Transcriptional profiling of depolarization-dependent phenotypic alterations in primary cultures of developing granule neurons.
Rat cerebellar granule neurons cultured in medium supplemented with elevated KCl are extensively used as a model to examine the coupling between neural activity and Ca(2+)-dependent gene expression. Elevated (25 mM) KCl is believed to mimic endogenous neural activity because it promotes depolarization and Ca(+2)-dependent survival and some aspects of maturation. By comparison, at least half of the granule neurons grown in standard medium containing 5 mM KCl undergo apoptosis beginning approximately 4 days in vitro. However, accumulating evidence suggests that chronic depolarization induces phenotypic abnormalities whereas growth in chemically defined medium containing 5 mM KCl more closely resembles the constitutive phenotype. To examine this, oligonucleotide microarrays and RT-PCR of selected mRNAs were used to compare transcription profiles of cultures grown in 5 mM and 25 mM KCl. In some cases, N-methyl-D-aspartate (NMDA) which, like elevated KCl, promotes long-term survival was also tested. Robust changes in several gene groups were observed and indicated that growth in elevated KCl: induces expression of mRNAs that are not normally observed; represses expression of mRNAs that should be present; maintains expression of mRNAs that are markers of immature neurons. Supplementation of the growth medium with NMDA instead of elevated KCl produces similar abnormalities. Altogether, these data indicate that growth in 5 mM KCl more closely mimics survival and maturation of granule neurons in vivo and should therefore be adopted in future studies.
Bui
Cuong J
CJ
Department of Neurosurgery, SUNY, Upstate Medical University, Syracuse, NY 13210, USA.
McGann
Alexandra C
AC
Middleton
Frank A
FA
Beaman-Hall
Carol M
CM
Vallano
Mary Lou
ML
D000818:Animals; D000831:Animals, Newborn; D019703:Calcineurin / Q000378:metabolism; D065095:Calcineurin Inhibitors; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology; D002454:Cell Differentiation / Q000502:physiology*; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects / Q000235:genetics; D002478:Cells, Cultured; D002525:Cerebellar Cortex / Q000166:cytology / Q000254:growth & development* / Q000378:metabolism; D016172:DNA Fingerprinting; D018690:Excitatory Amino Acid Agonists / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects / Q000235:genetics*; D008564:Membrane Potentials / Q000187:drug effects / Q000235:genetics; D016202:N-Methylaspartate / Q000494:pharmacology; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D010641:Phenotype; D011189:Potassium Chloride / Q000494:pharmacology*; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D015533:Transcriptional Activation / Q000187:drug effects / Q000502:physiology*
D065095:Calcineurin Inhibitors; D018690:Excitatory Amino Acid Agonists; D012333:RNA, Messenger; D016202:N-Methylaspartate; D011189:Potassium Chloride; D019703:Calcineurin; D002118:Calcium