Introduction

Addiction to several drugs and substances remains a critical health issue worldwide. According to the world drug report, Saudi Arabia had one of the highest reported drug seizure rates in 20111. As stated in several reports, the prevalence of drug addiction is high in Saudi Arabia, as are associated rates of hepatitis, HIV, crime, and socioeconomic decline (for review see2).

Illicit Pregabalin (Lyrica) use has recently become more common among young Saudis and worldwide3,4,5,6,7,8,9,10, likely secondary to lack of appropriate regulation. Pregabalin is an analogue of the neurotransmitter gamma-aminobutyric acid (GABA) that can bind with strong affinity to the α2δ subunit of pre-synaptic voltage-gated calcium channel receptor to decrease post-synaptic excitatory neurotransmitter release11. Pregabalin has a therapeutic value in the management of fibromyalgia, generalized anxiety disorder, diabetic neuropathy, partial epilepsy, and postherpetic neuralgia12,13,14. There remains controversy regarding the abuse potential of pregabalin in preclinical studies. In conditioned place preference (CPP) studies conducted in rats, pregabalin (up to 30 mg/kg) did not cause rewarding effects and did not change place preference15,16. However, these doses were small compared to higher doses used in previous case reports of pregabalin abuse4,5,6,7,8,9. Interestingly, pregabalin has been reported to cause euphoric effects as a side effect in participants of controlled clinical studies17,18,19,20.

Importantly, glutamate is the major excitatory neurotransmitter in the brain. The nucleus accumbens (NAc) is involved in the reinforcing and rewarding effects of several drugs of abuse21,22,23. Disturbances in glutamate homeostasis in the NAc have been shown to be associated with drug seeking behavior and chronic drug use24,25,26,27,28. The release of glutamate from prefrontal cortex (PFC) projections into the NAc has been shown to mediate drug seeking behavior in previous studies29,30. Blocking glutamatergic activation of mGlu131, mGlu532,33, AMPA receptors along with blocking the release of glutamate in the NAc was found to reduce reinstatement of self-administration for different drugs of abuse30,34,35,36. It is noteworthy that glutamate homeostasis is controlled by a number of glutamate receptors and transporters. One of these transporters is glutamate transporter type-1 (GLT-1) which can clear the majority of the synaptic glutamate37,38,39,40. Moreover, treatment with ceftriaxone (CEF), a well-known upregulator of GLT-1 expression, has been shown to oppose drug-seeking behavior induced, in part, by downregulation of GLT-1 following exposure to several drugs of abuse26,41,42,43,44,45. Therefore, in the present study we explored the abuse potential of different doses of pregabalin using CPP and assessed the potential mechanistic role of GLT-1 in pregabalin associated drug-seeking behavior by pretreatment with CEF.

Materials and Methods

Animals

Male BALB/c mice, weighing 25–30 g were obtained from King Fahd Medical Research Center (KFMRC, Jeddah, SA). Mice were housed in standard cages with controlled constant temperature (21 °C) and humidity (30%) on a 12:12 light-dark cycle. Animals had ad libitum intake of water and food. Mice were allowed to habituate for seven days before conducting any experiments. The experimental procedures of animals were ethically approved by the Research Ethics Committee at Taif University, in accordance with the Institutional Animal Care and Use Committee of the National Institutes of Health guide.

Drugs

Pregabalin was generously provided by Jamjoom Pharmaceuticals (Jeddah, KSA). Ceftriaxone was gifted by King Abdulaziz Hospital at Taif. All the drugs used in this study were reconstituted in sterile saline solution (0.9% NaCl).

Experimental design

The overall experimental design is presented in Fig. 1A.

Figure 1
figure 1

Experimental design of the CPP experiment 1 and 2 (A). Diagram of the CPP apparatus (B). Time spent in chamber 1 and 2 in the control group (C). No significant changes in time spent were found in chamber 1 and chamber 2 during the tested phases. Values shown as means ± S.E.M.

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Experiment 1: Mice were randomly assigned into five groups; Group 1: Control group (n = 8), mice in this group were treated with vehicle for 8 days. Group 2: Preg-30 group (n = 8), mice were injected with pregabalin (30 mg/kg, i.p.x 4) and vehicle for eight days during the acquisition phase. Group 3: Preg-60 group (n = 8), mice were injected with pregabalin (60 mg/kg, i.p.x 4) and vehicle for eight days during the acquisition phase. Group 4: Preg-90 group (n = 8), mice were injected with pregabalin (90 mg/kg, i.p.x 4) and vehicle for eight days during the acquisition phase. Group 5: Preg-120 group (n = 8), mice were injected with pregabalin (120 mg/kg, i.p.x 4) and vehicle for eight days during the acquisition phase. Mice were then examined for place preference following completing the conditioning training.

Experiment 2: Mice were randomly assigned into four groups; Group 1: C-C group (n = 8) Mice in this group were treated with vehicle in home cage as a 30 minutes pretreatment and vehicle for eight days during the acquisition phase. Group 2: CEF-C group (n = 8) Mice were injected with CEF (200 mg/kg, i.p.) in home cage as a 30 minutes pretreatment and then vehicle for eight days during the acquisition phase. Group 3: C-Preg group (n = 8) Mice were treated with vehicle in home cage as a 30 minutes pretreatment and then Pregabalin (60 mg/kg, i.p.x4) as well as vehicle for eight days during the acquisition phase. The dose of Pregabalin which induced CPP in the previous experiment was used in this study. Group 4: CEF-Preg group (n = 8) Mice were injected with CEF (200 mg/kg, i.p.) in home cage as a 30 minutes pretreatment and then Pregabalin (60 mg/kg, i.p.x4) as well as vehicle for eight days during the acquisition phase. Mice were then examined for place preference following completing the conditioning training.

Conditioned place preference paradigm

A custom made acrylic CPP apparatus (Fig. 1B) was used in this study as described in our previous work46. Briefly, this apparatus consists of two equal-sized conditioning chambers (35 cm × 35 cm × 50 cm), and one start box (10 cm × 15 cm × 10 cm) located outside of the CPP apparatus. The two conditioning chambers are distinguished by both tactile and visual cues. The interior walls of the first chamber are white in color with horizontal black stripes and textured walls (chamber 1). The interior walls of the other chamber (chamber 2) are black in color with vertical white stripes and smooth walls. The floor of chamber 1 is perforated with round holes. The floor of the other chamber is perforated with rectangle holes.

Habituation phase: The preconditioning day is considered as day one. On days one, two, and three each mouse was placed in the start box with door closed for 3 minutes. Then, the door was opened to let the mouse explore the conditioning chambers for 30 minutes. On day three, the animal exploration in both conditioning chambers was recorded by a digital camera fixed on the top of the apparatus. The time spent in both chambers was calculated using ANY-maze software (Stoelting, USA).

Conditioning phase: An un-biased CPP design has been used. Therefore, in each treatment group, half of the animals were randomly assigned to receive pregabalin and were placed in chamber 1, while the other half received this drug and were placed in chamber 2 during the conditioning phase (days four to eleven). Each mouse received intraperitoneal injection of specific treatment and were then located in the corresponding chamber with the door closed for 30 minutes session. On the following day, each mouse received vehicle and was located in the other chamber with the door closed for 30 minutes. The process was repeated until the completion of the eight conditioning sessions.

On day twelve, each mouse had free access to both chambers for 30 minutes. The time spent by the mice in both chambers was documented using digital camera (post-conditioning test) and counted by ANY-maze video tracking system.

Statistical analysis

Two-way repeated measure ANOVA, (Phase × Treatment) was used to analyze time spent, at two different timepoints (pre-test and post-test), in the conditioning chambers in response to the selected dose of pregabalin or saline. This analysis was selected based on previous published work46,47,48,49. When significant main interactions or effects were found, Newman-Keuls multiple comparisons were performed. All data were statistically analyzed by GraphPad Prism, using a 0.05 level of significance.

Results

Experiment 1

In this experiment, the abuse potential of different doses of pregabalin was assessed. No significant effect was revealed when mice were treated with saline in the conditioning training in phase [F (1, 7) = 0.1697, p = 0.6927], treatment [F (1,7) = 0.08970, p = 0.8965], and interaction either over phase and between treatments [F (1, 7) = 0.0006396, p = 0.9805; Fig. 1B]. After treatment with 30 mg/kg pregabalin throughout the conditioning phase, no significant effect was observed over phase [F (1, 7) = 5.282, p = 0.0551], or between treatments [F (1,7) = 0.02283, p = 0.8842], and interaction between phase and treatment [F (1, 7) = 0.01197, p = 0.9160; Fig. 2A].

Figure 2
figure 2

Time spent in pregabalin-paired chamber as compared to saline-paired chamber in Preg-30 group (A). No significant changes in time spent were found in the tested chambers. Time spent in pregabalin-paired chamber as compared to saline-paired chamber in Preg-60 group (B). A significant increase in time spent was found in pregabalin-paired chamber as compared to saline-paired chamber during post-test. A significant increase in time spent was found in pregabalin-paired chamber during post-test as compared to pre-test. Values shown as means ± S.E.M. ****p < 0.0001 compared to saline-paired chamber. (&&p < 0.01 compared to pre-test).

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No significant effect was observed between mice when treated with 60 mg/kg pregabalin throughout the conditioning training over phase [F (1, 9) = 2.215, p = 0.1709], however, we observed a significant effect between treatments [F (1,9) = 6.929, p = 0.0273], and an association between phase and treatments [F (1, 9) = 38.02, p = 0.0002]. Post-hoc analysis demonstrated that time spent was significantly elevated in in pregabalin-paired chamber in comparison with saline-paired chamber in the post-test (p < 0.0001; Fig. 2B), and time spent was significantly increased in pregabalin-paired chamber in the post-test compared to the pre-test (p < 0.01; Fig. 2B).

No significant effect was observed between mice when treated with 90 mg/kg of pregabalin during the conditioning training over phase [F (1, 8) = 1, p = 0.3466], but a significant effect was observed between treatments [F (1,8) = 7.195, p = 0.0278], and a significant association was observed over phase and between treatments [F (1, 8) = 6.036, p = 0.0395]. Post-hoc analysis revealed time spent in the pregabalin-paired chambers was significantly elevated in comparison to saline-paired chamber in the post-test (p < 0.05; Fig. 3A).

Figure 3
figure 3

Time spent in pregabalin-paired chamber as compared to saline-paired chamber in Preg-90 group (A). A significant increase in time spent was found in pregabalin-paired chamber as compared to saline-paired chamber during post-test. Time spent in pregabalin-paired chamber as compared to saline-paired chamber in Preg-120 group (B). No significant changes in time spent were found in the tested chambers. Values shown as means ± S.E.M. *p < 0.05 compared to saline-paired chamber.

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Administration of 120 mg/kg of pregabalin through the conditioning phase showed no significant change over phase [F (1, 10) = 0.8837, p = 0.3693] or between treatments [F (1,10) = 0.04083, p = 0.8439], and there was no considerable association between phase and treatment [F (1, 10) = 0.09504, p = 0.7642; Fig. 3B].

Experiment 2

In this experiment, the effects of CEF pretreatment on pregabalin (60 mg/kg) was assessed. When mice were treated with saline in home cage followed by saline treatment during the conditioning phase, no significant effect over phase [F (1, 5) = 5, p = 0.0756], between treatments [F (1,5) = 0.02791, p = 0.8739], or interaction between phase and treatment [F (1, 5) = 0.03735, p = 0.8544; Fig. 4A] were observed.

Figure 4
figure 4

Time spent in chamber 1 and 2 in the C-C group (A). No significant changes in time spent were found in chamber 1 and chamber 2 during the tested phases. Time spent in chamber 1 and 2 in CEF-C group (B). No significant changes in time spent were found in the tested chambers. Values shown as means ± S.E.M.

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When mice were treated with saline after 30 minutes of treatment with 200 mg/kg CEF, in home cages, no significant effect over phase [F (1, 7) = 1, p = 0.3506], between treatments [F (1,7) = 0.0.01810, p = 0.8968], or between phase and treatment [F (1, 7) = 0.7249, p = 0.4227; Fig. 4B] was observed throughout the conditioning phase.

Mice treated with saline in home cage followed by 60 mg/kg of pregabalin treatment during the conditioning phase; we did not observe any significant effect over phase [F (1, 6) = 1, p = 0.3559], however we did observe a significant effect between treatments [F (1, 6) = 7.605, p = 0.0330], and a significant interaction between phase and treatment [F (1, 6) = 39.63, p = 0.0007]. Post-hoc analysis demonstrated a significant increase in time spent in the pregabalin-paired chamber as compared to the saline-paired chamber in the post-test (p < 0.01; Fig. 5A, and time spent in pregabalin-paired chamber was significantly increased in the post-test compared to the pre-test (p < 0.05; Fig. 5A).

Figure 5
figure 5

Time spent in pregabalin-paired chamber as compared to saline-paired chamber in C-Preg group (A). A significant increase in time spent was found in pregabalin-paired chamber as compared to saline-paired chamber during post-test. A significant increase in time spent was found in pregabalin-paired chamber during post-test as compared to pre-test. Time spent in pregabalin-paired chamber as compared to saline-paired chamber in CEF-Preg group (B). No significant changes in time spent were found in the tested chambers. Values shown as means ± S.E.M. **p < 0.01 compared to saline-paired chamber. (&p < 0.05 compared to pre-test).

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Treatment with 60 mg/kg of pregabalin after 30 minutes of treatment with 200 mg/kg of CEF, in home cages, resulted in no significant effect over phase [F (1, 8) = 1, p = 0.3466], between treatments [F (1,8) = 0.07929, p = 0.7854], or between phase and treatment [F (1, 8) = 0.01454, p = 0.9070; Fig. 5B] throughout the conditioning phase.

Discussion

In the present study, we demonstrate for the first time, using a mice model of drug addiction, that pregabalin can induce CPP. These findings are in contrast to previous reports, however, the maximum tested dose of pregabalin was 30 mg/kg, which did not induce rewarding effects and did not change place preference15,16. Consistent with the previous findings from Andrews, et al., the dose of 30 mg/kg did not change place preference in our study. However, when the dose was increased to 60 mg/kg, a significant place preference was induced by pregabalin. This suggests a dose dependent effect of pregabalin’s rewarding effects. Interestingly, this effect is supported by previous controlled clinical studies17,18,19,20 showing that pregabalin can cause euphoric effects as a side effect in participants of these studies.

The drug seeking effects found in several drugs of abuse have been consistently reported to be mediated by glutamatergic mechanism. GLT-1, an astrocyte-specific excitatory amino acid transporter, which is responsible for glutamate homeostasis in the brain37. It had been previously demonstrated that downregulation of GLT-1 expression in the NAc was associated with continuous exposure to addicting drugs50,51,52. Interestingly, GLT-1 expression was found to be downregulated instantly in cocaine self-administration model53. Of note, the glutamatergic transmission is amplified as a result of increase in glutamate concentrations and decrease in glutamate uptake in the synapses54. Additionally, it has been observed that glutamate receptors such as mGlu-5 and N-Methyl-D-aspartate could be potentiated and activated by the spillover of glutamate which enhances drug seeking behavior54. In the present study, our results suggest that pregabalin at higher doses [60 mg, and 90 mg] may induce addiction partly by downregulating GLT-1 expression and thereby decreasing glutamate uptake at the synaptic cleft.

Treatment with CEF has been reported to prevent drug seeking behavior caused by, in part, decreased GLT-1 expression in methamphetamine, cocaine, ethanol, nicotine, and heroin dependence26,41,42,43,44,45 with the drug seeking associated with glutamate spillover secondary to GLT-1 downregulation25,55,56,57. Additionally, the normalization of GLT-1 expression, by CEF treatment, was associated with a decrease in drug-seeking behavior58,59. Therefore, pregabalin seeking at the addictive doses of [60 mg, and 90 mg] might be mediated by altering GLT-1 expression as the drug seeking effects of pregabalin was eliminated by CEF pretreatment in the present study. CEF has been demonstrated to have neuroprotective efficacy in many neurological disorders60,61 and can offer neuroprotective effects in drug addiction associated with glutamate excitotoxicity60,62,63. CEF can freely pass the blood brain barrier and enter the central nervous system to up-regulate GLT-1 making it an attractive potential therapeutic for future clinical use in antagonizing pregabalin-induced drug-seeking behavior60,62,63,64.

One limitation of the present study is that we did not demonstrate a mechanisms of action for CEF in antagonism of pregabalin-induced drug-seeking behavior. GLT-1 plays a central role in inflammatory mechanisms in the brain, which has previously been demonstrated to be associated with drug addition65,66,67. It has been previously reported that central administration of some neurotoxicants causes significant impairement in motor functions, increased neuroinflammation and increased drug addiction68,69,70. Post-treatment with CEF (200 mg/kg) significantly antagonized motor impairment, attenuated lipid peroxidation, restored endogenous antioxidant enzymes glutathione peroxidase and catalase, and decreased drug addiction71,72. Taken together, CEF-mediated antagonism of pregabalin-induced drug seeking like effects could promote restoration of glutamate homeostasis, and in this way modulate drug-seeking behavior. CEF could serve as a lead compound for developing treatment for pregabalin abuse as other pharmacological effects of this antibiotic could not be excluded.

Future studies are needed to investigate a mechanistic role for neuroinflamation in pregabalin abuse, as well as sex- and age-related mechanisms in pregabalin-induced neurochemical changes.