Glutamate Receptor Trafficking in Visual Development

描述（由申请人提供）：异常活动介导的谷氨酸能神经传递发育调节与弱视、立体视觉破坏和视网膜功能改变有关。因此，了解视觉发育过程中突触可塑性的分子基础是国家眼科研究所的核心任务。数据表明，肌凝蛋白VA （myo VA）移动肌动蛋白上的膜囊泡，并将谷氨酸受体（GRs）传递到突触。我们最近的数据表明，这一过程是由睁眼后的光驱动突触活动快速调节的。我们希望建立小鼠突变株flailer作为myo VA在突触发生中的机制研究模型，以及NMDA受体及其成熟支架复合物，突触后密度蛋白PSD-95和GKAP-95的视觉活动依赖性运输。Flailer小鼠表达一种融合基因，其中包含一种脑特异性G蛋白的启动子和前两个外显子，以及myo VA的c末端（Jones et al. 2000）。flailer蛋白似乎在中枢神经系统中起着显性负性myo VA的作用。Flailer小鼠具有与myo VA无突变体相似的神经异常，但与无突变体小鼠不同的是，纯合子Flailer小鼠存活并正常繁殖。翼片突变引起的功能变化尚未在突触水平上进行研究。我们打算确定(1)flailer蛋白和正常的myo VA是否存在于纯合子flailer小鼠的视网膜、浅表视觉、上丘层（sSC）和视觉皮层（VC）中，以及（II） flailer小鼠是否在睁眼后6小时内显示PSD-95/GKAP NR支架复合物到视觉突触的活动依赖性运输。我们将使用区分正常和flailer肌VA、PSD-95、GKAP-130和GKAP-95的抗体，对这些区域的匀浆和VC和sSC中富含树突状或全裂解蛋白的组分进行定量免疫印迹。如果flailer蛋白存在于视网膜中，我们将（ii）确定flailer和WT视网膜在睁眼后PSD-95的分布和神经节细胞对光的反应是否相似。视网膜功能将由我们的合作伙伴耶鲁大学的田娘博士进行分析。他将使用ERG、多电极阵列和全细胞贴片夹来研究视网膜神经节细胞对光的反应。我们将对睁眼前后经过PSD-95免疫细胞化学处理的视网膜进行定量共聚焦分析。最后，（IV）我们将利用sSC切片制备中谷氨酸受体电流的全细胞电压记录来确定，中央视觉通路谷氨酸受体功能在flailer小鼠大脑中是正常的。

DESCRIPTION (provided by applicant): Abnormal activity-mediated developmental regulation of glutamatergic neurotransmission is implicated in amblyopia, disruption of stereopsis and changes in retinal function. Thus, an understanding of molecular bases of synaptic plasticity during visual development is central to the mission of the National Eye Institute. Data suggest that myosin VA (myo VA) moves membrane vesicles on actin and delivers glutamate receptors (GRs) to synapses. Our recent data indicate that this process is rapidly regulated by light-driven synaptic activity following eye-opening. We hope to establish the mouse mutant strain flailer as a model for mechanistic studies of myo VA in synaptogenesis and for visual activity-dependent trafficking of the NMDA receptor and its mature scaffolding complex, the postsynaptic density proteins PSD-95 and GKAP-95. Flailer mice express a fusion gene containing the promoter and first two exons of a brain-specific G protein plus the C-terminal of myo VA (Jones et al. 2000). The flailer protein appears to act as a dominant-negative myo VA in the central nervous system. Flailer mice have neural abnormalities similar to those of myo VA null mutants, but unlike the null mice homozygous flailer mice survive and breed normally. Functional changes caused by the flailer mutation have not yet been explored at synaptic levels. We propose to determine (l) if flailer protein and normal myo VA are present in retina, superficial visual, layers of the superior colliculus (sSC), and visual cortex (VC) of homozygous flailer mice, and (II) if flailer mice show activity-dependent transport of the PSD-95/GKAP NR scaffolding complex to visual synapses within 6 hours of eye-opening. We will perform quantitative immunoblotting of homogenates of these regions and of fractions enriched for dendritic or whole-lysate protein from VC and sSC using antibodies that distinguish normal and flailer myo VA, PSD-95, GKAP-130 and GKAP-95. If flailer protein is present in retina, we will (Ill) determine whether flailer and WT retina are similar in the distribution of PSD-95 after eye-opening and in ganglion cell responses to light. Retinal function will be analyzed by our collaborator Dr. Niang Tian at Yale University. He will use ERG, mutielectrode arrays, and whole-cell patch clamping to study retinal ganglion cell responses to light. We will perform quantitative confocal analyses of retinas processed for PSD-95 immunocytochemistry before and after eye-opening. Finally, (IV) we will determine, central visual pathway glutamate receptor function is normal in flailer mouse brain using whole-cell voltage recordings of glutamate receptor currents in slice preparations of the sSC.