A knockin Ntsr1^Flp driver enables intersectional and systemic targeting of heterogeneous midbrain dopamine circuits

Precise genetic access to molecularly defined neuronal subpopulations is essential for dissecting circuit heterogeneity. We developed and validated a knockin neurotensin receptor 1 (Ntsr1)^Flp mouse line for intersectional targeting of Ntsr1-expressing neurons. Following delivery of Flp-dependent adeno-associated viral (AAV) reporters, robust recombination was observed throughout the midbrain and several other brain regions. A subset of Ntsr1⁺ neurons in the substantia nigra and ventral tegmental area lacked dopaminergic markers, indicating that midbrain Ntsr1 populations comprise both dopaminergic and non-dopaminergic neurons. Systemic delivery of Cre- and Flp-dependent reporters in complementary dual-recombinase configurations revealed configuration-dependent differences in dopaminergic targeting, which was supported by matched same-gene controls (Dat^Cre;Dat^Flp). Finally, a dual-recombinase-dependent taCaspase-3 construct enabled selective ablation of midbrain dopamine neurons in vivo, establishing Ntsr1^Flp as a versatile driver for scalable Boolean targeting.