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

摘要 内脏疼痛是出了名的难以治疗，通常在急慢性损伤/疾病消失后仍持续很长时间。 再明显不过了。在这个应用中，我们将探索一种新的、我们假设的多细胞外围电路 解释了慢性内脏疼痛的许多棘手的特征。 我们现在知道，上皮-神经元的通讯是广泛的，有许多种上皮细胞类型。 释放神经活性物质(如三磷酸腺苷、乙酰胆碱、5-羟色胺、谷氨酸)。这在结肠中尤为明显。 我们已经发现，通道视紫红质(ChR2)诱导的结肠上皮细胞激活产生高 结肠外源性初级传入神经元(EXPAN‘s)的频率爆发、表现性生理刺激和 诱导强健的行为反应(内脏运动反应(VMR)，一种经过验证的过敏性测试)。 基于这些发现，新的令人惊讶的数据表明，结肠上皮细胞也从 交感神经细胞；向结肠的交感神经投射的激活诱导大的、相位锁定的钙离子 上皮细胞中的信号。关闭环路，我们发现Expan的激活通过结直肠扩张(CRD) 在投射到结肠的节后交感神经元中诱导钙信号，而ChR2- Expan的诱导激活可诱导这些神经元中CFos的表达。这个多细胞电路 在内脏疼痛中起作用的初步数据进一步支持了炎症(急性和/或 慢性)与此回路所有部分中增加的信号相关。 因此，拟议的实验的目标是测试持续内脏的假设 超敏反应至少部分归因于上皮-扩张-交感神经回路的放大。 通过打断这个前馈回路的任何一条肢体来治疗疼痛是可能的(图1)。这一假设 将在3个目标中进行测试：目标1：确定在IBD(DSS)模型中是否诱导了持续性超敏反应 (葡聚糖硫酸钠)是由于上皮细胞信号和/或Expan兴奋性增加，目标2：确定 DSS诱导的炎症增加伸展神经激活椎前交感神经元的能力 交感神经节(PrSG)直接(通过PrSG内的突触)或间接(通过脊髓回路)，目标3 测定交感神经元驱动幼稚小鼠上皮细胞和DSS中的活动的能力 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.