https://www.nature.com/articles/s41593-022-01146-x Skip to main content Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript. Advertisement Advertisement Nature Neuroscience * View all journals * Search * My Account Login * Explore content * About the journal * Publish with us Subscribe * Sign up for alerts * RSS feed 1. nature 2. nature neuroscience 3. articles 4. article * Article * Published: 30 August 2022 Experimenters' sex modulates mouse behaviors and neural responses to ketamine via corticotropin releasing factor * Polymnia Georgiou^1,2,3, * Panos Zanos ORCID: orcid.org/0000-0002-1968-8648^2,4, * Ta-Chung M. Mou^2, * Xiaoxian An ORCID: orcid.org/0000-0002-1739-6342^2, * Danielle M. Gerhard^5^ nAff15, * Dilyan I. Dryanovski^2, * Liam E. Potter^2,6, * Jaclyn N. Highland^2,7, * Carleigh E. Jenne^2, * Brent W. Stewart^2,8, * Katherine J. Pultorak^8, * Peixiong Yuan^9, * Chris F. Powels ORCID: orcid.org/0000-0003-2483-6884^2, * Jacqueline Lovett^10, * Edna F. R. Pereira^11, * Sarah M. Clark^1,2, * Leonardo H. Tonelli^1,2, * Ruin Moaddel^10, * Carlos A. Zarate Jr^9, * Ronald S. Duman^5, * Scott M. Thompson ORCID: orcid.org/0000-0001-9844-9049^2,12 & * Todd D. Gould ORCID: orcid.org/0000-0003-1511-7183^1,2,13,14 Nature Neuroscience (2022)Cite this article * 54 Altmetric * Metrics details Subjects * Behavioural methods * Neuroscience Abstract We show that the sex of human experimenters affects mouse behaviors and responses following administration of the rapid-acting antidepressant ketamine and its bioactive metabolite (2R,6R) -hydroxynorketamine. Mice showed aversion to the scent of male experimenters, preference for the scent of female experimenters and increased stress susceptibility when handled by male experimenters. This human-male-scent-induced aversion and stress susceptibility was mediated by the activation of corticotropin-releasing factor (CRF) neurons in the entorhinal cortex that project to hippocampal area CA1. Exposure to the scent of male experimenters before ketamine administration activated CA1-projecting entorhinal cortex CRF neurons, and activation of this CRF pathway modulated in vivo and in vitro antidepressant-like effects of ketamine. A better understanding of the specific and quantitative contributions of the sex of human experimenters to study outcomes in rodents may improve replicability between studies and, as we have shown, reveal biological and pharmacological mechanisms. Access through your institution Buy or subscribe Your institute does not have access to this article Access options Access through your institution Access through your institution Change institution Buy or subscribe Subscribe to Nature+ Get immediate online access to the entire Nature family of 50+ journals $29.99 monthly Subscribe Subscribe to Journal Get full journal access for 1 year $59.00 only $4.92 per issue Subscribe All prices are NET prices. VAT will be added later in the checkout. Tax calculation will be finalised during checkout. Buy article Get time limited or full article access on ReadCube. $32.00 Buy All prices are NET prices. Additional access options: * Log in * Learn about institutional subscriptions Fig. 1: Mice manifest differential behavioral responses following exposure to male and female experimenter scent. [41593_2022_1146_Fig1_HTML] Fig. 2: The sex of the human experimenter influences antidepressant and electroencephalographic responses to KET. [41593_2022_1146_Fig2_HTML] Fig. 3: CRF mediates antidepressant responses to KET. [41593_2022_1146_Fig3_HTML] Fig. 4: CRF mediates electrophysiological responses to (2R,6R)-HNK. [41593_2022_1146_Fig4_HTML] Fig. 5: CRF-positive EC cells mediate aversion to male experimenters' scent. [41593_2022_1146_Fig5_HT] Data availability Data supporting the findings of this study are available within the paper and its Supplementary Information files. Source data are provided with this paper. Code availability Bonsai script was from the Neurophotometrics system manual at https:/ /static1.squarespace.com/static/60ff345fca665d50e1adc805/t/ 616103731b92d50b0f4d5833/1633747831997/Full+Length+Manual-2021.pdf References 1. Mogil, J. S. Laboratory environmental factors and pain behavior: the relevance of unknown unknowns to reproducibility and translation. Lab Anim. 46, 136-141 (2017). Article Google Scholar 2. Sorge, R. E. et al. Olfactory exposure to males, including men, causes stress and related analgesia in rodents. Nat. Methods 11, 629-632 (2014). CAS Article Google Scholar 3. Davis, H., Taylor, A. A. & Norris, C. Preference for familiar humans by rats. Psychon. Bull. Rev. 4, 118-120 (1997). Article Google Scholar 4. van Driel, K. S. & Talling, J. C. Familiarity increases consistency in animal tests. Behav. Brain Res. 159, 243-245 (2005). Article Google Scholar 5. Can, A. et al. The mouse forced swim test. J. Vis. Exp. (2012) https://doi.org/10.3791/3638 6. Planchez, B., Surget, A. & Belzung, C. Animal models of major depression: drawbacks and challenges. J. Neural Transm. 126, 1383-1408 (2019). CAS Article Google Scholar 7. Samuels, B. A. & Hen, R. in Mood and Anxiety Related Phenotypes in Mice: Characterization Using Behavioral Tests, Vol. 2 (ed. Gould, T. D.) Ch. XXX (Humana Press, 2011).. 8. Zanos, P. et al. NMDAR inhibition-independent antidepressant actions of ketamine metabolites. Nature 533, 481-486 (2016). CAS Article Google Scholar 9. Li, N. et al. mTOR-dependent synapse formation underlies the rapid antidepressant effects of NMDA antagonists. Science 329, 959-964 (2010). CAS Article Google Scholar 10. Autry, A. E. et al. NMDA receptor blockade at rest triggers rapid behavioural antidepressant responses. Nature 475, 91-95 (2011). CAS Article Google Scholar 11. Maeng, S. et al. Cellular mechanisms underlying the antidepressant effects of ketamine: role of a-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors. Biol. Psychiatry 63, 349-352 (2008). CAS Article Google Scholar 12. Highland, J. N., Zanos, P., Georgiou, P. & Gould, T. D. Group II metabotropic glutamate receptor blockade promotes stress resilience in mice. Neuropsychopharmacology 44, 1788-1796 (2019). CAS Article Google Scholar 13. Zanos, P. et al. (2R,6R)-Hydroxynorketamine exerts mGlu 2 receptor-dependent antidepressant actions. Proc. Natl Acad. Sci. USA 116, 6441-6450 (2019). CAS Article Google Scholar 14. Zanos, P. et al. (R)-Ketamine exerts antidepressant actions partly via conversion to (2R,6R)-hydroxynorketamine, while causing adverse effects at sub-anaesthetic doses. Br. J. Pharmacol. 176, 2573-2592 (2019). CAS Article Google Scholar 15. Lumsden, E. W. et al. Antidepressant-relevant concentrations of the ketamine metabolite (2R,6R)-hydroxynorketamine do not block NMDA receptor function. Proc. Natl Acad. Sci. USA 116, 5160-5169 (2019). CAS Article Google Scholar 16. Kim, J.-W. et al. Sustained effects of rapidly acting antidepressants require BDNF-dependent MeCP2 phosphorylation. Nat. Neurosci. 24, 1100-1109 (2021). CAS Article Google Scholar 17. Wulf, H. A., Browne, C. A., Zarate, C. A. & Lucki, I. Mediation of the behavioral effects of ketamine and (2R,6R) -hydroxynorketamine in mice by kappa opioid receptors. Psychopharmacology (Berl.) (2022) https://doi.org/10.1007/ s00213-022-06118-4 18. Pham, T. H. et al. Common neurotransmission recruited in (R,S) -ketamine and (2R,6R)-hydroxynorketamine-induced sustained antidepressant-like effects. Biol. Psychiatry 84, e3-e6 (2018). CAS Article Google Scholar 19. Chen, B. K. et al. Sex-specific neurobiological actions of prophylactic (R,S)-ketamine, (2R,6R)-hydroxynorketamine, and (2S,6S)-hydroxynorketamine. Neuropsychopharmacology 45, 1545-1556 (2020). CAS Article Google Scholar 20. Zanos, P. et al. Ketamine and ketamine metabolite pharmacology: insights into therapeutic mechanisms. Pharmacol. Rev. 70, 621 LP-621660 (2018). Article Google Scholar 21. Yao, N., Skiteva, O., Zhang, X., Svenningsson, P. & Chergui, K. Ketamine and its metabolite (2R,6R)-hydroxynorketamine induce lasting alterations in glutamatergic synaptic plasticity in the mesolimbic circuit. Mol. Psychiatry 23, 2066-2077 (2018). CAS Article Google Scholar 22. Aguilar-Valles, A. et al. Antidepressant actions of ketamine engage cell-specific translation via eIF4E. Nature 590, 315-319 (2021). CAS Article Google Scholar 23. Highland, J. N. et al. Hydroxynorketamines: pharmacology and potential therapeutic applications. Pharmacol. Rev. 73, 763 LP-763791 (2021). Article Google Scholar 24. Fukumoto, K. et al. Activity-dependent brain-derived neurotrophic factor signaling is required for the antidepressant actions of (2R,6R)-hydroxynorketamine. Proc. Natl Acad. Sci. USA 116, 297-302 (2019). CAS Article Google Scholar 25. Carreno, F. R. et al. Activation of a ventral hippocampus-medial prefrontal cortex pathway is both necessary and sufficient for an antidepressant response to ketamine. Mol. Psychiatry 21, 1298-1308 (2016). CAS Article Google Scholar 26. Hong, L. E. et al. Gamma and delta neural oscillations and association with clinical symptoms under subanesthetic ketamine. Neuropsychopharmacology 35, 632-640 (2010). Article Google Scholar 27. Smith, S. M. & Vale, W. W. The role of the hypothalamic-pituitary-adrenal axis in neuroendocrine responses to stress. Dialogues Clin. Neurosci. 8, 383-395 (2006). Article Google Scholar 28. Anisman, H., Lacosta, S., Kent, P., McIntyre, D. C. & Merali, Z. Stressor-induced corticotropin-releasing hormone, bombesin, ACTH and corticosterone variations in strains of mice differentially responsive to stressors. Stress 2, 209-220 (1998). CAS Article Google Scholar 29. Conti, L. H., Costello, D. G., Martin, L. A., White, M. F. & Abreu, M. E. Mouse strain differences in the behavioral effects of corticotropin-releasing factor (CRF) and the CRF antagonist a-helical CRF9-41. Pharmacol. Biochem. Behav. 48, 497-503 (1994). CAS Article Google Scholar 30. Riggs, L. M. et al. (2R,6R)-Hydroxynorketamine rapidly potentiates hippocampal glutamatergic transmission through a synapse-specific presynaptic mechanism.Neuropsychopharmacology 45 , 426-436 (2020). CAS Article Google Scholar 31. Riggs, L. M., Thompson, S. M. & Gould, T. D. (2R,6R) -Hydroxynorketamine rapidly potentiates optically-evoked Schaffer collateral synaptic activity. Neuropharmacology 214, 109153 (2022). CAS Article Google Scholar 32. Blank, T., Nijholt, I., Eckart, K. & Spiess, J. Priming of long-term potentiation in mouse hippocampus by corticotropin-releasing factor and acute stress: implications for hippocampus-dependent learning. J. Neurosci. 22, 3788-3794 (2002). CAS Article Google Scholar 33. Aldenhoff, J. B., Gruol, D. L., Rivier, J., Vale, W. & Siggins, G. R. Corticotropin releasing factor decreases postburst hyperpolarizations and excites hippocampal neurons. Science 221, 875-877 (1983). CAS Article Google Scholar 34. Wilson, D. A. et al. Cortical odor processing in health and disease. Prog. Brain Res. 208, 275-305 (2014). Article Google Scholar 35. Bitzenhofer, S. H., Westeinde, E. A., Zhang, H.-X. B. & Isaacson, J. S. Rapid odor processing by layer 2 subcircuits in lateral entorhinal cortex. eLife 11, e75065 (2022). CAS Article Google Scholar 36. Ferry, B., Herbeaux, K., Javelot, H. & Majchrzak, M. The entorhinal cortex is involved in conditioned odor and context aversions. Front. Neurosci. 9, 342 (2015). Article Google Scholar 37. Xu, W. & Wilson, D. A. Odor-evoked activity in the mouse lateral entorhinal cortex. Neuroscience 223, 12-20 (2012). CAS Article Google Scholar 38. Persson, B. M. et al. Lateral entorhinal cortex lesions impair odor-context associative memory in male rats. J. Neurosci. Res. 100, 1030-1046 (2022). CAS Article Google Scholar 39. Butler-Struben, H. M., Kentner, A. C. & Trainor, B. C. What's wrong with my experiment?: The impact of hidden variables on neuropsychopharmacology research. Neuropsychopharmacology (2022) https://doi.org/10.1038/s41386-022-01309-1 40. McBride, K., Slotnick, B. & Margolis, F. L. Does intranasal application of zinc sulfate produce anosmia in the mouse? An olfactometric and anatomical study. Chem. Senses 28, 659-670 (2003). CAS Article Google Scholar 41. Bruno, C. A. et al. pMAT: an open-source software suite for the analysis of fiber photometry data. Pharmacol. Biochem. Behav. 201 , 173093 (2021). CAS Article Google Scholar 42. Gagnon, R. C. & Peterson, J. J. Estimation of confidence intervals for area under the curve from destructively obtained pharmacokinetic data. J. Pharmacokinet. Biopharm. 26, 87-102 (1998). CAS Article Google Scholar Download references Acknowledgements We thank D. Sparta for providing the CRF-ires-cre founder mice. We thank the many volunteers participating in these experiments. Brain section schematics were created with BioRender.com. This work was supported by NIH grant no. MH107615 and VA Merit grant no. 1I01BX004062 (T.D.G.), NIH grant no. MH086828 (S.M.T.) and NIH grant no. MH093897 (R.S.D.). R.M. and C.A.Z. laboratories are supported by the NIH Intramural Research Program. The contents do not represent the views of the US Department of Veterans Affairs or the US government. Author information Author notes 1. Danielle M. Gerhard Present address: Department of Psychiatry, Weill Cornell Medicine, New York, NY, USA Authors and Affiliations 1. Veterans Affairs Maryland Health Care System, Baltimore, MD, USA Polymnia Georgiou, Sarah M. Clark, Leonardo H. Tonelli & Todd D. Gould 2. Department of Psychiatry, School of Medicine, University of Maryland, Baltimore, MD, USA Polymnia Georgiou, Panos Zanos, Ta-Chung M. Mou, Xiaoxian An, Dilyan I. Dryanovski, Liam E. Potter, Jaclyn N. Highland, Carleigh E. Jenne, Brent W. Stewart, Chris F. Powels, Sarah M. Clark, Leonardo H. Tonelli, Scott M. Thompson & Todd D. Gould 3. Department of Biology, University of Cyprus, Nicosia, Cyprus Polymnia Georgiou 4. Department of Psychology, University of Cyprus, Nicosia, Cyprus Panos Zanos 5. Department of Psychiatry, Yale University, New Haven, CT, USA Danielle M. Gerhard & Ronald S. Duman 6. Michigan Neuroscience Institute, University of Michigan, Ann Arbor, MI, USA Liam E. Potter 7. The Graduate Program in Toxicology, University of Maryland, Baltimore, MD, USA Jaclyn N. Highland 8. The Graduate Program in Neuroscience, University of Maryland, Baltimore, MD, USA Brent W. Stewart & Katherine J. Pultorak 9. Experimental Therapeutics and Pathophysiology Branch, Intramural Research Program, National Institute of Mental Health, National Institutes of Health, Bethesda, MD, USA Peixiong Yuan & Carlos A. Zarate Jr 10. Biomedical Research Center, National Institute on Aging, National Institutes of Health, Baltimore, MD, USA Jacqueline Lovett & Ruin Moaddel 11. Department of Epidemiology and Public Health, School of Medicine, University of Maryland, Baltimore, MD, USA Edna F. R. Pereira 12. Department of Physiology, School of Medicine, University of Maryland, Baltimore, MD, USA Scott M. Thompson 13. Department of Pharmacology, School of Medicine, University of Maryland, Baltimore, MD, USA Todd D. Gould 14. Department of Anatomy and Neurobiology, School of Medicine, University of Maryland, Baltimore, MD, USA Todd D. Gould Authors 1. Polymnia Georgiou View author publications You can also search for this author in PubMed Google Scholar 2. Panos Zanos View author publications You can also search for this author in PubMed Google Scholar 3. Ta-Chung M. Mou View author publications You can also search for this author in PubMed Google Scholar 4. Xiaoxian An View author publications You can also search for this author in PubMed Google Scholar 5. Danielle M. Gerhard View author publications You can also search for this author in PubMed Google Scholar 6. Dilyan I. Dryanovski View author publications You can also search for this author in PubMed Google Scholar 7. Liam E. Potter View author publications You can also search for this author in PubMed Google Scholar 8. Jaclyn N. Highland View author publications You can also search for this author in PubMed Google Scholar 9. Carleigh E. Jenne View author publications You can also search for this author in PubMed Google Scholar 10. Brent W. Stewart View author publications You can also search for this author in PubMed Google Scholar 11. Katherine J. Pultorak View author publications You can also search for this author in PubMed Google Scholar 12. Peixiong Yuan View author publications You can also search for this author in PubMed Google Scholar 13. Chris F. Powels View author publications You can also search for this author in PubMed Google Scholar 14. Jacqueline Lovett View author publications You can also search for this author in PubMed Google Scholar 15. Edna F. R. Pereira View author publications You can also search for this author in PubMed Google Scholar 16. Sarah M. Clark View author publications You can also search for this author in PubMed Google Scholar 17. Leonardo H. Tonelli View author publications You can also search for this author in PubMed Google Scholar 18. Ruin Moaddel View author publications You can also search for this author in PubMed Google Scholar 19. Carlos A. Zarate Jr View author publications You can also search for this author in PubMed Google Scholar 20. Ronald S. Duman View author publications You can also search for this author in PubMed Google Scholar 21. Scott M. Thompson View author publications You can also search for this author in PubMed Google Scholar 22. Todd D. Gould View author publications You can also search for this author in PubMed Google Scholar Contributions P.G. and T.D.G. were responsible for the overall experimental design. T.M.M. and K.J.P. performed the RNAscope experiments. T.M.M. performed automated RNAscope analysis with supervision by L.H.T. and S.M.C. D.M.G. and R.S.D. performed the independent FST replication at Yale University. C.F.P. and X.A. perfused mice and processed the brains for expression and cannula implantation confirmation. D.I.D. performed the whole-cell electrophysiology experiment with supervision from E.F.R.P. L.E.P. and P.G. performed analysis for the fiber photometry experiments. C.E.J. and B.W.S. performed qEEG surgeries. B.W.S. and P.G. performed qEEG data analysis. C.E.J. performed the FST. P.Z., J.N.H., P.G., B.W.S. and S.M.C. performed the PK studies (injections, euthanasia and tissue collection) and experiments utilizing single experimenters. R.M. and J.L. conducted bioanalytical quantitation of ketamine and metabolites. P.Y. and C.A.Z. performed the western blot experiments. S.M.T. helped design and analyze the electrophysiological experiments. P.G. conducted the experiments and their analysis unless otherwise noted. P.G. and T.D.G. outlined and wrote the paper, which was reviewed by all authors. Corresponding author Correspondence to Todd D. Gould. Ethics declarations Competing interests C.A.Z. is a co-inventor on a patent for the use of ketamine in major depression and suicidal ideation. P.Z., J.N.H., R.M., C.A.Z. and T.D.G. are co-inventors in patents or patent applications related to the pharmacology and use of (2R,6R)-HNK in the treatment of depression, anxiety, anhedonia, suicidal ideation and post-traumatic stress disorders. R.M. and C.A.Z. have assigned their patent rights to the US government but will share a percentage of any royalties that may be received by the government. P.Z., J.N.H. and T.D.G. have assigned their patent rights to the University of Maryland Baltimore but will share a percentage of any royalties that may be received by the University of Maryland Baltimore. T.D.G. has received research funding from Allergan and Roche Pharmaceuticals and has served as a consultant for FSV7 LLC, during the preceding 3 yr. All other authors declare no competing interests. Peer review Peer review information Nature Neuroscience thanks the anonymous reviewers for their contribution to the peer review of this work. Additional information Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Extended data Extended Data Fig. 1 Sex of human experimenter effects on stress-related behaviours. (a) Immobility time measured in the forced-swim test (FST) following saline injections by male and female experimenters in CD1 mice combined from all the experiments performed for the present manuscript where the mean immobility time of each experiment was used (n = 13 experiments; two-sided unpaired t-test, p = 0.007). (b,c) Escape failures following inescapable shock training in the learned helplessness paradigm in Swiss-Webster (CFW) mice handled by a male or a female experimenter (n = 50 mice; two-sided Kruskal-Wallis followed by correction with two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli for B; 11-15 trials q = 0.0099, 16-20 trials q = 0.021, 21-25 trials q = 0.004, 26-30 trials q = 0.0125 and two-sided chi-square test for C, p = 0.046). (d) Average sucrose preference over 48 hours following 10-days of chronic social defeat performed by a male and female experimenter (C57BL/6 J mice; n = 15, 16 mice; two-sided unpaired t-test; p = 0.016) and (e) time spent in light compartment in the light/dark box performed by male and female experimenters (CD1 mice; n = 10 experimenters; n = 20 mice /sex; two-sided unpaired t-test; p = 0.221). Data shown are mean +- S.E.M. * p < 0.05; ** p < 0.01. For detailed statistics information, see Supplementary Table 1. Source data Extended Data Fig. 2 Effects of the sex of human experimenter on the antidepressant responses to ketamine. (a) Immobility time in the forced-swim test (FST) 24 hours post- saline (SAL; 7.5 ml/kg) and ketamine (KET; 10 mg/kg) injections by a male and female experimenter in male CD1 mice (n = 9,8,9,8 mice; two-sided two-way ANOVA followed by |Holm-Sidak test; p = 0.023). (b) Immobility time in the FST 1-hour post-SAL or KET (10 mg/kg) injection by a male or female experimenter in female CD1 mice (n = 10 mice/experimenter/treatment group; two-sided Kruskal-Wallis followed by correction with two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli; q = 0.0096). (c) Immobility time 1-hour post-SAL (7.5 ml/kg) or KET (10 mg/kg) injection by a male or female experimenter in male CD1 mice pre-exposed to a 15-min swim session 24 hours prior to the FST (n = 10,9,10,9 mice; two-sided two-way ANOVA followed by |Holm-Sidak test; p = 0.024). (d) Total escape failures in the learned helplessness paradigm following SAL (7.5 ml/ kg) or KET (10 mg/kg) injections by male and female experimenters in male Swiss-webster (CFW) mice (n = 16,14,10,10 mice; two-sided two-way ANOVA followed by |Holm-Sidak test; p = 0.032). (e) Immobility scores in the FST 1-hour following SAL (7.5 ml/kg) or KET (10 mg/kg) injections for each individual experimenter in male CD1 mice, performed at a different institution (n = 2/experimenter). (f) KET dose-response (5, 10, 20, 40 mg/kg) in the FST 1-hr post-injection by a female experimenter in male CD1 mice (n = 9 mice/ dose; two-sided one-way ANOVA). Data are shown as mean +- S.E.M. * p < 0.05, **p < 0.01. For detailed statistics information, see Supplementary Table 1. Source data Extended Data Fig. 3 Sex of human experimenter effects on NMDAR inhibition dependent behavioural effects. Distance travelled per 5 min binned intervals in the open-field test in male CD1 mice that received no treatment (HAB; habituation) followed by injections of saline (SAL; 7.5 ml/kg) and then ketamine (KET; 10 mg/kg) administered by a male or female experimenter (n = 8 mice/experimenter/treatment group; two-sided RM two-way ANOVA with Geisser-Greenhouse correction). Immobility time in the forced-swim test 1-hour post-injection following administration of the (c) N-methyl-D-aspartate (NMDAR) receptor antagonist, MK-801 (0.03 mg/kg) (CD1 mice; n = 8,7,8,7 mice; two-sided two-way ANOVA; Treatment effect: p = 0.004), (d,e) ketamine metabolite, (2R,6R) -hydroxynorketamine (HNK; 10 and 50 mg/kg; CD1 mice; n = 2/ experimenter for D and; n = 8,9,8,8,9,8 mice and two-sided two-way ANOVA followed by Holm-Sidak test for E; SAL vs 10 mg/k - p = 0.038, SAL vs 50 mg/kg - p = 0.0006), and (f) the classical antidepressant desipramine (DSP; 20 mg/kg) vs SAL (7.5 ml/kg) injections by a male or female experimenters (CD1 mice; n = 10 mice/group; two-sided Kruskal-Wallis followed by correction with two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli; Male: p = 0.001, Female: p = 0.011). Data are shown as mean +- S.E.M. * p < 0.05; ** p < 0.01; *** p < 0.001. For detailed statistics information, see Supplementary Table 1. Source data Extended Data Fig. 4 Effects of experimenter scent on the antidepressant-like responses of ketamine. (a) Elimination of experimenter scent by administering saline (SAL; 7.5 ml/kg) or ketamine (KET; 10 mg/kg) within a biosafety cabinet and testing the mice in the forced-swim test (FST) 1-hour post-injection (CD1 mice; n = 8,7,8,7,8,7,8,7 mice; two-sided three-way ANOVA followed by Holm-Sidak test; p = 0.043). (b) Immobility time in mice tested in the FST 1-hour following injections of SAL (7.5 ml/kg) and KET (10 mg/kg) performed on a male worn t-shirt within the biosafety cabinet by a female experimenter (CD1 mice; n = 10 mice/group; two-sided two-way ANOVA followed by Holm-Sidak test; p = 0.011) Data are shown as mean +- S.E.M. * p < 0.05. For detailed statistics information, see Supplementary Table 1. Source data Extended Data Fig. 5 Effects of experimenter scent on the quantitative electroencephalographic oscillations. Effects of ketamine (KET; 10 mg/kg) administration by male and female experimenters on cortical quantitative electroencephalographic (qEEG) measurements in CD1 mice (n = 7 experimenters; n = 28-29 mice/sex) using the traditionally defined frequency bands (a) alpha (8-12 Hz), (b) beta (13-29 Hz), (c) delta (1-4 Hz), (d) theta (4-8 Hz), (e) gamma (30-100 Hz; two-sided Kruskal-Wallis followed by correction with two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli; Male: Baseline vs 10 q = 0.0121, vs 20 q = 0.0003, vs 30 q = 0.0003, vs 40 q = 0.0005, vs 50 q = 0.0009, vs 60 q = 0.0022; Female: Baseline vs 10 q = 0.057, vs 20 q = 0.0011, vs 30 q = 0.0009, vs 40 q = 0.0023, vs 50 q = 0.0124, vs 60 q = 0.0613; Male vs Female q = 0.0426), and (f) high frequency oscillations (100-160 Hz; two-sided Kruskal-Wallis followed by correction with two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli; Male: Baseline vs 10 q = 0.0315, vs 20 q = 0.0011, vs 30 q = 0.0004, vs 40 q = 0.0004, vs 50 p = 0.0004, vs 60 q = 0.0013; Female: Baseline vs 10 q = 0.0446, vs 20 q = 0.0074, vs 30 q = 0.0004, vs 40 q = 0.0011, vs 50 q = 0.0039, vs 60 p = 0.0539; Male vs Female p = 0.0342). Data are normalised to baseline, and the dashed vertical line indicates the time point of ketamine administration. Data are shown as mean +- S.E.M. ^+, ^# p < 0.05; ^++, ^## p < 0.01; ^+++, ### p < 0.001. Differences between ketamine response administered by male and female experimenters is indicated by *. Differences between baseline and ketamine is indicated by # for male experimenters and by + for female experimenters. For detailed statistics information, see Supplementary Table 1. Source data Extended Data Fig. 6 Effects of corticosterone on the antidepressant responses to ketamine. (a) Forced-swim test (FST) immobility measured in mice that received metyrapone (30, 50 and 70 mg/kg) prior to KET (10 mg/kg) and tested 1- and 24-hours later (CD1 mice; n = 9,8,8,8,8,8,8,8 mice; two-sided Kruskal-Wallis followed by correction with two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli; VEH-SAL vs VEK-KET p = 0.028, MET 30-SAL vs MET 30-KET p = 0.028, MET 50-SAL vs MET 50-KET p = 0.009, MET 70-SAL vs MET 70-KET p = 0.019 for 1 -hour; MET 70-SAL vs MET 70-KET p = 0.0021 for 24-hours). (b) Immobility time measured in the FST following saline (SAL; 7.5 ml/kg) or ketamine (KET; 10 mg/ kg) administration by male and female experimenters to male BALB/ cAnNCrl mice (n = 10 mice/treatment group; two-sided Kruskal-Wallis followed by correction with two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli for 1-hour; Male: SAL vs KET q = 0.0075, Female: SAL vs KET q = 0.0069 and two-sided two-way ANOVA for 24-hours; Treatment effect: p = 0.0024). Data are shown as mean +- S.E.M. * p < 0.05; ** p < 0.01; for non-parametric analysis ** q < 0.01. For detailed statistics information, see Supplementary Table 1. Source data Extended Data Fig. 7 Effects of the CRF1 antagonist, CP-154,526, on electroencephalographic measures following ketamine administration. Effects of the corticotropin-releasing factor 1 antagonist (CRF1), CP-154,526 (CP-526; 30 mg/kg) or vehicle (VEH; 1.5 ml/kg) prior to ketamine (KET; 10 mg/kg) administration by a male experimenter on cortical qEEG measurements in CD1 mice (n = 6 mice/treatment group) using the traditionally defined frequency bands (a) alpha (8-12 Hz), (b) beta (13-29 Hz), (c) delta (1-3 Hz), (d) theta power (4-8 Hz), (e ) gamma (30-100 Hz; two-sided Kruskal-Wallis followed by correction with two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli (pre-selected comparisons); VEH-KET vs CP-526-KET at 30 min q = 0.0439 and at 40 min q = 0.0518; Baseline 30 min vs 10 min VEH q = 0.0407, vs 10 min KET q = 0.0086, vs 20 min KET q = 0.0068, vs 30 min KET q = 0.0068, vs 40 min KET q = 0.0094, vs 50 min KET q = 0.052, vs 60 min KET q = 0.1150), and (f) high frequency oscillations (100-160 Hz). Data are normalised to baseline. The first dashed vertical line indicates the time point of VEH or CP-526 administration and the second dashed vertical line indicates the time point of KET administration. Data are shown as mean +- S.E.M. *, ^# p < 0.05, ***, ^### p < 0.001. Differences between mice pre-treated with CP-526 and VEH are indicated by *. Differences between the baseline and treatment in mice that received VEH prior to KET are indicated with #. For detailed statistics information, see Supplementary Table 1. Source data Extended Data Fig. 8 Effects of the combined CRF and (2R,6R) -hydroxynorketamine on field excitatory postsynaptic potentials in hippocampal slices in the SC-CA1 pathway. (a) Representative traces of Schaffer collateral-hippocampal CA1 (SC-CA1) field excitatory postsynaptic potentials (fEPSPs) and (b) quantification of fEPSPs slopes following SC-CA1 pathway stimulation during wash-in with Saline (SAL; n = 11 slices), corticotropin-releasing factor (CRF; 125 nM; n = 12 slices), (2R,6R) -hydroxynorketamine (HNK; 15 mM; n = 11 slices) or combined CRF (125 nM) + (2R,6R)-HNK (15 mM) (n = 11 slices; two-sided one-way ANOVA followed by Holm-Sidak test; SAL vs HNK p = 0.0053, SAL vs CRF+ HNK p = 0.0003, CRF vs HNK p = 0.0043, CRF vs CRF + HNK p = 0.0002). Data are shown as mean +- S.E.M. ** p < 0.01. For detailed statistics information, see Supplementary Table 1. Source data Extended Data Fig. 9 Identification of EC to CA1 projections. (a) Representative images of the injection site at ventral CA1 with retrograde conjugated cholera toxin and the (b,c) labelling in the anterior and posterior entorhinal cortex (EC). Representative RNAscope images from the EC revealing (d) DAPI labeling, (e) CRF transcript labelling, (f) CTb labelling and (g) the co-labelling between the tracer and CRF transcripts. (h) Quantification of RNA scope and tracer co-labelling at the anterior (aEC) and posterior EC (pEC) and the lateral (LEC) and medial EC (MEC) (n = 8-9 samples from 2 animals thus these findings were independently replicated twice). Source data Supplementary information Supplementary Information Supplementary Tables 1 and 2 and Figs. 1 and 2. Reporting Summary Supplementary Video Representative video clip assessing real-time place preference to male- and female-worn t-shirts. All t-shirts were purchased new, identical other than the size, and washed in a nonscented, hypoallergenic detergent before the experiment. T-shirts were worn for 24 h before collection. Control t-shirts were unworn. Mice show aversion to the chamber with the male worn t-shirt and preference to the chamber with the female worn t-shirt. Source data Source Data Fig. 1 Statistical source data. Source Data Fig. 2 Statistical source data. Source Data Fig. 3 Statistical source data. Source Data Fig. 4 Statistical source data. Source Data Fig. 5 Statistical source data. Source Data Extended Data Fig. 1 Statistical source data. Source Data Extended Data Fig. 2 Statistical source data. Source Data Extended Data Fig. 3 Statistical source data. Source Data Extended Data Fig. 4 Statistical source data. Source Data Extended Data Fig. 5 Statistical source data. Source Data Extended Data Fig. 6 Statistical source data. Source Data Extended Data Fig. 7 Statistical source data. Source Data Extended Data Fig. 8 Statistical source data. Source Data Extended Data Fig. 9 Statistical source data. Rights and permissions Reprints and Permissions About this article Verify currency and authenticity via CrossMark Cite this article Georgiou, P., Zanos, P., Mou, TC.M. et al. Experimenters' sex modulates mouse behaviors and neural responses to ketamine via corticotropin releasing factor. Nat Neurosci (2022). https://doi.org/ 10.1038/s41593-022-01146-x Download citation * Received: 20 December 2019 * Accepted: 14 July 2022 * Published: 30 August 2022 * DOI: https://doi.org/10.1038/s41593-022-01146-x Share this article Anyone you share the following link with will be able to read this content: Get shareable link Sorry, a shareable link is not currently available for this article. Copy to clipboard Provided by the Springer Nature SharedIt content-sharing initiative Access through your institution Buy or subscribe Access through your institution Change institution Buy or subscribe Advertisement Advertisement Explore content * Research articles * Reviews & Analysis * News & Comment * Videos * Current issue * Collections * Follow us on Twitter * Subscribe * Sign up for alerts * RSS feed About the journal * Aims & Scope * Journal Information * Journal Metrics * About the Editors * Our publishing models * Editorial Values Statement * Editorial Policies * Content Types * Web Feeds * Posters * Contact Publish with us * Submission Guidelines * For Reviewers * Submit manuscript Search Search articles by subject, keyword or author [ ] Show results from [All journals] Search Advanced search Quick links * Explore articles by subject * Find a job * Guide to authors * Editorial policies Nature Neuroscience (Nat Neurosci) ISSN 1546-1726 (online) ISSN 1097-6256 (print) nature.com sitemap Nature portfolio * About us * Press releases * Press office * Contact us * * * Discover content * Journals A-Z * Articles by subject * Nano * Protocol Exchange * Nature Index Publishing policies * Nature portfolio policies * Open access Author & Researcher services * Reprints & permissions * Research data * Language editing * Scientific editing * Nature Masterclasses * Nature Research Academies * Research Solutions Libraries & institutions * Librarian service & tools * Librarian portal * Open research * Recommend to library Advertising & partnerships * Advertising * Partnerships & Services * Media kits * Branded content Career development * Nature Careers * Nature Conferences * Nature events Regional websites * Nature Africa * Nature China * Nature India * Nature Italy * Nature Japan * Nature Korea * Nature Middle East Legal & Privacy * Privacy Policy * Use of cookies * Manage cookies/Do not sell my data * Legal notice * Accessibility statement * Terms & Conditions * California Privacy Statement Springer Nature (c) 2022 Springer Nature Limited Close Nature Briefing Sign up for the Nature Briefing newsletter -- what matters in science, free to your inbox daily. Email address [ ] Sign up [ ] I agree my information will be processed in accordance with the Nature and Springer Nature Limited Privacy Policy. Close Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing * *