RESUMO
Memories formed early in life are particularly stable and influential, representing privileged experiences that shape enduring behaviors. We show that exposing newly hatched C. elegans to pathogenic bacteria results in persistent aversion to those bacterial odors, whereas adult exposure generates only transient aversive memory. Long-lasting imprinted aversion has a critical period in the first larval stage and is specific to the experienced pathogen. Distinct groups of neurons are required during formation (AIB, RIM) and retrieval (AIY, RIA) of the imprinted memory. RIM synthesizes the neuromodulator tyramine, which is required in the L1 stage for learning. AIY memory retrieval neurons sense tyramine via the SER-2 receptor, which is essential for imprinted, but not for adult-learned, aversion. Odor responses in several neurons, most notably RIA, are altered in imprinted animals. These findings provide insight into neuronal substrates of different forms of memory, and lay a foundation for further understanding of early learning.
Assuntos
Caenorhabditis elegans/fisiologia , Vias Neurais , Neurônios/metabolismo , Animais , Bactérias/química , Comportamento Animal , Caenorhabditis elegans/crescimento & desenvolvimento , Fixação Psicológica Instintiva , Larva/fisiologia , Memória , Receptores de Amina Biogênica/metabolismo , Olfato , Tiramina/metabolismoRESUMO
Variability is a prominent feature of behavior and is an active element of certain behavioral strategies. To understand how neuronal circuits control variability, we examined the propagation of sensory information in a chemotaxis circuit of C. elegans where discrete sensory inputs can drive a probabilistic behavioral response. Olfactory neurons respond to odor stimuli with rapid and reliable changes in activity, but downstream AIB interneurons respond with a probabilistic delay. The interneuron response to odor depends on the collective activity of multiple neurons-AIB, RIM, and AVA-when the odor stimulus arrives. Certain activity states of the network correlate with reliable responses to odor stimuli. Artificially generating these activity states by modifying neuronal activity increases the reliability of odor responses in interneurons and the reliability of the behavioral response to odor. The integration of sensory information with network states may represent a general mechanism for generating variability in behavior.
Assuntos
Caenorhabditis elegans/fisiologia , Condutos Olfatórios , Animais , Comportamento Animal , Sinalização do Cálcio , Neurônios/metabolismo , OdorantesRESUMO
Foraging animals have distinct exploration and exploitation behaviors that are organized into discrete behavioral states. Here, we characterize a neuromodulatory circuit that generates long-lasting roaming and dwelling states in Caenorhabditis elegans. We find that two opposing neuromodulators, serotonin and the neuropeptide pigment dispersing factor (PDF), each initiate and extend one behavioral state. Serotonin promotes dwelling states through the MOD-1 serotonin-gated chloride channel. The spontaneous activity of serotonergic neurons correlates with dwelling behavior, and optogenetic modulation of the critical MOD-1-expressing targets induces prolonged dwelling states. PDF promotes roaming states through a Gαs-coupled PDF receptor; optogenetic activation of cAMP production in PDF receptor-expressing cells induces prolonged roaming states. The neurons that produce and respond to each neuromodulator form a distributed circuit orthogonal to the classical wiring diagram, with several essential neurons that express each molecule. The slow temporal dynamics of this neuromodulatory circuit supplement fast motor circuits to organize long-lasting behavioral states.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiologia , Neuropeptídeos/metabolismo , Serotonina/metabolismo , Transdução de Sinais , Animais , Comportamento Animal , Canais de Cloreto/metabolismo , AMP Cíclico/metabolismo , Neurônios/metabolismo , Receptores Acoplados a Proteínas G/metabolismoRESUMO
The GAL4-UAS system is a powerful tool for manipulating gene expression, but its application in Caenorhabditis elegans has not been described. Here we systematically optimize the system's three main components to develop a temperature-optimized GAL4-UAS system (cGAL) that robustly controls gene expression in C. elegans from 15 to 25 °C. We demonstrate this system's utility in transcriptional reporter analysis, site-of-action experiments and exogenous transgene expression; and we provide a basic driver and effector toolkit.
Assuntos
Caenorhabditis elegans/genética , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica , Engenharia Genética/métodos , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética , Animais , Animais Geneticamente Modificados , Defecação/genética , Herpesvirus Humano 1/genética , Microscopia de Fluorescência , Optogenética , TemperaturaRESUMO
Approaches for inhibiting and activating neurons are essential for understanding how neurons and neuronal circuits produce behavior. Optogenetics is a recently-developed technique which uses light to manipulate neuronal activity with temporal precision in behaving animals and is widely-used by neuroscience researchers. Optogenetics is also an excellent method to incorporate into an undergraduate neuroscience laboratory module because students can learn to conduct and analyze quantitative behavioral assays, reinforce their understanding of synaptic transmission, and investigate the genetic and neuronal basis of behavior. Here, we describe a module in which students use light to activate serotonergic neurons expressing the light-activated ion channel channelrhodopsin in wildtype and mutant Caenorhabditis elegans, and observe and analyze the effects on the movement behavior of the nematode. The methods described here provide a foundation which students can use to design and conduct additional experiments that may have never been done before. Thus, this laboratory module provides an opportunity for students to learn a state-of-the-art neuroscience technique, think about neuroscience on genetic, cellular and behavioral levels, and to develop an independent research project.
RESUMO
Recent progress in neuroscience has been facilitated by tools for neuronal activation and inactivation that are orthogonal to endogenous signaling systems. We describe here a chemical-genetic approach for inducible silencing of Caenorhabditis elegans neurons in intact animals, using the histamine-gated chloride channel HisCl1 from Drosophila and exogenous histamine. Administering histamine to freely moving C. elegans that express HisCl1 transgenes in neurons leads to rapid and potent inhibition of neural activity within minutes, as assessed by behavior, functional calcium imaging, and electrophysiology of neurons expressing HisCl1. C. elegans does not use histamine as an endogenous neurotransmitter, and exogenous histamine has little apparent effect on wild-type C. elegans behavior. HisCl1-histamine silencing of sensory neurons, interneurons, and motor neurons leads to behavioral effects matching their known functions. In addition, the HisCl1-histamine system can be used to titrate the level of neural activity, revealing quantitative relationships between neural activity and behavioral output. We use these methods to dissect escape circuits, define interneurons that regulate locomotion speed (AVA, AIB) and escape-related omega turns (AIB), and demonstrate graded control of reversal length by AVA interneurons and DA/VA motor neurons. The histamine-HisCl1 system is effective, robust, compatible with standard behavioral assays, and easily combined with optogenetic tools, properties that should make it a useful addition to C. elegans neurotechnology.
Assuntos
Caenorhabditis elegans/metabolismo , Canais de Cloreto/metabolismo , Proteínas de Drosophila/metabolismo , Histamina/farmacologia , Neurônios/metabolismo , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/genética , Canais de Cloreto/antagonistas & inibidores , Drosophila , Proteínas de Drosophila/antagonistas & inibidores , Técnicas Analíticas Microfluídicas , Modelos Neurológicos , Atividade Motora/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Técnicas de Patch-ClampRESUMO
Innate social behaviours emerge from neuronal circuits that interpret sensory information on the basis of an individual's own genotype, sex and experience. The regulated aggregation behaviour of the nematode Caenorhabditis elegans, a simple animal with only 302 neurons, is an attractive system to analyse these circuits. Wild social strains of C. elegans aggregate in the presence of specific sensory cues, but solitary strains do not. Here we identify the RMG inter/motor neuron as the hub of a regulated circuit that controls aggregation and related behaviours. RMG is the central site of action of the neuropeptide receptor gene npr-1, which distinguishes solitary strains (high npr-1 activity) from wild social strains (low npr-1 activity); high RMG activity is essential for all aspects of social behaviour. Anatomical gap junctions connect RMG to several classes of sensory neurons known to promote aggregation, and to ASK sensory neurons, which are implicated in male attraction to hermaphrodite pheromones. We find that ASK neurons respond directly to pheromones, and that high RMG activity enhances ASK responses in social strains, causing hermaphrodite attraction to pheromones at concentrations that repel solitary hermaphrodites. The coordination of social behaviours by RMG suggests an anatomical hub-and-spoke model for sensory integration in aggregation, and points to functions for related circuit motifs in the C. elegans wiring diagram.
Assuntos
Caenorhabditis elegans/fisiologia , Vias Neurais/fisiologia , Feromônios/fisiologia , Comportamento Social , Animais , Caenorhabditis elegans/citologia , Caenorhabditis elegans/efeitos dos fármacos , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Transtornos do Desenvolvimento Sexual , Comportamento Alimentar/efeitos dos fármacos , Comportamento Alimentar/fisiologia , Masculino , Modelos Neurológicos , Mutação , Vias Neurais/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Feromônios/farmacologia , Receptores de Neuropeptídeo Y/genética , Receptores de Neuropeptídeo Y/metabolismoRESUMO
Studies of C. elegans behavior have been crucial in identifying genetic pathways that control nervous system development and function, as well as basic principles of neural circuit function. Modern analysis of C. elegans behavior commonly relies on video recordings of animals, followed by automated image analysis and behavior quantification. Here, we describe two methods for recording and quantifying C. elegans behavior: a single-worm tracking approach that provides high-resolution behavioral data for individual animals and a multi-worm tracking approach that allows for quantification of the behavior of many animals in parallel. These approaches should be useful to a wide range of researchers studying the nervous system and behavior of C. elegans.
Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animais , Comportamento Animal/fisiologia , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Processamento de Imagem Assistida por Computador/métodos , Gravação em VídeoRESUMO
Foraging strategies emerge from genetically encoded programs that are similar across animal species. Here, we examine circuits that control a conserved foraging state, local search behavior after food removal, in Caenorhabditis elegans. We show that local search is triggered by two parallel groups of chemosensory and mechanosensory glutamatergic neurons that detect food-related cues. Each group of sensory neurons suppresses distinct integrating neurons through a G protein-coupled metabotropic glutamate receptor, MGL-1, to release local search. The chemosensory and mechanosensory modules are separate and redundant; glutamate release from either module can drive the full behavior. A transition from local search to global search over several minutes after food removal is associated with two changes in circuit function. First, the spontaneous activity of sensory neurons falls. Second, the motor pattern generator for local search becomes less responsive to sensory input. This multimodal, distributed short-term food memory provides robust control of an innate behavior.
Assuntos
Comportamento Apetitivo/fisiologia , Proteínas de Caenorhabditis elegans/metabolismo , Células Quimiorreceptoras/metabolismo , Mecanorreceptores/metabolismo , Neurônios/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Animais , Caenorhabditis elegans , Comportamento Alimentar , Neurônios/fisiologiaRESUMO
The development of the EGAD program and energy function for protein design is described. In contrast to most protein design methods, which require several empirical parameters or heuristics such as patterning of residues or rotamers, EGAD has a minimalist philosophy; it uses very few empirical factors to account for inaccuracies resulting from the use of fixed backbones and discrete rotamers in protein design calculations, and describes the unfolded state, aggregates, and alternative conformers explicitly with physical models instead of fitted parameters. This approach unveils important issues in protein design that are often camouflaged by heuristic-emphasizing methods. Inter-atom energies are modeled with the OPLS-AA all-atom forcefield, electrostatics with the generalized Born continuum model, and the hydrophobic effect with a solvent-accessible surface area-dependent term. Experimental characterization of proteins designed with an unmodified version of the energy function revealed problems with under-packing, stability, aggregation, and structural specificity. Under-packing was addressed by modifying the van der Waals function. By optimizing only three parameters, the effects of >400 mutations on protein-protein complex formation were predicted to within 1.0 kcal mol(-1). As an independent test, this modified energy function was used to predict the stabilities of >1500 mutants to within 1.0 kcal mol(-1); this required a physical model of the unfolded state that includes more interactions than traditional tripeptide-based models. Solubility and structural specificity were addressed with simple physical approximations of aggregation and conformational equilibria. The complete energy function can design protein sequences that have high levels of identity with their natural counterparts, and have predicted structural properties more consistent with soluble and uniquely folded proteins than the initial designs.
Assuntos
Modelos Moleculares , Conformação Proteica , Engenharia de Proteínas , Proteínas/química , Software , Dicroísmo Circular , Dobramento de Proteína , Proteínas/genética , Estatística como AssuntoRESUMO
Random search is a behavioral strategy used by organisms from bacteria to humans to locate food that is randomly distributed and undetectable at a distance. We investigated this behavior in the nematode Caenorhabditis elegans, an organism with a small, well-described nervous system. Here we formulate a mathematical model of random search abstracted from the C. elegans connectome and fit to a large-scale kinematic analysis of C. elegans behavior at submicron resolution. The model predicts behavioral effects of neuronal ablations and genetic perturbations, as well as unexpected aspects of wild type behavior. The predictive success of the model indicates that random search in C. elegans can be understood in terms of a neuronal flip-flop circuit involving reciprocal inhibition between two populations of stochastic neurons. Our findings establish a unified theoretical framework for understanding C. elegans locomotion and a testable neuronal model of random search that can be applied to other organisms.
RESUMO
The ability to evaluate variability in the environment is vital for making optimal behavioral decisions. Here we show that Caenorhabditis elegans evaluates variability in its food environment and modifies its future behavior accordingly. We derive a behavioral model that reveals a critical period over which information about the food environment is acquired and predicts future search behavior. We also identify a pair of high-threshold sensory neurons that encode variability in food concentration and the downstream dopamine-dependent circuit that generates appropriate search behavior upon removal from food. Further, we show that CREB is required in a subset of interneurons and determines the timescale over which the variability is integrated. Interestingly, the variability circuit is a subset of a larger circuit driving search behavior, showing that learning directly modifies the very same neurons driving behavior. Our study reveals how a neural circuit decodes environmental variability to generate contextually appropriate decisions.
Assuntos
Comportamento Animal/fisiologia , Caenorhabditis elegans/fisiologia , Dopamina/metabolismo , Comportamento Alimentar/fisiologia , Plasticidade Neuronal/fisiologia , Células Receptoras Sensoriais/fisiologia , Adaptação Fisiológica/fisiologia , Animais , Ácido Glutâmico/metabolismo , Interneurônios/metabolismo , Modelos BiológicosRESUMO
Electrostatics and solvation energies are important for defining protein stability, structural specificity, and molecular recognition. Because these energies are difficult to compute quickly and accurately, they are often ignored or modeled very crudely in computational protein design. To address this problem, we have developed a simple, fast, and accurate approximation for calculating Born radii in the context of protein design calculations. When these approximate Born radii are used with the generalized Born continuum dielectric model, energies calculated by the 10(6)-fold slower finite difference Poisson-Boltzmann model are faithfully reproduced. A similar approach can be used for estimating solvent-accessible surface areas (SASAs). As an independent test, we show that these approximations can be used to accurately predict the experimentally determined pK(a)s of >200 ionizable groups from 15 proteins.
Assuntos
Algoritmos , Computação Matemática , Proteínas/química , Simulação por Computador , Bases de Dados de Proteínas , Modelos Moleculares , Modelos Teóricos , Conformação Proteica , Solventes , Eletricidade Estática , Termodinâmica , MolhabilidadeRESUMO
Many biological functions are conserved, but the extent to which conservation applies to integrative behaviors is unknown. Vasopressin and oxytocin neuropeptides are strongly implicated in mammalian reproductive and social behaviors, yet rodent loss-of-function mutants have relatively subtle behavioral defects. Here we identify an oxytocin/vasopressin-like signaling system in Caenorhabditis elegans, consisting of a peptide and two receptors that are expressed in sexually dimorphic patterns. Males lacking the peptide or its receptors perform poorly in reproductive behaviors, including mate search, mate recognition, and mating, but other sensorimotor behaviors are intact. Quantitative analysis indicates that mating motor patterns are fragmented and inefficient in mutants, suggesting that oxytocin/vasopressin peptides increase the coherence of mating behaviors. These results indicate that conserved molecules coordinate diverse behavioral motifs in reproductive behavior.
Assuntos
Evolução Biológica , Proteínas de Caenorhabditis elegans/fisiologia , Caenorhabditis elegans/fisiologia , Neuropeptídeos/fisiologia , Ocitocina/fisiologia , Receptores Acoplados a Proteínas G/fisiologia , Comportamento Sexual Animal/fisiologia , Vasopressinas/fisiologia , Sequência de Aminoácidos , Animais , Células CHO , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/agonistas , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/farmacologia , Cricetinae , Humanos , Masculino , Neuropeptídeos/química , Neuropeptídeos/genética , Neuropeptídeos/farmacologia , Ocitocina/química , Ocitocina/genética , Ocitocina/farmacologia , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/genética , Reprodução , Vasopressinas/química , Vasopressinas/genética , Vasopressinas/farmacologiaRESUMO
Optogenetic approaches using light-activated proteins like Channelrhodopsin-2 (ChR2) enable investigating the function of populations of neurons in live Caenorhabditis elegans (and other) animals, as ChR2 expression can be targeted to these cells using specific promoters. Sub-populations of these neurons, or even single cells, can be further addressed by restricting the illumination to the cell of interest. However, this is technically demanding, particularly in free moving animals. Thus, it would be helpful if expression of ChR2 could be restricted to single neurons or neuron pairs, as even wide-field illumination would photostimulate only this particular cell. To this end we adopted the use of Cre or FLP recombinases and conditional ChR2 expression at the intersection of two promoter expression domains, i.e. in the cell of interest only. Success of this method depends on precise knowledge of the individual promoters' expression patterns and on relative expression levels of recombinase and ChR2. A bicistronic expression cassette with GFP helps to identify the correct expression pattern. Here we show specific expression in the AVA reverse command neurons and the aversive polymodal sensory ASH neurons. This approach shall enable to generate strains for optogenetic manipulation of each of the 302 C. elegans neurons. This may eventually allow to model the C. elegans nervous system in its entirety, based on functional data for each neuron.
Assuntos
Proteínas de Caenorhabditis elegans/biossíntese , Caenorhabditis elegans/metabolismo , Regulação da Expressão Gênica , Neurônios/metabolismo , Rodopsina/biossíntese , Animais , Comportamento Animal , Proteínas de Caenorhabditis elegans/genética , Perfilação da Expressão Gênica , Proteínas de Fluorescência Verde/metabolismo , Microscopia de Fluorescência/métodos , Modelos Biológicos , Modelos Genéticos , Plasmídeos/metabolismo , Regiões Promotoras Genéticas , Recombinases/metabolismo , Rodopsina/genética , TransgenesRESUMO
Homeostatic sensory systems detect small deviations in temperature, water balance, pH, and energy needs to regulate adaptive behavior and physiology. In C. elegans, a homeostatic preference for intermediate oxygen (O2) levels requires cGMP signaling through soluble guanylate cyclases (sGCs), proteins that bind gases through an associated heme group. Here we use behavioral analysis, functional imaging, and genetics to show that reciprocal changes in O2 levels are encoded by sensory neurons that express alternative sets of sGCs. URX sensory neurons are activated by increases in O2 levels, and require the sGCs gcy-35 and gcy-36. BAG sensory neurons are activated by decreases in O2 levels, and require the sGCs gcy-31 and gcy-33. The sGCs are instructive O2 sensors, as forced expression of URX sGC genes causes BAG neurons to detect O2 increases. Both sGC expression and cell-intrinsic dynamics contribute to the differential roles of URX and BAG in O2-dependent behaviors.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Guanilato Ciclase/classificação , Guanilato Ciclase/metabolismo , Oxigênio/metabolismo , Células Receptoras Sensoriais/classificação , Células Receptoras Sensoriais/fisiologia , Animais , Comportamento Animal , Caenorhabditis elegans/fisiologia , Proteínas de Caenorhabditis elegans/genética , Cálcio/metabolismo , Relação Dose-Resposta a Droga , Guanilato Ciclase/genética , Luz , Locomoção/efeitos dos fármacos , Locomoção/fisiologia , Mutação , Compostos Organometálicos/metabolismo , Oxigênio/farmacologia , Fenantrolinas/metabolismo , Inanição/metabolismoRESUMO
Recent advances in computational protein design have established it as a viable technique for the rational generation of stable protein sequences, novel protein folds, and even enzymatic activity. We present a new and object-oriented library of code, written specifically for protein design applications in C(++), called EGAD Library. The modular fashion in which this library is written allows developers to tailor various energy functions and minimizers for a specific purpose. It also allows for the generation of novel protein design applications with a minimal amount of code investment. It is our hope that this will permit labs that have not considered protein design to apply it to their own systems, thereby increasing its potential as a tool in biology. We also present various uses of EGAD Library: in the development of Interaction Viewer, a PyMOL plug-in for viewing interactions between protein residues; in the repacking of protein cores; and in the prediction of protein-protein complex stabilities.