Hu Y, Mitchell K M, Albahadily F N, Michaelis E K, Wilson G S
Department of Chemistry, University of Kansas, Lawrence 66045.
Brain Res. 1994 Oct 3;659(1-2):117-25. doi: 10.1016/0006-8993(94)90870-2.
The in vivo measurement of the rapid changes in the extracellular concentrations of L-glutamic acid in the mammalian brain during normal neuronal activity or following excessive release due to episodes of anoxia or ischemia has not been possible to this date. Current techniques for the measurement of the release of endogenous glutamate into the extracellular space of the central nervous system are relatively slow and do not measure the actual concentration of free glutamate in the extracellular space. An enzyme-based electrode with rapid response times (about 1 s) and high degree of sensitivity (less than 2 microM) and selectivity for L-glutamic acid is described in this paper. This electrode has both L-glutamate and ascorbate oxidase immobilized on its surface. The latter enzyme removes almost completely any interferences produced by the high levels of extracellular ascorbate present in brain tissue. The response of the electrode to glutamate and other potentially interfering substances was fully characterized in vitro and its selectivity, sensitivity and rapidity in responding to a rise in extracellular glutamate concentrations was also demonstrated in vivo. Placement of the electrode in the dentate gyrus of the hippocampus led to the detection of both KCl-induced release of L-glutamic acid and the release induced by stimulation of the axons in the perforant pathway. The development of this selective, sensitive and rapidly responding glutamate sensor should make it now possible to measure the dynamic events associated with glutamate neurotransmission in the central nervous system.
迄今为止，要在正常神经元活动期间或因缺氧或缺血发作导致过量释放后，对哺乳动物大脑中L - 谷氨酸细胞外浓度的快速变化进行体内测量是不可能的。目前用于测量内源性谷氨酸释放到中枢神经系统细胞外空间的技术相对较慢，并且无法测量细胞外空间中游离谷氨酸的实际浓度。本文描述了一种基于酶的电极，其对L - 谷氨酸具有快速响应时间（约1秒）、高灵敏度（小于2微摩尔）和选择性。该电极表面固定有L - 谷氨酸和抗坏血酸氧化酶。后一种酶几乎完全消除了脑组织中高浓度细胞外抗坏血酸产生的任何干扰。在体外对电极对谷氨酸和其他潜在干扰物质的响应进行了全面表征，并且在体内也证明了其对细胞外谷氨酸浓度升高的选择性、灵敏度和快速响应性。将电极放置在海马体齿状回中能够检测到KCl诱导的L - 谷氨酸释放以及穿通通路中轴突刺激诱导的释放。这种选择性、灵敏且快速响应的谷氨酸传感器的开发现在应该能够测量与中枢神经系统中谷氨酸神经传递相关的动态事件。
