5-HT neurons project to ZIR and PVT with collateral axons. A, A diagram shows AAV1-FLEX-EGFP-WPRE was injected into both DR and MnR of Sert-Cre mice for tracing 5-HT projections. B, A representative image showing EGFP-positive neurons in both DR and MnR. C, Dense EGFP-positive axons were detected in PVT. D, A representative image showing EGFP-positive axons in ZIR. E, A diagram shows that AAVrg-CAG-FLEX-tdTomato was injected into PVT and AAVrg-CAG-FLEX-EGFP-WPRE was injected into ZIR of Sert-Cre mice for retrogradely mapping raphe 5-HT neurons that project to both PVT and ZI. F, A representative image showing tdTomato-positive terminals in PVT. G, A representative image showing GFP-positive terminals in ZIR. H1–H2, Representative images showing both GFP- and tdTomato-positive neurons in DR. I1–I3, Representative images showing both GFP- and tdTomato-positive neurons in MnR. J, A bar graph showing ZI-projecting only, PVT-projecting only, and collateral-projecting 5-HT neurons in raphe nuclei from 5 mice. F(1.155,4.621) = 119.4, p = 0.0002; ns, no significance, ∗∗∗p < 0.001, One-way ANOVA followed by Bonferroni's comparisons test.