Mechanisms of activation of vagal afferent neurons

描述（由申请人提供）：该项目的长期目标是确定迷走神经传入神经元对能量稳态控制重要刺激的敏感性，并确定这些刺激激活传入信号通路的细胞机制。本研究将特别关注胆囊收缩素（CCK）、瘦素和游离脂肪酸（FFA）对迷走神经传入神经元的激活机制和相互作用程度。之所以选择这些物质，是因为有大量证据表明CCK、瘦素和FFA各自参与控制机体能量平衡，并且有证据表明这些物质之间存在系统性相互作用。已发表的报告以及提案中提出的初步数据表明，迷走神经事件亚群对CCK、瘦素和FFA敏感。初步结果进一步表明，在单个迷走神经传入神经元的激活中，这些物质之间存在重要的相互作用。在这个项目中，实验将主要集中在从成年大鼠结节神经节分离的迷走神经传入神经元上。该制剂中的神经元保留了体内完整迷走神经传入纤维的大部分（如果不是全部）特性。使用这种制剂可以设计出比在体内更精细和严格控制的神经反应的电、化学和药理学研究。该制剂将用于解决三个特定目的：1)单细胞Ca2+成像，逆行标记和免疫组织化学的组合，将用于建立离散迷走神经传入群体对CCK，瘦素和FFA敏感的神经目标。2)将利用药理学工具结合膜片钳电生理学和Ca2+成像来确定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上相互作用的细胞机制。3)瘦素、CCK和FFA除了急性激活和相互作用外，对这些神经元的反应性也有慢性和持久的影响，这一假设将被验证。单个迷走神经传入神经元对来自这些不同过程的信号作出反应和整合的程度在很大程度上仍未得到重视。在这一系列调查中发展起来的见解将使我们能够更好地重建这些重要调控信号的系统作用和相互作用。更好地了解这一重要的信号通路将有助于理解能量稳态和胃肠道功能，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.