Kristian Donner

Dark-adaptation processes in the rhodopsin rods of the frog's retina.

Abstract In the excised and opened frogs eye two phases of rod dark-adaptation can be distinguished. The slower phase, previously studied by us, depends on the rate of regeneration of rhodopsin. Supplementary evidence is here given based on measurements involving temperature changes of the eye during adaptation. The rate of regeneration, which has a Q10 of about 4.6, is thereby rapidly altered. This brings about quantitatively corresponding changes in the sensitivity of the single units studied. The initial, rapid phase of rod adaptation is found to depend on the decomposition of metarhodopsin in the rods, which shows the same time-course at different temperatures as the adaptation process. Here log threshold is found to be proportional to the amount of metarhodopsin. The evidence obtained in experiments at different temperatures further suggests that it is metarhodopsin II, which exists in a tautomeric equilibrium with metarhodopsin I, that causes this desensitization of the rods.

Visual performance of the toad (Bufo bufo) at low light levels: retinal ganglion cell responses and prey-catching accuracy.

The accuracy of toad snapping towards moving worm dummies under various levels of dim illumination (from absolute threshold to “moonlight”) was videorecorded and related to spike responses of retinal ganglion cells exposed to equivalent stimuli. Some toads (at ca. 16 °C) successfully snapped at dummies that produced only one photoisomerization per 50 rods per second in the retina, in good agreement with thresholds of sensitive retinal ganglion cells. One factor underlying such high sensitivity is extensive temporal summation by the ganglion cells. This, however, is inevitably accompanied by very long response latencies (around 3 s near threshold), whereby the information reaching the brain shows the dummy in a position where it was several seconds earlier. Indeed, as the light was dimmed, snaps were displaced successively further to the rear of the dummy, finally missing it. The results in weak but clearly supra-threshold illumination indicate that snaps were aimed at the advancing head as seen by the brain, but landed further backwards in proportion to the retinal latency. Near absolute threshold, however, accuracy was “too good”, suggesting that the animal had recourse to a neural representation of the regularly moving dummies to correct for the slowness of vision.

Visual adaptation of the rhodopsin rods in the frog's retina

1. The threshold of the discharge from single ganglion cells in the excised and opened frogs eye has been measured with on/off stimuli and test parameters that make it possible to activate the rhodopsin rods only. The test stimuli have been restricted to the central part of the receptive field, where no nervous reorganization can be observed with changes in the state of adaptation.

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.

The frequency of isomerization-like 'dark' events in rhodopsin and porphyropsin rods of the bull-frog retina.

1. The dark current and responses to dim flashes were recorded with the suction pipette technique from single rods in pieces of bull‐frog retina taken from either the dorsal porphyropsin or the ventral rhodopsin field. 2. The composition of visual pigment in the rods was determined by microspectrophotometry. Rods from the dorsal pieces contained 70‐88% porphyropsin523 mixed with rhodopsin502. The ventral rods contained almost pure rhodopsin, any possible admixture of porphyropsin being below the level of detectability (less than 5%). 3. In most cells, the responses to dim flashes were well fitted by a four‐stage linear filter model, with no systematic differences in the response kinetics of porphyropsin and rhodopsin rods. The amplitude of saturated responses varied between 8 and 55 pA and that of responses to single isomerizations between 0.4 and 3.5 pA. 4. In porphyropsin rods, discrete events similar to the response to one photoisomerization were clearly seen in complete darkness. The dark current amplitude histogram was fitted by a convolution of the probability densities for the Gaussian continuous noise component and the averaged dim‐flash response waveform. This allows estimation of the frequency and amplitude of discrete events and the standard deviation of the continuous component. The mean frequency of discrete dark events thus obtained from six porphyropsin cells was 0.057 rod‐1 s‐1 at 18 degrees C. 5. In rhodopsin rods, the dark current amplitude histogram appeared completely symmetrical, indicating that the frequency of discrete events must be lower than 0.005 rod‐1 s‐1 (except in one rod where it was 0.006 events rod‐1 s‐1). Per molecule of rhodopsin, the events are then at least 5 times rarer than reported for toad rhodopsin rods at the same temperature. 6. The low rate of isomerization‐like ‘dark’ events in bull‐frog rhodopsin rods shows, firstly, that results cannot be generalized across species even for rhodopsins which appear spectrally identical. Secondly, it suggests that these events need not (in an evolutionary sense) constitute an irreducible noise factor which must set the ultimate limit to the sensitivity of dark‐adapted vision. 7. The difference between porphyropsin and rhodopsin rods shows that, given (presumably) the same opsin, the pigment utilizing retinal2 and absorbing maximally at longer wavelengths produces more noise. The signal/noise ratio attained in the photoreceptor may be an important factor in the natural selection of visual pigments.

The dark-adaptation of single units in the frog's retina and its relation to the regeneration of rhodopsin.

Abstract The dark-adaptation of single units in excised and opened frogs eyes has been recorded with such test parameters that a nervous reorganization or changes in summation within the receptive fields can be excluded as explanations of the sensitivity increase observed. Further, using light of wave-length 500 nm cone contributions can be excluded after more than 40 min of dark-adaptation. It has thus been possible to measure the sensitivity changes caused by the rhodopsin rods. Because the regeneration of rhodopsin has also been measured, the significance of the regeneration process for the sensitivity changes can be analysed. It is found, that the log threshold is linearly related to the log rate of regeneration at each instant, when the threshold is expressed in terms of the relative number of quanta absorbed. Moreover, this relationship is the same as that observed between the increment threshold and the intensity of the corresponding adapting field.

Kinetics of long-lived rhodopsin photoproducts in the frog retina as a function of the amount bleached

Abstract Results are reported on measurements on the isolated, perfused frog retina of the decay of the long-lived photoproducts of rhodopsin as a function of the amount bleached. We find that the rates of decay of metarhodopsin II and III are increased when the amount bleached is reduced below about 15 per cent of the total. This leads to a decay process where very little (or finally no) metarhodopsin III is formed (bleaches smaller than 2 per cent). After small bleaches a restricted capacity for regeneration of rhodopsin is found In total, the isolated retina appears able to regenerate about 10 per cent of the total rhodopsin.

Chromophore switch from 11-cis-dehydroretinal (A2) to 11-cis-retinal (A1) decreases dark noise in salamander red rods

Dark noise, light‐induced noise and responses to brief flashes of light were recorded in the membrane current of isolated rods from larval tiger salamander retina before and after bleaching most of the native visual pigment, which mainly has the 11‐cis‐3,4‐dehydroretinal (A2) chromophore, and regenerating with the 11‐cis‐retinal (A1) chromophore in the same isolated rods. The purpose was to test the hypothesis that blue‐shifting the pigment by switching from A2 to A1 will decrease the rate of spontaneous thermal activations and thus intrinsic light‐like noise in the rod. Complete recordings were obtained in five cells (21°C). Based on the wavelength of maximum absorbance, λmax,A1= 502 nm and λmax,A2= 528 nm, the average A2 : A1 ratio determined from rod spectral sensitivities and absorbances was ∼0.74 : 0.26 in the native state and ∼0.09 : 0.91 in the final state. In the native (A2) state, the single‐quantum response (SQR) had an amplitude of 0.41 ± 0.03 pA and an integration time of 3.16 ± 0.15 s (mean ±s.e.m.). The low‐frequency branch of the dark noise power spectrum was consistent with discrete SQR‐like events occurring at a rate of 0.238 ± 0.026 rod−1 s−1. The corresponding values in the final state were 0.57 ± 0.07 pA (SQR amplitude), 3.47 ± 0.26 s (SQR integration time), and 0.030 ± 0.006 rod−1 s−1 (rate of dark events). Thus the rate of dark events per rod and the fraction of A2 pigment both changed by ca 8‐fold between the native and final states, indicating that the dark events originated mainly in A2 molecules even in the final state. By extrapolating the linear relation between event rates and A2 fraction to 0% A2 (100% A1) and 100% A2 (0% A1), we estimated that the A1 pigment is at least 36 times more stable than the A2 pigment. The noise component attributed to discrete dark events accounted for 73% of the total dark current variance in the native (A2) state and 46% in the final state. The power spectrum of the remaining ‘continuous’ noise component did not differ between the two states. The smaller and faster SQR in the native (A2) state is consistent with the idea that the rod behaves as if light‐adapted by dark events that occur at a rate of nearly one per integration time. Both the decreased level of dark noise and the increased SQR amplitude must significantly improve the reliability of photon detection in dim light in the presence of the A1 chromophore compared to the native (A2) state in salamander 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.

Retinal origins of the temperature effect on absolute visual sensitivity in frogs.

1. The absolute sensitivity of vision was studied as a function of temperature in two species of frog (Rana temporaria, 9‐21 degrees C, and Rana pipiens, 13‐28 degrees C). 2. Log behavioural threshold (measured as the lowest light intensity by which frogs trying to escape from a dark box were able to direct their jumping) rose near‐linearly with warming with a regression coefficient of 1.26 +/‐ 0.03 log units per 10 degrees C (Q10 = 18). Threshold retinal illumination corresponded to 0.011 photoisomerizations per rod per second (Rh* s‐1) at 16.5 degrees C. 3. The effect of dim backgrounds on jumping thresholds suggested ‘dark lights’ of 0.011 Rh* s‐1 at 16.5 degrees C and 0.080 Rh* s‐1 at 23.5 degrees C, corresponding to Q10 = 17. 4. Response thresholds of retinal ganglion cells were extracellularly recorded in the isolated eyecup of R. temporaria. The thresholds of the most sensitive cells when stimulated with large‐field steps of light were similar to the behavioural threshold and changed with temperature in a similar manner. 5. The decrease in ganglion cell step’ sensitivity with warming consisted of a decrease in summation time (by a factor of 2‐3 between 10 and 20 degrees C) and an increase in the threshold number of photoisomerizations (a decrease in ‘flash’ sensitivity, by a factor of 2‐5 over the same interval). No effect of temperature changes on spatial summation was found. 6. Frequency‐of‐response functions of ganglion cells indicated an 11‐fold increase in noise‐equivalent dark light between 10 and 20 degrees C (mean values in four cells 0.009 vs. 0.10 Rh* s‐1). 7. The temperature dependence of ganglion cell flash sensitivity could be strongly decreased with dim background illumination. 8. It is concluded that the desensitization of dark‐adapted vision with rising temperature is a retinal effect composed of shortened summation time and lowered flash sensitivity (increased numbers of photons required for a threshold response) in ganglion cells. The desensitization bears no simple relation to the apparent retinal noise increase.