The atypical 'hippocampal' glutamate receptor coupled to phospholipase D that controls stretch-sensitivity in primary mechanosensory nerve endings is homomeric purely metabotropic GluK2.

A metabotropic glutamate receptor coupled to phospholipase D (PLD-mGluR) was discovered in the hippocampus over three decades ago. Its pharmacology and direct linkage to PLD activation are well established and indicate it is a highly atypical glutamate receptor. A receptor with the same pharmacology is present in spindle primary sensory terminals where its blockade can totally abolish, and its activation can double, the normal stretch-evoked firing. We report here the first identification of this PLD-mGluR protein, by capitalizing on its expression in primary mechanosensory terminals, developing an enriched source, pharmacological profiling to identify an optimal ligand, and then functionalizing it as a molecular tool. Evidence from immunofluorescence, western and far-western blotting indicates PLD-mGluR is homomeric GluK2, since GluK2 is the only glutamate receptor protein/receptor subunit present in spindle mechanosensory terminals. Its expression was also found in the lanceolate palisade ending of hair follicle, also known to contain the PLD-mGluR. Finally, in a mouse model with ionotropic function ablated in the GluK2 subunit, spindle glutamatergic responses were still present, confirming it acts purely metabotropically. We conclude the PLD-mGluR is a homomeric GluK2 kainate receptor signalling purely metabotropically and it is common to other, perhaps all, primary mechanosensory endings.

The making of a proprioceptor: a tale of two identities.

Proprioception, the sense of body position in space, has a critical role in the control of posture and movement. Aside from skin and joint receptors, the main sources of proprioceptive information in tetrapods are mechanoreceptive end organs in skeletal muscle: muscle spindles (MSs) and Golgi tendon organs (GTOs). The sensory neurons that innervate these receptors are divided into subtypes that detect discrete aspects of sensory information from muscles with different biomechanical functions. Despite the importance of proprioceptive neurons in motor control, the developmental mechanisms that control the acquisition of their distinct functional properties and positional identity are not yet clear. In this review, we discuss recent findings on the development of mouse proprioceptor subtypes and challenges in defining them at the molecular and functional level.

Engineering mechanoreceptor feature selectivity.

Touch and proprioception rely on the discriminative abilities of distinct classes of mechanosensory neurons. In this issue of Neuron, two studies1,2 provide evidence that biomechanical mechanisms and ultrastructural cellular specializations are key contributors in defining mechanoreceptor stimulus threshold and selectivity.

Influencers in the Somatosensory System: Extrinsic Control of Sensory Neuron Phenotypes.

Somatosensory neurons in dorsal root ganglia (DRG) comprise several main subclasses: high threshold nociceptors/thermoceptors, high- and low-threshold mechanoreceptors, and proprioceptors. Recent years have seen an explosion in the identification of molecules that underlie the functional diversity of these sensory modalities. They also have begun to reveal the developmental mechanisms that channel the emergence of this subtype diversity, solidifying the importance of peripheral instructive signals. Somatic sensory neurons collectively serve numerous essential physiological and protective roles, and as such, an increased understanding of the processes that underlie the specialization of these sensory subtypes is not only biologically interesting but also clinically relevant.

Proprioception: A New Era Set in Motion by Emerging Genetic and Bionic Strategies?

The generation of an internal body model and its continuous update is essential in sensorimotor control. Although known to rely on proprioceptive sensory feedback, the underlying mechanism that transforms this sensory feedback into a dynamic body percept remains poorly understood. However, advances in the development of genetic tools for proprioceptive circuit elements, including the sensory receptors, are beginning to offer new and unprecedented leverage to dissect the central pathways responsible for proprioceptive encoding. Simultaneously, new data derived through emerging bionic neural machine-interface technologies reveal clues regarding the relative importance of kinesthetic sensory feedback and insights into the functional proprioceptive substrates that underlie natural motor behaviors.

MS and GTO proprioceptor subtypes in the molecular genetic era: Opportunities for new advances and perspectives.

Proprioceptive feedback from skeletal muscle is an integral element of motor control, yet the precise physiological roles of muscle spindle (MS) and Golgi tendon organ (GTO) sensory receptors have remained difficult to disentangle due to technical limitations. New insights into the molecular basis of MS and GTO afferent subtypes offers genetic opportunities to further our understanding of the distinct functional features of these proprioceptor classes, while at the same time revealing additional layers of complexity in the regulation of coordinated motor output.

An expansion of the non-coding genome and its regulatory potential underlies vertebrate neuronal diversity.

Proper assembly and function of the nervous system requires the generation of a uniquely diverse population of neurons expressing a cell-type-specific combination of effector genes that collectively define neuronal morphology, connectivity, and function. How countless partially overlapping but cell-type-specific patterns of gene expression are controlled at the genomic level remains poorly understood. Here we show that neuronal genes are associated with highly complex gene regulatory systems composed of independent cell-type- and cell-stage-specific regulatory elements that reside in expanded non-coding genomic domains. Mapping enhancer-promoter interactions revealed that motor neuron enhancers are broadly distributed across the large chromatin domains. This distributed regulatory architecture is not a unique property of motor neurons but is employed throughout the nervous system. The number of regulatory elements increased dramatically during the transition from invertebrates to vertebrates, suggesting that acquisition of new enhancers might be a fundamental process underlying the evolutionary increase in cellular complexity.

Molecular correlates of muscle spindle and Golgi tendon organ afferents.

Proprioceptive feedback mainly derives from groups Ia and II muscle spindle (MS) afferents and group Ib Golgi tendon organ (GTO) afferents, but the molecular correlates of these three afferent subtypes remain unknown. We performed single cell RNA sequencing of genetically identified adult proprioceptors and uncovered five molecularly distinct neuronal clusters. Validation of cluster-specific transcripts in dorsal root ganglia and skeletal muscle demonstrates that two of these clusters correspond to group Ia MS afferents and group Ib GTO afferent proprioceptors, respectively, and suggest that the remaining clusters could represent group II MS afferents. Lineage analysis between proprioceptor transcriptomes at different developmental stages provides evidence that proprioceptor subtype identities emerge late in development. Together, our data provide comprehensive molecular signatures for groups Ia and II MS afferents and group Ib GTO afferents, enabling genetic interrogation of the role of individual proprioceptor subtypes in regulating motor output.

Regulating muscle spindle and Golgi tendon organ proprioceptor phenotypes.

Proprioception is an essential part of motor control. The main sensory subclasses that underlie this feedback control system - muscle spindle and Golgi tendon organ afferents - have been extensively characterized at a morphological and physiological level. More recent studies are beginning to reveal the molecular foundation for distinct proprioceptor subtypes, offering new insights into their developmental ontogeny and phenotypic diversity. This review intends to highlight some of these new findings.

Airway Protection-A Role for Vagal P2RY1 Receptors.

Recent events bring the importance of respiratory health to the forefront of our collective attention. In this issue of Cell, a new study by Prescott and Umans et al. reveals how a dedicated laryngeal sensory motor reflex circuit protects our airways from aspirated foods or liquids.

A Role for Sensory end Organ-Derived Signals in Regulating Muscle Spindle Proprioceptor Phenotype.

Proprioceptive feedback from Group Ia/II muscle spindle afferents and Group Ib Golgi tendon afferents is critical for the normal execution of most motor tasks, yet how these distinct proprioceptor subtypes emerge during development remains poorly understood. Using molecular genetic approaches in mice of either sex, we identified 24 transcripts that have not previously been associated with a proprioceptor identity. Combinatorial expression analyses of these markers reveal at least three molecularly distinct proprioceptor subtypes. In addition, we find that 12 of these transcripts are expressed well after proprioceptors innervate their respective sensory receptors, and expression of three of these markers, including the heart development molecule Heg1, is significantly reduced in mice that lack muscle spindles. These data reveal Heg1 as a putative marker for proprioceptive muscle spindle afferents. Moreover, they suggest that the phenotypic specialization of functionally distinct proprioceptor subtypes depends, in part, on extrinsic sensory receptor organ-derived signals.SIGNIFICANCE STATEMENT Sensory feedback from muscle spindle (MS) and Golgi tendon organ (GTO) sensory end organs is critical for normal motor control, but how distinct MS and GTO afferent sensory neurons emerge during development remains poorly understood. Using (bulk) transcriptome analysis of genetically identified proprioceptors, this work reveals molecular markers for distinct proprioceptor subsets, including some that appear selectively expressed in MS afferents. Detailed analysis of the expression of these transcripts provides evidence that MS/GTO afferent subtype phenotypes may, at least in part, emerge through extrinsic, sensory end organ-derived signals.

Piezo2 is the principal mechanotransduction channel for proprioception.

Proprioception, the perception of body and limb position, is mediated by proprioceptors, specialized mechanosensory neurons that convey information about the stretch and tension experienced by muscles, tendons, skin and joints. In mammals, the molecular identity of the stretch-sensitive channel that mediates proprioception is unknown. We found that the mechanically activated nonselective cation channel Piezo2 was expressed in sensory endings of proprioceptors innervating muscle spindles and Golgi tendon organs in mice. Two independent mouse lines that lack Piezo2 in proprioceptive neurons showed severely uncoordinated body movements and abnormal limb positions. Moreover, the mechanosensitivity of parvalbumin-expressing neurons that predominantly mark proprioceptors was dependent on Piezo2 expression in vitro, and the stretch-induced firing of proprioceptors in muscle-nerve recordings was markedly reduced in Piezo2-deficient mice. Together, our results indicate that Piezo2 is the major mechanotransducer of mammalian proprioceptors.

The PDZ-domain protein Whirlin facilitates mechanosensory signaling in mammalian proprioceptors.

Mechanoreception is an essential feature of many sensory modalities. Nevertheless, the mechanisms that govern the conversion of a mechanical force to distinct patterns of action potentials remain poorly understood. Proprioceptive mechanoreceptors reside in skeletal muscle and inform the nervous system of the position of body and limbs in space. We show here that Whirlin/Deafness autosomal recessive 31 (DFNB31), a PDZ-scaffold protein involved in vestibular and auditory hair cell transduction, is also expressed by proprioceptive sensory neurons (pSNs) in dorsal root ganglia in mice. Whirlin localizes to the peripheral sensory endings of pSNs and facilitates pSN afferent firing in response to muscle stretch. The requirement of Whirlin in both proprioceptors and hair cells suggests that accessory mechanosensory signaling molecules define common features of mechanoreceptive processing across sensory systems.

Delta opioid receptors presynaptically regulate cutaneous mechanosensory neuron input to the spinal cord dorsal horn.

Cutaneous mechanosensory neurons detect mechanical stimuli that generate touch and pain sensation. Although opioids are generally associated only with the control of pain, here we report that the opioid system in fact broadly regulates cutaneous mechanosensation, including touch. This function is predominantly subserved by the delta opioid receptor (DOR), which is expressed by myelinated mechanoreceptors that form Meissner corpuscles, Merkel cell-neurite complexes, and circumferential hair follicle endings. These afferents also include a small population of CGRP-expressing myelinated nociceptors that we now identify as the somatosensory neurons that coexpress mu and delta opioid receptors. We further demonstrate that DOR activation at the central terminals of myelinated mechanoreceptors depresses synaptic input to the spinal dorsal horn, via the inhibition of voltage-gated calcium channels. Collectively our results uncover a molecular mechanism by which opioids modulate cutaneous mechanosensation and provide a rationale for targeting DOR to alleviate injury-induced mechanical hypersensitivity.

Etv1 inactivation reveals proprioceptor subclasses that reflect the level of NT3 expression in muscle targets.

The organization of spinal reflex circuits relies on the specification of distinct classes of proprioceptive sensory neurons (pSN), but the factors that drive such diversity remain unclear. We report here that pSNs supplying distinct skeletal muscles differ in their dependence on the ETS transcription factor Etv1 for their survival and differentiation. The status of Etv1-dependence is linked to the location of proprioceptor muscle targets: pSNs innervating hypaxial and axial muscles depend critically on Etv1 for survival, whereas those innervating certain limb muscles are resistant to Etv1 inactivation. The level of NT3 expression in individual muscles correlates with Etv1-dependence and the loss of pSNs triggered by Etv1 inactivation can be prevented by elevating the level of muscle-derived NT3-revealing a TrkC-activated Etv1-bypass pathway. Our findings support a model in which the specification of aspects of pSN subtype character is controlled by variation in the level of muscle NT3 expression and signaling.

Runx1 determines nociceptive sensory neuron phenotype and is required for thermal and neuropathic pain.

In mammals, the perception of pain is initiated by the transduction of noxious stimuli through specialized ion channels and receptors expressed by nociceptive sensory neurons. The molecular mechanisms responsible for the specification of distinct sensory modality are, however, largely unknown. We show here that Runx1, a Runt domain transcription factor, is expressed in most nociceptors during embryonic development but in adult mice, becomes restricted to nociceptors marked by expression of the neurotrophin receptor Ret. In these neurons, Runx1 regulates the expression of many ion channels and receptors, including TRP class thermal receptors, Na+-gated, ATP-gated, and H+-gated channels, the opioid receptor MOR, and Mrgpr class G protein coupled receptors. Runx1 also controls the lamina-specific innervation pattern of nociceptive afferents in the spinal cord. Moreover, mice lacking Runx1 exhibit specific defects in thermal and neuropathic pain. Thus, Runx1 coordinates the phenotype of a large cohort of nociceptors, a finding with implications for pain therapy.

A role for Runx transcription factor signaling in dorsal root ganglion sensory neuron diversification.

Subpopulations of sensory neurons in the dorsal root ganglion (DRG) can be characterized on the basis of sensory modalities that convey distinct peripheral stimuli, but the molecular mechanisms that underlie sensory neuronal diversification remain unclear. Here, we have used genetic manipulations in the mouse embryo to examine how Runx transcription factor signaling controls the acquisition of distinct DRG neuronal subtype identities. Runx3 acts to diversify an Ngn1-independent neuronal cohort by promoting the differentiation of proprioceptive sensory neurons through erosion of TrkB expression in prospective TrkC+ sensory neurons. In contrast, Runx1 controls neuronal diversification within Ngn1-dependent TrkA+ neurons by repression of neuropeptide CGRP expression and controlling the fine pattern of laminar termination in the dorsal spinal cord. Together, our findings suggest that Runx transcription factor signaling plays a key role in sensory neuron diversification.

Graded activity of transcription factor Runx3 specifies the laminar termination pattern of sensory axons in the developing spinal cord.

Different functional classes of dorsal root ganglion sensory neurons project their axons to distinct target zones within the developing spinal cord. To explore the mechanisms that link sensory neuron subtype identity and axonal projection pattern, we analyzed the roles of Runx and ETS transcription factors in the laminar targeting of sensory afferents. Gain- and loss-of-function studies in chick embryos reveal that the status of Runx3 expression is a major determinant of the dorso-ventral position of termination of proprioceptive and cutaneous sensory axons. In addition, the level of expression and/or activity of Runx3 in individual proprioceptive sensory neurons appears to specify whether their axons terminate in intermediate or ventral regions. Our findings suggest that the selectivity of Runx3 expression, and its level of activity, control sensory afferent targeting in the developing spinal cord.