No of Pages 9 06 September 2019 ARTICLE IN PRESS 2 Cheng Wang et al. / Neuroscience 418(2019)XXx-XXX EXPERIMENTAL PROCEDURES cage, and the food pellet was weighed again to obtain the Animals amount of food consumed Adult weight-matched (26-30 g)male and female C57BL/6 mice at the age of 6-8 weeks were housed 4-6 per cage Marble burying test under a 12 h light/dark cycle with food and water provided ad libitum(except before the novelty-suppressed feeding The marble burying test(Zhang et al., 2017)was performed test). Corn cobs were laid in the cage as bedding. All experi- 7 days after the novelty-suppressed feeding test. Twenty ments were conducted in a blind manner and in accordance marbles, with 4 x 5 arrangement, were put on cob bedding ith the institutional animal care and use committee of materials at a depth of 5 cm in home cage. Each mouse Peking University Health Science Center( LA2016061). All was left single in its home cage for 30 min before placed mice were handled for three days before behavioral experi in a random corner of the test apparatus facing the chamber ments 32 male and 36 female mice were used in behavioral wall. A marble with two-thirds or more in the cob bedding experiments Mice were randomly allocated to sham and was treated as a buried marble. the number of buried mar- lesion groups(16 male and 18 female mice in each group) bles was counted after 30 min For c-Fos staining, 6 male and 6 female mice were used Open field test Hippocampal lesioning 7 days after the marble burying test, each mouse was Mice were anesthetized with 1% pentobarbital sodium, and placed in a 60 x 60 x 60 cm open field chamber with 30 Ix positioned in a stereotaxic instrument( RWD Life Science illumination and allowed to explore freely for 5 min (Jiang Shenzhen, China): 0.4 ul of ibotenic acid(pH 7. 4, 10 mg et al., 2018). Locomotive activity was videotaped and the ml, Sigma-Aldrich, USA) was injected into bilateral ventral total distance traveled in the field was measured using the hippocampus(AP-3.40 mm, ML +3.0 mm, DV-32 mm SMART software(v2. 5.21, Panlab). The chamber was relative to Bregma)(Paxinos and Franklin, 2001) through a cleaned by 75% ethanol between tests 1-HL Hamilton microsyringe within 5 min, at a flow rate of 0.08 Hl/min. Sham lesion was performed by injecting the same amount of normal saline at the same flow rate After Elevated plus-maze test stereotaxic injection, the needle was left in place for another 5 min to allow for drug diffusion before slow withdrawal. Seven days after the open field test, mice performed the Mice were allowed to recover for 7 days before further elevated plus-maze test(Zheng et al., 2017). The maze consisted of two open(5 x 30 cm) and two closed arms (same size with 15 cm walls)and was placed 50 cm above the floor. In a room with 30 lx illumination each mouse was Novelty-suppressed feeding test placed onto the central area heading towards the same The timeline of behavioral experiments was shown in Fig. 1 open arm. Animal activity was videotaped and the time After recovery from surgery, mice were first examined in the spent in open arms and the percent of entries into open novelty-suppressed feeding test as previously reported arms in the following 5 min were assessed by hand-score (Zhang et al., 2017). The apparatus consisted of a chamber The maze was cleaned by 75% ethanol between tests (40 x 40 cm) filled with cob bedding materials at a depth of 5 cm. Under 300 Ix illumination, a standard chow pellet (5 g) was placed on a piece of white filter paper(10 cm in Estrus cycle identification diameter) positioned in the center of the arena. Each mouse was left single in a new cage for 30 min after 24 h food The estrus cycle of female mice consisted of proestrus estrus, metestrus and diestrus phases, and was defined deprivation before being placed in a random corner of the by vaginal cytology after each behavioral test. Proestrus chamber facing the chamber wall. The amount of time and estrus were allocated to high-estradiol phase(HEP), was recorded. After the first bite or a 10 min cut-off time while metestrus and diestrus were allocated to low- estradiol phase(LEP). Vaginal smears were obtained by the mouse was transferred to the prior new cage. A new sin gle food pellet, which was weighed in advance, was placed toothpicks scraping gently in vagina after each behavioral test. 95%alcohol was used to fix vaginal smears for in the cage. The mouse was allowed to eat the food pellet 15 min after drying in air for 15 min. Harris-Shorr staining for 5 min. After that the mouse was returned to its home was carried out to stain vaginal epithelial cells. Vaginal smears were put in Shorr stain solution for 10 min, then put in 95%, 95%, 100% and 100% alcohol for 1-2 min in turn, finally put in xylol for 10 min. After that, vaginal smears were sealed by neutral gum, dried in a 37C oven. Vaginal Fig. 1. Timeline of behavioral experiments. NSF: novelty-suppressed cytology was observed with a light microscope(Leica feeding test, MBT: marble burying test, OFT: open field test, EPM: ele DMI 4000B, Germany) as previously described(Nelsor ated plus-maze test. etal,1982)EXPERIMENTAL PROCEDURES Animals Adult weight-matched (26–30 g) male and female C57BL/6 mice at the age of 6–8 weeks were housed 4–6 per cage under a 12 h light/dark cycle with food and water provided ad libitum (except before the novelty-suppressed feeding test). Corn cobs were laid in the cage as bedding. All experi￾ments were conducted in a blind manner and in accordance with the Institutional Animal Care and Use Committee of Peking University Health Science Center (LA2016061). All mice were handled for three days before behavioral experi￾ments. 32 male and 36 female mice were used in behavioral experiments. Mice were randomly allocated to sham and lesion groups (16 male and 18 female mice in each group). For c-Fos staining, 6 male and 6 female mice were used. Hippocampal lesioning Mice were anesthetized with 1% pentobarbital sodium, and positioned in a stereotaxic instrument (RWD Life Science, Shenzhen, China); 0.4 μl of ibotenic acid (pH 7.4, 10 mg/ ml, Sigma-Aldrich, USA) was injected into bilateral ventral hippocampus (AP -3.40 mm, ML ± 3.0 mm, DV -3.2 mm relative to Bregma) (Paxinos and Franklin, 2001) through a 1-μL Hamilton microsyringe within 5 min, at a flow rate of 0.08 μl/min. Sham lesion was performed by injecting the same amount of normal saline at the same flow rate. After stereotaxic injection, the needle was left in place for another 5 min to allow for drug diffusion before slow withdrawal. Mice were allowed to recover for 7 days before further experiments. Novelty-suppressed feeding test The timeline of behavioral experiments was shown in Fig. 1. After recovery from surgery, mice were first examined in the novelty-suppressed feeding test as previously reported (Zhang et al., 2017). The apparatus consisted of a chamber (40 × 40 cm) filled with cob bedding materials at a depth of 5 cm. Under 300 lx illumination, a standard chow pellet (5 g) was placed on a piece of white filter paper (10 cm in diameter) positioned in the center of the arena. Each mouse was left single in a new cage for 30 min after 24 h food deprivation before being placed in a random corner of the chamber facing the chamber wall. The amount of time passed before the mouse approached and ate the pellet was recorded. After the first bite or a 10 min cut-off time, the mouse was transferred to the prior new cage. A new sin￾gle food pellet, which was weighed in advance, was placed in the cage. The mouse was allowed to eat the food pellet for 5 min. After that, the mouse was returned to its home cage, and the food pellet was weighed again to obtain the amount of food consumed. Marble burying test The marble burying test (Zhang et al., 2017) was performed 7 days after the novelty-suppressed feeding test. Twenty marbles, with 4 × 5 arrangement, were put on cob bedding materials at a depth of 5 cm in home cage. Each mouse was left single in its home cage for 30 min before placed in a random corner of the test apparatus facing the chamber wall. A marble with two-thirds or more in the cob bedding was treated as a buried marble. The number of buried mar￾bles was counted after 30 min. Open field test 7 days after the marble burying test, each mouse was placed in a 60 × 60 × 60 cm open field chamber with 30 lx illumination and allowed to explore freely for 5 min (Jiang et al., 2018). Locomotive activity was videotaped and the total distance traveled in the field was measured using the SMART software (v2.5.21, Panlab). The chamber was cleaned by 75% ethanol between tests. Elevated plus-maze test Seven days after the open field test, mice performed the elevated plus-maze test (Zheng et al., 2017). The maze consisted of two open (5 × 30 cm) and two closed arms (same size with 15 cm walls) and was placed 50 cm above the floor. In a room with 30 lx illumination, each mouse was placed onto the central area heading towards the same open arm. Animal activity was videotaped and the time spent in open arms and the percent of entries into open arms in the following 5 min were assessed by hand-score. The maze was cleaned by 75% ethanol between tests. Estrus cycle identification The estrus cycle of female mice consisted of proestrus, estrus, metestrus and diestrus phases, and was defined by vaginal cytology after each behavioral test. Proestrus and estrus were allocated to high-estradiol phase (HEP), while metestrus and diestrus were allocated to low￾estradiol phase (LEP). Vaginal smears were obtained by toothpicks scraping gently in vagina after each behavioral test. 95% alcohol was used to fix vaginal smears for 15 min after drying in air for 15 min. Harris–Shorr staining was carried out to stain vaginal epithelial cells. Vaginal smears were put in Shorr stain solution for 10 min, then put in 95%, 95%, 100% and 100% alcohol for 1–2 min in turn, finally put in xylol for 10 min. After that, vaginal smears were sealed by neutral gum, dried in a 37 °C oven. Vaginal cytology was observed with a light microscope (Leica DMI 4000B, Germany) as previously described (Nelson et al., 1982). Fig. 1. Timeline of behavioral experiments. NSF: novelty-suppressed feeding test, MBT: marble burying test, OFT: open field test, EPM: ele￾vated plus-maze test. 2 Cheng Wang et al. / Neuroscience 418 (2019) xxx–xxx NSC 19240 No of Pages 9 06 September 2019