Cognitive dysfunctions induced by a cholinergic blockade and Aβ25–35 peptide are attenuated by salvianolic acid B

Neuropharmacology 61 (2011) 1432e1440

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Cognitive dysfunctions induced by a cholinergic blockade and Ab25e35 peptide are attenuated by salvianolic acid B Dong Hyun Kima, c, Se Jin Parka, c, Jong Min Kima, c, Su Jin Jeond, Dae-Hoon Kimg, Young-Wuk Chog, Kun Ho Sond, Hyoung Jae Leeh, Jae-Hak Moonh, Jae Hoon Cheonge, Kwang Ho Kof, Jong Hoon Ryua, b, c, * a

Department of Life and Nanopharmaceutical Sciences, College of Pharmacy, Kyung Hee University, Hoeki-dong, Dongdaemoon-Ku, Seoul 130-701, South Korea Department of Oriental Pharmaceutical Science, College of Pharmacy, Kyung Hee University, Hoeki-dong, Dongdaemoon-Ku, Seoul 130-701, South Korea Kyung Hee East-West Pharmaceutical Research Institute, College of Pharmacy, Kyung Hee University, Hoeki-dong, Dongdaemoon-Ku, Seoul 130-701, South Korea d Department of Food and Nutrition, Andong National University, Andong 760-749, South Korea e Department of Pharmacy, Sahmyook University, Nowon-goo, Seoul 139-742, South Korea f Department of Pharmacology, College of Pharmacy, Seoul National University, San 56-1, Shillim-Dong, Kwanak-Gu, Seoul 151-742, South Korea g Department of Physiology, Biomedical Science Institute, Kyung Hee University, School of Medicine, Hoeki-dong, Dongdaemoon-Ku, Seoul 130-701, South Korea h Department of Food Science and Technology and Functional Food Research Center, Chonnam National University, Gwangju 500-757, South Korea b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 25 February 2010 Received in revised form 19 July 2011 Accepted 22 August 2011

Alzheimer’s disease (AD) is a neurodegenerative disorder associated with progressive cognitive and memory loss and neuronal cell death. Current therapeutic strategies for AD are very limited; thus, traditional herbal medicines or their active constituents receive much attention. The aim of this study was to investigate the cognitive enhancing effects of salvianolic acid B (SalB) isolated from Salvia miltiorrhiza and its ameliorating effects on various drug-induced amnesic models using the passive avoidance, Y-maze, and Morris water maze tasks. Drug-induced amnesia was induced by administering scopolamine, diazepam, muscimol, or amyloid-b (Ab)25e35 peptide. SalB (10 mg/kg, p.o.) was found to significantly reverse the cognitive impairments induced by scopolamine (1 mg/kg, i.p.) or Ab25e35 (10 nmol/5 ml, i.c.v.) injection. This ameliorating effect of SalB was antagonized by the GABAA receptor agonists, muscimol or diazepam, respectively. In addition, SalB alone was capable of improving cognitive performances. Furthermore, SalB (100 mM) was found to inhibit GABA-induced outward Cl
currents in single hippocampal CA1 neuron. These results suggest that the observed ameliorations of cholinergic dysfunction- or Ab25e35-induced memory impairment by SalB were mediated, in part, via the GABAergic neurotransmitter system after a single administration. Ó 2011 Elsevier Ltd. All rights reserved.

Keywords: Salvianolic acid B Memory Alzehimer’s disease GABAA receptor

1. Introduction Alzheimer’s disease (AD) is the most common age-related neurodegenerative disorder (Koo et al., 1999). Memory impairment is a cardinal symptom of AD and is believed to be derived, at least to some extent, from a central cholinergic neuronal dysfunction (Coyle et al., 1983; Daulatzai, 2010). Accordingly, an improved or repaired cholinergic function remains a target of AD therapy. Moreover, amyloid-b (Ab) peptide has been shown to have the potential to Abbrevations: AD, Alzheimer’s disease; SalB, salvianolic acid B; Ab, amyloid-b; GABA, g-aminobutyric acid; ACh, acetylcholine; AChE, acetylcholinesterase; BZ, benzodiazepine. * Corresponding author. Department of Oriental Pharmaceutical Science, College of Pharmacy, Kyung Hee University, #1 Hoeki-dong, Dongdeamoon-ku, Seoul 130-701, South Korea. Tel.: þ82 2 961 9230; fax: þ82 2 966 3885. E-mail address: [email protected] (J. H. Ryu). 0028-3908/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2011.08.038

induce oxidative stress and inflammation in the brain, and these have been postulated to play important roles in the pathogenesis of AD (McGeer and McGeer, 1999; Galasko and Montine, 2010). Therefore, any compound that improves cholinergic transmission or has anti-oxidative or anti-inflammatory properties is a candidate for AD therapy (Meunier et al., 2006; Saxena et al., 2008). It is generally acknowledged that cognition-enhancing agents should restore cholinergic neurotransmission either by directly activating its postsynaptic receptors or by indirectly overcoming their blockade by inhibiting acetylcholinesterase or stimulating acetylcholine release in the cholinergic blockade-induced memory impairment. The former anti-amnesic mechanism is induced by donepezil or tacrine, whereas the latter can be induced by GABAA receptor antagonists (Vazquez and Baghdoyan, 2003). However, GABAA receptor antagonists may exhibit anxiogenic and convulsant/proconvulsant activities that prevent their use in humans

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(Marcade et al., 2008). Because GABAA receptor antagonist could be beneficial in terms of memory enhancing, it should be of clinical usefulness for AD unless it has anxiogenic and convulsive activities. Salvianolic acid B (SalB) is a polyphenolic and one of the active components of Salvia miltiorrhiza Bunge. (Labiatae) (Liu et al., 2007). Previous studies have shown that SalB inhibits lipid peroxidation in the mitochondrial membrane of hepatic cells (Liu et al., 2001), and that it improves memory impairments induced by ischemiareperfusion injury (Du and Zhang, 1997). These findings suggest that SalB has potent anti-oxidative or anti-inflammatory activities. On the other hand, in a previous study, we found some tanshinone congeners isolated from S. miltiorrhiza have GABAA receptor antagonistic properties, and that they can ameliorate memory impairments induced by cholinergic dysfunction via their GABAergic actions (Kim et al., 2007a). However, no reports have been issued on the anti-amnesic or cognitive enhancing effects of SalB on cholinergic dysfunction. If SalB ameliorates cognitive impairments induced by chemicals such as scopolamine or Ab peptide or enhances cognitive functions, it would become a candidate means of enhancing memory and treating AD. In the present study, we investigated the anti-amnesic and cognitive enhancing effects of SalB on scopolamine- and Ab-induced memory impairment mouse models using the passive avoidance, the Y-maze, and the Morris water maze tasks. 2. Materials and methods 2.1. Animals Male CD1 mice (25e30 g, 7 weeks) and pregnant Sprague-Dawley (SD) rats were purchased from the Orient Co. Ltd., a branch of Charles River Laboratories (Seoul, Korea). Mice were housed 4 or 5 per cage, allowed access to water and food ad libitum, and maintained at a constant temperature (23  1  C) and humidity (60  10%) environment under a 12-h light/dark cycle (light on 07.30e19.30 h). Pregnant SD rats were housed individually and maintained in the same environment. Litters were culled to a maximum of 10 pups per litter by postnatal day 1. Animal treatment and maintenance were carried out in accordance with the Principles of Laboratory Animal Care (NIH publication No. 85-23, revised 1985) and the Animal Care and Use Guidelines of Kyung Hee University, Korea. All efforts were made to minimize the number of animals as well as suffering.

2.2. Materials Tacrine (9-amino-1, 2, 3, 4-tetrahydroacridine hydrochloride), (
) scopolamine hydrobromide, muscimol, flumazenil, bicuculline, Ab25e35 peptide, thiobarbituric acid, cresyl violet acetate, 3, 30 -diaminobenzidine, N-2-hydroxyethylpiperazineN0 -2-ethanesulphonic acid (HEPES), and ethylene glycol-bis (b-aminoethyl ether)N,N,N0 ,N0 -tetraacetic acid (EGTA) were purchased from the Sigma Chemical Co. (St. Louis, MO). Diazepam, a GABAA/benzodiazepine (BZ) binding site agonist, was obtained from DaeWon Pharmaceutical Company (Seoul, Korea). SalB (Fig. 1, 98.6% pure) isolated from S. miltiorrhiza Bunge was kindly donated by one of authors (K.H. Son). Zoletil 50Ò (tiletamine HCl 125 mg/5 ml þ zolazepam HCl 125 mg/5 ml) was purchased from Virbac laboratory (Carros, France). All other materials were of the highest grades available and were obtained from normal commercial sources.

Fig. 1. Structure of salvianolic acid B.

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2.3. Passive avoidance task Acquisition for and retention of passive avoidance behaviors were carried out in identical illuminated and non-illuminated boxes separated by a guillotine door (5  5 cm) (Gemini Avoidance System, San Diego, CA) as described elsewhere (Kim et al., 2007a). The illuminated compartment (20  20  20 cm) contained a 50 W bulb, and the floor of the non-illuminated compartment (20  20  20 cm) was composed of 2 mm stainless steel rods spaced 1 cm apart. The animals underwent 2 separated trials, an acquisition trial and 24 h later a retention trial. For the acquisition trial, a mouse was initially placed in the light compartment and the door between the two compartments was opened 10 s later. When the mouse entered the dark compartment, the door automatically closed and an electrical foot shock (0.5 mA, 3 s) was delivered through the grid floor. One hour before the acquisition trial, mice were administered SalB (2.5, 5, 10, or 20 mg/kg) or tacrine (10 mg/kg, p.o.) as a positive control. Thirty min before testing mice were treated with scopolamine (1 mg/kg, i.p.) or vehicle for the scopolamine-induced memory impairment study. A retention trial was conducted 24 h after the acquisition trial. Mouse was again placed in the light compartment and the latency to enter the dark compartment was recorded. When a mouse did not enter the dark compartment within 300 s, we concluded that the mouse had memorized the passive avoidance training after one acquisition trial. The control group received 10% Tween 80 vehicle solution instead of SalB or tacrine. In the case of GABAA receptor agonist-induced memory impairment, diazepam (1 mg/kg, i.p.) or muscimol (1 mg/kg, i.p.) was introduced to induce memory impairment instead of scopolamine (Kim et al., 2007a, 2008, 2009). Bicuculline (1 mg/kg, p.o.) or flumazenil (10 mg/kg, p.o.) were used as positive control. In a separate antagonism study, SalB (10 mg/kg, p.o.) treated mice were co-administered muscimol (0.5 mg/kg, i.p.) or diazepam (0.5 mg/kg, i.p.) and scopolamine (1 mg/kg, i.p.), 30 and 15 min prior to acquisition trials, respectively. To investigate the effect of SalB on memory in unimpaired animals, SalB alone was administered 1 h before the acquisition trial without administration of scopolamine, diazepam, or muscimol. To avoid a ceiling effect in unimpaired animals, the intensity of electrical foot shock was set at 0.25 mA (for 3 s) (Kim et al., 2007a). This lower intensity shock thus allowed for a behavioral window to see any potential enhancing effects of SalB. To investigate state dependency, SalB was administered 1 h before the acquisition and retention trials, respectively. Scopolamine, muscimol, or diazepam was also administered 30 min before the acquisition and retention trials. 2.4. Y-maze task The Y-maze which is designed for short term spatial memory is a three-arm maze with equal angles between all arms as described elsewhere (Kim et al., 2006). The maze floor and walls were constructed from dark opaque polyvinyl plastic. Mice were initially placed within one arm, and arm entry was recorded when total body of mouse entered an arm including returns into the same arm. The sequence and the number of arm entries were recorded manually for each mouse over an 8 min period. An alternation is counted when the mouse makes consecutive visits to the three different arms, i.e., ABC, CAB, or BCA but not BAB and is converted to a percentage by dividing the number of alternations by the total possible number of alternations (defined as the total number of arm entries minus two). Percentage of alternation was shown by the following equation: % Alternation ¼ [Number of alternations/(Total arm entries
2)  100]. In addition, same arm return (SAR) ratio was calculated by a ratio of same arm returns to the total arm entries. One hour before the test, mice were administered SalB (2.5, 5, 10, or 20 mg/kg, p.o) or tacrine (10 mg/kg, p.o.) as a positive control. After 30 min, memory impairment was induced by administering scopolamine (1 mg/kg, i.p). Control group animals received 10% Tween 80 solution instead of SalB or tacrine. Arms were cleaned between tests to remove odors and residues with water. 2.5. Morris water maze task The Morris water maze which is designed for hippocampal dependent short term and long-term spatial memory (Morris, 1984) is a circular pool (90 cm in diameter and 45 cm in height) with a featureless inner surface. The pool was filled to a depth of 30 cm with water containing 500 ml of milk (23  1  C). The tank was placed in a dimly lit, soundproof test room with various visual cues. The pool was conceptually divided into quadrants. A white platform (6 cm in diameter) was then placed in one of the pool quadrants and submerged 1 cm below the water surface so that it was invisible at water level. The first experimental day was dedicated to swimming training for 60 s in the absence of the platform. To test visible platform finding, small balloon was attached in the platform which was visible in the water surface level and all other visual cues were eliminated (Goodman et al., 2010). For four subsequent days, mice were given two training trials per day. In the case of invisible platform finding test, during the four subsequent training days, the mice were given two training trials per day with the platform in place. When a mouse located the platform, it was permitted to remain on it for 10 s. If the mouse did not locate the platform within 60 s, it was placed on the platform for 10 s. The animal was taken to its home cage and was allowed to dry up under an infrared lamp after each training trial. The time interval between each training trial was 30 min (Kim et al., 2006). During each training trial, the time taken to find the hidden platform (latency) was recorded using a video camera-based Ethovision System (Nodulus, Wageningen, The Netherlands). For each

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training trial, mice were placed in the water facing the pool wall at one of the pool quadrants in a different order each day. One day after the last training trial sessions, mice were subjected to a probe trial session in which the platform was removed from the pool, allowing the mice to swim for 60 s to search for it. A record was kept of the swimming time in the pool quadrant where the platform had previously been placed. SalB (10 mg/kg, p.o) or tacrine (10 mg/kg, p.o) as a positive control was given 1 h before the first training trial at every consecutive day for 4 days. Memory impairment was induced by scopolamine (1 mg/kg, i.p.) at 30 min after treatment of SalB. Control group received 10% Tween 80 solution only. To investigate the memory enhancing effects of SalB in unimpaired animals, mice were trained once a day and all other procedures were the same as described without scopolamine treatment.

lateral to midline at a depth of 2.5 mm from the skull surface at bregma) under anesthesia (a mixture of N2O and O2 (70:30) containing 2% isoflurane). After 5 min, the needle was removed using three intermediate steps with 1 min inter-step delay to minimize backflow, and mice were kept on a warm pad until awakened. Sham animals were injected in an identical manner with the same amount of sterile saline (5 ml). To investigate the anti-amnesic effect of a single SalB treatment against Ab25e35-induced memory impairments, mice received with Ab25e35 peptide were treated with SalB (1 or 10 mg/kg, p.o.) 1 h before the acquisition trial in the passive avoidance task and the Y-maze task at 7 days after Ab25e35 peptide injection. For the antagonism study, diazepam (0.5 mg/kg, i.p.) was administered 30 min after SalB (10 mg/kg, p.o). All other procedures were the same as described in the previous passive avoidance and Y-maze tasks.

2.6. Isolation of single hippocampal CA1 neurons

2.9. Statistics

Hippocampal CA1 neurons were dissociated using techniques described in detail (Cho et al., 2001; Lee et al., 2003). In brief, 10- to 15-day-old SD rats of both sexes were decapitated under Zoletil 50Ò anesthesia (50 mg/kg, i.p.). The brain was removed and transverse slices (400 mm thickness) were made using a vibratome (Series 1500, Vibratome, St. Louis, MO). The slices were pre-incubated in the incubation solution that had been well saturated with 95% O2 and 5% CO2 at room temperature for 30 min. The ionic composition of the incubation solution was composed of (in mM) NaCl 124, KCl 5, KH2PO4 1.2, NaHCO3 24, CaCl2 2.4, MgSO4 1.3, and glucose 10, and the pH was adjusted to 7.4 by continuous bubbling with 95% O2 and 5% CO2. Slices were treated with pronase (protease XIV, 2 mg/12 ml of the oxygenated incubation solution) for 40e60 min at 32  C and subsequently with thermolysin (protease X, 2 mg/12 ml) for 10e20 min at 32  C. Following the enzyme treatments the slices were kept in the incubation solution for 1 h. The hippocampal CA1 region was identified in a 60 mm culture dish coated with silicon using a binocular microscope (SZ-ST, Olympus, Tokyo, Japan), and then was micropunched out from the slices using an electrolytically polished injection needle. The pieces that had been micropunched-out were then mechanically dissociated in a different dish using fire-polished fine glass Pasteur pipettes in 35 mm plastic culture dishes (Falcon Primaria 3801, Becton Dickinson, Rutherford, New Jersey) filled with the standard external solution under an inverted phase-contrast microscope (CK-2, Olympus, Tokyo, Japan). The composition of the standard external solution was composed of (in mM) NaCl 150, KCl 5, CaCl2 2, MgCl2 1, glucose 10, and N-2-hydroxyethylpiperazine-N’-2-ethanesulphonic acid (HEPES) 10, and its pH was adjusted to 7.4 with tris-hydroxymethylaminomethane (Tris-base). The dissociated neurons usually adhered to the bottom of the dish within 20 min and remained viable for electrophysiological studies for up to 6 h after dissociation.

Values are expressed as means  S.E.M. For the passive avoidance test, data were analyzed by a KruskaleWallis non-parametric test. If the results were significant, each treatment group was compared by Dunn’s post hoc test. For the Y-maze tests and the probe trial of the Morris water maze test, data were analyzed by one-way analysis of variance (ANOVA) followed by the Student-NewmaneKeuls test for multiple comparisons. For the antagonism study, interactions between agonist and antagonist were analyzed separately by two-way ANOVA; pairwise comparisons for the assessment of the influence of antagonist on agonist effects were conducted using Bonferroni’s test. Escape latency in the Morris water maze task was analyzed using two-way repeated measures ANOVA followed by Bonferroni’s test with the day as one variable and treatment as a second. Electrophysiological data were analyzed by one-way ANOVA. In the case of comparing the time spent in target quadrant and swimming speed in the probe trial of memory enhancing effect of SalB, Student t-test was employed. Statistical significance was set at P < 0.05.

2.7. Electrophysiological recordings Electrical recordings were performed in the gramicidin-perforated patchrecording mode (Choi et al., 2008) under voltage-clamp conditions. Patch pipettes were prepared from glass capillaries (BF150-86-10, Sutter Instrument, Novato, CA) on a Flaming/Brown micropipette puller (P-97, Sutter Instrument, Novato, CA). The patch pipette was positioned on the neuron using a motorized micromanipulator (MP285, Sutter instrument, Novato, CA). The resistance between the recording electrode filled with the internal pipette solution and the reference electrode was 4e8 MU. The internal pipette solution used for gramicidin-perforated recordings were composed of (in mM) K-gluconate 143, KCl 2, HEPES 10, and EGTA 0.5, and the pH was adjusted to 7.2e7.3 with 1 N KOH. The patch pipette was filled with the internal solution containing gramicidin at the concentration of 60 mg/ml. SalB was dissolved in distilled water at 0.01 M as a stock solution. Drugs were added to external solutions at final concentrations provided in the text and the vehicle concentrations never exceeded 0.01%. Drugs were applied using a rapid application system termed the “Y-tube method” as described elsewhere (Min et al., 1996). The neurons were visualized with phase-contrast equipment on an inverted microscope (IX-70, Olympus, Tokyo, Japan). The hippocampal CA1 neurons were voltage-clamped at the holding voltage of 0 mV and were superfused with the external solution containing GABA at the concentration of 10 mM to induce GABA-activated Cl
currents. The external solution containing GABA was superfused every 2 min and the changes in currents were recorded. Electrical stimulation, current recording and filtration on currents (at 1 kHz) were obtained with an EPC-10 patch-clamp amplifier (HEKA Electronik, Lambrecht, Germany) linked to an IBM-compatible PC controlled by HEKA software. Data were digitized with a LIH 1600 board (HEKA Electronik, Lambrecht, Germany) and stored on a hard disk. The currents were monitored on a thermal linearcorder (WR3320-3CL, Graphtec Co., Yokohama, Japan). Leak sweeps were obtained at several intervals during the experiment by averaging 10 hyperpolarizing test pulses. Leak current was subtracted from the data sweeps by scaling the leak sweep to the data. All experiments were performed at room temperature (22e25  C). 2.8. Inctracerebroventricular injection of Ab25e35 peptide and drug administration protocol Ab25e35 peptide was dissolved in sterile saline (1 mM) in tubes, which were then sealed and incubated for 4 days at 37  C to cause the peptide to aggregate. Mice were injected with aggregated Ab25e35 peptide (10 nmol/5 ml, i.c.v.) into the right lateral ventricle at 1 ml/min (stereotaxic coordinates: 0.2 mm caudal to the bregma and 1.0 mm

3. Results 3.1. The effects of SalB alone on the passive avoidance or the Morris water maze tasks The step-through latency of groups treated with SalB alone in the passive avoidance task was significantly higher than that of the vehicle only control group [H (4) ¼ 12.269, P < 0.05] (Fig. 2A). Mean step-through latency in the SalB (10 mg/kg)-treated group was significantly longer than that in the vehicle only control group (P < 0.05, Fig. 2A). However, the memory effects of SalB on the passive avoidance task were not state-dependent [F (1, 18) ¼ 14.03, P < 0.05, Fig. 2B]. In the Y-maze task, a significant group effect was observed on spontaneous alternation behavior [F (4, 42) ¼ 3.756, P ¼ 0.011, Fig. 2C]. Spontaneous alternation in the SalB alone-treated group (10 and 20 mg/kg) was significantly higher compared to the vehicletreated control group (P < 0.05, Fig. 2C). However, it should be noted that SAR ratio [F (4, 42) ¼ 1.402, P > 0.05, Fig. 2D] and total arm entries [F (4, 42) ¼ 0.161, P > 0.05, Fig. 2E] were similar in all experimental groups. The effect of SalB (10 mg/kg, p.o.) on spatial long-term memory was evaluated using the Morris water maze task. Two-way repeated measures ANOVA revealed a significant difference between control and SalB-treated groups in escape latency [F (1, 18) ¼ 12.17, P < 0.05, Fig. 2G]. In the probe trial session, SalB-treated mice significantly spent more time in the target quadrant (i.e., the area where the platform was previously located) (P < 0.05, Fig. 2I). However, there were no significant differences in swim speed between control and SalB-treated groups during training and probe trials (Fig. 2H and J). Meanwhile, in the visible platform experiment, the escape latencies between groups were not different (Fig. 2F). 3.2. The effects of SalB on scopolamine-induced memory impairment A significant group effect was also observed on step-through latency in the SalB with scopolamine retention trial [H (6) ¼ 42.351, P < 0.05] (Fig. 3A). Mean step-through latency in the scopolamine-treated group was significantly shorter than that in the vehicle only control group (P < 0.05) and mean step-through

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Fig. 2. Effects of an administration of salvianolic acid B (SalB) alone in unimpaired mice. The passive avoidance test (A), state-dependency test (B), Y-maze test (C, spontaneous alternation; D, same arm return ratio; E, total entry), and the water maze task (F, visible platform finding; G, escape latency during 4 training days; H, swimming speed during 4 training days; I, percentage of time spent in the target quadrant (TQ) during the probe trial; J, swimming speed during the probe trial) were conducted in unimpaired mice. SalB (2.5, 5, 10, or 20 mg/kg, p.o.) or the same volume of 10% Tween 80 vehicle solution was administered 1 h before the acquisition trial of the passive avoidance task or the Y-maze task. In the state-dependency test, vehicle or SalB (10 mg/kg) was administered 1 h before both acquisition and retention trials. In the water maze task, SalB (10 mg/kg) or 10% Tween 80 solution was administered 1 h before the first training trial on consecutive days for the 4-day water maze task. Data represent the mean  S.E.M. (n ¼ 10/group). *P < 0.05 versus the vehicle control group.

latency in the SalB (10 and 20 mg/kg)-treated group was significantly greater than that in the scopolamine-treated group (P < 0.05). Furthermore, the shorter step-through latency time induced by scopolamine was significantly reversed by tacrine (P < 0.05, Fig. 3A). However, the ameliorating effects of SalB on the passive avoidance task were not state-dependent [F (2, 27) ¼ 6.152, P < 0.05, Fig. 3B]. In the Y-maze task, a significant group effect was observed on spontaneous alternation behavior [F (6, 63) ¼ 11.650, P < 0.05, Fig. 3C]. Spontaneous alternation in the scopolamine-treated group was significantly lower in the vehicle-treated control group (P < 0.05, Fig. 3C), and this reduced spontaneous alternation induced by scopolamine was significantly reversed by SalB (10 and 20 mg/kg, P < 0.05, Fig. 3C). However, no significant differences were observed between the SalB and tacrine-treated groups in terms of spontaneous alternation. In addition, SAR ratio [F (6, 63) ¼ 3.934, P < 0.05, Fig. 3D] and total arm entries [F (6, 63) ¼ 0.123, P > 0.05, Fig. 3E] were similar in all experimental groups. The effect of SalB (10 mg/kg, p.o.) on spatial learning was evaluated using the Morris water maze task. As shown in Fig. 3G, two-way repeated measures ANOVA revealed that there was main group effect on escape latency [F (3, 36) ¼ 8.506, P < 0.05]. During

the probe trial on day following the final day of training trial sessions, a significant group effect was observed on percentages of time spent in target quadrant [F (3, 36) ¼ 18.250, P < 0.05, Fig. 3I]. Percentage of time spent in the target quadrant in the scopolaminetreated group was significantly less than that in the vehicle-treated control group (P < 0.05, Fig. 3I). Furthermore, the reduction in percentage of time spent in the platform quadrant induced by scopolamine was significantly increased by SalB or tacrine (P < 0.05, Fig. 3I). However, no significant differences in the swimming speeds of both the training trials and the probe trial were observed between the groups (Fig. 3H and J). In the visible platform experiment, there were no significant differences between the groups in the ability to find visible platform (Fig. 3F). 3.3. The effects of SalB on GABAA receptor-activated Cl
currents To confirm the effect of SalB on the GABAergic neurotransmitter system, we examined the effect of SalB on GABAA receptor-activated Cl
currents. Under the experimental condition described above, GABA induced a rapidly activating outward current with gradual inactivation and rapid deactivation kinetics (Fig. 4A). Furthermore, this GABA response was sensitively blocked by the GABAA receptor

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Fig. 3. Effects of a single administration of salvianolic acid B (SalB) on scopolamine-induced memory impairment according to the passive avoidance test (A), state-dependency test (B), the Y-maze test (C, spontaneous alternation; D, same arm return ratio; E, total entry), and the water maze task (F, visible platform finding; G, escape latency during 4 training days; H, swimming speed during 4 training days; I, percentage of time spent in the target quadrant (TQ) during the probe trial; J, swimming speed during the probe trial). SalB (2.5, 5, 10, or 20 mg/kg, p.o.), tacrine (THA, 10 mg/kg, p.o.), or the same volume of 10% Tween 80 vehicle solution was administered 1 h before the acquisition trial in the passive avoidance task and the Y-maze task. Mice were also injected with scopolamine (1 mg/kg, i.p.) 30 min after drug or vehicle treatments. In the state-dependency test, all treatments were conducted before both acquisition and retention trials. In the Morris water maze test, a single treatment with SalB (10 mg/kg, p.o) or scopolamine was conducted first trial on each training trial session. Data represent the mean  S.E.M. (n ¼ 10/group). *P < 0.05 versus the vehicle control group. #P < 0.05 versus the scopolamine-treated group.

antagonist bicuculline (100 mM, data not shown), which indicated that this GABA-activated current is a GABAA receptor-activated Cl
current. We also measured the effect of SalB at various concentrations (0.1, 1, 10, and 100 mM) on GABA-activated Cl
currents. When hippocampal CA1 neurons were pretreated by superfusing them with external solution containing 10 mM of SalB, the peak amplitude of this GABA-activated Cl
current significantly decreased (Fig. 4A). When SalB superfusion was stopped, this GABAergic current gradually returned to the control level. Mean amplitude of this GABAergic current was significantly inhibited to 61 10% of the control by 10 mM of SalB (n ¼ 4, Fig. 4B) and to 42  8% by 100 mM of SalB (n ¼ 3, Fig. 4B), but this current was not significantly affected by 0.1 or 1 mM of SalB. 3.4. GABAA receptor agonists and SalB in the scopolamine-induced memory impairment model We investigated the effects of SalB on GABAA receptor agonist (including diazepam or muscimol) induced memory impairment

using the passive avoidance task. Significant group effects were observed in terms of step-through latencies in retention trials using muscimol [H (6) ¼ 25.708, P < 0.05, Fig. 5A] or diazepam [H (6) ¼ 29.196, P < 0.05, Fig. 5B]. Step-through latencies in the GABAA receptor agonist-treated groups were significantly shorter than in the vehicle only control group (P < 0.05). Moreover, the shorter step-through latency induced by GABAA receptor agonists was significantly reversed by SalB (10 mg/kg, P < 0.05). However, the ameliorating effects of SalB on the passive avoidance task were not state-dependent [F (2, 27) ¼ 8.762, P < 0.05, Fig. 5C; F (2, 27) ¼ 3.268, P < 0.05, Fig. 5D]. In order to determine whether the ameliorating effect of SalB on the cognitive dysfunction induced by scopolamine is exerted via the GABAergic neurotransmitter system, mice treated with SalB (10 mg/kg) were co-treated with scopolamine and sub-effective dose of muscimol (0.5 mg/kg) or diazepam (0.5 mg/kg). As shown in Fig. 5E and F, the increased latency induced by SalB was significantly decreased by muscimol (P < 0.05, Fig. 5E) or diazepam treatment

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significantly less than that of the sham group (P < 0.05). Moreover, the reduction in spontaneous alternation induced by Ab25e35 was significantly attenuated by SalB (10 mg/kg, P < 0.05, Fig. 6B), and this effect of SalB was blocked by diazepam (P < 0.05, Fig. 6E). Two-way ANOVA (using Ab25e35, Ab25e35 þ diazepam, Ab25e35 þ SalB, Ab25e35 þ SalB þ diazepam groups) revealed that there was significant interaction between SalB and diazepam in spontaneous alternation [F (1, 36) ¼ 11.26, P < 0.05], but not in SAR ratio [F (1, 36) ¼ 0.5129, P > 0.05, Fig. 6F]. Total number of entries was not changed by Ab25e35 or SalB (data not shown). 4. Discussion

Fig. 4. Effect of salvianolic acid B (SalB) on GABAA receptor-activated Cl
currents. Pretreatment with SalB inhibited GABAergic currents. (A) Representative current traces showing the inhibitory effect of SalB (10 mM) on GABAergic currents. Open and filled horizontal bars indicate the superfusion durations of GABA (10 mM) and SalB (10 mM), respectively. The holding potential was 0 mV. (B) Dose concentration curve showing the inhibitory effects of SalB (0.1, 1, 10, and 100 mM). Data represent the mean  S.E.M. (n ¼ 3e4/group). *P < 0.05 versus the vehicle control group.

(P < 0.05, Fig. 5F). Moreover, two-way ANOVA analysis revealed that there were significant interactions between SalB and muscimol using scopolamine þ vehicle, scopolamine þ SalB, scopolamine þ muscimol (or diazepam), scopolamine þ SalB þ muscimol (or diazepam) groups [F (1, 36) ¼ 20.538, P < 0.05] or SalB and diazepam [F (1, 36) ¼ 9.376, P < 0.05]. 3.5. The effect of single SalB administration on the memory impairment induced by Ab25e35 A significant group effect was observed in step-through latency during retention trials [H (3) ¼ 26.053, P < 0.05] of the passive avoidance task after the acute administration of SalB (Fig. 6A). Mean step-through latencies of the Ab25e35 (10 nmol/mouse) control group were significantly shorter than those of the sham control group (P < 0.05). However, the reduced step-through latency induced by Ab25e35 was significantly reversed by SalB (10 mg/kg, P < 0.05, Fig. 6A). Furthermore, this ameliorating effect of SalB was blocked by diazepam, a GABAA/BZ-site agonist (P < 0.05, Fig. 6D). Two-way ANOVA (using Ab25e35, Ab25e35 þ diazepam, Ab25e35 þ SalB, Ab25e35 þ SalB þ diazepam groups) revealed that there was significant interaction between SalB and diazepam [F (1, 36) ¼ 8.260, P < 0.05]. In the Y-maze task, a significant group effect was also observed on spontaneous alternation behavior [F (3, 28) ¼ 17.206, P < 0.05, Fig. 6B] and same arm return ratio [F (3, 28) ¼ 5.051, P < 0.05, Fig. 6C]. The level of spontaneous alternation shown by the Ab25e35 control group was

In the present study, we observed that SalB ameliorates the memory impairments induced by scopolamine or Ab25e35 peptide. Based on the findings of our antagonism and electrophysiological studies, we conclude that the memory-ameliorating properties of SalB are derived from a GABAA receptor blockade. GABAA/BZ receptor complex has been reported to control acetylcholine (ACh) release in the hippocampus (Imperato et al., 1993), and it has been suggested that GABAergic drugs might modulate memory formation by influencing cholinergic signaling (Linke et al., 1994; Nakagawa et al., 1995). GABAA receptor agonists (including muscimol) have been reported to cause various forms of memory impairment in rodents (Holt and Maren, 1999; Spanis et al., 1999; Ramirez et al., 2005). Furthermore, the anterograde amnesic effects of GABAA/BZ receptor agonists have been well established by many paradigms, including the passive avoidance test (Anglade et al., 1999; Salgueiro et al., 1997; Chapouthier and Venault, 2002). Likewise, the administration of GABAA receptor antagonist increased ACh release in the hippocampus and basal forebrain (Moor et al., 1998; Vazquez and Baghdoyan, 2003) and attenuated Ab-induced neurotoxicity (Lin and Jun-Tian, 2004), which suggests that GABAA receptor antagonist may be useful for AD therapy. We continue to work on the development of a new drug for AD therapy using GABAA receptor ligands, and during the course of this project, we found that oroxylin A is a candidate (Kim et al., 2007b). In the present study, we observed that SalB ameliorated scopolamine-induced memory impairments, which suggests that its beneficial effect might be associated with enhanced cholinergic signaling. Previously, we reported that tanshinone I and its congeners (diterpene quinones) from S. miltiorrhiza ameliorate memory impairment induced by scopolamine via GABAergic signaling (Kim et al., 2007a, 2009). Although the structure of SalB differs from other active compounds found in S. miltiorrhiza, we believe that SalB exerts its mnemonic activity via GABAA receptor signaling, because the available constituents from S. miltiorrhiza exhibit their anti-amnesic effects, in part, by GABAA receptor antagonism (Kim et al., 2007a). Initially, to identify the contribution of SalB on GABAergic neurotransmission, we examined whether SalB can inhibit GABAinduced Cl
currents in single hippocampal neuron. Interestingly, SalB was found to inhibit GABA-induced outward Cl
currents in a concentration-dependent manner (IC50 ¼ 65 mM). Therefore, we hypothesized that the memory-ameliorating activity of SalB in the scopolamine-induced amnesic animal model might be the result of the inhibition of GABA signaling. Accordingly, we examined whether SalB could improve memory impairment induced by muscimol (a GABAA receptor agonist) or diazepam (a GABAA/BZ-site agonist) using the passive avoidance task. As was expected, SalB reversed the step-through latency reductions induced by both muscimol and diazepam. These results suggest that associative memory related to the GABAergic neurotransmitter system is affected by SalB, and that the ameliorating effect of SalB on the scopolamine-induced cognitive dysfunctional state should be reversed by GABAergic receptor agonists. Thus, we conducted a blocking test using GABAergic

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Fig. 5. Effect of a single administration of salvianolic acid B (SalB) on GABAA receptor agonist-induced memory impairment. In GABAA receptor agonist (A, muscimol; B, diazepam)induced memory impairment model, SalB (2.5, 5, 10, or 20 mg/kg, p.o.), bicuculline (Bic, 1 mg/kg, p.o.), flumazenil (Flu, 10 mg/kg, p.o.) or the same volume of 10% Tween 80 vehicle solution was administered to mice 60 min before the acquisition trial. Mice were also injected with muscimol (Mus, 1 mg/kg, i.p.) or diazepam (Dia, 1 mg/kg, i.p.) 30 min after being treated with drugs or vehicle. Acquisition trial was carried out 30 min after a single treatment with diazepam or muscimol. Data represent the mean  S.E.M. (n ¼ 10/group). *P < 0.05, compared with the control group. #P < 0.05, compared with the muscimol- or diazepam-treated groups. Con, vehicle-treated control group. In the state-dependency test (C, muscimol; D, diazepam), all treatments were conducted as described in the Materials and Methods. Data represent the mean  S.E.M. (n ¼ 10/group). *P < 0.05, compared with the vehicle-treated control group. #P < 0.05, compared with muscimol (diazepam)-treated group. In the blocking test (E and F), SalB (10 mg/kg, p.o.) or the same volume of 10% Tween 80 solution was administered to mice 60 min before the acquisition trial, and 30 min later, mice were injected with scopolamine (Sco) (1 mg/kg, i.p.), and 15 min later, muscimol (E, 0.5 mg/kg, i.p.) or diazepam (F, 0.5 mg/kg, i.p.) were administered. Acquisition trial was carried out 15 min after muscimol or diazepam treatments. At 24 h after the acquisition trial, retention trial was carried out. Data represent the mean  S.E.M. (n ¼ 10/group). *P < 0.05, compared with the vehicle-treated control group. #P < 0.05, compared with scopolamine-treated group. $P < 0.05, compared with scopolamine plus SalB-treated group.

receptor agonists to determine whether the effect of SalB on GABAergic neurotransmitter system actually mediates the ameliorating effect of SalB on scopolamine-induced memory impairment. Interestingly, the ameliorating effect of SalB on scopolamine-induced

memory dysfunction was found to be blocked by both muscimol and diazepam. It can be raised that anxiolytic effect of GABAergic drugs including muscimol and diazepam might affect the results in the passive avoidance task, because this task is fear-motivated. However,

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Fig. 6. Anti-amnesic effects of acute salvianolic acid B (SalB) on Ab25e35-induced memory deficits as determined by the passive avoidance (A) and the Y-maze tasks (B, spontaneous alternation; C, same arm return ratio). The anti-amnesic effect of acute SalB on Ab25e35-induced memory deficits was blocked by diazepam (Dia, 0.5 mg/kg, i.p.) as determined by the passive avoidance (D) and Y-maze tasks (E, spontaneous alternation; F, same arm return ratio). SalB (1 or 10 mg/kg, p.o.) or the same volume of 10% Tween 80 solution was administered to mice 1 h before behavioral tasks. Latency times in the passive avoidance task (A and D), and spontaneous alternations (B and E) and same arm return ratio (C and F) in the Y-maze task are defined in Materials and Methods. Data represent the mean  S.E.M. (n ¼ 10/group). *P < 0.05 versus the sham group. #P < 0.05 versus the Ab25e35 control group. $P < 0.05 versus SalB plus Ab25e35-treated group.

it is likely that the step-through latency would not be affected by the muscimol or diazepam administration because the dose used in the present interaction study was sub-effective dose as observed in Fig. 5E and 5F. It is acknowledged that the risk of epilepsy would be increased if GABA inhibition is acutely inhibited (Yoshiike et al., 2008). However, the dose of SalB used in the present study was non-convulsive (data not shown). In addition, as observed in oroxylin A (Kim et al., 2007b), SalB alone exerted memory enhancing activities in normal naïve mice. Interestingly, SalB also enhanced spatial working memory in the Y-maze task. It is somewhat surprising that the performance could be enhanced in this task. Previously, it was suggested that SAR ratio might specifically applied to the degree of attentional difficulties in the Y-maze task (Wall and Messier, 2002). For the enhancement of learning and memory performances in the Y-maze task, mice must remember which arms has visited in a single session. Therefore, the variation of SAR ratio may affect the results of Y-maze task. In the scopolamine-induced memory impairment study, there was significant group effect in SAR ratio. Therefore, it can be speculated that the effect of SalB on SAR ratio may reflect the result of Y-maze task. In line with this conclusion, it can be also assumed that the effect of SalB on SAR ratio might affect the memory enhancing effect of SalB since SAR ratio was decreased by the administration of SalB. However, the differences between control and SalB-treated groups were not significant in this study. To clarify this, further researches will be needed. Recently, several groups have suggested that the activation of GABAA receptors protects neurons against the Ab toxicity observed in cell-based in vitro studies (Louzada et al., 2004; Lee et al., 2005). We also found that a GABAA receptor agonist, sinapic acid, protects neurons and ameliorates the cognitive dysfunction induced by Ab

in vivo (unpublished data). In the present study, we found that SalB acts as a GABAA receptor antagonist, as determined using behavioral and electrophysiological techniques, and that SalB attenuates Ab25e35-induced cognitive dysfunction by single administration. It remains unclear why SalB, a GABAA receptor antagonist, ameliorates Ab25e35-induced cognitive dysfunction. Yoshiike et al. (2008) reported that Ab peptide induces memory decline through the upregulation of GABA-mediated inhibition. Therefore, it can be speculated that SalB normalizes Ab peptide-induced the changes of GABAA receptor-mediated inhibition by maintaining balance and, in turn, reverses memory impairments. Further study is needed to clarify these issues. Summarizing, this study demonstrates that the memory impairments induced by cholinergic dysfunction caused by scopolamine or Ab25e35 peptide are alleviated by SalB due to its antagonism of GABAA receptors. Our findings and those of others (Durairajan et al., 2008; Liu et al., 2001; Du and Zhang, 1997) suggest that SalB has therapeutic potential for the administration of AD or dementia, because it antagonizes GABAA receptor signaling and has anti-oxidative and anti-inflammatory activities. Acknowledgments This research was supported by a grant funded by Korean Food and Drug Administration (S-06-02-2-CHM-230-0-B, 2006) and the Korea Science & Engineering Foundation (KOSEF) grants funded by the Korea government (MOST) (No. R13-2002-020-03002-0 (2007)). We thank Ike C. dela Peña (College of Pharmacy, Shamyook University), for constructive discussions on the manuscript and editorial input.

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