Role of Gprotein and SNAP-25 in NMDA vs mGluR presynaptic long-term plasticity

描述（由申请人提供）：在海马体和其他大脑区域中，突触的可塑性允许它根据其过去的激活历史（一种细胞记忆形式）改变其响应方式。突触的动态性质使得有可能通过持续增加突触的反应性（在长时程增强（LTP）中）或降低其强度（在长时程抑郁（LTD）中）来改变情景和空间记忆的形成。当记忆形成时，特定的神经元兴奋模式会产生突触强度的长期变化，这些模式在重新激活时会被回忆为记忆。在细胞水平，LTP/LTD的诱导涉及谷氨酸受体的多种亚型的激活，其介导或调节钙和其他第二信使的局部神经元内流/释放。N-甲基-D-天冬氨酸受体（NMDAR）对于LTP和LTD的突触前和突触后形式的诱导是至关重要的，而突触前II组代谢型谷氨酸受体（mGluRII）有助于递质释放的LTD的诱导。NMDAR-LTD和LTP的突触前成分改变神经递质释放的可能性，这种效应被认为与突触后神经元释放逆行信号有关。NMDAR和mGluRII激活完全不同的途径，各自导致LTD释放，或者两者由最终共同途径介导。 在囊泡融合过程中，SNARE蛋白共同介导囊泡神经递质的释放。我假设G？，通过mGluRII激活在突触前末端释放，结合突触体相关蛋白25 kD（SNAP-25）的C末端，并且这与NMDAR激活的逆行信使一氧化氮的释放结合以引起LTD。我们的实验室发现，从SNAP-25的C末端切割9个氨基酸的肉毒杆菌毒素A，和SNAP- 25的C末端的突触前输注，通过测量LTD的大小，在脑切片中使用场和全细胞电生理记录技术，并评估LTD对NMDAR和mGluRII拮抗剂的敏感性，我计划阐明每个受体如何有助于突触前LTD的诱导和表达。SNAP-25（SNAP-25 a），我建议测试的假设，有一个发育转变的主要形式的LTD在年轻的小鼠中表达，与SNAP-25 a的转变SNAP-25 b的表达相一致。最后，利用双光子激光扫描显微镜技术，我将从Schaffer侧支突触前释放位点的不同囊泡池中成像囊泡释放，以测试由NMDAR和mGluRIIs激活的生化级联在诱导突触前LTD中的作用。通过识别激活的NMDAR和mGluRIIs与SNAP-25之间的相互作用机制，我希望进一步了解递质释放的长期变化如何有助于记忆，并揭示改善疾病的机制，如阿尔茨海默氏症和癫痫，其中突触可塑性被改变。

DESCRIPTION (provided by applicant): In the hippocampus, and other brain areas, plasticity of a synapse allows it to change the way it responds based on its past history of activation, a form of cellular memory. The dynamic nature of a synapse makes it possible to alter episodic and spatial memory formation by persistently increasing responsiveness of synapses, in long-term potentiation (LTP), or decreasing their strength, in long-term depression (LTD). When memories form, particular patterns of neuronal excitation produce long-term changes in synaptic strengths, patterns that, when reactivated, are recalled as memories. At the cellular level, induction of LTP/LTD involve activation of multiple subtypes of glutamate receptors that mediate or regulate local neuronal influx/release of calcium and other second messengers. The N-Methyl-D-aspartic acid receptor (NMDAR) is crucial for the induction of presynaptic and postsynaptic forms of both LTP and LTD, while presynaptic group II metabotropic glutamate receptors (mGluRIIs) contribute to induction of LTD of transmitter release. Presynaptic components of NMDAR-LTD and LTP alter probability of neurotransmitter release, an effect believed to be associated with release of a retrograde signal from the postsynaptic neuron. Both NMDAR and mGluRII activate completely different pathways that each result in LTD of release or the two are mediated by a final common pathway. During vesicular fusion, SNARE proteins work together to mediate vesicular neurotransmitter release. I hypothesize that G¿?, liberated by mGluRII activation in the presynaptic terminal, binds the C-terminus of the synaptosomal associated protein 25kD (SNAP-25), and that this combines with NMDAR-activated release of the retrograde messenger nitric oxide to evoke LTD. Our laboratory discovered that botulinum toxin A, which cleaves 9 amino acids from the C-terminus of SNAP-25, and presynaptic infusion of the C-terminus of SNAP- 25, each occlude the expression of presynaptic LTD. By measuring the magnitude of LTD, using field and whole cell electrophysiology recording techniques in brain slices, and evaluating the sensitivity of LTD to NMDAR and mGluRII antagonists, I plan to elucidate how each receptor contributes to induction and expression of presynaptic LTD. Using NMDAR and mGluRII antagonists in brain slices from transgenic mice expressing an immature isoform of SNAP-25 (SNAP-25a), I propose to test the hypothesis that there is a developmental shift in the dominant form of LTD expressed in young mice that coincides with the shift from SNAP-25a to SNAP-25b expression. Finally, with two-photon laser scanning microscopy techniques, I will image vesicular release from distinct vesicle pools of Schaffer collateral presynaptic release sites, to test the role of biochemical cascades activated by NMDAR and mGluRIIs in induction of presynaptic LTD. Through identifying the mechanisms of interaction between activated NMDARs and mGluRIIs, and SNAP-25, I hope to further understanding of how long-term changes in transmitter release contribute to memory, and uncover mechanisms to ameliorate diseases such as Alzheimer's and epilepsy, where synaptic plasticity is altered.