GSE184370
The neurons that restore walking after paralysis [snRNA-seq]
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. Overall design: We used single-nucleus RNA sequencing (snRNA-seq) to profile the lumbar spinal cord of mice. We devised a progression of eight experimental conditions that captured the key therapeutic features of EESREHAB. We obtained high-quality transcriptomes from 82,093 nuclei that were evenly represented across 24 mice from all eight conditions. Mice were divided in three experimental groups: uninjured, SCI (no neurorehabilitation), and EESREHAB. At the end of the experimental period, mice were terminated to harvest fresh tissues or perfused and dissected. Half of the groups of mice underwent a terminal experimental condition immediately before being terminated, which is denoted by     final_condition. If more than one component was integrated in the terminal experimental condition, the additional component is denoted by  ::. Group 1 (uninjured) consisted of uninjured mice that did not perform a terminal behavioral task. Mice in Group 2 (SCI) received a contusion SCI and did not perform a terminal behavioral task. Mice in Group 3 (EESREHAB) received a contusion SCI and followed EESREHAB  for four weeks, but did not perform a terminal behavioral task.  Mice in Group 4 (SCI   EES::walking) received a contusion SCI and walked with EESON  for 30 minutes immediately before being terminated. Mice in Group 5 (EESREHAB   EES::walking) received a contusion SCI and followed EESREHAB  for four weeks, and walked with EESON  for 30 minutes immediately before being terminated. Mice in Group 6 (EESREHAB   EES) received a contusion SCI and and followed EESREHAB  for four weeks, and were stimulated for 30 minutes with EES just below motor threshold immediately before being terminated. Mice in Group 7 (EESREHAB   EES::cortex) received a contusion SCI and followed EESREHAB  for four weeks, and walked with optogenetic stimulation of the motor cortex for 30 minutes immediately before being terminated. Mice in Group 8 (EESREHAB   EES::cortex::walking) received a contusion SCI and followed EESREHAB  for four weeks, and walked with EESON  and optogenetic stimulation of the motor cortex for 30 minutes immediately before being terminated.