Cellular and neuronal circuit mechanisms involved in locomotor activity

项目摘要 运动是哺乳动物生存和活动的基本行为。运动包括 在相对的肢体之间以及对抗性肌肉之间协调和交替的节律性活动 相同的肢体。运动中枢模式发生器（CPG）是一种脊髓中间神经元网络，被认为是 产生运动模式和节奏，并直接激活运动神经元，进而激活外周神经元。 肌肉导致运动。我们最近报道了一组脊髓中间神经元，称为腹侧 脊髓小脑束（VSCT）神经元，是必要的和足够的运动行为，在新生儿和 成年老鼠VSCT神经元具有两种节律发生特性，即表现出Ih电流， 电耦合。此外，它们在同一脊髓节段内具有脊髓轴突侧支， 额外的节段。VSCT还与运动神经元轴突侧支接触，并且它们电耦合 至少在出生后的早期发育阶段。VSCT反过来与其他脊髓中间神经元连接 参与运动中枢模式发生器，通过它们的轴突侧支。我们公布的观察结果提供了 一个强大的基础，进一步剖析他们的细胞特征和他们的神经回路，他们形成与 其他脊髓神经元，包括运动神经元。我们假设VSCT的细胞特征和 与VSCT轴突侧支接触的脊髓神经元靶点将是运动行为的关键决定因素 对小鼠在目标1中，我们将研究VSCT中负责Ih电流的HCN通道的贡献， 确定我们通过初步结果确定的几种连接蛋白的作用， 运动节律发生中的关键神经元。为此，我们将利用条件性基因敲除小鼠、全细胞贴片 沿着药理学研究、免疫组织化学和病毒介导的通道 击倒。在目标2中，我们将研究VSCT与Chx10+，Sim1+和运动神经元的神经元连接。 我们将利用小鼠遗传学，行为测定， 生理学方法和病毒介导的方法来绘制一些关键的神经回路 在运动行为中。此外，我们将采用一种新的“清除”技术与双光子激光一起使用 共聚焦显微镜对新生和成年小鼠的整个脊髓进行成像，以揭示 在脊髓内以及在许多不同的脊髓中，VSCT与其潜在的神经元靶点之间存在着联系， 片段在目标3中，我们将确定是否有一组新的转录因子标记这类脊髓 中间神经元可以通过化学遗传学和光遗传学方法操纵， 类似运动的行为。总之，这套全面的实验将提供坚实的基础， 进一步加深了我们对脊髓运动网络的理解。

Project Summary Mammalian locomotion is an essential behavior for mobility and survival. Locomotion involves coordinated and alternating rhythmic activity between opposing limbs, as well as between antagonistic muscles of the same limb. The locomotor central pattern generator (CPG), a network of spinal interneurons, is thought to produce the locomotor pattern and rhythm and directly activate motor neurons, which in turn activate peripheral muscles resulting in movement. We have recently reported that a set of spinal interneurons, known as ventral spinocerebellar tract (VSCT) neurons, are both necessary and sufficient for locomotor behavior in neonatal and adult mice. VSCT neurons possess two rhythmogenic properties, namely the exhibit an Ih current and they are electrically coupled. Additionally, they possess spinal axon collaterals within the same spinal segment as well as extra-segmentally. VSCTs are also contacted by motor neuron axon collaterals and they are electrically coupled to them, at least during early postnatal development. VSCTs in turn, connect with other spinal interneurons involved in the locomotor central pattern generator, via their axon collaterals. Our published observations provide a strong foundation to dissect further their cellular characteristics and their neuronal circuits that they form with other spinal neurons, including motor neurons. We hypothesize that both cellular characteristics in VSCTs and the spinal neuronal targets contacted by VSCTs’ axon collaterals will be key determinants for locomotor behavior in mice. In Aim 1, we will study the contribution of the HCN channels responsible for the Ih current in VSCTs and determine the role of several connexins that we have identified through our preliminary results in their role as key neurons in locomotor rhythmogenesis. To do so, we will utilize conditional knock out mice, whole-cell patch clamp protocols along with pharmacological studies, immunohistochemistry, and virally-mediated channel knockdown. In Aim 2, we will study the neuronal connectivity of VSCTs with Chx10+, Sim1+ and motor neurons. We will investigate their functional role in these connections utilizing mouse genetics, behavioral assays, physiological approaches and viral-mediated approaches to map out some of the key neuronal circuits involved in locomotor behavior. Furthermore, we will employ a novel “clearing” technique together with 2-photon laser confocal microscopy to image the entire spinal cord of neonatal and adult mice to uncover the relationship between VSCTs and their potential neuronal targets within the spinal cord and across many different spinal segments. In Aim 3, we will establish whether a novel set of transcription factors marking this class of spinal interneurons can be manipulated by chemogenetic and optogenetic approaches and determine their involvement in locomotor-like behavior. In summary, this comprehensive set of experiments will provide a solid foundation to further our understanding of spinal locomotor networks.