Association of c-fos mRNA expression and excitotoxicity in primary cultures of mouse neocortical and cerebellar neurons.
The effect of excitatory amino acids (EAAs) on c-fos mRNA expression was studied in primary cultures of mouse cerebellar granule cells and in neocortical neurons after 2 and 7 days in vitro (div). In cultured granule cells at 2 and 7 div, and in cortical neurons at 2 div, exposure to low levels (< or = 10 microM) of a variety of EAAs (viz. glutamate [Glu], S-sulpho-L-cysteine [SC], N-methyl-D-aspartate [NMDA], alpha-amino-3-hydroxy-5-methyl-4-isoxazole [AMPA], and kainate [KA]) resulted in a transient increase in the level of c-fos mRNA which peaked at 30 min but returned to a basal level by 120 min. However, exposure of granule cells (7 div) to high levels (250 microM) of Glu, NMDA, KA, SC and of cortical neurons (7 div) to high levels (250 microM) of Glu, NMDA, KA, SC, or AMPA and to low levels (< or = 10 microM) of Glu and AMPA resulted in a delay in c-fos mRNA induction but a subsequent, progressive increase that was sustained for at least 240 min. Furthermore, this effect was accompanied by a dose-related increase in the release of the cytosolic enzyme, lactate dehydrogenase, used as an indicator of excitotoxicity. A ratio (Q240/30) for the steady-state levels of c-fos mRNA after 30 min and 240 min of exposure to EAAs was determined which showed that Q240/30 >2 correlated reproducibly with excitotoxic cell death, whereas a ratio of < or = 1 correlated with a nonexcitotoxic event. In both cell types at 7 div, coadministration of the selective NMDA receptor antagonist, DL(+/-)-2-amino-5-phosphonopentanoic acid (APV) with cytotoxic levels of Glu 1) protected against EAA-induced neurotoxicity and 2) exhibited a transient c-fos mRNA expression (Q240/30 values approximately 1). In contrast, the AMPA/KA receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), provided no protection against excitotoxicity and had no significant effect on the Glu-induced delay in c-fos mRNA expression. These results suggest that the Q240/30 c-fos mRNA ratio may 1) be used as a predictive index for excitotoxic neuronal death, 2) provide information on the identity of the receptor subtype mediating excitotoxicity in different brain cell types, and 3) aid in establishing the role of excitotoxicity during the development of neurons in vitro.
Griffiths
R
R
Neurochemistry Group, School of Biological and Medical Sciences, University of St. Andrews, Fife, Scotland. rg3@st-andrews.ac.uk
Malcolm
C
C
Ritchie
L
L
Frandsen
A
A
Schousboe
A
A
Scott
M
M
Rumsby
P
P
Meredith
C
C
D018750:6-Cyano-7-nitroquinoxaline-2,3-dione / Q000494:pharmacology; D000818:Animals; D016923:Cell Death; D002478:Cells, Cultured; D002525:Cerebellar Cortex / Q000166:cytology*; D002540:Cerebral Cortex / Q000166:cytology*; D003545:Cysteine / Q000031:analogs & derivatives / Q000494:pharmacology; D018690:Excitatory Amino Acid Agonists / Q000494:pharmacology*; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects; D016762:Genes, fos; D018698:Glutamic Acid / Q000494:pharmacology; D007608:Kainic Acid / Q000494:pharmacology; D051379:Mice; D016202:N-Methylaspartate / Q000494:pharmacology; D009419:Nerve Tissue Proteins / Q000096:biosynthesis / Q000235:genetics*; D009474:Neurons / Q000378:metabolism*; D016760:Proto-Oncogene Proteins c-fos / Q000096:biosynthesis; D012333:RNA, Messenger / Q000096:biosynthesis* / Q000235:genetics; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid / Q000494:pharmacology
D018690:Excitatory Amino Acid Agonists; D018691:Excitatory Amino Acid Antagonists; D009419:Nerve Tissue Proteins; D016760:Proto-Oncogene Proteins c-fos; D012333:RNA, Messenger; D018698:Glutamic Acid; D016202:N-Methylaspartate; D018750:6-Cyano-7-nitroquinoxaline-2,3-dione; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; C011119:S-sulphocysteine; D003545:Cysteine; D007608:Kainic Acid