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Optical and biophysical characterization of the vestibular periphery

前庭周边的光学和生物物理特征

基本信息

批准号：
9894783
负责人：
JONATHAN JAMES ART
金额：
$ 23.99万
依托单位：
UNIVERSITY OF ILLINOIS AT CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-04-01 至 2023-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9894783
关键词：
Address Anatomy Auditory Auditory system Autonomic nervous system disorders Axon Basilar Papilla Biological Biophysics Calculi Cell physiology Cells Closure by clamp Cochlea Crista ampullaris Data Diffusion Disease Distal Dizziness Dyes Electric Stimulation Electrodes Electrophysiology (science)Elements Endolymph Environment Epithelial Epithelium Equilibrium Face Fiber Fluorescence Functional disorder Future Geometry Goals Hair Cells Head Homologous Gene Image In Situ Individual Ions Labyrinth Light Lighting Location Measurement Measures Mechanical Stimulation Mechanics Membrane Potentials Microelectrodes Microscopy Modeling Nerve Optics Organ Parents Pathway interactions Perilymph Peripheral Phase Physiologic pulse Physiological Physiology Preparation Property Research Rotation Semicircular canal structure Sensory Hair Signal Transduction Structure Supporting Cell Surface Synapses Synaptic Cleft Synaptic Transmission Techniques Time Turtles Type I Hair Cell Type II Hair Cell Variant Vertigo Vestibular Hair Cells base biophysical properties electrical property experimental study innovation insight macula multi-photon multiphoton microscopy novel novel strategies otoconia reconstruction response signal processing statistics tongue papilla voltage voltage clamp voltage sensitive dye

项目摘要

Project Summary Prior research demonstrates that afferent responses from semicircular canal cristae and otolith organ maculae deviate from the coherent mechanical stimulation imparted by their overlying accessory structures. This implicates further processing by hair cells (HCs), primary afferents, and the HC - afferent synapse. Processing is complicated by the parallel modes of synaptic transmission between HCs and afferents, and the convergence of multiple HCs onto a single afferent. In the vestibular periphery progress towards describing variations in afferent discharge in terms of the time course of underlying voltage- and ion-sensitive conductances has been impeded by two major anatomical features of the vestibular epithelia: 1) access to HCs and afferents in their native bi-ionic (endolymph - perilymph) environment is mechanically impeded, so there are few in situ recordings to serve as controls for pathophysiology in recordings made from isolated cells or epithelial explants; and 2) analysis of integration at the level of a ramifying afferent is complicated by multiple HC convergence onto each, and the impossibility of using a single patch-electrode to space-clamp a distributed afferent arbor. To overcome the inaccessibility problem associated with obtaining physiological data for vestibular HCs in their native environment, we will measure voltages using slow, potentiometric (Nernstian) dyes, whose equilibrium partition (concentration) is voltage-dependent. These dyes will be superfused across the vestibular and auditory epithelia in a turtle half-head preparation. The voltage-dependent fluorescence of slow redistributive dyes will be measured using multiphoton microscopy (MPM), and calibrated using microelectrode recordings from HCs, afferents and supporting cells via the readily accessible perilymphatic space of the auditory papilla. The type I HC/calyceal afferent synapse is relatively compact electrically, but HC convergence onto a single afferent over distances of 10s to 100s of microns makes it difficult to address the passive and active properties of an afferent arbor. As a consequence, it remains problematic to characterize afferent integration using conventional electrophysiological techniques. We will examine afferent convergence by patch recording single afferents using electrodes filled with electrochromic voltage-sensitive dyes (VSDs). Steady-state depolarizations and hyperpolarizations of the afferent via the patch electrode will be used to optically characterize the passive cable properties of the ramifying afferent using lattice light sheet microscopy (LLSM). Pulses of current injected through the patch electrode, or electrical stimulation of the nerve - phase-locked to image acquisition on the LLSM - will be used to image and average orthodromic and antidromic AP propagation in the parent axon and throughout the afferent arbor. We propose novel approaches to make highly significant measures that are currently unavailable. By using optical and electrode recordings to characterize the cellular potentials and the afferent convergence, these experiments have the potential to make large and durable contributions to the field.

项目摘要以往的研究表明，半规管嵴和耳石器的传入反应黄斑偏离由其上覆的附属结构给予的连贯的机械刺激。这暗示了毛细胞（HC）、初级传入神经和HC -传入突触的进一步加工。处理 HC和传入神经之间的突触传递的平行模式，多个毛细胞到一个单一的传入。在前庭周围进展到描述的变化，传入放电的时间进程的基础电压和离子敏感电导已被前庭上皮的两个主要解剖学特征阻碍：1）进入HC和传入，天然双离子（内淋巴-外淋巴）环境受到机械阻碍，因此原位记录很少在由分离的细胞或上皮外植体制成的记录中用作病理生理学的对照;和2）在分支传入水平上的整合分析由于多个HC收敛到每个HC上而变得复杂，以及不可能使用单个膜片电极来空间夹持分布的传入心轴。克服与获得前庭HC在其自然状态下的生理数据相关的不可访问性问题环境中，我们将使用缓慢的电位（能斯特）染料测量电压，其平衡分配（浓度）是电压依赖性的。这些染料将被灌注穿过前庭和听觉上皮用乌龟的半个脑袋做准备缓慢再分配染料的电压依赖性荧光将是使用多光子显微镜（MPM）测量，并使用来自HC的微电极记录进行校准，传入和支持细胞通过容易接近的听乳头外淋巴间隙。的 I型HC/盏传入突触在电上相对紧凑，但HC会聚到单个传入突触上，在10微米到100微米的距离上，传入乔木因此，使用传统的方法来表征传入整合仍然是有问题的。电生理技术。我们将检查传入收敛补丁记录单个传入使用填充有电致变色电压敏感染料（VSD）的电极。稳态去极化和通过贴片电极的传入神经的超极化将用于光学表征无源电缆使用晶格光片显微镜（LLSM）的分支传入的特性。注入的电流脉冲贴片电极或神经的电刺激-锁相到LLSM上的图像采集-将用于成像和平均在母轴突和整个轴突中的顺向和逆向AP传播传入乔木我们提出了新的方法，使目前非常重要的措施，不可用.通过使用光学和电极记录来表征细胞电位和传入这些实验有可能为该领域做出巨大而持久的贡献。