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Do active cerebral neurons really use lactate rather than glucose?
Opinion
TRENDS in Neurosciences Vol.24 No.10 October 2001
Do active cerebral neurons really use lactate rather than glucose? Ching-Ping Chih, Peter Lipton, and Eugene L. Roberts, Jr Glucose has long been considered the substrate for neuronal energy metabolism in the brain. Recently, an alternative explanation of energy metabolism in the active brain, the astrocyte–neuron lactate shuttle hypothesis, has received attention. It suggests that during neural activity energy needs in glia are met by anaerobic glycolysis, whereas neuronal metabolism is fueled by lactate released from glia. In this article, we critically examine the evidence supporting this hypothesis and explain, from the perspective of enzyme kinetics and substrate availability, why neurons probably use ambient glucose, and not glial-derived lactate, as the major substrate during activity.
Ching-Ping Chih Eugene L. Roberts, Jr Geriatric Research, Education, and Clinical Center, and Research Office, Miami VA Medical Center, Miami, FL 33125, USA. Peter Lipton Dept of Physiology, University of Wisconsin School of Medicine, Madison, WI 53706, USA. Eugene L. Roberts, Jr* Dept of Neurology, University of Miami School of Medicine, Miami, FL 33136, USA. *e-mail: roberts@ neuron.med.miami.edu
The conventional view that glucose oxidation fuels most activity-associated energy metabolism in neurons (Fig. 1) has recently been challenged by the astrocyte–neuron lactate shuttle hypothesis (ANLSH)1–6, in which glial-produced lactate fuels neurons (Fig. 2). According to the ANLSH, neural activity increases the extracellular concentration of glutamate, whose uptake by glia stimulates Na+-K+ ATPase and glutamine synthetase activity. This stimulates glial anaerobic glycolysis (in this paper ‘anaerobic glycolysis’ refers to the conversion of glucose to lactate, and ‘glycolysis’ refers to the conversion of glucose to pyruvate). Glia then release lactate, and neurons use it to fuel their activity. In the next section, we critically examine the major experimental evidence used to support the ANLSH. Evidence used to support the ANLSH Glucose metabolism and glutamate cycling
Oxidative glucose metabolism and glutamate cycling: According to the ANLSH, glutamate cycling in glia drives all cerebral glucose metabolism, and glial glucose metabolism is entirely anaerobic. The hypothesis thus predicts that the rate at which metabolized glucose enters the neuronal tricarboxylic acid (TCA) cycle should equal the rate of glial glutamate cycling. Sibson et al.5 tested this prediction and found a 1:1 stoichiometry between oxidative glucose metabolism and glutamate cycling. Although this result was considered strong support for the http://tins.trends.com
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ANLSH (Ref. 2), neither the site of glucose uptake nor the mode of glial glucose metabolism could be determined, so the data are consistent with many different models of glucose utilization. For example, if as little as 6% of glial glucose metabolism is oxidative, then glutamate cycling would have needed only 50% of the measured glucose metabolism. This means that neurons could have consumed the other 50% of glucose aerobically. Thus, for the results of Sibson et al.5 to specifically support the ANLSH, glial glucose metabolism should be entirely anaerobic. Glial anaerobic glucose metabolism and glutamate cycling: The inference that this is the case comes from a study showing that uptake of exogenous glutamate was strongly associated with increased lactate production in cultured astrocytes7. However, the applicability of this finding to the brain in situ is questionable. Cultured cells, especially static cultures, such as those used by Pellerin et al.8, generally rely far more upon anaerobic glycolysis than do cells in situ. The cultured glia might have undergone this transformation. Furthermore, in other studies of cultured astroglia, glutamate uptake was accompanied by little or no lactate production and was driven by oxidative metabolism9–11. Thus, although the data of Sibson et al.5 are consistent with the ANLSH, they are also consistent with other models, including the conventional hypothesis. Comparison of glucose and lactate as energy substrates for neurons
According to the ANLSH, neurons use glial-derived lactate during activity. Thus, a crucial prediction is that neurons use lactate in preference to glucose, as both substrates are in the extracellular space at comparable concentrations (about 1 mM; Refs 12,13). Several lines of evidence have been used to support this prediction. Lactate dehydrogenase isozyme distribution: According to ANLSH proponents, neurons preferentially use lactate because lactate dehydrogenase-1 (LDH-1), the predominant isoform of LDH in neurons, is more suited to oxidize lactate than LDH-5 as a result of its lower Km for lactate14. However, a lower Vmax (Ref. 15) for, or abundance of, LDH-1 might well offset this Km difference, depending on lactate levels. More importantly, the Ki for the pyruvate product inhibition of LDH-1 (0.18 mM) is lower than that for LDH-5 (0.28 mM)16. Resting pyruvate levels of 0.1–0.2 mM in brain cells17 mean that LDH-1 will generally be more strongly inhibited than LDH-5, diminishing the likelihood that neurons use lactate during increased activity. Indeed, if in vitro kinetic data are applicable in vivo, the higher sensitivity of LDH-1 to pyruvate substrate inhibition and lactate product inhibition might help ensure metabolism of pyruvate through the TCA cycle rather
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Opinion
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Fig. 1. Steps leading to increased ATP production via oxidative energy metabolism in neurons during neural activity according to the conventional hypothesis: Na+ entry and K+ release during electrical activity initiate increased oxidative energy metabolism within neurons (1). By activating neuronal Na+-K+ ATPase in the plasma membrane, leading to increased levels of ADP, Pi, and AMP, and to decreased levels of ATP [shown partially at (2)]. These changes rapidly activate glycolysis (conversion of glucose to pyruvate) (3a), and the TCA cycle and mitochondrial oxidative phosphorylation (OP) (3b). This enhances the rate of ATP synthesis (4a and 4b), with the dominant effect being at the mitochondria. Activation of glycolysis lowers cell glucose levels (5), leading to an increased flux of glucose into neurons via the neuronal glucose transporter, GLUT-3 (6), which is localized in pre- and postsynaptic elements. The thick arrows emphasize that, according to the conventional hypothesis, neurons metabolize glucose to pyruvate, which enters the TCA cycle. Generated ATP is largely used to restore Na+/K+ balance via Na+-K+ ATPase. A product of the rapid increase in glycolysis is increased NADH/NAD+, H+, and increased cytoplasmic pyruvate. These changes drive the LDH reaction toward lactate production. These factors militate against utilization of lactate emanating from glia. In astrocytes, the uptake of glutamate (Glu) and Na+ via high affinity glutamate transporters activates oxidative glucose metabolism, which provides ATP for restoring Na+/K+ and synthesizing glutamine (Gln). The basic activation process is considered similar to that in the neuron. The conventional hypothesis does not ascribe any particular fraction of glucose metabolism to aerobic or anaerobic (lactate-producing) pathways.
than into the production of lactate, assisting in the full oxidation of glucose in highly aerobic cells such as neurons and cardiac cells18,19. Thus, a differential distribution of LDH-1 and LDH-5 does not imply that neurons are net users of lactate during activity. http://tins.trends.com
Lactate utilization by neurons: (1) Utilization by neuronal cultures and isolated nerve tissue. Astrocytes and neurons take up lactate and oxidize it into CO2 (Refs 20–26) and neurotransmitters27–30. Lactate supports low frequency neural activity in the absence of glucose31–33. However, none of these studies address whether active-neural tissue preferentially uses lactate. In one study, isolated nerve tissue produced more CO2 from lactate than from glucose when both substrates were present34, providing some support for the ANLSH. However, this study was carried out on unstimulated tissue, in which glycolysis is largely inhibited (see below), and the cell type that used lactate was not identified. Glia also readily metabolize lactate20 and could have accounted for the lactate utilization. (2) Utilization by photoreceptors. It was concluded that isolated photoreceptors only used lactate released from retinal glia (Müller cells), even when glucose was present35. Although results from the retina do not necessarily apply to brain, if they do this result might be supportive of the ANLSH. However, in their calculations the authors inadvertently failed to consider that only half the cell mass was composed of glia when glial cells and photoreceptors were incubated together (discussed in Ref. 19). This leads to a large overestimation of neuronal lactate consumption, negating the conclusion that neurons only use glial-derived lactate.
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Fig. 2. Steps leading to increased ATP production via oxidative energy metabolism in neurons during neural activity according to the astrocyte–neuron lactate shuttle hypothesis (ANLSH). The initiator of increased oxidative metabolism within the neuron is the uptake of glutamate and Na+ into glial cells via the high affinity glutamate transporter (1). This activates Na+-K+ ATPase and glutamine synthase in glia, leading to increased levels of ADP, Pi, and AMP, and to decreased levels of ATP (2a and 2b). This exclusively activates anaerobic glycolysis (3), which generates lactate (4). The lactate is transported across the glial membrane, and builds up in the extracellular space where the increased concentration gradient leads to its net transport into neurons. The increased lactate in the neurons is converted to pyruvate via LDH-1 (lactate dehydrogenase-1) (5), which enters the TCA cycle, and increases ATP production in the neurons via oxidative phosphorylation (OP) (6). The thick arrows emphasize that lactate produced via glycolysis in glia fuels oxidative metabolism in neurons. Decreased glucose levels in glia are restored from glucose in the blood and extracellular space via glucose transporter-1 (GLUT-1). The ANLSH postulates that the ATP that is used to remove 3 Na+ co-transported with 1 glutamate, and the ATP that is used to synthesize glutamine from 1 glutamate, are precisely regenerated by the 2 ATP produced by the anaerobic metabolism of 1 glucose molecule. Thus, the activity-associated increase in anaerobic glycolysis is equal to the increase in ‘glutamate cycling’. As noted in the figure, the ANLSH requires that ATP utilization from glutamate cycling activates anaerobic glycolysis only. It does not activate oxidative phosphorylation in glia. In addition, as noted in the figure, ATP utilization via the Na+,K+ pump in neurons does not activate neuronal glycolysis.
(3) Utilization of lactate in vivo. A study of rat cortex concluded that repetitive stimulation caused a larger decrease in extracellular lactate than in extracellular glucose, when lactate had been previously elevated ∼twofold by activity36. This appears to provide support for the ANLSH, but there http://tins.trends.com
are problems with the interpretation. Results from only one experiment were shown, without statistical analysis, and there was clearly observable intertrial variability (see Ref. 19). Even if the results are correct, the cell type using lactate was not determined; glia could have accounted for the lactate utilization. (4) Utilization of lactate during stress. A series of studies in hippocampal slices37,38 concluded that neurons require glial lactate during recovery from stress conditions. Although important, these studies might not provide strong support for the ANLSH for two reasons: first, 4-CIN (α-cyano-4-hydroxycinnamate), which was used to block plasmalemmal lactate transport, also blocks pyruvate entry into mitochondria39,40. This seriously compromises interpretation of the results. Second, the results describe obligatory lactate use during recovery from stress conditions, not during normal physiological activity. Metabolic conditions, including ATP levels, are greatly altered during anoxic and excitotoxic stress, so extrapolation of these results to normal physiology is not valid a priori. The next section offers a rationale for accepting the conventional (neuronal glucose utilization) hypothesis (Fig. 1). Evidence for glucose as the major substrate during neural activity
The crucial question in this section is whether activation of neuronal glycolysis occurs during
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activity in vivo, and whether it dominates metabolism of glial-generated lactate. Neuronal glycolysis is activated rapidly during neuronal activity
Neurons are well equipped to metabolize glucose in situ. Although neuronal axons and dendrites do not usually directly contact capillaries, glucose is readily available to neurons. Levels of brain glucose, which is generally evenly distributed between the intracellular and extracellular compartments41, greatly exceed the Km for hexokinase (0.04 mM; Ref. 42), even during neural stimulation when values might fall by 20–30% (Refs 36,43). Glucose transporters are abundant in synaptic membranes44,45. GLUT-3, the predominant glucose transporter in neurons, transports glucose seven times faster than GLUT-1, the main glial glucose transporter46. In addition, neurons have high levels of glycolytic enzymes47,48. Thus, neurons are well positioned to use glucose as an energy substrate. The glycolytic rate is tightly regulated by energy demand49. Hexokinase and phosphofructokinase are largely inhibited at rest49, but are rapidly activated by changes in adenine nucleotide and phosphate levels during increased ATP utilization49. Glycolytic rates rapidly increase in neurons when energy demand increases50–52. Thus, there is no apparent reason why neural activity in situ should activate glial glycolysis but not neuronal glycolysis, as proposed by the ANLSH (Fig. 2). Because oxygen utilization is elevated during the first 1–3 s of neural activity53,54, rapid increases in the supply of pyruvate to neuronal mitochondria are necessary to maintain oxidative metabolism. Activation of neuronal glycolysis requires only the transport of small molecules within neurons. By contrast, for glialproduced lactate to meet this need a multistep process, including glial-glutamate cycling, and lactate production and transport, must occur. It is thus probable that neuronal glycolysis is activated more rapidly. Increases in extracellular lactate lag behind neural activity
In contrast to glycolytic enzymes, LDH is not regulated by energy demand, so increases in lactate oxidation must be driven either by increases in extracellular lactate, and hence intracellular lactate, or by decreases in cytoplasmic pyruvate. (Changes in intracellular compartmentation, if present, could affect lactate oxidation55–58, however, any such changes are difficult to predict based on current knowledge.) Pyruvate levels increase very rapidly during neural activity59,60 suggesting that increases in lactate oxidation could only occur by elevating extracellular lactate. This is supported by studies in cultured cerebellar neurons, where elevating extracellular K+ at constant substrate concentrations stimulated glucose and pyruvate utilization but not lactate utilization52. Although http://tins.trends.com
extrapolation of these results to in vivo conditions might be questionable they support the conclusion that lactate utilization cannot be increased in the absence of an increase in extracellular lactate. Thus, it appears that extracellular lactate would have to increase for neuronal lactate to increase. These increases in lactate should be substantial. The monocarboxylate transporters do not show sigmoidal kinetics61 so that the 50% or greater increase in oxygen utilization normally accompanying high frequency activity62 would require at least a 50% increase in extracellular lactate to allow increased lactate influx to account for the new oxygen utilization rate. Substantial lactate increases, however, lag well behind peak neuronal activity36 and activityassociated increases in oxygen utilization53,54. For example, when a major afferent pathway was stimulated for 5 s in vivo36, lactate levels started to increase 10–12 s after termination of the evoked neural activity, and peaked at 60 s postactivity. These increases in extracellular lactate are far slower than the increases in oxygen utilization. Taken together, the considerations in this and the previous section strongly suggest that increased neural activity will be tightly coupled to increased neuronal glucose utilization rather than to increased metabolism of glial lactate. Lactate must compete with glucose during prolonged stimulation
ANLSH proponents have suggested that lactate might become a major substrate for neurons during prolonged stimulation36 because lactate increases above basal levels36,43,63,64. However, because the more rapidly activated glycolysis increases cytoplasmic pyruvate59, H+ (Ref. 65) and NADH levels49, it is not clear whether the observed 0.3–1.35 mM increases in extracellular lactate levels are enough to create conditions thermodynamically favorable for driving the LDH reaction toward pyruvate production rather than lactate production. Even if the reaction is thermodynamically feasible, LDH must compete with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) for NAD+. Similar to LDH, GAPDH is present in high concentrations in brain cells17. During increased glycolysis, the activity of GAPDH is increased by elevated glyceraldehyde-3-P levels66. Furthermore, as discussed above, LDH-1 is more easily inhibited by the elevated pyruvate. Thus, the metabolism of the elevated lactate will depend largely on the prevailing glycolytic rate, with rapid rates reducing lactate use. Summary
Although lactate is clearly produced during neural activity, and neurons can clearly use lactate as an energy source, studies have so far failed to show that neurons utilize glial-produced lactate as their principal energy substrate during activity, as proposed by the ANLSH. In our opinion, the characteristics of glycolytic enzymes and LDH, the
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measured metabolite changes, and the kinetics of extracellular lactate during neural activity point toward the direct utilization of glucose by neurons during activity. Lactate might be used by both glia and neurons after neural activity when glycolytic rates slow, and might provide some substrate during prolonged activity. It might also be important during recovery from pathological insults. References 1 Magistretti, P.J. and Pellerin, L. (1999) Cellular mechanisms of brain energy metabolism and their relevance to functional brain imaging. Philos. Trans. R. Soc. Lond. B Biol. Sci. 354, 1155–1163 2 Magistretti, P.J. et al. (1999) Energy on demand. Science 283, 496–497 3 Pellerin, L. et al. (1998) Evidence supporting the existence of an activity-dependent astrocyteneuron lactate shuttle. Dev. Neurosci. 20, 291–299 4 Rothman, D.L. et al. (1999) In vivo nuclear magnetic resonance spectroscopy studies of the relationship between the glutamate-glutamine neurotransmitter cycle and functional neuroenergetics. Philos. Trans. R. Soc. Lond. B Biol. Sci. 354, 1165–1177 5 Sibson, N.R. et al. (1998) Stoichiometric coupling of brain glucose metabolism and glutamatergic neuronal activity. Proc. Natl. Acad. Sci. U. S. A. 95, 316–321 6 Magistretti, P.J. (2000) Cellular bases of functional brain imaging: insights from neuronglia metabolic coupling. Brain Res. 886, 108–112 7 Pellerin, L. and Magistretti, P.J. (1994) Glutamate uptake into astrocytes stimulates aerobic glycolysis: a mechanism coupling neuronal activity to glucose utilization. Proc. Natl. Acad. Sci. U. S. A. 91, 10625–10629 8 Dickman, K.G. and Mandel, L.J. (1989) Glycolytic and oxidative metabolism in primary renal proximal tubule cultures. Am. J. Physiol. 257, C333–C340 9 McKenna, M.C. et al. (1996) Exogenous glutamate concentration regulates the metabolic fate of glutamate in astrocytes. J. Neurochem. 66, 386–393 10 Swanson, R.A. (1992) Astrocyte glutamate uptake during chemical hypoxia in vitro. Neurosci. Lett. 147, 143–146 11 Hertz, L. et al. (1998) Can experimental conditions explain the discrepancy over glutamate stimulation of aerobic glycolysis? Dev. Neurosci. 20, 339–347 12 McNay, E.C. and Gold, P.E. (1999) Extracellular glucose concentrations in the rat hippocampus measured by zero-net-flux: effects of microdialysis flow rate, strain, and age. J. Neurochem. 72, 785–790 13 Demestre, M. et al. (1997) Stimulated release of lactate in freely moving rats is dependent on the uptake of glutamate. J. Physiol. (London) 499, 825–832 14 Bittar, P.G. et al. (1996) Selective distribution of lactate dehydrogenase isoenzymes in neurons and astrocytes of human brain. J. Cereb. Blood Flow Metab. 16, 1079–1089 15 Nitisewojo, P. and Hultin, H.O. (1976) A comparison of some kinetic properties of soluble and bound lactate dehydrogenase isoenzymes at different temperatures. Eur. J. Biochem. 67, 87–94 16 Stambaugh, R. and Post, D. (1966) Substrate and product inhibition of rabbit muscle lactic dehydrogenase heart (H4) and muscle (M4) isozymes. J. Biol. Chem. 241, 1462–1467
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Definitive experiments to differentiate between the two hypotheses are needed. A crucial study will be to determine the locus of glycolytic metabolism during neural activity in situ. The purpose of this review has been to highlight the uncertainty in the evidence used to support the ANLSH, and to show that the existing evidence and theoretical considerations provide much credibility to the conventional hypothesis.
17 McIlwain, H. and Bachelard, H.S. (1985) Biochemistry and the Central Nervous System, 5th edn, Churchill Livingstone, New York 18 Cahn, R.D. et al. (1962) Nature and development of lactic dehydrogenases. Science 136, 962–969 19 Chih, C.P. et al. (2001) Comparison of glucose and lactate as substrates during NMDA-induced activation of hippocampal slices. Brain Res. 893, 143–154 20 McKenna, M.C. et al. (1993) Regulation of energy metabolism in synaptic terminals and cultured rat brain astrocytes: differences revealed using aminooxyacetate. Dev. Neurosci. 15, 320–329 21 McKenna, M.C. et al. (1998) Lactate transport by cortical synaptosomes from adult rat brain: characterization of kinetics and inhibitor specificity. Dev. Neurosci. 20, 300–309 22 Tildon, J.T. et al. (1993) Transport of L-lactate by cultured rat brain astrocytes. Neurochem. Res. 18, 177–184 23 Dringen, R. et al. (1995) Lactate transport in cultured glial cells. Dev. Neurosci. 17, 63–69 24 Dringen, R. et al. (1993) Uptake of L-lactate by cultured rat brain neurons. Neurosci. Lett. 163, 5–7 25 Waagepetersen, H.S. et al. (2000) A possible role of alanine for ammonia transfer between astrocytes and glutamatergic neurons. J. Neurochem. 75, 471–479 26 McKenna, M.C. et al. (1994) Energy metabolism in cortical synaptic terminals from weanling and mature rat brain: evidence for multiple compartments of tricarboxylic acid cycle activity. Dev. Neurosci. 16, 291–300 27 Waagepetersen, H.S. et al. (1998) Comparison of lactate and glucose metabolism in cultured neocortical neurons and astrocytes using 13CNMR spectroscopy. Dev. Neurosci. 20, 310–320 28 Hassel, B. and Brathe, A. (2000) Cerebral metabolism of lactate in vivo: evidence for neuronal pyruvate carboxylation. J. Cereb. Blood Flow Metab. 20, 327–336 29 Waagepetersen, H.S. et al. (1998) Metabolism of lactate in cultured GABAergic neurons studied by 13C nuclear magnetic resonance spectroscopy. J. Cereb. Blood Flow Metab. 18, 109–117 30 Schousboe, A. et al. (1997) Trafficking between glia and neurons of TCA cycle intermediates and related metabolites. Glia 21, 99–105 31 Schurr, A. et al. (1988) Lactate-supported synaptic function in the rat hippocampal slice preparation. Science 240, 1326–1328 32 Izumi, Y. et al. (1997) Endogenous monocarboxylates sustain hippocampal synaptic function and morphological integrity during energy deprivation. J. Neurosci. 17, 9448–9457 33 Roberts, E.L., Jr (1993) Glycolysis and recovery of potassium ion homeostasis and synaptic transmission in hippocampal slices after anoxia or stimulated potassium release. Brain Res. 620, 251–258
34 Larrabee, M.G. (1995) Lactate metabolism and its effects on glucose metabolism in an excised neural tissue. J. Neurochem. 64, 1734–1741 35 Poitry-Yamate, C.L. et al. (1995) Lactate released by Müller glial cells is metabolized by photoreceptors from mammalian retina. J. Neurosci. 15, 5179–5191 36 Hu, Y.B. and Wilson, G.S. (1997) A temporary local energy pool coupled to neuronal activity: fluctuations of extracellular lactate levels in rat brain monitored with rapid-response enzymebased sensor. J. Neurochem. 69, 1484–1490 37 Schurr, A. et al. (1997) Brain lactate is an obligatory aerobic energy substrate for functional recovery after hypoxia: further in vitro validation. J. Neurochem. 69, 423–426 38 Schurr, A. et al. (1999) An increase in lactate output by brain tissue serves to meet the energy needs of glutamate-activated neurons. J. Neurosci. 19, 34–39 39 Cox, D.D.W.G. et al. (1985) Effects of metabolic inhibitors on evoked activity and the energy state of hippocampal slices superfused in vitro. Exp. Brain Res. 57, 464–470 40 Halestrap, A.P. and Denton, R.M. (1974) Specific inhibition of pyruvate transport in rat liver mitochondria and human erythrocytes by cyano-4hydroxycinnamate. Biochem. J. 138, 313–316 41 Pfeuffer, J. et al. (2000) Extracellular-intracellular distribution of glucose and lactate in the rat brain assessed noninvasively by diffusion-weighted 1H nuclear magnetic resonance spectroscopy in vivo. J. Cereb. Blood Flow Metab. 20, 736–746 42 Lowry, O.H. and Passonneau, J.V. (1964) The relationships between substrates and enzymes of glycolysis in brain. J. Biol. Chem. 239, 31–42 43 Fray, A.E. et al. (1996) The mechanisms controlling physiologically stimulated changes in rat brain glucose and lactate: a microdialysis study. J. Physiol. (London) 496, 49–57 44 McCall, A.L. et al. (1994) Immunohistochemical localization of the neuron-specific glucose transporter (GLUT3) to neuropil in adult rat brain. Brain Res. 659, 292–297 45 Leino, R.L. et al. (1997) Ultrastructural localization of GLUT 1 and GLUT 3 glucose transporters in rat brain. J. Neurosci. Res. 49, 617–626 46 Vannucci, S.J. et al. (1997) Glucose transporter proteins in brain: delivery of glucose to neurons and glia. Glia 21, 2–21 47 Lai, J.C. et al. (1999) Hexokinase in astrocytes: kinetic and regulatory properties. Metab. Brain Dis. 14, 125–133 48 Cimino, M. et al. (1998) Expression of hexokinase mRNA in human hippocampus. Mol. Brain Res. 53, 297–300 49 Clarke, D.D. and Sokoloff, L. (1999) In Basic Neurochemistry, 1st ed., pp. 637–669, Lippincott–Raven, New York 50 Walz, W. and Mukerji, S. (1988) Lactate release from cultured astrocytes and neurons: a comparison. Glia 1, 366–370
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51 Wang, J.W. et al. (1994) Glutamate-induced intracellular acidification of cultured hippocampal neurons. J. Physiol. (London) 354, 163–172 52 Peng, L. et al. (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 53 Malonek, D. and Grinvald, A. (1996) Interactions between electrical activity and cortical microcirculation revealed by imaging spectroscopy: implications for functional brain mapping. Science 272, 551–554 54 Malonek, D. et al. (1997) Vascular imprints of neuronal activity: relationships between the dynamics of cortical blood flow, oxygenation, and volume changes following sensory stimulation. Proc. Natl. Acad. Sci. U. S. A. 94, 14826–14831 55 Gruetter, R. et al. (1998) Localized in vivo 13C-NMR of glutamate metabolism in the human
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brain: initial results at 4 Tesla. Dev. Neurosci. 20, 380–388 Bakken, I.J. et al. (1997) Lactate formation from [U-13C]aspartate in cultured astrocytes: compartmentation of pyruvate metabolism. Neurosci. Lett. 237, 117–120 Waagepetersen, H.S. et al. (1999) Synthesis of vesicular GABA from glutamine involves TCA cycle metabolism in neocortical neurons. J. Neurosci. Res. 57, 342–349 Sonnewald, U. et al. (1997) Mitochondrial heterogeneity in the brain at the cellular level. J. Cereb. Blood Flow Metab. 18, 231–237 Goldberg, N.D. et al. (1966) Effects of changes in brain metabolism on the levels of citric acid cycle intermediates. J. Biol. Chem. 241, 3997–4003 Ferrendelli, J.A. and McDougal, D.B., Jr (1971) The effect of audiogenic seizures on regional CNS energy reserves, glycolysis and citric acid cycle flux. J. Neurochem. 18, 1207–1220
The neurobiology of learning and memory: some reminders to remember Larry Cahill, James L. McGaugh and Norman M. Weinberger
61 Broer, S. et al. (1997) Comparison of lactate transport in astroglial cells and monocarboxylate transporter 1 (MCT 1) expressing Xenopus laevis oocytes. Expression of two different monocarboxylate transporters in astroglial cells and neurons. J. Biol. Chem. 272, 30096–30102 62 Lipton, P. (1973) Effects of membrane depolarization on nicotinamide nucleotide fluorescence in brain slices. Biochem. J. 136, 999–1009 63 Prichard, J. et al. (1991) Lactate rise detected by 1H NMR in human visual cortex during physiologic stimulation. Proc. Natl. Acad. Sci. U.S. A.88, 5829–5831 64 Fellows, L.K. and Boutelle, M.G. (1993) Rapid changes in extracellular glucose levels and blood flow in the striatum of the freely moving rat. Brain Res. 604, 225–231 65 Hochachka, P.W. and Mommsen, T.P. (1983) Protons and anaerobiosis. Science 219, 1391–1397 66 Williamson, J.R. (1965) In Control of energy metabolism (Chance, B. et al., eds), pp. 333–355, Academic Press, New York
related areas. For example, ‘cognitive neuroscientists’ might know nothing about LTP (long-term potentiation) or CREB (cAMP-response element binding protein), and ‘molecular neuroscientists’ might know nothing about the HERA (Hemispheric Encoding/Retrieval Asymmetry) model or ecphory (the interaction between a stored trace and retrieval conditions that creates the recollective experience). All of us, however, face the same essential problems in relating brain to memory; the problems faced by everyone from Hull1 to Hebb2, Köhler3 to Konorski4. To help ensure that our field moves forward rather than in circles, there are some key lessons that have been learned from more than a century of investigation into brain and memory that are worth remembering. Learning and memory cannot be simply assayed
We have learned much about the neurobiology of learning and memory in the past 100 years. We have also learned much about how we should, and should not, investigate these complex processes. However, with the rapid recent growth in the field and the influx of investigators not familiar with this past, these crucial lessons too often fail to guide the research of today. Here we highlight some major lessons gleaned from this wealth of experience. These include the need to carefully attend to the learning/performance distinction, to rely equally on synthetic as well as reductionistic thinking, and to avoid the seduction of simplicity. Examples in which the lessons of history are, and are not, educating current research are also given.
Larry Cahill* James L. McGaugh Norman M. Weinberger Center for the Neurobiology of Learning and Memory and Dept of Neurobiology and Behavior, Qureshey Research Laboratory, University of California, Irvine, CA, 92697-3800, USA. *e-mail: [email protected]
‘Those who cannot remember the past are condemned to repeat it.’ George Santayana 1863–1952
Not so long ago, all major investigators studying the neurobiology of learning and memory could easily meet in one small room. Today that would be impossible, as interest in the field has grown rapidly in recent years and is now the focus of investigators from many areas of neuroscience. However, because of this, researchers from different areas often know little about the most important issues in other closely http://tins.trends.com
No one has ever measured learning or memory. They can be only inferred from behavior. Therefore, in order to know what an animal (including a human) has learned, we must ask it carefully. For example, Liddell5 (a student of Pavlov) wanted to know what a sheep learned when it was trained to lift its leg off a shock pad upon hearing a tone. Had it learned a simple stimulus–response association: hear tone, lift leg? To find out, Liddell turned the sheep upside down, putting its head on the pad. When the tone was sounded, the sheep moved its head, not its leg. Understanding what the sheep had learned (a predictive relationship between two stimuli – a tone and a shock) required asking the sheep the appropriate question. Even a single Pavlovian conditioning trial creates learning that can be detected by careful behavioral testing. For example, after only a single pairing of two stimuli (e.g. a light followed by a shock) rats can learn not only that the light predicted shock, they can also learn about the length of temporal interval between the two stimuli even with an interval of over 50 seconds6. Because it involves simultaneous
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TRENDS in Neurosciences Vol.24 No.10 October 2001
Do active cerebral neurons really use lactate rather than glucose? Ching-Ping Chih, Peter Lipton, and Eugene L. Roberts, Jr Glucose has long been considered the substrate for neuronal energy metabolism in the brain. Recently, an alternative explanation of energy metabolism in the active brain, the astrocyte–neuron lactate shuttle hypothesis, has received attention. It suggests that during neural activity energy needs in glia are met by anaerobic glycolysis, whereas neuronal metabolism is fueled by lactate released from glia. In this article, we critically examine the evidence supporting this hypothesis and explain, from the perspective of enzyme kinetics and substrate availability, why neurons probably use ambient glucose, and not glial-derived lactate, as the major substrate during activity.
Ching-Ping Chih Eugene L. Roberts, Jr Geriatric Research, Education, and Clinical Center, and Research Office, Miami VA Medical Center, Miami, FL 33125, USA. Peter Lipton Dept of Physiology, University of Wisconsin School of Medicine, Madison, WI 53706, USA. Eugene L. Roberts, Jr* Dept of Neurology, University of Miami School of Medicine, Miami, FL 33136, USA. *e-mail: roberts@ neuron.med.miami.edu
The conventional view that glucose oxidation fuels most activity-associated energy metabolism in neurons (Fig. 1) has recently been challenged by the astrocyte–neuron lactate shuttle hypothesis (ANLSH)1–6, in which glial-produced lactate fuels neurons (Fig. 2). According to the ANLSH, neural activity increases the extracellular concentration of glutamate, whose uptake by glia stimulates Na+-K+ ATPase and glutamine synthetase activity. This stimulates glial anaerobic glycolysis (in this paper ‘anaerobic glycolysis’ refers to the conversion of glucose to lactate, and ‘glycolysis’ refers to the conversion of glucose to pyruvate). Glia then release lactate, and neurons use it to fuel their activity. In the next section, we critically examine the major experimental evidence used to support the ANLSH. Evidence used to support the ANLSH Glucose metabolism and glutamate cycling
Oxidative glucose metabolism and glutamate cycling: According to the ANLSH, glutamate cycling in glia drives all cerebral glucose metabolism, and glial glucose metabolism is entirely anaerobic. The hypothesis thus predicts that the rate at which metabolized glucose enters the neuronal tricarboxylic acid (TCA) cycle should equal the rate of glial glutamate cycling. Sibson et al.5 tested this prediction and found a 1:1 stoichiometry between oxidative glucose metabolism and glutamate cycling. Although this result was considered strong support for the http://tins.trends.com
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ANLSH (Ref. 2), neither the site of glucose uptake nor the mode of glial glucose metabolism could be determined, so the data are consistent with many different models of glucose utilization. For example, if as little as 6% of glial glucose metabolism is oxidative, then glutamate cycling would have needed only 50% of the measured glucose metabolism. This means that neurons could have consumed the other 50% of glucose aerobically. Thus, for the results of Sibson et al.5 to specifically support the ANLSH, glial glucose metabolism should be entirely anaerobic. Glial anaerobic glucose metabolism and glutamate cycling: The inference that this is the case comes from a study showing that uptake of exogenous glutamate was strongly associated with increased lactate production in cultured astrocytes7. However, the applicability of this finding to the brain in situ is questionable. Cultured cells, especially static cultures, such as those used by Pellerin et al.8, generally rely far more upon anaerobic glycolysis than do cells in situ. The cultured glia might have undergone this transformation. Furthermore, in other studies of cultured astroglia, glutamate uptake was accompanied by little or no lactate production and was driven by oxidative metabolism9–11. Thus, although the data of Sibson et al.5 are consistent with the ANLSH, they are also consistent with other models, including the conventional hypothesis. Comparison of glucose and lactate as energy substrates for neurons
According to the ANLSH, neurons use glial-derived lactate during activity. Thus, a crucial prediction is that neurons use lactate in preference to glucose, as both substrates are in the extracellular space at comparable concentrations (about 1 mM; Refs 12,13). Several lines of evidence have been used to support this prediction. Lactate dehydrogenase isozyme distribution: According to ANLSH proponents, neurons preferentially use lactate because lactate dehydrogenase-1 (LDH-1), the predominant isoform of LDH in neurons, is more suited to oxidize lactate than LDH-5 as a result of its lower Km for lactate14. However, a lower Vmax (Ref. 15) for, or abundance of, LDH-1 might well offset this Km difference, depending on lactate levels. More importantly, the Ki for the pyruvate product inhibition of LDH-1 (0.18 mM) is lower than that for LDH-5 (0.28 mM)16. Resting pyruvate levels of 0.1–0.2 mM in brain cells17 mean that LDH-1 will generally be more strongly inhibited than LDH-5, diminishing the likelihood that neurons use lactate during increased activity. Indeed, if in vitro kinetic data are applicable in vivo, the higher sensitivity of LDH-1 to pyruvate substrate inhibition and lactate product inhibition might help ensure metabolism of pyruvate through the TCA cycle rather
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Fig. 1. Steps leading to increased ATP production via oxidative energy metabolism in neurons during neural activity according to the conventional hypothesis: Na+ entry and K+ release during electrical activity initiate increased oxidative energy metabolism within neurons (1). By activating neuronal Na+-K+ ATPase in the plasma membrane, leading to increased levels of ADP, Pi, and AMP, and to decreased levels of ATP [shown partially at (2)]. These changes rapidly activate glycolysis (conversion of glucose to pyruvate) (3a), and the TCA cycle and mitochondrial oxidative phosphorylation (OP) (3b). This enhances the rate of ATP synthesis (4a and 4b), with the dominant effect being at the mitochondria. Activation of glycolysis lowers cell glucose levels (5), leading to an increased flux of glucose into neurons via the neuronal glucose transporter, GLUT-3 (6), which is localized in pre- and postsynaptic elements. The thick arrows emphasize that, according to the conventional hypothesis, neurons metabolize glucose to pyruvate, which enters the TCA cycle. Generated ATP is largely used to restore Na+/K+ balance via Na+-K+ ATPase. A product of the rapid increase in glycolysis is increased NADH/NAD+, H+, and increased cytoplasmic pyruvate. These changes drive the LDH reaction toward lactate production. These factors militate against utilization of lactate emanating from glia. In astrocytes, the uptake of glutamate (Glu) and Na+ via high affinity glutamate transporters activates oxidative glucose metabolism, which provides ATP for restoring Na+/K+ and synthesizing glutamine (Gln). The basic activation process is considered similar to that in the neuron. The conventional hypothesis does not ascribe any particular fraction of glucose metabolism to aerobic or anaerobic (lactate-producing) pathways.
than into the production of lactate, assisting in the full oxidation of glucose in highly aerobic cells such as neurons and cardiac cells18,19. Thus, a differential distribution of LDH-1 and LDH-5 does not imply that neurons are net users of lactate during activity. http://tins.trends.com
Lactate utilization by neurons: (1) Utilization by neuronal cultures and isolated nerve tissue. Astrocytes and neurons take up lactate and oxidize it into CO2 (Refs 20–26) and neurotransmitters27–30. Lactate supports low frequency neural activity in the absence of glucose31–33. However, none of these studies address whether active-neural tissue preferentially uses lactate. In one study, isolated nerve tissue produced more CO2 from lactate than from glucose when both substrates were present34, providing some support for the ANLSH. However, this study was carried out on unstimulated tissue, in which glycolysis is largely inhibited (see below), and the cell type that used lactate was not identified. Glia also readily metabolize lactate20 and could have accounted for the lactate utilization. (2) Utilization by photoreceptors. It was concluded that isolated photoreceptors only used lactate released from retinal glia (Müller cells), even when glucose was present35. Although results from the retina do not necessarily apply to brain, if they do this result might be supportive of the ANLSH. However, in their calculations the authors inadvertently failed to consider that only half the cell mass was composed of glia when glial cells and photoreceptors were incubated together (discussed in Ref. 19). This leads to a large overestimation of neuronal lactate consumption, negating the conclusion that neurons only use glial-derived lactate.
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Fig. 2. Steps leading to increased ATP production via oxidative energy metabolism in neurons during neural activity according to the astrocyte–neuron lactate shuttle hypothesis (ANLSH). The initiator of increased oxidative metabolism within the neuron is the uptake of glutamate and Na+ into glial cells via the high affinity glutamate transporter (1). This activates Na+-K+ ATPase and glutamine synthase in glia, leading to increased levels of ADP, Pi, and AMP, and to decreased levels of ATP (2a and 2b). This exclusively activates anaerobic glycolysis (3), which generates lactate (4). The lactate is transported across the glial membrane, and builds up in the extracellular space where the increased concentration gradient leads to its net transport into neurons. The increased lactate in the neurons is converted to pyruvate via LDH-1 (lactate dehydrogenase-1) (5), which enters the TCA cycle, and increases ATP production in the neurons via oxidative phosphorylation (OP) (6). The thick arrows emphasize that lactate produced via glycolysis in glia fuels oxidative metabolism in neurons. Decreased glucose levels in glia are restored from glucose in the blood and extracellular space via glucose transporter-1 (GLUT-1). The ANLSH postulates that the ATP that is used to remove 3 Na+ co-transported with 1 glutamate, and the ATP that is used to synthesize glutamine from 1 glutamate, are precisely regenerated by the 2 ATP produced by the anaerobic metabolism of 1 glucose molecule. Thus, the activity-associated increase in anaerobic glycolysis is equal to the increase in ‘glutamate cycling’. As noted in the figure, the ANLSH requires that ATP utilization from glutamate cycling activates anaerobic glycolysis only. It does not activate oxidative phosphorylation in glia. In addition, as noted in the figure, ATP utilization via the Na+,K+ pump in neurons does not activate neuronal glycolysis.
(3) Utilization of lactate in vivo. A study of rat cortex concluded that repetitive stimulation caused a larger decrease in extracellular lactate than in extracellular glucose, when lactate had been previously elevated ∼twofold by activity36. This appears to provide support for the ANLSH, but there http://tins.trends.com
are problems with the interpretation. Results from only one experiment were shown, without statistical analysis, and there was clearly observable intertrial variability (see Ref. 19). Even if the results are correct, the cell type using lactate was not determined; glia could have accounted for the lactate utilization. (4) Utilization of lactate during stress. A series of studies in hippocampal slices37,38 concluded that neurons require glial lactate during recovery from stress conditions. Although important, these studies might not provide strong support for the ANLSH for two reasons: first, 4-CIN (α-cyano-4-hydroxycinnamate), which was used to block plasmalemmal lactate transport, also blocks pyruvate entry into mitochondria39,40. This seriously compromises interpretation of the results. Second, the results describe obligatory lactate use during recovery from stress conditions, not during normal physiological activity. Metabolic conditions, including ATP levels, are greatly altered during anoxic and excitotoxic stress, so extrapolation of these results to normal physiology is not valid a priori. The next section offers a rationale for accepting the conventional (neuronal glucose utilization) hypothesis (Fig. 1). Evidence for glucose as the major substrate during neural activity
The crucial question in this section is whether activation of neuronal glycolysis occurs during
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activity in vivo, and whether it dominates metabolism of glial-generated lactate. Neuronal glycolysis is activated rapidly during neuronal activity
Neurons are well equipped to metabolize glucose in situ. Although neuronal axons and dendrites do not usually directly contact capillaries, glucose is readily available to neurons. Levels of brain glucose, which is generally evenly distributed between the intracellular and extracellular compartments41, greatly exceed the Km for hexokinase (0.04 mM; Ref. 42), even during neural stimulation when values might fall by 20–30% (Refs 36,43). Glucose transporters are abundant in synaptic membranes44,45. GLUT-3, the predominant glucose transporter in neurons, transports glucose seven times faster than GLUT-1, the main glial glucose transporter46. In addition, neurons have high levels of glycolytic enzymes47,48. Thus, neurons are well positioned to use glucose as an energy substrate. The glycolytic rate is tightly regulated by energy demand49. Hexokinase and phosphofructokinase are largely inhibited at rest49, but are rapidly activated by changes in adenine nucleotide and phosphate levels during increased ATP utilization49. Glycolytic rates rapidly increase in neurons when energy demand increases50–52. Thus, there is no apparent reason why neural activity in situ should activate glial glycolysis but not neuronal glycolysis, as proposed by the ANLSH (Fig. 2). Because oxygen utilization is elevated during the first 1–3 s of neural activity53,54, rapid increases in the supply of pyruvate to neuronal mitochondria are necessary to maintain oxidative metabolism. Activation of neuronal glycolysis requires only the transport of small molecules within neurons. By contrast, for glialproduced lactate to meet this need a multistep process, including glial-glutamate cycling, and lactate production and transport, must occur. It is thus probable that neuronal glycolysis is activated more rapidly. Increases in extracellular lactate lag behind neural activity
In contrast to glycolytic enzymes, LDH is not regulated by energy demand, so increases in lactate oxidation must be driven either by increases in extracellular lactate, and hence intracellular lactate, or by decreases in cytoplasmic pyruvate. (Changes in intracellular compartmentation, if present, could affect lactate oxidation55–58, however, any such changes are difficult to predict based on current knowledge.) Pyruvate levels increase very rapidly during neural activity59,60 suggesting that increases in lactate oxidation could only occur by elevating extracellular lactate. This is supported by studies in cultured cerebellar neurons, where elevating extracellular K+ at constant substrate concentrations stimulated glucose and pyruvate utilization but not lactate utilization52. Although http://tins.trends.com
extrapolation of these results to in vivo conditions might be questionable they support the conclusion that lactate utilization cannot be increased in the absence of an increase in extracellular lactate. Thus, it appears that extracellular lactate would have to increase for neuronal lactate to increase. These increases in lactate should be substantial. The monocarboxylate transporters do not show sigmoidal kinetics61 so that the 50% or greater increase in oxygen utilization normally accompanying high frequency activity62 would require at least a 50% increase in extracellular lactate to allow increased lactate influx to account for the new oxygen utilization rate. Substantial lactate increases, however, lag well behind peak neuronal activity36 and activityassociated increases in oxygen utilization53,54. For example, when a major afferent pathway was stimulated for 5 s in vivo36, lactate levels started to increase 10–12 s after termination of the evoked neural activity, and peaked at 60 s postactivity. These increases in extracellular lactate are far slower than the increases in oxygen utilization. Taken together, the considerations in this and the previous section strongly suggest that increased neural activity will be tightly coupled to increased neuronal glucose utilization rather than to increased metabolism of glial lactate. Lactate must compete with glucose during prolonged stimulation
ANLSH proponents have suggested that lactate might become a major substrate for neurons during prolonged stimulation36 because lactate increases above basal levels36,43,63,64. However, because the more rapidly activated glycolysis increases cytoplasmic pyruvate59, H+ (Ref. 65) and NADH levels49, it is not clear whether the observed 0.3–1.35 mM increases in extracellular lactate levels are enough to create conditions thermodynamically favorable for driving the LDH reaction toward pyruvate production rather than lactate production. Even if the reaction is thermodynamically feasible, LDH must compete with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) for NAD+. Similar to LDH, GAPDH is present in high concentrations in brain cells17. During increased glycolysis, the activity of GAPDH is increased by elevated glyceraldehyde-3-P levels66. Furthermore, as discussed above, LDH-1 is more easily inhibited by the elevated pyruvate. Thus, the metabolism of the elevated lactate will depend largely on the prevailing glycolytic rate, with rapid rates reducing lactate use. Summary
Although lactate is clearly produced during neural activity, and neurons can clearly use lactate as an energy source, studies have so far failed to show that neurons utilize glial-produced lactate as their principal energy substrate during activity, as proposed by the ANLSH. In our opinion, the characteristics of glycolytic enzymes and LDH, the
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measured metabolite changes, and the kinetics of extracellular lactate during neural activity point toward the direct utilization of glucose by neurons during activity. Lactate might be used by both glia and neurons after neural activity when glycolytic rates slow, and might provide some substrate during prolonged activity. It might also be important during recovery from pathological insults. References 1 Magistretti, P.J. and Pellerin, L. (1999) Cellular mechanisms of brain energy metabolism and their relevance to functional brain imaging. Philos. Trans. R. Soc. Lond. B Biol. Sci. 354, 1155–1163 2 Magistretti, P.J. et al. (1999) Energy on demand. Science 283, 496–497 3 Pellerin, L. et al. (1998) Evidence supporting the existence of an activity-dependent astrocyteneuron lactate shuttle. Dev. Neurosci. 20, 291–299 4 Rothman, D.L. et al. (1999) In vivo nuclear magnetic resonance spectroscopy studies of the relationship between the glutamate-glutamine neurotransmitter cycle and functional neuroenergetics. Philos. Trans. R. Soc. Lond. B Biol. Sci. 354, 1165–1177 5 Sibson, N.R. et al. (1998) Stoichiometric coupling of brain glucose metabolism and glutamatergic neuronal activity. Proc. Natl. Acad. Sci. U. S. A. 95, 316–321 6 Magistretti, P.J. (2000) Cellular bases of functional brain imaging: insights from neuronglia metabolic coupling. Brain Res. 886, 108–112 7 Pellerin, L. and Magistretti, P.J. (1994) Glutamate uptake into astrocytes stimulates aerobic glycolysis: a mechanism coupling neuronal activity to glucose utilization. Proc. Natl. Acad. Sci. U. S. A. 91, 10625–10629 8 Dickman, K.G. and Mandel, L.J. (1989) Glycolytic and oxidative metabolism in primary renal proximal tubule cultures. Am. J. Physiol. 257, C333–C340 9 McKenna, M.C. et al. (1996) Exogenous glutamate concentration regulates the metabolic fate of glutamate in astrocytes. J. Neurochem. 66, 386–393 10 Swanson, R.A. (1992) Astrocyte glutamate uptake during chemical hypoxia in vitro. Neurosci. Lett. 147, 143–146 11 Hertz, L. et al. (1998) Can experimental conditions explain the discrepancy over glutamate stimulation of aerobic glycolysis? Dev. Neurosci. 20, 339–347 12 McNay, E.C. and Gold, P.E. (1999) Extracellular glucose concentrations in the rat hippocampus measured by zero-net-flux: effects of microdialysis flow rate, strain, and age. J. Neurochem. 72, 785–790 13 Demestre, M. et al. (1997) Stimulated release of lactate in freely moving rats is dependent on the uptake of glutamate. J. Physiol. (London) 499, 825–832 14 Bittar, P.G. et al. (1996) Selective distribution of lactate dehydrogenase isoenzymes in neurons and astrocytes of human brain. J. Cereb. Blood Flow Metab. 16, 1079–1089 15 Nitisewojo, P. and Hultin, H.O. (1976) A comparison of some kinetic properties of soluble and bound lactate dehydrogenase isoenzymes at different temperatures. Eur. J. Biochem. 67, 87–94 16 Stambaugh, R. and Post, D. (1966) Substrate and product inhibition of rabbit muscle lactic dehydrogenase heart (H4) and muscle (M4) isozymes. J. Biol. Chem. 241, 1462–1467
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Definitive experiments to differentiate between the two hypotheses are needed. A crucial study will be to determine the locus of glycolytic metabolism during neural activity in situ. The purpose of this review has been to highlight the uncertainty in the evidence used to support the ANLSH, and to show that the existing evidence and theoretical considerations provide much credibility to the conventional hypothesis.
17 McIlwain, H. and Bachelard, H.S. (1985) Biochemistry and the Central Nervous System, 5th edn, Churchill Livingstone, New York 18 Cahn, R.D. et al. (1962) Nature and development of lactic dehydrogenases. Science 136, 962–969 19 Chih, C.P. et al. (2001) Comparison of glucose and lactate as substrates during NMDA-induced activation of hippocampal slices. Brain Res. 893, 143–154 20 McKenna, M.C. et al. (1993) Regulation of energy metabolism in synaptic terminals and cultured rat brain astrocytes: differences revealed using aminooxyacetate. Dev. Neurosci. 15, 320–329 21 McKenna, M.C. et al. (1998) Lactate transport by cortical synaptosomes from adult rat brain: characterization of kinetics and inhibitor specificity. Dev. Neurosci. 20, 300–309 22 Tildon, J.T. et al. (1993) Transport of L-lactate by cultured rat brain astrocytes. Neurochem. Res. 18, 177–184 23 Dringen, R. et al. (1995) Lactate transport in cultured glial cells. Dev. Neurosci. 17, 63–69 24 Dringen, R. et al. (1993) Uptake of L-lactate by cultured rat brain neurons. Neurosci. Lett. 163, 5–7 25 Waagepetersen, H.S. et al. (2000) A possible role of alanine for ammonia transfer between astrocytes and glutamatergic neurons. J. Neurochem. 75, 471–479 26 McKenna, M.C. et al. (1994) Energy metabolism in cortical synaptic terminals from weanling and mature rat brain: evidence for multiple compartments of tricarboxylic acid cycle activity. Dev. Neurosci. 16, 291–300 27 Waagepetersen, H.S. et al. (1998) Comparison of lactate and glucose metabolism in cultured neocortical neurons and astrocytes using 13CNMR spectroscopy. Dev. Neurosci. 20, 310–320 28 Hassel, B. and Brathe, A. (2000) Cerebral metabolism of lactate in vivo: evidence for neuronal pyruvate carboxylation. J. Cereb. Blood Flow Metab. 20, 327–336 29 Waagepetersen, H.S. et al. (1998) Metabolism of lactate in cultured GABAergic neurons studied by 13C nuclear magnetic resonance spectroscopy. J. Cereb. Blood Flow Metab. 18, 109–117 30 Schousboe, A. et al. (1997) Trafficking between glia and neurons of TCA cycle intermediates and related metabolites. Glia 21, 99–105 31 Schurr, A. et al. (1988) Lactate-supported synaptic function in the rat hippocampal slice preparation. Science 240, 1326–1328 32 Izumi, Y. et al. (1997) Endogenous monocarboxylates sustain hippocampal synaptic function and morphological integrity during energy deprivation. J. Neurosci. 17, 9448–9457 33 Roberts, E.L., Jr (1993) Glycolysis and recovery of potassium ion homeostasis and synaptic transmission in hippocampal slices after anoxia or stimulated potassium release. Brain Res. 620, 251–258
34 Larrabee, M.G. (1995) Lactate metabolism and its effects on glucose metabolism in an excised neural tissue. J. Neurochem. 64, 1734–1741 35 Poitry-Yamate, C.L. et al. (1995) Lactate released by Müller glial cells is metabolized by photoreceptors from mammalian retina. J. Neurosci. 15, 5179–5191 36 Hu, Y.B. and Wilson, G.S. (1997) A temporary local energy pool coupled to neuronal activity: fluctuations of extracellular lactate levels in rat brain monitored with rapid-response enzymebased sensor. J. Neurochem. 69, 1484–1490 37 Schurr, A. et al. (1997) Brain lactate is an obligatory aerobic energy substrate for functional recovery after hypoxia: further in vitro validation. J. Neurochem. 69, 423–426 38 Schurr, A. et al. (1999) An increase in lactate output by brain tissue serves to meet the energy needs of glutamate-activated neurons. J. Neurosci. 19, 34–39 39 Cox, D.D.W.G. et al. (1985) Effects of metabolic inhibitors on evoked activity and the energy state of hippocampal slices superfused in vitro. Exp. Brain Res. 57, 464–470 40 Halestrap, A.P. and Denton, R.M. (1974) Specific inhibition of pyruvate transport in rat liver mitochondria and human erythrocytes by cyano-4hydroxycinnamate. Biochem. J. 138, 313–316 41 Pfeuffer, J. et al. (2000) Extracellular-intracellular distribution of glucose and lactate in the rat brain assessed noninvasively by diffusion-weighted 1H nuclear magnetic resonance spectroscopy in vivo. J. Cereb. Blood Flow Metab. 20, 736–746 42 Lowry, O.H. and Passonneau, J.V. (1964) The relationships between substrates and enzymes of glycolysis in brain. J. Biol. Chem. 239, 31–42 43 Fray, A.E. et al. (1996) The mechanisms controlling physiologically stimulated changes in rat brain glucose and lactate: a microdialysis study. J. Physiol. (London) 496, 49–57 44 McCall, A.L. et al. (1994) Immunohistochemical localization of the neuron-specific glucose transporter (GLUT3) to neuropil in adult rat brain. Brain Res. 659, 292–297 45 Leino, R.L. et al. (1997) Ultrastructural localization of GLUT 1 and GLUT 3 glucose transporters in rat brain. J. Neurosci. Res. 49, 617–626 46 Vannucci, S.J. et al. (1997) Glucose transporter proteins in brain: delivery of glucose to neurons and glia. Glia 21, 2–21 47 Lai, J.C. et al. (1999) Hexokinase in astrocytes: kinetic and regulatory properties. Metab. Brain Dis. 14, 125–133 48 Cimino, M. et al. (1998) Expression of hexokinase mRNA in human hippocampus. Mol. Brain Res. 53, 297–300 49 Clarke, D.D. and Sokoloff, L. (1999) In Basic Neurochemistry, 1st ed., pp. 637–669, Lippincott–Raven, New York 50 Walz, W. and Mukerji, S. (1988) Lactate release from cultured astrocytes and neurons: a comparison. Glia 1, 366–370
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51 Wang, J.W. et al. (1994) Glutamate-induced intracellular acidification of cultured hippocampal neurons. J. Physiol. (London) 354, 163–172 52 Peng, L. et al. (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 53 Malonek, D. and Grinvald, A. (1996) Interactions between electrical activity and cortical microcirculation revealed by imaging spectroscopy: implications for functional brain mapping. Science 272, 551–554 54 Malonek, D. et al. (1997) Vascular imprints of neuronal activity: relationships between the dynamics of cortical blood flow, oxygenation, and volume changes following sensory stimulation. Proc. Natl. Acad. Sci. U. S. A. 94, 14826–14831 55 Gruetter, R. et al. (1998) Localized in vivo 13C-NMR of glutamate metabolism in the human
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brain: initial results at 4 Tesla. Dev. Neurosci. 20, 380–388 Bakken, I.J. et al. (1997) Lactate formation from [U-13C]aspartate in cultured astrocytes: compartmentation of pyruvate metabolism. Neurosci. Lett. 237, 117–120 Waagepetersen, H.S. et al. (1999) Synthesis of vesicular GABA from glutamine involves TCA cycle metabolism in neocortical neurons. J. Neurosci. Res. 57, 342–349 Sonnewald, U. et al. (1997) Mitochondrial heterogeneity in the brain at the cellular level. J. Cereb. Blood Flow Metab. 18, 231–237 Goldberg, N.D. et al. (1966) Effects of changes in brain metabolism on the levels of citric acid cycle intermediates. J. Biol. Chem. 241, 3997–4003 Ferrendelli, J.A. and McDougal, D.B., Jr (1971) The effect of audiogenic seizures on regional CNS energy reserves, glycolysis and citric acid cycle flux. J. Neurochem. 18, 1207–1220
The neurobiology of learning and memory: some reminders to remember Larry Cahill, James L. McGaugh and Norman M. Weinberger
61 Broer, S. et al. (1997) Comparison of lactate transport in astroglial cells and monocarboxylate transporter 1 (MCT 1) expressing Xenopus laevis oocytes. Expression of two different monocarboxylate transporters in astroglial cells and neurons. J. Biol. Chem. 272, 30096–30102 62 Lipton, P. (1973) Effects of membrane depolarization on nicotinamide nucleotide fluorescence in brain slices. Biochem. J. 136, 999–1009 63 Prichard, J. et al. (1991) Lactate rise detected by 1H NMR in human visual cortex during physiologic stimulation. Proc. Natl. Acad. Sci. U.S. A.88, 5829–5831 64 Fellows, L.K. and Boutelle, M.G. (1993) Rapid changes in extracellular glucose levels and blood flow in the striatum of the freely moving rat. Brain Res. 604, 225–231 65 Hochachka, P.W. and Mommsen, T.P. (1983) Protons and anaerobiosis. Science 219, 1391–1397 66 Williamson, J.R. (1965) In Control of energy metabolism (Chance, B. et al., eds), pp. 333–355, Academic Press, New York
related areas. For example, ‘cognitive neuroscientists’ might know nothing about LTP (long-term potentiation) or CREB (cAMP-response element binding protein), and ‘molecular neuroscientists’ might know nothing about the HERA (Hemispheric Encoding/Retrieval Asymmetry) model or ecphory (the interaction between a stored trace and retrieval conditions that creates the recollective experience). All of us, however, face the same essential problems in relating brain to memory; the problems faced by everyone from Hull1 to Hebb2, Köhler3 to Konorski4. To help ensure that our field moves forward rather than in circles, there are some key lessons that have been learned from more than a century of investigation into brain and memory that are worth remembering. Learning and memory cannot be simply assayed
We have learned much about the neurobiology of learning and memory in the past 100 years. We have also learned much about how we should, and should not, investigate these complex processes. However, with the rapid recent growth in the field and the influx of investigators not familiar with this past, these crucial lessons too often fail to guide the research of today. Here we highlight some major lessons gleaned from this wealth of experience. These include the need to carefully attend to the learning/performance distinction, to rely equally on synthetic as well as reductionistic thinking, and to avoid the seduction of simplicity. Examples in which the lessons of history are, and are not, educating current research are also given.
Larry Cahill* James L. McGaugh Norman M. Weinberger Center for the Neurobiology of Learning and Memory and Dept of Neurobiology and Behavior, Qureshey Research Laboratory, University of California, Irvine, CA, 92697-3800, USA. *e-mail: [email protected]
‘Those who cannot remember the past are condemned to repeat it.’ George Santayana 1863–1952
Not so long ago, all major investigators studying the neurobiology of learning and memory could easily meet in one small room. Today that would be impossible, as interest in the field has grown rapidly in recent years and is now the focus of investigators from many areas of neuroscience. However, because of this, researchers from different areas often know little about the most important issues in other closely http://tins.trends.com
No one has ever measured learning or memory. They can be only inferred from behavior. Therefore, in order to know what an animal (including a human) has learned, we must ask it carefully. For example, Liddell5 (a student of Pavlov) wanted to know what a sheep learned when it was trained to lift its leg off a shock pad upon hearing a tone. Had it learned a simple stimulus–response association: hear tone, lift leg? To find out, Liddell turned the sheep upside down, putting its head on the pad. When the tone was sounded, the sheep moved its head, not its leg. Understanding what the sheep had learned (a predictive relationship between two stimuli – a tone and a shock) required asking the sheep the appropriate question. Even a single Pavlovian conditioning trial creates learning that can be detected by careful behavioral testing. For example, after only a single pairing of two stimuli (e.g. a light followed by a shock) rats can learn not only that the light predicted shock, they can also learn about the length of temporal interval between the two stimuli even with an interval of over 50 seconds6. Because it involves simultaneous
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