谷氨酸在分离的小鼠皮层突触连接体的释放

Glutamate release from isolated synaptoneurosomes from the mouse cortex

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 detect the release of glutamate from the isolated synaptoneurosomes from the mouse cortex. Methods The mouse cortex was dissected, homogenized and filtered through three layers of 147 μm nylon meshes. The synaptoneurosomes were resuspended after centrifuged. An enzyme-linked fluorescence detection method was used to detect the glutamate release, which utilized glutamate dehydrogenase, and the reduction of nicotinamide adenine dinucleotide phosphate（NADP） to NADPH. The glutamate standard curve was set up and then the glutamate release from isolated synaptoneurosomes was tested and analyzed in three independent experiments. Results A good linear relationship was found between the glutamate concentrations and fluorescence values. The release of glutamate from synaptoneurosomes significantly increased after depolarization agent KC1 added and gradually reached a plateau. Glutamate release was increased more when Triton X-100 was added. The inactive synaptoneurosomes after one freeze-thaw cycle had a different curve of glutamate release. Conclusion The release of excitatory neurotransmitter glutamate from synaptoneurosomes of the mouse cortex isolated using the improved method is well studied.