Glutamate Receptor Trafficking in Visual Development

视觉发育中的谷氨酸受体贩运

• 批准号: 6706969
• 负责人: Martha Na Constantine-Paton
• 金额: $ 16.3万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-03-01 至 2006-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6706969
• 关键词: NMDA receptors; confocal scanning microscopy; developmental neurobiology; electrophysiology; eye movements; genetically modified animals; glutamate receptor; immunocytochemistry; laboratory mouse; myosins; neural plasticity; protein structure function; retinal ganglion; superior colliculus; synapses; synaptogenesis; visual cortex; visual feedback; visual fields; visual pathways; visual stimulus; voltage /patch clamp; western blottings

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.

描述（由申请人提供）：谷氨酸能神经传递的异常活动介导的发育调节与弱视、立体视觉破坏和视网膜功能变化有关。 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使命的核心。 数据表明，肌球蛋白 VA (myo VA) 移动肌动蛋白上的膜囊泡，并将谷氨酸受体 (GR) 传递至突触。 我们最近的数据表明，这一过程在睁眼后受到光驱动的突触活动的快速调节。 我们希望建立小鼠突变品系flailer作为突触发生中肌VA机制研究的模型，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的模型。 Flailer 小鼠表达含有启动子和脑特异性 G 蛋白的前两个外显子以及 myo VA C 末端的融合基因（Jones 等人，2000）。鞭打蛋白似乎在中枢神经系统中充当显性失活肌 VA。 鞭打小鼠具有与 myo VA 无效突变体类似的神经异常，但与无效小鼠不同的是，纯合鞭打小鼠能够正常生存和繁殖。 尚未在突触水平上探索由连枷突变引起的功能变化。 我们建议确定 (l) 纯合连枷小鼠的视网膜、浅表视觉、上丘层 (sSC) 和视皮层 (VC) 中是否存在连枷蛋白和正常肌 VA，以及 (II) 连枷小鼠是否在睁眼 6 小时内表现出 PSD-95/GKAP NR 支架复合物到视觉突触的活动依赖性转运。我们将使用区分正常和颤抖肌 VA、PSD-95、GKAP-130 和 GKAP-95 的抗体对这些区域的匀浆以及富含 VC 和 sSC 树突状或全裂解物蛋白的级分进行定量免疫印迹。 如果视网膜中存在 flailer 蛋白，我们将（III）确定 flailer 和 WT 视网膜在睁眼后 PSD-95 的分布以及神经节细胞对光的反应方面是否相似。 视网膜功能将由我们的合作者耶鲁大学的 Niang Tian 博士进行分析。 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）我们将使用 sSC 切片制剂中谷氨酸受体电流的全细胞电压记录来确定，连枷小鼠大脑中的中央视觉通路谷氨酸受体功能正常。