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Le canal Kv11.1, dont l’inhibition occasionne une prolongation de l’intervalle QT, est directement impliqué dans des cas d’effets secondaires cardiotoxiques. Depuis 2006, Santé Canada exige que les nouvelles molécules et leurs métabolites soient évalués en phase préclinique pour le risque d’allongement de l’intervalle QT. La méthode de référence évalue l’électrophysiologie des cardiomyocytes en culture lors d’une courte exposition au médicament (<30min). Bien que cette méthode soit la plus fiable actuellement, elle permet seulement d’identifier les molécules qui bloquent directement le passage des ions dans le pore du canal (effet aigu). La méthode HERG-BRET vise à identifier les molécules susceptibles d’interagir avec le canal Kv11.1 par le moyen d’une altération du trafic vésiculaire (effet chronique). Ce type d’interaction est considéré comme un biomarqueur de la capacité à bloquer l’activité de ce canal. L’étude présentée tente de déterminer si un test de localisation cellulaire de hERG basé sur le BRET permettra un criblage à haut débit et une meilleure évaluation de l’affinité d’interaction avec hERG, comparativement aux méthodes alternatives actuelles. Dans le modèle HERG-BRET, la protéine hERG fusionnée à la luciférase de renilla (donneur d’énergie) est exprimée dans une lignée cellulaire HEK293. Cette même lignée exprime également une protéine verte fluorescente modifiée (accepteur d’énergie) qui est ancrée à la membrane plasmique. L’échange d’énergie entre le donneur et l’accepteur est un indice de la localisation de hERG à la membrane plasmique. Les fluctuations de ratio BRET suite à une exposition de 16h à un composé pharmaceutique reflètent donc l’effet du composé sur la translocation de Kv11.1. Vingt-cinq composés pharmaceutiques déjà caractérisés dans la littérature scientifique ont été testés : 12 ont été classés comme chaperons pharmacologiques, 4 comme inhibiteurs du trafic, 1 comme inhibiteur ayant les deux effets mentionnés et 8 n’ont pas pu être classés. Le comportement du biosenseur à l’égard des composés testés suggère que la méthode HERG-BRET ne peut pas être utilisée seule pour évaluer le risque cardiotoxique des médicaments. Toutefois, elle peut fournir des informations complémentaires pertinentes quand à la nature de l’interaction entre un composé pharmaceutique et la sous unité hERG du canal Kv11.1. / The Kv11.1 channel is directly involved in cardiotoxic adverse effects since its inhibition is responsible for a prolongation of the QT interval. In 2006, Health Canada established a guideline that constrains drug developers to a preclinical evaluation of QT prolongation risks for new molecules and their metabolites. The gold standard method (patch-clamp) consists in electrophysiology measurements on cultured cardiomyocytes for a brief exposition to the tested compound (<30min). Even though this method is the most reliable, it only allows the identification of molecules that inhibit the channel by preventing ions from traveling through the pore (acute effect). The HERG-BRET method aims to identify molecules that can interact with Kv11.1 and alter its vesicular transport as a proxy for inhibiting the activity of the channel (chronic effects). This study attemps to determine if a BRET-based cellular localization assay will allow a high throughput screening and a better evaluation of the affinity of pharmaceuticals compounds with hERG, in comparison to alternative methods. In the HERG-BRET model, a fusion protein generated with the gene sequence for hERG and the one for the renilla luciferase (energy donor) is stably expressed in a HEK 293 cell line. The same cell line also stably expresses a green fluorescent protein (energy acceptor) that is anchored at the plasma membrane. The energy transfer that occurs between the donor and the acceptor suggests that hERG is located at the membrane. Variations of BRET ratios following a 16 hours inucabtion with a compound reflects the compound’s effects on Kv11.1’s translocation. Twenty-five compounds that have been previously characterized in the literature were tested: 12 were categorized as pharmacological chaperones, 4 as traffic inhibitors, 1 as an inhibitor that undergoes both effects and 8 remain uncategorized. The biosensor’s behavior towards the tested compounds suggests that the HERG-BRET method cannot be used alone to assess cardiotoxic liability, but it can bring interesting facts to our attention regarding the nature of the interaction between the hERG subunits of Kv11.1 and a tested compound.

Background potassium currents play an important role in the regulation of the resting membrane potential and excitability of mammalian neurons. Recently cloned two- pore domain potassium channels (K2p) are believed to underlie these currents. The roles of K2P channels in general anesthesia and neuroprotection have been proposed recently. In view of this, we investigated the ability of trichloroethanol (an active metabolite of the non-volatile general anesthetic cldoral hydrate, widely used as a pediatric sedative) to modulate the activity of human TREK-1 and TRAAK channels. We found that trichloroethanol potently activates both hTREK-1 and hTRAAK channels at pharmacologically relevant concentrations. The parent compound chloral hydrate was also found to augtnent the activity of both the channels reversibly. Studies with carboxy- terminal deletion mutants (hTREK-1A89, hTREK-1 A100 and hTREK-1 A1 19), suggested that C-terminal tail is not essential for the activation of TREK-1 by trichloroethanol. Our findings identify TREK-1 and TRCL4K channels as molecular targets for trichloroethanol and we propose that activation of both these channels might contribute to the CNS depressant effects of chloral hydrate. Another channel TASK-2, which is essentially absent in the human brain was also found to be potently activated by both trichloroethanol and chloral hydrate. In another series of experiments, we studied the effects of methyl xanthines caffeine and theophylline on hTREK-1 channels. Caffeine and theophylline are used for therapeutic purposes and frequently cause life-threatening convulsive seizures due to systemic toxicity. The mechanisms for the epileptogenicity of caffeine and theophylline are not clear. Recent experiments using knockout mice provided direct evidence for a role for TREK-1 in the control of epileptogenesis. We hypothesized that the epileptogenicity of caffeine and theophylline may be related to the inhibition of TREK-1 channels. We investigated this possibility and observed massive inhibition of TREK-1 channels at toxicologically relevant concentrations. Experiments with the mutant TREK-1 channel (S348A mutant) suggested the involvement of cANP/PKA pathway in the inhibition of TREK-1 channels by caffeine and theophylline. We suggest that inhibition of TREK-1 channels may contribute to the convulsive seizures induced by toxic levels of caffeine and theophylline. Local anesthetics exhibit their clinical effects not only by binding to voltage-gated sodium channels, but also by interacting with other ion channels such as potassium channels. Because of the physiological significance of TREK-1 channels and their abundant expression in peripheral sensory neurons, we investigated the effects of lidocaine to see whether its interaction with 'REK-1 channels contribute to the conduction blockade. Lidocaine caused dose-dependent inhibition of TREK-1channels and the inhibition was voltage-independent. Cytoplasmic C-terminal tail is critically required for lidocaine action. Inhibition of TREK-1 channels is achieved at concentrations for iiz vivo action and this effect may have implications for the clinically observed drug action of lidocaine.

The transmission of sex pheromone-mediated signals is essential for goldfish reproduction. However, the neural pathways underlying this reproductive signalling pathway in the goldfish brain is not well described. Lesioning experiments have shown previously that two brain areas, the preoptic area (POA) and the ventral telencephali pars ventralis (Vv) in particular, are important for reproduction. We used patch clamp electrophysiology to study the electrical activities of POA and Vv neurons. Based on the intrinsic properties of these neurons, we suggest there are five different functional classes of POA neurons and a single class of Vv neurons. In addition, by electrically stimulating the olfactory bulb (OB), we were able to show that this primary sensory structure makes monosynaptic glutamatergic connections with both POA and Vv neurons. While electrophysiology measures signalling events occurring at short time scales on the order of milliseconds to minutes, we were also interested in studying sex pheromone signalling in the goldfish brain over a long time scale. Thus, we describe changes in gene expression in male goldfish exposed to waterborne sex pheromones (17alpha,20beta dihydroxy-4-pregene-3-one and Prostaglandin-F2alpha) over 6 hours. We perform cDNA microarrays on Prostaglandin-F2alpha-treated fish to study the rapid modulation of transcription and define the signalling pathways affected. Our microarrays showed that 71 genes were differentially regulated (67 up and 4 down). Through gene ontology enrichment analysis, we found that these genes were involved in various biological processes such as RNA processing, neurotransmission, neuronal development, apoptosis, cellular metabolism and sexual reproduction. RT-PCRs were performed to validate our microarrays and to facilitate direct comparisons of the effects of the two sex pheromones, 17alpha,20beta dihydroxy-4-pregene-3-one and Prostaglandin-F2alpha. By combining electrophysiology and gene expression analyses, we were able to study sex-pheromone signalling on two different time scales. One short, occurring on the order of milliseconds to minutes, that involves electrical activities in the brain through the glutamatergic amino-3-hydroxy-5-methylisoxazole-4-propionate and N-methyl-D-aspartate receptors; and the other long occurring several hours later that involves changes in the gene expression levels of calmodulin and ependymin among other genes underlying neuroplasticity. Reproductive neuroplasticity in the goldfish may therefore require the activation of glutamatergic receptors which then activate downstream signals like calmodulin and ependymin to transform the sex pheromones-mediate signal into gene expression.

Molecular mechanism of Cannabinoid receptor 1 regulation by SGIP1 Abstract Src homology 3-domain growth factor receptor-bound 2-like endophilin interacting protein 1 (SGIP1) has been identified as an interacting partner of cannabinoid receptor 1 (CB1R). Their protein-protein interaction was confirmed by co-immunoprecipitation. SGIP1 hinders the internalization of activated CB1R and modulates its signaling in HEK293 cells. Employing whole-cell patch-clamp electrophysiology, we have shown that SGIP1 affects CB1R signaling in autaptic hippocampal neurons. Using a battery of behavioral tests in SGIP1 constitutive knock-out (SGIP1-/- ) and WT mice, we investigated the consequences of SGIP1 deletion on behavior regulated by the endocannabinoid system. In SGIP1-/- mice, exploratory levels, working memory and sensorimotor gating were unaltered. SGIP1-/- mice showed decreased anxiety-like and depressive-like behaviors. Fear extinction to tone was enhanced in SGIP1-/- females. Several cannabinoid tetrad behaviors were altered in the absence of SGIP1. SGIP1-/- males exhibited abnormal THC withdrawal behaviors. SGIP1 deletion also reduced acute nociception, and SGIP1-/- mice were more sensitive to antinociceptive effects of CB1R agonists and morphine. CB1R-SGIP1 interaction results in profound modification of CB1R...

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Dans la région CA1 de l’hippocampe, une population d’interneurones exprimant la somatostatine (SOM-INs) est reconnue pour une potentialisation à long terme (PLT) dépendante des récepteurs métabotropes du glutamate de type 1a (mGluR1a) à leurs synapses excitatrices provenant des cellules pyramidales (CP). Il a récemment été démontré que cette PLT est induite par l’apprentissage contextuel lié à la peur, illustrant l’importance de cette PLT des SOM-INs dans l’apprentissage et la mémoire. Cependant, l’implication du neuropeptide somatostatine (SST) dans cette PLT demeure inconnue. Dans la présente étude, le rôle de la SST dans la PLT dépendante des mGluR1a a été étudié, tout comme, l’effet de la somatostatine-14 (SST-14) exogène aux synapses excitatrices des SOM-INs. Pour ce faire, des souris transgéniques exprimant la « enhanced yellow fluorescent protein » (eYFP) sous le contrôle du promoteur de la SST ont été utilisées. Des enregistrements électrophysiologiques jumelés à une approche pharmacologique ont été réalisés sur ces souris. Les résultats suggèrent que la SST-14 exogène engendre une PLT persistante grâce aux récepteurs à la somatostatine 1-5 (SST1-5), aux synapses excitatrices des SOM-INs, mais n’affecte pas les synapses des CP ou bien des interneurones exprimant la parvalbumine (PV-INs). Cette potentialisation induite par SST-14 était indépendante des récepteurs à l’acide N-méthyl-D-aspartique (NMDAR) et mGluR1a, dépendante de l’activité synaptique concomitante et inhibée par le blocage des récepteurs GABAA. De plus, la PLT dépendante des récepteurs mGluR1a a été affectée par l’inhibition des SST1-5 ou bien par un traitement avec de la cystéamine suggérant un rôle pour de la SST endogène dans cette PLT. Nos résultats suggèrent que la SST endogène pourrait contribuer à la PLT hébbienne aux synapses excitatrices des SOM-INs en contrôlant l’inhibition GABAA. La SST aurait alors un rôle dans la modulation de la plasticité à long terme des SOM-INs qui pourrait être important dans la mémoire dépendante de l’hippocampe. / The CA1 region of the hippocampus includes a population of GABAergic interneurons expressing somatostatin (SOM-INs). This type of interneurons displays a long-term potentiation (LTP) dependant on type 1a metabotropic glutamate receptors (mGluR1a) at their excitatory synapses from pyramidal cells (PC). It was recently demonstrated that mGluR1a dependent LTP can be induce by contextual fear learning showing an important role of this LTP in learning and memory. However, the implication of the peptide somatostatin (SST) in this LTP remains unknown. In the present study, the role of SST in mGluR1 dependent LTP and the effect of exogenous somatostatin-14 (SST-14) onto excitatory synapses of SOM-INs were investigated. To do this, transgenic mice expressing enhanced yellow fluorescent protein (eYFP) under the control of the promoter of SST were used. Patch clamp recordings and pharmacological approaches were used with these mice. Results suggested that application of exogenous SST-14 induces a LTP through type 1-5 somatostatin receptors (SST1-5) of excitatory synapses of SOM-INs but does not affect synapses of PC or parvalbumin-expressing interneurons (PV-INs). This LTP induced by SST-14 was independent of N-methyl-D-aspartate receptor (NMDAR) and mGluR1a, activity dependent, and prevented by blocking GABAA receptors. Furthermore, mGluR1a dependent LTP was prevented by inhibition of SST1-5 and by depletion of SST by cysteamine treatment, suggesting a role of endogenous SST in LTP. Our results indicate that endogenous SST may contribute to Hebbian LTP at excitatory synapses by controlling GABAA inhibition. SST would then have a role in regulating SOM-INs LTP that may be important for hippocampus dependent memory processes.

The risks of damage to the developing nervous system of many chemicals are not known because these studies often require costly and time-consuming multi-generational animal experiments. Pluripotent stem cell-based systems can facilitate developmental neurotoxicity studies because disturbances in nervous system development can be modeled in vitro. In this study, neurons derived from embryonal carcinoma (EC) and induced pluripotent stem (iPS) cells, were first characterized to establish their suitability for developmental neurotoxicity studies. The EC stem cell line, TERA2.cl.SP-12, was differentiated into neurons that expressed voltage-gated sodium and potassium channels as well as ionotropic GABA and glutamate receptors. These cells could also fire action potentials when stimulated electronically. However spontaneous action potentials were not observed. In contrast, pre-differentiated neurons derived from iPS cells fired evoked and spontaneous action potentials. Furthermore, iPS cell-derived neurons also expressed a wide array of functional voltage- and ligand-gated ion channels. Antiepileptic drugs (AEDs) are associated with developmental neurotoxicity. These agents can cause congenital malformations, cognitive deficits and behavioral impairment in children as a result of in utero exposure. The impact of four major AEDs, namely phenobarbital, valproic acid, carbamazepine and lamotrigine, on cell viability, cell cycle and differentiation of TERA2.cl.SP-12 into neurons was studied. All AEDs tested reduced differentiating stem cell viability. Valproic acid and carbamazepine increased apoptosis and reduced cell proliferation. A brief exposure to phenobarbital, valproic acid and lamotrigine at the start of differentiation impaired the subsequent generation of neurons. Additionally, the effect of transient exposure to phenobarbital and carbamazepine on neuronal maturation of iPS-derived neurons was investigated. Exposure to both AEDs resulted in diminished membrane potentials and reduced the proportion of cells that were able to fire action potentials spontaneously in culture. The data from these studies suggest that impairments in proliferation, differentiation and maturation of neurons derived from human stem cells may be sensitive indicators of neurodevelopmental disruption by these drugs that can result from in utero exposure. Furthermore, these findings suggest that the use of human pluripotent stem cells and neurons derived from them can reduce the time, cost and the number of animals used in toxicological research.

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