Study rationale and design.

The same genetic background strain (R23E10;UAS-Chrimson) was used for optogenetic or pharmacologically-induced sleep. Flies were fed either all-trans retinal (ATR) or 4,5,6,7-tetrahyrdoisoxazolopyridin-3-ol (THIP) to promote either kind of sleep, which was assessed in three different ways: behavioral analysis, whole brain imaging, and gene expression changes. The comparisons made for each level of analysis are labelled A-E.

Optogenetic- and THIP-induced sleep have similar effects on sleep duration and consolidation.

A) Experimental regime for observing the effects of optogenetic activation and THIP provision (B). C) Sleep profile across 24 hours in the baseline condition (grey) and optogenetic activation condition (green). D) 3-day average of the 24-hour sleep profile of control (grey) and THIP fed (blue) flies. E) Daytime sleep consolidation scatterplot for optogenetic baseline and THIP control flies. F) Daytime sleep consolidation scatterplot for optogenetic- and THIP-induced sleep. G) Night-time sleep consolidation scatterplot for optogenetic baseline and THIP control flies. H) Night-time sleep consolidation scatterplot for optogenetic- and THIP-induced sleep. n = 87 for optogenetic activation across three replicates; n = 88 for –THIP, n = 85 for +THIP, across three replicates. Maximum bout duration possible is 720 minutes, or 12 hours. See Supplementary Figure 1 for summary histograms and Supplementary Table 1 for statistics.

Sleep architecture in optogenetic and THIP induced sleep.

A-B. Average number of sleep bouts in control (grey) and optogenetic activation (green) conditions in the day and night for both +ATR (A) and –ATR (B) fed flies. optogenetic-induced sleep results in an increase in the number of sleep bouts both during the day and the night, whereas red light alone has no effect. C-D. optogenetic activation (green) increases the average sleep duration during the day, but not the night when compared to controls (grey) in +ATR flies (C). D. –ATR flies show no difference in mean sleep bout duration during the day, but show a decrease in average bout duration during the night. THIP (blue) increases both the average number of sleep bouts (E) and the average duration of sleep bouts (F) during the day, but not the night, when compared to controls (grey). Analysis for a and b = Kruskal-Wallis test with Dunn’s multiple comparison correction. * = p<0.05, *** = p < 0.001, **** = p<0.0001. For e and f, analysis = Ordinary one-way ANOVA with Tukey correction for multiple comparisons. *** = p<0.001, **** = p <0.0001.

Brain imaging during optogenetic and THIP-induced sleep

A) Flies were mounted onto a custom-built holder that allowed a coronal visualization of the brain through the posterior side of the head. Perfusion of extracellular fluid (ECF) occurred throughout all experiments. A 617nm LED was delivered to the brain through the imaging objective during optogenetic experiments. During THIP experiments, 4% THIP in ECF was perfused onto the brain through a custom perfusion system. Behavior was recorded as the movement of flies on an air suspended ball. B) Left: Imaging was carried out across 18 z-slices, with a z-step of 6μm. Each z-plane spanned 667μm x 667μm, which was captured across 256 x 256 pixels. Right: A collapsed mask from one fly of neurons found to be active (green) in C alongside all identified regions of interest (ROIs, gray). C) Neural activity in an example fly brain, represented across cells (top) and as the population mean (middle) did not change following optogenetic-induced sleep (bottom). D) Neural activity in an example fly brain, represented across cells (top) and as the population mean (middle) showed an initial high level of activity in the baseline condition, which decreased when the fly fell asleep (bottom) following THIP exposure. The Y axis scale is standard deviation of the experiment mean, so the baseline is not absolute but rather reflects any difference with the overall experiment mean.

Brain activity and connectivity decreases during THIP-induced sleep.

A) Experimental protocol for behavioral responsiveness and brain imaging experiments. 5 mins of baseline condition were recorded, during which the exposed brain was perfused with extracellular fluid (ECF), followed by 5 mins of THIP perfusion. Following sleep induction, an additional 10 minutes of calcium activity was recorded, which was separated into ‘Early’ and ‘Mid’ sleep for analysis. Air puff stimuli were delivered to test for behavioral responsiveness. B) Mean behavioral response rate (% ± sem) to air puff stimuli over the course of an experiment (n = 6). Air puff delivery times are indicated by the solid dots. C) Percent neurons active (± sem) in non ATR-fed UAS:Chrimson / X; Nsyb:LexA/+; LexOp:nlsGCaMP6f / R23E10:Gal4 flies during wake, THIP-induced sleep, and recovery (n = 9; 3 flies were recorded post-waking). D) Correlation analysis (mean degree ± sem) of active neurons in (C). E) Collapsed mask of neurons active during wake, and both early and mid THIP sleep. F) Overlap in neural identities between wake and THIP-induced sleep in two example flies. Number indicates active neurons within each condition; same color code as in (E). G) Quantification of neural overlap data. Red dots indicate the flies shown in (F). n = 9 flies. All tests are one-way ANOVA with Dunnett’s multiple comparison test. ns = not significant, * = p<0.05, ** = p<0.01, **** = p < 0.001.

Metabolic processes are downregulated during THIP-induced sleep.

A) Schematic representation of the experimental set-up and samples processed using RNA-Sequencing. B) Venn diagram showing the gene expression overlap between flies that had been treated with THIP versus their control (shaded blue) and flies that had been treated with THIP in a sleep deprived background versus their control (shaded blue bars). The number of significant differentially-expressed genes in each category is indicated. C) Volcano plot representing the distribution of differentially expressed genes in the presence or absence of THIP. Genes that are significantly up/down regulated meeting a Log2Fold change of 0.58 and FDRq value of 0.05 are shown in red. Genes meeting the threshold for FDRq value only are shown in blue. Fold change only is shown in green. Those genes not meeting any predetermined criteria are shown in grey. D) Volcano plot representing the distribution of differentially expressed genes in the presence or absence of THIP in a sleep deprived background. Criteria as above (C). E) Schematic representation of Gene Ontology (GO) enrichment of biological process results. Colour coded to indicate parent and child terms for comparisons between groups highlighted above (C) - Left and (D) - Right). F) Bar chart representation of a subset of interesting significant GO pathway terms originating from the organic substance and primary metabolic processes for the dataset shown in (C). G) Comparison between significant gene hits obtained via RNA-Sequencing (Blue) and qRT-PCR (Grey) in response to THIP, represented by Log2Fold change values. See Supplemental Tables 2&3 and Supplementary Figures 1&2.

A variety of biological processes including axon guidance are upregulated during optogenetic-induced sleep.

A) Schematic representation of the experimental set-up and samples processed using RNA-Sequencing. B) Venn diagram showing the gene expression overlap between flies that experienced 10 hours of optogenetic-induced sleep (ZT10) compared to -ATR controls (ZT10) and those flies where optogenetic activation was restricted to 1 hour (ZT1) and compared to -ATR controls (ZT1). C) Volcano plot representing the distribution of differentially expressed genes resulting from optogenetic optogenetic activation for 10 hours versus control flies which were allowed to sleep spontaneously for 10 hours. Genes that are significantly up/down regulated meeting a Log2Fold change of 0.58 and FDRq value of 0.05 are shown in red. Genes meeting the threshold for FDRq value only are shown in blue. Fold change only in green. Those genes not meeting any predetermined criteria are shown in grey. D) Volcano plot representing the distribution of differentially expressed genes resulting from optogenetic activation for 1 hour versus control flies which were allowed to sleep spontaneously for 1 hour. Criteria as above (C). E) Schematic representation of Gene Ontology (GO) enrichment of biological process results. Color coded to indicate parent and child terms comparing flies that had been activated optogenetically for 10 hours versus flies which had been allowed to spontaneously sleep for the same duration. F) Bar chart representation of a subset of interesting significant GO pathway terms originating from the regulation of cellular processes and signalling biological processes. G) Comparison between significant gene hits obtained via RNA-Sequencing (Green) and qRT-PCR (Grey) in response to optogenetic sleep, represented by Log2Fold change values. See Supplemental Tables 4-7 and Supplementary Figures 3&4.

nAchRα subunit knockouts bidirectionally regulate >5min sleep as well as short sleep.

A. Average total day and night sleep duration (minutes±95% confidence intervals) in nAchRα knockout mutants, expressed as difference to their respective background controls (see Methods). α1, N=91; contro1 (X59w1118) = 93; α2, N=70; control (w1118ActinCas9) = 65; α3, N=43; (ActinCas9) =9; α4, N=87; (w1118ActinCas9) =98; α6, N=91; (w1118ActinCas9) =91; α7, N=94; (ActinCas9) =95. *P<0.05, ***P<0.001, ****P<0.0001 by t-test adjusted for multiple comparisons. B. Left two panels: sleep architecture for the same six knockout strains as in A (green), shown against their respective controls (black). Each datapoint is a fly. Right panels: cumulative short sleep (1-5min) expressed as a percentage of total sleep duration. Data are the from the same experiment as in A&B. Each datapoint is a fly. **P<0.01, ***P<0.001, ****P<0.0001 Man-Whitney U Test. All data were collected over three days and three nights and averaged.

AkhR knockdown decreases >5min sleep but not short sleep.

A,B. Total sleep (>5min) in UAS-AkhR:RNAi / R57C10-Gal4 flies (blue, N=126) compared to genetic controls (light grey: UAS-AkhR:RNAi / +, N= 124; dark grey: R57C10-Gal4 / +, N= 120). C. Sleep architecture (average bout duration versus bout number per fly) in data from A,B. D. Cumulative short sleep (1-5min, expressed as a % of total sleep) in UAS-AkhR:RNAi / R57C10-Gal4 flies (blue) compared to genetic controls (light grey: UAS-AkhR:RNAi / +; dark grey: R57C10-Gal4 / +). Wild-type background (+) is Canton-S(w1118). Each datapoint is a fly. ***P<0.001, ****P<0.0001 Man-Whitney U Test. ns, not significant. All data were collected over two days and two nights and averaged.

nAchRα subunit knockdowns in sleep-promoting neurons.

A. Average total day and night sleep duration (minutes±95% confidence intervals) in UAS-nAchRα RNAi / R23E10-Gal4 flies, expressed as difference to RNAi / + controls (all were also compared Gal4 / + controls, not shown). α1 N=60, Gal4 N=45, RNAi N=37; α2 N=21, Gal4 N=24, RNAi N=50; α3 N=33, Gal4 N=50, RNAi N=59; α4 N=92, Gal4 N=90, RNAi N=80; α5 N=94, Gal4 N=47, RNAi N=74; α6 N=44, Gal4 N=47, RNAi N=43; α7 N=32, Gal4 N=57, RNAi N=44. **P<0.01, ***P<0.001 by t-test adjusted for multiple comparisons. B. Total >5min sleep duration data for significant knoockdowns in (A). C. Cumulative short sleep (1-5min) expressed as a percentage of total sleep duration. Data are the from the same experiment as in A&B. Each datapoint is a fly. ***P<0.001, ****P<0.0001 Man-Whitney U Test. All data were collected over two days and two nights and averaged.

Gene Ontology (GO) enrichment analysis for THIP-induced sleep (related to Figure 6).

Significantly downregulated and upregulated GO categories for THIP-sleep (Table S2), listed from most enriched at the top. Broad GO categories are identified below.

Gene Ontology (GO) enrichment analysis for THIP-provisioned flies that were sleep deprived (related to Figure 6).

Significantly downregulated and upregulated GO categories for sleep deprived flies (Table S3), listed from most enriched at the top. Broad GO categories are identified below.

Gene Ontology (GO) enrichment analysis for optogenetic-induced sleep (related to Figure 7).

Significantly downregulated and upregulated GO categories for optogenetic-sleep (Table S4), listed from most enriched at the top. Broad GO categories are identified below.

Circadian-related genes uncovered in optogenetic-sleep dataset (related to Figure 7).

A. Zeitgeber (ZT) 10 timepoint was compared with ZT in to uncover potential circadian-regulated genes, in two separate datasets (-ATR and +ATR). 98 genes were shared between these datasets. B. Of the 98 shared genes, circadian-related processes were highly enriched. C. Expression levels of 7 circadian genes drawn from the two different datasets in A.

Summary of different Gene Ontogeny pathways engaged by optogenetic-induced sleep and THIP-induced sleep.

A. Either sleep induction method produces different levels of activity in the fly brain. We term optogenetic-induced sleep ‘active sleep’ because brain activity levels are not different than during wake. We term THIP-induced sleep ‘quiet sleep’ because neural activity is significant decreased already in the first 5 minutes. Both of these induced forms of sleep resemble sleep stages seen during spontaneous sleep in flies. B. Number of GO pathways engaged by either induced active or quiet sleep, separated by upregulated versus downregulated biological processes.

Waking activity levels of nAchRα knockout mutants.

Top: mutants are compared to their genetic background strain. Da1-7 = nAchRα1-7. Bottom: statistical tests for waking activity levels in each knockout compared to its genetic control, during bith day and night.