Identifying Defects in the ENS Connectome of Hirschsprung Disease Models

识别先天性巨结肠疾病模型的 ENS 连接组缺陷

• 批准号: 9755698
• 负责人: Kristen Michelle Smith-Edwards
• 金额: $ 6.12万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-03-01 至 2022-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9755698
• 关键词: Accounting; Action Potentials; Adult; Affect; Age; Anatomy; Behavior; Brain; Calcium; Cells; Child; Colon; Congenital Abnormality; Congenital Megacolon; Data; Defect; Development; Devices; Diagnosis; Disease model; Distal; Endothelin Receptor; Enteral; Enteric Nervous System; Equilibrium; Excision; Exhibits; Functional disorder; Gene Mutation; Growth Factor; Image; Immunohistochemistry; Impairment; Interstitial Cell of Cajal; Intestines; Ion Channel; Light; Live Birth; Measures; Mediating; Methods; Modeling; Monitor; Mus; Mutation; Nerve; Nervous System Physiology; Neurons; Operative Surgical Procedures; Pacemakers; Patients; Pattern; Pelvis; Phenocopy; Piebaldism; Population; Preparation; Proxy; Quality of life; Research; Signal Transduction; Smooth Muscle; Spinal Cord; Submucosa; Synapses; Techniques; Testing; Tissues; Visit; base; calcium indicator; cell motility; clinically relevant; connectome; experimental study; ganglion cell; improved; innovation; motility disorder; mouse model; movie; nerve supply; neurochemistry; neuroregulation; neurotransmission; new technology; novel; optogenetics; response; spatiotemporal; tool

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.

项目总结 先天性巨结肠症(HSCR)是一种出生缺陷，由许多基因突变引起，导致远端结肠 缺乏肠道神经系统(ENS)。患有HSCR的儿童通常通过手术切除 ENS缺失的远端肠道(称为无神经节细胞肠管)，希望剩余的ENS- 神经支配的结肠将显示正常的功能。然而，手术后高达50%的HSCR儿童患有 持续的问题，被认为是由于ENS神经支配的结肠的缺陷所致。许多研究都有 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HSCR患者和HSCR小鼠模型的结肠组织，增加了失衡的可能性 抑制性/兴奋性神经传递可能是术后持续肠运动障碍的潜在原因 做手术。为了改善HSCR的结肠动力障碍，我们首先需要更好地了解这些贡献 肌间神经元和ICC的特定亚型对结肠运动模式的影响，然后确定哪些ENS/ICC 电路在HSCR中被更改。我们的实验室使用了新开发的光遗传工具与 技术创新的体外制剂，保持内源性和外源性ENS电路的完整性，我们有 生成的初步数据描述了这些回路以及它们在 成年小鼠结肠。对于这项提议，我将在两个成熟的HSCR小鼠模型中使用这些技术 (花椒致死和Ret+/-)，以检验近端抑制和兴奋回路的中断 结肠导致异常的结肠运动行为。在目标1中，我将确定突触连接的变化 HSCR相关突变导致的固有ENS/ICC电路揭示了肌间神经元的哪些亚型 最受影响的是，这些变化与收缩模式的改变直接相关。目标2将决定 外源性副交感神经传入ENS/ICC回路及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，我将光遗传刺激(使用光激活离子通道， 通道视紫红质，ChR2)兴奋性和抑制性肠神经细胞确定手法的效果 兴奋/抑制音对HSCR小鼠模型运动模式的影响。总体而言，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 无论是使用外部电刺激或光遗传刺激对ENS神经支配的结肠进行神经调节， 能够纠正电路缺陷以恢复正常的排便功能。