- Email: [email protected]
Intravitreal kainic acid permanently eliminates off-pathways from chicken retina
Neurosc/ence Lette~, 36 (1983) 249-253 Elsevier Scientific Publishers Ireland Ltd.
249
INTIgAVlTREAL KAINIC ACID PERMANENTLY ELIMINATES OFF-PATHWAYS FROM CHICKEN RETINA
D.R. DVORAK and l.O. MORGAN
Department o f Behavioural Biology, Research School o f Biological Sciences, Australian National University, PO Box 473 Canberra City, ACT 2601 (Australia) (Received January 20th. 1983; Accepted February 22nd. 1983)
Key words: retina - kainic acid - OFF-responses - bipolar cells - amacrine cells -tectum
Kainic acid rapidly suppresses both ON- and OFF-visually evoked responses in the leCtUreafter injection into the vitreous of chicken eyes. One week after injection the OFF-responses were still suppressed, while ON-responses were detected of similar magnitude but of simplified form to those of control eyes. It is suggested that kainic acid selectively destroys OFF- rather than ON-bipolar cells, while the simplification of the ON-responses is due to the destruction of amacrine cells.
lntravitreal kainic acid causes species-specific patterns of cell death in the retinas of many vertebrates [6]. in avian retinas there is a hierarchy of cell death, such that most amacrine cells and two-thirds of the bipolar cells are destroyed by injections of around 40 nmol of kainic acid; these cells and horizontal cells are destroyed by injections of around 120 nmol of kainic acid; but the Muller glial cells, ganglion cells and photoreceptors survive exposure to as much as 600 nmol of kainic acid [3, 7, I I]. We have proposed that the pattern of toxicity results from an interaction of kainic acid with kainic acid-preferring receptors on horizontal cells and bipolar cells, depolarizing horizontal and OFF-bipolar cells, hyperpolarizing ON-bipolar cells, and ultimately killing those cells which are depolarized [5, 6]. Amacrine cell death could then be associated with abnormal activity in the OFF-bipolar cells resulting from their depolarization by kainic acid. If this hypothesis is correct, the retinal OFF-bipolar cells should be eliminated from retinal circuits, in this paper we show that intravitreal kainic acid eliminates visually-evoked responses in the optic tectum of the chicken within minutes of the injection. One week later, ON-responses recover, but the OFF-responses appear to be permanently abolished. Experiments were performed on 4-6-week-old chickens anaesthetized with 200/0 urethane (6-10 ml/kg, i.p.). The animal was secured in a head-holder, the optic tecturn exposed, and the eyelid and nictitating membrane of the contralateral eye removed. The pupil was dilated by topical application of 2% lignocaine. A micrcAitre syringe with a 26-gauge needle, mounted on a micro-manipulator was us0304-3940/83/0000--0000/$ 03.00 @) 1983 Elsevier Scientific Publishers Ireland Ltd.
250
ed for intravitreal injections during electrophysiological recordings. A 12 V tungsten-quartz-iodine f'dament lamp operated at 9 V was used as alight source, and stimuli (maximum intensity 7.5 log it-L) were delivered to the eye by means of a 3
p m e m i a l was measured becanse it is an exceUent indicator of summed
out-
put. Duff and Cohen [I] have shown that, in the pigeon, there is a close correspondence between the impulse activity of retinal ganglion cells and negative field potential components. Furthermore, in chickens, the vast majority of retinal ganglion cells project to the optic tectum. Histological procedures, and the methods for producing long-term lesions with intravitreal kalnic acid, have been previously described [3, 71. Stimulation of the control eye evoked a complex field potential in the contralateral tectum (Fig. ia, upper trace) which consisted of a pronounced ONresponse and a smaller OFF-response. At a given stimulus inteusity the latency and amplitude of the major peaks in each response showed little variability. Three minutes after the intravitreal injection of I0/d of 10 mM kainic acid, the tectal.......... !
]
!!i
I~ ~ " ~
..~ ~T~,I ",
2700
I Jr t00
,,~.r~,~
Fig. I, The effect of kainic acid on visually-evoked tectal field potentials. Test flashes of 500 msec duration (upl~'r signal) were presented at 30 sec intervals. Each response is the averaged field potential evoked by 6 stimulus repetitions, a: the upper record shows the control evoked potential, consisting of separate ON- and OFF-responses, The middle and lower traces show the short-term effects of a 10/d intravitreal injection o f I 0 mM kainic acid: within 5 min the visually-evoked responses were completely abolished, b: the long-term (one wee):) effects of kainic acid result in the total elimination o f OFF-responses, but ON-responses
appear
again.
251
evoked responses were markedly reduced in magnitude (Fig. la, middle trace) and were completely eliminated within 5 min of the injection (Fig. la, lower trace). Lower concentrations of kainic acid (0.1 and I raM) reduced but did not eliminate the tectal.evoked responses, suggesting that the dose-response curve for blocking
121
r due nged the rate of release of the photoreceptor transmitter, effects are consistent with the known effects of kainic acid on horizontal cells, and ON- and OFF,bipolar cells in fish [4, 9, 12]. The assumption of sindlar pharmacology at second-order retinal neurons in fish and chickens was strengthened by the observation that intravitreal injection of 2-amino-4-phosphonobutyric acid (2-APB) eliminated ON-tectalevoked responses. This compound mimics the effect of the photoreceptor transmitter on fish and mudpuppy ON-bipolar cells [12], and also blocks ON-responses in mudpuppy [131 and rabbit [81 ganglion cells, and in the rabbit lateral geniculate nucleus and striate cortex [10]. in our short-term experiments, the ON-responses were abolished while the OFF-responses were increased in size by 2-APB (10 ~1 of 200 raM). After a stable retinal lesion had been produced, I-4 weeks after the injection, OFF-responses could still not be evoked in the optic tectum by stimulation of the lesioned eye, but ON-responses were again prominent, though less complex (Fig. Ib). Both ON- and OFF-responses could be produced by stimulation of the control eye. The recovered ON-response could be eliminated by intravitreal injection of 2-APB, but there was no sign of an OFF-response, even though 2-APB significantly increased the magnitude of the OFF-responses in control retinas. Thus, despite the elimination of two-thirds of the bipolar cells and m o s t of the amacrine cells, ONpathways in the retina one week after an injection of kainic acid appeared to be sufficiently intact to transmit information to the optic tectum, whereas the OFFpathways had been irreversibly disrupted. The simplest interpretation consistent with the morphological data i~ that the OFF-bipolar cells had been eEminated by kainic acid. The morphological effects of intravitreal kainic acid on the retina were rapid and striking (Fig. 2). Within 15 min there was pronounced swelling and nuclear condensation in a population of bipolar cells, while others appeared normal or slightly hyperchromati¢. Amacrine cells were also clearly affected although the swelling and nuclear condensation was less advanced. There was also significant swelling of the inner and outer plexiferm layers, and the photoreceptor nuclei had been forced up outside the outer limiting membrane. Control injections had no observable shortterm morphological effects. Photoreceptor function did not seem to have been af1
I
v
~2
IONL I
opL
I INL
I
IPL
I I~CL
IPL
I
oF:
Fig. 2. Photographs o f semi-thin transverse sections through the central areas o f control retina, and retinas exposed to intravitreal kainic acid. a: control retina, 15 rain after an intravitreal injection of I0 pl of water. This retina was indistinguishable from uninjected control retinas morphologically and bioch~mically, b: retina ! 5 rain after an intravitreal injection of I0 #1 of 4 mM kainie acid. Note the pronounced swelling and pyknotic nuclei of cells in the outer half of the innc.-, nuclear layer (INL), in the region predominantly o~cupied by bipolar cells. Effects on cells in the ganglion cell layer (GCL), and in the inner half of the inner nuclear layer, in the region predominantly occupied by amacrine cells, were much less pronounced at this stage. Both ple~iform layers (Pl.s) were swollen and vacuolar° c: retina one week alter an inlravilreal injection of I0 ~1 of 4 mM kaini¢ acid. Nole the marked loss of bipolar and amacrine cells from the inner nuclear la)'er, and the marked decrea~ in thickness of the inner plexiform layer. ]'he outer part of Ihe retina appears relatively unaffected by the lesion. The reduction in cell numbers in the ganglion cell layer is probably due to destruction of the displaced amacrine cells. Scale bar = 50 pro.
fected, since at this stage, and indeed at all stages throughout these experiments, an a-wave in the electroretinogram was still detected (Dvorak and Morgan, unpublished). In agreement with previous results [3, 5-7, ! I], the retinal lesion had stabilized one week after the injection of kainic acid. Most amacrine cells had been destroyed, and the number of bipolar cells had been reduced, but most photoreceptors, horizontal cells and ganglion cells survived. The retinal lesions appeared to extend over the whole retina, and localized patches o f less or more severe lesioning were not seen.
While 2-APB can be used to eliminate ON-responses from the visual system for short periods, kainic acid appears to permanently abolish OFF-responses. The usefulness of the system is limited by the fact that amacrine cells are also eliminated, which may account for the simplified ON-tectal-evoked response, and by the fact that in many vertebrates, particularly mammals, many ganglion cells arc also
253
destroyed by intravitreal kainic acid [12, 14] (also Wienawa-Narkiewicz and Hughes, unpublished). But under conditions where enough ganglion cells survive, the specificity of the lesion ind,]~d by kainic acid should prove to be very u~ful for analyzing how ON- and OFF-inputs are integrated in higher visual centres to control visually-mediated behaviour. We are grateful to Dr. A. Hughes, Dr. W. Levick and Professor R.F. Mark for their helpful criticism of this manuscript, and to Dr. G. Stange, Pat Miethke and Margaret Canney for their skilled technical assistance. The manuscript was typed by Barbara Piper. I Duff, T.A. and Cohen, D.H., Retinal afferents to the pigeon optic tectum: discharge characteristics in response to whole field illumination, Brain Res., 92 (1975) 1-19. 2 Goto, M., Inomata, N.. Ono, H.. Saito K.-I. and Fukuda, H.. Changes of electroretinogram and neurochemical aspects of GABAergic neurons of retina after intraocular injection of kainic acid in rats. Brain Res., 211 (1981)305-314. 3 Ingham, C.A. and Morgan, I.G., Dose-dependent effects of intravitreal kainic acid on specific cell types in chicken retina, Neuroscience, in press. 4 Lasater, E.M. and Dowling, J.E., Carp horizontal ceils in culture respond selectively to t-glmamate and its agonists, Proc. natl. Acad. Sci. U.S.A.. 79 (1982) 936-940. 5 Morgan, I.G., Organisation of amacrine cell types which use different transmitters in chicken retina. In J.-P. Changeux et al. (Eds.) Molecular and Cellular Interactions Underlying Higher Brain Functions, Progress in Brain Research. Vol. 58. Elsevier Biomedical. Amsterdam, 1983, pp. 191-199. 6 Morgan. I.G., gainic acid as a tool in neurobiology. In G.J. Chader and N.N. Osborne ¢Eds.), Progress in Retinal Research, Vol. 2, Pergamon Press. Oxford, in press. 7 Morgan, I.G. attd Ingham, C.A., Kainic acid affects both plexifornl layers of ¢hk-ken retina, Neurosci. l.ett.. 21 (1981) 275-280° A Neal, M.J.. Cunningham, J.R., James, T.A., Joseph. M. and Collins, J.F., The effect of 2-amino-4-phosphonobutyri¢ (APB) on acetylcholine release from the rabbit retina: evidence for ONchannel input to cholinergic amacrine cells, Neuro~i. Left., 26 (1981) 301-305. 9 Rowe, J.S. and Ruddock, K.H., Depolarization of retinal horizontal cells by cxcitator~ ataino acid neurotransmitter agonists, Neurosci. Lett., 30 (1982) 257-262. I0 Schiller, P.H., Central connections of the retinal ON and OFF pathways, Nature (Lond.), 297 (1982) 580-583. I I Schwarcz, R. and Coyle, J.T., Kainic acid: Neurotoxic effects after intraocular injection, Invest. Ophthalmol., 16 (1977) 141-148. 12 Shiells, R.A., Falk, G. and Naghshineh, S., Action of glutamate and aspartate analogues on rod horizontal and bipolar cells, Nature (Lond.), 294 (1981) 592-594. 13 Slaughter, M.M. and Miller, R.F., 2-Amino-4-phosphonobutyric acid: a new pharmacological tool for retina research, Science, 211 (1981) 182-185. 14 Yazulla, S. and Kleinschmidt, J., The effects of intraocular injection of kainic acid on the synaptic organization of the goldfish retina, Brain Res., 182 (1980) 287-301.
249
INTIgAVlTREAL KAINIC ACID PERMANENTLY ELIMINATES OFF-PATHWAYS FROM CHICKEN RETINA
D.R. DVORAK and l.O. MORGAN
Department o f Behavioural Biology, Research School o f Biological Sciences, Australian National University, PO Box 473 Canberra City, ACT 2601 (Australia) (Received January 20th. 1983; Accepted February 22nd. 1983)
Key words: retina - kainic acid - OFF-responses - bipolar cells - amacrine cells -tectum
Kainic acid rapidly suppresses both ON- and OFF-visually evoked responses in the leCtUreafter injection into the vitreous of chicken eyes. One week after injection the OFF-responses were still suppressed, while ON-responses were detected of similar magnitude but of simplified form to those of control eyes. It is suggested that kainic acid selectively destroys OFF- rather than ON-bipolar cells, while the simplification of the ON-responses is due to the destruction of amacrine cells.
lntravitreal kainic acid causes species-specific patterns of cell death in the retinas of many vertebrates [6]. in avian retinas there is a hierarchy of cell death, such that most amacrine cells and two-thirds of the bipolar cells are destroyed by injections of around 40 nmol of kainic acid; these cells and horizontal cells are destroyed by injections of around 120 nmol of kainic acid; but the Muller glial cells, ganglion cells and photoreceptors survive exposure to as much as 600 nmol of kainic acid [3, 7, I I]. We have proposed that the pattern of toxicity results from an interaction of kainic acid with kainic acid-preferring receptors on horizontal cells and bipolar cells, depolarizing horizontal and OFF-bipolar cells, hyperpolarizing ON-bipolar cells, and ultimately killing those cells which are depolarized [5, 6]. Amacrine cell death could then be associated with abnormal activity in the OFF-bipolar cells resulting from their depolarization by kainic acid. If this hypothesis is correct, the retinal OFF-bipolar cells should be eliminated from retinal circuits, in this paper we show that intravitreal kainic acid eliminates visually-evoked responses in the optic tectum of the chicken within minutes of the injection. One week later, ON-responses recover, but the OFF-responses appear to be permanently abolished. Experiments were performed on 4-6-week-old chickens anaesthetized with 200/0 urethane (6-10 ml/kg, i.p.). The animal was secured in a head-holder, the optic tecturn exposed, and the eyelid and nictitating membrane of the contralateral eye removed. The pupil was dilated by topical application of 2% lignocaine. A micrcAitre syringe with a 26-gauge needle, mounted on a micro-manipulator was us0304-3940/83/0000--0000/$ 03.00 @) 1983 Elsevier Scientific Publishers Ireland Ltd.
250
ed for intravitreal injections during electrophysiological recordings. A 12 V tungsten-quartz-iodine f'dament lamp operated at 9 V was used as alight source, and stimuli (maximum intensity 7.5 log it-L) were delivered to the eye by means of a 3
p m e m i a l was measured becanse it is an exceUent indicator of summed
out-
put. Duff and Cohen [I] have shown that, in the pigeon, there is a close correspondence between the impulse activity of retinal ganglion cells and negative field potential components. Furthermore, in chickens, the vast majority of retinal ganglion cells project to the optic tectum. Histological procedures, and the methods for producing long-term lesions with intravitreal kalnic acid, have been previously described [3, 71. Stimulation of the control eye evoked a complex field potential in the contralateral tectum (Fig. ia, upper trace) which consisted of a pronounced ONresponse and a smaller OFF-response. At a given stimulus inteusity the latency and amplitude of the major peaks in each response showed little variability. Three minutes after the intravitreal injection of I0/d of 10 mM kainic acid, the tectal.......... !
]
!!i
I~ ~ " ~
..~ ~T~,I ",
2700
I Jr t00
,,~.r~,~
Fig. I, The effect of kainic acid on visually-evoked tectal field potentials. Test flashes of 500 msec duration (upl~'r signal) were presented at 30 sec intervals. Each response is the averaged field potential evoked by 6 stimulus repetitions, a: the upper record shows the control evoked potential, consisting of separate ON- and OFF-responses, The middle and lower traces show the short-term effects of a 10/d intravitreal injection o f I 0 mM kainic acid: within 5 min the visually-evoked responses were completely abolished, b: the long-term (one wee):) effects of kainic acid result in the total elimination o f OFF-responses, but ON-responses
appear
again.
251
evoked responses were markedly reduced in magnitude (Fig. la, middle trace) and were completely eliminated within 5 min of the injection (Fig. la, lower trace). Lower concentrations of kainic acid (0.1 and I raM) reduced but did not eliminate the tectal.evoked responses, suggesting that the dose-response curve for blocking
121
r due nged the rate of release of the photoreceptor transmitter, effects are consistent with the known effects of kainic acid on horizontal cells, and ON- and OFF,bipolar cells in fish [4, 9, 12]. The assumption of sindlar pharmacology at second-order retinal neurons in fish and chickens was strengthened by the observation that intravitreal injection of 2-amino-4-phosphonobutyric acid (2-APB) eliminated ON-tectalevoked responses. This compound mimics the effect of the photoreceptor transmitter on fish and mudpuppy ON-bipolar cells [12], and also blocks ON-responses in mudpuppy [131 and rabbit [81 ganglion cells, and in the rabbit lateral geniculate nucleus and striate cortex [10]. in our short-term experiments, the ON-responses were abolished while the OFF-responses were increased in size by 2-APB (10 ~1 of 200 raM). After a stable retinal lesion had been produced, I-4 weeks after the injection, OFF-responses could still not be evoked in the optic tectum by stimulation of the lesioned eye, but ON-responses were again prominent, though less complex (Fig. Ib). Both ON- and OFF-responses could be produced by stimulation of the control eye. The recovered ON-response could be eliminated by intravitreal injection of 2-APB, but there was no sign of an OFF-response, even though 2-APB significantly increased the magnitude of the OFF-responses in control retinas. Thus, despite the elimination of two-thirds of the bipolar cells and m o s t of the amacrine cells, ONpathways in the retina one week after an injection of kainic acid appeared to be sufficiently intact to transmit information to the optic tectum, whereas the OFFpathways had been irreversibly disrupted. The simplest interpretation consistent with the morphological data i~ that the OFF-bipolar cells had been eEminated by kainic acid. The morphological effects of intravitreal kainic acid on the retina were rapid and striking (Fig. 2). Within 15 min there was pronounced swelling and nuclear condensation in a population of bipolar cells, while others appeared normal or slightly hyperchromati¢. Amacrine cells were also clearly affected although the swelling and nuclear condensation was less advanced. There was also significant swelling of the inner and outer plexiferm layers, and the photoreceptor nuclei had been forced up outside the outer limiting membrane. Control injections had no observable shortterm morphological effects. Photoreceptor function did not seem to have been af1
I
v
~2
IONL I
opL
I INL
I
IPL
I I~CL
IPL
I
oF:
Fig. 2. Photographs o f semi-thin transverse sections through the central areas o f control retina, and retinas exposed to intravitreal kainic acid. a: control retina, 15 rain after an intravitreal injection of I0 pl of water. This retina was indistinguishable from uninjected control retinas morphologically and bioch~mically, b: retina ! 5 rain after an intravitreal injection of I0 #1 of 4 mM kainie acid. Note the pronounced swelling and pyknotic nuclei of cells in the outer half of the innc.-, nuclear layer (INL), in the region predominantly o~cupied by bipolar cells. Effects on cells in the ganglion cell layer (GCL), and in the inner half of the inner nuclear layer, in the region predominantly occupied by amacrine cells, were much less pronounced at this stage. Both ple~iform layers (Pl.s) were swollen and vacuolar° c: retina one week alter an inlravilreal injection of I0 ~1 of 4 mM kaini¢ acid. Nole the marked loss of bipolar and amacrine cells from the inner nuclear la)'er, and the marked decrea~ in thickness of the inner plexiform layer. ]'he outer part of Ihe retina appears relatively unaffected by the lesion. The reduction in cell numbers in the ganglion cell layer is probably due to destruction of the displaced amacrine cells. Scale bar = 50 pro.
fected, since at this stage, and indeed at all stages throughout these experiments, an a-wave in the electroretinogram was still detected (Dvorak and Morgan, unpublished). In agreement with previous results [3, 5-7, ! I], the retinal lesion had stabilized one week after the injection of kainic acid. Most amacrine cells had been destroyed, and the number of bipolar cells had been reduced, but most photoreceptors, horizontal cells and ganglion cells survived. The retinal lesions appeared to extend over the whole retina, and localized patches o f less or more severe lesioning were not seen.
While 2-APB can be used to eliminate ON-responses from the visual system for short periods, kainic acid appears to permanently abolish OFF-responses. The usefulness of the system is limited by the fact that amacrine cells are also eliminated, which may account for the simplified ON-tectal-evoked response, and by the fact that in many vertebrates, particularly mammals, many ganglion cells arc also
253
destroyed by intravitreal kainic acid [12, 14] (also Wienawa-Narkiewicz and Hughes, unpublished). But under conditions where enough ganglion cells survive, the specificity of the lesion ind,]~d by kainic acid should prove to be very u~ful for analyzing how ON- and OFF-inputs are integrated in higher visual centres to control visually-mediated behaviour. We are grateful to Dr. A. Hughes, Dr. W. Levick and Professor R.F. Mark for their helpful criticism of this manuscript, and to Dr. G. Stange, Pat Miethke and Margaret Canney for their skilled technical assistance. The manuscript was typed by Barbara Piper. I Duff, T.A. and Cohen, D.H., Retinal afferents to the pigeon optic tectum: discharge characteristics in response to whole field illumination, Brain Res., 92 (1975) 1-19. 2 Goto, M., Inomata, N.. Ono, H.. Saito K.-I. and Fukuda, H.. Changes of electroretinogram and neurochemical aspects of GABAergic neurons of retina after intraocular injection of kainic acid in rats. Brain Res., 211 (1981)305-314. 3 Ingham, C.A. and Morgan, I.G., Dose-dependent effects of intravitreal kainic acid on specific cell types in chicken retina, Neuroscience, in press. 4 Lasater, E.M. and Dowling, J.E., Carp horizontal ceils in culture respond selectively to t-glmamate and its agonists, Proc. natl. Acad. Sci. U.S.A.. 79 (1982) 936-940. 5 Morgan, I.G., Organisation of amacrine cell types which use different transmitters in chicken retina. In J.-P. Changeux et al. (Eds.) Molecular and Cellular Interactions Underlying Higher Brain Functions, Progress in Brain Research. Vol. 58. Elsevier Biomedical. Amsterdam, 1983, pp. 191-199. 6 Morgan. I.G., gainic acid as a tool in neurobiology. In G.J. Chader and N.N. Osborne ¢Eds.), Progress in Retinal Research, Vol. 2, Pergamon Press. Oxford, in press. 7 Morgan, I.G. attd Ingham, C.A., Kainic acid affects both plexifornl layers of ¢hk-ken retina, Neurosci. l.ett.. 21 (1981) 275-280° A Neal, M.J.. Cunningham, J.R., James, T.A., Joseph. M. and Collins, J.F., The effect of 2-amino-4-phosphonobutyri¢ (APB) on acetylcholine release from the rabbit retina: evidence for ONchannel input to cholinergic amacrine cells, Neuro~i. Left., 26 (1981) 301-305. 9 Rowe, J.S. and Ruddock, K.H., Depolarization of retinal horizontal cells by cxcitator~ ataino acid neurotransmitter agonists, Neurosci. Lett., 30 (1982) 257-262. I0 Schiller, P.H., Central connections of the retinal ON and OFF pathways, Nature (Lond.), 297 (1982) 580-583. I I Schwarcz, R. and Coyle, J.T., Kainic acid: Neurotoxic effects after intraocular injection, Invest. Ophthalmol., 16 (1977) 141-148. 12 Shiells, R.A., Falk, G. and Naghshineh, S., Action of glutamate and aspartate analogues on rod horizontal and bipolar cells, Nature (Lond.), 294 (1981) 592-594. 13 Slaughter, M.M. and Miller, R.F., 2-Amino-4-phosphonobutyric acid: a new pharmacological tool for retina research, Science, 211 (1981) 182-185. 14 Yazulla, S. and Kleinschmidt, J., The effects of intraocular injection of kainic acid on the synaptic organization of the goldfish retina, Brain Res., 182 (1980) 287-301.