Characterization of multiplex gene expression in raphe primary cultures optimized for serotonergic cells

M. CZESAK; F. REMES LENICOV; A. BURNS; P. R. ALBERT

Atlanta

Conferencia; 36th Annual Neuroscience Meeting; 2006

Society for Neuroscience

The serotonin system originates from neurons in the midbrain raphe nuclei that project extensively throughout the brain, and regulate behaviours associated with stress, anxiety and depression. Approximately 20,000 neurons in the rat raphe express a novel tryptophan hydroxylase isoform, TPH2, which mediates brain 5-HT synthesis (Walther et al., 2003) and 5-HT1A autoreceptors which negatively regulate their activity.  We have established an optimized protocol for primary cultures enriched in 5-HT neurons in order to investigate transcriptional mechanisms of 5-HT neuronal specification and differentiation.  Typically, embryonic midbrain cultures have yielded less than 1% 5-HT-positive neurons (200/75,000 cells, Rumajogee et al. 2002), a limitation for mechanistic studies.  Here, we characterize cultures in which ~8% of neurons were 5-HT immunopositive, and examine the feasibility of quantifying multiple RNA transcripts in these cultures for future regulation studies.  Rostral raphe cultures derived from E14-15 rat embryos were maintained in culture for 7 days, during which time processes are extended, and the cultures were then analyzed for serotonin markers using quantitative real-time PCR, immunohistochemistry and western blot analysis.  Serotonin marker RNAs included tryptophan hydroxylase (TPH2), 5-HT1A receptor and a transcriptional repressor of 5-HT1A expression NUDR/Deaf-1 (Lemonde et al., 2003; Czesak et al., 2006), with GAPDH as control.  The standard deviation of RNA levels within cultures was < 5% and between cultures it was <20%, indicating a consistent purity of 5-HT neurons. Furthermore, not all raphe neurons positive for 5-HT stained for TPH, thus, 5-HT-labeled cells that accumulate 5-HT in the absence of detectable TPH may do so via expression of 5-HT transporters. To address the purity of our raphe cultures we stained for GFAP (glial marker), tyrosine hydroxylase (dopaminergic), and GAD65/67 (GABAergic) and the % of cell labelled was 5, 1, and 54%, respectively.  Thus, we have established a highly consistent and reproducible primary culture system enriched in 5-HT neurons, and shown reproducible multiplex quantification of RNAs that will permit transcriptional studies of 5-HT neuronal differentiation.