Visualizing L-glutamate fluxes in acute hippocampal slices with glutamate oxidase-immobilized coverslips.
We used a glutamate oxidase (GluOx)-immobilized glass coverslip for reducing diffusional blur and improving the temporal resolution of visualizing L-glutamate fluxes in acute brain slices. The immobilization of GluOx on an avidin modified glass coverslips was achieved by optimized the amine coupling method. The GluOx coverslip was applied to the imaging of L-glutamate fluxes in acute hippocampal slices under hypoxia and KCl stimulation. A slice from mouse brain was loaded with horseradish peroxidase (HRP) and substrate DA-64, and placed on the GluOx coverslip for stimulation. The regional distribution of hypoxia-induced L-glutamate fluxes was analyzed. The maximum flux at 3 min after the onset of hypoxia increased in the order CA1>CA3>DG. The time-courses of the L-glutamate fluxes at CA1 and DG were biphasic, while that at CA3 decreased monotonously. The KCl-stimulated release of L-glutamate in the presence of the DL-TBOA uptake inhibitor was imaged. While no noticeable change was observed in the absence of DL-TBOA, L-glutamate fluxes in the presence of the inhibitor increased in the order CA1>CA3>DG, reflecting the effect of uptake processes. The present approach suppressed diffusional blur of the glutamate signal and improved the temporal resolution as compared with the BSA-HRP membrane method described earlier.
Okumura
Wataru
W
Department of Chemistry, College of Humanities and Sciences, Nihon University, Sakurajousui, Setagaya, Tokyo, Japan.
Moridera
Nobukatsu
N
Kanazawa
Ena
E
Shoji
Atushi
A
Hirano-Iwata
Ayumi
A
Sugawara
Masao
M
D000594:Amino Acid Oxidoreductases / Q000378:metabolism; D000818:Animals; D004800:Enzymes, Immobilized; D005969:Glutamate Dehydrogenase / Q000378:metabolism*; D018698:Glutamic Acid / Q000378:metabolism*; D006624:Hippocampus / Q000378:metabolism*; D006735:Horseradish Peroxidase; D000860:Hypoxia; D007700:Kinetics; D051379:Mice; D008853:Microscopy; D011189:Potassium Chloride
D004800:Enzymes, Immobilized; D018698:Glutamic Acid; D011189:Potassium Chloride; D006735:Horseradish Peroxidase; D000594:Amino Acid Oxidoreductases; D005969:Glutamate Dehydrogenase