Dose and time-dependent selective neurotoxicity induced by mephedrone in mice

Abstract

Mephedrone is a drug of abuse marketed as 'bath salts". There are discrepancies concerning its long-term effects. We have investigated the neurotoxicity of mephedrone in mice following different exposition schedules. Schedule 1: four doses of 50 mg/kg. Schedule 2: four doses of 25 mg/kg. Schedule 3: three daily doses of 25 mg/kg, for two consecutive days. All schedules induced, in some animals, an aggressive behavior and hyperthermia as well as a decrease in weight gain. Mephedrone (schedule 1) induced dopaminergic and serotoninergic neurotoxicity that persisted 7 days after exposition. At a lower dose (schedule 2) only a transient dopaminergic injury was found. In the weekend consumption pattern (schedule 3), mephedrone induced dopamine and serotonin transporter loss that was accompanied by a decrease in tyrosine hydroxylase and tryptophan hydroxylase 2 expression one week after exposition. Also, mephedrone induced a depressive-like behavior, as well as a reduction in striatal D2 density, suggesting higher susceptibility to addictive drugs. In cultured cortical neurons, mephedrone induced a concentration-dependent cytotoxic effect. Using repeated doses for 2 days in an elevated ambient temperature we evidenced a loss of frontal cortex dopaminergic and hippocampal serotoninergic neuronal markers that suggest injuries at nerve endings.

Figure 1. Effect of a mephedrone exposure (4 doses of 50 mg/kg, s.c. at 2 h interval) on dopamine transporter density, measured as [³H]WIN35428 binding in mouse striatum (A), or frontal cortex (B) and 5-HT transporter density, measured as [³H]paroxetine binding, in hippocampus (C) and frontal cortex (D).

Results are expressed as mean±S.E.M. from 8–10 animals. *p<0.05; **p<0.01 and ***p<0.001 vs. saline.

Figure 2. Effect of a mephedrone exposure (4 doses of 25 mg/kg, s.c. at 2 h interval) on dopamine transporter density, measured as [³H]WIN35428 binding in mouse striatum (A), or frontal cortex (B) and 5-HT transporter density, measured as [³H]paroxetine binding, in hippocampus (C) and frontal cortex (D).

Results are expressed as mean±S.E.M. from 8–10 animals. **p<0.01 vs. saline.

Figure 3. Effect of a mephedrone exposure (3 doses of 25 mg/kg, s.c. at 2 h interval for 2 days) on dopamine transporter density in mouse striatum (A), or frontal cortex (B) and on 5-HT transporter density in hippocampus (C) and frontal cortex (D).

Results are expressed as mean±S.E.M. from 8–10 animals. *p<0.05; **p<0.01 and ***p<0.001 vs. saline.

Figure 4. Effect of a mephedrone exposure (3 doses of 25 mg/kg s.c. at 2 h interval for 2 days) on total tryptophan hydroxylase TPH2 expression (A) and its phosphorylated form (B) in hippocampus.

Panel C shows a representative Western blot. Results are expressed as mean±S.E.M. from 8–10 animals. *p<0.05 and **p<0.01 vs. saline.

Figure 5. Effect of a mephedrone exposure (3 doses of 25 mg/kg s.c. at 2 h interval for 2 days) on total tryptophan hydroxylase 2 expression (A) and its phosphorylated form (B) in frontal cortex.

Panel C shows a representative Western blot. Results are expressed as mean±S.E.M. from 8–10 animals. *p<0.05 vs. saline.

Figure 6. Representative hippocampal expression of glial fibrilliary acidic protein (GFAP).

Sections of the dentate gyrus (×4 A, B; ×20, C, D) from mice exposed to saline (A, C) or mephedrone (3 doses of 25 mg/kg given subcutaneously for 2 days) (B, D). The animals were sacrificed 7 days after the last dose.

Figure 7. Effect of mephedrone on immobility time in mouse forced swim test.

Animals were randomly divided into two groups (12–16 animals/group) and administered subcutaneously with saline (5 ml/kg) or mephedrone (3 doses of 25 mg/kg for 2 days) and tested 1, 3 or 7 days after exposure. Each animal was recorded for 6 min and the total period of immobility (in seconds) was registered. Each mouse was used only once for each experimental session. Each bar represents mean±S.E.M. immobility time. *p<0.05 and ***p<0.001 vs saline (one-way ANOVA and post hoc Tukey test).