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Short-wavelength input to luminosity-type horizontal cells in the turtle retina
SHORT-WAVELENGTH INPUT TO LUMINOSITY-TYPE HORIZONTAL CELLS THE TURTLE RETINA IDO F'ERLMAN'
RICHARD
and
A.
IN
NORMANX
Laboratory of Neurophysiology, National Institute of Neurological and Communicative Disorders and Stroke, National Institutes of Health, Ekthesda. Maryland 20014, U.S.A. (Received 13 Norember 1978) Abstract-The spectral and spatial properties of the photoresponses of luminosity horizontal cells were studied intracellularly in the turtle retina. A bright, full field, short-wavelength stimulus elicited a response which hyperpolarized to an initial transient peak and then sagged to an intermediate level. This transient ON response could be eliminated by either decreasing the retinal area illuminated or by changing the color of the stimulus from green to red. The mechanism underlying the transient ON response had a large receptive field ( - 1.7 mm diameter) and a short-wavelength input, probably from green cones. These findings are consistent with the hypothesis that lateral interactions between chromaticity- and luminosity-type horizontal cells exist in the turtle retina.
were obtained by interposing narrow band interference filters in the light path. The intensity of the unattenuated light was 6.4 x 1OL5effective quanta (640 nm) set-! cm-’ (Normann and Perlman, 1979a). The stimulus intensities referred to in the text are the attenuation in log units by neutral density filters interposed in the light beam. Identification of L-type and C-type units was based upon criteria described elsewhere (Fuortes and Simon 1974; Saito, et cl., 1974). The receptive field size of these neurons are best characterized by their length constants. Since our study deals also with large responses which are not well characterized by a length constant (Lamb. 19761 we have defined the receptive field size of the horizontal cells as the spot diameter which evoked a response, the amplitude of which was 90% of the maximum response observed with the largest spot (3.2 mm diameter). Receptive field sizes were determined only for cells which exhibited a saturation in the response amplitude-spot diameter relationship.
INTRODUCTION
The neural interactions leading to the spatial and spectral properties of the photoresponses of luminosity horizontal cells (L-type) in the turtle retina have been extensively studied (Simon, 1973; Lamb, 1976; Fuortes and Simon, 1974; Yazulla, 1976). These studies have implicated two main sources of input to L-type horizontal cells; the primary input is mediated by red cones (Fuortes and Simon, 1974; Yazulla, 1976) with a secondary input originating from coupling between L-type units (Simon, 1973; Lamb, 1976). However, chromatic studies indicate that L-type units are not univariant for bright light stimulation (Fuortes et al., 1973; Fuortes and Simon, 1974). This additional input has been attributed to the green members of double cones (Fuortes and Simon 1973). We have investigated the origin of this additional short wavelength mechanism using intracellular recordings from luminosity and chromaticity (C-type) horizontal cells in the turtle retina. We suggest, on the basis of photoresponses elicited by colored stimuli of various diameters, that this mechanism may be the result of an interaction between L- and C-type horizontal cells. METHODS
The experimental apparatus and methods have been described in detail elsewhere (Normann and Perlman, 1979a). Isolated eyecups were prepared from the turtle Pseudemys scripta elegons, aerated with moist 95% Olr 5% CO2 and maintained at a constant temperature of 18’C. The stimuli used in this study were 5OOmsec pulses of light which illuminated retinal spots centered around the microelectrode. The diameter of the spot could be varied from 0.3 to 3.2 mm with an iris diaphragm. Monochromatic stimuli
’ Present address: Vision Research Laboratory, Hadassah University Hospital, Jerusalem, Israel.
RESULTS
The origin of the short-wavelength input into L-type horizontal cells was investigated in cells whose entire receptive field could be illuminated with our stimulator. L-type cells which met this requirement corresponded to the L-II units of Simon (1973) and the S.R.F. type of Normann and Perlman (1979b). The receptive field size of these neurons depended upon the intensity of the stimulus used to measure this parameter as noted by Lamb (1976). Figure 1 shows a series of responses from an L-type cell paired according to the intensity of the white test flash that evoked them (numbers on left). Each pair of traces compares responses elicited by moderate (1.05 mm) and large (2.4mm) spot diameters of the same intensity. For the dimmest stimuli (-6.3), the responses superpose. As the stimulus intensity was increased, an initial transient peak developed in the responses elicited by the large spot stimuli. However, very little change was observed during the plateau and OFF phases of the responses. These spatial effects were
903
60
mV-
;1
Fig. 1. Photoresponses of an L-II type horizontal cell paired according to the stimulus intensity used to evoke them. Each pair oi traces compares the photoresponses
evoked by white light stimuli attenuated by the ND filter indicated on the I& and covered either a medium size spot (I.05 mm diameter) or a large spot (1.3 mm diameter). The responses with the initial transient peak are those eli-
cited by the large soot stimuli. most pronounced for stimuli of moderate intensity and diminished for either dim or very bright stimuli. This figure suggests that the receptive field of this unit, measured either with dim stimuli or at the ptateau of the responses to bright stimuli. was I.05 mm or less, and smaller than that measured for the tran-
sient peak. Responses recorded in this cell to different spot diameters of constant intensity (log I = -4.1) were used to determine the receptive field size. A value of 1.5 mm was found for the peak component and 1.0 mm for the plateau (measured 450 msec after stimulus onset). Similar measurements from responses to dim flashes indicate a receptive field diameter of approximately 1.0 mm, a value equal to that measured at the plateau. In each of the thirteen L-type units studied with stimuli of moderate intensity, the receptive field diameter for the peak component was larger than that for the plateau. The mean values of receptive field diameters for the peak and plateau were 1.7 i: 0.4 and 1.1 2 O.ilmm, respectively. These data suggest that the photoresponses of L-type units arise from at least two mechanisms with different spatial properties. One mechanism which is revealed with small responses and at the plateau component of large responses has a smaller receptive field than the mechanism which underlies the transient ON response. The strong dependence of this latter mechanism on spot diameter is further illustrated in Fig. 2. Here the intensity of the medium size spot was approximately ten times brighter than that of the large spot which resulted in equal amplitude responses. However, no transient ON response was observed for the medium size spot (solid arrows). These two mechanisms can also be made apparent using chromatic stimulation as illustrated in Fig. 3. Large spot (3.2mm diameter) dim stimuli of longwavelength (700 nm) or short-wavelength (460 nm) light were matched in intensity to provide an equal L-type response. These responses were univariant
03
02
Set
Fig. 2. Photoresponses from the same cell used in Fig. I. The response marked by solid arrows was evoked by a moderate size spot (1.05 mm) which was approximately ten times brighter than the large spot stimulus used to evoke the response marked by open arrows. Even though the two responses have similar peak amplitude no transient OK response is observed in the response obtained with moderate size spot. (upper paired traces, Fig. 3). However. when the intensities of these matched stimuli were increased by approximately equal factors, the photoresponses fotlowed different time courses (lower paired traces); the responses to short-wavelength, like the response to large, bright white spots (Fig I) had an initial transient peak on the rising phase which was not observed in the responses to the long-wavelength stimuli. Similar to the area effect on the waveform, this chromatic effect was much less apparent during the plateau and falling phases of the photoresponses. Similar chromatic -ffects have been noted before (Fuortes et al., 1973; Fuortes and Simon, 1974). It could be argued that any small mismatch in the
intensities of the short- and long-wavelength stimuli may have produced the differences observed during the rising phase of the response. We have discounted __.-.I 460nm -2.1 -
1 700nm -4.6
- 1.1
-3.7
-0.6 v ---y
-3.2
Fig. 3. The spectral effects on the transient ON response of an L-type horizontaf cell are emphasized by recording the photoresponses elicited by large spot illumination (3.2 mm) of different colors. Each pair of traces describes the response to 460nm light and to 700nm stimulus. The smallest responses (top pair of traces) superpose and indicate univariance. As intensity is increased the response to the 46Onm flash develops an initial transient ON response not observed with 7OOnm light. The numbers on the left and right of the traces indicate the iog relative intensity of the 460nm stimulus and the 7OOnm stimulus respectively. The neutral density filters were calibrated for each wavelength.
Short-wavelength
905
input to luminosity-type horizontal cells
to have receptive field diameters greater than 1.6 mm. a value which agrees with the receptive field size measured for the initial transient peak observed in L-type horizontal cells. This agreement is consistent with the suggestion that L-type horizontal cells receive input from C-type units. Moreover, stimulation with a colored annulus (3.2 mm o.d.. 1.05 mm i.d.) resulted in a small (l-2 mV) depolarization in L-type units with red stimulation which was not observed with green stimulation. 30
mV
DISCUSSION
0
0.3
0.6
Set
Fig. 4. The intensity of the 7OOnm light was increased by an additional factor of 30 compared to the intensity in the bottom pair of traces in Fig. 3. The obtained photoresponse (solid arrows) started earlier and initially rose faster than the response to the brightest 460 nm stimulus (open arrows). The peak responses seen here for the two colored stimuli are similar, yet no transient ON response is seen in the response to the red light. this possibility by further increasing the intensity of the brightest red stimulus shown in Fig. 3 by a factor of 30, which provided an approximate match of the peak amplitudes of the responses to the colored stimuli, as shown in Fig. 4. Even though the response to red started earlier (solid arrows) and initially rose faster than that to the short wavelength flash (open arrows), no initial transient was observed in the red response. In this experiment, there was greater red cone excitation produced by the red stimulus, and, therefore, the plateau was more hyperpolarized and the OFF response was much slower. We have further investigated the spectral characteristics of the initial ON transient in L-type units by recording the photoresponses evoked by colored stimuli of different intensities. From these responses the intensity-response curves were obtained for both the peak and plateau phases. The action spectra at the peak and plateau of large responses were determined from the P’-log I curves by choosing an appropriate voltage (28 mV) as a criterion response. In Fig. 5 we have isolated the action spectrum of the transient mechanism (m) by subtracting the spectra measured for the plateau (0) from that measured for the peak (0). The spectrum for the plateau peaks around 650nm as expected from the predominance of red cone input during this component of the response. The difference spectrum peaks around 560 nm, indicating a larger contribution of green cone input during the transient component (Baylor and Hodgkin, 1973). We conclude that this initial transient is the result of a neuronal mechanism which has a large receptive field (Fig. 1) and which has short-wavelength input from presumably green cones (Figs 3 and 5). A likely candidate for such a mechanism may be the C-type horizontal cell. We have measured the receptive field properties of 3 R/G type cells using monochromatic stimuli. For both red and green light the dependence of response amplitude upon spot size was similar and indicated a receptive field diameter of about 1.8 mm. Saito et al. (1974) have measured the spatial properties of C-type units and found them
The waveform of the photoresponses recorded from L-type horizontal cells in the turtle retina can be selectively altered by changing the spatial and spectral properties of the light stimulus (Figs 1 and 3). The transient ON response observed with bright shortwavelength stimuli can be eliminated by either decreasing the size of the illuminated retinal spot or by changing the color of the stimulus to red. It is therefore concluded that the transient ON response is elicited by a mechanism which is characterized by a large receptive field (about 1.7mm diameter) and by a sensitivity to short-wavelength light which is higher than that of single red cones. This mechanism contributes to the L-type cell photoresponses in addition to the contribution due to red cone input and coupling between similar L-type units. Several explanations have been proposed that might account for some of the results described here.
I-
I-
I-
1 WAVELENGTH
(nm)
Fig. 5. Action-spectra for the transient peak (0) and piateau phase, measured 450msec after stimulus onset (0) obtained from large responses (28 mV criterion) recorded from an L-type horizontal cell. The increased sensitivitv to short-wavelength of the peak compared to the plateal is emphasized by plotting the difference spectrum between the two (a). The difference spectrum indicates that the transient ON response is due to a mechanism with peak sensitivity around 560 nm similar to green cones. The continuous curves were drawn through the data points by eye.
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One possibility is a voltage- and time-dependent conductance change such as has been suggested to exist in the plasma membrane of horizontal cells in the !Vecturus retina (Werblin, 1975). However, the results of Fig. 2 argue against this explanation. Increasing the intensity of the stimulus of a medium sized spot evoked a response that started earlier and had a faster initial rising phase than that obtained by a large spot of less intensity, yet no initial transient was observed. Another suggestion attributed the spectral origin of the transient ON response to input from the green members of double cones to the L-type cell (Fuortes and Simon, 1974). Although such an additional input is supported by spectral sensitivity experiments (Yazulla 1976) and anatomical studies (Leeper, 1978) it cannot account for the spatial properties of the transient mechanism. Double cones, like other cones, have relatively small receptive fields (Richter and Simon, 1974) and should have been maximally stimulated even with the smallest spot used in this study (0.3 mm). Therefore, a transient ON response should have been observed when the L-cells were exposed to bright green light that illuminated small retinal areas. However, in all the L-cells studied, transient ON responses could be elicited by bright green light only if the illuminated spot was bigger than 1.5 mm in diameter. Spectral effects, similar to those illustrated in Fig. 3. on the waveform of the S-potentials were also observed in the cat eye (Steinberg, 1969a, b: Niemeyer and Gouras, 1973). This phenomenon was attributed to an antagonistic input of either rods or blue cones to the L-units (Niemeyer and Gouras, 1973). Such an explanation does not hold in the turtle retina. The relatively large receptive field of the transient ON response excludes direct input from photoreceptors as the source of the phenomenon. The spatial and spectral properties of the transient ov response may arise from interactions between adjacent L-type and C-type horizontal cells. This interaction has to be a sign-preserving one because when the C-type units are depolarized or unaffected (due to red or dim green stimuli) the hyperpolarization of the L-unit is depressed, while in conditions where the C-units are significantly hyperpolarized (bright, full-field green stimuli) the L-units exhibit a very fast hyperpolarization. In addition, the interactions between the C- and L-units are time-dependent and are mainly expressed during the initial ON phase of the photoresponses. A similar interaction between two different types of luminosity cells has been proposed by Lasansky and Vallerga (1975) to account for a facillitatory surround observed in B-type horizontal cells in the retina of the tiger salamander. The C-type units exhibit the required properties of large receptive field (Saito et al., 1974) and high sensitivity to short-wavelength light (Fuortes and Simon, 1974; Ya~ulla, 1976). The extent of the spatial and spectral effects on the transient ON response depends upon two factors: (A) the strength of the interactions between the C- and L-type units and (B) the relative sizes of the receptive fields of the C-type inputs (arising from C- to C-type coupling) and of the L-type input (arising from L- to L-type coupling). In L-type units where the coupling with similar ceils extends over a smaller retinal area than the C-type receptive
RICHARD
.A.40~~4~
field. the spatial effect on the transient ON response is pronounced. In these units, the receptive field measured with dim stimuli or with red stimuli reveals the extent of the L- to L-type coupling while that measured with bright green stimuli describes the receptive field of the C-type input into the studied cell. In other L-type units, the differential effect of decreasing the spot size on the peak and plateau phases of the large responses was small. In these cells, the receptive field, measured with dim light, was very large and probably larger than that of the C-type unit. Thus, the additional input from C-type units was masked by the strong coupling between the L-units. However, chromatic stimuli did produce responses with different waveform; ON responses to red light were slower than those evoked by green light. It is therefore concluded that C-type to L-type interactions may exist in all luminosity-type horizontal cells in the turtle retina and that this interaction can account for the spatial, spectral and intensity effects described in this report.
REFERENCES
Baylor D. A. and Hodgkin A. L. (1973) Detection and resolution of visual sttmuli by turtle photoreceptors. J. Physiol. 234, 163-198. Fuortes M. G. F.. Schwartz E. A. and Simon E. J. (1973) Color-dependence of cone response in the turtle retina. J. Physiol. 234, 199-216. Fuortes M. G. F. and Simon E. J. (1974) Interactions ieading to horizontal ceil responses in the turtle retina. J. Physiol. 240, 177-198. Lamb T. D. (1976) Spatial properties of horizontal cell responses in the turtle retina. J. Physiol. 263, 239-255. Lasansky A. and Vallerga S. (1975) Horizontal cell responses in the retina of the larval tiger salamander. J. Physiol. 251, 145-165. Leeper H. F. (1978) Horizontal ceils of the turtle retina--II. Analysis of interconnections between photoreceptor cells and horizontal cells by light microscopy. J. camp. Neural.. 182, 795-810. Niemeyer G. and Gouras P. (1973) Rod and cone signals in S-potentials of the isolated perfused cat eye. Vision Res. 13, 1603-1612. Normann R. A. and Perlman I. (1979a) The effects of background illumination on the photoresponses of red and keen cones. J. Physiol. 286.491-507~ Normann R. A. and Perlman I. (1979b) Signal transmission from red cones to horizontal cells in the turtle retina. J. Physiol. 2S6, 509-524. Richter A. and Simon E. J. (1974) Electrical responses of double cones in the turtle retina. J. Physioi. 242, 673-683. Saito T.. Miller W. H. and Tomita T. (1974) C- and L-type horizontal cells in the turtle retina. Vision Rex 1-l. 119-123. Simon E. J. (1973) Two types of luminosity horizontal cells in the retina of the turtle. J. Physiol. 230, 199-211. Steinberg R. H. (1969a) Rod and cone contributions to S-potentials from the cat retina. Vision Res. 9. 1319-1330. Steinberg R. H. (1969b) Rod-cone interaction in S-potentials from the cat retina. Vision Res. 9, 1331-13-M. Werblin F. S. (1975) Anomalous rectification in horizontal cells. J. i’hysiol. 244, 639-657. Yazulla S. (1976) Cone input to horizontal cells in ths turtle retina. Vision Res. 16, 727-735.
RICHARD
and
A.
IN
NORMANX
Laboratory of Neurophysiology, National Institute of Neurological and Communicative Disorders and Stroke, National Institutes of Health, Ekthesda. Maryland 20014, U.S.A. (Received 13 Norember 1978) Abstract-The spectral and spatial properties of the photoresponses of luminosity horizontal cells were studied intracellularly in the turtle retina. A bright, full field, short-wavelength stimulus elicited a response which hyperpolarized to an initial transient peak and then sagged to an intermediate level. This transient ON response could be eliminated by either decreasing the retinal area illuminated or by changing the color of the stimulus from green to red. The mechanism underlying the transient ON response had a large receptive field ( - 1.7 mm diameter) and a short-wavelength input, probably from green cones. These findings are consistent with the hypothesis that lateral interactions between chromaticity- and luminosity-type horizontal cells exist in the turtle retina.
were obtained by interposing narrow band interference filters in the light path. The intensity of the unattenuated light was 6.4 x 1OL5effective quanta (640 nm) set-! cm-’ (Normann and Perlman, 1979a). The stimulus intensities referred to in the text are the attenuation in log units by neutral density filters interposed in the light beam. Identification of L-type and C-type units was based upon criteria described elsewhere (Fuortes and Simon 1974; Saito, et cl., 1974). The receptive field size of these neurons are best characterized by their length constants. Since our study deals also with large responses which are not well characterized by a length constant (Lamb. 19761 we have defined the receptive field size of the horizontal cells as the spot diameter which evoked a response, the amplitude of which was 90% of the maximum response observed with the largest spot (3.2 mm diameter). Receptive field sizes were determined only for cells which exhibited a saturation in the response amplitude-spot diameter relationship.
INTRODUCTION
The neural interactions leading to the spatial and spectral properties of the photoresponses of luminosity horizontal cells (L-type) in the turtle retina have been extensively studied (Simon, 1973; Lamb, 1976; Fuortes and Simon, 1974; Yazulla, 1976). These studies have implicated two main sources of input to L-type horizontal cells; the primary input is mediated by red cones (Fuortes and Simon, 1974; Yazulla, 1976) with a secondary input originating from coupling between L-type units (Simon, 1973; Lamb, 1976). However, chromatic studies indicate that L-type units are not univariant for bright light stimulation (Fuortes et al., 1973; Fuortes and Simon, 1974). This additional input has been attributed to the green members of double cones (Fuortes and Simon 1973). We have investigated the origin of this additional short wavelength mechanism using intracellular recordings from luminosity and chromaticity (C-type) horizontal cells in the turtle retina. We suggest, on the basis of photoresponses elicited by colored stimuli of various diameters, that this mechanism may be the result of an interaction between L- and C-type horizontal cells. METHODS
The experimental apparatus and methods have been described in detail elsewhere (Normann and Perlman, 1979a). Isolated eyecups were prepared from the turtle Pseudemys scripta elegons, aerated with moist 95% Olr 5% CO2 and maintained at a constant temperature of 18’C. The stimuli used in this study were 5OOmsec pulses of light which illuminated retinal spots centered around the microelectrode. The diameter of the spot could be varied from 0.3 to 3.2 mm with an iris diaphragm. Monochromatic stimuli
’ Present address: Vision Research Laboratory, Hadassah University Hospital, Jerusalem, Israel.
RESULTS
The origin of the short-wavelength input into L-type horizontal cells was investigated in cells whose entire receptive field could be illuminated with our stimulator. L-type cells which met this requirement corresponded to the L-II units of Simon (1973) and the S.R.F. type of Normann and Perlman (1979b). The receptive field size of these neurons depended upon the intensity of the stimulus used to measure this parameter as noted by Lamb (1976). Figure 1 shows a series of responses from an L-type cell paired according to the intensity of the white test flash that evoked them (numbers on left). Each pair of traces compares responses elicited by moderate (1.05 mm) and large (2.4mm) spot diameters of the same intensity. For the dimmest stimuli (-6.3), the responses superpose. As the stimulus intensity was increased, an initial transient peak developed in the responses elicited by the large spot stimuli. However, very little change was observed during the plateau and OFF phases of the responses. These spatial effects were
903
60
mV-
;1
Fig. 1. Photoresponses of an L-II type horizontal cell paired according to the stimulus intensity used to evoke them. Each pair oi traces compares the photoresponses
evoked by white light stimuli attenuated by the ND filter indicated on the I& and covered either a medium size spot (I.05 mm diameter) or a large spot (1.3 mm diameter). The responses with the initial transient peak are those eli-
cited by the large soot stimuli. most pronounced for stimuli of moderate intensity and diminished for either dim or very bright stimuli. This figure suggests that the receptive field of this unit, measured either with dim stimuli or at the ptateau of the responses to bright stimuli. was I.05 mm or less, and smaller than that measured for the tran-
sient peak. Responses recorded in this cell to different spot diameters of constant intensity (log I = -4.1) were used to determine the receptive field size. A value of 1.5 mm was found for the peak component and 1.0 mm for the plateau (measured 450 msec after stimulus onset). Similar measurements from responses to dim flashes indicate a receptive field diameter of approximately 1.0 mm, a value equal to that measured at the plateau. In each of the thirteen L-type units studied with stimuli of moderate intensity, the receptive field diameter for the peak component was larger than that for the plateau. The mean values of receptive field diameters for the peak and plateau were 1.7 i: 0.4 and 1.1 2 O.ilmm, respectively. These data suggest that the photoresponses of L-type units arise from at least two mechanisms with different spatial properties. One mechanism which is revealed with small responses and at the plateau component of large responses has a smaller receptive field than the mechanism which underlies the transient ON response. The strong dependence of this latter mechanism on spot diameter is further illustrated in Fig. 2. Here the intensity of the medium size spot was approximately ten times brighter than that of the large spot which resulted in equal amplitude responses. However, no transient ON response was observed for the medium size spot (solid arrows). These two mechanisms can also be made apparent using chromatic stimulation as illustrated in Fig. 3. Large spot (3.2mm diameter) dim stimuli of longwavelength (700 nm) or short-wavelength (460 nm) light were matched in intensity to provide an equal L-type response. These responses were univariant
03
02
Set
Fig. 2. Photoresponses from the same cell used in Fig. I. The response marked by solid arrows was evoked by a moderate size spot (1.05 mm) which was approximately ten times brighter than the large spot stimulus used to evoke the response marked by open arrows. Even though the two responses have similar peak amplitude no transient OK response is observed in the response obtained with moderate size spot. (upper paired traces, Fig. 3). However. when the intensities of these matched stimuli were increased by approximately equal factors, the photoresponses fotlowed different time courses (lower paired traces); the responses to short-wavelength, like the response to large, bright white spots (Fig I) had an initial transient peak on the rising phase which was not observed in the responses to the long-wavelength stimuli. Similar to the area effect on the waveform, this chromatic effect was much less apparent during the plateau and falling phases of the photoresponses. Similar chromatic -ffects have been noted before (Fuortes et al., 1973; Fuortes and Simon, 1974). It could be argued that any small mismatch in the
intensities of the short- and long-wavelength stimuli may have produced the differences observed during the rising phase of the response. We have discounted __.-.I 460nm -2.1 -
1 700nm -4.6
- 1.1
-3.7
-0.6 v ---y
-3.2
Fig. 3. The spectral effects on the transient ON response of an L-type horizontaf cell are emphasized by recording the photoresponses elicited by large spot illumination (3.2 mm) of different colors. Each pair of traces describes the response to 460nm light and to 700nm stimulus. The smallest responses (top pair of traces) superpose and indicate univariance. As intensity is increased the response to the 46Onm flash develops an initial transient ON response not observed with 7OOnm light. The numbers on the left and right of the traces indicate the iog relative intensity of the 460nm stimulus and the 7OOnm stimulus respectively. The neutral density filters were calibrated for each wavelength.
Short-wavelength
905
input to luminosity-type horizontal cells
to have receptive field diameters greater than 1.6 mm. a value which agrees with the receptive field size measured for the initial transient peak observed in L-type horizontal cells. This agreement is consistent with the suggestion that L-type horizontal cells receive input from C-type units. Moreover, stimulation with a colored annulus (3.2 mm o.d.. 1.05 mm i.d.) resulted in a small (l-2 mV) depolarization in L-type units with red stimulation which was not observed with green stimulation. 30
mV
DISCUSSION
0
0.3
0.6
Set
Fig. 4. The intensity of the 7OOnm light was increased by an additional factor of 30 compared to the intensity in the bottom pair of traces in Fig. 3. The obtained photoresponse (solid arrows) started earlier and initially rose faster than the response to the brightest 460 nm stimulus (open arrows). The peak responses seen here for the two colored stimuli are similar, yet no transient ON response is seen in the response to the red light. this possibility by further increasing the intensity of the brightest red stimulus shown in Fig. 3 by a factor of 30, which provided an approximate match of the peak amplitudes of the responses to the colored stimuli, as shown in Fig. 4. Even though the response to red started earlier (solid arrows) and initially rose faster than that to the short wavelength flash (open arrows), no initial transient was observed in the red response. In this experiment, there was greater red cone excitation produced by the red stimulus, and, therefore, the plateau was more hyperpolarized and the OFF response was much slower. We have further investigated the spectral characteristics of the initial ON transient in L-type units by recording the photoresponses evoked by colored stimuli of different intensities. From these responses the intensity-response curves were obtained for both the peak and plateau phases. The action spectra at the peak and plateau of large responses were determined from the P’-log I curves by choosing an appropriate voltage (28 mV) as a criterion response. In Fig. 5 we have isolated the action spectrum of the transient mechanism (m) by subtracting the spectra measured for the plateau (0) from that measured for the peak (0). The spectrum for the plateau peaks around 650nm as expected from the predominance of red cone input during this component of the response. The difference spectrum peaks around 560 nm, indicating a larger contribution of green cone input during the transient component (Baylor and Hodgkin, 1973). We conclude that this initial transient is the result of a neuronal mechanism which has a large receptive field (Fig. 1) and which has short-wavelength input from presumably green cones (Figs 3 and 5). A likely candidate for such a mechanism may be the C-type horizontal cell. We have measured the receptive field properties of 3 R/G type cells using monochromatic stimuli. For both red and green light the dependence of response amplitude upon spot size was similar and indicated a receptive field diameter of about 1.8 mm. Saito et al. (1974) have measured the spatial properties of C-type units and found them
The waveform of the photoresponses recorded from L-type horizontal cells in the turtle retina can be selectively altered by changing the spatial and spectral properties of the light stimulus (Figs 1 and 3). The transient ON response observed with bright shortwavelength stimuli can be eliminated by either decreasing the size of the illuminated retinal spot or by changing the color of the stimulus to red. It is therefore concluded that the transient ON response is elicited by a mechanism which is characterized by a large receptive field (about 1.7mm diameter) and by a sensitivity to short-wavelength light which is higher than that of single red cones. This mechanism contributes to the L-type cell photoresponses in addition to the contribution due to red cone input and coupling between similar L-type units. Several explanations have been proposed that might account for some of the results described here.
I-
I-
I-
1 WAVELENGTH
(nm)
Fig. 5. Action-spectra for the transient peak (0) and piateau phase, measured 450msec after stimulus onset (0) obtained from large responses (28 mV criterion) recorded from an L-type horizontal cell. The increased sensitivitv to short-wavelength of the peak compared to the plateal is emphasized by plotting the difference spectrum between the two (a). The difference spectrum indicates that the transient ON response is due to a mechanism with peak sensitivity around 560 nm similar to green cones. The continuous curves were drawn through the data points by eye.
9%
loo FERLWO
and
One possibility is a voltage- and time-dependent conductance change such as has been suggested to exist in the plasma membrane of horizontal cells in the !Vecturus retina (Werblin, 1975). However, the results of Fig. 2 argue against this explanation. Increasing the intensity of the stimulus of a medium sized spot evoked a response that started earlier and had a faster initial rising phase than that obtained by a large spot of less intensity, yet no initial transient was observed. Another suggestion attributed the spectral origin of the transient ON response to input from the green members of double cones to the L-type cell (Fuortes and Simon, 1974). Although such an additional input is supported by spectral sensitivity experiments (Yazulla 1976) and anatomical studies (Leeper, 1978) it cannot account for the spatial properties of the transient mechanism. Double cones, like other cones, have relatively small receptive fields (Richter and Simon, 1974) and should have been maximally stimulated even with the smallest spot used in this study (0.3 mm). Therefore, a transient ON response should have been observed when the L-cells were exposed to bright green light that illuminated small retinal areas. However, in all the L-cells studied, transient ON responses could be elicited by bright green light only if the illuminated spot was bigger than 1.5 mm in diameter. Spectral effects, similar to those illustrated in Fig. 3. on the waveform of the S-potentials were also observed in the cat eye (Steinberg, 1969a, b: Niemeyer and Gouras, 1973). This phenomenon was attributed to an antagonistic input of either rods or blue cones to the L-units (Niemeyer and Gouras, 1973). Such an explanation does not hold in the turtle retina. The relatively large receptive field of the transient ON response excludes direct input from photoreceptors as the source of the phenomenon. The spatial and spectral properties of the transient ov response may arise from interactions between adjacent L-type and C-type horizontal cells. This interaction has to be a sign-preserving one because when the C-type units are depolarized or unaffected (due to red or dim green stimuli) the hyperpolarization of the L-unit is depressed, while in conditions where the C-units are significantly hyperpolarized (bright, full-field green stimuli) the L-units exhibit a very fast hyperpolarization. In addition, the interactions between the C- and L-units are time-dependent and are mainly expressed during the initial ON phase of the photoresponses. A similar interaction between two different types of luminosity cells has been proposed by Lasansky and Vallerga (1975) to account for a facillitatory surround observed in B-type horizontal cells in the retina of the tiger salamander. The C-type units exhibit the required properties of large receptive field (Saito et al., 1974) and high sensitivity to short-wavelength light (Fuortes and Simon, 1974; Ya~ulla, 1976). The extent of the spatial and spectral effects on the transient ON response depends upon two factors: (A) the strength of the interactions between the C- and L-type units and (B) the relative sizes of the receptive fields of the C-type inputs (arising from C- to C-type coupling) and of the L-type input (arising from L- to L-type coupling). In L-type units where the coupling with similar ceils extends over a smaller retinal area than the C-type receptive
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field. the spatial effect on the transient ON response is pronounced. In these units, the receptive field measured with dim stimuli or with red stimuli reveals the extent of the L- to L-type coupling while that measured with bright green stimuli describes the receptive field of the C-type input into the studied cell. In other L-type units, the differential effect of decreasing the spot size on the peak and plateau phases of the large responses was small. In these cells, the receptive field, measured with dim light, was very large and probably larger than that of the C-type unit. Thus, the additional input from C-type units was masked by the strong coupling between the L-units. However, chromatic stimuli did produce responses with different waveform; ON responses to red light were slower than those evoked by green light. It is therefore concluded that C-type to L-type interactions may exist in all luminosity-type horizontal cells in the turtle retina and that this interaction can account for the spatial, spectral and intensity effects described in this report.
REFERENCES
Baylor D. A. and Hodgkin A. L. (1973) Detection and resolution of visual sttmuli by turtle photoreceptors. J. Physiol. 234, 163-198. Fuortes M. G. F.. Schwartz E. A. and Simon E. J. (1973) Color-dependence of cone response in the turtle retina. J. Physiol. 234, 199-216. Fuortes M. G. F. and Simon E. J. (1974) Interactions ieading to horizontal ceil responses in the turtle retina. J. Physiol. 240, 177-198. Lamb T. D. (1976) Spatial properties of horizontal cell responses in the turtle retina. J. Physiol. 263, 239-255. Lasansky A. and Vallerga S. (1975) Horizontal cell responses in the retina of the larval tiger salamander. J. Physiol. 251, 145-165. Leeper H. F. (1978) Horizontal ceils of the turtle retina--II. Analysis of interconnections between photoreceptor cells and horizontal cells by light microscopy. J. camp. Neural.. 182, 795-810. Niemeyer G. and Gouras P. (1973) Rod and cone signals in S-potentials of the isolated perfused cat eye. Vision Res. 13, 1603-1612. Normann R. A. and Perlman I. (1979a) The effects of background illumination on the photoresponses of red and keen cones. J. Physiol. 286.491-507~ Normann R. A. and Perlman I. (1979b) Signal transmission from red cones to horizontal cells in the turtle retina. J. Physiol. 2S6, 509-524. Richter A. and Simon E. J. (1974) Electrical responses of double cones in the turtle retina. J. Physioi. 242, 673-683. Saito T.. Miller W. H. and Tomita T. (1974) C- and L-type horizontal cells in the turtle retina. Vision Rex 1-l. 119-123. Simon E. J. (1973) Two types of luminosity horizontal cells in the retina of the turtle. J. Physiol. 230, 199-211. Steinberg R. H. (1969a) Rod and cone contributions to S-potentials from the cat retina. Vision Res. 9. 1319-1330. Steinberg R. H. (1969b) Rod-cone interaction in S-potentials from the cat retina. Vision Res. 9, 1331-13-M. Werblin F. S. (1975) Anomalous rectification in horizontal cells. J. i’hysiol. 244, 639-657. Yazulla S. (1976) Cone input to horizontal cells in ths turtle retina. Vision Res. 16, 727-735.