Identification of enteric nerve circuits controlling gut motility

控制肠道运动的肠神经回路的识别

• 批准号: 10203952
• 负责人: James J. Galligan
• 金额: $ 34.51万
• 依托单位: MICHIGAN STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-17 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10203952
• 关键词: Afferent Neurons; Antibodies; Anus; Autonomic nervous system; Biosensor; Blood flow; Cavia; Cells; Choline O-Acetyltransferase; Code; Colon; Cre driver; Cre-LoxP; Data; Dependovirus; Diamond; Dinucleoside Phosphates; Electrodes; Electrophysiology (science); Enteral; Enteric Nervous System; Enzymes; Esthesia; Ganglia; Gastrointestinal Motility; Gastrointestinal tract structure; Genes; Genetic; Goals; Individual; Interneurons; Intestines; Ion Channel; Knowledge; Large Intestine; Light; LoxP-flanked allele; Maps; Measurement; Measures; Mediating; Membrane Potentials; Methods; Microelectrodes; Motor; Mus; Muscle; Myenteric Plexus; Nerve; Nerve Endings; Nerve Fibers; Nervous System control; Neurons; Neurotransmitters; Nicotinic Acids; Nitric Oxide; Nitric Oxide Synthase; Nucleotides; Nutrient; Optics; Oral; Pathway interactions; Periodicity; Phenotype; Proteins; Purines; Reflex action; Relaxation; Rhodopsin; Serotonin; Side; Signaling Molecule; Site; Small Intestines; Smooth Muscle; Stomach; Synapses; Synaptic Transmission; Synaptic Vesicles; Techniques; Testing; Tracer; Varicosity; Vesicle; Water; absorption; base; cell motility; cholinergic; effective therapy; gastrointestinal; motility disorder; neurobiotin; neurochemistry; neuromuscular; neuromuscular transmission; neurophysiology; neuroregulation; neurotransmission; novel therapeutics; photoactivation; promoter; protein biomarkers; recombinase-mediated cassette exchange; selective expression

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.

项目概要 肠神经系统（ENS）是自主神经系统的半自主部分。 ENS 控制胃肠道运动功能、营养物质和水的吸收和分泌以及肠道感觉。这 控制这些重要功能的神经回路尚未完全绘制出来。在具体目标 1 中，我们将使用 针对囊泡核苷酸转运蛋白 (VNUT) 的抗体，这是嘌呤能神经识别的蛋白质标记 这些途径。嘌呤能神经传递在 ENS 中很重要，但没有数据描述 ENS 中的嘌呤能神经元或通路。这些将是重要的新数据，将扩大我们的知识 肠突触连接。在具体目标 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、通道视紫红质-2 (ChR2) 存在于特定的神经元亚型中。这将允许选择性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）以确定它们的特定突触连接和 这些神经元释放的神经递质。这些研究将在小鼠和豚鼠身上进行。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、硝酸 氧化物合酶，例如 5-HT）与含有编码 ChR2 的 floxed 基因的小鼠杂交。我们将 使用微电极电生理学测量兴奋性和抑制性连接电位（EJP、IJP） 确定具有兴奋性和抑制性突触的神经元的神经化学表型的技术 运动神经元供应纵向和环形肌肉层。在具体目标 3 中，我们还将使用 cre- lox 方法研究肌间神经节突触连接。我们还将使用电化学方法 测量肌间神经元 ATP 和一氧化氮 (NO) 的局部释放。我们将对个体进行光学刺激 神经节并从口腔侧和肛门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，以便可以识别记录的神经元 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。这些研究将确定负责的突触连接 周期性推进性结肠收缩。成功完成这些研究将确定突触 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。一个更多 完全了解这些途径将有助于识别肠道蠕动的神经控制缺陷和 肠道动力障碍的新药或基因治疗的鉴定。