Spontaneous activity in the developing cochlea

描述(申请人提供)：感觉系统发育中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感觉图的完善和稳定是重要的。在发育中的耳蜗，在听力开始之前，传入螺旋神经节神经元就会发生动作电位的爆发，这一活动已被追踪到内毛细胞(IHC)。尽管IHC能够在此期间产生钙离子动作电位，但启动这些事件所需的去极化刺激尚未确定。幼年啮齿动物耳蜗生毛细胞和邻近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成像进行可视化时，这种活动与组织的光学属性的变化一致，这表明可以非侵入性地监测这些事件。P2嘌呤能受体拮抗剂和缝隙连接抑制剂可阻断自发的电和光活动，提示ATP和缝隙连接/半通道参与了这些事件的启动。值得注意的是，在听力开始后，这种活动不再被观察到。在Corti发育器官中发现的自发的嘌呤能信号提出了许多新的问题，包括产生这种活动的机制，这种由ATP介导的信号在驱动传入放电中所起的作用，以及听力开始后这种活动消失的原因。我们假设，这些由ATP驱动的IHC的去极化负责启动发育听觉通路的活动。在维持适当的细胞-细胞相互作用的急性和培养的耳蜗中保存这种活动，为我们提供了一个前所未有的机会来理解导致这些强烈现象的机制。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。这些研究将评估特定的假设，即支持细胞内[Ca~(2+)]i的自发振荡同时触发内向电流和ATP的释放，从而去极化IHC。相关建议中概述的研究试图了解在发育中的耳蜗内支持细胞、毛细胞和传入树突中启动自发活动的机制。这种活动已被证明对脑干核团中靶神经元的存活、这些听觉神经元的生理特性以及这些区域的突触连接模式有深远的影响。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缝隙连接蛋白。由于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方面发挥作用，这里概述的研究可能有助于解释这些突变是如何导致耳聋的。此外，这些研究可能揭示一种机制，即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.