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Properties of the frog's retinal ganglion cells as revealed by substitution of chromatic stimuli
Vision Res.
Vol. 10,pp. 829-836. PergamonPress 1970. Printedin Great Britain.
PROPERTIES OF THE FROG’S RETINAL GANGLION CELLS AS REVEALED BY SUBSTITUTION OF CHROMATIC STIMULI1 H. SCHEIBNER and CH. BAUMANN Abteilung fiir Experimentelle Ophthalmologic (II. Physiologische Abteilung) des W. G. Kerckhoff-Instituts der Max-Planck-Gesellschaft, Bad Nauheim, Germany (Received 20 February 1970; in revised form 25 March 1970)
INTRODUCTION BEHAVIORAL studies have indicated that several frog species are in possession of a colour vision (BIRUKOW,1949; THOMAS,1956). From the standpoint of its visual pigments, the frog retina could well be able to process colour coded information since it contains beside the rod pigment rhodopsin, two cone pigments with absorption peaks at 502 nm and 575 nm (LIEBMANand ENTINE, 1968) and a visual pigment 433, (DARTNALL,1967) situated in the green rods. The latter receptors are known to function under mesopic conditions where pink rods, green rods, and cones contribute to the retinal response (DONNERand RUSHTON, 1959a, 1959b). As regards the neurophysiology of a complete colour vision, one might expect to find several independent photopic (or mesopic) mechanisms, of which the properties should be specifically related to the spectral absorption of the pigments mentioned. Our experiments were concerned with the processing of chromatic stimuli in the third neuron layer, the ganglion cells of the retina, where we looked for independent mechanisms. We used a method that is analogous to the visual colour match in calorimetry. In this, two visual fields are presented side by side, and the observer is asked to vary the colour of one field so that it appears indistinguishable from the other. The principle of colour matching can be modified in such a way that the stimuli are presented in succession and to the same retinal area, as was done by BONGARD,SMIRNOWand FRIEDRICH(1958) when investigating extra fovea1 colour vision. When working with animals, however, the criterion of indistinguishability has to be transferred to the responses recorded. In our experiments, a given coloured stimulus is quickly replaced by a second stimulus of different wavelength. By means of a microelectrode inserted into the ganglion cell layer, impulse discharges are obtained at the following times: (a) when the first stimulus is switched on, (b) when this stimulus is replaced by a second stimulus, and (c) when this second one is switched off. Our attention is particularly directed to the stage (b) when substitution takes place. Here, we distinguish two possibilities: (1) For a certain intensity ratio of the two successive stimuli, no new discharge is initiated. Following DONNER and RUSHTON (1959a), we call this event a “silent substitution”, and chose this non-appearance of discharge as a criterion of colour match. (2) Whenever the first stimulus is replaced by a second, a new discharge always occurs. This is taken to indicate the lack of silent substitution. The principle of this method applied to animals was first used by ISHIHARA (1906), later 1 Supported by the Deutsche Forschungsgemeinschaft. 829
830
H. SCIWBNERAND CN. BAUMANN
also by BONGARD (1955), BONGARD and SMIRNOW (1957, and, extensively, by DONNERand RUSHTON(1959a). Whereas Donner and Rushton aimed at retinal elements that could be silently substituted, we were primarily interested in ganglion cells that did not show silent substitution.
MATERIALS AND METHOIX Fisure I shows the s&me of the experimental set up (SCHEXBNBR and BA~RUNN,1969). The optical portion consists of four paths which originate from two 1SOWxenou lamps, and eventually ~~~the~~ua~froe~.paths~sndIIp~~~thePghts~fortha~on. ~~ofthapduiEhrp~~~andPu,thesebasmsarslinarrtiypdarizsdinpl~~~tocacb other, and supsipostd by a beam sphttintt cube. Tfie two beams then pass the pea fiber A, which acts as an ttnalyser. This anafyser may be in one of two fixed positions, which d&r by a rotation of 90”.In the
Xe - Lamp
Ro.
I. Scheme
of the experimental set up. Pt. Pu: polarixers, A: anaiyser, Sr: stop.
for a uuitary one. 3sam XVis superimposed to the stimulatmg lit&t at the last c&a; it serves illumiu&m or for bka&@. In front of the last cube, a stop Sr is mounted. This stop can be moved within a plane perpe&ii to the optical axis. By means of this movabk stop, the size of the stimulus on the retina can be consned and the position may be varkd. In all optical paths, there are neutmi and inte&rence fhters in order to vary the radiation ht respect to intensity and wavekraglh. These laftsrs as well as the knees are not drawn in Fig. 1. On the right side of Fig. 1, the perfusion chamber is schematically shown. Iu it, the isolated retina, receptors downward, is held between two nylon meshes whtk perfused by a suitttbk medium, For details, see Srcxz (l%l, 190, or BAUMANN (1967). P~n~~~ alloy after WAONBR,MACNICXIL and Wow (I%@& were used for extracelhdar record& of *hnptdsedischarges. Their diameter at the tip was I-3 qn. Immediately before the expe&knt, the tip wss pkti&ed; the resistance a@st Ringer solution was thtat kbout O$-141 M Q. TheamlitudGsofUltimpulsn~~wen.asaNlebot~60~Vand140ciV. Waterfrot3s Q&rnc csEdcnf0) were used in the exper&mts. The preparation and the procedure were as in carher experiments ~A~ and Scwaumxa, 1968) and are therefore report& o&y b&t& hem, The retina was isolated from the excised eye and mounted into the perfusion chamber. The electrode was inserted
Properties of the Frog’s Retinal Ganglion Cells
831
from above, the side of the inner limiting membrane. Stimulation with light took place through a quartz window from below, the receptor side. The diphasic nature of the spikes recorded showed that their origin were ganglion cells (GERNANDT,1948; BARLOW,1953a). After having recorded the first impulses, thestop Sr was moved in, and its optical image was placed concentrically around the tip of the electrode. For the first series of experiments, we chose a stop size such that its image on the retina had a dia. of 0.55 mm. According to BARLOW(1953b), the receptive fields in the frog retina are of about 0.5 mm dia. In this series, we employed a permanent background illumination of 2-2 lx or 0.8 lx. The second series of experiments was performed with a stop size which gave a light spot on the retina 0.35 mm dia. In this series, the entire retinae were bleached with monochromatic light before the recordings were taken. Intensity and duration of the bleaching light were such that about 95 per cent of the rhodopsin was bleached. This extent of bleaching was estimated by means of an equation that describes the kinetics of bleaching of rhodopsin in the isolated retina (BAUMANN,1966). The wavelength of the bleaching light was usually 578 nm, though in some cases light at 502 nm was used. Under the conditions observed, it is to be expected that practically no rhodopsin will regenerate after bleaching (BAUMANN,1970). Previous experiments had established (BAUMANNand SCHEIBNER,1968) that the scotopic system remains functionally eliminated if more than 40 per cent rhodopsin is bleached. This behaviour is contrasted to the recovery of the early receptor potential (GOLDSTEIN,1967), which indicates that the cone pigments do regenerate in the isolated retina when separated from its pigment epithelium. Thus, in this second series, we felt sure that only the photopic system was working, when, after a period of about 15 min following the end of bleaching, the impulses were recorded without permanent background illumination. This procedure was more favourable for recording and, moreover, revealed more ganglion cells that could not be silently substituted than the procedure of the first series. The duration of stimulation amounted altogether to 7 sec. Between set 3 and set 4, the first stimulus was substituted by a second one (see Introduction). For those wavelengths for which a silent substitution occurred, the spectral sensitivity function S, (h) was defined:
wherein I, (X,) means the intensity of the reference stimulus constant in wavelength, and Z(X) means the intensity of the stimulus variable inwavelength, both quantities taken at values for which silent substitution occurs. All intensities are expressed as relative numbers of light quanta. If a silent substitution can by no means be brought about, a description by means of a connected sensitivity function is not possible. In general, however, a different reference wavelength can be found that can be silently substituted. Thus, disconnected pieces of sensitivity functions are obtained. After RUSHTON(see, e.g. NAKA and RUSHTON, 1966a, 1966b; or SCHEIBNER and SCHMIDT, 1969), a transducing system that exhibits a (connected) invariant spectral sensitivity function may be called “univariant”. If a transducing system exhibits a disconnected spectral sensitivity function, it is in any case nof univariant. During the course of this study we found ganglion cells which showed what might be called a univariant stimulus-response behaviour over the whole spectrum, as well as other ganglion cells which did not show such behaviour. RESULTS
Figure 2 shows three spectral sensitivity functions which were measured against a permanent background illumination. The reference stimulus was of wavelength 502 nm (indicated by the arrow in Fig. 2). At this wavelength, the sensitivity is normalized to one, i.e. log sensitivity equals zero. All three ganglion cells could be silently substituted over the whole spectrum. Most of the ganglion cells investigated under these conditions showed such behaviour. In a few cases, however, there appeared a disconnected branch of sensitivity function in the blue wavelength region. Owing to the background illumination, these branches could not be determined in detail. A rough estimate of our data indicates that every fifth ganglion cell did not behave univariantly under the conditions of this first series. Figures 3 and 4 show results from retinae where about 95 per cent of rhodopsin had been bleached in advance so that the scotopic system remained functionally eliminated, even without background illumination. Figure 3 presents four ganglion cells that, over the whole spectrum, exhibited a stimulus-response behaviour that may be called univariant. The sensitivity is normalized to one at the wavelength of the reference stimulus, h, = 600 nm (arrow), i.e. log sensitivity equals zero.
832
H.SCHEIBNER
AND
Cn. BAUMANN
0.6
-56
Ro.
Bockground :n 0.8 Ix; o A 0.2 Ix; Stimulus tixa:033mm + Total duration of stimulotion~?
xenon SW
2. Spectral sensitivity functions of three u&&ant ganglion ceils measured under background illumination. 0.6 0.4 0.2
2%
Total
duration
I
??6
Fm. 3. Spectral sensitivity functions of four univariant *giion most of the rbodopsin.
ids measumd after bleaching
Figure 4 presents the results of four gan&ion cells that did not show univariance.With them, blue stimuli c&d not be silently substituted by red stimuli. For three cells, the break in univariance lies near 500 nm, for one cell near 550 nm (curve of It was difikuit to determine exactly the shape of the biue sensitivity branch. Some on c&s did not respond at al1 in a reproducible way to blue stimuli. Therefore, wecanpresent only
833
Properties of the Frog’s Retinal Ganglion Cells
06 i-
x cn” zf -J
t
i-6? 7.4
S,(450) -
T-2-
t
~
X [nm)
r,rboot
0
To
-
Se 76
-
F,4
-
s
o+ a
J,* ( is) I(X).
A,
=
600,450 nm
Bleaching liqht:c 502 nm,oaO578 nm Stzmulus .size:0,35mm Total durotlon of sttmulotion;7sec
FIG. 4. Spectral sensitivity functions of four non-univariant ganglion cells rneasurd after bleaching most of the rhodopsin.
one disconnected sensitivity branch in the blue region. Nevertheless, the break in univariance could be uniquely established. The reference wavelength for the sensitivity in the long wavelength region is 600 nm, for the ~nsit~tivity in the short wavelength region, 450 nm {arrows). The curves are not normalized at these wavelengths so that the absolute scatter of the measured values can be seen. A gross estimate indicates that every second ganglion cell did not behave univariantly under the conditions of this second series. DISCUSSION
Receptors containing one pigment only distinguish themselves by a univariant spectral stimulus-response behaviour. The same should be unlikely in the case of multipolar neurons such as retinal ganglion cells, if they receive signals from different classes of receptors. Nevertheless, many ganglion cells show a behaviour that may be called univariant, as can be seen from Figs. 2 and 3, although in a stricter sense univariance implies that silent substitution has to be held over a wide range of intensity. Since we were mainly interested in cases not exhibiting silent substitution, we did not test this aspect of univariance. A comparison proves that the curves of both Fig. 2 and Fig. 3 are in good agreement with Granit’s photopic dominator (C&WIT, 1942). This dominator peaks at 560 nm and shows a secondary maximum in the blue region (tabulated values are given by REUTER (1969)). Our curves exhibit some scatter in the peak wavelengths, which we do not consider to be significant; besides, a secondary peak in the blue region could not be verified. A similar deviation from the photopic dominator in the blue region is reported by CHAPMAN (1961). He determined the spectral sensitivity by means of thresholds using the latency of responses as a criterion.
834
H. SCHEIBNER ANDCH. BAUMANN
Jnvestigating the early receptor potential (ERP) of the isolated frog retina, GOLDSTEIK (1967) reported a spectral sensitivity function that resembles also the curves of Figs. 2 and 3. Goldstein attributed this ERP sensitivity to the cones. A univariant behaviour of a ganglion cell does not mean that this cell has to be fed from one type of receptors only. This conclusion is arrived at by DONNERand RUMTON (1959a, 1959b), DOUR (1959), and RWSHTON(1959). In other words, univariance is a necessary consequence of a retinal organization that connects a ganglion cell with only one receptor, but the converse is not true, i.e. one cannot infer from an observed univariance that such is the case. DONNEZR and RUSHTON(1959b) estabIished this conclusion experimentally by means of the Stiles-Crawford effect. In order to explain this rest&, RUSHTON(1959) postulated the existence of an “excitation pool” whose site is between the receptors and a ganglion cell. In spite of its univariant character, therefore, it is still pertinent to ask how the photopic dominator is determined from the contributions of the various receptor types. In view of the pigments found (LIEBMANand ENTINE, 1968), this question is still open (see also REUTER, 1969). The logical contraposition of Donner and Rushton’s conclusion tells us that a break in univariance is due either to the cooperation of more than one receptor type or to no cooperation at all. As far as there were reproducible responses in the blue region, the results of Fig. 4 confirm, therefore, the existence of an independent blue mechanism. After DONNER and REUTER(1962), REUTER(19691, and others, this blue mechanism originates in the green rods, the pigment of which exhibits its absorption maximum near 433 nm (DARTNALL,1967). The ganglion cell under consideration then receives information from green rods. As far as there were no unique responses in the blue region, the results of Fig. 4 suggest a retinal organization whereby this ganglion cell does not receive information from the green rods. After MUNTZ (1962), REUT~ (19691, and others, the blue mechanism reacts exclusively with “on” responses. On the other hand, CHAPMAN (1961) reported on findings that indicate pure “off” responses of the blue mechanism. It is difficult for us to decide in favour of one of these two opinions, as the substitution method is not particularly suited for that kind of problem.
REFERENCES B-w, H. B. (1953a).Won potcsntiatpfrum the fmg’s xetina.J. Ply&f. 119,58-f&?. BMLOW,H. B. (1953b). Swnawba and inhibition in the frogs mtina. J. Physcol. 119,69-8&. BALIMANN, C&I. (1966). Dcr EiidluB van Metarbd@n auf die Schpurpurbkichung in der isolierten Netzhaut. Vfston Res. 4,543, BAV~UNN,CH. (1967). ~~~8 und Wbchenfunktion in der isolii Froschnetzhaut, I. Die ~hp~~ I!#i&wrs Arch. ges. Physfaf. 293,444. BAUWNN, CR (197(f). Rmtkx.~ of rbodopsin in the isolated retina of the frog (Rmrrres&&z). yision Res. IO, 627-637. BAUMANN,Cx and ~SCHWW& H. (1968).The dark adaptation of sin@eunits in the isoMcd frog retina followingmal bkacb& of riiod&. Vi&wa &a. 8,1127-1138. Burrow, 0. (1949).Dii EntwWun8 des Ta8cs- und DV im A* dca &asfroschcs. 2. veigl. Physid. 31, 322-347. ml!302 z $. $53). C&.&wry in animafg (Russ.). C.R.Acad. sci. USSR 193,239-Z&!. !kstxow* M. S. fi937). Spe&ai sensitivity cum8 for mcqtton immuxed to single ffbFesbf tile &tic nerve of the frog. iY?iqi%*a5 336-341. BONCMRD, M. M., SWRNOW,M. 5. and l3u~~~~x, L. (1958).The four-dimensionalcolor space of the extra-fovea1 arwt of the hw eye. In NpL-sympaiwn on ViJkbaProbkvt~~of C&w, Tedfimo~~ 1957, Vol. X,327-330, Her k&jm~‘s StUmeq O&e, Lundon. CHAPMAN, R. M. (l%l). Spectral sensitivityof sin& neural units in the bullfrogretina. J. opt. Sot. Am. 51, 11024112.
Properties
of the Frog’s Retinal Ganglion
Cells
835
DARTNALL,H. J. A. (1967). The visual pigment of the green rods. Vision Res. 7, l-16. DONM?R, K. 0. (1959). The effect of a coloured adapting field on the spectral sensitivity of frog retinal elements. J. Physiol. 149, 318-326. DONNER, K. 0. and RUSHTON, W. A. H. (1959a). Retinal stimulation by light substitution. J. Physiof. 149,288-302, DONNER,K. 0. and RUSHTON,W. A. H. (1959b). Rod-cone interaction in the frog’s retina analyzed by the Stiles-Crawford effect and by dark adaptation. J. Physiol. 149, 303-317. DONNER, K. 0. and REUTER,T. (1962). The spectral sensitivity and photopigment of the green rods in the frog’s retina. Vision Res. 2, 357-372. GERNANDT,B. (1948). The form variations of the spikes recorded by a micro-electrode applied onto the mammalian retina. Acru physioi. stand. 15, 88-92. GOLDSIXIN,E. B. (1967). Early receptor potential of the isolated frog (Runa pipiens) retina. Vision Res. 7, 837-845. GRANIT, R. (1942). Colour receptors in the frog’s retina. Acfa physiol. stand. 3, 137-151. ISHIHARA,M. (1906). Versuch einer Deutung der phot~lektrischen ~hw~kun~n am Froschauge. Pptigers Arch. ges. Physiol. 114, 569-618. LIEBMAN,P. A. and ENTINE, G. (1968). Visual pigments of frog and tadpole (Runa pipiens). Vision Res. 8, 761-775. MUNTZ, W. R. A. (1962). Microelectrode recordings from the diencephalon of the frog (Rana pipiens), and a blue-sensitive system. J. ~earophysiof. 25,699-7 11. NAKA, K. I. and RUSHTON,W. A. H. (1966a). S-potentials from colour units in the retina of fish (Cyprinidue). J. Physiol. 185, 536-555. NAKA, K. I. and RUSHTON,W. A. H. (1966b). An attempt to analyse colour reception by electrophysiology. J. Physiol. 185, 556-586. REIX~R, T. (1969). Visual pigments and ganglion cell activity in the retinae of tadpoles and adult frogs (&ria temporuria L.). Acta 2001. Fenn. 122, l-64. RUS~~IQN,W. A. H. (1959). Excitation pools in the frog’s retina. J. Physiol. 149, 327-345. SCHEIBNER,H. and SCIIMIDT,B. (1969). Zum Begriff der spektralen visuellen Empfindlichkeit, mit elektroretinographischen Ergebnissen am Hund, V. Gruefis Arch. OphthaL 177,124-135. SCHEIBNER,H. and BALTMANN, Ch. (1969). ~lek~ophysiolo~sche Farbsi~~tersuchungen mittels Reizsubstitution. Ber. Dtsch. Ophthaf. Ges. 69, 124-126. SICKEL, W. (1961). Stoffwechsel und Funktion der isolierten Netzhaut. In Neurophysiologie und Psychophysik des visuellen Systems Symposium Freiburg 1960 (Hrg. R. JUNG und H. KORNHUBER),pp. 80-94. Springer, Berlin. Src~e~, W. (1966). The isolated retina maintained in a circulating medium. Combined optical and electrical inv~ti~tions of metabolic aspects of generation of the el~troretinog~. In CIinicaf E~ecfrorefi~ogruphy (edited by H. M. BUIUANand J. H. JACOBSON)pp. 115-124. Pergamon Press, Oxford. Trro~ns, E. (1956). Untersuchungen iiber den Helligkeits- und Farbensinn der Anuren. Zool. Juhrbuch 66, 129-178. WAGNER,H. G., MACNICHOL, E. F., JR. and WOLBARSH~‘, M. L. (1960). The response properties of single ganglion cells in the goldfish retina. J. gen. Physiof. 43, Supplement, 45-62.
Abstract-By means of the method of stimulus substitution, single ganglion cells of the isolated perfused retina of f&u escafenta were investigated. The scotopic system was eliminated (a) by a permanent background iliumination, (b) by bleaching more than POper cent of rhodopsin. The non-appearance of the impulse response during and after light substitution was taken as a criterion for an invariant spectral stimulus-response behaviour (univariance). Only some of the ganglion cells investigated showed this property. The spectral sensitivity of the other cells could be described only by several disconnected branches of functions. Therefore, it was concluded that there exist at least two photopic (or mesopic) mechanisms in the frog retina.
RiSsum&-Par la methode de substitution de stimulus, on Btudie des cellules gangliormaires tmiques de la retine iso& et perfused de Runa escuienta. On elimine le systeme scotopique (a) par un &z&rage permanent du fond et (b) en dt+colorant plus de POpour cent de la rhodopsine, La non-apparition de la reponse en impulsions pendant et apres la substitution de lumiere est prise comme criterium d’un comportement invariant de la response vis-a-vis du stimulus spectral (univariance). 11n’y a que certaines des cellules ganglionnaires Btudicies qui presentent cette proprittt5. Onhapeut d&ire la sensibilite spectrale des autres cellules qu’avec plusieurs branches de fonctions sans cormexions. On en deduit qu’il existe au moins deux mecanismes photopiques (ou mesopiques) dans la r&tine de grenouille.
836
H. SCHEIBNERAND
01. BAUMANN
B-Nach der Methode der Reizsubstitution wurden an der isolierten perfundierten Netzhaut von Rana escuhra einzelne Ganghenmll~ untersucht. Das skotopische System wurde ausgeschahet (a) durch eine permanente ~rnt~~d~~~ht~g, (b) durch Bleichung van mehr aIs 90 Prozent des Rhodopsins. Das Au&l&en der ~p~two~ bei der Substitution van Farbre&n wurde ah Kriterium fiir ein einheithches spektrafes ReizAntwort-Verhalten (Univarianz) gewertet. Nur em Teil der untersuchten ~Ganghenzehen zeigte diese Eigenschaft. Die spektrale Empfmdlichkeit der anderen Zellen Iieg sich nur durch mehrere nichsgende Teilfunktionen beschmiben. Hieraus wurde auf die Existenz van mindestens zwei photopischen (oder mesopischen) Mechanismen geschlossen.
Vol. 10,pp. 829-836. PergamonPress 1970. Printedin Great Britain.
PROPERTIES OF THE FROG’S RETINAL GANGLION CELLS AS REVEALED BY SUBSTITUTION OF CHROMATIC STIMULI1 H. SCHEIBNER and CH. BAUMANN Abteilung fiir Experimentelle Ophthalmologic (II. Physiologische Abteilung) des W. G. Kerckhoff-Instituts der Max-Planck-Gesellschaft, Bad Nauheim, Germany (Received 20 February 1970; in revised form 25 March 1970)
INTRODUCTION BEHAVIORAL studies have indicated that several frog species are in possession of a colour vision (BIRUKOW,1949; THOMAS,1956). From the standpoint of its visual pigments, the frog retina could well be able to process colour coded information since it contains beside the rod pigment rhodopsin, two cone pigments with absorption peaks at 502 nm and 575 nm (LIEBMANand ENTINE, 1968) and a visual pigment 433, (DARTNALL,1967) situated in the green rods. The latter receptors are known to function under mesopic conditions where pink rods, green rods, and cones contribute to the retinal response (DONNERand RUSHTON, 1959a, 1959b). As regards the neurophysiology of a complete colour vision, one might expect to find several independent photopic (or mesopic) mechanisms, of which the properties should be specifically related to the spectral absorption of the pigments mentioned. Our experiments were concerned with the processing of chromatic stimuli in the third neuron layer, the ganglion cells of the retina, where we looked for independent mechanisms. We used a method that is analogous to the visual colour match in calorimetry. In this, two visual fields are presented side by side, and the observer is asked to vary the colour of one field so that it appears indistinguishable from the other. The principle of colour matching can be modified in such a way that the stimuli are presented in succession and to the same retinal area, as was done by BONGARD,SMIRNOWand FRIEDRICH(1958) when investigating extra fovea1 colour vision. When working with animals, however, the criterion of indistinguishability has to be transferred to the responses recorded. In our experiments, a given coloured stimulus is quickly replaced by a second stimulus of different wavelength. By means of a microelectrode inserted into the ganglion cell layer, impulse discharges are obtained at the following times: (a) when the first stimulus is switched on, (b) when this stimulus is replaced by a second stimulus, and (c) when this second one is switched off. Our attention is particularly directed to the stage (b) when substitution takes place. Here, we distinguish two possibilities: (1) For a certain intensity ratio of the two successive stimuli, no new discharge is initiated. Following DONNER and RUSHTON (1959a), we call this event a “silent substitution”, and chose this non-appearance of discharge as a criterion of colour match. (2) Whenever the first stimulus is replaced by a second, a new discharge always occurs. This is taken to indicate the lack of silent substitution. The principle of this method applied to animals was first used by ISHIHARA (1906), later 1 Supported by the Deutsche Forschungsgemeinschaft. 829
830
H. SCIWBNERAND CN. BAUMANN
also by BONGARD (1955), BONGARD and SMIRNOW (1957, and, extensively, by DONNERand RUSHTON(1959a). Whereas Donner and Rushton aimed at retinal elements that could be silently substituted, we were primarily interested in ganglion cells that did not show silent substitution.
MATERIALS AND METHOIX Fisure I shows the s&me of the experimental set up (SCHEXBNBR and BA~RUNN,1969). The optical portion consists of four paths which originate from two 1SOWxenou lamps, and eventually ~~~the~~ua~froe~.paths~sndIIp~~~thePghts~fortha~on. ~~ofthapduiEhrp~~~andPu,thesebasmsarslinarrtiypdarizsdinpl~~~tocacb other, and supsipostd by a beam sphttintt cube. Tfie two beams then pass the pea fiber A, which acts as an ttnalyser. This anafyser may be in one of two fixed positions, which d&r by a rotation of 90”.In the
Xe - Lamp
Ro.
I. Scheme
of the experimental set up. Pt. Pu: polarixers, A: anaiyser, Sr: stop.
for a uuitary one. 3sam XVis superimposed to the stimulatmg lit&t at the last c&a; it serves illumiu&m or for bka&@. In front of the last cube, a stop Sr is mounted. This stop can be moved within a plane perpe&ii to the optical axis. By means of this movabk stop, the size of the stimulus on the retina can be consned and the position may be varkd. In all optical paths, there are neutmi and inte&rence fhters in order to vary the radiation ht respect to intensity and wavekraglh. These laftsrs as well as the knees are not drawn in Fig. 1. On the right side of Fig. 1, the perfusion chamber is schematically shown. Iu it, the isolated retina, receptors downward, is held between two nylon meshes whtk perfused by a suitttbk medium, For details, see Srcxz (l%l, 190, or BAUMANN (1967). P~n~~~ alloy after WAONBR,MACNICXIL and Wow (I%@& were used for extracelhdar record& of *hnptdsedischarges. Their diameter at the tip was I-3 qn. Immediately before the expe&knt, the tip wss pkti&ed; the resistance a@st Ringer solution was thtat kbout O$-141 M Q. TheamlitudGsofUltimpulsn~~wen.asaNlebot~60~Vand140ciV. Waterfrot3s Q&rnc csEdcnf0) were used in the exper&mts. The preparation and the procedure were as in carher experiments ~A~ and Scwaumxa, 1968) and are therefore report& o&y b&t& hem, The retina was isolated from the excised eye and mounted into the perfusion chamber. The electrode was inserted
Properties of the Frog’s Retinal Ganglion Cells
831
from above, the side of the inner limiting membrane. Stimulation with light took place through a quartz window from below, the receptor side. The diphasic nature of the spikes recorded showed that their origin were ganglion cells (GERNANDT,1948; BARLOW,1953a). After having recorded the first impulses, thestop Sr was moved in, and its optical image was placed concentrically around the tip of the electrode. For the first series of experiments, we chose a stop size such that its image on the retina had a dia. of 0.55 mm. According to BARLOW(1953b), the receptive fields in the frog retina are of about 0.5 mm dia. In this series, we employed a permanent background illumination of 2-2 lx or 0.8 lx. The second series of experiments was performed with a stop size which gave a light spot on the retina 0.35 mm dia. In this series, the entire retinae were bleached with monochromatic light before the recordings were taken. Intensity and duration of the bleaching light were such that about 95 per cent of the rhodopsin was bleached. This extent of bleaching was estimated by means of an equation that describes the kinetics of bleaching of rhodopsin in the isolated retina (BAUMANN,1966). The wavelength of the bleaching light was usually 578 nm, though in some cases light at 502 nm was used. Under the conditions observed, it is to be expected that practically no rhodopsin will regenerate after bleaching (BAUMANN,1970). Previous experiments had established (BAUMANNand SCHEIBNER,1968) that the scotopic system remains functionally eliminated if more than 40 per cent rhodopsin is bleached. This behaviour is contrasted to the recovery of the early receptor potential (GOLDSTEIN,1967), which indicates that the cone pigments do regenerate in the isolated retina when separated from its pigment epithelium. Thus, in this second series, we felt sure that only the photopic system was working, when, after a period of about 15 min following the end of bleaching, the impulses were recorded without permanent background illumination. This procedure was more favourable for recording and, moreover, revealed more ganglion cells that could not be silently substituted than the procedure of the first series. The duration of stimulation amounted altogether to 7 sec. Between set 3 and set 4, the first stimulus was substituted by a second one (see Introduction). For those wavelengths for which a silent substitution occurred, the spectral sensitivity function S, (h) was defined:
wherein I, (X,) means the intensity of the reference stimulus constant in wavelength, and Z(X) means the intensity of the stimulus variable inwavelength, both quantities taken at values for which silent substitution occurs. All intensities are expressed as relative numbers of light quanta. If a silent substitution can by no means be brought about, a description by means of a connected sensitivity function is not possible. In general, however, a different reference wavelength can be found that can be silently substituted. Thus, disconnected pieces of sensitivity functions are obtained. After RUSHTON(see, e.g. NAKA and RUSHTON, 1966a, 1966b; or SCHEIBNER and SCHMIDT, 1969), a transducing system that exhibits a (connected) invariant spectral sensitivity function may be called “univariant”. If a transducing system exhibits a disconnected spectral sensitivity function, it is in any case nof univariant. During the course of this study we found ganglion cells which showed what might be called a univariant stimulus-response behaviour over the whole spectrum, as well as other ganglion cells which did not show such behaviour. RESULTS
Figure 2 shows three spectral sensitivity functions which were measured against a permanent background illumination. The reference stimulus was of wavelength 502 nm (indicated by the arrow in Fig. 2). At this wavelength, the sensitivity is normalized to one, i.e. log sensitivity equals zero. All three ganglion cells could be silently substituted over the whole spectrum. Most of the ganglion cells investigated under these conditions showed such behaviour. In a few cases, however, there appeared a disconnected branch of sensitivity function in the blue wavelength region. Owing to the background illumination, these branches could not be determined in detail. A rough estimate of our data indicates that every fifth ganglion cell did not behave univariantly under the conditions of this first series. Figures 3 and 4 show results from retinae where about 95 per cent of rhodopsin had been bleached in advance so that the scotopic system remained functionally eliminated, even without background illumination. Figure 3 presents four ganglion cells that, over the whole spectrum, exhibited a stimulus-response behaviour that may be called univariant. The sensitivity is normalized to one at the wavelength of the reference stimulus, h, = 600 nm (arrow), i.e. log sensitivity equals zero.
832
H.SCHEIBNER
AND
Cn. BAUMANN
0.6
-56
Ro.
Bockground :n 0.8 Ix; o A 0.2 Ix; Stimulus tixa:033mm + Total duration of stimulotion~?
xenon SW
2. Spectral sensitivity functions of three u&&ant ganglion ceils measured under background illumination. 0.6 0.4 0.2
2%
Total
duration
I
??6
Fm. 3. Spectral sensitivity functions of four univariant *giion most of the rbodopsin.
ids measumd after bleaching
Figure 4 presents the results of four gan&ion cells that did not show univariance.With them, blue stimuli c&d not be silently substituted by red stimuli. For three cells, the break in univariance lies near 500 nm, for one cell near 550 nm (curve of It was difikuit to determine exactly the shape of the biue sensitivity branch. Some on c&s did not respond at al1 in a reproducible way to blue stimuli. Therefore, wecanpresent only
833
Properties of the Frog’s Retinal Ganglion Cells
06 i-
x cn” zf -J
t
i-6? 7.4
S,(450) -
T-2-
t
~
X [nm)
r,rboot
0
To
-
Se 76
-
F,4
-
s
o+ a
J,* ( is) I(X).
A,
=
600,450 nm
Bleaching liqht:c 502 nm,oaO578 nm Stzmulus .size:0,35mm Total durotlon of sttmulotion;7sec
FIG. 4. Spectral sensitivity functions of four non-univariant ganglion cells rneasurd after bleaching most of the rhodopsin.
one disconnected sensitivity branch in the blue region. Nevertheless, the break in univariance could be uniquely established. The reference wavelength for the sensitivity in the long wavelength region is 600 nm, for the ~nsit~tivity in the short wavelength region, 450 nm {arrows). The curves are not normalized at these wavelengths so that the absolute scatter of the measured values can be seen. A gross estimate indicates that every second ganglion cell did not behave univariantly under the conditions of this second series. DISCUSSION
Receptors containing one pigment only distinguish themselves by a univariant spectral stimulus-response behaviour. The same should be unlikely in the case of multipolar neurons such as retinal ganglion cells, if they receive signals from different classes of receptors. Nevertheless, many ganglion cells show a behaviour that may be called univariant, as can be seen from Figs. 2 and 3, although in a stricter sense univariance implies that silent substitution has to be held over a wide range of intensity. Since we were mainly interested in cases not exhibiting silent substitution, we did not test this aspect of univariance. A comparison proves that the curves of both Fig. 2 and Fig. 3 are in good agreement with Granit’s photopic dominator (C&WIT, 1942). This dominator peaks at 560 nm and shows a secondary maximum in the blue region (tabulated values are given by REUTER (1969)). Our curves exhibit some scatter in the peak wavelengths, which we do not consider to be significant; besides, a secondary peak in the blue region could not be verified. A similar deviation from the photopic dominator in the blue region is reported by CHAPMAN (1961). He determined the spectral sensitivity by means of thresholds using the latency of responses as a criterion.
834
H. SCHEIBNER ANDCH. BAUMANN
Jnvestigating the early receptor potential (ERP) of the isolated frog retina, GOLDSTEIK (1967) reported a spectral sensitivity function that resembles also the curves of Figs. 2 and 3. Goldstein attributed this ERP sensitivity to the cones. A univariant behaviour of a ganglion cell does not mean that this cell has to be fed from one type of receptors only. This conclusion is arrived at by DONNERand RUMTON (1959a, 1959b), DOUR (1959), and RWSHTON(1959). In other words, univariance is a necessary consequence of a retinal organization that connects a ganglion cell with only one receptor, but the converse is not true, i.e. one cannot infer from an observed univariance that such is the case. DONNEZR and RUSHTON(1959b) estabIished this conclusion experimentally by means of the Stiles-Crawford effect. In order to explain this rest&, RUSHTON(1959) postulated the existence of an “excitation pool” whose site is between the receptors and a ganglion cell. In spite of its univariant character, therefore, it is still pertinent to ask how the photopic dominator is determined from the contributions of the various receptor types. In view of the pigments found (LIEBMANand ENTINE, 1968), this question is still open (see also REUTER, 1969). The logical contraposition of Donner and Rushton’s conclusion tells us that a break in univariance is due either to the cooperation of more than one receptor type or to no cooperation at all. As far as there were reproducible responses in the blue region, the results of Fig. 4 confirm, therefore, the existence of an independent blue mechanism. After DONNER and REUTER(1962), REUTER(19691, and others, this blue mechanism originates in the green rods, the pigment of which exhibits its absorption maximum near 433 nm (DARTNALL,1967). The ganglion cell under consideration then receives information from green rods. As far as there were no unique responses in the blue region, the results of Fig. 4 suggest a retinal organization whereby this ganglion cell does not receive information from the green rods. After MUNTZ (1962), REUT~ (19691, and others, the blue mechanism reacts exclusively with “on” responses. On the other hand, CHAPMAN (1961) reported on findings that indicate pure “off” responses of the blue mechanism. It is difficult for us to decide in favour of one of these two opinions, as the substitution method is not particularly suited for that kind of problem.
REFERENCES B-w, H. B. (1953a).Won potcsntiatpfrum the fmg’s xetina.J. Ply&f. 119,58-f&?. BMLOW,H. B. (1953b). Swnawba and inhibition in the frogs mtina. J. Physcol. 119,69-8&. BALIMANN, C&I. (1966). Dcr EiidluB van Metarbd@n auf die Schpurpurbkichung in der isolierten Netzhaut. Vfston Res. 4,543, BAV~UNN,CH. (1967). ~~~8 und Wbchenfunktion in der isolii Froschnetzhaut, I. Die ~hp~~ I!#i&wrs Arch. ges. Physfaf. 293,444. BAUWNN, CR (197(f). Rmtkx.~ of rbodopsin in the isolated retina of the frog (Rmrrres&&z). yision Res. IO, 627-637. BAUMANN,Cx and ~SCHWW& H. (1968).The dark adaptation of sin@eunits in the isoMcd frog retina followingmal bkacb& of riiod&. Vi&wa &a. 8,1127-1138. Burrow, 0. (1949).Dii EntwWun8 des Ta8cs- und DV im A* dca &asfroschcs. 2. veigl. Physid. 31, 322-347. ml!302 z $. $53). C&.&wry in animafg (Russ.). C.R.Acad. sci. USSR 193,239-Z&!. !kstxow* M. S. fi937). Spe&ai sensitivity cum8 for mcqtton immuxed to single ffbFesbf tile &tic nerve of the frog. iY?iqi%*a5 336-341. BONCMRD, M. M., SWRNOW,M. 5. and l3u~~~~x, L. (1958).The four-dimensionalcolor space of the extra-fovea1 arwt of the hw eye. In NpL-sympaiwn on ViJkbaProbkvt~~of C&w, Tedfimo~~ 1957, Vol. X,327-330, Her k&jm~‘s StUmeq O&e, Lundon. CHAPMAN, R. M. (l%l). Spectral sensitivityof sin& neural units in the bullfrogretina. J. opt. Sot. Am. 51, 11024112.
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of the Frog’s Retinal Ganglion
Cells
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DARTNALL,H. J. A. (1967). The visual pigment of the green rods. Vision Res. 7, l-16. DONM?R, K. 0. (1959). The effect of a coloured adapting field on the spectral sensitivity of frog retinal elements. J. Physiol. 149, 318-326. DONNER, K. 0. and RUSHTON, W. A. H. (1959a). Retinal stimulation by light substitution. J. Physiof. 149,288-302, DONNER,K. 0. and RUSHTON,W. A. H. (1959b). Rod-cone interaction in the frog’s retina analyzed by the Stiles-Crawford effect and by dark adaptation. J. Physiol. 149, 303-317. DONNER, K. 0. and REUTER,T. (1962). The spectral sensitivity and photopigment of the green rods in the frog’s retina. Vision Res. 2, 357-372. GERNANDT,B. (1948). The form variations of the spikes recorded by a micro-electrode applied onto the mammalian retina. Acru physioi. stand. 15, 88-92. GOLDSIXIN,E. B. (1967). Early receptor potential of the isolated frog (Runa pipiens) retina. Vision Res. 7, 837-845. GRANIT, R. (1942). Colour receptors in the frog’s retina. Acfa physiol. stand. 3, 137-151. ISHIHARA,M. (1906). Versuch einer Deutung der phot~lektrischen ~hw~kun~n am Froschauge. Pptigers Arch. ges. Physiol. 114, 569-618. LIEBMAN,P. A. and ENTINE, G. (1968). Visual pigments of frog and tadpole (Runa pipiens). Vision Res. 8, 761-775. MUNTZ, W. R. A. (1962). Microelectrode recordings from the diencephalon of the frog (Rana pipiens), and a blue-sensitive system. J. ~earophysiof. 25,699-7 11. NAKA, K. I. and RUSHTON,W. A. H. (1966a). S-potentials from colour units in the retina of fish (Cyprinidue). J. Physiol. 185, 536-555. NAKA, K. I. and RUSHTON,W. A. H. (1966b). An attempt to analyse colour reception by electrophysiology. J. Physiol. 185, 556-586. REIX~R, T. (1969). Visual pigments and ganglion cell activity in the retinae of tadpoles and adult frogs (&ria temporuria L.). Acta 2001. Fenn. 122, l-64. RUS~~IQN,W. A. H. (1959). Excitation pools in the frog’s retina. J. Physiol. 149, 327-345. SCHEIBNER,H. and SCIIMIDT,B. (1969). Zum Begriff der spektralen visuellen Empfindlichkeit, mit elektroretinographischen Ergebnissen am Hund, V. Gruefis Arch. OphthaL 177,124-135. SCHEIBNER,H. and BALTMANN, Ch. (1969). ~lek~ophysiolo~sche Farbsi~~tersuchungen mittels Reizsubstitution. Ber. Dtsch. Ophthaf. Ges. 69, 124-126. SICKEL, W. (1961). Stoffwechsel und Funktion der isolierten Netzhaut. In Neurophysiologie und Psychophysik des visuellen Systems Symposium Freiburg 1960 (Hrg. R. JUNG und H. KORNHUBER),pp. 80-94. Springer, Berlin. Src~e~, W. (1966). The isolated retina maintained in a circulating medium. Combined optical and electrical inv~ti~tions of metabolic aspects of generation of the el~troretinog~. In CIinicaf E~ecfrorefi~ogruphy (edited by H. M. BUIUANand J. H. JACOBSON)pp. 115-124. Pergamon Press, Oxford. Trro~ns, E. (1956). Untersuchungen iiber den Helligkeits- und Farbensinn der Anuren. Zool. Juhrbuch 66, 129-178. WAGNER,H. G., MACNICHOL, E. F., JR. and WOLBARSH~‘, M. L. (1960). The response properties of single ganglion cells in the goldfish retina. J. gen. Physiof. 43, Supplement, 45-62.
Abstract-By means of the method of stimulus substitution, single ganglion cells of the isolated perfused retina of f&u escafenta were investigated. The scotopic system was eliminated (a) by a permanent background iliumination, (b) by bleaching more than POper cent of rhodopsin. The non-appearance of the impulse response during and after light substitution was taken as a criterion for an invariant spectral stimulus-response behaviour (univariance). Only some of the ganglion cells investigated showed this property. The spectral sensitivity of the other cells could be described only by several disconnected branches of functions. Therefore, it was concluded that there exist at least two photopic (or mesopic) mechanisms in the frog retina.
RiSsum&-Par la methode de substitution de stimulus, on Btudie des cellules gangliormaires tmiques de la retine iso& et perfused de Runa escuienta. On elimine le systeme scotopique (a) par un &z&rage permanent du fond et (b) en dt+colorant plus de POpour cent de la rhodopsine, La non-apparition de la reponse en impulsions pendant et apres la substitution de lumiere est prise comme criterium d’un comportement invariant de la response vis-a-vis du stimulus spectral (univariance). 11n’y a que certaines des cellules ganglionnaires Btudicies qui presentent cette proprittt5. Onhapeut d&ire la sensibilite spectrale des autres cellules qu’avec plusieurs branches de fonctions sans cormexions. On en deduit qu’il existe au moins deux mecanismes photopiques (ou mesopiques) dans la r&tine de grenouille.
836
H. SCHEIBNERAND
01. BAUMANN
B-Nach der Methode der Reizsubstitution wurden an der isolierten perfundierten Netzhaut von Rana escuhra einzelne Ganghenmll~ untersucht. Das skotopische System wurde ausgeschahet (a) durch eine permanente ~rnt~~d~~~ht~g, (b) durch Bleichung van mehr aIs 90 Prozent des Rhodopsins. Das Au&l&en der ~p~two~ bei der Substitution van Farbre&n wurde ah Kriterium fiir ein einheithches spektrafes ReizAntwort-Verhalten (Univarianz) gewertet. Nur em Teil der untersuchten ~Ganghenzehen zeigte diese Eigenschaft. Die spektrale Empfmdlichkeit der anderen Zellen Iieg sich nur durch mehrere nichsgende Teilfunktionen beschmiben. Hieraus wurde auf die Existenz van mindestens zwei photopischen (oder mesopischen) Mechanismen geschlossen.