Influence of peripheral and central protein loss on the wiring of auditory brainstem microcircuits

感官信息处理需要精确有序的拓扑神经网络。这种网络的发育布线是活动依赖性的，涉及突触细化，从而消除旺盛和加强剩余的投影。在哺乳动物听性脑干中，从斜方体内侧核（MNTB）到外侧上级橄榄（LSO）的甘氨酸能投射参与声音定位，是研究地形抑制投射成熟的理想模型。这项建议的目的是分析活动依赖的细化以及组织发生的一种新的方法，区分突触信号分子的功能在感觉器官（外周）和那些在大脑（中央）。通过这种方法，我们将克服以前研究的内在局限性，包括我们自己的研究，这些研究采用了系统性敲除小鼠（Cav1.3科斯，VIV1.3科斯），并且不能将观察到的损伤的来源精确定位到外周或CNS部位。在我们的方法中，外周和中枢蛋白质损失分别由耳铁蛋白科斯和脑干特异性Cav1.3Krox20 CKO例示。Otoferlin科斯缺乏从耳蜗内毛细胞释放的递质，因此剥夺了中枢听觉系统在外周产生的自发活动，而Cav1.3Krox20 CKO具有脑干特异性Ca 2+信号传导缺陷。我们假设，这两种KO类型显示受损的突触细化和组织发生，但缺陷是更微妙的Cav1.3Krox20 CKOs，因为在上升的听觉通路的开始的关键位置的Otoferlin。因此，我们提出，Otoferlin的损失比Cav1.3的现场损失具有更普遍和严重的影响。实验将包括体外MNTB神经元的电刺激和谷氨酸释放刺激，以确定MNTB输入到膜片钳LSO神经元上的强度和空间范围。定量分析将从机理上深入了解强化过程。还将评估听觉脑干核团的体积。在体内实验中，我们将分析外周蛋白损失对CNS中的尖峰活性的影响。我们将研究出生后第11天（听力开始）和第30 -50天（成年）的小鼠，这是迄今为止几乎没有解决的发育阶段。我们的研究将有助于阐明听觉脑干微回路的分子过程。通过将外周蛋白质丢失与中枢蛋白质丢失分开，我们也将阐明中枢听觉障碍的机制方面。

Sensory information processing requires precisely ordered topographic neural networks. Developmental wiring of such networks is activity-dependent and involves synaptic refinement, thereby eliminating exuberant and strengthening remaining projections. In the mammalian auditory brainstem, the glycinergic projection from the medial nucleus of the trapezoid body (MNTB) to the lateral superior olive (LSO), which is involved in sound localization, is a well-suited model to investigate the maturation of topographic inhibitory projections. The aim of this proposal is to analyze activity-dependent refinement as well as histogenesis by a novel approach that distinguishes between the function of synaptic signaling molecules in the sensory organ (peripheral) and those in the brain (central). By this means, we will overcome intrinsic limitations of previous studies, including our owns, which employed systemic knock-out mice (Cav1.3 KOs, Vglut3 KOs) and could not pinpoint the source of the observed impairments to the periphery or to CNS sites. In our approach, peripheral and central protein loss are exemplified by Otoferlin KOs and brainstem-specific Cav1.3Krox20 CKOs, respectively. Otoferlin KOs lack transmitter release from cochlear inner hair cells and thus deprive the central auditory system of spontaneous activity generated in the periphery, whereas Cav1.3Krox20 CKOs have brainstem-specific Ca2+-signaling defects. We hypothesize that both KO types show impaired synaptic refinement and histogenesis, yet defects are more subtle in Cav1.3Krox20 CKOs because of the pivotal position of Otoferlin at the beginning of the ascending auditory pathway. Thus, we propose that loss of Otoferlin has more general and severe effects than on-site loss of Cav1.3. Experiments will comprise electrical and glutamate-uncaging stimulation of MNTB neurons in vitro to determine the strength and spatial extent of MNTB inputs onto patch-clamped LSO neurons. Quantal analysis will give mechanistic insight into the strengthening process. Volume of auditory brainstem nuclei will also be assessed. In in vivo experiments, we will analyze the effect of peripheral protein loss on spiking activity in the CNS. We will investigate mice at postnatal day P11 (hearing onset) and P30-50 (adulthood), a developmental stage hardly tackled thus far. Our studies will help to elucidate the molecular processes governing the wiring of auditory brainstem microcircuits. By separating peripheral protein loss from central protein loss, we will also shed light on mechanistic aspects of central auditory disorders.