ASPARTATE TRANSMISSION

描述(由申请人提供)： 天冬氨酸与某些兴奋性海马通路的突触小泡有关，并由海马片和突触小体以钙依赖的方式释放。初步的释放研究和从其他实验方法获得的信息使天冬氨酸释放和功能的神经肽样工作模型得以构建，其中包括积累到不同的囊泡群体中，囊泡与主要位于活动区外的质膜融合，以及NMDA受体的扩散激活。天冬氨酸，而不是谷氨酸，可能产生一些NMDA受体激活的全局或突触外效应(细胞凋亡，神经发生的调节，轴突/树突状细胞生长的调节)。拟议的释放研究将利用大鼠海马区突触体。将开发一种方法，通过电穿孔技术将多肽和蛋白质引入突触体内，而不会显著干扰谷氨酸/天冬氨酸的释放。然后，为了区分先前工作提出的天冬氨酸释放的可能机制，将使用电穿孔方法将复合素抑制肽、NSF抑制肽、肉毒毒素A轻链和肉毒毒素B轻链引入突触体内。器官型海马片培养的全细胞膜片钳记录将验证天冬氨酸释放在特定条件下在兴奋性突触传递中发挥作用的假设。这些研究将探讨天冬氨酸和谷氨酸在释放机制和受体作用上的几个明显差异，包括从Schaffer侧支-连合途径释放天冬氨酸而不是从海马苔藓纤维释放天冬氨酸、低糖条件下天冬氨酸释放增加、重复活动时天冬氨酸释放增加和谷氨酸释放减少、天冬氨酸释放对梭菌毒素不敏感、天冬氨酸释放对P/Q型钙通道阻滞剂的敏感性降低，以及天冬氨酸对NMDA而不是AMPA受体的兴奋活性。拟议的研究将评估天冬氨酸在大脑中作为信号分子的潜在作用。它们在一定程度上需要开发新的实验方法，并涉及一些失败的风险。出于这些原因，探索性/发展性赠款支助机制似乎是适当的。该项目最终将阐明发育和神经退化过程，包括与癫痫发作和低血糖相关的病理生理学和病理学。

DESCRIPTION (provided by applicant): Aspartate is associated with the synaptic vesicles of certain excitatory hippocampal pathways and is released by hippocampal slices and synaptosomes in a calcium-dependent manner. Preliminary release studies and information available from other experimental approaches allowed construction of a neuropeptide-like working model for aspartate release and function that includes accumulation into a distinct vesicle population, vesicle fusion with the plasma membrane predominantly outside the active zone, and diffuse activation of NMDA receptors. Aspartate, rather than glutamate, may produce some of the global or extrasynaptic effects of NMDA receptor activation (apoptosis, regulation of neurogenesis, and regulation of axonal/dendritic growth). The proposed release studies will utilize rat hippocampal synaptosomes. A method will be developed to introduce polypeptides and proteins into the synaptosomes by an electroporation technique that does not disrupt glutamate/aspartate release significantly. Then, to discriminate among the possible mechanisms of aspartate release suggested by previous work, the electroporation method will be used to introduce complexin inhibitory peptide, NSF inhibitory peptide, botulinum toxin A light chain, and botulinum toxin B light chain into the synaptosomes. Whole cell patch clamp recordings in organotypic hippocampal slice cultures will test the hypothesis that aspartate release plays a role in excitatory synaptic transmission under certain conditions. These studies will exploit several apparent differences between aspartate and glutamate in their release mechanisms and receptor actions, including release of aspartate from the Schaffer collateral-commissural pathway but not from the hippocampal mossy fibers, increased aspartate under low glucose conditions, increased aspartate release and reduced glutamate release during repetitive activity, insensitivity of aspartate release to Clostridial toxins, lower sensitivity of aspartate release to a blocker of P/Q-type calcium channels, and agonist activity of aspartate upon NMDA but not upon AMPA receptors. The proposed studies will evaluate a potential role for aspartate as a signaling molecule in the brain. They require, in part, the development of new experimental approaches and involve some risk of failure. For these reasons, the Exploratory/Developmental Grant mechanism of support seems appropriate. This project will eventually shed light on developmental and neurodegenerative processes, including the pathophysiology and pathology associated with epileptic seizures and hypoglycemia.