Cellular and neuronal circuit mechanisms involved in locomotor activity

项目摘要 哺乳动物的运动是活动和生存所必需的行为。移动涉及到 在相对的四肢之间以及在对抗的肌肉之间协调和交替的节奏活动 有着相同的肢体。运动中枢模式发生器(CPG)，一个由脊髓中间神经元组成的网络，被认为是 产生运动模式和节奏，直接激活运动神经元，进而激活外周 导致运动的肌肉。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)神经元是新生儿和婴儿运动行为的必要条件和充分条件 成年小鼠。VSCT神经元具有两种节律特性，即呈现Ih电流和它们是 电耦合。此外，它们在同一脊椎节段内具有脊髓轴突侧支以及 超分段的。VSCT也被运动神经元轴突侧支联系，它们是电耦合的 对他们来说，至少在出生后早期发育期间是这样。VSCT依次与其他脊髓中间神经元连接 通过轴突侧支参与运动中枢模式生成器。我们发表的观察结果提供了 进一步剖析它们的细胞特征和它们形成的神经元回路的坚实基础 其他脊髓神经元，包括运动神经元。我们假设VSCT和VSCT的细胞特征 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 在老鼠身上。在目标1中，我们将研究在VSCT中负责Ih电流的HCN通道的贡献和 确定我们通过初步结果确定的几种连接蛋白的作用，它们的作用是 运动性节律发生中的关键神经元。为了做到这一点，我们将利用条件基因敲除小鼠，全细胞贴片 钳制方案以及药理学研究、免疫组织化学和病毒介导的通道 击倒对手。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、行为分析、 生理学方法和病毒介导的方法，以绘制涉及的一些关键神经元电路 在运动行为上。此外，我们还将使用一种新的“清除”技术和双光子激光。 用共聚焦显微镜对新生和成年小鼠的整个脊髓成像以揭示两者之间的关系 在脊髓内和许多不同的脊髓中VSCT与其潜在的神经元靶点之间 细分市场。在目标3中，我们将确定一组新的转录因子是否标志着这类脊髓 中间神经元可以通过化学发生和光发生的方法来操纵，并确定它们的参与 在类似运动的行为中。总而言之，这套全面的实验将为 进一步加深了我们对脊椎运动网络的理解。

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.