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.

项目摘要先前的研究表明，半圆形管骨和耳石器官的传入反应黄斑偏离其上覆的附件结构所赋予的相干机械刺激。这毛细胞（HC），主要传入和HC传入突触的进一步处理。加工 HCS和传入者之间的突触传播的平行模式以及收敛性使其复杂化。多个HC在单个传入上。在前庭外围方面的进展从基础电压和离子敏感电导的时间过程中，传入的放电一直是前庭上皮的两个主要解剖特征阻碍：1）访问其HCS和传入者机械阻碍了天然双离子（内晶 - 围膜）环境，因此在原位记录中很少用作由孤立细胞或上皮外植体的录音中的病理生理学的控制；和2）通过多个HC收敛到每个，分析在分析传入级别的集成级别是复杂的以及不可能使用单个斑块电极来空间钳A分布的传入植物植物。克服与在其本地获得前庭HC的生理数据相关的无法访问性问题环境，我们将使用缓慢的电位测量（Nernstian）染料测量电压，其平衡分区（浓度）是电压依赖性的。这些染料将在前庭和听觉上皮中被超级融化在乌龟半头准备中。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HCS的微电极记录进行校准通过听觉乳头的易于访问的围绕空间，传入和支撑单元。这 I型HC/Calyceal传入突触相对紧凑，但HC收敛到单个传入超过10至100秒的距离，很难解决一个被动和主动特性传入Arbor。结果，使用常规的传入集成表征传入集成仍然存在问题电生理技术。我们将通过使用补丁记录单个传入来检查传入收敛充满电染色电压敏感染料（VSD）的电极。稳态去极化和通过贴片电极的传入的超极化将用于光学地表征无源电缆使用晶格光片显微镜（LLSM）的分支传入的特性。电流注入的脉冲斑块电极或神经的电刺激 - 相锁在LLSM上的图像采集 - 将用于图像和平均正常粒子和抗抗胶粒AP在父轴突和整个过程中传入的Arbor。我们提出了新颖的方法来制定目前的高度重要措施不可用。通过使用光学和电极记录来表征细胞电位和传入收敛性，这些实验有可能对该领域做出大型耐用的贡献。