RESUMEN
Group II metabotropic glutamate (mGlu) receptor agonists were efficacious in randomized clinical research trials for schizophrenia and generalized anxiety disorder. The regional quantification of mGlu(2) and mGlu(3) receptors remains unknown. A selective and structurally novel mGlu(2/3) receptor agonist, 2-amino-4-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylic acid (LY459477) was tritiated and the distribution of mGlu(2) and mGlu(3) receptors was studied in transgenic mice lacking either mGlu(2), mGlu(3) or both receptors. LY459477 is an agonist with 1-2 nM potency for rodent and human mGlu(2) and mGlu(3) receptors. The functional selectivity of LY459477 was demonstrated by over 640-fold selectivity and the displacement binding selectivity was greater than 320-fold for all glutamate receptors except mGlu(6) (â¼230-fold). More than 1000-fold selectivity was demonstrated for all non-glutamate receptors known to be targeted by antipsychotic drugs. Like atypical antipsychotic drugs, LY459477 reversed in vitro electrophysiological effects of a serotonergic hallucinogen and behavioral effects of phencyclidine or amphetamine. There was virtually no binding of [(3)H]LY459477 to any brain region in mice with a deletion of both mGlu(2) and mGlu(3) receptors. Regions enriched in mGlu(2) receptors included the medial prefrontal cortex, select hippocampal regions, the medial mammillary nucleus, the medial habenula, and the cerebellar granular cell layer. Regions enriched in mGlu(3) receptors were the dorsolateral entorhinal cortex, the hippocampal CA1 field, the piriform cortex, the substantia nigra, the thalamic reticular nucleus, and primary sensory thalamic nuclei. These findings suggest [(3)H]LY459477 should be a useful tool to further define the role of mGlu(2) and mGlu(3) receptors throughout the brain with respect to major neuropsychiatric syndromes. This article is part of a Special Issue entitled 'Metabotropic Glutamate Receptors'.
Asunto(s)
Aminoácidos Dicarboxílicos , Encéfalo/metabolismo , Compuestos Bicíclicos con Puentes , Agonistas de Aminoácidos Excitadores , Receptores de Glutamato Metabotrópico/metabolismo , Animales , Células Cultivadas , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos ICR , Ratones Noqueados , Ensayo de Unión Radioligante/métodos , Ratas , Receptores de Glutamato Metabotrópico/genética , TritioRESUMEN
Alzheimer's disease (AD) may affect all cell types in the central nervous system. Astrocytes have rarely been investigated in the aged brain and the role of astrocytes in AD is poorly understood. In this study, we used acute brain slices from an amyloid-beta overexpressing double transgenic mouse line where astrocytes express the enhanced green fluorescent protein under the control of the glial fibrillary acidic protein promoter. Using the patch-clamp technique, we analyzed cell coupling and glutamate reactivity, two main features of astrocytes, in the living tissue of aged mice in an AD mouse model. We found large astrocytic networks in the aged (20 to 27 months) transgenic animals in the neocortex, but not in the hippocampus. In contrast, coupling was low in all brain regions of aged control mice. We furthermore noticed significant changes in the responses of astrocytes to glutamate. The expression of functional glutamate transporters and AMPA/kainate-type glutamate receptors increases in the amyloid-beta protein precursor overexpressing mice. Thus, exposure to amyloid-beta leads to altered astrocyte properties and this change might be beneficial to maintain synaptic function.
Asunto(s)
Envejecimiento/patología , Envejecimiento/fisiología , Enfermedad de Alzheimer/patología , Astrocitos/patología , Astrocitos/fisiología , Envejecimiento/genética , Enfermedad de Alzheimer/genética , Precursor de Proteína beta-Amiloide/genética , Animales , Humanos , Ratones , Ratones Transgénicos , Red Nerviosa/patología , Red Nerviosa/fisiologíaRESUMEN
The effects of amyloid-beta peptide (Aß) on astrocyte responses to activation of mGlu5 receptors have been investigated using calcium imaging. Pre-incubation with Aß1-40 peptide for up to 72 h produced a time- and concentration-dependent 2-4 fold enhancement in the magnitude of the intracellular calcium mobilization response to the group I metabotropic glutamate receptor agonist (S)-3,5-dihydroxyphenylglycine (DHPG). In contrast, pre-treatment with Aß1-40 did not alter the calcium responses induced by other G protein coupled- or ion channel-receptors. Aß 1-40-enhanced DHPG responses were blocked by the mGlu5 antagonist MPEP but not by inhibitors of voltage dependent calcium channels or by the AMPA/KA receptor antagonist CNQX. Up-regulation of mGlu5 coupled responses was associated with significant increases in astrocyte mGlu5 receptor-mRNA and-protein expression after preincubation with Aß . The changes observed in vitro were consistent with results obtained from human Alzheimer's disease (AD) patients.Immunostaining for mGlu5 receptors was increased on astrocytes which were colocalized with Aß plaques in hippocampal tissue from AD patients compared to age-matched controls. These results suggest that modulation of mGlu5 receptors in astrocytes could be an important mechanism in determining the progression of pathology in AD.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Astrocitos/metabolismo , Fragmentos de Péptidos/metabolismo , Receptor del Glutamato Metabotropico 5/metabolismo , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/metabolismo , Animales , Astrocitos/efectos de los fármacos , Calcio/metabolismo , Canales de Calcio/metabolismo , Células Cultivadas , Corteza Cerebral/metabolismo , Hipocampo/metabolismo , Humanos , Espacio Intracelular/metabolismo , Ratones Endogámicos C57BL , Persona de Mediana Edad , ARN Mensajero/metabolismo , Receptores AMPA/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Ácido Kaínico/metabolismo , Factores de Tiempo , Regulación hacia ArribaRESUMEN
Amyloid beta (Abeta) peptides are the major constituent of amyloid plaques, one of the hallmark pathologies of Alzheimer's disease. Accurate and precise quantitation of these peptides in biological fluids is a critical component of Alzheimer's disease research. The current most established assay for analysis of Abeta peptides in preclinical research is enzyme-linked immunosorbent assay (ELISA), which, although sensitive and of proven utility, is a multistep, labor-intensive assay that is difficult to automate completely. To overcome these limitations, the authors have developed and optimized simple, sensitive, homogeneous 384-well assays for Abeta1-42 and Abeta1-40 using AlphaScreen technology. The assays are capable of detecting Abeta peptides at concentrations <2 pg/mL and, using a final assay volume of 20 microL, routinely generate Z' values >0.85. The AlphaScreen format has the following key advantages: substantially less hands-on time to run, easier to automate, higher throughput, and less expensive to run than the traditional ELISA. The results presented here show that AlphaScreen technology permits robust, efficient, and cost-effective quantitation of Abeta peptides.
Asunto(s)
Enfermedad de Alzheimer/diagnóstico , Péptidos beta-Amiloides/aislamiento & purificación , Tamizaje Masivo/métodos , Fragmentos de Péptidos/aislamiento & purificación , Secuencia de Aminoácidos , Péptidos beta-Amiloides/análisis , Péptidos beta-Amiloides/inmunología , Animales , Anticuerpos Monoclonales/farmacología , Química Encefálica , Calibración , Células Cultivadas , Análisis Costo-Beneficio , Medios de Cultivo/química , Ensayo de Inmunoadsorción Enzimática/métodos , Femenino , Humanos , Tamizaje Masivo/economía , Ratones , Ratones Endogámicos C57BL , Modelos Biológicos , Datos de Secuencia Molecular , Fragmentos de Péptidos/análisis , Fragmentos de Péptidos/inmunología , Sensibilidad y Especificidad , Extractos de Tejidos/análisis , Extractos de Tejidos/químicaAsunto(s)
Analgésicos/farmacología , Dolor/tratamiento farmacológico , Enfermedades del Sistema Nervioso Periférico/tratamiento farmacológico , Receptores de Glutamato Metabotrópico/antagonistas & inhibidores , Analgésicos/uso terapéutico , Animales , Humanos , Dolor/metabolismo , Dolor/fisiopatología , Enfermedades del Sistema Nervioso Periférico/metabolismo , Enfermedades del Sistema Nervioso Periférico/fisiopatología , Receptores de Glutamato Metabotrópico/fisiologíaRESUMEN
Group II metabotropic glutamate (mGlu) receptor agonists, including (1S,2S,5R,6S)-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylate monohydrate (LY354740) and (-)-2-oxa-4-aminobicyclo[3.1.0]hexane-4,6-dicarboxylate (LY379268), have demonstrated efficacy in animal models of anxiety and schizophrenia, and LY354740 decreased anxiety in human subjects. Herein, we report the in vitro pharmacological profile and pharmacokinetic properties of another potent, selective, and structurally novel mGlu2/3 receptor agonist, (-)-(1R,4S,5S,6S)-4-amino-2-sulfonylbicyclo[3.1.0]hexane-4,6-dicarboxylic acid (LY404039) and provide comparisons with LY354740. Similar to LY354740, LY404039 is a nanomolar potent agonist at recombinant human mGlu2 and mGlu3 receptors (K(i) = 149 and 92, respectively) and in rat neurons expressing native mGlu2/3 receptors (Ki = 88). LY404039 is highly selective for mGlu2/3 receptors, showing more than 100-fold selectivity for these receptors, versus ionotropic glutamate receptors, glutamate transporters, and other receptors targeted by known anxiolytic and antipsychotic medications. Functionally, LY404039 potently inhibited forskolin-stimulated cAMP formation in cells expressing human mGlu2 and mGlu3 receptors. Electrophysiological studies indicated that LY404039 suppressed electrically evoked excitatory activity in the striatum, and serotonin-induced l-glutamate release in the prefrontal cortex; effects reversed by LY341495. These characteristics suggest LY404039 modulates glutamatergic activity in limbic and forebrain areas relevant to psychiatric disorders; and that, similar to LY354740, it works through a mechanism that may be devoid of negative side effects associated with current antipsychotics and anxiolytics. Interestingly, despite the slightly lower potency (approximately 2-5-fold) of LY404039 versus LY354740 in binding, functional, and electrophysiological assays, LY404039 demonstrated higher plasma exposure and better oral bioavailability in pharmacokinetic experiments. Collectively, the current data indicate that LY404039 may be valuable in the treatment of neuropsychiatric disorders, including anxiety and psychosis.
Asunto(s)
Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacocinética , Óxidos S-Cíclicos/farmacología , Óxidos S-Cíclicos/farmacocinética , Agonistas de Aminoácidos Excitadores/farmacología , Agonistas de Aminoácidos Excitadores/farmacocinética , Receptores de Glutamato Metabotrópico/efectos de los fármacos , Animales , Sitios de Unión , Compuestos Bicíclicos con Puentes/química , Compuestos Bicíclicos con Puentes/farmacocinética , Compuestos Bicíclicos con Puentes/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/química , Línea Celular , Corteza Cerebral/efectos de los fármacos , Colforsina/farmacología , AMP Cíclico/biosíntesis , Óxidos S-Cíclicos/química , Estimulación Eléctrica , Agonistas de Aminoácidos Excitadores/química , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Humanos , Hidrólisis , Técnicas In Vitro , Masculino , Fosfatidilinositoles/metabolismo , Ratas , Ratas Endogámicas F344 , Ratas Sprague-Dawley , Serotonina/farmacología , Antagonistas de la Serotonina/farmacologíaRESUMEN
The cytokine tumor necrosis factor-alpha (TNFalpha) induces Ca2+-dependent glutamate release from astrocytes via the downstream action of prostaglandin (PG) E2. By this process, astrocytes may participate in intercellular communication and neuromodulation. Acute inflammation in vitro, induced by adding reactive microglia to astrocyte cultures, enhances TNFalpha production and amplifies glutamate release, switching the pathway into a neurodamaging cascade (Bezzi, P., Domercq, M., Brambilla, L., Galli, R., Schols, D., De Clercq, E., Vescovi, A., Bagetta, G., Kollias, G., Meldolesi, J., and Volterra, A. (2001) Nat. Neurosci. 4, 702-710). Because glial inflammation is a component of Alzheimer disease (AD) and TNFalpha is overexpressed in AD brains, we investigated possible alterations of the cytokine-dependent pathway in PDAPP mice, a transgenic model of AD. Glutamate release was measured in acute hippocampal and cerebellar slices from mice at early (4-month-old) and late (12-month-old) disease stages in comparison with age-matched controls. Surprisingly, TNFalpha-evoked glutamate release, normal in 4-month-old PDAPP mice, was dramatically reduced in the hippocampus of 12-month-old animals. This defect correlated with the presence of numerous beta-amyloid deposits and hypertrophic astrocytes. In contrast, release was normal in cerebellum, a region devoid of beta-amyloid deposition and astrocytosis. The Ca2+-dependent process by which TNFalpha evokes glutamate release in acute slices is distinct from synaptic release and displays properties identical to those observed in cultured astrocytes, notably PG dependence. However, prostaglandin E2 induced normal glutamate release responses in 12-month-old PDAPP mice, suggesting that the pathology-associated defect involves the TNFalpha-dependent control of secretion rather than the secretory process itself. Reduced expression of DENN/MADD, a mediator of TNFalpha-PG coupling, might account for the defect. Alteration of this neuromodulatory astrocytic pathway is described here for the first time in relation to Alzheimer disease.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Astrocitos/metabolismo , Modelos Animales de Enfermedad , Ácido Glutámico/metabolismo , Factor de Necrosis Tumoral alfa/fisiología , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/metabolismo , Animales , Secuencia de Bases , Cartilla de ADN , Gliosis , Hipocampo/metabolismo , Hipocampo/patología , Inmunohistoquímica , Ratones , Ratones Transgénicos , Transducción de SeñalRESUMEN
Several lines of evidence suggest that mGlu1 metabotropic glutamate receptors may be involved in seizure disorders such as epilepsy. For example, the mGlu1 agonist DHPG produces limbic seizures and group I antagonists such as 4C3HPG and 4CPG are anticonvulsant when administered intracerebrally. The purpose of the present experiments was to characterize the anticonvulsant effects of the selective mGlu1 receptor antagonist LY456236 in mice and rats. In male and female DBA/2 mice, LY456236 produced a dose-related inhibition of sound-induced clonic-tonic seizures. In male CF1 mice, LY456236 produced a dose-related inhibition of tonic extensor seizures in the threshold electroshock model, and limbic seizures in the 6-Hz focal seizure model. However, this antagonist did not inhibit clonic seizures produced by pentylenetetrazol. In amygdala-kindled male Sprague-Dawley rats, LY456236 produced dose-related decreases in behavioral and electrographic seizures at threshold stimulus intensity. In addition, LY456236 produced a dose-related increase in the stimulus intensity required to produce generalized seizures. Taken together, the present results support the conclusion that mGlu1 receptor antagonists such as LY456236 may have clinical utility in the treatment of epilepsy and other seizure disorders.
Asunto(s)
Anticonvulsivantes/uso terapéutico , Antagonistas de Aminoácidos Excitadores/uso terapéutico , Receptores de Glutamato Metabotrópico/antagonistas & inhibidores , Convulsiones/tratamiento farmacológico , Estimulación Acústica/efectos adversos , Animales , Relación Dosis-Respuesta a Droga , Electrochoque/efectos adversos , Masculino , Ratones , Ratones Endogámicos DBA , Pentilenotetrazol , Quinazolinas/uso terapéutico , Ratas , Ratas Sprague-Dawley , Convulsiones/inducido químicamente , Convulsiones/etiología , Umbral Sensorial/fisiología , Factores de TiempoRESUMEN
G-protein-coupled receptors are well known for converting an extracellular signal into an intracellular response. Here we showed that the metabotropic glutamate receptor 5 (mGlu5) plays a dynamic intracellular role in signal transduction. Activation of endogenously expressed mGlu5 on striatal nuclear membranes leads to rapid, sustained calcium (Ca2+) responses within the nucleoplasm that can be blocked by receptor-specific antagonists. Extracellular ligands such as glutamate and quisqualate reach nuclear receptors via both sodium-dependent transporters and cystine glutamate exchangers. Inhibition of either transport system blocks radiolabeled agonist uptake as well as agonist-induced nuclear Ca2+ changes. Impermeable antagonists like LY393053 and LY367366 not only blocked [3H]quisqualate binding but also prevented nontransported agonists such as (RS)-3,5-dihydroxyphenylglycine from inducing intracellular Ca2+ changes in heterologous cells. In contrast, neither LY compound prevented quisqualate or glutamate from activating intracellular receptors leading to Ca2+ responses. Inasmuch as Ca2+ can enter the nucleoplasm via the nuclear pore complex or from the nuclear lumen, the presence of nuclear mGlu5 receptors appeared to amplify the latter process generating a faster nuclear response in heterologous cells. In isolated striatal nuclei, nuclear receptor activation results in the de novo appearance of phosphorylated CREB protein. Thus, activation of nuclear mGlu5 receptors initiates a signaling cascade that is known to alter gene transcription and regulate many paradigms of synaptic plasticity. These studies demonstrated that mGlu5 receptors play a dynamic role in signaling both on and off the plasma membrane.
Asunto(s)
Núcleo Celular/metabolismo , Neuronas/citología , Receptores de Glutamato Metabotrópico/química , Transporte Biológico , Calcio/metabolismo , Membrana Celular/metabolismo , Colorantes/farmacología , Cisteína/química , Citoplasma/metabolismo , Relación Dosis-Respuesta a Droga , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Inhibidores Enzimáticos/farmacología , Ácido Glutámico/química , Ácido Glutámico/metabolismo , Glicina/análogos & derivados , Glicina/farmacología , Inmunohistoquímica , Ligandos , Luz , Microscopía Fluorescente , Modelos Biológicos , Neuronas/metabolismo , Fosforilación , Propionatos/farmacología , Unión Proteica , Proteínas Recombinantes de Fusión/química , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Sodio/química , Fracciones Subcelulares/metabolismo , Tiofenos/farmacología , Factores de TiempoRESUMEN
The asymmetric synthesis and biological activity of (2S,1'S,2'R,3'R)-2-(2'-carboxy-3'-hydroxymethylcyclopropyl) glycine ((+)-3) is described. This novel C-3' substituted carboxy cyclopropyl glycine is a highly potent group 2 and group 3 mGluR agonist that has proven to be orally active in both fear potentiated startle (animal model for anxiety) and PCP-induced motor activation (animal model for psychosis) assays in rats.
Asunto(s)
Glicina/síntesis química , Receptores de Glutamato Metabotrópico/agonistas , Administración Oral , Animales , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , AMP Cíclico/biosíntesis , Miedo , Glicina/análogos & derivados , Glicina/química , Glicina/farmacología , Técnicas In Vitro , Masculino , Actividad Motora/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Reflejo de Sobresalto/efectos de los fármacos , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
This study investigated the distribution of metabotropic glutamate receptors (mGluRs) in meningeal and parenchymal microvasculature and in choroid plexus by means of Western blot analysis and immunohistochemistry. Western blot analysis demonstrated mGluR expression in both rat and human leptomeningeal tissues. In the rat, mGluR expression was developmentally regulated, with only mGluR2/3 showing expression at the embryonic day 19 developmental stage. In contrast, mGluR1 alpha, mGluR2/3, mGluR4a, and mGluR7 were expressed in leptomeninges from adult rats. Immunohistochemical analyses showed intense mGluR1 alpha immunoreactivity in the pia mater and blood vessels in the subarachnoid space and in the arachnoid layer of the meninges. mGluR2/3, mGluR4a, mGluR5, and mGluR7 were also expressed in meningeal microvasculature. In addition, the parenchymal microvasculature and choroid plexus were strongly immunoreactive for mGluR1 alpha, mGluR2/3, mGluR4a, mGluR5, and mGluR7. We used antibodies specific for phenotypic markers of microvascular and glial cells to characterize the cell type(s) immunopositive for mGluRs. Comparison of staining with anti-von Willebrand factor antibody and anti-mGluR antibodies revealed that mGluR immunoreactivity was present in cells that surrounded the luminal surface labeled by the endothelial cell marker. In these cells, smooth muscle actin and mGluR immunoreactivity overlapped, suggesting that, in addition to endothelial cells, pericytes within the microvasculature also express mGluRs. Furthermore, expression of mGluR1 alpha was also observed in pure pericyte cultures isolated from bovine retina. These data suggest that glutamate by means of activation of mGluRs may have a broad sphere of physiological influence in the brain which in addition to modulating synaptic transmission may also have a role in determining microvascular function and dysfunction.
Asunto(s)
Encéfalo/irrigación sanguínea , Circulación Cerebrovascular , Plexo Coroideo/irrigación sanguínea , Meninges/irrigación sanguínea , Ratas/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Animales , Vasos Sanguíneos/metabolismo , Western Blotting , Bovinos , Células Cultivadas , Humanos , Inmunohistoquímica , Masculino , Microcirculación , Pericitos/metabolismo , Ratas Sprague-DawleyRESUMEN
A series of novel group I metabotropic glutamate receptor (mGlu) antagonists have been designed on the basis of the 4-carboxyphenylglycine pharmacophore. The compounds are either mGlu1 receptor selective or equipotent for both mGlu1 and mGlu5 receptors and have IC(50) values ranging from 1 to 30 microM determined by phosphoinositide hydrolysis (PI) assay in vitro. All the compounds produced dose-dependent inhibition of group I mGlu receptor agonist (RS)-3,5-dihydroxyphenylglycine (DHPG)-induced limbic seizure responses in mice with ED(50) values ranging from 9 nmol for LY393053 to 138 nmol for LY339840 after intracerebroventricular injection and were more potent than the mGlu1 receptor antagonist 1-aminoindan-1,5-dicarboxylic acid (ED(50)=477 nmol). Further antagonist actions were also demonstrated in a model of (RS)-DHPG-induced PI hydrolysis in vivo such that LY367385 and the active cis isomer of LY393053 produced dose-dependent inhibition of PI responses in both cerebellum and hippocampus. Cis LY393053 also inhibited hippocampal PI responses when administered intraperitoneally at a dose of 30 mg/kg. These compounds define a new series of group I mGlu receptor antagonists which may serve as useful experimental tools.
Asunto(s)
Benzoatos , Antagonistas de Aminoácidos Excitadores/farmacología , Glicina/análogos & derivados , Glicina/farmacología , Receptores de Glutamato Metabotrópico/antagonistas & inhibidores , Alanina/análogos & derivados , Alanina/farmacología , Animales , Benzoatos/farmacología , Cerebelo/efectos de los fármacos , Cerebelo/metabolismo , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Agonistas de Aminoácidos Excitadores/farmacología , Glicina/química , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Concentración 50 Inhibidora , Ratones , Propionatos/farmacología , Ratas , Ratas Sprague-Dawley , Receptores de Glutamato Metabotrópico/agonistas , Receptores de Glutamato Metabotrópico/metabolismo , Resorcinoles/farmacología , Convulsiones/prevención & control , Relación Estructura-Actividad , Tiofenos/farmacología , Xantenos/farmacologíaRESUMEN
The asymmetric synthesis and biological activity of (2S,1'S,2'S,3'R)-2-(2'-carboxy-3'-methylcyclopropyl) glycine 7 and its epimer at the C3' center 6 are described. Compound 7 is a highly potent and selective agonist for group 2 metabotropric glutamate receptors (mGluRs). It is also systemically 4 orders of magnitude more active in the fear-potentiated startle model of anxiety in rats than the rigid constrained bicyclic system LY354740. Therefore, we have shown that high molecular complexity of conformationally constrained bicyclic systems is not a requirement to achieve highly selective and potent group 2 mGluRs agonists.
Asunto(s)
Ansiolíticos/síntesis química , Ciclopropanos/síntesis química , Glicina/síntesis química , Receptores de Glutamato Metabotrópico/agonistas , Animales , Ansiolíticos/química , Ansiolíticos/farmacología , Compuestos Bicíclicos con Puentes/química , Compuestos Bicíclicos con Puentes/farmacología , Línea Celular , Ciclopropanos/química , Ciclopropanos/farmacología , Glicina/análogos & derivados , Glicina/química , Glicina/farmacología , Ligandos , Modelos Moleculares , Ratas , Reflejo de Sobresalto/efectos de los fármacos , EstereoisomerismoRESUMEN
Striatal cholinergic interneurons were recorded from a rat slice preparation. Synaptic potentials evoked by intrastriatal stimulation revealed three distinct components: a glutamatergic EPSP, a GABA(A)-mediated depolarizing potential, and an acetylcholine (ACh)-mediated IPSP. The responses to group II metabotropic glutamate (mGlu) receptor activation were investigated on the isolated components of the synaptic potentials. Each pharmacologically isolated component was reversibly reduced by bath-applied LY379268 and ((2S,1'R,2'R,3'R)-2-(2,3-dicarboxylcyclopropyl)-glycine, group II agonists. In an attempt to define the relevance of group II mGlu receptor activation on cholinergic transmission, we focused on the inhibitory effect on the IPSP, which was mimicked and occluded by omega-agatoxin IVA (omega-Aga-IVA), suggesting a modulation on P-type high-voltage-activated calcium channels. Spontaneous calcium-dependent plateau-potentials (PPs) were recorded with cesium-filled electrodes plus tetraethylammonium and TTX in the perfusing solution, and measurements of intracellular calcium [Ca2+]i changes were obtained simultaneously. PPs and the concomitant [Ca2+]i elevations were significantly reduced in amplitude and duration by LY379268. The mGlu-mediated inhibitory effect on PPs was mimicked by omega-Aga-IVA, suggesting an involvement of P-type channels. Moreover, electrically induced ACh release from striatal slices was reduced by mGlu2 receptor agonists and occluded by omega-Aga-IVA in a dose-dependent manner. Finally, double-labeling experiments combining mGlu2 receptor in situ hybridization and choline acetyltransferase immunocytochemistry revealed a strong mGlu2 receptor labeling on cholinergic interneurons, whereas single-label isotopic in situ hybridization for mGlu3 receptors did not show any labeling in these large striatal interneurons. These results suggest that the mGlu2 receptor-mediated modulatory action on cell excitability would tune striatal ACh release, representing an interesting target for pharmacological intervention in basal ganglia disorders.