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Chromatic subclasses of frog retinal ganglion cells: Studies using black stimuli moving on a monochromatic background
034~.WXY x1 0404hY-IOSO? 00 0 Pergamon Press Ltd
Vi\wn Rc.wor< h Vol. ?I. pp. 4hY to 47X Printed m Great Bntaln
CHROMATIC SUBCLASSES OF FROG RETINAL GANGLION CELLS: STUDIES USING BLACK STIMULI MOVING ON A MONOCHROMATIC BACKGROUND URSULA GR~~SSER-CORNEHLSand RAMSEY McD. SAUNDERS*
Physiological Institute, Free University of Berlin, Arnimallee 22. 1000 Berlin 33, Federal Republic of Germany
(Received
5 December
1979)
Abstract-The
suprathreshold spectral responses of classes 1.2, 3 and 4 neurons of the frog’s retina to a black spot moving across monochromatic background fields of different wavelengths were determined under mesopic conditions by microelectrode recordings in the superficial layers of the optic tectum. This method revealed the existence of two subclasses of class I neurons, an a-type with a broad spectral response curve and a b-type with two maxima at 466 and 580 nm. Class 2 neurons also exhibited U- and b-types. In addition to the a- and b-subclasses, class 3 neurons exhibited a subclass with a narrow spectral response curve with a maximum at 5OOnm. The exponent of the velocity functions of the neurons was found to be greater when the response originated from cones than when it originated from rods. After neuronal adaptation, the green rods and the neuronal elements connected to them recovered much faster than the red rods and cones.
INTRODUCTION The retina of the frog
(Rum escdenta) contains two of rods and two types of cones. The two types of rods are known as “green rods” and “red rods” and they absorb light maximally at 433 and 502nm respectively. The two types of cones are known as “single cones” and “double cones”. The single cones, as the name indicates, consist of single elements. They absorb maximally at 578 nm. Each double cone has two elements, the principal element which has an absorption maximum of 578nm and the accessory element which has its absorption maximum at 502 nm (Dartnall, 1953, 1954; Liebman and Entine, 1968; Goldstein and Wolf, 1973; Knowles and Dartnall, 1977). The retinal afferents of the frog terminate in four separate layers in the superficial neuropil of the optic tectum. The neurons with axon endings in the different superficial layers of the optic tectum have different functional properties and are known according to the layer as class 1, 2, 3 and 4 neurons, class 1 being uppermost (Maturana er al, 1960; Griisser-Cornehls et al., 1964; Keating and Gaze, 1970). When a black spot moving across the excitatory receptive field (ERF) of a class I neuron (2-3” dia) is suddenly stopped within the ERF, the neuron discharges impulses continuously. The firing ceases when the light is turned off, but resumes when the light is turned on again. The class 2 neurons, which have an ERF size of 2.5-4” dia, behave like class 1 neurons; however, their response declines after a contrast stimulus is stopped within the ERF. The neuronal activation ceases when types
* Present address: Physics Department, University of the West Indies, St. Augustine, Trinidad. 469
the light is turned off and does not reappear when the light is turned on again. Class 3 neurons have a larger ERF (54”) and, unlike class 1 and 2 neurons, are activated by the onset and offset of a stationary large field light stimulus (on- off-neurons of Hartline, 1938). Finally, class 4 neurons have the largest receptive fields (8-10” dia). These are the off-neurons of Hartline exhibiting a rhythmic and sustained off response when a stationary light stimulus covering the ERF is turned off (for details see Griisser and Griisser-Cornehls, 1976). Griisser-Cornehls (1974, unpublished) observed that while some class 3 neurons displayed either a sustained “on” or sustained “off” response of 1-3 set duration under scotopic conditions, others displayed a short “on” and a sustained “off” response under the same conditions. Both neuronal classes, however, responded under photopic adaptation only with a short “on” and “off” burst. These observations suggested the existence of subclasses dependent on different receptor inputs. The existence of more than 4 classes of retinal ganglion cells was suggested by Cajal (1894) who could identify about I I different types of frog retinal ganglion cells from morphological criteria (branching of dendrites, cell size etc.). The work of BIckstram and Reuter (1975) and of Scheibner and Baumann (1970) and Scheibner et al. (1975) also indicated that the chromatic response properties, dependent on differing receptor input. might not be equal within the ganglion cells classified with black/white stimuli as class I, 2, 3 and 4 respectively. The purpose of the experiments described in the present report was therefore to prove the existence of subclasses of the 4 types of retinal neurons mentioned above. Moving stimuli rather than stationary stimuli were used. This method of stimulation compares
CIRsuA GROSSER-CORNEHLS and RAMSEY MrD. SALWXXS
470
closely with the type of stimulation the animal experiences in its environment. Class I and 2 neurons. moreover, do not normally respond to the onset and offset of diffuse stationary light stimuli (Maturana et al., 1960). Therefore, the application of black stimuli moving on a chromatic ba~k~ound or moving chromatic contrast stimuli was indicated. METHODS
Preparafion
Experiments were ~rform~d on 70 medium-sized adult frogs (Rana esc~~e~~~)~After anaesthesia with ethyl-m-aminobenzoate (MS 122 Sandoz), the skull above the optic tectum was removed, the dura opened and the brain covered with mineral oil. Each animal was immobilized by a lymphatic sac injection of OS-O.8 mg ~-tub~rarine chloride, covered with moist gauze and placed on a moist sponge with its head fixed in the center of a perimeter (Gr%sser and Dannen~rg” 1965). Stimulation The receptive fields of the retinal afferents of the optic tectum were stimulated by a physi~l~y btack spot 2.8” dia moving at constant velocities across chromatic background fields of 20” dia. The optical stimulator consisted of a tungsten lamp projection system (Sonnies, Freiburg i.Br., 1963) which was used with a filter wheel containing 16 mon~hr~mati~ filters (Balzers type} with peak transmissions in the range between 400 and 66Onm and a half b~d~dth of about 1Onm. Monochromatic light from the system was projected onto a di~u~ly reflecting white card situated in the stimulus window of a special purpose perimeter (Griisser and Dannenberg, 1965). The white card 28.5 cm from the frog’s eye contained a hole of 2.8” dia behind which a camera shutter system with matt black paper behind its aperture was situated. With this arr~gement (light trap), none or very tittle of the light projected onto the white card
in the region of the hole was reflected. Thus, OX had an absolute black or ‘physically black” surface. The energy reaching the cornea of the frog’s eye from each monochromatic titter was measured h? mounting a photomultipher (type RCA 4832) at eye position in the perimeter and measuring the photocurrents at the different wavelengths using a digital multimeter. To determine the energy incident on the retina, corrections were made for the spectral transmission of the ocular media. This was done using the method of Saunders (1977). Using a photomul~plier and digital multimeter, the photocurrents were measured at different wavelengths at the exit pupil of an integrating sphere, with and without the eye of the frog in the entrance pupil of the sphere. The percentage transmission (1”)at each wavelength was obtained from the relationship: T-
l
I
300
400
x 100
PI and P2 being the phot~urrents with and without the eye in the entrance pupil of the sphere. The results in Fig. 1 demonstrate that in freshly excised eyes the spectral transmission in the region between 400 and 700 nm is about 807; and is almost independent of the wavelength and therefore, in our experiments, no corrections were made for the spectral transmission of the ocular media. After making corrections for transmission losses, the retinal illumination was adjusted to 1.4 x 10’ photons mm-’ set- 1 at 551 nm (pupil dia 3.4 mm) by use of neutral density fibers. This is in the mesopic range for the frog (Blckstriim and Reuter, 1975). The response to constant quantum energy at different wavelengths rather than the threshold energy for stimulation was employed because of the neuronal adaptation to the moving stimulus (Griisser and Griisser-Comehls, 1973). This aspect will be discussed in more detail later. Considering the number of trials that must be made to achieve the threshold and the recovery time required between trials, the neuron
-f__-_.&
0
P,/P,
500
. --+___
-&---+
I
600
1 700
Wavelength Cnm) Fig. 1. The spectral transmission of the frog’s ocular media, T~~~~ and squares indicate the values from two different eyes. Curve drawn by hand.
Chromatic subclasses of frog retinal ganglion cells must be held for at least 3 hr to obtain a complete action spectrum using the latter method. Although occasionally a neuron can be held for this period or even longer, this is not generally the case. With the constant quantum energy method, a “spectral response curve” can be obtained in about 45 min allowing a recovery time of about 90 set between successive traverses of the ERF by the moving black spot. Three types of experiments were performed. In the first type the spot was moved at a constant velocity of 9.2” set-’ across various monochromatic background fields. The velocity was chosen because it lies in a velocity range in which strong responses are obtained from the retinal neurons, the responses at low and high velocities being very weak (Griisser et al., 1968). In the second type of experiment, velocities in the range of 0.46-46” see-’ were used, thereby enabling the response variations with velocity to be determined. In addition to the monochromatic backgrounds, chromatic backgrounds from broadband colour filters were used. These enabled measurements to be made under photopic as well as scotopic conditions. Finally, in the third type of experiment, the neuronal adaptation was investigated by moving the black spot to and fro at a velocity of 9.2” see-’ across the monochromatic background fields. When a stimulus is continuously moved to and fro across the receptive field, along the same path, the response to the first traverse is stronger than that to the second, which is stronger than the third etc. (Grusser and Griisser-Cornehls, 1973). The ratio of the second to the first traverse (i.e. backward movement to forward) 400
471
was determined. The neurons investigated were not direction sensitive. The spot was also moved to and fro until the neurons were completely adapted and the response magnitude after various recovery times was measured. Recording and data processing
Tungsten microelectrodes with electrolytically platinized tips of 1 to 3 pm dia were used to record the impulses of the afferent optic nerve fibers of the superficial tectal layers. The action potentials of single neurons were amplified by a Tectronix 122 amplifier and a 2A61 plug-in-unit of a Tektronix 565 oscilloscope. The movement of the black spot was recorded by potentiometers connected to the moving stage of the perimeter apparatus. The signal was fed into a 2A2 plug-in-unit of the oscilloscope. All signals were stored on a 7-channel-analog magnetic tape (Bell and Howell, VR 3200). Photographic recordings were made with a Tonnies Recordine camera (Tonnies K, 565). The data were later counted by hand and the average impulse rate calculated for the different wavelengths and conditions. For the statistical processing of the data, a Lint-8 digital computer was used, applying conventional statistical programs (mean, standard deviation, significance and regression line). RESULTS Responses to contrast stimuli moving on monochromatic background
The responses of 48 neurons recorded from the superficial layers of the optic tectum to a 2.8” black
500
589
526
601
551
578
Class
2
Neuron
Fig. 2. Original recording of the responses of a class 2 neuron to a black spot (2.8” dia) moving at a constant velocity of 9.2” set- ’ across various monochromatic background fields (20” dia). At each wavelength the stimulus movement is displayed above the action potentials. Note the dependence of the impulse pattern on the wavelength.
411
URSLILA GRUSSFK-CORNEHLS and RAMSEY M(,D. SAIINIWKS
spot moving at 9.2’ set _ ’ across the monochromatic background fields were quantitatively analysed. The units were first classified (class 1, 2, 3, 4) according to the procedures applied in earlier studies (Maturana t’r al.. 1960; Griisser and Griisser-Cornehls. 1976). Thereafter, the 2.8 black spot was moved every 90 set across a different monochromatic background field along the same path through the center of the respective ERF. Figure 2 shows responses recorded in such an experiment from a class 2 neuron. From data of this type the average impulse rates R were determined. By plotting R as a function of wavelength. we obtained “spectral response curves” for the different classes of retinal ganglion cells. These response curves were normalized (maximal response given a value of 100) for convenience. On the basis of their spectral response curves, several subclasses of the 4 main types of retinal ganglion cells were found. Class I neurons displayed two subclasses, one having a broad spectral response curve with three small maxima (Fig. 3a). the other
having two maxima, a dominant one at 466 nm and ;t weaker one at 580nm (Fig. 3b). In class 2 neurons as well, two subclasses were found. One type exhibited a broad spectra) response curve with three maxima similar to those found in part of class 1 neurons (Fig 3~). the other had two maxima at 466 and 580nm (Fig. 3d). Four subclasses of class 3 neurons were found. One type again had a broad spectral response curve with three maxima (Fig. 4a). Another group exhibited two maxima located approximately at 450 and 580nm respectively, one of which was dominant (Fig. 4b, c). Finally, a neuron type with a narrow spectral response curve was found having a single maximum at 500nm (Fig. 4d). To date we have only recorded from one class 4 neuron. This neuron had a double peak spectral response curve with maxima at 450 and 580 nm. To facilitate the differentiation of the subclasses on the basis of their spectral response curve. the following nomenclature is suggested: a-type for those neurons with a broad spectral response curve h-type
sbo
--T----i
600
700
Fig. 3. The spectral response curves of the two types of class I neurons (Figs 3a, b) and the two types of class 2 neurons (Figs 3c.d). The ordinate in each case represents the normalized average impulse frequency (imp’sec-‘) expressed as a percentage of the maximal response and the abscissa the wavelength (nm). The broken curves are the Dartnall nomogram curves for P 433 and P 578. The solid curves are drawn by hand. Vertical bars = SD
473
Chromatic subclasses of frog retinal ganglion cells for those with two maxima and c-type for those with a single maximum. For class 3 neurons, the b-type with a dominant maximum at 450nm will be referred to as 3bl and those b-type neurons with a dominant maximum at 580nm as class 3b2. The broken curves for the b- and c-types in Figs 3 and 4 are Dartnall nomogram curves for P,,,, PSo,, and P580r depending on the spectral region. We also determined the receptive field sizes of the a- and b-type neurons using monochromatic backgrounds and a 2.8 degree black moving contrast stimulus. Under these conditions the ERF sizes were found to be in the same range as described earlier for contrast stimuli moving on a black or white background (Maturana rt al., 1960; Griisser and GriisserCornehls, 1976). Dependence of the chromatic responses on the angular velocity of the moving spot
In the experiments described so far, the moving black spot had a velocity of 9.2” set-’ which is in the
400
500 Wavelength
so0 (“ml
midregion of the velocity response range of the neurons (Griisser et al., 1968). As the velocity of the stimulating spot was increased or decreased, the general shape of the chromatic response curve remained unchanged. The “velocity function”, however, changed quantitatively depending on background wavelength. This is seen best when the data are plotted in a double logarithmic coordinate system. The exponent c of the power function describing the relationship between stimulus angular velocity v and average impulse rate R during the traverse of the ERF, !? = k.vC impulses.sec-’
(I)
depended on the spectral nature of the background. For the neuron in Fig. 5 the exponent c was 0.97 for a 589nm background, 0.71 for a 492nm background and 0.65 for a 400nm background. To study the response at scotopic as well as photopic adaptation levels, we used 4 broad band chromatic filters and neutral density filters to shift the adaptation level
700 Wavelength
wlv
Fig. 4. The spectral response curves of four types of class 3 neurons. The ordinate in each case represents the normalized average impulse frequency (imp.sec-‘) expressed as a percentage of the maximal response and the abscissa the wavelength in nm. These neurons are classified as types 3a, 3b,, 36, and 3c. The broken curves are the Dartnall nomogram curves for P 433 and P 578. The solid curves are drawn by hand. Vertical bars = SD
473
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RAMSEY
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obtained in a class 3a neuron. but stmilar changes tn the exponent c were also found for the velocity !unction of class 2 neurons.
. 492nm 400nm 0,l J
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0,46 Velocity
,
I
45
46
(deg.
s-l)
Fig. 5. Velocity response functions of a class 30 neuron at wavelengths of 400. 492 and 589 nm. Impulse rates (imp’sec-‘) are displayed along the ordinate and the stimulus velocities (deg.sec -‘) along the abscissa. Mesopic conditions.
from the photopic into the scotopic range (Fig. 6). Under photopic illumination. the exponent c of equation (1) was always found to be larger than under scotopic conditions. The data shown in Fig. 6 were
r
Red
/ *- - /
/.
As described in earlier reports (Griisser and Griisser-Cornehls, 1973, 1976), a fast back and forth movement across the ERF leads in class 2 and class 3 neurons to a pronounced adaptation which is only in part of receptor origin and which is not caused by adaptation of the ganglion cell impulse generator (Griisser and Griisser-Cornehls, 1969). Neuronal activation can be measured quantitatively if a contrast stimulus is moved to and fro through the ERF and the ratio of the impulse rate of the second to the first traverse is measured. In the experiments performed. the time lag between the forward and backward motions i.e. between the exit of the stimulus from the ERF and its re-entry into the ERF. varied between 500 and 2OOOmsec. In Fig. X the ratio mentioned 13 plotted as a function of wavelength. This method reveals that a-type as well as b-type neurons change their spectral response characteristics in the course of neuronal adaptation. The responses to a contrast stimulus moving across backgrounds of 500 nm wavelength and longer were more sensitive to neuronal adaptation than the responses obtained when a blue background illumination was applied (Fig. 7). This difference was more pronounced in the cl-type than III the b-type neurons. When complete neuronal adaptation was achieved by continuous to and fro movement across the ERF. a similar difference in the recovery time of the rcsponses to contrast stimuli moving across the hluc and the red background was obtained. The recovery time for the short wavelengths was about 3Osec and for the longer wavelengths about 60 set for both dand h-type neurons. Figure 7 exhibits examples ol such recovery functions in class 3a neurons. These measurements of neuronal adaptation and recover! functions indicated in addition that the 90 set intervals between successive traverses of the ERF m measurement of spectral response curves fulfilled the condition that the receptive unit tested was ilt the same level of excitation before each measurement.
DISCUSSION 0 46 Velocity
(deg.
4.6
46
s-‘)
Fig. 6. Velocity response functions of a class 30 neuron under photopic (broken lines) and scotopic (solid lines) conditions. Impulse rates (imp.sec-‘) are shown along the ordinate and the stimulus velocity (deg.sec-‘) along the abscissa of each diagram. Blue (Corning 2-62) broad band filters were used. The photopic energy was in each case 1.7 x 10s photons nrnm2 set-’ and the scotopic energy 3 log units lower. Note the steeper increase in the velocity functions under photopic conditions.
The measurement of the “spectral response curves” of the classical neuron types in the frog’s retina with black spots moving on a monochromatic background revealed the existence of distinct subclasses of the retinal neuron types. In fact, the class 3 neurons which Grtisser-Cornehls observed to have a short “on” and sustained “off” response to white light stimuli under scotopic and mesopic conditions were found to be the class 3c neurons of our present study. The class 3c
475
Chromatic subclasses of frog retinal ganglion cells
I
Rana
esc.ulenla
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ib 3Class
9 Class
3b
2h and
neuram
500
600
/ 500
600
Wavelength
cnm)
Wavelength
cnm>
1
/
1
Fig, 7. (a) Neuronai adaptation of a-type neurons at various wavelengths. Along the ordinate, the neuronal response to the stimulus moving backwards through the receptive field is expressed as a percentage of the forward response, the pathway being identical. The wavelength in nm is indicated along the abscissa. These neurons were not directionally sensitive. Note the faster recovery (greater percentage neuronal response) in the blue spectra1 region. (b) Neuronal adaptation of b-type neurons at different wavelengths. Note again the faster recovery in the blue spectra1 region. (c) Recovery function after complete neuronal adaptation of class 3b neurons using 350 nm (so&d curves) and 578 nm (broken curves) as background fields. Identical symbols indicate the same neuron. The black spot was moved to and fro through the receptive field along the same path until the neuronal activation ceased (complete neuronal adaptation).
neurons exhibited the typical short “on” and “of?” bursts to the onset and offset of diffuse white stimuli under phatopic conditions. However, with our diffuse monochromatic stimuli (mesopic), a short “on” and a
sustained ‘WY’ response was obtained. At 500 nm the responses were longest (0.25 see for the “on” and 4 set for the “off”). fn addition, the different subclasses appear ta be closely correlated with the different his\.R ?I 4 b
tologicaal types described by Cajai (1894). This aspect. however, wili be dealt with in a subsequent publication (Griisser-Cornehls and Saunders, 1979 in prep aratian). The a-, b- and c-type response curves are not true action spectra, since they represent the neuronal response to constant quantum energy at different wavelengths and not the variation of sensitivity with wave-
476
IIRSI~LA
GKliSSER-C‘ORNtHLS
and
length. Generally the shape of a spectral response curve differs from that of the absorbance spectrum of a visual pigment. The spectral position of the respettive maxima. however. can be very close together if the response is dominated by that pigment. For example with h- and c-types. reasonable agreement with the appropriate receptor pigment was obtained, PdX3 and P,sc, for the h-type and P,,, for the c-type. Since the m~lximum in the blue region of the spectrum occurs at 450 or 466 nm. the nomogram curve for Pha3 was shifted to the right. The finding that the maximum in the blue region of the spectrum was shifted to the right is, however. in agreement with those of other authors who used diRerent experimental methods to ours (Gratiit. 1942: Donner and Rushton. 1959; Chapman, 1961: Muntz, 1962: Reuter. 1969). This shift is probably due to the influence of one or more other receptor types on the green rods. The data therefore suggest that neurons with an u-type response receive inputs from all receptor types of the frog’s retina. those with b-type mainly from the green rods and the single cones and those with c-type response mainly from the red rods. The different spectral response types probably play an important role in brightness and coiour detection in the frog. The broad spectral response curves of the cl-type units suggest that they are luminosity units (black--white detectors of DeVaiois. 1960). These units are probably important for brightness and contrast detection under mesopic and photopic conditions. One possible function of the accessory element of the double cone with its 500 nm pigment is to “fill in” the spectrum for brightness detection at photopic levels. In the absence of this unit. the main fun~tionai units under photopic conditions would be the green rods (433 nm) vvhich function at high intensity levels (Backstrom and Reuter, 1975) and the single cones (580 nm). The presence of an element with an absorption maximum in the midregion of the spectrum is desirable for iuminan~~ detection under these conditions. Units with b-type responses are probably the chromatic or colour detection units. However. colour detectors are generally expected to display a coiour opponency, the differential responses of neurons to the onset and offset of monochromati& stimuli in different spectra) regions (DeValois. 1965). According to Maturana et trl. (1960). class 1 and 2 neurons of Rona pipiers do not respond to the onset or offset of large field white light stimulation. The same is also true for R~JNJ ~~cuirrrro (Griisser et a/.. 1967). We have confirmed this finding for monochromatic stimuli with Ri.i~ esculemt. Thus colour opponency was not detected for class 1 or 2 neurons. Using smai) light spots of high intensity, Backstrom and Reuter (1975) have found, however. that some type of opponent activity in class 1 and 2 neurons in the isolated eyes of the frog occurs. They found a pure “on” response for the green rod input, but an “on” and “off” response for the cone input. This situation changes consider-
KAMSt’I
McD.
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lumRS
ably with moving stimuli. With black targets sm,~llct than 4” dia moving on a chromatic backgroutlL ;t real colour opponency is possible in class 1 mi ? neurons. since only the “off” response of the ~:onc input occurs which IS evoked by the leading cdgc of the target. The “on” response of the cone mechantsm. which would be elicited by the withdrawing edge: ot the target. falls well into the total neuronal adaptLitton time of the bipolar cells (Grusser and Grusser-!‘onehis. 1969). Consequently an “on” response ir ~)nl! elicited by the response of the green rods Although class 3 neurons respond to both the onstlt and cessation of diffuse white light as well as monochromatic light stimulation. only one class 3 neuron with very clear opponent properties was found m cxperiments in which the response of class 3 neuron\; to the onset and offset of monochromatic stimulation was tested. This neuron had inputs from green rods and single cones (b-type). The infrequent occurrence of class 3 opponent coiour neurons is in agreement with the findings of Backstrom and Reuter 0975). In their sample of neurons, most class 3 neurons showed an “on‘* and an “off” response for the input of hnrh receptor types (green rods and cones). Subsequently. in these neurons, even with moving targets, no real coiour opponency occurs. because only “off” responses are elicited by both receptor mechanisms with the leading edge of the moving dark spot. We suggest therefore that some kind of non-opponent coiour mechanism exists in the frog’s visual system. This, will be discussed in more detail in a following publication (Griisser-Cornehls and Saunders. 1979 in preparation). The c-type units are probably the scotopit luminosity units. These neurons may. in addition. play a role in colour vision at mesopic levels. A clear separation of class I -4 retinal ganglion cells into subclasses on the basis of their velocity function has not been possible so far. With monochromatic backgrounds the slope of the velocity function at 58Onm was always found to be steeper than that at 450 or 500 nm. The steeper slope of the velocity function at 580nm is attributed to the cones, the Hatter curves obtained at 450 and 5OOnm to the rods. At 5OOnm, however. there may be some input from the accessory element of the double cones. The broad band filters (red. green, yellow and blue) which permitted study under photopic as well as scotopic conditions yielded a similar result. In Fig. 7 the sharp increase in the impulse rate with velocity under photopic conditions is attributed to the cones, the flatter curves obtained under scotopic conditions are attributed to the rods (see also Grtisser-Cornehis and Wolynski. 19731. Thus. although both rods and cones detect movement. the cones are better able to differentiate velocities. The r~uuronal adaptation obtained by moving a spot to and fro through the receptive field along the same pathway is probably due to adaptive proLesses in several systems, the retinal receptors and the neuronal network connected with the ganglion cell (Grtisser
477
Chromatic subclasses of frog retinal ganglion cells
and Griisser-Cornehls, 1973). The shift in the spectral responsiveness of the a- and b-type neurons (Figs 8a, b) suggests that the green rods and the neuronal elements connected to them recover much faster than the red rods and the cones. This was confirmed by the experiment in which the neuronal response was determined at various periods after complete neuronal adaptation. The small difference in the response ratio of the a- and b-types in the blue region of the spectrum (6.5% relative to 50%) suggests that some separation into subclasses may be possible by this method. However, the determination of the spectral response curves is a more satisfactory method. The results with chromatic adaptation indicate the probable existence of receptor-specific bipolar cells, since neuronal adaptation does not take place at the receptor or ganglion cell level (Griisser-Cornehls et al., 1964). This opinion is supported by the fact that there exist two modes of signal transfer in the bipolar cells of the frog (Murakami and Shigematsu, 1970), one by electronic conduction and one by initiation and propagation of action potentials. Initiation and propagation of action potentials might occur in the green-rod bipolar cells.
CONCLUSION Thus with the use of moving colour contrasts, it is possible to isolate subclasses of the classical neuron types of the frog. Of the three types of experiments performed, the determination of the spectral response curves of the neurons was the most useful. This method revealed the existence of two subclasses of class 1 neurons, an a-type with a broad spectral response curve with three maxima and a b-type with two maxima at 4.50 and 580 nm. Class 2 neurons also exhibited a- and b-types. In addition to the a- and b-subclasses, class 3 neurons exhibited a subclass with a narrow spectral response curve with a maximum at 500 nm.
Acknowledgements-The investigations were sponsored by a grant of the “Deutsche Forschungsgemeinschaft” (Gr. 276/7.8, 12. 13). We wish to thank Professor Ch. Baumann and Professor O.-J. Griisser for their valuable comments on the manuscript. Miss H. Wolynski for her careful technical assistance and Mrs J. Dames for her help with the English translation. Mrs J. Vierkant-Glathe wrote the computer programs.
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Goldstein E. B. and Wolf B. M. (1973) Regeneration of the green rod pigment in the isolated frog retina. Vision Res. 13, 527-534.
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Granit R. and Wrede C. M. (1937) The electrical responses of light-adapted frog’s eyes to monochromatic stimuli. J. Physiol., Land. 89, 239-256.
Griisser O.-J. and Dannenberg H. (1965) Eine PerimeterApparatur zur Reizung mit bewegten visuellen Mustern. Pfliia. Arch. aes. Phvsiol. 285. 373-378.
G&&r, O.-J. and G&ser-Cornehls. U. (1969). Neurophysiologie des Bewegungssehens. Bewegungsempfindliche und richtungsempfindliche Neurone im visuellen System. Erg&. Physiol. 61, 178-265. Griisser O.-J. and Griisser-Cornehls U. (1973) Neuronal mechanisms of visual movement perception and some psychophysical and behavioural correlations. In Handbook of SensoryPhvsiology (Edited by Autrum H. rt al. VII, 333429. Springer, Berlin. Griisser O.-J. and Griisser-Cornehls U. (1976) Phvsioloev , of the anuran visual system. In Neurobiology qf the Frog, (Edited by Llin& R. and Precht W.). Springer, Berlin. Grtisser-Cornehls U. and Wolynski H. (1973) The dependence of the response of movement sensitive neurons of the anuran retina on the level of light-dark adaptation. Pjliigers. Arch. ges. Physiol. 343, Suppl., R90. Griisser O.-J., Finkelstein D. and Griisser-Cornehls U. (1968) The effect of stimulus velocity on the response of movement sensitive neurons of the frog’s retina. Pfliigers _s
Arch. ges. Physiol. 300. 49-66.
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Griisser-Cornehls U.. Griisser O.-J. and Bullock Th. H. (1964). Functional’ organisation of receptive fields of movement detecting neurons in the frog’s retina. Pfliigers. Arch. ges. Physiol. 279, 88-93.
Griisser O.-J., Grtisser-Cornehls U., Finkelstein D., Henn V., Patatschnik M. and Butenandt E. (1967) A quantitative analysis of movement detecting neurons in the frog’s retina. Pjiigers. Arch. ges. Physiol. 293, 10&106. Hartline H. K. (1938) The response of single nerve fibers of the vertebrate eye to illumination of the retina. Am. J. Physiol. 121, 400415.
Keating M. J. and Gaze R. M. (1970) Observations on the “surround” properties of the receptive fields of frog retinal ganglion cells. Q. JI. exp/ Physiol. 55, 129-142. Knowles A. and Dartnall H. J. A. (1977) The photobiology of vision. In The Eye. (Edited by Daveson H.) Vol. 2. Academic Press, New York. Liebman P. A. and Entine G. (1968) Visual pigments of frog and tadpole (Rana pipiens). Vision Res. 8, 761-775. Maturana H. R., Lettvin J. Y., McCulloch W. S. and Pitts W. H. (1960) Anatomy and physiology of vision in the frog. J. gen. Physiol. 43. 129-I 75. Muntz W. R. A. (1962) Microelectrode recordings from the diencephalon of the frog (Rana pipiens) and a blue-sensit-
ive system. J. Neuroph&iol. 25,’ 899-7 11. Murakami M. S. and Shieematsu Y. (1970) Oualitv of conduction mechanism in- bipolar ceils oi th‘e frdg retina. Vision Res. IO. I-IO. Reuter T. (1969) Visual pigments and ganglion cell activity in the retinae of tadpoles and adult frog. (Rana temporaria 1.) Acfa zoo/ Few. 122, l-64.
Saunders R. McD. (1977t The spectral responsiveness and the temporal frequency response (TFR) of cat optic tract and lateral geniculate neurons: Sinusoidal stimulation studies. I'ivim Rrc. 17. 285 ~191. Scheibner H. and Baumann Ch. (1970) Properties of the
frog’s retinal ganglion cells as revealed hq ahstitut!i)n I>I chromatic stimuli. Visio,l Res. IO. X?9~ X36. Scheibnrr i-1.. Hunold W. and Beraut M. (19751 ( *.)loui discrimination functions of the frog optic rectum ~KCJR:J C\CJJ/lWlcJ~ b’iS;iOJJ Rc\. 1s. 1 175 1 1 x0.
Vi\wn Rc.wor< h Vol. ?I. pp. 4hY to 47X Printed m Great Bntaln
CHROMATIC SUBCLASSES OF FROG RETINAL GANGLION CELLS: STUDIES USING BLACK STIMULI MOVING ON A MONOCHROMATIC BACKGROUND URSULA GR~~SSER-CORNEHLSand RAMSEY McD. SAUNDERS*
Physiological Institute, Free University of Berlin, Arnimallee 22. 1000 Berlin 33, Federal Republic of Germany
(Received
5 December
1979)
Abstract-The
suprathreshold spectral responses of classes 1.2, 3 and 4 neurons of the frog’s retina to a black spot moving across monochromatic background fields of different wavelengths were determined under mesopic conditions by microelectrode recordings in the superficial layers of the optic tectum. This method revealed the existence of two subclasses of class I neurons, an a-type with a broad spectral response curve and a b-type with two maxima at 466 and 580 nm. Class 2 neurons also exhibited U- and b-types. In addition to the a- and b-subclasses, class 3 neurons exhibited a subclass with a narrow spectral response curve with a maximum at 5OOnm. The exponent of the velocity functions of the neurons was found to be greater when the response originated from cones than when it originated from rods. After neuronal adaptation, the green rods and the neuronal elements connected to them recovered much faster than the red rods and cones.
INTRODUCTION The retina of the frog
(Rum escdenta) contains two of rods and two types of cones. The two types of rods are known as “green rods” and “red rods” and they absorb light maximally at 433 and 502nm respectively. The two types of cones are known as “single cones” and “double cones”. The single cones, as the name indicates, consist of single elements. They absorb maximally at 578 nm. Each double cone has two elements, the principal element which has an absorption maximum of 578nm and the accessory element which has its absorption maximum at 502 nm (Dartnall, 1953, 1954; Liebman and Entine, 1968; Goldstein and Wolf, 1973; Knowles and Dartnall, 1977). The retinal afferents of the frog terminate in four separate layers in the superficial neuropil of the optic tectum. The neurons with axon endings in the different superficial layers of the optic tectum have different functional properties and are known according to the layer as class 1, 2, 3 and 4 neurons, class 1 being uppermost (Maturana er al, 1960; Griisser-Cornehls et al., 1964; Keating and Gaze, 1970). When a black spot moving across the excitatory receptive field (ERF) of a class I neuron (2-3” dia) is suddenly stopped within the ERF, the neuron discharges impulses continuously. The firing ceases when the light is turned off, but resumes when the light is turned on again. The class 2 neurons, which have an ERF size of 2.5-4” dia, behave like class 1 neurons; however, their response declines after a contrast stimulus is stopped within the ERF. The neuronal activation ceases when types
* Present address: Physics Department, University of the West Indies, St. Augustine, Trinidad. 469
the light is turned off and does not reappear when the light is turned on again. Class 3 neurons have a larger ERF (54”) and, unlike class 1 and 2 neurons, are activated by the onset and offset of a stationary large field light stimulus (on- off-neurons of Hartline, 1938). Finally, class 4 neurons have the largest receptive fields (8-10” dia). These are the off-neurons of Hartline exhibiting a rhythmic and sustained off response when a stationary light stimulus covering the ERF is turned off (for details see Griisser and Griisser-Cornehls, 1976). Griisser-Cornehls (1974, unpublished) observed that while some class 3 neurons displayed either a sustained “on” or sustained “off” response of 1-3 set duration under scotopic conditions, others displayed a short “on” and a sustained “off” response under the same conditions. Both neuronal classes, however, responded under photopic adaptation only with a short “on” and “off” burst. These observations suggested the existence of subclasses dependent on different receptor inputs. The existence of more than 4 classes of retinal ganglion cells was suggested by Cajal (1894) who could identify about I I different types of frog retinal ganglion cells from morphological criteria (branching of dendrites, cell size etc.). The work of BIckstram and Reuter (1975) and of Scheibner and Baumann (1970) and Scheibner et al. (1975) also indicated that the chromatic response properties, dependent on differing receptor input. might not be equal within the ganglion cells classified with black/white stimuli as class I, 2, 3 and 4 respectively. The purpose of the experiments described in the present report was therefore to prove the existence of subclasses of the 4 types of retinal neurons mentioned above. Moving stimuli rather than stationary stimuli were used. This method of stimulation compares
CIRsuA GROSSER-CORNEHLS and RAMSEY MrD. SALWXXS
470
closely with the type of stimulation the animal experiences in its environment. Class I and 2 neurons. moreover, do not normally respond to the onset and offset of diffuse stationary light stimuli (Maturana et al., 1960). Therefore, the application of black stimuli moving on a chromatic ba~k~ound or moving chromatic contrast stimuli was indicated. METHODS
Preparafion
Experiments were ~rform~d on 70 medium-sized adult frogs (Rana esc~~e~~~)~After anaesthesia with ethyl-m-aminobenzoate (MS 122 Sandoz), the skull above the optic tectum was removed, the dura opened and the brain covered with mineral oil. Each animal was immobilized by a lymphatic sac injection of OS-O.8 mg ~-tub~rarine chloride, covered with moist gauze and placed on a moist sponge with its head fixed in the center of a perimeter (Gr%sser and Dannen~rg” 1965). Stimulation The receptive fields of the retinal afferents of the optic tectum were stimulated by a physi~l~y btack spot 2.8” dia moving at constant velocities across chromatic background fields of 20” dia. The optical stimulator consisted of a tungsten lamp projection system (Sonnies, Freiburg i.Br., 1963) which was used with a filter wheel containing 16 mon~hr~mati~ filters (Balzers type} with peak transmissions in the range between 400 and 66Onm and a half b~d~dth of about 1Onm. Monochromatic light from the system was projected onto a di~u~ly reflecting white card situated in the stimulus window of a special purpose perimeter (Griisser and Dannenberg, 1965). The white card 28.5 cm from the frog’s eye contained a hole of 2.8” dia behind which a camera shutter system with matt black paper behind its aperture was situated. With this arr~gement (light trap), none or very tittle of the light projected onto the white card
in the region of the hole was reflected. Thus, OX had an absolute black or ‘physically black” surface. The energy reaching the cornea of the frog’s eye from each monochromatic titter was measured h? mounting a photomultipher (type RCA 4832) at eye position in the perimeter and measuring the photocurrents at the different wavelengths using a digital multimeter. To determine the energy incident on the retina, corrections were made for the spectral transmission of the ocular media. This was done using the method of Saunders (1977). Using a photomul~plier and digital multimeter, the photocurrents were measured at different wavelengths at the exit pupil of an integrating sphere, with and without the eye of the frog in the entrance pupil of the sphere. The percentage transmission (1”)at each wavelength was obtained from the relationship: T-
l
I
300
400
x 100
PI and P2 being the phot~urrents with and without the eye in the entrance pupil of the sphere. The results in Fig. 1 demonstrate that in freshly excised eyes the spectral transmission in the region between 400 and 700 nm is about 807; and is almost independent of the wavelength and therefore, in our experiments, no corrections were made for the spectral transmission of the ocular media. After making corrections for transmission losses, the retinal illumination was adjusted to 1.4 x 10’ photons mm-’ set- 1 at 551 nm (pupil dia 3.4 mm) by use of neutral density fibers. This is in the mesopic range for the frog (Blckstriim and Reuter, 1975). The response to constant quantum energy at different wavelengths rather than the threshold energy for stimulation was employed because of the neuronal adaptation to the moving stimulus (Griisser and Griisser-Comehls, 1973). This aspect will be discussed in more detail later. Considering the number of trials that must be made to achieve the threshold and the recovery time required between trials, the neuron
-f__-_.&
0
P,/P,
500
. --+___
-&---+
I
600
1 700
Wavelength Cnm) Fig. 1. The spectral transmission of the frog’s ocular media, T~~~~ and squares indicate the values from two different eyes. Curve drawn by hand.
Chromatic subclasses of frog retinal ganglion cells must be held for at least 3 hr to obtain a complete action spectrum using the latter method. Although occasionally a neuron can be held for this period or even longer, this is not generally the case. With the constant quantum energy method, a “spectral response curve” can be obtained in about 45 min allowing a recovery time of about 90 set between successive traverses of the ERF by the moving black spot. Three types of experiments were performed. In the first type the spot was moved at a constant velocity of 9.2” set-’ across various monochromatic background fields. The velocity was chosen because it lies in a velocity range in which strong responses are obtained from the retinal neurons, the responses at low and high velocities being very weak (Griisser et al., 1968). In the second type of experiment, velocities in the range of 0.46-46” see-’ were used, thereby enabling the response variations with velocity to be determined. In addition to the monochromatic backgrounds, chromatic backgrounds from broadband colour filters were used. These enabled measurements to be made under photopic as well as scotopic conditions. Finally, in the third type of experiment, the neuronal adaptation was investigated by moving the black spot to and fro at a velocity of 9.2” see-’ across the monochromatic background fields. When a stimulus is continuously moved to and fro across the receptive field, along the same path, the response to the first traverse is stronger than that to the second, which is stronger than the third etc. (Grusser and Griisser-Cornehls, 1973). The ratio of the second to the first traverse (i.e. backward movement to forward) 400
471
was determined. The neurons investigated were not direction sensitive. The spot was also moved to and fro until the neurons were completely adapted and the response magnitude after various recovery times was measured. Recording and data processing
Tungsten microelectrodes with electrolytically platinized tips of 1 to 3 pm dia were used to record the impulses of the afferent optic nerve fibers of the superficial tectal layers. The action potentials of single neurons were amplified by a Tectronix 122 amplifier and a 2A61 plug-in-unit of a Tektronix 565 oscilloscope. The movement of the black spot was recorded by potentiometers connected to the moving stage of the perimeter apparatus. The signal was fed into a 2A2 plug-in-unit of the oscilloscope. All signals were stored on a 7-channel-analog magnetic tape (Bell and Howell, VR 3200). Photographic recordings were made with a Tonnies Recordine camera (Tonnies K, 565). The data were later counted by hand and the average impulse rate calculated for the different wavelengths and conditions. For the statistical processing of the data, a Lint-8 digital computer was used, applying conventional statistical programs (mean, standard deviation, significance and regression line). RESULTS Responses to contrast stimuli moving on monochromatic background
The responses of 48 neurons recorded from the superficial layers of the optic tectum to a 2.8” black
500
589
526
601
551
578
Class
2
Neuron
Fig. 2. Original recording of the responses of a class 2 neuron to a black spot (2.8” dia) moving at a constant velocity of 9.2” set- ’ across various monochromatic background fields (20” dia). At each wavelength the stimulus movement is displayed above the action potentials. Note the dependence of the impulse pattern on the wavelength.
411
URSLILA GRUSSFK-CORNEHLS and RAMSEY M(,D. SAIINIWKS
spot moving at 9.2’ set _ ’ across the monochromatic background fields were quantitatively analysed. The units were first classified (class 1, 2, 3, 4) according to the procedures applied in earlier studies (Maturana t’r al.. 1960; Griisser and Griisser-Cornehls. 1976). Thereafter, the 2.8 black spot was moved every 90 set across a different monochromatic background field along the same path through the center of the respective ERF. Figure 2 shows responses recorded in such an experiment from a class 2 neuron. From data of this type the average impulse rates R were determined. By plotting R as a function of wavelength. we obtained “spectral response curves” for the different classes of retinal ganglion cells. These response curves were normalized (maximal response given a value of 100) for convenience. On the basis of their spectral response curves, several subclasses of the 4 main types of retinal ganglion cells were found. Class I neurons displayed two subclasses, one having a broad spectral response curve with three small maxima (Fig. 3a). the other
having two maxima, a dominant one at 466 nm and ;t weaker one at 580nm (Fig. 3b). In class 2 neurons as well, two subclasses were found. One type exhibited a broad spectra) response curve with three maxima similar to those found in part of class 1 neurons (Fig 3~). the other had two maxima at 466 and 580nm (Fig. 3d). Four subclasses of class 3 neurons were found. One type again had a broad spectral response curve with three maxima (Fig. 4a). Another group exhibited two maxima located approximately at 450 and 580nm respectively, one of which was dominant (Fig. 4b, c). Finally, a neuron type with a narrow spectral response curve was found having a single maximum at 500nm (Fig. 4d). To date we have only recorded from one class 4 neuron. This neuron had a double peak spectral response curve with maxima at 450 and 580 nm. To facilitate the differentiation of the subclasses on the basis of their spectral response curve. the following nomenclature is suggested: a-type for those neurons with a broad spectral response curve h-type
sbo
--T----i
600
700
Fig. 3. The spectral response curves of the two types of class I neurons (Figs 3a, b) and the two types of class 2 neurons (Figs 3c.d). The ordinate in each case represents the normalized average impulse frequency (imp’sec-‘) expressed as a percentage of the maximal response and the abscissa the wavelength (nm). The broken curves are the Dartnall nomogram curves for P 433 and P 578. The solid curves are drawn by hand. Vertical bars = SD
473
Chromatic subclasses of frog retinal ganglion cells for those with two maxima and c-type for those with a single maximum. For class 3 neurons, the b-type with a dominant maximum at 450nm will be referred to as 3bl and those b-type neurons with a dominant maximum at 580nm as class 3b2. The broken curves for the b- and c-types in Figs 3 and 4 are Dartnall nomogram curves for P,,,, PSo,, and P580r depending on the spectral region. We also determined the receptive field sizes of the a- and b-type neurons using monochromatic backgrounds and a 2.8 degree black moving contrast stimulus. Under these conditions the ERF sizes were found to be in the same range as described earlier for contrast stimuli moving on a black or white background (Maturana rt al., 1960; Griisser and GriisserCornehls, 1976). Dependence of the chromatic responses on the angular velocity of the moving spot
In the experiments described so far, the moving black spot had a velocity of 9.2” set-’ which is in the
400
500 Wavelength
so0 (“ml
midregion of the velocity response range of the neurons (Griisser et al., 1968). As the velocity of the stimulating spot was increased or decreased, the general shape of the chromatic response curve remained unchanged. The “velocity function”, however, changed quantitatively depending on background wavelength. This is seen best when the data are plotted in a double logarithmic coordinate system. The exponent c of the power function describing the relationship between stimulus angular velocity v and average impulse rate R during the traverse of the ERF, !? = k.vC impulses.sec-’
(I)
depended on the spectral nature of the background. For the neuron in Fig. 5 the exponent c was 0.97 for a 589nm background, 0.71 for a 492nm background and 0.65 for a 400nm background. To study the response at scotopic as well as photopic adaptation levels, we used 4 broad band chromatic filters and neutral density filters to shift the adaptation level
700 Wavelength
wlv
Fig. 4. The spectral response curves of four types of class 3 neurons. The ordinate in each case represents the normalized average impulse frequency (imp.sec-‘) expressed as a percentage of the maximal response and the abscissa the wavelength in nm. These neurons are classified as types 3a, 3b,, 36, and 3c. The broken curves are the Dartnall nomogram curves for P 433 and P 578. The solid curves are drawn by hand. Vertical bars = SD
473
IJRSLLA
GR~~SSER-CORNEHLS
and
RAMSEY
Mc D.
S&r
NI)I.RS
obtained in a class 3a neuron. but stmilar changes tn the exponent c were also found for the velocity !unction of class 2 neurons.
. 492nm 400nm 0,l J
__
0,46 Velocity
,
I
45
46
(deg.
s-l)
Fig. 5. Velocity response functions of a class 30 neuron at wavelengths of 400. 492 and 589 nm. Impulse rates (imp’sec-‘) are displayed along the ordinate and the stimulus velocities (deg.sec -‘) along the abscissa. Mesopic conditions.
from the photopic into the scotopic range (Fig. 6). Under photopic illumination. the exponent c of equation (1) was always found to be larger than under scotopic conditions. The data shown in Fig. 6 were
r
Red
/ *- - /
/.
As described in earlier reports (Griisser and Griisser-Cornehls, 1973, 1976), a fast back and forth movement across the ERF leads in class 2 and class 3 neurons to a pronounced adaptation which is only in part of receptor origin and which is not caused by adaptation of the ganglion cell impulse generator (Griisser and Griisser-Cornehls, 1969). Neuronal activation can be measured quantitatively if a contrast stimulus is moved to and fro through the ERF and the ratio of the impulse rate of the second to the first traverse is measured. In the experiments performed. the time lag between the forward and backward motions i.e. between the exit of the stimulus from the ERF and its re-entry into the ERF. varied between 500 and 2OOOmsec. In Fig. X the ratio mentioned 13 plotted as a function of wavelength. This method reveals that a-type as well as b-type neurons change their spectral response characteristics in the course of neuronal adaptation. The responses to a contrast stimulus moving across backgrounds of 500 nm wavelength and longer were more sensitive to neuronal adaptation than the responses obtained when a blue background illumination was applied (Fig. 7). This difference was more pronounced in the cl-type than III the b-type neurons. When complete neuronal adaptation was achieved by continuous to and fro movement across the ERF. a similar difference in the recovery time of the rcsponses to contrast stimuli moving across the hluc and the red background was obtained. The recovery time for the short wavelengths was about 3Osec and for the longer wavelengths about 60 set for both dand h-type neurons. Figure 7 exhibits examples ol such recovery functions in class 3a neurons. These measurements of neuronal adaptation and recover! functions indicated in addition that the 90 set intervals between successive traverses of the ERF m measurement of spectral response curves fulfilled the condition that the receptive unit tested was ilt the same level of excitation before each measurement.
DISCUSSION 0 46 Velocity
(deg.
4.6
46
s-‘)
Fig. 6. Velocity response functions of a class 30 neuron under photopic (broken lines) and scotopic (solid lines) conditions. Impulse rates (imp.sec-‘) are shown along the ordinate and the stimulus velocity (deg.sec-‘) along the abscissa of each diagram. Blue (Corning 2-62) broad band filters were used. The photopic energy was in each case 1.7 x 10s photons nrnm2 set-’ and the scotopic energy 3 log units lower. Note the steeper increase in the velocity functions under photopic conditions.
The measurement of the “spectral response curves” of the classical neuron types in the frog’s retina with black spots moving on a monochromatic background revealed the existence of distinct subclasses of the retinal neuron types. In fact, the class 3 neurons which Grtisser-Cornehls observed to have a short “on” and sustained “off” response to white light stimuli under scotopic and mesopic conditions were found to be the class 3c neurons of our present study. The class 3c
475
Chromatic subclasses of frog retinal ganglion cells
I
Rana
esc.ulenla
5 Ciass
ib 3Class
9 Class
3b
2h and
neuram
500
600
/ 500
600
Wavelength
cnm)
Wavelength
cnm>
1
/
1
Fig, 7. (a) Neuronai adaptation of a-type neurons at various wavelengths. Along the ordinate, the neuronal response to the stimulus moving backwards through the receptive field is expressed as a percentage of the forward response, the pathway being identical. The wavelength in nm is indicated along the abscissa. These neurons were not directionally sensitive. Note the faster recovery (greater percentage neuronal response) in the blue spectra1 region. (b) Neuronal adaptation of b-type neurons at different wavelengths. Note again the faster recovery in the blue spectra1 region. (c) Recovery function after complete neuronal adaptation of class 3b neurons using 350 nm (so&d curves) and 578 nm (broken curves) as background fields. Identical symbols indicate the same neuron. The black spot was moved to and fro through the receptive field along the same path until the neuronal activation ceased (complete neuronal adaptation).
neurons exhibited the typical short “on” and “of?” bursts to the onset and offset of diffuse white stimuli under phatopic conditions. However, with our diffuse monochromatic stimuli (mesopic), a short “on” and a
sustained ‘WY’ response was obtained. At 500 nm the responses were longest (0.25 see for the “on” and 4 set for the “off”). fn addition, the different subclasses appear ta be closely correlated with the different his\.R ?I 4 b
tologicaal types described by Cajai (1894). This aspect. however, wili be dealt with in a subsequent publication (Griisser-Cornehls and Saunders, 1979 in prep aratian). The a-, b- and c-type response curves are not true action spectra, since they represent the neuronal response to constant quantum energy at different wavelengths and not the variation of sensitivity with wave-
476
IIRSI~LA
GKliSSER-C‘ORNtHLS
and
length. Generally the shape of a spectral response curve differs from that of the absorbance spectrum of a visual pigment. The spectral position of the respettive maxima. however. can be very close together if the response is dominated by that pigment. For example with h- and c-types. reasonable agreement with the appropriate receptor pigment was obtained, PdX3 and P,sc, for the h-type and P,,, for the c-type. Since the m~lximum in the blue region of the spectrum occurs at 450 or 466 nm. the nomogram curve for Pha3 was shifted to the right. The finding that the maximum in the blue region of the spectrum was shifted to the right is, however. in agreement with those of other authors who used diRerent experimental methods to ours (Gratiit. 1942: Donner and Rushton. 1959; Chapman, 1961: Muntz, 1962: Reuter. 1969). This shift is probably due to the influence of one or more other receptor types on the green rods. The data therefore suggest that neurons with an u-type response receive inputs from all receptor types of the frog’s retina. those with b-type mainly from the green rods and the single cones and those with c-type response mainly from the red rods. The different spectral response types probably play an important role in brightness and coiour detection in the frog. The broad spectral response curves of the cl-type units suggest that they are luminosity units (black--white detectors of DeVaiois. 1960). These units are probably important for brightness and contrast detection under mesopic and photopic conditions. One possible function of the accessory element of the double cone with its 500 nm pigment is to “fill in” the spectrum for brightness detection at photopic levels. In the absence of this unit. the main fun~tionai units under photopic conditions would be the green rods (433 nm) vvhich function at high intensity levels (Backstrom and Reuter, 1975) and the single cones (580 nm). The presence of an element with an absorption maximum in the midregion of the spectrum is desirable for iuminan~~ detection under these conditions. Units with b-type responses are probably the chromatic or colour detection units. However. colour detectors are generally expected to display a coiour opponency, the differential responses of neurons to the onset and offset of monochromati& stimuli in different spectra) regions (DeValois. 1965). According to Maturana et trl. (1960). class 1 and 2 neurons of Rona pipiers do not respond to the onset or offset of large field white light stimulation. The same is also true for R~JNJ ~~cuirrrro (Griisser et a/.. 1967). We have confirmed this finding for monochromatic stimuli with Ri.i~ esculemt. Thus colour opponency was not detected for class 1 or 2 neurons. Using smai) light spots of high intensity, Backstrom and Reuter (1975) have found, however. that some type of opponent activity in class 1 and 2 neurons in the isolated eyes of the frog occurs. They found a pure “on” response for the green rod input, but an “on” and “off” response for the cone input. This situation changes consider-
KAMSt’I
McD.
%l
lumRS
ably with moving stimuli. With black targets sm,~llct than 4” dia moving on a chromatic backgroutlL ;t real colour opponency is possible in class 1 mi ? neurons. since only the “off” response of the ~:onc input occurs which IS evoked by the leading cdgc of the target. The “on” response of the cone mechantsm. which would be elicited by the withdrawing edge: ot the target. falls well into the total neuronal adaptLitton time of the bipolar cells (Grusser and Grusser-!‘onehis. 1969). Consequently an “on” response ir ~)nl! elicited by the response of the green rods Although class 3 neurons respond to both the onstlt and cessation of diffuse white light as well as monochromatic light stimulation. only one class 3 neuron with very clear opponent properties was found m cxperiments in which the response of class 3 neuron\; to the onset and offset of monochromatic stimulation was tested. This neuron had inputs from green rods and single cones (b-type). The infrequent occurrence of class 3 opponent coiour neurons is in agreement with the findings of Backstrom and Reuter 0975). In their sample of neurons, most class 3 neurons showed an “on‘* and an “off” response for the input of hnrh receptor types (green rods and cones). Subsequently. in these neurons, even with moving targets, no real coiour opponency occurs. because only “off” responses are elicited by both receptor mechanisms with the leading edge of the moving dark spot. We suggest therefore that some kind of non-opponent coiour mechanism exists in the frog’s visual system. This, will be discussed in more detail in a following publication (Griisser-Cornehls and Saunders. 1979 in preparation). The c-type units are probably the scotopit luminosity units. These neurons may. in addition. play a role in colour vision at mesopic levels. A clear separation of class I -4 retinal ganglion cells into subclasses on the basis of their velocity function has not been possible so far. With monochromatic backgrounds the slope of the velocity function at 58Onm was always found to be steeper than that at 450 or 500 nm. The steeper slope of the velocity function at 580nm is attributed to the cones, the Hatter curves obtained at 450 and 5OOnm to the rods. At 5OOnm, however. there may be some input from the accessory element of the double cones. The broad band filters (red. green, yellow and blue) which permitted study under photopic as well as scotopic conditions yielded a similar result. In Fig. 7 the sharp increase in the impulse rate with velocity under photopic conditions is attributed to the cones, the flatter curves obtained under scotopic conditions are attributed to the rods (see also Grtisser-Cornehis and Wolynski. 19731. Thus. although both rods and cones detect movement. the cones are better able to differentiate velocities. The r~uuronal adaptation obtained by moving a spot to and fro through the receptive field along the same pathway is probably due to adaptive proLesses in several systems, the retinal receptors and the neuronal network connected with the ganglion cell (Grtisser
477
Chromatic subclasses of frog retinal ganglion cells
and Griisser-Cornehls, 1973). The shift in the spectral responsiveness of the a- and b-type neurons (Figs 8a, b) suggests that the green rods and the neuronal elements connected to them recover much faster than the red rods and the cones. This was confirmed by the experiment in which the neuronal response was determined at various periods after complete neuronal adaptation. The small difference in the response ratio of the a- and b-types in the blue region of the spectrum (6.5% relative to 50%) suggests that some separation into subclasses may be possible by this method. However, the determination of the spectral response curves is a more satisfactory method. The results with chromatic adaptation indicate the probable existence of receptor-specific bipolar cells, since neuronal adaptation does not take place at the receptor or ganglion cell level (Griisser-Cornehls et al., 1964). This opinion is supported by the fact that there exist two modes of signal transfer in the bipolar cells of the frog (Murakami and Shigematsu, 1970), one by electronic conduction and one by initiation and propagation of action potentials. Initiation and propagation of action potentials might occur in the green-rod bipolar cells.
CONCLUSION Thus with the use of moving colour contrasts, it is possible to isolate subclasses of the classical neuron types of the frog. Of the three types of experiments performed, the determination of the spectral response curves of the neurons was the most useful. This method revealed the existence of two subclasses of class 1 neurons, an a-type with a broad spectral response curve with three maxima and a b-type with two maxima at 4.50 and 580 nm. Class 2 neurons also exhibited a- and b-types. In addition to the a- and b-subclasses, class 3 neurons exhibited a subclass with a narrow spectral response curve with a maximum at 500 nm.
Acknowledgements-The investigations were sponsored by a grant of the “Deutsche Forschungsgemeinschaft” (Gr. 276/7.8, 12. 13). We wish to thank Professor Ch. Baumann and Professor O.-J. Griisser for their valuable comments on the manuscript. Miss H. Wolynski for her careful technical assistance and Mrs J. Dames for her help with the English translation. Mrs J. Vierkant-Glathe wrote the computer programs.
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