PreBotzinger Circuit in Respiratory Rhythm Generation

描述（由申请人提供）：越来越多的证据表明，节律产生电路的一个重要组成部分位于腹外侧延髓的一个离散区域，称为preBotzinger复核。然而，要理解波青格之前对呼吸的贡献，还需要对组成神经元的相关特性和网络相互作用进行全面的描述。本项目的目标是提供preBotzinger复合体中存在的呼吸神经元类型的合理完整描述，包括它们的放电模式，对选定传入输入激活的反应，神经递质（GABA，谷氨酸，甘氨酸）含量以及与其他preBotzinger神经元形成的突触连接模式。考虑到preBotzinger复合物的小尺寸（在成年大鼠中，它长约0.6毫米，直径约1.5毫米，包括树突），我们有能力提供这种分析。将采取三个具体目标。在Aim 1中，将使用细胞内或细胞外记录来对神经元的放电模式及其对迷走神经和喉上神经传入神经刺激的反应进行分类。记录下来的神经元将被注射染料。随后的免疫组织化学分析在光和超微结构水平将确定神经递质含量（GABA，甘氨酸，谷氨酸）和它们的轴突投射模式。在Aim 2中，preB6tzinger神经元之间的突触相互作用将通过互补的电生理和解剖学方法进行鉴定。在电生理方法中，峰值触发平均或相互关联方法将用于配对神经元记录。在解剖实验中，细胞内或细胞旁标记将用于染料标记2个不同功能组的细胞，以便随后对其突触相互作用进行光和超微结构分析。在Aim 3中，我们将使用已识别的神经元特性和连通性开发preB6tzinger呼吸网络的详细计算模型。要解决的假设是，preB6tzinger复合体内的神经元类型和突触相互作用足以在体内产生呼吸节律。

DESCRIPTION (provided by applicant): There is increasing evidence that an essential component of the rhythm generating circuitry is located within a discrete region of the ventrolateral medulla termed the preBotzinger complex. Nevertheless, an understanding of the preBotzinger contribution to breathing awaits a comprehensive description of the pertinent properties and network interactions of the constituent neurons. The goal of this project to provide a reasonably complete description of the respiratory neuron types present in the preBotzinger complex, including their discharge patterns, response to activation of selected afferent inputs, neurotransmitter (GABA, glutamate, glycine) content and pattern of synaptic connections formed with other preBotzinger neurons. Given the small size of the preBotzinger complex (in the adult rat it is approximately 0.6 mm long and about 1.5 mm in diameter, including dendrites), it is within our means to provide this analysis. Three Specific Aims will be undertaken. In Aim l, intracellular or extracellular recording will be used to classify neurons with respect to discharge pattern and their response to stimulation of vagus and superior laryngeal nerve afferents. The recorded neurons will then be injected with dye. Subsequent immunohistochemical analysis at the light and ultrastructural levels will identify the neurotransmitter content (GABA, glycine, glutamate) and their axonal projection patterns. In Aim 2, synaptic interactions between preB6tzinger neurons will be identified with complementary electrophysiological and anatomical approaches. In the electrophysiological approach, either spike triggered averaging or cross-correlation approaches will be used with paired neuronal recordings. In anatomical experiments intra- or juxtacellular labeling will be used to dye-label cells in 2 different functional groups for subsequent light and ultrastructural analysis of their synaptic interactions. In Aim 3, we will develop a detailed computational model of the preB6tzinger respiratory network using the identified neuronal properties and connectivity. The hypothesis to be addressed is that the neuronal types and synaptic interactions within the preB6tzinger complex are sufficient for respiratory rhythm generation in vivo.