RESUMEN

Genetic mutations of the Methyl-CpG-binding protein-2 (MECP2) gene underlie Rett syndrome (RTT). Developmental processes are often considered to be irrelevant in RTT pathogenesis but neuronal activity at birth has not been recorded. We report that the GABA developmental shift at birth is abolished in CA3 pyramidal neurons of Mecp2-/y mice and the glutamatergic/GABAergic postsynaptic currents (PSCs) ratio is increased. Two weeks later, GABA exerts strong excitatory actions, the glutamatergic/GABAergic PSCs ratio is enhanced, hyper-synchronized activity is present and metabotropic long-term depression (LTD) is impacted. One day before delivery, maternal administration of the NKCC1 chloride importer antagonist bumetanide restored these parameters but not respiratory or weight deficits, nor the onset of mortality. Results suggest that birth is a critical period in RTT with important alterations that can be attenuated by bumetanide raising the possibility of early treatment of the disorder.

Asunto(s)

Proteína 2 de Unión a Metil-CpG/fisiología , Neuronas/patología , Receptores de GABA-A/metabolismo , Síndrome de Rett/patología , Transmisión Sináptica/efectos de los fármacos , Ácido gamma-Aminobutírico/metabolismo , Animales , Bumetanida/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Plasticidad Neuronal , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Sistema Respiratorio/efectos de los fármacos , Síndrome de Rett/tratamiento farmacológico , Síndrome de Rett/genética , Síndrome de Rett/metabolismo , Inhibidores del Simportador de Cloruro Sódico y Cloruro Potásico/farmacología , Potenciales Sinápticos

RESUMEN

We report that the apical dendrites of CA3 hippocampal pyramidal neurons are increased during labor and birth in the valproate model of autism but not in control animals. Using the iDISCO clearing method, we show that hippocampal, especially CA3 region, and neocortical volumes are increased and that the cerebral volume distribution shifts from normal to lognormal in valproate-treated animals. Maternal administration during labor and birth of the NKCC1 chloride transporter antagonist bumetanide, which reduces [Cl-]i levels and attenuates the severity of autism, abolished the neocortical and hippocampal volume changes and reduced the whole-brain volume in valproate-treated animals. These results suggest that the abolition of the oxytocin-mediated excitatory-to-inhibitory shift of GABA actions during labor and birth contributes to the pathogenesis of autism spectrum disorders by stimulating growth during a vulnerable period.

Asunto(s)

Trastorno del Espectro Autista/tratamiento farmacológico , Trastorno del Espectro Autista/fisiopatología , Bumetanida/uso terapéutico , Hipocampo/metabolismo , Parto/metabolismo , Células Piramidales/metabolismo , Inhibidores del Simportador de Cloruro Sódico y Cloruro Potásico/uso terapéutico , Animales , Animales Recién Nacidos , Trastorno del Espectro Autista/inducido químicamente , Dendritas/efectos de los fármacos , Dendritas/metabolismo , Modelos Animales de Enfermedad , Femenino , GABAérgicos/farmacología , Embarazo , Células Piramidales/efectos de los fármacos , Ratas , Ratas Wistar , Ácido Valproico/farmacología

RESUMEN

We report that half striatal cholinergic interneurons are dual transmitter cholinergic and GABAergic interneurons (CGINs) expressing ChAT, GAD65, Lhx7, and Lhx6 mRNAs, labeled with GAD and VGAT, generating monosynaptic dual cholinergic/GABAergic currents and an inhibitory pause response. Dopamine deprivation increases CGINs ongoing activity and abolishes GABAergic inhibition including the cortico-striatal pause because of high [Cl-]i levels. Dopamine deprivation also dramatically increases CGINs dendritic arbors and monosynaptic interconnections probability, suggesting the formation of a dense CGINs network. The NKCC1 chloride importer antagonist bumetanide, which reduces [Cl-]i levels, restores GABAergic inhibition, the cortico-striatal pause-rebound response, and attenuates motor effects of dopamine deprivation. Therefore, most of the striatal cholinergic excitatory drive is balanced by a concomitant powerful GABAergic inhibition that is impaired by dopamine deprivation. The attenuation by bumetanide of cardinal features of Parkinson's disease paves the way to a novel therapeutic strategy based on a restoration of low [Cl-]i levels and GABAergic inhibition.

Asunto(s)

Neuronas Colinérgicas/metabolismo , Cuerpo Estriado/metabolismo , Potenciales Postsinápticos Inhibidores/efectos de los fármacos , Interneuronas/metabolismo , Enfermedad de Parkinson Secundaria/metabolismo , Ácido gamma-Aminobutírico/metabolismo , Acetilcolina/metabolismo , Acetilcolina/farmacología , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Animales , Bumetanida/farmacología , Cloruros/metabolismo , Colinérgicos/metabolismo , Colinérgicos/farmacología , Neuronas Colinérgicas/efectos de los fármacos , Neuronas Colinérgicas/patología , Cuerpo Estriado/efectos de los fármacos , Cuerpo Estriado/patología , Dopamina/deficiencia , Regulación de la Expresión Génica , Glutamato Descarboxilasa/genética , Glutamato Descarboxilasa/metabolismo , Humanos , Interneuronas/efectos de los fármacos , Interneuronas/patología , Transporte Iónico , Proteínas con Homeodominio LIM/genética , Proteínas con Homeodominio LIM/metabolismo , Ratones , Ratones Transgénicos , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Plasticidad Neuronal/efectos de los fármacos , Enfermedad de Parkinson Secundaria/genética , Enfermedad de Parkinson Secundaria/patología , Técnicas de Placa-Clamp , Inhibidores del Simportador de Cloruro Sódico y Cloruro Potásico/farmacología , Miembro 2 de la Familia de Transportadores de Soluto 12/genética , Miembro 2 de la Familia de Transportadores de Soluto 12/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas del Transporte Vesicular de Aminoácidos Inhibidores/genética , Proteínas del Transporte Vesicular de Aminoácidos Inhibidores/metabolismo , Ácido gamma-Aminobutírico/farmacología

RESUMEN

Tuberous sclerosis complex (TSC), caused by dominant mutations in either TSC1 or TSC2 tumour suppressor genes is characterized by the presence of brain malformations, the cortical tubers that are thought to contribute to the generation of pharmacoresistant epilepsy. Here we report that tuberless heterozygote Tsc1(+/-) mice show functional upregulation of cortical GluN2C-containing N-methyl-D-aspartate receptors (NMDARs) in an mTOR-dependent manner and exhibit recurrent, unprovoked seizures during early postnatal life (

Asunto(s)

Anticonvulsivantes/farmacología , Epilepsia/tratamiento farmacológico , Pirazoles/farmacología , Quinolonas/farmacología , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/genética , Esclerosis Tuberosa/tratamiento farmacológico , Proteínas Supresoras de Tumor/genética , Potenciales de Acción/efectos de los fármacos , Animales , Modelos Animales de Enfermedad , Electroencefalografía , Epilepsia/genética , Epilepsia/metabolismo , Epilepsia/patología , Regulación de la Expresión Génica , Heterocigoto , Humanos , Masculino , Ratones , Ratones Transgénicos , Microtomía , Neocórtex/efectos de los fármacos , Neocórtex/metabolismo , Neocórtex/patología , Técnicas de Placa-Clamp , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Transducción de Señal , Serina-Treonina Quinasas TOR/metabolismo , Técnicas de Cultivo de Tejidos , Esclerosis Tuberosa/genética , Esclerosis Tuberosa/metabolismo , Esclerosis Tuberosa/patología , Proteína 1 del Complejo de la Esclerosis Tuberosa , Proteínas Supresoras de Tumor/deficiencia

RESUMEN

P2X2 and P2X3 receptors expressed in mammalian sensory neurons participate in nociception. Cannabinoid receptors modulate nociceptive processing in various models of pain. They are also expressed in nociceptive sensory neurons. We have examined the effect of cannabinoids on the slow P2X2 and P2X2/3 receptors in the cells isolated from nodosal and dorsal root ganglia of rat. The study was carried out by means of the whole-cell patch clamp and rapid superfusion methods. We have found that both endogenous and synthetic cannabinoids (anandamide, WIN55,212-2, and (R)-(+)-methanandamide) inhibit the slow response to ATP mediated by P2X2 and P2X2/3 receptors in a majority of tested neurons. This inhibition was significant but only partial: anandamide (0.5-1 microM) inhibited the response to 51+/-21% of control. In the remaining minority of tested neurons, the response was transiently facilitated. The effect of cannabinoids appears to be mediated via cannabinoid CB(1) receptors: it was reversibly inhibited by selective CB(1) antagonist, SR141716A (10 microM). Introduction of cyclic AMP (0.5 mM) into the cell potently facilitated the inhibitory effect of cannabinoids: the ATP-activated current was inhibited to 13+/-10% of control. These data indicate that cannabinoids may inhibit nociceptive responses produced by P2X receptors.

Asunto(s)

Cannabinoides/farmacología , Neuronas Aferentes/fisiología , Receptores Purinérgicos P2/efectos de los fármacos , Receptores Purinérgicos P2/fisiología , Adenosina Trifosfato/fisiología , Animales , Ácidos Araquidónicos/farmacología , Benzoxazinas/farmacología , Bloqueadores de los Canales de Calcio/farmacología , Cannabinoides/antagonistas & inhibidores , Electrofisiología , Endocannabinoides , Ganglios Espinales/efectos de los fármacos , Ganglios Espinales/fisiología , Morfolinas/farmacología , Naftalenos/farmacología , Nociceptores , Ganglio Nudoso/efectos de los fármacos , Ganglio Nudoso/fisiología , Técnicas de Placa-Clamp , Piperidinas/farmacología , Alcamidas Poliinsaturadas/farmacología , Pirazoles/farmacología , Ratas , Receptores Purinérgicos P2X2 , Receptores Purinérgicos P2X3 , Rimonabant

RESUMEN

Despite almost forty years of widespread use of antidepressant drugs, their mode of action is still unknown. Hyperforin, a phloroglucinol derivative, is a major pharmacologically and therapeutically active constituent of Hypericum perforatum extract that is widely used as an herbal antidepressant drug. However, the mechanism or mechanisms of action of these naturally abundant, non-toxic extracts remain unclear. Enzymatically isolated patch-clamped rat central and peripheral neurons exposed to rapid changes in the composition of external medium (concentration clamp) were used in our experiments to investigate the modulation of the various voltage- and ligand-gated channels by hyperforin, as well as by other constituents of Hypericum perforatum. At nanomolar concentrations, hyperforin induced significant inhibition of various ion channels. In the case of P-type Ca2+ channels, we established that hyperforin acts via interaction with calmodulin or through calmodulin-activated pathways involving at least one second messenger. The results presented here indicate that multiple mechanisms and extract constituents may be involved in the antidepressant action of Hypericum extracts, and that they could also possess neuroprotective and analgesic effects.

Asunto(s)

Hypericum , Canales Iónicos/fisiología , Extractos Vegetales/farmacología , Células de Purkinje/efectos de los fármacos , Animales , Calcio/metabolismo , Bloqueadores de los Canales de Calcio/metabolismo , Canales de Calcio Tipo P/metabolismo , Calmodulina/metabolismo , Ganglios Espinales/citología , Guanosina 5'-O-(3-Tiotrifosfato)/farmacología , Hipocampo/citología , Activación del Canal Iónico/efectos de los fármacos , Potenciales de la Membrana/efectos de los fármacos , Técnicas de Placa-Clamp , Células de Purkinje/citología , Células de Purkinje/fisiología , Ratas , Ratas Wistar , Receptores AMPA/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Sodio/metabolismo , Canales de Sodio/metabolismo , omega-Agatoxina IVA/farmacología

RESUMEN

A new substance (R,S)-(3,4-dihydro-6,7-dimethoxyisoquinoline-1-yl)-2-cyclohexyl-N-(3,3-diphenylpropyl)-acetamide hydrochloride (BIIA388Cl), which demonstrates neuroprotective properties in animal models, was examined for its action on K(+) currents in acutely isolated rat hippocampal neurons using the patch-clamp/concentration clamp techniques in the whole-cell configuration. The delayed rectifier K(+)-current (I(DR)) was strongly inhibited by externally applied BIIA388Cl, while the transient A-current (I(A)) remained virtually unaffected. Block of I(DR) by the pre-applied BIIA388Cl was revealed as a rapid decay of the current indicating direct interaction of the drug with the open state of the channel. The removal of the block upon repolarization was also rapid (tau=22 ms). The dose-response relationship for the blocking action of BIIA388Cl revealed an IC(50) value of 300 nM for the peak I(DR), whereas the IC(50) value for I(DR) measured 300 ms after the onset of depolarization was 120 nM. The blocking action of BIIA388Cl on I(A) was at least 200 times less potent. These data allow us to conclude that BIIA388Cl is an effective and selective blocker of I(DR). This current is the main pathway for the loss of intracellular potassium by depolarized neurons. Selective obstruction of this pathway could be useful for neuroprotection.

Asunto(s)

Isoquinolinas/farmacología , Neuronas/fisiología , Fármacos Neuroprotectores/farmacología , Bloqueadores de los Canales de Potasio , Canales de Potasio con Entrada de Voltaje , Animales , Separación Celular , Canales de Potasio de Tipo Rectificador Tardío , Hipocampo/citología , Técnicas de Placa-Clamp , Canales de Potasio/fisiología , Ratas

RESUMEN

Whole-cell, patch-clamp recordings from acutely isolated cerebellar Purkinje neurons demonstrate a two-stage modulation of P-type high-voltage-activated (HVA) Ca2+ current by a constituent of St. John's wort, hyperforin (0.04-0.8 microM). The first stage of modulation was voltage dependent and reversible. It comprised slow-down of the activation kinetics and a shift in the voltage dependence of P-current to more negative voltages. Hyperforin (0.8 microM) shifted the maximum of the current/voltage (I/V) relationship by -8+/-2 mV. The second, voltage-independent stage of modulation was manifested as a slowly developing inhibition of P-current that could not be reversed within the period of study. Neither form of modulation was abolished by intracellular guanosine 5'-O-(2-thiodiphosphate) (GDPPS) or guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) or by strong depolarising pre-pulses, indicating that modulation via guanine nucleotide-binding proteins (G proteins) is not involved in the observed phenomenon. Calmidazolium (0.5 microM), an antagonist of the intracellular Ca2+-binding protein calmodulin significantly inhibited the hyperforin-induced shift of the IIV curve maximum and the slow-down of the activation kinetics. It did not, however, affect the delayed inhibition of P-current, indicating that the two stages of modulation are mediated by separate mechanisms.

Asunto(s)

Antibacterianos/farmacología , Canales de Calcio Tipo P/fisiología , Guanosina Difosfato/análogos & derivados , Activación del Canal Iónico/efectos de los fármacos , Células de Purkinje/fisiología , Terpenos/farmacología , Animales , Compuestos Bicíclicos con Puentes , Bloqueadores de los Canales de Calcio/farmacología , Guanosina 5'-O-(3-Tiotrifosfato)/farmacología , Guanosina Difosfato/farmacología , Hypericum , Potenciales de la Membrana/efectos de los fármacos , Técnicas de Placa-Clamp , Floroglucinol/análogos & derivados , Plantas Medicinales , Células de Purkinje/química , Ratas , Ratas Wistar , Sistemas de Mensajero Secundario/fisiología , Tionucleótidos/farmacología , omega-Agatoxina IVA/farmacología , omega-Conotoxina GVIA/farmacología

RESUMEN

Properties of excitatory postsynaptic currents during increased glutamate release were investigated by means of a whole-cell voltage-clamp in CA1 pyramidal neurons of rat hippocampal slices. Enhancement of transmitter release by 50 microM 4-aminopyridine or by elevated extracellular Ca2+ (up to 5 mM) resulted in a substantial increase in the peak excitatory postsynaptic current amplitude and in the significant stimulus-dependent prolongation of the excitatory postsynaptic current decay. The stronger the stimulus, the slower the excitatory postsynaptic current decay became. The pharmacologically isolated N-methyl-D-aspartate, but not alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid component of the excitatory postsynaptic current exhibited this phenomenon. The possible connection of such behaviour of the N-methyl-D-aspartate component to the loss of voltage control was tested in the following way: the peak of the N-methyl-D-aspartate component was enhanced under 50 microM 4-aminopyridine and then returned back to the control level by a low dose of D-2-amino-5-phosphonopentanoic acid. However, the decay of the decreased N-methyl-D-aspartate component remained slow suggesting another origin of the stimulus-dependent kinetics. Dihydrokainate, a non-competitive inhibitor of glutamate uptake, did not influence the kinetics of the N-methyl-D-aspartate component in control but induced its dramatic stimulus-dependent prolongation when applied on the background of a low dose of 4-aminopyridine (10 microM) which did not affect the decay by itself. We propose that the delayed stimulus-dependent kinetics of the N-methyl-D-aspartate component is due to the saturation of uptake mechanisms and subsequent activation of extrasynaptic N-methyl-D-aspartate receptors. Our present observations therefore support the hypothesis that N-methyl-D-aspartate receptors may play a role in the cross-talk between synapses by means of the transmitter spillover.

Asunto(s)

Ácido Glutámico/metabolismo , Hipocampo/fisiología , Receptores de N-Metil-D-Aspartato/fisiología , Transmisión Sináptica/fisiología , 4-Aminopiridina/farmacología , Animales , Calcio/metabolismo , Potenciales Postsinápticos Excitadores/fisiología , Hipocampo/metabolismo , Técnicas In Vitro , Cinética , N-Metilaspartato/fisiología , Concentración Osmolar , Ratas , Ratas Wistar

RESUMEN

Using an in situ patch clamp in hippocampal CA1 mini-slices, we measured excitatory postsynaptic currents (EPSC) by varying the strength of the stimulus applied to the axons of CA3 neurones. The kinetics of the EPSC was initially independent of the stimulus strength. Post-ischaemic potentiation of the EPSC was observed 60-80 min after brief periods (10 min) of anoxia/aglycaemia. The decay of the EPSC slowed significantly in most of the examined neurones. In 11 of 17 cells the EPSC kinetics became dependent on stimulus strength: a slower decay corresponded to a stronger stimulus. This effect was not abolished by N-methyl-D-aspartate (NMDA) or a non-NMDA receptor blocker (D-2-amino-5-phosphonovaleric acid or 6-cyano-7-nitroquinoxaline-2,3-dione respectively) indicating the polysynaptic nature of the modified EPSC: transient ischaemia led to the long-term recruitment of previously inactive, possibly latent NMDA synapses between CA1 neurones.

Asunto(s)

Isquemia Encefálica/fisiopatología , Hipocampo/fisiopatología , Receptores de N-Metil-D-Aspartato/fisiología , Sinapsis/fisiología , 6-Ciano 7-nitroquinoxalina 2,3-diona/farmacología , Animales , Ratas , Ratas Wistar

RESUMEN

When performed at increased external [Ca2+]/[Mg2+] ratio (2.5 mM/0.5 mM), temporary block of A1 adenosine receptors in hippocampus [by 8-cyclopentyltheophylline (CPT)] leads to a dramatic and irreversible change in the excitatory postsynaptic current (EPSC) evoked by Schaffer collateral/commissural (SCC) stimulation and recorded by in situ patch clamp in CA1 pyramidal neurons. The duration of the EPSC becomes stimulus dependent, increasing with increase in stimulus strength. The later occurring component of the EPSC is carried through N-methyl-D-aspartate (NMDA) receptor-operated channels but disappears under either the NMDA antagonist 2-amino-5-phosphonovaleric acid (APV) or the non-NMDA antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). These findings indicate that the late component of the SCC-evoked EPSC is polysynaptic: predominantly non-NMDA receptor-mediated SCC inputs excite CA1 neurons that recurrently excite each other by predominantly NDMA receptor-mediated synapses. These recurrent connections are normally silent but become active after CPT treatment, leading to enhancement of the late component of the EPSC. The activity of these connections is maintained for at least 2 hr after CPT removal. When all functional NMDA receptors are blocked by dizocilpine maleate (MK-801), subsequent application of CPT leads to a partial reappearance of NMDA receptor-mediated EPSCs evoked by SCC stimulation, indicating that latent NMDA receptors are recruited. Altogether, these findings indicate the existence of a powerful system of NMDA receptor-mediated synaptic contacts in SCC input to hippocampal CA1 pyramidal neurons and probably also in reciprocal connections between these neurons, which in the usual preparation are kept latent by activity of A1 receptors.

Asunto(s)

Calcio/farmacología , Hipocampo/fisiología , Antagonistas de Receptores Purinérgicos P1 , Células Piramidales/fisiología , Receptores de N-Metil-D-Aspartato/fisiología , Sinapsis/fisiología , Teofilina/análogos & derivados , 6-Ciano 7-nitroquinoxalina 2,3-diona/farmacología , Animales , Maleato de Dizocilpina/farmacología , Potenciales Evocados/efectos de los fármacos , Técnicas In Vitro , Magnesio/farmacología , Modelos Neurológicos , N-Metilaspartato/farmacología , Picrotoxina/farmacología , Cloruro de Potasio/farmacología , Células Piramidales/efectos de los fármacos , Ratas , Ratas Wistar , Receptores de N-Metil-D-Aspartato/efectos de los fármacos , Receptores Purinérgicos P1/fisiología , Sinapsis/efectos de los fármacos , Transmisión Sináptica/efectos de los fármacos , Teofilina/farmacología , Factores de Tiempo

RESUMEN

A1 adenosine receptors efficiently modulate the excitatory synaptic transmission in hippocampus. Here we report that in addition to previously known modulatory action on the synaptic efficacy, A1 adenosine receptors are also capable of regulating the relative contribution of N-methyl-D-aspartate receptor-mediated component of the excitatory postsynaptic current in CA3-CA1 excitatory synapses, in the rat. When applied externally, a selective A1 adenosine receptor antagonist, 8-cyclopentyl-1,3-dimethylxanthine, increases not only the amplitude of excitatory postsynaptic current but also the relative contribution of the N-methyl-D-aspartate receptor-mediated component of postsynaptic current recorded by in situ voltage clamp. This effect develops only at increased external Ca2+ concentration and also depends on the external Ca2+/Mg2+ ratio. The increased ratio of N-methyl-D-aspartate/non-N-methyl-D-aspartate components of excitatory postsynaptic current remains at a new level after the removal of 8-cyclopentyl-1,3-dimethylxanthine, even though the amplitude of excitatory postsynaptic current returns close to control value. These results indicate the existence of a mechanism that preferentially enhances the N-methyl-D-aspartate component of excitatory postsynaptic current when the A1 adenosine receptors are blocked and imprints the newly acquired ratio of corresponding excitatory postsynaptic current components.

Asunto(s)

Calcio/fisiología , Hipocampo/fisiología , Magnesio/fisiología , Receptores de Glutamato/fisiología , Receptores de N-Metil-D-Aspartato/fisiología , Receptores Purinérgicos P1/fisiología , 2-Amino-5-fosfonovalerato/farmacología , 6-Ciano 7-nitroquinoxalina 2,3-diona/farmacología , Adenosina/farmacología , Animales , Hipocampo/citología , Técnicas In Vitro , Canales Iónicos/efectos de los fármacos , Canales Iónicos/metabolismo , Canales Iónicos/fisiología , Técnicas de Placa-Clamp , Células Piramidales/efectos de los fármacos , Células Piramidales/fisiología , Ratas , Ratas Wistar , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Sinapsis/fisiología , Teofilina/análogos & derivados , Teofilina/farmacología

RESUMEN

The effects of phorbol 12,13-diacetate (PDAc) on evoked excitatory transmission were studied in neurons of the CA1 area of hippocampal slices of rats, using whole-cell voltage clamp of pyramidal neurons in situ and stimulation of the Schaffer collaterals. The application of PDAc (10 microM) increased the amplitude of the excitatory postsynaptic current (EPSC) and caused a lengthening of its decay, due to an increase in the contribution of the N-methyl-D-aspartate (NMDA) component to the total EPSC. The latter effect was depend upon the concentration of calcium in the extracellular medium. Experiments in which we separated the two components of the EPSCs by 6-cyano-7-nitroquinoxaline-2,3-dione and by 2-amino-5-phosphonopentanoic acid also demonstrated a more pronounced increase in the NMDA receptor-mediated current under PDAc. The effects of PDAc were markedly attenuated by the extracellular application of the protein kinase C inhibitor H-7 (300 microM), but not by intracellular perfusion with 20 mM of the same drug.

Asunto(s)

Hipocampo/metabolismo , Ésteres del Forbol/farmacología , Receptores de N-Metil-D-Aspartato/agonistas , Sinapsis/fisiología , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina , 2-Amino-5-fosfonovalerato/farmacología , 6-Ciano 7-nitroquinoxalina 2,3-diona/farmacología , Animales , Bicuculina/farmacología , Calcio/fisiología , Electrofisiología , Potenciales Evocados/efectos de los fármacos , Hipocampo/citología , Hipocampo/efectos de los fármacos , Técnicas In Vitro , Isoquinolinas/farmacología , Masculino , Piperazinas/farmacología , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/metabolismo , Células Piramidales/efectos de los fármacos , Ratas , Ratas Wistar , Sinapsis/efectos de los fármacos

RESUMEN

NMDA and non-NMDA receptor-mediated components of excitatory post-synaptic current (EPSC) were studied by in situ whole-cell voltage-clamp recordings in the CA1 field of rat hippocampus. We found that the amplitudes ratio of the NMDA to the non-NMDA components can be strongly increased by blocking A1 adenosine receptors. The necessary conditions for this effect are both, increased Ca2+ and lowered Mg2+ in the external medium. The so achieved increase in the NMDA/non-NMDA ratio of EPSC components is irreversible and no longer depends on the activity of A1 adenosine receptors.

Asunto(s)

Calcio/farmacología , Hipocampo/metabolismo , Magnesio/farmacología , N-Metilaspartato/metabolismo , Antagonistas de Receptores Purinérgicos P1 , Receptores de Glutamato/metabolismo , Sinapsis/fisiología , Animales , Electrofisiología , Hipocampo/efectos de los fármacos , Hipocampo/fisiología , Técnicas In Vitro , Canales Iónicos/efectos de los fármacos , Canales Iónicos/metabolismo , N-Metilaspartato/fisiología , Neuronas/efectos de los fármacos , Neuronas/fisiología , Técnicas de Placa-Clamp , Ratas , Ratas Wistar , Receptores de Glutamato/efectos de los fármacos , Sinapsis/efectos de los fármacos , Teofilina/análogos & derivados , Teofilina/farmacología

RESUMEN

Diadenosine polyphosphates (Ap4A and Ap5A) are present in secretory granules of chromaffin cells as well as in the rat brain synaptic terminals. Their contribution to the exocytosis of the total synaptosomal content is considerable, ranging from 7% to 12%. Ap4A and Ap5A are released from synaptosomes in a Ca(2+)-dependent manner. There are indications on the high affinity of diadenosine polyphosphates to P2 receptors, but their action on P1 receptors remains unclear. Here we report that both substances induce a blocking action on excitatory synaptic transmission in the rat hippocampus. This action is elicited via the A1 (subclass of P1) receptors and differs in some respects from the action of adenosine.

Asunto(s)

Fosfatos de Dinucleósidos/farmacología , Retroalimentación/efectos de los fármacos , Hipocampo/fisiología , Animales , Hipocampo/efectos de los fármacos , Técnicas In Vitro , Potenciales de la Membrana/efectos de los fármacos , Ratas , Ratas Wistar , Receptores Purinérgicos P1/efectos de los fármacos , Sinapsis/efectos de los fármacos , Transmisión Sináptica/efectos de los fármacos , Teofilina/análogos & derivados , Teofilina/farmacología

RESUMEN

Chloride current activated by nicotinic acetylcholine receptors (AChR) was examined in dialysed voltage-clamp neurons of Lymnaea stagnalis. Fast superfusion of acetylcholine (ACh) evoked an inward current rapidly rising to a peak followed by a decline due to desensitization. When adenosine triphosphate with Mg2+ (MgATP, 2-10 mM) was added intracellularly the peak of the ACh-induced current was increased and its decay was slowed down. ATP without Mg2+ did not affect desensitization. Mg2+ alone accelerated desensitization. Intracellular treatment with an inhibitor of ATP synthesis, sodium arsenate, increased the desensitization rate and decreased the peak current. MgATP after arsenate wash-out restored the initial characteristics of the response; a mixture of glycolytic substrates had a similar effect. A non-hydrolysable analogue of ATP, adenosine [gamma-thio]triphosphate mimicked ATP action after arsenate removal but was weaker; another non-hydrolysable analogue, adenylyl imidodiphosphate, did not affect desensitization at all. Intracellular treatment of the neurons with alkaline phosphatase accelerated current decay. The data suggest that a change in intracellular ATP concentration modulates AChR desensitization via an enzymatic process that might be phosphorylation of AChR or some associated protein(s). Involvement of Ca2+ homeostasis cannot be excluded. The results are compared with the data obtained on vertebrate tissues under conditions promoting phosphorylation.

Asunto(s)

Adenosina Trifosfato/metabolismo , Cloruros/metabolismo , Neuronas/metabolismo , Receptores Colinérgicos/metabolismo , Adenosina Trifosfato/análogos & derivados , Fosfatasa Alcalina/metabolismo , Animales , Arseniatos/farmacología , Electrofisiología , Cinética , Lymnaea , Magnesio/metabolismo , Neuronas/efectos de los fármacos

RESUMEN

1. The action of intracellular ATP on the fast transient outward K+ current (A-current) was studied in dialysed voltage-clamped Lymnaea stagnalis neurones. 2. When introduced intracellularly in millimolar concentrations ATP caused a shift of the steady-state inactivation curve along the voltage axis in the direction of positive potentials and decreased A-current at all test voltages. 3. Intracellular treatment with an inhibitor of ATP synthesis, sodium arsenate, led to the opposite changes. The action of arsenate was not reversed upon its removal. After wash-out of arsenate ATP restored the initial voltage dependence. 4. Addition of Mg2+ to the solution weakened the action of ATP in proportion to the Mg2+: ATP concentration ratio. On the other hand, in neurones pretreated with arsenate, Mg2+ did not affect the ATP action. 5. When a mixture of glycolytic substrates was applied after arsenate wash-out the activation and inactivation curves shifted towards positive voltages. A substrate of oxidative phosphorylation was ineffective in the same conditions. 6. Non-hydrolysable analogues of ATP, adenosine-5'-O-gamma-thiotriphosphate and adenylyl imidodiphosphate, did not mimic the ATP action. This means that the ATP effect is mediated by some enzymatic process(es). 7. Elevation of total cytosolic Ca2+ concentration as well as intracellular application of agents increasing intracellular free Ca2+ reduced A-current amplitude but failed to alter its voltage dependence. Therefore, ATP action cannot be related to activation of Ca2+ transport. 8. Treatment of the neurones with alkaline phosphatase evoked a shift of the inactivation voltage dependence towards hyperpolarizing potentials and increased the A-current amplitudes at all test voltages. 9. The data indicate that a change in intracellular ATP concentration modulates the A-current voltage dependence. The effect of ATP is probably the result of phosphorylation of a channel protein or some associated proteins, but lowering of free Mg2+ concentration cannot be excluded. The possible physiological significance of the phenomenon is discussed.

Asunto(s)

Adenosina Trifosfato/fisiología , Neuronas/fisiología , Potasio/fisiología , Adenosina Trifosfato/análogos & derivados , Fosfatasa Alcalina/farmacología , Animales , Arseniatos/farmacología , Calcio/metabolismo , Conductividad Eléctrica , Electrofisiología , Metabolismo Energético , Membranas Intracelulares/metabolismo , Lymnaea , Neuronas/metabolismo , Concentración Osmolar , Factores de Tiempo

RESUMEN

The effects of intracellular Mg2+ (2-8 mM) upon the transient outward current (the A-current) under normal conditions and under catechol-induced blockage were studied in molluscan neurons by using the voltage-clamp and intracellular dialysis techniques. Identified giant Lymnaea stagnalis L. neurons were investigated at room temperature (20-22 degrees C). When applied intracellularly, Mg2+ caused both time- and dose-dependent shifts of the voltage dependence of the steady-state activation and inactivation of the A-current to more negative membrane potentials. Upon external application, catechol suppressed (5-6 mM) or eliminated (9-10 mM) the A-currents, slowed down the current decay and shifted the activation and inactivation curves to more positive membrane voltages. Intracellular Mg2+ decreased the blocking ability of extracellularly applied catechol, whereas catechol antagonized the Mg2(+)-induced negative shift of the steady-state activation and inactivation curves of the A-currents.

Asunto(s)

Catecoles/farmacología , Magnesio/fisiología , Neuronas/fisiología , Animales , Técnicas In Vitro , Cinética , Lymnaea , Magnesio/farmacología , Potenciales de la Membrana/efectos de los fármacos , Neuronas/efectos de los fármacos