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Differential expression analysis in single-cell transcriptomics enables the dissection of cell-type-specific responses to perturbations such as disease, trauma, or experimental manipulations. While many statistical methods are available to identify differentially expressed genes, the principles that distinguish these methods and their performance remain unclear. Here, we show that the relative performance of these methods is contingent on their ability to account for variation between biological replicates. Methods that ignore this inevitable variation are biased and prone to false discoveries. Indeed, the most widely used methods can discover hundreds of differentially expressed genes in the absence of biological differences. To exemplify these principles, we exposed true and false discoveries of differentially expressed genes in the injured mouse spinal cord. Differential expression analysis of single-cell transcriptomics allows scientists to dissect cell-type-specific responses to biological perturbations. Here, the authors show that many commonly used methods are biased and can produce false discoveries.

Brain and spinal injury reduce mobility and often impair sensorimotor processing in the spinal cord leading to spasticity. Here, we establish that complete transection of corticospinal pathways in the pyramids impairs locomotion and leads to increased spasms and excessive mono- and polysynaptic low threshold spinal reflexes in rats. Treatment of affected forelimb muscles with an adeno-associated viral vector (AAV) encoding human Neurotrophin-3 at a clinically-feasible time-point after injury reduced spasticity. Neurotrophin-3 normalized the short latency Hoffmann reflex to a treated hand muscle as well as low threshold polysynaptic spinal reflexes involving afferents from other treated muscles. Neurotrophin-3 also enhanced locomotor recovery. Furthermore, the balance of inhibitory and excitatory boutons in the spinal cord and the level of an ion co-transporter in motor neuron membranes required for normal reflexes were normalized. Our findings pave the way for Neurotrophin-3 as a therapy that treats the underlying causes of spasticity and not only its symptoms. Injuries to the brain and spinal cord cause disability in millions of people worldwide. Physical rehabilitation can restore some muscle control and improve mobility in affected individuals. However, no current treatments provide long-term relief from the unwanted muscle contractions and spasms that affect as many as 78% of people with a spinal cord injury. These spasms can seriously hamper a person’s ability to carry out day-to-day tasks and get around independently. A few treatments can help in the short term but have side effects; indeed while Botox injections are used to paralyse the muscle, these also reduce the chances of useful improvements. As such, better therapies for muscle spasms are needed; especially ones that reduce spasms in the arms. Rats with injuries to the spinal cord between their middle to lower back typically develop spasms in their legs or tail, and rat models have helped scientists begin to understand why these involuntary movements occur. Now, Kathe et al. report that cutting one specific pathway that connects the brain to the spinal cord in anesthetised rats leads to the development of spasms in the forelimbs as well. Several months after the surgery, the rats had spontaneous muscle contractions in their forelimbs and walked abnormally. Further experiments showed that some other neural pathways in the rats became incorrectly wired and hyperactive and that this resulted in the abnormal movements. Next, Kathe et al. asked whether using gene therapy to deliver a protein that is required for neural circuits to form between muscles and the spinal cord (called neurotrophin-3) would stop the involuntary movements in the forelimbs. Delivering the gene therapy directly into the forelimb muscles of the disabled rats a day after their injury increased the levels of neurotrophin-3 in these muscles. Rats that received this treatment had fewer spasms and walked better than those that did not. Further experiments confirmed that this was because the rats’ previously hyperactive and abnormally wired neural circuits became more normal after the treatment. Together these results suggest that neurotrophin-3 might be a useful treatment for muscle spasms in people with spinal injury. There have already been preliminary studies in people showing that treatment with neurotrophin-3 is safe and well tolerated. Future studies are needed to confirm that it could be useful in humans.

A spinal cord injury disrupts communication between the brain and the circuits in the spinal cord that regulate neurological functions. The consequences are permanent paralysis, loss of sensation and debilitating dysautonomia. However, the majority of circuits located above and below the injury remain anatomically intact, and these circuits can reorganize naturally to improve function. In addition, various neuromodulation therapies have tapped into these processes to further augment recovery. Emerging research is illuminating the requirements to reconstitute damaged circuits. Here, we summarize these natural and targeted reorganizations of circuits after a spinal cord injury. We also advocate for new concepts of reorganizing circuits informed by multi-omic single-cell atlases of recovery from injury. These atlases will uncover the molecular logic that governs the selection of 'recovery-organizing' neuronal subpopulations, and are poised to herald a new era in spinal cord medicine. The authors summarize changes in circuits after spinal cord injury and current strategies to target these circuits in order to improve recovery, but also advocate for new concepts of reorganizing circuits informed by multi-omic single-cell atlases.

We present Augur, a method to prioritize the cell types most responsive to biological perturbations in single-cell data. Augur employs a machine-learning framework to quantify the separability of perturbed and unperturbed cells within a high-dimensional space. We validate our method on single-cell RNA sequencing, chromatin accessibility and imaging transcriptomics datasets, and show that Augur outperforms existing methods based on differential gene expression. Augur identified the neural circuits restoring locomotion in mice following spinal cord neurostimulation. The cell types affected by biological perturbations in complex tissues are uncovered by single-cell analysis.

Optoelectronic systems can exert precise control over targeted neurons and pathways throughout the brain in untethered animals, but similar technologies for the spinal cord are not well established. In the present study, we describe a system for ultrafast, wireless, closed-loop manipulation of targeted neurons and pathways across the entire dorsoventral spinal cord in untethered mice. We developed a soft stretchable carrier, integrating microscale light-emitting diodes (micro-LEDs), that conforms to the dura mater of the spinal cord. A coating of silicone–phosphor matrix over the micro-LEDs provides mechanical protection and light conversion for compatibility with a large library of opsins. A lightweight, head-mounted, wireless platform powers the micro-LEDs and performs low-latency, on-chip processing of sensed physiological signals to control photostimulation in a closed loop. We use the device to reveal the role of various neuronal subtypes, sensory pathways and supraspinal projections in the control of locomotion in healthy and spinal-cord injured mice. Optogenetics is applied to the entire mouse spinal cord.

Spinal cord injury (SCI) induces haemodynamic instability that threatens survival 1 – 3 , impairs neurological recovery 4 , 5 , increases the risk of cardiovascular disease 6 , 7 , and reduces quality of life 8 , 9 . Haemodynamic instability in this context is due to the interruption of supraspinal efferent commands to sympathetic circuits located in the spinal cord 10 , which prevents the natural baroreflex from controlling these circuits to adjust peripheral vascular resistance. Epidural electrical stimulation (EES) of the spinal cord has been shown to compensate for interrupted supraspinal commands to motor circuits below the injury 11 , and restored walking after paralysis 12 . Here, we leveraged these concepts to develop EES protocols that restored haemodynamic stability after SCI. We established a preclinical model that enabled us to dissect the topology and dynamics of the sympathetic circuits, and to understand how EES can engage these circuits. We incorporated these spatial and temporal features into stimulation protocols to conceive a clinical-grade biomimetic haemodynamic regulator that operates in a closed loop. This ‘neuroprosthetic baroreflex’ controlled haemodynamics for extended periods of time in rodents, non-human primates and humans, after both acute and chronic SCI. We will now conduct clinical trials to turn the neuroprosthetic baroreflex into a commonly available therapy for people with SCI. An epidural spinal cord stimulation system regulates blood pressure in the acute and chronic phases of spinal cord injury.

After spinal cord injury, tissue distal to the lesion contains undamaged cells that could support or augment recovery. Targeting these cells requires a clearer understanding of their injury responses and capacity for repair. Here, we use single nucleus RNA sequencing to profile how each cell type in the lumbar spinal cord changes after a thoracic injury in mice. We present an atlas of these dynamic responses across dozens of cell types in the acute, subacute, and chronically injured spinal cord. Using this resource, we find rare spinal neurons that express a signature of regeneration in response to injury, including a major population that represent spinocerebellar projection neurons. We characterize these cells anatomically and observed axonal sparing, outgrowth, and remodeling in the spinal cord and cerebellum. Together, this work provides a key resource for studying cellular responses to injury and uncovers the spontaneous plasticity of spinocerebellar neurons, uncovering a potential candidate for targeted therapy. Matson et al. performed single nucleus sequencing of the “spared” spinal cord tissue distal to an injury in mice. They found that spinocerebellar neurons expressed a pro-regenerative gene signature and showed axon outgrowth after injury.

A spinal cord injury (SCI) disrupts the neuronal projections from the brain to the region of the spinal cord that produces walking, leading to various degrees of paralysis. Here, we aimed to identify brain regions that steer the recovery of walking after incomplete SCI and that could be targeted to augment this recovery. To uncover these regions, we constructed a space–time brain-wide atlas of transcriptionally active and spinal cord-projecting neurons underlying the recovery of walking after incomplete SCI. Unexpectedly, interrogation of this atlas nominated the lateral hypothalamus (LH). We demonstrate that glutamatergic neurons located in the LH (LH Vglut2 ) contribute to the recovery of walking after incomplete SCI and that augmenting their activity improves walking. We translated this discovery into a deep brain stimulation therapy of the LH (DBS LH ) that immediately augmented walking in mice and rats with SCI and durably increased recovery through the reorganization of residual lumbar-terminating projections from brainstem neurons. A pilot clinical study showed that DBS LH immediately improved walking in two participants with incomplete SCI and, in conjunction with rehabilitation, mediated functional recovery that persisted when DBS LH was turned off. There were no serious adverse events related to DBS LH . These results highlight the potential of targeting specific brain regions to maximize the engagement of spinal cord-projecting neurons in the recovery of neurological functions after SCI. Further trials must establish the safety and efficacy profile of DBS LH , including potential changes in body weight, psychological status, hormonal profiles and autonomic functions. Whole-brain anatomical and activity surveys identify the lateral hypothalamus as a key driver of recovery from spinal cord injury, leading to a deep brain stimulation therapy that augments the recovery of walking in humans.

Recent advances in the development of in-body bioelectronic systems are providing new opportunities for the clinical management of various diseases and disorders. These emerging technologies are tailored to specific organs and are beginning to blend both diagnostic sensing and therapeutic actuation. The aim of these systems is to seamlessly integrate with the physiological environment, as illustrated by the diverse device strategies discussed throughout this article. Next generation modalities, such as optogenetics combining gene therapy with devices for photostimulation, are gaining popularity and offer advantages over existing therapeutic strategies. In this perspective, we explore the current state of technological developments, key challenges in the field and potential pathways for translating these innovations into clinical practice.

A spinal cord injury interrupts pathways from the brain and brainstem that project to the lumbar spinal cord, leading to paralysis. Here we show that spatiotemporal epidural electrical stimulation (EES) of the lumbar spinal cord 1 – 3 applied during neurorehabilitation 4 , 5 (EES REHAB ) restored walking in nine individuals with chronic spinal cord injury. This recovery involved a reduction in neuronal activity in the lumbar spinal cord of humans during walking. We hypothesized that this unexpected reduction reflects activity-dependent selection of specific neuronal subpopulations that become essential for a patient to walk after spinal cord injury. To identify these putative neurons, we modelled the technological and therapeutic features underlying EES REHAB in mice. We applied single-nucleus RNA sequencing 6 – 9 and spatial transcriptomics 10 , 11 to the spinal cords of these mice to chart a spatially resolved molecular atlas of recovery from paralysis. We then employed cell type 12 , 13 and spatial prioritization to identify the neurons involved in the recovery of walking. A single population of excitatory interneurons nested within intermediate laminae emerged. Although these neurons are not required for walking before spinal cord injury, we demonstrate that they are essential for the recovery of walking with EES following spinal cord injury. Augmenting the activity of these neurons phenocopied the recovery of walking enabled by EES REHAB , whereas ablating them prevented the recovery of walking that occurs spontaneously after moderate spinal cord injury. We thus identified a recovery-organizing neuronal subpopulation that is necessary and sufficient to regain walking after paralysis. Moreover, our methodology establishes a framework for using molecular cartography to identify the neurons that produce complex behaviours. Transcriptomic analysis following epidural electrical stimulation of the lumbar spinal cord during neurorehabilitation in mice identifies a population of neurons that orchestrates the restoration of walking following paralysis.