Characterization of rat rostral raphe primary cultures: multiplex quantification of serotonergic markers.
Previous reports establishing raphe cultures typically yield less than 1% serotonin (5-HT)-positive neurons and are impractical for transcriptional studies. In this study, we have established primary cultures enriched in 5-HT neurons and quantified the proportion of cells expressing serotonergic and non-serotonergic markers. We have also shown the feasibility of using the multiplex real-time PCR technique to measure the relative amounts of RNA for some of these markers. Rostral raphe cells derived from E13-15 rat embryos were cultured for 7 days and analyzed by quantitative immunofluorescence and western blot analysis. In these cultures, approximately 8% of neurons were immunopositive for serotonergic markers (5-HT or tryptophan hydroxylase (TPH)). The percentage of cells labeled for GFAP (glial marker), tyrosine hydroxylase (catecholaminergic), and GAD65/67 (GABAergic) was 5, 1, and 54%, respectively. Transcription factors REST/NRSF and Deaf-1 were present in 9 and 98% of cells, respectively. Multiplex quantitative RT-PCR (Q-PCR) analysis was done for TPH2, 5-HT1A receptor or Deaf-1 RNAs paired with GAPDH RNA as control. Using this approach, standard curves for each RNA were obtained over 200-fold concentration range of dilution with r2 values >0.99. The relative abundances determined by Q-PCR are consistent with the expression of TPH2>Deaf-1>5-HT1A receptor RNA in serotonergic raphe cells. The standard error of TPH2 RNA levels between cultures was <20%, indicating a consistent purity of 5-HT neurons. Thus, we have generated a highly consistent and reproducible model system that is enriched in 5-HT neurons and that will be valuable in future investigation of serotonergic regulation.
Czesak
Margaret
M
Ottawa Health Research Institute (Neuroscience), Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Canada K1H 8M5.
Burns
Ariel M
AM
Remes Lenicov
Federico
F
Albert
Paul R
PR
D000818:Animals; D015415:Biomarkers / Q000032:analysis / Q000378:metabolism; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000502:physiology; D002478:Cells, Cultured; D018507:Gene Expression Regulation, Developmental / Q000235:genetics; D009448:Neurochemistry / Q000379:methods*; D009474:Neurons / Q000737:chemistry* / Q000378:metabolism; D009687:Nuclear Proteins / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis* / Q000378:metabolism; D011903:Raphe Nuclei / Q000737:chemistry* / Q000166:cytology / Q000378:metabolism; D051381:Rats; D044282:Receptor, Serotonin, 5-HT1A / Q000235:genetics; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012701:Serotonin / Q000032:analysis* / Q000378:metabolism; D014157:Transcription Factors / Q000032:analysis / Q000378:metabolism; D014365:Tryptophan Hydroxylase / Q000235:genetics / Q000378:metabolism
D015415:Biomarkers; C485478:Deaf1 protein, rat; D009687:Nuclear Proteins; D012333:RNA, Messenger; D014157:Transcription Factors; D044282:Receptor, Serotonin, 5-HT1A; D012701:Serotonin; D014365:Tryptophan Hydroxylase; C504569:tph2 protein, rat