Glucose Oxidase-immobilized Glass Disks for Imaging of d-Glucose in Acute Brain Slices

Abstract

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...

Electroanalytical Studies of Immunoglobulin-Bound Glucose Oxidase

“…Gold and glass substrate surfaces were used to immobilize the IgG and GO molecules, because these surfaces are routinely used for adsorbing enzymes. [26][27][28][29] Surface-adsorbed GO on gold and glass substrates were similarly attempted but gave highly irreproducible results due to significant desorption of GO during the SECM experiments. Figure 3 shows the schematic of the SECM feedback mode used in probing a substrate surface physically adsorbed with IgG-bound GO immunocomplex.…”