关键词: 视网膜神经元, 膜片钳, 电压门控钾离子通道

Abstract: Objective Ion channels are the basis of neuronal signal processing and transmission, and the investigation of the selectivity, kinetics and regulation of varies ion channels on membrane of neurons is of great importance for evaluating the excitability and function of the cells. The characterizations of voltage-activated potassium channels were studied on newborn bovine retinal neurons in long-term cultures with whole-cell patch clamp technique. Methods Neurons from newborn bovine retina were isolated and cultured in vitro. The cells which cultured for 2, 4 or 6 weeks were studied morphologically by phase contrast microscope. Recording pipettes were pulled from borosilicate glass on the two-stage puller. When filled with intracellular solution, the resistances ranged from 2～5 MΩ. The neurons were incubated with standard bathing solution before recording. All voltage-clamp recordings were performed with Axopatch-1D patch-clamp amplifier using tight-seal whole-cell recording configuration under room temperature. Holding potential in the experiment was -70 mV and the test potentials ranged from -190 mV to +90 mV with the increment of 10mV. Whole cell patch clamp recordings were made on 58 neurons from each group. Selective inhibitors of Na+ channels(TTX), Ca²⁺ channels (CaCl₂) and K⁺ channels (TEA, 4-AP, CsCl and BaCl₂) were used to identify the currents. The data were obtained by Clampex software. Current amplitudes and current-voltage relationships were measured and plotted using PClamp 8.0 and Origin 6.0 software. χ² test and one-way ANOVA were used for statistical analysis by SPSS 11.0. Results Cultured neurons isolated from newborn bovine retina could survive in vitro, and had distinct morphological characteristics from co-cultured neuronal glial cells. As the culture went on, the number and length of neurite outgrowth enhanced, indicating the maturity of the cells. With the inhibition of Ca²⁺ and Na⁺ currents by CaCl₂ and TTX, two types of voltage-gated potassium channels were observed. A sustained outward rectifying potassium current was recorded at the depolarized potentials, and the activating threshold was -40 mV. It was suppressed by inducing 30 mmol TEA in the bathing solution. Its kinetics and pharmacology were similar to a delayed rectifier potassium current(I_K) observed in retinal neurons of other species. The average peak amplitude at +90 mV increased as the culture went on [2 weeks: (8.041±126.591) pA, 4 weeks: (549.760±67.673) pA, 6 weeks: (1 697.171±823.456) pA](P<0.05). There was no difference in the expression of I_K among the three groups, and the numbers of cells expressing I_K from each group are 44(2 weeks), 41(4 weeks), and 47(6 weeks) (n=58), respectively(P>0.05). Application of hyperpolarizing steps (-70 to -190 mV) exceeding 200 ms or longer in duration evoked an inward rectifier potassium current which could be partly inhibited by 10 mmol TEA, and be completely inhibited by 5 mmol CsCl or 0.5 mmol BaCl₂. It had a rectifier property as well. Such inward rectifier potassium current has electrophysiological properties identical to I_IR reported from retinal neurons of rat, monkey and human being previously. None of the cells cultured for 2 or 4 weeks expressed I_IR, and only 11 of the total 58 neurons cultured for 6 weeks exhibited such current as well. Conclusion The electrophysiological properties of cultured newborn bovine retinal neurons could be studied using whole-cell patch clamp technique. I_K is commonly expressed, and with the culture went on, the peak amplitude of which increased. Several long-term cultured neurons express an inwardly rectifier potassium current which is assumed to be specifically expressed in primate retinal neurons.

Key words: retinal neuron, whole cell patch clamp, voltage-gated potassium channel