From the Department of Neurobiophysics, University of Groningen, 9747 AG Groningen, The Netherlands
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Light adaptation in insect photoreceptors is caused by an increase in the cytosolic Ca2+ concentration. To better understand this process, we measured the cytosolic Ca2+ concentration in vivo as a function of adapting light intensity in the white-eyed blowfly mutant chalky. We developed a technique to measure the cytosolic Ca2+ concentration under conditions as natural as possible. The calcium indicator dyes Oregon Green 1, 2, or 5N (Molecular Probes, Inc., Eugene, OR) were iontophoretically injected via an intracellular electrode into a photoreceptor cell in the intact eye; the same electrode was also used to measure the membrane potential. The blue-induced green fluorescence of these dyes could be monitored by making use of the optics of the facet lens and the rhabdomere waveguide. The use of the different Ca2+-sensitive dyes that possess different affinities for Ca2+ allowed the quantitative determination of the cytosolic Ca2+ concentration in the steady state. Determining the cytosolic Ca2+ concentration as a function of the adapting light intensity shows that the Ca2+ concentration is regulated in a graded fashion over the whole dynamic range where a photoreceptor cell can respond to light. When a photoreceptor is adapted to bright light, the cytosolic Ca2+ concentration reaches stable values higher than 10 µM. The data are consistent with the hypothesis that the logarithm of the increase in cytosolic Ca2+ concentration is linear with the logarithm of the light intensity. From the estimated values of the cytosolic Ca2+ concentration, we conclude that the Ca2+-buffering capacity is limited. The percentage of the Ca2+ influx that is buffered gradually decreases with increasing Ca2+ concentrations; at cytosolic Ca2+ concentration levels above 10 µM, buffering becomes minimal.
Key words: phototransduction; light adaptation; calcium buffering; calcium homeostasis; fluorescent calcium indicators ![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The cytosolic free concentration of Ca2+ ions (Cai) 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 Cai 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, Cai 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
).
Cai 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 Cai 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, Cai 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, Cai 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 Cai. 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 Cai could not be studied in
this way.
To better understand the regulation of Cai under natural, physiological conditions, we developed a technique to directly measure Cai in the intact eye using fluorescent Ca2+ indicator dyes with varying affinity for Ca2+. We thus were able to estimate Cai as a function of adapting light intensity. We find that bright illumination of fly photoreceptors causes surprisingly high levels of Cai; i.e., exceeding 10 µM.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Preparation
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).
Electrophysiology
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 M 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.
Dye Filling
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).
Optical Setup
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.
Data Acquisition
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.
Photography
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).
|
Quantitative Data Analysis
To estimate Cai 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 Cai 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 Cai on the light intensity, we
calculated the expected fluorescence signal as a function of the
light intensity with the function F(Cai) = Caih/(Caih + Kdh). Since
our in vivo method does not allow a direct calibration of the indicators, we used the Kd values published by Haugland (1996):
OG1, Kd = 0.17 µM; OG2, Kd = 0.58 µM; OG5N, Kd = 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 Cai 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.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
A New Method to Measure Cytosolic Ca2+ Dynamics in Photoreceptors of Insect Compound Eyes In Vivo
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 Cai. Then, after a short delay (~3 ms), the emission very rapidly increases, indicating an abrupt rise in Cai (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).
|
The Effect of the Ca2+-indicator Dyes on the Membrane Potential
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 Cai (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.
Bright Light Causes Cai to Increase into the High Micromolar Range
Fluorescence measurements of cells injected with the
low affinity dye OG5N (Kd = 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; Kd = 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 Cai attains values where the high affinity dyes OG1 and OG2 are saturated. Because OG2 saturates at Cai
10 µM (Haugland, 1996
), Cai exceeds
this range during the peak observed with the low affinity dye OG5N.
|
Steady State Cai after Adaptation to Different Light Intensities
To measure the dye fluorescence with an acceptable signal to noise ratio, it is necessary to use very high light intensities. To assess Cai 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 Cai reached a stable plateau value and that diffusion of the Ca2+ ions had reached an equilibrium. We assume, therefore, that the subcellular distribution of Cai 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 Cai, 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 = Vmax*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 (Vmax).
Fig. 6 a presents the resulting peak and plateau values as a function of relative light intensity. The Vmax 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 Cai 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 Cai 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 Cai rises linearly with log I. To get a more quantitative picture of the changes of Cai caused by light adaptation, we have therefore tried to describe the dependency of Cai on the light intensity with a simple
power function: Cai(I) = Cai,da + a*I b; Cai,da denotes
here Cai 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 Cai is appropriate. As shown in Fig. 6 c, at
log I = 2, Cai 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 Cai, 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, Cai 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
Cai,da, the dark adapted Cai, 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.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Measuring Calcium in Insect Photoreceptors
We demonstrate in the present paper that it is possible to measure the light-induced changes of Cai 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.
Changes in Cai Induced by Light Stimulation
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 Cai to high concentrations.
In Drosophila (Hardie, 1996a
) and the drone (Walz et
al., 1994
), the increase in Cai is fast and consistently saturates high affinity dyes after ~200 ms. Using Mag-Indo-1, Hardie (1996a)
estimated that Cai reaches values up to 50 µM in isolated Drosophila photoreceptor
cells. Here we show that Cai 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 Cai reaches values exceeding 10 µM.
The adaptation experiments (Figs. 5 and 6) allow us
to estimate how Cai depends on the light intensity (Fig.
6 c). Quantitative measurements of Cai 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 Kd 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 Cai.
We conclude that Cai 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 Cai; this increase is spread
over a distance of at least 20 µm (Ukhanov and Payne,
1995
). Therefore, it seems likely that high values of Cai
are also reached in the cell bodies of Calliphora photoreceptor cells that have a diameter of ~10 µm.
Buffering of the Ca2+ Influx
The finding that Cai 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 (
Catot;
Fig. 7 a,
) and compare the resulting values with our
estimates of the increase in Cai (
Cai; Fig. 7 a, bold line).
The calculated values appear to be in good agreement
with the values of the light-induced Cai increase at log
I
2. Both curves have a similar slope at these high intensities.
|
However, at light intensities below log I = 2, Catot >
Cai, or the amount of Ca2+ entering the photoreceptor cells from the extracellular space is larger than the
increase of Cai (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 Binf =
Catot/
Cai is presented in Fig. 7 b as a function of light intensity, and in
Fig. 7 c as a function of Cai. Clearly, the buffering capacity is limited, and at Cai
10 µM (corresponding to
log I = 1.5) buffering becomes minor. The values of
10-20 obtained for the buffering coefficient at Cai
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 Binf. 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 Binf. However, if
Ca2+ buffering is assumed to be constant at a high buffering coefficient Binf throughout the light intensity
range studied, there would not be enough extracellular
Ca2+ to sustain a Ca2+ influx that causes Cai to rise to 20 µM. We calculate a maximal value for Binf 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 Cai (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 Cai values in bright light. This conclusion is not affected by the uncertainties in calculated values for Binf.
Another possible reason for the high discrepancy between the values for Binf 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 Cai
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 Catot/Cai = 1 + Btot/(Kd + Cai), where
Catot is the total Ca2+ concentration and Btot is the total
concentration of a buffer with dissociation constant Kd.
The buffering coefficient of the Ca2+ influx (Binf) was
calculated by Binf(I) = [Catot(I) Catot,da]/[Cai(I)
Cai,da], where Catot,da is the total Ca2+ concentration in
the dark. The smooth line in Fig. 7 c was obtained by
taking Btot = 18 µM and Kd = 0.77 µM.
Comparison with Other Cellular Processes Dependent on Calcium
Illumination of invertebrate photoreceptors with bright
light induces a rapid activation of mitochondrial respiration, presumably due to a rise in Cai (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 Cai 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 Cai 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 Cai levels.
A change in both the membrane potential and Cai 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, Cai 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., Cai > 10 µM; Fig. 7), the rise in light-dependent permeability
translates superlinearly into a rise in Cai. In addition,
high Cai possibly activates a K+ conductance (Weckström, 1989
), resulting in a reduced rise in membrane
potential.
![]() |
FOOTNOTES |
---|
Address correspondence to Johannes Oberwinkler, Department of Neurobiophysics, University of Groningen, Nijenborgh 4, NL-9747 AG Groningen, The Netherlands. Fax: +31 50 363-4740; E-mail: j.oberwinkler{at}bcn.rug.nl
Original version received 27 February 1998 and accepted version received 8 June 1998.
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.
![]() |
Abbreviations used in this paper |
---|
OG1, Oregon Green 1; OG2, Oregon Green 2; OG5N, Oregon Green 5N.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Autrum, H. 1979. Light and dark adaptation in invertebrates. In Handbook of Sensory Physiology. Vol. VII/6A. H. Autrum, editor. Springer Verlag, Berlin, Germany. 1-91. |
2. |
Babcock, D.F.,
J. Herrington,
P.C. Goodwin,
Y.B. Park, and
B. Hille.
1997.
Mitochondrial participation in the intracellular Ca2+ network.
J. Cell Biol.
136:
833-844
|
3. | Bader, C.R., F. Baumann, and D. Bertrand. 1976. Role of intracellular calcium and sodium in light adaptation in the retina of the honey bee drone (Apis mellifera L.). J. Gen. Physiol. 67: 475-491 [Abstract]. |
4. | Coles, J.A., and R.K. Orkand. 1985. Changes in sodium activity during light stimulation in photoreceptors, glia and extracellular space in drone retina. J. Physiol. (Camb.). 362: 415-435 [Abstract]. |
5. | Fein, A., and M. Tsacopoulos. 1988. Activation of mitochondrial oxidative metabolism by calcium ions in Limulus ventral photoreceptor. Nature. 331: 437-440 [Medline]. |
6. | Franceschini, N., and K. Kirschfeld. 1971. Les phénomènes de pseudopupille dans l'oeil composé de Drosophila. Kybernetik. 9: 159-182 [Medline]. |
7. | Gerster, U.. 1997. A quantitative estimate of flash-induced Ca2+- and Na+-influx and Na+/Ca2+-exchange in blowfly Calliphora photoreceptors. Vision Res 37: 2477-2485 [Medline]. |
8. | Hardie, R.C. 1985. Functional organization of the fly retina. In Progress in Sensory Physiology. Vol. 5. D. Ottoson, editor-in-chief. Springer Verlag, Berlin, Germany. 1-79. |
9. | Hardie, R.C.. 1991. Whole-cell recordings of the light-induced current in dissociated Drosophila photoreceptors: evidence for feedback by calcium permeating the light-sensitive channels. Proc. R. Soc. Lond. B Biol. Sci. 245: 203-210 . |
10. | Hardie, R.C.. 1995a. Photolysis of caged Ca2+ facilitates and inactivates but does not directly excite light-sensitive channels in Drosophila photoreceptors. J. Neurosci 15: 889-902 [Abstract]. |
11. | Hardie, R.C.. 1995b. Effects of intracellular Ca2+ chelation on the light response in Drosophila photoreceptors. J. Comp. Physiol. A Sens. Neural Behav. Physiol. 177: 707-721 [Medline]. |
12. |
Hardie, R.C..
1996a.
INDO-1 measurements of absolute resting and
light-induced Ca2+ concentration in Drosophila photoreceptors.
J.
Neurosci.
16:
2924-2933
|
13. | Hardie, R.C.. 1996b. Excitation of Drosophila photoreceptors by BAPTA and ionomycin: evidence for capacitative Ca2+ entry? Cell Calc. 20: 315-327 [Medline]. |
14. | Hardie, R.C., and B. Minke. 1994. Ca2+-dependent inactivation of light sensitive channels in Drosophila photoreceptors. J. Gen. Physiol. 103: 409-427 [Abstract]. |
15. | Haugland, R.P. 1996. Handbook of fluorescent probes and research chemicals. 6th ed. Molecular Probes Inc., Eugene, Oregon. 511 pp. |
16. | Hochstrate, P. 1991. Electrogenic Na+-Ca2+ exchange contributes to the light response of fly photoreceptors. Z. Naturforsch. 46c: 451-460. |
17. | Hochstrate, P., and A. Juse. 1991. Intracellular free calcium concentration in the blowfly retina studied by fura-2. Cell Calc. 12: 695-712 [Medline]. |
18. | Hofstee, C.A., and D.G. Stavenga. 1996. Calcium homeostasis in photoreceptor cells of Drosophila mutants inaC and trp studied with the pupil mechanism. Vis. Neurosci. 13: 257-263 [Medline]. |
19. | Howard, J.. 1984. Calcium enables photoreceptor pigment migration in a mutant fly. J. Exp. Biol. 113: 471-475 . |
20. | Howard, J., B. Blakeslee, and S.B. Laughlin. 1987. The intracellular pupil mechanism and photoreceptor signal:noise ratios in the fly Lucilia cuprina. Proc. R. Soc. Lond. B Biol. Sci. 231: 415-435 [Medline]. |
21. | Kirschfeld, K., and N. Franceschini. 1969. Ein Mechanismus zur Steuerung des Lichtflusses in den Rhabdomeren des Komplexauges von Musca. Kybernetik. 6: 13-22 [Medline]. |
22. | Kirschfeld, K., and K. Vogt. 1980. Calcium ions and pigment migration in fly photoreceptors. Naturwissenschaften. 67: 516-517 . |
23. | Laughlin, S.B. 1981. Neural principles in the peripheral visual systems of invertebrates. In Handbook of Sensory Physiology. Vol. VII/6B. H. Autrum, ed. Springer Verlag, Berlin, Germany. 133-280. |
24. | Laughlin, S.B., and R.C. Hardie. 1978. Common strategies for light adaptation in the peripheral visual systems of fly and dragonfly. J. Comp. Physiol. 128: 319-340 . |
25. | Matic, T., and S.B. Laughlin. 1981. Changes in the intensity- response function of an insect's photoreceptors due to light adaptation. J. Comp. Physiol. 145: 169-177 . |
26. | Minke, B., and Z. Selinger. 1996. The roles for trp and calcium in regulating photoreceptor function in Drosophila. Curr. Opin. Neurobiol. 6: 459-466 [Medline]. |
27. | Miyata, H., H.S. Silverman, S.J. Sollott, E.G. Lakatta, M.D. Stern, and R.G. Hansford. 1991. Measurement of mitochondrial free Ca2+ concentration in living single rat cardiac myocytes. Am. J. Physiol. 30: H1123-H1134 . |
28. | Mojet, M.H., J. Tinbergen, and D.G. Stavenga. 1991. Receptor potential and light-induced mitochondrial activation in blowfly photoreceptors mutant. J. Comp. Physiol. A Sens. Neural Behav. Physiol. 168: 305-312 . |
29. | Muijser, H.. 1979. The receptor potential of retinular cells of the blowfly Calliphora: the role of sodium, potassium and calcium ions. J. Comp. Physiol. 132: 87-95 . |
30. | Neher, E.. 1995. The use of fura-2 for estimating Ca buffers and Ca fluxes. Neuropharmacology. 34: 1423-1442 [Medline]. |
31. | O'Day, P.M., and M.P. Gray-Keller. 1989. Evidence for electrogenic Na+/Ca2+ exchange in Limulus ventral photoreceptors. J. Gen. Physiol 93: 473-492 [Abstract]. |
32. | Orkand, R.K., I. Dietzel, and J.A. Coles. 1984. Light-induced changes in extracellular volume in the retina of the drone, Apis mellifera. Neurosci. Lett. 45: 273-278 [Medline]. |
33. | Peretz, A., C. Sandler, K. Kirschfeld, R.C. Hardie, and B. Minke. 1994a. Genetic dissection of light-induced Ca2+ influx into Drosophila photoreceptors. J. Gen. Physiol. 104: 1057-1077 [Abstract]. |
34. | Peretz, A., E. Suss-Toby, A. Rom-Glas, A. Arnon, R. Payne, and B. Minke. 1994b. The light response of Drosophila photoreceptors is accompanied by an increase in cellular calcium: effects of specific mutations. Neuron. 12: 1257-1267 [Medline]. |
35. | Ranganathan, R., B.J. Bacskai, R.Y. Tsien, and C.S. Zuker. 1994. Cytosolic calcium transients: spatial localization and role in Drosophila photoreceptor cell function. Neuron. 13: 837-848 [Medline]. |
36. | Roebroek, J.G.H., and D.G. Stavenga. 1990. Insect pupil mechanisms. IV. Spectral characteristics and light intensity dependence in the blowfly, Calliphora erythrocephala. J. Comp. Physiol. A Sens. Neural Behav. Physiol. 166: 537-543 . |
37. | Rom-Glas, A., C. Sandler, K. Kirschfeld, and B. Minke. 1992. The nss mutation or lanthanum inhibits light-induced Ca2+ influx into fly photoreceptors. J. Gen. Physiol. 100: 767-781 [Abstract]. |
38. | Sandler, C., and K. Kirschfeld. 1988. Light intensity controls extracellular Ca2+ concentration in the blowfly retina. Naturwissenschaften. 75: 256-258 . |
39. | Sandler, C., and K. Kirschfeld. 1991. Light-induced extracellular calcium and sodium concentration changes in the retina of Calliphora: involvement in the mechanism of light adaptation. J. Comp. Physiol. A Sens. Neural Behav. Physiol. 169: 299-311 . |
40. | Sandler, C., and K. Kirschfeld. 1992. Light-induced changes in extracellular calcium concentration in the compound eye of Calliphora, Locusta and Apis. J. Comp. Physiol. A Sens. Neural Behav. Physiol. 171: 573-581 . |
41. | Selinger, Z., Y.N. Doza, and B. Minke. 1993. Mechanisms and genetics of photoreceptors desensitization in Drosophila flies. Biochim. Biophys. Acta. 1179: 283-299 [Medline]. |
42. | Stavenga, D.G., N. Franceschini, and K. Kirschfeld. 1984. Fluorescence of housefly visual pigment. Photochem. Photobiol. 40: 653-659 . |
43. | Stavenga, D.G., and J. Tinbergen. 1983. Light dependence of oxidative metabolism in fly compound eyes studied in vivo by microspectrofluorometry. Naturwissenschaften. 70: 618-620 . |
44. | Tsukahara, Y.. 1980. Effect of intracellular injection of EGTA and tetraethylammonium chloride on the receptor potential of locust photoreceptors. Photochem. Photobiol. 32: 509-514 [Medline]. |
45. | Ukhanov, K.Y., T.M. Flores, H.-S. Hsiao, P. Mohaparta, C.H. Pitts, and R. Payne. 1995. Measurement of cytosolic Ca2+ concentration in Limulus ventral photoreceptors using fluorescent dyes. J. Gen. Physiol. 105: 95-116 [Abstract]. |
46. | Ukhanov, K.Y., and R. Payne. 1995. Light activated calcium release in Limulus ventral photoreceptors as revealed by laser confocal microscopy. Cell Calc. 18: 301-311 [Medline]. |
47. | van Hateren, J.H.. 1984. Waveguide theory applied to optically measured angular sensitivities of fly photoreceptors. J. Comp. Physiol. A Sens. Neural Behav. Physiol. 154: 761-771 . |
48. | Walz, B.. 1992. Enhancement of sensitivity in photoreceptors of the honey bee drone by light and by Ca2+. J. Comp. Physiol. A Sens. Neural Behav. Physiol. 170: 605-613 [Medline]. |
49. | Walz, B., B. Zimmermann, and S. Seidl. 1994. Intracellular Ca2+ concentration and latency of light-induced Ca2+ changes in photoreceptors of the honeybee drone. J. Comp. Physiol. A Sens. Neural Behav. Physiol. 174: 421-431 . |
50. | Walz, B., O. Baumann, B. Zimmermann, and E.V. Ciriacy-Wantrup. 1995. Caffeine- and ryanodine-sensitive Ca2+-induced Ca2+ release from the endoplasmic reticulum in honeybee photoreceptors. J. Gen. Physiol 105: 537-567 [Abstract]. |
51. | Weckström, M.. 1989. Light and dark adaptation in fly photoreceptors: duration and time integral of the impulse response. Vision Res. 29: 1309-1317 [Medline]. |
52. | Ziegler, A., and B. Walz. 1989. Analysis of extracellular calcium and volume changes in the compound eye of the honeybee drone, Apis mellifera. J. Comp. Physiol. A Sens. Neural Behav. Physiol. 165: 697-709 . |