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Developmental expression of N-methyl-d-aspartate (NMDA)-induced neurotoxicity, NMDA receptor function, and the NMDAR1 and glutamate-binding protein subunits in cerebellar granule cells in primary cultures

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Abstract

Cerebellar granule cells maintained in vitro as primary cultures are a relatively homogeneous neuronal population that can be used to evaluate the developmental expression of neurotransmitter receptors and to assess their role in cell survival and degeneration. The toxicity induced by N-methyl-d-aspartate (NMDA) in granule cells maintained under partially depolarizing conditions and in the presence of physiologic extracellular concentrations of Mg2+ was greatest for the neurons maintained for 14 days in vitro (DIV). However, following NMDA receptor activation neurons as young as 5 DIV exhibited increases in the concentration of intracellular free Ca2+ which were as large as those achieved with cells at 8–9 or 13–14 DIV. The less mature neurons exhibited a “down-regulation” of responses to increasing concentrations of NMDA and the more mature cells maintained elevated intracellular Ca2+ levels during the inter-stimulus periods. Immunochemical analyses of the expression of the NMDA receptor-associated proteins NMDAR1 and glutamatebinding protein (GBP) in granule cells indicated a developmental increase in both proteins, albeit the pattern of expression of NMDAR1 was the more complex. No definite correlation has yet been established between toxicity induced by NMDA and the expression of these two proteins. Finally, although the developmental expression of nitric oxide synthase, an enzyme that catalyzes the formation of the potentially neurotoxic radicals nitric oxide and superoxide anion, increased progressively with the maturation of neurons in culture, an inhibitor of this enzyme did not protect neurons from NMDA-induced toxicity. Therefore, the developmental changes in granule cells that lead to increased vulnerability following excessive activation of NMDA receptors are not yet completely defined.

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References

  1. Choi, D. W., Koh, J., and Peters, S. 1988. Pharmacology of glutamate neurotoxicity in cortical cell culture: Attenuation by NMDA antagonists. J Neurosci. 8:85–196.

    Google Scholar 

  2. Choi, D. W. 1992. Bench to bedside: The glutamate connection. Science 258:241–243.

    PubMed  Google Scholar 

  3. Wieloch, T. 1985. Hypolycemia-induced neuronal damage prevented by an N-methyl-D-aspartate antagonist. Science 230:681–683.

    PubMed  Google Scholar 

  4. Meldrum, B. 1985. Possible therapeutic applications of antagonists of excitatory amino acid transmitters. Clin Sci. 68:113–122.

    PubMed  Google Scholar 

  5. Simon, R. P., Griffiths, T., Evans, M. C., Swan, J. H., and Meldrum, B. S. 1984. Blockade of N-methyl-D-aspartate receptors may protect against ischemic damage in the brain. Science 226:850–852.

    PubMed  Google Scholar 

  6. Isokawa, M. and Levesque, M. F. 1991. Increased NMDA responses and dendritic degeneration in human epileptic hippocampal neurons in slices. Neurosci. Lett. 132:212–216.

    PubMed  Google Scholar 

  7. Schwarcz, R., and Meldrum, B. 1985. Excitatory amino acid antagonists provide a therapeutic approach to neurological disorders. Lancet 2:140–143.

    PubMed  Google Scholar 

  8. Maragos, W. F., Greenamyre, J. T., Penny, J. B., and Young, A. B. Glutamate dysfunction in Alzheimer's disease: A hypothesis. 1987. Trends Neurosci. 10:65–68.

    Google Scholar 

  9. Lipton, S. A. 1994. HIV displays its coat of arms. Nature 367: 113–114.

    PubMed  Google Scholar 

  10. Lipton, S. A., Sucher, N. J., Kaiser, P. K., and Dreyer, E. B. 1991. Synergistic effects of HIV coat protein and NMDA receptor-mediated neurotoxicity. Neuron 7:111–118.

    PubMed  Google Scholar 

  11. Choi, D. W. 1987. Ionic dependence of glutamate neurotoxicity. J. Neurosci. 7:369–379.

    PubMed  Google Scholar 

  12. Benveniste, H., Jørgensen, M. B., Diemer, N. G., and Hansen, A. J. 1988. Calcium accumulation by glutamate receptor activation is involved in hippocampal cell damage after ischemia. Acta Neurol. Scand. 7:369–379.

    Google Scholar 

  13. Murphy, S. N., Thayer, S. A., and Miller, R. J. 1988. The effects of excitatory amino acids on intracellular calcium in single mouse striatal neurons in vitro. J. Neurosci. 7:4145–4158.

    Google Scholar 

  14. Mattson, M. P., Guthrie, P. B., and Kater, S. B. 1988. Intracellular messenger in the generation and degeneration of hippocampal neuroarchitecture. J. Neurosci. Res. 21:447–464.

    PubMed  Google Scholar 

  15. Burgoyne, R. D., Pearce, I. A., and Cambray-Deakin, M. 1988. N-Methyl-D-aspartate raises cytosolic calcium concentration in rat cerebellar granule cells in culture. Neurosci. Lett. 91:47–52.

    PubMed  Google Scholar 

  16. Courtney, M. J., Lambert, J. J., and Nicholls, D. G. 1990. The interactions between plasma membrane depolarization and glutamate receptor activation in the regulation of cytoplasmic free calcium in culture cerebellar granule cells. J. Neurosci. 10:3873–3879.

    PubMed  Google Scholar 

  17. Holopainen, I., Louve, M., Enkvist, M. O. K., and kerman, K. E. O. 1990. Coupling of glutamatergic receptors to changes in intracellular Ca2+ in rat cerebellar granule cells in primary culture. J. Neurosci. Res. 25:187–193.

    PubMed  Google Scholar 

  18. Coyle, J. T. and Puttfarcken, P. 1993. Oxidative stress, glutamate and neurodegenerative disorders. Science 262:689–695.

    PubMed  Google Scholar 

  19. Lipton, S. A., Choi, Y-B., Pan, Z-H., Lei, S. Z., Chen, H-S. V., Sucher, N. J., Loscalzo, J., Singel, D. J., and Stamler, J. S. 1993. A redox-based mechanism for the neuroprotective and neurodestructive effects of nitric oxide and related nitroso-compounds. Nature 364:626–632.

    PubMed  Google Scholar 

  20. Lafon-Cazal, M., Pietrl, S., Culcasi, M., and Bockaert, J. 1993. NMDA-dependent superoxide production and neurotoxicity. Nature 364:535–537.

    PubMed  Google Scholar 

  21. Lafon-Cazal, M., Culcasi, M., Gaven, F., Pietrl, S., and Bockaert, J. 1993. Nitric oxide, superoxide and peroxynitrite: putative mediators of NMDA-induced cell death in cerebellar granule cells. Neuropharmacol. 32:1259–1266.

    Google Scholar 

  22. Hewett, S. J., Csernansky, C. A., and Choi, D. W. 1994. Selective potentiation of NMDA-induced neuronal injury following induction of astrocytic iNOS. Neuron 13:487–494.

    PubMed  Google Scholar 

  23. Oh, S. M., and Betz, A. L. 1991. Interaction between free radicals and excitatory amino acids in the formation of ischemic brain edema in rats. Stroke 22:915–921.

    PubMed  Google Scholar 

  24. McCord, J. M. 1985. Oxygen-derived free radicals in the postischemic tissue injury, N. Engl. J. Med. 312:159–163.

    PubMed  Google Scholar 

  25. Dumuis, A., Sebben, M., Haynes, L., Pin, J.-P., and Bockaert, J. 1988. NMDA receptors activate the arachidonic acid cascade system in striatal neurons. Nature 336:68–70.

    PubMed  Google Scholar 

  26. Dumuis, A., Pin, J.-P., Oomagari, K., Sebben, M., and Bockaert, J. 1990. Arachidonic acid released from striatal neurons by joint stimulation of ionotropic and metabotropic quisqualate receptors. Nature 347:182–184.

    PubMed  Google Scholar 

  27. Needleman, P., Turk, J., Jakschik, B. A., Morrison, A. R., and Lefkowith, J. B. 1986. Arachidonic acid metabolism. Ann. Rev. Biochem. 55:69–102.

    PubMed  Google Scholar 

  28. Kappus, H. 1990. Biochemical mechanisms of chemical-induced lipid peroxidation. Pages 104–109,in Vigo-Pelfrey, C. (ed.), Membrane-Lipid Oxidation, Vol. II, CRC Press, Boca Raton, FL.

    Google Scholar 

  29. Bredt, D. S., and Snyder, S. H. 1990. Isolation of nitric oxide synthetase, a calmodulin-requiring enzyme. Proc. Natl. Acad. Sci. USA 87:682–685.

    PubMed  Google Scholar 

  30. Bredt, D. S., Hwang, P. M., Glatt, C. E., Lowenstein, C., Randall, R. R., and Snyder, S. H. 1991. Cloned and expressed nitric oxide synthase structurally resembles cytochrome P-450 reductase. Nature 351:714–718.

    PubMed  Google Scholar 

  31. Pou, S., Pou, W. S., Bredt, D. S., Snyder, S. H., and Rosen, G. R. 1992. Generation of superoxide by purified brain nitric oxide synthase. J. Biol. Chem. 267:24173–24176.

    PubMed  Google Scholar 

  32. Vaccario, F. M., Alho, H., Santi, M. R., and Guidotti, A. 1987. Coexistence of GABA receptors and GABA modulin in primary cultures of rat cerebellar granule cells. J. Neurosci. 7:65–76.

    PubMed  Google Scholar 

  33. Gallo, V., Ciotti, M. T., Aloisi, F., and Levi, G. 1982. Selective release of glutamate from cerebellar granule cells differentiating in culture. Proc. Natl. Acad. Sci. USA 79:7919–7923.

    PubMed  Google Scholar 

  34. Balázs, R., Jorgensen, O. S., and Hack, N. 1988. N-Methyl-D-Asparatate promotes the survival of cerebellar granule cells in culture. Neuroscience 27:437–451.

    PubMed  Google Scholar 

  35. Van der Valk, J. B. V., Resink, A., and Balazs, R. 1991. Membrane depolarization and the expression of glutamate receptors in cerebellar granule cells. Eur. J. Pharmacol. 201:247–250.

    PubMed  Google Scholar 

  36. Balázs, R., Resink, A., Hack, M., Van der Valk, J. B. V., Kumar, K. N., and Michaelis, E. K. 1992. NMDA treatment and K+-induced depolarization selectively promote the expression of an NMDA-preferring class of the ionotropic glutamate receptors in cerebellar granule neurones. Neurosci. Lett. 137:109–113.

    PubMed  Google Scholar 

  37. Cox, J. A., Felder, C. C., and Henneberry, R. C. 1990. Differential expression of excitatory amino acid receptor subtypes in cultured cerebellar neurons. Neuron 4:941–947.

    PubMed  Google Scholar 

  38. Nakanishi, S. 1992. Molecular diversity of glutamate receptors and implications for brain function, Science 258:597–603.

    PubMed  Google Scholar 

  39. Sprengel, R. and Seeburg, P. H. 1993. The unique properties of glutamate receptor channels. FEBS Lett. 325:90–94.

    PubMed  Google Scholar 

  40. Monyer, H., Sprengel, R., Schoepfer, R., Herb, A., Higuchi, M., Lomeli, H., Burnashev, N., Sakmann, B., and Seeburg, P. H. 1992. Heteromeric NMDA receptors: Molecular and function distinction of subtypes. Science 256:1217–1221.

    PubMed  Google Scholar 

  41. Kutsuwada, T., Kashiwabuchi, N., Mori, H., Sakimura, K., Kushiya, E., Araki, K., Meguro, H., Masaki, H., Kumanishi, T., Arakawa, M., and Mishina, M. 1992. Molecular diversity of the NMDA receptor channel. Nature 358:36–41.

    PubMed  Google Scholar 

  42. Wahlestedt, C., Golanov, E., Yamamoto, S., Yee, F., Ericson, H., Yoo, H., Inturrisi, C. E., and Reis, D. J. 1993. Antisense oligodeoxynucleotides to NMDA-R1 receptor channel protect cortical neurons from excitotoxicity and reduce focal ischaemic infarctions. Nature 363:260–263.

    PubMed  Google Scholar 

  43. Kumar, K. N., Babcock, K. K., Johnson, P. S., Chen, X., Eggeman, K. T., and Michaelis, E. K. 1994. Purification and pharmacological and immunochemical characterization of synaptic membrane proteins with ligand binding properties of N-methyl-D-aspartate receptors. J. Biol. Chem. 269:27384–27393.

    PubMed  Google Scholar 

  44. Mattson, M. P., Wang, H., and Michaelis, E. K. 1991. Developmental expression, compartmentalization, and possible role in excitotoxicity of a putative NMDA receptor protein in cultured hippocampal neurons. Brain Res. 565:94–108.

    PubMed  Google Scholar 

  45. Mattson, M. P., Kumar, K. N., Wang, H., Cheng, B., and Michaelis, E. K. 1993. Basic FGF regulates the expression of a functional 71 kKa NMDA receptor protein that mediates calcium influx and neurotoxicity in hippocampal neurons, J. Neurosci. 13: 4575–4588.

    PubMed  Google Scholar 

  46. Grynkiewicz, G., Poenie, M., Tsien, R. Y. 1985. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J. Biol. Chem. 260:3440–3450.

    PubMed  Google Scholar 

  47. Chen, J.-W., Cunningham, M. D., Galton, N. and Michaelis, E. K. 1988. Immune labelling and purification of a 71 kDa glutamate binding protein from brain synaptic membranes. J. Biol. Chem. 263:417–427.

    PubMed  Google Scholar 

  48. Park, K. B. and Labbé, R. G. 1989. Artifacts follwoing gold staining of Western-blotted membranes. Anal. Biochem. 180:55–58.

    PubMed  Google Scholar 

  49. Eaton, M. J., Chen, J.-W., Kumar, K. N., Cong, Y., and Michaelis, E. K. 1990. Immunochemical characterization of brain synaptic membrane glutamate-binding proteins. J. Biol. Chem. 265:16195–16204.

    PubMed  Google Scholar 

  50. Petralia, R. S., Yokotani, N., and Wenthold, R. J. 1994. Light and electron microscope distribution of the NMDA receptor subunit NMDAR1 in the rat nervous system using a selective anti-peptide antibody. J. Neurosci. 14:667–696.

    PubMed  Google Scholar 

  51. Schramm, M., Eimerl, S., and Costa, E. 1990. Serum and depolarizing agents cause acute neurotoxicity in culture cerebellar granule cells: Role of the glutamate receptor responsive to N-methyl-D-aspartate. Proc. Natl. Acad. Sci. USA 87:1193–1197.

    PubMed  Google Scholar 

  52. Driscoll, B. F., Law, M. J., and Crane, A. M. 1991. Cell damage associated with changing the medium of mesencephalic cultures in serum-free medium is mediated via N-Methyl-D-aspartate receptors. J. Neurochem. 56:1201–1206.

    PubMed  Google Scholar 

  53. Birch, P. J., Grossman, C. J., and Hayes, A. G. 1988. 6,7-Dinitroquinoxaline-2,3-dion and 6-nitro,7-cyano-quinoxaline-2,3-dion antagonise responses to NMDA in the rat spinal cord via an action at the strychnine-insensitive glycine receptor. Eur. J. Pharmacol. 156:177–180.

    PubMed  Google Scholar 

  54. Harris, K. M., and Miller, R. J. 1989. CNQX (6-cyano-7-nitro-quinoxaline-2,3-dione) antagonizes NMDA-evoked [3H]GABA release from cultured cortical neurons via an inhibitory action at the strychnine-insensitive glycine site. Brain Res. 489:185–189.

    PubMed  Google Scholar 

  55. Levi, G., Patrizio, M., and Gallo, V. 1991. Release of endogenous and newly synthesized glutamate and of other amino acids induced by non-N-methyl-D-aspartate receptor activation in cerebellar granule cell cultures. J. Neurochem. 56:199–206.

    PubMed  Google Scholar 

  56. Hollman, M., Boulter, J., Maron, C., Beasley, L., Sullivan, J., Pecht, G., and Heinemann, S. 1993. Zinc potentiates agonist-induced currents at certain splice variants of the NMDA receptor. Neuron 10:943–954.

    PubMed  Google Scholar 

  57. Bredt, D. S., and Snyder, S. H. 1989. Nitric oxide mediates glutamate-linked enhancement of cGMP levels in the cerebellum. Proc. Natl. Acad. Sci. USA 86:9030–9033.

    PubMed  Google Scholar 

  58. Dawson, V. L., Dawson, T. M., London, E. D., Bredt, D. S., and Snyder, S. H. 1991. Nitric oxide mediates glutamate neurotoxicity in primary cortical cultures. Proc. Natl. Acad. Sci. USA 88:6368–6371.

    PubMed  Google Scholar 

  59. Bredt, D. S., Hwang, P. M., and Snyder, S. H. 1990. Localization of nitric oxide synthase indicating a neural role for nitric oxide, Nature 347:768–770.

    PubMed  Google Scholar 

  60. Hecker, M., Mülsch, A., and Busse, R. 1994. Subcellular localization and characterization of neuronal nitric oxide synthase. J. Neurochem. 62:1524–1529.

    PubMed  Google Scholar 

  61. Cox, J. A., Lysko, P. G., and Hennberry, R. C. 1989. Excitatory amino acid neurotoxicity at the N-methyl-D-aspartate receptor in cultured neurons: role of the voltage-dependent magnesium block. Brain Res. 499:267–272.

    PubMed  Google Scholar 

  62. Novelli, A., Reilly, J. A., Lysko, P. G., and Henneberry, R. C. 1988. Glutamate becomes neurotoxic via the N-methyl-D-aspartate receptor when intracellular energy levels are reduced. Brain Res. 451:205–212.

    PubMed  Google Scholar 

  63. Resink, A., Boer, G. J., and Balazs, R. 1992. Treatment with excitatory amino acids or high K+ and NMDA receptors in cerebellar granule cells. Neuroreport 3:757–760.

    PubMed  Google Scholar 

  64. Ciardo, A., and Meldolesi, J. 1991. Regulation of intracellular calcium in cerebellar granule neurons: Effects of depolarization and of glutamatergic and cholinergic stimulation. J. Neurochem. 56:184–191.

    PubMed  Google Scholar 

  65. Rossi, D. J., and Slater, N. T. 1993. The developmental onset of NMDA receptor-channel activity during neuronal migration. Neuropharmacol. 32:1239–1248.

    Google Scholar 

  66. Farrant, M., Feldmeyer, D., Takahashi, T., and Cull-Candy, S. G. 1994. NMDA-receptor channel diversity in the developing cerebellum. Nature 368:335–339.

    PubMed  Google Scholar 

  67. Mattson, M. P., Guthrie, P. B., and Kater, S. B. 1989. A role for Na+-dependent Ca2+ extrusion in protection against neuronal excitotoxicity. FASEB J. 3:2519–2526.

    PubMed  Google Scholar 

  68. Andreeva, N., Khodorov, B., Stelmashook, E., Dragoe, E., Jr., and Victorov, I. 1991. Inhibition of Na+/Ca2+ exchange enhances delayed neuronal death elicited by glutamate in cerebellar granule cell cultures. Brain Res. 548:322–325.

    PubMed  Google Scholar 

  69. Aronica, E., Condorelli, D. F., Nicoletti, F., Dell'Albani, P., Amico, C., and Balázs, R. 1993. Metabotropic glutamate receptors in cultured cerebellar granule cells: Developmental profile. J. Neurochem. 60:559–565.

    PubMed  Google Scholar 

  70. Sheng, M., Cummings, J., Roldan, L. A., Yan, Y. N., Jan, L. Y. 1994. Changing subunit composition of heteromeric NMDA receptors during development of rat cortex. Nature 368:144–147.

    PubMed  Google Scholar 

  71. Brose, N., Gasic, G. P., Vetter, D. E., Sullivan, J. M. and Heinemann, S. F. 1993. Protein chemical characterization and immunocytochemical localization of the NMDA receptor subunit NMDA R1. J. Biol. Chem. 268:22663–22671.

    PubMed  Google Scholar 

  72. Cunningham, M. D., and Michaelis, E. K. 1990. Solubilization and partial purification of 3-((±)-2-carboxypiperazine-4-yl)-[1,2-3H]propyl-l-phosphonic acid recognition proteins from rat brain synaptic membranes. J. Biol. Chem. 265:7768–7778.

    PubMed  Google Scholar 

  73. Eggeman, K. T., Pal, R., Walsh, J., Kumar, K. N., and Michaelis, E. K. 1993. Immunochemical and immunohistochemical characterization of a synaptic membrane protein that binds the competitive antagonists of NMDA receptors. Neurosci Lett. 158:173–176.

    PubMed  Google Scholar 

  74. Babcock, K. A., Eggeman, K. T., Chen, X. and Michaelis, E. K. 1994. Purification and immunochemical characterization of a synaptic membrane glycine-binding protein, Neurosci. Abstr. 20:1123.

    Google Scholar 

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  1. Department of Pharmacology and Toxicology, University of Kansas, 5064 Malott Hall, 66045, Lawrence, KS

    Yue Xia, Ronald E. Ragan, E. E. Ching Seah, Mary L. Michaelis &  Elias K. Michaelis

  2. Higuchi Biosciences Center, University of Kansas, 66045, Lawrence, KS

    Yue Xia, Ronald E. Ragan, E. E. Ching Seah, Mary L. Michaelis &  Elias K. Michaelis

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Xia, Y., Ragan, R.E., Seah, E.E.C. et al. Developmental expression of N-methyl-d-aspartate (NMDA)-induced neurotoxicity, NMDA receptor function, and the NMDAR1 and glutamate-binding protein subunits in cerebellar granule cells in primary cultures. Neurochem Res 20, 617–629 (1995). https://doi.org/10.1007/BF01694545

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