No of Pages 9 06 September 2019 ARTICLE IN PRESS NEUROSCIENCE BR● RESEARCH ARTICLE Cheng Wang et al. /Neuroscience 418 (2019)XXx-XXX Ventral Hippocampus Modulates Anxiety-Like Behavior in Male But Not Female c57BL/6J Mice Cheng Wang, Yu Zhang, Shan Shao, Shuang Cui, You Wan. b, c, and Ming Yi b, * Neuroscience Research Institute, Peking University, 38 Xueyuan Road, Beiing 100083, China Department of Neurobiology, School of Basic Medical Sciences, Peking University, 38 Xueyuan Road, Bejing 100083, China Key Laboratory for Neuroscience, Ministry of Education/National Health Commission, Peking University, 38 Xueyuan Road, Beijing 100083, China Co-innovation Center of Neuroregeneration, Nantong University, Nantong 226001, China Abstract-Remarkable sex difference has been observed in emotional processing including anxiety. The hippocam pus, its ventral pole in particular, modulates anxiety like behavior in rodents. However, most researches have been per- formed in male animals only, leaving hippocampal modulation of anxiety in females poorly defined In the present study, we showed that excitotoxic lesioning of the ventral hippocampus with ibotenic acid produced anxiolytic effects in three behavioral tests(novelty-suppressed feeding, marble burying, and elevated-plus maze )in male but not female C57BL/6 J mice. Locomotion in the open field remained similar after lesioning in either sex More c-Fos-positive neu rons were observed in the ventral hippocampus in male than in female mice after exploration in an elevated plus maze, indicating stronger enrollment of this region in anxiety-like behavior in males. These results reveal significant biological sex difference in ventral hippocampal modulation on anxiety in mice and provide a new sight for anxiety modulation and hippocampal function. 2019 IBRo. Published by Elsevier Ltd. All rights reserved Key words: ventral hippocampus, anxiety, sex difference. INTRODUCTION Furthermore, local field potentials in the ventral hippocam- Anxiety disorders are prevalent( Kessler et al., 2010)and pus show increased correlation in theta-frequency cover 7. 3% of the population(Baxter et al., 2013). Signifi- oscillations with medial prefrontal cortex in anxiogenic cant sex difference has been observed in mood disorders. environments in mice(Adhikari et al., 2010). Optogenetic women are more likely than men to experience anxiety activation and inhibition of basolateral amygdala to ( Kessler et al., 1994: Craske and Stein, 2016)or depression ventral hippocampus projections increase and decrease (Nolen-Hoeksema and Girgus, 1994)from early adoles- anxiety-related behaviors in mice, respectively(Felix-Ortiz cence through adulthood. Brain regions in the limbic system et al., 2013). More recently, anxiety cells"have been dis underlie emotional processing(Rosen and Schulkin, 1998) covered in ventral hippocampal CA1 in mice (Jimenez In rodents, ventral hippocampus shows dense connection etal.2018) with affect-related regions including amygdala, prefrontal However, most of these studies were performed only in cortex and hypothalamus(Bannerman et al., 2004; Fanse male animals. To our knowledge, effects of ventral hippo- low and Dong, 2010). Excitotoxic lesioning of the ventral campal lesioning on anxiety in female rodents have not hippocampus in rats reduces anxiety-related behaviors in been reported Previous work has shown that males have elevated plus-maze and hyponeophagia tests( Kjelstrup larger hippocampal(Ruigrok et al., 2014; Meyer et al et al., 2002; Bannerman et al., 2003), whereas optogenetic 2017)and amygdala(Giedd et al., 1996: Goldstein et al activation of granule cells in the ventral dentate gyrus sup. 2001: Ruigrok et al., 2014)volumes, as well as greater presses innate anxiety in mice( Kheirbek et al., 2013 within-hemispheric connectivity(Ingalhalikar et al., 2014 than females in both mice and humans. The present study aims to explore potential sex difference in hippocampal modulation of anxiety, by examining anxiety-like behavior HEy L co in male and female C57BL/6 J China te+8613718823306. ing of the ventral hippocampus and assessing hippocampal sses:yuan@hsc.pku.edu.cn(YouWan)mingyi@hsc.pku neuronal activation with c-Fos staining after an anxious https://doi.org/10.1016/j-neuroscience.2019.08.032 0306-4522@ 2019 IBRO. Published by Elsevier Ltd. All rights reservedNEUROSCIENCE RESEARCH ARTICLE Cheng Wang et al. / Neuroscience 418 (2019) xxx–xxx Ventral Hippocampus Modulates Anxiety-Like Behavior in Male But Not Female C57BL/6 J Mice Cheng Wang, a Yu Zhang, a Shan Shao, a Shuang Cui, a You Wana,b,c,d,⁎ and Ming Yia,b,⁎⁎ a Neuroscience Research Institute, Peking University, 38 Xueyuan Road, Beijing 100083, China b Department of Neurobiology, School of Basic Medical Sciences, Peking University, 38 Xueyuan Road, Beijing 100083, China c Key Laboratory for Neuroscience, Ministry of Education/National Health Commission, Peking University, 38 Xueyuan Road, Beijing 100083, China d Co-innovation Center of Neuroregeneration, Nantong University, Nantong 226001, China Abstract—Remarkable sex difference has been observed in emotional processing including anxiety. The hippocam￾pus, its ventral pole in particular, modulates anxiety-like behavior in rodents. However, most researches have been per￾formed in male animals only, leaving hippocampal modulation of anxiety in females poorly defined. In the present study, we showed that excitotoxic lesioning of the ventral hippocampus with ibotenic acid produced anxiolytic effects in three behavioral tests (novelty-suppressed feeding, marble burying, and elevated-plus maze) in male but not female C57BL/6 J mice. Locomotion in the open field remained similar after lesioning in either sex. More c-Fos-positive neu￾rons were observed in the ventral hippocampus in male than in female mice after exploration in an elevated plus￾maze, indicating stronger enrollment of this region in anxiety-like behavior in males. These results reveal significant biological sex difference in ventral hippocampal modulation on anxiety in mice and provide a new sight for anxiety modulation and hippocampal function. © 2019 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: ventral hippocampus, anxiety, sex difference. INTRODUCTION Anxiety disorders are prevalent (Kessler et al., 2010) and cover 7.3% of the population (Baxter et al., 2013). Signifi- cant sex difference has been observed in mood disorders: women are more likely than men to experience anxiety (Kessler et al., 1994; Craske and Stein, 2016) or depression (Nolen-Hoeksema and Girgus, 1994) from early adoles￾cence through adulthood. Brain regions in the limbic system underlie emotional processing (Rosen and Schulkin, 1998). In rodents, ventral hippocampus shows dense connection with affect-related regions including amygdala, prefrontal cortex and hypothalamus (Bannerman et al., 2004; Fanse￾low and Dong, 2010). Excitotoxic lesioning of the ventral hippocampus in rats reduces anxiety-related behaviors in elevated plus-maze and hyponeophagia tests (Kjelstrup et al., 2002; Bannerman et al., 2003), whereas optogenetic activation of granule cells in the ventral dentate gyrus sup￾presses innate anxiety in mice (Kheirbek et al., 2013). Furthermore, local field potentials in the ventral hippocam￾pus show increased correlation in theta-frequency oscillations with medial prefrontal cortex in anxiogenic environments in mice (Adhikari et al., 2010). Optogenetic activation and inhibition of basolateral amygdala to ventral hippocampus projections increase and decrease anxiety-related behaviors in mice, respectively (Felix-Ortiz et al., 2013). More recently, “anxiety cells” have been dis￾covered in ventral hippocampal CA1 in mice (Jimenez et al., 2018). However, most of these studies were performed only in male animals. To our knowledge, effects of ventral hippo￾campal lesioning on anxiety in female rodents have not been reported. Previous work has shown that males have larger hippocampal (Ruigrok et al., 2014; Meyer et al., 2017) and amygdala (Giedd et al., 1996; Goldstein et al., 2001; Ruigrok et al., 2014) volumes, as well as greater within-hemispheric connectivity (Ingalhalikar et al., 2014), than females in both mice and humans. The present study aims to explore potential sex difference in hippocampal modulation of anxiety, by examining anxiety-like behavior in male and female C57BL/6 J mice with excitotoxic lesion￾ing of the ventral hippocampus and assessing hippocampal neuronal activation with c-Fos staining after an anxious experience. *Correspondence to: Key Laboratory for Neuroscience, Ministry of Education/National Health Commission, Peking University, 38 Xueyuan Road, Beijing 100083, China Tel.: +86 137 1882 3306. E-mail addresses: ywan@hsc.pku.edu.cn (You Wan) mingyi@hsc.pku. edu.cn (Ming Yi) https://doi.org/10.1016/j.neuroscience.2019.08.032 0306-4522/© 2019 IBRO. Published by Elsevier Ltd. All rights reserved. 1 NSC 19240 No of Pages 9 06 September 2019