Figure 4.

Confirmation of tamoxifen-induced targeting of the Vglut2 gene using tamoxifen-inducible DAT-Cre transgene and validation of intact midbrain DA system. (A) Schematic illustration of breeding strategy to generate Vglut2^{lx/lx;txDAT-Cre-wt} (txCtrl) and Vglut2^{lx/lx;txDAT-Cre-wt} (txKO). (B) Confirmation of Vglut2 gene targeting using the tamoxifen-inducible DAT-CReERT2 (txDAT-Cre) mouse line. Nested RT-PCR for β-actin, TH and Vglut2 from dissected VTA. Vglut2 wildtype band (500 bp) was observed in both txCtrl and txKO midbrain, while knockout (KO) band (KO; 250 bp) was only found in the gene targeted midbrain. β-actin and TH served as controls. (C) Number of TH positive neurons in the SN and VTA did not differ between txCtrl and txKO.Two-way ANOVA with Sidak post hoc for SN and VTA along three rostro-caudal section (txCtrl n = 3; txKO n = 3). (D) TH immunoreactivity in striatum and midbrain in txCtrl and txKO mice (scale bar 500 μm (striatum) and 50 μm (midbrain). (E,F) Cre-driven tdTom expression in ventral midbrain of eCtrl (E, top), eKO (E, bottom), txCtrl (F, top) and txKO (F, bottom) (scale bar 250 μm). Abbreviations: DStr, Dorsal striatum; NAcC, Nucleus accumbens core; NAcSh, Nucleus accumbens shell; SNc, Substantia nigra pars compacta; SNr, Substantia nigra pars reticulata; VTA, Ventral tegmental area; tdTom, tdTomato.