Experimenters' sex modulates mouse behaviors and neural responses to ketamine via corticotropin releasing factor

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.

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.

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.

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.

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.

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.

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.

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.

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 μM; n = 11 slices) or combined CRF (125 nM) + (2R,6R)-HNK (15 μM) (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.

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).

Fig. 1 ∣. Mice manifest differential behavioral responses following exposure to male and female experimenter scent.

a,b, Interaction time of experimentally naïve CD1 (P = 0.010 males, P = 0.008 females), BALB/cAnNCrl (P = 0.016), C57BL/6J (P = 0.008) (n = 8 experimenters per strain per sex; n = 32 mice per strain; two-sided paired t-test or Wilcoxon matched-pairs signed-rank test) (a) and anosmic CD1 mice with male versus female experimenter skin swabs (n = 8 experimenters per treatment group per sex; n = 16 mice per treatment group; two-sided Wilcoxon matched-pairs signed-rank test; SAL P = 0.008, ZnSO₄ P = 0.945) (b). c,d, Time spent interacting with male or female experimenter skin swabs versus control (water) swabs (CD1 mice; n = 6 experimenters per sex; n = 24 mice per sex; two-sided Wilcoxon matched-pairs signed-rank test; male swabs P = 0.031, female swabs P = 0.031) (c) and test versus habituation in the real-time place preference with experimenter and control t-shirts (CD1 mice; n = 8 experimenters per sex; n = 16 mice per sex; two-sided paired t-test; male t-shirts P < 0.001, female t-shirts P = 0.049) (d). e, Time spent in each arm of a Y-maze with male-, female- and water-scented swabs (CD1 mice; n = 8 experimenters per sex; n = 30 mice; two-sided repeated measures (RM) two-way ANOVA followed by Holm–Sidak correction, P = 0.023). f, Percentage change from habituation (CD1 mice; n = 8 experimenters per sex; n = 30 mice per sex, two-sided Kruskal–Wallis followed by correction with two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli; control versus male Q = 0.001, male versus female Q = 0.010). g, Representative heat maps of mice shown in h. h, Conditioned place preference/aversion (CPP and CPA) of mice with male-scent-paired and female-scent-paired compartments (CD1 mice; n = 8 mice per sex; two-sided Mann–Whitney U test for CPA, P = 0.038; and two-sided unpaired t-test for CPP, P = 0.831). i, ROC curve includes all data in this figure (P = 0.0031). j, Latency to groom in the presence of male or female experimenter skin swabs (CD1 mice; n = 13 experimenters per sex; n = 26 mice per sex; two-sided Kolmogorov–Smirnov test, P = 0.046). k, Latency to eat in the novelty-suppressed feeding test (CD1 mice; n = 7 experimenters per sex; n = 28 mice per sex; two-sided unpaired t-test, P = 0.009). l, Escape failures following inescapable shock training displayed by mice handled by male and female experimenters (CD1 mice; n = 8 experimenters per sex for male mice and n = 8 experimenters per sex for female mice; n = 40 female mice and n = 40 male mice per sex; two-sided RM two-way ANOVA, sex effect P = 0.048; and two-sided Mann–Whitney U test, P = 0.012). m, Contingency analysis of the number of stress-resilient and stress-prone mice (two-sided Fisher’s exact test, P = 0.0018). Data shown are mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001. For detailed statistical information, see Supplementary Table 1. VEH, vehicle.

Fig. 2 ∣. The sex of the human experimenter influences antidepressant and electroencephalographic responses to KET.

a–c, Immobility time in the FST performed 1 h after dosing (CD1 mice; pairs 1, 2 and 4: n = 10 per treatment group; pair 3: n = 9, 10, 10, 10; pair 5: n = 10, 8, 9, 9; two-sided two-way ANOVA followed by Holm–Sidak test for pairs 1 and 3 and two-sided unpaired t-test for pairs 2, 4 and 5; pair 1: P = 0.024; pair 2: P = 0.024; pair 3: P = 0.008; pair 4: P = 0.025; pair 5: P = 0.030) (a), sucrose preference following social defeat (C57BL/6J mice; n = 7–6 mice per treatment group; two-sided Kruskal–Wallis followed by correction with two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli; day 2: Q = 0.028; day 11: Q = 0.036) (b) and escape failures in the learned helplessness paradigm following administration of either KET or SAL by male versus female experimenter (CD1 mice; n = 7 per treatment group; two-sided two-way ANOVA followed by Holm–Sidak test; male SAL versus KET, P = 0.023; male KET versus female KET, P = 0.006) (c). d, Immobility time in the FST 1 h following either KET or SAL injections by male or female experimenters at another institution (CD1 mice; n = 8 experimenters per sex; n = 16 mice per treatment group; two-sided two-way ANOVA followed by Holm–Sidak test; P = 0.027). e, Brain concentrations of KET, norketamine (NK) and (2R,6R;2S,6S)-HNK following administration of KET (10 mg kg⁻¹; i.p.) by male or female experimenters (CD1 mice; n = 3 experimenters per sex; n = 12 mice per timepoint per sex). f, Immobility time in the FST 1 h following administration of (2R,6R)-HNK or SAL by male or female experimenters (CD1 mice; n = 8 experimenters per sex; n = 16 mice per treatment group; two-sided two-way ANOVA followed by Holm–Sidak test; P = 0.0008). g, Levels of GluA1 in ventral hippocampus. Blot images are cropped. The full gel images along with the molecular weight/size markers can be found in Supplementary Fig. 1 (CD1 mice; n = 8 experimenters per sex; n = 16 mice per treatment group; two-sided two-way ANOVA followed by Holm–Sidak test; P = 0.039). h–k, Representative qEEG local field potentials (LFPs) (h), spectrograms (i), fold change from baseline (CD1 mice; n = 7 experimenters per sex; n = 28–29 mice per treatment group; two-sided Mann–Whitney U test; P = 0.001) (j) and representative traces from 30–120 Hz following administration of KET by male or female experimenters (k). Data shown are mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001. For detailed statistical information, see Supplementary Table 1. Inj., injection.

Fig. 3 ∣. CRF mediates antidepressant responses to KET.

a–c, Effects of ICV administration of CRF before KET in the FST (CD1 mice; n = 20, 18, 20, 18 mice per treatment group; two-sided two-way ANOVA followed by Holm–Sidak test; P = 0.036) (a) and learned helplessness (LH) paradigm (CD1 mice; n = 12–13 mice per treatment group; two-sided Kruskal–Wallis followed by correction with two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli; VEH-KET versus CRF-KET, Q = 0.0086; CRF-SAL versus CRF-KET, Q = 0.0116) (b) and before (2R,6R)-HNK administration in the LH paradigm by a female experimenter (CD1 mice; n = 12, 12, 13, 11 mice per treatment group; two-sided Kruskal–Wallis followed by correction with two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli; Q = 0.033) (c). d–h, Escape failures (CD1 mice; n = 9, 10, 10, 10 mice per treatment group; two-sided two-way ANOVA followed by Holm–Sidak test; VEH-SAL versus VEH-KET, P = 0.006; VEH-KET versus CP-526-KET, P = 0.004) (d), immobility time (CD1 mice; n = 10 mice per treatment group; two-sided two-way ANOVA followed by Holm–Sidak test for 1 h, P = 0.021; and two-sided Kruskal–Wallis followed by correction with two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli for 24 h, Q = 0.026) (e), CSDS-induced sucrose preference deficits (C57BL/6J mice; n = 7 mice per treatment group; two-sided Kruskal–Wallis followed by correction with two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli for the treatment days; *VEH-KET versus VEH-VEH, ^#VEH-KET versus CP-526-VEH, ⁺VEH-KET versus CP-526-KET; day 1, VEH-KET versus CP-526-VEH, Q = 0.0237; day 2, VEH-KET versus VEH-VEH, Q = 0.0165; VEH-KET versus CP-526-KET, Q = 0.0288; VEH-KET versus CP-526-VEH, Q = 0.0086; day 3, VEH-KET versus VEH-VEH, Q = 0.0506; VEH-KET versus CP-526-KET, Q = 0.0336) (f) and skin swab preference (g) and preference index (h) following pretreatment with the CRF1 receptor antagonist CP-154,526 (CP-526) by a male experimenter (CD1 mice; n = 7 experimenters; n = 28 mice per treatment group; two-sided two-way ANOVA followed by Holm–Sidak test for g; control VEH versus CP-526, P = 0.0107; control VEH versus male VEH, P < 0.0001; male VEH versus male CP-154.526, P = 0.0107; and two-sided unpaired t-test for h, P = 0.0107). i–l, Representative spectrograms (i) and traces (j) from raw LFPs, fold change (CD1 mice; n = 6 mice per treatment group; two-sided unpaired t-test, P = 0.023) (k) and representative 30–120-Hz traces derived from qEEG recordings from mice treated with CP-526 before KET (l). m, Effects of CP-526 administration before KET in BALB/cAnNCrl mice by a female experimenter (n = 10 mice per treatment group; two-sided two-way ANOVA followed by Holm–Sidak test; VEH-SAL versus VEH-KET, P = 0.011; VEH-KET versus CP-526-KET, P = 0.0003). Data shown are mean ± s.e.m. *,^#,⁺P < 0.05; **P < 0.01; ***P < 0.001. For detailed statistical information, see Supplementary Table 1. PS, post-stress.

Fig. 4 ∣. CRF mediates electrophysiological responses to (2R,6R)-HNK.

a–d, Representative traces (a,c) and quantification (b,d) of fEPSP slopes following stimulation of the EC–hippocampal CA1 (EC–CA1) pathway following wash-in of (a,b) SAL and (2R,6R)-HNK (C57BL/6J mice; n = 8 slices per group; two-sided unpaired t-test) or (c,d) SAL, CRF, (2R,6R)-HNK or CRF + (2R,6R)-HNK in a separate experiment (C57BL/6J mice; n = 9, 8, 7, 9 slices per group; two-sided one-way ANOVA; CRF + HNK versus SAL, P = 0.0055; CRF + HNK versus CRF, P = 0.0495). e,f, Representative traces (e) and quantification (f) of fEPSP slopes from the EC–CA1 pathway during pre-wash-in with the CRF1 antagonist CP-154,526 and wash-in with CRF + (2R,6R)-HNK (C57BL/6J mice; n = 6, 6, 7, 7 slices per group; two-sided Kruskal–Wallis followed by correction with two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli; VEH versus CRF + HNK, Q = 0.012; VEH/CRF + HNK versus CP-526/CRF + HNK, Q = 0.008). g, Representative images from retrograde AAV-tdTomato injection in CRF-cre mice to the CA1 area and labeled projections in the EC (independently replicated in 3 animals). h, Experimental schematic. i, Representative traces of PSCs recorded from a CA1 stratum lacunosum moleculare (SLM) interneuron voltage clamped at −60 mV in response to optogenetic stimulation of the CRF-expressing EC–CA1 circuit before and after superfusion of the slices with DNQX + DL-AP5, and quantification of current amplitudes (CRF-cre mice; n = 7 cells from 4 animals). j, Experimental schematic and analysis of FST performance following the optogenetic stimulation of the CRF-expressing EC–CA1 circuit before KET administration in a biosafety cabinet (CRF-cre mice; n = 8, 10, 10, 9 mice; two-sided Kruskal–Wallis followed by correction with two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli for pre-selected comparisons; YPF KET versus ChR2 KET, Q = 0.021; ChR2 SAL versus ChR2 KET, Q = 0.046). Data shown are mean ± s.e.m. *P < 0.05; **P < 0.01. For detailed statistical information, see Supplementary Table 1. BS, baseline; rf, rhinal fissure; VS, ventral subiculum.

Fig. 5 ∣. CRF-positive EC cells mediate aversion to male experimenters’ scent.

a–d, Representative images (a) and quantification of CRH⁺ (b), Fos⁺ (c) and CRH⁺/Fos⁺ (d) cells in EC following exposure to experimenter scents (CD1 mice; n = 6 experimenters, n = 12 mice per group; two-sided extra-sum-of-squares; CRF: control versus male, P = 0.008; Fos/CRF: control versus male, P < 0.0001; male versus female, P = 0.0001). e,f, Schematic of the experiment and representative images (e), and quantification of the real-time place preference during optogenetic stimulation of the CRF EC–hippocampal CA1 (EC–CA1) pathway (CRF-Cre mice; n = 10, 14 mice; two-sided unpaired t-test, P = 0.022) (f). g,h, Experimental schematic and representative images (g), and quantification of the preference to the male experimenters’ scent compared with control following chemogenetic inhibition of the CRF EC–CA1 pathway (CRF-cre mice; n = 6, 7, 6, 6 mice; two-sided mixed-effects three-way RM ANOVA followed by correction with two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli; Water/mCherry-VEH versus Male/mCherry-VEH, P < 0.0001; Water/hM4Gi-VEH versus Male/hM4Gi-VEH, P = 0.007; Water/mCherry-CNO versus Male/mCherry-CNO, P = 0.017; Water/hM4Gi-CNO versus Male/hM4Gi-CNO, P = 0.007) (h). i,j, Experimental schematic and representative images (i) and quantification of the calcium transients of the soma of CRF EC–CA1 cells during a choice between male or female experimenter or water swabs using fiber photometry (CRF-cre; n = 6 mice; two-sided one-way ANOVA followed by Holm–Sidak test; control versus male, P < 0.0001; control versus female, P < 0.0001; male versus female, P < 0.0001) (j). Data for b,c and d are one-phase exponential decay curves representing the mean ±95% confidence intervals. Data for e–j are shown as mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001. For detailed statistical information, see Supplementary Table 1.