Modulation of Exocytosis and Excitability in Mature Auditory Brainstem Neurons

项目总结 哺乳动物的听觉脑干含有特殊的突触，可以保持精确的动作时间。 潜在的尖峰。我们建议研究其中的两个特化突触：大的持有突触的花瓣 斜方体内侧核(MNTB)和外侧的小Bouton型甘氨酸能突触 上橄榄(LSO)，通过MNTB主神经元联系。长期目标是确定 这两个关键突触的生物物理性质和结构/功能在计算 高频声音的轨迹。我们将在小鼠脑干切片上进行膜片钳记录，这些切片来自 类似成人的发育阶段，当小鼠完全获得听到声音和定位声音的微调能力时。 我们的初步数据显示，脑干突触的几个基本方面只有在出生后才成熟 第30天。因此，我们建议研究类成体的突触延迟、突触强度和短期可塑性 听觉突触。第一种假设是，MNTB内类似成体的花帽型神经末梢含有 异质和拥挤的活动区(AZ)，具有多个停靠的小泡，在 囊泡胞吐。我们将使用高分辨率对AZs进行详细的超微结构重建 电子断层扫描(ET)。我们计划找出促进短期排泄延迟的主要因素，例如 大的囊泡池大小，拥挤的AZs具有钙离子的扩散障碍和紧密的囊泡到钙通道 耦合。第二种假说是，LSO中甘氨酸释放的时间和强度在 由于释放概率和可随时释放池的大小的变化而导致的出生后发育 水泡。我们首次报道了来自LSO神经元的抑制性突触后电流是由 反映多个突触的突触前动作电位同步到达的预搏波 关于这一点。这使得我们第一次量化了甘氨酸能听觉突触的突触延迟。我们 还将检验这一假设，即尖峰刺激甘氨酸释放的时间精度依赖于 多量子胞吐。需要检验的第三个假设是，在出生后的发育过程中，LSO 甘氨酸能突触获得一个强大的钙依赖的囊泡募集机制。一个持续的稳定- 因此，甘氨酸在LSO神经元上的状态释放有效地阻止了它们对 兴奋性输入。我们的LSO初步数据显示，与Hold的花瓣相比，令人惊讶的是，这种成熟 甘氨酸能LSO突触减小其囊泡池大小，增加释放几率。使用共焦 显微镜和遗传编码的钙指示物，我们将成像甘氨酸能结节的钙内流，对于 首次使用ET以高分辨率描述了它们的3D超微结构。与我们的合作者一起，我们 将使用活体记录和计算机进一步验证和研究我们的结果的生理学相关性 模特儿。拟议的实验将启动对成熟的LSO突触结构/功能的新研究 LSO-Bouton型神经末梢的电子断层扫描、膜片钳记录和直接钙成像。

PROJECT SUMMARY The mammalian auditory brainstem contains specialized synapses that preserve the precise timing of action potential spikes. We propose to study two of these specialized synapses: the large calyx of Held synapse in the medial nucleus of the trapezoid body (MNTB) and the small bouton-type glycinergic synapses of the lateral superior olive (LSO), that are linked through the MNTB principal neuron. The long-term goal is to determine the biophysical properties and structure/function of these two pivotal synapses in the circuitry that computes the locus of high frequency sounds. We will perform patch clamp recordings in mouse brainstem slices from more adult-like stages of development, when mice fully acquire their fine-tuned ability to hear and localize sound. Our preliminary data show that several fundamental aspects of brainstem synapses mature only at postnatal day 30. We thus propose to study the synaptic delays, synaptic strength and short-term plasticity of adult-like auditory synapses. The first hypothesis is that adult-like calyx-type nerve terminals in the MNTB contain heterogeneous and crowded active zones (AZs) with multiple docked vesicles that produce ultrashort delays in vesicle exocytosis. We will perform detailed ultrastructural reconstructions of the AZs using high-resolution electron tomography (ET). We plan to identify the major factors that promote short exocytosis delays, such as a large vesicle pool size, crowded AZs with diffusional barriers for Ca2+ ions and tight vesicle-to-Ca2+-channel coupling. The second hypothesis is that the timing and strength of glycine release in the LSO change during postnatal development due to shifts in release probability and the size of the readily releasable pool of vesicles. We report for the first time that inhibitory postsynaptic currents from LSO neurons are preceded by a prespike waveform that reflects the synchronous arrival of the presynaptic action potentials at multiple synaptic boutons. This allowed us to quantify for the first time the synaptic delay of a glycinergic auditory synapse. We will also test the hypothesis that the temporal precision of spike-evoked glycine release relies on large multiquantal exocytosis. The third hypothesis to be tested is that during postnatal development the LSO glycinergic synapse acquires a robust Ca2+-dependent vesicle recruitment mechanism. A sustained steady- state release of glycine onto the LSO neurons thus effectively blocks their ability to fire spikes in response of excitatory inputs. Our preliminary LSO data show, surprisingly in contrast to the calyx of Held, that maturing glycinergic LSO synapses decrease their vesicle pool size and increase release probability. Using confocal microscopy and genetically encoded Ca2+ indicators, we will image Ca2+ influx at glycinergic boutons, and for the first time describe, using ET, their 3D ultrastructure at high resolution. Together with our collaborators we will further validate and study the physiological relevance of our results using in vivo recordings and computer modeling. The proposed experiments will launch new studies on mature LSO synapse structure/function using electron tomography, patch clamp recordings, and direct Ca2+ imaging of LSO bouton-type nerve terminals.