RESUMEN
The effect of "prophylactic" environmental stimulation on clinical symptoms and presynaptic defects in mice suffering from the experimental autoimmune encephalomyelitis (EAE) at the acute stage of disease (21⯱â¯1 days post immunization, d.p.i.) was investigated. In EAE mice raised in an enriched environment (EE), the clinical score was reduced when compared to EAE mice raised in standard environment (SE).Concomitantly, gain of weight and increased spontaneous motor activity and curiosity were observed, suggesting increased well-being in mice. Impaired glutamate exocytosis and cyclic adenosine monophosphate (cAMP) production in cortical terminals of SE-EAE mice were evident at 21⯱â¯1â¯d.p.i.. Differently, the 12â¯mM KCl-evoked glutamate exocytosis from cortical synaptosomes of EE-EAE mice was comparable to that observed in SE and EE-control mice, but significantly higher than that in SE-EAE mice. Similarly, the 12â¯mM KCl-evoked cAMP production in EE-EAE mice cortical synaptosomes recovered to the level observed in SE and EE-control mice. MUNC-18 and SNAP25 contents, but not Syntaxin-1a and Synaptotagmin 1 levels, were increased in cortical synaptosomes from EE-EAE mice when compared to SE-EAE mice. Circulating IL-1ß was increased in the spinal cord, but not in the cortex, of SE-EAE mice, and it did not recover in EE-EAE mice. Inflammatory infiltrates were reduced in the cortex but not in the spinal cord of EE-EAE mice. Demyelination was observed in the spinal cord; EE significantly diminished it. We conclude that "prophylactic" EE is beneficial to synaptic derangements and preserves glutamate transmission in the cortex of EAE mice. This article is part of the Special Issue entitled "Neurobiology of Environmental Enrichment".
Asunto(s)
Corteza Cerebral/metabolismo , Encefalomielitis Autoinmune Experimental/metabolismo , Encefalomielitis Autoinmune Experimental/terapia , Ambiente , Terminales Presinápticos/metabolismo , Animales , Conducta Animal , Antígeno CD146/metabolismo , Corteza Cerebral/patología , AMP Cíclico/metabolismo , Citocinas/metabolismo , Encefalomielitis Autoinmune Experimental/patología , Exocitosis/fisiología , Femenino , Ácido Glutámico/metabolismo , Vivienda para Animales , Inflamación/metabolismo , Inflamación/patología , Inflamación/terapia , Ratones Endogámicos C57BL , Terminales Presinápticos/patología , Distribución Aleatoria , Médula Espinal/metabolismo , Médula Espinal/patología , Proteína 25 Asociada a Sinaptosomas/metabolismo , Sinaptosomas/metabolismoRESUMEN
S 47445 is a positive modulator of glutamate AMPA-type receptors, possessing neurotrophic and enhancing synaptic plasticity effects as well as pro-cognitive and anti-stress properties. Here, the drug was assessed in the perinatal stress (PRS) rat model, known to have a high predictive validity with monoaminergic antidepressants. The effects of a chronic treatment (i.p.) with S 47445 were investigated on risk-taking, motivational and cognitive behavior. S 47445 (1 and 10â¯mg/kg) increased the exploration of the elevated-plus maze and light/dark box as well as the time spent grooming in the splash test, and improved social memory in PRS rats. Also, the effects of S 47445 were examined on the synaptic neurotransmission. The reduced depolarization-evoked glutamate release induced by PRS was corrected with S 47445 (10â¯mg/kg). Remarkably, the reduction in glutamate release induced by PRS and corrected by S 47445 chronic treatment was correlated with all the behavioral changes. S 47445â¯at 10â¯mg/kg also normalized the lower levels of synaptic vesicle-associated proteins in ventral hippocampus in PRS rats. Finally, S 47445 reversed the decrease of mGlu5 receptors, GR and OXTR induced by PRS. Collectively, in an animal model of stress-related disorders, S 47445 corrected the imbalance between excitatory and inhibitory neurotransmission by regulating glutamate-evoked release that is predictive of PRS behavioral alterations, and also normalized the reduction of trafficking of synaptic vesicles induced by PRS. These results support the interest of glutamatergic-based therapeutic strategies to alleviate stress-related disorders.
Asunto(s)
Benzoxazinas/farmacología , Cognición/efectos de los fármacos , Emociones/efectos de los fármacos , Ácido Glutámico/metabolismo , Efectos Tardíos de la Exposición Prenatal/prevención & control , Estrés Psicológico/metabolismo , Triazinas/farmacología , Animales , Femenino , Hipocampo/metabolismo , Masculino , Proteínas del Tejido Nervioso/metabolismo , Embarazo , Ratas , Receptor del Glutamato Metabotropico 5/metabolismo , Receptores de Glucocorticoides/metabolismo , Receptores de Oxitocina/metabolismoRESUMEN
BACKGROUND AND PURPOSE: Electrophysiological studies described potentiation of NMDA receptor function by metabotropic glutamate receptors (mGluRs) of group I occurring postsynaptically. Since release-enhancing NMDA receptors exist on noradrenergic terminals and group I mGluRs have recently been identified on these nerve endings, we have investigated if NMDA receptor-mGluR interactions also can occur at the presynaptic level. EXPERIMENTAL APPROACH: Rat hippocampus and human neocortex synaptosomes were labelled with [(3)H]noradrenaline and superfused with mGluR agonists and antagonists. NMDA-evoked [(3)H]noradrenaline release was produced by removal of external Mg(2+) or by simultaneous application of NMDA and AMPA in Mg(2+)-containing solutions. KEY RESULTS: The mGluR1/5 agonist 3,5-DHPG, inactive on its own, potentiated both the release of [(3)H]noradrenaline elicited by AMPA/NMDA/glycine and that evoked by NMDA/glycine following Mg(2+) removal. The effect of 3,5-DHPG on the AMPA/NMDA/glycine-induced release was insensitive to the mGluR1 antagonist CPCCOEt, but it was abolished by the mGluR5 antagonist MPEP; moreover, it was potentiated by the mGluR5 positive allosteric modulator DFB. When NMDA receptors were activated by Mg(2+) removal, both mGluR5 and mGluR1 contributed to the evoked release, the mGluR-mediated release being blocked only by CPCCOEt and MPEP in combination. Experiments with human neocortex synaptosomes show NMDA receptor-mGluR interactions qualitatively similar to those observed in rodents. CONCLUSIONS AND IMPLICATIONS: Group I mGluRs, both of the mGluR1 and mGluR5 subtypes, co-localize with NMDA receptors on noradrenergic terminals of rat hippocampus and human neocortex. Depending on the mode of activation, NMDA receptors exert differential permissive roles on the activation of presynaptic mGluR1 and mGluR5.
Asunto(s)
Terminales Presinápticos/fisiología , Receptor Cross-Talk/fisiología , Receptores de N-Metil-D-Aspartato/fisiología , Receptores Presinapticos/fisiología , Animales , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , Corteza Cerebral/fisiología , Cromonas/farmacología , Femenino , Glicina/análogos & derivados , Glicina/farmacología , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Hipocampo/fisiología , Humanos , Masculino , N-Metilaspartato/farmacología , Norepinefrina/metabolismo , Norepinefrina/farmacología , Terminales Presinápticos/efectos de los fármacos , Terminales Presinápticos/metabolismo , Unión Proteica/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Receptor Cross-Talk/efectos de los fármacos , Receptor del Glutamato Metabotropico 5 , Receptores AMPA/metabolismo , Receptores AMPA/fisiología , Receptores de Glutamato Metabotrópico/metabolismo , Receptores de Glutamato Metabotrópico/fisiología , Receptores de N-Metil-D-Aspartato/metabolismo , Receptores Presinapticos/metabolismo , Resorcinoles/farmacología , Sinaptosomas/efectos de los fármacos , Sinaptosomas/metabolismo , Sinaptosomas/fisiología , Tritio , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/farmacologíaRESUMEN
DM 232 and DM 235 are novel antiamnesic compounds structurally related to ampakines. The involvement of AMPA receptors in the mechanism of action of DM 232 and DM 235 was, therefore, investigated in vivo and in vitro. Both compounds (0.1 mg/kg(-1) i.p.) were able to reverse the amnesia induced by the AMPA receptor antagonist NBQX (30 mg/kg(-1) i.p.) in the mouse passive avoidance test. At the effective doses, the investigated compounds did not impair motor coordination, as revealed by the rota rod test, nor modify spontaneous motility and inspection activity, as revealed by the hole board test. DM 232 and DM 235 reversed the antagonism induced by kynurenic acid of the NMDA-mediated release of [(3)H]NA in the kynurenate test performed in rat hippocampal slices. This effect was abolished by NBQX. DM 232 increases, in a concentration dependent manner, excitatory synaptic transmission in the rat hippocampus in vitro. These results suggest that DM 232 and DM 235 act as cognition enhancers through the activation of the AMPA-mediated neurotransmission system.
Asunto(s)
Amnesia/tratamiento farmacológico , Nootrópicos/farmacología , Piperazinas/farmacología , Pirroles/farmacología , Receptores AMPA/fisiología , Amnesia/inducido químicamente , Animales , Reacción de Prevención/efectos de los fármacos , Cognición/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Electrofisiología/métodos , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Ácido Quinurénico/farmacología , Masculino , Ratones , N-Metilaspartato/farmacología , Nootrópicos/química , Piperazinas/química , Pirroles/química , Quinoxalinas/toxicidad , Ratas , Ratas Sprague-Dawley , Receptores AMPA/antagonistas & inhibidores , Receptores AMPA/metabolismoRESUMEN
CGP 36742 is a weak GABA(B) receptor antagonist. However, it improves cognitive performances at low doses; it blocks GABA(B) receptors potently and selectively on somatostatinergic terminals; it prevents kynurenate from antagonising NMDA-induced release of noradrenaline from rat brain slices potently. We here investigated whether and how somatostatin plays a role in the CGP 36742 activity. CGP 36742 increased the somatostatin-like immunoreactivity (SRIF-LI) release from hippocampal slices exposed to NMDA. In the kynurenate test with rat hippocampal slices SRIF-14 mimicked the effect of CGP 36742. CGP 36742 lost its activity in rats whose somatostatin content had been depleted with cysteamine. Exogenous SRIF-14 reverted kynurenate antagonism in somatostatin-depleted slices. L362855, an sst(5) receptor agonist, but not the selective sst(1)-sst(4) agonists, L797591, L779976, L796778 and L803087, displayed activity in the kynurenate test. The effects of CGP 36742, SRIF-14 and L362855 were antagonised by the sst(5)-preferring antagonist BIM-23056. The protein kinase C inhibitor GF 109203X prevented the reversal of the kynurenate antagonism by CGP 36742 or SRIF-14. In conclusion, by selectively blocking GABA(B) receptors on somatostatinergic terminals, CGP 36742 may disinhibit somatostatin release; the consequent activation of sst(5) receptors would potentiate the function of NMDA receptors coexisting with sst(5) receptors on noradrenergic neurons.
Asunto(s)
Cognición/efectos de los fármacos , Antagonistas de Aminoácidos Excitadores/farmacología , Antagonistas de Receptores de GABA-B , Receptores de N-Metil-D-Aspartato/agonistas , Somatostatina/metabolismo , Animales , Antagonistas del GABA/farmacología , Técnicas In Vitro , Masculino , Norepinefrina/metabolismo , Compuestos Organofosforados/farmacología , Proteína Quinasa C/metabolismo , Radioinmunoensayo , Ratas , Ratas Sprague-Dawley , Receptores AMPA/efectos de los fármacos , Receptores de Somatostatina/efectos de los fármacos , Receptores de Somatostatina/metabolismo , Sincalida/farmacología , Sinaptosomas/efectos de los fármacos , Sinaptosomas/metabolismoRESUMEN
Kynurenic acid is a tryptophan metabolite provided with antagonist activity on ionotropic glutamate and alpha7 nicotinic acetylcholine receptors. We noticed that in rats with a dialysis probe placed in the head of their caudate nuclei, local administration of kynurenic acid (30-100 nM) significantly reduced glutamate output. Qualitatively and quantitatively similar effects were observed after systemic administration of kynurenine hydroxylase inhibitors, a procedure able to increase brain kynurenate concentrations. Interestingly, in microdialysis studies, methyllycaconitine (0.3-10 nM), a selective alpha7 nicotinic receptor antagonist, also reduced glutamate output. In isolated superfused striatal synaptosomes, kynurenic acid (100 nM), but not methyllycaconitine, inhibited the depolarization (KCl 12.5 mM)-induced release of transmitter or previously taken-up [3H]-D-aspartate. This inhibition was not modified by glycine, N-methyl-D-aspartate or subtype-selective kainate receptor agents, while CNQX or DNQX (10 microM), two AMPA and kainate receptor antagonists, reduced kynurenic acid effects. Low concentrations of kynurenic acid, however, did not modify [3H]-kainate (high and low affinity) or [3H]-AMPA binding to rat brain membranes. Finally, because metabotropic glutamate (mGlu) receptors modulate transmitter release in striatal preparations, we evaluated, with negative results, kynurenic acid (1-100 nM) effects in cells transfected with mGlu1, mGlu2, mGlu4 or mGlu5 receptors. In conclusion, our data show that kynurenate-induced inhibition of glutamate release is not mediated by glutamate receptors. Nicotinic acetylcholine receptors, however, may contribute to the inhibitory effects of kynurenate found in microdialysis studies, but not in those found in isolated synaptosomes.
Asunto(s)
Encéfalo/efectos de los fármacos , Antagonistas de Aminoácidos Excitadores/farmacología , Ácido Glutámico/metabolismo , Ácido Quinurénico/farmacología , Terminales Presinápticos/efectos de los fármacos , Receptores de Glutamato/efectos de los fármacos , Receptores Nicotínicos/efectos de los fármacos , Animales , Encéfalo/citología , Encéfalo/metabolismo , Núcleo Caudado/citología , Núcleo Caudado/efectos de los fármacos , Núcleo Caudado/metabolismo , Agonistas de Aminoácidos Excitadores/farmacocinética , Espacio Extracelular/efectos de los fármacos , Espacio Extracelular/metabolismo , Ácido Quinurénico/metabolismo , Masculino , Microdiálisis , Terminales Presinápticos/metabolismo , Ratas , Ratas Wistar , Receptores de Glutamato/metabolismo , Receptores Nicotínicos/metabolismo , Transmisión Sináptica/efectos de los fármacos , Transmisión Sináptica/fisiología , Sinaptosomas/efectos de los fármacos , Sinaptosomas/metabolismo , Tritio/farmacocinéticaRESUMEN
NMDA receptors regulating hippocampal noradrenaline (NA) and striatal dopamine (DA) release have been compared using superfused synaptosomes prelabelled with the [(3)H]catecholamines. Both receptors mediated release augmentation when exposed to NMDA plus glycine. Quinolinic acid (100 microM or 1 mM) plus glycine (1 microM)-elicited [(3)H]NA, but not [(3)H]DA release. The NMDA (100 microM)-evoked release of [(3)H]NA and [(3)H]DA was similar and concentration-dependently enhanced by glycine or D-serine (0.1-1 microM); in contrast, the HIV-1 envelope protein gp120 potently (30-100 pM) enhanced the NMDA-evoked release of [(3)H]NA, but not that of [(3)H]DA. Gp120 also potentiated quinolinate-evoked [(3)H]NA release. Ifenprodil (0.1-0.5 microM) or CP-101,606 (0.1-10 microM) inhibited the NMDA plus glycine-evoked release of both [(3)H]catecholamines. Zinc (0.1-1 microM) was ineffective. Lowering external pH from 7.4 to 6.6 strongly inhibited the release of [(3)H]NA elicited by NMDA plus glycine, whereas the release of [(3)H]DA was unaffected. The protein kinase C inhibitors GF 109203X (0.1 microM) or H7 (10 microM) selectively prevented the effect of NMDA plus glycine on the release of [(3)H]NA. GF 109203X also blocked the release of [(3)H]NA induced by NMDA or quinolinate plus gp120. It is concluded that the hippocampal NMDA receptor and the striatal NMDA receptor are pharmacologically distinct native subtypes, possibly containing NR2B subunits but different splice variants of the NR1 subunit.
Asunto(s)
Cuerpo Estriado/metabolismo , Dopamina/metabolismo , Hipocampo/metabolismo , Norepinefrina/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Animales , Ácido Aspártico/metabolismo , Ácido Aspártico/farmacología , Cuerpo Estriado/química , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Glicina/metabolismo , Glicina/farmacología , Proteína gp120 de Envoltorio del VIH/farmacología , Hipocampo/química , Concentración de Iones de Hidrógeno , Masculino , N-Metilaspartato/metabolismo , N-Metilaspartato/farmacología , Piperidinas/farmacología , Proteína Quinasa C/antagonistas & inhibidores , Ácido Quinolínico/farmacología , Ratas , Ratas Sprague-Dawley , Receptores de N-Metil-D-Aspartato/agonistas , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Serina/metabolismo , Serina/farmacología , Sinaptosomas/química , Sinaptosomas/efectos de los fármacos , Sinaptosomas/metabolismo , Zinc/farmacologíaRESUMEN
The objective of this study was to investigate the effects of the HIV-1 envelope protein gp120 and its peptide fragments on the function of N-methyl-D-aspartate (NMDA) receptors mediating release of cholecystokinin (CCK) and somatostatin (SRIF). These are nonconventional NMDA receptors recently found to be activated by glycine or D-serine 'only'. The release of cholecystokinin-like immunoreactivity (CCK-LI) and of somatostatin-like immunoreactivity (SRIF-LI) elicited by 12 mM K+ from superfused rat neocortex synaptosomes was potently increased by gp120, its cyclic V3 loop and the linear V3 sequence BRU-C-34-A, but not by RP-135 (a central portion of BRU-C-34-A). The EC50 values of gp120 were 0.02 nM (CCK-LI release) and 0.01 nM (SRIF-LI release). The releasing effect of gp120 was prevented by blocking the glycine site or the ion channel of NMDA receptors, but not the glutamate recognition site; in addition, the gp120 effect was strongly inhibited by nanomolar concentrations of Zn2+ ions and by low micromolar concentrations of ifenprodil. It is concluded that gp120 acts as a very potent agonist at the glycine site of NMDA receptors sited on CCK- and SRIF-releasing nerve endings; the protein is able to activate the receptor channel in the absence of glutamate. Gp120 activates the receptors through its V3 loop as peptide fragments related to V3 retain near-maximal activity. The sensitivity of the gp120 effect to both Zn2+ and ifenprodil would not be incompatible with the idea that these NMDA receptors contain the triple subunit combination NR1/NR2A/NR2B.
Asunto(s)
Corteza Cerebral/metabolismo , Colecistoquinina/metabolismo , Proteína gp120 de Envoltorio del VIH/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Somatostatina/metabolismo , 2-Amino-5-fosfonovalerato/farmacología , Animales , Corteza Cerebral/citología , Corteza Cerebral/virología , Maleato de Dizocilpina/farmacología , Inhibidores Enzimáticos/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Proteína gp120 de Envoltorio del VIH/química , VIH-1 , Indoles/farmacología , Ácido Quinurénico/análogos & derivados , Ácido Quinurénico/farmacología , Masculino , Maleimidas/farmacología , Neuronas/metabolismo , Neuronas/virología , Nitroarginina/farmacología , Fragmentos de Péptidos/metabolismo , Ácidos Pipecólicos/farmacología , Piperazinas/farmacología , Piperidinas/farmacología , Potasio/farmacología , Estructura Terciaria de Proteína , Ratas , Ratas Sprague-Dawley , Transmisión Sináptica/efectos de los fármacos , Transmisión Sináptica/fisiología , Sinaptosomas/metabolismo , Zinc/farmacologíaRESUMEN
N-methyl-D-aspartate (NMDA) receptors exist on noradrenergic axon terminals and mediate enhancement of noradrenaline (NA) release. We here investigated modulation by somatostatin (SRIF, somatotropin release inhibiting factor) of the NMDA-induced release of NA using superfused hippocampal synaptosomes. The NMDA response was increased by SRIF-28 and SRIF-14, but not SRIF-28((1 - 14)), whereas the release of [(3)H]-NA elicited by alpha-amino-3-hydroxy-5-methylisoxazide-4-propionic acid (AMPA) was unaffected. SRIF-14 did not mimic glycine at the NMDA receptor but activated SRIF receptors sited on noradrenergic terminals. The SRIF-14 effect was blocked by pertussis toxin but mimicked by mastoparan, a G-protein activator. BIM-23056, but not Cyanamid 154806, antagonized the SRIF-14 effect. This effect was mimicked by L362855, a partial agonist at the sst(5) subtype, but not by the new selective sst(1) - sst(4) receptor agonists L797591, L779976, L796778 and L803087. Protein kinase C (PKC) inhibitors (H7, staurosporine, GF 209103X, cheleritrine and sphingosine) prevented the SRIF-14 effect, while phorbol 12-myristate 13-acetate enhanced the NMDA response. SRIF-14 permitted NMDA receptor activation in the presence of 1.2 mM Mg(2+) ions, both in hippocampal synaptosomes and slices. Blockade of inositol-1,4,5-trisphosphate (InsP(3)) receptors with heparin abolished the effect of SRIF-14. It is concluded that SRIF receptors, possibly of the sst(5) subtype, can exert a permissive role on NMDA receptors colocalized on hippocampal noradrenergic terminals: activation of sst(5) receptors is coupled to pertussis toxin-sensitive G proteins enhancing phosphoinositide metabolism with activation of InsP(3) receptors and PKC; NMDA receptor subunits might be phosphorylated with consequent removal of the Mg(2+) block in absence of depolarization.
Asunto(s)
Canales de Calcio/efectos de los fármacos , Antagonistas de Hormonas/farmacología , Magnesio/fisiología , Proteína Quinasa C/metabolismo , Receptores Citoplasmáticos y Nucleares/efectos de los fármacos , Receptores de N-Metil-D-Aspartato/agonistas , Somatostatina/farmacología , Animales , Electrofisiología , Activación Enzimática/efectos de los fármacos , Activadores de Enzimas/farmacología , Proteínas de Unión al GTP/metabolismo , Técnicas In Vitro , Receptores de Inositol 1,4,5-Trifosfato , Masculino , N-Metilaspartato/farmacología , Terminaciones Nerviosas/efectos de los fármacos , Terminaciones Nerviosas/fisiología , Péptidos/farmacología , Ratas , Ratas Sprague-Dawley , Sistema Nervioso Simpático/citología , Sistema Nervioso Simpático/efectos de los fármacos , Sistema Nervioso Simpático/fisiología , Sinaptosomas/efectos de los fármacos , Sinaptosomas/metabolismo , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/farmacologíaRESUMEN
Some cognition enhancers were previously shown to potently prevent antagonism of the N-methyl-D-aspartate (NMDA)-evoked release of norepinephrine (NE) brought about in slices of rat hippocampus by kynurenic acid, an endogenous NMDA receptor blocker. We have examined the impact of putative nootropic agents in the kynurenate test performed with slices of human cerebral cortex from patients undergoing neurosurgery. In slices of human neocortex, local application of NMDA evoked release of [3H]NE; the effect of NMDA was antagonized by several NMDA receptor antagonists, including kynurenic acid. The antagonism of the NMDA-evoked [3H]NE release produced by 300 microM kynurenate was potently (EC50 <10 microM) prevented by most of the nootropics tested, including aniracetam, oxiracetam, D-cycloserine, and the glutamate analog CR 2249 (but not its enantiomer CR 2361). Nicotine or tacrine (up to 10 microM) did not show any effect in the kynurenate test. Nicotine (30-100 microM) itself increased the release of [3H]NE; interestingly, the nicotine-evoked overflow was blocked not only by the nicotin receptor antagonist mecamylamine but also by NMDA receptor antagonists, suggesting an indirect mechanism mediated by glutamate/aspartate release. To conclude, the similarities between the data obtained here with human neocortex slices and those previously obtained in the rat indicate that the kynurenate test performed with rat brain slices may represent a useful biochemical assay to study cognition-enhancing drugs.
Asunto(s)
Antagonistas de Aminoácidos Excitadores/farmacología , Ácido Quinurénico/antagonistas & inhibidores , Neocórtex/efectos de los fármacos , Nootrópicos/farmacología , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Agonistas de Aminoácidos Excitadores/farmacología , Humanos , Técnicas In Vitro , Ácido Quinurénico/farmacología , N-Metilaspartato/farmacología , Neocórtex/metabolismo , Norepinefrina/metabolismoRESUMEN
Aniracetam, 1-(1,3-benzodioxol-5-yl-carbonyl)piperidine (1-BCP) and cyclothiazide, three compounds considered to enhance cognition through modulation of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors, were evaluated in the 'kynurenate test', a biochemical assay in which some nootropics have been shown to prevent the antagonism by kynurenic acid of the N-methyl-D-aspartate (NMDA)-evoked [3H]noradrenaline ([3H]NA) release from rat hippocampal slices. Aniracetam attenuated the kynurenate (100 microM) antagonism of the [3H]NA release elicited by 100 microM NMDA with high potency (EC50< or =0.1 microM). Cyclothiazide and 1-BCP were about 10 and 100 times less potent than aniracetam, respectively. The effect of aniracetam persisted in the presence of the AMPA receptor antagonist 6-nitro-7-sulphamoyl-benzo[f]quinoxaline-2,3-dione (NBQX) added at 5 microM, a concentration that did not affect NMDA receptors; in contrast, NBQX reduced the effect of 1-BCP and abolished that of cyclothiazide. The AMPA-evoked release of [3H]NA from hippocampal slices or synaptosomes was enhanced by cyclothiazide, less potently by 1-BCP and weakly by aniracetam. High concentrations of kynurenate (1 mM) antagonized the AMPA-evoked [3H]NA release in slices; this antagonism was attenuated by 1 microM cyclothiazide and reversed to an enhancement of AMPA-evoked [3H]NA release by 10 microM of the drug, but was insensitive to 1-BCP or aniracetam. It is concluded that aniracetam exerts a dual effect on glutamatergic transmission: modulation of NMDA receptor function at nanomolar concentrations, and modulation of AMPA receptors at high micromolar concentrations. As to cyclothiazide and 1-BCP, our data concur with the idea that both compounds largely act through AMPA receptors, although an NMDA component may be involved in the effect of 1-BCP.
Asunto(s)
Compuestos Heterocíclicos/farmacología , Hipocampo/efectos de los fármacos , Norepinefrina/metabolismo , Receptores AMPA/efectos de los fármacos , Receptores de N-Metil-D-Aspartato/efectos de los fármacos , Animales , Antihipertensivos/farmacología , Benzotiadiazinas/farmacología , Dioxoles/farmacología , Relación Dosis-Respuesta a Droga , Agonistas de Aminoácidos Excitadores/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Hipocampo/metabolismo , Técnicas In Vitro , Ácido Quinurénico/farmacología , Masculino , N-Metilaspartato/farmacología , Nootrópicos/farmacología , Piperidinas/farmacología , Pirrolidinonas/farmacología , Quinoxalinas/farmacología , Ratas , Ratas Sprague-Dawley , Receptores AMPA/fisiología , Receptores de N-Metil-D-Aspartato/fisiología , Tritio , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/farmacologíaRESUMEN
Recent results show that the HIV-1 protein gp120 can enhance N-methyl-D-aspartate receptor-mediated release of noradrenaline from CNS nerve endings. We now investigate the mechanism of this action, including the structural determinants of the gp120 effect and the nature of its binding sites. The N-methyl-D-aspartate-evoked release of [3H]noradrenaline from rat hippocampal synaptosomes was potentiated similarly by gp120 and gp160; gp41 was ineffective. The regions of gp120 involved appear to be outside the CD4-binding domain of the protein, because gp120 retained its activity after pretreatment with N-carbomethoxycarbonyl-D-prolyl-D-phenylalanine, a compound known to inhibit binding of gp120 to CD4 receptors. Moreover, sequences of gp120 critical for binding to CD4 did not mimic the effect of gp120. Preincubation of synaptosomes with anti-galactocerebroside antibodies did not affect gp120 activity. The protein effect was retained by peptides mimicking its V3 sequence, including the cyclic V3 "universal peptide" and the linear V3 sequence BRU-C-34-A, but not RP-135 (a central portion of BRU-C-34-A). The block of the N-methyl-D-aspartate-induced [3H]noradrenaline release by 7-chlorokynurenate, an antagonist at the N-methyl-D-aspartate receptor glycine site, was competitively reversed by glycine, by V3 and by BRU-C-34-A. When added with N-methyl-D-aspartate, V3 was three to four orders of magnitude more potent than glycine (EC50 values: about 20 pM and 150 nM, respectively) in enhancing [3H]noradrenaline release. Gp120 did not release glycine or serine from synaptosomes, thus excluding indirect actions through these agents. To conclude, gp120 may act following recognition by its V3 sequence of a high-affinity site possibly coincident with the glycine site of N-methyl-D-aspartate receptors present on hippocampal terminals of noradrenergic neurons. Considering the importance of N-methyl-D-aspartate receptor activation and of noradrenaline in cognitive processes, the effects of gp120 and V3 described here may be relevant to the pathology of AIDS dementia.
Asunto(s)
Proteína gp120 de Envoltorio del VIH/metabolismo , VIH-1 , Hipocampo/metabolismo , Norepinefrina/metabolismo , Receptores de Glicina/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Secuencia de Aminoácidos , Animales , Antígenos CD4/metabolismo , Galactosilceramidas/metabolismo , Proteínas gp160 de Envoltorio del VIH/fisiología , Proteína gp41 de Envoltorio del VIH/fisiología , Técnicas In Vitro , Masculino , Datos de Secuencia Molecular , Neuronas/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores de N-Metil-D-Aspartato/fisiologíaRESUMEN
Some putative cognition enhancers (oxiracetam, aniracetam and D-cycloserine) were previously shown to prevent the kynurenic acid antagonism of the N-methyl-D-aspartate (NMDA)-evoked norepinephrine (NE) release in rat hippocampal slices. This functional in vitro assay was further characterized in the present work. D-Serine, a glutamate coagonist at the NMDA receptor glycine site, concentration-dependently (EC50 approximately 0.1 microM) prevented the kynurenate (100 microM) block of the NMDA (100 microM)-evoked [3H]NE release. L-Serine was ineffective up to 10 microM. The gamma-aminobutyric acidB (GABA[B]) receptor antagonist CGP 36742, reported to improve cognitive performance, potently prevented the kynurenate antagonism. The activity of CGP 36742 (1 microM) appeared to be unaffected by 10 microM (-)-baclofen, a GABA(B) receptor agonist; furthermore, CGP 52432, a GABA(B) antagonist more potent than CGP 36742, but reportedly devoid of nootropic properties, was inactive in the "kynurenate test." The novel putative cognition enhancer CR2249, but not its enantiomer CR2361, also potently prevented the kynurenate antagonism. In contrast, linopirdine, nicotine and tacrine were inactive. In rat hippocampal synaptosomes glycine and D-cycloserine enhanced the NMDA-evoked [3H]NE release, whereas oxiracetam and CR2249 did not. These four compounds were all similarly effective in preventing kynurenate antagonism, both in slices and in synaptosomes. The NMDA potentiation caused by glycine (0.1-100 microM) was not affected by 100 microM oxiracetam, which suggested that drugs active in the "kynurenate test" may bind to sites different from the glycine site of the NMDA receptor. To conclude, the "kynurenate test" is an in vitro assay useful in the identification and characterization of putative cognition enhancers acting via NMDA receptors.
Asunto(s)
Cognición/efectos de los fármacos , Ácido Quinurénico/farmacología , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Animales , Técnicas In Vitro , Masculino , Norepinefrina/metabolismo , Compuestos Organofosforados/farmacología , Ácidos Pentanoicos/farmacología , Ratas , Ratas Sprague-Dawley , Serina/farmacologíaRESUMEN
The release of tritium from rat hippocampal synaptosomes prelabeled with [3H]noradrenaline ([3H]NA) or [3H]5-hydroxytryptamine ([3H]5-HT) and from rat neocortex synaptosomes prelabeled with [3H]choline and the release of endogenous GABA and glutamate from rat neocortex synaptosomes were monitored during superfusion with media containing varying concentrations of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) or kainic acid. Concentration-dependent release potentiations were elicited by both excitatory amino acids (EAAs) in all the transmitter systems investigated. The releases evoked by 100 microM AMPA were, in all cases, almost totally dependent on external Ca2+ and sensitive to 6.7-dinitroquinoxaline-2,3-dione (DNQX), indicating involvement of non-NMDA receptors. When cyclothiazide, a drug able to prevent desensitization of AMPA-preferring receptors, was added to the superfusion medium (at 1 or 10 microM) concomitantly with 100 microM AMPA or kainate, the EAA-evoked release of [3H]NA was significantly enhanced. Concanavalin A, a lectin thought to prevent desensitization of kainate-preferring receptors, had no effect (up to 10 microM) on the release of [3H]NA evoked by AMPA or kainate. The effect of cyclothiazide was lost if, after an 8-min pretreatment, the drug was removed just before the AMPA stimulus. When added concomitantly with the EAAs, cyclothiazide potentiated the release of [3H]5-HT elicited by AMPA and, less so, that evoked by kainate. Concanavalin A was ineffective. Neither cyclothiazide (1 or 10 microM) nor concanavalin A (3 or 10 microM) could affect the release of [3H]ACh or endogenous GABA provoked by 100 microM AMPA or kainate, suggesting that the receptors involved do not desensitize. Exposure of neocortex synaptosomes to AMPA or kainate concomitantly with cyclothiazide caused endogenous glutamate release that did not differ from that evoked by the EAAs alone. In contrast, the effects of AMPA and kainate were potentiated by concanavalin A. The activity of the lectin (3 microM) persisted when it was applied for 8 min and then removed before the AMPA or kainate (100 microM) pulse. When hippocampal synaptosomes prelabeled with [3H]NA were subjected to three subsequent AMPA (100 microM) stimuli (S1, S2 and S3), the release of [3H]NA decreased dramatically from S1 to S3 (S3/S1 = 0.14 +/- 0.04); a significant 'protection' of the AMPA effect was offered by 1 microM cyclothiazide (S3/S1 = 0.36 +/- 0.06). This value did not differ from the S3/S1 ratio (0.38 +/- 0.04) obtained in parallel experiments with 12 mM K+. The release evoked by high-K+ was insensitive to cyclothiazide. Finally, the effect of AMPA on the release of [3H]ACh did not respond to cyclothiazide also during three subsequent stimuli with 100 microM AMPA. To conclude: a) ionotropic non-NMDA receptors mediating enhancement of NA, 5-HT, ACh, GABA and glutamate release exist on the corresponding nerve terminals; b) the receptors present on noradrenergic and serotonergic neurons are AMPA-preferring receptors, whereas the glutamate autoreceptors resemble most the kainate-preferring subtype; the receptors mediating ACh and GABA release can not be subclassified at present; c) desensitization may not be a property of all non-NMDA ionotropic receptors. The receptors here characterized represent five models of native non-NMDA receptors suitable for pharmacological and molecular studies.
Asunto(s)
Receptores de Glutamato/metabolismo , Acetilcolina/metabolismo , Animales , Benzotiadiazinas/farmacología , Calcio/farmacología , Corteza Cerebral/metabolismo , Colina/metabolismo , Antagonistas de Aminoácidos Excitadores/farmacología , Ácido Glutámico/metabolismo , Hipocampo/metabolismo , Técnicas In Vitro , Ácido Kaínico/farmacología , Masculino , Norepinefrina/metabolismo , Potasio/farmacología , Quinoxalinas/farmacología , Ratas , Ratas Sprague-Dawley , Receptores AMPA/efectos de los fármacos , Receptores AMPA/metabolismo , Receptores AMPA/fisiología , Receptores de Glutamato/efectos de los fármacos , Receptores de Glutamato/fisiología , Receptores de Ácido Kaínico/efectos de los fármacos , Receptores de Ácido Kaínico/metabolismo , Receptores de Ácido Kaínico/fisiología , Serotonina/metabolismo , Sinaptosomas/metabolismo , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/farmacología , Ácido gamma-Aminobutírico/metabolismoRESUMEN
OBJECTIVE: To investigate the effect of HIV-1 gp120 on the function of glutamate receptors of the N-methyl-D-aspartate (NMDA) type in the human brain. DESIGN: The monitoring of neurotransmitter release from superfused isolated nerve endings is widely recognized as a technique appropriate for the study of neurotransmitter release and to attribute a precise localization to the site(s) of action of drugs able to modulate release. METHODS: Synaptosomes (pinched-off nerve endings) were prepared from fresh human brain tissue samples removed during neurosurgery, labelled with [3H]-noradrenaline and superfused at a rate of 0.5 ml/min with NMDA in the presence of gp41, gp160, gp120 or the V3 loop, with or without NMDA receptor antagonists. Fractions of superfusate were collected and measured for radioactivity. RESULTS: NMDA elicited a glycine-sensitive release of [3H]-noradrenaline from human brain synaptosomes. HIV-1 gp120 potentiated the NMDA (1 mM)-evoked [3H]-noradrenaline release (maximal effect approximately 110% at 1 nM). The release elicited by NMDA plus gp120 was prevented by the classical NMDA receptor antagonists dizocilpine or 7-chlorokynurenic acid, as well as by memantine. The potentiation by gp120 of the NMDA-evoked [3H]-noradrenaline release was mimicked by gp160 but not by gp41. The effect of gp120 was retained by the V3 loop. Finally, gp120 reversed (1 nM) and surmounted (10nM) the antagonism by 10 microM 7-chlorokynurenate of the NMDA-evoked [3H]-noradrenaline release. CONCLUSION: gp 120 binds directly through the V3 loop at noradrenergic axon terminals in human brain neocortex and may alter the function of presynaptic NMDA receptors mediating regulation of noradrenaline release.
Asunto(s)
Corteza Cerebral/metabolismo , Proteína gp120 de Envoltorio del VIH/farmacología , VIH-1 , Norepinefrina/metabolismo , Receptores de N-Metil-D-Aspartato/fisiología , Adulto , Anciano , Sitio Alostérico , Antagonistas de Aminoácidos Excitadores/farmacología , Femenino , Productos del Gen env/farmacología , Glicina/metabolismo , Proteína gp120 de Envoltorio del VIH/fisiología , Humanos , Ácido Quinurénico/análogos & derivados , Ácido Quinurénico/farmacología , Masculino , Persona de Mediana Edad , N-Metilaspartato/farmacología , Fragmentos de Péptidos/farmacología , Fragmentos de Péptidos/fisiología , Receptores de Glicina/antagonistas & inhibidores , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Receptores de N-Metil-D-Aspartato/metabolismo , Sinaptosomas/metabolismoRESUMEN
Oxiracetam, aniracetam and D-cycloserine, three putative cognition enhancers, were examined in a functional assay for NMDA receptors. Rat hippocampal slices or synaptosomes were labeled with [3H]noradrenaline and exposed to NMDA or glutamate in superfusion. NMDA (100 microM) elicited a remarkable rise (about 500%) in the release of [3H]noradrenaline from slices. The effect of NMDA was antagonized by the glutamate receptor blocker, kynurenic acid. The antagonism by 100 microM kynurenate was reduced by submicromolar concentrations of oxiracetam and totally reversed by 1 microM of the drug. The concentration-antagonism curve for kynurenic acid was shifted to the right in the presence of 0.2 or 1 microM oxiracetam. Aniracetam and D-cycloserine, as well as glycine and D-serine, behaved similarly to oxiracetam: all compounds, tested at 1 microM, reversed the antagonism by 100 microM kynurenate of the NMDA-evoked [3H]noradrenaline release. In superfused hippocampal synaptosomes, 100 microM NMDA or glutamic acid elicited the release of [3H]noradrenaline; the evoked release was enhanced by glycine, but not by oxiracetam. In this preparation 1 microM glycine or 1 microM oxiracetam prevented the antagonism by kynurenate of the NMDA- or the glutamate-evoked [3H]noradrenaline release. As kynurenic acid is an endogenous glutamate receptor antagonist whose brain levels are known to increase in conditions associated to cognitive deficits, it is proposed that the putative cognition enhancers tested may act in vivo by relieving the antagonism produced by excessive endogenous kynurenate.
Asunto(s)
Cognición/efectos de los fármacos , Hipocampo/efectos de los fármacos , Ácido Quinurénico/farmacología , Psicotrópicos/farmacología , Receptores de N-Metil-D-Aspartato/efectos de los fármacos , Animales , Cicloserina/farmacología , Glicina/farmacología , Técnicas In Vitro , Masculino , Norepinefrina/metabolismo , Pirrolidinas/farmacología , Pirrolidinonas/farmacología , Ratas , Ratas Sprague-Dawley , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidoresRESUMEN
Exposure of rat or human neocortical or hippocampal tissue to glutamate receptor agonists elicits as Ca(2+)-dependent, exocytotic-like release of previously accumulated [3H]noradrenaline through activation of both N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors colocalized on the noradrenergic axon terminals. Here we show that the NMDA (100 microM)-evoked release of [3H]noradrenaline from superfused thin layers of isolated rat hippocampal or cortical nerve endings was potentiated when the human immunodeficiency virus type 1 coat protein gp120 was added to the superfusion medium concomitantly with NMDA. The effect of gp120 (10 pM to 3 nM) on the 100 microM NMDA-evoked release of [3H]noradrenaline was concentration-dependent; the maximal effect (approximately 140% potentiation) was reached at 100 pM of gp120. The protein was inactive on its own. The [3H]noradrenaline release evoked by NMDA (100 microM)+gp120 (100 pM) was prevented by classical NMDA receptor antagonists, as well as by 10 microM memantine. Neither the release evoked by NMDA nor that elicited by NMDA+gp120 was sensitive to the nitric oxide synthase inhibitor NG-nitro-L-arginine, suggesting no involvement of nitric oxide. The [3H]noradrenaline release elicited by 100 microM AMPA was unaffected by gp120. The protein potentiated the release evoked by 100 microM glutamate; the effect of 100 pM gp120 was quantitatively identical to that of 1 microM glycine, with no apparent additivity between gp120 and glycine. The antagonism by 1 microM 7-chloro-kynurenic acid of the NMDA-induced [3H]noradrenaline release was reversed by glycine or gp120.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Corteza Cerebral/metabolismo , Proteína gp120 de Envoltorio del VIH/farmacología , Hipocampo/metabolismo , N-Metilaspartato/farmacología , Terminaciones Nerviosas/metabolismo , Norepinefrina/metabolismo , Animales , Corteza Cerebral/efectos de los fármacos , Sinergismo Farmacológico , Ácido Glutámico/farmacología , Hipocampo/efectos de los fármacos , Técnicas In Vitro , Masculino , Terminaciones Nerviosas/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Sinaptosomas/efectos de los fármacos , Sinaptosomas/metabolismoRESUMEN
The existence of a pharmacological heterogeneity among the glutamate receptors sensitive to (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) has been investigated in the adult rat central nervous system (CNS). AMPA stimulated [3H]noradrenaline ([3H]NA) release from hippocampal synaptosomes (pD2 = 4.58) and the production of cGMP in cerebellar slices (pD2 = 7.75). The AMPA effects in the two systems were tested against several glutamate receptor antagonists including 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), 6,7-dinitro-quinoxaline-2,3-dione (DNQX), L-glutamate diethylester (GDEE), gamma-D-glutamyl-glycine (GDGG) and gamma-D-glutamyl-aminomethylsulphonate (GAMS). In both systems the AMPA effect was equally sensitive to CNQX or DNQX. However, while the AMPA-evoked increase of [3H]NA release from presynaptic terminals was not affected by GAMS, GDGG or GDEE, the postsynaptic cGMP response was prevented by GDGG and GDEE. It is concluded that rat hippocampus and cerebellum possess, respectively, presynaptic and postsynaptic AMPA-sensitive receptors involved in different functions and endowed with diverse pharmacological properties.
Asunto(s)
Sistema Nervioso Central/metabolismo , Receptores AMPA/antagonistas & inhibidores , Animales , Sistema Nervioso Central/efectos de los fármacos , Cerebelo/efectos de los fármacos , Cerebelo/metabolismo , GMP Cíclico/metabolismo , Antagonistas de Aminoácidos Excitadores/farmacología , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Técnicas In Vitro , Masculino , Norepinefrina/administración & dosificación , Norepinefrina/metabolismo , Norepinefrina/farmacología , Ratas , Ratas Sprague-Dawley , Receptores de Glutamato/metabolismo , Sinaptosomas/efectos de los fármacos , Sinaptosomas/metabolismoRESUMEN
The effects of gamma-aminobutyric acid (GABA) on the spontaneous release of endogenous glutamic acid (Glu) or aspartic acid (Asp) and the effects of Glu on the release of endogenous GABA or [3H]GABA were studied in superfused rat cerebral cortex synaptosomes. GABA increased the outflow of Glu (EC50 17.2 microM) and Asp (EC50 18.4 microM). GABA was not antagonized by bicuculline or picrotoxin. Neither muscimol nor (-)-baclofen mimicked GABA. The effects of GABA were prevented by GABA uptake inhibitors and were Na+ dependent. Glu enhanced the release of [3H]GABA (EC50 11.5 microM) from cortical synaptosomes. Glu was not mimicked by the glutamate receptor agonists N-methyl-D-aspartic, kainic, or quisqualic acid. The Glu effect was decreased by the Glu uptake inhibitor D-threo-hydroxyaspartic acid (THA) and it was Na+ sensitive. Similarly to Glu, D-Asp increased [3H]GABA release (EC50 9.9 microM), an effect blocked by THA. Glu also increased the release of endogenous GABA from cortex synaptosomes. In this case the effect was in part blocked by the (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione, whereas the 6-cyano-7-nitroquinoxaline-2,3-dione-insensitive portion of the effect was prevented by THA. GABA increased the [3H]D-Asp outflow (EC50 13.7 microM) from hippocampal synaptosomes in a muscimol-, (-)-baclofen-, bicuculline-, and picrotoxin-insensitive manner. The GABA effect was abolished by blocking GABA uptake and was Na+ dependent.(ABSTRACT TRUNCATED AT 250 WORDS)