Absorbance spectra were recorded by microspectrophotometry from 39 different rod and cone types representing amphibians. reptiles, and fishes, with A1- or A2-based visual pigments and lambdamax ranging from 357 to 620 nm. The purpose was to investigate accuracy limits of putative universal templates for visual pigment absorbance spectra, and if possible to amend the templates to overcome the limitations. It was found that (1) the absorbance spectrum of frog rhodopsin extract very precisely parallels that of rod outer segments from the same individual, with only a slight hypsochromic shift in lambdamax, hence templates based on extracts are valid for absorbance in situ: (2) a template based on the bovine rhodopsin extract data of Partridge and De Grip (1991) describes the absorbance of amphibian rod outer segments excellently, contrary to recent electrophysiological results; (3) the lambdamax/lambda invariance of spectral shape fails for A1 pigments with small lambdamax and for A2 pigments with large lambdamax, but the deviations are systematic and can be readily incorporated into, for example, the Lamb (1995) template. We thus propose modified templates for the main "alpha-band" of A1 and A2 pigments and show that these describe both absorbance and spectral sensitivities of photoreceptors over the whole range of lambdamax. Subtraction of the alpha-band from the full absorbance spectrum leaves a "beta-band" described by a lambdamax-dependent Gaussian. We conclude that the idea of universal templates (one for A1- and one for A2-based visual pigments) remains valid and useful at the present level of accuracy of data on photoreceptor absorbance and sensitivity. The sum of our expressions for the alpha- and beta-band gives a good description for visual pigment spectra with lambdamax > 350 nm.

The weakest pulse of light a human can detect sends about 100 photons through the pupil and produces 10-20 rhodopsin isomerizations in a small retinal area. It has been postulated that we cannot see single photons because of a retinal noise arising from randomly occurring thermal isomerizations. Direct recordings have since demonstrated the existence of electrical 'dark' rod events indistinguishable from photoisomerization signals. Their mean rate of occurrence is roughly consistent with the 'dark light' in psychophysical threshold experiments, and their thermal parameters justify an identification with thermal isomerizations. In the retina of amphibians, a small proportion of sensitive ganglion cells have a performance-limiting noise that is low enough to be well accounted for by these events. Here we study the performance of dark-adapted toads and frogs and show that the performance limit of visually guided behaviour is also set by thermal isomerizations. As visual sensitivity limited by thermal events should rise when the temperature falls, poikilothermous vertebrates living at low temperatures should then reach light sensitivities unattainable by mammals and birds with optical factors equal. Comparison of different species at different temperatures shows a correlation between absolute threshold intensities and estimated thermal isomerization rates in the retina.

A visual pigment molecule in a retinal photoreceptor cell can be activated not only by absorption of a photon but also "spontaneously" by thermal energy. Current estimates of the activation energies for these two processes in vertebrate rod and cone pigments are on the order of 40-50 kcal/mol for activation by light and 20-25 kcal/mol for activation by heat, which has forced the conclusion that the two follow quite different molecular routes. It is shown here that the latter estimates, derived from the temperature dependence of the rate of pigment-initiated "dark events" in rods, depend on the unrealistic assumption that thermal activation of a complex molecule like rhodopsin (or even its 11-cis retinaldehyde chromophore) happens through a simple process, somewhat like the collision of gas molecules. When the internal energy present in the many vibrational modes of the molecule is taken into account, the thermal energy distribution of the molecules cannot be described by Boltzmann statistics, and conventional Arrhenius analysis gives incorrect estimates for the energy barrier. When the Boltzmann distribution is replaced by one derived by Hinshelwood for complex molecules with many vibrational modes, the same experimental data become consistent with thermal activation energies that are close to or even equal to the photoactivation energies. Thus activation by light and by heat may in fact follow the same molecular route, starting with 11-cis to all-trans isomerization of the chromophore in the native (resting) configuration of the opsin. Most importantly, the same model correctly predicts the empirical correlation between the wavelength of maximum absorbance and the rate of thermal activation in the whole set of visual pigments studied.

Literature data on light detection by cone and rod vision at absolute threshold are analysed in order (1) to decide whether the threshold performance of dark-adapted cone vision can, like that of rod vision, be consistently explained as limited by noise from a "dark light"; (2) to obtain comparable estimates of the dark noise and dark light of (foveal) cones and (peripheral) rods. The dark noise was estimated by a maximum-likelihood procedure from frequency-of-seeing data and compared with the dark light derived from increment-threshold functions. In both cone and rod vision, the estimated dark noise coincides with Poisson fluctuations of the estimated dark light if 17% (best estimate) of lambda max-quanta incident at the cornea produce excitations. At that fraction of quanta exciting, dark lights are equivalent to 112 isomerisations per sec in each foveal cone and 0.011 isomerisations per sec in each rod. It is concluded that (1) the threshold performance of dark-adapted cone as well as rod vision can be consistently described as noise-limited, but not by postulating a multi-quantum coincidence requirement for single receptors; (2) the underlying intrinsic activity in both the cone and the rod system is light-like as regards correspondence between noise effect and background adaptation effect. One possibility is that this activity is largely composed of events identical to the single-photon response, originating in the visual pigment, in cones as well as in rods.

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SUMMARY1. 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-' s-1 at 18°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-' s-I (except in one rod where it was 0-006 events rod-' s-). 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

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 (t p ) was somewhat steeper in rat (Q 10 ≈ 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 phototransduction cascade and its regulatory mechanisms are basically similar in all rod photoreceptors that have been studied (see . On the other hand, quantitative parameters of amplification, activation and deactivation kinetics, and light adaptation derived from the electrical responses to light differ so as to suggest important differences in the functioning of the phototransduction molecules in mammals and 'lower vertebrates' (commonly represented by amphibians). The rods of both classes can respond reliably to a single photon, but the initial amplification rate in mammalian rods is higher by two orders of magnitude and the response peaks at a much earlier time after photon absorption (Baylor et al. 1979b(Baylor et al. , 1984Matthews, 1991;Robinson et al. 1993;Kraft et al. 1993;Nikonov et al. 2000). Although mammalian rods, including those of humans, do have the capacity to light adapt, ...

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