RESUMO
Group II metabotropic glutamate receptors (mGlu2 and mGlu3 receptors) shape mechanisms of methamphetamine addiction, but the individual role played by the two subtypes is unclear. We measured methamphetamine-induced conditioned place preference (CPP) and motor responses to single or repeated injections of methamphetamine in wild-type, mGlu2-/-, and mGlu3-/-mice. Only mGlu3-/-mice showed methamphetamine preference in the CPP test. Motor response to the first methamphetamine injection was dramatically reduced in mGlu2-/-mice, unless these mice were treated with the mGlu5 receptor antagonist, MTEP. In contrast, methamphetamine-induced sensitization was increased in mGlu3-/-mice compared to wild-type mice. Only mGlu3-/-mice sensitized to methamphetamine showed increases in phospho-ERK1/2 levels in the nucleus accumbens (NAc) and free radical formation in the NAc and medial prefrontal cortex. These changes were not detected in mGlu2-/-mice. We also measured a series of biochemical parameters related to the mechanism of action of methamphetamine in naïve mice to disclose the nature of the differential behavioural responses of the three genotypes. We found a reduced expression and activity of dopamine transporter (DAT) and vesicular monoamine transporter-2 in the NAc and striatum of mGlu2-/-and mGlu3-/-mice, whereas expression of the DAT adaptor, syntaxin 1A, was selectively increased in the striatum of mGlu3-/-mice. Methamphetamine-stimulated dopamine release in striatal slices was largely reduced in mGlu2-/-, but not in mGlu3-/-, mice. These findings suggest that drugs that selectively enhance mGlu3 receptor activity or negatively modulate mGlu2 receptors might be beneficial in the treatment of methamphetamine addiction and associated brain damage.
Assuntos
Transtornos Relacionados ao Uso de Anfetaminas/metabolismo , Comportamento Animal/efeitos dos fármacos , Estimulantes do Sistema Nervoso Central/farmacologia , Condicionamento Clássico/efeitos dos fármacos , Metanfetamina/farmacologia , Receptores de Glutamato Metabotrópico/genética , Transtornos Relacionados ao Uso de Anfetaminas/fisiopatologia , Animais , Comportamento Animal/fisiologia , Modelos Animais de Doenças , Dopamina/metabolismo , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Masculino , Camundongos , Camundongos Knockout , Proteína Quinase 1 Ativada por Mitógeno/efeitos dos fármacos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/efeitos dos fármacos , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Neostriado/efeitos dos fármacos , Neostriado/metabolismo , Fosforilação , Córtex Pré-Frontal/efeitos dos fármacos , Córtex Pré-Frontal/metabolismo , Piridinas/farmacologia , Receptor de Glutamato Metabotrópico 5/antagonistas & inibidores , Receptores de Glutamato Metabotrópico/metabolismo , Sintaxina 1/efeitos dos fármacos , Sintaxina 1/metabolismo , Tiazóis/farmacologia , Proteínas Vesiculares de Transporte de Monoamina/metabolismoRESUMO
We investigated the effects of clozapine and haloperidol, drugs that are widely used in the treatment of schizophrenia, on gene expression in six cortical and subcortical brain regions of adult rats. Drug treatments started at postnatal day 85 and continued over a 12-week period. Ten animals received haloperidol (1 mg/kg bodyweight) and ten received clozapine (20 mg/kg bodyweight) orally each day. Ten control rats received no drugs. The ten genes selected for this study did not belong to the dopaminergic or serotoninergic systems, which are typically targeted by the two substances, but coded for proteins of the cytoskeleton and proteins belonging to the synaptic transmitter release machinery. Quantitative real-time PCR was performed in the prelimbic cortex, cingulate gyrus (CG1) and caudate putamen and in the hippocampal cornu ammonis 1 (CA1), cornu ammonis 3 (CA3) and dentate gyrus. Results show distinct patterns of gene expression under the influence of the two drugs, but also distinct gene regulations dependent on the brain regions. Haloperidol-medicated animals showed statistically significant downregulation of SNAP-25 in CA3 (p = 0.0134) and upregulation of STX1A in CA1 (p = 0.0133) compared to controls. Clozapine-treated animals showed significant downregulation of SNAP-25 in CG1 (p = 0.0013). Our results clearly reveal that the drugs' effects are different between brain regions. These effects are possibly indirectly mediated through feedback mechanisms by proteins targeted by the drugs, but direct effects of haloperidol or clozapine on mechanisms of gene expression cannot be excluded.
Assuntos
Antipsicóticos/farmacologia , Córtex Cerebral/efeitos dos fármacos , Clozapina/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Haloperidol/farmacologia , Neostriado/efeitos dos fármacos , Animais , Antipsicóticos/administração & dosagem , Região CA1 Hipocampal/efeitos dos fármacos , Região CA3 Hipocampal/efeitos dos fármacos , Clozapina/administração & dosagem , Giro Denteado/efeitos dos fármacos , Giro do Cíngulo/efeitos dos fármacos , Haloperidol/administração & dosagem , Masculino , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase em Tempo Real , Proteína 25 Associada a Sinaptossoma/efeitos dos fármacos , Sintaxina 1/efeitos dos fármacosRESUMO
Phosphatidylinositol 4,5-bisphosphate (PI 4,5-P(2)) on the plasma membrane is essential for vesicle exocytosis but its role in membrane fusion has not been determined. Here, we quantify the concentration of PI 4,5-P(2) as approximately 6 mol% in the cytoplasmic leaflet of plasma membrane microdomains at sites of docked vesicles. At this concentration of PI 4,5-P(2) soluble NSF attachment protein receptor (SNARE)-dependent liposome fusion is inhibited. Inhibition by PI 4,5-P(2) likely results from its intrinsic positive curvature-promoting properties that inhibit formation of high negative curvature membrane fusion intermediates. Mutation of juxtamembrane basic residues in the plasma membrane SNARE syntaxin-1 increase inhibition by PI 4,5-P(2), suggesting that syntaxin sequesters PI 4,5-P(2) to alleviate inhibition. To define an essential rather than inhibitory role for PI 4,5-P(2), we test a PI 4,5-P(2)-binding priming factor required for vesicle exocytosis. Ca(2+)-dependent activator protein for secretion promotes increased rates of SNARE-dependent fusion that are PI 4,5-P(2) dependent. These results indicate that PI 4,5-P(2) regulates fusion both as a fusion restraint that syntaxin-1 alleviates and as an essential cofactor that recruits protein priming factors to facilitate SNARE-dependent fusion.
Assuntos
Membrana Celular/metabolismo , Exocitose/fisiologia , Fusão de Membrana/fisiologia , Fosfatidilinositol 4,5-Difosfato/metabolismo , Proteínas SNARE/metabolismo , Vesículas Transportadoras/metabolismo , Animais , Membrana Celular/efeitos dos fármacos , Membrana Celular/ultraestrutura , Relação Dose-Resposta a Droga , Exocitose/efeitos dos fármacos , Lipossomos/metabolismo , Fusão de Membrana/efeitos dos fármacos , Microdomínios da Membrana/efeitos dos fármacos , Microdomínios da Membrana/metabolismo , Microdomínios da Membrana/ultraestrutura , Células PC12 , Fosfatidilinositol 4,5-Difosfato/farmacologia , Ratos , Sintaxina 1/efeitos dos fármacos , Sintaxina 1/metabolismo , Vesículas Transportadoras/efeitos dos fármacos , Vesículas Transportadoras/ultraestruturaRESUMO
Syntaxin and Munc18 are, in tandem, essential for exocytosis in all eukaryotes. Recently, it was shown that Munc18 inhibition of neuronal syntaxin 1 can be overcome by arachidonic acid, indicating that this common second messenger acts to disrupt the syntaxin-Munc18 interaction. Here, we show that arachidonic acid can stimulate syntaxin 1 alone, indicating that it is syntaxin 1 that undergoes a structural change in the syntaxin 1-Munc18 complex. Arachidonic acid is incapable of dissociating Munc18 from syntaxin 1 and, crucially, Munc18 remains associated with syntaxin 1 after arachidonic-acid-induced syntaxin 1 binding to synaptosomal-associated protein 25 kDa (SNAP25). We also show that the same principle operates in the case of the ubiquitous syntaxin 3 isoform, highlighting the conserved nature of the mechanism of arachidonic acid action. Neuronal soluble N-ethyl maleimide sensitive factor attachment protein receptors (SNAREs) can be isolated from brain membranes in a complex with endogenous Munc18, consistent with a proposed function of Munc18 in vesicle docking and fusion.