RESUMEN
The hippocampus is a plastic brain area that shows functional segregation along its longitudinal axis, reflected by a higher level of long-term potentiation (LTP) in the CA1 region of the dorsal hippocampus (DH) compared to the ventral hippocampus (VH), but the mechanisms underlying this difference remain elusive. Numerous studies have highlighted the importance of microglia-neuronal communication in modulating synaptic transmission and hippocampal plasticity, although its role in physiological contexts is still largely unknown. We characterized in depth the features of microglia in the two hippocampal poles and investigated their contribution to CA1 plasticity under physiological conditions. We unveiled the influence of microglia in differentially modulating the amplitude of LTP in the DH and VH, showing that minocycline or PLX5622 treatment reduced LTP amplitude in the DH, while increasing it in the VH. This was recapitulated in Cx3cr1 knockout mice, indicating that microglia have a key role in setting the conditions for plasticity processes in a region-specific manner, and that the CX3CL1-CX3CR1 pathway is a key element in determining the basal level of CA1 LTP in the two regions. The observed LTP differences at the two poles were associated with transcriptional changes in the expression of genes encoding for Il-1, Tnf-α, Il-6, and Bdnf, essential players of neuronal plasticity. Furthermore, microglia in the CA1 SR region showed an increase in soma and a more extensive arborization, an increased prevalence of immature lysosomes accompanied by an elevation in mRNA expression of phagocytic markers Mertk and Cd68 and a surge in the expression of microglial outward K+ currents in the VH compared to DH, suggesting a distinct basal phenotypic state of microglia across the two hippocampal poles. Overall, we characterized the molecular, morphological, ultrastructural, and functional profile of microglia at the two poles, suggesting that modifications in hippocampal subregions related to different microglial statuses can contribute to dissect the phenotypical aspects of many diseases in which microglia are known to be involved.
Asunto(s)
Plasticidad Neuronal , Masculino , Animales , RatonesRESUMEN
Microglia cells, resident immune cells of the brain, survey brain parenchyma by dynamically extending and retracting their processes. Cl- channels, activated in the cellular response to stretch/swelling, take part in several functions deeply connected with microglia physiology, including cell shape changes, proliferation, differentiation and migration. However, the molecular identity and functional properties of these Cl- channels are largely unknown. We investigated the properties of swelling-activated currents in microglial from acute hippocampal slices of Cx3cr1 +/GFP mice by whole-cell patch-clamp and imaging techniques. The exposure of cells to a mild hypotonic medium, caused an outward rectifying current, developing in 5-10 minutes and reverting upon stimulus washout. This current, required for microglia ability to extend processes towards a damage signal, was carried mainly by Cl- ions and dependent on intracellular Ca2+. Moreover, it involved swelling-induced ATP release. We identified a purine-dependent mechanism, likely constituting an amplification pathway of current activation: under hypotonic conditions, ATP release triggered the Ca2+-dependent activation of anionic channels by autocrine purine receptors stimulation. Our study on native microglia describes for the first time the functional properties of stretch/swelling-activated currents, representing a key element in microglia ability to monitor the brain parenchyma.
Asunto(s)
Adenosina Trifosfato/metabolismo , Comunicación Autocrina , Calcio/metabolismo , Canales de Cloruro/metabolismo , Hipocampo/citología , Microglía/metabolismo , Animales , Línea Celular , Activación del Canal Iónico , Ratones , Modelos Biológicos , Purinas/metabolismoRESUMEN
Among the strategies adopted by glioma to successfully invade the brain parenchyma is turning the infiltrating microglia/macrophages (M/MΦ) into allies, by shifting them toward an anti-inflammatory, pro-tumor phenotype. Both glioma and infiltrating M/MΦ cells express the Ca(2+)-activated K(+) channel (KCa3.1), and the inhibition of KCa3.1 activity on glioma cells reduces tumor infiltration in the healthy brain parenchyma. We wondered whether KCa3.1 inhibition could prevent the acquisition of a pro-tumor phenotype by M/MΦ cells, thus contributing to reduce glioma development. With this aim, we studied microglia cultured in glioma-conditioned medium or treated with IL-4, as well as M/MΦ cells acutely isolated from glioma-bearing mice and from human glioma biopsies. Under these different conditions, M/MΦ were always polarized toward an anti-inflammatory state, and preventing KCa3.1 activation by 1-[(2-Chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34), we observed a switch toward a pro-inflammatory, antitumor phenotype. We identified FAK and PI3K/AKT as the molecular mechanisms involved in this phenotype switch, activated in sequence after KCa3.1. Anti-inflammatory M/MΦ have higher expression levels of KCa3.1 mRNA (kcnn4) that are reduced by KCa3.1 inhibition. In line with these findings, TRAM-34 treatment, in vivo, significantly reduced the size of tumors in glioma-bearing mice. Our data indicate that KCa3.1 channels are involved in the inhibitory effects exerted by the glioma microenvironment on infiltrating M/MΦ, suggesting a possible role as therapeutic targets in glioma.
Asunto(s)
Canales de Potasio de Conductancia Intermedia Activados por el Calcio/metabolismo , Macrófagos/inmunología , Microglía/metabolismo , Animales , Encéfalo/metabolismo , Encéfalo/patología , Línea Celular Tumoral , Cromonas/farmacología , Quinasa 1 de Adhesión Focal/genética , Quinasa 1 de Adhesión Focal/metabolismo , Glioma/tratamiento farmacológico , Glioma/metabolismo , Glioma/patología , Humanos , Interleucina-4/farmacología , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/antagonistas & inhibidores , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/genética , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Microglía/citología , Morfolinas/farmacología , Fagocitosis/efectos de los fármacos , Fenotipo , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/metabolismo , Pirazoles/farmacología , Pirazoles/uso terapéutico , ARN Mensajero/metabolismoRESUMEN
Glioma patients commonly suffer from epileptic seizures. However, the mechanisms of glioma-associated epilepsy are far to be completely understood. Using glioma-neurons co-cultures, we found that tumor cells are able to deeply influence neuronal chloride homeostasis, by depolarizing the reversal potential of γ-aminobutyric acid (GABA)-evoked currents (EGABA). EGABA depolarizing shift is due to zinc-dependent reduction of neuronal KCC2 activity and requires glutamate release from glioma cells. Consistently, intracellular zinc loading rapidly depolarizes EGABA in mouse hippocampal neurons, through the Src/Trk pathway and this effect is promptly reverted upon zinc chelation. This study provides a possible molecular mechanism linking glioma invasion to excitation/inhibition imbalance and epileptic seizures, through the zinc-mediated disruption of neuronal chloride homeostasis.
Asunto(s)
Neoplasias Encefálicas/metabolismo , Cloruros/metabolismo , Glioma/metabolismo , Espacio Intracelular/metabolismo , Neuronas/metabolismo , Zinc/metabolismo , Animales , Técnicas de Cocultivo , Femenino , Glutamatos/metabolismo , Hipocampo/patología , Humanos , Masculino , Proteínas de Transporte de Membrana/metabolismo , Ratones Endogámicos C57BL , Modelos Biológicos , Receptor trkB/metabolismo , Simportadores/metabolismo , Ácido gamma-Aminobutírico/metabolismo , Familia-src Quinasas/metabolismo , Cotransportadores de K ClRESUMEN
We characterized the role of adenosine receptor (AR) subtypes in the modulation of glutamatergic neurotransmission by the chemokine fractalkine (CX3CL1) in mouse hippocampal CA1 neurons. CX(3)CL1 causes a reversible depression of excitatory postsynaptic current (EPSC), which is abolished by the A(3)R antagonist MRS1523, but not by A(1)R (DPCPX) or A(2A)R (SCH58261) antagonists. Consistently, CX3CL1-induced EPSC depression is absent in slices from A(3)R(-/-) but not A(1)R(-/-) or A(2A)R(-/-) mice. Further, A(3)R stimulation causes similar EPSC depression. In cultured neurons, CX3CL1-induced depression of AMPA current shows A(1)R-A(3)R pharmacology. We conclude that glutamatergic depression induced by released adenosine requires the stimulation of different ARs.
Asunto(s)
Región CA1 Hipocampal/inmunología , Región CA1 Hipocampal/metabolismo , Quimiocina CX3CL1/fisiología , Potenciales Postsinápticos Excitadores/inmunología , Inhibición Neural/inmunología , Receptores Purinérgicos P1/fisiología , Transmisión Sináptica/inmunología , Antagonistas del Receptor de Adenosina A1 , Antagonistas del Receptor de Adenosina A2 , Antagonistas del Receptor de Adenosina A3 , Animales , Región CA1 Hipocampal/ultraestructura , Células Cultivadas , Potenciales Postsinápticos Excitadores/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Inhibición Neural/genética , Técnicas de Cultivo de Órganos , Técnicas de Placa-Clamp , Terminales Presinápticos/inmunología , Terminales Presinápticos/metabolismo , Receptor de Adenosina A1/deficiencia , Receptor de Adenosina A1/fisiología , Receptor de Adenosina A3/deficiencia , Receptor de Adenosina A3/fisiología , Receptores de Adenosina A2/deficiencia , Receptores de Adenosina A2/fisiología , Receptores Purinérgicos P1/deficiencia , Receptores Purinérgicos P1/genética , Transmisión Sináptica/genéticaRESUMEN
The mRNA levels of NKCC1, an inwardly directed Na(+), K(+)-2Cl(-) cotransporter that facilitates the accumulation of intracellular Cl(-), and of KCC2, an outwardly directed K(+)-Cl(-) cotransporter that extrudes Cl(-), were studied in surgically resected brain specimens from drug-resistant temporal lobe (TL) epilepsy (TLE) patients. Quantitative RT-PCR analyses of the mRNAs extracted from the human TLE-associated brain regions revealed an up-regulation of NKCC1 mRNA and a down-regulation of KCC2 mRNA in the hippocampal subiculum, compared with the hippocampus proper or the TL neocortex, suggesting an abnormal transcription of Cl(-) transporters in the TLE subiculum. In parallel experiments, cell membranes isolated from the same TLE-associated brain regions were injected into Xenopus oocytes that rapidly incorporated human GABA(A) receptors into their surface membrane. The GABA currents elicited in oocytes injected with membranes from the subiculum had a more depolarized reversal potential (E(GABA)) compared with the hippocampus proper or the neocortex. The NKCC1 blocker bumetanide or a temperature decrease of 10 degrees C shifted the GABA-current E(GABA) more negative in oocytes injected with membranes from TLE hippocampal subiculum, matching the E(GABA) of TL neocortex-injected oocytes. We conclude that the anomalous expression of both Cl(-) transporters, NKCC1 and KCC2 [corrected] in TLE hippocampal subiculum probably causes altered Cl(-) transport in the "epileptic" neurons, as revealed in the microtransplanted Xenopus oocytes, and renders GABA aberrantly "exciting," a feature that may contribute to the precipitation of epileptic seizures.
Asunto(s)
Epilepsia del Lóbulo Temporal/genética , Epilepsia del Lóbulo Temporal/patología , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Simportadores del Cloruro de Sodio/genética , Ácido gamma-Aminobutírico/farmacología , Animales , Bumetanida/farmacología , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Regulación de la Expresión Génica , Humanos , Oocitos/efectos de los fármacos , Oocitos/metabolismo , Técnicas de Placa-Clamp , Inhibidores de los Simportadores del Cloruro de Sodio/farmacología , Xenopus laevisRESUMEN
Pharmacotherapeutic strategies have been difficult to develop for several forms of temporal lobe epilepsy, which are consequently treated by surgical resection. To examine this problem, we have studied the properties of transmitter receptors of tissues removed during surgical treatment. We find that when cell membranes, isolated from the temporal neocortex of patients afflicted with drug-resistant mesial temporal lobe epilepsy (TLE), are injected into frog oocytes they acquire GABA type A receptors (GABA(A)-receptors) that display a marked rundown during repetitive applications of GABA. In contrast, GABA(A)-receptor function is stable in oocytes injected with cell membranes isolated from the temporal lobe of TLE patients afflicted with neoplastic, dysgenetic, traumatic, or ischemic temporal lesions (lesional TLE, LTLE). Use-dependent GABA(A)-receptor rundown is also found in the pyramidal neurons of TLE neocortical slices and is antagonized by BDNF. Pyramidal neurons in cortical slices of a traumatic LTLE patient did not show GABA(A)-receptor rundown. However, the apparent affinity of GABA(A)-receptor in oocytes microtransplanted with membranes from all of the epileptic patients studied was smaller than the affinity of receptors transplanted from the nonepileptic brain. We conclude that the use-dependent rundown of neocortical GABA(A)-receptor represents a TLE-specific dysfunction, whereas the reduced affinity may be a general feature of brains of both TLE and LTLE patients, and we speculate that our findings may help to develop new treatments for TLE and LTLE.
Asunto(s)
Anticonvulsivantes/uso terapéutico , Resistencia a Medicamentos/fisiología , Epilepsia del Lóbulo Temporal/tratamiento farmacológico , Epilepsia del Lóbulo Temporal/fisiopatología , Receptores de GABA-A/metabolismo , Adolescente , Adulto , Anciano , Animales , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Membrana Celular/metabolismo , Niño , Electrofisiología , Epilepsia del Lóbulo Temporal/patología , Epilepsia del Lóbulo Temporal/cirugía , Femenino , Humanos , Masculino , Microinyecciones , Neuronas/citología , Neuronas/metabolismo , Oocitos/fisiología , Lóbulo Temporal/citología , Lóbulo Temporal/metabolismo , Xenopus laevis , Ácido gamma-Aminobutírico/metabolismoRESUMEN
The properties of gamma-aminobutyric acid (GABA) type A receptors (GABA(A) receptors) microtransplanted from the human epileptic brain to the plasma membrane of Xenopus oocytes were compared with those recorded directly from neurons, or glial cells, in human brains slices. Cell membranes isolated from brain specimens, surgically obtained from six patients afflicted with drug-resistant temporal lobe epilepsy (TLE) were injected into frog oocytes. Within a few hours, these oocytes acquired GABA(A) receptors that generated GABA currents with an unusual run-down, which was inhibited by orthovanadate and okadaic acid. In contrast, receptors derived from membranes of a nonepileptic hippocampal uncus, membranes from mouse brain, or recombinant rat alpha 1 beta 2 gamma 2-GABA receptors exhibited a much less pronounced GABA-current run-down. Moreover, the GABA(A) receptors of pyramidal neurons in temporal neocortex slices from the same six epileptic patients exhibited a stronger run-down than the receptors of rat pyramidal neurons. Interestingly, the GABA(A) receptors of neighboring glial cells remained substantially stable after repetitive activation. Therefore, the excessive GABA-current run-down observed in the membrane-injected oocytes recapitulates essentially what occurs in neurons, rather than in glial cells. Quantitative RT-PCR analyses from the same TLE neocortex specimens revealed that GABA(A)-receptor beta 1, beta 2, beta 3, and gamma 2 subunit mRNAs were significantly overexpressed (8- to 33-fold) compared with control autopsy tissues. Our results suggest that an abnormal GABA-receptor subunit transcription in the TLE brain leads to the expression of run-down-enhanced GABA(A) receptors. Blockage of phosphatases stabilizes the TLE GABA(A) receptors and strengthens GABAergic inhibition. It may be that this process can be targeted to develop new treatments for intractable epilepsy.
Asunto(s)
Encéfalo/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Epilepsia del Lóbulo Temporal/tratamiento farmacológico , Monoéster Fosfórico Hidrolasas/antagonistas & inhibidores , Receptores de GABA-A/efectos de los fármacos , Adulto , Encéfalo/metabolismo , Epilepsia del Lóbulo Temporal/etiología , Femenino , Humanos , Oocitos/metabolismo , Subunidades de Proteína , ARN Mensajero/análisis , Receptores de GABA-A/química , Receptores de GABA-A/metabolismoRESUMEN
Experiments were conducted in both HEK cells and cerebellar neurons to investigate whether CXC chemokine receptor 2 (CXCR2) is functionally coupled to GluR1. The co-expression of CXCR2 with GluR1 in HEK cells increased (i) the GluR1 "apparent" affinity for the transmitter; (ii) the GluR1 channel open probability; and (iii) GluR1 binding site cooperativity upon CXCR2 stimulation with CXC chemokine ligand 2 (CXCL2). The affinity of C-terminal-deleted GluR1 for glutamate (Glu) remained stable instead. Furthermore, CXCL2 increased the binding site cooperativity of AMPA receptors in rat cerebellar granule cells; and the amplitude of spontaneous excitatory postsynaptic current (sEPSCs) in Purkinje neurons (PNs). Our findings indicate that the coupling of CXCR2 with GluR1 may modulate glutamatergic synaptic transmission.
Asunto(s)
Sistema Nervioso Central/metabolismo , Quimiocinas CXC/metabolismo , Ácido Glutámico/metabolismo , Receptores AMPA/metabolismo , Receptores de Interleucina-8B/metabolismo , Sinapsis/metabolismo , Transmisión Sináptica/inmunología , Animales , Sitios de Unión/efectos de los fármacos , Sitios de Unión/inmunología , Células Cultivadas , Sistema Nervioso Central/inmunología , Corteza Cerebelosa/efectos de los fármacos , Corteza Cerebelosa/inmunología , Corteza Cerebelosa/metabolismo , Quimiocinas CXC/inmunología , Quimiocinas CXC/farmacología , ADN Complementario/genética , Relación Dosis-Respuesta a Droga , Antagonistas de Aminoácidos Excitadores/farmacología , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/inmunología , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/inmunología , Ácido Glutámico/farmacología , Humanos , Canales Iónicos/genética , Canales Iónicos/inmunología , Neuronas/efectos de los fármacos , Neuronas/inmunología , Neuronas/metabolismo , Ratas , Ratas Sprague-Dawley , Ratas Wistar , Receptores AMPA/genética , Receptores AMPA/inmunología , Receptores de Interleucina-8B/genética , Receptores de Interleucina-8B/inmunología , Sinapsis/inmunologíaRESUMEN
1. The influx of Zn2+ through the channels of fetal and adult mouse muscle nicotinic acetylcholine receptors (gamma- and epsilon-AChRs) and its effects on receptor function were studied in transiently transfected human BOSC 23 cells, by combining patch-clamp recordings with digital fluorescence microscopy. 2. ACh-induced whole-cell currents were reversibly reduced by external ZnCl2, with half-maximal inhibitory concentrations of 3 and 1 mM for gamma- and epsilon-AChRs, respectively. 3. Both gamma- and epsilon-AChR channels were permeable to Zn2+, as shown by fluorescence measurements using Zn2+-sensitive dyes. The fractional current carried by Zn2+ (Pf,Zn; 0.5 mM Zn2+ in Ca2+- and Mg2+-free medium) through gamma- and epsilon">-AChR channels was 1.7 and 4 %, respectively. 4. Pf,Zn increased with the concentration of ZnCl2, but was little affected by physiological concentrations of Ca2+ and Mg2+ in the external medium. 5. The conductance of ACh-evoked unitary events, measured by cell-attached or outside-out recordings, decreased when the patched membrane was exposed to ZnCl2 (1 or 3 mM). Simultaneous application of ACh and Zn2+ to the extra-patch membrane lengthened channel open duration (tau op) by 50%. No obvious increment of tau op was observed following exposure of inside-out patches to Zn2+. 6. The possible physiological relevance of zinc-induced modulation of AChR channels is discussed.
Asunto(s)
Canales Iónicos/metabolismo , Músculo Esquelético/metabolismo , Receptores Colinérgicos/metabolismo , Zinc/metabolismo , Animales , Línea Celular , Estimulación Eléctrica , Electrofisiología , Canales Iónicos/ultraestructura , Ratones , Microscopía Confocal , Músculo Esquelético/ultraestructura , Nicotina/farmacología , Agonistas Nicotínicos/farmacología , Técnicas de Placa-Clamp , Permeabilidad , Receptores Colinérgicos/ultraestructura , Espectrometría de FluorescenciaRESUMEN
The functional expression of the seven-transmembrane domain G protein-coupled chemokine receptor CXCR-4/fusin in rat nerve cell was demonstrated by staining with a polyclonal anti-CXCR-4 Ab, and by evaluating the calcium responses to the physiological agonist stromal-derived cell factor-1alpha (SDF-1alpha) in both cerebellar granule cells in culture and Purkinje neurons (PNs) in cerebellar slices. Cerebellar glial, granule and Purkinje cells showed a pronounced staining for CXCR-4. Furthermore, cultured granule cells exhibited Ca2+ transients elicited by the application of SDF-1alpha, both in cell bodies and in neuronal processes. Whole-cell patch-clamped PNs in cerebellar slices responded to SDF-1alpha application by a slow inward current followed by an increase of both intracellular Ca2+ level and spontaneous synaptic activity. In particular, the SDF-1alpha-induced slow inward current was considerably reduced by ionotropic glutamate receptor blockers, but developed fully in a medium in which synaptic transmission was inhibited, indicating that this current might be, at least in part, mediated by extrasynaptic glutamate, possibly released from the surrounding glial and/or nerve cells. Taken together, these findings indicate a functional involvement of CXCR-4 in the modulation of synaptic transmission, adding another member to the repertoire of the chemokine receptors exerting a neuromodulatory role in the cerebellum.
Asunto(s)
Quimiocinas CXC/farmacología , Células de Purkinje/fisiología , Transmisión Sináptica/efectos de los fármacos , 6-Ciano 7-nitroquinoxalina 2,3-diona/farmacología , Animales , Anticuerpos , Calcio/metabolismo , Células Cultivadas , Quimiocina CXCL12 , Electrofisiología , Antagonistas de Aminoácidos Excitadores/farmacología , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Microglía/química , Microglía/citología , Microglía/fisiología , Microscopía Confocal , Neuroinmunomodulación/fisiología , Neuronas/química , Neuronas/citología , Neuronas/fisiología , Células de Purkinje/química , Células de Purkinje/citología , Ratas , Ratas Wistar , Receptores CXCR4/análisis , Receptores CXCR4/inmunología , Transmisión Sináptica/fisiología , Tetrodotoxina/farmacologíaRESUMEN
Cultured cerebellar granule neurons are widely used as a cellular model to study mechanisms of neuronal cell death because they undergo programmed cell death when switched from a culture medium containing 25 mM to one containing 5 mM K(+). We have found that the growth-related gene product beta (GRObeta) partially prevents the K(+)-depletion-induced cell death, and that the neuroprotective action of GRObeta on granule cells is mediated through the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) type of ionotropic glutamate receptors. GRObeta-induced survival was suppressed by 6-cyano-7-nitroquinoxaline-2,3-dione, which is a specific antagonist of AMPA/kainate receptors; it was not affected by the inhibitor of N-methyl-D-aspartate receptors, 2-amino-5-phosphonopentanoic acid, and was comparable to the survival of granule cells induced by AMPA (10 microM) treatment. Moreover, GRObeta-induced neuroprotection was abolished when granule cells were treated with antisense oligonucleotides specific for the AMPA receptor subunits, which significantly reduced receptor expression, as verified by Western blot analysis with subunit-specific antibodies and by granule cell electrophysiological sensitivity to AMPA. Our data demonstrate that GRObeta is neurotrophic for cerebellar granule cells, and that this activity depends on AMPA receptors.
Asunto(s)
Apoptosis/efectos de los fármacos , Benzodiazepinas , Corteza Cerebelosa/efectos de los fármacos , Quimiocinas CXC , Factores Quimiotácticos/farmacología , Sustancias de Crecimiento/farmacología , Péptidos y Proteínas de Señalización Intercelular , Proteínas del Tejido Nervioso/fisiología , Fármacos Neuroprotectores/farmacología , Receptores AMPA/fisiología , 2-Amino-5-fosfonovalerato/farmacología , 6-Ciano 7-nitroquinoxalina 2,3-diona/farmacología , Animales , Ansiolíticos/farmacología , Corteza Cerebelosa/citología , ADN Complementario/genética , Regulación de la Expresión Génica/efectos de los fármacos , Interleucina-8/farmacología , Activación del Canal Iónico , Proteínas del Tejido Nervioso/efectos de los fármacos , Proteínas del Tejido Nervioso/genética , Oligodesoxirribonucleótidos Antisentido/farmacología , Potasio/farmacología , Potasio/fisiología , Ratas , Ratas Wistar , Receptores AMPA/efectos de los fármacos , Receptores AMPA/genética , Receptores de N-Metil-D-Aspartato/efectos de los fármacosRESUMEN
We report here that, in cultured cerebellar granule cells, the CXC chemokine GRObeta stimulates the signaling pathway of the extracellular signal-regulated kinases, and enhances both evoked and spontaneous postsynaptic currents in patch clamped Purkinje neurons from rat cerebellar slices. The GRObeta-induced enhancement of the excitatory post-synaptic currents evoked by stimulating the parallel fibres is blocked by the inhibitor of the extracellular signal-regulated kinases pathway PD98059, which also reduces both basal frequency of spontaneous post-synaptic currents and mean amplitude of evoked excitatory post-synaptic currents. Our results suggest that GRObeta modulates neurotransmitter release in the cerebellum through the activation of the extracellular signal-regulated kinases pathway.
Asunto(s)
Quimiocinas CXC , Factores Quimiotácticos/metabolismo , Inhibidores de Crecimiento/metabolismo , Sustancias de Crecimiento/metabolismo , Péptidos y Proteínas de Señalización Intercelular , Proteínas Quinasas Activadas por Mitógenos , Neurotransmisores/metabolismo , Células de Purkinje/enzimología , Animales , Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Células Cultivadas , Quimiocina CXCL1 , Inhibidores Enzimáticos/farmacología , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/fisiología , Flavonoides/farmacología , Proteína Quinasa 1 Activada por Mitógenos , Proteína Quinasa 3 Activada por Mitógenos , Técnicas de Cultivo de Órganos , Técnicas de Placa-Clamp , Células de Purkinje/citología , Ratas , Transducción de Señal/fisiología , Transmisión Sináptica/fisiología , Tetrodotoxina/farmacologíaRESUMEN
We examine some of the biological and physiological properties of the avian alpha6 neuronal nicotinic acetylcholine receptor (nAChR) subunit. We show here that, beginning at embryonic day 5, alpha6 mRNA is abundantly expressed in the developing chick neuroretina, where it coexists with other nicotinic receptor subunit mRNAs such as alpha3, beta2 and beta4. In contrast, alpha6 mRNA is absent from the optic tectum and from the peripheral ganglia. Despite numerous efforts, the alpha6 subunit has long failed the critical test of functional reconstitution. Here we use patch-clamp techniques and confocal laser microscopy to measure ACh-activated currents and nicotine-elicited Ca2+ transients in human BOSC 23 cells transfected with chick alpha6 in combination with other chick nAChR neuronal subunits. Heterologously expressed alpha6 and beta4 subunits form functional heteromeric nAChRs, which are permeable to Ca2+ ions and blocked by the nicotinic antagonist methyllycaconitine (10 microM). Likewise, ACh elicits measurable currents in cells transfected with alpha6 and beta2. Hill analysis of the dose-response curves in cells transfected with alpha3, beta4 and alpha6 cDNAs, suggests the assembly of functional alpha3beta4alpha6 receptor, with an apparent affinity for ACh threefold lower than alpha3beta4. Our results indicate that alpha6-containing nAChRs assemble in heterologous expression systems and are probably present in retinal cells.
Asunto(s)
Activación del Canal Iónico/fisiología , Neuronas/química , Receptores Nicotínicos/genética , Acetilcolina/farmacología , Animales , Embrión de Pollo , ADN Complementario , Humanos , Activación del Canal Iónico/efectos de los fármacos , Datos de Secuencia Molecular , Neuronas/fisiología , Nicotina/farmacología , Agonistas Nicotínicos/farmacología , Técnicas de Placa-Clamp , ARN Mensajero/análisis , Homología de Secuencia de Aminoácido , Transfección , Vías Visuales/química , Vías Visuales/citologíaRESUMEN
1. Combinations of cDNAs encoding mouse and chick nicotinic acetylcholine receptor (nAChR) subunits were transiently transfected into human BOSC 23 cells, and the expressed receptors were studied by simultaneously recording transmembrane currents and fluorescence transients using the whole-cell patch-clamp technique, and confocal microscopy with the Ca2+ indicator dye fluo-3. 2. The fractional Ca2+ current, Pf, of nAChRs was evaluated as the normalized ratio of nicotine-evoked fluorescence transient over total charge entering the cell (F/Q ratio). Mouse fetal muscle nAChR channels had a Pf, alphabetagammadelta value of 2.1 %. The substitution of the gamma subunit with the epsilon subunit resulted in a 2-fold increase in Pf (4.2 %). The difference in Ca2+ permeability was confirmed by determination of Ca2+/Cs+ permeability ratios. 3. Among the chick neuronal nAChRs tested, Pf,alpha3beta4 was 4.6 %, while Pf, alpha4beta4 and Pf,alpha4beta2 were 3.0 % and 2.9 %, respectively. 4. The amplitude of the current elicited by the activation of alpha3beta4 nAChRs increased as the external Ca2+ concentration was raised from 2 to 110 mM, whereas currents flowing through all other nAChRs tested were reduced to various extents. 5. Our findings indicate that the adult-type muscle nAChR (alphabetaepsilondelta) is more permeable to Ca2+ than the fetal-type (alphabetagammadelta), while ganglionic-like alpha3beta4 nAChR is more permeable to Ca2+ than the examined alpha4-containing nAChRs. The functional significance is discussed.
Asunto(s)
Canales de Calcio/fisiología , Calcio/metabolismo , Neuronas/metabolismo , Nicotina/farmacología , Receptores Nicotínicos/fisiología , Compuestos de Anilina , Animales , Calcio/farmacología , Canales de Calcio/efectos de los fármacos , Línea Celular , Membrana Celular/efectos de los fármacos , Membrana Celular/fisiología , Permeabilidad de la Membrana Celular , Pollos , Feto , Colorantes Fluorescentes , Humanos , Cinética , Sustancias Macromoleculares , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Ratones , Modelos Químicos , Músculo Esquelético/fisiología , Técnicas de Placa-Clamp , Multimerización de Proteína , Receptores Nicotínicos/biosíntesis , Proteínas Recombinantes/biosíntesis , Transfección , XantenosRESUMEN
We give here evidence that Purkinje neurons (PNs) of mouse cerebellar slices studied with patch clamp technique combined with laser confocal microscopy, respond to human IL-8 and GROalpha by (i) a cytosolic Ca2+ transient compatible with inositol (1,4,5) trisphosphate (InsP3) formation; (ii) an enhancement of the neurotransmitter release; and (iii) an impairment of the long-term depression of synaptic strength (LTD). It was also found the expression of IL-8 receptor type 2 in PN and granule cells by immunofluorescence, immunoblotting and RT-PCR analysis. Considered together these findings suggest that IL-8 and GROalpha may play a neuromodulatory role on mouse cerebellum.
Asunto(s)
Quimiocinas CXC , Factores Quimiotácticos/farmacología , Inhibidores de Crecimiento/farmacología , Sustancias de Crecimiento/farmacología , Péptidos y Proteínas de Señalización Intercelular , Interleucina-8/farmacología , Células de Purkinje/efectos de los fármacos , Animales , Animales Recién Nacidos , Calcio/metabolismo , Quimiocina CXCL1 , Citosol/metabolismo , Conductividad Eléctrica , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/fisiología , Colorantes Fluorescentes , Humanos , Técnicas In Vitro , Inositol 1,4,5-Trifosfato/metabolismo , Potenciación a Largo Plazo/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Compuestos Orgánicos , Células de Purkinje/metabolismo , Células de Purkinje/fisiología , Ratas , Ratas Wistar , Receptores de Quimiocina/metabolismo , Receptores de Interleucina/metabolismo , Receptores de Interleucina-8BRESUMEN
Fluorescence videomicroscopy was used to monitor changes in the cytosolic free Ca2+ concentration ([Ca2+]i) in the mouse muscle cell line C2Cl2 during in vitro myogenesis. Three different patterns of changes in [Ca2+]i were observed: (i) [Ca2+]i oscillations; (ii) faster Ca2+ events confined to subcellular regions (localized [Ca2+]i spikes) and (iii) [Ca2+]i spikes detectable in the entire myotube (global [Ca2+]i spikes). [Ca2+]i oscillations and localized [Ca2+]i spikes were detectable following the appearance of caffeine-sensitivity in differentiating C2Cl2 cells. Global [Ca2+]i spikes appeared later in the process of myogenesis in cells exhibiting coupling between voltage-operated Ca2+ channels and ryanodine receptors. In contrast to [Ca2+]i oscillations and localized [Ca2+]i spikes, the global events immediately stopped when cells were perfused either with a Ca2+-free solution, or a solution with TTX, TEA and verapamil. To explore further the mechanism of the global [Ca2+]i spikes, membrane currents and fluorescence signals were measured simultaneously. These experiments revealed that global [Ca2+]i spikes were correlated with an inward current. Moreover, while the depletion of the Ca2+ stores blocked [Ca2+]i oscillations and localized [Ca2+]i spikes, it only reduced the amplitude of global [Ca2+]i spikes. It is suggested that, during the earlier stages of the myogenesis, spontaneous and repetitive [Ca2+]i changes may be based on cytosolic oscillatory mechanisms. The coupling between voltage-operated Ca2+ channels and ryanodine receptors seems to be the prerequisite for the appearance of global [Ca2+]i spikes triggered by a membrane oscillatory mechanism, which characterizes the later phases of the myogenic process.
Asunto(s)
Calcio/metabolismo , Diferenciación Celular , Músculo Esquelético/fisiología , Ácido 3-piridinacarboxílico, 1,4-dihidro-2,6-dimetil-5-nitro-4-(2-(trifluorometil)fenil)-, Éster Metílico/farmacología , Adenosina Trifosfato/farmacología , Animales , Apamina/farmacología , Cafeína/farmacología , Diferenciación Celular/efectos de los fármacos , Línea Celular , Citosol/metabolismo , Ácido Egtácico/farmacología , Cinética , Potenciales de la Membrana/efectos de los fármacos , Ratones , Microscopía Confocal , Microscopía por Video , Músculo Esquelético/citología , Oscilometría , Cloruro de Potasio/farmacología , Tetraetilamonio , Compuestos de Tetraetilamonio/farmacología , Tetrodotoxina/farmacología , Verapamilo/farmacologíaRESUMEN
To study how subunit composition affects the functional properties of neuronal nicotinic acetylcholine receptors (nAChRs), we examined the behaviour of acetylcholine (ACh)-induced single-channel currents in human BOSC 23 cells transiently transfected with various subunit cDNA combinations. For all nAChRs examined (chick and rat alpha 3 beta 4, chick alpha 3 beta 2, alpha 4 beta 2, alpha 7 and alpha 8), expression levels were high enough to allow measurements of acetylcholine-evoked whole-cell currents and nicotine-elicited Ca2+ transients as well as the functional characterization of nAChR channels. Unitary acetylcholine-evoked events of alpha 8 nAChR had a slope conductance of 23 pS, whereas two conductance classes (19-23 and 32-45 pS) were identified for all other nAChR channels. The mean channel open times were significantly longer for homomeric alpha 7 and alpha 8 nAChRs (6-7 ms) than for heteromeric nAChRs (1-3 ms), with the exception of alpha 3 beta 4 nAChRs (8.4 ms for rat, 7 ms for chick). At least two species of heterologously expressed nAChRs (alpha 3 beta 4 and alpha 3 beta 2) exhibited single-channel characteristics similar to those reported for native receptors. The variety of nAChR channel conductance and kinetic properties encountered in human cells transfected with nAChR subunits contributes to the functional diversity of nAChRs in nerve cells.
Asunto(s)
Acetilcolina/farmacología , Neuronas/efectos de los fármacos , Receptores Nicotínicos/fisiología , Animales , Pollos , Humanos , Técnicas de Placa-Clamp , Ratas , Receptores Nicotínicos/efectos de los fármacos , TransfecciónRESUMEN
In mammals, receptors for the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) are divided into three pharmacological classes, which are denoted GABAA, GABAB, and GABAC. GABAC receptors are defined by their insensitivity to the GABAA receptor antagonist bicuculline and the GABAB receptor agonist (-)-baclofen. GABAC receptors probably are a heterogeneous group of proteins. The most extensively studied mammalian GABAC receptors are those found in neurons of the outer retina. These receptors are GABA-gated Cl- channels comprised of p subunits, of which there are two subtypes. The physiological functions served by GABAC receptors are largely unknown; to determine the functions, it would be useful to have GABAC-selective ligands. In a previous study, we found that isoguvacine, a GABAA-selective agonist, and 3-aminopropyl-(methyl)phosphinic acid (3-APMPA), a GABAB-selective agonist, show affinity for retinal GABAC receptors. In particular, 3-APMPA is an antagonist with low micromolar potency (Kb approximately 1 microM). Here, we report the synthesis and pharmacological characterization of (1,2,5,6-tetrahydropyridine-4-yl)methylphosphinic acid (TPMPA), a hybrid of isoguvacine and 3-APMPA designed to retain affinity for GABAC receptors but not to interact with GABAA or GABAB receptors. Electrical assays show that TPMPA is a competitive antagonist of cloned human mu 1 GABAC receptors expressed in Xenopus laevis oocytes (Kb approximately 2 microM). TPMPA is > 100-fold weaker as an inhibitor of rat brain GABAA receptors expressed in oocytes (Kb approximately 320 microM) and has only weak agonist activity on GABAB receptors assayed in rat hippocampal slices (EC50 approximately 500 microM). TPMPA should be a useful pharmacological probe with which to investigate GABAC receptor function in the outer retina and in any other areas of the nervous system in which these types of receptor are present.
Asunto(s)
Antagonistas del GABA/farmacología , Ácidos Fosfínicos/farmacología , Piridinas/farmacología , Receptores de GABA/efectos de los fármacos , Animales , Encéfalo/efectos de los fármacos , Encéfalo/ultraestructura , Diseño de Fármacos , Ácidos Fosfínicos/síntesis química , Piridinas/síntesis química , Ratas , Ratas Endogámicas ACI , Retina/efectos de los fármacos , Retina/ultraestructura , Xenopus laevisRESUMEN
Voltage-dependent magnesium block of N-methyl-D-aspartate-activated channels, and the N-methyl-D-aspartate component of excitatory synaptic currents were studied in CA3 pyramidal neurons of hippocampal slices from immature (postnatal day 3-8) and adult (postnatal day 25-60) rats. In all neurons studied the kinetics of single-channel openings in cell-attached and inside-out configurations was strongly modulated by extracellular Mg2+, in a voltage-dependent manner. No age-dependent difference in the Mg2+ sensitivity of N-methyl-D-aspartate channels was observed. At physiological concentrations of external Mg2+ (1.3 mM), N-methyl-D-aspartate components of excitatory synaptic currents measured from immature and adult rats displayed a similar voltage-dependence. In immature neurons (postnatal day 3-6), the N-methyl-D-aspartate component of excitatory postsynaptic currents decay time-course was a single-exponential with an average time-constant of about 300 ms. In neurons from older animals the decay was described by a double-exponential function with both a fast component (tau f, 54-130 ms) and a slow component (tau s, 275-400 ms). With age, the contribution of the fast component increased and the decay time-course of the N-methyl-D-aspartate component of excitatory postsynaptic currents accelerated. It is concluded that the Mg2+ block of N-methyl-D-aspartate channels in CA3 pyramidal neurons does not change with development, but the kinetic properties of N-methyl-D-aspartate receptor channels are developmentally regulated.