Ari Koskelainen

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 μm × 22 μm and had specific absorbance of 0.016 μm−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°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 (tp) was somewhat steeper in rat (Q10≈ 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°C was in the range 20–50 Rh* s−1 in both, compared with ca 4500 Rh* s−1 in rat rods at 36°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.

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.

On the relation between the photoactivation energy and the absorbance spectrum of visual pigments

We relate the collected experimental data on the minimum energy for photoactivation (E(a)) to the wavelengths of peak absorbance (lambda(max)) of 12 visual pigments. The E(a) values have been determined from the temperature-dependence of spectral sensitivity in the long-wavelength range. As shown previously, the simple physical idea E(a) =const. x (1/lambda(max)) (here termed the Stiles-Lewis-Barlow or SLB relation) does not hold strictly. Yet there is a significant correlation between E(a) and 1/lambda(max) (r(2)=0.73) and the regression slope obtained by an unbiased fit is 84% of the predicted value of the best SLB fit. The correlation can be decomposed into effects of A1 --> A2 chromophore change and effects of opsin differences. For a chromophore change in the same opsin, studied in two A1/A2 pigment pairs, the SLB relation holds nearly perfectly. In seven pigments having different opsins but the same (A2) chromophore, the correlation of E(a) and 1/lambda(max) remained highly significant (r(2)=0.78), but the regression coefficient is only 72% of the best SLB fit. We conclude that (1) when the chromophore is exchanged in the same opsin, the lambda(max) shift directly reflects the difference in photoactivation energies, (2) when the opsin is modified by amino acid substitutions, lambda(max) and E(a) can be tuned partly independently, although there is a dominant tendency for inverse proportionality. In four (A1) rhodopsins with virtually the same lambda(max), E(a) varied over a 4.5 kcal/mol range, which may be taken as a measure of the freedom for independent tuning. Assuming that low E(a) correlates with high thermal noise, we suggest that the leeway in lambda(max) - E(a) coupling is used by natural selection to keep E(a) as high as possible in long-wavelength-sensitive pigments, and that this is why the opsin-dependent E(a) (1/lambda(max))-relation is shallower than predicted.

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 (λmax) 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, 5,6,7) and test experimentally the predictions of an inverse relation between Ea and λmax. For rods and ‘red’ cones in the adult frog retina, we find no significant difference in Ea between the two pigments involved, although their λmax 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 λmax 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 λmax and Ea, but show that the A1 → A2 chromophore substitution decreases Ea.

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.

Calcium Sets the Physiological Value of the Dominant Time Constant of Saturated Mouse Rod Photoresponse Recovery

Background The rate-limiting step that determines the dominant time constant (τD) of mammalian rod photoresponse recovery is the deactivation of the active phosphodiesterase (PDE6). Physiologically relevant Ca2+-dependent mechanisms that would affect the PDE inactivation have not been identified. However, recently it has been shown that τD is modulated by background light in mouse rods. Methodology/Principal Findings We used ex vivo ERG technique to record pharmacologically isolated photoreceptor responses (fast PIII component). We show a novel static effect of calcium on mouse rod phototransduction: Ca2+ shortens the dominant time constant (τD) of saturated photoresponse recovery, i.e., when extracellular free Ca2+ is decreased from 1 mM to ∼25 nM, the τD is reversibly increased ∼1.5–2-fold. Conclusions We conclude that the increase in τD during low Ca2+ treatment is not due to increased [cGMP], increased [Na+] or decreased [ATP] in rod outer segment (ROS). Also it cannot be due to protein translocation mechanisms. We suggest that a Ca2+-dependent mechanism controls the life time of active PDE.

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.

Rod Phototransduction Determines the Trade-Off of Temporal Integration and Speed of Vision in Dark-Adapted Toads

Human vision is ∼10 times less sensitive than toad vision on a cool night. Here, we investigate (1) how far differences in the capacity for temporal integration underlie such differences in sensitivity and (2) whether the response kinetics of the rod photoreceptors can explain temporal integration at the behavioral level. The toad was studied as a model that allows experimentation at different body temperatures. Sensitivity, integration time, and temporal accuracy of vision were measured psychophysically by recording snapping at worm dummies moving at different velocities. Rod photoresponses were studied by ERG recording across the isolated retina. In both types of experiments, the general timescale of vision was varied by using two temperatures, 15 and 25°C. Behavioral integration times were 4.3 s at 15°C and 0.9 s at 25°C, and rod integration times were 4.2–4.3 s at 15°C and 1.0–1.3 s at 25°C. Maximal behavioral sensitivity was fivefold lower at 25°C than at 15°C, which can be accounted for by inability of the “warm” toads to integrate light over longer times than the rods. However, the long integration time at 15°C, allowing high sensitivity, degraded the accuracy of snapping toward quickly moving worms. We conclude that temporal integration explains a considerable part of all variation in absolute visual sensitivity. The strong correlation between rods and behavior suggests that the integration time of dark-adapted vision is set by rod phototransduction at the input to the visual system. This implies that there is an inexorable trade-off between temporal integration and resolution.

A novel Ca2+-feedback mechanism extends the operating range of mammalian rods to brighter light

A previously unidentified calcium-dependent mechanism contributes to light adaptation in mammalian rods.

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.