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Over the last thirty years, a growing number of studies showed that astrocytes play a pivotal role in the energy support to synapses. More precisely, astrocytes adjust energy production to neuronal energy needs through different mechanisms grouped under the term "neurometabolic coupling" (NMC). In this review we describe these mechanisms of coupling and how they involve astrocytes. From a physiological point of view, these mechanisms of coupling are particularly important to ensure normal synaptic functioning when neurons undergo rapid and repetitive changes in the firing rate such as during the sleep/wake transitions. Investigations into brain energy metabolism during the sleep/wake cycle have been mainly focused on glucose (Gluc) consumption and on glycogen metabolism. However, the recent development of substrate-specific biosensors allowed measurements of the variation in extracellular levels of glutamate, Gluc and lactate (Lac) with a time resolution compatible with sleep stage duration. Together with gene expression data these experiments allowed to better define the variations of energy metabolite regulation across the sleep/wake cycle. The aim of this review is to bring into perspective the role of astrocytes and NMC in the regulation of the sleep/wake cycle. The data reviewed also suggest an important role of the astrocytic network. In addition, the role of astrocytes in NMC mechanisms is consistent with the "local and use dependent" sleep hypothesis., (Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.)

This study examines the role of glucose and lactate as energy substrates to sustain synaptic vesicle cycling. Synaptic vesicle turnover was assessed in a quantitative manner by fluorescence microscopy in primary cultures of mouse cortical neurons. An electrode-equipped perfusion chamber was used to stimulate cells both by electrical field and potassium depolarization during image acquisition. An image analysis procedure was elaborated to select in an unbiased manner synaptic boutons loaded with the fluorescent dye N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl)pyridinium dibromide (FM1-43). Whereas a minority of the sites fully released their dye content following electrical stimulation, others needed subsequent K(+) depolarization to achieve full release. This functional heterogeneity was not significantly altered by the nature of metabolic substrates. Repetitive stimulation sequences of FM1-43 uptake and release were then performed in the absence of any metabolic substrate and showed that the number of active sites dramatically decreased after the first cycle of loading/unloading. The presence of 1 mM glucose or lactate was sufficient to sustain synaptic vesicle cycling under these conditions. Moreover, both substrates were equivalent for recovery of function after a phase of decreased metabolic substrate availability. Thus, lactate appears to be equivalent to glucose for sustaining synaptic vesicle turnover in cultured cortical neurons during activity.

Aquaporin 9 (AQP9) is a recently cloned water channel that is permeable to monocarboxylate, glycerol and urea. In rat, AQP9 has been found in testis and liver as well as in brain where its expression has been initially shown in glial cells in forebrain. However, the expression of AQP9 has not been investigated in the brainstem. The purpose of this study is to describe the distribution of AQP9-immunoreactive cells throughout the adult rat brain using reverse transcriptase-polymerase chain reaction (RT-PCR), Western blot and immunohistochemistry. We performed immunolabeling on brain from animals perfused with fixative and we show that AQP9 is expressed (i) in astrocytes in the glia limitans, in the white matter and in glial cells of the cerebellum, (ii) in the endothelial cells of pial vessels, and (iii) in specific groups of neurons. The neuronal AQP9 expression was almost exclusively observed in catecholaminergic cells including the adrenergic, noradrenergic and dopaminergic groups, but not in other monoaminergic neurons such as serotonergic or histaminergic cells. A slight labeling was also observed in non-catecholaminergic neurons localized in the paraventricular nucleus of the hypothalamus. These results indicate that AQP9 has a unique brain distribution with a preferential localization in catecholaminergic nuclei known to be involved in many cerebral functions. While the presence of AQP9 in glia limitans and in endothelial cells of the pial vessels could be related to water transport through the blood-brain barrier, its expression in neuronal cells, not directly involved in the osmoregulation, suggests that brain AQP9 could also be used as a metabolite channel since lactate and glycerol can be energy substrates for neurons.

The subdivisions of human inferior colliculus are currently based on Golgi and Nissl-stained preparations. We have investigated the distribution of calcium-binding protein immunoreactivity in the human inferior colliculus and found complementary or mutually exclusive localisations of parvalbumin versus calbindin D-28k and calretinin staining. The central nucleus of the inferior colliculus but not the surrounding regions contained parvalbumin-positive neuronal somata and fibres. Calbindin-positive neurons and fibres were concentrated in the dorsal aspect of the central nucleus and in structures surrounding it: the dorsal cortex, the lateral lemniscus, the ventrolateral nucleus, and the intercollicular region. In the dorsal cortex, labelling of calbindin and calretinin revealed four distinct layers.Thus, calcium-binding protein reactivity reveals in the human inferior colliculus distinct neuronal populations that are anatomically segregated. The different calcium-binding protein-defined subdivisions may belong to parallel auditory pathways that were previously demonstrated in non-human primates, and they may constitute a first indication of parallel processing in human subcortical auditory structures.

Recent evidence suggests that lactate could be a preferential energy substrate transferred from astrocytes to neurons. This would imply the presence of specific transporters for lactate on both cell types. We have investigated the immunohistochemical localization of two monocarboxylate transporters, MCT1 and MCT2, in the adult mouse brain. Using specific antibodies raised against MCT1 and MCT2, we found strong immunoreactivity for each transporter in glia limitans, ependymocytes and several microvessel-like elements. In addition, small processes distributed throughout the cerebral parenchyma were immunolabeled for monocarboxylate transporters. Double immunofluorescent labeling and confocal microscopy examination of these small processes revealed no co-localization between glial fibrillary acidic protein and monocarboxylate transporters, although many glial fibrillary acidic protein-positive processes were often in close apposition to elements labeled for monocarboxylate transporters. In contrast, several elements expressing the S100beta protein, another astrocytic marker found to be located in distinct parts of the same cell when compared with glial fibrillary acidic protein, were also strongly immunoreactive for MCT1, suggesting expression of this transporter by astrocytes. In contrast, MCT2 was expressed in a small subset of microtubule-associated protein-2-positive elements, indicating a neuronal localization. In conclusion, these observations are consistent with the possibility that lactate, produced and released by astrocytes (via MCT1), could be taken up (via MCT2) and used by neurons as an energy substrate.

The role of lactate in brain energy metabolism has recently received renewed attention. Although blood-borne monocarboxylates such as lactate poorly cross the blood-brain barrier in the adult brain, lactate produced within the brain parenchyma may be a suitable substrate for brain cells. Lactate dehydrogenase is crucial for both the production and utilization of lactate. In this article, we report the regional distribution of the messenger RNAs for lactate dehydrogenase isoforms 1 and 5 in the adult rat brain using in situ hybridization histochemistry with specific [alpha-(35)S]dATP 3' end-labeled oligoprobes. The autoradiographs revealed that the lactate dehydrogenase-1 messenger RNA is highly expressed in a variety of brain structures, including the main olfactory bulb, the piriform cortex, several thalamic and hypothalamic nuclei, the pontine nuclei, the ventral cochlear nucleus, the trigeminal nerve and the solitary tractus nucleus. In addition, the granular and Purkinje cell layers of the cerebellum showed a strong labeling. The neocortex (e.g., cingular, retrosplenial and frontoparietal cortices) often exhibits a marked laminar pattern of distribution of lactate dehydrogenase-1 messenger RNA (layers II/III, IV and VI being most strongly labeled). In contrast, expression of the lactate dehydrogenase-5 messenger RNA generally seemed more diffusely distributed across the different brain regions. Expression was particularly strong in the hippocampal formation (especially in Ammon's horn and dentate gyrus) and in the cerebral cortex, where no laminar pattern of distribution was observed. Overall, these data are consistent with the emerging idea that lactate is an important energy substrate produced and consumed by brain cells.

The distribution of pro-somatostatin-derived-peptide-positive profiles was examined by indirect immunohistofluorescence in nine post-mortem human brains (age 58-73 years). Three specific antisera were used for this study which recognize, respectively, somatostatin-28, somatostatin-28 (1-12) and somatostatin (1-14). Pro-somatostatin-derived-peptide-positive immunoreactive profiles were observed throughout the neuraxis. Cell bodies were found within archeo-, paleo- and neocortical areas, the subcortical white matter, in the nucleus accumbens, caudate nucleus and putamen, as well as in the hypothalamus, the reticular thalamic nucleus and the reticular formation of the brainstem. Fibers and terminals were seen in the same areas as well as in various thalamic nuclei, in the brainstem and spinal cord. Pro-somatostatin-derived-peptide-positive fibre tracts include the bed nucleus of the stria terminalis, the diagonal band of Broca, the stria medullaris, the inter-thalamic adhesion, the posterior commissure and the spinothalamic tract. Furthermore, differences between human and animal brains were noted and some somatostatin systems reported which may be implicated in certain human neuropathological states.

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 distribution of vasoactive intestinal peptide binding sites in the rat brain was examined by in vitro autoradiography on slide-mounted sections. A fully characterized monoiodinated form of vasoactive intestinal peptide (M-[125I]VIP) previously shown to maintain in the central nervous system the full biological activity of native vasoactive intestinal peptide was used for this study. In initial kinetic and pharmacological experiments the binding of M-[125I]vasoactive intestinal peptide to slide-mounted sections was shown to be time-dependent, saturable and reversible. Association of M-[125I]VIP specific binding was maximal within 90-120 minutes. Specific binding, corresponding to approximately 50% of total binding was saturable, of high affinity (Kd of 76.6 pM) and low capacity (fmol/mg prot range). Dissociation of M-[125I]VIP was maximal at 10 minutes. Unlabeled vasoactive intestinal peptide and the two structurally related peptides "peptide-histidine-isoleucine" (PHI) and secretin competed in a concentration-dependent manner for sites labeled by M-[125I]vasoactive intestinal peptide with the following rank order of potencies: vasoactive intestinal peptide greater than PHI greater than secretin. Vasoactive intestinal peptide receptors, as revealed by quantitative autoradiography, are present at various levels of the neuraxis. High densities were observed in olfactory bulb, cerebral cortex (highest in layers I, II, IV and VI), dentate gyrus, subiculum, various thalamic and hypothalamic nuclei, superior colliculus, locus coeruleus, area postrema, subependymal layer and pineal gland. Intermediate densities were found in the amygdala, nucleus accumbens, caudate-putamen, septum, bed nucleus of the stria terminalis, CA1 to CA4 fields of the hippocampus and central gray. No specific binding of M-[125I]vasoactive intestinal peptide was observed in white matter tracts such as corpus callosum, anterior commissure, medial forebrain bundle and fornix. The mapping of M-[125I]vasoactive intestinal peptide binding sites as revealed by autoradiography on slide-mounted sections indicates an association, although not exclusive, of vasoactive intestinal peptide receptors with brain regions involved in the processing of specific sensory inputs.

VIP, glucagon or several other neuropeptides were applied to intact retinae in vitro. VIP was found to be a very potent stimulator of cyclic AMP production in rabbit retina, whereas glucagon was efficient in pigeon retina. Attempts to modify the VIP-induced formation of cyclic AMP by somatostatin or dopamine-related drugs (neuroleptics) were not successful. These biochemical data may be correlated to some extent with the recent immunohistochemical localisation of VIP and glucagon in mammalian or avian retinae.

The stimulation by nicotine of intramural nerves and the role of ATP and vasoactive intestinal peptide (VIP) as inhibitory transmitters were studied in the isolated taenia of the guinea-pig caecum. Nicotine (4-32 microM) caused transient, concentration-dependent relaxations which were unaffected by atropine, prazosin or sotalol. Drugs with membrane-stabilizing activity, such as dl-propranolol (0.5 microM), d-propranolol (0.5 microM) or lidocaine (10 microM) antagonized the nicotine-induced relaxation without modifying the response to electrical field stimulation. Similar results were obtained with the cyclooxygenase inhibitor, indomethacin (2.8 microM). Nucleotide pyrophosphatase (0.5 U/ml), which hydrolyzes ATP to AMP, reversibly inhibited the response to nicotine but the response to field stimulation was not decreased. Nicotine evoked a calcium-dependent release of VIP, which was blocked by tetrodotoxin (1 microM), d-propranolol (0.5 microM) or, as previously shown, by apamin (0.2 microM). The finding that nicotine-induced relaxation was accompanied by the neuronal release of VIP is compatible with the possibility that VIP is an inhibitory transmitter but is not definitive evidence, since it could have been due to the stimulation of distinct populations of nerves by nicotine.

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.

Three types of voltage-sensitive Ca2+-channels, denominated T, N and L, have recently been identified in the nervous system. This classification is based on both the electrophysiological and pharmacological properties of each type of channel. The increase in free intracellular Ca2+ concentration that results from the opening of voltage-sensitive Ca2+-channels triggers various cellular processes. One such process is the depolarization-induced release of neurotransmitters. Owing to the rather selective sensitivity of each type of voltage-sensitive Ca2+-channel to certain antagonists, attempts have recently been carried out to determine which of the T, N or L channels mediates neurotransmitter release. In the present study we have applied such a strategy to the study of the release of vasoactive intestinal peptide from mouse cerebral cortical slices. The release of vasoactive intestinal peptide evoked by K+ 20 mM is inhibited in a concentration-dependent manner by Co2+, Ni2+ and Mn2+ while it remains unaffected by diltiazem 20 microM, nifedipine 10 microM, omega-conotoxin 1 microM and Cd2+ up to 100 microM. Such a pharmacological profile indicates that voltage-sensitive Ca2+-channels of the N and L types are not involved in the release of vasoactive intestinal peptide. These observations are in contrast to what has been described for amine release in the central nervous system, which appears to be mediated by voltage-sensitive Ca2+-channels of the N type.(ABSTRACT TRUNCATED AT 250 WORDS)