Smart-seq2 of spinal cord motoneurons in spinal cord injury model after dual electrical stimulation with specific frequencies
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ABSTRACT: Electrical stimulation can augment or modify neuronal function and can have therapeutic benefits for certain neurological disorders. There is evidence that enhancing spinal excitability with either epidural or transcutaneous stimulation can restore some volitional motor output after spinal cord injury (SCI). Lumbosacral epidural stimulation temporarily improves locomotor and autonomic function in both rodents and humans with SCI. When combined with overground locomotor training enabled by a weight-supporting device, epidural electrical stimulation (EES) promotes extensive reorganization of residual neural pathways that improves locomotion after stopping stimulation. However, the exact mechanism underlying the reconstruction of spinal cord neural circuits with electrical stimulation is not yet known. Thus, we developed a epidural electrical and muscle stimulation(EEMS) system at the interface of the spinal cord and muscle to mimic feedforward and feedback electrical signals in spinal sensorimotor circuits. Using methods of motor function evaluation, neural circuit tracing and neural signal recording, we discovered a unique stimulus frequency of 10-20 Hz under EEMS conditions that was required for structural and functional reconstruction of spinal sensorimotor circuits. Single-cell transcriptome analysis of EEMS activated motoneurons characterized molecular networks involved in spinal sensorimotor circuit reconstruction. This study provides insights into neural signal decoding during spinal sensorimotor circuit reconstruction, and indicates a technological approach for the clinical treatment of SCI.
Project description:The characteristics of electrical signals-mediated neural circuits reconstruction remain poorly understood. We initially developed a spinal-muscle synergistic bidirectional electrical stimulation (SBES) protocol to mimic feedforward and feedback electrical signals in spinal sensorimotor circuits. The results indicate that sensorimotor circuits could be precisely reassembled in structure and function levels by 10-20 Hz SBES. To gain mechanistic insights into the reassembly of the spinal sensorimotor circuits with 10-20 Hz SBES, we performed gene expression profiling analysis in motoneurons. Gene ontology (GO) analysis showed that one group of GO terms accounted for a large fraction of the upregulated transcripts. This group includes axon growth-related cellular functions, such as cell adhesion, regulation of cell growth, and cell projection assembly. Statistical analysis of the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways revealed sixteen signaling pathways were involved, two of which were related to the regulation of axonal regeneration (PI3K-Akt signaling pathway and cAMP signaling pathway). This study provides insights into neural signal decoding during spinal sensorimotor circuit reconstruction.
Project description:The goal of this study is to elucidate the influence of hemisection injury at thoracic spinal cord (T9) and epidural electrical spinal stimulation (L2-S1) on transcriptome of injured thoracic spinal cord. mRNA profiles of spinal cord at 5 days-post injury with or without epidural electrical spinal stimulation (L2-S1) and before injury were generated. Our study represents the detailed analysis of transcriptomes of injured spinal cord with biologic replicates, generated by RNA-seq technology.
Project description:Here, we show that epidural electrical stimulation (EES) of the lumbar spinal cord applied during neurorehabilitation (EESREHAB) restored walking in nine people with chronic spinal cord injury (SCI). This recovery involved a reduction of the metabolic activity in the lumbar spinal cord during walking. We hypothesized that this unexpected reduction reflects activity-dependent selection of specific neuronal subpopulations that become essential to walk after SCI. To identify these putative neurons, we modelled the technological and therapeutic features underlying EESREHAB in mice. We applied single-nucleus RNA sequencing and spatial transcriptomics to the spinal cord of these mice to chart a spatially-resolved molecular atlas of recovery from paralysis. We then employed cell type and spatial prioritization to uncover the neurons involved in the recovery of walking. A single population of excitatory interneurons nested within intermediate laminae emerged. Although these neurons were not necessary to walk before SCI, we demonstrate that they are essential to regain walking following SCI. In turn, augmenting their activity instantly phenocopied the recovery of walking enabled by EESREHAB. We thus identified a recovery-organizing neuronal subpopulation that is necessary and sufficient to regain walking after SCI. Moreover, our methodology establishes a framework to identify the neurons producing complex behaviours using molecular cartography.
Project description:Here, we show that epidural electrical stimulation (EES) of the lumbar spinal cord applied during neurorehabilitation (EESREHAB) restored walking in nine people with chronic spinal cord injury (SCI). This recovery involved a reduction of the metabolic activity in the lumbar spinal cord during walking. We hypothesized that this unexpected reduction reflects activity-dependent selection of specific neuronal subpopulations that become essential to walk after SCI. To identify these putative neurons, we modelled the technological and therapeutic features underlying EESREHAB in mice. We applied single-nucleus RNA sequencing and spatial transcriptomics to the spinal cord of these mice to chart a spatially-resolved molecular atlas of recovery from paralysis. We then employed cell type and spatial prioritization to uncover the neurons involved in the recovery of walking. A single population of excitatory interneurons nested within intermediate laminae emerged. Although these neurons were not necessary to walk before SCI, we demonstrate that they are essential to regain walking following SCI. In turn, augmenting their activity instantly phenocopied the recovery of walking enabled by EESREHAB. We thus identified a recovery-organizing neuronal subpopulation that is necessary and sufficient to regain walking after SCI. Moreover, our methodology establishes a framework to identify the neurons producing complex behaviours using molecular cartography.
Project description:Summary: Spinal cord injury (SCI) is a damage to the spinal cord induced by trauma or disease resulting in a loss of mobility or feeling. SCI is characterized by a primary mechanical injury followed by a secondary injury in which several molecular events are altered in the spinal cord often resulting in loss of neuronal function. Analysis of the areas directly (spinal cord) and indirectly (raphe and sensorimotor cortex) affected by injury will help understanding mechanisms of SCI. Hypothesis: Areas of the brain primarily affected by spinal cord injury are the Raphe and the Sensorimotor cortex thus gene expression profiling these two areas might contribute understanding the mechanisms of spinal cord injury. Specific Aim: The project aims at finding significantly altered genes in the Raphe and Sensorimotor cortex following an induced moderate spinal cord injury in T9.
Project description:Sensorimotor reflex circuits engage distinct neuronal subtypes, defined by precise connectivity, to transform sensation into compensatory behavior. Whether and how motor partner populations shape the subtype fate and connectivity of their pre-motor counterparts remains controversial. Here, we discovered that motor partners are dispensable for proper connectivity across an entire vestibular reflex circuit that stabilizes gaze. We first measured activity following vestibular sensation in pre-motor projection neurons after constitutive loss of their extraocular motor neuron partners.We observed normal responses and topography consistent with unchanged functional connectivity between sensory neurons and projection neurons. Next, we show that projection neurons remain anatomically and molecularly poised to connect appropriately with their motor partners. Lastly, we show that the transcriptional signatures of projection neuron subtypes develop independently of motor partners. Our findings comprehensively overturn a long-standing model: that connectivity in the circuit for gaze stabilization is retrogradely determined by motor partner-derived signals. By defining the contribution of motor neurons to canonical sensorimotor circuit assembly, our work speaks to comparable processes in spinal circuits and advances our understanding of general principles of neural development.
Project description:Summary: Spinal cord injury (SCI) is a damage to the spinal cord induced by trauma or disease resulting in a loss of mobility or feeling. SCI is characterized by a primary mechanical injury followed by a secondary injury in which several molecular events are altered in the spinal cord often resulting in loss of neuronal function. Analysis of the areas directly (spinal cord) and indirectly (raphe and sensorimotor cortex) affected by injury will help understanding mechanisms of SCI. Hypothesis: Areas of the brain primarily affected by spinal cord injury are the Raphe and the Sensorimotor cortex thus gene expression profiling these two areas might contribute understanding the mechanisms of spinal cord injury. Specific Aim: The project aims at finding significantly altered genes in the Raphe and Sensorimotor cortex following an induced moderate spinal cord injury in T9. Keywords: other
Project description:The fidelity of motor control requires the precise positional arrangement of motor pools and the establishment of synaptic connections between these pools. In the developing spinal cord, motor nerves project to specific target muscles and receive proprioceptive input from the muscles via the sensorimotor circuit. LIM-homeodomain transcription factors are known to successively restrict specific motor neuronal fates during neural development; however, it remains unclear to what extent they contribute to limb-based motor pools and locomotor circuits. Here, we showed in mice that deletion of Isl2 resulted in scattered motor pools, primarily in the median motor column and lateral LMC (LMCl) populations, and lacked Pea3 expression in the hindlimb motor pools, accompanied by reduced terminal axon branching and disorganized neuromuscular junctions. Transcriptomic analysis of Isl2-deficient spinal cords revealed that a variety of genes involved in motor neuron differentiation, axon development, and synapse organization were downregulated in hindlimb motor pools. Moreover, the loss of Isl2 impaired sensorimotor connectivity and hindlimb locomotion. Together, our studies indicate that Isl2 plays a critical role in organizing motor pool position and sensorimotor circuits in hindlimb motor pools.
Project description:Spinal cord injury disrupts ascending and descending neural signals causing sensory and motor dysfunction below the injury. Neuromodulation with electrical stimulation is used in both clinical and research settings to induce neural plasticity and improve functional recovery following injury. However, the mechanisms by which electrical stimulation affects recovery remain unclear. In this study we examined the effects of cortical electrical stimulation following injury on transcription at several levels of the central nervous system. We performed a unilateral cervical spinal contusion injury in rats and delivered stimulation for one week to the contralesional motor cortex to activate a descending motor tract.RNA was purified from bilateral subcortical white matter, and 3 levels of the spinal cord. Here we provide the complete data set in the hope that it will be useful for researchers studying electrical stimulation as a therapy to improve recovery from the deficits associated with spinal cord injury.
Project description:Astrocytes, the most abundant cells in the central nervous system, promote synapse formation and help refine neural connectivity. Although they are allocated to spatially distinct regional domains during development, it is unknown whether region-restricted astrocytes are functionally heterogeneous. Here we show that postnatal spinal cord astrocytes express several region-specific genes, and that ventral astrocyte-encoded Semaphorin3a (Sema3a) is required for proper motor neuron and sensory neuron circuit organization. Loss of astrocyte-encoded Sema3a led to dysregulated α−motor neuron axon initial segment orientation, markedly abnormal synaptic inputs, and selective death of α−but not of adjacent γ−motor neurons. Additionally, a subset of TrkA+ sensory afferents projected to ectopic ventral positions. These findings demonstrate that stable maintenance of a positional cue by developing astrocytes influences multiple aspects of sensorimotor circuit formation. More generally, they suggest that regional astrocyte heterogeneity may help to coordinate postnatal neural circuit refinement. 12 total samples consisting of three biological replicates each of flow sorted postnatal day 7 dorsal spinal cord astrocytes, ventral spinal cord astrocytes, dorsal SC non astrocytes, and ventral SC non astrocytes