Progress in Neuro-Psychopharmacology & Biological Psychiatry 49 (2014) 21–29

Contents lists available at ScienceDirect

Progress in Neuro-Psychopharmacology & Biological Psychiatry journal homepage: www.elsevier.com/locate/pnp

Resveratrol prevents impaired cognition induced by chronic unpredictable mild stress in rats Dexiang Liu a, Qingrui Zhang a, Jianhua Gu a, Xueer Wang c,a, Kai Xie a, Xiuying Xian a, Jianmei Wang b, Hong Jiang a, Zhen Wang b,⁎ a b c

Institute of Medical Psychology, Shandong University School of Medicine, 44#, Wenhua Xi Road, Jinan, Shandong 250012, PR China Institute of Physiology, Shandong University School of Medicine, 44#, Wenhua Xi Road, Jinan, Shandong 250012, PR China Institute of Bioscience, Luoyang Normal University, 71#, Longmen Road, Luoyang, Henan 471022, PR China

a r t i c l e

i n f o

Article history: Received 30 August 2013 Received in revised form 16 October 2013 Accepted 23 October 2013 Available online 31 October 2013 Keywords: Brain derived neurotrophic factor (BDNF) Chronic unpredictable mild stress (CUMS) Cognitive deficits Resveratrol

a b s t r a c t Depression is one of the most common neuropsychiatric disorders and has been associated with impaired cognition, as well as causing neuroendocrine systems and brain proteins alterations. Resveratrol is a natural polyphenol enriched in polygonum cuspidatum and has diverse biological activities, including potent antidepressantlike effects. The aim of this study was to determine whether resveratrol administration influences chronic unpredictable mild stress (CUMS)-induced cognitive deficits and explores underlying mechanisms. The results showed that CUMS (5 weeks) was effective in producing cognitive deficits in rats as indicated by Morris water maze and novel object recognition task. Additionally, CUMS exposure significantly elevated serum corticosterone levels and decreased BDNF levels in the prefrontal cortex (PFC) and hippocampus, accompanied by decreased phosphorylation of extracellular signal-regulated kinase (pERK) and cAMP response element-binding protein (pCREB). Chronic administration of resveratrol (80 mg/kg, i.p., 5 weeks) significantly prevented all these CUMS-induced behavioral and biochemical alterations. In conclusion, our study shows that resveratrol may be an effective therapeutic agent for cognitive disturbances as was seen within the stress model and its neuroprotective effect was mediated in part by normalizing serum corticosterone levels, up-regulating of the BDNF, pCREB and pERK levels. © 2013 Elsevier Inc. All rights reserved.

1. Introduction Stress is known to be one of the causal factors for development of major depression. Based on this observation, the chronic unpredictable mild stress (CUMS) animal model has been developed to mimic the development and progress of clinical depression (Willner, 1997). There is strong evidence that impaired cognition is a core element of major depression, and antidepressant treatment may ameliorate cognitive impairments in parallel to mood improvement of depressive patients (Airaksinen et al., 2004). With an increasing consensus, CUMS animals were also found to cause cognition impairment (McEwen, 2005). Chronic exposure to stress and stress hormones has an impact on brain structures and function involved in cognition and mental health. For example, glucocorticoids, produced by the stress-responsive hypothalamic–pituitary–adrenal (HPA) axis, are known to regulate Abbreviations: BDNF, brain derived neurotrophic factor; CUMS, chronic unpredictable mild stress; CREB, cAMP response element-binding protein; ERK, extracellular signal-regulated kinase; HPA, hypothalamic–pituitary–adrenal; RT-PCR, reverse transcription-polymerase chain reaction; PFC, prefrontal cortex; MWM, Morris water maze; NORT, novel object recognition task. ⁎ Corresponding author. Tel.: +86 531 88383902. E-mail address: [email protected] (Z. Wang). 0278-5846/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.pnpbp.2013.10.017

various brain functions, with well-described effects on human cognition (Lupien et al., 2007). In the same way, in animal, high circulating levels of glucocorticoids or following infusions of glucocorticoid receptor agonists into the hippocampus led to impaired memory retrieval processes (Roozendaal, 2002). In addition, stress-associated cognitive deficits were observed together with reduced synaptic proteins and neurotrophic factor expression. For example, brain derived neurotrophic factor (BDNF), as a member of the neurotrophin family, plays an important role in memory and synaptic plasticity. It has been shown that BDNF infusion ameliorates impairment of spatial learning and memory and long-term potentiation (LTP) caused by chronic stress (Radecki et al., 2005). Neurons in the prefrontal cortex (PFC) and hippocampus respond to repeated stress by showing atrophy and a down-regulation BDNF expression that is associated with memory impairment (McEwen, 2005). These data suggest that HPA axis and BDNF may be the potential target of antidepressants and participate in the molecular mechanism of stress-associated cognitive deficits. Resveratrol (trans-3,4′,5-trihydroxystilbene), is a phenolic compound enriched in polygonum cuspidatum and also found abundantly in the skin of red grapes and red wine. Several recent studies have demonstrated that resveratrol exerts a variety of pharmacological effects, including anti-inflammatory, antioxidant and antiapoptotic (Orsu et al., 2013;

22

D. Liu et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 49 (2014) 21–29

Zhang et al., 2010). Interestingly, accumulating evidence suggested that resveratrol acted as a powerful neuroprotective agent. It was demonstrated that resveratrol protected primary rat cortical neurons from oxidative stress-induced injury (Zhuang et al., 2003). It was also reported that resveratrol reversed the ethanol or Aβ-induced toxicity in the PC12 cells (Jang and Surh, 2003; Sun et al., 1997). Resveratrol abrogated alcohol-induced cognitive deficits and neuronal apoptosis (Tiwari and Chopra, 2013). Furthermore, the powerful neuroprotective effect of resveratrol has also been confirmed in neurodegenerative disorders, such as Alzheimer's disease, Parkinson's disease (Albani et al., 2009; Wang et al., 2006). Although there is only limited information about the antidepressant-like effect of resveratrol, the potential therapeutic value of resveratrol for depression has been increasingly recognized (Xu et al., 2010; Yu et al., 2013). In addition, resveratrol has low bioavailability, and this has been associated with its poor water solubility, its low stability against environmental stress, and its inability to reach a target site in the body to exert the desired health effect (Augustin et al., 2013). Further research is required in enhancing the delivery and bioavailability of resveratrol to obtain the desired biological effects. However, the potential neuroprotective effects of resveratrol against CUMS-induced cognitive deficits and the mechanisms remain to be clarified. In the present study, chronic administration of resveratrol significantly prevented the CUMS-cognitive disturbances, at least in part, by normalizing serum corticosterone levels, up-regulating of the BDNF, phosphorylation of extracellular signal-regulated kinase (pERK) and cAMP response element-binding protein (pCREB) levels in prefrontal cortex (PFC) and hippocampus. 2. Material and methods 2.1. Animals Male Wistar rats (180–200 g) were purchased from Laboratory Animal Center, Shandong University. Upon arrival, the animals were housed under standard laboratory conditions (temperature 20 ± 2 °C, 12 h:12 h light/dark cycle, lights on 0800 h), had free access to food and water and were allowed to habituate to the novel environment for 1 week. In the handling and care of all animals, the International Guiding Principles for Animal Research, as stipulated by the World Health Organization (1985) and as adopted by the Laboratory Animal Center at Shandong University were followed. All efforts were made to reduce the number of animal used and their suffering. 2.2. Drug administration and experimental groups All drugs used in the study were injected intraperitoneally (i.p.) in a total volume of 10 ml/kg. Resveratrol (Sigma, St. Louis, MO, USA) was dissolved in ethanol and diluted to the desired concentration on the day of experiment, and the final concentration of ethanol did not exceed 1% of the total volume. Rats received resveratrol (80 mg/kg) once daily for 5 weeks. Rats were randomly assigned to 4 groups, group I received vehicle (1% ethanol) and served as control; group II received resveratrol; group III was exposed to CUMS and received vehicle (1% ethanol); group IV were subjected to CUMS and received resveratrol. Drugs were administered between 9:00 a.m. and 10:00 a.m. once a day for 5 consecutive weeks. To habituate to i.p., all rats were administered saline (10 ml/kg) daily for three days prior to the experiment. Dose and route administration schedules of resveratrol used in the present experiment were chosen as based on previous results (Yu et al., 2013). 2.3. CUMS procedure Rats were subjected to CUMS for 5 weeks. The procedure of CUMS was performed as previously described (Jiang et al., 2013). In brief, the

CUMS protocol consisted of a variety of mild stressors: (1) food deprivation for 24 h, (2) water deprivation for 24 h, (3) exposure to noise for 3 h, (4) cage tilt (45°) for 7 h, (5) overnight illumination, (6) soiled cage for 24 h, and (7) forced swimming at 4 °C for 6 min. Stressors were administered in a semi-random manner, at any time of day. In this respect, the stress sequence was changed every week in order to make the stress procedure unpredictable. These stressors were randomly scheduled over a one-week period and repeated throughout the 5-week experiment. Control animals were housed in a separate room and had no contact with the stressed animals. After 5 weeks of CUMS, animals were submitted to the Morris water maze and Novel object recognition task 60 min after the last drug treatment. Rats were sacrificed following the behavioral test. Immediately after decapitation, serum samples were collected to measure corticosterone concentrations. Then brains were removed, and then the PFC and hippocampus were dissected according to the rat atlas and frozen at − 70 °C for further biochemical analysis. 2.4. Morris water maze (MWM) test 40 rats were used in the MWM. The spatial reference memory was assessed using MWM test as previously described with minor modifications (Morris, 1984). A black cylindrical tank (120 cm in diameter) was filled with water (21–24 °C), made opaque with the addition of atoxic acrylic black color. The tank was divided into 4 quadrants and a circular escape platform 10 cm in diameter was placed at a fixed position in the center of one of the four quadrants, the target quadrant. The platform was set 2 cm below the water level where rats could not see it directly. A digital camera was positioned above the centre of the tank and linked to a tracking system in order to record the performance of rat (SMART polyvalent video-tracking system, Panlab, Spain). Rats were allowed to swim freely for 60 s to become acclimatized to the apparatus before the test. From the next day, each rat performed four trials per day for 5 consecutive days to find the hidden platform. Each trial began by placing a rat into one of the four quadrants of the pool, facing the wall of the tank. The daily order of the entries into individual quadrants was fixed and all four quadrants were used once in a series of four trials every day. The time taken to escape onto the hidden platform (escape latency) was measured. Rats were given 60 s to find the hidden platform during each acquisition trial. If it failed to locate the platform within 60 s, it was guided onto the platform. The rat was allowed to stay on the platform for 20 s. Twenty-four hours after the last place navigation test, the probe test was performed to measure reference memory during which the platform was withdrawn. Each rat was released from the quadrant opposite to where the platform had been located and its behavior was monitored for 60 s. Time taken to reach the target quadrant and time spent in the target quadrant were recorded. 2.5. Novel object recognition task (NORT) This behavioral paradigm exploits the ability of the rats/mice to explore novel objects over familiar ones during simultaneous presentation and has been employed to evaluate recognition memory as described previously with minor modifications [20]. The object recognition task was conducted in a Plexiglas cage (60 × 40 × 40 cm) with an exchangeable floor. Two days before the experiment, the animals (40 rats, different from those used for MWM) were habituated to the empty experimental arena by allowing them to freely explore for 15 min/day. The objects to be discriminated were water-filled plastic bottles, 8 cm high × 5 cm in diameter, covered with white masking tape, and they were cleaned thoroughly between trials to ensure the absence of olfactory cues. The objects were tested with one independent group of rats and was verified that the rats employed the same time exploring each object.

D. Liu et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 49 (2014) 21–29

Exploration was defined as sniffing or touching the stimulus object with the nose and/or forepaws. Sitting on or walking around the objects was not considered exploratory behavior. During acquisition phase, rats were exposed to two different objects for 10 min but only the first 5 min, during which object discrimination is typically greatest, were later analyzed. One hour later (retrieval phase), one of the objects was replaced by a third, novel object of a similar size (retaining the same location), and the rats were allowed to explore them for 10 min. The new object and its position in the circular arena were counterbalanced across groups. The basic behavioral measurement was the mean time(s) spent exploring each object. Additionally, a discrimination ratio which represents the ability to discriminate the novel from familiar object: novel / (novel + familiar time) was calculated. 2.6. Measurement of serum corticosterone Following the NORT, rats were sacrificed by decapitation and serum samples were collected to measure corticosterone concentrations. Radioimmunoassay (RIA) of corticosterone was performed using [125I]-labeled corticosterone, antiserum and a standard solution in a kit obtained from Institute of Beijing North Biotechnology Academy (Beijing, China). The RIA was performed according to manufacturer's instruction. 2.7. Reverse transcription-polymerase chain reaction (RT-PCR) Total RNA was extracted from the PFC and hippocampus using the Trizol reagent (Gibco, Invitrogen) according to the manufacturer's instructions. RNA concentration was determined using a spectrophotometer (Bio-Rad. Labs) at 260 nm. Identical amounts of RNA (2 μg) were reversely transcribed into cDNA using a commercial RT-PCR kit (Fermentas, Vilnius, Lithuania) according to the manufacturer's instructions. cDNA was subsequently amplified by PCR with specific primers (BDNF: Forward 5′-AGC TGA GCG TGT GTG ACA GT-3′; Reverse 5′-ACC CAT GGG ATT ACA CTT GG-3′; β-actin: Forward 5′-TGG AAT CCT GTG GCA TCC ATG AAA C-3′; Reverse 5′-TAA AAC GCA GCT CAG TAA CAG TCC G-3′). PCR products, separated on a 1.2% agarose/TAE gel, were visualized by staining with ethidium bromide. The densitometric calculations of these values were normalized to β-actin. The intensity of bands was determined using Image-Pro Plus 6.0 software. 2.8. Western blot analysis Frozen tissue mentioned above was cut into small pieces and homogenized in ice-cold RIPA buffer containing protein inhibitors. The dissolved proteins were collected after centrifugation at 10,000 g for 10 min at 4 °C, and the supernatant was then collected. Protein concentration in the supernatant of tissue extracts was determined using a BCA protein assay kit (Pierce Biotechnology, Inc.). A quantity of 30–50 μg total protein was loaded onto a 4–20% gradient polyacrylamide gel, electrophoretically transferred to polyvinylidene difluoride membrane and probed with the following primary antibodies: BDNF (1:1000, Santa Cruz Biotechnology, Inc.), phospho-CREB(Ser133) (1:1000, Cell Signaling Tech.), CREB (1:1000, Cell Signaling Tech.), ERK1/2 (1:1000; Cell Signaling Tech.), and phospho-ERK1/2 (1:1000, Cell Signaling Tech.). β-actin (1:2000; Sigma-Aldrich) was used as an internal control. Secondary antibodies were horseradish peroxidase conjugated to goat/ mouse anti-rabbit IgG (1:8000, Sigma-Aldrich). The membranes were developed using an enhanced chemiluminescence detection system (Pierce, Rockford, IL). 2.9. Statistical analysis Quantitative data were presented as the mean ± SD. The data from the acquisition days in MWM were averaged for each rat (total

23

data / total number of trials per day) and analyzed using repeated measures ANCOVA. If the interaction between the day and treatment was significant, then one-way ANOVA using post-hoc Tukey–Kramer test for the treatment effect for each day was performed. Other data were analyzed statistically by two-way ANOVA for multiple comparisons with stress or drug, and post-hoc Tukey test was conducted to determine differences between specific groups. Differences were considered statistically significant if the p value was b 0.05. 3. Results 3.1. Effects of CUMS and resveratrol administration on MWM test The escape latencies of the four groups during acquisition training (5 days) are presented in Fig. 1A. All groups showed marked improvements in escape latencies over the 5 days of training [F(3,36) = 96.084, p b 0.001, repeated measures ANCOVA], indicating a memory for location of the escape platform. Repeated measures ANCOVA revealed no interaction between training days and groups [F(3,36) = 1.418, p N 0.05]. This suggests that all the rats effectively learned the task. In addition, there were significant differences between the control and stress groups for the escape latencies on day 4 (p b 0.05) and day 5 (p b 0.05). Administration of resveratrol significantly prevented such deficiencies on day 4 and day 5 (p b 0.05 and p b 0.01, respectively). After 5 days of acquisition training, rats learned to locate the escape platform position. To assess memory, on the sixth day the animal was placed in the pool for 60 s without the escape platform and the time required to reach and remain in the target quadrant where the escape platform was located during the training was recorded. ANOVA revealed the effects of stress [F(1, 36) = 6.868, p b 0.05], drug [F(1, 36) = 4.710, p b 0.05], and the stress × drug interaction [F(1, 36) = 6.843, p b 0.05] on the latency of reaching the position of the hidden platform. Post-hoc analysis indicated that the stressed rats spent more time reaching the position of the hidden platform (p b 0.01), compared with that of the control group. However, the stress + resveratrol group demonstrated an enhanced memory response, reaching the position of the hidden platform in a shorter latency as compared with the stress group (p b 0.01). Furthermore, ANOVA revealed the effects of stress [F(1, 36) = 4.217, p b 0.05], drug [F(1, 36) = 4.217, p b 0.05], and the stress × drug interaction [F(1, 36) = 6.371, p b 0.05] on the time in the target quadrant. Post-hoc analysis indicated that the stressed rats spent less time in the target quadrant (p b 0.05), compared with that of the control group. However, the stress + resveratrol group spent significantly more time in the target quadrant as compared with the stress group (p b 0.05) (Fig. 1B–C). These results showed that resveratrol improved the learning and memory performance of stress animals as indicated in the MWM test. 3.2. Effects of CUMS and resveratrol administration on NORT In order to evaluate effects of resveratrol on recognition memory, we analyzed the time each rat took to discriminate a novel object from a familiar one when both are presented simultaneously. Each group was analyzed separately by an observer blind to the treatment. Results of memory measurement were analyzed by two-way ANOVA (stress × drug). On the acquisition phase, all groups showed similar exploration times with the identical sample objects [F(3, 36) = 0.488, p N 0.05] (Fig. 2A). In the retrieval phase, as shown in Fig. 2B, ANOVA revealed the effects of stress [F(1, 36) = 6.833, p b 0.05], drug [F(1, 36) = 6.173, p b 0.05], and the stress × drug interaction [F(1, 36) = 4.761, p b 0.05] on exploration time in the novel object recognition test. Post-hoc analysis indicated that the stressed rats spent less time exploring the novel objects (p b 0.01), compared with that of the control group. Furthermore, treatment with resveratrol significantly increased the amount of time directed to novel

24

D. Liu et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 49 (2014) 21–29

A Escape Latency (sec)

100

Control Control+Res Stress Stress+Res

80 60 40

*

*

#

##

4

5

20 0 1

2

3

Days

B

Control

C

Control+Res

Time spent (s)

80

Stress

Stress+Res

Control Control+Res Stress Stress+Res

60

** ##

40

#

*

20

0 First arriving the platform

Spent in the target quadrant

Fig. 1. Effects of CUMS and resveratrol administration on spatial working memory performance in the Morris water maze test. (A) Rats at 5 weeks after CUMS were given 60 s to locate the hidden platform during each acquisition trial consisting of 4 trials per day for 5 days. Values presented indicate the escape latency (in seconds) in the navigation test trials. (B) Display of tracks of all groups on the sixth day, when the training platform (small blue circles) was removed and activity was monitored for 60 s. (C) The escape latency and percent time spent in the target quadrant in the spatial exploratory test was recorded and analyzed. Values represent the mean ± SD, n = 10, *p b 0.05, **p b 0.01 Stress VS the vehicle-treated, control group; #p b 0.05, ##p b 0.01 Stress + Res VS Stress.

object exploration during the retrieval phase in stress-treated rats, suggesting that resveratrol prevented the stress-induced memory deficits (p b 0.05). As shown in Fig. 2C, rats subjected to CUMS showed a decreased discrimination ratio when compared with control animals (p b 0.01), while resveratrol treatment was able to increase the discrimination ratio (p b 0.05). 3.3. Effects of CUMS and resveratrol on serum corticosterone levels It has been reported that CUMS exposure activated the HPA axis and produced alterations in serum corticosterone levels. Next, we measured the serum corticosterone levels to investigate the effects of resveratrol on the HPA axis. As shown in Fig. 3, ANOVA revealed the effects of stress [F (1, 36) = 17.740, p b 0.001], drug [F(1, 36) = 5.513, p b 0.05], and the stress × drug interaction [F(1, 36) = 4.432, p b 0.05] on serum corticosterone concentrations. Post-hoc analysis indicated that there

was a significant effect of CUMS exposure on serum corticosterone concentrations (p b 0.001). Moreover, resveratrol significantly prevented the altered serum corticosterone levels in stressed rats (p b 0.05). 3.4. Effects of CUMS and resveratrol on BDNF expression The content of BDNF within PFC and hippocampus was measured following administration of resveratrol as a means to assess the possible involvement of neurotrophic factors in the action of resveratrol. As shown in Figs. 4 and 5, ANOVA revealed the effects of stress [PFC: F (1, 12) = 10.859, p b 0.01; hippocampus: F (1, 12) = 13.975, p b 0.01], drug [PFC: F (1, 12) = 7.348, p b 0.05; hippocampus: F (1, 12) = 4.793, p b 0.05], and the stress × drug interaction [PFC: F (1, 12) = 7.022, p b 0.05; hippocampus: F (1, 12) = 6.146, p b 0.05] on BDNF mRNA, and ANOVA revealed the effects of stress [PFC: F (1, 16) = 6.863, p b 0.05; hippocampus: F (1, 16) = 7.015, p b 0.01], drug [PFC: F (1, 16) = 5.898, p b 0.05;

D. Liu et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 49 (2014) 21–29

Discrimanation ratio

Object A Object B

30 20 10 0

C

B

40

Exploration time (s)

Exploration time (Sec)

A

es t ro l Con ontrol+R C

1.0

50

Familar Novel

**

#

40 30 20 10 0

ss es S t r e t r ess + R S

25

es tr o l C o n o n tr o l + R C

es ss St r e t r e s s + R S

#

*

.8 .6 .4 .2 0.0

es tr o l C o n o n tr o l + R C

ss es S tr e tr e s s + R S

Fig. 2. Effects of CUMS and resveratrol administration on recognition memory performance in the novel object recognition task. (A) Bars depict the exploration time by four groups' rats during 5 min of exposure to objects A and B (acquisition phase). (B) Bars depict the exploration time by four group rats during 5 min of exposure to novel and familiar objects (retrieval phase). (C) Comparison of the exploration ratio (calculated as the difference in time spent exploring the novel and the familiar object and expressed as a ratio of the total time spent exploring both objects). Values represent the mean ± SD, n = 10 in each group. *p b 0.05, **p b 0.01 Stress VS the vehicle-treated, control group; #p b 0.05 Stress + Res VS Stress.

hippocampus: F (1, 16) = 5.273, p b 0.05], and the stress × drug interaction [PFC: F (1, 16) = 4.862, p b 0.05; hippocampus: F (1, 16) = 5.209, p b 0.05]on BDNF protein. Post-hoc analysis indicated that CUMS exposure significantly decreased BDNF mRNA in PFC (p b 0.01) (Fig. 4A) and hippocampus (p b 0.01) (Fig. 4B), as well as BDNF protein levels in PFC (p b 0.05) (Fig. 5A) and hippocampus (p b 0.05) (Fig. 5B) compared with the corresponding control rats. However, treatment with resveratrol significantly prevented CUMS-reduced BDNF mRNA and protein expression compared with the stressed rats (Figs. 4–5).

p b 0.05] on the level of phosphorylated protein levels of CREB (pCREB)/CREB ratios. Post-hoc analysis indicated that the pCREB/ CREB ratios in the PFC (p b 0.01) and hippocampus (p b 0.05) of stressed rats were significantly decreased as compared to the control rats. However, chronic administration of resveratrol effectively prevented the CUMS-induced increase of pCREB/CREB ratios in the PFC (p b 0.05 in PFC and p b 0.05 in hippocampus).

3.5. Effect of CUMS and resveratrol on CREB phosphorylation

We next investigated whether mitogen activated protein kinase (MAPK) pathways within the PFC and hippocampus is involved in the antidepressant-like effects of resveratrol. As shown in Fig. 7, ANOVA revealed the effects of stress [PFC: F (1, 12) = 12.051, p b 0.01; hippocampus: F (1, 12) = 20.413, p b 0.01], drug [PFC: F (1, 12) = 5.530, p b 0.05; hippocampus: F (1, 12) = 4.923, p b 0.05], and the stress × drug interaction [PFC: F (1, 12) = 4.851, p b 0.05; hippocampus: F (1, 12) = 8.781, p b 0.05] on the level of phosphorylated protein levels of ERK (p ERK)/ERK ratios. Post-hoc analysis indicated that p ERK/ERK ratios in different brain regions were reduced in stressed rats in the PFC (p b 0.01) and hippocampus (p b 0.01), while resveratrol treatment normalized its levels (p b 0.05 in PFC and p b 0.05 in hippocampus).

Corticosterone (ng/mL)

As shown in Fig. 6, ANOVA revealed the effects of stress [PFC: F (1, 12) = 22.611, p b 0.001; hippocampus: F (1, 12) = 7.201, p b 0.05], drug [PFC: F (1, 12) = 5.245, p b 0.05; hippocampus: F (1, 12) = 7.253, p b 0.05], and the stress × drug interaction [PFC: F (1, 12) = 8.685, p b 0.05; hippocampus: F (1, 12) = 5.274, 100

***

#

80 60 40

4. Discussion

20 0

es tr o l C o n o n tr o l + R C

3.6. Effects of CUMS and resveratrol on ERK1/2 phosphorylation

ss es S tr e tr e s s + R S

Fig. 3. Effects of CUMS and resveratrol administration on serum corticosterone. The results are expressed as the mean ± SD, n = 10 in each group. ***p b 0.001 Stress VS the vehicle-treated, control group; #p b 0.05 Stress + Res VS Stress.

The present study demonstrated that chronic administration of resveratrol significantly prevented deleterious effects of CUMS on the spatial working memory tested in MWW and on recognition memory tested in NORT. In addition, resveratrol treatment also prevented CUMS-induced elevated serum corticosterone levels. Furthermore, resveratrol treatment up-regulated the expressions of BDNF, pERK and pCREB in the PFC and hippocampus of stressed rats.

26

D. Liu et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 49 (2014) 21–29

A

B BDNF

β -actin

β -actin

#

**

.8

BDNF/β-actin (mRNA)

BDNF/β-actin (mRNA)

1.0

BDNF

.8 .6 .4 .2 0.0

es tr o l C o n o n tr o l + R C

.6 .4 .2 0.0

ss es S t r e t r es s+ R S

#

**

es tr o l C o n o n tr o l + R C

ss es S t r e t r es s+ R S

Fig. 4. Effects of CUMS and resveratrol administration on the BDNF mRNA levels in the prefrontal cortex (PFC) (A) and hippocampus (B). The expression of BDNF mRNA was determined by semi-quantitative RT-PCR, and each value was normalized to β-actin. The blot shown is a representative of results obtained from four separate experiments. Values represent the mean ± SD. **p b 0.01 Stress VS the vehicle-treated, control group; #p b 0.05 Stress + Res VS Stress. Band1: control group (1% ethanol); Band2: resveratrol (80 mg/kg); Band3: Stress + vehicle(1% ethanol); Band4: Stress + resveratrol (80 mg/kg).

In our study, resveratrol was tested in stress-impaired cognition using two paradigms indicative of different forms of memory, the MWW and NORT. MWM is a paradigm which tests cognitive efficiency, i.e. it tests whether treatment groups significantly differ from control in efficiency to find a hidden platform by observing and recording escape latency. Consistent with previous studies, the present data showed that rats exposed to CUMS had been demonstrated the prolonged escape latency and reduced percentage in the platform quadrant in the probe test as compared with vehicle-treated control groups, suggesting that rat exposure to CUMS produced a marked long-term deficit in learning and memory (Henningsen et al., 2009). Importantly, resveratrol treatment prevented these deficits as shown by the decreased escaping time and the increased probe test percentage in stressed rat, indicating that resveratrol prevented deficits in cognitive performance induced by stress. The data were also in accordance with previous studies showing resveratrol supplementation improved memory dysfunction resulting from hippocampal neuron loss such as in Alzheimer disease (Huang et al., 2012) and ischemic injury (Girbovan et al., 2012), attenuated high-fat diet-induced cognitive impairment and inflammation(Jeon et al., 2012).

A

.6

1

2

*

3

The NORT is a test of recognition memory based on the spontaneous preference of rodents for novel objects. Consistent with previous studies, our data showed that all groups explored the two identical objects to a similar extent, which indicates that rats had no spatial preference for either of the two object positions. In contrast, following a 1 h interval, CUMS treated rats tend to explore the novel objects to a lower extent than the familiar objects as compared with vehicle-treated control groups, suggesting that stress produced a marked short-term deficit in recognition memory. Interestingly, treatment with resveratrol resulted in a significantly greater novel object preference in stressed rats, suggesting that resveratrol prevented deficits in discriminative memory produced by stress. Further, stressed rats with or without resveratrol treatment did not interfere with the total object exploration time during the novel object tasting phase, which confirms a shift in relative time directed to the novel object. The possible mechanism for the impairment in cognitive produced by stress is HPA axis dysfunction. Steroid hormones can modulate neuronal transmission by a variety of mechanisms. For example, studies on animals have shown that repeated stress and chronically increased glucocorticoid levels have been reported to be associated with a

B

4

4

β -actin

β -actin

#

.6

*

#

.5

BDNF/β-actin

BDNF/β-actin

3

BDNF

.4 .3 .2 .1

es t rol Co n o nt r ol + R C

2

BDNF

.5

0.0

1

.4 .3 .2 .1

es ss Stre tres s+ R S

0.0

es tr o l C o n o n tr o l + R C

s ss S t r e t r e ss + R e S

Fig. 5. Effects of CUMS and resveratrol administration on the BDNF protein expression levels in the prefrontal cortex (PFC) (A) and hippocampus (B). The expression of BDNF was determined by western blotting, and β-actin was used to evaluate protein loading. The blot shown is a representative of results obtained from five separate experiments. Values represent the mean ± SD. *p b 0.05 Stress VS the vehicle-treated, control group; #p b 0.05 Stress + Res VS Stress. Band1: control (1% ethanol); Band2: resveratrol (80 mg/kg); Band3: Stress + vehicle(1% ethanol); Band4: Stress + resveratrol (80 mg/kg).

D. Liu et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 49 (2014) 21–29

A

1

2

B

4

4

CREB

1.0

.6 .4 .2

es tr o l C o n o n tr o l + R C

3

CREB

.8

0.0

2

p-CREB

#

**

1

p-CREB

pCREB/CREB

pCREB/CREB

1.0

3

#

*

.8 .6 .4 .2 0.0

es ss S tr e t r e s s + R S

27

es t r ol Con ontrol+R C

ss es Stre tress+R S

Fig. 6. Effects of CUMS and resveratrol administration on the CREB phosphorylation in the prefrontal cortex (PFC) (A) and hippocampus (B). The whole cell extracts were subjected to Western blot with antibodies against phosphorylated CREB (pCREB). Levels of total CREB were used to evaluate protein loading. The blot shown is a representative of results obtained from three separate experiments. Values represent the mean ± SD. *p b 0.05, **p b 0.01 Stress VS the vehicle-treated, control group; #p b 0.05 Stress + Res VS Stress. Band1: control (1% ethanol); Band2: resveratrol (80 mg/kg); Band3: Stress + vehicle (1% ethanol); Band4: Stress + resveratrol (80 mg/kg).

reduction in hippocampal volume, dendritic atrophy in CA3 region of hippocampus and reduced birth of new granule cells in the dentate gyrus of the hippocampus (Chen et al., 2010; McKittrick et al., 2000; Uno et al., 1989), and impaired learning and memory (Chen et al., 2010; Sapolsky et al., 1985). Moreover, some antidepressant drugs, such as antidepressants desipramine attenuated the stress-associated elevation in serum corticosterone levels (Yau et al., 2007) and cognitive disturbances (Bondi et al., 2008). Consistent with these previous findings, we found that the serum concentration of corticosterone in the stressed group was increased significantly compared to the control groups. Long-term treatment with resveratrol significantly prevented the concentration of corticosterone, which had been elevated by CUMS. This suggested that the protective effects of resveratrol on memory were accompanied by alterations in HPA axis. It has been shown that BDNF enhances normal memory (Alonso et al., 2002) and LTP (Figurov et al., 1996). Additionally, blocking tyrosine kinase B receptor (TrkB, a BDNF receptor) or applying antibodies against BDNF can block LTP in hippocampal slices indicating the vital role of BDNF in the expression of LTP (Figurov et al., 1996; Kang et al.,

A

1.0

1

2

**

3

1997). Importantly, there is increasing evidence that during stress, the expression of BDNF mRNA and protein levels is decreased (Aleisa et al., 2006; Gersner et al., 2010; Smith et al., 1995). In addition, reduced BDNF support is a significant factor in the pathogenesis of stressassociated cognitive deficits (McEwen, 2005). In accordance with this view, we found in the present study that chronic resveratrol treatment prevented the reduction of BDNF levels that was produced by the stress protocol. More importantly, these findings raise the possibility that resveratrol treatment may protect CUMS-induced cognitive deficits due to influencing BDNF. The ERK-CREB signal pathway is implicated in learning, memory, and neuroplasticity (Mazzucchelli and Brambilla, 2000; Sakamoto et al., 2011) and plays an important role in regulating many brain functions, including cell proliferation, differentiation, motility, apoptosis, survival and cellular responses to stress(Mebratu and Tesfaigzi, 2009; Roux and Blenis, 2004). ERK activation is necessary to induce the phosphorylation of the CREB and modulation of its transcriptional activity (Houslay and Kolch, 2000). Interestingly, phosphorylated CREB regulates the transcription of several genes that code for molecules

B

4

ERK

ERK

#

1.0

**

#

.8

pERK/ERK

pERK/ERK

4 p-ERK

.6 .4 .2

es tr o l C o n o n tr o l + R C

3

2

p-ERK

.8

0.0

1

.6 .4 .2

ss es S t r e t r es s + R S

0.0

es r ol Co n t o n t r o l + R C

es ss St r e t r e s s + R S

Fig. 7. Effects of CUMS and resveratrol administration on the ERK phosphorylation in the prefrontal cortex (PFC) (A) and hippocampus (B). The whole cell extracts were subjected to Western blot with antibodies against phosphorylated ERK1/2 (p-ERK1/2). Levels of total ERK1/2 were used to evaluate protein loading. The blot shown is a representative of results obtained in three separate experiments. Values represent the mean ± SD. **p b 0.01 Stress VS the vehicle-treated, control group; #p b 0.05 Stress + Res VS Stress. Band1: control (1% ethanol); Band2: resveratrol (80 mg/kg); Band3: Stress + vehicle (1% ethanol); Band4: Stress + resveratrol (80 mg/kg).

28

D. Liu et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 49 (2014) 21–29

involved in neuronal plasticity including tyrosine hydroxylase, BDNF, and neural cell adhesion molecule, involved in stress response and depression (Marsden, 2013; Qi et al., 2009). In fact, the increasing evidence suggests that the altered ERK-CREB signal pathway is involved in stress and stress-associated cognitive deficits. For example, chronic stress exposure caused the reduction in pERK and pCREB in the PFC, hippocampus and amygdala of rats thought to be involved in cognitive function (Chandran et al., 2013; First et al., 2011; Gronli et al., 2006; Xu et al., 2006). Moreover, chronic administration of antidepressants increases the expression, phosphorylation and function of ERK, CREB and its downstream target gene BDNF in the PFC, hippocampus, amygdale and other limbic brain regions(First et al., 2011; Nibuya et al., 1996; Thome et al., 2000), associated with reversed chronic stress-induced learning and memory deficits(First et al., 2013; Gumuslu et al., 2013). In accordance with the previous results, we found that CUMS decreased pERK/ERK, pCREB/CREB and BDNF levels in the PFC and hippocampus, and chronic resveratrol treatment prevented these reductions. These results indicated that resveratrol-induced increase in pERK and pCREB might be relevant to the increase in BDNF expression in CUMS rats. Thus, we hypothesize that the neuroprotective effects of resveratrol might be due to the increased BDNF expression through ERK-CREB pathways in the PFC and hippocampus in the stressed rats. In conclusion, the present study indicates that resveratrol may be effective in treating cognitive deficits in the CUMS model. The neuroprotective effects of resveratrol are possibly mediated by its modulatory action on the HPA axis function and its ability to prevent the alterations of pERK, pCREB and BDNF levels in PFC and hippocampus of stressed rats. Authors' contribution ZW was involved in study design, data interpretation and manuscript editing; DXL performed the majority of the laboratory work and contributed to the analysis of data and writing of the manuscript; QRZ, JHG, KX, JMW and HJ were responsible for the animal model; XEW and XYX were responsible for western blot. The authors have no conflict of interest to declare. Acknowledgments This work was supported by funding from National Natural Science Foundation of China (No. 81200879); Natural Science Foundation of Shandong Province (No. ZR2011HQ035, ZR2012HM021); Postdoctoral Science Foundation of China (2012M511514, 2013M 531610, 2013T60672); Independent Innovation Foundation of Shandong University (IIFSDU2012TS120, 2012TS123). The authors have no conflict of interest to declare. References Airaksinen E, Larsson M, Lundberg I, Forsell Y. Cognitive functions in depressive disorders: evidence from a population-based study. Psychol Med 2004;34:83–91. Albani D, Polito L, Batelli S, De Mauro S, Fracasso C, Martelli G, et al. The SIRT1 activator resveratrol protects SK-N-BE cells from oxidative stress and against toxicity caused by alpha-synuclein or amyloid-beta (1–42) peptide. J Neurochem 2009;110:1445–56. Aleisa AM, Alzoubi KH, Gerges NZ, Alkadhi KA. Chronic psychosocial stress-induced impairment of hippocampal LTP: possible role of BDNF. Neurobiol Dis 2006;22:453–62. Alonso M, Vianna MR, Depino AM, Mello e Souza T, Pereira P, Szapiro G, et al. BDNF-triggered events in the rat hippocampus are required for both short- and long-term memory formation. Hippocampus 2002;12:551–60. Augustin MA, Sanguansri L, Lockett T. Nano- and micro-encapsulated systems for enhancing the delivery of resveratrol. Ann N Y Acad Sci 2013;1290:107–12. Bondi CO, Rodriguez G, Gould GG, Frazer A, Morilak DA. Chronic unpredictable stress induces a cognitive deficit and anxiety-like behavior in rats that is prevented by chronic antidepressant drug treatment. Neuropsychopharmacology 2008;33:320–31. Chandran A, Iyo AH, Jernigan CS, Legutko B, Austin MC, Karolewicz B. Reduced phosphorylation of the mTOR signaling pathway components in the amygdala of rats exposed to chronic stress. Prog Neuropsychopharmacol Biol Psychiatry 2013;40:240–5.

Chen Y, Rex CS, Rice CJ, Dube CM, Gall CM, Lynch G, et al. Correlated memory defects and hippocampal dendritic spine loss after acute stress involve corticotropin-releasing hormone signaling. Proc Natl Acad Sci U S A 2010;107:13123–8. Figurov A, Pozzo-Miller LD, Olafsson P, Wang T, Lu B. Regulation of synaptic responses to high-frequency stimulation and LTP by neurotrophins in the hippocampus. Nature 1996;381:706–9. First M, Gil-Ad I, Taler M, Tarasenko I, Novak N, Weizman A. The effects of fluoxetine treatment in a chronic mild stress rat model on depression-related behavior, brain neurotrophins and ERK expression. J Mol Neurosci 2011;45:246–55. First M, Gil-Ad I, Taler M, Tarasenko I, Novak N, Weizman A. The effects of reboxetine treatment on depression-like behavior, brain neurotrophins, and ERK expression in rats exposed to chronic mild stress. J Mol Neurosci 2013;50:88–97. Gersner R, Toth E, Isserles M, Zangen A. Site-specific antidepressant effects of repeated subconvulsive electrical stimulation: potential role of brain-derived neurotrophic factor. Biol Psychiatry 2010;67:125–32. Girbovan C, Morin L, Plamondon H. Repeated resveratrol administration confers lasting protection against neuronal damage but induces dose-related alterations of behavioral impairments after global ischemia. Behav Pharmacol 2012;23:1–13. Gronli J, Bramham C, Murison R, Kanhema T, Fiske E, Bjorvatn B, et al. Chronic mild stress inhibits BDNF protein expression and CREB activation in the dentate gyrus but not in the hippocampus proper. Pharmacol Biochem Behav 2006;85:842–9. Gumuslu E, Mutlu O, Sunnetci D, Ulak G, Celikyurt IK, Cine N, et al. The effects of tianeptine, olanzapine and fluoxetine on the cognitive behaviors of unpredictable chronic mild stress-exposed mice. Drug Res (Stuttg) 2013;63:532–939. Henningsen K, Andreasen JT, Bouzinova EV, Jayatissa MN, Jensen MS, Redrobe JP, et al. Cognitive deficits in the rat chronic mild stress model for depression: relation to anhedonic-like responses. Behav Brain Res 2009;198:136–41. Houslay MD, Kolch W. Cell-type specific integration of cross-talk between extracellular signal-regulated kinase and cAMP signaling. Mol Pharmacol 2000;58:659–68. Huang TC, Lu KT, Wo YY, Wu YJ, Yang YL. Resveratrol protects rats from Abeta-induced neurotoxicity by the reduction of iNOS expression and lipid peroxidation. PLoS One 2012;6:e29102. Jang JH, Surh YJ. Protective effect of resveratrol on beta-amyloid-induced oxidative PC12 cell death. Free Radic Biol Med 2003;34:1100–10. Jeon BT, Jeong EA, Shin HJ, Lee Y, Lee DH, Kim HJ, et al. Resveratrol attenuates obesity-associated peripheral and central inflammation and improves memory deficit in mice fed a high-fat diet. Diabetes 2012;61:1444–54. Jiang H, Wang Z, Wang Y, Xie K, Zhang Q, Luan Q, et al. Antidepressant-like effects of curcumin in chronic mild stress of rats: involvement of its anti-inflammatory action. Prog Neuropsychopharmacol Biol Psychiatry 2013;47C:33–9. Kang H, Welcher AA, Shelton D, Schuman EM. Neurotrophins and time: different roles for TrkB signaling in hippocampal long-term potentiation. Neuron 1997;19:653–64. Lupien SJ, Maheu F, Tu M, Fiocco A, Schramek TE. The effects of stress and stress hormones on human cognition: implications for the field of brain and cognition. Brain Cogn 2007;65:209–37. Marsden WN. Synaptic plasticity in depression: molecular, cellular and functional correlates. Prog Neuropsychopharmacol Biol Psychiatry 2013;43:168–84. Mazzucchelli C, Brambilla R. Ras-related and MAPK signalling in neuronal plasticity and memory formation. Cell Mol Life Sci 2000;57:604–11. McEwen BS. Glucocorticoids, depression, and mood disorders: structural remodeling in the brain. Metabolism 2005;54:20–3. McKittrick CR, Magarinos AM, Blanchard DC, Blanchard RJ, McEwen BS, Sakai RR. Chronic social stress reduces dendritic arbors in CA3 of hippocampus and decreases binding to serotonin transporter sites. Synapse 2000;36:85–94. Mebratu Y, Tesfaigzi Y. How ERK1/2 activation controls cell proliferation and cell death: is subcellular localization the answer? Cell Cycle 2009;8:1168–75. Morris R. Developments of a water-maze procedure for studying spatial learning in the rat. J Neurosci Methods 1984;11:47–60. Nibuya M, Nestler EJ, Duman RS. Chronic antidepressant administration increases the expression of cAMP response element binding protein (CREB) in rat hippocampus. J Neurosci 1996;16:2365–72. Orsu P, Murthy BV, Akula A. Cerebroprotective potential of resveratrol through anti-oxidant and anti-inflammatory mechanisms in rats. J Neural Transm 2013;120:1217–23. Qi X, Lin W, Wang D, Pan Y, Wang W, Sun M. A role for the extracellular signal-regulated kinase signal pathway in depressive-like behavior. Behav Brain Res 2009;199:203–9. Radecki DT, Brown LM, Martinez J, Teyler TJ. BDNF protects against stress-induced impairments in spatial learning and memory and LTP. Hippocampus 2005;15:246–53. Roozendaal B. Stress and memory: opposing effects of glucocorticoids on memory consolidation and memory retrieval. Neurobiol Learn Mem 2002;78:578–95. Roux PP, Blenis J. ERK and p38 MAPK-activated protein kinases: a family of protein kinases with diverse biological functions. Microbiol Mol Biol Rev 2004;68: 320–44. Sakamoto K, Karelina K, Obrietan K. CREB: a multifaceted regulator of neuronal plasticity and protection. J Neurochem 2011;116:1–9. Sapolsky RM, Krey LC, McEwen BS. Prolonged glucocorticoid exposure reduces hippocampal neuron number: implications for aging. J Neurosci 1985;5: 1222–7. Smith MA, Makino S, Kvetnansky R, Post RM. Stress and glucocorticoids affect the expression of brain-derived neurotrophic factor and neurotrophin-3 mRNAs in the hippocampus. J Neurosci 1995;15:1768–77. Sun AY, Chen YM, James-Kracke M, Wixom P, Cheng Y. Ethanol-induced cell death by lipid peroxidation in PC12 cells. Neurochem Res 1997;22:1187–92. Thome J, Sakai N, Shin K, Steffen C, Zhang YJ, Impey S, et al. cAMP response element-mediated gene transcription is upregulated by chronic antidepressant treatment. J Neurosci 2000;20:4030–6.

D. Liu et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 49 (2014) 21–29 Tiwari V, Chopra K. Resveratrol abrogates alcohol-induced cognitive deficits by attenuating oxidative–nitrosative stress and inflammatory cascade in the adult rat brain. Neurochem Int 2013;62:861–9. Uno H, Tarara R, Else JG, Suleman MA, Sapolsky RM. Hippocampal damage associated with prolonged and fatal stress in primates. J Neurosci 1989;9:1705–11. Wang J, Ho L, Zhao Z, Seror I, Humala N, Dickstein DL, et al. Moderate consumption of Cabernet Sauvignon attenuates Abeta neuropathology in a mouse model of Alzheimer's disease. FASEB J 2006;20:2313–20. Willner P. Validity, reliability and utility of the chronic mild stress model of depression: a 10-year review and evaluation. Psychopharmacology (Berl) 1997;134:319–29. World Health Organization. International guiding principles for animal research. Chronicle 1985;39:51. Xu Y, Ku B, Tie L, Yao H, Jiang W, Ma X, et al. Curcumin reverses the effects of chronic stress on behavior, the HPA axis, BDNF expression and phosphorylation of CREB. Brain Res 2006;1122:56–64.

29

Xu Y, Wang Z, You W, Zhang X, Li S, Barish PA, et al. Antidepressant-like effect of trans-resveratrol: Involvement of serotonin and noradrenaline system. Eur Neuropsychopharmacol 2010;20:405–13. Yau JL, Noble J, Thomas S, Kerwin R, Morgan PE, Lightman S, et al. The antidepressant desipramine requires the ABCB1 (Mdr1)-type p-glycoprotein to upregulate the glucocorticoid receptor in mice. Neuropsychopharmacology 2007;32:2520–9. Yu Y, Wang R, Chen C, Du X, Ruan L, Sun J, et al. Antidepressant-like effect of trans-resveratrol in chronic stress model: behavioral and neurochemical evidences. J Psychiatr Res 2013;47:315–22. Zhang F, Liu J, Shi JS. Anti-inflammatory activities of resveratrol in the brain: role of resveratrol in microglial activation. Euro J Pharmacol 2010;636:1–7. Zhuang H, Kim YS, Koehler RC, Dore S. Potential mechanism by which resveratrol, a red wine constituent, protects neurons. Ann N Y Acad Sci 2003;993:276–86. [discussion 87-8].