Gastrodiae Rhizoma Ethanol Extract Enhances Pentobarbital-Induced Sleeping Behaviors and Rapid Eye Movement Sleep via the Activation of GABA A -ergic Transmission in Rodents

This research was designed to identify whether Gastrodiae Rhizoma ethanol extract (GREE) enhances pentobarbital-induced sleep via   γ-aminobutyric acid- (GABA-) ergic systems and modulated sleep architectures in animals. GREE (25, 50, and 100 mg/kg, p.o.) inhibited locomotor activity in mice, in a dose-dependent manner. GREE not only prolonged total sleep time, but also reduced sleep latency time in pentobarbital (42 mg/kg)-treated mice. Subhypnotic pentobarbital (28 mg/kg, i.p.) also increased the number of total sleeping animals in concomitant administration of GREE. GREE (100 mg/kg) alone reduced the count of sleep-wake cycles in electroencephalogram. Furthermore, GREE increased total sleep time and rapid eye movement (REM) sleep. From the in vitro experiments, GREE increased intracellular chloride level in primary cultured cerebellar granule cells. Protein expressions of glutamine acid decarboxylase (GAD) and GABAA receptors subtypes by western blot were increased. Therefore, our study suggested that GREE enhances pentobarbital-induced sleeping behaviors and increased REM via the activation of GABAA-ergic transmission in rodents.


Introduction
Many efforts for the treatment of insomnia have been made by European and oriental scientists from the herbs [1]. Some herbs have been useful for the treatment of mild insomnia [2]. Insomnia is one of the major public health concerns occurring roughly 10-30% in the world populations [3]. The drugs or remedies using treatment of insomnia are targeted to the -aminobutyric acid-(GABA A -) ergic systems [4]. GABA is the main inhibitory neurotransmitter of the CNS that plays the most important role in the balance of neuronal inhibition [5]. In particular, GABA A receptors have been relevant to control of sleep [6]. Additionally, GABA A receptors complex is contained in Cl − ionophore combined with GABA, barbiturates, benzodiazepines, ethanol, steroids, and picrotoxin binding sites [7]. Primary structures of GABA A receptors compose many kinds of subtypes such as (1-6) subunits, (1-4) subunits, (1-3) subunits, (1) subunits, and (1) subunits [8]. Pharmacological profiles and different behaviors of GABA A receptors agonists depend upon their subtypes [9]. Rhizoma of Gastrodia elata Blume (Orchidaceae) has been used traditionally to treat epilepsy, headache, dizziness, and convulsion in oriental countries [10]. The previous study has been focused on treatment of convulsive disorders, such as epilepsy on the central nervous system (CNS) for a long time [11,12]. GR contains many pharmacologically active compounds, including 4-hydroxybenzaldehyde, vanillin, gastrodin, p-hydroxybenzaldehyde, and vanillyl alcohol [13]. In particular, the sedative and hypnotic effects of gastrodin and NHBA [14] have been shown in human and animal models [15].
From these experiments, we focused on whether Gastrodiae Rhizoma ethanol extract (GREE) enhances pentobarbital-induced sleeping behaviors and modulates sleep architecture via GABA A -ergic systems in rodents.

Preparation of Gastrodiae Rhizoma Ethanol Extract.
Gastrodiae Rhizoma (GR) was purchased from the traditional market in Cheongju, Republic of Korea. The sample was identified by Professor Mi Kyeong Lee, who works at the Laboratory of Pharmacognosy, Chungbuk National University. The air-dried GR (1 kg) was extracted three times with 50% ethanol at room temperature. The sample was condensed under pressure to give a brown crude extract. After the extract was processed of centrifuge, the filtrate was condensed in vacuum using a rotary evaporator to give dried powder (500 g).

Animals.
All animals (4-week-old ICR mice and 8week-old Sprague-Dawley rats) were purchased from Samtako (Osan, Republic of Korea). The room temperature was maintained at 22 ± 2 ∘ C and at the relative humidity (50-52%) with a 12 h light/dark cycle in the animal room. Food and water were available ad libitum. All rodents were housed in acrylic cages (45 × 60 × 23 cm) and were kept at least 1 week for acclimation time. Experiments were carried out in accordance with the Principle of Laboratory Animal Care (NIH publication number 85-23, revised 1985) and the Animal Care and Use Guidelines of Chungbuk National University, Republic of Korea.

Measurement of Locomotor
Activity. Spontaneous locomotor activity was measured automatically with a tiltingtype ambulometer (AMB-10, O'Hara, Tokyo, Japan). Each group was processed to adaptation time for 10 min and then measured the movement for 1 h in the activity cage (20 cm in diameter and 18 cm in height) [16]. Groups were administered orally to GREE (25,50, and 100 mg/kg) and diazepam (2.0 mg/kg) before 30 min and 60 min, respectively. The measurement of locomotor activity was processed automatically for 1 h after sample administration [17].

Measurement of Pentobarbital-Induced Sleeping Behaviors.
All mice were fasted for a day. The experiments were carried out between 1:00 and 5:00 pm. Pentobarbital was diluted in 0.9% physiological saline. Muscimol and GREE were suspended in 0.01% DMSO. The groups were administered orally to GREE (25,50, and 100 mg/kg) and muscimol (0.2 mg/kg) before 1 h and 30 min, respectively, and then pentobarbital (42 mg/kg, i.p.) was injected to all mice (0.1 mL/10 g). The animal that stopped the movement after pentobarbital injection was moved carefully to another cage. The sleep latency was recorded as elapsed time between pentobarbital injection and loss of the reflex. The sleep was recorded as elapsed time between the loss and recovery righting movement. Mice which failed to induce sleep within 15 min after administration of pentobarbital were excluded from the experiments [18,19].

Implantation of the EEG Telemetric Transmitter in the
Rat. Rats were anesthetized by administering pentobarbital (50 mg/kg, i.p.). The animals were then placed on a pad and then secured in the stereotaxic apparatus under aseptic conditions. An incision was made in the part of the abdomen's midline, caudal to the cartilage, and the transmitter (Data Sciences International TA11CTA-F40, MN, USA) was placed in subcutaneously. It was attached to the skin with 3 sutures for stabilization. The periosteum was removed and hemostasis was achieved with sterile cotton. Two holes for positioning the electrodes were drilled in the skull surface (A: 2.0 (Bregma), L: 1.5; P: 7.0 (Bregma), L: 1.5 contralateral). Lines from the transmitter were connected subcutaneously and to the hole that made in the skull. The electrodes lines were anchored to the skull with screws and dental cement. It was fixed by a silk 4-0 suture. For a better recovery all rats were given an antibiotic (i.p.) (5-million-unit potassium penicillin-G injection, Kunwha Co., Republic of Korea).

Data
Collection. Following 7 days postsurgical recovery, rats were divided by control group and sample group. GREE (100 mg/kg) was suspended in 0.01% DMSO and administered orally 1 h before the recording. Telemetric recording of cortical EEG and activity were designed with a slight modification as previous reports [20]. The setting of EEG signal was amplified and arranged as 0.5-20.0 Hz in the transmitters. It was additionally set up −0.5/+0.5 volts/unit × 2 and was controlled by a Data Sciences International analog converter and passed to an AD converter (Eagle PC30, USA) saved via computer. The signal was digitized at a sampling rate of 128 Hz by an AD converter. Results of recording marked graphically were possible to express via the computer. An online fast Fourier transformation (FFT) was performed on the EEG data at 2 sec intervals during data acquisition (256 samples) after a Hanning window treatment. The FFT analysis generated power density values from 0.0 to 20.0 Hz at a resolution of 0.5 Hz. The FFT data were further averaged in the range of 0 to 20 Hz for every 10 sec. The sleep data and FFT results were saved on the hard disk every 10 sec for additional offline analysis. Movement of the animal in relation to the telemetry receiver generated transistor-transistor logic (TTL) pulses that were collected and counted as a measure of activity. Data were recorded between 11:00 am and 5:00 pm EEG. The activity was recorded simultaneously in each rat.

Data
Analysis. The states were scored visually from polygraphic recordings stored with SleepSign 2.1 software (KISSEI Comtec Co. Ltd, Matsumoto, Japan). Every 10 sec period was classified as amount of time in wakefulness, nonrapid eye movement (NREM), and rapid eye movement (REM) [21]. The software was classified as EEG signal according to following: wakefulness was identified by a high frequency. Recoding of NREM was focused on the presence of spindles interspersed with slow waves. EEG power during REM is significantly reduced in lower frequency -wave (0.75-4.0 Hz) and increased in the range of -wave activity (5.0-9.0 Hz, peak at 7.5 Hz). The time spent (min) in NREM, REM, total sleep time (NREM + REM), and counts of sleep-wake cycle were processed to get 6 h total for each rat. We also calculated the time of each recording spent in each sleep-wake state (wake, NREM, and REM). The absolute EEG power was calculated during wakefulness, NREM, and REM in 0.5 Hz bins from 0.5 to 20 Hz for the entire 6 h reading. The EEG power density was evaluated in 3 selected frequency bands for wakefulness, NREM, and REM ( -wave, -wave, and -wave (8.0 to 13.0 Hz)). The values were then exported to Microsoft Excel to calculate the amounts of each vigilance state.

Measurement of Intracellular Chloride
Influx. The intracellular Cl − concentration ([Cl − ]i) of cerebellar granule cells of rats was estimated using Cl − sensitive fluorescence probe MQAE according to the method of West and Molloy, with a slight modification [24]. After overnight incubation, the cells were washed three times in the buffer (pH 7.4) which contained 2.4 mM HPO 4 2− , 0.6 mM H 2 PO 4 − , 10 mM HEPES, 10 mM D-glucose, and 1 mM MgSO 4 . Fluorescence was monitored using excitation and excitation wavelength 320 nm and emission wave length 460 nm, respectively, in a microplate reader (SpectraMax M2e Multi-Mode, PA, USA) as described by previous report [25]. The data were calculated as the fluorescence / 0 on the basis of the Cl − ratio ( is the fluorescence as a function of each sample and 0 is the fluorescence without Cl − ions).

Western Blotting of Overexpression of GAD and GABA
Receptors Subunits. The proteins were extracted from the primary rat hypothalamic cell cultures. GREE was suspended in 0.01% DMSO and diluted, respectively, in culture medium to final concentrations of 10 and 100 g/mL. Control samples were treated with solvent alone at the same dilution as that used for drug treatment. The culture medium was completely replaced with fresh medium containing the appropriate drug. After treatment of pentobarbital and GREE, the cells were harvested and treated with cold lysis buffer (25 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM CaCl 2 , 1% Triton X-100, 1 mM PMSF, 10 L/mL aprotinin, 1 mM NaF, and 2 mM sodium orthovanadate). The extracts were centrifuged at 13,000 ×g at 4 ∘ C for 20 min, and the supernatant was taken. Protein concentrations were determined using the Bradford protein assay [26]. The samples were stored at −20 ∘ C.
Equal amounts of protein were loaded and electrophoresed on 10% SDS, polyacrylamide gel. Proteins were then transferred to PVDF membranes (Hybond-P, GE Healthcare, Amersham, UK) using a semidry transfer system. The blots were blocked for 1 h at room temperature with 5% (w/v) BSA (applied to all primary antibodies except for glyceraldehyde 3-phosphate dehydrogenase (GAPDH)) and 5.0% (w/v) skim milk (only applied to GAPDH) in tris-buffered saline solution (TBS) containing 0.1% Tween-20. Both specific rabbit polyclonal antibodies against GABA A receptors subunits and rabbit anti-GAD 65/67 polyclonal antibody at the appropriate dilution in TBST and 5.0% BSA (1 : 2,500 for all the primary antibodies used) were incubated for overnight at 4 ∘ C. Blots were washed and incubated with the horseradish peroxidaseconjugated secondary antibody (1 : 3,000 for goat anti-rabbit IgG) in TBST for 4 h at room temperature. The chemiluminescent substrate for the secondary antibody was developed using the ECL detection system (Roche Diagnostics, Mannheim, Germany).

Statistical Analysis.
All statistical analyses were calculated by Sigma Stat software (SPSS Inc., Chicago, IL, USA). The results were expressed as mean ± S.E.M. for the number of experiments. Statistical significances were compared between treated group and control group by Holm-Sidak test. Each experiment was repeated at least three times and yielded comparable results. Values with < 0.05 were considered significant.

Effects of GREE on Locomotor Activity in Mice.
Locomotor activity showed behavior change by diazepam and GREE. By treating diazepam (2.0 mg/kg) and GREE (25, 50, and 100 mg/kg) to mice, locomotor activity was significantly decreased (Figure 1).

Effects of GREE on Pentobarbital-Induced Sleeping
Behaviors in Mice. GREE (50 and 100 mg/kg) significantly decreased the sleep latency of sleep. However, 100 mg/kg GREE only increased total sleep duration significantly. Pretreatment of muscimol (0.2 mg/kg, i.p.), a positive control, 30 min before the administration of pentobarbital (42 mg/kg), also showed increase in the sleeping time and a decrease in the sleep latency of sleep ( Figure 2).   and prolonged the sleep duration induced by subhypnotic dosage of pentobarbital (28 mg/kg, i.p.). Similarly, muscimol group (0.2 mg/kg, i.p.) affected sleeping behavior induced by pentobarbital significantly (Table 1).

Effects of GREE on the Sleep-Wake Cycles.
After GREE (100 mg/kg, p.o.) treatment, sleep-wake cycles were observed in rats during 6 h. The sample group significantly decreased cycle counts compared to the control group (Figure 3).

Effects of GREE on the Sleep
Architectures. 100 mg/kg GREE was administered orally to the rat. It significantly increased total sleep and REM but decreased wakefulness. The experiment processed recording per 6 h periods (Figure 4).

Effects of GREE on EEG Power Density of REM and NREM
Sleep. During the measure, it showed a change of wakefulness, REM, and NREM by GREE (100 mg/kg, p.o.) ( Figure 5). GREE significantly increased -wave during the REM power density. However, -wave power was reduced during NREM power density.

Effects of GREE on Intracellular Cl − Influx in Primary Cultured Cerebellar Granule Cells. Primary cultured cerebellar neuronal cells enriched in granule cells were calculated.
The measured data is presented as the relative fluorescence / 0 , where 0 is the fluorescence without Cl − and directly proportional to intracellular Cl − concentration. Treatment of granule cells with GREE (0.1, 1, and 10 g/mL, resp.) produced an increase in intracellular Cl − influx. Interestingly, GREE at all dose concentrations showed significant elevated level of intracellular Cl − influx in this culture model. Pentobarbital at 10 M concentration as a positive control also increased the influx of Cl − in primary cultured of cerebellar granule cells ( Figure 6). 65/67 . The expression of GAD was examined with lower (10 g/mL) and higher (100 g/mL) GREE concentrations. Both concentrations affected the abundance of GAD. High protein content of GAD 65/67 was significantly detected by western blot for GREE treatment in primary cultured rat hypothalamic cells at both concentrations ( Figure 7).

Effects of GREE on the Expression of GABA Receptors
Subunits. GREE (10 g/mL) and pentobarbital (PENT 10 M) were treated in primary cultured rat hypothalamic cells for 1 h and were the amounts of -, -, and -subunits in the GABA A receptors. The 3 types of GABA A receptors showed all overexpressed protein levels in GREE. Pentobarbital was also increased all high protein levels except 5 and 3 via the relative values ( Figure 8). GREE has been traditionally used to treat convulsion, epilepsy, and tetanus showing sedative effects. Recent animal and clinical studies indicate that GABA A -ergic systems have been thought to be involved in the mechanisms of these inhibitory effects [27]. GREE and its phenolic compounds such as 4-hydroxybenzaldehyde, vanillin, and gastrodin showed anxiolytic-like effects in animal models through activation of GABA A -ergic neurons [28]. GREE also showed anticonvulsant effects on pentylenetetrazol-induced seizure [29]. The activation of GABA A receptors shows sedative, hypnotic, and anticonvulsant effects. Therefore, the activation of GABA A receptors induces sleep in the central nervous systems [30]. Therefore, we presume that anticonvulsive, neuronal firing [31]. To investigate the more precise mechanisms involved in prolongation of pentobarbital-induced sleeping time caused by GREE, Cl − influx was measured in primary cultured cerebellar granule cells after treatment of GREE. GREE increased intracellular Cl − influx. In addition, the activation of GAD is necessary for GABA synthesis [32] and also plays an important role in GABA A -ergic systems. GAD activation increases GABA A -ergic transmission because GABA is generated from glutamate by the action of GAD. We also confirmed that protein levels of GAD 65/67 by lower and higher dose of GREE were overexpressed in primary cultured hypothalamic cells. Furthermore, GABA A receptors possess different binding sites such as GABA, benzodiazepine, and barbiturates. Benzodiazepine agonists such as diazepam enhance the affinity of GABA A for its receptors and hence decrease locomotor activity. GABA A agonist, such as muscimol, directly interacted with the GABA binding site to activate induced sleeping [33]. Similarly, some herbs which interact with GABA A -ergic systems induce sedative-hypnotic effect [34,35]. GABA A receptors subunits were analyzed by overexpressed protein by pentobarbital and GREE in primary cultured rat hypothalamic cells. GREE produced significantly high protein expressions in all expression patterns of 4subunits ( 2, 3, 4, and 5), 2 -subunits ( 1 and 2), and -subunit ( 3) in GABA A receptors. Pentobarbital also increased high protein levels in all subunits except for 5 and 3. From these experiments, molecular targets of GREE are somewhat different from barbiturates, showing different protein overexpressions of GABA A receptors subtypes by western blotting. Further research is necessary to define the molecular targets of GREE.

Discussion
GABA A receptors agonists reduce wakefulness time and prolong the total REM and NREM sleep. However, these drugs sometimes decrease EEG activity in NREM sleep [36,37]. GREE prolonged significantly total sleep time and increased REM sleep except for NREM sleep. The previous research demonstrated that prolongation of REM and NREM sleep as well as special increase of slow waves in NREM plays important roles in the treatment of insomnia and sleep [38]. From our experiments, GREE increased REM sleep andwaves (slow waves) in REM sleep although it did not increase NREM sleep. It is suggesting that GREE improves the quality of sleep via GABA A -ergic systems.  Where there was significant variability, the individual values were compared using Holm-Sidak test. * * * < 0.005, compared with that of the control.  Figure 8: The effects of GREE on expression of GABA A receptors subunits. Immunoblots of lysed hypothalamic neuronal cells which were treated for 1 h following GREE are shown. GAPDH levels were needed for the normalization of the protein expression. Each column represents the mean with S.E.M. ( = 3). The significance of the effects of the compounds was assessed using analysis of variance (ANOVA). Where there was significant variability, the individual values were compared using Holm-Sidak test. * < 0.05, * * * < 0.005, compared with that of the control.

Conclusions
GREE prolonged pentobarbital-induced sleep behavior and EEG in rodents. It also activated GABA A receptors in GABA A -ergic systems. GREE increased the Cl − influx in the primary cerebellar cell culture and also increased the protein levels of GAD and GABA A receptors subtypes in the primary cultured hypothalamic cells. GREE showed somnogenic effects and might be useful for the treatment of insomnia via the activation of GABA A -ergic systems.