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

感觉信息处理需要精确有序的拓扑神经网络。这种网络的发育连接依赖于活动，涉及突触的精细化，从而消除了旺盛的神经并加强了剩余的投射。在哺乳动物听性脑干中，参与声音定位的斜方体内侧核(MNTB)到外侧上橄榄核(LSO)的甘氨酸能投射是研究地形图抑制投射成熟的理想模型。这项建议的目的是通过一种新的方法来分析活动依赖的精化以及组织发生，该方法区分了感觉器官(外周)和大脑(中央)中突触信号分子的功能。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，这些研究使用的是系统性基因敲除小鼠(Cav1.3 KO，Vlu3 KO)，并且无法准确地将观察到的损害的来源定位到周围或中枢神经系统部位。在我们的方法中，外周和中央的蛋白质丢失分别以Otoferlin KO和脑干特异性Cav1.3Krox20 CKO为例。Otoferlin CKO缺乏从耳蜗内毛细胞释放的递质，从而剥夺了中枢听觉系统在外周产生的自发活动，而Cav1.3Krox20 CKO具有脑干特异性的钙信号缺陷。我们推测，这两种类型的KO都显示出突触细化和组织发生受损，但Cav1.3Krox20 CKO的缺陷更为轻微，这是因为Otoferlin在上升听觉通路的开始处处于关键位置。因此，我们认为Otoferlin的损失比Cav1.3的现场损失具有更普遍和更严重的影响。实验将包括对体外MNTB神经元的电刺激和谷氨酸去除刺激，以确定MNTB输入到膜片钳LSO神经元的强度和空间范围。量化分析将使我们从机理上洞察强化过程。听觉脑干核团的体积也将被评估。在活体实验中，我们将分析外周蛋白丢失对中枢神经系统刺激性活动的影响。我们将在出生后第11天(听力开始)和P30-50(成年期)对小鼠进行研究，这是到目前为止还很难解决的发育阶段。我们的研究将有助于阐明控制听觉脑干微电路连接的分子过程。通过分离外周蛋白丢失和中枢蛋白丢失，我们还将阐明中枢性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.