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Fasting activated histaminergic neurons and enhanced arousal effect of caffeine in mice
Pharmacology, Biochemistry and Behavior 133 (2015) 164–173
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Fasting activated histaminergic neurons and enhanced arousal effect of caffeine in mice Yi-Qun Wang a,⁎,1, Rui Li a,b,c,1, Xu Wu b,c, Fen Zhu b,c, Yohko Takata d, Ze Zhang e, Meng-Qi Zhang f, Shan-Qun Li b,c,⁎⁎, Wei-Min Qu a,e,⁎⁎⁎ a
Department of Pharmacology and Shanghai Key Laboratory of Bioactive Small Molecules, School of Basic Medical Sciences, Fudan University, Shanghai, China Department of Respiratory Medicine, Zhongshan Hospital, Fudan University, Shanghai, China Clinical Center for Sleep Breathing Disorder and Snoring, Zhongshan Hospital, Fudan University, Shanghai, China d International Institute for Integrative Sleep Medicine (WPI-IIIS), University of Tsukuba, Ibaraki, Japan e The Institutes of Brain Science, Shanghai Medical College, Fudan University, Shanghai, China f State Key Laboratory of Medical Neurobiology, School of Basic Medical Sciences, Fudan University, Shanghai, China b c
a r t i c l e
i n f o
Article history: Received 1 February 2015 Received in revised form 24 March 2015 Accepted 9 April 2015 Available online 17 April 2015 Keywords: Caffeine EEG Fasted H1R Wakefulness
a b s t r a c t Caffeine, a popular psychoactive compound, promotes wakefulness via blocking adenosine A2A receptors in the shell of the nucleus accumbens, which projects to the arousal histaminergic tuberomammillary nucleus (TMN). The TMN controls several behaviors such as wakefulness and feeding. Fasting has been reported to activate the TMN histaminergic neurons to increase arousal. Therefore, we propose that caffeine may promote greater arousal under fasting rather than normal feeding conditions. In the current study, locomotor activity recording, electroencephalogram (EEG) and electromyogram recording and c-Fos expression were used in wild type (WT) and histamine H1 receptor (H1R) knockout (KO) mice to investigate the arousal effects of caffeine under fasting conditions. Caffeine (15 mg/kg) enhanced locomotor activity in fasted mice for 5 h, but only did so for 3 h in normally fed animals. Pretreatment with the H1R antagonist pyrilamine abolished caffeine-induced stimulation on locomotor activity in fasted mice. EEG recordings confirmed that caffeine-induced wakefulness for 3 h in fed WT mice, and for 5 h in fasted ones. A stimulatory effect of caffeine was not observed in fasted H1R KO mice. Furthermore, c-Fos expression was increased in the TMN under fasting conditions. These results indicate that caffeine had greater wakefulness-promoting effects in fasted mice through the mediation of H1R. © 2015 Elsevier Inc. All rights reserved.
1. Introduction Caffeine, a constituent of coffee and other beverages, is a globally used psychoactive for arousal and diminution of fatigue. Caffeine is an antagonist at both adenosine A1 (A1Rs) and A2A receptors (A2ARs) with similar affinities (Fredholm et al., 2011), and our previous studies
Abbreviations: A2ARs, adenosine A2A receptors; AAV, adeno-associated virus; EEG, electroencephalogram; EMG, electromyogram; H1R, histamine H1 receptor; KO, knockout; LH, lateral hypothalamic area; NAc, nucleus accumbens; NREM, non-rapid eye movement; REM, rapid eye movement; TMN, tuberomammillary nucleus; WT, wild type. ⁎ Correspondence to: Y.-Q. Wang, Department of Pharmacology, School of Basic Medical Sciences, Fudan University, Shanghai 200032, China. Tel.: +86 21 54237380; fax: +86 21 54237103. ⁎⁎ Correspondence to: S.-Q. Li, Department of Respiratory Medicine, Zhongshan Hospital, Fudan University, Shanghai 200032, China. ⁎⁎⁎ Correspondence to: W.-M. Qu, Department of Pharmacology, School of Basic Medical Sciences, Fudan University, Shanghai 200032, China. E-mail addresses: [email protected] (Y.-Q. Wang), [email protected] (S.-Q. Li), [email protected] (W.-M. Qu). 1 YQW and RL contributed equally to this work.
http://dx.doi.org/10.1016/j.pbb.2015.04.003 0091-3057/© 2015 Elsevier Inc. All rights reserved.
using global A1Rs and A2ARs genetic knockout (KO) mice indicated that caffeine's promotion of wakefulness was due to A2ARs blockade, but not A1Rs (Huang et al., 2005). We used site-specific gene manipulations, including A2AR KO mice based on the Cre/loxP technology in mice and focal RNA interference, to silence A2ARs expression in rats via local infection with adeno-associated virus (AAV) carrying shorthairpin RNA specific for the A2ARs mRNA; the A2ARs in the nucleus accumbens (NAc) shell are responsible for the wakefulness-promoting effect of caffeine (Lazarus et al., 2011). With AAV-encoding humanized Renilla green fluorescent protein to trace long axonal pathways, we found that A2ARs in the NAc shell projected to the arousal nuclei, including histaminergic tuberomamillary nucleus (TMN) (Zhang et al., 2013). Thus, we suggest that histaminergic neurons in the TMN may be involved in the arousal effect of caffeine. Histaminergic neurons primarily located in the TMN control several behaviors such as feeding and wakefulness (Panula and Nuutinen, 2013). Fasting has been reported to activate histaminergic neurons in the TMN and increase arousal, allowing goal-directed behavior such as obtaining food (Valdes et al., 2010). The TMN receives projections from orexin neurons in the lateral hypothalamic area (LH),
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which coordinate sleep-wakefulness and motivated behaviors such as food seeking, especially during fasting stress (Sakurai, 2003). This region also has extensive projections to the arousal nucleus, such as the dorsal raphe, laterodorsal tegmentum and cholinergic basal forebrain regions (Brown et al., 2002; Panula et al., 1989; Valdes et al., 2010). These observations suggest that the histaminergic TMN is critical for increased arousal under fasting conditions. Therefore, we speculate that caffeine enhances this wake-promoting effect under fasting conditions via manipulating the histaminergic system. H1 receptors (H1Rs) mediate arousal in the histaminergic system of the TMN (Lin et al., 1988; Monti et al., 1986; Tokunaga et al., 2009). Thus, we measured locomotor activity, recorded electroencephalogram (EEG) and electromyogram (EMG) and used immunohistochemical method in wild type (WT) and H1R KO mice to explore the mechanisms behind caffeine-induced stimulation under fasting conditions. We observed that the histaminergic system is involved in enhancing caffeine's effect on wakefulness in fasted mice.
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Cortical EEG and EMG signals were amplified, filtered (EEG, 0.5– 30 Hz; EMG, 20–200 Hz) and then digitized at a sampling rate of 128 Hz and recorded by using SleepSign (Kissei Comtec, Nagano, Japan) as described earlier (Huang et al., 2005; Qu et al., 2010; Wang et al., 2012, 2015). When completed, polygraphic recordings were automatically scored off-line at 4-s epochs as wakefulness, rapid eye movement (REM) and non-rapid eye movement (NREM) sleep by SleepSign according to standard criteria. Finally, defined sleep-wake stages were examined visually, and corrected when necessary. 2.5. Pharmacological treatments Caffeine (Wako, Osaka, Japan) and pyrilamine (Sigma-Aldrich, Saint Louis, MO, USA) were dissolved in sterile saline immediately before intraperitoneal (i.p.) administration to the mice at 09:00 or 08:00 on the experimental day at the dose of 15 mg/kg or 10 mg/kg. For baseline data, mice were given the same volume of vehicle at 09:00 or 08:00.
2. Materials and methods 2.1. Animals Male SPF inbred C57BL/6J mice, weighing 20–26 g (11–13 weeks old), were obtained from the Laboratory Animal Center, Chinese Academy of Sciences (Shanghai, China). Male H1R KO (Huang et al., 2006; Inoue et al., 1996) and their WT mice of the inbred C57BL/6 strain were generated from heterozygotes, weighing 20–26 g (11–13 weeks old), were maintained at Oriental Bioservice (Kyoto, Japan) and used in these experiments. Animals were housed at an ambient temperature of 22 ± 0.5°C at a relative humidity of 60 ± 2% and an automatically controlled 12 h light/12 h dark cycle (lights on at 07:00, illumination intensity ≈ 100 lux). Animals had ad libitum access to food and water. All experimental protocols were approved by the Animal Administrative Committee of Shanghai and Animal Care Committee of Osaka Bioscience Institute. Every effort was made to minimize the number of animals used and to minimize pain and discomfort experienced by the animals. 2.2. Fasting Mice were kept as depicted above. Food was removed from the cages of experimental mice at dark onset at 19:00. 2.3. Locomotor activity recordings Locomotor activity for an individual mouse was measured with a passive infrared sensor (Biotex, Kyoto, Japan) placed 17.5 cm above the floor of the recording cage (28 cm × 16.5 cm × 13 cm) as previously reported (Liu et al., 2012). 2.4. Polygraphic recordings and vigilance state analysis Under pentobarbital anesthesia (50 mg/kg, i.p.), mice were implanted with electrodes for polysomnographic EEG and EMG recordings. Two stainless steels screws (1 mm in diameter) were inserted through the skull (antero-posterior, + 1.0 mm; left-right, − 1.5 mm from the bregma or lambda) according to the mouse brain atlas; these screws served as EEG electrodes. Two Teflon-coated, insulated stainless steel wires were placed bilaterally into both trapezius muscles, and these served as the EMG electrodes. All electrodes were attached to a microconnector and fixed onto the skull using dental cement. After a 10-day recovery time, each mouse was transferred to a sound-proof recording chamber and connected to an EEG/EMG recording cable for a 4-day period of habituation to the experimental environment. Then the polygraphic recordings were recorded for 48 h in freely moving mice.
2.6. c-Fos immunohistochemistry Four groups of mice were studied. Each group was given caffeine (15 mg/kg, i.p.), or vehicle at 09:00, respectively, under fed or fasted conditions. Next 5 h after caffeine or vehicle administration, animals were anesthetized with 10% chloral hydrate and perfused via the heart with saline solution followed by ice-cold 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Brains were removed, post-fixed in 4% paraformaldehyde for 6 h and then immersed in 30% sucrose overnight. Thereafter, frozen sections of brain were cut in coronal planes, at a thickness of 30 μm with a freezing microtome (Microm HM 525, Thermoscientific, Germany). Sections were stored in a cryo-protectant solution at 20°C for histological analysis. Immunohistochemistry was performed on free-floating sections as described previously. In brief, sections were incubated with 0.3% hydrogen peroxide in 0.1 M phosphate-buffered saline (PBS, pH 7.4) for 15 min to quench endogenous peroxidase activity. After washing in PBS, sections were treated at room temperature in 3% normal donkey serum and 0.25% Triton X-100 in PBS (PBS-T) for 1 h, followed by primary rabbit anti-c-Fos (1:5000, CalBiochem, San Diego, CA, USA) antibody diluted in PBS-T with 0.02% sodium azide, overnight. After overnight incubation with the primary antisera, sections were rinsed and incubated for 2 h in biotinylated anti-rabbit secondary antiserum (Jackson ImmunoResearch, West Grove, PA, USA) at a dilution of 1:1000. All sections were then treated with avidin–biotin–peroxidase complex (1:1000, Vectastain ABC Elite kit, Vector Laboratories, Burlingame, CA, USA) for 1 h. The peroxidase reaction was visualized with 0.05% 3,3′diaminobenzidine (Sigma-Aldrich, Saint Louis, MO, USA) in 0.1 M PB and 0.01% hydrogen peroxide. After terminating the reaction with PBS-azide, sections were mounted, dehydrated and covered. Adjacent sections were incubated as controls, without the primary antibody to confirm a lack of non-specific staining. Sections were examined under bright-field illumination using an Olympus BX51 microscope (Japan). Images were captured with a digital camera (DP72, Olympus, Japan).
2.7. Statistical analysis All results are expressed as means ± SEM. For vigilance studies, amounts of sleep-wake states were expressed in minutes. The statistical significance of time course data for locomotor activity, sleep amount, stage transition, number of each stage bouts and mean duration was assessed with a two-tailed unpaired Student's t-test. Differences in drug treatment between fed and fasted mice were compared with a two-way ANOVA (with food and drug treatment as factors) followed by a post hoc Fisher's probable least-squares difference when appropriate. In all cases, p b 0.05 was the level of significance.
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3. Results 3.1. Caffeine prolonged locomotor activity in fasted mice As shown in Fig. 1A, the change in time course of locomotor activity revealed that caffeine at 15 mg/kg increased locomotor activity in fed mice for 3 h [09:00: t(34) = 4.63, p b 0.01; 10:00: t(34) = 8.80, p b 0.01; 11:00: t(34) = 4.56, p b 0.01] after administration at 09:00 compared with vehicle control. Fig. 1B summarizes locomotor activity in fasted mice treated with vehicle or caffeine. Compared with vehicle control, caffeine prolonged locomotor activity to 5 h [09:00: t(32) = 2.81, p b 0.01; 10:00: t(32) = 4.81, p b 0.01; 11:00: t(32) = 3.70, p b 0.01; 12:00: t(32) = 2.85, p b 0.01; 13:00: t(32) = 2.23, p b 0.01]. As the extended time of fasted WT mice is 2 h from 12:00 to 14:00, total locomotor activity was calculated for 3 h (during 09:00–12:00) and consecutive 2 h (during 12:00–14:00) in fed and fasted mice after caffeine injections (Fig. 1C and D). During the period of 09:00–12:00, caffeine significantly increased total amount of locomotor activity by 2.2-fold [t(34) = 7.90, p b 0.01] in fed mice and 1.6-fold [t(32) = 4.41, p b 0.01] in fasted mice, respectively, compared with the vehicle controls (Fig. 1C). During the subsequent 2-h period, caffeine increased locomotor activity by 2.9-fold [t(32) = 3.81, p b 0.05] in fasted mice but had no effects in fed animals (Fig. 1D). These results indicate that fasting prolonged caffeine-induced stimulation of locomotor activity in mice. 3.2. An H1R antagonist inhibited caffeine-induced potentiation on locomotor activity in fasted mice To investigate the contribution of H1R to the enhanced effect on locomotor activity of caffeine in fasted mice, we administered H1R antagonist pyrilamine (10 mg/kg) at 8:00, 1 h before caffeine administration.
This pretreatment had no effect in fed caffeine-treated mice (Fig. 2A and C). However, compared with vehicle control, pretreatment with pyrilamine significantly inhibited locomotor activity induced by caffeine in fasted mice shortened from 5 to 3 h (Fig. 2B and D). These results indicate that caffeine prolonged locomotor activity in fasted mice via H1R. 3.3. Caffeine increased wakefulness for 3 h in fed WT mice, while for 5 h in fasted WT mice Compared with vehicle control, caffeine markedly increased wakefulness in fed WT mice, and this was sustained for 3 h [09:00: t(10) = 5.43, p b 0.01; 10:00: t(10) = 17.57, p b 0.01; 11:00: t(10) = 4.32, p b 0.01] (Fig. 3A), whereas this effect was sustained for 5 h [09:00: t(9) = 3.22, p b 0.05; 10:00: t(9) = 11.17, p b 0.01; 11:00: t(9) = 3.02, p b 0.05; 12:00: t(9) = 3.45, p b 0.01; 13:00: t(9) = 2.86, p b 0.05] in fasted WT mice (Fig. 3B). These increases in wakefulness coincided with decreased NREM [fed mice: 09:00: t(10) = 5.61, p b 0.01; 10:00: t(10) = 14.89, p b 0.01; 11:00: t(10) = 3.94, p b 0.01; fasted mice: 09:00: t(9) = 3.24, p b 0.05; 10:00: t(9) = 11.45, p b 0.01; 11:00: t(9) = 3.04, p b 0.05; 12:00: t(9) = 4.85, p b 0.01; 13:00: t(9) = 3.74, p b 0.01] and REM sleep [fed mice: 10:00: t(10) = 7.79, p b 0.01; 11:00: t(10) = 3.21, p b 0.01; fasted mice: 10:00: t(9) = 3.59, p b 0.01; 11:00: t(9) = 2.62, p b 0.05] in both animal types after caffeine treatment (Fig. 3A and B). The sleep architecture during the subsequent period in both fed and fasted WT mice was not disrupted (Fig. 3A and B). The total time spent in wakefulness, NREM and REM sleep was calculated for 3 h (09:00–12:00) and a subsequent 2 h (12:00–14:00) in fed and fasted WT mice after caffeine administration. Fig. 3C shows that, during 09:00–12:00, the total amount of wakefulness after caffeine
Fig. 1. Fasting enhanced locomotor activity induced by caffeine in WT mice. Time course changes in locomotor activity in mice treated with caffeine (15 mg/kg) at 09:00 under fed (A) or fasted (B) conditions. Total locomotor activity during 3 h (09:00–12:00) (C) and 2 h (12:00–14:00) (D) in fed and fasted conditions after caffeine or vehicle administration. Values are means ± SEM, n = 17–18. *p b0.05 and **p b 0.01 indicate significant differences between caffeine-treated group and corresponding vehicle control (two-tailed unpaired Student's t-test).
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Fig. 2. Pyrilamine abolished fasting's augmentation of caffeine-induced locomotor activity in WT mice. Time course of locomotor activity in mice treated with vehicle (A, B) or pyrilamine (C, D) at 8:00, as indicated by the open arrow, and vehile or caffeine at 9:00, as indicated by the closed arrow, under fed or fasted conditions. Values are means ± SEM, n = 14–17. *p b 0.05 and **p b 0.01 indicate significant differences between caffeine-treated group and corresponding vehicle control (two-tailed unpaired Student's t-test).
injection increased in both fed [t(10) = 12.71, p b 0.01] and fasted WT mice [t(9) = 8.70, p b 0.01]. During the next 2 h, caffeine enhanced the amount of wakefulness by 1.0-fold in fasted mice [t(9) = 4.84, p b 0.01], but not in fed mice (Fig. 3D). The concomitant decreases in the total amount of NREM sleep in fed and fasted WT mice [fed mice: t(10) = 10.00, p b 0.01; fasted mice: t(9) = 8.98, p b 0.01] during 09:00–12:00, and in fasted mice [t(9) = 6.75, p b 0.01] during 12:00– 14:00 were observed (Fig. 3C and D). The amount of REM sleep was decreased after caffeine treatment in both fed [t(10) = 4.68, p b 0.01] and fasted [t(9) = 6.34, p b 0.01] WT mice during 3 h after caffeine administration (Fig. 3C). These results indicate that caffeine strengthened its arousal effects under fasting conditions. 3.4. Effects of caffeine on characteristics of sleep-wake episodes, numbers of wakefulness bouts and NREM sleep latency in fed and fasted WT mice To understand sleep architecture changes caused by caffeine under fasting conditions, we measured the mean duration and episode numbers, numbers of wakefulness bouts and NREM sleep latency during 09:00–12:00 in both fed and fasted WT mice, and during 12:00–14:00 in fasted WT mice. Compared to vehicle control, mean durations of wakefulness increased [fed mice: t(10) = 4.40, p b 0.05; fasted mice: t(9) = 3.16, p b 0.05] and of NREM sleep decreased [fed mice: t(10) = 2.59, p b 0.05; fasted mice: t(9) = 4.42, p b 0.01] during the 3 h after caffeine administration in both fed and fasted WT mice, but wakefulness increased more and NREM sleep decreased more in fasted mice compared to fed mice (Fig. 4A and B). During the next 12:00–14:00, fasting also induced greater mean duration of wakefulness after caffeine administration [t(9) = 2.79, p b 0.05]. Compared to vehicle controls, caffeine significantly decreased the episode numbers of wakefulness [fed mice: t(10) = 5.35, p b 0.01; fasted mice: t(9) = 4.98, p b 0.01], NREM [fed mice: t(10) = 5.42, p b 0.01; fasted mice: t(9) = 4.94, p b 0.01] and
REM sleep [fed mice: t(10) = 7.01, p b 0.01; fasted mice: t(9) = 4.44, p b 0.01] in both fed and fasted WT mice during 09:00–12:00 (Fig. 4A and B), but did not make any changes in episode numbers in fasted mice during 12:00–14:00 (Fig. 4C). EEG power spectra analysis revealed that caffeine induced the same change of EEG power density of NREM and REM sleep in fed and fasted WT mice during 09:00–12:00 (data not shown). These findings indicate that caffeine induced longer and stronger effect on wakefulness under fasted conditions. Compared with vehicle controls, the numbers of wakefulness bouts of 0–16 s [t(10) = 3.68, p b 0.01], 16–32 s [t(10) = 4.24, p b 0.01], 32–64 s [t(10) = 3.72, p b 0.01] and 64–128 s [t(10) = 2.77, p b 0.05] were decreased significantly after caffeine administration in fed WT mice during 09:00–12:00, but only reduced the bouts of 0–16 s [t(9) = 4.21, p b 0.01], 16–32 s [t(9) = 3.17, p b 0.05] and 32–64 s [t(9) = 3.36, p b 0.01] in the fasted WT mice during the same period (Fig. 4D and E). During the next 2 h, caffeine did not change the number of bouts in fasted WT mice (Fig. 4 F). As shown in Fig. 4G and H, during 09:00–12:00, the injection of caffeine remarkably prolonged NREM latency [fed mice: t(10) = 5.77, p b 0.01; fasted mice: t(9) = 8.72, p b 0.01], defined as the time from the vehicle or caffeine injection to the appearance of the first NREM sleep episode lasting for at least 20 s both in fed and fasted WT mice. Caffeine's effect in fasted mice was stronger than in fed mice. There is not significant change of NREM latency in the next 2 h after caffeine injection in fasted WT mice (Fig. 4I). The longer NREM sleep latency observed in caffeine-treated fasted WT mice suggests that fasting delays the initiation of NREM sleep. 3.5. Caffeine increased wakefulness for 3 h in fed and for 2 h in fasted H1R KO mice Compared with vehicle controls, caffeine markedly increased wakefulness after administration in fed H1R KO mice and was sustained for
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Fig. 3. Effects of fasting on sleep-wake profiles induced by caffeine in WT mice. (A, B) Time course changes in wakefulness, NREM and REM sleep in fed (A) and fasted (B) WT mice treated with caffeine. Circles represent the hourly mean amount of each stage. Open and closed circles represent profiles of vehicle and caffeine treatments, respectively. Horizontal filled and open bars on χ-axes indicate 12 h dark and 12 h light periods. (C, D) Total time spent in wakefulness, NREM and REM sleep during the 3 h (09:00–12:00) (C) or the 2 h (12:00–14:00) (D) in fed and fasted WT mice after administration of vehicle or caffeine. Open and filled bars depict vehicle and caffeine treatments, respectively. Values are means ± SEM (n = 5–7). *p b 0.05 and **p b 0.01 indicate significant differences between the caffeine-treated group and corresponding vehicle control (two-tailed unpaired Student's t-test).
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Fig. 4. Mean durations and episode numbers (A, B, C), numbers of NREM and REM sleep bouts (D, E, F) and NREM sleep latency (G, H, I) during 3 h (09:00–12:00) in fed mice and during 3 h (09:00–12:00) and 2 h (12:00–14:00) in fasted WT mice after administration of vehicle or caffeine. Open and filled bars depict vehicle and caffeine treatments, respectively. Values are means ± SEM (n = 5–7). *p b 0.05 and **p b 0.01 indicate significantly differences between caffeine-treated group and its corresponding vehicle control (two-tailed unpaired Student's t-test).
3 h [09:00: t(14) = 3.71, p b 0.01; 10:00: t(14) = 14.36, p b 0.01; 11:00: t(14) = 6.64, p b 0.01] (Fig. 5A). Whereas this enhanced effect of caffeine was shortened to 2 h in fasted H1R KO mice [09:00: t(8) = 3.48, p b 0.01; 10:00: t(8) = 17.21, p b 0.01] (Fig. 5B). These increases in wakefulness were concomitant with the decreases in NREM [fed mice: 09:00: t(14) = 3.85, p b 0.01; 10:00: t(14) = 12.95, p b 0.01; 11:00: t(14) = 6.90, p b 0.01; fasted mice: 09:00: t(8) = 3.59, p b 0.01; 10:00: t(8) = 18.79, p b 0.01] and REM sleep [fed mice: 10:00: t(14) = 25.80, p b 0.01; 11:00: t(14) = 3.83, p b 0.01; fasted mice: 10:00: t(8) = 5.20, p b 0.01] in both fed and fasted H1R KO mice after caffeine treatment (Fig. 5A and B). Moreover, the sleep architecture during the subsequent period in both fed and fasted ND mice was not disrupted (Fig. 5A and B).
The total time spent in wakefulness, NREM and REM sleep was calculated for 3 h in fed and fasted H1R KO mice after caffeine administration. As shown in Fig. 5C, the total amount of wakefulness increased by 1.1-fold [t(14) = 10.96, p b 0.01] and 1.0-fold [t(8) = 6.19, p b 0.01], respectively, in fed and fasted H1R KO mice during 3 h after caffeine injection. The total amount of NREM sleep was decreased in fed H1R KO mice by 80.3% [t(14) = 10.58, p b 0.01] and in fasted H1R KO mice by 69.9% [t(8) = 6.02, p b 0.01], respectively (Fig. 5C). The amount of REM sleep was also decreased after caffeine treatment both in fed and fasted H1R KO mice [fed mice: t(14) = 10.35, p b 0.01; fasted mice: t(8) = 5.91, p b 0.01] (Fig. 5C). During the next 2 h from 12:00 to 14:00, caffeine did not have any effects on the amount of wakefulness, NREM and REM sleep in neither fed nor fasted H1R KO mice (Fig. 5D). These
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Fig. 5. Effects of fasting on sleep-wake profiles induced by caffeine in H1R KO mice. (A, B) Time course changes in wakefulness, NREM and REM sleep in fed (A) and fasted (B) H1R KO mice treated with caffeine. Each circle represents the hourly mean amount of each stage. Open and closed circles represent profiles of vehicle and caffeine treatments, respectively. Horizontal filled and open bars on the χ-axes indicate 12 h dark and 12 h light periods, respectively. (C, D) Total time spent in wakefulness, NREM and REM sleep during the 3 h (09:00–12:00) (C) or the 2 h (12:00–14:00) (D) in fed and fasted H1R KO mice after administration of vehicle or caffeine. Open and filled bars depict profiles of vehicle and caffeine treatments, respectively. Values are means ± SEM (n = 5–8). **p b 0.01 indicates significant differences between the caffeine-treated group and its corresponding vehicle control (two-tailed unpaired Student's t-test).
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results indicate that through the mediation of H1R, caffeine enhanced its arousal effect under fasting conditions. 3.6. Effects of caffeine on characteristics of sleep-wake episodes, numbers of wakefulness bouts and NREM sleep latency in fed and fasted H1R KO mice The mean duration and episode number, numbers of wakefulness bouts and NREM latency for 3 h after caffeine administration in fed and fasted H1R KO mice were also analyzed. Compared to vehicle controls, mean durations of wakefulness were increased [fed mice: t(14) = 3.48, p b 0.05; fasted mice: t(8) = 3.00, p b 0.05] after caffeine administration in fed and fasted H1R KO mice during 09:00–12:00
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(Fig. 6A and B). Caffeine also significantly decreased the episode numbers of wakefulness [fed mice: t(14) = 5.53, p b 0.01; fasted mice: t(8) = 4.43, p b 0.01], NREM [fed mice: t(14) = 5.51, p b 0.01; fasted mice: t(8) = 4.33, p b 0.01] and REM sleep [fed mice: t(14) = 9.23, p b 0.01; fasted mice: t(8) = 7.23, p b 0.01] both in fed and fasted H1R KO mice (Fig. 6A and B). EEG power spectra analysis revealed that caffeine induced the same changes of EEG power density of NREM and REM sleep in the fed and fasted H1R KO mice (data not shown). These findings indicate that H1R mediated the longer and stronger effect on wakefulness under fasted conditions induced by caffeine. Compared with vehicle controls, the numbers of wakefulness bouts of 0–16 s [fed mice: t(14) = 8.65, p b 0.01; fasted mice: t(8) = 4.12,
Fig. 6. Mean durations and episode numbers (A, B), numbers of NREM and REM sleep bouts (C, D) and NREM sleep latency (E, F) during the 3 h in fed and fasted H1R KO mice after administration of vehicle or caffeine. Open and filled bars depict profiles for the vehicle and caffeine treatments, respectively. Values are means ± SEM (n = 5–8). *p b 0.05 and **p b 0.01 indicate significant differences between the caffeine-treated group and its corresponding vehicle control (two-tailed unpaired Student's t-test).
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p b 0.05] and 16–32 s [fed mice: t(14) = 2.66, p b 0.01; fasted mice: t(8) = 3.94, p b 0.01] were decreased significantly after caffeine administration in fed and fasted H1R KO mice during 9:00–12:00 (Fig. 6C and D). Also, caffeine induced a longer NREM latency in both fed and fasted H1R KO mice [fed mice: t(14) = 5.60, p b 0.01; fasted mice: t(8) = 6.72, p b 0.01] (Fig. 6E and F). These results suggested that fasting delays the initiation of NREM sleep after caffeine injection via H1R. 3.7. Effects of fasting on c-Fos expression in wake-promoting neurons in the TMN Fig. 7 A (a–h) depicts representative photomicrographs of c-Fos expression of the TMN in fed and fasted WT mice treated with vehicle or caffeine. Then, the number of c-Fos immunoreactive nuclei was analyzed. A two-way ANOVA confirmed that fasting had significantly effects as determined by the expression of c-Fos in the TMN [F(1,22) = 77.93, p b 0.01] (Fig. 7B). Compared with fed mice, further analysis showed that fasted mice given the vehicle increased the c-Fos expression in TMN (p b 0.01). However, the effects of caffeine on the expression of c-Fos were not obvious under fed or fasted conditions at 5 h after its administration compared with vehicle controls. Therefore, fasting activated the neurons in TMN, but caffeine is not sufficient to enhance the protein expression of c-Fos. 4. Discussion Histaminergic neurons in the TMN regulate various cerebral functions, such as wakefulness, drinking and feeding, sensation and motor activity (Masaki and Yoshimatsu, 2007; Panula and Nuutinen, 2013; Valdes et al., 2005). The immunohistochemistry results of c-Fos staining in our study showed that fasting induced more c-Fos positive neurons in the TMN. These results are in agreement with previous works (Valdes et al., 2010). Fasting also activates orexin neurons in the LH (Diano et al., 2003), and it coordinates sleep-wakefulness and motivated behaviors such as food seeking, especially during fasting stress (Sakurai, 2003, 2014). Orexin neuron-ablated mice did not have fasting-induced arousal, suggesting that orexin neurons are needed to evoke adaptive behavioral arousal during fasting (Akiyama et al., 2004). LH, as a feeding center, projects afferents to the histaminergic TMN (Huang et al., 2001; Sakurai, 1999). Histaminergic neurons in the TMN regulate wakefulness during which they discharge tonically and specifically (Vanni-Mercier et al., 2003). Histamine exerts its effects through four receptors (H1, H2, H3 and H4), which are primarily found in the brain. Previous research shows that potentiating brain histaminergic activity induces wakefulness and reduces sleep via regulation of the H1R (Lin et al.,
1988; Monti et al., 1986; Tokunaga et al., 2009). H1R antagonists can induce a robust sedative-hypnotic effect (Wang et al., 2015). The histaminergic and orexinergic systems are reciprocally connected in an excitatory fashion to regulate wakefulness, our group observed that wakefulness-promoting effect of orexin depends histaminergic neurons activation mediated through H1R (Huang et al., 2001). These findings suggest that fasting has an arousal effect that is dependent on activation of the histaminergic system via H1R. Caffeine is a central nervous system stimulant. In the cellular level, this drug is the inhibitor of adenosine receptor in the central nervous system. There are three kinds of adenosine receptors that are A1, A2 (including A2A and A2B subtype) and A3 receptor. A1Rs are coupled to Gi protein, which could effectively inhibit adenylate cyclase and some calcium channels (N type and Q type), through which to activate phospholipase and some potassium channels (Fredholm et al., 2005; Huang et al., 2014; Jacobson and Gao, 2006). On the contrary, A2ARs are coupled to Gs protein, which activate L type calcium channel and adenylate cyclase (Fredholm et al., 2005; Jacobson and Gao, 2006). We demonstrated previously that the wakefulness-promoting effect of caffeine is mediated by the A2AR, but not the A1R (Huang et al., 2005). Later, A2ARs in the shell region of the NAc were later reported to be responsible for the effect of caffeine on wakefulness (Lazarus et al., 2011). Likely, blockade of the massive GABAergic output of NAc shell neurons activates classical arousal centers, such as the LH and, the TMN, via direct or indirect projections from the NAc shell (Lazarus et al., 2011; Masaki and Yoshimatsu, 2007). Our data showed that caffeine increased wakefulness and locomotor activity in H1R antagonist-treated or H1R KO mice. Therefore, the NAc shell-LH-TMN pathway is not the most crucial pathway for the arousal effect of caffeine. With fasting condition, orexin was increased (Sakurai, 1999) and the TMN and LH were activated (Valdes et al., 2005), suggesting that this NAc shell-LH-TMN pathway is responsible for the enhanced arousal effect of caffeine. In addition, fasting also induces wakefulness via H1R. So histaminergic system is involved in the enhanced effect of caffeine in locomotor activity and wakefulness. 5. Conclusion These findings indicate fasting could enhance the wakefulnesspromoting effect of caffeine involved the mediation of H1R. Acknowledgements The authors thank Dr Zhi-Li Huang (Department of Pharmacology, School of Basic Medical Sciences, Fudan University) for his comments
Fig. 7. Effect of fasting on c-Fos immunoreactivity in the TMN. Representative photomicrographs of c-Fos immunostaining in the TMN. (A) Representative photomicrographs of c-Fos immunostaining in the TMN of a mouse treated with vehicle (a, c, e and g) and caffeine (b, d, f and h) under fed or fasted conditions. e, f, g and h (scale bars: 50 μm): high-magnification views of the rectangular areas marked in a, b, c and d (scale bars: 200 μm), respectively. (B) The number of c-Fos-immunoreactive neurons in TMN after vehicle or caffeine treatment. Values are means ± SEM (n = 6–8). **p b 0.01 indicates significant differences from the vehicle-treated fed mice (two-way ANOVA followed by a probable least-squares difference test). 3 V: third ventricle.
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and help in the preparation of the manuscript. This study was supported in part by grants-in-aid for scientific research from the National Natural Science Foundation of China (J1210041, 31171010, 31171049, 31121061, 31271164, 31471064, 81420108015), the National Basic Research Program of China (2011CB711000, 2015CB856401, 2009ZX09303-006), the Fundamental Research Funds for the Central Universities (10FX041), a key laboratory program of the Education Commission of Shanghai Municipality (ZDSYS14005), the Shanghai Committee of Science and Technology (13140903100, 14JC1400900, 13dz2260700), the Shanghai Leading Academic Discipline Project (B119), PhD, and the Programs Foundation of Ministry of Education of China (20110071110033).
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Pharmacology, Biochemistry and Behavior journal homepage: www.elsevier.com/locate/pharmbiochembeh
Fasting activated histaminergic neurons and enhanced arousal effect of caffeine in mice Yi-Qun Wang a,⁎,1, Rui Li a,b,c,1, Xu Wu b,c, Fen Zhu b,c, Yohko Takata d, Ze Zhang e, Meng-Qi Zhang f, Shan-Qun Li b,c,⁎⁎, Wei-Min Qu a,e,⁎⁎⁎ a
Department of Pharmacology and Shanghai Key Laboratory of Bioactive Small Molecules, School of Basic Medical Sciences, Fudan University, Shanghai, China Department of Respiratory Medicine, Zhongshan Hospital, Fudan University, Shanghai, China Clinical Center for Sleep Breathing Disorder and Snoring, Zhongshan Hospital, Fudan University, Shanghai, China d International Institute for Integrative Sleep Medicine (WPI-IIIS), University of Tsukuba, Ibaraki, Japan e The Institutes of Brain Science, Shanghai Medical College, Fudan University, Shanghai, China f State Key Laboratory of Medical Neurobiology, School of Basic Medical Sciences, Fudan University, Shanghai, China b c
a r t i c l e
i n f o
Article history: Received 1 February 2015 Received in revised form 24 March 2015 Accepted 9 April 2015 Available online 17 April 2015 Keywords: Caffeine EEG Fasted H1R Wakefulness
a b s t r a c t Caffeine, a popular psychoactive compound, promotes wakefulness via blocking adenosine A2A receptors in the shell of the nucleus accumbens, which projects to the arousal histaminergic tuberomammillary nucleus (TMN). The TMN controls several behaviors such as wakefulness and feeding. Fasting has been reported to activate the TMN histaminergic neurons to increase arousal. Therefore, we propose that caffeine may promote greater arousal under fasting rather than normal feeding conditions. In the current study, locomotor activity recording, electroencephalogram (EEG) and electromyogram recording and c-Fos expression were used in wild type (WT) and histamine H1 receptor (H1R) knockout (KO) mice to investigate the arousal effects of caffeine under fasting conditions. Caffeine (15 mg/kg) enhanced locomotor activity in fasted mice for 5 h, but only did so for 3 h in normally fed animals. Pretreatment with the H1R antagonist pyrilamine abolished caffeine-induced stimulation on locomotor activity in fasted mice. EEG recordings confirmed that caffeine-induced wakefulness for 3 h in fed WT mice, and for 5 h in fasted ones. A stimulatory effect of caffeine was not observed in fasted H1R KO mice. Furthermore, c-Fos expression was increased in the TMN under fasting conditions. These results indicate that caffeine had greater wakefulness-promoting effects in fasted mice through the mediation of H1R. © 2015 Elsevier Inc. All rights reserved.
1. Introduction Caffeine, a constituent of coffee and other beverages, is a globally used psychoactive for arousal and diminution of fatigue. Caffeine is an antagonist at both adenosine A1 (A1Rs) and A2A receptors (A2ARs) with similar affinities (Fredholm et al., 2011), and our previous studies
Abbreviations: A2ARs, adenosine A2A receptors; AAV, adeno-associated virus; EEG, electroencephalogram; EMG, electromyogram; H1R, histamine H1 receptor; KO, knockout; LH, lateral hypothalamic area; NAc, nucleus accumbens; NREM, non-rapid eye movement; REM, rapid eye movement; TMN, tuberomammillary nucleus; WT, wild type. ⁎ Correspondence to: Y.-Q. Wang, Department of Pharmacology, School of Basic Medical Sciences, Fudan University, Shanghai 200032, China. Tel.: +86 21 54237380; fax: +86 21 54237103. ⁎⁎ Correspondence to: S.-Q. Li, Department of Respiratory Medicine, Zhongshan Hospital, Fudan University, Shanghai 200032, China. ⁎⁎⁎ Correspondence to: W.-M. Qu, Department of Pharmacology, School of Basic Medical Sciences, Fudan University, Shanghai 200032, China. E-mail addresses: [email protected] (Y.-Q. Wang), [email protected] (S.-Q. Li), [email protected] (W.-M. Qu). 1 YQW and RL contributed equally to this work.
http://dx.doi.org/10.1016/j.pbb.2015.04.003 0091-3057/© 2015 Elsevier Inc. All rights reserved.
using global A1Rs and A2ARs genetic knockout (KO) mice indicated that caffeine's promotion of wakefulness was due to A2ARs blockade, but not A1Rs (Huang et al., 2005). We used site-specific gene manipulations, including A2AR KO mice based on the Cre/loxP technology in mice and focal RNA interference, to silence A2ARs expression in rats via local infection with adeno-associated virus (AAV) carrying shorthairpin RNA specific for the A2ARs mRNA; the A2ARs in the nucleus accumbens (NAc) shell are responsible for the wakefulness-promoting effect of caffeine (Lazarus et al., 2011). With AAV-encoding humanized Renilla green fluorescent protein to trace long axonal pathways, we found that A2ARs in the NAc shell projected to the arousal nuclei, including histaminergic tuberomamillary nucleus (TMN) (Zhang et al., 2013). Thus, we suggest that histaminergic neurons in the TMN may be involved in the arousal effect of caffeine. Histaminergic neurons primarily located in the TMN control several behaviors such as feeding and wakefulness (Panula and Nuutinen, 2013). Fasting has been reported to activate histaminergic neurons in the TMN and increase arousal, allowing goal-directed behavior such as obtaining food (Valdes et al., 2010). The TMN receives projections from orexin neurons in the lateral hypothalamic area (LH),
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which coordinate sleep-wakefulness and motivated behaviors such as food seeking, especially during fasting stress (Sakurai, 2003). This region also has extensive projections to the arousal nucleus, such as the dorsal raphe, laterodorsal tegmentum and cholinergic basal forebrain regions (Brown et al., 2002; Panula et al., 1989; Valdes et al., 2010). These observations suggest that the histaminergic TMN is critical for increased arousal under fasting conditions. Therefore, we speculate that caffeine enhances this wake-promoting effect under fasting conditions via manipulating the histaminergic system. H1 receptors (H1Rs) mediate arousal in the histaminergic system of the TMN (Lin et al., 1988; Monti et al., 1986; Tokunaga et al., 2009). Thus, we measured locomotor activity, recorded electroencephalogram (EEG) and electromyogram (EMG) and used immunohistochemical method in wild type (WT) and H1R KO mice to explore the mechanisms behind caffeine-induced stimulation under fasting conditions. We observed that the histaminergic system is involved in enhancing caffeine's effect on wakefulness in fasted mice.
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Cortical EEG and EMG signals were amplified, filtered (EEG, 0.5– 30 Hz; EMG, 20–200 Hz) and then digitized at a sampling rate of 128 Hz and recorded by using SleepSign (Kissei Comtec, Nagano, Japan) as described earlier (Huang et al., 2005; Qu et al., 2010; Wang et al., 2012, 2015). When completed, polygraphic recordings were automatically scored off-line at 4-s epochs as wakefulness, rapid eye movement (REM) and non-rapid eye movement (NREM) sleep by SleepSign according to standard criteria. Finally, defined sleep-wake stages were examined visually, and corrected when necessary. 2.5. Pharmacological treatments Caffeine (Wako, Osaka, Japan) and pyrilamine (Sigma-Aldrich, Saint Louis, MO, USA) were dissolved in sterile saline immediately before intraperitoneal (i.p.) administration to the mice at 09:00 or 08:00 on the experimental day at the dose of 15 mg/kg or 10 mg/kg. For baseline data, mice were given the same volume of vehicle at 09:00 or 08:00.
2. Materials and methods 2.1. Animals Male SPF inbred C57BL/6J mice, weighing 20–26 g (11–13 weeks old), were obtained from the Laboratory Animal Center, Chinese Academy of Sciences (Shanghai, China). Male H1R KO (Huang et al., 2006; Inoue et al., 1996) and their WT mice of the inbred C57BL/6 strain were generated from heterozygotes, weighing 20–26 g (11–13 weeks old), were maintained at Oriental Bioservice (Kyoto, Japan) and used in these experiments. Animals were housed at an ambient temperature of 22 ± 0.5°C at a relative humidity of 60 ± 2% and an automatically controlled 12 h light/12 h dark cycle (lights on at 07:00, illumination intensity ≈ 100 lux). Animals had ad libitum access to food and water. All experimental protocols were approved by the Animal Administrative Committee of Shanghai and Animal Care Committee of Osaka Bioscience Institute. Every effort was made to minimize the number of animals used and to minimize pain and discomfort experienced by the animals. 2.2. Fasting Mice were kept as depicted above. Food was removed from the cages of experimental mice at dark onset at 19:00. 2.3. Locomotor activity recordings Locomotor activity for an individual mouse was measured with a passive infrared sensor (Biotex, Kyoto, Japan) placed 17.5 cm above the floor of the recording cage (28 cm × 16.5 cm × 13 cm) as previously reported (Liu et al., 2012). 2.4. Polygraphic recordings and vigilance state analysis Under pentobarbital anesthesia (50 mg/kg, i.p.), mice were implanted with electrodes for polysomnographic EEG and EMG recordings. Two stainless steels screws (1 mm in diameter) were inserted through the skull (antero-posterior, + 1.0 mm; left-right, − 1.5 mm from the bregma or lambda) according to the mouse brain atlas; these screws served as EEG electrodes. Two Teflon-coated, insulated stainless steel wires were placed bilaterally into both trapezius muscles, and these served as the EMG electrodes. All electrodes were attached to a microconnector and fixed onto the skull using dental cement. After a 10-day recovery time, each mouse was transferred to a sound-proof recording chamber and connected to an EEG/EMG recording cable for a 4-day period of habituation to the experimental environment. Then the polygraphic recordings were recorded for 48 h in freely moving mice.
2.6. c-Fos immunohistochemistry Four groups of mice were studied. Each group was given caffeine (15 mg/kg, i.p.), or vehicle at 09:00, respectively, under fed or fasted conditions. Next 5 h after caffeine or vehicle administration, animals were anesthetized with 10% chloral hydrate and perfused via the heart with saline solution followed by ice-cold 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Brains were removed, post-fixed in 4% paraformaldehyde for 6 h and then immersed in 30% sucrose overnight. Thereafter, frozen sections of brain were cut in coronal planes, at a thickness of 30 μm with a freezing microtome (Microm HM 525, Thermoscientific, Germany). Sections were stored in a cryo-protectant solution at 20°C for histological analysis. Immunohistochemistry was performed on free-floating sections as described previously. In brief, sections were incubated with 0.3% hydrogen peroxide in 0.1 M phosphate-buffered saline (PBS, pH 7.4) for 15 min to quench endogenous peroxidase activity. After washing in PBS, sections were treated at room temperature in 3% normal donkey serum and 0.25% Triton X-100 in PBS (PBS-T) for 1 h, followed by primary rabbit anti-c-Fos (1:5000, CalBiochem, San Diego, CA, USA) antibody diluted in PBS-T with 0.02% sodium azide, overnight. After overnight incubation with the primary antisera, sections were rinsed and incubated for 2 h in biotinylated anti-rabbit secondary antiserum (Jackson ImmunoResearch, West Grove, PA, USA) at a dilution of 1:1000. All sections were then treated with avidin–biotin–peroxidase complex (1:1000, Vectastain ABC Elite kit, Vector Laboratories, Burlingame, CA, USA) for 1 h. The peroxidase reaction was visualized with 0.05% 3,3′diaminobenzidine (Sigma-Aldrich, Saint Louis, MO, USA) in 0.1 M PB and 0.01% hydrogen peroxide. After terminating the reaction with PBS-azide, sections were mounted, dehydrated and covered. Adjacent sections were incubated as controls, without the primary antibody to confirm a lack of non-specific staining. Sections were examined under bright-field illumination using an Olympus BX51 microscope (Japan). Images were captured with a digital camera (DP72, Olympus, Japan).
2.7. Statistical analysis All results are expressed as means ± SEM. For vigilance studies, amounts of sleep-wake states were expressed in minutes. The statistical significance of time course data for locomotor activity, sleep amount, stage transition, number of each stage bouts and mean duration was assessed with a two-tailed unpaired Student's t-test. Differences in drug treatment between fed and fasted mice were compared with a two-way ANOVA (with food and drug treatment as factors) followed by a post hoc Fisher's probable least-squares difference when appropriate. In all cases, p b 0.05 was the level of significance.
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3. Results 3.1. Caffeine prolonged locomotor activity in fasted mice As shown in Fig. 1A, the change in time course of locomotor activity revealed that caffeine at 15 mg/kg increased locomotor activity in fed mice for 3 h [09:00: t(34) = 4.63, p b 0.01; 10:00: t(34) = 8.80, p b 0.01; 11:00: t(34) = 4.56, p b 0.01] after administration at 09:00 compared with vehicle control. Fig. 1B summarizes locomotor activity in fasted mice treated with vehicle or caffeine. Compared with vehicle control, caffeine prolonged locomotor activity to 5 h [09:00: t(32) = 2.81, p b 0.01; 10:00: t(32) = 4.81, p b 0.01; 11:00: t(32) = 3.70, p b 0.01; 12:00: t(32) = 2.85, p b 0.01; 13:00: t(32) = 2.23, p b 0.01]. As the extended time of fasted WT mice is 2 h from 12:00 to 14:00, total locomotor activity was calculated for 3 h (during 09:00–12:00) and consecutive 2 h (during 12:00–14:00) in fed and fasted mice after caffeine injections (Fig. 1C and D). During the period of 09:00–12:00, caffeine significantly increased total amount of locomotor activity by 2.2-fold [t(34) = 7.90, p b 0.01] in fed mice and 1.6-fold [t(32) = 4.41, p b 0.01] in fasted mice, respectively, compared with the vehicle controls (Fig. 1C). During the subsequent 2-h period, caffeine increased locomotor activity by 2.9-fold [t(32) = 3.81, p b 0.05] in fasted mice but had no effects in fed animals (Fig. 1D). These results indicate that fasting prolonged caffeine-induced stimulation of locomotor activity in mice. 3.2. An H1R antagonist inhibited caffeine-induced potentiation on locomotor activity in fasted mice To investigate the contribution of H1R to the enhanced effect on locomotor activity of caffeine in fasted mice, we administered H1R antagonist pyrilamine (10 mg/kg) at 8:00, 1 h before caffeine administration.
This pretreatment had no effect in fed caffeine-treated mice (Fig. 2A and C). However, compared with vehicle control, pretreatment with pyrilamine significantly inhibited locomotor activity induced by caffeine in fasted mice shortened from 5 to 3 h (Fig. 2B and D). These results indicate that caffeine prolonged locomotor activity in fasted mice via H1R. 3.3. Caffeine increased wakefulness for 3 h in fed WT mice, while for 5 h in fasted WT mice Compared with vehicle control, caffeine markedly increased wakefulness in fed WT mice, and this was sustained for 3 h [09:00: t(10) = 5.43, p b 0.01; 10:00: t(10) = 17.57, p b 0.01; 11:00: t(10) = 4.32, p b 0.01] (Fig. 3A), whereas this effect was sustained for 5 h [09:00: t(9) = 3.22, p b 0.05; 10:00: t(9) = 11.17, p b 0.01; 11:00: t(9) = 3.02, p b 0.05; 12:00: t(9) = 3.45, p b 0.01; 13:00: t(9) = 2.86, p b 0.05] in fasted WT mice (Fig. 3B). These increases in wakefulness coincided with decreased NREM [fed mice: 09:00: t(10) = 5.61, p b 0.01; 10:00: t(10) = 14.89, p b 0.01; 11:00: t(10) = 3.94, p b 0.01; fasted mice: 09:00: t(9) = 3.24, p b 0.05; 10:00: t(9) = 11.45, p b 0.01; 11:00: t(9) = 3.04, p b 0.05; 12:00: t(9) = 4.85, p b 0.01; 13:00: t(9) = 3.74, p b 0.01] and REM sleep [fed mice: 10:00: t(10) = 7.79, p b 0.01; 11:00: t(10) = 3.21, p b 0.01; fasted mice: 10:00: t(9) = 3.59, p b 0.01; 11:00: t(9) = 2.62, p b 0.05] in both animal types after caffeine treatment (Fig. 3A and B). The sleep architecture during the subsequent period in both fed and fasted WT mice was not disrupted (Fig. 3A and B). The total time spent in wakefulness, NREM and REM sleep was calculated for 3 h (09:00–12:00) and a subsequent 2 h (12:00–14:00) in fed and fasted WT mice after caffeine administration. Fig. 3C shows that, during 09:00–12:00, the total amount of wakefulness after caffeine
Fig. 1. Fasting enhanced locomotor activity induced by caffeine in WT mice. Time course changes in locomotor activity in mice treated with caffeine (15 mg/kg) at 09:00 under fed (A) or fasted (B) conditions. Total locomotor activity during 3 h (09:00–12:00) (C) and 2 h (12:00–14:00) (D) in fed and fasted conditions after caffeine or vehicle administration. Values are means ± SEM, n = 17–18. *p b0.05 and **p b 0.01 indicate significant differences between caffeine-treated group and corresponding vehicle control (two-tailed unpaired Student's t-test).
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Fig. 2. Pyrilamine abolished fasting's augmentation of caffeine-induced locomotor activity in WT mice. Time course of locomotor activity in mice treated with vehicle (A, B) or pyrilamine (C, D) at 8:00, as indicated by the open arrow, and vehile or caffeine at 9:00, as indicated by the closed arrow, under fed or fasted conditions. Values are means ± SEM, n = 14–17. *p b 0.05 and **p b 0.01 indicate significant differences between caffeine-treated group and corresponding vehicle control (two-tailed unpaired Student's t-test).
injection increased in both fed [t(10) = 12.71, p b 0.01] and fasted WT mice [t(9) = 8.70, p b 0.01]. During the next 2 h, caffeine enhanced the amount of wakefulness by 1.0-fold in fasted mice [t(9) = 4.84, p b 0.01], but not in fed mice (Fig. 3D). The concomitant decreases in the total amount of NREM sleep in fed and fasted WT mice [fed mice: t(10) = 10.00, p b 0.01; fasted mice: t(9) = 8.98, p b 0.01] during 09:00–12:00, and in fasted mice [t(9) = 6.75, p b 0.01] during 12:00– 14:00 were observed (Fig. 3C and D). The amount of REM sleep was decreased after caffeine treatment in both fed [t(10) = 4.68, p b 0.01] and fasted [t(9) = 6.34, p b 0.01] WT mice during 3 h after caffeine administration (Fig. 3C). These results indicate that caffeine strengthened its arousal effects under fasting conditions. 3.4. Effects of caffeine on characteristics of sleep-wake episodes, numbers of wakefulness bouts and NREM sleep latency in fed and fasted WT mice To understand sleep architecture changes caused by caffeine under fasting conditions, we measured the mean duration and episode numbers, numbers of wakefulness bouts and NREM sleep latency during 09:00–12:00 in both fed and fasted WT mice, and during 12:00–14:00 in fasted WT mice. Compared to vehicle control, mean durations of wakefulness increased [fed mice: t(10) = 4.40, p b 0.05; fasted mice: t(9) = 3.16, p b 0.05] and of NREM sleep decreased [fed mice: t(10) = 2.59, p b 0.05; fasted mice: t(9) = 4.42, p b 0.01] during the 3 h after caffeine administration in both fed and fasted WT mice, but wakefulness increased more and NREM sleep decreased more in fasted mice compared to fed mice (Fig. 4A and B). During the next 12:00–14:00, fasting also induced greater mean duration of wakefulness after caffeine administration [t(9) = 2.79, p b 0.05]. Compared to vehicle controls, caffeine significantly decreased the episode numbers of wakefulness [fed mice: t(10) = 5.35, p b 0.01; fasted mice: t(9) = 4.98, p b 0.01], NREM [fed mice: t(10) = 5.42, p b 0.01; fasted mice: t(9) = 4.94, p b 0.01] and
REM sleep [fed mice: t(10) = 7.01, p b 0.01; fasted mice: t(9) = 4.44, p b 0.01] in both fed and fasted WT mice during 09:00–12:00 (Fig. 4A and B), but did not make any changes in episode numbers in fasted mice during 12:00–14:00 (Fig. 4C). EEG power spectra analysis revealed that caffeine induced the same change of EEG power density of NREM and REM sleep in fed and fasted WT mice during 09:00–12:00 (data not shown). These findings indicate that caffeine induced longer and stronger effect on wakefulness under fasted conditions. Compared with vehicle controls, the numbers of wakefulness bouts of 0–16 s [t(10) = 3.68, p b 0.01], 16–32 s [t(10) = 4.24, p b 0.01], 32–64 s [t(10) = 3.72, p b 0.01] and 64–128 s [t(10) = 2.77, p b 0.05] were decreased significantly after caffeine administration in fed WT mice during 09:00–12:00, but only reduced the bouts of 0–16 s [t(9) = 4.21, p b 0.01], 16–32 s [t(9) = 3.17, p b 0.05] and 32–64 s [t(9) = 3.36, p b 0.01] in the fasted WT mice during the same period (Fig. 4D and E). During the next 2 h, caffeine did not change the number of bouts in fasted WT mice (Fig. 4 F). As shown in Fig. 4G and H, during 09:00–12:00, the injection of caffeine remarkably prolonged NREM latency [fed mice: t(10) = 5.77, p b 0.01; fasted mice: t(9) = 8.72, p b 0.01], defined as the time from the vehicle or caffeine injection to the appearance of the first NREM sleep episode lasting for at least 20 s both in fed and fasted WT mice. Caffeine's effect in fasted mice was stronger than in fed mice. There is not significant change of NREM latency in the next 2 h after caffeine injection in fasted WT mice (Fig. 4I). The longer NREM sleep latency observed in caffeine-treated fasted WT mice suggests that fasting delays the initiation of NREM sleep. 3.5. Caffeine increased wakefulness for 3 h in fed and for 2 h in fasted H1R KO mice Compared with vehicle controls, caffeine markedly increased wakefulness after administration in fed H1R KO mice and was sustained for
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Fig. 3. Effects of fasting on sleep-wake profiles induced by caffeine in WT mice. (A, B) Time course changes in wakefulness, NREM and REM sleep in fed (A) and fasted (B) WT mice treated with caffeine. Circles represent the hourly mean amount of each stage. Open and closed circles represent profiles of vehicle and caffeine treatments, respectively. Horizontal filled and open bars on χ-axes indicate 12 h dark and 12 h light periods. (C, D) Total time spent in wakefulness, NREM and REM sleep during the 3 h (09:00–12:00) (C) or the 2 h (12:00–14:00) (D) in fed and fasted WT mice after administration of vehicle or caffeine. Open and filled bars depict vehicle and caffeine treatments, respectively. Values are means ± SEM (n = 5–7). *p b 0.05 and **p b 0.01 indicate significant differences between the caffeine-treated group and corresponding vehicle control (two-tailed unpaired Student's t-test).
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Fig. 4. Mean durations and episode numbers (A, B, C), numbers of NREM and REM sleep bouts (D, E, F) and NREM sleep latency (G, H, I) during 3 h (09:00–12:00) in fed mice and during 3 h (09:00–12:00) and 2 h (12:00–14:00) in fasted WT mice after administration of vehicle or caffeine. Open and filled bars depict vehicle and caffeine treatments, respectively. Values are means ± SEM (n = 5–7). *p b 0.05 and **p b 0.01 indicate significantly differences between caffeine-treated group and its corresponding vehicle control (two-tailed unpaired Student's t-test).
3 h [09:00: t(14) = 3.71, p b 0.01; 10:00: t(14) = 14.36, p b 0.01; 11:00: t(14) = 6.64, p b 0.01] (Fig. 5A). Whereas this enhanced effect of caffeine was shortened to 2 h in fasted H1R KO mice [09:00: t(8) = 3.48, p b 0.01; 10:00: t(8) = 17.21, p b 0.01] (Fig. 5B). These increases in wakefulness were concomitant with the decreases in NREM [fed mice: 09:00: t(14) = 3.85, p b 0.01; 10:00: t(14) = 12.95, p b 0.01; 11:00: t(14) = 6.90, p b 0.01; fasted mice: 09:00: t(8) = 3.59, p b 0.01; 10:00: t(8) = 18.79, p b 0.01] and REM sleep [fed mice: 10:00: t(14) = 25.80, p b 0.01; 11:00: t(14) = 3.83, p b 0.01; fasted mice: 10:00: t(8) = 5.20, p b 0.01] in both fed and fasted H1R KO mice after caffeine treatment (Fig. 5A and B). Moreover, the sleep architecture during the subsequent period in both fed and fasted ND mice was not disrupted (Fig. 5A and B).
The total time spent in wakefulness, NREM and REM sleep was calculated for 3 h in fed and fasted H1R KO mice after caffeine administration. As shown in Fig. 5C, the total amount of wakefulness increased by 1.1-fold [t(14) = 10.96, p b 0.01] and 1.0-fold [t(8) = 6.19, p b 0.01], respectively, in fed and fasted H1R KO mice during 3 h after caffeine injection. The total amount of NREM sleep was decreased in fed H1R KO mice by 80.3% [t(14) = 10.58, p b 0.01] and in fasted H1R KO mice by 69.9% [t(8) = 6.02, p b 0.01], respectively (Fig. 5C). The amount of REM sleep was also decreased after caffeine treatment both in fed and fasted H1R KO mice [fed mice: t(14) = 10.35, p b 0.01; fasted mice: t(8) = 5.91, p b 0.01] (Fig. 5C). During the next 2 h from 12:00 to 14:00, caffeine did not have any effects on the amount of wakefulness, NREM and REM sleep in neither fed nor fasted H1R KO mice (Fig. 5D). These
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Fig. 5. Effects of fasting on sleep-wake profiles induced by caffeine in H1R KO mice. (A, B) Time course changes in wakefulness, NREM and REM sleep in fed (A) and fasted (B) H1R KO mice treated with caffeine. Each circle represents the hourly mean amount of each stage. Open and closed circles represent profiles of vehicle and caffeine treatments, respectively. Horizontal filled and open bars on the χ-axes indicate 12 h dark and 12 h light periods, respectively. (C, D) Total time spent in wakefulness, NREM and REM sleep during the 3 h (09:00–12:00) (C) or the 2 h (12:00–14:00) (D) in fed and fasted H1R KO mice after administration of vehicle or caffeine. Open and filled bars depict profiles of vehicle and caffeine treatments, respectively. Values are means ± SEM (n = 5–8). **p b 0.01 indicates significant differences between the caffeine-treated group and its corresponding vehicle control (two-tailed unpaired Student's t-test).
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results indicate that through the mediation of H1R, caffeine enhanced its arousal effect under fasting conditions. 3.6. Effects of caffeine on characteristics of sleep-wake episodes, numbers of wakefulness bouts and NREM sleep latency in fed and fasted H1R KO mice The mean duration and episode number, numbers of wakefulness bouts and NREM latency for 3 h after caffeine administration in fed and fasted H1R KO mice were also analyzed. Compared to vehicle controls, mean durations of wakefulness were increased [fed mice: t(14) = 3.48, p b 0.05; fasted mice: t(8) = 3.00, p b 0.05] after caffeine administration in fed and fasted H1R KO mice during 09:00–12:00
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(Fig. 6A and B). Caffeine also significantly decreased the episode numbers of wakefulness [fed mice: t(14) = 5.53, p b 0.01; fasted mice: t(8) = 4.43, p b 0.01], NREM [fed mice: t(14) = 5.51, p b 0.01; fasted mice: t(8) = 4.33, p b 0.01] and REM sleep [fed mice: t(14) = 9.23, p b 0.01; fasted mice: t(8) = 7.23, p b 0.01] both in fed and fasted H1R KO mice (Fig. 6A and B). EEG power spectra analysis revealed that caffeine induced the same changes of EEG power density of NREM and REM sleep in the fed and fasted H1R KO mice (data not shown). These findings indicate that H1R mediated the longer and stronger effect on wakefulness under fasted conditions induced by caffeine. Compared with vehicle controls, the numbers of wakefulness bouts of 0–16 s [fed mice: t(14) = 8.65, p b 0.01; fasted mice: t(8) = 4.12,
Fig. 6. Mean durations and episode numbers (A, B), numbers of NREM and REM sleep bouts (C, D) and NREM sleep latency (E, F) during the 3 h in fed and fasted H1R KO mice after administration of vehicle or caffeine. Open and filled bars depict profiles for the vehicle and caffeine treatments, respectively. Values are means ± SEM (n = 5–8). *p b 0.05 and **p b 0.01 indicate significant differences between the caffeine-treated group and its corresponding vehicle control (two-tailed unpaired Student's t-test).
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p b 0.05] and 16–32 s [fed mice: t(14) = 2.66, p b 0.01; fasted mice: t(8) = 3.94, p b 0.01] were decreased significantly after caffeine administration in fed and fasted H1R KO mice during 9:00–12:00 (Fig. 6C and D). Also, caffeine induced a longer NREM latency in both fed and fasted H1R KO mice [fed mice: t(14) = 5.60, p b 0.01; fasted mice: t(8) = 6.72, p b 0.01] (Fig. 6E and F). These results suggested that fasting delays the initiation of NREM sleep after caffeine injection via H1R. 3.7. Effects of fasting on c-Fos expression in wake-promoting neurons in the TMN Fig. 7 A (a–h) depicts representative photomicrographs of c-Fos expression of the TMN in fed and fasted WT mice treated with vehicle or caffeine. Then, the number of c-Fos immunoreactive nuclei was analyzed. A two-way ANOVA confirmed that fasting had significantly effects as determined by the expression of c-Fos in the TMN [F(1,22) = 77.93, p b 0.01] (Fig. 7B). Compared with fed mice, further analysis showed that fasted mice given the vehicle increased the c-Fos expression in TMN (p b 0.01). However, the effects of caffeine on the expression of c-Fos were not obvious under fed or fasted conditions at 5 h after its administration compared with vehicle controls. Therefore, fasting activated the neurons in TMN, but caffeine is not sufficient to enhance the protein expression of c-Fos. 4. Discussion Histaminergic neurons in the TMN regulate various cerebral functions, such as wakefulness, drinking and feeding, sensation and motor activity (Masaki and Yoshimatsu, 2007; Panula and Nuutinen, 2013; Valdes et al., 2005). The immunohistochemistry results of c-Fos staining in our study showed that fasting induced more c-Fos positive neurons in the TMN. These results are in agreement with previous works (Valdes et al., 2010). Fasting also activates orexin neurons in the LH (Diano et al., 2003), and it coordinates sleep-wakefulness and motivated behaviors such as food seeking, especially during fasting stress (Sakurai, 2003, 2014). Orexin neuron-ablated mice did not have fasting-induced arousal, suggesting that orexin neurons are needed to evoke adaptive behavioral arousal during fasting (Akiyama et al., 2004). LH, as a feeding center, projects afferents to the histaminergic TMN (Huang et al., 2001; Sakurai, 1999). Histaminergic neurons in the TMN regulate wakefulness during which they discharge tonically and specifically (Vanni-Mercier et al., 2003). Histamine exerts its effects through four receptors (H1, H2, H3 and H4), which are primarily found in the brain. Previous research shows that potentiating brain histaminergic activity induces wakefulness and reduces sleep via regulation of the H1R (Lin et al.,
1988; Monti et al., 1986; Tokunaga et al., 2009). H1R antagonists can induce a robust sedative-hypnotic effect (Wang et al., 2015). The histaminergic and orexinergic systems are reciprocally connected in an excitatory fashion to regulate wakefulness, our group observed that wakefulness-promoting effect of orexin depends histaminergic neurons activation mediated through H1R (Huang et al., 2001). These findings suggest that fasting has an arousal effect that is dependent on activation of the histaminergic system via H1R. Caffeine is a central nervous system stimulant. In the cellular level, this drug is the inhibitor of adenosine receptor in the central nervous system. There are three kinds of adenosine receptors that are A1, A2 (including A2A and A2B subtype) and A3 receptor. A1Rs are coupled to Gi protein, which could effectively inhibit adenylate cyclase and some calcium channels (N type and Q type), through which to activate phospholipase and some potassium channels (Fredholm et al., 2005; Huang et al., 2014; Jacobson and Gao, 2006). On the contrary, A2ARs are coupled to Gs protein, which activate L type calcium channel and adenylate cyclase (Fredholm et al., 2005; Jacobson and Gao, 2006). We demonstrated previously that the wakefulness-promoting effect of caffeine is mediated by the A2AR, but not the A1R (Huang et al., 2005). Later, A2ARs in the shell region of the NAc were later reported to be responsible for the effect of caffeine on wakefulness (Lazarus et al., 2011). Likely, blockade of the massive GABAergic output of NAc shell neurons activates classical arousal centers, such as the LH and, the TMN, via direct or indirect projections from the NAc shell (Lazarus et al., 2011; Masaki and Yoshimatsu, 2007). Our data showed that caffeine increased wakefulness and locomotor activity in H1R antagonist-treated or H1R KO mice. Therefore, the NAc shell-LH-TMN pathway is not the most crucial pathway for the arousal effect of caffeine. With fasting condition, orexin was increased (Sakurai, 1999) and the TMN and LH were activated (Valdes et al., 2005), suggesting that this NAc shell-LH-TMN pathway is responsible for the enhanced arousal effect of caffeine. In addition, fasting also induces wakefulness via H1R. So histaminergic system is involved in the enhanced effect of caffeine in locomotor activity and wakefulness. 5. Conclusion These findings indicate fasting could enhance the wakefulnesspromoting effect of caffeine involved the mediation of H1R. Acknowledgements The authors thank Dr Zhi-Li Huang (Department of Pharmacology, School of Basic Medical Sciences, Fudan University) for his comments
Fig. 7. Effect of fasting on c-Fos immunoreactivity in the TMN. Representative photomicrographs of c-Fos immunostaining in the TMN. (A) Representative photomicrographs of c-Fos immunostaining in the TMN of a mouse treated with vehicle (a, c, e and g) and caffeine (b, d, f and h) under fed or fasted conditions. e, f, g and h (scale bars: 50 μm): high-magnification views of the rectangular areas marked in a, b, c and d (scale bars: 200 μm), respectively. (B) The number of c-Fos-immunoreactive neurons in TMN after vehicle or caffeine treatment. Values are means ± SEM (n = 6–8). **p b 0.01 indicates significant differences from the vehicle-treated fed mice (two-way ANOVA followed by a probable least-squares difference test). 3 V: third ventricle.
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and help in the preparation of the manuscript. This study was supported in part by grants-in-aid for scientific research from the National Natural Science Foundation of China (J1210041, 31171010, 31171049, 31121061, 31271164, 31471064, 81420108015), the National Basic Research Program of China (2011CB711000, 2015CB856401, 2009ZX09303-006), the Fundamental Research Funds for the Central Universities (10FX041), a key laboratory program of the Education Commission of Shanghai Municipality (ZDSYS14005), the Shanghai Committee of Science and Technology (13140903100, 14JC1400900, 13dz2260700), the Shanghai Leading Academic Discipline Project (B119), PhD, and the Programs Foundation of Ministry of Education of China (20110071110033).
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