Disease [20,21]. Many studies have shown that not only metabolic regulation but also brain functions such as emotional behavior, locomotor activity, and learning are all highly influenced by undernutrition during pregnancy [22]. As both PPARs and AMPK are activated under fasting conditions [23,24,25], it is expected that their function may be associated with undernutrution during gestation and/or LBW. We then hypothesized that adult offspring mice with embryonic undernutrition would exhibit changes in sleep architecture and/ or homeostasis. In this study we found that offspring mice display larger SWA during NREM sleep with normal sleep Title Loaded From File timing and duration, and an exaggerated rebound of SWA against SD.Augmented Sleep Pressure Model in MiceFigure 1. Birth body weight (A) and body weight changes during the lactation period (B) and after weaning (C) up to 8 weeks in offspring mice. Open bars and circles indicate AD mice. Closed bars and circles indicate DR mice. Data represent means 6 SEM (A; n = 22?3, B; n = 36?8, C; n = 16). **p,0.01 indicates a significant difference. doi:10.1371/journal.pone.0064263.gMaterials and Methods Animals and Experimental DesignExperiments were performed with Jcl:ICR mice (Slc Inc., Shizuoka, Japan). Mice (aged 10?2 weeks) were maintained on a 12-hour light/dark (L/D) cycle (lights on at 0900) at a controlled ambient temperature (2361uC). Virgin female mice were caged with male mice. Pregnancy was dated with vaginal plugs (day 0), and pregnant female mice were housed individually with ad libitum access to standard chow (MF, Oriental Yeast Co., LTD, Tokyo, Japan). On gestation day 12, pregnant female mice were randomly assigned to either control (fed ad libitum; AD) or dietary restriction (DR) Title Loaded From File groups. Food intake of DR mothers was restricted to 50 of controls from gestation days 12 to parturition. After parturition, litter size was equalized to ten in both AD and DR groups. Mothers received chow in the AD condition after parturition. Pups were weaned at 3 weeks, and fed standard chow AD afterwards up to the age of 8? weeks. All experiments were performed in male mice. The Animal Study Committee of Tokushima University (No. 12051) approved these experiments, and we performed them in accordance with Guidelines for the Care and Use of Animals approved by the Council of the Physiological Society of Japan.Sleep and Body Temperature RecordingsAdult offspring male mice were anesthetized with a cocktail of ketamine (100 mg/kg, Daiichi-Sankyo, Tokyo, Japan) and xylazine (25 mg/kg, Sigma, St. Louis, MO, USA). A telemetric device (TA10TA-F20; Data Sciences Int., USA) for recording body temperature and spontaneous activity was implanted in the peritoneal cavity. Stainless steel miniature screw electrodes (M1.7-3, Unique Medical, Tokyo, Japan) were implanted in the skull (1.0 mm anterior to the bregma, 1.0 mm right lateral to the midline; 2.5 mm posterior to the bregma, 2.5 mm left lateral to the midline) to record the EEG. Teflon-coated stainless steel wires (AS633, Cooner Wire, Chatsworth, CA, USA) were implanted in the neck muscles on both sides to record the electromyogram (EMG). After 2 weeks of recovery, the mice were transferred to plastic cages (20 cm624 cm630 cm) in a soundproof recording room and allowed 3 days of habituation. Thereafter, mice were connected through an electrical slip ring (T13EEG; Air Precision,Paris, France) to a polygraph (RM-6100; Nihon Kohden, Tokyo, Japan) with a computer flat cable, and th.Disease [20,21]. Many studies have shown that not only metabolic regulation but also brain functions such as emotional behavior, locomotor activity, and learning are all highly influenced by undernutrition during pregnancy [22]. As both PPARs and AMPK are activated under fasting conditions [23,24,25], it is expected that their function may be associated with undernutrution during gestation and/or LBW. We then hypothesized that adult offspring mice with embryonic undernutrition would exhibit changes in sleep architecture and/ or homeostasis. In this study we found that offspring mice display larger SWA during NREM sleep with normal sleep timing and duration, and an exaggerated rebound of SWA against SD.Augmented Sleep Pressure Model in MiceFigure 1. Birth body weight (A) and body weight changes during the lactation period (B) and after weaning (C) up to 8 weeks in offspring mice. Open bars and circles indicate AD mice. Closed bars and circles indicate DR mice. Data represent means 6 SEM (A; n = 22?3, B; n = 36?8, C; n = 16). **p,0.01 indicates a significant difference. doi:10.1371/journal.pone.0064263.gMaterials and Methods Animals and Experimental DesignExperiments were performed with Jcl:ICR mice (Slc Inc., Shizuoka, Japan). Mice (aged 10?2 weeks) were maintained on a 12-hour light/dark (L/D) cycle (lights on at 0900) at a controlled ambient temperature (2361uC). Virgin female mice were caged with male mice. Pregnancy was dated with vaginal plugs (day 0), and pregnant female mice were housed individually with ad libitum access to standard chow (MF, Oriental Yeast Co., LTD, Tokyo, Japan). On gestation day 12, pregnant female mice were randomly assigned to either control (fed ad libitum; AD) or dietary restriction (DR) groups. Food intake of DR mothers was restricted to 50 of controls from gestation days 12 to parturition. After parturition, litter size was equalized to ten in both AD and DR groups. Mothers received chow in the AD condition after parturition. Pups were weaned at 3 weeks, and fed standard chow AD afterwards up to the age of 8? weeks. All experiments were performed in male mice. The Animal Study Committee of Tokushima University (No. 12051) approved these experiments, and we performed them in accordance with Guidelines for the Care and Use of Animals approved by the Council of the Physiological Society of Japan.Sleep and Body Temperature RecordingsAdult offspring male mice were anesthetized with a cocktail of ketamine (100 mg/kg, Daiichi-Sankyo, Tokyo, Japan) and xylazine (25 mg/kg, Sigma, St. Louis, MO, USA). A telemetric device (TA10TA-F20; Data Sciences Int., USA) for recording body temperature and spontaneous activity was implanted in the peritoneal cavity. Stainless steel miniature screw electrodes (M1.7-3, Unique Medical, Tokyo, Japan) were implanted in the skull (1.0 mm anterior to the bregma, 1.0 mm right lateral to the midline; 2.5 mm posterior to the bregma, 2.5 mm left lateral to the midline) to record the EEG. Teflon-coated stainless steel wires (AS633, Cooner Wire, Chatsworth, CA, USA) were implanted in the neck muscles on both sides to record the electromyogram (EMG). After 2 weeks of recovery, the mice were transferred to plastic cages (20 cm624 cm630 cm) in a soundproof recording room and allowed 3 days of habituation. Thereafter, mice were connected through an electrical slip ring (T13EEG; Air Precision,Paris, France) to a polygraph (RM-6100; Nihon Kohden, Tokyo, Japan) with a computer flat cable, and th.
