可视脑片膜片钳技术前庭核团脑片的制备

The Preparation of Vestibular Nuclei Slices for Visual Patch Clamp Techniques

目的改良脑片制备方法，探讨影响脑片活性的实验因素。方法在国内外报道常用方法的基础上进行改良，制备含有前庭内侧核的脑干脑片，从肉眼观、镜下观和放电特性三个方面判断脑片活性，观察鼠龄、生理溶液、温度等因素对脑片活性的影响。结果建立了稳定的脑片制备方法。通过红外微分干涉相差技术可直接观察脑片表层下50～100μm神经元的立体影像，并可稳定地进行电生理记录；鼠龄越小神经元活性越易恢复，在成年鼠制备脑片时采取心脏灌注、使用高蔗糖溶液、较高温度预孵育等措施可提高脑片活性；孵育和记录温度直接影响神经元的兴奋性，一定范围内提高温度可提高神经元兴奋性，但会缩短恼片存活时间。结论改良的脑片制备方法简单可行，可为电生理研究提供大量具有良好活性的神经元，脑片制备方法的选择和实验环境的控制应根据所选用动物的年龄而调整。

Objective To approach a modified method of preparing brain slices, and to discuss the factors affecting the status of slices. Methods Brainstem slices with medial vestibular nuclei were made on the basis of reported methods, and the quality of slices was visually judged and via the light microscope observation and firing activities recordings. The effects of rats＇ age, physiological fluids and temperature upon slices health were observed. Resuits A simple and reliable preparation procedure of brain slices was established. The neurons were visualized by using infrared differential interference contrast technique and readily-- patched at a depth of 50 -- 100μm underneath the surface of slices. Brain slices were easy to obtain from juvenile animals, while performing intracardiac perfusion, using high--sucrose solution and pre--incubating slices at a warmer temperature were useful when prepared slices from adult rats. Within definite temperature rage, the excitability of neurons improved with the increasing of temperature during incubation and recording, while the survival time shortened. Conclusion Modified preparing methods could supply sufficient healthy neurons for electrophysiologic recordings. The preparation procedures and the controlling of physiological surroundings should be adjusted according to rats＇ age.