Figure 1

Identification of Frizzled-1 and β-catenin in primary mesencephalic neuronal cultures expressing the dopamine transporter (DAT). Enriched neuronal cultures derived from the mesencephalon of E14 rat embryos and maintained for 9-12 days in vitro (DIV) were used to identify the dopamine transporter (DAT), Frizzled-1 (Fzd-1) and β-catenin expression as compared to adult ventral midbrain (VM), using real time PCR, western blotting and immunocytochemistry, before and 24 h after MPTP/MPP⁺ insult. The values represent the means ± S.E. of three independent experiments, each performed in triplicate. Differences were analyzed by ANOVA followed by Newman-Keuls test, and considered significant when p < 0.05. A-B. Expression of TH (A) and DAT (B), before and after MPTP/MPP⁺. C-D: Western blotting of TH (C) and DAT (D) in neurons and VM, before and after MPTP/MPP⁺. E-F: Representative confocal images of TH⁺ neuron (in red, E) and dual staining with DAT (green, F) and TH (red), showing co-localization (orange) in SNpc neurons of the ventral midbrain.G-H: Frizzled-1 (Fzd-1) receptor mRNA (G) and protein (H) levels in DAT-expressing neuronal cultures and VM before and after MPTP/MPP⁺. I:. Dual staining with Fzd-1 (in green) and TH (in red) documenting co-localization (orange-to-yellow) of the two markers in purified neuron cultures. Note the distribution of Fzd-1 staining in TH⁺ processes and cell body. L-N. β-catenin mRNA (J) and protein levels (K-L) in DAT expressing neurons and VM. β-catenin⁺-IF signal (red, L) is mainly in the plasma membrane, well beneath the DAPI⁺ nucleus. *p < 0.05 compared to -MPTP/MPP⁺.