Developmental Regulation of Glutamate Receptor Function

描述（由申请人提供）：本提案的长期目标是了解视觉系统可塑性的机制。这些知识对于预防弱视和立体视觉的丧失以及创伤或中风后视力的恢复可能是至关重要的。视觉系统突触可塑性涉及谷氨酸受体（NR）的NMDA亚型，其由也与其他谷氨酸受体相互作用的蛋白质支撑。最近的一个假说是，由具有不同亚基组成的NR、NR支架蛋白（MAGUKs）和信号蛋白组成的两种不同的蛋白复合物在响应视觉系统活动时被不同地运输，并介导发育中突触的不同谷氨酸诱导的变化：早期系统携带富含NR 2B亚基和相关运输和信号分子的NR;在对光和眼开放（EO）的反应中，该系统被具有不同运输系统并携带富含NR 2A的NR的支架复合体所取代。为了验证这一假设，拟议的实验集中在表面视觉层的上级丘，这使得谷氨酸受体功能和突触细化的变化，以及生物化学研究的变化，突触蛋白复合物，可能是这些生理变化的基础。具体目的我是使用免疫共沉淀实验，以确定是否假设的受体MAGUK贩运复合物存在于体内时，他们提出了在突触竞争和细化。具体目标II是确定对EO的特异性反应是否与PSD-95的高树枝状水平紧密相关。PSD-95是MAGUK蛋白，其支撑成熟NR复合物并响应于EO而易位至突触区域。解剖学研究将确定EO诱导的新电流是否是树突出芽的结果，树突出芽与PSD-95结合蛋白有关。全细胞膜片钳将测试树突状PSD-95和精细化之间的紧密相关性，通过询问一旦PSD-95在眼睛重新闭合后降低至EO前水平，精细化是否丧失。具体目标III是使用NR 2A基因敲除和PSD-95基因敲除小鼠来尝试直接测试PSD-95/NR复合物对突触细化和响应EO发生的谷氨酸电流变化至关重要的假设。具体目标IV是构建一个杂合NR 2B/NR 2A NR亚基，这将允许确定PSD-95的生物学作用在多大程度上取决于其在突触定位特定NR的能力，而不是其刺激PSD-95介导的信号转导的能力。

DESCRIPTION (provided by applicant): The long-term objective of this proposal is to understand mechanisms of visual system plasticity. Such knowledge may prove vital to the prevention of amblyopia and the loss of stereopsis as well as to the restoration of vision following trauma or stroke. Visual system synaptic plasticity involves the NMDA subtype of glutamate receptor (NR), which is scaffolded by proteins that also interact with other glutamate receptors. A recent hypothesis is that two distinct protein complexes, consisting of NRs with different subunit compositions, NRscaffolding proteins (MAGUKs), and signaling proteins, are trafficked differently in response to visual system activity and mediate distinct glutamate-induced changes at developing synapses: an early system carries NRs enriched in the NR2B subunit and associated trafficking and signaling molecules; in response to light and eye- opening (EO), this system is replaced by a scaffolding complex with a different trafficking system and carrying NR2A-enriched NRs. To test this hypothesis, the proposed experiments focus on the superficial visual layers of the superior colliculus, which allow electrophysiological studies of changes in glutamate receptor function and synaptic refinement as well as biochemical studies of changes in synaptic protein complexes that may underlie these physiological changes. Specific Aim I is to use coimmunoprecipitation experiments to determine if the postulated receptor-MAGUK-trafficking complexes exist in vivo when they are proposed to act in synaptic competition and refinement. Specific Aim II is to determine if specific responses to EO are tightly linked to high dendritic levels of PSD-95. PSD-95 is the MAGUK protein that scaffolds the mature NR complex and that translocates to synaptic regions in response to EO. Anatomical studies will determine if new currents induced by EO are a consequence of dendritic sprouting, which has been associated with PSD-95-bound proteins. Whole-cell patch-clamping will test for a tight correlation between dendritic PSD-95 and refinement by asking if refinement is lost once PSD-95 is reduced to pre-EO levels after eye re-closure. Specific Aim III is to use NR2A knock-out and PSD-95 knockout mice to attempt to directly test the hypothesis that the PSD-95/NR complex is critical to synaptic refinement and to glutamate current changes that occur in response to EO. Specific Aim IV is to construct a hybrid NR2B/NR2A NR subunit that will allow a determination of the extent to which the biological roles of PSD-95 depend on its ability to localize specific NRs at the synapse as opposed to its ability to stimulate PSD-95-mediated signal transduction.