Polymodal Sensory Neurons Research Articles

Optogenetic Stimulation of Nociceptive Escape Behaviors in Drosophila Larvae.

In animals, noxious stimuli activate a neural process called nociception. Drosophila larvae perform a rolling escape locomotion behavior in response to nociceptive sensory stimuli. Noxious mechanical, thermal, and chemical stimuli each trigger this same escape response in larvae. The polymodal sensory neurons that initiate the rolling response have been identified based on the expression patterns of genes that are known to be required for nociception responses. The synaptic output of these neurons, known as class IV multidendritic sensory neurons, is required for behavioral responses to thermal, mechanical, and chemical triggers of the rolling escape locomotion. Importantly, optogenetic stimulation of the class IV multidendritic neurons has also shown that the activation of those cells is sufficient to trigger nociceptive rolling. Optogenetics uses light-activated ion channels expressed in neurons of interest to bypass the normal physiological transduction machinery so that the cell may be activated in response to light that is applied by the investigator. This protocol describes an optogenetic technique that uses channelrhodopsin-2 (ChR2) to activate larval nociceptors and trigger nociceptive rolling. First, we explain how to set up the necessary genetic crosses and culture the larval progeny. Next, we describe how to perform the optogenetic nociception assay on third-instar larvae.

Pain and itch coding mechanisms of polymodal sensory neurons

Pain and itch coding mechanisms in polymodal sensory neurons remain elusive. MrgprD+ neurons represent a major polymodal population and mediate both mechanical pain and nonhistaminergic itch. Here, we show that chemogenetic activation of MrgprD+ neurons elicited both pain- and itch-related behavior in a dose-dependent manner, revealing an unanticipated compatibility between pain and itch in polymodal neurons. While VGlut2-dependent glutamate release is required for both pain and itch transmission from MrgprD+ neurons, the neuropeptide neuromedin B (NMB) is selectively required for itch signaling. Electrophysiological recordings further demonstrated that glutamate synergizes with NMB to excite NMB-sensitive postsynaptic neurons. Ablation of these spinal neurons selectively abolished itch signals from MrgprD+ neurons, without affecting pain signals, suggesting a dedicated itch-processing central circuit. These findings reveal distinct neurotransmitters and neural circuit requirements for pain and itch signaling from MrgprD+ polymodal sensory neurons, providing new insights on coding and processing of pain and itch.

Multisensory integration by polymodal sensory neurons dictates larval settlement in a brainless cnidarian larva.

Multisensory integration (MSI) combines information from more than one sensory modality to elicit behaviours distinct from unisensory behaviours. MSI is best understood in animals with complex brains and specialized centres for parsing different modes of sensory information, but dispersive larvae of sessile marine invertebrates utilize multimodal environmental sensory stimuli to base irreversible settlement decisions on, and most lack complex brains. Here, we examined the sensory determinants of settlement in actinula larvae of the hydrozoan Ectopleura crocea (Cnidaria), which possess a diffuse nerve net. A factorial settlement study revealed that photo-, chemo- and mechanosensory cues each influenced the settlement response in a complex and hierarchical manner that was dependent on specific combinations of cues, an indication of MSI. Additionally, sensory gene expression over development peaked with developmental competence to settle, which in actinulae, requires cnidocyte discharge. Transcriptome analyses also highlighted several deep homological links between cnidarian and bilaterian mechano-, chemo-, and photosensory pathways. Fluorescent in situ hybridization studies of candidate transcripts suggested cellular partitioning of sensory function among the few cell types that comprise the actinula nervous system, where ubiquitous polymodal sensory neurons expressing putative chemo- and photosensitivity interface with mechanoreceptive cnidocytes. We propose a simple multisensory processing circuit, involving polymodal chemo/photosensory neurons and mechanoreceptive cnidocytes, that is sufficient to explain MSI in actinulae settlement. Our study demonstrates that MSI is not exclusive to complex brains, but likely predated and contextualized their evolution.

Nematode homologs of the sour taste receptor Otopetrin1 are evolutionarily conserved acid-sensitive proton channels.

Numerous taste receptors and related molecules have been identified in vertebrates and invertebrates. Otopetrin1 has recently been identified as mammalian sour taste receptor which is essential for acid sensation. However, whether other Otopetrin proteins are involved in PH-sensing remains unknown. In C. elegans, there are eight otopetrin homologous genes but their expression patterns and functions have not been reported so far. Through heterologous expression in HEK293T cells, we found that ceOTOP1a can be activated by acid in NMDG+ solution without conventional cations, which generated inward currents and can be blocked by zinc ions. Moreover, we found that Otopetrin channels are widely expressed in numerous tissues, especially in sensory neurons in the nematode. These results suggest that the biophysical characteristics of the Otopetrin channels in nematodes are generally conserved. However, a series of single gene mutations of otopetrins, which were constructed by CRISPR-Cas9 method, did not affect either calcium responses in ASH polymodal sensory neurons to acid stimulation or acid avoidance behaviors, suggesting that Otopetrin channels might have diverse functions among species. This study reveals that nematode Otopetrins are evolutionarily conserved acid-sensitive proton channels, and provides a framework for further revealing the function and mechanisms of Otopetrin channels in both invertebrates and vertebrates.

Pain and Itch Coding Mechanisms of Polymodal Sensory Neurons

Pain and Itch Coding Mechanisms of Polymodal Sensory Neurons

The Doublesex/Mab-3 domain transcription factor DMD-10 regulates ASH-dependent behavioral responses.

The Doublesex/Mab-3 Domain transcription factor DMD-10 is expressed in several cell types in C. elegans, including in the nervous system. We sought to investigate whether DMD-10 is required for normal neuronal function using behavioral assays. We found that mutation of dmd-10 did not broadly affect behavior. dmd-10 mutants were normal in several behavioral assays including a body bends assay for locomotion, egg laying, chemotaxis and response to gentle touch to the body. dmd-10 mutants did have defects in nose-touch responsiveness, which requires the glutamate receptor GLR-1. However, using quantitative fluorescence microscopy to measure levels of a GLR-1::GFP fusion protein in the ventral nerve cord, we found no evidence supporting a difference in the number of GLR-1 synapses or in the amount of GLR-1 present in dmd-10 mutants. dmd-10 mutants did have decreased responsiveness to high osmolarity, which, along with nose-touch, is sensed by the polymodal sensory neuron ASH. Furthermore, mutation of dmd-10 impaired behavioral response to optogenetic activation of ASH, suggesting that dmd-10 promotes neuronal signaling in ASH downstream of sensory receptor activation. Together our results suggest that DMD-10 is important in regulating the frequency of multiple ASH-dependent behavioral responses.

Sensory Glia Detect Repulsive Odorants and Drive Olfactory Adaptation

Glia are typically considered as supporting cells for neural development and synaptic transmission. Here, we report an active role of a glia in olfactory transduction. As a polymodal sensory neuron in C.elegans, the ASH neuron is previously known to detect multiple aversive odorants. We reveal that the AMsh glia, a sheath for multiple sensory neurons including ASH, cell-autonomously respond to aversive odorants via G-protein-coupled receptors (GPCRs) distinct from those in ASH. Upon activation, the AMsh glia suppress aversive odorant-triggered avoidance and promote olfactory adaptation by inhibiting the ASH neuron via GABA signaling. Thus, we propose a novel two-receptor model where the glia and sensory neuron jointly mediate adaptive olfaction. Our study reveals a non-canonical function of glial cells in olfactory transduction, which may provide new insights into the glia-like supporting cells in mammalian sensory procession.

Polymodal Responses in C. elegans Phasmid Neurons Rely on Multiple Intracellular and Intercellular Signaling Pathways

Animals utilize specialized sensory neurons enabling the detection of a wide range of environmental stimuli from the presence of toxic chemicals to that of touch. However, how these neurons discriminate between different kinds of stimuli remains poorly understood. By combining in vivo calcium imaging and molecular genetic manipulation, here we investigate the response patterns and the underlying mechanisms of the C. elegans phasmid neurons PHA/PHB to a variety of sensory stimuli. Our observations demonstrate that PHA/PHB neurons are polymodal sensory neurons which sense harmful chemicals, hyperosmotic solutions and mechanical stimulation. A repulsive concentration of IAA induces calcium elevations in PHA/PHB and both OSM-9 and TAX-4 are essential for IAA-sensing in PHA/PHB. Nevertheless, the PHA/PHB neurons are inhibited by copper and post-synaptically activated by copper removal. Neuropeptide is likely involved in copper removal-induced calcium elevations in PHA/PHB. Furthermore, mechanical stimulation activates PHA/PHB in an OSM-9-dependent manner. Our work demonstrates how PHA/PHB neurons respond to multiple environmental stimuli and lays a foundation for the further understanding of the mechanisms of polymodal signaling, such as nociception, in more complex organisms.

Ultrasound neuro-modulation chip: activation of sensory neurons in Caenorhabditis elegans by surface acoustic waves.

Ultrasound neuro-modulation has gained increasing attention as a non-invasive method. In this paper, we present an ultrasound neuro-modulation chip, capable of initiating reversal behaviour and activating neurons of C. elegans under the stimulation of a single-shot, short-pulsed ultrasound. About 85.29% ± 6.17% of worms respond to the ultrasound stimulation exhibiting reversal behaviour. Furthermore, the worms can adapt to the ultrasound stimulation with a lower acoustic pulse duration of stimulation. In vivo calcium imaging shows that the activity of ASH, a polymodal sensory neuron in C. elegans, can be directly evoked by the ultrasound stimulation. On the other hand, AFD, a thermal sensitive neuron, cannot be activated by the ultrasound stimulation using the same parameter and the temperature elevation during the stimulation process is relatively small. Consistent with the calcium imaging results, the tax-4 mutants, which are insensitive to temperature increase, do not show a significant difference in avoidance probability compared to the wild type. Therefore, the mechanical effects induced by ultrasound are the main reason for neural and behavioural modulation of C. elegans. With the advantages of confined acoustic energy on the surface, compatible with standard calcium imaging, this neuro-modulation chip could be a powerful tool for revealing the molecular mechanisms of ultrasound neuro-modulation.

Mechanosensation circuitry in Caenorhabditis elegans: A focus on gentle touch

Forward or reverse movement in Caenorhabditis elegans is the result of sequential contraction of muscle cells arranged along the body. In larvae, muscle cells are innervated by distinct classes of motorneurons. B motorneurons regulate forward movement and A motorneurons regulate backward movement. Ablation of the D motor neurons results in animals that are uncoordinated in either direction, which suggests that D motorneurons regulate the interaction between the two circuits. C. elegans locomotion is dictated by inputs from interneurons that regulate the activity of motorneurons which coordinate muscle contraction to facilitate forward or backwards movement. As C. elegans moves through the environment, sensory neurons interpret chemical and mechanical information which is relayed to the motor neurons that control locomotory direction. A mechanosensory input known as light nose touch can be simulated in the laboratory by touching the nose of the animal with a human eyebrow hair. The recoil reaction that follows from light nose touch appears to be primarily mediated by glutamate release from the polymodal sensory neuron ASH. Numerous glutamate receptor types are found in different neurons and interneurons which suggest that several pathways may regulate the aversive response. Based on the phenotypes of mutants in which neuropeptide processing is abolished, neuropeptides play a role in circuit regulation. The light touch response is also regulated by transient receptor channel proteins and degenerin/epithelial sodium channels which modulate the activity of sensory neurons involved in the nose touch response.

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.

Stimulus discrimination by the polymodal sensory neuron

Polymodal sensory neurons inform organisms about the nature of the physical world around them. The activity of these cells guide behaviors including the withdrawal from nocifensive stimuli such as intense heat or harsh force to feeling the comforting weight of a warm blanket. Molecular and genetic analysis of the channel proteins required for these divers behavioral responses have revealed an elaborate and disparate collection of channel proteins within the polymodal sensory neuron. Recent data supports that the biophysical traits of the channel proteins combined with the collection of channels activated during stimulation is sufficient to describe the nature of the stimulus. It is currently unclear what the functional arrangement of channel proteins are during perception. Specifically, are channel proteins arranged in parallel and function independently during perception, or are these channel proteins arranged in functional sensory networks. We propose a hierarchal functional arrangement of channels within polymodal sensory neurons that incorporates aspects of both parallel and serial arrangements of channel proteins.

Activated by Force

An amiloride-sensitive sodium channel transduces intense mechanical stimuli in Caenorhabditis elegans .

DEG/ENaCs Lead by a Nose: Mechanotransduction in a Polymodal Sensory Neuron

Degenerin/epithelial sodium channels (DEG/ENaCs) are luminaries of gentle touch in Caenorhabditis elegans. In this issue of Neuron, Geffeney etal. demonstrate that eponymous DEG-1 channels carry mechanotransduction currents in a polymodal neuron, where they act upstream of transient receptor potential (TRP) channels.

Evolution of a polymodal sensory response network

BackgroundAvoidance of noxious stimuli is essential for the survival of an animal in its natural habitat. Some avoidance responses require polymodal sensory neurons, which sense a range of diverse stimuli, whereas other stimuli require a unimodal sensory neuron, which senses a single stimulus. Polymodality might have evolved to help animals quickly detect and respond to diverse noxious stimuli. Nematodes inhabit diverse habitats and most nematode nervous systems are composed of a small number of neurons, despite a wide assortment in nematode sizes. Given this observation, we speculated that cellular contribution to stereotyped avoidance behaviors would also be conserved between nematode species. The ASH neuron mediates avoidance of three classes of noxious stimuli in Caenorhabditis elegans. Two species of parasitic nematodes also utilize the ASH neuron to avoid certain stimuli. We wanted to extend our knowledge of avoidance behaviors by comparing multiple stimuli in a set of free-living nematode species.ResultsWe used comparative behavioral analysis and laser microsurgery to examine three avoidance behaviors in six diverse species of free-living nematodes. We found that all species tested exhibit avoidance of chemo-, mechano- and osmosensory stimuli. In C. elegans, the bilaterally symmetric polymodal ASH neurons detect all three classes of repellant. We identified the putative ASH neurons in different nematode species by their anatomical positions and showed that in all six species ablation of the ASH neurons resulted in an inability to avoid noxious stimuli. However, in the nematode Pristionchus pacificus, the ADL neuron in addition to the ASH neuron contributed to osmosensation. In the species Caenorhabditis sp. 3, only the ASH neuron was required to mediate nose touch avoidance instead of three neurons in C. elegans. These data suggest that different species can increase or decrease the contribution of additional, non-ASH sensory neurons mediating osmosensation and mechanosensation.ConclusionThe overall conservation of ASH mediated polymodal nociception suggests that it is an ancestral evolutionarily stable feature of sensation. However, the finding that contribution from non-ASH sensory neurons mediates polymodal nociception in some nematode species suggests that even in conserved sensory behaviors, the cellular response network is dynamic over evolutionary time, perhaps shaped by adaptation of each species to its environment.

NEURONAL SUBSTRATES OF COMPLEX BEHAVIORS IN C. ELEGANS

A current challenge in neuroscience is to bridge the gaps between genes, proteins, neurons, neural circuits, and behavior in a single animal model. The nematode Caenorhabditis elegans has unique features that facilitate this synthesis. Its nervous system includes exactly 302 neurons, and their pattern of synaptic connectivity is known. With only five olfactory neurons, C. elegans can dynamically respond to dozens of attractive and repellent odors. Thermosensory neurons enable the nematode to remember its cultivation temperature and to track narrow isotherms. Polymodal sensory neurons detect a wide range of nociceptive cues and signal robust escape responses. Pairing of sensory stimuli leads to long-lived changes in behavior consistent with associative learning. Worms exhibit social behaviors and complex ultradian rhythms driven by Ca(2+) oscillators with clock-like properties. Genetic analysis has identified gene products required for nervous system function and elucidated the molecular and neural bases of behaviors.

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.

Mammalian TRPV4 (VR-OAC) directs behavioral responses to osmotic and mechanical stimuli in Caenorhabditis elegans.

All animals detect osmotic and mechanical stimuli, but the molecular basis for these responses is incompletely understood. The vertebrate transient receptor potential channel vanilloid subfamily 4 (TRPV4) (VR-OAC) cation channel has been suggested to be an osmo/mechanosensory channel. To assess its function in vivo, we expressed TRPV4 in Caenorhabditis elegans sensory neurons and examined its ability to generate behavioral responses to sensory stimuli. C. elegans ASH neurons function as polymodal sensory neurons that generate a characteristic escape behavior in response to mechanical, osmotic, or olfactory stimuli. These behaviors require the TRPV channel OSM-9 because osm-9 mutants do not avoid nose touch, high osmolarity, or noxious odors. Expression of mammalian TRPV4 in ASH neurons of osm-9 worms restored avoidance responses to hypertonicity and nose touch, but not the response to odorant repellents. Mutations known to reduce TRPV4 channel activity also reduced its ability to direct nematode avoidance behavior. TRPV4 function in ASH required the endogenous C. elegans osmotic and nose touch avoidance genes ocr-2, odr-3, osm-10, and glr-1, indicating that TRPV4 is integrated into the normal ASH sensory apparatus. The osmotic and mechanical avoidance responses of TRPV4-expressing animals were different in their sensitivity and temperature dependence from the responses of wild-type animals, suggesting that the TRPV4 channel confers its characteristic properties on the transgenic animals' behavior. These results provide evidence that TRPV4 can function as a component of an osmotic/mechanical sensor in vivo.

Genetics of chemotaxis and thermotaxis in the nematode Caenorhabditis elegans.

Molecular genetic analysis of chemotaxis and theramotaxis in Caenorhabditis elegans has revealed the molecular bases of olfaction, taste, and thermosensation, which, in turn, has demonstrated that sensory signaling in C. elegans is very similar to that in vertebrates. A cyclic nucleotide-gated channel (TAX-2/TAX-4) that is highly homologous to the olfactory and photoreceptor channels in vertebrates is required for taste and thermosensation, in addition to olfaction. A cation channel (OSM-9) that is closely related to a capsaicin receptor channel is required for olfactory adaptation in one olfactory neuron and olfactory sensation in the other olfactory neuron. A novel G alpha protein (ODR-3) is essential for olfactory responses in all olfactory neurons and aversive responses in a polymodal sensory neuron. A G protein-coupled seven-transmembrane receptor (ODR-10) is the first olfactory receptor whose ligand was elucidated. Using chemotaxis and thermotaxis as behavioral paradigms, neural plasticity including learning and memory can be studied genetically in C. elegans.

The C. elegans homeodomain gene unc-42 regulates chemosensory and glutamate receptor expression.

Genes that specify cell fate can influence multiple aspects of neuronal differentiation, including axon guidance, target selection and synapse formation. Mutations in the unc-42 gene disrupt axon guidance along the C. elegans ventral nerve cord and cause distinct functional defects in sensory-locomotory neural circuits. Here we show that unc-42 encodes a novel homeodomain protein that specifies the fate of three classes of neurons in the Caenorhabditis elegans nervous system: the ASH polymodal sensory neurons, the AVA, AVD and AVE interneurons that mediate repulsive sensory stimuli to the nematode head and anterior body, and a subset of motor neurons that innervate head and body-wall muscles. unc-42 is required for the expression of cell-surface receptors that are essential for the mature function of these neurons. In mutant animals, the ASH sensory neurons fail to express SRA-6 and SRB-6, putative chemosensory receptors. The AVA, AVD and AVE interneurons and RME and RMD motor neurons of unc-42 mutants similarly fail to express the GLR-1 glutamate receptor. These results show that unc-42 performs an essential role in defining neuron identity and contributes to the establishment of neural circuits in C. elegans by regulating the transcription of glutamate and chemosensory receptor genes.