A Knock-in Ntsr1-Flp Driver Enables Intersectional and Systemic Targeting of Heterogeneous Midbrain Dopamine Circuits

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 robust 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 Ntsr1Flp as a physiologically neutral, intersectionally compatible driver line supporting scalable Boolean targeting using local and systemic AAV strategies. Competing Interest Statement The authors have declared no competing interest.