The hair cell afferent nerve synapse is critical for audition. Loss of function of this synapse leads to hearing loss and deafness. This synapse is highly sensitive to noise induced, age related hearing loss as well as ototoxic agents. Fundamental questions remain to be addressed regarding synaptic specializations and mechanisms of regulation of presynaptic release and post synaptic integration. These questions require that several technically challenging problems be addressed. The purpose of this proposal is to develop and implement these technologies. The first problem is that enzymatic treatments used in either isolating cells or removing tectorial or otolithic membranes alter synaptic properties and can in many instances eliminate receptors.
Specific Aim (SA) 1 focusses on refining a nonenzymatic technique for removing tectorial membranes while maintaining functioning hair bundles. The technique involves using high divalent concentrations to soften the membrane and hair bundle connections to this membrane in order to eliminate mechanical perturbation of the hair bundle as the membrane is removed. The second issue is that present methods used to track membrane capacitance changes (as indicators of vesicular release) are limited in that they give only a steady-state response. Conductance changes, like calcium channel activation, compromise the capacitance measurement. SA 2 applies a dual sine wave technique, developed by Santos Sacchi, (2005) for measuring nonlinear capacitance in outer hair cells to estimate vesicle release. We will use the standard technology that we have mastered to compare the two techniques and determine the resolution and limitations of the dual sine wave method. This technique has the potential of allowing the kinetics of exocytosis to be determined directly and compared to the activation kinetics of the calcium current. The third obstacle is simultaneously recording from hair cells and primary afferent nerve. SA3 will develop this ability. We presently can record from each independently and maintain mechanical sensitivity of both the hair cell and synapse. We will then apply the capacitance techniques of SA2 with the recording additions of SA3 to directly measure EPSCs and correlate these amplitudes with the corresponding capacitance changes measured presynaptically. Together these technologies will allow us to determine the underlying mechanism responsible for multivesicular release. ? ? ?
