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We have previously reported that the pro-inflammatory cytokines tumor necrosis factor-α (TNFα) and interleukin-1β (IL-1β) induce profound modifications of the metabolic profile of astrocytes. The present study was undertaken to further characterize the effects of cytokines in astrocytes and to determine whether similar effects could also be observed in neurons. To do so, selected pro-inflammatory (IL-6 and interferon-γ, in addition to the above-mentioned TNFα and IL-1β) and anti-inflammatory cytokines (IL-4, IL-10, transforming growth factor-β1 and interferon-β) were applied to primary neuronal and astrocytic cultures, and key metabolic parameters were assessed. As a general pattern, we observed that pro-inflammatory cytokines increased glucose utilization in astrocytes while the anti-inflammatory cytokines IL-4 and IL-10 decreased astrocytic glucose utilization. In contrast, no significant change could be observed in neurons. When pairs of pro-inflammatory cytokines were co-applied in astrocytes, several additive or synergistic modifications could be observed. In contrast, IL-10 partially attenuated the effects of pro-inflammatory cytokines. Finally, the modifications of the astrocytic metabolism induced by TNFα + IL-1β and interferon-γ modulated neuronal susceptibility to an excitotoxic insult in neuron-astrocyte co-cultures. Together, these results suggest that pro- and anti-inflammatory cytokines differentially affect the metabolic profile of astrocytes, and that these changes have functional consequences for surrounding neurons., (© 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.)

Dopamine-induced changes in striatal gene expression are thought to play an important role in drug addiction and compulsive behaviour. In this study we report that dopamine induces the expression of the transcription factor CCAAT/Enhancer Binding Protein beta (C/EBP)-beta in primary cultures of striatal neurones. We identified the preprotachykinin-A (PPT-A) gene coding for substance P and neurokinin-A as a potential target gene of C/EBPbeta. We demonstrated that C/EBPbeta physically interacts with an element of the PPT-A promoter, thereby facilitating substance P precursor gene transcription. The regulation of PPT-A gene by C/EBPbeta could subserve many important physiological processes involving substance P, such as nociception, neurogenic inflammation and addiction. Given that substance P is known to increase dopamine signalling in the striatum and, in turn, dopamine increases substance P expression in medium spiny neurones, our results implicate C/EBPbeta in a positive feedback loop, changes of which might contribute to the development of drug addiction.

Glutamate was previously shown to enhance aerobic glycolysis i.e. increase glucose utilization and lactate production with no change in oxygen levels, in mouse cortical astrocytes by a mechanism involving glutamate uptake. It is reported here that a similar response is produced in both hippocampal and cerebellar astrocytes. Application of the cognitive-enhancing drug CX546 promoted further enhancement of glucose utilization by astrocytes from each brain area following glutamate exposure. alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors represent the purported molecular target of cognitive-enhancing drugs such as CX546, and the presence of AMPA receptor subunits GluR1-4 was evidenced in astrocytes from all three regions by immunocytochemistry. AMPA itself did not stimulate aerobic glycolysis, but in the presence of CX546, a strong enhancement of glucose utilization and lactate production was obtained in cortical, hippocampal and cerebellar astrocytes. The effect of CX546 was concentration-dependent, with an EC(50) of 93.2 microm in cortical astrocytes. AMPA-induced glucose utilization in the presence of CX546 was prevented by the AMPA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and the negative modulator GYKI 52466. In addition, the metabolic effect of CX546 in the presence of AMPA was mimicked by the AMPA receptor modulator cyclothiazide. Our data suggest that astrocyte energetics represents a novel target for cognitive-enhancing drugs acting as AMPA receptor modulators.

Glucocorticoids (GC) are considered as key modulators of glycogen homeostasis in peripheral tissues, but their role in the central nervous system has only partially been characterized. Exposure of primary cultures of cortical astrocytes to dexamethasone (DEX), a synthetic glucocorticoid, results in the reduction of noradrenaline (NA)-induced glycogen synthesis in a concentration-dependent manner with a IC50 of 4.88 nm and a maximum inhibition of 51%. Such an effect is mediated via glucocorticoid receptors (GRs), since it is mimicked by the glucocorticoid analogue RU28362 (100 nm) and prevented by the GR antagonist RU38486 (1 micro m). DEX does not act through alteration of signal transduction mechanisms, as cAMP formation induced by noradrenergic stimulation was unchanged. Moreover, glycogen synthesis was inhibited to the same extent when DEX was applied either together or only after a brief NA application. Neither [3H]2-deoxyglucose uptake nor lactate release was altered by DEX in the presence of NA, demonstrating that inhibition of glycogen synthesis is not a consequence of reduced glucose utilization or availability. Interestingly, enhancement of glycogen synthase activity induced by NA was reduced in the presence of DEX (-27%). These results suggest that GC could have a significant influence on neuroenergetics as they could modulate activity-related changes in brain glycogen metabolism.

Regulation of the expression of MCT1 and MCT2, two isoforms of the monocarboxylate transporter (MCT) family, was investigated in primary cultures of mouse cortical neurons. Under basal conditions, both MCT immunoreactivities (IR) were found in the cell soma and dendrites, although IR for MCT1 appeared less bright than for MCT2. Treatment of cultured cortical neurons with 100 microm noradrenaline (NA) led, after a few hours, to a striking enhancement in fluorescence intensity associated with MCT2 IR in the cell soma as well as in dendrites. In contrast, MCT1 IR was not altered by NA treatment. Western blot experiments performed on cultured neurons treated with NA confirmed that MCT2 protein expression was increased. Forskolin and dBcAMP also enhanced MCT2 expression, suggesting the implication of a cAMP-mediated pathway in the effect of NA. Surprisingly, neither NA, dBcAMP nor forskolin affected MCT2 mRNA expression. Application of cycloheximide, a protein synthesis inhibitor, prevented the enhancement of MCT2 IR, while the mRNA synthesis inhibitor actinomycin D also blocked the effect of NA on MCT2 IR levels. These results suggest that regulation of MCT2 expression in neurons by NA occurs at the translational level despite the requirement for an as yet unknown transcriptional step.

Regulation of calbindin and calretinin expression by brain-derived neurotrophic factor (BDNF) was examined in primary cultures of cortical neurons using immunocytochemistry and northern blot analysis. Here we report that regulation of calretinin expression by BDNF is in marked contrast to that of calbindin. Indeed, chronic exposure of cultured cortical neurons for 5 days to increasing concentrations of BDNF (0.1-10 ng/ml) resulted in a concentration-dependent decrease in the number of calretinin-positive neurons and a concentration-dependent increase in the number of calbindin-immunoreactive neurons. Consistent with the immunocytochemical analysis, BDNF reduced calretinin mRNA levels and up-regulated calbindin mRNA expression, providing evidence that modifications in gene expression accounted for the changes in the number of calretinin- and calbindin-containing neurons. Among other members of the neurotrophin family, neurotrophin-4 (NT-4), which also acts by activating tyrosine kinase TrkB receptors, exerted effects comparable to those of BDNF, whereas nerve growth factor (NGF) was ineffective. As for BDNF and NT-4, incubation of cortical neurons with neurotrophin-3 (NT-3) also led to a decrease in calretinin expression. However, in contrast to BDNF and NT-4, NT-3 did not affect calbindin expression. Double-labeling experiments evidenced that calretinin- and calbindin-containing neurons belong to distinct neuronal subpopulations, suggesting that BDNF and NT-4 exert opposite effects according to the neurochemical phenotype of the target cell.

The excitatory amino acid glutamate was previously shown to stimulate aerobic glycolysis in astrocytes by a mechanism involving its uptake through an Na+-dependent transporter. Evidence had been provided that Na+,K+-ATPase might be involved in this process. We have now measured the activity of Na+,K+-ATPase in cultured astrocytes, using ouabain-sensitive 86Rb uptake as an index. L-Glutamate increases glial Na+,K+-ATPase activity in a concentration-dependent manner with an EC50 = 67 microM. Both L- and D-aspartate, but not D-glutamate, produce a similar response, an observation that is consistent with an uptake-related effect rather than a receptor-mediated one. Under basal conditions, concentration-dependent inhibition of Na+,K+-ATPase activity in astrocytes by ouabain indicates the presence of a single catalytic site with a low affinity for ouabain (K0.5 = 113 microM), compatible with the presence of an alpha1 isozyme. On stimulation with glutamate, however, most of the increased activity is inhibited by low concentrations of ouabain (K0.5 = 20 nM), thus revealing a high-affinity site akin to the alpha2 isozyme. These results suggest that astrocytes possess a glutamate-sensitive isoform of Na+,K+-ATPase that can be mobilized in response to increased neuronal activity.

The sulfur-containing amino acid homocysteic acid (HCA) is present in and released from nervous tissue, exerting excitatory effects on neurons by predominantly activating NMDA receptors. It is interesting that HCA appears to be exclusively localized in glial cells, not in neurons. This profile of glial localization and excitatory action on neurons has led to the hypothesis that HCA could participate in intercellular communication in the brain as a "gliotransmitter." To test this hypothesis further, we searched for specific, receptor-mediated stimuli that could induce release of HCA from cultured astrocytes. For this reason we tested the effect of noradrenaline and vasoactive intestinal peptide, two transmitters known to interact with specific receptors on astrocytes, on the release of HCA from these cells. Noradrenaline and the beta-adrenergic agonist isoproterenol induced an efflux of HCA from astrocyte cultures. Further stressing the beta-adrenergic mediation of this effect is the blockade by atenolol of the HCA release evoked by isoproterenol. The stimulation of HCA release from astrocytes was not observed with the alpha-noradrenergic agonist methoxamine and with vasoactive intestinal peptide. These results taken together further strengthen the role of HCA as a gliotransmitter. Its efflux from glia could be controlled by noradrenaline, activating beta-adrenergic receptors on astrocytes. The present study provides the first evidence for an influence of beta-adrenergic receptor activation on the release of an excitatory amino acid from astrocytes and further supports the notion that glial-neuronal interactions play a role in synaptic transmission.

Previous reports have demonstrated that glutamate stimulates c-fos mRNA expression in primary cultures of mouse cerebral cortical neurons. We show here that vasoactive intestinal peptide (VIP) induces c-fos mRNA expression; however, this effect of VIP is completely inhibited by the noncompetitive NMDA receptor antagonist MK-801, therefore indicating that VIP stimulates c-fos expression in a glutamate-dependent manner. A similar effect was observed with pituitary adenylate cyclase-activating polypeptide27 (PACAP27). At the intracellular level, coactivation of protein kinases A and C mediates the glutamate-dependent stimulation of c-fos expression evoked by VIP, because either H-89 or staurosporin inhibits the effect of VIP as well as that of glutamate. These results point to a "biochemical AND gate" mechanism, which implies the obligatory activation of both protein kinases A and C in the transduction of c-fos expression. In summary, this article provides evidence that VIP and PACAP27 potentiate the effect of glutamate, the principal effector on c-fos expression, suggesting that both peptides can increase the "throughput" or "strength" of glutamate-containing circuits in the cerebral cortex.

In mouse cerebral cortical slices, noradrenaline (NA) potentiates cyclic AMP (cAMP) accumulation elicited by vasoactive intestinal peptide (VIP) through alpha 1-adrenergic receptors. This synergism is inhibited by indomethacin, and the prostaglandins E2 and F2 alpha mimic the effect of NA. In the present study, we observed that the synergism between VIP and NA is not inhibited by the protein kinase C inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) or the diacylglycerol-lipase inhibitor RHC 80267, thus further stressing the role of phospholipase A2 activation. Various neuroactive agents that potentiate the stimulatory effect of VIP on cAMP formation were also examined. As with NA, the potentiation by histamine and adenosine is inhibited by indomethacin. In contrast to NA, histamine, and adenosine, the synergistic interaction between phorbol esters and VIP on cAMP formation is abolished by H-7 but not by indomethacin. The potentiation by baclofen, a gamma-aminobutyric acidB receptor agonist, is partially inhibited by the 5-lipoxygenase inhibitor nafazatrom. The synergism between ouabain and VIP is reduced by H-7 but not by indomethacin and nafazatrom. These data indicate that the stimulation of cAMP formation elicited by VIP is under the modulation of various neuroactive agents that trigger diverse intracellular mechanisms to potentiate the effect of the peptide.

The modulation of 3,4-dihydroxyphenylethylamine (dopamine, DA) synthesis and release in rabbit retina in vitro by high K+; adenylate cyclase activators such as forskolin, 2-chloroadenosine, vasoactive intestinal polypeptide (VIP); and the putative DA autoreceptor agonist N-n-propyl-3-(3-hydroxyphenyl) piperidine (3-PPP) has been investigated. Incubation of retinas in 50 mM K+ resulted in the activation of tyrosine hydroxylase (TH). Activation did not require the presence of extracellular Ca2+. K+ 50 mM also induced a Ca2+-dependent release of DA. Forskolin 50 microM stimulated TH but 100 microM 2-chloroadenosine and 650 nM VIP did not. Individually, (+)-3-PPP, (-)-3-PPP, and (+/-)-3-PPP reduced DA synthesis and increased its release. The effects of (+/-)-3-PPP were dose-dependent and did not require the presence of extracellular Ca2+. The activation of TH induced by 50 mM K+, but not that induced by 50 microM forskolin, was abolished by 100 microM (+/-)-3-PPP.