Glucose is the primary, obligatory fuel for brain, and glucose metabolism-based assays have, therefore, been a cornerstone of non-invasive imaging and spectroscopic studies of brain function and disease for decades. In the late 1960s, metabolic studies using labeled tracers were initiated to identify brain fuel, define compartmentation of utilization of minor substrates by neurons and astrocytes, assess astrocyte-neuron metabolic interactions, and calculate glucose utilization rates. Enormous progress has been made in understanding function-metabolism relationships but the cellular contributions to brain energy metabolism and brain images are not yet clear. Under resting conditions, nearly all of the glucose is oxidized and the metabolic ratio of oxygen to glucose utilization is close to the theoretical maximum of 6 (i.e., 6O 2 + 1 glucose fi 6CO 2 + 6H 2 0), but during brain activation this metabolic ratio generally, but not always, falls even though oxygen delivery is adequate (reviewed by Cruz 2004, 2008). The basis for the preferential rise in non-oxidative metabolism of glucose during activation is not understood, and increased lactate production that exceeds its oxidation is inferred.The cellular origin and fate of lactate and the contribution of lactate to brain energetics in normal, activated brain are important, unresolved issues. In vivo experiments to define roles of endogenously-generated lactate are technically difficult, and the lack of consensus in the field is reflected by the diversity of the following current metabolic models. (i) Lactate generated by astrocytes during excitatory glutamatergic neurotransmission is hypothesized to be shuttled to neurons as a major fuel ( AbstractBrain 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...

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 glc 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. Keywords: brain activation, brain imaging, energetics, glucose utilization, glycogenolysis, sensory stimulation. The importance of quantifying metabolic fluxes induced by brain activation at a cellular level is emphasized by positron emission tomographic, magnetic resonance spectroscopic (MRS) and optical (infrared or fluorescence) studies of brain function in health and disease that rely on measurement of signals generated from endogenous or exogenous tracers metabolized by energy-producing pathways. Astrocytes are increasingly recognized as having essential roles in both signaling and energetics during brain activation, including modulation of neurotransmission via gliotransmitters, synthesis and cycling of amino acid neurotransmitters, regulation of extracellular glutamate and K + levels and blood flow

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.

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 ) caused perivascular labeling in the inferior colliculus, labeled the surrounding meninges, and Ab-labeledspecific blood vessels in the caudate and olfactory bulb and was deposited in cervical lymph nodes. Efflux of extracellular glucose, lactate, and Ab 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.

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.

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.

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.

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/L) or low (5.5 mmol/L) 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, and GAPDH in cerebral cortex, but actin level rose 24%. Cx43 was predominantly phosphorylated in control and diabetic samples, so reduced dye transfer is not due to overall dephosphorylation of Cx43. Astrocytic growth in high glucose reduced dye-labeled area by 75%, but 10 min 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 1h. Thus, modifications arising from oxidative-nitrosative stress, not altered connexin levels, may underlie 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 brain of STZ-diabetic rats.