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Photosensitivity of Primate Photoreceptors JULIE L. SCHNAPF Our ability to discriminate different wavelengths of light depends on the different spectral sensitivities of the three kinds of cones in our reti- nas. These spectral functions have been determined in rods and cones of macaque and human retinas by recording their photocurrents. The spectra obtained in these experiments provide the receptoral basis for human color- matching functions and luminosity functions. Results have been described in detail by Baylor et al. (1984, 1987) and Schnapf et al. (1987, 1988~. METHODS Using a suction pipette, photocurrent was recorded from single rods and cones of the monkey Macaca fasciculans. Psychophysical experiments (DeValois et al., 1974) suggest that this monkey's photoreceptors are similar to those of humans. A limited number of electrical recordings were also made in cones of a single human eye (Schnapf et al., 1987), and the results support that suggestion. Photoreceptors were illuminated by light incident perpendicular to the long axes of their outer segments so that pigment self-screening was neg- ligible. Assuming then that the quantal efficiency of bleaching is constant for the wavelengths used, the spectra obtained here should be proportional to the probability that a single photopigment will absorb light as a function of wavelength. Spectral sensitivity was obtained by the method of criterion response (Naka and Rushton, 1966; Baylor et al., 1984~. Brief flashes of light were applied at a test wavelength and a reference wavelength (500 nm) to determine the relative amount of light at the two wavelengths required to elicit responses of criterion size. The reciprocal of the flash photon densities 31

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32 JULIE Lo SCHNAPF of the two lights gave the relative spectral sensitivity. The responses of the photoreceptors appeared to obey the "principle of spectral univariance," depending only on the number of photons absorbed and not the wavelength (Naka and Rushton, 1966~. Results were obtained from 51 rods and cones in the macaque retina and 6 cones in a human retina. RESULTS Spectral Sensitivity Spectral sensitivities of the macaque photoreceptors are shown in Figure 1. The symbols plot the normalized average values obtained from the blue (o), green Gil, and red cones (o) and the rods (~) of 12 macaque monkeys. The curves near the cone spectra are drawn by eye, while the rod curve is the Dartnall nomogram with a peak sensitivity at 491 nm. The peaks of the cone spectra lie at about 430, 530, and 560 nm. Spectra obtained from human cones are shown in Figure 2. The points plot the average normalized spectra from five red cones (A) and one green cone (B). The smooth curves are the same as those drawn through the corresponding macaque spectra of Figure 1. Spectra obtained from the two species are indistinguishable. The amplitude and waveform of the light responses are also very similar for the two species. The spectra of individual cones varied little for cells within a class. For a given class of cone, the standard deviation of the positions of the spectra along the abscissa was less than 1.5 nm. (Spectral position was estimated by fitting a straight line to the descent of sensitivity at low wavenumber.) This amount of variability is similar to that estimated from human color- matching experiments (MacLeod and Webster, 1983; Neitz and Jacobs, 1986~. When plotted on a normalized wavenumber scale, where wavenumber is divided by the wavenumber of maximum absorption, the absorption spectra of the four macaque photoreceptors have a common shape. The action spectra shown here confirm this observation, although the normalized rod spectrum is slightly broader than that of the cones. It is not known what, if any, physical mechanism might be implied by this scaling principle. It appears, however, that the shape of the normalized spectrum is species specific, as the normalized action spectrum of photoreceptors of the ground squirrel differs significantly from that of the macaque (Kraft, 1988~. Color Matching How can the action spectra of the cones be compared to human color vision? Since the electrical response in a cone outer segment depends

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PHOTOSENSITIVITY OF PRIMATE PHOTORECEPTORS Wavenumber (pm~~) 2.6 2.4 2.2 2.0 1.8 33 1.6 1.4 1.2 1 1 1 1 1 1 1 1 o -1 Log S 2 3 4 - 5 6 _~] ~ 8=~ , Irk ~~ \ 400 500 600 700 800 900 Wavelength (nary) FIGURE 1 Spectral sensitivities of macaque photoreceptor. Points plot the average normalized sensitivities of 5 blue cones (o), 20 green cones God, 16 red cones (o), and 10 rods God, as a function of wavenumber. Wavelength scale above. SOURCE: Schnapf et al. (1988~.

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34 JULIE Lo SCHNAPF A Wavenumber (pm') t.2 1 1 1 1 1 1 1 ~ _ In, RED _~ ~ 1 1 1 1 1 1 1 1 2.6 2.4 2.2 2.0 1.8 1.6 1.4 n 1 Log S 2 3 4 5 B Wavonumber (lim-') 2.6 2.4 2.2 2.0 1.8 1.6 1.4 1.2 1 1 1 1 1 1 1 1 of 400 500 600 700 800 400 Wavelength (nary) ~0 GREEN 1 1 1 1 1 1 1 1 1 1 1 1 1 500 600 700 800 Wavelength tnm) _ FIGURE 2 Spectral sensitivity of human cones. Points plot the average normalized sensitivities of five red cones in A and one green code B as a function of wavenumber, wavelength above. The bars in A are the standard deviations. The curves are the same as those for the corresponding spectra in Figure 1. SOURCE: Schnapf et al. (1987~. Only on the number of photons absorbed and not wavelength, it follows that stimuli comprised of different combinations of wavelengths will be indistinguishable to us if the number of quantal adsorptions evoked in our cones is identical. The intensities of the component wavelengths required to equate quantal adsorptions elicited by physically different stimuli may be calculated from the spectral sensitivity functions of the three cones. The color-matching experiments of Stiles and Burch (1955) were used for the calculations. In these psychophysical experiments, observers ad- justed the intensities of long-, middle-, and short-wavelength "primaries" so that when the three lights were superimposed the stimulus looked iden- tical to a single "test" wavelength of unit intensity. The smooth curves in Figure 3 show the intensities of the primaries (incident on the cornea) plotted as a function of the wavelength of the test light. The symbols indicate the expected color-matching functions calculated from the spectral sensitivity measurements of the macaque cones of Figure 1. The macaque spectra were first adjusted to take into account differences in the way the light was applied in the two kinds of experiments. Specifically, the effects of light absorption by the human lens and macular pigment were taken into account as well as photopigment self-screening within the cone outer segments in the psychophysical experiment. The forms of the preretinal ab- sorption spectra were taken from Wyszecki and Stiles (1982~; self-screening was assumed to follow Beer's Law. The absolute magnitude of the lens,

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PHOTOSENSlT~ITY OF PRIMATE PHOTORECEPTORS 4 3 Relative Intensi ty 2 o 35 o _ ~ N~ t: _ ~ 400 500 600 700 Wavelength (nary) FIGURE 3 Color-matching functions. The smooth curves show the relative amount of light required of the three pnmanes to match a test light of unit intensity as a function of the wavelength of the test light (Stiles and Burch, 1955~. The points plot the predicted results based on the spectral sensitivity functions of the macaque cones in Figure 1 after co~Tectio~i for absorption by the lens and macular pigment and photopigment self-screening. The correction factors used were: macular pigment density at 460 nm, 0.29; lens density at 400 nm, 1.22; and peak axial photopigment density, 0.27. the wavelengths of the pIimanes are 444 (a), 526 (o) and 645 (o) nm. SOURCE: Baylor et al. (1987~. macular, and photopigment densities (given in Figure 3's caption) were chosen to give the best fit between the two sets of data. A good correspon- dence was obtained between the psychophysical and adjusted physiological measurements, supporting the idea that color-matching functions may be derived simply from the absorption characteristics of the cone pigments and that the macaque and human cone pigments are very similar.

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36 JULIE L SCHNAPF Luminosity Another reflection of the spectral sensitivity functions as seen in psy- chophysical experiments is luminosity. Luminosity describes the efficiency of light (incident on the cornea) in stimulating the visual system as a func- tion of wavelength. In light-adapted conditions the photopic luminosity function is thought to reflect a linear sum of the excitation of the red and green cones. Under dark-adapted conditions the scotopic function is thought to depend on the excitation of the rods. These two functions are given by the smooth curves in Figure 4. The continuous line is the photopic function of Vos (1978~. The open circles plot the weighted sum of the "corrected" spectral sensitivities of the red and green cones of the human retina (Figure 2~; the corrections were the same as those applied in Figure 3. The corrected spectra were weighted so that the green spec- trum contributed only about half as much (0.52) to the sum as did the red spectrum. This weighting was chosen to maximize the fit between the calculated and measured functions. The scotopic luminosity function of Crawford (1949) is shown by the dashed curve, and the filled symbols plot the "corrected" spectral sensitivity function of the macaque rods (Figure 1~. The correction factors here (given in Figure 4's caption) were chosen to give the best fit between the points and the curve. A good match was obtained between the two luminosity functions and the corresponding physiological results, consistent with the idea that the scotopic function is set by the filtered rod spectrum and that the photopic function is determined by the summed activity of the red and green cones. The scaling factor (0.52) weighting the contributions of the two cone spectra may reflect the relative numbers of the two cone types in the human eye or the relative strengths of their signals to higher-order neurons. Bleaching Cross Section The bleaching cross-sectional area, A, of a photopigment is a measure of the effective physical area over which a single pigment captures light. For steady light of intensity I, the product lA gives the expected rate of isomerization per pigment. This value has been previously estimated for rhodopsin by measuring, in the presence of steady bright light, the optical density of a dilute solution of rhodopsin as a function of time. The density falls exponential in time as the fraction of unbleached rhodopsin declines; the time constant of decay is (L4~-~. The estimated values of A for rhodopsin (for light of optimal wavelength) were in the range of 7-10 X 1o-~7 cm2 (Dartnall, 1972~.

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PHOTOSENSITIVITY OF PRIMATE PHOTORECEPTORS o 1 Log S l 2 ` - 5 6 37 / / ~',WQ / oW / \ ~ \ ~ \ \ \ \ \ \ 400 500 600 700 800 Wavelength ( rim ) FIGURE 4 Luminosity functions and comparison to rod and cone spectra. The continuous curve is the photopic luminosity function of Vos (1978~; the dashed curve is the scotopic luminosity function of Crawford (1949~. The symbols plot the correlated spectral sensitivities of the macaque rods (~) and the weighted sum of the human red and green cones (o). The cone spectra were corrected as in Figure 3. The corrections for the rods were: lens density at 400 nm, 1.54; and peak axial photopigment density, 0.35. SOURCE: Schnapf et al. (1988~. Similar density measurements for cone pigments are not available because of difficulties in pigment extraction. However, measurements of the photocurrent of single cones appear to provide an alternative method of estimating A. In the presence of steady bleaching light, the photocurrent was found to decline exponentially in time, with a time constant that was inversely related to I. These observations are consistent with the idea that the photocurrent is tracking the fraction of unbleached pigment in the cone outer segment. If so, A may be calculated from the intensity of the light and

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38 JULIE L SCHNAPF the time constant of decay. The mean value of A calculated from several cones was found to lie within the range of that estimated for rhodopsin. ACKNOWLEDGMENTS This work was supported by grant EY05750 from the National Eye Institute and by an award from Research to Prevent Blindness, Inc. REFERENCES Baylor, D.N, BJ. Nunn, and J.L. Schnapf 1984 The photocurrent, noise and spectral sensitivity of rods of the monkey Macaca fasciculans. Joumal of Physiology 357:575 607. 1987 Spectral sensitivity of cones of the monkey Macaca fasciculans Journal of Physiology 390:145-160. Crawford, B.H. 1949 The scotopic visibility function. Proceedings of ~hePhysical Society B62:321- 334. Dartnall, HJ.N 1972 Photosensitivity. Pp. 122-145 in Photochemistry of Vision, HJ.A. Dartnall, ed. New York: Springer-Verlag. DeValois, R., H.C. Morgan, M.C. Polson, OUR. Mead, and ELI. Hull 1974 Psychophysical studies on monkey vision I. Macaque luminosity and color vision tests. Vision Research 14:53 67. Kraft, TW. 1988 Transduction by cones in the golden-mantled ground squirrel, Citellus lateralis. Biophysical Joumal 53:474a. MacLeod, D.I.N, and M.N Webster 1983 Factors influencing the colour matches of normal observed. Pp. Sl-92 in Colour Vision, J.D. Mollon and LT. Sharpe, eds. London: Academic Press. Naka, HI., and W.A.H. Rushton 1966 S-potentials from colour units in the retina of fish (Cypnnidae). Joumal of Physiology 185:53~555. Neitz, J., and G.H. Jacobs 1986 Polymorphism of the long-wavelength cone in normal human colour vision. Nature 323:623 625. Schnapf, J.L, T.W. Kraft, and D.N Baylor 1987 Spectral sensitivity of human cone photoreceptors. Nature 325:439-441. Schnapf, J.L, T.W Kraft, BJ. Nunn, and D.N Baylor 1988 Spectral sensitivity of primate photoreceptors. Visual Neuroscience 1:255-261. Stiles, W.S., and J.M. Burch 1955 Interim report to the Commission Internationale de l'Eclairage, Zurich, 1955, on the National Physical Laborato~y's investigation of colour-matching (1955~. Optica Acta 2:168 181. Vos, J.J. 1978 Colorimetric and photometric properties of a 2 fundamental observer. Color Research and Applications 3:125-128. Wyszecki, G., and W.S. Stiles 1982 Color Science: Concepts and Methods, Quantitative Data and Formulae. New York: Wiley.