Spatial transcriptomics reveals unique gene expression changes in different brain regions after sleep deprivation.

Sleep deprivation has far-reaching consequences on the brain and behavior, impacting memory, attention, and metabolism. Previous research has focused on gene expression changes in individual brain regions, such as the hippocampus or cortex. Therefore, it is unclear how uniformly or heterogeneously sleep loss affects the brain. Here, we use spatial transcriptomics to define the impact of a brief period of sleep deprivation across the brain in male mice. We find that sleep deprivation induced pronounced differences in gene expression across the brain, with the greatest changes in the hippocampus, neocortex, hypothalamus, and thalamus. Both the differentially expressed genes and the direction of regulation differed markedly across regions. Importantly, we developed bioinformatic tools to register tissue sections and gene expression data into a common anatomical space, allowing a brain-wide comparison of gene expression patterns between samples. Our results suggest that distinct molecular mechanisms acting in discrete brain regions underlie the biological effects of sleep deprivation.

Sleep and memory: The impact of sleep deprivation on transcription, translational control, and protein synthesis in the brain.

In countries around the world, sleep deprivation represents a widespread problem affecting school-age children, teenagers, and adults. Acute sleep deprivation and more chronic sleep restriction adversely affect individual health, impairing memory and cognitive performance as well as increasing the risk and progression of numerous diseases. In mammals, the hippocampus and hippocampus-dependent memory are vulnerable to the effects of acute sleep deprivation. Sleep deprivation induces changes in molecular signaling, gene expression and may cause changes in dendritic structure in neurons. Genome wide studies have shown that acute sleep deprivation alters gene transcription, although the pool of genes affected varies between brain regions. More recently, advances in research have drawn attention to differences in gene regulation between the level of the transcriptome compared with the pool of mRNA associated with ribosomes for protein translation following sleep deprivation. Thus, in addition to transcriptional changes, sleep deprivation also affects downstream processes to alter protein translation. In this review, we focus on the multiple levels through which acute sleep deprivation impacts gene regulation, highlighting potential post-transcriptional and translational processes that may be affected by sleep deprivation. Understanding the multiple levels of gene regulation impacted by sleep deprivation is essential for future development of therapeutics that may mitigate the effects of sleep loss.

Spatial transcriptomics reveals unique gene expression changes in different brain regions after sleep deprivation.

Sleep deprivation has far-reaching consequences on the brain and behavior, impacting memory, attention, and metabolism. Previous research has focused on gene expression changes in individual brain regions, such as the hippocampus or cortex. Therefore, it is unclear how uniformly or heterogeneously sleep loss affects the brain. Here, we use spatial transcriptomics to define the impact of a brief period of sleep deprivation across the brain. We find that sleep deprivation induced pronounced differences in gene expression across the brain, with the greatest changes in the hippocampus, neocortex, hypothalamus, and thalamus. Both the differentially expressed genes and the direction of regulation differed markedly across regions. Importantly, we developed bioinformatic tools to register tissue sections and gene expression data into a common anatomical space, allowing a brain-wide comparison of gene expression patterns between samples. Our results suggest that distinct molecular mechanisms acting in discrete brain regions underlie the biological effects of sleep deprivation.

Sleep Modulates Alcohol Toxicity in Drosophila.

Alcohol abuse is a significant public health problem. While considerable research has shown that alcohol use affects sleep, little is known about the role of sleep deprivation in alcohol toxicity. We investigated sleep as a factor modulating alcohol toxicity using Drosophila melanogaster, a model for studies of sleep, alcohol, and aging. Following 24 h of sleep deprivation using a paradigm that similarly affects males and females and induces rebound sleep, flies were given binge-like alcohol exposures. Sleep deprivation increased mortality, with no sex-dependent differences. Sleep deprivation also abolished functional tolerance measured at 24 h after the initial alcohol exposure, although there was no effect on alcohol absorbance or clearance. We investigated the effect of chronic sleep deprivation using mutants with decreased sleep, insomniac and insulin-like peptide 2, finding increased alcohol mortality. Furthermore, we investigated whether pharmacologically inducing sleep prior to alcohol exposure using the GABAA-receptor agonist 4,5,6,7-tetrahydroisoxazolo(5,4-c)pyridin-3-ol (THIP) mitigated the effects of alcohol toxicity on middle-aged flies, flies with environmentally disrupted circadian clocks, and flies with short sleep. Pharmacologically increasing sleep prior to alcohol exposure decreased alcohol-induced mortality. Thus, sleep prior to binge-like alcohol exposure affects alcohol-induced mortality, even in vulnerable groups such as aging flies and those with circadian dysfunction.

Altered hippocampal transcriptome dynamics following sleep deprivation.

Widespread sleep deprivation is a continuing public health problem in the United States and worldwide affecting adolescents and adults. Acute sleep deprivation results in decrements in spatial memory and cognitive impairments. The hippocampus is vulnerable to acute sleep deprivation with changes in gene expression, cell signaling, and protein synthesis. Sleep deprivation also has long lasting effects on memory and performance that persist after recovery sleep, as seen in behavioral studies from invertebrates to humans. Although previous research has shown that acute sleep deprivation impacts gene expression, the extent to which sleep deprivation affects gene regulation remains unknown. Using an unbiased deep RNA sequencing approach, we investigated the effects of acute sleep deprivation on gene expression in the hippocampus. We identified 1,146 genes that were significantly dysregulated following sleep deprivation with 507 genes upregulated and 639 genes downregulated, including protein coding genes and long non-coding RNAs not previously identified as impacted by sleep deprivation. Notably, genes significantly upregulated after sleep deprivation were associated with RNA splicing and the nucleus. In contrast, downregulated genes were associated with cell adhesion, dendritic localization, the synapse, and postsynaptic membrane. Furthermore, we found through independent experiments analyzing a subset of genes that three hours of recovery sleep following acute sleep deprivation was sufficient to normalize mRNA abundance for most genes, although exceptions occurred for some genes that may affect RNA splicing or transcription. These results clearly demonstrate that sleep deprivation differentially regulates gene expression on multiple transcriptomic levels to impact hippocampal function.

Translating around the clock: Multi-level regulation of post-transcriptional processes by the circadian clock.

The endogenous circadian clock functions to maintain optimal physiological health through the tissue specific coordination of gene expression and synchronization between tissues of metabolic processes throughout the 24 hour day. Individuals face numerous challenges to circadian function on a daily basis resulting in significant incidences of circadian disorders in the United States and worldwide. Dysfunction of the circadian clock has been implicated in numerous diseases including cancer, diabetes, obesity, cardiovascular and hepatic abnormalities, mood disorders and neurodegenerative diseases. The circadian clock regulates molecular, metabolic and physiological processes through rhythmic gene expression via transcriptional and post-transcriptional processes. Mounting evidence indicates that post-transcriptional regulation by the circadian clock plays a crucial role in maintaining tissue specific biological rhythms. Circadian regulation affecting RNA stability and localization through RNA processing, mRNA degradation, and RNA availability for translation can result in rhythmic protein synthesis, even when the mRNA transcripts themselves do not exhibit rhythms in abundance. The circadian clock also targets the initiation and elongation steps of translation through multiple pathways. In this review, the influence of the circadian clock across the levels of post-transcriptional, translation, and post-translational modifications are examined using examples from humans to cyanobacteria demonstrating the phylogenetic conservation of circadian regulation. Lastly, we briefly discuss chronotherapies and pharmacological treatments that target circadian function. Understanding the complexity and levels through which the circadian clock regulates molecular and physiological processes is important for future advancement of therapeutic outcomes.

Translational changes induced by acute sleep deprivation uncovered by TRAP-Seq.

Sleep deprivation is a global health problem adversely affecting health as well as causing decrements in learning and performance. Sleep deprivation induces significant changes in gene transcription in many brain regions, with the hippocampus particularly susceptible to acute sleep deprivation. However, less is known about the impacts of sleep deprivation on post-transcriptional gene regulation. To identify the effects of sleep deprivation on the translatome, we took advantage of the RiboTag mouse line to express HA-labeled Rpl22 in CaMKIIα neurons to selectively isolate and sequence mRNA transcripts associated with ribosomes in excitatory neurons. We found 198 differentially expressed genes in the ribosome-associated mRNA subset after sleep deprivation. In comparison with previously published data on gene expression in the hippocampus after sleep deprivation, we found that the subset of genes affected by sleep deprivation was considerably different in the translatome compared with the transcriptome, with only 49 genes regulated similarly. Interestingly, we found 478 genes differentially regulated by sleep deprivation in the transcriptome that were not significantly regulated in the translatome of excitatory neurons. Conversely, there were 149 genes differentially regulated by sleep deprivation in the translatome but not in the whole transcriptome. Pathway analysis revealed differences in the biological functions of genes exclusively regulated in the transcriptome or translatome, with protein deacetylase activity and small GTPase binding regulated in the transcriptome and unfolded protein binding, kinase inhibitor activity, neurotransmitter receptors and circadian rhythms regulated in the translatome. These results indicate that sleep deprivation induces significant changes affecting the pool of actively translated mRNAs.

The CBP KIX domain regulates long-term memory and circadian activity.

BACKGROUND: CREB-dependent transcription necessary for long-term memory is driven by interactions with CREB-binding protein (CBP), a multi-domain protein that binds numerous transcription factors potentially affecting expression of thousands of genes. Identifying specific domain functions for multi-domain proteins is essential to understand processes such as cognitive function and circadian clocks. We investigated the function of the CBP KIX domain in hippocampal memory and gene expression using CBPKIX/KIX mice with mutations that prevent phospho-CREB (Ser133) binding. RESULTS: We found that CBPKIX/KIX mice were impaired in long-term memory, but not learning acquisition or short-term memory for the Morris water maze. Using an unbiased analysis of gene expression in the dorsal hippocampus after training in the Morris water maze or contextual fear conditioning, we discovered dysregulation of CREB, CLOCK, and BMAL1 target genes and downregulation of circadian genes in CBPKIX/KIX mice. Given our finding that the CBP KIX domain was important for transcription of circadian genes, we profiled circadian activity and phase resetting in CBPKIX/KIX mice. CBPKIX/KIX mice exhibited delayed activity peaks after light offset and longer free-running periods in constant dark. Interestingly, CBPKIX/KIX mice displayed phase delays and advances in response to photic stimulation comparable to wildtype littermates. Thus, this work delineates site-specific regulation of the circadian clock by a multi-domain protein. CONCLUSIONS: These studies provide insight into the significance of the CBP KIX domain by defining targets of CBP transcriptional co-activation in memory and the role of the CBP KIX domain in vivo on circadian rhythms.

Resetting the Aging Clock: Implications for Managing Age-Related Diseases.

Worldwide, individuals are living longer due to medical and scientific advances, increased availability of medical care and changes in public health policies. Consequently, increasing attention has been focused on managing chronic conditions and age-related diseases to ensure healthy aging. The endogenous circadian system regulates molecular, physiological and behavioral rhythms orchestrating functional coordination and processes across tissues and organs. Circadian disruption or desynchronization of circadian oscillators increases disease risk and appears to accelerate aging. Reciprocally, aging weakens circadian function aggravating age-related diseases and pathologies. In this review, we summarize the molecular composition and structural organization of the circadian system in mammals and humans, and evaluate the technological and societal factors contributing to the increasing incidence of circadian disorders. Furthermore, we discuss the adverse effects of circadian dysfunction on aging and longevity and the bidirectional interactions through which aging affects circadian function using examples from mammalian research models and humans. Additionally, we review promising methods for managing healthy aging through behavioral and pharmacological reinforcement of the circadian system. Understanding age-related changes in the circadian clock and minimizing circadian dysfunction may be crucial components to promote healthy aging.

Investigating Alcohol Behavior and Physiology Using Drosophila melanogaster.

Drosophila melanogaster, the fruit fly, is one of the most versatile models for biomedical studies due to the economical husbandry, rapid generation time, and the array of tools for spatial and temporal gene manipulation. The relatively short lifespan of Drosophila (60-80 days) and the high degree of molecular conservation across species make Drosophila ideal to study the complexities of aging. Alcohol is the most abused drug worldwide and alcohol use disorders represent a significant public health problem and economic burden to individuals and society. Stereotypical alcohol-induced behaviors and the underlying molecular mechanisms are conserved from flies to humans making Drosophila a practical model for investigating the development of alcohol-induced behaviors and alcohol pathologies. Here, we outline how to assemble an efficient and controlled alcohol vapor delivery system, the FlyBar, and review paradigms and protocols for the assessment of alcohol-induced behaviors and physiology in Drosophila including the loss-of-righting reflex, sedation, tolerance, alcohol metabolism, and gut permeability.

Aging and the clock: Perspective from flies to humans.

Endogenous circadian oscillators regulate molecular, cellular and physiological rhythms, synchronizing tissues and organ function to coordinate activity and metabolism with environmental cycles. The technological nature of modern society with round-the-clock work schedules and heavy reliance on personal electronics has precipitated a striking increase in the incidence of circadian and sleep disorders. Circadian dysfunction contributes to an increased risk for many diseases and appears to have adverse effects on aging and longevity in animal models. From invertebrate organisms to humans, the function and synchronization of the circadian system weakens with age aggravating the age-related disorders and pathologies. In this review, we highlight the impacts of circadian dysfunction on aging and longevity and the reciprocal effects of aging on circadian function with examples from Drosophila to humans underscoring the highly conserved nature of these interactions. Additionally, we review the potential for using reinforcement of the circadian system to promote healthy aging and mitigate age-related pathologies. Advancements in medicine and public health have significantly increased human life span in the past century. With the demographics of countries worldwide shifting to an older population, there is a critical need to understand the factors that shape healthy aging. Drosophila melanogaster, as a model for aging and circadian interactions, has the capacity to facilitate the rapid advancement of research in this area and provide mechanistic insights for targeted investigations in mammals.

Aging and circadian dysfunction increase alcohol sensitivity and exacerbate mortality in Drosophila melanogaster.

Alcohol abuse is a rising problem in middle-aged and older individuals resulting in serious health, family and economic consequences. Effective treatment necessitates the identification of factors influencing alcohol toxicity with aging. We investigated the interaction between aging, alcohol toxicity and circadian function using Drosophila as a model system. We found as wild type flies age, sensitivity to alcohol increases and circadian regulation of alcohol-induced behaviors weakens. Decreased circadian modulation is correlated with significantly greater alcohol sensitivity during the subjective day. The circadian clock modulates alcohol-induced mortality in younger flies with increased mortality following alcohol exposure at night. Older flies exhibit significantly longer recovery times following alcohol-induced sedation and increased mortality following binge-like or chronic alcohol exposure. Flies rendered arrhythmic either genetically or environmentally exhibit significantly increased alcohol sensitivity, longer recovery times and increased mortality. We hypothesize that the circadian clock phase specifically buffers behavioral and cellular alcohol sensitivity with this protection diminishing as the circadian clock weakens with age.

Drosophila: An Emergent Model for Delineating Interactions between the Circadian Clock and Drugs of Abuse.

Endogenous circadian oscillators orchestrate rhythms at the cellular, physiological, and behavioral levels across species to coordinate activity, for example, sleep/wake cycles, metabolism, and learning and memory, with predictable environmental cycles. The 21st century has seen a dramatic rise in the incidence of circadian and sleep disorders with globalization, technological advances, and the use of personal electronics. The circadian clock modulates alcohol- and drug-induced behaviors with circadian misalignment contributing to increased substance use and abuse. Invertebrate models, such as Drosophila melanogaster, have proven invaluable for the identification of genetic and molecular mechanisms underlying highly conserved processes including the circadian clock, drug tolerance, and reward systems. In this review, we highlight the contributions of Drosophila as a model system for understanding the bidirectional interactions between the circadian system and the drugs of abuse, alcohol and cocaine, and illustrate the highly conserved nature of these interactions between Drosophila and mammalian systems. Research in Drosophila provides mechanistic insights into the corresponding behaviors in higher organisms and can be used as a guide for targeted inquiries in mammals.

Role of proteasome-dependent protein degradation in long-term operant memory in Aplysia.

We investigated the in vivo role of protein degradation during intermediate (ITM) and long-term memory (LTM) in Aplysia using an operant learning paradigm. The proteasome inhibitor MG-132 inhibited the induction and molecular consolidation of LTM with no effect on ITM. Remarkably, maintenance of steady-state protein levels through inhibition of protein synthesis using either anisomycin or rapamycin in conjunction with proteasome inhibition permitted the formation of robust 24 h LTM. Our studies suggest a primary role for proteasomal activity in facilitation of gene transcription for LTM and raise the possibility that synaptic mechanisms are sufficient to sustain 24 h memory.

Differential role of calpain-dependent protein cleavage in intermediate and long-term operant memory in Aplysia.

In addition to protein synthesis, protein degradation or protein cleavage may be necessary for intermediate (ITM) and long-term memory (LTM) to remove molecular constraints, facilitate persistent kinase activity and modulate synaptic plasticity. Calpains, a family of conserved calcium dependent cysteine proteases, modulate synaptic function through protein cleavage. We used the marine mollusk Aplysia californica to investigate the in vivo role of calpains during intermediate and long-term operant memory formation using the learning that food is inedible (LFI) paradigm. A single LFI training session, in which the animal associates a specific netted seaweed with the failure to swallow, generates short (30min), intermediate (4-6h) and long-term (24h) memory. Using the calpain inhibitors calpeptin and MDL-28170, we found that ITM requires calpain activity for induction and consolidation similar to the previously reported requirements for persistent protein kinase C activity in intermediate-term LFI memory. The induction of LTM also required calpain activity. In contrast to ITM, calpain activity was not necessary for the molecular consolidation of LTM. Surprisingly, six hours after LFI training we found that calpain activity was necessary for LTM, although this is a time at which neither persistent PKC activity nor protein synthesis is required for the maintenance of long-term LFI memory. These results demonstrate that calpains function in multiple roles in vivo during associative memory formation.

Acute Sleep Deprivation Blocks Short- and Long-Term Operant Memory in Aplysia.

STUDY OBJECTIVES: Insufficient sleep in individuals appears increasingly common due to the demands of modern work schedules and technology use. Consequently, there is a growing need to understand the interactions between sleep deprivation and memory. The current study determined the effects of acute sleep deprivation on short and long-term associative memory using the marine mollusk Aplysia californica, a relatively simple model system well known for studies of learning and memory. METHODS: Aplysia were sleep deprived for 9 hours using context changes and tactile stimulation either prior to or after training for the operant learning paradigm, learning that food is inedible (LFI). The effects of sleep deprivation on short-term (STM) and long-term memory (LTM) were assessed. RESULTS: Acute sleep deprivation prior to LFI training impaired the induction of STM and LTM with persistent effects lasting at least 24 h. Sleep deprivation immediately after training blocked the consolidation of LTM. However, sleep deprivation following the period of molecular consolidation did not affect memory recall. Memory impairments were independent of handling-induced stress, as daytime handled control animals demonstrated no memory deficits. Additional training immediately after sleep deprivation failed to rescue the induction of memory, but additional training alleviated the persistent impairment in memory induction when training occurred 24 h following sleep deprivation. CONCLUSIONS: Acute sleep deprivation inhibited the induction and consolidation, but not the recall of memory. These behavioral studies establish Aplysia as an effective model system for studying the interactions between sleep and memory formation.

Chronic sleep deprivation differentially affects short and long-term operant memory in Aplysia.

The induction, formation and maintenance of memory represent dynamic processes modulated by multiple factors including the circadian clock and sleep. Chronic sleep restriction has become common in modern society due to occupational and social demands. Given the impact of cognitive impairments associated with sleep deprivation, there is a vital need for a simple animal model in which to study the interactions between chronic sleep deprivation and memory. We used the marine mollusk Aplysia californica, with its simple nervous system, nocturnal sleep pattern and well-characterized learning paradigms, to assess the effects of two chronic sleep restriction paradigms on short-term (STM) and long-term (LTM) associative memory. The effects of sleep deprivation on memory were evaluated using the operant learning paradigm, learning that food is inedible, in which the animal associates a specific netted seaweed with failed swallowing attempts. We found that two nights of 6h sleep deprivation occurring during the first or last half of the night inhibited both STM and LTM. Moreover, the impairment in STM persisted for more than 24h. A milder, prolonged sleep deprivation paradigm consisting of 3 consecutive nights of 4h sleep deprivation also blocked STM, but had no effect on LTM. These experiments highlight differences in the sensitivity of STM and LTM to chronic sleep deprivation. Moreover, these results establish Aplysia as a valid model for studying the interactions between chronic sleep deprivation and associative memory paving the way for future studies delineating the mechanisms through which sleep restriction affects memory formation.

Circadian Modulation of Alcohol-Induced Sedation and Recovery in Male and Female Drosophila.

Delineating the factors that affect behavioral and neurological responses to alcohol is critical to facilitate measures for preventing or treating alcohol abuse. The high degree of conserved molecular and physiological processes makes Drosophila melanogaster a valuable model for investigating circadian interactions with alcohol-induced behaviors and examining sex-specific differences in alcohol sensitivity. We found that wild-type Drosophila exhibited rhythms in alcohol-induced sedation under light-dark and constant dark conditions with considerably greater alcohol exposure necessary to induce sedation during the late (subjective) day and peak sensitivity to alcohol occurring during the late (subjective) night. The circadian clock also modulated the recovery from alcohol-induced sedation with flies regaining motor control significantly faster during the late (subjective) day. As predicted, the circadian rhythms in sedation and recovery were absent in flies with a mutation in the circadian gene period or arrhythmic flies housed in constant light conditions. Flies lacking a functional circadian clock were more sensitive to the effects of alcohol with significantly longer recovery times. Similar to other animals and humans, Drosophila exhibit sex-specific differences in alcohol sensitivity. We investigated whether the circadian clock modulated the rhythms in the loss-of-righting reflex, alcohol-induced sedation, and recovery differently in males and females. We found that both sexes demonstrated circadian rhythms in the loss-of-righting reflex and sedation with the differences in alcohol sensitivity between males and females most pronounced during the late subjective day. Recovery of motor reflexes following alcohol sedation also exhibited circadian modulation in male and female flies, although the circadian clock did not modulate the difference in recovery times between the sexes. These studies provide a framework outlining how the circadian clock modulates alcohol-induced behaviors in Drosophila and identifies sexual dimorphisms in the circadian modulation of alcohol behaviors.

Synchrony and desynchrony in circadian clocks: impacts on learning and memory.

Circadian clocks evolved under conditions of environmental variation, primarily alternating light dark cycles, to enable organisms to anticipate daily environmental events and coordinate metabolic, physiological, and behavioral activities. However, modern lifestyle and advances in technology have increased the percentage of individuals working in phases misaligned with natural circadian activity rhythms. Endogenous circadian oscillators modulate alertness, the acquisition of learning, memory formation, and the recall of memory with examples of circadian modulation of memory observed across phyla from invertebrates to humans. Cognitive performance and memory are significantly diminished when occurring out of phase with natural circadian rhythms. Disruptions in circadian regulation can lead to impairment in the formation of memories and manifestation of other cognitive deficits. This review explores the types of interactions through which the circadian clock modulates cognition, highlights recent progress in identifying mechanistic interactions between the circadian system and the processes involved in memory formation, and outlines methods used to remediate circadian perturbations and reinforce circadian adaptation.

Characterization of sleep in Aplysia californica.

STUDY OBJECTIVE: To characterize sleep in the marine mollusk, Aplysia californica. DESIGN: Animal behavior and activity were assessed using video recordings to measure activity, resting posture, resting place preference, and behavior after rest deprivation. Latencies for behavioral responses were measured for appetitive and aversive stimuli for animals in the wake and rest states. SETTING: Circadian research laboratory for Aplysia. PATIENTS OR PARTICIPANTS: A. californica from the Pacific Ocean. INTERVENTIONS: N/A. MEASUREMENTS AND RESULTS: Aplysia rest almost exclusively during the night in a semi-contracted body position with preferential resting locations in the upper corners of their tank. Resting animals demonstrate longer latencies in head orientation and biting in response to a seaweed stimulus and less frequent escape response steps following an aversive salt stimulus applied to the tail compared to awake animals at the same time point. Aplysia exhibit rebound rest the day following rest deprivation during the night, but not after similar handling stimulation during the day. CONCLUSIONS: Resting behavior in Aplysia fulfills all invertebrate characteristics of sleep including: (1) a specific sleep body posture, (2) preferred resting location, (3) reversible behavioral quiescence, (4) elevated arousal thresholds for sensory stimuli during sleep, and (5) compensatory sleep rebound after sleep deprivation.