Inglefield J R, Schwartz-Bloom R D
Department of Pharmacology, Duke University Medical Center, Durham, NC 27710, USA.
J Neurosci Methods. 1997 Aug 22;75(2):127-35. doi: 10.1016/s0165-0270(97)00054-x.
We have developed a method using UV laser-scanning confocal microscopy and the fluorescent chloride ion indicator, 6-methoxy-N-ethylquinolinium chloride (MEQ), to image GABA-mediated changes in intracellular chloride (Cli-) in individual neurons of the rat acute brain slice. After bath-loading slices with the cell-permeant form (reduced) of MEQ, there was intense fluorescence within neurons of diverse morphologies in the hippocampus, neocortex and cerebellum. MEQ fluorescence localized to the cytosolic compartment of both the somata and proximal dendrites. MEQ fluorescence was calibrated using the ionophores nigericin and tributyltin in the presence of varying extracellular Cl- concentrations. Neuronal MEQ fluorescence was inversely related to intracellular Cl-, with a Stern-Volmer constant of 16 M-1 (50% quench by 61 mM Cl-). Application of GABA in the perfusate produced a concentration-dependent decrease in MEQ fluorescence (EC50 = 40 microM) that was blocked in the presence of the Cl- channel antagonist, picrotoxin. Bath perfusion of hippocampal slices with modulators of the GABAA receptor, pentobarbital and diazepam, potentiated the GABA-mediated response by 85 and 44%, respectively. A regional comparison identified larger GABA responses for both cerebellar Purkinje and granule cells relative to pyramidal neurons of the hippocampus and neocortex and to hippocampal interneurons. Pressure ejection of the GABAA agonist, muscimol (40 microM), from a micropipet onto individual hippocampal neurons allowed the measurement of rapid responses (1-5 s), compared to those obtained with bath application. Thus, optical imaging of [Cl-]i using MEQ and UV-laser-scanning confocal microscopy provides investigators with a new method to study GABAA pharmacology in neighboring neurons and perhaps even in the soma versus dendrites simultaneously, within living brain slices.
我们开发了一种方法，利用紫外激光扫描共聚焦显微镜和荧光氯离子指示剂6-甲氧基-N-乙基喹啉氯化铵（MEQ），对大鼠急性脑片中单个神经元内GABA介导的细胞内氯离子（Cli-）变化进行成像。在用MEQ的细胞渗透性形式（还原型）对脑片进行孵育加载后，海马体、新皮层和小脑中形态各异的神经元内出现强烈荧光。MEQ荧光定位于胞体和近端树突的胞质区室。在不同的细胞外氯离子浓度下，使用尼日利亚菌素和三丁基锡离子载体对MEQ荧光进行校准。神经元的MEQ荧光与细胞内氯离子呈负相关，斯特恩-沃尔默常数为16 M-1（61 mM Cl-可使荧光淬灭50%）。在灌流液中施加GABA会导致MEQ荧光呈浓度依赖性降低（EC50 = 40 microM），在存在氯离子通道拮抗剂印防己毒素时这种降低会被阻断。用GABAA受体调节剂戊巴比妥和地西泮对海马体脑片进行灌流，分别使GABA介导的反应增强了85%和44%。区域比较发现，相对于海马体和新皮层的锥体神经元以及海马体中间神经元，小脑浦肯野细胞和颗粒细胞的GABA反应更大。从微量移液器向单个海马体神经元压力喷射GABAA激动剂蝇蕈醇（40 microM），与浴槽给药相比，可以测量快速反应（1 - 5秒）。因此，使用MEQ和紫外激光扫描共聚焦显微镜对[Cl-]i进行光学成像，为研究人员提供了一种新方法，可在活脑片中同时研究相邻神经元甚至胞体与树突中的GABAA药理学。
