Modulation of Exocytosis and Excitability in Mature Auditory Brainstem Neurons

成熟听觉脑干神经元胞吐作用和兴奋性的调节

• 批准号: 10471772
• 负责人: HENRIQUE Prado VON GERSDORFF
• 金额: $ 59.91万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-12-01 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10471772
• 关键词: 3-Dimensional; Action Potentials; Adult; Age-Months; Auditory; Axon; Binaural; Biophysical Process; Brain Stem; Buffers; Cell Nucleus; Cells; Cochlear nucleus; Computer Models; Confocal Microscopy; Contralateral; Coupled; Coupling; Crowding; Data; Development; Diffusion; Docking; Ear; Egtazic Acid; Electrodes; Exhibits; Exocytosis; Fire - disasters; Frequencies; Future; Glycine; Goals; Hearing; Hearing problem; Heterogeneity; Image; Ions; Ipsilateral; Laser Microscopy; Light; Link; Location; Measures; Medial; Membrane; Modeling; Mus; Nerve; Neurons; Output; Pharmacology; Physiological; Potassium Channel; Presynaptic Terminals; Probability; Reporting; Research; Resolution; Slice; Sound Localization; Spike Potential; Structure; Synapses; Synaptic Transmission; Techniques; Testing; Time; Transgenic Mice; Vesicle; Visualization; auditory pathway; binaural hearing; biophysical analysis; biophysical model; biophysical properties; channel blockers; electron tomography; experimental study; hearing impairment; improved; in vivo; insight; lateral superior olive; mature animal; neuronal cell body; patch clamp; postnatal; postnatal development; postsynaptic; preservation; presynaptic; reconstruction; recruit; response; sound; sound frequency; synaptic depression; trapezoid body; treatment strategy

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.

项目概要 哺乳动物的听觉脑干包含专门的突触，可以保持精确的动作时间 潜在的尖峰。我们建议研究其中两个专门的突触：Held 突触的大花萼 梯形体内侧核（MNTB）和外侧小布顿型甘氨酸突触 上橄榄核 (LSO)，通过 MNTB 主神经元连接。长期目标是确定 计算电路中这两个关键突触的生物物理特性和结构/功能 高频声音的轨迹。我们将对小鼠脑干切片进行膜片钳记录 类似于成年的发育阶段，此时小鼠完全获得了聆听和定位声音的微调能力。 我们的初步数据表明，脑干突触的几个基本方面仅在出生后成熟 第 30 天。因此，我们建议研究类成人的突触延迟、突触强度和短期可塑性 听觉突触。第一个假设是 MNTB 中的类似成人的萼型神经末梢包含 具有多个对接囊泡的异质且拥挤的活性区（AZ），可在 囊泡胞吐作用。我们将使用高分辨率对 AZ 进行详细的超微结构重建 电子断层扫描（ET）。我们计划确定促进短期胞吐作用延迟的主要因素，例如 囊泡池尺寸大、AZ 拥挤、Ca2+ 离子扩散屏障和囊泡与 Ca2+ 通道紧密 耦合。第二个假设是 LSO 中甘氨酸释放的时间和强度在 由于释放概率和易于释放的池的大小的变化而导致的出生后发育 囊泡。我们首次报道 LSO 神经元的抑制性突触后电流先于 反映突触前动作电位同步到达多个突触的预尖峰波形 纽扣。这使我们第一次能够量化甘氨酸听觉突触的突触延迟。我们 还将检验以下假设：尖峰诱发的甘氨酸释放的时间精度依赖于大 多量子胞吐作用。第三个要检验的假设是，在出生后发育过程中，LSO 甘氨酸突触获得了强大的 Ca2+ 依赖性囊泡招募机制。持续稳定的 甘氨酸的状态释放到 LSO 神经元上，从而有效地阻止了它们响应 兴奋性输入。我们的初步 LSO 数据显示，与 Held 的花萼形成鲜明对比的是，成熟的 甘氨酸能 LSO 突触减少了囊泡池的大小并增加了释放概率。使用共焦 显微镜和基因编码的 Ca2+ 指示剂，我们将在甘氨酸能 boutons 处对 Ca2+ 流入进行成像，并用于 首次使用 ET 描述其高分辨率的 3D 超微结构。我们与我们的合作者一起 将使用体内记录和计算机进一步验证和研究我们结果的生理相关性 造型。拟议的实验将利用成熟的 LSO 突触结构/功能开展新的研究 LSO 布顿型神经末梢的电子断层扫描、膜片钳记录和直接 Ca2+ 成像。