Brainstem circuits controlling gastrointestinal function

描述（由申请人提供）：来自胃肠道（GI）的感觉信息在传递到迷走神经背侧运动核（DMV）的运动神经元之前，由孤束核（NTS）的神经元感知和调节；这些核对迷走神经反射的协调至关重要。虽然肠道的迷走反射控制在基本的机制水平上被理解，但有许多因素（例如生物体在环境中的位置，一天中的时间，食物的味道，压力，疼痛，疾病中的细胞因子产生，激素背景等）可以从根本上改变摄食行为或胃肠道功能。消化功能与胃肠道状态的整合依赖于迷走神经携带的内脏传入数据。虽然已知几种神经递质通过NTS或DMV影响GI功能，但很少有研究调查这些核之间的突触接触。从我们实验室收集的数据表明，神经肽和激素可以通过NTS中神经元转导机制的差异门控极大地改变迷走神经传入输入的处理。我们的电生理学初步数据表明，迷走-迷走回路的阈下操作揭示了其他沉默的抑制性突触的反应。我们的免疫组织化学数据支持这种短期回路可塑性的概念，因为类似的操作允许检测其他隐藏的膜受体。我们的体内数据支持这种短期可塑性的概念，表明这种类型的反应强调了在需要时激活突触传递的一种普遍方式。本建议的总体目标是阐明这种类型的短期突触可塑性的基本机制与迷走神经传入事件的激活状态。基于我们的初步数据，我们提出了一个创新的假设，即迷走背复合体（DVC，即DMV和NTS）内抑制性神经传递的调节取决于NTS中间神经元的激活状态。我们将利用体外膜片钳技术结合荧光示踪方法对脑干DMV的gi投射神经元进行选择性记录；ii)在DMV体内微注射假定的神经递质以唤起和监测GI运动；iii)迷走脑干GI神经支配的组织学、免疫组织化学和形态学分析。我们提出的研究是基于两个具体目标。特异性目的1:DVC中的抑制性神经传递需要“启动”才能对神经调节剂做出反应。特别是，我们提出cAMP-Protein Kinase （PK）通路的激活是激活其他沉默突触中受体的必要先决条件。我们将通过以下假设来验证这一特定目标：(1):cAMP-PK通路的激活允许阿片样物质和/或胰腺多肽（PPs）调节其他无反应的抑制电流。(2): cAMP-PK通路的激活揭示了在DVC的gaba能末端无法检测到的肽受体。特异性目的2：内源性神经递质调节DVC抑制电流的能力取决于正在进行的迷走神经活动的水平。特别是，我们提出阿片类药物和PPs的作用取决于迷走神经是否受到刺激或未受到刺激，以及制剂是否来自喂食或禁食的动物。我们将通过以下假设来验证这一特定目标：(3)：体外DVC对阿片类药物或PPs的反应取决于喂养状态。(4)：迷走神经反射的激活或大鼠摄食状态的改变揭示了gaba能神经元和DVC末端的肽受体。(5)：体内DVC对外源性阿片类药物或PPs的反应取决于正在进行的迷走神经活动水平或大鼠的摄食状态。这项研究将提供一种创新的方式来观察迷走神经反射的调节。从这些研究中获得的数据将导致对神经调节剂对中枢迷走神经回路的短期调节的更好理解；对这些门控机制的理解可能使我们能够解释并最终治疗它们的病理生理后果。

DESCRIPTION (provided by applicant): Sensory information from the gastrointestinal (GI) tract is perceived and modulated by neurons in the nucleus of the tractus solitarius (NTS) before being transmitted to motoneurones of the dorsal motor nucleus of the vagus (DMV); these nuclei are essential for the coordination of vago-vagal reflexes. Although vago-vagal reflex control of the gut is understood at a basic mechanistic level, there are many factors (e.g. the organisms' place in the environment, time of day, taste of food, stress, pain, cytokine production in disease, hormonal background, etc.) that can radically alter feeding behavior or GI function. The integration of digestive function in relation to the state of the GI tract is dependent on visceral afferent data carried by the vagus nerve. Although several neurotransmitters are known to influence GI functions via either the NTS or the DMV, very few studies have investigated the synaptic contacts between these nuclei. Data collected from our laboratories suggest that neuropeptides and hormones can drastically alter the processing of vagal afferent input through differential gating of neuronal transduction mechanisms in the NTS. Our electrophysiological preliminary data suggest that subthreshold manipulations of vago-vagal circuits unmask responses in otherwise silent inhibitory synapses. Our immunohistochemical data support this concept of short-term circuit plasticity since similar manipulations allow the detection of otherwise concealed membrane receptors. Our in vivo data support this concept of short-term plasticity suggesting that this type of response highlights a generalized way of activating synaptic transmission as the need arises. The overall objective of this proposal is to elucidate the basic mechanisms of this type of short-term synaptic plasticity in relation to the activation state of the vagal afferents. Based on our preliminary data, we formulate the innovative hypothesis that the modulation of inhibitory neurotransmission within the dorsal vagal complex (DVC; i.e. DMV and NTS) depends on the state of activation of NTS interneurones. We will investigate the central hypothesis using i) in vitro patch-clamp techniques combined with fluorescent tracing methods to record selectively from GI-projecting neurons of the DMV in brainstem slices; ii) in vivo microinjections of putative neurotransmitters in the DMV to evoke and monitor GI motility; and iii) histological, immunohistochemical and morphological analyses of vagal brainstem GI innervation. The studies that we propose are based on two specific aims. Specific Aim 1: inhibitory neurotransmission in the DVC needs to be "primed" to respond to neuromodulators. In particular, we propose that the activation of the cAMP-Protein Kinase (PK) pathways is a necessary prerequisite for the activation of receptors in otherwise silent synapses. We will test this Specific Aim with the following hypotheses: (1): activation of the cAMP-PK pathway allows the modulation of otherwise unresponsive inhibitory currents by opioids and/or pancreatic polypeptides (PPs). (2): activation of the cAMP-PK pathway reveals otherwise undetectable peptide receptors in GABAergic terminals of the DVC. Specific Aim 2: the capacity of endogenous neurotransmitters to modulate inhibitory currents in the DVC is dependent upon the level of ongoing vagal activity. In particular, we propose that the effects of opioids and PPs depend upon whether the vagus nerve is stimulated or unstimulated or whether the preparation is from an animal that is fed or fasted. We will test this Specific Aim with the following hypotheses: (3): the in vitro DVC response to opioids or PPs depends upon the feeding status. (4): activation of vago-vagal reflexes or changes in the feeding status of the rat unmasks peptide receptors in GABAergic neurons and terminals in the DVC. (5): the in vivo DVC response to exogenously applied opioids or PPs depends upon the level of ongoing vagal activity or the feeding status of the rat. This research will provide an innovative way of viewing the modulation of vago-vagal reflexes. Data obtained from the pursuit of these studies will lead to an improved understanding of the short-term modulation of central vagal circuits by neuromodulators; the understanding of these gating mechanisms may allow us to explain, and eventually treat, their pathophysiological consequences.