350
Brain Research. 531 ! i991)) 35(~--354 FlscvicI
BRES 24362
Excitatory amino acid involvement in retinal
eration
Robert J. Ulshafer 1'2, David M. Sherry 2, Ralph Dawson Jr. 3 and David R. Wallace 3 Departments of i Ophthalmology, 2Neuroscience and 3Pharmacodynamics, University of Florida, Gainesville, FL (U. S. A.)
(Accepted 31 July 1990) Key words: Photoreceptor; Congenital blindness; Excitatory amino acid; Glutamate; Aspartate; Chicken; Hereditary retinal degeneration
Amino acid analysis using high-performance liquid chromatography demonstrated high levels of the excitatory amino acids, aspartate and glutamate, in the retinas of congenitally blind chicks at the time of photoreceptor degeneration. Concentrations of aspartate were about 2 times higher in blind chicks than in retinas of age-matched sighted chicks that were carriers for the genetic defect. Glutamate levels were similar in blind chicks and carriers at 1 day of age, but doubled and tripled sighted chick values at 1 week and 2 weeks of age in blind chick retinas. Light microscopic immunocytochemistry using antibodies that recognize aspartate and glutamate revealed increased levels of these two amino acids specifically in the photoreceptor layer of blind chicks. This report is the first to demonstrate high endogenous levels of excitatory amino acids associated with a hereditary degeneration of photoreceptor cells. Excitatory amino acids (EAA), such as aspartate (Asp) and glutamate (Glu), are believed to serve as neurotransmitters in a number of neural systems, including the vertebrate retina. In the retinas of a number of species, it is the photoreceptor cells where Glu and/or Asp have been found to meet many of the criteria characteristic of classical neurotransmitters. These criteria include a sodium-dependent high-affinity uptake 7, release upon stimulation 2, presence of synthetic enzymes, and mimic of action on post-synaptic membranes by agonist molecules 15. Amino acids such as Glu and Asp and others, however, are also excitotoxins that have been found to be associated with neuronal death in several human pathological conditions 8'9'14"~s'24. Exogenously applied or ingested E A A s 10'13'17A9'23'31 o r analogues of Glu or Asp, such as kainic acid 1'H'31, quisqualate ~6'2°, N-methyl-D-Asp31, RS-a-amino-3-hydroxy-5-methyl-4-isoxazoleacetic acid (AMPA) ~6, have been shown to induce retinal degeneration in all vertebrates studied to date. Although the majority of studies on E A A toxicity report pathological changes mediated through postsynaptic receptors on inner retinal neurons, degenerative changes have also been noted in photoreceptor cells. Sisk and Kuwabara 2-~ reported that degenerative changes in photoreceptor terminals and a loss of nuclei in the outer nuclear layer occur within 2 months following intravitreal injection of monosodium L-GIu. Injection of kainic acid into a chicken vitreous results in severe disruption of photoreceptor outer segments and terminals 2'21. These researchers also reported that kainic acid caused an
increase in size of the globe (weight, equatorial diameter and axial length). A genetic defect (termed rd, retinal degeneration) has been described in a strain of chickens resulting in total blindness at the time of hatching despite normal appearing retinal morphology 27"3°. After about a week po~thatch, rd photoreceptor cells begin to undergo degeneration 26'27. Cell death appears to be a secondary phenomenon resulting from lack of function/electrical activity of the photoreceptors. Following photoreceptor cell death. degeneration follows in the inner retinal layers. This chicken model of hereditary blindness has several of the pathological features of excitatory amino acid toxicity. These include degenerating photoreceptor outer segments and terminals, and later inner retinal neuronal degeneration 26-28 and enlarged globe 12. In an in vitro assay, Ulshafer and Meyer 28 reported that uptake mechanisms of Glu are present in the photoreceptor synaptosomal fraction of rd retinas although K~'-stimulated release of [3H]-Glu from this fraction is significantly lower than that obtained from sighted chicks. In light of all these findings in this strain of blind chicks, we examined endogenous levels of the EAAs Glu and Asp, comparing values or patterns to those present in sighted heterozygous clutchmates. Concentrations of Glu and Asp were measured by high-performance liquid chromatography (HPLC) and localized in light microscopic sections of retina by immunocytochemistry. Chicks homozygous for the rd mutation were obtained from breeding affected (rd/rd) adults. Heterozygous
Correspondence." R.J. Ulshafer, Department of Ophthalmology, Box J-284, J. Hillis Miller Health Center, University of Florida, Gainesville, FL 32610, U.S.A.
0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
351 sighted chicks were o b t a i n e d from either + / + x rd/rd crosses o r from rd/rd x rd/-F matings. Chicks retinas were isolated from 1-day-, 1-week- and 2-week-old specimens. Prior to sacrifice by decapitation, presence o r absence of sight was confirmed by behavioral exam. F o r H P L C analysis, retinas were placed in labeled polyvials and frozen in liquid nitrogen. Retinas were weighed prior to h o m o g e n i z a t i o n in 1.0 ml 0.1 M perchioric acid. The h o m o g e n a t e s were centrifuged for 1.5 rain in a microfuge tube and the resulting supernatant was diluted 1:10 with mobile phase. Retinal content of a m i n o acids was d e t e r m i n e d by H P L C separation and electrochemical detection of o-phthalaldehyde-derivitized amino acids as previously described 5'29. In o r d e r to d e t e r m i n e if high concentration of Glu and A s p were specifically associated with p h o t o r e c e p t o r cells, we p e r f o r m e d i m m u n o c y t o c h e m i c a l localization experiments. E y e c u p s were excised from animals at the same ages as described above, as well as at 3 weeks of age when p h o t o r e c e p t o r d e g e n e r a t i o n is well underway. The ante-
ASPARTATE
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rior third of the globe was cut and the vitreous r e m o v e d . W h o l e eyecups were then i m m e r s e d in 2 . 5 % glutaraldehyde in 0.1 M p h o s p h a t e buffer for 12 h, rinsed in buffer,
F--] CARRIER rd
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b Fig. 1. Asp (a) and Glu (b) content (mean + S.D.) as measured by HPLC in sighted and rd chick retinas at 1 day, 1 week and 2 weeks of age. Asp levels are significantly higher (P < 0.05) in rd retinas at all time points. Glu levels are significantly higher in rd retinas at l week (P < 0.05) and 2 weeks (P < 0.01) of age. n = 3-4 retinas per group per time point.
Fig. 2. Anti-Asp labeling in retinas of 1-week-old sighted (a) and blind (b,c) chicks. At one week of age in the normally sighted chick retina, labeling is seen throughout the retina hut is somewhat denser in photoreceptor cell layers (P) and horizontal cells (arrowheads) than in other retinal layers. However, in 1-week-old rd retinas (b), photoreceptor cells appear to stain more densely than other retinal cells. At 3 weeks of age (c), when photoreceptor degeneration is apparent in the rd chick retina, photoreceptor cells also label more densely. The magnification bar in (a) is equivalent to 150/~m and is representative for all photomicrographs in this figure and the next. No counterstain has been applied.
352
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Fig. 3. Anti-Glu labeling in retinas of 1-week-old sighted (a), 1-week-old blind (b), and 3-week-old blind (c) retinas. In the sighted bird (a), Glu antibodies stain the bipolar cells (B) and photoreceptor cells (P) heavier than other cell layers. Ganglion cells (G) also label relatively heavily with this antibody. In the rd retina (b) label density in the photoreceptor layer is as dense as that over the bipolar cells, while in (a) the bipolar cell layer stains heavier than photoreceptos. The 3-week-old rd retina (c) show signs of photoreceptor degeneration, most noticeable are the spaces between photoreceptor inner segments and nuclei (i.e. reduced numbers of cells). Anti-Glu labeling is very dense over the remaining photoreceptor cells while that over bipolar cells (and other retinal neurons) is no different than earlier time points or no different than that measured in sighted birds.
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Fig. 4. The ratio of staining density (mean optical density + S.D.) comparing photoreceptor cells to bipolar cells in rd chick and sighted carrier chick retinas, a: a-Asp labeling followed by silver intensification of gold-conjugated secondary antibodies. In carrier retinas labeld with a-Asp, photoreceptors label as heavily as bipolar cells (ratio ~1.0). In rd retinas photoreceptors label about 1.8-2.5 times heavier than bipolar cells, b: a-Glu labeling, bipolar cells stain almost twice as heavily as photoreceptors in sighted chicks (carriers) at all ages (PR/BP ratio ~0.6-0.7). No differences are seen in the ratio of photoreceptor: bipolar cell labeling fgr a-Glu at one day of age. However, at 1 week and 2 weeks of age, photoreceptors stain significantly darker than bipolar cells in rd retinas. Values represent optical density measurements for 3 animals in each group at each time point, 10 areas measured per animal. The ratio of 3-week-old rd specimens could not be accurately measured due to many empty spaces caused by missing or degenerating photoreceptor cells (see Fig. 3c).
353 overnight at 4 °C to secondary antibody (goat anti-rabbit) to which 15 nm gold particles had been conjugated. Gold particles were visualized by a silver intensification procedure modified after Danscher and Noorgard 4. For details of the procedure, see Sherry22. Sections were photographed on a Nikon Labophot microscope. Densitometric measurements were made over the various retinal layers on a Zeiss IBAS Imaging system. Sections from the 2 groups at all ages tested were reacted simultaneously in the same dilution of antiserum and other reagents. In order to control for any artifact that might account for differences seen between specimens, we calculated a ratio of photoreceptor label density to that over an equal area of bipolar cells (middle zone of the inner nuclear layer). Endogenous levels of Asp were higher in rd retinas than in retinas from sighted chicks at all ages examined (Fig. la). ANOVA performed on values obtained from HPLC revealed that differences between the two groups were at the P < 0.01 level and neither time (age within group) nor the interaction of group with time were important. Results obtained for Glu (Fig. lb) were similar except at one day of age when no significant difference (P > 0.05) was observed in Glu content between rd and carrier chicks. Glu concentrations were significantly elevated in rd chick retinas at 1 week and 2 weeks of age. a-Asp antiserum labeled virtually every cell in both sighted and blind chicks' retinas at relatively low densities, although photoreceptors and horizontal cells stained considerably darker than other cell types (Fig. 2). In the case of Asp immunoreactivity, rd photoreceptors (Fig. 2b) were significantly denser staining than those of sighted chicks (Fig. 2a) while labeling of bipolar cells and other retinal cells was virtually identical to that in the carrier group (Figs. 2 and 4a). This pattern was seen at all times examined. The ratio of photoreceptor:bipolar cell label optical density by anti-Asp was about 1.2 in sighted chicks at all ages studied. In blind chicks, it was 1.8-2.3 (Fig. 4). Antibodies recognizing Glu also stained all retinal cells in carrier chick retinas although bipolar cells stained darker than other retinal neurons (Fig. 3a). No difference was seen in Glu labeling between carrier and rd retinas at 1 day of age but 1 week (Fig. 3b) and older (Fig. 3c) rd chick retinas had significantly denser labeling photoreceptors (compared to bipolar cells) than sighted chicks (Figs. 3b and 4b). The photoreceptor to bipolar cell labeling density (PR/BP) was about 0.5 in carrier chicks while it was about 1.0 in 1 week-old rd retinas and 1.5 in
2-week-old chicks (Fig. 4b). In 3-week-old specimens label density appeared even darker in rd photoreceptor cells than in carrier photoreceptors, however, due to the degenerating cells (i.e. empty spaces) in this layer, densitometric measurements on both Asp and Glu immunoreactivity were not valid. Labeling of cells other than photoreceptors was comparable between the two groups at 3 weeks of age, as at earlier times. Immunocytochemical labeling results were remarkably similar to HPLC data. Both experiments showed that Asp levels were elevated in rd chicks at all ages and that Glu levels were significantly increased in the 1-week- and 2-week-old rd animals. Immunocytochemistry localized the differences between blind and sighted chicks specifically to the photoreceptor layers, since labeling density was not different in the inner retinal layers of rd and normal chicks. Photoreceptors of rd chicks do not appear morphologically different (at the EM level) than normals until about 1-2 weeks of age, the time when both Asp and Glu levels are elevated in rd photoreceptors. In light of the findings that rd photoreceptor synaptosomes are capable of taking up exogenously supplied Glu but fail to release it under depolarizing conditions 28, we speculate that in vivo Glu is continually synthesized in rd photoreceptors but due to lack of dark-induced depolarization, the cells cannot release it. Glu might then build up in photoreceptor cells and cause degenerative changes. Preliminary studies in our laboratory have shown that one enzyme involved in Glu synthesis (glutaminase) is increased in blind chick retinas. Because the increased content in Asp and Glu occur after the chicks are blind, we do not believe that the transduction defect is directly caused by an error in E A A metabolism. The secondary pathological changes that we noted in photoreceptor cells26-2s, however, are associated with increased levels of Asp and Glu. Because these changes resemble those produced in response to applied EAAs or agonists, it appears that photoreceptor degeneration in this model is caused or augmented by high endogenous levels of Asp and/or Glu via a novel (non-receptor-mediated) toxic mechanism. Subsequent degeneration of inner retinal neurons may ultimately be caused by release of E A A s by dying photoreceptor cells that kill the postsynaptic neurons via a receptor-mediated mechanism.
1 Abrams, L., Politi, E. and Adler, R., Differential susceptibility of isolated mouse retina neurons and photoreceptors to kainic acid toxicity.
2 Barrington, M., Sattayasi, J., Zappia, J. and Ehrlich, D., Excitatory amino acids interfere with normal eye growth in posthatch chick, Curt. Eye Res., 8 (1989) 781-792.
This work was supported by grants from the National Institutes of Health (RO1 EY04590 and P30 EY08571) and a non-restrictive grant to the Department of Ophthalmology from Research to Prevent Blindness, Inc. R.J.U. is a Research to Prevent Blindness Senior Scientific Investigator.
354 3 Copenhagen, D.R. and Jahr, G.E., Release of endogenous excitatory amino acids from turtle photoreceptors, Nature, 341 (1989) 536-539. 4 Danscher, G. and Noorgard, J.O.R., Light microscopic visualization of colloidal gold on resin-embedded tissue, J. Histochem. Cytoehem., 31: 1394-1398. 5 Dawson, R., Jr, Wallace, D.R. and Moldrum, M.J., Endogenous glutamate release from frontal cortex of adult and aged rats, Neurobiol. Aging, 10 (1989) 665-668. 6 De Nardis, R., Sattayasai, J., Zappia, J. and Ehrlich, D., Neurotoxic effects of kainic acid on developing chick retina, Dev. Neurosci., 10 (1988) 256-269. 7 Ehinger, G., [3H]-D-Aspartate accumulation in the retina of pigeon, guinea pig and rabbit, Exp. Eye Res., 33 (1981) 381-391. 8 Engelsen, B., Neurotransmitter glutamate: its clinical importance, Acta Neurol. Scand., 74 (1986) 337-355. 9 Greenamyre, J.T., The role of glutamate in neurotransmission and in neurologic disease, Arch. Neurol., 43 (1986) 1058-1063. 10 Hyndman, A.G. and Adler, R., Analysis of glutamate uptake and monosodium glutamate toxicity in neural retina monolayer cultures, Dev. Brain Res., 2 (1982) 303-314. 11 Ingham, C.A. and Morgan, I.G., Dose-dependent effects of intravitreal kainic acid on specific cell types in chicken retina, Neuroscience, 9 (1983) 165-181. 12 Lauber, J.K. and Oishi, T., Ocular responses of genetically blind chicks to the light environment and to lid suture, Curr. Eye Res., 8 (1989) 757-764. 13 Lucas, D.R. and Newhouse, J.P., The toxic effect of sodium L-glutamate on tile inner layers of the retina, Am. Med. Assoc. Arch. Ophthalmol., 58 (1957) 193-201. 14 Maragos, W.E, Greenamyre, J.T., Penney, J.B. and Young, A.B., Glutamate dysfunction in alzheimer's disease: an hypothesis, Trends Neurosci., 10 (1987) 65-68. 15 Miller, E E and Slaughter, M.M., Excitatory amino acid receptors of the retina: diversity of subtypes and conductance mechanisms, Trends Neurosci., 9 (1986) 211-217. 16 Morgan, I.G., AMPA is a powerful neurotoxin in the chicken retina, Neurosci. Lett., 79 (1987) 267-271. 17 Olney, J.W. and Ho, O., Brain damage in infant mice following oral intake of glutamate, aspartate or cysteine, Nature, 227 (1970) 609-610. 18 Perry, T.L., Glutamine, glutamate, and GABA in human diseases. In E. Kvamme (Ed.), Glutamine and Glutamate in Mammals, Vol. 2., CRC Press, Boca Raton, 1988, pp. 113-125. 19 Rogers, L.J,, Zappia, J.V. and Ehrlich, D., Visual deficits
20
21
22
23
24
25
26
27
28
29 30
31
following intraocular treatment of chicks with glutamate or kainic acid, Neurosci. Left., 57 (1985) 191-197. Sattayasai, J. and Ehrlich, D., Morphology of quisqualateinduced neurotoxicity in the chicken retina, Invest. Ophthalmol. Vis. Sci., 28 (1987), 106-117. Sattayasai, J., Zappia, J. and Ehrlich, D., Differential effects of excitatory amino acids on photoreceptors of the chick retina: an electron-microscopical study using the zinc-iodide-osmium technique, Vis. Neurosci., 2 (1989) 237-245. Sherry, D.M., Neuroactive amino acids in the cone photoreceptors of the Anolis carolinensis retina. Ph.D. Dissertation, University of Florida, Gainesville, FL.. Sisk, D.R. and Kuwabara, T., Histologic changes in the inner retina of albino rats following intravitreal injection of monosodium L-glutamate, Grae]'s Arch. Clin. Exp. Ophthalmol., 223 (1985) 250-258. Spencer, P.S., Ludolph, A., Dwivedi, M.P., Roy, D.N., Hugon, J. and Schaumburg, H.H., Lanthyrism: evidence for role of the neuroexcitatory amino acid BOAA, Lancet, (1986) 1066-1067. Storm-Mathisen, J., Leknes, A.K., Bore, At.T. Vaaland, J.L., Edminson, P., Haug, E-M.S. and Ottersen, O.P., First visualization of glutamate and GABA in neurones by immunocytochemistry, Nature, 301 (1983) 517-520. Ulshafer, R.J. and Allen, C.B., Hereditary retinal degeneration in the Rhode Island Red chicken: uttrastructural observations, Exp. Eye Res., 40 (1985) 865-877. Ulshafer, R.J., Allen, C.B., Dawson, W.W. and Wolf, E.D., Hereditary retinal degeneration in Rhode Island Red chickensl ERG and histology, Exp. Eye Res., 39 (1984) 123-135. Ulshafer, R.J. and Meyer, E.M., Studies on putative neurotransmitters in an animal model of heriditary blindness. In Hollyfield, Anderson and LaVail (Eds.), Degenerative Retinal Disorders: Clinical and Laboratory Investigations, Alan R. Liss, New York, 1987. Wallace, D.R. and Dawson, R., Decreased plasma taurine in aged rats, Gerontology, in press. Wolf, E.D., An inherited retinal abnormality in Rhode Island red chickens. In R.M. CLayton, J. Haywood, H.W. Reading and A. Wright (Eds.), Problems of Normal and Genetically Abnormal Retinas, Academic Press, London, 1982, pp. 249-252. Zeevalk, G.D. and Nicklas, W.J., Acute excitotoxieity in chick retina caused by the unusual amino acids B O A A and BMAA: effects of MK-801 and kynurenate, Neurosci. Lett., 102 (1989) 284-290.
Brain Research. 531 ! i991)) 35(~--354 FlscvicI
BRES 24362
Excitatory amino acid involvement in retinal
eration
Robert J. Ulshafer 1'2, David M. Sherry 2, Ralph Dawson Jr. 3 and David R. Wallace 3 Departments of i Ophthalmology, 2Neuroscience and 3Pharmacodynamics, University of Florida, Gainesville, FL (U. S. A.)
(Accepted 31 July 1990) Key words: Photoreceptor; Congenital blindness; Excitatory amino acid; Glutamate; Aspartate; Chicken; Hereditary retinal degeneration
Amino acid analysis using high-performance liquid chromatography demonstrated high levels of the excitatory amino acids, aspartate and glutamate, in the retinas of congenitally blind chicks at the time of photoreceptor degeneration. Concentrations of aspartate were about 2 times higher in blind chicks than in retinas of age-matched sighted chicks that were carriers for the genetic defect. Glutamate levels were similar in blind chicks and carriers at 1 day of age, but doubled and tripled sighted chick values at 1 week and 2 weeks of age in blind chick retinas. Light microscopic immunocytochemistry using antibodies that recognize aspartate and glutamate revealed increased levels of these two amino acids specifically in the photoreceptor layer of blind chicks. This report is the first to demonstrate high endogenous levels of excitatory amino acids associated with a hereditary degeneration of photoreceptor cells. Excitatory amino acids (EAA), such as aspartate (Asp) and glutamate (Glu), are believed to serve as neurotransmitters in a number of neural systems, including the vertebrate retina. In the retinas of a number of species, it is the photoreceptor cells where Glu and/or Asp have been found to meet many of the criteria characteristic of classical neurotransmitters. These criteria include a sodium-dependent high-affinity uptake 7, release upon stimulation 2, presence of synthetic enzymes, and mimic of action on post-synaptic membranes by agonist molecules 15. Amino acids such as Glu and Asp and others, however, are also excitotoxins that have been found to be associated with neuronal death in several human pathological conditions 8'9'14"~s'24. Exogenously applied or ingested E A A s 10'13'17A9'23'31 o r analogues of Glu or Asp, such as kainic acid 1'H'31, quisqualate ~6'2°, N-methyl-D-Asp31, RS-a-amino-3-hydroxy-5-methyl-4-isoxazoleacetic acid (AMPA) ~6, have been shown to induce retinal degeneration in all vertebrates studied to date. Although the majority of studies on E A A toxicity report pathological changes mediated through postsynaptic receptors on inner retinal neurons, degenerative changes have also been noted in photoreceptor cells. Sisk and Kuwabara 2-~ reported that degenerative changes in photoreceptor terminals and a loss of nuclei in the outer nuclear layer occur within 2 months following intravitreal injection of monosodium L-GIu. Injection of kainic acid into a chicken vitreous results in severe disruption of photoreceptor outer segments and terminals 2'21. These researchers also reported that kainic acid caused an
increase in size of the globe (weight, equatorial diameter and axial length). A genetic defect (termed rd, retinal degeneration) has been described in a strain of chickens resulting in total blindness at the time of hatching despite normal appearing retinal morphology 27"3°. After about a week po~thatch, rd photoreceptor cells begin to undergo degeneration 26'27. Cell death appears to be a secondary phenomenon resulting from lack of function/electrical activity of the photoreceptors. Following photoreceptor cell death. degeneration follows in the inner retinal layers. This chicken model of hereditary blindness has several of the pathological features of excitatory amino acid toxicity. These include degenerating photoreceptor outer segments and terminals, and later inner retinal neuronal degeneration 26-28 and enlarged globe 12. In an in vitro assay, Ulshafer and Meyer 28 reported that uptake mechanisms of Glu are present in the photoreceptor synaptosomal fraction of rd retinas although K~'-stimulated release of [3H]-Glu from this fraction is significantly lower than that obtained from sighted chicks. In light of all these findings in this strain of blind chicks, we examined endogenous levels of the EAAs Glu and Asp, comparing values or patterns to those present in sighted heterozygous clutchmates. Concentrations of Glu and Asp were measured by high-performance liquid chromatography (HPLC) and localized in light microscopic sections of retina by immunocytochemistry. Chicks homozygous for the rd mutation were obtained from breeding affected (rd/rd) adults. Heterozygous
Correspondence." R.J. Ulshafer, Department of Ophthalmology, Box J-284, J. Hillis Miller Health Center, University of Florida, Gainesville, FL 32610, U.S.A.
0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
351 sighted chicks were o b t a i n e d from either + / + x rd/rd crosses o r from rd/rd x rd/-F matings. Chicks retinas were isolated from 1-day-, 1-week- and 2-week-old specimens. Prior to sacrifice by decapitation, presence o r absence of sight was confirmed by behavioral exam. F o r H P L C analysis, retinas were placed in labeled polyvials and frozen in liquid nitrogen. Retinas were weighed prior to h o m o g e n i z a t i o n in 1.0 ml 0.1 M perchioric acid. The h o m o g e n a t e s were centrifuged for 1.5 rain in a microfuge tube and the resulting supernatant was diluted 1:10 with mobile phase. Retinal content of a m i n o acids was d e t e r m i n e d by H P L C separation and electrochemical detection of o-phthalaldehyde-derivitized amino acids as previously described 5'29. In o r d e r to d e t e r m i n e if high concentration of Glu and A s p were specifically associated with p h o t o r e c e p t o r cells, we p e r f o r m e d i m m u n o c y t o c h e m i c a l localization experiments. E y e c u p s were excised from animals at the same ages as described above, as well as at 3 weeks of age when p h o t o r e c e p t o r d e g e n e r a t i o n is well underway. The ante-
ASPARTATE
1.00
rior third of the globe was cut and the vitreous r e m o v e d . W h o l e eyecups were then i m m e r s e d in 2 . 5 % glutaraldehyde in 0.1 M p h o s p h a t e buffer for 12 h, rinsed in buffer,
F--] CARRIER rd
0 80
o 0
0.60
0.40
0.20
0.00 1 DAY
1 WEEK
2 WEEKS
a
I--q CARRIER IX-'R-'q rd
GLUTAMATE
600 5.00 4,00 o L~J
i
3.00
0
2.00 1.00 0.00 1 DAY
1 WEEK
2 WEEKS
b Fig. 1. Asp (a) and Glu (b) content (mean + S.D.) as measured by HPLC in sighted and rd chick retinas at 1 day, 1 week and 2 weeks of age. Asp levels are significantly higher (P < 0.05) in rd retinas at all time points. Glu levels are significantly higher in rd retinas at l week (P < 0.05) and 2 weeks (P < 0.01) of age. n = 3-4 retinas per group per time point.
Fig. 2. Anti-Asp labeling in retinas of 1-week-old sighted (a) and blind (b,c) chicks. At one week of age in the normally sighted chick retina, labeling is seen throughout the retina hut is somewhat denser in photoreceptor cell layers (P) and horizontal cells (arrowheads) than in other retinal layers. However, in 1-week-old rd retinas (b), photoreceptor cells appear to stain more densely than other retinal cells. At 3 weeks of age (c), when photoreceptor degeneration is apparent in the rd chick retina, photoreceptor cells also label more densely. The magnification bar in (a) is equivalent to 150/~m and is representative for all photomicrographs in this figure and the next. No counterstain has been applied.
352
dehydrated
through
ethyl a l c o h o l s and
embedded
in
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Fig. 3. Anti-Glu labeling in retinas of 1-week-old sighted (a), 1-week-old blind (b), and 3-week-old blind (c) retinas. In the sighted bird (a), Glu antibodies stain the bipolar cells (B) and photoreceptor cells (P) heavier than other cell layers. Ganglion cells (G) also label relatively heavily with this antibody. In the rd retina (b) label density in the photoreceptor layer is as dense as that over the bipolar cells, while in (a) the bipolar cell layer stains heavier than photoreceptos. The 3-week-old rd retina (c) show signs of photoreceptor degeneration, most noticeable are the spaces between photoreceptor inner segments and nuclei (i.e. reduced numbers of cells). Anti-Glu labeling is very dense over the remaining photoreceptor cells while that over bipolar cells (and other retinal neurons) is no different than earlier time points or no different than that measured in sighted birds.
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Fig. 4. The ratio of staining density (mean optical density + S.D.) comparing photoreceptor cells to bipolar cells in rd chick and sighted carrier chick retinas, a: a-Asp labeling followed by silver intensification of gold-conjugated secondary antibodies. In carrier retinas labeld with a-Asp, photoreceptors label as heavily as bipolar cells (ratio ~1.0). In rd retinas photoreceptors label about 1.8-2.5 times heavier than bipolar cells, b: a-Glu labeling, bipolar cells stain almost twice as heavily as photoreceptors in sighted chicks (carriers) at all ages (PR/BP ratio ~0.6-0.7). No differences are seen in the ratio of photoreceptor: bipolar cell labeling fgr a-Glu at one day of age. However, at 1 week and 2 weeks of age, photoreceptors stain significantly darker than bipolar cells in rd retinas. Values represent optical density measurements for 3 animals in each group at each time point, 10 areas measured per animal. The ratio of 3-week-old rd specimens could not be accurately measured due to many empty spaces caused by missing or degenerating photoreceptor cells (see Fig. 3c).
353 overnight at 4 °C to secondary antibody (goat anti-rabbit) to which 15 nm gold particles had been conjugated. Gold particles were visualized by a silver intensification procedure modified after Danscher and Noorgard 4. For details of the procedure, see Sherry22. Sections were photographed on a Nikon Labophot microscope. Densitometric measurements were made over the various retinal layers on a Zeiss IBAS Imaging system. Sections from the 2 groups at all ages tested were reacted simultaneously in the same dilution of antiserum and other reagents. In order to control for any artifact that might account for differences seen between specimens, we calculated a ratio of photoreceptor label density to that over an equal area of bipolar cells (middle zone of the inner nuclear layer). Endogenous levels of Asp were higher in rd retinas than in retinas from sighted chicks at all ages examined (Fig. la). ANOVA performed on values obtained from HPLC revealed that differences between the two groups were at the P < 0.01 level and neither time (age within group) nor the interaction of group with time were important. Results obtained for Glu (Fig. lb) were similar except at one day of age when no significant difference (P > 0.05) was observed in Glu content between rd and carrier chicks. Glu concentrations were significantly elevated in rd chick retinas at 1 week and 2 weeks of age. a-Asp antiserum labeled virtually every cell in both sighted and blind chicks' retinas at relatively low densities, although photoreceptors and horizontal cells stained considerably darker than other cell types (Fig. 2). In the case of Asp immunoreactivity, rd photoreceptors (Fig. 2b) were significantly denser staining than those of sighted chicks (Fig. 2a) while labeling of bipolar cells and other retinal cells was virtually identical to that in the carrier group (Figs. 2 and 4a). This pattern was seen at all times examined. The ratio of photoreceptor:bipolar cell label optical density by anti-Asp was about 1.2 in sighted chicks at all ages studied. In blind chicks, it was 1.8-2.3 (Fig. 4). Antibodies recognizing Glu also stained all retinal cells in carrier chick retinas although bipolar cells stained darker than other retinal neurons (Fig. 3a). No difference was seen in Glu labeling between carrier and rd retinas at 1 day of age but 1 week (Fig. 3b) and older (Fig. 3c) rd chick retinas had significantly denser labeling photoreceptors (compared to bipolar cells) than sighted chicks (Figs. 3b and 4b). The photoreceptor to bipolar cell labeling density (PR/BP) was about 0.5 in carrier chicks while it was about 1.0 in 1 week-old rd retinas and 1.5 in
2-week-old chicks (Fig. 4b). In 3-week-old specimens label density appeared even darker in rd photoreceptor cells than in carrier photoreceptors, however, due to the degenerating cells (i.e. empty spaces) in this layer, densitometric measurements on both Asp and Glu immunoreactivity were not valid. Labeling of cells other than photoreceptors was comparable between the two groups at 3 weeks of age, as at earlier times. Immunocytochemical labeling results were remarkably similar to HPLC data. Both experiments showed that Asp levels were elevated in rd chicks at all ages and that Glu levels were significantly increased in the 1-week- and 2-week-old rd animals. Immunocytochemistry localized the differences between blind and sighted chicks specifically to the photoreceptor layers, since labeling density was not different in the inner retinal layers of rd and normal chicks. Photoreceptors of rd chicks do not appear morphologically different (at the EM level) than normals until about 1-2 weeks of age, the time when both Asp and Glu levels are elevated in rd photoreceptors. In light of the findings that rd photoreceptor synaptosomes are capable of taking up exogenously supplied Glu but fail to release it under depolarizing conditions 28, we speculate that in vivo Glu is continually synthesized in rd photoreceptors but due to lack of dark-induced depolarization, the cells cannot release it. Glu might then build up in photoreceptor cells and cause degenerative changes. Preliminary studies in our laboratory have shown that one enzyme involved in Glu synthesis (glutaminase) is increased in blind chick retinas. Because the increased content in Asp and Glu occur after the chicks are blind, we do not believe that the transduction defect is directly caused by an error in E A A metabolism. The secondary pathological changes that we noted in photoreceptor cells26-2s, however, are associated with increased levels of Asp and Glu. Because these changes resemble those produced in response to applied EAAs or agonists, it appears that photoreceptor degeneration in this model is caused or augmented by high endogenous levels of Asp and/or Glu via a novel (non-receptor-mediated) toxic mechanism. Subsequent degeneration of inner retinal neurons may ultimately be caused by release of E A A s by dying photoreceptor cells that kill the postsynaptic neurons via a receptor-mediated mechanism.
1 Abrams, L., Politi, E. and Adler, R., Differential susceptibility of isolated mouse retina neurons and photoreceptors to kainic acid toxicity.
2 Barrington, M., Sattayasi, J., Zappia, J. and Ehrlich, D., Excitatory amino acids interfere with normal eye growth in posthatch chick, Curt. Eye Res., 8 (1989) 781-792.
This work was supported by grants from the National Institutes of Health (RO1 EY04590 and P30 EY08571) and a non-restrictive grant to the Department of Ophthalmology from Research to Prevent Blindness, Inc. R.J.U. is a Research to Prevent Blindness Senior Scientific Investigator.
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