Supplementary Figure 9: Characterization and quantification of rAAV-infected cerebellum of Tg(GAD67-GFP) and GFP negative animals (Related to Figure 6).
From: Cell type–specific manipulation with GFP-dependent Cre recombinase
a) Images of rAAV-CRE-DOGOPT and rAAV-FLEX-tdT-infected lobule V, immunostained for GFP (blue). (b) Quantification of anti-GFP immunostained sections. (c) Immunostaining of rAAV-infected lobule V for the molecular layer interneuron marker Parvalbumin (PV) (blue, top), or the Purkinje cell marker Calbindin (Cb) (blue, bottom). Scale bar for (a) and (c), 50 μm. (d) Quantification of tdT expression in different cell types. In densely infected areas, tdT+ was expressed in (mean +/- s.d.): 65 +/- 16 % of Purkinje Cells (108 Cb+ cells), 44 +/- 22 % molecular layer interneurons (394 PV+ cells) and 53 +/- 21 % of granular layer interneurons (99 GLIs, identified based on location and anatomy, not shown). n=4 animals. (e) Sagittal cerebellar section showing low leakage of FLEX-tdT in GFP negative brains. Arrowhead indicates a single tdT+ Purkinje cell in infected lobule V. Scale bar, 1 mm. (f) Example of tdT+ cells in GFP negative tissue. Left panel: Bergmann glia cell (BG). Scale bar, 50 μm. Top right: molecular layer interneuron (MLI) Scale bar in BG panel also applies to MLI panel, 50 μm. Bottom right: granule cell (GC). Scale bar, 30 μm. (g) Summary quantification of cell types (n=84 cells, 22 sections, 3 animals) labeled by tdT in GFP negative cerebellum infected with rAAV-CRE-DOGOPT and FLEX-tdT mix. MLI, molecular layer interneuron. PC, Purkinje cell. BG, Bergmann glia. Astro, astrocyte. GC, granule cell. Other, identities include oligodendrocyte, unipolar brush cell, NG2 cell. (h) CRE-DOGOPT system enables visualization of fine neuronal processes in the brain. Left panel: tdT expression in GFP+ Purkinje cells allows tracing of axons (arrowheads). Middle panel: higher magnification of Purkinje cells shows tdT labels fine dendritic processes, dendritic spines and synaptic boutons on cell bodies. Right panel: tdT-expression in GLI cell bodies (asterisks) and their axonal projections throughout the molecular layer (arrowheads). Scale bar, 30 μm. All images reported are gathered from or representative of 4 Tg(GAD-GFP)+ animals and 3 GFP negative animals. Scatter plots indicate median and interquartile range.
