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。内在的和 腰椎节律性中间神经元使用的局部机制及其如何被 椎管上运动中枢代表了我们对节律发生和运动理解的主要差距。 电路。这些信息在寻求治疗目标以治疗瘫痪的主要原因方面至关重要。 包括脊髓损伤、创伤性脑损伤和帕金森氏症。脊髓中间神经元表达 转录因子Shox2包括一组在小鼠体内可能导致节律的中间神经元。Shox2 成人腰椎切片中的中间神经元具有节律性离子电流，包括持续的内向电流。 电流，并与其他Shox2中间神经元建立功能性兴奋性联系。我们已经发现了一个子集 成人腰椎切片中Shox2中间神经元的自发性节律膜电位 震荡。固有的和局部的网络特性对于节律的启动和维持是必不可少的 脊髓薄片中神经元爆裂和Shox2中间神经元的其他模型的振荡允许研究 成年哺乳动物运动回路中涉及的特定机制。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，这种方法结合了 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，我们将检验Shox2中间神经元在成年小鼠腰髓中的主要假设 显示由持续钠电流和局部兴奋性突触关键介导的节律性放电 巨细胞旁外侧核的单突触兴奋性传入 脑干。在全细胞膜片钳实验中，我们将确定电压敏感电流(S)和基础电流 电压门控离子通道在单个Shox2中间神经元的节律性振荡中起关键作用。 此外，我们还将从药理学角度测试局部突触连接对振荡的贡献。 Shox2 Ins在腰椎切片中的性质。最后，单调投射的脊髓上核团 通过对Shox2的单突触限制性跨突触示踪，可以识别到Shox2的中间神经元 成年小鼠的中间神经元。这些解剖投影将在功能上进行光遗传学评估 成人腰椎切片。总而言之，这是识别和评估的重要第一步 Shox2中间神经元的节律产生和募集机制可能起治疗作用 脊髓运动回路完好无损但处于休眠状态的瘫痪的治疗靶点。

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.