Glutamine is the major precursor for GABA synthesis in rat neocortex in vivo following acute GABA-transaminase inhibition

Brain Research 919 (2001) 207–220 www.elsevier.com / locate / bres

Research report

Glutamine is the major precursor for GABA synthesis in rat neocortex in vivo following acute GABA-transaminase inhibition a, c b d, Anant B. Patel *, Douglas L. Rothman , Gary W. Cline , Kevin L. Behar * a

Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, CT, USA b Department of Internal Medicine, Yale University School of Medicine, New Haven, CT, USA c Departments of Neurology and Diagnostic Radiology, Yale University School of Medicine, New Haven, CT, USA d Department of Psychiatry, Yale University School of Medicine, New Haven, CT, USA Accepted 16 August 2001

Abstract The objective of the present study was to assess the degree to which astrocytic glutamine provides carbon for net synthesis of GABA in the rat neocortex in vivo. Isotopic labeling of GABA and glutamate from astrocytic glutamine was followed in halothane anesthetized and ventilated rats during an intravenous infusion of [2- 13 C]glucose. A net increase in GABA was achieved by administration of the GABA-transaminase inhibitor, gabaculine to suppress catabolism of GABA and recycling of 13 C label. 13 C Percentage enrichments of GABA, glutamate and glutamine were assessed in tissue extracts using 13 C-edited 1 H nuclear magneic resonance at 8.4 T. GABA levels increased 2.6 mmol / g at 2 h and 6.1 mmol / g at 5 h after gabaculine, whereas glutamate and glutamine decreased in toto by 5.6 mmol / g at 2 h and 3.1 mmol / g at 5 h. Selective enrichment of glutamine, glutamate, and GABA C3’s over other carbon positions was observed consistent with a precursor role for astrocytic glutamine. Between 1 h (control) and 3 h (gabaculine-treated) of [2- 13 C]glucose infusion, 13 C percentage enrichment increased in glutamine C3 (from 3.260.5 to 7.060.9%), glutamate C3 (from 1.860.5 to 3.460.9%), and GABA C3 (from 2.761.6 to 4.860.4%). The measured incremental [3- 13 C]GABA concentration (0.15 mmol / g) was close to the predicted value (0.13 mmol / g) that would be expected if the increase in GABA were produced entirely from glutamine compared to glutamate (0.07 mmol / g) based on the average precursor enrichments between 1 and 3 h. We conclude that glutamine is the major source of GABA carbon in the rat neocortex produced acutely following GABA-T inhibition by gabaculine in vivo.  2001 Elsevier Science B.V. All rights reserved. Theme: Neurotransmitters, modulators, transporters, and receptors Topic: GABA Keywords: g-Aminobutyric acid; Glutamic acid; Glutamine; Neuron–astrocyte interaction; Compartmentation; Substrate cycling; GABA-transaminase inhibitors; Gabaculine

1. Introduction g-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the mammalian central nervous system (CNS) [1,2] and is formed by the a-decarboxylation of

Abbreviations: GABA-T, GABA-transaminase; GAD, glutamate decarboxylase; GC–MS, gas chromatography–mass spectrometry; POCE, proton-observed carbon-13-edited; NMR, nuclear magnetic resonance *Corresponding authors. Magnetic Resonance Center, Yale University School of Medicine, New Haven, CT 06520, USA. Tel.: 11-203-7856199; fax: 11-203-785-6643. E-mail address: [email protected] (A.B. Patel).

glutamate in GABAergic neurons. GABA release by both vesicular and non-vesicular mechanisms is believed to be critical for maintaining normal inhibitory function [3,4]. The level of GABA appears to have a key role in maintaining normal GABA release [5]. Drugs, which raise GABA levels, have been shown in animals and humans to be effective anti epileptic medications, presumably through enhancing GABA release [6–8]. The regulation of cytosolic GABA concentration is complex involving control at the level of the synthesizing enzyme, glutamate decarboxylase (GAD) and possibly at sites along the pathways supplying precursor glutamate [9–12]. Levels of glutamate in GABAergic neurons appear to be lower than total tissue

0006-8993 / 01 / $ – see front matter  2001 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 01 )03015-3

208

A.B. Patel et al. / Brain Research 919 (2001) 207 – 220

levels. Selective immunostaining techniques show that GABA containing neurons generally have lower levels of glutamate immunoreactivity compared to other neurons [13,14]. 14 C-isotopic labeling studies of the rat brain by Patel et al. [15] suggested that a sizable fraction (|30%) of GABA is derived from a much smaller pool of glutamate than is measured in the whole tissue (i.e., ,3% of total glutamate or ,1 mmol / g). These results are consistent with findings in brain slices that GAD, which has a Km for glutamate of 0.2–1.2 mM, is probably not saturated with glutamate [16]. Thus, there appears to be a limited supply of endogenous glutamate available within the GABAergic neuron to maintain GABA levels when release demands are high. The source of the glutamate precursor used for the synthesis of GABA has been studied in vivo and in vitro. Early work by Van den Berg and co-workers using 14 C and 3 H isotopes suggested that glutamine was a precursor of GABA [17,18] laying the conceptual framework for a GABA neurotransmitter synthetic cycle involving glutamine. Subsequent studies using nerve terminal fractions [19], brain slices [16], cortical ‘GABAergic’ neurons [20–22], and 14 C-radiolabeled glutamine infused intraventricularly in vivo [23], have shown that glutamine is readily converted to GABA. However, glutamine may not be the only important precursor of GABA. Recent studies suggest that glutamate in the extracellular fluid (i.e., exogenous glutamate) may also be used in GABA synthesis based on the discovery of EAAC1 glutamate transporters on GABAergic neurons [24] and seizures in rats following manipulations that reduce EAAC1 protein [24– 27]. Thus, the quantitative significance of glutamine and glutamate as net precursors for GABA synthesis in vivo is far from clear. 13 C NMR has been used in studies of GABA metabolism in vitro [20,21,28] and in vivo [29,30]. By enriching specific carbons in glucose or other substrates (e.g., acetate) with 13 C the precursor pathways of GABA synthesis may in principle be determined by comparing the labeling patterns in GABA with glutamate and glutamine. A practical limitation in studies using [1- 13 C]glucose is that GABA is rapidly labeled through the glycolytic and TCA cycle pathways of the GABAergic neuron, obscuring labeling from exogenous sources. Therefore, it has not been possible to quantitatively separate the relative contributions of exogenous glutamate and glutamine to net GABA synthesis. In this study we have used a novel strategy for measuring the relative contributions of exogenous glutamate and glutamine to GABA synthesis in the cerebral cortex in vivo. 13 C NMR spectroscopy was used in conjunction with intravenously administered [2- 13 C]glucose to assess the quantitative role of astroglial glutamine as a precursor of GABA synthesis in the rat neocortex in vivo. [2- 13 C]Glucose specifically labels the astroglial TCA cycle intermediates, glutamate, and glutamine methylene carbon atoms (C-3 and C-2) through the catalytic actions of

pyruvate carboxylase and glutamine synthetase, which are expressed exclusively in astrocytes in mature brain [31– 33]. Neuronal glutamate is labeled secondarily from astrocytic glutamine by the action of phosphate-activated glutaminase (PAG); thus GABA labeled directly from glutamine or indirectly via neuronal glutamate will differ reflecting the contribution of each potential pathway to GABA synthesis. It should be noted that the relative flux through pyruvate carboxylase and pyruvate dehydrogenase limits the observed labeling of GABA and thus does not reflect the rate of GABA synthesis from glutamine. Net GABA synthesis was achieved by use of the GABAtransaminase inhibitor, gabaculine (3-amino-2,3dihydrobenzoic acid), to suppress the catabolism of GABA and recycling of the 13 C label.

2. Materials and methods

2.1. Animal preparation Two groups of male Sprague–Dawley rats fasted overnight, were studied. Control group (258613 g body wt.; mean6S.D., n54) infused with [2- 13 C]glucose for 1 h (n54) and 3 h (n54) (Fig. 1E) and group treated with gabaculine (264611 g, n55) infused with [2- 13 C]glucose for 3 h (n55) (Fig. 1E) and infused with [1- 13 C]glucose for 6 h (n53). All animals were anesthetized (induction) with 3% halothane in 30% oxygen and 67% nitrous oxide, tracheotomized and artificially ventilated. The femoral artery and either one or both (gabaculine treatment group) femoral veins were cannulated for the continuous monitoring of arterial blood pressure and gases and for the intravenous infusion of [2- 13 C]glucose and gabaculine, respectively. Halothane was reduced to 1% after completion of all surgery and animals were immobilized with D-tubocurarine-Cl, i.p. All physiological variables were maintained within normal limits throughout the experiments (Pa CO 2 535–42 mmHg; Pa O 2 .100 mmHg; pH 7.4060.05), and core body temperature was maintained |378C with a heating pad and a temperature-regulated circulating water bath. [2- 13 C]Glucose (99 atom%, Cambridge Isotopes) was dissolved in water and administered as a bolus of 48 mg in 0.27 ml in 15 s followed by a manual stepped reduction in infusion rate every 30 s for the next 8 min, whereupon the rate was constant until the time of in situ brain freezing. This protocol raises plasma glucose rapidly (,1 min) and maintains a constant level and enrichment thereafter [34]. In gabaculine-treated rats, gabaculine (100 mg / kg, i.v.) was administered after 1 h of the [2- 13 C]glucose or [113 C]glucose infusion and the glucose infusion was continued for 2 or 5 additional hours, respectively. Arterial blood samples were obtained periodically to measure plasma glucose concentration and isotopic enrichment,

A.B. Patel et al. / Brain Research 919 (2001) 207 – 220

209

Fig. 1. Qualitative depiction of different potential precursor pathways used for GABA synthesis during acute inhibition of GABA-T by gabaculine. (i) GABA synthesis from astrocytic glutamine (small astrocytic glutamate pool), (ii) GABA synthesis from exogenous glutamate derived from large neuronal glutamate pool, (iii) GABA synthesis from endogenous preexisting glutamate. Vcycle 5VGS 2Vcycle( Gln→GABA) , VGABA 5V GABA PAG 1VGT where Vcycle( Gln→GABA) 5 GABA–glutamine cycle rate, VGT 5GABA-T activity.

throughout the glucose infusion. At the end of the experiment, the brain was immediately frozen, in situ, in liquid nitrogen while mechanical ventilation was continued [35]. These experiments were approved by the Yale Animal Care and Use Committee.

2.2. Analysis of plasma extracts Plasma glucose concentrations were determined using Beckman Glucose Analyzer. Perchloric acid extracts of the plasma samples were prepared [36]. The 13 C enrichment of plasma glucose was determined using gas chromatog-

raphy–mass spectrometry (GC–MS) on a Hewlett-Packard (Palo Alto, CA, USA) 5890 gas chromatograph (HP-1 capillary column, 12 mm30.2 mm, 0.33 mm film thickness) interfaced to a Hewlett-Packard 5971 AMSD system (methane chemical ionization), as described by Cline et al. [37]. Ions with mass-to-charge ratios (m /z) of 331 and 332 were used to calculate the m11 enrichment of C2-glucose.

2.3. Analysis of brain extracts Ethanol extracts were prepared from frozen frontoparietal cortex (250–300 mg wet wt.) using a procedure

210

A.B. Patel et al. / Brain Research 919 (2001) 207 – 220

modified from that described by Dienel et al. [38]. The frozen cortical tissue was pulverized with ice-cold 0.1 M HCl in methanol (600 ml) using a glass homogenizer maintained in a dry-ice–ethanol bath. Following transfer to a wet-ice bath, 2 ml ice-cold ethanol solution (90% ethanol; 10% 100 mM phosphate buffer, pH 7) was added. The tissue was ground until no visible pieces remained and the solution remained in a wet-ice bath for 15 min with periodic mixing. The homogenate was centrifuged at |10000 g for 30 min at 48C. Following centrifugation the supernatant was removed and the pellet re-extracted as above from the point of adding 2 ml ethanol (60%) solution. Both supernatants were filtered (0.2 mm acrodisc syringe filter, Gelman Sciences, Ann Arbor, MI, USA), and passed through a 1-ml bed volume column of chelex100 resin (200–400 mesh, sodium form, pH 7; Bio-Rad Laboratories, Hercules, CA, USA). The samples were eluted from the column with 5 ml deionized water, neutralized and lyophilized. Following lyophilization, each sample was resuspended in a 2 H 2 O buffer (550 ml of 100 mM phosphate, pH 7, plus 4 mM TSP) for preliminary 1 H NMR analysis of the total sample before chromatographic separation. Following the initial 1 H NMR analysis, each sample was passed through an anion-exchange column to separate glutamate from glutamine and GABA. Pre-packed AG 1-X8 (200–400 mesh, 1.4 ml bed volume; Bio-Rad Laboratories) anion-exchange resin was converted from formate to the acetate form and washed thoroughly with deionized water. Glutamine and GABA were eluted with deionized water while glutamate was eluted with 2 M acetic acid. Both fractions were lyophilized and resuspended in buffered 2 H 2 O (550 ml of 100 mM phosphate buffer, 1 mM TSP) for NMR analysis.

2.4. NMR analysis NMR spectra were recorded at 8.4 T on an AM-360 wide-bore spectrometer operating at 360.13 MHz (Bruker Instruments, Billerica, MA, USA). Proton-observed, 13 Cedited NMR spectra of extracts were acquired under fully relaxed conditions as described [34]. From the initial extract spectra the absolute concentrations of glutamate and GABA were determined using a known concentration of TSP as an internal reference. The 13 C enrichments of C-3 and C-4 carbons of glutamate and glutamine, and C-2 and C-3 carbons of GABA were calculated from the ratio of the areas of these resonances in the POCE difference ( 13 C-labeled only) and the non-edited ( 12 C1 13 C) subspectrum. The concentration of [3- 13 C]glutamine was calculated from the product of the ratio of glutamine-to-glutamate [3- 13 CH 2 ] signal intensities in the POCE difference spectrum and the concentration of [3- 13 CH 2 ]glutamate. The concentration of [3- 13 CH 2 ]glutamate was calculated as the product of the total glutamate concentration and C-3 fractional enrichment. Total glutamine concentration was

calculated by dividing [3- 13 CH 2 ]glutamine concentration by the fractional enrichment of glutamine C-3. Prior to chromatography NOE-enhanced, 1 H-decoupled 13 C NMR spectra of brain extracts were acquired at 11.4 T ( 13 C frequency of 125 MHz) on an AM-500 NMR spectrometer (Bruker Instruments). Spectra were acquired fully relaxed (TR518.3 s, u 5908, total acquisition time of 20.4 h). NOE-enhanced, 1 H-decoupled 13 C NMR spectra of plasma extracts of blood samples obtained at the conclusion of each [2- 13 C]glucose infused experiment were also measured to determine potential scrambling of the label to other positions of the glucose molecule. The 13 C enrichment of glucose C-1 was determined through comparison of the C-1 and C-6 resonances in the 13 C spectrum (corrected for NOE and saturation) assuming C-6 represented natural abundance (1.1%).

2.5. Prediction of the source of precursor glutamate for GABA synthesis based on changes in percentage enrichment and concentrations of GABA, glutamate, and glutamine There are three potential sources of glutamate used in the synthesis of new GABA following GABA-T inhibition: (i) astroglial glutamine, (ii) exogenous glutamate released from glutamatergic neurons, and (iii) endogenous glutamate preexisting within the GABAergic neuron. These three pathways are schematically depicted in Fig. 1. A fourth potential pathway involving synthesis of GABA from polyamines (e.g., putrescine, spermine, and spermidine) is not considered here because the flux through this pathway appears to be very low relative to the pathway from glutamate in the adult rodent brain [39,40] and this pathway would not lead to any label incorporation into GABA. In order to determine the precursor source of the newly synthesized GABA after GABA-T inhibition we pre-labeled the glutamate and glutamine pools by beginning the infusion of the [2- 13 C]glucose 1 h prior to gabaculine administration (Fig. 2). The 13 C label in the internal positions (i.e., C-3 and C-2) of glutamate and GABA from [2- 13 C]glucose will arise from the metabolism of the labeled glucose in the astrocyte by action of pyruvate carboxylase and its conversion to glutamine labeled at C3 and C2. In contrast, metabolism of the [2- 13 C]glucose through pyruvate dehydrogenase in the TCA cycles of both astrocytes and neurons labels the C-5 carboxyl of glutamate which is lost as CO 2 in the next turn of the TCA cycle. Glutamine released from the astrocyte may be taken up by either GABAergic or glutamatergic neurons. In the glutamatergic neuron, the labeling of glutamate derived from C3 labeled glutamine will be diluted by exchange with the TCA cycle. Subsequently, C3 labeled glutamate in glutamatergic neurons may be released and taken up by the GABAergic neuron. In order to distinguish these pathways, the change in the concentration of [3- 13 C]GABA and total GABA concentration following

A.B. Patel et al. / Brain Research 919 (2001) 207 – 220

211

GABA-T inhibition were measured (trapping pool method) and compared to the 13 C labeling predicted if either: (i) astrocytic glutamine, (ii) exogenous glutamate, or (iii) endogenous glutamate served as sole precursor of the newly synthesized GABA. As described below, this method uses the measured percentage enrichment of C-3 of Glu, Gln and GABA and thus depends critically on the accuracy of the C-3 enrichment measurement. An independent assessment was made by using the percentage enrichment of C-3 and C-4 of glutamate or glutamine to predict the percentage enrichment ratios of C-3 to C-2 of GABA for different glutamate precursors for comparison with the measured ratio (ratiometry method).

2.5.1. Trapping pool method The trapping pool method is based on the assumption that gabaculine inhibits all further metabolism of GABA in the GABAergic neuron. Under these conditions the enrichment of any new GABA synthesized will be the same as that of its precursor. In order to determine the glutamate precursor source the percentage enrichment of glutamine or glutamate C-3 and the total increase in the concentration of GABA were used to calculate the predicted increase in the concentration of [3- 13 C]GABA assuming that all of the new GABA arose from one of these sources. For case (i), in which glutamine is the sole precursor for the incremental increase in GABA levels following GABA-T inhibition, the incremental [3- 13 C]GABA concentration is given by the following expression: ]] D[gaba3*] 5gln3* 3 D[gaba] (1) where D[gaba3*] is the change in [3- 13 C]GABA con]] centration, gln3* is the average percentage enrichment during the 2 h accumulation (average of glutamine C-3 enrichment at 1 h in the control and at 3 h in the gabaculine-treated rats), and D[gaba] is the change in the GABA concentration between the gabaculine-treated and control groups. Similarly, for case (ii), in which exogenous glutamate is the sole precursor of the incremental increase in GABA levels, the relationship is given by: ]] D[gaba3*] 5glu3* 3 D[gaba] (2)

Fig. 2. POCE NMR spectra of cortical extracts of control and gabaculine treated rats and schematic of experimental protocol. (A) Control, (B) gabaculine treated, (C) glutamine and GABA fraction from gabaculine treated rat, (D) glutamate fraction from gabaculine treated rat. For each pair of POCE spectra, the upper spectrum represents 13 C intensity while the lower spectrum total ( 13 C1 12 C) intensity. Peak labels refer to amino acids and carbon position. *Unassigned peak. (E) Experimental protocol scheme. Solid bar represents the [2- 13 C]glucose infusion time for the three experimental groups, while open bar depicts the period of gabaculine of treatment. The [2- 13 C]glucose infusion times in controls 1 and 2 were 1 and 3 h, respectively.

where the definitions of the terms are similar to Eq. (1). For case (iii), in which endogenous glutamate is the sole glutamate precursor for the synthesis of GABA, the incremental increase in the concentration of GABA was calculated by assuming that the percentage enrichment of glutamate C-3 (glu3) in the GABAergic neuron is equivalent to GABA C-3 (gaba3) and is given by: ]] D[gaba3*] 5gaba3* 3 D[gaba] (3) ]] where gaba3* is the average percentage enrichment of GABA C-3 at 1 h (control group) and at 3 h (gabaculinetreated group).

A.B. Patel et al. / Brain Research 919 (2001) 207 – 220

212

The incremental increase in the concentration of [3C]GABA can be obtained experimentally by the following expression:

2.6. Statistical analysis

13

D[gaba3*] 5 (gaba3*) 3 h 3 [gaba] 3 h 2 (gaba3*) 1 h 3 [gaba] 1 h

(4)

where (gaba3*) 1 h and (gaba3*) 3 h are the percentage enrichment of GABA C-3 at 1 h (control group) and 3 h (gabaculine-treated group), respectively; [gaba] 1 h and [gaba] 3 h are the concentrations of GABA at 1 h (control group) and 3 h (gabaculine group), respectively.

2.5.2. Ratiometry method In the ratiometry method the percentage enrichments of C-3 and C-4 of glutamine and glutamate were used to calculate the ratio of gaba3* / gaba2* that would be predicted to arise if glutamine, exogenous glutamate, or endogenous glutamate were to serve as the sole precursor for the incremental increase in GABA levels observed following inhibition of GABA-T. For the case in which glutamine serves as the sole precursor for the incremental increase in GABA levels, the ratio gaba3* / gaba2* is given by: ]] gaba3* gln3* 3 D[gaba] 1 gaba3* 3 [gaba] ]] 5 ]]]]]]]]]] (5) gaba2* ]] gln4* 3 D[gaba] 1 gaba2* 3 [gaba] ]] ]] where gln3* and gln4* represent the average percentage enrichments of glutamine C-3 and C-4 at 1 h of the [2- 13 C]glucose infusion in the control group and at 3 h in the gabaculine-treated group, respectively. The terms, gaba3* and gaba2*, represent the percentage enrichments of GABA C-3 and C-2, respectively, at 1 h of the [213 C]glucose infusion in the control group, D[gaba] is the incremental increase in concentration of GABA following gabaculine treatment, and [gaba] is the average concentration of GABA in the control group. A similar relationship was derived for the case in which exogenous glutamate serves as the sole precursor for GABA synthesis. However, to assess the case in which endogenous glutamate, i.e., glutamate in the GABAergic neuron, serves as the sole precursor for GABA synthesis, the following relationship was derived based on the assumption that the percentage enrichment of glutamate C-3 and C-4 present in the GABAergic neuron is the same as the percentage enrichment of GABA at C-3 and C-2, respectively. Thus, the expression relating these quantities is given by: ]] gaba3* gaba3* 3 D[gaba] 1 gaba3* 3 [gaba] ]] 5 ]]]]]]]]]]] (6) ]] gaba2* gaba2* 3 D[gaba] 1 gaba2* 3 [gaba] ]] ]] where gaba3* and gaba2* represent their average percentage enrichments at 1 h of [2- 13 C]glucose infusion in the control group and at 3 h in the gabaculine-treated group, respectively. The definitions of the other terms are given in Eq. (5).

The non-parametric, Mann–Whitney Test was used to determine the significance of differences among the different experimental groups for cerebral glutamate, glutamine and GABA concentrations and percent enrichments. This test was also carried out to determine the significance of differences of different predicted precursors (glutamate and glutamine) with the measured incremental [313 C]GABA concentration and gaba3* / gaba2* ratio.

3. Results

3.1. Effects of gabaculine on physiological variables In both the control and gabaculine-treated groups, arterial PO 2 .100 mmHg. No significant differences were noted between gabaculine-treated [MABP, 10468 mmHg; Pa CO 2 , 3568 mmHg; pH was 7.3560.08 (mean6S.D., n55)] and controls [MABP, 10668 mmHg; Pa CO 2 , 4068 mmHg; pH, 7.3460.06 (mean6S.D., n54)].

3.2. Plasma glucose concentration and percentage enrichment

13

C

Plasma glucose concentrations were increased rapidly by the [2- 13 C]glucose infusion from 5.660.6 to 11.661.5 mM (mean6S.D., n58) in the control group, and from 7.161.7 to 11.561.6 mM (mean6S.D., n55) in the gabaculinetreated rats. The percentage 13 C enrichment of [2- 13 C]glucose was 5469% (mean6S.D., n58) in controls and 5963% (mean6S.D., n55) in gabaculine-treated rats. The respective glucose levels and percentage 13 C enrichments remained relatively constant throughout the infusion period.

3.3. Effect of gabaculine on concentrations of cerebral amino acids No significant differences were revealed in the concentrations of GABA, glutamate, and glutamine in the 1 h and 3 h control groups (P.0.05; Mann–Whitney Test). Therefore, the glutamate, GABA, and glutamine concentration were calculated as the average of the 1 h and 3 h groups. Cortical GABA levels were increased significantly (P5 0.004; Mann–Whitney Test) in 2 h gabaculine-treated rats (3.7460.58 mmol / g, mean6S.D., n55) compared to controls (1.1260.34 mmol / g, n58) (Table 1). In contrast, cortical glutamate and glutamine levels were decreased significantly (P50.004; Mann–Whitney Test) in 2 h gabaculine-treated rats (8.2061.24 and 3.7661.06 mmol / g, respectively, mean6S.D., n55) compared to controls (10.7761.29 and 6.7561.06 mmol / g, respectively). After 5 h of gabaculine treatment, cortical GABA levels were

A.B. Patel et al. / Brain Research 919 (2001) 207 – 220

213

Table 1 Cortical concentrations (mmol / g) of cerebral glutamate, glutamine and GABA in control and gabaculine-treated rats following infusion of [2- 13 C]glucose

Glutamate Glutamine GABA

Control (n57)

Gabaculine (2 h, n55)

Gabaculine (5 h, n53)

10.7761.29 6.7561.06 1.1260.34

8.2061.24* 3.7661.06* 3.7460.58*

9.6160.18 3.9161.55* 8.0362.05*

Values are mean6S.D., n57 for control rats, and n55 and 3 for gabaculine treated rats for 2 and 5 h, respectively. *P,0.02 versus respective control value using the Mann–Whitney Test. Concentrations of amino were calculated using TSP as an internal reference.

elevated further (8.0362.05mmol / g, n53), whereas glutamine (3.9161.55 mmol / g, n53) levels remained significantly decreased (P50.017; Mann–Whitney Test) compared to control levels. However, there was no significant change (P50.138; Mann–Whitney Test) in the cortical glutamate concentration (9.6160.18 mmol / g, n53).

3.4. Comparison of the isotopic enrichments in control and gabaculine treated rats following [2 - 13 C] glucose infusion The percentage 13 C enrichments of glutamate and glutamine at C-3 and C-4 and GABA at C-2 and C-3 in gabaculine-treated rats are presented in Table 2. In control rats we observed an increase in the percentage enrichment with time (1 h and 3 h). In addition to labeling at C-3 in glutamate, glutamine and GABA (Fig. 2), we also observed labeling at C-4 of glutamate and glutamine and at C-2 of GABA, which is not expected for label incorporation through either pyruvate carboxylase or pyruvate dehydrogenase pathways using [2- 13 C]glucose as precursor. Acid extracts of the plasma of these animals revealed the presence of a small amount of labeling in glucose C-1 (1.2960.64% above natural abundance, mean6S.D., n5 4), suggesting that systemic label scrambling had occurred. As described in detail by Sibson et al. [41], the 13 C labeling of brain glutamate and glutamine at C-4 and GABA at C-2 from [2- 13 C]glucose can be attributed to a combination of label scrambling in the blood, presumably due to hepatic gluconeogenesis, and to the cerebral pentose phosphate cycle. In gabaculine treated rats the percentage enrichments of both glutamine C-3 (5.260.4 to 7.160.9%) and GABA C-3 (3.160.7 to 4.860.4%) increased significantly (P, Table 2 Percentage

13

0.05; Mann–Whitney Test) compared to controls. However, no significant (P50.22) change in the percentage 13 C enrichment of glutamate C-3 was observed (3.260.9%, control verses 3.260.2%, gabaculine-treated) between the two groups.

3.5. Evaluation of the source of precursor glutamate for GABA synthesis The selective enrichment of GABA C-3 over other carbon positions after administration of gabaculine during the [2- 13 C]glucose infusion suggests that astrocytic glutamine, either directly or indirectly through neuronal glutamate, serves as precursor of the glutamate used in GABA synthesis. Using the changes in the percentage enrichment and concentration of glutamine, GABA, and glutamate during the labeled glucose infusion, we calculated the predicted incremental changes in labeled GABA expected depending on precursor supply. The experimental and predicted increments in the concentration of [313 C]GABA are presented in Table 3. The predicted incremental change in the concentration of [3- 13 C]GABA (0.1360.02 mmol / g) assuming that glutamine is the sole precursor was not significantly different (P50.60; Mann– Whitney Test) from the experimentally measured increase (0.1560.04 mmol / g) in the [3- 13 C]GABA concentration. However, the predicted incremental change in concentration of [3- 13 C]GABA (0.0760.02 mmol / g) assuming that glutamate was the sole precursor is significantly less (P50.009; Mann–Whitney Test) than the experimental value (0.1560.04 mmol / g). These results suggest that glutamine is the major source of GABA produced acutely following inhibition of GABA-T by gabaculine. A similar conclusion was reached by use of the

C enrichment of cortical, glutamate, glutamine, and GABA in control and gabaculine-treated rats following infusion of [2- 13 C]glucose

Glutamate C3 Glutamate C4 Glutamine C3 Glutamine C4 GABA C2 GABA C3

Control (1 h, n54)

Control (3 h, n54)

Gabaculine (3 h, n55)

1.860.8 1.960.6 3.260.5 1.160.2 1.760.5 1.761.6

3.260.2 2.560.2 5.260.4 2.260.2 3.460.7 3.160.7

3.460.9 3.160.6 7.160.9* 2.760.2* 2.560.4 4.860.4*

All values (mean6S.D.) are expressed as atom percent enrichment with 13 C above natural abundance (1.1%) in control rats (n54), and gabaculine treated rats (n55). Percentage enrichments were determined from POCE spectra as described in Materials and methods. *P,0.05 versus respective control value using the Mann–Whitney Test.

A.B. Patel et al. / Brain Research 919 (2001) 207 – 220

214

Table 3 Measured and predicted, incremental [3- 13 C]GABA concentration (trapping pool approximation) and gaba3* / gaba2* ratio (ratiometry approximation) during acute GABA-T inhibition by gabaculine for 2 h following infusion of [2- 13 C]glucose in the rat cortex 1

D[3- 13 C]GABA (mmol / g) gaba3* / gaba2*

Measured

0.1560.04 1.9060.14

2

Predicted for

Glutamine

Glutamate exogenous

Glutamate endogenous

0.1360.02 1.9060.07

0.0760.02* 0.9560.14*

0.08 1.05

*P50.009 versus respective measured values by Mann–Whitney Test. 1 The measured increase in [3- 13 C]GABA (D[3- 13 C]GABA, n55) during (2 h) acute inhibition of GABA-T by gabaculine was calculated using Eq. (4). 2 Predicted values (n55) of D[3- 13 C]GABA for precursors glutamine, glutamate exogenous and glutamate endogenous were calculated using Eqs. (1), (2) and (3), respectively. Predicted gaba3* / gaba2* (n55) ratio for precursors glutamine and glutamate endogenous were calculated using Eqs. (5) and (6), respectively.

ratiometry method, which considers the changes in percentage enrichment of C-3 and C-4 of glutamine or glutamate and C-3 and C-2 of GABA. It is clear from Table 3 that the predicted ratio of gaba3* / gaba2* assuming that glutamine is the sole precursor (1.9060.07) was not significantly (P50.68; Mann–Whitney Test) different from the experimentally measured value (1.9060.14). However, when glutamate was considered as the sole precursor of GABA, the calculated ratio (0.9460.16) was considerably less (P50.009; Mann–Whitney Test) than the experimentally measured ratio (1.9060.07). Thus, these results also support the conclusion that glutamine is the major source of GABA produced during acute inhibition of GABA-T by gabaculine.

4. Discussion

4.1. Evidence for utilization of glutamine and glutamate in GABA synthesis in vivo The source of the glutamate precursor for GABA synthesis has been a subject of study in vivo and in vitro for over 30 years. Van den Berg and Garfinkel, as early as in 1971 [18], proposed that glutamine served as a precursor for GABA. Soon afterwards, Reubi et al. [42] reported that glutamine was an efficient precursor of released glutamate and GABA in pigeon brain slices. Intrastriatal administration in rats of methionine sulfoximine (MSO) to inhibit glutamine synthetase [43] or application of MSO or an inhibitor of neuronal glutaminase in rat brain slices [16] results in a major reduction in GABA synthesis suggesting that astroglial glutamine is a quantitatively important precursor of GABA. In neuron and astrocyte co-cultures 13 C-labeling of GABA from the astroglial substrate 13 Clabeled acetate is significantly decreased following MSO treatment [20], consistent with glutamine being an important GABA precursor. Although isotopic labeling studies in cultured neurons [77] and isolated nerve terminals [69] show that glutamine can serve as a precursor for GABA, the results from labeling experiments done in the intact brain have not clearly established its quantitative significance. Preece and Cerdan [30] compared multiplet patterns of GABA C-2 with glutamate and glutamine C-4

following infusions of [1- 13 C]glucose and [1,2- 13 C]acetate and concluded that GABA originated mainly from the ‘large’ pool of neuronal glutamate and not from the ‘small’ glial glutamate pool based on the similarity between the GABA C-2 and glutamate C-4 multiplets which differed from glutamine C-4. Similar labeling patterns and percentage enrichments between brain GABA and glutamate during infusions of [1- 13 C]glucose in vivo have also been reported by us and others [29,44,45]. A limitation of previous studies of GABA precursor sources in vivo is that the label, which enters GABA from exogenous precursors, could be further scrambled or diluted by metabolic processes in the GABAergic neuron. Thus, the 13 C labeling of GABA would not necessarily be the same as the labeling of its precursors. The labeling from [2- 13 C]glucose through astroglial-specific pyruvate carboxylase was used primarily to produce a difference in the label patterns of glutamate and glutamine [41,75], which permitted these precursors to be distinguished when incorporated into GABA. The deconvolution of label entry from [1- 13 C]glucose through pyruvate carboxylase and pyruvate dehydrogenase into glutamine and glutamate is not necessary in order to determine their contribution to net GABA synthesis. However, we have shown in non-gabaculine treated rats [41] that the much greater efficiency of labeling from [1- 13 C]glucose is due to the higher flux through pyruvate dehydrogenase compared to pyruvate carboxylase. Evidence for a role of exogenous glutamate in the synthesis of GABA has come from the discovery of EAAC1 (EAAT3) glutamate transporters on GABAergic neurons [24,46]. Administration of antisense oligonucleotides to block EAAC1 mRNA and transporter protein in rats or transgenic knockout of EAAC1 in mice induces seizures [25,26] and in hippocampus alters electrophysiological properties consistent with reduced GABAergic function [47]. The glutamate receptor agonist and epileptigen, kainic acid, decreases EAAC1 mRNA and protein [26] suggesting that exogenous glutamate may have a role in the regulation of GABA synthesis. Taken together, these studies indicate that the role of exogenous glutamate and glutamine as precursors for GABA synthesis is far from clear. In the present study we used the GABA-T inhibitor, gabaculine, to block further metabolism of GABA and trap

A.B. Patel et al. / Brain Research 919 (2001) 207 – 220

the isotopic label in GABA. The labeling of the GABA, which accumulated after the block, indicates that in the cerebral cortex glutamine is the major precursor for net GABA synthesis. Some implications of these findings, as well as the limitations of the method, are discussed below.

4.2. Evidence for the role of glutaminase in the synthesis of GABA Metabolic and enzymatic studies of cultured cortical neurons, which possess a GABAergic phenotype [82], show these cells to have the essential transport [76] and enzyme capacity to metabolize glutamine [81,82]. Glutamine metabolism in cortical ‘GABAergic’ neurons is catalyzed primarily by phosphate-activated glutaminase (PAG) [81], which is present at sufficient activity to support a rate of glutamine removal equal to GABA synthesis [83]. The rates of glutamine uptake and utilization reported in cortical GABAergic neurons of 1.8 nmol / min / mg protein [77] represents only about 12% of the maximal (Vmax -like) PAG activity of 14.9 nmol / min / mg protein. In addition to PAG, glutamine transaminase K and v-amidase, which together are capable of converting glutamine to a-ketoglutarate (a-KG), are present in cortical neurons [89–91], although the precise cellular location of these enzymes and their functional role(s) in vivo is not clearly known. Thus PAG alone could readily account for the rates of hydrolysis of glutamine in GABAergic neurons in vitro and the rate of glutamine-to-GABA cycling (Vcycle( Gln→GABA ) found in the present study. Although cortical GABAergic neurons possess significant PAG activity in vitro, immunohistochemical studies of presumed GABAergic cells in situ using antibodies against PAG have reported both PAG-positive [84–86] and PAG-negative [87,88] findings. The absence of PAG-like immunostaining of neocortical non-pyramidal neurons, which stained positively for GAD or GABA [88] or for peptides known to co-localize with GABA [87], prompted Kaneko et al. [87,88] to conclude that substrates other than glutamine (e.g., a-KG) provided precursors for GABA in these cells. It is important to note however that the lack of PAG-like immunostaining cannot be equated with the absence of enzyme activity, because the threshold for quantitation of immunostaining in terms of enzyme activity is not generally known. The inconsistency between the results of metabolic and inhibitor studies in vitro and in vivo on the one hand and in situ immunohistochemistry on the other is not clear. The utilization of astroglial precursors other than glutamine in GABA synthesis cannot be excluded in the present study because these substrates would also label GABA. However, if the 13 C-labeled precursor of GABA had arisen from an astroglial metabolite upstream of glutamine (e.g., a-KG), then we would expect the enrichment of GABA C3.Gln-C3, reflecting the more highly enriched smaller pools of astroglial TCA cycle intermediates and the large dilution obtained upon entry of the

215

label into the glutamine pool. This result was not observed suggesting that glutamine was the more likely precursor of GABA.

4.3. Metabolic cycles involved in GABA precursor synthesis It is now well established that glutamate, GABA and glutamine participate in substrate cycles between neurons and astrocytes [48]. Glutamate released from neurons is transported into astrocytes and converted to glutamine, which may serve as a precursor for the replenishment of neurotransmitter glutamate and GABA. Like glutamate, GABA is readily transported into astrocytes 1 [49–51], and its catabolism in the astrocytic TCA cycle results in the molar equivalent production of glutamate from a-ketoglutarate, which is then available for glutamine synthesis [52]. Recently, we showed that the glutamate / GABA– glutamine cycling flux was much greater than expected and was linearly related to glucose oxidative metabolism [41,53,54]. Thus, mass flows of GABA and glutamate between neurons and astrocytes are highly interrelated. The supply of glutamate precursors used in net GABA synthesis must arise ultimately from the exogenous substrates, glutamine and / or glutamate. While the necessary enzymes are present in brain for the synthesis of GABA from putrescine and other polyamines (e.g., spermine and spermidine) [39], the synthesis of GABA from these sources appear to be very low in the adult brain, in contrast to the developing brain or the retina where putrescine may be a quantitatively significant precursor [55]. Noto et al. [40] have estimated that under normal conditions |1% of total GABA synthesis in adult rat brain could be supplied by putrescine, whereas the majority (99%) arises from glutamate. Endogenous glutamate levels in GABAergic neurons appeared insufficient to account for the observed GABA synthesis (Table 3), consistent with the view that glutamate levels in GABAergic neurons are significantly lower than total tissue glutamate. As depicted in Fig. 2 (scheme i), to maintain total mass balance, the uptake of glutamine released from astrocytes to the extracellular space is divided between glutamatergic and GABAergic neurons. The rate of uptake into glutamatergic neurons is given by Vcycle( Gln→Glu) , where Vcycle 5VGS 2Vcycle( Gln→GABA) and Vcycle( Gln→GABA) is the rate of uptake of glutamine into GABAergic neurons. Based on the standard measurement error, Vcycle( Gln→GABA) constitutes at least 85% of net GABA synthesis after gabaculine treatment, so that shunting of glutamine carbon through the large neuronal glutamate pool in route to the GABAergic neuron could contribute at most 15% of the total GABA synthesis in the 1

GABA-positive immunolabeling is detected in cortical astrocytes of rat brain 24 h following treatment with g-vinyl GABA (K.L. Behar, M. Schwartz, work in preparation).

216

A.B. Patel et al. / Brain Research 919 (2001) 207 – 220

cortex. Thus, the rate of GABA synthesis will depend not only on GAD but also on the supply of astroglial precursors through GABA–glutamine cycling. To the extent that Vcycle( Gln→Glu) is partitioned through the GABAergic neuron, by uptake of exogenous glutamate released from glutamatergic neurons (Fig. 1, scheme ii), the activity of EAAC1 glutamate transporters may exert another level of control. An estimate of the contribution of GABA–glutamine cycling to the overall glutamate–glutamine cycling flux can be determined from the gabaculine-induced rise in GABA. Because GABA-T inhibition may be incomplete, the rise in GABA reflects a minimum value of the rate of GABA–glutamine cycling. The rate of GABA accumulation (0.02 mmol / min / g) is |5% of the glutamate– glutamine flux reported for the morphine-anesthetized rat cortex (0.44 mmol / min / g [54]) suggesting that the GABA–glutamine cycling flux represents a comparable fraction. Under non-inhibited conditions the relative amount of released GABA which is taken back up by the GABAergic neurons to the amount which is taken up by the astrocytes cannot be determined from this study, because the reuptake flow was not measured and gabaculine may alter the pathway fluxes of glutamate and GABA metabolism. However if the present findings apply to the non-inhibited state the rate of the GABA–glutamine cycle is a significant fraction of total GABA synthesis, on the order of 10–20%, and therefore is a significant pathway of GABA repletion.

4.4. Net supply of carbon skeletons for GABA synthesis during GABA-T inhibition The change in the amino acid levels following acute GABA-T inhibition is consistent with glutamine being the major direct precursor for the increase in GABA. Whereas the early decrease in the sum of Dglutamine1Dglutamate (25.6 mmol / g) 2 h after gabaculine treatment significantly exceeded the increase in GABA (12.6 mmol / g), the opposite pattern was observed at later times. Five hours after gabaculine treatment the net decrease in Dglutamine1Dglutamate compared to control (23.2 mmol / g) was less than the increase in GABA (16.1 mmol / g). Similar results have been reported by Pierard et al. [56], where it was shown that the total decrease in glutamine1glutamate leveled off at 2–3 h after gabaculine treatment in contrast to a steadily increasing GABA level. Such results would be expected if glutamine were the major precursor of the increased GABA carbon under these conditions. The gabaculine-induced block of GABA catabolism prevents the carbon skeletons from being returned to the astrocyte via the GABA / glutamine cycle, effectively trapping astrocyte-derived carbon in the GABA pool. Decreased levels of glutamate may reflect decreased PAG activity in glutamatergic neurons (i.e., large pool glutamate) due to a decreased supply of astroglial

glutamine, as glutamate released by the neurons is no longer replaced by the glutamate / glutamine cycle. Cortical glutamine concentration decreased substantially (by 3 mmol / g) after gabaculine-treatment. Glutamine in cerebral extracellular fluid (ECF), which can be considered a more direct precursor of neuronal glutaminase, also decrease after gabaculine treatment in rats [56]. The Km of PAG for glutamine (1.6–4 mM [79–80]) is higher than physiologically normal levels of ECF glutamine (|0.4 mM), suggesting that a reduction in ECF glutamine could reduce the rate of glutamine hydrolysis by this enzyme. The rate of the glutamate / glutamine cycle (0.44 mmol / g / min [54]), which is equivalent to the flux through PAG, is much less than the maximum capacity of this enzyme (Vmax |5 mmol / min / g measured in brain homogenate [79]) indicating that PAG is significantly inhibited in vivo. The rapid decrease in both glutamate and glutamine following gabaculine is consistent with the high glutamate–glutamine cycling flux reported in recent 13 C NMR studies [35,54,57]. The net increase in carbon skeletons that appears at later times suggests that the anaplerotic flux has increased in astrocytes. For example, the percentage 13 C enrichment of cortical glutamate C-3 after a 3 h infusion of [2- 13 C]glucose is similar in control (3.260.2%) and gabaculinetreated (3.460.9%) rats. In contrast the enrichment of both glutamine C-3 (7.060.9%) and GABA C-3 (4.860.4%) are significantly (P,0.05, Mann–Whitney Test) greater in gabaculine-treated compared to control (glutamine C-3, 5.260.4%; GABA C-3, 3.160.7%). Because 13 C labeling of glutamine C-3 and GABA C-3 arises primarily from anaplerosis (astrocytic pyruvate carboxylase), the results suggest that pyruvate carboxylase flux is increased at this time and may explain the discrepancy in carbon balance we observed following gabaculine treatment.

4.5. Effect of incomplete GABA trapping The primary assumption in the labeling analysis is that GABA-T inhibition by gabaculine was complete, effectively inhibiting further metabolism of GABA. Partial GABAT inhibition would allow further metabolism of GABA in the TCA cycle, resulting in the incorporation of GABA carbon into aKG, glutamate, and ultimately back into GABA. The effect of this recycling on the isotopic labeling pattern of GABA would be a dilution of the isotopic labeling at C3 and C2 relative to an exogenous precursor due to unlabeled carbon entering through pyruvate dehydrogenase [41]. If glutamine is the exogenous precursor, then the recycling will result in an underestimate of the actual percentage of GABA derived from glutamine because the dilution will make the labeling more similar to glutamate. The major complication presented by label entry from [1- 13 C]glucose into glutamine and glutamate for both ratiometry (C3 / C2) and trapping pool (D[3- 13 C]GABA) approaches will occur when GABA-T inhibition is incom-

A.B. Patel et al. / Brain Research 919 (2001) 207 – 220

217

plete, which we assumed to be complete in the derivation of the equations. The effect of incomplete GABA-T inhibition on GABA labeling permits shunting through the TCA cycle with a consequent loss of the C3 label (to C4 and CO 2 ) and asymmetry as depicted in the C3 / C2 ratio. To the extent that GABA-T inhibition is incomplete, the contribution of glutamine to GABA synthesis will be underestimated as glutamine has a larger C3 / C4 ratio and higher C3 enrichment than glutamate.

These studies show that an increased CNS level of GABA is beneficial in the treatment of epilepsy and may be also in other neurological disorders involving deficits in GABAergic function. Much interest has been focused on ways to enhance the GABA concentration in the CNS. The present findings suggest that the effectiveness of drugs in elevating GABA concentration will also depend on the availability of glutamate precursors supplied by the GABA–glutamine cycle.

4.6. Role of GAD isoforms and glutamate precursors in the regulation of GABA synthesis in vivo

Acknowledgements

The synthesis of GABA from glutamate is mediated by GAD which in brain exists as two major isoforms, GAD 67 and GAD 65 [58]. These two isoforms are the products of separate gene [59] and differ in their kinetic properties and subcellular localization [12,60]. GAD 67 is widely distributed throughout the GABAergic neuron, whereas GAD 65 is enriched in nerve terminals and is associated with synaptic vesicles [58]. Manor et al. [29] showed that a major decrease in GABA synthesis occurred under conditions that reduce GAD 67 but not GAD 65 protein [61], suggesting that GAD 67 mediated the majority of GABA synthesis in vivo. Results from studies of GAD 67 and GAD 65 knockout mice [62–64] have shown that GAD 67 is associated with the major pool of brain GABA. The present results extend these findings to suggest that glutamine is the major precursor of glutamate used in GAD 67 mediated synthesis of GABA, thus linking the synthesis of ‘cytoplasmic’ GABA to GABA–glutamine cycling. Although speculative, the reported findings that GAD 65 and EAAC1 glutamate transporter proteins are concentrated in GABAergic nerve terminals [24,78], and that seizures and altered GABA function develop in knockout models of either protein, suggests that GAD 65 could mediate vesicular synthesis of GABA from exogenous glutamate produced and released by glutamatergic neurons. Alternatively, the relative usage of exogenous glutamate and glutamine in GABA synthesis may vary with brain region with increased fractions of glutamate used in the hippocampus and cerebellum [65,66].

4.7. Implications of GABA–glutamine cycling for the pharmacotherapeutic use of GABA elevating drugs in epilepsy and affective disorders With the availability of new NMR spectroscopic editing pulse sequences it is now possible to measure brain GABA concentration non-invasively in human brain [67]. Occipital cortical GABA has been shown to be lower in patients with epilepsy and depressive and panic disorder, compared to healthy subjects [68–71]. GABA levels are responsive to anti-epileptic drugs such as vigabatrin, topiramate, or gabapentin and in epilepsy patients, seizure control improves with increasing GABA concentration [5–7,72–74].

The authors wish to acknowledge Ms. Bei Wang for the skillful preparation of animals used in the study and the discussion and support of Professor Robert G. Shulman. This work was supported by NIH grants R01 NS34813 and DK27121.

References [1] K. Krnjevic, Glutamate and g-aminobutyric acid in brain, Nature 228 (1970) 119–124. [2] D.A. McCormick, GABA as inhibitory neurotransmitter in human cerebral cortex, J. Neurophysiol. 62 (1989) 1018–1027. [3] J.D. Kocsis, R.H. Mattson, GABA levels in the brain: a target for new antiepileptic drugs, Neuroscientist 6 (1996) 326–334. [4] G.B. Richerson, H.L. Gaspary, Carrier-mediated GABA release: is there a functional role?, Neuroscientist 3 (1997) 151–157. [5] O.A. Petroff, F. Hyder, R.H. Mattson, D.L. Rothman, Topiramate increases brain GABA, homocarnosin, and pyrrolidone in patients with epilepsy, Neurology 52 (1999) 473–478. [6] Z. Liu, N. Seiler, C. Marescaux, A. Depaulis, M. Vergnes, Potentiation of g-vivnyl GABA (vigabatrin) effects by glycine, Eur. J. Pharmocol. 182 (1990) 109–115. [7] O.A. Petroff, D.L. Rothman, K.L. Behar, D. Lamoureux, R.H. Mattson, The effect of gabapentin on brain gamma-aminobutyric acid in patients with epilepsy, Ann. Neurol. 39 (1996) 95–99. [8] O.A. Petroff, D.L. Rothman, K.L. Behar, R.H. Mattson, Human brain gamma-aminobutyric acid (GABA) levels and seizure control following initiation of vigabatrin therapy, J. Neurochem. 67 (1996) 2399–2404. [9] H.F. Bradford, H.K. Ward, H.K. Thomas, Glutamine – a major substrate for nerve endings, J. Neurochem. 30 (1978) 1453–1459. [10] J.C. Szerb, Rate limiting steps in the synthesis of GABA and glutamate, in: M. Avoli, T.A. Reader, R.W. Dykes, P.W. Gloor (Eds.), Neurotransmitters and Cortical Function, Plenum Press, New York, 1988, pp. 153–166. [11] J. Litwak, M. Mercugliano, M.-F. Chesselet, G.A. Oltmans, Increased glutamic acid decarboxylase (GAD), mRNA, and GAD activity in cerebellar Purkinje cells following lesion induced increase in cell firing, Neurosci. Lett. 116 (1990) 179–183. [12] D.L. Martin, K. Rimvall, Regulation of g-aminobutyric acid synthesis in the brain, J. Neurochem. 60 (1993) 395–407. [13] J. Storm-Mathisen, A.K. Leknes, A.T. Bore, J. Vaalund, P. Edminsion, F.-M.S. Haug, O.P. Otterson, First visualization of glutamate and GABA in neurons by immunocytochemistry, Nature 301 (1983) 517–520. [14] O.P. Ottersen, J. Storm-Mathisen, Glutamate- and GABA-containing neurons in mouse and rat brain as demonstrated with new immunocytochemical technique, J. Comp. Neurol. 229 (1984) 374–392.

218

A.B. Patel et al. / Brain Research 919 (2001) 207 – 220

[15] A.J. Patel, A.L. Johnson, R. Balazs, Metabolic compartmentation of glutamate associated with the formation of g-aminobutyrate, J. Neurochem. 23 (1974) 1271–1279. [16] G. Battaglioli, D.L. Martin, GABA synthesis in brain slices is dependent on glutamine produced in the astrocytes, Neurochem. Res. 16 (1991) 151–156. [17] C.J. Van den Berg, L. Krzlic, P. Mela, H. Waelsch, Compartmentation of glutamate metabolism in brain. Evidence for the existence of two different tricarboxylic acid cycles in brain, Biochem. J. 113 (1969) 281–290. [18] C.J. Van den Berg, D. Garfinkel, A stimulation study of brain compartments: metabolism of glutamate and related substances in mouse brain, Biochem. J. 123 (1971) 211–218. [19] R.P. Shank, G.L. Campbell, Glutamine and alpha-ketoglutarate uptake and metabolism by nerve terminal enriched material from mouse cerebellum, Neurochem. Res. 7 (1982) 601–616. [20] U. Sonnewald, N. Westergaard, A. Schousboe, J.S. Svendsen, G. Unsgard, S.B. Petersen, Direct demonstration by [ 13 C]NMR spectroscopy that glutamine from astrocytes is a precursor for GABA synthesis in neurons, Neurochem. Int. 22 (1993) 19–29. [21] U. Sonnewald, I.S. Gribbestad, N. Westergaard, G. Nilsen, G. Unsgard, A. Schousboe, S.B. Petersen, Nuclear magnetic resonance spectroscopy: biochemical evaluation of brain function in vivo and in vitro, Neurotoxicology 15 (1994) 579–590. [22] H.S. Waagepetersen, I.J. Bakken, O.M. Larsson, U. Sonnewald, A. Schousboe, Metabolism of lactate in cultured GABAergic neurons studied by 13 C nuclear magnetic resonance, J. Cereb. Blood Flow Metab. 18 (1998) 109–117. [23] H.K. Ward, C.M. Thanki, H.F. Bradford, Glutamine and glucose as precursors of transmitter amino acids: ex vivo studies, J. Neurochem. 40 (1983) 855–860. [24] F. Conti, S. DeBiasi, A. Minelli, J.D. Rothstein, M. Melone, EAAC1, a high-affinity glutamate transporter, is localized to astrocytes and gabaergic neurons besides pyramidal cells in the rat cerebral cortex, Cereb. Cortex 8 (1998) 108–116. [25] J.D. Rothstein, M. Dykes-Hoberg, C.A. Pardo, L.A. Bristol, L. Jin, R.W. Kuncl, Y. Kanai, M.A. Hediger, Y. Wang, J.P. Schielke, D.F. Welty, Knockout of glutamate transporters reveals a major role for astroglial transport in excitotoxicity and clearance of glutamate, Neuron 16 (1996) 675–686. [26] M.T. Akabar, M. Rattray, R.J. Williams, N.W. Chong, M.S. Meldrum, Reduction of GABA and glutamate transporter messenger RNAs in the severe-seizure genetically epilepsy-prone rat, Neuroscience 85 (1998) 1235–1251. [27] M.S. Meldrum, M.T. Akabar, A.G. Chapman, Glutamate receptors and transporters in genetic and acquired models of epilepsy, Epilepsy Res. 36 (1999) 189–204. [28] R.S. Badar-Goffer, H.S. Bachelard, P.G. Morris, Cerebral metabolism of acetate and glucose studied by 13 C-NMR spectroscopy. A technique for investigating metabolic compartmentation in the brain, Biochem. J. 266 (1990) 133–139. [29] D. Manor, D.L. Rothman, G.F. Mason, F. Hyder, O.A. Petroff, K.L. Behar, The rate of turnover of cortical GABA from [1- 13 C]glucose is reduced in rats treated with the GABA-transaminase inhibitor vigabatrin (g-vinylGABA), Neurochem. Res. 21 (1996) 1031–1041. [30] N.E. Preece, S. Cerdan, Metabolic precursors and compartmentation of cerebral GABA in vigabatrin-treated rats, J. Neurochem. 67 (1996) 1718–1725. [31] A.C.H. Yu, J. Drejer, L. Hertz, A. Schousboe, Pyruvate carboxylase activity in primary cultures of astrocytes and neurons, J. Neurochem. 41 (1983) 1484–1487. [32] R.P. Shank, G.S. Bennett, S.O. Fretag, G.L.M. Campbell, Pyruvate carboxylase: an astrocyte specific enzyme implicated in the replenishment of amino acid neurotransmitter pools, Brain. Res. 329 (1985) 364–367. [33] M.D. Norenberg, A. Martinez-Hernandez, Fine structural localiza-

[34]

[35]

[36]

[37]

[38]

[39] [40]

[41]

[42]

[43]

[44]

[45]

[46]

[47]

[48] [49]

[50] [51]

tion of glutamine synthetase in astrocytes of rat brain, Brain. Res. 161 (1979) 303–310. S.M. Fitzpatrick, H.P. Hetherington, K.L. Behar, R.G. Shulman, The flux from glucose to glutamate in the rat brain in vivo as determined by 1 H observed, 13 C-edited NMR spectroscopy, J. Cereb. Blood Flow Metab. 10 (1990) 170–179. U. Ponten, R.A. Ratcheson, L.G. Salford, B.K. Siesjo, Optimal freezing conditions for cerebral metabolites in rats, J. Neurochem. 21 (1973) 1127–1138. G.F. Mason, D.L. Rothman, K.L. Behar, R.G. Shulman, NMR determination of TCA cycle rate and a-ketoglutarate / glutamate exchange rate in rat brain, J. Cereb. Blood Flow Metab. 12 (1992) 434–447. G.W. Cline, I. Magnusson, D.L. Rothman, K.F. Peterson, D. Laurent, G.I. Shulman, Mechanism of impaired insulin-stimulated muscle glucose metabolism in subjects with insulin-dependent diabetes mellitus, J. Chem. Invest. 99 (1997) 2219–2224. G.A. Dienel, N.F. Cruz, K. Mori, L. Sokoloff, Acid lability of metabolites of 2-deoxyglucose in rat brain: implications for estimates of kinetic parameters of deoxyglucose phosphorylation and transport between blood and brain, J. Neurochem. 54 (1990) 1440– 1448. N. Seiler, F.N. Bolkenius, Polyamine reutilization and turnover in brain, Neurochem. Res. 10 (1985) 529–544. T. Noto, H. Hashimoto, J. Nakao, H. Kamimura, T. Nakajima, Spontaneous release of g-aminobutyric acid formed from putrescine and its enhanced Ca21-dependent release by high K1 stimulation in the brains of freely moving rats, J. Neurochem. 46 (1986) 1877–1880. N.R. Sibson, G.F. Mason, J. Shen, G.W. Cline, Z. Herskovits, J.E.M. Wall, K.L. Behar, D.L. Rothman, R.G. Shulman, In vivo 13 C NMR measurement of neurotransmitter glutamate cycling, anaplerosis and TCA cycle flux in rat brain during [2- 13 C]glucose infusion, J. Neurochem. 757 (2001) 69–78. J.-C. Reubi, C. Van Der Berg, M. Cuenod, Glutamine as precursor for the GABA and glutamate transmitter pools, Neurosci. Lett. 10 (1978) 171–174. R.E. Paulsen, E. Odden, F. Fonnum, Importance of glutamine for g-aminobutyric acid synthesis in rat neostriatum in vivo, J. Neurochem. 51 (1988) 1294–1299. A. Lapidot, A. Gopher, Cerebral metabolic compartmentation. Estimation of glucose flux via pyruvate carboxylase / pyruvate dehydrogenase by 13 C NMR isotopomer analysis of D-[U- 13 C] glucose metabolites, J. Biol. Chem. 269 (1994) 27198–27208. R.P. Shank, G.C. Leo, H.R. Zielke, Cerebral metabolic compartmentation as revealed by nuclear magnetic resonance analysis of 13 D-[1- C]glucose metabolism, J. Neurochem. 61 (1993) 315–323. J.D. Rothstein, L. Martin, A.I. Levey, M. Dykes-Hoberg, L. Jin, D. Wu, N. Nash, R.W. Kuncl, Localization of neuronal and glial glutamate transporters, Neuron 13 (1994) 713–725. J. Sepkuty, C.U. Eccles, R.P. Lesser, M. Dykes-Hoberg, J.D. Rothstein, Molecular knockdown of neuronal glutamate transporter EAAT3 produces epilepsy and dysregulation of GABA metabolism, Abstr. Soc. Neurosci. 23 (1997) 1484. M. Erecinska, I.A. Silver, Metabolism and role of glutamate in mammalian brain, Prog. Neurobiol. 35 (1990) 245–296. A. Minelli, N.C. Brecha, C. Karschin, S. De Biasi, F. Conti, GAT-1, a high-affinity GABA plasma membrane transporter is localized to neurons and astroglia in the cerebral cortex, J. Neurosci. 15 (1995) 7734–7746. L.A. Borden, GABA transporter heterogeneity: pharmacology and cellular localization, Neurochem. Int. 29 (1996) 335–356. S. De Biasi, L. Vitellaro-Zuccarello, N.C. Brecha, Immunoreactivity for the GABA transporter-1 and GABA transporter-3 is restricted to astrocytes in the rat thalamus. A light and electron-microscopic immunolocalization, Neuroscience 83 (1998) 815–828.

A.B. Patel et al. / Brain Research 919 (2001) 207 – 220 [52] J. Bardakdjian, M. Tardy, C. Pimoule, P. Gonnard, GABA metabolism in cultured glial cells, Neurochem. Res. 4 (1979) 517–527. [53] N.R. Sibson, A. Dhankhar, G.F. Mason, K.L. Behar, D.L. Rothman, 13 R.G. Shulman, In vivo C NMR measurements of cerebral glutamine synthesis as evidence for glutamate–glutamine cycling, Proc. Natl. Acad. Sci. USA 94 (1997) 2699–2704. [54] N.R. Sibson, A. Dhankhar, G.F. Mason, D.L. Rothman, K.L. Behar, R.G. Shulman, Stoichiometric coupling of brain glucose metabolism and glutamatergic neuronal activity, Proc. Natl. Acad. Sci. USA 95 (1998) 316–321. [55] E.N. Yamasaki, V.D. Barbosa, F.G. De Mello, J.N. Hokoc, GABAergic system in the developing mammalian retina: dual sources of GABA at early stages of postnatal development, Int. J. Dev. Neurosci. 17 (1999) 201–213. [56] C. Pierard, M. Peres, P. Satabin, C.Y. Gueznnec, D. Lagarde, Effects of GABA-tarnsaminase inhibition on brain metabolism and amino acid compartmentation: an in vivo 2D 1 H-NMR spectroscopy coupled with microdialysis, Exp. Brain Res. 127 (1999) 321–327. [57] J. Shen, K.F. Petersen, K.L. Behar, P. Brown, T.W. Nixon, G.F. Mason, O.A. Petroff, G.I. Shulman, R.G. Shulman, D.L. Rothman, Determination of the rate of the glutamate / glutamine cycle in the human brain by in vivo 13 C NMR, Proc. Natl. Acad. Sci. USA 96 (1999) 8235–8240. [58] J.-J. Soghomonian, D.L. Martin, Two isoforms of glutamate decarboxylase: why?, Trends Pharmacol. Sci. 19 (1998) 500–505. [59] M.G. Erlander, A.J. Tobin, The structural and functional heterogeneity of glutamic acid decarboxylase: a review, Neurochem. Res. 16 (1991) 215–226. [60] D.L. Kaufman, C.R. Houser, A.J. Tobin, Two forms of the gaminobutyric acid synthetic enzyme glutamate decarboxylase have distinct intraneuronal distributions and cofactor interactions, J. Neurochem. 56 (1991) 720–723. [61] K. Rimvall, D.L. Martin, The level of GAD 67 protein is highly sensitive to small increases in intraneuronal gamma-aminobutyric acid levels, J. Neurochem. 62 (1994) 1375–1381. [62] H. Asada, Y. Kawamura, K. Maruyama, H. Kume, R.-G. Ding, F.Y. Ji, N. Kanbara, H. Kuzume, M. Sanbo, T. Yagi, K. Obata, Mice lacking the 65 kDa isoform of glutamic acid decarboxylase (GAD 65 ) maintain normal levels of GAD67 and GABA in their brains but are susceptible to seizures, Biochem. Biophys. Res. Commun. 229 (1996) 891–895. [63] H. Asada, Y. Kawamura, K. Maruyama, H. Kume, R.-G. Ding, F.Y. Ji, N. Kanbara, H. Kuzume, M. Sanbo, T. Yagi, K. Obata, Cleft palate and decreased brain g-aminobutyric acid in mice lacking the 67-kDa isoform of glutamic acid decarboxylase, Proc. Natl. Acad. Sci. USA 94 (1996) 6496–6499. [64] S.F. Kash, R.S. Johnson, L.H. Tecott, J.L. Noebels, R.D. Mayfield, D. Hanahan, S. Baekkeskov, Epilepsy in mice deficient in the 65-kDa isoform of glutamic acid decarboxylase, Proc. Natl. Acad. Sci. USA 94 (1997) 14060–14065. [65] J.P. Sepkuty, K.L. Behar, J.D. Rothstein, Molecular knockdown of the glutamate transporter EAAC1 reduces new GABA synthesis in rat hippocampus, Soc. Neurosci. Abstr., New Orleans, LA, 2000. [66] D.E. Bergles, C.E. Jahr, Glial contribution to glutamate uptake at Schaffer collateral-commissural synapses in the hippocampus, J. Neurosci. 18 (1998) 7709–7716. [67] D.L. Rothman, O.A. Petroff, K.L. Behar, R.H. Mattson, Localized 1 H NMR measurements of gamma-aminobutyric acid in human brain in vivo, Proc. Natl. Acad. Sci. USA 90 (1993) 5662–5666. [68] G. Sanacora, G.F. Mason, D.L. Rothman, K.L. Behar, F. Hyder, O.A. Petroff, R.M. Berman, D.S. Charney, J.H. Krystal, Reduced cortical gamma-aminobutyric acid levels in depressed patients determined by proton magnetic resonance spectroscopy, Arch. Gen. Psychiatry 56 (1999) 1043–1047. [69] R.P. Shank, G.L. Campbell, Glutamine and alpha-ketoglutarate

[70]

[71] [72]

[73]

[74]

[75]

[76] [77]

[78]

[79] [80]

[81]

[82]

[83]

[84]

[85]

[86]

[87]

[88]

219

uptake and metabolism by nerve terminal enriched material from mouse cerebellum, Neurochem. Res. 7 (1982) 601–616. O.A. Petroff, K.L. Behar, D.L. Rothman, New NMR measurements in epilepsy. Measuring brain GABA in patients with complex partial seizures, Adv. Neurol. 79 (1999) 939–945. S. Bonavita, F. Di Salle, G. Tedeschi, Proton MRS in neurological disorders, Eur. J. Radiol. 30 (1999) 125–131. R.H. Mattson, O.A. Petroff, D.L. Rothman, K.L. Behar, Vigabatrin: effect on brain GABA levels measured by nuclear magnetic resonance spectroscopy, Acta Neurol. Scand. Suppl. 162 (1995) 27–30. E.J. Novotny Jr., F. Hyder, M. Shevell, D.L. Rothman, GABA changes with vigabatrin in the developing human brain, Epilepsia 40 (1999) 462–466. O.M. Weber, A. Verhagen, C.O. Duc, D. Meier, K.L. Leenders, P. Boesiger, Effects of vigabatrin intake on brain GABA activity as monitored by spectrally edited magnetic resonance spectroscopy and positron emission tomography, Magn. Reson. Imaging 17 (1999) 417–425. T. Kanamatsu, Y. Tsukada, Effects of ammonia on the anaplerotic pathway and amino acid metabolism in the brain: an ex vivo 13 C NMR spectroscopic study of rats after administrating [2- 13 C]glucose with or without ammonium acetate, Brain Res. 841 (1999) 11–19. T.-Z. Su, G.W. Campbell, D.L. Oxender, Glutamine transport in cerebellar granule cells in culture, Brain Res. 757 (1997) 69–78. A.C. Yu, T.E. Fisher, E. Hertz, J.T. Tildon, A. Schousboe, L. Hertz, 14 Metabolic fate of [ C]-glutamine in mouse cerebral neurons in primary cultures, J. Neurosci. Res. 11 (1984) 351–357. Y. Kanai, P.G. Bhide, M. DiFiglia, M.A. Hediger, Neuronal highaffinity glutamate transport in the rat central nervous system, Neuroreport 6 (1995) 2357–2362. E.G. McGeer, P.L. McGeer, Localization of glutaminase in the rat neostriatum, J. Neurochem. 32 (1979) 1071–1075. A.M. Benjamin, Control of glutaminase activity in rat brain cortex in vitro: influence of glutamate, phosphate, ammonium, calcium and hydrogen ions, Brain Res. 208 (1981) 363–377. S. Hogstad, G. Svenneby, I.A. Torgner, E. Kvamme, L. Hertz, A. Schousboe, Glutaminase in neurons and astrocytes cultured from mouse brain: kinetic properties and effects of phosphate, glutamate, and ammonia, Neurochem. Res. 13 (1988) 383–388. A.C. Yu, E. Hertz, L. Hertz, Alterations in uptake and release rates for GABA, glutamate, and glutamine during biochemical maturation of highly purified cultures of cerebral cortical neurons, a GABAergic preparation, J. Neurochem. 42 (1984) 951–960. E. Kvamme, A. Schousboe, L. Hertz, I.A. Torgner, G. Svenneby, Developmental change of endogenous glutamate and gammaglutamyl transferase in cultured cerebral cortical interneurons and cerebellar granule cells, and in mouse cerebral cortex and cerebellum in vivo, Neurochem. Res. 10 (1985) 993–1008. H. Akiyama, T. Kaneko, N. Mizuno, P.L. McGeer, Distribution of phosphate-activated glutaminase in the human cerebral cortex, J. Comp. Neurol. 297 (1990) 239–252. J.H. Laake, Y. Takumi, J. Eidet, I.A. Torgner, B. Roberg, E. Kvamme, O.P. Ottersen, Postembedding immunogold labelling reveals subcellular localization and pathway-specific enrichment of phosphate activated glutaminase in rat cerebellum, Neuroscience 88 (1998) 1137–1151. S. Wurdig, P. Kugler, Histochemistry of glutamate metabolizing enzymes in the rat cerebellar cortex, Neurosci. Lett. 130 (1991) 165–168. T. Kaneko, Y. Nakaya, N. Mizuno, Paucity of glutaminase-immunoreactive nonpyramidal neurons in the rat cerebral cortex, J. Comp. Neurol. 322 (1992) 181–190. T. Kaneko, N. Mizuno, Glutamate-synthesizing enzymes in GABAergic neurons of the neocortex: a double immunofluorescence study in the rat, Neuroscience 61 (1994) 839–849.

220

A.B. Patel et al. / Brain Research 919 (2001) 207 – 220

[89] A.J.L. Cooper, M. Gross, The glutamine transaminase-omega-amidase system in rat and human brain, J. Neurochem. 28 (1977) 771–778. [90] A.J.L. Cooper, D.G. Abraham, A.S. Gelbard, J.C.K. Lai, C.K. Petito, High activities of glutamine transaminase K (dichlorovinylcystein b-lyase) and v-amidase in the choroid plexus of rat brain, J. Neurochem. 61 (1993) 1731–1741.

[91] T.K. Makar, M. Nedergaard, A. Preuss, L. Hertz, A.J.L. Cooper, Glutamine transaminase K and v-amidase activities in primary cultures of astrocytes and neurons and in embryonic chick forebrain: marked induction of brain glutamine transaminase K at time of hatching, J. Neurochem. 62 (1994) 1983–1988.