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目的分析兴奋性神经递质谷氨酸在体外分离的小鼠皮层突触连接体的释放。方法解剖分离小鼠的大脑皮层,组织经匀浆后使用3层147μm的尼龙网筛进行过滤,离心后重悬沉淀为突触连接体。谷氨酸释放检测实验根据谷氨酸脱氢酶酶促反应原理进行,利用多功能读数仪读取产物烟酰胺腺嘌呤二核苷酸磷酸(NADPH)的荧光值。采用谷氨酸标准品绘制标准曲线,使用制备的突触连接体进行谷氨酸释放检测,进行3次独立重复实验。结果谷氨酸标准品浓度与荧光值具有良好的线性关系。新鲜制备的突触连接体在加入去极化剂KCl后,释放谷氨酸显著增加并逐渐达到平台期,加入Triton X-100裂解突触连接体后释放谷氨酸明显增加。冻融1次的突触连接体失去活性,与新鲜制备的突触连接体的谷氨酸释放曲线不同。结论对小鼠皮层突触连接体的提取方法进行了改良,检测和分析了兴奋性神经递质谷氨酸在分离制备的小鼠皮层突触连接体的释放。
Objective To analyze the release of excitatory neurotransmitter glutamate in mouse cortical synaptosomes isolated in vitro. Methods The cerebral cortex of mice was dissected. The tissue was homogenized and filtered through a 3-layer nylon screen of 147μm. After centrifugation, the pellet was resuspended to a synaptic linker. Glutamate release assay was performed according to the principle of glutamate dehydrogenase enzymatic reaction, and the fluorescence value of the product nicotinamide adenine dinucleotide phosphate (NADPH) was read by a multi-function readout meter. Standard curves were prepared using glutamate standards, glutamate release assays were performed using the prepared synaptic linkers, and 3 independent replicates were performed. Results There was a good linear relationship between glutamate standard concentration and fluorescence value. The freshly prepared synaptic linker significantly increased the release of glutamate and gradually reached the plateau after the depolarizing agent KCl was added. The release of glutamate was significantly increased after the addition of Triton X-100 to cleave the synaptic linker. Synaptic linkers that freeze-thawed once lost activity, unlike the glutamate release profiles of freshly prepared synaptic linkers. Conclusion The extraction method of mouse cortical synaptic linker has been improved and the release of excitatory neurotransmitter glutamate in isolated cortical synaptosomes has been detected and analyzed.