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节律性模式运动输出可以完全由称为中央模式发生器（CPG）的专门脊髓（间）神经元回路产生。识别有助于CPG活动的中间神经元是理解启动持续模式化肢体运动所必需的脊髓神经元群体和通过感觉输入启动运动输出的脊髓神经元（即，反射环）与经由脊髓上下行输入启动运动的那些（即，自主循环），以及CPG神经元的相互连接如何导致有节奏的运动输出。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-已被建议以状态依赖的方式控制运动神经元的兴奋性，并修改运动节律的频率。胆碱能中间神经元群靠近中央管沿着几乎整个脊髓被描述为通过专门的突触支配脊髓运动神经元。这些胆碱能X层神经元（ChATX INs）是本提案的重点。 * 用于选择性激活不同神经元回路的光遗传学工具将在体内神经生理学实验室中用于成熟的鼠脊髓网络，该实验室具有在整合的立体定位脑和脊髓框架中研究功能成熟的小鼠脊髓的能力。在去大脑的小鼠中，运动输出可以通过几种方式产生，包括通过反射和自主回路。通过使用施加于ChATX IN的光学刺激，将研究它们产生运动输出或改变已经存在的运动活动以及自主或反射回路诱导的活动的输出的能力。将结合解剖学技术和活动依赖性标记使用电生理技术的光学刺激，以确定ChATX INS的功能作用和连接性。在拟定目标期间，概述了对5名本科生和2名研究生水平受训者的培训。*由于我的项目专门旨在研究成熟的哺乳动物网络，因此基本功能图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。通过定义ChATX IN神经元在运动中的作用，有可能在CPG网络内识别出一类新的兴奋性最后一级中间神经元，其与运动神经元直接连接。如果ChATX IN神经元的选择性激活导致持续的CPG输出，则ChATX IN神经元可能是可被视为CPG节律产生层神经元的第一群细胞。

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.**