Glutamate mediates acute glucose transport inhibition in hippocampal neurons - PubMed (original) (raw)

Glutamate mediates acute glucose transport inhibition in hippocampal neurons

Omar H Porras et al. J Neurosci. 2004.

Abstract

Although it is known that brain activity is fueled by glucose, the identity of the cell type that preferentially metabolizes the sugar remains elusive. To address this question, glucose uptake was studied simultaneously in cultured hippocampal neurons and neighboring astrocytes using a real-time assay based on confocal epifluorescence microscopy and fluorescent glucose analogs. Glutamate, although stimulating glucose transport in astrocytes, strongly inhibited glucose transport in neurons, producing in few seconds a 12-fold increase in the ratio of astrocytic-to-neuronal uptake rate. Neuronal transport inhibition was reversible on removal of the neurotransmitter and displayed an IC50 of 5 microm, suggesting its occurrence at physiological glutamate concentrations. The phenomenon was abolished by CNQX and mimicked by AMPA, demonstrating a role for the cognate subset of ionotropic glutamate receptors. Transport inhibition required extracellular sodium and calcium and was mimicked by veratridine but not by membrane depolarization with high K+ or by calcium overloading with ionomycin. Therefore, glutamate inhibits glucose transport via AMPA receptor-mediated sodium entry, whereas calcium entry plays a permissive role. This phenomenon suggests that glutamate redistributes glucose toward astrocytes and away from neurons and represents a novel molecular mechanism that may be important for functional imaging of the brain using positron emission tomography.

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Figures

Figure 1.

Glutamate inhibits glucose uptake by neurons. A, Imaged under phase contrast, living astrocytes were identified as flat polygonal sheets attached to the substrate, and neurons were defined as strongly birefringent bodies located on top of the astrocytic monolayer (top). These identification criteria were validated by costaining neurons for MAP1b (center) and astrocytes for GFAP (bottom). Scale bar, 10 μm. B, 6-NBDG uptake was measured by confocal microscopy in a single neuron (n) and a neighboring astrocyte (a). Scale bar, 10 μm. glu, Glutamate. At the time indicated (dashed line), 500 μ

glutamate was added to the culture. C, Hexose uptake rate as a function of glutamate concentration relative to the rate measured before glutamate addition (n > 13 neurons in 3-5 experiments for each concentration). Zero glutamate refers to mock addition of buffer. D, Inhibition of hexose uptake by 500 μ

glutamate in the absence and presence of 20 μ

CNQX.

Figure 2.

Sodium entry via AMPA receptors mediate neuronal glucose uptake inhibition. A, Hexose uptake by neurons was measured before and after the addition of 20 μ

AMPA. The top trace corresponds to simultaneous nonquantitative assessment of cytosolic calcium with the reciprocal dye Fura Red (n = 6 cells). B, Inhibition of hexose uptake elicited by AMPA alone (n = 24 cells, 7 exps.) in the presence of 20 μ

CNQX (n = 9 cells, 2 exps.) in a buffer in which Na+ was equimolarly replaced by _N-_methyl-

-glucamine (n = 15 cells, 3 exps.) and in the presence of 10 m

EGTA (n = 22 cells, 4 exps.). C, Hexose uptake by a neuron was measured before and after the addition of 75 μ

veratridine. The top trace represents the corresponding Fura Red signal. D, Inhibition of hexose uptake by veratridine (Vera) (6-NBDG; n = 18 cells, 4 exps.), 2 μ

ionomycin (Iono) (2-NBDG; n = 17 cells, 3 exps.), and 40 m

KCl(2-NBDG; n = 28 cells, 6 exps.).

Figure 3.

Glutamate (glu) inhibition of neuronal glucose uptake is fast and reversible. A, Linear functions were fitted to neuronal hexose uptake data before (open circles) and after (filled circles) the addition of 500 μ

glutamate. The intercept between the two curves was compared with the onset of the calcium deflection (top trace) to obtain the delay of transport inhibition. For this particular neuron, the delay was 24 sec. Inset, Frequency distribution for 21 neurons (7 exps.) exposed to glutamate. B, A neuron and a neighboring astrocyte were exposed to 500 μ

glutamate for 25 sec. Note that, after transient changes, transport rates returned to basal values in both cells. Data are representative of at least four independent experiments.

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Glutamate mediates acute glucose transport inhibition in hippocampal neurons - PubMed (original) (raw)