Increased Serotonergic Neurotransmission Is Not Responsible for the Anticompulsive Effect of Berberine in a Murine Model of Obsessive–Compulsive Disorder Pankaj V

716 Short report

Increased serotonergic neurotransmission is not responsible for the anticompulsive effect of berberine in a murine model of obsessive–compulsive disorder Pankaj V. Dixit, Gaurav Parihar, Dinesh K. Jain and Pooja Jaiswal

Berberine, an isoquinoline alkaloid, is being extensively p-chlorophenylamine (300 mg/kg, i.p. ¾ 3 days), a explored in several clinical trials for the treatment of tryptophan hydroxylase inhibitor and serotonin-depleting metabolic disorder and cancer. It is also reported to be a agent, completely blocked the effect of fluoxetine on potent inhibitor of prolyl oligopeptidase, which makes it marble burying, whereas it failed to alter the effect of a potential candidate for the treatment of neuropsychiatric berberine. In conclusion, the findings of the present disorders. We have previously shown the potential of investigation indicate that the anticompulsive berberine in the control of seizures in various murine and/or anxiolytic effect of berberine observed in the models of epilepsy, diabetes-induced memory dysfunction, present investigation may be attributed to its effect on and ethanol-induced hyperexcitability. We have now other neurotransmitter systems, such as the nitrergic or examined the effects of acute and subchronic (7 days) the dopaminergic system rather than to increased administration of berberine on a murine model of serotonin turnover in the brain. Behavioural Pharmacology obsessive–compulsive disorder – the marble-burying 23:716–721 c 2012 Wolters Kluwer Health | Lippincott behavior of male mice, because berberine administration is Williams & Wilkins. reported to increase brain monoamine levels – a desirable Behavioural Pharmacology 2012, 23:716–721 endpoint in the treatment of obsessive–compulsive disorder. The studies showed that an acute administration Keywords: fluoxetine, male, marble-burying behavior, mouse, p-chlorophenylamine of berberine [1–25 mg/kg, intraperitoneally (i.p.)] dose- dependently inhibited marble burying in male mice without Department of Pharmacology, College of Pharmacy, IPS Academy, Indore, India altering locomotor activity. This effect was retained Correspondence to Pankaj V. Dixit, Department of Pharmacology, College of after its subchronic administration. Furthermore, Pharmacy, IPS Academy, A.B. Road, Rajendrangar, Indore (MP) 452012, India E-mail: [email protected] coadministration of a subeffective dose of berberine (1 mg/kg) and fluoxetine (5 mg/kg, i.p.) significantly Received 3 May 2012 Accepted as revised 10 July 2012 reduced marble burying in mice. Pretreatment with

Introduction epilepsy, diabetes-induced memory dysfunction, ethanol- Berberine (5,6-dihydro-9,10-dimethoxy-benzo[g]-1,3- induced hyperexcitability, and inhibition of the motiva- benzodioxolo[5,6-a]quinolizinium) is an isoquinoline tional effects of ethanol in rodents (Bhutada et al., 2010a, alkaloid with a long history of medicinal use in both 2010b, 2011a, 2011b). These effects of berberine are Ayurvedic and Chinese medicine. Berberine possesses attributed to the modulation of several neurotransmitter multiple pharmacological effects and has promising prop- systems including glutamate (Yoo et al., 2006), serotonin erties as a drug for treatment of cardiovascular diseases, (Peng et al., 2007; Kulkarni and Dhir, 2008), and nitric diabetes, hyperlipidemia, cancer, diarrhea, and Alzhei- oxide (Kulkarni and Dhir, 2007). mer’s disease (Kulkarni and Dhir, 2010; Vuddanda et al., 2010). In fact, numerous clinical trials have substantiated Obsessive–compulsive disorder is characterized by per- these claims (Zeng et al., 2003; Tarrago et al., 2007; Tang sistent thoughts (obsessions), which are ego-dystonic et al., 2009), and many more are currently underway. and associated with seemingly purposeful behaviors (compulsions) (Rasmussen and Eisen, 1992). Only There is renewed interest in berberine because of its safe potent serotonin reuptake inhibitors are consistently administration in humans and beneficial effects in various effective in patients with obsessive–compulsive disorder neurodegenerative and neuropsychiatric disorders (Kong (El Mansari and Blier, 2006). Marble-burying behavior in et al., 2004; Peng et al., 2004; Yoo et al., 2006; Zhu and male mice is reported to be a valid paradigm for screening Qian, 2006; Kulkarni and Dhir, 2008). Recent evidence drugs with potential anticompulsive activity (Joel, 2006), suggests that berberine-containing herbal products pro- and it is being used successfully for this purpose (Uday duce anxiolytic, analgesic, antidepressant, and antiamne- et al., 2007; Umathe et al., 2009a, 2009b). sic effects, and have great potential in the treatment of drug addiction (Kulkarni and Dhir, 2010). We have It is reported that acute as well as chronic administration recently reported its potential in the treatment of of berberine in mice increased brain serotonin levels by

0955-8810 c 2012 Wolters Kluwer Health | Lippincott Williams & Wilkins DOI: 10.1097/FBP.0b013e328358477c

47 and 53%, respectively (Kulkarni and Dhir, 2008). berberine (0, 2, and 5 mg/kg, i.p., Â 7 days) and 30 min These observations are well supported by Peng et al. after the last dose, each mouse was subjected to the MBB (2007). In addition, berberine is also reported to block test. This was done to check whether the response to the monoamine oxidase-A isoform with IC50 values of berberine is altered after chronic dosing. More specifi- 126 mmol/l (Kong et al., 2001). Serotonergic dysfunction is cally, the aim was to check for precipitation of locomotor believed to be pivotal in obsessive–compulsive disorder toxicity, if any, after chronic dosing. and many antidepressant drugs have been found to be effective in its treatment. In experiment 3, a separate group of mice was pretreated with pCPA (300 mg/kg, i.p.) for 3 consecutive days, and Hence, on the basis of the above findings, we aimed to 24 h thereafter, either fluoxetine (10 mg/kg, i.p.) or screen the anticompulsive activity of berberine in male berberine (5 mg/kg, i.p.) was administered. Thirty mice using the marble-burying behavior paradigm. minutes later, each mouse was subjected to the marble- p-chlorophenylamine (pCPA), a tryptophan hydroxylase burying behavior test. In experiment 4, a group of mice inhibitor that produces a serotonin-depleted state in was treated with fluoxetine (5 mg/kg, i.p.) along with mice, was used to study the involvement of enhanced berberine (2 mg/kg, i.p.) 30 min before the behavioral serotonergic transmission in this effect of berberine. tests were carried out. Finally, we tested the influence of subeffective doses of fluoxetine and berberine administration in mice, as this For each of the above treatments, there was a separate experiment may confirm the findings with the pCPA control group, which received vehicle (10 ml/kg, i.p.). treatment protocol and strengthen the hypothesis. Separate groups (n = 5) of mice were used for each set of experiments. The doses of berberine and fluoxetine were based on our preliminary observations and reports Methods (Bhutada et al., 2010a; Uday et al., 2007), whereas the Subjects dose of pCPA was based on previous reports (Chiavegatto Adult male Swiss mice (22–25 g) were used in the present et al., 2001; Uday et al., 2007). The doses of berberine for study. Mice were group housed (5 per cage, 40 Â 28 Â chronic administration were selected from the results of 1 14 cm) and maintained at 23±2 C under a 12:12 h light/ experiment 1 such that one was subeffective and the dark cycle (lights on: 08:00–20:00 h), with free access to other was effective. standard rodent diet and tap water. Each experimental group included five mice. Behavioral studies were carried out between 09:00 and 14:00 h to minimize circadian Drugs and solutions influences. All the experiments were approved by the Berberine chloride and pCPA were purchased from Institutional Animal Ethics Committee (IAEC), consti- Sigma-Aldrich (St Louis, Missouri, USA). Fluoxetine tuted for the purpose of control and supervision of hydrochloride was a gift from Sun Pharmaceuticals experiments on animals by the Ministry of Environment Advanced Research Center (Vadodra, India). Berberine and Forests, Government of India, New Delhi, India. chloride was dissolved in distilled water and the pH was adjusted to 7.0 with a bicarbonate solution. pCPA was dissolved in 5% DMSO. All the control groups received Marble-burying behavior test appropriate vehicles. The marble-burying behavior test was carried out as described earlier (Umathe et al., 2009b). In brief, 20 small glass marbles (B10 mm) were arranged evenly spaced in Statistical analysis four rows of five in plastic cages (40 Â 28 Â 14 cm) Data were analyzed by one-way analysis of variance containing 5 cm thick saw dust bedding. The cage was (ANOVA), followed by Dunnett’s test for multiple covered by a transparent plastic sheet with line markings comparisons or two-way ANOVA, followed by Bonferroni’s (2 Â 2) and the apparatus was placed 2.0 m below a video multiple comparisons test. The results are expressed as camera. The luminance on the surface of test apparatus mean±SD. P value less than 0.05 was considered to be was 60 lx. Mice were tested individually for 30 min and statistically significant. unburied marbles were counted. The total number of line crossings measured during 30 min was considered as a Results measure of locomotor activity. Effects of acute and chronic treatment with berberine on marble-burying behavior Treatments One-way ANOVA showed that acute administration of In experiment 1, berberine [0, 1.5, 2, 5, 10, 15, 20, and berberine significantly decreased marble-burying behavior 25 mg/kg, intraperitoneally (i.p.)] or fluoxetine (0, 5, and [F(8, 36) = 4.52, P < 0.001] without significant alterations 10 mg/kg, i.p.) was administered 30 min before the in locomotor activity [F(8, 36) = 1.93, P = 0.083]. Further assessment of marble-burying behavior and locomotor analysis showed that the effect of berberine increased activity. In experiment 2, mice were treated with gradually from 5 up to 25 mg/kg, whereas at lower doses, it

Fig. 1

(a) 100 20 r2=0.9626

50 (b) 15 (c) 0 20 20 Percent reduction of Percent

marble-burying behavior 0.0 0.5 1.0 1.5 10 Log dose (berberine) 15 15 ∗ ∗∗ ∗∗ ∗∗∗ 10 ∗ 10 ∗ 5 5 5 Number of marbles buried 0 0 0 0 1 1.5 2 510 15 20 25 0 25 400

300 400 400 200 300 300 200 200 100 100 100 Number of line crossings 0 0 0 0 1 1.5 2 5 10 15 20 25 025 0510 Berberine (mg/kg, i.p.) Berberine (mg/kg, i.p.) Fluoxetine (mg/kg, i.p.)

Influence of (a) berberine (acute), (b) berberine (chronic), and (c) fluoxetine on marble-burying behavior and locomotor activity in mice (Inset: Log dose vs. percent response curve for berberine). Separate groups of mice were injected with vehicle [10 ml/kg, intraperitoneally (i.p.)] or increasing doses of berberine (1–25 mg/kg, i.p.) or fluoxetine (5–10 mg/kg, i.p.), and 30 min thereafter, each mouse was tested for both marble-burying behavior and locomotor activity. Each bar represents mean±SD of data from five mice. *P < 0.05, **P < 0.01, ***P < 0.001 versus respective vehicle control group (one-way analysis of variance, followed by Dunnett’s post-hoc test).

was ineffective (Fig. 1a). The effects of acute treatment None of these treatments influenced locomotor activity with berberine were maintained after its chronic adminis- significantly [pCPA pretreatment: F(1, 24) = 1.97, NS; tration [F(2, 12) = 4.16, P < 0.05], without significant drug treatment: F(2, 24) = 0.46, NS; pCPA Â drug treat- effects on locomotor activity [F(2, 12) = 2.03, NS]. The ment interaction: F(2, 24) = 2.44, NS] (Fig. 2b). higher chronic dose of berberine (5 mg/kg) was effective (P < 0.05), whereas the lower dose was not (Fig. 1b). Fluoxetine pretreatment (5 mg/kg) significantly en- Similarly, fluoxetine treatment also significantly decreased hanced the inhibitory influence of berberine (2 mg/kg) the marble-burying behavior [F(2, 14) = 5.26, P <0.025], on marble-burying behavior, without any action when without any significant effect on the concurrently recorded administered alone (P > 0.05). Two-way ANOVA indi- locomotor activity [F(2, 14) = 0.80, NS]. The analysis cated the effects of fluoxetine pretreatment [F(1, 14) = further indicated that fluoxetine was effective at a dose 34.04, P < 0.001] and berberine treatment [F(1, 16) = of 10 mg/kg (P < 0.05), but not at 5 mg/kg (Fig. 1c). 41.19, P < 0.001] and a significant fluoxetine pretreatment Â berberine treatment interaction [F(1, 16) = 14.38, P < 0.002]. None of these treatments significantly Effects of fluoxetine and p-chlorophenylamine affected locomotor activity [fluoxetine pretreatment: administration on the berberine-induced inhibition of F(1, 16) = 0.76, NS; berberine treatment: F(1, 16) = marble-burying behavior 4.06, NS; fluoxetine pretreatment Â berberine treatment Pretreatment of mice with pCPA (300 mg/kg, i.p., Â 3 interaction: F(1, 16) = 0.06, NS] (Fig. 2a). days) completely blocked the effects of fluoxetine on marble-burying behavior (P < 0.05), whereas it had no significant effect on the inhibition of marble burying by Discussion berberine(5mg/kg);pCPAalonehadnosignificanteffect. In this report, we show that berberine – an isoquinoline Two-way ANOVA showed a nonsignificant pCPA pre- alkaloid – dose-dependently attenuated marble-burying treatment effect [F(1, 24) = 1.77, NS] and pCPA Â drug behavior in mice, an effect that was comparable with that of treatment interaction [F(1, 24) = 0.10, NS], and a signifi- fluoxetine. In addition, this effect of berberine was cant drug treatment effect [F(2, 24) = 11.92, P <0.001]. maintained after its administration for 7 days. Pretreatment

Fig. 2

(a) (b) ∗∗∗ 20 ∗∗∗ 20

15 15 ∗ 10 10

5 ∗∗∗ 5

Number of marbles buried 0 0

400 400

300 300

200 200

100 100

Number of line crossings 0 0 Vehicle Berberine Vehicle Berberine Fluoxetine (10 ml/kg, i.p.) (2 mg/kg, i.p.) (10 ml/kg, i.p.) (5 mg/kg, i.p.) (10 mg/kg, i.p.)

Vehicle Fluoxetine pCPA

Effect of fluoxetine and p-chlorophenylamine (pCPA) pretreatments on the anticompulsive effect of berberine in mice; (a) pretreated with fluoxetine and (b) pretreated with pCPA. (a) Separate groups of mice were injected with vehicle [10 ml/kg, intraperitoneally (i.p.)] or fluoxetine (5 mg/kg, i.p.) 30 min before berberine (2 mg/kg, i.p.); 30 min thereafter, each mouse was tested for both marble-burying behavior and locomotor activity. (b) Separate groups of mice were injected with pCPA (300 mg/kg, i.p., Â 3 days) or vehicle (10 ml/kg, i.p., Â 3 days), and 24 h after the last dose, vehicle (10 ml/kg, i.p.), berberine (5 mg/kg, i.p.), or fluoxetine (10 mg/kg, i.p.) was administered; 30 min thereafter, each mouse was tested for both marble-burying behavior and locomotor activity. Each bar represents mean±SD of data from five mice. *P < 0.05, ***P < 0.001 versus respective control. Two-way analysis of variance, followed by the Bonferroni post-hoc test.

of mice with subeffective doses of fluoxetine and berberine whether marble burying measures anxiety or compulsivity; markedly reduced marble-burying behavior; however, pre- however, it is clear that marble burying is decreased by both treatment with pCPA, which blocked the effects of anxiolytic drugs and anticompulsive drugs. Therefore, our fluoxetine,failedtoreversetheinfluenceofberberineon results suggest that berberine may have antianxiety or marble-burying behavior in male mice. anticompulsive activity. Finally, although the marble- burying model may at times fail to discriminate between It is observed that mice do not avoid marbles when given anxiolytic and anticompulsive agents after acute adminis- the opportunity to do so, indicating their nonaversive tration, all drugs that modulate marble-burying behavior property. In addition, repeated exposure to marbles does after repeated exposure without altering locomotor activity not induce habituation, suggesting that this behavior is not have been found to be clinically useful in the treatment of related to novelty or fear (for a review, see Albelda and Joel, obsessive–compulsive disorder (Londei et al., 1998; Njung’e 2012). However, because the marbles are nonreactive, they and Handley, 1991). The anxiolytic actions of berberine are cannot provide the animal with the necessary stimuli to a well documented; hence, the observed effect of berberine natural ending of the investigation, and this ‘frustrated’ on marble burying observed in the present study indicates investigation leads to compulsive burying. Hence, although its anticompulsive potential and prompts further evaluation inhibition of object burying was originally suggested as a in other animal models of compulsivity. screening test for anxiolytic activity, the above findings and the reduction in burying behavior by serotonin reuptake Presuming increased serotonergic neurotransmission to be inhibitors suggest that this behavior may be related to the underlying reason for modulation of marble-burying by obsessive–compulsive disorder (for a review, see Albelda berberine, we tested the effect of a serotonin-depleting and Joel, 2012; Gyertyan et al., 1995; Millan et al., 2002; agent, pCPA, on the effect of berberine. Interestingly, the Thomas et al., 2009). For these reasons, we selected marble- effect of berberine was not altered by pCPA pretreatment. burying behavior as a paradigm to screen the antic- Next, we administered subeffective doses of fluoxetine ompulsive effect of berberine. It is still matter of debate and berberine in mice and found that they reduced the

Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited. 720 Behavioural Pharmacology 2012, Vol 23 No 7 marble-burying behavior effectively. Thus, the mechanism the previous findings and suggest further examination of of action of berberine appears intriguing. the effects of berberine in other preclinical models and in clinical studies. Acuteaswellaschronicadministrationofberberineis reported to increase the levels of monoaminergic neuro- Acknowledgements transmitters in the brain (Kulkarni and Dhir, 2008; Peng Conflicts of interest et al., 2007). However, Peng et al. (2004) reported that There are no conflicts of interest. berberine markedly decreased the levels of norepinephrine, dopamine, and serotonin, and increased the levels of homovanillic acid, vanillyl mandelic acid, and 5-hydroxyin- References Albelda N, Joel D. 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