Marios Chatzigeorgiou

Specific roles for DEG/ENaC and TRP channels in touch and thermosensation in C. elegans nociceptors

Polymodal nociceptors detect noxious stimuli, including harsh touch, toxic chemicals and extremes of heat and cold. The molecular mechanisms by which nociceptors are able to sense multiple qualitatively distinct stimuli are not well understood. We found that the C. elegans PVD neurons are mulitidendritic nociceptors that respond to harsh touch and cold temperatures. The harsh touch modality specifically required the DEG/ENaC proteins MEC-10 and DEGT-1, which represent putative components of a harsh touch mechanotransduction complex. In contrast, responses to cold required the TRPA-1 channel and were MEC-10 and DEGT-1 independent. Heterologous expression of C. elegans TRPA-1 conferred cold responsiveness to other C. elegans neurons and to mammalian cells, indicating that TRPA-1 is a cold sensor. Our results suggest that C. elegans nociceptors respond to thermal and mechanical stimuli using distinct sets of molecules and identify DEG/ENaC channels as potential receptors for mechanical pain.

C. elegans multi-dendritic sensory neurons: Morphology and function

PVD and FLP sensory neurons envelope the body of the C. elegans adult with a highly branched network of thin sensory processes. Both PVD and FLP neurons are mechanosensors. PVD is known to mediate the response to high threshold mechanical stimuli. Thus PVD and FLP neurons are similar in both morphology and function to mammalian nociceptors. To better understand the function of these neurons we generated strains lacking them. Behavioral analysis shows that PVD and FLP regulate movement under normal growth conditions, as animals lacking these neurons demonstrate higher dwelling behavior. In addition, PVD--whose thin branches project across the body-wall muscles--may have a role in proprioception, as ablation of PVD leads to defective posture. Moreover, movement-dependent calcium transients are seen in PVD, a response that requires MEC-10, a subunit of the mechanosensory DEG/ENaC channel that is also required for maintaining wild-type posture. Hence, PVD senses both noxious and innocuous signals to regulate C. elegans behavior, and thus combines the functions of multiple mammalian somatosensory neurons. Finally, strong mechanical stimulation leads to inhibition of egg-laying, and this response also depends on PVD and FLP neurons. Based on all these results we suggest that noxious signals perceived by PVD and FLP promote an escape behavior consisting of increased speed, reduced pauses and reversals, and inhibition of egg-laying.

tmc-1 encodes a sodium-sensitive channel required for salt chemosensation in C. elegans

Transmembrane channel-like (TMC) genes encode a broadly conserved family of multipass integral membrane proteins in animals. Human TMC1 and TMC2 genes are linked to human deafness and required for hair-cell mechanotransduction; however, the molecular functions of these and other TMC proteins have not been determined. Here we show that the Caenorhabditis elegans tmc-1 gene encodes a sodium sensor that functions specifically in salt taste chemosensation. tmc-1 is expressed in the ASH polymodal avoidance neurons, where it is required for salt-evoked neuronal activity and behavioural avoidance of high concentrations of NaCl. However, tmc-1 has no effect on responses to other stimuli sensed by the ASH neurons including high osmolarity and chemical repellents, indicating a specific role in salt sensation. When expressed in mammalian cell culture, C. elegans TMC-1 generates a predominantly cationic conductance activated by high extracellular sodium but not by other cations or uncharged small molecules. Thus, TMC-1 is both necessary for salt sensation in vivo and sufficient to generate a sodium-sensitive channel in vitro, identifying it as a probable ionotropic sensory receptor.

Analysis of NPR-1 Reveals a Circuit Mechanism for Behavioral Quiescence in C. elegans

Animals undergo periods of behavioral quiescence and arousal in response to environmental, circadian, or developmental cues. During larval molts, C. elegans undergoes a period of profound behavioral quiescence termed lethargus. Locomotion quiescence during lethargus was abolished in mutants lacking a neuropeptide receptor (NPR-1) and was reduced in mutants lacking NPR-1 ligands (FLP-18 and FLP-21). Wild-type strains are polymorphic for the npr-1 gene, and their lethargus behavior varies correspondingly. Locomotion quiescence and arousal were mediated by decreased and increased secretion of an arousal neuropeptide (PDF-1) from central neurons. PDF receptors (PDFR-1) expressed in peripheral mechanosensory neurons enhanced touch-evoked calcium transients. Thus, a central circuit stimulates arousal from lethargus by enhancing the sensitivity of peripheral mechanosensory neurons in the body. These results define a circuit mechanism controlling a developmentally programmed form of quiescence.

Lateral facilitation between primary mechanosensory neurons controls nose touch perception in C. elegans.

Summary The nematode C. elegans senses head and nose touch using multiple classes of mechanoreceptor neurons that are electrically coupled through a network of gap junctions. Using in vivo neuroimaging, we have found that multidendritic nociceptors in the head respond to harsh touch throughout their receptive field but respond to gentle touch only at the tip of the nose. Whereas the harsh touch response depends solely on cell-autonomous mechanosensory channels, gentle nose touch responses require facilitation by additional nose touch mechanoreceptors, which couple electrically to the nociceptors in a hub-and-spoke gap junction network. Conversely, nociceptor activity indirectly facilitates activation of the nose touch neurons, demonstrating that information flow across the network is bidirectional. Thus, a simple gap-junction circuit acts as a coincidence detector that allows primary sensory neurons to integrate information from neighboring mechanoreceptors and generate somatosensory perception.

Sensory Neurons Arouse C . elegans Locomotion via Both Glutamate and Neuropeptide Release

C. elegans undergoes periods of behavioral quiescence during larval molts (termed lethargus) and as adults. Little is known about the circuit mechanisms that establish these quiescent states. Lethargus and adult locomotion quiescence is dramatically reduced in mutants lacking the neuropeptide receptor NPR-1. Here, we show that the aroused locomotion of npr-1 mutants results from the exaggerated activity in multiple classes of sensory neurons, including nociceptive (ASH), touch sensitive (ALM and PLM), and stretch sensing (DVA) neurons. These sensory neurons accelerate locomotion via both neuropeptide and glutamate release. The relative contribution of these sensory neurons to arousal differs between larval molts and adults. Our results suggest that a broad network of sensory neurons dictates transitions between aroused and quiescent behavioral states.

Spatial Asymmetry in the Mechanosensory Phenotypes of the C. elegans DEG/ENaC Gene mec-10

DEG/ENaC channels have been broadly implicated in mechanosensory transduction, yet many questions remain about how these proteins contribute to complexes that sense mechanical stimuli. In C. elegans, two DEG/ENaC channel subunits are thought to contribute to a gentle touch transduction complex: MEC-4, which is essential for gentle touch sensation, and MEC-10, whose importance is less well defined. By characterizing a mec-10 deletion mutant, we have found that MEC-10 is important, but not essential, for gentle touch responses in the body touch neurons ALM, PLM, and PVM. Surprisingly, the requirement for MEC-10 in ALM and PLM is spatially asymmetric; mec-10 animals show significant behavioral and physiological responses to stimulation at the distal end of touch neuron dendrites, but respond poorly to stimuli applied near the neuronal cell body. The subcellular distribution of a rescuing MEC-10::GFP translational fusion was found to be restricted to the neuronal cell body and proximal dendrite, consistent with the hypothesis that MEC-10 protein is asymmetrically distributed within the touch neuron process. These results suggest that MEC-10 may contribute to only a subset of gentle touch mechanosensory complexes found preferentially at the proximal dendrite.

A Gap Junction Circuit Enhances Processing of Coincident Mechanosensory Inputs

Summary Electrical synapses have been shown to be important for enabling and detecting neuronal synchrony in both vertebrates [1–4] and invertebrates [5, 6]. Hub-and-spoke circuits, in which a central hub neuron is electrically coupled to several input neurons, are an overrepresented motif in the C. elegans nervous system [7] and may represent a conserved functional unit. The functional relevance of this configuration has been demonstrated for circuits mediating aggregation behavior [8] and nose touch perception [9]. Modeling approaches have been useful for understanding structurally and dynamically more complex electrical circuits [10, 11]. Therefore, we formulated a simple analytical model with minimal assumptions to obtain insight into the properties of the hub-and-spoke microcircuit motif. A key prediction of the model is that an active input neuron should facilitate activity throughout the network, whereas an inactive input should suppress network activity through shunting; this prediction was supported by cell ablation and in vivo neuroimaging experiments in the C. elegans nose touch circuit. Thus, the hub-and-spoke architecture may implement an analog coincidence detector enabling distinct responses to distributed and localized patterns of sensory input.

Rewiring neural circuits by the insertion of ectopic electrical synapses in transgenic C. elegans

Neural circuits are functional ensembles of neurons that are selectively interconnected by chemical or electrical synapses. Here we describe a synthetic biology approach to the study of neural circuits, whereby new electrical synapses can be introduced in novel sites in the neuronal circuitry to reprogram behaviour. We added electrical synapses composed of the vertebrate gap junction protein Cx36 between Caenorhabditis elegans chemosensory neurons with opposite intrinsic responses to salt. Connecting these neurons by an ectopic electrical synapse led to a loss of lateral asymmetry and altered chemotaxis behaviour. In a second example, introducing Cx36 into an inhibitory chemical synapse between an olfactory receptor neuron and an interneuron changed the sign of the connection from negative to positive, and abolished the animal’s behavioural response to benzaldehyde. These data demonstrate a synthetic strategy to rewire behavioural circuits by engineering synaptic connectivity in C. elegans.

Caenorhabditis elegans nicotinic acetylcholine receptors are required for nociception

Polymodal nociceptors sense and integrate information on injurious mechanical, thermal, and chemical stimuli. Chemical signals either activate nociceptors or modulate their responses to other stimuli. One chemical known to activate or modulate responses of nociceptors is acetylcholine (ACh). Across evolution nociceptors express subunits of the nicotinic acetylcholine receptor (nAChR) family, a family of ACh-gated ion channels. The roles of ACh and nAChRs in nociceptor function are, however, poorly understood. Caenorhabditis elegans polymodal nociceptors, PVD, express nAChR subunits on their sensory arbor. Here we show that mutations reducing ACh synthesis and mutations in nAChR subunits lead to defects in PVD function and morphology. A likely cause for these defects is a reduction in cytosolic calcium measured in ACh and nAChR mutants. Indeed, overexpression of a calcium pump in PVD mimics defects in PVD function and morphology found in nAChR mutants. Our results demonstrate, for the first time, a central role for nAChRs and ACh in nociceptor function and suggest that calcium permeating via nAChRs facilitates activity of several signaling pathways within this neuron.