Volume 26, Issue 9, Pages 1229-1234 (May 2016) Self-Exposure to the Male Pheromone ESP1 Enhances Male Aggressiveness in Mice  Tatsuya Hattori, Takuya Osakada, Ayaka Matsumoto, Naoki Matsuo, Sachiko Haga-Yamanaka, Takaya Nishida, Yuji Mori, Kazutaka Mogi, Kazushige Touhara, Takefumi Kikusui  Current Biology  Volume 26, Issue 9, Pages 1229-1234 (May 2016) DOI: 10.1016/j.cub.2016.03.029 Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 1 ESP1 in Conjunction with Unfamiliar Urine Enhances Male Aggression via the Vomeronasal Receptor V2Rp5 (A) Attack-bite frequency of ICR resident mice to various strains of intruders in the aggression test. ICR residents (n = 8) were repeatedly confronted with the following intruder mice: C57BL/6 (B6), A/J, BALB/c (BALB), BALB mice that had been castrated at 3 (3w Cast. BALB; 12–14 weeks) or 7 weeks of age (7w Cast. BALB; 12–15 weeks). The statistical significance was obtained by repeated-measures ANOVA with Bonferroni post hoc corrections, stimuli (F[4, 28] = 15.27, p < 0.01). ∗∗p < 0.01. No significant differences in the other behavioral parameters were observed (data not shown). (B) Attack-bite frequency of ICR resident mice pre-exposed to various stimuli when confronted with 3w-cast. BALB or 7w-cast. BALB intruders on the aggression test. ICR residents (n = 13) were exposed to various stimuli prior to encountering a 3w Cast. BALB (left panel) or 7w Cast. BALB intruder (right panel). The statistical significance was obtained by repeated-measures ANOVA with Bonferroni post hoc comparisons 3w Cast, F [6, 72] = 4.31, ∗∗p < 0.01: 7w Cast, F[1, 19] = 22.33, ∗∗p < 0.01. There were no significant differences in the other behavioral parameters (data not shown). See also Movie S1. (C) Attack-bite frequency of V2Rp5 gene-targeted B6 mice when confronted with 3w-castrated BALB intruders on the aggression test. V2Rp5 gene-targeted B6 mice (−/−; V2Rp5 null, +/−; V2Rp5 heterozygote, +/+; V2Rp5 wild-type) were pre-exposed to ESP1 or Tris-HCl control with or without intact BALB urine and then confronted with 3w Cast. BALB intruders V2Rp5 +/+ (n = 18), V2Rp5 +/− (n = 16), V2Rp5 −/− (n = 11). There were no significant differences in the other behavioral parameters (data not shown). The statistical significance was obtained by two-way repeated-measures ANOVA with Bonferroni post hoc corrections: genotype (F[2, 42] = 27.53, p < 0.01), pre-stimuli (F[3, 126] = 17.25, p < 0.01), genotype × stimulus interaction (F[6, 126] = 22.25, p < 0.01). Differences between each column: a versus b versus c, p < 0.05. Data are represented as mean ± SEM. In Figures 1A–1C, ESP1 or ESP1-secreting mice were shaded in boxes. Current Biology 2016 26, 1229-1234DOI: (10.1016/j.cub.2016.03.029) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 2 Self-Secreted ESP1 Increases Aggressiveness in an Auto-stimulatory Way (A) Attack-bite frequencies of B6 or BALB residents, which were pre-exposed to ESP1 or Tris-HCl control with or without intact BALB or B6 urine, when confronted with 3w-cast. BALB intruders in the aggression test (left) B6, n = 12; BALB, n = 12. The statistical significance was obtained by two-way repeated-measures ANOVA with Bonferroni post hoc corrections: resident strain (F[1, 22] = 0.01, p = 0.98), urine stimuli (F[2, 44] = 5.18, p < 0.05), resident strain × urine stimuli interaction (F[2, 44] = 39.37, p < 0.01). ∗p < 0.05. ∗∗p < 0.01. There was no significant difference in the other behavioral parameters (data not shown). (right) B6, n = 12; BALB, n = 12. The statistical significance was obtained by two-way repeated-measures ANOVA with Bonferroni post hoc corrections: resident strain (F[1, 22] = 0.815, p = 0.38), urine stimuli (F[2, 44] = 28.80, p < 0.01), resident strain × urine stimuli interaction, (F[2, 44] = 5.17, p < 0.05.) ∗p < 0.05. ∗∗p < 0.01. There was no significant difference in the other behavioral parameters (data not shown). See also Figures S1B and S2B. (B) Attack-bite frequencies of V2Rp5 gene-targeted BALB mice to 3w Cast. BALB intruders in the aggression test. V2Rp5 gene-targeted BALB mice self-secreting ESP1 were pre-exposed to ESP1 or Tris-HCl control with or without intact B6 urine and encountered 3w Cast. BALB intruders. V2Rp5 +/+ (n = 8), V2Rp5 +/− (n = 10), V2Rp5 −/− (n = 10). The statistical significance was obtained by two-way repeated-measures ANOVA with Bonferroni post hoc corrections: genotype (F[2, 25] = 8.15, p < 0.01), pheromone stimuli (F[3, 125] = 29.56, p < 0.01), genotype × stimulus interaction (F[6, 125] = 5.29, p < 0.01). There were no significant differences in the other behavioral parameters (data not shown). Data are represented as mean ± SEM. Differences between each column: x versus y versus z, p < 0.01. In (A) and (B), ESP1 or ESP1-secreting mice were shaded in boxes. See also Figure S3. Current Biology 2016 26, 1229-1234DOI: (10.1016/j.cub.2016.03.029) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 3 Reactivation of ESP1-Induced c-Fos-Positive Neurons by CNO in hM3Dqfos Males Induces Male Aggression (A) Images of c-Fos immunohistochemistry in BNST and MPA regions after injections of CNO or saline in ESP1-prestimulated hM3Dqfos male mice. Increases in c-Fos-positive neurons in BNST and MPA were observed in CNO-injected hM3Dqfos males that had been pre-exposed to ESP1 2 days prior to the injection. Enlarged views of dashed square areas are shown below each picture. Scale bars, 250 μm. (B and C) Quantification of c-Fos- immunoreactive (-ir) neurons in BNST (B) and MPA (C) in CNO- or saline-injected hM3Dqfos male mice. CNO-injected group, n = 8; saline-injected group, n = 8. In BNST, t(13.9) = 9.579, ∗∗p < 0.01, in MPA, t(11.9) = 16.177, ∗∗p < 0.01. (D) Attack-bite frequencies of CNO- or saline-injected ESP1-stimulated hM3Dqfos male mice on the aggression test. The expression of the hM3Dq receptor was induced by ESP1 prior to the behavior tests. hM3Dqfos double-transgenic male group (FT/M3, n = 12) and other genotype groups (FT/−, n = 10; −/M3, n = 10; −/−, n = 10). The statistical significance was obtained by two-way repeated-measures ANOVA with Bonferroni post hoc corrections: genotype (F[3, 38] = 20.85, p < 0.01), injections (F[1, 38] = 29.56, p < 0.01), genotype × injection interaction (F[3, 38] = 53.67, p < 0.01). There were no significant differences in the other behavioral parameters (data not shown). Differences between each column: ∗∗p < 0.01. Data are represented as mean ± SEM. See also Figure S4 and Movie S2. Current Biology 2016 26, 1229-1234DOI: (10.1016/j.cub.2016.03.029) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 4 Reactivation of ESP1-Induced c-Fos-Positive Neurons by CNO in hM3Dqfos Females Enhances Lordosis Behavior (A) c-Fos immunohistochemistry in VMH and PMCo regions after injections of CNO or saline in ESP1-prestimulated hM3Dqfos female mice. Increases in c-Fos-positive neurons in VMH and PMCo were observed in CNO-injected hM3Dqfos females that had been pre-exposed to ESP1 2 days prior to the injection. Enlarged views of dashed square areas are shown below each picture. Scale bar, 250 μm. (B and C) Quantification of c-Fos-immunoreactive (-ir) neurons in the VMH (B) and PMCo (C) of CNO- or saline-injected hM3Dqfos female mice. CNO-injected group, n = 10; saline-injected group, n = 9. In VMH; t(14.9) = 2.68, ∗p < 0.05, in PMCo; t(9.4) = 3.939, ∗∗p < 0.01. (D) Lordosis quotient in sexual behavior of CNO- or saline-injected ESP1-stimulated hM3Dqfos female mice. Expression of the hM3Dq receptor was induced by ESP1 prior to behavior tests. CNO-injected hM3Dqfos double-transgenic female group (FT/ M3; n = 8) and other groups (CNO-injected group FT/−, n = 6; −/M3, n = 6; −/−, n = 5; saline-injected group FT/M3, n = 8; FT/−, n = 6; −/M3, n = 5; −/−, n = 5). The statistical significance was obtained by two-way repeated-measures ANOVA with Bonferroni post hoc corrections: genotype (F[3, 39] = 0.51, p = 0.70) injections (F[1, 39] = 0.57, p = 0.50), genotype × injected agonist interaction (F[3, 39] = 65.61, p < 0.01). There was no significant difference in the other behavioral parameters (data not shown). Differences between each column: ∗p < 0.05, ∗∗p < 0.01. Data are represented as mean ± SEM. See also Movie S3. Current Biology 2016 26, 1229-1234DOI: (10.1016/j.cub.2016.03.029) Copyright © 2016 Elsevier Ltd Terms and Conditions