The goldfish electroretinogram: Relation between photopic spectral sensitivity functions and cone absorption spectra1

The goldfish electroretinogram: Relation between photopic spectral sensitivity functions and cone absorption spectra1

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THE GOLDFISH BETWEEN FUNCTIONS

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ELECTRORETINOGRAM:

PHOTOPIC AND

SPECTRAL

RELATION

SENSITIVITY

CONE ABSORPTION

SPECTRA I

DWIGHT A. BURKHARD1Hunter Laboratory of Psychology, Brown Uni,,ersity, Providence, Rhode Island (Received 1l Augu.s't 1965)

INTRODUCTION SINCE DEWAR and I'V[cKENDRICK (1874) opened tile subject almost a century ago, Spectral sensitivity measurements have enjoyed a prominent position in research on the electroretinogram (ERG). While the dependence of scotopic spectral sensitivity on the absorption spectrum of rod pigments is firmly established, the basis of the photopic electroretinal spectral sensitivity curve is still not well understood. Previous work has, however, supported the notion that several spectrally-distinct subsystems contribute to the photopic E R G of color-discriminating species. Four classes of observations may be cited: (1) Irregularities in the shape of the photopic curve (e.g. DE.~,~XE, ENROTH-CuGEt. L, GONGAV,,'ARE,NEYLAND and FORBES, 1958; TANSLEY, COPENHAVER and GUNKEL, 1961 ). (2) Results from "color-shift" experiments (Fo~,BES, DE',>E, NEYLAND and (JONGAW,-\RE, 1958; RIOt;S, JOH.XSOY and SCHICK, 1964). (3) Departures from the normal photopic curve in the case of human color-det'ecti~es (ARMINGYON, 1952; COPENHAVER and GU~XKEL, 1959). (4) Changes in the shape of the p h o t o p i c c u r v e a s a result of chromatic adaptation procedures (GRANIr, 1938; TANSLEY, COPENHA\ER and GU,XKEt., I961: Ct,',o,xlus, 1964:IKEDA, 1965). It has often been assumed that the spectral sensiti~ities of tt~e photopic subsystems correspond to the absorption spectra of different types o1 cone pigments. However, this assumption has not been rigorously tested since cone pigments have been difficult to extract (DARTNALL, 1960) and the indirect physiological approach through chromatic adaptation experiments has generally failed to adequately isolate the photopic subsystems (see CAvo.xIws, 1964). Without independent estimates of the subsystem spectral sensitivities, attempts to establish the exact manner by which these subsystems interact to determine the resultant photopic curve have been necessarily inconclusive. Recently, spectrophotometry o1" single receptors has provided measurements o" cone absorption spectra. Extensive study of goldfish cones has shown that they fall into three classes whose absorption spectra peak at about 450, 530, and 625 m# (M~,RKS, 1963: LIE~MAN and ENTINE, 1964); the absorption spectra of human cones also appear to be trimodally distributed (MARKS, DOBELLE and M.~,cNIc~oL, 1964: B~towN and \VALD, t This research was performed under the direction of Prot'essor Lorrin A. Riggs, and is basud on a dissertation submitted to the Graduate School of Brown University in 1965 in partial t'ulfilment of thc requirements for the Ph.D. degree in psychology. The author held a Predoctoral Fellowship from the United States Public Health Service. Much of the equipment used in the research ~as purchased through the aid o1 Public Health Service Grant No. NB 01453. 517

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1964). Microelectrode work ha> demonstrated the existence or" achromatic and chrc;:::a::c response mechanisms at both the horizontal and ganglion cell layers of the go[dt:tsh retina (WAGNER. MAcNICHOL and WOLBARSHT, 1960, 1963) and psychophysical experiments ha~e clearly shown that the goldfish is capable of hue discrimination (McCLEA~,~ and BERNSTEIN, t959). At the m o m e n t , it seems likely that the general mechanisms of color coding in the goldfish and primate visual systems may be qualitatively similar ( M A c N I ( H ~ t . , 1964). Since analysis at the cellular level has now outlined the f u n d a m e n t a l mechanisms of goldfish color vision, it is of considerable interest to examine the relation betv, een these mechanisms and the wavelength-dependent aspects of the etectroretinogram. Although the E R G of the excised fish eye deteriorates rapidly (DAY, 1915; GOURAS, 1960; HANYU and ALl, 1964), stable responses have been obtained from the intact goldfish eye (HANYU and ALl, 1963). The research reported here establishes the conditions for obtaining a photopic E R G and then examines the relation between photopic spectral sensitivity functions and the cone absorption spectra. METHOD Optical system The optical system, a two-channel Maxwellian-view system, is shown schematically in Fig. 1. All lenses were achromats. The sources (St and $2) were tungsten ribbon filaments. Lamp current was continuously monitored. A Bausch and Lomb grating monochromator provided spectral light with a nominal bandwidth of 10 mlt, Light could be interrupted at a filament image by an electromagnetic shutter, SH:, and in other experiments, a flicker vane, Vx, was used to provide intermittent stimulation. The vane was rotated by a d.c. motor which was mounted independently of the optical system and the preparation; rotation rate was monitored by a tachometer circuit. When desired, the flicker vane could be moved to V2 to provide intermittent stimulation from the second channel. The relative intensities of each channel were controlled by neutral metallic-film filters inserted at F~ and Fz. Light from the two channels was combined by a beam splitter, Sp, and then travelled a common path and entered a light-tight, electrically-shielded cage. The beam was directed to an eyepiece, Ep, which formed small (about 3 x 0.5 ram) filament images at the nodal point of the eye. Although the vertical dimension of the images approached the size of the pupillary diameter of the fish used in these experiments, slight encroachment of the image on the iris did not lead to stimulus variability since the goldfish pupil is immobile (HANYU and ALI, 1963).

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Relation Betv~een Photopic Spectral Sensiti,,ity Functions and Cone Absorption Spectra

5t9

The field produced by the monochromatic channel >`as rectangular, having an angular hcigilt of 71 and a width of 54 . The relati,,e energy output of this channel v, aa measured at 10 ml~ steps across the spectrum by means of a photomultiplier unit ( Photovolt Model 520M ) ~ hich had been calibrated against a thermopile. When the splitter, Sp, was not present, the maximum luminous flux at the cornea at 580 m,u v,as calculated from an illuminance measure made with a MacBeth llluminometer and found to be 4 • 10 -e Ira. Binocular matching re,,ealed that this channel pro,,ided a retinal illuminance or about 4.4 log photopic trolands at 580 m#. The field arising from the second channel was circular with a diameter of 37 . With the s21itter present, and a Farrand interference filter of 650 m# dominant wavelength inserted in this channvl, the luminous flux impinging on the goldfish cornea ~as about 3.5 • I0 a Im ~hich >,ould correspond to about 4"0 log photopic trolands if yielded by a human observer.

The preparation Common goldfish (Uarassius ~zuratus) about 10-15 cm long were kept in an aquarium under a regulated illumination cycle (12 h r o n , 12 hroff). A fish was prepared for experimentation by >`rapping it in netting. After receiving a 0.075 mg intramuscular injection of D-tubocurarine (Squibb), the animal was placed in a contoured plastic body clamp which held it firmly just behind the operculum. The body was in air, but a mouth tube ser,,ed to continuously circulate ~ater over the gills, and an auxilliary tube provided a jet of ~ater over the dorsal head region in order to keep the body and cornea moist. Water temperattr,-c >`a-, 24 - I:C. Se~eralexperiments ran for as long as 5 hr ~ithout apparent deterioration of the ERG.

Electrodes and recording system The electrodes consisted of chlorided platinum iridium alloy ~ires about 150 microns in dianaeter'. The insulated active electrode travelled within the bore of a hypodermic needle via a rack and pinioq gear mechanism. The needle penetrated the dorsal aspect of the sclera somewhat posterior to the region of the lens, and it >`as advanced into the vitreous and clamped into position. The electrode was then advanced until it protruded several millimeters, its tip being near the optic axis and several millimeters from the retinal surface. The indifferent electrode was placed in the nostril and the preparation was grounded by'a third electrode placed near the tail. The use of the vitreal rccordingsite was found to result in good long-term electrical stability, as well as a relatively high signal noise ratio. BRow>aandWxEsEC(1961)haveshov, nthat the vitreally-recorded ERG is fundamentally similar to the corneally-recorded ERG, and that the relative positions of the electrode and the retinal inaage may be varied considerably without markedly affect ng the amplitude of the response. The electrodes were connected to a cathode follower located inside the cage. The output ol the c.tthode follower was fed into a Grass P-6 preamptifier which was operated in the d.c. mode. The preamplifier output was capacitatively coupled to a 502 Tektronix dual-beam oscilloscope. The time constant of the recording system was 2 sec. Comparison of flash-evoked E RGs of the dark-adapted eye recorded ~ ith either this degree of capacitative coupling or with d.c. coupling, revealed no difference in the measured 5-~va~e amplitudes. Permanent records of the responses >`ere made by means of a C-I9 Tektronix oscilloscope camera equipped with Polaroid film.

R E L A T I V E T R A N S M I S S I O N OF THE O C U L A R M E D I A

Proce~ho'e A n i m a l s w e r e first a n e s t h e t i z e d by i n t r a p e r i t o n e a l i n j e c t i o n s o f N e m b u t a l .

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h a d b e e n e n u c l e a t e d a n d the o c u l a r a d n e x a r e m o v e d , an a p e r t u r e o f a b o u t 5 m m d i a m e t e r was c u t n e a r the p o s t e r i o r p o l e o f the g l o b e .

L i g h t was t h e n p a s s e d t h r o u g h tile eye in the

n o r m a l d i r e c t i o n . T h e b e a m i n c i d e n t at t h e c o r n e a w a s a b o u t 2 m m in d i a m e t e r , a n d u p o n r e a c h i n g the p l a n e o f t h e r e t i n a it was s m a l l e r t h a n the a p e r t u r e in the g l o b e . T h e light e m e r g i n g f r o m the eye f o r m e d a small aerial f i l a m e n t i m a g e w h i c h fell u p o n the face o f a 5 m m d i a m e t e r fiber o p t i c light guide. T h e o p p o s i t e e n d o f the light g u i d e w a s c o n n e c t e d in a l i g h t - t i g h t m a n n e r to the p h o t o t u b e o f a p h o t o m u l t i p l i e r unit ( P h o t o v o l t M o d e l 520M). T h e relative t r a n s m i s s i o n o f t h e m e d i a w a s o b t a i n e d by d e t e r m i n i n g tile s p e c t r a l d e n s i t y f u n c t i o n w i t h a n d w i t h o u t t h e eye in the s y s t e m . In an a t t e m p t to i n c r e a s e the a c c u r a c y o f m e a s u r e m e n t , the d e n s i t y s p e c t r a were m e a s u r e d by a m e t h o d r e s e m b l i n g the s t e p - b y - s t e p

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method of photometry: density differences were measured between successixe ~a~elengti~ pairs. In total, nineteen density differences were obtained with at least t'~o measurements per determination. The experiment was completed in about 30 rain and ~isual inspection or ~he eye after this interval indicated that little vitreous had escaped and the media still appeared clear. Resuhs and discussion Figure 2 shows the average relative transmission of the goldfish ocular media. In common with data from other species, the present determinations show that the ocular media of the goldfish preferentially absorb short-wavelength radiation. These data are in good quantitative agreement with measurements on transmission of the bovine eye (PITTS, 1959) and the rabbit eye (WE|slYGER, SCH,'4tDT, WILLIAMS, T|LLER, RUFFIN, GU~RR'," and H;,.~I, 1956). They are also similar to data on transmission of the isolated lenses of several fresh-water fish (KENNEDY and MILKMAN, 1956), a fact which suggests that the selective transmission of the goldfish ocular media may be primarily determined by the spectral transmittance of the lens.

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SPECTRAL

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OF THE

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Procedure Several experiments were performed with the dark-adapted eye in an attempt to evaluate the scotopic component of the goldfish E R G . Fish were allowed to dark-adapt for about 1} hr, following various experiments with the light-adapted eye. Responses were then obtained by stimulating the eyewith 20 msec flashes every 45 see. The short flash duration was selected to minimize light-adaptation from the test stimuli and to reduce the magnitude of the c-wave. The stimulus subtended 71 ° x 54°; twelve different wavelengths were used, and the relative energy of the flashes was appropriately adjusted so that responses to differSnt wavelengths obtained at comparable times were of approxima.tely equal amplitude. Intensity values were progressively increased in 0.3 log unit steps so that an intensity range of about 1.2 log units was covered at each wavelength.

Relation Bct~ecn Photopic Spectral Sensiti~it) Functions and (one Absorption Spcctr~t

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Re.sults Figure 3 shows typical electrical responses to light from various regions of the spec,:rum. The approximately-matched response amplitudes are in the region of the criterion amplitude used for the spectral sensitivity calculations. It may be seen that the response elicited under these conditions is simple: only the b-wave is in evidence although the goldfish eye generates all E R G waves under other conditions.

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Fie;. 3. Electroretinogramsofthedark-adaptedcye. Stimulus wavelength is indicated bythe number preceding the response. The ampl{tude of response at 580mlLis 51 l~V. The lowest trace is a record of the 20 msec flash which evoked the response at 675 mle. The vertical marks on this trace indicate intervals of 100 reset.

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DENSITY FIG. 4. Curves showing the relation between b-wave amplitude and the relative attenuatioil of the stimulus intensity. The number at the end of each curve denotes the stimulus wavelength. For each curve, the minimum density of 0.00 is based on a different reference energ? value. Figure 4 shows the relation between h-wave amplitude and intensity for the rel:tti\ely low-intensity flashes used in the spectral sensitivity experiments. After all ]ow-in:ensity determinations had been made, more intense stimuli were used on several occasions. With 20 msec flashes, the m a x i m u m o b t a i n a b l e b-wave amplitude was about 300 ,uV. When response amplitude was plotted over the entire stimulus range, the form of the b-wave amplitude-intensity curve was S-shaped, and thus resembled the curves previously reported for other species. The threshold intensity for a j u s t detectable a-wave was a b o u t l-log unit greater than the intensity level needed to evoke a 50/zV b-~vave.

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Follo~ing the conventional procedure for determining spectral sensitivity, the rela,~i~e energies necessary to produce a criterion amplitude of response (50/~V) ~ere determined b~ interpolation from the constructed amplitude-intensity cur~es. Best-fit procedures were not used; the data points were directly connected. The energy values were then corrected for transmission of the ocular media and quantized. The spectral sensitivity curve in Fig. 5 thus refers to log quantum sensitivity at the retina. LIF.BMaS(1965) has recently found that the visual pigment residing in the outer segments of goldfish rods has its maximum absorption at 525 m/t, and the smooth cur~e drawn in Fig. 5 has been derived from Darmali's nomogram (DARTNALL, 1953) on this basis. It may be seen that the spectral sensitivity curve is more complex than the absorption spectrum of the rod pigment; it diverges from the pigment curxe at both ends of the spectrum. 20

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Discttssiotl

There are several possible explanations for the lack of agreement between the rod absorption spectrum and the spectral sensitivity curve. The increased sensitivity in the blue region of the spectrum is not unexpected, for it is well known that electroretinal spectral sensitivity curves often show such an elevation in sensitivity (JOHNSON, 1958). Although large-area stimuli were used in our experiments, scattered light may still account for at least part of this effect. However, some fish retinae fluoresce strongly (GRANIT, 1955), and if this occurs in the goldfish retina it could also lead to an elevated sensitivity at short wavelengths. Some of the high sensitivity beyond 430 m/t might also be related to the c/s-peak of the rod absorption spectrum (see DA~TNALL, 1962, for discussion of this possibility). While the interpretation of the high sensitivity in the blue is somewhat unclear, it is quite likely that the elevated sensitivity seen at wavelengths greater than 540 mit may be attributed to the intrusion of photopic mechanisms related to the green- and red-sensitive cones. The apparent bimodal nature of the curve in Fig. 5 also suggests that photopic as well as scotopic mechanisms are contributing to the E R G . Since the scotopic system often tends to dominate the E R G s obtained from dark-adapted mixed retinas (GRANET, 1955,

Relation Bet~een Photopic Spectral Sensitivity F u n c t i o n s and Cone Absorption Spectra

523

1962), the present data suggest that a comparatively large fraction of the goldfish electroretinogram originates from the photopic system. EFFECT OF F L I C K E R

R A T E ON D E T E R M I N A T I O N S

OF S P E C T R A L S E N S I T I V I T Y

Procedure The effect of flicker rate on spectral sensitivity was investigated by finding the eye's relative spectral sensitivity to lights of 650 and 525 m/¢ at several flicker rates. The stimulus subtended 71 ~:,:54 ~and the light: dark ratio was 1:I. Flicker rates of'10, 15, 20,2:i. and 30 f s were used. Flicker rate was first set at 10 f s and the eye was exposed t'or 10 rain to continual flicker at 650 m,u. After this period of light-adaptation, a 250 msec sample 3f the response was recorded. The wavelength was then changed to 525 m/l, and 45 sec Iater another response sample was obtained. Tile wavelengths were alternated in an ab&~ order as frequency and intensity of the stimuli were progressively changed until an ascending intensity series had been completed for both wavelengths at all t'requencies. Tile peak-totrough response amplitude was measured. Median amplitude x~.as then plotted a~ainst relative energy. At each flicker rate, the relative energy at the cornea necessary to produce a criterion amplitude of response, was obtained by interpolation. If E,~s0 and E~es a;e the relative energy '~alues for tile 650 and 525 m/t stimuli, then their relative sensitixity ratio is expressed: Log Relative Sensitivity 650 5 2 5 : - L o g Ea.,_:, - - L o g E~a0. Results Sensitivity ratios computed from several criteria are shown in Fig. 6. In the case of two fish, dark-adapted spectral sensitivity data were available, and estimates of the relative sensitivity ratios obtained from these functions ha,,e been plotted near "'zero flicker rate" in Fig. 6. It may be seen that there i s a relatively large increase in the relative sensitivity ratio as a result of using an intermittent stimulus and or establishing a light-adapted state. However, this change of about l-log unit seems to occur even at low flicker rates, for there is little evidence o1" a systematic increase ill sensitivity from 10 to 30 f s. Od

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The independence of the relative sensitivity ratio on flicker rate abo~e I0 f s. provides the justification for concluding that 20 f s stimulation ~ill e,,oke a purely photopic ER(i (see below). The minimum flicker rate which is required to achieve the transition to a purcly photopic E R G varies from onc species to another• This transition frequency often seems to be positively correlated with the rod 'cone ratio of the retina in question (DODT and Wl ~-~u. 1953). Relatively low transition frequencies have been suggested by previous studies with fish (WOLF and WOLF, t936; S;aETICHI>,', 1953) and J,-xcoRs, JOXES, and DEVALOIS (1963) have found a low transition frequency for the squirrel monkey ERG. The low transition frequency implied by the present data, and the mesopic nature of the spectral sensitivity of the moderately dark-adapted eye, are consistent, since both observations seem to indicate that the photopic component of the goldfish E R G is relatively large. The high terminal C F F of the goldfish E R G (about 70 fs) found by HANYt.; and ALI (1963) also seems to point to this conclusion. PHOTOPIC SPECTRAL SENSITIVITY Procedure

Spectral sensitivity was determined at thirteen spectral points by using 20 f s stimuli (t:1 light :dark ratio) which subtended71 °:,:54 ° . After 10 min of adaptation to moderateintensity white light, the lowest intensity at 450 mlz was introduced and, several minutes later, a response sample was recorded. The wavelength setting was then changed to 475 mtz and, 45 sec thereafter, another response sample was obtained. The wavelength was progressively increased in twelve steps until 725 mll was reached. The relative intensity was then increased 0"3 log unit, and a descending wavelength series was run. This sequential procedure was continued until at least a l-log unit intensity range had been covered at each wavelength. Median amplitude-intensity functions were plotted by directly connecting the data points. A total of ten fish were used, and when possible, spectral sensitivity was separately calculated for response criteria of 40, 50, and 6 0 / V . 475

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Relation Bct~ecn Photc~pic Spectral Sensiti~it', Functions and Cone Absorption Spectra

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Re~tdfx Selected examples of the goldfish flicker response may be seen in Fig. 7 . . - k s in other species, the electroretinat flicker response is not simple. The small, brief positive deflection occurring near the trough of the waveform was consistently observed and m a y possibly be a n a l o g o u s to the fast photopic x-wave o f the h u m a n E R G (Aa,'4[XGTO~". 1952), but it remains to be studied in detail. The general form o f the amplitude-intensity curves obtained with fast flicker is illustrated in Fig. 8. N o obvious wavelength-correlated differences n the shape of these functions ~ e r e a p p a r e n t . The effect of choosing different criteria for the spectral sensitivity calculations was small. Differences in sensitivity c o m p u t e d for dif'erent I00

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criteria t40, 50, and 60 ,zV) averaged about -:0-03-log unit at any given ~a~etength. and a, one-sample runs test indicated that the differences were random with respect to wavelength= Accordingly, the average cur~e derived from the largest sample (n- 10 for the 50 /zV criterion) is presented in Fig. 9 as the best estimate of photopic spectral sensitivity. When based on relative energy at the cornea, the curve is relatively smooth and peaks at about 570 m,u. As may be seen in Fig. 9, the standard deviations of these data are about 0-15-log unit or less. The mean curve serves as a relatively good estimator of photopic sensitivity (or the standard errors of the means are all 0.06-log unit or less.

Discussion It appears that the maxima of the photopic and tile dark-adapted curves differ; the 560 m# submaximum of the dark-adapted curve occurs at about the same point as the peak of the photopic curve. However, the most conspicuous difference between the two curves occurs at long wavelengths: when the curves are normalized, the photopic curve displays considerably greater relative sensitivity for wavelengths greater than 600 m~L. These differences in spectral sensitivity due to light-adaptation a n d o r flicker stimulation demonstrate the well-known Purkinje Shift, and may be taken as presumptive physiological evidence for the existence of a duplex visual system in agreement with the known anatomical duplicity of the goldfish receptors (WALLS, 1942). It is of interest to note that the 570 m/.~ maximum of the gotdfish's photopic E R G curve, lies at a somewhat shorter wavelength than the spectral sensitivity maxima of both the L-type S potential (WAGNER, MACNICHOI. and WOLBARSHT, 1960) and the "luminosity-type" ganglion cells (MAcNIcHOL, WOLBARSHT and WAGNER, 196l). The light-adapted spectral sensitivity curve displays a shoulder around 450-475 mlt. It is likely that this may be a genuine property of bluesensitive elements of the photopic system as appears to be the case with other electroretinal photopic curves in the blue region (e.g. CAVONIt:S, 1964; JONES, POLSON and DEVALOIS, 1964). However, the possibility cannot yet be ruled out that at least part of this effect may be due to residual scotopic activity enhanced by either scatter of fluorescence. More intense sources, or a marked reduction of criterion will be necessary to extend sensitivity determinations farther into the violet region of the spectrum. It has been pointed out that several classes of previous experiments support the idea that photopic electroretinal spectral sensitivity is determined by several spectralty-distinct subsystems. The data presented here suggest that this is also the case with the goldfish E R G for it is clear that the photopic curve does not coincide with the absorption spectra of any of the three types of goldfish cones. In addition, evidence will be presented below for the separate existence of a red-sensitive mechanism. The vision literature is filled with suggestions that photopic spectral sensitivity may be accounted for by a general additive model of the type: V~.=bB~.--gG;.+rR~., where kS. represents the relative luminous efficiency of light of wavelength 5.; B~., Gx, and R~. represent the relative spectral sensitivities of three photopic subsystems B, G, and R at wavelength 5.; and b, g, and r represent weighting coefficients appropriate to each subsystem. It has been suggested that the terms B~., G,t, and R;. do in fact represent the absorption spectra of three cone photopigments. WALD (1964) has recently presented evidence that this photochemical assumption and the additive model, may adequately describe human psychophysical spectral sensitivity data, and he has reiterated the prevalent view that the weighting coefficients, b, g, and r, may reflect the relative incidence of the respective cone types in the retina. Since human photopic psychophysical, and modern E R G spectral, sensitivity measurements agree rather closely (e.g. CAVONI~_~S, 1964;

Relation Bet~vcen Photopic Spectral Sensitivity Functions and Cone Absorption Spectra

527

JOHNSON, RIGGS and SCHICK, 1965), it a p p e a r s that this theoretical anal,~sis may be equally a p p r o p r i a t e For spectral sensitivity cur~es d e m e d From E R G measurements. Since extensive d a t a are available on the a b s o r p t i o n spectra and the relative incidence of the goldfish cones, it seemed worthwhile to a t t e m p t a similar analysis of the p h o t o p i c electroretinal d a t a obtained here. In order to perform the analysis, the corneal-energy d a t a (Fig. 9) were corrected for transmission of the ocular media, and the resulting values were expressed in terms of q u a n t u m sensitivity. The t r a n s f o r m e d d a t a along with their s t a n d a r d errors, are plotted on a linear sensitivity scale in Fig. 10. It may be seen that the effect o f these t r a n s f o r m a t i o n s is to shift the peak o f the curve from a b o u t 570 m/z to 56(1 m/L. >.F-i.0

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LIEBMAN (1965) estimates that the three types o f cones occur in tile ratio of lOg:3r:lb. M a r k s ' s statements (MARKS, 1963) suggest a ratio o f a b o u t lOg:Sr:2b. These estimates were used to specify the ratio o f the weighting coefficients, r, g, and b; V;. values were then calculated From the formula: V;.=bB;--gG;.4-rR;.. The two normalized curves so obtained are shown in Fig. 10. Neither agrees closely with the data. A least-squares best-fit calculation in which no restrictions were placed on the values or signs o f the weighting coefficients indicated that the best-fitting theoretical curve is o b t a i n e d when the weighting coefficients a r e : g = 0 " 6 8 5 , r=0"520, a n d b = 0 " 0 1 7 . N o t e that tile ratio of the weighting coefficients is lOg:7"6r:O'25b, so in this scheme, the blue-sensitive cones make an inconsequential contribution to the theoretical spectral sensitivity curve. The best-fitting curve may be seen in Fig. 10. The d a t a are still not well described for the theoretical curve is too b r o a d and does not closely a p p r o a c h a m a x i m u m value o f 1.0. There are two major possibilities for the failure o f o u r theoretical curves to accurately describe the E R G curve: (I) the a b s o r p t i o n spectra used in our calculations may be

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inappropriate; (2) the additive model may not represent the E R G spcctraI sensiti~it~ mechanism. With regard to the first possibility, it should be emphasized that the data of Marks arid Liebman are both based on large samples and agree closely. In addition, JACOBSON ( 19641 has shown that there is an excellent agreement between the spectral sensitivity curves of some single units in the optic tectum and the reported absorption spectrum of the redsensitive cones. Nevertheless, some residual uncertainty may still exist as to the e,'~act absorption spectra of the goldfish cones, it is also possible that factors could be operating in tile present experiment which could serve to change the effective absorption spectrum of the cones. While oil droplets are not found in the teleost retina (WALLS, 1942), possible screening effects of accumulated photoproducts cannot be definitely excluded at present (GoLDSTEIN and WILLIAMS, 1965). On the other hand, very diverse spectral sensitivity curves may be obtained at different recording sites within tile goldfish visual system (e.g. WAGNER, MAcNICHOL and WOLBARSHT, 1960; JACOBSO>,', 1964) and a similar diversity has been found within the visual systems of other species (e.g. GRAYIT, 1947, 1962; DEVALOIS, 1960). This diversity does not seem to be the result of a comparable diversity in the types of cone photopigments; rather, it appears that different neural systems transform the relatively simple output of the receptors in a variety of complex ways. It is clear that the electroretinogram arises at a retinal region where considerable modification of the receptor output could already have occurred. For example, it has been reported that the spectral sensitivity of the E R G on-response and off-response may differ (HOWARTH, t961 : CHAPMAN,1964; IKEDA. 1965). It is thus possible that the failure of the theoretical curves to describe the spectral sensitivity function does not negate the fundamental dependence of the E R G curve on the measured absorption spectra, but rather indicates that the mechanism which serves to couple activity in the receptor layer with the E R G does not follow tile operations implied by the additive spectral sensitivity model. Future experiments should be devised to explore the nature of this coupling mechanism. Results of these experiments should indicate the relative importance of the two explanations proposed for the present spectral sensitivity data.

ACTION SPECTRUM OF A RED-SENSITIVE MECHANISM Procedure

The following procedure was used in an effort to uncover a photopic subsystem related to the red-sensitive cones. A 650 m/z interference filter was inserted in the second channel of the optical system, and a circular aperture was introduced to limit the field to 37 °. This field was then flickered at 20 f,s and the intensity level adjusted so that the response was about 40-60 ,uV. The intensity of this stimulus was subsequently held constant throughout the duration of the experiment. After the eye had adapted to the stimulus for I0 rain, a response sample was recorded. A steady, low-intensity background field of 540 mlt was then added; this field was obtained from the monochromatic channel and subtended 71°× 54 °. Thus, the flicker stimuli were now intermittent increments of 650 m/t on a steady background of 540 mlt. Responses were recorded at 45 sec intervals, and the background intensity was progressively increased in 0-3-log unit steps. The effect of intensity was investigated at a number of background field wavelengths. In practice, the background intensities and wavelengths were chosen so that approximately equal response amplitudes were produced at comparable times during the experiment.

Relation Betv,een Photopic Spectral Sensitivity Functions and Cone Absorption Spectra

529

Re'su/ts Data from these experiments were plotted as the median response amplitude t,3 the 650 m# flicker stimulus vs. relative a t t e n u a t i o n of the b a c k g r o u n d field, with b a c k g r o u n d field wavelength as the parameter (Fig. I l L [t may be seen that after the background exceeds a rather ill-defined threshold intensity, subsequent increases lead to progressive decrements in the flicker response. (It should be emphasized that the slope of these functions is \ e r r shallow and the smallest responses approach the noise level of the recording system.) An action spectrum may be defined as the function relating wavelength to the rehtti~e eqergy

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WAVELENGTH-mp FIG. 12. Action spectra of a red-sensitive mechanism of the goldfish retina. The smooth cur, c is the log absorption spectra of the red-sensitive cones (MARKS, 1963). The symbols refer to data obtained from different fish. The maxima of the action spectra have been adjusted to give an approximate best-fit with the pigment curve. The resulting maxima are: circles: 2-00: triangles: 2.03; squares: 2.13; inverted triangles: 2"13.

530

D'~'~IGHY .\. BURKH-XRD1

necessary to produce some constant response. In the present experiment, it is pos~,ible to derive an action spectrum by determining the relative energies o f the b a c k g r o u n d fields which are required to produce a criterion decrement in the 650 m u flicker response. Figure 12 shows the results from four experiments in which action spectra were obtained for a criterion decrement o f 15 uV. These d a t a are plotted in terms o f log q u a n t u m sensitivity at the retina, and the s m o o t h curve is the log a b s o r p t i o n spectrum of the red-sensitive cones as reported by MARKS (1963). Discussion

The o u t c o m e of this experirnent unequivocally d e m o n s t r a t e s that the photopic electror e t i n o g r a m o f the goldfish must receive influences from at least two spectrally-distinct subsystems. The fact that the data points tend to cluster a r o u n d the a b s o r p t i o n spectrum suggests that the action spectrum may depend largely on the light a b s o r b i n g properties of" the red-sensitive cones and, thus, reinforces the idea that the subsystem spectral sensitivites are closely related to the cone a b s o r p t i o n spectra. JACOBSON (1964) has previously found that the spectral sensitivity curves o f some units in the goldfish optic tectum, agree closely with the a b s o r p t i o n spectrum o f the red-sensitive cones. F o r m a l analysis o f the m e t h o d used here suggests that two necessary conditions for exact agreement between the action and a b s o r p t i o n spectra are: (1) The a d a p t a t i o n a l mechanisms o f the p h o t o p i c subsystems must be independent. (2) The 650 roll flicker stimulus must effectively activate only the red-sensitive cone system. (This requirement should be adequately satisfied since the ratio o f the a b s o r b a n c e s o f the red- and greensensitive cones is a b o u t 30:1 at 650 m/L. However, the appreciable overlap of the cone spectra at shorter wavelengths would suggest that this flicker d e m o d u l a t i o n method cannot be readily applied to derive the action spectra o f the green- or blue-sensitive cones.) It appears that the present techniqtte has the potential of d e t e r m i n i n g the exact action spectrum o f the red-sensitive mechanism, and that the success o f the technique may have i m p o r t a n t implications for the problem o f the effect o f c h r o m a t i c a d a p t a t i o n on the photopic electroretinogram. It is, therefore, desirable to increase the measurement precision of the experiment. In particular, the use of c o m p u t e r - a v e r a g i n g techniques may permit greater precision, especially in relation to responses o f relatively small amplitude. REFERENCES ARMINGTON, J. C. (1952). A component of the human electroretinogram associated with red color vision. J. opt. Soe. Am. 42, 393-401.

BROWN, K. T. and WIESEL,T. N. (196[). Analysis of the intraretinal electroretinogram in the intact cat eye. J. Physiol., Lond. 158, 229-256. BRowN, P. K. and WALD, G. (1964). Visual pigments in single rods and cones of the human retina. Science, N.Y. 144, 45-48. CAVONIUS,C. R. (1964). Color sensitive responses in the human flicker-ERG. Documenta ophth. 18, 101-113. CHAeMAN, R. M. (1964). Spectral sensitivity comparison of on- and off-responses of the frog electroretinogram. Vision Res. 4, 455-463. COPENHAVER, R. M. and GUNKEL, R. D. (1959). The spectral sensitivity of color-defective subjects determined by electroretinography. Archs Ophthal., N. Y. 62, 55-68. DARTNALL, H. J. A. (1953). The interpretation of spectral sensitivity curves. Br. reed. Bull. 9, 24--30. DARTNALL, H. J. A. (1960). Visual pigments of colour vision. Mechanisms o f Colottr Discrimination, edited by Y. GAHFRET, Pergamon Press, Oxford. DaRTNALL, H. J. A. (1962). The photobiology of visual processes. The Eye, edited by H. DAVSON, VO[. 2, Academic Press, New York. DAY, E. H. (1915). Photoelectric currents in the eye of the fish. Am. J. Physiol. 38, 369-398. DEANE, H. W., ENROTH-CUGELL, C., GONGAWARE, M. S., NEYLAND, M. and FORBES, A. (1958). Electroretinogram of fresh-water turtle: form and spectral sensitivity. J. Neurophysiol. 21, 45-61.

Relation Betv.een Photopic Spectral Sensitivity Functions and Cone Absorpth)n Spectra

531

DEVA LOIS, R. L. 11960). Color vision mechanisms in the monkey, d. gen. Physiol. 43. No. 6, Part 2, 115-128. DEWAR, ,i. and MCKENDRICK, 'i. G. ( [ 874}. On the physiological action of light. Trans. R. Soc. Edin5.27, 141-166. DODT, E. and WIRTH, A. (1953}. Differentiation betv, een rods and cones by flicker electroretinography in pigeon arid guinea pig. Acra physiol, sca~zd. 30, 80-89. GOLDSTEIN, E. B. and Vv'ILLZ:,~,ts,T. P.(1965}. Personal communication. GOURAS, P. (1960). Graded potentials of bream retina, d. Physiol., Lond. 1.52, 487-505. GRAN[T, R. (1938). Processes of adaptation in the vertebrate retina in light of recent photochemical and electrophysiological research. Dacumenta ophth. 1, 7-78. GRANIT, R. (I947). Sensory ;IJechanGms of the Re:inn. Oxford University Press, London. GRANIT, R.(1955). ReceprorsandSensoryPercept[o~t. Yale University Press, New Haven. GRANIT, R. {1962). Neurophysiology of the retina. The Eye, edited by H. DAvsox, Vol. 2, Academic Press, New York. H-x,'-,YU, l. and ALI, M. A.(1963). Flicker fusion frequency of electroretinogram in light-adapted gocifish at various temperatures. Science, N. Y. 140, 662 663. HA:'iYU, l. and ALI, M. A.(1964). Electroretinogram and its flicker fusion frequency at different temperatures in light-adapted salmon (Sahno salar). J. cell. romp. Phy'sioL 63, 309-321. HO'WARTH, C. I. (196I). On off interaction in the human electroretinogram. J. opt. Soc. Am. 51, 345 352. [KEDA, H. (1965). The spectral sensitivity of the pigeon (Columba l/via). V/s/or( Res. 5, 19 37. JAC{)Bs, G. H.,JONES, A. E. and DEVALoIS, R. L.(1963). Electroretinogram of the squirrel monkey, d. romp. physiol. Psycho/. 56, 405-409. JACOaSON, M. (1964}. Spectral sensitivity of single units in the optic tectum of the goldfish. Q. J, exp. Physiol. 49, 384-393. JOHNSON, E. P.(1958). The character of the b-wave in the humanelectroretinogram. ArchsOph:hal.,N.Y. 60', 565-591. JOHNSON, E. P., RIGGS, L. A. and SCHICK, A. M. L.(1966). Phase alternations of a barred pattern used as a stimulus to evoke photopic retinal potentials. Atn. Y. Ophthal. (In press.) JONES, A. E., POLSON, M. C. and DEVALoIS, R. L. (1964). Mangabey +v- and b-wave electroretinogram components: their dark-adapted luminosity functions. Science, N. Y. 146, 1486-1487. KENNED',', D. and MILKMAN, R. D. (1956). Selective light absorption by the lenses of lower vertebrates and its influence on spectral sensitivity. Biol. Bull 11 I, 375-386. [.IEt~,'aAN, P. A. (1965). Personal communication. I. IEt~',laX, P. A. and ENGINE, G. (1964). Sensitive low-light-level microspectrophotometer: detection of photosensitive pigments of retinal cones, d. opt. Soc. Am. 54, 1451-1458. MCCLEARY, R. A. and BE~NStEIN, J. J. (1959). A unique method for control of brightness cues in the ~tudy of color vision in fish. Physiol. ZoOI. 32, 284-292. MAcNtcHOL, E. F., Jr. (1964). Retinal mechanisms of color ,,ision. Vision Res. 4, 119-134. MAcNICHOL, E. F.,JR.,WOLBM
2

DWIGHI" ~. BL;RKHARD[ Abstract--Spectral sensitivity of the intact goldfish eye ~us measured by electrorc,tinograph? Experiments with the dark-adapted eye and experiments investigating the effect of flicker r?,te on spectral sensiti,,ity both sugge,,t that a comparatively large fraction or the goldfish ERG originates from the photopic s>stem. Flicker stimulation was used to measure photopic spectral sensitivity. These data ~ere analysed with regard to the absorption spectra of the goldfish cones. The photopic curve was not adequately derived from a conventional additive model and it therefore is suggested that a more complex mode of interaction may be operative. A special method was used to determine the action spectrum of a red-sensitive photopic mechanism. Thi, action spectrum seems to be highly correlated with the absorption spectrum of the red-sensiti~c cones. Rdsumd--On mesure par 61ectror6tinographie ta sensibilite spectrale de l'ceil intact du c~prin dor6. Les experiences sur l'ceil adapt6 /1 l'obscurite et celles sur l'effet du papillotement sur la sensibilitY, spectrale s'accordent ~ suggdrer qu'une fraction relativement importante de I'ERG du cyprin provient du systeme photopique. La stimulation papillotante a ser;i a mesurer la sensibilitt~ spectrale photopique. On compare ces rdsultats aux spectres d'absorption des cSnes du cyprin. On ne peut pas d6,river ta courbe photopique par le modele additif usuel et on suggere done qu'il se produit tin mode d'interaction plus complexe. On emploie une m~thode sp6ciale pour d6terminer le spectre d'action du mdcanisme photopique sensible au rouge. Ce spectre d'action semble en 6troite corrt!lation avec le spectre d'absorption des cSnes sensibles au rouge. Zusammenfassung--Die spektrale Empfindlichkeit eines unversehrten Goldfischauges wurde elektroretinographisch gemessen. Sowohl Versuche mit dem dunkeladaptierten Auge als auch Versuche, die den Einfluss der Flimmerfrequenz auf die spektrale Empfindlichkeit untersuchen, weisen darauf hin, dass ein verhtiltnism~issig grosser Tell des Goldfisch E R G aus dem photopischen System kommt. Flimmerlicht wurde benutzt, um die photopische spektrale Empfindlichkeit zu messen. Diese Daten wurden im Hinblick auf die Absorptionsspektren der Goldfischzapfen analysiert. Die photopische Kurve konnte aus einem konventionell additiven System nicht hinreichend abgeleitet werden. Deshalb ist anzunehmen, dass ein komplizierter Wechselwirkungsmechanismus vorliegt. Eine spezielle Methode wurde ben~itzt, um das Aktionsspektrum eines rotempfindlichen photopischen Mechanismus zu bestimmen. Dieses Aktionsspektrum scheint mit dem Absorptionsspektrum der rotempfindlichen Zapfen in engem Zusammenhang zu stehen. Pe3~oMe--I/hMepn~acb cneKTpa,qbHa~ qyBCTBHTe21bHOCTb HHTaKTHOFO l-.-IaBa 3OJ1OTOfl pbi6rH MeTO~IOM 3~qeKTpOpeTHHOrpaq~l~[. KaK 3KCFIep~iMeHTbI C TeMHoa~anTHpOBaHHbIM FJ'IaBOM, TaK |t 3KCFIepl4MeHTbl B KOTOEbtX Hcc~e~oBaJ'IOCb BJ'II4RHHeHaCTOTbI CBeTOBbtX MenbKaHHft Ha cneKTpanbHy~o qyBCTa~lTenbuocrb 3 a c r a a n m o r CqnTarb, ~rO cpaBHnTenbHO 60nbtuan qbpaKuna D P F 3onoTo/~ p b t 6 ~ i reHep~lpyeTca qboxoml~ecKo~i Ct4CTeMO~. ~[~J]R rlBMepeH~t~[ (~OTOFIHqeCKOf[ cneKTpaJlbHOfi ~tyBCTSHTenbHOCTH 6btna HCFIOTIb3OBaHa npepblBl4CTa~ CaeTOBafl CTI4MyJIRL[t4fl. ~Tl4 /IaHHble 6btJ'lH npoaHa2Hi3~ipOBaHbl C y~eTOM cnenTpa I'[OF.FIOLtleHH~t KOJ16OqeK 30.FIOTO~'i pbt6KH. ~OTOnH,~ecKafl KpHBaa He MOr,qa ~btMb BbtBe~[eHa a/leKBaTHO ~13 O~blqHO~ aVI~I,ITHBHO~t MozleIIH It 3TO 3acTaB£I~eT £1yMaTb, *tTO3~eCb Mo)KeT HMeTb MeCTO 60nee CJ'IO)KHbI~IxapaKzep B3a|tMO/Ie~ICTBH~. EbLq HCHOYlbBOBaH CHCUH&J'IhHbl~[ MeTO.~ LLqR Ol'[pe/Ie£IeHH~ ~KI.[HOHHOFO cneKTpa HyBCTBI.ITe.rlbHOFO K KpaCHOMy dpoTortn'.tec•oro MexaHHBMa. ~TOT aKHI4OHHbI~ crleKTp !4Meet BbICOKy~O Koppe.r/J;l[.IHIO CO CI'/eKTpOM rloFJiOl.IJeHl4~l qyBCTBtITe:qbHblX K KpaCHO,'4y ron6o~eK.