Imaging of L-glutamate fluxes in mouse brain slices based on an enzyme-based membrane combined with a difference-image analysis.
A time-resolved imaging method for visualizing L-glutamate release in mammalian brain slices is proposed by using an enzyme membrane combined with a difference-image analysis. The enzyme membrane is composed of L-glutamate oxidase and horseradish peroxidase incorporated into a bovine serum albumin matrix. L-Glutamate triggers an enzyme-coupling reaction to convert a redox substrate (DA-64) to Bindschedler's Green, which gives a green color signal. The difference-image analysis is based on calculating slopes of a signal versus time (t) plot in the time range from (t - 40 s) to (t + 40 s) for visualizing L-glutamate release in terms of its flux (in mol min(-1) cm(-2)). The method was applied to a time-resolved imaging of hippocampal distribution of ischemia-induced L-glutamate release in mouse brain slices. The image of L-glutamate distribution showed that the level and time courses of L-glutamate fluxes were neuronal region-dependent. The maximum flux of L-glutamate at CA1 was observed at 7.7 min after ischemia. The flux at 7.7 min increased in the order of CA1 approximately CA3 > DG. The time course of the L-glutamate flux in the CA1 region was biphasic and that in the DG region was modestly biphasic. In the CA3 region, such biphasic release of L-glutamate was not seen. The ischemia-induced L-glutamate flux was accelerated when Mg2+ was omitted from an extracellular solution. The present enzyme membrane-based approach provides a useful method for visualizing distribution of L-glutamate release in the brain slices during ischemia.
Department of Physics and Applied Physics and Department of Chemistry, College of Humanities and Sciences, Nihon University, Sakurajousui, Setagaya, Tokyo 156-8550, Japan. ahirano@chs.nihon-u.ac.jp
Ayumi
A
Hirano
Nobukatsu
N
Moridera
Mai
M
Akashi
Minoru
M
Saito
Masao
M
Sugawara
D000594:Amino Acid Oxidoreductases / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport; D001921:Brain / Q000378:metabolism*; D002545:Brain Ischemia / Q000378:metabolism; D002138:Calibration; D004800:Enzymes, Immobilized / Q000378:metabolism*; D018698:Glutamic Acid / Q000378:metabolism*; D006624:Hippocampus / Q000378:metabolism; D006735:Horseradish Peroxidase / Q000378:metabolism*; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D066298:In Vitro Techniques; D008274:Magnesium / Q000378:metabolism; D008567:Membranes, Artificial; D051379:Mice; D012710:Serum Albumin, Bovine; D013997:Time Factors
D004800:Enzymes, Immobilized; D008567:Membranes, Artificial; D012710:Serum Albumin, Bovine; D018698:Glutamic Acid; D006735:Horseradish Peroxidase; D000594:Amino Acid Oxidoreductases; C023061:L-glutamate oxidase; D008274:Magnesium