Impaired CO₂ sensitivity of astrocytes in a mouse model of Rett syndrome

Rett syndrome, a prototypical neurological disorder caused by loss of function of the transcriptional regulator methyl-CpG-binding protein 2 (MeCP2) gene, is associated with a severely disordered breathing pattern and reduced ventilatory CO(2) sensitivity. In a mouse model of Rett syndrome (MeCP2 knockout), re-introduction of the MeCP2 gene selectively in astrocytes rescues normal respiratory phenotype. In the present study we determined whether the metabolic and/or signalling functions of astrocytes are affected by testing the hypotheses that in conditions of MeCP2 deficiency, medullary astrocytes are unable to produce/release appropriate amounts of lactate or detect changes in [Image: see text]/[H(+)], or both. No differences in tonic or hypoxia-induced release of lactate from the ventral surface of the medulla oblongata or cerebral cortex in brain slices of MeCP2-knockout and wild-type mice were found. In brainstem slices of wild-type mice, respiratory acidosis triggered robust elevations in [Ca(2+)](i) in astrocytes residing near the ventral surface of the medulla oblongata. The magnitude of CO(2)-induced [Ca(2+)](i) responses in medullary astrocytes was markedly reduced in conditions of MeCP2 deficiency, whereas [Ca(2+)](i) responses to ATP were unaffected. These data suggest that (i) metabolic function of astrocytes in releasing lactate into the extracellular space is not affected by MeCP2 deficiency, and (ii) MeCP2 deficiency impairs the ability of medullary astrocytes to sense changes in [Image: see text]/[H(+)]. Taken together with the evidence of severely blunted ventilatory sensitivity to CO(2) in mice with conditional MeCP2 deletion in astroglia, these data support the hypothesis of an important role played by astrocytes in central respiratory CO(2)/pH chemosensitivity. KEY POINTS: Rett syndrome is a prototypical neurological disorder characterised by abnormal breathing pattern and reduced ventilatory CO(2) sensitivity. Medullary astrocytes are a crucial component of central CO(2)/pH chemosensitivity. . This study tested the hypotheses that methyl-CpG-binding protein 2 (MeCP2) deficient medullary astrocytes are (i) unable to produce/release appropriate amounts of lactate, and/or (ii) unable to sense changes in [Image: see text]/[H(+)]. . We found no differences in tonic or hypoxia-induced release of lactate from the ventral surface of the medulla oblongata or cerebral cortex between MeCP2-knockout and wild-type mice. . Respiratory acidosis triggered robust [Ca(2+)](i) responses in wild-type astrocytes residing near the ventral surface of the medulla oblongata. CO(2)-induced [Ca(2+)](i) responses in astrocytes were dramatically reduced in conditions of MeCP2 deficiency. . These data suggest that (i) ‘metabolic’ function of astrocytes in releasing lactate into the extracellular space is not affected by MeCP2 deficiency, and (ii) MeCP2 deficiency impairs the ability of medullary astrocytes to sense changes in [Image: see text]/[H(+)].

Hemichannel-mediated release of lactate

Funding This study was supported by The Wellcome Trust. A.V.G is a Wellcome Trust Senior Research Fellow (ref. 095064). S.S. research is funded by Moray Endowment Grant and Wellcome Trust ISSF-2 Grant 217SSV.Peer reviewedPublisher PD

The effect of general anaesthetics on brain lactate release

The effects of anaesthetic agents on brain energy metabolism may explain their shared neurophysiological actions but remain poorly understood. The brain lactate shuttle hypothesis proposes that lactate, provided by astrocytes, is an important neuronal energy substrate. Here we tested the hypothesis that anaesthetic agents impair the brain lactate shuttle by interfering with astrocytic glycolysis. Lactate biosensors were used to record changes in lactate release by adult rat brainstem and cortical slices in response to thiopental, propofol and etomidate. Changes in cytosolic nicotinamide adenine dinucleotide reduced (NADH) and oxidized (NAD+) ratio as a measure of glycolytic rate were recorded in cultured astrocytes. It was found that in brainstem slices thiopental, propofol and etomidate reduced lactate release by 7.4 ± 3.6% (P < 0.001), 9.7 ± 6.6% (P < 0.001) and 8.0 ± 7.8% (P = 0.04), respectively. In cortical slices, thiopental reduced lactate release by 8.2 ± 5.6% (P = 0.002) and propofol by 6.0 ± 4.5% (P = 0.009). Lactate release in cortical slices measured during the light phase (period of sleep/low activity) was ~25% lower than that measured during the dark phase (period of wakefulness) (326 ± 83 μM vs 430 ± 118 μM, n = 10; P = 0.04). Thiopental and etomidate induced proportionally similar decreases in cytosolic [NADH]:[NAD+] ratio in astrocytes, indicative of a reduction in glycolytic rate. These data suggest that anaesthetic agents inhibit astrocytic glycolysis and reduce the level of extracellular lactate in the brain. Similar reductions in brain lactate release occur during natural state of sleep, suggesting that general anaesthesia may recapitulate some of the effects of sleep on brain energy metabolism

COX-2-Derived Prostaglandin E2 Produced by Pyramidal Neurons Contributes to Neurovascular Coupling in the Rodent Cerebral Cortex

International audienceVasodilatory prostaglandins play a key role in neurovascular coupling (NVC), the tight link between neuronal activity and local cerebral blood flow, but their precise identity, cellular origin and the receptors involved remain unclear. Here we show in rats that NMDA-induced vasodilation and hemodynamic responses evoked by whisker stimulation involve cyclooxygenase-2 (COX-2) activity and activation of the prostaglandin E2 (PgE(2)) receptors EP2 and EP4. Using liquid chromatography-electrospray ionization-tandem mass spectrometry, we demonstrate that PgE(2) is released by NMDA in cortical slices. The characterization of PgE2 producing cells by immunohistochemistry and single-cell reverse transcriptase-PCR revealed that pyramidal cells and not astrocytes are the main cell type equipped for PgE2 synthesis, one third expressing COX-2 systematically associated with a PgE2 synthase. Consistent with their central role in NVC, in vivo optogenetic stimulation of pyramidal cells evoked COX-2-dependent hyperemic responses in mice. These observations identify PgE2 as the main prostaglandin mediating sensory-evoked NVC, pyramidal cells as their principal source and vasodilatory EP2 and EP4 receptors as their targets

Diversité neuronale néocorticale et influence du métabolisme sur l'activité cérébrale

Mes travaux de doctorat ont contribué à l'étude physiologique, moléculaire et morphologique de la diversité des interneurones inhibiteurs du néocortex et à l'étude des mécanismes d'influence du métabolisme sur l'activité neuronal. En clarifiant la diversité des neurones corticaux, cette étude a permis l identification et la caractérisation des interneurones GABAergiques co-exprimant le neuropeptide Y (NPY) et l enzyme neuronale de synthèse du monoxyde d azote (NO). Une meilleure connaissance de ces interneurones va considérablement faciliter l'étude de leur rôle au sein du couplage neurovasculaire et neurométabolique via le NPY et le NO. Dans ce travail nous avons également mis à jour un mécanisme de couplage métabo-neuronal par les canaux potassiques sensibles à l'ATP (KATP). L'apport en substrat énergétique, et notamment celui en lactate, ne va pas seulement répondre à une demande énergétique accrue des neurones mais va également exacerber leur activité via la fermeture de canaux KATPPARIS-BIUSJ-Biologie recherche (751052107) / SudocSudocFranceF

Classification of NPY-expressing neocortical interneurons

Neuropeptide Y (NPY) is an abundant neuropeptide of the neocortex involved in numerous physiological and pathological processes. Due to the large electrophysiological, molecular and morphological diversity of NPY-expressing neurons their precise identity remains unclear. To define distinct populations of NPY neurons we characterized, in acute slices of rat barrel cortex, 200 cortical neurons of layers I-IV by means of whole-cell patch-clamp recordings, biocytin labeling and single cell Reversed Transcriptase-Polymerase Chain Reaction (scRT-PCR) designed to probe for the expression of well established molecular markers for cortical neurons. To classify reliably cortical NPY neurons we used and compared different unsupervised clustering algorithms based on laminar location, electrophysiological and molecular properties. These classification schemes confirmed that NPY neurons are nearly exclusively gamma-aminobutyric acid (GABA)-ergic and consistently disclosed three main types of NPY-expressing interneurons. (1) Neurogliaform-like neurons exhibiting a dense axonal arbor, were the most frequent, superficial, and substantially expressed the neuronal isoform of nitric oxide synthase (NOS-I). (2) Martinotti-like cells characterized by an ascending axon ramifying in layer I co-expressed somatostatin (SOM) and were the most excitable type. (3) Among fast spiking (FS) and parvalbumin (PV) positive basket cells, NPY expression was correlated with pronounced spike latency. By clarifying the diversity of cortical NPY neurons, this study establishes a basis for future investigations aiming at elucidating their physiological roles

Brainstem hypoxia contributes to the development of hypertension in the spontaneously hypertensive rat

Systemic arterial hypertension has been previously suggested to develop as a compensatory condition when central nervous perfusion/oxygenation is compromised. Principal sympathoexcitatory C1 neurons of the rostral ventrolateral medulla oblongata (whose activation increases sympathetic drive and the arterial blood pressure) are highly sensitive to hypoxia, but the mechanisms of this O(2) sensitivity remain unknown. Here, we investigated potential mechanisms linking brainstem hypoxia and high systemic arterial blood pressure in the spontaneously hypertensive rat. Brainstem parenchymal PO(2) in the spontaneously hypertensive rat was found to be ≈15 mm Hg lower than in the normotensive Wistar rat at the same level of arterial oxygenation and systemic arterial blood pressure. Hypoxia-induced activation of rostral ventrolateral medulla oblongata neurons was suppressed in the presence of either an ATP receptor antagonist MRS2179 or a glycogenolysis inhibitor 1,4-dideoxy-1,4-imino-d-arabinitol, suggesting that sensitivity of these neurons to low PO2 is mediated by actions of extracellular ATP and lactate. Brainstem hypoxia triggers release of lactate and ATP which produce excitation of C1 neurons in vitro and increases sympathetic nerve activity and arterial blood pressure in vivo. Facilitated breakdown of extracellular ATP in the rostral ventrolateral medulla oblongata by virally-driven overexpression of a potent ectonucleotidase transmembrane prostatic acid phosphatase results in a significant reduction in the arterial blood pressure in the spontaneously hypertensive rats (but not in normotensive animals). These results suggest that in the spontaneously hypertensive rat, lower PO(2) of brainstem parenchyma may be associated with higher levels of ambient ATP and l-lactate within the presympathetic circuits, leading to increased central sympathetic drive and concomitant sustained increases in systemic arterial blood pressure

Lactate is an energy substrate for rodent cortical neurons and enhances their firing activity

International audienceGlucose is the mandatory fuel for the brain, yet the relative contribution of glucose and lactate for neuronal energy metabolism is unclear. We found that increased lactate, but not glucose concentration, enhances the spiking activity of neurons of the cerebral cortex. Enhanced spiking was dependent on ATP-sensitive potassium (K ATP ) channels formed with KCNJ11 and ABCC8 subunits, which we show are functionally expressed in most neocortical neuronal types. We also demonstrate the ability of cortical neurons to take-up and metabolize lactate. We further reveal that ATP is produced by cortical neurons largely via oxidative phosphorylation and only modestly by glycolysis. Our data demonstrate that in active neurons, lactate is preferred to glucose as an energy substrate, and that lactate metabolism shapes neuronal activity in the neocortex through K ATP channels. Our results highlight the importance of metabolic crosstalk between neurons and astrocytes for brain function