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

 描述(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神经纤维(ANF)肿胀、回缩和延迟退化的触发因素；然而，对其潜在机制知之甚少。这种兴奋性毒性似乎涉及谷氨酸受体，而其他系统的研究表明，突触后细胞内钙离子在介导导致慢性神经变性的兴奋性毒性中起着至关重要的作用。突触后钙离子也可介导动态平衡可塑性。目前尚不清楚钙信号是否或如何将兴奋性毒性与ANF的神经退行性变或保护联系起来。尽管所有ANF终末都表达谷氨酸受体，但它们对噪声诱导的突触病变和变性的敏感性不同。谷氨酸受体亚基在内耳中的作用值得关注，因为谷氨酸通过受体诱导的钙内流依赖于亚基的组成。该项目包括对耳蜗中依赖活动的突触可塑性的研究，因为我们的长期目标是识别突触损伤和修复的机制，这些机制可以在神经退化开始之前进行操作来防止或快速逆转损伤。我们已经证明，ANF末端在其AMPA型谷氨酸受体亚单位的补体方面彼此不同。我们假设，ANF终末谷氨酸受体亚单位表达的异质性是噪声诱导损伤易感性的关键决定因素。因此，我们正在研究噪声激活的亚基组成的变化。我们正在将受体亚单位的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，以比较内毛细胞在纵轴和纵轴上的位置。我们正在使用转基因小鼠来操纵谷氨酸能活动。我们以前使用超分辨率STED显微镜在2D中测量50 nm分辨率的突触结构。现在，我们首次在科尔蒂机构实施了20纳米分辨率的3D超分辨率风暴显微镜。我们现在能够用针对GluA2、GluA3和GluA4的亚基特异性抗体来测量AMPA受体亚基在突触内的组织。解剖测量将与功能记录相辅相成。在先前使用膜片钳技术的工作中，我们首次通过向ANF终端注入直流电流来测量ANF的兴奋性。在这里，射击行为的差异 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中实施了钙离子成像，这允许更少的侵入性，同时观察纤维上的活动。我们将使用钙离子成像来测试钙离子进入药理学的功能途径。了解ANF多样性是如何由谷氨酸受体亚单位和突触后钙离子形成的，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。

 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.