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 developed and validated a knock-in neurotensin receptor 1 (Ntsr1)Flpmouse line for intersectional targeting of Ntsr1-expressing neurons. Following delivery of Flp-dependent adeno-associated viral (AAV) reporters, robust recombination was observed throughout the 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 a Cre and Flp-dependent reporter in complementary dual-recombinase configurations revealed configuration-dependent differences in dopaminergic targeting. Cis-gene controls (DatCre;DatFlp) defined the maximal dopaminergic targeting ceiling and helped distinguish true non-dopaminergic targeting from recombinase-dependent off-target labeling. Finally, a dual-recombinase-dependent taCaspase-3 construct enabled selective ablation of midbrain dopamine neurons in vivo, establishing Ntsr1Flp as a versatile driver for scalable Boolean targeting.