Phencyclidine (PCP) and PCP derivatives are dissociative anesthetics of the arylcyclohexylamine class and they are used clinically in animals and humans as general anesthetics [1
]. Like other arylcyclohexylamines, PCP and PCP derivatives act as potent N
-methyl-d-aspartate glutamate receptor (NMDAR) inhibitors [3
], but they also work as a dopamine (DA) reuptake inhibitor [5
]. The latter effect of PCP and PCP derivatives contributes to increases of synaptic DA levels in the medial frontal cortex and striatum [5
], leading to a euphoric state. Due to the mind-altering effects of PCP and PCP derivatives, they have been sold for recreational and non-medical uses in illicit markets [2
]. In addition, the recreational and non-medical uses of PCP and PCP derivatives have emerged as a major problem because they cause severe adverse effects including abuse, trance-like ecstatic states, hallucinations, and violent behavior in humans [2
]. For these reasons, most countries control the use of PCP and PCP derivatives based on their abuse potential and hallucinogenic effects in humans [1
In order to circumvent legal restrictions for the abuse of PCP and PCP derivatives, a variety of novel PCP derivatives such as 4-methoxyphencyclidine (4-MeO-PCP) and 3-methoxyphencyclidine (3-MeO-PCP) have been newly synthesized and sold on global illicit drug markets [7
]. Based on structure–activity relationship studies, these novel PCP derivatives which are structurally similar to PCP also have the potential to cause neurobiological and psychopharmacological effects in vitro and in vivo [9
]. Addition of fluorine (a chemical element) to ring structures of drugs can influence the metabolism and distribution of the original drug molecules in the body and can dramatically change their biological activities [11
]. Recently, a study demonstrated that novel fluorinated PCP derivatives also have a high binding affinity to NMDAR in vitro and pharmacological efficacy in vivo [12
]. These findings suggest that fluorinated PCP derivatives may have the potential to induce euphoric effects and they could be illegally abused like other PCP derivatives. A kind of novel fluorinated PCP derivative, 1-(1-(4-fluorophenyl)cyclohexyl)piperidine (4′-F-PCP), was first synthesized in 2002 [13
]. Based on the pharmacological effects of other novel PCP derivatives, it seems that 4′-F-PCP has the potential to act as a bioactive molecule in the brain [11
], but the neurobiological and psychopharmacological effects of 4′-F-PCP have not yet been fully characterized.
The rewarding and reinforcing properties of psychostimulants are closely associated with the mesolimbic dopaminergic system, projecting from the ventral tegmental area (VTA) to the nucleus accumbens (NAc). In addition, the rewarding and reinforcing properties of psychostimulants such as cocaine, methamphetamine, and PCP have been well demonstrated through various behavioral assessments including open-field test, conditioned place preference (CPP), and intravenous (i.v.) drug self-administration paradigms [14
]. In previous studies, PCP and PCP derivatives produced robust increases in locomotor activity [17
] and CPP in rodents [18
]. Moreover, PCP and PCP derivatives were significantly self-administered in rodent and primate animal models [18
]. These findings suggest that PCP and PCP derivatives not only cause psychotomimetic effects [2
], but also produce psychological dependence with chronic use, due to their reward potential. However, there is currently no scientific evidence for the abuse potential of 4′-F-PCP.
Therefore, in this study, we demonstrated the abuse potential of 4′-F-PCP using multiple behavioral assessments and the mechanisms underlying addictive behaviors. We performed the open-field test to determine the psychomotor effects of 4′-F-PCP and the CPP test to examine the rewarding properties of 4′-F-PCP in mice. In addition, to determine the reinforcing effects of 4′-F-PCP, we performed self-administration studies under fixed ratio (FR) and progressive ratio (PR) schedules of reinforcement in rats. Finally, we evaluated the expression of DA-related proteins and extracellular signal-regulated kinase (ERK), cyclic AMP response-element binding protein (CREB), and Fos-family proteins in the NAc, a primary brain region mediating reward or reinforcing behavior, of 4′-F-PCP-self-administered rats.
In the present study, we demonstrated for the first time the abuse potential of 4′-F-PCP. The results showed that 4′-F-PCP significantly (1) increased locomotor and rotational activities in mice; (2) produced a drug-paired place preference in mice; (3) increased number of active lever-pressing responses and drug infusions under the FR and PR schedules of reinforcement in rats; (4) decreased the expression of DAT in the NAc of 4′-F-PCP-self-administered rats; (5) enhanced the expression of DAD1R in the NAc of 4′-F-PCP-self-administered rats; and (6) enhanced the expression of pERK, pCREB, c-Fos, FosB, and ΔFosB in the NAc of 4′-F-PCP-self-administered rats. Taken together, our findings suggest that 4′-F-PCP has an abuse potential, given its robust psychomotor, rewarding, and reinforcing properties, in part via the activation of DAergic neurotransmission and its downstream signaling pathways in the reward system of rodents.
Drug-induced increases in psychomotor activities are closely related to hyperstimulation of the mesolimbic DAergic reward system [26
]. Previous studies reported that administration of PCP or PCP derivatives increased locomotor and rotational activities in a dose-dependent manner [17
], and its psychomotor effects were blocked by antagonism of DAD1R activity [17
]. Similarly, administration of ketamine or methoxetamine, a kind of PCP-like substance, also increased psychomotor responses in a dose-dependent manner in rodents [29
]. Consistent with these findings, our results demonstrated that 10 mg/kg 4′-F-PCP administration, but not 1 or 3 mg/kg, significantly increased locomotor and rotational activities in mice. Taken together, these findings suggest that 4′-F-PCP is a potent psychoactive drug and produces psychobehavioral effects in a dose-dependent manner.
The CPP is another classic animal model used to evaluate the rewarding effect of contextual stimuli associated with exposure to addictive drugs [14
]. Previous studies demonstrated that repeated administration of PCP or PCP derivatives produced a positive preference [18
]. Consistent with these findings, our results demonstrated that 4′-F-PCP at a dose of 10 mg/kg produced a significant increase in preference in mice. Taken together, these findings suggest that 4′-F-PCP has a rewarding effect like PCP and other PCP derivatives. It is well-known that the PCP-induced rewarding effect is closely related to activation of the mesolimbic DAergic system [19
]. The structure–activity relationship studies demonstrated that PCP derivatives can act as DA reuptake inhibitors due to their structural similarity to PCP, thereby increasing extracellular DA levels in the brain [9
]. For example, Abiero et al. demonstrated that 4-MeO-PCP and 3-MeO-PCMo, which are structurally similar to PCP, increased DA concentration in the NAc of mice [18
]. Based on these findings, we speculate that 4′-F-PCP enhances psychomotor activities and produces the preference via increased DA levels in the NAc.
I.v. self-administration in rodents is a useful model for predicting the abuse liability of novel drugs in humans [15
]. Previous preclinical studies have demonstrated a dose–response relationship for the rate of PCP self-administration and level of drug intake in rats and monkeys under FR schedules of reinforcement [21
]. In addition, PCP derivatives such as 4-MeO-PCP, 3-MeO-PCMo, and ketamine were also self-administered in rats via activation of DAergic neurotransmission in the NAc [18
]. Consistent with these findings, our results demonstrated that 4′-F-PCP at a high dose of 1.0 mg/kg/infusion, but not 0.1 and 0.3 mg/kg/infusion, significantly increased the active lever-pressing responses for drug taking under FR1 and FR2 schedules of reinforcement. Taken together, these findings suggest that only a high dose of 4′-F-PCP can pharmacologically act as a positive reinforcer in the brain reward system, producing reinforcing effects in rats.
The progressive schedule of reinforcement in self-administration paradigms has been used to directly measure the strength of reinforcement (i.e., how hard the animal will work) of psychostimulants with abuse potential by increasing the response requirement for successive reinforcements [32
]. Previous self-administration studies demonstrated that various psychoactive drugs such as cocaine, PCP, and PCP derivatives showed a positive dose–response relationship under a PR schedule of reinforcement [18
]. Consistent with these findings, our results demonstrate that 1.0 mg/kg/infusion 4′-F-PCP, but not 0.1 or 0.3 mg/kg/infusion, self-administration under the PR schedules following the FR schedules significantly increased drug-taking behavior. The breakpoint of 1.0 mg/kg/infusion 4′-F-PCP was similar to those of 0.3 mg/kg/infusion 4-MeO-PCP and 3-MeO-PCMo, which are new PCP-derivative dissociative drugs [18
]. Moreover, the reinforcing efficacy of PCP derivatives varies from derivative to derivative [35
]. In particular, the addition of fluorine to the ring structure of a drug alters its psychopharmacological effects and biological activities [11
]. Based on these results, it could be speculated that a fluorine substitution in PCP also produces a relatively strong reinforcement, like other PCP derivatives, in a dose-dependent manner.
In general, a positive reinforcer (i.e., drugs) is provided to animals when they successfully accessed the drug-paired lever (active lever) in self-administration studies [15
]. However, unexpectedly, we found that number of inactive lever responses (not paired with drug) were also significantly increased compared to that of the saline control group during the FR2 (9th and 10th sessions) and PR schedules of reinforcement at 1.0 mg/kg/infusion 4′-F-PCP-self-administered rats. However, upon advanced analysis, the results demonstrated that the response ratio of active lever presses was over 74.1% during the FR2 schedules (8th session: 76.4%; 9th session: 68.1%; 10th session: 77.6%) and over 90.7% during the PR schedules. Based on the results of lever preference in the 1.0 mg/kg/infusion 4′-F-PCP group, the response to the drug-paired lever was well-reinforced in the 4′-F-PCP-self-administered rats. In reference to previous studies, administration of PCP or PCP derivatives commonly induces hallucinogenic effects and loss of balance with staggering behavior [1
]. Consistently, 4′-F-PCP (1.0 mg/kg/infusion) self-administered rats in this study showed staggering behavior during the FR2 and PR schedules of reinforcement. Taken together, it could be thought that non-specific responses to inactive lever were due to staggering behavior caused by 4′-F-PCP self-administration.
TH and DAT are well-known as modulators of DA concentrations in the reward system. TH, a rate-limiting enzyme, is involved in synthesis of DA, and DAT controls DA concentrations in the synaptic cleft and neurotransmission via reuptake of DA into the presynaptic terminals [38
]. Our results demonstrated that 4′-F-PCP self-administration decreased DAT expression in the NAc, but the expression of TH was not altered. Based on these findings, we speculate that 4′-F-PCP produces a reinforcing effect by inhibiting DA reuptake in DAergic terminals of the NAc, rather than increases in DA synthesis [38
]. However, acute administration of new synthetic PCP derivatives (4-MeO-PCP and 3-MeO-PCMo) or ketamine did not alter DAT expression in the NAc of mice [18
], suggesting that the expression and function of DAT can vary depending on experimental paradigm (type of exposed PCP derivatives, route of administration, dosing period, etc.). Moreover, a previous study reported that exposure to psychoactive drugs such as PCP derivatives and ketamine significantly increased DAD1R expression and decreased DAD2R expression in the NAc [18
]. Accordingly, we found that the DAD1R expression was significantly enhanced in the NAc of 4′-F-PCP-self-administered rats under the PR schedules of reinforcement. However, 4′-F-PCP did not alter DAD2R expression in the NAc. Taken together, these findings suggest that the stimulation of DAD1R rather than DAD2R may play an important role in the psychobehavioral properties of 4′-F-PCP.
ERK, a member of MAP kinase family, is a well-known downstream target of DAD1R-mediated signaling cascades involved in reward and behavioral changes due to drugs such as cocaine, amphetamine, and methamphetamine [40
]. ERK phosphorylation by external stimuli including drug exposure can activate transcription factors such as pCREB, c-Fos, and FosB/ΔFosB in the NAc [40
]. The activation and accumulation of these transcription factors in the NAc are implicated in neuroadaptation to drug abuse [41
]. These findings suggest that the activation of ERK, CREB, c-Fos, and FosB/ΔFosB have been closely linked to drug-induced reward and addiction [40
]. Consistent with these findings, our results demonstrated that 4′-F-PCP significantly enhanced pERK, pCREB, c-Fos, and FosB/ΔFosB levels in the NAc of self-administered rats. Taken together, these findings suggest that activation of ERK–CREB pathway and c-Fos, and FosB/ΔFosB accumulations in the NAc reflect 4′-F-PCP-induced neuroadaptations, which may contribute to the rewarding and reinforcing effects of 4′-F-PCP, leading to abuse.
However, the present study had a limitation in that we only evaluated the 4′-F-PCP-induced alternations in DAergic neurotransmission of the NAc. Others have demonstrated previously that PCP derivatives can affect glutamatergic functions by acting as potent NMDAR inhibitors [3
]; the altered glutamate system also influences dopaminergic neurotransmission in the reward system [23
]. Additionally, other brain regions such as the dorsal striatum, prefrontal cortex, and hippocampus also play important roles in the development of substance abuse [23
]. Thus, further studies are needed to investigate the brain-region-specific involvement of dopaminergic and glutamatergic neurotransmission in 4′-F-PCP abuse.
In summary, we demonstrated that 4′-F-PCP produces psychomotor hyperactivity, place preference, self-administration, and altered expression of DA-related proteins (DAT and DAD1R) and pERK, pCREB, c-Fos, and FosB/ΔFosB in the NAc. In conclusion, 4′-F-PCP produces psychopharmacological properties via activation of DAD1R-mediated neurotransmission in the reward system, providing compelling pre-clinical evidence of its abuse potential in humans. Moreover, these findings have important implications for the development of appropriate drug legislative measures and for predicting the potential for abuse of new synthetic PCP derivatives.