Circadian Variation of GABAergic Action in the Supracgiasmatic Nucleus Neurons.

本课题采用图像法直接测量细胞内Cl^-浓度的变化。将Cl 2-敏感性荧光染料MEQ（6-甲氧基-N-乙基喹啉碘化物）加载到大鼠脑切片。大鼠视交叉上核神经元在355 nm激发光下呈蓝色荧光。通过浴涂100 μ m GABA改变荧光强度。MEQ的荧光变化在某些情况下表明[Cl^-] i增加，在另一些情况下表明[Cl^-] i减少。为了获得最终结果，需要测量许多神经元。其次，我们评估MEQ荧光的变化是否实际上指示[Cl^-] i的变化，因为存在MEQ荧光也会根据伴随Cl^-或其他离子流入和/或流出的细胞体积变化而变化的问题。为了回答这个问题，我们将钙黄绿素加载到切片上，这样我们就可以在[Cl^-] i变化的同时测量细胞体积的变化。钙黄绿素对任何细胞内离子浓度变化或pH变化不敏感。它只对细胞体积变化敏感。结果表明，用MEQ荧光变化与钙黄绿素荧光变化的比值（F/F）来测量[Cl^-] i是可行<MEQ><calcein>的。最后，我们改进了用MEQ荧光法测定细胞内Cl^-绝对浓度的方法。在这种情况下，应将胞外Cl^-用其它阴离子代替，取标准曲线计算[Cl^-] i。为此，应使用最少淬灭细胞内MEQ的阴离子。甲基磺酸钾是最佳的阴离子。

In this project, change of intracellular Cl^- concentration was directly measured by imaging method. Cl^- sensitive fluorescent dye, MEQ (6-methoxy-N-ethylquinolinium iodide), was loaded to the rat brain slices. The neurons in rat suprachiasmatic nucleus showed blue fluorescence by excitation light of 355 nm. The fluorescence intensity was changed by the bath application of 100 um GABA. In some cases, the fluorescence change of MEQ indicated the increase of [Cl^-]_i, and in other cases, the decrease of [Cl^-]_i. To obtain the final result, it is necessary to measure many neurons. Second, we evaluated if the change in MEQ fluorescence actually indicated the change in [Cl^-]_i, because there was a question that MEQ fluorescence would also changed according to the cellular volume change accompanied with Cl^- or other ion influx and/or eflux. To answer this question, we loaded calcein to the slices so that we could measure the cell volume change simultaneously to [Cl^-]_i change. Calcein is insensitive to any intracellular ionic concentration change or pH change. It is sensitive only to cell volume change. The results indicates that pseudo-ratiometric measurement of [Cl^-]_i is feasible by taking the ratio of MEQ fluorescence change to calcein fluorescence change (F_<MEQ>/F_<calcein>). Finally, we improved the method to obtain the absolute intracellular Cl^- concentration by using MEQ fluorescence. In this case, extracellular Cl^- should be substituted by other anions to take the calibration curve to culculate [Cl^-]_i. For this purpose, the anion which least quenches the intracellular MEQ should be used. Metylsulfric acid potassium salt was found to be the best anion for this purpose.