新生大鼠海马神经元的急性分离与膜片钳技术

Rapid dissociation of hippocampal neurons from neonate rats by the whole-cell patch-clamp technique

目的:获得适用于膜片钳技术的单个海马神经元。方法:实验于2005-09/11在新乡医学院机能学研究室完成。应用酶消化及机械分离法,急性分离出生3～6d的SD大鼠海马神经元,在倒置显微镜下,选取已贴壁直径约10～15μm的锥体形神经元观察其急性分离效果和细胞活性状态。电生理学的鉴定采用全细胞膜片钳的模式记录电流反应,首先在大鼠海马CA1区锥体神经元诱发出内向离子流。在大鼠海马CA1锥体神经元诱发出内向离子流后,通过程控灌流系统在细胞外液中加1μmol/L的钠通道阻断剂河豚毒素灌流同一神经元,记录内向离子流的变化。结果:①该法可获得形态良好的单个海马神经元,海马锥体细胞具有锥形胞体穴10～20μm雪和厚墩的顶树突特征。活性较好的海马神经元熏胞体呈三角形或椭圆形,表面光亮,细胞膜完整、清晰的,立体感较强。一般有1个较长的顶树突和2个以上较短的基树突。②利用膜片钳技术内向电流被诱发,用1μmol/L河豚毒素完全阻断该电流,说明该内向电流为钠电流。结论:该法可获得形态和生理特性良好的单个海马神经元,证实该方法适用于海马神经元膜片钳研究,对深入探讨药物和毒物对海马离子通道及信号转导机制的作用有重要价值。

AIM： To get the single hippocampal neurons fitting the study of whole-cell patch-clamp technique. METHODS： The experiment was carried out in the Physiological Laboratory of Xinxiang Medical College from September to November 2005. Single hippocampal neurons of SD rat （postnatal 3-6 days） were dissociated by enzymatic dispersion of collagenase and mechanical separation technique. With inverted microscope, the adhered pyramidal neurons （10-15 pan in diameter） were selected to observe their acute dissociated effects and cytoactivity. The channel currents were recorded by using whole-cell configuration of patch-clamp technique to make the electrophysiological verification. Firstly, the inward ion currents were induced at the pyramidal neurons of hippocampus CA1 region in rats; Secondly, with the program control perfusion system, the same neuron was perfused with the extraeellular fluid adding sodium channel blocking agent tetrodotoxin （TTX, 1 μmol/L）, and the change of inward ion current was recorded. RESULTS： ① The single hippocampal neurons could be got by rapid dissociation. Hippocampal pyramidal cells presented the characters of conecell body （10-20 pan） and had ro-apical dendrite. Those hippocampal pyramidal cells with satisfactory cytoactivity were of the deltoid or elliptic cell body, vivid surface, complete and limpid cell envelope, as well as satisfactory stereo-feeling. Generally the cells included a long apical dendrite and over 2 shot radical dendrites. ② The inward currents were induced by whole-cell patch-clamp technique and could be hold-blocked with the 1 μmol/L TTX, which shown that the inward currents were sodium current. CONCLUSION： The single hippocampal neurons of good characteristics in both morphology and physiology can be obtained by rapid dissociation of hippocampal neurons from neonate rats fitting the study of whole-cell patch-clamp technique, which is an important way to study the effects of drugs and poisons on the ion channels of hippocampal neurons and the mechanism of signal transduction.