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YiftbnRes.Vol. 31,No. 3, pp. 567-576,1991
Printedin Great Britain.All rightsn~erved
Copyright Q 1991Rrgamon Press plc
ULTRAVIOLET PHOTORECEPTION IN CARP:
MICROSPECTROPHOTOMETRY AND BEHAVIORALLY
DETERMINED ACTION SPECTRA
CRAIG W. HAWRYSHYN’* and FERENC I. HAROSI~*~
‘Division of Biological Sciences, Section of Ecology and Systematics, Corson Hall, Cornell University,
Ithaca, NY 14853, *L&oratory of Sensory Physiology, Marine Biological Laboratory, Woods Hole, MA
02543 and 3~~~ent
of Physiology, Boston University School of Medicine, Boston, MA 02118, U.S.A.
(Receiued 3 March 1990; in revised form 10 July 1990)
Abstract-This
study demonstrates correlations between U.V. sensitivity and microspectrophotometric
absorption spectra determined sequentially for the same group of individuals. We used the heart-rate
~n~tioning
technique to measure spectral sensitivity of carp, a species known to have u.v.-sensitive
photor~pto~,
Mean spectral sensitivity (n = 3) determined with a spectrally-broad background (450 nm
long pass filter) revealed a small but consistent U.V. peak (.I,, of 380 nm) in addition to the other long
wavelength peaks. An intense blue-green background (490 nm) produced a more prominent U.V.peak (,I,,
of 4OOnm) when a 450 nm longpass filter was added to the background. Microspcctrophotometric
measurements of u.v.-sensitive photoreceptors from one individual, which belonged to the group used in
the spectral sensitivity experiments, revealed an average ,I,, of 377.5 nm (SD +4.5 nm, n = 5 ceils).
Bfeaching and dichroic measurements of these receptors ensured that we were examining typical vertebrate
visual pigments and not stable photoproducts. The mean spectral sensitivity points were compared with
the U.V.and blue-sensitive visual pigment absorption spectra. A linear subtractive model and ocular media
absorption were used in this comparison for the various photic conditions used in the heart-rate
conditioning experiments. The model successfully described the sensitivity of the test fish in two cases but
in a third case there was some discrepancy. The model generated curve was broader than the spectral
sensitivity of the u.v.-sensitive cone mechanism on the shortwave side even though the ocular media
corrections had been accounted for.
Ultraviolet photoreception
Ocular media transmission
Microspectrometry
Cone photoreceptors
Heart-rate conditioning
Spectral sensitivity
INTRODUC’MON
Most vertebrate visual pigments can absorb
ultraviolet (u.v.) radiation through /I-band
absorption. It is now evident, however, that
some vertebrates also possess u.v.-sensitive
visual pigments with a-band absorption in the
U.V. spectrum (Harosi & Hashimoto,
1983;
Avery, Bowmaker, Djagmoz & Downing, 1983;
Harosi, 1985; Bowmaker & Kunz, 1987). The
spectra1 nature of quanta1 absorption by the
u.v.-sensitive visual pigments is in~uen~d by
the presence of ocular media which may or may
not transmit U.V. radiation (Hawryshyn, Chou
& Beauchamp, 1985). Species occupying photic
environments hosting high ambient intensities
of U.V. radiation, usually possess U.V. ocular
*Present address, to which reprint requests should be addressed: Department of Biology, University of Victoria,
P.O. Box 1700, Victoria, B.C., Canada V8W 2Y2.
Carp (Cyprinus carpio)
filters. Species with u.v.-filtering media may
incur several selective advantages including protection from photic damage to photoreceptors,
and the reduction of excessive scattering and
chromatic aberration that could degrade image
contrast (Chou & Hawryshyn, 1987). Conversely, fish with more u.v.-transparent ocular
media (e.g. goldfish and rainbow trout) may
receive adequate levels of U.V. radiation for
photoreception. However, individual variability
in ocular media transmission can impose a
variable degree of narrowing on the U.V. peak
(Hawryshyn et al., 1985).
Three different comparative surveys have
shown that U.V. photosensitivity is especially
prevalent in birds (Chen & Goldsmith, 19861,
and in fish (Fukurotani & Hashimoto, 1984;
Harosi (8 Fukurotani, 1986). Several technical
approaches have been used to explore U.V.
photosensitivity in vertebrates: (1) MSP (microspectrometry) of single cones has identified
567
568
CRAIG W. HAWRYSHYN
and FERENC
I. HAROSI
u.v.-sensitive cone receptors in cyprinids (Avery
1983;
et al., 1983; Harosi & Hashimoto,
Harosi, 1985; Bowmaker & Kunz, 1987) and
cyprinidonts (Harosi & Fukurotani, 1986); (2)
ERG (electrophysiology) was used to measure
U.V. sensitivity in birds (Chen, Collins &
Goldsmith, 1984; Chen & Goldsmith, 1986); (3)
classical conditioning of behavior (Kreithen &
Eisner, 1978; Hawryshyn & Beauchamp, 1982,
1985; Hawryshyn, Arnold, Chaisson & Martin,
1989); (4) operant conditioning of behavior was
used to measure U.V. sensitivity and wavelength
discrimination in the near u.v.-spectrum in
fish and birds (Parrish, Ptacek & Kevin, 1984;
Neumeyer,
1985, 1986; Goldsmith,
1980;
Douglas, 1986).
For many vertebrate species, the identity of
photoreceptors mediating U.V. photosensitivity
is unclear. For example, goldfish exhibit a well
defined U.V. peak in its spectral sensitivity
(Hawryshyn & Beauchamp, 1985) and perform
well on near U.V. wavelength discrimination
tasks (Neumeyer, 1986), yet a u.v.-sensitive cone
photoreceptor remains unidentified. This may
be due to miniature long single cones and
miniature short single cones eluding the
recording beam. These cones remain the favored
candidates for the u.v.-sensitive receptors in
goldfish. MSP (Avery et al., 1983) and the use
of operant conditioning to examine spectral
sensitivity (Douglas, 1986) revealed U.V. sensitivity and u.v.-sensitive cone pigments in
another cyprinid species, the roach. However,
the full absorbance spectrum and dichroic properties are unknown parameters of the u.v.-sensitive cones. In a recent report by Harosi and
!Fukurotani (1986), a good correlation was
demonstrated between cone absorbances and
the response of horizontal cells (u.v.-sensitive
triphasic or tetraphasic) in several species of
fish.
In the present study, we measured the spectral
sensitivity and the absorbance spectrum of u.v.sensitive cones of carp from the same group.
Furthermore,
measurements
of the ocular
media transmission permitted a direct comparison of the u.v.-sensitive cone absorbance
spectrum with the spectral sensitivity.
MATERIALSAND METHODS
Heart -rate conditioning experiments
Animals. Seven carp (Cyprinus carpio) obtained from SP Engineering Technology (Salem,
Mass.) were used. Initially, three additional carp
were trained but these individuals lost their
conditioned responses at the beginning or
during the spectral sensitivity experiments. The
three that lost their conditioned responses were
discarded from the test group. They ranged in
size from 13.0 to 17.2 cm and 48.5 to 114.0 g. All
fish were kept in the holding facility at 20°C on
a 12HL/12HD cycle for at least a month prior
to conditioning. Fish were fed on alternate days
a mixed diet of live meal worms, frozen brine
shrimp and pellets.
Immobilization procedure and set -up of jish.
Fish were immobilized during each conditioning
and experimental session to prevent changes in
the position of the retinal image of the presented
stimuli, Each fish was first anesthetized with
sulfonate
(MS-222), at
tricaine methane
250 mg/l (immersion 2-5 min) and then immobilized with d-tubocurarine chloride (0.45 ,ug/g
body weight) injected into the dorsal musculature at several sites. The animal was held in a
Plexiglass restrainer, placed in a test tank (80 l),
and artificially irrigated with a 3 ml/set flow of
aerated water (all procedures and care of fish
were in compliance with the Cornell University
Council on Animal Care). The position of the
restrainer was adjusted so that the right eye of
the fish was 5 cm from the center of a circular
Albanene (Kuffler and Esser Co.) back projection screen (9 cm dia.) and the pupillary plane of
the eye was parallel with respect to the back
projection screen (see Fig. 1).
Stimuli. A three-channel optical system was
set up so that the light could be superimposed
from two background channels and a stimulus
channel on the back projection screen. The
wavelength of the stimuli was controlled by
22 narrow-band
(10 nm at half-max bandwidth) interference filters (Pornfret) spanning a
320-740 nm range. The transmission propertiesof
the filters were measured using a Perkin-Elmer
spectrophotometer.
Short- or long-pass cutoff
filters (Corion) were used in combination with
the interference filters to correct for transmission leaks outside the primary band-pass if
necessary. Irradiance measurements were made
for each stimulus filter combination with an
International Light Radiometer (Detector SEE
400 1360 calibrated by International Light Inc.)
with its probe positioned inside the test tank at
the center of the back projection screen. The
stimulus intensity was controlled by a u.v.-grade
Inconel-coated (Kodak) neutral density wedge
which was calibrated in 0.2 log unit steps for
each wavelength setting used during these
U.V. sensitivity in carp
569
Fig. 1. Top view of the experimental apparatus. AR = artificial respirator; BCH 1 = background channel
1; BCH2 = background channel 2; c = condensing lens; D = diaphragm; EKG = electrocardiogram
electrode and leads; ES =electronic shutter; FL = field lens; FR = fish restrainer; FT = filter tray;
H = light housing; IFW = interference filter wheel; NDW = neutral density wedge; PL = projection lens
system; PW = Pyrex optical window; S = light source, 250 W tungsten lamp; SC = stimulus channel;
SE = shock electrodes and leads; SM = photodiode stimulus monitor system; TF = test fish carp (Cvprinus
c&o); TT = test tank (flat black).
experiments. Additional u.v.-grade Inconelcoated (Corion) neutral density filters were used
in the stimulus channel as needed. Stimulus
duration was controlled by an electronic shutter
and was kept constant at 4sec during these
experiments. A 250 W tungsten lamp (EHJ
Spectra Lamps) powered by a Hewlett-Packard
24 V d.c., 10 A regulated power supply, provided the light for the stimulus channel.
The background channels also contained
250 W tungsten lamps and were powered by
24 V d.c., 5 A regulated power supplies. The
intensities of the background channels were
controlled by the same type of neutral density
filters as the stimulus channel and their spectral
composition was controlled by the same type of
short- and long-pass cutoff filters that were used
to correct for transmission leaks in the stimulus
channel. The absorption of the water between
the back projection screen and the fish’s eye
was determined by measuring the absorption
of 1 cm of the water using a Perkin-Elmer
spectrophotometer.
Conditioning protocol. After being immobilized and positioned in the test tank, the
fish were trained using the heart-rate conditioning technique (Beauchamp & Rowe, 1977;
Hawryshyn & Beauchamp, 1985). The purpose
of training was to elicit a conditioned decrease
of heart rate in response to detection of a light
stimulus. This was accomplished by presenting
a light stimulus to the fish, followed immediately by a low level (2-3 mA, 3-4 V r.m.s.)
electric shock delivered to the caudal peduncle.
The fish’s heart rate was monitored on a Grass
Polygraph using two-pin electrodes inserted just
distal to the pectoral fins. The response criterion
was chosen to be a stimulus period heart beat
interval that was 1.5 times the length of the
average pre-stimulus period (10 set) heartbeat
interval (see Fig. 2). During the conditioning
sessions, a moderate intensity white background
(for spectral characteristics of the backgrounds
see Fig. 5, right panel) was used, the stimulus
size was kept constant at 2.5 cm and the distance
between the fish’s eye and the back projection
screen was a constant 1Ocm. The conditioning
protocol consisted of repeatedly presenting a
bright 600 nm stimulus until the fish gave five
consecutive criterion responses and then presenting a series of stimuli of different wavelengths and intensities to achieve appropriate
levels of generalization. Each wavelength and
intensity combination was repeatedly presented
until two consecutive criterion responses were
given. At the end of each conditioning and
experimental session three blanks were presented to assure that responses were related to
CRAIGW. HAWRYSHYN
and FERENCI. HAR~X.I
570
Fig. 2. Conditioned EKG responses (arrows) of one fish to 340 nm stimuli. Threshold was determined
using a single ascending series of stimulus radiances. Each successive stimulus was 0.2 log units more
intense than the last. Three consecutive trials in a series are illustrated: top trace no. 1, subthreshold
stimulus; middle trace no. 2, threshold stimulus; bottom trace no. 3, suprathreshold stimulus. Threshold
was always preceded by at least three consecutive stimulus radiances of the series not giving a response,
and followed by at least one giving a response. A conditioned response was defined as an interbeat interval
during the stimulus which was at least 1.5 times the average interbeat interval in the 10 set preceding the
stimulus. Note: after training, shock followed only those trials on which criterion responses were measured
(after Hawryshyn & Reauchamp, 1985).
the light stimulus, and not to extraneous factors
such as sound or vibration.
Threshold determination. Threshold at a particular wavelength-background
condition was
determined by presenting a subthreshold stimulus intensity followed by increasing intensity
steps (0.2 log unit) until the test fish gave
two consecutive positive responses. Threshold
intensity was the first intensity level to
which the fish responded. To minimize the
incidence of false positives, the threshold intensity, the first positive response (i.e. criterion
bradycardia),
had to be preceeded by at
least three negative responses (three intensity
increments).
Spectral sensitivity experiments determined
the threshold of detection of a stimulus at
wavelengths varying from 320 to 740 nm for
each test fish. Test wavelength thresholds were
determined in random order to reduce any
effects of time of stimulus presentation on the
resultant spectral sensitivity curve.
Microspectrophotometric
Spectrophotometer.
trophotometer
measurements
The dichroic microspec(DMSP) described elsewhere
(Harosi & MacNichol, 1974; Harosi, 1982a,b)
was used in these experiments. It is a singlebeam photometer that simultaneously records
average and polarized transmitted fluxes as a
function of wavelength.
Spectral recordings. The cross-section of the
measuring light was a rectangle of ca 1 x 3 pm
in the specimen plane. The preparation, consisting of a quartz sandwich, was placed on the
sliding-gliding stage of the DMSP, and in
it transversely oriented photoreceptors
were
located under dim red background illumination.
The sample transmittance was usually recorded
in eight scans, whereas reference transmittance
was recorded in 8 or 16 scans through an
adjacent cell-free area (for technical details, see
Harosi, 1987).
RESULTS
MSP absorbance spectra
Absorbance spectra were measured from
cones located in the central regions of the carp
retina. Measurements shown in Fig. 3 were from
one individual (fish C-10 used in the spectral
sensitivity experiments). Except for the absence
U.V. sensitivity in carp
(A) UV-sensitive
1.1
cones
-
0.6
k
I;
300
+
+ + +>
+*+~&~++++++~+3+~+
I
I
,
350
400
450
I
I
I
I
I
I
1
I
I
I
I
550
600
650
300
350
400
450
500
550
600
650
600
650
++,
500
0
(B)
Blue-sensitive
cones
1.1
1.0
0.9
0.6
0.7
0.6
0.5
0.4
_..
300
350
400
450
500
550
400
Wavelength
450
500
5%
600
650
(nm)
Fig. 3. Absorbance spectra of cone photoreceptors in test carp C-10. (A) Ultraviolet-sensitive cones:
average of five single-cell prebleach absorption spectra. The solid line (in curves A-D) represents the
Fourier-smoothed spectrum (for technique, see Harosi, 1987). (B) Blue-sensitive cones: average of five
single-cell prebleach absorbance spectra. (C) Green-sensitive cones: prebleach absorbance spectrum of one
cell. (D) Red-sensitive cones: average of three single-cell prebleach absorbance spectra.
of miniature cones, photoreceptors in the carp
preparations were similar to those found in the
goldfish @tell dz Harosi, 1976). The u.v.-sensitive and blue-sensitive visual pigments were
located in short single cones. These cones were
indistinguishable from one another as viewed in
the light microscope. The numerical distribution
of the cone types examined is shown in Table 1.
Because the microspectrophotometer
was
adjusted to give optimal results at short
wavelengths, the u.v.- and blue-sensitive cones
Table 1. Visual pigments found in the cones of test carp
C-IO”
Cone type
u.v.-sensitive
Blue-sensitive
Green-sensitive
Red-sensitive
Number
of cells
found
Number
of cells
averaged
Peak of Fouriersmoothed curves
A,, (nm)
10
22
2
5
5
5
377.5
458.0
532.0
600.0
I
2
‘Note our search for photoreceptors was very much biased
toward short single cones and thus the frequency of
occurrence of the pigment type should not be construed
as being representative of the natural distribution.
yielded the clearest spectra. The green-sensitive
absorbance spectrum was somewhat noisier, but
we feel it to be acceptable since its I,,,,, compared favorably to previous estimates (J,,,
535 nm, Harosi, 1985). However, recordings
made on the red-sensitive cones showed
deviations from the expected 620 nm I,,, . We
have provided this absorbance spectrum to
demonstrate
the presence of red-sensitive
cones in this individual; in agreement with what
is expected for carp. An average absorbance
spectrum from five u.v.-sensitive cones had a
high amplitude a-band peaking at 377.5 nm
(Fig. 3).
Dichroism in u.v.-receptors
We performed transverse linear dichroism
measurements on u.v.-sensitive cones in the
bleached and unbleached states in order to test
the hypothesis that the u.v.-sensitive cones contain typical vertebrate visual pigments rather
than stable photoproducts
(Nolte & Brown,
1972) or sensitizing pigments (Kirschfeld,
CRAIGW. HAWRYSHYN
and FERENCI.
512
Franceschini & Minke, 1977) as seen in
insects. A high amplitude anisotropic response
in the cl-band of the unbleached
u.v.sensitive cones with a mean dichroic ratio of
2.8 (n = 3 cells) was observed (Fig. 4A).
When the u.v.-sensitive cones were bleached by
exposure to mon~hromatic
u.v., the dichroic
ratio was reduced close to unity (mean of
1.2, n = 3 cells, Fig. 4B). Clearly, these data
for transverse measurements
indicate that
u.v.-sensitive cones share the dichroic and
bleaching properties
of vertebrate
photoreceptors. Note, however, that these transverse
measurements do not test for the presence
of axial dichroism in cones nor do they give
any clues about the biophysical arrangements
involved in the orthogonal polarization sensitivity that was recendy documented in teleost
cone mechanisms (Hawryshyn & McFarland,
1987).
Spectral sensitivity
The spectral sensitivity of carp was measured
under three different background field conditions (curves in Fig. 5 arbitrarily displaced).
The different background adapting fields used in
these experiments were expected to produce a
6
g
3
004
0.03
lB’
Bleached
I
-0,021 1
’
’
’
’
’
’
’
300’ 350 400 450 500 550 600 650
A
Wavelength
(nm 1
Fig. 4. Linear dichroism in ultraviolet-sensitive cones in test
carp C-10. (A) Mean log,, linear dichroism of three unbleached u.v.-sensitive cones with a A,,,,, of 376.5 nm and a
dichroic ratio of 2.8. (B) Mean log,, linear dichroism of
three u.v.-sensitive cones after exposure to bleaching illuminations ,I,,,_ 400.0 nm and dichtoic ratio 1.2. Note that the
consequence of bleaching is the loss of transverse Iinear
dichroism.
HAROSI
I
I(A)
6-“cb;”, ,-
,a
400 500 600 700
Wovelength
400
500 600 700
(nm)
Fig. 5. Spectral sensitivtty of carp measured with different
chromatic backgrounds. Mean spectral sensitivity of several
indi~duals is shown on the left panel and relative power
spectra of the chromatic background is shown on the right
panel. Left panel-curve
A: mean (1 SEM) spectral sensitivity (n =4 carp C-IO, C-II, C-14, C-IS) using a 4SOnm
longpass plus 490 nm (lo nm half-max bandwidth) backgrounds (see right panel curve A). Curve B: mean spectral
sensitivity (n = 3 carp C-l, C-4, C-S) using a 450 nm long
pass background (see right panel curve B). Lines in curve B
have the same representation as curve A. Curve C: mean
spectral sensitivity (a = 2 carp C-14, C-15) using a white
background (see right panel curve C). Righrpanel+curve A.
background 1, a 250 W tungsten source operating at S A
with a 450 nm longpass filter and a 2.0 neutral density filter;
background 2, a 250 W tungsten sour= operating at 5 A
with a 490 nm (IO nm half-max bandwidth) and a 1.0
neutral density filter. Curve B: background I, a 250 W
tungsten source operating a S A with a 450 nm longpass
filter and a 2.0 neutral density filter. Curve C: background
1, a 250 W tungsten source operating at S A with a 2.2
neutral density filter.
variable degree of light adaptation in the u.v.sensitive cone mechanism. The mean spectral
sensitivity curves (of several individuals) are
shown on the left panel of Fig. 5 and the
chromatic background on the right panel.
Shortwave sensitivity varied with changes in the
intensity of U.V. radiation in the background.
When U.V. radiation was eliminated with the
450 nm longpass filter, shortwave sensitivity
increased. In addition, the narrow-band bluegreen background (490 nm, IO nm half-max
bandwidth) used in curve A appeared to produce the best defined U.V.peak. The addition of
the blue background reduces the sensitivity of
the blue-sensitive mechanism thus revealing the
U.V. peak. However, the U.V. peak was also
somewhat higher than the other peaks in curve
A compared to curve B.
573
U.V.sensitivity in carp
Ocular media transmission
Transmission
of light through the ocular
media of the carp eye was measured with an
integrating sphere spectrophotometric
technique (Hawryshyn
et al., 1985; Chou &
Hawryshyn, 1987). This technique measured the
transmission of the anterior part of the eye,
including the cornea, lens and some vitreous
humor. Figure 6 shows the average absorbance
of the five carp eyes (derived from different
animals used in the spectral sensitivity determinations). Corrections for ocular media transmission were made to absorption spectra of
cone pigments and illustrated in Fig. 7 along
with the spectral sensitivity points taken from
Fig. 5.
Correlation of spectral sensitivity
pigment absorption spectra
with visual
1.4
r
z
0.6-
0
z
z
06-
u
04
-
350
400
450
Wavelength
500
(nm
550
.
-*
Figure 7 illustrates the three corrected spectral sensitivity curves from Fig. 5. The output of
cone photoreceptors depends on a number of
principal parameters such as: absorption spectrum of the cone mechanism, spectral transmission of the ocular media, and neural
interactions, especially between spectrally opponent cone mechanisms. Many examples of
this type of interaction can be seen in electrophysiological and behavioral studies examining
red-green opponency in fish. Some recent evidence indicates that there may be an interaction
between the u.v.- and blue-sensitive cone types
in several cyprinid species. Wavelength discrimination experiments in goldfish (Neumeyer,
1985, 1986) have shown that there is good
discrimination in the shortwave part of the
6
I t
600
650
1
Fig. 6. Spectral absorbance of carp ocular media. The solid
line represents the average ocular media absorbance of five
carp eyes (carp C- 11, C- 14, C- 15)measured with an integrating sphere technique (refer to Hawryshyn et al., 1985).
.’
1
LOQ
Unit
IA
.
400
I
I
,
500
600
700
Wavelength
.
(nm)
Fig. 7. Correlation of ocular media corrected spectral
sensitivity and visual pigment absorption spectra. The data
for curves A-C were taken from Fig. 5. Curve A: the
triangles represent the mean sensitivity points (this is also
the case for curves B and C) while the dashed line represents
the u.v.- and blue-sensitive cone absorption spectrum corrected for ocular media absorption generated by the linear
subtractive model (see Results section for the k coefficients
used in the linear subtractive model). Curve B: the dashed
lines for the u.v.- and blue-sensitive cone mechanisms were
generated using the linear subtractive model (see Results
section for k coefficients). Curve C: the dashed line for the
blue-sensitive cone mechanism was generated using the
linear subtractive model (see the Results section for the k
coefficients).
spectrum indicating that the u.v.-sensitive cone
mechanism is an active participant in wavelength discrimination. Such performance depends on the interaction of at least two cone
types. Furthermore,
several studies have
demonstrated
the presence of u.v.-sensitive
tetraphasic horizontal cells in a variety of
cyprinid species (Hashimoto, Harosi, Ueki &
Fukurotani, 1988) although this has yet to be
shown for carp. This may be an important
consideration for fitting absorption spectra to
the u.v.- and blue-sensitive components of the
spectral sensitivity curve. It is conceivable that
opponent interactions may exist between the
u.v.- and blue-sensitive cone mechanisms and
that they may play a role in shaping the spectral
sensitivity of fish in the shortwave part of the
spectrum. A linear subtractive model was used
to produce absorption spectra (or cone output
spectra) to fit narrowed peaks of the u.v.- and
514
CRAIGW. HAWRYSHYN
and FERENC
I. HAROSI
blue-sensitive cone mechanisms (Sperling &
Harwerth, 1971; Douglas, 1986; Neumeyer,
1984). We suggest the hypothesis that inhibitory interactions occur between the u.v.- and
blue-sensitive cone mechanism that produce
narrowed spectral sensitivity peaks.
A linear subtractive model for receptor opponency, comparable to that used by Sperling
and Harwerth (1971), was employed to more
fully understand the deviation of the action
spectra from the u.v.-sensitive visual pigment
absorption spectrum. The inhibitory effect of
one cone mechanism on another (e.g. a u.v./b
opponency) can be described in the following
expressions:
S,, = t (k3 VPb - k4 VP, c );
where S,,. and S, are the sensitivities of the u.v.and blue-sensitive cone mechanisms, VP,, and
VPb represent the absorption of the u.v.- and
blue-sensitive cones respectively, taken from
Fig. 3; t is the ocular media transmittance; and
k,, k,, k3 and k4 are the strength constants. In
curve A, where shortwave narrowing of the u.v.sensitive cone mechanism is evident, the linear
subtractive model was successful in generating a
U.V. peak that was narrowed on the shortwave
limb. As the coefficient k, was increased relative
to k, the inhibitory effect was greatest on the
longwave limb. This is due to an asymmetry in
overlap of the blue-sensitive pigment spectrum
on the cc-band of the u.v.-sensitive pigment
(85% at 410 nm vs 78% at 340 nm). The dashed
line in Fig. 7A illustrates the best fit of modelled
absorption spectra (k, = 1.0, k, = 0.3, k3 = 0.25,
k, = 0.25) to the spectral sensitivity points.
Although the model fits well on the longwave
side of the u.v.-sensitive cone mechanism there
was a consistent discrepancy between ocular
media corrected absorption spectra generated
by the linear subtractive model and the spectral
sensitivity. This observation did not appear to
be related to u.v./b opponency or ocular media
absorption. Shortwave narrowing has been
observed in several species and laboratories and
it appears to be an important problem which
requires further study.
The model was successful in generating curves
that approximated the spectral sensitivity in
curve B and curve C. A fit was observed for
curve B when the coefficients were set to
k, = 0.9, k2 = 1.0, k, = 0.27 and k, = 0.27. Similarly, a fit was also observed for curve C when
the coefficients were set to k, = 0.9, k, = 1.3,
k, = 1.0 and k, = 0.63. While both u.v.- and
blue-sensitive mechanisms were evident in curve
B, the u.v.-sensitive mechanism appeared to be
absent in curve C, this curve being dominated
by the blue-sensitive cone absorption points.
This undoubtedly results from a more intense
U.V. content in the background used for determining the spectral sensitivity illustrated in
curve C.
DISCUSSION
This study relates the u.v.-sensitive cone absorption spectrum to the U.V.spectral sensitivity
of carp (Fig. 7). We initially compared the
ocular media-corrected u.v.-sensitive cone absorption spectrum with spectral sensitivity (a
test of cone mechanism independence); however, this revealed narrowing between the
mechanisms that reflected shortwave opponent
interactions. This coupled with previous work
suggests that the linear subtractive model could
provide a more realistic correlate of sensitivity.
The linear subtractive model generated curve
correlates well with the u.v.- and blue-sensitive
mechanism spectral sensitivity data (Fig. 7 curve
B). In Fig. 7C, only the blue-sensitive mechanism is apparent, although the model does
require a degree of inhibitory input from the
u.v.-sensitive mechanism to produce a reasonable correlation to the data. Collectively, these
data suggest the presence of a possible u.v./b
interaction, as evidenced by the narrowing
between cone mechanisms. Recent research has
revealed the presence of u.v.-sensitive triphasic
and tetraphasic horizontal cells. These cells have
been reported in some cyprinid species (Harosi
& Fukurotani, 1986), although not yet in carp.
A curious lack of correlation was observed
between the u.v.-sensitive absorption spectrum
(ocular media corrected) and the U.V. peak
action spectrum on the shortwave side of the
curve (Fig. 7A). Following correction of the U.V.
absorption spectrum for ocular media transmission shortwave narrowing of the U.V. sensitivity was evident. This was especially the case
when the blue-sensitive mechanism was light
adapted (Fig. 7A). This suggests that besides
light adaptation of the blue-sensitive mechanism, other processes may be occurring that
augment the sensitivity in the U.V. This is not
a new finding since data from Chen and
Goldsmith (1986) on the house sparrow show
shortwave narrowing of the U.V. mechanism in
U.V. sensitivity in carp
comparison with a I,,, of a hypothetical 370 nm
visual pigment absorption curve. A similar
observation has also been made in Tiger
Salamander rods (Cornwall, MacNichol &
Fein, 1984). As indicated previously, our initial
thoughts were that the shortwave narrowing of
the u.v.-sensitive mechanism could conceivably
be due to inhibitory interaction between the
u.v.- and blue-sensitive mechanism in the shortwave spectrum. However, the linear subtractive
model, over a wide range of k constants, could
not successfully demonstrate a correlation on
the shortwave side. Paradoxically, the shortwave narrowing occurs only when the monochromatic blue field (490 nm, 10 nm half-max
bandwidth) is added to the 450 nm longpass
field. The blue-sensitive mechanism decreases in
sensitivity while the u.v.-sensitive mechanism
increases, suggesting possible nonlinearity in
the opponent interactions.
It is not clear
what causes this shortwave narrowing when the
blue-mechanism is light adapted.
Most of the research on U.V. photoreception
in vertebrates has concentrated on passerine
birds and cyprinid fishes. It is not known
whether amphibians, reptiles or mammalian
species also possess U.V.photoreception. Recent
evidence by Hawryshyn et al. (1989) and
Bowmaker and Kunz (1987) has shown the
possible existence of an ontogenetic loss of U.V.
photosensitivity or cones in salmonids. This
observation of developmental losses of U.V.
sensitivity will further complicate our attempts
to survey the incidence of U.V.photoreception in
vertebrates. Because of this, our search for U.V.
photoreception should focus primarily on the
early life history of fish. The results of this paper
along with those on the horizontal cell recordings (Fukuortani & Hashimoto, 1984; Harosi &
Fukurotani, 1986; Hashimoto et al., 1988) collectively suggest a possible u.v./b opponency in
cyprinids and probably other vertebrate species.
The precise spectral nature of the opponency is
likely to be variable from one species to the next
since there is variance in the A,,,,, of the U.V.
mechanism. Species such as the Japanese date
have two shortwave cones with a A,,,,, of
350-370 nm and 405-415 nm, clearly differing
from carp which possesses a A,,,,,of 380 nm and
of 455 nm. Horizontal cells that are u.v.-sensitive are either tetraphasic showing differential
response to U.V. and blue stimuli or triphasic
that do not show differential responses to U.V.
and blue stimuli. Presumably, the possession of
tetraphasic cells (not yet seen in carp) would
515
augment the potential for wavelength discrimination in the U.V. spectrum. Data from
Neumeyer (1985, 1986) shows that cyprinids
have good discrimination in the shortwave
spectrum. Shortwave discrimination may permit
intraspecific communication
(Harosi,
1985,
photographic plates). It may also aid in the
detection of food items in the environment
such as zooplankton for fish or flowers and
berries for birds (see Burkhardt, 1982 for
discussion).
Acknowledgements-Thanks
to Margaret Arnold for technical assistance in this project. Thanks to Daryl Parkyn and
Luc Beaudet for reading the manuscript. This research was
supported by USPHS grant EY04876 to F.I.H., NATO
Postdoctoral Fellowship from the Natural Sciences and
Engineering Research Council of Canada to C.W.H. and
by a Cornell University Hatch Grant 403 to Dr W. N.
McFarland for equipment support.
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