(Received 4 July 1977)

Transcription

1 J. Physiol. (1978), 276, pp With 12 text figure Printed in Great Britain EFFECTS OF PICROTOXIN AND STRYCHNINE ON RABBIT RETINAL GANGLION CELLS: LATERAL INTERACTIONS FOR CELLS WITH MORE COMPLEX RECEPTIVE FIELDS BY J. H. CALDWELL,* N. W. DAW AND H. J. WYATTt From the Department of Physiology and Biophysics, Washington University School of Medicine, 660 South Euclid Avenue, St Louis, Missouri 63110, U.S.A. (Received 4 July 1977) SUMMARY 1. The effects of picrotoxin and strychnine were tested on the receptive fields of direction sensitive cells, orientation sensitive cells, local edge detectors, uniformity detectors and large field units in the rabbit retina. 2. Picrotoxin eliminated the direction specificity and size specificity of 'on-off' and 'on' directionally sensitive cells for both black and white objects. Picrotoxin also made 'on' directionally sensitive cells responsive to faster velocities. 3. Picrotoxin eliminated the orientation specificity of orientation sensitive cells, and changed the bar-flank arrangement of the receptive field into a centre surround arrangement. Thus, the orientation specificity is due to inhibitory rather than excitatory mechanisms. 4. Picrotoxin altered the speed sensitivity of large field units so that they responded to slow speeds as well as fast ones, like centre surround Y cells. 5. Strychnine abolished the size specificity of local edge detectors and changed their speed specificity so that they responded to faster speeds. 6. Picrotoxin changed a uniformity detector into a sustained on centre cell. 7. Strychnine did not affect the direction specificity of directionally sensitive cells, the orientation specificity of orientation sensitive cells, or the speed specificity of large field units. Picrotoxin did not affect the size specificity of local edge detectors. 8. Picrotoxin and strychnine usually had opposing effects on the transient responses of these units to spots and annuli. In general picrotoxin prolonged and enhanced these responses at both on and off, and strychnine shortened them. 9. The effect of these drugs for every type of ganglion cell with complex receptive field properties was to make the receptive field more simple. The orientation selective cells, large field cells, 'on' direction selective cells and uniformity detectors seem to be centre surround cells with special properties that are abolished by these drugs. The 'on-off' direction selective cells and local edge detectors still have on-off receptive fields, but in each case one of the drugs abolished the feature that was the basis for the cell's name. * Present address: Department of Physiology, University of Colorado School of Medicine, 4200 East Ninth Avenue, Denver, Colorado 80262, U.S.A. t Present address: State College of Optometry, SUNY, 100 East 24th Street, New York, New York, U.S.A.

2 278 J. H. CALDWELL, N. W. DAW AND H. J. WYATT INTRODUCTION A number of studies have been made on the effect of picrotoxin and strychnine on the retina (Adrian & Matthews, 1928; Straschill, 1968; Ames & Pollen, 1969; Burkhardt, 1972; Daniels, 1974; Kirby & Enroth-Cugell, 1976; Miller & Dacheux, 1977). The results, such as an increase in the spontaneous and driven activity in ganglion cells, and an increase in the size of some components of gross responses like the ERG and proximal negative response, are generally consistent with the concept that both picrotoxin and strychnine antagonize inhibitory transmitters, as they do in other parts of the nervous system. Picrotoxin is a likely antagonist to y-aminobutyric acid (GABA) and strychnine to glycine, or possibly taurine or 8i-alanine (Curtis, H6sli & Johnston, 1968; Takeuchi & Takeuchi, 1969; Curtis & Johnston, 1974; Krnjevid, 1974). In the mammalian retina, studies on the uptake of radioactive GABA and glycine have shown that these substances are taken up primarily into amacrine cells, if one ignores the uptake into glial cells (Ehinger, 1970; Ehinger & Falck, 1971; Bruun & Ehinger, 1972; Marshall & Voaden, 1975; Ehinger, 1976). The enzyme glutamic acid decarboxylase (GAD) is located in the inner plexiform layer (Kuriyama, Sisken, Haber & Roberts, 1968; Barber & Saito, 1976; Wood, McLaughlin & Vaughn, 1976). Putting these points together with the hypothesis that amacrine cells are responsible for the properties of the ganglion cells with more complex receptive fields (Dowling, 1968; Dubin, 1970), one might expect picrotoxin and/or strychnine to have dramatic effects on the receptive fields of these cells. We decided to investigate this question in the rabbit retina. 'On-off' directionally sensitive cells are common in the periphery of the rabbit retina (Barlow, Hill & Levick, 1964; Oyster, 1968) and orientation sensitive cells and local edge detectors are common in the visual streak (Levick, 1967). 'On' directionally sensitive cells, large field units and uniformity detectors can be found in reasonable percentages in both places (Levick, 1967; Oyster, 1968; Caldwell & Daw, 1978a). Moreover, the receptive fields of these various types of cell have been better characterized in the rabbit than in other animals (Barlow & Levick, 1965; Levick, 1967; Oyster, 1968; Wyatt & Daw, 1975). Pilot experiments have shown that workable results can be obtained recording from cells in the retina of the intact animal, and infusing the drugs into the arterial blood supply to the eye (Wyatt & Daw, 1976). METHODS The results are based on recordings from fifty-eight rabbits. Methods for recording from the ganglion cells and stimulating their receptive fields have been described (Caldwell & Daw, 1978a). Drug infusion. Picrotoxin and strychnine HCl (obtained from Sigma Chemical Company, St Louis) were infused via a cannula of PE 10 tubing which was inserted into the external maxillary artery and advanced until the tip was in the external carotid (Fig. 1). The lingual, internal carotid, and temporal arteries were ligated, although the temporal artery was inaccessible in some cases. Thus the drug was injected into the external carotid and carried into the internal maxillary, which supplies the retina and the lateral part of the face. Saline with heparin (10 u./ml.) was continuously infused (3 ml./hr) to keep the catheter from becoming blocked. At the end of some experiments a dye was infused via this catheter to confirm the distribution of the drug.

3 PICROTOXIN AND STRYCHNINE IN RETINA 279 For drug infusion, drugs dissolved in saline (1.6 mm picrotoxin or 2-7 mm strychnine) were substituted for saline. The volume of fluid in the catheter, from the syringe to the tip in the external carotid, was approximately 0-25 ml. The precise concentration of the drugs in the retina was unknown in the present experiments. The procedure was to add the drug at increasing rates (thus increasing the concentration in the blood stream) until an effect was seen, unless the effective rate was already known from other cells in the same animal. Cells were affected at the same rate of infusion in any one animal, but this rate of infusion was found to vary between 0 1 ml./min and 0 5 ml./min (occasionally less or more) from one animal to another, probably due to variations in the placement of the catheter Trachea Drug via catheter External maxillary Internal maxillary Lingual Temporal </Internal carotid Common carotid To eye Fig. 1. Schematic diagram of the major branches of the common carotid. The catheter was put into the external maxillary artery. The internal carotid, lingual, and temporal arteries were tied off. Thus the drug was injected directly into the blood flowing into the internal maxillary, which supplies part of the face and the eye. and the organization of the arteries of the animal. At the lower rates of infusion, with a blood flow of 10 ml./min in the artery where the drug was infused, the drugs were diluted by a factor of 100, to 16 EM for picrotoxin and 27 /M for strychnine. Presumably the concentration in the retina reached a maximum of rather less than this. Picrotoxin was often infused while recording from a direction selective cell as a means of testing and calibrating the preparation in terms of the rate of infusion and amount of infusion time necessary for an effect. With seventeen rabbits there was no indication of a drug effect for any cell even with a high rate of infusion (picrotoxin 0-8 fmole/min). These negative results are not included, because they were probably due to a poor placement of the catheter in the arterial system, and the fact that the internal carotid does contribute some of the blood supply to the retina in a small percentage of rabbits (Prince, 1964). Experimental procedure. Once a cell had been isolated and its receptive field plotted on the tangent screen, various tests were made to determine the cell type. Records were then taken for a pre-drug response. For example, for an orientation cell this consisted of accumulating several responses to a moving bar in the preferred and then in the orthogonal orientation. After the infusion of the drug was begun, records were taken almost continuously until an effect was obvious. Then saline was infused and records taken to observe the cell's recovery. Records were stored on magnetic tape and analysed later on a LINC computer. General comments. Since strychnine and picrotoxin have general systemic effects, blood pressure was monitored through a catheter in the femoral artery. Blood pressure changes occurred, but were variable and did not correlate with the neural effects. Moreover, the two drugs often caused the same increase in blood pressure but had different effects on the ganglion cells. The effects of picrotoxin reversed readily, usually after several minutes, although complete recovery usually required min. Strychnine required several hours for reversal, and a cell was often lost before this occurred. Moreover, once strychnine had been added, it was necessary to stop searching for cells for several hours while the retina recovered.

4 280 J. H. CALDWELL, N. TV. DA W AND H. J. WYATT It is worth noting that the cells which are described are only those for which the recording and preparation were excellent. This is especially important when the drug has no effect. Negative effects were considered reliable only when the drug altered other units which were recorded simultaneously or if the amount of drug and rate of infusion necessary for an effect were known from other results in the same animal. RESULTS There are at least fifteen physiologically different types of ganglion cells in the rabbit. Nine are centre surround, including one which is colour coded (Caldwell & Daw, 1978a). Six have complex receptive fields and are the subject of this study. The six complex ganglion cell types (Levick, 1967) are the 'on-off' direction selective, 'on' direction selective, orientation selective, local edge detector, large field, and uniformity cells. All of these have been studied under the effect of picrotoxin or strychnine. The results are summarized in Table 1 and will be described separately for each cell type. TABLE 1. 'On-off' direction selective 'On' direction selective Orientation selective Uniformity Large field Local edge detector Effects of picrotoxin and strychnine upon cells with more complex receptive fields Picrotoxin Eliminates direction selectivity Produces larger and more prolonged transients to a stationary spot Eliminates direction selectivity Eliminates size specificity Changes speed specificity Eliminates orientation selectivity Converts to a centre surround receptive field Produces larger and more prolonged transients to a stationary bar Eliminates inhibition Reveals excitation in centre Produces larger transients Changes speed specificity No change Strychnine No change in direction selectivity Transients become shorter No change in direction selectivity Speed specificity not tested No change in orientation selectivity Transients become shorter Not tested Produces smaller transients No change in speed specificity Eliminates size specificity Possible change in speed specificity 'On-off' direction selective cells Wyatt & Daw (1976) have shown that the direction selectivity of these cells is lost when picrotoxin is added to the arterial blood. This result was verified for thirty 'onoff ' direction selective cells in the present experiments. This loss ofdirection selectivity is not seen when strychnine is infused at a rate and concentration known to affect both other cell types and also other aspects of the response in these cells (see below). Picrotoxin also blocks a particular inhibitory response in these cells. A windmill stimulus (Werblin, 1972) rotating in the surround of the receptive field stimulates

5 PICROTOXIN AND STRYCHNINE IN RETINA 281 transient amacrine cells in the inner plexiform layer of the mudpuppy and inhibits 'on-off' ganglion cells in the mudpuppy. This stimulus also inhibits the response of ' on-off ' direction selective cells in the rabbit to a stationary or moving target. Picrotoxin reduced or abolished this inhibition. Control Picrotoxin,,,,^,,. Recovery Null + Preferred.1La 1-5 Control LLa hl...jjpl Picrotoxin. 1kt Recovery 1 25/sec L 1 seac Fig. 2. On-off direction selective unit. Picrotoxin abolished direction selectivity for both light/dark and dark/light edges. Responses to a white spot in the upper half of the Figure and to a black spot in the lower half. Receptive field plot in the centre. Responses in the preferred direction are on the right half of the Figure and responses in the null direction on the left half. Each traverse of the receptive field resulted in a double burst, one burst as the leading edge of the spot moved through the receptive field and a second burst for the trailing edge. Note that with picrotoxin the response in the null direction was also a double burst. Spot velocity was 70/sec in both directions. Elimination of direction selectivity by picrotoxin was demonstrated for both black and white spots. Direction selective units respond equally well to black or white spots (Barlow & Hill, 1963). An example of an 'on-off' direction selective cell responding to black and white spots before, during, and after picrotoxin is shown in Fig. 2. The size of each spot was made large enough so that the cell responded separately to the leading and trailing edge of the spot. The result was a double burst of action potentials each time the spot crossed the receptive field in the preferred direction. It is evident that direction selectivity was lost for both black and white spots during

6 282 J. H. CALDIWELL, N. W. DAW AND H. J. WYATT picrotoxin infusion, and that the response that appeared for movement in the null direction was a double burst similar to that in the preferred direction. Picrotoxin and strychnine had opposite effects on the on-off responses of the cells. Both the on and off responses are enhanced and prolonged by picrotoxin. With strychnine, the off response becomes extremely transient and the on response is relatively unchanged (Fig. 3). For other on-off direction selective cells both the on and the off transients were shortened by strychnine. Control LiFILL Picrotoxin L >IL Strychnine k Lt.Ai RA E je liiia, On Off 350/sec r 1 sec Fig. 3. On-off direction selective unit. Response to a stationary white spot. Picrotoxin increased and prolonged both the on and the off transients. Strychnine shortened the time course of the transients. Spot was centred on the receptive field and was approximately the same size as the receptive field (10 x 2 ). 'On' direction selective cells - The 'on' direction selective cell responds to only the leading edge of a white spot and the trailing edge of a black spot, giving a single burst each time the spot crosses the receptive field. The effect of picrotoxin was tested for both black and white spots as was done for the 'on-off ' direction selective cells described earlier. Again the size of the spots was made sufficiently large to separate the effects of the leading

7 PICROTOXIN AND STRYCHNINE IN RETINA 283 and trailing edges. With picrotoxin the directional selectivity for both black and white spots was abolished (Fig. 4), and the response for both spots was still only a single burst to the normally appropriate edge for the preferred and null directions. The loss of direction selectivity was observed for nine cells in this study. This confirms the report of Wyatt & Daw (1976), who also showed that size specificity, as well as direction selectivity, is lost during the application of picrotoxin. In addition, they found that the selectivity was unaffected by strychnine. Control ill Picrotoxin LU"L-AWAL - Recovery Null _ > MiL -La 3 degrees <:= Preferred Control Picrotoxin I 00/sec L Recovery 4 sec Fig. 4. On direction selective cell. Picrotoxin abolished direction selectivity for black and white spots. Same format as Fig. 2. Cell responded only to the leading edge ofa white spot (upper half of the Figure) and to the trailing edge of a black spot (lower half of the Figure). During picrotoxin the response in the null direction was also only to the leading edge of the white spot and the trailing edge of the black spot. Speed of the spot was 1.50/sec. in preferred direction and 1V/sec in null direction. The effect of picrotoxin upon speed selectivity was also tested. 'On-off' directionally selective cells respond to a wide range of speeds (<1/sec to > 100'/sec), but 'on' directionally selective cells respond only to slow target speeds (Oyster, 1968; Wyatt & Daw, 1975). Picrotoxin enhanced the response of three 'on' directionally selective cells at all speeds (Fig. 5): the cells responded to high speeds that were previously ineffective, and, in the cell shown, the speed causing the maximum rate of firing was shifted from 8 degrees/sec to about 80 degrees/sec. High speeds gave a response for both the preferred and null directions. This observation suggests that

8 284 J. H. CALDIWELL, N. TV. DA WV AND H. J. WYATT the restriction to slow target speeds is abolished by picrotoxin. Thus picrotoxin simultaneously abolishes size specificity and direction selectivity and changes speed specificity of these cells. 120 B ~~ID >~~~80 200: U,* 0F n P N 5dean Deg/sec C 8-5 deg/sec 25 deglsec 85 deglsec Control Li A, ILL Picrotoxin P N P N P N Fig. 5. On direction selective unit. Picrotoxin abolished direction selectivity and changed speed selectivity. A, stimulus was a spot moving in the preferred and null directions through the receptive field. B, average frequency of the response (continuous lines) and total action potentials per response (dashed lines) plotted versus target speed. Control (open symbols). Picrotoxin (filled symbols). The speed for the maximum frequency of firing was shifted from 80/see to 80'/sec by picrotoxin. As the speed increased the total number of action potentials continuously decreased due to the decreased time the stimulus spent in the receptive field. The arrows indicate the three speeds which are illustrated in C. Note that not only is the response increased at the higher speeds in C but that with picrotoxin the unit responds in both the preferred (P) and null (N) directions. The calibration scale for C is 100 spikes/see. Orientation selective cells For this type of ganglion cell in the rabbit retina there are only two preferred orientations, horizontal or vertical. The units are easily identified with a bar stimulus but are often difficult to map with small spots; they usually have a bar and flank arrangement of on and off regions. They can have either on or off centres, but the off centre units are encountered much mare frequently. Picrotoxin has a dramatic effect on the selectivity of these cells. Following an increase in spontaneous activity, cells exposed to picrotoxin respond increasingly well to bars in the orthogonal orientation (900 to the optimal or preferred orientation). The responses in the preferred and orthogonal orientations eventually become equal. These effects are illustrated in Fig. 6. After the cell recovered from picrotoxin, strychnine was infused. The spontaneous activity increased but orientation selectivity was unaffected. A loss of orientation selectivity with picrotoxin was seen in all ten

9 PICROTOXIN AND STRYCHNINE IN RETINA 285 cells tested (eight off centre, two on centre) for both black bars and white bars. Strychnine had no effect upon the selectivity of three out of four cells tested. With one cell, for which picrotoxin was not tested, strychnine did have a slight effect upon orientation selectivity, but the response in the orthogonal orientation remained small compared to that in the preferred orientation. 200/sec 2 sec Control L L Picrotoxin 1 _IaIi L -ll a Recovery a is +== +: Preferred Orthogonal Fig. 6. Off centre orientation selective cell. Picrotoxin abolished orientation selectivity. Unit preferred a horizontally orientated bar. Each traverse of the receptive field gave a double burst, the first burst as the bar entered the on flank and the second bust as the bar left the off centre and entered the other on flank. Speed of the bar was 5V/sec. The shape of the receptive field has also been studied with picrotoxin. The cell illustrated in Fig. 7 was off centre and preferred a vertically orientated bar. The receptive field had a bar-flank arrangement, in the sense that 'on' responses were not obtained in the upper or lower parts of the receptive field. Picrotoxin was infused and orientation selectivity was tested with moving vertical and horizontal bars. When the vertical and horizontal bar responses became equal, the receptive field was replotted with a small spot. The receptive field became symmetrical with a centre surround arrangement, The responses to stationary bars were tested on the same cell before and during picrotoxin. Both preferred and orthogonal orientations had

10 286 J. H. CALDWELL, N. W. DAW AND H. J. WYATT strong and roughly equal on and off responses.during exposure to the drug (Fig. 7B). 'On' centre orientation selective cells also have their orientation selectivity abolished by picrotoxin. The 'on' centre cells often have a high rate of spontaneous activity and have vague receptive fields when plotted with a spot. The cell in Fig. 8 had a spontaneous activity of about 30/sec in the control records. The cell was completely inhibited when a bar with the orthogonal orientation moved through the A 0 0 -: + A+ 0o O Od T Picrotoxin O :0 0 2C o '0 o 0 +_ I- 0 &:+ 0 0croox i B ControlI Picrotoxin _ L.. Li A i l : *. :tofo BI.-L L. 200/sec Fig. 7. Off centre orientation selective unit which preferred a vertical bar. All records labelled picrotoxin were taken when the unit had become non-selective for orientation. A, receptive field plotted with a stationary spot before and during picrotoxin. The receptive field changed from bar/flank to centre surround with picrotoxin. Cross hairs are provided, so that the two plots can be related to each other. B, responses to a stationary bar in the preferred orientation (top row) and the orthogonal orientation (bottom row). The small on response in the preferred, control record may have been due to a slight misplacement of the bar. receptive field. In the preferred orientation the bar inhibited the cell when it passed through the off flanks and excited the cell when it crossed the receptive field centre. When picrotoxin was infused, an orthogonally orientated bar had the same effect as a bar with the preferred orientation, and the responses to the bars were those that would be expected for a centre surround cell with an on centre. Off centre orientation 1 sec

11 PICROTOXIN AND STRYCHNINE IN RETINA 287 selective units are also normally inhibited by a moving bar with the orthogonal orientation, but the spontaneous activity is usually so low that the inhibition is not evident. Although strychnine did not affect orientation selectivity, it produced an extremely transient response to a stationary bar. Picrotoxin caused a more enhanced and longer response at both on and off. The contrasting effects of the two drugs are similar to the changes produced in 'on-off' direction selective cells in- response to stationary spots. Orthogonal Preferred L~L5~iMi _ih Control l i Picrotoxin ~h~ii _ L_ Recovery ] 1 00/sec Fig. 8. On centre orientation selective cell. Horizontal bar preferred. Picrotoxin abolished orientation selectivity. Note the bursts of 84 and 80 action potentials for the orthogonal orientation with picrotoxin, where there had previously been no response (zeros in the record above). The bursts with picrotoxin, as with all the records for the preferred orientation, occurred when the white bar crossed the receptive field centre. The inhibition on each side of the burst occurred when the bar crossed the off regions which flanked the centre. Unit was not completely back to normal when the recovery records were taken. Bar moved at 3.50/sec. The high rate of spontaneous activity (30/sec) is normal for this type of cell. Local edge detectors These cells have on-off receptive fields, low spontaneous activity, and respond only to small, slowly moving objects. Diffuse light or large objects have little effect. They have slowly conducting axons (Caldwell & Daw, 1978a) and are concentrated in the visual streak. Wyatt & Daw (1976) reported that the size specificity is abolished by strychnine but not by picrotoxin. The recordings of three local edge detectors in the present experiments confirm those findings and also show some increase in the response to fast moving objects with strychnine infusion. Both changes are illustrated in Fig. 9. The cell normally responded better to spots and narrow bars than to a wide bar larger than the receptive field, and better to slow than to fast target speeds. Picrotoxin

12 288 J. H. CALDWELL, N. f. DAW AND H. J. WYATT affected neither size specificity nor the responses to different speeds. Strychnine infusion resulted in large responses for the wide bar as well as the narrow bar, and the response at the faster speed was enhanced for both stimuli. 1 1 deg/sec 1 8 deg/sec I I II I b I I Narrow bar Wide bar Control ] 1 00/sec Picrotoxin. m6.ne AM. ALL, ---~~LL L Sti Vchnine LE -A L~.AL,. Slow TE F.a Fast ---a i. LE TE Fig. 9. Effects of picrotoxin and strychnine upon a local edge detector. Control records. Narrow bar - the first large burst of firing occurred when the leading edge (LE) of the bar slowly entered the receptive field and the second large burst occurred as the trailing edge (TE) slowly left the receptive field. The small third group of spikes occurred when the bar moved back through the receptive field relatively rapidly (indicating a speed specificity). Wide bar - stimulus conditions and movement were identical to those for the narrow bar except for bar width. The responses to the leading and trailing edge at the slow speed were much less than for the narrow bar (indicating size specificity) and there was no response at the fast speed. Picrotoxin recordings. The responses were similar to the controls. Note (1) the response of the wide bar was still much less than that of the narrow bar (no change in size specificity) and (2) the response at the fast speed was not affected for either bar (no change in speed specificity). Strychnine records. Size specificity was almost eliminated since the leading and trailing edge bursts of the wide bar were about the same as those of the narrow bar. Speed specificity was reduced since the responses at the fast speed were increased, relative to those at the slow speed. Large field cells These cells have a centre surround receptive field, but both Barlow et al. (1964) and Levick (1967) called them complex rather than centre surround cells. The complex feature which makes them unique is that, unlike all other cells with centre surround receptive fields, the large field cells do not respond to slow movements. Slow Fast

13 PICROTOXIN AND STRYCHNINE IN RETINA 289 Y cells, with which large field cells can be easily confused, respond to slow as well as fast movement (Caldwell & Daw, 1978 a). In fact, this is the only class of ganglion cell in the rabbit that does not respond to moderately slow movement. The effect of picrotoxin upon stationary and moving stimuli has been tested on two of these cells. The response to stationary stimuli was the same as that for the Y cells (Caldwell & Daw, 1978b): the centre (spot) and surround (annulus) responses A Spot Annulus Diffuse P, 200 B C 350/sec 1 sec, deg/sec 120 -A- ] 100/sec.X 0 X 40-0 I ~~~~~~~~~~~ Deg/sec Fig. 10. Large field unit. Effect of picrotoxin on stationary and moving stimuli. A, response to stationary contrast; C (control) and P (picrotoxin). Picrotoxin increased spontaneous activity. Spot (centre) and annulus (surround) responses increased slightly, and the off transient with diffuse light was especially increased. B, response to a moving spot. The total number of action potentials at the slow speeds was greatly enhanced by picrotoxin. Control (O), picrotoxin (0): for the picrotoxin points the spontaneous activity was subtracted. Arrow indicates the response at 20/sec, which is illustrated in C. The total number of action potentials in the response is indicated below the brackets. were slightly increased, but more noticeable was that the off transient response to diffuse light (due to the receptive field centre) became larger (Fig. IOA). The response to several speeds was tested (Fig. lob) and with picrotoxin the cell responded to both fast and slow speeds. The major change was in the total number of action potentials in the response at slow speeds. Thus, in the presence of picrotoxin, the cell resembled a normal Y cell in its response to speeds as well as to a stationary spot or annulus (see Caldwell & Daw, 1978a, Fig. 6). Strychnine has been tested on one large field unit. Strychnine abolished the transient on response from the surround (annulus) and reduced the transient off response from the centre (spot). The cell was still insensitive to slow movement with no sign of any change in speed specificity. P _ 10 PHY 276

14 290 J. H. CALD WELL, N. W. DAW AND H. J. WYATT Uniformity detectors These cells are unusual in having a moderately high rate of spontaneous activity which is inhibited by all stimuli, including increases or decreases in background illumination, and stationary or moving spots and bars of any shape and contrast. In the rabbit and cat these cells are encountered very rarely (about 1 %). Spot Annulus Control.wi.alJ.L_ A Picrotoxin L m ud JiLAME.f o 0~ 20/uc L o Fig. 11. Response of a uniformity cell to a spot and annulus before and during picrotoxin. The stippled area of the receptive field indicates the regions where a stationary spot caused inhibition (for both on and off). The spot was much smaller than this responsive region. The annulus, which lay entirely outside the stippled area, was also inhibitory, as was diffue light. Picrotoxin removed this inhibition and the central spot became excitatory. The piorotoxin records were taken when an on-off direction selective unit, which was simultaneously recorded, became non-selective for direction. The effect of picrotoxrin was obvious in the one cell tested (Fig. 11). Transient inhibition at on and off was the normal response to a small spot in the centre. With picrotoxin this inhibition was removed, and the spot gave a sustained on response. An annulus normally inhibited the cell as long as the light was on, with little or no off response occurring when the annulus was turned off. This inhibition completely disappeared with picrotoxin. Diffuse light had little effect in the control but produced a sustained on response during picrotoxin. The time course of the loss and recovery of the surround inhibition was faster than that of the centre inhibition; this has not been seen with picrotoxin or strychnine for other cells. DISCUSSION Specific effects of drugs have been found for each type of complex ganglion cell. These effects are probably produced by a block of the inhibitory actions of certain amacrine cells. This suggestion is based upon the following lines of reasoning, as amplified below. There is evidence that picrotoxin blocks GABA while strychnine

15 PICROTOXIN AND STRYCHNINE IN RETINA 291 blocks glycine and that these amino acids are contained in certain subgroups of amacrine cells in the rabbit. Previous electrophysiological results imply that these drugs are acting at the inner plexiform layer. Moreover, hypotheses about the location of the synapses responsible for the complexities of the receptive fields in the retina usually put the synapses in the inner plexiform layer. The convergence of these various lines of evidence and reasoning is very satisfactory. Evidence on the specificity of picrotoxin for GABA comes from a variety ofpreparations. At the crayfish neuromuscular junction picrotoxin is a non-competitive antagonist ofthe inhibitory transmitter GABA, both pre- and post-synaptically (Takeuchi & Takeuchi, 1969). For many locations in the mammalian central nervous system, pharmacological and chemical evidence indicates that GABA is a neuro-transmitter (Krnjevic, 1974); intracellular recordings in Deiter's nucleus have shown that both the neurally evoked i.p.s.p.s from cerebellar stimulation and the GABA induced hyperpolarization are antagonized by picrotoxin (Obata, Takeda & Shinozaki, 1970). In interneurones of lamprey spinal cord the conductance change due to GABA is noncompetitively antagonized by picrotoxin, and the conductance change due to glycine is unaffected by 20 /IM picrotoxin (Homma & Rovainen, 1978). Regional localization of glycine, similarity of the reversal potential for glycine action and for natural inhibition, and the glycine and i.p.s.p. dependence on different anions suggest that glycine is the neurotransmitter of certain spinal inhibitions (Werman, Davidoff & Aprison, 1968). Both i.p.s.p.s and glycine-caused hyperpolarization are blocked by strychnine whereas GABA-induced hyperpolarizaticn is not blocked by strychnine. However, antagonism of synaptic transmission by strychnine may not be a reliable indication that the transmitter is glycine. Diamond, Roper & Yasargil (1973) applied GABA, glycine, and strychnine onto the goldfish Mauthner cell. Strychnine blocked both the i.p.s.p. and iontophoretically applied glycine inhibition. The natural transmitter and glycine were, however, not equally sensitive to strychnine. Another complication is that cholinergic and possibly serotonin synapses may be affected by strychnine (Krnjevic, 1974). Landau (1967) studied the statistics of quantal release at the neuromuscular junction of rat diaphragm and found that strychnine had both pre- and post-synaptic effects. Strychnine had a curare-like action (65 #M strychnine = 1,UM curare) and also reduced the quantal content ( > 25 /M strychnine). The concentrations determined by Landau are an order of magnitude higher than the 2#M required for an effect in the rabbit retina (Ames & Pollen, 1969) and are probably higher than the effective concentrations in the present experiments. It is unlikely that we have used drug concentrations which cause non-specific effects. In the test experiments, the concentration of picrotoxin in the retina was probably less than 16,uM and that of strychnine less than 27 #M (see Methods). S. Homma & C. M. Rovainen (private communication) found that 20 /M picrotoxin had no effect upon glycine-induced conductance changes in the spinal cord. Strychnine has non-specific effects upon retinal neurones only at concentrations greater than IOfM (Daniels, 1974). When added intravenously to a cat at a dose of 2 2 umole/kg, strychnine has no effect on the spinal inhibition caused by GABA although it severely reduced the glycine inhibition (Curtis, Duggan & Johnston, 1971). This was over 10 times the total dose of strychnine in the present experiments. 10-2

16 292 J. H. CALDWELL, N. W. DAW AND H. J. WYATT Two techniques used to localize putative transmitters in the retina are autoradiography and enzyme localization. The uptake of [3H]GABA combined with autoradiography has shown that this amino acid is accumulated preferentially by amacrine cells of the rabbit (Ehinger & Falck, 1971; Ehinger, 1972; Marshall & Voaden, 1975; Ehinger, 1976). Label is also found in ganglion cells and Muller cells, but the ganglion cell uptake is non-specific, occurring for all amino acids (Ehinger, 1972). The Muller cell uptake is minimized if the experiment is done in vivo rather than in vitro (Ehinger, 1976). GABA is not taken up by horizontal cells of a variety of mammals (Marshall & Voaden, 1975). Another method is measurement of the activity of the synthetic enzyme, glutamic acid decarboxylase (GAD), in the various retinal layers. This activity is highest in the inner plexiform plus ganglion cell layers of the rabbit (Kuriyama et al. 1968). With an immunocytochemical technique for GAD in the rat retina, essentially all of the enzyme is found in the inner plexiform layer in distinct bands (Barber & Saito, 1976). With electron microscopy the cells containing this labelled enzyme antibody have been tentatively identified as amacrine cells (Wood et at. 1976). Uptake of [3H]glycine is primarily by amacrine cells in the rabbit (Ehinger & Falck, 1971; Ehinger, 1972, 1976), as well as in many other mammals (Bruun & Ehinger, 1974). The distribution or position of labelled cells in the inner nuclear layer was not identical to that for GABA uptake, suggesting that separate subgroups of amacrines take up and concentrate GABA and glycine. Electrophysiological evidence indicates that picrotoxin and strychnine act at the inner plexiform layer. The effects of GABA, strychnine, picrotoxin, and bicuculline (a GABA antagonist) have been studied with intracellular recording in the mudpuppy eyecup (Daniels, 1974; Miller & Dacheux, 1977). None of the drugs has any effect upon photoreceptors or most horizontal cells except at a high dose (> 100 /M strychnine) and with a time delay of at least 10 min. Both bicuculline and picrotoxin affected the depolarizing bipolars but not the hyperpolarizing bipolars; the converse was true for strychnine. The effect of GABA on the bipolar cell response is not clear: picrotoxin enhanced the bipolar response (R. F. Miller & R. F. Dacheux, private communication) but bicuculline reduced the response (Daniels, 1974). The amacrine cell responses were increased by both drugs. Some i.p.s.p.s in on-off ganglion cells in the mudpuppy were blocked by picrotoxin or by strychnine. An increased amacrine cell response due to picrotoxin and strychnine was also suggested by an increased proximal negative potential in the frog (Burkhardt, 1972); this is an extracellular potential considered to indicate amacrine cell activity (Burkhardt, 1970). This evidence on amino acid localization in the rabbit retina and the electrophysiological effects of picrotoxin and strychnine suggest that the drugs antagonize inhibitory transmission at the inner plexiform layer. Thus in discussing each of the complex cell types, we will assume that the effect is due to the block of certain amacrine cell output. Direction selective cells The direction selectivity for both the 'on' and 'on-off' type of cell is abolished by picrotoxin. The size specificity of both types is lost as well. Wyatt & Daw (1975) have proposed a model for the inhibition which produces both direction and size

17 PICROTOXIN AND STRYCHNINE IN RETINA 293 selectivity. The simultaneous loss of direction and size specificity is consistent with their model of a single inhibitory system. It is possible that speed specificity is also produced by this same system because this was also altered for 'on' direction selective cells in the present study. The failure of the 'on' directionally sensitive cells to respond to fast movement in the preferred direction could be due to inhibition extending in the preferred direction as well as the null direction. Wyatt & Daw (1975) showed that the inhibition does extend in the preferred direction: to explain the speed specificity of the 'on' directionally sensitive cells one needs to postulate that this inhibition is both powerful and rapidly decaying. We found no evidence that facilitation is important for the formation of direction selectivity. If facilitation were the overriding factor, then it might be expected that as a unit became non-selective with picrotoxin, the responses in the preferred direction would become smaller. This did not happen; the response always increased, which would suggest that inhibition is the most important mechanism for direction selectivity. Barlow & Levick (1965) also concluded that inhibition was much more important than facilitation, although they did have some evidence for a slight facilitatory effect. Wyatt & Daw (1975) found no evidence of facilitation in their study of direction selectivity in the rabbit retina. However, in cat cortex, Emerson & Gerstein (1977) have shown that both facilitation and inhibition can be important in creating the direction selectivity of simple cortical cells. It is not clear for cat cortex whether the inhibition of direction selective cells is due to the leading or trailing edge of the stimulus (Emerson & Gerstein, 1977). For the rabbit retina, however, inhibition of 'on-off' direction selective cells must be due to both the leading and the trailing edge. This is apparent from the inhibition that is created by the leading and trailing edges of a long narrow bar (black or white) that moves along its length in the null direction through the receptive field. This is supported by the response of the cell to movement in the null direction during picrotoxin infusion: the cell then responds to both the leading and trailing edge of a large spot (black or white) in the null direction. In other words, it responds to both light/dark and dark/light edges. The simplest explanation for this result is that both depolarizing and hyperpolarizing bipolar cells feed into the lateral inhibitory network responsible for directional sensitivity. The present experiments do not identify the postsynaptic member of the proposed GABA synapse, which could be a bipolar, amacrine, or ganglion cell. Postsynaptic inhibition of the ganglion cell can occur since directional ganglion cells in mudpuppy are hyperpolarized by a target moving in the null direction (Werblin, 1970) and picrotoxin blocks some of the i.p.s.p.s in mud-puppy on-off ganglion cells (Miller & Dacheux, 1977). Several types of mechanisms might be responsible for the transient responses of 'on-off' directionally selective cells to stationary spots. These include different preor postsynaptic effects, additional interneurones (excitatory or inhibitory) or direct input from both depolarizing and hyperpolarizing bipolar cells. Responses are still transient in the presence of picrotoxin, indicating that mechanisms other than GABAnergic inhibition contribute. However, GABA does have some effect because transients are prolonged by picrotoxin.

18 294 J. H. CALDWELL, N. W. DAW AND H. J. WYATT Orientation elective celil Orientation specificity in the retina can be accounted for by inhibition alone. The receptive field in the rabbit retina normally has a bar and flank arrangement. When lateral inhibition is blocked by picrotoxin, the shape of the receptive field becomes centre surround. The additional concentric surround was presumably always present but was only revealed when picrotoxin blocked the inhibition causing orientation specificity. a + b A... c Fig. 12. Proposed inhibitory region (stippled) which forms orientation selectivity for an on centre unit which prefers a moving or stationary vertical bar (a). In the rabbit retina even a bar (b) at 450 to the preferred will cause a response. However, a bar in the orthogonal orientation (c) does not even give a transient response, suggesting a rapidly acting and strong inhibition. A moving white bar in the preferred orientation will first inhibit the cell as it enters the stippled region, then cause a burst as it crosses the centre and again inhibit the cell as it moves through the other flank. A bar in the orthogonal orientation will strongly inhibit the cell as it moves through the entire receptive field. Two models have been proposed for orientation selectivity in the visual cortex. Hubel & Wiesel (1962) suggested that orientation selectivity of simple cells could be produced by excitatory mechanisms from the convergence of a line of centre surround inputs. They noted in the same paper that inhibition could also be used. The importance of inhibition for simple cells in cat cortex has been emphasized recently (Creutzfeldt & Ito, 1968; Henry & Bishop, 1972; Bishop, Coombs & Henry, 1973; Creutzfeldt, Kuhnt & Benevento, 1974; Henry, Dreher & Bishop, 1974). Experiments with bicuculline which were designed to block inhibition mediated by GABA, resulted in only a partial loss of orientation selectivity for some simple and complex

19 PICROTOXIN AND STRYCHNINE IN RETINA 295 cells in the cat cortex (Rose & Blakemore, 1974; Daniels & Pettigrew, 1975). In similar experiments Sillito found a reduced orientation selectivity for simple cells but a loss of orientation selectivity for complex cells (Sillito, 1975) and hypercomplex cells (Sillito & Versiani, 1976). It is possible that both excitation and inhibition are important for orientation in the cat cortex. In the retina, however, the effect of inhibition is dominant. A possible model for orientation specificity in the retina is schematically illustrated in Fig. 12. The example is an on centre cell that prefers a vertical bar. Hypothetical inhibitory regions (stippled) are on either side. The actual shape of these inhibitory areas could be measured, as has been done for directionally selective cells in the rabbit retina (Wyatt & Daw, 1975) or for simple cells in the cat cortex (Bishop et al. 1973). The orientation tuning of the cell would be determined by the shape of the inhibitory regions. For the rabbit the orientation tuning is quite broad; cells often respond to a bar tilted 450 from the preferred (Levick, 1967). Thus a bar at position (b) in Pig. 12 would stimulate the cell. The threshold for a bar in the orthogonal position (c in Fig. 12) would increase rapidly when it was longer than the centre region. When the inhibition is abolished by picrotoxin, all orientations will be equally effective. It should be noted that this model has points in common with the observations of Creutzfeldt et al. (1974), and is rather different from the model put forward by Levick (1967), who proposed that orientation selective cells might be missing the antagonistic surround on two sides of the centre. There is no evidence from our results that the surround is incomplete, either before or after the application of picrotoxin. The orientation selectivity is not the result of the absence of part of the antagonistic surround, but of the presence of an additional inhibitory mechanism. Gallego (1971) described a morphological type of amacrine cell commonly seen in whole mounts of silver stained rabbit retinas. These are large, stratified bipolar amacrines which cover an area of the form of a bow-tie. Their size was difficult to determine but a maximum diameter of 1 mm is reasonable. If the bow-tie amacrines are approximately this size, a single amacrine cell could easily span the entire inhibitory field as depicted in Fig. 12. These bow-tie amacrines probably have some orientation specificity purely by virtue of the shape of their dendritic arborization. Thus some of the specificity in this model already resides in a cell presynaptic to the ganglion cell. It is a result of the morphological shape of the amacrine cell. This is different from the view of the mechanism for direction selectivity. The model of Wyatt & Daw (1975) would predict that the amacrine cells causing the selectivity are not direction selective themselves, but that the selectivity is a result of the asymmetry of the connexions made onto the ganglion cell. However, the orientation selectivity could also result from inhibition from more circularly shaped amacrines in each flank of the receptive field. Uniformity detectors The single uniformity cell tested lost its normal inhibition by visual stimuli in the presence of picrotoxin. Specifically, the inhibition at on and off produced by a central spot was replaced by a sustained on response. Sillito (1975) has obtained results on similar cells in the cat cortex for which inhibition was also replaced by excitation

20 296 J. H.CALDWELL, N. W. DAW AND H. J. WYATT with the addition of a GABA antagonist. Presumably the excitatory effects were unmasked with the removal of inhibition. Large field cells The similarity between these cells and the Y cells has already been noted. Picrotoxin abolished the speed specificity which made the large field cell unusual. This suggests that the large field cell is organized like the Y cell with an additional input from a set of GABAnergic amacrine cells which make the large field cell insensitive to low target speeds. Strychnine had no effect upon the speed specificity of the one cell tested. However, the responses to a stationary spot and annulus were reduced by strychnine. This is similar to the effect of strychnine upon a Y cell (Caldwell & Daw, 1978b). Local edge detectors The size specificity of these cells is unaffected by picrotoxin but is eliminated by strychnine. Levick (1967) emphasized the many similarities between these cells and the 'on-off ' direction selective cells. The organizational basis for these similarities may be the same, but the drug effects argue against the use of the same set of amacrine cells. General conclusions The major assumption is that picrotoxin and strychnine are blocking neurotransmitters of amacrine cells. The histochemical and electrophysiological evidence described earlier suggests that GABA and glycine are likely to be transmitters of amacrine cells. Picrotoxin and strychnine antagonize these amino acids at synapses elsewhere in the central nervous system. As described in the Results, these drugs removed inhibition and changed specific properties of complex ganglion cell receptive fields; for each type of complex ganglion cell this resulted in a more simple receptive field. This suggests that amacrine inhibition produces the complex receptive fields of these ganglion cells. Dowling (1968) and Dubin (1970) have studied the inner plexiform layer in a variety of species. They concluded that the retinas with ganglion cells having complex receptive fields also have more amacrine synapses, including amacrine to amacrine and amacrine to bipolar synapses. The complex retinas also tend to have an inner plexiform layer that is highly stratified when stained (West, 1976). The concept that the complex receptive field of a ganglion cell depends upon amacrine cells was based originally on anatomical evidence. This is now supported by the combination of pharmacology and physiology that we have described. Dr Stanley Lang gave us invaluable assistance in measuring blood flow in the carotid artery. We are grateful to Kathy Kudray, Roy Auer and Judy Dodge for help in the course of the experiments and to Drs Carl Rovainen, Clyde Oyster and Mark Dubin for comments on the manuscript. The work was supported by a N.I.H. research grant EY

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