Neuronal anatomy, connectivity, and phenotypic innervation of the knee joint

项目摘要 识别神经元连接的模式对于理解功能和解剖电路至关重要， 介导疼痛感知。然而，关于关节组织中神经元的类型和分布的知识， 通常局限于传统的二维组织病理学和免疫组织病理学方法， 关于连接性和神经元表型的信息很少甚至没有。新技术 出现了允许跨突触电路分析和神经元放电的精确控制，包括 使用逆行转运的病毒载体（即，假型狂犬病病毒）和异源受体 activation.与此同时，神经元和血管模式的三维可视化已经被广泛应用。 通过组织清除技术结合细胞类型特异性荧光标记物来推进， 将细胞类型特异性Cre重组酶小鼠系与多种条件活化的报告基因杂交。 最后，单细胞RNA测序的出现允许将细胞表型扩展到分子水平。 水平不仅提高了分析分辨率，而且还提高了治疗靶向， 比以前更有可能。高分辨率空间转录组学的发展，人鱼， 允许scRNA-seq数据的关联和验证。在这种情况下，膝关节的骨关节炎是一种 作为丰富的遗传和手术模型，应用这些工具的最佳模型可用于正交 验证调查结果。此外，在临床前背景下，各种治疗方法，包括基因治疗， 治疗已被证明会影响疼痛测量，因此，它们构成了重要的干预措施。 验证在疾病状态下神经元中鉴定的分子变化。事实上，其中一些 目前正在进行临床试验的疗法增加了拟议的临床前发现的潜在转化影响 这里.最终，解剖学、3D和分子特征的结合将有助于翻译 在人体组织和活检中，同时最大限度地提高相关新治疗靶点的可能性。

PROJECT SUMMARY Identifying patterns of neuronal connectivity is critical for understanding functional and anatomical circuits that mediate pain perception. However, knowledge about the types and distribution of neurons in joint tissues have generally been limited to traditional 2-dimension histopathological and immunohistopathological approaches, and little to no information is available on connectivity and neuronal phenotypes. New technologies have emerged that allow for both trans-synaptic circuit analysis and precise control of neuronal firing, including the use of retrogradely transported viral vectors (i.e., pseudotyped rabies virus) and heterologous receptor activation. At the same time, 3-dimensional visualization of neuronal and vascular patterns have been advanced by tissue clearing techniques in conjunction with cell type specific fluorescent markers generated by intercrossing cell type specific Cre recombinase mouse lines with a variety of conditionally activated reporters. Finally, the advent of single cell RNA sequencing has allowed for extending cellular phenotyping to a molecular level that has not only increases analytic resolution, but also therapeutic targeting with greater disease specificity than previously possible. The development of high resolution spatial transcriptomics, i.e., MERFISH, allows for correlation and validation of scRNA-seq data. In this context, osteoarthritis of the knee joint is an optimal model for applying these tools as abundant genetic and surgical models are available for orthogonal validation of findings. Moreover, in the preclinical context, various therapeutic approaches including gene therapy have been shown to impact pain measures, and as such, they constitute an important interventional validation of molecular changes that are identified in neurons in the disease state. The fact that some of these therapies are now in clinical trial adds to the potential translational impact of the proposed preclinical findings here. Ultimately, the combination of both anatomic, 3-D, and molecular signatures will facilitate the translation into human tissues and biopsies, while maximizing the likelihood of relevant new therapeutic targets.