The Journal of Neuroscience, October 27, 2004 • 24(43):9669–9673 • 9669 Brief Communication Glutamate Mediates Acute Glucose Transport Inhibition in Hippocampal Neurons Omar H. Porras, Anitsi Loaiza, and L. Felipe Barros Centro de Estudios Cientı´ficos, Casilla 1469, Valdivia, Chile 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 astro- cytes 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 M, 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 ϩ 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. Key words: glucose; glutamate; neuron; membrane transport; 2-NBDG; 6-NBDG; AMPA Introduction tretti, 1994), which, together with the activation of astrocytic Energy consumption in the mammalian brain is supplied by the glycogen degradation (Shulman et al., 2001), may explain the oxidation of glucose. During the activation of discrete cortical large increase in local extracellular lactate that follows cortical areas, there is a correlative increase in local glucose uptake activation (Hu and Wilson, 1997). These observations support an (Sokoloff, 1999) that is consistent with the high energetic cost of alternative model of brain energy metabolism in which active ion gradient restoration (Attwell and Laughlin, 2001). This link neurons preferentially feed on lactate, which is produced by as- between neural activity and glucose uptake is exploited routinely trocytes from glucose and glycogen in response to neuronal glu- for research and clinical purposes by means of positron emission tamate (Magistretti et al., 1999). tomography (PET) scanning, a noninvasive technique that fol- Using real-time sugar transport assays, we were recently able lows the tissular uptake of a radioactive hexoses. to show that cultured astrocytes respond to glutamate by activat- Because 85% of the energy expenditure of the brain occurs in ing its glucose transporter GLUT1 on the order of seconds neurons (Attwell and Laughlin, 2001) and glucose is almost the (Loaiza et al., 2003). This mechanism was proposed to make only fuel used by the organ, the natural assumption has been that more glucose available to the astrocytic metabolism during and neurons must feed on glucose. Such a conventional notion is after neuronal activity. Exploiting the capability of these tech- consistent with the abundance of glucose transporters in these niques to resolve single cells, we set out to investigate whether a cells and the adequate electrical activity observed in cultured neu- similar mechanism was present in hippocampal neurons but rons grown in glucose-rich media. However, lactate is constitu- found that, in these cells, glutamate exerts an effect opposite to tively exported by astrocytes and can sustain firing in cultured that observed in astrocytes. neurons even more efficiently than glucose (Bouzier-Sore et al., 2003). Moreover, astrocytic lactate production is stimulated by Materials and Methods the excitatory neurotransmitter glutamate (Pellerin and Magis- Materials. Fura Red AM, calcein AM, hexoses [2-[N-(7-nitrobenz-2-oxa- 1,3-diazol-4-yl)amino]-2-deoxyglucose (2-NBDG) and 6-[N-(7- nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-6-deoxyglucose (6-NBDG)], Received May 14, 2004; revised Sept. 3, 2004; accepted Sept. 8, 2004. and pluronic acid were obtained from Molecular Probes (Eugene, OR). This work was supported by Fondo Nacional de Desarrollo Cientı´fico y Tecnolo´gico Grant 1020648. Centro de AMPA and CNQX were from Tocris-Cookson (Bristol, UK). Standard Estudios Cientı´ficos (CECS) is a Millennium Science Institute and is funded in part by Fundacio´n Andes, the Tinker chemicals, tissue culture reagents, veratridine, ionomycin, and anti- Foundation, and Empresas Compan˜ia Manufacturera de Papeles y Cartones. We thank our colleagues at CECS for discussionsandcriticalreadingofthismanuscript.Wealsothanktwoanonymousreviewersforhelpfulsuggestions. MAP1b (microtubule-associated protein 1b) were from Sigma (St. Louis, CorrespondenceshouldbeaddressedtoDr.LuisFelipeBarros,CentrodeEstudiosCientı´ficos,AvenidaArturoPrat MO). Anti-GFAP was from Dako (High Wycombe, UK), and FITC- 154, Casilla 1469, Valdivia, Chile. E-mail: [email protected]. conjugated and tetramethylrhodamine isothiocyanate-conjugated sec- DOI:10.1523/JNEUROSCI.1882-04.2004 ondary antibodies were from Sigma. Copyright © 2004 Society for Neuroscience 0270-6474/04/249669-05$15.00/0 Cell culture and immunodetection. Sprague Dawley rats were obtained 9670 • J. Neurosci., October 27, 2004 • 24(43):9669–9673 Porras et al. • Glutamate Inhibits Neuronal Glucose Transport from the Universidad Austral de Chile (Valdivia, Chile). Mixed cultures of neuronal and glial cells were prepared from 1- to 3-d-old neonatal as described by Loaiza et al. (2003). Briefly, cells were initially plated in MEM–10% fetal bovine serum media and maintained at 37°C in a humid atmosphere with 5% CO2 and 95% air. Two hours after plating, media was replaced by serum-free N1–MEM (MEM supplemented with 750 mg/ml glucose, 100 ␮M putrescine, 20 nM progesterone, 30 nM selenium dioxide, 100 ␮g of transferrin, 5 ␮g/ml insulin, 1 mM sodium pyruvate, 5 and 0.1% ovalbumin). Cells (10 ) were plated on 1 mg/ml poly-L-lysine- coated coverslips (25 mm). One-half of the culture media (N1–MEM) was removed and replaced with new media 4 d after plating. Cells were used for experiments within 10 d of being cultured. Immunocytochem- istry was performed using standard procedures described in detail previ- ously (Barros et al., 1995). Paraformaldehyde-fixed cells were stained with affinity-purified antipeptide antibodies at 2 ␮g/ml (MAP1b) or 4 ␮g/ml (GFAP). Single-cell fluorescent hexose uptake by confocal microscopy. Cells were imaged using an inverted Zeiss (Oberkochen, Germany) LSM 5 PASCAL, laser-scanning confocal microscope with 40ϫ (numerical aperture, 1.3) or 63ϫ (numerical aperture, 1.4) objectives. Pinhole was set to produce optical sections thinner than 2 ␮m. Control experiments showed that under our experimental conditions, dye bleaching was negligible. The uptake of the fluorescent hexoses 2-NBDG and 6-NBDG was assayed at room temperature (23–26°C) as described by Loaiza et al. (2003). Before transport measurements, culture medium was removed and coverslips were washed with Krebs’–Ringer’s–HEPES (KRH) buffer [in mM: 136 NaCl, 20 HEPES, 4.7 KCl, 1.25 MgSO4, and 1.25 CaCl2,pH 7.4] supplemented with 3.3 mM glucose. For some experiments, cells were then loaded for 30 min with 5 ␮M Fura Red AM. This allowed semiquantitative tracking of intracellular calcium. Five minutes before uptake, glucose in the medium was reduced to 0.5 mM to minimize competition with dye transport. Uptake was started by addition of 300 ␮M 2-NBDG or 6-NBDG, with the concentration chosen as the mini- mum capable of giving an adequate signal-to-noise ratio. Cultures were excited at 488 nm. 2-NBDG and 6-NBDG were imaged at 505–550 nm emission, and Fura Red was imaged simultaneously at Ͼ585 nm emis- Figure 1. Glutamate inhibits glucose uptake by neurons. A, Imaged under phase contrast, sion. Intracellular hexose concentration was calculated by comparing livingastrocyteswereidentifiedasflatpolygonalsheetsattachedtothesubstrate,andneurons intracellular fluorescence with the signal outside of the cells. 6-NBDG is were defined as strongly birefringent bodies located on top of the astrocytic monolayer (top). nonmetabolizable; thus, it directly reports transport. Because the effect of These identification criteria were validated by costaining neurons for MAP1b (center) and as- agonists and inhibitors was routinely assessed while the intracellular con- trocytes for GFAP (bottom). Scale bar, 10 ␮m. B, 6-NBDG uptake was measured by confocal centration of the sugar was Ͻ25–30% of the extracellular, the tracer is microscopy in a single neuron (n) and a neighboring astrocyte (a). Scale bar, 10 ␮m. glu, said to be measured under near zero-trans conditions. In the case of Glutamate. At the time indicated (dashed line), 500 ␮M glutamate was added to the culture. C, 2-NBDG, which can be phosphorylated by hexokinase to give fluorescent Hexose uptake rate as a function of glutamate concentration relative to the rate measured 2-NBDG-6-phosphate, the concentration of free intracellular sugar is before glutamate addition (n Ͼ 13 neurons in 3–5 experiments for each concentration). Zero