- Email: [email protected]
NMDA GluN2B receptors involved in the antidepressant effects of curcumin in the forced swim test
Progress in Neuro-Psychopharmacology & Biological Psychiatry 40 (2013) 12–17
Contents lists available at SciVerse ScienceDirect
Progress in Neuro-Psychopharmacology & Biological Psychiatry journal homepage: www.elsevier.com/locate/pnp
NMDA GluN2B receptors involved in the antidepressant effects of curcumin in the forced swim test Lin Zhang a, Tianyuan Xu a, Shuang Wang a, Lanqing Yu a, Dexiang Liu c, Renzhi Zhan a, b, Shu Yan Yu a, b,⁎ a b c
Department of Physiology, Shandong University, School of Medicine, Wenhuaxilu Road, Jinan, Shandong Province, 250012, PR China Shandong Provincial Key Laboratory of Mental Disorders, School of Medicine, Wenhuaxilu Road, Jinan, Shandong Province, 250012, PR China Institute of Psychology, Shandong University, School of Medicine, Wenhuaxilu Road, Jinan, Shandong Province, 250012, PR China
a r t i c l e
i n f o
Article history: Received 1 July 2012 Received in revised form 20 August 2012 Accepted 25 August 2012 Available online 31 August 2012 Keywords: Antidepressant Curcumin Forced swim test GluN2B NMDA receptor
a b s t r a c t The antidepressant-like effect of curcumin, a major active component of Curcuma longa, has been previously demonstrated in the forced swimming test. However, the mechanism of this beneficial effect on immobility scores, which is used to evaluate antidepressants, remains largely uncharacterized. The present study attempts to investigate the effects of curcumin on depressive-like behavior with a focus upon the possible contribution of N-methyl-D-aspartate (NMDA) subtype glutamate receptors in this antidepressant-like effect of curcumin. Male mice were pretreated with specific receptor antagonists to different NMDA receptor subtypes such as CPP, NVP-AAM077 and Ro25-6981 as well as to a partial NMDA receptor agonist, D-cycloserine (DCS), prior to administration of curcumin to observe the effects on depressive behavior as measured by immobility scores in the forced swim test. We found that pre-treatment of mice with CPP, a broad-spectrum competitive NMDA receptor antagonist, blocked the anti-immobility effect of curcumin, suggesting the involvement of the glutamate-NMDA receptors. While pretreatment with NVP-AAM077 (the GluN2A-preferring antagonist) did not affect the anti-immobility effect of curcumin, Ro25-6981 (the GluN2B-preferring antagonist) was found to prevent the effect of curcumin in the forced swimming test. Furthermore, pre-treatment with a sub-effective dose of DCS potentiated the anti-immobility effect of a sub-effective dose of curcumin in the forced swimming test. Taken together, these results suggest that curcumin shows antidepressant-like effects in mice and the activation of GluN2B-containing NMDARs is likely to play a predominate role in this beneficial effect. Therefore, the antidepressant-like effect of curcumin in the forced swim test may be mediated, at least in part, by the glutamatergic system. © 2012 Elsevier Inc. All rights reserved.
1. Introduction Depression is one of the most common life-threatening psychiatric disorders and imposes a substantial health burden for contemporary society (Nemeroff, 2007; Rosenzweig-Lipson et al., 2007). In recent years, traditional herbal medicines with antidepressant effects and high safety margins have become potential therapeutic tools in the treatment of depression (Nemeroff, 2007; Van der Watt et al., 2008). Curcumin, as the major biologically active constituent of Curcuma longa, exhibits an extensive array of pharmacological properties including anti-inflammatory, antioxidant, immunomodulatory and neuroprotective activities (Aggarwal and Harikumar, 2009; Maheshwari et al., 2006). More recently, some studies reported
Abbreviations: NMDA, N-methyl-D-aspartate; (DCS), D-cycloserine; LTP, Long-term potentiation; LTD, long-term depression; ANOVA, analysis of variance. ⁎ Corresponding author at: Department of Physiology, Shandong University, School of Medicine, Wenhuaxilu Road, Jinan, Shandong Province, 250012, PR China. Tel.: +86 531 88382037; fax: +86 531 88382502. E-mail address: [email protected] (S.Y. Yu). 0278-5846/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.pnpbp.2012.08.017
that acute or chronic treatment with curcumin could produce antidepressant-like activities by reducing the immobility times in behavioral despair tests in various animal models of depression (Xu et al., 2005a,b). It has been suggested that curcumin exerts its antidepressant effect by restoring monoaminergic function to normal levels which is congruent with established views, as most known studies involving antidepressants focus on monoamine receptors and their postreceptor actions (Kulkarni et al., 2008; Wang et al., 2008). However, the delay between the neurotransmission changes after antidepressant treatment and the eventual clinical effects suggest that other neurotransmitter systems may be involved in the therapeutic effects of antidepressants (Berton and Nestler, 2006). Curcumin has been demonstrated to be a multi-target natural compound which may modulate numerous pathways (Zhou et al., 2011). For example, it has been reported that curcumin could inhibit glutamate release from rat prefrontal cortex nerve terminals, a mechanism which is similar to the effects of the antidepressant fluoxetine (Lin et al., 2011). Therefore, it is clear that a more detailed understanding of the mechanisms underlying the antidepressant effect of curcumin, along with its comparison with other known antidepressants is warranted.
L. Zhang et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 40 (2013) 12–17
It is also important to note that psychopathological conditions, including depression- and anxiety-related disorders are frequently related to aversive emotional memory (Castaneda et al., 2008). Recently, several animal studies have demonstrated the effects of antidepressants on the extinction of aversive memories (Melo et al., 2012; Schulz et al., 2007); and it was suggested that a diminished capability for extinction of aversive memories could be related to the predominance of certain mood disorders (Bondi et al., 2008). Long-term potentiation (LTP) and long-term depression (LTD), the two most well characterized forms of synaptic plasticity, are thought to be the primary mechanism underlying the formation of new memories in behaving animals (Bliss and Collingridge, 1993). Within our laboratory, we have demonstrated that NMDAR-dependent LTP and LTD can be reliably induced in the amygdala, a critical structure considered to be involved with depression (Yu et al., 2008). Recent studies reported that curcumin could reverse the stress-induced learning and memory deficits in rats exposed to chronic unpredictable aversive stimulus (Dong et al., 2012; Xu et al., 2009). Therefore, we speculate that the antidepressant-like effects of curcumin may be related to the activation of glutamate-NMDA receptors, the crucial substrates that are thought to be potentiated in the extinction of aversive memories, and thus may improve the emotional disorders associated with depression. Collectively, the present study was designed to investigate the effects of curcumin on depressive-like behavior and the involvement of the glutamate system, particularly GluN2A and GluN2B NMDA receptors. To accomplish this goal, the antidepressant effects of curcumin were evaluated by using specific receptor antagonists in the forced swim test, a predictive model widely used for assessing antidepressant efficacy. 2. Materials and methods 2.1. Animals Male Kun-Ming mice weighing 22–25 g were obtained from Shandong University Animal Centre. All procedures in this study were approved by the Shandong University Animal Care and Use Committee and were performed in accordance with the National Institutes of Health guide to care and use of Laboratory animals. Mice were housed in groups of four per cage and maintained on a 12 h light/dark cycle (lights on 6:30 a.m.) with free access to standard laboratory food and water. Animals were allowed to acclimatize to the laboratory conditions for 7–8 days prior to the behavioral procedures which were carried out in the light phase. All efforts were made to minimize the pain and numbers of the animals used in these experiments. 2.2. Drugs and treatment Curcumin (Sigma, St. Louis, MO, USA), the GluN2A-preferring antagonist NVP-AAM077 (Novartis Pharma AG, Base, Switzerland) and GluN2B antagonist Ro25-6981 (Tocris Biosciences, Ellisville, Missouri, USA) were dissolved in 1 part DMSO: 2 parts physiological saline. In a separate series of experiments, NVP-AAM077 (1.2 mg/kg) was preinjected 45-min and Ro25-6981 (6 mg/kg) was pre-injected 30-min before the injection of curcumin (40 mg/kg). The broad-spectrum competitive NMDA receptor antagonist CPP (Sigma, St. Louis, MO, USA; 10 mg/kg) was dissolved in physiological saline (NaCl, 0.9%) and was administered 60-min before curcumin injection. Fluoxetine (Sigma, St. Louis, MO, USA; 30 mg/kg), a selective serotonin reuptake inhibitor, was dissolved in physiological saline (NaCl, 0.9%) and was used as a positive control for antidepressant action. In a separate series of experiments, a partial NMDA receptor agonist, D-cycloserine (DCS, Sigma, St. Louis, MO, USA; 15 mg/kg), was dissolved in physiological saline (NaCl, 0.9%) and was given to mice 30 min before the administration of a sub-effective dose of curcumin (10 mg/kg), and the test was carried out 45 min later.
13
All drugs were administered intraperitoneally (i.p.) at a volume of 10 ml/kg. Dose and route administration schedules of all drugs used in the present experiment were chosen as based on previous results — NVP-AAM077 and Ro 25‐6981 (Dalton et al., 2012; Fox et al., 2006), CPP (Dalton et al., 2012; Goosens and Maren, 2004) and D-cycloserine (Curlik and Shors, 2011; Walker et al., 2002). To habituate to the intraperitoneal injections, all mice were administered saline (10 ml/kg) daily for three days prior to the experiment. 2.3. Forced swim test Mice were subjected to the forced swim test similar to that described previously (Porsolt et al., 1977a). Briefly, mice were placed individually in a cylinder (height: 35 cm, diameter: 20 cm) containing 25 cm of water at 25 °C for two consecutive swim sessions. On the first day, the mice were exposed to 15 min of forced swim (training session) and then were removed and dried before being returned to cages. Twenty-four hours later, mice were placed in the cylinder again for a 5 min period (test session). The drugs and their corresponding vehicles were administered three times between these two swim sessions (23.5, 5, and 1 h before the 5 min swim). The 5-min test was scored for immobility time defined as floating with only small movement necessary to keep the head above water by an observer blind to the treatment condition of the animal. 2.4. Data analysis One-way analysis of variance (ANOVA) was used to establish differences among groups of animals treated with vehicle or drugs alone and was followed by the Newman–Keuls test for post-hoc comparisons. Other data were analyzed statistically by two-way ANOVA for multiple comparisons with pretreatment or co-administration, and followed by the Bonferroni test. All statistical procedures were performed on SPSS version 13.0. The results were expressed as mean ± SEM. Differences with P b 0.05 were considered statistically significant. 3. Results 3.1. Effects of curcumin and fluoxetine in the forced swim test The effects of curcumin (10, 20, 40 mg/kg) and fluoxetine (10, 20, 30 mg/kg) administrations on immobility time of mice in the forced swim test are presented in Fig. 1. The one-way ANOVA revealed significant differences in response to treatments with curcumin [F (3, 42) = 36.71, P b 0.01] and fluoxetine [F(3, 41) = 27.63, P b 0.01] on immobility time in the forced swim test. Post-hoc analysis indicated that administration of curcumin in doses of 20 and 40 mg/kg, or fluoxetine in doses of 20 and 30 mg/kg significantly decreased immobility duration compared to their respective vehicle treated groups. Such decreases in duration of immobility suggest an antidepressantlike effect of curcumin in this forced swim test. 3.2. Effect of NMDA receptor antagonist on the antidepressant-like effect of curcumin To assess possible involvement of the NMDA receptor in the antidepressant-like activity of curcumin, four separate groups of mice were pre-treated with the NMDA receptor antagonists CPP (10 mg/kg) or their respective vehicles prior to the administration of curcumin (40 mg/kg) or fluoxetine (30 mg/kg) for three consecutive times between the two swim sessions in the forced swim test. ANOVA showed a significant effect in the factors of pre-treatment [saline or CPP; F (1, 22)= 28.56, P b 0.01], treatment [curcumin or fluoxetine: F(2, 35)= 31.28, P b 0.01)] and the pre-treatment× treatment interaction [F(2, 35) =29.76, Pb 0.01)] on immobility time in the forced swim
14
L. Zhang et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 40 (2013) 12–17
Chemical structure of curcumin
Immobility time (s)
200
150
**
** **
**
100
50
0
0
10
20
40
Curcumin (mg/kg)
0
10
20
forced swim test, we used the GluN2A-prefering antagonist NVPAAM077 and the GluN2B antagonist Ro25-6981 to test for involvement of different NMDAR subtypes in subsequent experiments. Our previously results indicated that these two drugs exerted a preferential antagonism of GluN2A and GluN2B-containing receptors, respectively (Yu et al., 2010). NVP-AAM077 (1.2 mg/kg) was administered intraperitoneally at 45-min prior to the treatment of curcumin. The results of pre-treatment with NVP-AAM077 on reduction in immobility time elicited by curcumin (40 mg/kg) in the forced swim test are shown in Fig. 3. ANOVA revealed the effects of pre-treatment [saline or NVP-AAM077; F (1, 24) =0.61, P > 0.05], treatment [F(1, 24) = 26.87, P b 0.01], and the pre-treatment× treatment interaction [F(1, 24) = 0.49, P > 0.05] on immobility times in the forced swim test. Post-hoc analysis indicated that NVP-AAM077 pre-treatment did not exert any influence on the antidepressant-like effects elicited by curcumin as revealed from the duration of immobility scores in the forced swim test (P > 0.05). That is, NVP-AAM077 pre-treated mice displayed reductions in immobility times that did not differ significantly from curcumin-treated mice. These results indicated that blockade of GluN2A-containing receptors does not affect the antidepressant-like effects of curcumin.
30
Fluoxetine (mg/kg)
Fig. 1. Upper: the chemical structure of curcumin. Molecular weight: 368.38. Linear formula: [HOC6H3(OCH3)CH=CHCO]2CH2. Under: effects of administration of curcumin and fluoxetine on immobility times in the forced swimming test. Mice were treated with their vehicles, curcumin (10, 20, 40 mg/kg, i.p.) or fluoxetine (10, 20, 30 mg/kg, i.p.) three times between the two swim sessions. All values are presented as means±SEM (n=10–12). ** Pb 0.01, compared with their vehicle-treated group.
test. Post-hoc analyses indicated that pre-treatment of CPP significantly attenuated the decrease in immobility duration elicited by curcumin but not by fluoxetine in the forced swimming test while CPP-administration alone did not modify immobility time of mice compared to the control group (P >0.05) (Fig. 2). 3.3. Effect of GluN2A-containing NMDA receptor antagonist on the antidepressant-like effect of curcumin To determine whether distinct NMDA receptor subtypes differentially contribute to the antidepressant-like effects of curcumin in the
3.4. Effect of GluN2B receptor antagonist on the antidepressant-like effect of curcumin In a separate group of mice we next investigated whether GluN2B NMDA receptor activation is involved in the antidepressant-like effects induced by curcumin. These data showing the reversal of antidepressant-like effects of curcumin resulting from pre-treatment of mice with Ro25-6981 (6 mg/kg) in the forced swimming test are presented in Fig. 3. ANOVA revealed significant differences in the factors of pre-treatment [saline or Ro25-6981; F (1, 24)= 20.63, P b 0.01], treatment [F(1, 24) = 31.94, P b 0.01], and the pre-treatment× treatment interaction [F(1, 24)= 28.49, P b 0.01] on immobility times in the forced swim test. Post-hoc analyses indicated that, in contrast to the GluN2A receptor blockade, pre-treating mice with Ro25-6981 significantly attenuated the decrease in immobility elicited by curcumin in the forced swim test (P b 0.01). Collectively, these data showed that activation of GluN2B, but not the GluN2A-subunit containing NMDA receptors, played a predominate role in the antidepressant-like effects of curcumin in the forced swim test in mice. 200
150
**
**
**
100
50
0
Immobility time (s)
Immobility time (s)
200
150
**
**
100
50
0
Vehicle
+
+
+
+
-
-
Vehicle
+
+
+
+
-
-
Curcumin
+ -
+
-
+ -
+
Curcumin
Fluoxetine
-
NVP-AAM077
-
+
-
+ -
+ +
+ -
CCP
-
-
-
+
+
+
Ro25-6981
-
-
+
-
-
+
Fig. 2. Effects of pre-treatment with CPP (a competitive NMDA receptor antagonist, 10 mg/kg, i.p.) on curcumin (40 mg/kg, i.p.) and fluoxetine (30 mg/kg, i.p.) induced decrease in immobility times. All values are presented as means ± SEM (n = 10–12). ** P b 0.01, compared with their vehicle-treated group; # #P b 0.01, compared with the group treated with vehicle + curcumin or fluoxetine.
Fig. 3. Pre-treatment with Ro25-6981 (a specific GluN2B antagonist, 6 mg/kg, i.p.) but not with NVP-AAM077 (a GluN2A-prefering antagonist, 1.2 mg/kg, i.p.) prevent the decrease in immobility times induced by curcumin (40 mg/kg, i.p.). All values are presented as means±SEM (n=10–15). ** Pb 0.01, compared with their vehicle-treated group; ## Pb 0.01, compared with the group treated with vehicle+curcumin group.
L. Zhang et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 40 (2013) 12–17
Immobility time (s)
200
150
**
15
of pre-treatment [saline or curcumin; F(1, 22) = 22.42, P b 0.01], treatment [F(1, 22) = 18.10, P b 0.01], and the pre-treatment× treatment interaction [F(1, 22)= 17.90, P b 0.01] in the forced swim test. Post-hoc analyses indicated that pre-treatment with a sub-effective dose of curcumin elicited a significant synergistic effect when combined with a sub-effective dose of fluoxetine in the forced swimming test (P b 0.01).
100
4. Discussion 50
0
Vehicle DCS Curcumin
+ -
+ + -
+ +
+ +
Fig. 4. Effect of pre-treatment of a sub-effective dose of DCS (a partial NMDA receptor agonist, 15 mg/kg, i.p.) on the action of a sub-effective dose of curcumin (10 mg/kg, i.p.) in the forced swim test. All values are presented as means ± SEM (n = 10–12). ** P b 0.01, compared with their vehicle treated group; # #P b 0.01, compared with the group pre-treated with vehicle + curcumin group.
3.5. Effects of co-administration with sub-effective doses of curcumin and DCS on immobility time in mice The results obtained following co-administration of sub-effective doses of DCS (15 mg/kg) and curcumin (10 mg/kg) on immobility times in the forced swim test are shown in Fig. 4. ANOVA revealed significant differences in the factors of pre-treatment [saline or DCS; F(1, 22) = 21.16, P b 0.01], treatment [F(1, 22) = 19.86, P b 0.01], and the pre-treatment × treatment interaction [F(1, 21) = 25.18, P b 0.01] in the forced swim test. Post-hoc analyses indicated that pretreatment of mice with a sub-effective dose of DCS potentiated the anti-immobility effect of a sub-effective dose of curcumin in the forced swimming test (P b 0.01). 3.6. Effects of co-administration with sub-effective doses of curcumin and fluoxetine in the forced swimming test
The primary finding of this study was the demonstration of the important differences that exist between the roles of GluN2A and GluN2B NMDA receptor blockade in the antidepressant-like effects of curcumin in the forced swim test. Consistent with previous findings, our present results demonstrated that curcumin produces a significant decrease in the duration of immobility in mice, which was similar to the effects seen in response to treatment with fluoxetine. Specifically, we observed that blockade of GluN2B receptors with Ro25-6981, but not blockade of GluN2A receptors with NVP-AAM077, significantly impaired the antidepressant-like effects of curcumin in the forced swim test. 4.1. Forced swim test The forced swim test in mice is widely used to evaluate antidepressants as well as investigate the mechanisms underlying the effects of antidepressants in this animal model of depression (Porsolt et al., 1977b). During the test session, immobility time is interpreted as representing despair or a depression-like state which can be shortened by repeated antidepressant treatment. Thus, immobility duration is believed to provide a measure of the degree of “behavioral despair” exhibited by these animals. In the present study, animals treated with curcumin and fluoxetine show a dose-dependent decrease in their immobility durations in the forced swim test, as reported previously. However, we demonstrated that this effect was significantly blocked by pretreatment with CPP, a non-specific NMDA-receptor antagonist. Thus, the results suggest that NMDA receptors may be involved in the anti-depressant effect of curcumin in the forced swim test. 4.2. NMDA receptors in the antidepressant-like activity of curcumin
The interaction resulting from sub-effective doses of curcumin (10 mg/kg) with fluoxetine (10 mg/kg) in the forced swim test are presented in Fig. 5. ANOVA revealed significant differences in the factors
Immobility time (s)
200
150
** 100
50
0
Vehicle Curcumin Fluoxetine
+ -
+ +
+ + -
+ +
Fig. 5. Effect of co-administration of mice with sub-effective doses of curcumin (10 mg/kg, i.p.) and fluoxetine (10 mg/kg, i.p.) on immobility times. All values are presented as means ± SEM. (n = 10–12). ** P b 0.01, compared with their vehicle treated group. # #P b 0.01, compared with the group treated with vehicle + curcumin or fluoxetine.
In the present behavioral experiments, CPP alone did not modify immobility times of mice in the forced swimming test, but pretreatment with CPP essentially prevented the antidepressant effects of curcumin, suggesting a contribution of NMDA receptors to the effects of this herbal medicine. Additionally, we found that co-administration of sub-effective doses of curcumin and DCS produced a significant synergistic antidepressant effect in forced swim test. These results further reinforced the importance of NMDA receptors in the antidepressant-like effects of curcumin. Related findings with transgenic mice showed that over-expression of GluN2B NMDA receptor subunits in several forebrain areas, including the amygdala and hippocampus significantly accelerated conditioned fear extinction as compared with their wild-type controls (Tang et al., 1999). Similarly, our previous results as obtained with amygdala slice preparations demonstrated that activation of GluN2B subunits predominately contributed to the expression of NMDA receptordependent LTD (Yu et al., 2010), the most well characterized neural construct which is generally thought to play a critical role in the extinction of previously learned inappropriate responses (Dalton et al., 2011; Duffy et al., 2008; Kim et al., 2007). Moreover, recent electrophysiological evidence revealed that activation of receptors containing the GluN2B but not GluN2A subunit mediates the generation of LTD-like response (Izumi et al., 2006; Liu et al., 2004; Yang et al., 2005). Thus, GluN2B receptor-mediated LTD may play a specific role in the extinction of previously-acquired memories.
16
L. Zhang et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 40 (2013) 12–17
4.3. Distinct NMDA receptor subtypes in the antidepressant-like effects of curcumin To investigate further the dissociable mechanisms of NMDA receptor subunits on the effects of curcumin, mice were pre-treated with receptor antagonists specific for different NMDA receptor subtypes. Interestingly, we did not see any apparent reduction in antidepressant effects of curcumin in response to pretreatment with the GluN2A NMDA receptor antagonist NVP-AAM077 in the forced swim test suggesting that deficits in GluN2A subunits do not impair the antidepressant effects of curcumin. In contrast, it is interesting to note that we observed pretreated with Ro25-6981 before curcumin significantly increased immobility times during the test session. The higher levels of immobility observed in these mice after pretreatment with Ro25-6981 reflects the critical role of GluN2B receptors in the antidepressant-like effects of curcumin. Therefore, the data obtained from this behavioral test support the conclusion that activation of GluN2B-containing NMDARs represents the predominate pathway in the improvement of depressive-like behavior in mice resulting from curcumin. As mentioned above, the GluN2B NMDA receptor is strongly related to extinction of learning and memories, so the results reported here, in combination with previous studies, suggest that the antidepressant-like effectiveness of traditional herbal medicines, like curcumin, might be exhibited by promoting extinction of aversive memories mediated through a preferred GluN2B NMDA receptor subtype. 4.4. Synergistic antidepressant-like effect of curcumin and fluoxetine It should also be noted that pretreatment with CPP, a competitive NMDA receptor antagonist, did not prevent the antidepressant-like effect of fluoxetine in the forced swim test. Such results indicate these NMDA receptors are not involved in the effects of selective serotonin reuptake inhibitors in the treatment of depression. However, in our experiments, a sub-effective dose of curcumin potentiated the effects of a sub-effective dose of fluoxetine in the forced swimming test. These results suggest that although NMDA receptors, by themselves, may not be involved in the mechanism of fluoxetine action, a co-activation of NMDA and 5-HT receptors could produce a synergistic antidepressantlike effect in the forced swim test. This synergistic effect could be due to either a direct additive antidepressant effects by activation of NMDA and 5-HT receptors, or a result of an indirect pathway via Ca 2+, an important second messenger, science activation of postsynaptic NMDA receptors will increase the subsequent Ca2+ influx which may in turn lead to the activation of more 5-HT receptors. 5. Conclusion In summary, the results from our behavioral findings on depression as assessed in the forced swim test indicate that curcumin exerts an antidepressant-like effect and does so by activation of GluN2B NMDA receptor subunits. These findings provide one possible novel substrate by which herbal antidepressants may exert their beneficial effects in the treatment of depression. Additionally, the synergistic effect induced by co-administration with sub-effective doses of curcumin and fluoxetine hint at the possible interaction between glutamatergic and serotonergic systems in the antidepressant effects of the forced swim test. Further detailed molecular mechanisms underlying the antidepressant-like effects of curcumin, which are now under investigation in our laboratory, will provide further insight into this issue. Acknowledgments This study was supported by grants to Shu Yan Yu from the National Natural Science Foundation of China (NSFC81101006),
from the Excellent Young Scientist Foundation of Shandong Province (BS2010SW021), from the Independent Innovation Foundation of Jinan city (201202046) and from the Independent Innovation Foundation of Shandong University (IIFSDU2010TS091). References Aggarwal BB, Harikumar KB. Potential therapeutic effects of curcumin, the anti-inflammatory agent, against neurodegenerative, cardiovascular, pulmonary, metabolic, autoimmune and neoplastic diseases. Int J Biochem Cell Biol 2009;41: 40–59. Berton O, Nestler EJ. New approaches to antidepressant drug discovery: beyond monoamines. Nat Rev Neurosci 2006;7:137–51. Bliss TV, Collingridge GL. A synaptic model of memory: long-term potentiation in the hippocampus. Nature 1993;361:31–9. 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(2):320–31. Castaneda AE, Tuulio-Henriksson A, Marttunen M, Suvisaari J, Lönnqvist J. A review on cognitive impairments in depressive and anxiety disorders with a focus on young adults. J Affect Disord 2008;106:1-27. Curlik DM, Shors TJ. Learning increases the survival of newborn neurons provided that learning is difficult to achieve and successful. J Cogn Neurosci 2011;23(9): 2159–70. Dalton GL, Ma LM, Phillips AG, Floresco SB. Blockade of NMDA GluN2B receptors selectively impairs behavioral flexibility but not initial discrimination learning. Psychopharmacology 2011;216:525–35. Dalton GL, Wu DC, Wang YT, Floresco SB, Phillips AG. NMDA GluN2A and GluN2B receptors play separate roles in the induction of LTP and LTD in the amygdala and in the acquisition and extinction of conditioned fear. Neuropharmacology 2012;62:797–806. Dong S, Zeng Q, Mitchell ES, Xiu J, Duan Y, Li C, et al. Curcumin enhances neurogenesis and cognition in aged rats: implications for transcriptional interactions related to growth and synaptic plasticity. PLoS One 2012;7:e31211. Duffy S, Labrie V, Roder JC. D-serine augments NMDA-NR2B receptor-dependent hippocampal long-term depression and spatial reversal learning. Neuropsychopharmacology 2008;33:1004–18. Fox CJ, Russell KI, Wang YT, Christie BR. Contribution of NR2A and NR2B NMDA subunits to bidirectional synaptic plasticity in the hippocampus in vivo. Hippocampus 2006;16:907–15. Goosens KA, Maren S. NMDA receptors are essential for the acquisition, but not expression, of conditional fear and associative spike firing in the lateral amygdale. Eur J Neurosci 2004;20:537–48. Izumi Y, Auberson YP, Zorumski CF. Zinc modulates bidirectional hippocampal plasticity by effects on NMDA receptors. J Neurosci 2006;26:7181–8. Kim J, Lee S, Park K, Hong I, Song B, Son G, et al. Amygdala depotentiation and fear extinction. Proc Natl Acad Sci 2007;104:20955–60. Kulkarni SK, Bhutani MK, Bishnoi M. Antidepressant activity of curcumin: involvement of serotonin and dopamine system. Psychopharmacology (Berl) 2008;201:435–42. Lin TY, Lu CW, Wang CC, Wang YC, Wang SJ. Curcumin inhibits glutamate release in nerve terminals from rat prefrontal cortex: possible relevance to its antidepressant mechanism. Prog Neuropsychopharmacol Biol Psychiatry 2011;35:1785–93. Liu L, Wong LP, Pozza MF, Lingenhoehl K, Wang Y, Sheng M, et al. Role of NMDA receptor subtypes in governing the direction of hippocampal synaptic plasticity. Science 2004;304:1021–4. Maheshwari RK, Singh AK, Gaddipati J, Smiral RC. Multiple biological activities of curcumin: a short review. Life Sci 2006;78:2081–7. Melo TG, Izídio GS, Ferreira LS, Sousa DS, Macedo PT, Cabral A, et al. Antidepressants differentially modify the extinction of an aversive memory task in female rats. Prog Neuropsychopharmacol Biol Psychiatry 2012;37:33–40. Nemeroff CB. The burden of severe depression: a review of diagnostic challenges and treatment alternatives. J Psychiatr Res 2007;41:189–206. Porsolt RD, Le Pichon M, Jalfre M. Depression: a new animal model sensitive to antidepressant treatments. Nature 1977a;266:730–2. Porsolt RD, Pichon MLE, Jalfre M. Behavioral despair in mice: a primary screening test for antidepressant. Arch Int Pharmacodyn Ther 1977b;229:327–36. Rosenzweig-Lipson S, Beyer CE, Hughes ZA, Khawaja X, Rajarao SJ, Malberg JE, et al. Differentiating antidepressants of the future: efficacy and safety. Pharmacol Ther 2007;113:134–53. Schulz D, Buddenberg T, Huston JP. Extinction-induced “despair” in the water maze, exploratory behavior and fear: effects of chronic antidepressant treatment. Neurobiol Learn Mem 2007;87:624–34. Tang YP, Shimizu E, Dube GR, Rampon C, Kerchner GA, Zhuo M, et al. Genetic enhancement of learning and memory in mice. Nature 1999;401:25–7. Van der Watt G, Laugharne J, Janca A. Complementary and alternative medicine in the treatment of anxiety and depression. Curr Opin Psychiatry 2008;21:37–42. Walker DL, Ressler KJ, Lu KT, Davis M. Facilitation of conditioned fear extinction by systemic administration or intra-amygdala infusions of D-cycloserine as assessed with fear-potentiated startle in rats. J Neurosci 2002;22:2343–51. Wang R, Xu Y, Wu HL, Li YB, Li YH, Guo JB. The antidepressant effects of curcumin in the forced swimming test involve 5-HT1 and 5-HT2 receptors. Eur J Pharmacol 2008;578:43–50. Xu Y, Ku BS, Yao HY, Lin YH, Ma X, Zhang YH. The effects of curcumin on depressive-like behaviors in mice. Eur J Pharmacol 2005a;518:40–6.
L. Zhang et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 40 (2013) 12–17 Xu Y, Ku BS, Yao HY, Lin YH, Ma X, Zhang YH. Antidepressant effects of curcumin in the forced swim test and olfactory bulbectomy models of depression in rats. Pharmacol Biochem Behav 2005b;82:200–6. Xu Y, Lin D, Li S, Li G, Shyamala SG, Barish PA, et al. Curcumin reverses impaired cognition and neuronal plasticity induced by chronic stress. Neuropharmacology 2009;57: 463–71. Yang CH, Huang CC, Hsu KS. Behavioral stress enhances hippocampal CA1 long-term depression through the blockade of the glutamate uptake. J Neurosci 2005;25: 4288–93.
17
Yu SY, Wu DC, Liu L, Ge Y, Wang YT. Role of AMPA receptor trafficking in NMDA receptor-dependent synaptic plasticity in the rat lateral amygdala. J Neurochem 2008;106:889–99. Yu SY, Wu DC, Zhan RZ. GluN2B subunits of the NMDA receptor contribute to the AMPA receptor internalization during long-term depression in the lateral amygdala of juvenile rats. Neuroscience 2010;171:1102–8. Zhou H, Beevers CS, Huang S. The targets of curcumin. Curr Drug Targets 2011;12: 332–47.
Contents lists available at SciVerse ScienceDirect
Progress in Neuro-Psychopharmacology & Biological Psychiatry journal homepage: www.elsevier.com/locate/pnp
NMDA GluN2B receptors involved in the antidepressant effects of curcumin in the forced swim test Lin Zhang a, Tianyuan Xu a, Shuang Wang a, Lanqing Yu a, Dexiang Liu c, Renzhi Zhan a, b, Shu Yan Yu a, b,⁎ a b c
Department of Physiology, Shandong University, School of Medicine, Wenhuaxilu Road, Jinan, Shandong Province, 250012, PR China Shandong Provincial Key Laboratory of Mental Disorders, School of Medicine, Wenhuaxilu Road, Jinan, Shandong Province, 250012, PR China Institute of Psychology, Shandong University, School of Medicine, Wenhuaxilu Road, Jinan, Shandong Province, 250012, PR China
a r t i c l e
i n f o
Article history: Received 1 July 2012 Received in revised form 20 August 2012 Accepted 25 August 2012 Available online 31 August 2012 Keywords: Antidepressant Curcumin Forced swim test GluN2B NMDA receptor
a b s t r a c t The antidepressant-like effect of curcumin, a major active component of Curcuma longa, has been previously demonstrated in the forced swimming test. However, the mechanism of this beneficial effect on immobility scores, which is used to evaluate antidepressants, remains largely uncharacterized. The present study attempts to investigate the effects of curcumin on depressive-like behavior with a focus upon the possible contribution of N-methyl-D-aspartate (NMDA) subtype glutamate receptors in this antidepressant-like effect of curcumin. Male mice were pretreated with specific receptor antagonists to different NMDA receptor subtypes such as CPP, NVP-AAM077 and Ro25-6981 as well as to a partial NMDA receptor agonist, D-cycloserine (DCS), prior to administration of curcumin to observe the effects on depressive behavior as measured by immobility scores in the forced swim test. We found that pre-treatment of mice with CPP, a broad-spectrum competitive NMDA receptor antagonist, blocked the anti-immobility effect of curcumin, suggesting the involvement of the glutamate-NMDA receptors. While pretreatment with NVP-AAM077 (the GluN2A-preferring antagonist) did not affect the anti-immobility effect of curcumin, Ro25-6981 (the GluN2B-preferring antagonist) was found to prevent the effect of curcumin in the forced swimming test. Furthermore, pre-treatment with a sub-effective dose of DCS potentiated the anti-immobility effect of a sub-effective dose of curcumin in the forced swimming test. Taken together, these results suggest that curcumin shows antidepressant-like effects in mice and the activation of GluN2B-containing NMDARs is likely to play a predominate role in this beneficial effect. Therefore, the antidepressant-like effect of curcumin in the forced swim test may be mediated, at least in part, by the glutamatergic system. © 2012 Elsevier Inc. All rights reserved.
1. Introduction Depression is one of the most common life-threatening psychiatric disorders and imposes a substantial health burden for contemporary society (Nemeroff, 2007; Rosenzweig-Lipson et al., 2007). In recent years, traditional herbal medicines with antidepressant effects and high safety margins have become potential therapeutic tools in the treatment of depression (Nemeroff, 2007; Van der Watt et al., 2008). Curcumin, as the major biologically active constituent of Curcuma longa, exhibits an extensive array of pharmacological properties including anti-inflammatory, antioxidant, immunomodulatory and neuroprotective activities (Aggarwal and Harikumar, 2009; Maheshwari et al., 2006). More recently, some studies reported
Abbreviations: NMDA, N-methyl-D-aspartate; (DCS), D-cycloserine; LTP, Long-term potentiation; LTD, long-term depression; ANOVA, analysis of variance. ⁎ Corresponding author at: Department of Physiology, Shandong University, School of Medicine, Wenhuaxilu Road, Jinan, Shandong Province, 250012, PR China. Tel.: +86 531 88382037; fax: +86 531 88382502. E-mail address: [email protected] (S.Y. Yu). 0278-5846/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.pnpbp.2012.08.017
that acute or chronic treatment with curcumin could produce antidepressant-like activities by reducing the immobility times in behavioral despair tests in various animal models of depression (Xu et al., 2005a,b). It has been suggested that curcumin exerts its antidepressant effect by restoring monoaminergic function to normal levels which is congruent with established views, as most known studies involving antidepressants focus on monoamine receptors and their postreceptor actions (Kulkarni et al., 2008; Wang et al., 2008). However, the delay between the neurotransmission changes after antidepressant treatment and the eventual clinical effects suggest that other neurotransmitter systems may be involved in the therapeutic effects of antidepressants (Berton and Nestler, 2006). Curcumin has been demonstrated to be a multi-target natural compound which may modulate numerous pathways (Zhou et al., 2011). For example, it has been reported that curcumin could inhibit glutamate release from rat prefrontal cortex nerve terminals, a mechanism which is similar to the effects of the antidepressant fluoxetine (Lin et al., 2011). Therefore, it is clear that a more detailed understanding of the mechanisms underlying the antidepressant effect of curcumin, along with its comparison with other known antidepressants is warranted.
L. Zhang et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 40 (2013) 12–17
It is also important to note that psychopathological conditions, including depression- and anxiety-related disorders are frequently related to aversive emotional memory (Castaneda et al., 2008). Recently, several animal studies have demonstrated the effects of antidepressants on the extinction of aversive memories (Melo et al., 2012; Schulz et al., 2007); and it was suggested that a diminished capability for extinction of aversive memories could be related to the predominance of certain mood disorders (Bondi et al., 2008). Long-term potentiation (LTP) and long-term depression (LTD), the two most well characterized forms of synaptic plasticity, are thought to be the primary mechanism underlying the formation of new memories in behaving animals (Bliss and Collingridge, 1993). Within our laboratory, we have demonstrated that NMDAR-dependent LTP and LTD can be reliably induced in the amygdala, a critical structure considered to be involved with depression (Yu et al., 2008). Recent studies reported that curcumin could reverse the stress-induced learning and memory deficits in rats exposed to chronic unpredictable aversive stimulus (Dong et al., 2012; Xu et al., 2009). Therefore, we speculate that the antidepressant-like effects of curcumin may be related to the activation of glutamate-NMDA receptors, the crucial substrates that are thought to be potentiated in the extinction of aversive memories, and thus may improve the emotional disorders associated with depression. Collectively, the present study was designed to investigate the effects of curcumin on depressive-like behavior and the involvement of the glutamate system, particularly GluN2A and GluN2B NMDA receptors. To accomplish this goal, the antidepressant effects of curcumin were evaluated by using specific receptor antagonists in the forced swim test, a predictive model widely used for assessing antidepressant efficacy. 2. Materials and methods 2.1. Animals Male Kun-Ming mice weighing 22–25 g were obtained from Shandong University Animal Centre. All procedures in this study were approved by the Shandong University Animal Care and Use Committee and were performed in accordance with the National Institutes of Health guide to care and use of Laboratory animals. Mice were housed in groups of four per cage and maintained on a 12 h light/dark cycle (lights on 6:30 a.m.) with free access to standard laboratory food and water. Animals were allowed to acclimatize to the laboratory conditions for 7–8 days prior to the behavioral procedures which were carried out in the light phase. All efforts were made to minimize the pain and numbers of the animals used in these experiments. 2.2. Drugs and treatment Curcumin (Sigma, St. Louis, MO, USA), the GluN2A-preferring antagonist NVP-AAM077 (Novartis Pharma AG, Base, Switzerland) and GluN2B antagonist Ro25-6981 (Tocris Biosciences, Ellisville, Missouri, USA) were dissolved in 1 part DMSO: 2 parts physiological saline. In a separate series of experiments, NVP-AAM077 (1.2 mg/kg) was preinjected 45-min and Ro25-6981 (6 mg/kg) was pre-injected 30-min before the injection of curcumin (40 mg/kg). The broad-spectrum competitive NMDA receptor antagonist CPP (Sigma, St. Louis, MO, USA; 10 mg/kg) was dissolved in physiological saline (NaCl, 0.9%) and was administered 60-min before curcumin injection. Fluoxetine (Sigma, St. Louis, MO, USA; 30 mg/kg), a selective serotonin reuptake inhibitor, was dissolved in physiological saline (NaCl, 0.9%) and was used as a positive control for antidepressant action. In a separate series of experiments, a partial NMDA receptor agonist, D-cycloserine (DCS, Sigma, St. Louis, MO, USA; 15 mg/kg), was dissolved in physiological saline (NaCl, 0.9%) and was given to mice 30 min before the administration of a sub-effective dose of curcumin (10 mg/kg), and the test was carried out 45 min later.
13
All drugs were administered intraperitoneally (i.p.) at a volume of 10 ml/kg. Dose and route administration schedules of all drugs used in the present experiment were chosen as based on previous results — NVP-AAM077 and Ro 25‐6981 (Dalton et al., 2012; Fox et al., 2006), CPP (Dalton et al., 2012; Goosens and Maren, 2004) and D-cycloserine (Curlik and Shors, 2011; Walker et al., 2002). To habituate to the intraperitoneal injections, all mice were administered saline (10 ml/kg) daily for three days prior to the experiment. 2.3. Forced swim test Mice were subjected to the forced swim test similar to that described previously (Porsolt et al., 1977a). Briefly, mice were placed individually in a cylinder (height: 35 cm, diameter: 20 cm) containing 25 cm of water at 25 °C for two consecutive swim sessions. On the first day, the mice were exposed to 15 min of forced swim (training session) and then were removed and dried before being returned to cages. Twenty-four hours later, mice were placed in the cylinder again for a 5 min period (test session). The drugs and their corresponding vehicles were administered three times between these two swim sessions (23.5, 5, and 1 h before the 5 min swim). The 5-min test was scored for immobility time defined as floating with only small movement necessary to keep the head above water by an observer blind to the treatment condition of the animal. 2.4. Data analysis One-way analysis of variance (ANOVA) was used to establish differences among groups of animals treated with vehicle or drugs alone and was followed by the Newman–Keuls test for post-hoc comparisons. Other data were analyzed statistically by two-way ANOVA for multiple comparisons with pretreatment or co-administration, and followed by the Bonferroni test. All statistical procedures were performed on SPSS version 13.0. The results were expressed as mean ± SEM. Differences with P b 0.05 were considered statistically significant. 3. Results 3.1. Effects of curcumin and fluoxetine in the forced swim test The effects of curcumin (10, 20, 40 mg/kg) and fluoxetine (10, 20, 30 mg/kg) administrations on immobility time of mice in the forced swim test are presented in Fig. 1. The one-way ANOVA revealed significant differences in response to treatments with curcumin [F (3, 42) = 36.71, P b 0.01] and fluoxetine [F(3, 41) = 27.63, P b 0.01] on immobility time in the forced swim test. Post-hoc analysis indicated that administration of curcumin in doses of 20 and 40 mg/kg, or fluoxetine in doses of 20 and 30 mg/kg significantly decreased immobility duration compared to their respective vehicle treated groups. Such decreases in duration of immobility suggest an antidepressantlike effect of curcumin in this forced swim test. 3.2. Effect of NMDA receptor antagonist on the antidepressant-like effect of curcumin To assess possible involvement of the NMDA receptor in the antidepressant-like activity of curcumin, four separate groups of mice were pre-treated with the NMDA receptor antagonists CPP (10 mg/kg) or their respective vehicles prior to the administration of curcumin (40 mg/kg) or fluoxetine (30 mg/kg) for three consecutive times between the two swim sessions in the forced swim test. ANOVA showed a significant effect in the factors of pre-treatment [saline or CPP; F (1, 22)= 28.56, P b 0.01], treatment [curcumin or fluoxetine: F(2, 35)= 31.28, P b 0.01)] and the pre-treatment× treatment interaction [F(2, 35) =29.76, Pb 0.01)] on immobility time in the forced swim
14
L. Zhang et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 40 (2013) 12–17
Chemical structure of curcumin
Immobility time (s)
200
150
**
** **
**
100
50
0
0
10
20
40
Curcumin (mg/kg)
0
10
20
forced swim test, we used the GluN2A-prefering antagonist NVPAAM077 and the GluN2B antagonist Ro25-6981 to test for involvement of different NMDAR subtypes in subsequent experiments. Our previously results indicated that these two drugs exerted a preferential antagonism of GluN2A and GluN2B-containing receptors, respectively (Yu et al., 2010). NVP-AAM077 (1.2 mg/kg) was administered intraperitoneally at 45-min prior to the treatment of curcumin. The results of pre-treatment with NVP-AAM077 on reduction in immobility time elicited by curcumin (40 mg/kg) in the forced swim test are shown in Fig. 3. ANOVA revealed the effects of pre-treatment [saline or NVP-AAM077; F (1, 24) =0.61, P > 0.05], treatment [F(1, 24) = 26.87, P b 0.01], and the pre-treatment× treatment interaction [F(1, 24) = 0.49, P > 0.05] on immobility times in the forced swim test. Post-hoc analysis indicated that NVP-AAM077 pre-treatment did not exert any influence on the antidepressant-like effects elicited by curcumin as revealed from the duration of immobility scores in the forced swim test (P > 0.05). That is, NVP-AAM077 pre-treated mice displayed reductions in immobility times that did not differ significantly from curcumin-treated mice. These results indicated that blockade of GluN2A-containing receptors does not affect the antidepressant-like effects of curcumin.
30
Fluoxetine (mg/kg)
Fig. 1. Upper: the chemical structure of curcumin. Molecular weight: 368.38. Linear formula: [HOC6H3(OCH3)CH=CHCO]2CH2. Under: effects of administration of curcumin and fluoxetine on immobility times in the forced swimming test. Mice were treated with their vehicles, curcumin (10, 20, 40 mg/kg, i.p.) or fluoxetine (10, 20, 30 mg/kg, i.p.) three times between the two swim sessions. All values are presented as means±SEM (n=10–12). ** Pb 0.01, compared with their vehicle-treated group.
test. Post-hoc analyses indicated that pre-treatment of CPP significantly attenuated the decrease in immobility duration elicited by curcumin but not by fluoxetine in the forced swimming test while CPP-administration alone did not modify immobility time of mice compared to the control group (P >0.05) (Fig. 2). 3.3. Effect of GluN2A-containing NMDA receptor antagonist on the antidepressant-like effect of curcumin To determine whether distinct NMDA receptor subtypes differentially contribute to the antidepressant-like effects of curcumin in the
3.4. Effect of GluN2B receptor antagonist on the antidepressant-like effect of curcumin In a separate group of mice we next investigated whether GluN2B NMDA receptor activation is involved in the antidepressant-like effects induced by curcumin. These data showing the reversal of antidepressant-like effects of curcumin resulting from pre-treatment of mice with Ro25-6981 (6 mg/kg) in the forced swimming test are presented in Fig. 3. ANOVA revealed significant differences in the factors of pre-treatment [saline or Ro25-6981; F (1, 24)= 20.63, P b 0.01], treatment [F(1, 24) = 31.94, P b 0.01], and the pre-treatment× treatment interaction [F(1, 24)= 28.49, P b 0.01] on immobility times in the forced swim test. Post-hoc analyses indicated that, in contrast to the GluN2A receptor blockade, pre-treating mice with Ro25-6981 significantly attenuated the decrease in immobility elicited by curcumin in the forced swim test (P b 0.01). Collectively, these data showed that activation of GluN2B, but not the GluN2A-subunit containing NMDA receptors, played a predominate role in the antidepressant-like effects of curcumin in the forced swim test in mice. 200
150
**
**
**
100
50
0
Immobility time (s)
Immobility time (s)
200
150
**
**
100
50
0
Vehicle
+
+
+
+
-
-
Vehicle
+
+
+
+
-
-
Curcumin
+ -
+
-
+ -
+
Curcumin
Fluoxetine
-
NVP-AAM077
-
+
-
+ -
+ +
+ -
CCP
-
-
-
+
+
+
Ro25-6981
-
-
+
-
-
+
Fig. 2. Effects of pre-treatment with CPP (a competitive NMDA receptor antagonist, 10 mg/kg, i.p.) on curcumin (40 mg/kg, i.p.) and fluoxetine (30 mg/kg, i.p.) induced decrease in immobility times. All values are presented as means ± SEM (n = 10–12). ** P b 0.01, compared with their vehicle-treated group; # #P b 0.01, compared with the group treated with vehicle + curcumin or fluoxetine.
Fig. 3. Pre-treatment with Ro25-6981 (a specific GluN2B antagonist, 6 mg/kg, i.p.) but not with NVP-AAM077 (a GluN2A-prefering antagonist, 1.2 mg/kg, i.p.) prevent the decrease in immobility times induced by curcumin (40 mg/kg, i.p.). All values are presented as means±SEM (n=10–15). ** Pb 0.01, compared with their vehicle-treated group; ## Pb 0.01, compared with the group treated with vehicle+curcumin group.
L. Zhang et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 40 (2013) 12–17
Immobility time (s)
200
150
**
15
of pre-treatment [saline or curcumin; F(1, 22) = 22.42, P b 0.01], treatment [F(1, 22) = 18.10, P b 0.01], and the pre-treatment× treatment interaction [F(1, 22)= 17.90, P b 0.01] in the forced swim test. Post-hoc analyses indicated that pre-treatment with a sub-effective dose of curcumin elicited a significant synergistic effect when combined with a sub-effective dose of fluoxetine in the forced swimming test (P b 0.01).
100
4. Discussion 50
0
Vehicle DCS Curcumin
+ -
+ + -
+ +
+ +
Fig. 4. Effect of pre-treatment of a sub-effective dose of DCS (a partial NMDA receptor agonist, 15 mg/kg, i.p.) on the action of a sub-effective dose of curcumin (10 mg/kg, i.p.) in the forced swim test. All values are presented as means ± SEM (n = 10–12). ** P b 0.01, compared with their vehicle treated group; # #P b 0.01, compared with the group pre-treated with vehicle + curcumin group.
3.5. Effects of co-administration with sub-effective doses of curcumin and DCS on immobility time in mice The results obtained following co-administration of sub-effective doses of DCS (15 mg/kg) and curcumin (10 mg/kg) on immobility times in the forced swim test are shown in Fig. 4. ANOVA revealed significant differences in the factors of pre-treatment [saline or DCS; F(1, 22) = 21.16, P b 0.01], treatment [F(1, 22) = 19.86, P b 0.01], and the pre-treatment × treatment interaction [F(1, 21) = 25.18, P b 0.01] in the forced swim test. Post-hoc analyses indicated that pretreatment of mice with a sub-effective dose of DCS potentiated the anti-immobility effect of a sub-effective dose of curcumin in the forced swimming test (P b 0.01). 3.6. Effects of co-administration with sub-effective doses of curcumin and fluoxetine in the forced swimming test
The primary finding of this study was the demonstration of the important differences that exist between the roles of GluN2A and GluN2B NMDA receptor blockade in the antidepressant-like effects of curcumin in the forced swim test. Consistent with previous findings, our present results demonstrated that curcumin produces a significant decrease in the duration of immobility in mice, which was similar to the effects seen in response to treatment with fluoxetine. Specifically, we observed that blockade of GluN2B receptors with Ro25-6981, but not blockade of GluN2A receptors with NVP-AAM077, significantly impaired the antidepressant-like effects of curcumin in the forced swim test. 4.1. Forced swim test The forced swim test in mice is widely used to evaluate antidepressants as well as investigate the mechanisms underlying the effects of antidepressants in this animal model of depression (Porsolt et al., 1977b). During the test session, immobility time is interpreted as representing despair or a depression-like state which can be shortened by repeated antidepressant treatment. Thus, immobility duration is believed to provide a measure of the degree of “behavioral despair” exhibited by these animals. In the present study, animals treated with curcumin and fluoxetine show a dose-dependent decrease in their immobility durations in the forced swim test, as reported previously. However, we demonstrated that this effect was significantly blocked by pretreatment with CPP, a non-specific NMDA-receptor antagonist. Thus, the results suggest that NMDA receptors may be involved in the anti-depressant effect of curcumin in the forced swim test. 4.2. NMDA receptors in the antidepressant-like activity of curcumin
The interaction resulting from sub-effective doses of curcumin (10 mg/kg) with fluoxetine (10 mg/kg) in the forced swim test are presented in Fig. 5. ANOVA revealed significant differences in the factors
Immobility time (s)
200
150
** 100
50
0
Vehicle Curcumin Fluoxetine
+ -
+ +
+ + -
+ +
Fig. 5. Effect of co-administration of mice with sub-effective doses of curcumin (10 mg/kg, i.p.) and fluoxetine (10 mg/kg, i.p.) on immobility times. All values are presented as means ± SEM. (n = 10–12). ** P b 0.01, compared with their vehicle treated group. # #P b 0.01, compared with the group treated with vehicle + curcumin or fluoxetine.
In the present behavioral experiments, CPP alone did not modify immobility times of mice in the forced swimming test, but pretreatment with CPP essentially prevented the antidepressant effects of curcumin, suggesting a contribution of NMDA receptors to the effects of this herbal medicine. Additionally, we found that co-administration of sub-effective doses of curcumin and DCS produced a significant synergistic antidepressant effect in forced swim test. These results further reinforced the importance of NMDA receptors in the antidepressant-like effects of curcumin. Related findings with transgenic mice showed that over-expression of GluN2B NMDA receptor subunits in several forebrain areas, including the amygdala and hippocampus significantly accelerated conditioned fear extinction as compared with their wild-type controls (Tang et al., 1999). Similarly, our previous results as obtained with amygdala slice preparations demonstrated that activation of GluN2B subunits predominately contributed to the expression of NMDA receptordependent LTD (Yu et al., 2010), the most well characterized neural construct which is generally thought to play a critical role in the extinction of previously learned inappropriate responses (Dalton et al., 2011; Duffy et al., 2008; Kim et al., 2007). Moreover, recent electrophysiological evidence revealed that activation of receptors containing the GluN2B but not GluN2A subunit mediates the generation of LTD-like response (Izumi et al., 2006; Liu et al., 2004; Yang et al., 2005). Thus, GluN2B receptor-mediated LTD may play a specific role in the extinction of previously-acquired memories.
16
L. Zhang et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 40 (2013) 12–17
4.3. Distinct NMDA receptor subtypes in the antidepressant-like effects of curcumin To investigate further the dissociable mechanisms of NMDA receptor subunits on the effects of curcumin, mice were pre-treated with receptor antagonists specific for different NMDA receptor subtypes. Interestingly, we did not see any apparent reduction in antidepressant effects of curcumin in response to pretreatment with the GluN2A NMDA receptor antagonist NVP-AAM077 in the forced swim test suggesting that deficits in GluN2A subunits do not impair the antidepressant effects of curcumin. In contrast, it is interesting to note that we observed pretreated with Ro25-6981 before curcumin significantly increased immobility times during the test session. The higher levels of immobility observed in these mice after pretreatment with Ro25-6981 reflects the critical role of GluN2B receptors in the antidepressant-like effects of curcumin. Therefore, the data obtained from this behavioral test support the conclusion that activation of GluN2B-containing NMDARs represents the predominate pathway in the improvement of depressive-like behavior in mice resulting from curcumin. As mentioned above, the GluN2B NMDA receptor is strongly related to extinction of learning and memories, so the results reported here, in combination with previous studies, suggest that the antidepressant-like effectiveness of traditional herbal medicines, like curcumin, might be exhibited by promoting extinction of aversive memories mediated through a preferred GluN2B NMDA receptor subtype. 4.4. Synergistic antidepressant-like effect of curcumin and fluoxetine It should also be noted that pretreatment with CPP, a competitive NMDA receptor antagonist, did not prevent the antidepressant-like effect of fluoxetine in the forced swim test. Such results indicate these NMDA receptors are not involved in the effects of selective serotonin reuptake inhibitors in the treatment of depression. However, in our experiments, a sub-effective dose of curcumin potentiated the effects of a sub-effective dose of fluoxetine in the forced swimming test. These results suggest that although NMDA receptors, by themselves, may not be involved in the mechanism of fluoxetine action, a co-activation of NMDA and 5-HT receptors could produce a synergistic antidepressantlike effect in the forced swim test. This synergistic effect could be due to either a direct additive antidepressant effects by activation of NMDA and 5-HT receptors, or a result of an indirect pathway via Ca 2+, an important second messenger, science activation of postsynaptic NMDA receptors will increase the subsequent Ca2+ influx which may in turn lead to the activation of more 5-HT receptors. 5. Conclusion In summary, the results from our behavioral findings on depression as assessed in the forced swim test indicate that curcumin exerts an antidepressant-like effect and does so by activation of GluN2B NMDA receptor subunits. These findings provide one possible novel substrate by which herbal antidepressants may exert their beneficial effects in the treatment of depression. Additionally, the synergistic effect induced by co-administration with sub-effective doses of curcumin and fluoxetine hint at the possible interaction between glutamatergic and serotonergic systems in the antidepressant effects of the forced swim test. Further detailed molecular mechanisms underlying the antidepressant-like effects of curcumin, which are now under investigation in our laboratory, will provide further insight into this issue. Acknowledgments This study was supported by grants to Shu Yan Yu from the National Natural Science Foundation of China (NSFC81101006),
from the Excellent Young Scientist Foundation of Shandong Province (BS2010SW021), from the Independent Innovation Foundation of Jinan city (201202046) and from the Independent Innovation Foundation of Shandong University (IIFSDU2010TS091). References Aggarwal BB, Harikumar KB. Potential therapeutic effects of curcumin, the anti-inflammatory agent, against neurodegenerative, cardiovascular, pulmonary, metabolic, autoimmune and neoplastic diseases. Int J Biochem Cell Biol 2009;41: 40–59. Berton O, Nestler EJ. New approaches to antidepressant drug discovery: beyond monoamines. Nat Rev Neurosci 2006;7:137–51. Bliss TV, Collingridge GL. A synaptic model of memory: long-term potentiation in the hippocampus. Nature 1993;361:31–9. 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(2):320–31. Castaneda AE, Tuulio-Henriksson A, Marttunen M, Suvisaari J, Lönnqvist J. A review on cognitive impairments in depressive and anxiety disorders with a focus on young adults. J Affect Disord 2008;106:1-27. Curlik DM, Shors TJ. Learning increases the survival of newborn neurons provided that learning is difficult to achieve and successful. J Cogn Neurosci 2011;23(9): 2159–70. Dalton GL, Ma LM, Phillips AG, Floresco SB. Blockade of NMDA GluN2B receptors selectively impairs behavioral flexibility but not initial discrimination learning. Psychopharmacology 2011;216:525–35. Dalton GL, Wu DC, Wang YT, Floresco SB, Phillips AG. NMDA GluN2A and GluN2B receptors play separate roles in the induction of LTP and LTD in the amygdala and in the acquisition and extinction of conditioned fear. Neuropharmacology 2012;62:797–806. Dong S, Zeng Q, Mitchell ES, Xiu J, Duan Y, Li C, et al. Curcumin enhances neurogenesis and cognition in aged rats: implications for transcriptional interactions related to growth and synaptic plasticity. PLoS One 2012;7:e31211. Duffy S, Labrie V, Roder JC. D-serine augments NMDA-NR2B receptor-dependent hippocampal long-term depression and spatial reversal learning. Neuropsychopharmacology 2008;33:1004–18. Fox CJ, Russell KI, Wang YT, Christie BR. Contribution of NR2A and NR2B NMDA subunits to bidirectional synaptic plasticity in the hippocampus in vivo. Hippocampus 2006;16:907–15. Goosens KA, Maren S. NMDA receptors are essential for the acquisition, but not expression, of conditional fear and associative spike firing in the lateral amygdale. Eur J Neurosci 2004;20:537–48. Izumi Y, Auberson YP, Zorumski CF. Zinc modulates bidirectional hippocampal plasticity by effects on NMDA receptors. J Neurosci 2006;26:7181–8. Kim J, Lee S, Park K, Hong I, Song B, Son G, et al. Amygdala depotentiation and fear extinction. Proc Natl Acad Sci 2007;104:20955–60. Kulkarni SK, Bhutani MK, Bishnoi M. Antidepressant activity of curcumin: involvement of serotonin and dopamine system. Psychopharmacology (Berl) 2008;201:435–42. Lin TY, Lu CW, Wang CC, Wang YC, Wang SJ. Curcumin inhibits glutamate release in nerve terminals from rat prefrontal cortex: possible relevance to its antidepressant mechanism. Prog Neuropsychopharmacol Biol Psychiatry 2011;35:1785–93. Liu L, Wong LP, Pozza MF, Lingenhoehl K, Wang Y, Sheng M, et al. Role of NMDA receptor subtypes in governing the direction of hippocampal synaptic plasticity. Science 2004;304:1021–4. Maheshwari RK, Singh AK, Gaddipati J, Smiral RC. Multiple biological activities of curcumin: a short review. Life Sci 2006;78:2081–7. Melo TG, Izídio GS, Ferreira LS, Sousa DS, Macedo PT, Cabral A, et al. Antidepressants differentially modify the extinction of an aversive memory task in female rats. Prog Neuropsychopharmacol Biol Psychiatry 2012;37:33–40. Nemeroff CB. The burden of severe depression: a review of diagnostic challenges and treatment alternatives. J Psychiatr Res 2007;41:189–206. Porsolt RD, Le Pichon M, Jalfre M. Depression: a new animal model sensitive to antidepressant treatments. Nature 1977a;266:730–2. Porsolt RD, Pichon MLE, Jalfre M. Behavioral despair in mice: a primary screening test for antidepressant. Arch Int Pharmacodyn Ther 1977b;229:327–36. Rosenzweig-Lipson S, Beyer CE, Hughes ZA, Khawaja X, Rajarao SJ, Malberg JE, et al. Differentiating antidepressants of the future: efficacy and safety. Pharmacol Ther 2007;113:134–53. Schulz D, Buddenberg T, Huston JP. Extinction-induced “despair” in the water maze, exploratory behavior and fear: effects of chronic antidepressant treatment. Neurobiol Learn Mem 2007;87:624–34. Tang YP, Shimizu E, Dube GR, Rampon C, Kerchner GA, Zhuo M, et al. Genetic enhancement of learning and memory in mice. Nature 1999;401:25–7. Van der Watt G, Laugharne J, Janca A. Complementary and alternative medicine in the treatment of anxiety and depression. Curr Opin Psychiatry 2008;21:37–42. Walker DL, Ressler KJ, Lu KT, Davis M. Facilitation of conditioned fear extinction by systemic administration or intra-amygdala infusions of D-cycloserine as assessed with fear-potentiated startle in rats. J Neurosci 2002;22:2343–51. Wang R, Xu Y, Wu HL, Li YB, Li YH, Guo JB. The antidepressant effects of curcumin in the forced swimming test involve 5-HT1 and 5-HT2 receptors. Eur J Pharmacol 2008;578:43–50. Xu Y, Ku BS, Yao HY, Lin YH, Ma X, Zhang YH. The effects of curcumin on depressive-like behaviors in mice. Eur J Pharmacol 2005a;518:40–6.
L. Zhang et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 40 (2013) 12–17 Xu Y, Ku BS, Yao HY, Lin YH, Ma X, Zhang YH. Antidepressant effects of curcumin in the forced swim test and olfactory bulbectomy models of depression in rats. Pharmacol Biochem Behav 2005b;82:200–6. Xu Y, Lin D, Li S, Li G, Shyamala SG, Barish PA, et al. Curcumin reverses impaired cognition and neuronal plasticity induced by chronic stress. Neuropharmacology 2009;57: 463–71. Yang CH, Huang CC, Hsu KS. Behavioral stress enhances hippocampal CA1 long-term depression through the blockade of the glutamate uptake. J Neurosci 2005;25: 4288–93.
17
Yu SY, Wu DC, Liu L, Ge Y, Wang YT. Role of AMPA receptor trafficking in NMDA receptor-dependent synaptic plasticity in the rat lateral amygdala. J Neurochem 2008;106:889–99. Yu SY, Wu DC, Zhan RZ. GluN2B subunits of the NMDA receptor contribute to the AMPA receptor internalization during long-term depression in the lateral amygdala of juvenile rats. Neuroscience 2010;171:1102–8. Zhou H, Beevers CS, Huang S. The targets of curcumin. Curr Drug Targets 2011;12: 332–47.