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 ß-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).

    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).

    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).

    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).

    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).

    Smith, B. R. and Macdonald, S. J. (2020). Dissecting the Genetic Basis of Variation in Drosophila Sleep Using a Multiparental QTL Mapping Resource. Genes (Basel) 11(3). PubMed ID: 32168738

    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). .

    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).

    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).

    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).

    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).

    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).

    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).

    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).

    A semi-natural approach for studying seasonal diapause in Drosophila melanogaster reveals robust photoperiodicity

    The fruit fly Drosophila melanogaster survives thermally stressful conditions in a state of reproductive dormancy (diapause), manifested by reduced metabolic activity and arrested ovarian development in females. Unlike insects that rely primarily on photoperiodic stimuli to initiate the diapause program, in this species dormancy is regulated by low temperature and enhanced by shorter photoperiods. Overwintering phenotypes are usually studied under simple laboratory conditions, where animals are exposed to rectangular light-dark (LD) cycles at a constant temperature. This study sought to adopt more realistic diapause protocols by generating LD profiles that better mimic outdoor conditions. Experimental flies were subjected to semi-natural late autumn and summer days, while control females received the same amounts of light but in rectangular LD cycles (LD 8:16 and LD 15:9, respectively). Semi-natural autumnal days were found to induce a higher proportion of females to enter dormancy, while females in semi-natural summer days showed reduced diapause compared with their corresponding rectangular controls, generating an impressive photoperiodic response. In contrast, under rectangular light regimes, the diapause of Drosophila field lines exhibited minimal photoperiodicity. This semi-natural method reveals that D. melanogaster diapause is considerably more photoperiodic than previously believed and suggests that this seasonal response is best studied under simulated natural lighting conditions (Nagi, 2018).

    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).

    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).

    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).

    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).

    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).

    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).

    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 in Drosophila have identified the neuropeptides, including ion transport peptide (ITP), neuropeptide F (NPF), allatostatin A, short neuropeptide F, leucokinin and DH44, that regulate locomotor activity and sleep. However, given that DH31 complements the function of PDF in regulating free-running rhythmicity in the same clock cells, DH31 not only serves as one of the neuropeptides that regulates circadian rhythms but also might selectively influence PDF function in the regulation of free-running rhythms. Thus, these findings shed new light on the next steps required to improve understanding of the core neuropeptide regulatory mechanisms involved in the circadian rhythm (Goda, 2019).

    Circadian and feeding cues integrate to drive rhythms of physiology in Drosophila insulin-producing cells

    Circadian clocks regulate much of behavior and physiology, but the mechanisms by which they do so remain poorly understood. While cyclic gene expression is thought to underlie metabolic rhythms, little is known about cycles in cellular physiology. This study found that Drosophila insulin-producing cells (IPCs), which are located in the pars intercerebralis and lack an autonomous circadian clock, are functionally connected to the central circadian clock circuit via DN1 neurons. Insulin mediates circadian output by regulating the rhythmic expression of a metabolic gene (sxe2) in the fat body. Patch clamp electrophysiology reveals that IPCs display circadian clock-regulated daily rhythms in firing event frequency and bursting proportion under light:dark conditions. The activity of IPCs and the rhythmic expression of sxe2 are additionally regulated by feeding, as demonstrated by night feeding-induced changes in IPC firing characteristics and sxe2 levels in the fat body. These findings indicate circuit-level regulation of metabolism by clock cells in Drosophila and support a role for the pars intercerebralis in integrating circadian control of behavior and physiology (Barber, 2016).

    Circadian rhythms in neuronal activity propagate through output circuits

    Twenty-four hour rhythms in behavior are organized by a network of circadian pacemaker neurons. Rhythmic activity in this network is generated by intrinsic rhythms in clock neuron physiology and communication between clock neurons. However, it is poorly understood how the activity of a small number of pacemaker neurons is translated into rhythmic behavior of the whole animal. To understand this, a screen was carried out for signals that could identify circadian output circuits in Drosophila melanogaster. Leucokinin neuropeptide (LK) and its receptor (LK-R) were found to be required for normal behavioral rhythms. This LK/LK-R circuit connects pacemaker neurons to brain areas that regulate locomotor activity and sleep. These experiments revealed that pacemaker neurons impose rhythmic activity and excitability on LK- and LK-R-expressing neurons. Pacemaker neuron-dependent activity rhythms were also found in a second circadian output pathway controlled by DH44 neuropeptide-expressing neurons. It is concluded that rhythmic clock neuron activity propagates to multiple downstream circuits to orchestrate behavioral rhythms (Cavey, 2016).

    USP2-45 is a circadian clock output effector regulating calcium absorption at the post-translational level

    The mammalian circadian clock influences most aspects of physiology and behavior through the transcriptional control of a wide variety of genes, mostly in a tissue-specific manner. About 20 clock-controlled genes (CCGs) oscillate in virtually all mammalian tissues and are generally considered as core clock components. One of them is Ubiquitin-Specific Protease 2 (Usp2), whose status remains controversial, as it may be a cogwheel regulating the stability or activity of core cogwheels or an output effector. This study reports that Usp2 is a clock output effector related to bodily Ca2+ homeostasis, a feature that is conserved across evolution. Drosophila with a whole-body knockdown of the orthologue of Usp2, (Ubiquitin specific protease 2) predominantly die during pupation but are rescued by dietary Ca2+ supplementation. Usp2-KO mice show hyperabsorption of dietary Ca2+ in small intestine, likely due to strong overexpression of the membrane scaffold protein NHERF4, a regulator of the Ca2+ channel TRPV6 mediating dietary Ca2+ uptake. In this tissue, USP2-45 is found in membrane fractions and negatively regulates NHERF4 protein abundance in a rhythmic manner at the protein level. In clock mutant animals (Cry1/Cry2-dKO; see Drosophila Cryptochrome), rhythmic USP2-45 expression is lost, as well as the one of NHERF4, confirming the inverse relationship between USP2-45 and NHERF4 protein levels. Finally, USP2-45 interacts in vitro with NHERF4 and endogenous Clathrin Heavy Chain (see Drosophila Clathrin Heavy Chain). Taken together these data has led the authors to define USP2-45 as the first clock output effector acting at the post-translational level at cell membranes and possibly regulating membrane permeability of Ca2+ (Pouly, 2016).

    Molecular correlates of circadian clocks in fruit fly Drosophila melanogaster populations exhibiting early and late emergence chronotypes

    This study used a laboratory selection approach to raise populations of Drosophila melanogaster that emerge in the morning (early) or in the evening (late), and over 14 years of continued selection, clear divergence of their circadian phenotypes is reported. The molecular correlates of early and late emergence chronotypes were also assessed, and significant divergence is reported in transcriptional regulation, including the mean phase, amplitude and levels of period (per), timeless (tim), clock (clk) and vrille (vri) messenger RNA (mRNA) expression. Corroborating some of the previously reported light-sensitivity and oscillator network coupling differences between the early and the late populations, differences are also reported in mRNA expression of the circadian photoreceptor cryptochrome (cry) and in the mean phase, amplitude and levels of the neuropeptide pigment-dispersing factor (PDF). These results provide the first-ever direct evidence for divergent evolution of molecular circadian clocks in response to selection imposed on an overt rhythmic behavior and highlight early and late populations as potential models for chronotype studies by providing a preliminary groundwork for further exploration of molecular-genetic correlates underlying circadian clock-chronotype association (Nikhil, 2016).

    Drosophila Ionotropic Receptor 25a mediates circadian clock resetting by temperature

    Circadian clocks are endogenous timers adjusting behaviour and physiology with the solar day. 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 2-4 degrees C difference. 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.

    A homeostatic sleep-stabilizing pathway in Drosophila composed of the Sex Peptide receptor and its ligand, the myoinhibitory peptide

    Sleep, a reversible quiescent state found in both invertebrate and vertebrate animals, disconnects animals from their environment and is highly regulated for coordination with wakeful activities, such as reproduction. The fruit fly, Drosophila melanogaster, has proven to be a valuable model for studying the regulation of sleep by circadian clock and homeostatic mechanisms. This study demonstrates that the Sex peptide receptor (SPR) of Drosophila, known for its role in female reproduction, is also important in stabilizing sleep in both males and females. Mutants lacking either the SPR or its central ligand, myoinhibitory peptide (MIP), fall asleep normally, but have difficulty in maintaining a sleep-like state. This analyses have mapped the SPR sleep function to pigment dispersing factor (pdf) neurons, an arousal center in the insect brain. MIP downregulates intracellular cAMP levels in pdf neurons through the SPR. MIP is released centrally before and during night-time sleep, when the sleep drive is elevated. Sleep deprivation during the night facilitates MIP secretion from specific brain neurons innervating pdf neurons. Moreover, flies lacking either SPR or MIP cannot recover sleep after the night-time sleep deprivation. These results delineate a central neuropeptide circuit that stabilizes the sleep state by feeding a slow-acting inhibitory input into the arousal system and plays an important role in sleep homeostasis (Oh, 2014: PubMed).

    Drosophila DH31 neuropeptide and PDF receptor regulate night-onset temperature preference

    Body temperature exhibits rhythmic fluctuations over a 24 h period and decreases during the night, which is associated with sleep initiation. However, the underlying mechanism of this temperature decrease is largely unknown. Previous work has shown that Drosophila exhibit a daily temperature preference rhythm (TPR), in which their preferred temperatures increase during the daytime and then decrease at the transition from day to night (night-onset). Because Drosophila are small ectotherms, their body temperature is very close to that of the ambient temperature, suggesting that their TPR generates their body temperature rhythm. This study demonstrates that the neuropeptide diuretic hormone 31 (DH31) and pigment-dispersing factor receptor (PDFR) contribute to regulate the preferred temperature decrease at night-onset. PDFR and tethered-DH31 expression in dorsal neurons 2 (DN2s) restore the preferred temperature decrease at night-onset, suggesting that DH31 acts on PDFR in DN2s. Notably, it was previously shown that the molecular clock in DN2s is important for TPR. Although PDF (another ligand of PDFR) is a critical factor for locomotor activity rhythms, Pdf mutants exhibit normal preferred temperature decreases at night-onset. This suggests that DH31-PDFR signaling specifically regulates a preferred temperature decrease at night-onset. Thus, it is proposed that night-onset TPR and locomotor activity rhythms are differentially controlled not only by clock neurons but also by neuropeptide signaling in the brain (Goda, 2016).

    Body temperature rhythm (BTR) is fundamental for maintaining homeostasis, such as in generating metabolic energy and sleep. BTR is one of the most robust circadian outputs and can affect the peripheral clocks of mammals. The rhythmic patterns of BTR and locomotor activity rhythms are analogous. For instance, in diurnal mammals, both body temperature and locomotor activity increase during the daytime and decrease at night. Nonetheless, BTR and locomotor activity rhythms are regulated by different subsets of subparaventricular zone (SPZ) neurons, suggesting that these rhythms are controlled independently (Goda, 2016).

    Mammals are not the only examples of this phenomenon. Previous work has shown that Drosophila exhibit a daily temperature preference rhythm (TPR), in which their preferred temperatures increase during the daytime and then decrease at the transition from day to night (night-onset) (Kaneko, 2012). Because Drosophila are small ectotherms, their body temperature is very close to that of the ambient temperature, suggesting that their TPR generates their BTR. In Drosophila, TPR and locomotor activity rhythms are regulated by different subsets of clock neurons (Kaneko, 2012). There are ~150 central pacemaker cells in the fly brain, which are functionally homologous to mammalian suprachiasmatic nucleus (SCN) neurons. These pacemaker cells are approximately divided into lateral neurons [LNs; small ventral LNs (s-LNvs), large ventral LNs (l-LNvs), and dorsal lateral neurons (LNds)] and dorsal neurons (DNs; DN1, DN2, and DN3) based on their location and size. The clocks in DN2s are critical for regulating TPR during the daytime (Kaneko, 2012), but not for regulating locomotor activity rhythms (Goda, 2016 and references therein).

    Neuropeptides and their receptors have important roles in synchronizing circadian clocks. A class II G-protein-coupled receptor, pigment-dispersing factor receptor (PDFR), and its ligand (PDF) play important roles in synchronizing circadian clocks and are required for robust circadian locomotor activity in Drosophila. Notably, PDF and PDFR function in a similar manner to vasoactive intestinal peptide (VIP) and its receptor VPAC2 in mammals, both of which play important roles in the ability of clock neurons to regulate the rhythmicity and synchrony of both locomotor activity rhythms and BTRs (Goda, 2016).

    Recent reports have suggested that, in addition to PDF, diuretic hormone 31 (DH31) also activates PDFR based on in vitro experiments and a study that used brain imaging with bath-applied DH31. Moreover, it has been shown that DH31 is expressed in the posterior dorsal neurons 1 (DN1ps) and that it modulates sleep as a wake-promoting signal before dawn but does not affect locomotor activity rhythms in Drosophila. DH31 is a functional homolog of mammalian calcitonin gene-related peptide (CGRP), which mediates thermosensation and thermoregulation. However, it is unknown whether CGRP is involved in the regulation of BTR in mammals (Goda, 2016).

    This study demonstrates that DH31 and PDFR play important roles for TPR at night-onset. DN2s are the main clock cells for TPR, and the data suggest that DH31 binding to PDFR in DN2s regulates temperature preference decreases at night-onset, which is the first in vivo evidence that DH31 could function as a ligand of PDFR. Therefore, it is proposed that circadian locomotor activity and night-onset TPR are regulated by different neuropeptides that use the same receptor expressed in different clock cells (Goda, 2016).

    Both Dh31 and Pdfr mutants exhibited abnormal night-onset TPR, and t-DH31 and PDFR expression in DN2s are sufficient to control night-onset TPR, suggesting that DH31 could be a ligand of PDFR in DN2s. Unexpectedly, PDF, an important neuropeptide for locomotor activity, is not required for night-onset TPR. It suggests that PDF and DH31 appear to act on different subsets of PDFR-expressing clock cells to regulate locomotor activity rhythms and night-onset TPR, respectively. Together, the data suggest that locomotor activity and night-onset TPR are regulated through PDFR by different subsets of clock neurons using different neuropeptides. Nonetheless, because all Dh31, Pdf, and Pdfr mutants still maintain normal daytime TPR, the underlying molecular mechanisms of daytime TPR are still obscure (Goda, 2016).

    In humans, body temperature dramatically decreases at night, which is associated with sleep initiation. Although it suggests a relationship between BTR and sleep-wake cycles, the underlying mechanisms are largely unclear. A recent study suggested that DH31 mediates sleep-wake cycles and that DH31 secretion functions as a wake-promoting signal before dawn. Given that it was found that DH31 is required for night-onset TPR, DH31 could regulate both sleep-wake cycles and TPR with different timing (i.e., at night-onset for TPR and before dawn for sleep) (Goda, 2016).

    While t-DH31 expression in DN2s rescues the Dh31#51 phenotype, t-PDF expression in DN2s slightly rescues in comparison with UAS controls. This shows that t-DH31 in DN2s rescues more efficiently than t-PDF in DN2s. However, DH31 stimulates PDFR less efficiently than PDF in vitro. This suggests that either DH31 may more efficiently bind PDFR in DN2s than PDF or that DN2s may express another receptor of DH31 that also regulates night-onset TPR. Notably, although the DH31 receptor (DH31R) is a known receptor for DH31 in vitro, this study observed that DH31R is not expressed in DN2s and that the Dh31r mutant exhibited normal night-onset TPR. Therefore, it would be interesting to further explore whether additional receptors for DH31 in DN2s are involved in night-onset TPR. It has been previously shown that flies in the light prefer a 1°C higher temperature than in the dark (light-dependent temperature preference [LDTP]), thus demonstrating that light influences temperature preference behavior. Importantly, while LDTP is only affected by light, night-onset TPR is affected by both light and time (ZT10-ZT12 and ZT13-ZT15). Therefore, they are not controlled by the same pathway. In fact, PDFR expression using Clk4.5F-Gal4 rescues the abnormal LDTP phenotype of Pdfr5304 mutants, but it did not rescue night-onset TPR (Goda, 2016).

    DH31 is a functional homolog of mammalian CGRP. Importantly, CGRP is related to many physiological functions, such as temperature sensation, migraines, and chronic pain in mammals. CGRP has recently attracted attention for its role in the treatment of migraine attacks, which are associated with body-temperature fluctuations. Additionally, CGRP is expressed in the SCN, but its function in circadian rhythms is not entirely clear. Thus, the findings raise the possibility that CGRP may be involved in BTR regulation and may mediate the connection between the circadian clock and other physiological functions. Moreover, this research will not only expand current understanding of neuropeptidergic regulation, but may also ultimately facilitate a discovery of the fundamental mechanisms of BTR (Goda, 2016).

    Mated Drosophila melanogaster females consume more amino acids during the dark phase

    To maintain homeostasis, animals must ingest appropriate quantities, determined by their internal nutritional state, of suitable nutrients. In the fruit fly Drosophila melanogaster, an amino acid deficit induces a specific appetite for amino acids and thus results in their increased consumption. Although multiple processes of physiology, metabolism, and behavior are under circadian control in many organisms, it is unclear whether the circadian clock also modulates such motivated behavior driven by an internal need. Differences in levels of amino acid consumption by flies between the light and dark phases of the day:night cycle were examined using a capillary feeder assay following amino acid deprivation. Female flies exhibited increased consumption of amino acids during the dark phase compared with the light phase. Investigation of mutants lacking a functional period gene (per0), a well-characterized clock gene in Drosophila, found no difference between the light and dark phases in amino acid consumption by per0 flies. Furthermore, increased consumption of amino acids during the dark phase was observed in mated but not in virgin females, which strongly suggested that mating is involved in the rhythmic modulation of amino acid intake. Egg production, which is induced by mating, did not affect the rhythmic change in amino acid consumption, although egg-laying behavior showed a per0-dependent change in rhythm. Elevated consumption of amino acids during the dark phase was partly induced by the action of a seminal protein, sex peptide (SP), on the sex peptide receptor (SPR) in females. Moreover, the increased consumption of amino acids during the dark phase is induced in mated females independently of their internal level of amino acids. These results suggest that a post-mating SP/SPR signal elevates amino acid consumption during the dark phase via the circadian clock (Uchizono, 2017).

    Transient dysregulation of dopamine signaling in a developing Drosophila arousal circuit permanently impairs behavioral responsiveness in adults

    The dopamine ontogeny hypothesis for schizophrenia proposes that transient dysregulation of the dopaminergic system during brain development increases the likelihood of this disorder in adulthood. To test this hypothesis in a high-throughput animal model, this study transiently manipulated dopamine signaling in the developing fruit fly Drosophila melanogaster and examined behavioral responsiveness in adult flies. Either a transient increase of dopamine neuron activity or a transient decrease of dopamine receptor expression during fly brain development permanently impairs behavioral responsiveness in adults. A screen for impaired responsiveness revealed sleep-promoting neurons in the central brain as likely postsynaptic dopamine targets modulating these behavioral effects. Transient dopamine receptor knockdown during development in a restricted set of ~20 sleep-promoting neurons recapitulated the dopamine ontogeny phenotype, by permanently reducing responsiveness in adult animals. This suggests that disorders involving impaired behavioral responsiveness might result from defective ontogeny of sleep/wake circuits (Ferguson, 2017).

    Circadian rhythm in mRNA expression of the glutathione synthesis gene Gclc is controlled by peripheral glial clocks in Drosophila melanogaster

    Circadian coordination of metabolism, physiology, and behaviour is found in all living kingdoms. Clock genes are transcriptional regulators, and their rhythmic activities generate daily rhythms in clock-controlled genes which result in cellular and organismal rhythms. Insects provide numerous examples of rhythms in behaviour and reproduction, but less is known about control of metabolic processes by circadian clocks in insects. Recent data suggest that several pathways involved in protecting cells from oxidative stress may be modulated by the circadian system, including genes involved in glutathione (GSH) biosynthesis. Specifically, rhythmic expression of the gene encoding the catalytic subunit (Gclc) of the rate-limiting GSH biosynthetic enzyme was detected in Drosophila melanogaster heads. The aim of this study was to determine which clocks in the fly multi-oscillatory circadian system are responsible for Gclc rhythms. Genetic disruption of tissue-specific clocks in D. melanogaster revealed that transcriptional rhythms in Gclc mRNA levels occur independently of the central pacemaker neurons, because these rhythms persisted in heads of behaviourally arrhythmic flies with a disabled central clock but intact peripheral clocks. Disrupting the clock specifically in glial cells abolished rhythmic expression of Gclc, suggesting that glia play an important role in Gclc transcriptional regulation, which may contribute to maintaining homeostasis in the fly nervous system (Chow, 2017).

    Starvation promotes odor/feeding-time associations in flies

    Starvation causes a motivational state that facilitates diverse behaviors such as feeding, walking, and search. Starved Drosophila can form odor/feeding-time associations but the role of starvation in encoding of "time" is poorly understood. This study shows that the extent of starvation is correlated with the fly's ability to establish odor/feeding-time memories. Prolonged starvation promotes odor/feeding-time associations after just a single cycle of reciprocal training. Starvation is also show to be required for acquisition but is dispensable for retrieval of odor/feeding-time memory. Finally, even with extended starvation, a functional circadian oscillator is indispensable for establishing odor/feeding-time memories (Chouhan, 2017).

    Organization of circadian behavior relies on glycinergic transmission

    The small ventral lateral neurons (sLNvs) constitute a central circadian pacemaker in the Drosophila brain. They organize daily locomotor activity, partly through the release of the neuropeptide pigment-dispersing factor (PDF), coordinating the action of the remaining clusters required for network synchronization. Despite extensive efforts, the basic principles underlying communication among circadian clusters remain obscure. This study identified classical neurotransmitters released by sLNvs through disruption of specific transporters. Adult-specific RNAi-mediated downregulation of the glycine transporter or impairment of glycine synthesis in LNv neurons increased period length by nearly an hour without affecting rhythmicity of locomotor activity. Electrophysiological recordings showed that glycine reduces spiking frequency in circadian neurons. Interestingly, downregulation of glycine receptor subunits in specific sLNv targets impaired rhythmicity, revealing involvement of glycine in information processing within the network. These data identify glycinergic inhibition of specific targets as a cue that contributes to the synchronization of the circadian network (Frenkel, 2017).

    Anatomical and functional evidence indicates that classical neurotransmitters participate along with PDF in the output of the LNvs. In an attempt to define their classical neurotransmitter, this study manipulated the level of membrane or vesicular transporters of known neurotransmitter systems exclusively in this neuronal group. Downregulation of CG5549, the glycine transporter (dGlyT) responsible for recycling this transmitter from the extracellular space, triggered a consistent change in the endogenous period. This effect is specific, as downregulation of List (CG15088, the lithium-inducible SLC6 transporter) did not change any circadian parameter. On the other hand, downregulation of Drosophila Serine hydroxymethyltransferase CG3011 (dShmt; glycine hydroxymethyltransferase activity) also gives rise to period lengthening, consistent with an effect on the circadian clock as opposed to a general effect on LNv viability, which results in progressive loss of rhythmicity. Interestingly, it has been shown that expression of a tethered version of PDF in the LNvs in a condition where neurotransmitters cannot be released increases in almost an hour the endogenous circadian period, suggesting that neurotransmission contributes to rhythm acceleration, lending further support the findings showing that glycine plays such a role in the adult brain (Frenkel, 2017).

    Glycinergic inhibitory transmission plays a role in nociception and motor control in the brainstem and spinal cord. Glycine is also a modulator of neuronal excitation mediated by NMDA receptors at glutamatergic synapses in the central brain. This study found glycine in the Drosophila CNS (specifically, in a subset of circadian pacemakers) and found that it stops action potential firing in a postsynaptic target. Interestingly, at least some neurons of the suprachiasmatic nucleus (SCN) are inhibited in the presence of glycine, highlighting another layer of conservation among circadian clocks (Frenkel, 2017).

    Glycine receptors are part of the complex cys-loop receptor family of pentameric ligand-gated ion channels. Part of the complexity resides in their ability to assemble specific receptors depending on the subunits recruited, thus leading to both excitatory and inhibitory responses. Little is known about the glycinergic ones, particularly in the fruit fly. Initially, a single gene was reported as the putative glycine receptor GRD. To identify additional glycine subunits, an in silico approach was used. Three putative glycine receptor subunits share conserved features present in ligand-gated chloride channel ones. Interestingly, combined downregulation of those genes in DN1ps revealed they mediate, in part, the response to glycine. Additional subunits are also required to form other types of glycine receptors. GRD was shown to assemble functional GABA receptors in Xenopus oocytes only in the context of Lcch3, highlighting the underlying complexity. Grd is also involved in GABAergic transmission, as its downregulation in specific patterns attenuates the sleep-promoting effects of a GABA-A-R agonist. Thus, depending on the collection of subunits expressed in a particular neuron, GRD takes part of receptors that respond to different neurotransmitters (Frenkel, 2017).

    Several scenarios are envisioned accounting for the mismatch between depleting glycine in PDF neurons and the inability to respond to it in circadian targets. If other circadian neurons communicate time-related information through this fast neurotransmitter, downregulating glycine availability in LNvs would surely give rise to a different behavioral output than chronic downregulation of a subset of GlyR subunits in the entire circadian network. Additional non-circadian neurons could also be relevant in defining the properties of locomotor behavior, and the resulting unbalance (derived from inhibition of certain circadian clusters while the non-circadian remain active) would be the cause for the desynchronization; candidate neurons would be the pars intercerebralis. Alternatively, since the three subunits analyzed in this study do not assemble all functional GlyRs, a partial impairment of glycinergic transmission is ensured. In addition, neurons could express different spliced variants of each subunit hampering the efficiency of RNAi. In sum, modifying the stoichiometry among different receptor subunits in discrete neuronal subsets may alter the properties of native glycine receptors in an unpredictable manner (i.e., ligand affinity, ion conductance, and channel kinetics), influencing neuronal responsiveness to glycine and ultimately impinging on neuronal firing. Thus, behavioral complexity ultimately reflects the combination of functional changes in individual neuronal clusters and the orchestration resulting from network interactions (Frenkel, 2017).

    Drastically altering neuronal excitability triggered shortening or lengthening of the circadian period, depending on the cluster. Likewise, disrupting glycinergic transmission led to cluster-associated changes in the free-running period. Interestingly, downregulation of single receptor subunits in the DN1ps lengthened the period, further supporting their relevance and their ability to feed back onto the sLNvs. On the other hand, altering glycine transmission onto the LNds+5thsLNv gave rise to a shorter period, a phenotype also observed upon downregulation of all three subunits, in either the E-oscillator or the whole circadian network. Interestingly, a short period phenotype accompanied by deconsolidation of rhythmic activity is a hallmark of pdf01 mutants, in which the LNds are uncoupled from the sLNvs and run at a faster pace; these similarities open the provocative possibility that both PDF and glycine released from the sLNvs play a similar role onto the LNds. Altogether, these results support the notion that each cluster has a differential contribution to the dynamic operation of the circadian network (Frenkel, 2017).

    It has been suggested that electrical activity integrates phase information from endogenous oscillators within different regions of the SCN. In that model, the ventral SCN can shift the dorsal SCN and cause it to resynchronize to the new phase, and GABA is required for coupling those two regions. The fact that the sLNvs release an inhibitory neurotransmitter onto dorsal clusters is reminiscent of GABA’s role. In the SCN, Cl- reversal potential changes during the day in different circadian clusters, giving rise to either excitatory or inhibitory responses to GABA; if this were true in Drosophila, glycinergic responses could also change in a time-of-day fashion (Frenkel, 2017).

    It is not necessarily envisioned that glycine operates as a synchronizing cue to the molecular clocks, a role already shown to depend on PDF, but instead, it might coordinate the activity of independent clusters to provide coherence to the circadian network. Under this scenario, it is proposed the that sLNvs are acting as an orchestra conductor that relies on at least two batons: one fast inhibiting signal (glycine) and a slower excitatory one (PDF). Thus, the sLNv could operate as a time-of-day switch that rapidly turns off specific targets to keep the circadian network synchronized (Frenkel, 2017).

    SIK3-HDAC4 signaling regulates Drosophila circadian male sex drive rhythm via modulating the DN1 clock neurons

    The physiology and behavior of many organisms are subject to daily cycles. In Drosophila melanogaster the daily locomotion patterns of single flies are characterized by bursts of activity at dawn and dusk. Two distinct clusters of clock neurons-morning oscillators (M cells) and evening oscillators (E cells)-are largely responsible for these activity bursts. In contrast, male-female pairs of flies follow a distinct pattern, most notably characterized by an activity trough at dusk followed by a high level of male courtship during the night. This male sex drive rhythm (MSDR) is mediated by the M cells along with DN1 neurons, a cluster of clock neurons located in the dorsal posterior region of the brain. This study reports that males lacking Salt-inducible kinase 3 (SIK3) expression in M cells exhibit a short period of MSDR but a long period of single-fly locomotor rhythm (SLR). Moreover, lack of Sik3 in M cells decreases the amplitude of Period (Per) cycling in DN1 neurons, suggesting that SIK3 non-cell-autonomously regulates DN1 neurons' molecular clock. This study also shows that Sik3 reduction interferes with circadian nucleocytoplasmic shuttling of Histone deacetylase 4 (HDAC4), a SIK3 phosphorylation target, in clock neurons and that constitutive HDAC4 localization in the nucleus shortens the period of MSDR. Taking these findings together, it is concluded that SIK3-HDAC4 signaling in M cells regulates MSDR by regulating the molecular oscillation in DN1 neurons (Fujii, 2017).

    The physiology and behavior of most animals undergo daily oscillations, which are controlled by a small set of clock neurons in the brain. In mammals, a heterodimeric complex between CLOCK (CLK) and BMAL1 activates transcription of Period (Per1 and Per2) and Cryptochrome (Cry1 and Cry2) genes, and their protein products in turn inhibit the activity of CLK/BMAL1. Likewise, Drosophila heterodimeric complexes between CLK and CYCLE (CYC) activate the genes period (per) and timeless (tim), and their respective protein products repress CLK/CYC. These conserved negative-feedback loops, which include several kinases, produce rhythmic transcription profiles in numerous genes (Fujii, 2017).

    The Drosophila brain contains ∼150 clock neurons which are divided into seven clusters based on their anatomical locations and functional characteristics: the small and large ventral lateral neurons (sLNvs and lLNvs), the dorsal lateral neurons (LNds), the lateral posterior neurons (LPNs), and three dorsal neuron clusters (DN1-3). Some of these clock neurons have distinct functions in circadian locomotor behavior. Specifically, four sLNvs, also referred to as 'morning' (M) cells, express the neuropeptide pigment-dispersing factor (PDF) and control the timing of morning locomotor activity during light:dark (LD) cycles; these neurons are also the key pacemaker neurons in constant darkness (DD). The fifth, PDF−, sLNv and the LNds, referred to as 'evening' (E) cells, are required for the generation of the evening activity peak in LD cycles. Communication between various groups of cells within this interconnected neural network enhances the synchrony of molecular oscillation in each neuron (Fujii, 2017).

    Ventral lateral neuron (LNv)-derived PDF plays a critical role in regulating the molecular clock. Specifically, PDF participates in synchronization of clock neurons by up-regulating cAMP, which activates PKA, which in turn regulates the stability of PER and TIM in PDF receptor (PDFR)-expressing target neurons. Thus, ion status and is regu neurosecretory signaling: In well-fed flies, SIK3 is thought to be indirectly activated by insulin-likeavioral rhythms even when flies are kept in DD (Fujii, 2017).

    Locomotor activity is the best-characterized circadian behavior in Drosophila, but numerous other behaviors, such as courtship and mating, sleep, and feeding, are under strong circadian influence. Previously work has shown that male-female pairs of flies exhibit activity patterns strikingly distinct from those of singly kept males or females (i.e., single-fly locomotor rhythm or SLR) or same-sex pairs of flies. The activity pattern of male-female pairs, which is referred to as 'male sex-drive rhythm' (MSDR), is characterized by a trough at subjective dusk, followed by a sharp increase in male-driven courtship activity (especially 'following' behavior) that peaks during the subjective night. A functional molecular clock in both Pdf+ LNvs and DN1 neurons is necessary and sufficient for proper MSDR. However, few other cellular and molecular components contributing to MSDR have been identified to date. Specifically, information is lacking about both the molecular and cellular identity of downstream effectors of the main clock components that are important for MSDR (Fujii, 2017).

    This study reports the identification of two downstream effectors of the molecular clock that play distinct roles in MSDR and SLR. Using an RNAi screen for kinases, this study shows that Salt-inducible kinase 3 (SIK3) is a critical component for circadian behavior. Sik3 knockdown in subsets of clock neurons (DN1 neurons or Pdf+ LNvs) causes a short period of MSDR, whereas the period length of SLR is slightly shortened with Sik3 knockdown in DN1 neurons and is slightly elongated with Sik3 knockdown in sLNvs. This study also found that transcriptional activity of Histone deacetylase 4 (HDAC4) is regulated by SIK3 in a circadian manner. Finally, Sik3 reduction in Pdf+ LNvs reduces the amplitude of PER oscillation in DN1 neurons and shortens the length of the MSDR period, suggesting that SIK3-HDAC4 signaling plays an important role in the determination of MSDR period by modulating the intercellular communication between clock neurons (Fujii, 2017).

    SIK-HDAC (class IIa) signaling is evolutionarily conserved from worm to mammals, operating in a number of tissues, including the nervous system, liver, and muscle. In mice, SIK1-HDAC signaling is important for muscle integrity by regulating the activity of the transcription factor MEF2 (Stewart, 2013). In the fly, SIK3-HDAC4 signaling was shown to control the expression of lipolytic and gluconeogenic genes in the fat body (Choi, 2015). Furthermore, both Drosophila HDAC4 and MEF2 have been implicated in circadian rhythm, as has the related HDAC5 gene in mice. This paper has established a critical role for SIK3 in two circadian behaviors, single-fly locomotor activity and male sex drive, respectively (Fujii, 2017).

    MSDR is mediated through the activity of Pdf+ LNvs and DN1 neurons (Fujii, 2010). This study specifically targeted SIK3 in either group of circadian neurons using RNAi. Strikingly, SIK3 knockdown in LNvs shortened the period length of MSDR but slightly yet reproducibly lengthened that of SLR. The loss of PER rhythm amplitude observed specifically in the DN1 neurons and its apparent phase advance on the second or third day of constant conditions would fit with these observations. The advance would be symptomatic of DN1 neurons free-running with a short period and thus presumably explains the short-period MSDR. The loss of amplitude could indicate that a small subset of DN1 neurons runs at a different pace, perhaps explaining the slightly long period of the SLR. Indeed, both SLR and MSDR depend on sLNvs driving DN1 neurons. The broader M peak might also be an early sign that DN1 neurons are not as coherent, even under LD, because the DN1 neurons function downstream of the sLNvs to control morning anticipatory activity. The same short-period DN1 neurons might drive the M peak and MSDR. Unfortunately, the amplitude of the M peak in DD was too low to be able to determine whether it free-runs with a short period (Fujii, 2017).

    Knockdown of SIK3 in DN1 neurons shortened the period length of MSDR that is well correlated with shortened PER oscillations in DN1 neurons, and these flies show subtle but significantly shortened period length in SLR. Together, these findings suggest that SIK3 is a key component in molecular oscillator coupling between sLNvs and DN1 neurons and that its role is especially important for maintaining an appropriate MSDR period length. However, the possibility cannot be excluded that SIK3 also influences the period of the circadian pacemaker in a neuron-specific manner (i.e., in the DN1 neurons), as was proposed for SGG and CKII. It was also demonstrated that HDAC4 cycles in a SIK3-dependent fashion between the cytoplasm and the nucleus in the M cells (Fujii, 2017).

    Because M-cell restricted overexpression of phosphorylation-defective, constitutively nuclear-located HDAC43A, but not wild-type HDAC4, mimics the phenotype of flies lacking SIK3 in these cells, it is suggested that HDAC4 is a critical component for the transduction of the circadian intercellular signal from M cells to DN1 neurons. However, the function of SIK3 in oscillator coupling is unlikely to be mediated by HDAC4 in DN1 neurons, because most of these neurons do not express HDAC4. Another potential SIK3 phosphorylation target such as CREB or CRTC, which are implicated in cAMP-mediated signaling and the circadian clock, may play a role in the regulation of oscillator coupling in DN1 neurons for MSDR (Fujii, 2017).

    SIK3-dependent circadian shuttling of HDAC4 in sLNvs implies that the activity of SIK3 is under circadian control. How is SIK3 activity regulated in sLNvs? In fat cells (and rat adipocytes) SIK3 activity is dependent on nutrition status and is regulated indirectly through neurosecretory signaling: In well-fed flies, SIK3 is thought to be indirectly activated by insulin-like peptides (ILPs), whereas in starved flies, it is inhibited by adipokinetic hormone (AKH). SIK3 activity itself is regulated via phosphorylation by AKT1 (activated by ILPs) and cAMP-dependent protein kinase A (PKA) (activated by AKH). These kinases target distinct but overlapping sets of serine and threonine residues, and thus it appears that SIK3 activity is dependent on the particular phosphorylation pattern at these sites. Intriguingly, it has been reported that PDF stabilizes PER by increasing cAMP levels and PKA activity in Pdfr+ clock neurons (including M cells) at dawn, a time when HDAC4 is activated and translocated into the nucleus. PDF thus could be an indirect circadian regulator of SIK3 activity via PKA. However, the reduction of PDF in M cells did not shorten the MSDR period length, suggesting that PDF signaling probably does not regulate SIK3. Moreover, RNAi-mediated knockdown of MEF2, which regulates SIK3-HDAC in the mouse, had no effect on MSDR. Future experiments will be needed to investigate how SIK3 activity is regulated and how HDAC4 controls intercellular communications between M cells and DN1 neurons (Fujii, 2017).

    How does the lack of SIK3 in M cells (i.e., sLNvs) alter the robustness of PER cycling in some (DN1 neurons) but not other (sLNvs and LNds) PDFR-expressing clock cells? One possibility might be the manner by which sLNvs communicate with other clock cells. Functional and anatomical studies including GFP Reconstitution Across Synaptic Partners strongly suggest that at least some DN1 neurons are direct downstream targets of sLNvs, and hence accurately timed communication between these neurons likely occurs through synapses, which is proposed to rely on SIK3 function in LNvs. In contrast, autocrine (sLNvs) and paracrine (LNds) communication likely occurs via untargeted release of PDF, a process that is suggested not to be dependent on SIK3. The projections of sLNvs to DN1 neurons, in addition to PDF-containing dense core vesicles, harbor small clear vesicles that house classical neurotransmitters, raising the possibility that communication between sLNvs and DN1 neurons pertinent to the robust amplitude of PER oscillation in DN1 neurons is mediated by an as yet unidentified HDAC4-dependent signal. Moreover, DN1 neurons are probably heterogeneous in function, and thus it is quite likely that only some of these cells respond to the sLNv-derived and SIK3-HDAC4-dependent signal, whereas another either overlapping or entirely distinct group of DN1 neurons is responsive to the sLNv-derived PDF. In this context, it is worth noting that sLNvs also express the small neuropeptide F (sNPF). Moreover, a discrete requirement for both PDF-mediated and classical neurotransmitter signaling has been proposed for distinct aspects of SLR, and glycine in sLNvs was recently proposed to coordinate locomotor behavior and appears either to accelerate or to slow down circadian oscillators in specific neuronal groups. Future studies will be necessary to identify the LNv-derived signal that maintains the appropriate amplitude and speed of the clock in DN1 neurons to coordinate MSDR and SLR (Fujii, 2017).

    It is surprising that the loss of SIK3 in sLNvs results in a long SLR and a short MSDR, whereas the loss of SIK3 in the DN1 neurons shortens both SLR and MSDR, because in either case it appears that the DN1 neurons are disconnected from the sLNvs. One explanation could be that SIK3 is differentially modulated in different subpopulations of DN1 neurons by the sLNv synchronizing cue, thus resulting in DN1 desynchronization in flies lacking SIK3 in the sLNvs. However, when SIK3 is missing in DN1 neurons, they all adopt a short period by default (Fujii, 2017).

    In summary, this work unexpectedly reveals the existence of a SIK3-HDAC4 regulatory pathway that allows the M cells -- the critical circadian pacemaker neurons of the fly brain -- to control specific circadian neurons and behaviors. This pathway could prove particularly important in explaining how circadian behaviors can be differentially modulated in response to environmental conditions or internal states. Indeed, the ability to tune and prioritize specific behaviors in a daily manner to minimize energy expenditure and to maximize fitness and reproductive output is critical for animals. Given the strong neural and molecular homologies between the circadian system of fruit flies and mammals, it will be particularly interesting to determine whether the SIK-HDAC pathway is also active in VIP (vasoactive intestinal polypeptide-expressing) neurons of the mammalian suprachiasmatic nucleus and, if so, whether it also controls specific circadian behaviors (Fujii, 2017).

    Modulation of miR-210 alters phasing of circadian locomotor activity and impairs projections of PDF clock neurons in Drosophila melanogaster

    This study investigated the function of Drosophila miR-210 in circadian behaviour by misexpressing it within circadian clock cells. Manipulation of miR-210 expression levels in the PDF (pigment dispersing factor) positive neurons affected the phase of locomotor activity, under both light-dark conditions and constant darkness. PER cyclical expression was not affected in clock neurons, however, when miR-210 was up-regulated, a dramatic alteration in the morphology of PDF ventral lateral neuron (LNv) arborisations was observed. A transcriptomic analysis revealed that miR-210 overexpression affects the expression of several genes belonging to pathways related to circadian processes, neuronal development, GTPases signal transduction and photoreception. Collectively, these data reveal the role of miR-210 in modulating circadian outputs in flies and guiding/remodelling PDF positive LNv arborisations and indicate that miR-210 may have pleiotropic effects on the clock, light perception and neuronal development (Cusumano, 2018)

    Larks, owls, swifts, and woodcocks among fruit flies: differential responses of four heritable chronotypes to long and hot summer days

    This study examined the possibility to distinguish four extreme chronotypes among fruit flies and the possibility of the differential response of such chronotypes to light and heat stressors. Circadian rhythms of locomotor activity and sleep-wake pattern were tested in constant darkness, and four strains of fruit flies originating from three wild populations of Africa, Europe, and the USA were selected to represent four distinct chronotypes: "larks" (early morning and evening activity peaks), "owls" (late morning and evening peaks), "swifts" (early morning and late evening peaks), and "woodcocks" (late morning and early evening peaks). The circadian rhythms and sleep efficiency of the selected chronotypes were further tested under such extreme conditions as either long day (LD20:4 at 20 ° C) or a combination of LD20:4 with hot temperature (29 ° C). Despite the identity of such experimental conditions for four chronotypes, their circadian rhythms and sleep timing showed significantly distinct patterns of response to exposure to heat and/or long days. All two-way repeated measures analysis of variances yielded a significant interaction between chronotype and time of the day. It is concluded that an experimental study of heritable chronotypes in the fruit fly can facilitate a search for genetic underpinnings of individual variation in vulnerability to circadian misalignment, maladaptive sleep-wake behavior, and sleep disorders (Zakharenko, 2018).

    Allatostatin-C/AstC-R2 is a novel pathway to modulate the circadian activity pattern in Drosophila

    Seven neuropeptides are expressed within the Drosophila brain circadian network. Previous mRNA profiling suggested that Allatostatin-C (AstC) is an eighth neuropeptide and specifically expressed in dorsal clock neurons (DN1s). The results of this study show that AstC is, indeed, expressed in DN1s, where it oscillates. AstC is also expressed in two less well-characterized circadian neuronal clusters, the DN3s and lateral-posterior neurons (LPNs). Behavioral experiments indicate that clock-neuron-derived AstC is required to mediate evening locomotor activity under short (winter-like) and long (summer-like) photoperiods. The AstC-Receptor 2 (AstC-R2) is expressed in LNds, the clock neurons that drive evening locomotor activity, and AstC-R2 is required in these neurons to modulate the same short photoperiod evening phenotype. Ex vivo calcium imaging indicates that AstC directly inhibits a single LNd. The results suggest that a novel AstC/AstC-R2 signaling pathway, from dorsal circadian neurons to an LNd, regulates the evening phase in Drosophila (Diaz, 2019).

    Quantitative imaging of sleep behavior in Caenorhabditis elegans and larval Drosophila melanogaster

    Sleep is nearly universal among animals, yet remains poorly understood. Recent work has leveraged simple model organisms, such as Caenorhabditis elegans and Drosophila melanogaster larvae, to investigate the genetic and neural bases of sleep. However, manual methods of recording sleep behavior in these systems are labor intensive and low in throughput. To address these limitations, this study developed methods for quantitative imaging of individual animals cultivated in custom microfabricated multiwell substrates, and used them to elucidate molecular mechanisms underlying sleep. This paper describes the steps necessary to design, produce, and image these plates, as well as analyze the resulting behavioral data. Approaches are described for experimentally manipulating sleep. Following these procedures, after ~2 h of experimental preparation, it is possible to simultaneously image 24 C. elegans from the second larval stage to adult stages or 20 Drosophila larvae during the second instar life stage at a spatial resolution of 10 or 27 microm, respectively. Although this system has been optimized to measure activity and quiescence in Caenorhabditis larvae and adults and in Drosophila larvae, it can also be used to assess other behaviors over short or long periods. Moreover, with minor modifications, it can be adapted for the behavioral monitoring of a wide range of small animals (Churgin, 2019).

    Circadian clock properties and their relationships as a function of free-running period in Drosophila melanogaster

    The stability of circadian clock mechanisms under cyclic environments contributes to increased Darwinian fitness by accurately timing daily behavior and physiology. Earlier studies on biological clocks speculated that the timing of behavior and its accuracy are determined by the intrinsic period (tau) of the circadian clock under constant conditions, its stability, the period of the external cycle (T), and resetting of the clock by environmental time cues. However, most of these previous studies suffered from certain limitations, the major ones being a narrow range of examined tau values and a non-uniformity in the genetic background across the individuals tested. The data in this study rigorously test the following hypotheses by employing Drosophila melanogaster fruit flies with tau ranging from 17 to 30 h in a uniform genetic background. Tests were performed to see whether 1) precision (day-to-day stability of tau) is greater for clocks with tau close to 24 h; 2) accuracy (i.e., day-to-day stability of the phase relationship (psi), where psi is the duration between a phase of the rhythm and a phase of the external cycle) is greater for clocks with tau close to 24 h; 3) Psi is delayed with an increase in tau; and 4) Psi becomes more advanced with an increase in length of zeitgeber cycle (T). This study shows that precision is not always maximum for ~24-h clocks, but that accuracy is greatest when tau approximates T. Further, flies exhibit a delayed phase relationship with increasing tau and an advanced phase relationship under long T-cycles as compared with shorter T-cycles. Relationships between activity and rest durations are described and how these observations fit predictions from models of circadian entrainment. Overall, it is confirmed that accuracy and phase of entrained rhythm are governed by both intrinsic clock period and the length of the external cycle; however, it was found that the relationship between intrinsic period and precision does not fit previous predictions (Srivastava, 2019).

    Optimization of circadian responses with shorter and shorter millisecond flashes

    Recent work suggests that the circadian pacemaker responds optimally to millisecond flashes of light, not continuous light exposure as has been historically believed. It is unclear whether these responses are influenced by the physical characteristics of the pulsing. In the present study, Drosophila (n = 2199) were stimulated with 8, 16 or 120 ms flashes. For each duration, the energy content of the exposure was systematically varied by changing the pulse irradiance and the number of stimuli delivered over a fixed 15 min administration window (64 protocols surveyed in all). Results showed that per microjoule invested, 8 ms flashes were more effective at resetting the circadian activity rhythm than 16- and 120 ms flashes (i.e. left shift of the dose-response curve, as well as a higher estimated maximal response). These data suggest that the circadian pacemaker's photosensitivity declines within milliseconds of light contact. Further introduction of light beyond a floor of (at least) 8 ms leads to diminishing returns on phase-shifting (Kaladchibachi, 2019).

    Misregulation of Drosophila Myc disrupts circadian behavior and metabolism

    Drosophila Myc (dMyc) is highly conserved and functions as a transcription factor similar to mammalian Myc. Previous work has found that oncogenic Myc disrupts the molecular clock in cancer cells. This study demonstrates that misregulation of dMyc expression affects Drosophila circadian behavior. dMyc overexpression results in a high percentage of arrhythmic flies, concomitant with increases in the expression of clock genes cyc, tim, cry, and cwo. Conversely, flies with hypomorphic mutations in dMyc exhibit considerable arrhythmia, which can be rescued by loss of dMnt, a suppressor of dMyc activity. Metabolic profiling of fly heads revealed that loss of dMyc and its overexpression alter steady-state metabolite levels and have opposing effects on histidine, the histamine precursor, which is rescued in dMyc mutants by ablation of dMnt and could contribute to effects of dMyc on locomotor behavior. These results demonstrate a role of dMyc in modulating Drosophila circadian clock, behavior, and metabolism (Hsieh, 2019).

    Life at high latitudes does not require circadian behavioral rhythmicity under constant darkness

    Nearly all organisms evolved endogenous self-sustained timekeeping mechanisms to track and anticipate cyclic changes in the environment. Circadian clocks, with a periodicity of about 24 h, allow animals to adapt to day-night cycles. Biological clocks are highly adaptive, but strong behavioral rhythms might be a disadvantage for adaptation to weakly rhythmic environments such as polar areas. Several high-latitude species, including Drosophila species, were found to be highly arrhythmic under constant conditions. Furthermore, Drosophila species from subarctic regions can extend evening activity until dusk under long days. These traits depend on the clock network neurochemistry, and it has been previously proposed that high-latitude Drosophila species evolved specific clock adaptations to colonize polar regions. This study broadened this analysis to 3 species of the Chymomyza genus, which diverged circa 5 million years before the Drosophila radiation and colonized both low and high latitudes. C. costata, pararufithorax, and procnemis, independently of their latitude of origin, possess the clock neuronal network of low-latitude Drosophila species, and their locomotor activity does not track dusk under long photoperiods. Nevertheless, the high-latitude C. costata becomes arrhythmic under constant darkness (DD), whereas the two low-latitude species remain rhythmic. Different mechanisms are behind the arrhythmicity in DD of C. costata and the high-latitude Drosophila ezoana, suggesting that the ability to maintain behavioral rhythms has been lost more than once during drosophilid evolution and that it might indeed be an evolutionary adaptation for life at high latitudes (Bertolini, 2019).

    Circadian and Genetic Modulation of Visually-Guided Navigation in Drosophila Larvae

    Organisms possess an endogenous molecular clock which enables them to adapt to environmental rhythms and to synchronize their metabolism and behavior accordingly. Circadian rhythms govern daily oscillations in numerous physiological processes. Drosophila larvae have relatively simple nervous system compared to their adult counterparts, yet they both share a homologous molecular clock with mammals, governed by interlocking transcriptional feedback loops with highly conserved constituents. Larvae exhibit a robust light avoidance behavior, presumably enabling them to avoid predators and desiccation, and DNA-damage by exposure to ultraviolet light, hence are crucial for survival. Circadian rhythm has been shown to alter light-dark preference, however it remains unclear how distinct behavioral strategies are modulated by circadian time. To address this question, this study investigate the larval visual navigation at different time-points of the day employing a computer-based tracking system, which allows detailed evaluation of distinct navigation strategies. The results show that due to circadian modulation specific to light information processing, larvae avoid light most efficiently at dawn, and a functioning clock mechanism at both molecular and neuro-signaling level is necessary to conduct this modulation (Asirim, 2020).

    Cryptochrome-mediated phototransduction by modulation of the potassium ion channel β-subunit redox sensor

    Blue light activation of the photoreceptor Cryptochrome (Cry) evokes rapid depolarization and increased action potential firing in a subset of circadian and arousal neurons in Drosophila melanogaster. This study shows that acute arousal behavioral responses to blue light significantly differ in mutants lacking Cry, as well as mutants with disrupted opsin-based phototransduction. Light-activated Cry couples to membrane depolarization via a well conserved redox sensor of the voltage-gated potassium (K+) channel β-subunit (Kvβ) Hyperkinetic (Hk). The neuronal light response is almost completely absent in hk-/- mutants, but is functionally rescued by genetically targeted neuronal expression of WT Hk, but not by Hk point mutations that disable Hk redox sensor function. Multiple K+ channel α-subunits that coassemble with Hk, including Shaker, Ether-a-go-go, and Ether-a-go-go-related gene, are ion conducting channels for Cry/Hk-coupled light response. Light activation of Cry is transduced to membrane depolarization, increased firing rate, and acute behavioral responses by the Kvβ subunit redox sensor (Fogle, 2015).

    Acute behavioral arousal to blue light is significantly attenuated in CRY mutants. This study identified a redox signaling couple between blue light-activated CRY and rapid membrane depolarization via the redox sensor of Kvβ channel subunits coassembled with Kvα channel subunits. Additional unknown factors may act as intermediates between CRY and Hk. This finding provides in vivo validation of a very longstanding hypothesis that the highly conserved redox sensor of Kvβ subunit functionally senses cellular redox events to physiological changes in membrane electrical potential. Genetic loss of any single component functionally disrupts the CRY-mediated blue light response, which is functionally rescued by LNv restricted expression of their WT genes in the null backgrounds. Although little is known about the structural contacts between Kvβ and EAG subunits, Kvβ subunits make extensive physical contacts in a fourfold symmetric fashion in 1:1 stoichiometry with the T1 assembly domain of other coassembled tetrameric Kvα subunits that form the complete functional channel (Fogle, 2015).

    Application of Kvβ redox chemical substrate modulates voltage-evoked channel peak current, steady-state current, and inactivation in heterologously expressed α-β channels, which are reversed by fresh NADPH. These results indicate that measurements of channel biophysical properties can reflect the redox enzymatic cycle of Kvβ as these channel modulatory effects are absent in preparations that lack the expression of WT Kvβ subunits or express redox sensor mutant Kvβ subunits. Whether direct chemical redox reactions occur between CRY and Hk is unclear. For CRY, light or chemical reduction induces one-electron reduction of the FAD cofactor of CRY, whereas the reductive catalytic mechanism of AKRs (such as Hk) requires a hydride ion transferred from NADPH to a substrate carbonyl, then a solvent-donated proton reduces the substrate carbonyl to an alcohol. These differences in redox chemistry between CRY and Hk suggest that other intermediates, such as oxygen, are possibly required for redox coupling (Fogle, 2015).

    Spectroscopic analysis of animal and plant CRYs suggest that light activation causes reduction of the FAD oxidized base state. Light activation of Drosophila CRY also evokes conformational changes in the C terminus of CRY that clearly promotes CRY C-terminal access to proteolytic degradation and subsequent interactions with the Timeless clock protein, thus signaling degradation and circadian entrainment. However, all existing evidence suggests that light activated CRY-mediated circadian entrainment and membrane electrical phototransduction operate under different mechanisms, including their different activation thresholds and relative dependence on the C terminus of CRY. Further distinguishing the distinct mechanisms of the downstream effects of light-activated CRY, the light-induced conformational changes that couple CRY to ubiquitin ligase binding (thus causing circadian entrainment) occur in oxidized and reduced states of CRY and are unaffected in CRY tryptophan mutants that presumably are responsible for intraprotein electron transfer reactions following light-evoked reduction of the FAD cofactor. Another recent study shows that light- or chemical-evoked reduction of Drosophila CRY FAD is coupled to conformational changes of the CRY C terminus, along with reporting a surprising negative result that DPI has no effect on the reoxidation of the reduced anionic semiquinone of purified Drosophila CRY. DPI could hypothetically influence the electrophysiological light response by blocking the pentose phosphate pathway, which produces the Hk redox cofactor NADPH, but this does not explain the light dependence for DPI blocking the electrophysiological light response herein. The available evidence indicates that CRY-mediated light evoked membrane depolarization occurs independently of conformational changes in the CRY C-terminal domain but depends on redox changes in CRY, whereas CRY-mediated light evoked circadian entrainment depends on conformational changes in the CRY C-terminal domain and may or may not depend on CRY redox state (Fogle, 2015).

    Light-activated CRY evokes rapid membrane depolarization through the redox sensor of the Kvβ subunit Hk. A general role for circadian regulation of redox state coupled to membrane excitability has been described recently in mammalian suprachiasmatic neurons. Redox modulation of circadian neural excitability may be a well-conserved feature (Fogle, 2015).

    Control of sleep onset by Shal/Kv4 channels in Drosophila circadian neurons

    Sleep is highly conserved across animal species. Both wake- and sleep-promoting neurons are implicated in the regulation of wake-sleep transition at dusk in Drosophila However, little is known about how they cooperate and whether they act via different mechanisms. This study demonstrated that in female Drosophila, sleep onset was specifically delayed by blocking the Shaker cognate L channels (Shal, also known as voltage-gated K(+) channel 4, Kv4) in wake-promoting cells, including large ventral lateral neurons (l-LNvs) and pars intercerebralis (PI), but not in sleep-promoting dorsal neurons (DN1s). Delayed sleep onset was also observed in males by blocking Kv4 activity in wake-promoting neurons. Electrophysiological recordings show that Kv4 channels contribute A-type currents (IA) in LNvs and PI cells, but are much less conspicuous in DN1s. Interestingly, blocking Kv4 in wake-promoting neurons preferentially increased firing rates at dusk around ZT13, when the resting membrane potentials (RMPs) and firing rates were at lower levels. Furthermore, pigment-dispersing factor (PDF) is essential for the regulation of sleep onset by Kv4 in l-LNvs, and downregulation of PDF receptor (PDFR) in PI neurons advanced sleep onset, indicating Kv4 controls sleep onset via regulating PDF/PDFR signaling in wake-promoting neurons. It is proposed that Kv4 acts as a sleep onset controller by suppressing membrane excitability in a clock-dependent manner to balance the wake-sleep transition at dusk. These results have important implications for the understanding and treatment of sleep disorders such as insomnia (Feng, 2018).

    This is the first demonstration of the function of Kv4 in sleep regulation. This study has shown that Kv4 is important for night-time sleep in Drosophila, and is especially crucial to normal sleep onset. Pan-neuronal expression of DNKv4 leads to decreased night-time sleep, indicating a general sleep-promoting function of Kv4. The increased night-time sleep in Shal495-null mutant reveals a compensatory overexpression of Shaker. A transcription factor named Kruppel (Kr) was identified to be a central regulator of this process, consistent with the conclusion that the compensatory modulation occurs at the transcriptional level. However, the Kr expression is suggested to be a result from detecting Kv4 or Shaker/Kv1 conductance, since 4-Aminopyridine (4-AP) also increased Shaker RNA expression. In this study, no significant increase was seen in Shaker RNA expression in DNKv4 mutants suggesting that other transcription factor(s), rather than Kr, may be involved in regulating Shal/Kv4 and Shaker balance (Feng, 2018).

    Previous studies have shown that cyclin A and GABAA receptors (or RDL) in circadian neurons also regulate sleep latency. A molecule named WAKE interacts with RDL, and the cycling manner of WAKE promotes excitability oscillation of l-LNvs. Moreover, DN1 neurons may regulate wake-sleep transition at dusk in a clock-dependent manner. The results verified that Kv4-mediated IA in l-LNvs, and function-loss of Kv4 preferentially upregulated membrane excitability at dusk, although Kv4 expression was not rhythmic. Moreover, resting membrane potential (RMP) values exhibit circadian oscillation in l-LNvs, with depolarized RMPs at dawn. Thus, less Kv4 channels would be available for opening when RMPs are depolarized. This supports data showing that blocking Kv4 did not significantly increased firing rate when circadian neurons were hyperexcitable at dawn. Although RMPs and firing rate also exhibit circadian oscillation in DN1s, blocking Kv4 activity has no effects on sleep latency. It was further demonstrated that this may be caused by the near absence of Kv4-mediated IA in DN1s. Thus, Kv4 controls wake-sleep transition at dusk in subsets wake-promoting cells, but not in DN1s. The phases of Ca2+ waves recorded with a fine temporal resolution were approximately coincident with membrane excitability and RMPs oscillations in l-LNvs, but with slight (∼1-2 h) delay in peaks. In this study, DNKv4 preferentially reduced excitability at dusk without causing daily excitability oscillation shifts, and presumably would reduce Ca2+ peaks as well. But how Kv4 exactly regulates intracellular Ca2+ waves and coordination of electrical excitability and Ca2+ waves need further study (Feng, 2018).

    PI exerts its effects as downstream of clock neurons and is one of the key targets (direct or indirect) of PDF neurons, although PI cells do not express molecular clock machinery. Previous studies have suggested that EGFR/ERK signal in insulin-producing PI neurons plays important roles in regulating the consolidation and maintenance of sleep in Drosophila, indicating that PI cells act as wake-promoting neurons. These results are the first to demonstrate that the neuronal activities and PDF/PDFR signaling in subsets of PI cells (50y-GAL4 driver) participate in the regulation of sleep onset (Feng, 2018).

    In this study, defects in sleep initiation were presumed to be due to the hyperactivity of specific circadian neurons caused by blocking Kv4 activity. dTrpA1 experiments further supported this notion. However, a recent study concluded that reducing the Shaker/Kv1 current would decrease, rather than increase, the action potential discharge in dFB (dorsal FB; Pimentel, 2016). Depleting Shaker from dFB neurons could shift the interspike interval distribution toward longer values, making it more difficult to generate next spike. It is supposed that the opposite effect may be due to diverse types of Kv channels and different discharge and biophysical properties of neurons. For example, Kv4 is required for maintaining excitability in cultured neurons (Ping, 2011), but not in groups of neurons from dissected brain in this study. Whether driving DNKv4 expression in other sleep-promoting or wake-promoting neurons would cause parallel or opposite effects on sleep needs to be investigated. Neurons in EB, FB, or a subset MBNs driven by 201y-GAL4 could be tested in further studies (Feng, 2018).

    Previous work has provided a strong link between circadian periods with habitual sleep timing in human, and CRY1 is suggested to be associated with a familial form of delayed sleep phase disorder. However, no significant change was detected in circadian periods by blocking Kv4 in LNvs and DN1s, indicating that delayed sleep onset in the mutants was not due to changes in circadian periods. Blocking Kv4 in PI cells disrupted rhythmic activities in most flies, and a lengthened circadian period was detected in the rhythmic ones. Because PI is a circadian output region and does not express clock machinery, it is not likely that intrinsic circadian rhythm disorder contributes to the sleep onset phenotype caused by blocking Kv4 (Feng, 2018).

    Studies have provided evidence that links types of potassium channels to sleep phenotypes in Drosophila and human. For example, insomnia has been delineated in patients with neurologic disorders of Voltage-gated K+ channels (Kv) autoimmunity (or named Kv antibody syndrome). Sleep onset insomnia, defined as inability to fall asleep at the desired time, is observed in patients with neurodegenerative diseases and psychiatric disorders. Abnormal sleep timing and pattern have also been observed in Drosophila disease models. This study may provide a potential alternative therapy of sleep onset insomnia by targeting Kv4 channels (Feng, 2018).

    Shaw and Shal voltage-gated potassium channels mediate circadian changes in Drosophila clock neuron excitability

    Like in mammals, Drosophila circadian clock neurons display rhythms of activity with higher action potential firing rates and more positive resting membrane potentials during the day. This rhythmic excitability has been widely observed but, critically, its regulation remains unresolved. This study characterized and modeled the changes underlying these electrical activity rhythms in the lateral ventral clock neurons (LNvs). Currents mediated by the voltage-gated potassium channels Shaw (Kv3) and Shal (Kv4) oscillate in a circadian manner. Disruption of these channels, by expression of dominant negative (DN) subunits, leads to changes in circadian locomotor activity and shortens lifespan. LNv whole-cell recordings then show that changes in Shaw and Shal currents drive changes in action potential firing rate and that these rhythms are abolished when the circadian molecular clock is stopped. A whole-cell biophysical model using Hodgkin-Huxley equations can recapitulate these changes in electrical activity. Based on this model and by using dynamic clamp to manipulate clock neurons directly, the pharmacological block of Shaw and Shal can be rescued, restoring the firing rhythm, and thus demonstrating the critical importance of Shaw and Shal. Together, these findings point to a key role for Shaw and Shal in controlling circadian firing of clock neurons and show that changes in clock neuron currents can account for this. Moreover, with dynamic clamp the LNvs between morning-like and evening-like states of electrical activity can be switched. It is concluded that changes in Shaw and Shal underlie the daily oscillation in LNv firing rate (Smith, 2019).

    Calcitonin gene-related peptide neurons mediate sleep-specific circadian output in Drosophila

    Imbalances in amount and timing of sleep are harmful to physical and mental health. Therefore, the study of the underlying mechanisms is of great biological importance. Proper timing and amount of sleep are regulated by both the circadian clock and homeostatic sleep drive. However, very little is known about the cellular and molecular mechanisms by which the circadian clock regulates sleep. This study describes a novel role for Diuretic hormone 31 (DH31), the fly homolog of the vertebrate neuropeptide calcitonin gene-related peptide, as a circadian wake-promoting signal that awakens the fly in anticipation of dawn. Analysis of loss-of-function and gain-of-function Drosophila mutants demonstrates that DH31 suppresses sleep late at night. DH31 is expressed by a subset of dorsal circadian clock neurons that also express the receptor for the circadian neuropeptide pigment-dispersing factor (PDF). PDF secreted by the ventral pacemaker subset of circadian clock neurons acts on PDF receptors in the DH31-expressing dorsal clock neurons to increase DH31 secretion before dawn. Activation of PDF receptors in DH31-positive DN1 specifically affects sleep and has no effect on circadian rhythms, thus constituting a dedicated locus for circadian regulation of sleep. This study has identified a novel signaling molecule (DH31) as part of a neuropeptide relay mechanism for circadian control of sleep. The results indicate that outputs of the clock controlling sleep and locomotor rhythms are mediated via distinct neuronal pathways (Kunst, 2014).

    Vertebrate Calcitonin gene-related peptide (CGRP) has been implicated in controlling anxietyand stress response. While not previously addressed experimentally, the intimate relationship between stress, anxiety, and sleep suggests that CGRP might regulate sleep. Potentially relevant to this possibility is the recent observation that acute activation of CGRP signaling in zebrafish larvae increases spontaneous locomotor activity and decreases quiescence. This analysis of gain-of-function and loss-of-function mutant flies establishes that DH31, the Drosophila homolog of CGRP, is a negative regulator of sleep maintenance that awakens the animal in anticipation of dawn. This finding motivates investigation of a potential role for CGRP in the regulation of vertebrate sleep (Kunst, 2014).

    Using powerful tools for cell-specific neuronal manipulation, this study identified a highly restricted subset of DN1 circadian clock neurons that secrete DH31 late at night to awaken the fly in anticipation of dawn. Neuropeptides secreted from the circadian pacemaker in the mammalian SCN, such as prokinecticin 2 and cardiotrophin-like cytokine, are important clock outputs, but their cellular targets and molecular mechanisms remain unknown. In flies, PDF-expressing sLNv pacemaker neurons are known to be upstream of DN1s, sLNvs are most active around dawn, and PDF secretion from LNvs suppresses sleep at night. However, how PDF signals propagate out of the circadian clock network to regulate sleep remains unknown (Kunst, 2014).

    This study shows that PDF secreted by the sLNv pacemaker neurons activates PDFR in the DH31-expressing DN1s to increase neuronal activity and DH31 secretion late at night, thereby awakening the fly in anticipation of dawn. This is consistent with an earlier report demonstrating that flies lacking PDF or PDFR sleep more late at night. However, unlike DH31, PDF also promotes wake during the day. This suggests that PDF regulation of daytime sleep is mediated by neurons other than the DH31-expressing DN1s. PDFR is expressed by circadian clock neurons in addition to DN1s, and PDF signaling to these other clock neurons could be responsible for the wake-promoting effect of PDF during the day (Kunst, 2014).

    While PDF plays a key role in circadian timekeeping, neither constitutive activation of PDFR in the DH31-expressing DN1s nor manipulation of their secretion of DH31 affects free-running circadian rhythms. This establishes the PDF-to-DH31 neuropeptide relay as a novel sleep-specific output of the circadian pacemaker network, independent from and parallel to the outputs that drive circadian rhythms themselves. Since the basic cellular and molecular organization of vertebrate and insect circadian networks is conserved, this motivates the search for similar mechanisms in the SCN. Future studies are required to identify the neuronal targets of sleep-regulating DH31 signals and the cellular and molecular mechanisms by which DH31-R1 activation in these targets induces wake (Kunst, 2014).

    The acyl-CoA synthetase, pudgy, promotes sleep and is required for the homeostatic response to sleep deprivation

    The regulation of sleep and the response to sleep deprivation rely on multiple biochemical pathways. A critical connection is the link between sleep and metabolism. Metabolic changes can disrupt sleep, and conversely decreased sleep can alter the metabolic environment. There is building evidence that lipid metabolism, in particular, is a critical part of mounting the homeostatic response to sleep deprivation. This study evaluated an acyl-CoA synthetase, pudgy (pdgy), for its role in sleep and response to sleep deprivation. When pdgy transcript levels are decreased through transposable element disruption of the gene, mutant flies showed lower total sleep times and increased sleep fragmentation at night compared to genetic controls. Consistent with disrupted sleep, mutant flies had a decreased lifespan compared to controls. pdgy disrupted fatty acid handling as pdgy mutants showed increased sensitivity to starvation and exhibited lower fat stores. Moreover, the response to sleep deprivation is reduced when compared to a control flies. When the transcript levels for pdgy were decreased using RNAi, the response to sleep deprivation was decreased compared to background controls. In addition, when pdgy transcription is rescued throughout the fly, the response to sleep deprivation is restored. These data demonstrate that the regulation and function of acyl-CoA synthetase plays a critical role in regulating sleep and the response to sleep deprivation. Endocrine and metabolic signals that alter transcript levels of pdgy impact sleep regulation or interfere with the homeostatic response to sleep deprivation (Thimgan, 2018).

    Acute control of the sleep switch in Drosophila reveals a role for gap junctions in regulating behavioral responsiveness

    Sleep is a dynamic process in most animals, involving distinct stages that probably perform multiple functions for the brain. Before sleep functions can be initiated, it is likely that behavioral responsiveness to the outside world needs to be reduced, even while the animal is still awake. Recent work in Drosophila has uncovered a sleep switch in the dorsal fan-shaped body (dFB) of the fly's central brain, but it is not known whether these sleep-promoting neurons also govern the acute need to ignore salient stimuli in the environment during sleep transitions. This study found that optogenetic activation of the sleep switch suppressed behavioral responsiveness to mechanical stimuli, even in awake flies, indicating a broader role for these neurons in regulating arousal. The dFB-mediated suppression mechanism and its associated neural correlates requires innexin6 expression, suggesting that the acute need to reduce sensory perception when flies fall asleep is mediated in part by electrical synapses (Troup, 2018).

    Before sleep can begin to achieve any of its multiple putative functions, it would seem that behavioral responsiveness to the outside world first needs to be reduced. This is a fundamental yet poorly understood aspect of sleep in all animals. Mechanisms that regulate behavioral responsiveness are therefore likely to be involved during transitions from wakefulness to sleep, so it seems parsimonious that overlapping neuronal systems might govern both behavioral states. In this way, sleep onset has some similarity with selective attention in its capacity to gate or suppress perception rapidly. This study shows that gap junctions that are expressed in the sleep-promoting neurons of the dFB in the central fly brain are probably mediators for gating behavioral responsiveness in both awake and sleeping flies, as a consequence of acutely increased membrane potential in these neurons. Recent work has shown that sleep pressure increases the input resistance and membrane time constants in these same neurons (Donlea, 2014; Pimentel, 2016), so this seems to be a probable mechanism whereby increased sleep pressure impairs behavioral responsiveness via electrical channels, perhaps even before promoting and maintaining sleep - which may then occur via chemical (e.g. peptidergic) signaling from these neurons (Donlea, 2018). It is therefore possible that the R23E10 neurons, which have been recently characterized as comprising part of a 'sleep homeostat' in the fly brain, have two distinct arousal-related functions: to regulate behavioral responsiveness more generally -- and thus to promote sleep acutely -- and then to maintain sleep for the duration required by homeostatic demands. As increased arousal thresholds are a key criterion for sleep, it is of course difficult to disentangle these processes from each other. However, the data suggest that dFB-mediated suppression of behavioral responsiveness could be a prequel to sleep. First, on the basis of the current definitions of sleep, it does not make sense for sleep to precede changes in responsiveness, so either these processes are simultaneous or sleep functions succeed an initial increase in arousal thresholds that may already be evident during wakefulness (Troup, 2018).

    The experiments described in this study show that behavioral responsiveness can be suppressed by acutely activating the dFB even in awake flies, and that this effect has little inertia, at the levels of both behavior and electrophysiology. Sleep intensity was increased however upon prolonged dFB activation, suggesting a distinct cumulative effect. Indeed, at the deepest stage of sleep (when flies are least responsive to mechanical stimuli), removal of INX6 from the dFB had no effect on behavioral responsiveness, suggesting that other factors are involved at that stage for maintaining high arousal thresholds. When flies are closer to awakening again, or when they are engaged in lighter sleep stages, it is possible that INX6-mediated mechanisms come back into play to maintain sleep. In recent work, it has been shown that transitions in and out of sleep are associated with dFB activity, which is consistent with the view that a common system might be governing behavioral responsiveness during wakefulness as well as sleep (Troup, 2018).

    It is likely that other dFB neurons outside of the R23E10 circuit are also involved, as suggested by only partial overlap with INX6 labeling. However, without INX6, activated R23E10 neurons were unable to reduce behavioral responsiveness, suggesting a cell-autonomous effect. This study has shown that dFB activation is associated with increased local field potential (LFP) activity in the central brain, which could reflect increased synchronous neural firing. Without expression of INX6 in the R23E10 neurons, these induced LFP effects also disappear, suggesting that gap junctions are important for promoting synchronous activity in this circuit. This suggests a rapid mechanism through which the dFB might suppress behavioral responsiveness upon sleep onset, by for example producing synchronous low frequency activity that could interfere with ongoing sensory processing and integration in the central brain. Although acute activation of the R23E10 neurons alone is unlikely to occur in a natural context, electrical communication to other cells via gap junctions could support subtler levels of behavioral control while the flies are awake, which might consolidate into overall increased arousal thresholds as membrane potentials across the dFB increase with increasing sleep pressure. The possibility was not excluded that a fast-acting neuropeptide or neurotransmitter secreted from R23E10 neurons regulates behavioral responsiveness, but the data favor the view that a parallel channel involving electrical synapses exists. Future studies should uncover the extent of this gap-junction-coupled network, although antibody-labeling and dye-coupling experiments suggest that cells in the pars intercerebralis (PI) are probably involved. It also remains possible that dFB neurons are electrically coupled to each other, thereby generating LFP oscillations that are employed in the regulation of behavior. However, in dye-coupling experiments, no evidence was seen of labeling of another dFB cell, whereas multiple PI cells were labeled (Troup, 2018).

    It is not known whether electrical communication might be employed to promote sleep in other animal brains, although there is increasing evidence that gap junctions play an important role in the regulation of behavioral state and arousal in the mammalian brain. More generally, a role for gap junctions in sleep-promoting neurons also suggests a novel plasticity mechanism for regulating behavioral states. Whereas plasticity is typically viewed as a property of chemical synapses, the parallel electrical communication channel afforded by gap junctions could provide an alternate way of regulating sleep pressure, and thereby promoting sleep functions while ensuring that arousal thresholds are tightly linked to sleep need. Recent work has shown that transient electrical activation of the dFB neurons during fly brain development permanently impairs behavioral responsiveness in adult animals, further confirming the strong link between these neurons and the regulation of arousal in Drosophila. Different INX6 expression levels in the dFB may provide a mechanism for optimally linking behavioral responsiveness with sleep need. It will be interesting to see whether INX6 expression in the dFB correlates with the striking range of individual differences that were observed for sleep duration and behavioral responsiveness in wildtype flies, and whether INX6 expression in the dFB might be co-regulated alongside other proteins that have been shown to control neuronal excitability in this arousal circuit (Troup, 2018).

    Pleiotropic effects of loss of the Dα1 subunit in Drosophila melanogaster: Implications for insecticide resistance

    Nicotinic acetylcholine receptors (nAChRs) are a highly conserved gene family that form pentameric receptors involved in fast excitatory synaptic neurotransmission. The specific roles individual nAChR subunits perform in Drosophila melanogaster and other insects are relatively uncharacterized. Of the 10 D. melanogaster nAChR subunits, only three have described roles in behavioral pathways; Dα3 and Dα4 in sleep, and Dα7 in the escape response. Other subunits have been associated with resistance to several classes of insecticides. In particular, previous work has demonstrated that an allele of the Dα1 subunit is associated with resistance to neonicotinoid insecticides. This study used ends-out gene targeting to create a knockout of the Dα1 gene to facilitate phenotypic analysis in a controlled genetic background. This is the first report of a native function for any nAChR subunits known to be targeted by insecticides. Loss of Dα1 function was associated with changes in courtship, sleep, longevity, and insecticide resistance. While acetylcholine signaling had previously been linked with mating behavior and reproduction in D. melanogaster, no specific nAChR subunit had been directly implicated. The role of Dα1 in a number of behavioral phenotypes highlights the importance of understanding the biological roles of nAChRs and points to the fitness cost that may be associated with neonicotinoid resistance (Somers, 2017).

    Nicotinic acetylcholine receptors (nAChRs) belong to the Cys-loop receptor subfamily of ligand-gated ion channels (LGICs) that mediate the transduction of a chemical signal into an electrical signal. Activation in response to acetylcholine (ACh), the major excitatory neurotransmitter of the Drosophila melanogaster central nervous system, is on a micro- to submicrosecond timescale. nAChRs are expressed in a wide range of tissues, including but not limited to the nervous system. When expressed presynaptically, nAChRs can enhance neurotransmitter release, while postsynaptic expression mediates excitation. Like all LGICs, nAChRs are pentameric and can consist of several different subunits forming multiple receptor subtypes that vary in both their sensitivity to particular ligands and their permeability to particular cations. Individual subunits consist of a pore-forming transmembrane domain coupled to a large extracellular N-terminal domain, which forms the endogenous ligand-binding site with adjacent subunits. Upon ACh binding, a conformational change occurs opening the channel pore to permit the flow of cations (Somers, 2017).

    D. melanogaster has 10 nAChR subunits; however, only three of these have been associated with behavioral phenotypes, with specific roles described for Dα7 in the escape response and for both Dα3 and Dα4 in sleep behavior. Mutations in three D. melanogaster nAChR subunits confer resistance to two important classes of insecticides; Dα1 and Dβ2 to neonicotinoids, and Dα6 to spinosyns. Modification of an insecticide target protein can decrease the efficacy of an insecticide through altered affinity or pharmacological response. Modifications of this nature can provide a significant fitness boost to individuals in a population during insecticide treatment periods and can rapidly increase in frequency leading to control failures. However, allele frequencies will also be shaped by fitness costs if resistant mutations negatively impact reproductive output, either by reducing viability or the capacity to mate. This could be particularly true for alleles that directly modify the neonicotinoid-binding site as neonicotinoids occupy the same binding site as ACh. Loss-of-function mutants for the Dα1, Dα6, and Dβ2 genes are insecticide-resistant and viable. The viability phenotype suggests a level of functional redundancy among nAChR subunits. Questions remain as to whether individual subunits have other, nonredundant functions in controlling behavior or if the subtle pharmacological and physiological differences from compensating subunits manifests in altered behaviors. Impacts on mating behavior are of particular interest with respect to potential fitness costs (Somers, 2017).

    While invertebrate nAChR pharmacology and biochemistry with respect to insecticide binding has been examined in detail, little research has been devoted to the endogenous functions of these receptors. D. melanogaster is commonly used as a model insect system to study many facets of biology, including insecticide resistance. A large number of well-defined behavioral paradigms exist to investigate the roles of genes in traits, including sleep and cognition through to mating and auditory faculties (Somers, 2017).

    This study reports the creation of a Dα1 knockout mutant and a rescue system through transgene expression. This provided a consistent genetic background to analyze several behavioral paradigms. These reagents were used to identify roles for the D. melanogaster Dα1 subunit in mating, locomotion, and sleep, demonstrating the diverse pleiotropic influences that nAChRs can have on insect behavior. This research has relevance to the consideration of fitness costs that might be associated with resistance conferring mutations in Dα1 orthologs in pest insects, and the behavioral impact that exposure to neonicotinoids may have in beneficial species such as the western honey bee, Apis mellifera (Somers, 2017).

    To generate a Dα1 null mutant, a modified ends-out targeting scheme was used. A precise 57-kb deletion of the Dα1 genomic region was created, then validated by Southern blot and sequencing. This mutant is referred to as Dα1KO. Given that previously created Dα1 alleles are resistant to neonicotinoids (Perry, 2008), Dα1KO was screened on two different neonicotinoids (imidacloprid and nitenpyram) and was found to be highly resistant to both. Calculated LC50 values and resistance ratios were consistent with those measured for Dα1 mutants studied previously (Perry, 2008). Dα1 and orthologs in pest species are well-established targets for several different neonicotinoid insecticides. Heterologous expression and affinity chromatography studies have also implicated Dα1 in directly binding imidacloprid at a site that overlaps that normally occupied by the ligand, ACh. Therefore, these resistance data matched the prediction that a mutant with a genomic deletion of Dα1 would be resistant to neonicotinoid insecticides (Somers, 2017).

    The GAL4-UAS system was employed to create a phenotypic rescue in which a Dα1 cDNA clone was expressed in the Dα1KO background. Expression of the Dα1 clone using the pan-neuronal elav::GAL4 driver rescued sensitivity to both imidacloprid and nitenpyram. The reversion of the resistance phenotype indicates that the subunit expressed from the Dα1 transgene is assembling into functional nAChRs that bind these insecticides (Somers, 2017).

    The loss of Dα1 results in significant levels of resistance to neonicotinoids. However, the level of resistance is not of the same magnitude observed in the Dα6 knockout mutant, which is over 1000-fold resistant to spinosad. The Dα1KO mutant is still susceptible when exposed to a high enough dose, which may be due to expression of other neonicotinoid-sensitive nAChR subtypes. Mutations in the orthologs of Dα3 and Dβ1 have been identified in neonicotinoid-resistant strains of Nilaparvata lugens and Myzus persicae, respectively. While only one imidacloprid-binding site has been reported in adult D. melanogaster and other Dipteran and Lepidopteran species, multiple binding sites have been reported in several Hemipteran species. Unlike Hemipterans, Dipterans and Lepidopterans are holometabolous insects that undergo complete metamorphosis from larva to adult. It is possible that as yet undescribed imidacloprid-sensitive nAChR subtypes are expressed in the larval life stages. Another possibility is that a novel subtype is formed as a consequence of the loss of the Dα1 subunit that alters the mutant's sensitivity to neonicotinoids (Somers, 2017).

    RNAi knockdown of the Dα1 subunit resulted in defects in courtship and copulation behavior. Previous microarray studies, confirmed by RT-PCR, highlighted expression of Dα1 in both neuronal and reproductive tissues. Taken together, these lines of evidence suggested the potential for Dα1 to function in mating behavior (Somers, 2017).

    Mating behavior can be influenced by genetic background, for example expression of w is important for visual cues and misexpression of this gene can trigger male-male courtship. As the Dα1KO line was generated in the w1118 background, both the mutant and the control line have functionally null copies of w. Therefore, the w+ X chromosome from another isogenic line, RAL059, was used to replace the w1118 X chromosome present in both the Dα1KO mutant line and the w1118 background line. These lines will be referred to as the mutant and wild-type lines respectively (Somers, 2017).

    Courtship behavior was measured in terms of the latency of courtship initiation by males after female introduction into the mating chamber. Flies that failed to initiate courtship within the allowed 10-min period were given a maximum value. Wild-type males initiated courtship in every trial, regardless of the genotype of the female partner. Mutant males only initiated courtship 65% of the time with wild-type females and 79% of the time with mutant females. Mutant males also took significantly longer to initiate courtship than wild-type males when paired with either wild-type or mutant females (Somers, 2017).

    Copulation latency was also measured for the same flies. Mutant males rarely copulated within the 10-min period, only 15% of trials when paired with a wild-type female and only 3% when paired with a mutant female. Wild-type males were more successful, initiating copulation in 90% of the trials when paired with a wild-type female and in 56% of the trials when paired with a mutant female. No significant difference was observed in copulation latency of mutant males when paired with either wild-type or mutant females. In contrast, wild-type males did initiate copulation significantly faster with wild-type females compared to mutant females. This suggests that, unlike the latency of courtship initiation that is primarily influenced by the genotype of the male, both sexes contribute to the latency of copulation initiation (Somers, 2017).

    The rescue system was again employed to see if expression of a Dα1 transgene could rescue the courtship and copulation phenotypes observed for the Dα1KO mutant. Appropriate driver-only and UAS-only control flies were used for comparison to rescue flies. No discernible differences in courtship initiation were observed when wild-type males were crossed to control or rescue female flies. However, when crossed to wild-type female flies, male rescue flies were more successful in initiating courtship than male control flies. Male rescue flies also showed a significant decrease in courtship initiation when crossed to female wild-type compared to male control flies. Significant rescue of copulation initiation was also observed in both female and male rescue flies. Rescue flies were both more successful and faster at initiating copulation than the appropriate controls (Somers, 2017).

    It is clear from the data that Dα1 plays a role in Drosophila mating behavior; however, the underlying mechanism remains unknown. Defects in ACh signaling have previously been associated with abnormal mating behavior. Analysis of mosaic mutants, defective for cholinergic signaling, identified a neuropile in the mushroom body calyx critical for normal male courtship behavior. Male-specific, cholinergic neurons have also been identified in the abdominal ganglion, the disruption of which significantly decreased male fertility, potentially due to their innervation of the male reproductive system. The disruption of these neurons has been hypothesized to result in the uncoordinated or altered release of sperm, seminal fluid, and accessory proteins. The study, performed by Acebes (2004) used the presynaptic choline acetyltransferase marker to identify these neurons; however, the receptors receiving this signal were not identified. The data from mating experiments and expression analysis suggests that Dα1 may be one of the specific nAChR subunits expressed in this pathway. Another possibility to consider is a higher processing role for Dα1 in mating behavior. The phenotypes observed in the Dα1KO mutant are consistent with a defect in one or more sensory modalities. While there are specialized receptors responsible for detecting sensory stimuli, cholinergic signaling has been identified in connecting sensory circuitry to processing centers in the brain. Dα1 expression has previously been observed in the mushroom body calyx and lateral protocerebrum, which includes the lateral horn. Recently, the role of the lateral horn in locusts has been proposed to serve as a site for multimodal sensory integration. This may suggest that Dα1 plays a role in integrating multimodal courtship circuitry to higher sensory processing centers. Challenging Dα1KO mutants with sensory-specific behavioral paradigms and neuron-specific rescue could test this hypothesis (Somers, 2017).

    The possibility was explored that impaired locomotion may be contributing to the mating phenotype observed in the Dα1KO mutant. Over a 24-hr period, the Dα1KO mutant exhibited hyperactivity; however, it moved at a slower average speed. When average speed was binned in 3-hr intervals, the difference was significant during the middle two 3-hr bins of the day and the last three 3-hr bins of the night. Most importantly, there was no significant difference in average speed during the first 3-hr bin of the day, when courtship assays were performed, indicating that general locomotion deficits did not impact the measurement of mating behavior (Somers, 2017).

    The Dα1KO mutant also has an unusual pattern of sleep. Although there were no significant differences observed in total amount of sleep, mutant flies slept significantly less during the night, experiencing less sleep episodes of significantly shorter duration than wild-type controls. The total time of day sleep experienced by the mutant was significantly longer with a higher number of sleep episodes; however, there was no observable difference in episode length. Expression of the Dα1 transgene in the mutant background increased amount of sleep, both during the day and night. This increase was a result of longer sleep episodes, which may explain why both mutant and rescue flies both show less night sleep episodes. Rescue flies have less night sleep episodes due to the length of these episodes, whereas mutant flies may have trouble with sleep initiation and maintenance. The exceptional increase in episode length observed in rescue flies is likely due to nonnative expression of the transgene; however, it is clear Dα1 influences sleep (Somers, 2017).

    While activation of all cholinergic neurons inhibits sleep in flies, different neuronal groups can be wake-promoting or sleep-promoting. Individual nAChR subunits also appear to have different roles in sleep regulation. From this study, Dα1 seems to have net sleep-promoting effects, especially with sleep maintenance. The increase in daytime sleep observed in mutant flies may be a compensation mechanism to cope with the reduced amount of nighttime sleep. Further experiments are needed to determine whether the Dα1KO mutant has intact sleep homeostatic regulation, and whether loss of sleep affects sleep quality. Sleep deprivation has been linked to various detrimental effects, namely reduced life span and learning deficits (Somers, 2017).

    Longevity of Dα1KO mutants was measured, revealing a much shorter life span in the mutant compared to the wild-type. While longevity is not a direct measure of fitness, the mutants' reduced life span highlights the importance of the Dα1 subunit in D. melanogaster physiology. It is not clear if this effect is due to a single physiological role of Dα1, such as the subunits involvement in sleep, or cumulative effects of several impaired physiological roles that impact the mutant's longevity. In either case, it suggests that a resistance allele in the Dα1 subunit is likely to impact the fitness levels of the insect. It also supports the notion that sublethal exposures to insecticides that target receptor subunits orthologous to Dα1 encountered by beneficial insects, such as honey bees, are likely to affect their behavior in ways that may also impact their fitness (Somers, 2017).

    The Dα1KO mutant generated in this study provides a useful tool to study the role of Dα1 in insecticide resistance but, more significantly, to explore the endogenous roles of this gene (Somers, 2017).

    Based on prior evidence, it was expected that the Dα1KO mutant would be resistant to neonicotinoid insecticides (Perry, 2008). However, the data presented in this study allow a fresh evaluation of the value of Dα1 and orthologs in other species as insecticide targets. The ability of the Dα1 knockout flies to survive at all presents a potential issue for resistance evolution to compounds that target this receptor. Given that a wide range of mutations would lead to a total loss-of-function phenotype, such mutations will arise frequently, conferring insecticide resistance. The data presented in this study indicate that, looking beyond viability, there is a significant fitness cost associated with the total loss of Dα1 function, most obvious in terms of severe mating behavior defects, but possibly contributed to by the sleep and longevity phenotypes. Under optimal environmental conditions in the laboratory, large phenotypic differences between Dα1KO and control flies were observed. It is possible that the fitness cost associated with a null allele of this gene would be even greater under less ideal conditions experienced by natural populations. Therefore, while a nonsense mutation resulting in a resistance allele may be viable, it would likely be associated with fitness costs that would prevent it from persisting in the field. In contrast, mutations that may be null alleles have been found in Dα6 orthologs in spinosad-resistant insects in a number of species. Therefore, it is possible that the spectrum of mutations in Dα1 orthologs that can increase in frequency to confer insecticide resistance in a pest species are constrained by fitness costs (Somers, 2017).

    The potential impact of neonicotinoid insecticides on the behavior of beneficial insects, such as A. mellifera, has been intensively researched. Eleven nAChR subunits have been identified in A. mellifera, including a 1:1 ortholog of Dα1. While it cannot be assumed that the functional roles of the honey bee ortholog are identical to those of Dα1, it is likely to influence a range of behaviors that may be perturbed upon exposure to sufficient concentrations of neonicotinoid insecticides (Somers, 2017).

    The genetic resources that can be developed to study gene function in D. melanogaster, such as those described here, are extremely powerful. Research on nonmodel insects, both beneficial and pest species, is more challenging. Some functional analysis of Dα6 orthologs from pest species has been possible following the appropriate expression of these genes in a D. melanogaster Dα6 null mutant. A similar approach may be useful in the functional characterization of the orthologs of Dα1 (Somers, 2017).

    Insecticides can be powerful probes in neuroscience. To exert toxic effects, these chemicals bind to receptors that have crucial roles in neurotransmission. Research using pyrethroids and cyclodiene insecticides has been productive in elucidating the function of sodium channels and ligand-gated chloride channels, respectively. Similarly, the use of insecticides that target nAChRs has stimulated research on their function. The data demonstrates that such research will make a vital contribution in providing a detailed knowledge of the role neurotransmission in a wide range of behaviors. The variety of behavioral traits impacted by Dα1 loss-of-function indicates a high level of involvement of Dα1 in several behavioral neural circuits, which will require further investigation. Further analysis of Dα1 and the remaining members of the nAChR gene family with a range of paradigms is likely to reveal function in a wide range of insect behaviors (Somers, 2017).

    A new model to study sleep deprivation-induced seizure

    A relationship between sleep and seizures is well-described in both humans and rodent animal models; however, the mechanism underlying this relationship is unknown. Using Drosophila melanogaster mutants with seizure phenotypes, this study demonstrated that seizure activity can be modified by sleep deprivation. Seizure activity was evaluated in an adult bang-sensitive seizure mutant, stress sensitive B (sesB9ed4), and in an adult temperature sensitive seizure mutant seizure (seits1) under baseline and following 12 h of sleep deprivation. The long-term effect of sleep deprivation on young, immature sesB9ed4 flies was also assessed. Sleep deprivation increased seizure susceptibility in adult sesB9ed4 and seits1 mutants. Sleep deprivation also increased seizure susceptibility when sesB was disrupted using RNAi. The effect of sleep deprivation on seizure activity was reduced when sesB9ed4/+ flies were given the anti-seizure drug, valproic acid. In contrast to adult flies, sleep deprivation during early fly development resulted in chronic seizure susceptibility when sesB9ed4/+ became adults. These findings show that Drosophila is a model organism for investigating the relationship between sleep and seizure activity (Lucey, 2014).

    Aging induced endoplasmic reticulum stress alters sleep and sleep homeostasis
    Alterations in the quality, quantity and architecture of baseline and recovery sleep have been shown to occur during aging. Sleep deprivation induces endoplasmic reticular (ER) stress and upregulates a protective signaling pathway termed the unfolded protein response (UPR). The effectiveness of the adaptive UPR is diminished by age. Previously, it has been shown that endogenous chaperone levels alter recovery sleep in Drosophila melanogaster. This study reports that acute administration of the chemical chaperone sodium 4-phenylbutyrate (PBA) reduces ER stress and ameliorates age-associated sleep changes in Drosophila. PBA consolidates both baseline and recovery sleep in aging flies. The behavioral modifications of PBA are linked to its suppression of ER stress. PBA decreases splicing of X-box binding protein 1 (XBP1) and upregulation of phosphorylated elongation initiation factor 2α (p-eIF2α), in flies that were subjected to sleep deprivation. It was also demonstrated that directly activating ER stress in young flies fragments baseline sleep and alters recovery sleep. Alleviating prolonged/sustained ER stress during aging contributes to sleep consolidation and improves recovery sleep/ sleep debt discharge (Brown, 2014).

    With advancing age, humans demonstrate a number of sleep disruptions, which contribute to poor health consequences and accelerated senescence. The mechanisms that lead to these negative health outcomes, however, remain unclear. This study shows that both baseline and recovery sleep change with age and these alterations can be modulated by the chemical chaperone PBA. PBA is a low molecular weight fatty acid that has been shown to act as a chemical chaperone for misfolded or mislocalized proteins. It has also been shown to suppress ER-stress induced cell death without the aid of endogenous ER chaperones. It was also demonstrated in this study that directly inducing ER stress in young flies fragments baseline sleep and alters recovery sleep, phenocopying aged flies. Sleep fragmentation may contribute to accelerated aging and it has been suggested that an intervention that consolidates sleep may delay the aging process and increase life span (Brown, 2014).

    Aged flies exhibit less total baseline sleep and an altered homeostatic response in that they recover sleep over an extended 12 h period, whereas young flies recoup lost sleep within a much shorter window. Similar sleep disturbances are seen in humans and mammals, where aged individuals lose the ability to initiate and maintain nighttime sleep and recovery sleep after prolonged wakefulness is altered. Similar to this study, a significant age by time interaction was demonstrated, where young flies recover sleep in the first 4–5 h following sleep deprivation. Sleep deprived aged flies however, recover sleep over much a longer period. Variations in behavioral results compared with those existing in the literature are possibly due to our use of video analysis, which has been shown to be more accurate than DAMS in measurements of fly sleep, in particular daytime sleep and architecture (Brown, 2014).

    During the course of this study, an increase in locomotor activity in both young and aged flies treated with PBA was observed. A previous study found that PBA maintains locomotor vigor (activity) during aging in Drosophila. These results suggest that PBA is in fact increasing sleep and not merely reducing movement in the aged flies. Although total wake did not change in the young flies treated with PBA, wake became more consolidated. Not observing changes in total wake is possibly due to a ceiling effect in the young healthy flies. These results indicate that there is an age by drug interaction in the modification of sleep by PBA that remains to be elucidated (Brown, 2014).

    Aging also impairs quality control systems that are necessary for protein homeostasis. Earlier work has demonstrated that aging decreases BiP levels and increases pro-apoptotic factors such as CCAAT/enhancer-binding protein-homologous protein (CHOP) in the cerebral cortex of mice. Young flies experience less ER stress and their adaptive UPR is fully functional. Aged flies, on the other hand, are under chronic ER stress conditions with a diminished adaptive UPR. Therefore, basal ER stresses for the two groups are vastly different and likely contributes to the disparate responses to sleep deprivation. The young flies are readily able to handle acute sleep deprivation, whereas aged flies, already under stress, are overwhelmed by the secondary stressor. Addition of PBA has little effect on recovery sleep in young flies; however, PBA treatment ameliorates existing ER stress and suppresses further UPR induction in aged flies. As a result, treated aged flies display more efficient recovery sleep or are able to discharge sleep debt more efficiently than untreated aged flies (Brown, 2014).

    Although no significant differences were found in BiP expression in flies treated with PBA, XBP1 was significantly affected by PBA treatment. PBA not only reduces the amount of spliced XBP1, it also reduces the expression of the unspliced variant as well. PBA likely suppresses UPR induction by chaperoning unfolded proteins, so that BiP stays bound to IRE1 and PERK and delays or prevents its activation, even under the condition of sleep deprivation. Phospho-IF2α levels are significantly reduced in the PBA treated aged flies subjected to sleep deprivation. Changes in p-eIF2α can be directly linked to the behavioral changes in the aged flies given that increased p-eIF2α facilitates baseline and recovery sleep. The reduced eIF2α phosphorylation in PBA treated animals may account for the improved homeostatic response in the aged SD flies (Brown, 2014).

    This study also chemically induced ER stress in young flies and then subjected them to sleep deprivation. This acute tunicamycin treatment was found to decrease and fragment baseline sleep and phenocopy the recovery sleep behavior that is seen in the aged flies. There are two possible explanations for the tunicamycin results. First, tunicamycin activity leads to inhibition of glycosylation and general misfolding of proteins, which will ultimately result in ER stress. Secondly, tunicamycin could potentially prevent maturation of glycoproteins required for sleep maintenance. This data demonstrates that ER stress induces sleep fragmentation. Together with previous findings that sleep deprivation caused ER stress and activated the UPR, these results suggests that both processes are intimately linked and feedback on one another (Brown, 2014).

    PBA has been shown to significantly increase life span in Drosophila, purportedly through inhibition of HDAC activity. The moderate effects of PBA on sleep consolidation in young sssp1 mutant flies suggest that protein stabilization by PBA is only partially ameliorating defects in these animals. The decrease in sleep in the ctrl sss background strain treated with PBA supports the increase and consolidation of wake in the wild-type lines. It has been previously shown that induction of ER stress impairs waking in mice. The study speculates that application of PBA also promotes wake-active neuronal function and consolidates wake in the flies by reducing ER stress. The issue is not the accumulation of sleep debt, but the discharge of sleep debt that is impaired in the aged flies. By ameliorating ER stress and suppressing activation of the UPR, PBA allows for more efficient recovery sleep in the aged flies. Since PBA ameliorates ER stress by directly reducing aberrant protein load, treating Drosophila with PBA supplements chaperone activity and decreases the burden of misfolded proteins that occurs as a consequence of an external stressor, sleep deprivation, as well as normal aging (Brown, 2014).

    Aging alters circadian regulation of redox in Drosophila

    Circadian coordination of metabolism, physiology, and neural functions contributes to healthy aging and disease prevention. Clock genes govern the daily rhythmic expression of target genes whose activities underlie such broad physiological parameters as maintenance of redox homeostasis. Previously, it was reported that glutathione (GSH) biosynthesis is controlled by the circadian system via effects of the clock genes on expression of the catalytic (Gclc) and modulatory (Gclm) subunits comprising the glutamate cysteine ligase (GCL) holoenzyme. The objective of this study was to determine whether and how aging, which leads to weakened circadian oscillations, affects the daily profiles of redox-active biomolecules. Fly aging was found to be associated with altered profiles of Gclc and Gclm expression at both the mRNA and protein levels. Analysis of free aminothiols and GCL activity revealed that aging abolishes daily oscillations in GSH levels and alters the activity of glutathione biosynthetic pathways. Unlike GSH, its precursors and products of catabolism, methionine, cysteine and cysteinyl-glycine, were not rhythmic in young or old flies, while rhythms of the glutathione oxidation product, GSSG, were detectable. The study concludes that the temporal regulation of GSH biosynthesis is altered in the aging organism and that age-related loss of circadian modulation of pathways involved in glutathione production is likely to impair temporal redox homeostasis (Klichko, 2015).

    This study investigated a role for the circadian system in regulating redox in the context of organismal aging. Cellular redox homeostasis largely relies on the redox-active compound, glutathione, which is present at concentrations many fold higher compared to concentrations of other redox-active molecules. Previous studies have shown that the circadian clocks modulate the de novo synthesis of GSH via transcriptional control of GCLc and GCLm, the subunits that comprise the GCL holoenzyme. This study broadened the investigation of the relationship between clock and redox to determine the levels of other redox-active molecules, involved in glutathione synthesis and metabolism. Around the clock expression profiles of both cysteine and its precursor methionine were investigated (Klichko, 2015).

    As neither cysteine nor methionine exhibited evidence for diurnal rhythms in either young or old flies, it appears that the contribution of the trans-sulfuration pathway to glutathione homeostasis is not regulated by circadian clocks. Consistent with these findings, methionine was found to be arrhythmic in the study of the human metabolome of blood plasma and saliva although the analyses were performed with healthy but older (57-61 years) males, where age-dependent effects might have influenced the oscillations. In contrast, mouse hepatic metabolome and transcriptome studies revealed rhythmicity in metabolic sub-pathways, where oscillations in glutathione were ascertained by oscillations in its precursors, cysteine and methionine, albeit with a lower amplitude for the latter (Klichko, 2015).

    Analysis of the Drosophila heads revealed no cycling in the concentrations of Cys-Gly, consistent with the arrhythmic behavior of cysteine and methionine. Given that Cys-Gly also serves as a signature of glutathione degradation, interpretation of these results are somewhat tentative. Nevertheless, these results revealed no rhythmicity in cysteine, methionine and Cys-Gly, and suggest that, at least in flies, the pathways responsible for the supply of sulfur-containing precursors for glutathione synthesis are not regulated by the circadian clocks. It should be noted that the mammalian liver is a homogenous tissue with a strong food-entrained clock mechanism, while fly heads are enriched in nervous tissues with clocks entrained by light-dark cycles. Moreover recent analysis of the circadian transcriptome shows that liver possesses the highest number of rhythmic genes, while brain has the lowest (Klichko, 2015).

    Another important finding of this study is that the diurnal fluctuations in GSH levels were not followed by similar changes in the products of its degradation (Cys-Gly) and oxidation (GSSG). While Cys-Gly was completely arrhythmic, changes in GSSG profile did not mirror those observed for GSH. Even though both shared the same slow drop-off from ZT0 to ZT8 as well as the ZT8 trough, their peaks were quite distinct (ZT12 for GSSG and ZT20 for GSH). Also in old flies, a certain degree of rhythmicity is maintained for GSSG in contrast to the absence of any diurnal GSH patterns (Klichko, 2015).

    Another important finding of this study is that the rhythms in glutathione levels observed in young flies were lost in old flies, presumably due to the loss of diurnal fluctuations of GCLc, GCLm as well as GCL activity, in response to the weakening of the circadian clocks. In contrast, GSSG rhythms were largely preserved in older flies, suggesting that the daily changes in glutathione disulfide levels are supported by enzymatic reactions that are not under clock control (Klichko, 2015).

    Despite loss of circadian regulation, average daily levels of GSH remained unchanged during aging, while the levels of GSSG were slightly higher, mainly due to lesser drop in the early morning. In Drosophila, it has been established that whole body GSH levels were either relatively constant or slightly decreased during aging while GSSG rose 2-–3 fold. Similar age-related changes were documented in different mammalian tissues with the most significant reduction in GSH and accumulation of GSSG in the brain, indicative of a more pro-oxidative cellular environment. As such changes in GSH and GSSG were frequently associated with increases in enzyme activities related to GSH usage, the relatively steady glutathione concentrations observed in the heads of old flies could point to less efficient GSH utilization (Klichko, 2015).

    The rather unexpected finding of this study is that the expression of Gclc at both mRNA and protein levels significantly increased in the heads of old flies, and this increase was associated with about 25% higher average daily GCL activity. Despite this increase, the average daily levels of GSH remained unchanged suggesting a loss in GCL catalytic efficiency or an age-related increase in GSH utilization. One possible scenario is that the efficiency of GSH synthesis can be induced by oxidative stress, in part through the well-documented increase in H2O2 signaling that accompanies aging. For instance, post-translational control of γ-glutamylcysteine (γ-Glu-Cys) synthesis is influenced by oxidative stress, which can dramatically affect formation of GCL holoenzyme and its stabilization (Klichko, 2015).

    Consistent with induction of GCL by stress, it was previously reported that per-null mutants with disrupted clock displayed arrhythmic as well as elevated GCL activity, which was also reflected in arrhythmic and elevated ROS levels relative to the control. It should be noted that previous studies comparing GCL activity and GSH levels in young and old rats showed a decrease of both parameters in liver, while in aging brain and heart GSH decreased but GCL activity remained unchanged, pointing again to catalytic deficiency of the enzyme (Klichko, 2015).

    Other aminothiols that did not show cycling in young flies also remained arrhythmic in old flies, but displayed changes in their steady state levels. Consistent with previous reports, the amounts of Cys-Gly were ~50% higher in older flies. Cys-Gly, derived from the breakdown of glutathione, is required for GSH synthesis as a precursor of cysteine, but at the same time it is also a prooxidant generated during the catabolism of glutathione. The requirement of Cys-Gly for GSH synthesis justifies its increase with age, as the tissues require an increased supply of precursors for GSH biosynthesis in older flies. However, an increase in cysteine levels during aging was not observed. A more plausible explanation is that the increase in Cys-Gly is indicative of an increase in oxidative stress and GSH degradation. In agreement with this view, the average daily levels of methionine were about 35% lower in old flies suggesting the likelihood of an increase in oxidation of methionine to methionine sulfoxide by ROS rather than an increase in methionine consumption for cysteine biosynthesis. Together, these changes indicate a shift in redox homeostasis in the heads of older flies, consistent with the earlier reports in whole flies. Similar alterations in the redox components were also indicative of heightened oxidative stress in pathologies like systemic lupus erythematosus (Klichko, 2015).

    Neuron-specific overexpression of core clock genes improves stress-resistance and extends lifespan of Drosophila melanogaster

    Gene expression is much altered in aging. This study observed age-dependent decline of core clock genes' expression in the whole body of the fruit fly. It was hypothesized that inducible overexpression of clock genes (cry, per, tim, cyc and Clk) in the nervous system can improve healthspan of D. melanogaster. The lifespan of transgenic Drosophila was studied; life extension was shown for cry, per, cyc and tim genes. There were significant positive changes in the stress-resistance of flies overexpressing core clock genes in conditions of hyperthermia, hyperoxia, starvation and persistent lighting. The overexpression of per and cry restore circadian rhythms of locomotor activity. The results presented support the hypotheses that the compensation of circadian oscillator genes expression can improve the healthspan in Drosophila melanogaster (Solovev, 2019a).

    Circadian clock genes' overexpression in Drosophila alters diet impact on lifespan

    Diet restriction is one of the most accurately confirmed interventions which extend lifespan. Genes coding circadian core clock elements are known to be the key controllers of cell metabolism especially in aging aspect. The molecular mechanisms standing behind the phenomenon of diet-restriction-mediated life extension are connected to circadian clock either. This study investigated the effects of protein-rich and low-protein diets on lifespan observed in fruit flies overexpressing core clock genes (cry, per, Clk, cyc and tim). The majority of core clock genes being upregulated in peripheral tissues (muscles and fat body) on protein-rich diet significantly decrease the lifespan of male fruit flies from 5 to 61%. Nevertheless, positive increments of median lifespan were observed in both sexes, males overexpressing cry in fat body lived 20% longer on poor diet. Overexpression of per also on poor medium resulted in life extension in female fruit flies. Diet restriction reduces mortality caused by overexpression of core clock genes. Cox-regression model revealed that diet restriction seriously decreases mortality risks of flies which overexpress core clock genes. The hazard ratios are lower for flies overexpressing clock genes in fat body relatively to muscle-specific overexpression. The present work suggests a phenomenological view of how two peripheral circadian oscillators modify effects of rich and poor diets on lifespan and hazard ratios (Solovev, 2019).

    Sleep facilitates memory by blocking dopamine neuron-mediated forgetting

    Early studies from psychology suggest that sleep facilitates memory retention by stopping ongoing retroactive interference caused by mental activity or external sensory stimuli. Neuroscience research with animal models, on the other hand, suggests that sleep facilitates retention by enhancing memory consolidation. Recently, in Drosophila, the ongoing activity of specific dopamine neurons was shown to regulate the forgetting of olfactory memories. This study shows that this ongoing dopaminergic activity is modulated with behavioral state, increasing robustly with locomotor activity and decreasing with rest. Increasing sleep-drive, with either the sleep-promoting agent Gaboxadol or by genetic stimulation of the neural circuit for sleep, decreases ongoing dopaminergic activity, while enhancing memory retention. Conversely, increasing arousal stimulates ongoing dopaminergic activity and accelerates dopaminergic-based forgetting. Therefore, forgetting is regulated by the behavioral state modulation of dopaminergic-based plasticity. These findings integrate psychological and neuroscience research on sleep and forgetting (Berry 2015).

    While some memories are long-lasting, most others fade away and are forgotten. Why we forget, has been an intriguing and central question in psychology and neuroscience for more than a century. Even though forgetting is often thought of as a failure or limitation of the brain, recent studies support the view that forgetting is a biologically regulated function of the brain allowing optimal adaptability to an ever-changing environment. In the fruit fly Drosophila, it was recently shown that the very same set of dopamine neurons (DANs) that signal through one receptor to form aversive olfactory memories, also signal through a separate receptor after learning to forget these memories (Berry, 2012). However, it remains unclear whether this dopaminergic forgetting signal is constant and autonomous, or dynamic and regulated (Berry 2015).

    From fruit flies to humans, animals routinely alternate between highly active behavioral states and long states of immobility and quiescence called sleep. Despite the obvious disadvantages an inanimate state conveys to survival, sleep has been proposed to have critically important functions, including in memory and cognition. Since the earliest experimental studies of human memory, sleep shortly after learning has been shown to consistently lead to an increase in retention and thus less forgetting of many forms of memory including declarative and emotional memory in mammals and long-term courtship memory in Drosophila. However, there exists controversy as to how sleep benefits memory retention. Many studies in mammals support the idea that sleep benefits memory retention because it is accompanied by specific mechanisms, such as slow wave sleep, rapid eye movement (REM) sleep, and sharp-wave ripple-based memory replay, that increase memory retention by actively consolidating newly formed memories. Alternatively, it was proposed nearly a century ago and recently revisited, that sleep, or long periods of quiet wakefulness, benefit memory retention by muting experience-driven plasticity and new memory formation, thus reducing retroactive interference-based forgetting. In addition, this state of reduced neuronal activity might then allow consolidation to occur more efficiently, referred to as the "opportunistic consolidation" model. Thus, the essence of how exactly sleep benefits memory retention remains debated (Berry 2015).

    Previous studies have observed that, after promoting the acquisition of olfactory memories, a small set of DANs that innervate the mushroom body (MB) memory center, intriguingly, display synchronized and ongoing Ca2+-based activity after learning that causes the forgetting of early aversive olfactory memories in Drosophila(Berry, 2012). While this activity occurs as reoccurring bursts, it was noticed that the pattern of activity appeared temporally regulated, occurring in bouts. In order to understand how the DAN-based forgetting signal might be regulated, an in vivo imaging assay was developed allowing simultaneous monitoring of a fly's DAN Ca2+ activity, via GCaMP3.0 expression using TH-gal4 and behavior while walking on a ball supported by air. Focus was placed on two regions of the DAN processes that form synaptic connections to the MBs, referred to as neuropils, one that displays ongoing activity and belongs to the MV1 neuron and an adjacent control region belonging to the V1 neuron, which is relatively inactive. Remarkably, a 1-hr simultaneous recording of locomotion and DAN activity revealed that the MV1 neuropil displayed activity resembling the coarse temporal pattern of locomotor behavior. Ball rotation data was used to cluster time points into either behaviorally active or rest states, and MV1 neuropil activity was found to be robustly elevated during active states, whereas the V1 neuropil activity remained low in both states, but had a slight decrease during active states. Furthermore, the MV1 neuropil Ca2+ signal was strongly correlated with ball rotation, particularly in the lower frequency domains (frequency < 0.002 Hz, or ~1 cycle every 8 min or more, timescales consistent with that of locomotor bout structure). Finally, DAN activity was examined during stable transitions into and out of behaviorally active states ( by aligning transition segments of recordings across all animals. Interestingly, MV1 DAN activity robustly increased upon transition into active states, while, conversely, dropped during rest states. V1 activity remained low and was not significantly regulated with behavioral transitions. Together, these data, along with observations of synchronized activity between MV1 and another DAN, MP1, indicate that the ongoing activity from specific sets of DAN involved in forgetting, including MV1, is regulated with the behavioral state of the animal (Berry 2015).

    Given the strong correlation between DAN activity and locomotor activity, tests were performed to see whether DAN activation might promote locomotor activity, that is, whether DAN activity is upstream of locomotor circuits. Two prior studies found no role for these MB innervating DANs in regulating locomotor activity. When the synaptic output from these DANs was blocked with restricted expression in MV1, MP1, and V1 DANs to drive temperature-sensitiveUAS-shits1function, no decreased locomotor activity was seen between temperatures, although the experimental genotype exhibited less activity at high temperature compared to one but not both control genotypes. It was also noted from imaging experiments that locomotor activity occasionally occurs while the MV1 neuron is not active, thus further supporting that locomotor behavior does not require c150-gal4>DAN output. Furthermore, stimulation of these neurons, using UAS-trpA1, did not produce genotype specific and robust increases in locomotor activity. But similar to the blocking experiments, high temperature increased the locomotor activity of the two control genotypes (UAS-trpA1 and c150-gal4 alone). These data indicate that c150-gal4 DAN output is neither necessary nor sufficient to acutely drive locomotor activity. It is therefore concluded that the ongoing signal in MV1 is either downstream of locomotor behavior itself, or is regulated in parallel, by other brain areas that promote arousal and locomotor activity (Berry 2015).

    Given that the ongoing activity in MV1 was highest during behaviorally active states, the hypothesis was tested that reducing behavioral activity with increased sleep drive would reduce this ongoing activity. The GABAA agonist, Gaboxadol (or THIP), has been shown to specifically promote deep non-REM sleep in humans, while leaving REM sleep intact; sleep characteristics similar to those occurring during normal homeostatic regulation of sleep. Recently, it was shown that Gaboxadol also induces sleep in Drosophila. To confirm this, attempts were made to induce sleep in Drosophila by feeding flies various doses of Gaboxadol. Shortly after Gaboxadol feeding, long periods of quiescence, (>5 min) conventionally defined as 'sleep' in Drosophila, significantly increased during both day and night in a dose-dependent manner. Next, flies were fed Gaboxadol (0.1 mg/ml) for 1 day and then removed the drug to test whether these effects were reversible. Once again, Gaboxadol treatment increased sleep, occurring as bouts with increased duration, but remarkably, total sleep and bout duration actually decreased after drug removal compared to control fed flies. These data indicate that less sleep is needed in flies given Gaboxadol the prior day, suggestive of a homeostatic response. Finally, to test the arousability of flies fed Gaboxadol, a single mechanical stimulus was delivered every hour for 1 day followed by a day of drug treatment. Interestingly, the average-evoked activity, post-stimulus was significantly reduced with increasing Gaboxadol dosage. These data suggest that having Gaboxadol onboard increases arousal thresholds. Altogether, these data indicate that Gaboxadol, similar to effects on mammals, induces bona fide sleep in Drosophila, with hallmark characteristics that include reversible quiescence, homeostatic regulation, and increased arousal thresholds (Berry 2015).

    In order to observe the effects of Gaboxadol on DAN activity, varying concentrations of this sleep agent were perfused across the brain while performing in vivo imaging of MV1 activity and fly body movement was monitored in a recording chamber. Like walking on the ball, ongoing MV1 activity was also regulated with behavioral state in this assay, increasing during bouts of body movement. Remarkably, Gaboxadol perfusion rapidly and robustly attenuated both fly movement and MV1 activity at 0.01 and 0.1 mg/ml. Furthermore, it was found that the quiescent behavioral state and reduced MV1 activity was fully reversible with wash out, thus eliminating pharmacological-induced damage as a cause of decreased physical and DAN activity (Berry 2015).

    To rule out non-specific effects of Gaboxadol and extend these results, sleep drive was increased sharply by thermogenetic stimulation of the sleep circuit. Recent studies identified a dorsal fan shaped body (dfsb) circuit in the central brain, specifically represented in the R23E10-gal4 line, which acts as the effector arm of the sleep homeostat (Donlea; 2011 and Donlea; 2014b). Consistent with these studies, TrpA1-based stimulation of R23E10-gal4-expressing neurons caused a rapid and robust increase in daytime sleep followed by a negative sleep rebound the day after stimulation, confirming the dfsb circuit's role in homeostatic sleep regulation. In order to measure DAN activity in vivo while using the gal4-uas system to modulate the sleep circuit, a TH-lexA line was developed to express GCaMP3.0 in the MV1 and V1 DAN neurons, their associated MB neuropil regions, as well as DAN innervation of the anterior inferior medial protocerebrum (PR), a region also exhibiting ongoing activity like MV1 (Berry, 2012). While simultaneously measuring movement and DAN activity, before ('Pre'), during ('Treat'), and after ('Post') stimulation of dfsb neurons was recorded. As predicted, stimulation of the sleep circuit rapidly decreased fly behavioral activity and was accompanied with a robust decrease in MV1 and PR DAN activity, with no change in the control V1 region. Fly behavioral activity was partially restored and ongoing activity in MV1 and PR completely restored to pre-stimulation levels after stimulation of the sleep circuit was ceased. These results, along with those from Gaboxadol administration, indicate that increased sleep drive dramatically reduces the ongoing activity of DANs involved in forgetting (Berry 2015).

    Since ongoing MV1 activity is decreased with increasing sleep drive, it was hypothesized that acutely and reversibly increasing sleep drive specifically after learning would reduce DAN-mediated forgetting. To test this, sleep was modulated with Gaboxadol after aversive olfactory conditioning, where populations of flies learn to associate an odor with electric shock. Memory to this association is then tested in a T-maze, giving flies the choice between the trained odor and an unconditioned odor. Since memory from this kind of training decays quickly after training, attempts were made to increase the rate of Gaboxadol consumption and thus the rate of sleep onset, by increasing the hunger of flies via starvation prior to feeding. As was observed previously, flies fed Gaboxadol experienced more sleep than controls, and a 16-hr starvation period increased this effect. Furthermore, it was found that flies removed from Gaboxadol food 1 hr after learning partially returned to control sleep and activity levels by the sixth hour and completely by the eighth hour after learning, indicating that these time points were appropriate for testing memory retrieval. This Gaboxadol feeding protocol led to increased sleep during the period of memory retention. Remarkably, it was found that flies forced to sleep with Gaboxadol treatment after learning exhibited enhanced memory retention at both 6 and 8 hr. Similarly, sleep circuit stimulation after conditioning also rapidly and reversibly induced sleep and enhanced both 3- and 6-hr memory retention. Importantly, simultaneous stimulation of the dfsb sleep circuit and c150-gal4 DANs also led to strong sleep induction. Memory retention, in contrast, was markedly decreased, similar to that observed with stimulation of DANs alone. Therefore, sleep, after learning, loses its protective qualities when DAN signaling is artificially potentiated. This circuit level epistasis experiment indicates that DAN-mediated forgetting is downstream of sleep networks. Together, the data indicate that increased sleep and reduced arousal after learning reduces DAN-mediated forgetting of aversive olfactory memories (Berry 2015).

    If the DANs innervating the MB memory center are downstream of arousal circuitry, then they should respond to arousing stimuli. In fact, these neurons have already been shown to respond to many salient stimuli, including odors and electric shock and temperature changes. Since mechanical stimuli have been extensively used to arouse flies for sleep deprivation, airpuffs were delivered to the fly using a protocol shown to induce arousal in flies, while simultaneously recording fly movement and DAN activity. It was found that the DAN processes in all three areas (MV1, PR, V1) of the mushroom body neuropil exhibited robust responses to each airpuff. However, MV1 responsiveness was maintained across stimuli while the other regions showed attenuated responsiveness across stimuli. Importantly, both fly movement and ongoing MV1 activity continued at an elevated level just after stimulation, indicating a stimulus-induced elevation in arousal and MV1 DAN activity (Berry 2015).

    Next, it was reasoned that increasing the arousal after learning would accelerate DAN-mediated forgetting. To test this, a population arousal device was developed that allowed delivery of a mechanical stimuli (2-s stimulus every 1 min over 80 min) to flies in population vials after aversive olfactory learning. It was found that mechanical stimuli delivered for the first 80 min after learning significantly aroused populations of flies, leading to an overall increase in activity between each stimulus, with activity levels dropping back to control levels after treatment. Importantly, mechanical stimulation after learning caused a robust decrease in 3-hr memory for wild-type Canton-S flies. However, acquisition and immediate memory retrieval were not disrupted by prior mechanical stimulation, indicating that the stimuli must be delivered after learning to observe its disruptive effects. Remarkably, it was found that blocking neuronal output of c150-gal4DANs specifically during mechanical stimulation blocked the forgetting induced by this treatment. Therefore, these data indicate that increasing arousal after learning accelerates DAN-mediated forgetting (Berry 2015).

    The following conclusions are made from the data. First, after learning, the ongoing DA forgetting signal is not constant but instead is regulated with behavioral state. Thus, the forgetting signal does not chronically remove memories at a constant rate. Second, the ongoing forgetting signal is coupled directly to the arousal level of the animal, being suppressed with low levels of arousal such as with the state of sleep and being enhanced by activation of sensory pathways. As a result, forgetting decreases when flies rest or sleep and increases when flies are aroused by external stimuli (Berry 2015).

    DA is known to regulate various types of plasticity in mammals. In flies, DA has been shown to elicit presynaptic plasticity within the Kenyon cells of the MB memory center proposed to underlie learning. Additionally, it was previously found that DA after learning regulates forgetting (Berry, 2012), thus implicating a DA-based plasticity mechanism that weakens memories. Synthesizing these previous observations with the current data, it is proposed that the behavioral state-coupled DA signal, discovered in this study, regulates the plasticity of the memory system, making it malleable for memory updating so that memories of current events can be formed and old, unused memories can be forgotten. While it was found previously that different DA receptors underlie learning and forgetting, more work remains to distinguish the molecular cascades involved and the cellular events that underlie these forms of behavioral plasticity (Berry 2015).

    These findings add compelling mechanistic evidence to support the model that sleep, which begins with and is accompanied by inactivity or rest, benefits newly formed labile memories by reducing the level of plasticity induced by behavioral activity. Furthermore, as sleep progresses and arousal thresholds increase, DANs become less reactive to stimuli. Thus, the molecular/cellular model is congruent with early psychological models of sleep benefitting memory by muting the retroactive interference that causes forgetting. Nevertheless, the data do not eliminate the possibility that sleep-specific mechanisms exist that enhance memory consolidation, as often proposed from studies with mammalian systems. Mechanistically, the effects of sleep on memory consolidation and forgetting may operate in parallel and independently of one another or more intriguingly; they may operate in serial in a dependent fashion, with reduced forgetting being a prerequisite for sleep-facilitated consolidation, similar to the 'opportunistic consolidation' model proposed by Mednick, 2011 (Berry 2015).

    This study has observed that multiple DANs produce the ongoing DA signal, synchronized across the MB memory center and protocerebrum, that leads to forgetting of olfactory memories. It remains to be determined if this network activity is but one segment of a larger and more diffuse DA network that operates on memory types other than olfactory; whether there exist multiple, independent forgetting networks regulated by arousal levels; and whether forgetting of non-olfactory memories occurs through DA-based mechanisms or involves other neuromodulatory transmitters (Berry 2015).

    Manipulations of amyloid precursor protein cleavage disrupt the circadian clock in aging Drosophila

    Alzheimer’s disease (AD) is a neurodegenerative disease characterized by severe cognitive deterioration. While causes of AD pathology are debated, a large body of evidence suggests that increased cleavage of Amyloid Precursor Protein (APP) producing the neurotoxic Amyloid-β (Aβ) peptide plays a fundamental role in AD pathogenesis. One of the detrimental behavioral symptoms commonly associated with AD is the fragmentation of sleep-activity cycles with increased nighttime activity and daytime naps in humans. Sleep-activity cycles, as well as physiological and cellular rhythms, which may be important for neuronal homeostasis, are generated by a molecular system known as the circadian clock. Links between AD and the circadian system are increasingly evident but not well understood. This study examined whether genetic manipulations of APP-like (APPL) protein cleavage in Drosophila melanogaster affect rest-activity rhythms and core circadian clock function in this model organism. It was shown that the increased β-cleavage of endogenous APPL by the β-secretase (dBACE) severely disrupts circadian behavior and leads to reduced expression of clock protein PER in central clock neurons of aging flies. The study's data suggest that behavioral rhythm disruption is not a product of APPL-derived Aβ production but rather may be caused by a mechanism common to both α and β-cleavage pathways. Specifically, it was shown that increased production of the endogenous Drosophila Amyloid Intracellular Domain (dAICD) causes disruption of circadian rest-activity rhythms, while flies overexpressing endogenous APPL maintain stronger circadian rhythms during aging. In summary, this study offers a novel entry point toward understanding the mechanism of circadian rhythm disruption in Alzheimer's disease (Blake, 2015).

    Loss of rest-activity rhythms is a well-established early symptom of AD in humans. Because disruption of circadian rhythms is detrimental to neuronal homeostasis, it is important to understand relationships between AD and circadian rhythms at the cellular and molecular levels. To address this question, this study examined how manipulations of the fly ortholog of APP and its cleaving enzymes affect endogenous rest-activity rhythms and clock mechanism in Drosophila. Over-expression of dBACE was found to disrupt behavioral rest-activity rhythms, and this effect is most severe in aged flies suggesting an age-dependent mechanism. Furthermore, dBACE expression resulted in dampened oscillation of the core clock protein PER in central pacemaker neurons, which are master regulators of rest activity rhythms. Significantly reduced PER levels are observed in the sLNv and lLNv neurons of age 50d flies expressing dBACE in all clock cells (including glia), all neurons, or only in PDF-positive sLNv and lLNv neurons. These data suggest that manipulation of APP-cleavage by dBACE over-expression directly affects the oscillation of PER protein in central pacemaker neurons in a cell-autonomous manner. Since a functional clock mechanism in sLNv is necessary and sufficient to maintain free running activity rhythms, reduced oscillations of PER in these neurons could be responsible for the loss of activity rhythms in age 50d flies. Importantly, the decline in PER levels occurrs only in flies with manipulated dBACE, not in old control flies. This is in agreement with earlier findings that aging does not dampen PER oscillations in pacemaker neurons of wild type flies, while it reduces clock oscillations in peripheral clocks  (Blake, 2015).

    While this study reports that the loss of behavioral rhythms after manipulation of dBACE is associated with reduced expression of clock genes in the central pacemaker, other recent work shows that expression of human Aβ peptides leads to disruption of rest activity rhythms without interfering with PER oscillations in the central pacemaker. Even strongly neurotoxic Aβ peptides, such as Aβ42 arctic, do not cause rhythm disruption when expressed in central pacemaker neurons; rather, pan-neuronal expression is required. The fact that even the most neurotoxic Aβ peptides are not capable of dampening PER oscillation in pacemaker neurons suggests that Aβ production does not affect clock oscillations and that it is not Aβ production that causes the phenotype observed in this study upon over-expression of dBACE. This was confirmed by expression of KUZ, whose activity does not increase dAβ production; however, it also leads to disruption of rest-activity rhythms. Similar rhythm disruption by dBACE and KUZ suggests that an excess cleavage product of both pathways might be responsible for the disruption. Like in the mammalian APP cleavage pathway, in Drosophila cleavage of APPL by KUZ or dBACE results in a C-terminal fragment (CTF) that is subsequently cleaved by the ϒ-secretase resulting in the production of dAICD. Indeed, it was shown that expression of dAICD results in an age-dependent decline in rhythmic locomotor activity. As with dBACE and KUZ expression, dAICD expression causes weakening or complete loss of behavioral rhythms while age-matched control flies remain highly rhythmic. In this context, it is worth noting that α-secretase activators are considered for clinical trials to reduce Aβ production in AD patients. However, according to results in this study, this could lead to disruptions of circadian rhythms and sleep patterns thus negatively impacting the lives of patients and their caretakers (Blake, 2015).

    This study's data suggest that increased dAICD may be the proximal cause of decay in rest-activity rhythms. The role of AICD in AD is increasingly evident but poorly understood. AICD is able to enter the nucleus and has been implicated in transcriptional regulation that may affect cell death, neurite outgrowth and neuronal excitability. Interestingly, transgenic mice expressing AICD have increased activity of GSK-3, which in flies affects the circadian clock. Over-expression of GSK-3 in Drosophila leads to altered circadian behavior by hyper-phosphorylation of TIMELESS (TIM), a key circadian protein which forms dimers with PER that enter the nucleus and regulate the clock mechanism. Of further interest, increased GSK-3 activity has been implicated in AD, and in Drosophila, increased GSK-3 activity mediates the toxicity of Aβ peptides (Blake, 2015).

    Cleavage of APPL likely results in a significant decline in intact APPL, and this could be detrimental as APPL has neuroprotective effects. It was also recently shown that loss of full-length APPL induces cognitive deficits in memory. This study reports that flies over-expressing full-length APPL in central pacemaker neurons maintain stronger behavioral rest-activity rhythms during aging than control flies; however this effect is not observed when APPL is expressed pan-neuronally. This could be caused by negative effects of APPL when expressed in other unspecified neurons, or could be related to driver strength. Overall, the study suggests that the loss of full-length APPL might negatively affect circadian behavior by way of the central pacemaker neurons (Blake, 2015).

    Over-expression of dAICD induces a severe phenotype, disrupting rest-activity rhythms as early as age 5d when expressed in central pacemaker neurons and by age 35d with pan-neuronal expression. Taken together these results suggest that while loss of full-length APPL by over-expression of its secretases might negatively impact circadian behavior, the cleavage product dAICD induces the most severe behavioral rest-activity disruption. Interestingly, the observed effect is not likely a product of neurodegeneration as it was previously shown that dAICD has no effect on neurodegeneration, and this study shows that the pacemaker cells appear intact in pdf > dAICD flies. In addition, it was shown that dAICD, like the vertebrate AICD, can be found in the nucleus. Therefore, this study suggests that dAICD may directly or indirectly affect the expression of clock genes. This offers a novel entry point toward understanding the mechanism of circadian rhythm disruption in Alzheimer's disease (Blake, 2015).

    TARANIS functions with Cyclin A and Cdk1 in a novel arousal center to control sleep in Drosophila

    Sleep is an essential and conserved behavior whose regulation at the molecular and anatomical level remains to be elucidated. This study identifies Taranis (Tara), a Drosophila homolog of the Trip-Br (SERTAD) family of transcriptional coregulators, as a molecule that is required for normal sleep patterns. Through a forward-genetic screen, tara was isolated as a novel sleep gene associated with a marked reduction in sleep amount. Targeted knockdown of tara suggests that it functions in cholinergic neurons to promote sleep. tara encodes a conserved cell-cycle protein that contains a Cyclin A (CycA)-binding homology domain. Tara regulates CycA protein levels and genetically and physically interacts with CycA to promote sleep. Furthermore, decreased levels of Cyclin-dependent kinase 1 (Cdk1), a kinase partner of CycA, rescue the short-sleeping phenotype of tara and CycA mutants, while increased Cdk1 activity mimics the tara and CycA phenotypes, suggesting that Cdk1 mediates the role of Tare and CycA in sleep regulation. Finally, a novel wake-promoting role was described for a cluster of ∼14 CycA-expressing neurons in the pars lateralis (PL), previously proposed to be analogous to the mammalian hypothalamus. The study proposes that Taranis controls sleep amount by regulating CycA protein levels and inhibiting Cdk1 activity in a novel arousal center.

    Most animals sleep, and evidence for the essential nature of this behavior is accumulating. However, how sleep is controlled at a molecular and neural level is far from understood. The fruit fly, Drosophila, has emerged as a powerful model system for understanding complex behaviors such as sleep. Mutations in several Drosophila genes have been identified that cause significant alterations in sleep. Some of these genes were selected as candidates because they were implicated in mammalian sleep. However, others (such as Shaker and CREB) whose role in sleep was first discovered in Drosophila have later been shown to be involved in mammalian sleep, validating the use of Drosophila as a model system for sleep research. Since the strength of the Drosophila model system is the relative efficiency of large-scale screens, unbiased forward-genetic screens have been conducted to identify novel genes involved in sleep regulation. Previous genetic screens for short-sleeping fly mutants have identified genes that affect neuronal excitability, protein degradation, and cell-cycle progression. However, major gaps remain in understanding of the molecular and anatomical basis of sleep regulation by these and other genes (Afonso, 2015).

    Identifying the underlying neural circuits would facilitate the investigation of sleep regulation. The relative simplicity of the Drosophila brain provides an opportunity to dissect these sleep circuits at a level of resolution that would be difficult to achieve in the more complex mammalian brain. Several brain regions, including the mushroom bodies, pars intercerebralis, dorsal fan-shaped body, clock neurons, and subsets of octopaminergic and dopaminergic neurons, have been shown to regulate sleep. However, the recent discovery that Cyclin A (CycA) has a sleep-promoting role and is expressed in a small number of neurons distinct from brain regions suggests the existence of additional neural clusters involved in sleep regulation (Afonso, 2015).

    From an unbiased forward-genetic screen, this study discovered taranis (tara), a mutant that exhibits markedly reduced sleep amount. tara encodes a Drosophila homolog of the Trip-Br (SERTAD) family of mammalian transcriptional coregulators that are known primarily for their role in cell-cycle progression. TARA and Trip-Br proteins contain a conserved domain found in several CycA-binding proteins. This research shows that tara regulates CycA levels and genetically interacts with CycA and its kinase partner Cyclin-dependent kinase 1 (Cdk1) to regulate sleep. Furthermore, a cluster of CycA-expressing neurons in the dorsal brain was shown to lie in the pars lateralis (PL), a neurosecretory cluster previously proposed to be analogous to the mammalian hypothalamus, a major sleep center. Knockdown of tara and increased Cdk1 activity in CycA-expressing PL neurons, as well as activation of these cells, reduces sleep. Collectively, these data suggest that TARA promotes sleep through its interaction with CycA and Cdk1 in a novel arousal center (Afonso, 2015).

    From an unbiased forward genetic screen, this study has identified a novel sleep regulatory gene, tara. The data demonstrate that TARA interacts with CycA to regulate its levels and promote sleep. Cdk1 was also identified as a wake-promoting molecule that interacts antagonistically with TARA. Given the fact that TARA regulates CycA levels, the interaction between TARA and Cdk1 may be mediated by CycA. The finding that Cdk1 and CycA also exhibit an antagonistic interaction supports this view. The previous discovery that CycE sequesters its binding partner Cdk5 to repress its kinase activity in the adult mouse brain points to a potential mechanism, namely that TARA regulates CycA levels, which in turn sequesters and inhibits Cdk1 activity. TARA and its mammalian homologs (the Trip-Br family of proteins) are known for their role in cell-cycle progression. However, recent data have shown that Trip-Br2 is involved in lipid and oxidative metabolism in adult mice, demonstrating a role beyond cell-cycle control. Other cell-cycle proteins have also been implicated in processes unrelated to the cell cycle. For example, CycE functions in the adult mouse brain to regulate learning and memory. Based on the finding that CycA and its regulator Rca1 control sleep, it was hypothesized that a network of cell-cycle genes was appropriated for sleep regulation. The current data showing that two additional cell-cycle proteins, TARA and Cdk1, control sleep and wakefulness provide support for that hypothesis. Moreover, the fact that TARA and CycA, factors identified in two independent unbiased genetic screens, interact with each other highlights the importance of a network of cell-cycle genes in sleep regulation (Afonso, 2015).

    There are two main regulatory mechanisms for sleep: the circadian mechanism that controls the timing of sleep and the homeostatic mechanism that controls the sleep amount. This study has shown that TARA has a profound effect on total sleep time. TARA also affects rhythmic locomotor behavior. Since TARA is expressed in clock cells, whereas CycA is not, it is possible that TARA plays a non-CycA dependent role in clock cells to control rhythm strength. The finding that tara mutants exhibit severely reduced sleep in constant light suggests that the effect of TARA on sleep amount is not linked to its effect on rhythmicity. Instead, TARA may have a role in the sleep homeostatic machinery, which will be examined in an ongoing investigation (Afonso, 2015).

    To fully elucidate how sleep is regulated, it is important to identify the underlying neural circuits. This study has shown that activation of the CycA-expressing neurons in the PL suppresses sleep while blocking their activity increases sleep, which establishes them as a novel wake-promoting center. Importantly, knockdown of tara and increased Cdk1 activity specifically in the PL neurons leads to decreased sleep. A simple hypothesis, consistent with the finding that both activation of PL neurons and increased Cdk1 activity in these neurons suppress sleep is that Cdk1 affects neuronal excitability and synaptic transmission. Interestingly, large-scale screens for short-sleeping mutants in fruit flies and zebrafish have identified several channel proteins such as SHAKER, REDEYE, and ETHER-A-GO-GO and channel modulators such as SLEEPLESS and WIDE AWAKE. Thus, it is plausible that Cdk1 regulates sleep by phosphorylating substrates that modulate the function of synaptic ion channels or proteins involved in synaptic vesicle fusion, as has previously been demonstrated for Cdk5 at mammalian synapses (Afonso, 2015).

    Whereas the data mapped some of TARA-€™s role in sleep regulation to a small neuronal cluster, the fact that pan-neuronal tara knockdown results in a stronger effect on sleep than specific knockdown in PL neurons suggests that TARA may act in multiple neuronal clusters. PL-specific restoration of TARA expression did not rescue the tara sleep phenotype (data not shown), further implying that the PL cluster may not be the sole anatomical locus for TARA function. Given that CycA is expressed in a few additional clusters, TARA may act in all CycA-expressing neurons including those not covered by PL-Gal4. TARA may also act in non-CycA-expressing neurons. The data demonstrate that tara knockdown using Cha-Gal4 produces as strong an effect on sleep as pan-neuronal knockdown. This finding suggests that TARA acts in cholinergic neurons, although the possibility cannot be ruled out that the Cha-Gal4 expression pattern includes some non-cholinergic cells. Taken together, these data suggest that TARA acts in PL neurons as well as unidentified clusters of cholinergic neurons to regulate sleep (Afonso, 2015).

    Based on genetic interaction studies, tara has been classified as a member of the trithorax group genes, which typically act as transcriptional coactivators. However, TARA and Trip-Br1 have been shown to up- or downregulate the activity of E2F1 transcription factor depending on the cellular context, raising the possibility that they also function as transcriptional corepressors. Interestingly, TARA physically interacts with CycA and affects CycA protein levels but not its mRNA expression. These findings suggest a novel non-transcriptional role for TARA, although an indirect transcriptional mechanism cannot be ruled out. The hypothesis that TARA plays a non-transcriptional role in regulating CycA levels and Cdk1 activity at the synapse may provide an exciting new avenue for future research (Afonso, 2015).

    Exaggerated nighttime sleep and defective sleep homeostasis in a Drosophila knock-in model of human epilepsy

    Despite an established link between epilepsy and sleep behavior, it remains unclear how specific epileptogenic mutations affect sleep and subsequently influence seizure susceptibility. Recently, a fly knock-in model of human generalized epilepsy has been established that exhibits febrile seizures plus (GEFS+), a wide-spectrum disorder characterized by fever-associated seizing in childhood and lifelong affliction. GEFS+ flies carry a disease-causing mutation in their voltage-gated sodium channel (VGSC) gene and display semidominant heat-induced seizing, likely due to reduced GABAergic inhibitory activity at high temperature. This study shows that at room temperature the GEFS+ mutation dominantly modifies sleep, with mutants exhibiting rapid sleep onset at dusk and increased nighttime sleep as compared to controls. GEFS+ mutant sleep phenotypes were more resistant to pharmacologic reduction of GABA transmission by carbamazepine (CBZ) than controls, and were mitigated by reducing GABAA receptor expression specifically in wake-promoting pigment dispersing factor (PDF) neurons. These findings are consistent with increased GABAergic transmission to PDF neurons being mainly responsible for the enhanced nighttime sleep of GEFS+ mutants. This study reveals the sleep architecture of a Drosophila VGSC mutant that harbors a human GEFS+ mutation, and provides unique insight into the relationship between sleep and epilepsy (Petruccelli, 2015).

    The circadian clock gates sleep through time-of-day dependent modulation of sleep-promoting neurons

    Sleep is under the control of homeostatic and circadian processes, which interact to determine sleep timing and duration, but the mechanisms through which the circadian system modulates sleep are largely unknown. This study used adult-specific, temporally controlled neuronal activation and inhibition to identify an interaction between the circadian clock and a novel population of sleep-promoting neurons in Drosophila. In this study, transgenic flies expressed either dTRPA1, a neuronal activator, or Shibirets1, an inhibitor of synaptic release, in small subsets of neurons. Sleep, as determined by activity monitoring and video tracking, was assessed before and after temperature-induced activation or inhibition using these effector molecules. This study compared the effect of these manipulations in control flies and in mutant flies that lacked components of the molecular circadian clock. Adult-specific activation or inhibition of a population of neurons that projects to the sleep-promoting dorsal Fan-Shaped Body resulted in bidirectional control over sleep. Interestingly, the magnitude of the sleep changes were time-of-day dependent. Activation of sleep-promoting neurons was maximally effective during the middle of the day and night, and was relatively ineffective during the day-to-night and night-to-day transitions. These time-of-day specific effects were absent in flies that lacked functional circadian clocks. In conclusion, the circadian system functions to gate sleep through active inhibition at specific times of day. These data identify a mechanism through which the circadian system prevents premature sleep onset in the late evening, when homeostatic sleep drive is high (Cavanaugh, 2015).

    Context-specific comparison of sleep acquisition systems in Drosophila

    Sleep is conserved across phyla and can be measured through electrophysiological or behavioral characteristics. The fruit fly, Drosophila melanogaster, provides an excellent model for investigating the genetic and neural mechanisms that regulate sleep. Multiple systems exist for measuring fly activity, including video analysis and single-beam (SB) or multi-beam (MB) infrared (IR)-based monitoring. This study compared multiple sleep parameters of individual flies using a custom-built video-based acquisition system, and commercially available SB- or MB-IR acquisition systems. It was found that all three monitoring systems appear sufficiently sensitive to detect changes in sleep duration associated with diet, age, and mating status. It was also demonstrated that MB-IR detection appears more sensitive than the SB-IR for detecting baseline nuances in sleep architecture, while architectural changes associated with varying life-history and environment are generally detected across all acquisition types. Finally, video recording of flies in an arena allows measurement of the effect of ambient environment on sleep. These experiments demonstrate a robust effect of arena shape and size as well as light levels on sleep duration and architecture, and highlighting the versatility of tracking-based sleep acquisition. These findings provide insight into the context-specific basis for choosing between Drosophila sleep acquisition systems, describe a novel cost-effective system for video tracking, and characterize sleep analysis using the MB-IR sleep analysis. Further, the study also describes a modified dark-place preference sleep assay using video tracking, confirming that flies prefer to sleep in dark locations (Garbe, 2015).

    Anaplastic lymphoma kinase acts in the Drosophila mushroom body to negatively regulate sleep

    Though evidence is mounting that a major function of sleep is to maintain brain plasticity and consolidate memory, little is known about the molecular pathways by which learning and sleep processes intercept. Anaplastic lymphoma kinase (Alk), the gene encoding a tyrosine receptor kinase whose inadvertent activation is the cause of many cancers, is implicated in synapse formation and cognitive functions. In particular, Alk genetically interacts with Neurofibromatosis 1 (Nf1) to regulate growth and associative learning in flies. This study shows that Alk mutants have increased sleep. Using a targeted RNAi screen the negative effects of Alk on sleep was localized to the mushroom body, a structure important for both sleep and memory. Mutations in Nf1 produce a sexually dimorphic short sleep phenotype, and suppress the long sleep phenotype of Alk. Thus Alk and Nf1 interact in both learning and sleep regulation, highlighting a common pathway in these two processes (Bai, 2015).

    Though a few studies implicate Alk orthologs in regulating behaviors such as decision-making, cognition, associative learning and addiction, most functional studies demonstrate various developmental roles for Alk. This study acutely induce a long-sleep phenotype by taking advantage of a temperature-sensitive allele, Alkts, revealing that Alk regulates sleep directly rather than through developmental processes. Mutations in Nf1, a gene encoding a GAP that regulates the Ras/ERK pathway activated by ALK, also cause a sexually dimorphic short-sleep phenotype. Thus this study establishes a novel in vivo function for both Alk and Nf1 and shows they interact with each other to regulate sleep (Bai, 2015).

    Many downstream signaling pathways have been proposed for ALK, among them Ras/ERK, JAK/STAT, PI3K and PLCγ signaling. ERK activation through another tyrosine receptor kinase Epidermal growth factor receptor (EGFR) has been linked to increased sleep, while this study shows that Alk, a positive regulator of ERK, inhibits sleep. It is noted that ERK is a common signaling pathway targeted by many factors, and may have circuit- specific effects, with different effects on sleep in different brain regions. Indeed, neural populations that mediate effects of ERK on sleep have not been identified. The dose of ALK required for ERK activation might also differ in different circuits. Region-specific effects of Alk are supported by a GAL4 screen, in which down-regulation of Alk in some brain regions even decreased sleep. The overall effect, however, is to increase sleep, evident from the pan-neuronal knockdown. It was found that the mushroom body, a site previously implicated in sleep regulation and learning, requires Alk to inhibit sleep. Interestingly, the expression patterns of Alk and Nf1 overlap extensively in the mushroom body, suggesting that they may interact here to regulate both sleep and learning. However, it was previously shown that Alk activation in the mushroom body has no effect on learning. The mushroom body expression in that study was defined with MB247 and c772, both of which also had no effects on sleep when driving Alk RNAi. The spatial requirement for Nf1 in the context of learning has been disputed in previous studies with results both for and against a function in the mushroom body. The discrepancies between these studies could result from: 1) varied expression of different drivers within lobes of the mushroom body, with some not even specific to the mushroom body; 2) variability in the effectiveness and specificity of MB-Gal80 in combination with different GAL4s. It was confirmed that the MB-Gal80 manipulation eliminated all mushroom body expression and preserved most if not all other cells with 30Y, 386Y and c309. Future work will further define the cell populations in which Alk and Nf1 interact to affect sleep (Bai, 2015).

    A substantial sleep decrease was observed in Nf1 male flies compared to control flies. However, sleep phenotypes in Nf1 female flies are inconsistent. It is unlikely that unknown mutations on the X chromosome cause the short-sleeping phenotype because 7 generation outcrosses into the control iso31 background started with swapping X chromosomes in Nf1P1 and Nf1P2 male flies with those of iso31 flies. In support of a function in sleep regulation, restoring Nf1 expression in neurons of Nf1 mutants reverses the short sleep phenotype to long sleep in both males and females. This does not result from ectopic expression of the transgene as expressing the same UAS-Nf1 transgene in wild-type flies has no effect. It was hypothesized that Nf1 promotes sleep in some brain regions and inhibits it in others, and sub-threshold levels of Nf1, driven by the transgene in the mutant background, tilt the balance towards more sleep. As reported in this study, Alk also has differential effects on sleep in different brain regions, as does protein kinase A, thus such effects are not unprecedented. Severe sleep fragmentation was also observed in Nf1 mutants, which suggests that they have trouble maintaining sleep (Bai, 2015).

    The sex-specific phenotypes of Nf1 mutants may reflect sexually dimorphic regulation of sleep. A recently published genome-wide association study of sleep in Drosophila reported that an overwhelming majority of single nucleotide polymorphisms (SNPs) exhibit some degree of sexual dimorphism: the effects of ~80% SNPs on sleep are not equal in the two sexes. Interestingly, sex was found to be a major determinant of neuronal dysfunction in human NF1 patients and Nf1 knock-out mice, resulting in differential vision loss and learning deficits. The sex-dimorphic sleep phenotype in Nf1 flies provides another model to study sex-dimorphic circuits involving Nf1. Interestingly, a prevalence of sleep disturbances have recently been reported in NF1 patient, suggesting that NF1 possibly play a conserved function in sleep regulation (Bai, 2015).

    An attractive hypothesis for a function of sleep is that plastic processes during wake lead to a net increase in synaptic strength and sleep is necessary for synaptic renormalization. There is structural evidence in Drosophila to support this synaptic homeostasis hypothesis (SHY): synapse size and number increase during wake and after sleep deprivation, and decrease after sleep. However, little is known about the molecular mechanisms by which waking experience induces changes in plasticity and sleep. FMRP, the protein encoded by the Drosophila homolog of human fragile X mental retardation gene FMR1, mediates some of the effects of sleep/wake on synapses. Loss of Fmr1 is associated with synaptic overgrowth and strengthened neurotransmission and long sleep. Overexpressing Fmr1 results in dendritic and axonal underbranching and short sleep. More importantly, overexpression of Fmr1 in specific circuits eliminates the wake-induced increases in synapse number and branching in these circuits. Thus, up-regulation of FMR accomplishes a function normally associated with sleep (Bai, 2015).

    It is hypothesized that Alk and Nf1 similarly play roles in synaptic homeostasis. They are attractive candidates for bridging sleep and plastic processes, because: 1) Alk is expressed extensively in the developing and adult CNS synapses. In particular, both Alk and Nf1 are strongly expressed in the mushroom body, a major site of plasticity in the fly brain. 2) Functionally, postsynaptic hyperactivation of Alk negatively regulates NMJ size and elaboration. In contrast, Nf1 is required presynaptically at the NMJ to suppress synapse branching. 3) Alk and Nf1 affect learning in adults and they functionally interact with each other in this process. It is tempting to speculate that in Alk mutants, sleep is increased to prune the excess synaptic growth predicted to occur in these mutants. Such a role for sleep is consistent with the SHY hypothesis. The SHY model would predict that Alk flies have higher sleep need, which is expected to enhance rebound after sleep deprivation. While the data show equivalent quantity of rebound in Alk mutants, this study found that they fall asleep faster than control flies the morning after sleep deprivation, suggesting that they have higher sleep drive. Increased sleep need following deprivation could also be reflected in greater cognitive decline, but this has not yet been tested for Alk mutants. It is noted that Nf1 mutants have reduced sleep although their NMJ phenotypes also consist of overbranched synapses. It is postulated that their sleep need is not met and thus results in learning deficits. Clearly, more work is needed to test these hypotheses concerning the roles of Alk and Nf1 in sleep, learning, and memory circuits (Bai, 2015).

    Sleep in populations of Drosophila melanogaster

    The fruit fly Drosophila melanogaster is a diurnal insect active during the day with consolidated sleep at night. Social interactions between pairs of flies have been shown to affect locomotor activity patterns, but effects on locomotion and sleep patterns have not been assessed for larger populations. This study used a commercially available locomotor activity monitor (LAM25H) system to record and analyze sleep behavior. Surprisingly, it was found that same-sex populations of flies synchronize their sleep/wake activity, resulting in a population sleep pattern, which is similar but not identical to that of isolated individuals. Like individual flies, groups of flies show circadian and homeostatic regulation of sleep, as well as sexual dimorphism in sleep pattern and sensitivity to starvation and a known sleep-disrupting mutation (amnesiac). Populations of flies, however, exhibit distinct sleep characteristics from individuals. Differences in sleep appear to be due to olfaction-dependent social interactions and change with population size and sex ratio. These data support the idea that it is possible to investigate neural mechanisms underlying the effects of population behaviors on sleep by directly looking at a large number of animals in laboratory conditions (Liu, 2015).

    Sleep homeostasis and general anesthesia: Are fruit flies well rested after emergence from Propofol?

    Shared neurophysiologic features between sleep and anesthetic-induced hypnosis indicate a potential overlap in neuronal circuitry underlying both states. The authors explored the hypothesis that propofol anesthesia also dispels sleep pressure in the fruit fly. Propofol was administered by transferring flies onto food containing various doses of propofol. High-performance liquid chromatography was used to measure the tissue concentrations of ingested propofol. To determine whether propofol anesthesia substitutes for natural sleep, the flies were subjected to 10-h sleep deprivation (SD), followed by 6-h propofol exposure, and monitored for subsequent sleep. Oral propofol treatment was shown to cause anesthesia in flies as indicated by a dose-dependent reduction in locomotor activity and increased arousal threshold. Recovery sleep in flies fed propofol after SD was delayed until after flies had emerged from anesthesia. SD was also associated with a significant increase in mortality in propofol-fed flies. Together, these data indicate that fruit flies are effectively anesthetized by ingestion of propofol and suggest that homologous molecular and neuronal targets of propofol are conserved in Drosophila. However, behavioral measurements indicate that propofol anesthesia does not satisfy the homeostatic need for sleep and may compromise the restorative properties of sleep (Gardner, 2015).

    Identification of neurons with a privileged role in sleep homeostasis in Drosophila melanogaster

    Sleep is thought to be controlled by two main processes: a circadian clock that primarily regulates sleep timing and a homeostatic mechanism that detects and responds to sleep need. Whereas abundant experimental evidence suggests that sleep need increases with time spent awake, the contributions of different brain arousal systems have not been assessed independently of each other to determine whether certain neural circuits, rather than waking per se, selectively contribute to sleep homeostasis. This study found that flies sustained thermogenetic activation of three independent neurotransmitter systems promoted nighttime wakefulness. However, only sleep deprivation resulting from activation of cholinergic neurons was sufficient to elicit subsequent homeostatic recovery sleep, as assessed by multiple behavioral criteria. In contrast, sleep deprivation resulting from activation of octopaminergic neurons suppressed homeostatic recovery sleep, indicating that wakefulness can be dissociated from accrual of sleep need. Neurons that promote sleep homeostasis were found to innervate the central brain and motor control regions of the thoracic ganglion. Blocking activity of these neurons suppressed recovery sleep but did not alter baseline sleep, further differentiating between neural control of sleep homeostasis and daily fluctuations in the sleep/wake cycle. Importantly, selective activation of wake-promoting neurons without engaging the sleep homeostat impaired subsequent short-term memory, thus providing evidence that neural circuits that regulate sleep homeostasis are important for behavioral plasticity. Together, these data suggest a neural circuit model involving distinct populations of wake-promoting neurons, some of which are involved in homeostatic control of sleep and cognition (Seidner, 2015).

    Acetylcholine from visual circuits modulates the activity of arousal neurons in Drosophila

    Drosophila melanogaster's large lateral ventral neurons (lLNvs) are part of both the circadian and sleep-arousal neuronal circuits. In the past, electrophysiological analysis revealed that lLNvs fire action potentials (APs) in bursting or tonic modes and that the proportion of neurons firing in those specific patterns varies circadianly. This study provides evidence that lLNvs fire in bursts both during the day and at night and that the frequency of bursting is what is modulated in a circadian fashion. Moreover, lLNvs AP firing is not only under cell autonomous control, but is also modulated by the network, and in the process a novel preparation was developed to assess this. lLNv bursting mode was shown to rely on a cholinergic input because application of nicotinic acetylcholine receptor antagonists impairs this firing pattern. Finally, bursting of lLNvs depends was found to depend on an input from visual circuits that includes the cholinergic L2 monopolar neurons from the lamina. This work sheds light on the physiological properties of lLNvs and on a neuronal circuit that may provide visual information to these important arousal neurons (Muraro, 2015).

    ERK phosphorylation regulates sleep and plasticity in Drosophila

    Given the relationship between sleep and plasticity, this study examined the role of Extracellular signal-regulated kinase (ERK, Rolled in Drosophila) in regulating baseline sleep, and modulating the response to waking experience. Both sleep deprivation and social enrichment increase ERK phosphorylation in wild-type flies. The effects of both sleep deprivation and social enrichment on structural plasticity in the within the Pigment Dispersing Factor (PDF)-expressing ventral lateral neurons (LNvs) can be recapitulated by expressing an active version of ERK (UAS-ERKSEM) pan-neuronally in the adult fly using GeneSwitch (Gsw) Gsw-elav-GAL4. Conversely, disrupting ERK reduces sleep and prevents both the behavioral and structural plasticity normally induced by social enrichment. Finally, using transgenic flies carrying a cAMP response Element (CRE)-luciferase reporter it was shown that activating ERK enhances CRE-Luc activity while disrupting ERK reduces it. These data suggest that ERK phosphorylation is an important mediator in transducing waking experience into sleep (Vanderheyden, 2013).

    ERK plays a key role in regulating not only cell differentiation and proliferation during development, but is also critical for regulating long-term potentiation and plasticity related events in the fully developed adult. Recent studies have highlighted the important relationship between plasticity induced by waking-experience and sleep need. With that in mind, it was hypothesized that ERK may provide a molecular link between plasticity and sleep. Since, ERK phosphorylation has been previously correlated with sleep time following rhomboid mediated activation of EGFR, this study over-expressed an active version of ERK pan-neuronally in the adult fly and found a significant increase in sleep. ERK activation increased sleep during the day, was rapidly reversible, and was associated with increased activity during waking. In contrast, disrupting ERK signaling by feeding adults the MEK inhibitor SL327 decreased daytime sleep and lowered waking activity. Together these data indicate ERK activation plays a role in sleep regulation (Vanderheyden, 2013).

    As mentioned, inducing EGFR signaling resulted in an increase in sleep which seemed to correlate nicely with the increase in ppERK. Although these data strongly suggested that the increase in sleep was due to ppERK activation, ppERK was not directly manipulated. Thus these studies confirm and extend data demonstrating that directly activating ppERK can increase sleep. While the largest effects of sleep were obtained using a pan neuronal activation of EGFR signaling, it has also been reported that the EGFR induced increase in sleep could be mapped to the PI. Surprisingly, this study did not see any changes in sleep when ppERK was expressed in the PI using the same GAL4 drivers as a previous study. These data suggest that in the PI, EGFR activation may recruit additional factors along with ppERK to alter sleep. Such regulation may be particularly important for allowing ppERK to carry out multiple functions in various circuits as needed (Vanderheyden, 2013).

    In flies, sleep homeostasis is primarily observed during the subjective day. Similarly, social enrichment also produces increases in daytime sleep. Thus, the data indicate that modulating ERK activity in the adult produces a change in sleep during the portion of the circadian day during which sleep deprivation and social enrichment modify sleep time. Interestingly, ERK activation not only increases daytime sleep, but it also results in the proliferation of terminals in the wake-promoting LNvs. The ability of ERK activation to increase synaptic terminals is reminiscent of the change in synaptic markers and structural morphology that are independently observed following sleep deprivation and social enrichment. Interestingly, arouser mutants show both enhanced ethanol sensitivity and an increase in terminals from the LNvs through its activation by EGFR/ERK. Given that a well characterized function of ERK is to regulate synaptic morphology, the current results suggest that ERK activation may be a common mechanism linking waking experience, plasticity and sleep (Vanderheyden, 2013).

    As mentioned, ERK activation has been correlated with sleep time following rhomboid mediated activation of EGFR. Interestingly, in that study, ppERK was not detectable in cell bodies following rho mediated increases in sleep suggesting that, during rho activation, ppERK might be modifying sleep at the level of translation initiation. The current data extend these observations and provide genetic evidence that ERK activation may also play a role in regulating sleep and plasticity by activating gene transcription. That is, pan-neuronal expression of RSK, which retains ERK in the cytoplasm and prevents its nuclear translocation, results in a decrease in daytime sleep similar to that observed in wild type flies fed SL327. Although previous studies have established a link between CREB and sleep this study evaluated CRE-Luc solely as a reporter of transcriptional activation. The data indicates that the expression of UAS-ERKSEM increased CRE-Luc activity. In contrast, transgenic CRE-Luc reporter flies show reduced bioluminescence when crossed into a rl10a mutant background. Finally, flies fed SL327 also showed a reduction of CRE-Luc activity. These data are consistent with a recent report demonstrating that activating MEK increases bioluminescence in flies carrying a CRE-Luc reporter. Together these data suggest that ERK activation may alter plasticity and sleep, in part, by activating gene transcription (Vanderheyden, 2013).

    Interestingly, expressing UAS-ERKSEM in PDF neurons does not change the number of PDF-terminals and does not alter sleep time. This is in contrast to the effects of expressing UAS-ERKSEM pan-neuronally which increases both the number of PDF-terminals and increases sleep. These data suggest that ERK activation can either influence PDF neurons in a non-cell autonomous fashion or that ERK activation is required in multiple circuits to modulate plasticity. Indeed, it has been recently shown that increasing sleep by activating the dorsal Fan Shaped Body significantly reduces the number of PDF-terminals. Thus, PDF terminal number provides an accessible read-out of brain plasticity that can be used to elucidate molecular mechanisms linking sleep and plasticity at the circuit level (Vanderheyden, 2013).

    It is important to note that in flies there is a critical window of adult development that can influence sleep and learning. For example, 0-3 day old rut2080 mutants are able to respond to social enrichment with an increase in sleep but their older siblings (>3days) cannot. In other words, rut2080 mutants can exhibit higher or lower amounts of sleep as adults depending upon environmental context, not levels of rutabaga per se. Indeed, rutabaga mutants have been reported to have significant variations in sleep (both longer and shorter) compared to controls. Given that the environment can stably modify sleep during adult development, even in the absence of memory related genes, care must be taken when classifying a mutant as either long or short sleepers. It should be emphasize that the current experiments were designed to avoid making manipulations during this critical time window to avoid such confounds. However, it remains possible that ERK may modify sleep by activating additional downstream targets and/or by regulating translation initiation at the synapse (Vanderheyden, 2013).

    Recent studies have shown that waking experience, including both prolonged wakefulness and exposure to enriched environments, independently produce dramatic increases in both synaptic markers and structural morphology throughout the fly brain and that these changes are reversed during sleep. To date, most studies have evaluated mutations that disrupt synaptic plasticity to identify the molecular mechanisms linking sleep with plasticity. Given that ERK is a key molecule for the regulation of synaptic plasticity and long-term potentiation, this study evaluated its ability to alter both sleep and structural plasticity. The data indicate that both sleep deprivation and social enrichment independently increase ERK phosphorylation in wild-type flies. It is also reported that expressing an active version of ERK (UAS-ERKSEM) in the adult fly results in an increase in sleep and an increase in structural plasticity in the LNvs. These data suggest that ERK phosphorylation is an important mediator in transducing waking experience into sleep (Vanderheyden, 2013).

    Lowered insulin signalling ameliorates age-related sleep fragmentation in Drosophila

    Sleep fragmentation, particularly reduced and interrupted night sleep, impairs the quality of life of older people. Strikingly similar declines in sleep quality are seen during ageing in laboratory animals, including the fruit fly Drosophila. This study investigated whether reduced activity of the nutrient- and stress-sensing insulin/insulin-like growth factor (IIS)/TOR signalling network, which ameliorates ageing in diverse organisms, could rescue the sleep fragmentation of ageing Drosophila. Lowered IIS/TOR network activity improved sleep quality, with increased night sleep and day activity and reduced sleep fragmentation. Reduced TOR activity, even when started for the first time late in life, improved sleep quality. The effects of reduced IIS/TOR network activity on day and night phenotypes were mediated through distinct mechanisms: Day activity was induced by adipokinetic hormone, dFOXO, and enhanced octopaminergic signalling. In contrast, night sleep duration and consolidation were dependent on reduced S6K and dopaminergic signalling. These findings highlight the importance of different IIS/TOR components as potential therapeutic targets for pharmacological treatment of age-related sleep fragmentation in humans (Metaxakis, 2014).

    Sleep syndromes are highly prevalent in elderly humans and, with a continuing increase in life expectancy and a greater proportion of elderly people worldwide, effective treatments with fewer side effects are becoming increasingly needed. Sleep in flies shares striking similarities with sleep in humans, including an age-related reduction in sleep quality. This study used Drosophila to examine age-related sleep pathologies and to suppress these pathologies through genetic and pharmacological perturbation of insulin/IGF and TOR signaling (Metaxakis, 2014).

    This study has shown that the highly conserved IIS pathway, with roles in growth and development, metabolism, fecundity, stress resistance, and lifespan, also affects sleep patterns in Drosophila. Reduced IIS increases and consolidates night sleep, while decreasing day sleep and inducing day activity. Interestingly, dilp2-3 double mutant flies as well as flies with neuron or fat-body-specific down-regulation of IIS showed no obvious or only mild sleep phenotypes in a previous study, suggesting that a strong and/or systemic reduction in IIS activity may be necessary to induce the activity and sleep phenotypes. Consistently, dilp2-3 double mutant flies have very mild growth, lifespan, and metabolic phenotypes compared to the dilp2-3,5 triple mutant flies used in this study. Reduced IIS activity resulted in increased sleep consolidation in young flies. Importantly, reduced IIS ameliorated the age-related decline in sleep consolidation seen in wild-type flies, thus showing that it is malleable. Contrary to the increased sleep consolidation with reduced IIS, high calorie diets have been reported to accelerate sleep fragmentation. Furthermore, dietary sugar affects sleep pattern in flies. Taken together, these findings reveal a role of nutrition and metabolism in sleep regulation and age-related sleep decline in flies (Metaxakis, 2014).

    In humans, several studies suggest a link between nutrition and sleep. The amino acid tryptophan can promote sleep, possibly by affecting synthesis of the sleep regulators serotonin and melatonin. Also, the carbohydrate/fat content of the diet seemingly affects sleep parameters. However, most of these studies are based on correlational methods and small sample size, and it is not yet clear how diet affects sleep. Interestingly, sleep duration can affect metabolism, risk for obesity and diabetes, and even food preference. These findings associate sleep and metabolism; thus, manipulation of nutrient-sensing pathways, such as IIS and TOR signalling, may affect activity and sleep in humans (Metaxakis, 2014).

    The transcription factor FoxO is an important downstream mediator of IIS. In C. elegans all aspects of IIS are dependent on daf-16, the worm ortholog of foxO. In contrast, in Drosophila IIS-mediated lifespan extension is dependent on dfoxo, whereas several phenotypes of reduced IIS are dfoxo-independent. Activity and sleep were unaffected by the loss of dfoxo in wild-type flies. In contrast, under low IIS conditions loss of dfoxo specifically affected daytime behaviour, with night time behaviours unaffected. Reduced IIS therefore affects day and night sleep and activity through distinct mechanisms. It also uncouples the effects of IIS on lifespan and on night sleep consolidation, since dfoxo is essential for extended longevity of flies with reduced IIS. dFOXO has been previously shown to increase neuronal excitability, possibly via transcription of ion channel subunits or other (Metaxakis, 2014).

    It is suggested that a possible such regulator could be octopaminergic signalling, known to promote arousal in Drosophila. Octopamine, the arthropod equivalent of noradrenaline, regulates several behavioural/physiological processes, including glycogenolysis and fat metabolism, as well as synaptic and behavioural plasticity. Moreover, octopamine can affect sleep by acting on insulin-producing cells in the fly brain, thus linking IIS and sleep/activity. Indeed, this study found that IIS mutants have increased octopamine levels and, importantly, pharmacological inhibition of octopaminergic signalling reverted the increased day activity of IIS mutants. Noteworthy, mRNA expression of octopamine biosynthetic enzymes was not changed, but tyramine levels were significantly reduced, suggesting that increased translation, reduced degradation, or increased activity of the tyramine-β hydroxylase regulates octopamine levels in IIS mutants. In contrast to day activity, increased lifespan of IIS mutants was not affected by pharmacological inhibition of octopaminergic signalling, thus separating longevity from the day activity phenotype (Metaxakis, 2014).

    The effect of reduced IIS on day sleep/activity was mediated through AKH, the equivalent of human glucagon, an antagonist of. In flies, AKH coordinates the response to hunger through mobilizing energy stores and increasing food intake, as well as inducing a starvation-like hyperactivity. Loss of AKH receptor (AkhR) abrogated the increased activity of IIS mutants without affecting night sleep. These results demonstrate that day and night phenotypes of IIS mutants can be uncoupled, suggesting that the increased night sleep of IIS mutants is not just a compensatory consequence of increased day activity (Metaxakis, 2014).

    dilp2-3,5 mutants have increased octopamine levels, and loss of AkhR in the dilp2-3,5 mutant background reduced their octopamine level back to wild-type levels, suggesting that AkhR-mediated regulation of octopamine controls day hyperactivity in IIS mutants. In support of these findings, octopaminergic cells mediate the increased activity effect of AKH in other insects. Flies lacking dFOXO did not respond to chemically induced AKH release, suggesting that AKH affects activity through dFOXO. Therefore, it is suggested that dFOXO and AkhR act through overlapping mechanisms to enhance octopaminergic signalling and induce activity (Metaxakis, 2014).

    In flies, AkhR is highly expressed in fat body and its loss alters lipid and carbohydrate store levels. Therefore, AkhR might indirectly enhance octopaminergic signalling through alterations in lipid and carbohydrate metabolism. In support of this idea, lipid metabolism affects sleep homeostasis in flies. Additionally, AkhR expression in octopaminergic cells could regulate octopamine synthesis and release in flies. Interestingly, expression of AkhR is altered in dfoxo mutants, thus implicating dFOXO in AkhR regulation. Both are highly expressed in fat body, an important organ for metabolism in flies, and fat-body-specific insulin receptor may regulate AkhR function through dFOXO activation (Metaxakis, 2014).

    In larval motor neurons, dFOXO increases neuronal excitability and octopamine increases glutamate release, suggesting there is at least a spatial functional link between the two. Thus, together with a possible role in AkhR synthesis, dFOXO could act downstream of octopamine to increase activity (Metaxakis, 2014).

    To determine the mechanism underlying the IIS-dependent amelioration of age-related sleep decline, downstream components and genetic interactors of IIS were investigated. One such interactor that affects health and ageing is TORC1. TORC1 is a major regulator of translation, through S6K, 4E-BP, and of autophagy, through ATG1. Inhibiting TOR signalling, and thus translation, by rapamycin treatment in wild-type flies recapitulated the sleep features of IIS mutants, even in old flies. This rescue of sleep quality was blocked by ubiquitous expression of activated S6K, suggesting that reduced S6K activity is required for the rescue. These findings, together with previous results showing S6K to regulate hunger-driven behaviours, highlight the importance of S6K as a regulator of behaviour in flies. Thus, manipulating TOR signalling can improve sleep quality through S6K (Metaxakis, 2014).

    In mammals, rapamycin treatment has beneficial effects on behaviour throughout lifespan. Although complete block of TOR activity is detrimental for long-term memory, a moderate decrease through rapamycin treatment can improve cognitive function, abrogate age-related cognitive deterioration, and reduce anxiety and depression. Moreover, increased TOR activity throughout development is detrimental for neuronal plasticity and memory. In flies, rapamycin prevents dopaminergic neuron loss in mutants with parkinsonism. Although the role of TOR in brain function has not been well studied in flies, the advantageous effect of rapamycin in both mammalian brain function and sleep in flies may be mediated through common neurophysiological mechanisms (Metaxakis, 2014).

    Gene expression studies have suggested that protein synthesis is up-regulated during sleep, which may be an essential stage in macromolecular biosynthesis. Consistent with this, inhibiting protein synthesis in specific brain domains prolongs sleep duration in mammals, suggesting that sleep is maintained until specific levels of biosynthesis occur and aids in explaining the ubiquitously conserved need for sleep. Brief cycloheximide treatment has been shown to prolong night sleep and increased consolidation in flies, indicating an evolutionarily conserved role for protein synthesis inhibition on sleep regulation. Contrary to reduced IIS, cycloheximide reduced day activity, possibly due to the global effect of cycloheximide on protein synthesis or due to toxic defects in flies' physiology. Decreased protein synthesis rates may enhance the necessity for increased sleep duration, to allow sufficient synthesis of proteins and other macromolecules during sleep, allowing organisms to be healthy and functional during the day (Metaxakis, 2014).

    Alternatively, the effect of protein synthesis inhibition on night sleep could be the result of reduced expression of specific sleep regulators. This study found that DopR1 and dilp2-3,5 mutants share night phenotypes and that rapamycin did not affect sleep of DopR1 mutants, suggesting that TOR acts on dopaminergic signalling to affect night sleep. Reduced IIS elevated expression of DopR1, independently of dFOXO, in accordance with data from mammals. This effect may be feedback caused by down-regulation of dopaminergic signalling in IIS mutants, although not through direct regulation of DopR. Under normal physiological conditions, dopamine signalling is determined by the level of extracellular dopamine and the rate of DAT-mediated dopamine clearance from the synaptic cleft. The rate of dopamine clearance is dependent on the turnover rate of DAT and the number of functional transporters at the plasma membrane. This study found that reduced IIS and rapamycin treatment induced increased expression of DAT, suggesting an increased rate of dopamine clearance from the synaptic cleft, and thus a reduction in the amplitude of dopamine signalling, without changes in total dopamine levels. DAT function and IIS have recently been linked in mammals. DAT function increases upon insulin stimulation and is diminished on insulin depletion, through alterations in DAT membrane localization. However, IIS-dependent regulation of DAT subcellular localization in Drosophila has not yet been demonstrated. The current data suggest down-regulating dopaminergic signalling, either by loss of DopR1 or increasing DAT levels, is beneficial for sleep quality. In agreement with this it was shown that artificially increasing dopaminergic signalling, through short-term methamphetamine treatment, increases both day and night activity and reduces night sleep, and reverts the beneficial effect of reduced IIS on night behaviours. In mammals, cocaine administration, which enhances dopaminergic signalling, increases TOR activity. Also, rapamycin blocks cocaine-induced locomotor sensitization. Interestingly, cocaine stimulates S6K phosphorylation in rat brains, and this effect is blocked by rapamycin. Taken together, these results show that in flies and mammals dopaminergic and IIS/TOR signalling may interact in similar ways (Metaxakis, 2014).

    In conclusion, reduced IIS extends lifespan in diverse organisms. This study has have shown that it can also ameliorate age-related sleep fragmentation, but that the mechanisms by which it does so are distinct from those by which it extends lifespan. Reduced IIS affected day activity and sleep phenotypes through increased octopaminergic signalling, but enhanced octopaminergic signalling did not increase lifespan. Similarly, in Drosophila increased lifespan from reduced IIS requires dfoxo, but the night sleep phenotypes of IIS mutants were independent of this transcription factor. Reduced IIS thus acts through multiple pathways to ameliorate different aspects of loss of function during ageing. IIS links metabolism and behaviour through its components, such as S6K and dFOXO, which act through different neuronal circuits and neurons to affect sleep. The strong evolutionary conservation of these circuits and their functions suggests that pharmacological manipulation of IIS effectors could be beneficial in treatments of sleep syndromes in humans (Metaxakis, 2014).

    Ca-alpha1T, a fly T-type Ca(2+) channel, negatively modulates sleep

    Mammalian T-type Ca(2+) channels are encoded by three separate genes (Cav3.1, 3.2, 3.3). These channels are reported to be sleep stabilizers important in the generation of the delta rhythms of deep sleep, but controversy remains. The identification of precise physiological functions for the T-type channels has been hindered, at least in part, by the potential for compensation between the products of these three genes and a lack of specific pharmacological inhibitors. Invertebrates have only one T-type channel gene, but its functions are even less well-studied. Ca2+-channel protein α1 subunit T (Ca-alpha1T), the only Cav3 channel gene in Drosophila melanogaster, was cloned and expressed in Xenopus oocytes and HEK-293 cells and was confirmed to pass typical T-type currents. Voltage-clamp analysis revealed the biophysical properties of Ca-alpha1T show mixed similarity, sometimes falling closer to Cav3.1, sometimes to Cav3.2, and sometimes to Cav3.3. Ca-alpha1T was found to be broadly expressed across the adult fly brain in a pattern vaguely reminiscent of mammalian T-type channels. In addition, flies lacking Ca-alpha1T show an abnormal increase in sleep duration most pronounced during subjective day under continuous dark conditions despite normal oscillations of the circadian clock. Thus, this study suggests invertebrate T-type Ca(2+) channels promote wakefulness rather than stabilizing sleep (Jeong, 2015).

    T-type Ca2+ channels are a subfamily of voltage-dependent Ca2+ channels (VDCCs) that produce low-voltage-activated (LVA) Ca2+ currents implicated in NREM sleep in mammals. Three different genes encode the pore-forming alpha subunits of mammalian T-type channels, Cav3.1, 3.2, and 3.3. Of these, Cav3.1 and 3.3 are highly expressed in the thalamus, where the oscillations required for NREM sleep are generated. Mice lacking Cav3.1 show reduced delta-wave activity and reduced sleep stability, suggesting that mammalian T-type currents have a sleep-promoting or stabilizing function (Jeong, 2015).

    Unlike mammals, Drosophila melanogaster has only one T-type Ca2+ channel, Ca-α1T, which is also known as DmαG. A recent study found that motor neurons in flies lacking Ca-α1T show reduced LVA but also reduced high-voltage-activated (HVA) Ca2+ currents, suggesting that although Ca-α1T seems to be a genuine T-type channel, it may have interesting biophysical properties. Therefore a single isoform of Ca-α1T was cloned, it was expressed in Xenopus oocytes or HEK-293 cells, and its biophysical properties were compared with those of the rat T-type channel Cav3.1. Several Ca-α1T mutant alleles were generated, and a defect was generated in their sleep/wake cycles. Contrary to results in mammals, the fly T-type Ca2+ channel destabilizes sleep. It is anticipated that these findings will help clarify species-dependent differences in the in vivo functions of T-type Ca2+ channels, particularly their role in sleep physiology (Jeong, 2015).

    Ca-α1T is the largest T-type channel cloned to date, measuring 3205 amino acids. Electrophysiological characterization of Ca-α1T in Xenopus oocytes showed that Ca-α1T has the hallmark properties of a T-type channel: low-threshold activation at around -60 mV, a maximal current output at -20 mV, transient current kinetics elicited by a step-pulse protocol producing a “criss-crossing” pattern, and slow deactivation of tail currents. These biophysical properties are also consistent with previous studies that implicated Ca-α1T in low-voltage-activated (LVA) currents in both the central and peripheral nervous systems of the fly (Jeong, 2015 and references therein).

    Mammalian genomes contain three T-type Ca2+ channel genes (i.e., Cav3.1-3.3), while the fly genome contains only one. Therefore Ca-α1T was measured for some of the characteristics that distinguish the three mammalian channels. In terms of current kinetics, Ca-α1T is more similar to mammalian Cav3.1 and Cav3.2 than Cav3.3, which exhibits considerably slower kinetics. In terms of both its relative permeability to Ba2+ over Ca2+ and its sensitivity to nickel inhibition, Ca-α1T is most similar to Cav3.2 (Jeong, 2015).

    The three mammalian T-type Ca2+ channels, each with their own distinct biophysical properties, are expressed in largely complementary patterns of neurons throughout the brain, conferring considerable functional diversity. Areas of particularly strong expression include those important for the gating and processing of sensory inputs, motor control, learning and memory, as well as reward circuits (Talley, 1999). Using a GFP-tagged knock-in allele, this study reports that Ca-α1T is expressed broadly across the adult fly brain in structures reminiscent of the mammalian T-type Ca2+ channels. These include sensory neuropils (i.e., the optic and antennal lobes, the antennal mechanosensory and motor centers, the anterior ventrolateral protocerebrum, and the subesophageal zone), motor-associated neuropils (i.e., the central complex), and those associated with learning, memory, and reward (i.e., the mushroom bodies). It is still unclear, however, whether the different isoforms predicted to originate from the Ca-α1T locus will have different biophysical properties or different distributions around the brain (Jeong, 2015).

    Considering their broad expression, T-type knockout mice appear healthy and subtle mutant phenotypes emerge only upon close inspection. Sleep, in particular, has become a focal point in the search for a physiological function for the T-type channels. Mammalian T-type Ca2+ channels may act as sleep stabilizers and may help generate the burst firing necessary for the sleep oscillations of deep NREM sleep. Unfortunately, the three separate mammalian T-type genes all undergo alternative splicing to produce various channel isoforms that each have specific biophysical properties, neuroanatomical and subcellular localizations, and varying abilities to interact with other ion channels. All these variables and more combine to make it difficult if not impossible to define a precise physiological role in sleep for T-type channels as a group. Although Cav3.1 knockout mice lack the delta oscillations characteristic of deep sleep and show reduced total sleep, when the knockout is limited to the rostral midline thalamus, sleep is still reduced, but delta waves are mildly increased. Another more recent study showed that treatment with the T-type-specific channel blocker TTA-A2 enhances sleep and delta rhythms in wild type mice but not Cav3.1/Cav3.3 double knockout mice. In other words, manipulation of T-type channels can both enhance and reduce total sleep and deep delta-wave sleep depending on the experimental context (Jeong, 2015).

    Although perhaps an underestimate of the actual complexity of the situation, the subtlety of the phenotypes of the homozygous viable Cav3 mutant mice are often ascribed to functional compensation among the various Cav3.1-3 isoforms. It was therefore expected that a behavioral investigation of the sole fly T-type channel, Ca-α1T, would uncover less subtle sleep phenotypes. It was thus surprising to find, that despite its broad and relatively strong expression across adult fly brains, Ca-α1T-null mutants, like the Cav3.1-null mice, are homozygous viable and lack any overt phenotypes. Upon closer examination, however, it was observed that Ca-α1T-null mutants sleep more than controls, especially in constant darkness (Jeong, 2015).

    The reason for this relative specificity in the sleep phenotype caused by Ca-α1T loss-of-function to constant darkness is still unclear. Flies exhibit a burst of activity upon exposure to the early morning light but then sleep through most of the rest of the day. Since control flies show less sleep during subjective daytime under continuous darkness than under the light phase of light-dark conditions, it is clear that light exposure can also have sleep-promoting effects. Through a series of imaging experiments, Although dopamine (DA) is potently wake-promoting, light exposure can suppress this action of DA at least partly by causing the up-regulation of the inhibitory DA receptor D2R in PDF neurons, which are themselves wake-promoting. This modulation of the wake-promoting PDF neurons by light may help explain why the Ca-α1T loss-of-function phenotype is biased toward continuous dark conditions if Ca-α1T functions downstream of the PDF neurons. It would mean the responsible Ca-α1T-positive neurons are also modulated by light (Jeong, 2015 and references therein).

    It was possibe to replicate the increased sleep phenotype of Ca-α1T-null mutants via pan-neuronal knock-down of Ca-α1T, but it was not possible to further narrow the cause of this phenotype to a more specific neuronal subpopulation. This was in spite of numerous attempts with neuronal Gal4 driver lines ranging from broadly expressed enhancer traps and neurotransmitter Gal4 drivers to much more narrowly expressed neuropeptide drivers. This difficulty suggests Ca-α1T may function in novel sleep circuits (Jeong, 2015).

    In addition to their sleep phenotype, Ca-α1T-null mutants also have a circadian phenotype: an elongated circadian period and a reduction in rhythmic power. It is difficult to say, however, whether these altered circadian parameters are independent of or secondary to the sleep phenotype. Rhythmic power is proportional to the magnitude of the changes in activity level and the regularity with which they occur. Since the increased sleep observed in the Ca-α1T-null mutants does reduce the change in overall activity level between subjective day and subjective night, the increased sleep must also cause a reduction in rhythmic power (Jeong, 2015).

    The length of time animals spend sleeping is controlled by both the circadian clock and by a homeostatic drive to sleep that is proportional to time spent awake. Thus, most 'sleep mutants' described so far have had defects in one or the other—they are either circadian sleep mutants or homeostatic sleep mutants. After 24 hours of mechanically-induced sleep deprivation, it was observed that Ca-α1T-null mutants regain slightly more of their lost sleep than control flies, although the increase was not statistically significant. This suggests that, in addition to their circadian phenotype, Ca-α1T-null mutants may also have a slightly stronger homeostatic drive to sleep than controls. Although neither the circadian phenotype nor the homeostatic phenotype are particularly strong, together they produce a robust increase in sleep (Jeong, 2015).

    The 'three channel' compensation hypothesis in mice may yet turn out to be correct, but the current results in flies suggest that other factors -- isoform-specific differences, differences related to protein-protein interactions, or even something completely unforeseen -- may allow mice and flies lacking these broadly expressed and highly conserved ion channels to still function remarkably well. It will be interesting to see whether future studies focused on the technically demanding study of isoform-specific expression patterns and isoform-specific rescues in both mice and flies will clarify how T-type channels can at various times and in various contexts both enhance and reduce sleep (Jeong, 2015).

    Oh, Y., Jang, D., Sonn, J. Y. and Choe, J. (2013). Histamine-HisCl1 receptor axis regulates wake-promoting signals in Drosophila melanogaster. PLoS One 8: e68269. PubMed ID: 23844178

    Histamine-HisCl1 receptor axis regulates wake-promoting signals in Drosophila melanogaster

    Histamine and its two receptors, Histamine-gated chloride channel subunit 1 (HisCl1) and Ora transientless (Ort), are known to control photoreception and temperature sensing in Drosophila. However, histamine signaling in the context of neural circuitry for sleep-wake behaviors has not yet been examined in detail. Mutant flies were obtained with compromised or enhanced histamine signaling, and their baseline sleep was tested. Hypomorphic mutations in histidine decarboxylase (HDC), an enzyme catalyzing the conversion from histidine to histamine, caused an increase in sleep duration. Interestingly, hisCl1 mutants but not ort mutants showed long-sleep phenotypes similar to those in hdc mutants. Increased sleep duration in hisCl1 mutants was rescued by overexpressing hisCl1 in circadian pacemaker neurons expressing a neuropeptide pigment dispersing factor (PDF). Consistently, RNA interference (RNAi)-mediated depletion of hisCl1 in PDF neurons was sufficient to mimic hisCl1 mutant phenotypes, suggesting that PDF neurons are crucial for sleep regulation by the histamine-HisCl1 signaling. Finally, either hisCl1 mutation or genetic ablation of PDF neurons dampened wake-promoting effects of elevated histamine signaling via direct histamine administration. Taken together, these data clearly demonstrate that the histamine-HisCl1 receptor axis can activate and maintain the wake state in Drosophila and that wake-activating signals may travel via the PDF neurons (Oh, 2013).

    Using genetic and pharmacological methods to manipulate histamine signaling, this study shows that the HisCl1 receptor and its downstream signaling cascade regulate wake-evoking behavior in Drosophila, while Ort receptor does not show any sleep/wake regulatory function. Histamine promotes activity via the HisCl1 receptor. Reduced histamine in HDC mutants or loss of the HisCl1 receptor both show reduced activity and enhanced sleep. Additionally, the relevant signaling pathway downstream of the HisCl1 receptor may function in the PDF neurons. Finally, it was demonstrated that the histamine-HisCl1 receptor axis can activate and maintain wakefulness in PDF neurons (Oh, 2013).

    These data show the complete functional segregation of the two histamine receptors for the first time. Ort receptor is expressed in large monopolar cells (LMC), postsynaptic to photoreceptors in the lamina and is a major target of photoreceptor synaptic transmission in Drosophila. In contrast to Ort, HisCl1 receptor is not expressed in postsynaptic neurons of photoreceptors. It is expressed in lamina glia and shapes the LMC postsynaptic response of Ort signaling. Both Ort and HisCl1 receptor are involved in temperature-preference behaviors, but the major independent function of HisCl1 receptor remains elusive. This study showed that sleep regulation is a novel and independent function of HisCl1 receptor. Additionally, this finding is an important clue in understanding the functional evolution of the two histamine receptors in Drosophila (Oh, 2013).

    It is proposed that wake-activation by histamine signaling in Drosophila is similar to that found in mammals. hdc mutant flies have increased sleep durations compared to controls and a previous study showed that HDC-knockout mice have increased paradoxical sleep compared to controls. This suggests that the HDC enzyme has a common wake-promoting function in mammals and flies. However, the structures of histamine receptors differ between flies and mammals; the histamine receptors of Drosophila are histamine-gated chloride channels, whereas the mammalian histamine receptors belong to the rhodopsin-like G-protein-coupled receptor family. Currently, researchers are working to identify a metabotropic histamine receptor in Drosophila. Despite the structural differences of the mammalian and Drosophila receptors, they share a wake-activating function. This functional homology may be the result of evolution and provides a hint to find out the metabotropic histamine receptors in Drosophila (Oh, 2013).

    Surprisingly, functional conservations between flies and mammals are also found among the histamine receptor subtypes. The HisCl1 receptor has a wake-activating role, whereas the Ort receptor does not. This result parallels differences in the wake-activation roles of the H1 and H2 receptors in mammals: the H1 receptor can activate wakefulness, but the H2 receptor cannot. Thus, the data provide a more detailed understanding of the potential functional relationship between the HisCl1 and H1 receptors. A functional connection between the Ort receptor and the H2 receptor is also possible, since the two have little effect on sleep/wake regulation in their corresponding model systems. No auto-receptor of histamine has yet been found in Drosophila, suggesting that there may not be a Drosophila homolog for the mammalian H3 receptor. Further research should shed greater light on the evolutionary relationship between the histamine receptors of flies and mammals (Oh, 2013).

    Histamine signaling modulates the maintenance of wakefulness and controls light sensing, and it is speculated that a number of interactions are possible between these two different pathways. Previous studies on light-perception mechanisms showed that histamine mutants exhibit light-sensing defects. However, this study found that the sleep duration was increased in histamine signaling mutants compared to wild-type flies in constant darkness. Thus, the perception of light in the context of evoking wakefulness is independent of vision-related light perception in Drosophila. Further research will be required to definitively establish the relationship between light perception and sleep regulation (Oh, 2013).

    Previous studies revealed that the PDF neurons promote wakefulness in Drosophila. The current findings show that histamine signaling acts as a wake-promoting pathway in PDF neurons. The HisCl1 receptor is a chloride channel, which would be expected to inhibit the function of the neurons. However, since previous studies showed that chloride channels can activate the function of the neurons, hence the HisCl1 receptor might be an activator of the PDF neurons. The downstream signaling of histamine-HisCl1 receptor in PDF neurons should be further studied using genetic manipulation and electro-physiological methods (Oh, 2013).

    Orexin is a neuropeptide that acts as an important wake-activating neurotransmitter in mammals, as shown by the demonstration that defects in orexin synthesis can cause narcoleptic symptoms in human and animals. Orexin neurons activate wakefulness in the lateral hypothalamic area and the feedback loop between orexin neurons and monoaminergic neurons such as histaminergic and serotonergic neurons (tuberomammillary nucleus, TMN, and dorsal raphe nucleus, DR) controls wakefulness in the hypothalamus and the brain stem. Histamine receptors are essential for wake-activation by orexin treatment, indicating that orexin and histamine signaling constitute an interactive wake-activating system in mammals. However, orexin has not been found in Drosophila. A previous study suggested that the PDF neuropeptide functions similar to those of orexin in Drosophila (Parisky, 2008), potentially explaining many aspects of the wake-activation cascade in Drosophila. Histamine and orexin have similar wake-activating function, but mammalian histamine mutants do not show narcoleptic symptoms. This study has shown that histamine and one of its receptors, HisCl1, constitute an important wake-evoking axis in Drosophila. Moreover, it was demonstrated that histamine-signaling mutants cannot maintain wakefulness during the daytime, which is similar to the phenotype of orexin mutants in mammals. Hence, it is proposed that, in Drosophila, histamine may have a function similar to that of the mammalian orexin. Further research is required to establish the functional relationship between wake activation of histamine signaling in Drosophila and wake-promoting function of orexin and histaminergic system in mammals (Oh, 2013).

    Social experience is sufficient to modulate sleep need of Drosophila without increasing wakefulness

    Fruit flies exposed to highly enriched social environment are found to show increased synaptic connections and a corresponding increase in sleep. This study investigated if social environment comprising a pair of same-sex individuals could enhance sleep in the participating individuals. Same-sex pairs were maintained for a period of 1 to 4 days, and after separation, sleep of the previously socialized and solitary individuals was monitored under similar conditions. Males maintained in pairs for 3 or more days were found to sleep significantly more during daytime and showed a tendency to fall asleep sooner as compared to solitary controls (both measures together are henceforth referred to as "sleep-enhancement"). Sleep-enhancement was found to occur without any significant increase in wakefulness. Furthermore, while sleep-enhancement due to group-wise social interaction requires Pigment Dispersing Factor (PDF) positive neurons; PDF positive and Cryptochrome (CRY) positive circadian clock neurons and the core circadian clock genes are not required for sleep-enhancement to occur when males interact in pairs. Pair-wise social interaction mediated sleep-enhancement requires dopamine and olfactory signaling, while visual and gustatory signaling systems seem to be dispensable. These results suggest that socialization alone (without any change in wakefulness) is sufficient to cause sleep-enhancement in fruit fly D. melanogaster males, and that its neuronal control is context-specific (Lone, 2015). 

    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 was 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).

    Operation of a homeostatic sleep switch

    In Drosophila, a crucial component of the machinery for sleep homeostasis is a cluster of neurons innervating the dorsal fan-shaped body (dFB) of the central complex. dFB neurons in sleep-deprived flies tend to be electrically active, with high input resistances and long membrane time constants, while neurons in rested flies tend to be electrically silent. This study demonstrates state switching by dFB neurons, identifies dopamine as a neuromodulator that operates the switch, and delineates the switching mechanism. Arousing dopamine causes transient hyperpolarization of dFB neurons within tens of milliseconds and lasting excitability suppression within minutes. Both effects are transduced by Dop1R2 receptors and mediated by potassium conductances. The switch to electrical silence involves the downregulation of voltage-gated A-type currents carried by Shaker and Shab, and the upregulation of voltage-independent leak currents through a two-pore-domain potassium channel that was termed Sandman. Sandman is encoded by the CG8713 gene and translocates to the plasma membrane in response to dopamine. dFB-restricted interference with the expression of Shaker or Sandman decreases or increases sleep, respectively, by slowing the repetitive discharge of dFB neurons in the ON state or blocking their entry into the OFF state. Biophysical changes in a small population of neurons are thus linked to the control of sleep-wake state (Pimentel, 2016).

    Recordings were made from dFB neurons (which were marked by R23E10-GAL4 or R23E10-lexA-driven green fluorescent protein (GFP) expression) while head-fixed flies walked or rested on a spherical treadmill. Because inactivity is a necessary correlate but insufficient proof of sleep, the analysis was restricted to awakening, which is defined as a locomotor bout after >5 min of rest, during which the recorded dFB neuron had been persistently spiking. To deliver wake-promoting signals, the optogenetic actuator CsChrimson was expressed under TH-GAL4 control in the majority of dopaminergic neurons, including the PPL1 and PPM3 clusters, whose fan-shaped body (FB)-projecting members have been implicated in sleep control. Illumination at 630 nm, sustained for 1.5 s to release a bolus of dopamine, effectively stimulated locomotion. dFB neurons paused in successful (but not in unsuccessful) trials, and their membrane potentials dipped by 2-13 mV below the baseline during tonic activity. When flies bearing an undriven CsChrimson transgene were photostimulated, neither physiological nor behavioural changes were apparent. The tight correlation between the suppression of dFB neuron spiking and the initiation of movement might, however, merely mirror a causal dopamine effect elsewhere, as TH-GAL4 labels dopaminergic neurons throughout the brain. Because localized dopamine applications to dFB neuron dendrites similarly caused awakening, this possibility is considered remote (Pimentel, 2016).

    Flies with enhanced dopaminergic transmission exhibit a short-sleeping phenotype that requires the presence of a D1-like receptor in dFB neurons, suggesting that dopamine acts directly on these cells. dFB-restricted RNA interference (RNAi) confirmed this notion and pinpointed Dop1R2 as the responsible receptor, a conclusion reinforced by analysis of the mutant Dop1R2MI08664 allele. Previous evidence that Dop1R1, a receptor not involved in regulating baseline sleep, confers responsiveness to dopamine when expressed in the dFB indicates that either D1-like receptor can fulfill the role normally played by Dop1R2. Loss of Dop1R2 increased sleep during the day and the late hours of the night, by prolonging sleep bouts without affecting their frequency. This sleep pattern is consistent with reduced sensitivity to a dopaminergic arousal signal (Pimentel, 2016).

    To confirm the identity of the effective transmitter, avoid dopamine release outside the dFB, and reduce the transgene load for subsequent experiments, optogenetic manipulations of the dopaminergic system were replaced with pressure ejections of dopamine onto dFB neuron dendrites. Like optogenetically stimulated secretion, focal application of dopamine hyperpolarized the cells and suppressed their spiking. The inhibitory responses could be blocked at several nodes of an intracellular signalling pathway that connects the activation of dopamine receptors to the opening of potassium conductances: by RNAi-mediated knockdown of Dop1R2; by the inclusion in the patch pipette of pertussis toxin (PTX), which inactivates heterotrimeric G proteins of the Gi/o family; and by replacing intracellular potassium with caesium, which obstructs the pores of G-protein-coupled inward-rectifier channels. Elevating the chloride reversal potential above resting potential left the polarity of the responses unchanged, corroborating that potassium conductances mediate the bulk of dopaminergic inhibition (Pimentel, 2016).

    Coupling of Dop1R2 to Gi/o, although documented in a heterologous system, represents a sufficiently unusual transduction mechanism for a predicted D1-like receptor to prompt verification of its behavioural relevance. Like the loss of Dop1R2, temperature-inducible expression of PTX in dFB neurons increased overall sleep time by extending sleep bout length (Pimentel, 2016).

    While a single pulse of dopamine transiently hyperpolarized dFB neurons and inhibited their spiking, prolonged dopamine applications (50 ms pulses at 10 Hz, or 20 Hz optogenetic stimulation, both sustained for 2-10 min) switched the cells from electrical excitability (ON) to quiescence (OFF). The switching process required dopamine as well as Dop1R2, but once the switch had been actuated the cells remained in the OFF state-and flies, awake-without a steady supply of transmitter. Input resistances and membrane time constants dropped to 53.3 ± 1.8 and 24.0 ± 1.3% of their initial values (means ± s.e.m.), and depolarizing currents no longer elicited action potentials (15 out of 15 cells). The biophysical properties of single dFB neurons, recorded in the same individual before and after operating the dopamine switch, varied as widely as those in sleep-deprived and rested flies (Pimentel, 2016).

    Dopamine-induced changes in input resistance and membrane time constant occurred from similar baselines in all genotypes and followed single-exponential kinetics with time constants of 1.07-1.10 min. The speed of conversion points to post-translational modification and/or translocation of ion channels between intracellular pools and the plasma membrane as the underlying mechanism(s). In 7 out of 15 cases, recordings were held long enough to observe the spontaneous recommencement of spiking, which was accompanied by a rise to baseline of input resistance and membrane time constant, after 7-60 min of quiescence (mean ± s.e.m. = 25.86 ± 7.61 min). The temporary suspension of electrical output is thus part of the normal activity cycle of dFB neurons and not a dead end brought on by the experimental conditions (Pimentel, 2016).

    dFB neurons in the ON state expressed two types of potassium current: voltage-dependent A-type (rapidly inactivating) and voltage-independent non-A-type currents. The current-voltage (I-V) relation of iA resembled that of Shaker, the prototypical A-type channel: no current flowed below -50 mV, the approximate voltage threshold of Shaker; above -40 mV, peak currents increased steeply with voltage and inactivated with a time constant of 7.5 ± 2.1 ms (mean ± s.e.m.). Non-A-type currents showed weak outward rectification with a reversal potential of -80 mV, consistent with potassium as the permeant ion, and no inactivation (Pimentel, 2016).

    Switching the neurons OFF changed both types of potassium current. iA diminished by one-third, whereas inon-A nearly quadrupled when quantified between resting potential and spike threshold. The weak rectification of inon-A in the ON state vanished in the OFF state, giving way to the linear I-V relationship of an ideal leak conductance. dFB neurons thus upregulate iA in the sleep-promoting ON state. When dopamine switches the cells OFF, voltage-dependent currents are attenuated and leak currents augmented. This seesaw form of regulation should be sensitive to perturbations of the neurons' ion channel inventory: depletion of voltage-gated A-type (KV) channels (which predominate in the ON state) should tip the cells towards the OFF state; conversely, loss of leak channels (which predominate in the OFF state) should favour the ON state. To test these predictions, sleep was examined in flies carrying R23E10-GAL4-driven RNAi transgenes for dFB-restricted interference with individual potassium channel transcripts (Pimentel, 2016).

    RNAi-mediated knockdown of two of the five KV channel types of Drosophila (Shaker and Shab) reduced sleep relative to parental controls, while knockdown of the remaining three types had no effect. Biasing the potassium channel repertoire of dFB neurons against A-type conductances thus tilts the neurons' excitable state towards quiescence, causing insomnia, but leaves transient and sustained dopamine responses unaffected. The seemingly counterintuitive conclusion that reducing a potassium current would decrease, not increase, action potential discharge is explained by a requirement for A-type channels in generating repetitive activity of the kind displayed by dFB neurons during sleep. Depleting Shaker from dFB neurons shifted the interspike interval distribution towards longer values, as would be expected if KV channels with slow inactivation kinetics replaced rapidly inactivating Shaker as the principal force opposing the generation of the next spike. These findings identify a potential mechanism for the short-sleeping phenotypes caused by mutations in Shaker, its β subunit Hyperkinetic, or its regulator sleepless (Pimentel, 2016).

    Leak conductances are typically formed by two-pore-domain potassium (K2P) channels. dFB-restricted RNAi of one member of the 11-strong family of Drosophila K2P channels, encoded by the CG8713 gene, increased sleep relative to parental controls; interference with the remaining 10 K2P channels had no effect. Recordings from dFB neurons after knockdown of the CG8713 gene product, which this study termed Sandman, revealed undiminished non-A-type currents in the ON state and intact responses to a single pulse of dopamine but a defective OFF switch: during prolonged dopamine applications, inon-A failed to rise, input resistances and membrane time constants remained at their elevated levels, and the neurons continued to fire action potentials (7 out of 7 cells). Blocking vesicle exocytosis in the recorded cell with botulinum neurotoxin C (BoNT/C) similarly disabled the OFF switch. This, combined with the absence of detectable Sandman currents in the ON state, suggests that Sandman is internalized in electrically active cells and recycled to the plasma membrane when dopamine switches the neurons OFF (Pimentel, 2016).

    Because dFB neurons lacking Sandman spike persistently even after prolonged dopamine exposure, voltage-gated sodium channels remain functional in the OFF state. The difficulty of driving control cells to action potential threshold in this state must therefore be due to a lengthening of electrotonic distance between sites of current injection and spike generation. This lengthening is an expected consequence of a current leak, which may uncouple the axonal spike generator from somatodendritic synaptic inputs or pacemaker currents when sleep need is low (Pimentel, 2016).

    The two kinetically and mechanistically distinct actions of dopamine on dFB neurons-instant, but transient, hyperpolarization and a delayed, but lasting, switch in excitable state-ensure that transitions to vigilance can be both immediate and sustained, providing speedy alarm responses and stable homeostatic control. The key to stability lies in the switching behaviour of dFB neurons, which is driven by dopaminergic input accumulated over time. Unlike bistable neurons, in which two activity regimes coexist for the same set of conductances, dFB neurons switch regimes only when their membrane current densities change. This analysis of how dopamine effects such a change, from activity to silence, has uncovered elements familiar from other modulated systems: simultaneous, antagonistic regulation of multiple conductances; reduction of iA; and modulation of leak currents. Currently little is known about the reverse transition, from silence to activity, except that mutating the Rho-GTPase-activating protein Crossveinless-c locks dFB neurons in the OFF state, resulting in severe insomnia and an inability to correct sleep deficits. Discovering the signals and processes that switch sleep-promoting neurons back ON will hold important clues to the vital function of sleep (Pimentel, 2016).

    Postprandial sleep mechanics in Drosophila

    Food consumption is thought to induce sleepiness. However, little is known about how postprandial sleep is regulated. This study simultaneously measured sleep and food intake of individual flies and found a transient rise in sleep following meals. Depending on the amount consumed, the effect ranges from slightly arousing to strongly sleep inducing. Postprandial sleep was positively correlated with ingested volume, protein, and salt-but not sucrose-revealing meal property-specific regulation. Silencing of leucokinin receptor (Lkr) neurons specifically reduces sleep induced by protein consumption. Thermogenetic stimulation of leucokinin (Lk) neurons decreases whereas Lk downregulation by RNAi increases postprandial sleep, suggestive of an inhibitory connection in the Lk-Lkr circuit. A subset of non-leucokininergic cells proximal to Lkr neurons were identified to rhythmically increase postprandial sleep when silenced, suggesting that these cells are cyclically gated inhibitory inputs to Lkr neurons. Together, these findings reveal the dynamic nature of postprandial sleep (Murphy, 2016).

    Circadian neuron feedback controls the Drosophila sleep-activity profile

    Little is known about the ability of Drosophila circadian neurons to promote sleep. This study, using optogenetic manipulation and video recording, shows that a subset of dorsal clock neurons (DN1s) are potent sleep-promoting cells that release glutamate to directly inhibit key pacemaker neurons. The pacemakers promote morning arousal by activating these DN1s, implying that a late-day feedback circuit drives midday siesta and night-time sleep. To investigate more plastic aspects of the sleep program, the study used a calcium assay to monitor and compare the real-time activity of DN1 neurons in freely behaving males and females. It was found that DN1 neurons are more active in males than in females, consistent with the finding that male flies sleep more during the day. DN1 activity is also enhanced by elevated temperature, consistent with the ability of higher temperatures to increase sleep. These new approaches indicate that DN1s have a major effect on the fly sleep-wake profile and integrate environmental information with the circadian molecular program (Guo, 2016).

    Recurrent sleep fragmentation induces insulin and neuroprotective mechanisms in middle-aged flies

    Lack of quality sleep increases central nervous system oxidative stress and impairs removal of neurotoxic soluble metabolites from brain parenchyma. During aging poor sleep quality, caused by sleep fragmentation, increases central nervous system cellular stress. Currently, it is not known how organisms offset age-related cytotoxic metabolite increases in order to safeguard neuronal survival. Furthermore, it is not understood how age and sleep fragmentation interact to affect oxidative stress protection pathways. This study demonstrates that sleep fragmentation increases systems that protect against oxidative damage and neuroprotective endoplasmic reticulum molecular chaperones, as well as neuronal insulin and dopaminergic expression in middle-aged Drosophila males. Interestingly, even after sleep recovery the expression of these genes was still upregulated in middle-aged flies. Finally, sleep fragmentation generates higher levels of reactive oxygen species (ROS) in middle-aged flies and after sleep recovery these levels remain significantly higher than in young flies. The fact that neuroprotective pathways remain upregulated in middle-aged flies beyond sleep fragmentation suggests it might represent a strong stressor for the brain during later life (Williams, 2016).

    Glutamate is a wake-active neurotransmitter in Drosophila melanogaster

    In mammals, there is evidence that glutamate has a role as a wake-active neurotransmitter. So using video-based analysis of Drosophila behavior, a study was undertaken to examine if glutamate, which has been previously shown to have an excitatory role in neuromuscular junctions in Drosophila, may have a conserved wake-active role in the adult brain. Using 6- to 9-day-old female flies, the effect was examined of perturbations of the glutamatergic signaling on total wakefulness and wake bout architecture. Neuronal activity of glutamatergic neurons in the brains of adult flies was increased or decreased using Upstream Activating Sequence (UAS) NaChBac (a voltage-gated bacterial Na+ channel) and UAS EKO ('electrical knockout channel'), respectively. Neurotransmission was blocked from glutamatergic neurons in adult flies using the UAS-driven temperature-sensitive dynamin mutation shibirets. The behavior of flies was examined with loss of function mutations of individual subunits of brain-specific ionotropic glutamate receptors. Increasing the activity of glutamatergic neurons in the adult brain led to a significant increase in wakefulness compared to the control groups both in the daytime and nighttime and decreasing the activity of these same neurons reduced wakefulness in the nighttime. Blocking neurotransmitter release in glutamatergic neurons significantly reduced wake in the nighttime. The ionotropic receptor mutants had significantly less wake in the nighttime than their respective genetic background controls. The results show the following: glutamate is indeed a wake-active neurotransmitter in Drosophila; there is a major time of day effect associated with loss of glutamatergic neurotransmission; and it is a major wake-active neurotransmitter in the nighttime (Zimmerman, 2017).

    Sleep-dependent modulation of metabolic rate in Drosophila

    This study developed a system to simultaneously measure sleep and metabolic rate in individual Drosophila, allowing for interrogation of neural systems governing interactions between sleep and metabolic rate. Like mammals, metabolic rate in flies is reduced during sleep and increased during sleep deprivation suggesting sleep-dependent regulation of metabolic rate is conserved across phyla. The reduction of metabolic rate during sleep is not simply a consequence of inactivity because metabolic rate is reduced ~30 minutes following the onset of sleep, raising the possibility that CO2 production provides a metric to distinguish different sleep states in the fruit fly. It was determined that basal and sleep-dependent changes in metabolic rate are reduced in starved flies, suggesting that starvation inhibits normal sleep-associated effects on metabolic rate. Further, translin mutant flies that fail to suppress sleep during starvation demonstrate a lower basal metabolic rate, but this rate was further reduced in response to starvation, revealing that regulation of starvation-induced changes in metabolic rate and sleep duration are genetically distinct. Therefore, this system provides the unique ability to simultaneously measure sleep and oxidative metabolism, providing novel insight into the physiological changes associated with sleep and wakefulness in the fruit fly (Stahl, 2017).

    Circadian- and light-driven metabolic rhythms in Drosophila melanogaster

    Complex interactions of environmental cues and transcriptional clocks drive rhythmicity in organismal physiology. Light directly affects the circadian clock; however, little is known about its relative role in controlling metabolic variations in vivo. This study used high time-resolution sampling in Drosophila at every 2 h to measure metabolite outputs using a liquid-chromatography tandem mass spectrometry (LC-MS/MS) approach. Over 14% of detected metabolites oscillated with circadian periodicity under light-dark (LD) cycles. Many metabolites peaked shortly after lights-on, suggesting responsiveness to feeding and/or activity rather than the preactivity anticipation, as observed in previous transcriptomics analyses. Roughly 9% of measured metabolites uniquely oscillated under constant darkness (DD), suggesting that metabolite rhythms are associated with the transcriptional clock machinery. Strikingly, metabolome differences between LD and constant darkness were observed only during the light phase, highlighting the importance of photic input. Clock mutant flies exhibited strong 12-h ultradian rhythms, including 4 carbohydrate species with circadian periods in wild-type flies, but lacked 24-h circadian metabolic oscillations. A meta-analysis of these results with previous circadian metabolomics experiments uncovered the possibility of conserved rhythms in amino acids, keto-acids, and sugars across flies, mice, and humans and provides a basis for exploring the chrono-metabolic connection with powerful genetic tools in Drosophila (Rhoades, 2018).

    Regulation of sleep homeostasis by sexual arousal

    In all animals, sleep pressure is under continuous tight regulation. It is universally accepted that this regulation arises from a two-process model, integrating both a circadian and a homeostatic controller. This study has explored the role of environmental social signals as a third, parallel controller of sleep homeostasis and sleep pressure. It was shown that, in Drosophila melanogaster males, sleep pressure after sleep deprivation can be counteracted by raising their sexual arousal, either by engaging the flies with prolonged courtship activity or merely by exposing them to female pheromones (Beckwith, 2017).

    SIFamide and SIFamide receptor defines a novel neuropeptide signaling to promote sleep in Drosophila
    SIFamide receptor (SIFR) is a Drosophila G protein-coupled receptor for the neuropeptide SIFamide (SIFa). Although the sequence and spatial expression of SIFa are evolutionarily conserved among insect species, the physiological function of SIFa/SIFR signaling remains elusive. This study provides genetic evidence that SIFa and SIFR promote sleep in Drosophila. Either genetic ablation of SIFa-expressing neurons in the pars intercerebralis (PI) or pan-neuronal depletion of SIFa expression shortened baseline sleep and reduced sleep-bout length, suggesting that it caused sleep fragmentation. Consistently, RNA interference-mediated knockdown of SIFR expression caused short sleep phenotypes as observed in SIFa-ablated or depleted flies. Using a panel of neuron-specific Gal4 drivers, SIFR effects were further mapped to subsets of PI neurons. Taken together, these results reveal a novel physiological role of the neuropeptide SIFa/SIFR pathway to regulate sleep through sleep-promoting neural circuits in the PI of adult fly brains (Park 2014).

    This study provides new evidence that the neuropeptide SIFa and its G protein-coupled receptor SIFR are novel mediators for promoting sleep in Drosophila. Either genetic ablation of SIFa-expressing neurons or depletion of SIFa expression shortened baseline sleep and caused sleep fragmentation by decreasing sleep-bout length. Consistent with these observations, a recent study independently revealed a possible sleep promoting role of SIFa-expressing neurons in DD conditions. Using neuron-specific SIFR depletion, this study further mapped the sleep-promoting SIFR function to Dilp2-negative, SIFR-positive PI neurons (Park 2014).

    The PI in adult fly brain is homologous to the mammalian hypothalamus, the control center for neurotransmitter regulation. Several therapeutic targets for human sleep disorders are concentrated in the hypothalamus. For instance, dopaminergic neurons blocked by amphetamine-like drugs induce wake-promoting signals to cure narcolepsy. Benzodiazepine compounds increase gamma-aminobutryic acid (GABA)ergic neuronal transmission to enhance sleep-promoting signals to treat insomnia. Octopamine, which is similar to mammalian norepinephrine, has been identified as a wake-promoting molecule in Drosophila. When octopamine biosynthesis is compromised, flies exhibit enhanced sleep. On the other hand, octopamine promotes wakefulness in flies, particularly at night. Moreover, octopamine and OAMB, an octopamine receptor, act in Dilp2-expressing PI neurons to promote wakefulness through the cyclic AMP (cAMP) pathway. This is in contrast with the current finding that Dilp2-negative PI neurons are important for SIFR-dependent sleep promotion. Therefore, this study has defined a novel PI circuit that promotes sleep via the SIFa-SIFR signaling pathway (Park 2014).

    Additional genes have been identified as sleep regulators in the PI region of adult fly brain, including members of the rhomboid family, which are integral membrane proteases and star, a transmembrane cargo receptor. They process epidermal growth factor receptor (EGFR)- activating ligands, such as spitz, gurken, and keren, so that extracellular signal-regulated kinase (ERK) is activated by phosphorylation. When EGFR is activated, flies exhibit excessive sleep. Interestingly, depletion of rhomboid, one of the processors for the ligand of the EGF receptor in c767-Gal4 expressing PI neurons shortened sleep. Given that SIFR and rhomboid promote sleep in the same PI neurons, it might be possible that SIFR and rhomboid function together to regulate sleep through the EGFR-ERK signaling pathway. Not much is known about the SIFR in terms of its downstream effectors and how it exerts its physiological effects. In general, GPCR activates the protein kinase A (PKA)-cAMP pathway via Gs or Ca2+ through a Gq regulator. It was recently shown that lethality in flies with SIFR knock-down is rescued by the overexpression of dSTIM, one of the key regulators of store-operated Ca2+ entry. Furthermore, the nuclear factor of activated T cells (NFAT), a Ca2+-activated transcription factor, is regulated by SIFR in a Schneider 2 (S2) cell-based dsRNA screening, suggesting that sleep regulation by SIFR might involve Ca2+ signaling. Future studies will address which signaling pathways SIFR affects to regulate neuronal activity and sleep behavior (Park, 2014).

    Differential activation of immune factors in neurons and glia contribute to individual differences in resilience/vulnerability to sleep disruption

    Individuals frequently find themselves confronted with a variety of challenges that threaten their wellbeing. While some individuals face these challenges efficiently and thrive (resilient) others are unable to cope and may suffer persistent consequences (vulnerable). Resilience/vulnerability to sleep disruption may contribute to the vulnerability of individuals exposed to challenging conditions. With that in mind this study exploited individual differences in a fly's ability to form short-term memory (STM) following 3 different types of sleep disruption to identify the underlying genes. The analysis showed that in each category of flies examined, there are individuals that form STM in the face of sleep loss (resilient) while other individuals show dramatic declines in cognitive behavior (vulnerable). Molecular genetic studies revealed that Antimicrobial Peptides, factors important for innate immunity, were candidates for conferring resilience/vulnerability to sleep deprivation. Specifically, Metchnikowin (Mtk), drosocin (dro) and Attacin (Att) transcript levels seemed to be differentially increased by sleep deprivation in glia (Mtk), neurons (dro) or primarily in the head fat body (Att). Follow-up genetic studies confirmed that expressing Mtk in glia but not neurons, and expressing dro in neurons but not glia, disrupted memory while modulating sleep in opposite directions. These data indicate that various factors within glia or neurons can contribute to individual differences in resilience/vulnerability to sleep deprivation (Dissel, 2015).

    The timed depolarization of morning and evening oscillators phase shifts the circadian clock of Drosophila

    Phase response curves (PRCs) for light or temperature stimuli have been shown to be most valuable in understanding how circadian clocks are entrained to daily environmental cycles. Nowadays, PRC experiments in which clock neurons are manipulated in a temporally restricted manner by thermogenetic or optogenetic tools are also useful to comprehend clock network properties. In this study, specific clock neurons of Drosophila melanogaster were temporally depolarized by activating temperature-sensitive dTrpA1 channels to unravel their role in phase shifting the flies' activity rhythm. The depolarization of all clock neurons causes a PRC resembling the flies' light PRC, with strong phase delays in the first half of the subjective night and modest phase advances in its second half. However, the activation of the flies' pigment-dispersing factor (PDF)-positive morning (M) neurons (s-LNvs) only induces phase advances, and these reach into the subjective day, where the light PRC has its dead zone. This indicates that the M neurons are very potent in accelerating the clock, which is in line with previous observations. In contrast, the evening (E) neurons together with the PDF-positive l-LNvs appear to mediate phase delays. Most interestingly, the molecular clock (Period protein cycling) of the depolarized clock neurons is shifted in parallel to the behavior, and this shift is already visible within the first cycle after the temperature pulse. The cAMP response element binding protein B (CREB) was identified as a putative link between membrane depolarization and the molecular clock (Eck, 2016).

    The Circadian clock is a key driver of steroid hormone production in Drosophila

    Biological clocks allow organisms to anticipate daily environmental changes such as temperature fluctuations, abundance of daylight, and nutrient availability. Many circadian-controlled physiological states are coordinated by the release of systemically acting hormones, including steroids and insulin. Thus, hormones relay circadian outputs to target tissues, and disrupting these endocrine rhythms impairs human health by affecting sleep patterns, energy homeostasis, and immune functions. It is largely unclear, however, whether circadian circuits control hormone levels indirectly via central timekeeping neurons or whether peripheral endocrine clocks can modulate hormone synthesis directly. This study shows that perturbing the circadian clock, specifically in the major steroid hormone-producing gland of Drosophila, the prothoracic gland (PG), unexpectedly blocks larval development due to an inability to produce sufficient steroids. This is surprising, because classic circadian null mutants are viable and result in arrhythmic adults. Timeless and Period, both core components of the insect clock, are required for transcriptional upregulation of steroid hormone-producing enzymes. Timeless couples the circadian machinery directly to the two canonical pathways that regulate steroid synthesis in insects, insulin and PTTH signaling, respectively. Activating insulin signaling directly modulates Timeless function, suggesting that the local clock in the PG is normally synced with systemic insulin cues. Because both PTTH and systemic insulin signaling are themselves under circadian control, it is concluded that de-synchronization of a local endocrine clock with external circadian cues is the primary cause for steroid production to fail (Di Cara, 2016).

    The Drosophila clock neuron network features diverse coupling modes and requires network-wide coherence for robust circadian rhythms

    In animals, networks of clock neurons containing molecular clocks orchestrate daily rhythms in physiology and behavior. However, how various types of clock neurons communicate and coordinate with one another to produce coherent circadian rhythms is not well understood. This study investigated clock neuron coupling in the brain of Drosophila and demonstrates that the fly's various groups of clock neurons display unique and complex coupling relationships to core pacemaker neurons. Furthermore, coordinated free-running rhythms require molecular clock synchrony not only within the well-characterized lateral clock neuron classes but also between lateral clock neurons and dorsal clock neurons. These results uncover unexpected patterns of coupling in the clock neuron network and reveal that robust free-running behavioral rhythms require a coherence of molecular oscillations across most of the fly's clock neuron network (Yao, 2016).

    Temporal calcium profiling of specific circadian neurons in freely moving flies

    There are no general methods for reliably assessing the firing properties or even calcium profiles of specific neurons in freely moving flies. To this end, this study adapted a GFP-based calcium reporter to luciferase that was expressed in small subsets of circadian neurons. This Tric-LUC reporter allowed a direct comparison of luciferase activity with locomotor activity, which was assayed in the same flies with video recording. The LUC profile from activity-promoting E cells paralleled evening locomotor activity, and the LUC profile from sleep-promoting glutamatergic DN1s (gDN1s) paralleled daytime sleep. Similar profiles were generated by novel reporters recently identified based on transcription factor activation. As E cell and gDN1 activity is necessary and sufficient for normal evening locomotor activity and daytime sleep profiles, respectively, it is suggest that their luciferase profiles reflect their neuronal calcium and in some cases firing profiles in wake-behaving flies (Guo, 2017).

    Neuronal and glial clocks underlying structural remodeling of pacemaker neurons in Drosophila

    Ventral Lateral Neurons (LNvs), which are essential in the control of rest-activity cycles in Drosophila, undergo circadian remodeling of their axonal projections. This structural plasticity gives rise to changes in the degree of connectivity, which could provide a means of transmitting time of day information. Several neuronal types undergoing circadian remodeling hint to a differential effect of clock genes; while per mutants exhibited poorly developed axonal terminals giving rise to low complexity arbors, tim mutants displayed a characteristic hyper branching phenotype, suggesting these genes could be playing additional roles to those ascribed to core clock function. To shed light onto this possibility clock gene levels were altered through RNAi- mediated downregulation and expression of a dominant negative form exclusively in the adult LNvs. These experiments confirmed that the LNv clock is necessary to drive the remodeling process. The contribution of glia to the structural plasticity of the small LNvs was explored through acute disruption of their internal clock. Interestingly, impaired glial clocks also abolished circadian structural remodeling, without affecting other clock-controlled outputs. Taken together these data shows that both neuronal and glial clocks are recruited to define the architecture of the LNv projections along the day, thus enabling a precise reconfiguration of the circadian network (Herrero, 2017).

    Oscillatory brain activity in spontaneous and induced sleep stages in flies

    Sleep is a dynamic process comprising multiple stages, each associated with distinct electrophysiological properties and potentially serving different functions. While these phenomena are well described in vertebrates, it is unclear if invertebrates have distinct sleep stages. Local field potential (LFP) recordings were performed on flies spontaneously sleeping, and their brain activity was compared to flies induced to sleep using either genetic activation of sleep-promoting circuitry or the GABAA agonist Gaboxadol. A transitional sleep stage was found associated with a 7-10 Hz oscillation in the central brain during spontaneous sleep. Oscillatory activity is also evident when sleep-promoting neurons were acutely activated in the dorsal fan-shaped body (dFB) of Drosophila. In contrast, sleep following Gaboxadol exposure is characterized by low-amplitude LFPs, during which dFB-induced effects are suppressed. Sleep in flies thus appears to involve at least two distinct stages: increased oscillatory activity, particularly during sleep induction, followed by desynchronized or decreased brain activity (Yap, 2017).

    Chronic circadian misalignment results in reduced longevity and large-scale changes in gene expression in Drosophila

    Circadian clocks ensure that behavioral and physiological processes occur at optimal times of day and in the correct temporal order. It is becoming increasingly clear that chronic circadian misalignment (CCM), such as occurs in shift workers, has profound metabolic and cognitive consequences, but the proximate mechanisms connecting CCM with reduced organismal health are unknown. This study investigated the consequences of CCM in the powerful model system of the fruit fly, Drosophila melanogaster. Flies were subjected to daily 4-h phase delays in the light-dark schedule, and the Drosophila Activity Monitoring (DAM) system was used to continuously track locomotor activity and sleep while simultaneously monitoring fly lifespan. Consistent with previous results, exposing flies to CCM leads to a ~ 15% reduction in median lifespan in both male and female flies. Importantly, it was demonstrated that the reduced longevity occurs independent of changes in overall sleep or activity. To uncover potential molecular mechanisms of CCM-induced reduction in lifespan, whole body RNA-sequencing was conducted to assess differences in gene transcription between control and misaligned flies. CCM caused progressive, large-scale changes in gene expression characterized by upregulation of genes involved in response to toxic substances, aging and oxidative stress, and downregulation of genes involved in regulation of development and differentiation, gene expression and biosynthesis. Many of these gene expression changes mimic those that occur during natural aging, consistent with the idea that CCM results in premature organismal decline, however, genes involved in lipid metabolism are overrepresented among those that are differentially regulated by CCM and aging. This category of genes is also among the earliest to exhibit CCM-induced changes in expression, thus highlighting altered lipid metabolism as a potentially important mediator of the negative health consequences of CCM (Boomgarden, 2019).

    Most sleep does not serve a vital function: Evidence from Drosophila melanogaster

    Sleep appears to be a universally conserved phenomenon among the animal kingdom, but whether this notable evolutionary conservation underlies a basic vital function is still an open question. Using a machine learning-based video-tracking technology, a detailed high-throughput analysis of sleep was conducted in the fruit fly Drosophila melanogaster, coupled with a lifelong chronic and specific sleep restriction. These results show that some wild-type flies are virtually sleepless in baseline conditions and that complete, forced sleep restriction is not necessarily a lethal treatment in wild-type D. melanogaster. It was also shown that circadian drive, and not homeostatic regulation, is the main contributor to sleep pressure in flies. These results offer a new perspective on the biological role of sleep in Drosophila and, potentially, in other species (Geissmann, 2019).

    A Drosophila model of sleep restriction therapy for insomnia

    Insomnia is the most common sleep disorder among adults, especially affecting individuals of advanced age or with neurodegenerative disease. Insomnia is also a common comorbidity across psychiatric disorders. Cognitive behavioral therapy for insomnia (CBT-I) is the first-line treatment for insomnia; a key component of this intervention is restriction of sleep opportunity, which optimizes matching of sleep ability and opportunity, leading to enhanced sleep drive. Despite the well-documented efficacy of CBT-I, little is known regarding how CBT-I works at a cellular and molecular level to improve sleep, due in large part to an absence of experimentally-tractable animals models of this intervention. Guided by human behavioral sleep therapies, this study developed a Drosophila model for sleep restriction therapy (SRT) of insomnia. It was demonstrated that restriction of sleep opportunity through manipulation of environmental cues improves sleep efficiency in multiple short-sleeping Drosophila mutants. The response to sleep opportunity restriction requires ongoing environmental inputs, but is independent of the molecular circadian clock. This sleep opportunity restriction paradigm was applied to aging and Alzheimer's disease fly models; sleep impairments in these models are reversible with sleep restriction, with associated improvement in reproductive fitness and extended lifespan. This work establishes a model to investigate the neurobiological basis of CBT-I, and provides a platform that can be exploited toward novel treatment targets for insomnia (Belfer, 2019).

    Differential regulation of the Drosophila sleep homeostat by circadian and arousal inputs

    One output arm of the sleep homeostat in Drosophila appears to be a group of neurons with projections to the dorsal fan-shaped body (dFB neurons) of the central complex in the brain. However, neurons that regulate the sleep homeostat remain poorly understood. Using neurogenetic approaches combined with Ca(2+) imaging, synaptic connections were characterized between dFB neurons and distinct sets of upstream sleep-regulatory neurons. One group of the sleep-promoting upstream neurons is a set of circadian pacemaker neurons that activates dFB neurons via direct glutaminergic excitatory synaptic connections. Opposing this population, a group of arousal-promoting neurons downregulates dFB axonal output with dopamine. Co-activating these two inputs leads to frequent shifts between sleep and wake states. dFB neurons were shown to release the neurotransmitter GABA and inhibit octopaminergic arousal neurons. It is proposed that dFB neurons integrate synaptic inputs from distinct sets of upstream sleep-promoting circadian clock neurons, and arousal neurons (Ni, 2019).

    Network-specific synchronization of electrical slow-wave oscillations regulates sleep drive in Drosophila

    Slow-wave rhythms characteristic of deep sleep oscillate in the delta band (0.5-Hz) and can be found across various brain regions in vertebrates. Across phyla, however, an understanding of the mechanisms underlying oscillations and how these link to behavior remains limited. This study discovered compound delta oscillations in the sleep-regulating R network of Drosophila. The power of these slow-wave oscillations increases with sleep need and is subject to diurnal variation. Optical multi-unit voltage recordings reveal that single R neurons get synchronized by activating circadian input pathways. This synchronization depends on NMDA receptor (NMDAR) coincidence detector function, and an interplay of cholinergic and glutamatergic inputs regulates oscillatory frequency. Genetically targeting the coincidence detector function of NMDARs in R5, and thus the uncovered mechanism underlying synchronization, abolished network-specific compound slow-wave oscillations. It also disrupted sleep and facilitated light-induced wakening, establishing a role for slow-wave oscillations in regulating sleep and sensory gating. It is therefore proposed that the synchronization-based increase in oscillatory power likely represents an evolutionarily conserved, potentially 'optimal,' strategy for constructing sleep-regulating sensory gates (Raccuglia, 2019).

    In vertebrates, oscillatory electrical compound patterns are associated with fundamental brain functions and specific behaviors. Characteristic of vertebrate deep sleep are compound slow-wave oscillations in the delta band (0.5- Hz), which are thought to derive from the synchronization of neuronal activity. However, how specific neural networks contribute to generating compound oscillations and whether these oscillations represent a functional unit for sleep regulation largely remains unclear. This is partially due to methodological constraints, as readouts either focus on single-cell/unit or local compound potential recordings (electroencephalograms and local field potentials) that, confined by poor spatial resolution, do not permit for dissecting interactions of genetically identified multi-units (Raccuglia, 2019).

    Like vertebrates, invertebrates' sleep and behavior selection is sensitive to an animal's sleep need. However, in invertebrates, it is unknown whether electrical oscillations can gate specific behaviors and whether an electrophysiological sleep correlate, such as slow-wave oscillations, exists or is involved in sleep regulation. Local field potential (LFP) measurements in the Drosophila brain indicate that the frequency of large-scale compound neuronal activity is reduced during sleep, opening up the possibility that, comparable to vertebrates, slow oscillatory activity could be involved in mediating sleep. Yet such oscillations have not been identified, and it remains unknown which and how neural networks would generate slow-wave oscillations that could be crucial for sleep regulation (Raccuglia, 2019).

    This study made use of recent technological advancements in Drosophila melanogaster and combined targeted expression of a genetically encoded voltage indicator (GEVI) with that of optogenetic actuators. This all-optical electrophysiological approach bypasses common methodological constraints, allowing monitoring of multi-unit electrical patterns within a specific network and gaining of mechanistic insight into how sleep-relevant neural activity might be generated (Raccuglia, 2019).

    This study has discovered sleep-regulating, network-specific delta oscillations within the R network of the Drosophila ellipsoid body, which is situated at a crossroad involved in sleep regulation and sensory processing. Compound delta oscillations are not detectable in the morning (Zeitgeber time [ZT] 0-3) but become apparent, and increase in power, over the day. Activating circadian input pathways leads to multi-unit synchronization that depends on NMDA receptor (NMDAR)-mediated coincidence detection. Disrupting this synchronization and thus the emergence of compound delta oscillations affects the flies' sleep patterns and alters sensory gating during sleep. This study thus identified delta oscillations as an electrophysiological correlate of sleep pressure regulation in invertebrates and ties these oscillatory patterns to behavioral readouts. This work suggests that slow-wave oscillations and sleep could be fundamentally interconnected across phyla. Slow-wave oscillations may therefore potentially represent an evolutionarily conserved strategy for network mechanisms regulating internal states and sleep (Raccuglia, 2019).

    The data suggest that compound delta oscillations specific to the sleep-regulating R network are generated by circadian drive transduced via TuBu neurons. Optogenetic activation of TuBu neurons increases single-unit power and synchronizes R neurons, which should result in an increase of compound delta power and thus internal sleep drive. This is consistent with thermogenetic activation of TuBu neurons increasing the total amount of sleep in flies. High levels of TuBu neuron output could be generated by altering activity in sleep-modulating DN circadian clock neurons, which form direct connections with the TuBu neurons. Of note, following R synchronization mediated via activation of TuBu neurons, a prolonged hyperpolarization becomes apparent that typically can be observed following highly synchronized states (Raccuglia, 2019).

    The R network also receives excitatory input from Helicon cells, a potential source of cholinergic input. This study provides evidence that nAChRs act as prime candidates to provide concurrent depolarization required for NMDAR coincidence detection in R neurons. Analogous to the relationship between AMPARs and NMDARs in mammals, the interplay of nAChRs and NMDARs could provide a substrate for coincidence detection in Drosophila. By reducing the temporal variability of mixed synaptic inputs, NMDARs could promote the temporal summation of excitatory postsynaptic potentials, thus increasing the synchrony between R neurons. Indeed, rhythmic application of acetylcholine to R dendrites is sufficient to induce oscillations but only in the presence of functional NMDARs. Helicon cells also receive visual information and are part of a recurrent circuit mediating homeostatic sleep pressure regulation. Thus, R oscillatory activity is likely regulated via a complex interplay of sensory input, circadian rhythms, and homeostatic sleep pressure regulation (Raccuglia, 2019).

    These experiments further indicate that additional inhibitory input via glutamate-gated chloride channels could antagonize excitatory drives to retain a frequency within the delta range, potentially defining the time window for recurrent input. It is therefore proposed that the interplay between glutamatergic and cholinergic signals sets a frequency within the delta range (0.5-1. Hz), which permits temporal synchronization of single units to define the power and frequency of compound oscillatory activity and thus the strength of sleep drive (Raccuglia, 2019).

    Expression levels of NMDARs in R neurons have previously been associated with the regulation of sleep drive. However, the physiological contribution of NMDARs remained open. The current data suggest that NMDAR coincidence detection gates neuronal synchronization of delta-wave activity within the R network to increase the power of sleep-relevant compound oscillations. Indeed, at the single-cell level, expressing Mg2+ block-deficient NMDARs in R neurons led to irregular activity patterns, which could be at the basis of impaired synchronization and disrupted compound oscillations. It is therefore hypothesized that NMDAR-coincidence detection can lead to a state-dependent transition of activity patterns permitting synchronization. Disrupting synchronization by expressing Mg2+-block-deficient NMDARs interfered with multi-unit synchronization, directly altering the animals' sleep drive, sleep quality, and stimulus-induced wakening. This tightly links the mechanisms underlying network-specific delta-band oscillations to shaping animal behavior (Raccuglia, 2019).

    This study has identified slow-wave oscillations as an electrophysiological correlate of sleep pressure regulation involved in sensory gating in Drosophila. Interestingly, oscillatory activity in Drosophila R neurons is reminiscent of up- and down-states occurring at the level of mammalian cortical networks during deep sleep. It is thus hypothesized that the oscillations observed in this study are comparable to sleep-regulating thalamocortical oscillations as well as network-specific oscillations observed during sleep deprivation in vertebrates (local sleep). Thus, the R network could be functionally analogous to the thalamus, as network-specific synchronization of slow-wave activity within the thalamus plays a crucial role in maintaining sleep and sensory gating. Comparable to the potential function of network-specific oscillations during 'local sleep' in vertebrates, slow-wave oscillations within the flies' R network may well be involved in the homeostatic regulation of synaptic strength. It is thus suggested that oscillatory network synchronization may represent an evolutionarily selected optimal strategy for signaling sleep pressure as well as for the internal representation of sleepiness. One prediction from this analogy would be that slow-wave oscillations would also be detected in other Drosophila brain regions. Indeed, extracellular local field potential recordings suggest that overall activity in the fly brain does change during sleep. Future work should address whether network oscillations in the delta band can also be detected in other brain regions, for instance, the sleep-inducing fan-shaped bodies or the mushroom bodies. Additionally, whether cell-autonomous conductances contribute to sustained rhythmic activities of single R neuron remains an open question. This framework should pave the road for identifying evolutionarily conserved fundamental principles that link slow-wave oscillations as electrophysiological hallmarks of sleep to the neuronal processes underlying memory consolidation (Raccuglia, 2019).

    Dietary fatty acids promote sleep through a taste-independent mechanism

    Consumption of foods that are high in fat contribute to obesity and metabolism-related disorders. Dietary lipids are comprised of triglycerides and fatty acids, and the highly palatable taste of dietary fatty acids promotes food consumption, activates reward centers in mammals, and underlies hedonic feeding. Despite the central role of dietary fats in the regulation of food intake and the etiology of metabolic diseases, little is known about how fat consumption regulates sleep. The fruit fly, Drosophila melanogaster, provides a powerful model system for the study of sleep and metabolic traits, and flies potently regulate sleep in accordance with food availability. To investigate the effects of dietary fats on sleep regulation, fatty acids were supplemented into the diet of Drosophila and their effects on sleep and activity were measured. Flies fed a diet of hexanoic acid, a medium-chain fatty acid that is a by-product of yeast fermentation, slept more than flies starved on an agar diet. To assess whether dietary fatty acids regulate sleep through the taste system, sleep was assessed in flies with a mutation in the hexanoic acid receptor Ionotropic receptor 56D, which is required for fatty acid taste perception. These flies also sleep more than agar-fed flies when fed a hexanoic acid diet, suggesting the sleep promoting effect of hexanoic acid is not dependent on sensory perception. Taken together, these findings provide a platform to investigate the molecular and neural basis for fatty acid-dependent modulation of sleep (Sah Pamboro, 2019).

    Neuroanatomical details of the lateral neurons of Drosophila melanogaster support their functional role in the circadian system

    Drosophila melanogaster is a long-standing model organism in the circadian clock research. A major advantage is the relative small number of about 150 neurons, which built the circadian clock in Drosophila. A recent work focused on the neuroanatomical properties of the lateral neurons of the clock network. By applying the multicolor-labeling technique Flybow it was possible to identify the anatomical similarity of the previously described E2 subunit of the evening oscillator of the clock, which is built by the 5th small ventrolateral neuron (5th s-LNv ) and one ITP positive dorsolateral neuron (LNd ). These two clock neurons share the same spatial and functional properties. Both neurons were found innervating the same brain areas with similar pre- and postsynaptic sites in the brain. The anatomical findings support their shared function as a main evening oscillator in the clock network like also found in previous studies. A second quite surprising finding addresses the large lateral ventral PDF-neurons (l-LNv s). It was shown that the four hardly distinguishable l-LNv s consist of two subgroups with different innervation patterns. While three of the neurons reflect the well-known branching pattern reproduced by PDF immunohistochemistry, one neuron per brain hemisphere has a distinguished innervation profile and is restricted only to the proximal part of the medulla-surface. This neuron was named "extra" l-LNv (l-LNv x). The anatomical findings reflect different functional properties of the two l-LNv subgroups (Schubert, 2018).

    Reconfiguration of a multi-oscillator network by light in the Drosophila circadian clock

    The brain clock that drives circadian rhythms of locomotor activity relies on a multi-oscillator neuronal network. In addition to synchronizing the clock with day-night cycles, light also reformats the clock-driven daily activity pattern. How changes in lighting conditions modify the contribution of the different oscillators to remodel the daily activity pattern remains largely unknown. Data in Drosophila indicate that light readjusts the interactions between oscillators through two different modes. This study shows that a morning s-LNv > DN1p circuit works in series, whereas two parallel evening circuits are contributed by LNds and other DN1ps. Based on the photic context, the master pacemaker in the s-LNv neurons swaps its enslaved partner-oscillator-LNd in the presence of light or DN1p in the absence of light-to always link up with the most influential phase-determining oscillator. When exposure to light further increases, the light-activated LNd pacemaker becomes independent by decoupling from the s-LNvs. The calibration of coupling by light is layered on a clock-independent network interaction wherein light upregulates the expression of the PDF neuropeptide in the s-LNvs, which inhibits the behavioral output of the DN1p evening oscillator. Thus, light modifies inter-oscillator coupling and clock-independent output-gating to achieve flexibility in the network. It is likely that the light-induced changes in the Drosophila brain circadian network could reveal general principles of adapting to varying environmental cues in any neuronal multi-oscillator system (Chatterjee, 2018).

    Hub-organized parallel circuits of central circadian pacemaker neurons for visual photoentrainment in Drosophila

    Circadian rhythms are orchestrated by a master clock that emerges from a network of circadian pacemaker neurons. The master clock is synchronized to external light/dark cycles through photoentrainment, but the circuit mechanisms underlying visual photoentrainment remain largely unknown. This study reports that Drosophila has eye-mediated photoentrainment via a parallel pacemaker neuron organization. Patch-clamp recordings of central circadian pacemaker neurons reveal that light excites most of them independently of one another. Light-responding pacemaker neurons were shown to send their dendrites to a neuropil called accessary medulla (aMe), where they make monosynaptic connections with Hofbauer-Buchner eyelet photoreceptors and interneurons that transmit compound-eye signals. Laser ablation of aMe and eye removal both abolish light responses of circadian pacemaker neurons, revealing aMe as a hub to channel eye inputs to central circadian clock. Taken together, this study demonstrates that the central clock receives eye inputs via hub-organized parallel circuits in Drosophila (Li, 2018).

    Hierarchical control of Drosophila sleep, courtship, and feeding behaviors by male-specific P1 neurons

    Animals choose among sleep, courtship, and feeding behaviors based on the integration of both external sensory cues and internal states; such choices are essential for survival and reproduction. These competing behaviors are closely related and controlled by distinct neural circuits, but whether they are also regulated by shared neural nodes is unclear. This study investigated how a set of male-specific P1 neurons controls sleep, courtship, and feeding behaviors in Drosophila males. Mild activation of P1 neurons was sufficient to affect sleep, but not courtship or feeding, while stronger activation of P1 neurons labeled by four out of five independent drivers induced courtship, but only the driver that targeted the largest number of P1 neurons affected feeding. These results reveal a common neural node that affects sleep, courtship, and feeding in a threshold-dependent manner, and provide insights into how competing behaviors can be regulated by a shared neural node (Zhang, 2018).

    A wake-promoting circadian output circuit in Drosophila

    Circadian clocks play conserved roles in gating sleep and wake states throughout the day-night cycle. In the fruit fly Drosophila melanogaster, DN1p clock neurons have been reported to play both wake- and sleep-promoting roles, suggesting a complex coupling of DN1p neurons to downstream sleep and arousal centers. However, the circuit logic by which DN1p neurons modulate sleep remains poorly understood. This study shows that DN1p neurons can be divided into two morphologically distinct subsets. Projections from one subset surround the pars intercerebralis, a previously defined circadian output region. In contrast, the second subset also sends presynaptic termini to a visual processing center, the anterior optic tubercle (AOTU). Within the AOTU, DN1p neurons inhibit a class of tubercular-bulbar (TuBu) neurons that act to promote consolidated sleep. These TuBu neurons in turn form synaptic connections with R neurons of the ellipsoid body, a region linked to visual feature detection, locomotion, spatial memory, and sleep homeostasis. These results define a second output arm from DN1p neurons and suggest a role for TuBu neurons as regulators of sleep drive (Lamaze, 2018).

    A circadian output circuit controls sleep-wake arousal in Drosophila

    The Drosophila core circadian circuit contains distinct groups of interacting neurons that give rise to diurnal sleep-wake patterns. Previous work showed that a subset of dorsal neurons 1 (DN1s) are sleep-promoting through their inhibition of activity-promoting circadian pacemakers. This study shows that these anterior-projecting DNs (APDNs) also "exit" the circadian circuitry and communicate with the homeostatic sleep center in higher brain regions to regulate sleep and sleep-wake arousal. These APDNs connect to a small, discrete subset of tubercular-bulbar neurons, which are connected in turn to specific sleep-centric ellipsoid body (EB)-ring neurons of the central complex. Remarkably, activation of the APDNs produces sleep-like oscillations in the EB and affects arousal. The data indicate that this APDN-TuBusup-EB circuit temporally regulates sleep-wake arousal in addition to the previously defined role of the TuBu-EB circuit in vision, navigation, and attention (Guo, 2018).

    Neuronal reactivation during post-learning sleep consolidates long-term memory in Drosophila

    Animals consolidate some, but not all, learning experiences into long-term memory. Across the animal kingdom, sleep has been found to have a beneficial effect on the consolidation of recently formed memories into long-term storage. However, the underlying mechanisms of sleep dependent memory consolidation are poorly understood. This study shows that consolidation of courtship long-term memory in Drosophila is mediated by reactivation during sleep of dopaminergic neurons that were earlier involved in memory acquisition. Specific fan-shaped body neurons were identified that induce sleep after the learning experience and activate dopaminergic neurons for memory consolidation. Thus, this study provide a direct link between sleep, neuronal reactivation of dopaminergic neurons, and memory consolidation (Dag, 2019).

    The activity of DAN-aSP13s, which is essential for courtship memory acquisition, is also necessary during a discrete post-learning time window for LTM consolidation. Because neuronal reactivation occurs during sleep in rodents, it was hypothesized that post-learning activation of DAN-aSP13s involves a sleep-dependent mechanism. Using behavioral analysis and neuronal activity monitoring and perturbation approaches, thus study shows that DAN-aSP13s display an increased activity in freely behaving animals during sleep after a prolonged learning experience. This sleep is necessary for LTM consolidation, and it can be mediated by a specific class of sleep promoting neurons in the ventral layer of the fan shaped body (vFB). These vFB neurons consolidate courtship LTM in a discrete time window and provide an excitatory input to DAN-aSP13s. Thus, these data provide a causal link between sleep promoting neurons in the vFB, post-learning activation of dopaminergic neurons, and LTM consolidation (Dag, 2019).

    Based on these data, the following model is proposed for sleep-dependent consolidation of courtship LTM in Drosophila (see Post-learning activation of DAN-aSP13 neurons mediates LTM consolidation). During a prolonged learning experience, γKCs and DAN-aSP13s are repeatedly activated by olfactory and behavioral cues presented by an unreceptive female, respectively. Whereas prolonged wakefulness leads to an increase in homeostatic sleep drive in ellipsoid body neurons that in turn is conveyed to dFB, it is hypothesized that an extended learning experience generates a learning-dependent sleep drive that is transmitted to vFB. In turn, the vFB neurons enhance sleep after learning and provide an excitatory input back on DAN-aSP13s. It is thought that one potential site of a learning-dependent sleep drive are the MB neurons since they have been implicated in both memory formation and sleep regulation. Dopamine released upon DAN-aSP13 reactivation stimulates molecular processes in γKCs that are different from those engaged during STM acquisition and involve protein synthesis that is essential for LTM formation and persistence (Dag, 2019).

    Dopaminergic pathways are thought to convey information about whether an experience is rewarding or punishing and thus, worth remembering. Post-learning neuronal activity of the dopaminergic hippocampal inputs from the Ventral Tagmental Area (VTA) has been implicated in the consolidation of fear memory in rodents. Interestingly, this activity is critical during a discrete time window after learning. Post-learning activity of the VTA dopamine neurons has been also implicated in the reactivation during sleep of the hippocampal cells involved earlier in encoding of the spatial experience. This study shows that post-learning activation of DAN-aSP13s mediates the consolidation of courtship LTM in Drosophila. It is proposed that reactivation during sleep of the dopamine neurons that were previously active during memory acquisition ensures that spurious experiences are not admitted into LTM storage and thus only experiences that are either sufficiently salient or persistent become long-lasting memories. Specifically, reactivation of DAN-aSP13s during post-learning sleep enhances reactivation of the γKCs and cognate MBON-M6, which together with DAN-aSP13s form a recurrent circuit necessary for courtship memory acquisition. This study considers two hypotheses to account for this selectivity. During sleep, vFB neurons might selectively reactivate only the relevant DANs, or alternatively, they might activate all DANs but only the relevant subset is able to consolidate LTM. The selective-reactivation model would require some marker to distinguish which DANs were activated, whereas the selective-consolidation model would require a marker in the synapses of the γKCs that were earlier active during memory acquisition, for example translational regulator Orb2, which regulates translation upon neuronal activity during LTM consolidation (Dag, 2019).

    Activity of dopaminergic neurons regulates sleep-wake states in animals, including flies. Artificial activation of DAN-aSP13s has been shown to increase wakefulness. In contrast, the data presented in this study imply that activation of vFB neurons, although they activate DAN-aSP13s, do not promote wakefulness. These results suggest that activation of DAN-aSP13s by vFB neurons during sleep is qualitatively different from a direct optogenetic or thermogenetic activation used in previous studies. One potential explanation is that post-learning sleep involves activation of the vFB circuit, which provides both excitatory stimulus to DAN-aSP13s and inhibitory input to motor neurons. Another possibility is that post-learning sleep activates a subset od DAN-aSP13s that do not affect wakefulness (Dag, 2019).

    Thia study has identified a class of sleep-promoting neurons in the ventral layer of FB that are distinct from the well-studied sleep-promoting neurons in the dorsal layer of FB, which regulate sleep homeostasis. Given that vFB neurons enhance sleep and activate DAN-aSP13s for LTM consolidation, whereas dFB neurons are neither necessary nor sufficient for LTM consolidation, it is hypothesized that dFB and vFB neurons promote distinct components of sleep that have different functions. Homeostatic sleep is thought to facilitate memory encoding by downscaling synaptic weights and clearing metabolites from the brain accumulated during wakefulness. In contrast, the function of learning-dependent sleep might be to facilitate memory consolidation by strengthening synaptic connections that were engaged earlier during memory acquisition. Thus, the co-operation of homeostatic- and experience-dependent sleep would facilitate optimal conditions for learning new information and, if appropriate, incorporating it into long-term storage (Dag, 2019).

    Recent studies have implied that sleep in flies, as in humans and rodents, exhibits sleep stages characterized by distinct electrophysiological signatures. Interestingly, the signature of sleep that is induced by activation of the dFB neurons seems to have a simpler oscillatory pattern, recorded by local field potentials, than sleep that is induced by activation of the FB neurons comprising both dFB and vFB neurons. Thus, these data support the hypothesis that dFB and vFB neurons promote sleep with different properties and likely different functions (Dag, 2019).

    It is thought that sleep evolved in animals that are capable of complex learning which requires selective attention. Courtship learning is a multisensory form of learning that requires selective attention of a male to associate multiple learning cues presented by the mated female with the outcome of his own behavior. Accordingly, studies in bees have shown that sleep affects a complex form of learning such as spatial memory but has no role in the simple learning paradigm of proboscis extension. Hence, it would be interesting to investigate whether post-learning sleep is involved in the consolidation of other types of memory in Drosophila, such as the well-studied Pavlovian olfactory associative learning whereby animals associate an individual learning cue with a behavioral contingency (Dag, 2019).

    This work has established a functional link between a novel class of sleep-promoting neurons in the FB, post-learning reactivation of dopaminergic neurons and consolidation of courtship LTM. Moreover, the data suggest that sleep promoting vFB neurons mediate a learning-dependent regulation of sleep that is distinct from the homeostatic control which is facilitated by dFB neurons. Thus, this study uncovered a causal link between sleep-mediated neuronal reactivation and LTM consolidation in Drosophila. In addition, courtship LTM was establish in Drosophila as a tractable model to investigate the mechanisms that link learning-dependent sleep, neuronal reactivation and LTM consolidation (Dag, 2019).

    Sleep restores place learning to the adenylyl cyclase mutant rutabaga

    Sleep plays an important role in regulating plasticity. In Drosophila, the relationship between sleep and learning and memory has primarily focused on mushroom body dependent operant-learning assays such as aversive phototaxic suppression and courtship conditioning. In this study, sleep was increased in the classic mutant rutabaga (rut2080) and dunce (dnc1) by feeding them the GABA-A agonist gaboxadol (Gab). Performance was evaluated in each mutant in response to social enrichment and place learning, tasks that do not require the mushroom body. Gab-induced sleep did not restore behavioral plasticity to either rut2080 or dnc1 mutants following social enrichment. However, increased sleep restored place learning to rut2080 mutants. These data extend the positive effects of enhanced sleep to place learning and highlight the utility of Gab for elucidating the beneficial effects of sleep on brain functioning (Dissel, 2020).

    Morning and evening circadian pacemakers independently drive premotor centers via a specific dopamine relay

    Many animals exhibit morning and evening peaks of locomotor behavior. In Drosophila, two corresponding circadian neural oscillators-M (morning) cells and E (evening) cells-exhibit a corresponding morning or evening neural activity peak. Yet little is known of the neural circuitry by which distinct circadian oscillators produce specific outputs to precisely control behavioral episodes. This study shows that ring neurons of the ellipsoid body (EB-RNs) display spontaneous morning and evening neural activity peaks in vivo: these peaks coincide with the bouts of locomotor activity and result from independent activation by M and E pacemakers. Further, M and E cells regulate EB-RNs via identified PPM3 dopaminergic neurons, which project to the EB and are normally co-active with EB-RNs. These in vivo findings establish the fundamental elements of a circadian neuronal output pathway: distinct circadian oscillators independently drive a common pre-motor center through the agency of specific dopaminergic interneurons (Liang, 2019).

    Light-mediated circuit switching in the Drosophila neuronal clock network

    The circadian clock is a timekeeper but also helps adapt physiology to the outside world. This is because an essential feature of clocks is their ability to adjust (entrain) to the environment, with light being the most important signal. Whereas cryptochrome-mediated entrainment is well understood in Drosophila, integration of light information via the visual system lacks a neuronal or molecular mechanism. This study shows that a single photoreceptor subtype is essential for long-day adaptation. These cells activate key circadian neurons, namely the large ventral-lateral neurons (lLNvs), which release the neuropeptide pigment-dispersing factor (PDF). RNAi and rescue experiments show that PDF from these cells is necessary and sufficient for delaying the timing of the evening (E) activity in long-day conditions. This contrasts to PDF that derives from the small ventral-lateral neurons (sLNvs), which are essential for constant darkness (DD) rhythmicity. Using a cell-specific CRISPR/Cas9 assay, this study shows that lLNv-derived PDF directly interacts with neurons important for E activity timing. Interestingly, this pathway is specific for long-day adaptation and appears to be dispensable in equinox or DD conditions. The results therefore indicate that external cues cause a rearrangement of neuronal hierarchy, which contributes to behavioral plasticity (Schlichting, 2019a).

    Circadian clocks evolved as an adaptation to the continuous change of day and night and are believed to provide organisms a fitness advantage. The underlying molecular machinery includes a transcriptional-translational feedback loop, which generates oscillations of clock gene expression with an endogenous period close to 24 h. This period is approximately 24.2 h in humans, whereas a Drosophila period was reported to be 23.8 h. A key feature of circadian clocks is the ability to entrain to the 24 h environment. This means that the human clock has to be accelerated by about 0.2 h each day, whereas this Drosophila clock has to be slowed down to the same extent. To do so, clocks must integrate external cues, so-called zeitgebers, which are used to synchronize the molecular and physiological properties of the organism (Schlichting, 2019a).

    The most important zeitgeber is light. In mammals, a combination of the traditional photoreception pathway (rods and cones) and the circadian photoreceptor melanopsin in retinal ganglion cells allows for fine-tuning of clock synchronization. Similarly, Drosophila uses the visual system and possibly Rhodopsin 7 (Rh7) within the clock neurons as well as the circadian photoreceptor cryptochrome (CRY) for light synchronization. CRY-mediated entrainment is well understood in Drosophila, whereas less is known about the mechanism of entrainment via the visual system. It consists of seven eye structures: three ocelli, two Hofbauer-Buchner eyelets, and two compound eyes (Schlichting, 2019a).

    The compound eye consists of approximately 800 ommatidia, each harboring 8 photoreceptor cells (Rs): R1-6 are located in the periphery and span the whole depth of the ommatidium. These cells were previously shown to be important for motion vision and express Rhodopsin 1 (Rh1). In the center, R7 is located above R8. These cells have a complex expression pattern of Rh4 and/or Rh3 in R7 and Rh5 or Rh6 in R8. Although the mechanism of light transduction from the visual system to the central clock is still not completely understood, recent work indicates a special role for R8. These cells specifically target the sLNvs in standard conditions of 12 h light and 12 h darkness (LD 12:12). R8 photoreceptors additionally express and react to HisCl1 and can therefore not only act as photoreceptors but also as interneurons (Alejevski, 2109; Schlichting, 2019a and references therein).

    Recent electrophysiological results further suggest that the visual system is able to activate an array of circadian clock neurons, e.g., it can activate the small ventral-lateral neurons (sLNvs), an important center for morning (M) activity. Furthermore, the visual system increases neuronal firing in the large LNvs (lLNvs), the arousal center within the circadian network. The 5th sLNv and the NPF+ dorsal-lateral neurons (LNds), previously implicated as necessary for evening (E) activity, also increase their firing rates in response to visual system stimulation. In addition, the visual system activates several dorsal neurons (DNs), which were recently implicated in connecting the circadian clock to central brain sleep centers. These data suggest that visual input is integrated into the clock network in a parallel fashion, which contradicts a master-oscillator point of view. The latter posits that these are the pigment-dispersing factor (PDF)-expressing neurons (sLNvs and lLNvs), which receive light input and release PDF upon illumination, thereby adjusting their downstream target neurons to the LD cycle (Schlichting, 2019a).

    To investigate the impact of the visual input pathway at the behavioral and neuronal level, this study investigated fly behavior under long-day conditions. Long days cause plastic changes in fly behavior: in standard light-dark cycles (LD 12:12), flies show a bimodal activity pattern with a M anticipation peak around lights on and an E anticipation peak around lights off; this results in a phase relationship of approximately 12 h between the two peaks appropriately adjust their peak timings, i.e., their E peak is phase advanced compared to wild-type flies. Moreover, visual input appears to modulate PDF release from the lateral neurons, which in turn modulates cells important for E activity. In summary, complex interactions between CRY- and PDF-expressing neurons appear to be essential for the behavior under long days: expressing these proteins in different parts of the fly brain alters the behavior of flies and mimics the behavior of high-latitude species using Drosophila melanogaster as a model. It is still unknown, however, which receptors and which neuronal pathways are involved in this adjustment (Schlichting, 2019a).

    This study shows that R8 of the compound eyes is essential for long-day adaptation. These photoreceptor cells connect to the PDF-containing lLNvs and trigger the release of this neuropeptide. Using a cell-specific CRISPR/Cas9 strategy, it was demonstrated that light-mediated PDF directly signals to the PDF receptor (PDFR) on E cells and hence delays E activity. The data implicate a mammal-like structure of clock entrainment, with the visual system activating PDF-expressing clock neurons. These data further support a shift of PDF targets between LD and constant darkness (DD) conditions as well as a more quantitative reorganization of neuronal dominance within the clock network by changes in photoperiod (Schlichting, 2019a).

    The circadian clock is able to entrain to the changes of day and night, with light being the most important zeitgeber. The adaptation to summer-like days is especially important for insects, as they are prone to predator visibility and even more importantly desiccation. Therefore, the circadian clock has to be plastic and be able to adjust behavior in response to changing environments. For example, flies show an additional afternoon peak during summer days under semi-natural conditions, which is thought to be an escape response of the fly from heat. This study shows that Drosophila adjusts its behavior to extremely long photoperiods (LD 20:4) by delaying its E peak as reported previously. Even though this photoperiod can only be found in very northern countries, Drosophila melanogaster is still able to adjust to this extreme light condition, which exemplifies its ability to adapt to various environmental conditions. This delay allows the animal to reduce its activity during the unfavorable midday, when temperatures are highest. Most interestingly, this phenotype is easily visible even without temperature changes, underscoring the importance of light as a major entrainment cue (Schlichting, 2019a).

    A central finding is that flies lacking the compound eyes show an entrainment deficit, i.e., they have an advanced E peak under long-day conditions. Similarly, they show a reduction in M peak amplitude; this is likely due to a failure to properly activate PDF-expressing neurons. Using rhodopsin mutants and by manipulating specific photoreceptors using the GAL4/UAS system, only R8 of the compound eyes appears essential for this summer day response; R8 was previously implicated in the adaptation to nature-like light conditions (Schlichting, 2019a).

    Notably, even flies lacking all compound eyes significantly delay their E peak timing under long photoperiod conditions but to a much smaller extent. This indicates that other photoreceptors also contribute to the E peak delays under these conditions. One likely candidate is the HB-eyelet. Recent work has shown that this photoreceptor contributes to delaying the E peak under high-light-intensity conditions [48]. Similar mechanisms might apply under long days, which is supported by the strongly advanced E peak when rh5-GAL4 and rh6-GAL4-positive neurons, which includes the HB eyelets, were silenced or ablated (Schlichting, 2019a).

    lLNv arbors in the optic lobe are in close proximity to R8 termini, where they most likely interact via cholinergic interneurons in addition to the accessory medulla, which was recently shown to be important for light-evoked responses of the lLNvs. This interaction results in a change of neuronal bursting behavior and hence neuropeptide release. Indeed, this study shows that release of PDF from the lLNvs is necessary and sufficient for proper long-day adaptation (Schlichting, 2019a).

    These results are surprising given a recently published study on how the visual system is connected to the clock neuron network vv. It shows that the visual system can activate a broad spectrum of lateral and dorsal neurons; they include sLNvs, lLNvs, ITP+ LNds, and DN2s, among others. Ablation of PDF neurons left the other neurons responsive to visual input, suggesting a parallel model for clock synchronization, i.e., information from the visual system can be directly transferred to independent classes of clock neurons rather than only via PDF. This new pathway might be involved in the residual delay of E activity in pdf01 flies under long photoperiods, suggesting a potential PDF-independent contribution to long-day adaptation (Schlichting, 2019a).

    PDF stimulates different adenylate-cyclases and increases cAMP, which leads to the stabilization of PER and TIM and consequently a longer period or phase delay. Therefore, one view is that removing the compound eyes decreases PDF release from the lLNvs and phase advances the molecular clock in downstream target neurons like the LNds. This newly discovered 'visual system to LNd pathway' might also enhance CRY-mediated photoentrainment: CRY was shown to activate neurons upon stimulation, similar to the newly identified light activation of clock neuron pathway. Additional activation of the E cells could therefore contribute to the kinetics of TIM degradation, which was recently shown to be important for the phase advance of E activity under long-day conditions (Schlichting, 2019a).

    An intriguing inference of this work is that the principal targets of PDF must change with the environmental conditions. Previous work established the sLNvs as essential for DD rhythmicity, and recent work shows that these neurons are tightly coupled to the dorsal clock neurons in DD: speeding up the PDF neurons forced the DN1s to follow the short period of the sLNvs. In LD, however, this connection is much weaker, and the cell-type-specific CRISPR/Cas9 knockout strategy shows that it is the PDF-expressing lLNvs that communicate with the LNd neurons. The data show that the lLNv to LNd connection is important in LD conditions but does not affect DD behavior (Schlichting, 2019a).

    Importantly, the data not only indicate a qualitative shift of PDF targets between DD and LD but also suggest a quantitative shift of dominance, depending on photoperiod or the time of light exposure. In DD, the sLNvs are necessary for rhythmic behavior and show robust cycling in PER oscillations, whereas the lLNvs lose PER rhythms as early as the second day of DD. In equinox conditions, both groups may be relevant: PDF from either the sLNvs or lLNvs is sufficient for WT behavior, and only knockdown in both sets of neurons is able to reproduce the pdf01 mutant phenotype. In long photoperiods, however, PDF from the lLNvs is necessary and sufficient for proper entrainment, whereas the sLNvs do not contribute to E peak timing. The data therefore point to a profound circuit switch in response to photoperiod, analogous to the neurotransmitter switching that occurs in the mammalian paraventricular nucleus in response to long photoperiods (Schlichting, 2019a).

    A similar circuit reorganization might also occur in the principal mammalian brain clock neuron location, the suprachiasmatic nucleus (SCN). Light information from the visual system is transferred to cells in the ventral part of the SCN, which expresses VIP. VIP functions similarly to Drosophila PDF and is not only important for communication between different parts of the SCN but also essential for seasonal encoding. This is because VIP knockout mice show no change in peak width as measured by in vivo electrophysiological recordings in response to entrainment to different photoperiods]. This suggests that VIP is not only involved in relaying light information beyond the ventral SCN but also in the response to light duration as shown in this study for PDF in Drosophila. It will be interesting to see whether different VIP-expressing SCN neurons are involved in this response (Schlichting, 2019a).

    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. The 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 (Schlichting, 2019a).

    Neuronal activity in non-LNv clock cells is required to produce free-running rest:activity rhythms in Drosophila

    Circadian rhythms in behavior and physiology are produced by central brain clock neurons that can be divided into subpopulations based on molecular and functional characteristics. It has become clear that coherent behavioral rhythms result from the coordinated action of these clock neuron populations, but many questions remain regarding the organizational logic of the clock network. This study used targeted genetic tools in Drosophila to eliminate either molecular clock function or neuronal activity in discrete clock neuron subsets. Neuronal firing was found to be necessary across multiple clock cell populations to produce free-running rhythms of rest and activity. In contrast, such rhythms are much more subtly affected by molecular clock suppression in the same cells. These findings demonstrate that network connectivity can compensate for a lack of molecular oscillations within subsets of clock cells. It was further shown that small ventrolateral (sLNv) clock neurons, which have been characterized as master pacemakers under free-running conditions, cannot drive rhythms independent of communication between other cells of the clock network. In particular, an essential contribution of the dorsolateral (LNd) clock neurons was pinpointed and manipulations that affect LNd function were shown to reduce circadian rhythm strength without affecting molecular cycling in sLNv cells. These results suggest a hierarchical organization in which circadian information is first consolidated among one or more clock cell populations before accessing output pathways that control locomotor activity (Bulthuis, 2019).

    Sleep pressure regulates mushroom body neural-glial interactions in Drosophila

    Sleep is a behavior that exists broadly across animal phyla, from flies to humans, and is necessary for normal brain function. Recent studies in both vertebrates and invertebrates have suggested a role for glial cells in sleep regulatory processes. Changes in neural-glial interactions have been shown to be critical for synaptic plasticity and circuit function. This study tested the hypothesis that changes in sleep pressure alters neural-glial interactions. In the fruit fly, Drosophila melanogaster, sleep is known to be regulated by mushroom body (MB) circuits. The technique GFP Reconstitution Across Synaptic Partners (GRASP) was used to test whether changes in sleep pressure affect neural-glial interactions between MB neurons and astrocytes, a specialized glial cell type known to regulate sleep in flies and mammals. The MB-astrocyte GRASP signal was reduced after 24 h of sleep deprivation, whereas the signal returned to baseline levels following 72 h of recovery. Social enrichment, which increases sleep drive, similarly reduced the MB-astrocyte GRASP signal. No changes were observed in the MB-astrocyte GRASP signal over time-of-day, or following paraquat exposure or starvation. These data suggest that changes in sleep pressure are linked to dynamic changes in neural-glial interactions between astrocytes and neuronal sleep circuits, which are not caused by normal rest-activity cycles or stressors (Vanderheyden, 2019).

    Neuronal activity in non-LNv clock cells is required to produce free-running rest: activity rhythms in Drosophila

    Circadian rhythms in behavior and physiology are produced by central brain clock neurons that can be divided into subpopulations based on molecular and functional characteristics. It has become clear that coherent behavioral rhythms result from the coordinated action of these clock neuron populations, but many questions remain regarding the organizational logic of the clock network. This study used targeted genetic tools in Drosophila to eliminate either molecular clock function or neuronal activity in discrete clock neuron subsets. This study finds that neuronal firing is necessary across multiple clock cell populations to produce free-running rhythms of rest and activity. In contrast, such rhythms are much more subtly affected by molecular clock suppression in the same cells. These findings demonstrate that network connectivity can compensate for a lack of molecular oscillations within subsets of clock cells. It was further shown that small ventrolateral (sLNv) clock neurons, which have been characterized as master pacemakers under free-running conditions, cannot drive rhythms independent of communication between other cells of the clock network. In particular, this study has pinpointed an essential contribution of the dorsolateral (LNd) clock neurons and shows that manipulations that affect LNd function reduce circadian rhythm strength without affecting molecular cycling in sLNv cells. These results suggest a hierarchical organization in which circadian information is first consolidated among one or more clock cell populations before accessing output pathways that control locomotor activity (Bulthuis, 2019).

    A circadian output center controlling feeding:fasting rhythms in Drosophila

    Circadian rhythms allow animals to coordinate behavioral and physiological processes with respect to one another and to synchronize these processes to external environmental cycles. In most animals, circadian rhythms are produced by core clock neurons in the brain that generate and transmit time-of-day signals to downstream tissues, driving overt rhythms. The neuronal pathways controlling clock outputs, however, are not well understood. Furthermore, it is unclear how the central clock modulates multiple distinct circadian outputs. Identifying the cellular components and neuronal circuitry underlying circadian regulation is increasingly recognized as a critical step in the effort to address health pathologies linked to circadian disruption, including heart disease and metabolic disorders. Building on the conserved components of circadian and metabolic systems in mammals and Drosophila melanogaster, this study used a recently developed feeding monitor to characterize the contribution to circadian feeding rhythms of two key neuronal populations in the Drosophila pars intercerebralis (PI; the central neuroendocrine system), which is functionally homologous to the mammalian hypothalamus. Thermogenetic manipulations of PI neurons expressing the neuropeptide SIFamide (SIFa) as well as mutations of the SIFa gene degrade feeding:fasting rhythms. In contrast, manipulations of a nearby population of PI neurons that express the Drosophila insulin-like peptides (DILPs) affect total food consumption but leave feeding rhythms intact. The distinct contribution of these two PI cell populations to feeding is accompanied by vastly different neuronal connectivity as determined by trans-Tango synaptic mapping. These results for the first time identify a non-clock cell neuronal population in Drosophila that regulates feeding rhythms and furthermore demonstrate dissociable control of circadian and homeostatic aspects of feeding regulation by molecularly-defined neurons in a putative circadian output hub (Dreyer, 2019).

    At its core, the circadian system is made up of central clock neurons in the brain that keep time through the presence of cell-autonomous molecular clocks. To enact behavioral rhythms, these clock cells must be connected through output pathways to downstream neuronal populations that directly control behavioral outputs; therefore, a complete understanding of circadian regulation of behavior depends on the delineation of output circuitry. This study identified a population of SIFa+ neurons in the pars intercerebralis that comprises part of the output pathway controlling feeding:fasting rhythms in flies. Constitutive activation of these cells strongly compromises normal patterns of feeding behavior, including producing a substantial percentage of flies that feed arrhythmically. This study also pinpointed a specific contribution of SIFa peptide to feeding rhythms, as SIFa mutant and RNAi knockdown lines show similar reductions of feeding rhythm strength (Dreyer, 2019).

    The identification of a neuronal population and associated signaling molecule for the control of feeding:fasting rhythms should facilitate future studies aimed at further dissecting feeding output circuits, with the ultimate aim of tracing the pathway to motor neurons that directly control feeding. To that end, the trans-Tango analysis demonstrated that many neurons throughout the brain are postsynaptic to SIFa+ PI cells, including in areas such as the AL, which is involved in olfactory processing, and the SEZ, which is involved in gustatory processing and also contains feeding-related motor neurons. It will be of interest to more definitively determine the functional and neurochemical identity of postsynaptic neurons and to assess whether manipulations of SIFa receptor expression in these putative downstream output cells can recapitulate the feeding phenotypes observed following SIFa+ cell manipulations (Dreyer, 2019).

    A role for SIFa in feeding regulation is supported by a recent study that demonstrated that SIFa modulates olfactory processing under conditions of starvation. Flies normally show sensitized AL projection neuron responses to food odors following starvation, however, this sensitization is absent in flies in which SIFa expression has been reduced through RNAi mechanisms. Martelli (2017) also showed that SIFa+ cells exhibit increased activity in response to starvation, and that thermogenetic activation of SIFa+ cells increases food consumption in satiated flies. Their experiments suggest that SIFa tunes sensory responsiveness to food cues according to the energy status of the fly, which subsequently increases feeding propensity in energy-depleted states. The described effects in this study identify an additional function of SIFa in dictating temporal patterns of feeding (Dreyer, 2019).

    Interestingly, although the current findings of increased food consumption following SIFa+ cell activation are in line with those of Martelli (2017), this study found that feeding amount was also elevated in SIFa mutant flies, which is not predicted by a model in which SIFa peptide solely serves to increase appetitive and feeding behavior. This suggests that the exact nature of the regulation of feeding by SIFa is complex and may vary depending on environmental conditions and internal state. It is unclear why food consumption would be similarly affected by manipulations that eliminate SIFa peptide and those that hyperactivate SIFa+ cells, which should result in heightened SIFa signaling. One possibility is that constitutive SIFa+ cell activity could ultimately deplete SIFa stores, thus mimicking the SIFa mutant phenotype. Alternatively, feeding phenotypes may be affected differentially by SIFa mutations, which are present throughout development, compared to adult-specific thermogenetic activation. Regardless of whether acute SIFa signaling stimulates or inhibits food consumption, the fact that SIFa+ cell activation and reduction of SIFa signaling via mutations, cell ablation, or RNAi knockdown consistently degrade feeding:fasting rhythms provides strong evidence for a central contribution to the determination of the timing of feeding (Dreyer, 2019).

    Flies in which SIFa+ cells are constitutively activated or that lack SIFa peptide due to cellular ablation or mutation also exhibit significantly reduced rest:activity rhythms, which is consistent with previous results demonstrating weakened locomotor rhythms following ablation of these cells. This effect was most pronounced in SIFa>reaper flies, indicating a potential for additional neurotransmitters emanating from SIFa+ cells to contribute to the regulation of locomotor activity. The overlap of feeding:fasting and rest:activity disruption raises the question of whether SIFa cells independently regulate feeding and locomotor rhythms, or whether one of these is indirectly affected secondary to changes in the other. Feeding and locomotor activity are interconnected behaviors that usually coincide, as animals primarily feed during their active phase. Nevertheless, feeding and locomotor rhythms can be dissociated in both flies and mammals. For example, adipocyte-specific knockout of the mammalian clock gene Arntl attenuates feeding rhythms in mice while leaving rest:activity rhythms intact, and mutations in mammalian per1 and per2 genes have differential effects on the phasing of locomotor and feeding rhythms. A similar phenotype has been noted in flies, as cell-specific abrogation of the molecular clock in the Drosophila fat body, a peripheral metabolic tissue, selectively alters the phase and magnitude of feeding rhythms without changing cycles of rest and activity (Xu, 2008). More recently, it was shown that manipulations that downregulate DH44 signaling or silence neurons expressing the hugin peptide significantly degrade rest:activity rhythm strength in DD conditions but leave the strength of DD feeding:fasting intact. Taken together, these results confirm that feeding:fasting rhythms are under de facto circadian control and do not simply occur secondary to rest:activity rhythms. Because locomotor rhythm disruption can occur independent of changes in feeding behavior, it is concluded that the effects of the SIFa manipulations likely reflect direct feeding:fasting rhythm regulation (Dreyer, 2019).

    In addition to affecting rest:activity and feeding:fasting rhythms, adult-specific SIFa+ cell manipulations also resulted in high lethality, particularly in the case of adult-specific neuronal silencing. This suggests that SIFa+ cells perform some necessary function in the adult animal, though it seems that SIFa peptide itself is dispensable for survival, as mutants eclose at expected Mendelian ratios. Intriguingly, it was found that a substantial number of flies eclosed from genetic crosses that result in SIFa+ cell ablation during developmental stages due to expression of the apoptotic gene reaper. The lack of a lethality phenotype in SIFa+ ablated flies is perhaps due to compensatory changes in these flies that are not present following adult-specific manipulations. It is unclear whether the lethality phenotype is related to alterations in feeding behavior following SIFa+ cell manipulations, or whether other, yet unidentified contributions of SIFa+ cells are responsible, but as there is little evidence for SIFa expression in cells outside of the PI, it is likely that the phenotype stems from dysregulation of these cells (Dreyer, 2019).

    Together with previous findings, the current results add to a growing understanding of the PI in the control of circadian outputs. The PI is situated in a region of the Drosophila brain that is near the axon terminals of multiple groups of core clock cells, and previous work has shown anatomical and functional connections between clock cells and multiple PI populations, including those expressing DH44, SIFa and DILPs. These clock cell inputs could allow PI cells, which lack molecular clocks, to transmit circadian information to downstream output regions. Interestingly, the PI cell populations appear to differentially contribute to circadian outputs. As detailed above, DH44+ cells selectively regulate rest:activity rhythms while SIFa+ cells contribute to both rest:activity and feeding:fasting rhythms. DILP+ PI cells contribute to neither behavioral rhythm but instead have been shown to modulate circadian gene expression in the fat body. These results support the hypothesis that the PI is a circadian output hub that channels core clock input into anatomically distinct output pathways to coordinately regulate different circadian outputs (Dreyer, 2019).

    Though no effect of DILP+ cell manipulations on feeding:fasting rhythms were observed, changes in overall food intake following IPC activation were observed in two independent assays, which is consistent with a homeostatic role for these cells. The IPCs receive feedback from a range of circulating peptides and are also indirectly targeted by satiety signals secreted from the fat body integrating information regarding the nutritional status of a fly as one component of the intricate regulation of energy homeostasis. DILP+ neurons have also recently been shown to play a role in nutrient sensing in female flies, contributing to the modulation of reproductive dormancy by affecting overall feeding and maintaining females in a metabolically active state. Generally, IPC neuronal activity is regulated by feeding status, as the cells are more active in the fed versus starved state, which likely results in increased DILP secretion in fed flies. In turn, insulin/IGF signaling (IIS) is an integral regulator of growth and development and affects a range of physiological attributes including metabolism, reproduction, stress response, and aging (Dreyer, 2019).

    DILPs have also been directly implicated in regulating feeding behavior, with several studies demonstrating anorexigenic effects of increased DILP signaling, as well as of drosulfakinin peptides, which are an additional output of the IPCs and act as a satiety signal. These effects are in line with evidence demonstrating increased activation of IPCs and release of DILPs in the fed state. In contrast, it has also been shown that DILP+ cell silencing can result in hypophagia, as indicated by reduced fecal output, and that thermogenetic DILP+ cell activation can either stimulate or inhibit feeding depending on metabolic status. Thus, the role of the DILP+ PI cells in determining overall food consumption, similar to SIFa peptides, is likely complex. Given this, the finding of increased feeding following IPC stimulation, though counterintuitive, is not without precedent, and may occur as a result of an interaction between diet type, insulin signaling, and the metabolic condition of the flies, especially as they are exposed to a carbohydrate-only diet in the FLIC and CAFE assays. Alternatively, increased feeding could occur if DILPs are depleted by extended IPC activation. This possibility could be directly tested using recently-developed DILP2 reporter flies, which allow for sensitive measurements of circulating DILP2 levels (Dreyer, 2019).

    The contrasting effects of DILP+ and SIFa+ PI cell activation demonstrate dissociable control over homeostatic and circadian regulation of feeding by these two populations of PI cells. The results of the trans-Tango analyses provide a potential anatomical basis for this and suggest that DILP+ and SIFa+ PI cells rely on different signaling paradigms. Given their limited connectivity to other brain regions, the IPCs likely release DILPs systemically to act on target tissues, including the brain, via long-distance diffusion through the hemolymph. In contrast, SIFamidergic cells appear to act via direct synaptic connections to impact widespread brain areas. The differences in kinetics between these two signaling mechanisms could underlie the functional differences of these cell populations with respect to feeding regulation, with IPC activity reflecting overall energy status and therefore controlling homeostatic aspects of feeding, and SIFa cells regulating moment-to-moment feeding decisions and therefore controlling circadian patterns of feeding. In addition, the downstream connections of SIFa+ cells to central clock neurons, including l-LNvs and s-LNvs as well as LNds, implicates SIFa+ cells in feedback control of the central clock. Previous work found no alterations in central clock timing following SIFa+ cell ablation; however, as SIFa+ cells appear to lie at a crossroads of energetic signaling, it follows that they would have the capacity to relay that information back to the core clock and affect behavioral changes that are attuned to the circadian patterns of activity as necessary, perhaps under conditions in which food access is limited (Dreyer, 2019).

    Research into metabolic and feeding control continues to uncover a dense web of interconnected regulators, indicative of how integral proper nutrient signaling is to overall organismal health. The partial reduction of feeding rhythm strength ascribed to SIFa here leaves room for the discovery of additional signals affecting circadian feeding rhythms. Two promising neuropeptides that have been shown to affect feeding behaviors are short neuropeptide F and allatostatin A, both of which also have been associated with sleep regulation in flies. Characterizing the complete output circuit of circadian feeding behavior in flies will help identify the most important contributors to synchronized feeding patterns and increase understanding of the profound metabolic consequences of circadian disruption (Dreyer, 2019).

    Neuron-specific knockouts indicate the importance of network communication to Drosophila rhythmicity

    Animal circadian rhythms persist in constant darkness and are driven by intracellular transcription-translation feedback loops. Although these cellular oscillators communicate, isolated mammalian cellular clocks continue to tick away in darkness without intercellular communication. To investigate these issues in Drosophila, behavior as well as molecular rhythms were assayed within individual brain clock neurons while blocking communication within the ca. 150 neuron clock network. CRISPR-mediated neuron-specific circadian clock knockouts were also generated. The results point to two key clock neuron groups: loss of the clock within both regions but neither one alone has a strong behavioral phenotype in darkness; communication between these regions also contributes to circadian period determination. Under these dark conditions, the clock within one region persists without network communication. The clock within the famous PDF-expressing s-LNv neurons however was strongly dependent on network communication, likely because clock gene expression within these vulnerable sLNvs depends on neuronal firing or light (Schlichting, 2019b).

    Neuronal networks make myriad contributions to behavior and physiology. By definition, individual neurons within a network interact, and different networks also interact to coordinate specialized functions. For example, the visual cortex and motor output centers must coordinate to react properly to environmental changes. In a less immediate fashion, sleep centers and circadian clocks are intertwined to properly orchestrate animal physiology. The brain clock is of special interest: it not only times and coordinates physiology within neuronal tissues but also sends signals to the body to keep the entire organism in sync with the cycling external environment (Schlichting, 2019b).

    The small, circumscribed Drosophila clock network is ideal to address circadian communication issues. The comparable region in mammals, the suprachiasmatic nucleus, is composed of thousands of cells depending on the species. There are in contrast only 75 clock neurons per hemisphere in Drosophila. These different clock neurons can be divided into several subgroups according to their location within the fly brain. There are 4 lateral and three dorsal neuron clusters, which have different functions in controlling fly physiology (Schlichting, 2019b).

    The four small ventro-lateral neurons (sLNvs) are arguably the most important of the 75 clock neurons. This is because ablating or silencing these neurons abolishes rhythms in constant darkness (DD). They reside in the accessory medulla region of the fly brain, an important pacemaker center in many insects, and express the neuropeptide PDF. In addition, they are essential for predicting dawn. A very recent study suggests that the sLNvs are also able to modulate the timing of the evening (E) peak of behavior via PDF. The other ventral-lateral group, the four large-ventro-lateral neurons (lLNvs), also express PDF and send projections to the medulla, the visual center of the fly brain; they are important arousal neurons. Consistent with the ablation experiments mentioned above, the absence of pdf function or reducing PDF levels via RNAi causes substantial arrhythmic behavior in DD (Schlichting, 2019b).

    Other important clock neurons include the dorso-lateral neurons (LNds), which are essential for the timing of the E peak and adjustment to long photoperiods. Two other clock neuron groups, the lateral-posterior neurons (LPN