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The use of ion-sensitive electrodes and fluorescence imaging in hippocampal slices for studying pathological changes of intracellular Ca2+ regulation

  • P. Schubert
  • F. Keller
  • Y. Nakamura
  • K. Rudolphi
Part of the Journal of Neural Transmission book series (NEURAL SUPPL, volume 44)

Summary

The physiological regulation of the intracellular Ca2+ homeostasis and its pathological alteration has been studied in rat and gerbil hippocampal slices using ion-sensitive electrodes and the fluorescence imaging technique. The ischemia-induced intracellular Ca2+ rise, accentuated in the synaptic/dendritic layer of the vulnerable CA1 neurons was observed in vivo and could be replicated at an accellerated time course in the “ischemic” hippocampal slice superfused with unoxygenated, glucose-free medium. The intracellular Ca2+ loading, thought to be instrumental for the generation of postischemic nerve cell damage, seems to result from an increased Ca2+ release out of intracellular stores as well as from an enhanced synaptic Ca2+ influx. The latter is attributed to a depolarization-induced opening of the voltage-dependent Ca2+ channels and to an uncontrolled influx through “upregulated” NMDA receptor-operated channels. Such an ischemia-induced upregulation which is reported to occur physiologically by the activation of PKC, is reflected by the selective loss of the depressive control of the synaptic NMD A Ca2+ influx by adenosine. Ischemia also leads to a hypertrophy of astrocytes which may go along with an impairment of their physiological function to take up glutamate adding to the extracellular rise of the excitotoxic amino acids. A pathological activation of microglial cells and their transformation into macrophages, known to release oxygen radicals, may further add to neuronal damage. The observed neuroprotection by adenosine can be primarily ascribed to its limiting effect on a pathological membrane depolarization and its deleterious consequences. The more powerful neuroprotection by propentofylline, thought to act analogue to adenosine, seems to be achieved by additional mechanisms. This pharmacon depresses the ischemia-induced neuronal Ca2+ loading in vivo and in vitro, prevents the activation of astrocytes and interferes with the transformation as well as with the free radical formation of microglia-derived macrophages as demonstrated in complementary studies with fluorescence techniques on cell cultures.

Keywords

Microglial Cell Hippocampal Slice Cereb Blood Flow Voltage Sensitivity Long Term Poten 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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References

  1. Adams S, Harootunian A, Buechler Y, Taylor S, Tsien R (1991) Fluorescence ratio imaging of cyclic AMP in single cells. Nature 349: 694–697PubMedCrossRefGoogle Scholar
  2. Banati R, Gehrmann J, Schubert P, Kreutzberg G (1993) Cytotoxicity of microglia. Glia 7: 111–118PubMedCrossRefGoogle Scholar
  3. Banati R, Schubert P, Rothe G, Gehrmann J, Rudolphi K, Valet G, Kreutzberg G (1994) Modulation of intracellular formation of reactive oxygen intermediates in peritoneal macrophages and microglia/brain macrophages by propentophylline. J Cereb Blood Flow Metab 14: 145–149PubMedCrossRefGoogle Scholar
  4. Barbour B, Brew H, Attwell D (1988) Electrogenic glutamate uptake in glial cells is activated by intracellular potassium. Nature 335: 433–435PubMedCrossRefGoogle Scholar
  5. Ben-Ari Y, Aniksztejn L, Bregestovski P (1992) Protein kinase C modulation of NMDA currents: an important link for LTP induction. TINS 15: 333–339PubMedGoogle Scholar
  6. DeLeo J, Toth L, Schubert P, Rudolphi K, Kreutzberg G (1987) Ischemia-induced neuronal cell death, calcium accumulation and glial response in the hippocampus of the Mongolian gerbil and protection by the xanthine derivative HWA 285. J Cereb Blood Flow Metab 7: 745–752CrossRefGoogle Scholar
  7. Deri Z, Adam-Vizi V (1993) Detection of intracellular free Na+ concentration of synaptosomes by a fluorescent indicator. Na+-binding benzofuran isophthalate: the effect of veratridine, ouabain, and a-latrotoxin. J Neurochem 61: 818–825PubMedCrossRefGoogle Scholar
  8. Dunwiddie T, Lynch G (1978) Long-term potentiation and depression of synaptic responses in the hippocampus: localization and frequency dependency. J Physiol 276: 353–367PubMedGoogle Scholar
  9. Dux E, Schubert P, Kreutzberg GW (1992) Ultrastructural localization of calcium in ischemic hippocampal slices: the influence of adenosine and theophylline. J Cereb Blood Flow Cell Metab 12: 520–524CrossRefGoogle Scholar
  10. Gough AH, Taylor DL (1993) Fluorescence anisotropy imaging microscopy maps calmodulin binding during cellular contraction and locomotion. J Cell Biol 121: 1095–1107PubMedCrossRefGoogle Scholar
  11. Grynkiewicz G, Poenie M, Tsien RY (1985) A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 260: 3440–3450PubMedGoogle Scholar
  12. Heinemann U, Lux H, Gutnick M (1977) Free extracellular calcium and potassium during paroxysmal activity in cerebral cortex of the cat. Exp Brain Res 27: 237–243PubMedCrossRefGoogle Scholar
  13. Herron C, Lester R, Coan E, Collingridge G (1986) Frequency-dependent involvement of NMDA receptors in the hippocampus: a novel synaptic mechanism. Nature 322: 265–268PubMedCrossRefGoogle Scholar
  14. Kadoya F, Mitani A, Arai T, Kataoka K (1992) Effects of propentofylline on hypoxia- hypoglycemia induced calcium accumulation in gerbil hippocampal slices. J Cereb Blood Flow Metab 12: 301–305PubMedCrossRefGoogle Scholar
  15. Khalil RA, Morgan KG (1991) Imaging of protein kinase C distribution and translocation in living vascular smooth muscle cells. Circ Res 69: 1626–1631PubMedGoogle Scholar
  16. Mager R, Ferroni S, Schubert P (1990) Adensosine modulates a voltage-dependent chloride conductance in cultured hippocampal neurons. Brain Res 532: 58–62PubMedCrossRefGoogle Scholar
  17. Minta A, Kao J, Tsien R (1989) Fluorescent indicators for cytosolic calcium based on rhodamine and fluorescein chromophores. J Biol Chem 264: 8171–8176PubMedGoogle Scholar
  18. Mitani A, Yanase H, Sakai K, Wake Y, Kataoka K (1993) Origin of intracellular Ca2+ elevation induced by in vitro ischemia-like condition of hippocampal slices. Brain Res 601: 103–110PubMedCrossRefGoogle Scholar
  19. Monaghan T, Holets V, Toy D, Cotman C (1983) Anatomical distribution of four pharmacologically distinct 3H-L-glutamate binding sites. Nature 306: 176–178PubMedCrossRefGoogle Scholar
  20. Nowak L, Bregestovski P, Ascher P, Herbert A, Prochianz A (1984) Magnesium gates glutamate-activated channels in mouse central neurones. Nature 307: 462–465PubMedCrossRefGoogle Scholar
  21. Onodera H, Araki T, Kogure K (1989) Protein kinase C activity in the rat hippocampus after forebrain ischemia: autoradiographic analysis by 3H-phorbol 12,13-dibutyrate. Brain Res 481: 1–7PubMedCrossRefGoogle Scholar
  22. Ou-yang Y, Merllergard P, Siesjo BK (1993) Regulation of intracellular pH in single rat cortical neurons in vitro: a microspectrofluorometric study. J Cereb Blood Flow Metab 13: 827–840PubMedCrossRefGoogle Scholar
  23. Rudolphi K, Schubert P, Parkinson F, Fredholm B (1992) Neuroprotective role of adenosine in cerebral ischemia. TIPS 13: 439–445PubMedGoogle Scholar
  24. Schubert P, Banati R, Dux E, Gehrmann J, Rudolphi K, Kreutzberg G (1992) Reactive oxygen intermediates in microglial cells differentiating into brain macrophages: depression by propentophylline are relation to adenosine action. In: Krieglstein J, Oberpichler H (eds) Pharmacology of cerebral ischemia 1992. Wissenschaftliche Verlagsgesellschaft, Stuttgart, pp 461–467Google Scholar
  25. Schubert P, Keller F, Rudolphi K (1993) Depression of synaptic transmission and evoked NMDA Ca2+ influx in hippocampal neurons by adenosine and its blockade by LTP or ischemia. Drug Dev Res 28: 399–405CrossRefGoogle Scholar
  26. Schubert P, Mager R (1991) The critical input frequency for NMDA receptor-mediated neuronal Ca2+ influx depends on endogenous adenosine. Int J Purine Pyrimidine Res 2: 11–16Google Scholar
  27. Trussel LO, Jackson MB (1985) Adenosine-activated potassium conductance in cultured striatal neurons. Proc Natl Acad Sci 82: 4857–4861CrossRefGoogle Scholar
  28. Van Resandt R, Marsman H, Kaplan R, Davoust J, Stelzer H, Strieker R (1985) Optical fluorescence microscopy in three dimensions: microtomoscopy. J Microsc 138: 29–34CrossRefGoogle Scholar
  29. Verkman A (1990) Development and biological applications of chloride-sensitive fluorescent indicators. Am J Physiol 259: C375PubMedGoogle Scholar

Copyright information

© Springer-Verlag 1994

Authors and Affiliations

  • P. Schubert
    • 1
  • F. Keller
    • 1
  • Y. Nakamura
    • 1
  • K. Rudolphi
    • 1
    • 2
  1. 1.Department of NeuromorphologyMax-Planck-Institute for PsychiatryMartinsried, MunichFederal Republic of Germany
  2. 2.Hoechst AgFrankfurt a.M.Federal Republic of Germany

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