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

Garcia, F.; Villa, A.; Wong, J.; Fenno, L.; Leinninger, G.; Steele, A.

2026-03-12 neuroscience
10.64898/2026.03.11.711093 bioRxiv
Show 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.

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