A COMPARATIVE STUDY OF SPECTRAL SENSITIVITY I N TADPOLES AND

ADULT FROGS1

DONALD KENNEDY *-' The Biological Laboratories, Harvard University

THREE FIQURES

The role of photosensitive pigments in the visual process has been demonstrated most satisfactorily by experiments in which sensitivity (reciprocal of threshold) is measured as a function of wavelength. The spectral sensitivity curves expressing these data are then compared with the absorption spectra of extracted photopigments ; an agreement as to shape and position of the maximum constitutes strong evidence implicating the pigment in primary visual events. In the hu- man eye, for example, the peripheral sensitivity (dark adapted) matches well the absorption spectrum of the rod pigment rhodopsin, both having maxima at about 500m1.1 (Stiles and Smith, '44).

In the frog, Wald ('46) has demonstrated an interesting biochemical metamorphosis involving the visual pigments. The tadpole eye contains Vitamin A, and porphyropsin (ab- sorption maximum 522 Imp), a rod pigment with its carotenoid chromophore derived from Vitamin A, ; after metamorphosis, the frog - like other terrestrial vertebrates - is found to have Vitamin A, and rhodopsin. This situation thus presents an unparalleled opportunity to study both visual systems in the same animal species.

%Part of a thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biology, Harvard University.

Pre-doctoral research fellow of the National Science Foundation. Present address: Department of Zoology, Syracuse University, Syracuse-

New Pork. 155

156 DONALD KENNEDY

I n the present experiments, the electroretinogram was used to determine the spectral sensitivity of tadpoles and frogs. The electroretinogram is a slow potential which can be re- corded from the cornea of the eye; it represents the massed discharge of a number of retinal units whose identity is un- certain. Nevertheless, it has proven a satisfactory index of sensitivity if properly employed. Early work using this technique on the frog by Piper ( '11) and Chaffee and Hampson ('24) suggested the involvement of rhodopsin in rod sen- sitivity, but the state of adaptation was not kept constant. Granit and Munsterhjelm ( '37) and Granit and Wrede ('37) determined spectral sensitivity in dark and light adapted frogs respectively. These curves clearly demonstrate the Purkinje phenomenon, and give a good idea of the location of sensitivity maxima. Unfortunately, however, these authors presented the animals with a series of wavelengths of equal energy content, and then plotted the height of the response as a function of wavelength. It has been pointed out by Hecht and Pirenne ('40) that such curves do not actually represent the absorption spectra of visual pigments. The method as- sumes a linear relationship between stimulus intensity and the amplitude of the physiological response ; but the relationship is in fact mot linear, but sigmoid.

A second objective of the experiment reported here was to compare the photopic sensitivity of tadpoles and frogs with the existing chemical data on cone pigments. Wald, Brown and Smith ('55) have extracted iodopsin from the cones of the chicken retina ; this pigment is based upon the same carotenoid as that of rhodopsin, but it is combined with a different pro- tein. Although iodopsin is apparently only one of several cone pigments in the human retina (Wald, '45), it is not known whether one or several pigments determine cone sensitivity in other animals. Wald, Brown and Smith ('53) were also able to synthesize a second cone pigment, called cyanopsin, by combining the carotenoid derived from Vitamin A, with the protein from chicken cones. This pigment, which absorbs maximally at 620 mv, has never been extracted from a living

FRO0 AND TADPOLE SPECTRA 157

animal; but there is good reason to expect that it may be the cone pigment of animals which possess the Vitamin A2- porphyropsin visual system. Careful photopic sensitivity measurements were made on frogs and tadpoles in an attempt to provide answers to these questions. A preliminary report of some of the results has appeared elsewhere (Kennedy, '55).

METHODS

The frogs (Rarca pipiem) used in these experiments were injected with 2 cc of a solution of d-tubocurarine chloride (0.15 mg/cc). This caused paralysis but did not affect the retinal response. Stable electrical responses could be obtained from such preparations for as long as 24 hours. Tadpoles (same species) were anaesthetized with 3% Urethane.

The electroretinogram was recorded through cotton wick electrodes, one placed on the cornea and the second (an in- different electrode, grounded through the pre-amplifier) on the skin surface. The wicks connected through Ringer-filled pipettes to silver chloride electrodes. A capacity-coupled Grass P-4 preamplifier and oscilloscope were used.

The light source was a 300-watt tungsten filament lamp, operated with a voltage stabilizer. The intensity of the stimu- lus was varied with a pair of opposed annular wedges, built in the Jefferson Physics Laboratory of Harvard University and loaned through the courtesy of Professor George Wald. These provided an intensity range of six logarithmic units.

Monochromatic light was produced by the use of narrow- band interference filters (Baird Associates). These were com- bined with Corning and Wratten filters in order to remove unwanted bands of transmission. The transmission of all filter combinations was measured with a Beckman DU spec tropho tome t er.

Collimated light from the source was passed through the filters and a photographic shutter. A pair of 15 em focal length biconvex lenses focused the light, passed it through the wedges, and recollimated it on the other side. The beam was reflected

158 DONALD EENNEDY

downwards by a !mirror, and a third short-focus lens produced a spot of homogeneous intensity which filled the pupil of the preparation.

The optical system was calibrated by placing a thermopile in the position of the eye, and recording its output with a sensi- tive galvanometer. Measurements of intensity were made at a number of wedge settings for each wavelength used. At low intensities, below the sensitivity range of the thermopile, relative energies were measured with a photocell, amplifier and oscilloscope.

I n measuring spectral sensitivity, the large, cornea-positive potential of the electroretinogram (the b-wave) was used as an index of sensitivity. A certain b-wave amplitude was arbitrarily selected as a “criterion response’’ ; the stimulus intensity just necessary to elicit a b-wave of this amplitude was then found at each wavelength, and the reciprocal of that intensity value was plotted against the wavelength. In de- termining scotopic sensitivity, thoroughly dark adapted ani- mals were used, and intensities were kept low. Stimulus duration was 1/50 sec., and at least 30 seconds were allowed to intervene between stimuli. I n measurements of photopic sensitivity, animals were light adapted before the experiment. The response at some wavelength was then measured re- peatedly until sensitivity became constant. Determinations of sensitivity at each wavelength were then made while the eye was on the “cone plateau” of dark adaptation. I n all cases, determinations of sensitivity were made at one wavelength before and after the experiment to insure that sensitivity had remained constant during the period of measurement.

I n general, two determinations of spectral sensitivity were made on each animal, reversing the order of wavelength pre- sentation for the second experiment. An average of these two determinations was then used as the spectral sensitivity curve for that animal.

I n some experiments, animals were presented with eqoal- energy spectra, and the responses at each wavelength recorded

FROG AND TADPOLE SPECTRA 159

on film. This method is useful to verify the position of maxi- mum sensitivity, but does not yield a function comparable to actual spectral sensitivity curves.

RESULTS

Average spectral sensitivity curves for light and dark adapted frogs are shown in figure 1. In each case, these rep-

RELATIVE SENSITIVITY _.

0

400 500 6 00 70 0

W A V E L E N G T H - M).l

Fig. 1 Spectral sensitivity curves for dark and light adapted frogs (dotted lines), compared with Wald's absorption spectra for frog rhodopsin and chicken iodopsin (solid lines). Each curve is an average of data from six animals; standard error is indicated by vertical lines through the poinh.

resent the average of determinations on 6 animals; cht: standard error of each point is plotted. Absorption spectra of frog rhodopsin and of chicken iodopsin, both from the data of Wald, are included for comparison. (Wald, '55 ; Wald, Brown and Smith, '55).

160 DONALD KENNEDY

RESPONSE

(W

40 -

A 0-

"g LIGHT-ADAPTED FROG

EQUAL- ENERQY SPECTRUM

I I J 8 400 500 SO0 700

WAVELENGTH - M/L

Fig. 2 Responses of the frog's eye to stimulation at equal energies. A. Dark adapted; B. Light adapted. Stimulus duration 1/25 second; stimulus intensity in B 100 times that in A.

RELATIVE

S E N S l T l V l T

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I

10

RELATIVE S E N S l l l V l l

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5

I

4 DARK - ADAPTED

I I

I 0 I I I I I s

I

I I 1 500 600 700

WAVELENGTH - MP

4 LIGHT-ADAPTEO 1 TADPOLES - -/--

I I I 400 500 €00 700

0 WAVELENGTH - M j l

Fig. 3 Spcctral sensitivity curves for the tadpolc. A. Dark adapted, eompxretl wit11 Kald ' s absorption sprctrum for porpliyropsin (solid line). H. L ight nduptecl ; c.oiiip:ircd with absorption spectra for eyanopsin : I K ~ iodopsin (Aolid lines).

J l i l

162 DONALD KENNEDY

Agreement appears to be good between rod sensitivity and the absorption of rhodopsin, and between cone sensitivity and iodopsin absorption. The positions of the respective maxima are approximately the same, and the shapes of the curves closely similar. The major discrepancy occurs below 430 mp, where the lens begins to absorb selectively (Milkman and Kennedy, ’55).

The positions of the sensitivity maxima were verified by experiments in which the dark or light adapted animal was presented with an equal-energy spectrum, and the resulting responses at each wavelength photographed at equal ampli- fication. Figure 2 shows the amplitude of such responses plotted against wavelength for dark adapted (2 A) and light adapted (2 B) eyes.

Figure 3 shows spectral sensitivity curves for the tadpole’s eye, determined in the same way as those for the frog in figure 1. In the dark adapted eye, agreement is good with the absorp- tion of the rod pigment porphyropsin, though the high in- tensities necessary to elicit the electroretinogram in these animals apparently brings some photopic units into play, accounting f o r the excessive long-wavelength sensitivity. I n the light adapted tadpole (fig. 3 B), the maximum sensitivity is displaced far into the red-almost, but not quite, to the position of maximum absorption of cyanopsin.

DISCUSSION

These results affirm the agreement of scotopic sensitivity in the frog with the absorption of rhodopsin. Further, the sensitivity distribution of light adapted animals suggests that cone sensitivity is probably determined solely by the cone pigment iodopsin. Granit ( ’41 b), recording from single ganglion cells in the frog retina, found elements which showed narrow-band spectral sensitivity ; he called such elements “modulators. )’ Granit attributed their behavior to the pres- ence of several specific cone types, and felt that the light adapted spectral sensitivity function (the “photopic dom- inator”) represented the integral of this modulator activity.

FROG AND TADPOLE SPECTRA 163

The spectral sensitivity of these modulator elements, how- ever, was derived by procedures involving difference spectra between averaged curves, and selective adaptation. The com- plex, shifting discharge pattern of the retinal ganglion cell is well known (see K d e r , ’53), and it seems quite possible that such effects as those found by Granit could arise through inhibitory interaction between rod and cone pathways. It is doubtful whether the frog possesses a color-discrimination ability (Walls, ’42) ; thus there is no need to postulate such “primaries” to adhere to color vision theory. I n the case of the human eye, where a color discrimination ability obviously exists, spectral sensitivity data clearly show that iodopsin cannot be solely responsible for cone sensitivity. In the frog, however, the close agreement between iodopsin absorption and photopic sensitivity suggests that here one type of cone, with a single pigment, is functioning.

The experiments on tadpole spectral sensitivity support the conclusion, already reached on the basis of pigment extrac- tions, that the larval frog exchanges its porphyropsin for rhodopsin during metamorphosis. I n the course of these experiments, it appeared that the spectral sensitivity did not change to that characteristic of the adult animal until the very end of metamorphosis, when tail resorption had begun. A larger series of ani’mals would be necessary to determine the exact time at which the change occurs.

The results further suggest strongly that cyanopsin is the functional cone pigment of the tadpole. Granit (’41a) has presented evidence from microelectrode studies on the tench and the turtle that these animals, too, possess a strong red sensitivity. In the present data, the discrepancy between the maximum absorption of cyanopsin and the peak sensitivity of the light adapted tadpole’s eye amounts to about 10-15mu (see fig. 3B). The data of Wald reveal that the tadpole eye, in addition to its porphyropsin, contains a small amount of rhodopsin as well, resulting in a slight displacement of the absorption (maximum of rod-pigment extracts to shorter wave-

164 DONALD KENNEDY

lengths than those typical for pure porphyropsin. The dis- crepancy would not be noticeable in the scotopic sensitivity function (fig. 3A), since the shift is only a few mp. The presence of a small amount of iodopsin, however, would cause a marked shift in photopic sensitivity from the position ex- pected on the basis of pure cyanopsin. It is quite likely that this “impurity” of tadpole visual pigments lies behind the observed discrepancy.

SUMMARY

1. The data presented here verify the observation that in the metamorphosis from tadpole to frog, visual pigments be- longing to the Vitamin A, system are replaced by another pair whose carotenoid chromophores are related instead to Vitamin A,.

2. I n the adult frog, photopic sensitivity appears to depend solely upon the cone pigment iodopsin. This finding argues against the hypothesis of Granit that several different cone types combine to produce the photopic spectral sensitivity function.

3. I n the tadpole, photopic sensitivity data support the view that cyanopsin, a red-sensitive pigment heretofore known only from a laboratory synthesis, is actually a functional cone pigment in animals having the Vitamin A, visual system.

LITERATURE CITED

CHAFIEE, E. L., AND A. HAMPSON 1924 Effect of varying the wavelength of the stimulating light upon the electrical response of the retina. J. Opt. SOC. Am., 9: 1.

1941a A relation between rod and cone substances, based on photopic and scotopic spectra of Cyprinus, Tinca, Anguilla a d Testuao. Acta Physiol. Scand., 2 : 334.

1941b Color receptors of the frog’s retina. Acta Physiol. Scmd., 3: 137.

The electrical response of dark adapted frogs’ eyes to monochromatic stimuli. J. Physiol., 88: 436.

1937 The electrical responses of light adapted frogs ’ eyes to monochromatic stimuli. J. Physiol., 89 : 239.

1940 The sensibility of the nocturnal long-eared owl in the spectrum. J. Gen. Physiol., 2.3: 709.

GRANIT, R.

GRANIT, R., AND A. MUNSTERHJELM

GRANIT, R., AND C. M. WREDE

HECHT, S., AND M. H. PIRENNE

1937

FROG A K D TADl’OLE SPECTRA 165

KKSSEDY, D. 1955 Measurements of frog eleetrorotiiiograi1i. Am. J. Physiol., 183: 3.

I(ljFFLER, 8. \v. 1953 1)iSchargC pat terns alid fuiictional organization of maniuialiari retina. J. Neuropliysiol., 16‘ : 37.

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1956

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1963

19i2 T h e Vertebrate Eye and its Adnptive 1hli:ition. Crnnbrook Tiist., I3looiiificlil Hills, hficli. Pp. 490-494.