Publications

2023

Animals alter their behavior in manners that depend on environmental conditions as well as their developmental and metabolic states. For example, Celegans is quiescent during larval molts or during conditions of satiety. By contrast, worms enter an exploration state when removed from food. Sensory perception influences movement quiescence (defined as a lack of body movement), as well as the expression of additional locomotor states in Celegans that are associated with increased or reduced locomotion activity, such as roaming (exploration behavior) and dwelling (local search). Here we find that movement quiescence is enhanced, and exploration behavior is reduced in G protein-coupled receptor kinase grk-2 mutant animals. grk-2 was previously shown to act in chemosensation, locomotion, and egg-laying behaviors. Using neuron-specific rescuing experiments, we show that GRK-2 acts in multiple ciliated chemosensory neurons to control exploration behavior. grk-2 acts in opposite ways from the cGMP-dependent protein kinase gene egl-4 to control movement quiescence and exploration behavior. Analysis of mutants with defects in ciliated sensory neurons indicates that grk-2 and the cilium-structure mutants act in the same pathway to control exploration behavior. We find that GRK-2 controls exploration behavior in an opposite manner from the neuropeptide receptor NPR-1 and the neuropeptides FLP-1 and FLP-18. Finally, we show that secretion of the FLP-1 neuropeptide is negatively regulated by GRK-2 and that overexpression of FLP-1 reduces exploration behavior. These results define neurons and molecular pathways that modulate movement quiescence and exploration behavior.

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2022

The nematode Caenorhabditis elegans is one of the most widely studied organisms in biology due to its small size, rapid life cycle, and manipulable genetics. Research with C. elegans depends on labor-intensive and time-consuming manual procedures, imposing a major bottleneck for many studies, especially for those involving large numbers of animals. Here, we describe a general-purpose tool, WormPicker, a robotic system capable of performing complex genetic manipulations and other tasks by imaging, phenotyping, and transferring C. elegans on standard agar media. Our system uses a motorized stage to move an imaging system and a robotic arm over an array of agar plates. Machine vision tools identify animals and assay developmental stage, morphology, sex, expression of fluorescent reporters, and other phenotypes. Based on the results of these assays, the robotic arm selectively transfers individual animals using an electrically self-sterilized wire loop, with the aid of machine vision and electrical capacitance sensing. Automated C. elegans manipulation shows reliability and throughput comparable with standard manual methods. We developed software to enable the system to autonomously carry out complex protocols. To validate the effectiveness and versatility of our methods, we used the system to perform a collection of common C. elegansprocedures, including genetic crossing, genetic mapping, and genomic integration of a transgene. Our robotic system will accelerate C. elegans research and open possibilities for performing genetic and pharmacological screens that would be impractical using manual methods.

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The nematode C. elegans uses rhythmic muscle contractions (pumps) of the pharynx, a tubular feeding organ, to filter, transport, and crush food particles. A number of feeding mutants have been identified, including those with slow pharyngeal pumping rate, weak muscle contraction, defective muscle relaxation, and defective grinding of bacteria. Many aspects of these pharyngeal behavioral defects and how they affect pharyngeal function are not well understood. For example, the behavioral deficits underlying inefficient particle transport in ‘slippery’ mutants have been unclear. Here we use high speed video microscopy to describe pharyngeal pumping behaviors and particle transport in wild-type animals and in feeding mutants. Different ‘slippery’ mutants exhibit distinct defects including weak isthmus contraction, failure to trap particles in the anterior isthmus, and abnormal timing of contraction and relaxation in pharyngeal compartments. Our results show that multiple deficits in pharyngeal timing or contraction can cause defects in particle transport.

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2021

Gravity plays an important role in most life forms on Earth. Yet, a complete molecular understanding of sensing and responding to gravity is lacking. While there are anatomical differences among animals, there is a remarkable conservation across phylogeny at the molecular level. Caenorhabditis elegans is suitable for gene discovery approaches that may help identify molecular mechanisms of gravity sensing. It is unknown whether C. elegans can sense the direction of gravity.

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The interneurons ALA and RIS both regulate stress induced sleep in C. elegans but their roles in awake animal movement has been reported to differ. We describe the development of a motivated mobility-based assay that distinguishes between animals mutant for ALA function and those mutant for RIS function.

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Fatigue and sleepiness are widely observed but ill-understood responses to tissue injury. A new study in Caenorhabditis elegans illuminates how the innate immune system mediates injury-induced sleep, which may help in surviving the injury.

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Beyond the Symptom: The Biology of Fatigue took place on September 27-28, 2021.  This was a joint workshop organized by the Sleep Research Society and the Neurobiology of Fatigue Working Group of the NIH Blueprint Neuroscience Research Program, with support from the Blueprint Neuroscience Research Program.  The workshop was held via Zoom webinar from 10:00 am to 6:00 pm Eastern time on both days.

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2020

Many lines of evidence point to links between sleep regulation and energy homeostasis, but mechanisms underlying these connections are unknown. During Caenorhabditis elegans sleep, energetic stores are allocated to nonneural tasks with a resultant drop in the overall fat stores and energy charge. Mutants lacking KIN-29, the C. elegans homolog of a mammalian Salt-Inducible Kinase (SIK) that signals sleep pressure, have low ATP levels despite high-fat stores, indicating a defective response to cellular energy deficits. Liberating energy stores corrects adiposity and sleep defects of kin-29 mutants. kin-29 sleep and energy homeostasis roles map to a set of sensory neurons that act upstream of fat regulation as well as of central sleep-controlling neurons, suggesting hierarchical somatic/neural interactions regulating sleep and energy homeostasis. Genetic interaction between kin-29 and the histone deacetylase hda-4 coupled with subcellular localization studies indicate that KIN-29 acts in the nucleus to regulate sleep. We propose that KIN-29/SIK acts in nuclei of sensory neuroendocrine cells to transduce low cellular energy charge into the mobilization of energy stores, which in turn promotes sleep.

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Caenorhabditis elegans’ behavioral states, like those of other animals, are shaped by its immediate environment, its past experiences, and by internal factors. We here review the literature on C. elegansbehavioral states and their regulation. We discuss dwelling and roaming, local and global search, mate finding, sleep, and the interaction between internal metabolic states and behavior.

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Sleep and wakefulness are fundamental behavioral states of which the underlying molecular principles are becoming slowly elucidated. Transitions between these states require the coordination of multiple neurochemical and modulatory systems. In Caenorhabditis elegans sleep occurs during a larval transition stage called lethargus and is induced by somnogenic neuropeptides. Here, we identify two opposing neuropeptide/receptor signaling pathways: NLP-22 promotes behavioral quiescence, whereas NLP-2 promotes movement during lethargus, by signaling through gonadotropin-releasing hormone (GnRH) related receptors. Both NLP-2 and NLP-22 belong to the RPamide neuropeptide family and share sequence similarities with neuropeptides of the bilaterian GnRH, adipokinetic hormone (AKH) and corazonin family. RPamide neuropeptides dose-dependently activate the GnRH/AKH-like receptors GNRR-3 and GNRR-6 in a cellular receptor activation assay. In addition, nlp-22-induced locomotion quiescence requires the receptor gnrr-6. By contrast, wakefulness induced by nlp-2 overexpression is diminished by deletion of either gnrr-3 or gnrr-6. nlp-2 is expressed in a pair of olfactory AWA neurons and cycles with larval periodicity, as reported for nlp-22, which is expressed in RIA. Our data suggest that the somnogenic NLP-22 neuropeptide signals through GNRR-6, and that both GNRR-3 and GNRR-6 are required for the wake-promoting action of NLP-2 neuropeptides.

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An animal's behavioral and physiological response to stressors includes changes to its responses to stimuli. How such changes occur is not well understood. Here we describe a Caenorhabditis elegans quiescent behavior, post-response quiescence (PRQ), which is modulated by the C. elegans response to cellular stressors. Following an aversive mechanical or blue light stimulus, worms respond first by briefly moving, and then become more quiescent for a period lasting tens of seconds. PRQ occurs at low frequency in unstressed animals, but is more frequent in animals that have experienced cellular stress due to ultraviolet light exposure as well as in animals following overexpression of epidermal growth factor (EGF). PRQ requires the function of the carboxypeptidase EGL-21 and the calcium-activated protein for secretion (CAPS) UNC-31, suggesting it has a neuropeptidergic mechanism. Although PRQ requires the sleep-promoting neurons RIS and ALA, it is not accompanied by decreased arousability, and does not appear to be homeostatically regulated, suggesting that it is not a sleep state. PRQ represents a simple, tractable model for studying how neuromodulatory states like stress alter behavioral responses to stimuli.

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Complex extracellular structures exist throughout phylogeny, but the dynamics of their formation and dissolution are often opaque. One example is the pharyngeal grinder of the nematode Caenorhabditis elegans, an extracellular structure that ruptures bacteria during feeding. During each larval transition stage, called lethargus, the grinder is replaced with one of a larger size. Here, we characterize at the ultrastructural level the deconstruction of the larval grinder and the construction of the adult grinder during the fourth larval stage (L4)-to-adult transition. Early in L4 lethargus, pharyngeal muscle cells trans-differentiate from contractile to secretory cells, as evidenced by the appearance of clear and dense core vesicles and disruptions in sarcomere organization. This is followed, within minutes, by the dissolution of the L4 grinder and the formation and maturation of the adult grinder. Components of the nascent adult grinder are deposited basally, and are separated from the dissolving larval grinder by a visible apical layer. The complete grinder is a lamellated extracellular matrix comprised of five layers. Following grinder formation, pharyngeal muscle cells regain ultrastructural contractile properties, and muscle contractions resume. Our findings add to our understanding of how complex extracellular structures assemble and dissemble.

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Sleep is intertwined with metabolic function in vertebrates (Tsuneki et al. 2016; Herrera et al. 2017; Aalling et al. 2018; Wilms et al. 2018) and invertebrates (Kempf et al. 2019; Ki and Lim 2019; Yurgel et al. 2019; Grubbs et al. 2020), but the molecular underpinnings of this connection are not well understood. We recently reported that the salt inducible kinase (SIK) homolog KIN-29 is required in a subset of sensory neurons for the metabolic regulation of sleep in Caenorhabditis elegans (Grubbs et al. 2020). However, since our genetic manipulations made use of mutations that were present throughout the life of the animal, it is possible that the sleep defect of kin-29 mutants reflects a requirement for KIN-29 activity during development rather than during sleep. Indeed, because kin-29 is expressed throughout development as well as adulthood (Maduzia et al. 2005), and kin-29 mutants show altered expression of targets influential in larval development (Van Der Linden et al.2008), a role for kin-29 during development remained plausible. Distinguishing a developmental early role from a role during the time of sleep is important for constraining models for how KIN-29 regulates sleep.

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2019

During sleep, animals do not eat, reproduce or forage. Sleeping animals are vulnerable to predation. Yet, the persistence of sleep despite evolutionary pressures, and the deleterious effects of sleep deprivation, indicate that sleep serves a function or functions that cannot easily be bypassed. Recent research demonstrates sleep to be phylogenetically far more pervasive than previously appreciated; it is possible that the very first animals slept. Here, we give an overview of sleep across various species, with the aim of determining its original purpose. Sleep exists in animals without cephalized nervous systems and can be influenced by non-neuronal signals, including those associated with metabolic rhythms. Together, these observations support the notion that sleep serves metabolic functions in neural and non-neural tissues.

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Sleep is nearly universal among animals, yet remains poorly understood. Recent work has leveraged simple model organisms, such as Caenorhabditis elegans and Drosophila melanogaster larvae, to investigate the genetic and neural bases of sleep. However, manual methods of recording sleep behavior in these systems are labor intensive and low in throughput. To address these limitations, we developed methods for quantitative imaging of individual animals cultivated in custom microfabricated multiwell substrates, and used them to elucidate molecular mechanisms underlying sleep. Here, we describe the steps necessary to design, produce, and image these plates, as well as analyze the resulting behavioral data. We also describe approaches for experimentally manipulating sleep. Following these procedures, after ~2 h of experimental preparation, we are able to simultaneously image 24 C. elegans from the second larval stage to adult stages or 20 Drosophila larvae during the second instar life stage at a spatial resolution of 10 or 27 µm, respectively. Although this system has been optimized to measure activity and quiescence in Caenorhabditis larvae and adults and in Drosophila larvae, it can also be used to assess other behaviors over short or long periods. Moreover, with minor modifications, it can be adapted for the behavioral monitoring of a wide range of small animals.

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2018

Sleep during development is involved in refining brain circuitry, but a role for sleep in the earliest periods of nervous system elaboration, when neurons are first being born, has not been explored. Here we identify a sleep state in Drosophila larvae that coincides with a major wave of neurogenesis. Mechanisms controlling larval sleep are partially distinct from adult sleep: octopamine, the Drosophila analog of mammalian norepinephrine, is the major arousal neuromodulator in larvae, but dopamine is not required. Using real-time behavioral monitoring in a closed-loop sleep deprivation system, we find that sleep loss in larvae impairs cell division of neural progenitors. This work establishes a system uniquely suited for studying sleep during nascent periods, and demonstrates that sleep in early life regulates neural stem cell proliferation.

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Purpose: Fatigue is a common adverse effect among cancer patients undergoing external beam radiation therapy (EBRT), yet the underlying disease- and treatment-related factors influencing its development are poorly understood. We hypothesized that clinical, demographic, and treatment-related factors differentially affect fatigue and aimed to better characterize variables related to fatigue development in prostate cancer (PC) patients during EBRT.

Methods and materials: We identified a cohort of 681 patients with nonmetastatic PC undergoing a 6- to 9-week EBRT course. Patient fatigue scores (range, 0-3) were prospectively recorded by providers during treatment visits using standardized criteria. Clinical and demographic factors including age, race, EBRT details, disease staging, smoking status, comorbidities, urinary symptoms, employment status, weight, and concurrent medication use were assessed for their relationship to fatigue levels. Significant differences in fatigue severity by each variable at the beginning and end of EBRT were assessed by nonparametric means testing, and differences in the level of fatigue increase over the treatment course were assessed using an ordered logistic regression model.

Results: Significant increases in reported fatigue severity were seen in patients with age <60 years (P = .006), depressive symptoms (P < .001), and use of androgen deprivation therapy before radiation start (P = .04). In addition, the prescription of antiemetics before radiation start was associated with reduced fatigue severity (P = .03).

Conclusions: We identify factors associated with increased (young age, depressive symptoms, androgen deprivation therapy) and decreased (antiemetic prescription) fatigue in a large cohort of PC patients receiving EBRT. Continued investigation is needed to further elucidate clinical drivers and biological underpinnings of increased fatigue to guide potential interventions

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2017

During health, animal sleep is regulated by an internal clock and by the duration of prior wakefulness. During sickness, sleep is regulated by cytokines released from either peripheral cells or from cells within the nervous system. These cytokines regulate central nervous system neurons to induce sleep. Recent research in the invertebrates Caenorhabditis elegans and Drosophila melanogaster has led to new insights into the mechanism of sleep during sickness. Sickness is triggered by exposure to environments such as infection, heat, or ultraviolet light irradiation, all of which cause cellular stress. Epidermal growth factor is released from stressed cells and signals to activate central neuroendocrine cell(s). These neuron(s) release neuropeptides including those containing an amidated arginine(R)‐phenylalanine(F) motif at their C‐termini (RFamide peptides). Importantly, mechanisms regulating sickness sleep are partially distinct from those regulating healthy sleep. We will here review key findings that have elucidated the central neuroendocrine mechanism of sleep during sickness. Adaptive mechanisms employed in the control of sickness sleep may play a role in correcting cellular homeostasis after various insults. We speculate that these mechanisms may play a maladaptive role in human pathological conditions such as in the fatigue and anorexia associated with autoimmune diseases, with major depression, and with unexplained chronic fatigue.

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Stress-induced sleep (SIS) in Caenorhabditis elegans is important for restoration of cellular homeostasis and is a useful model to study the function and regulation of sleep. SIS is triggered when epidermal growth factor (EGF) activates the ALA neuron, which then releases neuropeptides to promote sleep. To further understand this behavior, we established a new model of SIS using irradiation by ultraviolet C (UVC) light. While UVC irradiation requires ALA signaling and leads to a sleep state similar to that induced by heat and other stressors, it does not induce the proteostatic stress seen with heat exposure. Based on the known genotoxic effects of UVC irradiation, we tested two genes, atl-1 and cep-1, which encode proteins that act in the DNA damage response pathway. Loss-of-function mutants of atl-1 had no defect in UVC-induced SIS but a partial loss-of-function mutant of cep-1gk138, had decreased movement quiescence following UVC irradiation. Germline ablation experiments and tissue-specific RNA interference experiments showed that cep-1 is required somatically in neurons for its effect on SIS. The cep-1(gk138) mutant suppressed body movement quiescence controlled by EGF, indicating that CEP-1 acts downstream or in parallel to ALA activation to promote quiescence in response to ultraviolet light.

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The roundworm C. elegans is a mainstay of aging research due to its short lifespan and easily manipulable genetics. Current, widely used methods for long-term measurement of C. elegans are limited by low throughput and the difficulty of performing longitudinal monitoring of aging phenotypes. Here we describe the WorMotel, a microfabricated device for long-term cultivation and automated longitudinal imaging of large numbers of C. elegans confined to individual wells. Using the WorMotel, we find that short-lived and long-lived strains exhibit patterns of behavioral decline that do not temporally scale between individuals or populations, but rather resemble the shortest and longest lived individuals in a wild type population. We also find that behavioral trajectories of worms subject to oxidative stress resemble trajectories observed during aging. Our method is a powerful and scalable tool for analysis of C. elegans behavior and aging.

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The roundworm Caenorhabditis elegans is widely used as a model for studying conserved pathways for fat storage, aging, and metabolism. The most broadly used methods for imaging fat in C. elegans require fixing and staining the animal. Here, we show that dark field images acquired through an ordinary light microscope can be used to estimate fat levels in worms. We define a metric based on the amount of light scattered per area, and show that this light scattering metric is strongly correlated with worm fat levels as measured by Oil Red O (ORO) staining across a wide variety of genetic backgrounds and feeding conditions. Dark field imaging requires no exogenous agents or chemical fixation, making it compatible with live worm imaging. Using our method, we track fat storage with high temporal resolution in developing larvae, and show that fat storage in the intestine increases in at least one burst during development.

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In response to environments that cause cellular stress, animals engage in sleep behavior that facilitates recovery from the stress. In Caenorhabditis elegans, stress-induced sleep(SIS) is regulated by cytokine activation of the ALA neuron, which releases FLP-13 neuropeptides characterized by an amidated arginine-phenylalanine (RFamide) C-terminus motif. By performing an unbiased genetic screen for mutants that impair the somnogenic effects of FLP-13 neuropeptides, we identified the gene dmsr-1, which encodes a G-protein coupled receptor similar to an insect RFamide receptor. DMSR-1 is activated by FLP-13 peptides in cell culture, is required for SIS in vivo, is expressed non-synaptically in several wake-promoting neurons, and likely couples to a Gi/o heterotrimeric G-protein. Our data expand our understanding of how a single neuroendocrine cell coordinates an organism-wide behavioral response, and suggest that similar signaling principles may function in other organisms to regulate sleep during sickness.

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2016

Sleeping animals do not move or feed and are less responsive. In Caenorhabditis elegans, a single neuron triggers sleep. A recent study shows that the neuron releases several neuropeptides — each with distinct sleep behavioral effects — to promote the collection of behaviors that is sleep.

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Rhythmic movements are ubiquitous in animal locomotion, feeding, and circulatory systems. In some systems, the muscle itself generates rhythmic contractions. In others, rhythms are generated by the nervous system or by interactions between the nervous system and muscles. In the nematode Caenorhabditis elegans, feeding occurs via rhythmic contractions (pumping) of the pharynx, a neuromuscular feeding organ. Here, we use pharmacology, optogenetics, genetics, and electrophysiology to investigate the roles of the nervous system and muscle in generating pharyngeal pumping. Hyperpolarization of the nervous system using a histamine-gated chloride channel abolishes pumping, and optogenetic stimulation of pharyngeal muscle in these animals causes abnormal contractions, demonstrating that normal pumping requires nervous system function. In mutants that pump slowly due to defective nervous system function, tonic muscle stimulation causes rapid pumping, suggesting tonic neurotransmitter release may regulate pumping. However, tonic cholinergic motor neuron stimulation, but not tonic muscle stimulation, triggers pumps that electrophysiologically resemble typical rapid pumps. This suggests that pharyngeal cholinergic motor neurons are normally rhythmically, and not tonically active. These results demonstrate that the pharynx generates a myogenic rhythm in the presence of tonically released acetylcholine, and suggest that the pharyngeal nervous system entrains contraction rate and timing through phasic neurotransmitter release.

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Nematodes such as Caenorhabditis elegans are heavier than water. When submerged in water, they settle to the bottom surface. Observations reveal that the animals do not lie flat on the bottom surface, but remain substantially suspended above the surface through continuous collisions with the surface, while maintaining their swimming gaits. Consequently, the swimming animals follow the bottom surface topography. When the bottom surface is inclined, the animals swim up or down along the incline. As the magnitude of the gravitational force can be easily estimated, this behaviour provides a convenient means to estimate the animal's propulsive thrust. The animals' tendency to follow the surface topography provides a means to control the swimmers' trajectories and direction of motion, which we demonstrate with a saw tooth-like ratchet that biases the animals to swim in a selected direction. The animals can also serve as surface topography probes since their residence time as a function of position provides information on surface features. Finally, we take advantage of surface following to construct a simple motility-based sorter that can sort animals based on genotype and state of health.

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The nematode Caenorhabditis elegans stops feeding and moving during a larval transition stage called lethargus and following exposure to cellular stressors. These behaviors have been termed “sleep-like states”. We argue that these behaviors should instead be called “sleep”. Sleep during lethargus is similar to sleep regulated by circadian timers in insects and mammals, and sleep in response to cellular stress is similar to sleep induced by sickness in other animals. Sleep in mammals and Drosophila shows molecular and functional conservation with C. elegans sleep. The simple neuroanatomy and powerful genetic tools of C. elegans have yielded insights into sleep regulation and hold great promise for future research into sleep regulation and function.

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2015

The new field of connectomics aims to obtain fine-grained anatomical connectivity data for vertebrate brains. A recent study highlights the types of experiments that will be necessary in order to draw conclusions about function from anatomical connectivity.

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Animal motility varies with genotype, disease, aging, and environmental conditions. In many studies, it is desirable to carry out high throughput motility-based sorting to isolate rare animals for, among other things, forward genetic screens to identify genetic pathways that regulate phenotypes of interest. Many commonly used screening processes are labor-intensive, lack sensitivity, and require extensive investigator training. Here, we describe a sensitive, high throughput, automated, motility-based method for sorting nematodes. Our method is implemented in a simple microfluidic device capable of sorting thousands of animals per hour per module, and is amenable to parallelism. The device successfully enriches for known C. elegans motility mutants. Furthermore, using this device, we isolate low-abundance mutants capable of suppressing the somnogenic effects of the flp-13 gene, which regulates C. elegans sleep. By performing genetic complementation tests, we demonstrate that our motility-based sorting device efficiently isolates mutants for the same gene identified by tedious visual inspection of behavior on an agar surface. Therefore, our motility-based sorter is capable of performing high throughput gene discovery approaches to investigate fundamental biological processes.

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Although small nematodes significantly impact human and animal health, agriculture, and ecology, little is known about the role of hydrodynamics in their life cycles. Using the nematode Caenorhabditis elegans as a model undulatory microswimmer, we have observed that animals are attracted to and swim along surfaces. The attraction to surfaces does not require mechanosensory neuron function. In dilute swarms, swimmers aggregate near surfaces. Using resistive force-based theory, symmetry arguments, and direct hydrodynamic simulations, we demonstrate that forces resulting from the interaction between the swimmer-induced flow field and a nearby surface cause a short-range hydrodynamic torque that stirs the swimmers towards the surface. When combined with steric forces, this causes locomotion along the surface. This surface attraction may affect nematode mate and food finding behaviour and, in the case of parasitic nematodes, may facilitate host penetration. Surface attraction must be accounted for when studying animals' responses to various stimuli, and suggests means of controlling undulatory microswimmers.

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Nearly half a century of neurobiological research using the nematode Caenorahbitis elegans has produced a remarkably detailed understanding of how genotype controls behavioral phenotype. However, the role of simple physical forces in regulating behavior has been understudied. Here, we review our recent observations of 3 behaviors of C. elegans suspended in solution that can be fully explained by the laws of mechanics. These behaviors are bordertaxis, the attraction toward solid surfaces; positive rheotaxis, the propensity to swim against the flow; and synchrophilia, the tendency of animals when close to each other to synchronize their gaits. Although these 3 behaviors are not directly regulated by the animal's nervous system, bordertaxis and rheotaxis require the animal to have an undulating gait. We conjecture that these behaviors are advantageous to the animals, and thus evolution may have favored microorganism that swim with an undulating gait.

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Electrophysiological recordings have enabled identification of physiologically distinct yet behaviorally similar states of mammalian sleep. In contrast, sleep in nonmammals has generally been identified behaviorally and therefore regarded as a physiologically uniform state characterized by quiescence of feeding and locomotion, reduced responsiveness, and rapid reversibility. The nematode Caenorhabditis elegans displays sleep-like quiescent behavior under two conditions: developmentally timed quiescence (DTQ) occurs during larval transitions, and stress-induced quiescence (SIQ) occurs in response to exposure to cellular stressors. Behaviorally, DTQ and SIQ appear identical. Here, we use optogenetic manipulations of neuronal and muscular activity, pharmacology, and genetic perturbations to uncover circuit and molecular mechanisms of DTQ and SIQ. We find that locomotion quiescence induced by DTQ- and SIQ-associated neuropeptides occurs via their action on the nervous system, although their neuronal target(s) and/or molecular mechanisms likely differ. Feeding quiescence during DTQ results from a loss of pharyngeal muscle excitability, whereas feeding quiescence during SIQ results from a loss of excitability in the nervous system. Together these results indicate that, as in mammals, quiescence is subserved by different mechanisms during distinct sleep-like states in C. elegans.

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Neuropeptides signal through G-protein coupled receptors (GPCRs) to regulate a broad array of animal behaviors and physiological processes. The Caenorhabditis elegans genome encodes approximately 100 predicted neuropeptide receptor GPCRs, but in vivo roles for only a few have been identified. We describe here a role for the GPCR FRPR-4 in the regulation of behavioral quiescence and locomotive posture. FRPR-4 is activated in cell culture by several neuropeptides with an amidated isoleucine-arginine-phenylalanine (IRF) motif or an amidated valine-arginine-phenylalanine (VRF) motif at their carboxy termini, including those encoded by the gene flp-13. Loss of frpr-4 function results in a minor feeding quiescence defect after heat-induced cellular stress. Overexpression of frpr-4 induces quiescence of locomotion and feeding as well as an exaggerated body bend posture. The exaggerated body bend posture requires the gene flp-13. While frpr-4 is expressed broadly, selective overexpression of frpr-4 in the proprioceptive DVA neurons results in exaggerated body bends that require flp-13 in the ALA neuron. Our results suggest that FLP-13 and other neuropeptides signal through FRPR-4 and other receptors to regulate locomotion posture and behavioral quiescence.

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Enhanced sleep in response to cellular stress is a conserved adaptive behavior across multiple species, but the mechanism of this process is poorly understood. Drosophila melanogaster increases sleep following exposure to septic or aseptic injury, and Caenorhabditis elegans displays sleep-like quiescence following exposure to high temperatures that stress cells. We show here that, similar to C. elegans, Drosophila responds to heat stress with an increase in sleep. In contrast to Drosophila infection-induced sleep, heat-induced sleep is not sensitive to the time-of-day of the heat pulse. Moreover, the sleep response to heat stress does not require Relish, the NFκB transcription factor that is necessary for infection-induced sleep, indicating that sleep is induced by multiple mechanisms from different stress modalities. We identify a sleep-regulating role for a signaling pathway involving FMRFamide neuropeptides and their receptor FR. Animals mutant for either FMRFamide or for the FMRFamide receptor (FR) have a reduced recovery sleep in response to heat stress. FR mutants, in addition, show reduced sleep responses following infection with Serratia marcescens, and succumb to infection at a faster rate than wild-type controls. Together, these findings support the hypothesis that FMRFamide and its receptor promote an adaptive increase in sleep following stress. Because an FMRFamide-like neuropeptide plays a similar role in C. elegans, we propose that FRMFamide neuropeptide signaling is an ancient regulator of recovery sleep which occurs in response to cellular stress.

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Caenorhabditis elegans is the simplest animal shown to sleep. It sleeps during lethargus, a larval transition stage. Behavior during lethargus has the sleep properties of a specific quiescent posture and elevated arousal threshold that are reversible to strong stimulation and of increased sleep drive following sleep deprivation. Genetic similarities between sleep regulation during C. elegans lethargus and sleep regulation in other animals point to a sleep state that was an evolutionarily ancestor to sleep both in C. elegans and other animals. Recent publications have shed light on key questions in sleep biology: First, How is sleep regulated? Second, How is sensory information gated during sleep? Third, How is sleep homeostasis mediated? Fourth, What is the core function of sleep?

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The new field of connectomics aims to obtain fine-grained anatomical connectivity data for vertebrate brains. A recent study highlights the types of experiments that will be necessary in order to draw conclusions about function from anatomical connectivity.

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2014

Collective motion is observed in swarms of swimmers of various sizes, ranging from self-propelled nanoparticles to fish. The mechanisms that govern interactions among individuals are debated, and vary from one species to another. Although the interactions among relatively large animals, such as fish, are controlled by their nervous systems, the interactions among microorganisms, which lack nervous systems, are controlled through physical and chemical pathways. Little is known, however, regarding the mechanism of collective movements in microscopic organisms with nervous systems. To attempt to remedy this, we studied collective swimming behavior in the nematode Caenorhabditis elegans, a microorganism with a compact nervous system. We evaluated the contributions of hydrodynamic forces, contact forces, and mechanosensory input to the interactions among individuals. We devised an experiment to examine pair interactions as a function of the distance between the animals and observed that gait synchronization occurred only when the animals were in close proximity, independent of genes required for mechanosensation. Our measurements and simulations indicate that steric hindrance is the dominant factor responsible for motion synchronization in C. elegans, and that hydrodynamic interactions and genotype do not play a significant role. We infer that a similar mechanism may apply to other microscopic swimming organisms and self-propelled particles.

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Bacteriophytochromes sense light in the near-infrared window, the spectral region where absorption by mammalian tissues is minimal, and their chromophore, biliverdin IXα, is naturally present in animal cells. These properties make bacteriophytochromes particularly attractive for optogenetic applications. However, the lack of understanding of how light-induced conformational changes control output activities has hindered engineering of bacteriophytochrome-based optogenetic tools. Many bacteriophytochromes function as homodimeric enzymes, in which light-induced conformational changes are transferred via α-helical linkers to the rigid output domains. We hypothesized that heterologous output domains requiring homodimerization can be fused to the photosensory modules of bacteriophytochromes to generate light-activated fusions. Here, we tested this hypothesis by engineering adenylate cyclases regulated by light in the near-infrared spectral window using the photosensory module of the Rhodobacter sphaeroides bacteriophytochrome BphG1 and the adenylate cyclase domain from Nostoc sp. CyaB1. We engineered several light-activated fusion proteins that differed from each other by approximately one or two α-helical turns, suggesting that positioning of the output domains in the same phase of the helix is important for light-dependent activity. Extensive mutagenesis of one of these fusions resulted in an adenylate cyclase with a sixfold photodynamic range. Additional mutagenesis produced an enzyme with a more stable photoactivated state. When expressed in cholinergic neurons in Caenorhabditis elegans, the engineered adenylate cyclase affected worm behavior in a light-dependent manner. The insights derived from this study can be applied to the engineering of other homodimeric bacteriophytochromes, which will further expand the optogenetic toolset.

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Background: The nematode Caenorhabditis elegans is widely used as a model for understanding the neuronal and genetic bases of behavior. Recent studies have required longitudinal assessment of individual animal's behavior over extended periods.

New method: Here we present a technique for automated monitoring of multiple worms for several days. Our method uses an array of plano-concave glass wells containing standard agar media. The concave well geometry allows worms to be imaged even at the edge of the agar surface and prevents them from burrowing under the agar. We transfer one worm or embryo into each well, and perform imaging of the array of wells using a single camera. Machine vision software is used to quantify size, activity, and/or fluorescence of each worm over time.

Results: We demonstrate the utility of our method in two applications: (1) quantifying behavioral quiescence and developmental rate in wild-type and mutant animals, and (2) characterizing differences in mating behavior between two C. elegans strains.

Comparison with existing method(s): Current techniques for tracking behavior in identified worms are generally restricted to imaging either single animals or have not been shown to work with arbitrary developmental stages; many are also technically complex. Our system works with up to 24 animals of any stages and is technically simple.

Conclusions: Our multi-well imaging method is a powerful tool for quantification of long-term behavioral phenotypes in C. elegans.

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A type III unattended sleep study performed on an 85-year-old man during his inpatient hospitalization after cardiac arrest reveals an increase in the average apnea length from 27.9 to 52.2 seconds after a routine therapy is initiated. The longest apnea after the intervention is 205.4 seconds. By demonstrating an adverse impact of a medical intervention, this case highlights the potential benefits and risks of a routine medical therapy.

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Among the most important decisions an animal makes is whether to engage in active movement and feeding behavior or to become quiescent. The molecular signaling mechanisms underlying this decision remain largely unknown. The nematode Caenorhabditis elegans displays sleep-like quiescence following exposures that result in cellular stress. The neurosecretory ALA neuron is required for this stress-induced recovery quiescence, but the mechanisms by which ALA induces quiescence have been unknown. We report here that quiescence induced by heat stress requires ALA depolarization and release of FMRFamide-like neuropeptides encoded by the flp-13 gene. Optogenetic activation of ALA reduces feeding and locomotion in a FLP-13-dependent manner. Overexpression of flp-13 is sufficient to induce quiescent behavior during normally active periods. We have here identified a major biological role for FMRFamide-like neuropeptides in nematodes, and we suggest that they may function in a similar capacity in other organisms.

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Degenerate networks, in which structurally distinct elements can perform the same function or yield the same output, are ubiquitous in biology. Degeneracy contributes to the robustness and adaptability of networks in varied environmental and evolutionary contexts. However, how degenerate neural networks regulate behavior in vivo is poorly understood, especially at the genetic level. Here, we identify degenerate neural and genetic mechanisms that underlie excitation of the pharynx (feeding organ) in the nematode Caenorhabditis elegans using cell-specific optogenetic excitation and inhibition. We show that the pharyngeal neurons MC, M2, M4, and I1 form multiple direct and indirect excitatory pathways in a robust network for control of pharyngeal pumping. I1 excites pumping via MC and M2 in a state-dependent manner. We identify nicotinic and muscarinic receptors through which the pharyngeal network regulates feeding rate. These results identify two different mechanisms by which degeneracy is manifest in a neural circuit in vivo.

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Sleep is recognized to be ancient in origin, with vertebrates and invertebrates experiencing behaviorally quiescent states that are regulated by conserved genetic mechanisms. Despite its conservation throughout phylogeny, the function of sleep remains debated. Hypotheses for the purpose of sleep include nervous-system-specific functions such as modulation of synaptic strength and clearance of metabolites from the brain, as well as more generalized cellular functions such as energy conservation and macromolecule biosynthesis. These models are supported by the identification of synaptic and metabolic processes that are perturbed during prolonged wakefulness. It remains to be seen whether perturbations of cellular homeostasis in turn drive sleep. Here we show that under conditions of cellular stress, including noxious heat, cold, hypertonicity, and tissue damage, the nematode Caenorhabditis elegans engages a behavioral quiescence program. The stress-induced quiescent state displays properties of sleep and is dependent on the ALA neuron, which mediates the conserved soporific effect of epidermal growth factor (EGF) ligand overexpression. We characterize heat-induced quiescence in detail and show that it is indeed dependent on components of EGF signaling, providing physiological relevance to the behavioral effects of EGF family ligands. We find that after noxious heat exposure, quiescence-defective animals show elevated expression of cellular stress reporter genes and are impaired for survival, demonstrating the benefit of stress-induced behavioral quiescence. These data provide evidence that cellular stress can induce a protective sleep-like state in C. elegans and suggest that a deeply conserved function of sleep is to mitigate disruptions of cellular homeostasis.

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The nematode Caenorhabditis (C.) elegans, a long time work horse for behavioral genetic studies of locomotion, has recently been studied for quiescent behavior. Methods previously established for the study of C. elegans locomotion are not well-suited for the study of quiescent behavior. We describe in detail two computer vision approaches to distinguish quiescent from movement bouts focusing on the behavioral quiescence that occurs during fourth larval stage lethargus, a transition stage between the larva and the adult. The first is the frame subtraction method, which consists of subtraction of temporally adjacent images as a sensitive way to detect motion. The second, which is more computationally intensive, is the posture analysis method, which consists of analysis of the rate of local angle change of the animal's body. Quiescence measurements should be done continuously while minimizing sensory perturbation of the animal.

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In molting animals, a cuticular extracellular matrix forms the first barrier to infection and other environmental insults. In the nematode Caenorhabditis elegans there are two types of cuticle: a well-studied collagenous cuticle lines the body, and a poorly-understood chitinous cuticle lines the pharynx. In the posterior end of the pharynx is the grinder, a tooth-like cuticular specialization that crushes food prior to transport to the intestine for digestion. We here show that the grinder increases in size only during the molt. To gain molecular insight into the structure of the grinder and pharyngeal cuticle, we performed a microarray analysis to identify mRNAs increased during the molt. We found strong transcriptional induction during the molt of 12 of 15 previously identified abu genes encoding Prion-like (P) glutamine (Q) and asparagine (N) rich PQN proteins, as well as 15 additional genes encoding closely related PQN proteins. abu/pqn genes, which we name the abu/pqn paralog group (APPG) genes, were expressed in pharyngeal cells and the proteins encoded by two APPG genes we tested localized to the pharyngeal cuticle. Deleting the APPG gene abu-14 caused abnormal pharyngeal cuticular structures and knocking down other APPG genes resulted in abnormal cuticular function. We propose that APPG proteins promote the assembly and function of a unique cuticular structure. The strong developmental regulation of the APPG genes raises the possibility that such genes would be identified in transcriptional profiling experiments in which the animals' developmental stage is not precisely staged.

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2013

Caenorhabditis elegans is the simplest animal shown to sleep. It sleeps during lethargus, a larval transition stage. Behavior during lethargus has the sleep properties of a specific quiescent posture and elevated arousal threshold that are reversible to strong stimulation and of increased sleep drive following sleep deprivation. Genetic similarities between sleep regulation during C. elegans lethargus and sleep regulation in other animals point to a sleep state that was an evolutionarily ancestor to sleep both in C. elegans and other animals. Recent publications have shed light on key questions in sleep biology: First, How is sleep regulated? Second, How is sensory information gated during sleep? Third, How is sleep homeostasis mediated? Fourth, What is the core function of sleep?

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Sleep homeostasis, which refers to the maintenance of sleep amount or depth following sleep deprivation, indicates that sleep and sleep-like states serve fundamental functions that cannot be bypassed [1]. Homeostasis of sleep-like behavior is observed during C. elegans lethargus, a 2-3 hr behavioral quiescent period that occurs during larval state transitions [2]. Here, we report a role for DAF-16/FOXO, a transcription factor that is active under conditions of stress [3], in the response to deprivation of lethargus quiescence. Forced locomotion during lethargus results in nuclear translocation of DAF-16. The formation of dauer larvae, a developmental state promoted by daf-16, is increased in response to quiescence deprivation. daf-16 mutants show an impaired homeostatic response to deprivation of lethargus quiescence and are hypersensitive to the lethal effects of forced locomotion during lethargus. DAF-16 expression in muscle cells, but not in neurons, is sufficient to restore a homeostatic response to deprivation of quiescence, pointing to a role for muscle in sleep homeostasis. These findings are relevant to clinical observations of altered metabolic signaling in response to sleep deprivation and suggest that these signaling pathways may act in nonneuronal tissue to regulate sleep behaviors.

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Neuropeptides have central roles in the regulation of homoeostatic behaviours such as sleep and feeding. Caenorhabditis elegans displays sleep-like quiescence of locomotion and feeding during a larval transition stage called lethargus and feeds during active larval and adult stages. Here we show that the neuropeptide NLP-22 is a regulator of Caenorhabditis elegans sleep-like quiescence observed during lethargus. nlp-22 shows cyclical mRNA expression in synchrony with lethargus; it is regulated by LIN-42, an orthologue of the core circadian protein PERIOD; and it is expressed solely in the two RIA interneurons. nlp-22 and the RIA interneurons are required for normal lethargus quiescence, and forced expression of nlp-22 during active stages causes anachronistic locomotion and feeding quiescence. Optogenetic stimulation of the RIA interneurons has a movement-promoting effect, demonstrating functional complexity in a single-neuron type. Our work defines a quiescence-regulating role for NLP-22 and expands our knowledge of the neural circuitry controlling Caenorhabditis elegans behavioural quiescence.

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Study objectives: To develop a method, called Caenorhabditis-in-Drop (CiD), encapsulating single worms in aqueous drops, for parallel analysis of behavioral quiescence in C. elegans nematodes.

Design: We designed, constructed, and tested a device that houses an array of aqueous droplets laden with individual worms. The droplets are separated and covered by immiscible, biocompatible oil. We modeled gas exchange across the aqueous/oil interface and tested the viability of the encapsulated animals. We studied the behavior of wild-type animals; of animals with a loss of function mutation in the cGMP-dependent protein kinase gene egl-4; of animals with a loss of function mutation in the gene kin-2, which encodes a cAMP-dependent protein kinase A regulatory subunit; of animals with a gain-of-function mutation in the gene acy-1, which encodes an adenylate cyclase; and of animals that express high levels of the EGF protein encoded by lin-3.

Measurements and results: We used CiD to simultaneously monitor the behavior of 24 worms, a nearly 5-fold improvement over the prior best methodology. In support of our gas exchange models, we found that worms remain viable on the chip for 4 days, past the 12-h period needed for observation, but show reduced longevity to that measured on an agar surface. Measurements of duration of lethargus quiescence and total leth-argus quiescence showed reduced amounts as well as reduced variability relative to prior methods. There was reduced lethargus quiescence in animals that were mutant for kin-2 and for acy-1, supporting a wake-promoting effect of PKA in C. elegans, but no change in lethargus quiescence in egl-4 mutants. There was increased quiescence in animals that expressed kin-2 in the nervous system or over-expressed EGF.

Conclusions: CiD is useful for the analysis of behavioral quiescence during lethargus as well as during the adult stage C. elegans. The method is expandable to parallel simultaneous monitoring of hundreds of animals and for other studies of long-term behavior. Using this method, we were successful in measuring, for the first time, quiescence in kin-2(ce179) and in acy-2(ce2) mutants, which are hyperactive. Our observations also highlight the impact of environmental conditions on quiescent behavior and show that longevity is reduced in CiD in comparison to agar surfaces.

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2011

Sleep disorders tend to be complex diseases, with multiple genes and environmental factors interacting to contribute to phenotypes. Our understanding of the genetic underpinnings of sleep disorders has benefited from recent genome-wide association studies (GWAS). We review principles underlying GWAS and discuss recent GWAS for restless legs syndrome and narcolepsy. These studies have identified four gene variants associated with restless legs syndrome (BTBD9, MEIS1, MAP2K5/LBXCOR1, and PTPRD) and two variants associated with narcolepsy (one in the T-cell receptor α locus and another between CPT1B and CHKB). These discoveries have opened new lines of research to understand the pathophysiology of these disorders. In addition to GWAS, we expect that new technologies, such as next-generation sequencing, and continued use of animal models will provide important contributions to our understanding of the genetic basis of sleep disorders.

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We demonstrate for the first time the dielectrophoretic trapping and manipulation of a whole animal, the nematode Caenorhabditis elegans. We studied the effect of the electric field on the nematode as a function of field intensity and frequency. We identified a range of electric field intensities and frequencies that trap worms without apparent adverse effect on their viability. Worms tethered by dielectrophoresis (DEP) exhibit behavioral responses to blue light, indicating that at least some of the nervous system functions are unimpaired by the electrical field. DEP is useful to dynamically tether nematodes, sort nematodes according to size, and separate dead worms from live ones.

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C. elegans is an attractive animal model for biomedical research and for studying animal locomotion. Electric fields offer a means to control nematodes’ motion and to apply remote, non-contact forces. We study the behavior of nematodes as a function of electric field intensity and frequency. The deliberate attraction of nematodes towards the negative pole of an electric field has been known for some time and dubbed electrotaxis.1 More recently, we have demonstrated that nematodes can be polarized with electric fields.2 

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Approximately one-fourth of the neurons in Caenorhabditis elegans adults are born during larval development, indicating tremendous plasticity in larval nervous system structure. Larval development shows cyclical expression of sleep-like quiescent behavior during lethargus periods, which occur at larval stage transitions. We studied plasticity at the neuromuscular junction during lethargus using the acetylcholinesterase inhibitor aldicarb. The rate of animal contraction when exposed to aldicarb is controlled by the balance between excitatory cholinergic and inhibitory GABAergic input on the muscle. During lethargus, there is an accelerated rate of contraction on aldicarb. Mutant analysis and optogenetic studies reveal that GABAergic synaptic transmission is reduced during lethargus. Worms in lethargus show partial resistance to GABA(A) receptor agonists, indicating that postsynaptic mechanisms contribute to lethargus-dependent plasticity. Using genetic manipulations that separate the quiescent state from the developmental stage, we show that the synaptic plasticity is dependent on developmental time and not on the behavioral state of the animal. We propose that the synaptic plasticity regulated by a developmental clock in C. elegans is analogous to synaptic plasticity regulated by the circadian clock in other species.

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The molecular pathways regulating sleep remain poorly understood. Studies in this issue demonstrate a role for Notch signaling in sleep regulation as well as stress response in both Caenorhabditis elegans and Drosophila.

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Among the most notable advances to the sleep research field in the past decade has been the addition of several new model systems that are amenable to genetic analysis. These include the fruit fly Drosophila melanogaster, the round worm Caenorhabditis elegans, and the Zebrafish Danio rerio. Though this field is young, research in these model systems has already yielded important contributions into our understanding of the regulation and function of sleep and sleep-like states. Looking forward, these model systems hold promise to contribute for many years to the discovery of new sleep regulators, to understanding the function of genes discovered in human genetic research, and to testing hypotheses regarding the function of sleep.

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2008

The past 10 years have seen new approaches to elucidating genetic pathways regulating sleep. The emerging theme is that sleep-like states are conserved in evolution, with similar signaling pathways playing a role in animals as distantly related as flies and humans. We review the evidence for the presence of sleep states in non-mammalian species including zebrafish (Danio rerio), fruitflies (Drosophila melanogaster) and roundworms (Caenorhabditis elegans). We describe conserved sleep-regulatory molecular pathways with a focus on cAMP and epidermal growth factor signaling; neurotransmitters with conserved effects on sleep and wake regulation, including dopamine and GABA; and a conserved molecular response to sleep deprivation involving the chaperone protein BiP/GRP78.

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Despite the prevalence of obesity and its related diseases, the signaling pathways for appetite control and satiety are not clearly understood. Here we report C. elegans quiescence behavior, a cessation of food intake and movement that is possibly a result of satiety. C. elegans quiescence shares several characteristics of satiety in mammals. It is induced by high-quality food, it requires nutritional signals from the intestine, and it depends on prior feeding history: fasting enhances quiescence after refeeding. During refeeding after fasting, quiescence is evoked, causing gradual inhibition of food intake and movement, mimicking the behavioral sequence of satiety in mammals. Based on these similarities, we propose that quiescence results from satiety. This hypothesized satiety-induced quiescence is regulated by peptide signals such as insulin and TGF-beta. The EGL-4 cGMP-dependent protein kinase functions downstream of insulin and TGF-beta in sensory neurons including ASI to control quiescence in response to food intake.

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Study objectives: To use video to determine the accuracy of the infrared beam-splitting method for measuring sleep in Drosophila and to determine the effect of time of day, sex, genotype, and age on sleep measurements.

Design: A digital image analysis method based on frame subtraction principle was developed to distinguish a quiescent from a moving fly. Data obtained using this method were compared with data obtained using the Drosophila Activity Monitoring System (DAMS). The location of the fly was identified based on its centroid location in the subtracted images.

Measurements and results: The error associated with the identification of total sleep using DAMS ranged from 7% to 95% and depended on genotype, sex, age, and time of day. The degree of the total sleep error was dependent on genotype during the daytime (P < 0.001) and was dependent on age during both the daytime and the nighttime (P < 0.001 for both). The DAMS method overestimated sleep bout duration during both the day and night, and the degree of these errors was genotype dependent (P < 0.001). Brief movements that occur during sleep bouts can be accurately identified using video. Both video and DAMS detected a homeostatic response to sleep deprivation.

Conclusions: Video digital analysis is more accurate than DAMS in fly sleep measurements. In particular, conclusions drawn from DAMS measurements regarding daytime sleep and sleep architecture should be made with caution. Video analysis also permits the assessment of fly position and brief movements during sleep.

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There are fundamental similarities between sleep in mammals and quiescence in the arthropod Drosophila melanogaster, suggesting that sleep-like states are evolutionarily ancient. The nematode Caenorhabditis elegans also has a quiescent behavioural state during a period called lethargus, which occurs before each of the four moults. Like sleep, lethargus maintains a constant temporal relationship with the expression of the C. elegans Period homologue LIN-42 (ref. 5). Here we show that quiescence associated with lethargus has the additional sleep-like properties of reversibility, reduced responsiveness and homeostasis. We identify the cGMP-dependent protein kinase (PKG) gene egl-4 as a regulator of sleep-like behaviour, and show that egl-4 functions in sensory neurons to promote the C. elegans sleep-like state. Conserved effects on sleep-like behaviour of homologous genes in C. elegans and Drosophila suggest a common genetic regulation of sleep-like states in arthropods and nematodes. Our results indicate that C. elegans is a suitable model system for the study of sleep regulation. The association of this C. elegans sleep-like state with developmental changes that occur with larval moults suggests that sleep may have evolved to allow for developmental changes.

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2006

Sleep disorders arise by an interaction between the environment and the genetic makeup of the individual but the relative contribution of nature and nurture varies with diseases. At one extreme are the disorders with simple Mendelian patterns of inheritance such as familial advanced sleep phase syndrome, and at the other extreme are diseases such as insomnia, which can be associated with a multitude of medical and psychiatric conditions. In this article, we review data on the relative contribution of genetic and environmental factors in the pathogenesis of various sleep disorders. The understanding of many of these disorders has been advanced by the study of sleep and circadian rhythms in model laboratory organisms. We summarize this model system research and how it relates to human sleep disorders. The current challenge in this field is the identification of susceptibility genetic loci for complex diseases such as obstructive sleep apnea. We anticipate such identification will increase our ability to assess risk for disease before symptom onset and by doing so will shift the focus from treatment to prevention of disease.

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cGMP-dependent protein kinases are key intracellular transducers of cell signaling. We identified a novel dominant mutation in the C. elegans egl-4 cGMP-dependent protein kinase (PKG) and show that this mutation causes increased normal gene activity although it is associated with a reduced EGL-4 protein level. Prior phenotypic analyses of this gain-of-function mutant demonstrated a reduced longevity and a reduced feeding behavior when the animals were left unperturbed. We characterize several additional phenotypes caused by increased gene activity of egl-4. These phenotypes include a small body size, reduced locomotion in the presence of food, a pale intestine, increased intestinal fat storage, and a decreased propensity to form dauer larvae. The multiple phenotypes of egl-4 dominant mutants are consistent with an instructive signaling role of PKG to control many aspects of animal physiology. This is among the first reported gain-of-function mutations in this enzyme of central physiological importance. In a genetic screen we have identified extragenic suppressors of this gain-of-function mutant. Thus, this mutant promises to be a useful tool for identifying downstream targets of PKG.

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The functions of sleep and what controls it remain unanswered biological questions. According to the two-process model, a circadian process and a homeostatic process interact to regulate sleep. While progress has been made in understanding the molecular and cellular functions of the circadian process, the mechanisms of the homeostatic process remain undiscovered. We use the recently established sleep model system organism Drosophila melanogaster to examine dynamic changes in gene expression during sleep and during prolonged wakefulness in the brain. Our experimental design controls for circadian processes by killing animals at three matched time points from the beginning of the consolidated rest period [Zeitgeber time (ZT) 14)] under two conditions, sleep deprived and spontaneously sleeping. Using ANOVA at a false discovery rate of 5%, we have identified 252 genes that were differentially expressed between sleep-deprived and control groups in the Drosophila brain. Using linear trends analysis, we have separated the significant differentially expressed genes into nine temporal expression patterns relative to a common anchor point (ZT 14). The most common expression pattern is a decrease during extended wakefulness but no change during spontaneous sleep (n = 114). Genes in this category were involved in protein production (n = 47), calcium homeostasis, and membrane excitability (n = 5). Multiple mechanisms, therefore, act to limit wakefulness. In addition, by studying the effects of the mechanical stimulus used in our deprivation studies during the period when the animals are predominantly active, we provide evidence for a previously unappreciated role for the Drosophila immune system in the brain response to stress.

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The ability to orient oneself in response to environmental cues is crucial to the survival and function of diverse organisms. One such orientation behavior is the alignment of aquatic organisms with (negative rheotaxis) or against (positive rheotaxis) fluid current. The questions of whether low-Reynolds-number, undulatory swimmers, such as worms, rheotax and whether rheotaxis is a deliberate or an involuntary response to mechanical forces have been the subject of conflicting reports. To address these questions, we use Caenorhabditis elegans as a model undulatory swimmer and examine, in experiment and theory, the orientation of C. elegans in the presence of flow. We find that when close to a stationary surface the animal aligns itself against the direction of the flow. We elucidate for the first time to our knowledge the mechanisms of rheotaxis in worms and show that rheotaxis can be explained solely by mechanical forces and does not require sensory input or deliberate action. The interaction between the flow field induced by the swimmer and a nearby surface causes the swimmer to tilt toward the surface and the velocity gradient associated with the flow rotates the animal to face upstream. Fluid mechanical computer simulations faithfully mimic the behavior observed in experiments, supporting the notion that rheotaxis behavior can be fully explained by hydrodynamics. Our study highlights the important role of hydrodynamics in the behavior of small undulating swimmers and may assist in developing control strategies to affect the animals' life cycles.

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2005

Objective: To describe seizure phenotypes associated with the hyperinsulinism/hyperammonemia syndrome (HI/HA), which is caused by gain of function mutations in the enzyme glutamate dehydrogenase (GDH).

Study design: A retrospective review of records of 14 patients with HI/HA.

Results: Nine patients had seizures as the first symptom of HI/HA, and six had seizures in the absence of hypoglycemia. No electroencephalogram (EEG) background abnormalities were identified. In four patients, EEG recordings during seizures in the setting of normal blood glucose contained generalized epileptiform discharges. EEGs of three of these patients showed 0.5- to 2-second generalized irregular spike-and-wave discharge at 3 to 6 Hz corresponding to eye blinks, eye rolling, or staring. The EEG of the fourth patient consisted of 20 seconds of generalized regular spike-and-wave discharge at 3 Hz in the clinical context of staring and unresponsiveness. In two patients, seizure control worsened with carbamezapine or oxcarbezapine treatment.

Conclusions: In patients with HI/HA, generalized seizures are common and can occur in the absence of hypoglycemia. The drugs carbamazepine and oxcarbazepine should be used with caution for treatment. Pathogenesis of epilepsy in these patients may be related to effects of GDH mutations in the brain, perhaps in combination with effects of recurrent hypoglycemia and chronic hyperammonemia.

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2004

Mutations in eat-2 and eat-18 cause the same defect in C. elegans feeding behavior: the pharynx is unable to pump rapidly in the presence of food. EAT-2 is a nicotinic acetylcholine receptor subunit that functions in the pharyngeal muscle. It is localized to the synapse between pharyngeal muscle and the main pharyngeal excitatory motor neuron MC, and it is required for MC stimulation of pharyngeal muscle. eat-18 encodes a small protein that has no homology to previously characterized proteins. It has a single transmembrane domain and a short extracellular region. Allele-specific genetic interactions between eat-2 and eat-18 suggest that EAT-18 interacts physically with the EAT-2 receptor. While eat-2 appears to be required specifically for MC neurotransmission, eat-18 also appears to be required for the function of other nicotinic receptors in the pharynx. In eat-18 mutants, the gross localization of EAT-2 at the MC synapse is normal, suggesting that it is not required for trafficking. These data indicate that eat-18 could be a novel component of the pharyngeal nicotinic receptor.

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2001

A 25-year-old woman with maple syrup urine disease (MSUD) developed generalized weakness over 1 week. She had severe leg and moderate arm weakness, areflexia, and distal sensory loss. Plasma branched-chain amino acid concentrations were elevated, reflecting an acute exacerbation of the disease. Electrodiagnostic studies indicated an acute axonal polyneuropathy and sural nerve biopsy revealed acute wallerian degeneration without inflammation. Peripheral neuropathy, although not identified previously as a clinical feature of MSUD, may become more common as chronic dietary restrictions and improved management of the disease allow survival into adulthood.

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Two patients with similar courses of neurologic impairment and subsequent recovery after cerebral air embolism complicating cardiac ablation procedures are described. Hyperbaric oxygen therapy, combined with aggressive resuscitative efforts, appears to have contributed to each patient's recovery.

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1995

We studied the control of pharyngeal excitation in Caenorhabditis elegans. By laser ablating subsets of the pharyngeal nervous system, we found that the MC neuron type is necessary and probably sufficient for rapid pharyngeal pumping. Electropharyngeograms showed that MC transmits excitatory postsynaptic potentials, suggesting that MC acts as a neurogenic pacemaker for pharyngeal pumping. Mutations in genes required for acetylcholine (ACh) release and an antagonist of the nicotinic ACh receptor (nAChR) reduced pumping rates, suggesting that a nAChR is required for MC transmission. To identify genes required for MC neurotransmission, we screened for mutations that cause slow pumping but no other defects. Mutations in two genes, eat-2 and eat-18, eliminated MC neurotransmission. A gain-of-function eat-18 mutation, ad820sd, and a putative loss-of-function eat-18 mutation, ad1110, both reduced the excitation of pharyngeal muscle in response to the nAChR agonists nicotine and carbachol, suggesting that eat-18 is required for the function of a pharyngeal nAChR. Fourteen recessive mutations in eat-2 fell into five complementation classes. We found allele-specific genetic interactions between eat-2 and eat-18 that correlated with complementation classes of eat-2. We propose that eat-18 and eat-2 function in a multisubunit protein complex involved in the function of a pharyngeal nAChR.

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1994

The pharynx of C. elegans, a model system for neural networks and for membrane excitability, has been chiefly studied by observing its behavior in normal worms, in mutant worms, and in worms lacking pharyngeal neurons. To complement this behavioral approach, we devised a method for recording currents produced by changes in pharyngeal muscle membrane potential. The electrical records, called electropharyngeograms, contain transients caused by pharyngeal muscle action potentials and by inhibitory synaptic transmission between pharyngeal neuron M3 and the muscle. Using the electropharyngeograms, we show that gamma-aminobutyric acid is not likely to be the M3 neurotransmitter, that synaptic transmission is present but abnormal in mutants lacking synaptotagmin, and that worms mutant in the eat-4 gene are defective for M3 function or transmission.

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