Intercellular metabolic compartmentation in the brain: past, present and future

Neurochemistry International 45 (2004) 285–296

Intercellular metabolic compartmentation in the brain: past, present and future Leif Hertz∗ College of Medical Sciences, China Medical University, Shenyang, PR China and Department of Pharmacology, University of Saskatchewan, Saskatoon, Saskatchewan, Canada Received 15 May 2003; received in revised form 1 August 2003; accepted 1 August 2003

Abstract The first indication of ‘metabolic compartmentation’ in brain was the demonstration that glutamine after intracisternal [14 C]glutamate administration is formed from a compartment of the glutamate pool that comprises at most one-fifth of the total glutamate content in the brain. This pool, which was designated ‘the small compartment,’ is now known to be made up predominantly or exclusively of astrocytes, which accumulate glutamate avidly and express glutamine synthetase activity, whereas this enzyme is absent from neurons, which eventually were established to constitute ‘the large compartment.’ During the following decades, the metabolic compartment concept was refined, aided by emerging studies of energy metabolism and glutamate uptake in cellularly homogenous preparations and by the histochemical observations that the two key enzymes glutamine synthetase and pyruvate carboxylase are active in astrocytes but absent in neurons. It is, however, only during the last few years that nuclear magnetic resonance (NMR) spectroscopy, assisted by previously obtained knowledge of metabolic pathways, has allowed accurate determination in the human brain in situ of actual metabolic fluxes through the neuronal tricarboxylic acid (TCA) cycle, the glial, presumably mainly astrocytic, TCA cycle, pyruvate carboxylation, and the ‘glutamate–glutamine cycle,’ connecting neuronal and astrocytic metabolism. Astrocytes account for 20% of oxidative metabolism of glucose in the human brain cortex and accumulate the bulk of neuronally released transmitter glutamate, part of which is rapidly converted to glutamine and returned to neurons in the glutamate–glutamine cycle. However, one-third of released transmitter glutamate is replaced by de novo synthesis of glutamate from glucose in astrocytes, suggesting that at steady state a corresponding amount of glutamate is oxidatively degraded. Net degradation of glutamate may not always equal its net production from glucose and enhanced glutamatergic activity, occurring during different types of cerebral stimulation, including the establishment of memory, may be associated with increased de novo synthesis of glutamate. This process may contribute to a larger increase in glucose utilization rate than in rate of oxygen consumption during brain activation. The energy yield in astrocytes from glutamate formation is strongly dependent upon the fate of the generated glutamate. © 2004 Elsevier Ltd. All rights reserved. Keywords: Astrocytes; Brain activation; Glucose metabolism; Glutamate; Neurons; Pyruvate carboxylation

1. Introduction Brain function depends upon co-ordinated function of a multitude of cell types: neurons, astrocytes, oligodendrocytes, microglial cells, choroid plexus epithelial cells, capillary endothelial cells, ependyma cells and tanycytes. With the exception of differentiation between gray and white matter, little or no attempt to separate metabolic events in various cell types from each other was made until after the middle of the 20th century. Two different approaches in the 1960s drew the attention to the fact that different cell types in the brain may carry out different metabolic processes: (i) ∗ Present address: RR 2, P.O. Box 245, Gilmour, Ontario, Canada, K0L 1W0. Tel.: +1-613-474-0537; fax: +1-613-474-0538. E-mail address: [email protected] (L. Hertz).

0197-0186/$ – see front matter © 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuint.2003.08.016

studies of ‘metabolic compartmentation’ in intact brain after administration of specific, radioactively labeled substrates, carried out in New York by Berl, Clarke, Lajtha and Waelsch (e.g., Berl et al., 1961; Waelsch et al., 1964; Berl and Frigyesi, 1969); and (ii) studies of metabolic events in isolated cells or groups of identical cells pioneered by Holger Hydén in Göteborg, Sweden, using carefully microdissected individual neurons and groups of glial cells (e.g., Hydén and Pigon, 1960; Hamberger, 1961a, b; Hamberger and Hyden, 1963). Cultures consisting of a single cell type were developed slightly later (Booher and Sensenbrenner, 1972) and used for metabolic studies (Hertz et al., 1973; Schousboe et al., 1977), and cell type-specific metabolic enzymes were discovered in intact brain tissue, most notably glutamine synthetase (Norenberg and Martinez-Hernandez, 1977) and pyruvate carboxylase (Shank et al., 1985). Both of these

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enzymes are absent in neurons but present in astrocytes. The demonstration of such a localization was not only instrumental in solidifying emerging concepts of the anatomical location of the two ‘metabolic compartments,’ which had been described, but it also served to legitimize the use of cells in primary cultures, which showed a similar enzyme distribution (Yu et al., 1983; Hertz and Schousboe, 1986). The era dominated by studies of metabolic compartmentation and of isolated cell types lasted until well into the 1990s. Although cell culture studies still can be useful, they may perhaps today be designated as ‘the past.’ This should not diminish the importance of the results obtained. On the contrary, the detailed information about actual metabolic fluxes in the intact functioning brain, obtained during the last decade, i.e., in the ‘present,’ following the development of highly sophisticated techniques, would not have been possible without the knowledge obtained in the past by studies of metabolic compartmentation, histochemical observations and investigations of isolated cell types. 2. The past: pathways of essential neuron–astrocyte interactions 2.1. Metabolic compartmentation Metabolic compartmentation in brain was first demonstrated when Berl et al. (1961) injected l-[14 C]glutamate intracisternally in the rat brain and within minutes observed that glutamine, formed from glutamate, displayed a five-fold higher specific activity, i.e., ratio between labeled and unlabeled compound (dpm/␮mol) than glutamate itself. The interpretation of this finding was that glutamine is exclusively formed from a small pool of glutamate, which rapidly accumulates exogenous glutamate and synthesizes glutamine. It was designated the ‘small’ compartment, because the glutamate pool giving rise to glutamine could constitute no more than one fifth of the total glutamate pool. When specific activity is determined in the total glutamate and glutamine pools in the tissue, the activity in the glutamine-forming glutamate pool, into which the precursor selectively entered, is ‘diluted with’ unlabeled glutamate from other pools, whereas similar dilution of glutamine cannot occur. Similar experiments with infusion of NaH14 CO3 showed also higher specific radioactivity of glutamine, and especially of aspartate, than of glutamate (Waelsch et al., 1964). This observation suggested that CO2 fixation leads to rapid labeling of oxaloacetate (ox.ac. in Fig. 1), the immediate precursor of aspartate, and that this oxaloacetate in turn is metabolized to ␣-ketoglutarate (␣-KG) and glutamate within the pool from which glutamine is formed. In contrast, glucose did not give rise to other than normal precursor–product relationships in the distribution of radioactive label, i.e., the specific activity in the obligatory precursor (glutamate) exceeded that of the product (glutamine) due to successive dilution of the label as it entered each metabolic pool in a

Fig. 1. Cartoon of metabolic compartmentation of glucose metabolism between neurons and astrocytes (not all intermediates are shown), with yield of ATP for individual reactions indicated by numbers. One molecule of glucose is glycolytically converted to two molecules of pyruvate in both neurons and astrocytes, leading to net generation of two molecules ATP by substrate phosphorylation and, under aerobic conditions, to formation of two ‘reducing equivalents’ (because there is one oxidation process during glycolysis), which after transfer to the mitochondria can generate another six ATP. Under anaerobic conditions, pyruvate is quantitatively converted to lactate (instead of the formation of ‘reducing equivalents’), and in the brain some lactate is formed even under aerobic conditions. The main metabolic fate of pyruvate is entry into the tricarboxylic acid (TCA) cycle. In neurons, pyruvate enters the TCA cycle exclusively via acetyl coenzyme A (ac.CoA), formed by oxidative decarboxylation, catalyzed by the pyruvate dehydrogenase complex (PDH) in a process generating three ATP per molecule pyruvate. Ac.CoA is metabolized in the TCA cycle after condensation with oxaloacetate (ox.ac.) to form citrate (citr), which is subsequently converted to ox.ac. during one turn of the cycle generating 12 ATP as indicated, for a total yield of 38 ATP per molecule glucose (generating two molecules of ac.CoA). However, no net formation of any TCA cycle intermediate occurs. In astrocytes pyruvate can in addition enter the TCA cycle by pyruvate carboxylation, generating one molecule of ox.ac., which condenses with ac.CoA, formed from another molecule of pyruvate to produce a new molecule of citr, from which ␣-ketoglutarate (␣-KG), the immediate precursor of glutamate (glu) is formed. Although ␣-KG as such might travel from astrocytes to neurons, it appears that in the brain it is mainly or exclusively converted in astrocytes via glu to glutamine (gln), which is transported in the glutamate–glutamine cycle to neurons, where it is hydrolyzed to glu. Formation of glu from glucose generates 14 ATP, but one ATP is required for pyruvate carboxylation and another ATP for glutamine synthesis, reducing the net gain to 12 ATP, i.e., approximately one third of the ATP yield by complete oxidation of glucose (Table 1). However, glu can also be degraded, probably mainly in astrocytes, via malate to pyruvate, generating nine ATP plus the energy residing in one molecule of pyruvate (Table 1). Moreover, the PDH-mediated flux in astrocytes exceeds the PC-mediated flux and thus the requirement for glutamate synthesis, and the remainder is oxidatively degraded exclusively for energy production in a similar manner as in neurons. In addition to carrying newly synthesized glu from astrocytes to neurons in the form of gln, the glutamate–glutamine cycle also serves to return transmitter glu to neurons after it has been accumulated in astrocytes (at the expense of one ATP per molecule of glu) for conversion to gln and return to neurons. Modified from Hertz et al. (1994).

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pathway. Glucose could accordingly be concluded to enter both compartments of glutamate equally well. During the next decade, several additional substrates were found to preferentially label the ‘small’ compartment, most notably acetate (Berl and Frigyesi, 1969), which enters the brain after intravenous administration, is taken up by astrocytes but not by neurons (Waniewski and Martin, 1998; Hertz and Dienel, 2002) and more recently has been used as a marker of astrocyte metabolism (Muir et al., 1986; Blüml et al., 2002; Lebon et al., 2002). The metabolic inhibitor, fluoroacetate is also taken up selectively by astrocytes, and it can be used as a specific inhibitor of oxidative metabolism in these cells (Muir et al., 1986; Fonnum et al., 1997). In addition, sophisticated simulation studies were carried out to determine the kinetics of metabolite flow in each of the two compartments as well as inter-compartmental fluxes (Garfinkel, 1966; Van den Berg and Garfinkel, 1971). Based on evidence that ␥-aminobutyric acid (GABA) was formed in one compartment, but predominantly degraded in a different compartment, it was concluded that the two compartments are connected by a flow of GABA from the large to the small compartment, and that this flow is compensated for by an identical flow of glutamine in the opposite direction (Fig. 1). The TCA cycle fluxes in the ‘large’; and the ‘small’ compartment of the rat brain were computed to be 1.25 and 0.3 ␮mol/min g wet weight, respectively, and the connecting GABA/glutamine flux to amount to 0.14 ␮mol/min g wet weight. Later, the presence of an analogous glutamate–glutamine cycle was suggested by Benjamin and Quastel (1975), and this cycle must have contributed to the connecting flux deduced by Van den Berg and Garfinkel (1971). The morphological correlates of each of the compartments remained a problem for a considerable length of time. Based on computer simulation studies, Garfinkel (1966) had concluded that the small compartment represented cells rich in mitochondria and in close contact with the intercellular space, and suggested that it might comprise nerve endings and perhaps some glial cells. Metabolic compartmentation is absent in the immature brain, and ontogenetic studies led to the proposal that the small compartment was present at birth and comprised glial cells and perhaps neuronal perikarya, whereas the large compartment corresponded to developing neuronal processes (Patel and Balazs, 1970). Although this appears to be the first suggestion of a mainly glial localization of the small compartment, it is not in keeping with a largely postnatal development of glial cells, and it is now obvious that both pools develop postnatally. That astrocytes account for most, and perhaps all, of the ‘small’ compartment was confirmed by the selectively astrocytic localization of glutamine synthetase, the enzyme forming glutamine from glutamate (Norenberg and Martinez-Hernandez, 1977) and of pyruvate carboxylase (Yu et al., 1983; Shank et al., 1985), the main enzyme catalyzing pyruvate carboxylation in brain (Patel, 1974). Since both of these enzymes are absent in neurons, all neuronal

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constituents must reside in the ‘large’ compartment. It is disputed whether glutamine synthetase is present in oligodendrocytes (Derouiche, 2004); pyruvate carboxylase activity has been reported in oligodendrocyte-enriched cultures and oligodendrocytic cell lines (Bal-Klara, 1989; Suormala et al., 2002), but no pyruvate carboxylase expression has been documented in oligodendrocytes in situ. 2.2. Studies of cell-specific preparations Based upon meticulous studies of metabolic rates in tumoral cells it was concluded by Hess (1961) that glial cells accounted for only 5% of the oxygen consumption rate in brain cortex. However, around the time when it was suggested that the ‘small’ compartment comprises glial cells, it had been shown in experiments using isolated normal astrocytes that these cells have relatively high rates of oxidative metabolism (Hamberger, 1961a, b; Hertz, 1966; Rose, 1967). This conclusion was corroborated during the following years (for tabulation, see Hertz, 1978), and it was shown that the intensity of oxidative metabolism in cultured astrocytes under optimum conditions (cells incubated in tissue culture medium and remaining attached to the culture flask) is approximately similar in neurons and astrocytes. This means that the contribution of astrocytes to oxidative metabolism in brain cortex in situ is likely to equal their contribution to total volume, which was assumed to be roughly one third in the brain cortex (Hertz and Schousboe, 1975; Hertz and Hertz, 1979). During the 1970s and 1980s, metabolic fluxes, enzyme activities and transmitter uptakes were determined in considerable detail, leading to the concept illustrated in Fig. 1 of metabolic interactions between neurons (N) and astrocytes (A). The Figure illustrates that both neurons and astrocytes metabolize glucose glycolytically to pyruvate (forming two molecules of pyruvate for each molecule of glucose), which can be either degraded via the tricarboxylic acid (TCA) cycle or further converted to lactate or alanine (Walz and Mukerji, 1988; Peng et al., 1994; Westergaard et al., 1993; Dienel and Hertz, 2001). Pyruvate enters the astrocytic TCA cycle by two different routes: (i) it can be oxidatively decarboxylated to acetyl coenzyme A (acetyl CoA [ac.CoA in the Figure]), and (ii) CO2 can be added in a pyruvate carboxylation process, leading to net synthesis of the TCA cycle constituent oxaloacetate (ox.ac.). Acetyl CoA formation is catalyzed by pyruvate dehydrogenase complex (PDH), and the acetyl moiety of acetyl CoA (a two-carbon unit) condenses with oxaloacetate (a four-carbon compound) in the TCA cycle to form citrate (a six-carbon compound), which during one turn of the TCA cycle gives rise to the production of two molecules of CO2 and is re-converted to oxaloacetate, which subsequently reacts with another molecule acetyl CoA, initiating another turn of the cycle, and so on. The turnover in the TCA cycle produces large amounts of energy (a maximum of 12 ATP per pyruvate molecule [assuming maximum efficacy of oxidative phosphorylation], to which should be

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L. Hertz / Neurochemistry International 45 (2004) 285–296

Table 1 ATP yield, oxygen (O2 ) consumption and net pyruvate (pyr) production (mol) during complete oxidative degradation of 1 mol of glucose (glc) to carbon dioxide (CO2 ) and water (H2 O), formation of glutamate (glu) from 1 mol of glc, complete oxidative degradation of 1 mol of glu without concomitant oxidation of the one molecule of pyruvate exiting the TCA cycle, complete oxidative degradation of 1 mol of pyr, and complete oxidative degradation of 1 mol of glc via glu and pyr Glc CO2 , H2 O b

ATPox ATPglyc f ATPred.eq g ATPtotal O2 Pyr

30 2 6 38 6

Glc glu 4c 2 6 12 2

Glu CO2 , H2 Oa

Pyr CO2 , H2 O

Glc CO2 , H2 O via glu, pyr

9a

15

9 1.5 1

15 2.5

28c,d ,e 2 6 36 6

a

Exclusive oxidation of the one mol of pyr exiting the TCA cycle. ATP production from pyruvate entry into the tricarboxylic acid (TCA) cycle and its oxidation in the cycle. c Two mol ATP have been subtracted reflecting ATP utilization during pyruvate carboxylation and glutamine synthesis. d Inclusive oxidation of the one mol of pyr exiting the TCA cycle. e Equals the sum of glc glu + glu CO , H O + pyr CO , H O. 2 2 2 2 f Net yield of ATP during glycolysis, disregarding oxidation of reducing equivalents formed during glycolysis and transferred to mitochondria for oxidation. g Maximum net yield of ATP during oxidation of reducing equivalents formed during glycolysis and transferred to mitochondria for oxidation. b

added the three molecules of ATP formed during pyruvate dehydrogenation [Fig. 1]). Another six molecules of ATP per molecule glucose are generated after transfer from the cytosol to the mitochondria of ‘reducing equivalents,’ formed in the cytosol during glycolysis (which includes one oxidative reaction), so that a total of 36 molecules of ATP can be generated oxidatively for each molecule of glucose (two molecules of pyruvate) degraded. This contrasts a net yield of two molecules ATP per molecule glucose formed by substrate phosphorylation during glycolysis (Table 1). The sole purpose of pyruvate oxidation via acetyl coenzyme A is the production of energy, and the operation of this pathway does not lead to either net synthesis or net degradation of any TCA cycle intermediate. Pyruvate carboxylation, catalyzed by pyruvate carboxylase (PC), is the major reaction in the brain giving rise to net synthesis of TCA cycle intermediates and their derivatives (an anaplerotic, i.e., pool-filling process), including the amino acid transmitters glutamate and GABA (Patel, 1974). No PC activity is found in neurons, which therefore do not synthesize glutamate and GABA from glucose on their own. In contrast, astrocytes express PC activity and carry out pyruvate carboxylation (Yu et al., 1983; Shank et al., 1985; Kaufman and Driscoll, 1992; Gamberino et al., 1997). Condensation of the newly synthesized molecule of oxaloacetate with acetyl CoA leads to net formation of one molecule citrate (citr in Fig. 1) from one molecule of glucose. In the TCA cycle citrate is converted to the five-carbon compound␣-ketoglutarate (␣-KG), a direct precursor of glutamate, by an oxidative decarboxylation. Two possibilities were suggested for conversion of astrocytic ␣-KG to neuronal glutamate: (i) ␣-KG may be transaminated in astrocytes to glutamate (glu) which is then carried to neurons in the glutamate–glutamine cycle after conversion to glutamine, which is re-converted to glutamate in neurons, or (ii) ␣-KG itself is transferred from astrocytes

to neurons and converted to glutamate in neurons. Only the former possibility is illustrated in the Fig. 1. Due to the transfer of two ‘reducing equivalents’ from the cytosol to the mitochondria as a result of the formation of two molecules of pyruvate from glucose and to the two oxidative decarboxylations occurring when pyruvate is converted to ␣-KG via acetyl CoA (Fig. 1), a maximum of 12 molecules of ATP are generated oxidatively for each molecule of glutamate synthesized, but the formation of oxaloacetate from pyruvate requires one molecule of ATP, as does glutamine formation from glutamate, reducing the net yield of ATP to 10 molecules of ATP, plus the two ATP molecules formed during glycolysis by substrate phosphorylation. Moreover, recent evidence indicates that glutamine uptake in neurons is dependent upon the sodium ion gradient, and accordingly might be energy requiring (Varoqui et al., 2000; Chaudhry et al., 2002). A total of two molecules of oxygen will be consumed (Table 1): one for the oxidation of the two ‘reducing equivalents’ and half a molecule (one oxygen atom) for each of the two oxidative decarboxylations. This contrasts the six molecules of oxygen required for complete oxidation of one molecule of glucose. Based on a low permeability of the blood–brain barrier to glutamate and glutamine it has long been postulated (e.g., Hertz et al., 1992) that net synthesis of glutamate must lead to a corresponding degradation of glutamate (a cataplerotic, i.e., pool-emptying process), although the two processes may be dissociated in time, e.g., leading to a temporary increase in glutamate pool size during enhanced glutamatergic activity (as will be discussed in Section 3.2). Conversion of glutamate to ␣-KG followed by decarboxylation of ␣-KG is pronounced in astrocytes, and it is increased at elevated concentrations of glutamate (Yu et al., 1982; Hertz et al., 1992; McKenna et al., 1996; Westergaard et al., 1996). In our hands, ␣-KG formation from glutamate has been found mainly to occur as an oxidative deamination, but other

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authors have reported that the process occurs as a transamination. The transamination can occur with oxaloacetate (generating aspartate) pyruvate (generating alanine) or with branched-chain ketoacids (generating branched chain amino acids). These amino acids can, in turn provide the nitrogen groups for formation of glutamate from ␣-KG (Yudkoff, 1997; Lieth et al., 2001), which does occur as a transamination (Hertz et al., 1992; Westergaard et al., 1996). In the calculation of oxygen utilization and ATP yield (Table 1) it will be assumed that the process occurs by transamination, which consumes no oxygen and generates no ATP. If it occurs as an oxidative deamination, another three ATP are formed. Both glutamine (Yu and Hertz, 1983) and glutamate (Hertz and Hertz, 2003) can maintain a constant rate of oxygen consumption in astrocytes when used as a metabolic substrate in the absence of glucose. Comparison of the rates of ␣-KG decarboxylation and of oxygen consumption in cultured astrocytes has indicated that glutamate at least under these conditions is completely oxidized, including oxidative metabolism of the one molecule of pyruvate exiting the TCA cycle (Hertz and Hertz, 2003). This does not mean that glutamate might not in the brain in situ also be degraded in neurons, a question that will be addressed in Section 3.1. To achieve complete oxidative degradation of glutamate, ␣-KG is in the astrocytic TCA cycle converted to the TCA cycle intermediate, malate, which exits from the cycle to form pyruvate (Fig. 1). Two oxidative decarboxylations are involved in this process (one of ␣-KG and the other of malate), and an additional oxidation and substrate phosphorylation between ␣-KG and malate leads to the formation of three more energy-rich phosphates. If glutamate formed by pyruvate carboxylation is oxidatively degraded, leading to the re-synthesis of one molecule of pyruvate without further oxidation of the pyruvate molecule, the amount of ATP generated equals the sum of glutamate formation from glucose and glutamate oxidation. This value is slightly larger than that formed by oxidation of half a molecule of glucose, because some ATP is generated during conversion of glucose to pyruvate. Since two molecules of pyruvate are consumed and one molecule of pyruvate is recovered (Fig. 1), the process represents ‘pyruvate recycling,’ i.e., entry of pyruvate into the TCA cycle followed by its exit from the cycle (Cerdan et al., 1990; Hassel et al., 1995). If the recovered molecule of pyruvate is also oxidized, almost the same amount of ATP is formed as by complete oxidative metabolism of one molecule of glucose (Table 1), the only difference being that the two molecules of ATP invested in the formation of oxaloacetate from pyruvate and glutamine from glutamate are not recovered. In contrast, if glutamate/glutamine is removed from the system or accumulates in the tissue, only a small fraction of the energy residing in the two molecules of pyruvate required for glutamate synthesis has been released, as was discussed above, and the oxygen consumption will be one third of that required for complete oxidation of glucose (Table 1).

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Large amounts of glutamate are also converted to glutamine in astrocytes (Yu et al., 1982; Hertz and Schousboe, 1986; McKenna et al., 1996). Subsequently, glutamine can be released from astrocytes and accumulated by neurons, where it is hydrolyzed to glutamate (Schousboe et al., 1979). These processes serve to return glutamate that has been accumulated by the potent and efficient glutamate transporters in astrocytes (Hertz et al., 1978; Drejer et al., 1982; Storm-Mathisen et al., 1992; Danbolt, 2001) to neurons in the glutamate–glutamine cycle. Uptake of glutamate into astrocytes and conversion of glutamate to glutamine each consumes one molecule of ATP. It has been postulated that this ATP must be generated by substrate phosphorylation during glycolysis (Magistretti et al., 1999), but the evidence in favor of this concept is weak (Peng et al., 2001). The functional importance of a glutamate–glutamine cycle, providing neurons with transmitter glutamate was confirmed in brain slices (Rothstein and Tabakoff, 1984) and in the intact retina (Pow and Robinson, 1994), where neuronal glutamate content and release are drastically reduced after administration of methionine sulfoximine (MSO), a rather specific inhibitor of glutamine synthetase. However, this effect is not necessarily exerted only on return of released transmitter glutamate from astrocytes to neurons, since ␣-KG, synthesized from glucose in astrocytes, may also be transaminated in the astrocytes to glutamate, which is then carried to neurons in the glutamate–glutamine cycle (Fig. 1). 3. The present: direct measurement of metabolic fluxes between astrocytes and neurons in living brain 3.1. Nuclear magnetic resonance (NMR) spectroscopy The event most clearly defining the beginning of the present era is the introduction slightly more than a decade ago of nuclear magnetic resonance spectroscopy for metabolic compartmentation studies in brain (Brainard et al., 1989; Cerdan et al., 1990; Badar-Goffer et al., 1992). This technique allows determination of the exact position of one or more labeled atom(s) within a given molecule, e.g., glutamate, GABA, aspartate, glutamine or lactate after administration of 13 C- or 15 N-labeled substrates. This ability, combined with the previously obtained knowledge that glutamine synthesis and pyruvate carboxylation are active in astrocytes but do not occur in neurons have allowed quantification of glial, probably mainly astrocytic, metabolism and of metabolic fluxes within and between compartments. However, immobilization of the head is required to achieve this, which presents no problem during determination of metabolic rates in humans. In some in vivo studies animals have been immobilized by anesthesia, which compromises the validity of the results obtained (Nakao et al., 2001). This problem has been overcome by combining in vivo injection of 13 C labeled substrates with ex vivo 13 C NMR spectroscopy of brain extracts (Hassel et al., 1995; Aureli

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et al., 1997). Similar techniques have been used in cultured cells and in brain slices. Labeling of glutamate from glucose or lactate in metabolic studies occurs both by net formation of the glutamate molecule from glucose in astrocytes as described above and by a rapid bi-directional exchange between ␣-ketoglutarate and glutamate, occurring both in neurons and in astrocytes (Fig. 1). When [1-13 C]glucose is used as the precursor, the latter reaction leads in the first turn of the TCA cycle to labeling in the [4-13 C] position of glutamate, a signal that is readily detectable due to a very high concentration of glutamate, especially in neurons (Storm-Mathisen et al., 1992; Pow and Robinson, 1994). Since most glutamate in the brain is cytosolic, its rate of exchange with ␣-ketoglutarate is likely to be limited by the rate of the malate aspartate shuttle carrying ␣-ketoglutarate from the mitochondrion to the cytosol (Gruetter et al., 1998, 2001; Gruetter, 2004), and at steady state it is similar to the rate of glucose oxidation. The rate of oxidative metabolism of glucose has been measured by determining incorporation of label from glucose into glutamate (Mason et al., 1995; Chen et al., 2001). However, due to a much lower glutamate content in astrocytes than in neurons (Storm-Mathisen et al., 1992), the astrocytic glutamate pool is rapidly equilibrated. For this reason, the continued incorporation of label almost exclusively reflects neuronal TCA cycle activity, whereas glial PDH-mediated flux is obscured. It can be estimated separately from incorporation of label into the C-2 position of aspartate, compared to that of incorporation into glutamate (Gruetter et al., 2001). Rates and extent of [4-13 C]glutamine labeling lag considerably behind those of glutamate, since glutamine only becomes labeled following its synthesis from glutamate in astrocytes. Accordingly, determination of glutamine synthesis by NMR is a direct manifestation of glial metabolism (Gruetter et al., 1994). Sibson et al. (1998) used the rate of glutamine synthesis as a measure of released transmitter glutamate followed by its accumulation into astrocytes and conversion to glutamine in the glutamate–glutamine cycle (Fig. 1). The calculated rate was almost identical to the rate of oxidative glucose utilization, for which reason they concluded that there is a 1:1 ratio between glutamatergic activity and rate of glucose oxidation. These authors ignored glial contributions to TCA cycle metabolism, which means that the total turnover rate in the TCA cycle might be underestimated. Moreover, NMR spectroscopy has shown that the rate of pyruvate carboxylation (leading to labeling of the C-2 position of glutamate from [1-13 C]glucose) corresponds to about one third of the rate of glutamine formation from glutamate (Lapidot and Gopher, 1994; Aureli et al., 1997; Gruetter et al., 2001). Unless de novo synthesis compensates for concomitant, stoichiometrically equivalent oxidation of transmitter glutamate accumulated into astrocytes, this fraction should not be included in the estimate of transmitter release, which accordingly may be severely overestimated. This point is important, because the postulated stoichiom-

Table 2 Metabolic fluxes (␮mol/min g wet weight) in human brain measured by nuclear magnetic resonance (NMR) spectroscopy Reference

PDHn

PC

PDHg

Vcycl

CMRglc

Gruetter et al. (2001) Blüml et al. (2002) Lebon et al. (2002)

0.57 0.70

0.09

0.15 0.13 0.14

0.26

0.41

0.32

PDHn : pyruvate dehydrogenase-mediated pyruvate flux in neurons (equals flux in neuronal TCA cycle); PC: pyruvate carboxylase-mediated flux (astrocyte-specific); PDHg : pyruvate dehydrogenase-mediated pyruvate flux in glial cells (astrocytes); Vcycl : flux via glutamate–glutamine cycle (includes PC, for which reason transport of glutamate from neurons to astrocytes equals Vcycl minus PC). Since two molecules pyruvate are formed from one molecule glucose PDHn + PC + PDHg (0.81 ␮mol/min g wet weight) equals approximately two times the metabolic rate of glucose.

etry between glutamate metabolism and transmitter release has been taken as support for the concept that astrocytic re-uptake of released glutamate and subsequent formation of glutamine, claimed to depend upon glycolytically derived energy (see Section 2.2) drives glucose metabolism in brain (Magistretti et al., 1999). A detailed mathematical analysis by Gruetter et al. (2001) established solid values for not only rate of pyruvate carboxylation and flux through the glutamate–glutamine cycle but also rates of oxidative metabolism in neurons and astrocytes (Table 2). Incorporation of label from [1-13 C]glucose was measured as a function of time into specific positions of a multitude of glucose metabolites, including glutamate, aspartate and glutamine. The rate of glutamine synthesis was calculated to be 0.26 ␮mol/min g wet weight and that of pyruvate carboxylation to be 0.09 ␮mol/min g wet weight. Subtraction of the rate of pyruvate carboxylation from that of glutamine formation yields a value of 0.17 ␮mol/min g wet weight, which is an estimate of transport of released transmitter glutamate from neurons to astrocytes. It corresponds to ∼40% of the rate of glucose utilization rather than the 100% reported by Sibson et al. (1998), and it is consistent with low rates of transfer of glutamate and GABA from neurons to astrocytes determined by Cruz and Cerdan (1999) in the rat brain (where all metabolic fluxes are about twice as high as in the human brain). The conclusion that one third of the trafficking in the glutamate–glutamine cycle represents glutamate newly synthesized from glucose in astrocytes (Lapidot and Gopher, 1994; Aureli et al., 1997; Gruetter et al., 2001) implies that the concept of the glutamate–glutamine cycle as a transfer of the 5-carbon units glutamate and glutamine between neighboring cells without mixing with the TCA cycle is far too simplistic. The rate for PDH-mediated flux in astrocytes in the study by Gruetter et al. (2001) consists of two components, i.e., one part (0.09 ␮mol/min g wet weight) equal to the rate of pyruvate carboxylation and reflecting de novo synthesis of glutamate from two molecules of pyruvate (Fig. 1), and a second component independent of pyruvate carboxylation (0.06 ␮mol/min g wet weight). The total astrocytic PDH-mediated flux thus amounts to 0.15 ␮mol/min g wet

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weight, which corresponds to 0.075 ␮mol of glucose/min g wet weight. The relative rate of pyruvate dehydrogenation in astrocytes is more than one-fourth of that in neurons (0.57 ␮mol/min g wet weight), and it constitutes 21% of the combined PDH-mediated fluxes in neurons and astrocytes (0.72 ␮mol/min g wet weight). This value is remarkably similar to the ∼20% of total TCA cycle activity which can be calculated from the computer simulation by Van den Berg and Garfinkel (1971) to occur in the small metabolic compartment, and it is slightly lower than the 29% which can be calculated from the values given by Cruz and Cerdan (1999) for the rat. It is also in relatively good agreement with the suggestion by Hertz and Schousboe (1975), based upon metabolic rates in preparations of isolated cells, that the relative contribution of astrocytes to the metabolic activity in brain equals their relative contribution to the volume. More important, virtually similar values have been deduced in human brain by NMR spectroscopy, using different modeling approaches (Table 2), i.e., determination of specific activities in either glutamate and glutamine (Lebon et al., 2002) or in bicarbonate (Blüml et al., 2002) from [13 C]acetate, which selectively enters astrocytes. The study by Lebon et al. (2002) also established that the role of transfer of ␣-KG from astrocytes to neurons at most is minor, compared to astrocytic formation of glutamate and glutamine and transfer via the glutamate–glutamine cycle (Fig. 1). Complete oxidative metabolism of 0.075 ␮mol glucose (0.15 ␮mol of pyruvate)/min g wet weight in astrocytes leads to a maximum ATP synthesis of 2.9 (38 mol ATP/mol glucose). As was discussed above, the fact that 0.09 ␮mol/min g wet weight is part of glutamate formation (in conjunction with carboxylation of another molecule of pyruvate) has extremely little effect on the net yield of ATP, if glutamate is also oxidatively degraded by these cells, including oxidation of the molecule of pyruvate exiting the TCA cycle (Table 1). If glutamate accumulates in the tissue, the total amount of ATP formed due to glutamate synthesis becomes reduced to 0.9 ␮mol/min g wet weight (Table 1). However, PDH-mediated pyruvate flux exceeded PC-mediated flux by 0.06 ␮mol/min g wet weight, providing another 1.14 ␮mol/min g wet weight of ATP, for a total of 2.0 ␮mol/min g wet weight. This value is two to three times more than the ATP production of 0.8 ␮mol/min g wet weight envisaged by Magistretti et al. (1999) as a result of glycolytic breakdown of virtually the entire glucose consumption (0.4 ␮mol/min g wet weight), but no oxidative metabolism in astrocytes. The NMR data analysis allows no distinction of the metabolic fate of glutamate, i.e., whether it is oxidatively degraded or not. However, a poor permeability of the blood brain barrier for glutamate and glutamine suggests that at steady state oxidation of glutamate equals its net synthesis. Formation of labeled lactate from [13 C]acetate and change in the labeling pattern of lactate from that of administered pyruvate has proven that pyruvate recycling does occur in the rat brain in vivo (Cerdan et al., 1990;

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Hassel et al., 1995), and in cultured astrocytes (Sonnewald et al., 1993). Cell culture experiments have also shown that lactate is formed from labeled glutamine, glutamate and aspartate in astrocytes, and that [3-13 C]glutamate is formed from [4-13 C]glutamate, an unequivocal indication that glutamate has left the TCA cycle and that generated pyruvate has re-entered the cycle (Waagepetersen et al., 2002 and references therein). In similar experiments using cerebral cortical neurons, no labeled lactate was formed from [13 C]glutamate, whereas small amounts of labeled lactate could be demonstrated in the case of the glutamatergic cerebellar granule neurons (Sonnewald et al., 1996). These findings, combined with the predominantly astrocytic uptake of extracellular glutamate (Danbolt, 2001) and the high rate of glutamate oxidation in cultured astrocytes (Yu et al., 1982; Hertz and Hertz, 2003), suggest that glutamate is oxidized in the brain in vivo and also that most of its oxidative degradation occurs in astrocytes. 3.2. Stimulation of brain activity During specific activation, such as visual stimulation, the cerebral rate of oxidative metabolism (CMRO2 ), measured by NMR spectroscopy, shows a moderate increase of 30% (Chen et al., 2001), which is less than the usual corresponding increase in metabolic rate of glucose utilization (CMRglc ). This observation is in agreement with previous observations made utilizing different methodologies and various types of stimulation (Fox and Raichle, 1986; Fox et al., 1988; Madsen et al., 1995, 1999), which consistently showed that CMRO2 increases less than CMRglc and cerebral blood flow (CBF). This phenomenon has been called ‘aerobic glycolysis,’ because it was believed that it reflected accumulation of lactate. This interpretation is partly correct, because many studies have shown an accumulation of lactate during activation of cerebral metabolism (e.g., Kuhr and Korf, 1988; Fellows et al., 1993; Taylor et al., 1994). However, accumulation of lactate in rat brain after generalized somatosensory stimulation can only explain about one half of the mismatch between CMRglc and CMRO2 (Madsen et al., 1999). Accordingly, either lactate may be removed from the activated area (Dienel and Hertz, 2001; Dienel and Cruz, 2003) or other incompletely oxidized products of glucose metabolism may have accumulated. It has been suggested that glutamate may be such a substance (Hertz and Fillenz, 1999), since two molecules pyruvate, corresponding to one molecule of glucose, are used for synthesis of one molecule glutamate, whereas the amount of oxygen consumed is only one third of that required for complete oxidative degradation of glucose (Table 1). In some situations, lactate is released to circulating blood. This is the case during spreading depression (Cruz et al., 1999), which leads to a large stimulation of energy metabolism in brain, probably secondary to an increase in active re-accumulation of K+ , as indicated by a massive, reversible increase in extracellular K+ (Hansen and Zeuthen,

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1981). However, in many conditions there is no net release of lactate to the circulation. This does not preclude that lactate may leave the stimulated area for adjacent regions of the brain, possibly through gap junction-mediated transport (Dienel and Cruz, 2003). Evidence that a glucose metabolite leaves the stimulated region has been provided by the observation that measurement of stimulated CMRglc with labeled glucose, which is metabolized, gives considerably lower values than measurement with labeled 2-deoxyglucose, which is trapped in the tissue after its initial phosphorylation (Collins et al., 1987; Ackermann and Lear, 1989). However, an enhanced de novo formation and accumulation of glutamate from glucose may also occur during brain activation. Recent studies by LaNoue have indicated a 70% increase in pyruvate carboxylation during an increase in glutamatergic activity in the retina (LaNoue, personal communication). These studies suggest that enhanced glutamatergic activity increases not only recycling in the glutamate–glutamine cycle of previously released transmitter glutamate but also de novo synthesis of glutamate from glucose. This situation is different from that during spreading depression, which is accompanied by an increase in CMRglc , and in accumulation of the astrocytic marker, acetate, but a decrease in pyruvate carboxylation (Dienel et al., 2001). If glutamate synthesis is stimulated as much in the human brain during glutamatergic activation as observed in the retina, it would mean an increase of pyruvate carboxylation of 0.06 ␮mol/min g wet weight (70% of 0.09 ␮mol/min g wet weight). In order to generate glutamate, the enhanced pyruvate carboxylation would be accompanied by a similar increase in PDH-mediated flux of pyruvate for a total of 0.12 ␮mol/min g wet weight. With a total pyruvate entry into the TCA cycle of 0.81 ␮mol/min g wet weight (Table 2), this would mean that ∼15% (0.12/0.81) of total pyruvate entry into the TCA cycle is ‘tied up’ in additional formation of glutamate. Provided no corresponding increase occurred in glutamate oxidation this would lead to a 10% larger increase in CMRglc than in CMRO2 (2/3 of 15%, reflecting that glutamate formation from glucose utilizes only one third of the amount of oxygen used for complete oxidation of the same amount of glucose [Table 1]). An increase in CMRO2 during a specific type of stimulation may comprise 30% of resting metabolism (as observed by Chen et al., 2001), that in CMRglc at the very most three times as much (90%). Since accumulation of lactate in the stimulated area may account for one half of the different degree of stimulation of CMRglc and CMRO2 (Madsen et al., 1999), an increase corresponding to ∼30% of resting CMRglc remains to be accounted for. The 10% larger increase in CMRglc than in CMRO2 due to glutamate production can explain approximately one third of this. Thus, together, lactate accumulation, lactate transfer away from the stimulated region, and glutamate accumulation in the region may account for the mismatch between stimulation of CMRglc and CMRO2 during many forms of brain activation.

Fig. 2. Contents of glutamate (A and B) and of glutamine (C and D) in the left (A and C) and right (B and D) hemispheres of the forebrain of 1-day-old chicks at different times after one-trial passive avoidance training as described in the text (䊏) compared with the contents after pre-training, preceding the actual aversive training experience (䊐). Results are means ± S.E.M. (when exceeding the symbols) of three individual experiments. From Hertz et al. (2003).

An increase of glutamate and glutamine pool sizes corresponding to the stimulation of pyruvate carboxylation observed by LaNoue has, however, been demonstrated in day-old chicks during one-trial passive avoidance training by exposure to a colored metal bead tainted with an aversively tasting compound (methylanthralinate) as well as to a differently colored bead, which was not tainted with the aversant (Hertz et al., 2003). Subsequently the chicks refuse to peck at untainted beads of the ‘aversive color,’ but peck freely at beads of the ‘non-aversive color.’ The aversive training leads to an immediate increase in glutamatergic activity reflected by glutamate release specifically in a left forebrain structure (Daisley et al., 1998) and to the changes in contents of glutamate and glutamine selective for this hemisphere shown in Fig. 2. Slightly later the changes in pool sizes are reversed. There is a concomitant, short-lasting decrease in glycogen content (Hertz et al., 2003), suggesting that glycogen was the main precursor for the increase in glutamate and glutamine pools. Such a role of glycogen might contribute to explaining why glycogen turnover in the resting brain is very low (Oz et al., 2003; Gruetter, 2004), whereas it is increased during neuronal activation (Swanson et al., 1992). However, the re-establishment of glycogen levels within 15 min after training (Hertz et al., 2003) suggests an increased glycogen formation from glucose. The increased synthesis of glutamate/glutamine appears to be a prerequisite for normal learning, since intracerebral administration specifically at the time when glutamate increases of either iodoacetate, inhibiting glycolysis and glycogenolysis, or fluorocitrate, inhibiting the astrocytic TCA cycle

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between citrate and ␣-KG, abolishes memory retention (O’Dowd et al., 1994; Hertz et al., 2003).

4. The future: NMR studies of brain activation and of glial contributions to neurological disease; compartmentation at the subcellular level Technological advances has made it possible to use NMR spectroscopy to study the effect of induced activity, for example during visual stimulation (Chen et al., 2001), but information is still lacking about the effect of stimulation on rate of pyruvate carboxylation and of trafficking in the glutamate–glutamine cycle in the intact brain. Such studies should include different types of well-defined stimulation, since the metabolic pattern is likely to be different for types of stimulation, e.g., increased glutamatergic activity versus stimulation of active ion transport. Conventional studies of metabolic fluxes and/or pool sizes in experimental animals may continue to provide additional information, especially if information can be obtained about glucose products leaving the activated structure. Studies of effects on metabolic compartmentation of such conditions as focal ischemia and exposure to neuroactive drugs are beginning to appear (Pascual et al., 1998; Håberg et al., 1998, 2001; Gruetter, 2004; Blüml et al., 2002), and they will probably show a dramatic increase in the future. So may studies of metabolism in other non-neuronal cell types. Due to their ubiquitous location in the brain astrocytes and oligodendrocytes are the non-neuronal cells most directly interacting with neurons, but many of the cell types at specific locations, e.g., choroid plexus epithelial cells, are also of crucial importance for brain function. Thus, choroid plexus epithelial cells continuously carry out active transport and may depend upon a high metabolic activity. The metabolic activities of other non-neuronal cells, e.g., microglia, is virtually unknown, although these cells, and their interactions with both neurons and astrocytes are of fundamental importance in neurological disease. Some of the most dramatic developments might occur in the understanding of neurological and mental disorders. There is increasing evidence suggesting astrocytic involvement in a multitude of neurological disorders, including dementias (Hertz, 2004). This applies to diseases of paramount clinical importance like Alzheimer’s disease, Parkinson’s disease and HIV-associated dementia as well as to rarer but equally devastating diseases like the prion diseases. Even mental disease, like affective disorders and schizophrenia, appear to involve glial cells. Both Alzheimer’s disease and affective disorders lead to changes in glucose phosphorylation rates in specific brain regions (Hoyer, 1986; Dunn et al., 2002), and Alzheimer’s disease may change the rate of pyruvate carboxylation (Hertz et al., 2000). The emphasis on NMR spectroscopy and related techniques should not lead to the impression that development of new and different methodologies should not be expected

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and is not necessary. First and foremost, these methods are macromethods, which only work at the cellular level because there are pronounced, and known, differences in metabolic pathways operating in neurons and astrocytes. However, both neurons and astrocytes (as well as other non-neuronal cells) are structurally complex. Histochemical studies have revealed important metabolic differences between different neuronal components and between different types of neurons. However, it is virtually unknown how these differences influence intercellular metabolic compartmentation, although emerging evidence suggests that the receptors expressed by a given neuron may be more important in this respect than the transmitter(s) released by the neuron (reviewed in Hertz and Dienel, 2002). Apart from suggestions that there may be different types of astrocytes, it is now becoming evident that a large part of the astrocytic volume is made up by a specific compartment, peripheral astrocytic processes (PAP) or filopodia, which express specific proteins in addition to such proteins as glutamine synthetase, which is expressed both in the peripheral and central parts of the cells (Derouiche and Frotscher, 2001; Wolff and Chao, 2004). The PAPs are too slender to contain mitochondria. They are, however, richly endowed with glycogen granules (Peters et al., 1991), and glycogen may function as a spatiotemporal, rechargeable energy reservoir, capable of rapid delivery of glycolytically derived energy and/or glucose equivalents for ion uptake and glutamate synthesis, followed by slower glycogen synthesis from glucose fueled by oxidatively derived energy. Such a notion is supported by the dependence of glucose phosphorylation by hexokinase on oxidatively derived energy (de Cesar and Wilson, 1998). The PAPs account for by far the major part of the astrocytic surface towards the extracellular fluid, towards other astrocytes, and towards neurons and other cells (Wolff and Chao, 2004), and their metabolic interactions with their own cell bodies, with processes of adjacent astrocytes, and with neurons remain to be established. References Ackermann, R.F., Lear, J.L., 1989. Glycolysis-induced discordance between glucose metabolic rates measured with radiolabeled fluorodeoxyglucose and glucose. J. Cereb. Blood Flow Metab. 9, 774–785. Aureli, T., Di Cocco, M.E., Calvani, M., Conti, F., 1997. The entry of [1-13 C]glucose into biochemical pathways reveals a complex compartmentation and metabolite trafficking between neurons: a study by 13 C-NMR glia and spectroscopy. Brain Res. 765, 218–227. Badar-Goffer, R.S., Ben-Yoseph, O., Bachelard, H.S., Morris, P.G., 1992. Neuronal-glial metabolism under depolarizing conditions: 13 C-NMR study. Biochem. J. 282, 225–230. Bal-Klara, A., 1989. Effects of some antidepressant drugs on the activity of glial cell enzymes in culture. Eur. J. Pharmacol. 161, 231–235. Benjamin, A., Quastel, J.H., 1975. Metabolism of amino acids and ammonia in rat brain cortex slices in vitro: a possible role of ammonia in brain function. J. Neurochem. 25, 197–206. Berl, S., Frigyesi, T.L., 1969. The turnover of glutamate, glutamine, aspartate and GABA labeled with [1-14 C]acetate in caudate nucleus, thalamus and motor cortex (cat). Brain Res. 12, 444–455.

294

L. Hertz / Neurochemistry International 45 (2004) 285–296

Berl, S., Lajtha, A., Waelsch, H., 1961. Amino acid and protein metabolism. VI. Cerebral compartments of glutamic acid metabolism. J. Neurochem. 7, 186–197. Blüml, S., Moreno-Torres, A., Shic, F., Nguy, C.H., Ross, B.D., 2002. Tricarboxylic acid cycle of glia in the in vivo human brain. NMR Biomed. 15, 1–5. Booher, J., Sensenbrenner, M., 1972. Growth and cultivation of dissociated neurons and glial cells from embryonic chick, rat and human brain in flask cultures. Neurobiology 2, 97–105. Brainard, J.R., Kyner, E., Rosenberg, G.A., 1989. 13 C nuclear magnetic resonance evidence for gamma-aminobutyric acid formation via pyruvate carboxylase in rat brain: ametabolic basis for compartmentation. J. Neurochem. 53, 1285–1292. Cerdan, S., Künnecke, B., Seelig, J., 1990. Cerebral metabolism of [1,2-13 C2]acetate as detected by in vivo and in vitro 13 C NMR. J. Biol. Chem. 365, 12916–12926. de Cesar, M.C., Wilson, J.E., 1998. Further studies on the coupling of mitochondrially bound hexokinase to intramitochondrially compartmented ATP, Generated by oxidative phosphorylation. Arch. Biochem. Biophys. 350, 109–117. Chaudhry, F.A., Schmitz, D., Reimer, R.J., Larsson, P., Gray, A.T., Nicoll, R., Kavanaugh, M., Edwards, R.H., 2002. J. Neurosci. 22, 62–72. Chen, W., Zhu, X.H., Gruetter, R., Seaquist, E.R., Adriany, G., Ugurbil, K., 2001. Study of tricarboxylic acid cycle flux changes in human visual cortex during hemifield visual stimulation using (1)H-(13)C MRS and fMRI. Magn. Reson. Med. 45, 349–355. Collins, R.C., McCandless, D.W., Wagman, I.L., 1987. Cerebral glucose utilization: comparison of [14 C]deoxyglucose and [6-14 C]glucose quantitative autoradiography. J. Neurochem. 49, 1564–1570. Cruz, F., Cerdan, S., 1999. Quantitative 13 C NMR studies of metabolic compartmentation in the adult mammalian brain. NMR Biomed. 12, 451–462. Cruz, N.F., Adachi, K., Dienel, G.A., 1999. Metabolite trafficking during K+ -induced spreading cortical depression: rapid efflux of lactate from cerebral cortex. J. Cereb. Blood Flow Metab. 19, 380–392. Daisley, J.N., Gruss, M., Rose, S.P.R., Braun, K., 1998. Passive avoidance training and recall are associated with increased glutamate levels in the intermediate medial hyperstriatum ventrale of the day-old chick. Neural Plast. 6, 53–61. Danbolt, N., 2001. Glutamate uptake. Prog. Neurobiol. 65, 1–105. Derouiche, A., 2004. The perisynaptic astrocyte process as a glial compartment—immunolabelling for glutamine synthetase and other glial markers. In: Hertz, L. (Ed.), Non-Neuronal cells of the Nervous System, Function and Dysfunction, vol. 1. Elsevier, Amsterdam. pp. 147–163. Derouiche, A., Frotscher, M., 2001. Peripheral astrocyte processes: monitoring by selective immunostaining for the actin-binding ERM proteins. Glia 36, 330–341. Dienel, G.A., Cruz, N.F., 2003. Neighborly interactions of metabolicallyactivated astrocytes in vivo. Neurochem. Int. 43, 339–354. Dienel, G.A., Hertz, L., 2001. Glucose and lactate metabolism during brain activation. J. Neurosci. Res. 66, 824–838. Dienel, G.A., Liu, K., Cruz, N.F., 2001. Local uptake of 14 C-labeled acetate and butyrate in rat brain in vivo during spreading cortical depression. J. Neurosci. Res. 66, 812–820. Drejer, J., Larsson, O.M., Schousboe, A., 1982. Characterization of l-glutamate uptake into and release from astrocytes and neurons cultured from different brain regions. Exp. Brain Res. 47, 259–269. Dunn, R.T., Kimbrell, T.A., Ketter, T.A., Frye, M.A., Willis, M.W., Luckenbaugh, D.A., Post, R.M., 2002. Principal components of the Beck Depression Inventory and regional cerebral metabolism in unipolar and bipolar depression. Biol. Psychiatry 51, 387–399. Fellows, L.K., Boutelle, M.G., Fillenz, M., 1993. Physiological stimulation increases nonoxidative glucose metabolism in the brain of the freely moving rat. J. Neurochem. 60, 1258–1263. Fonnum, F., Johnsen, A., Hassel, B., 1997. Use of fluorocitrate and fluoroacetate in the study of brain metabolism. Glia 21, 106–113.

Fox, P.T., Raichle, M.E., 1986. Focal physiological uncoupling of cerebral blood flow and oxidative metabolism during somatosensory stimulation in human subjects. Proc. Natl. Acad. Sci. U.S.A. 83, 1140–1144. Fox, P.T., Raichle, M.E., Mintun, M.A., Dence, C., 1988. Nonoxidative glucose consumption during focal physiologic neural activation. Science 241, 462–464. Gamberino, W.C., Berkich, D.A., Lynch, C.J., Xu, B., LaNoue, K.F., 1997. Role of pyruvate carboxylase in facilitation of synthesis of glutamate and glutamine in cultured astrocytes. J. Neurochem. 69, 2312–2325. Garfinkel, D.A., 1966. A simulation study of the metabolism and compartmentation in brain of glutamate, aspartate, the Krebs cycle and related metabolism. J. Biol. Chem. 241, 3918–3929. Gruetter, R., 2004. Principles of the measurement of neuro-glial metabolism using in vivo 13 C NMR spectroscopy. In: Hertz, L. (Ed.), Non-Neuronal cells of the Nervous System, Function and Dysfunction, vol. 2. Elsevier, Amsterdam. pp. 409–433. Gruetter, R., Novotny, E.J., Boulware, S.D., Mason, G.F., Rothman, D.L., Shulman, G.I., Prichard, J.W., Shulman, R.G., 1994. Localized 13 C NMR spectroscopy in the human brain of amino acid labeling from D-[1-13 C]glucose. J. Neurochem. 63, 1377–1385. Gruetter, R., Seaquist, E.R., Kim, S., Ugurbil, K., 1998. Localized in vivo 13 C NMR of glutamate metabolism in the human brain: initial results at 4 Tesla. Dev. Neurosci. 20, 380–388. Gruetter, R., Seaquist, E.R., Ugurbil, K., 2001. A mathematical model of compartmentalized neurotransmitter metabolism in human brain. Am. J. Physiol. Endocrinol. Metab. 282, E100–E112. Håberg, A., Qu, H., Haraldseth, O., Unsgard, G., Sonnewald, U., 1998. In vivo injection of [1-13 C]glucose and [1,2-13 C]acetate combined with ex vivo 13 C nuclear magnetic resonance spectroscopy: a novel approach to the study of middle cerebral occlusion in the rat. J. Cereb. Blood Flow Metab. 18, 1223–1232. Håberg, A., Qu, H., Bakken, I.J., Sande, L.M., White, L.R., Haraldseth, O., Unsgård, G., Aasly, J., Sonnewald, U., 2001. Differences in neurotransmitter synthesis and intermediary metabolism between glutamatergic and GABAergic neurons during 4 h of middle cerebral artery occlusion in the rat: the role of astrocytes in neuronal survival. J. Cereb. Blood Flow Metab. 21 (12), 1451–1463. Hamberger, A., 1961a. Oxidation of tricarboxylic acid cycle intermediates by nerve cell bodies and glial cells. J. Neurochem. 8, 31–35. Hamberger, A., 1961b. Difference between isolated neuronal and vascular glia with respect to respiratory intensity. Acta physiol. Scand. 58 (Suppl. 203), 1–52. Hamberger, A., Hyden, H., 1963. Inverse enzymatic changes in neurons and glia during increased function and hypoxia. J. Cell Biol. 16, 521– 525. Hansen, A.J., Zeuthen, T., 1981. Extracellular ion concentrations during spreading depression and ischemia in the rat brain cortex. Acta Physiol. Scand. 113, 437–445. Hassel, B., Sonnewald, U., Fonnum, F., 1995. Glial-neuronal interactions as studied by cerebral metabolism of [2-13 C]acetate and [1-13 C]glucose: an ex vivo 13 C NMR spectroscopic study. J. Neurochem. 64, 2773–2782. Hertz, E., Hertz, L., 1979. Polarographic measurement of oxygen uptake by astrocytes in primary cultures using the tissue culture flask as the respirometer chamber. In Vitro 15, 429–436. Hertz, L., 1966. Neuroglial localization of potassium and sodium effects on respiration in brain. J. Neurochem. 13, 1373–1387. Hertz, L., 1978. Energy metabolism of glial cells. In: Schoffeniels, E., Franck, G., Hertz, L., Tower, D.B. (Eds.), Dynamic Properties of Glial Cells. Pergamon Press, Oxford, pp. 121–132. Hertz, L. (Ed.), 2004. Non-Neuronal cells of the Nervous System, Function and Dysfunction, vol. 3. Elsevier, Amsterdam. Hertz, L., Dienel, G.A., 2002. Energy metabolism in the brain. Int. Rev. Neurobiol. 53, 1–101. Hertz, L., Fillenz, M., 1999. Does “the mystery of the extra glucose” during CNS activation reflect glutamate synthesis? Neurochem. Int. 34, 71–75.

L. Hertz / Neurochemistry International 45 (2004) 285–296 Hertz, L., Hertz, E., 2003. Determination of rate of glutamate-sustained oxygen consumption in primary cultures of astrocytes as a means to estimate cataplerotic TCA cycle flux. Neurochem. Int. 43, 355–361. Hertz, L., Schousboe, A., 1975. Ion and energy metabolism of the brain at the cellular level. Int. Rev. Neurobiol. 18, 141–211. Hertz, L., Schousboe, A., 1986. Role of astrocytes in compartmentation of amino acid and energy metabolism. In: Fedoroff, S, Vernadakis, A. (Eds.), Astrocytes, vol. 2. Academic Press, New York, pp. 179–208. Hertz, L., Dittmann, L., Mandel, P., 1973. K+ induced stimulation of oxygen uptake in cultured cerebral glial cells. Brain Res. 60, 517–520. Hertz, L., Schousboe, A., Boechler, N., Mukerji, S., Fedoroff, S., 1978. Kinetic characteristics of the glutamate uptake into normal astrocytes in cultures. Neurochem. Res. 3, 1–14. Hertz, L., Peng, L., Westergaard, N., Yudkoff, M., Schousboe, A., 1992. Neuronal-astrocytic interactions in metabolism of transmitter amino acids of the glutamate family. In: Schousboe, A., Diemer, N., Kofod, H. (Eds.), Drug Research Related to Neuroactive Amino Acids. Alfred Benzon Symposium 32. Munksgaard, Copenhagen, pp. 31–50. Hertz, L., Ocherashvili, E., O’Dowd, B., 1994. Glial cells. In: Ramachandran, V. (Ed.), Encyclopedia of Human Behavior. Academic Press, Orlando, pp. 429–439. Hertz, L., Yu, A.C.H., Kala, G., Schousboe, A., 2000. Neuronal-astrocytic and cytosolic mitochondrial metabolite metabolite trafficking during brain activation, hyperammonemia and energy deprivation. Neurochem. Int. 37, 83–102. Hertz, L., O’Dowd, B.S., Ng, K.T., Gibbs, M.E., 2003. Reciprocal changes in forebrain contents of glycogen and of glutamate/glutamine during early memory consolidation in the day-old chick. Brain Res. 994, 226–233. Hess, H.H., 1961. The rates of respiration of neurons and neuroglia in human cerebrum. In: Kety, S.S., Elkes J. (Eds.), Regional Neurochemistry. Pergamon, Oxford, pp. 200–212. Hoyer, S., 1986. Senile dementia and Alzheimer’s disease. Brain blood flow and metabolism. Prog. Neuropsychopharmacol. Biol. Psychiatry 10, 447–478. Hydén, H., Pigon, A., 1960. A cytophysiological study of the functional relationship between oligodendroglial cells and nerve cells of Deiters’ nucleus. J. Neurochem. 6, 57–72. Kaufman, E.E., Driscoll, B.F., 1992. Carbon dioxide fixation in neuronal and astroglial cells in culture. J. Neurochem. 58, 258–262. Kuhr, W.G., Korf, J., 1988. Extracellular lactic acid as an indicator of brain metabolism: continuous on-line measurement in conscious, freely moving rats with intrastriatal dialysis. J. Cereb. Blood Flow Metab. 8, 130–137. Kaufman, E.E., Driscoll, B.F., 1992. Carbon dioxide fixation in neuronal and astroglial cells in culture. J. Neurochem. 58, 258–262. Lapidot, A., Gopher, A., 1994. 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, 27198–27208. Lebon, V., Petersen, K.F., Cline, G.W., Shen, J., Mason, G.F., Dufour, S., Behar, K.L., Shulman, G.I., Rothman, D.L., 2002. Astroglial contribution to brain energy metabolism in humans revealed by 13 C nuclear magnetic resonance spectroscopy: elucidation of the dominant pathway for neurotransmitter glutamate repletion and measurement of astrocytic oxidative metabolism. J. Neurosci. 22, 1523–1531. Lieth, E., LaNoue, K.F., Berkich, D.A., Xu, B., Ratz, M., Taylor, C., Hutson, S.M., 2001. Nitrogen shuttling between neurons and glial cells during glutamate synthesis. J. Neurochem. 76, 1712–1723. Madsen, P.L., Hasselbalch, S.A.G., Hangman, L.P., Olsen, K.S., Bülow, J., Holm, S., Wildschøidtz, G., Paulson, O.B., Lassen, N.A., 1995. Persistent resetting of the cerebral oxygen/glucose uptake ratio by brain activation: evidence obtained by the Kety–Schmidt technique. J. Cereb. Blood Flow Metab. 15, 485–491. Madsen, P.L., Cruz, N.F., Sokoloff, L., Dienel, G.A., 1999. Cerebral oxygen/glucose ratio is low during sensory stimulation and rises above normal during recovery: excess glucose consumption during stimulation

295

is not accounted for by lactate efflux from or accumulation in brain tissue. J. Cereb. Blood Flow Metab. 19, 393–400. Magistretti, P.J., Pellerin, L., Rothman, D.L., Shulman, R.G., 1999. Energy on demand. Science 283, 496–497. Mason, G.F., Gruetter, R., Rothman, D.L., Behar, K.L., Shulman, R.G., Novotny, E.J., 1995. Simultaneous determination of the rates of the TCA cycle, glucose utilization, alpha-ketoglutarate/glutamate exchange, and glutamine synthesis in human brain by NMR. J. Cereb. Blood Flow Metab. 15, 12–25. McKenna, M.C., Sonnewald, U., Huang, X., Stevenson, J., Zielke, H.R., 1996. Exogenous glutamate concentration regulates the metabolic fate of glutamate in astrocytes. J. Neurochem. 66, 386–393. Muir, D., Berl, S., Clarke, D.D., 1986. Acetate and fluoroacetate as possible markers for glial metabolism in vivo. Brain Res. 380, 336– 340. Nakao, Y., Itoh, Y., Kuang, T.Y., Cook, M., Jehle, J., Sokoloff, L., 2001. Effects of anesthesia on functional activation of cerebral blood flow and metabolism. Proc. Natl. Acad. Sci. U.S.A. 98, 7593–7598. Norenberg, M.D., Martinez-Hernandez, A., 1977. Fine structural localization of glutamine synthetase in astrocytes of rat brain. Brain Res. 161, 303–310. O’Dowd, B.S., Gibbs, M.E., Sedman, G., Ng, K.T., 1994. Astrocytes implicated in the energizing of intermediate memory processes in neonate chicks. Cogn. Brain Res. 2, 93–102. Oz, G., Henry, P.G., Seaquist, E.R., Gruetter, R., 2003. Direct, noninvasive measurement of brain glycogen metabolism in humans. Neurochem. Int. 43, 323–329. Pascual, J.M., Carceller, F., Roda, J.M., Cerdan, S., 1998. Glutamate, glutamine and GABA as substrates for the neuronal and glial compartments after focal cerebral ischemia in rats. Stroke 29, 1048– 1056. Patel, M.S., 1974. The relative significance of CO2 fixing enzymes in the metabolism of rat brain. J. Neurochem. 22, 717–724. Patel, A.J., Balazs, R., 1970. manifestation of metabolic compartmentation during the maturation of the rat brain. J. Neurochem. 17, 955–971. Peng, L., Zhang, X., Hertz, L., 1994. High extracellular potassium concentrations stimulate oxidative metabolism in a glutamatergic neuronal culture and glycolysis in cultured astrocytes, but have no stimulatory effect in a GABAergic neuronal culture. Brain Res. 663, 168–172. Peng, L., Swanson, R.A., Hertz, L., 2001. Effects of l-glutamate, d-aspartate and monensin on glycolytic and oxidative glucose metabolism in mouse astrocyte cultures. Neurochem. Int. 38, 437–443. Peters, A., Palay, S.L., de Webster, H.F., 1991. The Fine Structure of the Nervous System: Neurons and Their Supporting Cells, third ed. Oxford University Press, New York, pp. 276–295. Pow, D.V., Robinson, S.R., 1994. Glutamate in some retinal neurons is derived solely from glia. Neuroscience 60, 355–366. Rose, S.P.R., 1967. Preparations of enriched fractions from cerebral cortexcontaining isolated, metabolically active neuronal and glial cells. Biochem. J. 102, 33–43. Rothstein, J., Tabakoff, B., 1984. Alteration of striatal glutamate release after glutamine synthetase inhibition. J. Neurochem. 43, 1438–1446. Schousboe, A., Svenneby, G., Hertz, L., 1977. Uptake and metabolism of glutamate in astrocytes cultured from dissociated mouse brain hemispheres. J. Neurochem. 29, 999–1005. Schousboe, A., Hertz, L., Svenneby, G., Kvamme, E., 1979. Phosphate activated glutaminase activity and glutamine uptake in astrocytes in primary cultures. J. Neurochem. 32, 943–950. Shank, R.P., Bennett, G.S., Freytag, S.O., Campbell, G.L., 1985. Pyruvate carboxylase: an astrocyte-specific enzyme implicated in the replenishment of amino acid neurotransmitter pools. Brain Res. 329, 364–367. Sibson, N.R., Dhankhar, A., Mason, G.F., Rothman, D.L., Behar, K.L., Shulman, R.G., 1998. Stoichiometric coupling of brain glucose metabolism and glutamatergic neuronal activity. Proc. Natl. Acad. Sci. U.S.A. 95, 316–321.

296

L. Hertz / Neurochemistry International 45 (2004) 285–296

Sonnewald, U., Westergaard, N., Petersen, S.B., Unsgård, G., Fonnum, F., Hertz, L., Schousboe, A., Petersen, S.B., 1993. NMR spectroscopic studies of 13 C acetate and 13 C glucose metabolism in neocortical astrocytes: evidence for mitochondrial heterogeneity. Dev. Neurosci. 15, 351–358. Sonnewald, U., White, L.R., Ødegård, E., Westergaard, N., Bakken, I.J., Aasly, J., Unsgård, G., Schousboe, A., 1996. MRS study of glutamate metabolism in cultured neurons/glia. Neurochem. Res. 21, 987– 993. Storm-Mathisen, J., Danbolt, N.C., Rothe, F., Torp, R., Zhang, N., Aas, J.E., Kanner, B.I., Langmoen, I., Ottersen, O.P., 1992. Ultrastructural immunocytochemical observations on the localization, metabolism and transport of glutamate in normal and ischemic brain tissue. Prog. Brain Res. 94, 225–241. Suormala, T., Wiesmann, U.N., Cruz, F., Wolf, A., Daschner, M., Limat, A., Fowler, B., Baumgartner, E.R., 2002. Biotin-dependent carboxylase activities in different CNS and skin-derived cells, and their sensitivity to biotin-depletion. Int. J. Vitam. Nutr. Res. 72, 278– 286. Swanson, R.A., Morton, M.M., Sagar, S.M., Sharp, F.R., 1992. Sensory stimulation induces local cerebral glycogenolysis: demonstration by autoradiography. Neuroscience 51, 451–461. Taylor, D.L., Richards, D.A., Obrenovitch, T.P., Symon, L., 1994. Time course of changes in extracellular lactate evoked by transient K+ -induced depolarization in the rat striatum. J. Neurochem. 62, 2368– 2374. Van den Berg, C.J., Garfinkel, D., 1971. A simulation study of brain compartments. Metabolism of glutamate and related substances in mouse brain. Biochem. J. 123, 211–218. Varoqui, H., Zhu, H., Yao, D., Ming, H., Erickson, J.D., 2000. Cloning and functional identification of a neuronal glutamine transporter. J. Biol. Chem. 275, 4049–4054.

Waagepetersen, H.S., Qu, H., Hertz, L., Sonnewald, U., Schousboe, A., 2002. Demonstration of pyruvate recycling in primary cultures of neocortical astrocytes but not in neurons. Neurochem. Res. 27, 1431– 1437. Waelsch, H., Berl, S., Rossi, C.A., Clarke, D.D., Purpura, D.D., 1964. Quantitative aspects of CO2 fixation in mammalian brain in vivo. J. Neurochem. 11, 717–728. Walz, W., Mukerji, S., 1988. Lactate release from cultured astrocytes and neurons: a comparison. Glia 1, 3666–3700. Waniewski, R.A., Martin, D.L., 1998. Preferential utilization of acetate by astrocytes is attributable to transport. J. Neurosci. 18, 5225–5233. Westergaard, N., Varming, T., Peng, L., Hertz, L., Schousboe, A., 1993. Uptake, release and metabolism of alanine in neurons and astrocytes in primary cultures. J. Neurosci. Res. 35, 540–545. Westergaard, N., Drejer, J., Schousboe, A., Sonnewald, U., 1996. Evaluation of the importance of transamination versus deamination in astrocytic metabolism of [U-13 C]glutamate. Glia 17, 160–168. Wolff, J.R., Chao, T.I., 2004. Cytoarchitectonics of non-neuronal cells in the central nervous system. In: Hertz, L. (Ed.), Non-Neuronal cells of the Nervous System, Function and Dysfunction, vol. 1, Elsevier, Amsterdam, pp. 1–51. Yu, A.C.H., Hertz, L., 1983. Metabolic sources of energy in astrocytes. In: Hertz, L., Kvamme, E., McGeer, E.G., Schousboe, A., (Eds.). Glutamine, Glutamate and GABA in the Central Nervous System. Alan R. Liss, New York, pp. 431–439. Yu, A.C.H., Schousboe, A., Hertz, L., 1982. Metabolic fate of (14C)−labelled glutamate in astrocytes. J. Neurochem. 39, 954–966. Yu, A.C.H., Drejer, J., Hertz, L., Schousboe, A., 1983. Pyruvate carboxylase activity in primary cultures of astrocytes and neurons. J. Neurochem. 41, 1484–1487. Yudkoff, M., 1997. Brain metabolism of branched-chain amino acids. Glia 21, 92–98.