Using Electrochemical Methods to Probe Neuronal Signaling in the GI Tract

描述(申请人提供)：使用钻石微电极的电化学方法将用于研究肠壁中的神经元信号通路。这些方法提供了高时间分辨率的释放和作用部位附近电活动神经递质局部浓度变化的直接测量。肠道神经元含有和释放许多神经递质，其中一些可以通过电化学方法检测到，特别是5-羟色胺(5-羟色胺)和一氧化氮(NO)。将这些技术扩展到周围神经系统的研究是一个有趣和重大的发展。许多功能性胃肠道疾病，如运动、吸收/分泌和感觉障碍，没有可识别的病理生理学基础，被认为与神经源性信号机制障碍有关。这项拟议的研究将提高我们对出生后肠道成熟期间胃肠道正常运动和分泌活动的理解。这些研究将导致发现导致疾病的功能性肠道疾病的神经元信号中年龄特定的病理生理变化。为此，我们将应用钻石微电极的电化学分析方法，研究两个功能问题：(I)能否在实验动物的小肠和结肠中检测到兴奋性神经递质5-羟色胺(5-HT)和抑制性神经递质一氧化氮(NO)，以及相关的控制释放和清除的神经药理学，以及(Ii)5-HT和NO信号通路如何随ENS的出生后成熟而变化？我们的目标是确定肠壁中5-羟色胺和一氧化氮的释放和清除的控制机制。我们将在体外对豚鼠的小肠(回肠和十二指肠)和结肠的肌间神经丛和粘膜下神经丛以及一种新的SERT-KO动物模型进行研究。我们还建议扩大研究范围，包括肌间神经丛释放的多巴胺(DA)和去甲肾上腺素(NE)。这些研究对儿科健康具有重要的相关性，将作为实验动物(豚鼠)年龄的函数进行，以了解出生后ENS的发育。通过高空间和时间分辨率的肠壁局部神经递质浓度的测量，可以更好地了解胃肠道健康和疾病中的神经信号通路。与公共健康相关：这项研究将调查控制肌肉功能的肠壁中神经元信号在出生后的发展。这将通过体外电化学测量释放和作用部位附近的神经递质浓度变化来实现。这些研究将为导致儿童胃肠动力障碍的潜在病理生理变化提供新的见解，这些障碍将持续到成年，并将导致功能性胃肠功能障碍在美国的流行。

DESCRIPTION (provided by applicant): Electrochemical methods with diamond microelectrodes will be used to investigate neuronal signaling pathways in the gut wall. These methods provide a direct measure of local concentration changes of electroactive neurotransmitters near the sites of release and action with high temporal resolution. Enteric neurons contain and release many neurotransmitters and some of these can be detected by electrochemical methods, in particular, serotonin (5-HT) and nitric oxide (NO). The extension of these techniques to the study of the peripheral nervous system constitutes an interesting and significant development. Many functional GI disorders, such as disturbances in motility, absorption/secretion and sensation that do not have an identifiable pathophysiological basis, are thought to be related to dysfunction in neurogenic signaling mechanisms. The proposed research will improve our understanding of the normal motility and secretory activities of the GI tract during postnatal maturation of the intestine. These studies will lead to the discovery of age specific pathophysiologic changes in neuronal signaling responsible for functional gut disorders that lead to disease. To this end, we will apply electrochemical methods of analysis with a diamond microelectrode, to investigate two functional questions: (i) can the excitatory neurotransmitter serotonin (5-HT) and the inhibitory neurotransmitter nitric oxide (NO) be measured in the small intestine and colon of test animals and can the associated neuropharmacology controlling release and clearance be understood, and (ii) how do the 5-HT and NO signaling pathways change with postnatal maturation of the ENS? Our goal is to identify the mechanisms controlling release and clearance of 5-HT and NO in the gut wall. We will conduct these studies in vitro in the myenteric and submucosal plexuses of the small intestine (ileum and duodenum) and colon of guinea pigs and a new SERT-KO rat animal model. We also propose to expand the scope of the studies to include dopamine (DA) and norepinephrine (NE) released in the myenteric plexus. These studies, which have significant relevance for pediatric health, will be performed as a function of test animal (guinea pig) age in order to learn about the postnatal ENS development. A better understanding of neural signaling pathways in GI health and disease can be gained through measurements of local neurotransmitter concentrations in the gut wall with high spatial and temporal resolution. PUBLIC HEALTH RELEVANCE: The research will investigate the postnatal development of neuronal signaling in the gut wall that controls muscular function. This will be accomplished through in vitro electrochemical measurements of neurotransmitter concentration changes made near the sites of release and action. These studies will provide new insights into potential pathophysiological changes responsible for pediatric GI motility disturbances that would persist into adulthood and would be responsible for the prevalence of functional GI disorders in the United States.