Mesopic background lights enhance dark-adapted cone ERG flash responses in the intact mouse retina: a possible role for gap junctional decoupling.

The cone-driven flash responses of mouse electroretinogram (ERG) increase as much as twofold over the course of several minutes during adaptation to a rod-compressing background light. The origins of this phenomenon were investigated in the present work by recording preflash-isolated (M-)cone flash responses ex vivo in darkness and during application of various steady background lights. In this protocol, the cone stimulating flash was preceded by a preflash that maintains rods under saturation (hyperpolarized) to allow selective stimulation of the cones at varying background light levels. The light-induced growth was found to represent true enhancement of cone flash responses with respect to their dark-adapted state. It developed within minutes, and its overall magnitude was a graded function of the background light intensity. The threshold intensity of cone response growth was observed with lights in the low mesopic luminance region, at which rod responses are partly compressed. Maximal effect was reached at intensities sufficient to suppress ∼ 90% of the rod responses. Light-induced enhancement of the cone photoresponses was not sensitive to antagonists and agonists of glutamatergic transmission. However, applying gap junction blockers to the dark-adapted retina produced qualitatively similar changes in the cone flash responses as did background light and prevented further growth during subsequent light-adaptation. These results are consistent with the idea that cone ERG photoresponses are suppressed in the dark-adapted mouse retina by gap junctional coupling between rods and cones. This coupling would then be gradually and reversibly removed by mesopic background lights, allowing larger functional range for the cone light responses.

Temperature dependence of dark-adapted sensitivity and light-adaptation in photoreceptors with A1 visual pigments: a comparison of frog L-cones and rods.

Flash responses of L-cones and rods were recorded as ERG mass potentials in the frog retina at different temperatures (2-25 degrees C). The purpose was to elucidate factors that make cones faster and less sensitive than rods, particularly the possible role of thermal activation of L-cone visual pigment in maintaining a "light-adapted" state even in darkness. Up to ca. 15 degrees C, cones and rods were desensitized roughly equally by warming (Q(10) approximately 2.2-2.7), retaining a 5-fold sensitivity difference. In this range, the cone/rod difference must depend on factors other than thermal activation of the visual pigment. Above 15 degrees C, cones showed an additional component of desensitization compared with rods, coupled to accelerated response shut-off. This behavior is consistent with light-adaptation from temperature-dependent intrinsic activity (dark light). The apparent dark light as measured by the minimum background intensities needed to affect sensitivity and/or kinetics increased by ca. 10-fold between 15 and 25 degrees C, whereas reported increases in visual-pigment activation rates over this range are less than 5-fold. We conclude that the dark state of frog L-cones above 15 degrees C may be largely set by thermal activation of the phototransduction machinery, but only part of the experimentally determined dark light can be ascribed to the visual pigment.

Mouse cone photoresponses obtained with electroretinogram from the isolated retina.

We characterize the dark-adapted photoresponses from mouse cones intact in the isolated retina, their virtually natural environment, by isolating pharmacologically the photoreceptor light responses from the electroretinogram (ERG). Due to the different photoresponse kinetics and sensitivity of rods and cones, the cone responses were readily attained by using a rod-saturating preflash. The stimulus wavelength (544 nm) was chosen to selectively stimulate the green sensitive ("M"-)pigment. Obtained responses were monophasic, showing fast kinetics (mean t(p)=51 ms) and low sensitivity (fractional single-photon response ca. 0.23%). The amplification coefficient of cones (4.6 s(-2)) was very close to that of rods (5.6 s(-2)), while the dominant time constant of recovery was clearly smaller for cones (33 ms) than for rods (160 ms).

Light responses and light adaptation in rat retinal rods at different temperatures.

Rod responses to brief pulses of light were recorded as electroretinogram (ERG) mass potentials across isolated, aspartate-superfused rat retinas at different temperatures and intensities of steady background light. The objective was to clarify to what extent differences in sensitivity, response kinetics and light adaptation between mammalian and amphibian rods can be explained by temperature and outer-segment size without assuming functional differences in the phototransduction molecules. Corresponding information for amphibian rods from the literature was supplemented by new recordings from toad retina. All light intensities were expressed as photoisomerizations per rod (Rh*). In the rat retina, an estimated 34% of incident photons at the wavelength of peak sensitivity caused isomerizations in rods, as the (hexagonally packed) outer segments measured 1.7 microm x 22 microm and had specific absorbance of 0.016 microm(-1) on average. Fractional sensitivity (S) in darkness increased with cooling in a similar manner in rat and toad rods, but the rat function as a whole was displaced to a ca 0.7 log unit higher sensitivity level. This difference can be fully explained by the smaller dimensions of rat rod outer segments, since the same rate of phosphodiesterase (PDE) activation by activated rhodopsin will produce a faster drop in cGMP concentration, hence a larger response in rat than in toad. In the range 15-25 degrees C, the waveform and absolute time scale of dark-adapted dim-flash photoresponses at any given temperature were similar in rat and toad, although the overall temperature dependence of the time to peak (t(p)) was somewhat steeper in rat (Q(10) approximately 4 versus 2-3). Light adaptation was similar in rat and amphibian rods when measured at the same temperature. The mean background intensity that depressed S by 1 log unit at 12 degrees C was in the range 20-50 Rh* s(-1) in both, compared with ca 4500 Rh* s(-1) in rat rods at 36 degrees C. We conclude that it is not necessary to assume major differences in the functional properties of the phototransduction molecules to account for the differences in response properties of mammalian and amphibian rods.

Measurement of thermal contribution to photoreceptor sensitivity.

Activation of a visual pigment molecule to initiate phototransduction requires a minimum energy, Ea, that need not be wholly derived from a photon, but may be supplemented by heat. Theory predicts that absorbance at very long wavelengths declines with the fraction of molecules that have a sufficient complement of thermal energy, and that Ea is inversely related to the wavelength of maximum absorbance (lambda(max)) of the pigment. Consistent with the first of these predictions, warming increases relative visual sensitivity to long wavelengths. Here we measure this effect in amphibian photoreceptors with different pigments to estimate Ea (refs 2, 5-7) and test experimentally the predictions of an inverse relation between Ea and lambda(max). For rods and 'red' cones in the adult frog retina, we find no significant difference in Ea between the two pigments involved, although their lambda(max) values are very different. We also determined Ea for the rhodopsin in toad retinal rods--spectrally similar to frog rhodopsin but differing in amino-acid sequence--and found that it was significantly higher. In addition, we estimated Ea for two pigments whose lambda(max) difference was due only to a chromophore difference (A1 and A2 pigment, in adult and larval frog cones). Here Ea for A2 was lower than for A1. Our results refute the idea of a necessary relation between lambda(max) and Ea, but show that the A1 --> A2 chromophore substitution decreases Ea.

Light adaptation of cone photoresponses studied at the photoreceptor and ganglion cell levels in the frog retina.

The sensitivity and time scale of the dominant (562 nm) cone system of the frog, Rana temporaria, were studied as functions of steady adapting illuminance (IB). Photoreceptor responses to brief flashes of light were recorded as aspartate-isolated ERG mass potentials from the isolated retina. The characteristics of the cone signal after transmission through the retina were derived from response thresholds and stimulus--intensity-response--latency functions for extracellularly recorded spike discharges of single ganglion cells in the eyecup. At 14 degrees C, the single-photon response of dark-adapted cones, extrapolated from ERG intensity-response functions, had an amplitude of 0.5% of the saturated response (Umax) and peaked at tp approximately 0.4 sec. Steady background illumination decreased both tp and flash sensitivity (SF), starting from apparent "dark lights" of, respectively, less than 10 (for time scale) and about 100 (for sensitivity) photoisomerisations per cone per second [P*sec-1]. From there upwards, two distinct ranges of background adaptation were apparent. Under moderate backgrounds (up to IB approximately 10(4) - 10(5) P*sec-1), sensitivity fell according to the relation SF alpha IB-0.64 and time scale shortened according to tp alpha IB-0.16. Under brighter backgrounds, from approx. 10(5) P*sec-1 up to the limit of our light source at 10(7) P*sec-1, the decrease in SF was significantly stronger than predicted by the Weber relation (SF alpha IB-1), while the decrease in tp levelled out and even tended to reverse. All these changes were virtually identical at the photoreceptor and ganglion cell levels, although the absolute time scale of cone signals apparent at the latter level was 2-fold longer. Our general conclusion is that photoreceptors have several distinct regimes for light adaptation, and traditional descriptions of functional changes (in sensitivity and kinetics) relevant to vision need to be restated with higher resolution, in view also of recent insights into the diversity of underlying mechanisms.

Regulation of intracellular pH in salamander retinal rods.

1. We measured intracellular pH (pHi) in rods isolated from the retina of the axolotl salamander, Ambystoma mexicanum, using the fluorescent indicator 2',7'-bis(carboxyethyl)-5(and -6)-carboxyfluorescein (BCECF). 2. The light exposures associated with data acquisition had no marked effect on pHi. There was no sharp change between the value obtained from the first exposure of dark-adapted rods and subsequent readings. Increasing the acquisition frequency from 1 to 10 min-1 either had no effect, or brought about a slow acidification, which was stopped or reversed when the low frequency was restored. 3. In nominally HCO3(-)-free solution at pH 7.5, the rods had a steady-state pHi of 7.09 +/- 0.02 (n = 46) and a buffering power (beta i) of 24 +/- 1 mM (pH unit)-1 (n = 48). The buffering power was virtually constant in the pH range 6.6-8.0. In the same range, pHi dependent linearly on perfusion pH (pHo) with regression coefficients of 0.4-0.5. 4. There were no significant differences between the inner and outer segment of intact rods as regards steady-state pHi or responses to experimental treatments. 5. Recovery from an intracellular acid load imposed by sodium propionate or an NH4Cl prepulse in nominally bicarbonate-free perfusate was completely blocked by decreasing the extracellular Na+ concentration to 7 mM, and slowed by 86% by applying 1 mM amiloride. 6. Introduction of 2% CO2-13 mM HCO3- caused an alkalinization that was often preceded by a transient acidification. Steady-state pHi was on average 0.1 pH units higher than in nominally bicarbonate-free solution. The mean acid extrusion rate, calculated on the assumption that CO2-HCO3- behaves as an open system, was 19% higher (31 +/- 2 mM h-1) than in a solution buffered only by Hepes (26 +/- 2 mM h-1). 7. In the presence of CO2-HCO3-, 100 microM 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) decreased the acid extrusion rate by 20% on average. Lowering the extracellular Cl-concentration to 7 mM raised pHi, but did not significantly affect the acid extrusion rate. 8. We conclude that retinal rods regulate pHi by both Na(+)-H+ exchange and mechanism(s) involving HCO3(-)-Cl- exchange. In the present conditions, the Na(+)-H+ exchanger appears as the dominant mechanism for acid extrusion.

pH changes in frog rods upon manipulation of putative pH-regulating transport mechanisms.

Rod intracellular pH (pHi) in the intact frog retina was measured fluorometrically with the dye 2',7'-bis(2-carboxyethyl)-5(and-6)-carboxyfluorescein under treatments chosen to affect putative pH-regulating transport mechanisms in the plasma membrane. The purpose was to relate possible pHi changes to previously reported effects on photoresponses. In nominally bicarbonate-free Ringer, application of amiloride (1 mM) or substitution of 95 mM external Na+ by K+ or choline triggered monotonic but reversible acidifications, consistent with inhibition of Na+/H+ exchange. Bicarbonate-dependent mechanisms were characterized as follows: (1) Replacing half of a 12 mM phosphate buffer by bicarbonate caused a sustained rise of pHi. (2) Subsequent application of the anion transport inhibitor 4,4'-diisothiocyanatostilbene-2',2'-disulphonic acid (DIDS, 0.2 mM) set off a slow acidification. (3) Substitution of external Cl- by gluconate (95 mM) caused a rapid pHi rise both in normal Na+ and low-Na+ perfusion. (4) This effect was inhibited by DIDS. The results support a consistent explanation of parallel electrophysiological experiments on the assumption that intracellular acidifications reduce and alkalinizations (in a certain range) augment photoresponses. It is concluded that both Na+/H+ exchange and bicarbonate transport control rod pHi, modulating the light-sensitive current. Part of the bicarbonate transport is by Na(+)-independent HCO3-/Cl- exchange, but a further Na(+)-coupled bicarbonate import mechanism is implicated.

Changes in retinal time scale under background light: observations on rods and ganglion cells in the frog retina.

The kinetics of rod responses to flashes and steps of light was studied as a function of background intensity (IB) at the photoreceptor and ganglion cell levels in the frog retina. Responses of the rod photoreceptors were recorded intracellularly in the eyecup and as ERG mass potentials across the isolated, aspartate-superfused retina. The kinetics of the retinally transmitted signal was derived from the latencies of ganglion cell spike discharges recorded extracellularly in the eyecup. In all states of adaptation the linear-range rod response to dim flashes could be modelled as the impulse response of a chain of low-pass filters with the same number of stages: 4 (ERG) or 4-6 (intracellular). Dark-adapted time-to-peak (tp, mean +/- SD) at 12 degrees C was 2.4 +/- 0.6 sec (ERG) or 1.7 +/- 0.4 sec (intracellular). Under background light, the time scale shortened as a power function of background intensity, I-bB with b = 0.19 +/- 0.03 (ERG) or 0.14 +/- 0.04 (intracellular). The latency-derived time scale of the rod-driven signal at the ganglion cell agreed well with that of the photoreceptor responses. The apparent underlying impulse response had tp = 2.0 +/- 0.7 sec in darkness and accelerated as I-bB with b = 0.17 +/- 0.03. The photoreceptor-to-ganglion-cell transmission delay shortened by 30% between darkness and a background delivering ca 10(4) photoisomerizations per rod per second. Data from the literature suggest that all vertebrate photoreceptors may accelerate according to similar power functions of adapting intensity, with exponents in the range 0.1-0.2. It is noteworthy that the time scale of human (foveal) vision in experiments on flicker sensitivity and temporal summation shortens as a power function of mean luminance with b approximately 0.15.

Spectral sensitivities of short- and long-wavelength sensitive cone mechanisms in the frog retina.

ERG mass photoreceptor responses were recorded across the isolated, aspartate-perfused retina of the frog, Rana temporaria, in order to determine spectral sensitivities of cones. Cone responses were distinguished from rod responses by their faster kinetics, and responses from different cone types were isolated by selective background adaptation. Our main finding is that of a novel short-wavelength sensitive cone population peaking at about 431 nm. Further, we find that the sensitivity spectrum of the dominant long-wavelength sensitive cone population fully accounts for the most common type of photopic ganglion cell spectrum. Both can be described by a nomogram with lambda max = 562 nm. This resolves a long-standing apparent conflict between cone absorbance spectra and ganglion cell sensitivities. Including the 502 nm cones previously described by microspectrophotometry, the frog possesses a collection of cones that could support trichromatic photopic vision.

Changes in the light-sensitive current of salamander rods upon manipulation of putative pH-regulating mechanisms in the inner and outer segment.

The light-sensitive current of dark-adapted rods isolated from the Ambystoma retina was recorded while either the inner or the outer segment (IS or OS) protruding from the suction pipette was exposed to treatments intended to reveal the physiological roles of pH-regulating transport mechanisms. Applied to the IS, both amiloride (presumed to block Na+/H+ exchange, 2 mM) and 4-4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) (presumed to block bicarbonate transport, 0.1 mM) generally abolished light sensitivity completely but reversibly, consistent with acidification of the IS. Yet, the circulating ("dark") current often persisted, implying that the OS was not acidified. Applied to the OS, amiloride depressed but DIDS increased the dark current and photoresponses. Given the fact that the current increases with rising OS-pHi, this suggests alkalinization, which could be due to DIDS inhibiting bicarbonate extrusion by HCO3-/Cl- exchangers in the OS. Consistent with this idea, replacing external Cl- by other anions increased the current as would be expected if HCO3-/Cl- exchange is reversed. We propose that the IS and OS manage their acid balances independently and with different sets of transport mechanisms. Acidosis in either compartment suppresses the photosensitivity of the rod, but by differing mechanisms.

pH regulation in frog cones studied by mass receptor photoresponses from the isolated retina.

Mass cone photoresponses were recorded across the aspartate-treated frog retina under treatments chosen to affect putative pH-regulating mechanisms. The saturated response amplitude (Umax) was found to be a monotonically increasing function of perfusion pH in the range 7-8, and thus presumably of intracellular pH (pHi). Accepting that Umax can be used as an index of pHi changes, two results indicate the importance of bicarbonate transport for preventing intracellular acidification: (1) bicarbonate-buffered (6 mM HCO3- + 6 mM HEPES) perfusate increased Umax compared with nominally bicarbonate-free perfusate (12 mM HEPES); (2) the anion transport blocker DIDS (0.1 mM) caused a strong decrease in the amplitude of photoresponses. Substitution of 95 mM chloride by gluconate in the perfusing fluid boosted photoresponses indicating that at least part of the bicarbonate transport involves HCO3-/Cl- exchange. Amiloride (2 mM) also caused a decrease of photoresponse amplitude, which suggests that Na+/H+ exchange contributes to pHi regulation. In all these respects, cones behaved similarly to rods. Cones differed from rods (in the intact retina) in that addition of 0.5 mM of the carbonic anhydrase inhibitor acetazolamide reduced (never augmented) photoresponses. The difference is considered in relation to the presence of carbonic anhydrase in cone, as opposed to rod, outer segments.

On the relation between ERG waves and retinal function: inverted rod photoresponses from the frog retina.

In rod mass receptor photoresponses recorded across the isolated frog retina, a paradoxical cornea-positive wave may precede the response of normal polarity. We present a model which shows that the light-induced decrease in rod current can give rise to inverted or biphasic ERG signals if the distal part (tip) of the rod outer segment responds more slowly and/or less sensitively than the proximal part (base). The condition is that current entering at the tip is represented with greater weight in the ERG. The model reproduces recorded ERG waveforms well. It further predicts that if there is a light-insensitive conductance in the tip membrane, ERG photoresponses may be non-recordable although current photoresponses are only slightly reduced. The model reveals a type of complexity in the relation between mass potentials and underlying physiological processes which has not previously received attention.

Rod phototransduction modulated by bicarbonate in the frog retina: roles of carbonic anhydrase and bicarbonate exchange.

1. Effects on rod phototransduction following manipulation of retinal CO2-HCO3- and H+ fluxes were studied in dark-adapted retinas of the frog and the tiger salamander. 2. Rod photoresponses to brief flashes of light were recorded from the isolated sensory retina as electroretinogram mass receptor potentials and from isolated rods by the suction-pipette technique. The experimental treatments were: (1) varying [CO2] + [HCO3-] in the perfusion fluid: (2) applying acetazolamide (AAA), which inhibits the enzyme carbonic anhydrase (CA); and (3) applying 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) which blocks exchange mechanisms transporting HCO3- across cell membranes. 3. The concentration of the internal transmitter of the rods, cyclic GMP, was biochemically determined from the rod outer segment layer of retinas that had been incubated in the same solutions as were used for perfusion in the electrophysiological experiments. 4. The introduction of 6 mM-sodium bicarbonate to replace half the buffer of a nominally CO2-HCO3(-)-free (12 mM-phosphate or HEPES, [Na+] constant) Ringer solution doubled the cyclic GMP concentration in the rod outer segment layer and increased the saturating response amplitude and the relative sensitivity of rods in the intact retina. 5. The introduction of 0.5 mM-AAA into bicarbonate-containing Ringer solution accelerated the growth of saturated responses and sensitivity. Incubation of the retina in AAA-bicarbonate Ringer solution elevated the concentration of cyclic GMP ninefold compared with the phosphate control. 6. No effects of switching to bicarbonate-AAA Ringer solution were observed in the photocurrent of isolated rods drawn into suction pipettes with only the outer segment protruding into the perfusion fluid. The target of AAA is probably the CA-containing Müller cell. 7. The introduction of DIDS into the perfusate (at normal pH 7.5) set off a continuous decay of photoresponses which finally abolished light sensitivity completely. The decay proceeded regardless of whether bicarbonate and AAA were present or not. 8. Rods that had lost their photosensitivity in DIDS recovered almost fully when the pH of the DIDS perfusate was raised to 8.5. They also recovered when DIDS was washed out with bicarbonate Ringer solution at constant pH (7.5). 9. It is proposed that all our treatments ultimately modulate the intracellular pH of the rods which is determined by the relative rates of H+ leakage and HCO3- transport into the cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Sulfhydryl binding reagents increase the conductivity of the light-sensitive channel and inhibit phototransduction in retinal rods.

The mechanisms by which sulfhydryl (SH-) binding reagents modulate the light-sensitive conductance of retinal rods were investigated by current recording from single rods, by patch clamp recording from the plasma membrane of the rod outer segment (ROS), and by biochemical study of their effects on the light-induced hydrolysis of cyclic GMP. The electrophysiology, as well as measurements of the reagents' ability to traverse the ROS plasma membrane, was done on amphibian (Rana and Ambystoma) rods, and the biochemistry on bovine rods. The main SH-reagents used were N-ethyl-maleimide (NEM) and iodoacetamide (IAA). Both transiently increased rod current, but part of the large current could not be turned off by light. After a few minutes' exposure, NEM, but not IAA, caused a continuous decay of the rod's light sensitivity. In patch-clamp recordings from the ROS plasma membrane, the reagents increased conductivity both in the presence and absence of cGMP, consistent with the observation that the drug-induced current increase in intact rods involved both light-sensitive and light-insensitive components. In vitro, NEM was found to be a powerful inhibitor of cGMP hydrolysis, which can explain the gradual loss of light sensitivity in the rod and could initially contribute to the increased dark current via elevated cGMP levels. Thus, SH-reagents act both by modifying the light-sensitive channel and by inhibiting phototransduction inside the rod.

Temperature-dependence of rod photoresponses from the aspartate-treated retina of the frog (Rana temporaria).

The effects of temperature changes on rod photoresponses were studied by recording the aspartate-isolated mass receptor potential in the dark-adapted retina of the frog Rana temporaria. The amplitude of saturating responses, indicating the magnitude of the dark current, increased linearly with temperature in the measured range 6-26 degrees C, extrapolating to zero dark current at 0 degrees C. Sensitivity was maximal around 18 degrees C but the decrease towards lower temperatures was shallow. The results show that rod phototransduction in the frog Rana temporaria is adapted to lower temperatures than in the tropical toad Bufo marinus. Responses to dim flashes were, approximately up to peak, well fitted by the same 'independent activation' model with four delay stages as have been found to best describe current responses from single toad rods. The kinetics (reciprocal time-to-peak) showed Arrhenius-type temperature-dependence with apparent activation energy 12.4 kcal mol-1 and Q10 = 2.1.

Transient sensitivity reduction and biphasic photoresponses observed when frog retinal rods are oxidized.

1. Rod photoresponses and the effects of oxidation have been studied by recording either the transretinal voltage in aspartate-treated retinas or the outer segment current of single rods. 2. Oxidizing conditions transiently decreased, reducing conditions increased sensitivity. 3. Biphasic photoresponses were seen when the level of oxidation was rising and also in some other sensitivity-depressing conditions. 4. A model is proposed which explains the biphasic responses in terms of sensitivity differences between the tip and the base of the rod outer segment.