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Circadian Variation of GABAergic Action in the Supracgiasmatic Nucleus Neurons.

上核神经元 GABA 能作用的昼夜变化。

基本信息

批准号：
12670056
负责人：
SAMEJIMA Michikazu
金额：
$ 1.86万
依托单位：
Hamamatsu University School of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12670056/
关键词：
INTRACELLULAR Cl^- CONCENTRATION SUPRACHIASMATIC NUCLEUS INHIBITORY SYNAPTIC TRANSMISSION Cl^-IMAGIN MEQ GABA

项目摘要

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.

在本项目中，通过成像方法直接测量细胞内氯离子浓度的变化。将对氯离子敏感的荧光染料MEQ(6-甲氧基-N-乙基喹啉碘)负载到大鼠脑片中。大鼠视交叉上核神经元在355 nm的激发光下呈蓝色荧光。用100um GABA浸浴，可改变荧光强度。在某些情况下，MEQ的荧光变化表明[Cl^-]i的增加，而在另一些情况下，[Cl^-]i的下降。为了获得最终的结果，需要测量许多神经元。其次，我们评估了MEQ荧光的变化是否真的反映了[Cl^-]i的变化，因为存在一个问题，即MEQ荧光也会随着伴随着Cl2-或其他离子内流和/或外流的细胞体积的变化而变化。为了回答这个问题，我们将钙黄绿素加载到切片上，以便我们可以同时测量细胞体积随[Cl^-]i变化的变化。钙黄绿素对任何细胞内离子浓度变化或pH变化都不敏感。它只对细胞体积的变化敏感。结果表明，用MEQ荧光变化量与钙黄绿素荧光变化量之比(F_&lt；MEQ&gt；/F_&lt；Calcein&gt；)进行[Cl~-]_i的赝比测量是可行的。最后，我们改进了利用MEQ荧光法获得细胞内绝对氯离子浓度的方法。在这种情况下，应该用其他阴离子取代胞外的氯离子，取校正曲线计算[Cl^-]_i。为此，应使用对胞内分子量猝灭最小的阴离子。甲基亚磺酸钾盐是最佳的阴离子。