Polymodal sensory perception drives settlement and metamorphosis of Ciona larvae.

The Earth's oceans brim with an incredible diversity of microscopic lifeforms, including motile planktonic larvae, whose survival critically depends on effective dispersal in the water column and subsequent exploration of the seafloor to identify a suitable settlement site. How their nervous systems mediate sensing of diverse multimodal cues remains enigmatic. Here, we uncover that the tunicate Ciona intestinalis larvae employ ectodermal sensory cells to sense various mechanical and chemical cues. Combining whole-brain imaging and chemogenetics, we demonstrate that stimuli encoded at the periphery are sufficient to drive global brain-state changes to promote or impede both larval attachment and metamorphosis behaviors. The ability of C. intestinalis larvae to leverage polymodal sensory perception to support information coding and chemotactile behaviors may explain how marine larvae make complex decisions despite streamlined nervous systems.

Neuronal gene expression in two generations of the marine parasitic worm, Cryptocotyle lingua.

Trematodes, or flukes, undergo intricate anatomical and behavioral transformations during their life cycle, yet the functional changes in their nervous system remain poorly understood. We investigated the molecular basis of nervous system function in Cryptocotyle lingua, a species of relevance for fisheries. Transcriptomic analysis revealed a streamlined molecular toolkit with the absence of key signaling pathways and ion channels. Notably, we observed the loss of nitric oxide synthase across the Platyhelminthes. Furthermore, we identified upregulated neuronal genes in dispersal larvae, including those involved in aminergic pathways, synaptic vesicle trafficking, TRPA channels, and surprisingly nitric oxide receptors. Using neuronal markers and in situ hybridization, we hypothesized their functional relevance to larval adaptations and host-finding strategies. Additionally, employing a behavior quantification toolkit, we assessed cercaria motility, facilitating further investigations into the behavior and physiology of parasitic flatworms. This study enhances our understanding of trematode neurobiology and provides insights for targeted antiparasitic strategies.

Ciona spp. and ascidians as bioindicator organisms for evaluating effects of endocrine disrupting chemicals: A discussion paper.

In context of testing, screening and monitoring of endocrine-disrupting (ED) type of environmental pollutants, tunicates could possibly represent a particularly interesting group of bioindicator organisms. These primitive chordates are already important model organisms within developmental and genomics research due to their central position in evolution and close relationship to vertebrates. The solitary ascidians, such as the genus Ciona spp. (vase tunicates), could possibly be extra feasible as ED bioindicators. They have a free-swimming, tadpole-like larval stage that develops extremely quickly (<20 h under favorable conditions), has a short life cycle (typically 2-3 months), are relatively easy to maintain in laboratory culture, have fully sequenced genomes, and transgenic embryos with 3D course data of the embryo ontogeny are available. In this article, we discuss possible roles of Ciona spp. (and other solitary ascidians) as ecotoxicological bioindicator organisms in general but perhaps especially for effect studies of contaminants with presumed endocrine disrupting modes of action.

Comprehensive analysis of locomotion dynamics in the protochordate Ciona intestinalis reveals how neuromodulators flexibly shape its behavioral repertoire.

Vertebrate nervous systems can generate a remarkable diversity of behaviors. However, our understanding of how behaviors may have evolved in the chordate lineage is limited by the lack of neuroethological studies leveraging our closest invertebrate relatives. Here, we combine high-throughput video acquisition with pharmacological perturbations of bioamine signaling to systematically reveal the global structure of the motor behavioral repertoire in the Ciona intestinalis larvae. Most of Ciona's postural variance can be captured by 6 basic shapes, which we term "eigencionas." Motif analysis of postural time series revealed numerous stereotyped behavioral maneuvers including "startle-like" and "beat-and-glide." Employing computational modeling of swimming dynamics and spatiotemporal embedding of postural features revealed that behavioral differences are generated at the levels of motor modules and the transitions between, which may in part be modulated by bioamines. Finally, we show that flexible motor module usage gives rise to diverse behaviors in response to different light stimuli.

Mesmerize is a dynamically adaptable user-friendly analysis platform for 2D and 3D calcium imaging data.

Calcium imaging is an increasingly valuable technique for understanding neural circuits, neuroethology, and cellular mechanisms. The analysis of calcium imaging data presents challenges in image processing, data organization, analysis, and accessibility. Tools have been created to address these problems independently, however a comprehensive user-friendly package does not exist. Here we present Mesmerize, an efficient, expandable and user-friendly analysis platform, which uses a Findable, Accessible, Interoperable and Reproducible (FAIR) system to encapsulate the entire analysis process, from raw data to interactive visualizations for publication. Mesmerize provides a user-friendly graphical interface to state-of-the-art analysis methods for signal extraction & downstream analysis. We demonstrate the broad scientific scope of Mesmerize's applications by analyzing neuronal datasets from mouse and a volumetric zebrafish dataset. We also applied contemporary time-series analysis techniques to analyze a novel dataset comprising neuronal, epidermal, and migratory mesenchymal cells of the protochordate Ciona intestinalis.

Neuropeptide repertoire and 3D anatomy of the ctenophore nervous system.

Ctenophores are gelatinous marine animals famous for locomotion by ciliary combs. Due to the uncertainties of the phylogenetic placement of ctenophores and the absence of some key bilaterian neuronal genes, it has been hypothesized that their neurons evolved independently. Additionally, recent whole-body, single-cell RNA sequencing (scRNA-seq) analysis failed to identify ctenophore neurons using any of the known neuronal molecular markers. To reveal the molecular machinery of ctenophore neurons, we have characterized the neuropeptide repertoire of the ctenophore Mnemiopsis leidyi. Using the machine learning NeuroPID tool, we predicted 129 new putative neuropeptide precursors. Sixteen of them were localized to the subepithelial nerve net (SNN), sensory aboral organ (AO), and epithelial sensory cells (ESCs), providing evidence that they are neuropeptide precursors. Four of these putative neuropeptides had a behavioral effect and increased the animals' swimming speed. Intriguingly, these putative neuropeptides finally allowed us to identify neuronal cell types in single-cell transcriptomic data and reveal the molecular identity of ctenophore neurons. High-resolution electron microscopy and 3D reconstructions of the nerve net underlying the comb plates confirmed a more than 100-year-old hypothesis of anastomoses between neurites of the same cell in ctenophores and revealed that they occur through a continuous membrane. Our work demonstrates the unique ultrastructure of the peptidergic nerve net and a rich neuropeptide repertoire of ctenophores, supporting the hypothesis that the first nervous system(s) evolved as nets of peptidergic cells.

Automated behavioural analysis reveals the basic behavioural repertoire of the urochordate Ciona intestinalis.

Quantitative analysis of animal behaviour in model organisms is becoming an increasingly essential approach for tackling the great challenge of understanding how activity in the brain gives rise to behaviour. Here we used automated image-based tracking to extract behavioural features from an organism of great importance in understanding the evolution of chordates, the free-swimming larval form of the tunicate Ciona intestinalis, which has a compact and fully mapped nervous system composed of only 231 neurons. We analysed hundreds of videos of larvae and we extracted basic geometric and physical descriptors of larval behaviour. Importantly, we used machine learning methods to create an objective ontology of behaviours for C. intestinalis larvae. We identified eleven behavioural modes using agglomerative clustering. Using our pipeline for quantitative behavioural analysis, we demonstrate that C. intestinalis larvae exhibit sensory arousal and thigmotaxis. Notably, the anxiotropic drug modafinil modulates thigmotactic behaviour. Furthermore, we tested the robustness of the larval behavioural repertoire by comparing different rearing conditions, ages and group sizes. This study shows that C. intestinalis larval behaviour can be broken down to a set of stereotyped behaviours that are used to different extents in a context-dependent manner.

Functional conserved non-coding elements among tunicates and chordates.

Non-coding regions with dozens to several hundred base pairs of extreme conservation have been found in all metazoan genomes. The distribution of these conserved non-coding elements (CNE) within and across genomes has suggested that many of them may have roles as transcriptional regulatory elements. A combination of bioinformatics and experimental approaches can be used to identify CNEs with regulatory activity in phylogenetically distant species. Nevertheless, the high divergent rate of genomic sequences of several organisms, such as tunicates, complicates the characterization of these conserved elements and very few examples really may prove their functional activity. We used a comparative approach to facilitate the identification of CNEs among distantly related or highly divergent species and experimentally demonstrated the functional significance of these novel CNEs. We first experimentally tested, in C. robusta and D. rerio transgenic embryos, the regulatory activity of conserved elements associated to genes involved in developmental control among different chordates (Homo sapiens and Danio rerio for vertebrates, Ciona robusta and Ciona savignyi for tunicates and Branchiostoma floridae for cephalochordates). Once demonstrated the cross-species functional conservation of these CNEs, the same gene loci were used as references to locate homologous regions and possible CNEs in available tunicate genomes. Comparison of tunicate-specific and chordate-specific CNEs revealed absence of conservation of the regulatory elements in spite of conservation of regulatory patterns, likely due to evolutionary specification of the respective developmental networks. This result highlights the importance of an integrative in-silico/in-vivo approach to CNEs investigation, encompassing both bioinformatics, essential for putative CNEs identification, and laboratory experiments, pivotal for the understanding of CNEs functionality.

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.

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.

Sensory neuron fates are distinguished by a transcriptional switch that regulates dendrite branch stabilization.

Sensory neurons adopt distinct morphologies and functional modalities to mediate responses to specific stimuli. Transcription factors and their downstream effectors orchestrate this outcome but are incompletely defined. Here, we show that different classes of mechanosensory neurons in C. elegans are distinguished by the combined action of the transcription factors MEC-3, AHR-1, and ZAG-1. Low levels of MEC-3 specify the elaborate branching pattern of PVD nociceptors, whereas high MEC-3 is correlated with the simple morphology of AVM and PVM touch neurons. AHR-1 specifies AVM touch neuron fate by elevating MEC-3 while simultaneously blocking expression of nociceptive genes such as the MEC-3 target, the claudin-like membrane protein HPO-30, that promotes the complex dendritic branching pattern of PVD. ZAG-1 exercises a parallel role to prevent PVM from adopting the PVD fate. The conserved dendritic branching function of the Drosophila AHR-1 homolog, Spineless, argues for similar pathways in mammals.

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.

A gap junction circuit enhances processing of coincident mechanosensory inputs.

Electrical synapses have been shown to be important for enabling and detecting neuronal synchrony in both vertebrates and invertebrates. 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 and may represent a conserved functional unit. The functional relevance of this configuration has been demonstrated for circuits mediating aggregation behavior and nose touch perception. Modeling approaches have been useful for understanding structurally and dynamically more complex electrical circuits. 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.

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.

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

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.

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.

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.

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.