New technologies for investigating the hair cell afferent fiber synapse

描述（申请人提供）：毛细胞传入神经突触对听力至关重要。这种突触功能的丧失会导致听力丧失和耳聋。该突触对噪音、年龄相关的听力损失以及耳毒性物质高度敏感。关于突触特化和突触前释放和突触后整合的调节机制的基本问题仍有待解决。这些问题需要解决几个技术上具有挑战性的问题。本提案的目的是开发和实现这些技术。第一个问题是，用于分离细胞或去除耳石膜或耳膜的酶处理会改变突触特性，并且在许多情况下会消除受体。特异性目的（SA） 1侧重于改进一种非酶技术，用于在保持毛束功能的同时去除被膜。该技术包括使用高二价浓度软化膜和与该膜的毛束连接，以消除当膜被移除时毛束的机械扰动。第二个问题是，目前用于跟踪膜电容变化（作为囊泡释放的指标）的方法是有限的，因为它们只给出稳态响应。电导变化，如钙通道激活，影响电容测量。SA 2采用Santos Sacchi（2005）开发的双正弦波技术，用于测量外毛细胞的非线性电容，以估计囊泡释放。我们将使用已经掌握的标准技术对两种技术进行比较，确定双正弦波法的分辨率和局限性。这项技术有可能直接测定胞吐的动力学，并与钙电流的激活动力学进行比较。第三个障碍是同时记录毛细胞和初级传入神经。SA3将发展这种能力。目前，我们可以独立地记录毛细胞和突触的机械灵敏度。然后，我们将应用SA2的电容技术与SA3的记录添加来直接测量EPSCs，并将这些振幅与突触前测量的相应电容变化相关联。这些技术将使我们能够确定多泡释放的潜在机制。

DESCRIPTION (provided by applicant): 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.