Identifying Defects in the ENS Connectome of Hirschsprung Disease Models

项目摘要 先天性巨结肠症（Hirschsprung's disease，HSCR）是一种由多个基因突变引起的先天性缺陷， 缺乏肠神经系统（ENS）。患有HSCR的儿童通常通过手术治疗， ENS缺失的远端肠（称为“无神经节肠”），希望剩余的ENS- 受神经支配的结肠将表现出正常的功能。然而，在手术后，高达50%的HSCR儿童 持续的问题，被认为是由于在ENS神经支配的结肠缺陷。大量研究 发现近端神经节细胞中抑制性肠神经元的数量不成比例地增加， 含有来自HSCR患者和HSCR小鼠模型的结肠组织，提高了不平衡的可能性。 在抑制性/兴奋性神经传递可能是持续性肠动力障碍的根本原因后， 手术为了改善HSCR中结肠动力障碍，我们首先需要更好地了解 的特定亚型的肌间神经元和ICC结肠动力模式，然后确定哪些ENS/ICC HSCR中的电路被改变。我们的实验室采用了新开发的光遗传学工具， 技术创新的离体制备，保持内在和外在ENS电路完整，我们有 产生的初步数据描述了这些电路和收缩的独特模式，他们产生的， 成年小鼠结肠。对于这个建议，我将使用这些技术在两个良好建立的小鼠模型的HSCR （花斑致死和Ret+/-），以检验近端抑制和兴奋回路中断的假设。 结肠导致结肠运动行为异常。在目标1中，我将识别突触连接的改变， 由于HSCR相关突变的内在ENS/ICC电路，揭示了肌间神经元的亚型， 受影响最大，并直接将这些变化与收缩力的改变模式相关联。目标2将决定 ENS/ICC回路的外源性副交感神经输入和由此产生的收缩反应是否异常， HSCR小鼠模型。最后，在目标3中，我将光遗传学刺激（使用光激活离子通道， channelrhodopsin，ChR 2）兴奋性和抑制性肠神经元，以确定操纵 在HSCR小鼠模型中，兴奋性/抑制性音调对运动模式的影响。总的来说，拟议的实验将 确定在HSCR小鼠模型中产生运动障碍的特定ENS/ICC电路缺陷，并确定 使用外部电刺激或光遗传学刺激对ENS神经支配的结肠进行神经调节， 能够纠正回路缺陷以使肠功能正常化。

PROJECT SUMMARY Hirschsprung’s disease (HSCR) is a birth defect caused by a number of gene mutations, resulting in distal colon that lacks an enteric nervous system (ENS). Children with HSCR are often treated by surgery to remove the distal bowel where the ENS is missing (called ‘aganglionic bowel’), with the hope that the remaining ENS- innervated colon will exhibit normal function. However, after surgery up to 50% of children with HSCR have ongoing problems that are thought to be due to defects in the ENS-innervated colon. Numerous studies have identified disproportionate increases in the number of inhibitory enteric neurons in proximal, ganglion cell- containing colon tissue from HSCR patients and HSCR mouse models, raising the possibility that an imbalance in inhibitory/excitatory neurotransmission may be the underlying cause of continued bowel dysmotility after surgery. In order to improve colon dysmotility in HSCR, we first need a better understanding of the contributions of specific subtypes of myenteric neurons and ICC to colon motility patterns, and then identify which ENS/ICC circuits are altered in HSCR. Our lab has employed newly developed optogenetic tools combined with a technically innovative ex vivo preparation that keeps intrinsic and extrinsic ENS circuits intact, and we have generated preliminary data describing these circuits and the distinct patterns of contractility they produce in the adult mouse colon. For this proposal, I will use these techniques in two well-established mouse models of HSCR (piebald lethal and Ret+/-) to test the hypothesis that disruption in inhibitory and excitatory circuits in the proximal colon leads to abnormal colon motility behavior. In Aim 1, I will identify alterations in synaptic connectivity in intrinsic ENS/ICC circuits due to HSCR-associated mutations, reveal which subtypes of myenteric neurons are most affected, and directly correlate these changes to altered patterns of contractility. Aim 2 will determine whether extrinsic parasympathetic input to ENS/ICC circuits and resulting contractile responses are abnormal in HSCR mouse models. Finally, in Aim 3, I will optogenetically stimulate (using the light-activated ion channel, channelrhodopsin, ChR2) excitatory and inhibitory enteric neurons to determine the effects of manipulating excitatory/inhibitory tone on motility patterns in HSCR mouse models. Overall, the proposed experiments will identify specific ENS/ICC circuit defects that produce dysmotility in mouse models of HSCR and determine whether neuromodulation of the ENS-innervated colon, using external electrical or optogenetic stimulation, is capable of correcting circuit defects to normalize bowel function.