Quantitative Immunohistochemistry of Glucose Transport Protein (Glut3) Expression in the Rat Hippocampus During Aging

The integral membrane protein Glut3 (45 kD) is the major glucose transporter in neurons and particularly abundant in regions of high synaptic density (Vannucci et al. 1997). In a previous report we documented that an age-related decay in neuron metabolism may be responsible for synaptic and mitochondrial deterioration and loss (Fattoretti et al. 1998). Therefore, we carried out a quantitative immunohistochemical study to assess Glut3 protein expression in rat hippocampus during aging. According to the guidelines of the Italian Ministry of Health regarding the use of laboratory animals, anesthetized rats were perfused with 4% paraformaldehyde in 0.1 M phosphate buffer (PB), pH 7.4. The hippocampi were excised from young (3 months) and old (28 months) female Wistar rats of our breed (n = 4 for each group). Pre-blocked, frozen free-floating sections (20 μm thick) were incubated in 1.5% normal goat serum, 0.03% Triton X-100 in 0.1 M PBS with the rabbit anti-Glut3 antiserum (1:1000; Alpha Diagnostic, San Antonio, TX) for 48 hr at 4C. Glut3 reactivity was detected with a biotinylated secondary antibody (1:200; Vector Laboratories, Burlingame, CA) and avidin–biotin–peroxidase complex (1:50; Vector) after treatment for 30 min with 0.3% hydrogen peroxide in PBS to quench endogenous peroxidase. The peroxidase reaction was performed with 0.05% 3,3-diaminobenzidine tetrahydrochloride (DAB) solution in PBS activated with 0.01% hydrogen peroxide for 10 min and stopped by several washes with PBS. Then the sections were mounted on gelatin-coated glass slides, dehydrated, delipidated in xylene, and coverslipped with DPX mountant (Figure 1). The sections were rinsed with PBS at each step, all reactions were performed at room temperature, and rabbit IgG was employed as negative control. Antiserum specificity was assessed by Western blotting analysis. The samples (20 μg protein/lane) and the molecular weight markers were electrophoresed by 10% SDS-PAGE and transferred to nitrocellulose membrane (0.45 μm; Bio-Rad Laboratories, Richmond, CA) by electroblotting (Matsumoto et al. 1995). The anti-Glut3 antibody was diluted 1:2000 and antibody binding was visualized using a chemiluminescence technique (Amersham; Poole, UK). Glut3 was identified as a marked band with a molecular weight of about 45 kD (Figure 2). Optical density measurements (ODs) were carried out by the Kontron Imaging System KS300 connected to a light microscope (Laborlux; Leitz, Wetzlar, Germany). Five 3100-μm² fields were measured on each section and three sections for each rat were analyzed. OD data were corrected for background by subtracting the measurements of their own negative control. Statistical comparisons were performed by one-way ANOVA and the Student–Newman–Keuls’ test (p > 0.05). Our quantitative immunohistochemical technique allowed us a more detailed localization of Glut3 protein in the discrete brain areas analyzed. Glut3 reactivity was intense in neuropil, axons, and dendrites, whereas nerve cell bodies were unstained. In old rats, Glut3 is significantly reduced by 46% in the inner molecular layer of the hippocampal dentate gyrus (iml) and by 34% in the mossy fibers (mf) of the CA3 sector (Figure 3). The CA1 stratum radiatum (sr) does not show any difference due to age. The present findings support an age-dependent decrease of Glut3 expression in old rat hippocampus and correlate with the age-related decline in learning and memory as well as local cerebral glucose utilization (Gage et al. 1984). The hippocampal formation, as a critical brain area involved in memory processing and retrieval, constitutes a preferential target for several histopathological alterations of the old and demented human brain, such as senile plaques and neurofibrillary tangles. The etiology of these hallmarks is unknown. However, metabolic impairment may represent an unfavorable condition predisposing to neuron dysfunction and disease. Our data support the concept that Glut3 reduction may be responsible for a decreased glucose supply which, in turn, leads to a significant loss of synapses, specifically in discrete areas closely depending on energy metabolism, e.g., the inner molecular layer above the dentate gyrus, mostly innervated by cholinergic fibers.

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Figure 1.

Distribution of immunoreactive Glut3 in hippocampal coronal section. Bar = 200 μm.

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Figure 2

Western blotting analysis. Glut3 protein expression of young (Lane 1) and old (Lane 2) rat hippocampal lysates.

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Figure 3

Statistical analysis of Glut3 optical density values (mean ± SEM) in discrete hippocampal regions in 3- and 28-month-old rats. ^∗ p > 0.001.