膜片钳技术在成年大鼠海马脑片应用的初步研究

Preliminary study of application of patch clamp technique in adult rat hippocampal slices

摘要:

目的：制备成年大鼠海马脑片，使其适应于膜片钳全细胞记录，并观察海马锥体神经元的电生理特性。方法：选200 g左右的成年SD大鼠，雌雄不拘，10%水合氯醛腹腔注射麻醉，四肢固定，剪开胸腔，夹闭下腔静脉，以0 ℃切片液进行心脏灌注，断头，取下全脑，置入0 ℃切片液中冰镇3 min，再修块，切片，厚度约350 μm，移入37 ℃预热的人工脑脊液中孵育1 h后，转入23 ℃水浴中孵育30 min备用。倒置相差显微镜和红外微分干涉相差显微镜观察脑片的神经元形态；全细胞膜片钳记录脑片神经元的电生理学特性。结果：海马CA1区神经元光泽度好，立体感强且突起明显；在进行膜片钳全细胞记录时，封接顺利，并且可以记录到电流和动作电位的变化图形。结论：本方法制备的成年大鼠脑片，可以耐受离体后缺血缺氧造成的损伤，保持电生理活性而适应膜片钳全细胞记录模式。

Abstract:

Objective：To prepare adult rat hippocampus slices for whole-cell patch clamp recording and to observe the electrophysio－logical properties of hippocampus pyramidal neurons. Methods：Adult male or female SD rats of 200 g in weight were selected and were narcotized by 10% chloral hydrate intraperitoneal injection. The limb was fixed，chest was cut open and inferior vena cava was clamped in all the rats. After cardiac perfusion by 0 ℃ slices liquid，the whole brain was removed and placed in 0 ℃ slice liquid for 3 min，then chopped into 350 μm thickness. The brain slices were transferred to 37 ℃ preheated artificial cerebrospinal fluid for 1 h and then transferred to 23 ℃ for 30 min. The neuronal morphology of brain slices were observed by using inverted phase contrast microscope and infrared differential interference phase contrast microscopy. Electrophysiological properties of brain slices neurons were recorded by whole-cell patch clamp. Results：Good gloss，stereoscopic and obvious neurite pyramidal neurons were observed in hippocampal CA1 region，which were easy to sealing. With the application of patch clamp technique，the changes of the currents and action potential of pyramidal neurons in CA1 region were recorded by the whole-cell patch clamp. Conclusions：The adult rat brain slices prepared by this method can withstand the damage caused by ischemia and hypoxia in vitro，remain electrophysiological properties and adapt to the whole-cell patch clamp recording.