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囊泡相关，并以钙依赖性方式由海马切片和突触体释放。初步释放研究和其他实验方法提供的信息允许构建天冬氨酸释放和功能的神经肽样工作模型，包括积累到一个不同的囊泡群体，囊泡融合与质膜主要在活性区外，和NMDA受体的扩散激活。天冬氨酸，而不是谷氨酸，可能会产生一些全球或突触外的NMDA受体激活的影响（细胞凋亡，神经发生的调节，轴突/树突生长的调节）。拟定的释放研究将使用大鼠海马突触体。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，该技术不会显著破坏谷氨酸/天冬氨酸的释放。然后，为了区分由先前工作提出的天冬氨酸释放的可能机制，电穿孔方法将用于将复合蛋白抑制肽、NSF抑制肽、肉毒杆菌毒素A轻链和肉毒杆菌毒素B轻链引入突触体中。全细胞膜片钳记录在器官型海马切片文化将测试的假设，天冬氨酸释放在兴奋性突触传递在一定条件下发挥作用。这些研究将利用天冬氨酸和谷氨酸在其释放机制和受体作用方面的几个明显差异，包括从Schaffer侧支-连合通路而不是从海马苔藓纤维释放天冬氨酸，在低葡萄糖条件下增加天冬氨酸，在重复活动期间增加天冬氨酸释放和减少谷氨酸释放，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较低，天冬氨酸对NMDA的激动剂活性，但对AMPA受体无激动剂活性。拟议的研究将评估天冬氨酸作为大脑中信号分子的潜在作用。它们在某种程度上需要开发新的实验方法，并有失败的风险。基于这些原因，探索/发展补助金的支助机制似乎是适当的。该项目最终将阐明发育和神经退行性过程，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.