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: 10672937
• 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/10672937
• 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; Experimental Models; Fire - disasters; Frequencies; Future; Glycine; Goals; Hearing; Hearing problem; Heterogeneity; Image; Invaded; Ions; Ipsilateral; Laser Microscopy; Light; Link; Location; Measures; Medial; Modeling; Mus; Nerve; Neurons; Output; Physiological; Potassium Channel; Presynaptic Terminals; Probability; Reporting; Research; Resolution; Slice; Sound Localization; Spike Potential; Structure; Synapses; Synaptic Membranes; 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; nano; patch clamp; pharmacologic; postnatal; postnatal development; postsynaptic; preservation; presynaptic; reconstruction; recruit; response; sound; sound frequency; synaptic depression; trapezoid body; treatment strategy; 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; Experimental Models; Fire - disasters; Frequencies; Future; Glycine; Goals; Hearing; Hearing problem; Heterogeneity; Image; Invaded; Ions; Ipsilateral; Laser Microscopy; Light; Link; Location; Measures; Medial; Modeling; Mus; Nerve; Neurons; Output; Physiological; Potassium Channel; Presynaptic Terminals; Probability; Reporting; Research; Resolution; Slice; Sound Localization; Spike Potential; Structure; Synapses; Synaptic Membranes; 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; nano; patch clamp; pharmacologic; 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，可保留精确的动作时间 潜在的尖峰。我们建议研究其中两个专门的突触：在 梯形体（MNTB）的内侧核和横向的小胸膜甘氨酸突触 通过MNTB主神经元连接的上橄榄（LSO）。长期目标是确定 电路中这两个关键突触的生物物理特性和结构/功能计算 高频声音的基因座。我们将从更多 当小鼠完全获得听力和本地化声音的微调能力时，成人的发展阶段。 我们的初步数据表明，脑干突触的几个基本方面仅在产后成熟 第30天。因此，我们建议研究成人样的突触延迟，突触强度和短期可塑性 听觉突触。第一个假设是MNTB中的成年花萼型神经末端包含 异质和拥挤的活动区（AZS），带有多个停靠囊泡，产生超平延迟 囊泡胞吐作用。我们将使用高分辨率进行AZS的详细超微结构重建 电子断层扫描（ET）。我们计划确定促进短胞吐延迟的主要因素，例如 大型囊泡池大小，拥挤的AZS，具有Ca2+离子的扩散屏障和紧密的囊泡至CA2+通道 耦合。第二个假设是LSO在LSO中释放的时机和强度变化 由于释放概率的变化和易于释放的池的大小而引起的产后发展 囊泡。我们首次报告说，LSO神经元的抑制性突触后电流先于 反映在多个突触处突触前作用电位同步到达的前波形 胸衣。这使我们能够首次量化甘油能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取决于大型 多功能胞吐作用。要检验的第三个假设是在产后发展期间 糖节功能突触获得了强大的Ca2+依赖性囊泡募集机制。持续的稳定 因此 兴奋性输入。我们的初步LSO数据显示，与Hold的花萼相反，该成熟 糖LSO突触降低了囊泡池的尺寸并增加了释放概率。使用共聚焦 显微镜和遗传编码的Ca2+指标，我们将在甘氨酸能量胸子上成像Ca2+涌入，对于 第一次使用ET描述其3D超微结构高分辨率。与我们的合作者一起 将进一步验证和研究结果使用体内记录和计算机的生理相关性 造型。提出的实验将使用有关成熟LSO突触结构/功能的新研究 LSO BOUTON型神经末端的电子断层扫描，贴片夹记录和直接CA2+成像。