DESCRIPTION (provided by applicant): This is a continuing study of synaptic transmission in vestibular organs with emphasis on the type I hair cell and its calyx ending, phylogenetically recent acquisitions only found in reptiles, birds and mammals. These structures are restricted to central/striolar zones in reptiles and birds. Their increasing importance in mammals is suggested by their distribution throughout the neuroepithelium of all organs. In addition, while type I and type II hair cells occur in approximately equal numbers in rodent cristae, type I hair cells predominate in the cristae of monkeys and possibly of humans. The peculiar structure and distinctive physiology of these structures raise problems as to how synaptic transmission is accomplished. 1) Type I hair cells have a distinctive basolateral current that may compromise synaptic transmission; 2) Housekeeping functions cannot be done by supporting cells; and 3) The geometry and electrophysiology of the calyx ending place unusual demands on the flow of synaptic currents to the spike encoder. We have only fragmentary knowledge as to how these problems are solved. Yet, because these structures become of increasing importance in mammals (including humans), such knowledge is crucial to our understanding as to how vestibular organs process information. Of clinical interest, the type I hair cell and/or its calyx ending are especially sensitive to aminoglycoside ototoxicity and age-related degeneration. Physiological studies will be done in the turtle posterior crista and will be integrated with morphological studies to be done in rats and turtles. There are three specific aims. 1) Hair cells: We will characterize synaptic transmission from type I hair cells by making whole- cell recordings from the calyx ending. The hypothesis is that neurotransmitter release from type I hair in low- frequency vestibular hair cells may differ from high-frequency auditory and vibratory organs. 2) Homeostasis: Both K+ ions and glutamate neurotransmitter are released from hair cells during transduction. In the case of type II hair cells, supporting cells serve to clear these substances. The presence of the calyx ending precludes supporting cells from acting in the same way for type I hair cells. We hypothesize that the type I hair cell and/or its ending subsume these housekeeping functions. 3) Postsynaptic mechanisms: We will explore how various ion channels and neurotransmitter receptors shape synaptic and spiking activity. The hypothesis to be tested is that the molecular organization of the calyx creates separate microdomains with discrete functions: synaptic transmission, spike initiation, and discharge regularity.

PUBLIC HEALTH RELEVANCE: Because type I hair cells and their calyx endings become of increasing importance in mammals (including humans), such knowledge is crucial to our understanding as to how vestibular organs process information. Of clinical interest, the type I hair cell and/or its calyx ending may be especially sensitive to aminoglycoside ototoxicity and age-related degeneration. Project Narrative Because type I hair cells and their calyx endings become of increasing importance in mammals (including humans), such knowledge is crucial to our understanding as to how vestibular organs process information. Of clinical interest, the type I hair cell and/or its calyx ending may be especially sensitive to aminoglycoside ototoxicityand age-related degeneration. __SpecificAimsTextDelimiter__ 1. Specific Aims Throughout vertebrate history, the vestibular labyrinth has been relatively conservative.7,121 Among the few evolutionary novelties are the type I hair cell and its associated calyx ending, which are only found in reptiles, birds and mammals.129,132,214 In reptiles and birds these structures are restricted to central/striolar zones. Their increasing importance in mammals is suggested by their distribution throughout the neuroepithelium of all organs. In addition, while type I and type II hair cells occur in approximately equal numbers in rodent cristae,46,63,126 type I hair cells predominate in the cristae of monkeys63,129,133 and of humans.143 Counting calyx and dimorphic fibers in mammals, 80-90% of all afferents have calyx endings innervating type I hair cells. While other calyx endings in the nervous system are presynaptic structures, the vestibular calyx is unique in being postsynaptic. Of clinical interest, the type I hair cell and its calyx ending are especially sensitive to aminoglycoside ototoxicity,89 and age-related degeneration.143 The peculiar structure and distinctive physiology of type I hair cells and their calyx endings raise questions as to the mechanisms of synaptic transmission between these structures. We divide the problem into three parts. To the extent possible, we will integrate morphological/molecular and physiological/pharmacological studies. The morphological studies will be done in the rat and, when necessary, extended to the turtle. Physiological work will be done in the turtle posterior crista where we have detailed information about spike discharge,19,20 the electrophysiology of type I and type II hair cells18,73 and both afferent92,94 and efferent neuro- transmission.93 All recordings will be made in a newly developed preparation, a split neuroepithelium which allows us to make whole-cell recordings (WCRs) from calyx endings. Morphological/molecular approaches include immunohistochemistry (IHC), RT-PCR and in situ hybridization. There are three specific aims. Specific Aim 1. Presynaptic mechanisms. Our hypothesis is that type I hair cells, given their peculiar morphology and electrophysiology, differ in their neurotransmitter release from auditory and vibratory hair cells and may be adapted to handle the low-frequency information (0 - 10 Hz) processed by vestibular organs. To be studied are: 1) the relation between hair-cell depolarization, quantal rate and multivesicular release; 2) the roles of quantal and non-quantal transmission; 3) the relative contributions of inner-face and outer-face synapses, receiving their inputs, respectively, from type I and type II hair cells; and 4) the response dynamics of synaptic activity evoked by hair-bundle stimulation at both low and high frequencies. The presynaptic and postsynaptic locations of the various Glu receptors will be studied by immunohistochemistry. Specific Aim 2. Homeostatic mechanisms. Glutamate (Glu) and K+ can accumulate in the extracellular space. In the case of type II hair cells, supporting cells function to clear these substances.68,157 The presence of the calyx ending precludes supporting cells from acting in the same way in type I hair cells. We hypothesize that transporters present in the type I hair cell, and/or the calyx ending, prevent the accumulation of K+ and glutamate in the elongated synaptic cleft between the hair cells and their calyx endings. Two mechanisms may be responsible for terminating neurotransmitter action: 1) receptor desensitization and 2) transmitter removal by EAATs. The relative roles of the two mechanisms will be studied by selective blocking individually and in tandem. KCC co-transporters, which are present in calyx endings, will be blocked to determine their effects on quantal and non-quantal transmission. The presynaptic and postsynaptic locations of EAATs will be studied by immunohistochemistry. Specific Aim 3. Postsynaptic mechanisms. The calyx ending has multiple functions, including synaptic transmission, action potential initiation and control of repetitive discharge. In addition, calyces have a complex organization with receptors, voltage-gated ion channels and auxiliary proteins organized into several discrete regions.130 Our hypothesis is that each of the many neurotransmitter receptors and ion channels plays a distinctive role in determining these functions. Neurotransmitter receptors. Postsynaptic Glu receptors will be defined both morphologically and physiologically. There is convincing evidence for AMPA receptors. NMDA receptors have been demonstrated by both IHC and physiologically; yet, their functional role is not understood. mGluRs will also be investigated because they may play an important role in the regulation of ionotropic glutamate receptors. Ion channels. By determining the impedance of the ending, we will be able to determine if it is electrotonically compact, which would imply that the complex spatial organization reflects molecular architecture, rather than the independent activation of the several voltage-gated processes present. In addition, we will explore how the various ion channels and neurotransmitter receptors shape synaptic transmission, spike generation and repetitive discharge.