Circadian photoreceptor CRYPTOCHROME promotes wakefulness under short winter-like days via a GABAergic circuitry

A cardinal symptom of seasonal affective disorder (SAD, also known as winter depression) is hypersomnolence, while the cause of this “winter sleepiness” is not known. Here we found that lack of the circadian photoreceptor cryptochrome (cry) leads to increased sleep under short winter-like days in fruit flies, reminiscent of the hypersomnolence in SAD. CRY functions in neurons that synthesize the major inhibitory neurotransmitter GABA, including the small ventral lateral neurons which are known to be circadian pacemakers, and down-regulates the GABAergic tone. This in turn leads to increased neural activity of the wake-promoting large ventral lateral neurons, a subset of circadian neurons that are inhibited by GABA-A receptor. CRY protein is known to be degraded by light, thus rendering CRY to be functional within this GABAergic circuitry to enhance wakefulness only under short day length. Taken together, we demonstrate a mechanism that specifically regulates wakefulness under short winter-like days, which may provide insights regarding the winter sleepiness in SAD.


Introduction
Seasonal affective disorder (SAD), also known as winter depression, is characterized by the onset of depression in fall/winter months and spontaneous remission in the spring/summer (Rosenthal et al., 1984).It is generally agreed that SAD is caused by lack of day light in the fall/winter months due to the short day length (or photoperiod), as bright light therapy is effective and most commonly used for treating the depression associated with SAD (Golden et al., 2005).The prevalence of SAD is ~1-10% world-wide, with symptoms lasting for approximately 40% of the year (Kurlansik and Ibay, 2012).About 64-80% of SAD patients report a winter increase in sleep, ranging from 30 min to 2 h longer in duration compared to controls, which is considered to be a distinguishing symptom in the characterization and diagnosis of SAD (Wescott et al., 2020).Currently almost nothing is known regarding the underlying mechanism of this winter hypersomnolence.This phenomenon of winter hypersomnolence in SAD patients implicates distinct mechanisms that regulate sleep under short winter-like photoperiods vs. longer non-winter-like photoperiods, as the sleep of these individuals appear to be selectively perturbed under short photoperiods.However, the mechanisms by which sleep duration is determined under different photoperiods have not been characterized.
Since the circadian clock is believed to be important for adaptations to seasonal changes in the environment and in particular, seasonal changes of photoperiod, we hypothesize that the circadian clock may also participate in regulating sleep duration under different photoperiods (Wood and Loudon, 2014).
To address our hypothesis, we tested fruit flies mutant for different circadian clock genes under a range of photoperiods.We found that flies lacking the circadian photoreceptor CRYPTOCRHOME (CRY) display increased sleep duration specifically under short photoperiods, similar to the winter hypersomnolence in SAD.
Genetic and pharmacological analysis identified that CRY is functioning in GABAergic neurons and acts upon GABA-A receptor to promote wakefulness.We further narrowed down the neural circuitry mediating the influences of CRY on sleep by demonstrating that cry deficiency increases the GABAergic tone and reduces calcium concentration in the wake-promoting large ventral lateral neurons (l-LNvs) which are known to be GABA-A+ (Hamasaka et al., 2005;Parisky et al., 2008).
Consistently, inhibiting these neurons increases sleep while activating them blocks the effects of cry deficiency on sleep.CRY likely functions in part in the GABAergic small ventral lateral neurons (s-LNvs), and lack of cry increases GABA level and the activity of these cells while impairing their GABA transmission suppresses the sleep phenotype of cry mutants (Allada and Chung, 2010).In summary, here we identify a potential role for CRY in down-regulating the GABAergic signaling from the s-LNvs to the l-LNvs specifically under short photoperiod.This in turn enhances the neural activity of the wake-promoting l-LNvs, resulting in increased wakefulness during short winter-like days.These findings reveal a mechanism underlying how sleep duration is determined under winter-like photoperiod, while disruptions of this regulatory system may be related to the winter hypersomnolence associated with SAD.

cry mutation increases sleep duration specifically under short photoperiods
We assessed the sleep of flies mutant for circadian clock gene period (per 0 ), timeless (tim 0 ), clock (clk jrk ), cycle (cyc 0 ) and cry (cry b ) under a range of photoperiods (Figure 1A and B) (Allada, 1998;Konopka and Benzer, 1971;Sehgal et al., 1994;Stanewsky, 1998).We found that cry b mutation, which is known to be a loss of function or severe hypomorphic allele, leads to increased sleep duration under 4 h light: 20 h dark condition (4L20D) and 8L16D but not under longer photoperiods (Stanewsky, 1998).
Since this phenotype recapitulates the winter hypersomnolence of SAD patients, we further characterized the effects of cry deficiency on sleep.We focused on sleep under 4L20D, as the extent of sleep increase is slightly larger than that of 8L16D.We also demonstrated that cry mutation lengthens sleep duration in both male and female flies, while waking activity is not significantly reduced in the mutants (Figure 1C and D).
This means the increased sleep in the mutants is not caused by defects of locomotor ability.We next examined the sleep architecture of these flies and found that cry mutation enhances sleep by extending the duration of average sleep bout rather than increasing sleep bout number, indicating that cry deficiency promotes sleep consolidation under short photoperiod (Figure 1E and F).Because CRY exerts influences on sleep/wakefulness in a gender-independent manner, we used male flies for the remainder of the study.In addition, we tested the effects of a cry knock-out allele (cry 0 ) on sleep under 4L20D and also observed significantly prolonged sleep duration, similar to cry b mutation (Figure 1-figure supplement 1) (Dolezelova et al., 2007).

GABA/GABA-A
Upon light activation, CRY binds to the clock protein TIM and results in its degradation, which is followed subsequently by degradation of CRY itself (Busza et al., 2004;Ceriani et al., 1999;Lin et al., 2001).Therefore, we first tested whether the influences of CRY on sleep/wakefulness requires TIM.We monitored sleep in flies mutant for both cry and tim, and found that the sleep duration of these double mutants are comparable to that of cry mutants (Figure 2-figure supplement 1).This indicates that CRY promotes wakefulness in a TIM-independent manner.
To identify anatomical substrates that mediate the effects of CRY on wakefulness, we employed the UAS/GAL4 system to knock down cry in different brain structures and cell types and verified that cry is indeed knocked down by assessing its mRNA level (Figure 2-figure supplement 2).We found that knocking down cry in GABAergic neurons using vesicular GABA transporter (VGAT)GAL4 or glutamic acid decarboxylase 1 (Gad1)GAL4 results in increased sleep duration under 4L20D but not 12L12D, similar to the cry mutant phenotype (Figure 2A-C; Figure 2-figure supplement 3A and B) (Deng et al., 2019).Given that Gad1GAL4 generated a more prominent sleep phenotype, we used this driver for the remainder of this study.
Since CRY appears to act in GABAergic neurons to promote wakefulness, we tested whether GABA is involved in this regulatory process.We first took a pharmacological approach and fed flies with drugs to inhibit GABA transaminase (ethanolamine-O-sulphate, EOS) or GABA transporter (nipecotic acid, NipA).Both of these treatments are known to increase GABA level, and indeed lengthens the sleep duration in WT flies (Ki and Lim, 2019;Leal and Neckameyer, 2002).cry mutation fails to enhance sleep when treated with NipA, while the extent of sleep increase caused by cry mutation is smaller when treated with EOS compared to the control (Figure 2D; Figure 2-figure supplement 3C and D).Similarly, knocking down cry in GABAergic neurons no longer lengthens sleep duration after NipA treatment (Figure 2E; Figure 2-figure supplement 3E).For further validation, we adopted a genetic approach.We found that knocking down Gad1, the synthetic enzyme for GABA, suppresses the long sleep phenotype of cry RNAi flies (Figure 2F; Figure 2-figure supplement 3F) (Jackson et al., 1990).In addition, we knocked down VGAT which encodes an essential transporter responsible for packing GABA into synaptic vesicles (Enell et al., 2007).cry deficiency fails to lengthen sleep duration when VGAT is knocked down (Figure 2G and H;.We verified that Gad1 and VGAT are indeed knocked down by measuring their mRNA levels (Figure 2-figure supplement 3I and J).As a control for the genetic interaction experiments, we co-expressed a GFP RNAi and found this does not significantly alter the sleep duration of cry RNAi flies (Figure 2-figure supplement 3K).These series of results indicate that CRY acts via GABA to promote wakefulness.
We next sought to identify GABA receptor that mediates the effects of CRY on sleep/wakefulness.We fed flies with agonists of GABA-A (THIP) and GABA-B receptor (SKF-97541) (Ki and Lim, 2019).Both drugs enhance sleep in WT, while cry mutation can increase sleep in flies fed with SKF-97541 but not THIP, implicating that CRY acts through GABA-A to promote wake (Figure 3A-C).Consistently, GABA-A receptor antagonist carbamezapine (CBZ) reduces sleep in WT flies while cry mutation fails to lengthen sleep duration after CBZ treatment (Figure 3D and E) (Agosto et al., 2008).We also treated cry RNAi flies with THIP or CBZ and found this treatment abolished the long sleep phenotype as well (Figure 3F-I).To validate that CRY modulates sleep/wakefulness by acting upon GABA-A receptor, we tested for genetic interaction between cry and Resistant to dieldrin (Rdl), the gene that encodes GABA-A receptor in flies (ffrench-Constant et al., 1993).We found that Rdl mutation (Rdl MDRR ) blocks the sleep-enhancing effect of cry RNAi (Figure 3J-L) (Agosto et al., 2008).These findings demonstrate that GABA-A receptor mediates the wake-promoting function of CRY.
In short, pharmacological and genetic approaches reveal that CRY acts in GABAergic neurons and impinges on GABA/GABA-A signaling to promote wakefulness.

CRY acts upon l-LNvs to promote wakefulness
Previous studies reported that GABA enhances sleep at least in part by activating RDL on the l-LNvs, thus inhibiting the activities of these cells and reducing the release of the arousal-promoting neuropeptide pigment dispersing factor (PDF) (Chung et al., 2009;Parisky et al., 2008).Therefore, we tested whether the l-LNvs mediate the effects of CRY on sleep/wakefulness.We first verified that the l-LNvs indeed receive projections from GABAergic neurons.We expressed a GFP-tagged synaptotagmin (syt-GFP) which labels axon terminals using Gad1GAL4, and GFP signal was observed at the l-LNv soma (Figure 4-figure supplement 1A) (Zhang et al., 2002).We employed the GFP Across Synaptic Partners (GRASP) method with enhanced specificity (t-GRASP) to check for synaptic connections between GABAergic neurons and l-LNvs (Shearin et al., 2018).Pre-mGRASP was expressed in the axon terminal of GABAergic neurons (using Gad1GAL4), whereas its t-GRASP partner post-mGRASP was expressed in the l-LNvs (using the driver PdfLexA) (Shang et al., 2008).GFP signal can be detected at the l-LNv soma, which means GABAergic neurons send synaptic projections to the l-LNvs (Figure 4 -figure supplement 1B).We further validated this connection using the trans-Tango technique which labels down-stream synaptic targets of GABAergic neurons with the HA tag, and we were able to observe HA expression in the l-LNvs (Figure 4-figure supplement 1C) (Talay et al., 2017).All in all, these results demonstrate that GABAergic neurons project to the l-LNvs and form synaptic connections.
We next examined the effects of cry mutation on GABA level at the l-LNv soma by immunostaining.As expected, GABA signal is significantly increased in cry mutants under 4L20D but not 12L12D (Figure 4A-D).Consistent with elevated GABA, GCaMP6m signal, an indicator of intracellular calcium concentration, is significantly reduced in the l-LNvs under 4L20D (Figure 4E and F) (Chen et al., 2013).This implies that the neural activity of these cells is down-regulated, possibly due to increased GABA signaling.
To test whether the long sleep phenotype in cry mutants is due to reduced activity of the l-LNvs, we electrically silenced these neurons by expressing an inward rectifying potassium channel Kir2.1 (Baines et al., 2001).This results in lengthened sleep duration under 4L20D, mimicking the sleep phenotype of cry mutants (Figure 4G;  (Hamada et al., 2008;Nitabach et al., 2006).These findings support the notion that cry mutation lengthens sleep duration by down-regulating the neural activity of the l-LNvs.Since it has been shown that the l-LNvs promote arousal by releasing PDF, we assessed whether PDF or its receptor PDFR is required by CRY to exert influence on sleep/wakefulness (Chung et al., 2009;Parisky et al., 2008).cry mutation fails to lengthen sleep duration on Pdf or Pdfr mutant background, indicating the necessity of PDF signaling in mediating the arousal-promoting function of CRY (Figure 4J and K; Figure 4-figure supplement 2D and E) (Hyun et al., 2005;Lear et al., 2005;Mertens et al., 2005;Renn et al., 1999).
To summarize, these results strongly suggest that CRY promotes wakefulness by reducing GABA signaling and thus increasing the activity of the l-LNvs, which may in turn lead to increased release of PDF.

CRY acts in the s-LNvs to promote wakefulness
To identify the subset of GABAergic neurons in which CRY functions to regulate wakefulness, we first examined the expression pattern of Gad1GAL4 by labeling the GAL4+ cells with a nuclear GFP (nls-GFP) (Shiga et al., 1996).While the l-LNvs are not GFP+, we noticed that the s-LNvs which are close to the l-LNvs and also express PDF appear to be GFP+ (Figure 5A and B).Because the s-LNvs are known to express CRY, we suspected that CRY may be acting in these s-LNvs to regulate the activity of the l-LNvs via GABA signaling (Benito et al., 2008;Yoshii et al., 2008).To test this idea, we knocked down cry in the s-LNvs using R6GAL4 while over-expressing dicer2 (dcr2) to enhance RNAi efficiency and observed a modest but significant lengthening of sleep duration (Figure 5C; Figure 5-figure supplement 1A) (Helfrich-Forster et al., 2007).However, when we adopted a PdfGAL80 to block the actions of GAL4 in the PDF neurons in Gad1GAL4/UAScryRNAi, this does not alter the long-sleep phenotype (Figure 5-figure supplement 1B) (Stoleru et al., 2004).
These series of results suggest that cry expression in the s-LNvs are necessary but not sufficient to maintain normal sleep/wakefulness.We were indeed able to detect GABA signal at the s-LNvs, while GABA intensity is enhanced in cry mutants under 4L20D but not 12L12D (Figure 5D-G).To further validate that the s-LNvs are GABAergic, we knocked down VGAT in these cells and observed a decrease of GABA intensity (Figure 5-figure supplement 2A and B).
Next, we examined the effects of cry deficiency on the activity level of the s-LNvs.We found that cry mutation increases calcium concentration in these cells under 4L20D, in stark contrast to that of the l-LNvs (Figure 5H and I).This strongly suggests that cry deficiency leads to elevated neural activity in the s-LNvs.Consistently, we observed increased sleep when we activated these cells using TrpA1, similar to the long-sleep phenotype of cry mutants (Figure 5J; Figure 5-figure supplement 3A).Because R6GAL4 is also expressed in several other cells in the brain, we combined PdfGAL80 to verify that the sleep-enhancing effect is caused by over-activation of the s-LNvs (Helfrich-Forster et al., 2007).Indeed, we no longer observed the long-sleep phenotype when GAL4 expression is inhibited in the PDF neurons by GAL80 (Figure 5J; Figure 5-figure supplement 3A).Moreover, we found that cry mutation can no longer exert effects on sleep when the s-LNvs are activated, further validating that the s-LNvs mediate the influences of CRY on sleep (Figure 5K; whether CRY and HK also function together in the s-LNvs to regulate arousal (Agrawal et al., 2017;Fogle et al., 2015).We found that while over-expressing hk in the s-LNvs of WT flies does not alter sleep duration under 4L20D, over-expressing it in cry mutants significantly increases sleep (Figure 5L; Figure 5-figure supplement 3C).This genetic interaction indicates that CRY and HK cooperate to regulate wakefulness, possibly by modulating the electric activity of the s-LNvs.
Next, we expressed syt-GFP in the s-LNvs and observed GFP signal at the soma of the l-LNvs, indicating that the s-LNvs send axonal terminals to the l-LNvs (Figure 6A).trans-Tango technique further demonstrated that the s-LNvs project to form synaptic connections with the l-LNvs (Figure 6B).Moreover, when we disrupted GABA transmission in the s-LNvs by knocking down VGAT or Gad1, this reduced GABA intensity in the l-LNvs, implicating that the s-LNvs release GABA onto the l-LNvs (Figure 6C-E).At the behavioral level, these manipulations supress the long-sleep phenotype of cry mutation (Figure 6F-I).
Taken together, these results suggest that cry deficiency in the s-LNvs results in increased neuronal activity and thus enhanced GABA release at the l-LNvs via direct synaptic projections, ultimately leading to decreased wakefulness and increased sleep.

Short photoperiod reduces GABA level at the l-LNvs
Our findings thus far point to an inhibitory role of CRY on the GABAergic tone, and this in turn removes the inhibition on the neural activities of the l-LNvs.Since CRY is degraded by light, we hypothesized that CRY should exert a stronger effect on GABA under short photoperiod (Emery et al., 1998).Consistently, GABA intensity does appear to be down-regulated at the l-LNvs under 4L20D vs. 12L12D (Figure 7A and     B).On the other hand, GABA intensity does not exhibit photoperiod-dependent alteration in the s-LNvs (Figure 7C and D).Presumably, this reduced GABA level at the l-LNvs under 4L20D can result in elevated activation of these cells and increased wakefulness.In line with this, WT flies display shortened sleep duration under 4L20D compared to 12L12D (Figure 7E and F).This sleep reduction is a result of decreased sleep bout length but not bout number, while wake activity is not altered by photoperiod (Figure 7G-I).These observations indicate that short photoperiod hampers sleep maintenance, similar to the effects of CRY.

Discussion
Previous studies have shown that CRY mediates light-induced electrical activity of the l-LNvs and acute arousal, which indicates that CRY can immediately act to promote wake and terminate sleep in response to light pulse (Fogle et al., 2015;Sheeba et al., 2008).At the molecular level, this is believed to be accomplished by a direct coupling of light-activated CRY with HK in the l-LNvs (Fogle et al., 2015).Here we found that CRY promotes extended wakefulness under short photoperiod by functioning as an inhibitor of GABAergic tone.In contrast to the previously characterized roles of CRY which are activated by light, we believe this novel function we identify here reflects a role for CRY in the dark.Under short photoperiods, CRY acts in the dark to inhibit GABA signaling and thus promote wakefulness, leading to reduced sleep during the dark phase compared to longer photoperiods.Indeed, CRY is an ideal signal for conveying photoperiodic information, for it is degraded by light and only accumulates during darkness (Emery et al., 1998).It can measure the length of the day/night and thus instruct downstream signaling components to modulate photoperiod-dependent processes.Consistent with this idea, the largest increases of sleep in cry mutants occur immediately after lights-off and prior to lights-on (Fig 1B), indicating that CRY functions to promote wakefulness during these time windows which are exactly the time windows that would be affected by photoperiod changes (i.e.there will be light during these time windows under longer photoperiods).In other words, CRY appears to act at the time of the day that is most sensitive to photoperiodic changes, which fits perfectly with the idea that it serves as an instructive signal of day/night length.
While EOS and NipA treatment lengthen sleep duration as previously reported, we are somewhat surprised by the observation that knocking down Gad1 or VGAT in GABAergic neurons also extends sleep duration (Ki and Lim, 2019;Leal and Neckameyer, 2002).We reason this may be due to some sort of over-compensation induced by chronic GABA deficiency to maintain excitation/inhibition balance, as previous studies have reported that the amplitude of glutamatergic current is substantially down-regulated in Gad1 mutant flies and increased in Gad1 over-expression flies (Featherstone et al., 2000), (Featherstone et al., 2002).
Consistent with this idea, we noticed when cry is knocked down in Gad1/VGAT RNAi flies, sleep duration is shortened and comparable to that of the controls.This is probably because cry depletion enhances the GABAergic tone, thus normalizing GABA signaling in these flies which results in normal sleep duration.
We acknowledge that THIP treatment leads to prominent lengthening of sleep duration, and thus it is possible in this case cry deficiency no longer increases sleep duration due to a ceiling effect rather than epistatic interaction.Nonetheless, considering that CBZ and rdl mutation also block the effect of cry deficiency on sleep duration, it is highly likely that GABA-A receptor mediates the influences of CRY on sleep.One caveat is that the rdl MD-RR mutation has been shown to diminish the desensitization of GABA-A receptor and is thus believed to be a gain-of-function allele, but here we found that it eliminates the effect of cry RNAi on sleep (Zhang et al., 1994).Since treatment with GABA-A agonist THIP leads to substantially increased sleep while under the same condition rdl MD-RR mutation does not appear to significantly alter sleep duration, we suspect that similar to Gad1/VGAT RNAi, chronic enhancement of GABA-A function associated with rdl MD-RR mutation may also trigger some kind of compensatory mechanism that counteracts this increased GABA-A activity.Consequently, the influences of cry RNAi on sleep are blocked.Of course, a lot more in depth characterizations are required to elucidate these issues.
The s-LNvs have been reported to receive GABAergic inputs possibly via GABA-B receptors, but have not been shown to be able to synthesize GABA (Dahdal et al., 2010;Hamasaka et al., 2005).Here we observed that Gad1GAL4 is expressed in these cells, and their GABA intensity is reduced when we use R6GAL4 to knock down VGAT in these cells.R6GAL4 drives prominent expression in the s-LNvs with very little if any expression in the l-LNvs, and weaker (and not very consistent) expression in several other neurons in the protocerebrum, pars intercerebralis and subesophageal area which are all believed to lie outside of the circadian neuron network (Helfrich-Forster et al., 2007).Therefore, we reason that the alteration of GABA signal associated with knocking down VGAT should arise from VGAT deficiency within the s-LNvs.Taken together, these results suggest that the s-LNvs are Gad1GAL4/UAScryRNAi flies.Therefore, we suspect that cry deficiency in other GABAergic neurons are also required for the long-sleep phenotype.Given that the s-LNvs are known to express CRY and appear to be GABAergic based on our findings here, we believe that CRY acts at least in part in the s-LNvs to promote wakefulness under short photoperiod.
The molecular mechanism by which CRY down-regulates the GABAergic tone remains unclear.Besides conducting light-driven depolarization via HK, CRY has also been shown to act in synergy with HK to prevent membrane input resistance from falling to a low level in larval salivary glands (Agrawal et al., 2017;Fogle et al., 2015).In contrast to previous studies, here we found that lack of CRY increases the activity of the s-LNvs.Instead of functioning in synergy with HK, CRY appears to act in the opposite direction of HK as over-expressing hk enhances the long-sleep phenotype caused by cry mutation.We reason that the coupling between CRY and HK as well as their influences on the electric activity in the s-LNvs may be different from that of the l-LNvs and larval salivary glands.Nonetheless, our results also support an interaction between CRY and HK to promote arousal.We suspect that CRY acts via HK to inhibit the activity of the s-LNvs, which results in decreased GABA release and dis-inhibition of the l-LNvs.Extensive further investigations will be needed to elucidate the mechanism by which CRY regulates the activity of the s-LNvs.
In conclusion, here we describe a CRY-controlled GABAergic circuitry involving the l-LNvs and the s-LNvs that adjusts sleep duration in adaptation to changes in day length and propose a mechanistic explanation regarding how this circuitry functions (Fig 7J).Under short photoperiods, more CRY accumulates and inhibits the activity of the GABAergic s-LNvs, leading to a dis-inhibition of the l-LNvs which can release more PDF and promote arousal.Under longer photoperiods, on the other hand, less CRY can accumulate and thus the s-LNvs will exert more inhibitory influences on the l-LNvs, leading to decreased release of PDF and wakefulness.Notably, almost all neurons in the mammalian pacemaker, the suprachiasmatic nucleus, are GABAergic, and GABA/GABA-A signaling have been shown to mediate neuronal coupling in response to photoperiod changes (Ono et al., 2021).We believe a similar GABAergic circuitry may exist in the mammalian system that adjusts sleep/wakefulness to photoperiodic changes.

Fly strains
All strains were obtained from Bloomington Drosophila Stock Center, Vienna Drosophila Resource Center and TsingHua Fly Center or as gifts from colleagues.
Except for Gad1GAL4, all neurotransmitter related GAL4 lines were generated in Dr.

Fly sleep monitoring and analysis
Flies were raised on standard cornmeal-yeast-sucrose medium and kept in 12L12D at 25°C until behavior monitoring.~3 to 4-day-old flies were entrained under different photoperiods at 25℃ for 4 days, and then their activities in the next 3 days were analyzed.Sleep is defined as 5 min consecutive inactivity.Sleep was analyzed with Counting Macro written in Excel (Microsoft) following previously published protocol (Pfeiffenberger et al., 2010).Flies were fed with agar-sucrose food (2% agar, 5% sucrose) during entire sleep monitoring.TrpA1 flies were raised at 21°C and baseline sleep was monitored at 21°C.Temperature was then raised at lights on to 29°C for further sleep monitoring.

Drug treatment
For pharmacological experiments, drugs were mixed in the fly food at the following concentrations.For nipecotic acid (10 mg/ml, Sigma) and EOS (10 mM, Sigma), drugs were fed during the entire sleep monitoring.For THIP (10 ug/ml, Sigma), SKF-97541 (10 ug/ml, Tocris) and CBZ (0.15 mg/ml, Sinopharm Chemical Reagent), drugs were fed for 1 day after baseline sleep monitoring.The same amount of solvent was added into the fly food as vehicle control.

RNA extraction and quantitative real-time PCR (qRT-PCR)
Approximately 50 5-day-old flies were collected and frozen immediately on dry ice.
Fly heads were isolated and homogenized in Trizol reagent (Life Technologies).Total RNA was extracted and qRT-PCR conducted following our previously published procedures (Bu et al., 2019).The following primers were used to quantify mRNA level, cry F: TGCAGGTACCAAGAATGTGG, R: GTCCACGTCCATCAGTTGC.

Figure 5 -
Figure 5-figure supplement 3B).Because CRY has been shown to regulate GABAergic.Although knocking down VGAT or Gad1 in these cells can suppress the long sleep phenotype of cry mutants, knocking down cry in these cells only lead to a modest lengthening of sleep duration.Moreover, inhibiting cry RNAi expression

Figure 2 -
Figure 2-figure supplement 2. Screening for anatomical substrates that mediate the effects of CRY on sleep/wakefulness.(A) Difference in sleep duration between male cry RNAi flies compared to GAL4 and UAS controls under 4L20D (n = 20-92).(B) Plots of relative mRNA abundance for cry determined by qRT-PCR in whole-head extracts of cryRNAi and control flies under 4L20D (n = 4).The value of the GAL4 control group was set to 1. Mann-Whitney test: compared to GAL4 control, *P < 0.05; compared to UAS control, #P < 0.05.G4, GAL4; U, UAS.

Figure 2 -
Figure 2-figure supplement 3. CRY promotes wakefulness via GABA signaling.(A, B) Sleep profile of male flies with cry knocked down in GABAergic neurons by VGATGAL4 (A) or Gad1GAL4 (B) monitored under 4L20D.(C, D) Sleep profile of male WT and cry mutant flies fed with NipA (D) or EOS (E) under 4L20D in Fig 2D.White box indicates light period while black box indicates dark period.(E) Sleep profile of cry RNAi and control flies fed with NipA under 4L20D in Fig 2E.(F) Sleep profile of male flies with cry and gad1 knocked down in GABAergic neurons monitored under 4L20D in Fig 2F.(G) Sleep profile of male flies with cry and VGAT knocked down in GABAergic neurons monitored under 4L20D in Fig 2G.(H) Sleep profile of cry mutant flies with VGAT knocked down in GABAergic neurons monitored under

Figure 4 -
Figure 4-figure supplement 1. GABAergic neurons project to and form synapses with the l-LNvs.(A) l-LNvs of flies expressing syt-GFP in GABAergic neurons using Gad1Gal4 maintained under 4L20D and immunostained with PDF antisera.(B) l-LNvs of flies expressing Pre-mGRASP in GABAergic neurons using Gad1Gal4 and t-GRASP in the l-LNvs using PdfLexA maintained under 4L20D.Brains are immunostained with PDF antisera.(C) l-LNvs of flies expressing trans-Tango in GABAergic neurons using Gad1Gal4 maintained under 4L20D and immunostained with HA and PDF antisera.G4, GAL4; U, UAS; LexAop, LexA operator.

Figure 4 -
Figure 4-figure supplement 2. CRY promote wakefulness by impinging on the excitability of l-LNv and PDF signaling.(A) Sleep profile of male flies expressing Kir2.1 in PDF neuron using PdfGAL4 and controls, monitored under 4L20D in Fig 4G.White box indicates light period while black box indicates dark period.(B) Sleep profile of male cry mutant flies expressing TrpA1 in the l-LNvs using c929GAL4 and relevant controls in Fig 4H, monitored under 4L20D and 29℃ to activate TrpA1.(C) Sleep profile of cry mutant flies expressing NachBac in the l-LNvs using c929GAL4 and relevant controls in Fig 4I, monitored under 4L20D.(D, E) Daily sleep duration of male Pdf 01 -cry b (D) and Pdfr han5304 ; cry b (E) mutants along with relevant controls in Fig 4J and K, respectively, monitored under 4L20D.

Figure 5 .
Figure 5. CRY acts in the s-LNvs to inhibit their neural activity and promote wakefulness.(A) Brains of male flies expressing nls-GFP driven by Gad1GAL4 maintained under 4L20D and immunostained with PDF antisera.Representative l-LNv and s-LNv are displayed.(B) Quantification of GFP signal intensity of PDF neuron (n = 20, 49, 43 cells).Two-tailed Student's t test: ***P < 0.001.(C) Daily sleep duration of flies with cry knocked down in the s-LNvs using R6GAL4 and relevant controls, monitored under 4L20D (n = 45, 60, 63, 34, 39 flies).(D-G) Brains from male WT and cry mutant flies dissected at ZT1, 7, 13, 19 under 4L20D (D) or 12L12D (F) are immunostained with GABA (green) and PDF (red) antisera, and representative s-LNvs are displayed.Merged signal is shown as yellow.Bar graphs represent normalized GABA

Figure 5 -
Figure 5-figure supplement 2. s-LNvs appear to be GABAergic neurons.(A) Brains from male flies with VGAT knocked down in the s-LNvs using R6GAL4 and control maintained under 4L20D and immunostained with GABA (green) and PDF (red) antisera.Representative s-LNvs are displayed.Merged signal is shown as yellow.(B) Bar graphs represent normalized GABA intensity in the s-LNvs with VGAT knocked down using R6GAL4 (n = 25, 32 cells).The average value of the control group is set to 1. Two-tailed Student's t-test, ***P < 0.001.The scale bar represents 15 µm.Error bars represent SEM.G4, GAL4; U, UAS.

Figure 5 -
Figure 5-figure supplement 3. CRY promotes wakefulness by impinging on the excitability of the s-LNvs.(A) Sleep profile of male flies expressing TrpA1 in the s-LNvs using R6GAL4 and relevant controls in Fig 5J, monitored under 4L20D and 29℃ to activate TrpA1.White box indicates light period while black box indicates dark period.(B) Sleep profile of male cry mutant flies expressing TrpA1 in the s-LNvs using R6GAL4 and relevant controls in Fig 5K, monitored under 4L20D and 29℃ to activate TrpA1.(C) Sleep profile of male cry mutant flies over-expressing HK in the s-LNvs using R6GAL4 and relevant controls in Fig 5L, monitored under 4L20D.Error bars represent SEM.G4, GAL4; G80, GAL80; U, UAS.

Figure 6
Figure 6.s-LNvs release GABA onto the l-LNvs.(A) l-LNvs of male flies expressing syt-GFP in the s-LNvs using R6GAL4 maintained under 4L20D and immunostained with PDF antisera.(B) l-LNvs of male flies expressing trans-Tango in the s-LNvs using R6GAL4 maintained under 4L20D and immunostained with HA and PDF antisera.(C) Brains from male flies with VGAT (top) or Gad1 (bottom) knocked down in the s-LNvs using R6GAL4 and controls maintained under 4L20D and immunostained with GABA (green) and PDF (red) antisera.Representative l-LNvs are displayed.Merged signal is shown as yellow.(D, E) Bar graphs represent normalized GABA intensity in the l-LNvs of flies with VGAT (D) (n = 29, 37 cells) or Gad1 (E) (n = 37, 51 cells) knocked down in the s-LNvs using R6GAL4, monitored under 4L20D.The average value of the control group is set to 1. Two-tailed Student's t-test: **P < 0.01, ***P < 0.001.(F) Daily sleep duration of male cry mutant flies with VGAT knocked down in the s-LNvs using R6GAL4 and relevant controls, monitored under4L20D (n = 31, 31, 32, 29, 43, 19 flies).For comparison between RNAi flies vs. UAS/GAL4 controls, one-way ANOVA with Bonferroni multiple comparison test was used: compared to GAL4 control, *P < 0.05, **P < 0.01; compared to UAS control, ###P < 0.01.For comparison between mutant vs. control, two-tailed Student's t-test was used: compared to WT background, &&&P < 0.001.(G) Sleep profile of male cry mutant flies with VGAT knocked down in the s-LNvs using R6GAL4 and relevant controls in F, monitored under 4L20D.(H) Daily sleep duration of male cry mutant flies with Gad1 knocked down in the s-LNvs using R6GAL4 and relevant controls, monitored under4L20D (n = 59, 59, 53, 60, 62, 45   flies).For comparison between RNAi flies vs. UAS/GAL4 controls, one-way ANOVA with