No of Pages 9 06 September 2019 ARTICLE IN PRESS Cheng Wang et al. / Neuroscience 418(2019)XXx-XXX acquisition settings were maintained for all subsequent ima MaeB□ Female ging of C-Fos o Sham For quantification of c-Fos expression, three slices were ■ Lesion ounted for ventral hippocampal CAl (AP -3.3 mm 3. 4 mm, and-3.5 mm relative to Bregma), basolateral amygdala(AP-1.5 mm, -1.6 mm, and-1.7 mm relative to Bregma) and prelimbic cortex(AP 1.9 mm, 1.8 mm, and 1.5 mm relative to Bregma), respectively. c-Fos expression was quantified for each of these brain regions by ImageJ (Version 1.52, National Institutes of Health, USA). The num- bers of c-Fos-positive neurons in left and right hemispheres LEP was pooled since no significant differences were observed (data not shown) 150 Statistical analysis All data were analyzed and plotted using the GraphPad Prism 6 software Data were expressed as mean t SEM(standard error of the mean). Males and females were analyzed sepa rately. The assumption of normality and equality of variance vas tested by Shapiro-Wilk test and F-test, respectively. All E6月088 data met the normal distribution and had equal variances in each group. Unpaired Student's t test was perfomed in males Two-way ANOVA with Bonferroni post hoc test was performed in females, with lesion and estrus cycle as the two factors Fig 3. Ventral hippocampal lesioning relieves anxiety-like behavior in Probability values of P <.05 were considered to represent sig the novelty-suppressed feeding test in male but not female mice. Bilat- nificant differences eral ventral hippocampal lesioning decreased feeding latency in the novelty-suppressed feeding test in male(A)but not female( B)mice despite significant influences from estrogen levels in females. Similar amount of food was consumed in two groups of male(C)and femal RESULTS (D)mice. "P<.05, "P<.01. "P<.001, unpaired t test or two-way ANOVA with Bonferronis test n 15/group for males, n 8/group for Reduced anxiety in male, but not female, mice females in HEP and LEP, respectively. HEP: high-estradiol phase. with ventral hippocampal lesions LEP: low-estradiol phase. To examine biological sex differences in hippocampal mod- ulation of anxiety-like behavior, we first performed excito- consumption were observed in either biological sex [male toxic lesions of bilateral ventral hippocampus by local t(28)=0.30, P=765, unpaired t test, Fig 3C; female injection of ibotenic acid. Fig. 2A and Fig. 2B showed sche- estrus cycle effect: F(1, 28)=0.3 33. P=. 568: lesion eff matic illustration of damages from male and female mice F(, 28)=0.05, P= 818; interaction- (r1, 28). g respectively. The lesion extended throughout bilateral ven- 899, two-way ANOVA with Bonferroni post hoc test tral hippocampus and included all cell fields, with minimal Fig. 3D], indicating similar levels of hunger and motivation extrahippocampal damages. 2 male and 4 female mice The same conclusion could be reached after food consump were excluded from analysis due to incorrect lesion site tion was normalized to body weight [male: t(28)=0.52, P resulting in 30 male and 32 female mice in final statistical analysis. Male and female mice had an average of 56% 0 25, P= 623: lesion effect: F(1. 28)=0.46, P=505 (40%-77%)and 60%(37%0-70%)bilateral tissue loss 607, unpaired t test; female: estrus cycle((. 22.5 action:F(, 28)=1.01, P=.323, two-way ANOVA with Bon- espectively. No significant differences of lesion size were ferroni post hoc test observed between males and females [t(6o)=1.07, P In the marble burying test, bilateral ventral hippocampal 291, unpaired t test]. excitotoxic lesion resulted in fewer buried marbles in male In the novelty-suppressed feeding test, bilateral ventral mice[ 28)=2.59, P=016, unpaired t test, Fig 4A], indicat hippocampal excitotoxic lesioning shortened the feeding ing decreased anxiety level. Again, we failed to observe latency in male mice [t(28)=2.21, P=.036, unpaired t test, such effects in female mice, despite a significant effect of Fig 3A], indicating reduced anxiety. By sharp contrast, in the estrus cycle [estrus cycle effect female mice, the same lesion did not produce anxiolytic P<.001: lesion effect: F( 28)=0.22, P=642; interaction: effects in this test [lesion effect: F( 28)=0.23, P=64 F(, 28)=2.55, P= 121, two-way ANOVA with Bonferroni interaction: F(1, 28)=0.38,P=541, Fig 3B], despite a sig post hoc test, Fig. 4B]. nificant effect of the estrus cycle [F(1. 28)=597.00 In the elevated plus-maze test, male mice spent more P<. 001, two-way ANOVA with Bonferroni post hoc test] time [t( 28)=0. 29, P=023, Fig. 5A] and exhibited more as previously reported(Mora et al., 1996: Seeman, 1997; entries into the open arms[t( 28)=2.53, P=018, unpaired Gangitano et al., 2009). No significant differences in food t test, Fig. 5C] after ventral hippocampal excitotoxic lesionsacquisition settings were maintained for all subsequent ima￾ging of c-Fos. For quantification of c-Fos expression, three slices were counted for ventral hippocampal CA1 (AP -3.3 mm, −3.4 mm, and − 3.5 mm relative to Bregma), basolateral amygdala (AP -1.5 mm, −1.6 mm, and − 1.7 mm relative to Bregma) and prelimbic cortex (AP 1.9 mm, 1.8 mm, and 1.5 mm relative to Bregma), respectively. c-Fos expression was quantified for each of these brain regions by ImageJ (Version 1.52, National Institutes of Health, USA). The num￾bers of c-Fos-positive neurons in left and right hemispheres was pooled since no significant differences were observed (data not shown). Statistical analysis All data were analyzed and plotted using the GraphPad Prism 6 software. Data were expressed as mean ± SEM (standard error of the mean). Males and females were analyzed sepa￾rately. The assumption of normality and equality of variance was tested by Shapiro–Wilk test and F-test, respectively. All data met the normal distribution and had equal variances in each group. Unpaired Student's t test was performed in males. Two-way ANOVA with Bonferroni post hoc test was performed in females, with lesion and estrus cycle as the two factors. Probability values of P < .05 were considered to represent sig￾nificant differences. RESULTS Reduced anxiety in male, but not female, mice with ventral hippocampal lesions To examine biological sex differences in hippocampal mod￾ulation of anxiety-like behavior, we first performed excito￾toxic lesions of bilateral ventral hippocampus by local injection of ibotenic acid. Fig. 2A and Fig. 2B showed sche￾matic illustration of damages from male and female mice, respectively. The lesion extended throughout bilateral ven￾tral hippocampus and included all cell fields, with minimal extrahippocampal damages. 2 male and 4 female mice were excluded from analysis due to incorrect lesion site, resulting in 30 male and 32 female mice in final statistical analysis. Male and female mice had an average of 56% (40%–77%) and 60% (37%–70%) bilateral tissue loss, respectively. No significant differences of lesion size were observed between males and females [t(60) = 1.07, P = .291, unpaired t test]. In the novelty-suppressed feeding test, bilateral ventral hippocampal excitotoxic lesioning shortened the feeding latency in male mice [t(28) = 2.21, P = .036, unpaired t test, Fig. 3A], indicating reduced anxiety. By sharp contrast, in female mice, the same lesion did not produce anxiolytic effects in this test [lesion effect: F(1, 28) = 0.23, P = .64; interaction: F(1, 28) = 0.38, P = .541, Fig. 3B], despite a sig￾nificant effect of the estrus cycle [F(1, 28) = 597.00, P < .001, two-way ANOVA with Bonferroni post hoc test] as previously reported (Mora et al., 1996; Seeman, 1997; Gangitano et al., 2009). No significant differences in food consumption were observed in either biological sex [male: t(28) = 0.30, P = .765, unpaired t test, Fig. 3C; female: estrus cycle effect: F(1, 28) = 0.33, P = .568; lesion effect: F(1, 28) = 0.05, P = .818; interaction: F(1, 28) = 0.02, P = .899, two-way ANOVA with Bonferroni post hoc test, Fig. 3D], indicating similar levels of hunger and motivation. The same conclusion could be reached after food consump￾tion was normalized to body weight [male: t(28) = 0.52, P = .607, unpaired t test; female: estrus cycle effect: F(1, 28) = 0.25, P = .623; lesion effect: F(1, 28) = 0.46, P = .505; inter￾action: F(1, 28) = 1.01, P = .323, two-way ANOVA with Bon￾ferroni post hoc test]. In the marble burying test, bilateral ventral hippocampal excitotoxic lesion resulted in fewer buried marbles in male mice [t(28) = 2.59, P = .016, unpaired t test, Fig. 4A], indicat￾ing decreased anxiety level. Again, we failed to observe such effects in female mice, despite a significant effect of the estrus cycle [estrus cycle effect: F(1, 28) = 108.80, P < .001; lesion effect: F(1, 28) = 0.22, P = .642; interaction: F(1, 28) = 2.55, P = .121, two-way ANOVA with Bonferroni post hoc test, Fig. 4B]. In the elevated plus-maze test, male mice spent more time [t(28) = 0.29, P = .023, Fig. 5A] and exhibited more entries into the open arms [t(28) = 2.53, P = .018, unpaired t test, Fig. 5C] after ventral hippocampal excitotoxic lesions. Fig. 3. Ventral hippocampal lesioning relieves anxiety-like behavior in the novelty-suppressed feeding test in male but not female mice. Bilat￾eral ventral hippocampal lesioning decreased feeding latency in the novelty-suppressed feeding test in male (A) but not female (B) mice, despite significant influences from estrogen levels in females. Similar amount of food was consumed in two groups of male (C) and female (D) mice. *P < .05, **P < .01, ***P < .001, unpaired t test or two-way ANOVA with Bonferroni's test. n = 15/group for males, n = 8/group for females in HEP and LEP, respectively. HEP: high-estradiol phase, LEP: low-estradiol phase. 4 Cheng Wang et al. / Neuroscience 418 (2019) xxx–xxx NSC 19240 No of Pages 9 06 September 2019