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. 2023 Oct 11;120(42):e2305950120. doi: 10.1073/pnas.2305950120

Fig. 3.

Fig. 3.

DREADD inhibition of MeApd-ERβ+ neurons disrupts receptivity-based, but not sex-based, preference in male mice. (A) A schematic diagram of bilateral AAV injection in the MeApd. (B) Representative image showing expression of hM4Di-mCherry in MeApd-ERβ+ neurons (scale = 500 µm). (C) A schematic diagram and the timeline for preference tests with DREADD inhibition. Male mice were injected with AAV and tested for receptivity- and sex-based preferences on three test days (4- to 5-d interval), with either saline (days 1 and 3) or CNO (day 2; 1 mg/kg) injections. (D) A schematic diagram of the receptivity-based (RF vs. XF) preference test and a representative heat map of body position in each test performed on three test days (4- to 5-d interval). The data were obtained from a hM4Di-injected animal. (E) Mean percentage (%) of time spent in the contact area of the RF cylinder during RF vs. XF preference tests for the hM4Di (red) and mCherry control (blue) groups. Values were obtained by dividing the RF duration by the total duration of time spent in the contact area of the RF and XF cylinders (Mean ± SEM, n = 11 for hM4Di and n = 9 for mCherry control, ****P < 0.0001, mCherry vs. hM4Di for the CNO condition). (F) Mean percentage (%) of time sniffing toward the RF cylinder during RF vs. XF tests for the hM4Di (red) and mCherry control (blue) groups. Values were obtained by dividing the RF sniffing duration by the total duration of sniffing toward RF and XF cylinders (Mean ± SEM, n = 11 for hM4Di and n = 9 for mCherry control, ****P < 0.0001 mCherry vs. hM4Di for the CNO condition). (G). A schematic diagram of the sex-based (RF vs. IM) preference test and a representative heat map of body position in each test performed on three test days (4- to 5-d interval). The data were obtained from a hM4Di-injected animal. (H) Mean percentage (%) of time spent in the contact area of the RF cylinder during RF vs. IM tests for the hM4Di (red) and mCherry control (blue) groups. Values were obtained by dividing the RF duration by the total duration of time spent in the contact area of RF and IM cylinders (Mean ± SEM, n = 11 for hM4Di and n = 9 for mCherry control). (I) Mean percentage (%) of time sniffing toward the RF cylinder during RF vs. IM tests for the hM4Di (red) and mCherry control (blue) groups. Values were obtained by dividing the RF sniffing duration by the total duration of sniffing toward RF and IMXF cylinders (Mean ± SEM, n = 11 for hM4Di and n = 9 for mCherry control). DREADD, designer receptors exclusively activated by designer drugs; AAV, adeno-associated virus; CNO, clozapine-N-oxide; MeA, medial amygdala; MeApd, MeA-posterodorsal; ER, estrogen receptor; RF, receptive female; XF, nonreceptive female; IM, intact male; SEM, standard error of the mean.