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

• 批准号: 9106802
• 负责人: Mark Allen Rutherford
• 金额: $ 46.3万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 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/9106802
• 关键词: AMPA Receptors; Acoustic Nerve; Action Potentials; Anatomy; Animals; Antibodies; Attention; 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; Shapes; Signal Transduction; Structure; Swelling; Synapses; Synaptic plasticity; System; Temporary Threshold Shift; Testing; Time; Transgenic Organisms; Work; base; cell type; density; excitotoxicity; hearing impairment; patch clamp; postsynaptic; presynaptic; prevent; public health relevance; receptor; repaired; research study; sample fixation; sound; synaptic function; transmission process

 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多地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，对潜在机制知之甚少。这种兴奋性毒性似乎涉及谷氨酸受体，并且在其他系统中的研究已经表明突触后细胞内Ca 2+在介导产生缓慢神经变性的兴奋性毒性中的关键作用。突触后Ca ~（2+）也可以介导稳态可塑性。目前尚不清楚Ca 2+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。虽然所有的ANF末梢都表达谷氨酸受体，但它们对噪声诱导的突触病和变性的易感性不同。谷氨酸受体亚单位在内耳中的作用值得关注，因为谷氨酸诱导的Ca 2+内流通过受体依赖于亚单位的组成。该项目包括对耳蜗中活动依赖性突触可塑性的研究，因为我们的长期目标是确定突触损伤和修复的机制，这些机制可以在神经退行性变发生之前进行操作以防止或快速逆转损伤。我们已经证明，ANF终端彼此不同的AMPA型谷氨酸受体亚单位的互补。我们推测，谷氨酸受体亚单位表达的异质性之间的ANF终端是一个重要的决定因素，对噪声引起的损害的易感性。因此，我们正在研究噪声激活的亚基组成的变化。我们正在比较受体亚基组成与突触前分子解剖学和重建内毛细胞（IHC）上的突触位置，以比较沿着轴轴和轴。我们用转基因老鼠来操纵神经元活动。我们以前采用超分辨率STED显微镜以50 nm的分辨率在2D中测量突触结构。我们现在实现，第一次在Corti器官，3D超分辨率风暴显微镜在20 nm的分辨率。我们现在能够测量突触内组织的AMPA受体亚单位与亚单位特异性抗体GluA 2，GluA 3，和GluA 4。解剖测量将辅以功能记录。在先前的工作与膜片钳技术，我们作出了第一次测量的心钠素兴奋性与直流注入到心钠素终端。在这里， 通过用染料填充记录的神经元，然后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中实施了Ca 2+成像，这允许更少的侵入性，同时观察跨纤维的活动。我们将使用Ca 2+成像来测试Ca 2+进入神经元的功能途径。了解谷氨酸受体亚单位和突触后Ca 2+如何塑造ANF多样性将为临床听力损失问题以及这种独特突触的基本机制提供新的视角。