RESUMEN
The current-passing pore of mammalian hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels is formed by subunit isoforms denoted HCN1-4. In various brain areas, antibodies directed against multiple isoforms bind to single neurons, and the current (I(h)) passed during hyperpolarizations differs from that of heterologously expressed homomeric channels. By contrast, retinal rod, cone, and bipolar cells appear to use homomeric HCN channels. Here, we assess the generality of this pattern by examining HCN1 and HCN4 immunoreactivity in rat retinal ganglion cells, measuring I(h) in dissociated cells, and testing whether HCN1 and HCN4 proteins coimmunoprecipitate. Nearly half of the ganglion cells in whole-mounted retinae bound antibodies against both isoforms. Consistent with colocalization and physical association, 8-bromo-cAMP shifted the voltage sensitivity of I(h) less than that of HCN4 channels and more than that of HCN1 channels, and HCN1 coimmunoprecipitated with HCN4 from membrane fraction proteins. Finally, the immunopositive somata ranged in diameter from the smallest to the largest in rat retina, the dendrites of immunopositive cells arborized at various levels of the inner plexiform layer and over fields of different diameters, and I(h) activated with similar kinetics and proportions of fast and slow components in small, medium, and large somata. These results show that different HCN subunits colocalize in single retinal ganglion cells, identify a subunit that can reconcile native I(h) properties with the previously reported presence of HCN4 in these cells, and indicate that I(h) is biophysically similar in morphologically diverse retinal ganglion cells and differs from I(h) in rods, cones, and bipolar cells.
Asunto(s)
Canales Catiónicos Regulados por Nucleótidos Cíclicos/metabolismo , Canales de Potasio/metabolismo , Isoformas de Proteínas/metabolismo , Células Ganglionares de la Retina/metabolismo , Animales , AMP Cíclico/metabolismo , Canales Catiónicos Regulados por Nucleótidos Cíclicos/genética , Humanos , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización , Técnicas de Placa-Clamp , Canales de Potasio/genética , Isoformas de Proteínas/genética , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Ratas , Células Ganglionares de la Retina/citologíaRESUMEN
The spike output of neural pathways can be regulated by modulating output neuron excitability and/or their synaptic inputs. Dopaminergic interneurons synapse onto cells that route signals to mammalian retinal ganglion cells, but it is unknown whether dopamine can activate receptors in these ganglion cells and, if it does, how this affects their excitability. Here, we show D(1a) receptor-like immunoreactivity in ganglion cells identified in adult rats by retrogradely transported dextran, and that dopamine, D(1)-type receptor agonists, and cAMP analogs inhibit spiking in ganglion cells dissociated from adult rats. These ligands curtailed repetitive spiking during constant current injections and reduced the number and rate of rise of spikes elicited by fluctuating current injections without significantly altering the timing of the remaining spikes. Consistent with mediation by D(1)-type receptors, SCH-23390 [R-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine] reversed the effects of dopamine on spikes. Contrary to a recent report, spike inhibition by dopamine was not precluded by blocking I(h). Consistent with the reduced rate of spike rise, dopamine reduced voltage-gated Na(+) current (I(Na)) amplitude, and tetrodotoxin, at doses that reduced I(Na) as moderately as dopamine, also inhibited spiking. These results provide the first direct evidence that D(1)-type dopamine receptor activation can alter mammalian retinal ganglion cell excitability and demonstrate that dopamine can modulate spikes in these cells by a mechanism different from the presynaptic and postsynaptic means proposed by previous studies. To our knowledge, our results also provide the first evidence that dopamine receptor activation can reduce excitability without altering the temporal precision of spike firing.
Asunto(s)
Potenciales de Acción/fisiología , Dopamina/metabolismo , Inhibición Neural/fisiología , Receptores de Dopamina D1/metabolismo , Células Ganglionares de la Retina/metabolismo , Transmisión Sináptica/fisiología , Potenciales de Acción/efectos de los fármacos , Animales , Células Cultivadas , AMP Cíclico/análogos & derivados , AMP Cíclico/metabolismo , Dextranos , Agonistas de Dopamina/farmacología , Antagonistas de Dopamina/farmacología , Femenino , Inhibición Neural/efectos de los fármacos , Técnicas de Trazados de Vías Neuroanatómicas , Trazadores del Tracto Neuronal , Técnicas de Cultivo de Órganos , Técnicas de Placa-Clamp , Ratas , Ratas Long-Evans , Receptores de Dopamina D1/agonistas , Células Ganglionares de la Retina/citología , Células Ganglionares de la Retina/efectos de los fármacos , Canales de Sodio/efectos de los fármacos , Canales de Sodio/metabolismo , Transmisión Sináptica/efectos de los fármacos , Visión Ocular/efectos de los fármacos , Visión Ocular/fisiologíaRESUMEN
Antisera directed against hyperpolarization-activated, cyclic nucleotide-sensitive (HCN) channels bind to somata in the ganglion cell layer of rat and rabbit retinas, and mRNA for different HCN channel isoforms has been detected in the ganglion cell layer of mouse retina. However, previous studies neither provided evidence that any of the somata are ganglion cells (as opposed to displaced amacrine cells) nor quantified these cells. We therefore tested whether isoform-specific anti-HCN channel antisera bind to ganglion cells labeled by retrograde transport of fluorophore-coupled dextran. In flat-mounted adult rat retinas, the number of dextran-backfilled ganglion cells agreed with cell densities reported in previous studies, and anti-HCN4 antisera bound to the somata of approximately 40% of these cells. The diameter of these somata ranged from 7 to 30 microm. Consistent with localization to cell membranes, the immunoreactivity formed a thin line that circumscribed individual somata. Optic fiber layer axon fascicles, and the proximal dendrites of some ganglion cells, also displayed binding of anti-HCN4 antisera. These results suggest that the response of some mammalian retinal ganglion cells to hyperpolarization may be modulated by changes in intracellular cAMP levels, and could thus be more complex than expected from previous voltage and current recordings.
Asunto(s)
Canales de Potasio/inmunología , Células Ganglionares de la Retina/inmunología , Animales , Recuento de Células/métodos , Dextranos/metabolismo , Femenino , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización , Ratas , Ratas Long-Evans , Células Ganglionares de la Retina/citologíaRESUMEN
Adult newts can regenerate their entire retinas following a complete removal of the original tissues. During retinal regeneration, ganglion cells differentiate first from the progenitor cells, and develop their capability of spike firing. In the present study, to understand the process of functional differentiation of ganglion cells, we investigated alterations of their voltage-gated sodium currents during retinal regeneration by a whole-cell patch-clamp technique. To minimize space clamp errors, sodium currents were recorded from neurite-free somata of presumptive ganglion cells that were mechanically isolated from living slices of regenerating retinas at different morphological stages. During retinal regeneration, the somatic sodium current density was increased 2.6-fold (48 to 123 pF/pA) and the half-activating voltage was shifted slightly to more hyperpolarizing membrane potentials (-10 to -13 mV), while steady-state inactivation was not changed obviously. Curve fitting analysis of currents revealed that the sodium current consists of two components with different inactivation time constants. During retinal regeneration, the ratio of slow to fast inactivating current component was increased 2.6-fold (0.11 to 0.29). These results suggest that the somatic sodium currents of ganglion cells may undergo modifications of their voltage dependence and kinetic properties during retinal regeneration. A small number of the presumptive ganglion cells in regenerating retinas with a segregating inner plexiform layer exhibited sodium currents comparable to those in the normal retina. This might suggest that maturational regulation of sodium channel function starts during a period of synaptic layer formation within the retina.
Asunto(s)
Diferenciación Celular/fisiología , Regeneración Nerviosa/fisiología , Células Ganglionares de la Retina/fisiología , Salamandridae/fisiología , Canales de Sodio/fisiología , Células Madre/fisiología , Potenciales de Acción/fisiología , Animales , Membrana Celular/fisiología , Vías Nerviosas/fisiología , Técnicas de Placa-Clamp , Salamandridae/anatomía & histología , Transmisión Sináptica/fisiologíaRESUMEN
Changes in the gap junctional coupling and maturation of voltage-activated Na(+) currents during regeneration of newt retinas were examined by whole-cell patch-clamping in slice preparations. Progenitor cells in regenerating retinas did not exhibit Na(+) currents but showed prominent electrical and tracer couplings. Cells identified by LY-fills were typically slender. Na(+) currents were detected in premature ganglion cells with round somata in the 'intermediate-II' regenerating retina. No electrical and tracer couplings were observed between these cells. Mature ganglion cells did not exhibit electrical coupling, but showed tracer coupling. On average, the maximum Na(+) current amplitude recorded from premature ganglion cells was roughly 2.5-fold smaller than that of mature ganglion cells. In addition, the activation threshold of the Na(+) current was nearly 11 mV more positive than that of mature cells. We provide morphological and physiological evidence showing that loss of gap junctions between progenitor cells is associated with ganglion cell differentiation during retinal regeneration and that new gap junctions are recreated between mature ganglion cells. Also we provide evidence suggesting that the loss of gap junctions correlates with the appearance of voltage-activated Na(+) currents in ganglion cells.
Asunto(s)
Uniones Comunicantes/fisiología , Lisina/análogos & derivados , Regeneración Nerviosa/fisiología , Retina/fisiología , Teofilina/análogos & derivados , Anfetaminas , Animales , Diferenciación Celular/fisiología , Conductividad Eléctrica , Colorantes Fluorescentes/metabolismo , Isoquinolinas/metabolismo , Lisina/metabolismo , Técnicas de Placa-Clamp , Retina/crecimiento & desarrollo , Células Ganglionares de la Retina/citología , Células Ganglionares de la Retina/fisiología , Salamandridae/fisiologíaRESUMEN
Using living slice preparations from newt retinas at different stages of regeneration, we examined the time course of appearance and maturation of neurotransmitter-induced currents with whole-cell patch-clamp methods. Neurons from which currents were recorded were identified by Lucifer Yellow fills. All progenitor cells examined at the regenerating retinas did not express any voltage-gated Na+ currents and responsiveness to excitatory amino acid analogues (AMPA and NMDA) and inhibitory amino acids (GABA and glycine). Voltage-gated Na+ currents were first detected in premature ganglion cells with round cell body located at the most proximal level of the 'intermediate-II' regenerating retina. AMPA- GABA- and glycine-induced currents were simultaneously observed in many premature ganglion cells expressing Na+ channels, but not all, suggesting that the onset of the Na+ channels is slightly earlier than that of excitatory and inhibitory amino acid receptors in regeneration. NMDA-evoked currents were first observed in the 'intermediate-III' regenerating retina just before the synaptogenesis. Pharmacological properties and reversal potential values of the excitatory and inhibitory amino acid responses did not change substantially between regenerating ganglion cells and mature ganglion cells, while rectification properties of current-voltage relations for AMPA and NMDA responses were somewhat different between them.