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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要本申请提出了一项开发研究计划来研究结构-功能哺乳动物前庭毛细胞中各个突触前复合体的关系。这些复合体结合可以表现出广泛的结构异质性的突触带，其功能意义这是未知的。突触前结构在初级毛细胞表型（即 I 型）中的分布 II）前庭上皮内，以及称为花萼（封装型）的独特树突特化 I 毛细胞），使得研究突触功能的传统方法不适合阐明各个突触位点的功能特征。光学生物传感器的研究进展病毒转导策略为检测和量化的新能力提供了基础单个突触释放神经递质。 iGluSnFR 是一种基因编码的膜结合谷氨酸传感器。当在突触后膜上表达时，它发出与以下值成比例的荧光信号：谷氨酸释放。最近的关键研究表明，当与 AAV9 衣壳和突触蛋白启动子、内耳传入神经元被转导并表达 iGluSnFR。这一战略指导将传感器连接到适当的突触后目标，以测量单个毛细胞的谷氨酸释放突触。当与突触带的记录后阐明策略相结合时，关联可以在谷氨酸释放和突触结构之间进行。该项目包括三个目标：1）优化前庭传入神经元的转导； 2）制定和优化记录策略以捕捉时间- 解决了各个突触位点的谷氨酸释放； 3）评估小鼠模型中的方法 I 型毛细胞的突触前功能急剧减弱。免疫组织化学处理接下来的光学记录将使免疫标记带与记录位点直接关联，功能区卷为其架构提供了代理。这些方法的发展提供了为未来的病理状况研究奠定基础，其病因被认为涉及突触病。这研究将使未来对正常和病理条件下突触功能的研究成为可能，并且为严格评估内耳功能障碍的新疗法提供了一个平台。