成年大鼠丘脑底核离体脑片的制备方法

A method for getting healthy cells of subthalamic nucleus from brain slice of adult rat

目的建立一种制备健康成年Sprague-Dawley(SD)大鼠丘脑底核(subthalamic nucleus,STN)离体脑片的方法。方法选取成年SD大鼠,体重280~450g,给予大鼠4℃氯化胆碱切片液主动脉灌流后取脑,取脑时间尽量控制在30s内,振动切片机制备含有STN的冠状脑片。同时采用其他保护脑片细胞活性的措施,如人工脑脊液中添加抗氧化剂,脑片制备全程注意保持低温冰浴状态等。利用碘化丙啶(propidium iodide,PI)染色法观察神经元活性。通过红外微分干涉相差显微镜(IR-DIC)观察脑片的神经元形态。全细胞膜片钳记录大鼠STN神经元的电生理学指标。结果 40倍镜下显示,大量STN神经元轮廓清晰、表面光滑、饱满、有立体感,PI染色为阴性。进行全细胞膜片钳记录时,封接顺利,获得的STN神经元电生理指标如下:静息膜电位为(-46.4±1.26)mV,膜电阻为(493.1±62.29)MΩ,有自发放电的神经元占记录细胞总数的43.8%(28/64),动作电位阈值为(-28.30±1.20)mV,动作电位幅度为(68.00±2.27)mV,动作电位半宽为(0.80±0.06)ms,基强度为(42.0±6.09)pA。结论本方法制备的成年SD大鼠STN离体脑片保证了STN神经元的活性,适用于诸如帕金森病等神经系统退行性疾病动物模型离体机制研究。

Objective To set up a protocol for preparing brain slices of subthalamic nucleus(STN)of adult rat.Methods Adult male or female Sprague-Dawley(SD)rats of 280-450 g in weight were arterially perfused by ice cold cutting solution,then quickly chopped into 300μm coronary brain slices containing STN.Sodium chloride was replaced by choline chloride in the cutting solution.The time of taking the brain was strictly controlled within 30 s.Antioxidants were added into incubation solution to keep the viability of the brain cells.The propidium iodide(PI)staining was used to detect the dead neurons.The neuronal morphology of brain slices were observed by using infrared differential interference phase contrast microscopy.Electrophysiological properties of STN neurons were recorded by whole-cell patch clamp.Results At high magnification,we found that the cell contour of STN neurons was clear.The surface was smooth,full,and stereo three-dimensional shape.The PI staining showed that these neurons were alive.The membrane potential amplitude of STN neurons was(-46.4±1.26)mV.Membrane resistance was(493.1±62.29)MΩ.In the total 64 recorded STN neurons,28 neurons showed spontaneous discharge.Action potential threshold was(-28.3±1.20)mV.Action potential amplitude was(68±2.27)mV.The half-width of action potential was(0.8±0.06)ms and hyperpolarization-activated cation current(I h)amplitude was(42.0±6.09)pA.Conclusions The adult rat brain slices of STN prepared by this method can maintain the viability of STN neurons,which is suitable for the study of the mechanism of degenerative diseases such as Parkinson s disease and other neurodegenerative diseases in the animal models.