background levels control the graded response domain for the photoreceptors Berkeley, California 94720, U.S.A. than 100 to 1.

Transcription

1 J. Phyaiol. (1978), 278, pp With 12 text-figure8 Printed in Great Britain THE RESPONSE PROPERTIES OF THE STEADY ANTAGONISTIC SURROUND IN THE MUDPUPPY RETINA BY LARRY N. THIBOS* AND FRANK S. WERBLIN From the Department of Electrical Engineering and Computer Sciences and the Electronics Research Laboratory, University of California, Berkeley, California 94720, U.S.A. (Received 20 September 1976) SUMMARY 1. The graded response of bipolar and ganglion cells to test flashes at the receptive field centre, spans only a limited portion of the test intensity domain: more than 90 % of the graded response range can be elicited by test flashes differing by less than 100 to In the presence of steady illumination of the receptive field surround, the absolute levels of log test intensities required to elicit 90 % of the graded response are increased (reset), but the relation in (1) still applies. 3. Each point in the receptive field surround, when illuminated, contributes to the resetting of the required centre test flash intensities by a weighting that decreases exponentially with distance from the centre. The space constant is 0-25 mm. 4. When the receptive field surround is fully covered with illumination, the centre test flash intensities required to elicit 90 % of the response range must be increased by about tenfold for each tenfold increase in surround intensity over a surround intensity domain of about 1000 to The absolute levels of surround and required centre test intensities are interrelated: when the receptive field surround is fully covered, a test flash with intensity equal to that of the surround elicits a half-maximal response. Thus, in the presence of a full field background, the bipolar potential is held near its half-maximum response potential. 6. The graded resetting of the required centre test flash intensities is well correlated with the graded increase in horizontal cell response as the surround intensity and area are varied. It is inferred that units with response and receptive field properties like those of the horizontal cells, when driven by surround illumination, act as interneurones to reset the relationship between required test flash intensity and response in bipolar and ganglion cells. INTRODUCTION Earlier studies in the retina of the mudpuppy have shown that there are at least three sites at which the sensitivity of the retinal output can be modified. Steady background levels control the graded response domain for the photoreceptors * Present address: Department of Physiology, John Curtin School of Medical Research, Canberra, Australia.

2 80 L. N. THIBOS AND F. S. WERBLIN through adaptive mechanisms within the photoreceptors themselves (Normann & Werblin, 1974). Then lateral interactions at the outer plexiform layer, presumably mediated by horizontal cells, elicited by steady annular illumination at the receptive field surround, control the sensitivity and entire response domain for the bipolar and more proximal cells to illumination at the receptive field centre (Werblin, 1974). Finally, lateral interactions at the inner plexiform layer, presumably mediated by amacrine cells and elicited by moving targets in the receptive field surround, control the sensitivity and response range of the ganglion cells to illumination at the receptive field centre (Werblin & Copenhagen, 1974). Other studies in a variety of animals are consistent with the findings in mudpuppy. Fain (1976) has shown that steady background levels control the response domain and sensitivity of rods in bufo marines. Kleinschmidt & Dowling (1975) showed that background illumination controls the response domain for photoreceptors in Gecko. Proximal to the photoreceptors, Green, Dowling, Siegel & Ripps (1975) have shown that background illumination which does not affect the photoreceptors in skate can alter the response domain for more proximal cells through 'network adaptation.' Miller & Dacheux (1976) have shown that horizontal cells are required to mediate the antagonistic surround in mudpuppy. Naka (1977) has characterized antagonistic interactions between horizontal and bipolar cells in fish which are consistent with the studies in the mudpuppy. This paper focuses upon the second level of response domain control: it is a study of the graded influence of steady surround illumination on the response domain of the bipolar and ganglion cells to test flashes at the receptive field centre. We reasoned that if steady surround illumination activates lateral interactions at the outer plexiform layer mediated by horizontal cells, then the graded antagonism measured in bipolar and ganglion cells should be closely correlated with the graded response of the horizontal cells. The results indicate a close correlation between lateral antagonism and horizontal cell activity. In the following paper (Thibos & Werblin, 1978) the graded properties of lateral interactions at the inner plexiform layer elicited by moving surround targets have been studied and correlated with the response properties of the amacrine cells. METHODS Recording Intracellular records were obtained using glass micropipettes described previously (Werblin, 1974). Extracellular ganglion cell recordings were obtained with insulated etched tungsten electrodes (Hubel, 1957). Electrode signals were amplified in a conventional manner and recorded on magnetic tape for later playback onto either a stripchart recorder or storage oscilloscope. A grounded Faraday cage was used to shield the preparation. Opticalstimulator Two independent light channels were used, one to present centre test flashes and one to provide surround illumination. Each channel was equipped with electronically controlled mechanical shutters with rise and fall times less than one msec. Light intensity was controlled separately in each channel by calibrated neutral density filters. Centre spots and surround annuli were formed on the retina by placing the appropriate aperture or opaque mask in that plane of the optical stimulator conjugate to the retina. Light from the two channels was combined with a beam splitter and directed into the Faraday cage by mirrors. Final imaging was

3 STEADY SURROUND ANTAGONISM done with a lens inside the cage. Optical alignment prior to the beam splitter ensured accurate centring of the test spot relative to the annular surround while fine adjustment of the mirrors after the beam splitter permitted alignment of the projected light patterns with the microelectrode. Light calibration The light intensity units used in this report refer to the amount of attenuation by neutral density filters of the tungsten source. Without neutral filters, i.e. zero log unit intensity, the total irradiance over the spectral band 400 nm-1000 nm was 7*5 mw/mm2 at the retina (United Detector Technology Optometer). The two light channels were calibrated separately and balanced using neutral density filters. The colour temperature of the tungsten was 2800 'K. The following calculation based on standard photometric principles (LeGrand, 1968) indicated that a -7 log unit intensity test flash delivered the equivalent of approximately 2 photons per rod per sec. The spectral emission curve for the tungsten (assuming black body radiation) was weighted by the spectral sensitivity curve for Necturuw rods (Fain & Dowling, 1973) and integrated over wavelength. The result was then normalized by the energy of a single quantum at 522 nm, (the wavelength of peak sensitivity) and corrected for rod packing density (estimated from scanning electron photomicrographs taken at 2800 magnification to be 6 x 103 rods/mm2). Protocol Animals were maintained in a dark temperature-controlled bath. The eye was excised, placed in a moist chamber, the cornea and lens removed, the vitreous humour drained and moist 02 passed over the preparation. All experiments were performed at room temperature in a light-tight Faraday cage without ambient illumination. After dissection the optical stimulator was brought into focus using a crosshair pattern. The electrode was lowered onto the vitreal side of the eyecup using a hydraulic microdrive. The stimulator was aligned with the electrode using a small spot of light on the peripheral retina to avoid adaptation of the central retina. At the beginning of the experiment the eyecup chamber was moved so as to place the electrode in the centre of the retina. Electronic circuits automatically controlled the sequence of light stimulation. Identification of cell type was accomplished using micro-electrode depth and time course of response to spots, annuli, windmills, and full field flashes (Werblin & Dowling, 1969; Werblin, 1974; Werblin & Copenhagen, 1974). Curve fitting Experimental data were fitted by the empirical expression described in Results using the following technique. Data were replotted on axes which were transformed in such a way that the model predicted a straight line. Then, if the data fell on a straight line it was inferred that the model fitted the data. This procedure was used because it permitted the use of standard linear regression techniques to determine model parameters and for testing the ability of the model to fit the data. The model to be proposed in Results has the form R = Rr"X IN/(IN + a.n) which in terms of log I is (1) R = 0 5 Rma,1[I +tanh 1 15 N (log I-log a)]. (2) The terms of this formula are response magnitude (R), maximum response (Rmax), intensity of the centre test flash (I), the intensity which elicits a half-maximal response (C-), and a constant (N). The graph of eqn. (2) is an S-shaped curve with a steepness that depends upon N and position along the log intensity axis that depends upon o-. The non-linear relation between R and log I may be linearized for regression purposes by transforming the variable R by the function g(r) defined by g(r) = tanh-1 (2R/Rma-1). (3) To verify that g(r) is linear with log I, apply (3) to (2) to give g(r) = I- I 5N log I 1-15N I og o-. (4) 81

4 82 L. N. THIBOS AND F. S. WVERBLIN Accordingly, to fit the experimental data with the model of (2), the measured responses were transformed by g(r) and plotted against log I. (To execute the transformation, Rmxwas determined by inspection of the raw data.) A least-squares regression of the transformed data was done by computer and the regression slope and intercept yielded values of parameters N andcr. Standard statistical tests of linearity were used routinely to ensure that the model adequately described the data. A second transformation of the data allowed a check on the value of Rmax used in the above regression procedure. If the logi variable is transformed by f(logi) = tanh1 15N(log I-log cr) (5) then eqn. (2) takes on the linear form R =0O5 RMax (1 +f(log I)). (6) Estimates of the parameters o and N were obtained from the first regression and then used to calculate f(logi). A regression of R against f(logi) yielded an estimate of R.,.. which was checked against the value determined previously by inspection of the raw data. The intensity span of a cell will be defined as the span of intensities, in log units, which corresponds to the response range from 005 R.. to 0-95 Rmax. A useful formula for the intensity span may be derived by substituting these values of R into eqn. (3) to give g(r)= -147 and g(r) = , respectively. Using these values in eqn. (4) gives Intensity span = log log '0.05 = 2-56/N. (7) RESULTS Effect of surround illumination on ganglion cells Graded response function of the receptive field centre The response of ganglion cells to test flashes at the receptive field centre is smoothly graded over a limited domain of log intensity, but the absolute domain is controlled by the level of fixed surround illumination. This study first defines how response magnitude is graded with intensity of test illumination at the centre of the receptive field. Then the effect of surround illumination on this response function is determined by presenting centre test flashes in the presence of fixed concentric annuli of graded size and intensity. The centre intensity-response function was measured by flashing spots of light, 0 4 mm in diameter, at the centre of the ganglion cell receptive field. Test flashes had sec duration, and were presented every 15 sec in a randomized series of intensities. a 1 The spot size was chosen to match the estimated size of the receptive field centre in mudpuppy (Werblin, 1970; Burkhardt, 1974) and the flash duration was selected to separate on and off responses unambiguously. Ganglion cell spikes were counted for 1 sec following the onset or termination of illumination. No background illumination was present so the responses began at scotopic levels (Normann & Werblin, 1974). Ganglion cells rarely showed spontaneous activity. In mudpuppy there appear to be two distinct classes of ganglion cells. Sustained cells generate a maintained discharge for the duration of centre illumination. Transient cells respond with a brief burst of activity at on and at off (Werblin & Dowling, 1969). The centre intensity-response function is different for the two classes as shown in Fig. 1. The span of log intensities which elicit graded responses is narrower for the transient than for the sustained cell, consistent with earlier work (Werblin & Copenhagen, 1974). The data in Fig. 1 were fitted by the method of least squares to the formula given

5 STEADY SURROUND ANTAGONISM 83 in eqn. 1 where the exponent, N, is a measure of the steepness of the curve on log I coordinates (see Methods). Eqn. (1) above is intended only as an empirical description of the data. It will be used in this paper to identify the interactions between cells, not to make inferences about the underlying synaptic mechanisms. The data for the cells in Fig. 1 are best fitted by eqn. (1) when N = Ps7 for the sustained cell and N = 3*4 for the transient cell. The response rises from 5 to 95 % A, 10 O) Co. 5 Co Log /centre 0n./ 0Co B Log /centre Fig. 1. Inltensity--response functions for a sustained (A) and a transient (B) ganglion cell. Both sets of data are fitted by the solid curves, which represent eqn. (1) with N = I1 7 in A and N = 3.4 in B. Response is spike count in a 1 see period following the test flash. Error bars are ± I s.e. with three or more response, determinations.

6 84 L. N. THIBOS AND F. S. WERBLIN of maximum in 1X5 log units for the sustained cell and 0-8 log units for the transient cell. Table 1 gives additional measurements of the intensity span for other ganglion cells. There was no consistent difference in the absolute intensity which elicited half maximal response for the two cell types. TABLE 1. Comparison of intensity spans of retinal cells. The parameter N was determined from intensity-response data by least-squares regression as described in Methods. The span of intensities which elicits graded responses from 5 to 95 % of maximum equals 2.56/N log units Cell type Bipolar Horizontal Ganglion Response type Depolarizing Depolarizing Hyperpolarizing Hyperpolarizing Hyperpolarizing Hyperpolarizing Hyperpolarizing Average Average Sustained on Sustained on Sustained on Sustained off Sustained off Average Transient on Transient on Transient on Transient on Transient off Transient off Average N Intensity span (log units) Graded effect of increasing surround intensity The effect of surround illumination on the centre response function was determined by presenting an annulus of fixed intensity and area, then measuring the graded response to centre test flashes in its presence. The surround annulus had 1 0 mm i.d. and 2-0 mm o.d., dimensions which were selected so that scatter into the 0-4 mm centre of the receptive field was minimized, while keeping the annulus within the boundaries of the 3 mm diameter retina. The surround illumination was presented for 7 sec. The 0-4 mm diameter centre test flash was presented for 1 sec starting 3 see after the surround appeared. This sequence was repeated every 15 see until the complete range of graded responses to centre flashes had been measured

7 STEADY SURROUND ANTAGONISM 85 It is shown below that surround illumination caused the intensity-response curves for the ganglion cells to shift to the right along the log intensity axis. We will claim that the shift is due to neurally-mediated interactions in the retina rather than to light scattered to the centre of the receptive field from the surround. The argument is based upon this observation: when the receptive field centre was dimly illuminated to simulate scatter from the surround, threshold was affected only if the centre illumination itself elicited a response. In the experiments below, surround illumination markedly affected threshold, but never elicited a response. Further support for neurally-mediated interaction initiated by surround illumination is given below in studies of the bipolar cells. There, surround illumination caused the membrane to polarize in a direction opposite to that of the centre response Ar _ ~ ~ S0A A (A)~~~~~~~~~~ 0)0 0u) cc ~~~~~ % (Apos daaotie ntedrk (fle cice)wr itdbyts q.0 )wt Log /cm Fig. 2. Effect of annular surrounds on the intensity-response function of a sustained 'on' ganglion cell. Log intensity of the surround is noted next to each curve. The response data obtained in the dark filledd circles) were fitted by test eqn. (1) with N = 1-2. This curve was then shifted to make least-squares fits with the remaining data sets. The curves shift about 1 log unit to the right for each log unit increase in annular illumination. Both transient and sustained ganglion cells were affected in the same way by the surround illumination. An example taken from a sustained cell is shown in Fig. 2. The intensity-response curve obtained in the dark (no surround) is shown by the filled circles which are best fitted by eqn. (1) when N = 1-2. The centre intensityresponse curve determined when a -3 log unit annulus was present is shown by the open circles in Fig. 2. The response curves obtained with a -3 log unit annulus may be described by the same template curve that describes responses in the dark when the template is shifted laterally along the log intensity axis. By fitting the response data with this shifted template, the effect of surround illumination can be characterized by the single parameter oc in eqn. (1). This measure is only valid for surround intensities below about -3 log units because, as shown below, the curves shift vertically as well as laterally for surrounds more intense than -3 log units. Data from five other

8 86 L. N. THIBOS AND F. S. WERBLIN sustained and eight transient cells, both for on and off responses, confirmed the result that for relatively dim surrounds the centre response function is translated only laterally along the log intensity axis, in agreement with earlier work in mudpuppy (Werblin & Copenhagen, 1974). When relatively bright surround annuli were used (greater than about -3 log units) the curves shifted in a complex way: the curves continued to shift laterally but the range of graded responses was also reduced as shown by the remaining data sets of Fig. 2. For the higher surround intensities the response curves could be fitted by the template, provided it was shifted vertically as well as laterally. For the most intense annulus used (zero log units) the vertical shift was greatest and the maximum response that could be elicited was only about half that which could be elicited in the dark. This reduction in the range of graded responses by bright annular surrounds was found for other sustained and transient ganglion cells and frequent checks with no surround indicated this effect was not due to long-term loss of responsiveness. No attempt was made to analyse separately the rod and cone contribution to the response curves of Fig. 2. The data of Fig. 2 describe the effect of steady surround illumination on the centre response over very broad limits of surround intensity. The results from other cells, both sustained and transient, were qualitatively similar although the absolute position of the graded centre response curve for different surround intensities varied from cell to cell. The variability might be due to inputs from different receptor populations (rods or cones) or differences in absolute threshold of individual preparations. In this report emphasis is placed on relative changes rather than absolute light levels in intensity-response curves. For most of these studies the centre response curves shifted laterally by about 0O5 to 1.0 log units for each log unit increased in surround intensity and retained their shape regardless of absolute intensity level. In the cat, Enroth-Cugell & Lennie (1975) have reported that the effect of steady surrounds is to shift the centre response function downward rather than laterally, but those authors measured only a limited portion of the response curves, excluding both high and low centre intensities. Under those conditions a lateral shift of full response curve can be interpreted as a downward shift. Therefore, there is insufficient data at present to say whether cat and mudpuppy cells are affected in the same way by steady surround illumination. Change of threshold with annulus area and intensity The annulus outer diameter was fixed at 2 1 mm and the i.d. was gradually reduced from 1P5 to 0-4 mm to permit extrapolation of the results to include the effect of full field background illumination. This scheme for increasing the area of annular stimulation was selected in an effort to restrict the effect of the surround stimulus to lateral interactions, uncontaminated by the effect of steady illumination of the receptive field centre. Threshold was taken to be the value of test flash intensity that gave a response on one of three trials. Thresholds for centre test flashes were determined in the same cell for several different combinations of annulus intensity and i.d. and the results are plotted as a function of intensity in Fig. 3A and again as a function of i.d. in Fig. 3B. The antagonistic effect of surround illumination was found to increase with both area

9 STEADY SURROUND ANTAGONISM 87 A / / / -3 -o -5 Co w 0) 0) 0 -j / / / /, 7/ o Log /surround On 0) 0) 0) 0j b 0) 0 -j Inside diameter (mm) Fig. 3. The effect of annulus size and intensity on the ganglion cell intensity-response function. The same data are plotted in two different ways. In A the abscissa is annulus log intensity. The annulus inside diameter (mm) is noted next to each set of data. In B the abscissa is inside diameter. The annulus log intensity is noted next to each set of data. The left ordinate is threshold intensity and right ordinate is log a, the intensity which elicits half-maximal response. The dotted line in A has the eqn. log a = log IsoundI -6

10 88 L. N. THIBOS AND F. S. WERBLIN and intensity. Different combinations of annulus parameters can lead to the same level of ganglion cell threshold. For these relatively dim annuli, threshold changed about 1 log unit for every log unit change in surround intensity, independent of annulus area. This is similar to the graded surround effect for more intense annuli (Fig. 2). The data of Fig. 3B indicate that threshold increases at a rate of between 0-5 and 1 0 log units for each 0 5 mm reduction in the annulus inner diameter, independent of intensity, suggesting that the annuli with larger inner diameters did not fill the entire receptive field surround. When the inner diameter was reduced, more of the receptive field surround was covered, causing greater ganglion cell antagonism ~ ~ ~ ~~~~~~0 0 I -20 C E Log 'centre Fig. 4. Intensity-response function for a bipolar cell. The ordinate is membrane potential at response plateau, abscissa is log test intensity. The solid curve is a leastsquares fit of text eqn. (1), with N = 1-2, In this experiment only surround intensities less than -3 log units were used. As noted above, under this condition the centre response curves would be expected to shift only laterally and so log ao was always 08 log units greater than log threshold. Using this relationship between log threshold and log o', the data in Fig. 3 shows that when the i.d. of the annulus approached zero, the value of log or approached the value of log surround intensity. Extrapolation from these data suggest that when the entire receptive field surround is illuminated, a test flash of intensity equal to that of the surround will elicit a half-maximal response in the ganglion cell. It is shown below that full field illumination will polarize the bipolar cell to a potential level at the half-maximal response. Effect of surround illumination in bipolar cells Graded antagonism in bipolar cells It has been previously suggested that the steady surround antagonism measured in ganglion cells is mediated by lateral interactions at the outer plexiform layer

11 STEADY SURROUND ANTAGONISM 89 (Werblin, 1974; Naka, 1977). If this is the case, bipolar cells should be affected by steady surround illumination in the same way as the ganglion cells described above. Experiments designed to test this notion are described below. They measure the graded effect of surround intensity and area upon the graded bipolar cell response. Both on-centre (depolarizing) and off-centre (hyperpolarizing) bipolars were studied. There was no measurable difference in their response properties as a function of centre and surround illumination. -17 > = -18 C 0 C co.o-19 E I-P^ Log Icentre Fig. 5. The effect of a steady annular surround on the bipolar intensity-response function. Filled circles are for no surround; open circles are for a -4 log unit annular surround. Text eqn. (8) was fitted to the filled circles and then translated laterally along the abscissa to fit the open circles. Bipolar cell receptive field centre intensity-response function The intensity-response function for bipolar cells was measured with a 04 mm spot at the receptive field centre flashed for 1 see every 7 see in a randomized series of intensities. The test spot diameter was chosen to stimulate maximally the centre of the receptive field and its duration permitted measurement of the initial transient and steady plateau of the response. When only the centre of the receptive field of a mudpuppy bipolar was illuminated, the response showed little overshoot; it reached a steady plateau level within about 100 msec (Werblin, 1974). This plateau level is plotted against log intensity in Fig. 4. The data points are best fitted by the method of least squares to the formula V = Vdark + Vmax JN/(IN + 0.N) (8) when N = 1-2. This value of N was the average for seven bipolar cells studied in detail (see Table 1). Thus the bipolar cells and the sustained ganglion cells (Fig. 1 A) have approximately the same intensity span. Graded effect of increasing surround intensity on the bipolar response The following expts. were designed to test whether steady surround illumination results in a repositioning of the intensity-response curve of the bipolar cell similar to

12 90 L. N. THIBOS AND F. S. WERBLIN that shown above (Fig. 2) for ganglion cells. The 0 4 mm test spot was presented at the centre of the bipolar cell receptive field every 7 see in a randomized series of intensities in the presence of various fixed levels of surround illumination. The surround was an annulus with 1P0 mm i.d. and 2-0 mm o.d., presented 2 see before the centre test spot. Fig. 5 shows the effect of a -4 log unit annular surround upon the graded centre response of a depolarizing bipolar cell. The annulus alone elicited no response, but when it was present, the intensity required to elicit a given centre response magnitude was increased. The effect of the annulus could be described by shifting the template for the centre response curve laterally by 0-7 log units along the abscissa, consistent -15- E A~~~~~~~ acl-20 Dak--3-0~~~~~~~~~~~~~~~~ C E -25 * Log /centre Fig. 6. Graded effect of surrounds on the bipolar intensity-response function. Surround log intensity is indicated next to each curve. Text eqn. (8) was fitted to the response data determined in the dark and then shifted to make least-squares fits with the sets of data at each surround level. with earlier results (Werblin, 1974). The lack of response to the annulus alone indicates that the lateral shift of the intensity-response curve was probably due primarily to neurally-mediated interactions rather than to light scattered onto the receptive field centre (Werblin, 1974). A similar experiment which shows antagonism graded with surround intensity is shown in Fig. 6. Limited recording time in bipolar cells precluded a detailed determination of the response function at each annulus intensity. Detailed studies of other cells indicated that the average value of N for the dark curves was 1 2 (see Table 1) and the corresponding template was fitted to the data of Fig. 6. Responses determined at each surround intensity indicate that the data points could be fitted by this template shifted laterally by between 0 6 and I 0 log units for each log unit increase in surround intensity. The relation between surround intensity and lateral shift of the intensityresponse curves is similar to that for the ganglion cells shown in Fig. 2.

13 STEADY SURROUND ANTAGONISM 91 Graded effect of surround area upon the centre response function The o.d. of the surround annulus was fixed at 20 mm and the i.d. was varied to assess the effect of surround area on the bipolar response curve. The response curves shown in Fig. 7 were obtained by the same procedure used in the previous experiments. As the annular area was increased, the centre intensity-response curve was shifted laterally to the right. The largest annulus (05 mm) filled the entire retina except the receptive field centre and in this case log or and log surround intensity were about equal. This suggests that the full-field response of the bipolar cell would be about midway between its dark and maximum potential levels as shown in previous experiments (Werblin, 1974). -13 E C W~~~~~~~~~ o 0. ~~~~~~0 A C0 I-10 Dark 1i E /surround Log / centre Fig. 7. Bipolar intensity-response function for different sized annular surrounds. Response functions were determined in the dark and with surround annuli of 1-0 mm and 0-5 mm i~d., as indicated next to each curve. The o.d. was fixed at 2.0 mm. For the surround with the largest area (0.5 mm i.d.) a response about half-maximal was evoked by a test flash with intensity equal to the surround intensity (shown by arrow). These results indicate that the portion of the full field background which falls within the receptive field surround positions the response function along the abscissa, so that the mid point of the curve is at the background intensity. The area of the background illumination which falls within the receptive field centre then elicits a steady half-maximal response. Therefore lateral interactions at the outer plexiform layer reposition the graded bipolar response so that it is kept from compressing against its response potential limits. The bipolar cell is then appropriately set for optimally signalling small changes above and below background, regardless of the absolute level of illumination. Effect of surround illumination in horizontal cells The annular backgrounds used in most of the above experiments had 1*0 mm i.d., which is larger than the estimated dendritic spread of bipolar or ganglion cells (Dowling & Werblin, 1969). Therefore lateral antagonistic activity elicited by surround

14 92 L. N. THIBOS AND F. S. WERBLIN illumination affects bipolars via lateral interneurones, probably the horizontal cells (Werblin and Dowling, 1969; Werblin, 1974). Therefore, horizontal cell activity might be related to the antagonism measured above for bipolar and ganglion cells. To test this, the response characteristics of horizontal cells were studied under conditions similar to those used for bipolar and ganglion cells in the following experiments. Intensity-response function Horizontal cells were stimulated by annular illumination of 1 0 mm i.d. and 2-0 mm o.d. placed concentric to and centred upon the receptive field, the same configuration used in the previous experiments. No centre illumination was present. Fig. 8 shows the curve of plateau responses to a series of 3 see flashes of the annulus presented in a random sequence plotted against stimulus intensities. The curve through these data is the best fit of eqn. (8). (In this case, the I in the eqn. would be annulus intensity.) The value of N is 07, indicating the intensity span of the cell is 3-7 log units, about the same as the average of other horizontal cells studied (see Table 1) E -40 CL I L / -30 lll Log 'surround Fig. 8. Intensity-response function for a horizontal cell. The ordinate is the plateau level of polarization due to annular stimuli and the abscissa is annulus log intensity. The solid curve is the best fit of text eqn. (8), with N = 0-7. Graded antagonism with annulus size and intensity The variation of horizontal cell response with both size and intensity was investigated using an annulus with 2-0 mm o.d. and variable i.d. The annulus intensity and i.d. were independently varied and chosen to match the values used in the bipolar and ganglion cell experiments described above. The measured plateau responses are presented as a function of intensity in Fig. 9A and again as a function of inner diameter in Fig. 9B. Responses were found to increase with both stimulus size and intensity, indicating that horizontal cells summate over most of the area of the retina, consistent with earlier results (Werblin, 1974). In an earlier study Werblin (1970) showed that horizontal cell responses to a small spot of light could be described by the equation. R = A exp(- xlxo), (9)

15 STEADY SURROUND ANTAGONISM 93 where x is the distance in mm between a small spot stimulus and the receptive field centre, x0 is a space constant equal to 0-25 mm, and A is an arbitrary constant. Under the assumption that the response to a disk of light is the integral of this weighting function over the region of the disk, measured horizontal cell responses and the reduction of bipolar responses by a disk background of increasing diameter were well described (Werblin, 1974). To test this assumption further, the weighting function of eqn. (9) was integrated (see Methods) over the region of the annuli used in the present experiments and the results are shown by the dotted curves in Fig. 9B. These theoretical curves make a reasonable fit to the data with the exception that the responses for the -3 log unit surround rise less steeply than predicted. -50 B -45 A ' - a, 0 0 ȧor.0 *N -.o -40 E 1.0 L ' Q o -\\ -6, -30 ~~~~~-30-7 AP-~~~~~~~~~~' * Log /surround Inside diameter (mm) Fig. 9. Horizontal cell response to annuli of different sizes and intensities. The ordinate is plateau level of polarization due to annular stimuli. A, data plotted as a function of intensity with annulus i.d. (mm) indicated next to each set of data. B, the same data plotted as a function of i.d., with log intensity noted next to each set of data. Dotted curves in B are predictions based on text eqn. (9).

16 94 L. N. THIBOS AND F. S. WERBLIN DISCUSSION Role for horizontal cells in lateral antagonism This study was begun with the notion that lateral interactions, initiated by steady surrounds, are mediated exclusively by horizontal cells at the outer plexiform layer of the retina. For any centre test flash with intensity within the graded response range of the bipolars, the antagonistic surround acts to reduce the response. Over the full range of graded response to centre test flash intensities, the S-shaped graded response curve is shifted bodily to the right along the log intensity axis by surround illumination. This has been shown qualitatively in an earlier report (Werblin, 1974), and an attempt was made here to acquire enough data to define the graded properties of surround antagonism. The graded effect of surround illumination is shown for the horizontal, bipolar and ganglion cells in Fig. 10A. The shift of the graded intensity-response curves, measured in terms of log oc are shown for the bipolar and ganglion cells; the response itself is plotted for the horizontal cells. These data are replotted from Fig. 2 for the ganglion cell, from Fig. 6 for the bipolar cell, and from Fig. 8 for the horizontal cell. The coincidence of the curves shows that the curve shift for the bipolar and ganglion cells, and the response potential for the horizontal cells span the same domain of intensity, and that the S-shaped curve relating annular intensity to shift or polarization is of roughly the same form. It is therefore reasonable, although not certain, that lateral interactions mediated by horizontal cells are responsible for the shifting of the response curves. The exclusive role of the horizontal cells in mediating the antagonistic surround is supported by the relationship of the curves shown in Fig. 10B. Here is plotted the graded increase in the strength of the antagonism as a function of increasing area (decreasing i.d.) of the annulus. The data are taken from Figs. 3A, 7 and 9A for the ganglion, bipolar, and horizontal cells, respectively. The coincidence of the shift of the bipolar and ganglion cell curves with the increase in potential of the horizontal cells suggests that cells with receptive field properties similar to those of the horizontal cells probably mediate lateral interactions initiated with steady annular surrounds. One objection to this assertion, might be that there is a population of sustained amacrine cells (Naka, 1977) acting at the inner plexiform layer that also affects the position of the bipolar and ganglion cell response curves. On rare occasions recordings have been made from cells resembling the sustained amacrines in the mudpuppy, but they appear to have an extremely steep graded intensity-response function spanning less than 1 log unit along the log intensity domain and have antagonistic receptive fields, unlike those of horizontal cells. Another objection might be that the measure of lateral interaction in terms of the shift of the intensity-response curves was chosen arbitrarily, and that an equally good measure might be the decrement in response measured in the bipolar and ganglion cells. We have measured the decrement in bipolar activity as a function of surround intensity, and the graded effect follows a curve similar to that plotted in Fig. IOA. Taken together, the curves in Fig. 10 suggest that cells with graded intensityresponse functions and receptive field properties similar to those of the horizontal

17 STEADY SURROUND ANTAGONISM 95 cells might mediate the lateral interactions described in this paper. The following sections derive a transfer function for receptor-to-bipolar activity and show how it is modified by lateral interactions. Finally, a mechanism for the antagonistic interactions is inferred from the results h A -2 Ẹ-I a, 0 a) 0 C o Cu I -35 I- -30 Ganglion. I Horizontal I I I Log annular intensity b 0 -J B E Z en CL 0o 0N en m Bipolar I Ganglion Horizontal No surround -5 b 0) 0 -j I Annular inside diameter (mm) Fig. 10. Lateral antagonism and horizontal cell response as a function of annular intensity (A) and area (B). Antagonism is measured in terms of the shift in the position of the response curves along the log intensity axis, given by the value of log a. A, increase in antagonism in bipolar and ganglion cells correlated with the increase in response in horizontal cells as annular intensity is increased; annulus dimensions: 2 mm outside; 1 mm inside. The curves show a similar increase in antagonism and response spanning the same domain of log intensity. B, increase in antagonism and horizontal cell response with increase in annular area (decrease in i.d.); outside diameter was 2-2 mm, abscissa shows inside diameter; annular intensity was -4 log units for the bipolar cell, but -3 log units for ganglion and horizontal cells; the bipolar curve has been shifted from -4 to -3 log units for comparison with the other curves. The curves in A and B indicate that the strength of lateral antagonism, as a function of annular intensity (A) or areas (B) has properties similar to the response of horizontal cells under the same conditions. I

18 96 L. N. THIBOS AND F. S. WERBLIN Approximation of the transfer curves relating receptor to bipolar response The responses of the receptor and bipolar cells can be correlated for each stimulus level to approximate the transfer curve relating steady receptor and bipolar cell potentials for each level of centre and surround illumination. A graphical method for deriving the transfer curve is shown in Fig. 11. It has been assumed here that, for the lowest light levels used, only the rods contribute to the change in synaptic input Bipolar potential Log Icentre Fig. 11. Graphical approximation of receptor-to-bipolar cell transfer function in the presence of various surround intensities. Quadrant 3 (Q3) shows the rod response as a function of log intensity; Q2 shows the bipolar responses, derived in Fig. 6, as a function of log intensity for four different surround levels. The bipolar potential is plotted against rod potential in Q I by graphically eliminating the common intensity axis. The transfer function in QI is non-linear, and is shifted bodily to the left along the receptor potential axis for increasing levels of fixed surround illumination. to the bipolars. At higher levels of stimulus intensity the cones may also contribute (Fain, 1975) to the response, but this derivation is based primarily upon activity at the lower light levels. Quadrant 3 in Fig. 11 contains the intensity-response curve for mudpuppy rods measured by Normann, & Werblin (1974). It has the form of Eqn. 8 with N = 0-7. The curves in quadrant 2 are based upon the measured bipolar responses in Fig. 6. They also obey the form of eqn. 8, but with N = 1-2. The curves appear to be shifted to the right (Fig. 6) with increasing surround intensity at low light levels.

19 STEADY SURROUND ANTAGONISM 97 Quadrant 1 shows the transfer curves relating rod to bipolar potential, derived by graphically eliminating the common intensity axes. For example, at a test flash intensity of -4 log units, the rod response is about half-maximal, and the bipolar response in the presence of a -3 log unit surround is given by the solid triangle about J up along the dotted curve. The vertical and horizontal lines in the figures show how the point represented by the solid triangle in Q1 was derived from these two responses at -4 log unit test intensity. Since recordings were not taken from bipolars and rods simultaneously, the relative position of the bipolar curves with respect to the rod curve used here is based upon the assumption that the position of the intensity response curve for all rods in the local population of rods driving the bipolar is the same, and that the test flash intensities which elicit just noticeable responses in rods also elicit just noticeable responses in bipolar cells (see Werblin (1971) for simultaneous measurements). -35 E Cu.Z -37 CI -17 -B Dark Receptor potential (mv) Fig. 12. Rod-bipolar transfer function. Curves extracted from Fig. 11 included approximate potentials for rod and bipolar responses derived in other experiments (see text). Bipolar response is an accelerating function of rod potential increasing tenfold for each 8 mv rod depolarization. A 1 log unit increase in surround illumination requires light-elicited rod response to increase by about 5 mv to maintain the same bipolar potential. Each of the points in Q2 were translated to Q2 by the method described above. They were then fitted by a transfer curve of the form labelled 'dark' by translating this curve to the left along the rod potential axis. The good fit of this template to the points indicates that the surround seems to leave the rod-bipolar transfer curve intact, but as the surround intensity is increased there is a concomitant increase in the light-elicited rod potential required to elicit a given bipolar potential level. These findings are consistent with a mechanism of lateral antagonism whereby the relationship between light-elicited rod response and the signal transmitted by the rod is 4 PHY 278

20 98 L. N. THIBOS AND F. S. WERBLIN reset with surround illumination. A possible site for such interaction would be at the receptor terminal where horizontal cells may feed back to depolarize the rods. However, there is at present no clear electrophysiological evidence for feedback to rods. If the lateral interactions were fed forward to the bipolar cell, then the transfer curves would be expected to shift vertically, downward along the bipolar potential axis. Some evidence for a vertical shift and feed-forward synapse is shown for lateral interactions between amacrine and ganglion cells by Thibos & Werblin (1978). Form of the rod-bipolar transfer curves Fig. 12 shows the transfer curves, derived in Fig. 10, replotted as a function of depolarization. The potential levels shown along the axes are based upon recordings from mudpuppy rods (Normann & Werblin, 1974) and bipolars (Werblin, 1974 and this paper). The curves are graded over a range of presynaptic (rod) potentials from about -45 to -30 mv and the postsynaptic (bipolar) potential increases about tenfold for each 8 mv presynaptic depolarization. TABLE 2. Comparison of transfer function characteristics for three different synapses: squid giant synapse, lamprey spinal cord, and mudpuppy rod-bipolar Squid Lamprey Mudpuppy Presynaptic potential range -40 to to to -30 Rate of postsynaptic increase per tenfold/lo mv tenfold/23 mv tenfold/8 mv presynaptic polarization The values in mudpuppy are compared with transfer characteristics measured directly in squid giant synapse (Katz & Miledi, 1967) and lamprey spinal cord (Martin & Ringham, 1975) in Table 2. The domain of presynaptic potentials in mudpuppy eliciting monotonically increasing postsynaptic potentials seems to fall near the potential domain of other preparations. Since the rod domain was tested with light, the total range of polarization over which the photoreceptors can drive the bipolars has not been defined. The transfer curves derived for the rod-bipolar synapse in the presence of surround illumination are also shown in Fig. 12. They indicate that the relation between lightelicited rod polarization and bipolar response is reset by about 5 mv along the rod potential axis for each log unit increase in surround intensity (the distance along the abscissa between the curves). This suggests that for each log unit increase in surround intensity the centre test flash must polarize the rod by an additional 5 mv to maintain the same bipolar potential. The research was sponsored by the National Institutes of Health (Eye Institute) Grant EY and (General Medical Sciences) Grant GM L.N.T. also received Fight for Sight SF 390 C-2 student fellowship. REFERENCES BURKHARDT, D. A. (1974). Sensitization and centre-surround antagonism in Necturus retina. J. Physiol. 236, DOWLING, J. E. & WERBLIN, F. S. (1969). Organization of the retina of the mudpuppy, Necturus maculosus. I. Synaptic structure. J. Neurophysiol. 32, ENROTH-CUGELL, C. & LENNIE, P. (1975). The control of retinal ganglion cell discharge by receptive field surrounds. J. Physiol. 247,

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