RESUMEN
Rearing rats in single cages from weaning until adulthood (social isolation) produces a number of behavioral and neurochemical alterations similar to those observed in psychoses such as schizophrenia. Also, a dysregulation of the endocannabinoid system has been implicated in schizophrenia. The aim of this study was to examine the effect of social isolation on changes to mRNA expression of 1) the cannabinoid receptor CB(1), 2) enzymes responsible for the synthesis of the endocannabinoids anandamide (N-acyl phosphatidylethanolamine-phospholipase D or NAPE-PLD) and 2-arachidonoyl-glycerol or 2-AG (diacylglycerol lipase or DAGL isozymes alpha and beta) and 3) enzymes that degrade endocannabinoids (fatty acid amide hydrolase/FAAH for anandamide, and monoacylglycerol lipase/MAGL for 2-AG). Twenty-one-day post natal rats were randomly housed individually, or in groups of 6, for 8 weeks. CB(1) receptor, DAGL(alpha) and DAGLbeta, MAGL and FAAH mRNA levels were measured in the brains using in situ hybridization histochemistry. CB(1) receptor, DAGL(alpha), DAGLbeta, MAGL and NAPE-PLD mRNA expression levels were significantly higher in a number of brain regions from socially isolated rats; particularly in the prefrontal regions, cortical layers and a number of thalamic regions. DAGLbeta mRNA was significantly higher in the substantia nigra and ventral tegmental area. FAAH mRNA expression was significantly lower in a number of prefrontal regions, the cortical layers and in the caudate putamen and other associated areas of socially isolated rats. Such differences in endocannabinoid system mRNA in brains of socially isolated rats compared to normal rats further supports the potential importance of the endocannabinoid system in psychotic disease states.
Asunto(s)
Química Encefálica/genética , Moduladores de Receptores de Cannabinoides/fisiología , Endocannabinoides , Regulación de la Expresión Génica/genética , Transducción de Señal/genética , Aislamiento Social/psicología , Estrés Psicológico/genética , Estrés Psicológico/metabolismo , Animales , Modelos Animales de Enfermedad , Masculino , Ratas , Ratas Sprague-Dawley , Estrés Psicológico/fisiopatología , Regulación hacia Arriba/genéticaRESUMEN
We recently identified LY2033298 as a novel allosteric potentiator of acetylcholine (ACh) at the M(4) muscarinic acetylcholine receptor (mAChR). This study characterized the molecular mode of action of this modulator in both recombinant and native systems. Radioligand-binding studies revealed that LY2033298 displayed a preference for the active state of the M(4) mAChR, manifested as a potentiation in the binding affinity of ACh (but not antagonists) and an increase in the proportion of high-affinity agonist-receptor complexes. This property accounted for the robust allosteric agonism displayed by the modulator in recombinant cells in assays of [(35)S]GTPgammaS binding, extracellular regulated kinase 1/2 phosphorylation, glycogen synthase kinase 3beta phosphorylation, and receptor internalization. We also found that the extent of modulation by LY2033298 differed depending on the signaling pathway, indicating that LY2033298 engenders functional selectivity in the actions of ACh. This property was retained in NG108-15 cells, which natively express rodent M(4) mAChRs. Functional interaction studies between LY2033298 and various orthosteric and allosteric ligands revealed that its site of action overlaps with the allosteric site used by prototypical mAChR modulators. Importantly, LY2033298 reduced [(3)H]ACh release from rat striatal slices, indicating retention of its ability to allosterically potentiate endogenous ACh in situ. Moreover, its ability to potentiate oxotremorine-mediated inhibition of condition avoidance responding in rodents was significantly attenuated in M(4) mAChR knockout mice, validating the M(4) mAChR as a key target of action of this novel allosteric ligand.
Asunto(s)
Acetilcolina/metabolismo , Antipsicóticos/farmacología , Unión Competitiva/efectos de los fármacos , Receptor Muscarínico M4/fisiología , Acetilcolina/farmacología , Regulación Alostérica/efectos de los fármacos , Regulación Alostérica/fisiología , Sitio Alostérico/efectos de los fármacos , Sitio Alostérico/fisiología , Animales , Antipsicóticos/química , Reacción de Prevención/efectos de los fármacos , Reacción de Prevención/fisiología , Línea Celular , Cuerpo Estriado/efectos de los fármacos , Cuerpo Estriado/metabolismo , Cricetinae , Cricetulus , Relación Dosis-Respuesta a Droga , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Técnicas In Vitro , Ratones , Ratones Noqueados , Modelos Moleculares , Análisis Multivariante , Antagonistas Muscarínicos/farmacocinética , N-Metilescopolamina/farmacocinética , Ácidos Nicotínicos/química , Ácidos Nicotínicos/farmacología , Parasimpatolíticos/farmacocinética , Fosforilación/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Quinuclidinil Bencilato/farmacocinética , Ensayo de Unión Radioligante/métodos , Ratas , Receptor Muscarínico M4/química , Receptor Muscarínico M4/deficiencia , Receptor Muscarínico M4/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Tiofenos/química , Tiofenos/farmacología , Tritio/metabolismo , Tritio/farmacocinéticaRESUMEN
The M4 muscarinic acetylcholine (ACh) receptor (mAChR) is a potential therapeutic target but characterized by a lack of subtype-selective ligands. We recently generated "designer receptors exclusively activated by a designer drug" (DREADDs), which contained mutations of two conserved orthosteric-site residues (Y113C/A203G in the M4 mAChR) that caused a loss of ACh activity but a gain in responsiveness to clozapine-N-oxide (CNO). The current study characterized the interactions of the wild type and the M4 DREADD with a range of agonists, antagonists, and the recently discovered M4 mAChR allosteric potentiator, 3-amino-5-chloro-6-methoxy-4-methyl-thieno[2,3-b]pyridine-2-carboxylic acid cyclopropylamide (LY2033298). LY2033298 displayed positive binding cooperativity with ACh, neutral cooperativity with the antagonist, [3H]quinuclidinyl benzilate, and agonism for activation of phosphorylated extracellular signal-regulated kinase (ERK) 1/2 at the wild-type M4 mAChR. LY2033298's cooperativity with clozapine or CNO was weakly positive with respect to binding but profoundly negative with respect to LY2033298 signaling. Although the DREADD mutations increased the binding and function of clozapine-like compounds, all other agonists lost the ability to activate the mutant; for the orthosteric agonists ACh and pilocarpine, this was due partly to a reduced affinity, whereas the affinity of LY2033298 or the atypical agonist 4-I-[3-chlorophenyl]carbamoyloxy)-2-butynyltrimethylammnonium chloride was unaltered. The interaction between LY2033298 and clozapine-like compounds reverted to neutral cooperativity on the DREADD, whereas LY2033298 caused a striking functional rescue of ACh potency and efficacy at the DREADD. These results provide conclusive evidence for the retention of a functional allosteric site on the M4 DREADD and highlight a role for residues Tyr113 and Ala203 in the transmission of cooperativity.
Asunto(s)
Ácidos Nicotínicos/metabolismo , Ácidos Nicotínicos/farmacología , Receptor Muscarínico M4/fisiología , Tiofenos/metabolismo , Tiofenos/farmacología , Acetilcolina/química , Acetilcolina/metabolismo , Acetilcolina/farmacología , Regulación Alostérica/fisiología , Sitio Alostérico/fisiología , Animales , Células CHO , Clozapina/análogos & derivados , Clozapina/química , Clozapina/metabolismo , Clozapina/farmacología , Cricetinae , Cricetulus , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Modelos Biológicos , Estructura Molecular , Mutación , Ácidos Nicotínicos/química , Ácidos Nicotínicos/genética , Fosforilación/efectos de los fármacos , Quinuclidinil Bencilato/metabolismo , Quinuclidinil Bencilato/farmacología , Ensayo de Unión Radioligante , Receptor Muscarínico M4/agonistas , Receptor Muscarínico M4/antagonistas & inhibidores , Transducción de Señal , Tiofenos/químicaRESUMEN
Noradrenaline is known to modulate memory formation in the mammalian hippocampus. We have examined how noradrenaline and selective beta-adrenoceptor (AR) agonists affect memory consolidation and how antagonists inhibit memory consolidation in the avian hippocampus. Injection of selective beta-AR agonists and antagonists at specific times within 30 min of a weakly or strongly reinforced, single-trial, bead discrimination learning test in 1-day-old chicks allowed us to determine the pattern of beta-AR involvement in hippocampal memory processing. Different beta-AR subtypes were recruited in temporal sequence after learning in the order beta(1), beta(3), and beta(2.) We provide evidence that the effect of manipulation of beta(1)-ARs by selective agonists and antagonists within 2.5 min of training parallels the action of NMDA receptor agonists and antagonists. Activation of beta(3)- and beta(2)-ARs facilitated memory but utilized different mechanisms: beta(3)-ARs by stimulating glucose uptake and metabolism, and beta(2)-ARs by increasing the breakdown of glycogen--with both metabolic events occurring in astrocytes and affecting intermediate memory. The different receptors are activated at different times within the lifetime of labile memory and within 30 min of learning. We have defined separate roles for the three beta-ARs in memory and demonstrated that the avian hippocampus is involved in learning and memory in much the same way as the hippocampus in the mammalian brain.
Asunto(s)
Catecolaminas/metabolismo , Metabolismo Energético/fisiología , Hipocampo/metabolismo , Memoria/fisiología , Receptores Adrenérgicos beta/metabolismo , Receptores de Glutamato/metabolismo , Agonistas Adrenérgicos beta/farmacología , Antagonistas Adrenérgicos beta/farmacología , Animales , Aves/fisiología , Catecolaminas/agonistas , Pollos , Metabolismo Energético/efectos de los fármacos , Agonistas de Aminoácidos Excitadores/farmacología , Glucosa/metabolismo , Glucogenólisis/efectos de los fármacos , Glucogenólisis/fisiología , Hipocampo/efectos de los fármacos , Aprendizaje/efectos de los fármacos , Aprendizaje/fisiología , Masculino , Mamíferos/fisiología , Memoria/efectos de los fármacos , Tiempo de Reacción/efectos de los fármacos , Tiempo de Reacción/fisiología , Receptores Adrenérgicos beta/efectos de los fármacos , Receptores de Glutamato/efectos de los fármacos , Especificidad de la Especie , Transmisión Sináptica/efectos de los fármacos , Transmisión Sináptica/fisiología , Factores de TiempoRESUMEN
The present study describes the isolation of the first neurotoxin (acantoxin IVa) from Acanthophis sp. Seram death adder venom and an examination of its activity at nicotinic acetylcholine receptor (nAChR) subtypes. Acantoxin IVa (MW 6815; 0.1-1.0 microM) caused concentration-dependent inhibition of indirect twitches (0.1 Hz, 0.2 ms, supramaximal V) and inhibited contractile responses to exogenous nicotinic agonists in the chick biventer cervicis nerve-muscle, confirming that this toxin is a postsynaptic neurotoxin. Acantoxin IVa (1-10 nM) caused pseudo-irreversible antagonism at skeletal muscle nAChR with an estimated pA2 of 8.36+/-0.17. Acantoxin IVa was approximately two-fold less potent than the long-chain (Type II) neurotoxin, alpha-bungarotoxin. With a pKi value of 4.48, acantoxin IVa was approximately 25,000 times less potent than alpha-bungarotoxin at alpha7-type neuronal nAChR. However, in contrast to alpha-bungarotoxin, acantoxin IVa completely inhibited specific [3H]-methyllycaconitine (MLA) binding in rat hippocampus homogenate. Acantoxin IVa had no activity at ganglionic nAChR, alpha4beta2 subtype neuronal nAChR or cytisine-resistant [3H]-epibatidine binding sites. While long-chain neurotoxin resistant [3H]-MLA binding in hippocampus homogenate requires further investigation, we have shown that a short-chain (Type I) neurotoxin is capable of fully inhibiting specific [3H]-MLA binding.