Effects of combined treatment with mephedrone and methamphetamine or 3,4-methylenedioxymethamphetamine on serotonin nerve endings of the hippocampus

Abstract

Aims: Mephedrone is a stimulant drug of abuse with close structural and mechanistic similarities to methamphetamine and 3,4-methylenedioxymethamphetamine (MDMA). Although mephedrone does not damage dopamine nerve endings it increases the neurotoxicity of amphetamine, methamphetamine and MDMA. The effects of mephedrone on serotonin (5HT) nerve endings are not fully understood, with some investigators reporting damage while others conclude it does not. Presently, we investigate if mephedrone given alone or with methamphetamine or MDMA damages 5HT nerve endings of the hippocampus.

Main methods: The status of 5HT nerve endings in the hippocampus of female C57BL mice was assessed through measures of 5HT by HPLC and by immunoblot analysis of serotonin transporter (SERT) and tryptophan hydroxylase 2 (TPH2), selective markers of 5HT nerve endings. Astrocytosis was assessed through measures of glial fibrillary acidic protein (GFAP) (immunoblotting) and microglial activation was determined by histochemical staining with Isolectin B4.

Key findings: Mephedrone alone did not cause persistent reductions in the levels of 5HT, SERT or TPH2. Methamphetamine and MDMA alone caused mild reductions in 5HT but did not change SERT and TPH2 levels. Combined treatment with mephedrone and methamphetamine or MDMA did not change the status of 5HT nerve endings to an extent that was different from either drug alone.

Significance: Mephedrone does not cause toxicity to 5HT nerve endings of the hippocampus. When co-administered with methamphetamine or MDMA, drugs that are often co-abused with mephedrone by humans, toxicity is not increased as is the case for dopamine nerve endings when these drugs are taken together.

Keywords: Bath salts; MDMA; Mephedrone; Methamphetamine; Neurotoxicity; Serotonin.

Fig. 1

Effects of mephedrone and methamphetamine on hippocampal levels of 5HT and 5HIAA. Mice were treated with mephedrone (Meph; 20 mg/kg) 30 min prior to each of the 4 injections of methamphetamine (Meth; 5 mg/kg) and sacrificed 2d later for determination of hippocampal levels of 5HT (A) and 5HIAA (B) by HPLC. Data are mean ± SEM for 6–11 mice per group. *p < 0.05, **p < 0.001, or ***p < 0.0001 vs controls and ^##p < 0.001 vs mephedrone alone (Tukey’s multiple comparison test).

Fig. 2

Effects of mephedrone and methamphetamine on hippocampal levels of SERT and TPH2. Mice were treated with mephedrone (Meph; 20 mg/kg) 30 min prior to each of the 4 injections of methamphetamine (Meth; 5 mg/kg) and sacrificed 2d later for determination of striatal levels of SERT (A) or TPH2 (B) by immunoblotting. Blots were quantified using ImageJ and data are mean ± SEM for 5–7 mice per group.

Fig. 3

Effects of mephedrone and MDMA on hippocampal levels of 5HT and 5HIAA. Mice were treated with mephedrone (Meph; 20 mg/kg) 30 min prior to each of the 4 injections of methamphetamine (MDMA; 20 mg/kg) and sacrificed 2d later for determination of hippocampal levels of 5HT (A) or 5HIAA (B) by HPLC. Data are mean ± SEM for 5–7 mice per group. **p < 0.001 or ***p < 0.0001 vs control and ^###p < 0.0001 vs mephedrone alone (Tukey’s multiple comparison test).

Fig. 4

Effects of mephedrone and MDMA on hippocampal levels of SERT and TPH2. Mice were treated with mephedrone (Meph; 20 mg/kg) 30 min prior to each of the 4 injections of MDMA (20 mg/kg) and sacrificed 2d later for determination of hippocampal levels of SERT (A) or TPH2 (B) by immunoblotting. Blots were quantified using ImageJ and data are mean ± SEM for 5–7 mice per group.

Fig. 5

Effects of mephedrone, methamphetamine and MDMA on hippocampal GFAP. Mice were treated with mephedrone (Meph; 20 mg/kg), methamphetamine (Meth; 5 mg/kg) or MDMA (20 mg/kg), or the combinations of mephedrone + methamphetamine or MDMA. When given in combination, mephedrone was given 30 min prior to each of the 4 injections of methamphetamine or MDMA. Mice were sacrificed 2d after treatment for determination of hippocampal GFAP by immunoblotting. Blots were quantified using ImageJ. Data are mean ± SEM for 5–7 mice in each group.

Fig. 6

Effects of mephedrone, methamphetamine or MDMA on hippocampal microglial activation. Mice were treated with mephedrone (Meph; 20 mg/kg), methamphetamine (Meth; 5 mg/kg) or MDMA (20 mg/kg), or the combinations of mephedrone + methamphetamine or MDMA. When given in combination, mephedrone was given 30 min prior to each of the 4 injections of methamphetamine or MDMA. Mice were sacrificed 2d after treatment for determination of hippocampal microglial activation using histochemical staining with ILB4 and representative results are shown for (A) control, (B) methamphetamine, (C) MDMA, (D) mephedrone, (E) mephedrone + methamphetamine and (F) mephedrone + MDMA. Numbers in the insets of each panel represent microglial counts and are the mean ± SEM for 5–7 mice per group. The calibration bar is 40 µm.