Modification of the procedure for dissociating rats' dorsal root ganglion cells used for patch clamp

Objective To modify the previous procedure for dissociating rats' dorsal root ganglion(DRG) cells used for whole-cell patch-clamp studies.Methods The rats' DRGs were obtained by microsurgical techniques,and then the DRG cells were acutely dissociated and acquired with optimized culture method including dual enzyme digestion and dual serum incubation.After a short-time of culture,the DRG neurons were observed under inverted microscope and those with normal morphologic characterstics were selected for the further studies.The basic electrophysiological membrane characteristics and sodium channel currents were recorded by whole-cell patch-clamp technique before and after the administration of TTX,respectively.Results The cultured DRG cells,which were acutely dissociated and acquired with optimized method,were morphologically intact with a round or oval shape and smooth clear cell membrane.The resting membrane potential was in a range of(55.22±4.39) mV.The slowly inactivated tetrodotoxin-resistant sodium currents were recorded after the administration of tetrodotoxin.The highest giga-seal successful rate(73.8%) with whole-cell patch-clamp was obtained in mid-diameter DRG neurons,which was significantly higher than that obtained from the small and large diameter neurons(P 0.05).Conclusions The optimized simple protocol,including dual enzyme digestion and dual serum incubation,can acquire morphologically intact rats' DRG neurons with a high separation efficiency.The dissociated mid-diameter DRG neurons under these proedures are suitable for patch clamp research.