Analysis of the vagal afferent innervation of the mouse colon

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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.