Whole-Brain Calcium Imaging in Drosophila during Sleep and Wake.

Genetically encoded calcium indicators (GECIs) allow for the noninvasive evaluation of neuronal activity in vivo, and imaging GECIs in Drosophila has become commonplace for understanding neural functions and connectivity in this system. GECIs can also be used as read-outs for studying sleep in this model organism. Here, we describe a methodology for tracking the activity of neurons in the fly brain using a two-photon (2p) microscopy system. This method can be adapted to perform functional studies of neural activity in Drosophila under both spontaneous and evoked conditions, as well as during spontaneous or induced sleep. We first describe a tethering and surgical procedure that allows survival under the microscopy conditions required for long-term recordings. We then outline the steps and reagents required for optogenetic activation of sleep-promoting neurons while simultaneously recording neural activity from the fly brain. We also describe the procedure for recording from two different locations-namely, the top of the head (e.g., to record mushroom body calyx activity) or the back of the head (e.g., to record central complex activity). We also provide different strategies for recording from GECIs confined to the cell body versus the entire neuron. Finally, we describe the steps required for analyzing the multidimensional data that can be acquired. In all, this protocol shows how to perform calcium imaging experiments in tethered flies, with a focus on acquiring spontaneous and induced sleep data.

Whole-Brain Electrophysiology and Calcium Imaging in Drosophila during Sleep and Wake.

Sleep is likely a whole-brain phenomenon, with most of the brain probably benefiting from this state of decreased arousal. Recent advances in our understanding of some potential sleep functions, such as metabolite clearance and synaptic homeostasis, make it evident why the whole brain is likely impacted by sleep: All neurons have synapses, and all neurons produce waste metabolites. Sleep experiments in the fly Drosophila melanogaster suggest that diverse sleep functions appear to be conserved across all animals. Studies of brain activity during sleep in humans typically involve multidimensional data sets, such as those acquired by electroencephalograms (EEGs) or functional magnetic resonance imaging (fMRI), and these whole-brain read-outs often reveal important qualities of different sleep stages, such as changes in frequency dynamics or connectivity. Recently, various techniques have been developed that allow for the recording of neural activity simultaneously across multiple regions of the fly brain. These whole-brain-recording approaches will be important for better understanding sleep physiology and function, as they provide a more comprehensive view of neural dynamics during sleep and wake in a relevant model system. Here, we present a brief summary of some of the findings derived from sleep activity recording studies in sleeping Drosophila flies and discuss the value of electrophysiological versus calcium imaging techniques. Although these involve very different preparations, they both highlight the value of multidimensional data for studying sleep in this model system, like the use of both EEG and fMRI in humans.

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

Sleep in mammals can be broadly classified into two different physiological categories: rapid eye movement (REM) sleep and slow-wave sleep (SWS), and accordingly REM and SWS are thought to achieve a different set of functions. The fruit fly Drosophila melanogaster is increasingly being used as a model to understand sleep functions, although it remains unclear if the fly brain also engages in different kinds of sleep as well. Here, we compare two commonly used approaches for studying sleep experimentally in Drosophila: optogenetic activation of sleep-promoting neurons and provision of a sleep-promoting drug, gaboxadol. We find that these different sleep-induction methods have similar effects on increasing sleep duration, but divergent effects on brain activity. Transcriptomic analysis reveals that drug-induced deep sleep ('quiet' sleep) mostly downregulates metabolism genes, whereas optogenetic 'active' sleep upregulates a wide range of genes relevant to normal waking functions. This suggests that optogenetics and pharmacological induction of sleep in Drosophila promote different features of sleep, which engage different sets of genes to achieve their respective functions.

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

Sleep in mammals can be broadly classified into two different physiological categories: rapid eye movement (REM) sleep and slow wave sleep (SWS), and accordingly REM and SWS are thought to achieve a different set of functions. The fruit fly Drosophila melanogaster is increasingly being used as a model to understand sleep functions, although it remains unclear if the fly brain also engages in different kinds of sleep as well. Here, we compare two commonly used approaches for studying sleep experimentally in Drosophila: optogenetic activation of sleep-promoting neurons and provision of a sleep-promoting drug, Gaboxadol. We find that these different sleep-induction methods have similar effects on increasing sleep duration, but divergent effects on brain activity. Transcriptomic analysis reveals that drug-induced deep sleep ('quiet' sleep) mostly downregulates metabolism genes, whereas optogenetic 'active' sleep upregulates a wide range of genes relevant to normal waking functions. This suggests that optogenetics and pharmacological induction of sleep in Drosophila promote different features of sleep, which engage different sets of genes to achieve their respective functions.

Neural Ensemble Fragmentation in the Anesthetized Drosophila Brain.

General anesthetics cause a profound loss of behavioral responsiveness in all animals. In mammals, general anesthesia is induced in part by the potentiation of endogenous sleep-promoting circuits, although "deep" anesthesia is understood to be more similar to coma (Brown et al., 2011). Surgically relevant concentrations of anesthetics, such as isoflurane and propofol, have been shown to impair neural connectivity across the mammalian brain (Mashour and Hudetz, 2017; Yang et al., 2021), which presents one explanation why animals become largely unresponsive when exposed to these drugs. It remains unclear whether general anesthetics affect brain dynamics similarly in all animal brains, or whether simpler animals, such as insects, even display levels of neural connectivity that could be disrupted by these drugs. Here, we used whole-brain calcium imaging in behaving female Drosophila flies to investigate whether isoflurane anesthesia induction activates sleep-promoting neurons, and then inquired how all other neurons across the fly brain behave under sustained anesthesia. We were able to track the activity of hundreds of neurons simultaneously during waking and anesthetized states, for spontaneous conditions as well as in response to visual and mechanical stimuli. We compared whole-brain dynamics and connectivity under isoflurane exposure to optogenetically induced sleep. Neurons in the Drosophila brain remain active during general anesthesia as well as induced sleep, although flies become behaviorally inert under both treatments. We identified surprisingly dynamic neural correlation patterns in the waking fly brain, suggesting ensemble-like behavior. These become more fragmented and less diverse under anesthesia but remain wake-like during induced sleep.SIGNIFICANCE STATEMENT When humans are rendered immobile and unresponsive by sleep or general anesthetics, their brains do not shut off - they just change how they operate. We tracked the activity of hundreds of neurons simultaneously in the brains of fruit flies that were anesthetized by isoflurane or genetically put to sleep, to investigate whether these behaviorally inert states shared similar brain dynamics. We uncovered dynamic patterns of neural activity in the waking fly brain, with stimulus-responsive neurons constantly changing through time. Wake-like neural dynamics persisted during induced sleep but became more fragmented under isoflurane anesthesia. This suggests that, like larger brains, the fly brain might also display ensemble-like behavior, which becomes degraded rather than silenced under general anesthesia.

A Paradoxical Kind of Sleep in Drosophila melanogaster.

The dynamic nature of sleep in many animals suggests distinct stages that serve different functions. Genetic sleep induction methods in animal models provide a powerful way to disambiguate these stages and functions, although behavioral methods alone are insufficient to accurately identify what kind of sleep is being engaged. In Drosophila, activation of the dorsal fan-shaped body (dFB) promotes sleep, but it remains unclear what kind of sleep this is, how the rest of the fly brain is behaving, or if any specific sleep functions are being achieved. Here, we developed a method to record calcium activity from thousands of neurons across a volume of the fly brain during spontaneous sleep and compared this to dFB-induced sleep. We found that spontaneous sleep typically transitions from an active "wake-like" stage to a less active stage. In contrast, optogenetic activation of the dFB promotes sustained wake-like levels of neural activity even though flies become unresponsive to mechanical stimuli. When we probed flies with salient visual stimuli, we found that the activity of visually responsive neurons in the central brain was blocked by transient dFB activation, confirming an acute disconnect from the external environment. Prolonged optogenetic dFB activation nevertheless achieved a key sleep function by correcting visual attention defects brought on by sleep deprivation. These results suggest that dFB activation promotes a distinct form of sleep in Drosophila, where brain activity appears similar to wakefulness, but responsiveness to external sensory stimuli is profoundly suppressed.