Joseph Christopher Holt - US grants

DESCRIPTION (provided by applicant): Vestibular organs, via their hair cells and afferent innervation, transmit information about the direction, speed, and magnitude of head and body movements, which are critical for maintaining our posture and stabilizing our gaze. The vestibular organs of nearly every vertebrate also receive a prominent efferent innervation which begins as a few hundred neurons within the brainstem and extensively collateralizes in the periphery to end as several thousands of bouton terminals on both hair cells and afferents. That this efferent innervation is positioned at such an early stage in the peripheral vestibular pathway suggests it is poised to modulate the initial processing of vestibular cues. Electrical stimulation of efferent neurons results in a diverse panel of profound, yet distinct, excitatory and inhibitory afferent responses where the kinetics of both activation and duration can vary from milliseconds to minutes. To date, three pharmacologically-distinct nicotinic ACh receptors, a muscarinic ACh receptor, at least two classes of potassium channels, and the release of calcium from hair cell internal stores (e.g., subsynaptic cisterns) are thought to underlie the different efferent responses. However, there is a clear gap in our knowledge in relating how and when these different efferent-mediated components impact the responses of vestibular afferents to their natural stimulus. To facilitate an understanding of efferent function in vestibular physiology, three major studies will be performed in the turtle semicircular canal where a strong pharmacological basis for some of the efferent actions has been established, and both hair cell and afferent morphophysiology have been adequately described. The specific aims are: (1) Identify the synaptic mechanisms underlying afferent responses to electrical activation of efferent fibers, (2) Establish how vestibular output is modified by electrical activation of efferent fibers, and (3) Characterize the morphophysiological properties of efferent neurons. To complete specific aims 1 and 2, sharp-electrode and patch clamp recordings will be made from primary afferents, afferent terminals, and hair cells during efferent and mechanical stimulation. Pharmacological agents will be applied to identify the receptors and downstream effectors as well as defining how each efferent synaptic mechanism impacts vestibular stimulation. Parallel immunohistochemical studies will be used to localize the different components implicated by our physiological and pharmacological experiments. For specific aim 3, sharp-electrodes will be used to record efferent activity and label single efferent fibers in a decerebrate preparation. Light and EM microscopy will be used to reconstruct terminal trees and to examine the distribution and synaptic structure of efferent terminals. These studies will provide insights into the mechanisms that the efferent system recruits to modulate afferent discharge as well as identifying synaptic processes that may be targeted pharmacologically for the treatment of diseases and functional defects of the peripheral vestibular apparatus.

Project Summary Vestibular organs, through their resident hair cells and afferent innervation, transmit information to the central nervous system about the direction, speed, and magnitude of head and body movements, which are necessary for maintaining posture, stabilizing gaze, and guiding navigational tasks. The vestibular organs are also endowed with a robust efferent innervation that begins as a few hundred neurons within the dorsal brainstem and extensively collateralizes in the periphery to end as thousands of bouton varicosities abutting hair cells and afferents. In mammals, activation of the efferent vestibular system (EVS) ultimately excites primary vestibular afferents along two distinct time scales. While acetylcholine (ACh) accounts for many EVS actions in other vertebrates, the synaptic mechanisms underlying afferent responses to EVS stimulation in mammals have not been identified. As a result, there is a clear gap in our knowledge in relating how the various EVS-mediated actions are initiated, and what impact they exert on the subsequent responses of vestibular afferents to natural stimuli. To facilitate an understanding of EVS function in mammalian vestibular physiology, three major directions will be pursued in the peripheral vestibular system of mice. The first specific aim will establish the pharmacological basis for the effects of EVS activation on spontaneous discharge of vestibular afferents. The second specific aim will specify EVS postsynaptic mechanisms required for these EVS actions by using transgenic animals where individual signaling components, implicated by our pharmacological data, are absent. Finally, the last specific aim will identify how the activation of each EVS synaptic mechanism modifies the responses of mammalian afferents to vestibular stimulation. To complete these specific aims, the discharge properties of primary vestibular afferents in the anesthetized mouse will be characterized during EVS activation with or without vestibular stimulation. Selective pharmacological agents will be applied to identify the receptors and downstream effectors and to determine how they impact both stimulation paradigms. To identify and localize specific signaling pathways, parallel electrophysiological and immunohistochemical studies will be performed in transgenic animals where the function of proteins, integral to the synaptic mechanisms implicated by the pharmacology in the first specific aim, are disrupted. The effects of EVS stimulation on afferent responses to vestibular stimulation will be characterized by pairing rotational and translational stimuli with EVS stimulation paradigms during pharmacological interrogation in both control and transgenic animals. These studies are significant as they will provide much needed insights into the diverse synaptic mechanisms that the EVS recruits to modulate afferent discharge in mammals. The data captured by this proposal is critical for probing the functional roles of the EVS in vestibular physiology as well as identifying novel synaptic processes that can be targeted pharmacologically for combatting vestibular dysfunction.