ASH Sensory Neurons Research Articles

Multilevel Modulation of a Sensory Motor Circuit during C. elegans Sleep and Arousal

Sleep is characterized by behavioral quiescence, homeostasis, increased arousal threshold, and rapid reversibility. Understanding how these properties are encoded by a neuronal circuit has been difficult, and no single molecular or neuronal pathway has been shown to be responsible for the regulation of sleep. Taking advantage of the well-mapped neuronal connections of Caenorhabditis elegans and the sleep-like states in this animal, we demonstrate the changed properties of both sensory neurons and downstream interneurons that mediate sleep and arousal. The ASH sensory neuron displays reduced sensitivity to stimuli in the sleep-like state, and the activity of the corresponding interneurons in ASH's motor circuit becomes asynchronous. Restoration of interneuron synchrony is sufficient for arousal. The multilevel circuit depression revealed provides an elegant strategy to promote a robust decrease in arousal while allowing for rapid reversibility of the sleep state.

Neuropeptides Amplify and Focus the Monoaminergic Inhibition of Nociception in Caenorhabditis elegans

Monoamines and neuropeptides interact to modulate most behaviors. To better understand these interactions, we have defined the roles of tyramine (TA), octopamine, and neuropeptides in the inhibition of aversive behavior in Caenorhabditis elegans. TA abolishes the serotonergic sensitization of aversive behavior mediated by the two nociceptive ASH sensory neurons and requires the expression of the adrenergic-like, Gαq-coupled, TA receptor TYRA-3 on inhibitory monoaminergic and peptidergic neurons. For example, TA inhibition requires Gαq and Gαs signaling in the peptidergic ASI sensory neurons, with an array of ASI neuropeptides activating neuropeptide receptors on additional neurons involved in locomotory decision-making. The ASI neuropeptides required for tyraminergic inhibition are distinct from those required for octopaminergic inhibition, suggesting that individual monoamines stimulate the release of different subsets of ASI neuropeptides. Together, these results demonstrate that a complex humoral mix of monoamines is focused by more local, synaptic, neuropeptide release to modulate nociception and highlight the similarities between the tyraminergic/octopaminergic inhibition of nociception in C. elegans and the noradrenergic inhibition of nociception in mammals that also involves inhibitory peptidergic signaling.

The C. elegans cGMP-dependent protein kinase EGL-4 regulates nociceptive behavioral sensitivity.

Signaling levels within sensory neurons must be tightly regulated to allow cells to integrate information from multiple signaling inputs and to respond to new stimuli. Herein we report a new role for the cGMP-dependent protein kinase EGL-4 in the negative regulation of G protein-coupled nociceptive chemosensory signaling. C. elegans lacking EGL-4 function are hypersensitive in their behavioral response to low concentrations of the bitter tastant quinine and exhibit an elevated calcium flux in the ASH sensory neurons in response to quinine. We provide the first direct evidence for cGMP/PKG function in ASH and propose that ODR-1, GCY-27, GCY-33 and GCY-34 act in a non-cell-autonomous manner to provide cGMP for EGL-4 function in ASH. Our data suggest that activated EGL-4 dampens quinine sensitivity via phosphorylation and activation of the regulator of G protein signaling (RGS) proteins RGS-2 and RGS-3, which in turn downregulate Gα signaling and behavioral sensitivity.

Strongly alkaline pH avoidance mediated by ASH sensory neurons in C. elegans

High pH is a noxious stimulus to animals, and their ability to avoid dangerously alkaline pH is critical for survival. However, the means by which they sense high pH has not been determined. The nematode Caenorhabditis elegans (C. elegans) avoids environmental pH above 10.5. In contrast, C. elegans mutants with structurally, developmentally, and/or functionally abnormal sensory cilia fail to avoid high pH, suggesting that sensory neurons in the cilia participate in sensing. Genetic rescue of the mutants indicates that ASH polymodal sensory neurons play a vital role in the process. Consistently, specific laser ablation of ASH neurons made animals insensitive to high pH. Furthermore, avoidance assays of other mutants also indicated that transient receptor potential vanilloid type (TRPV) ion channels encoded by osm-9 and ocr-2 are involved in sensing. Indeed, genetic rescue of osm-9 mutants by specifically expressing OSM-9 in ASH showed that TRPV channels play an essential role in sensing of high pH. Ca2+ imaging in vivo also revealed that ASH neurons were activated by high pH stimulation, but ASH of osm-9 or ocr-2 mutants were not. These results demonstrate that in C. elegans, high pH is sensed by ASH nociceptors through opening of OSM-9/OCR-2 TRPV channels.

CNP-1 (ARRD-17), a Novel Substrate of Calcineurin, Is Critical for Modulation of Egg-Laying and Locomotion in Response to Food and Lysine Sensation in Caenorhabditis elegans

Calcineurin is a Ca2+/calmodulin-dependent protein phosphatase involved in calcium signaling pathways. In Caenorhabditis elegans, the loss of calcineurin activity causes pleiotropic defects including hyperadaptation of sensory neurons, hypersensation to thermal difference and hyper-egg-laying when worms are refed after starvation. In this study, we report on arrd-17 as calcineurin-interacting protein-1 (cnp-1), which is a novel molecular target of calcineurin. CNP-1 interacts with the catalytic domain of the C. elegans calcineurin A subunit, TAX-6, in a yeast two-hybrid assay and is dephosphorylated by TAX-6 in vitro. cnp-1 is expressed in ASK, ADL, ASH and ASJ sensory neurons as TAX-6. It acts downstream of tax-6 in regulation of locomotion and egg-laying after starvation, ASH sensory neuron adaptation and lysine chemotaxis, that is known to be mediated by ASK neurons. Altogether, our biochemical and genetic evidence indicates that CNP-1 is a direct target of calcineurin and required in stimulated egg-laying and locomotion after starvation, adaptation to hyperosmolarity and attraction to lysine, which is modulated by calcineurin. We suggest that the phosphorylation status of CNP-1 plays an important role in regulation of refed stimulating behaviors after starvation and attraction to amino acid, which provides valuable nutritious information.

Odour concentration-dependent olfactory preference change in C. elegans

The same odorant can induce attractive or repulsive responses depending on its concentration in various animals including humans. However, little is understood about the neuronal basis of this behavioural phenomenon. Here we show that Caenorhabditis elegans avoids high concentrations of odorants that are attractive at low concentrations. Behavioural analyses and computer simulation reveal that the odour concentration-dependent behaviour is primarily generated by klinokinesis, a behavioural strategy in C. elegans. Genetic analyses and lesion experiments show that distinct combinations of sensory neurons function at different concentrations of the odorant; AWC and ASH sensory neurons have critical roles for attraction to or avoidance of the odorant, respectively. Moreover, we found that AWC neurons respond to only lower concentrations of the odorant, whereas ASH neurons respond to only higher concentrations of odorant. Hence, our study suggests that odour concentration coding in C. elegans mostly conforms to the labelled-line principle where distinct neurons respond to distinct stimuli.

Monoamines and neuropeptides interact to inhibit aversive behaviour in Caenorhabditis elegans.

Pain modulation is complex, but noradrenergic signalling promotes anti-nociception, with α(2)-adrenergic agonists used clinically. To better understand the noradrenergic/peptidergic modulation of nociception, we examined the octopaminergic inhibition of aversive behaviour initiated by the Caenorhabditis elegans nociceptive ASH sensory neurons. Octopamine (OA), the invertebrate counterpart of norepinephrine, modulates sensory-mediated reversal through three α-adrenergic-like OA receptors. OCTR-1 and SER-3 antagonistically modulate ASH signalling directly, with OCTR-1 signalling mediated by Gα(o). In contrast, SER-6 inhibits aversive responses by stimulating the release of an array of 'inhibitory' neuropeptides that activate receptors on sensory neurons mediating attraction or repulsion, suggesting that peptidergic signalling may integrate multiple sensory inputs to modulate locomotory transitions. These studies highlight the complexity of octopaminergic/peptidergic interactions, the role of OA in activating global peptidergic signalling cascades and the similarities of this modulatory network to the noradrenergic inhibition of nociception in mammals, where norepinephrine suppresses chronic pain through inhibitory α(2)-adrenoreceptors on afferent nociceptors and stimulatory α(1)-receptors on inhibitory peptidergic interneurons.

Dissecting the Serotonergic Food Signal Stimulating Sensory-Mediated Aversive Behavior in C. elegans

Nutritional state often modulates olfaction and in Caenorhabditis elegans food stimulates aversive responses mediated by the nociceptive ASH sensory neurons. In the present study, we have characterized the role of key serotonergic neurons that differentially modulate aversive behavior in response to changing nutritional status. The serotonergic NSM and ADF neurons play antagonistic roles in food stimulation. NSM 5-HT activates SER-5 on the ASHs and SER-1 on the RIA interneurons and stimulates aversive responses, suggesting that food-dependent serotonergic stimulation involves local changes in 5-HT levels mediated by extrasynaptic 5-HT receptors. In contrast, ADF 5-HT activates SER-1 on the octopaminergic RIC interneurons to inhibit food–stimulation, suggesting neuron-specific stimulatory and inhibitory roles for SER-1 signaling. Both the NSMs and ADFs express INS-1, an insulin-like peptide, that appears to cell autonomously inhibit serotonergic signaling. Food also modulates directional decisions after reversal is complete, through the same serotonergic neurons and receptors involved in the initiation of reversal, and the decision to continue forward or change direction after reversal is dictated entirely by nutritional state. These results highlight the complexity of the “food signal” and serotonergic signaling in the modulation of sensory-mediated aversive behaviors.

Microfluidic worm-chip for in vivo analysis of neuronal activity upon dynamic chemical stimulations

Conventional neuronal analysis at the single neuron level usually involves culturing of neurons in vitro and analysis of neuronal activities by electrophysiological or pharmacological methods. However, the extracellular environments of in vitro neuronal analysis cannot mimic the exact surroundings of the neurons. Here, we report a microfluidic worm-chip for in vivo analysis of neuronal activities upon dynamic chemical stimulations. A comb-shaped microvalve was developed to immobilize whole animal for high-resolution imaging of neuronal activities. Using a sequential sample introduction system, multiple chemical stimuli were delivered to an individual Caenorhabditis elegans nose tip based on programmed interface shifting of laminar flows. ASH sensory neuron responses to various stimuli in individual C. elegans were quantitatively evaluated, and mutants were significantly defective in neuronal responses to certain stimulus in comparison to others. Sensory reduction in the magnitude of the response to repetitive chemical stimulation with different durations was also found. Our study explored the possibility of real-time detection of neuronal activities in individual animals upon multiple stimulations.

The G Protein regulators EGL-10 and EAT-16, the Giα GOA-1 and the Gqα EGL-30 modulate the response of the C. elegansASH polymodal nociceptive sensory neurons to repellents

BackgroundPolymodal, nociceptive sensory neurons are key cellular elements of the way animals sense aversive and painful stimuli. In Caenorhabditis elegans, the polymodal nociceptive ASH sensory neurons detect aversive stimuli and release glutamate to generate avoidance responses. They are thus useful models for the nociceptive neurons of mammals. While several molecules affecting signal generation and transduction in ASH have been identified, less is known about transmission of the signal from ASH to downstream neurons and about the molecules involved in its modulation.ResultsWe discovered that the regulator of G protein signalling (RGS) protein, EGL-10, is required for appropriate avoidance responses to noxious stimuli sensed by ASH. As it does for other behaviours in which it is also involved, egl-10 interacts genetically with the Go/iα protein GOA-1, the Gqα protein EGL-30 and the RGS EAT-16. Genetic, behavioural and Ca2+ imaging analyses of ASH neurons in live animals demonstrate that, within ASH, EGL-10 and GOA-1 act downstream of stimulus-evoked signal transduction and of the main transduction channel OSM-9. EGL-30 instead appears to act upstream by regulating Ca2+ transients in response to aversive stimuli. Analysis of the delay in the avoidance response, of the frequency of spontaneous inversions and of the genetic interaction with the diacylglycerol kinase gene, dgk-1, indicate that EGL-10 and GOA-1 do not affect signal transduction and neuronal depolarization in response to aversive stimuli but act in ASH to modulate downstream transmission of the signal.ConclusionsThe ASH polymodal nociceptive sensory neurons can be modulated not only in their capacity to detect stimuli but also in the efficiency with which they respond to them. The Gα and RGS molecules studied in this work are conserved in evolution and, for each of them, mammalian orthologs can be identified. The discovery of their role in the modulation of signal transduction and signal transmission of nociceptors may help us to understand how pain is generated and how its control can go astray (such as chronic pain) and may suggest new pain control therapies.

The Monoaminergic Modulation of Sensory-Mediated Aversive Responses in Caenorhabditis elegans Requires Glutamatergic/Peptidergic Cotransmission

Monoamines and neuropeptides interact to modulate behavioral plasticity in both vertebrates and invertebrates. In Caenorhabditis elegans behavioral state or "mood" is dependent on food availability and is translated by both monoaminergic and peptidergic signaling in the fine-tuning of most behaviors. In the present study, we have examined the interaction of monoamines and peptides on C. elegans aversive behavior mediated by a pair of polymodal, nociceptive, ASH sensory neurons. Food or serotonin sensitize the ASHs and stimulate aversive responses through a pathway requiring the release of nlp-3-encoded neuropeptides from the ASHs. Peptides encoded by nlp-3 appear to stimulate ASH-mediated aversive behavior through the neuropeptide receptor-17 (NPR-17) receptor. nlp-3- and npr-17-null animals exhibit identical phenotypes and animals overexpressing either nlp-3 or npr-17 exhibit elevated aversive responses off food that are absent when nlp-3 or npr-17 are overexpressed in npr-17- or nlp-3-null animals, respectively. ASH-mediated aversive responses are increased by activating either Galpha(q) or Galpha(s) in the ASHs, with Galpha(s) signaling specifically stimulating the release of nlp-3-encoded peptides. In contrast, octopamine appears to inhibit 5-HT stimulation by activating Galpha(o) signaling in the ASHs that, in turn, inhibits both Galpha(s) and Galpha(q) signaling and the release of nlp-3-encoded peptides. These results demonstrate that serotonin and octopamine reversibly modulate the activity of the ASHs, and highlight the utility of the C. elegans model for defining interactions between monoamines and peptides in individual neurons of complex sensory-mediated circuits.

The C. elegans D2-like dopamine receptor DOP-3 decreases behavioral sensitivity to the olfactory stimulus 1-octanol.

We previously found that dopamine signaling modulates the sensitivity of wild-type C. elegans to the aversive odorant 1-octanol. C. elegans lacking the CAT-2 tyrosine hydroxylase enzyme, which is required for dopamine biosynthesis, are hypersensitive in their behavioral avoidance of dilute concentrations of octanol. Dopamine can also modulate the context-dependent response of C. elegans lacking RGS-3 function, a negative regulator of Gα signaling. rgs-3 mutant animals are defective in their avoidance of 100% octanol when they are assayed in the absence of food (E. coli bacterial lawn), but their response is restored when they are assayed in the presence of food or exogenous dopamine. However, it is not known which receptor might be mediating dopamine's effects on octanol avoidance. Herein we describe a role for the C. elegans D2-like receptor DOP-3 in the regulation of olfactory sensitivity. We show that DOP-3 is required for the ability of food and exogenous dopamine to rescue the octanol avoidance defect of rgs-3 mutant animals. In addition, otherwise wild-type animals lacking DOP-3 function are hypersensitive to dilute octanol, reminiscent of cat-2 mutants. Furthermore, we demonstrate that DOP-3 function in the ASH sensory neurons is sufficient to rescue the hypersensitivity of dop-3 mutant animals, while dop-3 RNAi knockdown in ASH results in octanol hypersensitivity. Taken together, our data suggest that dopaminergic signaling through DOP-3 normally acts to dampen ASH signaling and behavioral sensitivity to octanol.

Optical interrogation of neural circuits in Caenorhabditis elegans

The nematode Caenorhabditis elegans has a compact nervous system with only 302 neurons. Whereas most of the synaptic connections between these neurons have been identified by electron microscopy serial reconstructions, functional connections have been inferred between only a few neurons through combinations of electrophysiology, cell ablation, in vivo calcium imaging and genetic analysis. To map functional connections between neurons, we combined in vivo optical stimulation with simultaneous calcium imaging. We analyzed the connections from the ASH sensory neurons and RIM interneurons to the command interneurons AVA and AVD. Stimulation of ASH or RIM neurons using channelrhodopsin-2 (ChR2) resulted in activation of AVA neurons, evoking an avoidance behavior. Our results demonstrate that we can excite specific neurons expressing ChR2 while simultaneously monitoring G-CaMP fluorescence in several other neurons, making it possible to rapidly decipher functional connections in C. elegans neural circuits.

Acetylation Targets Mutant Huntingtin to Autophagosomes for Degradation

Huntington's disease (HD) is an incurable neurodegenerative disease caused by neuronal accumulation of the mutant protein huntingtin. Improving clearance of the mutant protein is expected to prevent cellular dysfunction and neurodegeneration in HD. We report here that such clearance can be achieved by posttranslational modification of the mutant Huntingtin (Htt) by acetylation at lysine residue 444 (K444). Increased acetylation at K444 facilitates trafficking of mutant Htt into autophagosomes, significantly improves clearance of the mutant protein by macroautophagy, and reverses the toxic effects of mutant huntingtin in primary striatal and cortical neurons and in a transgenic C. elegans model of HD. In contrast, mutant Htt that is rendered resistant to acetylation dramatically accumulates and leads to neurodegeneration in cultured neurons and in mouse brain. These studies identify acetylation as a mechanism for removing accumulated protein in HD, and more broadly for actively targeting proteins for degradation by autophagy.

Three Distinct Amine Receptors Operating at Different Levels within the Locomotory Circuit Are Each Essential for the Serotonergic Modulation of Chemosensation inCaenorhabditis elegans

Serotonin modulates behavioral plasticity in both vertebrates and invertebrates and in Caenorhabditis elegans regulates key behaviors, including locomotion, aversive learning and olfaction through at least four different 5-HT receptors. In the present study, we examined the serotonergic stimulation of aversive responses to dilute octanol in animals containing null alleles of these 5-HT receptors. Both ser-1 and mod-1 null animals failed to increase sensitivity to dilute octanol on food/5-HT, in contrast to wild-type, ser-4 or ser-7 null animals. 5-HT sensitivity was restored by the expression of MOD-1 and SER-1 in the AIB or potentially the AIY, and RIA interneurons of mod-1 and ser-1 null animals, respectively. Because none of these 5-HT receptors appear to be expressed in the ASH sensory neurons mediating octanol sensitivity, we identified a 5-HT(6)-like receptor, F16D3.7(SER-5), that was required for food/5-HT-dependent increases in octanol sensitivity. ser-5 null animals failed to increase octanol sensitivity in the presence of food/5-HT and sensitivity could be restored by expression of SER-5 in the ASHs. Similarly, the RNAi knockdown of ser-5 expression in the ASHs of wild-type animals also abolished 5-HT-dependent increases in octanol sensitivity, suggesting that SER-5 modulates the octanol responsiveness of the ASHs directly. Together, these results suggest that multiple amine receptors, functioning at different levels within the locomotory circuit, are each essential for the serotonergic modulation of ASH-mediated aversive responses.

Microfluidics for in vivo imaging of neuronal and behavioral activity in Caenorhabditis elegans

The nematode C. elegans is an excellent model organism for studying behavior at the neuronal level. Because of the organism's small size, it is challenging to deliver stimuli to C. elegans and monitor neuronal activity in a controlled environment. To address this problem, we developed two microfluidic chips, the 'behavior' chip and the 'olfactory' chip for imaging of neuronal and behavioral responses in C. elegans. We used the behavior chip to correlate the activity of AVA command interneurons with the worm locomotion pattern. We used the olfactory chip to record responses from ASH sensory neurons exposed to high-osmotic-strength stimulus. Observation of neuronal responses in these devices revealed previously unknown properties of AVA and ASH neurons. The use of these chips can be extended to correlate the activity of sensory neurons, interneurons and motor neurons with the worm's behavior.

Autophagy Genes Protect Against Disease Caused by Polyglutamine Expansion Proteins in Caenorhabditis elegans

Expanded polyglutamine (polyQ) proteins aggregate intracellularly in Huntington’s disease and other neurodegenerative disorders. The lysosomal degradation pathway, autophagy, is known to promote clearance of polyQ protein aggregates in cultured cells. Moreover, basal autophagy in neuronal cells in mice prevents neurodegeneration by suppressing the accumulation of abnormal intracellular proteins. However, it is not yet known whether autophagy genes play a role in vivo in protecting against disease caused by mutant aggregate-prone, expanded polyQ proteins. To examine this question, we used two models of polyQ-induced toxicity in C. elegans, including the expression of polyQ40 aggregates in muscle and the expression of a human huntingtin disease fragment containing a polyQ tract of 150 residues (Htn-Q150) in ASH sensory neurons. Here, we show that genetic inactivation of autophagy genes accelerates the accumulation of polyQ40 aggregates in C. elegans muscle cells and exacerbates polyQ40-induced muscle dysfunction. Autophagy gene inactivation also increases the accumulation of Htn-Q150 aggregates in C. elegans ASH sensory neurons and results in enhanced neurodegeneration. These data provide in vivo genetic evidence that autophagy genes suppress the accumulation of polyQ aggregates and protect cells from disease caused by polyQ toxicity.

Antagonistic sensory cues generate gustatory plasticity in Caenorhabditis elegans

Caenorhabditis elegans shows chemoattraction to 0.1-200 mM NaCl, avoidance of higher NaCl concentrations, and avoidance of otherwise attractive NaCl concentrations after prolonged exposure to NaCl (gustatory plasticity). Previous studies have shown that the ASE and ASH sensory neurons primarily mediate attraction and avoidance of NaCl, respectively. Here we show that balances between at least four sensory cell types, ASE, ASI, ASH, ADF and perhaps ADL, modulate the response to NaCl. Our results suggest that two NaCl-attraction signalling pathways exist, one of which uses Ca(2+)/cGMP signalling. In addition, we provide evidence that attraction to NaCl is antagonised by G-protein signalling in the ASH neurons, which is desensitised by the G-protein-coupled receptor kinase GRK-2. Finally, the response to NaCl is modulated by G-protein signalling in the ASI and ADF neurons, a second G-protein pathway in ASH and cGMP signalling in neurons exposed to the body fluid.

In vivo imaging of C. elegans ASH neurons: cellular response and adaptation to chemical repellents.

ASH sensory neurons are required in Caenorhabditis elegans for a wide range of avoidance behaviors in response to chemical repellents, high osmotic solutions and nose touch. The ASH neurons are therefore hypothesized to be polymodal nociceptive neurons. To understand the nature of polymodal sensory response and adaptation at the cellular level, we expressed the calcium indicator protein cameleon in ASH and analyzed intracellular Ca(2+) responses following stimulation with chemical repellents, osmotic shock and nose touch. We found that a variety of noxious stimuli evoked strong responses in ASH including quinine, denatonium, detergents, heavy metals, both hyper- and hypo-osmotic shock and nose touch. We observed that repeated chemical stimulation led to a reversible reduction in the magnitude of the sensory response, indicating that adaptation occurs within the ASH sensory neuron. A key component of ASH adaptation is GPC-1, a G-protein gamma-subunit expressed specifically in chemosensory neurons. We hypothesize that G-protein gamma-subunit heterogeneity provides a mechanism for repellent-specific adaptation, which could facilitate discrimination of a variety of repellents by these polymodal sensory neurons.

The DAF-7 TGF-beta signaling pathway regulates chemosensory receptor gene expression in C. elegans.

Regulation of chemoreceptor gene expression in response to environmental or developmental cues provides a mechanism by which animals can alter their sensory responses. Here we demonstrate a role for the daf-7 TGF-beta pathway in the regulation of expression of a subset of chemoreceptor genes in Caenorhabditis elegans. We describe a novel role of this pathway in maintaining receptor gene expression in the adult and show that the DAF-4 type II TGF-beta receptor functions cell-autonomously to modulate chemoreceptor expression. We also find that the alteration of receptor gene expression in the ASI chemosensory neurons by environmental signals, such as levels of a constitutively produced pheromone, may be mediated via a DAF-7-independent pathway. Receptor gene expression in the ASI and ASH sensory neurons appears to be regulated via distinct mechanisms. Our results suggest that the expression of individual chemoreceptor genes in C. elegans is subject to multiple modes of regulation, thereby ensuring that animals exhibit the responses most appropriate for their developmental stage and environmental conditions.