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Excitatory synaptic transmission is accompanied by a local surge in interstitial lactate that occurs despite adequate oxygen availability, a puzzling phenomenon termed aerobic glycolysis. In addition to its role as an energy substrate, recent studies have shown that lactate modulates neuronal excitability acting through various targets, including NMDA receptors and G-protein-coupled receptors specific for lactate, but little is known about the cellular and molecular mechanisms responsible for the increase in interstitial lactate. Using a panel of genetically encoded fluorescence nanosensors for energy metabolites, we show here that mouse astrocytes in culture, in cortical slices, and in vivo maintain a steady-state reservoir of lactate. The reservoir was released to the extracellular space immediately after exposure of astrocytes to a physiological rise in extracellular K(+) or cell depolarization. Cell-attached patch-clamp analysis of cultured astrocytes revealed a 37 pS lactate-permeable ion channel activated by cell depolarization. The channel was modulated by lactate itself, resulting in a positive feedback loop for lactate release. A rapid fall in intracellular lactate levels was also observed in cortical astrocytes of anesthetized mice in response to local field stimulation. The existence of an astrocytic lactate reservoir and its quick mobilization via an ion channel in response to a neuronal cue provides fresh support to lactate roles in neuronal fueling and in gliotransmission., (Copyright © 2015 the authors 0270-6474/15/354168-11$15.00/0.)

The glyoxalase system is the most important pathway for the detoxification of methylglyoxal (MG), a highly reactive dicarbonyl compound mainly formed as a by-product of glycolysis. MG is a major precursor of advanced glycation end products (AGEs), which are associated with several neurodegenerative disorders. Although the neurotoxic effects of MG and AGEs are well characterized, little is known about the glyoxalase system in the brain, in particular with regards to its activity in different neural cell types. Results of the present study reveal that both enzymes composing the glyoxalase system [glyoxalase-1 (Glo-1) and Glo-2] were highly expressed in primary mouse astrocytes compared with neurons, which translated into higher enzymatic activity rates in astrocytes (9.9- and 2.5-fold, respectively). The presence of a highly efficient glyoxalase system in astrocytes was associated with lower accumulation of AGEs compared with neurons (as assessed by Western blotting), a sixfold greater resistance to MG toxicity, and the capacity to protect neurons against MG in a coculture system. In addition, Glo-1 downregulation using RNA interference strategies resulted in a loss of viability in neurons, but not in astrocytes. Finally, stimulation of neuronal glycolysis via lentiviral-mediated overexpression of 6-phosphofructose-2-kinase/fructose-2,6-bisphosphatase-3 resulted in increased MG levels and MG-modified proteins. Since MG is largely produced through glycolysis, this suggests that the poor capacity of neurons to upregulate their glycolytic flux as compared with astrocytes may be related to weaker defense mechanisms against MG toxicity. Accordingly, the neuroenergetic specialization taking place between these two cell types may serve as a protective mechanism against MG-induced neurotoxicity.

Digital holographic microscopy (DHM) is a noninvasive optical imaging technique that provides quantitative phase images of living cells. In a recent study, we showed that the quantitative monitoring of the phase signal by DHM was a simple label-free method to study the effects of glutamate on neuronal optical responses (Pavillon et al., 2010). Here, we refine these observations and show that glutamate produces the following three distinct optical responses in mouse primary cortical neurons in culture, predominantly mediated by NMDA receptors: biphasic, reversible decrease (RD) and irreversible decrease (ID) responses. The shape and amplitude of the optical signal were not associated with a particular cellular phenotype but reflected the physiopathological status of neurons linked to the degree of NMDA activity. Thus, the biphasic, RD, and ID responses indicated, respectively, a low-level, a high-level, and an "excitotoxic" level of NMDA activation. Moreover, furosemide and bumetanide, two inhibitors of sodium-coupled and/or potassium-coupled chloride movement strongly modified the phase shift, suggesting an involvement of two neuronal cotransporters, NKCC1 (Na-K-Cl) and KCC2 (K-Cl) in the genesis of the optical signal. This observation is of particular interest since it shows that DHM is the first imaging technique able to monitor dynamically and in situ the activity of these cotransporters during physiological and/or pathological neuronal conditions.

Cerebral energy metabolism is a highly compartmentalized and complex process in which transcellular trafficking of metabolites plays a pivotal role. Over the past decade, a role for lactate in fueling the energetic requirements of neurons has emerged. Furthermore, a neuroprotective effect of lactate during hypoglycemia or cerebral ischemia has been reported. The majority of the current evidence concerning lactate metabolism at the cellular level is based on in vitro data; only a few recent in vivo results have demonstrated that the brain preferentially utilizes lactate over glucose. Using voltage-sensitive dye (VSD) imaging, beta-probe measurements of radiotracer kinetics, and brain activation by sensory stimulation in the anesthetized rat, we investigated several aspects of cerebral lactate metabolism. The present study is the first in vivo demonstration of the maintenance of neuronal activity in the presence of lactate as the primary energy source. The loss of the voltage-sensitive dye signal found during severe insulin-induced hypoglycemia is completely prevented by lactate infusion. Thus, lactate has a direct neuroprotective effect. Furthermore, we demonstrate that the brain readily oxidizes lactate in an activity-dependent manner. The washout of 1-[(11)C]L-lactate, reflecting cerebral lactate oxidation, was observed to increase during brain activation from 0.077 ± 0.009 to 0.105 ± 0.007 min(-1). Finally, our data confirm that the brain prefers lactate over glucose as an energy substrate when both substrates are available. Using [(18)F]fluorodeoxyglucose (FDG) to measure the local cerebral metabolic rate of glucose, we demonstrated a lactate concentration-dependent reduction of cerebral glucose utilization during experimentally increased plasma lactate levels.

During synaptic activity, the clearance of neuronally released glutamate leads to an intracellular sodium concentration increase in astrocytes that is associated with significant metabolic cost. The proximity of mitochondria at glutamate uptake sites in astrocytes raises the question of the ability of mitochondria to respond to these energy demands. We used dynamic fluorescence imaging to investigate the impact of glutamatergic transmission on mitochondria in intact astrocytes. Neuronal release of glutamate induced an intracellular acidification in astrocytes, via glutamate transporters, that spread over the mitochondrial matrix. The glutamate-induced mitochondrial matrix acidification exceeded cytosolic acidification and abrogated cytosol-to-mitochondrial matrix pH gradient. By decoupling glutamate uptake from cellular acidification, we found that glutamate induced a pH-mediated decrease in mitochondrial metabolism that surpasses the Ca(2+)-mediated stimulatory effects. These findings suggest a model in which excitatory neurotransmission dynamically regulates astrocyte energy metabolism by limiting the contribution of mitochondria to the metabolic response, thereby increasing the local oxygen availability and preventing excessive mitochondrial reactive oxygen species production.

Amyloid-beta (Abeta) peptides play a key role in the pathogenesis of Alzheimer's disease and exert various toxic effects on neurons; however, relatively little is known about their influence on glial cells. Astrocytes play a pivotal role in brain homeostasis, contributing to the regulation of local energy metabolism and oxidative stress defense, two aspects of importance for neuronal viability and function. In the present study, we explored the effects of Abeta peptides on glucose metabolism in cultured astrocytes. Following Abeta(25-35) exposure, we observed an increase in glucose uptake and its various metabolic fates, i.e., glycolysis (coupled to lactate release), tricarboxylic acid cycle, pentose phosphate pathway, and incorporation into glycogen. Abeta increased hydrogen peroxide production as well as glutathione release into the extracellular space without affecting intracellular glutathione content. A causal link between the effects of Abeta on glucose metabolism and its aggregation and internalization into astrocytes through binding to members of the class A scavenger receptor family could be demonstrated. Using astrocyte-neuron cocultures, we observed that the overall modifications of astrocyte metabolism induced by Abeta impair neuronal viability. The effects of the Abeta(25-35) fragment were reproduced by Abeta(1-42) but not by Abeta(1-40). Finally, the phosphoinositide 3-kinase (PI3-kinase) pathway appears to be crucial in these events since both the changes in glucose utilization and the decrease in neuronal viability are prevented by LY294002, a PI3-kinase inhibitor. This set of observations indicates that Abeta aggregation and internalization into astrocytes profoundly alter their metabolic phenotype with deleterious consequences for neuronal viability.

We have described previously a transcription-dependent induction of glycogen resynthesis by the vasoactive intestinal peptide (VIP) or noradrenaline (NA) in astrocytes, which is mediated by cAMP. Because it has been postulated that the cAMP-mediated regulation of energy balance in hepatocytes and adipocytes is channeled at least in part through the CCAAT/enhancer binding protein (C/EBP) family of transcription factors, we tested the hypothesis that C/EBP isoforms could be expressed in mouse cortical astrocytes and that their level of expression could be regulated by VIP, by the VIP-related neuropeptide pituitary adenylate cyclase-activating peptide (PACAP), or by NA. We report in this study that in these cells, C/EBP beta and C/EBP delta are induced by VIP, PACAP, or NA via the cAMP second-messenger pathway. Induction of C/EBP beta and -delta mRNA by VIP occurs in the presence of a protein synthesis inhibitor. Thus, c/ebp beta and c/ebp delta behave as cAMP-inducible immediate-early genes in astrocytes. Moreover, transfection of astrocytes with expression vectors selectively producing the transcriptionally active form of C/EBP beta, termed liver-enriched transcriptional activator protein, or C/EBP delta enhance the glycogen resynthesis elicited by NA, whereas an expression vector producing the transcriptionally inactive form of C/EBP beta, termed liver-enriched transcriptional inhibitory protein, reduces this resynthesis. These results support the idea that C/EBP beta and -delta regulate gene expression of energy metabolism-related enzymes in astrocytes.

Excitatory synaptic transmission is associated with changes in both extracellular and intracellular pH. Using mouse cortical neurons in primary cultures, we studied the sensitivity of glutamate-evoked release of 3H-arachidonic acid (3H-AA) to changes in extracellular pH (pHo) and related intracellular pH (pHi). As pHo was shifted from 7.2 to 7.8, the glutamate-evoked release of 3H-AA was enhanced by approximately threefold. The effect of alkaline pHo on the glutamate response was rapid, becoming significant within 2 min. 3H-AA release, evoked by both NMDA and kainate, was also enhanced by pHo alkalinization. NMDA- and kainate-induced increase in free intracellular Ca2+ was unaffected by changing pHo from 7.2 to 7.8, indicating that the receptor-induced Ca2+ influx is not responsible for the pHo sensitivity of the glutamate-evoked release of 3H-AA. Alkalinization of pHi obtained by incubating neurons in the presence of HCO3- or NH4 enhanced the glutamate-evoked release of 3H-AA, while pHi acidification obtained by blockade of Na+/H+ and Cl-/HCO3- exchangers decreased the glutamate response. Membrane-bound phospholipase A2 (mPLA2) activity was stimulated by Ca2+ in a pH-dependent manner, increasing its activity as pH was shifted from 7.2 to 7.8. This pH profile corresponds to the pH profile of the glutamate-, NMDA- and kainate-evoked release of 3H-AA. Taken together, these results indicate that the glutamate-evoked release of 3H-AA may be mediated by the pH-sensitive mPLA2. Since excitatory neurotransmission mediated by glutamate results in both pHo and pHi changes and since AA enhances glutamatergic neurotransmission at both pre- and postsynaptic levels, the data reported here reveals a possible molecular mechanism whereby glutamate can modulate its own signalling efficacy in a pH-dependent manner by regulating the release of AA.

Vasoactive intestinal peptide (VIP) and noradrenaline (NA) have been previously shown to promote glycogenolysis in mouse cerebral cortex (Magistretti, 1990). This action, which is fully expressed within a few minutes, is exerted on astrocytes (Sorg and Magistretti, 1991). In the present article, we report a second, temporally delayed, action of VIP or NA in primary cultures of mouse cerebral cortical astrocytes; thus, following glycogenolysis, an induction of glycogen resynthesis is observed, resulting, within 9 hr, in glycogen levels that are 6-10 times higher than those measured before the application of either neurotransmitter. This effect of VIP or NA is concentration dependent and, for NA, is mediated by adrenergic receptors of the beta subtype. The continued presence of the neurotransmitter is not necessary for this long-term effect, since pulses as short as 1 min result in the doubling of glycogen levels 9 hr later. The induction of glycogen resynthesis triggered by VIP or NA is dependent on protein synthesis, since both cycloheximide and actinomycin D abolish it entirely. The ability to elicit glycogenolysis is not sufficient per se to trigger the induction of glycogen resynthesis. Thus, two glycogenolytic agents such as methoxamine, an alpha 1-adrenergic agonist, and phorbol 12,13-dibutyrate, both acting via protein kinase C activation, are unable to induce glycogen resynthesis. This observation, taken together with the fact that dibutyryl-cAMP application also results in enhanced glycogen resynthesis, strongly suggests that the long-term effect of VIP or NA is mediated by the cAMP second-messenger pathway.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine promotes a concentration-dependent hydrolysis of 3H-glycogen newly synthesized from 3H-glucose by mouse cerebral cortical slices. The EC50 for this effect is 7 microM. Theophylline antagonizes the glycogenolysis induced by adenosine with an EC50 of 80 microM. The rank-order of potencies of adenosine agonists is adenosine 5'-cyclopropyl-carboxamide greater than 2-chloroadenosine much greater than N6-cyclohexyladenosine = adenosine, suggesting that adenosine promotes glycogenolysis via receptors of the A2 type. This contention is substantiated by the weak stereospecificity observed for the glycogenolytic action of D- and L-(phenylisopropyl)-adenosine. The glycogenolysis elicited by adenosine at 10 and 100 microM is inhibited by ouabain at 10 microM, a concentration of the cardiac glycoside not significantly affecting 3H-glycogen levels per se. Interestingly, the previously demonstrated glycogenolytic action of vasoactive intestinal peptide (Magistretti et al., 1981, 1984) and of norepinephrine (Quach et al., 1978) is also antagonized by ouabain. These results demonstrate the existence of a metabolic action of adenosine, which is sensitive to ouabain and appears to be mediated by A2 receptors. The concentrations at which adenosine promotes glycogenolysis are of the same order of magnitude as those reached extracellularly by the nucleoside during neuronal depolarization (Pull and McIlwain, 1972). This set of observations therefore supports the notion that adenosine plays a modulatory role in the coupling between neuronal activity and energy metabolism in the CNS.

The effect of increasing [K+]0 on 3H-glycogen levels was examined in mouse cerebral cortical slices. K+ stimulates in a time- and concentration-dependent manner the hydrolysis of 3H-glycogen. Over 70% of the maximal effect is reached within 30 sec and the EC50 for the glycogenolytic action of K+ is 11 mM. Significant 3H-glycogen hydrolysis occurs at 5-12 mM [K+]0, concentrations reached by the ion in the extracellular space during neuronal activity. The K+-evoked glycogenolysis is Ca2+-dependent, and is inhibited by Ca2+-channel blockers such as Ni2+ and Mn2+, but not by Cd2+, nifedipine, and omega-conotoxin. Furthermore, the effect of K+ is not enhanced by the Ca2+-channel agonist Bay K 8644. This type of pharmacological profile suggests that the activation of voltage-sensitive Ca2+ channels of the T subtype mediates the glycogenolytic action of K+. This set of observations suggests that K+ released in the extracellular space by active neurons may promote the mobilization of energy substrates and therefore play a role in the coupling between neuronal activity and energy metabolism.

We have examined the interactions, in eliciting cAMP accumulation, between vasoactive intestinal polypeptide (VIP) and the three monoamines norepinephrine (NE), histamine (HIS), and serotonin (5-hydroxytryptamine, 5-HT) in mouse cerebral cortical slices. We have observed that NE and HIS, but not 5-HT, act synergistically with VIP to increase cAMP levels. The rank-order of potency of several adrenergic agonists in potentiating the effect of VIP on cAMP levels is the following: epinephrine greater than NE greater than phenylephrine much greater than clonidine, with EC50 of 2.2, 5, and 10 microM, respectively (clonidine being only marginally effective). This pharmacological profile is characteristic of the activation of alpha 1-adrenergic receptors. This contention is substantiated by the observation that the potentiating effect of NE is antagonized by the selective alpha 1-adrenergic antagonist prazosin at nanomolar concentrations. The potentiating effect of HIS is mediated by H1-histaminergic receptors since it is antagonized by the selective H1-receptor antagonist mepyramine but not by cimetidine, a selective H2-receptor antagonist. The synergistic interaction between VIP and NE is also observed in the presence of the adenosine antagonist theophylline, thus discarding the possibility of a mediation of the synergism by adenosine released by VIP.(ABSTRACT TRUNCATED AT 250 WORDS)

In rodent cerebral cortex, vasoactive intestinal peptide (VIP) is contained in a homogeneous population of radially oriented bipolar interneurons. We have observed that 4-aminopyridine (4-AP), a K+-channel blocker, promotes a concentration- and Ca2+-dependent release of VIP from mouse cerebral cortical slices, with a significant effect already observed at 50 microM. Over 70% of VIP release elicited by 4-AP is blocked by 2 microM tetrodotoxin (TTX). Mepacrine, an inhibitor of phospholipase A2 (PLA2) activity and hence of arachidonic acid (AA) formation from membrane phospholipids, markedly inhibits (IC50 of approximately 15 microM) the release of VIP evoked by 4-AP. The inhibitory effect of mepacrine is not additive to that of TTX, thus indicating an involvement of PLA2 activation in the TTX-sensitive component of the 4-AP-evoked release. As a corollary, melittin (0.1-10 micrograms/ml), a PLA2 activator, promotes VIP release. Inhibition of AA metabolites of the lipoxygenase pathway by nordihydroguaiaretic acid, 5,8,11,14-eicosatetranoic acid, and caffeic acid results in a concentration-dependent inhibition of VIP release evoked by 4-AP. This set of observations indicates for the first time that the formation of AA metabolites of the lipoxygenase pathway plays a role in the release of a peptide in the mammalian CNS. Furthermore, these observations together with the previously reported potentiation by prostaglandins of the increase in cyclic AMP elicited by VIP in mouse cerebral cortex (Schaad et al., 1987) indicate that AA metabolites may act at both the presynaptic (lipoxygenase metabolites) and the postsynaptic (cyclooxygenase metabolites) levels to increase the "throughput" or "strength" of VIP-containing circuits in the rodent neocortex.