Abstract
Precise genetic access to molecularly defined neuronal subpopulations is essential for dissecting circuit heterogeneity. We report the development and validation of a knock-in neurotensin receptor 1 (Ntsr1)-FlpO mouse line enabling intersectional targeting of Ntsr1-expressing neurons. Following Flp-dependent adeno-associated viral (AAV) reporter delivery, we observed robtust recombination in the substantia nigra and ventral tegmental area, revealing that midbrain Ntsr1 populations include both dopaminergic and non-dopaminergic neurons. Systemic retro-orbital delivery of a Cre- and Flp-dependent Con/Fon reporter in complementary dual-recombinase configurations demonstrated orientation-dependent differences in dopaminergic targeting specificity. Cis-gene controls defined the maximal achievable dopaminergic ceiling and demonstrated that persistent non-dopaminergic populations exceed expectations from recombinase inefficiency alone. Finally, a dual-recombinase-dependent taCaspase-3 construct enabled intersectional ablation of midbrain dopamine neurons in vivo. Together, these findings establish Ntsr1(Flp) as a physiologically neutral, intersectionally compatible driver line supporting scalable Boolean targeting using local and systemic AAV strategies.