M. Carter Cornwall

Intra-retinal visual cycle required for rapid and complete cone dark adaptation

Daytime vision is mediated by retinal cones, which, unlike rods, remain functional even in bright light and dark-adapt rapidly. These cone properties are enabled by rapid regeneration of their pigment. This in turn requires rapid chromophore recycling that may not be achieved by the canonical retinal pigment epithelium visual cycle. Recent biochemical studies have suggested the presence of a second, cone-specific visual cycle, although its physiological function remains to be established. We found that the Müller cells in the salamander neural retina promote cone-specific pigment regeneration and dark adaptation that are independent of the pigment epithelium. Without this pathway, dark adaptation of cones was slow and incomplete. Notably, the rates of cone pigment regeneration by the retina and pigment epithelium visual cycles were essentially identical, suggesting a possible common rate-limiting step. Finally, we also observed cone dark adaptation in the isolated mouse retina.

Breaking the Covalent Bond— A Pigment Property that Contributes to Desensitization in Cones

Retinal rod and cone pigments consist of an apoprotein, opsin, covalently linked to a chromophore, 11-cis retinal. Here we demonstrate that the formation of the covalent bond between opsin and 11-cis retinal is reversible in darkness in amphibian red cones, but essentially irreversible in red rods. This dissociation, apparently a general property of cone pigments, results in a surprisingly large amount of free opsin--about 10% of total opsin--in dark-adapted red cones. We attribute this significant level of free opsin to the low concentration of intracellular free 11-cis retinal, estimated to be only a tiny fraction (approximately 0.1 %) of the pigment content in red cones. With its constitutive transducin-stimulating activity, the free cone opsin produces an approximately 2-fold desensitization in red cones, equivalent to that produced by a steady light causing 500 photoisomerizations s-1. Cone pigment dissociation therefore contributes to the sensitivity difference between rods and cones.

A Visual Pigment Expressed in Both Rod and Cone Photoreceptors

Rods and cones contain closely related but distinct G protein-coupled receptors, opsins, which have diverged to meet the differing requirements of night and day vision. Here, we provide evidence for an exception to that rule. Results from immunohistochemistry, spectrophotometry, and single-cell RT-PCR demonstrate that, in the tiger salamander, the green rods and blue-sensitive cones contain the same opsin. In contrast, the two cells express distinct G protein transducin alpha subunits: rod alpha transducin in green rods and cone alpha transducin in blue-sensitive cones. The different transducins do not appear to markedly affect photon sensitivity or response kinetics in the green rod and blue-sensitive cone. This suggests that neither the cell topology or the transducin is sufficient to differentiate the rod and the cone response.

Visual Cycle: Dependence of Retinol Production and Removal on Photoproduct Decay and Cell Morphology

The visual cycle is a chain of biochemical reactions that regenerate visual pigment following exposure to light. Initial steps, the liberation of all-trans retinal and its reduction to all-trans retinol by retinol dehydrogenase (RDH), take place in photoreceptors. We performed comparative microspectrophotometric and microfluorometric measurements on a variety of rod and cone photoreceptors isolated from salamander retinae to correlate the rates of photoproduct decay and retinol production. Metapigment decay rate was spatially uniform within outer segments and 50–70 times faster in the cells that contained cone-type pigment (SWS2 and M/LWS) compared to cells with rod-type pigment (RH1). Retinol production rate was strongly position dependent, fastest at the base of outer segments. Retinol production rate was 10–40 times faster in cones with cone pigments (SWS2 and M/LWS) than in the basal OS of rods containing rod pigment (RH1). Production rate was approximately five times faster in rods containing cone pigment (SWS2) than the rate in basal OS of rods containing the rod pigment (RH1). We show that retinol production is defined either by metapigment decay rate or RDH reaction rate, depending on cell type or outer segment region, whereas retinol removal is defined by the surface-to-volume ratio of the outer segment and the availability of retinoid binding protein (IRBP). The more rapid rates of retinol production in cones compared to rods are consistent with the more rapid operation of the visual cycle in these cells.

Opsin activation as a cause of congenital night blindness

Three different mutations of rhodopsin are known to cause autosomal dominant congenital night blindness in humans. Although the mutations have been studied for 10 years, the molecular mechanism of the disease is still a subject of controversy. We show here, using a transgenic Xenopus laevis model, that the photoreceptor cell desensitization that is a hallmark of the disease results from persistent signaling by constitutively active mutant opsins.

Role of noncovalent binding of 11-cis-retinal to opsin in dark adaptation of rod and cone photoreceptors.

Regeneration of visual pigments of vertebrate rod and cone photoreceptors occurs by the initial noncovalent binding of 11-cis-retinal to opsin, followed by the formation of a covalent bond between the ligand and the protein. Here, we show that the noncovalent interaction between 11-cis-retinal and opsin affects the rate of dark adaptation. In rods, 11-cis-retinal produces a transient activation of the phototransduction cascade that precedes sensitivity recovery, thus slowing dark adaptation. In cones, 11-cis-retinal immediately deactivates phototransduction. Thus, the initial binding of the same ligand to two very similar G protein receptors, the rod and cone opsins, activates one and deactivates the other, contributing to the remarkable difference in the rates of rod and cone dark adaptation.

Absorptance and spectral sensitivity measurements of rod photoreceptors of the tiger salamander, Ambystoma tigrinum.

The spectral sensitivity of the extracellularly-recorded photoresponse of isolated rods of the tiger salamander, Ambystoma tigrinum, was compared to the absorptance spectrum. Both measurements were made with the same optical system on the same portion of each cell to avoid errors that could occur when the two kinds of measurement were made under different conditions. The relative spectral sensitivity and absorptance spectrum were found to be in excellent agreement between 450 and 700 nm.

Movement of retinal along cone and rod photoreceptors

Single isolated photoreceptors can be taken through a visual cycle of light adaptation by bleaching visual pigment, followed by dark adaptation when supplied with 11-cis retinal. Light adaptation after bleaching is manifested by faster response kinetics and a permanent reduction in sensitivity to light flashes, presumed to be due to the presence of bleached visual pigment. The recovery of flash sensitivity during dark adaptation is assumed to be due to regeneration of visual pigment to pre-bleach levels. In previous work, the outer segments of bleached, light-adapted cells were exposed to 11-cis retinal. In the present work, the cell bodies of bleached photoreceptors were exposed. We report a marked difference between rods and cones. Bleached cones recover sensitivity when their cell bodies are exposed to 11-cis retinal. Bleached rods do not. These results imply that retinal can move freely along the cone photoreceptor, but retinal either is not taken up by the rod cell body or retinal cannot move from the rod cell body to the rod outer segment. The free transfer of retinal along cone but not along rod photoreceptors could explain why, during dark adaptation in the retina, cones have access to a store of 11-cis retinal which is not available to rods. Additional experiments investigated the movement of retinal along bleached rod outer segments. The results indicate that retinal can move along the rod outer segment, but that this movement is slow, occurring at about the same rate as the regeneration of visual pigment.

Physiological and Microfluorometric Studies of Reduction and Clearance of Retinal in Bleached Rod Photoreceptors

The visual cycle comprises a sequence of reactions that regenerate the visual pigment in photoreceptors during dark adaptation, starting with the reduction of all-trans retinal to all-trans retinol and its clearance from photoreceptors. We have followed the reduction of retinal and clearance of retinol within bleached outer segments of red rods isolated from salamander retina by measuring its intrinsic fluorescence. Following exposure to a bright light (bleach), increasing fluorescence intensity was observed to propagate along the outer segments in a direction from the proximal region adjacent to the inner segment toward the distal tip. Peak retinol fluorescence was achieved after ∼30 min, after which it declined very slowly. Clearance of retinol fluorescence is considerably accelerated by the presence of the exogenous lipophilic substances IRBP (interphotoreceptor retinoid binding protein) and serum albumin. We have used simultaneous fluorometric and electrophysiological measurements to compare the rate of reduction of all-trans retinal to all-trans retinol to the rate of recovery of flash response amplitude in these cells in the presence and absence of IRBP. We find that flash response recovery in rods is modestly accelerated in the presence of extracellular IRBP. These results suggest such substances may participate in the clearance of retinoids from rod photoreceptors, and that this clearance, at least in rods, may facilitate dark adaptation by accelerating the clearance of photoproducts of bleaching.

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.