Using suction electrodes, photocurrent responses to 100-ms saturating flashes were recorded from isolated retinal rods of the larval-stage tiger salamander (Ambystoma tigrinum). The delay period (7" c ) that preceded recovery of the dark current by a criterion amount (3 pA) was analyzed in relation to the flash intensity (If), and to the corresponding fractional bleach (R* 0 /R lol ) of the visual pigment; Rl/R lol was compared with R*/R lol , the fractional bleach at which the peak level of activated transducin approaches saturation. Over an approximately 8 In unit range of If that included the predicted value of R*/R lol , T c increased linearly with In If. Within the linear range, the slope of the function yielded an apparent exponential time constant (T C ) of 1.7 ± 0.2 s (mean ± S.D.). Background light reduced the value of T c measured at a given flash intensity but preserved a range over which T c increased linearly with In If-, the linear-range slope was similar to that measured in the absence of background light. The intensity dependence of T c resembles that of a delay (T d ) seen in light-scattering experiments on bovine retinas, which describes the period of essentially complete activation of transducin following a bright flash; the slope of the function relating T d and In flash intensity is thought to reflect the lifetime of photoactivated visual pigment (/?*) (Pepperberg et al., 1988; Kahlert et al., 1990). The present data suggest that the electrophysiological delay has a similar basis in the deactivation kinetics of / ? ' , and that T C represents T R >, the lifetime of R* in the phototransduction process. The results furthermore suggest a preservation of the "dark-adapted" value of T R * within the investigated range of background intensity.

After visual-pigment bleaching, single isolated rod photoreceptors of Ambystoma tigrinum recover their sensitivity to light when supplied with 11-cis-retinal from liposomes or with li-cis-retinal bound to interphotoreceptor retinoid-binding protein. Bleached rods do not recover sensitivity, or do so only very slowly, after exposure to 11-cis-retinol. The latter retinoid is "toxic" in that rods actually lose sensitivity in its presence. In contrast, bleached isolated cone cells recover sensitivity when either retinoid is supplied. It is suggested that the major pathway for rhodopsin regeneration during dark adaptation in the intact eye is transport of ll-cis-retinal from the pigment epithelium to the retina. The results also suggest that there may be separate pathways for visual-pigment regeneration in rods and cones during dark adaptation.Regeneration of visual pigment during dark adaptation or during maintained illumination requires retinoid isomerization from trans to cis form and conversion from alcohol to aldehyde before retinoid can be bound to opsin to reconstitute active pigment (1). The pigment epithelium (PE) has long been known to be involved in regeneration (2), implying a "visual cycle" that involves shuttling of retinoid from retina to PE and back again during cycles of light and dark. During light adaptation, there is indeed a progressive loss of retinoid from the retina, and an increase in the retinoid content of the PE. During darkness, the retinoid flow is reversed (3). Fulton and Rando (4) have presented strong evidence for localization of the retinoid-isomerizing system in PE rather than retina, but it remains uncertain which cells are involved in effecting the alcohol-to-aldehyde change that must take place during the visual cycle.We now report a difference in the use of retinol and retinal by rod and cone cells, and a "toxic" effect of retinol on rod cell function. We suggest that 11-cis-retinal is the major form of retinoid transported from PE to retina. Alternatively, ifthe alcohol form is transported it must be converted to the aldehyde in a cell type other than the rod photoreceptor (which may be cones, or possibly Muller cells).Pepperberg et al. (5) showed that the ordinarily permanent desensitization due to bleaching in isolated retina could be reversed by treatment with 11-cis-retinal. In their work, retinal was applied in ethanolic solution. Subsequently, it was shown (6) that liposomes could also be used to deliver retinoid. Our studies on isolated photoreceptors have employed both liposomes and interphotoreceptor retinoidbinding protein (IRBP). IRBP is a protein found at high concentration in the interphotoreceptor matrix, where its unique location and retinoid-binding properties (7,8) make it likely that it is involved in retinoid movement between retina and PE. In support of this, we demonstrate here the transfer of retinoid between IRBP and photoreceptor cells. MATERIALS AND METHODSIsolated photoreceptor cells from dark-adapted, larval tiger salamanders (Ambystoma tigrin...

The effect of light adaptation on the period of photocurrent saturation induced by a bright stimulating flash was examined in rod photoreceptors of the larval-stage tiger salamander (Ambystoma tigrinum). Using suction electrodes, photocurrent responses to brief flashes were recorded from single, isolated rods in the presence and absence of steady background illumination. Background light decreased the saturation period (T) measured at fixed flash intensity (fixed I/) and in this respect light-adapted the saturating response. Effects of the background on responses to weak (i.e. subsaturating) and bright flashes were compared with changes in a parameter, \[/ = e~A 77l «", where A7*is the decrease in saturation period, and where T R » is the slope of the line that relates r a n d ln/y in a given state of adaptation. Dark-and light-adapted responses to flash intensities if and / / , respectively, exhibited similar absolute peak photocurrent and falling-phase kinetics when if and / / satisfied the relation, if = i>(lf + I b T R '), where I b is the background intensity. It is argued that \p approximates the relative PDE//?* gain of transduction, i.e. the relative peak level of activated cGMP phosphodiesterase (PDE*) produced by a given, small amount of photoactivated visual pigment (R*). Interpreted on this view, the results imply that light adaptation derives largely from a decrease in PDE'/R* gain, rather than from the stimulation of guanylate cyclase activity. The data are consistent with the possibility that modulation of the lifetime of PDE* underlies the background dependence of \p.

A B ST RACT Visual pigment bleaching desensitizes rod photoreceptors greatly in excess of that due to loss of quantum catch. Whether this phenomenon also occurs in cone photoreceptors was investigated for isolated salamander red-sensitive cones. In parallel experiments, (a) visual pigment depletion by steps of bleaching light was measured by microspectrophotometry, and (b) flash sensitivity was measured by recording light-sensitive membrane current. In isolated cones, visual pigment bleaching permanently reduced flash sensitivity significantly below that due to the reduction in quantum catch, and there was little spontaneous recovery of visual pigment. The "extra" desensitization due to bleaching was most prominent up to bleaches of ~ 80% visual pigment and reached a level ~ 1 log unit beyond that due to loss of quantum catch. At higher bleaches, the effect of loss of quantum catch became more important. Bleaching did not greatly reduce the maximum lightsuppressible membrane current. A 99% reduction of the visual pigment permanently reduced the circulating current by only 30%. Visual pigment bleaching speeded up the kinetics of dim flash responses. All electrical effects of bleaching were reversed on exposure to 11-c/s retinal, which probably caused visual pigment regeneration. Light adaptation in photopic vision is known to involve significant visual pigment depletion. The present results indicate that cones operate with a maintained circulating current even after a large pigment depletion. It is shown how Weber/Fechner behavior may still be observed in photopic vision when the contributions of bleaching to adaptation are included.

Psychophysical experiments have shown an equivalence between sensitivity reduction by background light and by bleaches for the human scotopic system. We have compared the effects of backgrounds and bleaches on the light-sensitive membrane-current responses of isolated rod photoreceptors from the salamander Ambystoma tigrinum. The quantum catch loss was factored out from the desensitization due to bleaching to give the fraction of "extra" desensitization due to adaptation. For backgrounds, desensitization is well described by the Weber/Fechner equation. The extra desensitization after bleaches can also be described by the Weber/Fechner equation, if an "equivalent" background produced by bleaching is made linearly proportional to the fraction of pigment bleached. A background which produces an extra desensitization of a factor of two is equivalent to a fractional bleach of approximately 6%. Equivalent background and bleaching desensitizations were associated with similar reductions in circulating current. There is a linear relation between log flash sensitivity and decrease in circulating current. Equivalent background and bleaching desensitizations were associated with similar increases in cGMP phosphodiesterase and guanylate cyclase activity. These were inferred from membrane current changes after steps into lithium or IBMX solutions. There were also similar reductions in the integration times of dim flash responses for equivalent desensitizations produced by backgrounds and bleaches. These results suggest that the equivalence between background and bleaching found psychophysically may arise at the very earliest stages of visual processing and that these two processes of desensitization have similar underlying mechanisms.