EXCITATION AND EXCITOTOXICITY IN TYPE I COCHLEAR AFFERENTS: SYNAPTIC STRUCTURE AND FUNCTION

I 型耳蜗传入的兴奋和兴奋性毒性：突触结构和功能

基本信息

批准号：
9212800
负责人：
Mark Allen Rutherford
金额：
$ 40.62万
依托单位：
WASHINGTON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-03-01 至 2021-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9212800
关键词：
AMPA Receptors Acoustic Nerve Action Potentials Anatomy Animals Antibodies Attention Auditory Threshold Behavior Biological Assay Cessation of life Clinical Cochlea Complement Dependence Dyes Ear Electrophysiology (science)Exhibits Fiber Glutamate Receptor Glutamates Goals Hearing Heterogeneity Image Immunohistochemistry Individual Injection of therapeutic agent Inner Hair Cells Label Labyrinth Link Location Measurement Measures Mediating Microscopy Molecular Mus N-Methylaspartate Nerve Degeneration Nerve Fibers Neurons Noise Optics Organ of Corti Patch-Clamp Techniques Permeability Pharmacology Phenotype Positioning Attribute Predisposition Property Proteins Research Resolution Role Route Signal Transduction Structure Swelling Synapses Synaptic plasticity System Temporary Threshold Shift Testing Time Transgenic Organisms Work cell type density excitotoxicity experimental study hearing impairment patch clamp permissiveness postsynaptic presynaptic prevent public health relevance receptor repaired sample fixation sound synaptic function transmission process

项目摘要

DESCRIPTION (provided by applicant): Glutamate-induced excitotoxicity is increasingly recognized as the trigger for swelling, retraction, and delayed degeneration of auditory nerve fibers (ANFs) following moderate overexposure to sound; however, little is known about the underlying mechanisms. This excitotoxicity seems to involve glutamate receptors, and research in other systems has indicated the crucial role of postsynaptic intracellular Ca2+ in mediating the excitotoxicity that produces slow neurodegeneration. Postsynaptic Ca2+ can also mediate homeostatic plasticity. It is still unknown if or how Ca2+ signals link excitotoxicity to neurodegeneration or protection in ANFs. Although all ANF terminals express glutamate receptors, they differ in susceptibility to noise-induced synaptopathy and degeneration. The roles of glutamate receptor subunits in the inner ear deserve attention because glutamate-induced Ca2+ influx through receptors depends upon subunit composition. The project encompasses studies of activity-dependent synaptic plasticity in the cochlea because our long- term goal is to identify mechanisms of synaptic damage and repair that can be manipulated to prevent or rapidly reverse damage before the onset of neurodegeneration. We have already demonstrated that ANF terminals differ from each other in their complements of AMPA-type glutamate receptor subunits. We hypothesize that heterogeneity of glutamate receptor subunit expression among ANF terminals is a crucial determinant of susceptibility to noise-induced damage. Thus, we are studying noise-activated changes in subunit composition. We are comparing receptor subunit composition with presynaptic molecular anatomy and reconstructing synapse position on the inner hair cell (IHC) to compare along the modiolar-pillar and orthogonal axes. We are using genetically modified mice to manipulate glutamatergic activity. We previously employed superresolution STED microscopy to measure synaptic structures at 50 nm resolution in 2D. We now implement, for the first time in the organ of Corti, 3D superresolution STORM microscopy at 20 nm resolution. We are now able to measure the intrasynaptic organization of AMPA receptor subunits with subunit-specific antibodies to GluA2, GluA3, and GluA4. Anatomical measurements will be complemented with functional recordings. In prior work with the patch- clamp technique we made the first measurements of ANF excitability with direct current injection into ANF terminals. Here, differences in firing behavior will be compared with synaptic structure by filling recorded neurons with dye, followed by fixation and immunohistochemistry. We are implementing Ca2+ imaging in ANFs for the first time, which allows for less invasive, simultaneous observation of activity across fibers. We will use Ca2+ imaging to test for functional routes of Ca2+ entry pharmacologically. Understanding how ANF diversity is shaped by glutamate receptor subunits and postsynaptic Ca2+ will deliver new perspectives on questions of clinical hearing loss as well as the basic mechanisms underlying this unique synapse.

描述（由申请人提供）：谷氨酸诱导的兴奋性毒性越来越被认为是中度过度暴露于声音后听觉神经纤维（ANF）肿胀、回缩和延迟变性的触发因素；然而，人们对这种兴奋性毒性的潜在机制知之甚少。似乎涉及谷氨酸受体，其他系统的研究表明，突触后细胞内 Ca2+ 在介导产生缓慢的兴奋性毒性方面发挥着至关重要的作用。突触后 Ca2+ 还可以介导稳态可塑性，目前尚不清楚 Ca2+ 信号是否或如何将兴奋性毒性与 ANF 中的神经变性或保护联系起来。内耳中谷氨酸受体亚基的变化值得关注，因为谷氨酸诱导的 Ca2+ 通过受体的流入取决于该项目研究耳蜗中活动依赖性突触可塑性，因为我们的长期目标是确定突触损伤和修复的机制，以便在神经退行性疾病发生之前预防或快速逆转损伤。认为 ANF 末端谷氨酸受体亚基表达的异质性是 ANF 末端的关键决定因素因此，我们正在研究噪声激活的亚基组成变化，并将受体亚基组成与突触前分子解剖学进行比较，并重建内毛细胞 (IHC) 上的突触位置，以沿轴轴和轴进行比较。我们之前使用超分辨率 STED 显微镜以 50 nm 分辨率测量突触结构。 2D。我们现在首次在 Corti 器官中实现了 20 nm 分辨率的 3D 超分辨率 STORM 显微镜，我们现在能够使用 GluA2、GluA3 和 GluA4 的亚基特异性抗体来测量 AMPA 受体亚基的突触内组织。在之前的膜片钳技术工作中，我们首次使用直流电测量了 ANF 兴奋性。注入 ANF 终端的情况不同。我们将通过用染料填充记录的神经元，然后进行固定和免疫组织化学，将其与突触结构进行比较。我们首次在 ANF 中实施 Ca2+ 成像，这可以实现侵入性较小的同步观察纤维间的活动。测试 Ca2+ 进入药理学的功能途径。了解 ANF 多样性如何由谷氨酸受体亚基和突触后 Ca2+ 形成，将为临床听力损失以及听力损失问题提供新的视角。这种独特突触的基本机制。