Fig 1: Chemogenetic activation of hypophysiotropic TRH neurons increases TSH release. a–c Colocalization of Trh mRNA (a, magenta) and hM3D-mCherry (b, cyan) in the PVN 2 weeks after bilateral injection of AAV-TRH-hM3D-mCherry (PVN3D). Cells expressing Trh mRNA were positive for hM3D-mCherry (yellow arrowheads; c and d). Red arrowheads label hM3D-mCherry-positive cells without Trh mRNA expression; scale bar, 50 µm. e Plasma TSH concentrations 45 min after CNO administration to Trhr1 +/+ (Bl6) and littermate Trhr1 -/- mice without (PVNCon, gray) and with injection of AAV-TRH-hM3D-mCherry into the PVN (PVN3D, red). TSH plasma concentrations are shown as percentage of Bl6 for normalization, because of a slight but non-significant increase of TSH in Trhr1 -/- compared to Bl6 mice. Two-way ANOVA for genotype: F(1/20) = 5.3, p = 0.032; *p < 0.05 (Bonferroni post test); mean ± S.E.M.; n as indicated. f Relative change in Fos mRNA expression (2-??CT) in the pituitary 45 min after CNO administration to Bl6 and Trhr1 -/- mice without (PVNCon, gray) and with injection of AAV-TRH-hM3D-mCherry into the PVN (PVN3D, red). Two-way ANOVA for genotype: F(1/19) = 4.4, p = 0.049; *p < 0.05 (Bonferroni post test); mean ± S.E.M.; n as indicated. g and h Two weeks after bilateral injection of AAV-TRH-hM3D-mCherry (PVN3D) in the PVN of C57Bl/6 mice (PVN3D-Bl6) hM3D-mCherry-positive fibers were detected in the ME. g Colocalization of hM3D-mCherry with ppTRH in fibers of the ME (red, hM3D-mCherry; green, ppTRH; blue, DAPI; scale bar, 50 µm). h Colocalization of hM3D-mCherry with CRH in fibers of the ME (red, hM3D-mCherry; green, CRH; blue, DAPI; scale bar, 50 µm). i Plasma TSH concentrations 2 h after CNO stimulation of PVNCon-Bl6 (gray) and PVN3D-Bl6 (red) mice. *p = 0.0002 (two-tailed Student’s t-test); mean ± S.E.M.; n as indicated. j Plasma ACTH concentrations 2 h after CNO stimulation of PVNCon-Bl6 (gray) and PVN3D-Bl6 (red) mice. p = 0.11 (Student’s t-test); mean ± S.E.M; n as indicated. k–m No colocalization of hM3D-mCherry with vasopressin (green, j), oxytocin (green, k), or GnRH (green, l) in fibers of the ME. Red, hM3D-mCherry; blue, DAPI; scale bar, 50 µm