Cross-talk between the colon epithelium, colon afferents and sympathetic neurons regulate pain in the normal and inflamed colon

总结 内脏疼痛是出了名的难以治疗，通常在诱发性损伤/疾病消失后持续很长时间。 更长的明显。在这个应用程序中，我们将探索一种新的，多细胞的外围电路，我们假设 解释了慢性内脏疼痛的许多棘手特征。 我们现在知道上皮-神经元通讯是广泛存在的，有许多上皮细胞类型 释放神经活性物质（例如，ATP、ACh、5 HT、谷氨酸）。这在结肠中尤为明显 其中我们发现，通道视紫红质（ChR 2）诱导的结肠上皮细胞活化产生高水平的 结肠外源性初级传入神经元（ExPAN）的频率爆发，模拟生理刺激和 诱导强烈的行为反应（内脏反应（VMR），一种经验证的超敏反应测定）。 在这些发现的基础上，新的令人惊讶的数据表明，结肠上皮也接受来自 交感神经元;激活交感神经投射到结肠诱导大，相位锁定钙 在上皮细胞中的信号。闭合回路，我们发现通过结直肠扩张（CRD）激活ExPAN 在投射到结肠的节后交感神经元中诱导钙信号，并且ChR 2- ExPAN's的诱导激活诱导这些相同神经元中的cFos表达。这个多细胞回路 在内脏疼痛中起作用的初步数据进一步支持了这一点，这些数据表明，炎症（急性和/或 慢性）与该回路所有部分中的信号传导增加相关。 因此，所提出的实验的目标是检验持续内脏 超敏反应至少部分是由于上皮-ExPAN-交感神经回路中扩增， 通过破坏前馈回路的任何分支来治疗疼痛是可能的（图1）。这一假设 目的1：确定在IBD模型（DSS）中是否诱导持续性超敏反应 （葡聚糖硫酸钠））是由于增加的上皮信号传导和/或ExPAN兴奋性，目的2：确定是否 DSS诱导的炎症增加了ExpPANs激活椎前交感神经元的能力 交感神经节（PrSG）直接（通过PrSG中的突触）或间接（通过脊髓回路），目的3 确定交感神经元驱动未处理小鼠和DSS中上皮细胞活动的能力 IBD模型

SUMMARY Visceral pain is notoriously difficult to treat, often persisting long after the precipitating injury/disease is no longer evident. In this application we will explore a novel, multicellular peripheral circuit that we hypothesize explains many of the intractable features of chronic, visceral pain. We now know that epithelial-neuronal communication is widespread, with numerous epithelial cell types releasing neuroactive substances (e.g., ATP, ACh, 5HT, glutamate). This is particularly apparent in the colon where we have found that channelrhodopsin (ChR2) -induced activation of colon epithelial cells produces high frequency bursting of colon extrinsic primary afferent neurons (ExPAN’s), phenocopying physiologic stimuli and inducing robust behavioral responses (visceromotor responses (VMR), a validated assay of hypersensitivity). Building on these findings, new surprising data indicate colon epithelium also receives functional input from sympathetic neurons; activation of sympathetic projections to the colon induces large, phase-locked calcium signals in the epithelium. Closing the loop, we found that activation of ExPAN’s via colorectal distension (CRD) induces calcium signals in the post-ganglionic sympathetic neurons projecting to the colon, and that ChR2- induced activation of ExPAN’s induces cFos expression in these same neurons. That this multicellular circuit plays a role in visceral pain is supported further by preliminary data that shows that inflammation (acute and/or chronic) is correlated with increased signaling in all portions of this circuit. Thus, the goal of the proposed experiments is to test the hypothesis that persistent visceral hypersensitivity is due, at least in part, to amplification in an epithelial-ExPAN-sympathetic circuit such that it is possible to treat pain by breaking any limb of this feed-forward circuit (Fig.1). This hypothesis will be tested in 3 aims: Aim 1: Determine if persistent hypersensitivity induced in a model of IBD (DSS (dextran sulfate sodium)) is due to increased epithelial signaling and/or ExPAN excitability, Aim 2: Determine if DSS-induced inflammation increases the ability of ExPANs to activate sympathetic neurons in prevertebral sympathetic ganglion (PrSG) directly (via synapses in PrSG) or indirectly (via a spinal cord circuit) and, Aim 3 Determine the ability of sympathetic neurons to drive activity in epithelial cells in naïve mice and in the DSS model of IBD.