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Background Myotonia congenita (MC) is a common channelopathy affecting skeletal muscle and which is due to pathogenic variants within the CLCN1 gene. Various alterations in the function of the channel have been reported and we here illustrate a novel one. Methods A patient presenting the symptoms of myotonia congenita was shown to bear a new heterozygous missense variant in exon 9 of the CLCN1 gene (c.1010 T > G, p.(Phe337Cys)). Confocal imaging and patch clamp recordings of transiently transfected HEK293 cells were used to functionally analyze the effect of this variant on channel properties. Results Confocal imaging showed that the F337C mutant incorporated as well as the WT channel into the plasma membrane. However, in patch clamp, we observed a smaller conductance for F337C at −80 mV. We also found a marked reduction of the fast gating component in the mutant channels, as well as an overall reduced voltage dependence. Conclusion To our knowledge, this is the first report of a mixed alteration in the biophysical properties of hClC‐1 consisting of a reduced conductance at resting potential and an almost abolished voltage dependence., We describe a rare variant of the CLCN1 gene which was found in one family. Functional analysis demonstrated that the channel inserts normally into the membrane, but has a low conductance at −80 mV and a severely reduced voltage‐dependence.

Juvenile myoclonic epilepsy (JME), a lifelong disorder that starts during adolescence, is the most common of genetic generalized epilepsy syndromes. JME is characterized by awakening myoclonic jerks and myoclonic-tonic-clonic (m-t-c) grand mal convulsions. Unfortunately, one third of JME patients have drug refractory m-t-c convulsions and these recur in 70-80% who attempt to stop antiepileptic drugs (AEDs). Behavioral studies documented impulsivity, but also impairment of executive functions relying on organization and feedback, which points to prefrontal lobe dysfunction. Quantitative voxel-based morphometry (VBM) revealed abnormalities of gray matter (GM) volumes in cortical (frontal and parietal) and subcortical structures (thalamus, putamen, and hippocampus). Proton magnetic resonance spectroscopy (MRS) found evidence of dysfunction of thalamic neurons. White matter (WM) integrity was disrupted in corpus callosum and frontal WM tracts. Magnetic resonance imaging (MRI) further unveiled anomalies in both GM and WM structures that were already present at the time of seizure onset. Aberrant growth trajectories of brain development occurred during the first 2 years of JME diagnosis. Because of genetic origin, disease causing variants were sought, first by positional cloning, and most recently, by next generation sequencing. To date, only six genes harboring pathogenic variants (GABRA1, GABRD, EFHC1, BRD2, CASR, and ICK) with Mendelian and complex inheritance and covering a limited proportion of the world population, are considered as major susceptibility alleles for JME. Evidence on the cellular role, developmental and cell-type expression profiles of these six diverse JME genes, point to their pathogenic variants driving the first steps of brain development when cell division, expansion, axial, and tangential migration of progenitor cells (including interneuron cortical progenitors) sculpture subtle alterations in brain networks and microcircuits Frontiers during development. These alterations may explain "microdysgenesis" neuropathology, impulsivity, executive dysfunctions, EEG polyspike waves, and awakening m-t-c convulsions observed in JME patients.

The building of the brain is a multistep process that requires the coordinate expression of thousands of genes and an intense nucleocytoplasmic transport of RNA and proteins. This transport is mediated by karyopherins that comprise importins and exportins. Here, we investigated the role of the beta-importin, importin-8 (IPO8) during mouse cerebral corticogenesis as several of its cargoes have been shown to be essential during this process. First, we showed that Ipo8 mRNA is expressed in mouse brain at various embryonic ages with a clear signal in the sub-ventricular/ventricular zone (SVZ/VZ), the cerebral cortical plate (CP) and the ganglionic eminences. We found that acute knockdown of IPO8 in cortical progenitors reduced both their proliferation and cell cycle exit leading to the increase in apical progenitor pool without influencing the number of basal progenitors (BPs). Projection neurons ultimately reached their appropriate cerebral cortical layer, but their dendritogenesis was specifically affected, resulting in neurons with reduced dendrite complexity. IPO8 knockdown also slowed the migration of cortical interneurons. Together, our data demonstrate that IPO8 contribute to the coordination of several critical steps of cerebral cortex development. These results suggest that the impairment of IPO8 function might be associated with some diseases of neuronal migration defects.

Background: Given the continuous knowledge progression and the growing number of available antiepileptic drugs (AEDs), making appropriate treatment choices for patients with epilepsy is increasingly difficult. While published guidelines help for separate clinical aspects, patients with a combination of specific characteristics may escape proper guidance. This study aimed to determine the appropriateness of AEDs for particular clinical variables and to offer treatment recommendations for adult patients with epilepsy in a user-friendly format for practicing neurologists. Methods: Using the RAND/UCLA Appropriateness Method, the appropriateness of AEDs as initial/second mono-therapy and combination therapy was assessed in relation to selected clinical variables by a Belgian panel of 13 experts in epilepsy. Panel recommendations for particular patient profiles were determined by the outcome of these separate ratings. Results: The appropriateness outcome of individual AEDs was not substantially different between first and second mono-therapy; valproate was considered appropriate for all types of generalised and partial seizures. The outcome for combination therapy was highly dependent on the type of AED and seizures. With respect to co-morbidities and co-treatments, levetiracetam and pregabalin proved to have the least contra-indications. For the elderly and with respect to factors related to the female reproductive system the appropriateness of AEDs showed a more diffuse pattern. Although caution was deemed necessary for some combinations, the AEDs were never considered inappropriate regarding their drug interaction profile. Conclusions: The Epi-Scope (R) tool that displays appropriateness recommendations for highly specific, possibly complex cases, supports optimal treatment choices for adult patients with epilepsy in daily practice. (C) 2011 British Epilepsy Association. Published by Elsevier Ltd. All rights reserved.

The hypothalamic neuropeptide melanin-concentrating hormone (MCH) plays important roles in energy homeostasis, anxiety, and sleep regulation. Since the MCH receptor-1 (MCH-R1), the only functional receptor that mediates MCH functions in rodents, facilitates behavioral performance in hippocampus-dependent learning tasks, we investigated whether glutamatergic transmission in CA1 pyramidal cells could be modulated in mice lacking the MCH-R1 gene (MCH-R1−/−). We found that both α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-d-aspartate (NMDA) receptor-mediated transmissions were diminished in the mutant mice compared with their controls. This deficit was explained, at least in part, by a postsynaptic down-regulation of these receptors since the amplitude of miniature excitatory postsynaptic currents and the NMDA/AMPA ratio were decreased. Long-term synaptic potentiation (LTP) was also impaired in MCH-R1−/− mice. This was due to an altered induction, rather than an impaired, expression because repeating the induction stimulus restored LTP to a normal magnitude. In addition, long-term synaptic depression was strongly diminished in MCH-R1−/− mice. These results suggest that MCH exerts a facilitatory effect on CA1 glutamatergic synaptic transmission and long-term synaptic plasticity. Recently, it has been shown that MCH neurons fire exclusively during sleep and mainly during rapid eye movement sleep. Thus these findings provide a mechanism by which sleep might facilitate memory consolidation.

EFHC1, a gene mutated in juvenile myoclonic epilepsy, encodes EFHC1, a protein with three DM10 domains and one EF-hand motif. We recently demonstrated that this molecule is a microtubule-associated protein (MAP) implicated in neuronal migration. Because some controversies persist about the precise localization in the CNS, we studied the neuroanatomical distribution of EFHC1 in mature and developing mouse brain. In the adult, low mRNA expression was detected in several brain structures such as cortex, striatum, hippocampus and cerebellum. At E16, EFHC1 mRNA was shown to be expressed in cortex and not only in cells lining ventricles. Using a purified polyclonal antibody, EFHC1 staining was observed in all cortical layers, in piriform cortex, in hippocampus and in Purkinje cells of cerebellum. In the cortex, the majority of EFHC1 positive cells correspond to neurons, however some glial cells were also stained. In agreement with a previous study, we demonstrated strong EFHC1 expression in cilia of ependymal cells lining cerebral ventricles. Moreover, at E16, the protein was observed at the borders of brain ventricles, in choroid plexus, but also, although to a lesser extent, in piriform and neocortex. In these latter structures, the pattern of expression seems to correspond to the extensions of the radial glia fibers as demonstrated by BLBP immunostaining. Finally, we confirmed that EFHC1 was also expressed and co-localized with the mitotic spindle of neural stem cells. These results confirm that EFHC1 is a protein with a likely low expression level in mouse brain but detectable both in adult and embryonic brain supporting our previous data and hypothesis that EFHC1 could play an important role during brain development. As discussed, this opens the door to a new conceptual approach viewing some idiopathic generalized epilepsies as developmental diseases instead of classical channelopathies.

Mutations in the EFHC1 gene are linked to juvenile myoclonic epilepsy (JME), one of the most frequent forms of idiopathic generalized epilepsies. JME is associated with subtle alterations of cortical and subcortical architecture, but the underlying pathological mechanism remains unknown. We found that EFHC1 is a microtubule-associated protein involved in the regulation of cell division. In vitro, EFHC1 loss of function disrupted mitotic spindle organization, impaired M phase progression, induced microtubule bundling and increased apoptosis. EFHC1 impairment in the rat developing neocortex by ex vivo and in utero electroporation caused a marked disruption of radial migration. We found that this effect was a result of cortical progenitors failing to exit the cell cycle and defects in the radial glia scaffold organization and in the locomotion of postmitotic neurons. Therefore, we propose that EFHC1 is a regulator of cell division and neuronal migration during cortical development and that disruption of its functions leads to JME.

The present study aimed to test the hypothesis that mice lacking the MCHR1 receptor (Melanin-Concentrating Hormone Receptor-1) present an elevated vulnerability towards the neurobehavioural effects of d -amphetamine, presumably due to previously established up-regulations of dopamine D1 receptors in these mice. We examined the psychomotor effects of five once-daily injections of 1.5 and 3 mg/kg d -amphetamine (i.p.) or ten once-daily injections of 2.25 mg/kg d -amphetamine in knockout (KO) mice lacking the MCHR1 receptor. The first injection of d -amphetamine induced a greater psychomotor response amongst the KO mice at 2.25 and 3.0 mg/kg. On all subsequent d -amphetamine injections, KO mice still showed greater levels of psychomotor activity than the WT mice, but with no between-genotype difference in the rate of development of sensitization (similar slopes of the curves). Furthermore, 24 h after the last injection of 2.25 mg/kg d -amphetamine both genotypes exhibited a significant post-sensitization conditioned activity. Thus, MCHR1 receptors are likely not deeply involved in the mechanisms of induction of sensitization and related conditioned activity induced by d -amphetamine, albeit our results confirm a contribution of these receptors to the mechanisms of the acute effects of that drug, possibly via an inhibitory action on the dopaminergic mesolimbic system. Our results do not support the hypothesis of a functional contribution of MCHR1 receptors to the addictive effects of d -amphetamine.

This study was designed to document convulsant and neurotoxic properties of extracts of a tropical tree, Magnistipula butayei subsp. Montana , and to investigate the involvement of the glutamatergic system in these effects. Continuous behavioral observations and electroencephalographic (EEG) records were obtained after per os administration of an aqueous extract of Magnistipula (MBMAE) in rats. MBMAE (800 mg/kg) induced behavioral changes resembling motor limbic seizures: staring and head tremor, automatisms, forelimb clonic movements and violent tonic-clonic seizures leading to death in all animals. Concomitantly, important seizure activity that gradually evolved to epileptiform activity was recorded on the EEG. Moreover, c-Fos immunohistochemistry has revealed an increased c-Fos expression in the dentate gyrus and in piriform, peri- and entorhinal cortices 2 and 4 h after treatment. This expression pattern suggested that the mechanism of action for the MBMAE is similar to that observed in glutamate-induced models of epilepsy. The MBMAE increased cell death also in hippocampal cell cultures. Furthermore, the build-up of convulsive activity and epileptic discharges induced by MBMAE in rat were abolished by MK-801, an NMDA receptor antagonist. Our study suggests that MBMAE contains a potent toxin, with a powerful neurotoxic activity in rat, and corresponding to a new natural component(s) that act as an NMDA-mediated convulsant molecule.

A novel gene, EFHC1, mutated in juvenile myoclonic epilepsy (JME) encodes a protein with three DM10 domains of unknown function and one putative EF-hand motif. To study the properties of EFHC1, we expressed EGFP-tagged protein in various cell lines. In interphase cells, the fusion protein was present in the cytoplasm and in the nucleus with specific accumulation at the centrosome. During mitosis EGFP-EFHC1 colocalized with the mitotic spindle, especially at spindle poles and with the midbody during cytokinesis. Using a specific antibody, we demonstrated the same distribution of the endogenous protein. Deletion analyses revealed that the N-terminal region of EFHC1 is crucial for the association with the mitotic spindle and the midbody. Our results suggest that EFHC1 could play an important role during cell division.

Thiamine triphosphate (ThTP) is found in most organisms and may be an intracellular signal molecule produced in response to stress. We have recently cloned the cDNA coding for a highly specific mammalian 25-kDa thiamine triphosphatase. The enzyme was active in all mammalian species studied except pig, although the corresponding mRNA was present. In order to determine whether the very low ThTPase activity in pig tissues is due to the absence of the protein or to a lack of catalytic efficiency, we expressed human and pig ThTPase in E. coli as GST fusion proteins. The purified recombinant pig GST-ThTPase was found to be 2-3 orders of magnitude less active than human GST-ThTPase. Using site-directed mutagenesis, we show that, in particular, the change of Glu85 to lysine is responsible for decreased solubility and catalytic activity of the pig enzyme. Immunohistochemical studies revealed a distribution of the protein in pig brain very similar to the one reported in rodent brain. Thus, our results suggest that a 25-kDa protein homologous to hThTPase but practically devoid of enzyme activity is expressed in pig tissues. This raises the possibility that this protein may play a physiological role other than ThTP hydrolysis.

In order to investigate the physiological properties of the melanin-concentrating hormone (MCH) we have generated and used mice from which the MCH receptor 1 gene was deleted (MCHR1(Neo/Neo) mice). Complementary experimental approaches were used to investigate alterations in the learning and memory processes of our transgenic model. The ability of the knockout strain to carry out the inhibitory passive avoidance test was found to be considerably impaired although no significant differences were observed in anxiety levels. This impaired cognitive property prompted us to explore modifications in N-methyl D-aspartate (NMDA) responses in the hippocampus. Intracellular recordings of CA1 pyramidal neurons in hippocampal slices from the MCHR1(Neo/Neo) mice revealed significantly decreased NMDA responses. Finally, using in situ hybridization we found a 15% reduction in NMDAR1 subunit in the CA1 region. These results show for the first time a possible role for MCH in the control of the function of the NMDA receptor.

Juvenile Myoclonic Epilepsy (JME) accounts for almost 12% of all epilepsies and is one of the most frequent forms of genetic generalized epilepsies. Genetic studies have revealed that mutations in EFHC1 (EF-hand containing one) account for 3 to 9% of all cases around the world. This gene encodes a protein that is not an ion channel, and several studies have tried to find its cellular role. In this article, we review the various functions that have been proposed for this protein. Interestingly, all of them could affect brain development at different steps, suggesting that the developmental assembly of neural circuits may play a prominent role in JME. This article is part of a supplemental special issue entitled Juvenile Myoclonic Epilepsy: What is it Really?

Thiamine triphosphate (ThTP) is found in most organisms, but its biological role remains unclear. In mammalian tissues, cellular ThTP concentrations remain low, probably because of hydrolysis by a specific 25 kDa thiamine triphosphatase (ThTPase). The aim of the present study was to use quantitative PCR, for comparing the 25 kDa ThTPase mRNA expression in various mouse tissues with its enzyme activities. ThTPase mRNA was expressed at only a few copies per cell. The highest amount of mRNA was found in testis, followed by lung and muscle, while the highest enzyme activities were found in liver and kidney. The poor correlation between mRNA levels and enzyme activities might result either from tissue-specific post-transcriptional regulation of mRNA processing and/or translation or from the regulation of enzyme activities by post-translational mechanisms. Purified recombinant human ThTPase was phosphorylated by casein kinase II, but this phosphorylation did not modify the enzyme activity. However, the characterization of the 3′-untranslated mRNA region revealed a unique, highly conserved, 200-nucleotide sequence that might be involved in translational control. In situ hybridization studies in testis suggest a predominant localization of ThTPase mRNA in poorly differentiated spermatogenic cells. This is the first study demonstrating a cell-specific 25 kDa ThTPase mRNA expression, suggesting that this enzyme might be related to the degree of differentiation or the metabolic state of the cell.

Thiamine triphosphate (ThTP) is found in most living organisms and it may act as a phosphate donor for protein phosphorylation. We have recently cloned the cDNA coding for a highly specific mammalian 25 kDa thiamine triphosphatase (ThTPase; EC 3.6.1.28). As the enzyme has a high catalytic efficiency and no sequence homology with known phosphohydrolases, it was worth investigating its structure and catalytic properties. For this purpose, we expressed the untagged recombinant human ThTPase (hThTPase) in E. coli, produced the protein on a large scale and purified it to homogeneity. Its kinetic properties were similar to those of the genuine human enzyme, indicating that the recombinant hThTPase is completely functional. Mg2+ ions were required for activity and Ca2+ inhibited the enzyme by competition with Mg2+. With ATP as substrate, the catalytic efficiency was 10(-4)-fold lower than with ThTP, confirming the nearly absolute specificity of the 25 kDa ThTPase for ThTP. The activity was maximum at pH 8.5 and very low at pH 6.0. Zn2+ ions were inhibitory at micromolar concentrations at pH 8.0 but activated at pH 6.0. Kinetic analysis suggests an activator site for Mg2+ and a separate regulatory site for Zn2+. The effects of group-specific reagents such as Woodward's reagent K and diethylpyrocarbonate suggest that at least one carboxyl group in the active site is essential for catalysis, while a positively charged amino group may be involved in substrate binding. The secondary structure of the enzyme, as determined by Fourier-transform infrared spectroscopy, was predominantly beta-sheet and alpha-helix.

Thiamine triphosphate (ThTP) is present in low amounts in most organisms from bacteria to humans, but its biological role remains unknown. Escherichia coli grown aerobically in LB medium contain no detectable amounts of ThTP, but when they are transferred to M9 minimal medium with a substrate such as glucose or pyruvate, there is a rapid but transient accumulation of relatively high amounts of ThTP (about 20% of total thiamine). If a mixture of amino acids is present in addition to glucose, ThTP accumulation is impaired, suggesting that the latter may occur in response to amino acid starvation. To test the importance of ThTP for bacterial growth, we used an E. coli strain overexpressing a specific human recombinant thiamine triphosphatase as a glutathione S-transferase (GST) fusion protein (GST-ThTPase). Those bacteria were unable to accumulate measurable amounts of ThTP. On minimal medium supplemented with glucose, pyruvate, or acetate, they exhibited an intermediate plateau in cell growth compared with control bacteria expressing GST alone or a GST fusion protein unrelated to thiamine metabolism. These results suggest that the early accumulation of ThTP initiates a reaction cascade involved in the adaptation of bacteria to stringent conditions such as amino acid starvation. This is the first demonstration of a physiological role of this ubiquitous compound in any organism.

We evaluated the efficacy of vagus nerve stimulation (VNS) in Genetic Absence Epilepsy Rats from Strasbourg (GAERS), a validated model for absence epilepsy. In the first experiment, we investigated whether VNS applied at seizure onset can interrupt spike and wave discharges (SWD). In the second experiment, we investigated whether SWD are suppressed or shortened in duration when VNS is applied several hours per day. Both control and VNS groups underwent EEG and VNS electrode implantation. For the first experiment, a randomized crossover design was used. Stimuli (amplitude: 3 V; frequency: 30 Hz; pulse duration: 500 micros) were given when an SWD occurred on the EEG. The experiment was repeated the next day. In the second experiment, treated animals were stimulated (amplitude: 1.5 mA; frequency: 30 Hz; pulse duration: 500 micros; on/off time cycle: 30 s / 5 min) for 3h per day, during five consecutive days. In the first experiment, the duration of the SWD was increased on day 1, (P0.05). There was no difference in SWD duration on Day 2. In the second experiment, no significant differences could be found in number, duration and EEG frequency of SWD. VNS applied at the onset of an SWD can prolong the duration of SWD in GAERS. As a 5-day stimulation protocol had no effect, long-term VNS might be necessary to affect SWD.

The gene encoding the mouse melanin-concentrating hormone receptor 1 was isolated and its structural organization and flanking regions were characterized. The 3′ flanking region is marked by the presence of two polyadenylation signals but used with different frequencies. RNase protection and 5′ rapid amplification of cDNA ends (RACE) identified multiple transcription initiation sites between −150 and −203 bp upstream of the ATG initiation codon. Functional analysis of deletion mutants reveals a cell independent transcriptional activity localized between nucleotide −305 and −589. The proximal 1.5 kb region does not possess consensus TATA or CAAT boxes but has several consensus sequences for regulatory elements including USF, GATA, AP1, AP4, MyoD, GKLF and Ikaros that could explain the broad expression of the receptor.

Thiamine triphosphate (ThTP) is found in small amounts in most organisms from bacteria to mammals, but little is known about its physiological role. In vertebrate tissues, ThTP may act as a phosphate donor for the phosphorylation of certain proteins; this may be part of a new signal transduction pathway. We have recently characterized a highly specific 25-kDa thiamine triphosphatase (ThTPase) that is expressed in most mammalian tissues. The role of this enzyme may be the control of intracellular concentrations of ThTP. As the latter has been considered to be a neuroactive form of thiamine, we have studied the distribution of ThTPase mRNA and protein in rodent brain using in situ hybridization and immunohistochemistry. With both methods, we found the strongest staining in hippocampal pyramidal neurons, as well as cerebellar granule cells and Purkinje cells. Some interneurons were also labeled and many ThTPase mRNA-positive and immunoreactive cells were distributed throughout cerebral cortical gray matter and the thalamus. White matter was not significantly labeled. ThTPase immunoreactivity seems to be located mainly in the cytoplasm of neuronal perikarya. Immunocytochemical data using dissociated cultured cells from hippocampal and cerebellum showed that the staining was more intense in neurons than in astrocytes. The protein was rather uniformly located in the perikarya and dendrites, suggesting that ThTP and ThTPase may play a general role in neuronal metabolism rather than a specific role in excitability. There was no apparent correlation between ThTPase expression and selective vulnerability of certain brain regions to thiamine deficiency.

Several mono- and bisindole quaternary alkaloids isolated from the stem bark of Strychnos guianensis have recently been shown to be effective blockers of neuromuscular transmission in mice. In this study, we used a human clonal cell line (TE671) expressing muscle-type nicotinic acetylcholine receptors. The agonist carbamylcholine activated a receptor-mediated (86)Rb(+) efflux and this activation was antagonized by the indole alkaloids, the most active being bisindole bisquaternary compounds. The most effective antagonist, guiachrysine, had an IC(50) around 0.43 microM in the presence of 0.5 mM carbamylcholine, compared to 0.16 microM for d-tubocurarine, the most potent curarizing alkaloid. Guiaflavine and 5',6'-dehydroguiaflavine were slightly less effective. Monoindole compounds were 10 to 100 times less potent than bisindole alkaloids. Kinetic analysis showed that the inhibition of the carbamylcholine-dependent (86)Rb(+) efflux by guiaflavine was of mixed competitive and uncompetitive type. The competitive component (K(I)=0.21 microM) is presumably due to binding at the acetylcholine site, while the uncompetitive component (K'(I)=0.92 microM) may be due to open channel block.

When neuroblastoma cells were transferred to a medium of low (6 nM) thiamine concentration, a 16-fold decrease in total intracellular thiamine content occurred within 8 days. Respiration and ATP levels were only slightly affected, but addition of a thiamine transport inhibitor (amprolium) decreased ATP content and increased lactate production. Oxygen consumption became low and insensitive to oligomycin and uncouplers. At least 25% of mitochondria were swollen and electron translucent. Cell mortality increased to 75% within 5 days. [3H]PK 11195, a specific ligand of peripheral benzodiazepine receptors (located in the outer mitochondrial membrane) binds to the cells with high affinity (KD = 1.4 +/- 0.2 nM). Thiamine deficiency leads to an increase in both Bmax and KD. Changes in binding parameters for peripheral benzodiazepine receptors may be related to structural or permeability changes in mitochondrial outer membranes. In addition to the high-affinity (nanomolar range) binding site for peripheral benzodiazepine ligands, there is a low-affinity (micromolar range) saturable binding for PK 11195. At micromolar concentrations, peripheral benzodiazepines inhibit thiamine uptake by the cells. Altogether, our results suggest that impairment of oxidative metabolism, followed by mitochondrial swelling and disorganization of cristae, is the main cause of cell mortality in severely thiamine-deficient neuroblastoma cells.

We compared the thiamine and thiamine phosphate contents in the frontal, temporal, parietal, and occipital cortex of six patients with frontal lobe degeneration of the non-Alzheimer's type (FNAD) or frontotemporal dementia with five age-, postmortem delay-, and agonal status-matched control subjects. Our results reveal a 40-50% decrease in thiamine diphosphate (TDP) in the cortex of FNAD patients, whereas thiamine monophosphate was increased 49-119%. TDP synthesizing and hydrolyzing enzymes were unaffected. The activity of citrate synthase, a mitochondrial marker enzyme, was decreased in the frontal cortex of patients with FNAD, but no correlation with TDP content was found. These results suggest that decreased contents of TDP, which is essentially mitochondrial, is a specific feature of FNAD. As TDP is an essential cofactor for oxidative metabolism and neurotransmitter synthesis, and because low thiamine status (compared with other species) is a constant feature in humans, a nearly 50% decrease in cortical TDP content may contribute significantly to the clinical symptoms observed in FNAD. This study also provides a basis for a trial of thiamine, to improve the cognitive status of the patients.

Melanin-concentrating hormone (MCH) is highly expressed in the brain and modulates feeding behavior. It is also expressed in some peripheral tissues where its role remains unknown. We have investigated MCH function in human and mouse immune cells. RT-PCR analysis revealed a low expression of prepro-MCH and MCH receptor 1 (MCHR1) but not of MCHR2 transcript in tissular and peripheral blood immune cells. FACS and in vitro assay studies demonstrated that MCHR1 receptor expression on most cell types can trigger, in the presence of MCH, cAMP synthesis and calcium mobilization in peripheral blood mononuclear cells (PBMCs). Moreover, MCH treatment decreases the CD3-stimulated PBMC proliferation in vitro. Accordingly, our data indicate for the first time that MCH and MCHR1 may exert immunomodulatory functions.

In immature neurones, the steady-state intracellular Cl- concentration [Cl-](i) is generally higher than expected for passive distribution, and this is believed to be due to Na(+)-K(+)-2Cl(-) co-transport. Here, we show that N2a neuroblastoma cells, incubated in HEPES-buffered NaCl medium maintain a [Cl-](i) around 60 mm, two- to threefold higher than expected for passive distribution at a membrane potential of - 49 mV. When the cells were transferred to a Cl(-) -free medium, [Cl-](i) decreased quickly (t(1/2) < 5 min), suggesting a high Cl- permeability. When the intracellular ATP concentration was reduced to less than 1 mm by metabolic inhibitors, the initial rate of (36) Cl- uptake was strongly inhibited (60-65%) while steady-state [Cl-](i) decreased to 24 mm, close to the value predicted from the Nernst equilibrium. Moreover, after reduction of [ATP](i) and [Cl-](i) by rotenone, the subsequent addition of glucose led to a reaccumulation of Cl-, in parallel with ATP recovery. Internal bicarbonate did not affect Cl- pumping, suggesting that Cl-/HCO(3)(-) exchange does not significantly contribute to active transport. Likewise, Na(+) -K(+) -2Cl(-) co-transport also appeared to play a minor role: although mRNA for the NKCC1 form of the co-transporter was detected in N2a cells, neither the initial rate of (36)Cl- uptake nor steady-state [Cl-](i) were appreciably decreased by 10 microm bumetanide or replacement of external Na(+) by choline. These results suggest that a highly active ATP-dependent mechanism, distinct from Na(+) -K(+) -2Cl(-) co-transport, is responsible for most of the inward Cl- pumping in N2a cells.

Thiamine triphosphate (ThTP) is found at low concentrations in most animal tissues, and recent data suggest that it may act as a phosphate donor for the phosphorylation of some proteins. In the mammalian brain, ThTP synthesis is rapid, but its steady-state concentration remains low, presumably because of rapid hydrolysis. In this report we purified a soluble thiamine triphosphatase (ThTPase; EC3.6.1.28) from calf brain. The bovine ThTPase is a 24-kDa monomer, hydrolyzing ThTP with virtually absolute specificity. Partial sequence data obtained from the purified bovine enzyme by tandem mass spectrometry were used to search the GenBankTM data base. A significant identity was found with only one human sequence, the hypothetical 230-amino acid protein MGC2652. The coding regions from human and bovine brain mRNA were amplified by reverse transcription-PCR, cloned in Escherichia coli, and sequenced. The human open reading frame was expressed in E. coli as a GST fusion protein. Transformed bacteria had a high isopropyl-β-d-thiogalactopyranoside-inducible ThTPase activity. The recombinant ThTPase had properties similar to those of human brain ThTPase, and it was specific for ThTP. The mRNA was expressed in most human tissues but at relatively low levels. This is the first report of a molecular characterization of a specific ThTPase.

New genetic investigation techniques, including next-generation sequencing, epigenetic profiling, cell lineage mapping, targeted genetic manipulation of specific neuronal cell types, stem cell reprogramming, and optogenetic manipulations within epileptic networks are progressively unraveling the mysteries of epileptogenesis and ictogenesis. These techniques have opened new avenues to discover the molecular basis of epileptogenesis and to study the physiologic effects of mutations in epilepsy-associated genes on a multilayer level, from cells to circuits. This manuscript reviews recently published applications of these new genetic technologies in the study of epilepsy, as well as work presented by the authors at the genetic session of the XII Workshop on the Neurobiology of Epilepsy (WONOEP 2013) in Quebec, Canada. Next-generation sequencing is providing investigators with an unbiased means to assess the molecular causes of sporadic forms of epilepsy and has revealed the complexity and genetic heterogeneity of sporadic epilepsy disorders. To assess the functional impact of mutations in these newly identified genes on specific neuronal cell types during brain development, new modeling strategies in animals, including conditional genetics in mice and in utero knock-down approaches, are enabling functional validation with exquisite cell-type and temporal specificity. In addition, optogenetics, using cell-type-specific Cre recombinase driver lines, is enabling investigators to dissect networks involved in epilepsy. In addition, genetically encoded cell-type labeling is providing new means to assess the role of the nonneuronal components of epileptic networks such as glial cells. Furthermore, beyond its role in revealing coding variants involved in epileptogenesis, next-generation sequencing can be used to assess the epigenetic modifications that lead to sustained network hyperexcitability in epilepsy, including methylation changes in gene promoters and noncoding ribonucleic acid (RNA) involved in modifying gene expression following seizures. In addition, genetically based bioluminescent reporters are providing new opportunities to assess neuronal activity and neurotransmitter levels both in vitro and in vivo in the context of epilepsy. Finally, genetically rederived neurons generated from patient induced pluripotent stem cells and genetically modified zebrafish have become high-throughput means to investigate disease mechanisms and potential new therapies. Genetics has changed the field of epilepsy research considerably, and is paving the way for better diagnosis and therapies for patients with epilepsy.

Background Mutations in the proline-rich transmembrane protein 2 ( PRRT2 ) gene have been identified in patients with benign (familial) infantile convulsions (B(F)IC), infantile convulsions with choreoathetosis (ICCA) and paroxysmal dyskinesias (PDs). However it remains unknown whether PRRT2 mutations are causal in other epilepsy syndromes. After we discovered a PRRT2 mutation in a large family with ICCA containing one individual with febrile seizures (FS) and one individual with West syndrome, we analysed PRRT2 in a heterogeneous cohort of patients with different types of infantile epilepsy. Methods We screened a cohort of 460 patients with B(F)IC or ICCA, fever related seizures or infantile epileptic encephalopathies. All patients were tested for point mutations using direct sequencing. Results We identified heterozygous mutations in 16 individuals: 10 familial and 6 sporadic cases. All patients were diagnosed with B(F)IC, ICCA or PD. We were not able to detect mutations in any of the other epilepsy syndromes. Several mutation carriers had learning disabilities and/or impaired fine motor skills later in life. Conclusions PRRT2 mutations do not seem to be involved in the aetiology of FS or infantile epileptic encephalopathies. Therefore B(F)IC, ICCA and PD remain the core phenotypes associated with PRRT2 mutations. The presence of learning disabilities or neuropsychiatric problems in several mutation carriers calls for additional clinical studies addressing this developmental aspect in more detail.

A computer model of a thalamic network was used in order to examine the effects of an isolated augmentation in a low-threshold calcium current. Such an isolated augmentation has been observed in the reticular thalamic (RE) nucleus of the genetic absence epilepsy rat from the Strasbourg (GAERS) model of absence epilepsy. An augmentation of the low-threshold calcium conductance in the RE neurons (gTs) of the model thalamic network was found to lead to an increase in the synchronized firing of the network. This supports the hypothesis that the isolated increase in gTs may be responsible for epileptic activity in the GAERS rat. The increase of gTs in the RE neurons led to a slight increase in the period of the isolated RE neuron firing. In contrast, the low-threshold spike of the RE neuron remained relatively unchanged by the increase of gTs. This suggests that the enhanced synchrony in the network was primarily due to a phase shift in the firing of the RE neurons with respect to the thalamocortical neurons. The ability of this phase-shift mechanism to lead to changes in synchrony was further examined using the model thalamic network. A similar increase in the period of RE neuron oscillations was obtained through an increase in the conductance of the calcium-mediated potassium channel. This change was once again found to increase synchronous firing in the network.

Differential mRNA display was carried out to find genes that are differentially regulated in the brain of a rat strain with absence epilepsy, the genetic absence epilepsy rats from Strasbourg (GAERS). Among the 32 differentially displayed cDNA fragments actually cloned and sequenced, one shows 100% identity with the rat heavy chain ferritin (H-ferritin) mRNA. Northern blot analysis confirmed the up-regulation of the H-ferritin mRNA. Using dot blotting, a 40% increase in expression was reported in the subcortical forebrain of the adult GAERS, while cortex, brain stem, and cerebellum appeared unmodified. This change was not observed in the brain of 25-day-old rats, an age at which the epileptic phenotype is not present. By in situ hybridization, the enhanced expression was localized in the hippocampus. The increase in mRNA encoding H-ferritin was not immunodetected at the protein level by Western blotting. These results are not apparently related to the neural substrate of SWD or to the distribution of local increase in glucose metabolism previously described in the GAERS. It is hypothesized that the up-regulation of the H-ferritin mRNA is part of a mechanism protecting the hippocampus, a seizure-prone area, against a possible overactivation during absence seizures.

Tobacco smoking is considered a major risk factor for the development and progression of periodontal diseases (Haber, J. and Wattles, J. (1994). J. Periodontol., 64, 16-23). The purpose of this study was to determine the effects of nicotine on rat gingival fibroblasts (RGF) cultured in vitro. After ether anesthesia, rat gingival tissues were obtained from the attached gingiva of a Wistar rat. Small fragments of gingiva were maintained in culture in Petri dishes. Fibroblasts developing from these explants were collected to obtain monolayer cultures. After the fourth passage (T4), cells were supplemented with nicotine at various concentrations. Control and treated cells were examined under phase contrast or transmission electron microscopy. They were compared as regards their DNA content, mitochondrial activity, collagen and protein synthesis, and cell death by apoptosis or necrosis. Nicotine from 0.05 microM to 1 mM did not affect the DNA content or protein and collagen synthesis. At concentrations between 3 and 5 mM, growth was significantly diminished and the survival rate reduced. Ultrastructural analysis revealed dilated mitochondria and vacuolization in treated cells, suggestive of necrosis, but increased apoptosis was also revealed by cytometry. On the basis of this in vitro study, it appears that tobacco, through its component nicotine, may directly affect various functions of RGF.

A partial estrogen receptor-beta (ERbeta) cDNA had been previously cloned and sequenced in Japanese quail. The 3'- and 5'-rapid amplification of cDNA ends techniques were used here to identify a cDNA sequence of the quail ERbeta that contains a complete open reading frame. For the first time in an avian species, this cDNA sequence and the corresponding amino acid sequence are described. They are compared with the known ERbeta sequences previously described in mammals and with the ERalpha sequences identified in a selection of mammalian and avian species. The analysis by Northern blotting of the ERbeta mRNA expression in the brain and kidneys revealed the presence of several transcripts. The presence of ERbeta identified by reverse transcriptase-polymerase chain reaction demonstrated a widespread distribution quite different from the distribution of ERalpha. The complete neuroanatomical distribution of ERbeta mRNA as determined by in situ hybridization with 35S- and 33P-labeled oligoprobes is also presented. Transcripts are present in many nuclei implicated in the control of reproduction such as the medial preoptic nucleus, the nucleus striae terminalis, and the nucleus taeniae, the avian homologue of the amygdala. These data demonstrate the presence of ERbeta in a nonmammalian species and indicate that the (neuro)-anatomical distribution of this receptor type has been conserved in these two classes of vertebrates. The role of this receptor in the control of reproduction and other physiological processes should now be investigated.

Low voltage-activated calcium channels are thought to play a key role in the generation of spike and waves discharges characteristic of absence epilepsy. Therefore, the expression level of mRNA encoding calcium channel alpha1E and alpha1G subunits was measured in the brain of genetic absence epilepsy rats from Strasbourg (GAERS). Using quantitative RT-PCR and in situ hybridization, no difference was found in alpha1G mRNA expression between GAERS and control animals, while a decreased expression of alpha1E was seen in the cerebellum and the brain stem of the GAERS. This phenomenon was not observed in young animals when the epileptic phenotype is not expressed.

A partial estrogen receptor beta (ER-beta) cDNA was isolated from testicular quail RNA by RT-PCR with degenerate primers specific to the rat ER-beta sequence. A high expression of ER-beta was demonstrated by RT-PCR in the telencephalon, diencephalon, pituitary, testis and kidneys of male quail but little or no expression was detected in the cerebellum, pectoral muscle and adrenal gland. In situ hybridization with a 35S-labelled oligoprobe in sections through the preoptic area-rostral hypothalamus identified high expression in the medial preoptic nucleus, bed nucleus striae terminalis and nucleus taeniae. These data demonstrate the presence of an ER-beta in brain areas implicated in the control of reproduction in a non-mammalian species.

In order to isolate new G protein-coupled receptors expressed in the cerebral cortex, a set of degenerate oligonucleotides corresponding to the third and seventh transmembrane segment were synthetized. Their use in PCR on rat brain cortex mRNA amplified several cDNA fragments. One of them, a 526bp sequence, encoded for what was at that time an unknown G protein-coupled receptor. An oligonucleotide derived from the sequence was then used as a probe to isolate the receptor cDNA from a rat brain cDNA library. It encodes for a 353aa protein with seven transmembrane segments, three consensus N-glycosylation sites at the amino terminus and several potential phosphorylation sites in the intracellular loops. This protein shares 91% overall identity with a recently cloned human somatostatin-like receptor of 402aa named SLC-1. This suggests that we have cloned the rat orthologue of the human SLC-1. However, the extracellular N-terminus of the human receptor is 49 amino acids longer and shows 50% identity with the rat one. Because the human sequence was deduced from genomic DNA, we suspected the presence of an intron in the gene. This was confirmed by PCR using primers spanning the intron. On the basis of the sequence of a 128kb fragment of chromosome 22 encompassing the SLC-1 gene, we were able to deduce a corrected amino acids sequence for the human receptor. So both rat and human SLC-1 receptors are 353aa long, with three consensus N-glycosylation sites. They share 96% identity at the amino acid level and are encoded by a gene containing one intron in the coding sequence.

Chronic food restriction (FR) and maintenance of low body weight have long been known to increase the rewarding and motor-activating effects of addictive drugs. However, the neurobiological mechanisms through which FR potentiates drug reward remain largely unknown. Melanin-concentrating hormone (MCH) signaling could be one of these mechanisms since this peptide is involved in energy homeostasis and modulates mesolimbic dopaminergic transmission. The purpose of the present study was to test this hypothesis by investigating the impact of FR on amphetamine reward in wild-type (WT) and knockout mice lacking the melanin-concentrating hormone receptor-1 (MCHR1-KO). The rewarding effects of amphetamine (0.75-2.25 mg/kg, i.p.) were measured with the conditioned place preference (CPP) technique. The food of the mice was restricted to maintain their body weight at 80-85% of their free-feeding (FF) weight throughout the entire CPP experiment. Locomotor activity of the animals was recorded during the conditioning sessions. Our results show that locomotion of all the food-restricted mice treated with saline or amphetamine increased over the sessions whatever the genotype. On the place preference test, the amplitude of CPP induced by 0.75 mg/kg amphetamine was higher in food restricted WT mice than in free-fed WT mice and food restricted MCHR1-KO mice. However, FR did not affect amphetamine reward in MCHR1-KO mice. The present results indicate that MCH signaling could be involved in the ability of FR to increase amphetamine-induced CPP.

In the course of our investigations on Umutambasha in order to identify its convulsant principles, small quantities of monofluoroacetate were observed in stem bark, leaves, and fruits of this plant newly identified as Dichapetalum michelsonii Hauman. Conclusive evidence for a monofluoroacetate presence came from its isolation from the freeze-dried extract of stem bark. Three free unusual amino acids, named N -methyl- α -alanine, N -methyl- β -alanine, and 2,7-diaminooctan-1,8-dioic acid, described for the first time in a plant, and known trigonelline were also isolated from the stem bark of D. michelsonii . Structure elucidations were mainly achieved by spectroscopic methods ( 1 H-NMR, 2D-NMR, MS) and by comparison with authentic references. These unusual amino acids were detected by a fast, reliable TLC analysis in all our batches of Umutambasha, suggesting that they could be used for identification purposes in case of human or livestock intoxications. Finally, EEG recordings and behavioural observations performed in mice suggested that the convulsive patterns produced by Umutambasha are the consequence of monofluoroacetate presence in D. michelsonii.

Clinical data suggest that high-dose thiamine (vitamin B1) may have a mild beneficial effect in some patients with Alzheimer's disease (AD). Since this action could be related to a brain thiamine deficiency, we measured directly levels of free (nonphosphorylated) thiamine and its phosphate esters, thiamine monophosphate and thiamine diphosphate (TDP), and activities of three TDP-metabolizing enzymes (thiamine pyrophosphokinase, thiamine diphosphatase, and thiamine triphosphatase) in autopsied cerebral cortex of 18 patients with AD and 20 matched controls. In the AD group, mean levels of free thiamine and its monophosphate ester were normal, whereas levels of TDP were significantly reduced by 18 to 21% in all three cortical brain areas examined. Activities of the TDP-metabolizing enzymes were normal in the AD group, suggesting that decreased TDP is not due to altered levels of these enzymes. The TDP decrease could be explained by a cerebral cortical deficiency in AD of ATP, which is needed for TDP synthesis. Although the magnitude of the TDP reduction is slight, a chronic subclinical TDP deficiency could contribute to impaired brain function in AD and might provide the basis for the modest improvement by thiamine in cognitive status of some patients with AD.

Heterozygous mutations in Myoclonin1/EFHC1 cause juvenile myoclonic epilepsy (JME), the most common form of genetic generalized epilepsies, while homozygous F229L mutation is associated with primary intractable epilepsy in infancy. Heterozygous mutations in adolescent JME patients produce subtle malformations of cortical and subcortical architecture, whereas homozygous F229L mutation in infancy induces severe brain pathology and death. However, the underlying pathological mechanisms for these observations remain unknown. We had previously demonstrated that EFHC1 is a microtubule-associated protein (MAP) involved in cell division and radial migration during cerebral corticogenesis. Here, we show that JME mutations, including F229L, do not alter the ability of EFHC1 to colocalize with the centrosome and the mitotic spindle, but act in a dominant-negative manner to impair mitotic spindle organization. We also found that mutants EFHC1 expression disrupted radial and tangential migration by affecting the morphology of radial glia and migrating neurons. These results show how Myoclonin1/EFHC1 mutations disrupt brain development and potentially produce structural brain abnormalities on which epileptogenesis is established.

The epilepsies affect around 50 million people worldwide and constitute in 2012 the second neurological group of diseases. Anyone can suffer one type of epilepsy with more or less severe repeated seizures independently of age, race, or of socioeconomic or geographic situation. In Europe, the prevalence of epilepsy is of 8.2 per 1,000 corresponding to 6 million individuals with active epilepsy and around 15 million having experienced epilepsy during their lives (see [1]). Since almost two or even three decades now, the epileptologists repeatedly claim that in spite of the scientific progress in the field of basic neurosciences on the one hand and in clinical and neuropharmacological research on the other hand, 30–40 % of their patients remain incurable or at least drug-resistant. It took a long time to recognize the extreme diversity of the epileptic syndromes and to remain concentrated on the treatment of the seizures themselves. One of the symptoms of this is the ongoing use of the term ‘‘Epilepsy’’ in the singular form. For years, this attitude was clearly justified, since the antiepileptic drugs were efficient in at least half of the patients. Therefore, from the clinical point of view, considerable efforts have been developed to better identify and classify new epileptic syndromes and sometimes diseases. Truly, the principal symptom that links the different epileptic diseases is the seizure itself, a phenomenon due to the sudden occurrence in a particular area of the cerebral cortex of an epileptogenic circuit probably starting by hypersynchronous bioelectrical discharges in a set of cortical neurons then propagating in specific parts of the brain through synaptic connections. Therefore, for years, the therapeutic efforts tended to limit excitation and/or to favour inhibition considering epilepsy (i.e. ‘‘in singular’’) as a clear imbalance between these two phenomena. Although several experimental evidences illustrated that one can produce huge epileptogenic circuits by favouring GABAergic inhibitory inputs in some circumstances, one has to observe that this ‘‘old thought’’ is still hard to tackle among the clinical and also the pharmacological world. Since 1995, positional cloning strategies in multigenerational families with autosomal dominant transmission have revealed several genes (KCNQ2, KCNQ3, CHRNA4, CHRNA2, CHRNB2, SCN1B, SCN1A, SCN2A, GABRG2, GABRA1...) and numerous loci for febrile seizures and epilepsies. Most of these genes encode neuronal ion channel or neurotransmitter receptor subunits creating a first enthusiastic hypothesis that at least idiopathic generalized epilepsies could be considered as a new group of channelopathies. However, molecular approaches have revealed great genetic heterogeneity, with most genes remaining to be identified. One of the major challenges is now to understand phenotype–genotype correlations. Following these advances, it became more and more popular to think of the antiepileptic treatment in terms of their basic mechanisms of action, i.e. acting on Na or K channels or GABA transmission, etc...., as if a specific and precise molecular mechanism of action in given epileptic syndromes (childhood absence epilepsy, medial temporal lobe epilepsy, or the Dravet syndrome, etc....) were completely elucidated. We have to admit that this probably still constitutes an illusion. Today, the effort to clarify the effectiveness of a rationale of antiepileptic treatment on a (somewhat empirical) clinical basis and multidisciplinary T. Grisar (&) CHU, Department of Neurology, GIGA Neurosciences Research Centre, University of Liege, Liege, Belgium e-mail: tgrisar@ulg.ac.be

Background Thiamine (vitamin B1) is an essential molecule for all life forms because thiamine diphosphate (ThDP) is an indispensable cofactor for oxidative energy metabolism. The less abundant thiamine monophosphate (ThMP), thiamine triphosphate (ThTP) and adenosine thiamine triphosphate (AThTP), present in many organisms, may have still unidentified physiological functions. Diseases linked to thiamine deficiency (polyneuritis, Wernicke-Korsakoff syndrome) remain frequent among alcohol abusers and other risk populations. This is the first comprehensive study on the distribution of thiamine derivatives in human biopsies, body fluids and cell lines. Methodology and Principal Findings Thiamine derivatives were determined by HPLC. In human tissues, the total thiamine content is lower than in other animal species. ThDP is the major thiamine compound and tissue levels decrease at high age. In semen, ThDP content correlates with the concentration of spermatozoa but not with their motility. The proportion of ThTP is higher in humans than in rodents, probably because of a lower 25-kDa ThTPase activity. The expression and activity of this enzyme seems to correlate with the degree of cell differentiation. ThTP was present in nearly all brain and muscle samples and in ∼60% of other tissue samples, in particular fetal tissue and cultured cells. A low ([ThTP]+[ThMP])/([Thiamine]+[ThMP]) ratio was found in cardiovascular tissues of patients with cardiac insufficiency. AThTP was detected only sporadically in adult tissues but was found more consistently in fetal tissues and cell lines. Conclusions and Significance The high sensitivity of humans to thiamine deficiency is probably linked to low circulating thiamine concentrations and low ThDP tissue contents. ThTP levels are relatively high in many human tissues, as a result of low expression of the 25-kDa ThTPase. Another novel finding is the presence of ThTP and AThTP in poorly differentiated fast-growing cells, suggesting a hitherto unsuspected link between these compounds and cell division or differentiation.

The arguments supporting the existence of two molecular forms of the (Na + , K + )-ATPase in mammalian brain are reviewed. 1. Bulk isolated glial cells (Na + , K + )-ATPase activities are highly stimulated by K + ions between 5 and 20 mM. This phenomenon was reproduced with plasma membranes prepared from these cells but did not exist in neuronal (i.e. perikarial cells, neuronal membranes or synaptosomes) preparations. 2. This glial enzyme characteristic was not observed in young animals with histoenzymologically immature astrocytes. 3. The glial K + -dependent-paranitrophenylphosphatase (K + -pNPPase) was also activated by [K + ] o from 5 to 20 mM while no significant modification was shown for neuronal enzyme. 4. Kinetic analysis of the (Na + , K + )-ATPase activities, on the basis of the hysteretic model, demonstrated that the so-called physiological efficiency (V max /K m app) of glial enzyme was highly increased by [K + ] o from 5 to 20 mM. This phenomenon was not observed in neuronal preparations. These data indicated the existence in glial membranes of a (Na + , K + )-ATPase different from the neuronal enzyme and, because its particular structure, highly activated in presence of elevated [K + ] o (20 mM) the evolutionnary significance of this phenomenon is an active control of [K + ] o by glia.

To date, melanin-concentrating hormone (MCH) has been generally considered as peptide acting almost exclusively in the central nervous system. In the present paper, we revise the experimental evidence, demonstrating that MCH and its receptors are expressed by cells of the immune system and directly influence the response of these cells in some circumstances. This therefore supports the idea that, as with other peptides, MCH could be considered as a modulator of the immune system. Moreover, we suggest that this could have important implications in several immune-mediated disorders and affirm that there is a clear need for further investigation.

SUMMARY: The activity of carbonic anhydrase (CA), a glial enzyme, was measured in the epileptic cortex of audiogenic DBA/2 mice and of cats with a freeze lesion. In mice, the activity increased with age from birth to 24 days, but were always higher in audiogenic mice than in normal C57/BL mice, reflecting species differences. The difference between the two strains increased sharply from 25 to 40 days of age, after the period of maximal audiogenic susceptibility. Acetazolamide, a CA-specific inhibitor, greatly decreased the seizure severity score of DBA/ 2 mice after a single intraperitoneal (i.p.) administration (150 mg/kg). After 24 days of age, when CA activities were high, the effect of acetazolamide was less important, suggesting that the increased cortical CA activity might reflect a protective mechanism. In cats with a freeze lesion, no significant changes in CA activities were observed in the actively discharging primary and secondary foci as compared with the nonepileptogenic perifocal cortex and the control cortex of sham-operated animals. The results indicate that the cortex of genetically susceptible audiogenic mice has an increased CA activity. The hypothesis of an adaptive glial mechanism, relating to the age-dependent decrease of seizure susceptibility in DBA/2 mice, is postulated. RESUME L'activite de I'anhydrase carbonique (AC), une enzyme gliale, a ete mesuree dans des homogenats de cortex epileptique chez la souris audiogene DBA/2 et chez le chat porteur d'une lesion par le froid. Chez la souris, l'activite enzymatique augmente avec l'âge depuis la naissance jusqu'a 24 jours, mais est toujours superieure chez la souris audiogene par rapport a la souche controle C57/BL, refletant des differences genetiques. Entre 25 et 40 jours de vie postnatale, c'est a dire apres la periode de susceptibilite audiogenique maximale, la difference entre les deux souches s'accroit fortement. L'aceHazolamide, un inhibiteur specifique de l'AC, diminue de facon importante le score de severiteepileptique de la souris DBA/2 apres une administration intrapeitoneale unique (150 mg/kg). Cet effet de l'acetazolamide est moins marque lorsque l'activite AC est elevee, suggerant que l'augmentation de l'activite de l'AC apres 24 jours chez la souris DBA/2 pourrait constituer un meanisme protecteur. Chez le chat porteur d'une leion par le froid, aucune modification significative de l'activite enzymatique n'est observee dans les foyers epileptiques actifs primaires et secondares par rapport au cortex perifocal non epileptique et au cortex controle de chats temoins. Ces resultats indiquent l'existence d'une augmentation de l'activite de l'AC dans le cortex de souris genetiquement audiogeniques. L'hypothese d'un mecanisme glial adaptatif, en relation avec la disparition de la susceptibilite audiogenique avec l'âge chez la souris DBA/2, est evoquee. RESUMEN Se ha medido la actividad de la carbonico-anhidrasa (CA), una enzima glial, en la corteza epileptica de los ratones audiogenicos DBA/2 y en gatos con lesiones por congelacion. En las ratas la actividad se incrementa con la edad desde el nacimiento hasta los 24 dias pero fue siempre mas elevada en ratones audiogenicos que en ratones normales C57/BL, lo que refleja diferencias de especie. Las diferencias entre los dos tipos de raton se incrementaron abruptamente desde los 25 a los 40 dias de edad despues de un periodo de susceptibilidad audiogenica maxima. La acetazolamida, un inhibidor especifico de la CA, incremento la severidad de la frecuencia de ataques de los ratones DBA/2 despues de una inyeccion intraperitoneal unica (150 mg/kg). Despues del dia 24 de edad, cuando la actividad de la CA era elevada, el efecto de la acetazolamida fue menos importante, lo que sugiere que el incremento de la actividad cortical de la CA puede reflejar un mecanismo de proteccion. En gatos con lesiones por congelacion no se observaron cambios significativos en la actividad de la CA cuando se buscaron en la zona activamente primaria de descargas y en los focos secundarios, comparandolos con la corteza periofocal no-epileptogenica y con los animales control con tecnica operatoria similar (Sham). Los resultados indican que la corteza de ratones geneticamente susceptibles a crisis audiogenicas tiene un incremento de la actividad de la CA. Los autores postulan la existencia de un mecanismo adaptativo glial, en relation con una reduccion de la susceptibilidad para ataques edad-dependientes, en los ratones DBA/2. ZUSAMMENFASSUNG Die Aktivitat von Carboanhydrase (CA), einem glialen Enzym, wurde im epileptischen Kortex von audiogenen DBA/2-Mausen und Katzen mit Gefrierlasionen gemessen. Bei Mausen stieg die Aktivitat mit dem Alter von Geburt bis zum 24. Tag an, sie war bei audiogenen Mausen immer hohen als bei normalen C57/BL-Mausen. Der Unterschied zwischen den beiden Stammen stieg stark vom 25. zum 40. Lebenstag an, also nach der Periode der maximalen audiogenen Empfindlichkeit. Acetazolamid, ein spezifischer CA-Hemmer, minderte nach einer einzigen intraperitonealen Gabe (150 mg/kg) stark den Schweregrad-Rang von Anfallen von DBA/2-Mausen. Nach dem 24. Lebenstag, wenn die CA-Aktivitaten hoch waren, war die Wirkung von Acetazolamid weniger deutlich, was nahelegt, das die erhohte kortikale CA-Aktivitat einen protektiven Mechanismus wiedergibt. Bei Katzen mit Gefrierlasionen waren keine signifikanten CA-Aktivitatsanderungen bei aktiv entladenden primaren und sekundaren Foci zu sehen im Vergleich zum nichtepileptogenen perifokalen Kortex und zu scheinoperierten Kontrolltieren. Die Ergebnisse zeigen, das der Kortex von genetisch-audiogenen Mausen eine erhohte CA-Aktivitat hat. Es wird die Hypothese aufgestellt von einem adaptiven glialen Mechanismus, der alterabhangig zu einem Abfall der Anfallsempfindlichkeit bei DBA/2-Mausen fuhrt.

Growing amounts of data indicate involvement of the posterior hypothalamus in the regulation of sleep, especially paradoxical sleep (PS). Accordingly, we previously showed that the melanin-concentrating hormone (MCH)-producing neurons of the rat hypothalamus are selectively activated during a PS rebound. In addition, intracerebroventricular infusion of MCH increases total sleep duration, suggesting a new role for MCH in sleep regulation. To determine whether activation of the MCH system promotes sleep, we studied spontaneous sleep and its homeostatic regulation in mice with deletion of the MCH-receptor 1 gene (MCH-R1-/- vs. MCH-R1+/+) and their behavioural response to modafinil, a powerful antinarcoleptic drug. Here, we show that the lack of functional MCH-R1 results in a hypersomniac-like phenotype, both in basal conditions and after total sleep deprivation, compared to wild-type mice. Further, we found that modafinil was less potent at inducing wakefulness in MCH-R1-/- than in MCH-R1+/+ mice. We report for the first time that animals with genetically inactivated MCH signaling exhibit altered vigilance state architecture and sleep homeostasis. This study also suggests that the MCH system may modulate central pathways involved in the wake-promoting effect of modafinil.

The mRNA encoding prepro-Melanin concentrating hormone (ppMCH) is mainly expressed in the central nervous system but has also been detected at lower amount in many peripheral tissues including spleen and thymus. At the peptide level however, several forms of the precursor can be detected in these tissues and are sometimes expressed at similar levels compared to brain. In the present work, we have studied the in vitro action of a wide range of concentration (1 nM to 1 microM) of the different peptides encoded by ppMCH i.e. neuropeptide glycine-glutamic acid (NGE), neuropeptide glutamic acid-isoleucine (NEI), Melanin concentrating hormone (MCH) and the dipeptide NEI-MCH on peripheral blood mononuclear cells (PBMC) proliferation and cytokine production following anti-CD3 stimulation. Among them only MCH decreased PBMC proliferation with a maximal effect of 35% at 100 nM. Moreover as demonstrated by using ELISA, MCH significantly decreases IL-2 production by 25% but not IL-4, INF-gamma or TNF-alpha expression. Interestingly, exogenous IL-2 decreases significantly MCH-mediated inhibition, suggesting that it is an important downstream mediator of MCH action. Finally, we showed that after 7 to 9 days of incubation, MCH also inhibits proliferation of non-stimulated PBMC. Altogether, these data demonstrate that fully mature MCH modulates proliferation of anti-CD3 stimulated PBMC partially through regulation of IL-2 production.

Previous studies have shown that intraperitoneal injections of thioperamide, an imidazole-based H3 receptor inverse agonist that enhances histamine release in the brain, potentiate cocaine-induced hyperlocomotion. The present study examined the involvement of the histaminergic system in these effects of thioperamide in mice.We investigated whether immepip, a selective H3 agonist, could reverse the potentiating effects of thioperamide. Moreover, the non-imidazole H3 inverse agonist A-331440 was tested on the locomotor effects of cocaine. Using high-performance liquid chromatography with ultraviolet detection, cocaine plasma concentrations were measured to study potential drug-drug interactions between thioperamide and cocaine. Finally, thioperamide was tested on the locomotor effects of cocaine in histamine-deficient knockout mice in order to determine the contribution of histamine to the modulating effects of thioperamide.Thioperamide potentiated cocaine-induced hyperlocomotion in normal mice, and to a higher extent, in histamine-deficient knockout mice. A-331440 only slightly affected the locomotor effects of cocaine. Immepip did not alter cocaine-induced hyperactivity but significantly reduced the potentiating actions of thioperamide on cocaine's effects. Finally, plasma cocaine concentrations were more elevated in mice treated with thioperamide than in mice that received cocaine alone.The present results indicate that histamine released by thioperamide through the blockade of H3 autoreceptors is not involved in the ability of this compound to potentiate cocaine induced-hyperactivity. Our data suggest that thioperamide, at least at 10 mg/kg, increases cocaine-induced locomotion through the combination of pharmacokinetic effects and the blockade of H3 receptors located on non-histaminergic neurons.

The present study aimed at characterizing the acute and intermittent psychomotor responsiveness to cocaine in mice lacking the MCHR1 receptor, which is thought to modulate the mesocorticolimbic sytem functioning [Smith DG, Tzavara ET, Shaw J, Luecke S, Wade M, Davis R, et al. Mesolimbic dopamine super-sensitivity in melanin-concentrating hormone-1 receptor deficient mice. J Neurosci 2005;25:914–22]. On a first free-drug session, MCHR1-deficient mice exhibited significantly higher levels of locomotor activity elicited by the novelty of the test chambers than their wild-type counterparts. On the following day session, a first injection of 6 or 12 mg/kg cocaine induced comparable dose-related psychomotor activations in both genotypes, without significant difference in the relative increase in locomotion. Over the following eight once-daily test sessions, the slight psychomotor increase induced by 6 mg/kg was equivalent in both genotypes and constant over the sessions. At 12 mg/kg, cocaine induced a clear-cut incremental responsiveness to cocaine in both genotypes on the three first sessions; on the following sessions, only the wild-types displayed an incremental responsiveness until the last session, a sensitized effect that was confirmed for the wild-types but not for the knockouts on a subsequent sensitization test (cocaine challenge). Finally, the knockouts did not exhibit any sign of cocaine-conditioning (saline challenge), contrarily to the wild-types. It is speculated that MCHR1 may contribute to the neurobiological mechanisms of conditioned cocaine-induced psychomotor effects, possibly to those underpinning sensitization, and to a lesser extent to those sub-serving acute pharmacological cocaine action.

Item does not contain fulltext Thiamine triphosphate (ThTP) is found at low concentrations in most animal tissues and it may act as a phosphate donor for the phosphorylation of proteins, suggesting a potential role in cell signaling. Two mechanisms have been proposed for the enzymatic synthesis of ThTP. A thiamine diphosphate (ThDP) kinase (ThDP+ATP if ThTP+ADP) has been purified from brewer's yeast and shown to exist in rat liver. However, other data suggest that, at least in skeletal muscle, adenylate kinase 1 (AK1) is responsible for ThTP synthesis. In this study, we show that AK1 knockout mice have normal ThTP levels in skeletal muscle, heart, brain, liver and kidney, demonstrating that AK1 is not responsible for ThTP synthesis in those tissues. We predict that the high ThTP content of particular tissues like the Electrophorus electricus electric organ, or pig and chicken skeletal muscle is more tightly correlated with high ThDP kinase activity or low soluble ThTPase activity than with non-stringent substrate specificity and high activity of adenylate kinase.

Summary: Purpose: We present results obtained by computer modeling of the thalamic network and differential gene expression analysis in a rat strain with absence epilepsy, the genetic absence epilepsy rat from Strasbourg (GAERS). Methods: (a) Computer modeling used equations from the Hodgking–Huxley model with a circuit of 13 reticular thalamic (nRt) and 39 thalamocortical (TC) neurons; (b) gene-expression analysis using differential mRNA display (DD), in situ hybridization, Northern blotting, and competitive reverse transcriptase–polymerase chain reaction (RT-PCR). Results: (a) Computer modeling showed an increased network synchrony in the thalamic circuit as the value of conductance of low-voltage activated calcium channel (LVACC) is increased. (b) Using differential mRNA display, a 40% upregulation of the H-ferritin mRNA in the hippocampus was demonstrated. Looking for some candidate genes of the VACC family, no difference was found in the α1G mRNA expression between GAERS and control animals, whereas a decreased expression of the α1E subunit was observed in the cerebellum and the brainstem of the GAERS. This phenomenon was not observed in young animals when the epileptic phenotype is not expressed. Conclusions: The use of computer modeling appeared to be an efficient way to evaluate the impacts of electrophysiologic findings in vivo from single cells on an entire circuit. No clear single gene defect was revealed so far in GAERS. More information could arise from linkage analysis. However, some brain structures like hippocampus or cerebellum classically not known to be involved in the production of absence spike-and-wave discharges could in fact participate in the development of this phenotype.