Illicit bath salt formulations are not pure and forensic analyses reveal that they contain mixtures of various psychoactive ingredients (Spiller et al . 2011 ). In light of this fact and considering that individuals who abuse bath salts coabuse numerous other substances (Winstock et al . 2011 ; Miller and Stogner 2014 ), we expanded the study of β‐ketoamphetamine effects on the DA nerve ending by treating mice with combinations of methylone, MDPV, mephedrone, and methamphetamine. The results revealed that any combination of bath salt agents did not lead to neurotoxicity. On the other hand, MDPV potently and completely protected against methamphetamine neurotoxicity, whereas methylone, like mephedrone (Angoa‐Perez et al . 2013 ), significantly enhanced the toxic effects of methamphetamine. These results indicate that the non‐neurotoxic β‐ketoamphetamines can be differentiated as protectants or enhancers of neurotoxicity by virtue of their interaction with the DAT as non‐substrate blockers or substrates, respectively.

Based on the close chemical and pharmacological similarities shared by the amphetamines and β‐ketoamphetamines, we hypothesized that the β‐ketoamphetamines would cause neurotoxicity to monoamine nerve terminals in the striatum, hippocampus, and cortex like methamphetamine, amphetamine, and MDMA. Initial studies revealed the surprising finding that at least mephedrone did not cause damage to DA nerve endings, even when administered in a high‐dose binge regimen (Angoa‐Perez et al . 2012 ). Some studies have documented mild damage to 5‐HT nerve endings by mephedrone (Hadlock et al . 2011 ) and the toxicity of this drug can be unmasked to a small degree when given in high doses over a 2‐day span at elevated ambient temperature (Lopez‐Arnau et al . 2014 ; Martinez‐Clemente et al . 2014 ). In balance, a large number of emerging studies indicate that methylone, MDPV, and mephedrone do not appear to cause chronic depletions of DA, 5‐HT, or norepinephrine that would be indicative of neurotoxicity (Kehr et al . 2011 ; Angoa‐Perez et al . 2012 , 2014 ; Baumann et al . 2012 ; Motbey et al . 2012b ; Shortall et al . 2013b ). Failure to document a neurotoxic profile for mephedrone prompted the alternative hypothesis that its ability to block the uptake of DA by the DA transporter (DAT) would provide protection against methamphetamine neurotoxicity as is seen with more ‘classical’ DAT blockers (Schmidt and Gibb 1985 ; Pu et al . 1994 ). This prediction also proved incorrect when we found that mephedrone caused a significant enhancement of the neurotoxicity caused by methamphetamine, amphetamine, and MDMA (Angoa‐Perez et al . 2013 ).

Bath salt constituents are cathinone derivatives and are also referred to as β‐ketoamphetamines as they bear very close structural similarity to the amphetamines. In fact, their possession of a beta‐keto moiety is the only feature that differentiates them from their respective amphetamine congeners [e.g., 3,4‐methylenedioxymethamphetamine (MDMA) is the deketo form of methylone]. Not surprisingly, methylone, MDPV, and mephedrone share many of the pharmacological and behavioral characteristics commonly associated with the amphetamine psychostimulants to include increased locomotor activity (Huang et al . 2012 ; Lisek et al . 2012 ; Lopez‐Arnau et al . 2012 ; Marusich et al . 2012 , 2014 ; Motbey et al . 2012a ; Wright et al . 2012 ; Aarde et al . 2013a , b ; Fantegrossi et al . 2013 ; Gatch et al . 2013 ; Miller et al . 2013 ; Shortall et al . 2013b ; Varner et al . 2013 ), altered learning and memory (Motbey et al . 2012b ; den Hollander et al . 2014 ), disruptions in thermoregulation (Baumann et al . 2012 ; Aarde et al . 2013a ; Fantegrossi et al . 2013 ; Shortall et al . 2013a ; Lopez‐Arnau et al . 2014 ), induction of behavioral sensitization (Gregg et al . 2013a , b ), and the ability to serve as discriminative drug stimuli (Fantegrossi et al . 2013 ; Gatch et al . 2013 ; Varner et al . 2013 ). The abuse potential of these drugs has been affirmed in pre‐clinical studies that document their ability to support the formation of a conditioned place preference (Lisek et al . 2012 ; Karlsson et al . 2014 ), sustain self‐administration (Hadlock et al . 2011 ; Watterson et al . 2012 ; Aarde et al . 2013a , b ), and enhance intracranial self‐stimulation (Robinson et al . 2012 ; Watterson et al . 2012 ; Bonano et al . 2014 ). These drugs also elicit the release of dopamine (DA), serotonin (5‐HT), and norepinephrine and block the uptake of these monoamines by their respective transporters (Hadlock et al . 2011 ; Baumann et al . 2012 ; Eshleman et al . 2013 ; Marusich et al . 2014 ).

Methylone, 3,4‐methylenedioxypyrovalerone (MDPV), and mephedrone are synthetic psychoactive drugs that have received worldwide notoriety as components of ‘bath salts’. These agents are abused singly or with other psychoactive drugs like alcohol, cannabis, and 3,4‐methylenedioxymethamphetamine (Winstock et al . 2011 ; Miller and Stogner 2014 ). The high abuse potential of these illicit compounds and the clinically significant dangers associated with their intake has resulted in their classification as Schedule I compounds by the US Drug Enforcement Administration. These same drugs are also now banned by all member states of the European Monitoring Centre for Drugs and Drug Addiction. Despite regulatory efforts to restrict their distribution and sale, growing abuse of methylone, MDPV and mephedrone continues to be a significant public health, law enforcement, and societal concern (Miotto et al . 2013 ).

The effects of drug treatments on core body temperature over time were analyzed using two‐way anovas and post hoc comparisons were carried out using Bonferroni's test. Two‐way anovas were performed to analyze the dose effects of methylone, MDPV, and methamphetamine, and their combinations, on striatal levels of DA, DAT, TH, and GFAP. The effects of individual drug treatments on DA, DAT, TH, and GFAP content were also tested for significance by one‐way anova and post hoc comparisons were carried out with the Holm–Sidak test for multiple comparisons. The effects of MDPV on amphetamine, MDMA, and MPTP toxicity were analyzed with a one‐way anova and post hoc comparisons were carried out with the Holm–Sidak test for multiple comparisons. Differences were considered significant if p < 0.05. Because many of the treatments using multiple doses of methylone or MDPV in combination with methamphetamine resulted in such large numbers of planned comparisons, the outcomes of the statistical tests are described minimally in 3 (i.e., only p values presented) and detailed descriptions of all statistical outcomes are presented in Tables S1–S6. Likewise, the details of the statistical analyses of methylone, MDPV, and mephedrone (single and combined treatments) effects on body temperature are presented in Supporting information. All statistical analyses were carried out using GraphPad Prism version 6.01 for Windows (GraphPad Software, San Diego, CA, USA, www.graphpad.com ).

The effects of drug treatments on striatal DAT and TH levels, highly specific markers for striatal DA nerve endings, were determined by immunoblotting as an index of toxicity. GFAP levels were used as an index of nerve ending damage as described previously (Angoa‐Perez et al . 2012 ). Mice were killed by decapitation after treatment and striatum was dissected bilaterally. Tissue was stored at −80°C. Frozen tissue was disrupted by sonication in 1% sodium dodecyl sulfate at 95°C and insoluble material was sedimented by centrifugation. Protein was determined by the bicinchoninic acid method and equal amounts of protein (70 μg/lane) were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and then electroblotted to nitrocellulose. Blots were blocked in Odyssey blocking buffer (phosphate‐buffered saline) for 1 h at 23°C. Primary antibodies against DAT (1 : 1000), TH (1 : 1000), GFAP (1 : 2000) or GAPDH (1 : 10 000) were added to blots and allowed to incubate for 16 h at 4°C. Blots were washed three times in Tris‐buffered saline to remove unreacted antibodies and then incubated with IRDye secondary antibodies (1 : 4000) for 1 h at 23°C. Immunoreactive bands were visualized by enhanced fluorescence and the relative densities of TH‐, DAT‐, GFAP‐, and GAPDH‐reactive bands were determined by imaging with an Odyssey CLx Infrared Image System (LiCor Biosciences) and quantified using ImageJ software (NIH, Bethesda, MD, USA). TH, DAT and GFAP relative densities were normalized to GAPDH.

Mice were treated with methylone (30 mg/kg), MDPV (30 mg/kg), or mephedrone (40 mg/kg) using a binge‐like regimen comprised four injections with a 2‐h interval between each injection. This binge treatment regimen, when used to inject substituted amphetamines results in extensive DA nerve ending damage. Doses of all drugs used fall well within the range used in similar published studies (Angoa‐Perez et al . 2012 , 2013 ; Marusich et al . 2012 ; Fantegrossi et al . 2013 ; den Hollander et al . 2013 ; Karlsson et al . 2014 ). Independent groups of mice were treated with each drug separately or with all possible combinations of two of the drugs. For combination treatment of mice with methylone or MDPV with methamphetamine, mice were treated with varying doses of either β‐ketoamphetamine (4× – 10, 20, or 30 mg/kg) concurrent each injection of varying doses of methamphetamine (4× – 2.5, 5, or 10 mg/kg). The combination treatment of mephedrone + methamphetamine was carried out in a previous study (Angoa‐Perez et al . 2013 ) and was not repeated presently. Controls received injections of physiological saline on the same schedule used for the β‐ketoamphetamines and methamphetamine (alone or in combination). The effect of MDPV (4× – 30 mg/kg) on amphetamine (4× – 5 mg/kg)‐ and MDMA (4× – 20 mg/kg)‐induced damage to DA nerve endings was also tested. The latter drugs were administered using the same binge regimen described above for the β‐ketoamphetamines and methamphetamine. To determine if MDPV neuroprotection would extend to non‐amphetamine neurotoxins, mice were treated with MDPV (2× – 10 mg/kg) prior to each of two injections of MPTP (20 mg/kg). All injections were given via the i.p. route. Mice were killed 2 days after the last drug treatment when amphetamine‐associated neurotoxicity has reached maximum. Body temperature was monitored by telemetry using IPTT‐300 implantable temperature transponders from Bio Medic Data Systems, Inc. (Seaford, DE, USA). Temperatures were recorded non‐invasively every 20 min starting 60 min before the first drug injection and continuing for 9 h thereafter using the DAS‐5001 console system from Bio Medic Data Systems, Inc.

Female C57BL/6 mice (Harlan, Indianapolis, IN, USA) weighing 20–25 g at the time of experimentation were housed five per cage in large shoe‐box cages in a light (12‐h light/dark) and temperature‐controlled room. Female mice were used because they are known to be very sensitive to neuronal damage by the neurotoxic amphetamines and to maintain consistency with our previous studies of methamphetamine and β‐ketoamphetamine interactions (Angoa‐Perez et al . 2012 , 2013 , 2014 ). Mice had free access to food and water. The Institutional Care and Use Committee of Wayne State University approved the animal care and experimental procedures. All procedures were also in compliance with the NIH Guide for the Care and Use of Laboratory Animals and were conducted in compliance with ARRIVE guidelines.

The effect on body temperature of the highest doses of methylone (30 mg/kg) and methamphetamine (2.5 mg/kg) given in combination were measured and the results presented in Fig. 6 indicate that the main effects of time ( F (30,480) = 7.16, p < 0.0001) and treatment ( F (3,16) = 480.7, p < 0.0001) as well as their interaction ( F (90,480) = 6.77, p < 0.0001) were statistically significant. Compared to controls, methamphetamine ( p < 0.0001), methylone ( p < 0.0001), and their combination ( p < 0.0001) significantly increased body temperature. The effects of methamphetamine on body temperature also differed significantly from those of methylone ( p < 0.0001) and the combination of methamphetamine + methylone ( p < 0.0001). Finally, methylone effects on body temperature were significantly different from those caused by methamphetamine + methylone ( p < 0.01).

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.

Mice were treated with varying doses of methylone (4× – 10, 20 or 30 mg/kg) alone or in combination with 2.5 mg/kg methamphetamine and Fig. 5 shows the results of the effects of these treatments on DA (Fig. 5 a), DAT (Fig. 5 b), TH (Fig. 5 c), and GFAP (Fig. 5 d). The main effect of treatment on DA, DAT, TH, and GFAP was highly significant ( p < 0.01–0.0001). Methylone alone at any dose did not change the levels of any dependent variable. Methamphetamine (2.5 mg/kg) significantly changed the levels of DA ( p < 0.0001) and GFAP ( p < 0.01) and while DAT and TH were reduced by this low dose of methamphetamine, the changes were not significant. All doses of methylone significantly accentuated the effects of methamphetamine on DA, DAT, TH, and GFAP by comparison with controls ( p < 0.05–0.0001) with the exception of the 10 mg/kg dose, which did not significantly increase the methamphetamine effect on TH. Figure 5 also shows that methylone significantly enhanced methamphetamine effects on DA and DAT at all doses by comparison with methamphetamine alone. The enhancing effect of methylone was observed only at the 30 mg/kg dose in the case of TH but not for GFAP. Doses of methamphetamine higher than 2.5 mg/kg were not tested to avoid a floor effect on depletion of DA nerve ending markers. The specific outcomes for the statistical tests of all main effects and interactions as well as all post hoc comparisons for the data in Fig. 5 are presented in detail in Tables S5 and S6.

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.

The generality of the neuroprotective effects of MDPV was examined by testing it for protection against amphetamine‐, MDMA‐, and MPTP‐induced damage to DA nerve endings and the results are presented in Fig. 4 . Because MDPV does not change DA nerve ending markers (see Figs 1 and 2 ), data from groups treated with this drug alone are omitted from Fig. 4 . The main effect of drug treatment on striatal levels of DA (Fig. 4 a), DAT (Fig. 4 b) and TH (Fig. 4 c) was significant for amphetamine ( p < 0.009–0.0001), MDMA ( p < 0.0002–0.0001), and MPTP ( p < 0.0025–0.0001). Individually, all test drugs significantly decreased DA, DAT, and TH, and increases in GFAP by comparison to controls ( p < 0.05–0.0001) were observed after all drugs except MPTP. MDPV provided protection against each treatment by comparison to drug alone ( p < 0.05–0.0001), with a few exceptions. In most cases, the protective effect of MDPV was complete (i.e., drug + MDPV did not differ from control values). Exceptions were observed for amphetamine + MDPV, where DA levels were actually elevated above those of controls, for MDMA + MDPV effects on DAT and for MDMA + MDPV effects on TH, where protection was significant but partial. Although MDPV provided some protection against the MDMA‐induced reduction in TH, this effect did not reach statistical significance. MPTP alone did not elevate GFAP levels presently so it was not possible to observe a protective effect of MDPV on this dependent variable. The specific outcomes for the statistical tests of all main effects and interactions as well as all post hoc comparisons for the data in Fig. 4 are presented in detail in Tables S3 and S4.

The effects on body temperature of the highest doses of MDPV (30 mg/kg) given in combination with methamphetamine (10 mg/kg) were measured and the results are presented in Fig. 3 . The main effects of time ( F (30,480) = 7.861, p < 0.0001) and treatment ( F (3,16) = 157.1, p < 0.0001) as well as their interaction ( F (90,480) = 6.789, p < 0.0001) were statistically significant. When given alone, methamphetamine ( p < 0.0001) and MDPV ( p < 0.0001) caused significant increases in body temperature by comparison to controls. Combined treatment with methamphetamine + MDPV significantly increased body temperature by comparison to control ( p < 0.0001) and Fig. 3 also shows that MDPV did not reduce the hyperthermic effect of methamphetamine ( p > 0.05). The effects of MDPV on body temperature differed significantly from both methamphetamine alone ( p < 0.0001) and from methamphetamine + MDPV ( p < 0.0001). Taken together, these results indicate that MDPV protects against methamphetamine neurotoxicity without interfering with its hyperthermic effects.

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.

Mice were treated with MDPV and methamphetamine in varying doses of each drug alone or in combination and Fig. 2 shows the results of these treatments on DA (Fig. 2 a), DAT (Fig. 2 b), TH (Fig. 2 c), and GFAP (Fig. 2 d). The main effects of both drugs and their interactions on DA, DAT, TH, and GFAP were all highly statistically significant ( p < 0.01–0.0001). MDPV alone at any dose did not change the levels of any of the dependent variables, whereas with only one exception, all doses of methamphetamine alone significantly reduced DA, DAT, and TH and significantly increased GFAP ( p < 0.05–0.0001). The 2.5 mg/kg dose of methamphetamine alone did not lower TH levels significantly. All doses of MDPV provided complete protection against the effects of each dose of methamphetmine on all DA nerve ending markers and GFAP as evidenced by two different statistical comparisons. First, none of the MDPV + methamphetamine treatments differed from control at any dose combination for both drug. Second, with only a few exceptions, all MDPV + methamphetamine treatments were significantly different from methamphetamine alone for each respective dose of this drug ( p < 0.05–0.0001). The exceptions involved the lowest dose of methamphetamine. The specific outcomes for the statistical tests of all main effects and interactions as well as all post hoc comparisons for the data in Fig. 2 are presented in detail in Tables S1 and S2.

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.

Mice were treated with methylone (30 mg/kg), MDPV, (30 mg/kg) or mephedrone (40 mg/kg) singly or in all possible combinations of two of these drugs and the effects on striatal DA, DAT, and TH levels are presented in Fig. 1 . It can be seen that none of the drugs changed the levels from control of any marker for DA nerve endings to include DA (Fig. 1 a), DAT (Fig. 1 b) or TH (Fig. 1 c). Likewise, combined treatment with MDPV + mephedrone, methylone + mephedrone, or methylone + MDPV did not alter striatal levels of DA, DAT, or TH. The effects of these drugs singly or in combination on striatal GFAP are presented in Fig. 1 (d) and show that this marker for astrogliosis was not changed from control values by any treatment. In agreement with published accounts, each β‐ketoamphetamine alone and in all two‐drug combinations resulted in significant hyperthermia and these results are described in more detail in Figures S1–S3.

Discussion

The goal of the present work was to expand our previous studies of how mephedrone influences DA nerve ending damage caused by methamphetamine. Most pre‐clinical investigations of the neuropharmacological actions of the β‐ketoamphetamines have studied the effects of single drugs on the CNS. However, illicit bath salt formulations generally contain mixtures of psychoactive ingredients (Spiller et al. 2011) and individuals who abuse these drugs often coabuse other substances along with bath salts (Winstock et al. 2011; Miller and Stogner 2014). Therefore, a compelling rationale exists for testing bath salts in combination as well as with other psychostimulants like methamphetamine, amphetamine, and MDMA. Mephedrone, despite its close structural and pharmacological similarities to the neurotoxic amphetamines, does not cause neurotoxicity when administered to mice in a high‐dose binge regimen (Angoa‐Perez et al. 2012). Instead, this drug exerts the surprising property of enhancing the depletions in striatal DA, DAT, and TH caused by methamphetamine, amphetamine, and MDMA (Angoa‐Perez et al. 2013). These results raise at least two very interesting questions about the mechanisms of action of the β‐ketoamphetamines and their amphetamine congeners that cause damage to the CNS. First, if mephedrone shares with methamphetamine the ability to release DA, inhibit its uptake, and cause hyperthermia, factors that are thought to be essential for methamphetamine neurotoxicity, why is it not neurotoxic? Second, if mephedrone can block DA uptake via its interaction with the DAT, why does it not prevent methamphetamine‐induced neurotoxicity like other DAT blockers (Schmidt and Gibb 1985; Pu et al. 1994; Angoa‐Perez et al. 2013)? The answer to these questions may lie in the mechanisms by which mephedrone and other bath salt constituents interact with the DAT.

When given alone or in two‐drug combinations, mephedrone, MDPV, and methylone cause significant increases in body temperature. The most remarkable effect is associated with mephedrone which causes a profound hypothermia immediately after injection that reverts to hyperthermia after 30–40 min, in agreement with our previous studies with this drug (Angoa‐Perez et al. 2012, 2013). When methylone or MDPV are given with mephedrone, this hypothermic effect of mephedrone is retained and slightly exaggerated. Combined treatment with MDPV and methylone results in a 1–2°C steady increase in core body temperature that becomes evident within 15 min of treatment and persists for at least 8–9 h. Each of these drugs increases the synaptic levels of DA by stimulating release and blocking uptake (i.e., mephedrone, methylone) or by acting as a pure DAT antagonist (i.e., MDPV). Despite exerting significant hyperthermic effects overall, none of the β‐ketoamphetamines alone or in combination result in changes in striatal DA, DAT, TH, or GFAP that would be indicative of neurotoxicity. Taken together, these results leave open the question of why the β‐ketoamphetamines do not cause DA nerve ending damage despite exerting most of the effects that are considered important for methamphetamine‐induced neurotoxicity.

We found presently that methylone shares with mephedrone the ability to enhance methamphetamine‐induced damage to DA nerve terminals, whereas MDPV provides complete protection against neurotoxicity. This neuroprotective effect of MDPV extends to amphetamine and MDMA, and to MPP+, the neurotoxic metabolite of MPTP that is structurally unrelated to the neurotoxic amphetamines but which depends on uptake by the DAT to exert its damaging effects on DA neurons (Javitch et al. 1985). These differing effects of mephedrone, methylone, and MDPV on methamphetamine neurotoxicity cannot be explained by their effects on body temperature because neither interferes with the ability of methamphetamine to cause hyperthermia. Recent studies of the mechanisms by which β‐ketoamphetamines interact with the DAT offer significant insight into why these drugs have such divergent effects on neurotoxicity. The bath salts have been classified as substrates and non‐substrates based on whether or not they are transported by the DAT. Mephedrone and methylone, like methamphetamine, are substrates for DAT‐mediated uptake and they cause release of DA via carrier‐mediated exchange (Baumann et al. 2012; Cameron et al. 2013a,b; Eshleman et al. 2013). MDPV is not a substrate for transport and interacts with the DAT strictly as a blocker, like cocaine (Baumann et al. 2013; Cameron et al. 2013a,b; Eshleman et al. 2013; Kolanos et al. 2013; Simmler et al. 2013a). This dichotomy of interaction with the DAT by mephedrone and methylone on one hand and by MDPV on the other can explain their opposing effects on methamphetamine‐induced neurotoxicity. Mephedrone and methylone enhance the effects of methamphetamine, most likely by increasing the release of DA above that caused by either drug alone. This possibility has not yet been tested but is supported by prior results showing that treatments resulting in an increase in the releasable pool of DA significantly accentuate methamphetamine‐induced damage in DA nerve endings (Thomas et al. 2008, 2009). MDPV has an effect that is similar to more classical DAT blockers and protects against methamphetamine‐induced neurotoxicity. Compared to amfonelic acid (Schmidt and Gibb 1985; Pu et al. 1994) and nomifensine (Angoa‐Perez et al. 2013), which provide partial but significant protection, MDPV completely prevents the damaging effects of methamphetamine on DA nerve endings. By blocking DAT‐mediated transport (inward or outward), MDPV blocks methamphetamine‐induced efflux of DA (Simmler et al. 2013b). This property alone represents an important mechanism by which it protects against DA nerve ending damage caused by drugs that depend on inward DAT transport to exert their toxicity to include the neurotoxic amphetamines and MPTP (i.e., MPP+). MDPV may be one of the most powerful blockers of the DAT yet described (Baumann et al. 2013; Eshleman et al. 2013; Simmler et al. 2013a). It is also more effective than bupropion or methylphenidate in blocking methamphetamine‐induced DA release (Simmler et al. 2013b) and is the most potent drug identified to date for protecting against methamphetamine‐induced damage to DA nerve endings.

Methamphetamine is probably the prototypical neurotoxic amphetamine. Its ability to flood the synapse with DA, especially after binge administration (O'Dell et al. 1993), is thought to be the first step in a cascade that leads rapidly to mitochondrial dysfunction, enhanced excitatory neurotransmission, increases in glial reactivity and oxidative stress, nerve ending damage, and apoptosis (Halpin et al. 2014). The numerous facets of methamphetamine‐induced neurotoxicity have been studied in great detail over the past three decades, whereas the dangers associated with bath salts have emerged only recently. However, the β‐ketoamphetamines should offer new possibilities for achieving a better understanding of the mechanisms by which methamphetamine, amphetamine, and MDMA cause damage to monoamine nerve endings. Mephedrone and methylone cause little or no neurotoxicity yet they cause significant efflux of DA and inhibit its re‐uptake through their interactions with the DAT. The interactions of mephedrone and methylone with the DAT are very similar to those of methamphetamine and this leaves unanswered the question posed above of why they enhance the neurotoxicity of the amphetamines as opposed to offering protection. It could be predicted that mephedrone and methylone would dilute the effects of methamphetamine on DA release by substituting less toxic DAT substrates for a more toxic one. This does not appear to be the case.

Several other possibilities offer sources for speculation regarding enhanced neurotoxicity when mephedrone or methylone are combined with methamphetamine and include the following points. First, the β‐ketoamphetamines could alter the pharmacokinetics or metabolism of methamphetamine such that blood and brain drug levels are increased in amount and/or for longer periods of time over those seen after methamphetamine alone. The β‐keto group increases the polarity of mephedrone and methylone and reduces their relative penetration into the brain (Hill and Thomas 2011). It is therefore possible that methamphetamine‐induced alteration in the integrity of the blood–brain barrier (Northrop and Yamamoto 2012) could make it more permeable to the β‐ketoamphetamines. The net result of these possible effects would be similar to treatment with higher doses of both drugs. Second, at the level of the DA nerve ending, it is possible that the bath salts enhance methamphetamine toxicity because of increased release of DA over that seen after either drug alone. Methamphetamine collapses the pH gradient across the synaptic vesicle membrane, allowing DA leakage into the cytoplasm and subsequent efflux via reverse transport (Sulzer et al. 2005). Methylone and mephedrone release cytoplasmic DA via reverse transport through the DAT, but they differ significantly from methamphetamine in that they have little if any affinity for the vesicle monoamine transporter and thus their inhibition of uptake and stimulation of release from synaptic vesicles is far lower than that of methamphetamine (Eshleman et al. 2013). Because methylone and mephedrone do not likely release DA from synaptic vesicles, combined treatment with either mephedrone or methylone + methamphetamine could recruit greater numbers of DAT molecules to result in heightened DA efflux into the synapse because amphetamine‐induced release is greater when originating from both synaptic vesicle and cytoplasmic stores versus cytoplasmic stores only (Pifl et al. 1995). Third, mephedrone and methylone could enhance methamphetamine toxicity by inhibiting monoamine oxidase A. We have shown previously that the monoamine oxidase A inhibitor clorgyline significantly increases methamphetamine‐induced depletion of DA (Thomas et al. 2008) but it is not known if the bath salts inhibit monoamine oxidase. Ongoing studies in our laboratory are directed at achieving a better understanding of these possible mechanisms by which the β‐ketoamphetamines accentuate methamphetamine‐induced neurotoxicity.

From a strictly pre‐clinical, mechanistic perspective, MDPV has the potential to be an effective neuroprotectant in drug‐induced neurotoxicity and in neurodegenerative conditions such as Parkinson's disease, or for treatment of stimulant dependence. MDPV is not neurotoxic and is far more potent in preventing methamphetamine‐induced DA release than bupropion or methylphenidate as mentioned above (Simmler et al. 2013b). The latter two drugs have been tested as treatments for stimulant dependence (Tiihonen et al. 2007; Elkashef et al. 2008; Shoptaw et al. 2008) with mixed outcomes so far. However, as a powerful DAT antagonist, the abuse potential of MDPV is high in humans (Coppola and Mondola 2012) as well as in animal models of addiction (Watterson et al. 2012; Aarde et al. 2013b; Bonano et al. 2014; Karlsson et al. 2014) and this property alone would limit its use as a therapeutic agent. Despite its abuse potential in the illicit market, MDPV or a structural variant with lower abuse potential could have clinical effectiveness in treating substance abuse disorders as do methadone and buprenorphine, drugs that also possess high abuse potential in humans.