Berberine hydrochloride attenuates voluntary methamphetamine consumption and anxiety-like behaviors via modulation of oxytocin receptors in methamphetamine addicted rats

Accepted Manuscript Berberine hydrochloride attenuates voluntary methamphetamine consumption and anxiety-like behaviors via modulation of oxytocin receptors in methamphetamine addicted rats

Mahnaz Mesripour Alavijeh, Gholamhassan Vaezi, Mehdi Khaksari, Vida Hojati PII: DOI: Reference:

S0031-9384(18)30815-1 https://doi.org/10.1016/j.physbeh.2019.03.024 PHB 12505

To appear in:

Physiology & Behavior

Received date: Revised date: Accepted date:

21 September 2018 1 March 2019 23 March 2019

Please cite this article as: M.M. Alavijeh, G. Vaezi, M. Khaksari, et al., Berberine hydrochloride attenuates voluntary methamphetamine consumption and anxiety-like behaviors via modulation of oxytocin receptors in methamphetamine addicted rats, Physiology & Behavior, https://doi.org/10.1016/j.physbeh.2019.03.024

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ACCEPTED MANUSCRIPT Berberine hydrochloride attenuates voluntary methamphetamine consumption and anxiety-like behaviors via modulation of oxytocin receptors in methamphetamine addicted rats Mahnaz Mesripour Alavijeh1, Gholamhassan Vaezi1, Mehdi Khaksari2*, Vida Hojati1, 1

Department of Biology, Damghan Branch, Islamic Azad University, Damghan, Iran

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Corresponding author:

Corresponding author: Mehdi khaksari

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Addiction Research Center, Shahroud University of Medical Sciences, Shahroud, Iran

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Tel: +98- 23- 32395054 Fax: +98- 23- 32395054 [email protected]

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Abstract

Objective: Methamphetamine (METH) addiction is recognized as one of the major public health

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concerns, with no approved pharmacological agents for treatment. Berberine hydrochloride, an isoquinoline alkaloid in plants, induces antipsychotic and anxiolytic effects. Hence, we

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hypothesized that berberine may modulate the METH-induced rewarding effects. Materials and methods: In this study, three groups of rat including control (N= 10), METH +

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vehicle (N=10), and METH + berberine (N= 10) were kept in separate cages one day before expriments. METH (20 mg/L) was dissolved in tap water inside a bottle, while there was only

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tap water in the control bottle. Two groups received free METH solutions for two weeks (up to 12 mg/kg). Afterwards, they were abstianced for three weeks. Only one group received 100

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mg/kg/day of berberine. After three weeks, locomotor activity and anxiety (elevated plus maze test) were evaluated, then the two-bottles choice model was used for one week to evaluate drug

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preferences. Finally, the brain of rats was removed for evaluation of oxytocin receptor expression via immunofluorescence staining method. Results: The results showed that METH preference was lower in the berberine + METH group during drug intake compared to the METH group (P< 0.05). During withdrawal, berberine reduced anxiety-like behaviors (P< 0.05) and decreased locomotor activity versus the METH group (P< 0.001). Also, berberine increased numbers of oxytocin receptors in comparison with the METH group (P< 0.01). Conclusion: Considering the modulation of oxytocin receptors, berberine may be considered as a potential therapeutic agent for METH addiction.

ACCEPTED MANUSCRIPT Keywords: Berberine hydrocholoride, Methamphetamine addiction, Anxiety, Drug preference,

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Oxytocin receptor

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Introduction

Use of methamphetamine (METH) is increasing around the world. Following cannabis,

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amphetamine-type substances, such as METH, are recognized as the most common abused drugs throughout the globe (1). The prevalence of abused drugs has doubled over time. Medical

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complications, including psychiatric disorders and infectious diseases (e.g., hepatitis C virus and

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human immunodeficiency virus) are common among METH users (2-4). Outpatient and inpatient settings, psychosocial and cognitive-behavioral approaches are considered as the available treatment options for METH abuse (5, 6). Eventhough, various

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substances and compounds have been proposed and evaluated as therapeutic agents for METH abuse in animal and human models, no approved agents are suggeseted for METH addiction

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treatment, and rates of relapse are still high (7, 8).

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Though multitude mechanisms have been studied, the molecular mechanism which infelunce the addictive properties of chronic METH use (behavioural and emotional consequences) are remained unclear. According to previous studies, threre are strong relation between neuronal markers of social behavior, mood and stress regulation and drug reward/withdrawal markers (911) (12, 13). Hence, some consequences of METH use may be modified via neuropeptide oxytocin (OT) and its receptor (OTR) (14). In the hypothalamus, the magnocellular neurons of paraventricular (PVN) and supraoptic (SON) nuclei synthesize OT. These neurons are projected to the posterior pituitary gland. OT is stored in

ACCEPTED MANUSCRIPT the gland vesicles and excreted in the blood stream in order to lead peripheral impacts (15). In addition, oxytocinergic neurons project from the PVN and innervate brain regions such as the nucleus accumbens (NAc), septum, amygdal and the bed nucleus of striaterminalis, that correlated with drug-seeking behaviour, mood, fear and stress (16). The NAc is recognized as a prominent neural substrate, contributing to reward-related behaviors, particularly in the primary stages of drug abuse (17, 18). OT shows some attenuating impacts on

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psychostimulant and opiate addiction via its hippocampal receptors. Mühlethaler et al. indicated

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that OT raises firing rate of inhibitory neurons in the hippocampus. Some reaserches shows OT

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involvement in the effects of some drug abuse (19).

Berberine, an isoquinoline alkaloid from plants has been applied for medicinal applications in

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Chinese and India medicine for a long time (20). Berberine has potential to attenuate neuropsychiatric and neurodegenerative diseases. Some studies report that berberine exhibits

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anxiolytic (serotonergic system modulation), analgesic, antipsychotic (dopaminergic system modulation), antidepressant (adrenergic, serotonergic, nitrergic, dopaminergic, and sigma (21, 22),

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receptor modulation), antiamnesic (acetylcholinesterase enzyme inhibition) neuroprotective (23) and anticonvulsant activities (24).

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In addition, berberine can modulate psychomotor sensitization due to cocaine and nicotine via prevention of postsynaptic neuronal activation inside the central dopaminergic system (25).

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Furthermore, berberine leads to reduction of morphine sensitization by reducing the binding of N-methyl-D-aspartate (NMDA) and D1 dopamine receptors inside the cortex (26). Moreover, the

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locomotor stimulant activity of amphetamine is hindered by berberine (27), and Coptis japonica extract which contains berberine can inhibit conditioned place preference (CPP) due to morphine

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(28). Considering above mentioned facts, it can be hypothesized that the rewarding effects of METH are influenced by berberine through interactions in the OT system. Therefore, the objective of the current study was to evaluate the effects of berberine on the rewarding effects of METH.

Materials and methods Animals

ACCEPTED MANUSCRIPT This study was performed on male Wistar rats (200-250 g), provided from Pasteur Institute (Tehran, Iran). Before the experiments, one week was considered to adopt the animals to the new laboratory conditions. The rats were kept in transparent cages (one rat per cage) with wood chip bedding and had free access to pellet food and water. The conditions were similar in both rooms during the study and maintained constant conditions (temperature: 22 ± 2°C; humidity: 60 ± 5%). The animals were classified into three groups including: control (N= 10), METH + vehicle

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(N=10) and METH + berberine (N= 10). The protocols were followed in accordance with the

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international ethical standards. To determine the dosage of consumed METH, the rats were kept

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in separate cages one day before expriments.

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Two- bottle choice (TBC) paradigm

To determine voluntary METH use, the slightly modified TBC test was applied in animal

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craving models, following three withdrawal weeks (29). METH solutions (20 mg/L) was dissolved in tap water and prepared freshly every day, and the control bottle contained only tap

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water. To decrease the learning effects, the position of bottles was changed. Every day at 9:00 a.m., the volum of bottles were evaluated. Two groups of rats received METH solution for two

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weeks (up to 12 mg/kg). Afterwards, they were abandoned for three weeks but only one group that received METH solution was treated with 100 mg/kg/day of berberine hydrochloride(during

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withdrawal time) via oral gavage. Preference ratios (mL of consumed METH solution/total consumed mL from both bottles) was determined during one week after withdrawal period. Daily

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use of METH was expressed in mg/kg/day.

Open Field Locomotor Activity At the end of 5th week (after withdrawal from methamphetamine), the locomotor activity was evaluated, by the open field chamber. It was constructed of a 62 cm by 62 cm laminated wood floor surrounded by clear acrylic walls of 30 cm height. the floor of the open field was divided into 16 equal zones by four intersecting lines (that 4 squar zoon in center and Twelve squares are located in the environment(. that rats were placed on the open field chamber and allowed to

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that was equipped with a video camera above the center of the floor, Locomotor activity was monitored by a video tracking system using the openfildcal program(30). For five minutes, the rats had exposure to the open field. the number of lines crossed for each rat time and distance were measured at the center and on the side by the software, which was used to

Elevated Plus Maze: th

week. This maze is a plus shaped

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The elevated plus maze was performed at the end of the 5

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capture the motion of the rat. After each test the arena was cleaned with 90% alcohol solution.

apparatus with four arms at right angles to each other as described by Handley and Mithani (31). The two

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open arms lie across from each other measuring 25 x 5 x5 cm and perpendicular to two closed arms measuring 50 x 10 x 40 cm with a center platform (10 x 10 cm). The closed arms have a high wall (40

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cm) to enclose the arms whereas the open arms have no side wall. rats were placed in the central platform facing the closed arm and their behavior recorded for 5 min. based upon the early studies by Montgomery

(32). The criterion for arm visit was considered only when the animal decisively moved all its four limbs

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into an arm. The maze was cleaned with 5% ethanol after each trial . In this test, the animal's tendency

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(approach) towards enclosed and dark spaces and unconditioned fear (avoidance) of open spaces or heights are evaluated. To determine the percentage of time in the arms, the time spent in the

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arm (open or closed) was divided by the total time and multiplied by 100. Also, to measure the number of entries, the number of entries into the arm (open or closed) was divided by the total

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entry count .

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Tissue preparation

Immediately after the behavioral test, the animals were placed under anesthesia and trans cardiac perfusion was accomplished with 0.9% saline, continued by 4% paraformaldehyde in 0.1M phosphate buffer PH 7.4 (33). The brain tissues were embedded in paraffin after being extracted and

postfixed for 3 days using the same fixative. Afterwards, coronal sections (7 µm thicknesses), in accordance with the Paxinos atlas (accumbens: coronal sections, 2.7 mm anterior through 0.48 mm posterior to the bregma and hippocampus (CA1 area): between 3.3 mm and 4.2 mm posterior to the bregma) were prepared using different staining methods with a microtome(34). Measurement of oxytocin receptor immunoreactivity

ACCEPTED MANUSCRIPT Immunofluorescence staining was applied for identification of oxytocin receptor (OTR) activation on 7-µm tissue sections. After incubating the sections at 62°C for 20 minutes, they were rehydrated using descending alcohol series. Then, for 10 minutes, they were exposed to 10% hydrogen peroxide in methanol in order to reduce the activity of endogenous peroxidase. After washing in tris-buffer at pH of 7.4, autoclaving was applied for 11 minutes to retrieve antigens in citrate buffer (pH, 6 After a phase of washing, 10% normal goat serum was used to

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block the sections for 60 minutes. They were then incubated with anti-OTR antibodies with

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concentration of 1/100 (rabbit antibody against rat Abcam, UK) overnight at 4°C. After washing in

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triplicate with PBS, incubation was performed with fluorochrome-conjugated anti-rabbit secondary antibody with concentration of 1/1000 (Abcam, UK) for two hours in the dark to

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visualize the antigen. Then, the sections were counterstained for 5 minutes with DAPI in PBS to label the nucleus. After a washing step, the fluorescence signals from the accumbens and

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hippocampus fields, prepared from each slide of per animal) were detected with a fluorescence microscope (Labomed, USA, magnification 400×). The number of positive cells, as well as the

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total number of cells with image tool 2 software was also counted(35). The results are presented as the percentage of OTR-positive cells relative to the total cell count.

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F. Statistical Analysis

All data are reported as Mean ± SEM. The Kolmogorov– Smirnov test showed the normality of

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the distribution. In order to tell the differences among the groups, One-way analysis of variance (ANOVA) test was used. While, when the difference was significant, Dunnett's T3 or Scheffe's

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post hoc test was used to specify where the difference occurred. In the case of homogeneous variance, Scheffe's post hoc test was used; otherwise, we used Dunnett's T3 post hoc test. The

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level of significance was set at P≤ 0.05. All data analysis was performed by means of the SPSS software package (SPSS for Windows; SPSS Inc., Chicago, IL, USA; Version16.00).

Results: Two- bottle choice (TBC) paradigm: The amount of methamphetamine consumption (20 mg/L/24 h) in three groups of animals during a withdrawal period of 6 weeks shown in Figure 1A. According to results of Figure 1A that animals in the methamphetamine group have consumed higher amounts of methamphetamine, compared with control and berberine groups (P< 0.05). No significant

in difference

ACCEPTED MANUSCRIPT methamphetamine consumption can be found in berberine-traeted group and control group (P< 0.05). Figure 1B represents methamphetamine preference in three groups of animals during a withdrawal period of 6 weeks. The result of one-way ANOVA show that animals in the methamphetamine group had a greater preference for methamphetamine in comparison to control

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and berberine groups (P< 0.05).

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Open field:

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The analysis of total distance in open field at the end of 5th week shown in Figure 2A. Comparisons between groups show no significant differences in total distance locomotion.

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Figure 2B represents the analysis of central time/ total time (% C/ T time) percentage in the open field at the end of 5th week. It can be seen that compared with berberine-traeted (10.18 ± 1.06)

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and control groups (11.61 ± 1.44), methamphetamine-withdrawal group (6.47 ± 1.13) spent less time in the central area in open filed (P< 0.05).

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The analysis of central cross/ total cross (% C/ T cross) percentage in the open field at the end 5th week has been reported in Figure (2C). It can be seen that compared with berberine-treated

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(11.85 ± 1.33) and control groups (13.18 ± 1.92), methamphetamine-withdrawal group (7.33 ±

Elevated Plus Maze:

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1.31) has less cross in the central area in open filed (P< 0.05, Figure 3).

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The analysis of total number entries in open arm elevated plus maze in the open arm at the end of 5th week shown in Figure 4A. It can be seen that the number of open arm entries is greater in

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berberine-treated rats (3.78 ± 0.22) compared with the methamphetamine-withdrawal group (2.75 ± 0.313, P< 0.05). In addition, Time spend analysis in open arm elevated plus maze in the open arm at the end of 5th week has been presented in Figure 4B in control (19.75 ± 1.41), addicted (13 ± 1.43) and addiction berberine-treatment groups (18.33 ± 1.56). Berberine–treated rats show a larger time spent of the open arm compared with the methamphetamine-withdrawal group (P< 0.05). Figure 4C shows an analysis of the percentage of time spent in the open arms elevated plus maze in the open arm at the end of 5th week in control (6.58 ± 0.47), addicted (4.33 ± 0.48) and addiction treatment groups (6.11 ± 0.52). Berberine-treated rats show a higher percentage of time spent in the open arms compared with the methamphetamine-withdrawal

ACCEPTED MANUSCRIPT group (P< 0.05). Also Figure 5 show that methamphetamine-withdrawal group spend less time in the open arms compared with the control group. Percentage of oxytocin receptor in accumbens and hippocampus: The percentage of oxytocin receptor in accumbens shown in Figure (6,7). Results demonstrated the decrease amount of oxytocin receptor in the methamphetamine-addicted group (11.75 ± 1.18) compared with control group (19 ± 1.29, P<0.01), while, The percentage of oxytocin receptor

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improved in berberine treatment group (19.25 ± 1.49) in comparison with methamphetamine-

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addicted group (P< 0.01).

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Figure (8,9) shows the percentage of oxytocin receptor in the hippocampus. It represents that the oxytocin receptor decreases in the methamphetamine-addicted group (12 ± 1.78) compared with

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the control group (32.5 ± 2.5, P< 0.001). The percentage of oxytocin receptor in the hippocampus increased in berberine treatment group (30 ± 2.041) compared with the

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methamphetamine-addicted group (12 ± 1.78, P< 0.001) .

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Discussion

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Based on findings of the current study, administration of berberine in METH-addicted rats reduces motor activity, relapse-like and anxiety-like behaviors. Furthermore, berberine

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modulates OTR in NAc and hippocampus of the addicted rats. Althougth the molecular mechanism, through which berberine produces effects on motivation for drug-relapse responses

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in drug-addicted rats is unclear, one possible mechanism is that the oxytonergic system is considerably influenced by berberine.

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OTRs have been found in the hippocampus, NAc, and amygdala, which are regions of the brain involved in addictive behaviors and modulation of the drug effects (36, 37). Hypothalamic OT cells express DA receptors suggesting that dopamine may also mediate oxytocin release. In addition, pair bonding in monogamous prairie voles is dependent on dopamine interactions in the NAc; specifically, D2 receptors promote bonding and D1 receptors inhibit bonding(38).that DA may also mediate OT release. In addition,

pair bonding in monogamous prairie voles is

dependent on dopamine interactions in the NAc; specifically, D2 receptors promote bonding and D1 receptors inhibit bonding (39). Hence, the oxytonergic system may associated to the progression of addictive behaviors (40). Intracerebroventricular administration of OT inhibited

ACCEPTED MANUSCRIPT METH-induced place preference, that facilitated the elimination of METH-induced CPP and prevented its stress induced reinstatement in mouse model (41). This was mechanistically attributed to OT inhibition of improved DA adminstration in the striatum due to METH. In adition, OT antagonizes cocaine-induced enhance in DA utilization in the nucleus accumbens (42). Previous controversial data reveal that OT adminstration to central nucleus of rat amygdal has positive reinforcing with dose dependent maner in CPP test and reinforcing properties of OT

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may be owing to the modulation of the mesolimbic dopaminergic system ( MLDS) (43). It is

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demonsterated that the MLDS has a significant role concerninig reward processing mechanisms.

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Some investigations revealed a strong assosciation between dopaminergic systems and oxytocinergic. D2-D4 receptors of DA were positioned in the oxytocinergic neurons in the

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medial preoptic zone, paraventricular and supraoptic nucleus of hypothalamus(38). A study indicated that intracerebroventricular use of OTR antagonist results in reduction of DA agonist

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that influced DA release in the nucleus accumbens (44). Based on some studies, intraamygdaloid use of OT can increases the DA and dopamine, 3,4-dihydroxyphenylacetic acid

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(DOPAC ,main metabolite of DA) levels not only in the nucleus accumbens but also in the prelimbic medial prefrontal cortex (45). Some researches have mentioned a considerable

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decrease in the density of DA receptors of METH addiction in many brain regions. Furthermore, low levels of D2 receptors are known as a consequence of heavy and extended METH use (46,

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47). Indeed, toxic METH effects can be inhibited by antagonism of DA receptors. METHinduced behavioral sensitization is prevented by D1 and D2 receptor antagonists (48). Berberine

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has been shown as an antagonist in D1, D2-like receptors and its suppressed DA biosynthesis in the brain (49, 50). According to some studies, pre-treatment of berberine diminishes the

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development of locomotor effects in response to cocaine via modulating DA yeilding in the ventral tegmental area (VTA). It is demostrated that the mesolimbic system from the VTA to the NAc mediates the behavioral and reinforcing activity of cocaine (51). Considering the results of the present study, berberine can diminish anxiety-like behaviors. Althougth the major reason of the mechanism of berberine to induce these effects is unclear, one possible mechanism for this phenomenon is that the hypothalamic–pituitary–adrenal axis is considerably influenced by berberine. Corticotrophin-releasing factor (CRF) is the one of the major determining agents for dysphoria during drug withdrawal. CRF antagonists shows a preventive effects on both drug-seeking

ACCEPTED MANUSCRIPT behaviors and withdrawal-induced anxiety in rodent models (52). OT majorly inhibits the CRFmediated activation of the forebrain and hypothalamic pituitary adrenal axis (9, 53). Moreover, OT shows strong antidepressant-like and anxiolytic effects (20, 54). Another investigation on rats report that berberine can inhibit the enhancement of hypothalamic CRF expression after chronic morphine withdrawal (55). Hence, berberine leads to not only anti-anxiety effects directly via prevention of the hypothalamic CRF but also indirectly via increased OT.

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According to our results, berberin can attenuate rewaerd effect of METH. One possible

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mechanism is that berberin has ablility to block NMDA receptor. Considereing two previous

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studies, the NMDA receptors have been strongly implicated in the rewarding effects of METH and antagonists of these receptors have shown to block rewarding effects of METH (56, 57).

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Previous biochemical research have reported that the DA release is regulated by glutamate and NMDA receptors (58). Other study mentioned that drug-induced reinstatement of place

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preference may be closely associated to glutamatergic neurotransmission and independent of DA (59).

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NMDA receptor antagonism inhibits CPP caused by METH in mice. In addition, it was reported that berberine decreases the binding of NMDA receptors and inhibits NMDA receptor channel

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current in the brain (26). So, berberine may contribute to NMDA receptors regulation in the

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rewarding effects induced by METH.

Conclusion

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The authors from the current results can be concluded that berberine via modulation of OTR diminish relapse, drug preference and anxiety-like behaviors and it may be introuduced as a

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novel potential agent for the treatment of METH addiction.

Conflicts of interest: The authors declare that there are no conflicts of interest.

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Fig. 1: Methamphetamine consumption (A) and methamphetamine preference (B), in control, addicted and addicted treatment groups animals during a withdrawal period of 6 th weeks. Significantly different between addicted group with Control groups: • (P< 0.05), •• (P< 0. 01), ••• (P<0.001). Significantly different between addicted group with addicted treatment groups ♦ (P< 0.05), ♦♦ (P< 0. 01).

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Fig. 2: Total distance (A) , % C/T time spent (2B) and % C/T cross in open field(C) in control, addicted and addicted treatment groups animals at the end week 5 )after withdrawal from methamphetamine. Significantly different between addicted group with Control groups • (P< 0.05). Significantly different between addicted group with addicted treatment groups ♦(P< 0.05).

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Fig. 3: Open-Field Diagram in control, addicted and addicted treatment groups animals at the end week 5 )after withdrawal from methamphetamine).

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Fig. 4: Number entries in open arm and Figure (A), Time spent in open arm and Figure (B): % open arm time to total time spent in open arm elevated plus maze in open arm(C) at the end week 5 (after withdrawal from methamphetamine) in control, addicted and addicted treatment groups. Significantly different between addicted group with Control groups: • (P< 0.05), •• (P< 0. 01). Significantly different between addicted group with addicted treatment groups ♦(P<0.05).

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Fig.5: Elevated Plus Maze Diagram in control, addicted and addicted treatment groups animals at the end week 5 ) after withdrawal from methamphetamine). The arms A and C are open and the arms B and D are closed.

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Fig.6: Percentage of oxytocin receptor in accumbence in control, addicted and addicted treatment groups animals at the end week 5 )after withdrawal from methamphetamine). Significantly different between addicted group with Control groups •• (P<0. 01). Significantly different between addicted group with addicted treatment groups ♦♦ (P<0. 01).

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Fig.7: Photomicrographs of immunofluorescence staining of NAC Oxytocine receptor. A: Nuclei stained by PI. B:Primary antibody to oxytocin receptor. C: Merge PI positive cell. Magnification: 400×

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the end week 5 )after withdrawal from methamphetamine). between addicted group with Control groups••• (P<0.001. Significantly different between addicted group with addicted treatment groups ♦♦♦ (P<0.001).

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Fig.9: Photomicrographs of immunofluorescence staining of hippocampus Oxytocine receptor. A: Nuclei stained by PI. B:Primary antibody to oxytocin receptor. C: Merge PI positive cell. Magnification: 400×

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1- Berberine treatment decreased METH preference in addicted rat. 2- Berberine treatment decreased anxiety-like behaviors in addicted rat 3- Berberine treatment increased numbers of oxytocin receptors in the accumbens and hippocampus

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in addicted rat