Extended Data Fig. 5.
(a) Schematic representation of Aldh1a1-iCre transgenic line. Endogenous Aldh1a1 gene was targeted for insertion of a P2A peptide and iCre immediately following the peptide encoded by Exon 13. (b) Ratio of mCherry virally labelled cells co-staining for Aldh1a1 (n = 4 mice). (c) Substantia nigra pars compacta immunofluorescence staining from Aldh1a1-iCre mice injected with an AAV5-DIO-mCherry virus (n = 4 mice). Co-staining shows excellent efficiency and fidelity of iCre recombination, which is notably limited to TH+ cells in this region. White arrows: examples of mCherry and Aldh1a1 co-stained cells. Orange arrows: mCherry-expressing cells with undetectable Aldh1a1 staining, which were primarily localized to the dorsal and lateral SNc. Thresholds for intensity scaling and gamma changes were set for each individual channel to maximize visibility of stained cells. (d) Schematic representation of Anxa1-iCre transgenic line. (e) Ratios of virally labelled cells co-staining for Anxa1 protein (n = 4 mice), showing high fidelity of Cre recombination. (f) High magnification of immunofluorescence staining from Anxa1-iCre mice injected with an AAV1-CAG-FLEX-GCaMP6f virus (n = 4 mice) shows that recombination occurs in cells with both high Anxa1 (white arrow) and low Anxa1 (orange arrow), with ~10% of labelled cells showing undetectable Anxa1 protein (red arrows). Thresholds for intensity scaling and gamma changes were set for each individual channel to maximize visibility of stained cells. (g) High magnification of immunofluorescence staining from Anxa1-iCre mice injected with an AAV5-DIO-mCherry virus (n = 4 mice) confirms that recombination occurs in cells with both high Anxa1 protein staining (orange arrows) as well as low Anxa1 protein (white arrows), making it difficult to assess specificity using protein staining alone. Thresholds for intensity scaling and gamma changes were set for each individual channel to maximize visibility of stained cells. (h) IF staining of GFP and Aldh1a1 in Anxa1-iCre, TH-Flpo, RC::FrePe mice (n = 2 mice). Recombination by iCre and Flpo leads to GFP expression in Anxa1+ DA neurons. Co-staining with Aldh1a1 corroborates that Anxa1-iCre recombination is less broad than Aldh1a1 expression and confirms that viral labelling results were not due to insufficient viral delivery / diffusion (example cells with Aldh1a1 staining but no recombination shown with white arrows). Thresholds for intensity scaling and gamma changes were set for each individual channel to maximize visibility of stained cells.