Mephedrone does not damage dopamine nerve endings of the striatum, but enhances the neurotoxicity of methamphetamine, amphetamine, and MDMA

Abstract

Mephedrone (4-methylmethcathinone) is a β-ketoamphetamine stimulant drug of abuse with close structural and mechanistic similarities to methamphetamine. One of the most powerful actions associated with mephedrone is the ability to stimulate dopamine (DA) release and block its re-uptake through its interaction with the dopamine transporter (DAT). Although mephedrone does not cause toxicity to DA nerve endings, its ability to serve as a DAT blocker could provide protection against methamphetamine-induced neurotoxicity like other DAT inhibitors. To test this possibility, mice were treated with mephedrone (10, 20, or 40 mg/kg) prior to each injection of a neurotoxic regimen of methamphetamine (four injections of 2.5 or 5.0 mg/kg at 2 h intervals). The integrity of DA nerve endings of the striatum was assessed through measures of DA, DAT, and tyrosine hydroxylase levels. The moderate to severe DA toxicity associated with the different doses of methamphetamine was not prevented by any dose of mephedrone but was, in fact, significantly enhanced. The hyperthermia caused by combined treatment with mephedrone and methamphetamine was the same as seen after either drug alone. Mephedrone also enhanced the neurotoxic effects of amphetamine and 3,4-methylenedioxymethamphetamine on DA nerve endings. In contrast, nomifensine protected against methamphetamine-induced neurotoxicity. As mephedrone increases methamphetamine neurotoxicity, the present results suggest that it interacts with the DAT in a manner unlike that of other typical DAT inhibitors. The relatively innocuous effects of mephedrone alone on DA nerve endings mask a potentially dangerous interaction with drugs that are often co-abused with it, leading to heightened neurotoxicity.

Fig. 1

Effects of mephedrone on methamphetamine-induced reductions in striatal DA. Mice were treated with the indicated doses of mephedrone (MEPH) 30 min prior to each injection of 2.5 (•) or 5.0 mg/kg (■) methamphetamine (METH) and sacrificed 2d later for determination of striatal levels of DA by HPLC. Data are mean ± SEM for 5–7 mice per group. Some error bars were too small to exceed the size of the symbols and do not appear visible. ***p < 0.001 vs controls and ^#p < 0.01, ^##p < 0.001 or ^###p < 0.0001 vs the respective dose of methamphetamine (Tukey's multiple comparison test).

Fig. 2

Effects of mephedrone on methamphetamine-induced reductions in striatal DAT. Mice were treated with the indicated doses of mephedrone (MEPH) 30 min prior to each injection of 2.5 (●) or 5.0 mg/kg (■) methamphetamine (METH) and sacrificed 2d later for determination of striatal levels of DAT by immunoblotting (a). Blots were quantified using ImageJ and data are mean ± SEM for 10–12 mice per group (b). *p < 0.01 or ***p < 0.0001 vs control (C) and ^#p < 0.01 or ^##p < 0.001 vs the respective dose of methamphetamine (Tukey's multiple comparison test).

Fig. 3

Effects of mephedrone on methamphetamine-induced reductions in striatal TH. Mice were treated with the indicated doses of mephedrone (MEPH) 30 min prior to each injection of 2.5 (●) or 5.0 mg/kg (■) methamphetamine (METH) and sacrificed 2d later for determination of striatal levels of TH by immunoblotting (a). Blots were quantified using ImageJ and data are mean ± SEM for 10–12 mice per group (b). Some error bars were too small to exceed the size of the symbols and do not appear visible. **p < 0.001 or ***p < 0.0001 vs control (C) and ^#p < 0.01, ^##p < 0.001 or ^###p < 0.0001) vs the respective dose of methamphetamine (Tukey's multiple comparison test).

Fig. 4

Effects of mephedrone on methamphetamine-induced hyperthermia. Mice were treated with the indicated doses of mephedrone (MEPH) 30 min prior to each injection of 2.5 (a) or 5.0 mg/kg (b) methamphetamine (METH). Core temperatures were measured at 20 min intervals by telemetry starting 60 min before the first injection of methamphetamine. The 4 methamphetamine injections are indicated by the arrows resting on the x-axis. Data are expressed as mean body temperature of 6–8 mice per group. SEMs were always < 10% of the mean and are omitted for the sake of clarity.

Fig. 5

Effects of mephedrone on amphetamine- or MDMA-induced DA nerve ending neurotoxicity. Mice were treated with 20 mg/kg mephedrone (MEPH) 30 min prior to each injection of 5.0 mg/kg amphetamine (AMPH) or 20 mg/kg MDMA and sacrificed 2d after treatment for determination of striatal levels of (a) DA by HPLC. (b) DAT and (c) TH were determined by immunoblotting and blots were quantified using ImageJ. Representative immunoblots for DAT and TH are included as insets to panels (b) and (c) respectively and treatments for both panels are indicated by 1,5: control; 2,6: MEPH; 3: AMPH; 4: AMPH + MEPH; 7: MDMA; and 8: MDMA + MEPH. Data are mean ± SEM for 5–12 mice in each group. **p < 0.001 or ***p < 0.0001 vs control and ^#p < 0.01 or ^###p < 0.0001 vs AMPH or MDMA (Tukey's multiple comparison test).

Fig. 6

Effects of nomifensine on methamphetamine-induced DA nerve ending neurotoxicity. Mice were treated with 5.0 mg/kg nomifensine (NOM) 30 min prior to each injection of 5.0 mg/kg methamphetamine (METH) and sacrificed 2d later for determination of striatal levels of (a) DA by HPLC. (b) DAT and (c) TH were determined by immunoblotting and blots were quantified using ImageJ. Representative immunoblots for DAT and TH are included as insets to panels (b) and (c) respectfully. Data are mean plus SEM for 5–7 mice per group. ***p < 0.0001 vs control (C) and ^#p < 0.01 or ^##p < 0.001 vs methamphetamine alone (Tukey's multiple comparison test).