Nature Neuroscience

Single-cell transcriptomics reveals that glial cells integrate homeostatic and circadian processes to drive sleep–wake cycles

Single-cell transcriptomes of sleep–wake and circadian times

We performed scRNA-seq with 10x droplet microfluidics on adult Drosophila central brains (without optic lobes), sampled at distinct points of the sleep–wake cycle across different circadian times (Fig. 1a). These sample points can be (1) grouped according to four Zeitgeber (ZT) times to analyze the transcriptional correlates of circadian rhythms; (2) grouped by ‘sleep’ and ‘wakefulness’ states according to the animal’s vigilance status at the time of sample collection to examine sleep/wakefulness correlates; and (3) selected and ordered according to the fly’s level of sleep drive to determine the molecular changes associated with the sleep homeostat. To minimize the technical batch effects that may mask true biological responses, we applied demultiplexing based on natural variation between wild-type genotypes10. Specifically, instead of associating each batch to a different sleep or wakefulness state, we associated a different Drosophila genetic reference panel (DGRP)11 line to each behavioral condition (Fig. 1b and Supplementary Table 1). The full genome of all DGRP lines has been sequenced, thus they can be distinguished from one another according to their unique single-nucleotide polymorphisms (SNPs). Thanks to this natural genetic variation, RNA-seq data disclose the associated sleep or wakefulness states, which allows the pooling of brains from different behavioral conditions in a single batch (Fig. 1b and Methods). In addition to minimizing technical batch effects, we also counteracted potential genotype-specific effects by repeating the same conditions with different DGRP lines with similar sleep profiles in subsequent batches (Supplementary Table 1). To find the DGRP lines with the most similar sleep behavior, we carefully chose them by (1) preselecting 36 DGRP lines based on sleep architecture metrics previously reported in a large collection of DGRP lines12 and (2) thoroughly screening those 36 lines across several sleep parameters. The criteria for selection were (1) a robust amount of consolidated nighttime sleep and a low amount of daytime sleep, and (2) low between and within-genotype variability (Extended Data Fig. 1a). Principal component analysis (PCA) of these sleep parameters showed that the ten selected lines grouped together closer than the discarded lines (Extended Data Fig. 1b). Similar clustering of selected versus discarded lines was also visible when plotting the probabilities to transition from a wakefulness to a sleep state (pDoze) or vice versa (pWake), which indirectly assessed sleep drive and sleep depth in Drosophila13 (Extended Data Fig. 1c,d). The low variability between the sleep phenotype of the selected ten DGRP lines allowed us to repeat each condition with several lines in the same and in different batches (Supplementary Table 1). The success of this strategy is attested by the homogenous distribution of cells from different genotypes (Extended Data Fig. 2a), which blended within cell subtypes even without applying batch integration algorithms (Extended Data Fig. 2b).

Fig. 1: Sampling flies at sleep and wakefulness states for subsequent transcriptional profiling.
figure 1

a, Flies were sampled at four different ZT times and 11 different sleep or wakefulness states. Seven of the conditions were ordered according to accumulated sleep pressure. The corresponding downstream analyses of each of the three correlates are described in the corresponding figure (displayed in parentheses). b, In each of seven technical replicates (runs), each condition was linked to two or three DGRP lines. The link between condition and DGRP line was changed in every run. The flies’ central brains were dissected and the tissue was dissociated in a single tube, minimizing batch effects. Conditions were separated by demultiplexing the sequenced reads based on unique SNPs of DGRP lines.

Source data

To obtain an overview of the captured cell types, we first performed dimensionality reduction on all 106,762 cells combined and identified 214 clusters of cells (Fig. 2a). We annotated cells based on previously used marker genes in the fly brain cell atlas14,15, allowing us to successfully assign 22,988 cells to one of 25 known cell types (21.5%) (Fig. 2b and Supplementary Table 2), including five glial subtypes, Kenyon cells (KCs), clock neurons and cell types containing known sleep/wakefulness regulating circuits such as, non-protocerebral anterior medial (PAM) dopaminergic neurons (DANs)16, tyraminergic (Tyr) and octopaminergic (Oct) neurons17,18, and ellipsoid body (EB) ring neurons19,20. Another cell type involved in sleep21, the dorsal fan-shaped body (dFB) neuron, was annotated by correlating the previously published transcriptome of fluorescence-activated cell (FAC)-sorted dFB neurons14 with our gene expression data (Extended Data Fig. 3).

Fig. 2: Cell-type annotations in a single-cell atlas of the sleeping fruit fly.
figure 2

a, t-Distributed stochastic neighbor embedding (t-SNE) plot of the entire dataset of 106,762 single cells with annotated clusters and expression of key marker genes of glial and neuronal cell types used to annotate the clusters. b, Marker gene expression across most of the clusters annotated in a. 5-HT, 5-hydroxytryptamine (serotonergic) neuron; adPN, anterodorsal projection neuron; ALG, astrocyte-like glia; CXG, cortex glia; EG, ensheathing glia; PB, protocerebral bridge neuron; PG, perineurial glia.

Cycling of core circadian genes in clock neurons and glia

The expression levels of circadian clock gene transcripts cycle in a daily manner in clock neurons2. To validate our single-cell transcriptomic dataset, we asked whether the data accurately captured the cycling expression of core clock genes between the four sampled ZT time points. Indeed, as reported previously in clock neurons22, period (per) and timeless (tim) transcripts were expressed at higher levels in the early night compared to the early day, while the opposite applied to cryptochrome (cry) and Clock (Clk) mRNA (Fig. 3a). Beyond validating our dataset, we examined whether and how the clock genes were cycling in all remaining cell populations. In mammals, cycling of clock gene expression in cell populations other than the SCN has been reported, for example, in cortical regions23. Interestingly, we report that the expression and cycling of these genes is restricted specifically to clock neurons and glial cells (Fig. 3a,b and Extended Data Fig. 4a–e); this is consistent with previous immunostaining results for clock proteins in the fly brain24,25. To further confirm this, we tested the activity of the Clk regulatory network (regulon) across all cells by applying SCENIC26. This regulon was defined by the previously identified Clk binding element E-box sequence and its target genes, including tim, cry and vrille (vri)27. Our data show that the Clk regulon is only active in clock neurons and most glial subtypes, but not in other neurons (Fig. 3c), further indicating that core clock genes are expressed and cycle specifically in Drosophila clock neurons and glia. Interestingly, while Clk expression is restricted to the clock neurons and glia, other core clock genes, per, tim and Cycle (Cyc) are expressed across more cell types. The finding that no cell type expresses Clk without expression of other core circadian genes is consistent with the notion that Clk is a circadian master regulator28.

Fig. 3: Oscillating transcripts in neurons and glia.
figure 3

a,b, Circadian expression levels of the core clock genes per, tim, cry and Clk averaged across all neurons, some neuronal subtypes (a) and all glia and glial subtypes (b). The size of the dot indicates the fraction of cells in each group. Mean expression in each group was normalized to gene expression across the four ZT time points. The data in the line plot were plotted twice to better visualize the cycling patterns. c, Clk regulon and its activity across all cell types. d, Schematic of the template to detect cycling transcripts based on Borbély’s two-process model1. Examples of genes whose expression would match (yellow and orange) or not match (blue and red) the template are given. e, Heatmap of intersecting cycling genes between all annotated cell types with at least one shared gene relative to the total number of cycling genes (at least five genes, right bar plot) according to cell type. Genes with a JTK cycle Benjamini–Hochberg-corrected P < 0.05 were considered as significantly cycling.

Fly glia were previously associated with the expression of clock genes, especially astrocyte-like glia (ALGs)25 and perineurial glia (PG)29. Interestingly, our data showed that the molecular clock runs with different phases depending on the cell type. Specifically, the expression of tim and per mRNA is high throughout the night in clock neurons, while in PG and ensheathing glia (EG), its expression was already decreased at ZT20 (Fig. 3b and Extended Data Fig. 4a–e). In contrast, tim expression in astrocytes only peaks at ZT20. The delayed clock in astrocytes compared to neurons is also observed in the mouse SCN30. Taken together, the expression of key clock genes in glia in addition to clock neurons, suggests that these cells are directly involved in circadian regulation of rhythmic behaviors, including sleep.

Cyclers are more enriched in glia than neurons

Next, we comprehensively identified all transcripts (cyclers) oscillating in all cell types by applying the JTK cycle algorithm (Fig. 3d, Supplementary Table 3 and Methods)31. While the expression of the molecular clock was restricted to clock neurons and glial cells, most cell clusters (82.5%) had at least one cycler (Extended Data Fig. 4f) and 14 of the 19 annotated clusters had at least five cyclers (Fig. 3e). Like the analysis of core clock genes, glial cells stand out, this time by showing the highest number of cyclers, especially considering that they typically express fewer genes than neurons (Extended Data Fig. 4g). When comparing the overlap of cyclers between all cell types, we found that only between 20% and a maximum of 50% of cyclers are shared. We identified a higher number of shared cyclers between closely related cell types, such as the three KC subtypes, and particularly between neuropil-associated glial subtypes (ALGs and EGs) (Fig. 3e). Similarly, gene ontology (GO) analysis of cyclers showed that glial subtypes shared more similar signaling pathways compared to neuronal subtypes (Extended Data Fig. 4h). GO terms encompassed ‘mitochondrial electron transport, NADH to ubiquinone’, ‘sodium ion transport’, ‘chemical synaptic transmission’ and ‘extracellular region’, suggesting that many key functions of glia including metabolism, ionic homeostasis and gliotransmission, are affected by the circadian clock. In summary, these data suggest that different cell populations respond differently to process C, with glial cells being the most affected.

Sleep/wakefulness correlates differ between cell populations

Next, we asked whether we could detect transcript level changes across sleep/wakefulness states (referred to as sleep/wakefulness correlates), similarly to ZT times. We assigned each condition to a sleep or wakefulness group based on whether the animal had consolidated sleep or wakefulness before sampling (Figs. 1a and 4a and Methods). The sleep group included spontaneous sleep, recovery sleep after sleep deprivation (SD), yoked control (YC) and gaboxadol (GBX)-induced sleep32. We grouped spontaneous wakefulness and forced wakefulness (SD) to regress out the unwanted effects of ZT time and the potential stress induced by mechanical SD. Then, we performed differential expression analysis between the two groups for each cell population separately and asked whether transcriptomic changes between sleep and wakefulness states differ between distinct cell populations. We found that 46.7% of all clusters and 57.9% of annotated clusters had sleep/wakefulness correlates (Fig. 4b, Extended Data Fig. 5a,b and Supplementary Table 4). KCs and glia had the highest number of differentially expressed genes (DEGs) among the annotated cell types. Considering that glia express the lowest number of genes among all cell types (Extended Data Fig. 5b), these cells may be even more affected by sleep/wakefulness relative to their total expressed genes compared to neurons. Interestingly, most identified DEGs in each of the annotated cell types were unique to that cell type (Fig. 4b). On average only 12.8% or 13.5% of DEGs were shared within neuronal or glial subtypes, respectively. Like cyclers, the overlap of sleep/wakefulness-correlated transcripts was even smaller between neurons and glia, averaging between 3% and 4% only, with the exception of PAM DANs and protocerebral bridge (PB) neurons, which shared between 17% and 33% of their sleep/wakefulness-correlated genes with ALG and EG_1. This low overlap between closely related cell subtypes suggests that sleep–wake cycles affect their transcriptome in unique ways.

Fig. 4: The transcriptomes of glia and KCs change differently between sleep and wakefulness.
figure 4

a, Grouping of wakefulness–SD states and sleep states to compare them in differential expression analysis and a tree-based classifier. b, Heatmap of intersecting DEGs between all annotated cell types with at least one shared gene relative to the total number of DEGs according to cluster. The bottom bar plot shows the amount of total DEGs with an adjusted P < 0.05 and log fold change lower than −0.5 or greater than 0.5. The statistical method used was a Wilcoxon rank-sum test. P values were adjusted for multiple-comparisons testing using the Benjamini–Hochberg method. c, Classifying glial (top) and KC (bottom) subtypes into sleep or wakefulness labels revealed that performance was highest for the same subtype. di, For glia (d) and KCs (g), candidate genes were filtered by (1) removing the cell-type features from the sleep/wake features identified using separately trained Explainable Boosting Machines (EBMs) and (2) overlapping significant differential expression analysis (DEA) and EBM results. The volcano plots highlight a selection of the common genes between the two methods for all glia (e), EG_1 (f), all KC (h) and y-KC (i).

To further probe the differences between closely related KC and glial subtypes, we applied a complementary, independent approach to differential expression analysis. We asked whether a tree-based classifier could learn the transcriptome makeup of one cell subtype during the sleep and wakefulness states and subsequently, how accurately this model would perform in classifying cells of another closely related subtype into either of these states (Fig. 4c). This approach was applied to the KC and glial cell subtypes separately. To ensure that the classifier would discriminate between sleep and wakefulness states rather than cell identity, we excluded marker genes between either KC or glial subtypes up to the point that the subtypes merged into one another in the two-dimensional uniform manifold approximation and projection (UMAP) space (Extended Data Fig. 6a,b). The accuracy of the classifier is illustrated in a confusion matrix, where the color corresponds to the probability to accurately assign a sleep or wakefulness label per cell type. We found that the classifier performed better for the cell subtype it was trained on, than on other related ones (Fig. 4c). To ensure that the classifier’s improved performance was not driven by overfitting to the cell type (that is, learning cell identity features), we randomly shuffled the sleep and wakefulness labeling of cells within their subtype identity. Then, the classifier would not distinguish between the sleep and wakefulness states even in the same subtype, suggesting that the classifier truly learned sleep/wakefulness, rather than cell identity features (Extended Data Fig. 6c,d). Taken together, both the differential expression analysis and classification approach suggest that depending on their cell identity, different cell populations have different sleep/wakefulness correlates.

Next, we asked what kind of genes are altered between sleep and wakefulness in glia and KCs. To gain confidence in the candidate gene list, we first trained another classifier to detect cell identity features between glial and KC subtypes. Then, we subtracted those cell identity features from those used by the sleep–wakefulness classifier (Fig. 4d,g). To reduce the false positive rate further, we considered genes as significantly deregulated only if they remained after the filtering step above and if they were identified as significant in the differential expression analysis, leaving 30 and 39 sleep/wakefulness-correlated transcripts in glia and KCs, respectively (Fig. 4d,g). Among the filtered 39 sleep/wakefulness correlates of KCs, we found Hr38, an activity-dependent gene in insects33. To validate its increased expression during SD and wakefulness compared to sleep in KCs specifically, we performed fluorescence in situ hybridization (FISH) during sleep and SD. In accordance with our single-cell data, Hr38 mRNA in KCs increased expression after SD (Extended Data Fig. 7). The sleep/wakefulness correlates in glia included metabolism-related genes (CG9360, Cyp28a5, GstE12, GstE1, mdh1, Ssadh and Ugt86Dd), genes involved in protein synthesis and homeostasis (Rpl41, Cct2, CG34362, CG6770 and sm), and genes regulating the core circadian clock (CG31324 and vri) (Extended Data Fig. 5c and Supplementary Table 4). In contrast, sleep/wakefulness correlates in KCs included many genes involved in axon and synapse development and function: Ten-a, rho, pyd, hig, Gfrl, CG42674, Hasp and beat-IIIb (Extended Data Fig. 5c and Supplementary Table 4).

Template matching captures cell types involved in process S

While the sleep/wakefulness correlates detect how the transcriptome changes in a binary manner, we next assessed whether it also changed gradually with the level of sleep pressure (sleep drive correlates). We sampled flies at multiple sleep and SD states, which can be ordered according to their level of sleep drive, from 20 h of GBX-induced sleep to 20 h of SD (Fig. 5a). To detect transcripts whose expression correlated with the gradual increase of sleep drive, we adopted a feature selection method34. Like the JTK algorithm for the identification of cyclers, which calculates the correlations between measured gene expression and assumed sine waves, this method also asks whether gene expression changes according to a given pattern. More specifically, we tested whether the expression profile for each measured transcript across sleep drive states, significantly correlates with a predefined template. The template consisted of values from 0 to 1, where 0 corresponds to the condition of lowest sleep pressure and 1 to the highest. Another five conditions were assigned to continuous values between 0 and 1 according to the respective level of sleep pressure the animal experienced (Fig. 5a and Methods).

Fig. 5: Analysis of molecular correlates of sleep drive.
figure 5

a, Illustration of the sleep drive template based on Borbély’s two-process model1. Points of low and high sleep pressure are indicated. Examples of genes whose expression would match (blue and purple) or not match (red and yellow) the template are given. bg, Cluster map of all significant sleep drive correlates of dFB (b), Oct (c), Tyr (d) and non-PAM DANs (e). The gene expression of sleep drive correlates from non-PAM DANs did not correlate with the sleep drive template in PAM DANs (f) or all cells combined (pseudobulk) (g). A subset of significantly correlating sleep drive genes is labeled. The statistical test used was Pearson correlation. P values were adjusted for multiple tests using the Benjamini–Hochberg method. h, Heatmap of intersecting sleep drive correlates between all annotated cell types with at least one shared gene relative to the total number of sleep drive correlates according to cluster.

We found that 65.1% of all clusters and 68.4% of annotated clusters had at least one sleep drive correlate (Extended Data Fig. 8a and Supplementary Table 5). Interestingly, the four annotated clusters with the highest amount of sleep drive correlates were cell populations associated with sleep homeostasis, spearheaded with 121 correlates by dFB neurons (Fig. 5b and Extended Data Fig. 8a,b), which we previously annotated (Extended Data Fig. 3). Similarly, wakefulness-promoting Oct, Tyr and non-PAM DAN neurons each had more than 100 sleep drive correlates (Fig. 5c–e and Extended Data Fig. 8a,b). Plotting the gene expression of sleep drive correlates from low to high sleep pressure conditions showed clear correlating patterns for dFB, Oct, Tyr and non-PAM DAN neurons (Fig. 5c–e). In contrast, the related dopaminergic subtype of PAM neurons only had 14 correlates (Extended Data Fig. 8a,b). Furthermore, the expression of non-PAM DAN sleep drive correlates did not correlate with sleep pressure in PAM DAN neurons nor in all cells combined (pseudobulk) (Fig. 5f-g). Thus, in this study we demonstrated that the template-matching method was sufficiently sensitive and specific to capture sleep drive correlates of previously identified sleep circuits.

Analogous to asking whether cyclers and sleep/wakefulness correlates differed between cell populations, we next assessed whether molecular correlates of sleep drive varied depending on cell identity. We found that the specificity of sleep drive correlates to a cell type was even more pronounced compared to that of the sleep/wakefulness correlates and cyclers. The highest overlap across all clusters was merely 8% between abp-KC and clock or ring_2 and between astrocytes (ALGs) and Tyr neurons (Fig. 5h). These data suggested that different cells responded to process S in unique ways.

Similarly, GO analysis revealed little overlap of sleep drive correlates between cell types (Extended Data Fig. 8c). In dFB neurons, we found many sleep drive correlates that were involved in synaptic formation and function (unc-104, scramb1, dpr10, dpr19, syt1, sm, brp and CG8386). This is consistent with previous evidence linking neuronal activity of dFB neurons to levels of sleep pressure21. Our data also showed that the expression level of several mitochondria-related genes also correlated with sleep drive, including mRpL27, mRpL50, Sod2, CG32113 and Idh. This is in agreement with the notion that mitochondrial function in the dFB is critical for sleep homeostasis35.

R5 neurons have a high number of sleep drive correlates

We found that one subcluster of EB ring neurons (ring_2) had a substantial number of sleep drive correlates, while the other (ring_1) showed only a few (Fig. 5h). Therefore, we asked whether the previously identified sleep drive-regulating R5 neurons are part of the ring_2 subcluster. R5 neurons contain only approximately 32 cells in an adult fly brain19. To identify them, we first selected all EB ring neurons that were annotated based on previously used marker genes15 and repeated dimensionality reduction exclusively on the EB ring neuron cluster to specifically analyze its subclusters. This reclustering resulted in at least three separate subclusters expressing unique transcriptomic signatures (Fig. 6a). To identify R5 neurons among the three clusters, we focused on a subset of marker genes (Fig. 6b), whose expression pattern is known from T2A-Gal4 knock-in driver lines36,37. By crossing relevant driver lines to chemically tagged effectors, we visualized the expression pattern of multiple marker genes in the EB ring neuron subclusters (Fig. 6c). EB ring neuron subtypes can be distinguished by their projection patterns into the ring-shaped EB structure38. This allowed us to map the subtype identities to some of our single-cell EB ring neuron subclusters. Ring_C differentially expressed cry and pdfR (Fig. 6b), two genes that are expressed specifically in neurons projecting into the R5 ring (Fig. 6c). Therefore, we identified cluster ring_C as R5. Remarkably, we found a high number of sleep drive-correlated genes specifically in R5 neurons, while few to no genes were identified in the other two subclusters (Fig. 6d,e and Extended Data Fig. 8a,b). Many of these transcripts were mitochondrial genes, including mt:ATPase6 and Pink1, as confirmed by GO analysis (Extended Data Fig. 8c and Supplementary Table 5). This finding suggested that mitochondrial function in R5 neurons is important for the role of these neurons in sleep homeostasis. Interestingly, we found that a gene encoding a potassium channel ether-à-go-go (eag) correlated negatively with sleep drive in R5 neurons. Potassium channels, including Eag, reduce neuronal excitability39. This is consistent with the finding that the neuronal activity of R5 increases with the levels of sleep drive19.

Fig. 6: Identification of EB R5 neurons and sleep drive correlates across EB ring neuron subtypes.
figure 6

a, Top, t-SNE plot of two EB ring neuron subclusters. Bottom, reclustering of EB ring neurons into three subclusters, that is, ring_A, ring_B and ring_C. b, Top 20 DEGs between the three subclusters. c (i)–(v), Expression pattern and chemical tag staining of T2A-Gal4 driver lines of the genes boxed in b. Their morphology revealed that neurons in ring_C are EB R5 neurons. d, t-SNE displaying the number of sleep drive correlates at three-cluster resolution. e, Cluster map displaying the expression of sleep drive correlates for R5 neurons. A subset of significantly correlated sleep drive genes is labeled. f, t-SNE displaying the number of sleep drive correlates at a nine-cluster resolution.

Source data

Anatomic analyses showed that there were 11 subclusters of EB ring neurons40. Therefore, we asked whether our two bigger EB ring neuron clusters still contained multiple subpopulations and reclustered them into another eight subclusters. Coloring each cell by its assigned run showed that no clusters were dominated by one run, thereby confirming that our EB ring subclusters were indeed driven by different cell subtype identities rather than by technical batch effects (Extended Data Fig. 2c,d). Matching the sleep drive template to these subclusters revealed that two of them had a high number of correlating genes in addition to R5 (Fig. 6f). This is in line with studies that identified additional ring neuron subtypes apart from R5 that regulate sleep amount and sleep fragmentation20,41. Importantly, the remaining six subclusters had no or few genes correlating with sleep drive (Fig. 6e), demonstrating the specificity of the template-matching method. This reclustering analysis also highlighted the importance of examining homogeneous populations.

Clock neuron subtypes differ in sleep drive correlate numbers

Clock neurons are a heterogenous population consisting of four dorsal neuron subtypes (DN1a, DN1p, DN2 and DN3) and three lateral neuron (LN) subtypes (LNv, LNd and LPN)42. Previous anatomical analyses and single-cell transcriptomic data also suggested that many of these clock neuron subtypes, especially DN1p, can be further divided into smaller clusters22,43. Importantly, some clock neuron subtypes are involved in sleep regulation, while others are not44,45,46,47. We aimed to investigate if we could detect this discrepancy in our data. To this end, we first subclustered our 494 clock neurons using an approach similar to that used with the EB ring neurons. We found clear subclusters emerging in the dimensionality reduction (Fig. 7a and Extended Data Fig. 2e,f). Like subclusters of the EB ring neurons, clock neuron subclusters were driven by different cell identities rather than by technical batch effects (Extended Data Fig. 2e,f).

Fig. 7: Analysis of sleep drive correlates in clock neuron subtypes.
figure 7

a, Predicted annotations of clock neuron subtypes on our data based on training the scANVI model on data from Ma et al.22. Cluster annotation names were transferred from Ma et al.22. b, VGlut expression mapped onto our data; DN1p subtype clusters 18 (green) and 4 (red) are indicated. c, Plotting of our 494 clock neurons together with clock scRNA-seq atlas of Ma et al.22 split into 17 high-confidence annotated clusters. d, Our cells in blue highlighted in the UMAP space with data from Ma et al.22. e, Proportion of cells according to clock subtype for data from Ma et al.22, data from this study and published counts of cells from neuroanatomical studies. fh, Cluster map of all significant sleep drive correlates of 18:DN1p (f). Gene expression of the significant genes for 18:DN1p did not correlate with sleep drive in another DN1p subtype (g) or 14:DN3 (h).

To clearly annotate our clock neuron subclusters, we trained a semi-supervised model (single-cell annotation using variational inference (scANVI))48 on the 2,615 single-cell transcriptome profiles of the published clock neurons by Ma et al.22. The model was then used to predict the clock subcluster identities of our 494 clock neurons (Fig. 7a). Ma et al.22 annotated 17 clock neuron subclusters with high confidence. We found 14 of the 17 high-confidence subclusters in our data (Fig. 7a). We confirmed the expression of some cluster-specific genes (Pdf for s_LNv, CCHa1 for DN1a, AstA and Rh7 for DN1p, and vGlut for all clusters except for all LN clusters and 4:DN1p) (Fig. 7b and Extended Data Fig. 9a,d). We also tested the expression of the top five marker genes identified by Ma et al.22 across all 14 annotated clusters. Like their findings, we observed that within the same subtype of DN1p neurons, six subclusters with distinct gene expression signatures emerged (Extended Data Fig. 9e). Furthermore, most of our subclusters that had emerged using dimensionality reduction were clearly mapped to individual clock neuron subtypes, particularly the LNs and DN1p subclusters (Fig. 7a). These findings indicate that we successfully annotated our clock neuron subclusters.

However, the prediction was less successful when separating the 19:DN2 subcluster because it clustered together with the 6:DN1p and 18:DN1p subtypes (Fig. 7a). Furthermore, the predicted 14:DN3 cluster divided into multiple clusters (Fig. 7a), suggesting the existence of multiple DN3 subtypes that the study by Ma et al.22 may have missed. We propose that the larger number of DN3 neurons in our dataset (Fig. 7c,d) could be due to the difference in methodologies between the studies. Ma et al.22 FAC-sorted clock neurons by using the Clk856-Gal4 driver, which does not label most DN3 neurons. In contrast, our data were generated in an unbiased manner, that is, not by sorting a driver line. In accordance, the proportion of clock subtypes we yielded matches the number of cells that others have previously counted based on immunostaining of TIMELESS protein43,44 (Fig. 7e). In contrast, but as expected, the data sorted from the Clk856-Gal4 driver consisted of only a small proportion of DN3 cells. Interestingly, compared to the anatomical cell counts, we lacked LNs (Fig. 7e). This is probably because these cells may have been removed together with the optic lobe when we dissected the central brains, as LNs are located close to the border between the central brain and optic lobes.

Like the homogenous R5 subcluster, we next asked whether we could identify sleep drive correlates in the more homogenous, annotated clock neuron subclusters. However, while the template-matching method works well on homogenous populations, it still requires a minimum number of cells such that all template conditions are covered. This limitation resulted in the template matching to run only in three annotated clock subclusters, that is, 18:DN1p, 4:DN1p and 14:DN3. Interestingly, we identified significant sleep drive correlates only in the glutamatergic-positive cluster 18:DN1p, not in the glutamatergic-negative 4:DN1p subtype nor in 14:DN3 neurons (Fig. 7f–h). This is consistent with findings that glutamatergic DN1p neurons are involved in sleep/wakefulness regulation44,45. Among the sleep drive correlates of the 18:DN1p cluster are two genes that regulate the function of a potassium channel: slowpoke-binding protein (slob) and dyschronic (dysc) (Fig. 7f). This is consistent with the finding that the potassium channel (slowpoke) is important in glutamatergic DN1p neurons to regulate sleep quality49.

Homeostatic and circadian processes converge in glia

Previously, it was shown in flies that homeostatic and circadian processes interact indirectly by neuronal circuits connecting clock neurons with sleep homeostat circuits (EB ring neurons and dFB neurons)44,45,46,50,51. We asked whether there is a more direct interaction of the two processes in the same cell and whether one process affects a cell type more than the other one. To this end, we compared the number of sleep drive correlates with the number of cyclers by cell population and assigned each cell type to either process or to both simultaneously (Methods). Surprisingly, in most cases, we found that a given cell type was more affected by only one process (Fig. 8a,b). For example, dFB, Oct/Tyr, non-PAM DANs and R5 neurons had many sleep drive correlates but few circadian cyclers. On the other hand, cell types with many circadian correlates, for example, y-KCs, ab-KCs and PGs, had no sleep drive correlates. Remarkably, two subtypes of EB ring neurons, that is, R5 and ring_B were affected by either process in opposing ways, in accordance with previous findings, showing that R2/R4m neurons (probably part of ring_B) received circadian timing information from clock neurons51,52, while R5 neurons themselves encoded the sleep homeostat19. This suggested that the effect of either circadian or homeostatic process differs depending on the cell type and can vary even for closely related cell types. KC subtypes, except for abp-KCs, followed a pattern of high number of circadian cyclers, but few or no sleep drive correlates. Intriguingly, both the number of cyclers and sleep drive correlates were high in all glia with the exception of PGs, as opposed to few neuronal clusters with such high numbers. This demonstrated that a simultaneous convergence of both circadian and homeostatic processes takes place in glial cells, as their transcriptome is affected by both.

Fig. 8: Homeostatic and circadian processes converge on glial cells.
figure 8

a, Clusters assigned to cyclers (yellow, left) or sleep drive correlates (blue, right) visualized in the t-SNE. Middle, The t-SNE shows the merge of left and right t-SNE, highlighting the clusters assigned to both groups in green. b, Number of correlating genes with circadian or sleep drive template across all annotated clusters that have correlates for either process. The color indicates their assigned group (yellow, cyclers; blue, sleep drive correlates; green, both). c, Sleep amount 4 h after SD compared to the baseline sleep of the same fly in the same ZT time period before SD in flies expressing conditional knockout of vri (n = 30), tim (n = 47) and cry (n = 35) in pink and control flies (repo-Gal4>iso31 (n = 58); repo-Gal4>UAS-Cas9.P2 (n = 30); repo-Gal4>UAS-sgRNA-vri (n = 41); repo-Gal4>UAS-sgRNA-tim (n = 26); and repo-Gal4>UAS-sgRNA-cry (n = 41)) in purple. d, Sleep amount 4 h after SD compared to baseline sleep of the same fly in the same ZT time period before SD of flies expressing a dominant negative form of Cyc (n = 48) in pink and control flies (repo-Gal4>iso31 (n = 58)) in purple. c,d, The statistical method used was a Wilcoxon rank-sum test, with Bonferroni-corrected P value adjustment for multiple-comparisons testing. The boxplots indicate the minimum, median, maximum, and first and third quartiles. The error bars represent the first (third) quartile ± 1.5 times the interquartile range. Adjusted *P < 0.05, **P < 0.01, ***P < 0.001; NS, not significant.

Source data

Interestingly, we identified genes that regulate the core molecular clock, vri and CG31324, as sleep/wakefulness correlates in glia. Similarly, the expression levels of E23, a regulator of the circadian rhythm, correlated with the sleep drive in glia specifically. Thus, process S may directly influence the core clock machinery in glial cells. Next, we asked whether the disruption of the circadian clock, specifically in glia, would result in impairment of the sleep homeostat. We expressed a dominant negative form of Cyc53 or conditionally knocked out tim54, vri or cry55 specifically in glial cells, while leaving all neurons, including clock neurons, unaffected. To assess sleep homeostasis in these animals, we sleep-deprived flies for 12 h during the night and measured rebound sleep, a hallmark of sleep homeostasis, the following morning. Flies with a disrupted glial clock showed significantly reduced rebound sleep after SD compared to control flies (Fig. 8c,d). Interestingly, while disrupting expression of tim, vri and Cyc resulted in reduced sleep rebound, cry knockout did not. This is in accordance with cry participating in neither of the two core transcriptional–translational feedback loops in Drosophila. These data indicate that the glial clock is required for normal sleep homeostasis and suggest that processes S and C directly influence each other in glial cells to determine sleep–wake cycles.

Interface for browsing gene expression correlates

Our correlational analyses (cyclers, DEGs between sleep and wakefulness, and correlates of sleep drive) across all genes and clusters are accessible to explore visually within a user-friendly interface ( as a complement to Supplementary Tables 35. The expression of a gene of interest can be compared across cell types (Extended Data Fig. 10a) or in relation to other genes of the same cell type (Extended Data Fig. 10b).

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