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Brain Bioenergetics Bioenergetics Group and Neurochemical Group Joint Colloquium Organized by G. Brown (University College London) and C. Cooper (University College London Medical School), Edited by G. Brown (University College London) and Sponsored by Merck Sharpe and Dohme, The Wellcome Foundation and Hamamatsu Photonics UK Ltd. 65 I s t Meeting held at University of Lancaster, 13- I4 July I994 Energetics of the nerve terminal in relation to central nervous system function Maria Erecinska*§, David Nelson*, Marc Yudkofft and Ian A. Silver$ *Department of Pharmacology and tkpartment of Pediatrics, University of Pennsylvania, School of Medicine and Children's Hospital of Philadelphia, PA 19104, U.S.A. and $Department of Anatomy, School of Veterinary Science, University of Bristol. Bristol 852 8EJ, U.K. Introduction The main function of the mammalian central nervous system (CNS) is the generation, processing and transmission of impulses all of which require movements of ions down their concentration gradients. T o perform these activities, the key cations, Na+, K + and Ca2+,have to be maintained in electrochemical disequilibrium across the plasma membrane, a state which has to be rapidly reinstated before each functional cycle. Because maintenance of disequilibria requires the constant input of energy, 5 0 4 0 % of ATP produced in the CNS during rest, and 90% or more generated during enhanced activity, is consumed to support ion movements [ 11. Brain contains ATP (2-3pmoVg) and ADP (0.2-0.5 pmol/g) at an ATPIADP ratio of 8-10 and the additional energy reservoire, comprising creatine phosphate and creatine, at a total concentration of 10 pmol/g and a creatine phosphate] creatine ratio of 0.8-1.0 [l]. The key respiratory substrate is glucose, and under physiological conditions, with oxygen consumption rates of 1.6-5.4 pmol/g (depending on the animal), very little lactate is produced [l]. Thus brain relies heavily on glucose and oxygen to support its energy metabolism. These relationships are illustrated in Table 1 , which summarizes events occurring in hippocampal CA 1 neurons during short-term (8 min) ischaemia and subsequent recovery. It is Abbreviations used: CNS. central nervous system; [Na I,, intracellular [Na 1; TCA, tricarboxylicacid. $To whom correspondence should be addressed, at: Department of Pharmacology. University of Pennsylvania School of Medicine. Philadelphia, PA 19 104-6084. U.S.A. + + clear from the figures presented that glucose is exhausted very rapidly and that the lack of oxygen leads to massive movements of ions, with consequent collapse of their gradients. Internal K + concentration decreases and Na+, CaL+,H+ and CIconcentrations increase. However, this situation is swifily and almost completely reversed when oxygen and glucose are reintroduced, which indicates that neurons have powerful mechanisms that restore ion gradients and that aerobic ATP generation is able to support these processes. Studies of the mechanisms which control energy production and ion movements in whole brain are dificult. This has led to the development of several model systems in which conditions can be more readily manipulated and controlled. One such system is the preparation of nerve-ending particles (synaptosomes) which possesses many properties of intact neurons. This paper describes energetic properties of synaptosomes and the interrelationships between their energy level and production and ion movements and gradients. Energy level and production in synaptosomes Synaptosomes isolated from brains of nonanaesthetized animals and equilibrated with oxygen and glucose for 10-15 min contain ATP (1.405.6 nmol/mg of protein) and ADP (0.4-2.45 nmol/ mg of protein) at a ratio of 1.4-6 (range of values reported by different laboratories 2:l [2-81) and phosphocreatine (2.2-5.8 nmol/mg of protein [2,4,6- 101) and creatine ( 1 5-25 nmol/mg of protein [7-lo]) at a ratio of 0.4-0.5. Creatine phosphokinase immunoreactivity is concentrated in nerve terminals, at least in some regions of the brain [ 11 and brain mitochondria exhibit high activity of this 959 - I994 Biochemical Society Transactions Brain glucose levels, membrane potentials and intracellular ion concentrations in hippocampal CAI neurons during short-term low-flow ischaemia and recovery 960 All parameters were measured with microelectrodes. External concentrations of the ions of interest are as follows (in mM): Na’, 133 f 2.2; K + . 3.37f0.05; Ca2+. 1.45f0.20 CI-. 1 3 5 f 4 and H+ as pH, 7.34f0.02. Data taken from [20] and provided by M. Erecinska. D. Nelson, M. Yudkoff and I. A. Silver (unpublished work). Values are means f SD ( n = 12-104). Control lschaemia(8min) Recovery ( I0 min) 2.4fO.l -67.0f7.4 O.OfO.O 3.5 f 0.9 - 17.0f9.3 - 4 I .3 f 18.0 0.089f0.024 30.2f 11.3 0.354 f 0. I3 I 7.33f0.04 6.21 f0.45 6.94 f 0.22 25.5f2.3 72.3f9.6 33.7 f 6.4 83.6f3.9 37.1 f 11.3 76.8 f 8.2 24.7f5.8 67.2f8.1 38.7 f I I .7 Effect of enhanced ion movements on synaptosomal rates of energy generation All measurements were carried out at 37°C. The rates of lactate production were calculated from the difference in the level of this metabolite at ‘time zero’ (i.e. after 10 min of preincubation with glucose and Ca2’) and after 5 min of incubation either with (experimental) or without (control) the compounds indicated. Nucleotides were measured by h.p.1.c. on neutralized perchloric-acidextracts of synaptosomes quenched after 5 min incubation. The rates of ATP production were calculated assuming stoichiometric factors of 6 (0, uptake) and I (lactate generation) respectively. Values are meansf S.E.M. ( n = 3-8). Condition 0, uptake Lactate production (nmol/min per mg of protein) Control Veratridine (40pM) Veratridine (40 pM) + ouabain (I mM) Monensin ( I0 pM) Nigericin (5 pM) 5.20 f 0.22 16. I0 f I .03 4.72 f 0. I I I .40 f 0. I7 3.08 f 0.52 0.63 f 0.07 32.6 99.7 29.0 4 3 2 I .80 f 0.07 I. I3 f0.04 I .56 f 0.09 5.8 I f 0. I6 I .8 I f 0.09 2.95 f 0.32 8.58 f 0.78 18.2 f I .30 6.04 f I .08 69.7 73.2 26 8 0.72 f 0.05 0.43 f 0.07 I .2 I f 0.03 0.69 f 0.04 I I .20 f 0.44 enzyme [ l l ] . The rate of oxygen consumption ranges from 2 to 4nmoVmg of protein per min at 25-30°C and from 5.2 to 11 nmol/mg of protein per min at T C , and that for lactate production from 0.3 to 0.5 and from 0.8 to 2.7 nmol/mg of protein per min respectively at the same temperatures (Table 2) [ 3,6,8,12- 141. Thus oxidative phosphorylation provides over 90% of total ATP produced under aerobic conditions (see Table 2 for calculations). A comparison of the values measured in synaptosomes with those in whole brain indicates that both the content of the high-energy phosphate compounds and the glycolytic and mitochondria1 oxidative activity of the terminals are 5-10-fold lower than those in the intact organ. It has been proposed that this behaviour is a consequence of the heterogeneity of the preparation which, it was suggested, was composed of vesicles with differing degree of Volume 22 ATP production Glycolytic contribution (%) ATP (nmol/ mg of protein) ATP/ADP ‘intactness’ and different content of mitochondria, only some of which had high ATP/ADP ratios and the capacity to produce energy [6]. However, this conclusion was based on the finding that the overall ATP/ADP was only 2.18, whereas values as high as 5-6 have been obtained in studies by other authors (Table 2) [4,15]. Moreover, Leong et al. [ 161 reported that the activities of several enzymes of the tricarboxylic acid (TCA) cycle in synaptic mitochondria were at least 2-fold lower than in the non-synaptic organelles, which suggests that the intrinsic activity of the energy-producing pathways in the nerve endings may be lower than in cell bodies or glial cells. It should be remembered that small but constant loss of ATP from the terminals may also occur through exocytosis in the presence of calcium in the medium and contribute to the low nucleotide level. Brain Bioenergetics Although synaptosomes, in contrast to many intact cells, are able to transport and oxidize several intermediates of the TCA cycle [12], it is not clear at what concentration these molecules are present in vivo;thus glucose is likely to be the key physiological substrate. If this is true, two issues deserve comments: the transfer of NADH from the cytosol to the mitochondrion; and metabolism of pyruvate by pyruvate dehydrogenase. With respect to the first, it has been shown that inhibition of transamination either by amino-oxyacetate [17] or Bmethylene-aspartate [ 181 inhibits glucose oxidation and lowers the synaptosomal ATPlADP ratio while increasing lactate production and the lactatelpyruvate ratio. Because both treatments inhibit transamination reactions, which are a necessary component of the malate-aspartate shuttle, it has been suggested that the latter plays an important role in the transfer of reducing equivalents across the mitochondrial membrane. Interestingly, the same phenomenon was observed in the presence of 3-nitropropionate. an inhibitor of succinate dehydrogenase and a mitochondria1 poison, which lowers intrasynaptosomal aspartate level, most probably by depletion of oxaloacetate [ 191. With respect to the role of pyruvate dehydrogenase in synaptosomal energy production, this enzyme is regulated by a number of factors, one of which is Ca" [20]. It has been shown that synaptosomal pyruvate dehydrogenase is present largely (80-90%) in its active state and that an increase in internal [Ca" ] increases this proportion only slightly [21]. There is, moreover, some controversy with respect to the role of Ca" in synaptosoma1 pyruvate metabolism. Whereas Patel et al. [22] were unable to observe any stimulation of oxygen uptake by veratridine in the absence of external Ca" and with pyruvate as the respiratory substrate, no requirement for this cation under apparently identical conditions was seen in studies of other investigators [ 23,241. This suggests that CaL+ may not be a major regulator of pyruvate dehydrogenase in the nerve endings. One of the interesting issues in brain energy metabolism is to what extent other fuels can replace glucose as the energy source. Bradford and coworkers [25] have shown that glutamine is a major substrate for the nerve endings, and it is known that during in vivo hypoglycemia, the levels of glutamine and glutamate decrease and that of aspartate increases. Using "N-labelled amino acids, it was possible to demonstrate that transamination from glutamate to aspartate is very active [26,27] and that a series of reactions may operate in which gluta- mate produced from glutamine through the glutaminase reaction is transaminated to aspartate with the production of 2-oxoglutarate, which is then oxidized in the TCA cycle with the synthesis of ATP and regeneration of oxaloacetate. Consistent with this suggestion are the results of experiments with deuterated glutamine (Figure la) (M. Yudkoff D. Nelson, Y. Dai Khin and M. Erecinska, unpublished work) which show the rapid appearance of labelled succinate, malate and aspartate. From the extent and pattern of labelling, the rate of this segment of the TCA cycle in the presence of glucose was calculated to be 3.14-6.65 nmollmin per mg of protein (range of numbers derived from calculations from different labelled precursors) at 30°C. Using a similar approach but another labelled precursor, [3-13C]aspartate (Figure lb), the rate of segment between malate and 2-oxoglutarate was estimated to be somewhat smaller, 0.92-2.57 nmoll min per mg of protein. In contrast to the high activity of the aspartate transaminase reaction [27]. that of glutamate dehydrogenase is very slow [28]. which suggests that synaptosomes conserve glutamate. It is possibly significant that two endogenous constituents of the nerve cell, M&+ and polyamines, are powerful inhibitors of brain glutamate dehydrogenase [29]. Synaptosomal ion concentrations Synaptosomes contain potassium at a concentration of 45-65mM [10,13,30,31] and maintain a K + diffusion potential equivalent to - 50 to - 60 mV; this agrees well with estimates of the membrane potential from the distribution of XhRb [32]. Values derived from the distribution of the radioactive lipophylic cations are somewhat higher [33], most probably because of binding (andlor sequestration) of these probes to synaptosomal constituents. Measurements of intracellular [Na+1, "a'],, in synaptosomes (e.g. atomic absorption or distribution of zzNa) at physiological levels of the latter are more difficult to make because of the large contamination with the external fluid containing high concentrations of the cation. Hence, not surprisingly, the original calculations yielded figures for "a+], upwards of 50mM [13]. However, if the internal and trapped volume are measured simultaneously and the necessary corrections then made, estimated values range from 25 to 29mM at 130-140 mM NaCl in the medium [34,35], which is not much different from [Na'], in neurons in vivo. Recently, "a'], was determined from the fluorescence of an indicator, benzofuran isophthalate, I994 96 I Biochemical Society Transactions ~~ Diagram outlining pathways of tracers used to measure flux through the TCA cycle between 2-oxoglutarate and oxaloacetate (a) and oxaloacetate and 2-oxoglutarate (b) 962 In (0)~-[2,3,3,4,4-'H]glutaminewas used, and in (b). 1-[3-'~C]aspartate. A denotes 'H, identifies ' 'C. cytosol * Mitochondrion (4 Glucose+Pyruvme \ AGlutamine A Glutamine lsoatrate I ~ASUCClnnto 8 *Aspartate and reported to be 10.9 mM [ 361, i.e. considerably less than the figures above. Whether this disparity can be accounted for by the difference in methodology or whether the concentration of sodium in the nerve endings is indeed lower in the nerve cell body, cannot be decided at present. The advent of fluorescent indicators for CaL' has enabled estimates to be made of the free internal concentration of this cation in structures which are too small to be penetrated with microelectrodes. Although the number of studies using either Quin-2 or Fura-2 is very large, they do not differ significantly from the original estimates of Ashley et al. [ 371, Richards et al. [ 381 and Hansford and Castro [21]. The figures fall between 0.1 and 0.35 ,uM and depend to some extent on the state and/or quality Volume 22 'Oxaloacetate f \\ -+*Malate A \ 'cisAconitate I 1 \ \ \ '. Fumarate R '2sxoglutarate / / -SuccinateY of the synaptosomal preparation (see [ 391 and references therein). There are three other ions of interest for which some results are available. Measurements of intrasynaptosomal pH indicates that the concentration of protons inside the nerve ending is slightly higher than that in the external environment, with pH, values of 7.1-7.3 [ 14,38,40]. [Mg"], estimated with an entrapped indicator eriochrome blue was 0.3 mM at 1 mM external [MgL'] and no CaL+. With 1 mM CaL+ in the medium, the apparent [Mg"], declined to 0.2mM, and at 2mM to 0.1 mM. The concentration of free chloride calculated from changes in fluorescence of N-(6-methoxyquinolyl) acetoacetyl ester in synaptoneurosomes from rat brain was found to be 14 mM [41]. Brain Bioenergetics Relationships between ions and energy metabolism The key enzyme responsible for the maintenance of ion gradients in the brain is the ouabain-sensitive N a + / K + pump. This protein extrudes 3equiv. of Na' and accumulates 2equiv. of K' with concomitant hydrolysis of one ATP. The estimated maximal activity of this ATPase in synaptosomes is l60-200nmol/min per mg or protein at 37°C in a frozen-thawed preparation [ 421 and 260 nmol/min per mg in the membrane fraction [43]. Ultrastructural localization studies on whole brain confirm these in vitro results, in that they show intense ATPase-specific staining over the entire plasma membrane of the synaptic area [44]. Interestingly, these same areas are very reactive for cytochrome oxidase [45], which indicates that the maintenance and restitution of ionic balances are energetically costly. The K,,, of the Na'/K+ pump for K' in broken synaptosomes is low, 0.65 mM [42], which agrees well with the ECs,, values for the K + dependent stimulation of lactate generation and oxygen consumption (0.7- 1.5 mM) [24]. The K,,, for Na+ is considerably higher than that for K + , and values reported in the literature range from 10 1421 to 80mM 1431. Hrodsky and Guidotti [46] noted that Na' affinity of brain Na'/K'-ATPase was dependent on both isozyme and environment of the pump, the apparent dissociation constant being much greater in synaptosomes than in their membranes. There are two binding sites for ATP on the pump, with differing affinities: the K,,, on the catalytic site is low ( 10pM)whereas that on the regulatory site is much higher, > 0.5 mM [I]. It is evident from a comparison of the affinities for the three substrates that, under physiological conditions, the key factor bhich regulates the pump activity is [Na'],. llowever, the rather high K,,, for ATP on the regulatory site indicates that changes in concentration of the nucleotide may also be a contributory factor. particularly in some parts of the neuron, such as the synapse, where the levels of high-energy phosphate compounds may be lower. Direct measurements of the ouabain-sensitive ""Rb influx in the presence of amytal (an inhibitor of the respiratory chain), either with or without glucose 1.241. confirm this supposition and show that a decrease in pump activity can occur at an early stage of limitation in ATP generation. In non-stimulated synaptosomes incubated with 5 mM K', the rate of ""Rb influx is 9.8 nmoV min per mg of protein at 30°C: [24]. Addition of ouabain under the same conditions decreases the - rate of oxygen uptake by 0.89 nmol/min per mg of protein, which gives an Rb/O, ratio of 11.5 and a Rb/ATP stoichiometry of 2, in agreement with the known properties of the pump. Stimulation of pump activity markedly increases the rate of K' uptake [ 3 11 and simultaneously raises the rate of synaptosoma1 energy synthesis (Table 2) [ 14,24,47]. Increase in [Na'li, such as occurs after opening by veratridine of the voltage-dependent Na -channels, stimulates oxygen consumption by 2-5-fold [ 3,6,8,24]. This rise is completely prevented by addition of ouabain (Table 2), which indicates that enhanced ion movements consume under such conditions the overwhelming proportion of the ATP produced. Glycolysis, albeit activated, contributes only marginally to total energy generation (Table 2). Interestingly, when the activity of phosphofructokinase (the rate-controlling enzyme of glycolysis) is independently stimulated by an increase in intrasynaptosomal pH caused by addition of the ionophore monensin (which exchanges Na+ for H+), the glycolytic contribution to overall ATP synthesis becomes much greater (Table 2) [ 141. The role of glycolysis in supporting the N a + / K+-ATPase is crucial when oxygen becomes limiting and the rate of oxidative phosphorylation declines. This is underscored by experiments which show that the rate of anaerobic K + emux, and hence its loss from synaptosomes, is > 2-fold faster in the absence of glucose [31]. Although there is some evidence [20] that the pump uses the ATP produced by glycolysis in preference to that supplied by oxidative phosphorylation. when the nucleotide level falls beyond a critical value, high rates of lactate generation become insensitive to the action of ouabain 1241. In addition to constant uphill movements of Na' and K'. synaptosomes also maintain a large electrochemical gradient for Ca2+. There are two mechanisms that expel CaL+ from the nerve endings: the Ca" pump and the Na'/Ca'+ exchanger 1481. The former is fueled directly by ATP and exchanges CaL+ for 1-2 11'. The latter removes one CaL' ion from inside against three Na' ions entering from outside; as three Ka+ ions are pumped out by the Na+/K+-ATPase per each ATP hydrolysed, the energetic cost of the coupled process is ICa'+ per ATP. The pump functions predominantly under low Ca" loads, whereas the exchange predominates at higher loads [ 491. The rate of Ca2+ influx under non-stimulated conditions is 0.5-10 nmol/min per mg of protein at 30-37°C [SO], which corresponds to the same rate of ATP utilization, or 0.1-0.2 nmol OL consumed/min per + I994 963 Biochemical Society Transactions 964 mg of protein. This amount is too small to be detected experimentally. Upon membrane depolarization and opening of the voltage-dependent channels, Caz+ entry pathways can be activated by as much as 3-&fold [SO], which would increase the rate of energy utilization to 3-6nmoVmin per mg of protein. However, the maximal increase in the oxygen consumption which this would cause is only 1 nmol/min per mg of protein, which is small compared with that caused by movements of ions through the Na+ pump. This may explain why a rise in [Caz+],induced by administration of veratridine, monensin or nigericin has little effect on synaptosomal respiration [ 14,24,47]. - Concluding remarks Like any model system, the synaptosomal preparation has advantages and pitfalls. Nevertheless, when care is taken to purify vesicles with a high ATP/ ADP ratio, several properties of neurons can be conveniently studied. Those discussed herein include ion levels, movements and gradients and the relationships between these parameters and energy expenditure. A similarity between the results obtained in this preparation in vitro and those in brain in vivo makes it a valid model system for the study of nerve-cell metabolism. The authors’ research cited in this review was supported by grants NS28329 and NS27889 from the National Institutes of Health (LISA.). 1 Erecinska, M. and Silver, I. A. (1989) J. Cereb. Hlood Flow Metab. 9,2-10 2 1)e Helleroche. J. S. and Bradford. H. F. (1972) J. Neurochem. 19,585-602 3 Harvey, S. A. K., Hooth. H. F. G. and Clark, J. H. (1983) Hiochem. J. 212,289-295 4 Kauppinen, H. A,. McMahon, H. and Nicholls. I). <;. ( 1988) Neuroscience 27. 175- 182 5 Kauppinen, K.A. and Nicholls, 1). G. ( 1 986)J. Neurochem. 47. 1804- 1869 6 Kyriazi, 11. T. and Hasford, H. E. (1980) J. Neurochem. 47.5 12-528 7 Hafalowska, U., Erecinska, M. and Wilson, 1). F. ( 1980)J. Neurochem. 34,1180- 1 186 8 Scott, 1. A. and Nicholls. L). G. (1980) Hiochem. 186, 21-33 9 Harvey, S. A. K.. Hooth, H. F. G. and Clark, J. H. (1982) Hiochem. 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(1989) Trends Neurosci. 12. 94- 101 46 Hrodsky. J. I,. and Cuidotti. <;. (1990) Am. J. I’hysiol. 258. C803-CX11 47 Erecinska, M., Nelson, 11.. Dagani. F.$Deas. J. and Silver. 1. A. (1993) J. Neurochem. 61, 1356- 1368 48 Hlaustein, M. P. (1988) Trends Neurosci. 11,438-443 49 Sanchez-Armass, S. and Hlaustein, M. 1’. (1987) Am. J. I’hysiol. 252, C595-Ch03 50 Hlaustein, M. P. (1975) J. I’hysiol. (Idondon) 247. 617-655 Received 20 May 1994 From the synaptosome to the intact brain Risto A. Kauppinen NMR Research Group, Department of Biochemistry and Biotechnology,A. I. Virtanen Institute, University of Kuopio, P.O. Box 1627, FIN-70620 Kuopio, Finland Introduction Ikain function is centered at the synapse and, consequently, substantial scientific efforts have been directed towards exploring synaptic function. In neurochemical research, it has been customary to divide neuronal parts of the synapse into two. Since the early 1960s. ‘pinched-off presynaptic nerve terminals or ‘synaptosomes’ have been extensively used as a model of the presynaptic neuron [ 11. Our present understanding of nerve-terminal metabolism and bioenergetics [2], neurotransmitter release and uptake [ 3 ] , and electrophysiology is largely based on studies carried on synaptosomes. One of the interesting issues within neuroscience is the role(s) of 1.-glutamic acid. As the most abundant cerebral transmitter, glutamate mediates a majority of fast excitatory impulses in the cerebral cortex. and is therefore strongly involved in integrated brain function [4].On the other hand, activation of postsynaptic glutamate receptors [ 51 evidently causes neuronal degeneration during brain energy failure, for example, following ischaemia [6,7]. In this paper, mechanisms of glutamate release from synaptosomes are discussed with major emphasis on their energy and CaL’ dependency and the contribution of various intraterminal glutamate pools to this release [XI. This discussion will be extended towards compartmentation of the transmitter glutamate in the intact cerebral cortex in the light of previous studies using ‘ € I and ‘€I{’.’C}n.m.r. spectroscopy [g-ll]. The aim in the latter part will be to weight the relevance of synaptosome studies to the conditions in an intact brain preparaAbbreviations used: [Ca’+I,. intracellular ICa” J; CI’MG. Carr-l’urcell-Meiboom-Gill; NAA, N-acetylaspartate. tion with special reference to the origin of ‘excitotoxic’ glutamate during severe energy failure. Bioenergetics of synaptosomes Virtually all ( > 90%) of the synaptosomes metabolizing glucose isolated from guinea-pig cerebral cortex contain functioning mitochondria [ 121. IJnstimulated nerve terminals respire in the absence of glucose or in the presence of a glycolytic inhibitor, such as iodoacetate, at rates which are not different from those determined in the presence of glucose. Oxidation of endogenous non-glucose substrates is, however, inefficient in maintaining the ATPIADP ratio, which is reduced by 50% in the absence of glucose [ 121. ‘Resting’ plasma or mitochondrial membrane potentials are not affected by iodoacetate [ 121. Interestingly enough, it has been reported that ATP levels in primary culture of cortical astrocytes are strictly supported through aerobic glucose metabolism, with minor contribution from mitochondria1 synthesis [ 131. In the absence of glucose, astrocytic ATP levels and plasma-membrane potential collapse precipitously. Processes of astrocytes reside perisynaptically and contribute both to the maintenance of ionic and transmitter homeostasis in the extracellular space [ 141. Assuming that a similar type of glucose dependency prevails in the brain in &YO, shortage of exogenous glucose would primarily affect energy levels in astrocytes. On the other hand, astrocytes contain the majority of cerebral glycogen [ 1.51. Synaptosomal energy state is highly sensitive to ‘ischaemia-like’ conditions similar to the intact brain [ 161. When oxidative phosphorylation is inhibited, either by a protonophore or by blocking cytochrome oxidase, a precipitous drop of ATP and I994 965