-LNvs responded with increased firing at night and no net response during the day, whereas altering Qsm, NKKC, or Shaw levels abolished these day/night differences. Finally, coexpression of ShawOX and NKCCRNAi in a qsm mutant background restored LL-induced behavioral arrhythmicity and wild-type neuronal activity patterns, suggesting that the three genes operate in the same pathway. It is proposed that Qsm affects both daily and acute light effects in l-LNvs probably acting on Shaw and NKCC (Buhl, 2016).

    All organisms are subject to predictable but drastic daily environmental changes caused by the earth's rotation around the sun. It is critical for the fitness and well-being of an individual to anticipate these changes, and this anticipation is done by circadian timekeeping systems (clocks). These regulate changes in behavior, physiology, and metabolism to ensure they occur at certain times during the day, thereby adapting the organism to its environment. The circadian system consists of three elements: the circadian clock to keep time, inputs that allow entrainment, and outputs that influence physiology and behavior. Like a normal clock, circadian clocks run at a steady pace (24 h) and can be reset. In nature this environmental synchronization is done via daily light and temperature cycles, food intake, and social interactions (Buhl, 2016).

    In Drosophila the central clock comprises 75 neuron pairs grouped into identifiable clusters that subserve different circadian functions. The molecular basis of the circadian clock is remarkably conserved from Drosophila to mammals. This intracellular molecular clock drives clock neurons to express circadian rhythms in electrical excitability, including variation in membrane potential and spike firing. Clock neurons are depolarized and fire more during the day than at night, and circadian changes in the expression of clock-controlled genes encoding membrane proteins such as ion channels and transporters likely contribute to these rhythms. Such cyclical variations in activity play a critical role in synchronizing different clock neurons and conveying circadian signals to other parts of the nervous system and body. Furthermore, they provide positive feedback to the molecular clock, which can dampen rapidly without such feedback (Buhl, 2016).

    Light resets the circadian clock every morning to synchronize the clock to the environment via Timeless (Tim) degradation after activation of the blue-light photoreceptor Cryptochrome (Cry), Quasimodo (Qsm), and potentially also visual photoreceptors. Qsm acts either independently or downstream of Cry and also is able to affect clock protein stability in Qsm-negative neurons by an unknown non-cell-autonomous mechanism (Chen, 2011). Recently Cry has been shown to regulate clock neuron excitability via the redox sensor of the Hyperkinetic voltage-gated potassium (KV)-β subunit (Hk) (Fogle, 2015), and this study asked if Qsm affects the clock neurons in a similar way (Buhl, 2016).

    Membrane potential is important for control of circadian behavior, and manipulation of Shaw and the Narrow Abdomen (NA) channels, both of which are expressed and function within clock neurons influence neuronal electrical activity, the circadian clock, and clock-controlled behavior in both flies and mice. The firing rate is a key component in mammalian circadian rhythmicity and can be regulated by regional and circadian expression of the sodium potassium chloride cotransporter NKCC, which switches the effects of GABA from inhibitory to excitatory across the day (Buhl, 2016).

    This study shows that down-regulation or overexpression of the three membrane proteins encoded by the genes qsm, Shaw, and NKCC leads to rhythmicity in constant illumination (LL) and that these genes interact. All three genes are expressed in the well-characterized pigment-dispersing factor (Pdf)- and Cry-positive large ventral lateral neurons (l-LNv), which are important for arousal and light input to the clock. Whole-cell recordings of l-LNvs were used to characterize their physiological properties and acute light effects across the day, and Qsm was found to help set the circadian state of clock neurons and modify their response to light, possibly by acting via Shaw and NKCC (Buhl, 2016).

    Light is the dominant circadian zeitgeber that resets the molecular clock. This study determined how light affects membrane excitability via the membrane proteins Qsm, Shaw, and NKCC. Previously it was shown that Qsm contributes to circadian clock light input with down-regulation in all clock neurons (tim-gal4) resulting in robust rhythmic behavior in LL (Chen, 2011). This study shows that overexpression (qsmOX) also results in robust LL rhythmicity, but with predominantly ~13-h periods, suggesting a more robust morning oscillator that is normally weakened in DD conditions. Manipulating the expression levels of both the potassium channel Shaw and the ion cotransporter NKCC also resulted in LL rhythmicity, whereas several visual system mutants behaved like wild-type flies and became arrhythmic. These experiments show that the membrane proteins encoded by qsm, Shaw, and NKCC control rhythmic behavior in LL. Furthermore, the rescue of wild-type behavior and neurophysiological properties by reciprocal changes of Qsm and Shaw and by the simultaneous reduction of Qsm and NKCC suggests that Qsm interacts genetically and perhaps directly with Shaw and NKCC (Buhl, 2016).

    Clock neurons are more depolarized and fire more during the daytime, and circadian changes in the expression of clock-controlled genes such as ion channels and transporters are likely to play a part. Contributing to this rhythm is a sodium leak current mediated by NA that recently has been shown to depolarize Drosophila clock neurons. This study shows that Shaw, NKCC, and Qsm also contribute to daily electrical activity rhythms: overexpression and RNAi knock-down of qsm and Shaw compared with NKCC resulted in opposing phenotypes. Interestingly, qsmOX or ShawOX and NKCCRNAi promote the less active nighttime state, whereas qsmRNAi, ShawRNAi, and NKCCOX push the neurons into the more depolarized daytime state, eliminating the acute day/night differences in all cases. Previous work has shown that Shaw regulates circadian behavior and that, in agreement with the current findings, Shaw regulates membrane potential and firing in Drosophila motoneurons. NKCC activity is electrically neutral but increases the intracellular Cl- concentration so that the GABAA receptor opens in response to GABA but, as a consequence, Cl- presumably exits the cell down its electrochemical gradient, thereby depolarizing the membrane potential so that GABA effectively becomes an excitatory neurotransmitter. The current data show that in Drosophila a similar mechanism occurs, which is consistent with potential NKCC enrichment in l-LNv at dawn. The mechanism setting the neuronal state to either daytime or nighttime via Qsm, Shaw, and NKCC is likely to be predominantly cell-autonomous, because all components have been shown to act or to be expressed in the l-LNv. Although in an earlier study using qsm-gal4 lines qsm expression was not detected in the l-LNv, these lines may not report expression faithfully in all qsm cells. Now, the finding that qsm RNA is enriched in the l-LNv, combined with the strong effects of two qsm-RNAi lines on l-LNv electrical properties presented in this study, indicates that qsm is endogenously expressed in these neurons (Buhl, 2016).

    Physiological studies are limited to the Pdf-expressing l-LNv neurons. These neurons are unlikely candidates for driving behavioral rhythms in LL, and previous work has shown that qsm knockdown in Pdf neurons (s-LNv and l-LNv) does not result in robust LL rhythmicity. Therefore the effects of light on the electrical properties of l-LNv reported here do not necessarily explain the LL rhythmicity observed after manipulating qsm, Shaw, and NKCC in all clock neurons. However, the electrophysiological results using tim-gal4 show that Qsm, Shaw, and NKCC could fulfill similar functions in other clock neurons, including those crucial for LL rhythmicity (e.g., LNd and DN1). Additionally, or alternatively, the manipulated l-LNv could generate signals interfering with normal network function, resulting in the observed rhythmic LL behavior (Buhl, 2016).

    Although qsm is a clock-controlled gene, the acute blue-light effects that were observed are too fast to be mediated by transcriptional changes. Therefore, a more direct membrane-localized mechanism is favored in which rapid light-dependent posttranslational changes of Qsm alter the activity of Shaw and NKCC. Because (i) Cry is required for light-dependent Tim degradation in l-LNv, (ii) changing the Qsm level has no effect on Cry levels, and (iii) qsmOX triggers Tim degradation in the absence of Cry, the most likely explanation for the results reported in this study is that, in addition to activating Hk, Cry acts upstream of Qsm, which in turn regulates the activity of Shaw and NKCC. It is assumed that Qsm is activated by light because a light pulse at night rapidly increases protein levels. Qsm is an extracellular zona-pellucida (ZP) membrane-anchored protein, and it is hypothesized that after light exposure the extracellular ZP domain is cleaved at a conserved furin protease cleavage site, a form of posttranslational processing typical for ZP-domain proteins. It is also possible that Qsm signals to Shaw and NKCC in both membrane-bound and cleaved forms. For example, at night membrane-bound Qsm could block NKCC, whereas light-induced cleavage could release this block, and the freed extracellular part could inactivate Shaw. This mechanism is reminiscent to the mechanism by which the GPI-anchored extracellular protein Sleepless increases Shaker (KV1 channel) activity for regulating Drosophila sleep (Buhl, 2016).

    How Qsm-induced changes in clock neuron activity influence the molecular clock remains an open question. Recent work shows that, in addition to the canonical degradation via Cry and Jetlag, Tim is also degraded via a Cul-3 and neuronal activity-dependent pathway in DD that has been implicated in mediating phase delays in the circadian clock. In contrast to this activity-dependent Cul-3 pathway, the light responses in the current study depend on Cry. Therefore a model is favored in which the combined functions of Qsm, Shaw, and NKCC contribute to the canonical Cry- and Jetlag-dependent Tim-degradation pathway (Buhl, 2016).

    In conclusion, this study demonstrates that Qsm affects both daily and acute light responses of l-LNvs, and therefore (Qsm) presumably contributes to light-input to the Drosophila circadian clock. Qsm possibly signals downstream of Cry and acts on Shaw and NKCC to change clock neuronal activity in response to light (Buhl, 2016).

    Cycles of circadian illuminance are sufficient to entrain and maintain circadian locomotor rhythms in Drosophila

    Light at night disrupts the circadian clock and causes serious health problems in the modern world. This study shows that newly developed four-package light-emitting diodes (LEDs) can provide harmless lighting at night. To quantify the effects of light on the circadian clock, the concept of circadian illuminance (CIL) was employed. CIL represents the amount of light weighted toward the wavelengths to which the circadian clock is most sensitive, whereas visual illuminance (VIL) represents the total amount of visible light. Exposure to 12 h:12 h cycles of white LED light with high and low CIL values but a constant VIL value (conditions hereafter referred to as CH/CL) can entrain behavioral and molecular circadian rhythms in flies. Moreover, flies re-entrain to phase shift in the CH/CL cycle. Core-clock proteins are required for the rhythmic behaviors seen with this LED lighting scheme. Taken together, this study provides a guide for designing healthful white LED lights for use at night, and proposes the use of the CIL value for estimating the harmful effects of any light source on organismal health (Cho, 2016).

    Role of the circadian clock in the statistics of locomotor activity in Drosophila

    In many animals the circadian rhythm of locomotor activity is controlled by an endogenous circadian clock. Using custom made housing and video tracking software in order to obtain high spatial and temporal resolution, wthe statistical properties of the locomotor activity of wild type and two clock mutants of Drosophila melanogaster were studied. This study showed that the distributions of activity and quiescence bouts for the clock mutants in light-dark conditions (LD) are very different from the distributions obtained when there are no external cues from the environment (DD). In the wild type these distributions are very similar, showing that the clock controls this aspect of behavior in both regimes (LD and DD). Furthermore, the distributions are very similar to those reported for Wistar rats. For the timing of events important differences were observed, quantified by how the event rate distributions scale for increasing time windows. For the wild type these distributions can be rescaled by the same function in DD as in LD. Interestingly, the same function has been shown to rescale the rate distributions in Wistar rats. On the other hand, for the clock mutants it is not possible to rescale the rate distributions, which might indicate that the extent of circadian control depends on the statistical properties of activity and quiescence (Cascallares, 2018).

    Daily rewiring of a neural circuit generates a predictive model of environmental light

    Behavioral responsiveness to external stimulation is shaped by context. This study examined how sensory information can be contextualized, by examining light-evoked locomotor responsiveness of Drosophila relative to time of day. Light elicits an acute increase in locomotion (startle) that is modulated in a time-of-day-dependent manner: startle is potentiated during the nighttime, when light is unexpected, but is suppressed during the daytime. The internal daytime-nighttime context is generated by two interconnected and functionally opposing populations of circadian neurons-LNvs generating the daytime state and DN1 as generating the nighttime state. Switching between the two states requires daily remodeling of LNv and DN1a axons such that the maximum presynaptic area in one population coincides with the minimum in the other. It is proposed that a dynamic model of environmental light resides in the shifting connectivities of the LNv-DN1a circuit, which helps animals evaluate ongoing conditions and choose a behavioral response (Song, 2021).

    Alterations of neuronal activity, rather than morphology, are usually considered the cause of cognitive flexibility. The mechanism that this study describes relies on physical cellular restructuring. What are the advantages of a system like this? While near-instantaneous electrical activity is the basic language of neurons, many behaviors and internal states occur on much longer time scales. Morphological remodeling is a slow process, aligning with functions that change over the course of hours. In support of this view, changes in neuronal morphology have been found to underlie appetite, sexual experience, and foraging history. Although seemingly wasteful, physical remodeling may be particularly useful for encoding relatively stable states, due to presumably high energetic barrier (Song, 2021).

    Understanding the mechanisms of circuit state transitions may help clarify the etiology of mood disorders like depression or bipolar disorder. These disorders are characterized by excessive or insufficient transitions between extreme states and, as such, may reflect a collapse of organizational principles that permit flexible circuit function. It is generally unknown how behavioral states can be stable across long time scales while also being able to undergo flexible transitions. Motifs from the LNv-DN1a circuit illustrate one solution to this apparent contradiction. LNvs and DN1as are arranged in a mutually inhibitory system, which may help ensure stability, consistency, and accuracy over long time scales. In the absence of external influences, reciprocal inhibition can stabilize a winner-take-all steady state. Structural plasticity is a potential way to overcome rigidity by providing a molecular mechanism to surmount electrical inhibition (Song, 2021).

    In this model, the molecular oscillations of the circadian clock direct oscillations in cell shape through molecular effectors such as Rho1, ultimately leading to changes in behavior. Rho1 overexpression experiments suggest that neuronal morphology causally influences behavior. However, it cannot be ruled out that high levels of Rho1 cause off-target effects that have not been accounted for. Previous work used conditional methods to determine that Rho1 regulates daily LNv remodeling rhythms, but this study used constitutive Rho1 overexpression (to avoid using high temperatures required for the conditional experiment), and thus cannot exclude the possibility of developmental confounds (Song, 2021).

    It is possible, if not likely, that other circadian neuronal populations also contribute to generating predictions about light. In this study, the optogenetic silencing phenotypes of LNvs and DN1as together recapitulate the phenotype produced by network-wide loss of clock function. However, the native circuit signal may be built by the cooperative action of multiple subpopulations with overlapping tuning. Hints of subpopulation cooperativity are apparent in the data: there was a stronger phenotype when PDF was knocked down in both small and large LNvs, compared to small LNvs alone. Glycine was recently found to be a fast, inhibitory neurotransmitter that is co-released from s-LNvs in addition to the neuropeptide PDF. While a necessary and sufficient role for the neuropeptide PDF in the behavioral assay, it cannot be ruled out that glycine instead mediates the fast inhibition seen during ex vivo calcium imaging. Furthermore, this study focused on two time points, but other populations may be more influential during other times of day. In support of this theory, it was recently found that lateral dorsal neurons also show axon remodeling rhythms centered around the evening, a time point that this study did not address. Because the other clock neuron subpopulations are active at different times, and recurrent inhibition is prevalent in the fly circadian network, it is likely that LNv-DN1a interactions are necessary, but not sufficient, for circadian network operations in the context described in this study (Song, 2021).

    What this study calls 'startle' is commonly referred to as 'masking' because acute reactivity to lights-on and lights-off can distort measurements of circadian rhythmicity. For this reason, most assays of circadian function are done in unchanging environmental conditions (e.g., constant darkness). This study showed that startle to lights-on can also be an informative indicator of internal timekeeping. The conditional experiments using optogenetics suggest that clock neuron activity contextualizes light on an ongoing basis. However, because the experimental animals were exposed to daytime light during the habituation period, the possibility that LNv and DN1a activity is also required during entrainment cannot be excluded. Another consideration is that light resets the molecular clock. One hour of nighttime light can advance or delay circadian rhythms, which is apparent in the timing of rest and activity on subsequent days. It is unknown if this phase-shifting phenomenon affects the acute reactivity observed in this study (Song, 2021).

    Circadian clocks have evolved in the context of consistent light schedules, and this predictability has been relatively unchallenged across evolutionary history. Ubiquitous artificial lighting introduces new strains on the circadian system. Misalignment between internal rhythms and the external world can have profound consequences on health and cognition. The feeling of jetlag demonstrates the acute physical and mental burden of when internal clocks are in conflict with the external world. Chronic misalignment, such as in the case of night-shift workers, causes increased rates of cancer. The use of electronic devices before bedtime has been linked to delays in sleep onset and reductions in sleep quality. The system this study described in Drosophila presents a model to understand the acute consequences of circadian misalignment (Song, 2021).

    A predictive nervous system enables continual evaluation of reality relative to context. One result of this is that a fixed stimulus can evoke a multitude of behaviors depending on an animal's history, needs, and external context. This study shows how the Drosophila circadian system creates a dynamic internal reference for what environmental conditions should be. Many of the motifs observed are conserved: Mammalian circadian clock neuron subpopulations are also active at different times and are linked by recurrent inhibition, suggesting that mammalian temporal estimation may operate using similar principles. The paradigm that this study developed offers opportunities to understand the interface between internal models and sensory evidence. Circadian neurons are sensitive to environmental inputs: can they autonomously compute prediction error? They communicate with downstream dopaminergic populations: are those analogous to mammalian midbrain dopaminergic neurons whose activities reflect prediction error? It is proposed that flies assign valence to experienced environmental conditions, a computation that uses an internal model generated through circuit remodeling (Song, 2021).

    Neuropeptides PDF and DH31 hierarchically regulate free-running rhythmicity in Drosophila circadian locomotor activity

    Neuropeptides play pivotal roles in modulating circadian rhythms. Pigment-dispersing factor (PDF) is critical to the circadian rhythms in Drosophila locomotor activity. This study demonstrates that diuretic hormone 31 (DH31) complements PDF function in regulating free-running rhythmicity using male flies. It was determined that Dh31 loss-of-function mutants (Dh31#51) showed normal rhythmicity, whereas Dh31(#51);Pdf01 double mutants exhibited a severe arrhythmic phenotype compared to Pdf-null mutants (Pdf01). The expression of tethered-PDF or tethered-DH31 in clock cells, posterior dorsal neurons 1 (DN1ps), overcomes the severe arrhythmicity of Dh31(#51);Pdf01double mutants, suggesting that DH31 and PDF may act on DN1ps to regulate free-running rhythmicity in a hierarchical manner. Unexpectedly, the molecular oscillations in Dh31(#51);Pdf01 mutants were similar to those in Pdf01 mutants in DN1ps, indicating that DH31 does not contribute to molecular oscillations. Furthermore, a reduction in Dh31 receptor (Dh31r) expression resulted in normal locomotor activity and did not enhance the arrhythmic phenotype caused by the Pdf receptor (Pdfr) mutation, suggesting that PDFR, but not DH31R, in DN1ps mainly regulates free-running rhythmicity. Taken together, this study identifies a novel role of DH31, in which DH31 and PDF hierarchically regulate free-running rhythmicity through DN1ps (Goda, 2019).

    This study has demonstrated a novel function of DH31 in regulating Drosophila locomotor activity rhythms. Dh31#51 mutants maintained a robust free-running rhythm, whereas Dh31#51;Pdf01 double-mutant flies exhibited a severe disruption of their free-running rhythm compared to Pdf01 mutants. These findings suggest that Dh31#51 mutants maintain a robust free-running rhythm because the primary factor, PDF, can sustain a strong rhythm. ~40% of Pdf01 single-mutant flies exhibited a preserved rhythmic state, which is because DH31 can partially support free-running rhythmicity. Thus, the severe disruptions of free-running rhythm in Pdf01 and Dh31#51 double-mutant flies is likely caused by the loss of both pathways (Goda, 2019).

    PDF is secreted from the main circadian neurons, LNvs, and acts on other clock cells through PDFR to synchronize and maintain robust molecular rhythms. PDF expression from LNvs in Dh31#51;Pdf01 mutants restored rhythmicity, in contrast to tethered-PDF (t-PDF) expression in LNvs, indicating that an autoreceptor of PDF signals in LNvs is not sufficient to maintain rhythmicity. Instead, t-PDF expression in DN1ps restored rhythmicity, suggesting that PDF signaling in DN1ps is sufficient to maintain robust free-running rhythmicity. Recently, the responsiveness to PDF was shown to be strongly altered for 24 h via RalA GTPase in sLNvs28. Therefore, it is expected that the continuous activation of PDFR by t-PDF generates rhythmic downstream signaling in PDFR-expressing neurons (Goda, 2019).

    Molecular oscillations in DN1s were strongly dampened in Pdf01 mutants compared with WT flies. These data are consistent with previous studies in which the molecular oscillations of PER in Pdf01 mutants held under DD conditions were dampened in DN1s10 and the genetic manipulation of the circadian clocks in PDF-positive cells altered the molecular rhythms in DN1ps. Furthermore, Pdfr expression in DN1ps has been reported to prevent the arrhythmic phenotype in Pdfr5304 mutants. These findings support the idea that PDF is secreted from LNvs and acts on DN1ps to regulate free-running rhythmicity (Goda, 2019).

    Furthermore, it was shown that t-DH31 expression in DN1ps rescued the Pdf01 and Dh31#51 double-mutant phenotypes, which suggests that DH31 acts on DN1ps to regulate rhythmicity. Although it has been suggested that DH31 release might increase at dawn and that DH31-mRNA expression levels oscillate for 24 h, how t-DH31 expression causes rhythmic behavioral output remains unclear. Because DH31 can modestly activate PDFR in vitro, it cannot be excluded that t-DH31 overexpression might simply activate PDFR in DN1ps instead of the intrinsic PDF signals, thereby restoring locomotor activity rhythms in the flies. However, the rhythmicity of Dh31#51;Pdf01 mutants overexpressing t-DH31 in tim-Gal4-expressing neurons or R18H11-Gal4-expressing DN1ps only reached levels similar to that of the Pdf01 single-mutant flies. Therefore, DH31 likely acts on DN1ps separately from the PDF pathway (Goda, 2019).

    Although it has been shown that DH31 is expressed in a subset of DN1ps, DH31 expression using R18H11-Gal4 did not rescue the Pdf01 and Dh31#51 double-mutant phenotypes, suggesting that DH31 expression in R18H11-Gal4-expressing neurons is insufficient to maintain rhythmicity. Instead, DH31 is expressed in DN1as and DH31 expression in tim-Gal4-expressing neurons rescued the phenotype, which suggests that DH31 expression in clock neurons maintains rhythmicity. That said, given that DH31 is expressed in nonclock neurons and that tim-Gal4 is expressed in nonclock cells, it cannot be excluded that DH31 expression in nonclock neurons might play a role in rescuing the severe phenotype of Dh31#51;Pdf01 mutants. Alternatively, although DH31 expression in LNvs was not detectable via anti-DH31 antibody staining, a recent RNA-seq analysis detected Dh31 gene expression in both LNvs and DN1s. Therefore, DH31 expression from LNvs may potentially act on DN1s to support locomotor activity rhythms (Goda, 2019).

    In summary, it is proposed that PDF and DH31 regulate free-running rhythms in a hierarchical fashion in DN1ps. As t-DH31 or t-PDF expression in DN1ps resulted in a similar level of rhythmicity as that observed in flies expressing t-DH31 or t-PDF, respectively, in tim-Gal4-expressing neurons, DN1ps are at least one of the important clock cells that regulate free-running rhythmicity (Goda, 2019).

    Given that Dh31#51;Pdf01 mutants exhibited severe arrhythmicity in free-running rhythm, it is speculated that the severe arrhythmic phenotype might be a result of abnormal molecular oscillations. However, the molecular oscillations of Dh31#51;Pdf01 mutants were similar to those of Pdf01 mutants. Therefore, the molecular mechanisms by which DH31 regulates free-running rhythms still remain unclear. Importantly, the peak of VRI expression in LNds in Dh31#51 was at ZT 19, which was delayed compared with those of WT flies and the other mutants. The data suggested that DH31 is involved in the regulation of molecular oscillations in LNds. Because LNds are the evening pacemaker, the delayed VRI oscillations in LNds might be associated with the longer period of free-running rhythm in Dh31#51 (Goda, 2019).

    Recently, the intracellular calcium rhythms in each clock cell were reported to be nonsynchronous and associated with morning and evening peaks in locomotor activity. DH31 signaling may possibly contribute to the downstream output that controls molecular rhythms in pacemaker processes, such as intracellular calcium rhythms. Given that PDF from sLNvs regulates strong molecular rhythms in DN1ps and generates robust free-running rhythms under constant conditions, DH31 may help maintain vigorous output signals downstream of the molecular clocks in DN1ps (Goda, 2019).

    Recently work has shown that both Dh31r1/Df mutants and flies undergoing Dh31r knockdown in their neurons showed normal rhythmicity in the locomotor activity rhythm25. In contrast to Dh31#51;Pdf01 double mutants, Pdfr5304;Dh31r1/Df double mutants did not enhance the arrhythmicity observed in Pdfr single mutants, which suggests that Dh31r does not complement PDFR function; thus, Dh31r does not function as a receptor for DH31 in this context. Given that Dh31r1/Df flies showed a strong abnormality in the TPR phenotype25, it is more likely that Dh31r does not play an important role in locomotor activity rhythms. However, Dh31r1/Df4 mutants are not null25, and it cannot be excluded that a small amount of residual Dh31r might drive robust locomotor activity rhythms with the PDF pathway (Goda, 2019).

    Which receptors might function with DH31 to regulate free-running rhythmicity? Given that DH31 can activate PDFR in vitro, bath applications of DH31 can activate LNvs via PDFR19 and DH31 can function as a ligand of PDFR in TPR at the onset of night, PDFR may function as a receptors for both DH31 and PDF in the regulation of free-running rhythmicity. However, because the arrhythmicity of Pdfr5304 mutants was not as severe as that of Dh31#51;Pdf01 mutants, PDFR does not appear to act as a receptor for DH31 in this context (Goda, 2019).

    Both Dh31r and PDFR are class II G-protein coupled receptors (GPCRs), which also include Hector and Diuretic hormone 44 receptors 1 and 2 (DH44R1 and DH44R2, respectively). Interestingly, the DH44R1 and DH44R2 ligand DH44 has been implicated in circadian output circuits. Therefore, although there is no evidence from in vitro or in vivo experiments, these receptors might nevertheless function as receptors for DH31 to regulate free-running rhythmicity (Goda, 2019).

    Orchestration of neuropeptides regulates locomotor activity rhythms in species ranging from flies to mammals The orchestration of neuropeptides is critical for regulating circadian clock functions in species that range from flies to mammals. In mammals, several neuropeptides, including vasoactive intestinal polypeptide (VIP), arginine vasopressin (AVP) and neuromedin S (NMS), are expressed in the SCN, which is the center for circadian clock control. The hierarchy of neuropeptide signaling contributes to circadian function in the SCN. Several recent studies i