ASPARTATE TRANSMISSION

天门冬氨酸传输

• 批准号: 6818936
• 负责人: J. VICTOR NADLER
• 金额: $ 17.81万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-07-01 至 2006-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6818936
• 关键词: AMPA receptors; NMDA receptors; aspartate; biological signal transduction; brain cell; central nervous system disorders; developmental neurobiology; electrophysiology; electroporation; exocytosis; glutamates; high performance liquid chromatography; hippocampus; hypoglycemia; laboratory rat; method development; mossy fiber; neural transmission; neurotransmitter transport; organ culture; pathologic process; synaptosomes; voltage /patch clamp

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.

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相关，并由海马切片和突触体以钙依赖性方式释放。初步释放研究和其他实验方法提供的信息允许构建用于天冬氨酸释放和功能的类似神经肽的工作模型，包括积累到不同的囊泡群体中、囊泡与主要在活性区之外的质膜融合以及 NMDA 的扩散激活受体。天冬氨酸而不是谷氨酸可能会产生 NMDA 受体激活的一些全局或突触外效应（细胞凋亡、神经发生的调节和轴突/树突生长的调节）。拟议的释放研究将利用大鼠海马突触体。将开发一种通过电穿孔技术将多肽和蛋白质引入突触体的方法，该技术不会显着破坏谷氨酸/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络合蛋白抑制肽、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。器官型海马切片培养物中的全细胞膜片钳记录将检验天冬氨酸释放在某些条件下在兴奋性突触传递中发挥作用的假设。这些研究将利用天冬氨酸和谷氨酸在释放机制和受体作用方面的几个明显差异，包括从谢弗侧支连合途径释放天冬氨酸，但不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，增加天冬氨酸释放并减少天冬氨酸释放。重复活动期间谷氨酸释放，天冬氨酸释放对梭菌毒素不敏感，天冬氨酸释放对 P/Q 型钙阻滞剂的敏感性较低通道，以及天冬氨酸对 NMDA 而非 AMPA 受体的激动剂活性。拟议的研究将评估天冬氨酸作为大脑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，并涉及一定的失败风险。由于这些原因，探索性/发展性拨款支持机制似乎是合适的。该项目最终将揭示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。