It has been known that RA, one of major constituents of
Primary insomnia which is characterized by difficulty in initiating and maintaining sleep, causes significant psychological distress. It can be lifelong traits or may be acquired secondary to arousal caused by psychological stress. In 2002, approximately 10% of the world’s population was suffered from insomnia (Paparrigopoulos
GABA which is one of the inhibitory neurotransmitters plays an important role on sleep in the central nervous systems (CNS). GABA-ergic neurons in the rostral hypothalamus are activated during both rapid eye movement (REM) and non-rapid eye movement (NREM) sleep. The POAH consists mainly of inhibitory neurons that release GABA and has been found to be an effective sleep-enhancing site (McGinty and Szymusiak, 2003). The GABA receptors have pentameric structures assembled from five subunits (each with four membrane-spanning domains) selected from multiple polypeptide classes (α, β, γ, δ, etc). GABA appears to interact at two sites between and units triggering chloride channel opening with resulting membrane hyperpolarization (DaSettimo
RA was purchased from Sigma-Aldrich Co (St. Louis, MO, USA). Muscimol (Tocris Bioscience, Bristol, UK) and dimethyl sulfoxide (Amresco Solon, Ohio, USA) was purchased respectively. Sodium pentobarbital (100 mg/2 ml) and diazepam (10 mg/2 ml) were respectively purchased through Hanlim Pharm. Co., Ltd. and Samjin Pharm (Seoul, Korea). Fetal bovine serum (FBS), Dulbecco’s Modified Eagle medium, neurobasal A medium, Trypsin-EDTA and Penicilline-Streptomycin was purchased from GIBCO (Grand island, NY, USA). N-(ethoxycarbonyl methyl)-6-methyl quinolinium bromide (MQAE) and cytosine beta-D-arabinofuranoside was purchased from Sigma-Aldrich Co. Specific polyclonal antibodies on the GABAA receptors subunits of the GAD65/67 extracted from rabbits and anti-rabbit immunoglobulin G-horseradish peroxidase was purchased from Abcam Inc. in Cambridge, UK. Chemiluminescent HRP substrate was purchased from Millipore Corporation (Billerica, MA, USA).
ICR mice (20–24 g) and Sprague Dawley (SD) rats (220–250 g) were purchased from Samtako (Osan, Korea). Rodents were kept in acrylic cages of 45×60×23 cm. The water and feed were supplied enough not to disappear. Temperature and humidity has been maintained 22 ± 2°C and 50–52% respectively, and the animal room to change light and darkness automatically was used. Rodents went through an adjustment period of one week prior to the experiment, and the experiment was carried out between 10:00 a.m. and 5:00 p.m. The animal experiments were conducted according to National institute of Health Guide for Care and Use of Laboratory Animals (NIH publication No. 85-23, revised 1985) and the Animal Care and Use Guidelines of Chungbuk National University (Cheongju, Korea).
Locomotor activity which is spontaneous movement was measured using a tilting type ambulometer (O’Hara AMB-10 in Tokyo, Japan). RA (0.5, 1.0 and 2.0 mg/kg) and diazepam (2.0 mg/kg) were dissolved in 0.9% physiological saline and administered orally to the mice 1 hour and 30 minutes prior to experiment respectively. Each mouse was adapted for 10 minutes prior to the measurement in the activity cage which is diameter 20 cm, height 18 cm (Park
The 12 mice were used in a group. Fasting was conducted 24 hours before the test, and the experiment was carried out between 1:00 p.m. and 5:00 p.m. Pentobarbital sodium (42 mg/kg or 28 mg/kg), muscimol (0.2 mg/kg) and RA (0.5, 1.0 and 2.0 mg/kg) were dissolved in 0.9% physiological saline. RA (0.5, 1.0 and 2.0 mg/kg) and muscimol (0.2 mg/kg) were orally administrated respectively 1 hour and 30 minutes before the experiment, and then pentobarbital sodium (42 mg/ kg) was administered intraperitoneally (0.1 ml/10 g). After administration of pentobarbital sodium, mice that do not appear stereotactic reflection were moved to another empty cage. The period from the administration of pentobarbital sodium to not appearing stereotactic reflection was measured in sleep latency and the period from falling into the sleep to appearing stereotactic reflection was measured in total sleeping time. When mice treated with pentobarbital sodium did not sleep within 15 minutes, these were excluded from the experiment (Wolfman
Rat was anesthetized by administration of pentobarbital (50 mg/kg) into the abdominal cavity. After checking anesthesia, the hair of the head portion was removed and placed on a pad that has a fixed stereotaxic apparatus. The scalp made an incision with a scalpel and splayed the incision under that skin. The transmitter (Data Sciences International TA11CTAF40, MN, USA) inserted, and the two lines of the seven lines of the transmitter was fixed under the skin. The periosteum to show skull was removed, and the blood was wiped with sterile cotton. The two holes were made in the skull with a drill (A: 2.0 [Bregma], L: 1.5; P: 7.0 [Bregma], L: 1.5 contra-lateral) (Paxinos
After recovery period, RA (2.0 mg/kg) dissolved in 0.9% physiological saline was orally administered 1 hour before the measurement. EEG measurements were a little changed from the previous study (Sanford
Sleep data was stored using the Sleep-Sign 2.1 software (KISSEI Comtec Co. Ltd., Matsumoto, Japan). Data have been classified as wakefulness, non-rapid eye movement (NREM) sleep and rapid eye movement (REM) sleep every 10 seconds (Tokunaga
The primary culture of hypothalamus was carried out using 8 days-old SD rats (Ma
The intracellular Cl− influx of the hypothalamic cells was measured by using MQAE that is the Cl− sensitive fluorescence probe (West and Molloy, 1996). After treated with 10 μM (final concentration) MQAE and incubated overnight, cells were washed using the pH 7.4 buffer including 10 mM HEPES, 2.4 mM HPO42−, 0.6 mM H2PO4−, 10 mM D-glucose and 1.0 mM MgSO4 three times (Ma
RA (2.0 mg/kg) and diazepam (2.0 mg/kg) were orally administered 1 hour and 30 minutes before each time in the mice. After sample processing, the hypothalamus taking off was homogenized with 4°C lysis buffer (25 mM Tris-HCl/pH 7.4, 150 mM NaCl, 1.0 mM CaCl2, 1.0% Triton X-100, 1.0 mM PMSF, 10 μl/ml aprotinin, 1.0 mM NaF and 2.0 mM sodium ortho-vanadate). After homogenization, lysis buffer was centrifuged for 15 min at 4°C, 13,000 rpm, and the supernatant was collected. Protein concentration was calculated using the Bradford protein assay method (Fanger, 1987). The amount of protein calculated was put in 10% SDS-polyacrylamide gel. And, it was electrophoresed. Proteins in a gel by using the PVDF membrane (Hybond-P GE Healthcare, Amersham, UK) were transferred. The membrane transferred was blocked for 1 hour and at room temperature by using the 5% (w/v) BSA (all primary antibodies) dissolved in the tris-buffered saline solution including 0.1% Tween-20 (TBST). The membrane was washed with TBS including 3% Tween-20 (TBST) three times. The specific polyclonal antibody for the GABAA receptors and GAD65/67 was diluted in 1: 2500 and made with 5.0% BSA and TBST. After it was attached to the membrane, it was incubated overnight at 4°C. After washing, and the Membrane with PBS three times, the membrane adding horseradish peroxidase-conjugated secondary antibody (1:3,000 for goat anti-rabbit IgG) made of TBST was incubated for 4 hours at room temperature. After washing with TBST three times, proteins in membrane were taken using ECL solution (Roche Diagnostics, Mannheim, Germany).
All statistical analysis was performed with SigmaStat software (SPSS Inc., Chicago, USA). Experimental results are shown as mean ± SEM. When compared to the control group and the sample group, Significance was evaluated by analysis of variance (ANOVA). If there is a significant difference, values were compared respectively with Student’s t-test. However, in sub-hypnotic pentobarbital-induced sleep, the number of falling asleep/total was compared by using Chi-square test. It was considered that
When compared to the control group and the group treated with RA, RA (0.5 mg/kg) reduced about 19.4%, RA (1.0 mg/ kg) reduced about 39.6% and RA (2.0 mg/kg) reduced about 49.8% of the locomotor activity (Fig. 2). A group administering diazepam (2.0 mg/kg) as the positive control decreased about 58.2%, and it showed the most significant reduction.
When compared to the control group, RA (0.5, 1.0 and 2.0 mg/kg) decreased the sleep latency. RA (0.5 mg/kg) decreased approximately 10.7%, RA (1 mg/kg) decreased approximately 12.5% and RA (2.0 mg/kg) decreased approximately 14.0% (Fig. 3A). However, RA (2.0 mg/kg) only increased approximately 22.4% of the total sleeping time after administrating the hypnotic dose of pentobarbital (42 mg/kg) (Fig. 3B). Muscimol (0.2 mg/kg) used as a positive control not only decreased about 34.7% of the sleep latency but also increased about 33.3% of the total sleeping time after injection of pentobarbital.
RA (0.5, 1.0 and 2.0 mg/kg) improved the sleep time. However, when compared to the control group, RA (2.0 mg/kg) increased roughly 31.5%, and the significant change was identified in RA (2.0 mg/kg) only (Table 1). Muscimol (0.2 mg/ kg), a positive control increased roughly 33.6% and showed the biggest significant change in this result. But, there were not significant changes in the number of mice falling asleep although both RA (0.5, 1.0 and 2.0 mg/kg) and muscimol (0.2 mg/kg) improved it.
After administrating RA (2.0 mg/kg) to rat, sleep/wake cycles were measured for 6 hours. RA (2.0 mg/kg) group significantly decreased about 30.2% of sleep/wake cycles counts more than these of control group (Fig. 4). Also, a little significant change was identified.
After RA (2.0 mg/kg) was injected to the rat orally, the sleep architectures (wake, NREM sleep, REM sleep) were recorded. When compared to control group respectively, RA (2.0 mg/kg) not only decreased wake and REM sleep but also increased total sleep and NREM sleep (Fig. 5). Namely, Wake and REM sleep decreased approximately 52.7% and 39.7%, and total sleep and NREM sleep increased approximately 3.9% and 10.9%. Particularly, NREM sleep significantly increased.
RA (2.0 mg/kg) changed wake, total sleep, NREM sleep and REM sleep (Fig. 5). In EEG power density, although the changes of wake and REM sleep existed, there were not the significant changes (Fig. 6A, 6B). However, of NREM sleep, the power density of δ-wave increased about 9.0% and that of α-wave decreased about 15.7% (Fig. 6C). So, the increase of δ-wave and the decrease of α-wave in NREM sleep is significant.
Intracellular Cl− influx of hypothalamus was measured by MQAE. The data shows the relative fluorescence F/F0, that its value was calculated with F value that treated with sample and was measured and F0 value that treated with control and was measured. RA (0.1, 1.0 and 10.0 μg/ml, respectively) increased in intracellular Cl− influx of hypothalamus in a dose dependent manner (Fig. 7). In other words, when compared to control group, RA (0.1, 1.0 and 10.0 μg/ml) increased roughly 30.7%, 47.0% and 55.2% respectively. Intracellular Cl− influx of pentobarbital (10 μM), positive control also increased roughly 74.2%. All the groups treated with RA (0.1 μM, 1.0 μM and 10.0 μM) and pentobarbital showed the significant changes.
The expression of
RA (2.0 mg/kg) and diazepam (2.0 mg/kg) was treated in primary cultured hypothalamic cells of rat and incubated for 1 hour to give the reaction time. When compared to the control, RA (2.0 mg/kg) increased in the expression of α 3, α 4, α 5, β 2 and γ 3 subunits except β1 (Fig. 9). In other words, RA (2.0 mg/kg) increased about 24.8% in α 3, 24.5% in α 4, 48.9% in α 5, 17.8% in β2 and 12.7% in γ3. Also, diazepam (2.0 mg/kg) used as a positive control also increased about 15.2% in α 3, 12.2% in α 4, 32.8% in α 5, 9.1% in β2 and 5.2% in γ3 in the expression of subunits same as being expressed by RA.
The intracellular Cl− influx is increased in the primary cultured hypothalamic cells of rats. GABAA receptors are the ligand-gated ion channels. When GABAA receptor agonists bind to their binding site, Cl− ion channels open and enter into cells. So, the cells become hyperpolarized state, inducing the sleep. GABAA receptors agonists increase GABA-induced Cl− influx. Particularly, it has been known barbiturates including the pentobarbital activate the channel directly (Cottrell
Multiple subunits of several of these classes have been characterized,
This work was conducted during the research year of Chungbuk National University in 2015, and by a grant from the National Research Foundation (NRF) - South Korea by Korean Government (MSIP: MRC, 2008-0062275).