Synapse-specific trapping of SNARE machinery proteins in the anesthetized Drosophila brain.

General anesthetics disrupt brain network dynamics through multiple pathways, in part through post-synaptic potentiation of inhibitory ion channels as well as pre-synaptic inhibition of neuroexocytosis. Common clinical general anesthetic drugs, such as propofol and isoflurane, have been shown to interact and interfere with core components of the exocytic release machinery to cause impaired neurotransmitter release. Recent studies however suggest that these drugs do not affect all synapse subtypes equally. We investigated the role of the presynaptic release machinery in multiple neurotransmitter systems under isoflurane general anesthesia in the adult female Drosophila brain using live-cell super resolution microscopy and optogenetic readouts of exocytosis and neural excitability. We activated neurotransmitter-specific mushroom body output neurons (MBONs) and imaged presynaptic function under isoflurane anesthesia. We found that isoflurane impaired synaptic release and presynaptic protein dynamics in excitatory cholinergic synapses. In contrast, isoflurane had little to no effect on inhibitory GABAergic or glutamatergic synapses. These results present a distinct inhibitory mechanism for general anesthesia, whereby neuroexocytosis is selectively impaired at excitatory synapses, while inhibitory synapses remain functional. This suggests a presynaptic inhibitory mechanism that complements the other inhibitory effects of these drugs.Significance Statement General anesthetics are promiscuous drugs that act on a variety of pre-synaptic and post-synaptic proteins. Yet they produce a common endpoint - loss of behavioral responsiveness - in all animals. Using optogenetic techniques to measure functional readouts in identified neurons in the Drosophila brain, we have found that the volatile anesthetic isoflurane impairs neurotransmitter release from excitatory synapses, and that this is associated with immobilization of release machinery proteins. Inhibitory synapses were unaffected. This suggests a level of presynaptic specificity to the anesthetic's mechanism of action which complements the other known effects on synaptic function, and potentially explains how some of these drugs might work to produce the common endpoint termed general anesthesia.

Multivariate classification of multichannel long-term electrophysiology data identifies different sleep stages in fruit flies.

Identifying different sleep stages in humans and other mammals has traditionally relied on electroencephalograms. Such an approach is not feasible in certain animals such as invertebrates, although these animals could also be sleeping in stages. Here, we perform long-term multichannel local field potential recordings in the brains of behaving flies undergoing spontaneous sleep bouts. We acquired consistent spatial recordings of local field potentials across multiple flies, allowing us to compare brain activity across awake and sleep periods. Using machine learning, we uncover distinct temporal stages of sleep and explore the associated spatial and spectral features across the fly brain. Further, we analyze the electrophysiological correlates of microbehaviors associated with certain sleep stages. We confirm the existence of a distinct sleep stage associated with rhythmic proboscis extensions and show that spectral features of this sleep-related behavior differ significantly from those associated with the same behavior during wakefulness, indicating a dissociation between behavior and the brain states wherein these behaviors reside.

Time to Wake Up! The Ongoing Search for General Anesthetic Reversal Agents.

How general anesthetics work remains a topic of ongoing study. A parallel field of research has sought to identify methods to reverse general anesthesia. Reversal agents could shorten patients' recovery time and potentially reduce the risk of postoperative complications. An incomplete understanding of the mechanisms of general anesthesia has hampered the pursuit for reversal agents. Nevertheless, the search for reversal agents has furthered understanding of the mechanisms underlying general anesthesia. The study of potential reversal agents has highlighted the importance of rigorous criteria to assess recovery from general anesthesia in animal models, and has helped identify key arousal systems (e.g., cholinergic, dopaminergic, and orexinergic systems) relevant to emergence from general anesthesia. Furthermore, the effects of reversal agents have been found to be inconsistent across different general anesthetics, revealing differences in mechanisms among these drugs. The presynapse and glia probably also contribute to general anesthesia recovery alongside postsynaptic receptors. The next stage in the search for reversal agents will have to consider alternate mechanisms encompassing the tripartite synapse.

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 in Drosophila during Sleep and Wake.

Sleep studies in Drosophila melanogaster rely mostly on behavioral read-outs to support molecular or circuit-level investigations in this model. Electrophysiology can provide an additional level of understanding in these studies to, for example, investigate changes in brain activity associated with sleep manipulations. In this protocol, we describe a procedure for performing multichannel local field potential (LFP) recordings in the fruit fly, with a flexible system that can be adapted to different experimental paradigms and situations. The approach uses electrodes containing multiple recording sites (16), allowing the acquisition of large amounts of neuronal activity data from a transect through the brain while flies are still able to sleep. The approach starts by tethering the fly, followed by positioning it on an air-supported ball. A multichannel silicon probe is then inserted laterally into the fly brain via one eye, allowing for recording of electrical signals from the retina through to the central brain. These recordings can be acquired under spontaneous conditions or in the presence of visual stimuli, and the minimal surgery promotes long-term recordings (e.g., overnight). Sleep and wake can be tracked using infrared cameras, which allow for the measurement of locomotive activity as well as microbehaviors such as proboscis extensions during sleep. The protocol has been optimized to promote subject survivability, which is an important factor when performing long-term (∼16-h) recordings. The approach described here uses specific recording probes, data acquisition devices, and analysis tools. Although it is expected that some of these items might need to be adapted to the equipment available in different laboratories, the overall aim is to provide an overview on how to record electrical activity across the brain of behaving (and sleeping) flies using this kind of approach and technology.

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.

Multivariate classification of multichannel long-term electrophysiology data identifies different sleep stages in fruit flies.

Sleep is observed in most animals, which suggests it subserves a fundamental process associated with adaptive biological functions. However, the evidence to directly associate sleep with a specific function is lacking, in part because sleep is not a single process in many animals. In humans and other mammals, different sleep stages have traditionally been identified using electroencephalograms (EEGs), but such an approach is not feasible in different animals such as insects. Here, we perform long-term multichannel local field potential (LFP) recordings in the brains of behaving flies undergoing spontaneous sleep bouts. We developed protocols to allow for consistent spatial recordings of LFPs across multiple flies, allowing us to compare the LFP activity across awake and sleep periods and further compare the same to induced sleep. Using machine learning, we uncover the existence of distinct temporal stages of sleep and explore the associated spatial and spectral features across the fly brain. Further, we analyze the electrophysiological correlates of micro-behaviours associated with certain sleep stages. We confirm the existence of a distinct sleep stage associated with rhythmic proboscis extensions and show that spectral features of this sleep-related behavior differ significantly from those associated with the same behavior during wakefulness, indicating a dissociation between behavior and the brain states wherein these behaviors reside.

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.

Sleep-promoting neurons remodel their response properties to calibrate sleep drive with environmental demands.

Falling asleep at the wrong time can place an individual at risk of immediate physical harm. However, not sleeping degrades cognition and adaptive behavior. To understand how animals match sleep need with environmental demands, we used live-brain imaging to examine the physiological response properties of the dorsal fan-shaped body (dFB) following interventions that modify sleep (sleep deprivation, starvation, time-restricted feeding, memory consolidation) in Drosophila. We report that dFB neurons change their physiological response-properties to dopamine (DA) and allatostatin-A (AstA) in response to different types of waking. That is, dFB neurons are not simply passive components of a hard-wired circuit. Rather, the dFB neurons intrinsically regulate their response to the activity from upstream circuits. Finally, we show that the dFB appears to contain a memory trace of prior exposure to metabolic challenges induced by starvation or time-restricted feeding. Together, these data highlight that the sleep homeostat is plastic and suggests an underlying mechanism.

Anesthesia: Synaptic power failure.

One of the greatest unresolved mysteries in medicine relates to the molecular and neuronal mechanisms through which general anesthetics abolish perception. A new study in mice with mutations affecting mitochondrial complex 1 suggests that anesthetic-disruption of cellular energetics impairs endocytosis to alter synaptic function.

Balancing Prediction and Surprise: A Role for Active Sleep at the Dawn of Consciousness?

The brain is a prediction machine. Yet the world is never entirely predictable, for any animal. Unexpected events are surprising, and this typically evokes prediction error signatures in mammalian brains. In humans such mismatched expectations are often associated with an emotional response as well, and emotional dysregulation can lead to cognitive disorders such as depression or schizophrenia. Emotional responses are understood to be important for memory consolidation, suggesting that positive or negative 'valence' cues more generally constitute an ancient mechanism designed to potently refine and generalize internal models of the world and thereby minimize prediction errors. On the other hand, abolishing error detection and surprise entirely (as could happen by generalization or habituation) is probably maladaptive, as this might undermine the very mechanism that brains use to become better prediction machines. This paradoxical view of brain function as an ongoing balance between prediction and surprise suggests a compelling approach to study and understand the evolution of consciousness in animals. In particular, this view may provide insight into the function and evolution of 'active' sleep. Here, we propose that active sleep - when animals are behaviorally asleep but their brain seems awake - is widespread beyond mammals and birds, and may have evolved as a mechanism for optimizing predictive processing in motile creatures confronted with constantly changing environments. To explore our hypothesis, we progress from humans to invertebrates, investigating how a potential role for rapid eye movement (REM) sleep in emotional regulation in humans could be re-examined as a conserved sleep function that co-evolved alongside selective attention to maintain an adaptive balance between prediction and surprise. This view of active sleep has some interesting implications for the evolution of subjective awareness and consciousness in animals.

Tracking Single Molecule Dynamics in the Adult Drosophila Brain.

Super-resolution microscopy provides valuable insight for understanding the nanoscale organization within living tissue, although this method is typically restricted to cultured or dissociated cells. Here, we develop a method to track the mobility of individual proteins in ex vivo adult Drosophila melanogaster brains, focusing on a key component of the presynaptic release machinery, syntaxin1A (Sx1a). We show that individual Sx1a dynamics can be reliably tracked within neurons in the whole fly brain, and that the mobility of Sx1a molecules increases following conditional neural stimulation. We then apply this preparation to the problem of general anesthesia, to address how different anesthetics might affect single molecule dynamics in intact brain synapses. We find that propofol, etomidate, and isoflurane significantly impair Sx1a mobility, while ketamine and sevoflurane have little effect. Resolving single molecule dynamics in intact fly brains provides a novel approach to link localized molecular effects with systems-level phenomena such as general anesthesia.

Integrated information structure collapses with anesthetic loss of conscious arousal in Drosophila melanogaster.

The physical basis of consciousness remains one of the most elusive concepts in current science. One influential conjecture is that consciousness is to do with some form of causality, measurable through information. The integrated information theory of consciousness (IIT) proposes that conscious experience, filled with rich and specific content, corresponds directly to a hierarchically organised, irreducible pattern of causal interactions; i.e. an integrated informational structure among elements of a system. Here, we tested this conjecture in a simple biological system (fruit flies), estimating the information structure of the system during wakefulness and general anesthesia. Consistent with this conjecture, we found that integrated interactions among populations of neurons during wakefulness collapsed to isolated clusters of interactions during anesthesia. We used classification analysis to quantify the accuracy of discrimination between wakeful and anesthetised states, and found that informational structures inferred conscious states with greater accuracy than a scalar summary of the structure, a measure which is generally championed as the main measure of IIT. In stark contrast to a view which assumes feedforward architecture for insect brains, especially fly visual systems, we found rich information structures, which cannot arise from purely feedforward systems, occurred across the fly brain. Further, these information structures collapsed uniformly across the brain during anesthesia. Our results speak to the potential utility of the novel concept of an "informational structure" as a measure for level of consciousness, above and beyond simple scalar values.

A deep sleep stage in Drosophila with a functional role in waste clearance.

Sleep is a highly conserved state, suggesting that sleep's benefits outweigh the increased vulnerability it brings. Yet, little is known about how sleep fulfills its functions. Here, we used video tracking in tethered flies to identify a discrete deep sleep stage in Drosophila, termed proboscis extension sleep, that is defined by repeated stereotyped proboscis extensions and retractions. Proboscis extension sleep is accompanied by highly elevated arousal thresholds and decreased brain activity, indicative of a deep sleep state. Preventing proboscis extensions increases injury-related mortality and reduces waste clearance. Sleep deprivation reduces waste clearance and during subsequent rebound sleep, sleep, proboscis extensions, and waste clearance are increased. Together, these results provide evidence of a discrete deep sleep stage that is linked to a specific function and suggest that waste clearance is a core and ancient function of deep sleep.

A conserved role for sleep in supporting Spatial Learning in Drosophila.

Sleep loss and aging impair hippocampus-dependent Spatial Learning in mammalian systems. Here we use the fly Drosophila melanogaster to investigate the relationship between sleep and Spatial Learning in healthy and impaired flies. The Spatial Learning assay is modeled after the Morris Water Maze. The assay uses a "thermal maze" consisting of a 5 × 5 grid of Peltier plates maintained at 36-37°C and a visual panorama. The first trial begins when a single tile that is associated with a specific visual cue is cooled to 25°C. For subsequent trials, the cold tile is heated, the visual panorama is rotated and the flies must find the new cold tile by remembering its association with the visual cue. Significant learning was observed with two different wild-type strains-Cs and 2U, validating our design. Sleep deprivation prior to training impaired Spatial Learning. Learning was also impaired in the classic learning mutant rutabaga (rut); enhancing sleep restored learning to rut mutants. Further, we found that flies exhibited a dramatic age-dependent cognitive decline in Spatial Learning starting at 20-24 days of age. These impairments could be reversed by enhancing sleep. Finally, we find that Spatial Learning requires dopaminergic signaling and that enhancing dopaminergic signaling in aged flies restored learning. Our results are consistent with the impairments seen in rodents and humans. These results thus demonstrate a critical conserved role for sleep in supporting Spatial Learning, and suggest potential avenues for therapeutic intervention during aging.

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.

Oscillations in the central brain of Drosophila are phase locked to attended visual features.

Object-based attention describes the brain's capacity to prioritize one set of stimuli while ignoring others. Human research suggests that the binding of diverse stimuli into one attended percept requires phase-locked oscillatory activity in the brain. Even insects display oscillatory brain activity during visual attention tasks, but it is unclear if neural oscillations in insects are selectively correlated to different features of attended objects. We addressed this question by recording local field potentials in the Drosophila central complex, a brain structure involved in visual navigation and decision making. We found that attention selectively increased the neural gain of visual features associated with attended objects and that attention could be redirected to unattended objects by activation of a reward circuit. Attention was associated with increased beta (20- to 30-Hz) oscillations that selectively locked onto temporal features of the attended visual objects. Our results suggest a conserved function for the beta frequency range in regulating selective attention to salient visual features.