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Photometry in goldfish by electrophysiological recording: Comparison of criterion response method with heterochromatic flicker photometry
PHOTOMETRY IN GOLDFISH BY ELECTROPHYSIOLOGICAL RECORDING: COMPARISON OF CRITERION RESPONSE METHOD WITH HETEROCHROMATIC FLICKER PHOTOMETRY D. REGAS. N. A. M. SCHELLART.H. SPEKREIJSEand T. J. 1. P. VAN DES BERG Department of Communication. University of Keels. Keels. Staffordshire, England and Laboratory of Medical Physics. University of Amsterdam, Amsterdam. The Netherlands (Rewired
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.Ibstract-In everyday vision the eye commonly functions while its state of adaptation is continuously changing rather than being held constant as in most scientific studies. IVe measured spectral luminosity curves for goldfish by recording ERG and tectal evoked response. Continuously changing the state of adaptation had a large effect at tectal level, but comparatively little at ERG level. Wtth changing adaptation the relation between light intensity and response amphtude showed gross hysteresis and nonlinearity at rectal level. This was not so marked at ERG level. and not at all at either level when adaptation was not changing. The widely-used criterion response method showed severe limitations when adaptation was changing. Heterochromatic flicker photometry was a more accurate. and considerably more sensitive (DOI 10: unit) way of measuring the spectral lumtnosity curve. ERG and TER gave similar luminosity curves. These curves did not depend on flicker frequency (3.5-20 Hz). adaptanon level (over 2.0 log units) or harmontc component. The luminosity curve was fitted by absorption spectra for cone photopigments: the red pigment dominated, and the blue contribution was negligible.
I\TRODCCTIOS In studies of colour vision it is of first importance to distinguish between the effects of stimulus wavelength and the effects of stimulus intensity. For this purpose it is necessary to have some agreed method of equating the intensities of differently-coloured stimuli according to visual efficiency rather than, for example, energy. For this the criterion response method (C.R. method) and the method of heterochromatic flicker photometry (H.F.P.) are used in electrophysiological studies of colour vision. In human electrophysiology both methods have been used (Cavonius. 1965; Armington, 1966; Riggs, Johnson and Schick, 1966; Regan. 1970a; Padmos and Van Norren, 1971) and heterochromatic flicker photometry has been reported to be markedly the more precise method (Regan. 1970a). In animal studies there seems to have been no comparative assessment of the two methods. and the criterion response approach has been most commonly adopted (DeValois and Jacobs, 1963; Sperling and Harwerth, 1971; Padmos and Van Norren, 1971). In goldfish this method has been used in ERG (Burckhardt. 1966, 1965; Schellart. Spekreijse and Van den Berg, 1971) horizontal cell (Spekreijse, Wagner and Wolbarsht, 1972) ganglion cell (Wagner. MacNichol and Wolbarsht. 1960; Beauchamp and Lovasik. 1973) and tectal recordings (Jacobson, 1964; Yager. Buck and Duncan. 1971). The H.F.P. method is to alternate a monochromatic light with a standard white reference light. The intensity of the coloured light is then varied until the electrophysiological response amplitude is a minimum (Regan 1970a). The visual efficiency of the monochromatic light is defined as inversely proportional to the intensity that gives this minimum response. This mea-
sure of visual efficiency is closely analogous to the psychophysical way in which, in human vision. the CIE defines the luminance of a light (Wyszecki and Stiles, 1967; Walsh, 193). In the CR. method the amplitude of the slectrophysiological response is plotted versus the intensity of a monochromatic stimulus. The visual effectiveness of a light is now defined as inversely proportional to the stimulus intensity required to elicit an (arbitrary) criterion response amplitude. It is clear that the visual effectiveness of light measured by the H.F.P. and CR. method may be quite different quantities and may relate differently to behavioral estimates. The two methods are not interchangeable even from a methodological point of view. For example. if there is a linear relation between stimulus intensity and response amplitude (e.g. curve A, Fig. 1) then a unique value of intensity gives the criterion response. and furthermore a sensitivity function measured by the C.R. method is independent of the choice of criterion level. If the response saturates (e.g. curve B, Fig. l), then the method is imprecise for high criterion response levels. For a non-monotonic function such as curve C in Fig. I several values of intensity correspond to a single criterion response level. In such a situation the C.R. method is inappropriate. This article evaluates the applicability of the H.F.P. and C.R. methods for the electrophysiological determination of the spectral luminosity curve. For this purpose the photopically evoked potentials derived from the eye (electroretinogram. ERG) and from the surface of the tectum (tectal evoked response. TER) of goldfish were chosen, because these responses can be maintained for a sufficiently long period. The common goldfish is an animal known to have good colour
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Fig. 1. This figure depicts hypothetical stimulus intensity versus response amplitude curves. Curve A iI1ustrates a linear relation, curve B saturation and curve C is an example of a non-monotonic relation. In situation B a criterion response method is usable only for levels below saturation. In situation C it is aimost impossible to determine an unambiguous criterion.
vision &kCIeary and Bernstein, 1959; Cronly-Dillon and Mum, 1965; Mum and Cron~y-Dillon, 1966: YaWzower and Bitterman, 1965).
METHODS Animal
preparation
AND
APPARATUS
and elecnoph~siological
recordiny
Common goldfish (Curassius auratus) 15-25 cm long were used. They were firmly held in a perspex tank with one eye. immediately behind a plane glass window, located on the optic axis of the visual stimulator. Anaesthesia was maintained by continuously circulating aerated water with 30-100 mg, I MS-222 (tricaine. Sandoz) over the gills. The temperature of this solution was stabilized at lg-22°C and maintained constant to within @j’C throughout an experiment. Part of the skull of the anaesthetized fish was removed and fatty tissue sucked away so as to expose the tectal lobes. The active tectal electrode was placed just medial to the projection of the optic axis of the stimulated (Ieft) eye on the surfaee of the contralateral tectal lobe (Jacobson and Gaze, 1964).The reference electrode was placed in contact with the exposed skull on the ipsilateral side, The ERG electrode was inserted about 3 mm through the sclera in the vitreous just behind the ora sertata. TER electrodes were platinumiridium wires of @3 mm dia insulated with varnish. ERG electrodes were varnished platinum-iridium wires electrolytically sharpened to a tip diameter of 0.1 mm. The tip was exposed over a length of 1 mm. Stimulation
A beamsplitter (BS) directed collimated light from a 500W xenon arc (X) into two beams (top half Fig. 2). Both interference (I) filters (Baltzers with a half width of 10 nm) and ND filters (Kodak Wratten) could be placed in either beam. Each beam was brought to a focus at an opaque light chopper driven by a pen motor (PM). In one beam an ND wedge (W) was placed immediately in front of the chopper to vary the intensity continuously over a -2-log unit range. The two beams were reunited at a second beamspiittsr and focussed (spot diameter 7 mm) onto a small piece of ping-pong ball diffuser (D) placed immediately in front of the fish’s cornea. The two light choppers were square wave driven by an oscillator (osc) at a frequency of F Hz. The phase relation (4) between the choppers could be continuously adjusted
Fig. 2. Schematic representation of optical stimulator (top half) and of data reduction equipment (bottom half). X-xenon lamp: BS-beam splitters: f-interference filters; ND-neutral density filters; W-motor driven neutral density wedge: PM I, PM L-light choppers mounted on penmotors; D-ping-pong ball diffuser. &c-sine and cosine generator with third variable phase output o,; A-power amplifier; Ml, M2. M3, ~f~multipljers; sq-squaring device; TQG-tuned quadrature oscillator; R-recorder.
through 360’. With the two choppers in antiphase the stimulus alternated between the two wavelengths 2F times per second. In general this alternation was accompanied by changes in light intensity. Sigqnalanal,vsis It is well known that a periodic F Hz waveform can be described as the sum of a series of pure sinusoida waveforms (harmonics) whose frequencies are multiples of the repetition frequency F. In our experiments the first harmonic component (F Hz) in the ERG and TER to periodic stimulation was extracted by separately multiplying (&fl and M2) the electrophysiological signals with a sine and cosine of frequency f (bottom half Fig. 2). The two products of multiplication t.u and _v)were then processed [fx-’ f J)*] to give the amplitude of the first harmonic component. After smoothing, this amplitude was displayed as a running average (Regan, 1966). For OK present experiments this form of display had the advantage of giving a continuous indication of the w+ayin which response amplitude varied when the stimulus was changed. The amplitude of the second harmonic (2FHz) component of the response was measured in a similar way to that ofthe fun~mentai con-ponent. Sines and cosines offrequency 2F were obtained by squaring (Sq) sin 2 x Fr and then feeding the frequency-doubled wave to a tuned quadrature oscillator (TQO). Usually the second harmonic component was relatively small (less than 20 per cent) in the ERG, but could be much larger in the TER. sometimes even larger than the fundamental component. RESCLTS
This section describes the photopically evoked potentials recorded from electrodes placed within the
Photometry
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udaptatim~ is mrch grearrt for TER thm ERG. The traces show amplitudes of Fig. 3. Contpresstcr the 10 Hz components of ERG and TER evoked by a 650 nm 10 Hz square wave modulated (Lociper cent) light stimulus. During this stimulation mean intensitT,was abruptly decreased by 0,610g unit and then abruptly restored to the initial level of Z W.m -_ Both ERG and TER gave a strong transient response immediately followin, 0 the decrease and the increase of intensity. However. though the ERG’s final steady amplitude strongly depended on the new steady intensity, the TER’s tinal amplitude was hardly affected bv the steady level of intensity. In other words. the ERG gave a strong sustained response to intensity. but the TER gave a weak sustained response (response expressed
as a percentage). and on ths tectum (TER) of common goldfish. Firstlv two methods for determining relative spectral luminosity curves are compared: the criterion response method and the heterochromatic flicker photometry method. Secondly the spectral luminosity curves are presented. These curves were determined by both methods for the ERG. and by the heterochromatic flicker photometry method for the TER.
eye (ERG)
Figure JA shows the amplitude of the IO-Hz component of the ERG during a continuous change of mean stimulus intensitv. Each trace of Fig. -1.4was produced by a 1.9~log unit increase of stimulus intensity followed by a 1.9-log unit decrease. The complete increase and decrease took 54 sec. The two superposed
A CO~IPARISOS METHOD
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ASD HETEROCHROMATIC PHOTOMETRY METHOD
FLICKER
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When stimulus intensity is abruptly changed. the amplitude of either ERG or TER takes some time to reach a new steady value. Figure 3 illustrates the time course of these adaptive changes. In this figure the amplitudes of the lo-Hz component of ERG and TER elicited by a IO-Hz squarewave-modulated (100 per cent) light are plotted while mean stimulus intensity was abruptly increased and then abruptly decreased. For an abrupt intensity step of 0.6 log, ERG amplitude followed roughly the change in intensity. and the adaptive change was 90 per cent complete after about 50 sec. This in contrast to the TER’s behaviour where a brisk initial increase in the amplitude was followed by a slow adaptive change. After the initia1 transient was completed. the final level of response amplitude was such that the effect of the change in stimulus intensity was reduced. This slow adaptive change was 90 per cent complete only after roughly 150sec. Moreover some hysteresis was evident in the TER in that the initial and final readings at 0.0 log units intensity were dissimilar. Since the sustained adaptive changes are much smaller for ERG than TER, this finding suggests that the locus ofcompressive adaptation is mainly central to ERG generation.
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Fig. 4 i\mplitude of the IOHz component of ERG (Fig. A) and TER (Fig. B) elicited by a 650 nm IO Hz square wave modulated (100 per cent) light whose mean intensity was continuously first increased and then decreased by 1.9 log unit within Ssec. Log intensity was triangularly modulated in time. The highest intensity was 5 W.m-‘. For both ERG and TER two superimposed recordings are shown. Figure -lC is a plot of the IOHz component of the TER as a function of the mean intensity of a 650 nm stimulus. After each change in mean intensity response amplitude was measured when the responw reached a steady level. The direction in which intensity was varied step-by-step is indicated with arrows.
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Fig. 5. Amplitude of the 20Hz component of the ERG (Fig. A) and TER (Fig. B) evoked by a IOHz square wave modulated monochromatic (650 nm) light whose mean intensity was continuously changed by 1.9 log unit as in Fig. JA and B. Two samples of each curve are shown.
Fig, 6. Amplitude of the 1OHz component of the TER ehcned by a IO Hz square-wave modulated (100 per cent) monochromatic (650nm) light, when mean light intensity was progressively changed at different rates. A complete increase and decrease of I.9 log units in mean light &ensity took 8Osec (left hand curve), 409~ (mid curve) and 20~ (right hand curve). The highest intensity used is 5 W.m’-‘. Two samples of each curse 3re shown.
The latter is not true for the XHz (second harmonic) ERG component which shows marked non-monotonic behaviour and hysteresis (Fig. where the criterion response method can conveniently 5A). be used for the fundamental component of the electroFigure 4B shows the IO-Hz component of the TER physiological response. elicited by a similar stimulus to that used in Fig. -I.-! for the ERG. The curve is quite non-monotonic. As Heterochromatic Picker photometry method stimulus intensity was progressively increased, reA monochromatic light (650nm) and a standard sponse amplitude first rose to a maximum, then fell to white light were alternated at a frequency of IOHz. The relative timing of the two stimuli was first a minimum, then rose again. An equal and opposite reduction of intensity did not reverse the changes in re- adjusted to give minimum response amplitude. Figure sponse amplitude; Fig. 4B shows marked hysteresis. 7 shows how the amplitude of the W-Hz response Also the 20-Hz component of the TER shows very component of the TER then varied it5 a function non-monotonic behaviour and hysteresis (Fig. 5B). of the red light’s intensity. The data of Fig. 7A were obtained when the intensity was changed conTherefore. both fundamental and second harmonic components of the TER should be taken into account tinuously, those of Figs. 7B and C with the intensity when using the TER for photometric measurement. of the monochromatic beam changed in staircase fashFigure 6 shows that the marked non-monotonicit) ion. In Figs. 7B and C two sets of points are shown, one for increasing and one for decreasing intensities. and hysteresis of the TER shown in Fig. 4B occurred Both sets of points have a sharp minimum. The posalso when mean intensity was changed by 1.9 log units ition of this minimum is slightly different (@I log in 10. 20 and 40 sec. Figure 4C is a plot of the amplitude of the IO-Hz units) when intensity is increased from very low values than when intensity is decreased from very component of the TER vs stimulus intensity. Response high values. Figure 7C illustrates how this hysteresis amplitude was measured by the step-by-step method after allowing sufficient time for response amplitude to effect is reduced and in addition how the minimum is sharpened when the light intensity is varied over reach a steady level. The curve is not so markedly nonmonotonic as that of Fig. 4B nor does it show such a smaller range (+030 log units). Whereas the ERG always showed one unique minimarked hysteresis. mum_ the TER had sometimes more than one miniFigures 3, 4, j and 6 show that the relationship mum. In those cases, however, one of the TER’s between TER amplitude and stimulus jntensity minima was clearly deepest and this was the setting depends on the way in which intensity is changed. we recorded. The minima for the fundamental and Since the relationship between response amplitude md second harmonic components fell within a range of intensity exhibits a non-monotonic behaviour and hys0.2log units. Moreover these minima always cointeresis in all these situations, the criterion response method is inappropriate for TER recordings. It follows cided with the ERG minimum. The heterochromatic flicker photometry method that this is also the case for the criterion response allowed the minimum to be measured to within method allied to feedback control of stimulus intensity as introduced by Padmos and Van Norren (1972). The 0.01 log units. Over a 5-hr experiment. the minimum point never altered by more than 0.2 log units. Long situation is quite different for the ERG recordings. curves are monotonic.
Photometry in goldrish
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Fig. 7. ,Amplitude of 10 Hz component in fhe TER elicited by a 10 Hz alternation between a monochromatic light and a standard white light plotted as a function of the mean intensity of the monochromatic beam. The reiatibe timing of the two stimuli was such as to gise minimum response amplitude. The ‘curve of Fig. .A was obtained when intensity of a 650nm beam was changed continuously. the data of Fig. B and C were obtained with step-by-step changes of mean intensit? of a 475 nm beam, The direction of the intensity change is indicated \sith arrows. The data of Fy. .-I are from another goldtish than those of Fig. B and C.
term drifts of the minimum caused by changes in the physiological condition of the preparation were much more marked for the TER than for the ERG. If the relative timing (phase difference) of monochromatic test light and comparison white light was changed, then the minimum might be both displaced and made more shallow. We checked the seriousness of this effect by measuring the minimum point of TER for three phase dil?erences benveen test and white stimuli (Fig. 8). Figure 8 shows that the balance point did not vary by more than O-1log units when the relative timing was altered oser a range of 50’. This held for both ERG and TER. Moreover for
frequencies up to 10 Hz both ERG and TER minima often diRered by no more than a few tens of degrees (Fig. 9). For all colours. the phase difference for minimum response was 1SO” to within a few tens of degrees. This is quite different for human evoked potentials for which large ~olour-dependent phase shifts have been reported (Regan, 1970b). Therefore in goldfish, since the error in finding the correct relative timing is unlikely to exceed 50’. then the error in the relative luminous efficiency-determined by heterochromatic flicker photometry-will be no more than 0.2 log units.
Fig. 9. ,Amplitude of the 10Hz component of the TER to the same stimulus as of Fig. 7B but for different relative timings of the monochromatic test and white comparison light. The phase differences are 135’. 150’ and 158’ respectively. The 1%’ data are also depicred in Fig. 7B.
Fig. 9. Response amplitude of the 10 Hz component of ERG (dashed line) and TER (continuous line) as a function of the phase difference between test (620nm) and white reference beam. The mean intensit? of the monochromatic beam was 2 W.m-’
Conclusion When stimulus intensity is continuously varied, the amplitude of the goldfish TER depends on whether stimulus intensity is decreasing or increasing and on how rapidly it is changing. Consequently the criterion response method is not a suitable way of using the TER as an index of the visual effectiveness of differently coloured lights. ERG amplitude is much less affected than TER amplitude by the direction and rate at which stimulus intensity is changed. However, these effects are not entirely absent so that the criterion response method should be applied with caution even for the ERG. SPECTR.AL LCSIISOSITY
CURVES
Figure 10 shows relative spectral luminosity curves for the TER determined with the H.F.P. method. White and monochromatic lights were alternated at a rate of 2.5, 10 and 20 Hz respectively. For each stimnlus frequency the response was anaIysed into first harmonic and second harmonic frequency components. which are represented in Fig. 10 by + and x respectively. At each stimulus frequency, first and second har-
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Fig. I I. Spectral luminosity curves for the fundamerital component of the ERG and the simultaneously recorded PER determined with the heterochromatic flicker photometry method. The ( x ) data were obtained with stimulus alternation of 10Hz. the (8) data with l0Hz and the i-k) data with j Hz. The 20 and j Hz data are from the same goldfish.
manic components of the TER gave simiiar results. The discrepancy never exceeded O-2 Iog units, a value comparable with the maximum scatter of individual settings over the 5 hr of an experiment. Average response waveforms were also recorded. Although the response waveform changed in the neiz+bourhood of the minimum, it was clear that the amplitude of the response fell to a minimum at a point close to that of the fun~mental and second harmonic minima. Figure 1I shows that both the ERG and TER spectral luminosity curves were little afkcted by stimulus alternation frequency. This holds over the range of frequencies investigated (2.5-20 Hz; seealso Fig. 10). Figure 12 compares the shapes of the relative spectra1 luminosity curves for the first harmonic of the ERG and TER. At the three levels of light a~p~tion
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Fig, 10. Specaai luminosity curves measured with TER by the method of heterochromatic flicker photometry. White and monochromatic lights were alternated at rates of 10, 10 and 1.5 Hz respectively. For each stimulus frequency the response was analysed into first harmonic ( +) and second harmonic component ( x ).
Fig. 13. Spectral luminosity cur\es for rhe first harmonic component of ERG ( x ) and simultaneousir-recorded TER (+) obtained at three levels of light adaptation over a range of 2.0 log: units. White and monochromatic beams were alternated at a rate of lOHa. Thz top and bottom sets of data are from the same goldfish.
Photomerry
in goldfijh
used. ths correspondsnce between the ERG and TER curves is within @35 log units (see also Fig. 11). Uoreover. a ?&log unit change in adaptation Irvel had littls effect on the shape of th2 curves. DISCI_ SSIOU
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vision the eye’s state of adaptation is / / / Ii continuously changing. so that methods of studying 7.90 500 600 400 vision in this situatron might well be at least as relzvant wavelength [nmi to normal vision as the rssults of experiments where Fig. 13. Relative spectral luminosity curves of goldfish adaptation is carefully held constant. We find that when adaptation level is changed continuously the determined with the criterion response method i x) and electrophysiological responses recorded from a high the hetsrochromatic Rickrr photometry method (+). The level in goldfish visual system (the tectum) show gross criterion response method could only be used with the ERG. Both ERG and YER data w2re used to calculate hysteresis and nonlin2arlt~. Therrfore. th2 criterion rethe other luminosity curve. Both sets of data are corrected sponse method is inappropriate for measuring relative for ocular absorption. Algebraic summation of the absorpspectral sensitivity at tectal level. However, ths method tion spectra of the three goldfish cone-pigments resulted of heterochromatic Bicker photometry gives precise in a fit of the luminosity data on a least square basis results even when adaptation is continuously changgiven by the continuous curw. With the absorption spectra normalized. the weightint factor for the “red” pigment ing. since problems due to hysteresis and dynamic behaviour are much less than with the criterion re- is 0.52. for the “green” pigment 0.22 and for the -‘blue” pigment 043. sponse method. On the other hand, in the commonly-employed but perhaps less realistic situation where adaptation level heterochromatic Bicker photometry method. Furtheris allowed sufficient time to settle down before recording any response. hystzresis and nonlinearities are more, since HFP is a comparison method. it has the advantage that it is not the actual size of the response, much smaller. The methods of criterion response and but the position of th2 minimum that is the measure. heterochromatic flicker photometry then give similar Therefore effects which restrict the accuracy and reproresults. Ths high sensitivity of electrophysioiogical heter- ducibility of electrophysiological measurements, such achromatic flicker photometry is inherent to the as vaiiatton in the physiological condition of the prepmethod. As discussed previousiy (Regan, 197Oa) re- aration. changes in the electrical proper&s at the recording site, hysteresis and non-monotonicity of response variability and certain measurement errors sponse curves, are of less importance in heterochromahave less effect when th2 eye is sirnultaneousl~stimulated with test and standard stimuli than when the eye tic flicker photometry. However, under appropriate conditions both is stimulated sequentially by different monochromatic methods can give directly comparable results. This is lights. In addition. the noise of the recording is of less illustrated in Fig. 13, where the spectral luminosity importance in hzterochromatic flicker photometry btlcause the effective stimulus parameter is ~o~[~~~r~~~~curves of goldfish determined on the basis of either the criterion response or the heterochromatic flicker phocfeprli(fractional change of intensity during a stimulus cycle). Starting in a condition that the effective modutometry method are presented. The ERG data lation depth is near zero. a doubling of the test beam’s obtained with the criterion response method are indiintensity (03 log units increase) results in an effective cated with x ‘s. The f’s stand for ERG and TER data modulation depth of about 33 per cent. In response to determined with the heterochromatic flicker photosuch an increase of modulation depth the amplituds of metry method. Both sets of data agree closely. They th2 electrophysiological signal can rise to considerably have been corrected for ocular absorption [Burckmore than 33 per cent of maximum amplitude due to hardt 1966). Since the absorption spectra of the cone comprzssive nonlinearity (saturation). For example a pigments of goldfish are known (Liebman and Entine, doubling of stimulus intensity causes response ampli19M; Marks, 19653 and since there is evidence that tude in Fig. 7B to rise from near-zero to 75 per cent retinal processing is substantially linear, at any rate of its maximum value. In contrast. the zffective stimuperipherally (Spekreijse and Norton. 1970: Norton. lus parameter in the critsrion response method is the Spekreijse. Wagner and WoIbarsh~ 1970; Spekreijse absolute intensity of the light. In general, a given and van den Berg. 1971; Schellart and Spekreijse, change of absolute intensity produces a weaker r2- 1972). we thought it of interest to see whether the sponse than a corresponding change of modulation luminosity functions of goldfish could be fitted with an depth. For example, in Fig. 4C before saturation algebraic summation of the absorption spectra of the occurs response amplitude changes by a verv small three cone pigments. The result of such a fit on a leastpercentage (about 70 per cent), when intensity’is dousquare basis IS presented by the full curve shown in bled. Fig. 13. Not only for this fit, but also for the other Thus in most cases. in order to change response luminosity functions presented in this paper. we found amplitude by a given number of microvolts a much that no significant contribution of the blue cone pigsmallsr change of stimulus intensity is required when ment was needed. Furthermore, when the maxima of the heterochromatic flicker photometry method is the absorption spectra were normalized. the contribuused. Consequently, for a given accuracv of electrophytion of the red pigment was always about four times siological measurement the correspondmg accuracy of higher than that of the green cone pigment. Also in electroph~siolog~cal photometry is higher for the man a negligible contribution of the blue pigment is In ever@ay
needed to fit the luminositv function determined by the method of hetsrochroma& flicker photometry. In view of the firm basis for three cone pigments in goldfish retina the absence of the “blue” spectrum in the luminosit!- function might seem surprising. Houecer, the per cent blue spectra found in both microspectrophotometrical (Marks. 1965) and ganglion cell studies (Spekreijse el Eli.. 1972) is as low as 5 to 7 per cent. Values of the same order are also derived for the cone action spectra (Tomita, Kaneko. Muraksmi and Pautler, 1967) and horizontal cell responses (Spekreijx and Norton, 1970) in carp retina. Granted weighting of the contributions of the cone systems on the basis of the actual number of cones, such a low value is within the accuracy of our data.
(I) This paper compares the relation between light intensity and electrophysiological responses in common goldfish in conditions where (a) the eye’s level of adaptation was changing continuously and (b) the eye’s level of adaptation was not changing. We present relative spectral luminosity curves for the common goldfish measured by recording the electroretinogram (ERG) and tectal evoked response (TER). (2) Two methods were compared: the heterochromatic flicker photometry method and the criterion response method. The first method is analogous to the way in which luminance is defined in photometry. (3) With continuously changing adaptation, heterochromatic flicker photometry and criterion response method gave rather similar estimates of spectral sensitivity for ERG recording. However, the heterochromatic flicker photometry method was more accurate. (4) When adaptation was changing continuously, the criterion response method had severe limitations when used with TER recording. This was due both to complex adaptive behaviour and to hysteresis. These effects invalidate the criterion response method for determining the relative spectral luminosity under changing conditions by tectal recording in goldfish. Although smaller, such effects could sometimes also be observed for the ERG. These findings suggest that in spite of the widespread use of the CR method in electrophysiological studies of colour vision, the method should be regarded with caution. i5) With the heterochromatic flicker photometry method ERG and TER had closely similar (within 0.2 log units) relative spectral luminosity curves. The shape of these curves did not vary with flicker frequency (from 2.5 to 20Hz), adaptation level (over a range of 2.0 log units), nor with the harmonic component considered (first and second harmonic). (6) The sensitivity of the heterochromatic flicker photometry method, assessed by the sensitivity of setting the response minimum, could be as high as @OI log units. Accuracy was lower, though over an experimental time of some 5 hr the setting varied by no more than 0.2 log units for the TER and even less for the ERG. (7) The heterochromatic flicker photometry method always gave a unique minimum for the fundamental ERG component. For the TER there was either (a) one
unique minimum, or (b) when there were several minima one of them was not only cleaflv deepest, but also coincided with the unique ERG mmlmum.
(S) Although the minimum of the hererochromattc Hicker photometry method is affected by the relative timing (relative phase) of. the two alrrrnating beams. the setting remains within 0.1 log units over a 50. change of relative phase. (9) The spectral luminosit\ curves :::c discussed in terms of trlchomacv of vision and titted with the absorption spectra otthe three goldfish cone pigments. In the fit the red cone pigment dominates. .-lc6r~o~vl~dge~~~erlrs--Travel from Amsterdam and stay ar Keele for H. Spckreijsr. N. ;\. ht. Schelhrt and T. 1. T. P van den Berg was funded by the European Training Programme in Brain and Behaviour Research. D. Regan was supported by rhe Medical Research Courxil who provided part of the equipment. We thank R. F. Cdr:uright for invaluable technical assistance.
REFERESCES
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1, 12_$-233.
Beauchamp R. D. and LobasiL J. V. ( 1973!Blue mechanism response of single goldfish optic fibers. J. .Vncrophrsio/. 36, 9X-939. Burckhardt D. A. (1966) The goldfish eiectroretinogram: relation between photopic spectral sensitivity functions and cone absorption spectra. Vision Rzi. 6, 517-532. Burckhardt D. A. I 1965)Cone action specrrs: evidence from the eoldfish electroretinogram. Visiorl KG. 8. 339-353. CavoGus C. R. (1965) Evoked response o
Jacobson M. and Gaze M. (1961): Types oi bisual response from single units in the optic tectum and optic nerve of the goldfish. Q. JI exp. Physioi. 49, 199-209. Liebman P. A. and Entine G. f 1964) Sensitive low-light-level microspectrophotometer: detection of photosensitive pigments of retinal cones. J. opt. Sot. .4m. 54, 1151-1459. Marks W. B. (1965) Visual pigments of single goldfish cones. J. Phvsiol.. LotId. 178. 14-3’.McCleary R. .A.and Bernstein J. (1959) A unique method for control of brightness cues in rhe stud) of color vision in fish. Physiol. 2001.32, 33-l-292. Muntz W. and Cronlv-Dillon J. R. (19661 Color discrimination in goldfish. .-km. Behnc. 14; 311-166. Xorton A. L., Spekreijse H.. Wolbarsht 31. L. and Wagner H. G. (196S)Receptivetield organisation ofthe S-potential. Science, It’ash. 160, 10’1-1032. Padmos P. and Van Norren D. (1971) Cone spectral sensitivity and chromatic adaptation as rehealed by human Ricker-eiectroretinography. F.&ion Res. 11. 2742. Padmos P. and Van Norren D. (1972) The vector voltmeter as a tool fo measure electroretinogram +ectral sensitivity and dark adautation. I~esr. Ookthaln?of. 9, 783-739. Reean D. (19661 Some characte&cs of average steadyState and transient responses evoked b\ modulated light. Elecnoei~cr~h. c/in. ,Vetrroohvsiol. 20, 2%X8. Regan D. (197Ga) An obje&i;e method of measuring the relative spectral luminosity curve in man. J. opt. Sot. .qm. 60, 856459. Regan D. (1970b) Evoked porentiai and psychophysical COTrelates of changes in stimulus colour ar.d intensity. C’isior! Rex 10, 16.;- 17’3
Photometry in goldfish Riggs L. A., Johnson E. P. and Schick A. M. L. (1966) Electrical responses of the human eye to changes in wavelength of the stimulating light. J. opt. Sot. Am. 56. 16211627. Schellart N. A. M. and Spekreijse H. (1972) Dynamic characteristics of retinal ganglion cell responses in goldfish. J. qen. Physiol. 59, l-21. Schellart N. A. M.. Spekreijse H. and Van den Berg T. J. T. P. (1971) Influence of temperature on retinal ganglion cell response and ERG of goldfish. .I. Physiol.. Land. (in press). Spekreijse H. and Norton A. L. (1970) The dynamic characteristics ofcolor-coded S-potentials. 1. gen. Physiol. 56, l12. Spekreijse H., Wagner H. G. and Wolbarsht M. L. (1972) Spectral and spatial coding of ganglion cell responses in goldfish retina. J. Nelrroohvsiol. 35. 73-86. SGkreijse H. and Van den ‘Berg T. J. T. P. (1971) Interaction between color and spatial coded processes converging to retinal ganglion cells in goldfish. J. Physiol., Land. 215, 6’9-692.
807
Sperling H. G. and Harwerth R. S. (1971) Red green cone interactions in the increment threshold spectral sensitivity of p&rates. Science 172. 180-18-t. Tomita T., Kaneko A.. Murakami M. and Pautler E. L. (1967) Spectral response curves of single cones in the carp. vision Res. 7, 5 19-53 1. Wagner H. G., MacNichol E. F. Jr. and Wolbarsht M. L. (1960) The response properties of single ganghon cells in the goldfish retina. J. qen. Physiol. 48, mppl. G-62. Walsh-G. W. T. (1953) Photometry. Constabie, London. Wvszecki G. and Stiles W. S. (1967) Co/our Science. Wilev. ‘New York. Yager D., Buck S. and Duncan I. (1971) Effects of temperature on the visually evoked tectal potential and brightness perception in gold&h. Vision Res. 11, 849-860. Yarczower and Bitterman (1965) Stimulus generalization in the goldfish. In Stimulus Generakation (Edited by Mosotfsky D.), pp. 139-192. Stanford University Press, Stanford. I
6 Srprembrr
197-t; in recisedfbrm
27 Yocembrr
1971)
.Ibstract-In everyday vision the eye commonly functions while its state of adaptation is continuously changing rather than being held constant as in most scientific studies. IVe measured spectral luminosity curves for goldfish by recording ERG and tectal evoked response. Continuously changing the state of adaptation had a large effect at tectal level, but comparatively little at ERG level. Wtth changing adaptation the relation between light intensity and response amphtude showed gross hysteresis and nonlinearity at rectal level. This was not so marked at ERG level. and not at all at either level when adaptation was not changing. The widely-used criterion response method showed severe limitations when adaptation was changing. Heterochromatic flicker photometry was a more accurate. and considerably more sensitive (DOI 10: unit) way of measuring the spectral lumtnosity curve. ERG and TER gave similar luminosity curves. These curves did not depend on flicker frequency (3.5-20 Hz). adaptanon level (over 2.0 log units) or harmontc component. The luminosity curve was fitted by absorption spectra for cone photopigments: the red pigment dominated, and the blue contribution was negligible.
I\TRODCCTIOS In studies of colour vision it is of first importance to distinguish between the effects of stimulus wavelength and the effects of stimulus intensity. For this purpose it is necessary to have some agreed method of equating the intensities of differently-coloured stimuli according to visual efficiency rather than, for example, energy. For this the criterion response method (C.R. method) and the method of heterochromatic flicker photometry (H.F.P.) are used in electrophysiological studies of colour vision. In human electrophysiology both methods have been used (Cavonius. 1965; Armington, 1966; Riggs, Johnson and Schick, 1966; Regan. 1970a; Padmos and Van Norren, 1971) and heterochromatic flicker photometry has been reported to be markedly the more precise method (Regan. 1970a). In animal studies there seems to have been no comparative assessment of the two methods. and the criterion response approach has been most commonly adopted (DeValois and Jacobs, 1963; Sperling and Harwerth, 1971; Padmos and Van Norren, 1971). In goldfish this method has been used in ERG (Burckhardt. 1966, 1965; Schellart. Spekreijse and Van den Berg, 1971) horizontal cell (Spekreijse, Wagner and Wolbarsht, 1972) ganglion cell (Wagner. MacNichol and Wolbarsht. 1960; Beauchamp and Lovasik. 1973) and tectal recordings (Jacobson, 1964; Yager. Buck and Duncan. 1971). The H.F.P. method is to alternate a monochromatic light with a standard white reference light. The intensity of the coloured light is then varied until the electrophysiological response amplitude is a minimum (Regan 1970a). The visual efficiency of the monochromatic light is defined as inversely proportional to the intensity that gives this minimum response. This mea-
sure of visual efficiency is closely analogous to the psychophysical way in which, in human vision. the CIE defines the luminance of a light (Wyszecki and Stiles, 1967; Walsh, 193). In the CR. method the amplitude of the slectrophysiological response is plotted versus the intensity of a monochromatic stimulus. The visual effectiveness of a light is now defined as inversely proportional to the stimulus intensity required to elicit an (arbitrary) criterion response amplitude. It is clear that the visual effectiveness of light measured by the H.F.P. and CR. method may be quite different quantities and may relate differently to behavioral estimates. The two methods are not interchangeable even from a methodological point of view. For example. if there is a linear relation between stimulus intensity and response amplitude (e.g. curve A, Fig. 1) then a unique value of intensity gives the criterion response. and furthermore a sensitivity function measured by the C.R. method is independent of the choice of criterion level. If the response saturates (e.g. curve B, Fig. l), then the method is imprecise for high criterion response levels. For a non-monotonic function such as curve C in Fig. I several values of intensity correspond to a single criterion response level. In such a situation the C.R. method is inappropriate. This article evaluates the applicability of the H.F.P. and C.R. methods for the electrophysiological determination of the spectral luminosity curve. For this purpose the photopically evoked potentials derived from the eye (electroretinogram. ERG) and from the surface of the tectum (tectal evoked response. TER) of goldfish were chosen, because these responses can be maintained for a sufficiently long period. The common goldfish is an animal known to have good colour
+
i
ND
l-l
r-l
Fig. 1. This figure depicts hypothetical stimulus intensity versus response amplitude curves. Curve A iI1ustrates a linear relation, curve B saturation and curve C is an example of a non-monotonic relation. In situation B a criterion response method is usable only for levels below saturation. In situation C it is aimost impossible to determine an unambiguous criterion.
vision &kCIeary and Bernstein, 1959; Cronly-Dillon and Mum, 1965; Mum and Cron~y-Dillon, 1966: YaWzower and Bitterman, 1965).
METHODS Animal
preparation
AND
APPARATUS
and elecnoph~siological
recordiny
Common goldfish (Curassius auratus) 15-25 cm long were used. They were firmly held in a perspex tank with one eye. immediately behind a plane glass window, located on the optic axis of the visual stimulator. Anaesthesia was maintained by continuously circulating aerated water with 30-100 mg, I MS-222 (tricaine. Sandoz) over the gills. The temperature of this solution was stabilized at lg-22°C and maintained constant to within @j’C throughout an experiment. Part of the skull of the anaesthetized fish was removed and fatty tissue sucked away so as to expose the tectal lobes. The active tectal electrode was placed just medial to the projection of the optic axis of the stimulated (Ieft) eye on the surfaee of the contralateral tectal lobe (Jacobson and Gaze, 1964).The reference electrode was placed in contact with the exposed skull on the ipsilateral side, The ERG electrode was inserted about 3 mm through the sclera in the vitreous just behind the ora sertata. TER electrodes were platinumiridium wires of @3 mm dia insulated with varnish. ERG electrodes were varnished platinum-iridium wires electrolytically sharpened to a tip diameter of 0.1 mm. The tip was exposed over a length of 1 mm. Stimulation
A beamsplitter (BS) directed collimated light from a 500W xenon arc (X) into two beams (top half Fig. 2). Both interference (I) filters (Baltzers with a half width of 10 nm) and ND filters (Kodak Wratten) could be placed in either beam. Each beam was brought to a focus at an opaque light chopper driven by a pen motor (PM). In one beam an ND wedge (W) was placed immediately in front of the chopper to vary the intensity continuously over a -2-log unit range. The two beams were reunited at a second beamspiittsr and focussed (spot diameter 7 mm) onto a small piece of ping-pong ball diffuser (D) placed immediately in front of the fish’s cornea. The two light choppers were square wave driven by an oscillator (osc) at a frequency of F Hz. The phase relation (4) between the choppers could be continuously adjusted
Fig. 2. Schematic representation of optical stimulator (top half) and of data reduction equipment (bottom half). X-xenon lamp: BS-beam splitters: f-interference filters; ND-neutral density filters; W-motor driven neutral density wedge: PM I, PM L-light choppers mounted on penmotors; D-ping-pong ball diffuser. &c-sine and cosine generator with third variable phase output o,; A-power amplifier; Ml, M2. M3, ~f~multipljers; sq-squaring device; TQG-tuned quadrature oscillator; R-recorder.
through 360’. With the two choppers in antiphase the stimulus alternated between the two wavelengths 2F times per second. In general this alternation was accompanied by changes in light intensity. Sigqnalanal,vsis It is well known that a periodic F Hz waveform can be described as the sum of a series of pure sinusoida waveforms (harmonics) whose frequencies are multiples of the repetition frequency F. In our experiments the first harmonic component (F Hz) in the ERG and TER to periodic stimulation was extracted by separately multiplying (&fl and M2) the electrophysiological signals with a sine and cosine of frequency f (bottom half Fig. 2). The two products of multiplication t.u and _v)were then processed [fx-’ f J)*] to give the amplitude of the first harmonic component. After smoothing, this amplitude was displayed as a running average (Regan, 1966). For OK present experiments this form of display had the advantage of giving a continuous indication of the w+ayin which response amplitude varied when the stimulus was changed. The amplitude of the second harmonic (2FHz) component of the response was measured in a similar way to that ofthe fun~mentai con-ponent. Sines and cosines offrequency 2F were obtained by squaring (Sq) sin 2 x Fr and then feeding the frequency-doubled wave to a tuned quadrature oscillator (TQO). Usually the second harmonic component was relatively small (less than 20 per cent) in the ERG, but could be much larger in the TER. sometimes even larger than the fundamental component. RESCLTS
This section describes the photopically evoked potentials recorded from electrodes placed within the
Photometry
301
in goldfish
1 / I
TE?
I
1
1
udaptatim~ is mrch grearrt for TER thm ERG. The traces show amplitudes of Fig. 3. Contpresstcr the 10 Hz components of ERG and TER evoked by a 650 nm 10 Hz square wave modulated (Lociper cent) light stimulus. During this stimulation mean intensitT,was abruptly decreased by 0,610g unit and then abruptly restored to the initial level of Z W.m -_ Both ERG and TER gave a strong transient response immediately followin, 0 the decrease and the increase of intensity. However. though the ERG’s final steady amplitude strongly depended on the new steady intensity, the TER’s tinal amplitude was hardly affected bv the steady level of intensity. In other words. the ERG gave a strong sustained response to intensity. but the TER gave a weak sustained response (response expressed
as a percentage). and on ths tectum (TER) of common goldfish. Firstlv two methods for determining relative spectral luminosity curves are compared: the criterion response method and the heterochromatic flicker photometry method. Secondly the spectral luminosity curves are presented. These curves were determined by both methods for the ERG. and by the heterochromatic flicker photometry method for the TER.
eye (ERG)
Figure JA shows the amplitude of the IO-Hz component of the ERG during a continuous change of mean stimulus intensitv. Each trace of Fig. -1.4was produced by a 1.9~log unit increase of stimulus intensity followed by a 1.9-log unit decrease. The complete increase and decrease took 54 sec. The two superposed
A CO~IPARISOS METHOD
Crirrriotl
OF CRITERIOS
r i’
RESPOSE
ASD HETEROCHROMATIC PHOTOMETRY METHOD
FLICKER
resporlsr method
When stimulus intensity is abruptly changed. the amplitude of either ERG or TER takes some time to reach a new steady value. Figure 3 illustrates the time course of these adaptive changes. In this figure the amplitudes of the lo-Hz component of ERG and TER elicited by a IO-Hz squarewave-modulated (100 per cent) light are plotted while mean stimulus intensity was abruptly increased and then abruptly decreased. For an abrupt intensity step of 0.6 log, ERG amplitude followed roughly the change in intensity. and the adaptive change was 90 per cent complete after about 50 sec. This in contrast to the TER’s behaviour where a brisk initial increase in the amplitude was followed by a slow adaptive change. After the initia1 transient was completed. the final level of response amplitude was such that the effect of the change in stimulus intensity was reduced. This slow adaptive change was 90 per cent complete only after roughly 150sec. Moreover some hysteresis was evident in the TER in that the initial and final readings at 0.0 log units intensity were dissimilar. Since the sustained adaptive changes are much smaller for ERG than TER, this finding suggests that the locus ofcompressive adaptation is mainly central to ERG generation.
i I ERG
/ 1 / / / ,I ,
Fig. 4 i\mplitude of the IOHz component of ERG (Fig. A) and TER (Fig. B) elicited by a 650 nm IO Hz square wave modulated (100 per cent) light whose mean intensity was continuously first increased and then decreased by 1.9 log unit within Ssec. Log intensity was triangularly modulated in time. The highest intensity was 5 W.m-‘. For both ERG and TER two superimposed recordings are shown. Figure -lC is a plot of the IOHz component of the TER as a function of the mean intensity of a 650 nm stimulus. After each change in mean intensity response amplitude was measured when the responw reached a steady level. The direction in which intensity was varied step-by-step is indicated with arrows.
SO’
B TE
Fig. 5. Amplitude of the 20Hz component of the ERG (Fig. A) and TER (Fig. B) evoked by a IOHz square wave modulated monochromatic (650 nm) light whose mean intensity was continuously changed by 1.9 log unit as in Fig. JA and B. Two samples of each curve are shown.
Fig, 6. Amplitude of the 1OHz component of the TER ehcned by a IO Hz square-wave modulated (100 per cent) monochromatic (650nm) light, when mean light intensity was progressively changed at different rates. A complete increase and decrease of I.9 log units in mean light &ensity took 8Osec (left hand curve), 409~ (mid curve) and 20~ (right hand curve). The highest intensity used is 5 W.m’-‘. Two samples of each curse 3re shown.
The latter is not true for the XHz (second harmonic) ERG component which shows marked non-monotonic behaviour and hysteresis (Fig. where the criterion response method can conveniently 5A). be used for the fundamental component of the electroFigure 4B shows the IO-Hz component of the TER physiological response. elicited by a similar stimulus to that used in Fig. -I.-! for the ERG. The curve is quite non-monotonic. As Heterochromatic Picker photometry method stimulus intensity was progressively increased, reA monochromatic light (650nm) and a standard sponse amplitude first rose to a maximum, then fell to white light were alternated at a frequency of IOHz. The relative timing of the two stimuli was first a minimum, then rose again. An equal and opposite reduction of intensity did not reverse the changes in re- adjusted to give minimum response amplitude. Figure sponse amplitude; Fig. 4B shows marked hysteresis. 7 shows how the amplitude of the W-Hz response Also the 20-Hz component of the TER shows very component of the TER then varied it5 a function non-monotonic behaviour and hysteresis (Fig. 5B). of the red light’s intensity. The data of Fig. 7A were obtained when the intensity was changed conTherefore. both fundamental and second harmonic components of the TER should be taken into account tinuously, those of Figs. 7B and C with the intensity when using the TER for photometric measurement. of the monochromatic beam changed in staircase fashFigure 6 shows that the marked non-monotonicit) ion. In Figs. 7B and C two sets of points are shown, one for increasing and one for decreasing intensities. and hysteresis of the TER shown in Fig. 4B occurred Both sets of points have a sharp minimum. The posalso when mean intensity was changed by 1.9 log units ition of this minimum is slightly different (@I log in 10. 20 and 40 sec. Figure 4C is a plot of the amplitude of the IO-Hz units) when intensity is increased from very low values than when intensity is decreased from very component of the TER vs stimulus intensity. Response high values. Figure 7C illustrates how this hysteresis amplitude was measured by the step-by-step method after allowing sufficient time for response amplitude to effect is reduced and in addition how the minimum is sharpened when the light intensity is varied over reach a steady level. The curve is not so markedly nonmonotonic as that of Fig. 4B nor does it show such a smaller range (+030 log units). Whereas the ERG always showed one unique minimarked hysteresis. mum_ the TER had sometimes more than one miniFigures 3, 4, j and 6 show that the relationship mum. In those cases, however, one of the TER’s between TER amplitude and stimulus jntensity minima was clearly deepest and this was the setting depends on the way in which intensity is changed. we recorded. The minima for the fundamental and Since the relationship between response amplitude md second harmonic components fell within a range of intensity exhibits a non-monotonic behaviour and hys0.2log units. Moreover these minima always cointeresis in all these situations, the criterion response method is inappropriate for TER recordings. It follows cided with the ERG minimum. The heterochromatic flicker photometry method that this is also the case for the criterion response allowed the minimum to be measured to within method allied to feedback control of stimulus intensity as introduced by Padmos and Van Norren (1972). The 0.01 log units. Over a 5-hr experiment. the minimum point never altered by more than 0.2 log units. Long situation is quite different for the ERG recordings. curves are monotonic.
Photometry in goldrish
c
Fig. 7. ,Amplitude of 10 Hz component in fhe TER elicited by a 10 Hz alternation between a monochromatic light and a standard white light plotted as a function of the mean intensity of the monochromatic beam. The reiatibe timing of the two stimuli was such as to gise minimum response amplitude. The ‘curve of Fig. .A was obtained when intensity of a 650nm beam was changed continuously. the data of Fig. B and C were obtained with step-by-step changes of mean intensit? of a 475 nm beam, The direction of the intensity change is indicated \sith arrows. The data of Fy. .-I are from another goldtish than those of Fig. B and C.
term drifts of the minimum caused by changes in the physiological condition of the preparation were much more marked for the TER than for the ERG. If the relative timing (phase difference) of monochromatic test light and comparison white light was changed, then the minimum might be both displaced and made more shallow. We checked the seriousness of this effect by measuring the minimum point of TER for three phase dil?erences benveen test and white stimuli (Fig. 8). Figure 8 shows that the balance point did not vary by more than O-1log units when the relative timing was altered oser a range of 50’. This held for both ERG and TER. Moreover for
frequencies up to 10 Hz both ERG and TER minima often diRered by no more than a few tens of degrees (Fig. 9). For all colours. the phase difference for minimum response was 1SO” to within a few tens of degrees. This is quite different for human evoked potentials for which large ~olour-dependent phase shifts have been reported (Regan, 1970b). Therefore in goldfish, since the error in finding the correct relative timing is unlikely to exceed 50’. then the error in the relative luminous efficiency-determined by heterochromatic flicker photometry-will be no more than 0.2 log units.
Fig. 9. ,Amplitude of the 10Hz component of the TER to the same stimulus as of Fig. 7B but for different relative timings of the monochromatic test and white comparison light. The phase differences are 135’. 150’ and 158’ respectively. The 1%’ data are also depicred in Fig. 7B.
Fig. 9. Response amplitude of the 10 Hz component of ERG (dashed line) and TER (continuous line) as a function of the phase difference between test (620nm) and white reference beam. The mean intensit? of the monochromatic beam was 2 W.m-’
Conclusion When stimulus intensity is continuously varied, the amplitude of the goldfish TER depends on whether stimulus intensity is decreasing or increasing and on how rapidly it is changing. Consequently the criterion response method is not a suitable way of using the TER as an index of the visual effectiveness of differently coloured lights. ERG amplitude is much less affected than TER amplitude by the direction and rate at which stimulus intensity is changed. However, these effects are not entirely absent so that the criterion response method should be applied with caution even for the ERG. SPECTR.AL LCSIISOSITY
CURVES
Figure 10 shows relative spectral luminosity curves for the TER determined with the H.F.P. method. White and monochromatic lights were alternated at a rate of 2.5, 10 and 20 Hz respectively. For each stimnlus frequency the response was anaIysed into first harmonic and second harmonic frequency components. which are represented in Fig. 10 by + and x respectively. At each stimulus frequency, first and second har-
12
i
0.8 i c
20Hr
*
.
f
500
Fig. I I. Spectral luminosity curves for the fundamerital component of the ERG and the simultaneously recorded PER determined with the heterochromatic flicker photometry method. The ( x ) data were obtained with stimulus alternation of 10Hz. the (8) data with l0Hz and the i-k) data with j Hz. The 20 and j Hz data are from the same goldfish.
manic components of the TER gave simiiar results. The discrepancy never exceeded O-2 Iog units, a value comparable with the maximum scatter of individual settings over the 5 hr of an experiment. Average response waveforms were also recorded. Although the response waveform changed in the neiz+bourhood of the minimum, it was clear that the amplitude of the response fell to a minimum at a point close to that of the fun~mental and second harmonic minima. Figure 1I shows that both the ERG and TER spectral luminosity curves were little afkcted by stimulus alternation frequency. This holds over the range of frequencies investigated (2.5-20 Hz; seealso Fig. 10). Figure 12 compares the shapes of the relative spectra1 luminosity curves for the first harmonic of the ERG and TER. At the three levels of light a~p~tion
7
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400 500
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Fig, 10. Specaai luminosity curves measured with TER by the method of heterochromatic flicker photometry. White and monochromatic lights were alternated at rates of 10, 10 and 1.5 Hz respectively. For each stimulus frequency the response was analysed into first harmonic ( +) and second harmonic component ( x ).
Fig. 13. Spectral luminosity cur\es for rhe first harmonic component of ERG ( x ) and simultaneousir-recorded TER (+) obtained at three levels of light adaptation over a range of 2.0 log: units. White and monochromatic beams were alternated at a rate of lOHa. Thz top and bottom sets of data are from the same goldfish.
Photomerry
in goldfijh
used. ths correspondsnce between the ERG and TER curves is within @35 log units (see also Fig. 11). Uoreover. a ?&log unit change in adaptation Irvel had littls effect on the shape of th2 curves. DISCI_ SSIOU
0.01
vision the eye’s state of adaptation is / / / Ii continuously changing. so that methods of studying 7.90 500 600 400 vision in this situatron might well be at least as relzvant wavelength [nmi to normal vision as the rssults of experiments where Fig. 13. Relative spectral luminosity curves of goldfish adaptation is carefully held constant. We find that when adaptation level is changed continuously the determined with the criterion response method i x) and electrophysiological responses recorded from a high the hetsrochromatic Rickrr photometry method (+). The level in goldfish visual system (the tectum) show gross criterion response method could only be used with the ERG. Both ERG and YER data w2re used to calculate hysteresis and nonlin2arlt~. Therrfore. th2 criterion rethe other luminosity curve. Both sets of data are corrected sponse method is inappropriate for measuring relative for ocular absorption. Algebraic summation of the absorpspectral sensitivity at tectal level. However, ths method tion spectra of the three goldfish cone-pigments resulted of heterochromatic Bicker photometry gives precise in a fit of the luminosity data on a least square basis results even when adaptation is continuously changgiven by the continuous curw. With the absorption spectra normalized. the weightint factor for the “red” pigment ing. since problems due to hysteresis and dynamic behaviour are much less than with the criterion re- is 0.52. for the “green” pigment 0.22 and for the -‘blue” pigment 043. sponse method. On the other hand, in the commonly-employed but perhaps less realistic situation where adaptation level heterochromatic Bicker photometry method. Furtheris allowed sufficient time to settle down before recording any response. hystzresis and nonlinearities are more, since HFP is a comparison method. it has the advantage that it is not the actual size of the response, much smaller. The methods of criterion response and but the position of th2 minimum that is the measure. heterochromatic flicker photometry then give similar Therefore effects which restrict the accuracy and reproresults. Ths high sensitivity of electrophysioiogical heter- ducibility of electrophysiological measurements, such achromatic flicker photometry is inherent to the as vaiiatton in the physiological condition of the prepmethod. As discussed previousiy (Regan, 197Oa) re- aration. changes in the electrical proper&s at the recording site, hysteresis and non-monotonicity of response variability and certain measurement errors sponse curves, are of less importance in heterochromahave less effect when th2 eye is sirnultaneousl~stimulated with test and standard stimuli than when the eye tic flicker photometry. However, under appropriate conditions both is stimulated sequentially by different monochromatic methods can give directly comparable results. This is lights. In addition. the noise of the recording is of less illustrated in Fig. 13, where the spectral luminosity importance in hzterochromatic flicker photometry btlcause the effective stimulus parameter is ~o~[~~~r~~~~curves of goldfish determined on the basis of either the criterion response or the heterochromatic flicker phocfeprli(fractional change of intensity during a stimulus cycle). Starting in a condition that the effective modutometry method are presented. The ERG data lation depth is near zero. a doubling of the test beam’s obtained with the criterion response method are indiintensity (03 log units increase) results in an effective cated with x ‘s. The f’s stand for ERG and TER data modulation depth of about 33 per cent. In response to determined with the heterochromatic flicker photosuch an increase of modulation depth the amplituds of metry method. Both sets of data agree closely. They th2 electrophysiological signal can rise to considerably have been corrected for ocular absorption [Burckmore than 33 per cent of maximum amplitude due to hardt 1966). Since the absorption spectra of the cone comprzssive nonlinearity (saturation). For example a pigments of goldfish are known (Liebman and Entine, doubling of stimulus intensity causes response ampli19M; Marks, 19653 and since there is evidence that tude in Fig. 7B to rise from near-zero to 75 per cent retinal processing is substantially linear, at any rate of its maximum value. In contrast. the zffective stimuperipherally (Spekreijse and Norton. 1970: Norton. lus parameter in the critsrion response method is the Spekreijse. Wagner and WoIbarsh~ 1970; Spekreijse absolute intensity of the light. In general, a given and van den Berg. 1971; Schellart and Spekreijse, change of absolute intensity produces a weaker r2- 1972). we thought it of interest to see whether the sponse than a corresponding change of modulation luminosity functions of goldfish could be fitted with an depth. For example, in Fig. 4C before saturation algebraic summation of the absorption spectra of the occurs response amplitude changes by a verv small three cone pigments. The result of such a fit on a leastpercentage (about 70 per cent), when intensity’is dousquare basis IS presented by the full curve shown in bled. Fig. 13. Not only for this fit, but also for the other Thus in most cases. in order to change response luminosity functions presented in this paper. we found amplitude by a given number of microvolts a much that no significant contribution of the blue cone pigsmallsr change of stimulus intensity is required when ment was needed. Furthermore, when the maxima of the heterochromatic flicker photometry method is the absorption spectra were normalized. the contribuused. Consequently, for a given accuracv of electrophytion of the red pigment was always about four times siological measurement the correspondmg accuracy of higher than that of the green cone pigment. Also in electroph~siolog~cal photometry is higher for the man a negligible contribution of the blue pigment is In ever@ay
needed to fit the luminositv function determined by the method of hetsrochroma& flicker photometry. In view of the firm basis for three cone pigments in goldfish retina the absence of the “blue” spectrum in the luminosit!- function might seem surprising. Houecer, the per cent blue spectra found in both microspectrophotometrical (Marks. 1965) and ganglion cell studies (Spekreijse el Eli.. 1972) is as low as 5 to 7 per cent. Values of the same order are also derived for the cone action spectra (Tomita, Kaneko. Muraksmi and Pautler, 1967) and horizontal cell responses (Spekreijx and Norton, 1970) in carp retina. Granted weighting of the contributions of the cone systems on the basis of the actual number of cones, such a low value is within the accuracy of our data.
(I) This paper compares the relation between light intensity and electrophysiological responses in common goldfish in conditions where (a) the eye’s level of adaptation was changing continuously and (b) the eye’s level of adaptation was not changing. We present relative spectral luminosity curves for the common goldfish measured by recording the electroretinogram (ERG) and tectal evoked response (TER). (2) Two methods were compared: the heterochromatic flicker photometry method and the criterion response method. The first method is analogous to the way in which luminance is defined in photometry. (3) With continuously changing adaptation, heterochromatic flicker photometry and criterion response method gave rather similar estimates of spectral sensitivity for ERG recording. However, the heterochromatic flicker photometry method was more accurate. (4) When adaptation was changing continuously, the criterion response method had severe limitations when used with TER recording. This was due both to complex adaptive behaviour and to hysteresis. These effects invalidate the criterion response method for determining the relative spectral luminosity under changing conditions by tectal recording in goldfish. Although smaller, such effects could sometimes also be observed for the ERG. These findings suggest that in spite of the widespread use of the CR method in electrophysiological studies of colour vision, the method should be regarded with caution. i5) With the heterochromatic flicker photometry method ERG and TER had closely similar (within 0.2 log units) relative spectral luminosity curves. The shape of these curves did not vary with flicker frequency (from 2.5 to 20Hz), adaptation level (over a range of 2.0 log units), nor with the harmonic component considered (first and second harmonic). (6) The sensitivity of the heterochromatic flicker photometry method, assessed by the sensitivity of setting the response minimum, could be as high as @OI log units. Accuracy was lower, though over an experimental time of some 5 hr the setting varied by no more than 0.2 log units for the TER and even less for the ERG. (7) The heterochromatic flicker photometry method always gave a unique minimum for the fundamental ERG component. For the TER there was either (a) one
unique minimum, or (b) when there were several minima one of them was not only cleaflv deepest, but also coincided with the unique ERG mmlmum.
(S) Although the minimum of the hererochromattc Hicker photometry method is affected by the relative timing (relative phase) of. the two alrrrnating beams. the setting remains within 0.1 log units over a 50. change of relative phase. (9) The spectral luminosit\ curves :::c discussed in terms of trlchomacv of vision and titted with the absorption spectra otthe three goldfish cone pigments. In the fit the red cone pigment dominates. .-lc6r~o~vl~dge~~~erlrs--Travel from Amsterdam and stay ar Keele for H. Spckreijsr. N. ;\. ht. Schelhrt and T. 1. T. P van den Berg was funded by the European Training Programme in Brain and Behaviour Research. D. Regan was supported by rhe Medical Research Courxil who provided part of the equipment. We thank R. F. Cdr:uright for invaluable technical assistance.
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