When light is absorbed within the outer segment of a vertebrate photoreceptor, the conformation of the photopigment rhodopsin is altered to produce an activated photoproduct called metarhodopsin II or Rh(*). Rh(*) initiates a transduction cascade similar to that for metabotropic synaptic receptors and many hormones; the Rh(*) activates a heterotrimeric G protein, which in turn stimulates an effector enzyme, a cyclic nucleotide phosphodiesterase. The phosphodiesterase then hydrolyzes cGMP, and the decrease in the concentration of free cGMP reduces the probability of opening of channels in the outer segment plasma membrane, producing the electrical response of the cell. Photoreceptor transduction can be modulated by changes in the mean light level. This process, called light adaptation (or background adaptation), maintains the working range of the transduction cascade within a physiologically useful region of light intensities. There is increasing evidence that the second messenger responsible for the modulation of the transduction cascade during background adaptation is primarily, if not exclusively, Ca(2+), whose intracellular free concentration is decreased by illumination. The change in free Ca(2+) is believed to have a variety of effects on the transduction mechanism, including modulation of the rate of the guanylyl cyclase and rhodopsin kinase, alteration of the gain of the transduction cascade, and regulation of the affinity of the outer segment channels for cGMP. The sensitivity of the photoreceptor is also reduced by previous exposure to light bright enough to bleach a substantial fraction of the photopigment in the outer segment. This form of desensitization, called bleaching adaptation (the recovery from which is known as dark adaptation), seems largely to be due to an activation of the transduction cascade by some form of bleached pigment. The bleached pigment appears to activate the G protein transducin directly, although with a gain less than Rh(*). The resulting decrease in intracellular Ca(2+) then modulates the transduction cascade, by a mechanism very similar to the one responsible for altering sensitivity during background adaptation.

A 10 μm spot of argon laser light was focused onto the outer segments of intact mouse rods loaded with fluo‐3, fluo‐4 or fluo‐5F, to estimate dark, resting free Ca2+ concentration ([Ca2+]i) and changes in [Ca2+]i upon illumination. Dye concentration was adjusted to preserve the normal physiology of the rod, and the laser intensity was selected to minimise bleaching of the fluorescent dye. Wild‐type mouse rods illuminated continuously with laser light showed a progressive decrease in fluorescence well fitted by two exponentials with mean time constants of 154 and 540 ms. Rods from transducin α‐subunit knock‐out (Trα–/–) animals showed no light‐dependent decline in fluorescence but exhibited an initial rapid component of fluorescence increase which could be fitted with a single exponential (τ∼1–4 ms). This fluorescence increase was triggered by rhodopsin bleaching, since its amplitude was reduced by pre‐exposure to bright bleaching light and its time constant decreased with increasing laser intensity. The rapid component was however unaffected by incorporation of the calcium chelator BAPTA and seemed therefore not to reflect an actual increase in [Ca2+]i. A similar rapid increase in fluorescence was also seen in the rods of wild‐type mice just preceding the fall in fluorescence produced by the light‐dependent decrease in [Ca2+]i. Dissociation constants were measured in vitro for fluo‐3, fluo‐4 and fluo‐5F with and without 1 mm Mg2+ from 20 to 37 °C. All three dyes showed a strong temperature dependence, with the dissociation constant changing by a factor of 3–4 over this range. Values at 37 °C were used to estimate absolute levels of rod [Ca2+]i. All three dyes gave similar values for [Ca2+]i in wild‐type rods of 250 ± 20 nm in darkness and 23 ± 2 nm after exposure to saturating light. There was no significant difference in dark [Ca2+]i between wild‐type and Trα–/– animals.

The vertebrate visual system can operate over a large range of light intensities. This is possible in part because the sensitivity of photoreceptors decreases approximately in inverse proportion to the background light intensity. This process, called photoreceptor light adaptation, is known to be mediated by a diffusible intracellular messenger, but the identity of the messenger is still unclear. There has been considerable speculation that decreased cytoplasmic Ca2+ concentration (Cai2+) may play a role in light adaptation, and recent experiments in which Ca2+ buffer was incorporated into rod-cells have supported this notion. The extent of the contribution of calcium, however, remains unresolved. We now show that light-dependent changes in sensitivity in amphibian photoreceptors can be abolished by preventing movements of Ca2+ across the outer-segment plasma membrane. These experiments demonstrate that light adaptation in photoreceptors is mediated in cones primarily, and in rods perhaps exclusively, by changes in Cai2+.

SUMMARY1. In order to study the role of cytoplasmic calcium concentration (Ca2+) in rod photoreceptor light adaptation, we have attempted to prevent light-induced changes in Ca 2' by minimizing calcium fluxes across the outer segment plasma membrane. This was achieved by exposing the outer segment to a low-Ca2 , 0-Na+ solution, in which sodium was replaced with either guanidinium or lithium and the external calcium concentration (Ca2+) was reduced to micromolar levels.2. With guanidinium and 1-3 ,sM-Cao , the circulating current in darkness was maintained for a period of at least 15 s, consistent with approximate stability of Ca 2+ With Li+ rather than guanidinium most of the initial current was suppressed, but the residual current was again relatively stable.3. During prolonged exposures (> 30 s) to low-Ca2 , 0-Na+ solution followed by dim illumination, the circulating current did not remain constant but slowly increased. Incorporation of calcium buffer into the cytoplasm greatly reduced the rate of change of current, consistent with the idea that the increase arose from a gradual decrease in Ca2+.4. Light responses of rods exposed to low-Ca2+, 0-Na+ solution in darkness were altered in a characteristic manner. Although the initial rising phase of the light response was little changed, the peak amplitude of the response was larger and occurred later, and the response decayed more slowly than in control. The response-intensity relation was steepened and was shifted towards lower intensities both for flashes and for steps of light. The normal sag in the response to steps disappeared, and the waveform of the step response could be predicted to a close approximation from the integral of the dim flash response.5. Presentation of background illumination in Ringer solution produced a marked acceleration of the response to a subsequent bright flash. No such acceleration was observed if the background was given in low-Ca2+, 0-Na+ solution.6. The results described in paragraphs 4 and 5 indicate that, under conditions expected to minimize changes in Ca2+, all manifestations of light adaptation disappear, and the rod simply sums the effects of incident photons with an invariant integration time.t To whom correspondence should be addressed. G. L. FAIN AND OTHERS7. Exposure of a light-adapted rod to low-Ca2 , 0-Na+ solution altered the responses to superimposed test flashes in much the same way as for rods in darkness. The initial rising phases in low-Ca2+, 0-Na+ solution were unchanged, but the responses were larger, reached peak later and decayed more slowly. Nevertheless, when the low-Ca2+, 0-Na+ solution was presented on adapting backgrounds of increasing intensity, the time-to-peak of responses shortened in a graded manner.8. Extinction of the background in low-Ca2 , 0-Na+ solution elicited large lightsuppressible currents, presumably as a result of increased cytoplasmic cyclic GMP concentration. The rate of increase of these currents was graded with the intensity of the prior background.9. The results described in para...

A spot confocal microscope based on an argon ion laser was used to make measurements of cytoplasmic calcium concentration (Ca2+ i) from the outer segment of an isolated rod loaded with the fluorescent calcium indicator fluo-3 during simultaneous suction pipette recording of the photoresponse. The decline in fluo-3 fluorescence from a rod exposed to saturating illumination was best fitted by two exponentials of approximately equal amplitude with time constants of 260 and 2,200 ms. Calibration of fluo-3 fluorescence in situ yielded Ca2+ i estimates of 670 ± 250 nM in a dark-adapted rod and 30 ± 10 nM during response saturation after exposure to bright light (mean ± SD). The resting level of Ca2+ i was significantly reduced after bleaching by the laser spot, peak fluo-3 fluorescence falling to 56 ± 5% (SEM, n = 9) of its value in the dark-adapted rod. Regeneration of the photopigment with exogenous 11-cis-retinal restored peak fluo-3 fluorescence to a value not significantly different from that originally measured in darkness, indicating restoration of the dark-adapted level of Ca2+ i. These results are consistent with the notion that sustained activation of the transduction cascade by bleached pigment produces a sustained decrease in rod outer segment Ca2+ i, which may be responsible for the bleach-induced adaptation of the kinetics and sensitivity of the photoresponse.

Response properties of isolated mouse olfactory receptor cells were investigated using the suction pipette technique. Cells were exposed to the odour cineole or to solutions of modified ionic content by rapidly changing the solution superfusing the cilia. All experiments were performed at 37°C. Mouse olfactory receptor cells displayed a steep dependence of action potential frequency on stimulus concentration, a 3‐fold increase in stimulus concentration often saturating the firing frequency at 200‐300 Hz. The receptor current increased more gradually with increasing cineole concentration and did not saturate within the 100‐fold range of cineole concentrations applied. When stimulated for 30 s with a low odour concentration, cells responded with sporadic spike firing. Higher concentrations led to the generation of a large receptor current at the onset of stimulation which returned to baseline levels within a few seconds, accompanied during its rising phase by a short burst of action potentials. Thereafter an oscillating response pattern was observed during the remainder of the stimulus, consisting of repetitive increases in receptor current of around 1 s duration accompanied by short bursts of action potentials. Olfactory adaptation was studied by comparing the responses to two closely spaced odour stimuli. The response to the second odour stimulus recovered to 80% of its original magnitude when the cell was superfused with Ringer solution during the 5 s interval between odour exposures. In contrast, exposure to a choline‐substituted low Na+ solution between odour stimuli had two effects. First, the receptor current response to the first odour stimulus did not terminate as quickly as in the presence of Na+, suggesting the presence of a Na+‐Ca2+ exchanger. Second, the response to the second stimulus only recovered to 55% of its original magnitude, demonstrating the involvement of Na+‐Ca2+ exchange in the recovery of sensitivity in mouse olfactory receptor cells following stimulation.

SUMMARY1. Light adaptation has been studied in isolated red-sensitive cone photoreceptors of the salamander, using suction pipette recordings of circulating current.2. In the presence of background illumination, the response to incremental dim flashes became desensitized according to the Weber-Fechner law. The recovery phase of the flash response was accelerated significantly, although the time-to-peak was reduced only slightly, and for dim backgrounds the rising phase was unaltered.3. The role of cytoplasmic calcium concentration, Ca? , in mediating cone adaptation was investigated by minimizing light-induced changes in Cai , either by incorporating calcium buffer into the cytoplasm or by exposing the outer segment to low-Ca2+, O-Na+ solution. Both treatments appeared to slow dramatically or even to eliminate the onset of light adaptation in the cone.4. When the low-Ca2+, O-Na+ solution was presented in darkness, responses to subsequent illumination were affected in a characteristic manner: (i) the response-intensity relation was steepened and shifted to lower intensities, (ii) the response to a step of light could be predicted by integration and compression of the flash response, and (iii) the flash sensitivity declined steeply as a function of background intensity. 5. After extended exposure of the cone to bright backgrounds, the sensitivity in darkness failed to return to its original level. The flash response kinetics were faster and more biphasic than for dark-adapted responses or for responses desensitized to a comparable degree by exposure to steady background illumination.6. The results indicate that, in cones isolated from the pigment epithelium, the primary factor influencing the adaptational state of the cell is the cytoplasmic concentration of free calcium, but that at high intensities the effects of pigment bleaching are likely to be significant.

Simultaneous measurements of photocurrent and outer segment Ca2+ were made from isolated salamander cone photoreceptors. While recording the photocurrent from the inner segment, which was drawn into a suction pipette, a laser spot confocal technique was employed to evoke fluorescence from the outer segment of a cone loaded with the Ca2+ indicator fluo-3. When a dark-adapted cone was exposed to the intense illumination of the laser, the circulating current was completely suppressed and fluo-3 fluorescence rapidly declined. In the more numerous red-sensitive cones this light-induced decay in fluo-3 fluorescence was best fitted as the sum of two decaying exponentials with time constants of 43 ± 2.4 and 640 ± 55 ms (mean ± SEM, n = 25) and unequal amplitudes: the faster component was 1.7-fold larger than the slower. In blue-sensitive cones, the decay in fluorescence was slower, with time constants of 140 ± 30 and 1,400 ± 300 ms, and nearly equal amplitudes. Calibration of fluo-3 fluorescence in situ from red-sensitive cones allowed the calculation of the free-Ca2+ concentration, yielding values of 410 ± 37 nM in the dark-adapted outer segment and 5.5 ± 2.4 nM after saturating illumination (mean ± SEM, n = 8). Photopigment bleaching by the laser resulted in a considerable reduction in light sensitivity and a maintained decrease in outer segment Ca2+ concentration. When the photopigment was regenerated by applying exogenous 11-cis-retinal, both the light sensitivity and fluo-3 fluorescence recovered rapidly to near dark-adapted levels. Regeneration of the photopigment allowed repeated measurements of fluo-3 fluorescence to be made from a single red-sensitive cone during adaptation to steady light over a range of intensities. These measurements demonstrated that the outer segment Ca2+ concentration declines in a graded manner during adaptation to background light, varying linearly with the magnitude of the circulating current.