Analysis of the vagal afferent innervation of the mouse colon

关于结肠状态的信息通过两个传入通路传递到中枢神经系统：脊髓传入通路和迷走传入通路。来自结肠的脊髓传入通路已经被深入研究，因为它们参与了对导致疼痛的伤害性刺激的感知。内脏器官的迷走神经传入神经支配是神经系统的一个进化上古老的分支，其功能在大多数器官中已被很好地理解。然而，结肠的迷走神经传入神经支配却很少受到重视。最近发表的研究结果表明，结肠的迷走传入神经支配是肠道微生物群和大脑之间相互作用的重要渠道，并激活胆碱能抗炎途径。然而，目前缺乏支持这些作用的直接证据，结肠迷走神经传入神经支配结构的信息也是如此。我们的初步数据表明，结肠的迷走神经支配比以前怀疑的更广泛，它可以检测微生物群组成和免疫系统激活的变化。因此，我们提出了两个目标，以阐明结肠迷走神经传入神经支配的解剖和生理。目标1. 描述结肠迷走神经传入支配的神经解剖学特征。使用迷走神经传入轴突的逆行标记，我们将量化有多少细胞体的神经元在结状神经节（NG）支配近端和远端结肠，并确定是否有许多NG神经元支配这两个网站。我们将利用顺行追踪来确定迷走神经传入终末在结肠粘膜和平滑肌层中的定位。定位这些传入终端将提供洞察哪些生理参数是最有可能被监测的迷走神经传入神经支配的结肠。 目标2. 确定迷走神经传入神经元在检测结肠稳态破坏中的作用。我们将研究肠道微生物生态失调和结肠炎症是否影响迷走神经传入神经元的活动，使用电生理学和神经元激活的免疫组织化学标记物，c-fos免疫反应。如果我们观察到结肠炎或微生物生态失调导致NG神经元和下游神经元的激活，我们将使用膜片钳确定涉及的离子机制，并切断迷走神经以确定迷走传入神经元的外周末梢是否对这些反应至关重要。我们还将尝试确定所涉及的微生物或宿主衍生的介质。 拟议的研究将是第一次全面分析迷走神经传入神经支配的结肠，并将促进肠脑轴的作用，结肠稳态和介导微生物对大脑的影响的机制研究。

Information about the state of the colon is conveyed to the central nervous system by two afferent pathways: the spinal afferent pathway and the vagal afferent pathway. Spinal afferent pathways from the colon have been intensely studied due to their involvement in the perception of noxious stimuli that contribute to pain. The vagal afferent innervation of visceral organs is an evolutionarily ancient branch of the nervous system whose functions are well understood in most organs. However, much less attention has been paid to the vagal afferent innervation of the colon. Recently published findings have suggested the vagal afferent innervation of the colon is a vital conduit of interactions between the gut microbiota and the brain, and in activating the cholinergic anti-inflammatory pathway. However, direct evidence in support of these roles is currently lacking, as is information on the structure of the vagal afferent innervation of the colon. Our preliminary data suggest that the vagal innervation of the colon is more extensive than previously suspected and that it detects changes in microbiota composition and immune system activation. We therefore propose two aims to characterise the anatomy and physiology of the vagal afferent innervation of the colon. Aim 1. Characterise the neuroanatomy of the vagal afferent innervation of the colon. Using retrograde labelling of vagal afferent axons, we will quantify how many cell bodies of neurons in the nodose ganglia (NG) innervate the proximal and distal colon and determine whether many NG neurons innervate both sites. We will utilize anterograde tracing to determine the localization of vagal afferent terminals in the colonic mucosa and smooth muscle layers. The localisation of these afferent terminals will provide insight into what physiological parameters are most likely to be monitored by the vagal afferent innervation of the colon. Aim 2. Determine the role of vagal afferent neurons in detecting disruptions of colonic homeostasis. We will examine whether gut microbial dysbiosis and colonic inflammation affect the activity of vagal afferent neurons using electrophysiology and an immunohistochemical marker of neuronal activation, c-fos immunoreactivity. If we observe that colitis or microbial dysbiosis leads to activation of NG neurons and downstream neurons, we will identify the ionic mechanisms involved using patch clamp, and will sever the vagus nerve to determine whether the peripheral terminals of vagal afferent neurons are essential for these responses. We will also attempt to identify the microbial or host-derived mediators involved. The proposed studies will be the first comprehensive analysis of the vagal afferent innervation of the colon and will facilitate mechanistic studies of the role of the gut-brain axis in colonic homeostasis and in mediating microbial influences on the brain.