Neurochemical Research

, Volume 28, Issue 10, pp 1517–1524 | Cite as

Epilepsy, Neurodegeneration, and Extracellular Glutamate in the Hippocampus of Awake and Anesthetized Rats Treated with Okadaic Acid

  • Nadia Ramírez-Munguía
  • Gabriela Vera
  • Ricardo Tapia


We have previously shown that the intrahippocampal microinjection of okadaic acid (OKA), a potent inhibitor of serine/threonine protein phosphatases, induces epileptic seizures, neuronal death, and the hyperphosphorylation of the NR2B subunit of the N-methyl-d-aspartate (NMDA) receptor. We administered OKA by reverse microdialysis in the hippocampus of awake and halothane-anesthetized rats, with simultaneous collection of microdialysis fractions and recording of the EEG activity, and subsequent histological analysis. OKA produced intense behavioral and persistent EEG seizure activity in the awake rats but not in the anesthetized animals, and did not significantly alter the extracellular concentration of glutamate and aspartate detected in the microdialysis fractions. One day after the experiment a remarkable neurodegeneration of CA1 hippocampal region was observed in both the awake and the anesthetized rats. We conclude that the OKA-induced epilepsy cannot be ascribed to increased extracellular glutamate, but to an increased sensitivity of NMDA receptor. We propose that halothane protected against the epilepsy because it blocks NMDA receptor overactivation, and that the neurodegeneration of CA1 region is independent of this overactivation and due probably to alterations of cytoskeletal proteins consequent to the OKA-induced hyperphosphorylation.

Okadaic acid microdialysis hippocampus extracellular glutamate neurodegeneration epilepsy 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Choi, D. W. 1988. Glutamate neurotoxicity and diseases of the nervous system. Neuron 1:623-634.PubMedGoogle Scholar
  2. 2.
    Meldrum, B. 1991. Excitotoxicity and epileptic brain damage. Epilepsy Res. 10:55-61.PubMedGoogle Scholar
  3. 3.
    Tapia, R., Medina-Ceja, L., and Peña, F. 1999. On the relationship between extracellular glutamate, hyperexcitation and neurodegeneration, in vivo. Neurochem. Int. 34:23-31.PubMedGoogle Scholar
  4. 4.
    Peña, F. and Tapia, R. 1999. Relationships among seizures, extracellular amino acid changes, and neurodegeneration induced by 4-aminopyridine in rat hippocampus: A microdialysis and electroencephalographic study. J. Neurochem. 72:2006-2014.PubMedGoogle Scholar
  5. 5.
    Peña, F. and Tapia, R. 2000. Seizures and neurodegeneration induced by 4-aminopyridine in rat hippocampus in vivo: Role of glutamate-and GABA-mediated neurotransmission and of ion channels. Neuroscience 101:547-561.PubMedGoogle Scholar
  6. 6.
    Massieu, L., Morales-Villagrán, A., and Tapia, R. 1995. Accumulation of extracellular glutamate by inhibition of its uptake is not sufficient for inducing neuronal damage: An in vivo microdialysis study. J. Neurochem. 64:2262-2272.Google Scholar
  7. 7.
    Massieu, L. and Tapia, R. 1997. Glutamate uptake impairment and neuronal damage in young and aged rats in vivo. J. Neurochem. 69:151-1160.PubMedGoogle Scholar
  8. 8.
    Ben-Ari, Y., Aniksztejn, L., and Bregestovski, P. 1992. Protein kinase C modulation of NMDA currents: An important link for LTP induction. Trends Neurosci. 15:333-339.PubMedGoogle Scholar
  9. 9.
    Raymond, L. A., Blackstone, C. D., and Huganir, R. L. 1993. Phosphorylation of amino acid neurotransmitter receptors in synaptic plasticity. Trends Neurosci. 16:147-153.PubMedGoogle Scholar
  10. 10.
    Westphal, R. S., Tavalin, S. J., Lin, J. W., Alto, N. M., Fraser, I. D. C., Langeberg, L. K., Sheng, M., and Scott, J. D. 1999. Regulation of NMDA receptors by an associated phosphatasekinase signaling complex. Science 285:93-96.PubMedGoogle Scholar
  11. 11.
    Cerne, R., Rusin, K. I., and Randic, M. 1993. Enhancement of the N-methyl-D-aspartate response in spinal dorsal horn neurons by cAMP-dependent protein kinase. Neurosci. Lett. 161:124-128.PubMedGoogle Scholar
  12. 12.
    Greengard, P., Jen, J., Nairn, A. C., and Stevens, C. F. 1991. Enhancement of the glutamate response by cAMP-dependent protein kinase in hippocampal neurons. Science 253:1135-1138.PubMedGoogle Scholar
  13. 13.
    Snyder, G. L., Fienberg, A. A., Huganir, R. L., and Greengard, P. 1998. A dopamine/D1 receptor/protein kinase A/dopamine-and cAMP-regulated phosphoprotein (Mr 32 kDa)/protein phosphatase-1 pathway regulates dephosphorylation of the NMDA receptor. J. Neurosci. 18:10297-10303.PubMedGoogle Scholar
  14. 14.
    Wyllie, D. J. A. and Nicoll, R. A. 1994. A role for protein kinases and phosphatases in the Ca2+-induced enhancement of hippocampal AMPA receptor-mediated synaptic responses. Neuron 13:635-643.PubMedGoogle Scholar
  15. 15.
    Wang, L.-Y., Salter, M. W., and MacDonald, J. F. 1991. Regulation of kainate receptors by CAMP-dependent protein kinase and phosphatases. Science 253:1132-1135.PubMedGoogle Scholar
  16. 16.
    Wang, L.-Y., Orser, B. A., Brautigan, D. L., and MacDonald, J. F. 1994. Regulation of the NMDA receptors in cultured hippocampal neurons by protein phosphatase 1 and 2 A. Nature 369:230-232.PubMedGoogle Scholar
  17. 17.
    Lieberman, D. N. and Mody, I. 1994. Regulation of NMDA channel function by endogenous Ca2+-dependent phosphatase. Nature 369:235-239.PubMedGoogle Scholar
  18. 18.
    Tachibana, K., Scheuer, P. J., Tsukitani, Y., Kikuchi, H., Van Engen, D., Clardy, J., Gopichand, Y., and Schmitz, J. 1981. Okadaic acid, a cytotoxic polyether from two marine sponges of the genus Hallichondria. J. Am. Chem. Soc. 103:2469-2471.Google Scholar
  19. 19.
    Bialojan, C. and Takai, A. 1988. Inhibitory effect of a marine-sponge toxin, okadaic acid, on protein phosphatases: Specificity and kinetics. Biochem. J. 256:283-290.PubMedGoogle Scholar
  20. 20.
    Cohen, P., Holmes, C. B. F., and Tsukitani, Y. 1990. Okadaic acid: A new probe for the study of cellular regulation. Trends Biochem. Sci. 15:98-102.PubMedGoogle Scholar
  21. 21.
    Tapia, R., Peña, F., and Arias, C. 1999. Neurotoxic and synaptic effects of okadaic acid, an inhibitor of protein phosphatases. Neurochem. Res. 24:1423-1430.PubMedGoogle Scholar
  22. 22.
    Arias, C., Becerra-Garcia, F., Arrieta, I., and Tapia, R. 1998. The protein phosphatase inhibitor okadaic acid induces heat-shock protein expression and neurodegeneration in rat hippocampus in vivo. Exp. Neurol. 153:242-254.PubMedGoogle Scholar
  23. 23.
    Arias, C., Montiel, T., Peña, F., Ferrera, P., and Tapia, R. 2002. Okadaic acid induces epileptic seizures and hyperphosphorylation of the NR2B subunit of the NMDA receptor in rat hippocampus in vivo. Exp. Neurol. 177:284-291.PubMedGoogle Scholar
  24. 24.
    Colwell, C. S. and Levine, M. S. 1995. Excitatory synaptic transmission in neostriatal neurons: Regulation by cyclic AMP-dependent mechanism. J. Neurosci. 15:1704-1713.PubMedGoogle Scholar
  25. 25.
    Sim, A. T. R., Lloyd, H. G. E., Jarvie, P. E., Morrisom, M., Rostas, J. A. P., and Dunkley, P. R. 1993. Synaptosomal amino acid release: Effect of inhibiting protein phosphatases with okadaic acid. Neurosci. Lett. 160:181-184.PubMedGoogle Scholar
  26. 26.
    Ganel, R. and Crosson, C. E. 1998. Modulation of human glutamate transporter activity by phorbol ester. J. Neurochem. 70:993-1000.PubMedGoogle Scholar
  27. 27.
    Paxinos, G. and Watson, C. 1982. The Rat Brain in Stereotaxic Coordinates, Academic Press, Sidney.Google Scholar
  28. 28.
    Morales-Villagrán, A. and Tapia, R. 1996. Preferential stimulation of glutamate release by 4-aminopyridine in rat striatum in vivo. Neurochem. Int. 28:35-40.PubMedGoogle Scholar
  29. 29.
    Salazar, P., Montiel, T., Brailowsky, S., and Tapia, R. 1994. Decrease of glutamate decarboxylase activity after in vivo cortical infusion of γ-aminobutyric acid. Neurochem. Int. 24:363-368.PubMedGoogle Scholar
  30. 30.
    Keifer, J. C., Baghdoyan, H. A., Becker, L., and Lydic, R. 1994. Halothane decreases pontine acetylcholine release and increases EEG spindles. Neuroreport 5:577-580.PubMedGoogle Scholar
  31. 31.
    Farber, N. E., Poterack, A. K., and Schmeling, W. T. 1997. Dexmedetomidine and halothane produce similar alterations in electroencephalographic and electromyographic activity in cats. Brain Res. 774:131-141.PubMedGoogle Scholar
  32. 32.
    Leonard, A. S. and Hell, J. W. 1997. Cyclic AMP-dependent protein kinase and protein kinase C phosphorylate N-methyl-D-aspartate receptors at different sites. J. Biol. Chem. 272:12107-12115.PubMedGoogle Scholar
  33. 33.
    Raman, I. M., Tong, G., and Jahr, C. E. 1996. β-Adrenergic regulation of synaptic NMDA receptors by cAMP-dependent protein kinase. Neuron 16:415-421.PubMedGoogle Scholar
  34. 34.
    Hemmings, H. C. Jr., and Adamo, A. I. 1996. Activation of endogenous protein kinase C by halothane in synaptosomes. Anesthesiology 84:652-662.PubMedGoogle Scholar
  35. 35.
    Wakasugi, M., Hirota, K., Roth, S. H., and Ito, Y. 1999. The effects of general anesthetics on excitatory and inhibitory synaptic transmission in area CA1 of the rat hippocampus in vitro. Anesth. Analg. 88:676-680.PubMedGoogle Scholar
  36. 36.
    Kirson, E. D., Yaari, Y., and Perouanski, M. 1998. Presynaptic and postsynaptic actions of halothane at glutamatergic synapses in the mouse hippocampus. Br. J. Pharmacol. 124:1607-1614.PubMedGoogle Scholar
  37. 37.
    Perouanski, M., Kirson, E. D., and Yaari, Y. 1998. Mechanism of action of volatile anesthetics: Effects of halothane on glutamate receptors in vitro. Toxicol. Lett. 100–101:65-69.Google Scholar
  38. 38.
    Frizelle, H. P., Moriarty, D. C., and O'Connor, J. J. 1999. The combined effects of halothane and lamotrigine on excitatory post-synaptic potentials and use-dependent block in the rat dentate gyrus in vitro. Anesth. Analg. 89:496-501.PubMedGoogle Scholar
  39. 39.
    Martin, D. C., Plagenhoef, M., Abraham, J., Dennison, R. L., and Aronstam, R. S. 1995. Volatile anesthetics and glutamate activation of N-methyl-D-aspartate receptors. Biochem. Pharmacol. 49:809-817.PubMedGoogle Scholar
  40. 40.
    McFarlane, C., Warner, D. S., and Dexter, F. 1995. Interactions between NMDA and AMPA glutamate receptor antagonists during halothane anesthesia in the rat Neuropharmacology 34:659-663.PubMedGoogle Scholar
  41. 41.
    Arendt, T., Holzer, M., Fruth R., Brückner, M. K., and Gärtner, U. 1995. Paired helical filament-like phosphorylation of tau, deposition of β/A4-amyloid and memory impairment in rat induced by chronic inhibition of phosphatase 1 and 2 A. Neuroscience 69:691-698.PubMedGoogle Scholar
  42. 42.
    Arias, C., Sharma, N., Davies, P., and Shafit-Zagardo, B. 1993. Okadaic acid induces early changes in microtubule-associated protein 2 and tau phosphorylation prior to neurodegeneration in cultured cortical neurons. J. Neurochem. 61:673-682.PubMedGoogle Scholar
  43. 43.
    Sacher, M., Athlan, E. S., and Mushynski, W. E. 1992. Okadaic acid induces the rapid and reversible disruption of the neurofilament network in rat dorsal root ganglion neurons. Biochem. Biophys. Res. Commun. 186:524-530.PubMedGoogle Scholar
  44. 44.
    Shea, T. B., Paskevich, P. A., and Beermann, M. L. 1993. The protein phosphatase inhibitor okadaic acid increases axonal neurofilaments and neurite caliber, and decreases axonal microtubules in NB2a/d1 cells. J. Neurosci. Res. 35:507-521.PubMedGoogle Scholar
  45. 45.
    Malchiodi-Albedi, F., Petrucci, T. C., Picconi, B., Iosi, F., and Falchi, M. 1997. Protein phosphatase inhibitors induce modification of synaptic structure and tau hyperphosphorylation in cultured rat hippocampal neurons. J. Neurosci. Res. 48:425-438.PubMedGoogle Scholar
  46. 46.
    Merrick, S. E., Trojanowski, J. Q., and Lee, V. M.-Y. 1997. Selective destruction of stable microtubules and axons by inhibitors of protein/serine phosphatases in cultured human neurons (NT2 N cells). J. Neurosci. 17:5726-5737.PubMedGoogle Scholar
  47. 47.
    Fernández, M. T., Zitko, V., Gascón, S., and Novelli, A. 1991. The marine toxin okadaic acid is a potent neurotoxin for cultured cerebellar neurons. Life Sci. 49:PL157-PL162.PubMedGoogle Scholar
  48. 48.
    Fernández, M. T., Zitko, V., Gascón, S., Torreblanca, A., and Novelli, A. 1993. Neurotoxic effect of okadaic acid, a seafood-related toxin, on cultured cerebellar neurons. Ann. NY Acad. Sci. 679:260-269.PubMedGoogle Scholar

Copyright information

© Plenum Publishing Corporation 2003

Authors and Affiliations

  • Nadia Ramírez-Munguía
    • 1
  • Gabriela Vera
    • 1
  • Ricardo Tapia
    • 1
  1. 1.Departamento de Neurociencias, Instituto de Fisiología CelularUnversidad Nacional Autónoma de MéxicoMéxico

Personalised recommendations