Reinnervation of inner hair cells following excitotoxic trauma

描述（由申请人提供）：为了实验研究兴奋毒性损伤耳蜗的神经再生和突触发生，我们开发了一种器官型耳蜗外植体，其中Corti器官的一部分和相应的螺旋神经节部分被完整地切除，保持正常的形态和突触相互作用。用高水平谷氨酸激动剂短暂处理外植体可导致内毛细胞(IHC) - I型螺旋神经节神经元（SGN）突触的兴奋毒性变性，但不影响毛细胞或SGN的活力。突触再生，但恢复的神经支配是异常的：突触数量减少，单个SGN轴突接触多个ihc。在所有这些方面，体外模型模拟了在体内噪音或谷氨酸兴奋毒性损伤后观察到的情况。外源性神经营养因子- BDNF或NT-3 -显著改善恢复：ihc上的突触数量增加，突触数量增加，单个轴突对多个ihc的神经支配减少。在Aim 1中，我们定量比较了BDNF和NT-3在延长恢复期时促进再生的能力，并试图通过将其扩展到老年动物来改进我们的模型。我们在目标2-4中的核心实验集使用分子遗传学方法，包括转基因的使用

DESCRIPTION (provided by applicant): To experimentally investigate reinnervation and synaptogenesis in excitotoxically-damaged cochleae, we developed an organotypic cochlear explant in which a portion of the organ of Corti and corresponding portion of the spiral ganglion are removed intact, maintaining normal morphology and synaptic interactions. Briefly treating the explant with high levels of glutamate agonists results in excitotoxic degeneration of inner hair cell (IHC) - type I spiral ganglion neuron (SGN) synapses but does not affect hair cell or SGN viability. The synapses regenerate but the restored innervation is aberrant: the number of synapses is reduced, and individual SGN axons contact multiple IHCs. In all these respects, the in vitro model mimics what has been observed following noise or glutamatergic excitotoxic damage in vivo. Exogenous neurotrophins - BDNF or NT-3 - significantly improve recovery: the number of synapses on IHCs is increased, synapse number is increased, and innervation of multiple IHCs by single axons is reduced. In Aim 1, we quantitatively compare the ability of BDNF and NT-3 to promote regeneration with an extended recovery period and seek to improve our model by extending it to older animals. Our core set of experiments in Aims 2-4 use molecular genetic approaches, including the use of transgenic mice, to test specific hypotheses, suggested by our preliminary data, regarding the function of neurotrophins in recovery and reinnervation of IHCs after excitotoxic trauma. In Aim 2 we test whether NT-3, the endogenous neurotrophin, acts in a highly spatially restricted manner to maintain synapses on individual IHCs. We will delete NT-3 from a small number of IHCs or inhibit TrkC function in a small number of SGNs and quantitatively compare these with their unmodified neighbors. In Aim 3, we replace NT-3 with BDNF to test the hypothesis that NT-3 has a distinctive function in maintaining IHCSGN synapses and BDNF can't substitute. Finally, in Aim 4, we use p75NTR knockout mice to test the hypothesis that the neurotrophin receptor p75NTR promotes reinnervation after excitotoxic trauma. We will also assay post-trauma expression of p75NTR and putative ligands and test a specific mechanism: whether p75NTR promotes reinnervation by upregulating NT-3.