Skip to main content
SpringerLink
Log in

Effect of serum on intracellular calcium homeostasis and survival of primary cortical and hippocampal CA1 neurons following brief glutamate treatment

  • Published:
Metabolic Brain Disease Aims and scope Submit manuscript

Abstract

Glutamate neurotoxicity was studied in primary neuronal cultures prepared from rat cerebral cortex and hippocampal CA1 sector. Neurons were cultivated with 5% native horse serum and then exposed to 0.1 or 1.0 mM glutamate for 5 min. Subsequently, neurons were allowed to recover for 24 hours either in the presence or in the absence of 5% native horse serum. In the absence of serum, neurons showed morphological signs of degeneration and exhibited marked loss of vitality as tested by vital staining and release of lactate dehydrogenase (LDH). In contrast, when neurons were cultivated in the presence of serum, no degenerative changes were seen and the neurons survived. Heat inactivated serum did not prevent neuronal death but addition of basic fibroblast growth factor (bFGF) or transforming growth factor-ß1 (TGF-ß1) had the same protective effect as native serum. Measurements of intracellular calcium activity ([Ca2+]i) with the indicator dye fura-2 revealed a sharp increase during glutamate exposure. In the absence of serum, [Ca2+]i returned to near control within 5 min but it secondarily increased after 1 hour to almost the same level as during glutamate exposure. This delayed increase was more pronounced in CA1 than in cortical neurons, it correlated linearly with the initial rise during glutamate exposure, and it was greatly reduced in the presence of serum. These observations suggest that glutamate neurotoxicityin vitro is a function of the delayed and not of the primary rise of intracellular calcium activity, and that trophic factors prevent neurotoxicity by attenuating this delayed response.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

Abbreviations

BSS:

Basal Salt Solution

[Ca2+]i :

intracellular calcium activity

MOPS:

3-[N-Morpholino]propanesulfonic acid

EGTA:

ethylene glycol-bis (ß-aminoethylether) N, N, N′, N′-tetraacetic acid

bFGF:

basic fibroblast growth factor

(TGF-ß1):

transforming growth factor-ß1

LDH:

lactate dehydrogenase

References

  • Abe K., Iri Y., Takayanagi M., and Saito H. (1992). Involvement of protein kinase activation in neurotrophic effects of basic fibroblast growth factor in cultured brain neurons.Jpn. J. Pharmacol. 56:563–566.

    Google Scholar 

  • Akaneya Y., Enokido Y., Takahashi M., and Hatanaka H. (1993).In vitro model of hypoxia: basic fibroblast growth factor can rescue cultured CNS neurons from oxygen-deprived cell death.J. Cereb. Blood Flow Metab. 13:1029–1032.

    PubMed  Google Scholar 

  • Baimbridge K.G., and Miller J.J. (1982). Immunohistochemical localization of calcium-binding protein in the cerebellum, hippocampal formation and olfactory bulb of the rat.Brain Res. 245:223–229.

    Article  PubMed  Google Scholar 

  • Ben-Ari Y. (1985). Limbic seizure and brain damage produced by kainic acid: mechanisms and relevance to human temporal lobe epilepsy.Neuroscience.14:375–403.

    Article  PubMed  Google Scholar 

  • Benveniste H., Drejer J., Schousboe A., and Diemer N.H. (1984). Elevation of the extracellular concentrations of glutamate and L-aspartate in rat hippocampus during transient cerebral ischemia monitored by intracerebral microdialysis.J. Neurochem. 43:1369–1374.

    PubMed  Google Scholar 

  • Bonni A., Frank D. A., Schindler C., and Greenberg M. E. (1993). Characterization of a pathway for ciliary neurotrophic factor signaling to the nucleus.Science.262:1575–1579.

    PubMed  Google Scholar 

  • Cheng B., Barger S. W., and Mattson M. P. (1994). Staurosporine, K-252a, and K-252b stabilize calcium homeostasis and promote survival of CNS neurons in the absence of glucose.J. Neurochem. 62:1319–1329.

    PubMed  Google Scholar 

  • Cheng B., McMahon D.G., and Mattson M.P. (1993). Modulation of calcium current, intracellular calcium levels and cell survival by glucose deprivation and growth factors in hippocampal neurons.Brain Res. 607:275–285.

    Article  PubMed  Google Scholar 

  • Choi D.W. (1985). Glutamate neurotoxicity in cortical cell culture is calcium dependent.Neurosci. Lett. 58:293–297.

    Article  PubMed  Google Scholar 

  • Choi D.W. (1988). Calcium-mediated neurotoxicity: relationship to specific channel types and role in ischemic damage.Trends Neurosci. 11:465–469.

    Article  PubMed  Google Scholar 

  • Choi D.W., Maulucci-Gedde M., and Kriegstein A.R. (1987). Glutamate neurotoxicity in cortical cell culture.J. Neurosci. 7:357–368.

    PubMed  Google Scholar 

  • Collazo D., Takahashi H., and McKay R.D. (1992). Cellular targets and trophic functions of neurotrophin-3 in the developing rat hippocampus.Neuron. 9:643–656.

    Article  PubMed  Google Scholar 

  • Crumrine R.C., Dubyak, G., and LaManna J.C. (1990). Decreased protein kinase C activity during cerebral ischemia and after reperfusion in the adult rat.J. Neurochem. 55:2001–2007.

    PubMed  Google Scholar 

  • Dienel G.D., and Pulsinelli W.A. (1986). Uptake of radiolabeled ions in normal and ischemia-damaged brain.Ann. Neurol. 19:465–472.

    Article  PubMed  Google Scholar 

  • Dux E., Oschlies U., Wiessner C., and Hossmann K.A. (1992). Glutamate-induced ribosomal disaggregation and ultrastructural changes in rat cortical neuronal culture: Protective effect of horse serum.Neurosci. Lett. 141:173–176.

    Article  PubMed  Google Scholar 

  • Faden A.I., and Simon R.P. (1988). A potential role for excitotoxins in the pathophysiology of spinal cord injury.Ann. Neurol. 23:623–626.

    Article  PubMed  Google Scholar 

  • Freese A., Finklestein S.P., and DiFiglia M. (1992). Basic fibroblast growth factor protects striatal neuronsin vitro from NMDA-receptor mediated excitotoxicity.Brain Res. 575:351–355.

    Article  PubMed  Google Scholar 

  • Griffiths T., Evans M.C., and Meldrum B.S. (1984). Status epilepticus: the reversibility of calcium loading and acute neuronal pathological changes in the rat hippocampus.Neuroscience.12:557–567.

    Article  PubMed  Google Scholar 

  • Gross C.E., Bednar M.M., Howard D.B., and Sporn M.B. (1993). Transforming growth factor-ß1 reduces infarct size after experimental cerebral ischemia in a rabbit model.Stroke 24:558–562.

    PubMed  Google Scholar 

  • Hahn J.S., Aizenman E., and Lipton S.A. (1988). Central mammalian neurons resistant to glutamate toxicity are made sensitive by elevated extracellular calcium; toxicity blocked by the N-methyl-D-aspartate antagonist MK-801.Proc. Natl. Acad. Sci. USA.85:6556–6560.

    PubMed  Google Scholar 

  • Hara H., Onodera H., Yoshidomi M., Matsuda Y., and Kogure, K. (1990). Staurosporine, a novel protein kinase C inhibitor, prevents postischemic neuronal damage in the gerbil and rat.J. Cereb. Blood Flow Metab. 10:646–653.

    PubMed  Google Scholar 

  • Harafuji H., and Ogawa Y. (1980). Re-examination of the apparent binding constant of ethylene glycol bis (ß-aminoethyl ether)-N, N, N′, N′-tetraacetic acid with calcium around neutral pH.J. Biochem. Tokyo.87:1305–1312.

    PubMed  Google Scholar 

  • Hayes R.I., Jenkins L.W., Lyeth B.G., Balster R.L., Robinson S.E., Clifton G.L., and Stubbins J.F. (1988). Pretreatment with phencyclidine, an N-methyl-D-aspartate antagonist, attenuates long-term behavioural deficits in the rat produced by traumatic brain injury.J. Neurotrauma.5:259–274.

    PubMed  Google Scholar 

  • Iacopino A.M., Christakos S., German D., Sonsalla P.K., and Altar C.A. (1992). Calbindin-D28K-containing neurons in animal models of neurodegeneration: possible detection from excitotoxicity.Mol. Brain Res. 13:251–261.

    Article  PubMed  Google Scholar 

  • Ip N.Y., Li Y., Yancopoulos G.D., and Lindsay R.M. (1993). Cultured hippocampal neurons show responses to BDNF, NT-3, and NT-4, but not NGF.J. Neurosci. 13:3394–3405.

    PubMed  Google Scholar 

  • Jorgensen M.B., and Diemer N.H. (1982). Selective neuron loss after cerebral ischemia in the rat: possible role of transmitter glutamate.Acta Neurol. Scand. 66:536–546.

    PubMed  Google Scholar 

  • Koike T., Martin D.P., and Johnson E.M. (1989). Role of Ca2+ channels in the ability of membrane depolarization to prevent neuronal death induced by trophic-factor deprivation: evidence that levels of internal Ca2+ determine nerve growth factor dependence of sympathetic ganglion cells.Proc. Natl. Acad. Sci. USA.86:6421–6425.

    PubMed  Google Scholar 

  • Lehmann A. (1987). Pharmacologic protection against the toxicity of N-methyl-D-asparate in immature rat cerebellar slices.Neuropharmacology.26:1751–1761.

    Article  PubMed  Google Scholar 

  • Maiese K., Boniece I., DeMeo D., and Wagner J.A. (1993). Peptide growth factors protect against ischemia in culture by preventing nitric oxide toxicity.J. Neurosci. 13:3034–3040.

    PubMed  Google Scholar 

  • 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 

  • Mattson M.P., Rychlik B., Chu C., and Christakos S. (1991). Evidence for calcium-reducing and excito-protective roles for the calcium-binding protein calbindin-D28K in cultured hippocampal neurons.Neuron 6:41–51.

    Article  PubMed  Google Scholar 

  • Miyamoto M., Murphy T.H., Schnaar R.L., and Coyle J.T. (1989). Antioxidants protect against glutamate-induced cytotoxicity in a neuronal cell line.J. Pharmacol. Exp. Ther. 250:1132–1140.

    PubMed  Google Scholar 

  • Nicotera P., Bellomo G., and Orrenius S. (1990). The role of Ca2+ in cell killing.Chem. Res. Toxicol. 3: 484–494.

    Article  PubMed  Google Scholar 

  • Nozaki K., Finklestein S.P., and Beal M.F. (1993). Basic fibroblast growth factor protects against hypoxia-ischemia and NMDA neurotoxicity in neonatal rats.J. Cereb. Blood Flow Metab. 13:221–228.

    PubMed  Google Scholar 

  • Prehn J.H.M., Backhauß C., and Krieglstein J. (1993a). Transforming growth factor-ß1 prevents glutamate neurotoxicity in rat neocortical cultures and protects mouse neocortex from ischemic injuryin vivo.J. Cereb. Blood Flow Metab. 13:521–525.

    PubMed  Google Scholar 

  • Prehn J.H.M., Peruche B., Unsicker K., and Krieglstein J. (1993b). Isoform-specific effects of transforming growth factors-β on degeneration of primary neuronal cultures induced by cytotoxic hypoxia or glutamate.J. Neurochem. 60:1665–1672.

    PubMed  Google Scholar 

  • Raley-Susman K.M., and Lipton P. (1990).In vitro ischemia and protein synthesis in the rat hippocampal slice: the role of calcium and NMDA receptor activation.Brain Res. 515:27–38.

    Article  PubMed  Google Scholar 

  • Randall R.D., and Thayer S.A. (1992). Glutamate-induced calcium transient triggers delayed calcium overload and neurotoxicity in rat hippocampal neurons.J. Neurosci. 12:1882–1895.

    PubMed  Google Scholar 

  • Rothman S.M. (1983). Synaptic activity mediates death of hypoxic neurons.Science.220:536–537.

    PubMed  Google Scholar 

  • Rothman S.M., and Olney J.W. (1986). Glutamate and the pathophysiology of hypoxic-ischemic brain damage.Ann. Neurol. 19:105–111.

    Article  PubMed  Google Scholar 

  • Sequier J.M., Hunziker W., Andressen C., and Celio M.R. (1990). Calbindin D-28K protein and mRNA localization in the rat brain.Eur. J. Neurosci. 2:1118–1126.

    PubMed  Google Scholar 

  • Shigeno T., Mima T., Takakura K., Graham D.I., Kato G., Hashimoto Y., and Furukawa S. (1991). Amelioration of delayed neuronal death in the hippocampus by nerve growth factor.J. Neurosci. 11:2914–2919.

    PubMed  Google Scholar 

  • Siesjö B. K. (1981). Cell damage in the brain: a speculative synthesis.J. Cereb. Blood Flow Metab. 1:155–185.

    PubMed  Google Scholar 

  • Siesjö B.K., Memezawa H., and Smith M.L. (1991). Neurocytotoxicity: pharmacological implications.Fundam. Clin. Pharmacol. 5:755–767.

    PubMed  Google Scholar 

  • Sloviter R.S. (1989). Calcium-binding protein (calbindin-D28K) and parvalbumin immunocytochemistry: localization in the rat hippocampus with specific reference to the selective vulnerability of hippocampal neurons to seizure activity.J. Comp. Neurol. 280:183–196.

    Article  PubMed  Google Scholar 

  • Snyder S.H., and Bredt D.S. (1991). Nitric oxide as a neuronal messenger.Trends Pharmacol. 12:125–128.

    Article  Google Scholar 

  • Tymianski M., Charlton M.P., Carlen P.L., and Tator C. H. (1993a). Secondary Ca2+ overload indicates early neuronal injury which precedes staining with viablility indicators.Brain Res. 607:319–323.

    Article  PubMed  Google Scholar 

  • Tymianski M., Wallace M.C., Spigelman I., Uno M., Carlen P.L., Tator C.H., and Charlton M.P. (1993b). Cell-permeant Ca2+ chelators reduce early excitotoxic and ischemic neuronal injuryin vitro andin vivo. Neuron.11: 221–235.

    Article  PubMed  Google Scholar 

  • Ullrich A., and Schlesinger J. (1990). Signal transduction by receptors with tyrosine kinase activity.Cell. 61:203–212.

    Article  PubMed  Google Scholar 

  • Widmer H.R., Knusel B., and Hefti F. (1992). Stimulation of phosphatidylinositol hydrolysis by brain-derived neurotrophic factor and neurotrophin-3 in rat cerebral cortical neurons developing in culture.J. Neurochem. 59: 2113–2124.

    PubMed  Google Scholar 

  • Wroblewski F., and La Due J.S. (1955). Lactic dehydrogenase activity in blood.Proc. Soc. Exp. Biol. Med. 90:210–213.

    PubMed  Google Scholar 

  • Yamamori T. (1992). Molecular mechanisms for generation of neural diversity and specificity: roles of polypeptide factors in development of postmitotic neurons.Neurosci. Res. 12:545–582.

    Article  PubMed  Google Scholar 

  • Zhang Y., Tatsuno T., Carney J.M., and Mattson M.P. (1993). Basic FGF, NGF, and IGFs protect hippocampal and cortical neurons against iron-induced degeneration.J. Cereb. Blood Flow Metab. 13:378–388.

    PubMed  Google Scholar 

  • Zivin J.A., Kochhar A., and Saitoh T. (1990). Protein phosphorylation during ischemia.Stroke 21:III117-III121.

    PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Max-Planck-Institute for Neurological Research, Department of Experimental Neurology, Cologne, Germany

    A. Uto, E. Dux & K. -A. Hossmann

Authors
  1. A. Uto
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. E. Dux
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. K. -A. Hossmann
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Uto, A., Dux, E. & Hossmann, K.A. Effect of serum on intracellular calcium homeostasis and survival of primary cortical and hippocampal CA1 neurons following brief glutamate treatment. Metab Brain Dis 9, 333–345 (1994). https://doi.org/10.1007/BF02098880

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF02098880

Key words

Search

Navigation