Light adaptation of red cones and L1-horizontal cells in the turtle retina: effect of the background spatial pattern

Vision Rrs. Vol. 21, No. 5, pp. 6854%. 1981 Printed in Great Britain. All rights mend

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0042&X39/87 $3.00 + 0.00 1987 Pcrgamon Journals Ltd

LIGHT ADAPTATION OF RED CONES AND Ll-HORIZONTAL CELLS IN THE TURTLE RETINA: EFFECT OF THE BACKGROUND SPATIAL PATTERN AVIRAN ITZHAKI and ILBOF%RLMAN* Department of Physiology, Faculty of Medicine and the Rappaport Family Institute for Research in the Medical Sciences, Technion-Israel Institute of Technology, Haifa, Israel (Received 17 July 1986; in revised form 28 October 1986)

Ahstraet-Electrical coupling allow red cones and Ll-horizontal cells to respond to light stimuli illuminating remote retinal loci. The contribution of the spatial pattern of background light to flash sensitivity of red cones and Ll-horizontal cells was studied intracellularly in the turtle retina. Lateral spread of background adaptation was observed in the cones. The sensitivity of the cones was related to the steady hyperpolarization of the cells irrespective of the background’s spatial pattern. Light sensitivity of Ll-horizontal cells mainly depended upon the background illuminating their dendritic field. Unlike red cones, the horizontal cells responded differently to spot and annular backgrounds regardless of the steady hyperpolarization induced. Light adaptation

Cellular coupling

Cones

Horizontal cells

INTRODUCTION

The process of light-adaptation has been extensively studied in the vertebrate retina. Electrophysiological studies in different retinas attribute the major control of visual sensitivity to mechanisms located within the photoreceptors (Dowling and Ripps, 1970; Normann and Werblin, 1974; Kleinschmidt and Dowling, 1975; Fain, 1976; Normann and Perlman, 1979a). Intracellular recordings from the turtle retina showed that the cone sensitivity to light was correlated with the intensity of the background light in a manner similar to the Weber-Fechner law defined psychophysically for human subjects (Normann and Anderton, 1983). The photoreceptors in the vertebrate retina are not independent units. Electrical coupling between adjacent cones causes lateral spread of current between cells when a potential difference exists between them. Thus, the photoresponse of a single cone depends not only on light absorption in its own outer segment but also on light ilhuninating its neighbours (Baylor, Fuortes and G’Bryan, 1971; Baylor and Hodgkin, 1973;

*To whom reprint requests should be addressed.

De&viler and Hodgkin, 1979). Electrical coup ling was also demonstrated anatomically and electrophysiologically in the horizontal cell layer (Naka and Rushton, 1967; Yamada and Ishikawa, 1965; Stell, 1967; Kaneko, 1971). These interactions presumably mediate the large receptive fields of the horizontal cells (Simon, 1973; Lamb, 1976; Normann and Perlman, 1979b; Byzov and Shura-Bura, 1983) which are considerably larger than the retinal areas providing direct input from photoreceptors to these cells (Leeper, 1978). The electrical interactions in the photoreceptor and horizontal cell layers may constitute a pathway by which the sensitivity of a cell may be affected by background light illuminating remote retinal loci. This notion is supported by studies in Necturus (Burkhardt, 1974) and turtle retina (Itzhaki and Perlman, 1984) which demonstrated the phenomenon of surround enhancement in horizontal cells. Electrophysiological studies in the retina of the snapping turtle showed that background adaptation could spread laterally in the cone network (Green and Copenhaqen, 1984). In order to investigate the above hypothesis we recorded the photoresponses of red cones and L-type horizontal cells in the turtle eyecup preparation. The sensitivity of the impaled cells to light was determined with test flashes of

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different sizes during adaptation to background illumination of different intensities and spatial patterns. In this study only Ll-type horizontal cells were included and compared to red cones to avoid the spatial complications of the center _..---..-A dnrdgcmsm r--‘r----I_-_ iouna I...-_l m I.. L,+rype IO * a- ‘surrouno norizontal cells (Piccolino, Neyton and Gerschenfeld, 1981). It was found that the cone sensitivity to light depended on its own state of adaptation and on the state of adaptation of its neighbours. The cone sensitivity was best correlated with the steady hyperpolarization induced by the background light irrespective of its spatial pattern. The horizontal cells showed summation of adaptation withing an area smaller than their receptive fields. No unique relationship between desensitization and membrane potential could be demonstrate for Ll-ho~zontal cells. An annular background illumination evoked a large amplitude hyperpolarization but had little effect on the cell’s sensitivity to light. While, a small spot background elicited steady hyperpolarization of small amplitude but had a large desensitizing effect. It is suggested that electrical coupling may serve for lateral spread of adaptation in the cone network. The characteristics of the horizontal ceil network may contribute to the processing of spatial info~ation and allow ceiis to respond digerentiy to backgrounds with different spatial pattern.

METHODS

The experiments were performed on the eyecup preparation of the swamp turtle Clemmys caspica. In our experience, the properties of the photoresponses recorded intracelluiarly from cones and horizontal cells in the retina of this turtle are vex similar to those rgnnrtcd r-- --before for the retina of the turtle Pseudemys scripta elegans (Baylor et al., 1971; Baylor and Hodgkin, 1973, 197% Normann and Perlman, 1979a, b). The eyecup was prepared as previously described (Itzhaki and Perlman, 1984). After enucleation, the eye was hemisected and the vitreous was carefully removed. The preparation was placed in a light-shielded Faraday cage where it was aerated with a moist mixture of 95% Or and 5% COZ to keep it from drying. Intracellular recordings were achieved with giass micropipettes fiiied with 3M potassium acetate, with resistance of 2~M~. The signal from the microelectrode was passed through a d.c. microprobe system (WPI M707),

displayed on an oscilloscope (Tektronix 5 113) and recorded by an FM tape-recorder (Teat). The signals were also fed into a pen recorder (Gould 2200) in order to verify stability of the recordings. The data from the tape-recorder .~~___ >f_lrl_. , ,... -- A---u . TXconverter for anaiysis were aigiuzea DYan by a Commodore 8032 computer. Signals of small amplitude were averaged by the computer. Sensitivity to light was calculated from responses which were within the linear range of the impaled cell by dividing the peak amplitude of the response by the quanta1 content (calculated for a unit retina1 area illuminated) of the Aash used to elicit this response. Sensitivity data were expressed in ~V/quantum/~m*. The photostimulation system consisted of two beams originating from a single light source (100 W tungsten-iodide lamp). Each beam could be independently modified with regards to its intensity, color and spatial pattern, One beam served for test flash stimuli and its diameter ranged from 40 to 2770 ,um. The background light either illuminated retinal spots of different diameters (ranging from 60 to 2800 pm) or annuli of constant outer diameter (2800pm) but different inner diameter (90 to 1030 pm). In all the experiments reported here the diameter of the test flash used to determine sensitivity was kept smaiier than the diameter of the spot backgrounds and the inner diameter of the annular ones. The test flash duration was set at 50 msec by pulse generator (WPI 1800 series). For the experiments reported here both the test flashes and the background light were monochromatic light with peak at 650 nm. The intensities of the test and background beams and the attenuations of the “neutral” density filters were measured with a calibrated photodiode (United Detector Technology). The unattenuated intensities of the test and background beams were found to be identical having a value at 650 nm of 6.73 log quanta/sec/pm2.

RESULTS

The first series of experiments were designed to determine the dependence of sensitivity to light on diffuse background illumination for red cones and Ll-horizontal cells. In these experiments the retinal area illuminated by the ---background was 2800 pm in diameter and that illuminated by the test light was 2770 ,um in diameter. In order to allow comparison between red cones and horizontal cells and between cells

Background pattern and retinal adaptation

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Fig. 1. Representative experiment of light-adaptation in LI-horizontal cell. Dark-adapted photoresponses elicited by flashes of different intensities were recorded before and after background illumination. The amplitude of the dark-adapted photoresponse evoked by a supersaturating stimuhts is detined as V,,. The horizontal bar at the top trace marks the duration of the background light (log I = 5.41 quanta/ ~/~rn2). The parameter d.c. describes the steady h~~Ia~tion induced by the ~ckground light relative to the dark-adapted level (dashed line). Calibration bars have a height of 15 mV and a length of 20 sec.

studied in different experiments, sensitivity loss was calculated relative to the dark-adapted state and the background strength was defined by its steady state effect on the membrane potential. It has been shown before that cones and horizontal cells in the turtle retina respond to background illumination by a transient hyperpolarization which is related to the background intensity (for intensities below saturation) followed by partial recovery to a steady level. The steady level is usually half way between the peak of the transient response to the background and the dark-adapted membrane potential (Normann and Perlman, 1979a, b). Thus, the level of steady membrane hy~rpola~zation induced by the background light may serve as a useful index for the effective strength of the background on the studied cell. Since different cells were characterized by photoresponses of different amplitude, the steady membrane hyperpolarization was normalized to the maximal response that could be obtained from the cell in the dark-adapted state. Figure 1 illustrates these definitions. The responses of a horizontal cell to large field stimuli of different intensities were first recorded in the dark-adapted state and the maximal response (I’,,& was found. Then, a background light was turned on inducing a transient hyperpolarization followed by partial recovery. After l-2 min the membrane potential stabilized and the steady level of hy~~ola~~tion relative to the dark-adapted level (dc.) could be determined. The photoresponses elicited by flashes of increasing intensities were recorded. After termination of the background dark-adapted photoresponses were recorded as a control, In the light- and dark-adapted states sensitivity to light was determined from “linear” range responses by averaging about 20 photoresponses

evoked by dim test flashes (not shown in Fig. 1). The data described in this report were obtained from experiments on 40 red cones and 42 Ll-horizontal cells. Using a large spot (2.77 mm diameter) test flash in the darkadapted state the red cones had an average of 18.0 f 3.1 mV maximal photoresponse (SD) and average flash sensitivity of 50.4 + 41.1 ~V/quantum/~m2 (SD). Under the same conditions, the Ll-horizontal cells had an average maximal response of 47.4 + 4.9 mV (SD) and average flash sensitivity of 348.6 + 178.3 ~V~quantum/~m* (SD). Figure 2 shows the desensiti~ng effect of large field (2800 pm diameter) backgrounds on 12 red

Fig. 2. The relationship between the log loss in sensitivity to light and the steady h~~la~~tion induced by background lights for 12 red cones (solid squares) and 17 L1 -horizontal ceils (open squares). Different background intensities were used ranging from 2.51 to 6.73 log quanta/ set/pm’. To allow comparison between different cells the steady hyperpolarixation was normalized to the maximal response of the cell. These data were obtained with large diameter (2770rm) test flashes delivered during large diameter (2800 pm) background illumination. The continuous and dashed lines described the data of red cones and Ll-horizontal cells respectively.

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AVIRAN Irzm~l

cones (solid squares) and 17 Ll-horizontal cells (open squares). Sensitivity was measured with large diametqr (2770 pm) test flashes. The desensitizing effects of the background lights were calculated in log units by dividing the sensitivity measured in the dark-adapted state by the value determined during background illumination. The 9 data points, grouped in the upper right side of Fig. 2, were obtained during illumination with the brightest background available in the system (log I = 6.73.quan~/~c/~m*). This back~ound was bright enough to bleach about 90% of the cone’s visual pigment as estimated from the data reported by Hodgkin and O’Bryan (1977) for turtle cones. Therefore, there was additional loss in sensitivity of about 1 log unit above the loss expected from the mechanisms of light adaptation. The continuous and dashed lines were drawn through the red cones data and the horizontal cells data respectively by linear regression. Only data from backgrounds that produced less than 50% bleaching were used to calculate the regression lines. The relationships between sensitivity loss and steady hyperpolarization are: for red cones (continuous line) log (S,/S,> = 4.694* (d.c./V,,) + 0.017 (1) for LI-horizontai ceil (dashed iinej log (~~~~,j = 4.591* (d.c.,‘v,,) - 0.004. (2) S, and S, are respectively the sensitivity to light of the studied cell in the dark- and light-adapted conditions; d.c. is the steady hyperpolarization induced by the background light relative to the dark-adapted membrane potential and V,,,,, is the amplitude of the maximal dark-adapted response elicited by a large diameter flash of su~rsaturating intensity. The two lines fit the corresponding data points for backgrounds having intensity range of about 4 log units. The data presented in Fig. 2 show that for large diameter flashes the desensitizing effects of large field backgrounds on both red cones and Ll-horizontal cells can be directly related to the steady hyperpolarization induced by the back~ounds. Furthermore, under these conditions sensitivity loss in the horizontal cells can be accurately predicted from the effects of the backgrounds on the red cones. The contribution of the spatial pattern of the L-,1 _^_^___A .1u. .L, E.-L* *__*:*:..:4.. VI -I C;GlID ,,I,” .WUJ ..^^ uacn&Iuullu 1llC 11~111 i3cIlarllvlly studied with stimuli illuminating retinal areas considerably smaller than the receptive fields of the cells. Sensitivity values calculated from re-

and IDO PERLMAN

sponses evoked with such test flashes do not reflect the true sensitivity of the cell because part of the signal generated in the impaled cell is shunted to its neighbours. We, therefore, defined in these experiments the apparent sensi.l-~lr- C_-.-- ...__ __. >.I-. wiry rrom JUSK recoraaole photoresponses (about 0.5 mV in amplitude). It was calculated by dividing the amplitude of the photoresponse by the quanta1 content (calculated for a unit retinal area> of the test flash. The apparent sensitivity data could be used to determine the effects of background illumination on retinal cells because comparison was made between different conditions using the same test flashes. No attempt was made to characterize absolutely the light adaptation properties of these cells with the small spot flashes. In all the following experiments the background intensities were kept below the level which produced significant bleaching of the visual pigments in order to avoid the cont~bution of bleaching to sensitivity losses. Figure 3 shows the effect of enlarging the retinal area illuminated by a constant intensity (4.55 log quanta/sec/pm2) background on the apparent sensitivity (A) and steady hyperpolarization (B) of three red cones measured with light stimuli of 4Opm diameter. For each I,._ cone tamerent syrnbois describe diRerent ceilsj the sensitivity and steady hy~~ola~zation measured during each spot background were normalized to the corresponding values measured for the background of largest diameter. The sensitivity to light gradually decreased as the diameter of the retinal area illuminated by the background light was increased until a relatively constant value was reached for background diameter of about 2OOpm. Further increase in the background diameter did not produced significant change in the sensitivity of the cells [Fig. 3(A)]. The steady hyperpolarization induced by the background lights show similar dependency on the background diameter [Fig. 3(B)]. Similar findings were observed in 13 red cones. In all the red cones studied the receptive field properties were dete~ined in the dark-adapted state using flashes of constant intensity but different diameter. The length constants were determined according to Lamb (1976) and were found to be in the range of 25-45 pm. The size of the **..*-r:..* L,.. cswn~~s~iw _*.l-_**A L-Y-rl_ rn;cpuvc C-I-l* IIGIUJ.-.** wus _^.._ ruugmy mm mc test flash size above which further increase in the spot diameter did not cause any increase in the amplitude of the photoresponse. The range

Background pattern and retinal adaptation

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BACKG&ND

DIAMETER

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05 BACKGROUND

DIAMETER

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eight red cones were studied (A) and with background intensity of 5.41 log quanta/sec/~m* seven red cones were studied (B). A linear relationship was found for the red cones between the log loss in apparent sensitivity and the normalized steady hyperpolarization induced by the background irrespective of its spatial pattern. The slope of the line fitted to the data points was smaller for the dimmer background (3.646 in A compared to 6.030 in B). This difference probably did not reflect changes in the receptive field properties of the red cones induced by the level of background illumination. The length constants of the cones’ receptive fields were determined by measuring the amplitude of the photoresponses as a function of the diameter of the test flash (Lamb, 1976; Perhnan et al., 1985).

7

28

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Fig. 3. The effect of enlarging the diameter of a constant intensity (4.55 log quanta/sec/pm2) background light on the sensitivity to light (A) and steady hyperpolarization (B) in three red cones. Different symbols describe different cells. The sensitivity was measured with 4Opm diameter test flashes. The apparent sensitivity and steady hyperpolarization measured at each background size were normalized to the corresponding values measured during illumination with background of largest diameter (2800 pm).

of receptive fields diameter was 150-200 pm. The similarity between the receptive field sizes of red cones and the size of retinal area in which background adaptation may summate suggest that the underlying mechanism of these two phenomena may be similar. In order to further investigate this possibility the same small diameter (40 pm) flashes were used while applying background illumination of either annular or spot patterns. All annuli had the same large outer diameter (28OOpm) and different inner diameter (from 90 to 1030 pm). The background spot pattern ranged from 60 to 28OOpm in diameter. The effective strength of each background pattern was defined by the steady hyperpolarization elicited by it. Figure 4 illustrates the loss in apparent sensitivity above the dark-adapted value caused by spot backgrounds (solid symbols) and annular ones (open symbols). Two different background intensities were used to construct Fig. 4. With a background intensity of 4.55 log quanta/set/pm*

4

1

0.1

NORMALIZED

a2 STEADY

a3 HYPERPOLAR!ZATIC

C

Fig. 4. The relationship beween log sensitivity loss (relative to the dark-adapted state) and normalized steady hyperpolarization induced by background light of different patterns in red cones. Eight red cones were studied with background intensity of 4.55 log quanta/sec/~m* (A) and 7 red cones were studied with background intensity of 5.41 log quanta/sec/~m* (B). Sensitivity data were obtained with 40 pm diameter stimuli. Background light was either a spot (solid symbols) of different diameter (60, 120, 200, 540, 1020,280O pm) or an annulus (open symbols) with constant outer diameter (2800 pm) and different inner diameter (90, 150, 530, 103Opm). The straight lines through the data points were obtained with linear regression procedure.

AWMN ITZHAKIand IDO PERLMAN

690

Table 1. Comparing the measurements of stray light produced by the different background patterns to their desensitizing effects measured in 5 red cones

Background pattern Spot dia. 28OOycm Spot dia. 1020pm Spot dia. 54Opm Spot dia. 2OOpm Spot dia. 120 pm Spot dia. 6Opm Annulus i.d. 90 pm Annulus id. 150 pm Annulus i.d. 530 pm Annulus i.d. 1030 urn

Stray light measurements 19Opm hole 45 pm hole (mv) (mv) 5.8 5.3 5.1 4.1 4.5 3.3 1.0 0.9 0.55 0.35

105 100 96 72 33 5.5 55 26 6.4 4.0

Background effect d.c.1 V,, log &i/s, 1.09 & 0.29 1.11 kO.29 1.11 Ifro.30 0.99 : 0.38 0.86 f 0.43 0.51 + 0.28 0.83 f 0.14 0.61 * 0.10 0.44*0.18 0.36kO.17

0.27 + 0.08 0.27 + 0.08 0.26 + 0.08 0.26 z 0. I1 0.23 f 0.10 0.15 +0.09 0.25 + 0.13 0.17+0.05 0.11 + 0.05 0. IO f 0.03

All backgrounds were of constant intensity (log I = 4.55 quanta/set/pm’). Total light (direct and stray) transmitted through 2 different hole sizes drilled in an opaque cover was measured for each backgroundpattern with a photodiode.The test flash used for the sensitivity measurements was of 40 pm diameter.

No significant differences were observed between the dark- and light-adapted states. Similar findings on the effect of steady illumination on receptive field properties have been recently reported for rods in the retina of the salamander (Attwell et al., 1985). The data in Fig. 4 suggested that the cone sensitivity to light during background illumination depended upon the state of adaptation of the impaled cell and its neighbors. The ..* . . conrnourion of stray iight to these findings was investigated by measuring the light intensity of the different background patterns with a photodiode covered with an opaque cover in which a small hole was drilled. Two hole sizes were used. One of 45 pm diameter to estimate the total light (direct and stray) reaching the impaled cone itself. The other had a diameter of 190 pm and was used to determine the light illuminating the entire receptive field of the cone. In order to mimic the experimental conditions a piece of turtle retina was placed on the opaque cover. In Table 1 the light-intensities (given in mV) of all the different background patterns measured through the two holes sizes are compared with the average desensitizing effect of the same background patterns on five cones studied. The photodiode is operating in the photovoltaic mode and therefore the potential measured is directly related to light intensity. With the small hole measurements the intensity of light produced by the smallest diameter (6Opm) background was about 3 times larger than that of the ..,....1..1 .WLLU . ..rl. rl.... ,-..ll,.4 :,,,, .a:..--*,, I, .I alll‘uluJ LUG 5111411G51 L‘1‘1~‘ ULILII‘GL~I (“AI. 2800 pm, i.d. 90 pm). However, sensitivity loss and normalized steady hyperpolarization were larger when measured during this annular back-

ground compared with the values obtained during the small spot background. Even when the inner diameter of the annular background was enlarged to 530pm a similar discrepancy between light measurements and sensitivity data were found when compared to the smallest diameter (60 pm) background. In contrast, measurements of light through the large hole were parallel to the sensitivity data measured in the red cones. These data indicated that even if stray ii&t contributed to iight adaptation of red cones, it could not be solely responsible for the observed effects of the different background patterns of the cones. Thus, light illuminating other cones within the receptive field of the impaled cell must have contributed to the adaptive state of the studied cone. The receptive fields of Ll-horizontal cells are considerably larger than those of red cones. In order to obtain meaningful photoresponses the test flash diameter was increased to 110 pm. Figure 5 illustrates, for three cells (different symbols), the effect of enlarging the diameter of the background spot on the apparent sensitivity measured with 110pm stimuli (A) and steady hyperpolarization (B). For each cell the apparent sensitivity and steady hyperpolarization were normalized to the values measured during background illumination of largest diameter. In the horizontal cells a reduction in sensitivity was observed when the background diameter was increased from 120 to 540 pm. For all 14 horizontal cells studied the background size which :_-I __^_J .L, ,,..,:r:.*r.. ..I.. cI”LIJI”,.,....:A IILUUGCU LUGI--,-r Ua‘)gFJL 3c.‘JlU”l&JI..“, LVDJ.w43 erably smaller than the receptive fields of the cells measured in the dark-adapted state. In our preparation the receptive field size for excitation

Background pattern and retinal adaptation

3

L

SACKORO”~D DIAMETER

2 mm)

BACKGf?O”~D

2 (mm)

DIAMETER

Fig. 5. The effect of enlarging the diameter of the background light (fog I = 4.55 quanta/sec/~m*) on the sensitivity (A) and steady hyperpolarization (B) of 3 Llhorizontal cells. Different symbols describe different cells. Sensitivity was measured with 110 gm diameter test flashes. The apparent sensitivity and steady hyperpolarization measured at each background were normalized to the values obtained during iarge diameter (28OOpmj background illumination.

(estimated from the dependency of the photoresponse amplitude on the flash diameter) of Ll-horizontal cells was larger than 2.2mm in diameter. While, the retinal area over which summation of background adaptation was evident was about 0.5 mm in diameter. In most cells (9 out of 14) further increase in the background diameter caused an increase in the sensitivity [e.g. Fig. 5(A), solid circles]. This is an electrophysiological expression of the phenomenon termed “surround enhancement” that has been previously described (Burkhardt, 1974; Itzhaki and Perlman, 1984). The phenomenon of surround enhancement observed with test flashes of 110 pm diameter was most apparent in cells with relatively small receptive field and was almost undetectable in cells characterized by receptive field diameter larger than 2800 pm. The relationship between the steady hyperpolarization and the background diameter >:m___> c__- .l_,-. 00srrvea _I__-._> c__ rL_ arnerea rrom indl ior inr: appUHii sensitivity. As the background diameter was increased from 120 to 2800 pm the steady hyperpolarization increased monotonically [Fig.

691

S(B)]. This differential effect of the background size on the apparent sensitivity and steady hyperpolarization was observed in all 14 L lhorizontal cells studied. The data presented in Fig. 5 suggest that the direct relationship be-______ ____:r:_.:._. I___ ~ween sensunwy loss aiid IXXiiiC3liZ~~ SkZbj; hyperpolarization which was found for red cones (Figs 3 and 4) fails to describe the behavior of Ll-horizontal cells under backgrounds of different spatial patterns. Figure 6 shows the effects of the background spatial pattern on the loss in apparent sensitivity (above the dark-adapted state) for 12 Ll-horizontal cells. For all the cells, sensitivity was measured with a test flash of 110 pm diameter. The background intensity was kept at 4.55 log quanta@c/~m* at the retinal surface. The background was either a spot (solid triangles) with diameter range of 120-2800 pm or an annular pattern (open triangles) with outside diameter of 2800 pm and inside diameter that ranged from 150 to 1030 ,um. In the same figure, corresponding data from eight red cones are presented. These data points were obtained with the same test flash and background parameters as those of the horizontal cells. Sensitivity loss induced by spot backgrounds in the cones are presented by the solid circles and those measured during annuiar backgrounds by the open circies.

NORMALIZED

STEADY

WVPERPOLARIZATION

Fig. 6. The relationship between log loss in apparent sensitivity (relative to the dark-adapted state) and the normalized steady hyperpolarization induced by background light of constant intensity (4.55 log quanta/sec/~m*) and different spatial patterns. The data points were obtained from 8 red cones (circles) and IZLl-horizontal cells (triangles). Sensitivity measurements were done with I10 pm diameter test gashed. Spot background illuminations (solid svmbolsj had diameter 120. ___, 2Otl _5M. 2RIKIum -_- ______of __ ___, __, 10211. ____, ----r----. -,------ I --Annularbackgrounds(opensymbols)had outer diameter of 28OOpm and inner diameter of 150, 530, 1030pm. The continuous line describes the data points obtained from the red cones.

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AVIRAN ITZHAKI and ILKIPERLMAN

The straight line through the red cone data is described by the following relationship i0g(s,p;j

=

4.449* (d.c./ V,,,,) + 0.046. (3)

Thus, with test flashes that illuminated almost the entire receptive fields of the red cones the dependency of sensitivity loss upon the normalized steady hyperpolarization was similar to the one calculated for large diameter test flashes and backgrounds [Fig. 2, equation (l)]. Figure 6 clearly demonstrates an apparent lack of dependency in the cones-horizontal cells interactions caused by changing the background pattern. In these test flash and background conditions the loss in apparent sensitivity of Ll-horizontal cells is not directly related to that of the red cones. Furthermore, while the red cone sensitivity to light depends on the steady hyperpolarization induced by the background light irrespective of its pattern. The horizontal cells are more desensitized by spot backgrounds compared to annuli regardless of the level of steady hyperpolarization induced by them. The data presented in Fig. 5 indicate that summation of adaptation in Ll-horizontal cells is limited to a retinal area with diameter of about 5OOpm. In order to further test the red cone-horizontal cell relationship a test flash illuminating retinal area of 470 pm diameter was used. This test flash size was chosen because

it illuminated the entire receptive field of the red cones (Baylor et al., 1971 Baylor and Hodgkin, 1973; Detwiler and Hodgkin, 1979) and most of the cones providing direct synaptic input to the horizontal cells (Leeper, 1978). In Fig. 7 sensitivity loss (above the dark-adapted level) measured in 14 red cones (A) and 16 Ll-horizontal cells (B) is plotted as a function of the normalized steady hyperpolarization induced by background illuminations of different patterns and intensities. The background patterns were spots with diameter of either 540 or 1020 or 2800 pm. The annular backgrounds had outer diameter of 2800 pm and inner diameter of either 530 or 1030,~m. The desensitizing effect of all the backgrounds on red cones [Fig. 7(A)] irrespective of intensity and spatial pattern (large spot: circles; small spot: triangles; annuli: diamonds) was directly related to the steady hyperpolarization induced by it. The straight line was drawn through the red cone data by linear regression. It is described by the following relationship log&,/S,) = 4.837* (d.c./V,,)

+ 0.015. (4)

The straight line describing the effect of the backgrounds on the red cone was redrawn in Fig. 7(B). If the Ll-horizontal cells just follow the red cones than the data points describing the desensitizing effects of the different backgrounds on horizontal cells should have clus-

20

.

/1

15 I

.

Fig. 7. The relationship between log loss in sensitivity (relative to the dark-adapted state) and the normalized steady hyperpolarization induced by background light of different intensities and spatial patterns. Sensitivity was measured with 470 pm diameter stimuli. (A) Data from 14 red cones measured during background illumination of large spot (2800 pm, open circles); small spot (540 or 1020pm, open triangles) or annulus (o.d. 28OOpm, i.d. 530 or 1030pm. open diamonds). The line was obtained with linear regression. (B) Data from 16 Ll-horizontal cells measured during the same background patterns as the red cones. Large spot backgrounds (open circles), small spot backgrounds (solid triangles) and annular ones (solid diamonds). The straight line describes the red cones data and was redrawn from A.

Background pattern and retinal adaptation

tered around this line. This was not the case. Backgrounds with annular pattern (solid diamonds) produced large hyperpolarizations and small sensitivity losses. While, backgrounds covering small retinal spots (solid triangles) produced small hyperpoiarizations and very large sensitivity losses. Only with large field backgrounds (open circles) the horizontal cells’ data clustered around the straight line. The data presented in Figs 6 and 7 indicated that no unique relationship between sensitivity loss and steady hyperpolarization could be derived for Ll-horizontal cells when the spatial pattern of the background was changed. Yet, for red cones sensitivity loss could be accurately predicted from the steady hyperpolarization irrespective of the background pattern. The red cones constitute the major input to Ll-horizontal cells in the retina of the turtle Pseudemys scripta elegans (Fuortes and Simon, 1974). Since the retina of the turtle Clemmys caspica used in this study is very similar to the one of the Pseudemys scripta elegans, the effects of backgrounds of constant intensity but different patterns were compared between these two cell types. The average effects of the background (log Z = 4.55 quanta/set/pm*) patterns on 12 red cones and i4 Li-horizontai ceiis are compared in Table 2. The sensitivity data included in Table 2 were obtained with test flashes of 470 pm diameter. For large field (2800 pm diameter) background the loss in sensitivity and the normalized steady hyperpolarization measured in the horizontal cells were similar to the corresponding values obtained from the red cones. During continuous illumination with the annular patterns the sensitivity loss in the horizontal cells followed that of the red cones but

693

the normalized steady hyperpolarization was considerably larger in the horizontal cells compared to the cones. With small field backgrounds the horizontal cells exhibited a larger loss in sensitivity and a smaller value for relative steady hyperpoiarization when compared to the red cones. DISCUSSION

The data presented in this study indicate that the light sensitivity of a single red cone during background illumination depends upon the adaptation state of its own outer segment as well as upon the state of adaptation of its neighbours (Figs 3 and 4). Similar lateral spread of background ----~~~~- ~~~adaptation in the cone network has been observed in the retina of the snapping turtle (Green and Copenhagen, 1984). Measurements of the light intensity, produced by the different background patterns, and comparing these measurements to the desensitizing effects of the same backgrounds on the cones indicated that stray light could not be solely responsible for the observed effects (Table 1). The size of retinal area over which summation of background adaptation was evident was similar to the receptive field for excitation of the cones .-. (Fig. 3). it is, therefore, possibie that both phenomena arise from the same retinal mechanism, namely the electrical coupling between adjacent cones (Baylor er al., 1971; Baylor and Hodgkin, 1973; Detwiler and Hodgkin, 1979). Thus, electrical coupling in the cone network may serve as a neural pathway responsible for the adaptation pool of the cone system defined psychophysically in humans (Rushton, 1965). The loss in sensitivity of red cones produced by any background configuration could be ac-

Table 2. The effects of the background pattern on normalized steady hyperpolarization sensitivity observed in red cones and Ll-horizontal cells Red cones

Ll-horizontal d.c./ V_ N

and loss in

cells

Background pattern

d.c./ I’,,,,.

N

Spot dia. 2800 pm Spot dia. 1020pm Spot dia. 54Oym Annulus o.d. 28OOpm i.d. 1030pm Annulus o.d. 28OOpm i.d. 530pm

0.267 f 0.062 0.255 f0.069 0.261 f 0.064

8 6 10

1.345 f 0.359 1.279f 0.377 1.274 f 0.234

0.288 f 0.044 0.193 f 0.028 0.118*0.050

9 7 13

1.399 f 0.235 1.779 * 0.330 1.830 * 0.300

0.074 f 0.036

9

0.324 f 0.159

0.191 f 0.014

9

0.355 f 0.188

0.079 * 0.040

9

0.424 * 0.105

0.248 f 0.025

9

0.503 f 0.168

log vs,

log &IS,

The background intensity was kept constant at 4.55 log quanta/set/pm’. Test flash diameter for sensitivity measurements was 470 pm. The total number of cells used to construct this table was 12 red cones and 14 horizontal cells. However, not all of these cells had complete data with all the different background patterns. The number of cells used to calculate each set of parameters is denoted in the columns marked N.

694

AWRAN ITZHAKIand IDO PERLMAN

predicted from the steady hyperpolarization induced by it, provided that no sibilant bleaching occurred (Figs 2, 4, 6, and 7). A similar relationship has been previously noted to exist for cones in the retinas of the turtle ~seudemys scripiff eiegans (Bayior ana Hodgkin, 1974) and of snapping turtle (Green and Copenhagen, 1984). Based on the data presented here, it can not be concluded that the steady membrane potential of the red cone directly determines its sensitivity to light. However, the loss in sensitivity observed during annular backgrounds indicates that membrane hyperpolarization induced mainly by current spread from adjacent cells may play a role in controlling light sensitivity of the impaled cell. The transduction mechanism from light absorption by the photopigment to a change in membrane potential involves a cascade of enzymes related to cyclic GMP metabolism (Liebman and Pugh, 1981; Stryer, 1986). It may be hypothesized that some of these enzymes are closely associated with the plasma membrane and their activity may be modified by the membrane potential of the cone outer segment. This hypothesis is supported by experiments done on the snapping turtle retina, where lateral spread of adaptation has been demonstrated in the _^^ _. cone network (Green and Copenhagen, IYL(4) but not in the rod network (Copenhagen and Green, 1985). In cones, the visual pigment is located on discs which are formed by infoldings of the plasma membrane while in the rods the discs are physically separated from the cell membrane. Therefore, a change in the potential of the plasma membrane may modify the transduction mechanism in the cones but not in the rods. The Ll-horizontal cells are also characterized by electrical coupling between adjacent cells (Yamada and Ishikawa, 1965; Naka and Rushton, 1967; Stell, 1967; Kaneko, 1971). However, unlike the situation in the cones, the horizontal cells did not exhibit lateral spread of background adaptation over the entire receptive field. Summation of adaptation was limited to a retinal area of about 500 pm in diameter (Fig. 5). This value is considerably smaller than the receptive field sizes (2200 to more than 2800 pm diameter) of the cells but is similar in magnitude to the size of retinal area containing all the red __..*n ilcl”‘U& hn‘r;rrr “IL+ZQI X--t JJL@l)rUi, ,....,...+:a &,“L,L4&13 ..n..tn,.,n I^_a b”II&J L” “‘E horizontal cell (Leeper, 1978). These findings suggest that the sensitivity to light of an horizontal cell is mainly determined by the adaptacurately

tion state of the cones within its dendritic field. In the Ll-horizontal cells no unique relationship between sensitivity loss and normalized steady hy~~ola~zation could be obtained. With large diameter test fIashes and back-~_-__.-~-*a.- I-_ *_-- :_. --__L.~-.2L_. _cI_..l__-*cI grounas me iog loss m sensluviry 01 norizontdcir cells was linearly related to the steady hyperpolarization in a manner similar to that found in the red cones (Fig. 2). However, when test flashes of small diameter were used (smaller than the receptive field size of the horizontal cells) an annular background induced a large steady hyperpolarization accompanied by a small loss in sensitivity, while, a small diameter spot of background elicited a small steady hyperpolarization with large loss in sensitivity (Figs 6 and 7). Thus, a clear distinction between the red cones and the Ll-horizontal cells was revealed when the spatial pattern of the background was changed. In the cones the sensitivity loss and steady hyperpolarization could not reveal information about the background’s pattern because they were directly related. In the Ll-horizontal cells annular and spot backgrounds could be accurately identified by compa~ng the loss in sensitivity to the steady hy~~la~zation. The data shown in Fig. 7(B) can be qualitatively expiained by the red cone’s data [Fig. 7(A)] considering the relative intensities of small spot, Iarge spot and annular backgrounds needed to elicit a given steady hyperpolarization in the horizontal cells. However, quantitative examination of the data obtained for a given background intensity shows that this trivial explanation does not hold (Table 2). Sensitivity loss in the horizontal cells followed that of the cones for large spot and annular backgrounds but was significantly larger than that of the cones when small spot background was applied. These observations are also represented in Fig. 5 where maximal sensitivity loss is obtained for background diameter of about 0.5 mm while further enlargements of the background diameter result in sensitivity increase despite further increase in the steady hyperpolarization of the cells. These findings are equivalent to the phenomenon termed “surround enhancement” which was described psychophysically for human cone vision ~W~theimer, 1967) and was also observed el~trophysiologically in horizonc-1 -I,, fm..,tL....l* iar VZI1S tIJu,nli~l”r, 10?A. I=,+, 1’r,E.,t: Ihu‘OrU,...A Ol‘UD.!..s-.... fG‘1111(111, 1984). The data presented here cannot be explained by amplification of horizontal cell’s photo-

Background pattern and retinal adaptation

responses due to su~ound illusion (Byzov et al., 1977). The loss in sensitivity measured in Ll-horizontal cells during large diameter (2800 pm) or annular backgrounds followed that of the red cones (Table 2). Furthermore, we couid not obtain increases in sensitivity reiative to the dark-adapted state even when very dim annular backgrounds were used. The data can neither be accounted for by the negative feedback pathway from peripheral horizontal cells onto the central cones. The involvement of this mechanism predicts a decrease in the cone steady hyperpolarization and an increase in its sensitivity to light when the feedback pathway is maximally activated with large spot background. This was not the case as illustrated in Figs 3,6 and 7 and in a previous report (Itzhaki and Perlman, 1984). It is still possible that the feedback from horizontal cells may modify the synaptic transmission from the red cones in a manner that will not manifest in the membrane potential of the cones but will modify the transmitter release mechanism. The difference described in Fig. 7 and Table 2 between red cones and Ll-horizontal cells indicate that the effects of the background spatial pattern may arise from mechanisms located in the horizontal cells layer. A plausible hypothesis assumes that the sensitivity to iight of horizontal cells during back~ound ill~ination is controlled by two mechanisms. The major determinant of sensitivity is the adaptation state of the cones which have direct synaptic input to the cell. However, it can be modified by changes in the effective coupling within the horizontal cell network. This latter mechanism may arise from the direct effect of the background light on the coupling resistance between adjacent cells or indirectly from changes in the membrane resistance of the horizontal cells induced by the different background patterns. The resistance of horizontal cells during background illumination is determined by a conductance decrease in the synaptic membrane and by a voltage dependent conductance increase in nonsynaptic membrane (Werblin, 1975; Byzov et al., 1977). Small and large spot backgrounds of the same intensity produce similar effects on the synaptic membrane of the horizontal cells because both illuminate most of the cones within the dendritic field of the cell. However, the small spot back____~__l t._>.._.. _-_-II__ _r__.-. L.._--___1-_1_ grouna inauces a smaller sreaay nyperpolanzation and hence a voltage dependent conductance increase of lesser magnitude than does the large spot background. The net result is a larger

695

membrane resistance of, the illuminated horizontal cells during small spot background compared to large spot one. Therefore, a larger shunting effect by the peripheral horizontal cells on the central ones, is expected to exist during smaii spot compared to large spot background illumination. Thus, the signal elicited by a small test flash in the central horizontal cells during small spot background spreads laterally more than during large diameter background and the sensitivity calculated is smaller than expected from that of the red cones. According to the above hypothesis the minimum in the sensitivity-background diameter relationship observed, in the turtle, for medium size background (Fig. 5) is due to larger than expected desensitization of the horizontal cells and not due to sensitivity enhancing mechanisms activated by surround illumination. The data presented in this study suggest a role for electrical coupling to light adaptation and information processing in the distal turtle retina. In the cone network, current spread from peripheral cells illuminated by the background light contribute to the flash sensitivity of the central cell. This mechanism prevents the cones from differentiating between different patterns of backgrounds which elicit similar steady h~~oia~zation. In the ii-horizontai ceii network the electrical coupling mainly contribute to processing of spatial information. The sensitivity of the Ll-horizontal cells is mainly dependent upon the direct synaptic input from the red cones. However, changes in the effective coupling between horizontal cells may lead to modulation of their response to the same input from the cones. As a result, the horizontal cells can differentiate between a spot and an annular background and can exhibit a phenomenon similar to surround enhancement. Acknowfedgement-This work was supported by the National Eye Institute grant EY-03423 USPHS to I. Perlman. REFERENTS

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