Female mice show decreased sexual motivation after mating completion.

(A) Schematic of female self-paced mating assay. The behavior apparatus was divided by a wall with a hole small enough for only the female subject to go through. The female subject was allowed to freely choose between “interaction” with the male intruder in the larger room or “isolation” in the smaller room.

(B) Raster plots of time spent in the interaction zone (gray). The onset of mating completion, start and the endpoint of the experiment is shown in yellow, blue and red respectively.

(C – F) Raster plots of time spent in the interaction zone (gray) around the onset of the first female to male sniffing, male mounting, intromission and mating completion.

(G) Representative heatmap showing the time spent in the behavior apparatus during a control trial or a sexual interaction trial. The sexual interaction trial was further divided into pre- and post-mating completion.

(H–I) Number of transitions between the room per 10 minutes (H) and the average time spent in the isolation zone (I) during the control trial, and pre- and post-mating completion.

(J–M) Number of female to male sniffing (J), male mounting (K), intromission (L) and female self-grooming (M) per 10 minutes during pre- and post-mating completion.

All data are shown as mean ± 95% confidence interval and were analyzed by two-way RM ANOVA with Tukey HSD post-hoc test (FWER = 0.05) (H and I) or Wilcoxon signed-rank test (J, K, L and M). n = 9 (D–K). *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant.

Brain wide analysis of cells responding to mating completion in the female brain.

(A) Overview of the experiment pipeline. After the activity labeling using TRAP, brain tissue was collected and cleared using SHIELD. The cleared tissue was imaged using a light-sheet microscope. The dataset was registered to a reference brain atlas and the number of tdTomato+ cells were quantified using ClearMap and ilastik.

(B) Representative image showing cell segmentation.

(C) Density of tdTomato+ cells per mm3 in larger brain regions. CTXpl: Cortical plate, CTXsp: Cortical subplate, STR: Striatum, PAL: Pallidum, VERM: Vermal regions, HEM: Hemispheric regions, TH: Thalamus, HY: Hypothalamus, MBsen: Midbrain, sensory related, MBmot: Midbrain, motor related, MBsta: Midbrain, behavioral state related, P: Pons.

(D) Heatmap showing density of tdTomato+ cells per mm3 in subregions. Each column indicates data from one subject. Each row indicates data from one subregion. MPN: Medial preoptic nucleus, MPA: Medial preoptic area, BNST: Bed nuclei of the stria terminalis, LPO: Lateral preoptic area, VLPO: Ventrolateral preoptic nucleus, MeA: Medial amygdaloid nucleus, CoA: cortical amygdaloid nucleus, BMP: Basomedial amygdaloid nucleus, posterior part, BLA: Basolateral amygdaloid nucleus, anterior part, BLP: Basolateral amygdaloid nucleus, posterior part, VMH: Ventromedial hypothalamic nucleus, Arc: Arcuate hypothalamic nucleus, DM: Dorsomedial hypothalamic nucleus, LH: Lateral hypothalamic area, SCh: Suprachiasmatic nucleus, PMV: Premamillary nucleus, ventral part, AO: Anterior olfactory area, GENv: Geniculate group, ventral thalamus, PS: Parastrial nucleus.

(E-H) Representative coronal hemisphere image showing expression of tdTomato. Images from Appetitive group are shown in left column, images from Completion group are shown in right. Scale bar 500 μm.

(I) Density of tdTomato+ cells per mm3 in a subset of subregions. The full list of brain regions is shown in Figure S2.

All data are shown as mean ± 95% confidence interval and were analyzed by Student’s t-test with Benjamini/Hochberg correction (C: FDR = 0.05, I: FDR = 0.10). Detailed statistical values are shown in Table 2 and Table 3. Appetitive (green), n = 4. Completion (orange), n = 5. *p < 0.05, ns, not significant.

p-values were adjusted for post-hoc test/multiple comparison.

Related to Figure 2C.

Student’s t-test with Benjamini/Hochberg correction (FDR = 0.05).

Related to Figure 2I.

Student’s t-test with Benjamini/Hochberg correction (FDR = 0.10).

Mating completion signal is strongly represented in the MPOA.

(A) Schematic of in vivo calcium imaging from MPOA Vgat and Vglut2 neurons. AAVDj EF1a-DIO-GCaMP6s was injected into the MPOA of Vgat-Cre or Vglut2-Cre female mice. A GRIN lens was placed above the MPOA following virus injection.

(B) Calcium activity was imaged from a female subject during a free moving mating assay with a sexually experienced male intruder.

(C) Representative maximum projected image of the field of view during calcium imaging. Vgat: 87 ± 28 cells, n = 6. Vglut2: 68 ± 42 cells, n = 4.

(D) Representative coronal section showing GCaMP6s expression and GRIN lens placement.

(E) Representative raster plot showing behavior events during the mating assay and a heatmap showing calcium activity from cells imaged during the assay. Behaviors quantified: female to male sniffing (Sniff), female self-grooming (Self-groom), male to female anogenital sniffing (Ano. sniff), male mounting (Mount), male intromission (Intromission) and mating completion (Completion).

(F) Heatmap showing peri-event calcium activity around behavior onset. Cells are ordered by the average response after mating completion.

(G) Line plot showing average calcium activity around behavior onset. Vgat neurons, blue. Vglut cells, orange.

(H) Average calcium response magnitude following behavior onset per cell.

(I) Proportion of cells which responded positively or negatively to behavior onset (>2δ).

All data are shown as mean ± 95% confidence interval and were analyzed by Student’s t-test with Bonferroni correction (H) and chisquare test with Bonferroni correction (I). *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant.

Heterogenous response properties to sexual behaviors in the MPOA.

(A) Heatmap showing peri-event calcium activity around behavior onset of cells that were categorized based on response magnitude. S: Sniff, M: Mount, C: Completion.

(B) Line plot showing average calcium activity around behavior onset. Sniff cells, blue. Mount cells, green. Completion cells, magenta.

(C) Venn diagram showing the number of cells with positive and negative response in each cell type. (>2δ).

(D) Average calcium response magnitude following behavior onset per cell in each cell types.

(E) Representative field of view showing spatial location of cells with different response properties to sexual behavior in Vgat (left) and Vglut2 (right) cells.

All data are shown as mean ± 95% confidence interval and were analyzed by Student’s t-test with Bonferroni correction (D). *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant.

MPOA inhibitory neurons display prolonged activity late after the onset of mating completion in female mice.

(A) Heatmap showing peri-event calcium activity around mating completion onset for mating completion-responding cells in a longer time scale.

(B) Line plot showing average calcium activity of Vgat and Vglut2 population around behavior onset.

(C) The decay length for mating completion-responding cells from Vgat and Vglut2 population. (D–G) Cluster based analysis of the activity pattern of the mating completion-cells.

(D) Heatmap showing peri-event calcium activity around mating completion onset sorted by cluster.

(E) Representative calcium activity traces of cells from each cluster.

(F) The decay length for mating completion-responding cells from each cluster.

(G) Average z-scored response for mating completion-responding cells from each cluster.

(H–K) Cluster based analysis of the activity pattern of the mating completion-cells in Vgat and Vglut2 population.

(H–I) Heatmap showing peri-event calcium activity of Vgat (H) and Vglut2 (I) cells around male ejaculation onset sorted by cluster.

(J) Line plot showing average calcium activity of Vgat and Vglut2 cells in each cluster around behavior onset.

(K) Proportion of Vgat and Vglut2 cells in each cluster over Vgat and Vglut2 cells in all clusters. Statistical results are shown next to the cluster name. The proportion of type 3 cells in Vgat population was significantly larger than in Vglut2 (p<0.05). The proportion of type 1 cells in Vglut2 population was significantly larger than in Vgat (p<0.001).

(L) Representative field of view showing spatial location of cells with different response properties after mating completion in Vgat (left) and Vglut2 (right) cells

All data are shown as mean ± 95% confidence interval and were analyzed by Student’s t-test (C), two-way ANOVA with post-hoc Tukey HSD test (F and G), and chisquare test with Bonferroni correction (K). (C and K) *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant. (F and G) a vs. b: p < 0.001. b vs. c: p < 0.05. a vs. c: p < 0.001.

Activation of a subpopulation of MPOA labeled following mating is sufficient to suppress female sexual behavior.

(A) Schematic of selective labeling of MPOA neurons using TRAP2. AAV5 EF1a-DIO-hM3Dq-mCherry or AAV5 EF1a-DIO-eYFP as control was injected into the MPOA of the female TRAP2 subjects. After incubation of the virus, the female subjects went through procedures to TRAP cells in MPOA (further described in C).

(B) Schematic of pharmaco-genetic activation of MPOA neurons during a female self-paced mating assay. Female subjects were primed with hormone before the experiment day. On the experiment day, subjects were injected i.p. with CNO to activate TRAP cells, or saline as control.

30 minutes after the injection, the male intruder was placed into the apparatus and the animal behavior was recorded for 2 hrs.

(C) After incubation of the virus, the female subjects were administered with hormones, then received an ejaculation from the male intruder or only interacted without mating. Immediately after, the subjects were administrated with 4-hydroxytamoxifen (4-OHT).

(D) Representative image showing TRAP cells MPN from Completion-eYFP, Appetitive-hM3Dq and Completion-hM3Dq group. Further histological analysis is shown in Figure S7. Scale bar, 100 μm.

(E) Raster plots of time spent in the interaction zone the female self-paced mating assay (Completion-eYFP: blue. Appetitive-hM3Dq: green, Completion-hM3Dq: orange).

(F–M) Number of zone transitions per 10 minutes (F), average time in isolation zone (G), number of female-to-male sniff (H), female self-grooming (I), male mount (J), male intromission (K), the receptive score (number of mount episodes with intromissions/number of all mount episodes) (L).

(M) Schematic illustration summarizing how MPOA neurons suppress female sexual motivation after the completion of mating.

All data are shown by thin lines for individual subjects and thick lines for mean. The data were analyzed by Wilcoxon rank sum test with Bonferroni correction (F–L). Completion-eYFP: n = 6, Appetitive-hM3Dq: n = 7, Completion-hM3Dq: n = 8. *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant.

Sexual experience increase sexual motivation in female mice, related to Figure 1.

(A) The number of entries in the isolation zone per 10 minutes (B) and the average time spent in the isolation zone between virgin animals and sexually experienced animals. All data are shown as mean ± 95% confidence interval and were analyzed by Mann-Whitney U test (A and B). Virgin: n = 6, SE: n = 6. *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant.

Brain-wide analysis of Completion-responding cells in the female brain related to Figure 2 and Table 3.

Density of tdTomato+ cells in each brain region from Completion group and Appetitive group. Statistical significances are shown above each column. The regions are sorted from the top left to bottom right by the qvalue and then the difference in the density between Completion group and Appetitive group. Detail of statistical values and full name of acronyms are shown in Table 3. Appetitive, n = 4. Completion, n = 5. Student’s t-test with Benjamini/Hochberg correction (FDR = 0.10), *p < 0.05; ns, not significant.

Mating completion labeled cells are composed of a subset of excitatory and inhibitory cells in the MPOA.

(A) Brain sections went through multiplexed in situ RNA hybridization chain reaction (HCR) to identify cell type markers of MPOA and an immediate early gene Fos. Vgat, Vglut2, Esr1, Gal, Calcr, Nts, Prlr were used as cell type markers.

(B) Schematic of tissue sampling for Fos expression analysis in the MPOA. Wild type female subjects were administered with hormones before the experiment day. On the experiment day, male intruders entered the female home cage. Male intruders were removed immediately after the completion of mating (Completion group) or during the appetitive phase (Appetitive group). Brain tissue was harvested 20 minutes after the male exit.

(C) Representative coronal section showing Fos mRNA expression in the MPOA of Appetitive and Completion group.

(D) Proportion of Fos+ cells in each brain subregion. MPN: medial preoptic nucleus, MPA: medial preoptic area, BNST: bed nucleus of stria terminalis, LPO: lateral preoptic area, vLPO: ventral lateral preoptic area.

(E) Representative coronal section showing expression of 7 genes: Fos, Vgat, Vglut2, Esr1, Gal, Calcr, Nts.

(F–H) Expanded image of area highlighted in yellow in (E). (F) Expression of Fos, Vgat and Slc17a6. (G) Expression of Fos, Vgat and Gal. (H) Expression of Fos, Calcr and Nts. Colored arrow indicates cells expressing given gene. Scale bar, 50 μm.

(I) Proportion of Vgat+ or Vglut2+ and Fos+ cells in the MPN of Appetitive and Completion group.

(J) Proportion of Vgat+ Fos+ or Vglut2+ Fos+ and cell type marker expressing cells in the MPN of Appetitive and Completion group.

(K) UMAP plot of MPN cells.

(L) Disc plot showing proportion of gene expressing cell and the number of copies per cell for each gene and cluster. The proportion of cells from each group are shown on the right.

(M) Proportion of Fos+ cells from Appetitive and Completion group in each cluster.

All data are shown as mean ± 95% confidence interval and were analyzed by Student’s t-test with Bonferroni correction (D, E, K and N). Appetitive group, n = 5. Completion group, n = 5. *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant.

Lens placement and field of view of in vivo imaging experiment related to Figure 3, Figure 4, Figure 5.

(A) Schematic illustration showing position of GRIN lens placement from each animal. Positions shown on the left hemisphere are from Vglut2 animals, the ones on the right hemisphere are from Vgat animals.

(B) Field of view and region of interest (ROI) for each animal. The subject ID and the number of ROI in each FOV are shown above.

Distribution of subjects per cluster related to Figure 4.

Cells that had significant positive or negative response (>2δ) to each behavior onset were categorized as positive- or negative-responding cells respectively. Outer ring indicates the mouse line Vgat or Vglut2. The inner ring indicates each subject.

Distribution of subjects per cluster related to Figure 5.

Cells that had significant positive or negative response (>2δ) to each behavior onset were categorized as positive- or negative-responding cells respectively. Outer ring indicates the mouse line Vgat or Vglut2. The inner ring indicates each subject.

Histological analysis of TRAP labeling related to Figure 6.

(A) Histological analysis of the distribution of TRAP labeled cells. Representative coronal section showing eYFP or hM3Dq-mCherry expression in the MPOA. Scale bar, 100 μm.

(B–D) Number of TRAP labeled cells (B), number of TRAP labeled cells in the MPN (C) and the proportion of cells in the MPN among all the cells (D). Data from Completion-eYFP and Completion-hM3Dq were grouped into Completion group. Appetitive group, n = 7. Completion group, n = 14.

(E) In situ RNA hybridization analysis of the excitatory and inhibitory marker gene expression in the MPN. Appetitive-hM3Dq and Completion-hM3Dq groups were used for analysis. Representative coronal section showing mCherry, Vgat and Vglut2 expression in the MPOA. Enlarged image indicated by the yellow box, is shown on the right.

(F–H) Number of Vgat+ TRAP labeled cells in the MPN (F), number of Vglut2+ TRAP labeled cells in the MPN (G) and the proportion of TRAP labeled cells that express Vgat or Vglut2 (H). Appetitive group, n = 6. Completion group, n = 7.

(I) Antibody analysis of c-Fos expression after reactivation of mating completion ensemble. Representative coronal section showing mCherry and c-Fos expression in the MPOA. Enlarged image indicated by the yellow box, is shown on the right.

(J–M) Number of TRAP labeled cells in the MPN (J), number of c-Fos+ TRAP labeled cells in the MPN (K), the accuracy score (proportion of c-Fos+ TRAP labeled cells among all TRAP labeled cells) (L) and the efficiency score (proportion of c-Fos+ TRAP labeled cells among all c-Fos+ cells) (M). Appetitive group, n = 6. Completion group, n = 8.

All data are shown by thin lines for individual subjects and thick lines for mean. The data were analyzed by Student’s t-test (B, C, D, F, G, H, J, K, L, M), Student’s relative t-test with Bonferroni correction (H). *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant.

MPOACompletion cells supress female sexual behavior in home cage mating assay but not other behaviors, related to Figure 6.

(A) Schematic of pharmaco-genetic activation of MPOA neurons during a home cage mating assay. Female subjects were primed with hormone before the experiment day. On the experiment day, subjects were injected i.p. with CNO to activate TRAP cells 30 minutes before the experiment. Animal behavior was recorded for 90 minutes.

(B) The male intruder was placed into the home cage of the female subject.

(C–F) Number of female-to-male sniff (C), male mounting (D), male intromission (E) and the receptive score (number of mounting episodes with intromissions over all mounting episodes).

(G) Schematic of conditional place preference (CPP) assay to evaluate the valence of activation of MPOA neurons. A two-chamber behavior apparatus, one room with vertical stripes and one room with horizontal stripes. On the first day animals were placed in the behavior apparatus to test their initial preference to each room for 30 minutes (Pre-test). On the conditioning days, the animals were placed in one side of the room where they were i.p. injected with either saline as control or CNO. CNO was injected 4 hours after the saline injection. On the last day, the animals were placed in the behavior apparatus to test their preference 30 minutes (Post-test).

(H–J) The difference in the time spent in the CNO zone (Time spent in the CNO zone in Post-test – Pre-test) (H), the difference in the number of CNO zone entries (Entries in Post-test – Pre-test)

(I) and the difference in velocity of the animal (Velocity in Post-test – Pre-test) (J).

(K) Representative image of pup retrieval test. Female subject was i.p. injected with saline as control or CNO in their home cage. 30-minutes after injection, 3x 7w-old pup was placed in the corner of the home cage. Animal behavior was examined for 10 minutes.

(L–O) The number of pup retrievals (L), the number of nest building (M), the number of pup grooming (N) and the number of pup sniffing (O).

All data are shown by thin lines for individual subjects and thick lines for mean. The data were analyzed by Mann–Whitney U test with Bonferroni correction (C–F, H–J) and Wilcoxon rank sum test (L–O) with Bonferroni correction. Completion-eYFP: n = 6, Appetitive-hM3Dq: n = 7, Completion-hM3Dq: n = 8. *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant.