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. 2023 Oct 13;49(2):443–454. doi: 10.1038/s41386-023-01739-5

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© The Author(s) 2023

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 2 — A 4 days after beginning CNO treat consumption, 62.5% of DREADD+ males (n = 7) partially or fully stopped consuming CNO treats compared to 12.5% of female DREADD+ mice (n = 9). 100% of DREADD+ males partially or fully stopped consuming the treats after 5 days compared to 25% of DREADD+ females. B Weekly body weight measurements across 4 weeks of CNO in male mice (2-way RM ANOVA; F_Time(1.345,16.14) = 4.716, p = 0.039; F_genotype(1,12) = 3.201, p = 0. 099; F_{time*genotype}(4,48) = 2.480, p = 0.056; n = 7 per group). C 4 weeks of CNO significantly reduced body weight (unpaired t-test, t₍₁₁₎ = 2.638, p = 0.023; n = 6–7) and D thymus weights (unpaired t-test; t₍₁₁₎ = 2.638, p = 0.023, n = 4–5) in DREADD+ males compared to controls. E Weekly body weight measurements across 4 weeks of CNO in female mice (2-way RM ANOVA; F_time(2.478,34.70) = 4.938, p = 0.009; F_genotype(1,14) = 0.751, p = 0.401; F_{time*genotype}(4,56) = 0.964, p = 0.435; n = 7–9). F 4 weeks of CNO did not induce overall body weight change (unpaired t-test, t₍₁₄₎ = 0.952, p = 0.357, n = 7–9) or (G) affect thymus weights (unpaired t-test; t₍₈₎₌0.721, p = 0.492; n = 3–7) in female DREADD+ mice compared to controls. H Representative images of c-Fos immunostaining in the PVN and (I) central amygdala. Dashed lines indicate region of interest used for quantification. J Quantification of c-Fos immunoreactivity in the PVN (2-way ANOVA; F_sex(1,10) = 22.77, p = 0.0008; F_genotype(1,10) = 20.68, p = 0.001, F_sex*genotype(1,10) = 14.15, p = 0.004; n = 3–4). DREADD+ males had a significantly higher proportion of c-Fos immunoreactivity compared to controls (p = 0.0007) and DREADD+ females (p = 0.0003) while there were no significant differences in DREADD+ females compared to controls (p = 0.943). K Quantification of c-Fos immunoreactivity in the CeA (2-way ANOVA; F_sex(1,12) = 7.037, p = 0.021; F_genotype(1,12) = 34.16, p < 0.0001; F_sex*genotype(1,12) = 3.831, p = 0.074). DREADD+ males had a significantly higher proportion of c-Fos immunoreactivity (p = 0.0007) compared to controls and DREADD+ females (p = 0.03) while there were no significant differences in c-Fos immunoreactivity between DREADD+ females and controls (p = 0.073). L Representative images of HA immunostaining in the PVN and (M) CeA in male and female DREADD+ mice. By visual inspection of expression patterns, no apparent differences were noted. (***p < 0.001, *p < 0.05, @ main effect of genotype).