Antagonistic circuits mediating infanticide and maternal care in female mice

Abstract

In many species, including mice, female animals show markedly different pup-directed behaviours based on their reproductive state^1,2. Naive wild female mice often kill pups, while lactating female mice are dedicated to pup caring^3,4. The neural mechanisms that mediate infanticide and its switch to maternal behaviours during motherhood remain unclear. Here, on the basis of the hypothesis that maternal and infanticidal behaviours are supported by distinct and competing neural circuits^5,6, we use the medial preoptic area (MPOA), a key site for maternal behaviours^7-11, as a starting point and identify three MPOA-connected brain regions that drive differential negative pup-directed behaviours. Functional manipulation and in vivo recording reveal that oestrogen receptor α (ESR1)-expressing cells in the principal nucleus of the bed nucleus of stria terminalis (BNSTpr^ESR1) are necessary, sufficient and naturally activated during infanticide in female mice. MPOA^ESR1 and BNSTpr^ESR1 neurons form reciprocal inhibition to control the balance between positive and negative infant-directed behaviours. During motherhood, MPOA^ESR1 and BNSTpr^ESR1 cells change their excitability in opposite directions, supporting a marked switch of female behaviours towards the young.

Extended Data Fig. 1:. Ablation or chemogenetic inhibition of MPOA^Esr1 cells induces infanticide in SW virgin female mice

(a) Experimental design to ablate MPOA^Esr1 cells. (b) Experimental timeline. (c, d) Left showing representative histology images of Esr1 staining (magenta) in MPOA and BNSTpr after DT injection in a mCherry female of 6 mice(c) and a DTR female of 8 mice (d). Right showing raster plots of pup-directed behaviors in mCherry (c) and DTR females (d) before and after i.p. injection of DT. *Remove wounded pups and stop recording. (e) Number of mCherry and DTR virgin females that attack, ignore or retrieve pups before and after DT injection. Fisher’s exact test for comparing behaviors (attack vs. no attack) between groups before or after DT injection. McNemar’s test for comparing behaviors (attack vs. no attack) between pre-DT and after-DT within a group. * p < 0.05. **p < 0.01. (f, g) Latency to attack pup (f) and investigate pup (g) before and after DT injection in mCherry and DTR females. If the behavior of interest is not observed during the entire session, the latency is 600 s. Error bars: ± SEM. Mixed-effects analysis followed with multiple comparisons test. ****p < 0.0001. n = 6 mice for mCherry group, n=8 mice for DTR group. (h) Experimental design to chemogenetically inhibit MPOA^Esr1 cells. (i) Experimental timeline. (j, k) Left showing representative histology images of mCherry (j) and hM4Di-mCherry (k) expression of 6 mice each group in MPOA. Right showing raster plots of pup-directed behaviors in mCherry (j) and hM4Di females (k) after i.p. injection of saline or CNO. *Remove wounded pups and stop recording. (l) Number of mCherry and hM4Di virgin females that attack, ignore or retrieve pups after saline or CNO injection. Fisher’s exact test for comparing behaviors (attack vs. no attack) between groups after saline or CNO injection. McNemar’s test for comparing behaviors (attack vs. no attack) between saline and CNO injections within a group. * p < 0.05. **p < 0.01. (m, n) Latency to attack pup (m) and investigate pup (n) after saline or CNO injection in mCherry and hM4Di females. Error bars: ± SEM. Mixed-effects analysis (m) and Two-way RM ANOVA (n) followed with multiple comparisons test. ****p < 0.0001. n = 6 mice for each group. Brain illustrations in (a) and (h) are produced based on reference atlas from https://atlas.brain-map.org/. See Source Data Extended Data Fig. 1 for detailed values and statistics.

Extended Data Fig. 2:. MPOA-connecting cells across brain regions and their overlap with Infanticide and maternal care induced c-Fos.

(a) Experimental design to trace MPOA-connecting cells throughout the brain using Ai6 female mice and high titer (> 1×10¹³ vg/mL) AAV1-hSyn-Cre. (b and c) Images from a representative animal of 4 mice showing the primary injection site in the MPOA (b) and MPOA-connecting cells in various brain areas (c). Scale bars: 1 mm (b) and 200 μm (c). (d) Distribution of MPOA-connecting neurons in various brain regions. All regions containing over 1% of total labeled cells are shown. n = 4. Error bars: SEM. (e-g) Representative images of 3 mice each group of 18 regions showing baseline (e), infanticide-induced (f), and maternal behavior-induced (g) c-Fos and zsGreen expression in Ai6 female mice injected with AAV1-hSyn-Cre into the MPOA. Baseline c-Fos are from females left undisturbed in the home cage. Brain illustrationsare produced based on reference atlas from https://atlas.brain-map.org/. (h) The density of c-Fos expressing cells in each MPOA-connecting brain region in control, infanticidal and maternal female mice. n = 3 mice for each group. One-way ANOVA followed with multiple comparisons test. *p < 0.05, **p < 0.01, Error bars: ±SEM. (i) The total number of Fos+Tracer+ and Fos-Trace+ cells per 100 Tracer+ cells in each MPOA-connecting brain region in control, infanticidal and maternal female mice. n = 3 mice for each group. Fisher’s exact test is based on unnormalized total numbers of Fos+Tracer+ and Fos-Trace+ cells of each group. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. See Source Data Extended Data Fig. 2 for detailed values and statistics.

Extended Data Fig. 3:. Pup-directed behaviors after chemogenetic activation of MPOA-connecting cells in the VMHvl, SUM, PVT, PVN, or PMv in female mice

(a) Experimental design to chemogenetically activate various MPOA-connecting regions. (b) Experimental timeline. (c1, d1, e1, f1, g1) Representative histology images of 8 mice showing hM3Dq-mCherry expression in the VMHvl (c1), SUM (d1), PVT (e1), PVN (f1), and PMv (g1) after injecting AAV1-Syn-Cre in MPOA and AAV2-hSyn-DIO-hM3Dq-mCherry into each of the regions. Scale bars: 200 μm. (c2, d2, e2, f2, g2) Representative raster plots showing pup-directed behaviors after saline or CNO injection into animals expressing hM3Dq-mCherry in MPOA-connecting cells in the VMHvl (c2), SUM (d2), PVT (e2), PVN (f2), and PMv (g2). Each raster lasts 10 min. (c3, d3, e3, f3, g3) Pup-directed attack after saline or CNO injection in female mice that express hM3Dq-mCherry in MPOA-connecting cells in the VMHvl (c3), SUM (d3), PVT (e3), PVN (f3), and PMv (g3). Each circle represents one mouse. n = 8 for each group. (c4, d4, e4, f4, g4) Duration of pup investigation between saline- and CNO-injected days in female mice that express hM3Dq-mCherry in MPOA-connecting cells in the VMHvl (c4), SUM (d4), PVT (e4), PVN (f4), and PMv (g4). Each gray line represents one animal. The colored line represents the group average. Error bars: ± SEM. Wilcoxon matched-pairs signed rank test (c4) or Two-tailed Paired t test (d4, e4, f4, g4). **p<0.01. n = 8 for each group. (c5, d5, e5, f5, g5) Duration of pup grooming between saline- and CNO-injected days in female mice that express hM3Dq-mCherry in MPOA-connecting cells in the VMHvl (c5), SUM (d5), PVT (e5), PVN (f5), and PMv (g5). Figure conventions as in c4-g4. (c6, d6, e6, f6, g6) Latency to first pup attack between saline- and CNO-injected days in female mice that express hM3Dq-mCherry in MPOA-connecting cells in the VMHvl (c6), SUM (d6), PVT (e6), PVN (f6), and PMv (g6). Figure conventions as in c4-g4. See Source Data Extended Data Fig. 3 for detailed values and statistics.

Extended Data Fig. 4:. Optogenetic activation of BNSTpr^MPOA neurons elicits infanticide, induces real-time place preference, and reduces anxiety in female mice

(a) Experimental design to optogenetically activate BNSTpr^MPOA cells. (b) Experimental timeline. (c) Light delivery protocol. (d and f) Representative raster plots showing pup-directed behaviors during sham and 2 mW light stimulation in virgin female mice expressing GFP (d) or ChR2-EYFP (f) in BNSTpr^MPOA cells. # Remove wounded pup(s) and introduce a new pup. (e and g) PETH of attack pup probability in virgin female mice expressing GFP (e) and virgin female mice expressing ChR2-EYFP (g) in BNSTpr^MPOA cells following sham or light stimulation. Only trials with female-pup contact are included for analysis. Left black dash line: Light on. Right red dash line: light off. (h) Percentage of animals that attacked pups in GFP and ChR2 group. Fisher’s exact test for comparison between GFP and ChR2 group. McNemar’s test for comparison between sham and different laser intensity within ChR2 group. *p < 0.05, **p < 0.01, ****p < 0.0001. n = 8 mice for GFP control, n = 11 mice for ChR2 virgin group (sham, 0–1, 2–3), n = 10 mice for ChR2 virgin group (>3). (i) Percentage of trials showing pup attack. Each dot represents one mouse. Error bars: ± SEM. Mixed-effects analysis followed with multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. n = 8 mice for GFP control, n = 11 mice for ChR2 virgin group (sham, 0–1, 2–3), n = 10 mice for ChR2 virgin group (>3). (j) Averaged latency to attack pup upon encountering the pup following sham or light stimulation. Error bars: ± SEM. Mixed-effects analysis followed with multiple comparisons test. ****p < 0.0001. n = 8 mice for GFP control, n = 11 mice for ChR2 virgin group (sham, 0–1, 2–3), n = 10 mice for ChR2 virgin group (>3). (k-l) Representative tracking results during the RTPP test (k) and the time spent in sham or 3 mW stimulation chamber (l). n = 8 mice. Paired t test. *p < 0.05. Error bars: SEM. (m-n) Representative tracking results during an EPM test (m) and the time spent in open arms (n) with sham or 3 mW laser stimulation. n = 8 mice. Paired t test. *p < 0.05. Error bars: SEM. See Source Data Extended Data Fig. 4 for detailed values and statistics.

Extended Data Fig. 5:. Additional characterization of BNSTpr Esr1 cells and their activation-induced infanticide in SW female mice.

(a) Representative coronal sections of 3 mice each group showing Esr1 immunostaining in MPOA and BNSTpr in a male (top), a virgin female (middle) and a mother (bottom). Scale bars: 500 μm. (b, c) Number of Esr1 positive cells in the MPOA (b) and BNSTpr (c) in male, virgin female, and mother mice. n = 3 mice for each group. One-way ANOVA followed with multiple comparisons test. **p < 0.01, ****p < 0.0001. Error bars: ± SEM. (d) Representative images of 3 mice showing overlap between Esr1 (red) and infanticide-induced c-Fos (green) in the BNSTpr. Right shows the enlarged view of the boxed area. Scale bars: 100 μm (left) and 10 μm (right). (e) Representative images of 3 mice showing the overlap between Esr1 (red) and zsGreen (green) in the BNSTpr in an Ai6 animals injected with AAV1-hSyn-Cre at the MPOA. Right shows the enlarged view of the boxed area. Scale bars: 100 μm (left) and 10 μm (right). (f) Quantification of overlap between MPOA-connected cells, Esr1 and the infanticide-induced c-Fos in the BNSTpr. n = 3 mice for each group, Error bars: ± SEM. (g) Experimental design to optogenetically activate BNSTpr^Esr1 cells in non-infanticidal SW virgin females. (h) Experimental timeline. (i) Representative image of 4 mice showing ChR2 expression (green) in the BNSTpr and fiber tracks (while boxes). Scale bar: 1 mm. (j) Representative raster plots showing pup-directed behaviors during sham and 2 mW light stimulation in a non-infanticidal SW virgin female (top) and a mother (bottom) expressing ChR2-EYFP in BNSTpr^Esr1 cells. # Replace a wounded pup. (k) Percentage of animals that attack pups. n = 5 mice for ChR2 virgin group, n = 4 mice for ChR2 mother group. McNemar’s test for comparison between sham and different laser intensity within each ChR2 group. (l) Percentage of trials showing pup attack. Each dot represents one mouse. Mean ± SEM. Mixed-effects analysis followed with multiple comparisons test. **p < 0.01, ***p < 0.001, ****p < 0.0001. n = 5 mice for ChR2 virgin group, n = 4 mice for ChR2 mother group. (m) Average latency to attack pup upon encountering the pup following sham or light stimulation. Error bars: ± SEM. Mixed-effects analysis followed with multiple comparisons test. ***p < 0.001, ****p < 0.0001. n = 5 mice for ChR2 virgin group, n = 4 mice for ChR2 mother group. See Source Data Extended Data Fig. 5 for detailed values and statistics.

Extended Data Fig. 6:. Chemogenetic inhibition of BNSTpr^Esr1 neurons promotes maternal behavior in non-hostile virgin females and mothers

(a) Representative raster plots showing pup-directed behaviors in non-hostile non-maternal mCherry and hM4Di virgin females after saline or CNO injection. (b) Percentage of mCherry and hM4Di virgin females that ignore, retrieve or attack pups after saline or CNO injection. Fisher’s exact test between mCherry and hM4Di group. (c, d) Latency to retrieve pup (c) and pup investigation duration (d) after saline or CNO injection in non-hostile non-maternal virgin mCherry and hM4Di females. Error bars: ± SEM. n = 6 mice for mCherry group; n = 5 mice for hM4Di group. Mixed-effects analysis followed with multiple comparisons test. **p < 0.01. (e) Representative raster plots showing various pup-directed behaviors in lactating mCherry and hM4Di females after saline or CNO injection. (f) All mCherry and hM4Di lactating females retrieved pups after saline or CNO injection. (g) All 5 pups were retrieved in the 10-min testing period in mCherry and hM4Di lactating females after either saline or CNO injection. n = 9 mice for mCherry group; n = 11 mice for hM4Di group. (h) Latency to retrieve the first pup and all five pups in mCherry and hM4Di lactating females after saline or CNO injection. Error bars: SEM. n = 9 mice for mCherry group; n = 11 mice for hM4Di group. Mixed-effects analysis followed with multiple comparisons test. **p < 0.01. See Source Data Extended Data Fig. 6 for detailed values and statistics.

Extended Data Fig. 7:. Histology and light-induced c-Fos in the MPOA^Esr1 and BNSTpr^Esr1 terminal manipulation experiments

(a) Experimental design to optogenetically inactivate MPOA^Esr1 inputs to BNSTpr. (b) Representative histology of 6 mice showing ArchT expression (red) in the MPOA (left), and ArchT expression terminals and optic fiber tracks in the BNSTpr (right). (c) Experimental design to optogenetically activate MPOA^Esr1 inputs to the BNSTpr. (d) Representative histology of 7 mice showing ChrimsonR (red) and c-Fos (white) in the MPOA after delivering 5 mW pulsed yellow light to the right side of the BNSTpr for 10 min. (e) Representative histology of 3 mice showing ChR2 (green), hM4Di (red), and c-Fos (white) in the MPOA after delivering 10 min 5 mW pulsed blue light to the right side of the BNSTpr 30 min after CNO injection. (f) Number of c-Fos+ cells in the right MPOA in ChrimsonR and ChR2+Gi group. Error bars: ± SEM. Unpaired t test, ****p<0.0001. n =7 mice for ChrimsonR group, n =3 for ChR2+Gi group. (g) Experimental design to optogenetically inactivate BNSTpr^Esr1 inputs to MPOA. (h) Representative histology of 6 mice showing ArchT (red) in the BNSTpr (left), and ArchT expressing axons and optic fiber tracks in the MPOA (right). (i) Experimental design to optogenetically activate BNSTpr^Esr1 inputs to MPOA. (j) Representative histology of 5 mice showing ChrimsonR (red) and c-Fos (white) in the BNSTpr after delivering 5 mW pulsed yellow light to the right side of the MPOA for 10 min. (k) Representative histology of 4 mice showing ChR2 (green), hM4Di (red) and c-Fos (white) in BNSTpr after delivering 10 min 5 mW pulsed blue light to the right MPOA 30 min after CNO injection. (l) Number of c-Fos+ cells in the right BNSTpr in ChrimsonR and ChR2+Gi group. Error bars: ± SEM. Mann Whitney test, *p<0.05. n =5 mice for ChrimsonR group, n =4 for ChR2+Gi group. See Source Data Extended Data Fig. 7 for detailed values and statistics.

Extended Data Fig. 8:. Verification of ArchT-mediated terminal inhibition

(a and d) Experimental design to examine the efficacy of ArchT-mediated inhibition of MPOA^Esr1 → BNSTpr^Esr1 (a) and BNSTpr^Esr1 → MPOA^Esr1 (d) projections. (b and e) Representative blue light pulse evoked IPSCs from BNSTpr^Esr1 (b) and MPOA^Esr1cells (e) with (yellow) and without (black) simultaneous 5 mW yellow light delivery. (c and f) The amplitude of oIPSCs of BNSTpr^Esr1 (c) and MPOA^Esr1cells (f) with and without 5 mW yellow light delivery. Error bars: ± SEM. Paired t-test (c) and Wilcoxon matched-pairs signed rank test (f), **p<0.01. n = 4 BNSTpr^Esr1 cells, n = 8 MPOA^Esr1 cells. See Source Data Extended Data Fig. 8 for detailed values and statistics.

Extended Data Fig. 9:. Fiber photometry recording of GFP-expressing BNSTpr^Esr1 and MPOA^Esr1 cells and GCaMP6f-expressing BNSTpr^MPOA cells during pup interaction

(a) Fiber photometry setup. (b and d) Viral construct and targeted brain regions. Brain illustrations are produced based on reference atlas from https://atlas.brain-map.org/. (c and e) Representative histological images of 4 mice showing GFP (green) expression and fiber tracks (white box) in BNSTpr (c) and MPOA (e). (f) Experimental timeline. (g, k) Representative GFP recording (ΔF/F) traces of BNSTpr^Esr1 (g) and MPOA^Esr1 (k) cells during pup interaction in a hostile SW virgin female (g1, k1) and a mother (g2, k2). Color shades indicate various behaviors. (h, l) PETHs of GFP signal (Z-scored ΔF/F) of BNSTpr^Esr1 (h) and MPOA^Esr1 (l) cells aligned to the first pup contact in hostile virgin females (h1, l1), and mothers (h2, l2). n = 4 mice. Solid lines indicate the onset on first pup contact; dashed black lines indicate the mean duration of first pup contact. Shades: ± SEM. (i, m) PETHs of GFP signal (Z-scored ΔF/F) of BNSTpr^Esr1 (i) and MPOA^Esr1 (m) cells aligned to the onset of pup approach in hostile virgin females (i1, m1), and mothers (i2, m2). n = 4 mice for each group. Blue dashed lines indicate the onset of pup approach; green dashed lines indicate the mean latency to pup investigation; the red and black dashed lines in i1 and m1 indicate the mean latency to attack and stop attacking pups, respectively; the magenta and black dashed lines in i2 and m2 indicate the mean latency to retrieve and stop retrieving pups, respectively. Shades: ± SEM. (j, n) Mean AUC of Z-scored ΔF/F signal of BNSTpr^Esr1 (j) and MPOA^Esr1 (n) cells during various pup-directed behaviors in hostile virgin females and mothers. Two-way RM ANOVA (j) and Mixed-effects analysis (n) followed with multiple comparisons test. n = 4 mice for each group. Error bars: ± SEM. (o) Experimental design. (p) A representative histological image of 9 mice showing GCaMP6f (green) expression and the fiber track in BNSTpr (white lines). (q) Experimental timeline. (r) Representative GCaMP6f recording (ΔF/F) traces of BNSTpr^MPOA cells during pup interaction in a hostile SW virgin female (r1) and a mother (r2). Color shades indicate various behaviors. (s-v) PETHs of GCaMP6f signal (Z-scored ΔF/F) of BNSTpr^MPOA cells aligned to the onset of various behaviors in hostile virgin females (s1-v1), and mothers (s2-v2). n = 6 mice. Solid lines indicate the onset on each behavior; dashed black lines indicate the end of each behavior. Shades: ± SEM. (w) Mean AUC of Z-scored ΔF/F signal of BNSTpr^MPOA cells during the first pup contact. Paired t-test. **p < 0.01. Error bars: SEM. n = 6 mice. (x, y) Mean AUC of Z-scored ΔF/F signal of BNSTpr^MPOA cells during pre-pup period and various pup-directed behaviors in hostile virgin females and mothers. Two-way RM ANOVA followed with multiple comparisons test. **p < 0.01, ****p < 0.0001, mean ± SEM. n = 6 mice. See Source Data Extended Data Fig. 9 for detailed values and statistics.

Extended Data Fig. 10:. Simultaneous recording of MPOA^Esr1 and BNSTpr^Esr1 cells and comparing infanticide and maternal behavior-induced c-Fos between strains.

(a) Fiber photometry setup. (b) Viral construct and targeted brain regions. Brain illustrations are produced based on reference atlas from https://atlas.brain-map.org/. (c, d) Representative histological images of 3 mice showing GCaMP6f (green) expression and fiber tracks (white boxes) in MPOA (c) and BNSTpr (d). (e) Experimental timeline. (f) Representative GCaMP6f recording (ΔF/F) traces of MPOA^Esr1 (black) and BNSTpr^Esr1 (purple) cells during pup interaction in a hostile SW virgin female (f1) and a mother (f2). Color shades indicate various behaviors. (g) PETHs of GCaMP6f signal (Z-score ΔF/F) of MPOA^Esr1 (black) and BNSTpr^Esr1 (purple) cells aligned to the onset of pup approach in hostile virgin females (g1), and mothers (g2). n = 3 mice for each group. Blue dashed lines indicate the onset of pup approach; green dashed lines indicate the mean latency to pup investigation; the red and black dashed lines in g1 indicate the mean latency to attack and stop attacking pups, respectively; the magenta and black dashed lines in g2 indicate the mean latency to retrieve and stop retrieving pups, respectively. Shades: ± SEM. (h) Mean AUC of Z-scored ΔF/F signal of MPOA^Esr1 (black) and BNSTpr^Esr1 (purple) cells during pup approach, investigation, attack, and retrieval in hostile virgin females (h1) and mothers (h2). Two-way RM ANOVA (h1) and Mixed-effects analysis (h2) followed with multiple comparisons test. n = 3 mice for each group, *p < 0.05, ***p < 0.001, mean ± SEM. (i) BNSTpr^Esr1 and MPOA^Esr1 response ratio in hostile virgin females and mothers during pup interaction period. The red dashed line indicates 1. n = 3 mice for each group, Paired t-test. *p < 0.05, mean ± SEM. (j) Representative histological images of 3 mice in each group showing c-Fos expression in MPOA and BNSTpr of a C57BL/6 virgin maternal female (left), a SW virgin maternal female (middle), and a SW infanticidal female (right). (k, l) Number of c-Fos+ cells in MPOA (k) and BNSTpr (l) in C57BL/6 virgin maternal, SW virgin maternal and SW infanticidal female mice. n=3 for each group, ***p < 0.001, ****p < 0.0001. Error bars: ± SEM. One-way ANOVA followed with multiple comparisons test. See Source Data Extended Data Fig. 10 for detailed values and statistics.

Fig. 1:. Functional screening of brain regions relevant for negative pup-directed behaviors in females

(a) Experimental design to identify infanticide-activated MPOA-connecting cells in female mice. (b) The density of c-Fos expressing cells in MPOA-connecting brain regions in female mice after infanticide or left undisturbed (control). n = 3 mice/group. (c) Total number of Fos+Tracer+ and Fos-Tracer+ cells per 100 Tracer+ cells in MPOA-connecting brain regions across all infanticide and control females. (d) Experimental design to chemogenetically activate MPOA-connecting brain regions. (e) Experimental timeline. (f) Representative histology images showing hM3Dq-mCherry expression (red) in BNSTpr^MPOA cells. Blue: DAPI. (g) Representative raster plots showing pup-directed behaviors after saline or CNO injection in mCherry and hM3Dq mice. (h) The number of female mice that attack or not after saline or CNO injection in mCherry and hM3Dq mice. (i-k) Investigating pup duration (i), grooming pup duration (j), and latency to attack pups (k) after saline or CNO injection in mCherry and hM3Dq mice. Each line represents one animal. If no behavior of interest is observed during the test, the latency is 600 s. (l-q) Chemogenetic activation of MeApd^MPOA cells increases infanticide and pup grooming. Figure conventions as in f-k. All error bars: SEM. (c) Fisher’s exact test based on raw Fos+Tracer+ and Fos-Tracer+ cell numbers for each brain region. (h, n) McNemar’s test for saline vs. CNO comparison. Fisher’s exact test for mCherry vs. hM3Dq compraison. Two-way RM ANOVA (b, I, o, p), or Mixed-effects analysis (j, k, q) followed with multiple comparisons test. All tests are two-sided. **p < 0.01; ***p < 0.001; ****p < 0.0001. See Source Data Fig. 1 for detailed values and statistics for all plots.

Fig. 2:. BNSTpr^Esr1 neurons are sufficient and necessary for female infanticide.

(a) Experimental design. (b) Representative histology showing ChR2-EYFP expression. White lines: fiber tracks. (c) Experimental timeline. (d, f, h) Raster plots showing pup-directed behaviors during sham and light stimulation. # Replace a pup. (e, g, i) Post-event histograms (PETHs) of attack pup probability aligned to sham and light onset. Only trials with female-pup contact were included. Dashed lines mark light period. (j) Percentage of animals that attacked pups. (k) Percentage of trials showing pup attack. Only trials with female-pup contact were included. (l) Average latency to attack pup upon pup encounter following sham or light stimulation. (m) Experimental design to inhibit BNSTpr^Esr1 cells. Brain illustration is based on reference atlas from https://atlas.brain-map.org/. (n) Representative histology showing hM4Di-mCherry expression. (o) Experimental timeline. (p) Raster plots showing pup-directed behaviors after CNO or saline injection. *Remove wounded pups and stop recording. (q) Percentage of spontaneously infanticidal virgin females that attack, ignore or retrieve pups after saline or CNO injection. (r, s) Latency to attack (r) and retrieve pups (s) after saline or CNO injection. Latency equals to 600 s if the behavior doesn’t occur during the test. (k, l, r, s) Each dot or line represents one animal. Shades in (e, g, i) and error bars in (k, l, r, s): ±SEM. (j, q) Fisher’s exact test for between animal comparisons; McNemar’s test for within animal comparisons. (k, l, r, s) Mixed-effects analysis followed with multiple comparisons test. All tests are two-sided. *p< 0.05, **p<0.01, ***p < 0.001. ****p < 0.0001. (e, g, i, j-l) n = 8 (GFP), 8 (ChR2 virgin) and 6 (ChR2 mother) mice. (q-s) n = 8 (mCherry) and 9 (hM4Di) mice. See Source Data Fig. 2 for detailed values and statistics.

Fig. 3:. Mutual inhibition between BNSTpr^Esr1 and MPOA^Esr1 cells

(a) Anterograde tracing of BNSTpr^Esr1 cells. (b) Representative GFP expression in BNSTpr^Esr1 cells. (c) Representative GFP-expressing BNSTpr^Esr1 terminals (green) in the MPOA and Esr1 staining (magenta). (d) Representative GFP-expressing BNSTpr^Esr1 terminals in various regions. (e) The brain-wide projection density of BNSTpr^Esr1 cells, normalized by BNSTpr fluorescence intensity. n = 4 mice. (f-j) MPOA^Esr1 cell projection patterns. n = 4 mice. Figure conventions as in a-e. (k, s) Schematics of ChR2-assisted circuit mapping of BNSTpr^Esr1→MPOA^Esr1 (k) and MPOA^Esr1→BNSTpr^Esr1 (s) pathways and representative histological images. Brain atlas are reproduced based on reference atlas from atlas.brain.org. (l, t) The synaptic response patterns of MPOA^Esr1 cells to BNSTpr^Esr1 terminal activation (l, 22 cells/3 mice) and BNSTpr^Esr1 cells to MPOA^Esr1 terminal activation (t, 35 cells/3 mice). (m, u) Representative oIPSCs from MPOA^Esr1 (m) and BNSTpr^Esr1 cells (u) with different blockers. (n, v) oIPSC amplitude with different blockers. Mixed-effects analysis followed with multiple comparisons test. ***p < 0.001, ****p < 0.0001. n = 16 cells/4 mice (n); 13 cells/5 mice (v). (o, w) oIPSC amplitude of MPOA^Esr1 (o) and BNSTpr^Esr1 cells (w) after applying TTX, 4-AP, and gabazine mixture. n = 36 cells/8 mice (o) and 23 cells/9 mice (w). (p, x) The number of MPOA^Esr1 (p) and BNSTpr^Esr1 cells (x) having a residual oIPSC >50 or <50 pA after applying TTX, 4-AP, and gabazine mixture. (q, y) Representative oIPSCs of MPOA^Esr1 (q) and BNSTpr^Esr1 cells (y) before and after applying strychnine, a glycine receptor antagonist. (r, z) Amplitude of oIPSCs of MPOA^Esr1 (r) and BNSTpr^Esr1 cells (z) before and after applying strychnine. Two-sided paired signed rank test, *p<0.05. n = 8 cells/4 mice (r); 9 cells/4 mice (z). All error bars: SEM. See Source Data Fig. 3 for detailed values and statistics.

Fig. 4:. BNSTpr^Esr1 and MPOA^Esr1 cells antagonize each other functionally through their reciprocal projections.

(a) Experimental design to optogenetically inactivate MPOA^Esr1→BNSTpr pathway. (b) Raster plots showing pup-directed behaviors in mCherry and ArchT females during sham or 5 mW continuous yellow light delivery. *Remove wounded pups and stop recording. (c) Number of mCherry and ArchT females that attack, ignore or retrieve pups during sham or light stimulation. (d, e) Latency to attack (d) and investigate pup (e) during sham and light delivery in mCherry and ArchT females. The latency equals to 600s if the behavior of interest doesn’t occur during the test. n = 6 mice/group. (f) Experimental design to optogenetically activate BNSTpr^Esr1→MPOA pathway with or without BNSTpr^Esr1 chemogenetic inhibition. (g) Raster plots showing pup-directed behaviors in various groups during sham or light delivery. *Remove wounded pups and stop recording. (h) Number of females in each group that attack, ignore, or retrieve pups during sham or light delivery. (i, j) Latency to attack (i) and investigate pup (j) during sham or light delivery in control and test females. n = 7 mice/group. (k-o) Optogenetic inactivation of BNSTpr^Esr1→MPOA pathway in spontaneously infanticidal virgin SW females suppresses infanticide. Figure conventions as in a-e. n = 6 mice/group. (p-t) Optogenetic activation of BNSTpr^Esr1→MPOA pathway suppresses maternal behaviors. Figure conventions as in f-j. n = 6 (mCherry), 5(ChrimsonR), and 5(ChR2+Gi) mice. All error bars: ± SEM. (c, h) Fisher’s exact test for comparing behavior outcomes (attack vs. not attack) between control and test groups with the same light treatment. McNemar’s test for comparison between light and sham trials within a group. (d, e, i, j) Mixed-effects analysis followed with multiple comparisons test. All tests are two-sided. *p < 0.05; **p < 0.01; ****p < 0.0001. See Source Data Fig. 4 for detailed values and statistics.

Fig. 5:. Distinct responses of BNSTpr^Esr1 and MPOA^Esr1 cells during female infanticide and maternal care.

(a) Fiber photometry setup. (b, d) Viral construct and targeted brain regions. Brain illustration is produced based on reference atlas from https://atlas.brain-map.org/. (c) A representative image of 4 mice showing the fiber track in BNSTpr (white line), the overlap between Esr1 staining (red) and GCaMP6f (green). Right shows the enlarged view of the boxed area. Bar graph showing the percentage of GCaMP6f cells expressing Esr1. n = 4 recording mice. (e) Representative GCaMP6f expression (green) and fiber track (white line) in the MPOA of 4 mice. (f, g) Experimental timelines. (h) Representative ΔF/F traces of BNSTpr^Esr1 cells during pup interaction in a hostile virgin female (h1) and a mother (h2). (i-l) PETHs of Z-scored ΔF/F of BNSTpr^Esr1 cells aligned to the onset of various behaviors. (m) Average area under the curve (AUC) of Z-scored ΔF/F during the first pup contact. (n-o) Mean AUC of Z-scored ΔF/F during various pup-directed behaviors in different groups. (p) Representative ΔF/F traces of MPOA^Esr1 cells during pup interaction. (q-t) PETHs of Z-scored ΔF/F of MPOA^Esr1 cells aligned to the onset of various pup-directed behaviors. (u) Mean AUC of Z-scored ΔF/F during the first pup contact. (v-w) Mean AUC of Z-scored ΔF/F during various pup-directed behaviors in different groups. All error bars and shades of PETHs: SEM. Solid and dashed lines in PETH plots indicate the onset and offset of behaviors, respectively. (m) Two-sided paired t-test. (u) RM one-way ANOVA with multiple comparisons test. (n, o) Two-way RM ANOVA with multiple comparisons test. (v, w) Mixed-effects analysis with multiple comparisons test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. n = 7 (i-o) and 5 (q-w) mice/group. See Source Data Fig. 5 for detailed values and statistics.

Fig. 6:. BNSTpr^Esr1 and MPOA^Esr1 cell excitability varies with females’ reproductive state and genetic background

(a) Representative recording traces of MPOA^Esr1 cells. (b) F-I curves of MPOA^Esr1 cells from hostile virgin (red, 25 cells/3 mice), maternal virgin (green, 24 cells/3 mice), and lactating (blue, 26 cells/3 mice) SW females. (c) Representative recording traces of BNSTpr^Esr1 cells. (d) F-I curves of BNSTpr^Esr1 cells from SW virgin females (orange, 57 cells/6 mice) and mothers (blue, 51 cells/9 mice). Inset: F-I curves of BNSTpr^Esr1 cells from hostile (red, 36 cells/3 mice) and maternal virgin SW females (green, 21 cells/3 mice). (e) Maximum AP number of MPOA^Esr1 and BNSTpr^Esr1 cells from hostile virgin (red, MPOA: 25 cells/3 mice; BNSTpr: 36 cells/3 mice), maternal virgin (green, MPOA: 24 cells/3 mice; BNSTpr: 21 cells/3 mice), and lactating (blue, MPOA: 24 cells/3 mice; BNSTpr: 51 cells/9 mice) SW females with maximally 250 pA injected current. (f) Representative traces showing spiking patterns of type I and II BNSTpr^Esr1 cells. (g) The number of type I and II BNSTpr^Esr1 cells in C57BL/6 (3 mice) and SW (6 mice) virgin females. (h) Representative recording traces of type II BNSTpr^Esr1 cells from C57BL/6 and SW virgin females and mothers. (i) F-I curves of BNSTpr^Esr1 cells recorded from C57BL/6 (virgin: 43 cells/3 mice; mother: 31 cells/3 mice) and SW (virgin: 57 cells/6 mice; mother: 51 cells/9 mice) females. (j) A cartoon summary of the antagonism between the infanticide and maternal circuits and theirs changes with reproductive states. All error bars: ± SEM. (b, d, i) Mixed-effects analysis with multiple comparisons test. (e) Mann Whitney test (Hostile virgin and Mother) or Unpaired t test (Maternal virgin). (g) Fisher’s exact test. All tests are two-sided. **p < 0.01, ***p < 0.001, ****p < 0.0001. See Source Data Fig. 6 for detailed values and statistics.