DIFFERENT METABOLISM OF GLUTAMATERGIC AND GABAERGIC COMPARTMENTS IN SUPERFUSED HIPPOCAMPAL SLICES CHARACTERIZED BY NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY J. M. N. DUARTE, a,b R. A. CUNHA a * AND R. A. CARVALHO a,b a Centre for Neurosciences of Coimbra, Institute of Biochemistry, Fac- ulty of Medicine, University of Coimbra, 3004-504 Coimbra, Portugal b Department of Biochemistry, University of Coimbra, Apartado 3126, 3001-401 Coimbra, Portugal Abstract—We investigated intermediary metabolism using 13 C- glucose and 13 C-acetate tracers followed by 13 C-nuclear mag- netic resonance (NMR) isotopomer analysis in rat hippocampal slice preparations, the most widely used preparation for elec- trophysiological studies. Slices displayed a stable metabolic activity over a wide range of superfusion periods in the absence or presence of 50 M 4-aminopyridine (4AP), which triggers an intermittent burst-like neuronal firing. This caused an increase of tricarboxylic acid (TCA)-related amino acids (glutamate, as- partate and GABA) and shortened the time required to reach metabolic and isotopic steady state (3 h in the presence of 4AP and 7 h in its absence). 13 C-NMR isotopomer analysis revealed an increase in TCA flux in astrocytes and in GABA compart- ments greater than in putative glutamatergic neurons and the fitting of these data further indicated that the metabolic network in GABAergic and glutamatergic compartments has a different design and reacts differently to the stimulation by the presence of 4AP. These results show that 13 C-isotopomer analysis allows estimating metabolic parameters/fluxes under both steady- and non-steady-state metabolic conditions in hippocampal slices, opening the possibility of combining electrophysiological and metabolic studies in the same preparation. © 2006 IBRO. Pub- lished by Elsevier Ltd. All rights reserved. Key words: hippocampal slices, 13 C-NMR, intermediary me- tabolism, isotopomer analysis. 13 C-nuclear magnetic resonance (NMR) spectroscopy is a powerful tool to investigate intermediary metabolism since it is able to simultaneously detect 13 C incorporation into molecules and the positions of 13 C incorporation within the same molecule (isotopomers). It has been used for studies of brain intermediary metabolism both in vitro and in vivo (e.g. Cerdán et al., 1990; Ebert et al., 2003; García-Espi- nosa et al., 2004; Gruetter, 2002; Künnecke et al., 1993; Zwingmann and Leibfritz, 2003), allowing following of the fate of labeling from 13 C-enriched substrates through par- ticular metabolic pathways. This has allowed demonstrat- ing that glucose is the main metabolic fuel although the energy requirements of cerebral tissue can be satisfied by the oxidation of other substrates such as ketone bodies (Badar-Goffer et al., 1990; Cerdán et al., 1990; Künnecke et al., 1993; Melo et al., 2006), lactate (Bouzier et al., 2000; Hassel and Brathe 2000; Tyson et al., 2003) and even fatty acids (Ebert et al., 2003; Kuge et al., 1995). Brain metabolism has been mainly investigated in the brain or in cultured brain cells, but scarcely in hippocampal slices (Cohen et al., 1984; Whittingham et al., 1984; Bra- dler et al., 1991; Ben-Yoseph et al., 1993; Schurr et al., 1999), which are the gold standard for electrophysiological studies since slices preserve the anatomy of neuronal circuits and synaptic properties of excitability and plasticity (Bahr et al., 1995). However, the physiological perfor- mance of hippocampal slices ultimately depends on its metabolic dynamics, which has been poorly studied. As would be expected from its ability to endure prolonged periods of electrical activity, it has already been shown that hippocampal slices are metabolically competent (e.g. Whittingham et al., 1984; Bradler et al., 1991; Schurr et al., 1999), but no detailed characterization of the intermediary metabolism of this preparation has yet been carried out. Therefore, to set the basis for future parallel electrophys- iological and metabolic studies, the present work intends to characterize the metabolic profile of the superfused hip- pocampal slice preparation by 13 C-NMR isotopomer anal- ysis after labeling with [U- 13 C]glucose and [2- 13 C]acetate, to evaluate both neuronal and astrocytic metabolic com- partments (Cerdán et al., 1990; Melo et al., 2006). EXPERIMENTAL PROCEDURES Reagents [U- 13 C]glucose (99%), sodium [2- 13 C]acetate (99%) and 2 H 2 O (99.9%) were purchased from Isotec Inc. (Miamisburg, OH, USA). 4-Aminopyridine (4AP) was purchased from Alomone Laborato- ries (Jerusalem, Israel). Solutions of 2 HCl (20% w/w) and NaO 2 H (40% w/w) and other common reagents (highest purity available) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Carbo- gen (gas mixture of 95% O 2 +5 % CO 2 ) was purchased to Linde Sogás (Lisbon, Portugal). Preparation and superfusion of hippocampal slices All experiments were conducted according to EU guidelines on ethical use of experimental animals (86/609/EEC), with particular care to minimize both animal suffering and the number of animals *Corresponding author. Tel: +351-239-820190; fax: +351-239-822776. E-mail address: racunha@clix.pt (R. A. Cunha). Abbreviations: ACS, acyl-CoA synthetase; Cre, creatine; LDH, lactate dehydrogenase (E.C. 1.1.1.27); NMR, nuclear magnetic resonance; PC, pyruvate carboxylase (E.C. 6.4.1.1); PCA, perchloric acid; PCre, phosphocreatine; PDH, pyruvate dehydrogenase (E.C. 1.2.4.1); TCA, tricarboxylic acid; Y, anaplerotic flux; 4AP, 4-aminopyridine. Neuroscience 144 (2007) 1305–1313 0306-4522/07$30.00+0.00 © 2006 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2006.11.027 1305