Modulation of Exocytosis and Excitability in Mature Auditory Brainstem Neurons

描述(申请人提供)：在哺乳动物听觉脑干中计算声源定位的电路中，握持的神经末梢的大花瓣是一个关键元件。从这个突触输出的动作电位的精确时间被认为是 这项任务。然而，在成熟动物中，由于很难记录和可视化大量髓鞘的成年脑干中的神经元，因此在高频放电过程中调节和保存动作电位时序的突触机制还不是很清楚。我们的实验室率先在小鼠发育的更成熟和成体阶段的脑干切片上进行了膜片钳录音，当它们完全获得听到和定位声音的微调能力时。第一个要检验的假说是，在保持的突触的成熟花瓣中观察到的自发微小兴奋性突触后电流(MEPSCs)的大幅度是由于高度同步的多量子释放形式。我们认为，这些大的和多量子的mEPSC是随着突触的成熟，钙通道与停靠的小泡之间的耦合逐渐紧密的自然结果。我们将以3-5纳米的分辨率重建成熟花萼的活动区，并将这一信息与我们在单个囊泡胞吐水平上的量化分析相关联。因此，对听觉突触结构和功能关系的新见解将被揭示。需要检验的第二个假设是，在更成熟突触的刺激序列中，由突触前钙储存库的开放触发的对储备池中的囊泡的快速依赖的招募，会产生一过性的EPSC增强，或者说是晚期强直性反弹。我们将通过使用药理学方法来验证这一假设，这些方法要么阻止钙浓度的增加，要么阻止钙储存释放钙。有待检验的第三个假设是，突触后MNTB主细胞树突在缩短兴奋性突触后电位(EPSP)衰减中起主要作用。我们假设，诱发的EPSP的这种“树突速度”提高了高频刺激传入纤维触发的突触后棘波的精确度。最后，为了研究成体样MNTB主细胞及其树突的被动和主动特性，我们将使用膜片钳和钙离子成像技术。这将使我们能够建立真实的计算机模型，说明MNTB细胞如何整合突触输入和激发动作电位，以保持传入声音信号的时序。

DESCRIPTION (provided by applicant): The large calyx of Held nerve terminal is a pivotal element in the circuitry that computes sound source localization in the mammalian auditory brainstem. Precise timing of action potential output from this synapse is thought to be central for this task. However, the synaptic mechanisms that modulate and preserve action potential timing during high frequency firing are not well understood in mature animals because of the difficultly of recording and visualizing neurons in the heavily myelinated adult brainstem. Our lab has pioneered patch clamp recordings in brainstem slices from more mature and adult-like stages of mouse development, when they fully acquire their fine-tuned ability to hear and localize sound. The first hypothesis to be tested is that the large amplitude of spontaneous miniature excitatory postsynaptic currents (mEPSCs) observed in mature calyx of Held synapses is due to a highly synchronous form of multiquantal release. We suggest that these large and multiquantal mEPSCs are a natural consequence of the progressively tighter coupling of Ca2+ channels to docked vesicles as the synapse matures. We will reconstruct the active zones of mature calyces at a resolution of 3-5 nm and correlate this information with our quantal analysis at the single vesicle exocytosis level. Novel insights into the structure and function relationship of auditory synapses will thus be revealed. The second hypothesis to be tested is that a fast Ca2+-dependent recruitment of vesicles from a reserve pool, triggered by the opening of presynaptic Ca2+ stores, produces a transient EPSC enhancement, or a late-tetanic rebound, during a stimulus train in more mature synapses. We will test this hypothesis by using pharmacological approaches that either block increases in Ca2+ concentration or block the Ca2+ stores from releasing Ca2+. The third hypothesis to be tested is that postsynaptic MNTB principal cell dendrites play a major role in shortening the excitatory postsynaptic potential (EPSP) decay. We hypothesize that this "dendritic speeding" of the evoked EPSP enhances the precision of postsynaptic spikes triggered by afferent fiber stimulation at high frequencies. Finally, to study the passive and active properties of the adult-like MNTB principal cells and their dendrites we will use patch-clamp and Ca2+ imaging techniques. This will allow us to build realistic computer models of how the MNTB cell integrates synaptic inputs and fires action potentials to preserve the timing of incoming sound signals.