Mechanisms of activation of vagal afferent neurons

描述（由申请人提供）：本项目的长期目标是确定迷走神经传入神经元对控制能量稳态的重要刺激的敏感性，并确定这些刺激激活该传入信号通路的细胞机制。在这个建议中的调查将具体集中在胆囊收缩素（CCK），瘦素，和游离脂肪酸（FFA）对迷走神经传入神经元的激活和相互作用的程度的机制。选择这些物质是因为有大量证据表明CCK、瘦素和FFA各自参与身体能量平衡的控制，并且有证据表明这些物质之间存在全身相互作用。已发表的报告以及提案中提出的初步数据表明，迷走神经传入神经亚群对CCK、瘦素和FFA敏感。初步结果进一步表明，在激活单个迷走神经传入神经元中，这些物质之间存在重要的相互作用。在这个项目中，实验将主要集中在迷走神经传入神经元分离成年大鼠结状神经节。在这种制备的神经元保留大部分，如果不是全部，归因于完整的迷走传入纤维在体内的属性。使用这种制剂能够设计出比体内更精细和更严格控制的神经元反应的电、化学和药理学研究。该制备将用于解决三个具体目标：1）将利用单细胞Ca 2+成像、逆行标记和免疫组织化学的组合来建立对CCK、瘦素和FFA敏感的离散迷走神经传入群体的神经支配靶点。2)结合膜片钳电生理学和Ca2+成像的药理学工具将被用来确定CCK，瘦素和FFA激活迷走神经传入神经元的细胞机制，并确定这些个体刺激在迷走神经传入神经元水平相互作用的细胞机制。3)假设，除了急性激活和相互作用，瘦素，CCK，FFA也有慢性和持久的影响，这些神经元的反应将被测试。单个迷走神经传入神经元对这些不同过程的信号的反应和整合程度在很大程度上仍然没有得到重视。在这一系列的调查中发展的见解将能够更好地重建这些重要的监管信号的系统性行动和相互作用。更好地理解这一重要的信号通路将有助于理解能量稳态和GI功能，并有助于设计更好的健康治疗干预措施。

DESCRIPTION (provided by applicant): The long-term goals of this project are to define the sensitivity of vagal afferent neurons to stimuli that are important to the control of energy homeostasis and to determine the cellular mechanisms by which these stimuli activate this afferent signaling pathway. The investigations in this proposal will specifically focus on the mechanisms of activation and degree of interaction of cholecystokinin (CCK), leptin, and free fatty acids (FFA) on vagal afferent neurons. These substances were chosen because there is abundant evidence that CCK, leptin, and FFA each participates in the control of body energy balance, and evidence exists that there are systemic interaction between these substances. Published reports, as well as preliminary data presented in the proposal, indicate that subpopulations of vagal afferents are sensitive to CCK, leptin, and FFA. Preliminary results further indicate that there are important interactions between these substances in the activation of individual vagal afferent neurons. In this project experiments will concentrate primarily on vagal afferent neurons isolated from adult rat nodose ganglia. The neurons in this preparation retain most, if not all, of the properties ascribed to intact vagal afferent fibers in vivo. The use of this preparation enables the design of much more refined and tightly controlled electrical, chemical, and pharmacological investigations of neuronal responses than are possible in vivo. This preparation will be used to address three specific aims: 1) A combination of single cell Ca2+ imaging, retrograde labeling, and immunohistochemistry, will be utilized to establish the innervation targets of discrete vagal afferent populations sensitive to CCK, leptin, and FFA. 2) Pharmacological tools combined with patch clamp electrophysiology and Ca2+ imaging will be utilized to determine the cellular mechanisms by which CCK, leptin, and FFA activate vagal afferent neurons, and to determine the cellular mechanisms by which these individual stimuli interact at the level of the vagal afferent neuron. 3) The hypothesis that in addition to acute activation and interactive effects, leptin, CCK, and FFA also have chronic and enduring effects on the responsiveness of these neurons will be tested. The degree to which individual vagal afferent neurons respond to and integrate signals from these disparate processes largely remains unappreciated. The insights developed in this series of investigations will enable a better reconstruct of the systemic actions and interactions of these important regulatory signals. A better appreciation of this important signaling pathway will contribute to the understanding of energy homeostasis and GI function, and aid in the design of therapeutic interventions for better health.