Mechanisms of rhythm generation and recruitment in mammalian locomotor-related spinal interneurons

摘要 节律是运动的一个重要特征，由脊髓中的中间神经元产生。内在和 腰椎节律性中间神经元采用的局部机制以及它们如何被 脊髓上运动中枢代表了我们对节律发生和运动的理解中的主要空白。 电路这一信息对于寻求治疗瘫痪的主要原因的治疗目标至关重要 包括脊髓损伤、创伤性脑损伤和帕金森病。脊髓中间神经元表达 转录因子Shox 2包括小鼠中的一组致节律性中间神经元。Shox2 在成人腰段脊髓切片的中间神经元具有节律性离子电流，包括持续向内 电流，并与其他Shox2中间神经元进行功能性兴奋性连接。我们发现一个子集 Shox 2中间神经元在成人腰段脊髓切片显示自发节律性膜电位 振荡内在的和局部的网络特性是启动和维持节奏的基本条件 在脊髓切片中的神经元爆发和Shox2中间神经元的其他模型中的振荡允许研究 成年哺乳动物运动回路的具体机制。该提案探讨了如何 Shox2中间神经元的振荡是使用一种创新的方法产生和招募的， 全细胞膜片钳电生理学、跨突触束追踪和光遗传学。有了这个组合 方法，我们将测试总体假设，Shox 2中间神经元在成年小鼠的腰脊髓 显示由持续钠电流和局部兴奋性突触介导的节律性放电 连接和招募的单突触兴奋性输入从外侧旁巨细胞核在 脑干在全细胞膜片钳实验中，我们将确定电压敏感电流和潜在的 电压门控离子通道在个体Shox2中间神经元中的节律振荡中起关键作用。 此外，我们将进一步测试局部突触连接对振荡的贡献。 腰椎切片中Shox2 IN的特性。最后，单突触投射的棘上核 将通过来自Shox 2的单突触限制性跨突触追踪来鉴定腰椎Shox 2中间神经元。 成年小鼠的中间神经元。这些解剖投影将在光遗传学中进行功能评估。 成人腰椎切片。总之，这是确定和评价 Shox 2中间神经元的节律产生和募集机制， 治疗脊髓运动回路保持完整但处于休眠状态的瘫痪的靶点。

ABSTRACT Rhythm is a critical feature of locomotion and is generated by interneurons in the spinal cord. The intrinsic and local mechanisms employed by lumbar spinal rhythmogenic interneurons and how they are recruited by supraspinal locomotor centers represent major gaps in our understanding of rhythmogenesis and locomotor circuitry. This information is crucial in the pursuit of therapeutic targets to treat the leading causes of paralysis including spinal cord injury, traumatic brain injury, and Parkinson’s Disease. Spinal interneurons expressing the transcription factor Shox2 include a group of putatively rhythmogenic interneurons in the mouse. Shox2 interneurons in the adult lumbar spinal slice possess rhythmogenic ionic currents, including persistent inward currents, and make functional excitatory connections to other Shox2 interneurons. We have found that a subset of Shox2 interneurons in the adult lumbar spinal slice displays spontaneous rhythmic membrane potential oscillations. Intrinsic and local network properties are essential for the initiation and maintenance of rhythmic oscillations in other models of neuronal bursting and Shox2 interneurons in the spinal slice allow for the study of the specific mechanisms involved in the adult mammalian locomotor circuitry. This proposal explores how oscillations in Shox2 interneurons are generated and recruited using an innovative approach that combines whole cell patch clamp electrophysiology, transsynaptic tract tracing, and optogenetics. With this combinatorial approach, we will test the overarching hypothesis that Shox2 interneurons in the lumbar spinal cord of adult mice display rhythmic firing that is critically mediated by persistent sodium current and local excitatory synaptic connections and recruited by monosynaptic excitatory input from the lateral paragigantocellular nucleus in the brainstem. In whole-cell patch clamp experiments, we will identify the voltage sensitive current(s) and underlying voltage-gated ion channels critically involved in rhythmic oscillations in individual Shox2 interneurons. Additionally, we will pharmacologically test the contributions of the local synaptic connections to oscillatory properties in Shox2 INs in the lumbar spinal slice. Lastly, the supraspinal nuclei which monosynaptically project to lumbar Shox2 interneurons will be identified by monosynaptic restricted transsynaptic tracing from Shox2 interneurons in the adult mouse. These anatomical projections will be functionally evaluated optogenetically in the adult lumbar spinal slice. Together, this represents essential first steps in identifying and evaluating mechanisms of rhythm generation in and recruitment of Shox2 interneurons which may serve as therapeutic targets for the treatment of paralysis in which spinal locomotor circuits are left intact, but dormant.