Identification of enteric nerve circuits controlling gut motility

项目摘要 肠神经系统(ENS)是自主神经系统的一个半自治分支。美国国税局 控制胃肠运动功能，营养物质和水分的吸收和分泌，以及肠道感觉。这个 控制这些重要功能的神经回路并没有完全被绘制出来。在特定的目标1中，我们将使用 针对嘌呤能神经识别蛋白质标记物的囊泡核苷酸转运体抗体 这些小路。嘌呤能神经传递在ENS中很重要，但没有数据描述 鼻内窥镜下的嘌呤能神经元或通路。这些将是重要的新数据，将拓宽我们的知识 肠内突触连通性。在特定目标2中，我们将使用cre-lox技术和腺相关病毒 (AAV9)转导肌间神经元表达光激活离子通道视紫红质-2 (ChR2)在特定的神经元亚型中。这将允许选择性地激活特定功能类别的神经元 (中间神经元、运动神经元、感觉神经元)，以确定它们特定的突触连接和 这些神经元释放的神经递质。这些研究将在老鼠和豚鼠身上进行。我们将使用 Cre驱动小鼠，其中Cre由神经元亚型特异性启动子(胆碱乙酰转移酶、嘌呤、一氧化氮)驱动 氧化物合成酶，例如，5-羟色胺)与含有编码ChR2的FLOXED基因的小鼠杂交。我们会 微电极电生理学测量兴奋性和抑制性连接电位 兴奋性和抑制性突触神经元的神经化学表型测定技术 运动神经元供应纵肌层和环肌层。在具体目标3中，我们还将使用cre- LOX法研究肌间神经节中的突触连接。我们还将使用电化学方法来 测量肌间神经元局部释放的三磷酸腺苷和一氧化氮(NO)。我们将对个人进行光学刺激 神经节，并从口腔和肛门一侧的肌间神经元进行细胞内记录 刺激。我们将使用充满神经生物素的微电极，以便记录的神经元可以在 随后的免疫组织化学研究以确定接受突触的神经元的神经化学表型 来自光刺激神经元的输入。这些研究将确定突触连接负责 周期性推进性结肠收缩。这些研究的成功完成将确定突触 神经化学鉴定的肌间神经元亚群之间的联系。这些连接控制 肠道平滑肌的协调收缩和松弛导致肠道内容物的推进。A更多 对这些通路的完整理解将有助于识别神经控制肠道运动和 肠动力障碍的新药或基因疗法的鉴定。

Project Summary The enteric nervous system (ENS) is a semi-autonomous division of the autonomic nervous system. The ENS controls gastrointestinal motor function, absorption and secretion of nutrients and water and gut sensation. The nerve circuits controlling these important functions are incompletely mapped. In specific aim 1, we will use an antibody against the vesicular nucleotide transporter (VNUT), a protein marker for purinergic nerves to identify these pathways. Purinergic neurotransmission is important in the ENS but there are no data describing the purinergic neurons or pathways in the ENS. These will be important new data that will broaden our knowledge of enteric synaptic connectivity. In specific aim 2 we will use cre-lox technology and adeno-associated virus (AAV9) transduction of myenteric neurons to express the light-activated ion channel, channel rhodopsin-2 (ChR2) in specific neuronal subtypes. This will allow selective activation of specific functional classes of neurons (interneurons, motorneurons, sensory neurons) to determine their specific synaptic connections and the neurotransmitters that these neurons release. These studies will be done in mice and guinea pigs. We will use cre driver mice where cre is driven by neuron-subtype specific promoter (choline acetyltransferase, purines, nitric oxide synthase, 5-HT, for example) crossed with mice containing the floxed gene encoding ChR2. We will measure excitatory and inhibitory junction potentials (EJPs, IJPs) using microelectrode electrophysiology techniques to determine the neurochemical phenotype of neurons synapsing with excitatory and inhibitory motorneurons supplying the longitudinal and circular muscle layers. In specific aim 3, we will also use the cre- lox approach to study synaptic connections in myenteric ganglia. We will also use electrochemical methods to measure local release of ATP and nitric oxide (NO) from myenteric neurons. We will optically stimulate individual ganglia and make intracellular recordings from myenteric neurons on the oral and anal sides of the site of stimulation. We will use microelectrodes filled with neurobiotin so the recorded neurons can be identified in subsequent immunohistochemical studies to identify the neurochemical phenotype of neurons receiving synaptic input from the optically stimulated neurons. These studies will identify synaptic connections responsible for periodic propulsive colonic contractions. Successful completion of these studies will identify synaptic connections between neurochemically identified subsets of myenteric neurons. These connections control coordinated contractions and relaxation of gut smooth muscle leading to propulsion of gut content. A more complete understanding of these pathways will aid in identifying deficits in neural control of gut motility and identification of new drug or genetic treatments of gut motility disorders.