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 本申请提出了一项开发研究计划，以调查结构功能 哺乳动物前庭毛细胞中各个突触前复合物的关系。这些复合物 结合可以表现出广泛建筑异质性的突触丝带，功能意义 这是不知道的。主要毛细胞表型中突触前体系结构的分布（即I型 ii）在前庭上皮中，以及独特的树突专业化，称为花萼（封装类型 I毛细胞），使调查突触功能的传统方法不适合阐明 单个突触部位的功能特征。光学生物传感器开发的最新进展 病毒转导策略为检测和量化的新功能奠定了基础 神经递质在单个突触处释放。 Iglusnfr是一种遗传编码的膜结合 谷氨酸传感器。当在突触后膜上表达时，它会发出与成比例的荧光信号 谷氨酸释放。最近的关键调查表明，用AAV9 CAPSID打包和 突触蛋白启动子，内耳传入神经元被转导并表达IglusnFR。该策略指导 适当的突触后靶标的传感器，用于测量单个毛细胞释放谷氨酸的传感器 突触。结合策略以阐明突触色带的策略 可以在谷氨酸释放和突触结构之间进行。该项目包括以下三个目标：1）优化 前庭传入神经元转导； 2）制定并优化记录策略以捕获时间 - 从单个突触部位释放了谷氨酸释放； 3）评估鼠标模型中的方法 I型毛细胞中的突触前功能大大减弱。免疫组织化学处理 遵循光学记录将使免疫标记的丝带与记录位点的直接关联， 色带量提供了其架构的代理。这些方法的开发提供了 基金会未来的病理状况研究，其病因涉及突触病。这 调查将使未来对正常和病理状况下突触功能的研究，以及 为内耳功能障碍的新型治疗方法进行严格评估提供了一个平台。