3,4-Methylenedioxypyrovalerone prevents while methylone enhances methamphetamine-induced damage to dopamine nerve endings: β-ketoamphetamine modulation of neurotoxicity by the dopamine transporter

Abstract

Methylone, 3,4-methylenedioxypyrovalerone (MDPV), and mephedrone are psychoactive ingredients of 'bath salts' and their abuse represents a growing public health care concern. These drugs are cathinone derivatives and are classified chemically as β-ketoamphetamines. Because of their close structural similarity to the amphetamines, methylone, MDPV, and mephedrone share most of their pharmacological, neurochemical, and behavioral properties. One point of divergence in their actions is the ability to cause damage to the CNS. Unlike methamphetamine, the β-ketoamphetamines do not damage dopamine (DA) nerve endings. However, mephedrone has been shown to significantly accentuate methamphetamine neurotoxicity. Bath salt formulations contain numerous different psychoactive ingredients, and individuals who abuse bath salts also coabuse other illicit drugs. Therefore, we have evaluated the effects of methylone, MDPV, mephedrone, and methamphetamine on DA nerve endings. The β-ketoamphetamines alone or in all possible two-drug combinations do not result in damage to DA nerve endings but do cause hyperthermia. MDPV completely protects against the neurotoxic effects of methamphetamine while methylone accentuates it. Neither MDPV nor methylone attenuates the hyperthermic effects of methamphetamine. The potent neuroprotective effects of MDPV extend to amphetamine-, 3,4-methylenedioxymethamphetamine-, and MPTP-induced neurotoxicity. These results indicate that β-ketoamphetamine drugs that are non-substrate blockers of the DA transporter (i.e., MDPV) protect against methamphetamine neurotoxicity, whereas those that are substrates for uptake by the DA transporter and which cause DA release (i.e., methylone, mephedrone) accentuate neurotoxicity. METH (a) enters DA nerve endings via the DAT, causes leakage of DA into the cytoplasm and then into the synapse via DAT-mediated reverse transport. Methylone (METHY) and mephedrone (MEPH; b), like METH, are substrates for the DAT but release DA from cytoplasmic pools selectively. When METH is combined with METHY or MEPH (c), DA efflux and neurotoxicity are enhanced. MDPV (d), which is a non-substrate blocker of the DAT, prevents METH uptake and efflux of DA. Therefore, bath salts that are substrates for the DAT and release DA (METHY, MEPH) accentuate METH neurotoxicity, whereas those that are non-substrate blockers of the DAT (MDPV) are neuroprotective.

Keywords: dopamine nerve ending; dopamine transporter; neurotoxic amphetamines; neurotoxicity; β-ketoamphetamines.

Fig. 1

Effects of mephedrone (4′ – 40 mg/kg), methylone (4′ –30 mg/kg), and MDPV (4′ – 30 mg/kg) alone or in combination on DA nerve endings of the striatum. Mice were treated with mephedrone (MEPH; 4′ – 40 mg/kg), MDPV (4′ – 30 mg/kg), or methylone (MTHY; 4′ – 30 mg/kg) singly or in the indicated two-drug combinations and the levels of DA (a), dopamine transporter (DAT) (b), tyrosine hydroxylase (TH), (c) and glial fibrillary acidic protein (GFAP) (d) were determined 2 days after treatment. Controls were injected with physiological saline on the same binge schedule used for the β-ketoamphetamines. DA levels were determined by HPLC and are reported as % control. Relative pixel densities for immunoblots of DAT, TH, and GFAP were quantified using ImageJ, normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and expressed as relative band density by comparison to the respective control. Data are expressed as mean ± SEM for n = 4–5 mice per group.

Fig. 2

Effects of MDPV (4′ – 10, 20, or 30 mg/kg) on methamphetamine (2.5, 5, or 10 mg/kg)-induced neurotoxicity to DA nerve endings. Mice were treated with MDPV (4′ – 10, 20 or 30 mg/kg), methamphetamine (Meth; 2.5, 5 or 10 mg/kg), or their combination in the indicated doses and the levels of DA (a), dopamine transporter (DAT) (b), tyrosine hydroxylase (TH) (c), and glial fibrillary acidic protein (GFAP) (d) were determined 2 days after treatment. Controls were injected with physiological saline on the same binge schedule used for MDPV and methamphetamine. DA levels were determined by HPLC and are reported as % control. Relative pixel densities for immunoblots of DAT, TH, and GFAP were quantified using ImageJ, normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and expressed as relative band density by comparison with the respective control. Data are means for n = 4–8 mice per group. SEM bars (< 5% of means) and symbols indicating p values are omitted from the figure for the sake of clarity. Specific details of all statistical comparisons for the data in this figure are included in Tables S1 and S2.

Fig. 3

Effects of MDPV (4′ – 30 mg/kg), methamphetamine (4′ –10 mg/kg), and their combination on core body temperature. Mice were treated with MDPV (4′ – 30 mg/kg), methamphetamine (4′ –10 mg/kg), or their combination and body temperature was measured via telemetry at 20-min intervals starting 40 min before the first drug injection and continuing for 560 min during drug treatments. Controls were injected with physiological saline on the same binge schedule used for MDPV and methamphetamine. Data are means for n = 5–6 mice per group. SEM bars (< 5% of means) and symbols indicating p values are omitted from the figure for the sake of clarity.

Fig. 4

Effects of MDPV (4′ – 30 mg/kg) on amphetamine (4′ – 5 mg/ kg)-, 3,4-methylenedioxymethamphetamine (MDMA) (4′ – 20 mg/kg)-, and MPTP (2′ – 20 mg/kg)-induced neurotoxicity to DA nerve endings. Mice were treated with MDPV (MV; 4′ – 30 mg/kg) in combination with amphetamine (AM; 4′ – 5 mg/kg), MDMA (MD; 4′ –20 mg/kg) or MPTP (MP; 2′ – 20 mg/kg) and the levels DA (a), dopamine transporter (DAT) (b), tyrosine hydroxylase (TH) (c), and glial fibrillary acidic protein (GFAP) (d) were determined 2 days after treatment. Controls were injected with physiological saline on the same binge schedule used for all drugs. DA levels were determined by HPLC and are reported as % control. Relative pixel densities for immunoblots of DAT, TH and GFAP were quantified using ImageJ, normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and expressed as relative band density by comparison with the respective control. Data are mean ± SEM for n = 5–6 mice per group. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 by comparison with untreated controls. ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 and ^####p < 0.0001 by comparison with AMPH, MDMA or MPTP alone. Specific details of all statistical comparisons for the data in this figure are included in Tables S3 and S4.

Fig. 5

Effects of methylone (4′ – 10, 20 or 30 mg/kg) on methamphetamine (4′ – 2.5 mg/kg)-induced neurotoxicity to DA nerve endings. Mice were treated with methylone (4′ – 10, 20, or 30 mg/ kg) alone or in combination with methamphetamine (4′ – 2.5 mg/kg) and the levels DA (a), dopamine transporter (DAT) (b), tyrosine hydroxylase (TH) (c), and glial fibrillary acidic protein (GFAP) (d) were determined 2 days after treatment. Controls were injected with physiological saline on the same binge schedule used for all drugs. DA levels were determined by HPLC and are reported as % control. Relative pixel densities for immunoblots of DAT, TH and GFAP were quantified using ImageJ, normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and expressed as relative band density by comparison with the respective control. Data are mean ± SEM for n = 4–6 mice per group. **p < 0.01, ***p < 0.001 and ****p < 0.0001 by comparison with untreated controls. ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 and ^####p < 0.0001 by comparison with methamphetamine alone. Specific details of all statistical comparisons for the data in this figure are included in Tables S5 and S6.

Fig. 6

Effects of methylone (4′ 30 mg/kg) and methamphetamine (4′ – 2.5 mg/kg) and their combination on core body temperature. Mice were treated with methylone (4′ – 30 mg/kg), methamphetamine (4′ – 2.5 mg/kg), or their combination and body temperature was measured via telemetry at 20-min intervals starting 40 min before the first drug injection and continuing for 560 min during drug treatments. Controls were injected with physiological saline on the same binge schedule used for MDPV and methamphetamine. Data are means for n = 5–8 mice per group. SEM bars (< 5% of means) and symbols indicating p values are omitted from the figure for the sake of clarity.