Light-evoked Decrease Research Articles

Near-ultraviolet light perceived by the retina generates the signal suppressing melatonin synthesis in the chick pineal gland—an involvement of NMDA glutamate receptors

Exposure of dark-adapted chicks to near ultraviolet (UV-A) light significantly decreased melatonin (MEL) content and the activity of serotonin N-acetyltransferase (AA-NAT; the penultimate and key regulatory enzyme in MEL production) in the pineal glands. Significant reduction in MEL level and AA-NAT activity was also found in pineals of animals whose heads were covered with black opaque tape, an observation suggesting that in the chicken UV-A light perceived by the eyes alone is capable of affecting MEL synthesis in the pineal gland. Covering the chick's eyes, in addition to the head, totally blocked the studied UV-A action. Although SCH 23390 (a selective D1-dopamine receptor antagonist), injected directly into both eyes at a dose of 10 nmol/eye, prevented the decline in pineal AA-NAT activity produced by retinal illumination with white light, the drug did not modify the UV-A light-evoked decrease in the enzyme activity. MK-801 (a selective antagonist of NMDA glutamate receptors; 1 nmol/eye) abolished the suppressive action of UV-A light on pineal AA-NAT activity, but it was inactive in the case of white light. Intraocularly injected sulpiride and CNQX (selective antagonists of D2-dopamine and AMPA/kainite glutamate receptors, respectively) had no effect on the actions of both UV-A and white light (acting on the eyes only) on pineal AA-NAT activity. It is concluded that in the chick retinally perceived UV-A light generates a signal which suppresses MEL production in the pineal gland. At the level of the retina, such signal does not involve dopamine, but is dependent on the stimulation of NMDA glutamate receptors.

Near-ultraviolet radiation suppresses melatonin synthesis in the chicken retina: A role of dopamine

Effects of near-ultraviolet radiation (UV-A; 325–390 nm, peak at 365 nm) on melatonin content and activity of serotonin N-acetyltransferase (AA-NAT; a key regulatory enzyme in melatonin biosynthesis) were examined in the retina of chickens. Acute exposure of dark-adapted animals to UV-A light produced a marked decline in melatonin content and AA-NAT activity of the retina. The magnitude of the observed changes was dependent upon duration of the light pulse and age of chickens, with 1–2-week old birds being more sensitive to UV-A action than 6–7-week old ones. The decrease in the nocturnal AA-NAT activity evoked by a 5-min UV-A pulse gradually deepened during the first 30 min after the return of chickens to constant darkness, then the enzyme activity began to rise, reaching nearly complete restoration within 2.5 hr. Systemic administration to chickens of α-methyl- p-tyrosine (an inhibitor of catecholamine synthesis; 0.3 g/kg) blocked the suppressive effect of UV-A light on retinal AA-NAT activity. Haloperidol, sulpiride (blockers of D2-family of dopamine (DA) receptors) and 2-chloro-11-(4-methylpiperazino)dibenz[ b,f]oxepin (an antagonist of D 4-DA receptors), given intraocularly (1–100 nmol/eye) prevented the UV-A light-evoked decrease in AA-NAT activity in the chicken retina in a dose-dependent manner, while raclopride (300 nmol/eye), an antagonist of D 2/D 3-DA receptors, was ineffective. In dark-adapted chickens exposure to UV-A light increased the DA content of the retina. It is concluded that UV-A radiation, similar to visible light, potently suppresses melatonin biosynthesis in the retina of chicken, with a D 4-dopaminergic signal playing the role of an intermediate in this action.

Effects of hypoxia and post-hypoxic recovery on chick retinal pigment epithelium potentials and light-evoked responses in vitro

Purpose. To determine the cellular mechanisms involved in the hypoxia-induced alteration of the retinal pigment epithelium (RPE) potentials and the light-evoked responses of the RPE in chicks. In addition, to determine the mechanisms involved in the recovery of the RPE during the post-hypoxic period.Methods. In vitro preparations of chick retina-RPE-choroid were studied by potassium-selective microelectrodes placed in the subretinal space. In addition, single-barrel microelectrodes were used to obtain intracellular recordings from the RPE cells. The perfusate was bubbled continuously with 95% oxygen and 5% carbon dioxide for the control condition and replaced by 95% nitrogen and 5% carbon dioxide to induce hypoxia.Results. Hypoxia induced a significant reduction of the trans-tissue potential which was found to result from the depolarization of the apical membrane of the RPE. This depolarization was induced by an increase of subretinal [K+]o. The c-wave was also markedly decreased or abolished during hypoxia. There were two phases of post-hypoxic recovery: an initial small increase in the trans-tissue potential resulting from a basal membrane depolarization followed by an apical membrane hyperpolarization. The trans-tissue potential and the c-wave also were supernormal in two phases during this post-hypoxic period. The c-wave amplitude was temporarily elevated (263.7 ± 77.4% of pre-hypoxic control) because of the enhanced trans-epithelial c-wave and without a light-evoked decrease in subretinal [K+]o.Conclusions. The trans-tissue potential and the c-wave were markedly decreased during hypoxia. During the post-hypoxic period, both potential recovered with transient supernormalities in two phases. The results suggested that the hypoxic changes resulted directly from changes of the RPE membranes and indirectly from a change in the subretinal [K+]o but were not mediated by the light-evoked decrease in subretinal [K+]o. Curr. Eye Res. 17: 384–391, 1998.

The origin of slow PIII in frog retina: current source density analysis in the eyecup and isolated retina.

The objective of this research was to determine the sources and sinks of current underlying the slow PIII component of the electroretinogram. Current source density analysis of the ERG evoked by diffuse light flashes was performed in eyecup and isolated retinas of frog. Blockade of synaptic transmission with aminophosphonobutyric + kynurenic acids simplified the CSD profiles through the retina. In addition to the photoreceptor source/sink pair, there was evidence for a major slow PIII source near the outer limiting membrane, a major sink near the inner limiting membrane, and a small source near the inner plexiform layer. Addition of Ba2+ abolished the slow PIII source/sinks, and it left only the photoreceptor source and sink. The results support the idea that slow PIII originates through K+ spatial buffering by Müller cells. Specifically, the light-evoked decrease in [K+]o in the subretinal space causes a primary K+ efflux from Müller cells (current source) and a primary K+ influx at the Müller cell endfeet (current sink). A decrease in [K+]zero in the proximal retina, caused by diffusion of K+ to the subretinal space, results in K+ efflux (the current source) at the inner plexiform layer.

Light-dependent hydration of the space surrounding photoreceptors in the cat retina.

We have studied the effect of retinal illumination on the concentration of the extracellular space marker tetramethylammonium (TMA+) in the dark-adapted cat retina using double-barreled ion-selective microelectrodes. The retina was loaded with TMA+ by a single intravitreal injection. Retinal illumination produced a slow decrease in [TMA+]o, which was maximal in amplitude in the most distal portion of the space surrounding photoreceptors, the subretinal space. The light-evoked decrease in [TMA+]o was considerably slower and of a different overall time course than the light-evoked decrease in [K+]o, also recorded in the subretinal space. [TMA+]o decreased to a peak at 38 s after the onset of illumination, then slowly recovered towards the baseline, and transiently increased following the offset of illumination. It resembled the light-evoked [TMA+]o decreases previously recorded in the in vitro preparations of frog (Huang & Karwoski, 1990, 1992) and chick (Li et al., 1992, 1994) but was considerably larger in amplitude, 22% compared with 7%. As in frog, where it was first recorded, the light-evoked [TMA+]o decrease is considered to originate from a light-evoked increase in the volume of the subretinal space (or subretinal hydration). A mathematical model accounting for [TMA+]o diffusion predicted that the volume increase underlying the response was 63% on average and could be as large as 95% and last for minutes. The estimated volume increase was then used to examine its effect on K+ concentration in the subretinal space. We conclude that a light-dependent hydration of the subretinal space represents a significant physiological event in the intact cat eye, which should affect the organization of the interphotoreceptor matrix, and the concentrations of all ions and metabolites located in the subretinal space.

Calcium gradients and light-evoked calcium changes outside rods in the intact cat retina.

We have studied light-evoked changes in extracellular Ca2+ concentration ([Ca2+]o) in the intact cat eye using ion-sensitive double-barreled microelectrodes. Two prominent changes in Ca2+ concentration were observed that differed in retinal location. There was a light-evoked increase in [Ca2+]o, accompanied by brief ON and OFF transients, which was maximal in the inner plexiform layer and was not further studied. There was an unexpected sustained light-evoked decrease in [Ca2+]o, of relatively rapid onset and offset, which was maximal in the distalmost region of the subretinal space (SRS). [Ca2+]o in the SRS was 1.0 mM higher than in the vitreous humor during dark adaptation and this transretinal gradient disappeared during rod-saturating illumination. After correcting for the light-evoked increase in the volume of the SRS, an increase in the total Ca2+ content of the SRS during illumination was revealed, which presumably represents the Ca2+ released by rods. To explain the light-evoked [Ca2+]o changes, we used the diffusion model described in the accompanying paper (Li et al., 1994b), with the addition of light-dependent sources of Ca2+ at the retina/retinal pigment epithelium (RPE) border and rod outer segments. We conclude that a drop in [Ca2+]o around photoreceptors, which persists during illumination and reduces a transretinal Ca2+ gradient, is the combined effect of the light-evoked SRS volume increase, Ca2+ release from photoreceptors, and an unidentified mechanism(s), which is presumably Ca2+ transport by the RPE. The relatively rapid onset and offset of the [Ca2+]o decrease remains unexplained. These steady-state shifts in [Ca2+]o should have significant effects on photoreceptor function, especially adaptation.

Basolateral membrane Cl- and K+ conductances of the dark-adapted chick retinal pigment epithelium.

1. We characterized the basolateral membrane Cl- and K+ conductances of the dark-adapted chick neural retina-retinal pigment epithelium (RPE)-choroid preparation. Conventional microelectrodes were used to measure apical (V(ap)) and basolateral (Vba) membrane voltage, and double-barreled Cl- and K+ selective microelectrodes were used to follow the time course and magnitude of ion concentration changes outside the basolateral (basal) membrane. 2. In response to a fivefold decrease in basal [Cl-]o, Vba rapidly depolarized by 6.4 +/- 0.7 (SE) mV, and the apparent resistance of the basolateral membrane (Rba) increased. The Cl- channel blocker 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) suppressed the Vba depolarization by 40% and blocked the Rba increase. Estimates of the relative Cl- conductance (transference number, TCl) from the DIDS-sensitive component of the Cl- diffusion potential gave an average value for TCl of 0.22 +/- 0.03. 3. Further evidence for a Cl- conductance was obtained by measuring changes in intracellular Cl- activity (aCli) induced by transtissue current. Depolarizing Vba elevated aiCl, whereas hyperpolarizing Vba had the opposite effect, consistent with conductive Cl- movement across the basal membrane. TCl estimated from these data averaged 0.23 +/- 0.02. 4. In response to a sixfold increase in basal [K+]o, Vba depolarized 6.1 +/- 0.8 mV. The amplitude of this K+ diffusion potential was inhibited 44 and 67% by 5 and 10 mM Ba2+, respectively. TK was estimated to be 0.61 +/- 0.05. 5. The rapid c-wave membrane hyperpolarizations in response to the light-evoked decrease in subretinal [K+]o were used to calculate the equivalent resistances of the apical membrane (R(ap)), basolateral membrane (Rba), and the paracellular shunt pathway (Rs). They were 152 +/- 10, 615 +/- 38, and 138 +/- 7 omega.cm2 (n = 11 tissues), respectively. From these data the equivalent electromotive force for the basal (Eba) and apical (Eap) membranes were estimated to be -45 +/- 2 and -77 +/- 1 mV, respectively. This estimate of Eba is in the range of that predicted from our estimates of TCl and TK, indicating that, in the dark-adapted chick retina, the resting conductance of the basal membrane can largely be accounted for by the Cl- and K+ conductances described here.

Light-evoked expansion of subretinal space volume in the retina of the frog

The retina of the frog was superfused with a Ringer solution containing impermeant "probe" cations and anions. Light-evoked concentration changes in these probe ions were measured in the subretinal space (SRS) with ion-selective microelectrodes. A decrease in probe ion concentration was found, and several observations suggest that this is caused by a light-evoked expansion of the SRS. The probe ion decrease was not seen in the isolated retina; thus, the pigment epithelial (PE) cells are important for its generation. Pharmacological studies suggest that K+ channels in the PE cells are important--perhaps the PE cells shrink in response to the light-evoked decrease in SRS [K+]. The light-evoked decrease of SRS volume may be important in the understanding of SRS solute concentrations, retina-PE adhesivity, photoreceptor-PE cell interactions, and the interphotoreceptor matrix.

Spatial buffering of extracellular potassium by Müller (glial) cells in the toad retina

We examined the role of Müller (glial) cells in buffering light-evoked changes in extracellular K+ concentration, [K+]o, in the isolated retina of the toad, Bufo marinus. We found evidence for two opposing Müller cell current loops that are generated by a light-evoked increase in [K+]o in the inner plexiform layer. These current loops, which are involved in the generation of the M-wave of the electroretinogram (ERG), prevent the accumulation of K+ in the inner plexiform layer by transporting K+ both to vitreous and to distal retina. In addition, under dark-adapted conditions, we found evidence for a Müller cell current loop that is generated by a light-evoked decrease in [K+]o in the receptor layer. This current loop, which is involved in the generation of the slow PIII component of the ERG, helps to buffer the light-evoked decrease in [K+]o throughout distal retina by transporting K+ from vitreous. The spatial buffering fluxes of K+ can be abolished by blocking Müller cell K+ conductance with 200 microM Ba2+. The separate contributions of the M-wave and slow PIII currents to Müller cell spatial buffering were isolated by various pharmacological treatments that were designed to enhance or suppress light-evoked activity in specific retinal neurons. Our results show that Müller cell K+ currents not only buffer light-evoked increases in [K+]o, but also buffer light-evoked decreases in [K+]o, and thereby diminish any deleterious effects upon neuronal function that could arise in response to large changes in [K+]o in the plexiform layers. Moreover, our results emphasize that spatial buffering currents generate many components of the electroretinogram.

Origin of negative potentials in the light-adapted ERG of cat retina

1. The light-adapted diffuse-flash electroretinogram (ERG) in the cat exhibits two prominent negative components: a sustained negative potential during illumination and a negative-going OFF response. We investigated their intraretinal origins and found that the sustained component originates from the rod photoreceptors, whereas the OFF response represents a combination of the return to base line of the rod-receptor potential, the offset of PII (rod and cone), and a cone-dependent OFF response originating proximal to the photoreceptors at very high background levels. 2. The ERG, evoked in response to diffuse illumination of the light- and dark-adapted cat retina, was recorded between a chlorided silver wire in the vitreous and a plate behind the eye. Extracellular field potentials were recorded simultaneously with a microelectrode placed intraretinally at different retinal depths. 3. The sustained negative potential and the negative OFF response were not the M-wave ON and OFF responses of proximal retina, despite an overall resemblance in form and time course: 1) the M-wave was spatially tuned, whereas the ERG components were not; 2) tetrodotoxin (TTX) (3.8-microM vitreal concentration) did not alter the M-wave, but it reduced the ERG OFF response; 3) picrotoxin (0.14 mM, after TTX) enhanced the M-wave but did not affect the negative ERG; and 4) 2-amino-4-phosphonobutyric acid (APB; 0.95 mM) removed the M-wave ON response, and aspartate (43 mM) removed the M-wave OFF response, in addition, while the sustained negative potential persisted. 4. The sustained negative potential was not slow PIII, the neural retinal component of the c-wave, a Müller cell response to the photoreceptor-dependent light-evoked decrease in subretinal extracellular K+ concentration [( K+]o). Although Ba2+ (repeated injections of 4-5 mM), a K+ conductance blocker, eliminated slow PIII, it did not remove the sustained negative potential. We concluded that the sustained negative potential was a photoreceptor potential, and the spectral sensitivity of the response indicated that it arose from rods. 5. The contribution of the rod-receptor potential to the ERG depended on background illumination. It was a sustained potential for a range of backgrounds near and 1 or 2 log units above the illumination that saturates rod-driven responses in cat (8.2 log quanta.deg-2.s-1). At lower background intensities, it appeared only as a dip between the b- and c-waves, the b-wave trough, which also was present in fully dark-adapted responses.(ABSTRACT TRUNCATED AT 400 WORDS)

Light-evoked increases in [K+]o in proximal portion of the dark-adapted cat retina.

1. The scotopic threshold response (STR) and slow negative response are negative-going potentials in the dark-adapted electroretinogram (ERG) of the cat eye that originate proximal to the photoreceptors and are present at threshold and with dim illuminations. The present paper examines the hypothesis that these events are associated with Müller cell responses to potassium released by proximal retinal neurons by measuring light-dependent changes in extracellular K+ concentration [( K+]o, in proximal retina. 2. Extracellular field potentials and changes in [K+]o, evoked in response to diffuse illumination of the dark-adapted retina, were recorded with a double-barreled K+-sensitive microelectrode placed in the retina at different depths. The vitreal ERG was recorded at the same time. 3. The dynamic range of the light-evoked increases in [K+]o, recorded in proximal retina from threshold to saturation, was strikingly similar to that of the STR and slow negative response. The threshold for the [K+]o increase was near that of the most sensitive ganglion cells, and the response saturated approximately 2.4 log units below rod saturation. Also, onset latencies and rates of rise for the increases in [K+]o and the field potentials in proximal retina followed similar functions of intensity. 4. The depth distribution of the light-evoked [K+]o increases resembled that of the STR. The increases in [K+]o were largest and fastest in proximal retina where the STR was largest. This was true both for very low intensity stimuli, below the threshold for PII (DC-component and b-wave) in distal retina, and for stimulus intensities near STR saturation, where PII (DC-component) was present. For stimuli of intensities near rod saturation, when the b-wave was present in distal retina, there was a small fast increase in [K+]o at light onset in distal retina and a slower increase in proximal retina. Both increases were truncated by spread of the large light-evoked decrease in [K+]o in subretinal space that is known to cause the c-wave in the ERG and slow PIII in neural retina. 5. The duration of the increases in [K+]o in proximal retina was more sustained than the proximal field potentials. In response to stimuli of 2-4 s, the field potential began to recover toward base line after about 300 ms, whereas the increases in [K+]o remained at maximal levels until stimulus offset.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of DIDS on the chick retinal pigment epithelium. I. Membrane potentials, apparent resistances, and mechanisms

While little is known about the transport properties of the retinal pigment epithelium (RPE) basal membrane, mechanisms for anion movement across the basal membrane appear to be present (Miller and Steinberg, 1977; Hughes et al., 1984; Miller and Farber, 1984). This work examines the electrophysiological effects of the anion conductance blocker, 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) on the basal membrane of an in vitro preparation of chick retina-RPE-choroid. DIDS (10-125 microM), added to the choroidal bath, decreased the transtissue potential by decreasing the potential across the RPE. Intracellular RPE recordings showed that DIDS affected the membrane potential in 2 phases, initially hyperpolarizing the basal membrane and then, after prolonged exposure, depolarizing the apical membrane. Resistance assessment by transtissue current pulses and intracellular c-wave recordings suggested that DIDS increased basal membrane resistance (Rba) during the first phase and increased apical membrane resistance (Rap) during the second phase. Measurements of intracellular Cl- activity (aiCl) showed that Cl- was actively accumulated by the chick RPE since it was distributed above equilibrium across both the apical and basal membranes. Perfusion of the basal membrane with 50 microM DIDS significantly increased aiCl-. The DIDS-induced basal membrane hyperpolarization, apparent increase in Rba, and increase in aiCl- are all consistent with Cl- -conductance blockade. During the second phase, apical membrane responsiveness to the light-evoked decrease in subretinal [K+]o (Oakley, 1977) was reduced an average of 58%. This finding, given the second-phase apical membrane depolarization and apparent increase in Rap, is consistent with a decrease in apical membrane K+ conductance.(ABSTRACT TRUNCATED AT 250 WORDS)

Measurement of potassium turnover in rod photoreceptors in toad isolated retina using ion-selective microelectrodes.

Ion-selective microelectrodes (ISMs) were used to measure the turnover of intracellular K+ (Ki+) in rods in the isolated retina of the toad, Bufo marinus. The light-evoked hyperpolarization of rods decreases their passive K+ efflux, which in combination with active K+ uptake, decreases extracellular K+ concentration, Ko+.Rb+ substitutes for K+ in these processes. The turnover of Ki+ was measured as Rb+ and K+ were exchanged, using ISMs that were approximately five times more sensitive to Rb+ than to K+. When Ko+ was replaced by Rbo+, the light-evoked decrease in K+ efflux produced only a small change in ISM voltage, delta VISM, owing to the background of Rbo+. As Rbi+ replaced Ki+, the efflux shifted from K+ to Rb+ and delta VISM grew in amplitude. After loading the rods with Rbi+, Rbo+ was replaced by Ko+. The light-evoked decrease in Rb+ efflux lead transiently to a large delta VISM, since the change in Rbo+ was superimposed upon a background of Ko+. As Ki+ replaced Rbi+, the amplitude of delta VISM declined. When measured using this technique, the turnover of Ki+ was 95% complete in approximately 15 min. In low Ca2+ solutions, transmembrane fluxes of K+ (Rb+) increased and turnover of Ki+ occurred more rapidly. During background illumination, transmembrane fluxes of K+ (Rb+) decreased and turnover of Ki+ was slowed. These experiments have provided independent corroboration of earlier observations concerning rod K+ fluxes. This ISM-based technique also may be useful in measuring K+ turnover in other cell types.

Ba2+ unmasks K+ modulation of the Na+-K+ pump in the frog retinal pigment epithelium.

This paper presents electrophysiological evidence that small changes in [K+]o modulate the activity of the Na+-K+ pump on the apical membrane of the frog retinal pigment epithelium (RPE). This membrane also has a large relative K+ conductance so that lowering [K+]o hyperpolarizes it and therefore increases the transepithelial potential (TEP). Ba2+, a K+ channel blocker, eliminated these normal K+-evoked responses; in their place, lowering [K+]o evoked an apical depolarization and TEP decrease that were blocked by apical ouabain or strophanthidin. These data indicate that Ba2+ blocked the major K+ conductance(s) of the RPE apical membrane and unmasked a slowing of the normally hyperpolarizing electrogenic Na+-K+ pump caused by lowering [K+]o. Evidence is also presented that [K+]o modulates the pump in the isolated RPE under physiological conditions (i.e., without Ba2+). In the intact retina, light decreases subretinal [K+]o and produces the vitreal-positive c-wave of the electroretinogram (ERG) that originates primarily in the RPE from a hyperpolarization of the apical membrane and TEP increase. When Ba2+ was present in the retinal perfusate, the apical membrane depolarized in response to light and the TEP decreased so that the ERG c-wave inverted. The retinal component of the c-wave, slow PIII, was abolished by Ba2+. The effects of Ba2+ were completely reversible. We conclude that Ba2+ unmasks a slowing of the RPE Na+-K+ pump by the light-evoked decrease in [K+]o. Such a response would reduce the amplitude of the normal ERG c-wave.

Extracellular K+ activity changes related to electroretinogram components. I. Amphibian (I-type) retinas.

Electroretinographic (ERG) and extracellular potassium activity measurements were carried out in superfused eyecup preparations of several amphibians. Light-evoked changes in extracellular K+ activity were characterized on the bases of depth profile analysis and latency measurements and through the application of pharmacological agents that have selective actions on the retinal network. Three different extracellular potassium modulations evoked at light onset were identified and characterized according to their phenomenological and pharmacological properties. These modulations include two separable sources of light-evoked increases in extracellular K+: (a) a proximal source that is largely post-bipolar in origin, and (b) a distal source that is primarily or exclusively of depolarizing bipolar cell origin. The pharmacological properties of the distal extracellular potassium increase closely parallel those of the b-wave. A distal light-evoked decrease in extracellular potassium appears to be associated with the slow PIII potential, based on a combination of simultaneous intracellular Müller cell recordings and extracellular ERG and potassium activity measurements before and during pharmacological isolation of the photoreceptor responses. The extracellular potassium activity increases are discussed with respect to the Müller cell theory of b-wave generation.

Reaccumulation of [K+]o in the toad retina during maintained illumination.

Using K+-selective microelectrodes, [K+]o was measured in the subretinal space of the isolated retina of the toad, Bufo marinus. During maintained illumination, [K+]o fell to a minimum and then recovered to a steady level that was approximately 0.1 mM below its dark level. Spatial buffering of [K+]o by Müller (glial) cells could contribute to this reaccumulation of K+. However, superfusion with substances that might be expected to block glial transport of K+ had no significant effect upon the reaccumulation of K+. These substances included blockers of gK (TEA+, Cs+, Rb+, 4-AP) and a gliotoxin (alpha AAA). Progressive slowing of the rods' Na+/K+ pump (perhaps caused by a light-evoked decrease in [Na+]i) also could contribute to this reaccumulation of K+ by reducing the uptake of K+ from the subretinal space. As evidence for a major contribution by this mechanism, treatments designed to prevent such slowing of the pump reversibly blocked reaccumulation. These treatments included superfusion with 2 microM ouabain, or lowering [K+]o, PO2, or temperature. It is likely that such treatments inhibit the pump, increase [Na+]i, and attenuate any light-evoked decrease in [Na+]i. The results are consistent with the following hypothesis. At light onset, the decrease in rod gNa will reduce the Na+ influx and the resulting rod hyperpolarization will reduce the K+ efflux. In combination with these reduced passive fluxes, the continuing active fluxes will lower both [K+]o and [Na+]i, which in turn will inhibit the pump. In support of this hypothesis, the solutions to a pair of coupled differential equations that model changes in both [K+]o and [Na+]i match quantitatively the time course of the observed changes in [K+]o during and after maintained illumination for all stimuli examined.

Changes in apical [K+] produce delayed basal membrane responses of the retinal pigment epithelium in the gecko.

We describe here a new retinal pigment epithelium (RPE) response, a delayed hyperpolarization of the RPE basal membrane, which is initiated by the light-evoked decrease of [K+]o in the subretinal space. This occurs in addition to an apical hyperpolarization previously described in cat (Steinberg et al., 1970; Schmidt and Steinberg, 1971) and in bullfrog (Oakley et al., 1977; Oakley, 1977). Intracellular and extracellular potentials and measurements of subretinal [K+]o were recorded from an in vitro preparation of neural retina-RPE-choroid from the lizard Gekko gekko in response to light. Extracellularly, the potential across the RPE, the transepithelial potential (TEP), first increased and then decreased during illumination. Whereas the light-evoked decrease in [K+]o predicted the increase in TEP, the subsequent decrease in TEP was greater than predicted by the reaccumulation of [K+]o. Intracellular RPE recordings showed that a delayed hyperpolarization generated at the RPE basal membrane produced the extra TEP decrease. At light offset, the opposite sequence of membrane potential changes occurred. RPE responses to changes in [K+]o were studied directly in the isolated gecko RPE-choroid. Decreasing [K+]o in the apical bathing solution produced first a hyperpolarization of the apical membrane, followed by a delayed hyperpolarization of the basal membrane, a sequence of membrane potential changes identical to those evoked by light. Increasing [K+]o produced the opposite sequence of membrane potential changes. In both preparations, the delayed basal membrane potentials were accompanied by changes in basal membrane conductance. The mechanism by which a change in extracellular [K+] outside the apical membrane leads to a polarization of the basal membrane remains to be determined.

Effects of the rod receptor potential upon retinal extracellular potassium concentration.

It has been hypothesized that the light-evoked rod hyperpolarization (the receptor potential) initiates the light-evoked decrease in extracellular potassium ion concentration, [K+]o, in the distal retina. The hypothesis was tested using the isolated, superfused retina of the toad, Bufo marinus; the receptor potential was recorded intracellularly from red rods, and [K+]o was measured in the photoreceptor layer with K+-specific microelectrodes. In support of the hypothesis, variations in stimulus irradiance or duration, or in retinal temperature, produced qualitatively similar effects on both the receptor potential and the decrease in [K+]o. A mechanism for the relationship between the receptor potential and the decrease in [K+]o was suggested by Matsuura et al. (1978. Vision Res. 18:767-775). In the dark, the passive efflux of K+ out of the rod is balanced by an equal influx of K+ fromthe Na+/K+ pump. The light-evoked rod hyperpolarization is assumed to reduce the passive efflux, with little effect on the pump. Thus, the influx will exceed the efflux, and [K+]o will decrease. Consistent with this mechanism, the largest and most rapid decrease in [K+]o was measured adjacent to the rod inner segments, where the Na+/K+ pump is most likely located; in addition, inhibition of the pump with ouabain abolished the decrease in [K]o more rapidly than the rod hyperpolarization. Based upon this mechanism, Matsuura et al. (1978) developed a mathematical model: over a wide range of stimulus irradiance, this model successfully predicts the time-course of the decrease in [K+]o, given only the time-course of the rod hyperpolarization.

Potassium and the photoreceptor-dependent pigment epithelial hyperpolarization.

Light-evoked changes in pigment epithelial cell membrane potentials and retinal extracellular potassium ion concentration, [K+]0, were measured in an in vitro frog retina-pigment epithelium-choroid preparation. Light stimuli hyperpolarized the apical membrane of the pigment epithelium. Through an electrical shunt pathway connecting the apical and basal membranes, the basal membrane also hyperpolarized, but to a lesser degree than the apical membrane. This differential hyperpolariation of the two membranes increased the transepithelial potential (TEP). This increase in TEP was shown to be the major voltage source of the c-wave of the electroretinogram (ERG). Direct measurement of [K+]0 in the distal retina, made with K+-specific microelectrodes, showed a light-evoked decrease in [K+]0 having an identical time course to the apical hyperpolarization. There was a linear relationship between the light-evoked change in TEP and the logarithm of [K+]0. This exact relationship was also found when the apical membrane was perfused directly with solutions of varying [K+]0. The change in TEP associated with the ERC c-wave, therefore, was explained solely by the response of the pigment epithelium to the light-evoked decrease in [K+]0 in the distal retina.