Sleep drive reconfigures wake-promoting clock circuitry to regulate adaptive behavior.

Circadian rhythms help animals synchronize motivated behaviors to match environmental demands. Recent evidence indicates that clock neurons influence the timing of behavior by differentially altering the activity of a distributed network of downstream neurons. Downstream circuits can be remodeled by Hebbian plasticity, synaptic scaling, and, under some circumstances, activity-dependent addition of cell surface receptors; the role of this receptor respecification phenomena is not well studied. We demonstrate that high sleep pressure quickly reprograms the wake-promoting large ventrolateral clock neurons to express the pigment dispersing factor receptor (PDFR). The addition of this signaling input into the circuit is associated with increased waking and early mating success. The respecification of PDFR in both young and adult large ventrolateral neurons requires 2 dopamine (DA) receptors and activation of the transcriptional regulator nejire (cAMP response element-binding protein [CREBBP]). These data identify receptor respecification as an important mechanism to sculpt circuit function to match sleep levels with demand.

Introduction

Circadian rhythms help animals synchronize motivated behaviors with salient events such as when food or mates are available or when it is time to forage or sleep [1]. However, the optimal time of day to engage in particular behaviors can vary depending upon seasonal and environmental factors, which can, under some circumstances, place competing behaviors in conflict (e.g., foraging versus risk of predation; sleep versus mating success) [2-4]. The mechanisms regulating the timing of competing behaviors are not well understood.

Central and peripheral clocks can be synchronized by sleep and environmental cues such as light, temperature, food, social, and interactions [5-9]. Interestingly, recent evidence indicates that clock neurons do not act in a hierarchical manner but rather regulate behavior as a distributed network [10-15]. Although the regulation of these networks is complex, the neuropeptide pigment dispersing factor (PDF) and its receptor (PDFR) play a prominent role in synchronizing oscillations in the clock network [13,16]. Indeed, PDF can influence the timing of behavior by differentially staggering the timing of activity peaks in diverse neuronal groups [16,17]. Thus, the PDFR is well suited for regulating the timing of competing behaviors.

In mammals, exposure to short- and long-day photoperiods, which mimic naturally occurring seasonal changes, results in respecification of transmitters and their receptors [18,19]. Receptor respecification is a form of plasticity that, like Hebbian and homeostatic plasticity, may be employed to alter circuit function in response to changing environmental demands [19]. Importantly, sleep circuitry is plastic and can change through developmental and in response to environmental factors (e.g., starvation, predation risk, and mating status) [3,20-27]. Surprisingly, it remains unknown whether receptor respecification plays a role in modulating sleep plasticity.

In the Drosophila brain, there are approximately 150 clock neurons that are divided into 6 groups [28]. PDF is expressed in both the large and small ventral lateral neurons (l-LNvs and s-LNvs); In contrast to other clock neurons, the l-LNvs are not believed to express PDFR [13,29-31]. Given the role that PDF plays in coordinating the timing of diverse neuronal groups important for adaptive behavior, we hypothesized that under some circumstances, the PDFR could be respecified to help regulate the timing of competing behaviors. Indeed, we find that the PDFR is respecified in the l-LNvs for the first approximately 48 h after eclosion, when sleep drive is highest. Gain and loss of function experiments reveal that in young flies, PDFR expression is associated with increased waking and early mating success. Importantly, the PDFR can be reestablished in adult l-LNvs through prolonged sleep disruption. The most common forms of respecification alter the polarity of the synapse to alter the function of the circuit [19]. In contrast, our data suggest an additional type of respecification in which an input pathway into a circuit can be turned on and off, without changing the sign of the synapse (excitatory/inhibitory). These data identify receptor respecification as an important mechanism to sculpt circuit function to match sleep need with environmental demands.

Results

PDFR is expressed in l-LNvs in young flies

Sleep is highest in young animals during a critical period of brain development when neuronal plasticity is high [26,32]. As previously described, flies were collected and sexed using C02 anesthesia on the day they eclosed, placed into tubes, and sleep was quantified during their first full day of adult life. Sleep is highest during the first 48 h after eclosion (day 0, day1) and then reaches stable mature adult levels by approximately day 3 (Fig 1A and 1B). The increased sleep observed during these approximately 48 h is important for the development of circuits that maintain adaptive behavior into adulthood [33,34]. How neurons in sleep circuitry change during this period has not been explored. The l-LNvs promote waking behavior through both dopamine (DA) and octopamine (Oa) signaling (19,22–24); thus, we hypothesized that one or both of these pathways might be down-regulated during this early developmental period of high sleep. To test this hypothesis, we used live brain imaging in l-LNvs expressing the reporter Epac1 camps to define cAMP response properties [30,31,35,36]. Contrary to our hypothesis, neither DA- or Oa-induced cAMP responses changed as the flies matured (Figs 1C and 1D and S1). Interestingly, we did observe PDF-induced cAMP responses in l-LNvs in the first 48 h of adulthood (Fig 1E and 1F), while they were predominantly absent in mature adult l-LNvs, consistent with previous reports [30,31]. To determine if this transient PDF sensitivity is regulated at the receptor level, expression of the PDFR was examined directly using Pdfr-myc, a tagged receptor genetic construct under the natural PDF promoter [29]. As anticipated, detection of MYC antibody staining is high on day 0 and not detectable on day 5 of adulthood (Fig 1G), revealing transient expression of the receptor. Finally, we examined an adjacent group of clock neurons, the s-LNvs [37]. Responses to PDF in s-LNvs are present at the beginning of adulthood and then decrease in amplitude over the first approximately 48 h of adulthood. In contrast to the l-LNvs, sensitivity to PDF in the s-LNvs persists into mature adulthood (S1 Fig). Together, these data indicate that the PDFR is transiently expressed in wake-promoting l-LNvs in young flies to support waking when sleep drive is highest.

Fig 1

PDFR is expressed in l-LNvs of young flies.

(A, B) Sleep is elevated in young male flies following eclosion and reaches stable adult values in 3-day-old flies (n = 35–93 flies/age; one-way ANOVA F[5,472] = 81.34, for age, p = 3.7E-63). (C–E) FRET ratio measurements in Pdf-GAL4>UAS-Epac1 flies in response to DA (3e-3M), Oa (3e-3M), and PDF (1e-6M) (n = 5–15 ROI. Each ROI represents 2 to 4 l-LNvs). (F) The amplitude of l-LNv responses to PDF decreases with age (PDF amplitude); (n = 13–24 ROI/age; one-way ANOVA for age F[5,93] = 19.86, p = 3.3E-13). (G) GFP expression in LNv neurons (Pdf-GAL4; UAS-gfp). (H) Immunohistochemistry reveals coexpression of PDF (red) and myc (green) in 0-day-old P[acman] Pdfr-myc70 flies, which is not observed on day 5. *p < 0.05, modified Bonferroni test. Data underlying this figure can be found in S1 Data. DA, dopamine; FRET, Förster Resonance Energy Transfer; GFP, green fluorescent protein; l-LNv, large ventral lateral neuron; Oa, octopamine; PDF, pigment dispersing factor; PDFR, pigment dispersing factor receptor; ROI, region of interest.

Expression of PDFR in l-LNvs alters behavior in young flies

During the day, sleep is highest during the midday siesta and is reduced in the hours preceding lights out (Fig 1A) [26,33,34]. We have operationally defined the 2-h period before lights out as the wake maintenance zone (WMZ) based upon the observation that sleep rebound is absent or dramatically reduced when flies are released into recovery during this time window [33,38]. The ability to maintain waking in the face of high sleep drive suggests that this window of time is protected for important waking behaviors [39]. With that in mind, we hypothesized that flies lacking the PDFR would sleep more than genetic controls during the WMZ. Pdfr-null mutant (Pdfr) flies were outcrossed to Cs flies for 5 generations. To avoid handling of flies on the day they eclosed, Pdfr and Cs flies were plated on juice plates for 4 h to lay eggs, and then L1 larvae were put into individual glass tubes and monitored. Sleep was assessed in male flies that eclosed between Zeitgeber time (ZT1) and ZT4. As seen in Fig 2A and 2C on the day of eclosion, Pdfr null mutants sleep significantly more than their genetic controls during the WMZ. To determine whether the change in sleep was due to expression of the PDFR in the l-LNvs, we expressed wild-type Pdfr (UAS-Pdfr) using the c929-GAL4 driver in a Pdfr mutant background. Since c929-GAL4 is expressed in other peptidergic neurons [40], we combined c929-GAL4 with cry-Gal80, which targets the GAL4 inhibitor GAL80 to all CRY+ neurons including the l-LNvs to suppress the PDFR rescue [41]. As seen in Fig 2B and 2D, sleep remained elevated during the WMZ in Pdfr;c929/+;cryGAL80/+ (green) and Pdfr;UAS-Pdfr/+ (purple) parental controls as expected. In contrast, waking was rescued during the WMZ in Pdfr;c929/UAS-Pdfr flies (red), and this increase in waking was prevented when the expression of UAS-Pdfr was blocked in clock cells (Pdfr;c929/ UAS-Pdfr;cryGAL80/+,blue). We verified the effectiveness of cry-GAL80 using a UAS-GFP reporter (S2 Fig). To further exclude the possibility that expression of UAS-Pdfr in other peptidergic neurons outside the l-LNvs altered waking, we rescued the expression UAS-Pdfr in a Pdfr mutant background using Pdf-GAL4, which targets only LNv neurons. As seen in S2 Fig, sleep was reduced in Pdfr;Pdf-GAL4/UAS-Pdfr compared to parental controls. Finally, we asked whether the inability of Pdfr mutants to stay awake during the WMZ was due to the absence of Pdfr in the l-LNvs. As seen in Fig 2E, Dcr2;929-GAL4/UAS-Pdfr flies slept significantly longer during the WMZ than either Dcr2;c929-GAL4/+ or UAS-Pdfr/+ parental controls. Together, these data indicate that PDFR in the l-LNv promotes waking in young flies when sleep drive is high.

Fig 2

Expression of PDFR in l-LNvs regulates sleep in young flies.

(A) Sleep traces of Pdfr mutants and Cs controls on day 0. (B) Sleep traces for Pdfr; c929-GAL4; UAS-Pdfr (rescue, green), Pdfr; UAS-Pdfr/+, Pdfr; c929-GAL4/+; Cry-Gal80/+, and Pdfr; c929-GAL4/UAS-Pdfr; Cry-Gal80 (n = 26–31/genotype). (C) Quantification of sleep during the WMZ of flies shown in (A). Cs flies sleep less during the WMZ than Pdfr mutants (n = 26/genotype; t test, p < 0.05); (D) Quantification of sleep during the WMZ of flies shown in (B). Pdfr; c929-GAL4; UAS-Pdfr sleep less than parental controls; ANOVA F[3,109] = 6.33, p = 5.4E-4; n = 22–31. (E) Sleep is increased in Dcr2; c929-GAL4/UAS-PdfrRNAi flies on day 0 compared to Dcr2; c929-GAL4/+ and UAS-Pdfr/+ parental controls (ANOVA; F[2,279] = 12.00, p = 1.04E-5; n = 26–28). Data underlying this figure can be found in S2 Data. l-LNv, large ventral lateral neuron; PDFR, pigment dispersing factor receptor; WMZ, wake maintenance zone.

Although the respecification of the PDFR in l-LNvs supports waking in young flies, it is unclear whether the observed changes impact ecologically relevant behaviors. Inspired by the observation that the male pectoral sandpipers that sleep the least during breeding season sire more offspring [3], we assayed mating success in flies with and without PDFR. As above, we began by evaluating Pdfr mutants and their genetic controls (Cs). Following eclosion, male flies were individually paired with a wild-type virgin female fly at ZT4 for 20 h, and the pairings that produce offspring were tabulated. As seen in Fig 3A, approximately 80% of pairings with Cs males resulted in offspring, while only 25% of pairings with Pdfr mutants were successful on day 0. Moreover, mating success was also reduced when Pdfr was knocked down in c929-GAL4 expressing cells (Fig 3B). Importantly, the deficit in mating success observed in Pdfr mutants on day 0 was rescued by expressing wild-type PDFR using c929-GAL4 (Fig 3C). Previous studies have shown that the expression of PDFR in the dorsal lateral (LNd) neurons, a different set of clock neurons, promotes mating behavior in mature males [42]. However, no changes in mating success were observed in 2-day-old Pdfr mutants or in Pdfr;c929/UAS-Pdfr rescue flies compared to genetic controls (Fig 3A–3C).

Fig 3

Role for PDFR in l-LNvs in mating success.

(A) Cs flies produce more offspring than Pdfr mutants (n = 30/genotype); χ2 = 12.13, p = 0.0004. (B) Dcr2; c929-GAL4/UAS-Pdfr flies produce fewer vials with offspring compared to Dcr2; c929-GAL4/+ and +/UAS-PdfrRNAi parental controls; (n = 30/genotype); χ2 = 21.3, p = 0.00002 (C) Pdfr; c92-GAL4; UAS-Pdfr (rescue, green) flies produce more offspring than Pdfr; UAS-Pdfr/+ (purple), Pdfr; c929-GAL4/+; CryGal80/+ (red), and Pdfr; c929-GAL4/UAS-Pdfr; Cry-Gal80 (blue) parental controls (n = 30/genotype) χ2 = 8.95, p = 0.029. (D) Mating competition assay scheme on day 1. (E) Cs males outcompeted white-eyed challenger flies compared to Pdfr mutants (t test, p < 0.001, n = 3 sets of 20 flies/genotype). (F) Pdfr; c929-GAL4/+; UAS-Pdfr/+ males outcompeted white-eyed challengers compared to Pdfr; UAS-Pdfr, Pdfr/+; c929-GAL4/+; Cry-Gal80/+, or Pdfr; c929-GAL4/UAS-Pdfr; Cry-Gal80/+ controls (ANOVA F[3,13] = 15.01, p = 4.9−4; n = 3 sets of 20 flies/line). (G) Dcr2; c929-GAL4/UAS-Pdfr RNAi flies displayed reduced mating success compared to Dcr2; c929-GAL4/+ and +/UAS-Pdfr RNAi control flies (ANOVA, F[2,8] = 8.10, p = 0.019, n = 43–55). *p < 0.05, modified Bonferroni test. Data underlying this figure can be found in S3 Data. l-LNv, large ventral lateral neuron; PDFR, pigment dispersing factor receptor; RNAi, RNA interference.

To further determine whether expression of PDFR in the l-LNvs was important for mating success, we utilized a competition assay in which we rescued PDFR in a Pdfr mutant background. In this assay, one red-eyed male and one white-eyed male were combined with a white-eyed female for 2 h at the beginning of the WMZ at ZT10 on day 1. Successful mating of the red-eyed male was determined by female progeny with eye color (Fig 3D). Consistent with the data presented above, Pdfr males sired fewer offspring than the Cs controls (Fig 3E). Despite the fact that white flies show impaired courtship [43,44], white-eyed males sired more offspring than red-eyed Pdfr;c929/+, Pdfr; UAS-Pdfr, and Pdfr;c929/UAS-Pdfr;cryGAL80/+ controls (Fig 3F). In contrast, male flies expressing the Pdfr in l-LNvs (Pdfr;c929/UAS-Pdfr) sired more red-eyed progeny on day 1 (Fig 3F). To determine whether the deficit in mating success in Pdfr mutants was due to loss of PDFR in the l-LNvs, we evaluated Dcr2;929-GAL4/UAS-Pdfr flies. As seen in Fig 3G, Dcr2; c929>; UAS-Pdfr lines reduced mating success compared to Dcr2; c929/+ and UAS-PdfrRNAi/+ parental controls. Therefore, the expression of the PDF receptor in the l-LNvs is associated with successful mating in early adulthood when sleep pressure is high.

Respecification of PDFR in l-LNvs modulates adult behavior

Given that the expression of the PDFR in the l-LNvs confers advantages to the young fly, we wondered why its expression would then be turned off on days 2 to 3 of adult life. To gain further insight into this question, we evaluated behavior in 5-day-old flies ectopically expressing the PDFR in the l-LNv using a specific split-GAL4 driver (GRSS000645, l-LNv-GAL4). Daytime sleep was modestly reduced in l-LNv-GAL4>UAS-Pdfr flies compared to l-LNv-GAL4/+ and UAS-Pdfr/+ parental controls (Fig 4A). As a negative control, we evaluated sleep in adult flies while expressing UAS-Pdfr in the l-LNvs. Not surprisingly, expressing UAS-Pdfr in the l-LNvs did not alter sleep in adult flies (Fig 4B). Previous studies have shown that mutations that confer resistance in one environmental setting may increase the vulnerability of individuals in alternate settings [45]. Thus, we hypothesized that increased waking could sufficiently alter energy demands to make adult flies expressing PDFR in the l-LNvs vulnerable to starvation. To test this hypothesis, we starved flies and examined survival. As above, we examine the impact of starvation when the PDFR was overexpressed or knocked down in the l-LNvs. As seen in Fig 4C, survival was shorter in l-LNv>UAS-Pdfr compared to l-LNv/ + and UAS-Pdfrwt/+ parental controls. Astonishingly, l-LNv-GAL4>UAS-Pdfr flies showed improved survival compared to both parental controls flies (Fig 4D). Although knocking down Pdfr attenuated starvation induced waking, sleep was reduced in both pdf mutants and w genetic controls during the first 18 h of starvation (S4 Fig). However, waking activity was significantly lower in pdf mutants, and they survived significantly longer compared to w controls (S4 Fig). Together, these data indicate that the ability of pdf to influence adaptive behavior during starvation extends beyond modifying sleep time.

Fig 4

Behavioral consequences of PDFR expression in l-LNvs.

(A) Daytime sleep in 5-day-old l-LNv-GAL4>; UAS-Pdfr/+ flies and parental controls (ANOVA[,92] = 5.7 p = 0.004; n = 30–32/genotype). (B) Sleep in Dcr2; c929-GAL4/UAS-Pdfr flies and parental controls (ANOVA[,150] = 17.24 p = 1.86E-07; n = 40–60/genotype; *p < 0.05, modified Bonferroni test). (C) Kaplan–Meier analysis reveals % survival during starvation in l-LNv-GAL4>; UAS-Pdfr flies and parental controls (n = 3 replicates of 10–16/genotype, χ2 = 19.55, df = 2, p-value < 0.0001). (D) Kaplan–Meier analysis reveals % survival during starvation in Dcr2; c929-GAL4/UAS-Pdfr flies and parental controls (n = 3 replicates of 10–16/genotype, χ2 = 23.35, df = 2, p-value < 0.00001). Data underlying this figure can be found in S4 Data. l-LNv, large ventral lateral neuron; n.s., not significant; PDFR, pigment dispersing factor receptor.

The increased survival seen in starved l-LNv-GAL4>UAS-Pdfr flies suggested that the genetic program that activates the PDFR in the l-LNvs may be reactivated in mature adults during conditions of high sleep drive. Short periods of starvation (approximately 12 h) increase waking without activating sleep drive presumably to maintain cognition during foraging [45,46]. However, longer periods of starvation (approximately 20 h) are able to activate homeostatic mechanisms [22]. Thus, we hypothesized that starvation would lead to the respecification of the PDFR in the l-LNvs. As seen in Fig 5A, PDF responses in the l-LNvs of mature adult flies are restored following starvation when compared to age-matched, nonstarved siblings. To determine how much time was required for starvation to respecify the PDFR in the l-LNvs, we evaluated the time course of PDFR respecification in the l-LNvs. Interestingly, starvation-induced restoration of PDF sensitivity in l-LNvs requires a similar duration as reported for the activation of homeostatic drive (Fig 5B). These data suggest that the respecification of the PDFR in the l-LNvs is to help flies maintain wakefulness during starvation. With that in mind, we hypothesized that blocking the expression of the PDFR in the l-LNvs would result in more sleep during starvation. Indeed, Dcr2; c929>; UAS-Pdfr flies slept more than parental controls between 21 h of starvation, when homeostatic drive begins, and 32 h of starvation prior to the point when flies begin dying (S5 Fig). In summary, starvation reduces sleep resulting in a buildup of sleep pressure, which may mimic the conditions present in early adulthood that lead to PDFR respecification in l-LNvs.

Fig 5

Sleep pressure induces PDFR expression in mature l-LNvs.

(A) Normalized FRET ratio during PDF application in l-LNvs from starved (n = 10) and fed (n = 16) Pdf-GAL4>UAS-Epac1 flies. (B)The amplitude of l-LNv responses to PDF is observed in 5-day-old Pdf-GAL4>UAS-Epac1 flies following 21–24 h of starvation. Data are shown for 8 h bins (ANOVA F[7,71] = 9.08, p = 9.8E-8; ROI = 7–20). (C) Sleep in Cs flies on sleep restriction day 2 and recovery. (D) Normalized FRET ratio during PDF application in l-LNvs recorded from Pdf-GAL4>UAS-Epac1 flies during sleep restriction sleep deprivation and bang controls (n = 12–25 ROI/genotype). (E) Immunohistochemistry of PDF (red) and myc (green) in 5-day-old sleep-restricted P[acman] Pdfr-myc70 flies. (F) The amplitude of l-LNv responses to PDF in l-LNvs during baseline, sleep restriction, and recovery (ANOVA F[4,81] = 8.00, p = 1.94E-4, n = 9–24 ROI/condition). (G) Sleep rebound in Dcr2; c929-GAL4/UAS-Pdfr flies and parental controls (ANOVA F[2,147] = 6.37, p = 2.22E-3; n = 43–55). *p < 0.05, modified Bonferroni test. Data underlying this figure can be found in S5 Data. FRET, Förster Resonance Energy Transfer; l-LNv, large ventral lateral neuron; n.s., not significant; PDF, pigment dispersing factor; PDFR, pigment dispersing factor receptor; ROI, region of interest.

Starvation is an indirect method to increase sleep pressure. With that in mind, we asked whether sleep deprivation would also result in the respecification of the PDFR in the l-LNvs in mature, adult flies. As seen in Fig 5D, l-LNvs respond physiologically to PDF following sleep deprivation in 5-day-old flies. Although total sleep deprivation is the most common method for increasing sleep drive in the laboratory, it seems unlikely that circumstances in the natural environment would keep an animal awake continuously for 12 h or more. In contrast, sleep consolidation is more easily disrupted and, perhaps, more likely to be impacted by a variety of environmental conditions [21,23]. Thus, we hypothesized that interrupting sleep consolidation would be sufficient to respecify the PDFR in the l-LNvs. A variety of manipulations that increase sleep drive (e.g., memory consolidation and activating the dorsal fan-shaped body) increase average daytime sleep bout duration to >22 min/bout [47,48]. Thus, we disrupted sleep consolidation by presenting a mechanical stimulus to the flies for 1 min every 15 min for 48 h. As seen in Fig 5C, this protocol modestly disrupted sleep and did not result in a compensatory sleep rebound. To determine if the lack of a sleep rebound was due to the respecification of PDFR, we examined the l-LNvs physiologically and histologically. As seen in Fig 5D, PDF responses in the l-LNvs of mature adult flies are restored following 48 h of sleep restriction. To determine if the mechanical stimulus alone would respecify the PDFR in the l-LNvs, siblings were exposed the same amount of stimulation (approximately 190 min) as sleep-restricted siblings but during the biological day when sleep debt does not accrue [26,49]. As expected, mechanical stimulation in the absence of sleep restriction did not respecify the PDFR in the l-LNvs (Fig 5D). To confirm the physiological data, PDFR was examined directly using Pdfr-myc [29]. MYC antibody staining in the l-LNvs is clearly visible in mature adult flies following sleep restriction but is not observed in nondisturbed age-matched controls (Fig 5E). Next, we asked how much sleep restriction was required for the respecification of the PDFR. As seen in Fig 5F, PDF sensitivity becomes apparent after 24 to 48 h of sleep restriction, is sustained during the first day of recovery, and then dissipates. Finally, we asked whether knocking down the PDFR in the l-LNvs would modulate sleep homeostasis following sleep disruption. As seen in Fig 5G, Dcr2;c929/+>UAS-Pdfr flies slept significantly more following sleep restriction than Dcr2;c929/+ and UAS-Pdfr/+ parental controls. These data indicate that PDFR can be respecified to mitigate against the effects of sleep pressure in the context of sleep disruption.

Nejire modulates PDFR in both young and mature l-LNvs

The PDFR is transiently expressed in the l-LNvs of young flies and can be respecified again in mature adults in response to certain environmental perturbations. Thus, we asked whether these seemingly different conditions invoke the same mechanisms to activate the expression of PDFR in the l-LNvs. To begin, we conducted an RNA interference (RNAi) screen of transcription factors that are known to be expressed in LNvs [50]. We crossed UAS-RNAi lines with pdf-GAL4;UAS-Epac and monitored PDF sensitivity in both l-LNvs and s-LNvs in young flies on day 0. As mentioned above, s-LNvs display persistent expression of the PDFR in both young and mature adults. Thus, we hypothesized that by monitoring both cell types, we could distinguish between regulatory elements specific to the transient pathway in l-LNvs. We also examined DA responses to discriminate between transcription factors specifically involved in the PDF pathway and those common to other GPCR signaling pathways. As seen in Fig 6A, knocking down Drosophila cAMP response element-binding protein (CREB) (nejire) or Suppressor of Under-Replication (SuUR) ablated PDF sensitivity in l-LNvs on day 0, while other transcription factors left the sensitivity of the l-LNvs to PDFR largely intact. The amplitude of DA responses was not altered by nejire, SuUR, or any other RNAi lines, revealing that the roles of nejire and SuUR are specific to the PDF pathway in this context (S6A Fig). PDF sensitivity in the s-LNvs was not ablated by RNAi knockdown of nejire (S6 Fig). Interestingly, nejire also plays a role in the respecification of the PDFR in the l-LNvs in mature adults following sleep restriction (Fig 6B). As in young flies, the panel of RNAi lines did not alter DA responses in the l-LNvs (S6 Fig). To further evaluate the role of nejire in the respecification of the PDFR in mature adults, we expressed wild-type nejire (UAS-nejire) or UAS-nejire using Pdf-GA4; UAS-Epac. We hypothesized that the overexpression of nejire would restore PDFR sensitivity to the l-LNvs in well-rested mature adults and that knocking down nejire would block the respecification of the PDFR in the l-LNvs during sleep restriction. Indeed, the sensitivity of the l-LNvs to PDF was restored in well-rested mature adults by overexpressing UAS-nejire. Conversely, the respecification of the PDFR to the l-LNvs during sleep restriction was blocked by UAS-nejire (Fig 6D). Together, these data reveal that conditional PDFR expression in l-LNvs shares common mechanisms in both young flies and mature adults.

Fig 6

nejire regulates PDFR respecification in l-LNvs of both young and mature flies.

(A) The amplitude of l-LNv responses to PDF on day 0 in Pdf-GAL4>UAS-Epac1 flies crossed to UAS-RNAi lines of the depicted transcription factors (ANOVA; F[8,127] = 11.1, p = 1.26E-11, *p < 0.05, modified Bonferroni test, n are listed below the x-axis). (B) PDF amplitude in control and 5-day-old sleep restricted Pdf-GAL4>UAS-Epac1 flies compared to Pdf-GAL4>UAS-Epac1 flies expressing UAS-nej (n = 6–19 neurons/genotype). (C) In the absence of sleep loss, the l-LNvs of Pdf-GAL4>UAS-Epac1/UAS-nej respond to PDF while age-matched Pdf-GAL4>UAS-Epac1 do not (t test, DF 34–1, t = 22.6, p = 3.74e-5, n = 9–26 neurons/genotype). (D) Data quantified from (B). (t test, DF 40–1, t = 17.8, p = 4.10e-4, n = 16–26). (E) The amplitude of l-LNv responses to PDF in l-LNvs on day 0 in Pdf-GAL4>UAS-Epac1 flies crossed to UAS-RNAi lines of the depicted cell surface receptors (ANOVA F[10,105] = 5.38, p = 2.8−6 p < 0.05, modified Bonferroni test, n are listed below the x-axis). (F) The amplitude of l-LNv responses to PDF in l-LNvs on day 5 in sleep-restricted Pdf-GAL4>UAS-Epac1 flies crossed to UAS-RNAi lines of the depicted cell surface receptors (ANOVA F[10,179] = 7.04, p = 3.3E-9 p < 0.05, modified Bonferroni test, n are listed below the x-axis). Data underlying this figure can be found in S6 Data. FRET, Förster Resonance Energy Transfer; l-LNv, large ventral lateral neuron; PDF, pigment dispersing factor; PDFR, pigment dispersing factor receptor.

Finally, we asked whether similar mechanisms were used by young and mature adults for the activation of nejire. To identify cell surface receptors that might interact with nejire, we once again consulted a database of genes known to be enriched in the LNvs [50]. We then conducted a targeted RNAi screen to evaluate PDF sensitivity in young flies and mature adults following sleep restriction (Figs 6E and 6F and S6). Given that we only evaluated 1 RNAi line to evaluate each receptor, these results should be viewed cautiously.

Nonetheless, these data suggest the possibility that DopEcR, Dop1R2, and InR may play a role in the respecification of the PDFR in both young flies and mature adults during sleep restriction.

Discussion

In Drosophila, PDF differentially coordinates the activity of diverse neuronal groups to optimize behavioral output with the prevailing environmental conditions [16,51]. One of the most successful topics in circadian neurobiology today has been the use of circuit mapping to decipher the logic used by the clock to regulate such rhythmic behavior [10,11,13,52,53]. Indeed, a number of studies highlight the role of PDF in coordinating the activity of downstream circuits [16,41,54]. In this manuscript, we replicate previous finding that the Pdfr is not normally expressed in the wake-promoting l-LNvs of healthy adults [13,29,30]. However, we now show that high sleep pressure quickly reprograms the l-LNvs to express the Pdfr. The addition of a new signaling input into the circuit, through expression of the Pdfr in the l-LNvs, is associated with increased waking and early mating success. Importantly, these data demonstrate that the constellation of neurons that express Pdfr is not constant and points to a novel type of plasticity that can be used by the clock to coordinate behavioral output.

A growing number of studies indicate that sleep regulatory mechanisms are plastic and can be harnessed to match an individual’s sleep need with environmental demands [21,46,55]. Although Hebbian and synaptic plasticity modulate circuit function in a variety of contexts, these forms of plasticity may not be well suited to sculpt the balance of sleep and wake-promoting circuits at specific times of day [56]. In contrast, receptor respecification is a form of plasticity that may allow an individual to engage in adaptive waking behaviors at optimal circadian times while still allowing the animals to obtain needed sleep at other times [19]. Indeed, our data indicate that the PDFR is transiently expressed in wake-promoting clock neurons during the first approximately 48 h of adult life when sleep drive is high. The associated increase in waking is confined to a small portion of the circadian day and supports mating success and mating competition. In contrast, the response properties of the l-LNvs to the global wake-promoting transmitters Oa and DA remains unchanged [57]. Interestingly, when sleep is disrupted in 5-day-old adults, the PDFR is once again expressed in the l-LNvs. Thus, targeted receptor respecification may be an effective strategy that can be used to support important, species-specific behaviors during conditions of high sleep drive without substantially disrupting the ability of the animal to obtain needed sleep.

Our data indicate that there is a strong relationship between sleep drive and the respecification of the PDFR in a subset of clock neurons. That is, while the l-LNvs are unresponsive to PDF in mature adults [30], the l-LNvs display robust responses to PDF following sleep deprivation, sleep restriction, and prolonged starvation. Importantly, no changes in the response properties of the l-LNvs were observed when the animals were exposed to the mechanical stimulus in the absence of sleep restriction. Interestingly, the response properties of the l-LNvs was not visible until the second day of sleep restriction indicating that low amounts of sleep drive are not sufficient to respecify the PDFR. Consistent with this hypothesis, short durations of starvation induce episodes of waking that are not compensated by a sleep rebound [58] and do not change the response properties of the l-LNvs to PDF. In contrast, after approximately 20 h of starvation, a time when flies begin to display a sleep rebound, the l-LNvs begin to respond to PDF. These data suggest that the PDFR may be respecified in the l-LNvs to, for example, help sleepy animals stay awake long enough to support a basal level of foraging. Indeed, knocking down the Pdfr in clock neurons results in a larger sleep rebound following sleep restriction. Increased sleep in many circumstances may be maladaptive since it would likely limit the opportunity to forage or mate [59]. Together, these data support the hypothesis that the PDFR is expressed to assist waking behaviors during conditions of high sleep drive.

Given that high sleep drive can negatively impact male sexual behavior (Chen and colleagues [55]), it is curious that the PDFR is not typically expressed in the l-LNvs of healthy adults. However, previous studies have shown that genes that confer resilience to specific environmental challenges can be deleterious in other circumstances (Donlea and colleagues [45]). Indeed, the exogenous expression of PDFR in the l-LNvs during adulthood reduced survival during prolonged starvation. These data suggest that the normal down-regulation of PDFR expression in l-LNvs of healthy adults may be advantageous in that it removes potentially excessive behavioral drives that could deplete valuable resources. Indeed, genetically preventing PDFR expression in l-LNvs during starvation extended survival.

Although sleep drive does not change the response properties of the l-LNvs to DA, our data suggest that changes in dopaminergic tone may play a role in the respecification of the PDFR in the l-LNvs. Specifically, knocking down specific DA receptors in the l-LNvs prevents the respecification of the PDFR in both young flies and sleep-restricted 5-day-old adults. Although the precise dopaminergic neurons have not yet been identified, the PPL2 dopaminergic neurons project to the l-LNvs to promote wakefulness [40,41] and may play a role in the expression of the PDFR in l-LNvs. In addition to DA receptors, our data identify a role of the transcription factor nejire (cAMP response element-binding protein) in promoting the expression of the PDFR during conditions of high sleep drive. Interestingly, nejire plays a role in circadian function where it has been suggested to allow cross-talk between circadian transcription and the transcriptional regulation of other important processes such as sleep, metabolism, and memory formation [60,61].

Previous studies have shown that activity-dependent respecification of receptors in mammals can occur in adult neurons in response to >1 week of sustained increases in sensory activity [18,19]. The most common forms of respecification alter the polarity of the synapse to alter the function of the circuit [19]. Our data suggest an additional type of respecification in which an input pathway into a circuit can be turned on and off, without changing the sign of the synapse (excitatory/inhibitory). Presumably, turning on an input pathway may be a rapid first step to balance the impact of sustained activity in opposing circuits (e.g., sleep versus wake). However, enhancing the activity of a circuit may create a positive feedback loop, which can destabilize the system and lead to adverse consequences. Indeed, while the respecification of the PDFR in the l-LNvs can improve mating success during high sleep drive, it also results in early lethality during starvation. Understanding how sleep drive modulates respecification plasticity in other sleep regulatory circuits may provide critical insight into the role that sleep plays in maintaining adaptive behavior in an ever changing environment.

Materials and methods

Flies

Flies were cultured at 25°C with 50% to 60% relative humidity and kept on a diet of yeast, dark corn syrup, and agar. Newly eclosed males were collected and entrained 4 to 7 days in a 12-h:12-h light:dark (LD) cycle, unless otherwise specified. RNAi stocks were obtained from VDRC and TRiP stock centers. DopEcR , Dop1R1, Dop1R2 , D2R, InR, NPFR, Oamb, TkR86C, Oa2, Cry, Mael , dimm , mamo , cac , achintya, SuUR, nejire, nejire. Other stocks used were c929(dimm)-GAL4; PDF-GAL4; lLNv -GAL4 (G. Rubin, H. Dione, A. Nern); UAS-nejire, pdf, w [37]. All other UAS lines and GAL4 lines have been described previously: Pdfr-null mutant (Pdfr); UAS-Pdfr; w; UAS-Epac1camps50A [, w, Pdf-GAL4(M) and UAS-Pdfr [62]. c929-GAL4; cry-GAL80/UAS-GFP flies and P[acman] pdfr-myc70 flies [ were used for immunolabeling.

Sleep

Sleep was measured as described previously [26]. In short, individual flies were placed into approximately 65 mm tubes, which were then placed into Trikinetics Drosophila Activity Monitoring System (www.Trikinetics.com, Waltham, Massachusetts). Locomotor activity was monitored using an infrared beam and was assessed using 1-min time bins. Sleep has been defined as periods of quiescence lasting 5 min or longer [26].

Mating success

Mating success assay consisted of putting 1 virgin female in a vial with a single male of the genotype to be evaluated. Each pair of flies was placed into a vial at ZT4 on the day of eclosion, and then the male was removed at ZT24. Mating success was determined days later through visual inspection of viable offspring (pupae, larvae, etc.). Females from vials that produced no offspring were subsequently provided several males to test for her reproduction viability.

Mating competition

A mating competition assay was also carried out using 2 males who compete for 1 female. In each tube, 1 white-eyed male and 1 red-eyed male of varying PDFR levels competed to mate with a white-eyed female. The 2 competing males were added to a vial simultaneously with a mature virgin female, just prior to the WMZ (ZT10) and left in the vial until the end of WMZ (ZT12). Successful mating of the red-eyed male was determined by female progeny with eye color. Twenty competitions were set up for each genotype and repeated 3 times. Only competitions resulting in progeny were used for analysis.

Sleep restriction

Disruption of sleep was performed similarly as previously described [38,63]. Flies were placed into individual 65 mm tubes and a sleep-nullifying apparatus (SNAP), which mechanically disrupted sleep for 1 min every 15 min for 24 to 48 hours, which both reduced and fragmented sleep. For sleep deprivation, the SNAP was activated once every 8 s for the duration of the experiment. Sleep homeostasis was calculated for each individual as a ratio of the minutes of sleep gained above baseline during the 48 h of recovery divided by the total minutes of sleep lost during 12 h of sleep deprivation.

Starvation

For starvation experiments, adult flies loaded into Trikinetics tubes containing 1% agar, which provides water but not nutrients. Flies’ behavior was monitored until being evaluated for imaging or for survival experiments. The duration of starvation is noted in the text.

Physiology

Methods generally followed those of Klose and colleagues [31]. Flies were removed from DAM monitors, and glass tubes were placed on ice for approximately 5 min. Three to 4 flies were pinned onto a sylgaard dissection dish and were dissected in cold calcium-free HL3 (Stewart and colleagues [64]). Dissected brains were transferred onto a polylysine treated dish (35 3 10 mm Falcon polystyrene) containing 3 ml of 1.5 mM calcium HL3. Two to 4 brains were assayed concurrently, typically a mutant line and its genetic controls. Image capture and x,y,z stage movements were controlled using SLIDEBOOK 5.0 (Intelligent Imaging Innovations, Denver, CO, USA), which controlled a Prior H105Plan Power Stage through a Prior ProScanII. Multiple YFP/CFP ratio measurements were recorded in sequence from region of interest (ROI) in each hemi-segment of each brain in the dish. Each ROI comprised 2 to 4 l-LNvs. Following baseline measurements, 1 ml of saline containing various concentrations of either PDF, DA, or OA (Sigma-Aldrich, St. Louis, MO, USA) was added to the bath (dilution factor of 1/4). We tested normality in the data using the Shapiro–Wilk test in SigmaPlot (Systat Software, San Jose, CA, USA); maximum amplitude values were used to perform ANOVA analyses followed by post hoc Tukey tests.

Immunocytochemistry

Whole flies were fixed in 4% PFA for several hours, and brains were then dissected in ice-cold PBS and incubated overnight with the following primary antibodies: mouse anti-PDF, (5F10, 1,10 dilution, Hybridoma Bank, University of Iowa), chicken anti-myc (GFP-1020; 1:1,000), and anti-GFP. Secondary antibodies were Alexa 488 and 633 conjugated at a dilution 1:200. Brains were mounted on polylysine-treated slides in Vectashield H-1000 mounting medium. Confocal stacks were acquired with a 0.5-μm slice thickness using an Olympus FV1200 laser scanning confocal microscope and processed using ImageJ.

Statistics

All comparisons were done using a Student t test or, if appropriate, ANOVA and subsequent planned comparisons using modified Bonferroni test unless otherwise stated. All statistically different groups are defined as *p < 0.05.

(A, B) Response of l-LNvs to DA and Oa in Pdf-GAL4>UAS-Epac1 flies from day 0 to day 5+ (n = 4–15 hemi-segments per age, ANOVA F[5,36] = 6.08, p = 0.96 and ANOVA F[5,54] = 8.93, p = 0.90, respectively). (C) Normalized FRET ratio in s-LNvs before and during PDF exposure on day 0 (n = 6) and day 5 (n = 7). (D) PDF response amplitude in s-LNvs on day 0 to day 5+ (ANOVA F[5,76] = 13.31, p = 3.75E-9 n = 8–20 hemi-segments per age). *p < 0.05, modified Bonferroni test. Data underlying this figure can be found in S7 Data. DA, dopamine; FRET, Förster Resonance Energy Transfer; l-LNv, large ventral lateral neuron; Oa, octopamine; PDF, pigment dispersing factor; s-LNv, small ventral lateral neuron.

(TIF)

Click here for additional data file.

(A) Immunohistochemistry for PDF and GFP reveals the expression of GFP in the l-LNvs of c929-GAL4/UAS-gfp flies but not in the brains of c929-GAL4/UAS-gfp; Cry-Gal80 flies. (B) Pdfr; PDF>/UAS-Pdfr flies exhibit more waking during the WMZ than Pdfr; Pdf-GAL4/+ and Pdfr;UAS-Pdfr/+ parental controls (ANOVA F[2,91] = 4.63, p = 0.01 n = 41–64 flies/genotype) flies. Data underlying this figure can be found in S8 Data. GFP, green fluorescent protein; l-LNv, large ventral lateral neuron; PDF, pigment dispersing factor; WMZ, wake maintenance zone.

(TIF)

Click here for additional data file.

CD8 GFP expression using l-LNv–specific split Gal4 driver GRSS000645.

(A) CNS with overlay traced reveals cell bodies and optic lobe projections of l-LNvs in the left hemi-segment of a brain. (B) Four cell bodies and projections of l-LNvs of right hemi-segment. Z-stack projections with 2 μm steps. Scale bar: 15 μm. GFP, green fluorescent protein; l-LNv, large ventral lateral neuron.

(TIF)

Click here for additional data file.

(A) Sleep was reduced in both pdf mutants and w genetic controls during the first 18 h of starvation (data presented as change from baseline; (n = 20–22 flies/genotype, p > 0.05). (B) During the first 18 h of starvation, waking activity was significantly lower in pdf mutants compared to w controls (p < 0.05). (C) Kaplan–Meier analysis reveals % survival during starvation in pdf01 (n = 25) flies and w1118 (n = 24) controls (χ2 = 6.20, df = 1, p = 0.01). Data underlying this figure can be found in S9 Data. n.s., not significant.

(TIF)

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(A) Sleep (minutes) during 48 h of starvation in Dcr2; c929-GAL4/UAS-pdfr flies (n = 24), Dcr2; c929-GAL4/+ (n = 18), and +/UAS-Pdfr (n = 32) control flies. ZT, Zeitgeber time.

(TIF)

Click here for additional data file.

(A) The amplitude of l-LNv responses to DA on day 0 in Pdf-GAL4>UAS-Epac1 flies coexpressing RNAi lines for the depicted transcription factors (ANOVA F[8,92] = 1.04, p = 0.42; n is as indicated beneath each bin). (B) The amplitude of s-LNvs responses in Pdf-GAL4>UAS-Epac1 flies coexpressing RNAi lines for the depicted transcription factors neurons on day 0 (ANOVA F[8,112] = 9.36, p = 1.19E-9). (C) The amplitude of s-LNvs responses to DA on day 0 in Pdf-GAL4>UAS-Epac1 flies coexpressing RNAi lines for the depicted cell surface receptors (ANOVA F[10,108] = 0.79, p = 0.63; n is as indicated beneath each bin). (D) The amplitude of l-LNvs responses to DA following sleep restriction in 5-day-old Pdf-GAL4>UAS-Epac1 flies coexpressing RNAi lines for the depicted cell surface receptors (ANOVA F[10,159] = 0.42, p = 0.94; n is as indicated beneath each bin). Data underlying this figure can be found in S9 Data. DA, dopamine; l-LNv, large ventral lateral neuron; PDF, pigment dispersing factor; RNAi, RNA interference; s-LNv, small ventral lateral neuron.

(TIF)

Click here for additional data file.

(A, B) Sleep in minutes/hours for 0-, 1-, and 3-day-old flies maintained on a 12:12 LD schedule. (C–E) FRET ratio measurements in Pdf-GAL4>UAS-Epac1 flies in response to DA, Oa, and PDF. (F) The amplitude of l-LNv responses to PDF. DA, dopamine; FRET, Förster Resonance Energy Transfer; LD, light:dark; l-LNv, large ventral lateral neuron; Oa, octopamine; PDF, pigment dispersing factor.

(XLSX)

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(A) Sleep in minutes/hours in Pdfr5504, Cs, Pdfr5504; c929-GAL4; UAS-Pdfr (rescue, green), Pdfr5504; UAS-Pdfr/+, Pdfr5504; c929-GAL4/+; Cry-Gal80/+, and Pdfr5504; c929-GAL4/UAS-Pdfr; Cry-Gal80.

(XLSX)

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(A–C) % of vials with offspring for Cs, Dcr2; c929-GAL4/UAS-Pdfr, Dcr2; c929-GAL4/+, +/UAS-PdfrRNAi, Pdfr; c92-GAL4; UAS-Pdfr, Pdfr; UAS-Pdfr/+, Pdfr; c929-GAL4/+; CryGal80/+, and Pdfr; c929-GAL4/UAS-Pdfr; Cry-Gal80. (D) Mating competition assay scheme on day 1. (E–G) % of vials with red eyes.

(XLSX)

Click here for additional data file.

(A) Daytime sleep in l-LNv-GAL4>; UAS-Pdfr/+, l-LNv-GAL4/+, UAS-Pdfr/+ Dcr2; c929-GAL4/UAS-Pdfr, c929-GAL4/+, and UAS-Pdfr/+ flies (C) % of surviving flies each hour during starvation in l-LNv-GAL4>; UAS-Pdfr/+, l-LNv-GAL4/+, UAS-Pdfr/+.

(XLSX)

Click here for additional data file.

(A) Traces of normalized FRET ratio during PDF application in l-LNvs from starved and fed Pdf-GAL4>UAS-Epac1 flies. (B)The amplitude of l-LNv responses to PDF in 5-day-old Pdf-GAL4>UAS-Epac1 flies following 21–24 h of starvation. Data are shown for 8 h bins. (C) Sleep in minute/hour in Cs flies maintained on a 12:12 LD schedule during sleep restriction day 2 and during recovery. (D) Traces of normalized FRET ratio during PDF application in l-LNvs recorded from Pdf-GAL4>UAS-Epac1 flies during sleep restriction sleep deprivation and bang. (F) The amplitude of l-LNv responses to PDF in l-LNvs during baseline, sleep restriction, and recovery. (G) Sleep rebound expressed as a difference with baseline sleep in Dcr2; c929-GAL4/UAS-Pdfr, Dcr2; c929-GAL4/+, and UAS-Pdfr/+ flies. FRET, Förster Resonance Energy Transfer; LD, light:dark; l-LNv, large ventral lateral neuron; PDF, pigment dispersing factor.

(XLSX)

Click here for additional data file.

(A)The amplitude of l-LNv responses to PDF on day 0 in Pdf-GAL4>UAS-Epac1 flies crossed to UAS-RNAi lines of the labeled transcription factors. (B) Trace of PDF responses in Pdf-GAL4>UAS-Epac1 during baseline, in sleep-restricted flies, and Pdf-GAL4>UAS-Epac1 flies expressing UAS-nej. (C, D) The amplitude of l-LNv responses in Pdf-GAL4>UAS-Epac1/UAS-nej and age-matched Pdf-GAL4>UAS-Epac1 flies. (E) The amplitude of l-LNv responses to PDF Pdf-GAL4>UAS-Epac1 flies crossed to UAS-RNAi lines of the listed cell surface receptors. (F) The amplitude of l-LNv responses to PDF in sleep restricted. l-LNv, large ventral lateral neuron; PDF, pigment dispersing factor.

(XLSX)

Click here for additional data file.

(A, B) Response of l-LNvs to DA and Oa in Pdf-GAL4>UAS-Epac1 flies from day 0 to day 5+. (C) Normalized FRET ratio in s-LNvs before and during PDF exposure on day 0 and day 5. (D) PDF response amplitude in s-LNvs on day 0 to day 5+. DA, dopamine; FRET, Förster Resonance Energy Transfer; l-LNv, large ventral lateral neuron; Oa, octopamine; PDF, pigment dispersing factor; s-LNv, small ventral lateral neuron.

(XLSX)

Click here for additional data file.

(B) Sleep during the waking during the WMZ in Pdfr; Pdf-GAL4/+ Pdfr;UAS-Pdfr/+ and Pdfr; PDF>/UAS-Pdfr flies. WMZ, wake maintenance zone.

(XLSX)

Click here for additional data file.

(A) Sleep in pdf mutants and w flies during the first 18 h of starvation (data presented as change from baseline. (B) Waking activity during the first 18 h of starvation. (C) Surviving flies each hour during starvation.

(XLSX)

Click here for additional data file.

(A) The amplitude of l-LNv responses to DA on day 0 in Pdf-GAL4>UAS-Epac1 flies coexpressing RNAi lines for the listed transcription factors. (B) The amplitude of s-LNvs responses in Pdf-GAL4>UAS-Epac1 flies coexpressing RNAi lines for the listed transcription factors neurons on day 0. (C) The amplitude of s-LNvs responses to DA on day 0 in in Pdf-GAL4>UAS-Epac1 flies coexpressing RNAi lines for the depicted cell surface receptors. (D) The amplitude of l-LNvs responses to DA following sleep restriction in 5-day-old Pdf-GAL4>UAS-Epac1 flies coexpressing RNAi lines for the listed cell surface receptors. DA, dopamine; l-LNv, large ventral lateral neuron; RNAi, RNA interference; s-LNv, small ventral lateral neuron.

(XLSX)

Click here for additional data file.

3 Sep 2020

Dear Dr Shaw,

Thank you for submitting your manuscript entitled "Sleep-drive reconfigures clock circuitry to regulate adaptive behavior" for consideration as a Research Article by PLOS Biology.

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26 Oct 2020

Dear Dr Shaw,

Thank you very much for submitting your manuscript "Sleep-drive reconfigures clock circuitry to regulate adaptive behavior" for consideration as a Research Article at PLOS Biology. Your manuscript has been evaluated by the PLOS Biology editors, an Academic Editor with relevant expertise, and by several independent reviewers.

As you will see from their detailed responses, the reviewers are enthusiastic about the study in principle, but raise a number of concerns that would need to be addressed to bolster the conclusions drawn in this study. In particular, a concern was raised with the fact that the potential promiscuity of PDFR makes it hard to know whether the effects are acting via PDF or other signals. The reviewers also questioned the reliance on the C929 driver, which is expressed in regions outside the lLNv, thereby making it difficult to say whether the effects are lLNv-specific, and would like additional mechanistic insights.

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REVIEWS:

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Reviewer #4: No

Reviewer #1: In this study, Klose and Shaw describe a striking phenomenon in which an important GPCR for circadian timekeeping (PdfR) is expressed transiently within wake promoting clock neurons (the l-LNvs) during early adulthood to promote late day wakefulness during a developmental window characterized by high levels of sleep. Remarkably, they show that the expression of PdfR is reinstated during times of extended sleep disruption. The authors also examine the fitness implications for this phenomenon and address the relationships between sleep disruption and l-LNv PdfR expression in detail. They also go on to implicate genes mediating the context dependency of PdfR expression in the l-LNvs. These results are highly significant and of great interest. The following points, all modest or minor, might be considered by the authors to improve this very interesting contribution to the field:

1.) What about the PDF neuropeptide? PdfR is a type II GPCR, a class of receptors known for their promiscuity. Are the effects of PdfR signaling in the l-LNvs during times of high juvenile sleep drive and during prolonged sleep disruption mediating PDF signals or signals from some other peptide? It's surprising not to see the Han5304 experiments repeated for Pdf01 mutants. The results of this analysis would be interesting either way and the central conclusion would not depend on the outcome, but the absence of Pdf01 data here produces a bit of an itch that could be easily scratched.

2.) The authors should provide a more detailed rationale for focusing on the 2-h time-window just before dusk as a time corresponding "high sleep-drive." This might confuse some readers, as this is the window in which flies show anticipatory evening activity. Readers might therefore be predisposed to expect low sleep drive here. The idea that this is specific to sleepy young flies could be clarified.

3.) The description of the methods for gauging mating success should be more detailed. Without more detail is it difficult to gauge the quality of the experimental design and the appropriateness of the statistical analysis.

4.) There are no error bars shown on figure depicting mating success data (3A-C), where these experiments replicated?

5.) Although the survival results depicted in Figs. 4C and D are quite striking and clear, a Kaplan-Meier survival analysis should be done for the statistical analysis on these data.

6.) In Fig. 5D, the baseline sleep has clear MYC signal, which is not is what is reported in the results. Is this a labeling error (i.e., are PDF and and MYC labels switched)?

7.) The data shown in supplemental figure S3 seems critical and should be placed in the main figures (Fig. 5).

8.) On Page 2, the four citations (10-13) provided for the notion that circadian timekeeping is a distributed network property neglect two important studies at the expense of a review and a study of questionable relevance (Yao et al. 2014 Science Vol. 343, p. 1516 and Yao et al. 2016 Cell Reports Vol. 13, p.2873).

9.) The use of the split GAL4 specific for the l-LNvs is fantastic, but it's used only in a subset of the experiments. The authors should consider using it alongside all c929-GAL4 experiments, as c929 is widely expressed throughout the brain.

10.) Page 8, line 11, "factor" should probably be "factors."

11.) Page 9, line 1: I don't think it's accurate to call PDF "the main output of the clock" at this point (it also brings the itch of the absence of PDF peptide data back).

12.) Page 19, line 2: "~20" should be "~20h."

13.) I'm not entirely sure about the term "respecification" here. Might there be a better term or phrase?

14.) "Data Table S1 does not match the figures presented in the manuscript.

15.) The text on the histogram in Fig. 2D is difficult to read.

Reviewer #2: This is an interesting manuscript that presents evidence for a dynamic expression of Drosophila PDFR in a specific group of circadian neurons - the lLNvs - known to regulate arousal. PDFR expression in adults is limited to the first couple of days following eclosion, when sleep is high. Also, PDFR expression can be reactivated in case of sleep restriction. PDFR appears to ensure wakefulness in the evening, and thus improves mating in young adult and survival from starvation. The last figure of the paper presents a mini-screen for genes that might contribute to PDFR signaling. This part of the manuscript is quite preliminary, but otherwise, overall, this appears to be an important and for the most part solid study.

My main suggestion to improve this manuscript would be to more clearly establish that the genes identified in figure 6 participate in PDFR signaling and sleep regulation. Do these genes indeed affect sleep similarly to PDFR, do they genetically interact with PDFR?. Also, I do not understand why downregulation of dopamine receptors decrease PDFR responses, but oddly do not reduce sensitivity of the lLNvs to dopamine. This needs to be explained.

I also have a couple of concerns about specific experiments:

1) Is rescue of fig 2b statistically significant? Why is it much weaker than with c929-GAL4? PDF-GAL4 is a strong driver

2) I would suggest that the authors overexpress PDFR in lLNvs of wild-type flies with c929-GAL4 and PDF-GAL4 to ensure that this does not result in gain of functions that could confound the rescue experiments.

3) Why do control flies sleep so little in the RNAi experiments on fig. 2E? Are these flies defective for juvenile sleep regulation, or is there considerable variations between genetic backgrounds? This brings me to wonder how carefully genetic backgrounds are controlled. Are all flies backcrossed to CS? This was not explained in the material and methods or in the main text.

Minor comments:

1) Is "receptor respecification" a commonly used term? It would be helpful to define this as it is used throughout the manuscript

2) The first sentence of the last paragraph of p.2 should be rephrased, as it sounds as if sleep is an environmental cues. It is also not clear to me how sleep synchronizes central and peripheral clocks

4) The first sentence of p.3 needs references

5) My understanding is that only about half of circadian neurons express PDFR, based on work by the Shafer and Taghert labs (Im et al., 2010, 2011 and Yao and Shafer 2014). The relevant statement at the end of the introduction should be corrected.

6) The H on figure 1 is missing.

7) I would indicate on page 4 that there is a noticeable reduction in PDFR sensitivity in sLNv

8) Cry-gal80 is only expressed in CRY+ neurons. The relevant statement at the bottom of p.4 needs to be corrected.

9) On p.4, the authors say that they hypothesized PDFR would be there to promote wakefulness in the evening. While indeed this was the case, the authors could explain better why they thought this would be the case. It seems to me PDFR could have been there to promote sleep.

10) Panels shown on figure 3 are also present on figure S2

11) Second paragraph, p.5, correct "PDF" to "PDFR"

12) Figure 5E. PDf amplitude is not a clear label for the y-axis. Also, it would be clearer to spell out day 1 and day 2 rather then just having 1 or 2 on the x-axis

Reviewer #3: The authors present convincing data that Pdfr expression and receptivity to PDF changes with age in the large LNv, and that perturbations in adult sleep or feeding can induce Pdfr expression in the lLNv. The authors also find that the behavioral effects of loss of Pdfr appear different in young flies than phenotypes previously reported in older adults.

Major Comments:

1. It's not clear whether the physiologically relevant changes in PDFR expression/ PDFR signaling are mainly limited to the large LNv or if other cell groups may contribute. The authors rely primarily on C929-GAL4 to argue for function in the lLNv group, but C929 is expressed outside of the lLNv. The use of cryGAL80 with C929 is helpful but may not limit function to the lLNv given that it blocks expression in other clock neuron groups. Instead, pdfGAL80 would be a much cleaner reagent.

2. In addition, the authors describe a split driver that is specific to the lLNv—the expression pattern and specificity of this line should be documented and functional analysis performed to confirm pdfr function in the lLNv.

3. Young pdf5304 flies sleep more and have less success at mating. If sleep and mating success are related, would other types of wake or sleep promotion (pharmacological, thermogenetic) in young wild type flies also affect mating chances? Also, are pdf5304flies healthy, or can their reduced success at mating/increased sleep be explained as a sickness phenotype?

Other specific comments:

1. Experimental details for assaying Day 0 behavior were included for Figure 2 but not Figure 1A-B. Please clarify method.

2. For Figure 1G, it would be helpful to compare representative images between Day 0 and Day 5. Are there any other differences observed in PDFR expression between these ages? The findings in S1c-d suggest PDFR expression in the sLNv might decrease as well. If there are notable increases/decreases in PDFR levels in other groups, this should be quantified.

3. Statistical significance indicated in some graphs/panels but not others (eg. Figure 3A-C). Please also clarify statistical method used for Figure 3A-C.

4. Previous reports have demonstrated a role for PDF/PDFR in adult sleep regulation (Parisky 2008, Chung 2009), including increased sleep in light phase in PDF/PDFR mutants. The authors should relate their current findings to these published data. A comparison in phenotypes between Day 0 and Day 5 for your backcrossed strains would be informative.

5. The methods relating to starvation related assays (Fig 4C,D; Fig 5A; Fig S3A) were not clearly described.

6. The format of Figure S3a is confusing given that some of the timepoints have an n of 0, thus PDF amplitude was not determined. Additional timepoints should be added and/or data should be binned differently.

7. The methods should more clearly describe the differences between the 'sleep deprivation' and 'sleep restriction' protocols (Fig 5C).

8. Figure 5D, it is difficult to identify PDF+ MYC- large LNv in the baseline images shown. Higher resolution images should be provided with labels to indicate large LNv, ideally comparing a full cluster of large LNv for both baseline and sleep-restricted conditions.

9. neijere has previously been implicated in regulating CLK-CYC transcription activity- does nej RNAi affect pdfGAL4/ epac expression?

10. It is not clear which UAS-pdfr RNAi strain(s) were used.

11. "Receptor respecification is a form of plasticity that, like Hebbian and homeostatic plasticity, may be employed to alter circuit function in response to changing environmental demands [16]"

Can the authors elaborate more on receptor respecification? I find the term a bit confusing - it suggests that one receptor is changed, respecified, to another receptor. But here it seems that a receptor appears in a circuit where it hasn't been previously observed. Also, the appearance of a new receptor should coincide with a change in presynaptic neurotransmitters. Can the authors speculate on which presynaptic inputs have changed?

12. After the sleep restriction protocol that restricts fly sleep to bouts of 15 minutes, flies do not show increased rebound sleep. Is their sleep architecture (bout lengths, bout numbers) also unchanged?

Also, during the one minute sleep deprivation, how many times did the SNAP device move?

13. Fig 4C,D - survival curves are usually represented as Kaplan Meier plots and tested for significance using a logrank test. Can the authors reanalyze their data this way?

Reviewer #4: Comments on "Sleep drive reconfigures clock circuitry…"

This is a promising paper with a set of interesting ideas woven together. It piggy backs onto the outstanding work of Kayser et al. (Sehgal lag) and more recently Matt Kayser from his own lab. They showed that very young flies, days 1-3 approximately after eclosion, sleep considerably more than more mature adults - like human babies. And the more recent work explores the molecular underpinnings of the initial observations. This paper takes off from these observations and finds that the expression of the famous PDF receptor (PDFR) in a well-studied group of circadian neurons is regulated in a similar fashion, namely, PDFR is expressed in the large ventral lateral neurons (l-LNvs) in very young adult flies but then disappears from these cells by day 5. This receptor is known to be expressed in many places in the fly brain including in the l-LNv's neighboring cells, the small ventral lateral neurons (s-LNvs), where it is present all the time in adult flies. The paper then goes on the explore the role of this receptor and how its expression is regulated. They argue that it is expressed in response to a number of stresses including (a particular version of) sleep deprivation, and they identify a transcription factor likely responsible for its synthesis as well as some receptors expressed in the l-LNvs that may be upstream of transcription factor activation.

Comments:

1) From the outset the paper is difficult to follow. The framing and beginning of the paper leads the reader to believe that PDFR expression in l-LNvs must contribute to sleep (since these very young flies sleep more), and yet it does the opposite. Indeed, these neurons have been shown to contribute to daytime activity and therefore would be expected to inhibit sleep. The presence of the receptor causes them to do this only more so it seems to me. A clear definitive sentence at the end of the first paragraph of the Results saying something like this and leading into paragraph 2 would be helpful.

2) The paper relies heavily on the han (PDFR) mutant strain and I think a single RNAi. Also, the key figure 2A should be done in parallel with the pdf01 mutant strain. The results should be very similar, no? And Figure 2A is missing the data from ZT0-ZT4. Nonetheless, it looks like the WT has morning anticipation here but not in Fig. 1. How come? And I'd like to see the % sleep change at each ZT value as well as activity plots and their % change at each ZT. Since the l-LNvs have been shown to contribute to daytime activity in LD, a contribution to activity and therefore some inhibition of sleep is not too surprising - as mentioned above. So is this really specific for some "wake-maintenance zone?" And to what extent is this attributable specifically to this PDFR expression; two RNAis here are essential. And this is an issue throughout the paper, where single RNAs appear to be frequently sufficient to draw a conclusion. (Have at least two RNAis been used to validate the transcription factors identified near the end of the paper?)

3) tPDF and uas-PDFR could also be expressed in l-LNvs (specific gal4 is much better than c929) and in a pdf01 background. This would make the expression data more convincing. This would be much easier for the authors than a direct PDFR RNA assay from purified l-LNvs, which is another alternative.

4) The numbers for live imaging are extremely low (5 neurons? That's maybe 2 brains). How many neurons react (1-4) and how reproducibly?

5) In Fig. 3B, why are the parental controls so low compared to CantonS in Fig. 3A? If statistics are done on all of the data in Fig. 3A and 3B together, would the PDFR mutant statistically differ from the parental controls of the RNAi experiment?

6) How often were the mating success experiments done? No error bars indicate n=1 (M&M also not clear). Also, the data are not very convincing, the WT controls in Fig. 3B are probably not significantly different from the han mutant in Fig 3A. How do the authors explain the index of 0? This suggests too many constructs rather than an effect of PDFR knockdown (pdf01 and han mutants are not sterile). The PDFR rescue should be done with lLNv-GAL4.

7) Labeling of images in Fig 5D is inverted.

8) How can a knockdown of dopamine receptors have no effect on the reactivity of the cell to dopamine? Once again a second RNAi is necessary to make this surprising claim.

9) I don't think the lifetime experiments (Fig. 4) are very compelling. I think of this more as manipulating activity than sleep. It makes sense that starving a more hyperactive fly is worse than a less active fly. Can this be done in parallel with another hyperactive strain?

10) The expression by different modalities is for sure interesting. The reader should learn if only this very special way to sleep deprive upregulates receptor expression or if a more traditional deprivation strategy can do the same thing.

11) Can we see the time of day data that underlies the decreased sleep (and presumably increased activity) in Fig. 5F, the sleep and activity profiles? Are the flies just generally more active, or is there some interesting time of day information? Perhaps the upregulation is just a stress-induced flight or fight response? A bit like enhancing foraging in response to starvation.

12) What's the sleep phenotype in the manipulations of Fig 6? Do nej knockdown flies phenocopy the absence of PDFR?

26 Feb 2021

Submitted filename: Klose and Shaw Response to Reviewers feb 24.docx

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9 Apr 2021

Dear Dr Shaw,

Thank you very much for submitting a revised version of your manuscript "Sleep-drive reconfigures wake-promoting clock circuitry to regulate adaptive behavior" for consideration as a Research Article at PLOS Biology. This revised version of your manuscript has been evaluated by the PLOS Biology editors, the Academic Editor and three of the original reviewers.

The reviews are appended below my signature. As you will see, reviewer 1 is satisfied with the revision, however reviewers 2 and 3 have a number of lingering concerns and note points requiring additional discussion and explanation. Most notably, both reviewers 2 and 3 have commented that it would be valuable to add data from pdf01 flies, even if this doesn’t phenocopy the pdfr mutants. Having discussed the reviewer comments with the Academic Editor, we think that it would be important for your revision to include this pdf01 data - whether they phenocopy Pdfr or not - and to discuss the implications of your findings. Given that this point has now been highlighted by all the reviewers, we think that this will be a question in the mind of many readers.

We are pleased to offer you the opportunity to address the remaining points from the reviewers in a revised version that we anticipate should not take you very long. We expect the revision in a month, however please do let us know if you need an extension to perform the additional pdf01 studies requested by the reviewers. We would be happy to accommodate such a request, as we think it is important that this data is added. We will then assess your revised manuscript and your response to the reviewers' comments and we may consult the reviewers again.

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REVIEWS:

Reviewer #1: The authors have responded directly to the majority of the (all minor) concerns I expressed in my first review. I have no further concerns. It's an interesting, clear, and significant study.

Reviewer #2: Most of my concerns have been addressed. There is one lingering issue. I had realized that figure 2E showed sleep during WMZ. The value for controls in this panel are around 25-40, but on panel 2C CS shows a value of about 85. Those are rather big differences between different control flies. Where is this variability coming from? Is this a genetic background issue? Was pdfr-5304 backcrossed to CS? Controls for genetic backgrounds are good within figures, and a genetic rescue for the pdfr mutant phenotype is presented, so this does not seem to be a major concern, but some explanations are needed.

Minor: The last paragraph of the results on p.9 contains a fragment of sentence that needs to be removed.

I was also asked to look closely at the responses to the comments made by reviewer#4. Again, I think the authors did a good job responding to these comments.

1) One issue that was brought up not just by reviewer #4 but also reviewer #1 is whether pdf0 phenocopies pdfr. This is a fair comment, and I agree with the authors' reply that pdf and pdfr mutants do not necessarily phenocopy each other, due to promiscuity of the pdfr receptor. However, this will be a question in the mind of many readers, and I would suggest that authors show pdf0 mutant data if they have it.

2) I would like to comment on the issue of the need for two RNAis, which was brought up by reviewer #4 and #1. For pdfr phenotypes, I feel that a single RNAi phenocopying the pdfr mutant is sufficient, particularly since the authors can rescue this mutant. For the screen, when only one RNAi line is used, some words of caution would be worth adding.

Reviewer #3: The authors have addressed my major comments--I have one remaining issue that I think would be beneficial for them to address. The authors have nicely clarified in their comments to the reviewers the definition of respecification. I suggest that they provide this level of clarity when they first introduce the concept in the paper and make clear the distinction with other examples in the literature.

With regards to reviewer 4's comment on pdf01, the authors make note of differences between pdf01 and pdfr mutants and their frustration with pdf01. I do think it would be valuable to include pdf01 data even if negative as it would highlight those differences and provide some insight into the relevant ligand for pdfr. Nonetheless the authors should make reference to the differences between pdf01 and pdfr mutants when discussing potential mechanisms by which pdfr might be functioning, i.e., pdfr may not be necessarily be working via PDF activation.

10 May 2021

Submitted filename: Response to Reviewers.docx

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25 May 2021

Dear Dr Shaw,

Thank you for submitting your revised Research Article entitled "Sleep-drive reconfigures wake-promoting clock circuitry to regulate adaptive behavior" for publication in PLOS Biology. I have now obtained advice from the Academic Editor.

We think that the revised manuscript has, for the most part, addressed the concerns of the reviewers in the last round of review. However, we agree with the reviewers that it would be important to tone down statements regarding the RNAi screen on cell surface receptors, given that these are based on experiments with one RNAi line for each gene. Without using more than one RNAi line or transgene rescue, we think that you should not conclude positive effects nor negative effects from this screen, as there is the possibility that these effects were caused by off-target interactions or due to ineffective knockdown. The last paragraph of the results section should therefore be substantially revised, or even removed.

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9 Jun 2021

Submitted filename: Response to Reviewers.docx

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15 Jun 2021

Dear Dr Shaw,

On behalf of my colleagues and the Academic Editor, Bing Ye, I am pleased to say that we can in principle offer to publish your Research Article "Sleep-drive reconfigures wake-promoting clock circuitry to regulate adaptive behavior" in PLOS Biology, provided you address any remaining formatting and reporting issues. These will be detailed in an email that will follow this letter and that you will usually receive within 2-3 business days, during which time no action is required from you. Please note that we will not be able to formally accept your manuscript and schedule it for publication until you have made the required changes.

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