No of Pages 9 06 September 2019 ARTICLE IN PRESS 6 Cheng Wang et al. / Neuroscience 418(2019)XXx-XXX Male B□ Female Human neuroimaging studies have revealed significantly stronger activation of hippocampus and amygdala under stress(Seo et al., 2011), as well as stronger correlation between trait anxiety and white matter tract integrity of the E58 temporal lobe(Montag et al., 2012), in males than females In the present study, we observed higher expression of C- Fos in vCA1, basolateral amygdala and prelimbic cortex in both male and female mice after the elevated-plus maze test, indicating enrollment of these brain regions in task induced anxiety. However, we noticed significantly greater HEP c-Fos expression in vCAl in male than in female mice despite similar levels of anxiety in the elevated plus-maze Fig. 6. Ventral hippocampal lesioning does not affect locomotive activity in test(Fig. 7C). VCA1 and basolateral amygdala have robust the open field test. Bilateral ventral hippocampal lesioning did not affect di reciprocal connections (O'Donnell and Grace, 1995 tance traveled in the open field in male(A)or female( B)mice. 'P<. 05, *P<.01, P<.001, unpaired t test or two-way ANOVA with Bonferronis Pikkarainen et al., 1999)and participate in anxiety modula est n= 15/group for males, n= 8/group for females in HEP and LEP, tion in mice(Felix-Ortiz et al., 2013). It is possible that VCA1 spectively. HEP: high-estradiol phase, LEP: low-estradiol phase is more strongly modulated by basolateral amygdala in male than in female mice after anxiety tests. Both ventral hippocampus and basolateral amygdala exert inhibi show that excitotoxic lesioning of ventral hippocampus tion on the hypothalamic-pituitary-adrenocortical(HPA) yields anxiolysis in novelty-suppressed feeding, marble axis (Jacobson and Sapolsky, 1991; Bhatnagar et al burying and elevated plus-maze tests in male C57BU/6 J mice 2004), and disinhibition of the HPA axis induces secretion Figs. 3-5), consistent with previous reports(Bannerman et al of glucocorticoids and initiates numerous physiological 2002: McHugh et al., 2004). By sharp contrast, the same effects(Herman and Cullinan, 1997; Herbert et al., 2006) lesion did not affect anxiety in female mice Female HPA axis shows relative inability of adaptation. Sex hormones are important factors underlying biological demonstrated by deficits in glucocorticoid receptor regula- sex difference in anxiety. The fluctuation of estrogen in the tion in female but not male rats following chronic stress menstrual cycle, concomitant with alteration in levels of pro- (Bourke et aL., 2013). The deficits may result from distinct gesterone, androgens and their metabolites, increases sus- ventral hippocampus enrollment in anxiety in different ceptibility of women to develop affective disorders( Roca sexes. However, we need to note that the present stud et al., 2003; Walf and Frye, 2006: Sahingoz et al., 2011). checks c-Fos expression only in the elevated plus-maze Our findings indicate that the estrus cycle has a robust test, thus could not exclude the possibility of a test-specific effect on anxiety level in C57BL/6 J mice, showing low phenomenon unless other anxiety tests are carried out. and high levels of anxiety in high- and low-estradiol phases, The weaker hippocampal involvement in anxiety-like respectively(Figs. 3-5). This is consistent with a number of behavior in female mice indicates alternative neural sub previous studies using the marble burying test in Wistar strate of anxiety modulation in females. One possible candi- (Fernandez-Guasti and Picazo, 1992; Schneider and Popik, date is the prefrontal cortex. Neuroimaging studies have 2007)and Long-Evans rats(Llaneza and Frye, 2009), the shown stronger enrollment of prefrontal areas in anxiety elevated-plus maze test in Sprague-Dawley(Mora et al. tests in women(Hakamata et al., 2009: Marumo et al 1996: Diaz-Veliz et al., 1997), Wistar(Marcondes et al 2009: Seo et al., 2017). However, we did not observe stron 2001)and Long-Evans rats(Walf and Frye, 2007), and ger activation in the prelimbic cortex in our study(Fig. 71), he elevated T-maze test in Wistar rats(Gouveia Jr. et al which might be caused by different subregion included: only 2004). The hippocampus shows high-level expression of the prelimbic area was examined in the present study, sex hormone receptors. Systemic, intra-hippocampal and whereas neuroimaging studies included all prefrontal intra-amygdala administration of estradiol all produces subregions. anxiolysis in rats(Frye and Walf, 2004; Walf and Frye Neurons in VCAl are heterogenous and different VCA1 2006). Estradiol binds to estrogen receptors a and B, which subpopulations project to different downstream targets such are widely distributed in hippocampus, amygdala, hypotha as amygdala, infralimbic cortex and hypothalamus lamus and other regions in rodents and humans(Shughrue (Fanselow and Dong, 2010). Several studies have demon- et al., 1997; Osterlund et al., 2000), and exerts its anxiolytic strated hippocampal modulation of anxiety behavior by tem- effect at least partly through upregulating brain-derived neu- poral and reversible manipulation of ventral hippocampal rotrophic factor expression in the brain(Gourley et al., 2008; circuits. Inhibiting ventral hippocampal inputs to medial pre- Deltheil et al., 2009: Bath et al., 2012). However, the pre- frontal cortex elicits anxiolysis(Padilla-Coreano et al., 2016: sent study carefully differentiated between high and low Parfitt et al., 2017), while inhibiting inputs to lateral septum estrogen level phases in the female, but did not observe dif- (Parfitt et al., 2017)or activating inputs to lateral hypothala ferent lesioning effects in either phase(Figs. 3-5).These mus(Jimenez et al., 2018)elicits anxiogenic effects in mice findings indicate that the biological sex difference of hippo- These seemingly conflicting data are consistent with the campal modulation of anxiety in the present study is inde finding that distinct sub-populations of ventral CA1 pyrami of estre dal neurons target different downstream regionsshow that excitotoxic lesioning of ventral hippocampus yields anxiolysis in novelty-suppressed feeding, marble burying and elevated plus-maze tests in male C57BL/6 J mice (Figs. 3-5), consistent with previous reports (Bannerman et al., 2002; McHugh et al., 2004). By sharp contrast, the same lesion did not affect anxiety in female mice. Sex hormones are important factors underlying biological sex difference in anxiety. The fluctuation of estrogen in the menstrual cycle, concomitant with alteration in levels of pro￾gesterone, androgens and their metabolites, increases sus￾ceptibility of women to develop affective disorders (Roca et al., 2003; Walf and Frye, 2006; Sahingoz et al., 2011). Our findings indicate that the estrus cycle has a robust effect on anxiety level in C57BL/6 J mice, showing low and high levels of anxiety in high- and low-estradiol phases, respectively (Figs. 3-5). This is consistent with a number of previous studies using the marble burying test in Wistar (Fernandez-Guasti and Picazo, 1992; Schneider and Popik, 2007) and Long–Evans rats (Llaneza and Frye, 2009), the elevated-plus maze test in Sprague–Dawley (Mora et al., 1996; Diaz-Veliz et al., 1997), Wistar (Marcondes et al., 2001) and Long–Evans rats (Walf and Frye, 2007), and the elevated T-maze test in Wistar rats (Gouveia Jr. et al., 2004). The hippocampus shows high-level expression of sex hormone receptors. Systemic, intra-hippocampal and intra-amygdala administration of estradiol all produces anxiolysis in rats (Frye and Walf, 2004; Walf and Frye, 2006). Estradiol binds to estrogen receptors α and β, which are widely distributed in hippocampus, amygdala, hypotha￾lamus and other regions in rodents and humans (Shughrue et al., 1997; Osterlund et al., 2000), and exerts its anxiolytic effect at least partly through upregulating brain-derived neu￾rotrophic factor expression in the brain (Gourley et al., 2008; Deltheil et al., 2009; Bath et al., 2012). However, the pre￾sent study carefully differentiated between high and low estrogen level phases in the female, but did not observe dif￾ferent lesioning effects in either phase (Figs. 3-5). These findings indicate that the biological sex difference of hippo￾campal modulation of anxiety in the present study is inde￾pendent of estrogen levels. Human neuroimaging studies have revealed significantly stronger activation of hippocampus and amygdala under stress (Seo et al., 2011), as well as stronger correlation between trait anxiety and white matter tract integrity of the temporal lobe (Montag et al., 2012), in males than females. In the present study, we observed higher expression of c￾Fos in vCA1, basolateral amygdala and prelimbic cortex in both male and female mice after the elevated-plus maze test, indicating enrollment of these brain regions in task￾induced anxiety. However, we noticed significantly greater c-Fos expression in vCA1 in male than in female mice, despite similar levels of anxiety in the elevated plus-maze test (Fig. 7C). vCA1 and basolateral amygdala have robust reciprocal connections (O'Donnell and Grace, 1995; Pikkarainen et al., 1999) and participate in anxiety modula￾tion in mice (Felix-Ortiz et al., 2013). It is possible that vCA1 is more strongly modulated by basolateral amygdala in male than in female mice after anxiety tests. Both ventral hippocampus and basolateral amygdala exert inhibi￾tion on the hypothalamic–pituitary-adrenocortical (HPA) axis (Jacobson and Sapolsky, 1991; Bhatnagar et al., 2004), and disinhibition of the HPA axis induces secretion of glucocorticoids and initiates numerous physiological effects (Herman and Cullinan, 1997; Herbert et al., 2006). Female HPA axis shows relative inability of adaptation, demonstrated by deficits in glucocorticoid receptor regula￾tion in female but not male rats following chronic stress (Bourke et al., 2013). The deficits may result from distinct ventral hippocampus enrollment in anxiety in different sexes. However, we need to note that the present study checks c-Fos expression only in the elevated plus-maze test, thus could not exclude the possibility of a test-specific phenomenon unless other anxiety tests are carried out. The weaker hippocampal involvement in anxiety-like behavior in female mice indicates alternative neural sub￾strate of anxiety modulation in females. One possible candi￾date is the prefrontal cortex. Neuroimaging studies have shown stronger enrollment of prefrontal areas in anxiety tests in women (Hakamata et al., 2009; Marumo et al., 2009; Seo et al., 2017). However, we did not observe stron￾ger activation in the prelimbic cortex in our study (Fig. 7I), which might be caused by different subregion included: only the prelimbic area was examined in the present study, whereas neuroimaging studies included all prefrontal subregions. Neurons in vCA1 are heterogenous and different vCA1 subpopulations project to different downstream targets such as amygdala, infralimbic cortex and hypothalamus (Fanselow and Dong, 2010). Several studies have demon￾strated hippocampal modulation of anxiety behavior by tem￾poral and reversible manipulation of ventral hippocampal circuits. Inhibiting ventral hippocampal inputs to medial pre￾frontal cortex elicits anxiolysis (Padilla-Coreano et al., 2016; Parfitt et al., 2017), while inhibiting inputs to lateral septum (Parfitt et al., 2017) or activating inputs to lateral hypothala￾mus (Jimenez et al., 2018) elicits anxiogenic effects in mice. These seemingly conflicting data are consistent with the finding that distinct sub-populations of ventral CA1 pyrami￾dal neurons target different downstream regions Fig. 6. Ventral hippocampal lesioning does not affect locomotive activity in the open field test. Bilateral ventral hippocampal lesioning did not affect dis￾tance traveled in the open field in male (A) or female (B) 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. 6 Cheng Wang et al. / Neuroscience 418 (2019) xxx–xxx NSC 19240 No of Pages 9 06 September 2019