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: 9974250
• 负责人: HENRIQUE Prado VON GERSDORFF
• 金额: $ 65.59万
• 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/9974250
• 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.

项目摘要 哺乳动物的听觉脑干包含专门的突触，它能保持动作的精确时间 潜在的尖峰我们建议研究其中的两种特殊的突触： 斜方体内侧核（MNTB）和外侧核小的终扣型甘氨酸能突触 上级橄榄（LSO），通过MNTB主神经元连接。长期目标是确定 这两个关键突触的生物物理特性和结构/功能，在电路中，计算 高频声音的位置。我们将进行膜片钳记录在小鼠脑干切片从更多 成年阶段的发展，当老鼠完全获得他们的微调能力听到和定位声音。 我们的初步数据显示，脑干突触的几个基本方面只在出生后才成熟 第30天因此，我们建议研究成人样神经元的突触延迟、突触强度和短时程可塑性 听觉突触第一种假设是MNTB中的成人样萼型神经末梢含有 具有多个对接囊泡的异质且拥挤的活性区（AZ）， 囊泡胞吐作用。我们将使用高分辨率的显微镜对AZ进行详细的超微结构重建。 电子断层成像（ET）。我们计划确定促进短胞吐延迟的主要因素，如 大的囊泡池尺寸、具有Ca 2+离子扩散屏障的拥挤的AZs和紧密的囊泡-Ca 2+通道 偶合器.第二个假设是LSO中甘氨酸释放的时间和强度在LSO中变化。 由于释放概率的变化和易于释放的药物库的大小， 囊泡我们首次报道了LSO神经元的抑制性突触后电流之前有一个 反映突触前动作电位同步到达多个突触的峰前波形 饰扣这使我们能够第一次量化甘氨酸能听觉突触的突触延迟。我们 还将测试的假设，即尖峰诱发甘氨酸释放的时间精度依赖于大 多量子胞吐作用第三个有待检验的假设是，在出生后的发育过程中， 甘氨酸能突触获得一个强大的钙依赖性囊泡募集机制。持续稳定的- 因此，甘氨酸在LSO神经元上的状态释放有效地阻断了它们响应于 兴奋性输入我们初步的LSO数据显示，令人惊讶的是，与Held的花萼相反， 甘氨酸能LSO突触减小它们的囊泡池大小并增加释放概率。使用共聚焦 显微镜和遗传编码的Ca 2+指示剂，我们将图像Ca 2+流入甘氨酸能终扣， 第一次用ET以高分辨率描述了它们的三维超微结构。与我们的合作伙伴一起， 将进一步验证和研究我们的结果的生理相关性，使用体内记录和计算机 建模拟议的实验将启动对成熟LSO突触结构/功能的新研究， 电子断层扫描，膜片钳记录，和直接的钙离子成像LSO bouton型神经末梢。