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Goldfish spectral sensitivity: Identification of the three cone mechanisms in heart-rate conditioned fish using colored adapting backgrounds
NM?-6989
79 l?Ol-1195SOXO
0
GOLDFISH SPECTRAL SENSITIVITY: IDENTIFICATION OF THE THREE CONE MECHANISMS IN HEART-RATE CONDITIONED FISH USING COLORED ADAPTING BACKGROUNDS R. D. BEAUCHAMP,J. S. Row
and L. A. O’REILLY
Departments of Optometry and Biology. University of Waterloo, Waterloo, Ontario. Canada (Receiced 24 Ocrobur 1978; in rerised/orm
19 April
1979)
Abstract-Increment threshold curves were measured for curarized goldfish using a conditioned heartrate procedure. Chromatic backgrounds were utilized to enhance the sensitivity of one goldfish cone mechanism relative to the other two. Sensitivity peaks were at 4%. 530 and 630nm against yellow. red-blue and blue-green backgrounds. respectively. The short- and mid-wave peaks agreed well with the appropriate goldfish cone difference spectrum, but the long-wave peak was narrower on the left shoulder and wider on the right shoulder than the corresponding cone difference spectrum. Against the bluegreen background. a prominent short-wave peak attributed to beta band absorption by the red-sensitive cones was observed. The magnitude of aberrant high sensitivity noted for 400 and 420 stimuli was dependent on the amount of short-wave energy in the background; highest aberrance was noted for yellow backgrounds. and lowest for blue or red-blue backgrounds.
lNTRODUCTlON The goldfish is a useful animal in which to study color vision because its three cone pigments have widely separated sensitivity peaks at 455, 530 and 625 nm (Liebman and Entine, 1964; Marks, 1965; Harosi and MacNichol, 1974). Therefore. the cone mechanisms contributing to a neural or behavioral response can be identified readily by noting the location of peaks in a spectral sensitivity curve. For example, in psychophysical measurements of spectral sensitivity with a tungsten-white background, the goldfish increment threshold curve had a prominent short-wave peak at about 455 nm, and the peak had the same shape as the difference spectrum of the blue-absorbing cone pigment (Beauchamp and Rowe, 1977). The 455 nm peak indicated that the psychophysical response at short wavelengths originated in blue-sensitive cones. Two other components were noted in the increment threshold curve measured against a tungsten-white background. There was a mid-wave shoulder to the short-wave peak and, as well, there was a long-wave peak at about 660 nm. The mid- and long-wave components were narrower than the corresponding cone absorption spectra, which suggested an influence of antagonistic interaction between red- and greensensitive cone mechanisms (Wagner et al., 1960: Daw, 1968; Beauchamp and Lovasik, 1973). In the present paper, the origin of the mid- and long-wave components observed with the tungstenwhite background is clarified. Bright chromatic backgrounds have been used to reduce selectively the sensitivity of two of the three goldfish cone mechanisms, leaving the third cone mechanism to dominate the psychophysical response over a wider-than-normal range of the spectrum (Stiles, 1959: Yager, 1969: Beauchamp and Lovasik, 1973). The procedure allowed us to observe the consequence of reduced “A
19 12-74
antagonistic interactions on the shape of the increment threshold curve. In addition, further information was gathered on the high aberrant sensitivity previously noted at the short-wave end of the spectrum (Yager, 1967; Muntz and Northmore, 1970; Beauchamp and Rowe, 1977). >lETHODS Details of the methods are presented in a recent. closelyrelated paper (Beauchamp and Rowe, 1977). Subjects
and conditions
Ten common goldfish (C’arassius aurarus) weighing from 29 to 64 g (9.5 to 12.5 cm. standard length) were kept for at least a week prior to experimentation.at about 2O’C on a 16 hr light, 8 hr dark cycle. The white fluorescent light reflected from surfaces facing the holding tanks measured 1.1-1.3 log ft-L using an SE1 meter. Preparation
and apparatus
Fish were immobilized with 0.0012mg per gram body weight D-tubocurarine chloride (Abbott Laboratories. Montreal, P.Q.) injected into the dorsal musculature. Our previous work showed that the immobilizing drug does not change the shape of spectral sensitivity curves or reduce absolute sensitivity (Beauchamp and Rowe, 1977). Fish were held in moulded plastic restrainers. positioned in an 8.25 gallon test tank filled with 20°C tap water, and respired with aerated water at 2.5 ml/set. The left eye of the fish was located at the center and 5.5 cm away from an Albanene back-projection screen. A three channel optical system on the side of the screen remote from the fish was used to produce a circular 102. background field. and a circular 21.5’ spot superimposed on the background and concentric with it. The spot stimulus was presented for durations of between 3 and 5 set controlled with an electronic shutter. Two separate background channels permitted the superposition on the screen of two backgrounds having different chromatic properties: for example, a red background plus
1295
R. D. BF.ALCH.~YP. J. S. ROWE and L. A. O’REILL~
1’96
a blue background. or the presentation of a single chromatic background. The wavelength composition of the background light. which originated in 250 W tungstenhalogen bulbs. was varied using selected short-wave pass and long-wave pass interference filters (Optics Technology, Palo ;\lto. Calif.). The wavelength of the monochromatic spot stimuli was controlled with IS narrow band (100 A at half maximum transmission) interference filters (Baird ,\tomic. Bedford. Mass.) covering the spectrum in 20nm intervals from 400 to 7-10 nm. Background luminances were varied using Wratten No, 96 neutral density hlters, while spot luminances were adJusted with two adjacent Inconel-coated circular neutral density wedges (Eastman-Kodak. Rochester. N.Y.). The transmission characteristics of the narrow band interference tilters were measured on a Unicam spectrophotometer. The spectral distribution of energy in the colored backgrounds produced by the long- and short-wave pass interference filters was measured by placing the probe of an Alphametrics Radiometer against, the back projection screen on the side of the fish’s eye. A reading was obtained for each of the I8 narrow band interference filters. then corrections were made for their known relative transmissions. The spectral composition of the backgrounds used in this study is shown in Fig. I.
Thresholds were measured using the same condttioned heart-rate procedure successfully employed in previous work (Bsauchamp and Rowe. 1977). During training of the conditioned response. suprathreshold spot stimuli at 600nm were presented to the fish. with each stimulus followed immediately by a mild 300 msec shock to the flanks of the fish (4.0 i: 0.5 mA. 2.35-3.0 V r.m.s.1. After l-29 pairings of stimulus and shock. the heart-rate of the fish decreased markedi! during the 3-5 set duration of the stimulus. Heart-rate was monitored with external silver disc electrodes held snuggly against the fish by the plastic restrainers. leaving the fish undamaged for further training and testing at intervals of weeks after initial training. Wavelength and luminance generalization of the response resulted from trials with 400. 500. 600 and 700 nm stimuli presented in irregular sequence over a I.5 log unit range of luminances. When a reliable heart-rate decrease wits establtshed for stimuli at all wavelength-luminance combinations. the conditioned response was used to determine increment thresholds as outlined below. The threshold at each wavelength was determined using a single ascending series of stimulus luminances. each successive stimulus 0.1 log unit more intense than the last. This method was chosen in order to keep the number of shocks given to a fish at 50 to 75 in a given day. while obtaining a complete spectral sensitivity curve from 100 to 7-tOnm in 20nm steps, The precision of the thresholds obtained from a single ascending series. discussed below (Variability of thresholds), is clearly adequate for our present purposes. Threshold was defined as the stimulus luminance when the first of at least two consecutive conditioned responses was observed on successive trials. A conditioned response was defined as an interbeat interval during the stimulus uhich was at least 1.5 times the average interbeat interval in the IOsec preceding the stimulus. Shock was delivered following every stimulus in which a
Table
I. Variability
of
Fish Mean Standard Range
error:
n = 20
neutral
density
conditioned response was noted. but not on sub-threshold trials in which there was not a criterion heart-rate decrease. The absence of shock at sub-threshold stimuli did not. as one might suspect, raise the luminance value of thresholds. A series of 20 thresholds measured at the same wavelength gave notably consistent results (see below). Also. experiments designed to lower the threshold by shocking at luminance values 0.2 and 0.4 log units lower than the threshold established using our procedure. failed to do so (Beauchamp and O’Reilly. unpublished). Thresholds were obtained in sequence from short-. medium- and long-wave regions of the spectrum to minimize possible time-related influences on the shape of the spectral sensitivity curves. A frequent presentation oi shutter activation with light blocked guarded against conditioning to extraneous cues,
Since a single ascending series of luminances was used to establish a threshold at a given wavelength, no measure of within-fish variability was availabile for a single rhreshold value. To provide an estimate of threshold variability at one wavelength. five fish were trained in the usual manner. then. for each fish. 20 consecutive threshold determinations were made at the same wavelength. 600nm. The mean. standard error of the mean, and range of threshotd values are presented in Table I. The thresholds for 20 separate ascending series are notably consistent for all five fish. This finding is not altogether surprising considering the large 0.2 log unit increments in luminance which were used tn the ascending series to establish thresholds. The between fish variability. discussed further below and presented in Table 2. were comparably low. RESULTS
total of thirteen increment threshold curves were measured from six fish. Bright chromatic backgrounds were used to enhance the relative sensitivity in turn of red-, blue- and green-sensitive cone mechanisms. A
The
red nrechanism:
blue-green
background
The sensitivity of the red-sensitive cone mechanism was enhanced relative to that of the green- and bluesensitive cone mechanisms by adapting with a bright blue-green light. The log relative quanta1 spectral energy distribution of the blue-green background is shown in Fig. IA. This background was produced by placing a 500nm short-wave pass cutoff filter. and a 5jOnm short-wave pass cutoff filter. in a tungstenwhite beam. With the cutoff filters absent. the tungsten-white beam measured 2.15 log It-L at the back projection screen using an SE1 meter in the position of the fish’s eye. The mean sensitivities for three fish measured against the blue-green background are shown in Fig. ZA. At four wavelengths. 680 nm to 740nm. the means calculated from nine determinations, instead of three, are also shown. The six additional threshold readings at each of these four wavelengths were taken from the experiments represented in Figs 3 and 4. The
values 600 nm
for
20 threshold
determinations
0,
N I-
Nl2
N,,
2.77 0.03 2.63.0
3.0 I 0.03 I&3.2
3.19 0.03 3.G3.4
2.76 0.04 2.13.0
at
N,j 2.69 0.06 _._ 7 ,_’ 1._1
Goldfish
spectral sensitivity
>
1
c
‘*-._
‘\._. ,f
400 500 600 WAVELENGTH
700
700
600 hm)
tnm)
means shown in Fig. 2A have been fitted with an estimated mean curve by drawing a smooth curve through the points by eye. The mean curve had three features: (i) a prominent peak at 630’nm + 5 nm. (ii) a trough in sensitivity at about XXI nm and (iii) a rising sensitivity below 500 nm. The sensitivity is still rising at 400 nm, the shortest stimulus wavelength tested. A less prominent feature of the curve noted by an astute observer was a possible notch or shoulder to the longwave peak at about 700nm. The three main features of the mean curve also characterized the increment threshold curves of the three individual fish contributing to the means.
Fig. 2. (A) Red mechanism response (blue-green background). Solid triangles (A): means for three determinations. Solid circles (01: means for nine determinations. Continuous line: estimated mean red mechanism curve. (B) The correspondence of the three sets of spectral sensitivity values to the estimated mean curve (blue-green background). (C) Comparison of estimated mean red mechanism curve from (A) above (continuous line) to difference spectrum of goldfish 625 nm max. cone pigment ibroken line) and 625 nm max., retinene-2 pigment nomogram of Ebrey
Table
Mean
and Honig
(dotted
line).
same scale, and are therefore presented in Table 2. The standard errors shown in Column 2. Table 3, are unadjusted. and hence include variability resulting from differences in absolute sensitivity among the fish. To provide a better measure of the variability of points about the estimated mean curve after discounting differences in absolute sensitivity, an adjusted standard error was calculated in the following man-
The standard errors associated with the means in Fig. ?A were too small to represent graphically on the
Fig. ?A. n = 3 Adjusted Mean SE SE
500 WAVELENGTH
Fig. I. Log relative quanta1 energy of colored adapting backgrounds: (A) blue-green background; (B) top line: blue-green plus I.2 log ft-L tungsten-white; bottom line: I.2 log ft-L tungsten-white alone: (C) yellow background; (D) red-blue background.
i
1-I
t
D
O('
400
1297
ner. The estimated mean curve taken from Fig. ZA was fit to each of the three sets of data points making up the means. Then the three individual curves so
2. Means and standard
Fig. 3. n = 3 Adjusted SE SE
errors
Fig. 4. )I = 3 Adjusted Mean SE SE
Fig. 6. ,I = 4 Adjusted Mean SE SE
400 420 440 460 480
I.25 I.10 I.12 0.85 0.76
0.15 0.07 0.07 0.06 0. I 6
0.14 0.06 0.09 0.06 0.16
I.28 I.16 1.17 0.65 0.97
0.26 0.17 0.26 0.11 0.18
0.11 0.06 0.13 0.07 0.07
3.73 2.90 2.82 2.89 2.87
0.37 .0.07 0. I 5 0.20 0.08
0.45 0.70 0.03 0.08 0.10
1.75 1.80 I.92 2.00 2.08
0. I 1 0.06 0.00 0.1 I 0.06
0.1 I 0.03 0.03 0.09 0.07
500 520 S-IO 560 580
0.69 0.87 1.26 I.30 1.55
0.13 0.14 0. I 4 0.05 0.06
0.13 0.1-t 0. I 3 0.07 0.06
0.98 I.01 1.14 I.10 I .42
0.07 0.04 0.09 0.08 0.17
0.07 0.13 0. I 3 0.07 0.05
2.5 I 2.10 1.83 I .30 I.41
0.07 0.10 0.07 0. I 2 0.1 I
0.06 0.09 0.06 0.16 0.08
2.36 2.29 2.47 2.13 1.29
0.00 0.09 0.08 0.10 0.06
0.03 0.07 0.05 0.08 0.08
600 610 640 660 680
I.50 I .76 1.69 I .63 1.58
0.1 I 0.06 0.09 0.06 0.06
0.1 I 0.06 0.08 0.07 0.05
I .43 I .66 I .J3 I.55 I .49
0.06 0.15 0.15 0.14 0.14
a.08 0.04 0.01 0.09 0.08
I.14 I.19 I.41 I .43 I.31
0.1 I 0. I 2 0.06 0. I 2 0.10
0.06 0.02 0.10 0.02 0. I 5
I .96 I .63 I.22 I.24 1.17
0.11 0.08 0.09 0.1 I 0.08
0.10 0.10 0.08 0.08 0.05
700 720 740
1.06 0.75 0.74
0.10 0.06 0.34
0.09 0.06 0.32
0.9 I I.00 0.51
0.15 0.15 0.10
0.04 0.09 0.17
0.95 0.53 0.18
0.10 0.28 0.1 I
0.05 0.16 0.08
0.84 0.80 0.37
9.15 0.07 0.08
0.13 0.05 0.09
R. D.
I198 2
2 0 ’
I
1 1 1 1 I
1 1 1 1 1 1 1 1 1 1 1 1 .
-Li s ; 3 5
J. S. Row
B~~ICHMP.
A/
: c) &gy;,(;
,,*a**:--T&A
_
‘$ ?? ‘0
I>. _ ‘I ,““““1”‘1”‘1’ 430
500 WAVELENGTH
700
600 :nm)
Fiu-_.-. : Location oi long-ware peak: Dotted hnc: at 660 nm using a 1.2 log ft-L tungsten-uhite background. Solid line: at 6!0nm using :I I.1 log ft-L tungsten-Nhitc plus a bluegccn background. Solid triangles (AI: means determinations. Continuous line: cur\e from Dotted line: curIt from Beauchamp and Ro~e 19771
for
three
Fig. ?A. (Frg
-IA.
established were brought to the same position on the log relative sensitivity scale by adding an appropriate correction to each data point. With the three curves now superimposed, the variability about the mean curve of the adjusted points was calculated. These values. shown in Column 3. Table 2. differ only a little. in the case of the blue-green background experiments. from the unadjusted standard errors shown in Column 7, because the three fish contributing to the mean had almost identical absolute sensitivities. However. in later experiments to be described in this paper. subjects often differed in absolute sensitivity. and the adjusted standard error. calculated in the same way as described above. may more faithfully represent the fit to the estimated mean curve. As previously noted. the three characteristics of the mean red mechanism curve shown in Fig. 2A were descriptive of the curves from the three component curves: all had a peak at about 630nm. a trough at about 5OOnm and a rising sensitivity at wavelengths shorter than 500nm (Fig. 2B). However. the position of the trough varied somewhat for the three fish from 480 to 520nm. and the steepness of the rise in sensitivity was also distinctive. The largest rise in sensitivity from the trough to the position at 100nm was 1.09 log unit, while the smallest rise was 0.38 log unit. .A comparison of the estimated mean curve shown in Fig. 2A to two curves of interest is seen in Fig. 2C. The mean curve had the same wavelength of peak sensitivity at about 630nm as does the difference spectrum for the goldfish red-absorbing cone pigment (Harosi and MacNichol, 197-I). Hovvever. it was wider than the pigment difference spectrum on the right shoulder of the curve, and narrower on the left shoulder. A better fit on the right shoulder is provided by the pigment nomogram for a retinene-2 pigment peaking at 630 nm (Ebrey and Honig. 1977). but the left shoulder discrepancy remains. hUr1011
(J/ ~Oll~/-~Ur'e pdS
In a recent and closely-related publication (Beauchamp and Rowe. 1977). the mean spectral sensitivity curve for seven fish. measured against a I.2 log ft-L tungsten-white background. was characterized by a long-wave component which peaked at 660nm (redrawn in Fig. 3. broken line). The 660nm location of the peak is of interest
and L. 4. O‘REILL~
because it is remote irom the 62 nm position prcdieted from the absorption spectrum of the redabsorbing cones (Marks. 1965: H~osi and LiacS~chol. 197-t). A likely explanation for the longwave shift of the peak depends on the antagonistic action of green mechanism activity. aroused strongly by wavelengths close to 520 nm and progressively less so ;tt longer wavelengths. At the ganglion cell level in goldfish. such antagonistic green mechanism activity csn lower the sensitivity for responses originating in rsdsensitive cones (Wagner c’t trl.. 1960: Davv. 196s: Beauchamp and Lovasik. 19731. To test the antagonistic action explanation of the 660 nm peak Iocation. the spectral sensitivity curve against a 1.3 log ft-L tungsten-white background. 3s in the previous work. was msllsured once again. but this time a bright blue-green background was superimposed on the white in an attempt to depress green mechanism antagonism. The blue-green background used was the same as that described in the previous section of this paper. The spectral composition of the tungsten-whtte background and the composite tungsten-white with superimposed blue-green background are shown in Fig. IB. Note that the composite w hits i blue-green background has a higher quanta1 energy than the white alone only below 560nm and hence will lower the sensitivities of green and blue cone mechanisms. while influencing the red mechanism sensitivity verv little. The solid symbols in Fig. 3 Indicate the mean sensetivities for three fish when the white + blue-green background was used. The peak now falls at 630nm instead of at 660 nm. The points above 540 nm fall on ths estimated mean curve for the red mechanism taken from Fig. ?A. These results point to green mechanism antagonism as the c;1use of the shifted 660nm location of peak sensitivity when a tungstenwhite background was used alone. Another feature of interest in the composite white + blue-green curve of Fig. 3 was the absence of a clearly defined trough in sensitivity at 500 nm like that seen for the blue-green condition in Fig. 2A. The absence of the trough was associated with the add!tion of the I.3 log ft-L tungsten-white background which was the only difference between the two stimulus conditions. A comparison of the absolute sensttivities for the two mean curves in Fig. 2A and Fig. ? revealed that the latter was 0.4 log units less sensitive. As one might have predicted. the tungsten-vvhitt background, with a preponderance of long-wave energy (Beauchamp and Rowe, 1977). has acted to lower the red mechanism sensttivitv. This suggests that the high points at GO. 500 and ;lOnm in Fig. 3. obscuring the trough. may result irom green mechanism activity which vvas not present when the red mechanism was more sensitive. as was the case in the conditions of Fig. ?A-\. The hiue wYh7,1;s/tr: ,w/lo~r~ huc/qrorri!tl The relative sensitivtty of the blue mechanism was enhanced using a bright yellow adapting background. The yellow background resulted from a 500 nm longwave pass cutoff filter, and a 550 nm long-wave pass cutoff filter, placed in a 2.14 log it-L tungsten-white beam. The log relative quanta1 spectral energy distribution of the background is shown in Fig. 1C.
Goldfish
spectral sensitivity reduced
0.50 log tion. We absolute and the
400
500 WAVELENGTH
700
600 (nml
Fig. 4. Blue mechanism response (yellow background): Solid triangles (A): means for three determinations. Shortwave curve: difference spectrum for goldfish 455 nm max. cone pizsent. Long-wave curve: taken from Fig. 2A. The mean sensitivities for three fish using the yellow background are presented in Fig. 4. The blue mechanism component of the curve under these conditions was about 1.5 log unit more sensitive than the red mechanism component. Between 420 and 520 nm, the mean sensitivities were well-described by the different spectrum for the blue-absorbing cone pigment (Harosi and 1MacNichol. 1974). At 540 and 560 nm the sensitivity was somewhat higher than predicted from the pigment absorption curve, which may indicate the intrusion of some green mechanism activity. Above 560nm. the means fall on the red mechanism curve taken from Fig. 2A. Of interest in Fig. 4 was the high sensitivity at 400 nm, also noted when using a 1.2 log ft-L tungstenwhite background (Beauchamp and Rowe. 1977). However. the aberrance was markedly greater against the yellow background than against the white. For the yellow background. the mean 400nm sensitivity was 1.13 log unit above that.predicted from the pigment absorption curve, while against the white background the mean aberrance was 0.53 log unit. The fact that aberrant high blue sensitivity at 400 nm persists. and was in fact greater. when the red and green mechanism sensitivities were depressed by means of a yellow background, makes it difficult to attribute the aberrance to activity originating in red or’ greensensitive cones. For example, the high point at 400 nm cannot be caused by beta band absorption by the red-sensitive pigment. A feature of the high aberrance at 400nm was the great range of values noted in individual goldfish. Figure 5A illustrates the range observed for the three fish tested with a yellow background: the smallest aberrance was 0.35 log unit, while the largest was 1.95 log unit. An aberrance as high as 2.70 log units was noted in an early experiment (Beauchamp. 1978: Beauchamp and Rowe, unpublished). Where the 400 nm point was very high. the 420 and 440 nm sensitivities sometimes fell above the pigment absorption curve also. When a white background was used, the aberrance was smaller and restricted to the 400nm response (Fig. 5B). Addimg a I.2 log ft-L tungstenwhite background on top of the yellow background
1299
high aberrance at 400nm to values about unit typical of the white background condifound no significant correlation between the sensitivity of the blue mechanism response degree of 100 nm aberrance.
The green mechanism was enhanced in sensitivity relative to the red and blue mechanisms by adapting with two superimposed colored backgrounds. In one background channel, a blue background was formed by placing a 450 nm short-wave pass cutoff filter in a 2.15 log ft-L tungsten-white beam. In a second background channel, a red background was produced by placing a 650 nm long-wave pass cutoff filter in a 2.15 log ft-L tungsten-white beam. The two backgrounds superimposed on the back projection screen gave a cyan-appearing background, the spectral composition of which is shown in Fig. ID. From 400 to 640nm. the mean sensitivities obtained with a red-blue background were well described by the Ebrey and Honig (1977) nomogram for a retinene-2 pigment with peak sensitivity at 53Onm (Fig. 6). The difference spectrum for the green-absorbing pigment also provided a satisfactory tit, although it was a little wide on the right shoulder. Above 660nm the points fell on the red mechanism curve taken from Fig. 2A. An interpretive problem arises when using spectral sensitivity curves in goldfish to identify green mechanism activity. because goldfish rods have a peak sensitivity at 522 nm. similar to that of the green-sensitive cones at 530 nm. Our evidence that the activity shown in Fig. 6 originates in green-sensitive cones. and not rods, comes from parallel neurophysiological work. When recording from optic nerve fibers. the rod and green-sensitive cone responses can be differentiated by the sign of the response, excitatory or inhibitory
I
’
’
’
’
I
’
’
0400
500
WAVELENGTH
Fig. 5. Variability in aberrant high sensitivity at 400 and 420 nm: (A) against yellow background: (B) against I.2 log h-L tungsten-white background. Curves: difference spectrum for goldfish 455 nm max. cone pigment.
R. D. BE.~LCH~UP. J. S.
1300
Rour
and L. A. O‘RHLLI
the curve and wider than it on the right shoulder. The narrowing on the left shoulder may result from residual ant~~o~isrn from green-sensitive shoulder of
cones. The close proximity
400
600
500 WAVELENGTH
700 inm;
Fig. 6. Green mechanism response (red-blue background). Filled triangles (A): means for four deterrni~~t~on~. Open circles (01: values for fish with 0.5 log ft-L tungsten-white added to red-blur background. Midwave curve: Ebrey and Honig retincne-:! 530 nm max. nomogram. Long-wave curve taken from Fig. ?A.
(Ra~nauld.
1972; Beauchamp
and Daw,
1972). Using
similar red-blue adapting conditions in the optic fiber work, the activity of green-sensitive cones. and not rods. was observed (Beauchamp and Lovasik. 1973). There is no reason to believe that, with the same stimulus conditions. the psychophysical response does not also originate with green-sensitive cones. As a precaution, one of the four fish tested with the redblue background had, in addition, a third background superimposed on the others, a 0.5 log ft-L tungstenwhite background. This ensured that photopic conditions prevailed. The results, similar to those for the other three fish, are shown with open symbols in Fig. 6. A final point of interest in Fig. 6 is the absence for this condition of noteworthy high aberrant sensitivity at the blue end of the spectrum. Seemingly, blue backgrounds or white backgrounds containing a shortwave component seem to act to reduce high blue aberrance. DiSCUSSlON
Three cone mechanisms were characterized psychophysically from spectral sensitivity curves measured against colored adapting backgrounds. A long-wave peak at 630nm was obtained when using a btuegreen background. a mid-wave peak at 530 nm when uing a red-blue background, and a short-wave peak at 46Onm with a yellow background. Since these peaks were in the same iocation as those of the difference spectra of the three goldfish cone photopigments (‘Marks. 1965: Harosi and MacNichol. 1974). an origin in red-, green- and blue-sensitive goldfish cones (Stell and Harosi. 1976: Marc and Sperling. 1976) was indicated. The shape of the short-wave peak matched that of the corresponding cone absorption curve. except at 4OOnm. the shortest wavelength tested, where the sensitivity was higher than predicted from the absorption curve. The mid-wave peak was only slightly wider than the corresponding pigment curve on the right shoulder. and the nomogram of Ebrey and Honig (1977) also provided a satisfactory fit. The long-wave peak, however, was markedly narrower than the pigment absorption curve on the left
of the green-sensitive
ac-
cessory member of the goldfish double cone to the red-sensitive principal member tStel1 and Harosi. 1976: >farc and Spetling. 1976) may provide a particularly etficient inhibitory pathway, the influence of which persists even when green-sensitive cone sensitivity is reduced with a blue-green background. The widening on the right shoulder of the long-wave peak compared to the pigment ditTerence spectrum has no ready explanation. Bowmaker { 1975) has demonstrated that the absorbance of visual pigments in the intact retina is greater at longer wavelengths than the absorbance of the pigments in solution. Perhaps. in a similar fashion the spectral sensitivity of the goldfish. mediated by the intact retina and measured psychophysically. is higher at long wavelengths than predicted from the difference spectra of single photoreceptors measured microsp~trophotom~tr~call~. Somewhat enigmatically. the retinene-2 nomogram of Ebrey and Honig (1977) provided a much better fit on the right shoulder of the long-wave peak than did the emptrtcafiy determined pigment difference spectrum. An alternative point of view is provided if one interprets the discontinuity evident in fig 7A at 7M)nm as a shoulder to the long:wave peak. Then, one might be reasonably satisfied with the fit of the difference spectra for pomts below 7OOnm, and for the shoulder of the curve above 700 nm, another spectral mechanism, as yet unspecified, coufd be proposed. Such a hypothetical spectral mechanism, for which there is little evidence at present, might relate to the puzzling longwave sensitivity encountered by Naka and Rushton (1966). Red-yrrm
mrchunisni
inreracrions
Against a I.2 tog ft-L tungsten-white background. the goldfish increment threshold curve was charactetized by a prominent long-wave peak at 66Onm (Beauchamp and Rowe, 1977). When a blue-green background was added to the tungsten-white, the long-wave peak became wider and shifted.to 630 nm. This result suggests that the 660 nm location with the tungsten-white alone was a result of green-sensitive cone inhibition on red-sensitive cones. When green mechanism sensitivity was reduced by adding the blue-green background, the green mechanism inhibitory influence was diminished, and the long-wave peak more nearly corresponded with the photopigment absorption spectrum. This explanation is consistent with our understanding of red-green interactions measured at the ganglion cell level in goldfish iWagner et al., 1960; Daw, 1968; Beauchamp and Lovasik, 1973), and is similar to ef%cts demonstrated in rhesus monkey (Sperling and Harwerth, 1971). .ihsertce of blur-yreen umqotlisric itueruction Beauchamp and Lovasik (I 973) reported that optic fiber responses originating in blue-sensitive cones had the same sign, inhibitory or excitatory, as responses initiated by green-sensitive cones. The response at a given wavelength seemed to be that of the most sensitive cone mechanism. blue or green. with no evidence of addition in sensitivity of blue and green mechan-
Goldfish
spectral
isms. The psychophy,sical results presented in this paper confirm this view: both the short- and midwave curves are well fit by their respective pigment difference spectra. There is no evidence of a narrowing on the right shoulder of the short-wave curve. or of a narrowing on the left shoulder of the mid-wave curve, both of which would have been predicted had blue and green mechanisms been mutually antagonistic. Nor is there a marked widening of the short- and mid-wave peaks which summation of blue and greensensitive cone activity should have caused. Apparently the blue-sensitive cones, unlike the red- and greensensitive cones, function independently or nearly so. Similarly. in rhesus monkey, Sperling and Harwerth (1971) found the blue-sensitive c3nes acted independently. This work. as was earlier neurophysiological work (Beauchamp and Lovasik, 1973). is inconsistent with the report of Spekreijse et al. (1972) that blue mechanism activity is of opposite sign to that of green mechanism activity. The results of Spekreijse er al. (1972). however, might be a consequence of’ attributing to blue-sensitive cones responses which in fact resulted from beta band stimulation of red-sensitive cones. As the discussion will elaborate below, a prominent short-wave peak caused by absorption in the beta band of the red-sensitive pigment is noted when bluesensitive cone sensitivity is reduced. Beta band actioity
When using a blue-green adapting background, a rising sensitivity below 460 nm was noted. Because of the background used, which would act t’o reduce blue and green mechanism sensitivity. this short-wave peak was unlikely to be due to blue- or green-sensitive cone activity. The peak was reminiscent of the beta band peaks noted for similar background conditions in optic fiber work (Beauchamp and Lovasik, 1973). Subsequent experiments (Beauchamp and O’Reilly, in preparation) have confirmed that the short-wave peak seen with a blue-green adapting background is a result of beta band absorption by the red-sensitive pigment. Changes in the intensity of a blue adapting background influenced the beta band short-wave sensitivity very little, while alterations in the intensity of a red background moved the long-wave peak and the beta band short-wave peak up and down in synchrony. The beta band of the goldfish red-sensitive pigment is particularly prominent because of the longwave location at 625 nm of the retinene-2 alpha peak. However, even in primates, where the retinene-1 peaks are located at shorter wavelengths. shorbwave responses originating in beta band absorption by the red-sensitive pigment have recently been reported (Monasterio and Gouras, 1977). This may explain the purple, or red-blue, appearance sometimes reported by humans for short-wave stimuli. High aberrant
blue sensitivity
A short-wave peak of a different origin is observed when employing a yellow background. The high short-wave sensitivity seen at 4OOnm and sometimes 420 and 440 nm as well, cannot be a result of activity originating in red- or green-sensitive cones because these are depressed by the yellow background. Moreover, the magnitude of the aberrance at 4OOnm is
sensiti\iq
1301
dependent on the energy of the background at the blue end of the spectrum. When blue light is altogether absent from the background, as in the yellow background condition. the aberrance is at its highest. When there is an intermediate level of energy at the blue end. as in the tungsten-white 1.2 log ft-L condition, the aberrance is small. For a bright blue background condition. as in the red-blue adapting background experiments. there is no evidence of aberrant high blue sensitivity whatsoever. These findings have led us to hypothesize that absorption of scattered light by blue-sensitive cones or rods are responsible for the high blue aberrant sensitivity. The photoreceptors involved are likely those in the 40’ ring of the retina in the far periphery not adapted by our 100” background. For yellow and red backgrounds. little ‘blue light is available to scatter into the far periphery. The blue-sensitive cones or rods will be relatively dark-adapted and sensitive to any scattered light from the stimulus. In blue-green or white background conditions where there is blue light in the background, this will scatter into the far periphery and light-adapt the photoreceptors there. For these backgrounds there will be reduced sensitivity to scattered light from spot stimuli. Thus. the spectral composition of the background will determine the sensitivity to scattered light of far peripheral photoreceptors. Whether or not there is scattered stimulus light to activate the far peripheral receptors will depend on the wavelength of the spot stimulus. The intensity of scattering in an optical medium containing small particles is inversely proportional to the fourth power of the wavelength of the light. Hence the greatest amount. of scatter will arise from the 4OOnm spot stimulus, much less from the 420 nm stimulus and still less for stimulus spots with wavelengths at 31Onm and above. This accounts for the fact that the greatest high sensitivity aberrance occurred for the 400 nm stimulus, with less aberrance for the 420 nm stimulus and little or none for 440 nm stimuii and above. Our data thus far are consistent with the scatter hypothesis, but we have not yet attempted to measure the proposed scattered light directly. Also. we have not eliminated the possibility that fluorescence stimulated by 4OOnm light (rather than scatter of the 4OOnm light) is the source of far peripheral retinal stimul8tion. An interesting aspect of the high aberrant sensitivity at short wavelengths is the great variability noted amongst individual fish. We are not sure if this results from variability in our location of the eye in experiments (scatter off the optic disc is a possibility). or from differences in the optical system of the fish (opacities in the optic media or the size of the lens, for example). or from some other cause. Shape of the spectral sensiticity
curce
An integrating principle emphasized by this work is that the shape of the spectral sensitivity curve is determined by the balance of sensitivity existing among the three cone mechanisms. As proposed in the yupper envelope model” of Stiles (1959) and Sperling and Harwerth (I 97 I ). threshold at a given wavelength was determined by the most sensitive of the three cone mechanisms operating at that wavelength. At the short-wave end of the spectrum, for example. the most
R. D. BULCHUIP.
1302
1. S. Rows
sensitive cone mechanism normally was the blue mechanism, acting free of interactions with the other two cone mechanisms. However. a red-blue background favoured a green mechanism response and a blue-green background favoured a response originating in beta band absorption by the red mechanism. At the long-wave end of the spectrum. threshold was influenced by antagonistic interaction between green and red cone mechanisms; with an excitatory input from one cone mechanism, red or green, and an inhibitory input from the other. Thus the linear subtractive process between red- and green-sensitive cone mechanisms described by Sperling and Harwerth (1971) to account for rhesus monkey long-wave spectral sensitivity, is applicable to goldfish spectral sensitivity data as well. Again, the composition of chromatic adapting backgrounds was important in the case of long-wave sensitivity, determining the relative strength of the inhibitory and excitatory influence operating at each wavelength. Hence, as others have demonstrated in the past (Yager. 1969: Yager. 1974: Muntz and Northmore. 1971). the spectra) energy distribution of adapting backgrounds may strongly influence the shape of spectral sensitivity curves. In fact, there will be a unique spectral sensitivity curve ior each chromatic background. of
the
three
Nonetheless, cone
all curves
mechanism
will
sensitivity
be composed curves
de-
in this paper; the short-wave mechanism apparently independent of the other two, and the mid- and long-wave mechanisms mutually antagonistic. The parallels of these results with those of Sperling and Harwerth (1971) working with rhesus monkey are striking. scribed
Acknorr,ledgemmrs-This work was supported by funds from NRC (Canada) and MRC (Canada). Thanks to Olga Vrablic and Sherry1 DiCiccio for technical assistance.
REFERENCES Beauchamp R. D. (1978) Color vision in goldfish: A comparison of psychophysical and neurophysiological findings. In Visual Psrcho~h~sics (Edited by Armington J. C. a& Krauskopf J). Academic Press, New York.Beauchamu R. D. and Daw N. W. (1972) Rod and cone input to’single goldfish optic nerve fibers. Vision Res. 12. 1201-1212. Beauchamp R. D. and Lovasik J. V. (1973) Blue mechanism response of single goldfish optic fibers. J. ,veurophysiol. 36, 925-939.
and L. r\. O‘REILL~
Beauchamp R. D. and Rowe J. S. (IY’7) Goldfish spectral sensitivity: il conditioned heart rate measure in restrained or curarized fish. Vision Rzr. 17. 617-621. Bowmakcr J. K. (1975) Spectral sensitivity and visual pigment absorbance. b’isioti Rtis. 13. 783-792. Daw N. W. (1968) Color-coded gang!ion cells in the goldfish re!ina: extension of their receptive Relds by means of new stimuli. J. Physiol. 197. 567-592. Ebrey T. G. and Honig B. (1977) New wavelength &pendent visual pigment nomograms. t’ision R~s. 17. l-17-151. Harosi F. 1. and MacNichol E. F. (I9741Visual pigments oi goldfish cones. J. yen Physiol. 63, 219-303. Liebman P. .A. and Entine G. (19631 Sensitive low-lightlevel microspectrophotometer: detection of photosensitive pigments of retinal cones. J. opr. Sot. .Im. 54. l-tjl-l-t.i9. Marc R. E. and Sperlmg H. G. ( 1976) Color receptor identities of goldfish cones. Scinicr 191. Zt7--189. Marks W. G. (1965) Visual pigments of single goldfish cones. J. PlrJsiol. 178. l-I-32. hfonasterio F. &I. de and Gouras P. (1977) Responses of macaque ganglion cells to far violet lights. Visiotl Rex 17. 1117-l 156. Muntz W. R. A. and Northmore D. P. M. (1970) Vision and visual pigments in a fish Scurdiriiu.s rr~rhrophrl~u/~~~u.s (the ruddl. C’ision Rex 10. 281-298. .Muntz W. R. A. and Northmore D. P. LI. (1971) The independence of the photppic receptor slstems underlying visual thresholds in a teleost. Vision R?s. 11. 861-876. Naka K. I. and Rushton W. .A. H. (19661 S-potentials from colour units in the retina of fish (CJprrnidm). J. Plrysiol.
185. 53&SSj. Raynauld J. P. (1972) Goldfish retina: sign of the rod input in opponent color ganglion cells. Scirnce 177. S&85. Spekreijse H.. Wagner H. G. and Wolbarsht M. L. 11972) Spectral and spatial coding of ganglion cell responses in goldfish retina. J. Neurophysiol. 35. 75-86. Sperling H. G. and Harwerth R. S. (1971) Red-green cone interactions in the increment-threshold spectral sensitivity oi primates. Science 172. 18&l 81. Stell W. R. and Harosi F. 1. (1976) Cone structure and visual pigment content in the retina of the goldfish.
Vision Res. 16. 647-657. Stiles W. S. (1959) Color vision: the approach through increment-threshold sensitivity Proc. mm. Acad. Sci. U.S.A. 45. IWI 14. Wagner H. G.. MacNichol E. F. Jr and Wolbarsht M. L. (1960) The response properties of single ganglion cells in the goldfish retina. J. yen. Physiol. l8 (Suppl.). J-562. Yager D. (1967) Behavioral measures and theoretical analysis of spectral sensitivity and spectral saturation in the goldfish Carussius uuratus. Vision Rrs. 7. 707-727. Yager D. (1969) Behavioral measures of spectral sensitivity in the goldfish following chromatic adaptation. Vision Rex 9. 179-l 86. Yager D. (197-t) Effects of chromatic adaptation on saturation discrimination in goldfish. Vision Res. 14. 1089 IC9-l.
79 l?Ol-1195SOXO
0
GOLDFISH SPECTRAL SENSITIVITY: IDENTIFICATION OF THE THREE CONE MECHANISMS IN HEART-RATE CONDITIONED FISH USING COLORED ADAPTING BACKGROUNDS R. D. BEAUCHAMP,J. S. Row
and L. A. O’REILLY
Departments of Optometry and Biology. University of Waterloo, Waterloo, Ontario. Canada (Receiced 24 Ocrobur 1978; in rerised/orm
19 April
1979)
Abstract-Increment threshold curves were measured for curarized goldfish using a conditioned heartrate procedure. Chromatic backgrounds were utilized to enhance the sensitivity of one goldfish cone mechanism relative to the other two. Sensitivity peaks were at 4%. 530 and 630nm against yellow. red-blue and blue-green backgrounds. respectively. The short- and mid-wave peaks agreed well with the appropriate goldfish cone difference spectrum, but the long-wave peak was narrower on the left shoulder and wider on the right shoulder than the corresponding cone difference spectrum. Against the bluegreen background. a prominent short-wave peak attributed to beta band absorption by the red-sensitive cones was observed. The magnitude of aberrant high sensitivity noted for 400 and 420 stimuli was dependent on the amount of short-wave energy in the background; highest aberrance was noted for yellow backgrounds. and lowest for blue or red-blue backgrounds.
lNTRODUCTlON The goldfish is a useful animal in which to study color vision because its three cone pigments have widely separated sensitivity peaks at 455, 530 and 625 nm (Liebman and Entine, 1964; Marks, 1965; Harosi and MacNichol, 1974). Therefore. the cone mechanisms contributing to a neural or behavioral response can be identified readily by noting the location of peaks in a spectral sensitivity curve. For example, in psychophysical measurements of spectral sensitivity with a tungsten-white background, the goldfish increment threshold curve had a prominent short-wave peak at about 455 nm, and the peak had the same shape as the difference spectrum of the blue-absorbing cone pigment (Beauchamp and Rowe, 1977). The 455 nm peak indicated that the psychophysical response at short wavelengths originated in blue-sensitive cones. Two other components were noted in the increment threshold curve measured against a tungsten-white background. There was a mid-wave shoulder to the short-wave peak and, as well, there was a long-wave peak at about 660 nm. The mid- and long-wave components were narrower than the corresponding cone absorption spectra, which suggested an influence of antagonistic interaction between red- and greensensitive cone mechanisms (Wagner et al., 1960: Daw, 1968; Beauchamp and Lovasik, 1973). In the present paper, the origin of the mid- and long-wave components observed with the tungstenwhite background is clarified. Bright chromatic backgrounds have been used to reduce selectively the sensitivity of two of the three goldfish cone mechanisms, leaving the third cone mechanism to dominate the psychophysical response over a wider-than-normal range of the spectrum (Stiles, 1959: Yager, 1969: Beauchamp and Lovasik, 1973). The procedure allowed us to observe the consequence of reduced “A
19 12-74
antagonistic interactions on the shape of the increment threshold curve. In addition, further information was gathered on the high aberrant sensitivity previously noted at the short-wave end of the spectrum (Yager, 1967; Muntz and Northmore, 1970; Beauchamp and Rowe, 1977). >lETHODS Details of the methods are presented in a recent. closelyrelated paper (Beauchamp and Rowe, 1977). Subjects
and conditions
Ten common goldfish (C’arassius aurarus) weighing from 29 to 64 g (9.5 to 12.5 cm. standard length) were kept for at least a week prior to experimentation.at about 2O’C on a 16 hr light, 8 hr dark cycle. The white fluorescent light reflected from surfaces facing the holding tanks measured 1.1-1.3 log ft-L using an SE1 meter. Preparation
and apparatus
Fish were immobilized with 0.0012mg per gram body weight D-tubocurarine chloride (Abbott Laboratories. Montreal, P.Q.) injected into the dorsal musculature. Our previous work showed that the immobilizing drug does not change the shape of spectral sensitivity curves or reduce absolute sensitivity (Beauchamp and Rowe, 1977). Fish were held in moulded plastic restrainers. positioned in an 8.25 gallon test tank filled with 20°C tap water, and respired with aerated water at 2.5 ml/set. The left eye of the fish was located at the center and 5.5 cm away from an Albanene back-projection screen. A three channel optical system on the side of the screen remote from the fish was used to produce a circular 102. background field. and a circular 21.5’ spot superimposed on the background and concentric with it. The spot stimulus was presented for durations of between 3 and 5 set controlled with an electronic shutter. Two separate background channels permitted the superposition on the screen of two backgrounds having different chromatic properties: for example, a red background plus
1295
R. D. BF.ALCH.~YP. J. S. ROWE and L. A. O’REILL~
1’96
a blue background. or the presentation of a single chromatic background. The wavelength composition of the background light. which originated in 250 W tungstenhalogen bulbs. was varied using selected short-wave pass and long-wave pass interference filters (Optics Technology, Palo ;\lto. Calif.). The wavelength of the monochromatic spot stimuli was controlled with IS narrow band (100 A at half maximum transmission) interference filters (Baird ,\tomic. Bedford. Mass.) covering the spectrum in 20nm intervals from 400 to 7-10 nm. Background luminances were varied using Wratten No, 96 neutral density hlters, while spot luminances were adJusted with two adjacent Inconel-coated circular neutral density wedges (Eastman-Kodak. Rochester. N.Y.). The transmission characteristics of the narrow band interference tilters were measured on a Unicam spectrophotometer. The spectral distribution of energy in the colored backgrounds produced by the long- and short-wave pass interference filters was measured by placing the probe of an Alphametrics Radiometer against, the back projection screen on the side of the fish’s eye. A reading was obtained for each of the I8 narrow band interference filters. then corrections were made for their known relative transmissions. The spectral composition of the backgrounds used in this study is shown in Fig. I.
Thresholds were measured using the same condttioned heart-rate procedure successfully employed in previous work (Bsauchamp and Rowe. 1977). During training of the conditioned response. suprathreshold spot stimuli at 600nm were presented to the fish. with each stimulus followed immediately by a mild 300 msec shock to the flanks of the fish (4.0 i: 0.5 mA. 2.35-3.0 V r.m.s.1. After l-29 pairings of stimulus and shock. the heart-rate of the fish decreased markedi! during the 3-5 set duration of the stimulus. Heart-rate was monitored with external silver disc electrodes held snuggly against the fish by the plastic restrainers. leaving the fish undamaged for further training and testing at intervals of weeks after initial training. Wavelength and luminance generalization of the response resulted from trials with 400. 500. 600 and 700 nm stimuli presented in irregular sequence over a I.5 log unit range of luminances. When a reliable heart-rate decrease wits establtshed for stimuli at all wavelength-luminance combinations. the conditioned response was used to determine increment thresholds as outlined below. The threshold at each wavelength was determined using a single ascending series of stimulus luminances. each successive stimulus 0.1 log unit more intense than the last. This method was chosen in order to keep the number of shocks given to a fish at 50 to 75 in a given day. while obtaining a complete spectral sensitivity curve from 100 to 7-tOnm in 20nm steps, The precision of the thresholds obtained from a single ascending series. discussed below (Variability of thresholds), is clearly adequate for our present purposes. Threshold was defined as the stimulus luminance when the first of at least two consecutive conditioned responses was observed on successive trials. A conditioned response was defined as an interbeat interval during the stimulus uhich was at least 1.5 times the average interbeat interval in the IOsec preceding the stimulus. Shock was delivered following every stimulus in which a
Table
I. Variability
of
Fish Mean Standard Range
error:
n = 20
neutral
density
conditioned response was noted. but not on sub-threshold trials in which there was not a criterion heart-rate decrease. The absence of shock at sub-threshold stimuli did not. as one might suspect, raise the luminance value of thresholds. A series of 20 thresholds measured at the same wavelength gave notably consistent results (see below). Also. experiments designed to lower the threshold by shocking at luminance values 0.2 and 0.4 log units lower than the threshold established using our procedure. failed to do so (Beauchamp and O’Reilly. unpublished). Thresholds were obtained in sequence from short-. medium- and long-wave regions of the spectrum to minimize possible time-related influences on the shape of the spectral sensitivity curves. A frequent presentation oi shutter activation with light blocked guarded against conditioning to extraneous cues,
Since a single ascending series of luminances was used to establish a threshold at a given wavelength, no measure of within-fish variability was availabile for a single rhreshold value. To provide an estimate of threshold variability at one wavelength. five fish were trained in the usual manner. then. for each fish. 20 consecutive threshold determinations were made at the same wavelength. 600nm. The mean. standard error of the mean, and range of threshotd values are presented in Table I. The thresholds for 20 separate ascending series are notably consistent for all five fish. This finding is not altogether surprising considering the large 0.2 log unit increments in luminance which were used tn the ascending series to establish thresholds. The between fish variability. discussed further below and presented in Table 2. were comparably low. RESULTS
total of thirteen increment threshold curves were measured from six fish. Bright chromatic backgrounds were used to enhance the relative sensitivity in turn of red-, blue- and green-sensitive cone mechanisms. A
The
red nrechanism:
blue-green
background
The sensitivity of the red-sensitive cone mechanism was enhanced relative to that of the green- and bluesensitive cone mechanisms by adapting with a bright blue-green light. The log relative quanta1 spectral energy distribution of the blue-green background is shown in Fig. IA. This background was produced by placing a 500nm short-wave pass cutoff filter. and a 5jOnm short-wave pass cutoff filter. in a tungstenwhite beam. With the cutoff filters absent. the tungsten-white beam measured 2.15 log It-L at the back projection screen using an SE1 meter in the position of the fish’s eye. The mean sensitivities for three fish measured against the blue-green background are shown in Fig. ZA. At four wavelengths. 680 nm to 740nm. the means calculated from nine determinations, instead of three, are also shown. The six additional threshold readings at each of these four wavelengths were taken from the experiments represented in Figs 3 and 4. The
values 600 nm
for
20 threshold
determinations
0,
N I-
Nl2
N,,
2.77 0.03 2.63.0
3.0 I 0.03 I&3.2
3.19 0.03 3.G3.4
2.76 0.04 2.13.0
at
N,j 2.69 0.06 _._ 7 ,_’ 1._1
Goldfish
spectral sensitivity
>
1
c
‘*-._
‘\._. ,f
400 500 600 WAVELENGTH
700
700
600 hm)
tnm)
means shown in Fig. 2A have been fitted with an estimated mean curve by drawing a smooth curve through the points by eye. The mean curve had three features: (i) a prominent peak at 630’nm + 5 nm. (ii) a trough in sensitivity at about XXI nm and (iii) a rising sensitivity below 500 nm. The sensitivity is still rising at 400 nm, the shortest stimulus wavelength tested. A less prominent feature of the curve noted by an astute observer was a possible notch or shoulder to the longwave peak at about 700nm. The three main features of the mean curve also characterized the increment threshold curves of the three individual fish contributing to the means.
Fig. 2. (A) Red mechanism response (blue-green background). Solid triangles (A): means for three determinations. Solid circles (01: means for nine determinations. Continuous line: estimated mean red mechanism curve. (B) The correspondence of the three sets of spectral sensitivity values to the estimated mean curve (blue-green background). (C) Comparison of estimated mean red mechanism curve from (A) above (continuous line) to difference spectrum of goldfish 625 nm max. cone pigment ibroken line) and 625 nm max., retinene-2 pigment nomogram of Ebrey
Table
Mean
and Honig
(dotted
line).
same scale, and are therefore presented in Table 2. The standard errors shown in Column 2. Table 3, are unadjusted. and hence include variability resulting from differences in absolute sensitivity among the fish. To provide a better measure of the variability of points about the estimated mean curve after discounting differences in absolute sensitivity, an adjusted standard error was calculated in the following man-
The standard errors associated with the means in Fig. ?A were too small to represent graphically on the
Fig. ?A. n = 3 Adjusted Mean SE SE
500 WAVELENGTH
Fig. I. Log relative quanta1 energy of colored adapting backgrounds: (A) blue-green background; (B) top line: blue-green plus I.2 log ft-L tungsten-white; bottom line: I.2 log ft-L tungsten-white alone: (C) yellow background; (D) red-blue background.
i
1-I
t
D
O('
400
1297
ner. The estimated mean curve taken from Fig. ZA was fit to each of the three sets of data points making up the means. Then the three individual curves so
2. Means and standard
Fig. 3. n = 3 Adjusted SE SE
errors
Fig. 4. )I = 3 Adjusted Mean SE SE
Fig. 6. ,I = 4 Adjusted Mean SE SE
400 420 440 460 480
I.25 I.10 I.12 0.85 0.76
0.15 0.07 0.07 0.06 0. I 6
0.14 0.06 0.09 0.06 0.16
I.28 I.16 1.17 0.65 0.97
0.26 0.17 0.26 0.11 0.18
0.11 0.06 0.13 0.07 0.07
3.73 2.90 2.82 2.89 2.87
0.37 .0.07 0. I 5 0.20 0.08
0.45 0.70 0.03 0.08 0.10
1.75 1.80 I.92 2.00 2.08
0. I 1 0.06 0.00 0.1 I 0.06
0.1 I 0.03 0.03 0.09 0.07
500 520 S-IO 560 580
0.69 0.87 1.26 I.30 1.55
0.13 0.14 0. I 4 0.05 0.06
0.13 0.1-t 0. I 3 0.07 0.06
0.98 I.01 1.14 I.10 I .42
0.07 0.04 0.09 0.08 0.17
0.07 0.13 0. I 3 0.07 0.05
2.5 I 2.10 1.83 I .30 I.41
0.07 0.10 0.07 0. I 2 0.1 I
0.06 0.09 0.06 0.16 0.08
2.36 2.29 2.47 2.13 1.29
0.00 0.09 0.08 0.10 0.06
0.03 0.07 0.05 0.08 0.08
600 610 640 660 680
I.50 I .76 1.69 I .63 1.58
0.1 I 0.06 0.09 0.06 0.06
0.1 I 0.06 0.08 0.07 0.05
I .43 I .66 I .J3 I.55 I .49
0.06 0.15 0.15 0.14 0.14
a.08 0.04 0.01 0.09 0.08
I.14 I.19 I.41 I .43 I.31
0.1 I 0. I 2 0.06 0. I 2 0.10
0.06 0.02 0.10 0.02 0. I 5
I .96 I .63 I.22 I.24 1.17
0.11 0.08 0.09 0.1 I 0.08
0.10 0.10 0.08 0.08 0.05
700 720 740
1.06 0.75 0.74
0.10 0.06 0.34
0.09 0.06 0.32
0.9 I I.00 0.51
0.15 0.15 0.10
0.04 0.09 0.17
0.95 0.53 0.18
0.10 0.28 0.1 I
0.05 0.16 0.08
0.84 0.80 0.37
9.15 0.07 0.08
0.13 0.05 0.09
R. D.
I198 2
2 0 ’
I
1 1 1 1 I
1 1 1 1 1 1 1 1 1 1 1 1 .
-Li s ; 3 5
J. S. Row
B~~ICHMP.
A/
: c) &gy;,(;
,,*a**:--T&A
_
‘$ ?? ‘0
I>. _ ‘I ,““““1”‘1”‘1’ 430
500 WAVELENGTH
700
600 :nm)
Fiu-_.-. : Location oi long-ware peak: Dotted hnc: at 660 nm using a 1.2 log ft-L tungsten-uhite background. Solid line: at 6!0nm using :I I.1 log ft-L tungsten-Nhitc plus a bluegccn background. Solid triangles (AI: means determinations. Continuous line: cur\e from Dotted line: curIt from Beauchamp and Ro~e 19771
for
three
Fig. ?A. (Frg
-IA.
established were brought to the same position on the log relative sensitivity scale by adding an appropriate correction to each data point. With the three curves now superimposed, the variability about the mean curve of the adjusted points was calculated. These values. shown in Column 3. Table 2. differ only a little. in the case of the blue-green background experiments. from the unadjusted standard errors shown in Column 7, because the three fish contributing to the mean had almost identical absolute sensitivities. However. in later experiments to be described in this paper. subjects often differed in absolute sensitivity. and the adjusted standard error. calculated in the same way as described above. may more faithfully represent the fit to the estimated mean curve. As previously noted. the three characteristics of the mean red mechanism curve shown in Fig. 2A were descriptive of the curves from the three component curves: all had a peak at about 630nm. a trough at about 5OOnm and a rising sensitivity at wavelengths shorter than 500nm (Fig. 2B). However. the position of the trough varied somewhat for the three fish from 480 to 520nm. and the steepness of the rise in sensitivity was also distinctive. The largest rise in sensitivity from the trough to the position at 100nm was 1.09 log unit, while the smallest rise was 0.38 log unit. .A comparison of the estimated mean curve shown in Fig. 2A to two curves of interest is seen in Fig. 2C. The mean curve had the same wavelength of peak sensitivity at about 630nm as does the difference spectrum for the goldfish red-absorbing cone pigment (Harosi and MacNichol, 197-I). Hovvever. it was wider than the pigment difference spectrum on the right shoulder of the curve, and narrower on the left shoulder. A better fit on the right shoulder is provided by the pigment nomogram for a retinene-2 pigment peaking at 630 nm (Ebrey and Honig. 1977). but the left shoulder discrepancy remains. hUr1011
(J/ ~Oll~/-~Ur'e pdS
In a recent and closely-related publication (Beauchamp and Rowe. 1977). the mean spectral sensitivity curve for seven fish. measured against a I.2 log ft-L tungsten-white background. was characterized by a long-wave component which peaked at 660nm (redrawn in Fig. 3. broken line). The 660nm location of the peak is of interest
and L. 4. O‘REILL~
because it is remote irom the 62 nm position prcdieted from the absorption spectrum of the redabsorbing cones (Marks. 1965: H~osi and LiacS~chol. 197-t). A likely explanation for the longwave shift of the peak depends on the antagonistic action of green mechanism activity. aroused strongly by wavelengths close to 520 nm and progressively less so ;tt longer wavelengths. At the ganglion cell level in goldfish. such antagonistic green mechanism activity csn lower the sensitivity for responses originating in rsdsensitive cones (Wagner c’t trl.. 1960: Davv. 196s: Beauchamp and Lovasik. 19731. To test the antagonistic action explanation of the 660 nm peak Iocation. the spectral sensitivity curve against a 1.3 log ft-L tungsten-white background. 3s in the previous work. was msllsured once again. but this time a bright blue-green background was superimposed on the white in an attempt to depress green mechanism antagonism. The blue-green background used was the same as that described in the previous section of this paper. The spectral composition of the tungsten-whtte background and the composite tungsten-white with superimposed blue-green background are shown in Fig. IB. Note that the composite w hits i blue-green background has a higher quanta1 energy than the white alone only below 560nm and hence will lower the sensitivities of green and blue cone mechanisms. while influencing the red mechanism sensitivity verv little. The solid symbols in Fig. 3 Indicate the mean sensetivities for three fish when the white + blue-green background was used. The peak now falls at 630nm instead of at 660 nm. The points above 540 nm fall on ths estimated mean curve for the red mechanism taken from Fig. ?A. These results point to green mechanism antagonism as the c;1use of the shifted 660nm location of peak sensitivity when a tungstenwhite background was used alone. Another feature of interest in the composite white + blue-green curve of Fig. 3 was the absence of a clearly defined trough in sensitivity at 500 nm like that seen for the blue-green condition in Fig. 2A. The absence of the trough was associated with the add!tion of the I.3 log ft-L tungsten-white background which was the only difference between the two stimulus conditions. A comparison of the absolute sensttivities for the two mean curves in Fig. 2A and Fig. ? revealed that the latter was 0.4 log units less sensitive. As one might have predicted. the tungsten-vvhitt background, with a preponderance of long-wave energy (Beauchamp and Rowe, 1977). has acted to lower the red mechanism sensttivitv. This suggests that the high points at GO. 500 and ;lOnm in Fig. 3. obscuring the trough. may result irom green mechanism activity which vvas not present when the red mechanism was more sensitive. as was the case in the conditions of Fig. ?A-\. The hiue wYh7,1;s/tr: ,w/lo~r~ huc/qrorri!tl The relative sensitivtty of the blue mechanism was enhanced using a bright yellow adapting background. The yellow background resulted from a 500 nm longwave pass cutoff filter, and a 550 nm long-wave pass cutoff filter, placed in a 2.14 log it-L tungsten-white beam. The log relative quanta1 spectral energy distribution of the background is shown in Fig. 1C.
Goldfish
spectral sensitivity reduced
0.50 log tion. We absolute and the
400
500 WAVELENGTH
700
600 (nml
Fig. 4. Blue mechanism response (yellow background): Solid triangles (A): means for three determinations. Shortwave curve: difference spectrum for goldfish 455 nm max. cone pizsent. Long-wave curve: taken from Fig. 2A. The mean sensitivities for three fish using the yellow background are presented in Fig. 4. The blue mechanism component of the curve under these conditions was about 1.5 log unit more sensitive than the red mechanism component. Between 420 and 520 nm, the mean sensitivities were well-described by the different spectrum for the blue-absorbing cone pigment (Harosi and 1MacNichol. 1974). At 540 and 560 nm the sensitivity was somewhat higher than predicted from the pigment absorption curve, which may indicate the intrusion of some green mechanism activity. Above 560nm. the means fall on the red mechanism curve taken from Fig. 2A. Of interest in Fig. 4 was the high sensitivity at 400 nm, also noted when using a 1.2 log ft-L tungstenwhite background (Beauchamp and Rowe. 1977). However. the aberrance was markedly greater against the yellow background than against the white. For the yellow background. the mean 400nm sensitivity was 1.13 log unit above that.predicted from the pigment absorption curve, while against the white background the mean aberrance was 0.53 log unit. The fact that aberrant high blue sensitivity at 400 nm persists. and was in fact greater. when the red and green mechanism sensitivities were depressed by means of a yellow background, makes it difficult to attribute the aberrance to activity originating in red or’ greensensitive cones. For example, the high point at 400 nm cannot be caused by beta band absorption by the red-sensitive pigment. A feature of the high aberrance at 400nm was the great range of values noted in individual goldfish. Figure 5A illustrates the range observed for the three fish tested with a yellow background: the smallest aberrance was 0.35 log unit, while the largest was 1.95 log unit. An aberrance as high as 2.70 log units was noted in an early experiment (Beauchamp. 1978: Beauchamp and Rowe, unpublished). Where the 400 nm point was very high. the 420 and 440 nm sensitivities sometimes fell above the pigment absorption curve also. When a white background was used, the aberrance was smaller and restricted to the 400nm response (Fig. 5B). Addimg a I.2 log ft-L tungstenwhite background on top of the yellow background
1299
high aberrance at 400nm to values about unit typical of the white background condifound no significant correlation between the sensitivity of the blue mechanism response degree of 100 nm aberrance.
The green mechanism was enhanced in sensitivity relative to the red and blue mechanisms by adapting with two superimposed colored backgrounds. In one background channel, a blue background was formed by placing a 450 nm short-wave pass cutoff filter in a 2.15 log ft-L tungsten-white beam. In a second background channel, a red background was produced by placing a 650 nm long-wave pass cutoff filter in a 2.15 log ft-L tungsten-white beam. The two backgrounds superimposed on the back projection screen gave a cyan-appearing background, the spectral composition of which is shown in Fig. ID. From 400 to 640nm. the mean sensitivities obtained with a red-blue background were well described by the Ebrey and Honig (1977) nomogram for a retinene-2 pigment with peak sensitivity at 53Onm (Fig. 6). The difference spectrum for the green-absorbing pigment also provided a satisfactory tit, although it was a little wide on the right shoulder. Above 660nm the points fell on the red mechanism curve taken from Fig. 2A. An interpretive problem arises when using spectral sensitivity curves in goldfish to identify green mechanism activity. because goldfish rods have a peak sensitivity at 522 nm. similar to that of the green-sensitive cones at 530 nm. Our evidence that the activity shown in Fig. 6 originates in green-sensitive cones. and not rods, comes from parallel neurophysiological work. When recording from optic nerve fibers. the rod and green-sensitive cone responses can be differentiated by the sign of the response, excitatory or inhibitory
I
’
’
’
’
I
’
’
0400
500
WAVELENGTH
Fig. 5. Variability in aberrant high sensitivity at 400 and 420 nm: (A) against yellow background: (B) against I.2 log h-L tungsten-white background. Curves: difference spectrum for goldfish 455 nm max. cone pigment.
R. D. BE.~LCH~UP. J. S.
1300
Rour
and L. A. O‘RHLLI
the curve and wider than it on the right shoulder. The narrowing on the left shoulder may result from residual ant~~o~isrn from green-sensitive shoulder of
cones. The close proximity
400
600
500 WAVELENGTH
700 inm;
Fig. 6. Green mechanism response (red-blue background). Filled triangles (A): means for four deterrni~~t~on~. Open circles (01: values for fish with 0.5 log ft-L tungsten-white added to red-blur background. Midwave curve: Ebrey and Honig retincne-:! 530 nm max. nomogram. Long-wave curve taken from Fig. ?A.
(Ra~nauld.
1972; Beauchamp
and Daw,
1972). Using
similar red-blue adapting conditions in the optic fiber work, the activity of green-sensitive cones. and not rods. was observed (Beauchamp and Lovasik. 1973). There is no reason to believe that, with the same stimulus conditions. the psychophysical response does not also originate with green-sensitive cones. As a precaution, one of the four fish tested with the redblue background had, in addition, a third background superimposed on the others, a 0.5 log ft-L tungstenwhite background. This ensured that photopic conditions prevailed. The results, similar to those for the other three fish, are shown with open symbols in Fig. 6. A final point of interest in Fig. 6 is the absence for this condition of noteworthy high aberrant sensitivity at the blue end of the spectrum. Seemingly, blue backgrounds or white backgrounds containing a shortwave component seem to act to reduce high blue aberrance. DiSCUSSlON
Three cone mechanisms were characterized psychophysically from spectral sensitivity curves measured against colored adapting backgrounds. A long-wave peak at 630nm was obtained when using a btuegreen background. a mid-wave peak at 530 nm when uing a red-blue background, and a short-wave peak at 46Onm with a yellow background. Since these peaks were in the same iocation as those of the difference spectra of the three goldfish cone photopigments (‘Marks. 1965: Harosi and MacNichol. 1974). an origin in red-, green- and blue-sensitive goldfish cones (Stell and Harosi. 1976: Marc and Sperling. 1976) was indicated. The shape of the short-wave peak matched that of the corresponding cone absorption curve. except at 4OOnm. the shortest wavelength tested, where the sensitivity was higher than predicted from the absorption curve. The mid-wave peak was only slightly wider than the corresponding pigment curve on the right shoulder. and the nomogram of Ebrey and Honig (1977) also provided a satisfactory fit. The long-wave peak, however, was markedly narrower than the pigment absorption curve on the left
of the green-sensitive
ac-
cessory member of the goldfish double cone to the red-sensitive principal member tStel1 and Harosi. 1976: >farc and Spetling. 1976) may provide a particularly etficient inhibitory pathway, the influence of which persists even when green-sensitive cone sensitivity is reduced with a blue-green background. The widening on the right shoulder of the long-wave peak compared to the pigment ditTerence spectrum has no ready explanation. Bowmaker { 1975) has demonstrated that the absorbance of visual pigments in the intact retina is greater at longer wavelengths than the absorbance of the pigments in solution. Perhaps. in a similar fashion the spectral sensitivity of the goldfish. mediated by the intact retina and measured psychophysically. is higher at long wavelengths than predicted from the difference spectra of single photoreceptors measured microsp~trophotom~tr~call~. Somewhat enigmatically. the retinene-2 nomogram of Ebrey and Honig (1977) provided a much better fit on the right shoulder of the long-wave peak than did the emptrtcafiy determined pigment difference spectrum. An alternative point of view is provided if one interprets the discontinuity evident in fig 7A at 7M)nm as a shoulder to the long:wave peak. Then, one might be reasonably satisfied with the fit of the difference spectra for pomts below 7OOnm, and for the shoulder of the curve above 700 nm, another spectral mechanism, as yet unspecified, coufd be proposed. Such a hypothetical spectral mechanism, for which there is little evidence at present, might relate to the puzzling longwave sensitivity encountered by Naka and Rushton (1966). Red-yrrm
mrchunisni
inreracrions
Against a I.2 tog ft-L tungsten-white background. the goldfish increment threshold curve was charactetized by a prominent long-wave peak at 66Onm (Beauchamp and Rowe, 1977). When a blue-green background was added to the tungsten-white, the long-wave peak became wider and shifted.to 630 nm. This result suggests that the 660 nm location with the tungsten-white alone was a result of green-sensitive cone inhibition on red-sensitive cones. When green mechanism sensitivity was reduced by adding the blue-green background, the green mechanism inhibitory influence was diminished, and the long-wave peak more nearly corresponded with the photopigment absorption spectrum. This explanation is consistent with our understanding of red-green interactions measured at the ganglion cell level in goldfish iWagner et al., 1960; Daw, 1968; Beauchamp and Lovasik, 1973), and is similar to ef%cts demonstrated in rhesus monkey (Sperling and Harwerth, 1971). .ihsertce of blur-yreen umqotlisric itueruction Beauchamp and Lovasik (I 973) reported that optic fiber responses originating in blue-sensitive cones had the same sign, inhibitory or excitatory, as responses initiated by green-sensitive cones. The response at a given wavelength seemed to be that of the most sensitive cone mechanism. blue or green. with no evidence of addition in sensitivity of blue and green mechan-
Goldfish
spectral
isms. The psychophy,sical results presented in this paper confirm this view: both the short- and midwave curves are well fit by their respective pigment difference spectra. There is no evidence of a narrowing on the right shoulder of the short-wave curve. or of a narrowing on the left shoulder of the mid-wave curve, both of which would have been predicted had blue and green mechanisms been mutually antagonistic. Nor is there a marked widening of the short- and mid-wave peaks which summation of blue and greensensitive cone activity should have caused. Apparently the blue-sensitive cones, unlike the red- and greensensitive cones, function independently or nearly so. Similarly. in rhesus monkey, Sperling and Harwerth (1971) found the blue-sensitive c3nes acted independently. This work. as was earlier neurophysiological work (Beauchamp and Lovasik, 1973). is inconsistent with the report of Spekreijse et al. (1972) that blue mechanism activity is of opposite sign to that of green mechanism activity. The results of Spekreijse er al. (1972). however, might be a consequence of’ attributing to blue-sensitive cones responses which in fact resulted from beta band stimulation of red-sensitive cones. As the discussion will elaborate below, a prominent short-wave peak caused by absorption in the beta band of the red-sensitive pigment is noted when bluesensitive cone sensitivity is reduced. Beta band actioity
When using a blue-green adapting background, a rising sensitivity below 460 nm was noted. Because of the background used, which would act t’o reduce blue and green mechanism sensitivity. this short-wave peak was unlikely to be due to blue- or green-sensitive cone activity. The peak was reminiscent of the beta band peaks noted for similar background conditions in optic fiber work (Beauchamp and Lovasik, 1973). Subsequent experiments (Beauchamp and O’Reilly, in preparation) have confirmed that the short-wave peak seen with a blue-green adapting background is a result of beta band absorption by the red-sensitive pigment. Changes in the intensity of a blue adapting background influenced the beta band short-wave sensitivity very little, while alterations in the intensity of a red background moved the long-wave peak and the beta band short-wave peak up and down in synchrony. The beta band of the goldfish red-sensitive pigment is particularly prominent because of the longwave location at 625 nm of the retinene-2 alpha peak. However, even in primates, where the retinene-1 peaks are located at shorter wavelengths. shorbwave responses originating in beta band absorption by the red-sensitive pigment have recently been reported (Monasterio and Gouras, 1977). This may explain the purple, or red-blue, appearance sometimes reported by humans for short-wave stimuli. High aberrant
blue sensitivity
A short-wave peak of a different origin is observed when employing a yellow background. The high short-wave sensitivity seen at 4OOnm and sometimes 420 and 440 nm as well, cannot be a result of activity originating in red- or green-sensitive cones because these are depressed by the yellow background. Moreover, the magnitude of the aberrance at 4OOnm is
sensiti\iq
1301
dependent on the energy of the background at the blue end of the spectrum. When blue light is altogether absent from the background, as in the yellow background condition. the aberrance is at its highest. When there is an intermediate level of energy at the blue end. as in the tungsten-white 1.2 log ft-L condition, the aberrance is small. For a bright blue background condition. as in the red-blue adapting background experiments. there is no evidence of aberrant high blue sensitivity whatsoever. These findings have led us to hypothesize that absorption of scattered light by blue-sensitive cones or rods are responsible for the high blue aberrant sensitivity. The photoreceptors involved are likely those in the 40’ ring of the retina in the far periphery not adapted by our 100” background. For yellow and red backgrounds. little ‘blue light is available to scatter into the far periphery. The blue-sensitive cones or rods will be relatively dark-adapted and sensitive to any scattered light from the stimulus. In blue-green or white background conditions where there is blue light in the background, this will scatter into the far periphery and light-adapt the photoreceptors there. For these backgrounds there will be reduced sensitivity to scattered light from spot stimuli. Thus. the spectral composition of the background will determine the sensitivity to scattered light of far peripheral photoreceptors. Whether or not there is scattered stimulus light to activate the far peripheral receptors will depend on the wavelength of the spot stimulus. The intensity of scattering in an optical medium containing small particles is inversely proportional to the fourth power of the wavelength of the light. Hence the greatest amount. of scatter will arise from the 4OOnm spot stimulus, much less from the 420 nm stimulus and still less for stimulus spots with wavelengths at 31Onm and above. This accounts for the fact that the greatest high sensitivity aberrance occurred for the 400 nm stimulus, with less aberrance for the 420 nm stimulus and little or none for 440 nm stimuii and above. Our data thus far are consistent with the scatter hypothesis, but we have not yet attempted to measure the proposed scattered light directly. Also. we have not eliminated the possibility that fluorescence stimulated by 4OOnm light (rather than scatter of the 4OOnm light) is the source of far peripheral retinal stimul8tion. An interesting aspect of the high aberrant sensitivity at short wavelengths is the great variability noted amongst individual fish. We are not sure if this results from variability in our location of the eye in experiments (scatter off the optic disc is a possibility). or from differences in the optical system of the fish (opacities in the optic media or the size of the lens, for example). or from some other cause. Shape of the spectral sensiticity
curce
An integrating principle emphasized by this work is that the shape of the spectral sensitivity curve is determined by the balance of sensitivity existing among the three cone mechanisms. As proposed in the yupper envelope model” of Stiles (1959) and Sperling and Harwerth (I 97 I ). threshold at a given wavelength was determined by the most sensitive of the three cone mechanisms operating at that wavelength. At the short-wave end of the spectrum, for example. the most
R. D. BULCHUIP.
1302
1. S. Rows
sensitive cone mechanism normally was the blue mechanism, acting free of interactions with the other two cone mechanisms. However. a red-blue background favoured a green mechanism response and a blue-green background favoured a response originating in beta band absorption by the red mechanism. At the long-wave end of the spectrum. threshold was influenced by antagonistic interaction between green and red cone mechanisms; with an excitatory input from one cone mechanism, red or green, and an inhibitory input from the other. Thus the linear subtractive process between red- and green-sensitive cone mechanisms described by Sperling and Harwerth (1971) to account for rhesus monkey long-wave spectral sensitivity, is applicable to goldfish spectral sensitivity data as well. Again, the composition of chromatic adapting backgrounds was important in the case of long-wave sensitivity, determining the relative strength of the inhibitory and excitatory influence operating at each wavelength. Hence, as others have demonstrated in the past (Yager. 1969: Yager. 1974: Muntz and Northmore. 1971). the spectra) energy distribution of adapting backgrounds may strongly influence the shape of spectral sensitivity curves. In fact, there will be a unique spectral sensitivity curve ior each chromatic background. of
the
three
Nonetheless, cone
all curves
mechanism
will
sensitivity
be composed curves
de-
in this paper; the short-wave mechanism apparently independent of the other two, and the mid- and long-wave mechanisms mutually antagonistic. The parallels of these results with those of Sperling and Harwerth (1971) working with rhesus monkey are striking. scribed
Acknorr,ledgemmrs-This work was supported by funds from NRC (Canada) and MRC (Canada). Thanks to Olga Vrablic and Sherry1 DiCiccio for technical assistance.
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and L. r\. O‘REILL~
Beauchamp R. D. and Rowe J. S. (IY’7) Goldfish spectral sensitivity: il conditioned heart rate measure in restrained or curarized fish. Vision Rzr. 17. 617-621. Bowmakcr J. K. (1975) Spectral sensitivity and visual pigment absorbance. b’isioti Rtis. 13. 783-792. Daw N. W. (1968) Color-coded gang!ion cells in the goldfish re!ina: extension of their receptive Relds by means of new stimuli. J. Physiol. 197. 567-592. Ebrey T. G. and Honig B. (1977) New wavelength &pendent visual pigment nomograms. t’ision R~s. 17. l-17-151. Harosi F. 1. and MacNichol E. F. (I9741Visual pigments oi goldfish cones. J. yen Physiol. 63, 219-303. Liebman P. .A. and Entine G. (19631 Sensitive low-lightlevel microspectrophotometer: detection of photosensitive pigments of retinal cones. J. opr. Sot. .Im. 54. l-tjl-l-t.i9. Marc R. E. and Sperlmg H. G. ( 1976) Color receptor identities of goldfish cones. Scinicr 191. Zt7--189. Marks W. G. (1965) Visual pigments of single goldfish cones. J. PlrJsiol. 178. l-I-32. hfonasterio F. &I. de and Gouras P. (1977) Responses of macaque ganglion cells to far violet lights. Visiotl Rex 17. 1117-l 156. Muntz W. R. A. and Northmore D. P. M. (1970) Vision and visual pigments in a fish Scurdiriiu.s rr~rhrophrl~u/~~~u.s (the ruddl. C’ision Rex 10. 281-298. .Muntz W. R. A. and Northmore D. P. LI. (1971) The independence of the photppic receptor slstems underlying visual thresholds in a teleost. Vision R?s. 11. 861-876. Naka K. I. and Rushton W. .A. H. (19661 S-potentials from colour units in the retina of fish (CJprrnidm). J. Plrysiol.
185. 53&SSj. Raynauld J. P. (1972) Goldfish retina: sign of the rod input in opponent color ganglion cells. Scirnce 177. S&85. Spekreijse H.. Wagner H. G. and Wolbarsht M. L. 11972) Spectral and spatial coding of ganglion cell responses in goldfish retina. J. Neurophysiol. 35. 75-86. Sperling H. G. and Harwerth R. S. (1971) Red-green cone interactions in the increment-threshold spectral sensitivity oi primates. Science 172. 18&l 81. Stell W. R. and Harosi F. 1. (1976) Cone structure and visual pigment content in the retina of the goldfish.
Vision Res. 16. 647-657. Stiles W. S. (1959) Color vision: the approach through increment-threshold sensitivity Proc. mm. Acad. Sci. U.S.A. 45. IWI 14. Wagner H. G.. MacNichol E. F. Jr and Wolbarsht M. L. (1960) The response properties of single ganglion cells in the goldfish retina. J. yen. Physiol. l8 (Suppl.). J-562. Yager D. (1967) Behavioral measures and theoretical analysis of spectral sensitivity and spectral saturation in the goldfish Carussius uuratus. Vision Rrs. 7. 707-727. Yager D. (1969) Behavioral measures of spectral sensitivity in the goldfish following chromatic adaptation. Vision Rex 9. 179-l 86. Yager D. (197-t) Effects of chromatic adaptation on saturation discrimination in goldfish. Vision Res. 14. 1089 IC9-l.