RESUMEN
BACKGROUND AND PURPOSE: Both cannabinoid CB1 and adenosine A2A receptors (CB1 receptors and A2A receptors) control synaptic transmission at corticostriatal synapses, with great therapeutic importance for neurological and psychiatric disorders. A postsynaptic CB1 -A2A receptor interaction has already been elucidated, but the presynaptic A2A receptor-mediated control of presynaptic neuromodulation by CB1 receptors remains to be defined. Because the corticostriatal terminals provide the major input to the basal ganglia, understanding the interactive nature of converging neuromodulation on them will provide us with novel powerful tools to understand the physiology of corticostriatal synaptic transmission and interpret changes associated with pathological conditions. EXPERIMENTAL APPROACH: Pharmacological manipulation of CB1 and A2A receptors was carried out in brain nerve terminals isolated from rats and mice, using flow synaptometry, immunoprecipitation, radioligand binding, ATP and glutamate release measurement. Whole-cell patch-clamp recordings were made in horizontal corticostriatal slices. KEY RESULTS: Flow synaptometry showed that A2A receptors were extensively co-localized with CB1 receptor-immunopositive corticostriatal terminals and A2A receptors co-immunoprecipitated CB1 receptors in these purified terminals. A2A receptor activation decreased CB1 receptor radioligand binding and decreased the CB1 receptor-mediated inhibition of high-K(+) -evoked glutamate release in corticostriatal terminals. Accordingly, A2A receptor activation prevented CB1 receptor-mediated paired-pulse facilitation and attenuated the CB1 receptor-mediated inhibition of synaptic transmission in glutamatergic synapses of corticostriatal slices. CONCLUSIONS AND IMPLICATIONS: Activation of presynaptic A2A receptors dampened CB1 receptor-mediated inhibition of corticostriatal terminals. This constitutes a thus far unrecognized mechanism to modulate the potent CB1 receptor-mediated presynaptic inhibition, allowing frequency-dependent enhancement of synaptic efficacy at corticostriatal synapses.
Asunto(s)
Ácido Glutámico/metabolismo , Receptor de Adenosina A2A/metabolismo , Receptor Cannabinoide CB1/metabolismo , Receptores Presinapticos/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Encéfalo/metabolismo , Encéfalo/fisiología , Masculino , Ratones Noqueados , Ratas Wistar , Sinapsis/metabolismo , Transmisión SinápticaRESUMEN
Basal ganglia are a network of interconnected nuclei, involved in motor control, goal-directed behaviors and procedural learning. Basal ganglia process information from the cerebral cortex through three main pathways. The striatum is the input nucleus of the direct (cortico-striato-nigral) and indirect (cortico-striato-pallido-subthalamo-nigral) pathways while the subthalamic nucleus (STN) is the input structure of the hyperdirect (cortico-subthalamo-nigral) pathway. Despite the fact that the hyperdirect pathway constitutes a central part of most of basal ganglia models, experimental studies concerning its synaptic transmission and plasticity are still lacking. This is mainly because in vitro brain slices do not preserve the hyperdirect pathway. Here, we address this by developing a hyperdirect pathway brain slice where cortico-subthalamo-nigral connections were preserved. We characterized the transmission properties and its monosynaptic features between the frontal cortex and the STN, and between the STN and the substantia nigra pars reticulata (SNr), the output nucleus of the hyperdirect pathway. Cortical stimulation evoked monosynaptic glutamatergic events in STN neurons with a mean latency of 11.3 ms and a mean amplitude of 21 pA. STN stimulations evoked monosynaptic glutamatergic events in SNr neurons with a mean latency of 2.5 ms and a mean amplitude of 116 pA. This brain slice also preserved a part of the direct and indirect pathways such as the cortico-striatal connection. This novel slice configuration containing the hyperdirect pathway is a useful tool to better understand the transmission and plasticity in this pathway and hence the physiology and the pathophysiology of basal ganglia.
Asunto(s)
Ganglios Basales/fisiología , Lóbulo Frontal/fisiología , Vías Nerviosas/fisiología , Sustancia Negra/fisiología , 6-Ciano 7-nitroquinoxalina 2,3-diona/farmacología , Animales , Animales Recién Nacidos , Ganglios Basales/anatomía & histología , Ganglios Basales/citología , Simulación por Computador , Estimulación Eléctrica , Antagonistas de Aminoácidos Excitadores/farmacología , Técnicas In Vitro , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Modelos Neurológicos , Técnicas de Placa-Clamp , Ratas , Ratas Sprague-Dawley , Tiempo de Reacción/efectos de los fármacos , Transmisión Sináptica/efectos de los fármacos , Valina/análogos & derivados , Valina/farmacologíaRESUMEN
Dopaminergic neurons of the substantia nigra pars compacta play a key role in the modulation of basal ganglia and provide a reward-related teaching signal essential for adaptative motor control. They are generally considered as a homogenous population despite several chemical and electrophysiological heterogeneities, which could underlie different preferential patterns of activity and/or different roles. Using whole-cell patch-clamp recordings in juvenile rat brain slices, we observed that the evoked activity of dopaminergic neurons displays variable spike frequency adaptation patterns. The intensity of spike frequency adaptation decreased during post-natal development. The adaptation was associated with an increase in the initial firing frequency due to faster kinetics of the afterhyperpolarization component of the spike. Adaptation was enhanced when small conductance calcium-activated potassium (SK) channels were blocked with bath application of apamine. Lastly, spike frequency adaptation of the evoked discharge was associated with more irregularity in the spontaneous firing pattern. Altogether these results show a developmental heterogeneity and electrophysiological maturation of substantia nigra dopaminergic neurons.
Asunto(s)
Potenciales de Acción/fisiología , Adaptación Fisiológica/fisiología , Neuronas Dopaminérgicas/fisiología , Sustancia Negra/fisiología , Animales , Femenino , Inmunohistoquímica , Masculino , Técnicas de Cultivo de Órganos , Técnicas de Placa-Clamp , Ratas , Ratas Sprague-Dawley , Sustancia Negra/crecimiento & desarrolloRESUMEN
Corticostriatal projections constitute the major inputs to basal ganglia, an ensemble of sub-cortical nuclei involved in the learning of cognitive-motor sequences in response to environmental stimuli. Besides striatal output neurons (medium-sized spiny neurons, MSNs) in charge of the detection of cortical activity, three main classes of interneurons (GABAergic, cholinergic and nitric oxide (NO)-synthase interneurons) tightly regulate the corticostriatal information transfer. Despite the crucial role of NO on neuronal signaling and synaptic plasticity, little is known about corticostriatal synaptic transmission and plasticity at the level of striatal neuronal nitric oxide synthase (nNOS) interneurons. Using a corticostriatal rat brain slice preserving the connections between the somatosensory cortex and the striatal cells, we have explored the synaptic transmission between the cerebral cortex and striatal nNOS interneurons and their capability to develop activity-dependent long-term plasticity based on the quasi-coincident cortical and striatal activities (spike-timing dependent plasticity, STDP). We have observed that cortical pyramidal cells activate monosynaptically and very efficiently the striatal nNOS interneurons. In addition, nNOS interneurons are able to develop strong bidirectional long-term plasticity, following STDP protocols. Indeed, the strength of cortically-evoked response at nNOS interneurons varied as a function of time interval between pre- and postsynaptic activations (Deltat=t(post)-t(pre)). For Deltat<0, excitatory post-synaptic currents (EPSCs) were depressed, peaking at a delay of -25 ms. For Deltat>0, EPSCs depressed for 0
Asunto(s)
Potenciales de Acción/fisiología , Interneuronas/metabolismo , Neostriado/metabolismo , Plasticidad Neuronal/fisiología , Óxido Nítrico Sintasa de Tipo I/metabolismo , Corteza Somatosensorial/metabolismo , Transmisión Sináptica/fisiología , Animales , Comunicación Celular/fisiología , Interneuronas/citología , Potenciación a Largo Plazo/fisiología , Depresión Sináptica a Largo Plazo/fisiología , Neostriado/citología , Vías Nerviosas/citología , Vías Nerviosas/metabolismo , Óxido Nítrico/metabolismo , Técnicas de Cultivo de Órganos , Técnicas de Placa-Clamp , Ratas , Ratas Sprague-Dawley , Transducción de Señal/fisiología , Corteza Somatosensorial/citologíaRESUMEN
Nucleotides are signaling molecules involved in variety of interactions between neurons, between glial cells as well as between neurons and glial cells. In addition, ATP and other nucleotides are massively released following brain insults, including inflammation, and may thereby be involved in mechanisms of cerebral injury. Recent concepts have shown that in astrocytes intercellular communication through gap junctions may play an important role in neuroprotection. Therefore, we have studied the effects of nucleotides on gap junction communication in astrocytes. Based on measurement of intercellular dye coupling and recording of junctional currents, the present study shows that ATP (10-100 microM) induces a rapid and a concentration-dependent inhibition of gap junction communication in cultured cortical astrocytes from newborn mice. Effects of agonists and antagonists of purinergic receptors indicate that the inhibition of gap junctional communication by ATP mainly involves the stimulation of metabotropic purinergic 1 (P2Y(1)) receptors. Pretreatment with the pro-inflammatory cytokine interleukin-1beta (10 ng/ml, 24 h), which has no effect by itself on gap junctional communication, increases the inhibitory effect of ATP and astrocytes become sensitive to uridine 5'-triphosphate (UTP). As indicated by the enhanced expression of P2Y(2) receptor mRNA, P2Y(2) receptors are responsible for the increased responses evoked by ATP and UTP in interleukin-1beta-treated cells. In addition, the effect of endothelin-1, a well-known inhibitor of gap junctional communication in astrocytes was also exacerbated following interleukin-1beta treatment. We conclude that ATP decreases intercellular communication through gap junctions in astrocytes and that the increased sensitivity of gap junction channels to nucleotides and endothelin-1 is a characteristic feature of astrocytes exposed to pro-inflammatory treatments.
Asunto(s)
Adenosina Trifosfato/farmacología , Astrocitos/fisiología , Comunicación Celular/efectos de los fármacos , Uniones Comunicantes/fisiología , Interleucina-1/farmacología , Adenosina Difosfato/farmacología , Animales , Astrocitos/citología , Astrocitos/efectos de los fármacos , Comunicación Celular/fisiología , Células Cultivadas , Corteza Cerebral/citología , Conexina 43/metabolismo , Cuerpo Estriado/citología , Sinergismo Farmacológico , Conductividad Eléctrica , Endotelina-1/farmacología , Ratones , Ratas , Uridina Trifosfato/farmacologíaRESUMEN
Gap junctional communication (GJC) is a typical feature of astrocytes proposed to contribute to the role played by these glial cells in brain physiology and pathology. In acutely isolated hippocampal slices from rat (P11-P19), intercellular diffusion of biocytin through gap junction channels was shown to occur between hundreds of cells immuno-positive for astrocytic markers studied in the CA1/CA2 region. Single-cell RT-PCR demonstrated astrocytic mRNA expression of several connexin (Cx) subtypes, the molecular constituent of gap junction channels, whereas immunoblotting confirmed that Cx43 and Cx30 are the main gap junction proteins in hippocampal astrocytes. In the brain, astrocytes represent a major target for endothelins (Ets), a vasoactive family of peptides. Our results demonstrate that Ets decrease the expression of phosphorylated Cx43 forms and are potent inhibitors of GJC. The Et-induced effects were investigated using specific Et receptor agonists and antagonists, including Bosentan (Tracleer trade mark ), an EtA/B receptor antagonist, and using hippocampal slices and cultures from EtB-receptor-deficient rats. Interestingly, the pharmacological profile of Ets effects did not follow the classical profile established in cardiovascular systems. The present study therefore identifies Ets as potent endogenous inhibitory regulators of astrocyte networks. As such, the action of these peptides on astrocyte GJC might be involved in the contribution of astrocytes to neuroprotective processes and have a therapeutic potential in neuropathological situations.
Asunto(s)
Astrocitos/fisiología , Endotelinas/fisiología , Uniones Comunicantes/fisiología , Hipocampo/fisiología , Animales , Animales Modificados Genéticamente , Astrocitos/efectos de los fármacos , Bosentán , Células Cultivadas , Antagonistas de los Receptores de la Endotelina B , Endotelinas/antagonistas & inhibidores , Endotelinas/deficiencia , Uniones Comunicantes/efectos de los fármacos , Hipocampo/efectos de los fármacos , Técnicas In Vitro , Ratas , Ratas Wistar , Receptor de Endotelina B/deficiencia , Sulfonamidas/farmacologíaRESUMEN
The main neuronal population of the striatum is composed of the medium spiny neurones (MSNs). In fact several sub-populations of MSNs can be distinguished according to the striatal compartment (striosomes and matrix) to which they belong, their afferents and their sites of projection, their biochemical markers and their morphologies. However, these cells are generally described as an electrophysiological homogeneous population. Using brain slices from the rat and whole cell patch clamp recordings, we show that at P(15) 28% of the MSNs display a spike frequency adaptation. While the mean frequency adaptation ratio for non-adapting cells was 1.07+/-0.01 it reached 2.66+/-0.09 in adapting MSNs and the incidence of this frequency adaptation phenotype appeared to be stable during post-natal development. Single-cell RT-PCR analysis of mRNAs for mu opioid receptors, enkephalin and substance P precursors suggested that adapting MSNs are present in both striatal compartments as well as in the direct and indirect pathways of the matrix. Adapting neurones were also distinguished from non-adapting cells by a lower membrane time constant, a higher AP threshold, a reduced delay to the first spike and a higher initial firing rate. Micro-domains differing by their magnitude of adaptation could be distinguished within the spike frequency adaptation process.A subgroup of MSNs exists, showing a marked spike frequency adaptation together with other distinct properties, such as shorter delay to first spike and membrane time constant, and higher initial frequency and action potential threshold. In conclusion, when strong cortical inputs are delivered in coincidence, adapting MSNs could not only transmit faster the first AP but also exert a sort of cutoff of the transmission due to their spike frequency adaptation process.
Asunto(s)
Potenciales de Acción , Cuerpo Estriado/fisiología , Neuronas/fisiología , Adaptación Fisiológica , Animales , Cuerpo Estriado/citología , Electrofisiología , Femenino , Masculino , Potenciales de la Membrana , Técnicas de Placa-Clamp , Ratas , Ratas Wistar , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Gap junctions are widely expressed in the various cell types of the central nervous system. These specialized membrane intercellular junctions provide the morphological support for direct electrical and biochemical communication between adjacent cells. This intercellular coupling is controlled by neurotransmitters and other endogenous compounds produced and released in basal as well as in pathological situations. Changes in the expression and the function of connexins are associated with number of brain pathologies and lesions suggesting that they could contribute to the expansion of brain damages. The purpose of this review is to summarize data presently available concerning gap junctions and the expression and function of connexins in different cell types of the central nervous system and to present their physiopathological relevance in three major brain dysfunctions: inflammation, epilepsy and ischemia.
Asunto(s)
Sistema Nervioso Central/metabolismo , Conexinas/metabolismo , Uniones Comunicantes/metabolismo , Animales , Astrocitos/citología , Astrocitos/metabolismo , Astrocitos/patología , Encefalopatías/metabolismo , Encefalopatías/patología , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patología , Sistema Nervioso Central/química , Sistema Nervioso Central/patología , Conexinas/biosíntesis , Conexinas/fisiología , Epilepsia/metabolismo , Epilepsia/patología , Uniones Comunicantes/fisiología , Humanos , Inflamación/metabolismo , Inflamación/patología , Neuronas/citología , Neuronas/metabolismo , Neuronas/patologíaRESUMEN
Sphingosine-1-phosphate (S1P) is a potent lysophospholipid mediator mostly released by activated platelets. It is involved in several functions in peripheral tissues, but its effects in the central nervous system are poorly documented. Therefore, we have examined the effects of S1P on the proliferation of striatal astrocytes from the mouse embryo. These cells have been found to express mRNAs for the S1P receptors, Edg-1 and Edg-3. S1P stimulated thymidine incorporation and induced activation of extracellular signal-regulated kinases (Erks). Both effects were prevented by U0126, an Erk kinase inhibitor. The S1P-evoked activation of Erk1 was totally blocked in astrocytes pretreated with a combination of either phorbol ester (24 h) and LY294002, or phorbol ester (24 h) and pertussis toxin (PTX). Each individual treatment only partially inhibited Erk1 activation. This suggests that several separate mechanisms mediate this process, one involving protein kinase C and another involving Gi/Go proteins and phosphatidylinositol 3-kinase. In contrast, the stimulatory effect of S1P on astrocyte proliferation was totally blocked by either PTX or LY294002, but not by a downregulation of protein kinase C. S1P dramatically inhibited the evoked production of cyclic AMP, a response that was impaired by PTX. Finally, S1P stimulated the production of inositol phosphates and increased intracellular calcium by mobilization from thapsigargin-sensitive stores. These latter effects were mainly insensitive to PTX. Probably, Gi/Go protein activation and phosphoinositide hydrolysis are early events that regulate the activation of Erks by S1P. Altogether, these observations show that astrocytes are targets for S1P. Their proliferation in response to S1P could have physiopathological consequences at sites of brain lesions and alterations of the blood-brain barrier.
Asunto(s)
Astrocitos/efectos de los fármacos , Proteínas I-kappa B , Lisofosfolípidos , Neostriado/efectos de los fármacos , Receptores Acoplados a Proteínas G , Transducción de Señal/efectos de los fármacos , Esfingosina/metabolismo , Esfingosina/farmacología , Animales , Astrocitos/metabolismo , Lesiones Encefálicas/metabolismo , Lesiones Encefálicas/fisiopatología , Calcio/metabolismo , División Celular/efectos de los fármacos , División Celular/fisiología , Células Cultivadas/efectos de los fármacos , Células Cultivadas/metabolismo , AMP Cíclico/metabolismo , Proteínas de Unión al ADN/genética , Inhibidores Enzimáticos/farmacología , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/antagonistas & inhibidores , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Gliosis/metabolismo , Gliosis/fisiopatología , Proteínas Inmediatas-Precoces/genética , Líquido Intracelular/efectos de los fármacos , Líquido Intracelular/metabolismo , Ratones , Proteínas Quinasas Activadas por Mitógenos/efectos de los fármacos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Inhibidor NF-kappaB alfa , Neostriado/embriología , Neostriado/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/metabolismo , ARN Mensajero/metabolismo , Receptores de Superficie Celular/genética , Receptores Lisofosfolípidos , Transducción de Señal/fisiología , Esfingosina/análogos & derivadosRESUMEN
Sphingosine-1-phosphate (S1P) is a potent lysophospholipid mediator mostly released by activated platelets. It is involved in several functions in peripheral tissues, but its effects in the central nervous system are poorly documented. Therefore, we have examined the effects of S1P on the proliferation of striatal astrocytes from the mouse embryo. These cells have been found to express mRNAs for the S1P receptors, Edg-1 and Edg-3. S1P stimulated thymidine incorporation and induced activation of extracellular signal-regulated kinases (Erks). Both effects were prevented by U0126, an Erk kinase inhibitor. The S1P-evoked activation of Erk1 was totally blocked in astrocytes pretreated with a combination of either phorbol ester (24 h) and LY294002, or phorbol ester (24 h) and pertussis toxin (PTX). Each individual treatment only partially inhibited Erk1 activation. This suggests that several separate mechanisms mediate this process, one involving protein kinase C and another involving Gi/Go proteins and phosphatidylinositol 3-kinase. In contrast, the stimulatory effect of S1P on astrocyte proliferation was totally blocked by either PTX or LY294002, but not by a downregulation of protein kinase C. S1P dramatically inhibited the evoked production of cyclic AMP, a response that was impaired by PTX. Finally, S1P stimulated the production of inositol phosphates and increased intracellular calcium by mobilization from thapsigargin-sensitive stores. These latter effects were mainly insensitive to PTX. Probably, Gi/Go protein activation and phosphoinositide hydrolysis are early events that regulate the activation of Erks by S1P. Altogether, these observations show that astrocytes are targets for S1P. Their proliferation in response to S1P could have physiopathological consequences at sites of brain lesions and alterations of the blood-brain barrier.
Asunto(s)
Astrocitos/efectos de los fármacos , División Celular/efectos de los fármacos , Gliosis/metabolismo , Proteínas I-kappa B , Lisofosfolípidos , Neostriado/efectos de los fármacos , Receptores Acoplados a Proteínas G , Esfingosina/metabolismo , Esfingosina/farmacología , Animales , Astrocitos/metabolismo , Lesiones Encefálicas/metabolismo , Lesiones Encefálicas/fisiopatología , Calcio/metabolismo , División Celular/fisiología , Células Cultivadas/efectos de los fármacos , Células Cultivadas/metabolismo , AMP Cíclico/metabolismo , Proteínas de Unión al ADN/genética , Inhibidores Enzimáticos/farmacología , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/antagonistas & inhibidores , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Gliosis/fisiopatología , Proteínas de Unión al GTP Heterotriméricas/antagonistas & inhibidores , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Proteínas Inmediatas-Precoces/genética , Líquido Intracelular/efectos de los fármacos , Líquido Intracelular/metabolismo , Ratones , Proteínas Quinasas Activadas por Mitógenos/efectos de los fármacos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Inhibidor NF-kappaB alfa , Neostriado/embriología , Neostriado/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfatidilinositoles/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/metabolismo , ARN Mensajero/metabolismo , Receptores de Superficie Celular/genética , Receptores Lisofosfolípidos , Esfingosina/análogos & derivadosAsunto(s)
Encéfalo/metabolismo , Conexinas/genética , Perfilación de la Expresión Génica/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Animales , Secuencia de Bases , Cartilla de ADN/genética , Técnicas In Vitro , Técnicas de Placa-Clamp , ARN Mensajero/genética , ARN Mensajero/metabolismo , RatasRESUMEN
Electrical coupling by gap junctions is an important form of cell-to-cell communication in early brain development. Whereas glial cells remain electrically coupled at postnatal stages, adult vertebrate neurons were thought to communicate mainly via chemical synapses. There is now accumulating evidence that in certain neuronal cell populations the capacity for electrical signaling by gap junction channels is still present in the adult. Here we identified electrically coupled pairs of neurons between postnatal days 12 and 18 in rat visual cortex, somatosensory cortex, and hippocampus. Notably, coupling was found both between pairs of inhibitory neurons and between inhibitory and excitatory neurons. Molecular analysis by single-cell reverse transcription-PCR revealed a differential expression pattern of connexins in these identified neurons.
Asunto(s)
Encéfalo/fisiología , Conexinas/genética , Neuronas/fisiología , Animales , Comunicación Celular , Uniones Comunicantes/fisiología , Hipocampo/fisiología , Técnicas In Vitro , Microscopía de Interferencia , Técnicas de Placa-Clamp , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Corteza Somatosensorial/fisiología , Corteza Visual/fisiologíaRESUMEN
The effects of anandamide and the cannabinoid receptor agonists WIN 55212-2 and CP 55940 on the evoked formation of cyclic AMP were compared in cultured neurons and astrocytes from the cerebral cortex and striatum of mouse embryos. The three compounds inhibited the isoproterenol-induced accumulation of cyclic AMP in neuronal cells, and these responses were blocked by the selective CB1 receptor antagonist SR 141716A. The three agonists were more potent in cortical than striatal neurons. Interestingly, WIN 55212-2, CP 55940 and anandamide also inhibited the isoproterenol-evoked accumulation of cyclic AMP in astrocytes but, in contrast to WIN 55212-2 and CP 55940, anandamide was much more potent in striatal than cortical astrocytes. Inhibition was prevented by pertussis toxin pretreatment, but not blocked by SR 141716A. Therefore, G-protein-coupled receptors, distinct from CB1 receptors, are involved in these astrocytic responses. Moreover, specific binding sites for [3H]-SR 141716A were found in neurons but not astrocytes. Furthermore, using a polyclonal CB1 receptor antibody, staining was observed in striatal and cortical neurons, but not in striatal and cortical astrocytes. Taken together, these results suggest that glial cells possess G-protein-coupled receptors activated by cannabinoids distinct from the neuronal CB1 receptor, and that glial cells responses must be taken into account when assessing central effects of cannabinoids.
Asunto(s)
Ácidos Araquidónicos/farmacología , Astrocitos/química , Bloqueadores de los Canales de Calcio/farmacología , AMP Cíclico/metabolismo , Morfolinas/farmacología , Naftalenos/farmacología , Receptores de Droga/metabolismo , Agonistas Adrenérgicos beta/farmacología , Animales , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Benzoxazinas , Células Cultivadas , Corteza Cerebral/citología , Cuerpo Estriado/citología , Endocannabinoides , Proteínas de Unión al GTP/metabolismo , Isoproterenol/farmacología , Ratones , Neuronas/química , Neuronas/citología , Neuronas/efectos de los fármacos , Toxina del Pertussis , Piperidinas/farmacología , Alcamidas Poliinsaturadas , Pirazoles/farmacología , Ratas , Receptores de Cannabinoides , Receptores de Droga/agonistas , Receptores de Droga/análisis , Rimonabant , Tritio , Factores de Virulencia de Bordetella/farmacologíaRESUMEN
Indo-1 and fluo-3 imaging techniques were used to investigate the role of gap junctions in the changes in cytosolic calcium concentrations ([Ca2+]i) induced by several receptor agonists. Subpopulations of confluent cultured astrocytes from the rat striatum were superfused with submaximal concentrations of endothelin-1 (Et1) and the alpha 1-adrenergic and muscarinic receptor agonists, methoxamine and carbachol, respectively. 2. Combined binding and autoradiographic studies indicated that all striatal astrocytes possess binding sites for Et1. In contrast, alpha 1-adrenergic and muscarinic binding sites were found to be heterogeneously distributed. In agreement with these findings, Et1 induced fast calcium responses in all cells while only subsets of striatal astrocytes responded to the application of methoxamine or carbachol. 3. Halothane, heptanol and octanol, which are commonly used as gap junction inhibitors, drastically reduced the amplitude of Et1-induced calcium responses. In contrast, 18-alpha-glycyrrhetinic acid (alpha GA) used at a concentration known to block gap junction permeability in astrocytes had no significant effect on the amplitude of these calcium responses. 4. As demonstrated by quantitative and topological analysis, Et1 application similarly increased [Ca2+]i levels in all astrocytes in both the absence and presence of alpha GA. 5. In control conditions, subpopulations of cells responding to methoxamine or carbachol exhibited two main types of calcium responses which differed in their shape and kinetic characteristics. In the presence of alpha GA the number of cells responding to these receptor agonists was significantly reduced. Indeed, responses characterized by their long latency, slow rise time and weak amplitude disappeared in the presence of alpha GA while responses with short latency and fast rise time were preserved. 6. These results indicate that permeable gap junction channels tend to attenuate the pharmacological and functional heterogeneity of populations of astrocytes, while their inhibition restricts calcium responses in astrocytes expressing high densities of transmitter receptors coupled to phospholipase C.
Asunto(s)
Astrocitos/metabolismo , Comunicación Celular/fisiología , Uniones Comunicantes/fisiología , Neostriado/metabolismo , Animales , Astrocitos/citología , Astrocitos/efectos de los fármacos , Autorradiografía , Calcio/metabolismo , Comunicación Celular/efectos de los fármacos , Células Cultivadas , Endotelina-1/metabolismo , Uniones Comunicantes/efectos de los fármacos , Inmunohistoquímica , Cinética , Microscopía Fluorescente , Neostriado/citología , Neostriado/efectos de los fármacos , Ratas , Receptores Adrenérgicos alfa 1/efectos de los fármacos , Receptores Adrenérgicos alfa 1/metabolismo , Receptores de Endotelina/efectos de los fármacosRESUMEN
Two main characteristics of astrocytes are their elaborated intracellular calcium signaling and their high degree of intercellular communication mediated by gap junctional channels. In these cells a number of studies have contributed to demonstrate that the combination of these two properties provides a basis for a long-range signaling system within the brain. Intercellular calcium signaling, also termed calcium waves, allows astrocytes to communicate with each other and to interact with adjacent neurons. Most of the intra- and inter-cellular events involved in the initiation and propagation phases of this process has now been identified. This sequence of events includes the permeability of gap junction channels, which at the time-scale for calcium waves propagation, are likely permeated rather than closed by Ca2+ and/or related signaling molecules like IP3. In addition, in some studies an external component have been reported to participate to the propagation process. Finally, the control of the spread of intercellular calcium signaling has been demonstrated to occur at several levels including phospholipase C, IP3 receptors, intracellular Ca2+ stores, and cytoplasmic Ca2+ buffering. Accordingly, normal and pathological situations that affect one or several of these steps can be predicted to influence on astrocytic calcium waves.
Asunto(s)
Astrocitos/fisiología , Calcio/fisiología , Comunicación Celular/fisiología , Espacio Extracelular/metabolismo , Uniones Comunicantes/fisiología , Transducción de Señal/fisiología , Animales , HumanosRESUMEN
Astrocytes are characterized by extensive intercellular communication mediated primarily by gap junction channels composed of connexin43. To examine this junctional protein in astrocytic functions, astrocytes were cultured from embryonic mice with a null mutation in the connexin43 gene (Reaume et al.: Science 267:1831-1834, 1995). Using anti-Cx43 antibodies, immunoblotting and immunostaining indicated that homozygous null astrocytes were devoid of Cx43. They are also deficient in intercellular dye transfer. Astrocytes cultured from heterozygous embryos express significantly lower Cx43 compared to wild type, and their dye coupling is reduced. Markers of glial differentiation, such as glial fibrillary acidic protein and S100, appeared similar in all genotypes. Measurement of intercellular calcium concentration following mechanical stimulation of confluent astrocytes revealed that the number of cells affected by a rise in intracellular calcium was reduced in homozygous cultures compared to wild type. In fact, the calcium response in homozygous astrocytes was similar to that observed in wild-type astrocytes in the presence of a gap junction blocker. The growth rate of astrocytes lacking Cx43 was reduced compared to wild-type astrocytes. These results suggest that gap junctional intercellular communication mediated by Cx43 is not critical for astrocyte differentiation but is likely involved in the regulation of intercellular calcium signaling and cell growth.
Asunto(s)
Astrocitos/fisiología , División Celular/fisiología , Conexina 43/deficiencia , Uniones Comunicantes/fisiología , Transducción de Señal/fisiología , Animales , Calcio/metabolismo , Células Cultivadas , Conexina 43/genética , Inmunohistoquímica , Ratones , Ratones MutantesRESUMEN
The effect of (R)-methandamide, a chiral analog of the endogenous cannabimimetic anandamide, was investigated on calcium signalling in cultured rat astrocytes loaded with Indo-1. Pretreatment of astrocytes with (R)-methandamide resulted in the inhibition of calcium responses induced by endothelin-1 or glutamate. Test of the filling level of internal calcium stores and biochemical assays of phospholipase C activity suggested that this inhibition resulted from the depletion of internal pools. This effect occurred in a reversible time- and dose-dependent manner, and was prevented by treating the cells with pertussis toxin (PTX) but was not reproduced by similar concentrations of arachidonic acid. Altogether, these observations demonstrate that this stable analog of anandamide controls calcium signalling in astrocytes through a PTX-sensitive mechanism which leads to the depletion of fast mobilizable internal stores.
Asunto(s)
Ácidos Araquidónicos/farmacología , Astrocitos/efectos de los fármacos , Calcio/metabolismo , Animales , Células Cultivadas/efectos de los fármacos , RatasRESUMEN
The mechanisms involved in the initiation and the propagation of intercellular calcium signaling (calcium waves) were studied in cultured rat astrocytes. The analysis of calcium waves, induced either by mechanical stimulation or by focal application of ionomycin, indicated that initiation was dependent on the presence of external calcium. In addition, pharmacological experiments indicate that intercellular propagation required PLC activation, integrity of IP3-sensitive internal calcium stores, and functional gap junctions. An extracellular action of ATP or glutamate and participation of voltage-dependent Ca2+ channels were tested by using enzymatic degradation, receptor antagonists, and channel blockers, respectively. Because neither the speed of propagation nor the extent of the calcium waves was affected by these treatments, these alternate mechanisms were excluded from playing a role in intercellular calcium signaling. Biochemical assays and focal applications of several agonists (methoxamine, carbachol, glutamate) of membrane receptors to neurotransmitters and peptides (endothelin 1) demonstrated that their ability to trigger regenerative calcium waves depended on phospholipase C activity and inositol phosphate production. Thus, in rat astrocytes, initiation and propagation of calcium waves involve a sequence of intra- and intercellular steps in which phospholipase C, inositol trisphosphate, internal calcium stores, and gap junction channels play a critical role. The identification of these different events allows us to determine several targets at which the level of long-range signaling in astrocytes may be controlled.
Asunto(s)
Astrocitos/fisiología , Calcio/fisiología , Uniones Comunicantes/fisiología , Transducción de Señal/fisiología , Animales , Recuento de Células/efectos de los fármacos , Células Cultivadas , Femenino , Ionomicina/farmacología , Ratones , Ratones Endogámicos , Embarazo , RatasAsunto(s)
Encéfalo/metabolismo , Lípidos de la Membrana/metabolismo , Neuronas/metabolismo , Fosfatidiletanolaminas/metabolismo , Receptores de Droga/metabolismo , Animales , Ácidos Araquidónicos/metabolismo , Ácidos Araquidónicos/farmacología , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Encéfalo/citología , Encéfalo/efectos de los fármacos , Calcio/fisiología , Canales de Calcio/efectos de los fármacos , Células Cultivadas , Endocannabinoides , Ionomicina/farmacología , Neuronas/efectos de los fármacos , Fosfatidiletanolaminas/farmacología , Hidrolasas Diéster Fosfóricas/metabolismo , Alcamidas Poliinsaturadas , Ratas , Receptores de Cannabinoides , Receptores de Droga/efectos de los fármacos , Veratridina/farmacologíaRESUMEN
Understanding the mechanisms involved in the biogenesis of N-arachidonoylethanolamine (anandamide) and N-palmitoylethanolamine is important in view of the possible role of these lipids as endogenous cannabinoid substances. Anandamide (which activates cannabinoid CB1 receptors) and N-palmitoylethanolamine (which activates a CB2-like receptor subtype in mast cells) may both derive from cleavage of precursor phospholipid, N-acylphosphatidylethanolamine (NAPE), catalyzed by Ca(2+)-activated D-type phosphodiesterase activity. We report here that the de novo biosynthesis of NAPE is enhanced in a Ca(2+)-dependent manner when rat cortical neurons are stimulated with the Ca(2+)-ionophore ionomycin or with membrane-depolarizing agents such as veratridine and kainate. This reaction is likely to be mediated by a neuronal N-acyltransferase activity, which catalyzes the transfer of an acyl group from phosphatidylcholine to the ethanolamine moiety of phosphatidylethanolamine. In addition, we show that Ca2+-dependent NAPE biosynthesis is potentiated by agents that increase cAMP levels, including forskolin and vasoactive intestinal peptide. Our results thus indicate that NAPE levels in cortical neurons are controlled by Ca2+ ions and cAMP. Such regulatory effect may participate in maintaining a supply of cannabimimetic N-acylethanolamines during synaptic activity, and prime target neurons for release of these bioactive lipids.