Glutamate Receptor Trafficking in Visual Development

描述(申请人提供)：谷氨酸能神经传递的异常活动介导的发育调节与弱视、立体视障碍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对国家眼科研究所的任务至关重要。数据表明，肌球蛋白VA(MyoVA)移动肌动蛋白上的膜小泡，并将谷氨酸受体(GRs)传递到突触。我们最近的数据表明，这一过程迅速受到睁开眼睛后光驱动的突触活动的调节。我们希望建立小鼠突变株Flailer作为研究myo VA在突触发生中的机制以及NMDA受体及其成熟支架复合体、突触后密度蛋白PSD-95和GKAP-95的视觉活性依赖性运输的模型。Flailer小鼠表达包含大脑特异性G蛋白的启动子和前两个外显子以及myo VA的C末端的融合基因(Jones等人。2000)。Flailer蛋白似乎在中枢神经系统中扮演着显性负向肌VA的角色。Flailer小鼠的神经异常与myo VA缺失突变小鼠相似，但与空白小鼠不同，纯合子Flailer小鼠存活并正常繁殖。突触突变引起的功能变化还没有在突触水平上进行研究。我们建议确定(L)Flailer蛋白和正常myo VA是否存在于纯合子Flailer小鼠的视网膜、浅层视觉、上丘(SSc)和视觉皮质(VC)中，以及(Ii)Flailer小鼠是否在睁开眼睛后6小时内显示PSD-95/GKAP NR支架复合体活性依赖的运输到视觉突触。我们将对这些区域的匀浆以及来自VC和SSC的富含树突状或完整裂解蛋白的部分进行定量免疫印迹，使用区分正常和转折型Myo VA、PSD-95、GKAP-130和GKAP-95的抗体。如果Flailer蛋白存在于视网膜中，我们将(Ill)确定Flailer和WT视网膜在睁开眼睛后PSD-95的分布以及神经节细胞对光的反应是否相似。我们的合作者耶鲁大学的田娘博士将对视网膜功能进行分析。他将使用ERG、多电极阵列和全细胞膜片钳来研究视网膜神经节细胞对光的反应。我们将对睁眼前后进行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切片制备的谷氨酸受体电流的全细胞电压记录来确定中枢视觉通路谷氨酸受体功能在颤动小鼠脑中是正常的。

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.