Reinnervation of inner hair cells following excitotoxic trauma

描述（由申请人提供）：为了实验研究兴奋性毒性损伤耳蜗中的神经再支配和突触发生，我们开发了一种器官型耳蜗外植体，其中Corti器官的一部分和螺旋神经节的相应部分被完整地去除，保持正常的形态和突触相互作用。简单地说，用高水平的谷氨酸激动剂处理外植体导致内毛细胞（IHC）-I型螺旋神经节神经元（SGN）突触的兴奋性毒性变性，但不影响毛细胞或SGN活力。突触再生，但恢复的神经支配是异常的：突触的数量减少，单个SGN轴突接触多个IHC。在所有这些方面，体外模型模拟了体内噪声或神经元兴奋性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。在目标1中，我们定量比较了BDNF和NT-3促进再生的能力，并延长了恢复期，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中的核心实验组使用分子遗传学方法，包括使用转基因技术。 小鼠，以测试特定的假设，建议我们的初步数据，关于功能， 神经营养因子在兴奋性中毒性创伤后IHC恢复和神经再支配中的作用在目标2中，我们测试 NT-3是一种内源性神经营养因子，以高度空间限制的方式发挥作用，以维持单个IHC上的突触。我们将从少量IHC中删除NT-3或在少量IHC中抑制TrkC功能。 SGN的数量，并定量比较这些与它们的未修改的邻居。在目标3中， 用BDNF替代NT-3，以验证NT-3在维持IHCSGN突触中具有独特功能而BDNF不能替代的假设。最后，在目标4中，我们使用p75 NTR敲除小鼠来检验神经营养因子受体p75 NTR促进兴奋性毒性创伤后神经再支配的假设。我们还将检测创伤后p75 NTR和假定配体的表达，并测试一种特定的机制：p75 NTR是否通过上调NT-3促进神经再支配。

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.