The Interactive Fly
Zygotically transcribed genes
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 (http://www.neuron.org/cgi/content/full/32/4/657/DC1). 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).
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).
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).
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, 48 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).
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 PittendrighDaan 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 ME 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 unimodaldiurnal to bimodalnocturnal 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).
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).
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 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).
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).
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).
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).
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) neuronsâa brain region involved in locomotor control and implicated in sleep and sleep homeostasisâarborize 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).
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)
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).
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).
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).
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).
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).
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).
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).
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 sleepâarousal regulation but are rather important for maintaining circadian rhythmicity under DD (reviewed by Helfrich-FÃ¶rster, 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).
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).
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).
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).
The model organism 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 regimesâincluding 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).
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).
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).
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).
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 2
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).
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).
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-GAL4induced 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).
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 2â3 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)
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 (Knust, 2014).
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).
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).
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).
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).
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 (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).
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).
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 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).
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).
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).
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).
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).
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).
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).
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).
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).
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.
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).
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).
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).
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).
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 45 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).
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 2Â3 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).
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).
Alzheimers 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).
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 TARAâs 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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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 otherthey 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 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).
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).
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
(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).
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).
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).
Sleep rebound - the increase in sleep that follows sleep deprivation (SD) - is a hallmark of homeostatic sleep regulation. This, this study developed an efficient thermogenetic method of inducing SD in Drosophila that produces a strong rebound. To develop a thermogenetic method of SD suitable for screening, different populations of wake-promoting neurons labeled by Gal4 drivers were thermogenetically stimulated. Thermogenetic activation of neurons marked by the c584-Gal4 driver produces both strong sleep loss and a substantial rebound that is more subject to genetic regulation and more consistent within genotypes than rebound following mechanical or caffeine-induced SD. This driver was then used to induce SD in a screen of 1,741 mutagenized lines generated by the Drosophila Gene Disruption Project. Flies were subjected to 9 h of SD during the dark period and released from SD 3 h before lights-on. Recovery was measured over the 15 h following SD. Following identification of lines with reduced sleep rebound, baseline sleep and sleep depth was characterized before and after SD. Two lines were identified that consistently exhibit a blunted increase in the duration and depth of sleep after thermogenetic SD. Neither of the two genotypes has reduced total baseline sleep. Statistical analysis across all screened lines shows that genotype is a strong predictor of recovery sleep, independent from effects of genotype on baseline sleep. These data show that rebound sleep following thermogenetic SD can be genetically separated from sleep at baseline. This suggests that genetically controlled mechanisms of sleep regulation not manifest under undisturbed conditions contribute to sleep rebound following thermogenetic SD (Dubowy, 2016).
In the fruit fly Drosophila melanogaster, as in mammals, acute exposure to a high dose of ethanol leads to stereotypical behavioral changes beginning with increased activity, followed by incoordination, loss of postural control, and eventually, sedation. The mechanism(s) by which ethanol impacts the CNS leading to ethanol-induced sedation and the genes required for normal sedation sensitivity remain largely unknown. This study identified the gene apontic (apt), an Myb/SANT-containing transcription factor that is required in the nervous system for normal sensitivity to ethanol sedation. Using genetic and behavioral analyses, it was shown that apt mediates sensitivity to ethanol sedation by acting in a small set of neurons that express Corazonin (Crz), a neuropeptide likely involved in the physiological response to stress. The activity of Crz neurons regulates the behavioral response to ethanol, as silencing and activating these neurons affects sedation sensitivity in opposite ways. Furthermore, this effect is mediated by Crz, as flies with reduced crz expression show reduced sensitivity to ethanol sedation. Finally, both apt and crz were found to be rapidly upregulated by acute ethanol exposure. Thus, two genes and a small set of peptidergic neurons were identified that regulate sensitivity to ethanol-induced sedation. It is proposed that Apt regulates the activity of Crz neurons and/or release of the neuropeptide during ethanol exposure (McClure, 2013).
This work identifies apt and crz that mediate the fly's sensitivity to ethanol-induced sedation. Flies with reduced expression of either gene display dramatically decreased ethanol sedation sensitivity; thus, both apt and crz normally promote ethanol sedation. Normal sensitivity to ethanol sedation requires apt expression in neurons during two distinct life stages, metamorphosis and adulthood. Apt function in a subset of crz-expressing neurons (approximately 6 of the 12-16 crz-expressing cells) is necessary and sufficient for normal sensitivity to ethanol sedation. Acute manipulations of the activity of crz neurons led to altered ethanol sedation sensitivity, demonstrating that these neurons play an active role in regulating the behavioral response to ethanol-induced sedation. The neuropeptide Crz is also involved in ethanol sedation, as flies with reduced crz expression, specifically during adulthood, show dramatically decreased ethanol sedation sensitivity. Finally, in response to acute ethanol exposure the expression of both apt and crz are rapidly upregulated during ethanol exposure. It is hypothesized that the Apt-Crz system, functioning in a very small group of neurosecretory cells, may be an early target of ethanol in the fly brain whose function is crucial for normal sensitivity to ethanol (McClure, 2013).
How does Apt regulate ethanol-induced sedation? Although Apt's role could be to regulate the expression of crz, no such function was observed. It is thus postulated that Apt functions to regulate the activity of crz neurons and/or neuropeptide release. For example, Apt, acting as a transcription factor, could regulate the transcription of proteins required for synthesis, packaging, and/or release of Crz and possibly other neuropeptides. Alternatively, Apt could regulate the expression of proteins required for synapse formation. In support of these two possibilities, apt mutant embryos were observed to have defective synaptic transmission at the neuromuscular junction, as well as fewer numbers of active zones within motoneurons, indicating a presynaptic defect (McClure, 2013).
Another possible function for Apt in regulating ethanol sedation behavior may be found in its neuronal requirement during metamorphosis, a time of intense remodeling to construct the adult CNS. During embryogenesis, Apt functions in multiple morphogenetic processes, including tracheal, head, CNS, and heart morphogenesis, as well as border cell migration. It is therefore possible that during metamorphosis Apt establishes proper development and neuronal connectivity of the adult CNS, and in particular the Crz neurons. However, this possibility seems somewhat unlikely given that the adult CNS in apt13-66 flies appeared normal, as was the number and morphology of Crz-expressing neurons. Additionally, it was found that adult-specific expression of apt in neurons was necessary for normal sedation sensitivity. However, there may be subtle defects in the adult CNS of apt mutant flies, which were not detected, that could contribute to their altered sedation sensitivity (McClure, 2013).
Apt shows highest sequence conservation with the human FSBP, a negative regulator of transcription of the gamma chain of fibrinogen (see Starz-Gaiano, 2008). Sequence conservation between apt and FSBP is observed within the DNA-binding domain. Interestingly, moderate alcohol consumption in humans has been known to exert a cardioprotective effect, in part by lowering levels of circulating Fibrinogen. The mechanism for how alcohol consumption regulates Fibrinogen is currently unknown, but in light of the current findings it is speculated that it may occur at the level of transcription. Ethanol exposure in flies was found to acutely upregulate apt expression. A similar situation may occur in humans, whereby alcohol consumption could upregulate the transcription of FSBP, ultimately leading to negative regulation of the gamma chain of fibrinogen and lowered levels of circulating Fibrinogen, which in turn would provide cardioprotection (McClure, 2013).
The observations implicate crz-expressing neurons in the regulation of ethanol sedation behavior, a function not previously attributed to these neurons. This study demonstrated that adult-specific silencing of crz neurons significantly reduced ethanol sedation sensitivity, while increasing their activity resulted in the opposite phenotype, an increase in ethanol sedation sensitivity. Based on the observation that inhibiting crz expression also reduced ethanol sedation sensitivity, it is believed that the phenotypes associated with crz neuronal manipulations reflect changes in the release of the neuropeptide Crz and activation of its signaling pathway. However, a few Crz neurons also express the short Neuropeptide F (sNPF). sNPF is considered to be a multifunctional neuropeptide due to its broad expression in diverse neuronal types, and its possible role in crz-expressing neurons and in ethanol sedation sensitivity has not been excluded. However, the observation that Apt function is required in a subset of crz neurons to promote ethanol sedation behavior, firmly establishes the importance of these neurons in mediating the behavioral response to ethanol. The data also suggest that the function of both genes, crz and apt, overlaps in a small set of neurons likely located in the pars lateralis (PL) to mediate the behavioral response to ethanol-induced sedation (McClure, 2013).
It has been hypothesized that Crz is released in response to various types of stress in insects (Veenstra, 2009; Boerjan, 2010), and that this could explain its pleiotropic effects. This hypothesis was bolstered by a recent study showing that flies deficient in Crz are resistant to metabolic, osmotic, and oxidative stress, as measured by survival (Zhao, 2010). In addition, Crz plays a role in stress physiology through its association with well characterized stress hormones. For instance, crz-expressing neurons in the PL also express receptors for two diuretic hormones, DH44 and DH31. By virtue of receptor similarity, DH44 and DH31 are related to corticotrophin-releasing factor (CRF) and calcitonin-gene related peptide (CGRP), respectively, both of which mediate the mammalian physiological and behavioral responses to stress. Interestingly, both CRF and CGRP act to inhibit secretion of GnRH in the mammalian hypothalamus. This is significant because Crz is thought to be the homolog of mammalian GnRH, and suggests that analogous regulation occurs in Drosophila (Cazzamali, 2002). It is thus possible that in flies a stress signal or the animal's stress status may be relayed to Crz neurons and alters their function. Thus, based on its functional and molecular associations with stress physiology, it is tempting to speculate that the role of Crz signaling in ethanol sedation sensitivity is related to a stress response. A previous study has shown that stress, in the form of heat shock, induces tolerance to a subsequent ethanol exposure, and that ethanol tolerance relies on the gene hangover, a large nuclear zinc-finger protein, that mediates various other stress responses (Scholz, 2005). In addition, several genes related to stress responses have been shown to be upregulated by ethanol exposure in transcriptional profiling studies, including nearly half of all Drosophila heat shock protein genes, as well as genes involved in the regulation of oxidative stress and aging. Importantly, a maladaptive response to stress has been shown in humans to be a major and common element contributing to drug addiction. Finally, an increase in ethanol self-administration has been observed in animal models with physical, social, and emotional stress. In light of these findings, it will be interesting to further explore the role of Crz and its function in stress physiology and the regulation of ethanol-related behaviors (McClure, 2013).
Neuropeptides are diverse signaling molecules that mediate a broad spectrum of physiological and behavioral processes. Several studies have linked neuropeptides to behavioral responses to ethanol. For instance, one of the first ethanol sensitivity mutants described in Drosophila, amnesiac, encodes a neuropeptide homologous to the vertebrate pituitary adenylate cyclase-activating peptide. In addition, mice lacking either neuropeptide Y (NPY), a neuromodulator abundantly expressed in many regions of the CNS, or its Y1 receptor subtype, display increased ethanol consumption and resistance to ethanol sedation, whereas animals overexpressing NPY show the opposite behavioral phenotypes. Neuropeptide F (NPF), the sole member of the NPY family in Drosophila, and its receptor NPFR1, has similarly been shown to mediate the fly's sensitivity to ethanol-induced sedation. Finally, flies with neuronal perturbations in the insulin signaling pathway displayed increased ethanol sedation sensitivity. These studies, implicating the neuropeptide Crz in sensitivity to ethanol sedation, suggest that neuropeptides are important regulators of the behavioral response to ethanol, and it would therefore be interesting to survey all known Drosophila neuropeptides and their downstream signaling components for possible role(s) in ethanol-related behaviors (McClure, 2013).
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date revised: 25 June 2016
Zygotically transcribed genes
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