RESUMEN
Astrocytes express voltage-gated calcium channels (VGCCs) that are upregulated in the context of the reactive astrogliosis occurring in several CNS pathologies. Moreover, the ability of selective calcium channel blockers to inhibit reactive astrogliosis has been revealed in a variety of experimental models. However, the functions and regulation of VGCC in astrocytes are still poorly understood. Interestingly, protein kinase C epsilon (PKCepsilon), one of the known regulators of VGCC in several cell types, induces in astrocytes a stellated morphology similar to that associated to gliosis. Thereby, here we explored the possible regulation of VGCC by adenovirally expressed PKCepsilon in astrocytes. We found that PKCepsilon potently increases the mRNA levels of two different calcium channel alpha(1) subunits, Ca(V)1.2 (L-type channel) and Ca(V)2.1 (P/Q-type channel). The mRNA upregulation was followed by a robust increase in the corresponding peptides. Moreover, the new calcium channels formed as a consequence of PKCepsilon activation are functional, since overexpression of constitutively-active PKCepsilon increased significantly the calcium current density in astrocytes. PKCepsilon raised currents carried by both L- and P/Q-type channels. However, the effect on the P/Q-type channel was more prominent since an increase of the relative contribution of this channel to the whole cell calcium current was observed. Finally, we found that PKCepsilon-induced stellation was significantly reduced by the specific L-type channel blocker nifedipine, indicating that calcium influx through VGCC mediates the change in astrocyte morphology induced by PKCepsilon. Therefore, here we describe a novel regulatory pathway involving VGCC that participates in PKCepsilon-dependent astrocyte activation.
Asunto(s)
Astrocitos/metabolismo , Encéfalo/metabolismo , Canales de Calcio/genética , Canales de Calcio/metabolismo , Proteína Quinasa C-epsilon/genética , Proteína Quinasa C-epsilon/metabolismo , Animales , Animales Recién Nacidos , Astrocitos/efectos de los fármacos , Encéfalo/citología , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio/efectos de los fármacos , Canales de Calcio Tipo L/genética , Canales de Calcio Tipo L/metabolismo , Canales de Calcio Tipo N/genética , Canales de Calcio Tipo N/metabolismo , Células Cultivadas , Vectores Genéticos , Gliosis/genética , Gliosis/metabolismo , Ratones , Ratones Endogámicos C57BL , Técnicas de Placa-Clamp , ARN Mensajero/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Transfección , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/fisiologíaRESUMEN
Vertebrates can detect light intensity changes in vastly different photic environments, in part, because postreceptoral neurons undergo "network adaptation." Previous data implicated dopaminergic, cAMP-dependent inhibition of retinal ganglion cells in this process yet left unclear how this occurs and whether this occurs in darkness versus light. To test for light- and dopamine-dependent changes in ganglion cell cAMP levels in situ, we immunostained dark- and light-adapted retinas with anti-cAMP antisera in the presence and absence of various dopamine receptor ligands. To test for direct effects of dopamine receptor ligands and membrane-permeable protein kinase ligands on ganglion cell excitability, we recorded spikes from isolated ganglion cells in perforated-patch whole-cell mode before and during application of these agents by microperfusion. Our immunostainings show that light, endogenous dopamine, and exogenous dopamine elevate ganglion cell cAMP levels in situ by activating D1-type dopamine receptors. Our spike recordings show that D1-type agonists and 8-bromo cAMP reduce spike frequency and curtail sustained spike firing and that these effects entail protein kinase A activation. These effects resemble those of background light on ganglion cell responses to light flashes. Network adaptation could thus be produced, to some extent, by dopaminergic modulation of ganglion cell spike generation, a mechanism distinct from modulation of transmitter release onto ganglion cells or of transmitter-gated currents in ganglion cells. Combining these observations with results obtained in studies of photoreceptor, bipolar, and horizontal cells indicates that all three layers of neurons in the retina are equipped with mechanisms for adaptation to ambient light intensity.
Asunto(s)
Adaptación Ocular/fisiología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Diclororribofuranosil Benzoimidazol/análogos & derivados , Dopamina/metabolismo , Red Nerviosa/metabolismo , Células Ganglionares de la Retina/metabolismo , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Potenciales de Acción/efectos de la radiación , Animales , Bloqueadores de los Canales de Calcio/farmacología , Separación Celular , AMP Cíclico/metabolismo , Oscuridad , Diclororribofuranosil Benzoimidazol/farmacología , Dopamina/farmacología , Agonistas de Dopamina/farmacología , Antagonistas de Dopamina/farmacología , Carpa Dorada , Técnicas In Vitro , Luz , Red Nerviosa/efectos de los fármacos , Técnicas de Placa-Clamp , Estimulación Luminosa , Fotoperiodo , Tiempo de Reacción/efectos de los fármacos , Tiempo de Reacción/fisiología , Receptores de Dopamina D1/agonistas , Receptores de Dopamina D1/antagonistas & inhibidores , Receptores de Dopamina D1/metabolismo , Células Ganglionares de la Retina/citología , Células Ganglionares de la Retina/efectos de los fármacos , Células Ganglionares de la Retina/efectos de la radiación , Tionucleótidos/farmacologíaRESUMEN
We propose a mathematical model of rabbit retinal horizontal cell based on the ionic current mechanisms. Five types of ionic currents in rabbit retinal horizontal cell, I(Na), I(Ca), I(Kv), I(A) and I(Ka), are described by Hodgkin-Huxley type equations based on voltage clamp measurements. In simulation the model reproduced similar responses to voltage and current clamp experiments. Under the current clamp experiment a repetitive action potential was found on A-type rabbit horizontal cells. Our result suggests that the repetitive action potential is generated by an interaction Of I(Ca) and I(Kv).
Asunto(s)
Canales Iónicos/fisiología , Modelos Neurológicos , Conejos/fisiología , Retina/fisiología , Potenciales de Acción/fisiología , Animales , Canales de Calcio/fisiología , Simulación por Computador , Conductividad Eléctrica , Electrofisiología , Técnicas de Placa-Clamp , Canales de Potasio/fisiología , Retina/citología , Canales de Sodio/fisiologíaRESUMEN
In the vertebrate retina, the rod bipolar cells make reciprocal synapses with amacrine cells at the axon terminal. Amacrine cells may perform a fine control of the transmitter release from rod bipolar cells by means of GABAergic synapses acting on different types of GABA receptors. To clarify this possibility GABA-induced currents were recorded by the patch-clamp whole cell method in rod bipolar cells enzymatically dissociated from the mouse retina. All cells tested showed a desensitising chloride-sensitive GABA-induced current. When GABA 30 microM was applied in presence of 100 microM biccuculine, a blocker of the GABA(A) receptors, a slow-desensitising component of the current still remains. This current was blocked when GABA 30 microM was applied in presence of 100 microM 3-aminopropylphosphonic acid, an antagonist of the GABA(C) receptors. The current mediated by GABA(C) receptors showed an EC50 of less that 5 microM; the ionic current through the GABA(A) receptor showed an EC50 of ca. 30 microM. Two pieces of evidence demonstrated that the GABA(C)-mediated current was localised at the axon terminal of rod bipolar cells: (1) cells lacking the axon terminal only showed the biccuculine-sensitive GABA-induced current; and (2) after mechanical section of the axon terminal, bipolar cells lost the slow-desensitising component of the GABA-induced current. We conclude that the rod bipolar cells express two types of ionotropic GABA receptors, and that the high sensitive GABA(C) receptors are mainly localised at the level of the axon terminal and therefore may contribute to the modulation of the transmitter release from the rod bipolar cell.
Asunto(s)
Axones/fisiología , Receptores de GABA/fisiología , Células Fotorreceptoras Retinianas Bastones/fisiología , Sinapsis/fisiología , Ácido gamma-Aminobutírico/fisiología , Animales , Axones/efectos de los fármacos , Bicuculina/farmacología , Técnicas In Vitro , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Ratones , Técnicas de Placa-Clamp , Terminales Presinápticos/efectos de los fármacos , Terminales Presinápticos/fisiología , Propilaminas/farmacología , Receptores de GABA-A/fisiología , Células Fotorreceptoras Retinianas Bastones/efectos de los fármacos , Sinapsis/efectos de los fármacos , Ácido gamma-Aminobutírico/farmacologíaRESUMEN
In the vertebrate retina, the bipolar cell makes reciprocal synapses with amacrine cells at the axon terminal. It has been postulated that amacrine cells may control the transmitter release from bipolar cells by modulating their calcium currents (ICa). To clarify this possibility calcium currents were studied in bipolar cells of the mouse retina using a slice preparation. ICa was identified by voltage clamp protocols, ionic substitution and pharmacological tools. Depolarization to -30 mV from a holding voltage of -80 mV induced an inward current consisting of an initial transient and a long-lasting sustained component. The transient component was inactivated by holding the membrane at more positive voltages. Addition of 100 microM nifedipine suppressed the sustained component, leaving the transient component almost intact. The sustained component was enhanced when external solution contained 0.1 microM Bay K 8644 or when the external Ca2+ was substituted by equimolar Ba2+. Omega-conotoxin (10 microM omega-ctxn GVIA) did not alter either component. We concluded that the transient component is a low-voltage activated T-type ICa, while the sustained component is a high-voltage activated L-type ICa. T-type ICa was recorded in all cells tested, while L-type ICa was found only in cells that retained axon terminals ramifying in the inner plexiform layer. Thus, it is highly likely that L-type ICa is generated at the axon terminal and contributes to the transmitter release from the bipolar cell. The present results confirm that in addition to the T-type ICa that had been previously described, bipolar cells of the mammalian retina also contain L-type ICa similar to the one that has been reported in bipolar cells of the goldfish. The use of retinal slice preparation allowed us to record this current that was not seen previously in the dissociated mouse bipolar cells.
Asunto(s)
Canales de Calcio/fisiología , Calcio/metabolismo , Retina/fisiología , Ácido 3-piridinacarboxílico, 1,4-dihidro-2,6-dimetil-5-nitro-4-(2-(trifluorometil)fenil)-, Éster Metílico/farmacología , Animales , Calcio/farmacocinética , Agonistas de los Canales de Calcio/farmacología , Bloqueadores de los Canales de Calcio/farmacología , Cobalto/farmacología , Dihidropiridinas/farmacología , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Ratones , Ratones Endogámicos , Técnicas de Cultivo de Órganos , Técnicas de Placa-Clamp , Péptidos/farmacología , Retina/química , Tetraetilamonio/farmacología , omega-Conotoxina GVIARESUMEN
The intensity of the immunohistochemical reaction (IIR) against the alpha species of protein kinase C (PKC) was quantified in the rod bipolar cells (RBC) of the goldfish retina using of image analysis. Retinae incubated in control Ringer solution showed similar IIR in both the soma and the axon terminal (IIR-ratio approximately 1). Activation of PKC induces the 'transport' of the enzyme to the synaptic terminal of RBC and an increase in the IIR-ratio. In the present report, the effect of retinal neurotransmitters on the IIR-ratio and the time course of PKC transport was studied.
Asunto(s)
Isoenzimas/metabolismo , Proteína Quinasa C/metabolismo , Proteínas Quinasas/metabolismo , Retina/citología , Células Fotorreceptoras Retinianas Bastones/citología , Células Fotorreceptoras Retinianas Bastones/enzimología , Animales , Axones/enzimología , Axones/ultraestructura , Calcio/farmacología , Dopamina/farmacología , Carpa Dorada , Inmunohistoquímica , Técnicas In Vitro , Isoenzimas/análisis , Cinética , Terminaciones Nerviosas/enzimología , Terminaciones Nerviosas/ultraestructura , Nifedipino/farmacología , Potasio/farmacología , Proteína Quinasa C/análisis , Proteína Quinasa C-alfa , Proteínas Quinasas/análisis , Serotonina/farmacología , Sinapsis/enzimología , Sinapsis/ultraestructura , Péptido Intestinal Vasoactivo/farmacología , Ácido gamma-Aminobutírico/farmacologíaRESUMEN
Intracellular and patch-clamp recordings have been used to characterize GABA-activated channels in axonless horizontal cells (ALHC) of the rabbit retina. In our intracellular recordings on an everted eyecup preparation, GABA depolarized the horizontal cells (HC), diminished their light response amplitude and slowed the response rise time. Glycine showed similar effects on the HC light responses. In our whole cell patch-clamp recordings on dissociated ALHC, all HCs responded to 3 microM GABA but none to glycine, even at 100 microM. Dose-response relationship for GABA gave EC50 values around 10 microM and Hill slopes of 1.3. Whole-cell current-voltage (I-V) relationships of GABA-activated currents reversed close to the predicted Cl- equilibrium potential. Partial replacement of intracellular Cl- with isothetionate shifted the GABA reversal potential to a more negative value. Muscimol (30 microM), a GABAA agonist mimicked the effect of GABA, but baclofen (30 microM), a GABAB agonist and cis-aminocaprionic acid (30 microM), a GABAC agonist did not elicit any effect on ALHC. Responses to GABA were blocked by the GABAA receptor antagonist bicuculline (10 microM) and picrotoxin (100 microM). According to our results, we conclude that ALHC express GABA receptors coupled to ion channels, and they correspond to GABAA receptor subtypes.
Asunto(s)
Glicina/farmacología , Conejos/fisiología , Retina/efectos de los fármacos , Ácido gamma-Aminobutírico/farmacología , Animales , Relación Dosis-Respuesta a Droga , Conductividad Eléctrica , Electrofisiología , Canales Iónicos/fisiología , Técnicas de Placa-Clamp , Estimulación Luminosa , Receptores de GABA/fisiología , Retina/citología , Retina/fisiologíaRESUMEN
The purpose of the present study was to elucidate the physiological mechanisms that determine the activation of protein kinase C (PKC) in rod bipolar cells (RBC) of mouse and goldfish. The localization of PKC in RBC was examined using immunoreactivity (IR) against the alpha species of the enzyme. After incubating the whole retina or dissociated cells in control or test solutions, PKC-IR was performed on retinal transverse sections or on isolated cells. Cell depolarization induced the transport of the PKC to the synaptic terminal of RBC. The transport of the enzyme was also induced upon incubating dissociated cells in a solution containing phorbol esters. Enzyme transport was inhibited when the isolated retina was incubated in solutions containing GABA or nifedipine. We conclude that calcium and diacylglycerol, which contribute to the activation of PKC in RBC, induce transport of the enzyme to the synaptic terminal where it is presumed to play its functional role.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Terminales Presinápticos/enzimología , Células Fotorreceptoras Retinianas Bastones/enzimología , Animales , Axones/enzimología , Axones/ultraestructura , Calcio/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/análisis , Diglicéridos/farmacología , Carpa Dorada , Inmunohistoquímica , Ratones , Nifedipino/farmacología , Terminales Presinápticos/efectos de los fármacos , Terminales Presinápticos/ultraestructura , Células Fotorreceptoras Retinianas Bastones/citología , Ácido gamma-Aminobutírico/farmacologíaRESUMEN
Axonless horizontal cells were enzymatically dissociated from the retinae of adult rabbits. Whole-cell patch-clamp recordings were made on dissociated cells and voltage- and ligand-induced currents were studied. When membrane potential was measured in the current-clamp configuration, current pulses injected into the cell induced repetitive action potentials. When the cells were depolarised by bath application of kainic acid (KA, 30 microM), a train of fast-repetitive action potential was evoked. Also, a slow long-lasting calcium action potential kept the cells depolarised long after the cessation of the KA application. These findings indicate for the first time that horizontal cells of the mammalian retina are able to produce trains of action potentials.