Biology subjects

Keene, A. C.

Publications and source records attributed to Keene, A. C..

66 records · Page 4Linked to original sources

bioRxiv2019

Sleep regulates the glial engulfment receptor Draper to promote Wallerian degeneration

Sleep, a universal behavior, is critical for diverse aspects of brain function. Chronic sleep disturbance is associated with numerous health consequences, including neurodegenerative disease and cognitive decline. Neurite damage due to apoptosis, trauma, or genetic factors is a common feature of aging, and clearance of damaged neurons is essential for maintenance of brain function. In the central nervous system, damaged neurites are cleared by Wallerian degeneration, in which activated microglia and macrophages engulf damaged neurons. The fruit fly Drosophila melanogaster provides a powerful model for investigating the relationship between sleep and Wallerian degeneration. Several lines of evidence suggest that glia influence sleep duration, sleep-mediated neuronal homeostasis, and clearance of toxic substances during sleep, raising the possibility that glial engulfment of damaged axons is regulated by sleep. To explore this possibility, we axotomized olfactory receptor neurons and measured the effects of sleep loss or gain on the clearance of damaged neurites. Mechanical sleep deprivation impaired the clearance of damaged neurites, whereas the sleep-promoting drug gaboxadol accelerated clearance. In sleep-deprived animals, multiple markers of glial activation were delayed, including activation of the JAK/STAT pathway, upregulation of the cell corpse engulfment receptor Draper, and innervation of the antennal lobe by glial membranes. These markers were all enhanced when sleep was induced in gaboxadol-treated flies. Taken together, these findings reveal a critical role for sleep in regulation glial activation and engulfment following axotomy, providing a platform for further investigations of the molecular mechanisms underlying sleep-dependent modulation of glial function and neurite clearance.\n\nHighlightsO_LISleep deprivation impairs Wallerian degeneration in fruit flies.\nC_LIO_LIPharmacological induction of sleep accelerates Wallerian degeneration.\nC_LIO_LISleep promotes innervation surrounding damaged neurites by phagocytic glia.\nC_LIO_LISleep increases levels of the glial activation markers Draper and Stat92E.\nC_LI

neuroscience↗

bioRxiv2019

Dietary fatty acids promote sleep through a taste-independent mechanism

Consumption of foods that are high in fat contributes to obesity and metabolism-related disorders that are increasing in prevalence and present an enormous health burden throughout the world. Dietary lipids are comprised of triglycerides and fatty acids, and the highly palatable taste of dietary fatty acids promotes food consumption, activates reward centers in mammals, and underlies hedonic feeding. Despite a central role of dietary fats in the regulation of food intake and the etiology of metabolic diseases, little is known about how fat consumption regulates sleep. The fruit fly, Drosophila melanogaster, provides a powerful model system for the study of sleep and metabolic traits, and flies potently regulate sleep in accordance with food availability. To investigate the effects of dietary fats on sleep regulation, we have supplemented fatty acids into the diet of Drosophila and measured their effects on sleep and activity. We found that feeding flies a diet of hexanoic acid, a medium-chain fatty acid that is a by-product of yeast fermentation, promotes sleep by increasing the number of sleep episodes. This increase in sleep is dose-dependent and independent of the light-dark cues. Diets consisting of other fatty acids, including medium- and long-chain fatty acids, also increase sleep, suggesting many fatty acid types promote sleep. To assess whether dietary fatty acids regulate sleep through the taste system, we assessed sleep in flies with a mutation in the hexanoic acid receptor Ionotropic receptor 56d, which is required for fatty acid taste perception. We found that these flies also increase their sleep when fed a hexanoic acid diet, suggesting the sleep promoting effect of hexanoic acid is not dependent on sensory perception. Overall, these results define a role for fatty acids in sleep regulation, providing a foundation to investigate the molecular and neural basis for fatty acid-dependent modulation of sleep duration.

neuroscience↗

bioRxiv2019

Drosophila insulin-like peptide 2 mediates dietary regulation of sleep intensity

Sleep is a nearly universal behavior that is regulated by diverse environmental and physiological stimuli. A defining feature of sleep is a homeostatic rebound following deprivation, where animals compensate for lost sleep by increasing sleep duration and/or sleep depth. Fruit flies exhibit robust recovery sleep following deprivation and represent a powerful model to study neural circuits regulating sleep homeostasis. Numerous neuronal populations have been identified in modulating sleep homeostasis as well as depth, raising the possibility that recovery sleep is differentially regulated by environmental or physiological processes that induce sleep deprivation. Here, we find that unlike most pharmacological and environmental manipulations commonly used to restrict sleep, starvation potently induces sleep loss without a subsequent rebound in sleep duration or depth. We find that both starvation and a sucrose-only diet result in reduced metabolic rate and increased sleep depth, suggesting that dietary yeast protein is essential for normal sleep depth and homeostasis. Finally, we find that Drosophila insulin like peptide 2 (Dilp2) is required for starvation-induced changes in sleep depth without regulating the duration of sleep. Remarkably, Dilp2 mutant flies require rebound sleep following sleep deprivation, suggesting Dilp2 underlies resilience to sleep loss. Together, these findings reveal innate resilience to starvation-induced sleep loss and identify distinct mechanisms that underlie starvation-induced changes in sleep duration and depth.\n\nAuthor SummarySleep is nearly universal throughout the animal kingdom and homeostatic regulation represents a defining feature of sleep, where animals compensate for lost sleep by increasing sleep over subsequent time periods. Despite the robustness of this feature, surprisingly little is known about how recovery-sleep is regulated in response to different types of sleep deprivation. Fruit flies provide a powerful model for investigating the genetic regulation of sleep, and like mammals, display robust recovery sleep following deprivation. Here, we find that unlike most stimuli that suppress sleep, sleep deprivation by starvation does not require a homeostatic rebound. These findings appear to be due to flies engaging in deeper sleep during the period of partial deprivation, suggesting a natural resilience to starvation-induced sleep loss. This unique resilience to starvation-induced sleep loss is dependent on Drosophila insulin-like peptide 2, suggesting a critical role for insulin signaling in regulating interactions between diet and sleep homeostasis.

neuroscience↗

bioRxiv2019

An adult brain atlas reveals broad neuroanatomical changes in independently evolved populations of Mexican cavefish

A shift in environmental conditions impacts the evolution of complex developmental and behavioral traits. The Mexican cavefish, Astyanax mexicanus, is a powerful model for examining the evolution of development, physiology, and behavior because multiple cavefish populations can be compared to an extant and ancestral-like surface population of the same species. Many behaviors have diverged in cave populations of A. mexicanus, and previous studies have shown that cavefish have a loss of sleep, reduced stress, an absence of social behaviors, and hyperphagia. Despite these findings, surprisingly little is known about the changes in neuroanatomy that underlie these behavioral phenotypes. Here, we use serial sectioning to generate a brain atlas of surface fish and three independent cavefish populations. Volumetric reconstruction of serial-sectioned brains confirms convergent evolution on reduced optic tectum volume in all cavefish populations tested. In addition, we quantified volumes of specific neuroanatomical loci within several brain regions, which have previously been implicated in behavioral regulation, including the hypothalamus, thalamus, and habenula. These analyses reveal an expansion of the hypothalamus across all three cavefish populations relative to surface fish, as well as subnuclei-specific differences within the thalamus and habenulae. Taken together, these analyses support the notion that changes in environmental conditions are accompanied by neuroanatomical changes in brain structures associated with behavior. This atlas provides a resource for comparative neuroanatomy of additional brain regions and the opportunity to associate brain anatomy with evolved changes in behavior.

neuroscience↗

bioRxiv2019

Stable transgenesis in Astyanax mexicanus using the Tol2 transposase system.

Astyanax mexicanus is a well-established and widely used fish model system for evolutionary and developmental biology research. These fish exist as surface forms that inhabit rivers and 30 different populations of cavefish. The establishment of A. mexicanus as an emergent model organism for understanding the evolutionary basis of development and behavior has been accelerated by an increasing availability of genomic approaches to identify genotype-phenotype associations. Despite important progress in the deployment of new technologies, deep mechanistic insights into A. mexicanus evolution and development have been limited by a lack of transgenic lines commonly used in genetic model systems. Here, we expand the toolkit of transgenesis by characterizing two novel stable transgenic lines that were generated using the highly efficient Tol2 system, commonly used to generate transgenic zebrafish. A stable transgenic line consisting of the zebrafish ubiquitin promoter fused to eGFP expressed eGFP ubiquitously throughout development in a surface population of Astyanax. To define specific cell-types, we injected fish with a Cntnap2-mCherry construct that labels lateral line mechanosensory neurons in zebrafish. Strikingly, both constructs appear to label the predicted cell types, suggesting many genetic tools and defined promoter regions in zebrafish are directly transferrable to cavefish. The lines provide proof-of-principle for the application of Tol2 transgenic technology in A. mexicanus. Expansion on these initial transgenic lines will provide a platform to address broadly important problems in the quest to bridge the genotype to phenotype gap.

developmental biology↗

bioRxiv2018

RNAlater and flash freezing storage methods nonrandomly influence observed gene expression in RNAseq experiments

RNA-sequencing is a popular next-generation sequencing technique for assaying genome-wide gene expression profiles. Nonetheless, it is susceptible to biases that are introduced by sample handling prior gene expression measurements. Two of the most common methods for preserving samples in both field-based and laboratory conditions are submersion in RNAlater and flash freezing in liquid nitrogen. Flash freezing in liquid nitrogen can be impractical, particularly for field collections. RNAlater is a solution for stabilizing tissue for longer-term storage as it rapidly permeates tissue to protect cellular RNA. In this study, we assessed genome-wide expression patterns in 30 day old fry collected from the same brood at the same time point that were flash-frozen in liquid nitrogen and stored at -80{degrees}C or submerged and stored in RNAlater at room temperature, simulating conditions of fieldwork. We show that sample storage is a significant factor influencing observed differential gene expression. In particular, genes with elevated GC content exhibit higher observed expression levels in liquid nitrogen flash-freezing relative to RNAlater-storage. Further, genes with higher expression in RNAlater relative to liquid nitrogen experience disproportionate enrichment for functional categories, many of which are involved in RNA processing. This suggests that RNAlater may elicit a physiological response that has the potential to bias biological interpretations of expression studies. The biases introduced to observed gene expression arising from mimicking many field-based studies are substantial and should not be ignored.

genomics↗

bioRxiv2018

Long term impact of intensive post graduate laboratory training at the Cold Spring Harbor Neurobiology of Drosophila summer course

Intensive postgraduate courses provide an opportunity for junior and senior level scientists to learn concepts and techniques that will advance their training and research programs. It is commonly assumed that short intensive courses have positive impacts within fields of research; however, these assumptions are rarely tested. Here we describe the framework of a long running postgraduate summer course at Cold Spring Harbor and attempt to quantify the impact made over its history. For over three decades, the Drosophila Neurobiology: Genes, Circuits & Behavior Summer Course at Cold Spring Harbor Laboratories (CSHL) has provided participants with intense instruction on a wide variety of topics and techniques in integrative neuroscience using Drosophila as a model organism. Students are introduced to the latest approaches for studying nervous system development, activity and connectivity, as well as complex behaviors and diseases. The course has a long history of successful alumni, many of whom describe participation in the course as foundational to their training. Student surveys of recent participants indicate a high level of satisfaction, improved career outcomes, and direct impact on publications. Analysis of student success reveals that over 64% of participants obtain independent faculty positions. Further, we describe ongoing efforts to enhance diversity and encourage access to scientific research at undergraduate-focused institutions. Together, our findings suggest that laboratory-intensive postgraduate courses provide a highly effective mechanism for scientific training that has lasting positive impacts on trainees.

scientific communication and education↗

bioRxiv2018

Orchestration of Drosophila post-feeding physiology and behavior by the neuropeptide leucokinin

Behavior and physiology are orchestrated by neuropeptides acting as neuromodulators and/or circulating hormones. A central question is how these neuropeptides function to coordinate complex and competing behaviors. The neuropeptide leucokinin (LK) modulates diverse functions, including circadian rhythms, feeding, water homeostasis, and sleep, but the mechanisms underlying these complex interactions remain poorly understood. Here, we delineate the LK circuitry that governs homeostatic functions that are critical for survival. We found that impaired LK signaling affects diverse but coordinated processes, including regulation of stress, water homeostasis, locomotor activity, and metabolic rate. There are three different sets of LK neurons, which contribute to different aspects of this physiology. We show that the calcium activity of abdominal ganglia LK neurons (ABLKs) increases specifically following water consumption, but not under other conditions, suggesting that these neurons regulate water homeostasis and its associated physiology. To identify targets of LK peptide, we mapped the distribution of the LK receptor (Lkr), mined brain single-cell transcriptome dataset for genes coexpressed with Lkr, and utilized trans-synaptic labeling to identify synaptic partners of LK neurons. Lkr expression in the brain insulin-producing cells (IPCs), gut, renal tubules and sensory cells, and the post-synaptic signal in sensory neurons, correlates well with regulatory roles detected in the Lk and Lkr mutants. Furthermore, these mutants and flies with targeted knockdown of Lkr in IPCs displayed altered expression of insulin-like peptides (DILPs) in IPCs and modulated stress responses. Thus, some effects of LK signaling appear to occur via DILP action. Collectively, our data suggest that the three sets of LK neurons orchestrate the establishment of post-prandial homeostasis by regulating distinct physiological processes and behaviors such as diuresis, metabolism, organismal activity and insulin signaling. These findings provide a platform for investigating neuroendocrine regulation of behavior and brain-to-periphery communication.

neuroscience↗

bioRxiv2018

The role of gene flow in rapid and repeated evolution of cave related traits in Mexican tetra, Astyanax mexicanus

Understanding the molecular basis of repeated evolved phenotypes can yield key insights into the evolutionary process. Quantifying the amount of gene flow between populations is especially important in interpreting mechanisms of repeated phenotypic evolution, and genomic analyses have revealed that admixture is more common between diverging lineages than previously thought. In this study, we resequenced and analyzed nearly 50 whole genomes of the Mexican tetra from three blind cave populations, two surface populations, and outgroup samples. We confirmed that cave populations are polyphyletic and two Astyanax mexicanus lineages are present in our dataset. The two lineages likely diverged [~]257k generations ago, which, assuming 1 generation per year, is substantially younger than previous mitochondrial estimates of 5-7mya. Divergence of cave populations from their phylogenetically closest surface population likely occurred between [~]161k - 191k generations ago. The favored demographic model for most population pairs accounts for divergence with secondary contact and heterogeneous gene flow across the genome, and we rigorously identified abundant gene flow between cave and surface fish, between caves, and between separate lineages of cave and surface fish. Therefore, the evolution of cave-related traits occurred more rapidly than previously thought, and trogolomorphic traits are maintained despite substantial gene flow with surface populations. After incorporating these new demographic estimates, our models support that selection may drive the evolution of cave-derived traits, as opposed to the classic hypothesis of disuse and drift. Finally, we show that a key QTL is enriched for genomic regions with very low divergence between caves, suggesting that regions important for cave phenotypes may be transferred between caves via gene flow. In sum, our study shows that shared evolutionary history via gene flow must be considered in studies of independent, repeated trait evolution.

evolutionary biology↗

bioRxiv2017

A subset of sweet-sensing neurons identified by IR56d are necessary and sufficient for fatty acid taste

Fat represents a calorically potent food source that yields approximately twice the amount of energy as carbohydrates or proteins per unit of mass. The highly palatable taste of FAs promotes food consumption, activates reward circuitry, and is thought to contribute to hedonic feeding underlying many metabolism-related disorders. Despite a role in the etiology of metabolic diseases, little is known about how dietary fats are detected by the gustatory system to promote feeding. Previously, we showed that a broad population of sugar-sensitive taste neurons expressing Gustatory Receptor 64f (Gr64f), is required for reflexive feeding responses to both FAs and sugars. Here, we report a genetic silencing screen to identify specific populations of taste neurons that mediate fatty acid taste. We find neurons identified by expression of Ionotropic Receptor 56d (IR56d) are necessary and sufficient for reflexive feeding response to FAs. Functional imaging reveals that IR56d-expressing neurons are responsive to short- and medium-chain FAs. Silencing IR56d neurons selectively abolishes fatty acid taste, while their activation is sufficient to drive feeding responses. Analysis of co-expression with Gr64f identifies two subpopulations of IR56d-expressing neurons. While physiological imaging reveals that both populations are responsive to FAs, IR56d/Gr64f neurons are activated by medium-chain FAs and are sufficient for reflexive feeding responses to FA. Moreover, flies can discriminate between sugar and FAs in an aversive taste memory assay, indicating that FA taste is a unique modality in Drosophila. Taken together, these findings localize fatty acid taste within the Drosophila gustatory center and provide an opportunity to investigate discrimination between different categories of appetitive tastants.\n\nAuthors SummaryFat represents a calorically potent food source that yields approximately twice the amount of energy as carbohydrates or proteins per unit of mass. Dietary lipids are comprised of both triacylglycerides and FAs, and growing evidence suggests that it is the free FAs that are detected by the gustatory system. The highly palatable taste of FAs promotes food consumption, activates reward centers in mammals, and is thought to contribute to hedonic feeding that underlies many metabolism-related disorders. Despite a role in the etiology of metabolic diseases, little is known about how dietary fats are detected by the gustatory system to promote feeding. We have identified a subset of sugar-sensing neurons in the fly that also respond to medium-chain FAs and are necessary and sufficient for behavioral responses to FAs. Further, we find that despite being sensed by shared neuron populations, flies can differentiate between the taste of sugar and FAs, fortifying the notion that FAs and sugar represent distinct taste modalities in flies.

neuroscience↗

bioRxiv2017

Sleep-Dependent Modulation Of Metabolic Rate In Drosophila

Dysregulation of sleep is associated with metabolic diseases, and metabolic rate is acutely regulated by sleep-wake behavior. In humans and rodent models, sleep loss is associated with obesity, reduced metabolic rate, and negative energy balance, yet little is known about the neural mechanisms governing interactions between sleep and metabolism. We have developed a system to simultaneously measure sleep and metabolic rate in individual Drosophila, allowing for interrogation of neural systems governing interactions between sleep and metabolic rate. Like mammals, metabolic rate in flies is reduced during sleep and increased during sleep deprivation suggesting sleep-dependent regulation of metabolic rate is conserved across phyla. The reduction of metabolic rate during sleep is not simply a consequence of inactivity because metabolic rate is reduced [~]30 minutes following the onset of sleep, raising the possibility that CO2 production provides a metric to distinguish different sleep states in the fruit fly. To examine the relationship between sleep and metabolism, we determined basal and sleep-dependent changes in metabolic rate is reduced in starved flies, suggesting that starvation inhibits normal sleep-associated effects on metabolic rate. Further, translin mutant flies that fail to suppress sleep during starvation demonstrate a lower basal metabolic rate, but this rate was further reduced in response to starvation, revealing that regulation of starvation-induced changes in metabolic rate and sleep duration are genetically distinct. Therefore, this system provides the unique ability to simultaneously measure sleep and oxidative metabolism, providing novel insight into the physiological changes associated with sleep and wakefulness in the fruit fly.\n\nSignificance statementMetabolic disorders are associated with sleep disturbances, yet our understanding of the mechanisms underlying interactions between sleep and metabolism remain limited. Here, we describe a novel system to simultaneously record sleep and metabolic rate in single Drosophila. Our findings reveal that uninterrupted sleep bouts of 30 minutes or greater are associated with a reduction in metabolic rate providing a physiological readout of sleep. Use of this system, combined with existing genetic tools in Drosophila, will facilitate identification of novel sleep genes and neurons, with implications for understanding the relationship between sleep loss and metabolic disease.

neuroscience↗

bioRxiv2017

Hypocretin underlies the evolution of sleep loss in the Mexican cavefish

The duration of sleep varies dramatically between species, yet little is known about genetic bases or evolutionary factors driving this variation in behavior. The Mexican cavefish, Astyanax mexicanus, exists as surface populations that inhabit rivers, and multiple independently derived cave populations with convergent evolution on sleep loss. The number of Hypocretin/Orexin (HCRT)-positive hypothalamic neurons is increased significantly in cavefish, and HCRT is upregulated at both the transcript and protein levels. Pharmacological or genetic inhibition of HCRT signaling increases sleep duration in cavefish without affecting sleep in surface fish, suggesting enhanced HCRT signaling underlies sleep loss in cavefish. Ablation of the lateral line or starvation, manipulations that selectively promote sleep in cavefish, inhibit hcrt expression in cavefish while having little effect in surface fish. These findings provide the first evidence of genetic and neuronal changes that contribute to the evolution of sleep loss, and support a conserved role for HCRT in sleep regulation.

genomics↗