Developmental Regulation of Glutamate Receptor Function

谷氨酸受体功能的发育调节

• 批准号: 6681801
• 负责人: Martha Na Constantine-Paton
• 金额: $ 36万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-08-08 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6681801
• 关键词: NMDA receptors; biological signal transduction; developmental neurobiology; electrophysiology; genetically modified animals; immunoprecipitation; laboratory mouse; laboratory rat; nerve /myelin protein; neural plasticity; superior colliculus; synapses; visual pathways; voltage /patch clamp

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.

描述(申请人提供)：本方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丧失以及在创伤或中风后恢复视力至关重要。视觉系统突触的可塑性涉及谷氨酸受体(NR)的NMDA亚型，该亚型由也与其他谷氨酸受体相互作用的蛋白质构成。最近的一个假说是，两种不同的蛋白质复合体，由具有不同亚基组成的NRs、NRscaffolding Proteins(MAGUK)和信号蛋白组成，根据视觉系统的活动而被不同的交通运输，并在发育中的突触介导不同的谷氨酸诱导的变化：早期系统携带富含NR2B亚基的NRs以及相关的运输和信号分子；为了响应光和睁眼(EO)，该系统被具有不同运输系统的支架复合体所取代，该支架复合体携带富含NR2A的NRs。为了验证这一假设，拟议的实验重点放在上丘的表层视觉上，这使得可以对谷氨酸受体功能和突触细化的变化进行电生理研究，以及对可能导致这些生理变化的突触蛋白复合体的变化进行生化研究。具体目的I是利用免疫共沉淀实验来确定假设的受体-Maguk运输复合体在被提议作用于突触竞争和精炼时是否在体内存在。具体目标二是确定对EO的特定反应是否与PSD-95的高树突状水平密切相关。PSD-95是一种Maguk蛋白，它作为成熟的NR复合体的支架，在EO的作用下移位到突触区域。解剖学研究将确定EO诱导的新电流是否是树突萌发的结果，树突萌发与PSD-95结合蛋白有关。全细胞膜片钳技术将通过询问一旦PSD-95在重新闭眼后降至EO前的水平，是否会失去精化，以测试树突PSD-95与精细化之间的紧密相关性。具体目的III是使用NR2A基因敲除和PSD-95基因敲除小鼠来尝试直接测试PSD-95/NR复合体对突触细化至关重要的假设，并尝试谷氨酸化EO反应中发生的电流变化。具体目的是构建一个NR2B/NR2ANR亚基，以确定PSD-95的生物学作用在多大程度上取决于它在突触定位特定NRs的能力，而不是它刺激PSD-95介导的信号转导的能力。