Publications

Sleep is a behavior that is conserved throughout the animal kingdom. Yet, despite extensive studies in humans and animal models, the exact function or functions of sleep remain(s) unknown. A complicating factor in trying to elucidate the function of sleep is the complexity and multiplicity of neuronal circuits that are involved in sleep regulation. It is conceivable that distinct sleep-regulating circuits are only involved in specific aspects of sleep and may underlie different sleep functions. Thus, it would be beneficial to assess the contribution of individual circuits in sleep’s putative functions. The intricacy of the mammalian brain makes this task extremely difficult. However, the fruit fly Drosophila melanogaster, with its simpler brain organization, available connectomics, and unparalleled genetics, offers the opportunity to interrogate individual sleep-regulating centers. In Drosophila, neurons projecting to the dorsal fan-shaped body (dFB) have been proposed to be key regulators of sleep, particularly sleep homeostasis. We recently demonstrated that the most widely used genetic tool to manipulate dFB neurons, the 23E10-GAL4 driver, expresses in 2 sleep-regulating neurons (VNC-SP neurons) located in the ventral nerve cord (VNC), the fly analog of the vertebrate spinal cord. Since most data supporting a role for the dFB in sleep regulation have been obtained using 23E10-GAL4, it is unclear whether the sleep phenotypes reported in these studies are caused by dFB neurons or VNC-SP cells. A recent publication replicated our finding that 23E10-GAL4 contains sleep-promoting neurons in the VNC. However, it also proposed that the dFB is not involved in sleep regulation at all, but this suggestion was made using genetic tools that are not dFB-specific and a very mild sleep deprivation protocol. In this study, using a newly created dFB-specific genetic driver line, we demonstrate that optogenetic activation of the majority of 23E10-GAL4 dFB neurons promotes sleep and that these neurons are involved in sleep homeostasis. We also show that dFB neurons require stronger stimulation than VNC-SP cells to promote sleep. In addition, we demonstrate that dFB-induced sleep can consolidate short-term memory (STM) into long-term memory (LTM), suggesting that the benefit of sleep on memory is not circuit-specific. Finally, we show that dFB neurons are neurochemically heterogeneous and can be divided in 3 populations. Most dFB neurons express both glutamate and acetylcholine, while a minority of cells expresses only one of these 2 neurotransmitters. Importantly, dFB neurons do not express GABA, as previously suggested. Using neurotransmitter-specific dFB tools, our data also points at cholinergic dFB neurons as particularly potent at regulating sleep and sleep homeostasis.

Sleep is a behavior that is conserved throughout the animal kingdom. Yet, despite extensive studies in humans and animal models, the exact function or functions of sleep remain(s) unknown. A complicating factor in trying to elucidate the function of sleep is the complexity and multiplicity of neuronal circuits that are involved in sleep regulation. It is conceivable that distinct sleep-regulating circuits are only involved in specific aspects of sleep and may underlie different sleep functions. Thus, it would be beneficial to assess the contribution of individual circuits in sleep’s putative functions. The intricacy of the mammalian brain makes this task extremely difficult. However, the fruit fly Drosophila melanogaster, with its simpler brain organization, available connectomics, and unparalleled genetics offers the opportunity to interrogate individual sleep-regulating centers. In Drosophila, neurons projecting to the dorsal Fan-Shaped Body (dFB) have been proposed to be key regulators of sleep, particularly sleep homeostasis. We recently demonstrated that the most widely used genetic tool to manipulate dFB neurons, the 23E10-GAL4 driver, expresses in two sleep-regulating neurons (VNC-SP neurons) located in the Ventral Nerve Cord (VNC), the fly analog of the vertebrate spinal cord. Since most data supporting a role for the dFB in sleep regulation have been obtained using 23E10-GAL4, it is unclear whether the sleep phenotypes reported in these studies are caused by dFB neurons or VNC-SP cells. A recent publication replicated our finding that 23E10-GAL4 contains sleep-promoting neurons in the VNC. However, it also proposed that the dFB is not involved in sleep regulation at all, but this suggestion was made using genetic tools that are not dFB-specific and a very mild sleep deprivation protocol. In this study, using a newly created dFB-specific genetic driver line, we demonstrate that the majority of 23E10-GAL4 dFB neurons can promote sleep when activated and that these neurons are involved in sleep homeostasis. We also show that dFB neurons require stronger stimulation than VNC-SP cells to promote sleep. In addition, we demonstrate that dFB-induced sleep can consolidate Short-Term Memory (STM) into Long-Term Memory (LTM), suggesting that the benefit of sleep on memory is not circuit-specific. Finally, we show that dFB neurons are neurochemically heterogeneous and can be divided in 3 populations. Most dFB neurons express both glutamate and acetylcholine, while a minority of cells express only one of these two neurotransmitters. Importantly, dFB neurons do not express GABA, as previously suggested. Using neurotransmitter-specific dFB tools, our data also points at cholinergic dFB neurons as particularly potent at regulating sleep and sleep homeostasis.Competing Interest StatementThe authors have declared no competing interest.

The relationship between sleep and memory is an active topic of investigation. In this context, we demonstrate that enhancing sleep restores memory to flies with ablated Mushroom Bodies (MB), a key memory center; this is consistent across several memory assays. Mapping the underlying circuitry reveals circadian modulation of a subset of Dopaminergic neurons (DANs) that modulate aversive learning. Using imaging, we show that MB-ablation disrupts, and sleep restores the time of day these neurons are most responsive. Knocking down the receptor for the clock output signal, Pigment-dispersing factor (Pdfr), in this subset of DANs restores memory to MB-ablated flies. Crucially, MB-ablation does not result in memory impairments in the absence of a functioning clock. Our results reveal neuromodulation’s key role in cognitive restoration, where sleep aids memory in damaged brains, but a functioning clock unexpectedly hinders this process.Competing Interest StatementThe authors have declared no competing interest.

Sleep is a complex and plastic behavior regulated by multiple brain regions and influenced by numerous internal and external stimuli. Thus, to fully uncover the function(s) of sleep, cellular resolution of sleep-regulating neurons needs to be achieved. Doing so will help to unequivocally assign a role or function to a given neuron or group of neurons in sleep behavior. In the Drosophila brain, neurons projecting to the dorsal fan-shaped body (dFB) have emerged as a key sleep-regulating area. To dissect the contribution of individual dFB neurons to sleep, we undertook an intersectional Split-GAL4 genetic screen focusing on cells contained within the 23E10-GAL4 driver, the most widely used tool to manipulate dFB neurons. In this study, we demonstrate that 23E10-GAL4 expresses in neurons outside the dFB and in the fly equivalent of the spinal cord, the ventral nerve cord (VNC). Furthermore, we show that 2 VNC cholinergic neurons strongly contribute to the sleep-promoting capacity of the 23E10-GAL4 driver under baseline conditions. However, in contrast to other 23E10-GAL4 neurons, silencing these VNC cells does not block sleep homeostasis. Thus, our data demonstrate that the 23E10-GAL4 driver contains at least 2 different types of sleep-regulating neurons controlling distinct aspects of sleep behavior.

Sleep is a very important behavior observed in almost all animals. Importantly, sleep is subject to both circadian and homeostatic regulation. The circadian rhythm determines the daily alternation of the sleep-wake cycle, while homeostasis mediates the rise and dissipation of sleep pressure during the wake and sleep period. As an important kinase, dbt plays a central role in both circadian rhythms and development. We investigated the sleep patterns of several ethyl methanesulfonate-induced dbt mutants and discuss the possible reasons why different sleep phenotypes were shown in these mutants. In order to reduce DBT in all neurons in which it is expressed, CRISPR-Cas9 was used to produce flies that expressed GAL4 in frame with the dbt gene at its endogenous locus, and knock-down of DBT with this construct produced elevated sleep during the day and reduced sleep at night. Loss of sleep at night is mediated by dbt loss during the sleep/wake cycle in the adult, while the increased sleep during the day is produced by reductions in dbt during development and not by reductions in the adult. Additionally, using targeted RNA interference, we uncovered the contribution of dbt on sleep in different subsets of neurons in which dbt is normally expressed. Reduction of dbt in circadian neurons produced less sleep at night, while lower expression of dbt in noncircadian neurons produced increased sleep during the day. Importantly, independently of the types of neurons where dbt affects sleep, we demonstrate that the PER protein is involved in DBT mediated sleep regulation.

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.

Sleep plays an important role in regulating plasticity. In Drosophila, the relationship between sleep and learning and memory has primarily focused on mushroom body dependent operant-learning assays such as aversive phototaxic suppression and courtship conditioning. In this study, sleep was increased in the classic mutant rutabaga (rut2080) and dunce (dnc1) by feeding them the GABA-A agonist gaboxadol (Gab). Performance was evaluated in each mutant in response to social enrichment and place learning, tasks that do not require the mushroom body. Gab-induced sleep did not restore behavioral plasticity to either rut2080 or dnc1 mutants following social enrichment. However, increased sleep restored place learning to rut2080 mutants. These data extend the positive effects of enhanced sleep to place learning and highlight the utility of Gab for elucidating the beneficial effects of sleep on brain functioning.

Humans spend nearly a third of their life sleeping, yet, despite decades of research the function of sleep still remains a mystery. Sleep has been linked with various biological systems and functions, including metabolism, immunity, the cardiovascular system, and cognitive functions. Importantly, sleep appears to be present throughout the animal kingdom suggesting that it must provide an evolutionary advantage. Among the many possible functions of sleep, the relationship between sleep, and cognition has received a lot of support. We have all experienced the negative cognitive effects associated with a night of sleep deprivation. These can include increased emotional reactivity, poor judgment, deficit in attention, impairment in learning and memory, and obviously increase in daytime sleepiness. Furthermore, many neurological diseases like Alzheimer’s disease often have a sleep disorder component. In some cases, the sleep disorder can exacerbate the progression of the neurological disease. Thus, it is clear that sleep plays an important role for many brain functions. In particular, sleep has been shown to play a positive role in the consolidation of long-term memory while sleep deprivation negatively impacts learning and memory. Importantly, sleep is a behavior that is adapted to an individual’s need and influenced by many external and internal stimuli. In addition to being an adaptive behavior, sleep can also modulate plasticity in the brain at the level of synaptic connections between neurons and neuronal plasticity influences sleep. Understanding how sleep is modulated by internal and external stimuli and how sleep can modulate memory and plasticity is a key question in neuroscience. In order to address this question, several animal models have been developed. Among them, the fruit fly Drosophila melanogaster with its unparalleled genetics has proved to be extremely valuable. In addition to sleep, Drosophila has been shown to be an excellent model to study many complex behaviors, including learning, and memory. This review describes our current knowledge of the relationship between sleep, plasticity, and memory using the fly model.

Antimicrobial peptides act as a host defense mechanism and regulate the commensal microbiome. To obtain a comprehensive view of genes contributing to long-term memory we performed mRNA sequencing from single Drosophila heads following behavioral training that produces long-lasting memory. Surprisingly, we found that Diptericin B, an immune peptide with antimicrobial activity, is upregulated following behavioral training. Deletion and knock down experiments revealed that Diptericin B and another immune peptide, Gram-Negative Bacteria Binding Protein like 3, regulate long-term but not short-term memory or instinctive behavior in Drosophila. Interestingly, removal of DptB in the head fat body and GNBP-like3 in neurons results in memory deficit. That putative antimicrobial peptides influence memory provides an example of how some immune peptides may have been repurposed to influence the function of nervous system.

To test the hypothesis that sleep can reverse cognitive impairment during Alzheimer s disease, we enhanced sleep in flies either co-expressing human amyloid precursor protein and Beta-secretase (APP:BACE), or in flies expressing human tau. The ubiquitous expression of APP:BACE or human tau disrupted sleep. The sleep deficits could be reversed and sleep could be enhanced when flies were administered the GABA-A agonist 4,5,6,7-tetrahydroisoxazolo-[5,4-c]pyridine-3-ol (THIP). Expressing APP:BACE disrupted both Short-term memory (STM) and Long-term memory (LTM) as assessed using Aversive Phototaxic Suppression (APS) and courtship conditioning. Flies expressing APP:BACE also showed reduced levels of the synaptic protein discs large (DLG). Enhancing sleep in memory-impaired APP:BACE flies fully restored both STM and LTM and restored DLG levels. Sleep also restored STM to flies expressing human tau. Using live-brain imaging of individual clock neurons expressing both tau and the cAMP sensor Epac1-camps, we found that tau disrupted cAMP signaling. Importantly, enhancing sleep in flies expressing human tau restored proper cAMP signaling. Thus, we demonstrate that sleep can be used as a therapeutic to reverse deficits that accrue during the expression of toxic peptides associated with Alzheimer s disease.