RESUMO
The nervous system evaluates environmental cues and adjusts motor output to ensure navigation toward a preferred environment. The nematode Caenorhabditis elegans navigates in the thermal environment and migrates toward its cultivation temperature by moving up or down thermal gradients depending not only on absolute temperature but on relative difference between current and previously experienced cultivation temperature. Although previous studies showed that such thermal context-dependent opposing migration is mediated by bias in frequency and direction of reorientation behavior, the complete neural pathways-from sensory to motor neurons-and their circuit logics underlying the opposing behavioral bias remain elusive. By conducting comprehensive cell ablation, high-resolution behavioral analyses, and computational modeling, we identified multiple neural pathways regulating behavioral components important for thermotaxis, and demonstrate that distinct sets of neurons are required for opposing bias of even single behavioral components. Furthermore, our imaging analyses show that the context-dependent operation is evident in sensory neurons, very early in the neural pathway, and manifested by bidirectional responses of a first-layer interneuron AIB under different thermal contexts. Our results suggest that the contextual differences are encoded among sensory neurons and a first-layer interneuron, processed among different downstream neurons, and lead to the flexible execution of context-dependent behavior.
Assuntos
Comportamento Animal/fisiologia , Caenorhabditis elegans/fisiologia , Interneurônios/fisiologia , Navegação Espacial/fisiologia , Termorreceptores/fisiologia , Animais , Técnicas de Observação do Comportamento , Locomoção/fisiologia , Vias Neurais/fisiologia , Temperatura , Sensação Térmica/fisiologiaRESUMO
In the past 15 years, optogenetic methods became invaluable tools in neurobiological research but also in general cell biology. Most prominently, optogenetic methods utilize microbial rhodopsins to elicit neuronal de- or hyperpolarization. However, other optogenetic tools have emerged that allow influencing neuronal function by different approaches. In this chapter we describe the use of photoactivated adenylyl cyclases (PACs) as modulators of neuronal activity. Using Caenorhabditis elegans as a model organism, this chapter shows how to measure the effect of PAC photoactivation by behavioral assays in different tissues (neurons and muscles), as well as their significance to neurobiology. Further, this chapter describes in vitro cyclic nucleoside-3',5'-monophosphate measurements (cNMP) to characterize new PACs in C. elegans.
Assuntos
Adenilil Ciclases , Optogenética , Adenilil Ciclases/genética , Animais , Caenorhabditis elegans/genética , Neurônios , Optogenética/métodos , Rodopsinas MicrobianasRESUMO
Sleep requires sleep-active neurons that depolarize to inhibit wake circuits. Sleep-active neurons are under the control of homeostatic mechanisms that determine sleep need. However, little is known about the molecular and circuit mechanisms that translate sleep need into the depolarization of sleep-active neurons. During many stages and conditions in C. elegans, sleep requires a sleep-active neuron called RIS. Here, we defined the transcriptome of RIS and discovered that genes of the epidermal growth factor receptor (EGFR) signaling pathway are expressed in RIS. Because of cellular stress, EGFR directly activates RIS. Activation of EGFR signaling in the ALA neuron has previously been suggested to promote sleep independently of RIS. Unexpectedly, we found that ALA activation promotes RIS depolarization. Our results suggest that ALA is a drowsiness neuron with two separable functions: (1) it inhibits specific behaviors, such as feeding, independently of RIS, (2) and it activates RIS. Whereas ALA plays a strong role in surviving cellular stress, surprisingly, RIS does not. In summary, EGFR signaling can depolarize RIS by an indirect mechanism through activation of the ALA neuron that acts upstream of the sleep-active RIS neuron and through a direct mechanism using EGFR signaling in RIS. ALA-dependent drowsiness, rather than RIS-dependent sleep bouts, appears to be important for increasing survival after cellular stress, suggesting that different types of behavioral inhibition play different roles in restoring health. VIDEO ABSTRACT.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiologia , Fator de Crescimento Epidérmico/metabolismo , Neurônios/fisiologia , Transdução de Sinais , Sono/fisiologia , AnimaisRESUMO
cGMP plays a role in sensory signaling and plasticity by regulating ion channels, phosphodiesterases, and kinases. Studies that primarily used genetic and biochemical tools suggest that cGMP is spatiotemporally regulated in multiple sensory modalities. FRET- and GFP-based cGMP sensors were developed to visualize cGMP in primary cell culture and Caenorhabditis elegans to corroborate these findings. While a FRET-based sensor has been used in an intact animal to visualize cGMP, the requirement of a multiple emission system limits its ability to be used on its own as well as with other fluorophores. Here, we demonstrate that a C. elegans codon-optimized version of the cpEGFP-based cGMP sensor FlincG3 can be used to visualize rapidly changing cGMP levels in living, behaving C. elegans We coexpressed FlincG3 with the blue-light-activated guanylyl cyclases BeCyclOp and bPGC in body wall muscles, and found that the rate of change in FlincG3 fluorescence correlated with the rate of cGMP production by each cyclase. Furthermore, we show that FlincG3 responds to cultivation temperature, NaCl concentration changes, and sodium dodecyl sulfate in the sensory neurons AFD, ASEL/R, and PHB, respectively. Intriguingly, FlincG3 fluorescence in ASEL and ASER decreased in response to a NaCl concentration upstep and downstep, respectively, which is opposite in sign to the coexpressed calcium sensor jRGECO1a and previously published calcium recordings. These results illustrate that FlincG3 can be used to report rapidly changing cGMP levels in an intact animal, and that the reporter can potentially reveal unexpected spatiotemporal landscapes of cGMP in response to stimuli.
Assuntos
GMP Cíclico/metabolismo , Transferência Ressonante de Energia de Fluorescência/métodos , Proteínas de Fluorescência Verde/metabolismo , Optogenética/métodos , Animais , Caenorhabditis elegans , Células Cultivadas , Proteínas de Fluorescência Verde/genética , Guanilato Ciclase/genética , Guanilato Ciclase/metabolismo , Opsinas/genética , Opsinas/metabolismo , Imagem Óptica/métodos , Células Receptoras Sensoriais/citologia , Células Receptoras Sensoriais/metabolismoRESUMO
In recent years, optogenetic methods became invaluable tools, particularly in neurobiological research. Most prominently, optogenetic methods utilize microbial rhodopsins to elicit neuronal de- or hyperpolarization. However, other optogenetic tools have emerged that allow influencing neuronal function by different approaches. In this chapter we describe the use of photoactivated adenylyl cyclases (PACs) as modulators of neuronal activity. Using Caenorhabditis elegans as a model organism, this chapter shows how to measure the effect of PAC photoactivation by behavioral and electrophysiological assays, as well as their significance to neurobiology.
Assuntos
Adenilil Ciclases/biossíntese , Neurônios/fisiologia , Optogenética , Animais , Caenorhabditis elegans , Ativação Enzimática/efeitos da radiação , Luz , Técnicas de Patch-Clamp , Processos Fotoquímicos , Proteínas de Protozoários/biossíntese , Transdução de SinaisRESUMO
C. elegans is used extensively as a model system in the neurosciences due to its well defined nervous system. However, the seeming simplicity of this nervous system in anatomical structure and neuronal connectivity, at least compared to higher animals, underlies a rich diversity of behaviors. The usefulness of the worm in genome-wide mutagenesis or RNAi screens, where thousands of strains are assessed for phenotype, emphasizes the need for computational methods for automated parameterization of generated behaviors. In addition, behaviors can be modulated upon external cues like temperature, O(subscript)2(/subscript) and CO(subscript)2(/subscript) concentrations, mechanosensory and chemosensory inputs. Different machine vision tools have been developed to aid researchers in their efforts to inventory and characterize defined behavioral "outputs". Here we aim at providing an overview of different worm-tracking packages or video analysis tools designed to quantify different aspects of locomotion such as the occurrence of directional changes (turns, omega bends), curvature of the sinusoidal shape (amplitude, body bend angles) and velocity (speed, backward or forward movement).
Assuntos
Caenorhabditis elegans/fisiologia , Locomoção , Filmes Cinematográficos , Software , Animais , Processamento de Imagem Assistida por Computador , Locomoção/fisiologia , Microscopia/métodosRESUMO
The electrophysiological method we present is based on a solid supported membrane (SSM) composed of an octadecanethiol layer chemisorbed on a gold coated sensor chip and a phosphatidylcholine monolayer on top. This assembly is mounted into a cuvette system containing the reference electrode, a chlorinated silver wire. After adsorption of membrane fragments or proteoliposomes containing the membrane protein of interest, a fast solution exchange is used to induce the transport activity of the membrane protein. In the single solution exchange protocol two solutions, one non-activating and one activating solution, are needed. The flow is controlled by pressurized air and a valve and tubing system within a faraday cage. The kinetics of the electrogenic transport activity is obtained via capacitive coupling between the SSM and the proteoliposomes or membrane fragments. The method, therefore, yields only transient currents. The peak current represents the stationary transport activity. The time dependent transporter currents can be reconstructed by circuit analysis. This method is especially suited for prokaryotic transporters or eukaryotic transporters from intracellular membranes, which cannot be investigated by patch clamp or voltage clamp methods.
Assuntos
Eletrofisiologia/métodos , Proteínas de Membrana/química , Membranas Artificiais , Proteolipídeos/química , Adsorção , Eletrofisiologia/instrumentação , Ouro/química , Proteínas de Membrana/metabolismo , Proteolipídeos/metabolismo , Compostos de Sulfidrila/químicaRESUMO
BACKGROUND: Nociception generally evokes rapid withdrawal behavior in order to protect the tissue from harmful insults. Most nociceptive neurons responding to mechanical insults display highly branched dendrites, an anatomy shared by Caenorhabditis elegans FLP and PVD neurons, which mediate harsh touch responses. Although several primary molecular nociceptive sensors have been characterized, less is known about modulation and amplification of noxious signals within nociceptor neurons. First, we analyzed the FLP/PVD network by optogenetics and studied integration of signals from these cells in downstream interneurons. Second, we investigated which genes modulate PVD function, based on prior single-neuron mRNA profiling of PVD. RESULTS: Selectively photoactivating PVD, FLP, and downstream interneurons via Channelrhodopsin-2 (ChR2) enabled the functional dissection of this nociceptive network, without interfering signals by other mechanoreceptors. Forward or reverse escape behaviors were determined by PVD and FLP, via integration by command interneurons. To identify mediators of PVD function, acting downstream of primary nocisensor molecules, we knocked down PVD-specific transcripts by RNAi and quantified light-evoked PVD-dependent behavior. Cell-specific disruption of synaptobrevin or voltage-gated Ca(2+) channels (VGCCs) showed that PVD signals chemically to command interneurons. Knocking down the DEG/ENaC channel ASIC-1 and the TRPM channel GTL-1 indicated that ASIC-1 may extend PVD's dynamic range and that GTL-1 may amplify its signals. These channels act cell autonomously in PVD, downstream of primary mechanosensory molecules. CONCLUSIONS: Our work implicates TRPM channels in modifying excitability of and DEG/ENaCs in potentiating signal output from a mechano-nociceptor neuron. ASIC-1 and GTL-1 homologs, if functionally conserved, may denote valid targets for novel analgesics.