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.

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Glucose metabolism is the major pathway of energy production in the mature brain. 1 The relationship between neuronal activity, especially glutamatergic neuronal acitivity, 2,3 and energy consumption has attracted attention concerning the energetic basis of brain activity. For the in vivo mapping of brain activity, functional magnetic resonance imaging (fMRI), positron-emission tomography (PET) and magnetic resonance spectroscopy (MRS) are the dominant methods. [4][5][6] On the other hand, for in vitro imaging of the spatial and temporal distribution of glucose in brain slices, which are one of the most common models of the brain, a few methods have been reported, including autoradiography with radio-labeled glucose and related compounds, 7-10 31 P NMR, 11 and the measurements of intrinsic optical signals.12,13 Although these methods have been utilized for elucidating glucose metabolisms in brain slices or cultured slices, the use of radio-labeled compounds is hampered by ease of handling and safety. The intrinsic optical signal measures any cell swelling, i.e., volume changes, rather than D-glucose, itself. 14,15 In the present paper, extending our previous approach 15,16 to glucose systems, we describe an enzyme-based imaging method for D-glucose in acute brain slices. Biotinylated glucose oxidase (bGOD) is immobilized on a glass disk by the avidinbiotin technique. Brain slices are placed on the bGOD disks, followed by stimulation with a solution containing a stimulant, horse radish peroxidase (HRP) and an oxidation dye DA-64. Glucose triggers an enzyme reaction and oxidizes the substrate DA-64 to Bindschedler's Green (BG) according to D-glucose + O2 + H2O gluconic acid + H2O2 bGOD ⎯ ⎯⎯ → H2O2 + 2H + + DA-64Bindschedler's Green + 2H2OBy applying a differential image analysis, glucose fluxes at various neuronal regions of mouse hippocampal slices are imaged. The response of the bGOD disks to D-glucose is discussed in terms of the principle, concentration dependence and selectivity. The bGOD disks are then applied to in vitro imaging of D-glucose fluxes in acute mouse hippocampal slices under a chemical stimulation. Since bGOD is first immobilized on the disk, the diffusion of bGOD into a brain slice is avoided. Consequently, chemical perturbation of the system will be minimized. Humanities and Sciences, Nihon University, Sakurajousui, Setagaya, Japan A biotinylated glucose oxidase (bGOD)-immobilized glass disk was prepared for visualizing D-glucose fluxes in acute brain slices. A mouse hippocampal slice was placed on the bGOD disk and stimulated with a stimulant solution containing horseradish peroxidase (HRP) and a substrate DA-64, followed by capturing digital images of Bindschedler's Green (BG), an oxidized form of DA-64, with a CCD camera. The bGOD membranes responded proportionally to Dglucose, ranging from 2.0 to 5.0 mM. Sucrose, GABA, L-glutamic acid, L-aspartic acid, glycine, acetylcholine and Lascorbic acid at 10 mM did not cause any responses. The D-glucose fluxes in mouse hippocampal slices st...