Gic system responsiveness in adult offspring mice. The influence of caffeine on immobility time (A) in the forced swim test. The effect of caffeine on latency to the onset of sleep (B) 10781694 and EEG delta/theta ratios in NREM sleep (C). Gene expression related to the regulation of adenosine signaling in the hypothalamus (D) and cortex (E). Open bars and circles indicate AD mice. Closed bars and circles indicate DR mice. Arrows indicate the injection time (ZT0). Data represent means 6 SEM (A; n = 9, B, C; n = 3, D, E; n = 6?). **p,0.01 and *p,0.05 indicate a significant difference. doi:10.1371/journal.pone.0064263.gEffects of CaffeineCaffeine is a pan-antagonist at adenosine receptors. As DR mice are considered to exhibit larger sleep pressure and adenosinergic function is well documented to be critical for sleep homeostasis [7,8,9], we hypothesized that adenosine or related systems would be involved in the behavioral and sleep changes in DR mice. A single i.p. injection of caffeine significantly elevated swimming behavior in DR mice in the forced swim test (Figure 7A). Although caffeine lengthened the latency to sleep in both AD and DR mice as reported in a previous study [27], differences in SWA were not Title Loaded From File observed (Figure 7B, C). There were also no significant changes in adenosine A1 receptor (ADORA1, encoded by Adora1), adenosine A2a receptor (ADORA2, encoded by Adora2), adenosine kinase (ADK, encoded by Adk), or adenosine deaminase (ADA, encoded by Ada) mRNA expression in either hypothalamus or cortex (Figure 7D, E).DiscussionStudies in animal models have shown that early undernutrition has a great impact on developmental programming, causing permanent changes in a variety of physiological functions including higher brain function [22]. We found in this study for the first time that sleep homeostasis is also affected by prenatal nutritional condition. Our DR adult offspring mice born with LBW showed an enhancement of SWA during NREM sleep, which is accepted to be a parameter of sleep pressure or sleep intensity. DR mice also Title Loaded From File displayed an exaggerated rebound of SWA against a 6-hour SD. Although SWA during NREM sleep is well known to be proportional to the length or activity of preceding wakefulness [1,2], DR mice did not exhibit significant changes in the structure of their sleep-wake cycle, and their spontaneous activity was rather decreased. These data indicate that the enhanced SWA in DR mice is caused by alterations not precedingAugmented Sleep Pressure Model in Micewakefulness but sleep homeostatic function itself, and that DR mice have exaggerated homeostatic sleep function. As DR mice have a higher threshold for waking by external stimuli (such as cage shaking and lights-off conditions), with normal timing and duration of sleep, we believe that DR mice actually exhibit a greater sleep pressure. The mRNA expression of Ppara and Hmgcs2, a main target gene of PPARa [28,29,30], was increased in the fetal DR mice, indicating that they were under a fasting condition. In fact, we observed a marked decrease in blood glucose in the fetal DR mice. The enhancement of ketogenesis was observed in the fetal DR mice not only in the liver but also in the brain. At 8? weeks old, the DR adult offspring mice showed small but significant increases in the mRNA expression of Ppara and Cpt1c in the hypothalamus. Cpt1 is another target gene of PPARa, and its activation indicates enhanced lipid b-oxidation in mitochondria [31]. It is suggested that hypothalam.Gic system responsiveness in adult offspring mice. The influence of caffeine on immobility time (A) in the forced swim test. The effect of caffeine on latency to the onset of sleep (B) 10781694 and EEG delta/theta ratios in NREM sleep (C). Gene expression related to the regulation of adenosine signaling in the hypothalamus (D) and cortex (E). Open bars and circles indicate AD mice. Closed bars and circles indicate DR mice. Arrows indicate the injection time (ZT0). Data represent means 6 SEM (A; n = 9, B, C; n = 3, D, E; n = 6?). **p,0.01 and *p,0.05 indicate a significant difference. doi:10.1371/journal.pone.0064263.gEffects of CaffeineCaffeine is a pan-antagonist at adenosine receptors. As DR mice are considered to exhibit larger sleep pressure and adenosinergic function is well documented to be critical for sleep homeostasis [7,8,9], we hypothesized that adenosine or related systems would be involved in the behavioral and sleep changes in DR mice. A single i.p. injection of caffeine significantly elevated swimming behavior in DR mice in the forced swim test (Figure 7A). Although caffeine lengthened the latency to sleep in both AD and DR mice as reported in a previous study [27], differences in SWA were not observed (Figure 7B, C). There were also no significant changes in adenosine A1 receptor (ADORA1, encoded by Adora1), adenosine A2a receptor (ADORA2, encoded by Adora2), adenosine kinase (ADK, encoded by Adk), or adenosine deaminase (ADA, encoded by Ada) mRNA expression in either hypothalamus or cortex (Figure 7D, E).DiscussionStudies in animal models have shown that early undernutrition has a great impact on developmental programming, causing permanent changes in a variety of physiological functions including higher brain function [22]. We found in this study for the first time that sleep homeostasis is also affected by prenatal nutritional condition. Our DR adult offspring mice born with LBW showed an enhancement of SWA during NREM sleep, which is accepted to be a parameter of sleep pressure or sleep intensity. DR mice also displayed an exaggerated rebound of SWA against a 6-hour SD. Although SWA during NREM sleep is well known to be proportional to the length or activity of preceding wakefulness [1,2], DR mice did not exhibit significant changes in the structure of their sleep-wake cycle, and their spontaneous activity was rather decreased. These data indicate that the enhanced SWA in DR mice is caused by alterations not precedingAugmented Sleep Pressure Model in Micewakefulness but sleep homeostatic function itself, and that DR mice have exaggerated homeostatic sleep function. As DR mice have a higher threshold for waking by external stimuli (such as cage shaking and lights-off conditions), with normal timing and duration of sleep, we believe that DR mice actually exhibit a greater sleep pressure. The mRNA expression of Ppara and Hmgcs2, a main target gene of PPARa [28,29,30], was increased in the fetal DR mice, indicating that they were under a fasting condition. In fact, we observed a marked decrease in blood glucose in the fetal DR mice. The enhancement of ketogenesis was observed in the fetal DR mice not only in the liver but also in the brain. At 8? weeks old, the DR adult offspring mice showed small but significant increases in the mRNA expression of Ppara and Cpt1c in the hypothalamus. Cpt1 is another target gene of PPARa, and its activation indicates enhanced lipid b-oxidation in mitochondria [31]. It is suggested that hypothalam.
