Chemogenetic afferent modulation to understand spinal cord circuit function and plasticity post injury

项目总结 脊髓损伤(SCI)会导致终生神经损害，目前还没有 有效的治疗。这一提议的前提是最近的工作证明了传入 刺激配合跑步机训练可以加强站立、行走和意志控制 在人类和动物模型中。因此，了解这些机制至关重要。 通过传入刺激来推动运动的改善。可以识别哪些传入的工具 是加强恢复的必要和充分条件，并可促进表征 有益的神经可塑性，是迫切需要的。我们的长期目标是为 选择性传入调制，并将其应用于对潜在机制的剖析 从脊髓损伤中恢复。这项资助的目的是确定哪组传入神经是重要的 以及脊髓环路如何改变以促进康复。为了实现选择性调制， 并启用遗传追踪，我们将使用设计者受体，由 可以调节特定神经元群体兴奋性的特制药物(DREADD)。至 准确量化改进，我们将使用深度学习来分析大型运动学数据 布景。我们的初步数据显示DREADDS在大直径DRG中有很强的表达 神经元，它们被CNO激活可以兴奋或抑制H反射，而激活 脊髓损伤后跑步机训练中的兴奋性DREADDS可改善步态。我们的主要假设 在跑台训练过程中，兴奋性DREADD(HM3Dq)是否激活了大传入神经元 会促进恢复，而抑制DREADDS(HM4Di)会抑制恢复。四个分项- 假说将测试康复是否通过增加对运动的传入投射来调节 神经元，或2)抑制性中间神经元；或通过3)网状脊髓和4)本征脊髓的萌发 电路。我们的具体目标是确定DREADD在(AIM)中的选择性表达 1)所有大直径(本体感觉和触觉)神经元和(目标2)大本体感觉 只有传入才能促进恢复。这些目的的基本原理是，传入刺激是 假设通过选择性兴奋大直径感觉神经传入(LDSA)起作用 这两者都在当地驱动运动池，并促进本征和脊髓外输入。到目前为止，它已经 无法确定哪些传入神经是在电击后招募的 刺激，或在直径相似的传入神经之间进行选择。这项工作的意义在于 在确定康复是否完全由本体感觉轴突或 本体感觉和触觉传入的结合，并揭示了 脊髓的功能可塑性。

PROJECT SUMMARY Spinal cord injury (SCI) causes life-long neurological impairment, and there is currently no effective treatment. The premise of this proposal is recent work demonstrating that afferent stimulation paired with treadmill training can enhance standing, stepping, and volitional control in humans and animal models. Therefore, it is critically important to understand the mechanisms by which afferent stimulation drives motor improvement. Tools that can identify which afferents are necessary and sufficient to enhance recovery, and that can facilitate characterization of the helpful neural plasticity, are urgently needed. Our long-term goal is to develop approaches for selective afferent modulation, and apply them to the dissection of the mechanisms underlying recovery from SCI. The objective of this grant is to identify which sets of afferents are important for recovery and how spinal circuits change to facilitate it. To achieve selective modulation of afferents and enable genetic tracing we will use Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) that can modulate excitability in specific populations of neurons. To accurately quantify improvement, we will use Deep Learning to analyze large kinematic data sets. Our preliminary data shows strong expression of DREADDs in large diameter DRG neurons, that their activation by CNO can excite or inhibit the H-reflex, and that activation of excitatory DREADDs during treadmill training post-SCI improves stepping. Our main hypothesis is that activation of large afferents by the excitatory DREADD (hM3Dq) during treadmill training will enhance recovery, whereas inhibitory DREADDs (hM4Di) will suppress recovery. Four sub- hypotheses will test whether recovery is mediated by increased afferent projection onto 1) motor neurons, or 2) inhibitory interneurons; or by sprouting of 3) reticulospinal and 4) propriospinal circuits. Our Specific Aims are to determine whether selective expression of DREADDs in (Aim 1) all large diameter (proprioceptive and tactile) neurons and (Aim 2) large proprioceptive afferents only can enhance recovery. The rationale for these aims is that afferent stimulation is hypothesized to work through selective excitation of large diameter sensory afferents (LDSA) that both drive motor pools locally and facilitate proprio- and surpraspinal input. To date, it has not been possible to definitively determine which afferents were recruited after electrical stimulation, or to select between afferents of similar diameter. The significance of this work lies in determining whether recovery is mediated exclusively by proprioceptive axons or a combination of proprioceptive and tactile afferents, and uncovering the mechanisms of functional plasticity in the spinal cord.