用毛细管电泳-激光诱发荧光技术检测清醒吗啡戒断大鼠导水管周围灰质微透析液中谷氨酸和精氨酸的含量

清醒动物脑微透析技术能够用于动态观察某一特定核团中生物活性物质变化及其与行为的关系,结合高效毛细管电泳-激光诱发荧光对衍生后的透析样品进行检测,使这一技术更趋完善。本实验用荧光素异硫氰酸酯(FITC)与痕量氨基酸样品进行衍生,通过适当增加衍生温度,能明显缩短衍生时间,衍生效果与传统的室温下衍生16h无明显差别;进一步确定30℃水浴反应5h是较理想的FITC与痕量氨基酸的衍生条件。在此优化衍生条件下,成功检测了清醒吗啡戒断大鼠脑导水管周围灰质(periaqueductal gray matter,PAG)微透析液中谷氨酸(glutamate,Glu)和精氨酸(arginine,Arg)含量变化。结果表明,非吗啡依赖和吗啡依赖大鼠脑PAG中L-精氨酸(L-arginine,L-Arg)和L-谷氨酸(L-glutamate,L-Glu)含量无明显差别,纳洛酮催促戒断后的第一个10min内PAG中的L-Arg和L-Glu含量显著增加,分别比戒断前增加了63％和105％,10min后含量逐渐下降,这种变化趋势与同时观察的吗啡戒断评分变化相一致。

Application of combination of capillary electrophoresis with laser-induced fluorescence: measurement of glutamate and arginine in PAG microdialytes of conscions morphine-withdrawal rats

Microdialysis technique in free-moving animals can be used to monitor continuously the changes of many extracellular neurotransmitters in certain brain areas and study the relationship between neurotransmitter and functions. Using detection of capillary electrophoresis combined with laser-induced fluorescence (CE-LIF) further improves the above-mentioned technique. In the present study, fluorescein isothiocyanate (FITC) was used to derivatizate amino acid in very low concentration. We found that increasing derivatization temperature could shorten derivatization time and that the derivatizative efficiency was not different from that when experiment was performed under the traditional derivatization condition (room temperature for 16 h). We also got an optimized condition of amino acid derivatization with FITC at 30 degrees C water bath for 5 h. Using the optimized condition of amino acid derivatization, we investigated the changes in L-arginine (L-Arg) and L-glutamate (L-Glu) concentration in periaqueductal gray matter (PAG) microdialytes of free-moving morphine-withdrawal rats. The results indicated that there was no significant difference in the concentration of L-Arg and L-Glu in PAG between non-dependent and dependent rats. The concentration of L-Arg and L-Glu in PAG increased by 63% and 105%, respectively, in the first 10 min after naloxone-precipitated withdrawal and then declined gradually. These changes were in correspondence with the scores of morphine withdrawal symptom.