Glucocorticoids and endocannabinoids in vagal complex

描述（由申请人提供）：本研究拨款提案中的实验将确定内源性大麻素（eCB）配体和糖皮质激素诱导的迷走神经背运动核（DMV）中eCBs释放的细胞和突触效应。DMV中的神经元调节副交感神经对大部分膈下脏器的输出，因此对摄食、消化、葡萄糖和胰岛素分泌以及其他代谢功能具有关键的控制作用。它们的活动在很大程度上是由突触对DMV的输入控制的，而DMV是由局部释放的化学物质和循环激素调节的。大麻素、香草素和糖皮质激素对DMV神经元的调节已被提出；当集中使用这些化合物时，会深刻地改变副交感神经功能。几种eCB配体，被认为以逆行方式从细胞膜释放，激活大麻素1型受体（CB1R）和瞬时受体电位香草素1型（TRPV1）。在DMV中，TRPV1的激活增强了神经递质释放，而CB1R倾向于抑制突触。这两种效应都是通过突触前末端受体的激活而发生的。初步证据表明eCB配体从DMV神经元释放，糖皮质激素或去极化可诱导这种释放。因此，欧洲央行在DMV中的活动可能会调节TRPV1和CB1R的活动。释放的eCB配体的类型，大多数eCB配体对突触活性的影响，以及eCB在DMV中释放的触发或机制都不知道。我们将使用脑干切片中DMV神经元的全细胞膜片钳记录来确定eCB配体对DMV细胞活性的影响，并将使用药理学和生化方法确定该区域细胞释放的化合物。实验将以三个具体目标为指导：1)区分eCB配体对DMV中CB1R和TRPV1的影响；2)确定eCB参与介导糖皮质激素对DMV局部电路的快速影响；3)明确糖皮质激素作用的细胞通路。我们将验证以下假设：eCBs以特定和可预测的空间、时间、功能相关和活性依赖模式改变DMV中的TRPV1和CBR1活性；糖皮质激素通过作用于DMV神经元上的膜结合G蛋白偶联受体，诱导eCB从DMV细胞释放。基于eCB系统的药物正在被研究用于治疗各种神经系统疾病，包括与进食、消化和肥胖相关的疾病。糖皮质激素在某些系统中释放出ecb，被广泛使用，压力刺激也会释放糖皮质激素。这些研究结果对于预测和理解这些化合物如何相互作用并影响副交感神经功能至关重要。由于糖皮质激素升高患者迷走神经系统中控制eCB水平的益处，可能存在转化益处。内源性大麻素化合物在脑干最负责维持器官系统功能的区域具有特别复杂的作用，许多基于改变大脑中这些化合物的药物疗法正在开发中，但不知道它们在这一关键大脑区域的作用。这项研究拨款提案中的实验将确定这些药物的特性，它们是如何被糖皮质激素释放的，以及它们对神经元有什么作用。这些研究的结果对于预测和理解这些化合物如何相互作用影响消化功能至关重要，它们也将适用于多种其他神经系统。

DESCRIPTION (provided by applicant): The experiments in this research grant proposal will identify the cellular and synaptic effects of endogenous cannabinoid (eCB) ligands and of glucocorticoid-induced release of eCBs in the dorsal motor nucleus of the vagus (DMV). Neurons in the DMV regulate parasympathetic output to most of the subdiaphragmatic viscera and therefore critically control feeding, digestion, glucose and insulin secretion, and other metabolic functions. Their activity is largely controlled by synaptic input to the DMV, which is modulated by locally released chemicals and circulating hormones. Regulation of DMV neurons by cannabinoids, vanilloids, and glucocorticoids has been suggested; when applied centrally these compounds profoundly alter parasympathetic function. Several eCB ligands, which are thought to be released from cell membranes in a retrograde fashion, activate both cannabinoid type 1 receptors (CB1R) and transient receptor potential vanilloid type 1 (TRPV1). In the DMV, activation of TRPV1 enhances neurotransmitter release, whereas CB1R tends to inhibit synapses. Both effects occur by activation of receptors on presynaptic terminals. Preliminary evidence suggests that eCB ligands are released from DMV neurons, and that glucocorticoids or depolarization can induce this release. Thus, eCB activity in the DMV may modulate both TRPV1 and CB1R activity. Neither the type(s) of eCB ligands released, the effects of most eCB ligands on synaptic activity, nor the trigger or mechanism of eCB release in the DMV are known. We will use whole-cell patch-clamp recordings from DMV neurons in brainstem slices to identify effects of eCB ligands on cellular activity in the DMV, and will also identify the compounds released by cells in the area using pharmacological and biochemical methods. The experiments will be guided by three specific aims: 1) Differentiate effects of eCB ligands on CB1R and TRPV1 in the DMV; 2) Determine the eCB involvement in mediating rapid effects of glucocorticoids on local circuitry in the DMV; and 3) Identify the cellular pathway of the glucocorticoid effect. We will test the hypotheses that eCBs alter TRPV1 and CBR1 activity in the DMV in specific and predictable spatial, temporal, functionally relevant, and activity-dependent patterns, and that glucocorticoids induce eCB release from DMV cells by acting at membrane-bound G protein-coupled receptors on DMV neurons. Drugs based on the eCB system are being investigated for therapeutic use in a variety of nervous system pathologies, including disorders related to feeding, digestion, and obesity. Glucocorticoids, which release eCBs in some systems, are widely prescribed, and are also released by stressful stimuli. Results of these studies will be critical to predicting and understanding how these compounds interact with each other and affect parasympathetic function. Possible translational benefits also exist because of the benefit in controlling eCB levels in the vagal system of patients with elevated glucocorticoids. PUBLIC HEALTH RELEVANCE Endogenous cannabinoid compounds have particularly complex actions in the area of the brainstem most responsible for maintaining functions of organ systems, and a number of drugs therapies based on altering these compounds in the brain are being developed without knowledge about their effects in this critical brain area. The experiments in this research grant proposal will identify the characteristics of these drugs, how they are released by glucocorticoid hormones, and what actions they have on the neurons. Results of these studies will be critical for predicting and understanding how these compounds interact to affect digestive functions, and they will also be applicable to multiple other neuronal systems.