From the * Department of Physiology, Boston University School of Medicine, Boston, Massachusetts 02118; and Marine Biological Laboratory, Woods Hole, Massachusetts 02543
The ability of scallop hyperpolarizing photoreceptors to respond without attenuation to repetitive
flashes, together with their low light sensitivity, lack of resolvable quantum bumps and fast photoresponse kinetics,
had prompted the suggestion that these cells may be constitutively in a state akin to light adaptation. We here
demonstrate that their photocurrent displays all manifestations of sensory adaptation: (a) The response amplitude to a test flash is decreased in a graded way by background or conditioning lights. This attenuation of the response develops with a time constant of 200-800 ms, inversely related to background intensity. (b) Adapting stimuli shift the stimulus-response curve and reduce the size of the saturating photocurrent. (c) The fall kinetics of the
photoresponse are accelerated by light adaptation, and the roll-off of the modulation transfer function is displaced to higher frequencies. This light-induced desensitization exhibits a rapid recovery, on the order of a few
seconds. Based on the notion that Ca mediates light adaptation in other cells, we examined the consequences of
manipulating this ion. Removal of external Ca reversibly increased the photocurrent amplitude, without affecting light sensitivity, photoresponse kinetics, or susceptibility to background adaptation; the effect, therefore, concerns
ion permeation, rather than the regulation of the visual response. Intracellular dialysis with 10 mM BAPTA did
not reduce the peak-to-plateau decay of the photocurrent elicited by prolonged light steps, not the background-induced compression of the response amplitude range and the acceleration of its kinetics. Conversely, high levels of buffered free [Ca]i (10 µM) only marginally shifted the sensitivity curve ( = 0.3 log) and spared all manifestations of light adaptation. These results indicate that hyperpolarizing invertebrate photoreceptors adapt to light,
but the underlying mechanisms must utilize pathways that are largely independent of changes in cytosolic Ca. The
results are discussed in terms of aspects of commonalty to other ciliary sensory receptor cells.
The eyes of some invertebrate organisms display the remarkable feature of a double retina (Dakin, 1910),
composed of a proximal layer of depolarizing photoreceptors, which possess a microvilli-covered light-sensitive lobe similar to that found in most invertebrates, and a distal layer of hyperpolarizing photoreceptors
(Gorman and McReynolds, 1969
; Mpitsos, 1973
). In
the latter, the light-sensing organelles are modified ciliary appendages (Miller, 1958
) analogous to the outer
segment of rods and cones (Tokuyasu and Yamada,
1959
; Eakin, 1965
). In a previous report we used patch-electrode recording to characterize the properties of
the light-sensitive conductance in enzymatically isolated ciliary photoreceptors from two species, Lima scabra and Pecten irradians (Gomez and Nasi, 1994
); subsequently, we demonstrated that these cells, like rods and
cones, use the cyclic GMP cascade for transduction,
whereas the inositol 1,4,5-trisphosphate (IP3)1/Ca signalling pathway appears not to play a role in the activation of the light-sensitive conductance (Gomez and Nasi
1995
). In addition to underscoring an essential divergence in the visual excitation mechanisms across photoreceptor cell lines, these results raise questions about
light adaptation, which in all other known photoreceptors is chiefly controlled by cytosolic calcium levels (reviewed by Fain and Matthews, 1990
; Fein and Szuts,
1982
): in addition to the absence of light-dependent
IP3-mediated Ca release, no changes of Ca fluxes accompany the photocurrent in these cells (e.g., unlike
rods and cones), because their light-sensitive conductance is highly selective for potassium ions (Cornwall
and Gorman 1983
; Gomez and Nasi, 1994
). The possibility that the light response of ciliary photoreceptors
may not be coupled to changes in intracellular Ca could
simply point to a lack of normal adaptation processes. This prospect is not to be dismissed a priori, if one considers that the biological function of the distal retina in
these organisms is to initiate a discharge of action potentials that triggers a defensive reflex when light is
abruptly decreased (as when an approaching predator
casts a shadow in the animal's visual field). This requires the ability to respond to continuous illumination, so that the inactivation of voltage-gated Ca channels (which occurs at the "dark" resting potential) is
tonically removed by the hyperpolarizing receptor potential; in this way, the system is constantly primed to
respond to light dimming, producing a rebound excitation when the membrane depolarizes. As such, this
scheme may not necessarily benefit from a gain-control
mechanism. An alternative possibility is that adaptation
does occur, and the lack of calcium involvement may
be indicative of a novel regulatory mechanism for the
light response.
Little information is available on the modulation of
the photoresponse in hyperpolarizing invertebrate visual cells. In one of their seminal descriptions of the
physiology of the dual retina of Pecten, McReynolds and
Gorman (1970) reported that when ciliary photoreceptors were stimulated with pairs of flashes, the response to the second light suffered no attenuation even with
inter-stimulus intervals as short as 5 s; by contrast, rhabdomeric cells required minutes to regain responsiveness
after the first flash. Although this phenomenon may indicate that ciliary cells lack the capability to desensitize,
one cannot rule out the possibility that light-adaptation does occur, but recovers rapidly. In fact, Cornwall and
Gorman (1979)
briefly reported that the response to a
test flash was reduced in amplitude when the stimulus
was superimposed on a steady light. In the present report, we examined systematically the effect of background
and conditioning lights on photoresponse sensitivity and
kinetics; we have found that all the manifestations of
light adaptation are present in ciliary photoreceptors,
with similar characteristics to those of other visual cells.
We then ascertained the effect of manipulations of calcium on light adaptation and determined that regulation of the photoresponse must use a pathway that is
largely calcium-independent.
Specimens of Pecten irradians were obtained from the Aquatic Resources Division of the Marine Biological Laboratory (Woods Hole, MA) and used immediately. The protocol for obtaining viable isolated hyperpolarizing photoreceptors by enzymatic dispersion of the retina has been described previously (Gomez and
Nasi, 1994). Cells were plated in a recording chamber continuously superfused with artificial sea water (ASW) containing 480 mM NaCl, 10 mM KCl, 49 mM MgCl2, 10 mM CaCl2, 10 mM HEPES,
and 5 mM glucose, pH 7.8 (NaOH). In 0-Ca ASW, calcium was replaced by magnesium. Patch electrodes were fabricated with thin-wall (1.5 mm o.d. 1.1 mm i.d.) borosilicate capillary tubing (type
7052; Garner Glass, Claremont, CA) pulled to a 2-3 µm tip diameter and fire-polished immediately before use. Their resistance,
measured in ASW, was 2-4 M
. The "intracellular" filling solution contained 100 mM KCl, 200 mM K-aspartate or K-glutamate,
12 mM NaCl, 5 mM Na2ATP, 5 mM MgCl2, 10 mM HEPES, 1 mM
EGTA, 300 mM sucrose, and 200 µM GTP, pH 7.3 (KOH). In
some experiments, instead of EGTA a higher concentration (10 mM) of the rapid Ca chelator BAPTA (1,2-bis(2-aminophenoxy) ethane-N,N,N
,N
-tetraacetic acid) was used to provide stronger calcium buffering. In others, CaCl2 was added to the internal solution to yield a free concentration of 10 µM, as determined by
the program Chelator (kindly provided by Dr. Theo Schoenmakers, University of Nijmegen, The Netherlands). The calculated
concentration of free Mg was 0.99 mM for the internal solutions
containing EGTA, and 0.94 mM for the solution containing
BAPTA. In all recordings series resistance errors were electronically corrected (maximum residual error <2 mV). Currents were
low-pass filtered with a Bessel 4-pole filter, using a cutoff frequency of 500-1,000 Hz and digitized on-line at 2-3 kHz sampling rate, 12-bit resolution by an analogue/digital computer interface board (2821; Data Translation, Marlboro, MA). All experiments were conducted at room temperature (22-24°C).
Light stimuli were provided by two independent photostimulators: one consisted of a halogen-quartz light source which delivered light to the preparation from above, through the microscope condenser (Nasi, 1991a, b
; Nasi and Gomez, 1992a
). The
other one utilized a Xenon arc lamp (Photon Technologies Inc.,
South Brunswick, NJ) coupled by a fiber optics light-guide to the
epifluorescence port of the inverted microscope and stimulated
the cells from below, through the objective (Gomez and Nasi,
1994
). The application of light stimuli was controlled by a microprocessor-based programmable stimulator (Stim 6; Ionoptix, Milton, MA) and electromechanical shutters and drivers (Vincent
Associates, Rochester, NY). To provide sufficiently bright test
flashes to saturate the photoresponse under light-adapted conditions, broad-band light was used in many of the experiments
(515-670 nm); its effective intensity was calibrated in vivo in several cells (n = 9), by comparing it to monochromatic stimulation
(500 nm peak, 10 nm half-width; Ditric Optics, Hudson, MA), as
previously described (Gomez and Nasi, 1994
). In the experiments described below, light intensity is expressed either in
terms of flux of equivalent photons at 500 nm, or by specifying
the relative attenuation with respect to a known standard (i.e.,
log10[I/Io], where Io is the maximum light intensity). Calibrated
neutral-density filters (Melles Griot, Irvine, CA) provided controlled attenuation. For continuous modulation of the intensity
of the light beam, an adjustable attenuator was used (Meadowlark Optics, Longmont, CO); this consisted of a liquid crystal variable retarder placed between two crossed polarizers, controlled
by an arbitrary waveform generator (Hewlett Packard Mod.
33120A) which was programmed to modulate the light sinusoidally, with a defined depth and frequency. Monochromatic light
(580 nm) was used for this purpose. During experimental manipulations the cells were illuminated through a near-infrared long-pass filter (
> 780 nm; Andover Corp., Salem, NH) and viewed
with a Newvicon TV camera (Mod. WV-1550; Panasonic, Secaucus, NJ). The infrared illuminator was turned off for several minutes before testing light responses.
Why the Susceptibility to Light Adaptation Is at Issue
To appreciate why the presence of light-adaptation
mechanisms in ciliary photoreceptors has been called
into question, it is helpful to compare some salient features of their photoresponses to those of rhabdomeric
cells. In rhabdomeric photoreceptors of many species
illumination with dim light evokes discrete waves of depolarization (or inward current, if measured under
voltage clamp); statistical analysis reveals that these discrete waves, or "quantum bumps" originate from individual absorbed photons (Yeandle, 1958). When the
photoreceptors are light adapted, the size of the bumps
is dramatically reduced, an effect that accounts in part
for the resulting desensitization (Wong, 1978
). Fig. 1 A
shows an example of light-induced current fluctuations
recorded in an isolated rhabdomeric photoreceptor
from Pecten. Membrane current was recorded at
50
mV in the dark, or during 10-s steps of dim light, the intensity of which was progressively increased. In ciliary
photoreceptors, by contrast, quantum bumps cannot
be resolved even at the lowest stimulating intensities (Fig.
1 B): in this case the brighter lights gave rise to a small
sustained outward photocurrent with a smooth time
course, lacking any resolvable discrete events. Similar
results were obtained in three additional cells.
The minute size of single-photon currents is consistent with the observation that the light sensitivity of ciliary photoreceptors is substantially lower than that of
their rhabdomeric counterparts (McReynolds and Gorman, 1970; Gomez and Nasi, 1994
). Fig. 2 A directly
compares families of responses to flashes of increasing intensity measured under voltage clamp in the two types
of photoreceptors; in part (B) of the same figure, the
normalized stimulus-response relations are plotted in
semi-logarithmic coordinates, revealing a relative shift
of
2.5 log units along the abscissa. Similar results
were obtained in five additional cells of each class (see also Gomez and Nasi, 1994
). On the assumption that
the amount of rhodopsin is comparable (as suggested
by measurements of the early receptor current; unpublished observations), one can calculate the relative transduction gain in rhabdomeric vs. ciliary cells taking into
account the amplitude of their respective macroscopic light-induced conductances, as well as the unitary conductance (48 and 26 pS, respectively) and the mean
open times of single-channels (Nasi and Gomez, 1992a
;
Gomez and Nasi, 1994
); the estimated number of channels activated per isomerized rhodopsin turns out to be at least 100-fold lower in ciliary cells. In addition, the
photocurrent of ciliary cells also displays considerably
more rapid kinetics than that of rhabdomeric cells
from the same preparation (see also McReynolds and
Gorman, 1970
). Reduced quantal response size, low
sensitivity, and fast kinetics are characteristic features of the behavior of a light-adapted photoreceptor. In
fact, it has been suggested that ciliary cells may be constitutively in a state akin to light-adaptation (McReynolds, 1976
). This called for demonstrating whether
normal light-adaptation mechanisms are indeed operative in these cells.
Demonstration of Adaptation
We first determined the effect of background light on
the responses elicited by test flashes of fixed intensity.
Fig. 3 shows recordings in a cell stimulated every 2.5 s;
after the first two responses, a background light was
turned on for 14 s. Four different background intensities were tested. In the presence of the sustained light
step, the incremental responses to the test flashes grew smaller, the amount of reduction being directly related
to background intensity. The test response amplitude
rapidly recovered after termination of the adapting
light. The decrease in light response cannot be accounted for by direct occlusive effects of the two lights
(namely, the reduced number of light-dependent channels that remain available during background illumination), because in the presence of the adapting light the
total current amplitude at the peak of the flash responses was lower than that attained when the flashes
were presented alone (n = 3).
To examine systematically the adapting effects of sustained illumination, the full sensitivity curve of the
flash response was determined with a standard background-adaptation protocol. The top row of Fig. 4 A
shows recordings obtained in a ciliary photoreceptor
voltage-clamped at 20 mV and stimulated with 100-ms flashes of light, the intensity of which was increased at
0.3 log increments. After this dark-adapted series, a
steady background light was turned on, and a similar
intensity series was administered. The procedure was
repeated, increasing the intensity of the background light by one log unit each time. At the end a second
control series was run to verify that the dark-adapted responsiveness recovered (peak amplitudes were within
10% of control) so that the photocurrent reduction in
the presence of the background light could not be attributed to response run-down. In Fig. 4 B, the peak
amplitude of the photocurrent is plotted as a function
of log attenuation of the test light for each of the four
background conditions. Sigmoidal functions were least-squares fitted to the data to estimate the saturating
photocurrent amplitude (S) and the half-maximal stimulus intensity (
). The resulting curves exhibit two essential features of light adaptation: (a) as the background light intensity is increased, the asymptotic value
of the response amplitude is reduced and (b) the stimulus-response curves are progressively shifted in the positive direction along the abscissa. We next examined
whether this desensitization was accompanied by changes in photocurrent kinetics. A remarkable feature observed when brief flashes are used to stimulate ciliary
photoreceptors is the similarity in the time course of
the responses as the light intensity is varied over a substantial range, spanning over 1.5 log units; this can be
appreciated in the second row of Fig. 4 A, in which the
photocurrent records in each family have been normalized and superimposed. There is a barely noticeable acceleration of the rising phase of the response (which
becomes discernible at higher temporal resolution),
but the overall shape of the response changes very little. Such near-constancy of kinetics justifies comparing across the four background conditions, because it makes
comparisons independent of which particular trace is
selected from each family. Fig. 4 C shows the superimposed, normalized half-saturating photocurrents of each
group. As the intensity of the background illumination is increased, the response kinetics become faster; the
effect is particularly pronounced in the decay phase,
whereas the acceleration of the rising phase is comparatively more modest. Complete intensity series in the
presence of different background lights were measured in four other cells, with similar results.
The faster kinetics of the flash response in the presence of a background light reflects the trade-off of
speed for sensitivity as the ambient illumination level is
varied, a well-known phenomenon in other classes of
photoreceptors (Fain and Cornwall, 1994). A convenient quantification of this process entails measuring the temporal modulation transfer function (MTF) at
different states of adaptation. Fig. 5 A shows the responses to 20-s steps of 580 nm light, whose intensity
was modulated sinusoidally with a depth of 15% and a
frequency that varied between 0.125 Hz and 6 Hz. In
the recordings on the left, the light was dim, and produced a maximum response <5% of the saturating
photocurrent. As the frequency was increased, the modulation of the response was quickly lost, and above 2 Hz
only the mean photocurrent is seen (last 4 sweeps).
Subsequently, the cell was exposed to a steady background that caused
0.5 log desensitization; the test
light was made brighter by 1.2 log units in order to produce an incremental response of comparable size as the
dark adapted response (right). In the light-adapted state
the photoreceptor was capable of tracking more rapid
temporal fluctuations in light intensity, and the response modulation faded only for the highest frequency (e.g.,
6 Hz). The transfer function is shown in Fig. 5 B: the response modulation is plotted as a function of frequency
in double-logarithmic coordinates. The 50% drop-off
occurred at
0.75 Hz for the dark-adapted measurement, whereas with light adaptation it was attained at
2.5 Hz. Similar observations were obtained in a total
of eight cells; the average shift in the 50% roll off frequency with light adaptation was 248 ± 64% SD.
Time Course of the Development of Adaptation and Its Recovery
When background illumination is turned on, the process of adjusting the transduction gain must require a
defined amount of time. In Fig. 3, above, the amplitude
of the first flash response after the onset of the background light was already reduced, and no further
changes occurred in subsequent responses. This suggests that light adaptation developed more rapidly than
the inter-stimulus interval; the time resolution afforded
by this protocol could not be increased substantially,
because reduction in the temporal lag between flashes
would result in response overlap. On the other hand,
the observation that the time course of the photocurrent elicited by brief flashes changes little when the
stimulus intensity is varied over a wide range (e.g., Fig.
4 A) indicates that adaptation must require at least a
few hundred milliseconds to become manifest. As a
consequence, one would predict that with more prolonged light steps the kinetics of the photocurrent
should change as a function of light intensity. Such a
comparison is presented in Fig. 6. In part A (left) a photoreceptor was stimulated with 100-ms flashes of increasing intensity, spanning 2.4 log; on the right, the
protocol was repeated in the same cell, but duration of
the stimulus was increased to 2,800 ms. To better compare the time course of the photocurrent at different
light intensities, the responses in each family of traces
were normalized with respect to their peak amplitude,
and superimposed in part B of the figure. Whereas the currents evoked by flashes are almost superimposable
(except for a slight acceleration in the rising phase with
brighter lights), those evoked by the sustained steps
show the progressive development of a relaxation to a
reduced plateau level (the differences in the rising
phase reflect chiefly the integration time of the cell). Similar results were replicated in three other cells.
To quantify the time course of the development of
light adaptation, we used a more complex protocol,
which provided a substantially finer time resolution. A
pair of identical test flashes, 100-ms in duration, were
presented separated by 4 s, a sufficient temporal lag to
allow any desensitization by the first light to dissipate (see also Fig. 7 below). On successive trials a steady
background light was turned on during the period between the two test flashes, preceding the second one by
an interval that varied between 300 and 2,700 ms. The
second flash response, therefore, was always superimposed on the adapting background. As shown in Fig. 7,
the amplitude of this incremental response decreased
monotonically as the interval between background onset and test flash delivery was increased; this behavior
reflects the gradual development of desensitization induced by the adapting light. The constancy of the responses to the first flash indicates that no response run-down occurred during the experiment. The entire sequence was repeated in the same cell, increasing the intensity of the background light at 0.6 log increments.
On the right side of the same figure, the peak amplitude of the incremental response is plotted as a function of temporal lag, for the various conditions. Each
set of data points was least-squares fitted by a single exponential function. As one would expect, the asymptotic level of response reduction, which measures the
extent of desensitization, is directly related to the background light intensity. In addition, a striking change
occurred in the exponential relaxation, which reflects the time course of the onset of light adaptation: as the
intensity of the background light was increased, the
time constant of the process decreased, in the range
800-200 ms (n = 3).
The photoresponse amplitude changes, the shift in
sensitivity, and the acceleration of kinetics induced by
background lights, demonstrated in Figs. 1-5, clearly
indicate that all salient manifestations of light adaptation are present in ciliary photoreceptors. This conclusion implies that the previously mentioned observation
by McReynolds and Gorman (1970), concerning the
unabated responsiveness to closely spaced light stimuli,
must be due to a very rapid recovery of sensitivity, rather
than lack of desensitization. We corroborated this notion by examining the time course of dark adaptation,
using the triple-stimulus protocol illustrated in Fig. 8: a
photoreceptor was stimulated with a 100-ms test flash,
which served as a reference, then exposed to a 3-s
adapting light of the same intensity; recovery of sensitivity was measured by presenting another 100-ms test flash at different intervals, in the range of 300-4,200
ms, after the termination of the adapting light. A period of darkness, 1 min in duration, was interposed between trials to allow any residual desensitization to dissipate completely. In the left part of the figure one can
appreciate that the responses to both the pre-test and
the adapting lights are superimposable, indicating that
no drift in responsiveness occurred during the experiment. During the 3-s light step a pronounced decay of
the photocurrent occurred, chiefly reflecting the adaptation process; when the test flash was delivered shortly
afterwards, the response was greatly depressed, but as
the dark interval was progressively lengthened the response increased monotonically. The peak amplitude
of the test photocurrent is plotted in the right side of
the figure as a function of time elapsed since termination of the adapting light, and fitted to a two-time constant exponential function. Recovery was
99% complete in about 4 s (n = 3). Considering that the light intensity employed was nearly saturating, these results fully
account for the apparent failure to desensitize the photoresponse with pairs of stimuli separated by a few seconds. Similar procedures were used with dimmer lights;
in this case the recovery process followed a single-exponential function, lacking the rapid phase (data not shown).
Effects of Calcium Manipulations on Light Adaptation and Recovery
A long-standing tenet in photoreceptor physiology is
that the modulation of sensitivity is controlled by changes
in intracellular Ca levels. A simple demonstration of
this proposition entails superfusing cells with a solution
lacking Ca. In rhabdomeric cells from several species
this treatment transiently enhances the response amplitude and slows down its kinetics, an effect that has been attributed to the sensitization of the transduction cascade (Limulus : Millecchia and Mauro, 1969; Balanus :
Brown et al., 1970
; Apis : Raggenbass, 1983
). In ciliary
photoreceptors 0-Ca ASW increases the amplitude of
the light response but has no significant effect on the
intensity-response relation (not shown), nor on the kinetics of the photocurrent (Gomez and Nasi, 1995
).
Because extracellular Ca and Mg were previously shown
to block the light-dependent conductance in these cells
(Gomez and Nasi 1994
; Nasi and Gomez, 1996
), the larger responses measured in 0-Ca indicate relief from
blockage, rather than a change in the state of adaptation. To further confirm this contention, we examined
the effect of Ca removal on the desensitization induced
by background lights of different intensities.
Fig. 9 shows the results of an experiment in which an
adapting light step of variable intensity was presented
for 5.5 s, and a fixed test flash was superimposed on it
during the plateau phase of the response. The incremental response to the flash decreased monotonically
as the background intensity was increased. This dependency was not significantly different in 10 mM vs. 0 Ca
(n = 5). Exposure to Ca-free solution also failed to affect the recovery from light adaptation. In Fig. 10, a triple-stimulus protocol similar to that of Fig. 8 was used,
but with dimmer lights designed to optimize the detection of changes in sensitivity. As the interval elapsed
since the termination of the adapting light was increased, the response to the test flash recovered its amplitude; this recovery, however, followed a similar time
course in the presence and in the absence of extracellular Ca (n = 2).
The insensitivity of ciliary photoreceptors to removal
of extracellular Ca stands in striking contrast with the
effects observed in rhabdomeric photoreceptors. A possible explanation could be that such manipulation simply fails to change [Ca]i significantly, because in these
cells (a) light-dependent channels are highly selective
for potassium (Gomez and Nasi, 1994),(b) internal Ca release is unlikely to occur, as these cells are unresponsive to IP3 and unaffected by IP3 antagonists (Gomez
and Nasi, 1995
), and (c) voltage clamping forestalls any
contribution by voltage-sensitive Ca channels (Cornwall and Gorman, 1979
). An alternative strategy, therefore, is to directly alter intracellular Ca by dialysis via the patch pipette. In a previous report, we demonstrated
that the exchange between the pipette solution and the
cytosol is rapid and efficient: equilibration occurs with
a time constant on the order of 30 s, and the dialysate
unambiguously reaches the site of phototransduction
(Gomez and Nasi, 1995
). We first examined the effects
of strongly buffering intracellular calcium on light adaptation.
The left side of Fig. 11 A shows a standard background adaptation procedure in a control cell voltage
clamped at 30 mV with an electrode filled with the
standard intracellular solution containing 1 mM EGTA;
light intensity series were measured in the dark-adapted state and in the presence of steady illumination. The
adapting light reduced the saturating photoresponse
amplitude to 84% and shifted the sensitivity curve
(
= 0.73 log); in addition, the response kinetics
were significantly accelerated (Fig. 11 B, left). The same
effects occurred in a quantitatively comparable manner in the presence of 10 mM intracellular BAPTA, as
shown by the panels on the right: the background-
induced response reduction was 80%, and
was 0.74 log. Similar results were obtained in 10 cells (5 in each
condition). We also compared the effects of different
intensities of background illumination in the presence or absence of intracellular BAPTA. In Fig. 12 a cell dialyzed with 10 mM BAPTA was exposed to a 5-s light step
of increasing intensity, upon which a fixed 100-ms test
flash was superimposed. The incremental response decreased in size as the background was made brighter; this is shown in greater detail in the panel on the right.
In Fig. 12 B the average normalized amplitude of the
test response, pooled for several control cells and several BAPTA-treated cells, is plotted as a function of the
intensity of the adapting light. Again, there is no indication of any systematic change in susceptibility to adaptation in the presence of the calcium chelator.
A final criterion is to compare the peak-to-plateau decay of the photocurrent elicited by a sustained light in
cells treated with 10 mM BAPTA vs. 1 mM EGTA. Because the relaxation reflects in part the development of
adaptation, if Ca changes play an important role in the
process, the decay phase should be attenuated by the high concentration of the Ca chelator. Fig. 13 illustrates
such a comparison. Light stimuli of increasing intensity
were presented for a period of 3 s, separated by a 1-min
interval of dark adaptation. The ratio of peak amplitude vs. plateau is plotted in the bottom graph of the
figure, and shows no significant differences at any of
the light intensities used. The converging results obtained with different approaches strongly indicate that
chelating internal Ca has little effect on light response
in ciliary cells; by contrast, identical treatments administered to rhabdomeric cells from the same retina profoundly influence the amplitude sensitivity and kinetics
of the photocurrent (data not shown).
A complementary approach is to induce an elevation
of intracellular Ca; we used 10 µM free Ca in the electrode solution, a concentration than in rhabdomeric
cells causes a loss of sensitivity of at least 3 orders of
magnitude, and a marked acceleration of the response
kinetics. Fig. 14 A compares the intensity-response function averaged for six control cells (1 mM EGTA, no
added Ca) and six cells internally perfused with 10 µM
Ca2+. In the high-Ca condition the sensitivity curve
shifted marginally, by 0.3 log. This shift, however, is
quite modest by comparison to that induced by background illumination and is equivalent to the desensitization produced by an adapting light
20-fold dimmer than the half-saturating intensity. Fig. 14 B shows a
comparison of the desensitization of the response to a
fixed test flash in control conditions vs. elevated intracellular Ca, as a function of background light intensity.
In the presence of high [Ca]i the cells retained the full
dynamic range of modulation by the adapting light;
and the background light intensity required to reduce
the size of the incremental photoresponse by 50% was
only slightly lower (by 0.49 log).
The results presented above indicate that changes of
ambient illumination modulate the photocurrent sensitivity and kinetics in Pecten ciliary photoreceptors, in a
way that resembles in all respects the light adaptation
process found in other photoreceptor types, both vertebrate and invertebrate. However, cytosolic calcium appears to have little influence on this modulatory process, in sharp contrast with its pivotal role in several
crucial aspects of light adaptation in other visual cells.
In amphibian rods a decrease in intracellular Ca, such
as it occurs during illumination (Yau and Nakatani,
1985; McNaughton et al., 1986
; Ratto et al., 1988
),
modulates, via Ca-binding proteins, the activity of guanylate cyclase (Koch and Stryer, 1988
), thus partially restoring the dark current and light responsiveness (reviewed by Fain and Matthews, 1990
). In addition, Ca
also regulates the lifetimes of activated rhodopsin (and,
consequently, stimulation of PDE; Kawamura, 1993
), the catalytic activity of rhodopsin (Lagnado and Baylor,
1994
), and the affinity of the channels for cGMP (Hsu
and Molday, 1993
). These coordinated functions decrease sensitivity and speed-up the dark-current recovery during background illumination. In rhabdomeric photoreceptors of Limulus and Apis, where light causes
an increase in internal Ca (Brown and Blinks, 1974
;
Walz et al., 1994
), the photoresponse is transiently enhanced by lowering [Ca]o (Millecchia and Mauro, 1969
;
Raggenbass, 1983
) or by intracellular application of Ca
chelators (Lisman and Brown, 1975
; Bader et al., 1976
), whereas direct Ca injection causes desensitization (Lisman and Brown, 1972
; Bader et al. 1976
; Fein and
Charlton, 1977
). The mechanisms by which Ca mediates some key aspects of light adaptation in rhabdomeric photoreceptors are presently poorly understood, but the possible involvement of Ca-triggered protein
phosphorylation mediated by a protein kinase C (PKC)
was recently suggested by observations on a Drosophila
mutant, inaC, which lacks an eye-specific PKC (Smith et
al., 1991
) and displays reduced desensitization by background illumination (Hardie et al., 1993
).
We should emphasize that the present observations
in ciliary visual cells point to the existence of a powerful
alternative pathway for light adaptation but do not necessarily rule out all involvement of Ca under physiological conditions: for example, manipulations of cytosolic
calcium could be rendered ineffective by the loss of
some required soluble co-factor, as a result of cell dialysis. In principle, it should be possible to prevent such
wash-out effects by using the perforated-patch variant
of whole-cell clamp (Horn and Marty, 1988). Although
this approach has been successfully applied to other
isolated molluscan photoreceptors (Nasi and Gomez,
1992b
), control of internal Ca becomes problematic, because the best characterized pore-forming agents used
for this purpose, such as Nystatin and Amphotericin B,
only allow small monovalent ions to exchange between
pipette and cytosol.
The nature of the Ca-independent pathway that modulates the photoresponse in Pecten ciliary photoreceptors is presently unknown. Possible leads are provided
by experiments in which the light-sensitive conductance
was directly stimulated by intracellular dialysis with
cGMP: this treatment decreased the response to subsequent test flashes, as one would expect if light and
cGMP tap the same effector (Gomez and Nasi, 1995);
however, the observed decrease in responsiveness appeared to exceed the reduction in available channels,
and the residual photocurrent displayed faster kinetics, suggesting that the nucleotides may have also desensitized the cell. Possible mechanisms that may mediate
such an effect include protein phosphorylation by a cyclic-nucleotide-dependent kinase (e.g., PKG). In vertebrate rods two small phosphopeptides are phopsphorylated in a light-dependent manner (Polans et al.,
1979
); this phosphorylation has been shown to be regulated by cyclic nucleotides (Hermolin et al., 1982
)
probably by a cAMP-dependent protein kinase (Hamm,
1990
). Cyclic-nucleotide-dependent protein phosphor-ylation was indeed proposed to contribute to light adaptation (Hermolin et al., 1982
). A precedent for a
negative feedback loop in which the ligand for the
channels that underlie the receptor potential also down-regulates the transduction process via protein phosphorylation has recently been demonstrated: in olfactory
cells that possess two separate transduction pathways,
cAMP (one of the internal transmitters that gates odorant-dependent channels) also activates a PKA which, in
turn, inhibits the stimulus-induced increase in intracellular cAMP. Conversely, a PKC is involved in the termination of the IP3-mediated signal (Boekhoff and Breer, 1992
).
The present data raise the question of whether a regulatory scheme that operates independently of cytosol-ic Ca changes is unique to molluscan hyperpolarizing
photoreceptors or if it may be shared by other visual
cells. In spite of the powerful case for Ca mediation of
adaptation in vertebrate rods, during the course of the
last few years evidence has been emerging that some manifestations of light adaptation occur independently
of Ca changes: for instance, an adapting light is capable
of inducing an acceleration of the rising phase of the
photoresponse, even under conditions in which cytosolic
Ca is manipulated over a wide range of concentrations (Nicol and Bownds, 1989). Conversely, the acceleration
of flash response recovery induced by background illumination does not obtain if an equivalent change of
[Ca]i is imposed in the dark (Gray-Keller and Detwiler,
1996
). Furthermore, the state of phosphorylation of
light dependent channels, which determines their responsiveness to cyclic nucleotides, is controlled by endogenous phosphatases in a manner that is insensitive
to Ca over a concentration range spanning 4 orders of
magnitude (Gordon et al., 1992
). Thus, the presence
of alternative modulatory mechanisms of visual transduction may be of wide generality. Isolation of modulatory mechanisms that operate in a Ca-independent way
is difficult in most photoreceptor cells because Ca
changes are usually a natural concomitant of the photoresponse; in the case of invertebrates, they may in
fact be inextricably linked to the visual excitation process (Bolsover and Brown, 1985
; Werner et al., 1992
;
Shin, Richard and Lisman, 1993). In this respect, ciliary
invertebrate photoreceptors provide a uniquely advantageous situation, because the two processes can be easily uncoupled.
Original version received 15 October 1996 and in revised form 30 December 1996.
Address correspondence to Dr. Enrico Nasi, Department of Physiology, Boston University School of Medicine, 80 E. Concord Street, Boston, MA 02118. Fax: 617-638-4273; E-mail: enasi{at}acs.bu.edu
1 Abbreviations used in this paper: ASW, artificial sea water; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,NThis work was supported by National Institutes of Health grant RO1 EY07559.