European Journal of Pharmacology 546 (2006) 74–81 www.elsevier.com/locate/ejphar Repeated administration of the substituted amphetamine p-methoxyamphetamine produces reductions in cortical 5-HT transporter binding but not 5-HT content, unlike 3,4-methylenedioxyamethamphetamine ⁎ Paul D. Callaghan a,b, Kirsten Farrand a, Abdallah Salem a, , Patrick Hughes a, Lynette C. Daws a,b, Rodney J. Irvine a a Discipline of Pharmacology, School of Medical Sciences, Medical School North, The University of Adelaide, Adelaide, South Australia, 5005, Australia b Department of Physiology, The University of Texas Health Science Center at San Antonio, TX, USA Received 30 May 2006; received in revised form 13 July 2006; accepted 17 July 2006 Available online 25 July 2006 Abstract Worldwide growth in p-methoxyamphetamine (PMA) usage amongst ‘ecstasy’ users indicates a proportionally greater incidence of acute toxicity compared to 3,4-methylenedioxymethamphetamine (MDMA). While longer-term use of MDMA appears to produce degeneration of 5- hydroxytryptamine (5-HT, serotonin) neurons, PMA effects are poorly understood. The aim of this study was to determine the effect of repeated PMA administration on two indices of 5-HT axonal degeneration, cortical brain 5-HT transporter (SERT) density and 5-HT/5-hydroxyindolacetic acid (5-HIAA) content. Treatment of male rats once daily for 4 days (10 or 20 mg/kg) with PMA or MDMA resulted in significant reductions (20 mg/kg: 53% and 23% of vehicle treatment respectively) in [3H]-paroxetine binding (SERT density) one week after final drug administration. When rats were housed at a higher ambient temperature (28 °C vs. 22 °C) for 6 h after dosing, no additive effect was seen for either drug. A more intensive dosing regimen (10 or 20 mg/kg twice daily for 4 days) was used to examine PMA/MDMA effects on cortical 5-HT content. Two weeks after MDMA treatment, significant reductions in cortical 5-HT content (20 mg/kg: 39% of vehicle treatment) were seen. However, PMA did not alter cortical 5-HT content, yet reduced cortical 5-HIAA content (20 mg/kg: 72% of vehicle treatment). These data suggest PMA has severe long- term implications clinically for alteration of 5-HT neurotransmission that may differ from MDMA, but may not necessarily be interpreted as a degeneration of 5-HT fibres. © 2006 Elsevier B.V. All rights reserved. Keywords: Para-methoxyamphetamine; 3,4-methylenedioxymethamphetamine; Serotonin transporter; Neurodegeneration; 5-HT 1. Introduction structure of these compounds, sold under the collective name “ecstasy”, are most commonly based on the amphetamine Recreational use of substituted amphetamines has grown molecule, with the most widespread example being 3,4- exponentially since the mid 1980s, leading to a proliferation of methylenedioxymethamphetamine (MDMA). Of considerable compounds possessing a unique mixture of euphoric and concern is the significant risk when using ‘ecstasy’ of acute stimulant properties, popular within ‘dance party culture’. The toxicity, resulting from a severe hyperthermic response, which can lead to rhabdomyolysis and coma (Green et al., 2003). Additionally, drugs from this class have varying abilities to induce degeneration of monoaminergic systems within the ⁎ Corresponding author. Tel.: +61 8 8303 4327; fax: +61 8 8224 0685. E-mail address: [email protected] (A. Salem). brain, which is thought to be responsible for cognitive deficits URL: http://www.adelaide.edu.au/health/pharm/staff/asalem.html seen with regular ‘ecstasy’ use clinically (Green et al., 2003; (A. Salem). Parrott, 2001). 0014-2999/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2006.07.011 P.D. Callaghan et al. / European Journal of Pharmacology 546 (2006) 74–81 75 p-Methoxyamphetamine (PMA) is a substituted amphetamine examine the effects of PMA at more pharmacologically relevant that is usually sold as ‘ecstasy’,andhasbeenlinkedwithan doses, and also in other brain regions relevant to understanding apparent higher incidence of acute hyperthermia and toxicity than the cognitive deficits seen in regular substituted amphetamine seen for MDMA (Byard et al., 2002; White et al., 1997). While users. initially appearing in Canada in 1973 (Cimbura, 1974), its use has The primary aim of this study was to determine if repeated only become widespread since 1994 in Australia, where it is PMA treatment does indeed alter indices of 5-HT and/or responsible for most ‘ecstasy’-related deaths (Byard et al., 2002; dopamine presynaptic terminal viability. First, rats were treated Felgate et al., 1998). More recently, PMA use has been reported in once per day for 4 days with vehicle, PMA or MDMA (10 or USA and Europe where again an increased incidence of acute 20 mg/kg) and one week after the final dose, [3H]-paroxetine morbidity has been noted (Becker et al., 2003; Chodorowski et al., binding was measured in cortical membrane preparations. This 2002; Dams et al., 2003; Galloway and Forrest, 2002; Johansen measure has been shown to be a sensitive measure of 5-HT et al., 2003; Kraner et al., 2001; Lora-Tamayo et al., 2004; presynaptic terminal loss (Battaglia et al., 1987). Additionally, Refstad, 2003; Voorspoels et al., 2002). MDMA effects on neurodegeneration of 5-HT fibres are poten- These acute effects of both PMA and MDMA are mediated tiated by elevated ambient temperature during drug adminis- by their interaction with either the 5-hydroxytryptamine (5-HT, tration (Malberg and Seiden, 1998). This is of particular serotonin) transporter (SERT) and/or the dopamine transporter relevance due to the high ambient temperature in environments (DAT) to evoke release of 5-HT and/or dopamine, and inhibit where “ecstasy” is used recreationally. In order to examine if neurotransmitter uptake from presynaptic sites within the cen- any additive effect of ambient temperature was seen, animals tral nervous system (Callaghan et al., 2005; Daws et al., 2000; were maintained at an ambient temperature of either 22 °C Freezer et al., 2005; Gough et al., 2002; Green et al., 2003; (room temperature) or 28 °C for 6 h after each PMA, MDMA or Kaminskas et al., 2002; McKenna and Peroutka, 1990; Menon vehicle dose. The once daily treatment regimen was chosen as et al., 1976). Both the acute euphoric effects and detrimental MDMA has been shown previously to alter cortical [3H]- effects of hyperthermia and serotonergic syndrome are all paroxetine binding with this regimen (O'Shea et al., 1998). thought to be due to the acute actions of these drugs on 5-HT Additionally, the pharmacokinetic parameters of the drugs vary neurotransmission (Green et al., 1995). However the distinctly considerably (Farre et al., 2004; Kitchen et al., 1979; Lim and different neurodegenerative effects resulting from long term Foltz, 1988), and a single daily dose would simplify the inter- usage of these drugs appear far more insidious. MDMA pro- pretation of any effect of the 6 h alteration in ambient environ- duces dose dependent selective degeneration of 5-HT axonal ment after each dose of drug. terminals as little as one week after cessation of use in rodents If there were indeed a loss in presynaptic monoaminergic and primates (Battaglia et al., 1988, 1987; Hewitt and Green, terminals with PMA treatment, it would be expected, like 1994; Lew et al., 1996; O'Hearn et al., 1988; O'Shea et al., MDMA, that there would be a corresponding loss in neuro- 1998; Sabol et al., 1996; Scanzello et al., 1993; Schmidt, 1987). transmitter and subsequent metabolites. As such, cortical This has been demonstrated in rats using indices of 5-HT axonal changes in dopamine/3,4-dihydroxyphenylacetic acid degeneration, such as a reduction in forebrain 5-HT, the 5-HT (DOPAC) and 5-HT/5-HIAA content were measured. However, metabolite 5-hydroxyindolacetic acid (5-HIAA) and reduction for this experiment a higher and more frequent dosing regimen in [3H]-paroxetine binding (a selective ligand for SERT). These was chosen, based on MDMA treatments that have been pre- effects are demonstrated indirectly in human subjects who reg- viously reported to produce maximal effects (Commins et al., ularly use ‘ecstasy’ through reductions in cerebrospinal fluid 1987; O'Hearn et al., 1988). Rats were treated twice daily for (CSF) levels of the 5-HIAA, reduced markers of 5-HT terminals 4 days (10 or 20 mg/kg), then two weeks after the final dose, using PET ligands and various cognitive deficits (McCann cortex was removed and frozen for later measurement of 5-HT/ et al., 1999, 1994; Morgan, 2000; Parrott, 2001; Vollenweider 5-HIAA/dopamine/DOPAC by high-performance liquid chro- et al., 2002). matography assay with electrochemical detection (HPLC-ED). In contrast, the potential for PMA to induce degeneration of monoaminergic fibres within the central nervous system is not 2. Materials and methods clear. An initial study by Steele and co-workers showed that one week after a high dose of PMA (80 mg/kg, twice daily for 2.1. Animals 4 days), forebrain 5-HT and 5-HIAA concentrations were sig- nificantly reduced (Steele et al., 1992). However, the interpre- All experiments were approved by the institutional animal tation of this finding is complicated by the dose used being well care and use committee at the University of Adelaide, and were in excess of the acute LD50 for PMA (Nichols et al., 1975; in strict accordance with the NHMRC guidelines for the Care Steele et al., 1992). Findings from our laboratory indicate re- and Use of Laboratory Animals. Male Sprague–Dawley rats peated PMA treatment resulted in reductions in hippocampal 250–400 g, were obtained from Laboratory Animal Services SERT binding and synaptosomal 5-HT uptake, but not 5-HT (University of Adelaide, Australia). They were housed in content (Callaghan et al., in press). This was in contrast to the groups of three under a 12:12 light/dark cycle, at a constant effects of repeated MDMA treatment, which resulted in a re- temperature of 20±2 °C; food and water were available ad duction in hippocampal SERT binding, synaptosomal 5-HT libitum.