RESUMEN
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
Visual deprivation impedes refinement of neuronal function in higher visual centers of mammals. It is often assumed that visual deprivation has minimal effect, if any, on neuronal function in retina. Here we report that dark rearing reduces the light-evoked responsiveness of inner retinal neurons in young mice. We also find that 1 to 2 weeks after eye opening, there is a surge (>4-fold) in the frequency of spontaneous excitatory and inhibitory synaptic events in ganglion cells. Dark rearing reversibly suppresses this surge, but recovery takes >6 days. Frequency changes are not accompanied by amplitude changes, indicating that synaptic reorganization is likely to be presynaptic. These findings indicate there is a degree of activity-dependent plasticity in the mammalian retina that has not been previously described.
Asunto(s)
Retina/crecimiento & desarrollo , Privación Sensorial/fisiología , Sinapsis/fisiología , Visión Ocular/fisiología , Animales , Oscuridad , Potenciales Evocados Visuales/fisiología , Ojo/crecimiento & desarrollo , Ratones , Ratones Endogámicos C57BL , Retina/fisiología , Células Ganglionares de la Retina/fisiologíaRESUMEN
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
Ganglion cells responding only transiently to changes in illumination are found in many different vertebrate retinas. The interactions underlying formation of these transient responses are still poorly understood. Two recently proposed hypotheses are (1) functional inhibitory pathways are necessary for transient response production, and (2) direct inhibition of the ganglion cell has little effect on its output. Here, we examine these conclusions by using cell-attached patch-clamp recordings of spiking, whole-cell recordings of synaptic currents, and computer modeling. We found that picrotoxin (a GABA(A) and GABA(C) receptor antagonist), bicuculline (a GABA(A) receptor antagonist), and strychnine (a glycine receptor antagonist), applied either singly or in combination, always failed to convert transient responses to sustained responses. Application of the GABA(B) antagonist CGP35348 in the presence of picrotoxin and strychnine also failed to convert transient responses into sustained responses. Whole-cell recordings of synaptic currents at various holding potentials indicated that direct inhibitory inputs to ganglion cells limit the duration of net excitation, implying that direct inhibition does act to truncate the ganglion cell spiking response. Computer simulations using spiking and synaptic data from combined cell-attached and whole-cell recordings supported this interpretation. We conclude that inhibitory pathways are not required for generation of transient responses, but these pathways do serve to modulate transient ganglion cell spiking responses. We find that this modulation occurs, in part, via inhibitory inputs directly to the ganglion cell.
Asunto(s)
Potenciales de Acción/fisiología , Ambystoma/fisiología , Células Ganglionares de la Retina/fisiología , Potenciales de Acción/efectos de los fármacos , Animales , Electrofisiología , Antagonistas del GABA/farmacología , Técnicas de Placa-Clamp , Estimulación Luminosa , Receptores de GABA/fisiología , Receptores de Glicina/antagonistas & inhibidores , Células Ganglionares de la Retina/efectos de los fármacos , Estricnina/farmacología , Ácido gamma-Aminobutírico/fisiologíaRESUMEN
The zebrafish has recently joined the ranks of Drosophila and C. elegans as a tractable model for genetic screens (Fishman, 1999). Zebrafish grow fast, can be kept in large numbers in a small space, and are efficiently mutagenized and screened. Genomic resources are made available at an increasing pace. These days, a mutation can be mapped and cloned in a matter of months. Because a mutant hunt is intrinsically unbiased in terms of the classes of genes that will be tagged, it holds the unique potential to discover novel genes or, in our era of genome sequencing, to identify novel functions for known genes. Zebrafish display dozens of innate behaviours in response to light, of which the optomotor and the optokinetic responses are the most widely studied (Brockerhoff et al. 1995; Easter & Nicola, 1996). Their retinae are crisply layered following the typical vertebrate pattern, and the retinal layers are tiled in an almost crystalline fashion by mosaics of different cell types. Electroretinograms are recorded routinely and therefore, not surprisingly, zebrafish are now also being used for a genetic approach to the visual system.
Asunto(s)
Retina/fisiología , Pez Cebra/genética , Pez Cebra/fisiología , Animales , VertebradosRESUMEN
The processing of signals by integrative neurons in the retina and CNS relies strongly on inhibitory synaptic inputs, principally from GABAergic and glycinergic neurons that serve primarily to hyperpolarize postsynaptic neurons. Recent evidence indicates that the neuron-specific K-Cl cotransporter 2 (KCC2) is the major chloride extrusion system permitting hyperpolarizing inhibitory responses. It has been hypothesized that depolarizing GABA responses observed in immature neurons are converted to hyperpolarizing responses in large part by the expression of KCC2 during the second week of postnatal development. The cell-specific localization and developmental expression of KCC2 protein have been examined in relatively few neural tissues and have never been studied in retina, of which much is known physiologically and morphologically about inhibitory synaptic circuits. We examined the localization of KCC2 in adult rat retina with immunohistochemical techniques and determined the time course of its postnatal expression. KCC2 expression was localized in horizontal cells, bipolar cells, amacrine cells, and, most likely, ganglion cells, all of which are known to express GABA receptor subtypes. Developmentally, KCC2 expression in the retina increased gradually from postnatal day 1 (P1) until P14 in the inner retina, whereas expression was delayed in the outer plexiform layer until P7 but reached its adult level by P14. These data support the hypothesis that the function of KCC2 is intimately involved in GABAergic synaptic processing. Furthermore, the delayed temporal expression of KCC2 in the outer plexiform layer indicates that GABAergic function may be differentially regulated in retina during postnatal development and that GABA may produce depolarizing responses in the outer plexiform layer at times when it generates hyperpolarizing responses in the inner plexiform layer.
Asunto(s)
Envejecimiento/metabolismo , Proteínas Portadoras/genética , Regulación del Desarrollo de la Expresión Génica , Retina/metabolismo , Simportadores , Animales , Animales Recién Nacidos , Proteínas Portadoras/análisis , Cloruros/metabolismo , Inmunohistoquímica , Neuronas/citología , Neuronas/metabolismo , Potasio/metabolismo , Ratas , Ratas Long-Evans , Receptores de GABA/análisis , Receptores de Glicina/análisis , Retina/citología , Retina/crecimiento & desarrollo , Cotransportadores de K ClRESUMEN
Rapid and reliable detection of randomly occurring small amplitude synaptic events resulting from activation of different classes of ligand-gated receptors is a difficult task. Here, we describe and characterize an amplitude threshold algorithm, written as an IGOR Pro procedure, which detects events as well as characterizes their amplitudes and kinetics. The program was developed to analyze recording traces that each contained both excitatory (glutamate-mediated) and inhibitory (GABA and glycine-mediated) events. By using differences in kinetics, the program could discriminate between the two different classes of events. In summary, the program has the following strengths: (1) it is generally applicable to circumstances in which different populations of elementary events occur concurrently, a drawback of methods that employ matched filtering techniques, (2) it is relatively insensitive to drifts in baseline, and (3) it generates user-accessible arrays of the timing, amplitude and kinetic parameters of the detected events, making customized statistical analysis of event characteristics easily executable.
Asunto(s)
Potenciales Postsinápticos Excitadores/fisiología , Células Ganglionares de la Retina/fisiología , Diseño de Software , Transmisión Sináptica/fisiología , Animales , RatonesRESUMEN
We have studied GABAergic synaptic transmission in retinal ganglion cells and hippocampal pyramidal cells to determine, at a cellular level, what is the effect of the targeted disruption of the gene encoding the synthetic enzyme GAD65 on the synaptic release of gamma-aminobutyric acid (GABA). Neither the size nor the frequency of GABA-mediated spontaneous inhibitory postsynaptic currents (IPSCs) were reduced in retina or hippocampus in GAD65-/- mice. However, the release of GABA during sustained synaptic activation was substantially reduced. In the retina both electrical- and K(+)-induced increases in IPSC frequency were depressed without a change in IPSC amplitude. In the hippocampus the transient increase in the probability of inhibitory transmitter release associated with posttetanic potentiation was absent in the GAD65-/- mice. These results indicate that during and immediately after sustained stimulation the increase in the probability of transmitter release is not maintained in GAD65-/- mice. Such a finding suggests a decrease in the size or refilling kinetics of the releasable pool of vesicles, and various mechanisms are discussed that could account for such a defect.
Asunto(s)
Glutamato Descarboxilasa/fisiología , Isoenzimas/fisiología , Neuronas/metabolismo , Ácido gamma-Aminobutírico/metabolismo , Animales , Técnicas In Vitro , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Neuronas/enzimología , Sinapsis/enzimología , Sinapsis/metabolismoRESUMEN
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
Although most CNS neurons require sodium action potentials (Na-APs) for normal stimulus-evoked release of classical neurotransmitters, many types of retinal and other sensory neurons instead use only graded potentials for neurotransmitter release. The physiological properties and information processing capacity of Na-AP-producing neurons appear significantly different from those of graded potential neurons. To classify amacrine cells in this dichotomy, we investigated whether Na-APs, which are often observed in these cells, are required for functional light-evoked release of inhibitory neurotransmitters from these cells. We recorded light-evoked inhibitory postsynaptic currents (IPSCs) from retinal ganglion cells, neurons directly postsynaptic to amacrine cells, and applied TTX to block Na-APs. In control solution, TTX application always led to partial suppression of the light-evoked IPSC. To isolate release from glycinergic amacrine cells, we used either bicuculline, a GABAA receptor antagonist, or picrotoxin, a GABAA and GABAC receptor antagonist. TTX application only partially suppressed the glycinergic IPSC. To isolate release from GABAergic amacrine cells, we used the glycine receptor blocker strychnine. TTX application only partially suppressed the light-evoked GABAergic IPSC. Glycinergic and GABAergic amacrine cells did not obviously differ in the usage of Na-APs for release. These observations, in conjunction with previous studies of other retinal neurons, indicate that amacrine cells, taken as a class, are the only type of retinal neuron that uses both Na-AP-dependent and -independent modes for light-evoked release of neurotransmitters. These results also provide evidence for another parallel between the properties of retinal amacrine cells and olfactory bulb granule cells.
Asunto(s)
Glicina/metabolismo , Retina/metabolismo , Canales de Sodio/fisiología , Ácido gamma-Aminobutírico/metabolismo , Potenciales de Acción/fisiología , Ambystoma , Animales , Bicuculina/farmacología , Antagonistas del GABA/farmacología , Glicinérgicos/farmacología , Técnicas In Vitro , Estimulación Luminosa , Picrotoxina/farmacología , Retina/citología , Bloqueadores de los Canales de Sodio , Estricnina/farmacología , Tetrodotoxina/farmacologíaRESUMEN
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
Spontaneous inhibitory and excitatory postsynaptic currents (sIPSCs and sEPSCs) were identified and characterized with whole cell and perforated patch voltage-clamp recordings in adult mouse retinal ganglion cells. Pharmacological dissection revealed that all cells were driven by spontaneous synaptic inputs mediated by glutamate and gamma-aminobutyric acid-A (GABAA) receptors. One-half (7/14) of the cells also received glycinergic spontaneous synaptic inputs. Both GABAA and glycine receptor-mediated sIPSCs had rise times (10-90%) of < 1 ms. The decay times of the GABAA receptor-mediated sIPSCs were comparable with those of the glycine receptor-mediated sIPSCs. The average decay time constant for monoexponentially fitted sIPSCs was 63.2 +/- 74.1 ms (mean +/- SD, n = 3278). Glutamate receptor-mediated sEPSCs had an average rise time of 0.50 +/- 0.20 ms (n = 109) and an average monoexponential decay time constant of 5.9 +/- 8.6 ms (n = 2705). Slightly more than two-thirds of the spontaneous synaptic events were monoexponential (68% for sIPSCs and 76% for sEPSCs). The remainder of the events was biexponential. The amplitudes of the spontaneous synaptic events were not correlated with rise times, suggesting that the electrotonic filtering properties of the neurons and/or differences in the spatial location of synaptic inputs could not account for the difference between the decay time constants of the glutamate and GABAA/glycine receptor-mediated spontaneous synaptic events. The amplitudes of sEPSCs were similar to those recorded in tetrodotoxin (TTX), consistent with the events measured in control saline being the response to the release of a single quantum of transmitter. The range of the sIPSC amplitudes in control saline was wider than that recorded in TTX, consistent with some sIPSCs being evoked by presynaptic spikes having an average quantal size greater than one. The rates of sIPSCs and sEPSCs were determined under equivalent conditions by recording with perforated patch electrodes at potentials at which both types of event could be identified. Two groups of ganglion cell were observed; one group had an average sEPSCs/sIPSCs frequency ratio of 0.96 +/- 0.77 (n = 28) and another group had an average ratio of 6.63 +/- 0.82 (n = 7). These findings suggest that a subset of cells is driven much more strongly by excitatory synaptic inputs. We propose that this subset of cells could be OFF ganglion cells, consistent with the higher frequency of spontaneous action potentials found in OFF ganglion cells in other studies.
Asunto(s)
Potenciales Postsinápticos Excitadores/fisiología , Células Ganglionares de la Retina/fisiología , Transmisión Sináptica/fisiología , 2-Amino-5-fosfonovalerato/análogos & derivados , 2-Amino-5-fosfonovalerato/farmacología , 6-Ciano 7-nitroquinoxalina 2,3-diona/farmacología , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Factores de Edad , Animales , Bicuculina/farmacología , Cadmio/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Antagonistas del GABA/farmacología , Glicinérgicos/farmacología , Cinética , Ratones , Ratones Endogámicos C57BL , Inhibición Neural/efectos de los fármacos , Inhibición Neural/fisiología , Tiempo de Reacción/fisiología , Receptores de GABA-A/fisiología , Receptores de Glicina/fisiología , Células Ganglionares de la Retina/química , Estricnina/farmacología , Transmisión Sináptica/efectos de los fármacos , Tetrodotoxina/farmacología , Factores de Virulencia de Bordetella/farmacologíaRESUMEN
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
Glutamate, the neurotransmitter released by photoreceptors, excites horizontal cells and OFF-type bipolar cells by activating ionotropic receptors. This study investigated an additional action of glutamate in which it modulates a voltage-gated ion channel in horizontal cells. We find that glutamate and APB (2-amino-4-phosphonobutyrate) produce a delayed and moderately prolonged suppression of an inward rectifier current (IRK+). This effect is proposed to occur via an APB-sensitive metabotropic glutamate receptor (mGluR) because common agonists for the ionotropic or APB-insensitive mGluRs are ineffective and the APB-insensitive receptor antagonist alpha-methyl-4-carboxyphenylglycine (MCPG) does not block the actions of glutamate or APB. 8-Br-cGMP, 1-methyl-3-isobutylxanthine (IBMX), and atrial natriuretic peptide (ANP) but not 8-Br-cAMP mimic the suppression of IRK+. The effects of glutamate and APB are blocked by protein kinase inhibitors including Rp-8-pCPT-cGMPS, H-8, and H-7 as well as by ATPgammaS. We hypothesize that the APB receptor suppresses IRK+ via upregulation of cGMP and subsequent activation of a cGMP-dependent protein kinase. This pathway is likely regulated by an ATP-dependent phosphorylation. This is a novel signaling pathway for mGluRs and indicates that at least two distinct APB-activated pathways exist in the retina. Functionally, this APB receptor-mediated action found in horizontal cells would provide a means by which spatially restricted changes of glutamate, produced by local illumination of photoreceptors, could regulate IRK+ and consequently the response properties of these neurons. This would serve to adapt selectively retinal regions stimulated by small regions of the visual world.
Asunto(s)
GMP Cíclico/fisiología , Activación del Canal Iónico/efectos de los fármacos , Canales de Potasio/fisiología , Receptores de Glutamato Metabotrópico/fisiología , Sistemas de Mensajero Secundario/fisiología , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacología , 1-Metil-3-Isobutilxantina/farmacología , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/farmacología , Aminobutiratos/farmacología , Animales , Factor Natriurético Atrial/farmacología , Benzoatos/farmacología , GMP Cíclico/análogos & derivados , GMP Cíclico/farmacología , Inhibidores Enzimáticos/farmacología , Ácido Glutámico/farmacología , Glicina/análogos & derivados , Glicina/farmacología , Ictaluridae , Isoquinolinas/farmacología , Modelos Neurológicos , Técnicas de Placa-Clamp , Células Fotorreceptoras/efectos de los fármacos , Células Fotorreceptoras/fisiología , Canales de Potasio/efectos de los fármacos , Tionucleótidos/farmacologíaRESUMEN
Quinine increases the conductance of hemi-gap junctions in horizontal cells. We investigated the mechanisms of alkalinization and the hypothesis that quinine-induced alkalinization produced these conductance increases. We found that quinine-induced alkalinizations were not blocked by cobalt, amiloride, or DIDS. Therefore, this suggests that the alkalinization is not likely due to net proton flux through opened hemi-gap channels nor is it likely due to an action on Cl-/HCO3- exchanger or Na+/H+ exchanger, both of which are known to regulate pHi in the horizontal cells. Quinine increased hemi-gap conductance even when cells were recorded with patch pipets containing up to 80 mM HEPES. We conclude that quinine-induced alkalinization cannot account solely for the hemi-gap junctional conductance increases.
Asunto(s)
Bagres/fisiología , Uniones Comunicantes/efectos de los fármacos , Quinina/farmacología , Retina/efectos de los fármacos , Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico/farmacología , Amilorida/farmacología , Animales , Cobalto/farmacología , Conductividad Eléctrica , Electrofisiología , Uniones Comunicantes/fisiología , Concentración de Iones de Hidrógeno/efectos de los fármacos , Técnicas de Placa-Clamp , Retina/citología , Retina/fisiologíaRESUMEN
Propagated waves of excitation in developing neural tissues may be a critical feature of maturation. Recent findings shed new light on the mechanisms underlying these waves.
Asunto(s)
Retina/fisiología , Transducción de Señal/fisiología , Animales , Retina/citologíaRESUMEN
The passive electrical properties of 17 ON-OFF retinal ganglion cells were derived from electrophysiological recordings. The parameters for each cells' equivalent model were obtained from the transient current responses to small step changes in clamp potential. Thirteen of the cells could be adequately approximated by a spherical soma connected to an equivalent dendritic cable. Estimates for the cell input conductance (GN), membrane time constant (tau m), the dendritic-to-soma conductance ratio (rho), and the normalized electrotonic length (L) were obtained (mean +/- standard deviation, n = 13): GN = 580 +/- 530 pS, tau m = 97 +/- 72 ms, rho = 2.8 +/- 2.8, and L = 0.34 +/- 0.13. Series resistance averaged 32 +/- 11 M omega. The mean of the derived soma diameters was 18 +/- 6 microns and the mean diameter and length of the equivalent cables were 1.4 +/- 0.6 and 470 +/- 90 microns, respectively. The average of the specific membrane conductances, 1.67 +/- 1.08 S/cm2, corresponded to a membrane resistivity of 60 k omega. cm2. Computer simulations of synaptic inputs were performed on a representative model, with an electrode at the soma and using the worst-case configuration, in which all synaptic inputs were confined to the tips of the dendrites. We draw three conclusions from the modeling: (1) Under voltage clamp, fast spontaneous EPSCs would be significantly attenuated and slowed while the time course of the slower, light-evoked non-NMDA and NMDA EPSCs would be minimally distorted by dendritic filtering. (2) Excitatory synaptic reversal potentials can be accurately determined under voltage clamp. (3) In the absence of GABAergic and glycinergic inhibition, the efficacy at the soma of excitatory conductance changes is essentially independent of their dendritic location. The specific membrane resistivity appears to represent a good compromise between having a small membrane time constant and minimal EPSP attenuation.
Asunto(s)
Células Ganglionares de la Retina/fisiología , Sinapsis/fisiología , Animales , Electrofisiología , Agonistas de Aminoácidos Excitadores/farmacología , Potenciales de la Membrana/fisiología , N-Metilaspartato/farmacología , Técnicas de Placa-Clamp , Células Ganglionares de la Retina/efectos de los fármacos , UrodelosRESUMEN
Recent studies have revealed that excitation of specific nerve pathways can produce localized changes of pH in nervous tissue. It is important to determine both how these pH changes are generated and, even more importantly, how the excitability of neurons in the localized areas are affected. Evidence indicates that activation of both gamma-aminobutyric acid (GABA) and L-glutamate receptor channels in inhibitory and excitatory pathways, respectively, can raise extracellular pH (pHo) and lower intracellular pH (pHi). At the target location, it has been shown that several types of voltage-gated ion channels in neurons were modified by a change in pHi. These studies, taken together, enable us to hypothesize that intracellular hydrogen ions (H+) might function as neuromodulatory factors, like other types of intracellular second messengers. This hypothesis was tested by using horizontal cells enzymatically dissociated from catfish retina. We found that the high-voltage-activated (HVA) Ca2+ current, inward rectifier K+ current and hemi-gap junctional current are modulated by a change in intracellular H+ concentration, and that L-glutamate suppresses the HVA Ca2+ current by raising the intracellular H+ concentration. These observations support the hypothesis that intracellular H+, acting as a second messenger, governs neuronal excitability via modulation of ionic channel activity. This article reviews recent studies of ours and others on the effect of pHi upon neuronal function.
Asunto(s)
Neuronas/fisiología , Animales , Humanos , Concentración de Iones de Hidrógeno , Canales Iónicos/fisiologíaRESUMEN
1. Light-evoked input-output characteristics of ganglion cells in dark-adapted tiger salamander retina were studied in the slice preparation using patch-clamp techniques. Excitatory postsynaptic currents (EPSCs), isolated by blocking inhibitory inputs and evoked by a range of light stimulus intensities, were recorded under whole-cell voltage clamp. Spike responses, evoked by the same light intensities, were recorded extracellularly from the same cells using the cell-attached patch-clamp technique. 2. When N-methyl-D-aspartate (NMDA) receptor-mediated input was blocked by the competitive NMDA antagonist DL-2-amino-5-phosphonoheptanoate (AP7), light-evoked EPSC amplitude and peak firing rate were reduced at all light intensities. In both cases, the data obtained in the presence of AP7 scaled linearly to control data, indicating that NMDA and non-NMDA receptors are activated in the same proportions across the entire 2 log unit stimulus response range of these ganglion cells. 3. The relationship between light-evoked spike frequency and light-evoked EPSC amplitude was linear. The slope of the light-evoked synaptic current-spike frequency relationship was close to the slope of the injected current-spike frequency relationship, indicating that synaptic current and injected current drive spiking in a similar manner. The linearity of the synaptic current-spike frequency relationship was not compromised when NMDA input was blocked by AP7. 4. Light-evoked voltage responses, recorded under whole-cell current clamp, revealed that the average membrane potential during a spike response was depolarized only slightly with increased firing rate. Once the membrane potential surpassed spike threshold, it was maintained by the voltage-gated, spike-generating conductances at a depolarized plateau upon which action potentials were fired. The potential of this plateau varied only slightly with spike frequency. We conclude that the voltage control exerted by the spike-generating currents in ganglion cells prevents a substantial response-dependent decrease in the electrical driving force of the excitatory currents, obviating the need for the voltage-independent synaptic efficacy provided by the combination of NMDA and non-NMDA inputs.