Mechanosensory Neurons Research Articles

The cellular basis of mechanosensation in mammalian tongue.

Mechanosensory neurons that innervate the tongue provide essential information to guide feeding, speech, and social grooming. We use invivo calcium imaging of mouse trigeminal ganglion neurons to identify functional groups of mechanosensory neurons innervating the anterior tongue. These sensory neurons respond to thermal and mechanical stimulation. Analysis of neuronal activity patterns reveal that most mechanosensory trigeminal neurons are tuned to detect moving stimuli across the tongue. Using an unbiased, multilayer hierarchical clustering approach to classify pressure-evoked activity based on temporal response dynamics, we identify five functional classes of mechanosensory neurons with distinct force-response relations and adaptation profiles. These populations are tuned to detect different features of touch. Molecular markers of functionally distinct clusters are identified by analyzing cluster representation in genetically marked neuronal subsets. Collectively, these studies provide a platform for defining the contributions of functionally distinct mechanosensory neurons to oral behaviors crucial for survival in mammals.

Functional specializations of Drosophila primary mechanosensory neurons.

Understanding how forces activate primary mechanosensors, and how neurons encode detailed features of mechanical stimuli, has proved difficult. This is due in part to the difficulty of recording activity from identifiable mechanosensors in situ while delivering controlled stimuli.

A dedicate sensorimotor circuit enables fine texture discrimination by active touch.

Active touch facilitates environments exploration by voluntary, self-generated movements. However, the neural mechanisms underlying sensorimotor control for active touch are poorly understood. During foraging and feeding, Drosophila gather information on the properties of food (texture, hardness, taste) by constant probing with their proboscis. Here we identify a group of neurons (sd-L neurons) on the fly labellum that are mechanosensitive to labellum displacement and synapse onto the sugar-sensing neurons via axo-axonal synapses to induce preference to harder food. These neurons also feed onto the motor circuits that control proboscis extension and labellum spreading to provide on-line sensory feedback critical for controlling the probing processes, thus facilitating ingestion of less liquified food. Intriguingly, this preference was eliminated in mated female flies, reflecting an elevated need for softer food. Our results propose a sensorimotor circuit composed of mechanosensory, gustatory and motor neurons that enables the flies to select ripe yet not over-rotten food by active touch.

BAM15 Relieves Neurodegeneration in Aged Caenorhabditis elegans and Extends Lifespan.

BAM15 was recently screened as a protonophore uncoupler specifically for the mitochondrial membrane but not the plasma membrane. It is equally as potent as FCCP, but less toxic. Previously, mitochondrial uncoupling via DNP alleviates neurodegeneration in the nematode Caenorhabditis elegans during aging. Therefore, we investigated whether BAM15 uncouplers could phenotypically and functionally reduce neuronal defects in aged nematodes. We observed green fluorescence protein-tagged mechanosensory neurons and performed touch and chemotaxis assays during aging. Wild-type animals treated with both 50 µM BAM15 and 10 µM DNP showed reduced mechanosensory neuronal defects during aging, which correlates with the maintenance of touch responses and short-term memory during aging. Uncoupler mutant ucp-4 also responded the same way as the wild-type, reducing neurodegeneration in 50 µM BAM15 and 10 µM DNP-treated animals compared to the DMSO control. These results suggest that 50 µM BAM15 alleviates neurodegeneration phenotypically and functionally in C. elegans during aging, potentially through mitochondrial uncoupling. In accordance with the preserved neuronal shape and function in aged C. elegans, 50 µM BAM15 extended the mean lifespan of both wild-type and ucp-4 mutants.

Spontaneous activity in whisker-innervating region of neonatal mouse trigeminal ganglion

Spontaneous activity during the early postnatal period is thought to be crucial for the establishment of mature neural circuits. It remains unclear if the peripheral structure of the developing somatosensory system exhibits spontaneous activity, similar to that observed in the retina and cochlea of developing mammals. By establishing an ex vivo calcium imaging system, here we found that neurons in the whisker-innervating region of the trigeminal ganglion (TG) of neonatal mice generate spontaneous activity. A small percentage of neurons showed some obvious correlated activity, and these neurons were mostly located close to one another. TG spontaneous activity was majorly exhibited by medium-to-large diameter neurons, a characteristic of mechanosensory neurons, and was blocked by chelation of extracellular calcium. Moreover, this activity was diminished by the adult stage. Spontaneous activity in the TG during the first postnatal week could be a source of spontaneous activity observed in the neonatal mouse barrel cortex.

Science, Medicine, and the Anesthesiologist

Science, Medicine, and the Anesthesiologist

TRPV channel nanchung and TRPA channel water witch form insecticide-activated complexes

We have previously shown that insect vanilloid-type transient receptor potential (TRPV) channels Nanchung (Nan) and Inactive (Iav) form complexes, which can be over-stimulated and eventually silenced by commercial insecticides, afidopyropen, pymetrozine and pyrifluquinazon. Silencing of the TRPV channels by the insecticides perturbs function of the mechano-sensory organs, chordotonal organs, disrupting sound perception, gravitaxis, and feeding. In addition to TRPV channels, chordotonal organs express an ankyrin-type transient receptor potential (TRPA) channel, Water witch (Wtrw). Genetic data implicate Wtrw in sound and humidity sensing, although the signaling pathway, which links Wtrw to these functions has not been clearly defined. Here we show that, in heterologous system, Nan and Wtrw form calcium channels, which can be activated by afidopyropen, pymetrozine and an endogenous agonist, nicotinamide. Analogous to Nan-Iav heteromers, Nan forms the main binding interface for afidopyropen, whereas co-expression of Wtrw dramatically increases its binding affinity. Pymetrozine competes with afidopyropen for binding to Nan-Wtrw complexes, suggesting that these compounds have overlapping binding sites. Analysis of Drosophila single-nucleus transcriptomic atlas revealed co-expression of nan and wtrw in audio- and mechanosensory neurons. The observation that Nan can form insecticide-sensitive heteromers with more than one type of TRP channels, raises a possibility that Nan may partner with some other TRP channel(s). In addition, we show that Wtrw can be activated by plant-derived reactive electrophiles, allyl isothiocyanate and cinnamaldehyde, defining new molecular target for these repellents.

Synaptic branch stability is mediated by non-enzymatic functions of MEC-17/αTAT1 and ATAT-2

Microtubules are fundamental elements of neuronal structure and function. They are dynamic structures formed from protofilament chains of α- and β-tubulin heterodimers. Acetylation of the lysine 40 (K40) residue of α-tubulin protects microtubules from mechanical stresses by imparting structural elasticity. The enzyme responsible for this acetylation event is MEC-17/αTAT1. Despite its functional importance, however, the consequences of altered MEC-17/αTAT1 levels on neuronal structure and function are incompletely defined. Here we demonstrate that overexpression or loss of MEC-17, or of its functional paralogue ATAT-2, causes a delay in synaptic branch extension, and defective synaptogenesis in the mechanosensory neurons of Caenorhabditis elegans. Strikingly, by adulthood, the synaptic branches in these animals are lost, while the main axon shaft remains mostly intact. We show that MEC-17 and ATAT-2 regulate the stability of the synaptic branches largely independently from their acetyltransferase domains. Genetic analyses reveals novel interactions between both mec-17 and atat-2 with the focal adhesion gene zyx-1/Zyxin, which has previously been implicated in actin remodelling. Together, our results reveal new, acetylation-independent roles for MEC-17 and ATAT-2 in the development and maintenance of neuronal architecture.

Strongly Truncated Dnaaf4 Plays a Conserved Role in Drosophila Ciliary Dynein Assembly as Part of an R2TP-Like Co-Chaperone Complex With Dnaaf6.

Axonemal dynein motors are large multi-subunit complexes that drive ciliary movement. Cytoplasmic assembly of these motor complexes involves several co-chaperones, some of which are related to the R2TP co-chaperone complex. Mutations of these genes in humans cause the motile ciliopathy, Primary Ciliary Dyskinesia (PCD), but their different roles are not completely known. Two such dynein (axonemal) assembly factors (DNAAFs) that are thought to function together in an R2TP-like complex are DNAAF4 (DYX1C1) and DNAAF6 (PIH1D3). Here we investigate the Drosophila homologues, CG14921/Dnaaf4 and CG5048/Dnaaf6. Surprisingly, Drosophila Dnaaf4 is truncated such that it completely lacks a TPR domain, which in human DNAAF4 is likely required to recruit HSP90. Despite this, we provide evidence that Drosophila Dnaaf4 and Dnaaf6 proteins can associate in an R2TP-like complex that has a conserved role in dynein assembly. Both are specifically expressed and required during the development of the two Drosophila cell types with motile cilia: mechanosensory chordotonal neurons and sperm. Flies that lack Dnaaf4 or Dnaaf6 genes are viable but with impaired chordotonal neuron function and lack motile sperm. We provide molecular evidence that Dnaaf4 and Dnaaf6 are required for assembly of outer dynein arms (ODAs) and a subset of inner dynein arms (IDAs).

Synaptophysin is a selective marker for axons in human cutaneous end organ complexes

BackgroundSmall clear synaptic-like vesicles fill axon terminals of mechanoreceptors. Their functional significance is controversial and probably includes release of neurotransmitters from afferent axon terminals. Synaptophysin, a major protein of the synaptic vesicle membrane, is present in presynaptic endings of the central and peripheral nervous systems. It is also expressed in mechanosensory neurons which extend into skin forming sensory corpuscles. Nevertheless, synaptophysin occurrence in these structures has never been investigated. MethodsHere we used immunohistochemistry to detect synaptophysin in adult human dorsal root ganglia, cutaneous Meissner and Pacinian corpuscles and Merkel cell-neurite complexes from foetal to elderly period. Moreover, we analyzed whether synaptophysin co-localizes with the mechano-gated protein PIEZO2. ResultsSynaptophysin immunoreactivity was observed in primary sensory neurons (36 ± 6%) covering the entire soma size ranges. Axons of Meissner’s and Pacinian corpuscles were positive for synaptophysin from 36 and 12 weeks of estimated gestational age respectively, to 72 years old. Synaptophysin was also detected in Merkel cells (from 14 weeks of estimated gestational age to old age). Additionally in adult skin, synaptophysin and PIEZO2 co-localized in the axon of Meissner and Pacinian corpuscles, Merkel cells as well as in some axons of Merkel cell-neurite complexes. ConclusionPresent results demonstrate that a subpopulation of primary sensory neurons and their axon terminals forming cutaneous sensory corpuscles contain synaptophysin, a typical presynaptic vesicle protein. Although the functional relevance of these findings is unknown it might be related to neurotransmission mechanisms linked to mechanotransduction.

Focused ultrasound excites action potentials in mammalian peripheral neurons in part through the mechanically gated ion channel PIEZO2

Neurons of the peripheral nervous system (PNS) are tasked with diverse roles, from encoding touch, pain, and itch to interoceptive control of inflammation and organ physiology. Thus, technologies that allow precise control of peripheral nerve activity have the potential to regulate a wide range of biological processes. Noninvasive modulation of neuronal activity is an important translational application of focused ultrasound (FUS). Recent studies have identified effective strategies to modulate brain circuits; however, reliable parameters to control the activity of the PNS are lacking. To develop robust noninvasive technologies for peripheral nerve modulation, we employed targeted FUS stimulation and electrophysiology in mouse ex vivo skin-saphenous nerve preparations to record the activity of individual mechanosensory neurons. Parameter space exploration showed that stimulating neuronal receptive fields with high-intensity, millisecond FUS pulses reliably and repeatedly evoked one-to-one action potentials in all peripheral neurons recorded. Interestingly, when neurons were classified based on neurophysiological properties, we identified a discrete range of FUS parameters capable of exciting all neuronal classes, including myelinated A fibers and unmyelinated C fibers. Peripheral neurons were excited by FUS stimulation targeted to either cutaneous receptive fields or peripheral nerves, a key finding that increases the therapeutic range of FUS-based peripheral neuromodulation. FUS elicited action potentials with millisecond latencies compared with electrical stimulation, suggesting ion channel–mediated mechanisms. Indeed, FUS thresholds were elevated in neurons lacking the mechanically gated channel PIEZO2. Together, these results demonstrate that transcutaneous FUS drives peripheral nerve activity by engaging intrinsic mechanotransduction mechanisms in neurons [B. U. Hoffman, PhD thesis, (2019)].

Central projections of cercal giant interneurons in the adult field cricket, Gryllus bimaculatus.

The structures of neurons, such as dendrites and axonal projections, are closely related to their response properties and their specific functions in neural circuits. Identified neurons, having genetically determined morphological features and pre- and postsynaptic partners, play significant roles in specific behaviors. Giant interneurons (GIs) are identified in the terminal abdominal ganglion of the cricket as mechanosensory projection neurons and are sensitive to airflow stimulation of the cerci. GIs are classified into ventral GIs (vGIs) or dorsal GIs (dGIs) depending on the location of their axons running within the connective nerve cord. Based on their response properties to airflow, vGIs are presumed to be involved in triggering the wind-elicited escape response, whereas dGIs are thought to be airflow direction-encoding neurons. The previous findings regarding airflow sensitivity point to possible differences in the morphology of the central projections that may correspond to their neural functions. However, the detailed morphologies of the GIs in the cephalic and thoracic ganglia of adult crickets remain unclear. In this study, we stained six GIs, namely, GI 8-1 (medial giant interneuron, MGI), 9-1 (lateral giant interneuron, LGI), 9-2, 9-3, 10-2, and 10-3, using intracellular iontophoretic or pressure injection of dyes. Staining revealed remarkable differences in the axonal branching patterns between vGIs and dGIs. The dGIs were further divided into subgroups based on the profiles of their axon collaterals and projection sites in the brain. The anatomical differences between the GIs' central projections seemed to be related to their information encodement and behavioral functions.

A human coronavirus OC43-derived polypeptide causes neuropathic pain.

Human coronaviruses have been recently implicated in neurological sequelae by insufficiently understood mechanisms. We here identify an amino acid sequence within the HCoV‐OC43 p65‐like protein homologous to the evolutionarily conserved motif of myelin basic protein (MBP). Because MBP‐derived peptide exposure in the sciatic nerve produces pronociceptive activity in female rodents, we examined whether a synthetic peptide derived from the homologous region of HCoV‐OC43 (OC43p) acts by molecular mimicry to promote neuropathic pain. OC43p, but not scrambled peptides, induces mechanical hypersensitivity in rats following intrasciatic injections. Transcriptome analyses of the corresponding spinal cords reveal upregulation of genes and signaling pathways with known nociception‐, immune‐, and cellular energy‐related activities. Affinity capture shows the association of OC43p with an Na+/K+‐transporting ATPase, providing a potential direct target and mechanistic insight into virus‐induced effects on energy homeostasis and the sensory neuraxis. We propose that HCoV‐OC43 polypeptides released during infection dysregulate normal nervous system functions through molecular mimicry of MBP, leading to mechanical hypersensitivity. Our findings might provide a new paradigm for virus‐induced neuropathic pain.

Ubiquitin ligase activity inhibits Cdk5 to control axon termination.

The Cdk5 kinase plays prominent roles in nervous system development, plasticity, behavior and disease. It also has important, non-neuronal functions in cancer, the immune system and insulin secretion. At present, we do not fully understand negative regulatory mechanisms that restrict Cdk5. Here, we use Caenorhabditis elegans to show that CDK-5 is inhibited by the RPM-1/FSN-1 ubiquitin ligase complex. This atypical RING ubiquitin ligase is conserved from C. elegans through mammals. Our finding originated from unbiased, in vivo affinity purification proteomics, which identified CDK-5 as a putative RPM-1 substrate. CRISPR-based, native biochemistry showed that CDK-5 interacts with the RPM-1/FSN-1 ubiquitin ligase complex. A CRISPR engineered RPM-1 substrate 'trap' enriched CDK-5 binding, which was mediated by the FSN-1 substrate recognition module. To test the functional genetic relationship between the RPM-1/FSN-1 ubiquitin ligase complex and CDK-5, we evaluated axon termination in mechanosensory neurons and motor neurons. Our results indicate that RPM-1/FSN-1 ubiquitin ligase activity restricts CDK-5 to control axon termination. Collectively, these proteomic, biochemical and genetic results increase our understanding of mechanisms that restrain Cdk5 in the nervous system.

Sexually dimorphic peripheral sensory neurons regulate copulation duration and persistence in male Drosophila

Peripheral sensory neurons are the gateway to the environment across species. In Drosophila, olfactory and gustatory senses are required to initiate courtship, as well as for the escalation of courtship patterns that lead to copulation. To be successful, copulation must last long enough to ensure the transfer of sperm and seminal fluid that ultimately leads to fertilization. The peripheral sensory information required to regulate copulation duration is unclear. Here, we employed genetic manipulations that allow driving gene expression in the male genitalia as a tool to uncover the role of these genitalia specific neurons in copulation. The fly genitalia contain sex-specific bristle hairs innervated by mechanosensory neurons. To date, the role of the sensory information collected by these peripheral neurons in male copulatory behavior is unknown. We confirmed that these MSNs are cholinergic and co-express both fru and dsx. We found that the sensory information received by the peripheral sensory neurons from the front legs (GRNs) and mechanosensory neurons (MSNs) at the male genitalia contribute to the regulation of copulation duration. Moreover, our results show that their function is required for copulation persistence, which ensures copulation is undisrupted in the presence of environmental stress before sperm transfer is complete.

Noncanonical necrosis in 2 different cell types in a Caenorhabditis elegans NAD+ salvage pathway mutant.

Necrosis was once described as a chaotic unregulated response to cellular insult. We now know that necrosis is controlled by multiple pathways in response to many different cellular conditions. In our pnc-1 NAD+ salvage deficient Caenorhabditis elegans model excess nicotinamide induces excitotoxic death in uterine-vulval uv1 cells and OLQ mechanosensory neurons. We sought to characterize necrosis in our pnc-1 model in the context of well-characterized necrosis, apoptosis, and autophagy pathways in C. elegans. We confirmed that calpain and aspartic proteases were required for uv1 necrosis, but changes in intracellular calcium levels and autophagy were not, suggesting that uv1 necrosis occurs by a pathway that diverges from mec-4d-induced touch cell necrosis downstream of effector aspartic proteases. OLQ necrosis does not require changes in intracellular calcium, the function of calpain or aspartic proteases, or autophagy. Instead, OLQ survival requires the function of calreticulin and calnexin, pro-apoptotic ced-4 (Apaf1), and genes involved in both autophagy and axon guidance. In addition, the partially OLQ-dependent gentle nose touch response decreased significantly in pnc-1 animals on poor quality food, further suggesting that uv1 and OLQ necrosis differ downstream of their common trigger. Together these results show that, although phenotypically very similar, uv1, OLQ, and touch cell necrosis are very different at the molecular level.

An in vivo assay to study locomotion in Caenorhabditis elegans

Adaptation in the sensory-mechanical loop during locomotion is a powerful mechanism that allows organisms to survive in different conditions and environments. Motile animals need to alter motion patterns in different environments. For example, crocodiles and other animals can walk on solid ground but switch to swimming in water beds. The nematode Caenorhabditis elegans also shows adaptability by employing thrashing behaviour in low viscosity media and crawling in high viscosity media. The mechanism that enables this adaptability is an active area of research. It has been attributed previously to neuro-modulation by dopamine and serotonin. This study introduces an experimental assay to physiologically investigate the neuronal mechanisms of modulation of locomotion by dopamine. The technique is utilized to test gait switching while imaging the mechanosensory dopaminergic neurons PDE. Results revealed their role to be not limited to touch sensation, but to sensing surrounding environment resistance as well. The significance of such characterization is improving our understanding of dopamine gait switching which gets impaired in Parkinson's disease.-A locomotion pattern switching system was devised to allow studying this process in vivo in the nematode C. elegans.-This system allowed the study of dopaminergic neurons PDE response as the worms switched from crawling to swimming.

Cnidarian hair cell development illuminates an ancient role for the class IV POU transcription factor in defining mechanoreceptor identity.

Although specialized mechanosensory cells are found across animal phylogeny, early evolutionary histories of mechanoreceptor development remain enigmatic. Cnidaria (e.g. sea anemones and jellyfishes) is the sister group to well-studied Bilateria (e.g. flies and vertebrates), and has two mechanosensory cell types - a lineage-specific sensory effector known as the cnidocyte, and a classical mechanosensory neuron referred to as the hair cell. While developmental genetics of cnidocytes is increasingly understood, genes essential for cnidarian hair cell development are unknown. Here, we show that the class IV POU homeodomain transcription factor (POU-IV) - an indispensable regulator of mechanosensory cell differentiation in Bilateria and cnidocyte differentiation in Cnidaria - controls hair cell development in the sea anemone cnidarian Nematostella vectensis. N. vectensis POU-IV is postmitotically expressed in tentacular hair cells, and is necessary for development of the apical mechanosensory apparatus, but not of neurites, in hair cells. Moreover, it binds to deeply conserved DNA recognition elements, and turns on a unique set of effector genes - including the transmembrane receptor-encoding gene polycystin 1 - specifically in hair cells. Our results suggest that POU-IV directs differentiation of cnidarian hair cells and cnidocytes via distinct gene regulatory mechanisms, and support an evolutionarily ancient role for POU-IV in defining the mature state of mechanosensory neurons.

FlyWire: online community for whole-brain connectomics.

Due to advances in automated image acquisition and analysis, whole-brain connectomes with 100,000 or more neurons are on the horizon. Proofreading of whole-brain automated reconstructions will require many person-years of effort, due to the huge volumes of data involved. Here we present FlyWire, an online community for proofreading neural circuits in a Drosophila melanogaster brain, and explain how its computational and social structures are organized to scale up to whole-brain connectomics. Browser-based 3D interactive segmentation by collaborative editing of a spatially chunked supervoxel graph makes it possible to distribute proofreading to individuals located virtually anywhere in the world. Information in the edit history is programmatically accessible for a variety of uses such as estimating proofreading accuracy or building incentive systems. An open community accelerates proofreading by recruiting more participants and accelerates scientific discovery by requiring information sharing. We demonstrate how FlyWire enables circuit analysis by reconstructing and analysing the connectome of mechanosensory neurons.

An Autonomous Molecular Bioluminescent Reporter (AMBER) for Voltage Imaging in Freely Moving Animals.

Genetically encoded reporters have greatly increased our understanding of biology. While fluorescent reporters have been widely used, photostability and phototoxicity have hindered their use in long-term experiments. Bioluminescence overcomes some of these challenges but requires the addition of an exogenous luciferin limiting its use. Using a modular approach, Autonomous Molecular BioluminEscent Reporter (AMBER), an indicator of membrane potential is engineered. Unlike other bioluminescent systems, AMBER is a voltage-gated luciferase coupling the functionalities of the Ciona voltage-sensing domain (VSD) and bacterial luciferase, luxAB. When co-expressed with the luciferin-producing genes, AMBER reversibly switches the bioluminescent intensity as a function of membrane potential. Using biophysical and biochemical methods, it is shown that AMBER switches its enzymatic activity from an OFF to an ON state as a function of the membrane potential. Upon depolarization, AMBER switches from a low to a high enzymatic activity state, showing a several-fold increase in the bioluminescence output (ΔL/L). AMBER in the pharyngeal muscles and mechanosensory touch neurons of Caenorhabditis elegans is expressed. Using the compressed sensing approach, the electropharingeogram of the C. elegans pharynx is reconstructed, validating the sensor in vivo. Thus, AMBER represents the first fully genetically encoded bioluminescent reporter without requiring exogenous luciferin addition.