Determinants of developing efferent synapses in the mammalian cochlea

描述(申请人提供)：在发育中的哺乳动物耳蜗中，内毛细胞(IHC)暂时和外毛细胞(OHC)永久地接受起源于听觉脑干的传出神经。这种胆碱能输入是由A9/A10烟碱型乙酰胆碱受体(NAChRs)介导的，并由于随后激活钙依赖的钾通道而受到抑制。在成熟的耳蜗体中，传出输入调节内耳毛细胞的活动，从而显著影响耳蜗的敏感度和频率选择性，很可能起到声音保护的作用。在发育过程中，IHC传出神经被认为影响听神经纤维的活动，从而可能影响听觉通路的成熟过程。为了使传出信息充分发挥作用，在发育过程中需要正确设置传出突触。我们认为，除了A9/A10nAChRs外，A1nAChR是在肌肉中发现的一个亚单位，在毛细胞中具有功能，可能在传出突触的形成和/或维持中发挥作用。这一假说是基于我们的发现，即除了A9/a10nAChRs介导的反应外，在发育中的小鼠IHC中，存在尼古丁介导的反应，该反应被A1 nAChR特异性阻滞剂抑制。在目标1中，将描述新发现的尼古丁介导的反应。由于A9/a10nAChRs不对尼古丁产生反应，这意味着存在其他nAChRs。在急性切除的小鼠耳蜗准备中，将从IHC进行膜片钳记录，以确定尼古丁敏感电流的表达时间线。我们将使用nAChR亚型选择性药理学药物来确定介导反应的亚基，并进一步检验A1 nAChR亚基是受体的一部分的假设。为了测试尼古丁敏感的nAChR是否参与传出突触传递，我们将测量传出突触电流是否部分通过该受体介导，并将使用特定的荧光毒素研究其亚细胞定位是否与传出突触后结构相一致。此外，尼古丁敏感反应的存在将在OHC中进行测试。在目标2中，我们将使用内耳缺乏A1 nAChR表达的小鼠模型，以测试其可能的功能。我们将测试在没有A1的情况下尼古丁介导的反应是否会丢失。如果是这样的话，我们将得出结论，A1是尼古丁敏感的AChR的关键元件。免疫组织化学和荧光毒素标记将被用来测试A1 nAChR的缺失是否与传出神经模式的变化有关。使用电生理学方法，我们将研究在没有A1的情况下，功能性传出突触是否正常发育。在突触维持中的潜在作用也将被研究。如果A1型肌肉nAChR在毛细胞传出突触形成/维持中的作用得到证实，这将为进一步破译潜在的分子机制提供重要的关键。对神经肌肉连接处突触形成和维持的广泛表征可以帮助我们理解这些过程在内耳中如何工作。

DESCRIPTION (provided by applicant): In the developing mammalian cochlea, the inner hair cells (IHCs) transiently and the outer hair cells (OHCs) permanently receive efferent innervation originating in the auditory brain stem. This cholinergic input is mediated by a9/a10nicotinic acetylcholine receptors (nAChRs) and is inhibitory due to the subsequent activation of calcium-dependent potassium channels. In the mature cochlea, efferent inputs modulate OHCs activity, thereby significantly influencing sensitivity and frequency selectivity of the cochlea, and most likely playing a role in sound protection. During development, IHC efferents are thought to affect auditory nerve fiber activity and thereby possibly influencing maturation processes in the auditory pathway. For efferent inputs to function adequately, efferent synapses need to be set up properly during development. Here we propose that in addition to a9/a10 nAChRs, the a1 nAChR, a subunit found in muscle, is functional in hair cells and may play a role in efferent synapse formation and/or maintenance. This hypothesis is based on our finding that in addition to the response mediated by a9/a10nAChRs, in developing mouse IHCs a nicotine-mediated response exists that is inhibited by a blocker specific for the a1 nAChR. In Aim 1 the newly found nicotine- mediated response will be characterized. Since a9/a10nAChRs do not respond to nicotine, this implies the existence of other nAChRs. In acutely excised mouse cochlear preparations, patch-clamp recordings will be performed from IHCs to determine the timeline of expression of the nicotinic-sensitive current. We will use nAChR subtype selective pharmacological agents to identify subunits mediating the response and to further test the hypothesis that the a1 nAChR subunit is part of the receptor. To test if the nicotine-sensitive nAChR is involved in efferent synaptic transmission, we will measure whether efferent synaptic currents are partially mediated through this receptor and we will study whether its subcellular localization coincides with efferent postsynaptic structures using specific fluorescent toxins. Additionally, the presence of a nicotinic-sensitive response will be tested in OHCs. In Aim 2, we will use a mouse model lacking a1 nAChR expression in the inner ear, to test for its possible function. We will test whether the nicotine-mediated response is lost in the absence of a1. If so, we will conclude that a1 is a key element of the nicotine-sensitive AChR. Immunohistochemistry and labeling with fluorescent toxin markers will be used to test if the lack of the a1 nAChR is associated with changes in efferent innervation pattern. Using electrophysiological measures, we will investigate whether functional efferent synapses develop normally in the absence of a1. A potential role in synapse maintenance will also be examined. If a role for the muscle type a1 nAChR is confirmed for hair cell efferent synapse formation/maintenance, this will provide an important key for further deciphering the under- lying molecular mechanisms. The extensive characterization of synapse formation and maintenance at the neuromuscular junction can then aid our understanding on how these processes might work in the inner ear.