The goal of the proposed research is to study the neuroelectric events that accompany immature avian (pigeon) vestibular hair cell regeneration and synaptic reinnervation of regenerated hair cells by primary afferents and efferents following streptomycin (SM) ototoxicity. The semicircular canals (SCCs) will be studied. Basolateral ionic currents in dissociated cells (supporting and hair cells) and action potentials on primary afferents will be studied in vitro and in vivo, respectively. In one set of experiments, cells will be dissociated from the SCC cristae during 3 time periods (1-10, 15-25, and 55-65 days) following the end of SM treatment. Thus, selective sampling of the basolateral ionic currents can occur from: supporting (progenitor) cells before and after differentiation, hair cells before and after specialization into type I and type II hair cells and type I hair cells from either single or multiple hair cell calyxes. Therefore, one can study the sequence of basolateral ionic channel expression as a progenitor cell differentiates into a type II and/or subsequently into a type I hair cell. In a second set of studies, using the same time windows, spontaneous and driven discharge will be recorded from one group of awake animals using rotatory stimulation and a separate group of anesthetized animals using mechanical stimulation. In the latter group, an axon in each horizontal ampullary nerve will be injected with an neuronal tracer and it and its terminals reconstructed. The difference in time of regeneration of the two types of hair cells, and reinnervation by single and multiple hair cell calyxes will be used as a tool to study the contribution of these components to calyx only, dimorph and bouton only afferent unit responses. A comparison of the responses from awake and anesthetized animals during the three time windows will elucidate the role of the efferents in afferent responses during hair cell regeneration and reinnervation. Solitary cells and cell clusters will be studied using whole cell current and voltage clamp techniques. Primary afferents will be studied using single unit extracellular or intracellular recording techniques. Reconstruction of labeled dendritic terminals will be achieved using laser scanning confocal microscopy. Correlations will be drawn between discharge properties and morphology of partially and fully reinnervated single hair cell calyx, multiple hair cell calyx, dimorph and bouton afferents. It has been estimated that 17 million people in the U. S. suffer from premature hair cell loss (Corwin, 1992). Evidence exists that in some species hair cells can regenerate. Little is known about the physiology of vestibular supporting cells (putative hair cell progenitors - Specific Aim 1 (SA1)) or regenerating vestibular hair cells (SA2) or about changes, if any, in the action potential discharge of primary afferents as afferent and efferent terminals reconnect to regenerated type II then type I hair cells (SA3) in different regions of the neuroepithelium (SA4). These issues are directly or indirectly addressed by the specific aims of this proposal.
