Molecular and spatial mapping of bladder nociceptors during development and matur

描述(由申请人提供)：骨盆疼痛状况使人虚弱，治疗选择有限。造成这一问题的一个主要问题是相对缺乏对持续性内脏疼痛条件背后的因素的了解--在躯体疼痛(例如，皮肤、骨骼肌、骨源性疼痛)领域存在着更大的研究努力。这个nGUDMAP分子解剖学项目的目标是绘制支配小鼠膀胱的主要伤害性感受器神经元的主要结构和分子特征。该项目与泌尿生殖系统疼痛有关，例如间质性膀胱炎/痛性膀胱综合征。这些实验将集中在在TRPV1报告小鼠中发现的伤害性神经元，以加深对它们的结构、与膀胱的联系以及它们的“分子指纹”(RNA图谱)的理解。这项研究将在发育中和成熟的小鼠身上进行，以捕捉膀胱控制发生重大变化的时间段。在AIM 1中，将生成胚胎和出生后小鼠膀胱内TRPV1阳性轴突的拓扑图，提供可以叠加到现有GUDMAP数据上的高分辨率位置图。在AIM 2中，将应用深度测序技术来表征这些TRPV1神经元的特定群体的转录组。首先，解剖特定的背根神经节(DRG)，用荧光激活细胞分选技术(FACS)分离TRPV1阳性和阴性神经元。这将允许对它们的总RNA进行测序和表征，构建这些伤害性感受器的发育和成熟的分子图谱。通过应用最新的生物信息学分析，这将(I)识别含有内脏伤害性感受器的DRG的转录本，(Ii)通过组织取样前的显微注射逆行追踪方法，表征投射膀胱的伤害性感受神经元的转录组。这些数据将为未来关于泌尿生殖系统伤害性感受器的发育和成熟的假说驱动的研究提供丰富的知识来源，并可能应用于疾病模型的研究。

DESCRIPTION (provided by applicant): Pelvic pain conditions are debilitating and treatment options limited. A major issue contributing to this problem is the relative lack of understanding o factors underlying persistent visceral pain conditions - a much larger research effort exists in th area of somatic pain (e.g., pain of skin, skeletal muscle, bone origin). The goal of this nGUDMAP molecular anatomy project is to map the primary structural and molecular features of a major population of nociceptor neurons that innervate the urinary bladder of the mouse. This project is relevant to genitourinary pain, such as interstitial cystitis/painful bladder syndrome. The experiments will focus on nociceptive neurons, identified in a Trpv1 reporter mouse, to develop a deeper understanding of their structure and connections with the bladder, and their 'molecular fingerprint' (RNA profile). The study will be performed on developing and mature mice, to capture the periods of time when major changes occur in bladder control. In AIM 1, a topological map of Trpv1-positive axons within the embryonic and postnatal mouse bladder will be generated, providing a high-resolution positional map that can be superimposed onto existing GUDMAP data. In AIM 2, deep sequencing techniques will be applied to characterize the transcriptome of particular populations of these Trpv1 neurons. First, specific dorsal root ganglia (DRG) will be dissected and Trpv1- positive and -negative neurons separated using fluorescence activated cell sorting (FACS). This will allow their total RNA to be sequenced and characterized, building a molecular map of the development and maturation of these nociceptors. By applying the latest bioinformatics analyses this will (i) identify transcripts uniqe to DRG that contain visceral nociceptors, and (ii) by micro-injecting retrograde tracing methods prior to tissue sampling, characterize the transcriptome of bladder-projecting nociceptive neurons. This data will provide a rich source of knowledge to seed future hypothesis-driven studies on development and maturation of urogenital nociceptors, as well as potential application to studies on disease models.