Determination of Glucose Utilization Rates in Cultured Astrocytes and Neurons with [14C]deoxyglucose: Progress, Pitfalls, and Discovery of Intracellular Glucose Compartmentation.

2-Deoxy-D-[14C]glucose ([14C]DG) is commonly used to determine local glucose utilization rates (CMRglc) in living brain and to estimate CMRglc in cultured brain cells as rates of [14C]DG phosphorylation. Phosphorylation rates of [14C]DG and its metabolizable fluorescent analog, 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG), however, do not take into account differences in the kinetics of transport and metabolism of [14C]DG or 2-NBDG and glucose in neuronal and astrocytic cells in cultures or in single cells in brain tissue, and conclusions drawn from these data may, therefore, not be correct. As a first step toward the goal of quantitative determination of CMRglc in astrocytes and neurons in cultures, the steady-state intracellular-to-extracellular concentration ratios (distribution spaces) for glucose and [14C]DG were determined in cultured striatal neurons and astrocytes as functions of extracellular glucose concentration. Unexpectedly, the glucose distribution spaces rose during extreme hypoglycemia, exceeding 1.0 in astrocytes, whereas the [14C]DG distribution space fell at the lowest glucose levels. Calculated CMRglc was greatly overestimated in hypoglycemic and normoglycemic cells because the intracellular glucose concentrations were too high. Determination of the distribution space for [14C]glucose revealed compartmentation of intracellular glucose in astrocytes, and probably, also in neurons. A smaller metabolic pool is readily accessible to hexokinase and communicates with extracellular glucose, whereas the larger pool is sequestered from hexokinase activity. A new experimental approach using double-labeled assays with DG and glucose is suggested to avoid the limitations imposed by glucose compartmentation on metabolic assays.

Organ Distribution of 13N Following Intravenous Injection of [13N]Ammonia into Portacaval-Shunted Rats.

Ammonia is neurotoxic, and chronic hyperammonemia is thought to be a major contributing factor to hepatic encephalopathy in patients with liver disease. Portacaval shunting of rats is used as an animal model to study the detrimental metabolic effects of elevated ammonia levels on body tissues, particularly brain and testes that are deleteriously targeted by high blood ammonia. In normal adult rats, the initial uptake of label (expressed as relative concentration) in these organs was relatively low following a bolus intravenous injection of [13N]ammonia compared with lungs, kidneys, liver, and some other organs. The objective of the present study was to determine the distribution of label following intravenous administration of [13N]ammonia among 14 organs in portacaval-shunted rats at 12 weeks after shunt construction. At an early time point (12 s) following administration of [13N]ammonia the relative concentration of label was highest in lung with lower, but still appreciable relative concentrations in kidney and heart. Clearance of 13N from blood and kidney tended to be slower in portacaval-shunted rats versus normal rats during the 2-10 min interval after the injection. At later times post injection, brain and testes tended to have higher-than-normal 13N levels, whereas many other tissues had similar levels in both groups. Thus, reduced removal of ammonia from circulating blood by the liver diverts more ammonia to extrahepatic tissues, including brain and testes, and alters the nitrogen homeostasis in these tissues. These results emphasize the importance of treatment paradigms designed to reduce blood ammonia levels in patients with liver disease.

Aerobic glycolysis during brain activation: adrenergic regulation and influence of norepinephrine on astrocytic metabolism.

Aerobic glycolysis occurs during brain activation and is characterized by preferential up-regulation of glucose utilization compared with oxygen consumption even though oxygen level and delivery are adequate. Aerobic glycolysis is a widespread phenomenon that underlies energetics of diverse brain activities, such as alerting, sensory processing, cognition, memory, and pathophysiological conditions, but specific cellular functions fulfilled by aerobic glycolysis are poorly understood. Evaluation of evidence derived from different disciplines reveals that aerobic glycolysis is a complex, regulated phenomenon that is prevented by propranolol, a non-specific ß-adrenoceptor antagonist. The metabolic pathways that contribute to excess utilization of glucose compared with oxygen include glycolysis, the pentose phosphate shunt pathway, the malate-aspartate shuttle, and astrocytic glycogen turnover. Increased lactate production by unidentified cells, and lactate dispersal from activated cells and lactate release from the brain, both facilitated by astrocytes, are major factors underlying aerobic glycolysis in subjects with low blood lactate levels. Astrocyte-neuron lactate shuttling with local oxidation is minor. Blockade of aerobic glycolysis by propranolol implicates adrenergic regulatory processes including adrenal release of epinephrine, signaling to brain via the vagus nerve, and increased norepinephrine release from the locus coeruleus. Norepinephrine has a powerful influence on astrocytic metabolism and glycogen turnover that can stimulate carbohydrate utilization more than oxygen consumption, whereas ß-receptor blockade 're-balances' the stoichiometry of oxygen-glucose or -carbohydrate metabolism by suppressing glucose and glycogen utilization more than oxygen consumption. This conceptual framework may be helpful for design of future studies to elucidate functional roles of preferential non-oxidative glucose utilization and glycogen turnover during brain activation. Aerobic glycolysis, the preferential up-regulation of glucose utilization (CMRglc ) compared with oxygen consumption (CMRO2 ) during brain activation, is blocked by propranolol. Epinephrine release from the adrenal gland stimulates vagus nerve signaling to the locus coeruleus, enhancing norepinephrine release in the brain, and regulation of astrocytic and neuronal metabolism to stimulate CMRglc more than CMRO2 . Propranolol suppresses CMRglc more than CMRO2 .

Biochemical, Metabolic, and Behavioral Characteristics of Immature Chronic Hyperphenylalanemic Rats.

Phenylketonuria and hyperphenylalanemia are inborn errors in metabolism of phenylalanine arising from defects in steps to convert phenylalanine to tyrosine. Phe accumulation causes severe mental retardation that can be prevented by timely identification of affected individuals and their placement on a Phe-restricted diet. In spite of many studies in patients and animal models, the basis for acquisition of mental retardation during the critical period of brain development is not adequately understood. All animal models for human disease have advantages and limitations, and characteristics common to different models are most likely to correspond to the disorder. This study established similar levels of Phe exposure in developing rats between 3 and 16 days of age using three models to produce chronic hyperphenylalanemia, and identified changes in brain amino acid levels common to all models that persist for ~16 h of each day. In a representative model, local rates of glucose utilization (CMRglc) were determined at 25-27 days of age, and only selective changes that appeared to depend on Phe exposure were observed. CMRglc was reduced in frontal cortex and thalamus and increased in hippocampus and globus pallidus. Behavioral testing to evaluate neuromuscular competence revealed poor performance in chronically-hyperphenylalanemic rats that persisted for at least 3 weeks after cessation of Phe injections and did not occur with mild or acute hyperphenylalanemia. Thus, the abnormal amino acid environment, including hyperglycinemia, in developing rat brain is associated with selective regional changes in glucose utilization and behavioral abnormalities that are not readily reversed after they are acquired.

Contributions of glycogen to astrocytic energetics during brain activation.

Glycogen is the major store of glucose in brain and is mainly in astrocytes. Brain glycogen levels in unstimulated, carefully-handled rats are 10-12 µmol/g, and assuming that astrocytes account for half the brain mass, astrocytic glycogen content is twice as high. Glycogen turnover is slow under basal conditions, but it is mobilized during activation. There is no net increase in incorporation of label from glucose during activation, whereas label release from pre-labeled glycogen exceeds net glycogen consumption, which increases during stronger stimuli. Because glycogen level is restored by non-oxidative metabolism, astrocytes can influence the global ratio of oxygen to glucose utilization. Compensatory increases in utilization of blood glucose during inhibition of glycogen phosphorylase are large and approximate glycogenolysis rates during sensory stimulation. In contrast, glycogenolysis rates during hypoglycemia are low due to continued glucose delivery and oxidation of endogenous substrates; rates that preserve neuronal function in the absence of glucose are also low, probably due to metabolite oxidation. Modeling studies predict that glycogenolysis maintains a high level of glucose-6-phosphate in astrocytes to maintain feedback inhibition of hexokinase, thereby diverting glucose for use by neurons. The fate of glycogen carbon in vivo is not known, but lactate efflux from brain best accounts for the major metabolic characteristics during activation of living brain. Substantial shuttling coupled with oxidation of glycogen-derived lactate is inconsistent with available evidence. Glycogen has important roles in astrocytic energetics, including glucose sparing, control of extracellular K(+) level, oxidative stress management, and memory consolidation; it is a multi-functional compound.

Reduced clearance of proteins labeled with diisopropylfluorophosphate in portacaval-shunted rats.

Portacaval shunting is a model for hepatic encephalopathy that causes chronic hyperammonemia, disruption of metabolic, signaling, and neurotransmitter systems, and progressive morphological changes. Exposure of cultured cells to ammonia raises intralysosomal pH and inhibits proteolysis, and the present study tested the hypothesis that proteolytic capacity is diminished in portacaval-shunted rats. Proteins were labeled in vivo with tracer doses of diisopropylfluorophosphate (DFP) and clearance of label was assayed. This approach labeled proteins independent of protein synthesis, which is reported to be altered in shunted rats, and avoided complications arising from re-utilization of labeled amino acids that causes underestimation of degradation rate. Characterization of DFP labeling showed that protein labeling was fast, about 50% of the label was released during a 24 h interval, labeling by DFP metabolites was negligible, inhibition of brain acetylcholinesterase was not detectable, and labeling by [(3)H]- and [(14)C]DFP was equivalent. To assay degradative capacity, proteins were first labeled with [(3)H]DFP, followed by labeling with [(14)C]DFP that was given 24 or 72 h later. The (3)H/(14)C ratio in each animal was used as a relative measure of removal of (3)H-labeled proteins. (3)H/(14)C ratios were generally significantly higher in portacaval-shunted rats than in controls, consistent with reduced proteolytic capacity. Assays of amino acid incorporation into brain protein generally replicated literature reports, supporting the conclusion that protein synthesis unlikely to be markedly inhibited and amino acid recycling influences calculated protein synthesis rates in shunted rats. Therapeutic strategies to reduce ammonia level would help normalize lysosomal functions and protein and lipid turnover.

The unfolded protein response to endoplasmic reticulum stress in cultured astrocytes and rat brain during experimental diabetes.

Oxidative-nitrosative stress and inflammatory responses are associated with endoplasmic reticulum (ER) stress in diabetic retinopathy, raising the possibility that disturbances in ER protein processing may contribute to CNS dysfunction in diabetics. Upregulation of the unfolded protein response (UPR) is a homeostatic response to accumulation of abnormal proteins in the ER, and the present study tested the hypothesis that the UPR is upregulated in two models for diabetes, cultured astrocytes grown in 25mmol/L glucose for up to 4weeks and brain of streptozotocin (STZ)-treated rats with diabetes for 1-7months. Markers associated with translational blockade (phospho-eIF2α and apoptosis (CHOP), inflammatory response (inducible nitric oxide synthase, iNOS), and nitrosative stress (nuclear translocation of glyceraldehyde-3-phosphate dehydrogenase, GAPDH) were not detected in either model. Nrf2 was present in nuclei of low- and high-glucose cultures, consistent with oxidative stress. Astrocytic ATF4 expression was not altered by culture glucose concentration, whereas phospho-IRE and ATF6 levels were higher in low- compared with high-glucose cultures. The glucose-regulated chaperones, GRP78 and GRP94, were also expressed at higher levels in low- than high-glucose cultures, probably due to recurrent glucose depletion between feeding cycles. In STZ-rat cerebral cortex, ATF4 level was transiently reduced at 4months, and p-IRE levels were transiently elevated at 3months. However, GRP78 and GRP94 expression was not upregulated, and iNOS, amyloid-ß, and nuclear accumulation of GAPDH were not evident in STZ-diabetic brain. High-glucose cultured astrocytes and STZ-diabetic brain are relatively resistant to diabetes-induced ER stress, in sharp contrast with cultured retinal Müller cells and diabetic rodent retina.

Regional registration of [6-(14)C]glucose metabolism during brain activation of α-syntrophin knockout mice.

α-Syntrophin is a component of the dystrophin scaffold-protein complex that serves as an adaptor for recruitment of key proteins to the cytoplasmic side of plasma membranes. α-Syntrophin knockout (KO) causes loss of the polarized localization of aquaporin4 (AQP4) at astrocytic endfeet and interferes with water and K(+) homeostasis. During brain activation, release of ions and metabolites from endfeet is anticipated to increase perivascular fluid osmolarity, AQP4-mediated osmotic water flow from endfeet, and metabolite washout from brain. This study tests the hypothesis that reduced levels of endfoot AQP4 increase retention of [(14)C]metabolites during sensory stimulation. Conscious KO and wild-type mice were pulse-labeled with [6-(14)C] glucose during unilateral acoustic stimulation or bilateral acoustic plus whisker stimulation, and label retention was assayed by computer-assisted brain imaging or analysis of [(14)C]metabolites in extracts, respectively. High-resolution autoradiographic assays detected a 17% side-to-side difference (p < 0.05) in inferior colliculus of KO mice, not wild-type mice. However, there were no labeling differences between KO and wild-type mice for five major HPLC fractions from four dissected regions, presumably because of insufficient anatomical resolution. The results suggest a role for AQP4-mediated water flow in support of washout of metabolites, and underscore the need for greater understanding of astrocytic water and metabolite fluxes.

Reduced gap junctional communication among astrocytes in experimental diabetes: contributions of altered connexin protein levels and oxidative-nitrosative modifications.

Experimental diabetes increases production of reactive oxygen-nitrogen species and inhibits astrocytic gap junctional communication in tissue culture and brain slices from streptozotocin (STZ)-diabetic rats by unidentified mechanisms. Relative connexin (Cx) protein levels were assessed by Western blotting using extracts from cultured astrocytes grown in high (25 mmol/liter) or low (5.5 mmol/liter) glucose for 2-3 weeks and STZ-diabetic rat brain. Chemiluminescent signals for diabetic samples were normalized to those of controls on the same blot and same protein load. Growth in high glucose did not alter relative Cx26 level, whereas Cx30 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were reduced by ∼30%, and Cx43 increased ∼1.9-fold. In the inferior colliculus of STZ-diabetic rats, Cx30 and Cx43 levels in three of four rats were half those of controls, whereas GAPDH and actin were unaffected. Diabetes did not affect levels of Cx30, Cx43, or GAPDH in cerebral cortex, but actin level rose 24%. Cx43 was predominantly phosphorylated in control and diabetic samples, so the reduced dye transfer is not due to overall dephosphorylation of Cx43. Astrocytic growth in high glucose reduced the dye-labeled area by 75%, but 10 min of treatment with dithiothreitol restored normal dye transfer. In contrast, nitric oxide donors inhibited dye transfer among astrocytes grown in low glucose by 50-65% within 1 hr. Thus, modifications arising from oxidative-nitrosative stress, not altered connexin levels, may underlie the reduced dye transfer among severely hyperglycemic cultured astrocytes, whereas both oxidative-nitrosative stress and regionally selective down-regulation of connexin protein content may affect gap junctional communication in the brains of STZ-diabetic rats.

Astrocytic gap junctional communication is reduced in amyloid-ß-treated cultured astrocytes, but not in Alzheimer's disease transgenic mice.

Alzheimer's disease is characterized by accumulation of amyloid deposits in brain, progressive cognitive deficits and reduced glucose utilization. Many consequences of the disease are attributed to neuronal dysfunction, but roles of astrocytes in its pathogenesis are not well understood. Astrocytes are extensively coupled via gap junctions, and abnormal trafficking of metabolites and signalling molecules within astrocytic syncytia could alter functional interactions among cells comprising the neurovascular unit. To evaluate the influence of amyloid-beta on astrocyte gap junctional communication, cultured astrocytes were treated with monomerized amyloid-ß(1-40) (1 µmol/l) for intervals ranging from 2 h to 5 days, and the areas labelled by test compounds were determined by impaling a single astrocyte with a micropipette and diffusion of material into coupled cells. Amyloid-ß-treated astrocytes had rapid, sustained 50-70% reductions in the area labelled by Lucifer Yellow, anionic Alexa Fluor® dyes and energy-related compounds, 6-NBDG (a fluorescent glucose analogue), NADH and NADPH. Amyloid-ß treatment also caused a transient increase in oxidative stress. In striking contrast with these results, spreading of Lucifer Yellow within astrocytic networks in brain slices from three regions of 8.5-14-month-old control and transgenic Alzheimer's model mice was variable, labelling 10-2000 cells; there were no statistically significant differences in the number of dye-labelled cells among the groups or with age. Thus amyloid-induced dysfunction of gap junctional communication in cultured astrocytes does not reflect the maintenance of dye transfer through astrocytic syncytial networks in transgenic mice; the pathophysiology of Alzheimer's disease is not appropriately represented by the cell culture system.

Hyperglycaemia and diabetes impair gap junctional communication among astrocytes.

Sensory and cognitive impairments have been documented in diabetic humans and animals, but the pathophysiology of diabetes in the central nervous system is poorly understood. Because a high glucose level disrupts gap junctional communication in various cell types and astrocytes are extensively coupled by gap junctions to form large syncytia, the influence of experimental diabetes on gap junction channel-mediated dye transfer was assessed in astrocytes in tissue culture and in brain slices from diabetic rats. Astrocytes grown in 15-25 mmol/l glucose had a slow-onset, poorly reversible decrement in gap junctional communication compared with those grown in 5.5 mmol/l glucose. Astrocytes in brain slices from adult STZ (streptozotocin)-treated rats at 20-24 weeks after the onset of diabetes also exhibited reduced dye transfer. In cultured astrocytes grown in high glucose, increased oxidative stress preceded the decrement in dye transfer by several days, and gap junctional impairment was prevented, but not rescued, after its manifestation by compounds that can block or reduce oxidative stress. In sharp contrast with these findings, chaperone molecules known to facilitate protein folding could prevent and rescue gap junctional impairment, even in the presence of elevated glucose level and oxidative stress. Immunostaining of Cx (connexin) 43 and 30, but not Cx26, was altered by growth in high glucose. Disruption of astrocytic trafficking of metabolites and signalling molecules may alter interactions among astrocytes, neurons and endothelial cells and contribute to changes in brain function in diabetes. Involvement of the microvasculature may contribute to diabetic complications in the brain, the cardiovascular system and other organs.

Trafficking of glucose, lactate, and amyloid-beta from the inferior colliculus through perivascular routes.

Metabolic brain imaging is widely used to evaluate brain function and disease, and quantitative assays require local retention of compounds used to register changes in cellular activity. As labeled metabolites of [1- and 6-(14)C]glucose are rapidly released in large quantities during brain activation, this study evaluated release of metabolites and proteins through perivascular fluid flow, a pathway that carries solutes from brain to peripheral lymphatic drainage sites. Assays with [3,4-(14)C]glucose ruled out local oxidation of glucose-derived lactate as a major contributor of label loss. Brief infusion of [1-(14)C]glucose and D-[(14)C]lactate into the inferior colliculus of conscious rats during acoustic stimulation labeled the meninges, consistent with perivascular clearance of [(14)C]metabolites from interstitial fluid. Microinfusion of Evans blue albumin and amyloid-beta(1-40) (Abeta) caused perivascular labeling in the inferior colliculus, labeled the surrounding meninges, and Abeta-labeled-specific blood vessels in the caudate and olfactory bulb and was deposited in cervical lymph nodes. Efflux of extracellular glucose, lactate, and Abeta into perivascular fluid pathways is a normal route for clearance of material from the inferior colliculus that contributes to underestimates of brain energetics. Convergence of 'watershed' drainage to common pathways may facilitate perivascular amyloid plaque formation and pathway obstruction in Alzheimer's disease.

Astrocytes are poised for lactate trafficking and release from activated brain and for supply of glucose to neurons.

Brain is a highly-oxidative organ, but during activation, glycolytic flux is preferentially up-regulated even though oxygen supply is adequate. The biochemical and cellular basis of metabolic changes during brain activation and the fate of lactate produced within brain are important, unresolved issues central to understanding brain function, brain images, and spectroscopic data. Because in vivo brain imaging studies reveal rapid efflux of labeled glucose metabolites during activation, lactate trafficking among astrocytes and between astrocytes and neurons was examined after devising specific, real-time, sensitive enzymatic fluorescent assays to measure lactate and glucose levels in single cells in adult rat brain slices. Astrocytes have a 2- to 4-fold faster and higher capacity for lactate uptake from extracellular fluid and for lactate dispersal via the astrocytic syncytium compared to neuronal lactate uptake from extracellular fluid or shuttling of lactate to neurons from neighboring astrocytes. Astrocytes can also supply glucose to neurons as well as glucose can be taken up by neurons from extracellular fluid. Astrocytic networks can provide neuronal fuel and quickly remove lactate from activated glycolytic domains, and the lactate can be dispersed widely throughout the syncytium to endfeet along the vasculature for release to blood or other brain regions via perivascular fluid flow.

Selective astrocytic gap junctional trafficking of molecules involved in the glycolytic pathway: impact on cellular brain imaging.

To assess the specificity of metabolite trafficking among gap junction-coupled astrocytes, we developed novel, real-time, single-cell enzymatic fluorescence assays to assay cell-to-cell transfer of unlabeled glycolytic intermediates and report (i) highly restricted transfer of glucose-6-phosphate (P) and two analogs, deoxyglucose (DG)-6-P, and 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-DG-6-P, compared with DG and 2- and 6-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-DG, (ii) extensive junctional diffusion of glyceraldehyde-3-P, NADH, and NADPH plus three anionic fluorescent dyes used as internal standards for transfer assays, and (iii) stimulation of gap junctional communication by increased intracellular Na(+) that also evokes metabolic responses in nearby coupled astrocytes. Thus, dye transfer does not predict gap junctional permeability of endogenous metabolites. Intracellular retention of flux-regulating compounds (e.g. glucose-6-P) may be necessary for local metabolic control, whereas 'syncytial sharing' may dissipate the work load on peri-synaptic astrocytes. Imaging of brain functional activity depends on local accumulation of exogenous or endogenous signals, and DG-6-P is trapped in the cell where it is phosphorylated, whereas rapid dispersal of cytoplasmic NAD(P)H and labeled glucose metabolites throughout the astrocytic syncytium can interfere with cellular assessment of neuron-astrocyte relationships in autoradiographic, fluorescence microscopic, and magnetic resonance spectroscopic studies.

Exchange-mediated dilution of brain lactate specific activity: implications for the origin of glutamate dilution and the contributions of glutamine dilution and other pathways.

The magnitude of metabolic activation is greatly underestimated in autoradiographic studies using [1- or 6-14C]glucose compared to parallel assays with [14C]deoxyglucose indicating that most of the label corresponding to the additional [14C]glucose consumed during activation compared to rest is quickly released from activated structures. Label could be lost by net release of [14C]lactate from brain or via lactate exchange between blood and brain. These possibilities were distinguished by comparison of glucose and lactate specific activities in arterial blood and brain before, during, and after generalized sensory stimulation and during spreading cortical depression. Over a wide range of brain lactate concentrations, lactate specific activity was close to the theoretical maximum, i.e. half that of [6-14C]glucose, indicating that exchange-mediated dilution of lactate is negligible and that efflux of [14C]lactate probably accounts for most of the label loss. Low lactate dilution also indicates that dilution of glutamate C4 fractional enrichment in [13C]glucose studies, currently ascribed predominantly to lactate exchange, arises from other unidentified pathways or factors. Alternative explanations for glutamate dilution (presented in Supporting Information) include poorly labeled amino acid pools and oxidative metabolism of minor substrates in astrocytes to first dilute the astrocytic glutamine pool, followed by dilution of glutamate via glutamate-glutamine cycling.

Imaging brain activation: simple pictures of complex biology.

Elucidation of biochemical, physiological, and cellular contributions to metabolic images of brain is important for interpretation of images of brain activation and disease. Discordant brain images obtained with [(14)C]deoxyglucose and [1- or 6-(14)C]glucose were previously ascribed to increased glycolysis and rapid [(14)C]lactate release from tissue, but direct proof of [(14)C]lactate release from activated brain structures is lacking. Analysis of factors contributing to images of focal metabolic activity evoked by monotonic acoustic stimulation of conscious rats reveals that labeled metabolites of [1- or 6-(14)C]glucose are quickly released from activated cells as a result of decarboxylation reactions, spreading via gap junctions, and efflux via lactate transporters. Label release from activated tissue accounts for most of the additional [(14)C]glucose consumed during activation compared to rest. Metabolism of [3,4-(14)C]glucose generates about four times more [(14)C]lactate compared to (14)CO(2) in extracellular fluid, suggesting that most lactate is not locally oxidized. In brain slices, direct assays of lactate uptake from extracellular fluid demonstrate that astrocytes have faster influx and higher transport capacity than neurons. Also, lactate transfer from a single astrocyte to other gap junction-coupled astrocytes exceeds astrocyte-to-neuron lactate shuttling. Astrocytes and neurons have excess capacities for glycolysis, and oxidative metabolism in both cell types rises during sensory stimulation. The energetics of brain activation is quite complex, and the proportion of glucose consumed by astrocytes and neurons, lactate generation by either cell type, and the contributions of both cell types to brain images during brain activation are likely to vary with the stimulus paradigm and activated pathways.

Astrocyte activation in vivo during graded photic stimulation.

Astrocytes have important roles in control of extracellular environment, de novo synthesis of neurotransmitters, and regulation of neurotransmission and blood flow. All of these functions require energy, suggesting that astrocytic metabolism should rise and fall with changes in neuronal activity and that brain imaging can be used to visualize and quantify astrocytic activation in vivo. A unilateral photic stimulation paradigm was used to test the hypothesis that graded sensory stimuli cause progressive increases in the uptake coefficient of [2-(14)C]acetate, a substrate preferentially oxidized by astrocytes. The acetate uptake coefficient fell in deafferented visual structures and it rose in intact tissue during photic stimulation of conscious rats; the increase was highest in structures with monosynaptic input from the eye and was much smaller in magnitude than the change in glucose utilization (CMR(glc)) by all cells. The acetate uptake coefficient was not proportional to stimulus rate and did not correlate with CMR(glc) in resting or activated structures. Simulation studies support the conclusions that acetate uptake coefficients represent mainly metabolism and respond to changes in metabolism rate, with a lower response at high rates. A model portraying regulation of acetate oxidation illustrates complex relationships among functional activation, cation levels, and astrocytic metabolism.

Astrocytic connexin distributions and rapid, extensive dye transfer via gap junctions in the inferior colliculus: implications for [(14)C]glucose metabolite trafficking.

The inferior colliculus has the highest rates of blood flow and metabolism in brain, and functional metabolic activity increases markedly in response to acoustic stimulation. However, brain imaging with [1- and 6-(14)C]glucose greatly underestimates focal metabolic activation that is readily detected with [(14)C]deoxyglucose, suggesting that labeled glucose metabolites are quickly dispersed and released from highly activated zones of the inferior colliculus. To evaluate the role of coupling of astrocytes via gap junctions in dispersal of molecules within the inferior colliculus, the present study assessed the distribution of connexin (Cx) proteins in the inferior colliculus and spreading of Lucifer yellow from single microinjected astrocytes in slices of adult rat brain. Immunoreactive Cx43, Cx30, and Cx26 were heterogeneously distributed; the patterns for Cx43 and Cx 30 differed and were similar to those of immunoreactive GFAP and S100beta, respectively. Most Cx43 was phosphorylated in resting and acoustically stimulated rats. Dye spreading revealed an extensive syncytial network that included thousands of cells and perivasculature endfeet; with 8% Lucifer yellow VS and a 5-min diffusion duration, about 6,100 astrocytes (range 2,068-11,939) were labeled as far as 1-1.5 mm from the injected cell. The relative concentration of Lucifer yellow fell by 50% within 0.3-0.8 mm from the injected cell with a 5-min diffusion interval. Perivascular dye labeling was readily detectable and often exceeded dye levels in nearby neuropil. Thus, astrocytes have the capability to distribute intracellular molecules quickly from activated regions throughout the large, heterogeneous syncytial volume of the inferior colliculus, and rapid trafficking of labeled metabolites would degrade resolution of focal metabolic activation.

A glycogen phosphorylase inhibitor selectively enhances local rates of glucose utilization in brain during sensory stimulation of conscious rats: implications for glycogen turnover.

Glycogen is degraded during brain activation but its role and contribution to functional energetics in normal activated brain have not been established. In the present study, glycogen utilization in brain of normal conscious rats during sensory stimulation was assessed by three approaches, change in concentration, release of (14)C from pre-labeled glycogen and compensatory increase in utilization of blood glucose (CMR(glc)) evoked by treatment with a glycogen phosphorylase inhibitor. Glycogen level fell in cortex, (14)C release increased in three structures and inhibitor treatment caused regionally selective compensatory increases in CMR(glc) over and above the activation-induced rise in vehicle-treated rats. The compensatory rise in CMR(glc) was highest in sensory-parietal cortex where it corresponded to about half of the stimulus-induced rise in CMR(glcf) in vehicle-treated rats; this response did not correlate with metabolic rate, stimulus-induced rise in CMR(glc) or sequential station in sensory pathway. Thus, glycogen is an active fuel for specific structures in normal activated brain, not simply an emergency fuel depot and flux-generated pyruvate greatly exceeded net accumulation of lactate or net consumption of glycogen during activation. The metabolic fate of glycogen is unknown, but adding glycogen to the fuel consumed during activation would contribute to a fall in CMR(O2)/CMR(glc) ratio.

Functional imaging of focal brain activation in conscious rats: impact of [(14)C]glucose metabolite spreading and release.

Labeled glucose and its analogs are widely used in imaging and metabolic studies of brain function, astrocyte-neuron interactions, and neurotransmission. Metabolite shuttling among astrocytes and neurons is essential for cell-cell transfer of neurotransmitter precursors and supply and elimination of energy metabolites, but dispersion and release of labeled compounds from activated tissue would reduce signal registration in metabolic labeling studies, causing underestimation of focal functional activation. Processes and pathways involved in metabolite trafficking and release were therefore assessed in the auditory pathway of conscious rats. Unilateral monotonic stimulation increased glucose utilization (CMR(glc)) in tonotopic bands in the activated inferior colliculus by 35-85% compared with contralateral tissue when assayed with [(14)C]deoxyglucose (DG), whereas only 20-30% increases were registered with [1- or 6-(14)C]glucose. Tonotopic bands were not evident with [1-(14)C]glucose unless assayed during halothane anesthesia or pretreatment with probenecid but were detectable with [6-(14)C]glucose. Extracellular lactate levels transiently doubled during acoustic stimulation, so metabolite spreading was assessed by microinfusion of [(14)C]tracers into the inferior colliculus. The volume of tissue labeled by [1-(14)C]glucose exceeded that by [(14)C]DG by 3.2- and 1.4-fold during rest and acoustic activation, respectively. During activation, the tissue volume labeled by U-(14)C-labeled glutamine and lactate rose, whereas that by glucose fell 50% and that by DG was unchanged. Dispersion of [1-(14)C]glucose and its metabolites during rest was also reduced 50% by preinfusion of gap junction blockers. To summarize, during brain activation focal CMR(glc) is underestimated with labeled glucose because of decarboxylation reactions, spreading within tissue and via the astrocyte syncytium, and release from activated tissue. These findings help explain the fall in CMR(O2)/CMR(glc) during brain activation and suggest that lactate and other nonoxidized metabolites of glucose are quickly shuttled away from sites of functional activation.