Respiratory interneuron circuit plasticity: promoting recovery of diaphragm function after spinal cord injury

项目摘要/摘要（最多30行） 脊髓中间神经元（IN）在中枢神经系统回路的连接和功能中起着不可或缺的作用。重要的是， 脊髓IN群体主要参与可塑性机制，负责介导有限程度的 脊髓损伤（SCI）后自发发生的功能恢复。因此，关键问题 需要解决的问题是：治疗干预是否可以进一步招募这些脊髓神经元， 电路，以强大地恢复失去的功能后，SCI？如果是的话，通过什么样的回路可塑性模式， 是否发生脊髓内募集？为了在R 01续订申请中解决这个非常重要的主题，我们的目标是 检查治疗性刺激轴突再生是否可以通过 形成膈前IN（PP-IN）中继回路，以促进颈脊髓损伤后膈肌功能的恢复。 大多数SCI病例发生在颈脊髓，导致持续的呼吸困难。 与死亡率相关的功能障碍、对机械通气的依赖、一系列疾病， 呼吸道感染，生活质量大大降低。膈肌由膈神经直接控制 神经元（PhMN）位于颈脊髓水平C3、C4和C5。PhMN是单突触激活的 由位于喙侧呼吸群（rVRG）的脊髓上脑干神经元引起。颈椎脊髓损伤导致 下行rVRG纤维的轴突切断、备用PhMN的去神经支配和沉默以及膈肌麻痹。 我们已经证明，全身给予血脑屏障可渗透的PTEN拮抗剂， PAP肽可使脊髓损伤后rVRG轴突再生，从而显著恢复膈肌功能 功能令人兴奋的是，我们的研究结果表明，PAP诱导的rVRG轴突再生促进了大量的神经再生。 通过形成多突触PP-IN中继电路来部分该隔膜恢复。具体来说，我们假设 可以募集节段间PP-IN以将来自再生rVRG轴突的球脊髓输入中继到PhMN。 在目标1研究中，我们将确定选择性沉默C3-至-C5投射节段间PP- INs影响C2后PAP诱导的rVRG轴突再生刺激的膈肌功能恢复 脊髓半切为此，我们将使用一种创新的双病毒载体化学基因沉默方法， 用单突触和多突触逆行和顺行rVRG / PP-IN / PhMN回路追踪。在目标2中， 我们将确定治疗性刺激C1至C5投射PP-IN轴突的再生是否跨越 SCI损伤促进C2半切后横膈膜的恢复，再次使用化学基因沉默策略。 在目标3中，我们将把这项工作扩展到临床相关的颈挫伤SCI模型，开始， 评估靶向节段间PP-IN可塑性促进呼吸恢复的翻译潜力。 在这个创新和高度重要的项目中，我们将：（1）确定脊髓IN人群是否 可以响应轴突再生的治疗性刺激而有益地被募集到新的神经回路中; （2）揭示PP-IN可塑性促进颈脊髓损伤后呼吸功能恢复的机制。

Project Summary / Abstract (30-line maximum) Spinal cord interneurons (INs) play indispensable roles in CNS circuit connectivity and function. Importantly, spinal IN populations are centrally involved in plasticity mechanisms responsible for mediating the limited degree of functional recovery that can occur spontaneously after spinal cord injury (SCI). Therefore, critical questions that need to be addressed are: Can therapeutic interventions further recruit these spinal cord INs into remodelled circuits to robustly restore lost function following SCI? If so, by which modes of circuit plasticity can this spinal cord IN recruitment occur? To address this highly important topic in this R01 Renewal application, we aim to examine whether therapeutically stimulating axon regeneration can promote respiratory circuit plasticity via the formation of pre-phrenic IN (PP-IN) relay circuits to drive recovery of diaphragm function after cervical SCI. A majority of SCI cases occur in the cervical spinal cord, resulting in persistent diaphragmatic respiratory dysfunction that is associated with mortality, dependence on mechanical ventilation, a host of morbidities such as respiratory infections, and greatly reduced quality of life. Diaphragm is directly controlled by phrenic motor neurons (PhMNs) located at cervical spinal cord levels C3, C4 and C5. PhMNs are monosynaptically activated by supraspinal brainstem neurons located in rostral Ventral Respiratory Group (rVRG). Cervical SCI results in the axotomy of descending rVRG fibers, denervation and silencing of spared PhMNs, and diaphragm paralysis. We have demonstrated that systemic administration of a blood brain barrier-permeable PTEN antagonist peptide (PAP) can regenerate rVRG axons after cervical SCI, resulting in significant restoration of diaphragm function. Excitingly, our findings suggest that PAP-induced rVRG axon regeneration is promoting a substantial portion this diaphragm recovery via formation of polysynaptic PP-IN relay circuitry. Specifically, we hypothesize that intersegmental PP-INs can be recruited to relay bulbospinal input from regenerating rVRG axons to PhMNs. In Aim 1 studies, we will determine whether selectively silencing C3-to-C5 projecting intersegmental PP- INs impacts recovery of diaphragm function stimulated by PAP-induced rVRG axon regeneration after C2 hemisection SCI. To do so, we will use an innovative dual-viral vector chemogenetic silencing approach, coupled with monosynaptic and polysynaptic retrograde and anterograde rVRG / PP-IN / PhMN circuit tracing. In Aim 2, we will determine whether therapeutically stimulating regeneration of C1-to-C5 projecting PP-IN axons across a SCI lesion promotes diaphragm recovery after C2 hemisection, again using a chemogenetic silencing strategy. In Aim 3, we will extend this work to the more clinically-associated cervical contusion SCI model to begin to assess the translational potential of targeting intersegmental PP-IN plasticity to promote respiratory recovery. In this innovative and highly-significant project, we will: (1) determine whether spinal cord IN populations can be beneficially recruited into new neural circuits in response to therapeutic stimulation of axon regeneration; and (2) uncover PP-IN plasticity mechanisms capable of promoting respiratory recovery following cervical SCI.