Seven-week old male C57Bl/6 mice (Central Laboratory Animals Inc., Korea) (n=104) were housed in a temperature- and humidity-controlled environment under 12-hour light-dark cycles (7:00 AM to 7:00 PM) with free access to chow and water.

All protocols were approved by the Institutional Animal Care and Use Community of Kyung Hee University (KHMC-IACUC: 12-009) and thus conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals. All tests were conducted between the hours of 9:00 and 18:00.

RP was purchased from Kyung Hee Hanyak (Korea). A voucher specimen was deposited. Dried RP was boiled at 80℃ in 70% ethanol for 1 hour then repeated for 40 more minutes. The combined filtrate was evaporated on a rotary evaporator under reduced pressure and freeze-dried to yield 28% (w/w) of the original product. For experimental use, crude extracts were completely dissolved in DW at a concentration of 10 µg/ml (w/v).

SPS-CF was adapted from a method previously described [9]. Briefly, training consisted of daily EFS delivery for 5 days. Sessions began with 60 s of chamber adaptation (JD-SI-11 Shuttle Box, Jeungdo B&P), followed by 10 s of 80 dB white noise cue and chamber illumination. An EFS of 1 mA was delivered during the last 2 s of cue signals. On the sixth day, mice suffered SPS by 2 hours of immobilization, 20 minutes of forced swimming and exposure to ether until the loss of consciousness. For the next 7 days, mice were left undisturbed in their home cages.

Freezing following exposure to the shock chamber and white noise were observed as measurements of acquired fear for context and cue, respectively. Contextual freezing was measured in the shock chamber for 5 minutes. For the measurement of cued freezing, mice were placed in a novel chamber shaped like an equilateral triangular prism (base length 24 cm; height 57 cm; height 35 cm) for 3 minutes, followed by 3 minutes of the 80 dB white noise. Activity was videotaped for later analysis.

Y maze consisted with three corridors joined at the center at equal angles. Alternation was defined as a sequential visit to each arm without repetition of either of the two previous arms. After placing a mouse in the maze, movements were recorded for 5 minutes. Percent alternation was calculated as the number of correct alternations per total arm visits minus 2 [12].

Spontaneous locomotion was measured in a plexiglass box in a dimly lit room. Locomotor activity of the mice placed in the box was traced for 30 minutes. Distance moved in the open field was analyzed with Smart 3.0 videotracking software (Panlab, Spain).

Anxiety-like behaviors was measured by the elevated plus maze. The apparatus was a cross-shaped maze which has open arms and closed arms on each side, which were elevated 60 cm above the floor. For test, mice were placed in the center region with its head pointing toward an open arm. The movement of mice through the maze was recorded for 5 minutes. Time spent in open arms was analyzed using Smart software.

The novelty-suppressed feeding test compares the strength of a mouse's fear for open environments against its desire to feed; our version of the test was adapted from a method previously described [13]. Before testing, mice were deprived of food in the home cage for 24 hours. During testing, a food pellet was placed in the center of the test box, and a mouse was lowered to a corner of the box. The latency until the mouse bit the pellet was measured for 10 minutes. Any sniffing or touching the pellet was ignored. If the mouse had not acquired the pellet within 10 minutes, feeding latency was recorded as 10 minutes. After this, mice were returned to their home cages, and the latency to acquire a food pellet was observed.

Long-term spatial memory was measured by performance on the Morris water maze. Testing was performed in a water tank (80 cm diameter) filled to 0.5 cm above a platform with tap water made opaque by white paint. For 5 days, mice were trained to locate the submerged platform according to symbols placed around the walls. Each training day included 4 sessions, each starting in a different quadrant. Mice were given 60 seconds to locate the platform, and were allowed to remain on it for 10 s before being returned to the home cage. If mouse was unable to escape within 60 seconds, it was guided to the platform. On the sixth day, probe test was performed without the platform, during which the time spent in the target quadrant was measured for 60 seconds.

For tissue collection, mice were deep anesthetized, decapitated and each hippocampus was rapidly dissected and stored at −80℃ until use. Immunoblotting was performed as described previously [3]. In brief, the hippocampus was denatured in lysis buffer and the protein concentration was determined by BCA method (Bio-rad, CA, USA). Then it was resolved by SDS-PAGE and transferred to the PVDF membrane. Primary antibodies (anti-BAG1; sc-377454, anti-beta actin; sc−517582, Santa Cruz Biotech. Dallas TX) were diluted to a 1:1000 concentration ratio and applied overnight at 4℃. After an hour of incubation in 1:1000 diluted secondary antibody, bands were visualized by ECL solution (Thermo Fisher Scientific, MA) and quantified with a chemiluminiscence detector (Davinch Chemi imaging system, CellTagen, Korea) and the ImageJ image analysis software (NIH, USA).

Norharmane (P1121) and 3,4,5-trimethoxycinnamic acid (TMCA, T1104) were purchased from TCI (Japan). Tenuifolin (CFN98157) and 3,6,disinapoyl sucrose (DISS; CFN90578) were purchased from ChemFaces (Wuhan, China). The HPLC analysis was performed on an Agilent 1100 series liquid chromatograph with diode array detector (DAD) interfaced with an Agilent Chem. Station for the data analysis. The column was a 4.6 mm ID×250 mm Zorbax Eclipse Plus C18 column (5 µm pore size; Agilent, CA) with a Security GuardTM guard cartridge (3.0×4.0 mm, Phenomenex, CA). The mobile phase was composed of 0.1% phosphoric acid in water and acetonitrile using the following linear gradient program: 0~20 min, 10~40% ; 20~30 min, 40~50%. Chromatography was carried out in gradient mode using a flow rate of 1.2 ml/min at 25℃ and was detected at various UV wavelength of individual standards and polygalae radix sample. Injection volume is 10 µL. The stock solutions of norharmane, DISS, and TMCA were prepared at 0.1 mM in DMSO (16.8, 75.5, 23.8 16.8 µg/ml, respectively). The stock solution of tenuifolin was prepated at 1 mM in DMSO (680.4 µg/ml). Polygalae radix extract powder was dissolved in DMSO at 20 mg/ml.

SPSS ver. 20 was used for statistical analysis. Between-group comparisons were made using one-and two-way ANOVA. Tukey's HSD test was performed following significant results. Alpha was set at p<0.05.

After a week of habituation, mice in the cohort 1 and 2 were assigned to four groups: 1) vehicle + non-stress (VeN); 2) RP 0.1 mg/kg + non-stress (RPN); 3) vehicle + SPS-CF (VeS); 4) RP 0.1 mg/kg + SPS-CF (RPS). In cohort 3, mice were assigned to five groups: 1) vehicle + SPS-CF (RPS0); 2) RP 0.1 mg/kg + SPS-CF (RPS0.1); 3) RP 1 mg/kg + SPS-CF (RPS1); 4) RP 10 mg/kg + SPS-CF (RPS10); 5) RP 100 mg/kg + SPS-CF (RPS100). RP was administered by oral gavage during the SPS-CF exposure and undisturbed period for total 13 days. Vehicle group was fed the same volume of DW. All behavioral procedures started at day 14 (Fig. 1).

Fear memory was measured by the intensity of freezing to context and auditory cue (Fig. 2). By exposure to the context, freezing response was higher in SPS-CF mice than unstressed mice both after vehicle (VeN vs VeS, p<0.001) and RP (RPN vs RPS, p<0.001) treatment (Fig. 2A). Among SPS-CF mice, freezing time was lower in RP-treated relative to vehicle-treated (VeS vs RPS, p<0.01). The freezing response to context exposure was lower in all dose ranges [0.1 mg/kg (p=0.004), 1 mg/kg (p=0.011), 10 mg/kg (p<0.001) and 100 mg/kg (p=0.002)] relative to untreated (Fig. 2B).

Freezing response to auditory cue demonstrated a significant effect of SPS-CF before and after the presentation of auditory cue (p<0.001), but RP had no effect on cue-induced fear reduction in SPS-CF mice (VeS vs RPS, p=0.55) (Fig. 2C). Freezing response to auditory cue after incremental doses of RP was non-significant (p=0.61) (Fig. 2D). These results showed a selective effect of RP on contextual conditioned fear reduction but not on cue-induced conditioned fear in SPS-CF mice.

Because the reduction of contextual fear response by RP could be a consequence of cognitive impairments, spatial reference and working memory was measured in the Morris water maze and Ymaze, respectively (Fig. 3).

The latency to locate the hidden platform in Morris water maze was delayed by SPS-CF (VeN vs VeS, p<0.001) (Fig. 3A). RP improved the latency in SPS-CF mice (VeS vs RPS, p<0.001). On the probe trial in Morris water maze, SPS-CF mice spent less time in the target quadrant than unstressed mice whether in RP-(VeN vs VeS, p<0.05) or RP+ (RPN vs RPS, p<0.01) conditions (Fig. 3B). RP had no effect on time spent in the target quadrant relative to unstressed (VeN vs RPN, p=0.593) or SPS-CF mice (VeN vs RPS, p=0.895). Percent alternation in the Y-maze, as a measurement of working memory, demonstrated no effect by SPS-CF (p=0.42) and RP (p=0.84) treatment (Fig. 3C). There was no difference in the swimming speed among experimental groups.

Because the performance in Morris water maze and Y-maze depends on activities in the maze, spontaneous locomotion was measured in the open field. SPS-CF and RP had no effect on travel distance, which meant our cognitive measurement was not confounded by activity changes (Fig. 3D). Based on these findings, RP's reduced contextual fear memory without disrupting spatial reference and working memories.

Because the reduction of contextual fear response by RP could be a consequence of anxiolytic effect, anxiety-like behaviors were measured in the elevated plus maze and novelty suppressed feeding paradigm (Fig. 4).

Both SPS-CF and RP treatment had no effect on open arms time in the elevated plus maze (Fig. 4A). Similarly, the latency to feed in novelty suppressed feeding paradigm were non-significant (Fig. 4B). In their homecages, which is a familiar environment, all mice acquired a food pellet within 5 seconds. Based on these findings, reduced contextual fear was not a consequence of anxiolytic effect.

The expression of BAG1 was measured in the SPS-CF and RP treated mice (Fig. 5). BAG1 is a regulatory protein of GR which has the role of increasing stress resistance. Compared with unstressed mice, the hippocampal BAG1 expression was reduced in SPS-CF mice (VeN vs VeS, p=0.027). Compared with SPS-CF mice, RP treatment to SPS-CF mice increased the hippocampal BAG1 expression (VeS vs RPS, p=0.045).

The presence of norharmane, DISS, TMCA, and tenuifolin, which are the known key components of RP extract, was tested using the HPLC analysis. To measure the retention time, 10 µl of the stock solution of each molecules were injected. The retention time of norharmane, DISS, TMCA and tenuifolin was 8.613 minute measured at 254 nm, 13.15 minute measured at 330 nm, 18.68 minutes measured at 310 nm and 21.38 minute measured at 202 nm, respectively. In the RP extract, peaks of norharmane (Fig. 6A), DISS (Fig. 6B), TMCA (Fig. 6C) were identified. But the peak of tenuifolin (Fig. 6D) was not detected.