Experimentally induced active and quiet sleep engage non-overlapping transcriptional programs in Drosophila

There is increasing evidence that sleep is a complex phenomenon in most animals, comprising of distinct stages that are characterized by dramatically different physiological processes and brain activity signatures (Jaggard et al., 2021; Van De Poll and van Swinderen, 2021). This suggests that different sleep stages, such as rapid eye movement (REM) and slow-wave sleep (SWS) in humans and other mammals (Dijk et al., 1990), are accomplishing distinct functions that are nevertheless collectively important for adaptive behavior and survival (Tononi and Cirelli, 2014). While REM and SWS appear to be restricted to a subset of vertebrates (e.g., mammals, birds, and possibly some reptiles; Siegel, 2011; Shein-Idelson et al., 2016; Rattenborg et al., 2019), a broader range of animals, including invertebrates, demonstrate evidence of ‘active’ vs. ‘quiet’ sleep (Jaggard et al., 2021; Van De Poll and van Swinderen, 2021; Libourel and Herrel, 2016). Whether active and quiet sleep represent evolutionary antecedents of REM and SWS, respectively, remains speculative (Jaggard et al., 2021; Van De Poll and van Swinderen, 2021). However, during active sleep, although animals are less responsive, brain recordings reveal a level of neural activity that is similar to wakefulness, in contrast to quiet sleep, which is characterized by significantly decreased neural activity in invertebrates (Yap et al., 2017; Tainton-Heap et al., 2021) as well as certain fish (Leung et al., 2019), mollusks (Iglesias et al., 2019), and reptiles (Shein-Idelson et al., 2016).

Although it is likely that even insects such as fruit flies and honeybees sleep in distinct stages (Sauer et al., 2003; van Alphen et al., 2013), sleep studies using the genetic model Drosophila melanogaster still mostly measure sleep as a single phenomenon, defined by 5 min (or more) of inactivity (Shaw et al., 2000; Hendricks et al., 2000). As sleep studies increasingly employ Drosophila to investigate molecular and cellular processes underpinning potential sleep functions, this simplified approach to measuring sleep in flies carries the risk of overlooking different functions accomplished by distinct kinds of sleep. Sleep physiology and functions are increasingly being addressed in the fly model by imposing experimentally controlled sleep regimes, either pharmacologically or via transient control of sleep-promoting circuits by using opto- or thermogenetic tools (Shafer and Keene, 2021). Yet, there is little knowledge available on whether these different approaches are producing qualitatively similar sleep. For example, sleep can be induced genetically in flies by activating sleep-promoting neurons in the central complex (CX) – a part of the insect brain that has been found to be involved in multimodal sensory processing (Wolff and Rubin, 2018). In particular, the dorsal fan-shaped body (dFB) of the CX has been proposed to serve as a discharge circuit for the insect’s sleep homeostat, whereby increased sleep pressure (e.g., due to extended wakefulness) alters the physiological properties of dFB neurons, causing them to fire more readily and thereby promote decreased behavioral responsiveness (Troup et al., 2018) and thus sleep (Donlea et al., 2011; Donlea et al., 2014; Donlea et al., 2018). Crucially, activation of sleep-promoting circuits including the dFB has been shown to be sleep-restorative (Tainton-Heap et al., 2021; Dissel et al., 2015), but confusingly, brain recordings during optogenetically induced sleep, via electrophysiology, as well as whole-brain calcium imaging techniques, reveal wake-like levels of brain activity (Yap et al., 2017; Tainton-Heap et al., 2021). This suggests that some approaches to optogenetically induced sleep might be promoting a form of sleep akin to the ‘active’ sleep stage detected during spontaneous sleep (Yap et al., 2017; Tainton-Heap et al., 2021).

An alternate way to induce sleep in Drosophila is by feeding flies drugs that have been designed to treat insomnia in humans, such as the GABA-agonist 4,5,6,7-tetrahyrdoisoxazolopyridin-3-ol (THIP), also known as gaboxadol (Harrison, 2007). Several studies have shown that THIP-induced sleep in flies is also restorative and achieves key functions ranging from memory consolidation to cellular repair and waste clearance (Dissel et al., 2015; Berry et al., 2015; Stanhope et al., 2020; van Alphen et al., 2021). This pharmacological approach centered on GABA function has a solid foundation based on better-understood sleep processes: in mammals, many sleep-inducing drugs also target GABA receptors, and this class of drugs tends to promote SWS (Lundahl et al., 2012). In contrast, there are no obvious drugs that promote REM sleep, although local infusion of cholinergic agonists (e.g., carbachol) to the brainstem has been shown to induce REM-like states in cats (Mitler and Dement, 1974).

In this study, we compare THIP-induced sleep with optogenetically induced sleep in Drosophila using behavior, brain activity, and transcriptomics. To ensure the validity of our comparisons, we performed all of our experiments in the same genetic background, employing a canonical Gal4 strain that expresses a transgenic cation channel in a sleep-promoting circuit: R23E10-Gal4>UAS-Chrimson (Jenett et al., 2012; Klapoetke et al., 2014). When these flies are fed all-trans-retinal (ATR) and then exposed to red light, they are put to sleep optogenetically. When these flies are instead fed THIP, they are put to sleep pharmacologically. By using the same genetic background, we were thus able to contrast the effects of either kind of sleep at the level of behavior, brain activity, and gene expression (Figure 1). We questioned how similar either form of induced sleep was.

Figure 1

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One of the key advantages of studying sleep in Drosophila is that this versatile model provides a variety of reliable approaches for inducing and controlling sleep. By being able to induce sleep on demand, either genetically or pharmacologically, researchers have been able to manipulate sleep as an experimental variable, and in this way be better able to assess causality when probing potential sleep functions. However, this approach has often sidestepped the question of whether different sleep induction methods are equivalent or whether distinct forms of sleep might be engaged by different genetic or pharmacological treatments. In mammals, GABA agonists typically promote SWS, which has been associated with cellular homeostasis and repair process in the brain (Tononi and Cirelli, 2014; Xie et al., 2013). In contrast, drugs targeting acetylcholine receptors, such as carbachol, have been found to promote brain states more reminiscent of REM sleep (Kubin, 2001; Torterolo et al., 2016). Although these drugs all induce sedative (or dissociative) states, they are clearly producing dissimilar forms of sleep in mammals, with likely different functions or consequences for the brain. In Drosophila, evidence suggests that the GABA agonist THIP promotes a form of deep or ‘quiet’ sleep, which may be functionally analogous to mammalian SWS (Yap et al., 2017; van Alphen et al., 2021; Kirszenblat and van Swinderen, 2015). In contrast, optogenetic activation of the dFB as well as other neurons may promote a form of active sleep, which we have suggested could be achieving some similar sleep functions as REM (Van De Poll and van Swinderen, 2021). THIP-induced sleep in flies has been associated with waste clearance from the brain (van Alphen et al., 2021), just as SWS has been associated with clearance of waste metabolites via the mammalian brain’s glymphatic system (Xie et al., 2013). Such functional homology suggests that the transcriptional changes we uncovered for THIP-induced sleep in Drosophila might also be relevant for mammalian SWS, with these largely centered on reduced metabolic processes and stress regulation (Muheim et al., 2019). In contrast, except for the upregulation of cholinergic signaling (Vazquez and Baghdoyan, 2001), there is little to compare to test hypotheses potentially linking active sleep in flies with REM sleep in mammals, except for the potential upregulation of cholinergic signaling. Even this cholinergic connection seems odd, seeing that the predominant excitatory neurotransmitters are reversed in the brains of insects and mammals: glutamate in mammals and acetylcholine in insects (Smarandache-Wellmann, 2016). Additionally, only nicotinic receptor subunits were identified in our analyses, whereas muscarinic receptors have been more commonly associated with REM sleep in mammals (Bourgin et al., 1995; Kashiwagi and Hayashi, 2018). Nevertheless, it is clear from our results that optogenetic-induced active sleep upregulates the expression of multiple nicotinic acetylcholine subunits, and that knocking these out individually has profound (and opposing) effects on sleep architecture in flies, and that a subset might function intrinsically within sleep-promoting neurons. This supports other studies showing a role for nAchRα’s in sleep regulation (Shi et al., 2014; Dai et al., 2021). It will be especially interesting in future brain imaging studies to see whether a knockout such as nAchRα3 is eliminating one kind of sleep (e.g., active sleep) as predicted by our behavioral data, and whether this is associated with any functional consequences. Similarly, it will be telling to see whether the opposite sleep phenotypes observed in nAchRα1, for example, result in a distinct class of functional consequences. A previous study has shown that nAchRα1 knockout animals have significantly shorter survivorship compared to controls, with flies dying almost 20 d earlier (Somers et al., 2017). One reason could be because of impaired or insufficient deep sleep functions (e.g., brain waste clearance; van Alphen et al., 2021). The nAchRα knockouts provide an opportunity to further examine mutant animals potentially lacking either kind of sleep, although this will have to be confirmed by brain imaging or electrophysiology.

We found little similarity between two different approaches to inducing sleep in flies at the level of gene expression as well as acute effects on brain activity. It may, however, not be surprising that these entirely different sleep induction methods produce dissimilar physiological effects. After all, one method requires flies to ingest a drug, which then must make its way to the brain, while the other method directly activates a subset of neurons in the central brain, as well as in the ventral nerve cord (Jones et al., 2023). Yet both methods yield similarly increased sleep duration profiles and consolidated sleep architecture (Figure 2). One underlying assumption with focusing on sleep duration as the most relevant metric for understanding sleep function in Drosophila is that sleep is a unitary phenomenon in the fly model, meaning that primarily one set of functions and one form of brain activity are occurring when flies sleep. There is now substantial evidence that this is unlikely to be true, and that like other animals flies probably also experience distinct sleep stages that accomplish different functions (Yap et al., 2017; Tainton-Heap et al., 2021; van Alphen et al., 2021; van Alphen et al., 2013; Wiggin et al., 2020; Gong et al., 2022). This does not mean that these functions are mutually exclusive; for example, both THIP provision and neuronal activation of central brain circuits have been found to promote memory consolidation in Drosophila (Dissel et al., 2015). Indeed, it seems reasonable to propose that different sleep stages could be synergistic, accomplishing a variety of homeostatic functions that might be required for adaptive behaviors in an animal. Our results suggest that THIP provision promotes a ‘quiet sleep’ stage in flies, which induces a brain-wide downregulation of metabolism-related genes. This is consistent with studies in flies showing that metabolic rate is decreased in longer sleep bouts, especially at night, and that this is recapitulated by THIP-induced sleep (Stahl et al., 2017). Our findings also align with a previous study showing that mitochondrial metabolic processes are upregulated in the absence of sleep (Kempf et al., 2019). One argument for why metabolism-related genes are downregulated during THIP-induced sleep might be that flies are starved (because they are sleeping more, and thus perhaps not eating). However, flies induced to sleep by optogenetic activation are also sleeping more (indeed, their average sleep bouts are longer), yet these did not reveal a similar downregulation of metabolic processes. Another view might be that our sampling was done after flies had achieved 10 hr of induced sleep, so sleep functions might have already been achieved by that time. Thus, we might not be uncovering genes required for achieving ‘quiet’ sleep functions as much as identifying exactly the opposite: genetic pathways that have been satisfied by 10 hr of induced quiet sleep. Other studies using THIP to induce sleep have examined longer time frames (e.g., 2 d; Dissel et al., 2015), so it remains unclear whether changes in gene regulation relate to sleep functions that have been achieved or that are still being engaged. For our transcriptomic study, both sleep induction protocols were conducted during the day (thus in presumably well-rested flies) to deliberately avoid any sleep pressure confounds. Since the dFB has been proposed as a regulator of sleep homeostasis (Donlea et al., 2011; Donlea et al., 2014; Donlea et al., 2018), one could question why its prolonged activation did not incur a deep sleep debt, which might have been detectable at a transcriptomic level by some overlap with our SD controls. That we did not see this suggests induced daytime active sleep might not accrue any sleep debt. We have shown previously that daytime sleep is qualitatively different than night-time sleep in flies, which is ‘deeper’ (van Alphen et al., 2013). It is, for example, possible that most quiet sleep happens at night in flies, so a daytime regime of induced active or quiet sleep might not incur any sleep debt for either. Following this logic, our RNA analyses simply revealed the transcriptomic changes resulting from either manipulation in well-rested flies, with the key result being that there are zero shared genes after 10 daytime hours of either manipulation.

In contrast to THIP, our optogenetic manipulation induced a brain-wide upregulation of a variety of neural mechanisms unrelated to metabolic processes, suggesting a different kind of sleep was engaged. Although many studies have shown that the R23E10-Gal4 circuit is sleep promoting (e.g., Troup et al., 2018; Donlea et al., 2014; Gong et al., 2022), and we have shown that acute activation of these neurons produces a form of active sleep (Tainton-Heap et al., 2021), it seems unlikely that active sleep regulation is limited to the dFB neurons alone (Jones et al., 2023). Other circuits in the fly central brain are also sleep-promoting, including in the ellipsoid body (Liu et al., 2016) and the ventral fan-shaped body (vFB) (Lei et al., 2022), although it remains unknown whether activation of these other circuits also promotes an active sleep stage or whether a similar transcriptome might be engaged by these alternate approaches to optogenetic sleep induction in flies. This again highlights a variant of the same problem we have uncovered in this study comparing pharmacology with optogenetics: different circuit-based approaches could all be increasing sleep duration but achieving entirely different functions by engaging distinct transcriptomes and thus different sleep functions. How many different kinds of functions are engaged by sleep remains unclear: is it roughly two functional categories linked to quiet and active sleep, or is it a broader range of subcategories that are not so tightly linked to these obviously different brain activity states?

A compelling argument could nevertheless be made for two kinds of sleep in most animals, with two distinct sets of functions (Jaggard et al., 2021; Kirszenblat and van Swinderen, 2015). Most animals have been shown to require a form of ‘quiet’ sleep to ensure survival, suggesting that these might encompass an evolutionarily conserved set of cellular processes that promote neural health and development (Zimmerman et al., 2008a), and that operate best during periods of behavioral quiescence. Nematode worms thus experience a form of quiet sleep when they pause to molt (‘lethargus’) into different life stages during their development (Raizen et al., 2008) or when cellular repair processes are needed following environmental stress (Hill et al., 2014). In flies, quiet sleep seems to be similarly required for neuronal repair (Stanhope et al., 2020) or waste clearance (van Alphen et al., 2021), and there is evidence that glia might play a key role in these cellular homeostatic processes in flies (Stanhope et al., 2020), as well as other animals (Artiushin and Sehgal, 2020). Thus, SWS in mammals and birds might present a narrow neocortical view of a more ancient set of sleep functions centered on quiescence and decreased metabolic rate. Indeed, neural quiescence is also a feature of SWS, both at the level of pulsed inhibition (down-states) and in other parts of the brain beyond the cortex (Jaggard et al., 2021). Similar to findings in flies that are induced into a quiet sleep stage with THIP (Stahl et al., 2017), metabolic rate also decreases during SWS in mammals (Caron and Stephenson, 2010). In contrast, metabolic rate is similar to waking during REM sleep in mammals (Maquet, 1995), suggesting an alternate set of functions not linked to cellular homeostasis. What might these functions be, and could some of these be conserved between active sleep in invertebrates and REM sleep in mammals? A REM-like sleep stage has now been identified in a variety of invertebrate species, including cephalopods (Iglesias et al., 2019) and jumping spiders (Rößler et al., 2022). In humans, REM sleep has been implicated in emotion regulation (Hutchison and Rathore, 2015), and cognitive disorders where emotions are dysregulated, such as depression, are often associated with REM sleep dysfunction (Harrington et al., 2018). While it is not evident how to study emotions in insects (but see Anderson and Adolphs, 2014), it could be argued that arousal systems more generally are employed to detect prediction errors and thereby promote learning (Seth and Friston, 2016). Thus, we and others have suggested that REM sleep might be crucial for optimizing prediction, and attention, and learning (Van De Poll and van Swinderen, 2021; Kirszenblat and van Swinderen, 2015; Hobson, 2009), and this may involve different kinds of homeostatic mechanisms centered on brain circuits rather than cells. Whether active sleep in flies plays a similar homeostatic role remains to be determined. Our finding that optogenetically induced sleep in Drosophila upregulates different nAchRα subunits is consistent with new findings that these subunits regulate appetitive memories in flies (Pribbenow et al., 2022) and that cholinergic systems more generally underpin learning and memory in this animal (Barnstedt et al., 2016). Yet learning and memory in flies clearly also benefit from quiet sleep, as evidenced by multiple studies using THIP as a sleep-promoting agent (Dissel et al., 2015; Berry et al., 2015; Melnattur et al., 2021). One view consistent with our findings and previous studies is that both kinds of sleep are crucial for optimal behavior: quiet sleep for cellular homeostasis and active sleep for circuit-level homeostasis. Manipulating these separately, alongside the non-overlapping pathways engaged by either kind of sleep, should help further disambiguate the functions potentially associated with these distinct sleep stages.

All data generated or analysed during this study are included in the manuscript and supporting files. The data and analysis tools underpinning this study (brain imaging as well as RNA sequencing) are available online via Dryad: https://doi.org/10.5061/dryad.1rn8pk10x.

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Author details

1. Niki Anthoney

   Queensland Brain Institute, The University of Queensland, Brisbane, Australia

   Contribution

   Data curation, Investigation, Visualization, Methodology, Writing – review and editing

   Competing interests

   No competing interests declared

2. Lucy Tainton-Heap

   Queensland Brain Institute, The University of Queensland, Brisbane, Australia

   Contribution

   Software, Formal analysis, Validation, Investigation, Visualization, Methodology

   Competing interests

   No competing interests declared

3. Hang Luong

   School of BioSciences, The University of Melbourne, Melbourne, Australia

   Contribution

   Formal analysis, Investigation, Methodology

   Competing interests

   No competing interests declared

4. Eleni Notaras

   Queensland Brain Institute, The University of Queensland, Brisbane, Australia

   Contribution

   Formal analysis, Investigation, Methodology

   Competing interests

   No competing interests declared

5. Amber B Kewin

   Queensland Brain Institute, The University of Queensland, Brisbane, Australia

   Contribution

   Investigation, Methodology

   Competing interests

   No competing interests declared

6. Qiongyi Zhao

   Queensland Brain Institute, The University of Queensland, Brisbane, Australia

   Contribution

   Data curation, Software, Formal analysis, Investigation, Methodology

   Competing interests

   No competing interests declared

7. Trent Perry

   School of BioSciences, The University of Melbourne, Melbourne, Australia

   Contribution

   Resources, Supervision, Methodology

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-8045-0487

8. Philip Batterham

   School of BioSciences, The University of Melbourne, Melbourne, Australia

   Contribution

   Resources, Supervision, Methodology

   Competing interests

   No competing interests declared

9. Paul J Shaw

   Department of Neuroscience, School of Medicine, Washington University in St. Louis, St Louis, United States

   Contribution

   Conceptualization, Resources, Funding acquisition, Investigation, Methodology, Project administration, Writing – review and editing

   Competing interests

   No competing interests declared

10. Bruno van Swinderen

    Queensland Brain Institute, The University of Queensland, Brisbane, Australia

    Contribution

    Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Investigation, Visualization, Methodology, Writing – original draft, Project administration, Writing – review and editing

    For correspondence

    b.vanswinderen@uq.edu.au

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0001-6552-7418

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