Spontaneous activity in the developing cochlea

描述（由申请人提供）：已经证明，发育中的感觉系统的自发活动对于投射神经元的生长和存活以及大脑中感觉图的细化和稳定是重要的。在发育中的耳蜗中，在听力开始之前，传入螺旋神经节神经元中发生动作电位的爆发，这种活动已经追溯到内毛细胞（IHC）。虽然IHC能够在此期间产生Ca 2+动作电位，但启动这些事件所需的去极化刺激尚未确定。全细胞记录从IHC和支持细胞位于邻近的IHC在离体耳蜗从年轻的啮齿动物揭示了自发的内向电流，能够诱导大的去极化的存在。当使用IR/DIC成像可视化时，该活动与组织的光学性质的变化一致，表明可以非侵入性地监测这些事件。自发的电和光学活动被P2嘌呤受体拮抗剂和间隙连接抑制剂阻断，表明ATP和间隙连接/半通道参与启动这些事件。值得注意的是，这种活动在听力开始后不再观察到。在发育中的Corti器官中自发嘌呤能信号的发现提出了许多新的问题，这些问题涉及负责产生这种活动的机制，这种ATP介导的信号在驱动传入放电中的作用，以及听力发作后活动消失的原因。我们假设这些ATP驱动的去极化的IHC负责启动活动，在发展听觉通路。在维持适当细胞-细胞相互作用的急性耳蜗和培养耳蜗中保存这种活性为我们提供了前所未有的机会来了解这些强大现象的机制。我们建议使用IR/DIC和共聚焦荧光成像，光解，以及全细胞和细胞外记录，以调查支持细胞和毛细胞在发展中的Corti器官的自发活动的机制。这些研究将评估的具体假设，即自发振荡[Ca 2 +]i内支持细胞触发内向电流和释放ATP，去极化IHCs.Relevance-在这项建议中概述的研究旨在了解负责启动自发活动的机制，支持细胞，毛细胞，传入树突在发育中的耳蜗。这种活动已被证明对脑干核中靶神经元的存活、这些听觉神经元的生理特性以及这些区域中突触连接的模式具有深远的影响。大多数先天性耳聋是由连接蛋白26突变引起的，连接蛋白26是一种由耳蜗支持细胞高度表达的间隙连接蛋白。我们的初步结果表明，连接蛋白半通道可能在支持细胞释放ATP中发挥作用，这里概述的研究可能有助于解释这些突变如何导致耳聋。此外，这些研究可能揭示了一种机制，通过这种机制，在没有声音的情况下，可以在传入神经中诱导活动，这可能与耳鸣等人类状况直接相关。

DESCRIPTION (provided by applicant): Spontaneous activity in developing sensory systems has been shown to be important for the growth and survival of projection neurons as well as the refinement and stabilization of sensory maps in the brain. In the developing cochlea, bursts of action potentials occur in afferent spiral ganglion neurons prior to the onset of hearing, activity that has been traced to inner hair cells (IHCs). Although IHCs are capable of generating Ca2+ action potentials during this period, the depolarizing stimulus required to initiate these events has not been identified. Whole-cell recordings from IHCs and supporting cells located adjacent in IHCs in ex vivo cochleas from young rodents revealed the presence of spontaneous inward currents that were capable of inducing large depolarizations. This activity was coincident with changes in the optical properties of the tissue when visualized using IR/DIC imaging, indicating that these events can be monitored non- invasively. Spontaneous electrical and optical activity was blocked by P2 purinergic receptor antagonists and gap junction inhibitors, suggesting that ATP and gap junctions/hemichannels are involved in initiating these events. Remarkably, this activity is no longer observed after the onset of hearing. This discovery of spontaneous purinergic signaling in the developing organ of Corti raises many new questions about the mechanisms responsible for producing this activity, the role that this ATP-mediated signaling plays in driving afferent firing, and the cause of the disappearance of the activity after hearing onset. We hypothesize that these ATP driven depolarizations of IHCs are responsible for initiating activity in developing auditory pathways. The preservation of this activity in both acute and cultured cochleas in which appropriate cell- cell interactions are maintained provides us with an unprecedented opportunity to understand the mechanisms responsible for these robust phenomena. We propose to use IR/DIC and confocal fluorescence imaging, photolysis, and both whole cell and extracellular recording to investigate the mechanisms underlying spontaneous activity in supporting cells and hair cells in the developing organ of Corti. These studies will evaluate the specific hypothesis that spontaneous oscillations in [Ca2+]i within supporting cells triggers both inward currents and the release of ATP that depolarizes IHCs.Relevance The studies outlined in this proposal seek to understand the mechanisms responsible for initiating spontaneous activity in supporting cells, hair cells, and afferent dendrites in the developing cochlea. This activity has been shown to have a profound influence on survival of target neurons in brainstem nuclei, the physiological properties of these auditory neurons, and the pattern of synaptic connectivity in these regions. Most congenital forms of deafness result from mutations in connexin 26, a gap junction protein highly expressed by cochelar supporting cells. As our preliminary results suggest that connexin hemichannels may play a role in ATP release from supporting cells, the studies outlined here may help explain how these mutations lead to deafness. Furthermore, these studies may reveal one mechanism by which activity can be induced in afferent nerves in the absence of sound, which may have direct relevance to human conditions such as tinnitus.