EFFERENT INNERVATION OF IDENTIFIED VESTIBULAR AFFERENTS

本研究的目的是研究外周前庭器官的传出神经支配。了解神经递质和脑干传出神经元向外周迷路的投射将有助于我们理解前庭传出系统在健康和疾病中的作用。最近的研究表明，前庭传出核的神经元在其递质组成和外周靶点上是不均匀的。这种异质性改变了我们长期以来认为前庭传入是一个非特异性系统的观点。本研究的目的是根据鼠鼠的脑干位置、投射模式、外周末梢、神经递质和受体亚型来定义鼠鼠的传出神经元亚群。具体目的是：1)通过将逆行和顺行示踪剂与递质免疫组织化学相结合来表征脑干传出神经元的解剖和神经化学亚群；2)在光镜和电镜水平上确定外周传出神经元的形态终止细节，并通过确定其递质受体亚型来确定其分子水平上的神经化学细节。脑干的传出神经元是通过向周围前庭器官注射逆行示踪剂来识别的。同时用免疫组织化学方法对几种假定的神经递质（乙酰胆碱、降钙素基因相关肽、一氧化氮合酶、脑啡肽、三磷酸腺苷等）的抗体进行鉴定。在外周，将检查肽能、嘌呤能和毒蕈碱传递的受体亚型分布，以试图具体理解传入神经对传出刺激的缓慢反应。通过细胞外辣根过氧化物酶注射或calretinin免疫组织化学鉴定前庭传入事件。外周传出末梢的神经支配模式是通过中央注射生物素化的葡聚糖胺来确定的。电镜免疫组织化学方法被用来根据它们在感觉上皮中的末端来描述化学上不同的传出钮扣，也就是说，根据传入事件的类别（花萼、二形或钮扣）和它们支配的区域（中枢或外周区）来描述。目的是产生一个关于传出神经的结构基础的知识体系，以及它们与传入神经的关系，从中可以推导出生理学和药理学上可检验的假设。

The purpose of this grant is to investigate the efferent innervation of the peripheral vestibular apparatus. Knowledge of the neurotransmitters and the projections from the brainstem efferent neurons to the peripheral labyrinth will help us understand the role of the vestibular efferent system in health and disease. Recent studies have suggested that neurons in the vestibular efferent nucleus are heterogeneous in their transmitter composition and in their peripheral targets. Such heterogeneity changes our long-held view that vestibular efferents are a nonspecific system. The goal of this study is to define subpopulations of efferent neurons in the chinchilla based upon their brain stem locations, their projection patterns, their peripheral endings, their neurotransmitters, and their receptor subtypes. Specific aims are: 1) to characterize anatomical and neurochemical subpopulations of brainstem efferent neurons by combining retrograde and anterograde tracers with transmitter immunohistochemistry, and 2) to determine details of the morphological terminations of efferents in the periphery, at both the light and electron microscopic levels, and details of their neurochemistry at the molecular level by determining their transmitter receptor subtypes. Efferent neurons in the brainstem are identified by means of retrograde tracer injections into the peripheral vestibular apparatus. They are simultaneously characterized immunohistochemically with antibodies to several putative neurotransmitters (acetylcholine, calcitonin gene- related peptide, nitric oxide synthase, met-enkephalin, adenosine triphosphate, and others). In the periphery, the distribution of receptor sub-types for peptidergic, purinergic and muscarinic transmission will be examined in an attempt to specifically understand the slow response of afferents to efferent stimulation. Vestibular afferents are identified by means of extracellular horseradish peroxidase injections or calretinin immunohistochemistry. The innervation patterns of efferent terminals in the periphery is determined by injections of biotinylated dextran amine centrally. Electron microscopic immunohistochemical methods are used to characterize chemically distinct efferent boutons according to their terminations in the sensory epithelium, that is, by the class of afferents (calyx, dimorphic or bouton) and by the region (central or peripheral zones) that they innervate. The intent is to produce a body of knowledge about the structural basis of the efferents, and their relationship to the afferents, from which physiologically and pharmacologically testable hypotheses can be derived.