Yves Charnay

A mutation in a case of early onset narcolepsy and a generalized absence of hypocretin peptides in human narcoleptic brains

We explored the role of hypocretins in human narcolepsy through histopathology of six narcolepsy brains and mutation screening of Hcrt, Hcrtr1 and Hcrtr2 in 74 patients of various human leukocyte antigen and family history status. One Hcrt mutation, impairing peptide trafficking and processing, was found in a single case with early onset narcolepsy. In situ hybridization of the perifornical area and peptide radioimmunoassays indicated global loss of hypocretins, without gliosis or signs of inflammation in all human cases examined. Although hypocretin loci do not contribute significantly to genetic predisposition, most cases of human narcolepsy are associated with a deficient hypocretin system.

Evidence supporting the existence of an activity-dependent astrocyte-neuron lactate shuttle

Mounting evidence from in vitro experiments indicates that lactate is an efficient energy substrate for neurons and that it may significantly contribute to maintain synaptic transmission, particularly during periods of intense activity. Since lactate does not cross the blood-brain barrier easily, blood-borne lactate cannot be a significant source. In vitro studies by several laboratories indicate that astrocytes release large amounts of lactate. In 1994, we proposed a mechanism whereby lactate could be produced by astrocytes in an activity-dependent, glutamate-mediated manner. Over the last 2 years we have obtained further evidence supporting the notion that a transfer of lactate from astrocytes to neurons might indeed take place. In this article, we first review data showing the presence of mRNA encoding for two monocarboxylate transporters, MCT1 and MCT2, in the adult mouse brain. Second, by using monoclonal antibodies selectively directed against the two distinct lactate dehydrogenase isoforms, LDH1 and LDH5, a specific cellular distribution between neurons and astrocytes is revealed which suggests that a population of astrocytes is a lactate ‘source’ while neurons may be a lactate ‘sink’. Third, we provide biochemical evidence that lactate is interchangeable with glucose to support oxidative metabolism in cortical neurons. This set of data is consistent with the existence of an activity-dependent astrocyte-neuron lactate shuttle for the supply of energy substrates to neurons.

Selective Distribution of Lactate Dehydrogenase Isoenzymes in Neurons and Astrocytes of Human Brain

In vertebrates, the interconversion of lactate and pyruvate is catalyzed by the enzyme lactate dehydrogenase. Two distinct subunits combine to form the five tetrameric isoenzymes of lactate dehydrogenase. The LDH-5 subunit (muscle type) has higher maximal velocity (Vmax) and is present in glycolytic tissues, favoring the formation of lactate from pyruvate. The LDH-1 subunit (heart type) is inhibited by pyruvate and therefore preferentially drives the reaction toward the production of pyruvate. There is mounting evidence indicating that during activation the brain resorts to the transient glycolytic processing of glucose. Indeed, transient lactate formation during physiological stimulation has been shown by 1H-magnetic resonance spectroscopy. However, since whole-brain arteriovenous studies under basal conditions indicate a virtually complete oxidation of glucose, the vast proportion of the lactate transiently formed during activation is likely to be oxidized. These in vivo data suggest that lactate may be formed in certain cells and oxidized in others. We therefore set out to determine whether the two isoforms of lactate dehydrogenase are localized to selective cell types in the human brain. We report here the production and characterization of two rat antisera, specific for the LDH-5 and LDH-1 subunits of lactate dehydrogenase, respectively. Immunohistochemical, immunodot, and western-blot analyses show that these antisera specifically recognize their homologous antigens. Immunohistochemistry on 10 control cases demonstrated a differential cellular distribution between both subunits in the hippocampus and occipital cortex: neurons are exclusively stained with the anti-LDH1 subunit while astrocytes are stained by both antibodies. These observations support the notion of a regulated lactate flux between astrocytes and neurons.

Neuronal expression of the NADPH oxidase NOX4, and its regulation in mouse experimental brain ischemia.

Ischemia-induced neuronal damage has been linked to elevated production of reactive oxygen species (ROS) both in animal models and in humans. NADPH oxidase enzymes (NOX-es) are a major enzymatic source of ROS, but their role in brain ischemia has not yet been investigated. The present study was carried out to examine the expression of NOX4, one of the new NADPH oxidase isoforms in a mouse model of focal permanent brain ischemia. We demonstrate that NOX4 is expressed in neurons using in situ hybridization and immunohistochemistry. Ischemia, induced by middle cerebral artery occlusion resulted in a dramatic increase in cortical NOX4 expression. Elevated NOX4 mRNA levels were detectable as early as 24 h after the onset of ischemia and persisted throughout the 30 days of follow-up period, reaching a maximum between days 7 and 15. The early onset suggests neuronal reaction, while the peak period corresponds to the time of neoangiogenesis occurring mainly in the peri-infarct region. The occurrence of NOX4 in the new capillaries was confirmed by immunohistochemical staining. In summary, our paper reports the presence of the ROS producing NADPH oxidase NOX4 in neurons and demonstrates an upregulation of its expression under ischemic conditions. Moreover, a role for NOX4 in ischemia/hypoxia-induced angiogenesis is suggested by its prominent expression in newly formed capillaries.

Anorexia induced by activation of serotonin 5-HT4 receptors is mediated by increases in CART in the nucleus accumbens

Anorexia nervosa is a growing concern in mental health, often inducing death. The potential neuronal deficits that may underlie abnormal inhibitions of food intake, however, remain largely unexplored. We hypothesized that anorexia may involve altered signaling events within the nucleus accumbens (NAc), a brain structure involved in reward. We show here that direct stimulation of serotonin (5-hydroxytryptamine, 5-HT) 4 receptors (5-HT4R) in the NAc reduces the physiological drive to eat and increases CART (cocaine- and amphetamine-regulated transcript) mRNA levels in fed and food-deprived mice. It further shows that injecting 5-HT4R antagonist or siRNA-mediated 5-HT4R knockdown into the NAc induced hyperphagia only in fed mice. This hyperphagia was not associated with changes in CART mRNA expression in the NAc in fed and food-deprived mice. Results include that 5-HT4R control CART mRNA expression into the NAc via a cAMP/PKA signaling pathway. Considering that CART may interfere with food- and drug-related rewards, we tested whether the appetite suppressant properties of 3,4-N-methylenedioxymethamphetamine (MDMA, ecstasy) involve the 5-HT4R. Using 5-HT4R knockout mice, we demonstrate that 5-HT4R are required for the anorectic effect of MDMA as well as for the MDMA-induced enhancement of CART mRNA expression in the NAc. Directly injecting CART peptide or CART siRNA into the NAc reduces or increases food consumption, respectively. Finally, stimulating 5-HT4R- and MDMA-induced anorexia were both reduced by injecting CART siRNA into the NAc. Collectively, these results demonstrate that 5-HT4R-mediated up-regulation of CART in the NAc triggers the appetite-suppressant effects of ecstasy.

Distribution of enkephalin-immunoreactive cell bodies in relation to serotonin-containing neurons in the raphe nuclei of the cat: Immunohistochemical evidence for the coexistence of enkephalins and serotonin in certain cells

In this study we have examined the distribution of enkephalin-like immunoreactive (ELI) cell bodies in the cat raphe nuclei pallidus (NRP), obscurus (NRO), magnus (NRM), pontis and dorsalis (NRD) after intraventricular administration of colchicine. All the raphe nuclei examined were observed to contain ELI cell bodies along their whole caudorostral extent. By comparing consecutive sections treated separately with anti-5-HT and enkephalin-antiserum it was observed that certain 5-HT cells in each raphe nucleus contain ELI material. A quantitative estimation was attempted. In NRP and NRO approximately half of the total immunoreactive neuronal population appeared to be immunoreactive for both 5-HT and the enkephalins. In NRM the proportion would be one-third, whereas it seemed almost negligible in NRD. Among the 5-HT cells, approximately two-thirds might be ELI in NRP and NRO, and one-half in NRM.

Frontocortical 5-HT4 receptors exert positive feedback on serotonergic activity: Viral transfections, subacute and chronic treatments with 5-HT4 agonists

BACKGROUND We recently identified a facilitory control exerted by serotonin4 (5-HT4) receptors on the in vivo firing activity of dorsal raphe nucleus (DRN) serotonergic (5-HT) neurons. However, these findings were based on acute administrations of 5-HT4 receptor agonists and antagonists, which were active only in a subpopulation of 5-HT neurons. We had no evidence that this influence was significant when considering the entire DRN, nor if it was persistent after chronic treatments. In addition, the poor distribution of 5-HT4 receptors within the DRN raised the question of the neuroanatomical bases underlying this control. METHODS AND RESULTS Here we show that the subacute intraperitoneal (IP) injection of the 5-HT4 receptor agonists prucalopride (2.5 mg/kg) and RS 67333 (1.5 mg/kg) 30 minutes before the beginning of recordings augment the mean firing rate of DRN neurons by 40% and 66%, respectively. These increases remain stable when the compounds are administered continuously during 3 and 21 days; the effects of the 3-day treatment are blocked by the 5-HT4 receptor antagonist GR 125487 (1000 microg/kg, intravenous [i.v.]). In addition, stereotaxic microinjections of herpes simplex viruses, transformed to overexpress 5-HT4 receptors, increase DRN 5-HT neuronal mean activity when performed in the medial prefrontal cortex (mPFC) but not in the striatum or in the hippocampus. CONCLUSIONS This finding suggests the existence of a 5-HT(4)-dependent activation of DRN that may involve the mPFC, unveiling the 5-HT4 receptor as a putative player in the physiopathology of several disorders related to central 5-HT dysfunction.

Distribution of substance P-like immunoreactivity in the spinal cord and dorsal root ganglia of the human foetus and infant.

Using the indirect immunofluorescence method, the distribution of substance P-like-immunoreactivity was studied in spinal cord and dorsal root ganglia of 25 human foetuses ranging from 12 to 29 weeks of gestational age. The spinal cord and dorsal root ganglia of three infants (1 day-, 2 and 4 month-old) were also investigated as a post-natal reference. On the whole, the substance P distribution patterns seen in infants were already visible throughout most of foetal life. The highest density of substance P-like-immunoreactive fibres was localized over the superficial layers of the dorsal grey horn. Punctiform immunofluorescence was often found over the white matter especially in the funiculi dorsalis et lateralis. In the ventral horn, substance P immunoreactive fibres were few and far between in the grey matter and were only detected from foetal stage 16 weeks. In addition, longitudino-frontal sections through the dorsal regions revealed repetitive arrangements of substance P-like-immunoreactive fibres along the whole spinal cord. In dorsal root ganglia only a few immunoreactive cells were observed. These findings demonstrate the wide and early occurrence of substance P-like-immunoreactivity in the human foetus spinal cord and dorsal root ganglia. They suggest that the development of the substance P neuronal system begins early in ontogenesis and is regionally differentiated.

Evidence for the presence of enkephalin in catecholaminergic neurones of cat locus coeruleus

The distribution of enkephalin (Enk) and tyrosine hydroxylase (TH) immunoreactivity in the cat locus coeruleus (LC) has been studied with indirect immunofluorescence technique. After intratissular injection of colchicine numerous enkephalin-containing cell bodies were seen throughout the rostrocaudal extent of the LC complex. Comparison of 8 micrometers-thick consecutive sections treated with antiserum to Enk of TH, a specific marker for catecholaminergic neurons, shows that most cells containing TH also present Enk immunoreactivity. This study provides the first evidence for the coexistence of enkephalin and catecholamine in CNS neurons.

Inhibition of post-ischemic brain injury by clusterin overexpression.

(Fig. 1e). In contrast, there was no prion transport in Caco-2 cultures without M cells (n = 9) (Fig. 1e), except for traces of infectivity (< 1 logLD50) in one case. Transport of prion infectivity by Raji B cells in M-cell–containing cocultures is extremely unlikely, since Raji B cells are restricted to the basolateral compartment (Fig. 1c) and are not capable of traversing the epithelial monolayer to the apical compartment. These findings indicate that M-cell differentiation is necessary and sufficient for active transepithelial prion transport in vitro. M-cell–dependent uptake of foreign antigens or particles is known to be followed by rapid transcytosis directly to the intraepithelial pocket, where key players of the immune system, such as macrophages, dendritic cells and lymphocytes, are located. Because at least some of these immune cells have been shown to be crucially involved in the process of neuroinvasion, prions might exploit the M-cell–dependent transcytosis to gain access to the immune system. These findings indicate that M cells are a plausible candidate for the mucosal portal of prion infection.