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。这些知识可能对预防弱视和立体视觉丧失以及创伤或中风后视力的恢复至关重要。视觉系统突触可塑性涉及谷氨酸受体（NR）的NMDA亚型，该亚型由与其他谷氨酸受体相互作用的蛋白质构成。最近的一项假设是，两种不同的蛋白质复合物，由具有不同亚基组成的nrrs组成，NRscaffolding蛋白（MAGUKs）和信号蛋白，在视觉系统活动中以不同的方式运输，并在发育中的突触中介导不同的谷氨酸诱导的变化：早期系统携带NR2B亚基丰富的nrrs以及相关的运输和信号分子；为了应对光线和开阔视野（EO），该系统被一个具有不同运输系统的脚手架复合物所取代，并携带富含nr2a的nr。为了验证这一假设，所提出的实验集中在上丘的表面视觉层，这允许对谷氨酸受体功能和突触精细的变化进行电生理研究，以及对突触蛋白复合物变化的生化研究，这些变化可能是这些生理变化的基础。具体目的1是使用共免疫沉淀实验来确定假设的受体- maguk转运复合物是否存在于体内，当它们被提议在突触竞争和细化中起作用时。特异性目的II是确定对EO的特异性反应是否与高树突水平的PSD-95紧密相关。PSD-95是支撑成熟NR复合物的MAGUK蛋白，并在EO反应中易位到突触区域。解剖研究将确定EO诱导的新电流是否是树突发芽的结果，树突发芽与psd -95结合蛋白有关。全细胞贴片夹紧将通过询问在复闭后PSD-95降低到eo前水平后是否会失去精细化来测试树突PSD-95与精细化之间的紧密相关性。具体目标III是使用NR2A敲除和PSD-95敲除小鼠，试图直接验证PSD-95/NR复合物对突触精细和EO响应中发生的谷氨酸电流变化至关重要的假设。具体目的IV是构建一个NR2B/NR2A NR亚基杂交，以确定PSD-95的生物学作用在多大程度上取决于其在突触中定位特定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.