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The mechanism of rapid energy supply to the brain, especially to accommodate the heightened metabolic activity of excited states, is not well-understood. We explored the role of glycogen as a fuel source for neuromodulation using the noradrenergic stimulation of glia in a computational model of the neural-glial-vasculature ensemble (NGV). The detection of norepinephrine (NE) by the astrocyte and the coupled cAMP signal are rapid and largely insensitive to the distance of the locus coeruleus projection release sites from the glia, implying a diminished impact for volume transmission in high affinity receptor transduction systems. Glucosyl-conjugated units liberated from glial glycogen by NE-elicited cAMP second messenger transduction winds sequentially through the glycolytic cascade, generating robust increases in NADH and ATP before pyruvate is finally transformed into lactate. This astrocytic lactate is rapidly exported by monocarboxylate transporters to the associated neuron, demonstrating that the astrocyte-to-neuron lactate shuttle activated by glycogenolysis is a likely fuel source for neuromodulation and enhanced neural activity. Altogether, the energy supply for both astrocytes and neurons can be supplied rapidly by glycogenolysis upon neuromodulatory stimulus., Competing Interests: The authors have declared that no competing interests exist.

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.

Brain glycogen levels are dynamically regulated by certain neurotransmitters, including noradrenaline (NA) and vasoactive intestinal peptide (VIP). In particular, glycogen synthesis involves activation by NA and VIP of the transcription factors C/EBPbeta and -delta as well as the induction of glycogen synthase. Glycogen accumulation is found in a variety of neuropathological conditions, including reactive astrocytosis after CNS lesions, as well as in Alzheimer's disease. Protein targeting to glycogen (PTG) belongs to a family of proteins that play a key role in glycogen synthesis in peripheral tissues. In this study, we report the presence of PTG mRNA in adult mouse brain, as well as in astrocytes, a non-neuronal cell type that contains most of brain glycogen. Using primary cultures of mouse cortical astrocytes, we observed that NA leads to time- and concentration-dependent induction of PTG mRNA expression. This effect, concomitant to an enhancement of glycogen synthesis in these cells, depends on the activation of beta(1)-adrenergic receptors. Induction of PTG mRNA expression was mimicked by the adenylate cyclase activator forskolin and by dibutyryl cAMP, suggesting the involvement of the cAMP-dependent signal transduction cascade. Among other neuroactive substances known to elevate glycogen levels in astrocytes, VIP had a comparable effect to that of noradrenaline, whereas insulin and glutamate were without effect on PTG mRNA expression. These data suggest that increased PTG expression by neurotransmitters such as noradrenaline and VIP could represent a major event leading to enhancement of glycogen levels in astrocytes., (Copyright 2000 Wiley-Liss, Inc.)

The entorhinal cortex and the amygdala are interconnected structures of the limbic system in which paroxysmal activity occurs during temporal lobe epilepsy. Conflicting evidence shows that noradrenaline (i) inhibits the spreading to other parts of the limbic system of paroxysmal activity generated in the amygdala or the entorhinal cortex, but also (ii) increases glutamatergic transmission in the basolateral amygdala. Given our previous work on the inhibitory effect of noradrenaline on entorhinal cortex neurons, we developed an in vitro slice preparation to study the synaptic transmission in the basolateral amygdala and its modulation by noradrenaline. Noradrenaline reduced the fast excitatory postsynaptic potential (EPSP) by approximately 40% at 100 microM and the slow EPSP by approximately 50% at 50 microM. A similar effect was obtained with the alpha2-agonist UK 14304 at 100 and 50 microM respectively. In contrast, the beta-agonist isoproterenol increased the fast EPSP by approximately 40% at 100 microM and the slow EPSP by approximately 20% at 50 microM. Accordingly, the effect of noradrenaline on the EPSPs was blocked by the alpha2-antagonist yohimbine (10 microM) but not by the alpha1-antagonist prazosine (10 microM) and the beta-antagonist propranolol (10 microM). Noradrenaline (50-100 microM) was ineffective on most (14/16) of the isolated inhibitory postsynaptic potentials (IPSPs). These experiments provide evidence that noradrenaline inhibits the excitatory synaptic response of basolateral amygdala neurons. A pharmacological analysis revealed that the noradrenergic modulation of the excitatory transmission in the basolateral amygdala can be dissected into a predominant alpha2-adrenoreceptor-mediated inhibition and a beta-adrenoreceptor-mediated excitation.

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.

The entorhinal cortex is a gateway to the hippocampus; it receives inputs from several cortical associative areas as well as subcortical areas. Since there is evidence showing that noradrenaline reduces the epileptic activity generated in the entorhinal cortex, we have examined the action of noradrenaline in the superficial layer of the entorhinal cortex, which is the main source of afferents to the hippocampus. In a previous study we showed that noradrenaline hyperpolarized layer II entorhinal cortex neurons and reduced global synaptic transmission via alpha 2-adrenoreceptors. Here we present a detailed analysis of the effect of noradrenaline on membrane resistance and on the pharmacologically isolated postsynaptic potentials in layer II entorhinal cortex neurons of mice. Noradrenaline (50 microM) hyperpolarized most layer II entorhinal cortex neurons. This hyperpolarization corresponded to an outward current with a reversal potential following the Nernst equilibrium potential for potassium. The hyperpolarizing effect of noradrenaline was blocked by 10 microM yohimbine. These observations suggest that noradrenaline activates a potassium conductance via an alpha 2-adrenoreceptor. Noradrenaline (10-50 microM) reversibly reduced the amplitude of the pharmacologically isolated excitatory potentials mediated by both NMDA and alpha-amino-3-hydroxy-5-methyl-isoxazole-propionic acid (AMPA) receptors, the former being more strongly affected. Again this effect was blocked by 10 microM yohimbine. In contrast, GABAA-mediated synaptic transmission was virtually unaffected by noradrenaline. Thus, noradrenaline appears to strongly inhibit the glutamate-mediated synaptic transmission in the entorhinal cortex without affecting inhibitory post-synaptic potentials. These observations suggest that alpha 2-adrenergic receptor agonists may exert a beneficial effect in the control of hyperexcitability in temporal lobe epilepsy.

The effects of noradrenaline (NA) on synaptic responses in layer II of the entorhinal cortex (EC) were studied in normal and spontaneously epileptic mutant mice tottering using intracellular recording in a slice preparation. Neither the membrane properties of neurones nor the responses to NA differed between normal and tottering mice. NA (50 microM) hyperpolarized most (29/54) of the neurones via alpha 2 adrenergic receptors. Synaptic responses of EC neurones were complex. NA (10-100 microM) reduced all the components of the synaptic response in a concentration-dependent and reversible manner. The pharmacological properties of the inhibitory effect of NA were characterised and quantified on one component of the complex synaptic response, the fast excitatory postsynaptic potential. The effect of NA was mimicked by the alpha 2 agonist UK 14,304 and blocked by the alpha 2 antagonist yohimbine. It is concluded that NA can inhibit via an alpha 2 receptor-mediated action synaptic responses in the superficial layers of the EC.

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)

In recent years evidence has accumulated indicating the presence of functional receptors for most neurotransmitters on astrocytes. In particular, receptors coupled to adenylate cyclase have been demonstrated, in primary astrocyte cultures, for vasoactive intestinal peptide (VIP), noradrenaline (NA) and adenosine. Here we provide, in primary cultures of cerebral cortical astrocytes prepared from neonatal mice, a detailed characterization of a cAMP-dependent process elicited by VIP, NA and adenosine, i.e. the hydrolysis of glycogen. The EC50s for the glycogenolytic effect of VIP, NA and adenosine are 3, 20 and 800 nM, respectively. The initial rate of glycogen hydrolysis is, in nmol/mg prot/min, 9.1 for VIP and 7.5 for NA. The effect of NA is predominantly mediated by beta-adrenoceptors, although an alpha 1-adrenergic component, acting most likely through protein kinase C activation, is also present. The action of VIP is mimicked by peptides sharing sequence homologies such as PHI and secretin. Glutamate, GABA, carbachol and the peptides NPY and somatostatin do not influence glycogen levels. The glycogen content of the cultures can be markedly increased by anabolic factors present in fetal calf serum, by high (e.g. 25 mM) glucose in the medium and by 48-h pretreatment of the cultures with dibutyryl cAMP. These results indicate that the glycogen content of astrocytes is under the dynamic control of various factors, including certain neurotransmitters. They also further stress the notion of a functional interaction between neurons and glial cells aimed at maintaining local energy metabolism homeostasis.

There is growing evidence that two, or possibly more, neurotransmitters can coexist within the same neurone. In particular, the presence of a peptide and a biogenic amine has been demonstrated in the same terminals of central and peripheral neurones. These findings have led to the hypothesis that neurotransmitters, coexisting within the same neurones, can interact at pre- or postsynaptic sites in a functionally coordinated manner. However, interactions between neurotransmitters contained in distinct neuronal systems terminating within the same region of the central nervous system (CNS) can be envisaged. We have examined this last possibility in the cerebral cortex, an area of the CNS where the two neurotransmitters vasoactive intestinal polypeptide and noradrenaline are contained in separate neuronal systems and where they both stimulate the formation of cyclic AMP. We report here that vasoactive intestinal polypeptide and noradrenaline act synergistically to stimulate the formation of cyclic AMP and that this synergistic interaction is antagonized by the specific alpha-adrenergic antagonist phentolamine.

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.

Among several effects, ethanol (EtOH) interferes with membrane fluidity and lipid-protein interactions. As proteins are influenced by surrounding lipids, the activity of membrane-bound enzymes such as adenylate cyclase (AC) could be modulated by EtOH, as shown in potentiating, at toxic concentrations, the stimulating effect of hormones or neurotransmitters. We have also found that EtOH potentiates in a dose-dependent manner (EC50 = 100 mM) the cAMP production elicited by vasoactive intestinal peptide (VIP), already noticeably at 70 mM, without affecting basal cAMP levels (up to 400 mM). Propanol produces a similar potentiation, whereas methanol was inactive. Butanol (200 mM) displays toxic effects. The potentiation induced by EtOH is similar for peptide- (VIP) or monoamine- (noradrenaline) stimulated cAMP formation, suggesting a primary action at a interaction between VIP and NA in stimulating cAMP formation.

The effects of norepinephrine (NE) on in vitro [3H]glycogenolysis were assessed in slices of cerebral cortex from mice whose cortical noradrenergic innervation had been severely reduced by intracisternal 6-hydroxydopamine (6-OHDA) injections. A supersensitive response to NE was observed, as demonstrated by a decrease in the EC50 of the catecholamine in the lesioned mice from 533 +/- 88 nM to 39.3 +/- 7.9 nM. This supersensitive response, observed two weeks after the lesion, was post-synaptic since isoproterenol, a beta-adrenergic agonist not accumulated by pre-synaptic uptake mechanisms, also gave an equally supersensitive response.

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)

The glycogenolytic action of norepinephrine (NE) was examined in the tottering mouse, a spontaneously epileptic mutant which presents a noradrenergic hyperinnervation of various CNS areas, including the cerebral cortex. The potency and efficacy of NE in promoting glycogenolysis were markedly decreased in cerebral cortical slices prepared from homozygous tottering (tg/tg) when compared to control C57BL/6j (+/+) mice, indicating a sub-sensitive response to a cellular action of NE. The metabolic nature of this adaptive change suggests that an impaired capacity of NE in mobilizing energy substrates may be related to the expression of the epileptic symptomatology in this mutant.

We have previously shown that vasoactive intestinal peptide (VIP) and noradrenaline (NA) interact synergistically to increase cyclic AMP levels in mouse cerebral cortical slices. The pharmacological mechanism of this synergism is the potentiation by NA, through alpha 1 adrenergic receptors, of the stimulatory effect of VIP on cAMP formation. A similar interaction has been confirmed in guinea pig cerebral cortex and in discrete nuclei of the rat hypothalamus. Furthermore VIP and NA interact synergistically to depress the spontaneous activity of identified neurons in rat neocortex. At the cellular level, this synergistic interaction suggests that VIP- and NA-containing neuronal systems may converge, at least in part, on the same target cells to increase cAMP levels in the cerebral cortex. At the molecular level, the interaction may occur at various steps in signal transduction, between receptors, intramembrane transduction processes or intracellular effector mechanisms. Here we report that the alpha 1-adrenergic potentiation of the increases in cAMP elicited by VIP involves the formation of arachidonic acid metabolites and is mimicked by prostglandins F2 alpha and E2.

Neurotransmitter-specific anatomical techniques have provided a tool to define the morphological constraints within which a given neurotransmitter will exert its cellular actions. Biochemical and electrophysiological approaches have revealed the nature of these cellular actions for several neurotransmitters. Furthermore, by using purified preparations and tissue cultures a certain degree of resolution has been achieved by which the cell type, where a neurotransmitter's effect takes place, can be determined. In this article we review these aspects for noradrenaline and vasoactive intestinal peptide, two neurotransmitters of the cerebral cortex contained within neuronal systems that present strikingly different morphologies. Nevertheless, noradrenaline and vasoactive intestinal peptide share certain cellular actions and can interact synergistically. The experimental evidence accumulated to date indicates that noradrenaline- and vasoactive intestinal peptide-containing neurons can influence three general cell types of the cerebral cortex, i.e. (i) other neurons, (ii) astrocytes and (iii) cells of the vasculature. This diversity in cellular partners supports the notion that noradrenaline and vasoactive intestinal peptide can be released from neurons at conventional synapses as well as at extrasynaptic sites, thus suggesting the co-existence of two modes of release within the same neuron.