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Magnolol, a major bioactive constituent of the bark of Magnolia officinalis, induces sleep via the benzodiazepine site of GABAA receptor in mice
Neuropharmacology 63 (2012) 1191e1199
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Magnolol, a major bioactive constituent of the bark of Magnolia officinalis, induces sleep via the benzodiazepine site of GABAA receptor in mice Chang-Rui Chen a,1, Xu-Zhao Zhou a,1, Yan-Jia Luo a, Zhi-Li Huang a, b, c, *, Yoshihiro Urade d, Wei-Min Qu a, c, * a
Department of Pharmacology, Fudan University, Shanghai, 200032, PR China State Key Laboratory of Medical Neurobiology, Shanghai Medical College, Fudan University, Shanghai, 200032, PR China c Institute of Brain Science, Fudan University, Shanghai, 200032, PR China d Department of Molecular Behavioral Biology, Osaka Bioscience Institute, 6-2-4 Furuedai, Suita, Osaka 565-0874, Japan b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 29 March 2012 Received in revised form 13 June 2012 Accepted 14 June 2012
Magnolol (6,60 ,7,12-tetramethoxy-2,20 -dimethyl-1-beta-berbaman, C18H18O2), an active ingredient of the bark of Magnolia officinalis, has been reported to exert potent anti-epileptic effects via the GABAA receptor. The receptor also mediates sleep in humans and animals. The aim of this study was to determine whether magnolol could modulate sleep behaviors by recording EEG and electromyogram in mice. The results showed that magnolol administered i.p. at a dose of 5 or 25 mg/kg could significantly shorten the sleep latency, increase the amount of non-rapid eye movement (non-REM, NREM) and rapid eye movement (REM) sleep for 3 h after administration with an increase in the number of NREM and REM sleep episodes. Magnolol at doses of 5 and 25 mg/kg increased the number of bouts of wakefulness but decreased their duration. On the other hand, magnolol increased the number of state transitions from wakefulness to NREM sleep and subsequently from NREM sleep to wakefulness. Immunohistochemical study showed that magnolol increased c-Fos expression in the neurons of ventrolateral preoptic area, a sleep center in the anterior hypothalamus, and decreased c-Fos expression in the arousal tuberomammillary nucleus, which was located in the caudolateral hypothalamus. The sleep-promoting effects and changes in c-Fos induced by magnolol were reversed by flumazenil, an antagonist at the benzodiazepine site of the GABAA receptor. These results indicate that magnolol increased NREM and REM sleep via the GABAA receptor. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: EEG GABAA Magnolol Sleep VLPO TMN
1. Introduction Insomnia is a highly prevalent condition characterized by inability to sleep or a total lack of sleep. Insomnia afflicts 35% of the general population worldwide. About 10e15% of insomnia patients are diagnosed with moderate to severe disorders. This gives rise to emotional distress, daytime fatigue, and loss of productivity (Buckner et al., 2008). Among pharmacotherapeutic agents, nonbenzodiazepine hypnotics (Z-drugs) are the first line of
Abbreviations: EEG, electroencephalogram; BZ, benzodiazepines; Flu, flumazenil; Mag, magnolol; VLPO, ventrolateral preoptic area; TMN, tuberomammillary nucleus. * Corresponding authors. Department of Pharmacology and State Key Laboratory of Medical Neurobiology, Shanghai Medical College, Fudan University, Shanghai 200032, PR China. Tel.: þ86 21 54237043. E-mail addresses: [email protected] (Z.-L. Huang), quweimin@ fudan.edu.cn (W.-M. Qu). 1 C.R.C. and X.Z.Z equally contributed to this work. 0028-3908/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neuropharm.2012.06.031
management for insomnia, followed by benzodiazepines (BZ), amitryptiline, and antihistamines. The traditional BZ and Z-drugs modulate the GABAA receptors (Rudolph and Knoflach, 2011). Diazepam and zolpidem significantly increase total non-rapid eye movement (non-REM, NREM) sleep, but they can also cause a remarkable decrease in delta power activity during NREM sleep (van Lier et al., 2004). The discrepancy between the increase in sleep continuity and the reduction of power in the lower EEG frequencies caused by BZ in humans is consistent with findings in mice (Tobler et al., 2001). The adverse effects of BZ include decreased psychomotor performance (e.g. next-day drowsiness), tolerance, dependency, and withdrawal symptoms. These have limited their use, indicating that there is still a need for hypnotics that do not produce dependence or detrimental daytime consequences such as sedation and drowsiness. Over the past decades, the identification of separable key functions of GABAA receptor subtypes has suggested that receptor subtype-selective compounds could overcome the limitations of classical BZ (Rudolph and Knoflach, 2011).
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Fig. 1. Chemical structure of magnolol.
Magnolol (6,60 ,7,12-tetramethoxy-2,20 -dimethyl-1-beta-berbaman, C18H18O2, see Fig. 1 for chemical structure) is the major bioactive constituent of magnolia bark. Magnolol increases the threshold of NMDA-induced seizures (Lin et al., 2005). It also delays the onset of myoclonic jerks and generalized clonic seizures and decreases seizure stage and mortality (Chen et al., 2011). Magnolol has also been found to prolong the duration of sleeping time induced by pentobarbital in mice (Ma et al., 2009). These data indicate that magnolol may have sleep-promoting effects. The GABAA receptor is the main inhibitory neurotransmitter receptor in the CNS. The fast-inhibitory activity of GABA is mediated by the GABAA receptor. It has been reported that magnolol increases the number of binding sites and affinity of the GABAA receptor for GABA and then enhances chloride influx (Ai et al., 2001; Ma et al., 2009; Squires et al., 1999; Alexeev et al., 2012). These findings suggest that magnolol might enhance the inhibitory action of GABA on GABAA receptors by binding to the sites in GABAA receptor. We hypothesized that the GABAA receptor might be responsible for the sleep-promoting effects of magnolol. In the present study, we showed that magnolol shortened sleep latency, increased the number of bouts of NREM, and the amount of NREM and REM sleep. Immunostaining showed that magnolol increased c-Fos expression in neurons of the ventrolateral preoptic nucleus (VLPO), which is one of the sleep centers, and decreased it in the arousal histaminergic tuberomammillary nucleus (TMN). These findings suggest that magnolol may have applications in the treatment of insomnia. 2. Methods and materials 2.1. Animals Male SPF inbred C57BL/6J mice (weighing 20e28 g, 11e13 weeks old) were obtained from the Laboratory Animal Center, Chinese Academy of Sciences (Shanghai, China). The animals were housed individually at a constant temperature (24 0.5 C) with a relative humidity of 60 2% on an automatically controlled 12 h light/dark cycle (lights on at 7:00 a.m.), and they had free access to food and water. All efforts were made to minimize animal suffering and to use only the number of animals required for the production of reliable scientific data. All experiments were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. 2.2. Chemicals Magnolol was purchased from the National Center for Safety Evaluation of Drugs (Beijing, China). It was shown to be 98% pure by high-performance liquid chromatography. Rabbit polyclonal anti-c-Fos antibody was purchased from Abcam (Cambridge, MA). Biotinylated donkey anti-rabbit IgG and avidinebiotin-peroxidase came from Vector Laboratories (CA); flumazenil and 3,30 -diamino-benzidine-tetrahydrochloride (DAB) from SigmaeAldrich (SigmaeAldrich, St. Louis, MO, U.S.). Magnolol and flumazenil were dissolved in saline with 5% dimethyl-sulfoxide (DMSO). 2.3. Polygraphic recordings and vigilance state analysis Under pentobarbital anesthesia (50 mg/kg, i.p.), mice were chronically implanted with electrodes for polysomnographic recordings of EEG and electromyogram (EMG). Two stainless steel screws (1 mm in diameter) were inserted through the skull into the cortex (antero-posterior, þ1.0 mm; lefteright, 1.5 mm from bregma or lambda) according to the atlas of Franklin and Paxinos (1997). These
served as EEG electrodes. Two insulated stainless steel, teflon-coated wires were bilaterally placed into both trapezius muscles. These served as EMG electrodes. All electrodes were attached to a microconnector and fixed onto the skull with dental cement. The EEG and EMG recordings were carried out by means of a slip ring designed so that the behavioral movement of the mice would not be restricted. After a 10-day recovery period, the mice were housed individually in transparent barrels and habituated to the recording cable for 3e4 days before polygraphic recording. All mice that were subjected to EEG recordings received vehicle and drug treatment on 2 consecutive days. On day 1, the mice were treated with vehicle (intraperitoneally) at 21:00 and the recordings made on that day served as the baseline data. On day 2, mice were treated with magnolol (intraperitoneally, in a volume of 10 ml/kg body weight) at 21:00, and EEG/EMG signals were recorded for 24 h. 2.4. Analysis of vigilance state The EEG/EMG signals were amplified and filtered (EEG, 0.5e30 Hz; EMG, 20e200 Hz), then digitized at a sampling rate of 128 Hz, and recorded using SLEEPSIGN software as described before (Oishi et al., 2008; Qu et al., 2008). The vigilance states were automatically classified off-line in 4 s epochs into REM sleep, NREM sleep, and wakefulness by SLEEPSIGN, according to the standard criteria (Qu et al., 2010). As a final step, defined sleepewake stages were examined visually and corrected if necessary. 2.5. Pharmacological treatments Magnolol was prepared as described above immediately before use and administered i.p. at 21:00 on the day of the experiment at a dose of 1, 5 or 25 mg/kg (n ¼ 5e7). All drugs were freshly prepared prior to use, and an injection volume (10 ml/kg) was kept constant for in vivo experiments. For baseline data, mice were injected i.p. with vehicle (10 ml/kg). To test receptor mechanisms, 30 min before the injection of magnolol (25 mg/kg), mice were pretreated with flumazenil (Flu) i.p. at 0.5 or 1 mg/kg. Diazepam at 6 mg/kg was injected as a positive control. 2.6. c-Fos immunohistochemistry Nine groups of mice were used. One group was treated with vehicle; and the others were injected i.p. with magnolol at doses of 1, 5, and 25 mg/kg and diazepam at 6 mg/kg. To test the receptor mechanisms, four groups of mice were used: Flumazenil (Flu) 1 mg/kg þ vehicle, Flu 1 mg/kg þ magnolol 25 mg/kg, Flu 0.5 mg/ kg þ magnolol 25 mg/kg, and Flu 1 mg/kg þ diazepam 6 mg/kg groups. Mice were pretreated with Flu, and then the magnolol 25 mg/kg was given after 30 min. At 60 min after the administration of magnolol, the animals were anesthetized with 10% chloral hydrate and perfused via the heart with saline solution followed by icecold 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (pH 7.4). Their brains were then removed, post-fixed in 4% PFA for 6 h, and immersed in 30% sucrose overnight. Thereafter, frozen sections were cut at 30 mm in coronal planes by use of a freezing microtome (Leica Microsystems, Wetzlar, Germany). The sections were stored in a cryoprotectant solution at 20 C for histological analysis. Immunohistochemistry was performed in accordance with the free-floating method described earlier (Chen et al., 2011). Sections were fixed in 4% PFA for 10 min and incubated with 0.3% H2O2 for 15 min to quench the endogenous peroxidase activity. The sections were next placed in blocking solution containing 10% normal goat serum with 0.3% Triton X-100 in 0.01 M phosphate-buffered saline (PBS, pH 7.2) for 30 min at 37 C and then incubated at 4 C for 24 h with a rabbit polyclonal antibody against c-Fos at a 1:5000 dilution in PBS containing 0.3% Triton X-100. On the second day, the sections were incubated with a 1:1000 dilution of biotinylated donkey antirabbit secondary antibodies for 30 min followed by a 1:200 dilution of avidinebiotin-peroxidase for 1 h at 37 C. The peroxidase reaction was visualized with 0.05% DAB in 0.1 M phosphate buffer and 0.01% H2O2. Sections were mounted, dehydrated, and cover slipped. As controls, adjacent sections were incubated without primary antibody to confirm that no non-specific staining had occurred. Digital images were viewed and captured using the Olympus DP 72 microscope (Olympus, Tokyo, Japan). Figures were assembled and adjusted for brightness and contrast in Adobe Photoshop (Adobe Systems Inc, San Jose, CA, U.S.). 2.7. Statistical analysis All data were expressed as the mean SEM (n ¼ 5e7). Statistical analysis was performed with SPSS 17.0 (SPSS Inc., Chicago, IL, U.S.). Time-course of the hourly amounts of each stage, histograms of the amounts of sleep and wakefulness, the number of transitions between sleep and wakefulness, and the number and duration of bouts of sleep and wakefulness were analyzed using the paired t-test, with each animal serving as its own control. For sleep latency, the total number of each vigilance stage during the 3 h immediately following drug treatment, one-way repeated measures analysis of variance (ANOVA) was performed followed by the Fisher probable least-squares difference (PLSD) test to determine whether the differences among groups were statistically significant. For the number of c-Fos immunoreactive neurons, one-way or two-way ANOVA was used, followed by PLSD test. The significance level was set at P < 0.05 for all statistical tests.
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3. Results 3.1. Effects of magnolol on the total amount of sleep and on sleep latency To determine the effects of magnolol on sleepewake profiles, magnolol was injected i.p. into mice at 21:00 at a dose of 1, 5, or 25 mg/kg and the sleepewake patterns of the animals were examined by recording their EEG and EMG. Diazepam was given at 6 mg/kg as a positive control. Typical examples of polygraphic recordings and corresponding hypnograms illustrated the effects of magnolol on the sleepewake profiles of an individual mouse (Fig. 2A). During the period of 21:00 to 00:00, the control mouse spent more time awake than the magnolol-treated mouse. The magnolol-treated mouse started to sleep within several minutes and spent more time in sleep than the control mouse (Fig. 2A). Magnolol significantly decreased the latency to NREM sleep (F[3,20] ¼ 3.0, P < 0.05), which was defined as the time from the
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injection to the appearance of the first NREM sleep episode lasting for at least 20 s. The sleep latency was 30 5.1 min in the mice treated with magnolol at 25 mg/kg. This was significantly shorter than the 84.5 20 min for mice given vehicle only (Fig. 2B). The short sleep latency indicates that magnolol accelerates the initiation of NREM sleep. Time-course changes in NREM sleep showed that the sleeppromoting effects of magnolol at 25 mg/kg lasted for 3 h (Fig. 2C). Magnolol given at 25 mg/kg increased the amount of NREM sleep during the first, second, and third hours to 5.6-, 3.3-, and 3-fold relative to vehicle control, respectively. This enhancement of NREM sleep was concomitant with an increase in REM sleep and a decrease in wakefulness (data not shown) during the second and third hours after the administration of magnolol. There was no further disruption of the sleep architecture during the subsequent period. Similar time-course profiles were observed at the lower dose of 5 mg/kg, but the effect on sleep was slighter, lasting about 2 h after the injection. Magnolol at 1 mg/kg did not affect sleep profiles (data not shown).
Fig. 2. Sleep-stage distributions produced in mice by i.p. administration of magnolol. (A) Typical examples of polygraphic recordings and corresponding hypnograms in mouse treated with vehicle or magnolol at 25 mg/kg. (B) Effect of magnolol and diazepam on NREM sleep latency. Open and filled bars show the profiles for the respective baseline day (vehicle) and experimental day (magnolol or diazepam injection). (C) Time-course changes produced by the i.p. administration of magnolol at 25 mg/kg. Each circle represents the hourly mean SEM of NREM and REM sleep. Open and filled circles indicate the baseline and experimental day profiles, respectively. Magnolol at 25 mg/kg was given at 21:00. The horizontal filled and open bars on the X-axes indicate the 12 h dark and 12 h light periods, respectively. (D) Total time spent in NREM and REM sleep over the course of 3 h after the administration of magnolol. Open and filled bars show the profiles of the respective baseline day (vehicle) and experimental day (magnolol or diazepam injection). Values are mean SEM (n ¼ 5e7). *P < 0.05, **P < 0.01, relative to vehicle control as assessed by one-way ANOVA, followed by PLSD test.
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We calculated the total amount of time spent in NREM and REM sleep and in wakefulness during the 3 h following magnolol and diazepam injection (Fig. 2D). For vehicle treated groups, there was no statistical difference in amounts of NREM (F[3,20] ¼ 0.107, P > 0.05) and REM (F[3,20] ¼ 1.23, P > 0.05) sleep. ANOVA analysis revealed that magnolol increased NREM (F[3, 20] ¼ 4.8, P < 0.01) and REM sleep (F[3, 20] ¼ 3.2, P < 0.01). Magnolol given at 5 and 25 mg/ kg significantly increased the total amounts of NREM sleep during 3 h period by 2.3- and 4-fold, respectively, and decreased the total amount of wakefulness by 23% and 35%, relative to vehicle control. Magnolol given at 5 and 25 mg/kg significantly increased the total amounts of REM sleep during that 3 h period from 0.79 0.45 min and 0.40 0.19 min to 1.81 0.58 min and 5.77 1.31 min, respectively. However, magnolol at 1 mg/kg did not affect the cumulative amount of NREM and REM sleep or that of wakefulness during the 3 h following injection. Diazepam at 6 mg/kg increased the total amount of NREM sleep by 54% and decreased that of wakefulness by 22% during that 3 h period relative to the vehicle control (P < 0.01). However, it did not increase REM sleep during the 3 h following injection relative to vehicle control. These results clearly indicate that magnolol increases the amounts of both NREM and REM sleep.
3.2. Effects of flumazenil on the sleep-promoting effects of magnolol To determine whether the GABAergic system might be involved in the somnogenic effects of magnolol, mice were pretreated with flumazenil, an antagonist at the benzodiazepine site of the GABAA receptor. The magnolol-induced decreases in the duration of sleep latency and increases in the total duration of NREM sleep (25 mg/ kg) were found to be completely antagonized by flumazenil relative to vehicle treated group (Fig. 3A and B). Fig. 3A summarizes time-courses of the hourly amounts of NREM, REM sleep and wakefulness. Flumazenil at 1 mg/kg completely antagonized the increase in NREM and REM sleep caused by magnolol at 25 mg/kg. As shown in Fig. 3B, flumazenil at 1 mg/kg completely antagonized the magnolol-induced increases in the total amounts of NREM and REM sleep during 3 h period (magnolol 25 mg/kg), but flumazenil 0.5 mg/kg partly abolished the increased amounts of NREM sleep and completely blocked the increased amounts of REM induced by magnolol. The injection of flumazenil alone at dose of 1 mg/kg did not produce significant changes in NREM sleep (data not shown). These results indicate that the induction of sleep caused by magnolol is mediated by the benzodiazepine site of the GABAA receptor.
Fig. 3. Sleepewake profiles produced by magnolol in mice pretreated with flumazenil. (A) Time course changes in NREM and REM sleep and waking after pretreatment with flumazenil (Flu, 1 mg/kg, i.p. at 20:30) and injection of magnolol (Mag, 25 mg/kg, i.p. at 21:00) or vehicle in mice. Each circle represents the hourly mean amount of each stage. Open and closed circles represent the profiles of vehicle and drug treatments. The horizontal filled and open bars on the X-axes indicate the 12 h dark and 12 h light periods, respectively. (B) Total time spent in waking, NREM sleep, and REM sleep over the course of 3 h after the Mag at 25 mg/kg in mice pretreated with Flu. Open and filled bars show the profiles for the vehicle-treated day and experimental day (Flu and Mag injection). Values are means SEM (n ¼ 5e7). *P < 0.05, **P < 0.01, relative to vehicle control, as assessed by two-tailed paired t test.
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3.3. Effects of magnolol on characteristics of sleepewake episodes and power density To better understand the sleepewake profile caused by magnolol, we determined the distribution of bouts of NREM sleep and wakefulness as a function of bout or episode duration (Fig. 4). As shown in Fig. 4A, there were no statistical differences in the episode number (F[3,20] ¼ 1.05, P > 0.05) and mean duration (F[3,20] ¼ 1.13, P > 0.05) of wake, the episode number (F[3,20] ¼ 1.08, P > 0.05) and mean duration (F[3,20] ¼ 1.65, P > 0.05) of NREM sleep among the vehicle treatment groups. Magnolol at 5 and 25 mg/kg increased the number of bouts of NREM bouts by 1.5- and 2.3-fold, and the number of episodes of wakefulness by 1.8- and 2.2-fold, but it decreased the mean duration of wakefulness 73% and 72%, respectively. Magnolol at 25 mg/kg also increased the number of REM bouts by 8.3-fold. However, the mean duration of NREM and REM sleep was not altered during the 3 h period following injection (Fig. 4A). Similar changes were seen in positive group of diazepam 6 mg/kg. When we determined the sleep bout distribution during the 3 h following magnolol injection, the magnolol (5 and 25 mg/ kg) increased the number of prolonged bouts of NREM sleep (128e512 s, 64e512 s, respectively) relative to the vehicle injection (Fig. 4B). Magnolol at 25 mg/kg prolonged bouts of REM ranging in duration from 16 to 128 s, and magnolol at 5 mg/kg had no effect on REM sleep (Fig. 4B). Similarly, diazepam 6 mg/kg increased the
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number of bouts of NREM sleep that ranged in duration from 16 to 128 s over that of the vehicle control. These results suggest that magnolol increased the total number of NREM sleep bouts, but did not extend the duration. As a result, the numbers of state transitions from wakefulness (W) to NREM (N) and from N to W were increased by 2.4- and 2.1-fold, respectively, during the 3 h following injection with magnolol at 25 mg/kg. No change in the number of transitions from N to REM (R) or in the number of transitions from R to W was observed (Fig. 5A). As shown in Fig. 5A,B, vehicle or vehicle þ vehicle treatment did not influence stage transition number from wake to NREM (F[1,11] ¼ 1.76, P > 0.05), and NREM to wake (F[1,11] ¼ 1.6, P > 0.05) for the groups treated with magnolol or flumazenil þ magnolol. Magnolol was found to change the number and duration of episodes of wakefulness and NREM sleep and the number of transitions between these two states. These alterations were completely abolished by flumazenil at 1 mg/kg (Figs. 4A, 4B, 5B), further supporting the hypothesis that magnolol promotes sleep via GABAA receptors. We then determined the EEG power spectra during NREM sleep in mice. The power of each 0.5 Hz bin was first averaged across the sleep stages individually and then normalized by calculating the percentage of each bin from the total power (0e24.5 Hz) of the individual animal. As shown in Fig. 5C, there were no significant differences in EEG power density of NREM sleep between the
Fig. 4. Characteristics of sleepewake episodes produced by the administration of magnolol at 5 or 25 mg/kg, diazepam 6 mg/kg, and flumazenil pretreatment followed by magnolol at 25 mg/kg. (A) Total number and mean duration of waking, NREM, and REM bouts in a 3 h period. (B) Changes in the numbers of NREM and REM bouts across different ranges of episode duration over the course of 3 h after the administration magnolol at 5 or 25 mg/kg, flumazenil 1 mg/kg þ magnolol 25 mg/kg, and diazepam at 6 mg/kg. Open and filled bars show the profiles for the respective baseline day (vehicle) and experimental day (magnolol, flumazenil þ magnolol or diazepam). Values are means SEM (n ¼ 5e7). *P < 0.05, **P < 0.01, two-tailed paired t test.
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Fig. 5. Sleepewake state transitions during the 3 h following the administration of (A) Magnolol or (B) Flumazenil þ magnolol. Open and filled bars show the profiles for the respective baseline day (vehicle) and experimental day (magnolol or flumazenil þ magnolol). W, N, and R represent the stages for wakefulness, NREM sleep, and REM sleep, respectively. Values are means SEM (n ¼ 5e7), *P < 0.05, two-tailed paired t test. EEG power density of NREM sleep after the administration of (C) Magnolol or (D) Flumazenil þ magnolol. There was no essential difference in EEG power density during NREM sleep between magnolol and vehicle or between flumazenil þ magnolol treatment and the vehicle control, respectively.
magnolol treatment and the vehicle control. These results suggest that magnolol induced a form of NREM sleep very similar to physiological NREM sleep.
3.4. Effects of magnolol on c-Fos expression in the VLPO and TMN An immunohistochemical study was performed to determine the parts of the brain involved in the sleep-promoting effects of magnolol. Fig. 6A, B shows representative photomicrographs of cFos expression of the VLPO in mice treated with vehicle or magnolol 25 mg/kg. When the number of c-Fos-immunoreactive neurons in the VLPO was taken into account, magnolol and diazepam significantly increased the number of c-Fos-immunoreactive neurons expressed in the VLPO (F[4,25] ¼ 33.6, P < 0.001). The number of Fos-positive cells in the VLPO was found to be 3.67 0.6 in saline treated group. Magnolol at 5 and 25 mg/kg increased the number of Fos-positive cells in the VLPO to 20 2.4 and 23.7 2.0, respectively (Fig. 6E). Fig. 6C, D shows representative photomicrographs of c-Fos expression in the TMN of mice treated with vehicle or magnolol at 25 mg/kg. Magnolol significantly decreased the expression of c-Fos in the TMN (F[4,25] ¼ 16.5, P < 0.001). The expression of c-Fos was
15.8 1.9 in the vehicle control and 7.5 1.0 and 3.8 1.3 in mice treated with magnolol at 5 and 25 mg/kg, respectively (Fig. 6F). Similar changes were observed in the positive group of diazepam 6 mg/kg. However, magnolol at 1 mg/kg did not affect the expression of c-Fos in the VLPO or TMN. These findings indicate that magnolol activated the VLPO sleep center and inhibited the TMN wake center.
3.5. Effects of flumazenil on altered c-fos immunoreactivity induced by magnolol in the VLPO and TMN Fig. 6GeJ shows representative photomicrographs of c-Fos expression in the VLPO and TMN. In subjects pretreated with flumazenil at 1 mg/kg followed by vehicle, magnolol at 25 mg/kg or diazepam at 6 mg/kg delivered by injection induced a change in cFos expression in the VLPO (F[2,20] ¼ 22.64, P < 0.001) and TMN (F[2,20] ¼ 10.9, P < 0.001). Pretreatment with flumazenil at 1 mg/kg completely antagonized the change in the number of Fos-positive cells caused by magnolol given at 25 mg/kg (P > 0.05) and diazepam at 6 mg/kg (P > 0.05) in the VLPO and TMN (Fig. 6K, L). However, flumazenil at 0.5 mg/kg only partially blocked the change in the number of Fos-positive cells caused by magnolol (P < 0.01).
Fig. 6. Magnolol increased the number of Fos-positive cells in the VLPO and decreased expression of Fos in TMN. Magnolol was found to (AeB) increase the number of Fos-positive cells in the VLPO and (CeD) decrease the expression in the TMN. Low- and high-power photomicrographs representative of the (A, a, B, b) VLPO and (C, c, D, d) TMN, respectively. Fos-positive cells were counted. Scale bars: c and d, 400 mm; C and D, 100 mm. Mean number of Fos-positive cells in the (E) VLPO and (F) TMN of vehicle-, magnolol- and diazepamtreated groups, respectively. (GeJ) pretreatment with FLU at 1 mg/kg blocked (H, h). The increase in the number of Fos-positive cells in the VLPO, and (J, j) The decrease of it in the TMN caused by magnolol at 25 mg/kg. Mean number of Fos-positive cells in the VLPO (K) and (L) TMN of flumazenil (0, 0.5, 1 mg/kg) pretreated groups. Flumazenil at 0.5, 1 mg/kg dose-dependently blocked the increase in the number of Fos-positive cells in the VLPO and the decrease caused by magnolol at 25 mg/kg or diazepam 6 mg/kg. Each value represents the mean SEM (n ¼ 6). **P < 0.01, significantly different from the vehicle or flumazenil 1 mg/kg treated group.
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These findings indicate that the GABAA receptor mediates the ability of magnolol to activate the VLPO sleep center and inhibit the activity of the TMN arousal system. 4. Discussion The present study clearly showed that magnolol can shorten sleep latency and increase the amounts of NREM and REM sleep. Magnolol also increased the number of NREM sleep episodes and the number of transitions between wakefulness and NREM sleep states. Flumazenil, a competitive inhibitor of the activity at the BZ recognition site on the GABAA/BZ ligandereceptor complex, reversed the sleep-promoting effects of magnolol. These results suggest that magnolol might share a similar mechanism of action with BZ. GABA is the most abundant inhibitory neurotransmitter in the mammalian central nervous system. It plays a key role in neuronal firing patterns and in the activity of neuronal networks. The modulators of GABAA receptors have been the focus of several therapies aimed at insomnia (Zammit, 2007). Several in vivo and in vitro studies support our findings that magnolol might elicit sleep-promoting effects by acting on the GABAA receptor. Magnolol was found to decrease the release of many neurotransmitters, such as acetylcholine (Ach) and 5-hydroxytryptamine (5-HT) (Kuribara et al., 2000; Tsai et al., 1995; Xu et al., 2008). However, 5-HT and cholinergic receptor subtype antagonists could not block the inhibitory effects of magnolol on 5-HT or Ach release (Kuribara et al., 2000; Tsai et al., 1995). Therefore, the effects of magnolol on GABA receptor seem to be primary, while its impact on other neurotransmission pathways might be secondary. GABAA receptors are ligand-gated chloride channels assembled from a family of 16 homologous subunits including a1e6, b1e3, g1e3, d, ε, p, and q. The most widely distributed GABAA receptors in the human brain are composed of two a1, two b2, and one g2 subunit (Enna and Mohler, 2007; Hadingham et al., 1995; Olsen and Sieghart, 2008; Ymer et al., 1990). The GABAA receptor can be modulated by allosteric regulators such as the classical agonist BZ. The pharmacological properties of GABAA receptors are largely determined by their subunit composition (Rudolph et al., 2001). BZ action requires the presence of both a and g subunits, but not b subunit (Wafford et al., 1993; Wieland et al., 1992). Magnolol most efficiently modulates GABAA receptors containing a and b subunits (Ai et al., 2001; Taferner et al., 2011), but not g subunit (Ai et al., 2001). In addition, magnolol also selectively up-regulates the expression of the GABAA receptor a subunit, elevates both the number of binding sites and the affinity of [3]H-muscimol’s binding to GABAA receptor a-subunit, and increases Cl influx (Ai et al., 2001; Squires et al., 1999). However, magnolol showed no effect on the abundance of g subunits (Ai et al., 2001; Ma et al., 2009). These results suggest that hypnotic effect of magnolol might be mediated by the GABAA receptor a subunit. EEG delta wave activity has been used as an indicator of the depth of NREM sleep (Tobler et al., 2001). Although diazepam and magnolol promote sleep, diazepam suppresses the EEG delta wave activity of NREM sleep, whereas mognolol does not. Why the effects of magnolol on sleep are different from those of benzodiazepines still remains unknown. Probably, they act at different GABAA receptor a subunits. Diazepam binds to GABAA receptors containing a1, a2, a3, and a5 subunits, but its ability to inhibit REM sleep and to alter EEG spectra density was found to be mediated by a2-containing GABAA receptors (Kopp et al., 2004), but not by a1- or a3containing GABAA receptors (Kopp et al., 2003; Tobler et al., 2001). It was reported that magnolol most efficiently modulated receptors containing a1, a2, a3 and a5 subunits (Ai et al., 2001; Taferner et al., 2011). In addition, magnolol increased the number of binding sites
and the binding affinity on a1b2g2 and a1b2 combinations in in vitro saturation binding assays (Ai et al., 2001). Honokiol, another active ingredient of the Chinese herb houpo also does not alter the EEG power density of NREM sleep but increases NREM sleep without effects on REM sleep (Qu et al., 2012). Honokiol enhanced chloride currents through GABAA receptors of seven different subunit compositions and was most effective at those containing a3b2 > a2b2 > a1b2 > a1b1 subunits (Taferner et al., 2011), indicating that magnolol and honokiol may more effective on a1 and a3, respectively, than a2-containing receptors, and thus have less influence on EEG delta activity of NREM sleep. Taken together, these findings indicate that sleep promotion by magnolol, honokiol and benzodiazepine is mediated by different GABAA receptor subunits. No alterations of honokiol and magnolol on delta power density of NREM sleep suggest that they may be suitable for the treatment of insomnia because they increase sleep similar to physiological sleep. Fos expression can be utilized as biomarker of neural activation by magnolol. The VLPO is a crucial site for the regulation of sleep. The VLPO contains one essential population of sleep-promoting neurons, which are more active during sleep, as indicated by the expression of c-Fos (Gong et al., 2000; Lu et al., 2000; Saper et al., 2005; Sherin et al., 1996). GABAergic neurons are found to be clustered in the region of the VLPO. They make up roughly 80% of the neurons of the VLPO (Sherin et al., 1998). The sleep-positive neurons in the VLPO innervate the histaminergic neurons in the TMN (Chou et al., 2002; Sherin et al., 1996). These histaminergic cones are closely linked to the transition between arousal and NREM sleep (Huang et al., 2006, 2007). Histaminergic output from the TMN is thought to play an important role in the mediation of forebrain arousal (Lin et al., 1986; Monti, 1993). Inhibition of these neurons by the VLPO may play a major role in causing sleep. In the present study, we observed that magnolol increased c-Fos protein expression in the VLPO and decreased expression in the TMN, indicating that the hypnotic effects of magnolol may be mediated by activation of the sleep center VLPO and inhibiting the neurons of the TMN. Conflicts of interest Except as noted in the acknowledgment, all authors declare that no financial support or compensation has been received from any individual or corporate entity for any research or professional service at any time during the past 3 years. All authors declare no conflict of interest. Acknowledgments We are grateful to Dou Yin and to Yi-Ying Li for excellent technical support. This study was supported in part by grants-in-aid for scientific research from National Natural Science Foundation of China (30970955, 30901797, 31070957, 31171049, 31171010, 31121061), Shanghai Committee of Science and Technology (10XD1400400, 10441901600), National Basic Research Program of China (2009CB5220004, 2011CB711000), Shanghai Leading Academic Discipline Project (B119), China National Science and Technology Major Project for Drug Discovery (2009ZX09303-006), and Program of Basic and Applied Researches for Innovations in Bio-oriented Industry of Japan. References Ai, J., Wang, X., Nielsen, M., 2001. Honokiol and magnolol selectively interact with GABAA receptor subtypes in vitro. Pharmacology 63, 34e41. Alexeev, M., Grosenbaugh, D.K., Mott, D.D., Fisher, J.L., 2012. The natural products magnolol and honokiol are positive allosteric modulators of both synaptic and extra-synaptic GABA(A) receptors. Neuropharmacology 62, 2507e2514.
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Neuropharmacology journal homepage: www.elsevier.com/locate/neuropharm
Magnolol, a major bioactive constituent of the bark of Magnolia officinalis, induces sleep via the benzodiazepine site of GABAA receptor in mice Chang-Rui Chen a,1, Xu-Zhao Zhou a,1, Yan-Jia Luo a, Zhi-Li Huang a, b, c, *, Yoshihiro Urade d, Wei-Min Qu a, c, * a
Department of Pharmacology, Fudan University, Shanghai, 200032, PR China State Key Laboratory of Medical Neurobiology, Shanghai Medical College, Fudan University, Shanghai, 200032, PR China c Institute of Brain Science, Fudan University, Shanghai, 200032, PR China d Department of Molecular Behavioral Biology, Osaka Bioscience Institute, 6-2-4 Furuedai, Suita, Osaka 565-0874, Japan b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 29 March 2012 Received in revised form 13 June 2012 Accepted 14 June 2012
Magnolol (6,60 ,7,12-tetramethoxy-2,20 -dimethyl-1-beta-berbaman, C18H18O2), an active ingredient of the bark of Magnolia officinalis, has been reported to exert potent anti-epileptic effects via the GABAA receptor. The receptor also mediates sleep in humans and animals. The aim of this study was to determine whether magnolol could modulate sleep behaviors by recording EEG and electromyogram in mice. The results showed that magnolol administered i.p. at a dose of 5 or 25 mg/kg could significantly shorten the sleep latency, increase the amount of non-rapid eye movement (non-REM, NREM) and rapid eye movement (REM) sleep for 3 h after administration with an increase in the number of NREM and REM sleep episodes. Magnolol at doses of 5 and 25 mg/kg increased the number of bouts of wakefulness but decreased their duration. On the other hand, magnolol increased the number of state transitions from wakefulness to NREM sleep and subsequently from NREM sleep to wakefulness. Immunohistochemical study showed that magnolol increased c-Fos expression in the neurons of ventrolateral preoptic area, a sleep center in the anterior hypothalamus, and decreased c-Fos expression in the arousal tuberomammillary nucleus, which was located in the caudolateral hypothalamus. The sleep-promoting effects and changes in c-Fos induced by magnolol were reversed by flumazenil, an antagonist at the benzodiazepine site of the GABAA receptor. These results indicate that magnolol increased NREM and REM sleep via the GABAA receptor. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: EEG GABAA Magnolol Sleep VLPO TMN
1. Introduction Insomnia is a highly prevalent condition characterized by inability to sleep or a total lack of sleep. Insomnia afflicts 35% of the general population worldwide. About 10e15% of insomnia patients are diagnosed with moderate to severe disorders. This gives rise to emotional distress, daytime fatigue, and loss of productivity (Buckner et al., 2008). Among pharmacotherapeutic agents, nonbenzodiazepine hypnotics (Z-drugs) are the first line of
Abbreviations: EEG, electroencephalogram; BZ, benzodiazepines; Flu, flumazenil; Mag, magnolol; VLPO, ventrolateral preoptic area; TMN, tuberomammillary nucleus. * Corresponding authors. Department of Pharmacology and State Key Laboratory of Medical Neurobiology, Shanghai Medical College, Fudan University, Shanghai 200032, PR China. Tel.: þ86 21 54237043. E-mail addresses: [email protected] (Z.-L. Huang), quweimin@ fudan.edu.cn (W.-M. Qu). 1 C.R.C. and X.Z.Z equally contributed to this work. 0028-3908/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neuropharm.2012.06.031
management for insomnia, followed by benzodiazepines (BZ), amitryptiline, and antihistamines. The traditional BZ and Z-drugs modulate the GABAA receptors (Rudolph and Knoflach, 2011). Diazepam and zolpidem significantly increase total non-rapid eye movement (non-REM, NREM) sleep, but they can also cause a remarkable decrease in delta power activity during NREM sleep (van Lier et al., 2004). The discrepancy between the increase in sleep continuity and the reduction of power in the lower EEG frequencies caused by BZ in humans is consistent with findings in mice (Tobler et al., 2001). The adverse effects of BZ include decreased psychomotor performance (e.g. next-day drowsiness), tolerance, dependency, and withdrawal symptoms. These have limited their use, indicating that there is still a need for hypnotics that do not produce dependence or detrimental daytime consequences such as sedation and drowsiness. Over the past decades, the identification of separable key functions of GABAA receptor subtypes has suggested that receptor subtype-selective compounds could overcome the limitations of classical BZ (Rudolph and Knoflach, 2011).
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Fig. 1. Chemical structure of magnolol.
Magnolol (6,60 ,7,12-tetramethoxy-2,20 -dimethyl-1-beta-berbaman, C18H18O2, see Fig. 1 for chemical structure) is the major bioactive constituent of magnolia bark. Magnolol increases the threshold of NMDA-induced seizures (Lin et al., 2005). It also delays the onset of myoclonic jerks and generalized clonic seizures and decreases seizure stage and mortality (Chen et al., 2011). Magnolol has also been found to prolong the duration of sleeping time induced by pentobarbital in mice (Ma et al., 2009). These data indicate that magnolol may have sleep-promoting effects. The GABAA receptor is the main inhibitory neurotransmitter receptor in the CNS. The fast-inhibitory activity of GABA is mediated by the GABAA receptor. It has been reported that magnolol increases the number of binding sites and affinity of the GABAA receptor for GABA and then enhances chloride influx (Ai et al., 2001; Ma et al., 2009; Squires et al., 1999; Alexeev et al., 2012). These findings suggest that magnolol might enhance the inhibitory action of GABA on GABAA receptors by binding to the sites in GABAA receptor. We hypothesized that the GABAA receptor might be responsible for the sleep-promoting effects of magnolol. In the present study, we showed that magnolol shortened sleep latency, increased the number of bouts of NREM, and the amount of NREM and REM sleep. Immunostaining showed that magnolol increased c-Fos expression in neurons of the ventrolateral preoptic nucleus (VLPO), which is one of the sleep centers, and decreased it in the arousal histaminergic tuberomammillary nucleus (TMN). These findings suggest that magnolol may have applications in the treatment of insomnia. 2. Methods and materials 2.1. Animals Male SPF inbred C57BL/6J mice (weighing 20e28 g, 11e13 weeks old) were obtained from the Laboratory Animal Center, Chinese Academy of Sciences (Shanghai, China). The animals were housed individually at a constant temperature (24 0.5 C) with a relative humidity of 60 2% on an automatically controlled 12 h light/dark cycle (lights on at 7:00 a.m.), and they had free access to food and water. All efforts were made to minimize animal suffering and to use only the number of animals required for the production of reliable scientific data. All experiments were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. 2.2. Chemicals Magnolol was purchased from the National Center for Safety Evaluation of Drugs (Beijing, China). It was shown to be 98% pure by high-performance liquid chromatography. Rabbit polyclonal anti-c-Fos antibody was purchased from Abcam (Cambridge, MA). Biotinylated donkey anti-rabbit IgG and avidinebiotin-peroxidase came from Vector Laboratories (CA); flumazenil and 3,30 -diamino-benzidine-tetrahydrochloride (DAB) from SigmaeAldrich (SigmaeAldrich, St. Louis, MO, U.S.). Magnolol and flumazenil were dissolved in saline with 5% dimethyl-sulfoxide (DMSO). 2.3. Polygraphic recordings and vigilance state analysis Under pentobarbital anesthesia (50 mg/kg, i.p.), mice were chronically implanted with electrodes for polysomnographic recordings of EEG and electromyogram (EMG). Two stainless steel screws (1 mm in diameter) were inserted through the skull into the cortex (antero-posterior, þ1.0 mm; lefteright, 1.5 mm from bregma or lambda) according to the atlas of Franklin and Paxinos (1997). These
served as EEG electrodes. Two insulated stainless steel, teflon-coated wires were bilaterally placed into both trapezius muscles. These served as EMG electrodes. All electrodes were attached to a microconnector and fixed onto the skull with dental cement. The EEG and EMG recordings were carried out by means of a slip ring designed so that the behavioral movement of the mice would not be restricted. After a 10-day recovery period, the mice were housed individually in transparent barrels and habituated to the recording cable for 3e4 days before polygraphic recording. All mice that were subjected to EEG recordings received vehicle and drug treatment on 2 consecutive days. On day 1, the mice were treated with vehicle (intraperitoneally) at 21:00 and the recordings made on that day served as the baseline data. On day 2, mice were treated with magnolol (intraperitoneally, in a volume of 10 ml/kg body weight) at 21:00, and EEG/EMG signals were recorded for 24 h. 2.4. Analysis of vigilance state The EEG/EMG signals were amplified and filtered (EEG, 0.5e30 Hz; EMG, 20e200 Hz), then digitized at a sampling rate of 128 Hz, and recorded using SLEEPSIGN software as described before (Oishi et al., 2008; Qu et al., 2008). The vigilance states were automatically classified off-line in 4 s epochs into REM sleep, NREM sleep, and wakefulness by SLEEPSIGN, according to the standard criteria (Qu et al., 2010). As a final step, defined sleepewake stages were examined visually and corrected if necessary. 2.5. Pharmacological treatments Magnolol was prepared as described above immediately before use and administered i.p. at 21:00 on the day of the experiment at a dose of 1, 5 or 25 mg/kg (n ¼ 5e7). All drugs were freshly prepared prior to use, and an injection volume (10 ml/kg) was kept constant for in vivo experiments. For baseline data, mice were injected i.p. with vehicle (10 ml/kg). To test receptor mechanisms, 30 min before the injection of magnolol (25 mg/kg), mice were pretreated with flumazenil (Flu) i.p. at 0.5 or 1 mg/kg. Diazepam at 6 mg/kg was injected as a positive control. 2.6. c-Fos immunohistochemistry Nine groups of mice were used. One group was treated with vehicle; and the others were injected i.p. with magnolol at doses of 1, 5, and 25 mg/kg and diazepam at 6 mg/kg. To test the receptor mechanisms, four groups of mice were used: Flumazenil (Flu) 1 mg/kg þ vehicle, Flu 1 mg/kg þ magnolol 25 mg/kg, Flu 0.5 mg/ kg þ magnolol 25 mg/kg, and Flu 1 mg/kg þ diazepam 6 mg/kg groups. Mice were pretreated with Flu, and then the magnolol 25 mg/kg was given after 30 min. At 60 min after the administration of magnolol, the animals were anesthetized with 10% chloral hydrate and perfused via the heart with saline solution followed by icecold 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (pH 7.4). Their brains were then removed, post-fixed in 4% PFA for 6 h, and immersed in 30% sucrose overnight. Thereafter, frozen sections were cut at 30 mm in coronal planes by use of a freezing microtome (Leica Microsystems, Wetzlar, Germany). The sections were stored in a cryoprotectant solution at 20 C for histological analysis. Immunohistochemistry was performed in accordance with the free-floating method described earlier (Chen et al., 2011). Sections were fixed in 4% PFA for 10 min and incubated with 0.3% H2O2 for 15 min to quench the endogenous peroxidase activity. The sections were next placed in blocking solution containing 10% normal goat serum with 0.3% Triton X-100 in 0.01 M phosphate-buffered saline (PBS, pH 7.2) for 30 min at 37 C and then incubated at 4 C for 24 h with a rabbit polyclonal antibody against c-Fos at a 1:5000 dilution in PBS containing 0.3% Triton X-100. On the second day, the sections were incubated with a 1:1000 dilution of biotinylated donkey antirabbit secondary antibodies for 30 min followed by a 1:200 dilution of avidinebiotin-peroxidase for 1 h at 37 C. The peroxidase reaction was visualized with 0.05% DAB in 0.1 M phosphate buffer and 0.01% H2O2. Sections were mounted, dehydrated, and cover slipped. As controls, adjacent sections were incubated without primary antibody to confirm that no non-specific staining had occurred. Digital images were viewed and captured using the Olympus DP 72 microscope (Olympus, Tokyo, Japan). Figures were assembled and adjusted for brightness and contrast in Adobe Photoshop (Adobe Systems Inc, San Jose, CA, U.S.). 2.7. Statistical analysis All data were expressed as the mean SEM (n ¼ 5e7). Statistical analysis was performed with SPSS 17.0 (SPSS Inc., Chicago, IL, U.S.). Time-course of the hourly amounts of each stage, histograms of the amounts of sleep and wakefulness, the number of transitions between sleep and wakefulness, and the number and duration of bouts of sleep and wakefulness were analyzed using the paired t-test, with each animal serving as its own control. For sleep latency, the total number of each vigilance stage during the 3 h immediately following drug treatment, one-way repeated measures analysis of variance (ANOVA) was performed followed by the Fisher probable least-squares difference (PLSD) test to determine whether the differences among groups were statistically significant. For the number of c-Fos immunoreactive neurons, one-way or two-way ANOVA was used, followed by PLSD test. The significance level was set at P < 0.05 for all statistical tests.
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3. Results 3.1. Effects of magnolol on the total amount of sleep and on sleep latency To determine the effects of magnolol on sleepewake profiles, magnolol was injected i.p. into mice at 21:00 at a dose of 1, 5, or 25 mg/kg and the sleepewake patterns of the animals were examined by recording their EEG and EMG. Diazepam was given at 6 mg/kg as a positive control. Typical examples of polygraphic recordings and corresponding hypnograms illustrated the effects of magnolol on the sleepewake profiles of an individual mouse (Fig. 2A). During the period of 21:00 to 00:00, the control mouse spent more time awake than the magnolol-treated mouse. The magnolol-treated mouse started to sleep within several minutes and spent more time in sleep than the control mouse (Fig. 2A). Magnolol significantly decreased the latency to NREM sleep (F[3,20] ¼ 3.0, P < 0.05), which was defined as the time from the
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injection to the appearance of the first NREM sleep episode lasting for at least 20 s. The sleep latency was 30 5.1 min in the mice treated with magnolol at 25 mg/kg. This was significantly shorter than the 84.5 20 min for mice given vehicle only (Fig. 2B). The short sleep latency indicates that magnolol accelerates the initiation of NREM sleep. Time-course changes in NREM sleep showed that the sleeppromoting effects of magnolol at 25 mg/kg lasted for 3 h (Fig. 2C). Magnolol given at 25 mg/kg increased the amount of NREM sleep during the first, second, and third hours to 5.6-, 3.3-, and 3-fold relative to vehicle control, respectively. This enhancement of NREM sleep was concomitant with an increase in REM sleep and a decrease in wakefulness (data not shown) during the second and third hours after the administration of magnolol. There was no further disruption of the sleep architecture during the subsequent period. Similar time-course profiles were observed at the lower dose of 5 mg/kg, but the effect on sleep was slighter, lasting about 2 h after the injection. Magnolol at 1 mg/kg did not affect sleep profiles (data not shown).
Fig. 2. Sleep-stage distributions produced in mice by i.p. administration of magnolol. (A) Typical examples of polygraphic recordings and corresponding hypnograms in mouse treated with vehicle or magnolol at 25 mg/kg. (B) Effect of magnolol and diazepam on NREM sleep latency. Open and filled bars show the profiles for the respective baseline day (vehicle) and experimental day (magnolol or diazepam injection). (C) Time-course changes produced by the i.p. administration of magnolol at 25 mg/kg. Each circle represents the hourly mean SEM of NREM and REM sleep. Open and filled circles indicate the baseline and experimental day profiles, respectively. Magnolol at 25 mg/kg was given at 21:00. The horizontal filled and open bars on the X-axes indicate the 12 h dark and 12 h light periods, respectively. (D) Total time spent in NREM and REM sleep over the course of 3 h after the administration of magnolol. Open and filled bars show the profiles of the respective baseline day (vehicle) and experimental day (magnolol or diazepam injection). Values are mean SEM (n ¼ 5e7). *P < 0.05, **P < 0.01, relative to vehicle control as assessed by one-way ANOVA, followed by PLSD test.
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We calculated the total amount of time spent in NREM and REM sleep and in wakefulness during the 3 h following magnolol and diazepam injection (Fig. 2D). For vehicle treated groups, there was no statistical difference in amounts of NREM (F[3,20] ¼ 0.107, P > 0.05) and REM (F[3,20] ¼ 1.23, P > 0.05) sleep. ANOVA analysis revealed that magnolol increased NREM (F[3, 20] ¼ 4.8, P < 0.01) and REM sleep (F[3, 20] ¼ 3.2, P < 0.01). Magnolol given at 5 and 25 mg/ kg significantly increased the total amounts of NREM sleep during 3 h period by 2.3- and 4-fold, respectively, and decreased the total amount of wakefulness by 23% and 35%, relative to vehicle control. Magnolol given at 5 and 25 mg/kg significantly increased the total amounts of REM sleep during that 3 h period from 0.79 0.45 min and 0.40 0.19 min to 1.81 0.58 min and 5.77 1.31 min, respectively. However, magnolol at 1 mg/kg did not affect the cumulative amount of NREM and REM sleep or that of wakefulness during the 3 h following injection. Diazepam at 6 mg/kg increased the total amount of NREM sleep by 54% and decreased that of wakefulness by 22% during that 3 h period relative to the vehicle control (P < 0.01). However, it did not increase REM sleep during the 3 h following injection relative to vehicle control. These results clearly indicate that magnolol increases the amounts of both NREM and REM sleep.
3.2. Effects of flumazenil on the sleep-promoting effects of magnolol To determine whether the GABAergic system might be involved in the somnogenic effects of magnolol, mice were pretreated with flumazenil, an antagonist at the benzodiazepine site of the GABAA receptor. The magnolol-induced decreases in the duration of sleep latency and increases in the total duration of NREM sleep (25 mg/ kg) were found to be completely antagonized by flumazenil relative to vehicle treated group (Fig. 3A and B). Fig. 3A summarizes time-courses of the hourly amounts of NREM, REM sleep and wakefulness. Flumazenil at 1 mg/kg completely antagonized the increase in NREM and REM sleep caused by magnolol at 25 mg/kg. As shown in Fig. 3B, flumazenil at 1 mg/kg completely antagonized the magnolol-induced increases in the total amounts of NREM and REM sleep during 3 h period (magnolol 25 mg/kg), but flumazenil 0.5 mg/kg partly abolished the increased amounts of NREM sleep and completely blocked the increased amounts of REM induced by magnolol. The injection of flumazenil alone at dose of 1 mg/kg did not produce significant changes in NREM sleep (data not shown). These results indicate that the induction of sleep caused by magnolol is mediated by the benzodiazepine site of the GABAA receptor.
Fig. 3. Sleepewake profiles produced by magnolol in mice pretreated with flumazenil. (A) Time course changes in NREM and REM sleep and waking after pretreatment with flumazenil (Flu, 1 mg/kg, i.p. at 20:30) and injection of magnolol (Mag, 25 mg/kg, i.p. at 21:00) or vehicle in mice. Each circle represents the hourly mean amount of each stage. Open and closed circles represent the profiles of vehicle and drug treatments. The horizontal filled and open bars on the X-axes indicate the 12 h dark and 12 h light periods, respectively. (B) Total time spent in waking, NREM sleep, and REM sleep over the course of 3 h after the Mag at 25 mg/kg in mice pretreated with Flu. Open and filled bars show the profiles for the vehicle-treated day and experimental day (Flu and Mag injection). Values are means SEM (n ¼ 5e7). *P < 0.05, **P < 0.01, relative to vehicle control, as assessed by two-tailed paired t test.
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3.3. Effects of magnolol on characteristics of sleepewake episodes and power density To better understand the sleepewake profile caused by magnolol, we determined the distribution of bouts of NREM sleep and wakefulness as a function of bout or episode duration (Fig. 4). As shown in Fig. 4A, there were no statistical differences in the episode number (F[3,20] ¼ 1.05, P > 0.05) and mean duration (F[3,20] ¼ 1.13, P > 0.05) of wake, the episode number (F[3,20] ¼ 1.08, P > 0.05) and mean duration (F[3,20] ¼ 1.65, P > 0.05) of NREM sleep among the vehicle treatment groups. Magnolol at 5 and 25 mg/kg increased the number of bouts of NREM bouts by 1.5- and 2.3-fold, and the number of episodes of wakefulness by 1.8- and 2.2-fold, but it decreased the mean duration of wakefulness 73% and 72%, respectively. Magnolol at 25 mg/kg also increased the number of REM bouts by 8.3-fold. However, the mean duration of NREM and REM sleep was not altered during the 3 h period following injection (Fig. 4A). Similar changes were seen in positive group of diazepam 6 mg/kg. When we determined the sleep bout distribution during the 3 h following magnolol injection, the magnolol (5 and 25 mg/ kg) increased the number of prolonged bouts of NREM sleep (128e512 s, 64e512 s, respectively) relative to the vehicle injection (Fig. 4B). Magnolol at 25 mg/kg prolonged bouts of REM ranging in duration from 16 to 128 s, and magnolol at 5 mg/kg had no effect on REM sleep (Fig. 4B). Similarly, diazepam 6 mg/kg increased the
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number of bouts of NREM sleep that ranged in duration from 16 to 128 s over that of the vehicle control. These results suggest that magnolol increased the total number of NREM sleep bouts, but did not extend the duration. As a result, the numbers of state transitions from wakefulness (W) to NREM (N) and from N to W were increased by 2.4- and 2.1-fold, respectively, during the 3 h following injection with magnolol at 25 mg/kg. No change in the number of transitions from N to REM (R) or in the number of transitions from R to W was observed (Fig. 5A). As shown in Fig. 5A,B, vehicle or vehicle þ vehicle treatment did not influence stage transition number from wake to NREM (F[1,11] ¼ 1.76, P > 0.05), and NREM to wake (F[1,11] ¼ 1.6, P > 0.05) for the groups treated with magnolol or flumazenil þ magnolol. Magnolol was found to change the number and duration of episodes of wakefulness and NREM sleep and the number of transitions between these two states. These alterations were completely abolished by flumazenil at 1 mg/kg (Figs. 4A, 4B, 5B), further supporting the hypothesis that magnolol promotes sleep via GABAA receptors. We then determined the EEG power spectra during NREM sleep in mice. The power of each 0.5 Hz bin was first averaged across the sleep stages individually and then normalized by calculating the percentage of each bin from the total power (0e24.5 Hz) of the individual animal. As shown in Fig. 5C, there were no significant differences in EEG power density of NREM sleep between the
Fig. 4. Characteristics of sleepewake episodes produced by the administration of magnolol at 5 or 25 mg/kg, diazepam 6 mg/kg, and flumazenil pretreatment followed by magnolol at 25 mg/kg. (A) Total number and mean duration of waking, NREM, and REM bouts in a 3 h period. (B) Changes in the numbers of NREM and REM bouts across different ranges of episode duration over the course of 3 h after the administration magnolol at 5 or 25 mg/kg, flumazenil 1 mg/kg þ magnolol 25 mg/kg, and diazepam at 6 mg/kg. Open and filled bars show the profiles for the respective baseline day (vehicle) and experimental day (magnolol, flumazenil þ magnolol or diazepam). Values are means SEM (n ¼ 5e7). *P < 0.05, **P < 0.01, two-tailed paired t test.
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Fig. 5. Sleepewake state transitions during the 3 h following the administration of (A) Magnolol or (B) Flumazenil þ magnolol. Open and filled bars show the profiles for the respective baseline day (vehicle) and experimental day (magnolol or flumazenil þ magnolol). W, N, and R represent the stages for wakefulness, NREM sleep, and REM sleep, respectively. Values are means SEM (n ¼ 5e7), *P < 0.05, two-tailed paired t test. EEG power density of NREM sleep after the administration of (C) Magnolol or (D) Flumazenil þ magnolol. There was no essential difference in EEG power density during NREM sleep between magnolol and vehicle or between flumazenil þ magnolol treatment and the vehicle control, respectively.
magnolol treatment and the vehicle control. These results suggest that magnolol induced a form of NREM sleep very similar to physiological NREM sleep.
3.4. Effects of magnolol on c-Fos expression in the VLPO and TMN An immunohistochemical study was performed to determine the parts of the brain involved in the sleep-promoting effects of magnolol. Fig. 6A, B shows representative photomicrographs of cFos expression of the VLPO in mice treated with vehicle or magnolol 25 mg/kg. When the number of c-Fos-immunoreactive neurons in the VLPO was taken into account, magnolol and diazepam significantly increased the number of c-Fos-immunoreactive neurons expressed in the VLPO (F[4,25] ¼ 33.6, P < 0.001). The number of Fos-positive cells in the VLPO was found to be 3.67 0.6 in saline treated group. Magnolol at 5 and 25 mg/kg increased the number of Fos-positive cells in the VLPO to 20 2.4 and 23.7 2.0, respectively (Fig. 6E). Fig. 6C, D shows representative photomicrographs of c-Fos expression in the TMN of mice treated with vehicle or magnolol at 25 mg/kg. Magnolol significantly decreased the expression of c-Fos in the TMN (F[4,25] ¼ 16.5, P < 0.001). The expression of c-Fos was
15.8 1.9 in the vehicle control and 7.5 1.0 and 3.8 1.3 in mice treated with magnolol at 5 and 25 mg/kg, respectively (Fig. 6F). Similar changes were observed in the positive group of diazepam 6 mg/kg. However, magnolol at 1 mg/kg did not affect the expression of c-Fos in the VLPO or TMN. These findings indicate that magnolol activated the VLPO sleep center and inhibited the TMN wake center.
3.5. Effects of flumazenil on altered c-fos immunoreactivity induced by magnolol in the VLPO and TMN Fig. 6GeJ shows representative photomicrographs of c-Fos expression in the VLPO and TMN. In subjects pretreated with flumazenil at 1 mg/kg followed by vehicle, magnolol at 25 mg/kg or diazepam at 6 mg/kg delivered by injection induced a change in cFos expression in the VLPO (F[2,20] ¼ 22.64, P < 0.001) and TMN (F[2,20] ¼ 10.9, P < 0.001). Pretreatment with flumazenil at 1 mg/kg completely antagonized the change in the number of Fos-positive cells caused by magnolol given at 25 mg/kg (P > 0.05) and diazepam at 6 mg/kg (P > 0.05) in the VLPO and TMN (Fig. 6K, L). However, flumazenil at 0.5 mg/kg only partially blocked the change in the number of Fos-positive cells caused by magnolol (P < 0.01).
Fig. 6. Magnolol increased the number of Fos-positive cells in the VLPO and decreased expression of Fos in TMN. Magnolol was found to (AeB) increase the number of Fos-positive cells in the VLPO and (CeD) decrease the expression in the TMN. Low- and high-power photomicrographs representative of the (A, a, B, b) VLPO and (C, c, D, d) TMN, respectively. Fos-positive cells were counted. Scale bars: c and d, 400 mm; C and D, 100 mm. Mean number of Fos-positive cells in the (E) VLPO and (F) TMN of vehicle-, magnolol- and diazepamtreated groups, respectively. (GeJ) pretreatment with FLU at 1 mg/kg blocked (H, h). The increase in the number of Fos-positive cells in the VLPO, and (J, j) The decrease of it in the TMN caused by magnolol at 25 mg/kg. Mean number of Fos-positive cells in the VLPO (K) and (L) TMN of flumazenil (0, 0.5, 1 mg/kg) pretreated groups. Flumazenil at 0.5, 1 mg/kg dose-dependently blocked the increase in the number of Fos-positive cells in the VLPO and the decrease caused by magnolol at 25 mg/kg or diazepam 6 mg/kg. Each value represents the mean SEM (n ¼ 6). **P < 0.01, significantly different from the vehicle or flumazenil 1 mg/kg treated group.
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These findings indicate that the GABAA receptor mediates the ability of magnolol to activate the VLPO sleep center and inhibit the activity of the TMN arousal system. 4. Discussion The present study clearly showed that magnolol can shorten sleep latency and increase the amounts of NREM and REM sleep. Magnolol also increased the number of NREM sleep episodes and the number of transitions between wakefulness and NREM sleep states. Flumazenil, a competitive inhibitor of the activity at the BZ recognition site on the GABAA/BZ ligandereceptor complex, reversed the sleep-promoting effects of magnolol. These results suggest that magnolol might share a similar mechanism of action with BZ. GABA is the most abundant inhibitory neurotransmitter in the mammalian central nervous system. It plays a key role in neuronal firing patterns and in the activity of neuronal networks. The modulators of GABAA receptors have been the focus of several therapies aimed at insomnia (Zammit, 2007). Several in vivo and in vitro studies support our findings that magnolol might elicit sleep-promoting effects by acting on the GABAA receptor. Magnolol was found to decrease the release of many neurotransmitters, such as acetylcholine (Ach) and 5-hydroxytryptamine (5-HT) (Kuribara et al., 2000; Tsai et al., 1995; Xu et al., 2008). However, 5-HT and cholinergic receptor subtype antagonists could not block the inhibitory effects of magnolol on 5-HT or Ach release (Kuribara et al., 2000; Tsai et al., 1995). Therefore, the effects of magnolol on GABA receptor seem to be primary, while its impact on other neurotransmission pathways might be secondary. GABAA receptors are ligand-gated chloride channels assembled from a family of 16 homologous subunits including a1e6, b1e3, g1e3, d, ε, p, and q. The most widely distributed GABAA receptors in the human brain are composed of two a1, two b2, and one g2 subunit (Enna and Mohler, 2007; Hadingham et al., 1995; Olsen and Sieghart, 2008; Ymer et al., 1990). The GABAA receptor can be modulated by allosteric regulators such as the classical agonist BZ. The pharmacological properties of GABAA receptors are largely determined by their subunit composition (Rudolph et al., 2001). BZ action requires the presence of both a and g subunits, but not b subunit (Wafford et al., 1993; Wieland et al., 1992). Magnolol most efficiently modulates GABAA receptors containing a and b subunits (Ai et al., 2001; Taferner et al., 2011), but not g subunit (Ai et al., 2001). In addition, magnolol also selectively up-regulates the expression of the GABAA receptor a subunit, elevates both the number of binding sites and the affinity of [3]H-muscimol’s binding to GABAA receptor a-subunit, and increases Cl influx (Ai et al., 2001; Squires et al., 1999). However, magnolol showed no effect on the abundance of g subunits (Ai et al., 2001; Ma et al., 2009). These results suggest that hypnotic effect of magnolol might be mediated by the GABAA receptor a subunit. EEG delta wave activity has been used as an indicator of the depth of NREM sleep (Tobler et al., 2001). Although diazepam and magnolol promote sleep, diazepam suppresses the EEG delta wave activity of NREM sleep, whereas mognolol does not. Why the effects of magnolol on sleep are different from those of benzodiazepines still remains unknown. Probably, they act at different GABAA receptor a subunits. Diazepam binds to GABAA receptors containing a1, a2, a3, and a5 subunits, but its ability to inhibit REM sleep and to alter EEG spectra density was found to be mediated by a2-containing GABAA receptors (Kopp et al., 2004), but not by a1- or a3containing GABAA receptors (Kopp et al., 2003; Tobler et al., 2001). It was reported that magnolol most efficiently modulated receptors containing a1, a2, a3 and a5 subunits (Ai et al., 2001; Taferner et al., 2011). In addition, magnolol increased the number of binding sites
and the binding affinity on a1b2g2 and a1b2 combinations in in vitro saturation binding assays (Ai et al., 2001). Honokiol, another active ingredient of the Chinese herb houpo also does not alter the EEG power density of NREM sleep but increases NREM sleep without effects on REM sleep (Qu et al., 2012). Honokiol enhanced chloride currents through GABAA receptors of seven different subunit compositions and was most effective at those containing a3b2 > a2b2 > a1b2 > a1b1 subunits (Taferner et al., 2011), indicating that magnolol and honokiol may more effective on a1 and a3, respectively, than a2-containing receptors, and thus have less influence on EEG delta activity of NREM sleep. Taken together, these findings indicate that sleep promotion by magnolol, honokiol and benzodiazepine is mediated by different GABAA receptor subunits. No alterations of honokiol and magnolol on delta power density of NREM sleep suggest that they may be suitable for the treatment of insomnia because they increase sleep similar to physiological sleep. Fos expression can be utilized as biomarker of neural activation by magnolol. The VLPO is a crucial site for the regulation of sleep. The VLPO contains one essential population of sleep-promoting neurons, which are more active during sleep, as indicated by the expression of c-Fos (Gong et al., 2000; Lu et al., 2000; Saper et al., 2005; Sherin et al., 1996). GABAergic neurons are found to be clustered in the region of the VLPO. They make up roughly 80% of the neurons of the VLPO (Sherin et al., 1998). The sleep-positive neurons in the VLPO innervate the histaminergic neurons in the TMN (Chou et al., 2002; Sherin et al., 1996). These histaminergic cones are closely linked to the transition between arousal and NREM sleep (Huang et al., 2006, 2007). Histaminergic output from the TMN is thought to play an important role in the mediation of forebrain arousal (Lin et al., 1986; Monti, 1993). Inhibition of these neurons by the VLPO may play a major role in causing sleep. In the present study, we observed that magnolol increased c-Fos protein expression in the VLPO and decreased expression in the TMN, indicating that the hypnotic effects of magnolol may be mediated by activation of the sleep center VLPO and inhibiting the neurons of the TMN. Conflicts of interest Except as noted in the acknowledgment, all authors declare that no financial support or compensation has been received from any individual or corporate entity for any research or professional service at any time during the past 3 years. All authors declare no conflict of interest. Acknowledgments We are grateful to Dou Yin and to Yi-Ying Li for excellent technical support. This study was supported in part by grants-in-aid for scientific research from National Natural Science Foundation of China (30970955, 30901797, 31070957, 31171049, 31171010, 31121061), Shanghai Committee of Science and Technology (10XD1400400, 10441901600), National Basic Research Program of China (2009CB5220004, 2011CB711000), Shanghai Leading Academic Discipline Project (B119), China National Science and Technology Major Project for Drug Discovery (2009ZX09303-006), and Program of Basic and Applied Researches for Innovations in Bio-oriented Industry of Japan. References Ai, J., Wang, X., Nielsen, M., 2001. Honokiol and magnolol selectively interact with GABAA receptor subtypes in vitro. Pharmacology 63, 34e41. Alexeev, M., Grosenbaugh, D.K., Mott, D.D., Fisher, J.L., 2012. The natural products magnolol and honokiol are positive allosteric modulators of both synaptic and extra-synaptic GABA(A) receptors. Neuropharmacology 62, 2507e2514.
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