Spontaneous activity in the developing cochlea

描述（由申请人提供）：发展中的感觉系统的自发活动已被证明对投射神经元的生长和存活以及大脑中感觉地图的细化和稳定是重要的。在发育中的耳蜗中，传入螺旋神经节神经元在听力开始之前就会发生动作电位的爆发，这种活动可以追溯到内毛细胞（IHCs）。尽管ihc能够在此期间产生Ca2+动作电位，但尚未确定启动这些事件所需的去极化刺激。来自幼鼠离体耳蜗的ihc和邻近ihc的支持细胞的全细胞记录显示，存在自发的内向电流，能够诱导大的去极化。当使用IR/DIC成像时，这种活动与组织光学特性的变化一致，表明这些事件可以非侵入性地监测。自发的电和光学活性被P2嘌呤能受体拮抗剂和间隙连接抑制剂阻断，这表明ATP和间隙连接/半通道参与了这些事件的启动。值得注意的是，这种活动在听力开始后不再被观察到。在Corti发育器官中自发嘌呤能信号的发现提出了许多新的问题，包括产生这种活性的机制，这种atp介导的信号在驱动传入放电中的作用，以及听力发作后活性消失的原因。我们假设这些ATP驱动的ihc去极化负责启动听觉通路的活动。这种活性在急性耳蜗和人工耳蜗中得到保存，其中维持了适当的细胞-细胞相互作用，为我们提供了一个前所未有的机会来理解这些强大现象的机制。我们建议利用IR/DIC和共聚焦荧光成像、光解以及全细胞和细胞外记录来研究Corti发育器官中支持细胞和毛细胞自发活动的机制。这些研究将评估一个特定的假设，即支持细胞内[Ca2+]i的自发振荡触发内向电流和ATP的释放，从而使ihc去极化。本提案中概述的研究旨在了解在发育中的耳蜗中支持细胞、毛细胞和传入树突启动自发活动的机制。这种活动已被证明对脑干核中目标神经元的存活、这些听觉神经元的生理特性以及这些区域的突触连接模式具有深远的影响。大多数先天性耳聋是由连接蛋白26的突变引起的，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的ATP释放中发挥作用，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在没有声音的情况下传入神经活动被诱导的一种机制，这可能与耳鸣等人类疾病直接相关。

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.