Fig. 3

Chemogenetic activation of LC-NE signaling increases CS-elicited freezing in threat-conditioned animals. a Top: Timeline indicating habituation (Day 1), mild conditioning (0.4 mA US) (Day 2), expression test (Day 3), and drug-free test (Day 4) phases. Bottom: Schematic depicting hM3Dq or mCherry virus injection and CNO treatment strategy. b Representative IHC images show robust and selective targeting of hM3Dq-HA to DBH⁺ LC neurons. (Red = HA; Green = DBH; Yellow = co-localization). Scale bars: top three panels = 500 µm, middle and bottom six panels = 100 µm. c Systemic injection of CNO (1.0 mg/kg) prior to the expression test significantly enhanced freezing in the trained hM3Dq group (n = 9) compared with the trained mCherry group (n = 7; left panel, one-way ANOVA, F (2,20) = 69.54, ****P < 0.0001, Tukey’s MCS, ***P < 0.001). No differences were observed between groups during a CNO-free test (center panel). A difference in CS-elicited freezing was observed in trained animals between CNO- and CNO-free tests (two-way RM ANOVA test, Interaction: F (2, 20) = 16.62, ****p < 0.0001; Training X virus: F (2, 20) = 55.64, ****p < 0.0001, Time (CNO versus CNO-free): F (1,20) = 43.45, ****p < 0.0001; Sidak MCS, CNO versus CNO-free: hM4Di untrained, p = n.s., hM3Dq trained, ****p < 0.0001, mCherry trained, *p < 0.05). All error bars indicate mean ± SEM. *p < 0.05, ***p < 0.001, ****p < 0.0001