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• W2000501648 abstract "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, ∼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." @default.
• W2000501648 created "2016-06-24" @default.
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• W2000501648 date "2007-08-01" @default.
• W2000501648 modified "2023-10-09" @default.
• W2000501648 title "Characterization of rat rostral raphe primary cultures: Multiplex quantification of serotonergic markers" @default.
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• W2000501648 doi "https://doi.org/10.1016/j.jneumeth.2007.04.002" @default.
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