Effect of Glutamine and GABA on[U-13C]Glutamate Metabolism in CerebellarAstrocytes and Granule Neurons

Hong Qu,1–3 Jon R. Konradsen,1 Marike van Hengel,1 Saskia Wolt,1 andUrsula Sonnewald1*1Department of Clinical Neuroscience, Norwegian University of Science and Technology, Trondheim, Norway2Department of Physics, Norwegian University of Science and Technology, Trondheim, Norway3MR-Center, SINTEF-UNIMED, Trondheim, Norway

To probe the effect of glutamine and GABA on metabo-lism of [U-13C]glutamate, cerebellar astrocytes were in-cubated with [U-13C]glutamate (0.5 mM) in the presenceand absence of glutamine (2.5 mM) or GABA (0.2 mM). Itcould be shown that consumption of [U-13C]glutamatewas decreased in the presence of glutamine and releaseof labeled aspartate and [1,2,3-13C]glutamate decreasedas well, whereas the concentrations of these metabolitesincreased inside the cells. Glutamine decreased energyproduction from [U-13C]glutamate presumably by substi-tuting for glutamate as an energy substrate. No addi-tional effect was seen in the presence of both glutamineand GABA. When cerebellar granule neurons were incu-bated with [U-13C]glutamate (0.25 mM) and GABA(0.05 mM), less [U-13C]glutamate was used for energyproduction than in controls. Because the barbituratethiopental did not elicit such response (Qu et al., 2000,Neurochem Int 37:207–215) it appears that GABA alsohas a metabolic function in the glutamatergic cerebellargranule neurons in contrast to the astrocytes. J. Neuro-sci. Res. 66:885–890, 2001. © 2001 Wiley-Liss, Inc.

Key words: cerebellar astrocytes; cerebellar granuleneurons; glutamate; glutamine; GABA; MR spectroscopy

Glutamate is the main excitatory neurotransmitter inthe brain (Fonnum, 1984) and all glutamate is formedfrom glucose within the CNS itself (Gruetter et al., 1994)because glutamate does not readily cross the blood–brainbarrier (Oldendorf, 1971; Smith et al., 1987; Hawkins etal., 1995). Glutamate is synthesized from 2-oxoglutarateby transamination either with alanine, aspartate or one ofthe branched chain amino acids leucine, isoleucine andvaline (for review see Yudkoff, 1997), and can also beformed from glutamine by phosphate-activated glutami-nase (PAG). Glutamate is accumulated into vesicles to ahigh concentration and released to the synapses bycalcium-dependent exocytosis upon the arrival of an ac-tion potential. As a high extracellular concentration ofglutamate is also neurotoxic, high-affinity glutamate trans-porters are essential for terminating synaptic transmissionand for maintaining a low extracellular glutamate concen-

tration. Five structurally distinct subtypes of glutamatetransporters (GLT-1, GLAST, EAAC1, EAAT4, andEAAT5) have been identified and characterized by mo-lecular cloning, all being Na1-dependent and electro-genic. GLT-1 is most commonly expressed in glial cellsand EAAC1 in neurons (for reviews, see Tanaka, 2000;Attwell, 2000). The transmembrane gradients of Na1 andK1 provide the driving force for the transport. It is gen-erally accepted that astrocytes have more efficient uptakesystems for glutamate than neurons (Schousboe et al.,1977; Erecinska et al., 1990; Gegelashvili and Schousboe,1998). The capacity of such uptake systems could beregulated by various factors some of which are releasedfrom neurons (Drejer et al., 1983; Gegelashvili et al.,1997), and it was shown that the expression of GLASTand GLT-1 in astrocytes in the brain was down regulatedafter glutamatergic denervation (Levy et al., 1995). Fur-thermore exposure to glutamate for prolonged periods oftime led to an increased capacity for glutamate uptake andan even more pronounced expression of GLAST proteinin cultured astrocytes (Gegelashvili et al., 1996).

Once taken up into the cells, glutamate is also ametabolite. Isolated or cultured brain cells metabolize glu-tamate well (Yu et al., 1982; Tildon and Roeder, 1984,Sonnewald et al., 1997). It has also been shown thatglutamate and glutamine are oxidized in brain in vivo(Pascual et al., 1998; Zielke et al., 1998). In astrocytesmetabolites, such as glutamine, aspartate, and lactate, canbe formed from glutamate and can be released for uptakeby neurons (for review, see Sonnewald et al., 1997).

Contract grant sponsor: Research Council of Norway; Contract grantnumber: SIP 782008.01; Contract grant sponsor: SINTEF Unimed Foun-dation; Contract grant sponsor: NorFA (Normox).

Marike van Hengel and Saskia Wolt are currently on leave from theMedical Faculty, the Vrije University of Amsterdam, Netherlands.

*Correspondence to: Prof. U. Sonnewald, Department of Clinical Neuro-science, Faculty of Medicine, Olav Kyrresgt. 3, N-7489 Trondheim, Nor-way. E-mail: ursula.sonnewald@medisin.ntnu.no

Received 17 August 2001; Revised 20 August 2001; Accepted 21 August2001

Journal of Neuroscience Research 66:885–890 (2001)

© 2001 Wiley-Liss, Inc.

The aim of the present study was to investigate theeffects of glutamine and GABA on [U-13C]glutamatemetabolism in cerebellar astrocytes and of GABA on[U-13C]glutamate metabolism in cerebellar granuleneurons.

MATERIALS AND METHODS

Materials

Plastic tissue culture dishes were purchased from NuncA/S (Roskilde, Denmark), fetal bovine serum from Seralab Ltd.(Sussex, UK) and culture medium from GIBCO BRL, LifeTechnologies (Roskilde, Denmark). NMRI mice were pur-chased from Møllegaard Breeding Center (Copenhagen, Den-mark). [U-13C]glutamate (98% enriched) and 99.9% D2O werefrom Cambridge Isotopes Laboratories (Woburn, MA), andethylene glycol from Merck (Darmstadt, Germany). All otherchemicals were of the purest grade available from regular com-mercial sources.

Cell Cultures

These experiments were approved by the NTNU AnimalCare and Use Committee.

Cerebellar astrocytes were cultured as described earlier(Hertz et al., 1989). Briefly, cerebella were taken from 7-day-old mice and passed through Nitex nylon netting (80 mm poresize) into Dulbecco’s minimum essential medium (DMEM)containing 20% (v/v) fetal bovine serum (FBS). Medium waschanged 2 days after plating and subsequently twice a weekgradually changing to 10% FBS. From the beginning of the thirdweek dibutyryl-cAMP was added to the medium to promotethe morphological differentiation of astrocytes for 1 week. Ex-periments were carried out on 3-week-old astrocytes. Mediumwas removed and replaced by DMEM without FBS, containing0.5 mM [U-13C]glutamate 6 mM glucose with or without2.5 mM glutamine or 0.2 mM GABA. After 4 hr, the mediumwas removed and cells were washed with 0.9% saline andextracted with 70% ethanol (v/v). For detailed description ofextraction and protein quantification see Qu et al. (1999).

Cerebellar granule cells were isolated and cultured fromcerebella of 7-day-old mice, after mild trypsinization of thetissue followed by trituration in a DNase solution containing atrypsin inhibitor from soybeans (Schousboe et al., 1989). Cellswere used for experiments after 1 week in culture. Medium wasremoved and replaced by DMEM without glutamine and FBS,containing 0.25 mM [U-13C]glutamate and 3 mM glucose inthe presence or absence of GABA (50 mM). After 2 h, themedium was removed, cells were washed with 0.9% saline andextracted with 70% ethanol (v/v). For detailed experimentalprocedures and MR and GC/MS analysis see Qu et al. (2000).

MR Spectroscopy and HPLC

Proton decoupled 125.5 MHz 13C MR spectra wereobtained on a Bruker DRX-500 spectrometer with the sametechnical parameters as Qu et al. (1999).

Amino acids in the cell extracts were quantified by highperformance liquid chromatography (HPLC) analysis on aHewlett Packard 1100 system (Agilent, USA) with fluorescencedetection, after derivatization with o-phthaldialdehyde (Geddesand Wood, 1984).

Data Analysis

Relevant peaks from glucose, glutamate, glutamine,glutathione, aspartate, and lactate in MR spectra were inte-grated, and the amounts were quantified from the integrals ofthe peak areas, using ethylene glycol as an internal standard.Results are presented as mean 6 SEM. Differences amonggroups were analyzed statistically using one-way ANOVAfollowed by the post-hoc test (astrocytes only); P , 0.05 wasconsidered significant.

The amount of [U-13C]glutamate taken up during theincubation time was calculated as: (the amount added to themedium 2 the amount left in the medium) divided by theamount of protein. The % other pathways was calculated as: theamount of [U-13C]glutamate taken up minus the sum of all thelabel synthesized from [U-13C]glutamate in media and cell ex-tracts divided by the amount of [U-13C]glutamate taken up.

RESULTSTypical spectra of cell extracts from cerebellar astro-

cytes (Fig. 1A) and neurons (Fig. 1B) incubated with[U-13C]glutamate are shown in Figure 1. As seen in thespectra, labeled glutamine (only in astrocytes), glutathioneand aspartate formed from [U-13C]glutamate werepresent. [1,2,3-13C]Glutamate (doublet in the C-3 posi-tion), formed via the TCA cycle, was also observed inaddition to [U-13C]glutamate (apparent triplet in the C-3position). For detailed isotopomer analysis see Qu et al.(1999).

Table I lists the amounts of metabolites in the mediaformed from [U-13C]glutamate. In cerebellar astrocytesthe amount of [U-13C]glutamate removed from the me-dium was decreased in the presence of unlabeled glu-

Fig. 1. 13C MR spectra from cultures of cerebellar astrocytes (A) andneurons (B). Peak assignment: 1, glutamine C-3; 2, glutamate C-3 inglutathione, 3, glutamate C-3; 4, glutamine C-4; 5, glutamate C-4 inglutathione; 6, glutamate C-4; 7, aspartate C-3. The peak in the middleof glutamate C-3 in neurons is truncated.

886 Qu et al.

tamine as well as with glutamine plus GABA. No addi-tional effect of GABA was observed. The amount of[U-13C]aspartate and [1,2,3-13C]glutamate was also de-creased in the presence of glutamine and glutamine plusGABA. [U-13C]Alanine was observed in one out of threesamples in the control cultures, and more was detected inthe presence of glutamine and glutamine plus GABA.There were no differences between the glutamine andglutamine plus GABA groups. [2,3-13C]Lactate can onlybe formed via the so called pyruvate recycling pathwayand thus entry of [1,2-13C]acetyl CoA from pyru-vate synthesized from [U-13C]glutamate into the TCAcycle. This form of lactate was present in astrocytes,but not neurons. In cerebellar neurons no differenceswere observed between groups (results from Qu et al.,2000). [U-13C]Alanine was not observed in cerebellarneurons.

The amount of metabolites formed from[U-13C]glutamate in the extracts of cerebellar astrocytesand neurons is shown in Table II. In cerebellar astrocytesthe amounts of [U-13C]aspartate and [1,2,3-13C]glutamatewere increased in the presence of glutamine and glutamineplus GABA. The amount of labeled glutathione was de-creased in the presence of glutamine and glutamine plusGABA. The 13C label (67.6%) from [U-13C]glutamate wasnot observed in the spectra of media or cell extracts and wasthus consumed for other pathways, presumably energy pro-duction in the astrocytes. The amount of [U-13C]glutamateconsumed for these pathways was decreased in the pres-ence of glutamine and glutamine plus GABA. The samewas observed for cerebellar neurons in the presence ofGABA (Table II). No differences in the amounts of 13Clabeled metabolites, however, were observed in thecerebellar granule neurons in the presence of GABA(results from Qu et al., 2000). This might be due to the

fact that small differences in the amounts of metabolitesmight not reach statistical significance due to individualvariability that is circumvented by calculations of ratios.[1,2-13C]glutamate, formed from [U-13C]glutamate inthe second turn via the TCA cycle, was larger incerebellar neurons than astrocytes, indicating less cy-cling in astrocytes than neurons.

The total amounts of metabolites in the cell extractsof cerebellar astrocytes are listed in Table III. The amountsof aspartate and glutamine were increased in the presenceof glutamine and glutamine plus GABA. The concentra-tion of glutathione, however, was decreased. The onlydifference between the groups receiving glutamine andglutamine plus GABA was a larger amount of GABA inthe glutamine plus GABA group.

The GC/MS results from cell extracts showed thatlabeling of GABA took place in the cerebellar granuleneurons. In control cells [U-13C]GABA was found to be8.6 6 2.7% (n 5 4) of the total GABA pool (results fromQu et al., 2000). When cells were incubated in the pres-ence of unlabeled GABA the amount of unlabeled GABAinside the cells increased and the labeling became essen-tially undetectable. This labeling was only observed byGC/MS because this method is more sensitive than MRspectroscopy.

DISCUSSIONGlutamate, the main excitatory neurotransmitter, is a

potential neurotoxin (Olney, 1971; Choi, 1988). In thiscontext astrocytes play an important role in removal ofextracellular glutamate (Gegelashvili and Schousboe,1998). After uptake by astrocytes, glutamate can be di-rectly converted to glutamine, catalyzed by cytosolic glu-tamine synthetase, which is localized only in glia (Noren-

TABLE I. 13C Amount (nmol/mg Protein) in Metabolites From Lyophilized Cell Media of Cerebellar Astrocytes and Neurons AfterIncubation With [U-13C]Glutamate Under Various Conditions*

Astrocytes Neuronsc

Controln 5 3

Glutaminen 5 3

Glutamine1 GABA

n 5 3Controln 5 3

GABAn 5 4

[U-13C]glu removed 7273 6 637 5287 6 408a 4654 6 362a 2443 6 149 2334 6 71[U-13C]lactate 565 6 68 638 6 56 577 6 29 66 6 6 72 6 8[1,2-13C]lactate 45 6 3 57 6 6 55 6 4 ND ND[2,3-13C]lactate 69 6 10 70 6 2 69 6 3 ND ND[U-13C]aspartate 198 6 22 99 6 33a 162 6 46a 46 6 1 51 6 1[1,2,3-13C]glutamate 338 6 30 111 6 31c 101 6 25a 104 6 3 115 6 8[U-13C]glutamine 873 6 98 930 6 122 821 6 16 — —[1,2,3-13C]glutamine 75 6 41 185 6 27 154 6 5 — —[U-13C]alanine 22b 58 6 4 51 6 3 ND ND

*All astrocytes were incubated with [U-13C]glutamate (0.5 mM) with or without 2.5 mM glutamine or 2.5 mM glutamine plus 0.2 mM GABA for 4 hras described in Materials and Methods. Neurons were incubated with [U-13C]glutamate (0.25 mM) with or without 50 mM GABA for 2 hr as describedin Materials and Methods. The C-3 resonance was used for 13C MR determination, except for lactate and aspartate, where the C-2 resonance was used.Results are presented as mean 6SEM. Superscript indicates statistical differences as determined by one-way ANOVA followed by the post-hoc test formultiple comparisons (P , 0.05 was considered significant).aSignificantly different from controls in cerebellar astrocytes.bQuantified in one out of three samples. ND, not detected.cValues from Qu et al., 2000.

Glutamate Metabolism in Cerebellar Cultures 887

berg and Martinez-Hernandez, 1979) and requires ATP.Glutamate can also be incorporated into polyamines andpeptides, for example, the dipeptide gGluCys, which iscombined with glycine by glutathione synthetase to gen-erate glutathione (Meister, 1974). Incorporation of gluta-mate into proteins has been reported to be 10% of the totalamount of glutamate added to the incubation medium ofastrocytes (Waniewski and Martin, 1986). The prerequi-site for glutamate metabolism in the TCA cycle is theconversion of glutamate to 2-oxoglutarate, which canoccur either by deamination or transamination. In thelatter case aspartate and alanine can be formed. Contro-versy exists about which pathway is quantitatively moresignificant (Yudkoff et al., 1991; Farinelli and Nicklas,1992; Westergaard et al., 1996).

Pyruvate RecyclingTo be metabolized completely in the TCA cycle,

glutamate has to be taken out and reenter the TCA cycle.Malate and oxaloacetate can be formed from glutamate via2-oxoglutarate, where malate can be converted into pyru-vate by malic enzyme, whereas oxolacetate can be con-verted to pyruvate by the concerted action of phos-phoenolpyruvate carboxykinase and pyruvate kinase. Thereentry of pyruvate into the TCA cycle in the form ofacetyl CoA is named ‘pyruvate recycling.’ Pyruvate recy-cling in brain was first reported by Cerdan et al. (1990).Such recycling of pyruvate was, however, not observedusing [U-13C]glucose in rabbits (Lapidot and Gopher,1994). The formation of lactate from astrocytic TCA cycleintermediates has been demonstrated in mouse brain (Has-sel et al., 1995). Later the presence of pyruvate recyclingwas reported in astrocytes both in vivo (Håberg et al.,1998) and in vitro (Sonnewald et al., 1996; Håberg et al.,1998). Also in the present study evidence for pyruvaterecycling in the formation of [2,3-13C]lactate was seen inastrocytes, but not in neurons, in agreement with an earlierstudy by Bakken et al. (1998). The physiological signifi-cance of such recycling, however, remains uncertain.

[U-13C]Glutamate and Glutamine in AstrocytesThe metabolic fate of exogenous glutamate was

shown to be concentration dependent in astrocytes (Mc-Kenna et al., 1996). At low concentrations the directconversion was dominant, and the amount of glutamatemetabolized via the TCA cycle increased progressively asthe incubation concentration increased from 0.1 mM to0.5 mM. Aspartate and lactate formation from glutamateoccurred only in astrocytes incubated with glutamate con-centration $0.2 mM. In the present study 0.5 mM[U-13C]glutamate was used in the incubation medium of

TABLE III. The Amount (nmol/mg Protein) of Metabolites inCerebellar Astrocytes*

Controln 5 3

Glutaminen 5 3

Glutamine 1 GABAn 5 3

Glutathione 100 6 9 53 6 11a 57 6 10a

Aspartate 54 6 6 135 6 19a 145 6 22a

Glutamate 403 6 40 348 6 37 403 6 69Glutamine 108 6 19 264 6 46a 262 6 38a

Alanine 11 6 1 38 6 9a 25 6 2GABA ND ND 22 6 3

*Astrocytes were incubated with [U-13C]glutamate (0.5 mM) with orwithout 2.5 mM glutamine or 2.5 mM glutamine plus 0.2 mM GABA for4 hr as described in Materials and Methods. Cell contents were analyzed byHPLC. Results are represented as mean 6 SEM. Superscript indicatesstatistical differences as determined by one-way ANOVA followed bypost-hoc test for multiple comparisons (P , 0.05 was considered signifi-cant).aSignificantly different from controls.

TABLE II. Content of 13C (nmol/mg Protein) in Metabolites From Lyophilized Cell Extracts of Cerebellar Astrocytes and NeuronsAfter Incubation With [U-13C]Glutamate Under Various Conditions*

Astrocytes Neuronsd

Controln 5 3

Glutaminen 5 3

Glutamine1 GABA

n 5 3Controln 5 3

GABAn 5 3

[U-13C]aspartate 32 6 2 80 6 10a 72 6 5a 36 6 6 37 6 8Labeled glutathione 63 6 3 41 6 4a 40 6 5a 29b 34 6 3[U-13C]glutamate 223 6 11 206 6 29 188 6 17 212 6 5 226 6 15[1,2,3-13C]glutamate 34 6 2 66 6 3a 57 6 4a 62 6 7 59 6 5[U-13C]glutamine 63 6 11 45 6 5 45 6 10 — —[1,2-13C]glutamate NQ NQ NQ 20 6 4 20 6 2% other pathways 67.6 6 2.1 53.8 6 1.4a 52.2 6 1.6a 83.6 6 1.8 76.7 6 0.2c

*Astrocytes were incubated with [U-13C]glutamate (0.5 mM) with or without 2.5 mM glutamine or 2.5 mM glutamine plus 0.2 mM GABA for 4 hr asdescribed in Materials and Methods. Neurons were incubated with [U-13C]glutamate (0.25 mM) with or without 50 mM GABA for 2 hr as described inQu et al., 2000. The C-3 resonance was used for 13C MR determination except that the C-4 glutamate resonance of glutathione and the C-4 resonanceof [U-13C]glutamine were used. Results are represented as mean 6 SEM. Superscript indicates statistical differences as determined by one-way ANOVAfollowed by the post-hoc Test for multiple comparisons (P , 0.05 was considered significant).aSignificantly different from controls in astrocytes.bQuantified in one out of three samples. NQ, not quantifiable.cSignificantly different from controls in neurons.dValues from Qu et al., 2000.

888 Qu et al.

cerebellar astrocytes to be able to evaluate the effects ofglutamine and GABA on TCA cycle activity and directconversions of glutamate in astrocytes.

Glutamine transporters have been identified on cer-ebellar granule neurons (Su et al., 1997; Varoqui et al.,2000) and on cerebellar astrocytes ( J. Albrecht, personalcommunication). The increased intracellular glutamineconcentration in the astrocytes in the presence of extra-cellular glutamine showed that glutamine, indeed, enteredthe cells. Even though the presence of PAG has not beenshown in vivo in astrocytes, it is present in the culturedcells because [U-13C]glutamine was metabolized exten-sively in cortical astrocytes (Sonnewald et al., 1996). In thepresent study it could be shown, that glutamine had aneffect on [U-13C]glutamate metabolism in the cerebellarastrocytes. Most strikingly, consumption of [U-13C]-glutamate was decreased in the presence of glutamine andrelease of labeled aspartate and [1,2,3-13C]glutamate de-creased, whereas the concentrations of these metabolitesincreased inside the cells. Thus it appears likely that glu-tamate generated from glutamine substituted for exoge-nous glutamate and reduced the consumption of[U-13C]glutamate. This is confirmed by the increasedconcentrations of glutamine, aspartate and alanine in thepresence of glutamine. The glutamate concentration was,however, unchanged pointing toward a rapid turnover. Ithas been shown earlier that [U-13C]glutamine is metabo-lized via the TCA cycle (Sonnewald et al., 1996) and canthus be used for energy production. In the present studyglutamine decreased the amount of [U-13C]glutamate me-tabolized in other pathways. This is in agreement with astudy by Sonnewald et al. (1996) where it was shown thatglutamate was metabolized in the TCA cycle to a largerextent than glutamine. Surprisingly glutathione concen-tration and labeling decreased in the presence of glu-tamine. Thus it appears that glutathione is preferentiallysynthesized from exogenous glutamate because the totalglutamate concentration was unchanged and an un-changed glutathione concentration should be expected.The reduced amount of glutathione points toward a com-partmentation of metabolism as has been demonstratedearlier (Sonnewald et al., 1998; Qu et al., 1999).

[U-13C]Glutamate and GABA in CerebellarAstrocytes and Neurons

GABA is released into the extracellular space byGABAergic neurons in a Ca21 dependent manner. Afterits release GABA can be transported back into theGABAergic neurons, into glutamatergic neurons or betransported into the surrounding astrocytes (Yu and Hertz,1982; Radian et al., 1990). Transporters are located onneurons and astrocytes (Borden, 1996; Schousboe, 2000)and the enzymes responsible for conversion of GABA tosuccinate are also located in both cell types (Larsson andSchousboe, 1990). Furthermore, GABAA receptors arepresent on neurons and astrocytes (Bovolin et al., 1992).When astrocytes were incubated with [U-13C]glutamate,glutamine and GABA, no differences in consumption andmetabolism were detected compared to the cells incubatedwith [U-13C]glutamate and glutamine alone. When, how-

ever, cerebellar granule neurons were incubated with[U-13C]glutamate and GABA less [U-13C]glutamate wasused for energy production compared to the cells incu-bated with [U-13C]glutamate alone. This could either bedue to the fact that GABA can be converted to succinateand can thus enter the TCA cycle for energy production,alternatively activation of GABAA receptors might causethe observed effect. The fact that the barbiturate thiopentalelicited no effect on the distribution of [U-13C]glutamateinto different pathways (Qu et al., 2001) indicates that thedecrease is caused by metabolism of GABA via the TCAcycle. Barbiturates are known to bind to GABAA recep-tors, and cause changes in conformation and ion ho-meostasis (Hertz, 1979; Frenkel et al., 1990; Franks andLieb, 1994).

Extracellular GABA had an effect on [U-13C]-glutamate metabolism and [U-13C]glutamate had an effecton GABA synthesis in cerebellar granule neurons. Surpris-ingly, [U-13C]GABA was detected in these glutamatergiccells (Qu et al., 2000) and the entry of extracellular GABAwas clearly seen in the dilution of label.

The role of GABA in metabolism is far from clear.Both a metabolic and neurotransmitter function has beenascribed to GABA (Martin and Barke, 1998) and com-partmentation of GABA metabolism has been shown inGABAergic cortex neurons (Waagepetersen et al., 2001).In the present study it could be shown that GABA also hada metabolic function in the glutamatergic cerebellar gran-ule neurons, but not in the astrocytes.

ACKNOWLEDGMENTThe excellent technical assistance of Bente Urfjell is

greatly appreciated.

REFERENCESAttwell D. 2000. Brain uptake of glutamate: food for thought. J Nutr

130:1023S–1025S.Bakken IJ, White LR, Aasly J, Unsgard G, Sonnewald U. 1998. [U-13C]

aspartate metabolism in cultured cortical astrocytes and cerebellar granuleneurons studied by NMR spectroscopy. Glia 23:271–277.

Borden LA. 1996. GABA transporter heterogeneity: pharmacology andcellular localization. Neurochem Int 29:335–356.

Bovolin P, Santi MR, Puia G, Costa E, Grayson D. 1992. Expressionpatterns of gamma-aminobutyric acid type A receptor subunit mRNAs inprimary cultures of granule neurons and astrocytes from neonatal ratcerebella. Proc Natl Acad Sci USA 89:9344–9348.

Cerdan S, Kunnecke B, Seelig J. 1990. Cerebral metabolism of [1,2-13C2]acetate as detected by in vivo and in vitro 13C NMR. J Biol Chem265:12916–12926.

Choi DD. 1988. Glutamate neurotoxicity and diseases of the nervoussystem. Neuron 1:623–634.

Drejer J, Larsson OM, Schousboe A. 1983. Characterization of uptake andrelease processes for D- and L-aspartate in primary cultures of astrocytesand cerebellar granule cells. Neurochem Res 8:231–243.

Erecinska M, Silver IA. 1990. Metabolism and role of glutamate in mam-malian brain. Prog Neurobiol 35:245–296.

Farinelli SE, Nicklas WJ. 1992. Glutamate metabolism in rat corticalastrocyte cultures. J Neurochem 58:1905–1915.

Fonnum F. 1984. Glutamate: a neurotransmitter in mammalian brain.J Neurochem 42:1–11.

Franks NP, Lieb WR. 1994. Molecular and cellular mechanisms of generalanesthesia. Nature 367:607–614.

Glutamate Metabolism in Cerebellar Cultures 889

Frenkel C, Duch DS, Urban BW. 1990. Molecular actions of pentobarbitalisomers on sodium channels from human brain cortex. Anesthesiology72:640–649.

Geddes JW, Wood JD. 1984. Changes in the amino acid content of nerveendings (synaptosomes) induced by drugs that alter the metabolism ofglutamate and g-aminobutyric acid. J Neurochem 42:16–24.

Gegelashvili G, Schousboe A. 1998. Cellular distribution and kinetic prop-erties of high-affinity glutamate transporters. Brain Res Bull 45:233–238.

Gegelashvili G, Danbolt NC, Schousboe A. 1997. Neuronal soluble factorsdifferentially regulate the expression of the GLT1 and GLAST glutamatetransporters in cultured astroglia. J Neurochem 69:2612–2615.

Gegelashvili G, Civenni G, Racagni G, Danbolt NC, Schousboe I,Schousboe A. 1996. Glutamate receptor agonists up-regulate glutamatetransporter GLAST in astrocytes. Neuroreport 8:261–265.

Gruetter R, Novotny EJ, Boulware SD, Mason GF, Rothman DL, Shul-man GI, Prichard JW, Shulman RG. 1994. Localized 13C NMR spec-troscopy in the human brain of amino acid labeling from D-[1-13C]glucose. J Neurochem 63:1377–1385.

Hassel B, Sonnewald U. 1995. Glial formation of pyruvate and lactate fromTCA cycle intermediates: implications for the inactivation of transmitteramino acids? J Neurochem 65:2227–2234.

Hawkins RA, DeJoseph MR, Hawkins PA. 1995. Regional brain gluta-mate transport in rats at normal and raised concentrations of circulatingglutamate. Cell Tissue Res 281:207–214.

Hertz L. 1979. Inhibition by barbiturates of an intense net uptake ofpotassium into astrocytes. Neuropharmacology 18:629–632.

Hertz L, Juurlink BHJ, Hertz E, Fosmark H, Schousboe A. 1989. Prepa-ration of primary cultures of mouse (rat): astrocytes. In: Shahar A, DeVellis J, Vernadakis A, Haber B, editors. A dissection and tissue culturemanual for the nervous system. New York: Alan R Liss, Inc. p 105–108.

Håberg A, Qu H, Bakken IJ, Sande LM, White LR, Haraldseth O, UnsgårdG, Aasly J, Sonnewald U. 1998. Pyruvate recycling in brain studied byNMR spectroscopy. Dev Neurosci 20:389–398.

Lapidot A, Gopher A. 1994. Cerebral metabolic compartmentation. Esti-mation of glucose flux via pyruvate carboxylase/pyruvate dehydrogenaseby 13C NMR isotopomer analysis of D-[U-13C]glucose metabolites. J BiolChem 269:27198–27208.

Larsson OM, Schousboe A. 1990. Kinetic characterization of GABA-transaminase from cultured neurons and astrocytes. Neurochem Res15:1073–1077.

Levy LM, Lehre KP, Walaas SI, Storm-Mathisen J, Danbolt NC. 1995.Down-regulation of glial glutamate transporters after glutamatergic de-nervation in the rat brain. Eur J Neurosci 7:2036–2041.

Martin DL, Barke KE. 1998. Are GAD65 and GAD67 associated withspecific pools of GABA in brain? Perspect Dev Neurobiol 5:119–129.

McKenna MC, Sonnewald U, Huang X, Stevenson J, Zielke HR. 1996.Exogenous glutamate concentration regulates the metabolic fate of glu-tamate in astrocytes. J Neurochem 66:386–393.

Meister A. 1974. Glutathione synthesis. Enzymes 10:671–697.Norenberg MD, Martinez-Hernandez A. 1979. Fine structural localization

of glutamine synthetase in astrocytes of rat brain. Brain Res 161:303–310.Oldendorf WH. 1971. Brain uptake of radiolabeled amino acids, amines

and hexoses after arterial injection. Am J Physiol 221:1629–1635.Olney JW. 1971. Glutamate-induced neuronal necrosis in the infant mouse

hypothalamus. An electron microscopic study. J Neuropathol Exp Neurol30:75–90.

Pascual JM, Carceller F, Roda JM, Cerdan S. 1998. Glutamate, glutamine,and GABA as substrates for the neuronal and glial compartments afterfocal cerebral ischemia in rats. Stroke 29:1048–1056.

Qu H, Le Maire T, van der Gaag M, Sonnewald U. 2001. The effect ofthiopental on [U-13C]glutamate metabolism in cultured cerebellar astro-cytes. Neurosci Lett 304:141–144.

Qu H, Waagepetersen HS, van Hengel M, Wolt S, Dale O, Unsgård G,Sletvold O, Schousboe A, Sonnewald U. 2000. Effects of thiopental on

transport and metabolism of glutamate in cultured cerebellar granuleneurons. Neurochem Int 37:207–215.

Qu H, Færø E, Jørgensen P, Dale O, Gisvold SE, Unsgård G, SonnewaldU. 1999. Decreased glutamate metabolism in cultured astrocytes in thepresence of thiopental. Biochem Pharmacol 58:1075–1080.

Radian R, Ottersen OP, Storm-Mathisen J, Castel M, Kanner BI. 1990.Immunocytochemical localization of the GABA transporter in rat brain.J Neurosci 10:1319–1330.

Schousboe A. 2000. Pharmacological and functional characterization ofastrocytic GABA transport: a short review. Neurochem Res 25:1241–1244.

Schousboe A, Meier E, Drejer J, Hertz L. 1989. Preparation of primarycultures of mouse (rat) cerebellar granule cells. In: Shahar A, de-Vellis J,Vernadakis A, Haber B, editors. A dissection and tissue culture manual forthe central nervous system. New York: Alan R Liss, Inc. p 203–206.

Schousboe A, Svenneby G, Hertz L. 1977. Uptake and metabolism ofglutamate in astrocytes cultured from dissociated mouse brain hemi-spheres. J Neurochem 29:999–1005.

Smith QR, Momma S, Aoyagi M, Rapoport SI. 1987. Kinetics of neutralamino acid transport across the blood-brain barrier. J Neurochem 49:1651–1658.

Sonnewald U, Hertz L, Schousboe A. 1998. Mitochondrial heterogeneityin the brain at the cellular level. J Cereb Blood Flow Metab 18:231–237.

Sonnewald U, Westergaard N, Schousboe A. 1997. Glutamate transportand metabolism in astrocytes. Glia 21:56–63.

Sonnewald U, Westergaard N, Jones P, Taylor A, Bachelard HS, Schous-boe A. 1996. Metabolism of [U-13C5] glutamine in cultured astrocytesstudied by NMR spectroscopy: first evidence of astrocytic pyruvaterecycling. J Neurochem 67:2566–2572.

Su TZ, Campbell GW, Oxender DL. 1997. Glutamine transport in cere-bellar granule cells in culture. Brain Res 757:69–78.

Tanaka K. 2000. Functions of glutamate transporters in the brain. NeurosciRes 37:15–19.

Tildon JT, Roeder LM. 1984. Glutamine oxidation by dissociated cells andhomogenates of rat brain: kinetics and inhibitor studies. J Neurochem42:1069–1076.

Varoqui H, Zhu H, Yao D, Ming H, Erickson JD. 2000. Cloning andfunctional identification of a neuronal glutamine transporter. J Biol Chem275:4049–4054.

Waagepetersen HS, Sonnewald U, Gegelashvili G, Larsson OM, SchousboeA. 2001. Metabolic distinction between vesicular and cytosolic GABA incultured GABAergic neurons using 13C magnetic resonance spectroscopy.J Neurosci Res 63:347–355.

Waniewski RA, Martin DL. 1986. Exogenous glutamate is metabolized toglutamine and exported by rat primary astrocyte cultures. J Neurochem47:304–313.

Westergaard N, Drejer J, Schousboe A, Sonnewald U. 1996. Evaluation ofthe importance of transamination versus deamination in astrocytic me-tabolism of [U-13C]glutamate. Glia 17:160–168.

Yu AC, Hertz L. 1982. Uptake of glutamate, GABA, and glutamine into apredominantly GABA-ergic and a predominantly glutamatergic nerve cellpopulation in culture. J Neurosci Res 7:23–35.

Yu AC, Schousboe A, Hertz L. 1982. Metabolic fate of 14C-labelledglutamate in astrocytes. J Neurochem 39:954–966.

Yudkoff M. 1997. Brain metabolism of branched-chain amino acids. Glia21:92–98.

Yudkoff M, Nissim I, Nelson D, Lin ZP, Erecinska M. 1991. Glutamatedehydrogenase reaction as a source of glutamic acid in synaptosomes.J Neurochem 57:153–160.

Zielke HR, Collins RM, Baab PJ, Huang Y, Zielke CL, Tildon JT. 1998.Compartmentation of [14C]glutamate and [14C]glutamine oxidative me-tabolism in the rat hippocampus as determined by microdialysis. J Neu-rochem 71:1315–1320.

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