RESUMEN
Modulation of Ca(2+) within cells is tightly regulated through complex and dynamic interactions between the plasma membrane and internal compartments. In this study, we exploit in vivo imaging strategies based on genetically encoded Ca(2+) indicators to define changes in perikaryal Ca(2+) concentration of intact photoreceptors. We developed double-transgenic zebrafish larvae expressing GCaMP3 in all cones and tdTomato in long-wavelength cones to test the hypothesis that photoreceptor degeneration induced by mutations in the phosphodiesterase-6 (Pde6) gene is driven by excessive [Ca(2+)]i levels within the cell body. Arguing against Ca(2+) overload in Pde6 mutant photoreceptors, simultaneous analysis of cone photoreceptor morphology and Ca(2+) fluxes revealed that degeneration of pde6c(w59) mutant cones, which lack the cone-specific cGMP phosphodiesterase, is not associated with sustained increases in perikaryal [Ca(2+)]i. Analysis of [Ca(2+)]i in dissociated Pde6ß(rd1)mouse rods shows conservation of this finding across vertebrates. In vivo, transient and Pde6-independent Ca(2+) elevations ('flashes') were detected throughout the inner segment and the synapse. As the mutant cells proceeded to degenerate, these Ca(2+) fluxes diminished. This study thus provides insight into Ca(2+) dynamics in a common form of inherited blindness and uncovers a dramatic, light-independent modulation of [Ca(2+)]i that occurs in normal cones.
Asunto(s)
Señalización del Calcio , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/deficiencia , Células Fotorreceptoras Retinianas Conos/patología , Células Fotorreceptoras Retinianas Bastones/enzimología , Células Fotorreceptoras Retinianas Bastones/patología , Proteínas de Pez Cebra/deficiencia , Pez Cebra/metabolismo , Animales , Animales Modificados Genéticamente , Muerte Celular/efectos de la radiación , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/metabolismo , Electrorretinografía , Luz , Ratones , Mutación/genética , Células Fotorreceptoras Retinianas Conos/efectos de la radiación , Células Fotorreceptoras Retinianas Bastones/efectos de la radiación , Proteínas de Pez Cebra/metabolismoRESUMEN
AIM: To provide insight into the principles of operation of electronic apex locators, determine optimal measuring parameters of the impedance ratio method, and to evaluate its accuracy. METHODOLOGY: Electrical impedance was measured ex vivo on 14 extracted human teeth using a QuadTech 1920 precision impedance analyzer. A file electrode was inserted into the root canal; the second electrode was placed in the saline solution surrounding the tooth. Measurements were performed in a frequency range from 20 Hz to 1 MHz, and repeated with different distances of the file tip from the major apical foramen. The measured impedances were analysed as a function of distance of the file tip to the major apical foramen. Parameters (e.g. measurement frequencies, impedance ratio value) that would result in optimal working length determination were evaluated. RESULTS: The optimal determination of the major apical foramen position was obtained at frequencies of 5 kHz and 0.5 kHz, 10 kHz and 0.5 kHz, and 5 kHz and 1 kHz, for the impedance ratios 0.73 (95% CI: -0.33 to 1.74 mm), 0.66 (95% CI: -0.34 to 1.81 mm) and 0.79 (95% CI: -0.33 to 1.58 mm) respectively. The limit of +/-0.5 mm was attained in 86% of all measurements. Standard deviations decreased as the average measured distance approached and extended beyond the major apical foramen. CONCLUSIONS: With the obtained optimal measuring parameters, the impedance ratio method determined position of the major foramen within +/-0.5 mm. Accuracy varied depending on the set of frequencies used for evaluation as well as on the selected impedance ratio.
Asunto(s)
Cavidad Pulpar/anatomía & histología , Tratamiento del Conducto Radicular/instrumentación , Ápice del Diente/anatomía & histología , Anciano , Impedancia Eléctrica , Electrónica Médica/instrumentación , Humanos , Persona de Mediana Edad , Odontometría/instrumentación , Reproducibilidad de los ResultadosRESUMEN
Four-electrode impedance spectra of relaxed and contracted muscle biceps brachii were analyzed in an adult human subject over the frequency range from 300 Hz to 75 kHz. A feasibility of the principal component analysis of bioimpedance measurement for the evaluation of skeletal muscle contractile state was examined. The principal components score plots show a data grouping of the impedance spectra from the two muscle groups. The classification was performed using a soft independent modeling of class analogy (SIMCA) method. The data set comprised 32 samples (16 samples of contracted muscle and 16 samples of relaxed muscle). The leave-one-out test of the classification yields about 80% of correctly classified samples (11 samples for contracted and 15 samples for relaxed muscle).
Asunto(s)
Contracción Muscular/fisiología , Relajación Muscular/fisiología , Músculo Esquelético/fisiología , Análisis Espectral/métodos , Adulto , Impedancia Eléctrica , Humanos , Masculino , Análisis Multivariante , Análisis de Componente PrincipalRESUMEN
The performance of a monolithic instrumentation amplifier used as an interface for a four-electrode bioimpedance measurement is examined with a commercially available impedance meter based on an auto-balancing bridge. The errors due to particularities in the input stage of the impedance meter, when used without a front-end, were several orders of magnitude higher than the measured quantity. The analysis was performed on an electrical circuit model of the skin and electrodes over a frequency range of 20 Hz to 1 MHz. The achieved accuracy with balanced electrode impedances for the frequencies up to 100 kHz can be below 0.2% for impedance magnitude and 0.1 degrees for impedance phase, which is within the specified basic accuracy range of the LCR-meter used for the measurements. At frequencies above 100 kHz the errors are increasing and are higher than the LCR-meter's basic accuracy. This study indicates that use of an instrumentation amplifier as a front-end with the particular LCR-meter can significantly improve the measurement accuracy of the four-electrode bioimpedance measurement at low frequencies.
Asunto(s)
Amplificadores Electrónicos , Impedancia Eléctrica , Algoritmos , Electrodos , Respuesta Galvánica de la Piel/fisiología , HumanosRESUMEN
Vertebrate photoreceptors consist of strictly delimited subcellular domains: the outer segment, ellipsoid, cell body and synaptic terminal, each hosting crucial cellular functions, including phototransduction, oxidative metabolism, gene expression and transmitter release. We used optical imaging to explore the spatiotemporal dynamics of Ca(2+) signaling in non-outer segment regions of rods and cones. Sustained depolarization, designed to emulate photoreceptor activation in the darkness, evoked a standing Ca(2+) gradient in tiger salamander photoreceptors with spatially-averaged intracellular Ca(2+) concentration within synaptic terminals of approximately 2 microM and lower (approximately 750 nM) intracellular calcium concentration in the ellipsoid. Measurements from axotomized cell bodies and isolated ellipsoids showed that Ca(2+) enters the two compartments via both local L-type Ca(2+) channels and diffusion. The results from optical imaging studies were supported by immunostaining analysis. L-type voltage-operated Ca(2+) channels and plasma membrane Ca(2+) ATPases were highly expressed in synaptic terminals with progressively lower expression levels in the cell body and ellipsoid. These results show photoreceptor Ca(2+) homeostasis is controlled in a region-specific manner by direct Ca(2+) entry and diffusion as well as Ca(2+) extrusion. Moreover, quantitative measurement of intracellular calcium concentration levels in different photoreceptor compartments indicates that the dynamic range of Ca(2+) signaling in photoreceptors is approximately 40-fold, from approximately 50 nM in the light to approximately 2 microM in darkness.
Asunto(s)
Señalización del Calcio/fisiología , Calcio/metabolismo , Espacio Extracelular/metabolismo , Células Fotorreceptoras/citología , Células Fotorreceptoras/metabolismo , Animales , Antimicina A/análogos & derivados , Antimicina A/farmacología , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio Tipo L/metabolismo , Señalización del Calcio/efectos de los fármacos , ATPasas Transportadoras de Calcio/metabolismo , Proteínas de Transporte de Catión/metabolismo , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Espacio Extracelular/efectos de los fármacos , Inmunohistoquímica/métodos , Modelos Biológicos , Nifedipino/farmacología , ATPasas Transportadoras de Calcio de la Membrana Plasmática , Potasio/farmacología , Tiempo de Reacción/efectos de los fármacos , Células Fotorreceptoras Retinianas Conos/efectos de los fármacos , Células Fotorreceptoras Retinianas Bastones/efectos de los fármacos , UrodelosRESUMEN
Ligand-gated ion channels (ionotropic receptors) link to the cortical cytoskeleton via specialized scaffold proteins and thereby to appropriate signal transduction pathways in the cell. We studied the role of filamentous actin in the regulation of Ca influx through glutamate receptor-activated channels in third-order neurons of salamander retina. Staining by Alexa-Fluor 488-phalloidin, to visualize polymerized actin, we show localization of filamentous actin in neurites, and the membrane surrounding the cell soma. With Ca(2+) imaging we found that in dissociated neurons, depolymerization of filamentous actin by latrunculin A, or cytochalasin D significantly reduced glutamate-induced intracellular Ca(2+) accumulation to 53+/-7% of control value. Jasplakinolide, a stabilizer of filamentous actin, by itself slightly increased the glutamate-induced Ca(2+) signal and completely attenuated the inhibitory effect when applied in combination with actin depolymerizing agents. These results indicate that in salamander retinal neurons the actin cytoskeleton regulates Ca(2+) influx through ionotropic glutamate receptor-activated channels, suggesting regulatory roles for filamentous actin in a number of Ca(2+)-dependent physiological and pathological processes.
Asunto(s)
Actinas/fisiología , Ambystoma/metabolismo , Calcio/metabolismo , Citoesqueleto/fisiología , Ácido Glutámico/toxicidad , Retina/metabolismo , Células Ganglionares de la Retina/metabolismo , Actinas/metabolismo , Animales , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Células Cultivadas , Citocalasina D/farmacología , Interpretación Estadística de Datos , Depsipéptidos/farmacología , Técnicas In Vitro , Microscopía Confocal , Receptores de Glutamato/efectos de los fármacos , Receptores de Glutamato/metabolismo , Retina/citología , Células Ganglionares de la Retina/efectos de los fármacos , Tiazoles/farmacología , TiazolidinasRESUMEN
A commercial variable-capacitance micromachined accelerometer was validated for muscle belly radial displacement measurement. The displacement was calculated by the acceleration data being integrated twice and was compared with the results obtained simultaneously by an accurate mechanical displacement sensor based on an optical encoder. The aim of the investigation was to evaluate the accuracy and precision of an accelerometer for tensiomyography, which is a method for the detection of skeletal muscle contractile properties on the basis of muscle belly radial displacement. A hundred measurements at a bandwidth of 2300 Hz were performed. It was shown that the accuracy and precision in determination of the maximum displacement and the time of the maximum displacement from the calculated curve were satisfactory, in spite of the standard deviation of the twice-integrated acceleration growing approximately linearly with time. The results were accurate enough since the elapsed time from the beginning of the integration was small (less than 75 ms). The measured maximum displacement ranges were between 9.2 and 10.2 mm. The mean relative error was less than 1% (SD = 0.02mm) for the maximum displacement and about 1% (SD = 0.6 ms) for the time to maximum displacement. The accuracy of the half-relaxation time determination was more uncertain because of the relatively high relative error of -2.4% (SD = 3 ms). Results showed that a commercial micromachined accelerometer could be suitable for the measurement of muscle belly radial displacement and used for development of a future miniaturised and flexible system for the measurement of similar displacements.
Asunto(s)
Contracción Muscular/fisiología , Músculo Esquelético/fisiología , Aceleración , Humanos , Miografía/instrumentación , Miografía/métodos , Reproducibilidad de los ResultadosRESUMEN
Synaptic transmission at the photoreceptor synapse is characterized by continuous release of glutamate in darkness. Release is regulated by the intracellular calcium concentration ([Ca2+]i). We here examined the physiological properties of exocytosis in tiger salamander (Ambystoma tigrinum) retinal rods and cones. Patch-clamp capacitance measurements were used to monitor exocytosis elicited by a rapid and uniform increase in [Ca2+]i by photolysis of the caged Ca2+ compound NP-EGTA. The amplitude of flash-induced increases in membrane capacitance (Cm) varied monotonically with [Ca2+]i beyond approximately 15 microM. The following two types of kinetic responses in Cm were recorded in both rods and cones: 1) a single exponential rise (39% of cells) or 2) a double-exponential rise (61%). Average rate constants of rapid and slow exocytotic responses were 420 +/- 168 and 7.85 +/- 5.02 s-1, respectively. The rate constant for the single exponential exocytotic response was 17.5 +/- 12.4 s-1, not significantly different from that of the slow exocytotic response. Beyond the threshold [Ca2+]i of approximately 15 microM, the average amplitude of rapid, slow, and single Cm response were 0.84 +/- 0.35, 0.82 +/- 0.20, and 0.70 +/- 0.23 pF, respectively. Antibodies against synaptotagmin I, a vesicle protein associated with fast exocytosis, strongly stained the synaptic terminal of isolated photoreceptors, suggesting the presence of fusion-competent vesicles. Our results confirm that photoreceptors possess a large rapidly releasable pool activated by a low-affinity Ca2+ sensor whose kinetic and calcium-dependent properties are similar to those reported in retinal bipolar cells and cochlear hair cells.
Asunto(s)
Proteínas de Unión al Calcio , Ácido Egtácico/análogos & derivados , Exocitosis , Ácido Glutámico/metabolismo , Células Fotorreceptoras Retinianas Conos/fisiología , Células Fotorreceptoras Retinianas Bastones/fisiología , Transmisión Sináptica , Animales , Calcio/metabolismo , Técnicas de Cultivo de Célula , Inmunohistoquímica , Glicoproteínas de Membrana/análisis , Microscopía Confocal , Proteínas del Tejido Nervioso/análisis , Técnicas de Placa-Clamp , Fotólisis , Terminales Presinápticos/fisiología , Retina/fisiología , Células Fotorreceptoras Retinianas Conos/química , Células Fotorreceptoras Retinianas Conos/metabolismo , Células Fotorreceptoras Retinianas Bastones/química , Células Fotorreceptoras Retinianas Bastones/metabolismo , Vesículas Sinápticas/fisiología , Sinaptotagmina I , Sinaptotagminas , UrodelosRESUMEN
The system N transporter SN1 has been proposed to mediate the efflux of glutamine from cells required to sustain the urea cycle and the glutamine-glutamate cycle that regenerates glutamate and gamma-aminobutyric acid (GABA) for synaptic release. We now show that SN1 also mediates an ionic conductance activated by glutamine, and this conductance is selective for H(+). Although SN1 couples amino acid uptake to H(+) exchange, the glutamine-gated H(+) conductance is not stoichiometrically coupled to transport. Protons thus permeate SN1 both coupled to and uncoupled from amino acid flux, providing novel mechanisms to regulate the transfer of glutamine between cells.
Asunto(s)
Sistemas de Transporte de Aminoácidos/metabolismo , Animales , Astrocitos/metabolismo , Ácido Glutámico/metabolismo , Glutamina/metabolismo , Activación del Canal Iónico , Protones , Xenopus , Ácido gamma-Aminobutírico/metabolismoRESUMEN
At least twice daily our retinas move between a light adapted, cone-dominated (photopic) state and a dark-adapted, color-blind and highly light-sensitive rod-dominated (scotopic) state. In between is a rather ill-defined transitional state called the mesopic state in which retinal circuits express both rod and cone signals. The mesopic state is characterized by its dynamic and fluid nature: the rod and cone signals flowing through retinal networks are continually changing. Consequently, in the mesopic state the retinal output to the brain contained in the firing patterns of the ganglion cells consists of information derived from both rod and cone signals. Morphology, physiology, and psychophysics all contributed to an understanding that the two systems are not independent but interact extensively via both pooling and mutual inhibition. This review lays down a rationale for such rod-cone interactions in the vertebrate retinas. It suggests that the important functional role of rod-cone interactions is that they shorten the duration of the mesopic state. As a result, the retina is maintained in either in the (rod-dominated) high sensitivity photon counting mode or in the second mode, which emphasizes temporal transients and spatial resolution (the cone-dominated photopic state). Experimental evidence for pre- and postsynaptic mixing of rod and cone signals in the retina of the clawed frog, Xenopus, is shown together with the preeminent neuromodulatory role of both light and dopamine in controlling interactions between rod and cone signals. Dopamine is shown to be both necessary and sufficient to mediate light adaptation in the amphibian retina.
Asunto(s)
Dopamina/fisiología , Células Fotorreceptoras Retinianas Conos/fisiología , Células Fotorreceptoras Retinianas Bastones/fisiología , Sinapsis/fisiología , Animales , Adaptación a la Oscuridad/fisiología , Uniones Comunicantes/fisiología , Uniones Comunicantes/ultraestructura , Terminales Presinápticos/fisiología , Terminales Presinápticos/ultraestructura , Células Fotorreceptoras Retinianas Conos/ultraestructura , Células Fotorreceptoras Retinianas Bastones/ultraestructura , Sinapsis/ultraestructura , XenopusRESUMEN
Brain-derived neurotrophic factor (BDNF) can potentiate synaptic release at newly developed frog neuromuscular junctions. Although this potentiation depends on extracellular Ca(2+) and reflects changes in acetylcholine release, little is known about the intracellular transduction or calcium signaling pathways. We have developed a video assay for neurotrophin-induced potentiation of myocyte twitching as a measure of potentiation of synaptic activity. We use this assay to show that BDNF-induced synaptic potentiation is not blocked by cadmium, indicating that Ca(2+) influx through voltage-gated Ca(2+) channels is not required. TrkB autophosphorylation is not blocked in Ca(2+)-free conditions, indicating that TrkB activity is not Ca(2+) dependent. Additionally, an inhibitor of phospholipase C interferes with BDNF-induced potentiation. These results suggest that activation of the TrkB receptor activates phospholipase C to initiate intracellular Ca(2+) release from stores which subsequently potentiates transmitter release.
Asunto(s)
Factor Neurotrófico Derivado del Encéfalo/farmacología , Calcio/metabolismo , Neuronas Motoras/fisiología , Fibras Musculares Esqueléticas/efectos de los fármacos , Fibras Musculares Esqueléticas/fisiología , Potenciales de Acción/fisiología , Animales , Cadmio/farmacología , Calcio/farmacocinética , Canales de Calcio/fisiología , Inhibidores Enzimáticos/farmacología , Estrenos/farmacología , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/fisiología , Indoles/farmacología , Contracción Muscular/efectos de los fármacos , Contracción Muscular/fisiología , Oocitos/fisiología , Células PC12 , Inhibidores de Fosfodiesterasa/farmacología , Fosforilación , Fosfotirosina/análisis , Pirrolidinonas/farmacología , Ratas , Receptor trkB/fisiología , Sinapsis/enzimología , Tapsigargina/farmacología , Fosfolipasas de Tipo C/antagonistas & inhibidores , Fosfolipasas de Tipo C/metabolismo , Tirosina/metabolismo , XenopusRESUMEN
We investigated the role of caffeine-sensitive intracellular stores in regulating intracellular calcium ([Ca(2+)](i)) and glutamatergic synaptic transmission from rod photoreceptors. Caffeine transiently elevated and then markedly depressed [Ca(2+)](i) to below prestimulus levels in rod inner segments and synaptic terminals. Concomitant with the depression was a reduction of glutamate release and a hyperpolarization of horizontal cells, neurons postsynaptic to rods. Caffeine did not affect the rods' membrane potentials indicating that caffeine likely acted via some mechanism(s) other than a voltage-dependent deactivation of the calcium channels. Most of caffeine's depressive action on [Ca(2+)](i), on glutamate release, and on I(Ca) in rods can be attributed to calcium release from stores: (1) caffeine's actions on [Ca(2+)](i) and I(Ca) were reduced by intracellular BAPTA and barium substitution for calcium, (2) other nonxanthine store-releasing compounds, such as thymol and chlorocresol, also depressed [Ca(2+)](i), and (3) the magnitude of [Ca(2+)](i) depression depended on basal [Ca(2+)](i) before caffeine. We propose that caffeine-released calcium reduces I(Ca) in rods by an as yet unidentified intracellular signaling mechanism. To account for the depression of [Ca(2+)](i) below rest levels and the increased fall rate of [Ca(2+)](i) with higher basal calcium, we also propose that caffeine-evoked calcium release from stores activates a calcium transporter that, via sequestration into stores or extrusion, lowers [Ca(2+)](i) and suppresses glutamate release. The effects of store-released calcium reported here operate at physiological calcium concentrations, supporting a role in regulating synaptic signaling in vivo.
Asunto(s)
Cafeína/farmacología , Calcio/fisiología , Terminales Presinápticos/fisiología , Células Fotorreceptoras Retinianas Bastones/fisiología , Transmisión Sináptica/fisiología , Ambystoma , Animales , Bario/farmacología , Canales de Calcio/fisiología , Quelantes/farmacología , Cresoles/farmacología , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Electrofisiología , Técnicas In Vitro , Cinética , Modelos Neurológicos , Terminales Presinápticos/efectos de los fármacos , Células Fotorreceptoras Retinianas Bastones/efectos de los fármacos , Rianodina/farmacología , Transmisión Sináptica/efectos de los fármacos , Timol/farmacología , Xenopus laevisRESUMEN
The amino acid glutamine has a central role in nitrogen metabolism. Although the molecular mechanisms responsible for its transport across cell membranes remain poorly understood, classical amino acid transport system N appears particularly important. Using intracellular pH measurements, we have now identified an orphan protein related to a vesicular neurotransmitter transporter as system N. Functional analysis shows that this protein (SN1) involves H+ exchange as well as Na+ cotransport and, under physiological conditions, mediates glutamine efflux as well as uptake. Together with the pattern of SN1 expression, these unusual properties suggest novel physiological roles for system N in nitrogen metabolism and synaptic transmission.
Asunto(s)
Sistemas de Transporte de Aminoácidos Neutros , Proteínas Portadoras/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana , Neurotransmisores/metabolismo , Nitrógeno/metabolismo , Transmisión Sináptica/fisiología , Secuencia de Aminoácidos , Animales , Astrocitos/metabolismo , Astrocitos/ultraestructura , Encéfalo/metabolismo , Encéfalo/ultraestructura , Proteínas Portadoras/genética , Línea Celular , Clonación Molecular , Glutamina/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Inmunohistoquímica , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Neurotransmisores/genética , Especificidad de Órganos , Ratas , Alineación de Secuencia , Sodio/metabolismo , Sinapsis/metabolismoRESUMEN
We studied the responses of rod photoreceptors that were elicited with light flashes or sinusoidally modulated light by using intracellular recording. Dark-adapted Xenopus rod photoreceptors responded to sinusoidally modulated green lights at temporal frequencies between 1 Hz and 4 Hz. In normal Ringer's solution, 57% of the rods tested could follow red lights that were matched for equal rod absorbance to frequencies >5 Hz, indicating an input from red-sensitive cones. Quinpirole (10 microM), a D2 dopamine agonist, increased rod-cone coupling, whereas spiperone (5 microM), a selective D2 antagonist, completely suppressed it. D1 dopamine ligands were without effect. Neurobiotin that was injected into single rods diffused into neighboring rods and cones in quinpirole-treated retinas but only diffused into rods in spiperone-treated retinas. A subpopulation of rods (ca. 10% total rods) received a very strong cone input, which quickened the kinetics of their responses to red flashes and greatly increased the bandpass of their responses to sinusoidally modulated light. Based on electron microscopic examination, which showed that rod-rod and cone-cone gap junctions are common, whereas rod-cone junctions are relatively rare, we postulate that cone signals enter the rod network through a minority of rods with strong cone connections, from which the cone signal is further distributed in the rod network. A semiquantitative model of coupling, based on measures of gap-junction size and distribution and estimates of their conductance and open times, provides support for this assumption. The same network would permit rod signals to reach cones.
Asunto(s)
Receptores de Dopamina D2/fisiología , Células Fotorreceptoras Retinianas Conos/fisiología , Células Fotorreceptoras Retinianas Bastones/fisiología , Xenopus/fisiología , Animales , Biotina/análogos & derivados , Agonistas de Dopamina/farmacología , Antagonistas de Dopamina/farmacología , Antagonistas de los Receptores de Dopamina D2 , Uniones Comunicantes/fisiología , Masculino , Microinyecciones , Microscopía Electrónica , Estimulación LuminosaRESUMEN
Differential localization of calcium channel subtypes in divergent regions of individual neurons strongly suggests that calcium signaling and regulation could be compartmentalized. Region-specific expression of calcium extrusion transporters would serve also to partition calcium regulation within single cells. Little is known about selective localization of the calcium extrusion transporters, nor has compartmentalized calcium regulation within single neurons been studied in detail. Sensory neurons provide an experimentally tractable preparation to investigate this functional compartmentalization. We studied calcium regulation in the outer segment (OS) and inner segment/synaptic terminal (IS/ST) regions of rods and cones. We report these areas can function as separate compartments. Moreover, ionic, pharmacological, and immunolocalization results show that a Ca-ATPase, but not the Na+/K+, Ca2+ exchanger found in the OSs, extrudes calcium from the IS/ST region. The compartmentalization of calcium regulation in the photoreceptor outer and inner segments implies that transduction and synaptic signaling can be independently controlled. Similar separation of calcium-dependent functions is likely to apply in many types of neuron.
Asunto(s)
Calcio/metabolismo , Células Fotorreceptoras Retinianas Bastones/metabolismo , Segmento Externo de la Célula en Bastón/metabolismo , Animales , ATPasas Transportadoras de Calcio/metabolismo , Calmodulina/antagonistas & inhibidores , Membrana Celular/enzimología , Concentración de Iones de Hidrógeno , Membranas Intracelulares/metabolismo , Lantano/farmacología , Intercambiador de Sodio-Calcio/metabolismo , Distribución Tisular , UrodelosRESUMEN
We related rod to horizontal cell synaptic transfer to glutamate release by rods. Simultaneous intracellular records were obtained from dark-adapted rod-horizontal cell pairs. Steady-state synaptic gain (defined as the ratio of horizontal cell voltage to rod voltage evoked by the same light stimulus) was 3.35 +/- 0.60 for dim flashes and 1.50 +/- 0.03 for bright flashes. Under conditions of maintained illumination, there was a measurable increment of horizontal cell hyperpolarization for each light-induced increment of rod hyperpolarization over the full range of rod voltages. In separate experiments we studied glutamate release from an intact, light-responsive photoreceptor layer, from which inner retinal layers were removed. Steady light reduced glutamate release as a monotonic function of intensity; spectral sensitivity measures indicated that we monitored glutamate release from rods. The dependence of glutamate release on rod voltage was well fit by the activation function for a high-voltage-activated, dihydropyridine-sensitive L-type calcium current, suggesting a linear dependence of glutamate release on [Ca]i in the synaptic terminal. A simple model incorporating this assumption accounts for the steady-state gain of the rod to horizontal cell synapse.
Asunto(s)
Calcio/fisiología , Dihidropiridinas/farmacología , Ácido Glutámico/metabolismo , Células Fotorreceptoras/fisiología , Células Fotorreceptoras Retinianas Bastones/fisiología , Sinapsis/fisiología , Animales , Conductividad Eléctrica , Luz , Masculino , Modelos Neurológicos , Células Fotorreceptoras Retinianas Bastones/metabolismo , Células Fotorreceptoras Retinianas Bastones/efectos de la radiación , Xenopus laevisRESUMEN
We examined the contribution of two intrinsic voltage-dependent calcium channels to the light-evoked responses of a non-spiking retinal neuron, the horizontal cell (HC). HC's isolated from the Xenopus retina were studied by the whole cell version of the patch clamp. In a mixture of agents which suppressed Na- and K-dependent currents, we identified a transient, low voltage-activated Ca current suppressed by Ba2+ and blocked by Ni2+ (T-type) and a sustained, high voltage-activated, dihydropyridine-sensitive Ca current that was enhanced by Ba2+ (L-type). We made simultaneous intracellular recordings from rods and HC's in the intact, dark-adapted Xenopus retina. Under certain stimulus conditions, transient oscillations appeared in HC responses but were absent in rod light-evoked waveforms. One type of transient was seen at relatively hyperpolarized potentials (< -45 mV), was enhanced by Sr2+ and inhibited by Ni2+. It thus appears to depend on a T-type Ca-current. A second type of oscillation was seen to be superimposed on a prolonged depolarizing wave following light off in the HC and as spike-like depolarizations in rods. These oscillations were enhanced by Ba2+ and Sr2+, but blocked by the dihydropyridine, nifedipine, indicating their dependence on an L-type calcium conductance. All calcium-dependent oscillations were suppressed by 0.05-0.5 mM Co2+. Suppression of glutamate neurotransmission with CNQX or kynurenate, or glycine neurotransmission with strychnine, enhanced the HC oscillations.
Asunto(s)
Adaptación Ocular/fisiología , Canales de Calcio/fisiología , Calcio/metabolismo , Activación del Canal Iónico/fisiología , Retina/citología , 6-Ciano 7-nitroquinoxalina 2,3-diona/farmacología , Animales , Electrofisiología , Antagonistas de Aminoácidos Excitadores/farmacología , Ácido Glutámico/farmacología , Glicina/farmacología , Glicinérgicos/farmacología , Activación del Canal Iónico/efectos de los fármacos , Ligandos , Masculino , Potenciales de la Membrana/fisiología , Periodicidad , Estimulación Luminosa , Retina/química , Retina/fisiología , Estimulación Química , Estricnina/farmacología , Xenopus laevisRESUMEN
1. Water compartmentalization in the turtle cerebellum subject to media of different osmolalities was quantified by combining extracellular diffusion analysis with wet weight and dry weight measurements. The diffusion analysis also determined the tortuosity of the extracellular space. 2. Isolated cerebella were immersed in normal, oxygenated physiological saline (302 mosmol kg-1), hypotonic saline (238 mosmol kg-1) and a series of hypertonic salines (up to 668 mosmol kg-1). The osmolality was varied by altering the NaCl content. 3. Extracellular volume fraction and tortuosity of the granular layer of the cerebellum were determined from measurements of ionophoretically induced diffusion profiles of tetramethylammonium, using ion-selective microelectrodes. The volume fraction was 0.22 in normal saline, 0.12 in hypotonic medium and 0.60 in the most hypertonic medium. Tortuosity was 1.70 in the normal saline, 1.79 in the hypotonic and 1.50 in the most hypertonic saline. 4. The water content, defined as (wet weight-dry weight)/wet weight, of a typical isolated cerebellum (including granular, Purkinje cell and molecular layers) was 82.9%. It increased to 85.2% in hypotonic saline and decreased to 80.1% in the most hypertonic saline. 5. Measurements of extracellular volume fraction and water content were combined to show that hypotonic solutions caused water to move from the extracellular to the intracellular compartment while hypertonic solutions caused water to move from the intracellular to extracellular compartment, with only a relatively small changes in total water in both cases. 6. These results suggest the use of the isolated turtle cerebellum as a model system for studying light scattering or diffusion-weighted magnetic resonance imaging.
Asunto(s)
Cerebelo/metabolismo , Espacio Extracelular/metabolismo , Presión Osmótica , Tortugas/metabolismo , Agua/metabolismo , Animales , Compartimentos de Líquidos Corporales , Cerebelo/efectos de los fármacos , Difusión , Potenciales Evocados , Espacio Extracelular/efectos de los fármacos , Técnicas In Vitro , Imagen por Resonancia Magnética , Microelectrodos , Modelos Biológicos , Concentración Osmolar , Presión Osmótica/efectos de los fármacos , Compuestos de Amonio Cuaternario/farmacología , Dispersión de Radiación , Cloruro de Sodio/farmacologíaRESUMEN
It has been proposed that the depolarizing responses of chromaticity horizontal cells (C-HCs) to red light depend on a feedback signal from luminosity horizontal cells (L-HCs) to short-wavelength-sensitive cones in the retinas of lower vertebrates. In this regard we studied the C-HCs of the Xenopus retina. C-HCs and L-HCs were identified by physiological criteria and then injected with neurobiotin. The retina then was incubated with peanut agglutinin, which stains red-but not blue-sensitive cones. Electron microscopic examination revealed that L-HCs contact all cone classes, whereas C-HCs contact only blue-sensitive cones. Simultaneous recordings from C-HC/L-HC pairs established that when the L-HC was saturated by a steady bright red light, C-HCs alone responded to a superimposed blue stimulus. In response to red test flashes, the C-HC response was delayed by approximately 30 msec with respect to the L-HC response. Isolated HCs of both subtypes were examined by whole-cell patch clamp. Both responded to kainate with sustained inward currents and to quisqualate or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) with desensitizing currents from a negative holding potential; i.e., both have AMPA-type glutamate receptors. gamma-Aminobutyric acid or glycine opened a chloride channel in the L-HC, whereas the C-HC was unresponsive to either inhibitory amino acid. Since glycine has been shown to abolish selectively the depolarizing response of the C-HC, this finding and other pharmacological data strongly implicate the L-HC in the underlying circuit. Moreover, because the C-HC does not respond to gamma-aminobutyric acid, the neurotransmitter of the L-HC, by elimination, a feedback synapse from L-HC to blue cone is the most plausible mechanism for the creation of depolarizing responses in C-HCs.
Asunto(s)
Comunicación Celular/fisiología , Potenciales Evocados Visuales/fisiología , Neuronas/fisiología , Retina/citología , Retina/fisiología , Animales , Cobalto/farmacología , Potenciales Evocados Visuales/efectos de la radiación , Retroalimentación , Glicina/farmacología , Luz , Masculino , Neuronas/efectos de la radiación , Técnicas de Placa-Clamp , Receptores de Glutamato/metabolismo , Retina/efectos de la radiación , Células Fotorreceptoras Retinianas Conos/citología , Xenopus , Ácido gamma-Aminobutírico/farmacologíaRESUMEN
We studied the responses of isolated and intact luminosity-type horizontal cells (L-HC) in the Xenopus retina to L-glutamate (L-glu) and its analogs. Isolated L-HCs studied with whole-cell patch clamp responded to L-glu, kainate (KA), AMPA, or quisqualate (quis) with inward currents from a holding potential of -60 mV, associated with a conductance increase. The current elicited by KA was relatively large and sustained, whereas AMPA or quis evoked a desensitizing current. Coapplication of quis and KA resulted in a smaller current and conductance change than that evoked by a pulse of either alone at the same concentration. This finding suggests that the L-HC has a single subtype of glutamate receptor that responds to both quis and KA. Prior exposure to dopamine enhanced the KA-evoked current about twofold. In the superfused eyecup we found that L-HC responses to quinoxalinediones (CNQX or DNQX) and to L-glu, KA, AMPA, and quis varied as a function of adaptational state. When driven exclusively by either cones or by rods, CNQX/DNQX hyperpolarized the L-HC and reduced its light response, without altering response kinetics, indicating that both rods and cones communicate with L-HCs at ionotropic glutamatergic synapses. Under mesopic conditions, however, as CNQX or DNQX reduced cone input, the rod input to the L-HC increased up to fivefold in magnitude and had slowed kinetics. The depolarizing response of the L-HC to L-glu, AMPA, or quis was relatively small and transient under photopic conditions, but was much larger and sustained when the eyecup was dark adapted. The D1 dopamine antagonist SCH 23390 potentiated the response to quis. In contrast, responses to KA were largest in light-adapted eyecups, were potentiated by a D1 dopamine agonist, SKF 38393, and were reduced by SCH 23390. We hypothesize that the segregated populations of glutamate receptors in the L-HC opposite cone and rod synaptic endings can be separately modulated to respond differentially to the native transmitter, glutamate. In photopic and mesopic states the dominant cone input tonically inhibits rod to L-HC communication. This inhibition appears to occur at the postsynaptic membrane and may be mediated by second messengers.