Esr1+ cells in the ventromedial hypothalamus control female aggression

Abstract

As an essential means of resolving conflicts, aggression is expressed by both sexes but often at a higher level in males than in females. Recent studies suggest that cells in the ventrolateral part of the ventromedial hypothalamus (VMHvl) that express estrogen receptor-α (Esr1) and progesterone receptor are essential for male but not female mouse aggression. In contrast, here we show that VMHvl^Esr1+ cells are indispensable for female aggression. This population was active when females attacked naturally. Inactivation of these cells reduced female aggression whereas their activation elicited attack. Additionally, we found that female VMHvl contains two anatomically distinguishable subdivisions that showed differential gene expression, projection and activation patterns after mating and fighting. These results support an essential role of the VMHvl in both male and female aggression and reveal the existence of two previously unappreciated subdivisions in the female VMHvl that are involved in distinct social behaviors.

Figure 1. Esr1⁺ neurons in the VMHvl of female mice are preferentially activated during fighting and mating

(a–d) left: Representative images showing the expression of Esr1 (red) and c-Fos (green) in the VMHvl of (a) a virgin or (b) a lactating female mouse that attacked a juvenile male mouse, (c) a lactating female mouse that attacked an adult male mouse and (d) a virgin female mouse that mated with an adult male. Insets show the boxed areas. Scale bars: 150 μm and 20 μm (insets). Middle: The percentage of neurons in the VMHvl that expressed c-Fos after various stimulus conditions. Unpaired t-test. Right: The percentage of Esr1⁺ neurons in all VMHvl neurons (gray) and the percentage of c-Fos⁺ neurons expressing Esr1 in the VMHvl after fighting (red) or mating (blue). Paired t-test. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001. Data are presented as means ± s.e.m. The N for each group is indicated on the bar.. [AU Query: OK?] See Supplementary Table 2 for detailed statistics. See also Supplementary Figure 1.

Figure 2. Responses of Esr1⁺ population in the female VMHvl during fighting and mating

(a) Light paths of the fiber photometry recording setup. (b) Viral constructs and implantation scheme. (c) Representative images showing mCherry (red), GCaMP6f (green), Nissl (blue) and optical fiber tract (yellow dashed line). Scale bar: 300 μm. (d–i) (left): Representative normalized GCaMP6f (black) and mCherry (red) traces during interaction with (d) an object and (e–i) various social stimuli introduced into the home cage of the test female. Colored shades mark behavioral episodes. Green: investigation; Blue: mounted; Red: attack, Yellow: pup retrieval. (d) (right): The velocity of the recorded animal did not correlate with GCaMP6f signal. Pearson product-moment correlation. Inset in (d) and right panels in (e–i) showing representative PETHs of GCaMP6f signal aligned to the onset of various behaviors. (j–k) The peak ΔF/F of GCaMP6f (black) and mCherry (red) signals during various behaviors in virgin (j) and lactating (k) females. (l–n) Population PETHs (left) and the peak ΔF/F (right) of GCaMP6f signals aligned to the onset (l, n) or offset (m) of various behaviors. j, k, l, m and n: paired t-test. ^*p < 0.05, ^**p < 0.01, ^***p < 0.01. Data are presented as means ± s.e.m.. [AU Query: OK?] See Supplementary Table 2 for detailed statistics. See also Supplementary Figure 3–5.

Figure 3. The activity of Esr1⁺ neurons in the VMHvl is necessary for female aggression

(a) Viral constructs and a representative histological image showing the expression of hM4Di-mCherry (red) at the injection sites. Scale bar: 300 μm. (b) Experimental schedule. (c) In vitro whole-cell patch clamp recording from a representative Esr1⁺ neuron in the VMHvl (Left, scale bar: 10 μm). Right shows changes in the cell’s spontaneous spiking activity after 10 μM CNO application. Horizontal and vertical scale bars: 30 s and 20 mV. The result was replicated in two hM4Di-mCherry expressing cells. (d) Behavioral results of representative test and control animals. Scale bar: 60 s. (e) Reduction in aggression was reproduced in multiple CNO injected days. (f, g) Attack durations towards (f) juvenile male and (g) adult male intruders were significantly decreased after CNO injection in hM4Di group but not in control group. (h–j) No significant change was observed in (h) duration of investigation of a juvenile male intruder, (i) the time spent to retrieve four scattered pups and (j) the average movement velocity in the hM4Di group after CNO injection in comparison to saline injection. e–j only include animals with over 10% of infected cells in the VMHvl. (k) The percentage of cells that were infected in the VMHvl was significantly correlated with the amount of decrease in attack duration towards a juvenile male intruder after CNO injection. k includes all animals with attack duration over 20 s on saline days regardless of the percentage of infected cells. e and h: Two-way repeated measure ANOVA followed by Holm-Sidak post-hoc multiple comparisons; f, g, i and j: Paired t-test; k: Pearson product-moment correlation. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001. Data are presented as means ± s.e.m.. [AU Query: OK?] See Supplementary Table 2 for detailed statistics. See also Supplementary Figure 6.

Figure 4. Activation of the Esr1⁺ neurons in the VMHvl is sufficient to induce attack in virgin female mice

(a) Viral constructs and a histological picture showing the ChR2-EYFP expression (green) at the injection sites. Scale bar: 300 μm. (b) Images showing the expressions of ChR2-EYFP (green), Esr1 (red), c-Fos (yellow), Nissl (blue) and their overlaps. Scale bar: 20 μm. (c) The percentage of all VMHvl neurons expressing Esr1 (black) and the percentage of ChR2-EYFP⁺ neurons expressing Esr1 (green). (d) Experimental schedule. (e) The percentage of VMHvl cells expressing light-induced c-Fos in the anterior vs. posterior VMHvl of all tested sites with over 10% of light induced c-Fos. (f) Representative raster plots illustrating progressive behavioral changes towards a female intruder with increased light intensity. (g, h) Increase in (g) duration of investigation and (h) duration of attack towards an adult female or male intruder during light-on period in comparison to sham-on period in animals with preferential posterior VMHvl activation. (i) The minimal light intensity that is required to elicit close investigation is lower than that to elicit attack against either a male or a female intruder. (j) The duration of investigation (left) and attacks (right) did not differ during light-on and sham-on periods in the control group. g, h and j, paired t-test; i, Two-way repeated measure ANOVA followed by Holm-Sidak post-hoc multiple comparisons; ^*p < 0.05, ^**p < 0.01, ^***p < 0.001. Data are presented as means ± s.e.m.. [AU Query: OK?] See Supplementary Table 2 for detailed statistics. See also Supplementary Figure 7–10.

Figure 5. Electrophysiological responses of female VMHvl neurons during encounters with adult male and juvenile intruders

(a) Raster plots (top) and PETHs (bottom) aligned to the onsets of various behavioral episodes for a juvenile-excited cell. (b) Average PETHs of the firing rate from 18 juvenile-excited cells. (c) Firing rates of juvenile-excited cells before juvenile introduction (base) and when the animal investigated, attacked or stayed away from (no contact) the juvenile. b and c only show juvenile-excited cells with attacking juvenile episode. (d) Example and (e–f) population responses of adult male-excited cells. Figure conventions as in (a–c). d–f only show male-excited cells with being mounted episodes. (g, h) Distributions of the firing rate changes (g) during attack and being mounted or (h) during investigating juvenile and investigating an adult male across all the recorded cells with both behaviors. (i) Left, a representative histological image showing the electrode tract. Scale bar: 300 μm. Right, the correlation between mediolateral distance of the electrode and the percentage of recorded male-excited cells among all male-excited and juvenile-excited cells. c, f: One-way ANOVA followed by Holm-Sidak post-hoc multiple comparisons; g, h, i: Pearson product-moment correlation; ^*p < 0.05, ^**p < 0.01, ^***p < 0.001. Data are presented as means ± s.e.m.. [AU Query: OK?] See Supplementary Table 2 for detailed statistics. See also Supplementary Figure 11.

Figure 6. Topographical organization of fighting- and mating-related cells in the female VMHvl

(a) Cytoplasmic + nuclear (red) and nuclear only (green) c-fos induced by two sequential behavioral episodes. Insets show the enlarged images of the boxed areas. Blue: Hoechst. Scale bars: 150 μm and 20 μm (insets). (b) Percentage of neurons expressing nuclear c-fos that also express cytoplasmic c-fos. One-way ANOVA followed by Tukey’s multiple comparisons. **p < 0.01, ***p < 0.001. Data are presented as means ± s.e.m.. [AU Query: OK?] See Supplementary Table 2 for detailed statistics. See also Supplementary Figure 12.

Figure 7. Female VMHvl has anatomically and molecularly distinct subdivisions

(a) Nissl staining illustrating the boundary (yellow arrows) between female VMHpvlm and VMHpvll. Scale bars (left and right): 300 and 150 μm. (b) Overlay of Esr1 (red) and ZsGreen (green) in the VMHvl of a female Vglut2-ires-Cre × Ai6 mouse (left) and a female Vgat-ires-Cre × Ai6 mouse (right). Scale bar: 300 μm. (c) The percentage of Esr1⁺ (red), Vglut2⁺ (green, left) and Vgat⁺ (green, right) cells in the VMHpvll and VMHpvlm and the percentage of Esr1⁺ cells that overlap with Vglut2⁺ or Vgat⁺ cells (yellow). (N = 3 animals for each group). (d) Object investigation, fighting or mating induced c-Fos (green) in the VMHvl in virgin female mice. Scale bar = 150 μm. (e) Average number of c-Fos⁺ cells/section in the VMHpvlm and VMHpvll following object investigation (black), fighting (red) or mating (blue) in female mice. N = 3–7. paired t-test. *p < 0.05, **p < 0.01, ***p < 0.001. (f) Experimental scheme. (g) A brain section from a Vglut2-ires-Cre × Ai6 mouse after microdissection of VMHdm, VMHpvll and VMHpvlm. Scale bar: 300 μm. (h) RNAseq results of all samples from various VMHvl subregions mapped onto the principal component (PC) space. (i) Average normalized counts of each gene in the VMHpvlm (x-axis) and VMHpvll (y-axis). Red and blue dots represent genes with significantly biased expression (Benjamini and Hochberg method, p< 0.05, >1.2×, log2 scale). (j) In situs of 5 genes at the VMHvl that are indicated in (i). Scale bars (top and bottom): 300 and 150 μm. Data are presented as means ± s.e.m.. [AU Query: OK?] See Supplementary Table 2 for detailed statistics. See also Supplementary Figure 13.

Figure 8. VMHpvlm and VMHpvll in female mice have distinct downstream targets

(a, b) Experimental scheme for anterograde tracing. (c) The two projection sites of the VMHvl (red boxes) that are shown in (d). Images from Allen Brain Atlas. (d) Projection patterns in the AVPV and PAG in animals with primary infection in the whole VMHvl (left), VMHpvlm (middle) and VMHpvll (right). Scale bars (top to bottom): 150, 300 and 300 μm. Top bar graphs show the distributions of infected neurons in the VMHvl along the medial-lateral axis. (e) Experimental scheme of retrograde tracing. (f) Percentage of neurons in the VMHplvm or VMHpvll that were retrogradely labeled from AVPV (left) or PAG (right). Paired t-test. *p < 0.05. (g, h) Left large image shows neurons in the VMHvl that are retrogradely labeled (red) from (g) AVPV or (h) PAG. Insets show the injection sites. Scale bars: 150 μm and 300 μm (insets); Right images show the overlap between the CTB labeling (red) and Esr1 (green). Scale bar: 20 μm. (i) c-fos mRNA expression in the AVPV or PAG after various testing conditions. Scale bars: 300 μm. (j) Average number of c-fos expressing cells per section in the AVPV (left) and PAG (right) after various testing conditions. One-way ANOVA followed by Tukey’s multiple comparisons. ***p < 0.001. Data are presented as means ± s.e.m. [AU Query: OK?] See Supplementary Table 2 for detailed statistics. See also Supplementary Figure 14.