High-frequency transmission in the superior olivary complex: molecular regulation by presynaptic proteins

许多听觉脑干神经元能够在长时间内保持高带宽、高频率的放电。例如，兴奋性（谷氨酸能）和抑制性（甘氨酸能）神经元投射到侧上橄榄（LSO），通过耳间强度检测形成声音定位的基础，驱动它们的突触后目标神经元高达每秒600次。我们已经证明，在持续的刺激下，这些突触的快速突触传递非常强劲，这可以通过抗疲劳和低故障率来证明。这些特征使它们与“传统的”突触区别开来，后者的抑制更深刻，频率也低得多，从抑制状态中恢复的效率也低得多。显然，听觉突触利用非常有效的囊泡补充来不知疲倦地编码声音。突触性能差异的潜在分子机制尚不清楚。我们希望通过关注突触前释放机制中的四个关键蛋白来分析高频突触传递到LSO神经元的分子调控，这些蛋白似乎是传递物释放速度和保真度的决定因素（Bassoon, Munc13-1, Synaptotagmin2, CAPS1）。因果关系将通过特定的基因缺失来证明。为了避免系统性基因缺失所固有的问题，我们将进行空间限制性基因消融，这是最近通过产生具有候选分子的封闭基因的转基因小鼠而实现的。时间和空间限制基因沉默将通过病毒转染的立体定向基因传递（Cre重组酶）来实现。作为基准，我们还将分析海马结构中的两种突触类型，即从内嗅皮层到齿状回的谷氨酸能CA3-CA1连接和gaba能连接。我们将量化基本突触传递以及突触可塑性与一系列既定的刺激方案。我们假设，突触前释放机制中的突触特异性特征体现了强大的高频传输和对突触疲劳的抵抗力。我们还认为，这四种候选蛋白的贡献方式仅与它们的同类蛋白部分重叠，从而实现适度的冗余。组织学调查包括光镜和电镜免疫组织化学将补充我们的生理分析。总之，我们期望更好地理解高频神经传递的潜在机制，并阐明一些关键参与者在突触功能中的作用。我们的项目也将有助于理解各种神经元系统中突触信息传递的一般方面。

Many auditory brainstem neurons are able to maintain high-bandwidth, high-frequency firing over prolonged periods of time. For example, excitatory (glutamatergic) and inhibitory (glycinergic) neurons projecting to the lateral superior olive (LSO), which form the basis of sound localization via interaural intensity detection, drive their postsynaptic target neurons up to ca. 600 times per second. We have shown that fast synaptic transmission at these synapses is remarkably robust during sustained stimulation as evidenced by resistance to fatigue and low failure rates. These features set them apart from "conventional" synapses, which depress more profoundly and at significantly lower frequency, and recover much less efficiently from their depressed state. Apparently, auditory synapses employ very efficient vesicle replenishment to indefatigably encode sound. The underlying molecular mechanisms for the differences in synaptic performance are unknown. We want to analyze the molecular regulation of high-frequency synaptic transmission to LSO neurons by focusing on four key proteins in the presynaptic release machinery which appear to be determinants of the speed and fidelity of transmitter release (Bassoon, Munc13-1, Synaptotagmin2, CAPS1). Causal relationship will be demonstrated by specific gene deletion. To circumvent problems immanent to systemic gene deletion, we will perform spatially restricted gene ablation, which became available only very recently through the generation of transgenic mice with floxed genes for the candidate molecules. Temporally and spatially restricted gene silencing will be pursued via stereotaxic gene delivery (Cre recombinase) through viral transfection. As a benchmark, we will also analyze two synapse types in the hippocampal formation, namely the glutamatergic CA3-CA1 connection and the GABAergic connection from the entorhinal cortex to the dentate gyrus. We will quantify basic synaptic transmission as well as synaptic plasticity with a battery of established stimulus protocols. We hypothesize that robust high-frequency transmission and resistance to synaptic fatigue are manifested by synapse-specific characteristics in the presynaptic release machinery. We also suggest that the four candidate proteins contribute in a manner that overlaps only partially with their peer proteins, thereby achieving a moderate degree of redundancy. Histological investigations comprising light and electron microscopic immunohistochemistry will complement our physiological analyses. In total, we expect to better understand the underlying mechanisms for high frequency neurotransmission and to elucidate the role of some key players in synapse function. Our project shall also contribute to comprehending general aspects of synaptic information transfer in various neuronal systems.