125

Journal of Physiology (1992), 456, pp. 125-142 With 10 figures Printed in Great Britain

RECEPTIVE FIELD ORGANIZATION OF RETINAL GANGLION CELLS IN THE SPASTIC MUTANT MOUSE

BY CHARLENE STONE AND LAWRENCE H. PINTO From the Department of Neurobiology and Physiology, Northwestern University, Evanston, IL 60208, USA

(Received 19 July 1991) SUMMARY

1. We examined the receptive field properties of retinal ganglion cells in the isolated, superfused retinae of spastic mutant mice (B6C3Fe-spa/spa) that did not have the retinal degeneration (rd) phenotype. Glycine receptor density in the spastic mutant is greatly reduced in all areas of the CNS that have been examined. Phenotypically normal litter-mates were used as controls. Radial sections from the retinae of both spastic and normal animals were examined with light and electron microscopy and no differences were observed. The planimetric density of the cell bodies in the inner nuclear layer did not differ between the normal and mutant animals, about 400 cm-2. The absolute dark-adapted sensitivity of spastic ganglion cells was greater (271 + 69-0 impulses quanta-' rod-') than that of normal ganglion cells (47 7 + 10-4 impulses quanta-' rod-'; P < 0-01). 2. Extracellular recordings of retinal ganglion cell responses to circular and annular stimuli, centred on the receptive field, were used to construct peristimulus-time histograms. In normal retinae, an annular stimulus elicited a response that was characteristic of the surround response mechanism of receptive fields with antagonistic centre-surround organization. In the mutant retina, annular stimuli did not elicit a surround-type response; instead, a centre-type response was recorded. 3. Illumination of the receptive field periphery attenuated centre-type responses in ganglion cells from both spastic and normal retinae. Centred circular stimuli of various areas (14, 35, 78, 122, 235, 783 deg2) were presented to the receptive fields. For mutant and normal ganglion cells, the response to the largest stimulus was smaller than that to an intermediate-sized stimulus. 4. The effect of strychnine, a glycine receptor antagonist, on the response to circular stimuli was examined. Very low concentrations of strychnine attenuated the light response in mutant retinae (apparent inhibitory binding constant KI = 8-1 x 10-13 M). In normal animals, the light response was also attenuated by strychnine, but the apparent K3 was much higher (apparent K, = 1 x 10-7 M). 5. In normal ganglion cells, the sustained component of the light response was much more attenuated by strychnine than was the transient component. Interestingly, ganglion cells from spastic retinae did not exhibit a sustained component, even at stimulus luminances that evoked responses near threshold. MS 9569

C. STONE AND L. H. PINTO 6. These results suggest that the generation of surround-type responses may depend upon a glycine receptor or that a glycine receptor may lie in the pathway of the surround-response mechanism and the mechanism generating the sustained component of the centre response. Perhaps the expression of a novel retinal form of the glycine receptor, which is extremely sensitive to strychnine, is unmasked by the spastic mutation. 126

INTRODUCTION

Several lines of evidence suggest that glycine is a mammalian retinal neurotransmitter. Strychnine-sensitive currents induced by glycine were observed in cultured rodent ganglion cells (Tauck, Frosch & Lipton, 1988). Similar currents were recorded in bipolar cells (Suzuki, Tachibana & Kaneko, 1990) isolated from the mouse retina. In the cat retina, high-affinity [3H]glycine uptake labelled about 10 % of cone bipolar cells and 50 % of amacrine cells, mostly small-field amacrines (Jiger & Wissle, 1987). Pourcho & Goebel (1985) found four subtypes of amacrine cells accumulate glycine: A3, A4, A7(AII), and A8. In addition, glycine receptors have been localized to both sublaminae of the inner plexiform layer (Jiger & Wassle, 1987), the site of synaptic input to the ganglion cells. In the cat, rod bipolars do not synapse directly onto ganglion cells but have the AII(A7) amacrine cell interposed (Kolb & Nelson, 1983; Wdssle, Schafer-Trenkler & Voigt, 1986). For the off-centre ganglion cell, the synapse between the ganglion cell and the AII amacrine is glycinergic and presumably is required for signal transfer under scotopic conditions. A mutant mouse, spastic, is characterized by reduced density of glycine receptors (glyR) in the central nervous system (Becker, Hermans-Borgmeyer, Schmitt & Betz, 1986). In the spastic mouse, [3H]strychnine binding in spinal cord homogenates indicates that glyR density is 20 % of the value for normal litter-mates (Becker et al. 1986). Although glyR density is reduced in the mutant, ligand affinity and subunit structure appear to be unaffected. A similar reduction in glyR density has been reported in other areas of the CNS (White & Heller, 1982), including the retina (Lorry, 1985). Becker et al. (1986) suggest that the spastic mutation involves a regulatory, not a structural, gene. We investigated the effect of reduced glyR density associated with this mutation on receptive field characteristics of retinal ganglion cells. We compared extracellularly recorded light responses of ganglion cells in retinae isolated from spastic mice and their normal litter-mates, before, during and after the administration of strychnine. To increase the probability of observing alterations in receptive field properties in the spastic mutant, we recorded under scotopic conditions. We found a reduction in the strength of the surround response mechanism in the mutant retina. In addition, strychnine sensitivity in the mutant was enhanced, suggesting that a novel isoform of the glycine receptor may be expressed in the mutant retina. METHODS

Animals Heterozygous, hybrid breeder pairs of mice (B6C3Fe-spa/ +; derived from a cross between B6C3Fe-spa/spa and B6C3Fe- + / +) were obtained from the Jackson Laboratory (Bar Harbor, ME, USA). Pups were tested for the spastic phenotype at three weeks of age; homozygous spa/spa

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pups displayed tremor and increased righting time (White & Heller, 1982). Normal litter-mates (+ / -) were used as controls. Members of the C3HFe parent strain all carry the mutant allele at the retinal degeneration (rd) locus (LaVail, 1981). Therefore, all experimental animals were screened for retinal degeneration by observing pupillary constriction in response to a dim full-field white light using an infrared-sensitive television camera (Mitchiner, Pinto & Vanable, 1976) and animals with retinal degeneration were excluded from the current study. Experimental animals were two to three months old, with the exception of two experiments performed on retinae from normal mice on postnatal day 14 (PND14). We found PND14 to be the youngest possible age for ganglion cell recordings in this study. This is the youngest age at which the spa8tic phenotype can be identified. Moreover, at this age the normal mouse retina expresses opsin at adult levels (Bowes, van Veen & Farber, 1988) and can reliably be screened for retinal degeneration (LaVail, 1981). We also performed control experiments using the standard +/+ C57B1/6J mice and found no difference in receptive field size or organization when compared to our hybrid control. We therefore concluded that the hybrid control was adequate.

Preparation of retina Mice were dark-adapted for 4-8 h prior to the experiment (Balkema & Pinto, 1982). After cervical dislocation under dim red illumination, the eyes were removed and placed in oxygenated mouse Ringer solution containing (mM): NaCl, 120; MgCl2, 0-6; CaCl2, 1-9; KCl, 7-5; NaHCO3, 24; HEPES, 2-4; dextrose, 11 (Po2 570 mmHg, Pco 40 mmHg; pH 74; 290 mosmol kg-') at room temperature (Balkema, Mangini & Pinto, 1983). The retinal dissection was performed using infrared illumination and a dissecting microscope equipped with infrared image converters. The lens was removed through a slit cut across the cornea. The sclera-retinal pigment epithelium was grasped at the corner of the slit and was gently torn to pull it away from the retina, isolating the retina with minimal stretching or contact with instruments. The retina was cut in half with scissors, transferred to a small chamber (volume 0 5 ml) fashioned on a microscope slide, and loosely pinned with the vitreal surface up. This chamber was placed on the stage of a compound microscope and superfused (oxygenated mouse Ringer solution, 2 ml min-', 35 + 0 5 00). The retina was observed with infrared illumination and an infrared-sensitive television camera (RCA T1005, Lancaster, PA, USA). Strychnine (Sigma, St Louis, MO or American Tokyo Kasei, Portland, OR, USA) was applied in the superfusate, using aliquots of a stock solution that were stored at -20 00 in the dark and were thawed for immediate (< 3 h) use. Strychnine was applied only once per retina.

Recording We recorded extracellularly from ganglion cells that were 200-500 ,um (corresponding to 7-17 deg) from the optic disc using tungsten-in-glass electrodes (Merrill & Ainsworth, 1972). Action potentials were displayed on an oscilloscope and played over an audio monitor. The spikes triggered a transistor-transistor logic (TTL) converter and these TTL pulses were collected by an IBM-AT computer. A trial consisted of at least twenty presentations of the stimulus (1 s); the responses (gathered in 10 ms bins) were averaged, smoothed by fitting a 21st order polynomial function (Savitzky & Golay, 1964), and used to generate peristimulus time histograms. For an oncentre cell, the light response was calculated as the number of extra spikes in the response (i.e. by subtracting from the area under the response during the stimulus the area from an equal time of dark discharge). For off-centre cells, we made similar calculations for the off-discharge or the deficit of spikes in the response (by subtracting the area under the response during the stimulus from the area of an equal time of dark discharge). We used the larger of these two measures for an individual off-centre cell. Recording protocol Experiments using spastic retinae were always preceded on the same day by experiments using normal retinae, and the same solutions, stimulus conditions, and electrode were used for both.

Visual stimuli Two types of visual stimuli were used; (1) circular spots and annuli and (2) sinusoidal gratings (Stone & Pinto, 1991). Both were focused upon the photoreceptor outer segments using the microscope condenser (0415 numerical aperture). The flashing circular stimuli were provided by moveable masks mounted on an optic bench with a 60 W incandescent source and neutral density filters. An

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interference filter produced a stimulus of 660 nm that was visible to an infrared-sensitive camera. There was no steady background illumination. Sinusoidal gratings (2 Hz temporal frequency) were generated by a programmable pattern generator (Neuroscientific Inc., Farmingdale, NY, USA) that provided Z-axis modulation to a display oscilloscope (P31 phosphor, Tektronix 608, Beaverton, OR, USA). The grating pattern subtended about 35 deg on the retina. The mean retinal illumination was 20-2 cd m-2 (this value is just above the inflexion point of the increment sensitivity curve generated from ganglion cell responses in the normal isolated mouse retina; Balkema et al. 1983). The grating was presented to the receptive field at various contrasts for several spatial frequencies and the amplitude of the fundamental component of the response was calculated to generate a spatial frequency tuning curve. Receptive field mapping and classification Receptive fields were mapped using small flashing spots (diameter 3 deg, 4 Hz). Flashing circular or annular stimuli of various sizes were then centred in the receptive field. Anatomy Animals of normal and spastic phenotype were deeply anaesthetized with ether prior to intracardiac perfusion with buffered 2% paraformaldehyde and 2-5 % glutaraldehyde. The eyes were removed, bisected, and prepared for electron microscopy (LaVail & Battelle, 1975). Thick sections were first examined using light microscopy and an area 1 mm from the optic nerve was selected for thin sectioning and further examination with low power (4600 x ) electron microscopy. The planimetric density of cell bodies in the inner nuclear layer was measured from mosaic electron micrographs (Fisher, 1979). These measurements were corrected for non-centred sections (Coupland, 1968) and section thickness (Abercrombie, 1946), including the Floderus modification (Floderus, 1944). RESULTS

Anatomy We examined semi-thin plastic sections from four animals of normal and spastic phenotype for cytoarchitecture and pattern of retinal layering. This examination did not reveal any alterations in the spastic mutant retina. For four animals of each phenotype, we performed an electron microscopic examination of the cellular morphology. We examined the thickness of the retinal layers, integrity of the cellular nuclei and mitochondria, appearance of the cytoplasm, morphology of the photoreceptor outer segments, and searched for the presence of conventional and ribbon synapses and their associated postsynaptic structures in the plexiform layers (Williams, Gherson, Fisher & Pinto, 1985a). We found no differences in these features between the mutant and the normal. In addition, for one animal of each phenotype, we measured the planimetric density (Fisher, 1979) of cell bodies in the inner nuclear layer. The values obtained were similar for the mutant and normal retinae (360/1000 mm2 and 400/1000 mm2, respectively). We also confirmed that the overall morphology of the retinal pigment epithelium (basal infoldings, apical processes, melanosomes, lysosomes, and nuclei) appeared normal in the mutant animal (Williams, Pinto &Gherson, 1985 b). Thus, the spastic mutation does not alter the overall layering and cytoarchitecture of the retina. Types of cells recorded In normal retinae (n = 39) we recorded from ganglion cells with the following types of receptive fields: on-centre (n = 41; 47 %), off-centre (n = 38; 44%), on-off (n = 8; 9%). Using hand-held stimuli, we did not find cells that were directionally selective. The fraction of cells of each type is about the same as that observed in

129 GANGLION CELLS IN SPASTIC MUTANT RETINAE recordings from optic nerve axons in the anaesthetized mouse (Balkema & Pinto, 1982). In spastic retinae (n = 21), we also recorded on-centre (n = 26; 66%), offcentre (n = 10; 26 %), and on-off (n = 3; 8 %) ganglion cells; however, the surround appeared to be reduced in the two former types. All of the ganglion cells studied in both Spastic

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Fig. 1. Peri-stimulus-time histograms from representative on-centre ganglion cells in normal (left) and spastic (right) retinae. The upper histograms were collected in response to a spot of light (3 deg diam; 660 nm) applied to the centre of the receptive field; the lower histograms display the response to a centred annulus (8 deg i.d.; 25 deg o.d.; 660 nm) of the same luminance. Note that the annular stimulus evoked a centre-type response in the mutant. In this and in subsequent figures, the stimulus duration is indicated by the bar above the histograms.

phenotypes discharged in the dark at a rate of 27 + 3 (S.E.M.) spikes s-' with a range of 6-85 spikes s-l. The absolute dark-adapted sensitivity was greater for spastic ganglion cells (271 + 69-0 impulses quanta-' rod-'; n = 28) than for normal ganglion cells (477+10-4 impulses quanta-' rod-'; n = 34; P < 001). The dark-adapted sensitivity for normal ganglion cells in this study is comparable to that measured in intact mice (Balkema et al. 1983). Receptive field organization The finding that the retinal layering and cytoarchitecture were normal in the mutant does not preclude alterations in receptive field organization of the ganglion cells. We therefore compared receptive field organization in retinae from normal and spastic mice by applying circular and annular stimuli. We examined peri-stimulustime (PST) histograms constructed from the responses to centred circular and annular stimuli. On-centre cells from both normal and spastic retinae responded to the circular stimulus with a centre-type response: a transient increase in firing rate at light onset (Fig. 1) and a decrease at light offset. The normal on-centre cell exhibited a surround-type (Kuffler, 1953) response to annular stimuli: a decrease in

C. STONE AND L. H. PINTO firing rate at light onset (Fig. 1) and an increase in firing rate at light offset. In contrast, the mutant retina generated a centre-type response to the same annular stimulus (Fig. 1). No spastic ganglion cell (n = 31) produced a surround-type response to annular stimuli of any size, although 70 % of normal on-centre cells did 130

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0 200 400 200 400 600 800 800 600 0 Stimulus area (deg2) Stimulus area (deg2) Fig. 2. Amplitude of responses evoked by equiluminous circular stimuli centred on the receptive field as a function of stimulus area (14, 35, 78, 122, 235, 366, 783 deg2) for ganglion cells from a normal (left) and spastic (right) retina. The area-response function for the mutant displays the same increase in response for small stimuli and decrease for large stimuli observed in the control. .

.

0

(n = 38). Off-centre ganglion cells were also studied in both phenotypes (n = 33 for controls; n = 10 for spastic). In off-centre cells, most (79%) receptive fields from normal retinae exhibited a surround-type response, while receptive fields in the mutant retina exhibited no surround-type response to annular stimulation. Since annular stimuli never elicited a surround-type response in ganglion cells from the mutant retina, we tested for the presence of another manifestation of the surround: attenuation of centre-type responses. Equiluminous circular stimuli of seven different areas were presented to the receptive field and the amplitude of the response was measured. The response amplitude was plotted against stimulus area (Fig. 2). For both normal (n = 4) and mutant (n = 6) retinae, there was an intermediate-sized stimulus (about 100 deg2, for the retinal eccentricity used in these experiments) that evoked the largest response; stimuli larger than the optimum size evoked smaller responses. This attenuation of the response by larger stimuli is believed to arise because 'outlying regions antagonize the central region and the threshold therefore rises when these are included' (Barlow, Fitzhugh & Kuffler, 1957, p. 341). By this criterion, although not when annuli were used, surround antagonism was found in the receptive fields of both normal and mutant ganglion cells. Transient character of the response waveform We compared the waveforms of the responses of ganglion cells from normal (n = 79) and spastic (n = 36) retinae. For normal ganglion cells (Fig. 3A), the response was less transient with less luminous stimuli, as has been previously observed for cat ganglion cells (Cleland & Enroth-Cugell, 1968). The responses of ganglion cells from

131 GANGLION CELLS IN SPASTIC MUTANT RETINAE spastic retinae (Fig. 3B), however, were very transient for all stimulus luminances. It is unlikely that this difference resulted from a difference in ganglion cell subtype. The majority of normal ganglion cells are of the X subtype (Stone & Pinto, 1992), and eleven of thirteen spastic ganglion cells tested were of the X subtype, as determined by linearity of spatial summation using sinusoidal gratings (EnrothCugell et al. 1983). It will be shown below that strychnine application selectively attenuates the sustained component of the response of normal ganglion cells. Spastic

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Effect of light adaptation We were interested in whether the altered surround responses observed in the dark-adapted retina of the spastic mutant would also be observed under lightadapted conditions. Experiments with light adaptation are difficult in the isolated retina preparation, since photopigment regeneration is incomplete. To overcome this difficulty, we used sinusoidal grating stimuli and maximized the likelihood of observing a surround contribution to the response by using a mean luminance just above the inflexion point of the increment sensitivity curve generated from ganglion cell responses in normal isolated mouse retina (Balkema et al. 1983). We generated spatial frequency tuning curves and examined the amplitude of the curves at spatial frequencies approximately one log unit below the spatial frequency eliciting the maximal response (Stone & Pinto, 1992). An attenuation of the amplitude of the fundamental component in this lower spatial frequency portion of the tuning curve

C. STONE AND L. H. PINTO is believed to indicate the antagonism of the receptive field surround (Linsenmeier, Frishman, Jakiela & Enroth-Cugell, 1982; Enroth-Cugell, Robson, Schweitzer & Watson, 1983). As a measure of the surround contribution, we calculated the ratio of the amplitude of the fundamental component at the lowest spatial frequency to the amplitude of the fundamental at the peak of the tuning curve, defined as the low 132

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Fig. 4. The distribution of low spatial frequency attenuation factors for ganglion cells in normal (top) and spastic (bottom) retinae. This factor, which is a measure of the surround contribution to the response to a drifting grating stimulus (see text for definition), was not distributed differently for the two phenotypes.

spatial frequency attenuation factor. The distribution of this factor was similar for both phenotypes (Fig. 4). In both normal (n = 18) and spastic (n = 15) retinae, for most cells the ratio was close to 1D0, indicating that there was little attenuation of the response at low spatial frequencies and little contribution from the antagonistic surround. Therefore, experiments under light-adapted conditions, using sinusoidal grating stimuli, were not useful in quantifying the contribution of the receptive field surround in the isolated mouse retina. Effect of strychnine The inability to isolate surround-type responses to annular stimuli in the spastic mutant retina suggested that these responses depend upon a glycinergic pathway. We therefore examined the effect of strychnine, a glycine receptor antagonist, on ganglion cell responses to centred circular and annular stimuli in normal mice. The responses of the normal retina to both circular (Fig. 5) and annular (Fig. 6) stimuli

GANGLION CELLS IN SPASTIC MUTANT RETINAE 133 were attenuated during strychnine application. This attenuation was reversible for strychnine concentrations up to 100 nm, although the surround-type response to the annulus frequently exhibited poor recovery. The attenuation of the response to a circular stimulus increased with concentration of strychnine (see p. 136). A given A Normal

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Fig. 5. Effect of strychnine on the response of on-centre cells to a centred spot of light (3 deg diam; 660 nm) in normal (A) and spastic (B) retinae. PST histograms were collected in Ringer solution, during application of 10 nm-strychnine, and again in Ringer solution. Strychnine caused the amplitude of the light response to be attenuated by only 50 % in the normal retina, but eliminated the light response in the mutant retina.

concentration of strychnine produced approximately the same attenuation of the responses to both circular and annular stimuli. Strychnine also altered the discharge rate of ganglion cells in the dark. About half of the cells responded to strychnine with an increase and half with a decrease in dark discharge rate. No consistent change was associated with on- or off-centre cells. We also recorded from retinae isolated from normal mice on postnatal day (PND) 14. Ganglion cell responses to centred spots and annuli at PND 14 were indistinguishable from those of adult retinae (data not shown). In addition, the effect of strychnine on the light response of PND 14 retinae was indistinguishable from that upon adult retinae. Strychnine exerted differential effects on the transient and sustained components of the light response of ganglion cells in normal retinae. The transient was relatively

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134

insensitive to strychnine at a concentration that substantially attenuated the sustained component (Fig. 7). Recovery of the sustained component after wash-out in normal Ringer solution was usually incomplete. We quantified this differential effect by separately integrating the area under the transient and sustained portions (@B

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3 0 1 2 3 Time (s) Fig. 6. Effect of strychnine on the response of on-centre cells to annular stimulation (4 deg i.d.; 25 deg o.d.; 660 nm) in normal (A) and spastic (B) retinae (same cells as in Fig. 5). PST histograms were collected in Ringer solution, during application of 10 nM-strychnine, and again in Ringer solution. The response of the mutant to the annular stimulus was 1

2

eliminated by this concentration of strychnine.

of the response waveform, before and during the application of strychnine. The shoulder of the plateau of the response was considered the boundary between the transient and sustained response components (see shaded portions of top histogram, Fig. 7A). Although we integrated the area under the entire transient component, we integrated the area under only the final half of the sustained component, to obtain a value that was relatively uncontaminated by the transient component. We plotted the relative amplitude of the transient component of the response to a centred spot stimulus in strychnine against the relative amplitude of the sustained component (Fig. 7B); most of the data points lay above the identity line, indicating that the sustained component was more affected than the transient component. Both on- and

GANGLION CELLS IN SPASTIC MUTANT RETINAE 135 off-centre cells were affected similarly, although there may have been a somewhat greater affect of strychnine for on-centre cells. Thus, the sustained component (1) is more sensitive to strychnine in normal ganglion cells and (2) is absent in responses from spastic ganglion cells.

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Fig. 7. Effect of strychnine application on sustained and transient components of the light response of the normal retina. A, PST histograms from an on-centre cell before, during and after strychnine (10 nm) show that the sustained component is decreased during strychnine while the transient component is only slightly affected. B, the relative amplitude of the transient component is plotted against the relative amplitude of the sustained component for both on-centre (0) and off-centre (A) cells. Note that these data fall above the identity line, indicating that the transient component was less affected by strychnine.

Strychnine had different effects on the on- and off-components of the light response of on-off cells (n = 5) from normal retinae. The discharge at light offset (offdischarge) was attenuated by strychnine (10 nM) while the discharge at light onset (on-discharge) was unchanged or enhanced (Fig. 8). If the spastic mutation results solely in the reduction of glycine receptors with normal properties (Becker et al. 1986), then application of strychnine should have as little effect upon the light responses of mutant retinae as it has upon the ventral root reflex in the mutant spinal cord (Biscoe & Duchen, 1986). Surprisingly, the light response to both circular and annular stimuli in the mutant retina was completely abolished by concentrations of strychnine that only modestly attenuated the responses of normal retinae. This is shown in Figs 5B and 6B for 10 nM-strychnine. Attenuation of the light response in the mutant was reversible for strychnine concentrations up to 10 nM.

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Concentration dependence of strychnine effects For the effect of strychnine on responses to a centred spot stimulus we fitted a curve of the form: Percentage response =

1 + ([strychnine]/app K,)m

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Fig. 8. Effect of strychnine on the light response of an on-off ganglion cell from a normal k'tina. PST histograms of an on-off cell before, during, and after the application of 10 nM strychnine. The off-discharge is attenuated during strychnine application but the ondischarge is little changed.

to the data (DeLean, Munson & Rodbard, 1978) for the mutant and normal retina (Fig. 9), where m is the slope of the linear portion of the sigmoidal curve, and 'app K1' is the concentration producing a half-maximal attenuation in the response amplitude. The apparent K, differed between the two phenotypes by almost five log units (apparent K, = 1 x 10-7 M for normal; 8- x 1013M for mutant). There was no significant difference in the slopes of the two curves. In initial experiments to determine the concentration dependence of the attenuation of the light responses by strychnine, we noticed that very low concentrations had a profound effect on the mutant retina. Therefore, we took special precautions to ensure that uncontaminated solutions containing known concentrations of strychnine reached the retina. This was done by arranging two containers for the delivery of superfusate. At the beginning of the experiment both of these containers were filled with normal mouse Ringer solution. We recorded the

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Fig. 9. Plot of mean percentage attenuation of the amplitude of the light response as a function of strychnine concentration; normal retinae (0), spastic retinae (A). The error bars indicate the standard error of the mean and the horizontal line represents no change due to strychnine. The curves show the results of fitting an equation of the form: 100 Percentage

response

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+ ([strychnine]/app

K)m'

to the data, and demonstrate that the mutant ganglion cells strychnine by approximately five log units. The apparent K3 was 1

are more sensitive to x 10-7 M for the normal

and 8-1 10-13 M for the spastic retinae. x

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Fig. 10. Effect of very low strychnine concentration (01 and 1 0 pM) on the response (circular stimulus; 3 deg; 660 nm) of an on-centre cell from a spastic mutant. For explanation of two control histograms, see text.

137

C. STONE AND L. H. PINTO light response while solution was flowing from the first container and noted the size of the response (Fig. 10). We then superfused the retina with the Ringer solution from the second container, recorded a second response to the same stimulus and measured its size (Fig. 10; control 2). Only if the amplitudes of these two control responses agreed within 10 % did we continue the experiment. While the retina was superfused from the second container, a small volume of strychnine stock solution was pipetted into it and mixed well. The response was again recorded (Fig. 10) and further aliquots of strychnine were added to the container. Finally, we checked for reversibility by recording the response while strychnine-free Ringer solution flowed from the first container (Fig. 10). These experiments demonstrate that strychnine concentrations as low as 1 pM attenuated the response in the mutant by nearly 50 % (Fig. 10). 138

DISCUSSION

Our experiments revealed the receptive field organization was altered in spastic ganglion cells. No surround-type response was observed in the mutant to annular stimuli that did evoke surround-type responses in the normal retina. However, the surround mechanism in the mutant was capable of attenuating responses generated by the centre mechanism. These results suggest that generation of surround-type responses depends on a glycinergic synapse. In addition, very low concentrations of strychnine (ca 1 pM) attenuated the light response in the spastic retina. This result was surprising because the spastic mutation has been shown to result in reduced glycine receptor density in the brain, spinal cord (White & Heller, 1982; Becker et al. 1986) and retina (Lorry, 1985). This increased sensitivity is not consistent with a simple decrease in glycine receptor density and suggests that the mutant retina expresses an isoform of the glycine receptor with greater strychnine affinity than found in the normal retina. There are two manifestations of the surround mechanism in receptive fields having centre-surround organization. The first is to generate a surround-type response, for example, an on-centre cell responds to an annulus with a decrease in firing rate at light onset and an increase in firing rate at light offset (Kuffler, 1953). A second manifestation of the surround is an attenuation or antagonism of the excitatory centre response. An example of this in an on-centre cell is the diminution of the ondischarge evoked by a centred circular stimulus by simultaneous application of a stimulus to the receptive field periphery (Barlow, Fitzhugh & Kuffler, 1957). In the mutant retina, a surround-type response could not be elicited by stimulating the periphery of the receptive field; instead, a centre-type response was observed. This suggests that the surround response requires a glycinergic synapse that is absent in the mutant. In contrast, attenuation of the centre response by stimuli that fell on the periphery of the receptive field (Fig. 2) occurred in the mutant, suggesting that this manifestation of the surround does not require a glycinergic synapse. The apparent K1 for strychnine was five log units lower in the mutant retina than in the normal retina. The increased strychnine sensitivity observed in the mutant retina is in contrast with findings in the spinal cord (Biscoe & Duchen, 1986). In the spinal cord, responses were altered by the same concentration of strychnine in both mutant and normal animals, and reduced glycine receptor density was a sufficient

139 GANGLION CELLS IN SPASTIC MUTANT RETINAE explanation for the result. The increased sensitivity to strychnine in the retina of the spastic mutant cannot be explained solely by a reduction in glycine receptor density, although it is not inconsistent with reduced density. The increased sensitivity may be due to the presence of a strychnine binding site with higher affinity in the mutant retina than in the normal retina. Such high affinity strychnine binding could occur in two possible ways: (1) by increased expression in the mutant of a glycine receptor with high strychnine affinity that is expressed minimally in the normal retina or (2) by expression of a novel glycine receptor in the mutant, through a mechanism such as alternative splicing of gene products that does not occur in the normal retina. The present experiments cannot distinguish between these possibilities. The present study cannot eliminate two alternate explanations for the decrease in the apparent K1 in the mutant. First, the existence of an amplification step between ligand binding to the glycine receptor and the physiological response could generate ' spare receptors' and these could be eliminated in the mutant, leading to an apparent increase in the potency of strychnine. Second, if the spastic mutation resulted in reduced glycine concentration in the synaptic cleft, the apparent potency of strychnine would be increased. Since the current study did not attempt to measure glycine receptor occupancy or glycine concentration, these explanations cannot be excluded. Strychnine exerted differential effects on components of the light response. In onand off-centre cells, the sustained component was attenuated by strychnine while the transient component was relatively strychnine insensitive. In on-off cells, strychnine attenuated the off-discharge only. These results indicate that the sustained component of the light response and the off-discharge of on-off cells depend upon a glycinergic synapse. In contrast, the transient component of on- and off-centre cells, and the on-discharge of on-off cells are not sensitive to strychnine and therefore do not depend on a glycinergic synapse. Chen & Linsenmeier (1989) reported that sustained and transient response components of off-centre retinal ganglion cells in the cat were pharmacologically distinct. We expected the responses of spastic ganglion cells to imitate responses from normal ganglion cells during strychnine application. Consistent with this expectation, we observed that the sustained component of the light response was more sensitive to strychnine in normal ganglion cells and was absent in responses from the mutant. In addition, the surround-type response of normal ganglion cells to annular stimulation exhibited poor recovery following moderate (1-100 nm) concentrations of strychnine, and this surround-type response was absent in the mutant. Thus, strychnine application to normal retinae mimicked the effect of the spastic mutation. A number of studies using various preparations have found differences in the effect of strychnine upon the classes of ganglion cells: X and Y, or on- and off-centre cells. In the superfused cat eye-cup 2-40 tM strychnine attenuated the response to an annulus superimposed upon a steady spot for on-centre X cells, but had no effect on the light response of X-off-centre or Y cells (Saito, 1981; Saito, 1983). However, studies utilizing ionophoresis of strychnine in the intact cat retina (Ikeda & Sheardown, 1983; Muller, Wassle & Voigt, 1988) did not reveal this distinction between X and Y cells. Instead, these investigators reported an effect of strychnine for off-centre ganglion cells only. In a superfused rabbit retina preparation (Jensen,

C. STONE AND L. H. PINTO 1991), strychnine enhanced the surround response in off-centre cells. In the intact rabbit retina, Caldwell & Daw (1978) found a strychnine-induced decrease in the transient light response of all ganglion cell classes. Results of the current study showed attenuation by strychnine of all light responses of all ganglion cells studied. There may be several reasons for the differences between our results and those found in other studies. High concentrations (> 100 /M) of strychnine are known to have non-specific effects upon other neurotransmitter receptors (Choi & Fischbach, 1981; Barron & Guth, 1987). Strychnine (20 /tM) was reported to block GABAinduced currents in patch-clamp recordings of isolated mouse ganglion cells although, in concentrations below 5 /M, strychnine was a specific blocker of glycine-induced currents (Tauck et al. 1988). Ionophoretic application of strychnine will invariably result in an elevated concentration near the tip of the pipette, and may result in effects that are not specific for the glyR. Vitreal application of strychnine will result in an unknown intraretinal concentration. None of these problems occurred in the present study, since we superfused both photoreceptor and vitreal surfaces and used known concentrations of strychnine that exceeded 10 nm in only a few instances. Some differences can be expected, a priori, between the effect of strychnine application on normal retinas and the effect of the spastic mutation. The spastic mutant may exhibit developmental compensation for reduced glyR density. For example, in the spinal cord, GABA receptor density is greater in spastic mutants than in normal litter-mates (Biscoe & Fry, 1982). It is also possible that the spastic mutation affects a subpopulation of glyRs and/or that strychnine affects a subpopulation of glyRs. This would complicate comparison of the physiological effects of the two methods (i.e. strychnine or the spastic mutation) of reducing glycinergic input to the ganglion cells. The spastic mutant may be of use for understanding the neuronal circuitry of the retina. Identification of the site of action of the spastic gene may provide information useful in understanding the mechanism for surround-type responses and the mechanism generating the sustained component of the light response. Our results (Fig. 9) indicate that the spastic mutant retina may express an altered molecular form of the glycine receptor, and the retina may therefore also be useful in understanding the molecular mechanism for the spastic mutation. 140

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