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

描述(申请人提供)：在海马体和其他大脑区域，突触的可塑性使其能够根据过去的激活历史改变反应方式，这是细胞记忆的一种形式。突触的动态性质使其有可能通过在长时程增强(LTP)中持续增加突触的反应性或在长期抑郁(LTD)中降低突触的强度来改变情节和空间记忆的形成。当记忆形成时，特定的神经元兴奋模式会导致突触强度的长期变化，当重新激活时，这些模式就会被召回为记忆。在细胞水平上，LTP/LTD的诱导涉及多种谷氨酸受体亚型的激活，这些受体介导或调节局部神经元钙离子和其他第二信使的内流/释放。N-甲基-D-天冬氨酸受体(NMDAR)在LTP和LTD突触前和突触后形式的诱导中起关键作用，而突触前II类代谢性谷氨酸受体(MGluRII S)则参与LTD递质释放的诱导。NMDAR-LTD和LTP的突触前成分改变了神经递质释放的可能性，这一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的释放，或者两者由最终的共同途径介导。在囊泡融合过程中，SNARE蛋白共同作用，介导囊泡神经递质的释放。我推测，突触前终末mGluRII激活释放的G？与突触小体相关蛋白25kD(SNAP-25)的C末端结合，并与NMDAR激活的逆行信使一氧化氮的释放相结合，从而诱发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和突触前注入SNAP-25的C末端都阻断了突触前LTD的表达。通过测量LTD的大小，利用脑片现场和全细胞电生理记录技术，以及评估LTD对NMDAR和mGluRII拮抗剂的敏感性，我计划阐明每个受体是如何参与突触前LTD的诱导和表达的。在表达未成熟SNAP-25(SNAP-25a)的转基因小鼠的脑片中使用NMDAR和mGluRII拮抗剂，我建议验证这样的假设，即在年轻小鼠中表达的LTD的主要形式存在发育转变，这与SNAP-25a向SNAP-25b表达的转变相吻合。最后，利用双光子激光扫描显微镜技术，我将从不同的Schaffer侧支突触前释放部位的囊泡池中成像，以测试NMDAR和mGluRII激活的生化级联在诱导突触前LTD中的作用。通过确定激活的NMDAR和mGluRII之间的相互作用机制，以及SNAP-25，我希望进一步了解递质释放的长期变化如何有助于记忆，并揭示改善阿尔茨海默氏症和癫痫等突触可塑性改变的疾病的机制。

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.