Shedding light on balance: Interrogating individual synapses within vestibular epithelia

阐明平衡：询问前庭上皮内的单个突触

• 批准号: 10593864
• 负责人: LARRY F HOFFMAN
• 金额: $ 23.4万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-02-01 至 2025-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10593864
• 关键词: Acoustic Trauma; Affect; Afferent Neurons; Aging; Architecture; Attenuated; Binding; Biosensor; Capsid; Characteristics; Cochlea; Code; Communication; Complex; Coupled; Craniocerebral Trauma; Development; Dimensions; Disease; Dose; Encapsulated; Epithelium; Equilibrium; Etiology; Evaluation; Exhibits; Experimental Models; Face; Fluorescence; Foundations; Functional disorder; Future; Genotype; Glutamates; Hair Cells; Head Movements; Health; Heterogeneity; Histologic; Immunohistochemistry; Individual; Investigation; Labyrinth; Light; Measures; Mediating; Membrane; Methodology; Methods; Modification; Morphology; Mus; Neurotransmitters; OTOF gene; Optics; Outcome; Pathologic; Pathology; Pattern; Peripheral; Phenotype; Physiological; Postsynaptic Membrane; Proxy; Recovery; Reflex action; Reporter; Research; Research Design; Saccades; Signal Transduction; Site; Space Flight; Structure; Structure-Activity Relationship; Subcellular structure; Synapses; Synapsins; Synaptic Receptors; Testing; Time; Transfection; Tubulin; Type I Hair Cell; Vestibular Hair Cells; Viral; Work; analog; cochlear synaptopathy; experience; experimental study; hidden hearing loss; high resolution imaging; method development; mosaic; mouse model; neurotransmitter release; novel; ototoxicity; postsynaptic; presynaptic; promoter; public health relevance; response; ribbon synapse; segregation; sensor; superresolution microscopy; synaptic function; temporal measurement; tool

Project Summary The present application proposes a developmental research plan to investigate the structure-function relationship of individual presynaptic complexes in mammalian vestibular hair cells. These complexes incorporate synaptic ribbons that can exhibit broad architectural heterogeneity, the functional significance of which is not known. The distribution of presynaptic architectures in the primary hair cell phenotypes (i.e. types I and II) within vestibular epithelia, and the unique dendritic specialization known as the calyx (encapsulating type I hair cells), render traditional methods of investigating synaptic function inappropriate for elucidating the functional characteristics of individual synaptic sites. Recent advances in the development of optical biosensors and viral transduction strategies provide the foundation for novel capabilities to detect and quantify neurotransmitter release at individual synapses. iGluSnFR is a genetically encoded, membrane bound glutamate sensor. When expressed at the postsynaptic membrane, it emits a fluorescent signal proportional to glutamate release. Recent key investigations demonstrated that when packaged with an AAV9 capsid and a synapsin promoter, inner ear afferent neurons are transduced and iGluSnFR is expressed. This strategy directs the sensor to the appropriate postsynaptic targets for measuring glutamate release from individual hair cell synapses. When coupled with strategies for post-recording elucidation of synaptic ribbons an association between glutamate release and synapse structure can be made. The project includes three Aims to: 1) optimize transduction in vestibular afferent neurons; 2) develop and optimize recording strategies to capture the time- resolved glutamate release from individual synaptic sites; and 3) evaluate the methods in a mouse model for which presynaptic function in type I hair cells is drastically attenuated. Immunohistochemical processing following optical recording will enable the direct association of immunolabeled ribbons with the recording sites, for which ribbon volume provides a proxy of its architecture. The development of these methods provides the foundation future studies of pathologic conditions whose etiologies are proposed to involve synaptopathies. This investigation will enable future investigations of synaptic function in normal and pathologic conditions, and provide a platform for the rigorous evaluation of novel treatments of inner ear dysfunction.

项目摘要 本申请提出了一个研究结构-功能的发展研究计划 哺乳动物前庭毛细胞中单个突触前复合体的关系。这些复合体 整合可展示广泛结构异质性的突触功能区，其功能意义 这一点还不清楚。初级毛细胞表型(即I型)突触前构筑的分布 和ii)在前庭上皮细胞内，以及被称为花萼(包膜型)的独特的树突特化 I毛细胞)，使得传统的研究突触功能的方法不适合阐明 单个突触部位的功能特征。光学生物传感器的发展近况 病毒转导策略为新的检测和量化能力提供了基础 神经递质在单个突触的释放。IGluSnFR是一种遗传编码的膜结合蛋白 谷氨酸传感器。当在突触后膜上表达时，它发出的荧光信号与 谷氨酸的释放。最近的关键调查表明，当包装上AAV9衣壳和 突触素启动子、内耳传入神经元被转导并表达iGluSnFR。这一战略将指导 将传感器定位于适当的突触后靶点以测量单个毛细胞的谷氨酸释放 突触。当结合记录后阐明突触带的策略时， 谷氨酸的释放和突触结构之间可能存在联系。该项目包括三个目标：1)优化 前庭传入神经元的传导；2)开发和优化记录策略，以捕捉时间- 从单个突触位置分解谷氨酸释放；以及3)在小鼠模型中评估这些方法 I型毛细胞中的哪种突触前功能明显减弱。免疫组织化学处理 随后的光学记录将使得免疫标记的条带能够与记录位置直接关联， 其带状体积为其体系结构提供了代理。这些方法的发展提供了 为未来对其病因可能涉及突触病变的病理条件的研究奠定基础。这 研究将使未来在正常和病理条件下研究突触功能成为可能，以及 为严格评估内耳功能障碍的新疗法提供了一个平台。