Light Dependence of Calcium and Membrane Potential Measured in Blowfly Photoreceptors In Vivo

The cytosolic free concentration of Ca²⁺ ions (Ca_i) is one of the most important regulation factors in biological cells, influencing a great number of cellular processes. This holds particularly for insect photoreceptor cells, where Ca_i has been shown to play a key role in the regulation of the light sensitivity (Bader et al., 1976; Autrum, 1979; Muijser, 1979; Tsukahara, 1980; Walz, 1992). More specifically, Ca_i has been implicated in the control of numerous cellular processes in fly photoreceptors; e.g., in the modulation of the light-activated ion channels (Hardie, 1991, 1995a, 1995b; Hardie and Minke, 1994), the activation of the Na⁺/Ca²⁺ exchanger (Hardie, 1995a, 1995b), the regulation of many enzymes involved in the transduction cascade (Selinger et al., 1993; Minke and Selinger, 1996), the activation of mitochondria (Fein and Tsacopoulos, 1988; Mojet et al., 1991), and the migration of pigment granules in the photoreceptor cells (Kirschfeld and Vogt, 1980; Howard, 1984; Hofstee and Stavenga, 1996).

Ca_i has been reported to rise in insect photoreceptor cells during light stimulation (Howard, 1984; Hardie, 1991, 1996a; Peretz et al., 1994b; Ranganathan et al., 1994; Walz et al., 1994). In fly photoreceptors, the main part of this increase in Ca_i is caused by the influx of extracellular Ca²⁺ through the light-activated channels (Howard, 1984; Hardie, 1991, 1996a; Hardie and Minke, 1994; Peretz et al., 1994b; Ranganathan et al., 1994). Therefore, in an intact eye, Ca_i will not only depend on processes inside the photoreceptors themselves, but also on the ionic conditions in the extracellular space. With respect to Ca²⁺, these can vary considerably (Sandler and Kirschfeld, 1988, 1991; Ziegler and Walz, 1989; Rom-Glas et al., 1992; Peretz et al., 1994a).

In the past, Ca_i of insect photoreceptors and its dynamic regulation have been measured either in isolated ommatidia (Peretz et al., 1994b; Ranganathan et al., 1994; Hardie, 1995a, 1996a, 1996b) or in slice preparations of the retina superfused with Ringer solutions (Coles and Orkand, 1985; Hochstrate and Juse, 1991; Walz et al., 1994). Both of these techniques are likely to strongly influence the extracellular ion concentrations and hence to affect Ca_i. In an alternative approach, the light dependence of the Ca²⁺ homeostasis in insect photoreceptors has been studied via measurements of the Ca²⁺ concentration in the extracellular space (Sandler and Kirschfeld, 1988, 1991, 1992; Rom-Glas et al., 1992; Peretz et al., 1994a); however, the possibly strong influence of intracellular Ca²⁺ buffering (Hardie, 1996a) and of Ca²⁺ release from intracellular stores (Walz et al., 1995; Hardie, 1996b) on Ca_i could not be studied in this way.

To better understand the regulation of Ca_i under natural, physiological conditions, we developed a technique to directly measure Ca_i in the intact eye using fluorescent Ca²⁺ indicator dyes with varying affinity for Ca²⁺. We thus were able to estimate Ca_i as a function of adapting light intensity. We find that bright illumination of fly photoreceptors causes surprisingly high levels of Ca_i; i.e., exceeding 10 μM.

All experiments were performed on female blowflies (Calliphora vicina, white-eyed mutant chalky) taken from a laboratory culture. The mutant chalky was chosen because screening pigments and a functional pupil mechanism are lacking. The animals were immobilized with wax and a small hole was cut in the cornea that was immediately sealed with silicon grease. A silver wire was placed as reference electrode in the same eye. The intactness of the optics of the eye was checked before and after preparation by inspecting the deep pseudopupil (Franceschini and Kirschfeld, 1971). The animal was placed in a holder that allowed adjustment of its orientation. The holder with the animal was then positioned on the micromanipulator-controlled stage of a Leitz Orthoplan epi-fluorescence microscope (Leitz, Wetzlar, Germany).

Conventional electronic equipment was used to measure the intracellular membrane potential and to pass current through the electrode (Axoclamp 2A, operated in bridge mode; Axon Instruments, Foster City, CA). The electrodes were pulled on a P-87 (Brown and Flaming; Sutter Instruments, Co., Novato, CA) from borosilicate glass (1.5 mm o.d., 0.86 mm i.d.; Clark Electromedical Instruments, Reading, UK), and their tip was filled with a solution containing 5 mM of calcium indicator dye (Oregon Green 1, 2, or 5N; Molecular Probes, Inc., Eugene, OR, in the following abbreviated as OG1, OG2, and OG5N, respectively)¹ in 0.1 M KCl. The shank was then backfilled with 0.1 M KCl solution. The electrodes had a resistance of 150–250 MΩ in the tissue. The procedure of electrical recording was as follows. First, the tip of the electrode was adjusted at the optical axis of the objective, at a level 150 μm below the focal plane. The stage of the microscope with the fly in the holder was then moved under the objective so that the electrode penetrated the eye through the hole at a level 150 μm below the corneal surface and the fly was advanced so far that a penetrated cell was approximately coaxial with the objective.

After impalement, the cell was dye-filled by applying pulses of −1.2 to −2.2 nA at 0.5 Hz (50% duty cycle). The process of filling lasted at most 5 min, but was usually complete after 1–2 min. Sometimes no current was necessary, because cells filled simply by diffusion of the dye from the tip of the electrode. The filling of a cell was immediately apparent from the distinct fluorescence emerging from one of the facet lenses. As outlined in results, excessive concentrations of the dye induced alterations of the electrical response of the photoreceptor cells. Therefore, as a precaution, the process of dye filling was checked in regular intervals by visually judging the intensity of the fluorescence and filling was stopped when the intensity of the fluorescence reached values sufficient for optical recordings. Recordings of cells that were subsequently found to display alterations in their peak–plateau transitions (indicative of excessive additional Ca²⁺ buffering) were rejected (see results).

Two light sources, a 75-W xenon lamp and a 100-W halogen lamp, delivered the test and adapting light beams, respectively. Shutters (Uniblitz; Vincent Associates, Rochester, NY; rise time <3 ms) and grey filters controlled the light flux in both light paths independently. A 50% mirror combined the beams, which then passed the microscope's fluorescence cube (Leitz DM 510; i.e., blue excitation causing green emission). A 10× objective (NA 0.25; Spindler & Hoyer) projected the blue illumination onto the fly eye. The green emission was measured by a photomultiplier (R928; Hamamatsu Corp., Bridgewater, NJ). A small diaphragm (diameter 0.2 mm) in the image plane was adjusted so that only the fluorescence emerging from the brightly shining facet lens was selected. The background due to a distinct autofluorescence of the cornea thus was minimized.

The signals from the electrode amplifier and the photomultiplier were filtered at 2 kHz (3343; Krohn-Hite Corp., Avon, MA) and sampled at 5 kHz per channel by a CED 1401 interface (Cambridge Electronic Design Limited, Cambridge, UK). Further processing of the data was performed off-line.

After filling a cell with OG1, the fly was placed in a fluorescence microscope (Nikon Diaphot; Nikon Inc., Melville, NY) equipped with a F-601M camera (Nikon Inc.) containing a black and white film (SFX; 200 ASA pushed to 800 ASA; Ilford Imaging Limited, Mobberley, UK). The blue (477 nm)-induced green (>510 nm) fluorescence was photographed with a dry objective (4×, NA 0.1, Spindler & Hoyer; see Fig. 1,a) as well as a water immersion objective (SW25, NA 0.6; Leitz; see Fig. 1, c and d). In Fig. 1,a, a halogen light source delivered additional side illumination for recognition of the eye and facet pattern. To identify the stained cell, the eye was first illuminated for 5 s with 380 nm light for creating the highly fluorescent visual pigment state M′ (Stavenga et al., 1984). Then the green (546 nm)-induced red emission (>580 nm) was photographed (water immersion SW25; Fig. 1 d).

To estimate Ca_i quantitatively as a function of the adapting light, we first adapted the photoreceptor cells for 5 s to a given light intensity, and then probed the fluorescence with a bright test flash. The fluorescence signal at the beginning of the test flash thus represents the Ca_i signal due to the adapting light. At the end of the test flash, the signal is dominated by the Ca²⁺ influx caused by the much brighter test flash.

Because we used nonratiometric Ca²⁺ indicators, it was necessary to ensure that changes in dye concentration (caused by bleaching or by active transport out of the cell) did not corrupt the measurements. When using the high affinity dyes OG1 and OG2, we therefore took the difference in the fluorescence signal between the beginning and the end of the fluorescence trace for the quantitative analysis. Any change in the magnitude of the fluorescence signal at the end of the test flash (i.e., when the dye is saturated) indicated that the concentration of the dye changed. This procedure was not possible for the data from OG5N because, due to the low affinity of OG5N for Ca²⁺, the signal does not saturate. We therefore took the difference between the initial fluorescence of the photoreceptor cell adapted to different light intensities and the initial fluorescence signal of the dark-adapted photoreceptor cell. This method requires regular checks for changes in the magnitude of the fluorescence signal from the dark-adapted photoreceptor.

Because the magnitude of the fluorescence signal of our single wavelength dyes depends on the concentration of the dye, we normalized the data to compare data from different cells. The quantitative values from a single cell describing the influence of the adapting light were normalized between the value of the lowest adaptation intensity and the value of the highest adaptation intensity, and subsequently plotted as a function of the light intensity.

To estimate the dependence of Ca_i on the light intensity, we calculated the expected fluorescence signal as a function of the light intensity with the function F(Ca_i) = Ca_i^h/(Ca_i^h + K_d^h). Since our in vivo method does not allow a direct calibration of the indicators, we used the K_d values published by Haugland (1996): OG1, K_d = 0.17 μM; OG2, K_d = 0.58 μM; OG5N, K_d = 20 μM. Hill coefficients were taken equal to 1, except for OG5N, for which Hill coefficients <1 have been repeatedly reported (e.g., Ukhanov et al., 1995). We used a value of 0.7, derived from fitting the data published for Calcium Green 5N (Haugland, 1996). Using this function, we calculated the expected fluorescence as a function of the light intensity, for functions of Ca_i depending on the light intensity. The resulting functions of fluorescence depending on the light intensity were normalized (again between the value for the lowest light intensity and the value for the highest light intensity; i.e., between log I = −3 and log I = 2) to allow comparison with the measured data.

The preferred method for recording the membrane potential of individual insect photoreceptors in intact animals is to insert an electrode through a small hole in the cornea and to subsequently impale a photoreceptor cell. Here we demonstrate that this technique can also be used to inject calcium indicator dyes into a penetrated cell. Fig. 1,a shows an eye of a blowfly where one cell was dye-filled, photographed through a dry objective. One facet lens clearly shines up. Neutralizing the cornea by using a water immersion objective (Kirschfeld and Franceschini, 1969) allows examination of the subcellular distribution of the dye, because it is then possible to focus on the tips of the rhabdomeres and the cell bodies; Fig. 1,b depicts this optical situation diagrammatically (for a detailed account of the anatomy of the fly retina, see Hardie, 1985). Fig. 1,c shows the blue-induced green fluorescence of the stained cell. Both the soma and rhabdomere of one of the photoreceptor cells fluoresce, indicating that the dye is distributed throughout that photoreceptor cell and that part of the excited fluorescence is efficiently guided by the rhabdomere. To visualize the localization of the stained cell within the ommatidial lattice of the fly's eye, we exploited the bright red fluorescence of the M′ state of the visual pigments (Stavenga et al., 1984, see materials and methods) when illuminated with green light. The green-induced red fluorescence (Fig. 1,d) of the same part of the retina as in Fig. 1 c shows the rhabdomere of the stained cell (arrow), an R5 cell, and the regular pattern of fluorescing rhabdomeres; of course, the green-induced red fluorescence of the dye is much weaker than the blue-induced green fluorescence.

Under the physiological optical conditions used in the experiments, light emitted from the rhabdomere leaves the eye within an angle of 1–2° (van Hateren, 1984), while the fluorescence coming from the cell body is expected to radiate from the cornea within an angle of ∼11° (assuming a diameter of the cell of ∼10 μm and a focal distance of 50 μm of the facet lens). Because the objective aperture is ∼14°, the photomultiplier samples a mixture of light emitted by the rhabdomere and the cell body; however, the ratio of the amount of light sampled from the two cellular compartments depends on the precise alignment of the investigated cell's visual axis with the microscope objective. This inevitably varied from one recording to another.

From such a dye-filled cell, we recorded simultaneously the light-induced changes in the membrane potential and in the accompanying fluorescence, using the low affinity dye OG5N (Fig. 2). As in all experiments presented here, the cell was dark adapted for 1 min before and between the recordings. Illumination causes, after a delay of a few milliseconds, a rapid depolarization of the cell membrane, reaching a peak after ∼10 ms (Fig. 2,a); subsequently, the receptor potential levels off to a plateau value (Fig. 2,b). The blue-induced green fluorescence appears to follow a similar, although somewhat slower, time course. First, during the opening of the shutter, the fluorescence signal rises to an initial plateau, the dark level. This is the sum of tissue autofluorescence and fluorescence of the dye due to resting Ca_i. Then, after a short delay (∼3 ms), the emission very rapidly increases, indicating an abrupt rise in Ca_i (Fig. 2,c). The peak occurs after ∼100 ms, and the subsequent decrease to a plateau of the fluorescence distinctly lags that of the receptor potential (Fig. 2 d).

All Ca²⁺ indicator dyes are also Ca²⁺ buffers. Increasing the intracellular Ca²⁺ buffering capacity by introducing the dyes can considerably alter the dynamics and the regulation of Ca_i (e.g., Neher, 1995). Because fly photoreceptors are thought to react sensitively to changes in the Ca²⁺ homeostasis (Muijser, 1979; Hardie, 1995b), we checked for changes in the waveform of the membrane potential due to loading with the dyes. The cell of Fig. 3 spontaneously filled with OG2; i.e., without the need to apply current. The first measurement was taken ∼1 min after impalement of the cell, the next after 4 and 6 min. The fluorescence signal (Fig. 3, c and d) increased with time, indicating that the cell progressively took up more of the dye. After light-off, the time course of the afterdepolarization became prolonged. The afterdepolarization is at least partially caused by the Na⁺/Ca²⁺ exchanger (Hochstrate, 1991), which in Calliphora photoreceptor cells can generate currents stronger than 1 nA (Gerster, 1997). Therefore, the prolongation of the afterpotential indicates that the Na⁺/ Ca²⁺ exchanger extrudes more Ca²⁺ when the dye concentration increases. This is in line with an increased buffering of Ca²⁺ ions by the dye, which leads to an increase in the total concentration of Ca²⁺ at comparable concentrations of free Ca²⁺. We consistently found that the dyes prolonged the duration of the afterdepolarization, even at concentrations that were difficult to detect photometrically. However, the waveform of the receptor potential during light-on remained virtually unchanged (Fig. 3, a and b), suggesting that the increased dye concentration did not appreciably affect the phototransduction process.

Generally, the effect of the dye on the membrane potential during the light stimulus was inconspicuous, but peak values sometimes increased by a few millivolts after filling the cell; in some cases, the peak to plateau transition of the membrane potential at the onset of light stimulation was accelerated after dye filling. When cells were filled too much, the typical reduction of the peak–plateau transition, caused by a substantial increase in Ca²⁺ buffering (Bader et al., 1976; Muijser, 1979; Tsukahara, 1980; Walz et al., 1994), could be observed. These cells were rejected.

In addition to the fluorescence from the dyes, we measured the tissue autofluorescence from an eye of which no cell was injected with a Ca²⁺ indicator. This tissue autofluorescence remained essentially constant upon illumination (Fig. 3, e and f). Nevertheless, occasionally a very slight, transient increase in autofluorescence could be noticed in this background (Fig. 3,f, *), probably due to the light-induced, transient redox changes of the flavoproteins in the photoreceptor mitochondria (Stavenga and Tinbergen, 1983; Mojet et al., 1991). Sometimes, such a small increase in fluorescence also was observed when measuring from a cell filled with the high affinity dyes (i.e., in Fig. 3 d, *). This small increase might also be attributable to the observed increase of the autofluorescence, that is, to transient changes in the redox state of the flavoproteins. In the processed experiments, this variation in the background signal was fully negligible compared with the light-induced changes in dye fluorescence.

Fluorescence measurements of cells injected with the low affinity dye OG5N (K_d = 20 μM; Haugland, 1996) yielded somewhat variable results. The time to peak ranged from 100 ms to almost 1 s; the time required for reaching a stable plateau is 2–4 s. This variability might be the result of slight differences in the alignment of the investigated cells. Because the Ca²⁺ influx occurs in the rhabdomeres, and the Ca²⁺ ions diffuse from the rhabdomere into the cell body rather slowly (Ranganathan et al., 1994), a variation in the ratio of light sampled from the rhabdomere and from the cell body could cause a variation in the observed time course of the fluorescence signal.

No peak in the fluorescence signal was observed when high affinity dyes (OG1 or OG2; K_d = 0.16 and 0.58 μM, respectively; Haugland, 1996) were used (Fig. 4, a and b). The fluorescence signal then increased monotonically towards a stable plateau that was reached after 100–400 ms. These findings are fully consistent with the difference in affinity for Ca²⁺ of the dyes used, suggesting that Ca_i attains values where the high affinity dyes OG1 and OG2 are saturated. Because OG2 saturates at Ca_i ≈ 10 μM (Haugland, 1996), Ca_i exceeds this range during the peak observed with the low affinity dye OG5N.

To measure the dye fluorescence with an acceptable signal to noise ratio, it is necessary to use very high light intensities. To assess Ca_i at moderate and intermediate intensities, we employed a double pulse paradigm, where an adapting light stimulus was followed by a bright test flash. We adapted the photoreceptors for 5 s at a given intensity, and then probed the fluorescence with a short (0.2–0.5 s) test flash. An adaptation time of 5 s was considered sufficient because both the stability of the membrane potential and the fluorescence measurements indicated that after 5 s Ca_i reached a stable plateau value and that diffusion of the Ca²⁺ ions had reached an equilibrium. We assume, therefore, that the subcellular distribution of Ca_i in the cytosol is fairly homogeneous after 5 s.

Fig. 5 shows an example of such an experiment. The high affinity dye OG1 was used. The fluorescence signal at the beginning of the bright test flash increases with increasing adapting light intensity in the low intensity range, but it saturates at high intensities. Again, we confirmed that this is caused by saturation of the dye using the low affinity dye OG5N. This dye reports an increase in Ca_i, even up to the highest intensities used (see below).

We repeated the experiment of Fig. 5 with all three dyes (OG1, OG2, and OG5N) in nine cells (three for each dye) from six animals. Fig. 6 summarizes the experiments. To correct for the different absolute sensitivities of the different cells, the V/log I curve of the peak receptor potential of each cell was fitted separately to a logistic function: V = V_max*Iⁿ/(Iⁿ + 1) (Laughlin, 1981). The light intensity I is taken here relative to the light intensity that causes a half maximal peak depolarization; this intensity was assigned the value log I = 0. Furthermore, the potential values were normalized to the maximal peak depolarization (V_max). Fig. 6,a presents the resulting peak and plateau values as a function of relative light intensity. The V_max values ranged for the peak from 60 to 82 mV (average 72 ± 6 mV SD) and for the plateau from 19 to 40 mV (average 30 ± 6 mV SD); the exponent n for the peak values ranged from 0.40 to 0.47 (average 0.44 ± 0.02 SD) and for the plateau values from 0.42 to 0.58 (average 0.50 ± 0.05 SD). The V/log I curves appeared to be homogeneous and are fully consistent with similar measurements reported in the literature (Laughlin and Hardie, 1978; Matic and Laughlin, 1981; Sandler and Kirschfeld, 1988; Roebroek and Stavenga, 1990); this suggests again that the dyes did not seriously affect the membrane potential. The fluorescence signals measured during the test flashes were evaluated quantitatively as described in materials and methods. Fig. 6 b shows the resulting dependency of the fluorescence signal on the adapting light intensity for the three different dyes.

Obviously, the results for the high affinity dyes OG1 and OG2 are quite different from those for OG5N (Fig. 6 b). While the signals obtained with OG1 or OG2 both show saturation, the signal obtained with OG5N increases with light intensity even up to the highest intensities used. In addition, while the signals of OG1 and OG2 already show a pronounced increase at the lowest intensities, with OG5N this occurs only at log I ≥ 0. The differences between OG1 and OG2 are rather inconspicuous. Mainly, OG1 seems to become activated on average at intensities half a log-unit lower than OG2, as can be seen from the leftward shift in the activation curves of OG1 with respect to the curve of OG2. Taken together, the important findings of these experiments are (a) that Ca_i is regulated over the whole intensity range where the photoreceptor can respond to light, and (b) that OG1 and OG2 are saturated at intensities about one log-unit above the intensity for half-maximal activation of the peak membrane potential. This demonstrates that, at bright adaptation intensities, the plateau values of Ca_i exceed 10 μM, the saturation value of OG2 (Haugland, 1996).

The slope of the fluorescence vs. log I plots is almost linear in the low intensity region for OG1, in the region between log I = −2 to log I = 0.5 for OG2 and in the high intensity region for OG5N. This seems to imply that log Ca_i rises linearly with log I. To get a more quantitative picture of the changes of Ca_i caused by light adaptation, we have therefore tried to describe the dependency of Ca_i on the light intensity with a simple power function: Ca_i(I) = Ca_i,da + a*I  ^b; Ca_i,da denotes here Ca_i in the dark-adapted state, assumed to be 0.16 μM, the value found for Drosophila (Hardie, 1996a). The experimental data were then fitted by taking a = 2.5 μM and b = 0.5, resulting in the curve shown in Fig. 6,c; the bold lines in Fig. 6,b represent the simulated fluorescence values, calculated as described in materials and methods. The similarity between measured and simulated data suggests that the function chosen for describing Ca_i is appropriate. As shown in Fig. 6,c, at log I = 2, Ca_i equals 25 μM. We are well aware that the accuracy of this approach is limited. With slight variations of the parameters a and b, reasonably good fits can still be obtained while yielding considerably different values for Ca_i, especially for high light intensities. It was no longer possible to obtain a good fit between the simulated fluorescence functions and our data when the parameter a was chosen smaller than 2 or the parameter b smaller than 0.5. Yet even with this combination of parameters, Ca_i at log I = 2 is still estimated to reach 20 μM. Therefore, the values of Fig. 6 c can be considered to be a conservative estimate. The value for Ca_i,da, the dark adapted Ca_i, influences the simulated fluorescence curves negligibly, and it was thus not possible to estimate it with our data. We therefore used 0.16 μM, the value measured in Drosophila (Hardie, 1996a), throughout the simulations.

We demonstrate in the present paper that it is possible to measure the light-induced changes of Ca_i in photoreceptor cells in the intact eye of flies by using fluorescent Ca²⁺ indicators. The fluorescence signal can be measured simultaneously with the light-induced receptor potential. Dye filling causes alterations of the membrane potential (Fig. 3, a and b) that appear to be consistent and at least qualitatively explainable with the buffer action of the dyes. An increase in buffer capacity leads to an increased amount of Ca²⁺ ions entering the cell before a given concentration is reached. This in turn causes the Na⁺/Ca²⁺ exchanger to be activated for a longer period to extrude the extra load of Ca²⁺; the afterdepolarization is therefore prolonged.

Increasing the intracellular buffer capacity by introducing Ca²⁺ buffers normally induces pronounced changes in the light response. The peak to plateau transition is diminished and the response kinetics are slowed down (Bader et al., 1976; Muijser, 1979; Tsukahara, 1980; Walz et al., 1994; Hardie, 1995b). We also observed these effects at high dye loads. However, usually this could be avoided and we carefully checked for these alterations of the waveform. Since the membrane potential is a sensitive measure of photoreceptor function, we conclude that our manipulations have not, or at most weakly, influenced the Ca²⁺ homeostasis during the light response.

Bright light stimulation of insect photoreceptors in isolated ommatidia (Peretz et al., 1994b; Ranganathan et al., 1994; Hardie, 1996a) or perfused eye slices (Walz et al., 1994) rapidly increases Ca_i to high concentrations. In Drosophila (Hardie, 1996a) and the drone (Walz et al., 1994), the increase in Ca_i is fast and consistently saturates high affinity dyes after ∼200 ms. Using Mag-Indo-1, Hardie (1996a) estimated that Ca_i reaches values up to 50 μM in isolated Drosophila photoreceptor cells. Here we show that Ca_i reaches similar values in photoreceptor cells of Calliphora in vivo (Figs. 2 and 4). The saturation of OG2 upon bright illumination indicates that in Calliphora Ca_i reaches values exceeding 10 μM.

The adaptation experiments (Figs. 5 and 6) allow us to estimate how Ca_i depends on the light intensity (Fig. 6,c). Quantitative measurements of Ca_i with fluorescent indicators are often complicated by the fact that the indicators have different properties in the cytoplasm of cells than in solutions. However, since the K_d values of the indicators only have been reported to increase when the indicator is brought into the cytosol (e.g., Hardie, 1996a), it is unlikely that we overestimated Ca_i. We conclude that Ca_i reaches values at least up to 20 μM when illuminated with the brightest intensity used in this study. Surprisingly, these high values are not only reached during short and local Ca²⁺ peaks, but are maintained after several seconds of light adaptation, implying that these high concentrations are sustained during prolonged periods. The fluorescence signal at high light intensities reaches a stable level after at most 2–4 s (Figs. 2–4), showing that the distribution of Ca²⁺ ions then is in a steady state. In Limulus, only the R-lobe shows a dramatic increase in Ca_i; this increase is spread over a distance of at least 20 μm (Ukhanov and Payne, 1995). Therefore, it seems likely that high values of Ca_i are also reached in the cell bodies of Calliphora photoreceptor cells that have a diameter of ∼10 μm.

The finding that Ca_i reaches very high values is supported by measurements of changes of the extracellular calcium concentration with Ca²⁺-selective electrodes (Sandler and Kirschfeld, 1988). In the drone, the glia cells do not take up Ca²⁺ (Coles and Orkand, 1985) and volume changes in the retina are small (Orkand et al., 1984; Ziegler and Walz, 1989). Assuming that this is also the case in the blowfly, and that there is no substantial Ca²⁺ release from internal stores (Ranganathan et al., 1994; Hardie, 1996a), we can calculate from the decrease in extracellular calcium measured by Sandler and Kirschfeld (1988, their Fig. 1,c) the amount of Ca²⁺ entering the photoreceptors (ΔCa_tot; Fig. 7,a, ▪) and compare the resulting values with our estimates of the increase in Ca_i (ΔCa_i; Fig. 7 a, bold line). The calculated values appear to be in good agreement with the values of the light-induced Ca_i increase at log I ≥ 2. Both curves have a similar slope at these high intensities.

However, at light intensities below log I = 2, ΔCa_tot > ΔCa_i, or the amount of Ca²⁺ entering the photoreceptor cells from the extracellular space is larger than the increase of Ca_i (Fig. 7,a). This difference can be explained by assuming that the Ca²⁺ influx is buffered; e.g., by uptake in organelles or binding to proteins. The buffering coefficient B_inf = ΔCa_tot/ΔCa_i is presented in Fig. 7,b as a function of light intensity, and in Fig. 7 c as a function of Ca_i. Clearly, the buffering capacity is limited, and at Ca_i ≥ 10 μM (corresponding to log I = 1.5) buffering becomes minor. The values of 10–20 obtained for the buffering coefficient at Ca_i ≲ 1 μM are more than an order of magnitude lower than the estimates for photoreceptors of Drosophila (Hardie, 1996a) or Limulus (O'Day and Gray-Keller, 1989). We note that the obtained buffering values are subject to a number of uncertainties. First, mismatching the stimulation intensities of Sandler and Kirschfeld's (1988) measurements with respect to our experiments might have affected the estimation of the values for B_inf. In addition, our calculations assume that there is no extracellular Ca²⁺ buffering; the buffering coefficients for the Ca²⁺ influx would be underestimated by the factor of extracellular Ca²⁺ buffering if this assumption does not hold. Also, if the assumptions of a constant extracellular volume and the noninvolvement of the glial cells in the Ca²⁺ homeostasis do not hold, this will obviously modify the quantitative values of B_inf. However, if Ca²⁺ buffering is assumed to be constant at a high buffering coefficient B_inf throughout the light intensity range studied, there would not be enough extracellular Ca²⁺ to sustain a Ca²⁺ influx that causes Ca_i to rise to 20 μM. We calculate a maximal value for B_inf of 11 under those conditions by dividing the extracellular Ca²⁺ concentration (1.4 mM; Sandler and Kirschfeld, 1991) with the product of the maximal value of Ca_i (20 μM) and the ratio of intracellular to extracellular volume (6.3; Sandler and Kirschfeld, 1991). We assume again that the following conditions hold: (a) there is no extracellular Ca²⁺ buffering, (b) the glial cells do not participate in the Ca²⁺ homeostasis, and (c) there is no substantial Ca²⁺ release from internal stores. Therefore, the important point that we wish to emphasize here is that the ratio of buffered to free calcium very likely is not constant and this limitation of the buffering capacity results in high Ca_i values in bright light. This conclusion is not affected by the uncertainties in calculated values for B_inf.

Another possible reason for the high discrepancy between the values for B_inf derived here and those reported by Hardie (1996a) is the different method. The estimate of Hardie (1996a) is based on the ratio of influx (measured by integrating the current) to free Ca_i measured with optical methods. Any Ca²⁺ extruded in the period of integrating the current (possibly by the Na⁺/Ca²⁺ exchanger) contributes to the buffering, while in our approach this Ca²⁺ reappears in the extracellular space, and thus does not contribute to the buffering. Therefore our values are necessarily lower than the estimate made by Hardie (1996a).

Probably, there are many different buffer mechanisms with different affinities and capacities. However, it is possible to calculate the parameters of a single, equivalent buffer from the values of Fig. 7,c. We have fitted the data points of Fig. 7,c to a simple buffer model, taking Ca_tot/Ca_i = 1 + B_tot/(K_d + Ca_i), where Ca_tot is the total Ca²⁺ concentration and B_tot is the total concentration of a buffer with dissociation constant K_d. The buffering coefficient of the Ca²⁺ influx (B_inf) was calculated by B_inf(I) = [Ca_tot(I) − Ca_tot,da]/[Ca_i(I) − Ca_i,da], where Ca_tot,da is the total Ca²⁺ concentration in the dark. The smooth line in Fig. 7 c was obtained by taking B_tot = 18 μM and K_d = 0.77 μM.

Illumination of invertebrate photoreceptors with bright light induces a rapid activation of mitochondrial respiration, presumably due to a rise in Ca_i (Fein and Tsacopoulos, 1988). In the white-eyed blowfly mutant chalky, illumination causes a rapid change in the redox state of mitochondrial flavoproteins (Stavenga and Tinbergen, 1983). A comparison of the intensity dependence of this process (Mojet et al., 1991) with the present calcium measurements shows that the transient shift in the redox state of flavoproteins occurs when Ca_i levels rise above ∼1 μM. At these concentrations, the mitochondria are indeed likely to take up considerable amounts of Ca²⁺ (e.g., Miyata et al., 1991; Babcock et al., 1997).

The pupil mechanism of wild-type fly photoreceptors, consisting of pigment granules migrating inside the cell soma, also has a distinct dependence on Ca²⁺ influx (Kirschfeld and Vogt, 1980; Howard, 1984; Hofstee and Stavenga, 1996). The measurements of the intensity dependence of this system, together with that of the receptor potential (Roebroek and Stavenga, 1990), also show that the pupil gets activated at Ca_i levels ≥ 1 μM. In addition to increasing the signal to noise ratio of the receptor potential at high light intensities (Howard et al., 1987), the function of the pupil in wild-type photoreceptors may be to avoid very high Ca_i levels.

A change in both the membrane potential and Ca_i is caused by the same underlying event; i.e., a change in the permeability of the light-activated channels. Curiously, whereas the plateau membrane potential saturates at log I ≈ 1–2, Ca_i shows a continuous rise with intensity (Fig. 6). Apparently, the light-dependent permeability increases with illumination intensity even at the brightest light intensities. Because buffering becomes minimal with large calcium loads (i.e., Ca_i > 10 μM; Fig. 7), the rise in light-dependent permeability translates superlinearly into a rise in Ca_i. In addition, high Ca_i possibly activates a K⁺ conductance (Weckström, 1989), resulting in a reduced rise in membrane potential.

We thank J. Land and H.L. Leertouwer for technical assistance. Drs. U. Gerster, J. Tinbergen, and M. Weckström gave helpful comments on an earlier version of this manuscript.

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