Vagal Neurons & Gastric Function by Hypocretin

描述(申请人提供)：迷走神经背侧核(DMV)向胃肠道(GI)提供副交感神经运动输出，与孤束核(NTS)一起在胃运动和顺应性的反射控制中起着不可或缺的作用。此外，这些核团中的每一个都接受下丘脑外侧区含下丘脑(HCRT)神经元和室旁核(PVN)含催产素(OT)神经元的下行投射，两者共同提供了GI活动的中枢控制手段和协调消化过程与摄食行为的重要途径。这项研究的一个主要目的是确定HCRT肽和OT在DMV的细胞和突触作用，并确定它们如何与这些肽能输入对胃相关功能产生更广泛的影响有关。为了实现这一点，我们将进行活体实验，并从具有明确GI投射的逆行标记的DMV神经元中获得脑片和细胞培养的膜片钳记录。在目标1中，我们将在麻醉大鼠身上进行微量注射实验，并结合免疫组织化学研究，确定HCRT在DMV中作用的部位和细胞底物，以刺激胃运动功能。还将检查胃对HCRT反应的药理学敏感性，以确定所涉及的迷走神经传出通路的性质。在目标2中，脑片上的斑片记录将与NTS的电刺激相结合，以确定沐浴应用HCRT或OT对已识别的GI投射的DMV神经元的膜特性和突触反应的影响。然后，将使用单细胞逆转录聚合酶链式反应来确定每个采样神经元的神经化学。单个细胞对HCRT或OT的反应将与其特定的GI靶点和化学表型相关，以检验以下假设：这些肽能信号通过调节节前迷走神经运动神经元功能亚群的兴奋性而对胃运动和张力产生相反的影响。在目标3中，我们将获得培养中已鉴定的胃肠道投射的DMV神经元的全细胞记录，以表征这些细胞中的电压依赖性钙(Ca2+)电导，并确定HCRT是否通过调节特定通道类型的钙内流来调节神经化学上不同的节前神经元的兴奋性。这项拟议的研究将增加我们对控制胃相关功能的下丘脑机制的理解，这样做可能会导致设计新的干预措施，用于有效地控制体重和治疗与胃动力和胃排空改变相关的消化障碍，如胃停滞或非溃疡性消化不良。

DESCRIPTION (provided by applicant): The dorsal motor nucleus of the vagus (DMV) provides the parasympathetic motor output to the gastrointestinal (GI) tract and plays an integral role, along with the nucleus of the tractus solitarius (NTS), in the reflex control of gastric motility and compliance. In addition, each of these nuclei receive descending projections from hypocretin (HCRT)- containing neurons in lateral hypothalamus and oxytocin (OT)-containing neurons of the paraventricular nucleus (PVN) that together provide both the means for central control of GI activity and an important route for coordinating digestive processes with feeding behavior. A major objective of the proposed research is to define the cellular and synaptic actions of HCRT peptides and OT in the DMV and ascertain how they relate to the more global influence that these peptidergic inputs have on gastric-related functions. To accomplish this, we will conduct in vivo experiments and obtain patch clamp recordings in brain slices and cell culture from retrogradely labeled DMV neurons with defined GI projections. In Aim 1, we will conduct microinjection experiments in anesthethized rats together with immunohistochemical studies to determine the sites and cellular substrates where HCRTs act in the DMV to stimulate gastric motor function. The pharmacological sensitivity of gastric responses to HCRT will also be examined to determine the nature of the vagal efferent pathways involved. In Aim 2, patch recordings in slices will be used in conjunction with electrical stimulation in NTS to determine the effects of bath application of HCRTs or OT on the membrane properties and synaptic responses of identified GI-projecting DMV neurons. The neurochemistry of each sampled neuron will then be defined using single cell reverse transcription -polymerase chain reaction. Responses of individual cells to HCRT or OT application will be correlated with their specific GI target and chemical phenotype to test the hypothesis that these peptidergic signals exert opposing effects on gastric motility and tone by modulating the excitability of functionally distinct subpopulations of preganglionic vagal motor neurons. In Aim 3, we will obtain whole-cell recordings from identified GI-projecting DMV neurons in culture to characterize the voltage dependent calcium (Ca 2+) conductances in these cells and determine whether HCRTs regulate excitability of neurochemically distinct groups of preganglionic neurons by modulating Ca 2+ influx through specific channels types. The proposed research will increase our understanding of hypothalamic mechanisms that control gastric-related functions, and in so doing may lead to the design of new interventions for effective weight management and treatment of digestive disorders such as gastric stasis or non-ulcer dyspepsia associated with alterations in gastric motility and stomach emptying.