Neighborly interactions of metabolically-activated astrocytes in vivo

Neurochemistry International 43 (2003) 339–354

Neighborly interactions of metabolically-activated astrocytes in vivo Gerald A. Dienel∗ , Nancy F. Cruz Department of Neurology, Slot 500, University of Arkansas for Medical Sciences, 4301 W. Markham St., Shorey Bldg., Room 7S15, Little Rock, AR 72205, USA Received 18 October 2002; received in revised form 18 December 2002; accepted 19 December 2002

Abstract Metabolic responses of brain cells to a stimulus are governed, in part, by their enzymatic specialization and interrelationships with neighboring cells, and local shifts in functional metabolism during brain activation are likely to be influenced by the neurotransmitter system, subcellular compartmentation, and anatomical structure. Selected examples of functional activation illustrate the complexity of metabolic interactions in working brain and of interpretation of changes in brain lactate levels. The major focus of this article is the disproportionately higher metabolism of glucose compared to oxygen in normoxic brain, a phenomenon that occurs during activation in humans and animals. The glucose utilized in excess of oxygen is not fully explained by accumulation of glucose, lactate, or glycogen in brain or by lactate efflux from brain to blood. Thus, any lactate derived from the excess glucose could not have been stoichiometrically exported to and metabolized by neighboring neurons because oxygen consumption would have otherwise increased and matched that of glucose. Metabolic labeling of tricarboxylic acid cycle-derived amino acids increased during brief sensory stimulation, reflecting a rise in oxidative metabolism. Brain glycogen is mainly in astrocytes, and its level falls throughout the stimulus and early post-activation interval. Glycogenolysis cannot be accounted for by lactate accumulation or oxidation; there must be rapid product clearance. Glycogen restoration is slow and diversion of glucose from oxidative pathways for its re-synthesis could reduce the global O2 /glucose uptake ratio; astrocytes could downshift this ratio for up to an hour after 5 min stimulus. Morphological studies of astrocytes reveal a paucity of cytoplasm and organelles in the fine processes that surround synapses and form gap junction connections with neighboring astrocytes. Specialized regions of astrocytes, e.g. their endfeet and thin peripheral lamellae, are likely to have compartmentalized metabolic activities. Anatomical constraints imposed upon the fine processes might require preferential utilization of glycolysis to satisfy their energy demands, but rapid lactate clearance would then be essential, since its accumulation would inhibit glycolysis. Gap junctional connections between neighboring astrocytes provide a mechanism for rapid metabolite spreading via the astrocytic syncytium and elimination of by-products. Local structure–function relationships need to be incorporated into experimental models of neuron–astrocyte and astrocyte–astrocyte interactions in working brain. © 2003 Elsevier Science Ltd. All rights reserved. Keywords: Astrocytes; Sensory stimulation; Glycolysis; Glycogen; Lactate

1. Introduction Activation of metabolism in the specific anatomical pathways involved in carrying out a defined task is a hallmark of brain function, but the dynamic interactions between neurons and astrocytes in the complex environment of the intact brain during ‘resting’ conditions (i.e. when no specific stimulus is provided to a conscious normal subject) and during activation (induced by one or more stimuli to increase information processing by brain cells) are not well understood. The specialized capabilities of brain cells have has been recognized since Lowry’s pioneering micro-analytical studies that measured enzyme activities and levels of intermediary metabolites in single, dissected cells (Lowry et al., 1956; Lowry and ∗

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Passonneau, 1972; Kato and Lowry, 1973; Lowry, 1990), and metabolic heterogeneity of brain mitochondria, cells, and structures has been extensively characterized (Balázs and Cremer, 1972; Berl et al., 1975; Clark and Lai, 1989; Lai and Clark, 1989; Sokoloff, 1986, 1996; Borowsky and Collins, 1989a,b; Hamprecht and Dringen, 1995; Wiesinger, 1995; Robinson et al., 1998; Hertz et al., 1999, 2000; Hertz and Dienel, 2002). Local rates of synthesis of ATP, neurotransmitters, and other essential compounds must match the demands arising from changes in neuronal signaling activity, and it is well established that the energy needs of the brain are satisfied by oxidative metabolism of glucose, its obligatory fuel. However, recent studies have brought attention to the possibility that glycolytic metabolism of glucose to lactate might support brain work and that glucose-derived metabolites synthesized within brain might serve as alternative fuel during activation. A number of critical issues,

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including the possibility of preferential generation and use of lactate by astrocytes or neurons, oxidative metabolism of glutamate by astrocytes, and metabolite fluxes through various transporters, are still unresolved, and current concepts of neuron–astrocyte metabolic interactions are the subject of considerable debate (Magistretti and Pellerin, 1999; Magistretti et al., 1999; Attwell and Laughlin, 2001; Chih et al., 2001; Dienel and Hertz, 2001; Gjedde and Marrett, 2001; Gjedde et al., 2002). Recent studies have reported a shift in the stoichiometric relationship between the cerebral metabolic rates of oxygen (CMRO2 ) and glucose (CMRglc ) in normal, normoxic working brain in vivo, with a disproportionate rise in CMRglc compared to CMRO2 during activation (Fox and Raichle, 1986; Fox et al., 1988; Madsen et al., 1995a, 1999; Fujita et al., 1999). These data suggest that specific cellular functions or subcellular structures might preferentially use glycolysis to fulfill their energy needs during activation. However, if lactate is produced in the activated tissue, it cannot have been oxidized in the area of interest because otherwise CMRO2 would have increased in parallel with CMRglc . To help integrate this new perspective into the discussion of neuron–astrocyte interactions during functional activation, this article reviews selected studies of functional activation in conscious subjects (humans and rats) in which the ratio CMRO2 /CMRglc is either maintained or falls during stimulation. A few examples are chosen to illustrate the complexity of issues related to changes in CMRO2 /CMRglc , interpretation of lactate production, utilization, and release from activated tissue, and understanding of shifts in glucose utilization in working brain. The analysis involves consideration of anatomical characteristics and enzymatic specialization of astrocytes, the putative energy demands of the fine astrocytic processes, and glycogen turnover. We suggest a ‘working hypothesis’ in which glycogen turnover and metabolite spreading within the astrocyte syncytium and to the vasculature can contribute to shifts in CMRO2 /CMRglc .

ditions. For example, certain types of visual stimuli do increase CMRO2 in activated tissue (Marrett et al., 1995; Hoge et al., 1999; Vafaee et al., 1999), and the rise in CMRO2 is frequency-dependent (Vafaee et al., 1999). In contrast, other activation paradigms caused disproportionately greater increases in the CMRglc and CBF compared to changes, if any, in CMRO2 in normal, normoxic subjects (Fox and Raichle, 1986; Fox et al., 1988; Madsen et al., 1995a; Fujita et al., 1999), suggesting that (1) the glucose consumed in excess of oxygen might be converted to lactate or used in non-oxidative biosynthetic reactions, and (2) the energy required for brain activation might be less than that calculated by assuming that CMRglc reflects oxidative metabolism (Fox and Raichle, 1986; Fox et al., 1988; Madsen et al., 1995a). Results from two studies in conscious humans that used different stimuli and methodologies to measure CBF and metabolism are compared in Table 1. The CMRO2 /CMRglc ratio obtained in primary visual cortex during rest was much lower and the percent decrease in the CMRO2 /CMRglc ratio during activation was greater (Table 1A) compared to the corresponding values obtained with global activation (Table 1B). Three findings are of particular interest: (1) the low ratio is detectable in a local region and also in the brain as a whole, (2) increased efflux of lactate to blood during stimulation equivalent to 5–7% of glucose influx suggests greater lactate production during activation, and (3) the low CMRO2 /CMRglc ratio was long-lasting and persisted for at least 40 min after only 10 min of increased sensory-cognitive activity (Madsen

(A) Activation of primary visual cortexa Blood flow (ml/(100 g min)) 54 171 CMRO2 (␮mol/(100 g min)) CMRglc (␮mol/(100 g min)) 42 CMRO2 /CMRglc 4.1

81b 179 63b 2.8

1.5 1.05 1.51 0.68

2. Preferential rise in glucose compared to oxygen consumption in normoxic working brain

(B) Global cerebral activationc Blood flow (ml/(100 g min)) CMRO2 (␮mol/(100 g min)) CMRglc (␮mol/(100 g min)) CMRlac (␮mol/(100 g min)) CMRO2 /CMRglc

58.0b 140.5 26.1d −4.08d 5.4d

1.15 0.99 1.12 1.47 0.89

2.1. Stimulus dependence of the CMRO2 /CMRglc ratio The relationships between cerebral blood flow (CBF) and oxygen and glucose utilization vary with physiological state. Under resting steady-state conditions nearly all of the glucose utilized by normal brain is oxidized, and the ratio of oxygen to glucose utilization is somewhat less than the theoretical maximum of 6 due to continuous biosynthetic reactions and low rates of efflux of some compounds from brain (Siesjö, 1978). Increased energy demands arising from sensory stimulation and cognitive and motor activity stimulate blood flow and metabolism, and the stoichiometric relationship between CMRO2 and CMRglc can be maintained or altered, depending on the experimental con-

Table 1 Effects of activation on glucose and oxygen utilization in human brain Rest

50.6 142.5 23.3 −2.78 6.1

Stimulation

Stimulation/ rest

a Local rates of blood flow and metabolism were measured with positron emission tomography during the resting and stimulated state in each subject; stimulation of primary visual cortex was achieved by viewing an annular reversing checkerboard (data from Fox et al. (1988)). b Mean values are shown for each condition (P < 0.001 vs. rest). c Global rates of cerebral blood flow and metabolism were determined in each subject by measurement of A-V differences across the brain for 133 Xe (desaturation assay) and three metabolites before and during a Wisconsin card sorting text combined with moderate psychologic harassment by pacing the test; rates were calculated as flow times arteriovenous difference and a negative value indicates efflux from the brain (data from Madsen et al. (1995a)). Abbreviations: CMR, cerebral metabolic rate; lac, lactate; glc, glucose. d Mean values are shown for each condition (P < 0.05).

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et al., 1995a). The apparent stimulus dependence of changes in the CMRO2 /CMRglc ratio during activation underscores the importance of evaluating the influence of the specific stimulus (type, intensity, and duration) and the anatomical pathway(s) involved, to try to account for metabolic demand as well as the metabolic capacities of activated cells (Gjedde et al., 2002). When the CMRO2 /CMRglc ratio falls during activation, any lactate that might have been produced and contributed to the differential increase in CMRglc over and above that corresponding to CMRO2 could not have been oxidized within the region of interest (ranging from visual cortex to whole brain, Table 1), otherwise CMRO2 would have increased in parallel to match the rise in CMRglc . Thus, products of glucose metabolism equivalent to the glucose consumed in excess of oxygen must either accumulate or be quickly removed from the activated tissue, and the model in which astrocytes are portrayed as lactate producers that supply stoichiometric amounts of lactate as a fuel for oxidation by nearby neurons during excitatory neurotransmission (Magistretti and Pellerin, 1999; Magistretti et al., 1999) cannot broadly apply to activation. If lactate is a major product of the excess glucose metabolized in activated brain and if lactate is quickly cleared from the activated tissue, mechanisms for rapid transport of lactate need to be incorporated into current models of brain activation. Interpretation of changes in brain lactate concentration and lactate efflux from brain is, however, complicated, as illustrated in the following examples. 2.2. CMRO2 /CMRglc ratio and lactate production, accumulation, and clearance An increase in tissue lactate concentration is frequently interpreted in terms of higher glycolytic activity, but it is important to keep in mind that a change in lactate level can be the net result of many processes, including the glycolytic rate (conversion of glucose to pyruvate) relative to the rates of the malate–aspartate shuttle activity (to transfer reducing equivalents into mitochondria), pyruvate dehydrogenase complex, and tricarboxylic acid cycle; the cytoplasmic pH and oxidation–reduction state; the rate of glycogenolysis in astrocytes; and the rate of lactate efflux from tissue. The lactate/pyruvate ratio is about 13:1 in normal brain and the brain pyruvate level (0.050–0.1 mM; Siesjö, 1978) is approximately equal to the Km of pyruvate dehydrogenase (PDH) for pyruvate (Ksiezak-Reding et al., 1982). Thus, increased flux into the oxidative pathway could be expected to be associated with a rise in pyruvate concentration (and activation of the PDH complex), and pyruvate levels do increase during metabolic activation (Lowry et al., 1964; Duffy et al., 1972; Hawkins et al., 1973). If the cytoplasmic redox state and pH are kept constant, doubling of pyruvate content should increase lactate level two-fold, which is within the range frequently observed during brain activation. On the other hand, a sharp rise in glycolytic rate (exceeding that of oxidative metabolism) or activation of

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metabolic demand in regions of cells that are devoid of mitochondria would be expected to enhance lactate production and release. Lactate transport occurs by facilitated diffusion of lactate plus a proton (Halestrap and Price, 1999), and Juel (2001) noted “it has never been reported that a monocarboxylic acid transporter (MCT) isoform is capable of selectively moving lactate in one direction; in fact, one study has demonstrated that the transporters behave symmetrically. . . ”. The direction of lactate/H+ transport is downhill along their concentration gradients (Juel, 2001). Intracellular lactate accumulation would eventually inhibit its synthesis and since trans-membrane transport of lactate is much faster than metabolism-driven uptake into cultured neurons and astrocytes (Dienel and Hertz, 2001), rapid efflux of lactate from activated cells and clearance of lactate from the activated tissue is likely to be essential to maintain a high glycolytic rate. 2.2.1. Brain lactate levels do not necessarily predict either the metabolic status of tissue or the magnitude of lactate fluxes across the blood–brain barrier Three examples illustrate the fact that metabolic activation can have very different effects on brain lactate accumulation and lactate efflux from brain to blood; interpretation of changes in lactate levels is fraught with difficulty unless much more information is available. (1) An intraperitoneal injection of ammonia causes brain tissue lactate levels to double and stimulates release of lactate from brain to blood in quantities equal to about 15% of glucose influx into brain (Table 2A). In the ammonia-injected rats, oxygen and glucose utilization increased in parallel by about 35%; the (A-V)O2 /(A-V)glc ratios were 7.4 and 7.3 in control and ammonia-injected rats, respectively (Hawkins et al., 1973). The rats were starved for 48 h prior to the experiments, and Hawkins et al. (1973) suggested that ketone bodies might account for the additional fuel for oxidation. Close examination of these data reveals that the mean brain lactate concentration in the ammonia-treated rats was actually lower than mean lactate levels in arterial or cerebral venous blood; nevertheless, there was still a substantial outflow of lactate from brain to blood against an apparent concentration gradient (Table 2A), suggesting that there must have been a cellular compartment with high brain to blood lactate and/or pH gradients. Because ammonia diffusing from blood into brain first enters the astrocytic endfeet where it is converted to glutamine by an ATP-requiring enzyme (Cooper et al., 1979) astrocytic endfeet might have an especially high metabolic demand, and they might have higher lactate levels compared to whole tissue average. Brain glycogen levels fall after the ammonia load (Hawkins et al., 1973), and glycogen and glycogen phosphorylase immunoreactivity are localized in pericapillary astrocytes and their endfeet (Phelps, 1972, 1975; Richter et al.,

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Table 2 Blood and brain metabolite levels and arteriovenous differences across brain of conscious adult rats during metabolic activation Experimental condition

Concentration Glucose

Lactate

Lactate fluxa (% glucose influx)

challengeb

(A) Acute ammonia Before ammonia injection Arterial (A) blood (mM) Cerebral venous (V) blood (mM) A-V difference (mM) Brain (␮mol/g)

4–5 min after ammonia injection Arterial blood (mM) Cerebral venous blood (mM) A-V difference (mM) Brain (␮mol/g)

5.10 4.53

2.64 2.68

0.57 0.75

−0.04 1.25

5.74 4.97 0.77c 1.05d

3.20 3.43 −0.23d 2.39d

−4

−15c

(B) Spreading cortical depressione,f Arterial bloode (mM) 7.32 0.62 A-V differencee (mM) Brain (␮mol/g)f 1.2

0.87 −0.27c 8.5d

−22c

(C) Generalized sensory stimulationg Before stimulation Arterial blood (mM) 6.81 A-V difference (mM) 0.68 Brain (␮mol/g) 2.8

0.50 −0.08 1.0

−6

After 5 min of stimulation Arterial blood (mM) A-V difference (mM) Brain (␮mol/g)

7.81c 0.60c 3.1

1.96c 0.02c 1.9c

+2c

a Calculation of glucose equivalents of lactate flux is based on 2 mol of lactate per mole glucose. A negative arteriovenous (A-V) difference across rat brain indicates efflux from brain. Mean values are tabulated for each condition. b Data from Hawkins et al. (1973). c P < 0.05. d P < 0.01 versus control. e Data from Cruz et al. (1999). Efflux of 14 C-labeled lactate from brain during spreading depression was detectable within 2 min after an intravenous pulse of [6-14 C]glucose, and between 2 and 8 min after the pulse of [14 C]glucose the efflux of [14 C]lactate from brain was equivalent to that of unlabeled lactate indicating rapid equilibration with glycolytic intermediates and efflux of lactate derived from blood-borne glucose (Cruz et al., 1999). f Data from Adachi et al. (1995). g Data from Madsen et al. (1999).

1996). Glycogen can be converted to lactate (Dringen et al., 1993; Hamprecht and Dringen, 1994, 1995) suggesting that glycogenolysis in endfeet might contribute to the lactate released to blood. Thus, substantial lactate release to blood against an apparent concentration gradient occurred when there was glycogenolysis and a higher-than-normal (A-V)O2 /(A-V)glc ratio. Thus, lactate efflux does not predict the relationship between glucose and oxygen metabolism, and a small astrocytic compartment, the endfeet, is likely to be a major site of metabolic activation. (2) During spreading cortical depression the cerebral cortex is heterogeneously activated and the average brain lac-

tate level rose eight-fold; brain lactate level was much higher than that of arterial blood and the efflux of lactate to blood was equivalent to about 22% of the glucose uptake into brain (Table 2B). When compared to data in Table 2A these results suggest a barrier that limits lactate efflux to blood. Autoradiographs of brains labeled with [6-14 C]glucose failed to register the 50% increase in CMRglc detected with [14 C]deoxyglucose (Adachi et al., 1995; Cruz et al., 1999) in spite of the large increase in labeled and unlabeled lactate recovered in brain during spreading depression, indicating substantial release of products of glucose metabolism from activated cerebral cortex. Lactate release to blood accounted for only about half of this underestimation, indicating lactate spreading within brain. (3) During normal physiological sensory stimulation of the rat, brain lactate level doubled in association with a slight uptake of lactate into brain from blood (Table 2C). Both CMRO2 and CMRglc increased but the ratio CMRO2 /CMRglc fell during activation; metabolic changes in these rats are described in more detail in Section 3. Taken together the data in Table 2 illustrate the substantial capacity of adult rat brain to very quickly export lactate to blood even though the blood–brain barrier restricts its passage. Relatively small changes in brain lactate level (e.g. a doubling) do not predict either the magnitude or direction of changes in the CMRO2 /CMRglc ratio or of lactate flux across the blood brain barrier. Subcellular heterogeneity of astrocytic metabolic response to functional challenge might have a high impact on local lactate levels and fluxes. If lactate accumulates in brain tissue due to transport barriers it probably also spreads quickly within the brain.

3. Metabolic changes arising from brain activation 3.1. Requirements for metabolic balance studies Most previous experiments to assess quantitative relationships between CBF, CMRO2 , and CMRglc during brain activation have been carried out in human subjects using local or global methods, and animal studies are required to analyze metabolic changes in brain tissue. Unfortunately, local CMRO2 cannot be readily determined in small experimental animals, and arteriovenous (A-V) differences across the brain for major substrates (glucose, oxygen, lactate) must, therefore, be measured in conjunction with parallel analysis of brain metabolism and metabolite levels. Investigation of metabolic changes during brain activation requires determination of local metabolic balances and includes quantitative analysis of oxygen and glucose metabolism, blood flow, tissue metabolite levels, fluxes of carbon from glucose and other metabolites into various pathways within brain, and efflux of metabolites from brain to blood. Evaluation of net

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conversion of glucose to lactate also requires an estimate of lactate efflux from the activated tissue to blood or other brain regions. Additionally, as many pathways as possible must be quantitatively taken into account because many small but additive changes in metabolic fluxes in different pathways contribute to CMRglc relative to CMRO2 . Ideally, studies are carried out in conscious experimental subjects to avoid the complications introduced by use of anesthetics. Anesthesia reduces consciousness, and it might alter the regulatory steps and the type and magnitude of energy-requiring and energy-producing processes associated with information processing and physiological activation. In fact, anesthesia does cause disproportionate changes in blood flow and metabolism in different stations of the same sensory pathway compared to the unanesthetized state (Nakao et al., 2001). In practice, the above goals are quite difficult to achieve due to the extensive analysis required. Furthermore, most metabolic studies require product trapping for the duration of the experimental period and some metabolites, such as lactate and CO2 , are diffusible and not readily retained in activated tissue; trapping of products of glucose metabolism in the amino acid pools is, therefore, not necessarily equivalent to total glucose utilization. Many experimental studies have restricted objectives and assay levels of only a few metabolites such as glucose and lactate; the conclusions that can be drawn from them are limited. 3.2. Metabolic changes in brain during generalized sensory stimulation 3.2.1. Arteriovenous differences across rat brain As a first step in an investigation of metabolic changes during brain activation we developed and studied an in vivo animal model in which the oxygen/glucose uptake ratio fell during generalized sensory stimulation of the conscious rat (Madsen et al., 1995b, 1999; Cruz and Dienel, 2002; Dienel et al., 2002). In brief, the non-fasted male rats were very carefully handled and sequestered in a shelter for the 3 h recovery interval after preparative surgery to implant blood sampling catheters. Sensory stimulation consisted of removal of the shelter and 5 min of gentle bilateral brushing of the body (head, whiskers, face, back, paws, and tail) with a soft paint brush to produce changes in neural activity and metabolism throughout cerebral cortex, after which the rats were placed back into the shelter for 15 min recovery. Arteriovenous differences for oxygen, glucose, and lactate were assayed across the brain, and brains were then frozen in situ. The tissue samples corresponded as closely as possible to the structures drained by the venous system that was sampled when A-V differences across brain were determined, and metabolic responses to sensory stimulation were analyzed in well-mixed powders to provide average values weighted in proportion to the size, flow, and metabolism of component brain structures. Metabolic rates are calculated as the product of blood flow and A-V difference, and because oxygen and glucose

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levels are measured in timed pairs of arterial and venous samples changes in the ratio (A-V)O2 /(A-V)glc are equivalent to those in CMRO2 /CMRglc . During stimulation when cerebral blood flow increased, the arteriovenous difference for oxygen was reduced compared to rest and recovery, whereas that for glucose (glc) remained approximately constant (Fig. 1A). Thus, extraction of glucose into brain must have changed in proportion to the rise in blood flow, whereas oxygen uptake did not, causing the (A-V)O2 /(A-V)glc ratio to fall (Fig. 1B). The mean steady-state oxygen/glucose uptake ratio in resting, sequestered rats averaged 6.1, but after abrupt exposure to an open environment and gentle brushing the ratio fell to 5.0, then rose to 7.7 after termination of stimulation (Fig. 1B). During activation, there was also a slight uptake of lactate into brain from blood (Table 2C). The higher-than-expected uptake ratio during recovery suggest oxidation of endogenous substrates in brain, possibly lactate and glycogen which were metabolized during this period (see below and Fig. 2A). A fall in (A-V)O2 /(A-V)glc ratio can also be produced by simply removing the shelter without physical stimulation, and pretreatment with propranolol blocks this change (Schmalbruch et al., 2002). The oxygen/glucose brain uptake ratio occasionally exceeded 6.0 in the first arteriovenous sample drawn from sequestered resting rats, raising the possibility that rapid oxidation of endogenous substrates might have accounted for an increase in oxidative metabolism during the initial moments of blood sampling, perhaps due to a startle or stress response. Data from our original study (Madsen et al., 1999) were, therefore, re-examined in more detail to determine if blood sampling might have triggered consumption of brain glucose, lactate, or glycogen, or if brain lactate levels rose due to glycogenolysis (Dringen et al., 1993). Brains of 14 resting, sequestered rats were funnel frozen immediately after a single arteriovenous sample and analyzed. A plot of brain metabolite levels as a function of (A-V)O2 in each rat (Fig. 1C) shows that the brain glycogen, glucose, and lactate levels were relatively stable over a wide range of oxygen/glucose arteriovenous difference ratios but glycogen levels tended to be more variable than those of glucose and lactate. Similar results were obtained when all data from the resting rats in the Madsen et al. (1999) study were plotted (not shown), indicating that multiple sampling of cerebral venous blood in the resting rat did not measurably influence brain glucose or lactate levels. In a follow-up study (Cruz and Dienel, 2002), onset of glycogenolysis was assessed by clearance of 14 C from glycogen pre-labeled with [14 C]glucose. At 30 s after initiation of generalized sensory stimulation the 14 C recovered in glycogen tended to be lower (16%, P = 0.059) than unstimulated brain, suggesting that glycogen turnover starts soon after functional activation. Brain glycogen levels in resting rats were too variable to permit detection of glycogenolysis, if any, when the oxygen/glucose uptake ratio exceeded 6.0, but oxidation of endogenous brain glucose or lactate during a startle

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Fig. 1. Oxygen and glucose uptake into brain before, during and after generalized sensory stimulation of the conscious rat. (A) Arterial and cerebral venous concentrations of oxygen and glucose in a representative rat. Timed pairs of arterial and venous blood samples were drawn at rest when the rat was sequestered in a shelter, during generalized somatosensory stimulation by gentle brushing of the body, and during recovery from stimulation after the rat was returned to the shelter. (B) Mean changes in the ratios of oxygen to glucose uptake; values are means of 8–39 rats per group and vertical bars represent 1 S.D. (C) Glycogen, glucose, and lactate levels in dorsal cerebral cortex of resting, sequestered rats that were funnel frozen immediately after a single arteriovenous sample to determine the ratio of oxygen to glucose uptake. Each set of metabolite levels is from a separate rat and is plotted as a function of the arteriovenous difference ratio; linear regression lines are shown for each metabolite. Glycogen was assayed in HCl digests and underestimate the true values by about 50% (Cruz and Dienel, 2002). Data are from Madsen et al. (1999).

response is ruled out since their concentrations did not fall when (A-V)O2 /(A-V)glc exceeded 6 (Fig. 1C). 3.2.2. Major glucose carbon pools Cellular demands associated with increased neurotransmission might lead to transient changes in the glucosederived carbohydrates and amino acids, which together form a large pool of carbon in brain, i.e. about 15 ␮mol carbo-

hydrate/g plus another 25 ␮mol/g in the aggregate amino acid pool (Fig. 2A). Our initial (Madsen et al., 1999) and metabolic labeling (Dienel et al., 2002) studies of the major brain carbohydrate carbon pools in brain showed that glucose levels were nearly constant during rest, activation, and recovery from stimulation, whereas lactate levels rose during activation then normalized within 15 min after stimulation ceased, and glycogen levels progressively fell throughout

Fig. 2. Effect of generalized sensory stimulation on metabolite levels and labeling in dorsal cerebral cortex. (A) Levels of brain metabolites were measured in ethanol (carbohydrates) or perchloric acid (amino acids) extracts of dorsal cerebral cortex from funnel-frozen brain. (B) Incorporation of labeled glucose into intermediary metabolites during and after brain activation. Brains were sampled by funnel freezing at 5 min after a pulse intravenous injection of [6-14 C]glucose given during rest, at onset of 5 min of stimulation by gentle brushing of the body, or during recovery from stimulation at 10 min after termination of the stimulus. (C) Ratios of specific activities of purified metabolites. Values are means of six rats per group and vertical bars represent 1 S.D.; data are from Dienel et al. (2002). Abbreviations are as follows: Glc, glucose; Lac, lactate; Gln, glutamine; GABA, ␥-aminobutyric acid; Glu, glutamate; Asp, aspartate; Ala, alanine; Ser, serine.

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the activation and recovery period (Fig. 2A). The net increase in the quantity of lactate at the end of the stimulation period (1.1 ␮mol/g) corresponded to 10–15% of the lactate that could have been produced from the glucose consumed, and the lactate recovered in brain was estimated to account for only about half of the glucose consumed in excess of oxygen (Madsen et al., 1999). During stimulation there was a small but statistically significant increase in glutamate level and a larger fall in aspartate level; alanine content increased during activation and recovery (Fig. 2A). An unexpected discovery was the presence of extremely high glycogen levels, 10–12 ␮mol/g wet weight, in brains of carefully handled resting, sequestered rats (Fig. 2A). Glycogen levels in ethanol-insoluble fractions were twice those obtained in separate portions of the same samples analyzed after the routine HCl digestion procedure, and were even higher than those in perchloric acid-insoluble fractions (Cruz and Dienel, 2002). Thus, brain glycogen levels are several-fold higher than values generally reported, suggesting that astrocytes have an unrecognized and quantitatively important role in the energetics of brain activation. Pool size changes reflect metabolic shifts as the brain responds to demands of functional activation, and restoration of the glycogen stores in astrocytes was particularly slow. 3.2.3. Metabolic labeling of pathways of glucose metabolism Labeling of brain with [6-14 C]glucose during different stages of activity was used to identify changes in major pathways of glucose metabolism (Dienel et al., 2002). Overall glucose utilization in the dorsal cerebral cortex as assessed by label trapping in brain metabolites increased by at least 25% during stimulation (Fig. 2B), a value smaller than the 44% rise calculated from arteriovenous differences and the increase in blood flow (Madsen et al., 1999). This underestimation of the true extent of stimulation arises, in part, from calculations using plasma integrated specific activity which exceeds that of the true precursor pool in brain (and therefore reduces the calculated rate) and, perhaps, loss of diffusible [14 C]metabolites from activated tissue which causes underestimation of product formation. Labeling of lactate increased during stimulation, whereas labeling of glycogen doubled after cessation of stimulation compared to rest and activation; the fraction of total 14 C recovered in these two metabolites was, however, very low (Fig. 2B). In sharp contrast to the ‘non-oxidized’ products of glucose, about 35% of the total 14 C recovered in metabolites was incorporated into glutamate at all stages of activity, exceeding that into other amino acids. Glutamate labeling rose by 50% during stimulation (Fig. 2B), reflecting increased oxidative metabolism of glucose concurrent with the disproportionate increase in glucose compared to oxygen utilization. For [6-14 C]glucose-derived label to be trapped in glutamate via the very extensive and rapid transaminase exchange reaction between ␣-ketoglutarate and glutamate, the labeled carbon must first pass through the mitochondrial pyruvate dehy-

drogenase and isocitrate dehydrogenase steps, thereby producing NADH which is oxidized via the respiratory chain. Labeling of GABA and alanine also increased during stimulation, and all changes in amino acid labeling normalized within 15 min during recovery (Fig. 2B). Thus, during activation most 14 C derived from [6-14 C]glucose and retained in brain was recovered in amino acids derived from oxidative pathways. Comparison of specific activities of metabolites derived from a common precursor can help to identify the source of the metabolite, evaluate compartmentation, and perhaps suggest the fate of a metabolite. For example, the specific activity of brain lactate was approximately half that of [6-14 C]glucose during rest, activation, or recovery, indicating that brain lactate was mainly derived from blood-borne glucose at all stages of activity (Fig. 2C). This finding is of particular interest because the specific activity of glycogen was very low (Fig. 2A and B), and in cultured astrocytes glycogen is converted mainly to lactate which is exported to the culture medium (Dringen et al., 1993; Hamprecht and Dringen, 1994, 1995). The high specific activity of brain lactate suggests the possibility that any lactate derived from glycogen in vivo does not mix with lactate derived from blood-borne glucose and must have left the activated tissue, otherwise lactate specific activity should have been markedly depressed (and lactate levels should have risen). The outer tiers of glycogen would be expected to be labeled with high specific activity glucose and there are, therefore, some uncertainties arising from the exact timing of the fluxes of carbon from different sources (blood glucose versus glycogen) during activation. Alanine and lactate are both synthesized from pyruvate; alanine relative specific activity was half that of lactate (Fig. 2C), and alanine specific activity rose during stimulation, indicating pyruvate compartmentation. Also, the contrast in glutamine and GABA labeling relative to glutamate is consistent with the lag in labeling of glutamine due to slower incorporation via the glutamate–glutamine cycle (Sibson et al., 1997) and suggests differential fluxes of glucose into the respective glutamate precursor pools, perhaps with more rapid labeling of the smaller GABA pool. 3.3. Complexity of metabolic changes during brain activation 3.3.1. Oxidative metabolism is predominant even though CMRO2 /CMRglc falls Physiological activation of the normal, conscious rat by gentle brushing of the body to activate cerebral cortex causes both CMRglc and CMRO2 to rise. In spite of the differential increase in CMRglc compared to CMRO2 , oxidative metabolism of glucose predominates, and glutamate is the major compound labeled by [6-14 C]glucose. The glucose consumed in excess of oxygen is not fully explained by lactate and glycogen accumulation in brain (Fig. 2A) or lactate efflux to blood (Table 2C), but if the remainder of the

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‘excess glucose’ is due to lactate formation, this lactate must have been transported to other brain regions, since it did not cross the blood–brain barrier. During and after stimulation, labeling of oxidative and biosynthetic pathways changed in neurons (reflected by GABA and perhaps glutamate) and astrocytes (reflected by glycogen) with different temporal profiles, reflecting cellular heterogeneity of metabolism. 3.3.2. Astrocytic glycogen turnover could influence the CMRO2 /CMRglc ratio The roles of glycogen in brain energy metabolism and its fine-structure localization in astrocytes are not well understood (Hamprecht and Dringen, 1994, 1995). Many studies have emphasized the importance of astrocytes and their store of glycogen to supply energy substrates for use by neurons or other brain cells during pathophysiological conditions, including glucose deprivation (Swanson and Choi, 1993; Wender et al., 2000); hypoxia, hypoglycemia, or ischemia (Siesjö, 1978); and peroxide disposal, which involves activation of the pentose phosphate shunt pathway (Rahman et al., 2000). Fewer studies have addressed glycogen turnover in vivo during normal physiological activation (Swanson, 1992; Swanson et al., 1992; Kong et al., 2002; Cruz and Dienel, 2002; Fig. 2). Very high glycogen levels in resting brain (Kong et al., 2002; Cruz and Dienel, 2002) reveal the capability for rapid and sustained responses to high glucose demand by astrocytes. The magnitude of stimulus-induced glycogen utilization, calculated from the loss of glycogen (3 ␮mol/g) during 5 min stimulation was estimated to be about half that of blood-borne glucose in the activated cerebral cortex or 1.4 times the incremental increase in CMRglc above CMRO2 during activation (Dienel et al., 2002). One of the major unresolved issues in our studies is the metabolic fate of the glycogen degraded during activation and recovery. If glycogen were quantitatively converted to lactate and exported from the cell, as observed in tissue culture (Dringen et al., 1993), what is the fate of the 6 ␮mol/g lactate that could have been produced from glycogen at an average rate of 1.2 ␮mol/(g min) during activation? If oxidized, CMRO2 should have increased considerably above that related to (A-V)glc , and if glycogen metabolites were oxidized then more of the glucose taken up into brain from blood should have been converted to other non-oxidized compounds. If lactate derived from glycogen or glucose were exported to blood it should have been readily detected, but, in fact, the opposite was observed; (A-V)lac was slightly positive indicating uptake (Table 2C). Could all of this lactate have spread to other brain regions, and if so, by what mechanism? These questions will be discussed in Section 4.2. Segregated pyruvate/lactate pools suggest separate glycolytic pathways to extract energy from blood-borne glucose and stored glucose (glycogen), and raise experimentally-challenging questions for future studies designed to evaluate functional metabolic contributions of astrocytes and astrocyte–neuron interactions.

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Glycogen re-synthesis could depress the CMRO2 /CMRglc ratio during recovery from brain activation because glycogen level is restored quite slowly compared to normalization of other compounds (Fig. 2A) and the non-oxidative biosynthetic activity will contribute to a prolonged disproportionate uptake of glucose into brain compared to oxygen. For example, at 15 min after termination of 5 min of sensory stimulation the level of glycogen was still 4 ␮mol/g below the control value (Cruz and Dienel, 2002). During the subsequent more extended recovery period (which was not assayed in our studies) diversion of 10% of the glucose utilized by brain to restore glycogen would reduce the O2 /glucose uptake ratio from 5.9 to 5.5 and could cause a mismatch of glucose compared to oxygen uptake for as long as almost an hour after termination of the brief stimulus. Linkage of glycogenolysis to the ␤-adrenergic stress response pathways (see discussion and references cited in Cruz and Dienel, 2002) raises the interesting possibility that the low resting CMRO2 /CMRglc ratio, 4.1, observed by Fox et al. (1988) (Table 1) might have arisen, in part, from stressor anxiety-dependent glycogenolysis in the human subjects prior to the experiment, followed by gradual re-synthesis of glycogen during the experimental period when control ‘resting’ CMRO2 /CMRglc was determined. To summarize, astrocytic glycogen could make a substantial contribution to energy metabolism during brain activation, use of this energy source is not detectable by the usual CMRglc assay procedures, e.g. (A-V) determination or labeled tracer methods, and the recent history of the subject and astrocytic metabolic activities are factors that can influence the global O2 /glucose uptake ratio. 4. Local structure–function relationships: astrocyte morphology and metabolism 4.1. Fine processes of astrocytes The likelihood of metabolic compartmentation within an astrocyte underscores the importance of taking into account the complex anatomical and functional relationships between the fine processes of astrocytes and synaptic structures of nearby neurons in the energetics of brain activation and metabolite trafficking. Fluorescent dye-injected astrocytes in lightly-fixed brain slices permit visualization of the densely-branched structures of the astrocytes and complex interactions with adjacent neurons and blood vessels (Blümcke et al., 1995). The detailed analysis by Bushong et al. (2002) of the domains occupied by protoplasmic astrocytes emphasize the distinct astrocyte territories, the large astrocytic volume (66,000 ␮m3 ), and the elaborate, fine structure of distal processes that can interact with an estimated 140,000 synapses; these properties are not detected by immunostaining against glial fibrillary acidic protein (GFAP), and in fact, GFAP staining reveals only 15% of the astrocytic volume (Fig. 3A, left panel). The border zones

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Fig. 3. Fine processes occupy a large fraction of the astrocytic volume. (A) Astrocytes in rat hippocampus were labeled by filling of single astrocytes in fixed tissue slices by injection of a fluorescent dye and immunostained with an anti-glial fibrillary acidic protein (GFAP) antibody. The left top panel shows the limited extent of GFAP labeling (green) compared to Lucifer yellow (red), which reveals the extensive volume of the cell; yellow indicates the overlay of the labeled cytoskeleton on the dye-filled volume. The lower left panel shows a three-dimensional projection through the same volume as in the upper panel. The right panel illustrates the domains of four adjacent astrocytes (red and green). The pseudocolored yellow ribbons represent the pixels with red and green colocalization and illustrate regions of interface between neighboring protoplasmic astrocytes. Figures were reprinted from Bushong et al. (2002) with the permission of the Society for Neuroscience (Copyright 2002 by the Society for Neuroscience). (B) A cultured astrocyte double labeled with antibodies against glial fibrillary acidic protein (red fluorescence) against ezrin (green fluorescence), an actin-binding protein. The ezrin-immunoreactive filopodia and microvilli allow visualization of the fine peripheral processes of the astrocytes. Reprinted from Derouiche and Frotscher (2001) by permission of Wiley-Liss Inc., a subsidiary of John Wiley & Sons Inc. (Copyright 2001, Wiley-Liss).

in which adjacent astrocytes form interactive boundaries (Fig. 3A, right panel) appear to delineate interactive regions at which neighboring astrocytes communicate. The fine lamellate peripheral processes and filopodia of astrocytes are dispersed throughout the neuropil and surround synapses, and they can also be visualized by staining with antibodies against actin-binding proteins, ezrin and radixin (Derouiche and Frotscher, 2001). The distribution of these markers on

fine processes appears to emerge from, but is discontinuous with the main GFAP-containing processes of the astrocyte (Fig. 3B). Because ezrin and radixin are associated with actin, Derouiche and Frotscher (2001) suggest a role in motility and morphological plasticity of astrocytes and their thin processes (Theodosis and Poulain, 1993; Oliet et al., 2001; Safavi-Abbasi et al., 2001; Hatton, 1999, 2002), which they think might represent separate subcellular compartment

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characterized by paucity of cytoplasm and organelles. Distal astrocytic structures are also labeled by antibodies to glutamine synthetase (Norenberg and Martinez-Hernandez, 1979; Derouiche and Frotscher, 1991; Derouiche and Ohm, 1994) and glutamate transporters (Derouiche and Rauen, 1995; Derouiche, 1997). Thus, fine astrocytic processes appear to be adaptive, highly metabolically-active zones, with a large surface area that surrounds capillaries and synapses and contacts other astrocytes. 4.2. Functional energy metabolism in fine processes of astrocytes The paucity of cytoplasm and lack of organelles in the fine lamina of the peripheral astrocytic processes that are 50–100 nm wide (Derouiche and Frotscher, 2001) raises the intriguing question as to how the local energy demands associated with rapid, excitatory neurotransmission (e.g. K+ uptake, sodium-dependent glutamate transport, glutamate and glutamine synthesis, and calcium signaling) are satisfied. Depending on stimulus intensity and duration, rapid but limited metabolism of glucose might be essential, but it must be coupled to immediate lactate export in order to maintain high glycolytic rates, because otherwise lactate and hydrogen ion accumulation in the cytosol of fine processes would lead to subsequent feedback inhibition of glycolytic enzymes (Halestrap and Price, 1999; Juel, 2001). It is also well established that glutamate, lactate, glucose, and probably other energy substrates are oxidized by astrocytes (Yu et al., 1982; Eriksson et al., 1995; Sonnewald et al., 1993; McKenna et al., 1996; Hutson et al., 1998; Lieth et al., 2001) and these compounds should also contribute to satisfying the energy demands of activated cells. The synaptosomal fraction contains about half of the hexokinase in brain (Wilson, 1972) and neurons are well poised to utilize glucose during activation, although supplementation with other substrate fuel is not ruled out. The in vivo metabolic fate of various substrates is a particularly difficult issue to address experimentally, and local metabolic activity in astrocytic processes that surround active synapses or capillaries within the complex architecture of the brain is unlikely to be approximated by in vitro experiments that administer bio-active agents to the entire dorsal surface of a monolayer of metabolically more-or-less competent astrocytes grown in the absence of neurons. These difficulties are not remedied by use of metabolic inhibitors that compel the use of alternative pathways, or the use of high loads of alternative substrates that distort normal metabolic preferences. More refined techniques must be developed to analyze compartmentation of functional metabolism in the thinnest and most distal astrocytic processes to determine whether glycolysis and glycogenolysis might be favored over mitochondrial metabolism. If cytoplasm is limiting in fine astrocytic processes, are energy reserves available for rapid metabolic activation, and could glycogen be a fuel for these structures? Ultrastructural

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analysis has demonstrated the predominant localization of glycogen granules in astrocytes, the association of glycogen particles with endoplasmic reticulum and secondary lysosomes, widespread distribution of glycogen throughout the neuropil (including localization in processes located among synapses, in pericapillary domains, and in endfeet), and accumulation of glycogen in astrocytic structures during prolonged anesthesia and methionine sulphoximine treatment (Phelps, 1972, 1975; Cataldo and Broadwell, 1986; Peters et al., 1991). These studies are supported by electron microscopic studies of glycogen phosphorylase immunoreactivity in which the enzyme is localized mainly to astrocytes with diffuse staining throughout the cytoplasm and processes ensheathing capillaries, and in the fine processes and lamellae adjacent to synaptic structures (Richter et al., 1996). Glycogen particles appear to be small enough, with diameters ranging from 10 to 44 nm, depending on staining method (Cataldo and Broadwell, 1986; Wender et al., 2000) to reside within the thin 50–100 nm wide lamellae (Derouiche and Frotscher, 2001) of fine astrocytic processes. If glycogen were an important energy source in astrocytic processes, then it is likely to be converted to lactate due to the lack of nearby mitochondria, and elimination of lactate from the extracellular space would be essential. The presumptive functional importance of astrocytic filopodia to brain activation requires structure–function analysis relationships of energy-providing and utilizing systems and metabolite transporters. Compartmentation of a portion of glycogen metabolism in distal processes remote from those surrounding capillaries would provide a mechanism to segregate blood-borne [14 C]glucose-derived labeled lactate from glycogen-derived lactate that is much less labeled. 4.3. Does astrocyte gap junction connectivity have a major role in metabolite trafficking? Rapid spreading and loss of labeled products of [6-14 C]glucose within and from activated structures in brain in vivo is suggested by failure of [6-14 C]glucose to register the magnitude of increase of CMRglc observed during physiological activation of visual (Collins et al., 1987) and auditory (Dienel and Hertz, 2001) structures with [14 C]deoxyglucose. Mechanisms of clearance of metabolites from functionally-activated cells are not known but might involve movement through astrocytic gap junctions and redistribution within the astrocytic syncytium. Preliminary studies in our laboratory showed that spreading of [14 C]glucose and its labeled products during microinfusion into brain of conscious ‘resting’ rats is reduced by about half by prior infusion of gap junction inhibitors (Dienel et al., 2001). The potential for rapid, extensive metabolite dispersal and metabolite release to capillaries via the astrocytic syncytium is illustrated by studies in which the heterogeneous connectivity of hippocampal astrocytes was revealed by infusing biocytin into astrocytes in brain slices

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Fig. 4. Gap junction-coupled hippocampal astrocytes. Single astrocytes were labeled with biocytin during electrophysiological recording in hippocampal slices from the rat and were visualized in fixed sections by immunocytochemistry. Labeling of astrocytes in CA1 (A) and CA3 (B) regions of hippocampus after biocytin injection into a single cell in the stratum radiatum of CA1 or CA3. Note the lack of labeling in the pyramidal neuronal layer (pyr) and the staining of astrocytic structures (endfeet) along blood vessels of varying sizes. Heterogeneity of cell–cell coupling in the CA1 and CA3 hippocampus was quantified by comparing the number of labeled cells per injection (C). Abbreviations are as follows: or, stratum oriens; pyr, stratum pyramidale; rad, stratum radiatum; mol, stratum molecular; WC, whole cell. Reprinted from D’Ambrosio et al. (1998) by permission of the Society for Neuroscience (Copyright 1998, Society for Neuroscience).

Fig. 5. Hypothetical schematic model illustrating some metabolic interactions between working brain cells. (A) [14 C]Glucose preferentially labels the “large” glutamate compartment, presumably mainly neuronal, resulting in a glutamine/glutamate specific activity ratio <1.0 (Bal´azs and Cremer, 1972; Berl et al., 1975; Fig. 2B). The glutamate–glutamine cycle provides a mechanism for transfer of carbon between neurons and astrocytes, with slower labeling of the glutamine pool (Sibson et al., 1997). In pulse labeling experiments the specific activity of 14 C-labeled lactate indicates that it is derived mainly from blood-borne glucose (Adachi et al., 1995; Dienel et al., 2002), but might be synthesized in either or both neurons and astrocytes. Lactate is also expected to arise from glycogenolysis in astrocytes (Dringen et al., 1993) but its fate is uncertain. If the glycogen degraded during brain activation were oxidized, then (A-V)O2 /(A-V)glc should have increased above 6.0 during activation; instead this ratio fell (Fig. 1). Glycogen has low specific activity (Fig. 2B), and if glycogen-derived lactate were retained in brain cells the specific activity of brain lactate should have fallen, but it remained constant (Fig. 2C); these data suggest that products of glycogenolysis were cleared from activated tissue. Because the specific activity of brain lactate was always half that of blood-borne brain glucose and alanine was half that of lactate (Fig. 2C), there is segregation of (at least two) major pyruvate/lactate pools, one from blood-borne glucose, another diluted by unlabeled endogenous metabolites. Likely metabolite clearance mechanisms include extracellular fluid (ECF), perhaps via paravascular flow (Rennels et al., 1985) and rapid transfer of material via astrocytic gap junctional connections, which could lead to widespread and heterogeneous dispersal of metabolites within the astrocytic syncytium and to capillaries (see Figs. 3 and 4). Abbreviations are as follows: glc, glucose; pyr, pyruvate; lac, lactate; glu, glutamate; gln, glutamine; TCA, tricarboxylic acid. (B) An expanded view showing one excitatory synapse surrounded by fine processes of two astrocytes, both of which connect to capillaries, one

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(Konietzko and Muller, 1994; D’Ambrosio et al., 1998). In CA1 hippocampus a large number of widely-dispersed astrocytes were labeled from a single cell in a tissue sample with a cross-section area of about 1 mm2 ; many of the junction-coupled astrocytes had visible endfeet localized on capillaries (Fig. 4A). Less extensive labeling was observed in CA3 hippocampal astrocytes (Fig. 4B), with most CA3 cells linked to about 20 others compared to more than 50 junction-coupled cells in the CA1 region (Fig. 4C). In addition, autocellular gap junctions (connections between different regions of the same cell) are also prevalent on the filopodia of cultured astrocyte (Wolff et al., 1998), suggesting the possibility of delivery and dispersal of energy metabolites and signaling molecules between functional domains of the same cell. Metabolite spreading via the astrocytic syncytium could be a mechanism for rapid supply of energy metabolites from adjacent cells in the network and also elimination of by-products from activated tissue to blood or other brain regions (Fig. 5). Lactate in extracellular fluid, whether produced by neurons or astrocytes, could be taken up into nearby unactivated astrocytes and quickly dispersed, oxidatively metabolized, or cleared. If metabolite spreading does occur via gap junctions, the fluxes of various compounds through these pores could be substantial and they would be directed down metabolite concentration gradients away from activated cells. For example if CMRglc in an activated structure were 1.15 ␮mol/(g min) and 1 ␮mol/(g min) were completely oxidized to CO2 and water, efflux of metabolically-produced water would be six times that of CMRO2 or 6 ␮mol/(g min). Metabolic water clearance is essential (for osmotic reasons), and might involve aquaporin water channels, such as those in astrocytes and their endfeet (Nielsen et al., 1997; Rash et al., 1998). If about 13% of the glucose consumed, 0.15 ␮mol/(g min), were not oxidized but instead converted to lactate and cleared from activated tissue, 0.3 ␮mol lactate/(g min) must be transported away. If during the same interval 3 ␮mol glycogen/g is consumed and converted to lactate, another 6 ␮mol lactate are produced/g. Averaged over a 5 min period, an additional 1.2 ␮mol lactate/(g min) must be cleared, for a total of 1.5 ␮mol lactate/(g min). Thus, during brief activation, lac-

directly and the other indirectly via gap junction coupling to two other astrocytes. Metabolite clearance from an active perisynaptic zone through the astrocytic syncytium to capillaries is illustrated. For simplicity, many critical aspects of these processes are not included in the figure, including glucose fluxes and various transporters. Spatial separation of distal filopodia from mitochondria might lead to lactate synthesis in fine processes, perhaps from glucose and glycogen; based on phosphorylase immunohistochemistry, some glycogen symbols are included in fine fibers as “food for thought” (see text, Sections 3.3.2 and 4.2). Immediate removal of lactate from activated structures is suggested because lactate metabolism in neurons and astrocytes is much slower than its trans-membrane transport and its accumulation would slow or block glycolysis by end-product feedback inhibition (Halestrap and Price, 1999; Juel, 2001; Dienel and Hertz, 2001).

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tate clearance rates from glucose plus glycogen could exceed the rate of glucose influx across the blood–brain barrier to match CMRglc . The mechanisms of glucose delivery, the cellular origin of lactate produced during activation, routes and rates of clearance of metabolic by-products, and the possible roles of gap junctions in these processes are important areas for innovative future work.

5. Concluding comments An implication of the disproportionate increase in CMRglc compared to CMRO2 during brain activation is that a fraction of the products of glucose metabolism are not locally oxidized even though there is a concomitant increase in oxidative metabolism and increased trapping of products of blood-borne glucose in the tricarboxylic acid cycle (Figs. 1, 2, and 5A). Although some lactate does accumulate in activated tissue, the amount recovered does not fully explain the glucose/oxygen uptake mismatch. The magnitude of energy utilization during brain activation is underestimated because it does not take into account metabolism of endogenous energy stores, such as glycogen. The situation is quite complicated because lactate can be derived from glucose and glycogen, and much of the glycogen carbon lost during activation cannot be accounted for as lactate or CMRO2 ; there must be rapid product clearance. If lactate is a major contributor to the fall in the CMRO2 /CMRglc ratio, lactate is not further metabolized within or released to blood from the large volume of tissue sampled by arteriovenous differences, i.e. the dorsal cerebral cortex in the rat. A model of stoichiometric, directed transfer of lactate from astrocytes to neighboring neurons for subsequent oxidation is not consistent with these experimental observations. Instead, there is probably rapid spreading of lactate and perhaps other metabolites from the activated area by various unidentified mechanisms, including paravascular flow, distribution within the astrocytic syncytium, and efflux to blood and other brain regions. Dispersal routes (Fig. 5B) must be extensive enough to very quickly remove glucose metabolites not only from the local site but also from the entire activated structure, the cerebral cortex in our studies. Due to their large energy stores and the complex structures of their endfeet and fine processes, astrocytes have the potential to influence the global oxygen/glucose utilization ratio by various mechanisms. This potential raises compelling questions regarding the cellular origin of lactate, the location(s) of glycogen turnover, metabolic fate of glycogen, and role of metabolite trafficking via interand intracellular gap junctions during activation of energy metabolism. These factors can impact interpretation of overall metabolic status of the brain, and novel methods need to be developed to evaluate functional activities of different structures of astrocytes in their normal environment within which they matured and formed intricate interactions with their neighbors.

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Acknowledgements This work was supported, in part, by NIH grant NS36728.

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