Identification of factors underlying spiral ganglion neurons??? dynamic range

描述（由申请人提供）：听觉系统的一个惊人特征是其宽广的动态范围-听觉系统可以探测到从水滴到喷气发动机的声音而不会造成损害。在听觉系统的第一个神经处理阶段，不同组的螺旋神经节神经元（sgn）被认为代表不同的动态范围。鉴于内毛细胞的动态范围被认为在很大程度上是均匀的，传入纤维如何获得这种不均匀的动态范围仍有待解决。最近的研究提供证据表明，传入纤维之间动态范围的差异至少部分源于IHC/传入突触。本研究假设了动作电位（AP）产生机制对动态范围的额外贡献。在具体目标1中，我们将确定在传入纤维中触发AP所需的注入电流大小是否不同，从而测试突触后阈值的差异，作为建立动态范围的一种手段。为此，我们将在单个传入纤维的钮扣上进行电流钳记录，并测量触发AP所需的最小电流注入。AP触发电流阈值的广泛分布将支持突触电流在传入纤维之间可能以不同的方式传播和整合以触发AP。在具体目标2中，我们将确定AP产生机制是否确实有助于在传入光纤中创建异构动态范围。为此，将记录来自单个传入纤维的兴奋性突触后电流（EPSCs）和ap，同时将记录的纤维接触的IHC去极化到一系列电压。本实验使我们能够确定传入纤维在EPSCs（突触前和突触后机制的输出）水平上的动态范围，以及在SGN输出水平上的动态范围，即AP放电率。通过比较EPSC的动态范围下界和上界与AP放电率的动态范围，我们将确定突触和AP产生机制在传入纤维之间形成异质动态范围的各自贡献。在具体目标3中，我们将确定传入纤维中是否存在分子谱的变化。我们特别感兴趣的是哪些特定分子参与了AP的产生机制，这些机制在建立sgn的异质动态范围中发挥作用。我们将通过qRT-PCR在单细胞水平检测候选分子的mRNA表达。候选通道包括电压门控钠（Nav）通道，它负责AP的产生，是设置神经元兴奋性的主要决定因素。子单元?1~3和Src家族激酶，已知它们调节Nav通道的性质。我们将使用基因芯片阵列以无偏倚的方式比较螺旋神经节成分，重点关注那些建立兴奋性的分子。该研究结果将通过提供sgn的输入-输出功能来促进我们对听觉神经生理学领域的理解。它还将揭示sgn异质性的可能分子机制，这对于正常的听觉功能和寻找听力缺陷的临床解决方案至关重要。

DESCRIPTION (provided by applicant): One astonishing feature of the auditory system is its broad dynamic range - the auditory system detects sounds from a water droplet to jet engine without causing damage. At the first neural processing stage of the auditory system, different groups of spiral ganglion neurons (SGNs) are thought to represent distinct dynamic ranges. Given that the dynamic range of inner hair cells is thought to be largely homogeneous, how afferent fibers acquire such heterogeneous dynamic ranges remains to be resolved. Recent studies provide evidence that the difference in dynamic range among afferent fibers originates, at least partially, at IHC/afferent synapses. Here, the proposed study hypothesizes an additional contributor to dynamic range by action potential (AP) generation mechanisms. In Specific Aim 1, we will determine whether the size of injected current required to fire an AP is variable among afferent fibers, thus testing for differences in postsynaptic threshold as a means of establishing dynamic range. For this, we will perform a current-clamp recording on single afferent fiber's bouton, and measure the minimum current injection required to fire an AP. A broad distribution of current threshold for AP firing would support that synaptic current may be differently propagated and integrated to fire AP among afferent fibers. In Specific Aim 2, we will determine whether AP generation mechanisms indeed contribute in creating heterogeneous dynamic range among afferent fiber. For this, excitatory post- synaptic currents (EPSCs) as well as APs from a single afferent fiber will be recorded while depolarizing the IHC contacted by the recorded fiber to a sequence of voltages. This experiment allows us to determine the afferent fiber's dynamic range at the level of EPSCs (output of pre- and post-synaptic mechanisms) as well as its dynamic range at the level of SGN output, AP firing rate. By comparing the lower and upper bounds of dynamic ranges by EPSC with those by AP firing rate, we will determine the respective contributions of synaptic and AP generation mechanisms in creating heterogeneous dynamic range among afferent fibers. In Specific Aim 3, we will determine whether there are variations in molecular profiles among afferent fibers. We are particularly interested in what specific molecules are involved in AP generation mechanisms that play a role in establishing heterogeneous dynamic range of SGNs. We will examine mRNA expression of candidate molecules at single-cell level by qRT-PCR. Candidates include voltage-gated sodium (Nav) channels, which are responsible for AP generation and one major determinant for setting the excitability of neurons as well as Nav channel ?-subunits ?1~3 and Src family kinases, which are known to modulate the property of Nav channels. We will use a gene chip array to compare spiral ganglia constituents in nonbiased manner focusing on those molecules that establish excitability. Outcome of the proposed study will advance our understanding in the field of auditory neurophysiology by providing input-output functions of SGNs. It will also reveal possible molecular players underlying heterogeneity of SGNs, which are important for proper auditory functions and for finding clinical solutions for hearing deficits.