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Gaba and the lateral spread of tonic activity in frog retina
0041-6989’79
0501-05Ol
y)?.OOO
GABA AND THE LATERAL SPREAD OF TONIC ACTIVITY IN FROG RETINA RICHARD D. MOONEY’ Vision Laboratory. Department of Psychology. University of Minnesota. Minneapolis. U.S.A. (Received
5 June
1978)
Abstract-The presence of GABA in association with horizontal cells of the frog retina suggests it may play a role in signal transmission by these cells. The actions of GABA and chemical treatments antagonistic to its effects were studied in the perfused frog eyecup on sensitization. a tonic. lateral effect produced by the horizontal cells. It was observed that GABA facilitated and bicuculline and chloride-free solutions depressed sensitization. Thus. GABA appears to be involved in lateral signal processing in the outer plexiform layer. Key Words-GABA: tions in the retina
sensitization: intraretinal recording: horizontal cell circuits: frog: lateral interac-
INTRODUCIION The center-surround organization of bipolar cell receptive fields in the retinas of amphibians results
in part from lateral conduction of tonic peripheral activity through networks of horizontal cells (Burkhardt, 1974; Matsumoto and Naka. 1972: Werblin and Dowling. 1969). Recent histochemicai work in amphibians suggests that gamma-aminobutyric acid (GABA) may have some function in synaptic transmission by horizontal cells. Endogenous stores of GABA and its main synthetic enzyme have been found in the region of the frog retina which contains mainly horizontal cells (Graham, 1972). Some horizontal cells incorporate exogenously applied GABA (Voaden et aI., 1974). One of the intermediate enzymes associated with degradation of GABA ultimately to succinate, succinic semialdehyde dehydrogenase, has been localized in the region of photoreceptor terminals where close contact between receptors, bipolar, and horizontal cells exists (Moore and Gruberg, 1974). The purpose of this report is to show that tonic, laterally conducted signals through horizontal cells are changed by chemical treatments which are known to alter the efficacy of GABA-sensitive synapses. The tonic lateral activity which was studied is the sensitization effect which is present in bipolar and ganglion cells of the mudpuppy (Burkhardt. 1974; Karwoski and Burkhardt, 1976; Werblin, 1974) and the proximal negative response (PNR) of the mudpuppy and frog (Burkhardt, 1974). This effect is based on a stimulus configuration used extensively in psy chophysics (see, for example, Westheimer, 1965) to compare the effectiveness of small and large adapting spots. When the small adapting spot is present, the amplitude of a response evoked by a small centered test flash is depressed. Subsequent enlargement of the I Reprint requests should be sent fo: R. Mooney. Department of M.C.D. Biology. University of Colorado. Boulder, CO 80309. U.S.A.
spot causes an increase in the response to the test flash. The enhancement of the response to the test flash which is produced by expanding the adaptive field is the sensitization effect. The extent of spatial summation which is involved in sensitization is similar to the extent of horizontal cell receptive fields in the mudpuppy and greatly exceeds the receptive fields of receptors or bipolar cells. Thus. adapting
sensitization probably arises from the influence of horizontal cells on receptors or bipolar cells (Burkhardt, 1974).
METHODS Isolated
retina-eyecup preparations of frogs (Ranu maintained in a perfusion system by an oxygenated Ringer’s solutidn buffered at pH 7.5. Solutions of different ionic composition or to which certain chemicals had been added could be switched into the perfusion system during stable electrical recording from the retina Micropipette electrodes (about I pm tip diameter) were filled with the Ringer’s solution and used to record retinal potentials. Records of light-evoked responses were stored on magnetic tape after d.c. amplification. Other details of the preparation and recording system have been described elsewhere (Mooney, 1978). The PNR was used to monitor sensitization. The PNR is a localized extracrllular. graded potential found maximally in the region of the inner plexiform layer of all vertebrate classes (Burkhardt and Mooney, 1977). It has been well characterized in the frog and mudpuppy (Burkhardt. 1970; Proenza and Burkhardt, 1973). The PNR was selected here because it could be recorded in most prep arations for many hours. and it showed high and relatively constant contrast sensitivity to light flashes during this time. Such stability was necessary for reliable observation of the effects of chemical treatments. A four-channel optical stimulator produced the stimulus shown schematically in Fig. l(A). Here the test flash (a 0.10 mm spot) which was used to evoke a PNR is presented on a steady, central. adapting background (a 025mm spot). The adapting illumination in the surround is an annulus (0.48 mm inner diameter, 1.3 mm outer diameter) concentric with the central adapting spot and rest flash. pipiens)
501
were
502
RKHARD D. M’IOO\EI
Fig. I. A. Schematic representation of the stimuius For sensitization seen in cross section. Intensity (actually retinal iiluminance) is expressed here on a scale with 10.0 log units arbitrartly set to equal 3360 lu.:m’. The diffuse background (1.1 log units) covers the entire eyecup. The central background (5.4 log units) is 0.25 mm in diameter and is also continuously present. The test flash. presented every 30 sec. is 0.10 mm in diameter and has an intensity which produces a small response in the presence of the diffuse and central backgrounds (see text). The steady annulus is 0.48 mm inner diameter and 1.3 mm outer diameter. and its intensity is the independent variable. B. Sensitization of PNR and on/off ganglion cell. Negative polarity is up in this and all successive figures. The 400 msec test flash at an intensity of 52log units produced a very small PNR and no spikes in the presence of the 3.5 log unit annulus (lowest response). As the annulus intensity was increased with all other stimulus parameters fixed. both on- and off-responses of the PNR and ganglion cell increased. Thus. at the higher intensities. the annulus produces a lateral spread of activity which influences responses at its center.
The intensity of the adapting annulus could be varied independently of the central adapting spot. The intensity of the annulus was typically increased in fixed steps every 24 min while the test flash was presented every 30 set to evoke a PNR. After a series of steps in annulus intensity, the annulus was extinguished to verify that the PNR to the test flash on the cen:ral adapting spot was the same as it was before the series. The change in PNR amplitude as a function of annulus intensity describes the sensitization effect. After a control sensitization function was thus obtained. chemicals were introduced into the perfusate. When responses were well stabilized, about 15 min later. the sensitization trials were repeated. The intensity of the test flash was adjusted so that the response amplitude produced by the flash on the central adapting spot was ap proximately the same for each sensitization series. This provides a common base point (labeled “B” in Figs 2 and 3) for all sensitization functions in each preparation. By referencing the sensitization curves to the same amplitude response, the effects produced by the chemicals represent changes in sensitization and not simply changes in the PNR. RESL!LTS
Figure l(B) shows a typical example of sensitization over a limited range of annulus intensity for the stimulus represented in Fig l(A). In this preparation, the graded PNR was recorded simultaneously with spikes from an on/off ganghon cell. In the presence
of the central background and a dim annulus (3.5 log units. see legend for photometric scaling), the test flash evoked a very small PNR and produced no spikes from the ganglion cell. In this case the PNR amplitude was the same as when no annulus had been present. Raising the annulus intensity in discrete steps caused the PNR to increase in amplitude (both on and off responses) and produced a similar sensitization of the on/off spike discharge. The effect on the ganglion cell is particularly striking The increased adapting light in the region surrounding the cell’s receptive field center has altered the cell’s output from no response to a fairly vigorous response. Figure 2 shows a comparison of the sensitizing effects of annuli on a preparation during perfusion with Ringer’s solution in the control condition (left) and then during exposure to a 0.1 mg/ml GABA solution (right). For the lowest traces, marked “B”, the annular background is absent, and the intensity of the test flash on the central adapting spot was adjusted to give a response of about 0.1 mV. As the annulus intensity was then increased (upper traces). sensitization of the PNR in the presence of GABA increased by a greater extent than that of the control condition. The effects of chemicals on sensitization were studied on 70 preparations. In 24 retinas a systematic procedure was used to gather data for quantitative com-
GABA and the lateral spread of tonic activity control
503
GABA
109 annulus intensity /%A
5.5
4.9
4.5
3.9
B
Fig. 2. Effects of GABA on sensitization. A l.Osec flash is indicated by lines below the responses. Test flash intensity was 5.5 log units during the control series (right column) and 6.0 log units during treatment with 0.1 mg,‘ml GABA (left column) to produce approximately 0.1 mV responses in the bottom responses during which no annulus was present (level B). The annulus intensity was increased in steps, and the test flash presented I min after each annulus increment. This figure shows that sensitization is enhanced by GABA solutions. parisons. Some of these results are presented graphically in Fig. 3. Eight levels of annulus intensity were presented in ascending order with four identical test flashes at each level. The final three responses at each annulus intensity were measured, and the median amplitude value was plotted as a function of annulus intensity. Sensitization is identified in these graphs as a gradual increase of response amplitude up to a
maximum with increasing annulus intensity. Response amplitudes eventually start to decline at higher annulus intensities (above 5.5 log units in Fig. 3). This is most likely a result of stray light from the annulus depressing the center’s sensitivity (Burkhardt, 1974), for it can be seen in all the curves of Fig. 3 independently of response amplitude and whether or not sensitization is present at lower intensities.
.
control
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ANNULUS
INTENSITY
Fig. 3. Effects of various chemical treatments on sensitization. In each preparation the test Rash intensity was adjusted to equate the responses at level B (no annulus). Thus. the extent of sensitization. as revealed by the increase in response amplitude. may be directly compared between conditions in each preparation. (A) GABA at 0.1 mg/ml (filled squares) enhances sensitization. The GABA curve rises more abruptly and peaks at a higher amplitude than the control curve (filled circles). Bicuculline N-methyl bicuculline iodide) at 0.01 mg/ml depressed sensitization below the control level (open squares). After return to Ringer’s solution, sensitization recovered (open circles). Test flash intensities in log units were: control 5.3. GABA 5.7, bicuculline 5.2 wash 5.4. (B) Chloride-free solution (open squares) strongly depressed sensitization from control levels (filled circles). but return to normal Ringer’, solution does not produce recovery of sensitization in this preparation. Test flash intensities were: control 5.5. chloride-free 6.0, wash 5.5. (C) &uanidinopropionic acid at 0.1 mg/ml (open squares) enhanced sensitization. Test gash intensity was 5.5 log units for all curves. Y.R. 19,5--c
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RICHARD D. kf00hEI
Figure 3(A) confirms and extends the findings in Fig 2. Here a 0.1 mg, ml GABA solution increased sensitization in a preparation in which the control effect is already strong. In 17 preparations which showed normal control sensitization functions. GABA solutions between 0.05 and 0.2 mg’ml produced a 10-120°~ (average 409,) increase in the peak value of the sensitization curve. For unknown reasons. not all eyecups with otherwise normal ERG. PNR. and ganglion cell responses showed sensitization effects. as was also found earlier (Burkhardt, t974). and most of these were not tested further. In several of these retinas. however. GABA solutions of 0.1 mgiml clearly produced sensitization effects to the annulus, and in the other cases. the effect was still absent after the application of GABA. At GABA concentrations above 0.5 mg’ml. the PNR often gradually decreased in amplitude. and quantitative study was not feasible. However, the simple procedure of expanding the central background spot from 0.25 to 1.0 mm gave a quick evaluation of the extent of sensitization. This test showed that the higher concentration solutions did not enhance sensitization and often reduced it. Following the GABA series of Fig. 3(A). a solution (0.01 mg/ml) of bicuculline. a potent antagonist of GABA in the frog retina (Mooney. 1978). was introduced and markedly decreased sensitization below control levels. After the bicuculline was removed from the perfusing solution. sensitization returned (open circles). This effect was found consistently for bicuculline solutions at this concentration or stronger and for 0.05-0.1 mg/ml solutions of picrotoxin, another GABA antagonist. On the assumption that permeability to chloride ions might be involved in the effects of GABA and bicuculline seen in Fig. 3(A). experiments were conducted in which chloride levels were reduced. In Fig. 3(B). sensitization was tested during depletion of chloride ion (chloride was repiaced with sulfate ions, and the solution was osmotically balanced with sucrose). As seen with bicucul~ne. sensitization is reduced or abolished by chloride-free solutions. This change was seen consistently in over 20 preparations. After restoration of the standard chloride level (114 mM), the response in Fig. 3(B) was normal in every respect except that sensitization did not recover. This was typical of other preparations which had been exposed to chloride-free solutions for long periods of time. Sensitization was also severely reduced or abolished to shorter exposures of chloridefree solution and to partially depleted solutions which lacked 66:,; or 7%; chloride ion. In these cases, recovery was possible following return to the normal Ringers. The effect of ~-guanidinopropio~ic acid, an amino acid analogue of GABA, was similar to that of GABA: at 0.1 mg/ml it enhanced sensitization in six preparations (Fig. 3C). and at 0.5 mg/ml it decreased sensitization. It could also be substituted for GABA at equal concentrations without producing any further change in the sensitization level achieved during exposure to the GABA solution. Glycine seemed to have little consistent effect, except at high concentrations (above 0.5 mg/ml), which depressed sensitization. Likewise, no consistent effects of strychnine. a
gfycine antagonist in the frog retina (Mooney. 197~1. could be identified. ,The failure of the lower concentration (0.1 mg,mll glycine solutions to enhance sensitization was examined thoroughly in tive preparations in which sensitization had been reversibly increased by the 0.1 mg ml GABA solutions. Again. no noticeable effect of glytine was found. These trials constituted a control of the depressive effects of GABA which reduced the PNR amplitude and necessitated increasing the test flash intensity to obtain matched responses to start each sensitization series. The amplitude of the PNR was depressed also by glycine comparably to the effects of GABA. and yet there was no change in sensitization starting from the matched initial responses. Thus, the increase in sensitization which was found during the application of GABA solutions is probably unrelated to the procedure of increasing the test flash intensity to match the initial responses.
DiSCCSSIOS
The essential interaction in sensitization is an antagonism of the centra1 adapting spot by the steady annulus. The result of this combination is found at least as early in the visual pathway as the bipolar cells (Burkhardt, 1974: Werblin. 1974). The spatial extent that is involved suggests that the horizontal cells must provide the surround component by either feedback (Baylor et al., 1971) or feedforward (Werblin and Dowting, 1969) pathways. The sensitivity change produced by horizontal ceils in bipolar cells is seen at successive levels as a change in sensitivity of the PNR and on/off ganglion cells (Burkhardt, 1971). If there are no subsequent interactions between the steady annulus and either the central adapting spot or test spot. then sensitization of the PNR can be used as an index of the change mediated at the outer ‘plexiform layer. Several control experiments were done to assess whether there were any subsequent tonic interactions in the PNR. At the light levels which produced sensitization, a steady annulus in the absence of the central adapting spot had no measurable effect on the PNR. However, at higher annulus intensities (above %56.0 log units), stray light from the annulus was appreciable and adapted the response evoked at its center. Secondly, no measurements of sensitization were made for several minutes after the central adapting spot was turned on and at least 1 min had passed after the annulus intensity had been incremented. At this time there was no measureable contribution of either steady spot or annulus to the recorded potential, and the amplitude of the PNR to Hashes 30 set apart was essentially constant. Thus, no tonic centersurround effects are evident in the PNR, and the steady annulus does not appear to interact with the test spot. These findings are in agreement with results in the pigeon (Brooks and Holden. 1974). and suggest that the tonic interactions between the central spot and annulus must occur distally to the PNR. Therefore, a change in sensitivity of the PNR due to sensitization is a reasonable indication of the qualitative change in sensitivity established at the outer plexiform laver. *
GABA
and the
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lateral spread of tonic acrivir)
The method used here fo measure sensitization was chosen to diminish the possibility that chemical effects on the PNR itself would alter its properties as an index of sensitivity. Control and chemically treated trials were compared on the basis of equivalent initial (and final) responses to the test flash on only the central adapting spot. Changes in light intensity of the size needed to match response amplitudes did not change the magnitude of sensitization in control trials. In part this was probably because the PNR’s were only about half peak amplitude at the greatest extent of sensitization. In addition, test spots of 0.1 mm, which were used here, have the same proportionate effect on PNR amplitude in control and chemically treated trials (Mooney. 1978). Thus. the PNR can be used to provide a faithful index of sensitivity changes due to sensitization for both control and chemical treatments. In summary. this paper shows that sensitization is enhanced by lower concentrations of GABA. In contrast, glycine did not enhance sensitization. while the GABA-antagonist bicuculline and chloride-free solutions produce a decrease in sensitization. Higher concentrations of all the amino acids tested also reduce sensitization. but this may only reflect the rather fragile nature of the effect (Burkhardt, 1974). In the mudpuppy, it has recently been shown that horizontal cells and the surrounds of bipolar cells are very sensitive to chloride levels and are inactivated by chloridefree solutions (Miller and Dacheux. 1976). This is a possible basis for the effects found here. but the site or sites of action or mechanisms for the effects of the chemicals are not directly revealed by these experiments. The present findings do suggest that GABA, which is known to be sequestered in horizontal cells of the frog. can participate in lateral interactions mediated by these cells. Further study of the effects of chemicals on receptors and horizontal and bipolar cells is now indicated to show more precisely how GABA is utilized in the outer retina.
Mooney R. D. (1978) GABA-mediated control of transient signals in the inner retina. Brain Rrs. I45 97-115. Moore C. L. and Gruberg E. R. (1974) The distribution of succinic semialdehyde dehydrogenase in the brain and retina of the tiger salamander (.-lruh~sto~~~u riyrinum). Brain Rrs. 67. 467178. Proenza L. IM. and Burkhardt D. A. (1973) Proximal negative response and retinal sensitivity in the mudpuppy. Nrcrurus mcu/osus. J. Nrurophxsiol. 36. 502-j 18. Voaden M. J.. Marshall J. and Murani N. (1974) The uptake of ‘H-gamma-aminobutyric acid and ‘H-glycine by the isolated retina of the frog. Brain Rex. 67. 115-132. Werblin F. S. (I9741 Control of retinal sensitivity II: lateral interactions at the outer plexiform layer. J. yen. Physio/. 63. 62-87.
dckno~ledyrrrlenrs-This work was supported in part by grants from the National Institutes of Health (EY 00406) and from the Graduate School of the University of Minnesota to Dr D. A. Burkhardt. I am most grateful for Dr Burkhardt’s-support and guidance in this study.
Werblin F. S. and Dowling J. E. (1969) Organization of the retina of the mudpuppy. Necturus n~aculosu~ II: Intracellular recording. J. Neurophysiol. 32. 339-345. Westheimer G. (1965) Spatial interaction in the human retina during scotopic vision. J. P~WJ~.. Land. 181. 88 l-894.
REFERESCES Baylor D. .A.. Fourres M. C. F. and O’Bryan P. M. 11971) Receptive fields of cones in the retina of the turrle. J.
Physiol..
Land. 214. 265294.
Brooks B. and Holden A. L. (19711 Centre and surround influence in the proximal negative response of the pigeon retina. J. Phvsiol.. Land. 239. 15-29. Burkhardt D. A. (19701 Proximal negative response of frog retina. J. SrurophJsiol. 33. 405-X0. Burkhardt D. A. (1974) Sensitization and centre-surround antagonism in .Vrcrurrts retina. J. Ph.r.siol.. Land. 236. 593610. Burkhardt D. A. and Mooney R. D. (1977) Electrical responses in the inner retina of teleosts and reptiles. Vision Rer. 17. 151-156. Graham L. T. Jr (1972) Intraretinal distribution of GABA content and GAD acrivity. Brain Rrs. 36. 476-479. Karwoski C. J. and Burkhardt D. A. (1976) Ganglion cell responses of the mudpuppy retina to-flashing and moving stimuli. Vision Res. 16. 1483-1496. Matsumoto N. and Naka K.-I. (1972) Identification of intracellular responses in the frog retina. Brain Rrs. 42. 59-7 1. Miller R. F. and Dacheux R. F. (1976) Synaptic organization and ionic basis of on and off channels in mudpuppy retina I. Intracellular analysis 0; iiioriue-sensitive elcctrogenic properties of receptors. horizontal cells. bipolar cells. and amacrine cells. J. ym. Phpiol. 67.
639-659.
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GABA AND THE LATERAL SPREAD OF TONIC ACTIVITY IN FROG RETINA RICHARD D. MOONEY’ Vision Laboratory. Department of Psychology. University of Minnesota. Minneapolis. U.S.A. (Received
5 June
1978)
Abstract-The presence of GABA in association with horizontal cells of the frog retina suggests it may play a role in signal transmission by these cells. The actions of GABA and chemical treatments antagonistic to its effects were studied in the perfused frog eyecup on sensitization. a tonic. lateral effect produced by the horizontal cells. It was observed that GABA facilitated and bicuculline and chloride-free solutions depressed sensitization. Thus. GABA appears to be involved in lateral signal processing in the outer plexiform layer. Key Words-GABA: tions in the retina
sensitization: intraretinal recording: horizontal cell circuits: frog: lateral interac-
INTRODUCIION The center-surround organization of bipolar cell receptive fields in the retinas of amphibians results
in part from lateral conduction of tonic peripheral activity through networks of horizontal cells (Burkhardt, 1974; Matsumoto and Naka. 1972: Werblin and Dowling. 1969). Recent histochemicai work in amphibians suggests that gamma-aminobutyric acid (GABA) may have some function in synaptic transmission by horizontal cells. Endogenous stores of GABA and its main synthetic enzyme have been found in the region of the frog retina which contains mainly horizontal cells (Graham, 1972). Some horizontal cells incorporate exogenously applied GABA (Voaden et aI., 1974). One of the intermediate enzymes associated with degradation of GABA ultimately to succinate, succinic semialdehyde dehydrogenase, has been localized in the region of photoreceptor terminals where close contact between receptors, bipolar, and horizontal cells exists (Moore and Gruberg, 1974). The purpose of this report is to show that tonic, laterally conducted signals through horizontal cells are changed by chemical treatments which are known to alter the efficacy of GABA-sensitive synapses. The tonic lateral activity which was studied is the sensitization effect which is present in bipolar and ganglion cells of the mudpuppy (Burkhardt. 1974; Karwoski and Burkhardt, 1976; Werblin, 1974) and the proximal negative response (PNR) of the mudpuppy and frog (Burkhardt, 1974). This effect is based on a stimulus configuration used extensively in psy chophysics (see, for example, Westheimer, 1965) to compare the effectiveness of small and large adapting spots. When the small adapting spot is present, the amplitude of a response evoked by a small centered test flash is depressed. Subsequent enlargement of the I Reprint requests should be sent fo: R. Mooney. Department of M.C.D. Biology. University of Colorado. Boulder, CO 80309. U.S.A.
spot causes an increase in the response to the test flash. The enhancement of the response to the test flash which is produced by expanding the adaptive field is the sensitization effect. The extent of spatial summation which is involved in sensitization is similar to the extent of horizontal cell receptive fields in the mudpuppy and greatly exceeds the receptive fields of receptors or bipolar cells. Thus. adapting
sensitization probably arises from the influence of horizontal cells on receptors or bipolar cells (Burkhardt, 1974).
METHODS Isolated
retina-eyecup preparations of frogs (Ranu maintained in a perfusion system by an oxygenated Ringer’s solutidn buffered at pH 7.5. Solutions of different ionic composition or to which certain chemicals had been added could be switched into the perfusion system during stable electrical recording from the retina Micropipette electrodes (about I pm tip diameter) were filled with the Ringer’s solution and used to record retinal potentials. Records of light-evoked responses were stored on magnetic tape after d.c. amplification. Other details of the preparation and recording system have been described elsewhere (Mooney, 1978). The PNR was used to monitor sensitization. The PNR is a localized extracrllular. graded potential found maximally in the region of the inner plexiform layer of all vertebrate classes (Burkhardt and Mooney, 1977). It has been well characterized in the frog and mudpuppy (Burkhardt. 1970; Proenza and Burkhardt, 1973). The PNR was selected here because it could be recorded in most prep arations for many hours. and it showed high and relatively constant contrast sensitivity to light flashes during this time. Such stability was necessary for reliable observation of the effects of chemical treatments. A four-channel optical stimulator produced the stimulus shown schematically in Fig. l(A). Here the test flash (a 0.10 mm spot) which was used to evoke a PNR is presented on a steady, central. adapting background (a 025mm spot). The adapting illumination in the surround is an annulus (0.48 mm inner diameter, 1.3 mm outer diameter) concentric with the central adapting spot and rest flash. pipiens)
501
were
502
RKHARD D. M’IOO\EI
Fig. I. A. Schematic representation of the stimuius For sensitization seen in cross section. Intensity (actually retinal iiluminance) is expressed here on a scale with 10.0 log units arbitrartly set to equal 3360 lu.:m’. The diffuse background (1.1 log units) covers the entire eyecup. The central background (5.4 log units) is 0.25 mm in diameter and is also continuously present. The test flash. presented every 30 sec. is 0.10 mm in diameter and has an intensity which produces a small response in the presence of the diffuse and central backgrounds (see text). The steady annulus is 0.48 mm inner diameter and 1.3 mm outer diameter. and its intensity is the independent variable. B. Sensitization of PNR and on/off ganglion cell. Negative polarity is up in this and all successive figures. The 400 msec test flash at an intensity of 52log units produced a very small PNR and no spikes in the presence of the 3.5 log unit annulus (lowest response). As the annulus intensity was increased with all other stimulus parameters fixed. both on- and off-responses of the PNR and ganglion cell increased. Thus. at the higher intensities. the annulus produces a lateral spread of activity which influences responses at its center.
The intensity of the adapting annulus could be varied independently of the central adapting spot. The intensity of the annulus was typically increased in fixed steps every 24 min while the test flash was presented every 30 set to evoke a PNR. After a series of steps in annulus intensity, the annulus was extinguished to verify that the PNR to the test flash on the cen:ral adapting spot was the same as it was before the series. The change in PNR amplitude as a function of annulus intensity describes the sensitization effect. After a control sensitization function was thus obtained. chemicals were introduced into the perfusate. When responses were well stabilized, about 15 min later. the sensitization trials were repeated. The intensity of the test flash was adjusted so that the response amplitude produced by the flash on the central adapting spot was ap proximately the same for each sensitization series. This provides a common base point (labeled “B” in Figs 2 and 3) for all sensitization functions in each preparation. By referencing the sensitization curves to the same amplitude response, the effects produced by the chemicals represent changes in sensitization and not simply changes in the PNR. RESL!LTS
Figure l(B) shows a typical example of sensitization over a limited range of annulus intensity for the stimulus represented in Fig l(A). In this preparation, the graded PNR was recorded simultaneously with spikes from an on/off ganghon cell. In the presence
of the central background and a dim annulus (3.5 log units. see legend for photometric scaling), the test flash evoked a very small PNR and produced no spikes from the ganglion cell. In this case the PNR amplitude was the same as when no annulus had been present. Raising the annulus intensity in discrete steps caused the PNR to increase in amplitude (both on and off responses) and produced a similar sensitization of the on/off spike discharge. The effect on the ganglion cell is particularly striking The increased adapting light in the region surrounding the cell’s receptive field center has altered the cell’s output from no response to a fairly vigorous response. Figure 2 shows a comparison of the sensitizing effects of annuli on a preparation during perfusion with Ringer’s solution in the control condition (left) and then during exposure to a 0.1 mg/ml GABA solution (right). For the lowest traces, marked “B”, the annular background is absent, and the intensity of the test flash on the central adapting spot was adjusted to give a response of about 0.1 mV. As the annulus intensity was then increased (upper traces). sensitization of the PNR in the presence of GABA increased by a greater extent than that of the control condition. The effects of chemicals on sensitization were studied on 70 preparations. In 24 retinas a systematic procedure was used to gather data for quantitative com-
GABA and the lateral spread of tonic activity control
503
GABA
109 annulus intensity /%A
5.5
4.9
4.5
3.9
B
Fig. 2. Effects of GABA on sensitization. A l.Osec flash is indicated by lines below the responses. Test flash intensity was 5.5 log units during the control series (right column) and 6.0 log units during treatment with 0.1 mg,‘ml GABA (left column) to produce approximately 0.1 mV responses in the bottom responses during which no annulus was present (level B). The annulus intensity was increased in steps, and the test flash presented I min after each annulus increment. This figure shows that sensitization is enhanced by GABA solutions. parisons. Some of these results are presented graphically in Fig. 3. Eight levels of annulus intensity were presented in ascending order with four identical test flashes at each level. The final three responses at each annulus intensity were measured, and the median amplitude value was plotted as a function of annulus intensity. Sensitization is identified in these graphs as a gradual increase of response amplitude up to a
maximum with increasing annulus intensity. Response amplitudes eventually start to decline at higher annulus intensities (above 5.5 log units in Fig. 3). This is most likely a result of stray light from the annulus depressing the center’s sensitivity (Burkhardt, 1974), for it can be seen in all the curves of Fig. 3 independently of response amplitude and whether or not sensitization is present at lower intensities.
.
control
3
Cl-d.fici.nt
0 rash
LOG
ANNULUS
INTENSITY
Fig. 3. Effects of various chemical treatments on sensitization. In each preparation the test Rash intensity was adjusted to equate the responses at level B (no annulus). Thus. the extent of sensitization. as revealed by the increase in response amplitude. may be directly compared between conditions in each preparation. (A) GABA at 0.1 mg/ml (filled squares) enhances sensitization. The GABA curve rises more abruptly and peaks at a higher amplitude than the control curve (filled circles). Bicuculline N-methyl bicuculline iodide) at 0.01 mg/ml depressed sensitization below the control level (open squares). After return to Ringer’s solution, sensitization recovered (open circles). Test flash intensities in log units were: control 5.3. GABA 5.7, bicuculline 5.2 wash 5.4. (B) Chloride-free solution (open squares) strongly depressed sensitization from control levels (filled circles). but return to normal Ringer’, solution does not produce recovery of sensitization in this preparation. Test flash intensities were: control 5.5. chloride-free 6.0, wash 5.5. (C) &uanidinopropionic acid at 0.1 mg/ml (open squares) enhanced sensitization. Test gash intensity was 5.5 log units for all curves. Y.R. 19,5--c
3X
RICHARD D. kf00hEI
Figure 3(A) confirms and extends the findings in Fig 2. Here a 0.1 mg, ml GABA solution increased sensitization in a preparation in which the control effect is already strong. In 17 preparations which showed normal control sensitization functions. GABA solutions between 0.05 and 0.2 mg’ml produced a 10-120°~ (average 409,) increase in the peak value of the sensitization curve. For unknown reasons. not all eyecups with otherwise normal ERG. PNR. and ganglion cell responses showed sensitization effects. as was also found earlier (Burkhardt, t974). and most of these were not tested further. In several of these retinas. however. GABA solutions of 0.1 mgiml clearly produced sensitization effects to the annulus, and in the other cases. the effect was still absent after the application of GABA. At GABA concentrations above 0.5 mg’ml. the PNR often gradually decreased in amplitude. and quantitative study was not feasible. However, the simple procedure of expanding the central background spot from 0.25 to 1.0 mm gave a quick evaluation of the extent of sensitization. This test showed that the higher concentration solutions did not enhance sensitization and often reduced it. Following the GABA series of Fig. 3(A). a solution (0.01 mg/ml) of bicuculline. a potent antagonist of GABA in the frog retina (Mooney. 1978). was introduced and markedly decreased sensitization below control levels. After the bicuculline was removed from the perfusing solution. sensitization returned (open circles). This effect was found consistently for bicuculline solutions at this concentration or stronger and for 0.05-0.1 mg/ml solutions of picrotoxin, another GABA antagonist. On the assumption that permeability to chloride ions might be involved in the effects of GABA and bicuculline seen in Fig. 3(A). experiments were conducted in which chloride levels were reduced. In Fig. 3(B). sensitization was tested during depletion of chloride ion (chloride was repiaced with sulfate ions, and the solution was osmotically balanced with sucrose). As seen with bicucul~ne. sensitization is reduced or abolished by chloride-free solutions. This change was seen consistently in over 20 preparations. After restoration of the standard chloride level (114 mM), the response in Fig. 3(B) was normal in every respect except that sensitization did not recover. This was typical of other preparations which had been exposed to chloride-free solutions for long periods of time. Sensitization was also severely reduced or abolished to shorter exposures of chloridefree solution and to partially depleted solutions which lacked 66:,; or 7%; chloride ion. In these cases, recovery was possible following return to the normal Ringers. The effect of ~-guanidinopropio~ic acid, an amino acid analogue of GABA, was similar to that of GABA: at 0.1 mg/ml it enhanced sensitization in six preparations (Fig. 3C). and at 0.5 mg/ml it decreased sensitization. It could also be substituted for GABA at equal concentrations without producing any further change in the sensitization level achieved during exposure to the GABA solution. Glycine seemed to have little consistent effect, except at high concentrations (above 0.5 mg/ml), which depressed sensitization. Likewise, no consistent effects of strychnine. a
gfycine antagonist in the frog retina (Mooney. 197~1. could be identified. ,The failure of the lower concentration (0.1 mg,mll glycine solutions to enhance sensitization was examined thoroughly in tive preparations in which sensitization had been reversibly increased by the 0.1 mg ml GABA solutions. Again. no noticeable effect of glytine was found. These trials constituted a control of the depressive effects of GABA which reduced the PNR amplitude and necessitated increasing the test flash intensity to obtain matched responses to start each sensitization series. The amplitude of the PNR was depressed also by glycine comparably to the effects of GABA. and yet there was no change in sensitization starting from the matched initial responses. Thus, the increase in sensitization which was found during the application of GABA solutions is probably unrelated to the procedure of increasing the test flash intensity to match the initial responses.
DiSCCSSIOS
The essential interaction in sensitization is an antagonism of the centra1 adapting spot by the steady annulus. The result of this combination is found at least as early in the visual pathway as the bipolar cells (Burkhardt, 1974: Werblin. 1974). The spatial extent that is involved suggests that the horizontal cells must provide the surround component by either feedback (Baylor et al., 1971) or feedforward (Werblin and Dowting, 1969) pathways. The sensitivity change produced by horizontal ceils in bipolar cells is seen at successive levels as a change in sensitivity of the PNR and on/off ganglion cells (Burkhardt, 1971). If there are no subsequent interactions between the steady annulus and either the central adapting spot or test spot. then sensitization of the PNR can be used as an index of the change mediated at the outer ‘plexiform layer. Several control experiments were done to assess whether there were any subsequent tonic interactions in the PNR. At the light levels which produced sensitization, a steady annulus in the absence of the central adapting spot had no measurable effect on the PNR. However, at higher annulus intensities (above %56.0 log units), stray light from the annulus was appreciable and adapted the response evoked at its center. Secondly, no measurements of sensitization were made for several minutes after the central adapting spot was turned on and at least 1 min had passed after the annulus intensity had been incremented. At this time there was no measureable contribution of either steady spot or annulus to the recorded potential, and the amplitude of the PNR to Hashes 30 set apart was essentially constant. Thus, no tonic centersurround effects are evident in the PNR, and the steady annulus does not appear to interact with the test spot. These findings are in agreement with results in the pigeon (Brooks and Holden. 1974). and suggest that the tonic interactions between the central spot and annulus must occur distally to the PNR. Therefore, a change in sensitivity of the PNR due to sensitization is a reasonable indication of the qualitative change in sensitivity established at the outer plexiform laver. *
GABA
and the
505
lateral spread of tonic acrivir)
The method used here fo measure sensitization was chosen to diminish the possibility that chemical effects on the PNR itself would alter its properties as an index of sensitivity. Control and chemically treated trials were compared on the basis of equivalent initial (and final) responses to the test flash on only the central adapting spot. Changes in light intensity of the size needed to match response amplitudes did not change the magnitude of sensitization in control trials. In part this was probably because the PNR’s were only about half peak amplitude at the greatest extent of sensitization. In addition, test spots of 0.1 mm, which were used here, have the same proportionate effect on PNR amplitude in control and chemically treated trials (Mooney. 1978). Thus. the PNR can be used to provide a faithful index of sensitivity changes due to sensitization for both control and chemical treatments. In summary. this paper shows that sensitization is enhanced by lower concentrations of GABA. In contrast, glycine did not enhance sensitization. while the GABA-antagonist bicuculline and chloride-free solutions produce a decrease in sensitization. Higher concentrations of all the amino acids tested also reduce sensitization. but this may only reflect the rather fragile nature of the effect (Burkhardt, 1974). In the mudpuppy, it has recently been shown that horizontal cells and the surrounds of bipolar cells are very sensitive to chloride levels and are inactivated by chloridefree solutions (Miller and Dacheux. 1976). This is a possible basis for the effects found here. but the site or sites of action or mechanisms for the effects of the chemicals are not directly revealed by these experiments. The present findings do suggest that GABA, which is known to be sequestered in horizontal cells of the frog. can participate in lateral interactions mediated by these cells. Further study of the effects of chemicals on receptors and horizontal and bipolar cells is now indicated to show more precisely how GABA is utilized in the outer retina.
Mooney R. D. (1978) GABA-mediated control of transient signals in the inner retina. Brain Rrs. I45 97-115. Moore C. L. and Gruberg E. R. (1974) The distribution of succinic semialdehyde dehydrogenase in the brain and retina of the tiger salamander (.-lruh~sto~~~u riyrinum). Brain Rrs. 67. 467178. Proenza L. IM. and Burkhardt D. A. (1973) Proximal negative response and retinal sensitivity in the mudpuppy. Nrcrurus mcu/osus. J. Nrurophxsiol. 36. 502-j 18. Voaden M. J.. Marshall J. and Murani N. (1974) The uptake of ‘H-gamma-aminobutyric acid and ‘H-glycine by the isolated retina of the frog. Brain Rex. 67. 115-132. Werblin F. S. (I9741 Control of retinal sensitivity II: lateral interactions at the outer plexiform layer. J. yen. Physio/. 63. 62-87.
dckno~ledyrrrlenrs-This work was supported in part by grants from the National Institutes of Health (EY 00406) and from the Graduate School of the University of Minnesota to Dr D. A. Burkhardt. I am most grateful for Dr Burkhardt’s-support and guidance in this study.
Werblin F. S. and Dowling J. E. (1969) Organization of the retina of the mudpuppy. Necturus n~aculosu~ II: Intracellular recording. J. Neurophysiol. 32. 339-345. Westheimer G. (1965) Spatial interaction in the human retina during scotopic vision. J. P~WJ~.. Land. 181. 88 l-894.
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