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, 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.
journal_name
J Neurosci Methodsjournal_title
Journal of neuroscience methodsauthors
Czesak M,Burns AM,Remes Lenicov F,Albert PRdoi
10.1016/j.jneumeth.2007.04.002subject
Has Abstractpub_date
2007-08-15 00:00:00pages
59-67issue
1eissn
0165-0270issn
1872-678Xpii
S0165-0270(07)00176-8journal_volume
164pub_type
杂志文章abstract：BACKGROUND:Events that instigate disease may involve biochemical events distinct from changes in the steady-state levels of proteins. Even chronic degenerative disorders appear to involve changes such as post-translational modifications. NEW METHOD:We have begun a series of proteomics analyses on proteins that have be...
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journal_title：Journal of neuroscience methods
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journal_title：Journal of neuroscience methods
pub_type： 杂志文章
doi：
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journal_title：Journal of neuroscience methods
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journal_title：Journal of neuroscience methods
pub_type： 杂志文章
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journal_title：Journal of neuroscience methods
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journal_title：Journal of neuroscience methods
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journal_title：Journal of neuroscience methods
pub_type： 杂志文章
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更新日期：2016-05-01 00:00:00
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journal_title：Journal of neuroscience methods
pub_type： 杂志文章
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journal_title：Journal of neuroscience methods
pub_type： 杂志文章
doi：10.1016/j.jneumeth.2006.04.025
更新日期：2006-11-15 00:00:00
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journal_title：Journal of neuroscience methods
pub_type： 杂志文章
doi：10.1016/j.jneumeth.2013.04.008
更新日期：2013-07-15 00:00:00
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journal_title：Journal of neuroscience methods
pub_type： 杂志文章
doi：10.1016/j.jneumeth.2007.10.018
更新日期：2008-03-15 00:00:00
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journal_title：Journal of neuroscience methods
pub_type： 杂志文章
doi：10.1016/j.jneumeth.2015.11.028
更新日期：2016-03-01 00:00:00
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journal_title：Journal of neuroscience methods
pub_type： 杂志文章
doi：10.1016/j.jneumeth.2010.09.005
更新日期：2011-01-15 00:00:00
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journal_title：Journal of neuroscience methods
pub_type： 杂志文章
doi：10.1016/j.jneumeth.2008.04.029
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journal_title：Journal of neuroscience methods
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journal_title：Journal of neuroscience methods
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journal_title：Journal of neuroscience methods
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