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High vulnerability of GABA-immunoreactive neurons to kainate in rat retinal cultures: correlation with the kainate-stimulated cobalt uptake
Brain Research 823 Ž1999. 33–41
Research report
High vulnerability of GABA-immunoreactive neurons to kainate in rat retinal cultures: correlation with the kainate-stimulated cobalt uptake Young Hee Yoon a , Kyung Hee Jeong a , Myong Ja Shim a , Jae-Young Koh b
b,c,)
a Department of Ophthalmology, UniÕersity of Ulsan, College of Medicine, 388-1 Poongnap-Dong Songpa-Gu, Seoul 138-040, South Korea National CreatiÕe Research InitiatiÕe Center for the Study of CNS Zinc, UniÕersity of Ulsan, College of Medicine, 388-1 Poongnap-Dong Songpa-Gu, Seoul 138-040, South Korea c Department of Neurology, UniÕersity of Ulsan, College of Medicine, 388-1 Poongnap-Dong Songpa-Gu, Seoul 138-040, South Korea
Accepted 8 December 1998
Abstract Like other areas of the central nervous system, the retina is highly vulnerable to ischemia. In particular, neurons in the inner nuclear layer, including g-amino butyric acid ŽGABA.-ergic amacrine neurons, are highly vulnerable. Since excitotoxicity is likely a major mechanism of ischemic retinal injury, using rat retinal cell culture, we examined whether GABAergic retinal neurons are differentially vulnerable to particular excitotoxins. The neuronal population as a whole, identified by anti-microtubule associated protein-2 ŽMAP-2. immunocytochemistry, was equally vulnerable to kainate, but more resistant to N-methyl-D-aspartate ŽNMDA. than cultured cortical neurons. Compared to Thy-1 immunoreactive neurons, GABA immunoreactive neurons were more vulnerable to kainate, but more resistant to NMDA neurotoxicity. Double staining of cultures with anti-GABA immunocytochemistry and the kainate-stimulated cobalt uptake method, revealed a close correlation between the two. However, unlike in other neuronal cells, there was no clear correlation between GluR2 immunoreactivity and the cobalt staining. The heightened vulnerability of GABAergic neurons to kainate, as compared to the general neuronal population, may be due to the calcium-permeable AMPArkainate receptors they have, as identified functionally by the kainate-stimulated cobalt uptake staining. Since these neurons are preferentially injured in ischemia, AMPArkainate receptor-mediated neurotoxicity may contribute significantly to ischemic retinal injury. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Ischemia; Glutamate; N-methyl-D-aspartate; Neuronal death; Ganglion neurons
1. Introduction As in other regions of the central nervous system ŽCNS., the retina is highly vulnerable to ischemic insults. Cessation of blood flow to the retina for only 60 min in animals results in functional changes as demonstrated by loss of the b-wave of the electroretinogram w1x. More prolonged ischemia induces irreversible neuronal injury in the retina w2,3x. Particularly, amacrine and horizontal neurons in the inner nuclear layer, some of which contain GABA andror acetylcholine w4x, are highly vulnerable to ischemic insults w3,5x. Although the mechanistic basis of their high vulnerability to ischemia is currently unknown, it is possible that glutamate receptor-mediated neurotoxic)
Corresponding author. Department of Neurology, University of Ulsan, College of Medicine, 388-1 Poongnap-Dong Songpa-Gu, Seoul 138-040, S outh K orea. F ax: q 82-2-474-4691; E -m ail: [email protected]
ity, one of the major mechanisms proposed for ischemic neuronal injury w2,6–8x, may underlie this phenomenon. Of the two types of ionotropic glutamate receptors, the NMDA receptor has been shown to play a disproportionately large role in brief intense insults w9x, likely reflecting its high calcium permeability w10,11x. By contrast, the AMPAr kainate receptor may mediate neuronal death in more prolonged insults w12x due to their relatively low calcium permeability w11x. Although most central neurons are more vulnerable to the NMDA receptor-mediated neurotoxicity, some neurons are atypically more vulnerable to the AMPArkainate receptor-mediated one. Evidence suggests that ischemia-vulnerable retinal interneurons may belong to this group of neurons w13–15x. Amacrine and horizontal neurons in the inner nuclear layer are interneurons that contain GABA, acetylcholine, dopamine or other neurotransmitters w16,17x. Their counterparts in the forebrain, GABAergic neurons in cerebral cortex w18x and acetylcholinergic neurons in basal fore-
0006-8993r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 8 . 0 1 3 3 6 - 5
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brain w19x, are also highly vulnerable to kainate neurotoxicity, but relatively resistant to NMDA neurotoxicity. In addition, striatal and cortical interneurons containing NADPH-diaphorasernitric oxide synthase ŽNOS., somatostatin, or parvalbumin, also exhibit the same characteristics w20–23x. It has been suggested that these neurons may lack one form of AMPArkainate receptor subunits, GluR2 ŽGluR-B., the presence of which limits calcium permeability of the AMPArkainate receptor w24,25x. The absence of GluR2, therefore, markedly increases calcium permeability of AMPArkainate receptors. Thus, it has been speculated that the high calcium permeability of GluR2-lacking AMPArkainate receptors underlie the phenomenon of heightened vulnerability of these interneurons to nonNMDA type neurotoxins such as kainate w26x. Supporting this idea, these neurons lack immunoreactivity to anti-
GluR2r3 antibody, and exhibit increased cobalt uptake upon stimulation with kainate, an indicator of high calcium permeability of these receptors w27,28x. In the present study, we have examined in primary rat retinal cultures, whether GABAergic neurons exhibit heightened vulnerability to kainate neurotoxicity, and whether the basis for this is due to the presence of calcium-permeable AMPArkainate receptors.
2. Materials and methods 2.1. Retinal cell culture Primary cultures were generated from retinas of newborn Žpostnatal day 1 or 2. Sprague–Dawley rats. Retinas
Fig. 1. Three cell types in retinal culture. ŽA. A phase-contrast photomicrograph Ž200 = . of retinal cell culture ŽDIV10.. Neurons with processes Žsee also B. are lying on background flat cells, mostly astrocytes Žsee C.. ŽB. A photomicrograph of rat retinal cell culture ŽDIV10. immunocytochemically stained with anti-MAP2 antibody. Only cells with neuronal morphology bearing long processes were stained. ŽC. Anti-GFAP-stained retinal culture. Flat background cells in A were immunoreactive, indicating their astrocytic origin. ŽD. A phase-contrast photomicrograph of retinal culture showing small round cells that form a rosette-like structure Žarrow.. These cells are likely photoreceptor cells. Scale bars, 100 mm.
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were isolated and placed in Hanks balanced salt solution ŽHBSS, Gibco. lacking Ca2q and Mg 2q and mechanically dissociated into single cells by trituration with fire-polished Pasteur pipettes. Dissociated cells were plated on poly-Llysine-coated 24-well plates Ž3 retinas per plate.. Plating medium was based on Eagle’s minimum essential medium ŽMEM, Gibco. supplemented with 10% heat-inactivated fetal bovine serum and 25 mM KCl. These retinal cultures were maintained at 378C in a humidified 5% CO 2 incubator, and used for study after 8–10 days in vitro. Retinal cultures were exposed for 24 h to excitotoxins in Eagle’s MEM. Sham wash control cultures with the same MEM did not exhibit any neuronal degeneration up to 72 h after the media change. 2.2. Immunocytochemistry [18,23] Cultures were fixed for 60 min at room temperature in 1% glutaraldehyde in 0.1 M PBS buffer, and incubated with primary antibodies wanti-MAP-2 Ž1r500, Sigma., anti-GFAP Ž1r1000, Sigma., anti-Thy-1 Ž1r1000, Chemicon., and anti-GABA Ž1r1000, Sigma. overnight at 48C. The cultures were then treated with biotinylated secondary antibodies for 60 min at room temperature. Cells were processed for immunoperoxidase staining using avidin– horse radish peroxidase and diaminobenzidine ŽDAB.. 2.3. Assessment of cell death Retinal neuronal death was morphologically assessed under the microscope after immunocytochemical staining. Number of surviving cells was counted in five 200 = fields in each well; the fields were randomly chosen before the counting. Usually, the total number of counted neurons in 5 fields of controls ranged from 610 to 680 Ž640 " 30. for MAP-2Žq. neurons, from 240–340 for GABAŽq. Ž313 " 32. and Thy-1Žq. Ž266 " 20. neurons, respectively. Neurons with intact cell bodies and proximal neurites were counted as surviving, whereas neurons with severe cell body damage and neurite disruption, or cell debris were excluded. Each number was normalized to the mean number of surviving neurons in control sister cultures with sham wash alone as 100% to yield percent of surviving neurons. 2.4. Kainate-stimulated Co 2 q uptake method [29] Retinal cultures were incubated at room temperature for 30 min in the low sodium uptake buffer containing 5 mM CoCl 2 and 100 mM kainate. After thorough washout with the same buffer, cultures were treated with 0.12% Na 2 S for 5 min, and then fixed with 4% paraformaldehyde. The
Fig. 2. NMDA and kainate induce neuronal death. ŽA. Data represent percent of surviving neurons Žmean"S.E.M., ns 4. after 24 h exposure to varying concentrations of NMDA. ŽB. Data represent percent of surviving neurons Žmean"S.E.M., ns 4. after 24 h exposure to varying concentrations of kainate.
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cobalt image was developed by adding 0.1% AgNO 3 every 15 min Ž3 times.. Then cultures were washed with PBS several times to halt the reaction, and examined under the microscope. About 50–60% Ž57.3 " 1.6%, n s 5. of neurons in retinal cultures were stained with the kainatestimulated cobalt-uptake method.
3. Results In rat retinal cultures, three morphologically distinct cell types were readily identifiable by the light microscopic examination. Cells bearing long processes were stained with anti-MAP-2 antibody indicating their neuronal
Fig. 3. Resistance of GABAŽq. neurons to NMDA neurotoxicity. ŽA, B. Retinal cultures stained with anti-GABA antibody; sham wash ŽA. and after 24 h exposure to 300 mM NMDA ŽB.. Little loss of GABAŽq. neurons was seen. ŽC, D. Retinal cultures stained with anti-Thy-1 antibody. Substantial loss of Thy-1Žq. neurons was seen after 24 h exposure to 300 mM NMDA ŽD. as compared with sham wash control ŽC.. Arrows denote cell debris stained with anti-Thy-1 antibody. These debris were not counted. Scale bar, 100 mm. ŽE. Percent of surviving Thy-1Žq. Žsquare. and GABAŽq. Žcircle. neurons Žmean " S.E.M., n s 4. in sister retinal cultures after 24 h exposure to varying concentrations of NMDA. Note that GABAŽq. neurons are somewhat less vulnerable to NMDA neurotoxicity. Asterisks denote difference from each other Ž p - 0.05, two tail t test with Bonferroni correction for 4 comparisons..
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Fig. 3 Žcontinued..
identity w30x, and flat background cells with anti-GFAP-antibody indicating their astrocytic identity w31x. The small round cells without processes were likely photoreceptor cells as they tended to form a rosette-like structure ŽFig. 1.. Exposure of retinal cultures for 24 h to varying concentrations of NMDA or kainate induced concentration-dependent loss of total neuronal population identified with MAP-2 immunocytochemistry ŽFig. 2. without injuring astrocytes. Compared with cultured cortical neurons ŽLD50 ’s: 21 mM to kainate and 16 mM to NMDA. w32x, retinal neurons as a whole exhibited about equal vulnerability to kainate but significantly lower vulnerability to NMDA; the LD50 ’s were about 25 and 200 mM, respectively ŽFig. 2A and B.. We next examined the possibility that GABAŽq. neurons may exhibit differential vulnerability to NMDA and kainate, as compared with Thy-1Žq. ganglion neurons w33x. Exposure of retinal cultures for 24 h to NMDA resulted in a concentration-dependent death of Thy-1Žq. neurons, and to a much lesser degree, GABAŽq. neurons ŽFig. 3.. The LD50 of NMDA neurotoxicity to Thy-1Žq. ganglion neurons with 24 h exposure was around 200 mM, whereas that to GABAŽq. neurons were greater than 1 mM ŽFig. 3E.. Hence, GABAŽq. neurons were more resistant to NMDA neurotoxicity than Thy-1Žq. ganglion neurons. Of note, unlike in animal studies, anti-Thy-1 antibody mainly stained neuronal cell bodies and some proximal dendrites, which suggests that neurites of ganglion neurons may not be fully developed.
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By contrast, GABAŽq. neurons were more vulnerable to kainate neurotoxicity than were Thy-1Žq. ganglion neurons ŽFig. 4.. While 24 h exposure to 30 mM kainate killed only about 40% of Thy-1Žq. ganglion neurons, it almost completely destroyed GABAŽq. neurons. The LD50 of kainate to Thy-1Žq. neurons was around 40 mM, whereas that to GABAergic neurons was estimated to be about 20 mM ŽFig. 4C.. To examine the possibility that heightened vulnerability of GABAŽq. neurons comes from the high calcium permeability of the unconventional AMPArkainate receptors, retinal cultures were stained with the kainate-stimulated cobalt uptake method. This revealed that almost all GABAŽq. neurons Ž97.3 " 2.8%, n s 4. were stained with the kainate-stimulated cobalt uptake method ŽFig. 5.. Some cobaltŽq. neurons were not immunoreactive to GABA Ž43.6 " 9.4%, n s 4.. The combined data do, however, indicate that the majority of cobaltŽq. neurons in retinal cultures are GABAergic in nature. Like GABAŽq. neurons, cobaltŽq. neurons were also highly vulnerable to kainate neurotoxicity; only 30 mM kainate killed about 90% of cobaltŽq. neurons with 24 h exposure ŽFig. 5E.. Finally, we examined the possibility that cobaltŽq. neurons may not express the GluR2 subunit as their cortical counterparts w27x. However, most neurons Žabout 80– 90%. were stained with the specific anti-GluR2 antibody ŽFig. 6A.. As a consequence, subsequent staining of the same culture with the kainate-stimulated cobalt uptake method showed that the majority of cobaltŽq. neurons were GluR2 immunoreactive ŽFig. 6B; thick arrows.. Although drawing a line between GluR2Žq. and Žy. immunoreactivity was difficult because of the continuous spectrum of staining density, of 13.9 " 2.3% Ž n s 5. of neurons with very low level GluR2Žy. immunoreactivity, a similar fraction Žabout 50%. was stained with the cobalt method. In Fig. 6, two cobaltŽy. neuron with low level GluR2 immunoreactivity are presented Žthin arrows..
4. Discussion The present results have demonstrated that retinal neurons as a whole are equally vulnerable to kainate neurotoxicity but substantially more resistant to NMDA neurotoxicity than are cultured cortical neurons. This suggests a possibility that AMPArkainate receptor-mediated neurotoxicity may play a relatively more significant role in ischemic neuronal injury in retina than in cerebral cortex w34x. Furthermore, among cultured retinal neurons, GABAŽq. neurons in particular, as compared with Thy1Žq. ganglion neurons, exhibit unusually high vulnerability to kainate neurotoxicity but relative resistance to NMDA-induced neurotoxicity. Although Thy-1 immunoreactivity has been shown to be specific for retinal gan-
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Fig. 4. Heightened vulnerability of GABAŽq. neurons to kainate neurotoxicity. ŽA. Retinal cultures stained with anti-GABA antibody after 24 h exposure to 30 mM kainate. Severe loss of GABAŽq. neurons was seen as compared with sham wash control ŽFig. 3A.. ŽB. Retinal cultures stained with anti-Thy-1 antibody. Little loss of Thy-1Žq. neurons was seen after 24 h exposure to 30 mM kainate ŽB. as compared with sham wash control ŽFig. 3C.. Scale bar, 100 mm. ŽC. Percent of surviving Žmean " S.E.M., n s 4. Thy-1Žq. Žsquare. and GABAŽq. Žcircle. neurons in sister retinal cultures after 24 h exposure to varying concentrations of kainate. Note that GABAŽq. neurons are more vulnerable to kainate neurotoxicity. Asterisks denote difference from each other Ž p - 0.05, two tail t test with Bonferroni correction for 4 comparisons..
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glion neurons, a study suggest that it may stain other cell types after the injury w35x. Hence, the result obtained in the present study may have underestimated the vulnerability of Thy-1Žq. neurons, although we could not find additional cell types stained with anti-Thy-1 antibody after excitotoxic injury. The fact that GABAergic neurons in the inner nuclear layer are those with high vulnerability to ischemia w13x, further supports the idea that the AMPArkainate receptor-mediated neurotoxicity plays a significant role in retinal ischemia. The mechanism of the heightened vulnerability of GABAŽq. retinal neurons in culture may be similar to that of their cortical counterparts. As in cortical cultures w27x, the majority of GABAŽq. neurons in retinal cultures were stained with the kainate-stimulated cobalt uptake
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method w29x, a histochemical way to functionally identify neurons containing the AMPArkainate receptors with high calcium permeability w24,25x. Cobalt permeates the AMPArkainate receptor-operated channels with high calcium permeability, whereas it may not permeate those with low calcium permeability. Thus, at a functional level, cobalt uptake in GABAŽq. retinal neurons likely reflects the presence of the unconventional calcium-permeable AMPArkainate receptors on these neurons. In addition to GABAŽq. neurons, cholinergic neurons in the basal forebrain are also stained with the cobalt uptake method, and are highly vulnerable to kainate w19x. Since cholinergic amacrine neurons in the retina are also highly vulnerable to ischemia w15x, it is tempting to speculate that the common denominator of ischemia-vulnerable neurons in
Fig. 5. Correlation between GABA immunocytochemistry and kainate-stimulated cobalt uptake. ŽA, B. Photomicrographs of retinal cultures after staining with the cobalt uptake method, without ŽA. or with ŽB. addition of kainate during the incubation. ŽC, D. Photomicrographs of an identical field of a retinal culture stained with anti-GABA antibody ŽC. and subsequently with the kainate-stimulated cobalt uptake method ŽD.. Note that almost all GABAŽq. neurons were cobalt-stained Žthick arrows.. Some GABAŽy. neurons were additionally cobalt-stained Žthin arrows.. ŽE. Like GABAŽq. neurons, cobalt-stained neurons are highly vulnerable to kainate. Bars represent percent of surviving cobaltŽq. neurons Žmean " S.E.M., n s 4. after 24 h exposure to 30 or 50 mM kainate. Almost complete loss of cobaltŽq. neurons were seen in both conditions. Asterisks denote difference from control Ž p - 0.05, two tail t test with Bonferroni correction for 2 comparisons..
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Fig. 5 Žcontinued..
retina may be the presence of the calcium-permeable AMPArkainate receptors on those neurons. At molecular levels, the basis of high calcium permeability of certain AMPArkainate receptors has been at-
tributed to the lack of GluR2 subunits. The absence of GluR2 subunit has been shown to markedly increase the calcium permeability of the AMPArkainate receptor-operated channels w24,25x, indicating that the GluR2 subunit is responsible for the low calcium permeability of the ordinary AMPArkainate receptors. In cortical culture, there is a strong correlation between the lack of GluR2r3 immunoreactivity and the presence of kainate-stimulated cobalt uptake w27x. However, since anti-GluR2 immunocytochemistry stained most neurons in the current retinal cultures and cobaltŽq. neurons were mainly a subset of GluR2Žq. neurons, and since some GluR2Žy. neurons were cobaltŽy., we could not find a clear correlation between GluR2 immunoreactivity and the kainate-stimulated cobalt uptake. It seems possible that not an absolute but a relative deficiency of GluR2, in relation to other AMPArkainate receptor subunits ŽGluR1, R3, R4., may underlie the cobalt uptake in retinal neurons. Alternatively, calcium permeable channels comprised of kainate receptor subunits w36x may play a role in this case. Further studies may be needed to examine these possibilities. Regardless of the molecular basis, GABAŽq. neurons in retinal cultures are highly vulnerable to kainate neurotoxicity, probably due to the presence of AMPArkainate receptors with high calcium permeability. Since several lines of evidence suggest significant involvement of AMPArkainate receptors in retinal ischemia, antagonism at these receptors may be effective in ameliorating the resultant retinal neuron loss, especially that of GABAŽq. neurons in the inner nuclear layer.
Fig. 6. GluR2 immunocytochemistry and the cobalt staining. A retinal culture was stained immunocytochemically with a specific anti-GluR2 antibody ŽA. and then with the kainate-stimulated cobalt uptake method ŽB.. Note that the majority of process-bearing cells were stained with anti-GluR2 antibody in ŽA., and consequently, in this field, all cobaltŽq. neurons were GluR2Žq. Žthick arrows.. A few cobaltŽy. neurons exhibited low level of GluR2 immunoreactivity Žthin arrows.. Scale bar, 100 mm.
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Acknowledgements This study was supported by Asan Foundation ŽYHY. and Creative Research Initiatives of the Korean Ministry of Science and Technology ŽJYK.. References w1x F. Block, M. Schwarz, The b-wave of the electroretinogram as an index of retinal ischemia, Gen. Pharm. 30 Ž1998. 281–287. w2x Y.H. Yoon, M.F. Marmor, Dextromethorphan protects retina against ischemic injury in vivo, Arch. Ophthalmol. 107 Ž1989. 409–411. w3x W.F. Hughes, Quantitation of ischemic damage in rat retina, Exp. Eye Res. 53 Ž1991. 573–582. w4x E. Strettoi, R.H. Masland, The organization of the inner nuclear layer of the rabbit retina, J. Neurosci. 15 Ž1995. 875–888. w5x E.R. Buchi, Cell death in the rat retina after a pressure-induced ischemia-reperfusion insult: An electron microscopic study: I. Ganglion cell layer and inner nuclear layer, Exp. Eye Res. 55 Ž1992. 605–613. w6x D.W. Choi, Glutamate neurotoxicity and diseases of the nervous system, Neuron 1 Ž1988. 623–634. w7x S.M. Rothman, J.W. Olney, Glutamate and the pathophysiology of hypoxic–ischemic brain damage, Ann. Neurol. 19 Ž1986. 105–111. w8x G.H. Bresnick, Excitotoxins: a possible new mechanism for the pathogenesis of ischemic retinal damage, Arch. Ophthalmol. 107 Ž1989. 339–341. w9x D.W. Choi, Excitotoxic cell death, J. Neurobiol. 23 Ž1992. 1261– 1276. w10x A.B. MacDermott, M.L. Mayer, G.L. Westbrook, S.J. Smith, J.L. Barker, NMDA-receptor activation increases cytoplasmic calcium concentration in cultured spinal cord neurons, Nature 321 Ž1986. 519–522. w11x L.M. Canzoniero, S.L. Sensi, D.M. Turetsky, M.F. Finely, D.W. Choi, J.E. Huettner, Glutamate receptor-mediated calcium entry in neurons derived from P19 embryonal carcinoma cells, J. Neurosci. Res. 45 Ž1996. 226–236. w12x J. Koh, M.P. Goldberg, D.M. Hartley, D.W. Choi, Non-NMDA receptor-mediated neurotoxicity in cortical culture, J. Neurosci. 10 Ž1990. 693–705. w13x N.N. Osborne, A.J. Herrera, The effect of experimental ischemia and excitatory amino acid agonists on the GABA and serotonin immunoreactivity in the rabbit retina, Neuroscience 59 Ž1994. 1071–1081. w14x L. Abrams, L.E. Politi, R. Adler, Differential susceptibility of isolated mouse retinal neurons and photoreceptors to kainic acid toxicity: In vitro studies, Invest. Ophthalmol. Vis. Sci. 30 Ž1989. 2300–2308. w15x N.N. Osborne, A. Larsen, N.L. Barnett, Influence of excitatory amino acids and ischemia on rat retinal choline acetyl transferasecontaining cells, Invest. Ophthalmol. Vis. Sci. 36 Ž1995. 1692–1700. w16x R.H. Masland, Cell mosaics and neurotransmitters, in: D.M. Albert, F.A. Jakobiec ŽEds.., Principles and Practice of Ophthalmology, Saunders, Philadelphia, 1994, pp. 384–398. w17x C.J. Pycock, Retinal neurotransmission, Surv. Ophthalmol. 29 Ž1985. 355–365. w18x E.S. Tecoma, D.W. Choi, GABAergic neocortical neurons are resistant to NMDA receptor-mediated injury, Neurology 39 Ž1989. 676– 682.
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Research report
High vulnerability of GABA-immunoreactive neurons to kainate in rat retinal cultures: correlation with the kainate-stimulated cobalt uptake Young Hee Yoon a , Kyung Hee Jeong a , Myong Ja Shim a , Jae-Young Koh b
b,c,)
a Department of Ophthalmology, UniÕersity of Ulsan, College of Medicine, 388-1 Poongnap-Dong Songpa-Gu, Seoul 138-040, South Korea National CreatiÕe Research InitiatiÕe Center for the Study of CNS Zinc, UniÕersity of Ulsan, College of Medicine, 388-1 Poongnap-Dong Songpa-Gu, Seoul 138-040, South Korea c Department of Neurology, UniÕersity of Ulsan, College of Medicine, 388-1 Poongnap-Dong Songpa-Gu, Seoul 138-040, South Korea
Accepted 8 December 1998
Abstract Like other areas of the central nervous system, the retina is highly vulnerable to ischemia. In particular, neurons in the inner nuclear layer, including g-amino butyric acid ŽGABA.-ergic amacrine neurons, are highly vulnerable. Since excitotoxicity is likely a major mechanism of ischemic retinal injury, using rat retinal cell culture, we examined whether GABAergic retinal neurons are differentially vulnerable to particular excitotoxins. The neuronal population as a whole, identified by anti-microtubule associated protein-2 ŽMAP-2. immunocytochemistry, was equally vulnerable to kainate, but more resistant to N-methyl-D-aspartate ŽNMDA. than cultured cortical neurons. Compared to Thy-1 immunoreactive neurons, GABA immunoreactive neurons were more vulnerable to kainate, but more resistant to NMDA neurotoxicity. Double staining of cultures with anti-GABA immunocytochemistry and the kainate-stimulated cobalt uptake method, revealed a close correlation between the two. However, unlike in other neuronal cells, there was no clear correlation between GluR2 immunoreactivity and the cobalt staining. The heightened vulnerability of GABAergic neurons to kainate, as compared to the general neuronal population, may be due to the calcium-permeable AMPArkainate receptors they have, as identified functionally by the kainate-stimulated cobalt uptake staining. Since these neurons are preferentially injured in ischemia, AMPArkainate receptor-mediated neurotoxicity may contribute significantly to ischemic retinal injury. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Ischemia; Glutamate; N-methyl-D-aspartate; Neuronal death; Ganglion neurons
1. Introduction As in other regions of the central nervous system ŽCNS., the retina is highly vulnerable to ischemic insults. Cessation of blood flow to the retina for only 60 min in animals results in functional changes as demonstrated by loss of the b-wave of the electroretinogram w1x. More prolonged ischemia induces irreversible neuronal injury in the retina w2,3x. Particularly, amacrine and horizontal neurons in the inner nuclear layer, some of which contain GABA andror acetylcholine w4x, are highly vulnerable to ischemic insults w3,5x. Although the mechanistic basis of their high vulnerability to ischemia is currently unknown, it is possible that glutamate receptor-mediated neurotoxic)
Corresponding author. Department of Neurology, University of Ulsan, College of Medicine, 388-1 Poongnap-Dong Songpa-Gu, Seoul 138-040, S outh K orea. F ax: q 82-2-474-4691; E -m ail: [email protected]
ity, one of the major mechanisms proposed for ischemic neuronal injury w2,6–8x, may underlie this phenomenon. Of the two types of ionotropic glutamate receptors, the NMDA receptor has been shown to play a disproportionately large role in brief intense insults w9x, likely reflecting its high calcium permeability w10,11x. By contrast, the AMPAr kainate receptor may mediate neuronal death in more prolonged insults w12x due to their relatively low calcium permeability w11x. Although most central neurons are more vulnerable to the NMDA receptor-mediated neurotoxicity, some neurons are atypically more vulnerable to the AMPArkainate receptor-mediated one. Evidence suggests that ischemia-vulnerable retinal interneurons may belong to this group of neurons w13–15x. Amacrine and horizontal neurons in the inner nuclear layer are interneurons that contain GABA, acetylcholine, dopamine or other neurotransmitters w16,17x. Their counterparts in the forebrain, GABAergic neurons in cerebral cortex w18x and acetylcholinergic neurons in basal fore-
0006-8993r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 8 . 0 1 3 3 6 - 5
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brain w19x, are also highly vulnerable to kainate neurotoxicity, but relatively resistant to NMDA neurotoxicity. In addition, striatal and cortical interneurons containing NADPH-diaphorasernitric oxide synthase ŽNOS., somatostatin, or parvalbumin, also exhibit the same characteristics w20–23x. It has been suggested that these neurons may lack one form of AMPArkainate receptor subunits, GluR2 ŽGluR-B., the presence of which limits calcium permeability of the AMPArkainate receptor w24,25x. The absence of GluR2, therefore, markedly increases calcium permeability of AMPArkainate receptors. Thus, it has been speculated that the high calcium permeability of GluR2-lacking AMPArkainate receptors underlie the phenomenon of heightened vulnerability of these interneurons to nonNMDA type neurotoxins such as kainate w26x. Supporting this idea, these neurons lack immunoreactivity to anti-
GluR2r3 antibody, and exhibit increased cobalt uptake upon stimulation with kainate, an indicator of high calcium permeability of these receptors w27,28x. In the present study, we have examined in primary rat retinal cultures, whether GABAergic neurons exhibit heightened vulnerability to kainate neurotoxicity, and whether the basis for this is due to the presence of calcium-permeable AMPArkainate receptors.
2. Materials and methods 2.1. Retinal cell culture Primary cultures were generated from retinas of newborn Žpostnatal day 1 or 2. Sprague–Dawley rats. Retinas
Fig. 1. Three cell types in retinal culture. ŽA. A phase-contrast photomicrograph Ž200 = . of retinal cell culture ŽDIV10.. Neurons with processes Žsee also B. are lying on background flat cells, mostly astrocytes Žsee C.. ŽB. A photomicrograph of rat retinal cell culture ŽDIV10. immunocytochemically stained with anti-MAP2 antibody. Only cells with neuronal morphology bearing long processes were stained. ŽC. Anti-GFAP-stained retinal culture. Flat background cells in A were immunoreactive, indicating their astrocytic origin. ŽD. A phase-contrast photomicrograph of retinal culture showing small round cells that form a rosette-like structure Žarrow.. These cells are likely photoreceptor cells. Scale bars, 100 mm.
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were isolated and placed in Hanks balanced salt solution ŽHBSS, Gibco. lacking Ca2q and Mg 2q and mechanically dissociated into single cells by trituration with fire-polished Pasteur pipettes. Dissociated cells were plated on poly-Llysine-coated 24-well plates Ž3 retinas per plate.. Plating medium was based on Eagle’s minimum essential medium ŽMEM, Gibco. supplemented with 10% heat-inactivated fetal bovine serum and 25 mM KCl. These retinal cultures were maintained at 378C in a humidified 5% CO 2 incubator, and used for study after 8–10 days in vitro. Retinal cultures were exposed for 24 h to excitotoxins in Eagle’s MEM. Sham wash control cultures with the same MEM did not exhibit any neuronal degeneration up to 72 h after the media change. 2.2. Immunocytochemistry [18,23] Cultures were fixed for 60 min at room temperature in 1% glutaraldehyde in 0.1 M PBS buffer, and incubated with primary antibodies wanti-MAP-2 Ž1r500, Sigma., anti-GFAP Ž1r1000, Sigma., anti-Thy-1 Ž1r1000, Chemicon., and anti-GABA Ž1r1000, Sigma. overnight at 48C. The cultures were then treated with biotinylated secondary antibodies for 60 min at room temperature. Cells were processed for immunoperoxidase staining using avidin– horse radish peroxidase and diaminobenzidine ŽDAB.. 2.3. Assessment of cell death Retinal neuronal death was morphologically assessed under the microscope after immunocytochemical staining. Number of surviving cells was counted in five 200 = fields in each well; the fields were randomly chosen before the counting. Usually, the total number of counted neurons in 5 fields of controls ranged from 610 to 680 Ž640 " 30. for MAP-2Žq. neurons, from 240–340 for GABAŽq. Ž313 " 32. and Thy-1Žq. Ž266 " 20. neurons, respectively. Neurons with intact cell bodies and proximal neurites were counted as surviving, whereas neurons with severe cell body damage and neurite disruption, or cell debris were excluded. Each number was normalized to the mean number of surviving neurons in control sister cultures with sham wash alone as 100% to yield percent of surviving neurons. 2.4. Kainate-stimulated Co 2 q uptake method [29] Retinal cultures were incubated at room temperature for 30 min in the low sodium uptake buffer containing 5 mM CoCl 2 and 100 mM kainate. After thorough washout with the same buffer, cultures were treated with 0.12% Na 2 S for 5 min, and then fixed with 4% paraformaldehyde. The
Fig. 2. NMDA and kainate induce neuronal death. ŽA. Data represent percent of surviving neurons Žmean"S.E.M., ns 4. after 24 h exposure to varying concentrations of NMDA. ŽB. Data represent percent of surviving neurons Žmean"S.E.M., ns 4. after 24 h exposure to varying concentrations of kainate.
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cobalt image was developed by adding 0.1% AgNO 3 every 15 min Ž3 times.. Then cultures were washed with PBS several times to halt the reaction, and examined under the microscope. About 50–60% Ž57.3 " 1.6%, n s 5. of neurons in retinal cultures were stained with the kainatestimulated cobalt-uptake method.
3. Results In rat retinal cultures, three morphologically distinct cell types were readily identifiable by the light microscopic examination. Cells bearing long processes were stained with anti-MAP-2 antibody indicating their neuronal
Fig. 3. Resistance of GABAŽq. neurons to NMDA neurotoxicity. ŽA, B. Retinal cultures stained with anti-GABA antibody; sham wash ŽA. and after 24 h exposure to 300 mM NMDA ŽB.. Little loss of GABAŽq. neurons was seen. ŽC, D. Retinal cultures stained with anti-Thy-1 antibody. Substantial loss of Thy-1Žq. neurons was seen after 24 h exposure to 300 mM NMDA ŽD. as compared with sham wash control ŽC.. Arrows denote cell debris stained with anti-Thy-1 antibody. These debris were not counted. Scale bar, 100 mm. ŽE. Percent of surviving Thy-1Žq. Žsquare. and GABAŽq. Žcircle. neurons Žmean " S.E.M., n s 4. in sister retinal cultures after 24 h exposure to varying concentrations of NMDA. Note that GABAŽq. neurons are somewhat less vulnerable to NMDA neurotoxicity. Asterisks denote difference from each other Ž p - 0.05, two tail t test with Bonferroni correction for 4 comparisons..
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Fig. 3 Žcontinued..
identity w30x, and flat background cells with anti-GFAP-antibody indicating their astrocytic identity w31x. The small round cells without processes were likely photoreceptor cells as they tended to form a rosette-like structure ŽFig. 1.. Exposure of retinal cultures for 24 h to varying concentrations of NMDA or kainate induced concentration-dependent loss of total neuronal population identified with MAP-2 immunocytochemistry ŽFig. 2. without injuring astrocytes. Compared with cultured cortical neurons ŽLD50 ’s: 21 mM to kainate and 16 mM to NMDA. w32x, retinal neurons as a whole exhibited about equal vulnerability to kainate but significantly lower vulnerability to NMDA; the LD50 ’s were about 25 and 200 mM, respectively ŽFig. 2A and B.. We next examined the possibility that GABAŽq. neurons may exhibit differential vulnerability to NMDA and kainate, as compared with Thy-1Žq. ganglion neurons w33x. Exposure of retinal cultures for 24 h to NMDA resulted in a concentration-dependent death of Thy-1Žq. neurons, and to a much lesser degree, GABAŽq. neurons ŽFig. 3.. The LD50 of NMDA neurotoxicity to Thy-1Žq. ganglion neurons with 24 h exposure was around 200 mM, whereas that to GABAŽq. neurons were greater than 1 mM ŽFig. 3E.. Hence, GABAŽq. neurons were more resistant to NMDA neurotoxicity than Thy-1Žq. ganglion neurons. Of note, unlike in animal studies, anti-Thy-1 antibody mainly stained neuronal cell bodies and some proximal dendrites, which suggests that neurites of ganglion neurons may not be fully developed.
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By contrast, GABAŽq. neurons were more vulnerable to kainate neurotoxicity than were Thy-1Žq. ganglion neurons ŽFig. 4.. While 24 h exposure to 30 mM kainate killed only about 40% of Thy-1Žq. ganglion neurons, it almost completely destroyed GABAŽq. neurons. The LD50 of kainate to Thy-1Žq. neurons was around 40 mM, whereas that to GABAergic neurons was estimated to be about 20 mM ŽFig. 4C.. To examine the possibility that heightened vulnerability of GABAŽq. neurons comes from the high calcium permeability of the unconventional AMPArkainate receptors, retinal cultures were stained with the kainate-stimulated cobalt uptake method. This revealed that almost all GABAŽq. neurons Ž97.3 " 2.8%, n s 4. were stained with the kainate-stimulated cobalt uptake method ŽFig. 5.. Some cobaltŽq. neurons were not immunoreactive to GABA Ž43.6 " 9.4%, n s 4.. The combined data do, however, indicate that the majority of cobaltŽq. neurons in retinal cultures are GABAergic in nature. Like GABAŽq. neurons, cobaltŽq. neurons were also highly vulnerable to kainate neurotoxicity; only 30 mM kainate killed about 90% of cobaltŽq. neurons with 24 h exposure ŽFig. 5E.. Finally, we examined the possibility that cobaltŽq. neurons may not express the GluR2 subunit as their cortical counterparts w27x. However, most neurons Žabout 80– 90%. were stained with the specific anti-GluR2 antibody ŽFig. 6A.. As a consequence, subsequent staining of the same culture with the kainate-stimulated cobalt uptake method showed that the majority of cobaltŽq. neurons were GluR2 immunoreactive ŽFig. 6B; thick arrows.. Although drawing a line between GluR2Žq. and Žy. immunoreactivity was difficult because of the continuous spectrum of staining density, of 13.9 " 2.3% Ž n s 5. of neurons with very low level GluR2Žy. immunoreactivity, a similar fraction Žabout 50%. was stained with the cobalt method. In Fig. 6, two cobaltŽy. neuron with low level GluR2 immunoreactivity are presented Žthin arrows..
4. Discussion The present results have demonstrated that retinal neurons as a whole are equally vulnerable to kainate neurotoxicity but substantially more resistant to NMDA neurotoxicity than are cultured cortical neurons. This suggests a possibility that AMPArkainate receptor-mediated neurotoxicity may play a relatively more significant role in ischemic neuronal injury in retina than in cerebral cortex w34x. Furthermore, among cultured retinal neurons, GABAŽq. neurons in particular, as compared with Thy1Žq. ganglion neurons, exhibit unusually high vulnerability to kainate neurotoxicity but relative resistance to NMDA-induced neurotoxicity. Although Thy-1 immunoreactivity has been shown to be specific for retinal gan-
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Fig. 4. Heightened vulnerability of GABAŽq. neurons to kainate neurotoxicity. ŽA. Retinal cultures stained with anti-GABA antibody after 24 h exposure to 30 mM kainate. Severe loss of GABAŽq. neurons was seen as compared with sham wash control ŽFig. 3A.. ŽB. Retinal cultures stained with anti-Thy-1 antibody. Little loss of Thy-1Žq. neurons was seen after 24 h exposure to 30 mM kainate ŽB. as compared with sham wash control ŽFig. 3C.. Scale bar, 100 mm. ŽC. Percent of surviving Žmean " S.E.M., n s 4. Thy-1Žq. Žsquare. and GABAŽq. Žcircle. neurons in sister retinal cultures after 24 h exposure to varying concentrations of kainate. Note that GABAŽq. neurons are more vulnerable to kainate neurotoxicity. Asterisks denote difference from each other Ž p - 0.05, two tail t test with Bonferroni correction for 4 comparisons..
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glion neurons, a study suggest that it may stain other cell types after the injury w35x. Hence, the result obtained in the present study may have underestimated the vulnerability of Thy-1Žq. neurons, although we could not find additional cell types stained with anti-Thy-1 antibody after excitotoxic injury. The fact that GABAergic neurons in the inner nuclear layer are those with high vulnerability to ischemia w13x, further supports the idea that the AMPArkainate receptor-mediated neurotoxicity plays a significant role in retinal ischemia. The mechanism of the heightened vulnerability of GABAŽq. retinal neurons in culture may be similar to that of their cortical counterparts. As in cortical cultures w27x, the majority of GABAŽq. neurons in retinal cultures were stained with the kainate-stimulated cobalt uptake
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method w29x, a histochemical way to functionally identify neurons containing the AMPArkainate receptors with high calcium permeability w24,25x. Cobalt permeates the AMPArkainate receptor-operated channels with high calcium permeability, whereas it may not permeate those with low calcium permeability. Thus, at a functional level, cobalt uptake in GABAŽq. retinal neurons likely reflects the presence of the unconventional calcium-permeable AMPArkainate receptors on these neurons. In addition to GABAŽq. neurons, cholinergic neurons in the basal forebrain are also stained with the cobalt uptake method, and are highly vulnerable to kainate w19x. Since cholinergic amacrine neurons in the retina are also highly vulnerable to ischemia w15x, it is tempting to speculate that the common denominator of ischemia-vulnerable neurons in
Fig. 5. Correlation between GABA immunocytochemistry and kainate-stimulated cobalt uptake. ŽA, B. Photomicrographs of retinal cultures after staining with the cobalt uptake method, without ŽA. or with ŽB. addition of kainate during the incubation. ŽC, D. Photomicrographs of an identical field of a retinal culture stained with anti-GABA antibody ŽC. and subsequently with the kainate-stimulated cobalt uptake method ŽD.. Note that almost all GABAŽq. neurons were cobalt-stained Žthick arrows.. Some GABAŽy. neurons were additionally cobalt-stained Žthin arrows.. ŽE. Like GABAŽq. neurons, cobalt-stained neurons are highly vulnerable to kainate. Bars represent percent of surviving cobaltŽq. neurons Žmean " S.E.M., n s 4. after 24 h exposure to 30 or 50 mM kainate. Almost complete loss of cobaltŽq. neurons were seen in both conditions. Asterisks denote difference from control Ž p - 0.05, two tail t test with Bonferroni correction for 2 comparisons..
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Fig. 5 Žcontinued..
retina may be the presence of the calcium-permeable AMPArkainate receptors on those neurons. At molecular levels, the basis of high calcium permeability of certain AMPArkainate receptors has been at-
tributed to the lack of GluR2 subunits. The absence of GluR2 subunit has been shown to markedly increase the calcium permeability of the AMPArkainate receptor-operated channels w24,25x, indicating that the GluR2 subunit is responsible for the low calcium permeability of the ordinary AMPArkainate receptors. In cortical culture, there is a strong correlation between the lack of GluR2r3 immunoreactivity and the presence of kainate-stimulated cobalt uptake w27x. However, since anti-GluR2 immunocytochemistry stained most neurons in the current retinal cultures and cobaltŽq. neurons were mainly a subset of GluR2Žq. neurons, and since some GluR2Žy. neurons were cobaltŽy., we could not find a clear correlation between GluR2 immunoreactivity and the kainate-stimulated cobalt uptake. It seems possible that not an absolute but a relative deficiency of GluR2, in relation to other AMPArkainate receptor subunits ŽGluR1, R3, R4., may underlie the cobalt uptake in retinal neurons. Alternatively, calcium permeable channels comprised of kainate receptor subunits w36x may play a role in this case. Further studies may be needed to examine these possibilities. Regardless of the molecular basis, GABAŽq. neurons in retinal cultures are highly vulnerable to kainate neurotoxicity, probably due to the presence of AMPArkainate receptors with high calcium permeability. Since several lines of evidence suggest significant involvement of AMPArkainate receptors in retinal ischemia, antagonism at these receptors may be effective in ameliorating the resultant retinal neuron loss, especially that of GABAŽq. neurons in the inner nuclear layer.
Fig. 6. GluR2 immunocytochemistry and the cobalt staining. A retinal culture was stained immunocytochemically with a specific anti-GluR2 antibody ŽA. and then with the kainate-stimulated cobalt uptake method ŽB.. Note that the majority of process-bearing cells were stained with anti-GluR2 antibody in ŽA., and consequently, in this field, all cobaltŽq. neurons were GluR2Žq. Žthick arrows.. A few cobaltŽy. neurons exhibited low level of GluR2 immunoreactivity Žthin arrows.. Scale bar, 100 mm.
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