Mature spinal networks in rodents: electrophysiological investigations combined with optogenetic methods to define neuron and circuit function

哺乳动物运动所需的有节奏的有模式的运动输出完全可以由一种名为中央模式生成器(CPG)的特殊脊髓(中间)神经元电路产生。识别参与CPG活动的中间神经元对于了解启动持续的模式肢体运动所必需的脊髓神经元以及通过感觉输入(即反射环)启动运动输出的脊髓神经元与通过棘上下行输入(即自主环)启动运动的脊髓神经元之间的联系以及CPG神经元之间的相互连接如何导致有节奏的运动输出是至关重要的。 使用乙酰胆碱作为神经递质的中间神经元-CPG的胆碱能神经元成分-被认为以一种状态依赖的方式控制运动神经元的兴奋性，并改变运动节律的频率。中央管附近沿几乎整个脊髓的胆碱能中间神经元群已被描述为通过特殊的突触来支配脊髓运动神经元。这些胆碱能神经元(ChATX INS)是这一提议的重点。 用于选择性激活不同神经元电路的光遗传学工具将在体内神经生理学实验室的成熟小鼠脊髓网络中使用，该实验室有能力在整合的脑和脊柱立体定位框架中研究功能成熟的小鼠脊髓。在去大脑的小鼠中，运动输出可以通过几种方式产生，包括通过反射和自愿环路。通过将光刺激应用于ChATX INS，将研究它们产生运动输出或改变已有的运动活动以及自愿或反射环诱导活动的输出的能力。电生理技术的光刺激将与解剖技术和活性依赖标记相结合，以确定ChATX INS的功能作用和连接性。在拟议的目标中，概述了对五名本科生和两名研究生的培训。 由于我的项目专门旨在研究成熟的哺乳动物网络，基本功能图谱将连接来自简化系统(例如，新生动物)的知识和来自人类的知识。通过确定ChATX IN神经元在运动中的作用，有可能在CPG网络中识别出一类与运动神经元直接联系的兴奋性末级中间神经元。ChATX IN神经元可能是第一批细胞，如果它们的选择性激活导致持续的CPG输出，则可以被视为CPG节律生成层神经元。

The rhythmic patterned motor output required for locomotion in mammals can be produced entirely by a specialized spinal (inter)neuronal circuit called the central pattern generator (CPG). Identification of interneurons that contribute to CPG activity is pivotal understanding spinal neuronal populations that are necessary to initiate sustained patterned limb movements and those spinal neurons that initiate motor output via sensory-input (i.e., reflex loops) vs. those that initiate locomotion via supraspinal descending input (i.e., voluntary loops), and how the interconnections of CPG neurons lead to rhythmic motor output. Interneurons using acetylcholine as a neurotransmitter - cholinergic neuronal components of the CPG - have been suggested to control motoneuronal excitability in a state-dependent manner and to modify the frequency of the locomotor rhythm. A cholinergic interneuron population near the central canal along nearly the entire spinal cord has been described to innervate spinal motoneurons by specialized synapses. These cholinergic lamina X neurons (ChATX INs) are the focus of this proposal. Optogenetic tools for selective activation of distinct neuronal circuits will be used in mature, murine spinal networks in an in vivo neurophysiology laboratory with capabilities for studying functionally mature mouse spinal cords in an integrated stereotaxic brain and spinal frame. In decerebrated mice, motor output can be generated in several ways, including via reflex and voluntary loops. By the use of optical stimulation applied to the ChATX INs, their ability to generate locomotor output or to modify already existing locomotor activity and the output of voluntary or reflex loop induced activity will be investigated. Optical stimulation with electrophysiological techniques will be used in combination with anatomical techniques and activity dependent labelling to determine the functional roles and connectivity of ChATX INs. During the proposed objectives, training of five undergraduate students and two graduate level trainees is outlined. As my program specifically aims to study mature, mammalian networks, the basic functional maps will bridge knowledge derived from simplified systems (for example, neonatal animals) and those derived from humans. By defining the role of ChATX IN neurons in locomotion, it may be possible to identify a novel class of excitatory last order interneurons within the CPG network with direct connections to motoneurons The ChATX IN neurons may be the first population of cells which could be viewed as CPG rhythm generation layer neurons if their selective activation leads to sustained CPG output.