Neurotrophins and Neuropeptides in Colon and Bladder Hypersensitivity

描述（由申请人提供）：炎症性肠病患者经常经历膀胱感觉反应性增加，反映神经源性膀胱过度活动。这种内脏症状重叠涉及到膀胱传入神经元敏感的背根神经节（DRG）的神经元交叉激活。该项目的长期目标是了解结肠和膀胱感觉通路之间复杂的神经元相互作用，并确定调节结肠炎引起的膀胱感觉超敏反应的介质。我们的出版物和初步数据表明，脑源性神经营养因子(BDNF)/TrkB系统在膀胱活动的调节中具有突出作用。在这个更新应用中，我们假设磷脂酶c - γ （PLC?）-钙（Ca2+）通路是膀胱传入神经元内源性BDNF/TrkB增加的独特下游，并在结肠炎期间通过Ca2+依赖的神经活性化合物转录和翻译后调节在膀胱传入激活中发挥不可或缺的作用。为了解决这一假设，提出了三个相互关联的具体目标，以检查PLC的监管机制和目标。-Ca2+途径，包括Ca2+/钙调素依赖性蛋白激酶（CaMK）II， camp反应元件结合蛋白（CREB），降钙素基因相关肽（CGRP）和小脑素1前体（Cbln1）在结肠炎前和期间膀胱传入神经元。在AIM 1中，我们将描述一系列调节Ca2+动员的成分(磷脂酶C？在结肠炎第7天和第21天，膀胱传入神经元中的电压门控Ca2+通道（主要是n型通道Cav2.2）和Ca2+依赖性神经元激活（CaMKII和CREB）。这将在分子（mRNA和蛋白质）和功能（细胞内Ca2+记录和电生理）水平上完成。在第二目标中，我们会探讨PLC？结肠炎期间膀胱传入神经元内源性BDNF激活-Ca2+通路。为此，我们将使用一种新开发的具有良好特征的BDNF+/-大鼠菌株。在AIM 3中，我们将结合分子生物学，药理学，神经化学和行为测试来检查BDNF-Ca2+轴在结肠炎期间膀胱过度活动中的功能作用。我们将描述Ca2+依赖通路如何参与CGRP和Cbln1的表达，以及它们如何调节结肠炎期间膀胱传入神经元的过度活跃和调节膀胱排尿参数。对于上述研究，我们将利用各种体内(转基因动物，鞘内给药，行为研究和离体/体外（DRG外植体，分离的DRG神经元培养和转染）系统。大鼠结肠内灌注三硝基苯磺酸（TNBS）可引起局部结肠炎症。由于一些Ca2+通路的小分子拮抗剂正在进行治疗其他疼痛症状的临床试验，我们预计目前的系统研究将为形成治疗内脏超敏反应的治疗策略提供见解。

DESCRIPTION (provided by applicant): Patients with inflammatory bowel disease often experience increased sensory responsiveness in the urinary bladder reflecting neurogenic bladder overactivity. This visceral symptom overlap involves neuronal cross-activation in the dorsal root ganglia (DRG) where the bladder afferent neurons are sensitized. The long- term goal of this project has been to understand the complex neuronal interaction between colonic and bladder sensory pathways, and to identify mediators that regulate bladder sensory hypersensitivity as a result of colitis. Our publications and preliminary data have shown that the brain-derived neurotrophic factor (BDNF)/TrkB system has a prominent role in the regulation of bladder activity. In this renewal application, we hypothesize that the phospholipase C-gamma (PLC?)-calcium (Ca2+) pathways are unique downstream of the increased endogenous BDNF/TrkB in bladder afferent neurons, and play an integral role in bladder afferent activation by Ca2+-dependent transcriptional and posttranslational regulation of neuroactive compounds during colitis. To address this hypothesis, three interrelated Specific Aims are proposed to examine the regulatory mechanism and the targets of the PLC?-Ca2+ pathways including Ca2+/calmodulin-dependent protein kinase (CaMK)II, cAMP-response element binding protein (CREB), calcitonin gene-related peptide (CGRP), and cerebellin 1 precursor (Cbln1) in bladder afferent neurons before and during colitis. In AIM 1, we will characterize the expression profiles of a series of components regulating Ca2+ mobilization (phospholipase C?, InsP3R-1, voltage-gated Ca2+ channels predominantly the N-type channel Cav2.2) and Ca2+-dependent neuronal activation (CaMKII and CREB) in bladder afferent neurons at 7 days and 21 days of colitis. This will be done at the molecular (mRNA and protein) and functional (intracellular Ca2+ recording and electrophysiology) levels. In AIM 2, we will examine the regulatory mechanism by which the PLC?-Ca2+ pathways are activated by endogenous BDNF in bladder afferent neurons during colitis. For this purpose, we will use a newly developed yet well-characterized BDNF+/- rat strain. In AIM 3, we will combine molecular biological, pharmacological, neurochemical, and behavioral tests to examine the functional role of the BDNF-Ca2+ axis in bladder hyperactivity during colitis. We will characterize how the Ca2+-dependent pathways are involved in CGRP and Cbln1 expression, and how they regulate bladder afferent neuronal hyperactivity and modulate bladder micturition parameters during colitis. For studies proposed above, we will utilize a variety of in vivo (transgenic animals, intrathecal delivery of drugs, behavioral studies and ex vivo/in vitro (DRG explants, isolated DRG neuron culture and transfection) systems. The localized colonic inflammation will be induced by intracolonic instillation of tri-nitrobenzene sulfonic acid (TNBS) in rat. As several small molecule antagonists of the Ca2+ pathways are under clinical trials in treatment of other pain symptoms, we anticipate that the current systematic studies will provide insights into forming therapeutic strategies in the treatment of visceral hypersensitivity.