Hereditary sensory neuropathy type IA (HSN1A) is a rare neurodegenerative condition caused by dominant mutations in the Serine Palmitoyl Transferase Long Chain base subunit 1 (SPTLC1) gene. There is no treatment available. Allele-specific silencing by antisense oligonucleotides (ASOs) to preferentially silence the mutant transcripts has shown therapeutic promise for dominant gain-of-function genetic disorders. In this study, we validated an allele-specific ASO therapy to selectively silence mutant SPTLC1 (p.S331F) in a disease mouse model carrying a heterozygous p.S331F mutation (S331F mice). Gapmer ASOs, targeting the S331F variant in either 2'-O-methyl (2'-OMe), locked nucleic acid (LNA) or 2'-O-methoxy ethyl (MOE) chemistries, were first studied in cultured mouse skin fibroblasts. The candidate ASOs in LNA or MOE were further evaluated in vivo. Single subcutaneous injection of ASOs into neonatal or adult S331F mice achieved over 90% mutant transcript silencing in liver and dorsal root ganglia (DRG). Weekly subcutaneous injections of LNA-ASOs, either unconjugated or conjugated with N-acetylgalactosamine (GalNAc), into S331F mice showed GalNAc-LNA-ASOs to be more efficient than unconjugated LNA-ASOs at reducing mutant transcripts in liver, DRG and sciatic nerve, without affecting wild-type transcripts. GalNAc-LNA-ASOs also resulted in significantly reduced blood levels of 1-deoxysphingoid bases (1-deoxySL), neurotoxic metabolites used as biomarkers in HSN1A patients. Transcriptomic studies in DRG demonstrated mitochondrial pathway involvement in the pathological changes observed in S331F mice. Quantitative RT-PCR confirmed the differentially expressed genes between S331F and wild-type mice. Furthermore, these aberrantly expressed genes in S331F mice were reversed by GalNAc-LNA-ASO treatment. Our data provide necessary in vivo evidence as proof of concept for ASO-mediated mutant-allele-specific silencing as a therapeutic approach for SPTLC1-related HSN1.

Reach and grasp are critical components of skilled mammalian motor control and their detailed analysis in rodents has been key to deepening our understanding of prehension in the context of health and disease. However, most studies investigating these behaviours focus on isolating forelimb movements with little regard to the whole-body movements that are key for effective behaviour. To address this issue, we designed a novel behavioural approach to investigate reach and grasp during whole-body, vertical locomotion in mice. Using a customizable transparent climbing surface, we show that our behavioural approach can extract key kinematic features of climbing. Mouse climbing gait reflects aspects of quadrupedal locomotion, showing similar phase dependencies on increasing speed, including reduced stance (i.e. grasp) time and duty factor. Analysis of multi-limb coordination indicated that climbing revolves around anti-phasic forepaw movements with less consistency in interlimb coordination in the hindpaws. Fore- and hindpaws also differed in their reach trajectories and velocity profiles. The flexibility of this approach also allows for tailored climbing configurations, which we use to show that mice can adapt to and overcome vertical obstacles. By leveraging naturalistic climbing, our modular behavioural approach enables investigation of complex prehensile behaviours and facilitates new study into the neural circuits underlying whole-body skilled motor control. ### Competing Interest Statement The authors have declared no competing interest.

Sensory systems are shaped in postnatal life by the refinement of synaptic connectivity. In the dorsal horn of the spinal cord, somatosensory circuits undergo postnatal activity-dependent reorganization, including the refinement of primary afferent A-fiber terminals from superficial to deeper spinal dorsal horn laminae which is accompanied by decreases in cutaneous sensitivity. Here, we show in the mouse that microglia, the resident immune cells in the CNS, phagocytose A-fiber terminals in superficial laminae in the first weeks of life. Genetic perturbation of microglial engulfment during the initial postnatal period in either sex prevents the normal process of A-fiber refinement and elimination, resulting in an altered sensitivity of dorsal horn cells to dynamic tactile cutaneous stimulation, and behavioral hypersensitivity to dynamic touch. Thus, functional microglia are necessary for the normal postnatal development of dorsal horn sensory circuits. In the absence of microglial engulfment, superfluous A-fiber projections remain in the dorsal horn, and the balance of sensory connectivity is disrupted, leading to lifelong hypersensitivity to dynamic touch.

In the spinal cord dorsal horn, sensory circuits undergo remarkable postnatal reorganisation, including refinement of primary afferent A-fibres in the superficial layers, accompanied by decreased cutaneous sensitivity. Here we show a physiological role of microglia necessary for normal development of dorsal horn sensory circuits and tactile sensitivity. In the absence of microglial engulfment, superfluous A-fibre projections persist, leading to lifelong hypersensitivity to dynamic touch.

Single-cell RNA sequencing data can unveil the molecular diversity of cell types. Cell type atlases of the mouse spinal cord have been published in recent years but have not been integrated together. Here, we generate an atlas of spinal cell types based on single-cell transcriptomic data, unifying the available datasets into a common reference framework. We report a hierarchical structure of postnatal cell type relationships, with location providing the highest level of organization, then neurotransmitter status, family, and finally, dozens of refined populations. We validate a combinatorial marker code for each neuronal cell type and map their spatial distributions in the adult spinal cord. We also show complex lineage relationships among postnatal cell types. Additionally, we develop an open-source cell type classifier, SeqSeek, to facilitate the standardization of cell type identification. This work provides an integrated view of spinal cell types, their gene expression signatures, and their molecular organization.

ABSTRACT Single cell sequencing is transforming many fields of science but the vast amount of data it creates has the potential to both illuminate and obscure underlying biology. To harness the exciting potential of single cell data for the study of the mouse spinal cord, we have created a harmonized atlas of spinal cord transcriptomic cell types that unifies six independent and disparate studies into one common analysis. With the power of this large and diverse dataset, we reveal spinal cord cell type organization, validate a combinatorial set of markers for in-tissue spatial gene expression analysis, and optimize the computational classification of spinal cord cell types based on transcriptomic data. This work provides a comprehensive resource with unprecedented resolution of spinal cord cell types and charts a path forward for how to utilize transcriptomic data to expand our knowledge of spinal cord biology.

Spinal sensorimotor networks integrate sensory information into the ongoing locomotor program, allowing adaptation of motor behavior to the external world. This review summarizes sensorimotor research to date, and highlights how recently identified spinal sensorimotor interneurons have unveiled the task-specificity of local spinal networks in mammalian systems.

The exteroceptive somatosensory system is important for reflexive and adaptive behaviors and for the dynamic control of movement in response to external stimuli. This review outlines recent efforts using genetic approaches in the mouse to map the spinal cord circuits that transmit and gate the cutaneous somatosensory modalities of touch, pain, and itch. Recent studies have revealed an underlying modular architecture in which nociceptive, pruritic, and innocuous stimuli are processed by distinct molecularly defined interneuron cell types. These include excitatory populations that transmit information about both innocuous and painful touch and inhibitory populations that serve as a gate to prevent innocuous stimuli from activating the nociceptive and pruritic transmission pathways. By dissecting the cellular composition of dorsal-horn networks, studies are beginning to elucidate the intricate computational logic of somatosensory transformation in health and disease.

Animals depend on sensory feedback from mecha-nosensory afferents for the dynamic control of movement. This sensory feedback needs to be selectively modulated in a task-and context-dependent manner. Here, we show that inhibitory interneurons (INs) expressing the ROR beta orphan nuclear receptor gate sensory feedback to the spinal motor system during walking and are required for the production of a fluid locomotor rhythm. Genetic manipulations that abrogate inhibitory RORb IN function result in an ataxic gait characterized by exaggerated flexion movements and marked alterations to the step cycle. Inactivation of ROR beta in inhibitory neurons leads to reduced presynaptic inhibition and changes to sensory-evoked reflexes, arguing that the RORb inhibitory INs function to suppress the sensory transmission pathways that activate flexor motor reflexes and interfere with the ongoing locomotor program.

Sensory circuits in the dorsal spinal cord integrate and transmit multiple cutaneous sensory modalities including the sense of light touch. Here, we identify a population of excitatory interneurons (INs) in the dorsal horn that are important for transmitting innocuous light touch sensation. These neurons express the ROR alpha (RORα) nuclear orphan receptor and are selectively innervated by cutaneous low threshold mechanoreceptors (LTMs). Targeted removal of RORα INs in the dorsal spinal cord leads to a marked reduction in behavioral responsiveness to light touch without affecting responses to noxious and itch stimuli. RORα IN-deficient mice also display a selective deficit in corrective foot movements. This phenotype, together with our demonstration that the RORα INs are innervated by corticospinal and vestibulospinal projection neurons, argues that the RORα INs direct corrective reflex movements by integrating touch information with descending motor commands from the cortex and cerebellum.

Light mechanical stimulation of hairy skin can induce a form of itch known as mechanical itch. This itch sensation is normally suppressed by inputs from mechanoreceptors; however, in many forms of chronic itch, including alloknesis, this gating mechanism is lost. Here we demonstrate that a population of spinal inhibitory interneurons that are defined by the expression of neuropeptide Y::Cre (NPY::Cre) act to gate mechanical itch. Mice in which dorsal NPY::Cre-derived neurons are selectively ablated or silenced develop mechanical itch without an increase in sensitivity to chemical itch or pain. This chronic itch state is histamine-independent and is transmitted independently of neurons that express the gastrin-releasing peptide receptor. Thus, our studies reveal a dedicated spinal cord inhibitory pathway that gates the transmission of mechanical itch.

Key points Brainstem descending pathways control the balance of excitation and inhibition in spinal sensory networks. In adult rodents, descending inhibition is targeted to spinal neurons with a strong afferent C fibre input. Descending inhibitory control matures slowly; the first postnatal weeks are characterized by greater descending facilitation than inhibition. We report that, in contrast to adults, brainstem descending facilitation of spinal sensory neurons in young rats (postnatal day 21) is targeted to A fibre inputs. The selective inhibition of C fibre inputs observed in adults is absent at postnatal day 21. In both young and adult rats, descending inhibition or facilitation is correlated with the excitability of individual neurons, as measured by ‘wind‐up’ to repeated C fibre stimulation. The facilitation of A fibre input in early life is likely to enhance innocuous, tactile sensory inputs to the dorsal horn in the critical early postnatal weeks and thus promote activity‐dependent development of sensory networks. Abstract Brainstem descending control is crucial in maintaining the balance of excitation and inhibition in spinal sensory networks. In the adult, descending inhibition of spinal dorsal horn circuits arising from the brainstem rostroventral medial medulla (RVM) is targeted to neurons with a strong nociceptive C fibre input. Before the fourth postnatal week, the RVM exerts a net facilitation of spinal networks but it is not known if this is targeted to specific dorsal horn neuronal inputs. As the maturation from descending facilitation to inhibition occurs only after C fibre central synaptic maturation is complete, we hypothesized that RVM facilitation in young animals is targeted to A fibre afferent inputs. To test this, the RVM was stimulated while recording dorsal horn neuronal activity in vivo under isoflurane anaesthesia at postnatal day (P) 21 and P40 (adult). Electrical thresholds for A and C fibre evoked activity, spike counts and wind‐up characteristics at baseline and during RVM stimulation (10–100 µA, 10 Hz) were compared. In adults, RVM stimulation selectively increased the threshold for C fibre evoked activity while at P21, it selectively decreased the threshold for A fibre evoked activity and these effects were correlated to the wind‐up characteristics of the neuron. Thus, the postnatal shift in RVM control of dorsal horn circuits is not only directional but also modality specific, from facilitation of A fibre input in the young animal to inhibition of nociceptive C input in the adult, with additional contextual factors. The descending control of spinal sensory networks serves very different functions in young and adult animals.

Inhibitory neurons in the spinal cord perform dedicated roles in processing somatosensory information and shaping motor behaviors that range from simple protective reflexes to more complex motor tasks such as locomotion, reaching and grasping. Recent efforts examining inhibition in the spinal cord have been directed toward determining how inhibitory cell types are specified and incorporated into the sensorimotor circuitry, identifying and characterizing molecularly defined cohorts of inhibitory neurons and interrogating the functional contribution these cells make to sensory processing and motor behaviors. Rapid progress is being made on all these fronts, driven in large part by molecular genetic and optogenetic approaches that are being creatively combined with neuroanatomical, electrophysiological and behavioral techniques.

Developing brain circuits are shaped by postnatal sensory experience, but little is known about this process at the level of the spinal cord. Here we review the mechanisms by which cutaneous sensory input drives the maturation of spinal sensory circuits. Newborn animals are highly sensitive to tactile input and dorsal horn circuits are dominated by low threshold A fiber inputs. We show that this arises from the absence of the functional, targeted glycinergic inhibition of tactile activity that emerges only in the second week of life. Selective block of afferent C fibers in postnatal week 2 delays the maturation of glycinergic inhibition and maintains dorsal horn circuits in a neonatal state. We propose that in the newborn strong tactile A fiber input facilitates activity‐dependent synaptic strengthening in the dorsal horn, but that this ends with the arrival of nociceptive C fiber spinal input that drives the maturation of targeted glycinergic inhibition.

Sensory circuits are shaped by experience in early postnatal life and in many brain areas late maturation of inhibition drives activity-dependent development. In the newborn spinal dorsal horn, activity is dominated by inputs from low threshold A fibers, whereas nociceptive C-fiber inputs mature gradually over the first postnatal weeks. How this changing afferent input influences the maturation of dorsal horn inhibition is not known. We show an absence of functional glycinergic inhibition in newborn dorsal horn circuits: Dorsal horn receptive fields and afferent-evoked excitation are initially facilitated by glycinergic activity due, at least in part, to glycinergic disinhibition of GAD67 cells. Glycinergic inhibitory control emerges in the second postnatal week, coinciding with an expression switch from neonatal α2 homomeric to predominantly mature α1/β glycine receptors (GlyRs). We further show that the onset of glycinergic inhibition depends upon the maturation of C-fiber inputs to the dorsal horn: selective block of afferent C fibers in postnatal week 2, using perisciatic injections of the cationic anesthetic QX-314, lidocaine, and capsaicin, delays the maturation of both GlyR subunits and glycinergic inhibition, maintaining dorsal neurons in a neonatal state, where tactile responses are facilitated, rather than inhibited, by glycinergic network activity. Thus, glycine may serve to facilitate tactile A-fiber–mediated information and enhance activity-dependent synaptic strengthening in the immature dorsal horn. This period ceases in the second postnatal week with the maturation of C-fiber spinal input, which triggers postsynaptic changes leading to glycinergic inhibition and only then is balanced excitation and inhibition achieved in dorsal horn sensory circuits.

Neonatal responses to peripheral cutaneous sensory stimuli appear hyperexcitable compared to those of the adult, at both behavioural and cellular levels. Little is known, however, of the mechanisms involved in the maturation of this sensory circuitry over the postnatal period. I hypothesise that the excitability of neonatal networks is due to immature local and descending inhibitory control of spinal circuits. To test this I have examined the maturation of descending and local inhibitory spinal circuitry using immunohistochemical staining in the dorsal horn, in vivo electrophysiological recordings of dorsal horn neurons and stimulation of brainstem descending pathways. Firstly, I mapped the development of spinal glycinergic circuitry over the first three postnatal weeks using immunohistochemical staining of glycinergic terminals and receptors. Results show a clear shift in expression pattern from deep dorsal horn staining of both glycinergic terminals and receptors in the neonate, to selective expression in lamina III by the third postnatal week. I then characterised the functional development of glycinergic inhibition of spinal sensory pathways at a cellular level using in vivo extracellular recordings of dorsal horn neurons in neonatal and adolescent rats in the presence of the glycine receptor antagonist strychnine. Results illustrate an absence of glycinergic inhibition of sensory stimuli until postnatal day 21 and a facilitatory role of glycine in the transmission of low-threshold stimuli in the neonatal spinal cord. Finally, I examined the descending influence of the rostroventral medulla on dorsal horn neuronal activity over postnatal development. Results indicate that the influence of descending control shifts dramatically from predominantly excitatory in early development, to predominantly inhibitory at a later stage in life. In conclusion, there is significant postnatal modulation of segmental and descending influences on spinal networks in the postnatal period, both of which are likely to contribute to the maturation of cutaneous sensory spinal processing.

Brainstem–spinal cord connections play an essential role in adult pain processing, and the modulation of spinal pain network excitability by brainstem nuclei is known to contribute to hyperalgesia and chronic pain. Less well understood is the role of descending brainstem pathways in young animals when pain networks are more excitable and exposure to injury and stress can lead to permanent modulation of pain processing. Here we show that up to postnatal day 21 (P21) in the rat, the rostroventral medulla of the brainstem (RVM) exclusively facilitates spinal pain transmission but that after this age (P28 to adult), the influence of the RVM shifts to biphasic facilitation and inhibition. Graded electrical microstimulation of the RVM at different postnatal ages revealed a robust shift in the balance of descending control of both spinal nociceptive flexion reflex EMG activity and individual dorsal horn neuron firing properties, from excitation to inhibition, beginning after P21. The shift in polarity of descending control was also observed following excitotoxic lesions of the RVM in adult and P21 rats. In adults, RVM lesions decreased behavioural mechanical sensory reflex thresholds, whereas the same lesion in P21 rats increased thresholds. These data demonstrate, for the first time, the changing postnatal influence of the RVM in spinal nociception and highlight the central role of descending brainstem control in the maturation of pain processing.