RESUMO
BACKGROUND: The main difficulties of whole-cell patch clamP are cell culture and whole-call current recording. OBJECTIVE: To reduce the difficulty of patch-clamp experiment by combining acute cell isolation with current separation technique. METHODS: A total of 40 wistar rats aged 4-7 days, irrespective of genders, were selected. The pallium of wistar rats were cut into slices with 400-600 μm, rested for 1 hour in artificial cerebrospinal fluid with gas mixture, simultaneously put in oxygen. The brain tissues were placed into artificial cerebrospinal fluid containing 16 u/mL(type X) and 2 u/mL(type XIV) proteinase, incubated for 60 minutes, and cleared the digestive enzyme. Under the whole-cell voltage clamp mode, the potential was hold at -80mV, depolarizing pulse stimulation from -60 mV to 60 mV, 10 mV step and 160 ms width. The total Tran membranes current was recorded, and then 70 mmol/LCsCI, 70mmol/L CsF was 11 μmol/L Na~+ channel blockers tetrodotoxin, 30 mmol/L tetraethylammonium, 1 mmol/L 4-AP was successively added into extracellular fluid. The inward sodium current, transient outward potassium current and delayed rectifier potassium current was recorded, and then the result was analyzed using clampfit. MAIN OUTCOME MEASURES:①Cell morphological observation; ②Whole-ceil current recording;③Inward sodium currentrecording; ④Outward potassium current recording.RESULTS AND CONCLUSION: The cell had clearly three-dimensional structure, smooth surface and whole neurodendrite or neuraxon. The cell viability could maintained for 8-10 hours at 25 ℃ temperature. The added 11 tetrodotoxin in extracellular fluid could block sodium Current. The outward potassium current could be blocked by tetraethylammonium with 30 mmol/L and 4-AP with 1 mmol/L. The calls harvested by modified rapid dissociation have good functions. Using current separation method, only specific blockers are needed, without extracellular fluid or electrode solution replacing, which can record the inward sodium current, transient outward potassium current, as well as delayed rectifier potassium current. This method can obviously improve the work efficiency than traditional one.