اخبار و مقالات
Heroin Addict Relat Clin Probl 2020; 22(x): xx-xx
HEROIN ADDICTION & RELATED CLINICAL PROBLEMS
Berberis integerrima and berberine attenuate morphine reward memory and restore the reduction of CSF serotonin levels following morphine conditioned place preference in rats
Sadegh Sabeghi1, 2, Mahdi Zahedi-Khorasani1, 2, Ali Ghanbari1, Ali Khaleghian3, Fatemeh Doroodi1,2 and Hossein Miladi-Gorji1, 2
1. Research Center of Physiology, Semnan University of Medical Sciences, Semnan, Iran.
2. Department of Physiology, School of Medicine, Semnan University of Medical Sciences, Semnan, Iran.
3. Department of Biochemistry, School of Medicine, Semnan University of Medical Sciences, Semnan, Iran.
Background: Berberis integerrima is a well-known plant in traditional medicine, and has strong medicinal properties. Methods: The present study was designed to examine the effects of Berberis integerrima and its active constituent, ber- berine, on the morphine-induced conditioned place preference (CPP) and the levels of serotonin in the cerebrospinal fluid (CSF) after an extinction period in rats. In this study, adult male Wistar rats received injections of Berberis integerrima and berberine interperitoneally to allow assessment of the rewarding effects of morphine using a CPP paradigm. CSF serotonin levels were assessed after a 5-day extinction period by using high-performance liquid chromatography (HPLC). Results: The results showed that Berberis integerrima and berberine significantly attenuated the acquisition, expression, extinction and reinstatement of morphine CPP. While Berberis integerrima and berberine alone did not produce saline CPP, rats receiving Berberis integerrima and berberine during the extinction period showed a significantly higher level of serotonin in the CSF. Conclusions: We conclude that Berberis integerrima extract and berberine attenuated memories of morphine reward and lowered the reinstatement of morphine CPP after an extinction period in rats. It also restored the reduction of CSF serotonin levels induced by morphine CPP in rats. It must therefore be concluded that, as an adjunct therapy, Berberis integerrima may offer sunstantial benefits through its capacity to prevent relapse in opiate-addicted individuals.
Key Words: Morphine; Berberis integerrima; Berberine; Conditioned place preference; Serotonin
Conditioned place preference (CPP) is an ex- perimental test for studying the rewarding effects of drugs, including opiates in laboratory animals. In this test, there is a strong association between the reward- ing effects of the drug and the drug-taking environ- ment or drug-associated environmental cues . Re- lapses could occur after drug withdrawal in response to cues expressed in the environment, which may lead to drug-seeking following a period of extinction of drug use. This means that environmental stimuli are able to reinstate CPP , while extinction learning
attenuates that association . The results of CPP showed that serotonergic innervation arising from the dorsal raphe to the nucleus accumbens (NAc) may play an important role in the rewarding effects of acute morphine [7, 38]. It has been shown that mor- phine withdrawal limited dopamine  and seroto- nin (5-HT) transmission . Also, the CPP induced by apomorphine resulted in a fall in caudate and NAc serotonin levels . Relapse after morphine with- drawal is a clinical problem in addicted individuals . Thus, the prevention of drug-induced reinforce- ment and motivation effects by using an experimental test of CPP may prove beneficial in preventing the
restoration of relapse. An accumulating body of evi- dence suggests that medicinal plants are unlimited resources to be drawn on in treating drugs. Differ- ent species of barberry grow in different parts of the world, such as Iran, China, central Asia, and many countries in Europe, Africa, and America. Various parts of the barberry plant, including its root, bark, leaf, and fruit, have shown quite a variety of phar- macological properties, and have been used as a folk medicine for a long time in Iran and other communi- ties . The fruits of barberry shrubs have been used for their dietary and and pharmaceutical benefits . Numerous studies have shown that berberine, as an isoquinoline alkaloid, is responsible for many of the beneficial effects of barberry [16, 21]. Experimental studies have shown the anti-inflammatory , an- tioxidant, , antidiabetic [6, 9], antihypertensive,
vasodilatory , antiallodynic , antinociception
 and memory improvement  properties of bar- berry extract (Berberis integerrima – black barberry and Berberis vulgaris – red barberry). There have been quite a number of other studies showing that both berberine [14, 39] and Berberis vulgaris  are able to attenuate the acquisition and reinstatement of morphine-induced CPP and locomotor sensitization after repeated exposure to morphine. A few studies have investigated the effects of Berberis vulgaris and berberine on morphine CPP in mice. Conversely, the effects of Berberis integerrima have never been investigated previously. Berberis integerrima is a well-known plant in traditional medicine because it possesses strong medicinal properties arising from its high levels of pharmaceutically active ingredients; these include antioxidants, flavonoids, and anti-free radical elements, as well as various alkaloids includ- ing berberine, which may be considered its most im- portant one [3, 6, 32]. Until the present study, the role of Berberis integerrima in being able to determine morphine-induced reinforcement and motivation ef- fects, and in affecting serotonin levels in the cerebro- spinal fluid (CSF) has, however, remained unknown. Aim: In the present study we have examined the ef- fects of Berberis integerrima and its active constitu- ent, berberine, on the acquisition and expression, ex- tinction and reinstatement of morphine-induced CPP and also CSF serotonin levels following the extinc- tion phase in rats displaying CPP. It is important to note that wild samples of black barberry (Berberis in- tegerrima) belong to the Berberidaceae family, which is endemic both in Asia and Europe, but most abun- dantly in the mountainous northern and northeastern regions of Iran .
Adult male Wistar rats (200-250 gr) were housed in cages made subject to a 12-h light/dark cy- cle at 22±24 °C, and had ad libitum access to food and water. The experimental protocol was approved by the Ethical Review Board of Semnan University of Medical Sciences (Semnan, Iran, IR.SEMUMS. REC.2019.305). All of the experimental procedures were conducted in accordance with the National In- stitutes of Health's Guide for the Care and Use of Laboratory Animals, Drugs and Preparation of the Extract.
Morphine sulfate (Temad, Iran) dissolved with saline and injected subcutaneously (s.c.) at a dose of 5 mg/kg. Berberine hydrochloride was purchased from Sigma-Aldrich Co., St Louis, MO. Pure Berberis in- tegerrima powder was purchased from Adonis Gol Darou Co., Iran. According to the instructions pro- vided by the latter, the aqueous extract of Berberis integerrima fruit had been extracted from 100 g of the powder of fruits in boiling water (1,000 ml, for 15 min); it was then filtered, concentrated, and dried in a vacuum. Both the drugs derived from Berberis integerrima and berberine itself were dissolved in sa- line and then injected intraperitoneally (ip) at doses of 100 and 10 mg/kg, respectively, applying the re- sults of a pilot study. In the present study, no adverse effects were observed following the injection of Ber- beris integerrima and berberine.
2.2. Conditioning apparatus and paradigm
The CPP paradigm was used to study the re- warding effects of morphine as described in our labo- ratory results and those of other laboratories . In brief, the specially designed apparatus was made from wood, the main features being two distinct chambers (A and B) (30 cm × 30 cm× 40 cm), separated from each other by a neutral chamber (30 × 15 × 40 cm), and a removable door. Each chamber of A and B had a black background or a white one; the two chambers differed in the pattern displayed, which, however, in both cases consisted of alternating white and black stripes (vertical or horizontal). The time spent in each chamber and the distance travelled to the condition- ing chambers were recorded by a 3CCD camera (Pa- nasonic Inc., Japan), which was placed above the CPP box. Both parameters were analysed using Ethovision software – a video tracking system for the automation
of behavioural experiments. The CPP paradigm con- sisted of a 5-day schedule with three distinct phases: pre-conditioning, conditioning and post-condition- ing, in all three cases followed by extinction and rein- statement phases.
2.2.1. Pre-conditioning phase
On day 1, each rat was allowed access to all three chambers in a drug-free state for a total of 10 min. Time spent in each chamber and the rat's move- ments were recorded (Pre-test day). Animals were then randomly assigned to one of the two chambers for place conditioning.
2.2.2. Conditioning phase
In this phase, rats received either morphine or equal volumes of saline in one or other chamber on days 2, 3, and 4, and placed in the A or the B chamber (randomly) for 45 min immediately after injection, so that half of the rats received the drug in chamber A and the other half in chamber B. The conditioning tri- als were carried out twice per day with an interval of 6 h for saline-morphine pairing in an alternated morn- ing/afternoon design.
2.2.3. Post-conditioning phase
This phase was carried out on day 5 in a drug- free state as a conditioning test. The removable door was removed and each rat was allowed access to the entire apparatus for 10 min. The time spent in each chamber was recorded for each rat. The conditioning score was calculated as the time spent in the drug- paired chamber minus the time spent in the saline- paired chamber. Total distance travelled for each ani- mal was also considered as the locomotor activity for each study group.
2.3. Experimental protocols
We first evaluated the effect of each dose of Ber- beris integerrima and berberine alone during condi- tioning days on place preference in the saline CPP. Rats were divided into three groups (n=7): Saline- Saline, Berberis integerrima-Saline and Berberine- Saline. On conditioning days, the rats received daily ip injections of saline, Berberis integerrima and ber- berine, and were placed in the CPP apparatus for 45 min immediately after saline injection. On day 5, rats were exposed to the CPP test without any injection, and the saline CPP score was recorded for 10 min. The following experiments were then conducted.
2.3.1. Experiment 1: Effects of Berberis integerrima and berberine on the acquisition of morphine-induced CPP
At first rats were divided into three groups (n=8): control (Saline-treated rats), Berberis inte- gerrima-treated rats and berberine-treated rats. On conditioning days, the rats received daily ip injec- tions of saline, Berberis integerrima and berberine 30 min prior to morphine injection. Rats were placed in the CPP apparatus for 45 min immediately after mor- phine injection. On day 5, rats were exposed to the CPP test without any injection, and both CPP score and distance travelled were recorded for 10 min (Fig- ure 1A).
2.3.2. Experiment 2: Effects of Berberis integerrima and berberine on the expression of morphine-induced CPP
In this experiment, three groups of rats (n = 8 for each group) received ip injections of saline, Ber- beris integerrima and berberine 30 min prior to the conditioning test (held on day 5) and CPP scores and distance travelled were recorded for 10 min (Figure 1B).
2.3.3. Experiment 3: Effects of Berberis integerrima and berberine during the extinction period on the mainte- nance of morphine-induced CPP and the CSF serotonin levels
In this experiment, three groups of rats (n = 8 for each group) received ip injections of saline, Ber- beris integerrima and berberine 30 min prior to saline injection on days 6, 7, and 8 during the extinction pe- riod, and were placed in the drug-paired compartment for 45 min immediately after saline injection. The rats then allowed to rest for 9-10 days without any injec- tions. They were then tested for the CPP score and distance travelled for a 10-min period without any in- jection on day 11 (Extinction test) (Figure 1C).
To investigate the levels of serotonin in the CSF, a further group of rats with the function of saline- treated control group, was given saline injection and placed in the CPP apparatus, without CPP trials or testing.
Immediately after the assessment of extinction (on day 10), the CSF was drawn from cisterna mag- na, as described previously  in anaesthetized rats. The sample was collected in a microtube and kept at
-80°C until serotonin assay. Analysis of the samples was performed by high-performance liquid chroma- tography (HPLC) with the Agilent 1200 Series LC System, as described previously .
2.3.4. Experiment 4: Effects of Berberis integerrima and berberine on the reinstatement of morphine-induced CPP
After a 5-day extinction period and an extinc- tion test, three groups of rats (n = 8 for each group) received ip injections of saline, Berberis integerrima and berberine 15 min prior to the ip injection of an in- effective dose of morphine (1 mg/kg) on day 12 in our
laboratory, as described previously . The groups of rats were then tested 15 min after morphine injection for the CPP score and distance travelled for a 10-min period (Figure 1D).
2.4. Data analysis
Data are expressed as the mean ± SEM. Statisti- cal analyses were performed using one-way ANOVA followed by post-hoc analyses, including Tukey's test for multiple comparisons and, in some cases, includ- ing the Paired sample t-test. Statistical differences were considered significant at P<0.05.
The results of the effects of Berberis integerrima and berberine on the acquisition of saline-induced CPP in rats are illustrated in Figure 2. One-way ANOVA revealed no significant differences between the groups as regards the score and distance travelled during pre-conditioning and post-conditioning. The
results showed that rats receiving Berberis integerri- ma and berberine showed no place preference in the saline CPP for each during the acquisition other than pre-conditioning score, using the paired sample t-test.
3.1. Berberis integerrima and berberine attenuated the acquisition and expression of morphine- induced CPP in rats.
The results of the acquisition and expression of morphine-induced CPP are illustrated in Figure 3A and 4A. A one-way ANOVA revealed a signifi- cant group effect for the acquisition (F2, 21=21.99, P=0.0001) and expression (F2, 21=39.22, P=0.0001)
phases. Rats receiving Berberis integerrima and ber- berine showed a decrease in place preference for morphine both during its acquisition (in both cases,
P=0.0001) and expression (in both cases, P=0.0001) phases than saline-treated (control) rats. It did not af- fect the pre-conditioning score (Figures 3A and 4A) or the distance travelled (Figsures 3B and 4B) in the acquisition and expression phases of morphine- induced CPP. Thus, Berberis integerrima and berber- ine shortened the acquisition and expression phases of morphine-induced CPP in rats.
3.2. Effects of Berberis integerrima and berberine during the extinction period on the maintenance of morphine-induced CPP and serotonin levels in the CSF
The results of the extinction of morphine- induced CPP are illustrated in Figures 5A and B. One-way ANOVA revealed no significant differences between the groups for the score or the distance trav- elled, whether in the pre-conditioning, post-condi- tioning or extinction phases. The difference between the conditioning scores for the post-conditioning and then the extinction phases was calculated by applying the Paired sample t-test. The results showed that all three groups of rats receiving saline (P=0.0001), Ber- beris integerrima (P=0.001) and berberine (P=0.001), respectively, showed a greater fall in place preference for morphine during the extinction phase than during the post-conditioning phase. In this way, a 5-day ex-
tinction period extinguished morphine-induced CPP in all three groups of rats.
One-way ANOVA revealed a significant group effect for the CSF serotonin levels (F3, 26=10.79, P=0.0001) during the extinction of morphine-induced CPP (Figure 6). Post hoc comparisons showed that serotonin levels in the CSF were lower in morphine/ saline-treated rats than in saline/saline ones during the extinction phase of morphine-induced CPP (P=0.045). Rats receiving Berberis integerrima (P=0.0001) and berberine (P=0.008) showed an increase in the CSF serotonin levels than morphine/saline-treated rats. Also, rats receiving Berberis integerrima showed an increase in the levels of serotonin in the CSF than sa- line/saline-treated ones (P=0.034). Thus, during the extinction phase, Berberis integerrima and berberine restored the lowering of the levels of serotonin in the CSF arising from a morphine-induced CPP in rats.
3.3. Berberis integerrima and berberine following morphine challenge partly attenuated the reinstatement of morphine CPP
The results of the reinstatement of a morphine- induced CPP are illustrated in Figures 7A and B. One-way ANOVA revealed no significant differ- ences between the groups for the scores recorded for the pre-conditioning, post-conditioning and extinc-
tion phases, but there was a significant group effect in achieving the reinstatement of morphine-induced CPP (F2, 21=22.43, P=0.0001). In addition, there were no significant differences between distances travelled during the process of morphine CPP, as shown in Figure 7B.
Post hoc comparisons showed that rats receiving Berberis integerrima and berberine experienced a fall in place preference for morphine following morphine challenge than control rats (in both cases, P=0.0001). The difference between the scores for post- conditioning and extinction and, using the same technique, the difference between the scores for ex- tinction and reinstatement were compared by apply- ing the Paired sample t-test. All three groups of rats receiving saline, Berberis integerrima and berberine, respectively, showed a fall in place preference for morphine during the extinction than the post-condi- tioning phase (all, P=0.0001). Saline-treated (control) rats showed a greater increase in place preference for morphine following morphine challenge than control rats in the reinstatement phase (P=0.0001), but rats receiving Berberis integerrima and berberine during the reinstatement phase showed a lower morphine-in- duced CPP compared with their own reference group in the extinction phase (in both cases, P=0.001). Thus, during the reinstatement phase, Berberis integerrima and berberine showed a lower morphine-induced CPP
following morphine challenge.
The results of our study indicated that the in- jection of Berberis integerrima and berberine before morphine injection during conditioning trials lowered the acquisition of morphine-induced CPP. Also, prior to the conditioning test, a single injection of Berberis integerrima and berberine reduced the expression of morphine-induced CPP. In addition, neither Berberis integerrima nor berberine alone produced any place preference in the saline CPP group. As a result, the rewarding effects of morphine could merely be at- tenuated by the Berberis integerrima and berberine. Our finding is in accordance with previous studies showing that the injection of berberine decreased the acquisition and expression of CPP induced by ethanol
 and morphine  in mice. This study provides new evidence for an active role of Berberis integerri- ma in determining morphine-induced CPP. A previous study, however, showed that the injection of aqueous extract of Berberis vulgaris (red barberry) lowered the acquisition of morphine-induced CPP . Our
findings indicated the negative effects of Berberis integerrima on the motivational and rewarding prop- erties of morphine. From existing data, it seems that these effects are exerted through berberine, given that berberine is one of the main alkaloids (if not the most important of them) present in Berberis integerrima . The mechanism by which Berberis integerrima and/or berberine attenuated the morphine-induced CPP in the present study is not clear. Previous studies have shown that the brain-derived neurotrophic fac- tor (BDNF) synthesized in the dopaminergic neurons
 and an increase in BDNF expression may be in- volved in morphine-induced reward memory . On the other hand, it was shown that both BDNF release
 and the expression of morphine CPP  are mediated by mesolimbic, NMDA-type glutamate re- ceptors. It has also been shown that berberine lowered BDNF mRNA expression  and inhibited NMDA receptors . Thus, this effect of Berberis integerri- ma and/or berberine on morphine CPP may be due to their inhibitory effects on BDNF expression and NMDA receptors.
At this point it should be noted that no signifi- cant differences emerged between the distances trav- elled during the process of morphine CPP, so indi- cating that neither Berberis integerrima extract nor berberine had any effect on motor activity. Thus, the greater preference for morphine detected in rats ex- hibiting CPP was not directly due to an increase in motor activity.
We found that a 5-day extinction period extin- guished morphine-induced CPP in all three groups of rats receiving saline, Berberis integerrima and berber- ine, respectively. The same declining trend was ob- served in the extinction of CPP, showing that a period of 5 days was enough to extinguish the reward mem- ory. Despite these findings, it cannot be concluded that the administration of Berberis integerrima and/ or berberine was ineffective in reducing morphine CPP. An additional experiment should now be con- ducted to verify data that are still uncertain. Also, we found that rats showing morphine-induced CPP, and receiving Berberis integerrima and berberine during the extinction period, displayed a greater increase in the levels of serotinin in the CSF than others show- ing morphine CPP but receiving only saline. What is more, we have observed that rats exhibiting morphine CPP after a 5-day extinction period showed a fall in CSF serotonin levels, which is consistent with previ- ous studies showing that morphine withdrawal , the extinction of morphine-induced CPP and induc- tion of sensitization  lowered 5-HT activity. It fol-
lows that Berberis integerrima and berberine during the extinction period successfully restored the reduc- tion of serotonin levels in the CSF due to morphine- induced CPP in rats. Our findings support the previ- ous conclusion that berberine increased 5-HT levels in the hippocampus and frontal cortex . Berberis integerrima, unlike berberine, after a period of extinc- tion showed higher levels of serotonin in rats display- ing morphine-induced CPP than saline/saline-treated ones, so showing that other biologically active com- pounds besides berberine must be involved in Berb- eris integerrima – an issue that now calls for further investigation.
Another interesting finding in our study was that a single injection of Berberis integerrima and/or ber- berine before morphine challenge produced a sharper fall in the conditioning score than that in control rats, which is consistent with previous results showing that Berberis vulgaris  and berberine  prevented the reinstatement of morphine-induced CPP.
Although the underlying mechanisms are not well known, it seems that the inhibition of dorsal raphe-5-HT neurons [23, 35] and the higher concen- trations of the corticotropin-releasing factor (CRF)
 could trigger the reinstatement of drug-seeking behaviour and may increase vulnerability to drug re- lapse. It is noteworthy that an increased 5-HT, though dependent on the receptor subtype, exerts its effects . It seems that higher levels of serotonin in rats receiving Berberis integerrima and berberine during the extinction period may attenuate the reinstatement of morphine CPP after morphine challenge. Previous studies showed that berberine raised 5-HT levels , while lowering hypothalamic CRF expression . It is also more likely that the effects of Berberis inte- gerrima are exerted through the mechanisms men- tioned above, which now need further investigation.
An additional finding was that the conditioning score after morphine challenge was still higher than after a period of extinction, indicating that a single in- jection of Berberis integerrima and/or berberine in the reinstatement test partly attenuated the reinstatement of morphine CPP induced by a priming injection in rats. It is possible that a single injection of Berberis integerrima and berberine was insufficient to achieve further attenuation of CPP after morphine challenge. Clearly, new research methods are now needed to ex- plore this effect.
This study provides novel evidence that Berberis integerrima extract and berberine attenuated the ac- quisition, expression, maintenance and reinstatement of morphine CPP after a 5-day extinction period in rats. Besides this discovery, an increased CSF sero- tonin level following the taking of Berberis integerri- ma extract and berberine is probably responsible for the reduction of morphine reinstatement. Given the medicinal, health benefits and nutritional values of Berberis integerrima, it must be concluded that this naturally occurring herb may prove to offer a useful adjunctive therapeutic strategy for preventing relapse in opiate-addicted individuals. This suggestion obvi- ously requires further research.
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We would like to thank the Research Centre of Physi- ology, Semnan University of Medical Sciences, Semnan, Iran for its support.
Role of the funding source
The author(s) disclosed receipt of the following fi- nancial support for the research, authorship, and/or pub- lication of this article: This work was supported by grants from Semnan Medical Sciences Universities (Semnan, Iran, 1537). This article is part of the thesis of Mr. Sadegh Sabeghi for the degree of Master of Sciences in Physiol- ogy.
All authors were responsible for the study design. S.S, A.G, A.K and F.D performed the experiments. H.MG and M.ZK, supervised the project. H.MG analysed the data and wrote the manuscript. All authors reviewed and then approved the definitive version of the manuscript.
Conflict of interest
All authors have no conflict of interest.
Authors confirm that the study submitted was con- ducted according to the WMA Declaration of Helsinki – [Come sempre, è un dash, non un trattino.] Ethical Princi- ples for Medical Research Involving Human Subjects. The study has received IRB review/approval.
It is the policy of this Journal to provide a free re- vision of English for Authors who are not native English speakers.
Received September 23, 2020 - Accepted November 30, 2020