Development of excitatory V2a connectivity within spinal circuits

描述（由申请方提供）：所有脊椎动物物种在发育过程中都会经历身体大小、肌肉组织、神经元和网络特性的实质性变化。尽管有这些变化，运动行为必须继续适当地产生。为了在整个发育过程中有效地维持运动，脊髓中的运动回路可能稳定了对运动神经元驱动行为的兴奋性驱动。运动回路如何完成这项任务尚不清楚。然而，更好地了解这一过程可以提供宝贵的洞察力，发展障碍，导致中断运动网络连接和兴奋性。因此，本建议的目标是定义脊髓运动前兴奋性驱动通过发育进行修改以产生一致的运动输出的机制。斑马鱼模型系统非常适合于研究发育中的运动行为。它们的透明性使得能够前所未有地在体内进入发育中的运动回路，包括神经元和突触连接的纵向成像以及神经元活动和运动输出的记录。在斑马鱼，在其他脊椎动物中，脊髓运动前兴奋性驱动的主要来源来自于V2 a神经元。重要的是，在早期，胚胎和后来，幼虫阶段的最背侧定位和最早出生的V2 a细胞（dV 2as）和“初级”运动神经元（pMNs）被激活，尽管在相同类型的强烈运动的大小和电特性的脊柱人口的戏剧性变化。这些观察结果表明，dV 2as在强运动期间驱动pMNs的作用在发育期间得以维持。该提案将解决dV 2as如何通过这些发育变化来维持连接并稳定突触驱动到pMN以支持网络功能。在目标1中，我们将首先考虑如何通过轴突和突触分布和稳定性的形态学评估来建立和维持突触接触。在目标2中，我们将通过在发育的不同阶段进行全细胞膜片钳记录来检查dV 2a-pMN连接的功能成熟。我们的试验数据表明，pMN大小的增加（从而降低输入电阻）伴随着兴奋性驱动的增加。这些实验将有助于确定这种关系是否可以通过加强单个dV 2a细胞连接或增加新的连接来解释。最后，在目标3中，我们将研究突触后兴奋性在设置突触前强度中的指导作用，通过外源性表达离子通道来降低或增加pMN的兴奋性。总之，本提案中概述的实验将提供关键的洞察力， 在发育过程中负责稳定可识别的运动回路功能的形态和功能机制。

DESCRIPTION (provided by applicant): All vertebrate species experience substantial changes in body size, musculature, neuronal and network properties during development. Despite these changes, motor behaviors must continue to be generated appropriately. In order to efficiently maintain movements throughout development, motor circuits in the spinal cord presumably stabilize excitatory drive to the motoneurons driving behavior. How motor circuits accomplish this task is unknown. However, a better understanding of this process could provide invaluable insight into developmental disorders that result from disruptions in motor network connectivity and excitability. Therefore, the goal of this proposal is to define the mechanisms by which spinal premotor excitatory drive is modified through development to generate consistent motor output. The zebrafish model system is ideally suited for the investigation of developing motor behaviors. Their transparency enables unprecedented in vivo access to developing motor circuitry, including longitudinal imaging of neurons and synaptic connections and recordings of neuronal activity and motor output. In zebrafish, as in other vertebrates, the major source of spinal premotor excitatory drive arises from glutamatergic V2a interneurons. Critically, at early, embryonic and later, larval stages the most dorsally located and earliest born V2a cells (dV2as) and 'primary' motoneurons (pMNs) are activated during the same types of strong movements despite dramatic changes in the size and electrical properties of spinal populations. These observations suggest that the role of the dV2as in driving pMNs during strong movements is maintained during development. This proposal will address how dV2as maintain connectivity and stabilize synaptic drive to pMNs through these developmental changes to support network function. In Aim 1, we will first consider how synaptic contacts are made and maintained by morphological assessment of axon and synapse distribution and stability. In Aim 2, we will examine the functional maturation of the dV2a-pMN connection by performing whole-cell patch clamp recordings at distinct stages of development. Our pilot data suggest that increases in pMN size (and thus decreases in input resistance) are accompanied by increases in excitatory drive. These experiments will help determine if this relationship can be explained by the strengthening of individual dV2a cell connections or the addition of new connections. Finally, in Aim 3 we will investigate the instructional role of postsynaptic excitability in setting presynapti strength by decreasing or increasing the excitability of pMNs through exogenous expression of ion channels. Together, the experiments outlined in this proposal will provide key insight into the morphological and functional mechanisms responsible for stabilizing function in an identifiable motor circuit during development.