In the previous experiments, we reported that ethanol extract of Gastrodiae Rhizoma, the dried tuber of Gastrodia ElataBlume (Orchidaceae) increased pentobarbital-induced sleeping behaviors. These experiments were undertaken to know whether 4-hydroxybenzaldehyde (4-HBD), is one of the major compounds of Gastrodiae Rhizoma increases pentobarbital-induced sleeping behaviors and changes sleep architectures via activating GABA _A -ergic systems in rodents. 4-HBD decreased locomotor activity in mice. 4-HBD increased total sleep time, and decreased of sleep onset by pentobarbital (28 mg/kg and 40 mg/kg). 4-HBD showed synergistic effects with muscimol (a GABA _A receptor agonist), shortening sleep onset and enhancing sleep time on pentobarbital-induced sleeping behaviors. On the other hand, 4-HBD (200 mg/kg, p.o.) itself significantly inhibited the counts of sleepwake cycles, and prolonged total sleep time and non-rapid eye movement (NREM) in rats. Moreover, 4-HBD increased intracellular Cl ⁻ levels in the primary cultured cerebellar cells. The protein levels of glutamic acid decarboxylase (GAD) and GABA _A receptors subunits were over-expressed by 4-HBD. Consequently, these results demonstrate that 4-HBD increased NREM sleep as well as sleeping behaviors via the activation of GABA _A -ergic systems in rodents. Insomnia is a common clinical condition characterized by difficulty in sleep onset or maintaining sleep. 1 The current pharmacological treatments for insomnia patients are primarily benzodiazepines and non-benzodiazepines by activating GABA _A receptors and GABA _A -ergic systems. Recently, many kinds of phytomedicines have been introduced and focused on insomnia and anxiety because of modulating GABA _A -ergic systems. 2 Gastrodiae Rhizomahas traditionally been used to treat headaches, myocardial infarction, atherosclerosis, hyperlipidemia, hypertension, dementia, gout and epilepsy. 3 Phytochemical studies of this plant have revealed the presence of several phenolic compounds, including 4-hydroxybenzyl alcohol, 4-hyroxybenzaldehyde (4-HBD), vanillin, vanillyl acohol, β -sitosterol and gastrodin. 4 4-HBD is a major active constituent of Gastrodiae Rhizoma, which is an effective anxiolytic agent via GABA _A -ergic systems in the central nervous systems. 5 In addition, 4-HBD inhibits GABA transaminase (GABA-T) and succinic semialdehyde dehydrogenase in GABA _A -ergic systems. 6 In addition, N ⁶ -(3-methoxyl-4-hydroxybenzyl) adenine riboside, which was originally isolated from Gastrodia elataincreased GABA levels, activating GAD. 7 Many pharmacological evidences suggest that traditional Chinese medicine interacts with GABA _A receptors. This receptor, which functions as a chloride ion channel, is activated by the inhibitory neurotransmitter GABA. Thus, GABA _A -ergic drugs have induced sedative-hypnotic effects in animals and humans. 8 GABA _A receptors complex has specific binding sites: GABA, barbiturates, benzodiazepines, ethanol, steroids and picrotoxin. Basic subunits are composed to α (1~6), β (1~3) and γ (1~3). 9 These binding sites are triggering chloride channel open with resulting membrane hyperpolarization. 10 Base on previous results, it should be very interested in whether 4-HBD, a major component of Gastrodiae Rhizoma also augments pentobarbital-induced sleeping behaviors and modulates sleep architectures in the rodents. The possible mechanisms were involved via the GABA _A -ergic systems. Reagents and chemicals − 4-HBD, n-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE) and cytosine β -D-arabinofuranoside were purchased from Sigma-Aldrich Co. (St Louis, MO, USA). Muscimol, pentobarbital and diazepam were used following company respectively: Tocris (Cookson, Avonmouth, UK or Ellisville, MO, USA), Hanlim Pharm. Co., Ltd. (Seoul, Korea) and Samjin Pharm. (Seoul, Korea). Fetal bovine serum (FBS) and DMEM were obtained from GIBCO (Grand Island, NY, USA). The specific rabbit polyclonal antibodies against GABA _A receptors subunits, GAD _65/67 and the corresponding conjugated anti-rabbit immunoglobulin G-horseradish peroxidase were obtained from Abcam Inc. (Cambridge, UK). Chemiluminescent HRP substrate was purchased from Millipore Co. (Billerica, MA, USA). Animals − The animals used for experiments were 4-weeks old ICR male mice and 8-weeks old male Sprague Dawley rats weighing 20 - 25 g and 300 - 310 g respectively (purchased from Samtako, Osan, Korea). All rodents were housed in acrylic cages (45 × 60 × 23 cm), and were kept at least 1 week for acclimation time. The room condition was maintained at 22 ± 2 ℃, relative humidity (50 - 52%), and a 12-h light/dark cycle with fed ad libitum . This experiment was performed in accordance with the Animal Care and Use Guidelines of Chungbuk National University, Korea. Locomotor activity measurement − The spontaneous locomotor activity was measured by a tilting-type ambulometer (AMB-10, O’Hara, Tokyo, Japan) for 1 h. 11 . Diazepam (2 mg/kg, p.o.) and 4-HBD (50, 100 and 200 mg/kg, p.o.) dissolved in distilled water were administered 30 min and 60 min prior to the experiment, respectively. Pentobarbital-induced sleeping behaviors measurement − All mice were fasted for a day, and all experiments were carried out between 1:00 and 5:00. Pentobarbital was diluted in 0.9% physiological saline. 4-HBD (50, 100 and 200 mg/kg) and muscimol (0.2 mg/kg) were orally administered before 60min and 15min, respectively, and then pentobarbital (42 mg/kg) was injected intraperitoneally. After treatment of pentobarbital, mice were moved to another cage. The sleep latency was recorded as elapse time after pentobarbital injection. The sleep was recorded as the time between the elapse and the righting of animals. The mice that failed sleep within 15 min were excluded from the experiments. 12 EEG telemetry transmitter implantation and data collection − After administration of pentobarbital (50 mg/kg, i.p.), rats were placed on a pad in the stereotaxic apparatus under aseptic conditions. Transmitters (Data Sciences International TA11CTA-F40, MN, USA) were implanted under the skin after the scalp incision. In detail, the skull periosteum was removed, and then drilled the two holes for insert of electric lines. (A: 2.0 [Bregma], L: 1.5; P: 7.0 [Bregma], L: 1.5 contra-lateral). The lines of transmitter were connected skull and subcutaneously. Dental cement was used to fix the electric lines to the skull. The incision parts were sew up by a silk 4 - 0 suture. Antibiotic were given to all rats after surgery (5 million unit potassium penicillin-G Injection, Kunwha, KOREA). After the implantation of transmitter, rats were given recovery time for a week. 4-HBD (200 mg/kg, p.o) was administered to rats. All signals were transmitted by AD converter (Eagle PC30, USA), and stored in the computer. Results of recording graphically shown were also possible to express by the computer. The FFT analysis generated power density values to 0~20.0 Hz with a resolution of 0.5 Hz. FFT data was also issued a mean in the range of 0~20.0 Hz for every 10 sec. EEG data in all rats were recorded from 11:00 am to 5:00 pm. 13 Data analysis − Sleep cycle was saved visually by Sleep-Sign 2.1software (KISSEI Comtec Co Ltd, Matsumoto, Japan). Data were classified into wakefulness, nonrapid eye movement (NREM) and rapid eye movement (REM) for every 10 sec. 14 Wakefulness states and NREM states were found in high frequency and slow wave respectively.-wave (0.75~4.0 Hz) and -wave (5.0~9.0 Hz, peak at 7.5 Hz) increased in low waves of EEG during REM sleep. Counts of wakefulness, NREM, REM and total sleep time (NREM+REM) were recorded to each rat for 6 h. The EEG power was set up 0.5~20.0 Hz in 0.5 Hz bins. Sleep architecture was evaluated in the three waves range of, and (8.0~13.0 Hz). 15 Data was calculated as relative value by the Microsoft Excel. Cell culture − Primary cultures of cerebellar enriched in granule cells were experimented by 7 - 8 days rats. 16 Cerebellar granule cells were seeded amounts of 1.0 × 10 ⁵ cells in per 96-well dishes coated with poly-L-lysine (50 μg/mL; Sigma, St. Louis, MO, USA). DMEM used for cell cultures contained 10% fetal bovine serum, glutamine (2.0 mM), gentamicin (100 μg/mL), antibiotic-antimycotic solution (10 μg/mL; Sigma) and potassium chloride (25 mM). Cells were incubated at 37 ℃ in a humidified atmosphere of 5% CO ₂ /95% air.After 16 h for cell cultures, 96-well plates were added into cytosine arabinofuranoside (final concentration 10 μM; Sigma) for inhibit of nonneuronal cells growth. Measurement of intracellular Cl⁻ influx − MQAE (a sensitive fluorescent substance for Cl ⁻ ) was used for measurement of Cl ⁻ influx of cerebellum cells of rats, and it was based on the method of West and Molloy. 17 After treatment of MQAE for overnight, 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 . The fluorescence data was measured according to excitation wavelength 320 nm and emission wavelength 460 nm by Elisa Reader (SpectraMax M2e Multi-Mode, PA, USA). 18 The data was calculated by F/F ₀ on the basis of the ratio of Cl ⁻ data. F is the fluorescence of each sample, and F ₀ is the fluorescence without Cl ⁻ ions. Western blottings − Protein samples were extracted from the cell cultures of the rat hypothalamus. 4-HBD (final concentration, 10 and 100 g/ml) was dissolve in 0.01% DMSO. The control sample was treated in the same solvent as that used in the 4-HBD treatment. Cell cultures were replaced with fresh medium periodically. After treatment of pentobarbital or 4-HBD, 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 μl/mL aprotinin, 1 mM NaF and 2 mM sodium ortho-vanadate]. Extracts were recovered supernatant after centrifugation at 13,000 × g at 4 ℃ for 20 minutes. Protein concentration was measured using the Bradford protein analysis and stored at −20 ℃. 19 The same amounts of protein put in 10% SDS-polyacrylamide gel, and then was loaded the electrophoresis. The protein was transferred to PVDF membranes (Hybond-P, GE Healthcare, Amersham, UK) using semidry transfer system. The blots were blocked for 1 h at room temperature with 5.0% (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 ℃. After washing with TBST, the blots were treated 1: 3,000 dilution of a secondary antibody at room temperature for 4 hours (goat anti-rabbit, IgG). A chemical for detecting the secondary antibody was performed by using the light emitting substrate is the ECL system (Roche Diagnostics, Mannheim, Germany). Statistical analysis − All statistical analyses were calculated using the Sigma Stat software (SPSS Inc., Chicago, IL, USA).The average for experiments data was calculated as mean ± S.E.M. Statistical significances were compared with control group by Holm-sidak test. All experiments were repeated at least three times for obtain comparable results. P < 0.05 was judged as the value of the significance. Effects of 4-HBD on locomotor activity in mice − 4-HBD (100 and 200 mg/kg) significantly decreased locomotor activity. Diazepam 2 mg/kg, as a positive control also decreased locomotor activity ( Fig. 1 ) From these preliminary experiments, we suggest that 4-HBD might be sedative. Effects of 4-HBD on pentobarbital-induced sleeping behaviors in mice − 4-HBD (50, 100 and 200 mg/kg) significantly reduced sleep latency time. 4-HBD (200 mg/kg, i.p) and muscimol (0.2mg/kg, i.p) significantly enhanced pentobarbital (42 mg/kg, i.p)-induced sleeping time in mice ( Fig. 2 ). We suggest that 4HBD could reduce sleep latency, and increase total sleep. Similar results were obtained by diazepam. Effects of 4-HBD on sleep onset by sub-hypnotic dosage of pentobarbital in mice − 4-HBD (100 and 200 mg/kg) increased the number of sleeping mice, and total sleep time in sub-hypnotic dose (28 mg/kg) of pentobarbital. Muscimol (a GABA agonist), as a positive control also the number of sleeping mice and prolonged the sleep time ( Table 1 ). We suggest that 4-HBD would interact with GABA _A receptors. Effects of 4-HBD on sleep-wake cycles − 4-HBD (200 mg/kg) significantly reduced the sleep-wake cycles ( Fig. 3 ). It means that wakefulness can be reduced by 4HBD. Effects of 4-HBD on sleep architectures − After EEG analysis, we found that 4-HBD (200 mg/kg) significantly prolonged total sleep time and especially NREM (slow wave sleep) sleep. 4HBD also decreased wakefulness ( Fig. 4 ). Effects of 4-HBD on EEG power density − 4-HBD increaseddelta waves in REM sleep ( Fig. 5 B). However, waves in NREM sleep and wakefulness showed no changes by 4-HBD ( Fig. 5 A, 5 C). So, 4-HBD increases slow waves sleep during REM sleep. Effects of 4-HBD on intracellular Cl⁻ influx in primary cultured cerebellar granule cells − 4-HBD (1.0 μM) increased intracellular Cl ⁻ influx, resulting hyperpolarization of neuronal cell membrane. Pentobarbital (10 μM) also significantly increased intracellular Cl ⁻ influx in primary cultured granule cells ( Fig. 6 ). Effects of 4-HBD on expression of GAD_65/67 − The expression of GAD _65/67 was induced by 4-HBD (1.0 and 10 μM) in the primary hypothalamic neuron cells of rats ( Fig. 7 ). We suggest that 4-HBD activates GAD _65/67 . Effects of 4-HBD on expression of GABA_A receptors subunits − From these experiments, the activations of GABA _A receptors subtypes were measured by western blotting. All subtypes of GABA _A receptors except γ subtype were overexpressed by 4-HBD (10 μM). Pentobarbital, as a positive control also showed similar patterns ( Fig. 8 ). The sedative/hypnotic effects of Gastrodiae Rhizoma have been shown in human and animal models. 20 Recent study has reported that N(6)-(3-methoxyl-4-hydroxybenzyl) adenine from Gastrodiae Rhizoma increases GABA _A -ergic systems, such as activating GAD enzyme, increasing GABA levels and overexpressing GABA receptors. 7 , 21 So, it was demonstrated that Gastrodiae Rhizoma would be useful for the treatment of insomnia. 21 It has been evidenced which 4-HBD, an active compound of Gastrodiae Rhizomais neuroprotective. 21 Based on previous studies, we focused on the hypnotic effect with 4-HBD, as an ultimate goal of the experiment. We first found that the locomotor activity was inhibited by 4-HBD. 4-HBD also increased the number of sleep animals and total sleep in sub-hypnotic (28 mg/kg) pentobarbital-induced sleeping behaviors, and shortened sleep onset and increased total sleep in hypnotic (42 mg/kg) pentobarbital-induced sleep. Moreover, 4-HBD decreased sleep/wake cycles by the measurement of EEG. 4-HBD also potentiated pentobarbital-induced sleep behaviors in combination with muscimol (a GABA agonist). Muscimol and other GABA _A agonists cause potentiation of Cl ⁻ influx when administered with pentobarbital or other GABA _A agonists. We presumed that 4-HBD enhances the hypnotic effects via GABA _A -ergic systems in the CNS. Furthermore, the sleep architectures by EEG after oral administration of 4-HBD were analyzed in rats. 4-HBD increased total sleep and NREM sleep. Especially, delta waves in REM sleep were enhanced. 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. It has been well known that GABA _A receptors agonists, such as barbiturates and benzodiazepine decrease wakefulness, and increase REM and NREM. 22 Slow waves of NREM plays an important role in sleep quality. 23 δ-Waveswere associated in REM sleep. To define GABA _A -ergic mechanisms, intracellular chloride influx, and GAD and GABAA receptors activations were measured in the primary cultured cells. Intracellular influx of Cl ⁻ was increased by 4-HBD, causing hyperpolarization of the neuronal membrane and reducing neuronal firing. 24 In addition, GAD is the enzyme for decarboxylase, and plays an important role for GABA synthesis from glutamate in the GABAA-ergic systems., 25 High expression of GAD _65/67 protein levels by 4-HBD was found in the hypothalamic cell of rats. The expressions GABA _A receptors subunits in primary cultured hypothalamic neuronal cells were also measured. All subunits except γ3, α and β sub-units of GABA _A receptors were overexpressed. The most abundant GABA _A receptors submits composition, α ₁ β ₂ γ ₂ is present in most brain regions, and these submits are related to the hypnotic/sedative effects of GABA _A receptors. 4-HBD induced of α- and β-subunitsprotein over-expressions of GABA _A receptors. However, pentobarbital a positive control, induced all the αβγ subtypes in these experiments. From these experiments, 4-HBD enhanced the sedative-hypnotic effect in pentobarbital-induced sleep. 4-HBD, itself decreased sleepwake cycles, and increased NREM sleep. 4-HBD activated GABA _A receptors chloride channel complex, increasing intracellular chloride, activating GAD and GABA _A receptors subtypes. It is concluded that 4-HBD, one of component of Gastrodiae Rhizoma would be useful for the treatment of insomnia.