Internal perfusion of isolated CNS neurons and the analysis of neuronal receptor potential by concentration-clamp technique.

在急性分离的成年无脊椎动物和脊椎动物标本、心肌和平滑肌的外周神经元上进行了电生理学研究。用培养的胎鼠和小鼠细胞对单个中枢神经系统神经元进行了研究。其他研究涉及使用从幼年大鼠和成年豚鼠身上急性分离的海马锥体神经元。然而，以前的方法分离的神经元缺乏远端树突结构，胞体的原始形状丢失。因此，一种分离具有保存完好的形态结构的中枢神经系统神经元的技术尚未建立。在这里，我们成功地分离出了形态结构保存完好的中枢神经系统神经元。这些分离的神经元的生理功能(例如，电压依赖和药物门控的腔体)也被很好地保存下来。例如，幼年和成年大鼠海马区锥体细胞对所有兴奋性氨基酸(谷氨酸、海人酸、奎斯奎酸和NMDA)和抑制性氨基酸(甘氨酸、牛磺酸、GABA)都很敏感。加入阈值浓度的甘氨酸(10^~(-6)~(-6))可显著增强NMDA的反应。此外，分离的CNS神经元电压依赖性钠通道的离子选择性顺序为：Li&GT；Na&GT；肼&GT；甲双胍&GT；甲基胍和单甲胺。其排列顺序与心室细胞和鱿鱼巨轴突十分相似。

Electrophysiological studies have been performed on acutely isolated peripheral neurons from adult invertebrate and vertebrate preparations, heart muscles and smooth muscles. Investigations on single CNS neurons have been performed using cultured cells from fetal rats and mice. Other studies have involved the use of hippocampal pyramidal neurons dissociated acutely from both infant rats and adult guinea pogs. However, the neurons isolated by previous techniques lacked the distal dendric structures, and the original shape of soma was lost. Thus, a technique for isolating CNS neurons with well-preserved morphological structures has not been established. Here we have succeeded in isolating CNS neurons with a well-preserved morphological structure. The physiological functions (e.g., voltage-dependent and drug-gated chammels) in these isolated neurons were also well preserved. For example, isolated pyramidal cells from young and adult rat hippocampus were sensitive to all excitatory amino acids (glutamate, kainate, quisqualate and NMDA) and inhibitory amino acids (glycine, taurine, GABA). The NMDA response was dramatically potentiated by adding glycine at threshold concentration (10^<-6>M). In addition, the ionic selectivity of voltage-dependent Na channel of isolated CNS neurons was in the order of Li > Na > hydrazine > formamidine > guanidine > methylguanidine > monomethylamine. The order was quite similar to that in heart ventricle cell and squid giant axon.