Primary Cultured Brain Cells as Screening Methods for Natural Products Acting on Glutamatergic Neurons

일차배양 뇌세포를 이용한 글루타메이트성 신경에 작용하는 천연물의 검색방법

  • Published : 1995.08.01

Abstract

Primary cultures of rat cortical and chicken embryonic brain cells were employed to establish a reliable screening method for natural products blocldng or enhancing glutamate-induced neurotoxicity. Exposure of primary cultured rat cortical cells or chicken embryonic brain cells to high dose of glutamate resulted in the fragmentation of neutites and consequent neuronal death. The level of cytoplasmic lactate dehydrogenase(LDH), indicator for cell survival in cultures, was significantly reduced at exposure to glutamate. For the practical application of the methods, series of concentrations of plants extracts and positive control were applied prior to the glutamate insult on primary cultures of rat cortical and chicken embryonic, brain cells. Relative LDH level in cells was measured for the estimation of the effect of the test materials on the glutamatergic neurons. The validity of the present screening method for natural products acting on glutamatergic neurons was examined with dextromethorphan, a known glutamatergic antagonist. The treatment of 100 $\mu{M}$ dextromethorphan prevented the reduction of LDH in rat cortical and chicken embryonic brain cells caused by glutamate insult keeping 60% and 90% of LDH level in normal control, respectively. Above results indicate that primary cultures of rat cortical and chicken embryonic brain cells could be proper systems for the screening of potential natural agents acting on glutamatergic, neurons. Between the two types of cultures, primary culture of chicken embryonic brain cells seemed to be a better system for the primary screening, since it is technically easier and economical compared to that of rat cortical cells.

Keywords

References

  1. Neurotransmitters, Drugs and Disease Webster,R.A.;Jordan,C.C.
  2. Clin. Res. v.288 Neurochemical characteristics of early and late onset types of Alzheimer's disease Rossor,M.N.;Iversen,L.L.;Reynolds,G.P.;Mountjoy,C.Q.;Roth,M.
  3. FASEB J. v.2 Enhancement of learning and memory in mice by a benzodiazepine antagonist Lal,H.;Kumar,B.;Froster,M.J.
  4. Brain Res. v.452 Regional distribution of pre- and post- synaptic glutamatergic function in Alzheimer's disease Cowburn,R.;Hardy,J.;Roberts,P.;Briggs,R.
  5. Proc. Natl. Acad. Sci. v.87 Differential alterations of cortical glutamatergic binding sites in senile dementia of the Alzheimer type Chalmers,D.T.;Dewar,D.;Graham,D.I.;Brooks,D.N.;Mcclloch,J.
  6. TiPS. v.12 Role of NMDA receptors in memory Izquierdo,I.
  7. J. Neurochem. v.53 Direct evidence that cytotoxicity in cultured neurons is mediated via N-methyl-D-aspartate(NMDA) as well as non-NMDA receptors Frandsen,A.;Drejer,J.;Schousboe,A.
  8. J. Neurosci. v.17 In vitro neurotoxicity of excitatory acid analogues during cerebellar development Garthwaite,G.;Garthwaite,J.
  9. Neurosci. v.18 Reversible and irrerversible neuronal damage caused by excitatory amino acid analogues in rat cerebellar slices Hajos,F.;Garthwaite,G.;Garthwaite,J.
  10. Neurosci. v.27 Ca²+ - dependent and Ca²+ -independent glutamate release, energy status and cytosolic free Ca²+ concentration on isolated nerve terminals following metabolic inhibition: Possible relevance to hypoglycemia and anoxia Kauppisen,R.A.;McMahon,H.T.;Nicholls,De.G.
  11. Neurosci. Lett. v.58 Glutamate neurotoxicity in cortical cell culture is calcium dependent Choi,D.W.
  12. Yakhak Hoeji v.24 Studies on the effects of ginseng extract on chicken embryonic nerve and muscle cells Kim,Y.C.;Kim,E.K.
  13. J. Neurosci. Methods v.20 Quantitative determinations of glutamate mediated cortical neuronal injury in cell culture by lactate dehydrogenase efflux assay Choi,D.W.;Koh,J.Y.
  14. Neuron Glutamate neurotoxicity and diseases of the nervous system Choi,D.W.
  15. Brain Res. v.403 Dextrophan and dextromethorphan attenuate glutamate neurotoxicity Choi,D.W.
  16. Annu. Rev. Pharmacol. Toxicol. v.29 The excitatory amino acid receptors: Their classes, pharmacology, and distinct properties in the function of the central nervous system Monaghan,D.T.;Bridges,R.J.;Cotman,C.W.
  17. TiPS. v.11 Excitatory amino acid neurotoxicity and neurodegenerative disease Meldrum,B.;Garthwaite,J.