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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. 1 — A Corticosterone levels were measured in response to 4 CNO doses in DREADD+ males (2-way RM ANOVA; F_time(1.698,15.28) = 15.91, p < 0.001; F_dose(3,9) = 2.933, p = 0.092; F_time*dose(9,27) = 1.278, p = 0.293; n = 3–4). Corticosterone was elevated in response to 5 mg/kg CNO compared to 0.25 mg/kg CNO at 60 mins (p = 0.020) and 180 mins (p = 0.041) post-injection. B Area under the curve analysis of total corticosterone release did not show a significant effect of CNO dose (1-way ANOVA; F_dose(3,9) = 2.998, p = 0.088; n = 3–4). C CNO dose did not significantly affect corticosterone levels at the HPA axis recovery timepoint in DREADD+ males (1-way ANOVA; F_dose(3,9) = 2.403; p = 0.135; n = 3–4). D Corticosterone responses in DREADD+ females were significantly affected by CNO dose (2-way RM ANOVA; F_dose(3,13) = 18.28, p < 0.0001; F_time(2.066,26.86) = 55.10, p < 0.0001; F_time*dose(9,39) = 6.684, p < 0.0001; 120 mins post-injection: 5 mg/kg vs 0.5 mg/kg p = 0.009; 5 mg/kg vs 0.25 mg/kg p = 0.005; 180 mins post-injection: 5 mg/kg vs 1 mg/kg, p = 0.007; 5 mg/kg vs. 0.5 mg/kg p = 0.013; 5 mg/kg vs 0.25 mg/kg p = 0.0008; n = 3–6). E CNO dosing significantly affected the total amount of corticosterone released in DREADD+ females (1-way ANOVA; F_dose(3,13) = 17.04, p < 0.0001; 0.25 mg/kg vs. 1.0 mg/kg p = 0.021; 0.25 mg/kg vs. 5.0 mg/kg p = 0.0001; 0.5 mg/kg vs. 5.0 mg/kg p = 0.0006, 1.0 mg/kg vs. 5.0 mg/kg p = 0.013; n = 3–6). F 5 mg/kg CNO significantly elevated corticosterone at the HPA axis recovery timepoint in DREADD+ females (1-way ANOVA; F_dose(3,13) = 13.51, p = 0.0003; 5 mg/kg vs 1.0 mg/kg p = 0.001; 5 mg/kg vs. 0.5 mg/kg p = 0.005; 5 mg/kg vs 0.25 mg/kg p = 0.0005; n = 3–6). (**** p < 0.0001, ***p < 0.001, **p < 0.01, ^##p < 0.01, *p < 0.05, ^p < 0.05, ^#p < 0.05, @ main effect of genotype). *An example week of the variable-dose CNO treat paradigm is shown in Table S1.