The neurons that restore walking after paralysis [spatial transcriptomics]

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. 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.本研究证实，在神经康复期间实施腰脊髓硬膜外电刺激（epidural electrical stimulation, EES，该联合治疗方案缩写为EESREHAB），可使9名慢性脊髓损伤（spinal cord injury, SCI）患者恢复行走能力。该恢复过程伴随行走时腰脊髓代谢活动的降低。我们推测，这一意外的代谢降低现象，反映了特定神经元亚群的活动依赖性筛选——这类神经元在脊髓损伤后成为行走功能恢复所必需的细胞群体。 为鉴定这些潜在的关键神经元，我们在小鼠模型中复现了EESREHAB的技术与治疗特征。我们对该模型小鼠的脊髓组织开展单细胞核RNA测序（single-nucleus RNA sequencing）与空间转录组学分析，绘制了解剖空间分辨率下的瘫痪恢复分子图谱。随后通过细胞类型与空间优先级分析，挖掘出参与行走功能恢复的神经元群体。 研究发现，仅存在一群定位于脊髓中间板层的兴奋性中间神经元。尽管这类神经元在脊髓损伤前并非行走功能所必需，但我们证实其在脊髓损伤后对行走能力的恢复至关重要。进一步实验表明，增强该神经元群体的活动，可即时模拟EESREHAB所促成的行走恢复效果。据此，我们鉴定出一类可组织恢复过程的神经元亚群，其在脊髓损伤后既是行走恢复所必需，亦足以单独促成行走功能的恢复。 此外，本研究的方法学框架可通过分子图谱绘制，用于鉴定调控复杂行为的神经元群体。我们借助单细胞核RNA测序（single-nucleus RNA sequencing, snRNA-seq）对小鼠腰脊髓组织进行转录组分析。我们设计了包含8种实验条件的递进方案，以捕捉EESREHAB的核心治疗特征。最终从8组共24只小鼠的82093个细胞核中获得了高质量转录组数据，各组细胞核占比均一。 小鼠被分为3大实验组：未损伤组、脊髓损伤组（未接受神经康复）以及EESREHAB组。实验周期结束后，对小鼠实施安乐死以获取新鲜组织，或经灌注固定后进行解剖取材。部分小鼠在安乐死前需完成一项终端实验操作，该操作记为final_condition；若终端实验包含多个操作组件，则额外组件以::分隔。 第1组（未损伤组）：未接受脊髓损伤且未执行终端行为任务的小鼠。 第2组（脊髓损伤组）：接受脊髓挫伤损伤且未执行终端行为任务的小鼠。 第3组（EESREHAB组）：接受脊髓挫伤损伤并接受4周EESREHAB治疗，但未执行终端行为任务的小鼠。 第4组（脊髓损伤+EES::walking组）：接受脊髓挫伤损伤，且在安乐死前30分钟接受EESON辅助行走的小鼠。 第5组（EESREHAB+EES::walking组）：接受脊髓挫伤损伤并完成4周EESREHAB治疗，且在安乐死前30分钟接受EESON辅助行走的小鼠。 第6组（EESREHAB+EES组）：接受脊髓挫伤损伤并完成4周EESREHAB治疗，且在安乐死前30分钟接受低于运动阈值的硬膜外电刺激30分钟的小鼠。 第7组（EESREHAB+EES::cortex组）：接受脊髓挫伤损伤并完成4周EESREHAB治疗，且在安乐死前30分钟接受运动皮层光遗传刺激辅助行走的小鼠。 第8组（EESREHAB+EES::cortex::walking组）：接受脊髓挫伤损伤并完成4周EESREHAB治疗，且在安乐死前30分钟同时接受EESON与运动皮层光遗传刺激辅助行走的小鼠。