Author for correspondence: Esa Meririnne, Drug Research Unit, Department of Mental Health and Alcohol Research, National Public Health Institute, Mannerheimintie 166, FIN 00300 Helsinki, Finland (fax +358 9 4744 8553, e-mail email@example.com).
Abstract: 1-Benzylpiperazine (also known as ‘Legal X’, ‘Legal E’, or ‘A2’) is a psychoactive compound increasingly encountered on the clandestine market. Previous experimental data suggest that the compound possesses addictive properties. In the present study, we used the conditioned place preference method in the rat to test whether 1-benzylpiperazine possesses rewarding properties. Furthermore, the mechanisms of the 1-benzylpiperazine reward were investigated using selected dopamine and serotonin receptor antagonists. 1-Benzylpiperazine (1.25, 5, and 20 mg/kg) induced dose-dependently place preference. This place preference was attenuated by the antagonists SCH23390 (0.2 mg/kg; dopamine D1-like receptors) and MDL72222 (1.0 mg/kg; serotonin3 receptors), but not by raclopride (0.8 mg/kg; dopamine D2-like receptors) or ketanserin (2 mg/kg; preferentially serotonin2 receptors). Our results show that 1-benzylpiperazine possesses rewarding properties in the rat, which suggests the compound to be susceptible to human abuse. The brain dopaminergic and serotonergic systems appear to be involved in the 1-benzylpiperazine reward.
1-Benzylpiperazine (1-benzyl-1,4-DIAZA-cyclohexane, also known as ‘Legal X’, ‘Legal E’, or ‘A2’) is a psychoactive compound increasingly encountered on the clandestine market. The compound is actively marketed on drug-culture related websites, where users' experiences and opinions regarding its effects, dosage, and intake manners can be found (e.g., htpp://www.erowid.org or http://www.lycaeum.org). In Europe, 1-benzylpiperazine has been found in capsules sold as ‘a synthetic stimulant’ (de Boer et al. 2001). A fatal brain oedema after the intake of 1-benzylpiperazine with ecstasy in Switzerland, and several cases of abuse in Sweden has been reported (Balmelli et al. 2001; Wikström et al. 2004). In the U.S., nearly 18,000 tablets and 600 kg powder of 1-benzylpiperazine have been confiscated since the year 2000 (Drug Enforcement Administration 2004). Although 1-benzylpiperazine has been recently placed under statutory control at least in Sweden and the U.S. (Drug Enforcement Administration 2004; Wikström et al. 2004), it is still legal and readily available in several countries.
Experimental data show that there are notable similarities between 1-benzylpiperazine and psychostimulant-like drugs of abuse. 1-Benzylpiperazine's mechanism of action in the rat brain involves the elevation of extracellular dopamine and serotonin (DA and 5-HT, respectively) levels, which is a common feature for psychostimulants such as amphetamine and cocaine (Baumann et al. 2005). In line with this, a recent study with Rhesus monkeys shows that 1-benzylpiperazine shares a discriminative stimulus with that of amphetamine and, when administered alone, it can maintain self-administration (Fantegrossi et al. 2004). In former addicts 1-benzylpiperazine and dexamphetamine yielded indistinguishable subjective effects, and both drugs were considered desirable (Campbell et al. 1973).
Taken together, the accumulating evidence suggests that 1-benzylpiperazine could possess addictive properties. We studied here whether it possesses rewarding properties using the place preference method in the rat. Compounds prone to human abuse are known to be capable of inducing place preference (Carr et al. 1989; Tzschentke 1998). Furthermore, given 1-benzylpiperazine's mechanism of action, the involvement of the DA- and 5-HTergic systems in the 1-benzylpiperazine reward was examined using the receptor antagonists SCH 23390 (DA D1-like receptors; Bourne 2001), raclopride (DA D2-like receptors; Hall et al. 1988), MDL 72222 (5-HT3 receptors; Hoyer et al. 1994), and ketanserin (preferentially 5-HT2 receptors; Hoyer et al. 1994; Jerman et al. 2001).
Materials and Methods
Animals. Adult male Han:Wistar rats weighing 200–300 g were used in the study. The rats were obtained from Harlan Nederland B.V., Horst, the Netherlands, at least 1 week prior to the experiments, and they were housed two per Macrolon III-type cage (18×33×15 cm) in a temperature-controlled room (22±2 °) with a light cycle of 12 hr. The lights were on from 8:00 a.m. to 8:00 p.m, during which time all the experiments were conducted. The animals had free access to standard laboratory chow and tap water. The local institutional animal care and use committee, or the chief veterinarian of the county administrative board approved the experiments, and they were conducted in compliance with the European Communities Council Directive of 24 November 1986 (86/609/EEC).
Conditioned place preference. The place preference experiments were conducted using the apparatus and procedure essentially as described previously (Meririnne et al. 2001 & 2005).
Apparatus. The conditioned place preference test was conducted in eight identical rectangular boxes (60×30×45 cm) made of polyvinyl chloride. Each box was divided into two compartments of equal size by a separating wall with a guillotine door (8×6 cm). Both compartments were covered with loose-fitting transparent plastic lids. One compartment was black with a smooth floor and small drops of 2% acetic acid added in both back corners, and the other was white with wire mesh on the floor and no acetic acid added. Thus, the compartments differed in three modalities: visual, tactile, and olfactory. The boxes were placed in a quiet dim room with white noise present for masking external sounds.
Experimental procedure. The place preference procedure consisted of three phases:
1Preconditioning phase (days 1–3): The guillotine door of the box was open, and the rats were equitably placed in the black or white compartment and left to explore freely both compartments for 15 min. (900 sec.). On the 3rd day the times spent in both compartments were measured with a stopwatch. According to this preconditioning time, the rats were assigned to treatment groups with the less-preferred compartment (preconditioning time <450 sec.) serving as the drug-paired compartment. If the preconditioning time for a rat was less than 180 sec. in either compartment, the rat was excluded from further testing. In each treatment group, approximately half of the rats were assigned to the black compartment as the drug-paired compartment, while the other half were assigned to the white compartment as the drug-paired compartment (±1 rat when n≤10; ±2 rats when n>10).
2Conditioning phase (days 4–6): During the conditioning phase the guillotine door was closed. The rats experienced two conditioning sessions each day. In the 1st session the rats received one or two injections of vehicle, after which they were immediately confined to the vehicle-paired compartment (opposite the drug-paired compartment) for 40 min. After an interval of at least 90 min., the 2nd session of the day began; the rats received one or two injections of drugs or vehicle, after which they were immediately confined to the drug-paired compartment for 40 min.
3Postconditioning phase (day 7): The guillotine door was opened, the rats were placed in the drug-paired compartment, and they were allowed to move freely between the compartments. The time the rats spent in the drug-paired compartment (postconditioning time) was measured for 15 min. Shift in preference for the drug-paired compartment induced by conditioning (postconditioning time – preconditioning time) served as the measure of reward.
Drugs and treatments. 1-Benzylpiperazine was obtained from Fluka Chemie GmbH (Buchs, Switzerland). SCH 23390 hydrochloride (SCH; D 054), raclopride L-tartrate (RAC; R 121), ketanserin tartrate (KET; S 006), and MDL 72222 (MDL; T 102) were obtained from RBI, Natick, MA, USA. 1-Benzylpiperazine, KET, and MDL were dissolved in a small volume of distilled water acidified with a drop of 2 M hydrochloric acid (1-benzylpiperazine and KET) or glacial acetic acid (MDL). The pH was then adjusted as near to physiological level as possible with 2 M NaOH (approximately 7 with 1-benzylpiperazine, and 5 with KET and MDL), and the solutions were brought to volume with distilled water. SCH and RAC were dissolved in saline (0.9% NaCL). Care was taken to prepare the corresponding vehicles exactly in the same way. The doses were calculated as free base, and the drugs were administered intraperitoneally at a volume of 1 ml/kg.
First, we investigated whether 1-benzylpiperazine can induce place preference: the rats received only vehicle or 1-benzylpiperazine at the doses of 1.25 mg, 5 and 20 mg/kg during the conditioning phase. These doses were chosen on the basis of results from our preliminary experiments (Kajos et al. 2003). Second, the involvement of the brain DA- and 5-HTergic systems was evaluated using the respective receptor antagonists. DA D1-like receptor antagonist SCH (0.2 mg/kg) was administered 10 min., DA D2-like antagonist RAC (0.8 mg/kg) 40 min., 5-HT2A antagonist KET (2.0 mg/kg) 60 min., and 5-HT3 antagonist MDL (1.0 mg/kg) 30 min. prior to 1-benzylpiperazine treatment. According to previous studies, these doses and intervals should be behaviourally relevant (Ågmo et al. 1993; Hoffman & Donovan 1995; Herges & Taylor 1998; McMahon & Cunningham 2001; Meririnne et al. 2001; Vickers et al. 2001; Kankaanpääet al. 2002). At this dose the possible affinity of SCH for 5-HT2 receptors appears to be of no significance (Bischoff et al. 1986). KET, which shows relatively good selectivity for 5-HT2A receptors over other 5-HT receptors (Hoyer et al. 1994; Jerman et al. 2001), also has some affinity for α1-adrenergic and H1-histaminergic receptors (the respective affinities being 18 and 9 times lower than that for 5-HT2 receptors; Leysen et al. 1985).
Statistics. Statistical analyses were performed using one-way ANCOVA (dependent factor: shift in preference; independent factor: drug dose; covariant: preconditioning time). Post hoc comparisons were made with Bonferroni's test adjusted for appropriate number of comparisons. Paired and two-sample t-tests were used when appropriate. All data are expressed as mean±S.E.
1-Benzylpiperazine induced a dose-dependent increase in place preference; there was a significant difference between the doses [F(3,33)=7.55, p=0.001, ANCOVA] (fig. 1). At the highest dose of 20 mg/kg the postconditioning time in the drug-paired compartment exceeded the average time between the two compartments (450 sec.; the postconditioning time was measured for 900 sec.); i.e., the initially less-preferred compartment (preconditioning time <450 sec.) had now become the preferred compartment (postconditioning time >450 sec.). Thus, 1-benzylpiperazine is not only capable of inducing increase in preference, but also of causing an actual place preference for the environment paired with drug effects.
The four DA and 5-HT antagonists, or their corresponding vehicles, were tested with the 1-benzylpiperazine dose of 10 mg/kg (fig. 2). When given with vehicle, 1-benzylpiperazine seemed to induce reliably an increase in place preference; in all four groups there was a significant difference between postconditioning and preconditioning times [P<0.005 in all test groups, paired t-test]. DA D1-like receptor antagonist SCH and 5-HT3 antagonist MDL attenuated 1-benzylpiperazine-induced place preference; there was a significant difference between the vehicle or antagonist pretreatment [F(1,16)=6.83, P=0.019 with SCH, and F(1,20)=5.92, P=0.025 with MDL, ANCOVA]. Instead, DA D2-like receptor antagonist RAC and 5-HT2A antagonist KET appeared to be ineffective; in both groups there was a significant difference between the preconditioning and postconditioning times [P=0.001 with RAC, and P=0.011 with KET, paired t-test]. When tested alone, none of the antagonists induced place preference or aversion in our experimental set-up. Because we have demonstrated this previously with SCH, RAC, and MDL (Kankaanpääet al. 2002; Meririnne et al. 2005), it was considered adequate to ascertain their inefficacy with a small number of rats and without a corresponding control group.
In the present study 1-benzylpiperazine induced place preference in the rat, which is a common feature of drugs abused by human beings (Carr et al. 1989; Tzschentke 1998). This is in agreement with previous studies; 1-benzylpiperazine has a mechanism of action in the brain similar to that of psychostimulant drugs of abuse (Baumann et al. 2005), it can maintain self-administration in monkeys (Fantegrossi et al. 2004), its discriminative stimulus is similar to that of amphetamine in monkeys as well as in man (Campbell et al. 1973; Fantegrossi et al. 2004), and finally, it produces subjective and physiological effects comparable to those of amphetamine in human beings (Campbell et al. 1973). Thus, these experimental data together quite congruently suggest that 1-benzylpiperazine is susceptible to human abuse.
The DA D1-like receptor antagonist SCH, though not the DA D2-like receptor antagonist RAC, attenuated 1-benzylpiperazine-induced place preference, which indicates involvement of the brain DAergic system in the 1-benzylpiperazine reward. This is in agreement with the general view on the importance of the DAergic system in psychostimulant reward (Bardo 1998). More specifically, the DAergic mechanisms of the 1-benzylpiperazine-induced place preference seem to be somewhat similar to those of cocaine-like DA uptake inhibiting drugs; but perhaps separable from those of amphetamine-like DA releasing drugs, which induce place preference sensitive to both DA D1- and D2-blockade (Spyraki et al. 1982a & b; Tzschentke 1998). This may be considered unexpected, given the recent findings that 1-benzylpiperazine releases DA from nerve terminals in an amphetamine-like manner (Baumann et al. 2005), though the drug is also reported to affect the DA uptake process (Tekes et al. 1987). While this complex issue awaits further clarification, it is important to note that although only one RAC dose of 0.8 mg/kg was tested in our study, such relatively high doses have been effective previously in our experimental set-up, as well as in other studies (Hoffman & Donovan 1995; Meririnne et al. 2001 & 2005).
The 5-HTergic system appears to be also involved in 1-benzylpiperazine reward; the 5-HT3 receptor antagonist MDL attenuated 1-benzylpiperazine-induced place preference. Our finding augments the previous neurochemical findings showing the 5-HTergic component in 1-benzylpiperazine's mechanism of action (Tekes et al. 1987; Baumann et al. 2005). This action of MDL may be mediated via the DAergic system; 5-HT3 receptors are probably related to controlling the elevated DAergic tone in reward-related brain loci (Chen et al. 1991; Tanda et al. 1995; De Deurwaerdere et al. 1998). In contrast, 5-HT2 receptor blockade by KET did not attenuate the 1-benzylpiperazine-induced place preference. Only one relatively high dose of 2 mg/kg was tested, but comparable doses have previously prevented behavioural effects induced by the 5-HT2A/C receptor agonist 2,5-dimethoxy-4-iodoamphetamine (DOI; Vickers et al. 2001) and cocaine (Herges & Taylor 1998, McMahon & Cunningham 2001); thus, it appears unlikely that the inefficacy of KET could be solely attributed to inadequate dose. Furthermore, when we tested the higher dose of 3 mg/kg alone, this resulted in place aversion (data not shown) thereby ruling out further experiments with these higher doses.
In conclusion, our results show that 1-benzylpiperazine induced place preference in the rat, which indicates that the compound possesses rewarding properties, and thus abuse potential. In view of our data and the previous evidence, it appears advisable to consider placing 1-benzylpiperazine under statutory control. Furthermore, this 1-benzylpiperazine-induced place preference was attenuated by SCH and MDL, though not by RAC or KET, indicating the involvement of the brain DA- and 5-HTergic systems. It is interesting that DA D1-like and 5-HT3 receptors are involved in the 1-benzylpiperazine reward, whereas DA D2-like and 5-HT2 receptors are not. There is no straightforward explanation for this diversity, and the clarification of the complex interplay of neuronal systems behind the 1-benzylpiperazine reward awaits further studies.
The Yrjö Jahnsson Foundation, Helsinki, Finland, supported financially this work.