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Spectral sensitivity curves of diurnal squirrels
Vision
Ret.
vol.
1. pp.
Is&165.
SPECTRAL
Pwpnroa
Reu
1961.
Primedin Chat Britain.
SENSITIVITY CURVES SQUIRRELS
OF DIURNAL
KATHARINE TANSLEY, R. M. COPHNHAVERand R. D. GUNKEL -lnuitutoof
Inuituta
sariar,U.S.
Matgand. (&e&d
Ahmct-Speural
saaaicivity curva
IS July 1960) we38
OolBQuQad for four spa&
of ground
forthctwomaxima wcmtosoma4xtesttan&gon&ic. ThcpoSiblcttatumofthaaatwo tn&m&maisdirna#dinthatigbtofnaprtworkontlle~ttwtrYbrtracft cxtract&bfromth8mtimtoftha a*amongmySquirml. Tharewas~evidencefora f~~r~~~~450ad460m~atlrrstinsomtofthe spcciainvutbgtmd. R(rd-_on a d&crmi& la courbes de scnsibilit& spcctm& pour quatre csp&ces d’&urcuihterrestres,dcspWsd’&um@arbotW&etune eap&c de “chipmunk”, en Lea rttincs de utilisant comma crit&htm unc vakur constante de l%lcotror&inogram. nt que des c&es, bien que la structure touta cu asp&a nc pa&dent apparemm r&inicmte dcs doux csp&cs arboricoks ditRre queiqw peu de cc& des espbceJ terrestres et du “chigm~unk”. 11n’y a pas traced’&t Fwkinjc. Lescowbes de sensibilit6 spcctrale duseptapbcase fasem&nt tout08 bcawmup; elk prhentcnt dcux maximum, I’un vers S35 ma l’autre vers 490 m#; ks hauteurs relatives de ces maximum varient dune cxp&icncc P l’autrc. sans qu’on puisse d&erminer les causes de ces variations. L’adaptation par la lumi&e bkuc abaissc le maximum de 490 m# tandis quc l’adaptation par des lum&rcs vcrte ou orange obaiasc celui de 535 mr. 11scmbkrait que la ticanismes rcsponsabks de ces deux maximum soimt en quclque facon antagonistcs. La nature possibie de ces dcux m&anismas cst d&cut& il la lurni&e de travaux ricmts sur les pigments photoscnsibks quc I’on extrait de la r&me da l’ecumtil gris commun. Dam ccrtaincs des up&es Ctudi&s tout au moms, il pour& exista un autre mtcanisme dont le maximum de schsibilitC se situerait entrc 450 et 440 mfl. zuuwclllbuup_Dii spcktraie EmpfWlichkcit wurde bci vier Exemplaren des BodenEichhiWcbens, zwei Exemplaren dcs Baum-EichhUrnchens und einem gcstreiften Eichhomchen untcrsucht. indem die zur Eneugung e&s konstantm Ekktroretinogramms notwcndigc Lichtintenaitat ermittelt wurdc. Alle drei Arten bcsitzen nugenscheinlich nur Zapfen in der Netzhaut, obwohl die Netzhautstruktur der b&den BaumEichhomchen etwas von der du Boden Eichharnchen und dcs gestrciften Eichhijmchens abweicht. Ein Purkinjc-Effekt hat sich nicht gezcigt. Die spckfralen Empfmdlichkeitskurven warcn bci alkn siekn Exemplaren schr Bhnlich. Sic zcigten &ei Maxima, eina bei etwa 535 mg, das andem bei ctsa 490 mv. Die relative H&he dcr kiden Maxima Indcrtc sich von e&m Expmknmt zum an&m; die Ursa&m dicser Veranderung konnten nicht bestimmt wcrckn. Adaptation an blaucs Licht verkleinert das Maximum bci 490 rnp, wghrend Adaptation an mines oder oranges Licht dasjenigc bci 535 m/r vcrkkinert. Es konnte gczcigt warden, dass die fur die b&den Maxima verantwortlichen Mechanismen bis zu einem gcwissen Grade antagonistisch warm. Die Mechanismen werden im Hinblick auf Untcrsuchungen diskutiert, die ktirzlich an den aus dcr NetzIs4
Spectral Sensitivity Curves of Diurnal Squirrels
155
haut des gcwBhnlichen graucn Eichhijmchens extrahiertcn lichtcmpfindlichen Stoffen durchgefiihrt wurden. Einige Hinwcise auf eincn weitercn Mechanismus, dcr zwischen 450 und 460 rnp am empfindlichsten ist, wurden mindcstens in einipn der untersuchten
Exemplaregefunden. IN PREVIOUSinvestigations on the spectral sensitivity of two species of the squirrel family, the gray squirrel, Sciurus carolinensis leucqtis and the souslik or European ground squirrel, Cite&s citellus, one of us (ARDEN and TANSLEY, 1955a, b) recorded curves with their maxima around 530 rnp. In the case of the gray squirrel the curve was an unexpectedly narrow one reminiscent of one of GRANIT’Smodulator curves.1 The mean souslik curve, on the other hand, was much broader on the short-wave side of the maximum reflecting a raised blue sensitivity which was not present in all experiments. It seemed possible that we were dealing with a second visual mechanism maximally sensitive to the shorter wavelengths and rather capricious in its manifestations, Again we could not be certain whether the different sensitivity curves we found reflected a true difference between the tree squirrels and the ground squirrels, so that it seemed desirable to repeat the experiments with other species, a more satisfactory method of recording the spectral sensitivity curves and a greater number of individual animals from each species. MATERIALS
AND
METHODS
The experiments were done on four species of ground squirrel: Cirellus beecheyi (four animals), Cirellus rridecemlinearus (two animals), Citelfus variegarus buckleyi (four animals) and Citeilus lateralis (four animals); on two tree squirrel species, the gray, Sciurus carolinensis leucoris (four animals) and the American red, Tamiosciurus hudsonicus loquax (two animals); and on one chipmunk species, Euramias amoenus lureiventris (two animals). Histological preparations were made of the eyes of all species with the exception of the gray squirrel which had a?ready been examined (ARDEN and TANSLEY, 1955a). The animal was removed from its cage in a nylon fish-net and given 20% urethane solution intraperitoneally, the usual dose being 2 g/kg body weight. After the animal was anaesthetized the eyes were treated with 0.5% proparacaine hydrochloride (Ophthaine) to produce topical anaesthesia of the cornea and with 10% phenylephrine (Neosynephrine) viscous to dilate the pupil. The cornea1 electrode was incorporated in a specially made plastic contact glass and was a silver wire; the contact glass was filled with methyl cellulose saline. The indifferent electrode was a small silver plate (an EEG electrode disc) held in position on a shaved area of the forehead, which had previously been smeared with electrode jelly, by means of a rubber band round the upper jaw. Some of these electrodes were photosensitive and care was taken to shield them from the stimulating light. The animal was grounded by means of a bulldog clip wrapped in saline-soaked cotton wool attached to one ear. The light stimulus was provided by a xenon arc and double interference filters as dsscribed by COPENHAVERand GUNKEL (1959). Three additional filters were used with maximum transmissions at 480, 509 and 545 rnp respectively. Neutral density filters were used to regulate the stimulus intensity as in Copenhaver and Gunkel’s investigation. Most of the experiments were done with the light focused on the cornea so that it would be in Maxwellian view. In a few cases field sizes of 1” and 60” were also used. The intensity produced at the eye without neutral or color filters in the light beam was 7500 lux. The earliest experiments were done with the 32 per set flickering stimulus used in human ’
GRANIT
(1947).
156
KATHARINE TANSLEY, R. M. COPENHAVER AND R. D. GUNKCL
observations by Copenhaver and Gunkel, but this type of stimulus was found to be of no advantage and for most of the experiments a light gash of l/4 set duration was used. For observations taken during light adaptation the stimulus was presented once a second; in dark-adapted experiments the stimuli were not given oftener than once a minute. For the color-adaptation experiments a special light source was contrived consisting of a standard 100 W Sylvania zirconium concentrated arc lamp provided with condensers and a filter holder. So that there should be no interference with the stimulating beam the adaptation light was concentrated and brought to the pupillary area of the contact glass by means of a curved Zeiss transilluminator tip of about 2 mm diameter. This tip in close proximity to the cornea acted like a point source and gave. maximum illumination of the retina. Inconel neutral density filters (Bausch and Lomb) were used to vary the illumination when required. The color filters used were Eastman Kodak’s Wratten 22 (orange), Wrattcn 58 (green), and Wratten 47 (blue). Their dominant wavelengths were 595, 534 and 480 rnp respectively. The ordinary light-adapted experiments were done with the room lights on giving a level of 170 to 240 lux at the animars eye. In each experiment records of the response to each colored stimulus were taken starting with a cheek point at 53 I rnk, then working through the spectral range usually starting at the red end, and finally checking back to 531 rnp at the end of the run. Runs were always made under both dark- and light-adapted conditions. In addition, in some experiments runs were made with an adapting color shining into the eye. For these the adapting light was used for 5 min before readings were begun and was kept on during the run, the spectral stimuius being superimposed on the adapting light. In the earlier experiments, the crystalline lens was removed from the experimental eye (usually the left eye) at the end of the experiment; it was then mounted in a special holder and its transmission at each stimulating wavelength measured by substituting a photocell for the eye. Then the lens and holder were removed and the measurements repeated using the interference tilters alone. This procedure also provided a calibration for the xenon arc light source. In the later experiments the lens was extracted before recording began so that no correction for its spectral absorption was required. In all the species used for these experiments the lens is more or less yellow in color so that it was necessary either to correct for its transmission or to make the readings in its absence. Histology
When recording was complete, the upper part of the animal was intravitally ftxed through the ascending aorta after washing out with isotonic saline. The fixative used was either Zenker’s, Kolmer’s, or Bouin’s fluid. The control eyes were then removed and left overnight in the fixative. Embedding was in par&n after removal of the lens and cornea and sections were cut at 6 jr; they were stained with haematoxylin and eosin, the azan mixture or by Feulgen’s method. Histo fogy
RESULTS
The retinae of all seven species used in these experiments showed the general characteristics of a cone retina. Fig. 1 shows a section through the central area of the retina of the ground squirrel, Citeh beecheyi, showing a thin outer nuclear layer indicating a relatively small number of visual cells per unit area, with unusually thick inner nuclear and ganglion cell layers. It was found that the two species of tree squirrel, Sciurus carolinensis kucotis and
Fro. I. The ground squirrel retina. Section through the central area of the retina of CiteffrtJ &ee+&-~i. Note the exceptionally thick ganglion cell layer. Haematoxylin and eosin; Bouin. - 285. The following numbering applies to Figs. I,2 and 3: 1, pigment epithelium; 2, cones; 3, outer nuclear layer; 4, outer fiber layer; 5, inner nuclear layer; 6, inner fiber layer; 7, ganglion cell layer; 8, optic nerve fiber layer.
FIG;. 2. The squirrel visual cells. Section through the outer retina of Scirr~~r caroiinc~tsit lertcoris. Note the double layer of cones. Bleached: Azan : Kolmer ’ 700.
If... .“,. ,, 1Sh,
FIG. 3. The ground $&d
visual c&k. L!kctionthrough th&outer retina of Cite/h larerdis. acidhaunatoxylin; Zcnker. x 775.
Note the sir&e layer of cones. Mallo~y’s $ho&t?twtic
Spectral Sensitivity Curves of Diurnal Squirrels
157
Tamiosciurus hudronicus loquax, both had the double row of cones already described for Sciurus carolinensis (ARDEN and TANSLEY, 1955a). A high power view of the outer part of
the retina of this species is shown in Fig. 2. The retina of another ground squirrel, Citellus has also been described by ARDEN and TANSLEY (1955b) and the four Cite//us species used in the present investigation showed just the same picture, as abo did the one chipmunk used, Eutamias amoenus luteiventris. A high power view of the outer retina of Citellus lateralis is shown in Fig. 3. cireks,
FIG. 4. The pure-cone electroretinogram. Two electroretinograms of Cite//us beecheyi after removal of the lens. Dark-adapted; stimulus wavelength 531 rnp; no neutral filter; stimulus duration I /4 sec.
Electrophysiological
Fig. 4 shows a pair of typical electroretinograms as recorded on the Grass encephalograph. These demonstrate the initial negative (downwards) o-wave, the second positive b-wave (upwards) and the striking positive off-effect. The negativity recorded after the b-wave is partly, but probably not wholly, an artifact due to the pen’s overshooting. All eight channels of the encephalograph were used with a different amplification on each. It was found that excursions above about 10 mm were unreliable and those below 1 mm could not be read accurately. By using eight different amplifications, a much wider range of responses could be analysed. Most of the results analysed here are based on the amplitude of the b-wave (measured from the trough of the a-wave), but curves were also constructed for the responses of the a-wave and off-effect. It was found, however, that these did not
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240
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220. 200 180
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I40
-
I20
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On
1.0
20 LOG
30
I
FIG. 5. Intensity-response curves. A group of representative intensity-response curves from a dark-adapted Cirellus beecheyi after removal of the lens. Single flash l/4 set stimulus. Ordinates: amplitude of the b-wave in JIV. Abscissae: log stimulus-intensity. L
KATHARINETAWSLBY,R. M.
158
COPENHAVER AND
R, D. GIJNKEL
difFcr sign&antly from those obtained from the b-wave and they are not, therefore, considcrcd separatciy. The spcctrai sc&ivity curves wcrc ca&tiatcd in the general way dcscribcd by COPENHAVSRand GUNXEL(1959). ~n~~zy-~~~ curves, mostly on b-wave ~p~t~c, were plotted for each wave&g& us& in a run and the neutral fBcr density rcquircd to give a constant rcsponsc dctcrmincd. This density was corrcctcd for di&cnccs in energy value for each spectral stimulus and for an equal quantum intensity spectrum; this correction incl‘llded that for the crystalline lens in those experiments in which it was not extracted. A typical group of intensity-response curves from the short-wave part of the spectrum caicuiatcd for one run with the animal whose ci~trorc~o~~ is given in Fig. 4 is shown in Fig. 5. Those from the rest of the spectrum were very similar. For this particular run criterion values of 300, 200, 100 and 50 pV wcrc chosen for constz-ucting the spectral sensitivity curves, but it
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I
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1
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/
~~~ii~ 400
j j \u, !
so
500
600
450
600
FIG. 6. Spwtml sensitivitycurves. Two spectralsensitivitycurves from two dWmnt NM in one c$qmimmt on a dark-&p&d Cite&a latmiis. A. Fit in, 250ak’ aitkfm. 8. Stcand run, 100pV criterion. Or&a&es: pcrosnurscsensitivity.Absciww: wwekngth in mp (equal
qilantum intensity spa%unl). was found that there was no sign&ant di&encc between the curves for low and those for high criterion values (ARMINGTON, 19%). The density at each wavckngth was then detcrmined as a percentage of the density at the most effective wavelength and the points plotted against wavelength. To begin with, the curves were made by simply joining the individual points, but later, as a constant pattern emerged, the curves were drawn freehand. No sign&ant dil%rcncc was found between the curves for the different species, The curves were ncariy aH found to bc double humped; occasionally the humps were nearly of the same height but more often one or other was markedly higher. Fig. 6 shows two typical curves, both from the same animal and taken under the same experimental conditions. None of our different experimental conditions was found regularly to produce one type of curve rather than the other. It was thought, for instance, that the “biue” hump might be due to scattered fight. In order to test this point curves taken with a smdl (I”) field during dark adaptation, conditions which should favor stimulation of the retinal periphery by scattcrcd light, were compared with ones taken with a large (50”) f&d in light adaptation, but there was no evidence that the former conditions favored the “blue” hump
159
Spectral Sensitivity Curves of Diurnal Squirkls
or that the latter favored the “green”. Again it was considered possible that the presence of the yellow lens might protect a “blue” mechanism and so produce a higher relative sensitivity to the short wavelengths. It was for this reason that we originally removed the lens halfway through an experiment, repeating all the previous runs with an aphakic eye. Although the removal of the lens made it possible to get readings at shorter wavelengths than before, it had no effect on the relative sensitivities. No consistent difference was found between those curves taken during dark adaptation and those taken in light adaptation. There was therefore no evidence of a Purkinje shift in any of the species examined.
900
500
600
FIG. 7. Spectral sensitivity curve. Mean spectrai sensitivity curve from all the experiments on Citellus lateralis.
Ordinates:
Abscissae: percentage sensitivity. quantum intensity spectrum).
wavelength
in rnp (equal
The two different types of curve illustrated in Fig. 6 were found in about equal numbers so that the mean spectral sensitivity curve for this species was nearly symmetrical with the two humps of about the same magnitude (Fig. 7). A fair proportion of the spectral sensitivity curves showed a distinct, albeit often very minor hump on the descending slo.pe between 450 and 460 rnp (Fig. 11). When a mean curve for all the species investigated was drawn there was a distinct inflexion in this position which is outside the experimental error. The data for this mean curve (Fig. 8) are given in Table 1. It was calculated from the results of all the runs in which there was no evidence of a general change in sensitivity while the records were being made. It includes results in light- and dark-adaptation, with small, large and normal fields, using the amplitude of the b-wave, u-wave and off-effect, using high and low criterion values and for eyes with and without thecrystalline lens. It was calculated from the records of 248 runs in all. As a rule, our color-adaptation depressed the general sensitivity of the retina, but when the results were calculated as percentage of the maximum it was found that blue-adaptation depressed the hump at 490 to 500 rnp in all three experiments on Cirellus beecheyi, in one out of two experiments on Citellus lateralis, in one experiment on Citeilus tridecemlineatus, in one out of two experiments on Citellus variegatus, in one experiment on Tamiosci’urus huhonicus and in one experiment on Eutamias amoenus. Green- or orange-adaptation depressed the hump at 535 rnp in all three experiments on Citeflus beecheyi, in two experiments on Citeilus tridecemiineatus, in four experiments on Citelius lateralis, in two
160
KATHARINE TANSLHY, R. M. COPENHAVER AND R. D. GUNUL
TABLE 1 No. of
Widows
dnll
-4
I
610 583 558 545 531 s23 509 iii 473 456 442 419 404
/ : 1
Mm
sensitivity
(%I
iit 199 247 248 2: 248 143 248 32 z 113 24 8
experiments on Cirdus variegatus and in one experiment on Ta&o&nr~ &u&Mcw, but had no effect in one experiment on EutMuirr rlnrocm(s. The results ofoneof&6 ~XJW%WS on Citelbu kcheyi are shown in Fig. 9. In this figure the v&e at 531 rnp for the ordi~uy white-light-adapted sensitivity curve wo3 taken as 100 pet cent. For the cup toti with the blue-adapting light the value at 531 rnp was againtaken as 100 per CGILf,wbiie for the curve taken with the green-pskptiag light tbc value at 473 rnp was taken as 1IS par cent, the figure for this point on the original white &ht-adapted curve; the values for the other wavelengths were calculated accordingly.
Spectral Sensitivity Curves of Diurnal Squirrels
161
The electrophysiological results were analysed for us by the Biometrics Branch of the National Institute of Neurological Diseases and Blindness. It was concluded (I) that our data for the seven species were homogeneous and could legitimately be combined, and (2) that our mean spectral sensitivity curve (Fig. 8) was a good description of the response data as a whole. LA
Curve
Blue Bleach Green Blroch
_____
1
......‘..
IOC
50
-I
400
600
FIG. 9. The effect of color-adaptation. Three spectral sensitivity curves from one experiment on Citellus beecheyi. “.““““’ effect light-adapted curve; - - -effect of blue-adaptation; of green-adaptation. Ordinates: percentage sensitivity. Abscissae: wavelength in mp (equal quantum intensity spectrum). DISCUSSION
The histological examinations indicate that all Cite//usspecies and at least the one chipmunk species used have a similar retinal structure with a single layer of cones, and that in the central retina there is probably a one-to-one relationship between these cones and the ganglion cells and, therefore, the optic nerve fibers. In the tree squirrels it seems likely that the cones always form a double layer and, since the ganglion cell layer is nowhere as thick in these species as it is in the central retina of the ground squirrel, it is improbable that there is a separate optic nerve fiber for each cone in any part of the retina. A similar retinal structure has been described by ROCHON-DUVIGNEAUD (1943) for the European red squirrel, Sciurus vulgaris. He recognized two groups of visual ceils in this animal, but he used tangential sections and mistook the outer segments of the inner row of cones for rods. All the physiological evidence points to these being pure-cone retinae (TANSLEY, 1957). The suggestion that a high blue sensitivity might be characteristic of the ground squirrels as opposed to the tree squirrels was not confirmed by the results of the present investigation where no significant difference was found between the sensitivity curves of the two groups. The shape of the spectral sensitivity curves came as a surprise. The long-wave part of the mean curve is in reasonable agreement with this part of the curves published by Arden and
162
KATHARINE TANSLEY, R. M. COPI!NWAVER AND R. D. GUNKEL
TansIcy for the gray squirrel and CireuuE c~te~~~and with that for the dif&rence spectrum of the photolabile pigment found by WWLE (1955) in the living retina of the gray squirrel. However, the second peak at about 495 rn~ was quite unexpected. It is true that a capriczious high Mua sensitivity was found by Arden and Tans@ in Ore&s citehs but their points in the relevant part of the spectrum between r(80 and 545 rnp were too few and their stlrndard error too great to make a satisfactory characterization of this part of the curve possible. Although Arden and Tandey did not suspect a second rise in sensitivity in their gray squirrels, if mean values are calculated for their pubtished points there is some evidence for an inflexion on their curve at about 490 rn# aithough it is not very marked.
I-
I
We made many attempts to elucidate the nature of the m~hani~ responsible for the “biue” paak and were satishd that it was not due to scattend Ii@. ft did not baeome more prominent in dark-adaptation nor if low crit&a were used. The only way in whi& we were able Co a&et the &ape of the curve in a predictable way was by using c&red adapting tights while the EM&S were b&g taken. Then we found that a b~~~~g light depressed the “blue” peak and a green- or orange-adapting light depmsmd tk “mn” one. This suggests that both puaks are rather closely associated with photosensitive mechanisms of different spec.tral sensftivities (fig. 9). Although thu two peaks are of roughly the same height in the mean curve, the curves for individual experiments usually showed one or other tQ be the higher (Fig. 6) and this suggested th+ the two mechanisms might b in some sort of rivalry. In the hop of throwing some light on this idea all the curves were divid& into two groups, one in wfrich the values at 523 and 531 rn& were the hi+@ and a saoond in which the highest values ware fou& to be at wavelengths shorter than 523 rnp. Wktn the means of these two groups w plot&& separWly the two curves shown in Fig, 10 were obtained. These show that when one peak is high the other does indaed tend to be very much red-d thus indicating some degreeof rivairy between the mechanisms responsible for the two peaks.
Spectral Sensitivity Curves of Diurnal Squirrels
163
In trying to obtain more information about the mechanism responsible for the “blue” peak one must remember that the position of its maximum sensitivity is likely to be at a rather shorter wavelength than the 495 rnp shown in Fig. 8 or the 490 rnp shown in Fig. 10. Even the curve in Fig. 10 is probably pulled over somewhat towards longer wavelengths by the effect of the “green” mechanism and, in fact, if one plots the difference between the two curves in Fig. 10 for the shorter wavelengths the peak of the resultant curve falls at about 480 rnp. Recently DARTNALL (1960) has studied extracts of the gray squirrel retina and has found it to contain a rhodopsm with a maximum absorption at 502 rnp. This pigment on bleaching produced, in addition to the normal retinene, a photoproduct with its maximum absorption at 480 rnp. In considering the bearing of this result-on the spectra1 sensitivity
FIG. 11. Spectral sensitivity curve. Spectral sensitivity curve from a light-adapted Circllus beecheyi showing high values between 450 and 460 rnp. Ordinates: percentage sensitivity. Abscissae: wavelength in rnp (equal quantum intensity spectrum).
function found by Arden and Tansley and on the difference spectrum found by Weale in living squirrels Dartnall suggests that in viva rhodopsin in the squirrel cones may bleach to a product having its maximum absorption at 480 ml and which is itself photosensitive. He further suggests that the spectra1 sensitivity function with its maximum at around 530 rnp “may be mediated by a mechanism depending on the difference between the light absorbed by the rhodopsin at 502 rnp and that absorbed by the 480 photoproduct”. He points out that if this is indeed the case a truly threshold sensitivity function would be expected to reproduce the 502 curve alone. None of our records was taken at this level since the electroretinogram is too insensitive a criterion to be useable at the absolute threshold. He also suggests that prolonged adaptation to long-wavelength light should result in a spectra1 sensitivity function which follows the form of the broad band 480 curve. The orange filter we used for adaptation had too broad a transmission band for adequate testing of this point. If sufficient adaptation was used entirely to eliminate the peak at 531mp the whole retina1 sensitivity was so depressed that satisfactory records were unobtainable even in the shortwave part of the spectrum. In the light of Dartnall’s findings a very tentative explanation of the spectral sensitivity curves found in this investigation might be that the 535 rnp peak is the result of interaction of some sort between two photosensitive substances with maxima at
164
KATHARINE
TANSLEK, R.
M.
COPENHAWR
AND 9,
D.
GUNKEL
SO2and 480 rn& and that, in addition, the spectral sensitivity of the 480 pigment IS responsible for the peak in this region. If, further, these pigments may be formed from one another, as Dartnall believes, this would explain the apparent rivalry of the mechanisms responsible, for our two peaks for the more of one pigment was present the less of the other. In the mean curve of Fig. 8 there is a distinct inflexion around 450 to 460 rn& which, although small, is outside the experimental error. In 37 per cent of all the experimeats in which records down to 442 m,u were obtainable the readings at 456 rnp were high, occasionally as high as or even higher than is shown in Fig. Il. In the remaining 63 per cent there was no evidence whatever of any special activity in this region. All but one of the curves with enhanced values at 456 rn@ were obtained from tight-adapted eyes. In the paper already quoted, Dartnall points out that interaction between possible activity mediated by the 480 pi~ent and that mediated by the 502 pigment might also give a spectral sensitivity function with a maximum at 448 mp. Spectral sensitivity curves obtained by e~~trophysio~ogi~ai methods with maxima or humps in the blue, usually capricious in their appearance, have often been reported in the literature. GRANIT (1947) in one of his figures shows a curve obtained from the guinea-pig retina by means of his microelectrode technique in which the maximum is at about 460 rnp with a secondary hump at about 535 mp. DODT and ELENIUS(1956) working on single-fiber responses from the rabbit retina obtained some curves in which the maximum sensitivity is at 460 mp. In two of these there is evidence of a secondary maximum near 500 rnp but in a third the secondary maximum is ptobabiy at a rather longer wavelength. ELENIUS (1958) shows two spectral sensitivity curves also from the rabbit obtained by means of the eiectroretinogram in which there was an enhanced sensitivity at 480 m,u. In both guinea-pig~nd rabbit the predominant visual pigment is, of course, rhodopsin. In this connexion it is worth mentioning ~NGVAR’S(1959) paper on the cortex of the cat (afso with a predominantly rhodopsin-containing retinal in which he suggests that there may be two scotopic mechanisms, one with maxima at about 460 and 530 rn& which is in rivalry with a second whose maximum is at 490 mp. lngvar found these mechanisms to be inherently utistable. Although the mean curve of Fig. 8 shows no irregularity in the long-wave region of the spectrum, a comparison of this curve with Weale’s for the gray squirrel does show a slight discrepancy at about 560 rnp which might indicate a further mechanism of some sort active in this spectral region, but the evidence is certainly not convincing enough to warrant drawing any conclusions on this point. It has been argued that spectral sensitivity curves obtained by means of intarfinnce lifters can show odd peaks which are reaily artefacts due to the s+ shape of the transmission bands of some filters of this type. This is not like& to be a corn~~~~ in the present investigation since Copenhaver and Gunkei got pcrf%ctly smooth phatopic senaitivity curves from human subjects with normal vision using the same apparatus and all but three of the fiiters we have used here. However, in order to test this point we did do two experiments on the dark-adapted rabbit and in these we obtained spectral sensitivity curves which satisfactorily fitted the rhodopsin absorption curve, SUMMARY 1. Using the e~ectroretino~am as the criterion, spectral sensivity curves were obt&ed from seven different species of the squirrel family. 2. Histological e~amjnatjon showed that a11these species have pure-cone retinae.
Spectral Sensitivity Curves of Diurnal Squirrels
165
3. The curves showed two maxima at about 535 and 490 rnp respectively. There was evidence from color-adaptation experiments that the two maxima are associated with two mechanisms with different spectral sensitivities. 4. The possible nature of the mechanisms responsible for these two maxima is discussed. 5. There was some evidence for a further mechanism with its maximum activity between 450 and 460 m,u. REFERENCES ARDEN, G. B. and TANSLEY, K. (1955a). squirrel (Sciwrc~ curolinenis). J. fhysiol.
The spectral sensitivity curve of the pure-cone retina of the gray 127, 592-602.
ARDEN. G. B. and TANSLEY, K. (1955b). The spectral sensitivity curve of the pure-cone retina of the souslik (CireNlr, cite//us). J. Physiol. 130, 225-232. ARMINGTON, J. C. (1955). Amplitude of response and relative spectral sensitivity of the human electroretinogram.
J. opr. Sot. Amer. 45, 1058-1064.
COPENHAVER, R. M. and GUNKEL, R. D. (1959).
The spectral sensitivity of color-def&tive subjects deterArch. Ophrh. 62. 55-68. DARTNALL, H. J. A. (1960). Visual pigment from a pure-cone retina. Ncrure. Lond., H&475-479. DODT, E. and ELENIUS. V. (1956). Spektrale Sensitivitiit einzelner Elemente der Kaninchennetzhaut. t’fliig. Arch. ges. Physiol. 262, 301-306. ELENIUS, V. (1958). Recovery in the dark of the rabbit’s electroretinogram. Acta physiol. stand. 44, suppl. 150. GRANIT, R. (1947). Sensory mechunisms ofthe retino. Cumberlege and Oxford Univ. Press, London. INCVAR. D. H. (1959). Spectral sensitivity as measured in cerebral visual centers. Acruphysiol. scund. 46, mined by electroretinography.
A.M.A.
suppl. 159. ROCHON-DUVIGNEAUD, A. (1943). Les yeux et la vision des VerrdbrPs. Masson, Paris. TANSLEY. K. (1957). Some observations on mammalian cone electroretinograms. Bib/. ophthal. 48, 7-14. WEALE. R. A.’ (1955). Bleaching experiments on eyes of living gray squirrels (Sciww curolinensis leucotis). J. Physiol. 127, 587-591.
Ret.
vol.
1. pp.
Is&165.
SPECTRAL
Pwpnroa
Reu
1961.
Primedin Chat Britain.
SENSITIVITY CURVES SQUIRRELS
OF DIURNAL
KATHARINE TANSLEY, R. M. COPHNHAVERand R. D. GUNKEL -lnuitutoof
Inuituta
sariar,U.S.
Matgand. (&e&d
Ahmct-Speural
saaaicivity curva
IS July 1960) we38
OolBQuQad for four spa&
of ground
forthctwomaxima wcmtosoma4xtesttan&gon&ic. ThcpoSiblcttatumofthaaatwo tn&m&maisdirna#dinthatigbtofnaprtworkontlle~ttwtrYbrtracft cxtract&bfromth8mtimtoftha a*amongmySquirml. Tharewas~evidencefora f~~r~~~~450ad460m~atlrrstinsomtofthe spcciainvutbgtmd. R(rd-_on a d&crmi& la courbes de scnsibilit& spcctm& pour quatre csp&ces d’&urcuihterrestres,dcspWsd’&um@arbotW&etune eap&c de “chipmunk”, en Lea rttincs de utilisant comma crit&htm unc vakur constante de l%lcotror&inogram. nt que des c&es, bien que la structure touta cu asp&a nc pa&dent apparemm r&inicmte dcs doux csp&cs arboricoks ditRre queiqw peu de cc& des espbceJ terrestres et du “chigm~unk”. 11n’y a pas traced’&t Fwkinjc. Lescowbes de sensibilit6 spcctrale duseptapbcase fasem&nt tout08 bcawmup; elk prhentcnt dcux maximum, I’un vers S35 ma l’autre vers 490 m#; ks hauteurs relatives de ces maximum varient dune cxp&icncc P l’autrc. sans qu’on puisse d&erminer les causes de ces variations. L’adaptation par la lumi&e bkuc abaissc le maximum de 490 m# tandis quc l’adaptation par des lum&rcs vcrte ou orange obaiasc celui de 535 mr. 11scmbkrait que la ticanismes rcsponsabks de ces deux maximum soimt en quclque facon antagonistcs. La nature possibie de ces dcux m&anismas cst d&cut& il la lurni&e de travaux ricmts sur les pigments photoscnsibks quc I’on extrait de la r&me da l’ecumtil gris commun. Dam ccrtaincs des up&es Ctudi&s tout au moms, il pour& exista un autre mtcanisme dont le maximum de schsibilitC se situerait entrc 450 et 440 mfl. zuuwclllbuup_Dii spcktraie EmpfWlichkcit wurde bci vier Exemplaren des BodenEichhiWcbens, zwei Exemplaren dcs Baum-EichhUrnchens und einem gcstreiften Eichhomchen untcrsucht. indem die zur Eneugung e&s konstantm Ekktroretinogramms notwcndigc Lichtintenaitat ermittelt wurdc. Alle drei Arten bcsitzen nugenscheinlich nur Zapfen in der Netzhaut, obwohl die Netzhautstruktur der b&den BaumEichhomchen etwas von der du Boden Eichharnchen und dcs gestrciften Eichhijmchens abweicht. Ein Purkinjc-Effekt hat sich nicht gezcigt. Die spckfralen Empfmdlichkeitskurven warcn bci alkn siekn Exemplaren schr Bhnlich. Sic zcigten &ei Maxima, eina bei etwa 535 mg, das andem bei ctsa 490 mv. Die relative H&he dcr kiden Maxima Indcrtc sich von e&m Expmknmt zum an&m; die Ursa&m dicser Veranderung konnten nicht bestimmt wcrckn. Adaptation an blaucs Licht verkleinert das Maximum bci 490 rnp, wghrend Adaptation an mines oder oranges Licht dasjenigc bci 535 m/r vcrkkinert. Es konnte gczcigt warden, dass die fur die b&den Maxima verantwortlichen Mechanismen bis zu einem gcwissen Grade antagonistisch warm. Die Mechanismen werden im Hinblick auf Untcrsuchungen diskutiert, die ktirzlich an den aus dcr NetzIs4
Spectral Sensitivity Curves of Diurnal Squirrels
155
haut des gcwBhnlichen graucn Eichhijmchens extrahiertcn lichtcmpfindlichen Stoffen durchgefiihrt wurden. Einige Hinwcise auf eincn weitercn Mechanismus, dcr zwischen 450 und 460 rnp am empfindlichsten ist, wurden mindcstens in einipn der untersuchten
Exemplaregefunden. IN PREVIOUSinvestigations on the spectral sensitivity of two species of the squirrel family, the gray squirrel, Sciurus carolinensis leucqtis and the souslik or European ground squirrel, Cite&s citellus, one of us (ARDEN and TANSLEY, 1955a, b) recorded curves with their maxima around 530 rnp. In the case of the gray squirrel the curve was an unexpectedly narrow one reminiscent of one of GRANIT’Smodulator curves.1 The mean souslik curve, on the other hand, was much broader on the short-wave side of the maximum reflecting a raised blue sensitivity which was not present in all experiments. It seemed possible that we were dealing with a second visual mechanism maximally sensitive to the shorter wavelengths and rather capricious in its manifestations, Again we could not be certain whether the different sensitivity curves we found reflected a true difference between the tree squirrels and the ground squirrels, so that it seemed desirable to repeat the experiments with other species, a more satisfactory method of recording the spectral sensitivity curves and a greater number of individual animals from each species. MATERIALS
AND
METHODS
The experiments were done on four species of ground squirrel: Cirellus beecheyi (four animals), Cirellus rridecemlinearus (two animals), Citelfus variegarus buckleyi (four animals) and Citeilus lateralis (four animals); on two tree squirrel species, the gray, Sciurus carolinensis leucoris (four animals) and the American red, Tamiosciurus hudsonicus loquax (two animals); and on one chipmunk species, Euramias amoenus lureiventris (two animals). Histological preparations were made of the eyes of all species with the exception of the gray squirrel which had a?ready been examined (ARDEN and TANSLEY, 1955a). The animal was removed from its cage in a nylon fish-net and given 20% urethane solution intraperitoneally, the usual dose being 2 g/kg body weight. After the animal was anaesthetized the eyes were treated with 0.5% proparacaine hydrochloride (Ophthaine) to produce topical anaesthesia of the cornea and with 10% phenylephrine (Neosynephrine) viscous to dilate the pupil. The cornea1 electrode was incorporated in a specially made plastic contact glass and was a silver wire; the contact glass was filled with methyl cellulose saline. The indifferent electrode was a small silver plate (an EEG electrode disc) held in position on a shaved area of the forehead, which had previously been smeared with electrode jelly, by means of a rubber band round the upper jaw. Some of these electrodes were photosensitive and care was taken to shield them from the stimulating light. The animal was grounded by means of a bulldog clip wrapped in saline-soaked cotton wool attached to one ear. The light stimulus was provided by a xenon arc and double interference filters as dsscribed by COPENHAVERand GUNKEL (1959). Three additional filters were used with maximum transmissions at 480, 509 and 545 rnp respectively. Neutral density filters were used to regulate the stimulus intensity as in Copenhaver and Gunkel’s investigation. Most of the experiments were done with the light focused on the cornea so that it would be in Maxwellian view. In a few cases field sizes of 1” and 60” were also used. The intensity produced at the eye without neutral or color filters in the light beam was 7500 lux. The earliest experiments were done with the 32 per set flickering stimulus used in human ’
GRANIT
(1947).
156
KATHARINE TANSLEY, R. M. COPENHAVER AND R. D. GUNKCL
observations by Copenhaver and Gunkel, but this type of stimulus was found to be of no advantage and for most of the experiments a light gash of l/4 set duration was used. For observations taken during light adaptation the stimulus was presented once a second; in dark-adapted experiments the stimuli were not given oftener than once a minute. For the color-adaptation experiments a special light source was contrived consisting of a standard 100 W Sylvania zirconium concentrated arc lamp provided with condensers and a filter holder. So that there should be no interference with the stimulating beam the adaptation light was concentrated and brought to the pupillary area of the contact glass by means of a curved Zeiss transilluminator tip of about 2 mm diameter. This tip in close proximity to the cornea acted like a point source and gave. maximum illumination of the retina. Inconel neutral density filters (Bausch and Lomb) were used to vary the illumination when required. The color filters used were Eastman Kodak’s Wratten 22 (orange), Wrattcn 58 (green), and Wratten 47 (blue). Their dominant wavelengths were 595, 534 and 480 rnp respectively. The ordinary light-adapted experiments were done with the room lights on giving a level of 170 to 240 lux at the animars eye. In each experiment records of the response to each colored stimulus were taken starting with a cheek point at 53 I rnk, then working through the spectral range usually starting at the red end, and finally checking back to 531 rnp at the end of the run. Runs were always made under both dark- and light-adapted conditions. In addition, in some experiments runs were made with an adapting color shining into the eye. For these the adapting light was used for 5 min before readings were begun and was kept on during the run, the spectral stimuius being superimposed on the adapting light. In the earlier experiments, the crystalline lens was removed from the experimental eye (usually the left eye) at the end of the experiment; it was then mounted in a special holder and its transmission at each stimulating wavelength measured by substituting a photocell for the eye. Then the lens and holder were removed and the measurements repeated using the interference tilters alone. This procedure also provided a calibration for the xenon arc light source. In the later experiments the lens was extracted before recording began so that no correction for its spectral absorption was required. In all the species used for these experiments the lens is more or less yellow in color so that it was necessary either to correct for its transmission or to make the readings in its absence. Histology
When recording was complete, the upper part of the animal was intravitally ftxed through the ascending aorta after washing out with isotonic saline. The fixative used was either Zenker’s, Kolmer’s, or Bouin’s fluid. The control eyes were then removed and left overnight in the fixative. Embedding was in par&n after removal of the lens and cornea and sections were cut at 6 jr; they were stained with haematoxylin and eosin, the azan mixture or by Feulgen’s method. Histo fogy
RESULTS
The retinae of all seven species used in these experiments showed the general characteristics of a cone retina. Fig. 1 shows a section through the central area of the retina of the ground squirrel, Citeh beecheyi, showing a thin outer nuclear layer indicating a relatively small number of visual cells per unit area, with unusually thick inner nuclear and ganglion cell layers. It was found that the two species of tree squirrel, Sciurus carolinensis kucotis and
Fro. I. The ground squirrel retina. Section through the central area of the retina of CiteffrtJ &ee+&-~i. Note the exceptionally thick ganglion cell layer. Haematoxylin and eosin; Bouin. - 285. The following numbering applies to Figs. I,2 and 3: 1, pigment epithelium; 2, cones; 3, outer nuclear layer; 4, outer fiber layer; 5, inner nuclear layer; 6, inner fiber layer; 7, ganglion cell layer; 8, optic nerve fiber layer.
FIG;. 2. The squirrel visual cells. Section through the outer retina of Scirr~~r caroiinc~tsit lertcoris. Note the double layer of cones. Bleached: Azan : Kolmer ’ 700.
If... .“,. ,, 1Sh,
FIG. 3. The ground $&d
visual c&k. L!kctionthrough th&outer retina of Cite/h larerdis. acidhaunatoxylin; Zcnker. x 775.
Note the sir&e layer of cones. Mallo~y’s $ho&t?twtic
Spectral Sensitivity Curves of Diurnal Squirrels
157
Tamiosciurus hudronicus loquax, both had the double row of cones already described for Sciurus carolinensis (ARDEN and TANSLEY, 1955a). A high power view of the outer part of
the retina of this species is shown in Fig. 2. The retina of another ground squirrel, Citellus has also been described by ARDEN and TANSLEY (1955b) and the four Cite//us species used in the present investigation showed just the same picture, as abo did the one chipmunk used, Eutamias amoenus luteiventris. A high power view of the outer retina of Citellus lateralis is shown in Fig. 3. cireks,
FIG. 4. The pure-cone electroretinogram. Two electroretinograms of Cite//us beecheyi after removal of the lens. Dark-adapted; stimulus wavelength 531 rnp; no neutral filter; stimulus duration I /4 sec.
Electrophysiological
Fig. 4 shows a pair of typical electroretinograms as recorded on the Grass encephalograph. These demonstrate the initial negative (downwards) o-wave, the second positive b-wave (upwards) and the striking positive off-effect. The negativity recorded after the b-wave is partly, but probably not wholly, an artifact due to the pen’s overshooting. All eight channels of the encephalograph were used with a different amplification on each. It was found that excursions above about 10 mm were unreliable and those below 1 mm could not be read accurately. By using eight different amplifications, a much wider range of responses could be analysed. Most of the results analysed here are based on the amplitude of the b-wave (measured from the trough of the a-wave), but curves were also constructed for the responses of the a-wave and off-effect. It was found, however, that these did not
260
> a
-
260
-
240
-
220. 200 180
-
160
-
I40
-
I20
-
On
1.0
20 LOG
30
I
FIG. 5. Intensity-response curves. A group of representative intensity-response curves from a dark-adapted Cirellus beecheyi after removal of the lens. Single flash l/4 set stimulus. Ordinates: amplitude of the b-wave in JIV. Abscissae: log stimulus-intensity. L
KATHARINETAWSLBY,R. M.
158
COPENHAVER AND
R, D. GIJNKEL
difFcr sign&antly from those obtained from the b-wave and they are not, therefore, considcrcd separatciy. The spcctrai sc&ivity curves wcrc ca&tiatcd in the general way dcscribcd by COPENHAVSRand GUNXEL(1959). ~n~~zy-~~~ curves, mostly on b-wave ~p~t~c, were plotted for each wave&g& us& in a run and the neutral fBcr density rcquircd to give a constant rcsponsc dctcrmincd. This density was corrcctcd for di&cnccs in energy value for each spectral stimulus and for an equal quantum intensity spectrum; this correction incl‘llded that for the crystalline lens in those experiments in which it was not extracted. A typical group of intensity-response curves from the short-wave part of the spectrum caicuiatcd for one run with the animal whose ci~trorc~o~~ is given in Fig. 4 is shown in Fig. 5. Those from the rest of the spectrum were very similar. For this particular run criterion values of 300, 200, 100 and 50 pV wcrc chosen for constz-ucting the spectral sensitivity curves, but it
!
t
I
I
/
I
!
1
!
/
~~~ii~ 400
j j \u, !
so
500
600
450
600
FIG. 6. Spwtml sensitivitycurves. Two spectralsensitivitycurves from two dWmnt NM in one c$qmimmt on a dark-&p&d Cite&a latmiis. A. Fit in, 250ak’ aitkfm. 8. Stcand run, 100pV criterion. Or&a&es: pcrosnurscsensitivity.Absciww: wwekngth in mp (equal
qilantum intensity spa%unl). was found that there was no sign&ant di&encc between the curves for low and those for high criterion values (ARMINGTON, 19%). The density at each wavckngth was then detcrmined as a percentage of the density at the most effective wavelength and the points plotted against wavelength. To begin with, the curves were made by simply joining the individual points, but later, as a constant pattern emerged, the curves were drawn freehand. No sign&ant dil%rcncc was found between the curves for the different species, The curves were ncariy aH found to bc double humped; occasionally the humps were nearly of the same height but more often one or other was markedly higher. Fig. 6 shows two typical curves, both from the same animal and taken under the same experimental conditions. None of our different experimental conditions was found regularly to produce one type of curve rather than the other. It was thought, for instance, that the “biue” hump might be due to scattered fight. In order to test this point curves taken with a smdl (I”) field during dark adaptation, conditions which should favor stimulation of the retinal periphery by scattcrcd light, were compared with ones taken with a large (50”) f&d in light adaptation, but there was no evidence that the former conditions favored the “blue” hump
159
Spectral Sensitivity Curves of Diurnal Squirkls
or that the latter favored the “green”. Again it was considered possible that the presence of the yellow lens might protect a “blue” mechanism and so produce a higher relative sensitivity to the short wavelengths. It was for this reason that we originally removed the lens halfway through an experiment, repeating all the previous runs with an aphakic eye. Although the removal of the lens made it possible to get readings at shorter wavelengths than before, it had no effect on the relative sensitivities. No consistent difference was found between those curves taken during dark adaptation and those taken in light adaptation. There was therefore no evidence of a Purkinje shift in any of the species examined.
900
500
600
FIG. 7. Spectral sensitivity curve. Mean spectrai sensitivity curve from all the experiments on Citellus lateralis.
Ordinates:
Abscissae: percentage sensitivity. quantum intensity spectrum).
wavelength
in rnp (equal
The two different types of curve illustrated in Fig. 6 were found in about equal numbers so that the mean spectral sensitivity curve for this species was nearly symmetrical with the two humps of about the same magnitude (Fig. 7). A fair proportion of the spectral sensitivity curves showed a distinct, albeit often very minor hump on the descending slo.pe between 450 and 460 rnp (Fig. 11). When a mean curve for all the species investigated was drawn there was a distinct inflexion in this position which is outside the experimental error. The data for this mean curve (Fig. 8) are given in Table 1. It was calculated from the results of all the runs in which there was no evidence of a general change in sensitivity while the records were being made. It includes results in light- and dark-adaptation, with small, large and normal fields, using the amplitude of the b-wave, u-wave and off-effect, using high and low criterion values and for eyes with and without thecrystalline lens. It was calculated from the records of 248 runs in all. As a rule, our color-adaptation depressed the general sensitivity of the retina, but when the results were calculated as percentage of the maximum it was found that blue-adaptation depressed the hump at 490 to 500 rnp in all three experiments on Cirellus beecheyi, in one out of two experiments on Citellus lateralis, in one experiment on Citeilus tridecemlineatus, in one out of two experiments on Citellus variegatus, in one experiment on Tamiosci’urus huhonicus and in one experiment on Eutamias amoenus. Green- or orange-adaptation depressed the hump at 535 rnp in all three experiments on Citeflus beecheyi, in two experiments on Citeilus tridecemiineatus, in four experiments on Citelius lateralis, in two
160
KATHARINE TANSLHY, R. M. COPENHAVER AND R. D. GUNUL
TABLE 1 No. of
Widows
dnll
-4
I
610 583 558 545 531 s23 509 iii 473 456 442 419 404
/ : 1
Mm
sensitivity
(%I
iit 199 247 248 2: 248 143 248 32 z 113 24 8
experiments on Cirdus variegatus and in one experiment on Ta&o&nr~ &u&Mcw, but had no effect in one experiment on EutMuirr rlnrocm(s. The results ofoneof&6 ~XJW%WS on Citelbu kcheyi are shown in Fig. 9. In this figure the v&e at 531 rnp for the ordi~uy white-light-adapted sensitivity curve wo3 taken as 100 pet cent. For the cup toti with the blue-adapting light the value at 531 rnp was againtaken as 100 per CGILf,wbiie for the curve taken with the green-pskptiag light tbc value at 473 rnp was taken as 1IS par cent, the figure for this point on the original white &ht-adapted curve; the values for the other wavelengths were calculated accordingly.
Spectral Sensitivity Curves of Diurnal Squirrels
161
The electrophysiological results were analysed for us by the Biometrics Branch of the National Institute of Neurological Diseases and Blindness. It was concluded (I) that our data for the seven species were homogeneous and could legitimately be combined, and (2) that our mean spectral sensitivity curve (Fig. 8) was a good description of the response data as a whole. LA
Curve
Blue Bleach Green Blroch
_____
1
......‘..
IOC
50
-I
400
600
FIG. 9. The effect of color-adaptation. Three spectral sensitivity curves from one experiment on Citellus beecheyi. “.““““’ effect light-adapted curve; - - -effect of blue-adaptation; of green-adaptation. Ordinates: percentage sensitivity. Abscissae: wavelength in mp (equal quantum intensity spectrum). DISCUSSION
The histological examinations indicate that all Cite//usspecies and at least the one chipmunk species used have a similar retinal structure with a single layer of cones, and that in the central retina there is probably a one-to-one relationship between these cones and the ganglion cells and, therefore, the optic nerve fibers. In the tree squirrels it seems likely that the cones always form a double layer and, since the ganglion cell layer is nowhere as thick in these species as it is in the central retina of the ground squirrel, it is improbable that there is a separate optic nerve fiber for each cone in any part of the retina. A similar retinal structure has been described by ROCHON-DUVIGNEAUD (1943) for the European red squirrel, Sciurus vulgaris. He recognized two groups of visual ceils in this animal, but he used tangential sections and mistook the outer segments of the inner row of cones for rods. All the physiological evidence points to these being pure-cone retinae (TANSLEY, 1957). The suggestion that a high blue sensitivity might be characteristic of the ground squirrels as opposed to the tree squirrels was not confirmed by the results of the present investigation where no significant difference was found between the sensitivity curves of the two groups. The shape of the spectral sensitivity curves came as a surprise. The long-wave part of the mean curve is in reasonable agreement with this part of the curves published by Arden and
162
KATHARINE TANSLEY, R. M. COPI!NWAVER AND R. D. GUNKEL
TansIcy for the gray squirrel and CireuuE c~te~~~and with that for the dif&rence spectrum of the photolabile pigment found by WWLE (1955) in the living retina of the gray squirrel. However, the second peak at about 495 rn~ was quite unexpected. It is true that a capriczious high Mua sensitivity was found by Arden and Tans@ in Ore&s citehs but their points in the relevant part of the spectrum between r(80 and 545 rnp were too few and their stlrndard error too great to make a satisfactory characterization of this part of the curve possible. Although Arden and Tandey did not suspect a second rise in sensitivity in their gray squirrels, if mean values are calculated for their pubtished points there is some evidence for an inflexion on their curve at about 490 rn# aithough it is not very marked.
I-
I
We made many attempts to elucidate the nature of the m~hani~ responsible for the “biue” paak and were satishd that it was not due to scattend Ii@. ft did not baeome more prominent in dark-adaptation nor if low crit&a were used. The only way in whi& we were able Co a&et the &ape of the curve in a predictable way was by using c&red adapting tights while the EM&S were b&g taken. Then we found that a b~~~~g light depressed the “blue” peak and a green- or orange-adapting light depmsmd tk “mn” one. This suggests that both puaks are rather closely associated with photosensitive mechanisms of different spec.tral sensftivities (fig. 9). Although thu two peaks are of roughly the same height in the mean curve, the curves for individual experiments usually showed one or other tQ be the higher (Fig. 6) and this suggested th+ the two mechanisms might b in some sort of rivalry. In the hop of throwing some light on this idea all the curves were divid& into two groups, one in wfrich the values at 523 and 531 rn& were the hi+@ and a saoond in which the highest values ware fou& to be at wavelengths shorter than 523 rnp. Wktn the means of these two groups w plot&& separWly the two curves shown in Fig, 10 were obtained. These show that when one peak is high the other does indaed tend to be very much red-d thus indicating some degreeof rivairy between the mechanisms responsible for the two peaks.
Spectral Sensitivity Curves of Diurnal Squirrels
163
In trying to obtain more information about the mechanism responsible for the “blue” peak one must remember that the position of its maximum sensitivity is likely to be at a rather shorter wavelength than the 495 rnp shown in Fig. 8 or the 490 rnp shown in Fig. 10. Even the curve in Fig. 10 is probably pulled over somewhat towards longer wavelengths by the effect of the “green” mechanism and, in fact, if one plots the difference between the two curves in Fig. 10 for the shorter wavelengths the peak of the resultant curve falls at about 480 rnp. Recently DARTNALL (1960) has studied extracts of the gray squirrel retina and has found it to contain a rhodopsm with a maximum absorption at 502 rnp. This pigment on bleaching produced, in addition to the normal retinene, a photoproduct with its maximum absorption at 480 rnp. In considering the bearing of this result-on the spectra1 sensitivity
FIG. 11. Spectral sensitivity curve. Spectral sensitivity curve from a light-adapted Circllus beecheyi showing high values between 450 and 460 rnp. Ordinates: percentage sensitivity. Abscissae: wavelength in rnp (equal quantum intensity spectrum).
function found by Arden and Tansley and on the difference spectrum found by Weale in living squirrels Dartnall suggests that in viva rhodopsin in the squirrel cones may bleach to a product having its maximum absorption at 480 ml and which is itself photosensitive. He further suggests that the spectra1 sensitivity function with its maximum at around 530 rnp “may be mediated by a mechanism depending on the difference between the light absorbed by the rhodopsin at 502 rnp and that absorbed by the 480 photoproduct”. He points out that if this is indeed the case a truly threshold sensitivity function would be expected to reproduce the 502 curve alone. None of our records was taken at this level since the electroretinogram is too insensitive a criterion to be useable at the absolute threshold. He also suggests that prolonged adaptation to long-wavelength light should result in a spectra1 sensitivity function which follows the form of the broad band 480 curve. The orange filter we used for adaptation had too broad a transmission band for adequate testing of this point. If sufficient adaptation was used entirely to eliminate the peak at 531mp the whole retina1 sensitivity was so depressed that satisfactory records were unobtainable even in the shortwave part of the spectrum. In the light of Dartnall’s findings a very tentative explanation of the spectral sensitivity curves found in this investigation might be that the 535 rnp peak is the result of interaction of some sort between two photosensitive substances with maxima at
164
KATHARINE
TANSLEK, R.
M.
COPENHAWR
AND 9,
D.
GUNKEL
SO2and 480 rn& and that, in addition, the spectral sensitivity of the 480 pigment IS responsible for the peak in this region. If, further, these pigments may be formed from one another, as Dartnall believes, this would explain the apparent rivalry of the mechanisms responsible, for our two peaks for the more of one pigment was present the less of the other. In the mean curve of Fig. 8 there is a distinct inflexion around 450 to 460 rn& which, although small, is outside the experimental error. In 37 per cent of all the experimeats in which records down to 442 m,u were obtainable the readings at 456 rnp were high, occasionally as high as or even higher than is shown in Fig. Il. In the remaining 63 per cent there was no evidence whatever of any special activity in this region. All but one of the curves with enhanced values at 456 rn@ were obtained from tight-adapted eyes. In the paper already quoted, Dartnall points out that interaction between possible activity mediated by the 480 pi~ent and that mediated by the 502 pigment might also give a spectral sensitivity function with a maximum at 448 mp. Spectral sensitivity curves obtained by e~~trophysio~ogi~ai methods with maxima or humps in the blue, usually capricious in their appearance, have often been reported in the literature. GRANIT (1947) in one of his figures shows a curve obtained from the guinea-pig retina by means of his microelectrode technique in which the maximum is at about 460 rnp with a secondary hump at about 535 mp. DODT and ELENIUS(1956) working on single-fiber responses from the rabbit retina obtained some curves in which the maximum sensitivity is at 460 mp. In two of these there is evidence of a secondary maximum near 500 rnp but in a third the secondary maximum is ptobabiy at a rather longer wavelength. ELENIUS (1958) shows two spectral sensitivity curves also from the rabbit obtained by means of the eiectroretinogram in which there was an enhanced sensitivity at 480 m,u. In both guinea-pig~nd rabbit the predominant visual pigment is, of course, rhodopsin. In this connexion it is worth mentioning ~NGVAR’S(1959) paper on the cortex of the cat (afso with a predominantly rhodopsin-containing retinal in which he suggests that there may be two scotopic mechanisms, one with maxima at about 460 and 530 rn& which is in rivalry with a second whose maximum is at 490 mp. lngvar found these mechanisms to be inherently utistable. Although the mean curve of Fig. 8 shows no irregularity in the long-wave region of the spectrum, a comparison of this curve with Weale’s for the gray squirrel does show a slight discrepancy at about 560 rnp which might indicate a further mechanism of some sort active in this spectral region, but the evidence is certainly not convincing enough to warrant drawing any conclusions on this point. It has been argued that spectral sensitivity curves obtained by means of intarfinnce lifters can show odd peaks which are reaily artefacts due to the s+ shape of the transmission bands of some filters of this type. This is not like& to be a corn~~~~ in the present investigation since Copenhaver and Gunkei got pcrf%ctly smooth phatopic senaitivity curves from human subjects with normal vision using the same apparatus and all but three of the fiiters we have used here. However, in order to test this point we did do two experiments on the dark-adapted rabbit and in these we obtained spectral sensitivity curves which satisfactorily fitted the rhodopsin absorption curve, SUMMARY 1. Using the e~ectroretino~am as the criterion, spectral sensivity curves were obt&ed from seven different species of the squirrel family. 2. Histological e~amjnatjon showed that a11these species have pure-cone retinae.
Spectral Sensitivity Curves of Diurnal Squirrels
165
3. The curves showed two maxima at about 535 and 490 rnp respectively. There was evidence from color-adaptation experiments that the two maxima are associated with two mechanisms with different spectral sensitivities. 4. The possible nature of the mechanisms responsible for these two maxima is discussed. 5. There was some evidence for a further mechanism with its maximum activity between 450 and 460 m,u. REFERENCES ARDEN, G. B. and TANSLEY, K. (1955a). squirrel (Sciwrc~ curolinenis). J. fhysiol.
The spectral sensitivity curve of the pure-cone retina of the gray 127, 592-602.
ARDEN. G. B. and TANSLEY, K. (1955b). The spectral sensitivity curve of the pure-cone retina of the souslik (CireNlr, cite//us). J. Physiol. 130, 225-232. ARMINGTON, J. C. (1955). Amplitude of response and relative spectral sensitivity of the human electroretinogram.
J. opr. Sot. Amer. 45, 1058-1064.
COPENHAVER, R. M. and GUNKEL, R. D. (1959).
The spectral sensitivity of color-def&tive subjects deterArch. Ophrh. 62. 55-68. DARTNALL, H. J. A. (1960). Visual pigment from a pure-cone retina. Ncrure. Lond., H&475-479. DODT, E. and ELENIUS. V. (1956). Spektrale Sensitivitiit einzelner Elemente der Kaninchennetzhaut. t’fliig. Arch. ges. Physiol. 262, 301-306. ELENIUS, V. (1958). Recovery in the dark of the rabbit’s electroretinogram. Acta physiol. stand. 44, suppl. 150. GRANIT, R. (1947). Sensory mechunisms ofthe retino. Cumberlege and Oxford Univ. Press, London. INCVAR. D. H. (1959). Spectral sensitivity as measured in cerebral visual centers. Acruphysiol. scund. 46, mined by electroretinography.
A.M.A.
suppl. 159. ROCHON-DUVIGNEAUD, A. (1943). Les yeux et la vision des VerrdbrPs. Masson, Paris. TANSLEY. K. (1957). Some observations on mammalian cone electroretinograms. Bib/. ophthal. 48, 7-14. WEALE. R. A.’ (1955). Bleaching experiments on eyes of living gray squirrels (Sciww curolinensis leucotis). J. Physiol. 127, 587-591.