可视法脑片膜片钳技术观察大鼠前庭内侧核神经元毒蕈碱样胆碱能受体的电生理特性

Electrophysiological characteristics of muscarinic cholinergic receptor in rat medial vestibular nucleus neurons by visual patch clamp technique

目的建立大鼠前庭内侧核脑片可视法膜片钳实验技术,探讨前庭内侧核神经元毒蕈碱样胆碱能受体(muscarinic cholinergic receptor,M 受体)介导电流的生物学特性。方法选用15只Wistar 大鼠用于制备前庭内侧核脑片,应用红外微分干涉相差(infrared differential interferencecontrast,IR-DIC)技术结合电荷耦合式感光成像(charge coupled device-camera,CCD-camera)系统,在可视法膜片钳全细胞记录模式下对20个正常功能状态的前庭内侧核神经元 M 受体的通道电流性质进行观察和分析。结果可视法脑片膜片钳技术可对神经元直接进行准确定位和功能状态的筛选。前庭内侧核神经元给予毒草碱后电流-电压(I-V)曲线斜率增加,毒蕈碱引发效应电流的反转电位为(-88.4±4.9)mV(±s,下同),表明 M 受体去极化效应是由钾电导的减少所介导;M 受体介导电流的电压敏感性测试显示:毒蕈碱引发的效应曲线呈线性关系,反转电位为(-86.7±3.5)mV,提示毒蕈碱所阻断的钾电流为非电压敏感性的漏钾电流。结论可视法脑片膜片钳实验技术克服了盲法脑片膜片钳技术的缺陷,提高了神经元封接的成功率。通过对前庭内侧核神经元 M 受体通道电流性质的分析,进一步揭示毒蕈碱样胆碱能机制的兴奋性调节作用,为临床抗胆碱药物的应用提供新思路。

Objective To establish the visual patch clamp whole-cell recording technique and study the properties and functional significance of muscarinic receptor-mediated currents in rat medial vestibular nucleus neurons （MVNn）. Methods Brain slices containing the MVN were prepared from fifteen Wistar rats. By combining infrared differential interference contrast （IR-DIC） technique and CCD-Camera system with visual patch clamp whole-cell recording technique, twenty healthy neurons were located and muscarinic receptor-mediated currents in rat MVNn were observed and analyzed. Results Visual patch clamp technique can be used to make direct localization and to make sure of active neuron. In MVNn, a comparison of the current-voltage relationships before and during the application of muscarine, which revealed an increase in the slope of the Ⅰ-Ⅴ curve and the reversal potential for this response lay at （ - 88. 4 ± 4. 9） mV （x ± s） , indicates that the activation of muscarinic cholinergic receptors leads to a decrease in potassium current. The test in the voltage sensitivity of the muscarine-induced response, which showed that the effect had a linear current-vohage relationship and reversed at （ - 86. 7 ± 3. 5 ） mV, indicates that the potassium current blocked by muscarine corresponds to the voltage-insensitive leak potassium current. Conclusions Visual patch clamp technique, which was considered better than blind patch clamp technique, can improve the success of sealing process. By the analysis of muscarinic receptor-mediated currents, the data provide support that muscarinic cholinergic mechanisms play a prominent role in the modulation of the excitability of MVNn and also offer a new idea for the efficacy of anticholinergic drugs.