Repeated Administration of the Substituted Amphetamine P

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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).

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 significantly 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. All efforts were made to minimise pain or discomfort to uptake and 5-HT content. As such, further studies are needed to the animals during experimental procedures. 76 P.D. Callaghan et al. / European Journal of Pharmacology 546 (2006) 74–81

2.2. Experiment 1: Repeated dosing of PMA or MDMA: Effect on resuspended in 10 vol of Tris buffer (50 mM Tris, 120 mM NaCl 5-HT transporter binding ([3H]-paroxetine binding) in cortex and 5 mM KCl, pH 7.4). The resultant membrane preparation was incubated with 1 nM [3H]-paroxetine (Amersham, UK; Animals received subcutaneous injections of vehicle (saline), concentration is ∼10 timesNKd) for 60 min at room temperature MDMA or PMA (10 or 20 mg/kg) once per day (at 10 am) for (Habert et al., 1985). Non-specific binding was assessed by co- 4 days. Half of each drug-treatment group was maintained at incubation of [3H]-paroxetine with 10 μM Fluoxetine (Alpha- 22 °C for 6 h after drug administration, while remaining animals pharm, Australia). Membranes were collected in Whatman GF- were maintained at 28 °C for 6 h after drug administration. One B glass fibre filters (pretreated with 0.05% polyethylimine; week after treatment, rats were sacrificed by cervical dislocation, Sigma, Aust.) using vacuum manifolds, then placed in vials the brains removed and the cortex dissected over ice. Cortical containing scintillation fluid (CytoScint; ICN, Aust.), and samples were then homogenised using an Ultramax tissue counted in a Beckman LS 3801 liquid scintillation counter. homogeniser, in 20 vol of isotonic sucrose buffer (0.32 M), and Protein concentration was determined using the bicincho- centrifuged at 2000 g for 15 min at 4 °C (Beckman J2-21 ninic acid (BCA) protein assay (Pierce Chemical company, centrifuge, California, USA). The resulting supernatant was 1984), calibrated using 20–100 μg/ml bovine serum albumin, centrifuged at 30,000 g for 20 min at 4 °C, and the pellet and measurement of absorbance at 562 nm using a Hitachi spectrophotometer.

2.3. Experiment 2: Repeated dosing of PMA or MDMA: Effect on cortical 5-HT and dopamine content

Animals received subcutaneous injections of vehicle (saline), MDMA or PMA (10 or 20 mg/kg) twice daily for 4 days. Animals were maintained at an ambient temperature of 22 °C during the experiment. Two weeks after the final dose, rats were sacrificed, the brains removed, and the cortex dissected over ice. Samples were then homogenised at 4 °C using a Ultramax tissue homogeniser in 10 volumes of 0.5 M perchloric acid. The internal standards, N-methyl-5-HT (for 5-HT) and dihydroxybenzoic acid (DHBA, for dopamine) were used. Homogenates were centrifuged at 9000 g for 10 min. The supernatant was stored at −80 °C for detection of 5-HT/dopamine and their metabolites by HPLC- ED (Bioanalytical systems LC-4B amperometric detector, USA). Samples were injected onto a Beckman reverse phase ODS C18 column (5 μm particles, 4.6×25 cm) at a flow rate of 0.8 ml/min (using a LC 1500 HPLC pump, ICI Instruments, Australia). The mobile phase consisted of 0.1 M Na2HPO4, 0.1 mM ethylene- diamine tetra-acetic acid (EDTA), 0.01 mM octane-sulphonic acid in 12.5% methanol (pH 3.8). The detector applied a +0.7 V potential compared to a Ag/AgCl reference electrode (RE-4, Bioanalytical Systems, USA), with a sensitivity of 2 nA current.

2.4. Drugs

3 Fig. 1. Changes in [ H]-paroxetine binding to cortical membranes two weeks PMA HCl and MDMA HCl were obtained from the Aus- after repeated treatment (once daily for 4 days) with PMA or MDMA. Data are expressed as mean±S.E.M. fmol/mg protein (n values for each group are shown tralian Government Analytical Laboratories (Sydney, Austra- in brackets at the base of each bar). All data were analysed by two-way ANOVA lia). Serotonin HCl, 5-HIAA, Dopamine HCl, N-methyl-5-HT, (drug effect and temperature), with Bonferroni post hoc test. There was a bicinchoninic acid, dihydroxybenzoic acid, EDTA and poly- significant effect of drug (F4,47 =31.66), temperature (F1,47 =7.02) but not drug ethylimine were purchased from the Sigma Chemical Co. (St. 3 effect×temperature (F4,47 =0.14). A: Cortical [ H]-paroxetine binding in Louis, MO, USA). Octane sulphonic acid was obtained from animals held at 22 °C for 6 h after drug administration. Both PMA and 3 MDMA at all doses significantly reduced receptor binding compared to vehicle Aldrich (Milwaukee, WI, USA). [ H]-paroxetine was obtained treatment. ***Pb0.001 All drug treatments cf. vehicle; ##Pb0.01 PMA (10 and from Amersham (UK). CytoScint was obtained from ICN 20 mg/kg) and MDMA (10 mg/kg) cf. MDMA (20 mg/kg). B: Cortical [3H]- (Aust.). Fluoxetine was obtained from Alphapharm (Aust). paroxetine binding in animals held at 28 °C for 6 h after drug administration. Both PMA and MDMA at all doses significantly reduced receptor binding 2.5. Statistics compared to vehicle treatment. ***Pb0.001 PMA (20 mg/kg) and MDMA (10 and 20 mg/kg) cf. vehicle treatment; **Pb0.01 PMA (10 mg/kg) cf. vehicle treatment; ##Pb0.01 PMA (10 mg/kg) cf. MDMA (20 mg/kg); #Pb0.05 PMA All data were expressed as mean±S.E.M. The n values in all (20 mg/kg) and MDMA (10 mg/kg) cf. MDMA (20 mg/kg). statistical analyses refer to the number of animals used in each P.D. Callaghan et al. / European Journal of Pharmacology 546 (2006) 74–81 77 experiment. For multiple comparisons, one way or two-way (Fig. 1A, two way ANOVAwith Bonferroni's post hoc test). In ANOVA tests were used, with Tukey's and Bonferroni post hoc the group of rats treated at an ambient temperature of 28 °C, tests, respectively. Individual analyses used are outlined for both PMA and MDMA treatments at both doses were each experiment, below. Significance was set at Pb0.05. significantly different from the vehicle treatment group, and again the MDMA (20 mg/kg) treatment was significantly 3. Results different compared to both PMA doses and MDMA (10 mg/ kg) treatment (Fig. 1B, two way ANOVA with Bonferroni's 3.1. PMA and MDMA effects on [3H] paroxetine binding in post hoc test). cortical membrane preparations 3.2. PMA and MDMA effects on cortical 5-HT and dopamine Treatment of rats once per day for 4 days with either PMA content (10 or 20 mg/kg) or MDMA (10 or 20 mg/kg) resulted in a significant reduction in [3H]-paroxetine binding to cortical Two weeks after final dosing, MDMA significantly membranes 2 weeks after the final drug treatment (Fig. 1A, B). reduced cortical 5-HT content at both doses (10 mg/kg: 63± There was a significant effect of drug treatment (F4,47 =31.66, 8%; 20 mg/kg: 39±4% cf. vehicle treatment; Fig. 2A), while P b0.001; two way ANOVA), temperature (F1,47 =7.02, PMA had no significant effect compared to vehicle (one way P b0.05), but not drug treatment×temperature (F4,47 =0.14). ANOVA with Tukey's post hoc test). Both PMA (10 and Post hoc analyses indicated all PMA and MDMA treatments to 20 mg/kg: 67±9% and 72±4% respectively cf. vehicle significantly reduce cortical [3H]-paroxetine binding with res- treatment, Fig. 2B) and MDMA (10 and 20 mg/kg: 68±3% pect to vehicle treatment (Fig. 1A and B, two way ANOVA and 51±5% respectively cf. vehicle treatment, Fig. 2B) with Bonferroni's post hoc test). In the group of rats treated at appeared to have maximal effects on the reduction of cortical an ambient temperature of 22 °C, MDMA (20 mg/kg) also 5-HIAA content. Neither PMA nor MDMA at both doses reduced binding to a significantly greater extent compared to significantly altered cortical dopamine content (Fig. 2C) or both PMA doses (10 and 20 mg/kg) and MDMA (10 mg/kg) DOPAC content (Fig. 2D).

Fig. 2. Cortical 5-HT and dopamine concentration in animals two weeks after repeated treatment (twice daily for 4 days) with PMA and MDMA. All data are represented as mean±S.E.M. (n values for each group are shown in brackets at the base of each bar). Data were analysed by one way ANOVAwith Tukey's post hoc test. A: Cortical 5-HT concentration was significantly reduced (***Pb0.001 cf. control) after MDMA treatment (10 and 20 mg/kg), but not PMA treatment. B: Cortical 5-HIAA concentration was significantly reduced by PMA and MDMA treatment (*Pb0.05, **Pb0.01, ***Pb0.001 cf. control). C: Cortical dopamine concentration was unaltered by PMA or MDMA treatment. D: Cortical DOPAC concentration was unaltered by PMA or MDMA treatment. 78 P.D. Callaghan et al. / European Journal of Pharmacology 546 (2006) 74–81

4. Discussion The once daily drug treatment also allowed us to test if an increase in ambient temperature to 28 °C for a 6 h period after This study provides evidence that repeated PMA treatment in injection could potentiate any effect on SERT binding. This rats does lead to alterations in aspects of serotonergic neuro- shorter period of ambient temperature change than previously transmission, as seen with MDMA and other substituted am- tested was thought to model more closely the time period that phetamines. Repeated PMA administration reduced cortical human subjects would be exposed to higher ambient temperature [3H]-paroxetine binding (as could be expected if there was when taking ‘ecstasy’ in nightclubs. Interestingly, the increase in indeed degeneration of cortical 5-HT fibres). However, an the ambient temperature to 28 °C for 6 h after drug administration increase in ambient temperature for a 6 h period after drug did not potentiate the reduction in [3H]-paroxetine binding seen administration did not potentiate the effects on [3H]-paroxetine for either drug. However, the elegant study of Malberg and Seiden binding seen. Surprisingly, using a more frequent dosing regi- has shown after a single MDMA dose (20 mg/kg), a shift in men, cortical 5-HT content was unaltered by PMA treatment, ambient temperature to 28 °C was great enough to significantly yet cortical 5-HIAA content was significantly reduced. As has reduce cortical 5-HT and 5-HIAA concentrations two weeks post been shown extensively in prior studies (Green et al., 2003; treatment, while no effect was seen at the same dose given at an McKenna and Peroutka, 1990), profound reductions were seen ambient temperature of 22 °C (Malberg and Seiden, 1998). The in both cortical [3H]-paroxetine binding and 5-HT/5-HIAA difference in findings may be explained by variations in content after repeated MDMA treatment. However, increased experimental design compared with the current study. Malberg ambient temperature for a 6 h period after drug administration and Seiden housed animals at the chosen ambient temperature for was also unable to significantly change the effects of MDMA 24 h after the one dose of drug given, as opposed to 6 h in the treatments on alterations of [3H]-paroxetine binding. current study (for each of the 4 days of drug administration). The As was expected from previous studies, all MDMA treatment use of a once daily dosage regimen for this experiment was in part groups displayed significant reductions in cortical 5-HT/5-HIAA necessitated by the considerable differences in metabolism content, and cortical [3H]-paroxetine binding to SERT. This is between the two drugs. PMA is metabolised to its primary indicative of degeneration of a subset of 5-HT fibres within metabolite, p-hydroxyamphetamine, and mostly excreted with particular brain regions including cortex, that have been shown 24 h (Kitchen et al., 1979). While p-hydroxyamphetamine is using markers including those chosen in this study (Battaglia et potent amine releasing agent, inhibitor of amine uptake and al., 1988, 1987; Bogen et al., 2003; Callaghan et al., in press; indirect noradrenergic agonist (Kaminskas et al., 2002; Raiteri et Callahan et al., 2001; Commins et al., 1987; Hewitt and Green, al., 1977), it is no longer present in blood or brain 6 h after a 1994; Lew et al., 1996; O'Hearn et al., 1988; O'Shea et al., 1998; 10 mg/kg dose of PMA (Kaminskas et al., 2002). However, the Sabol et al., 1996; Scanzello et al., 1993; Schmidt, 1987). Both non-linear pharmacokinetics and longer half life of MDMA the measures of regional SERT binding and indolamine content metabolism would mean the plasma concentration of MDMA were chosen as they have been previously used extensively to would be unlikely to return to baseline if multiple daily dosing indicate the neurotoxic potential of many other substituted am- was used (Farre et al., 2004; Lim and Foltz, 1988). As such, using phetamines (Huang et al., 1992; Johnson and Nichols, 1991; a daily dosing regimen, both parent drugs should have returned to Nichols et al., 1990; Sprague et al., 1996). The reductions seen in near baseline concentrations for the subsequent day's dosing. our study in cortical [3H]-paroxetine binding were comparable in A second, more aggressive, twice daily treatment regimen magnitude to those seen for both MDMA doses in previous was chosen to examine the effects of PMA or MDMA treatment studies in rats (Battaglia et al., 1988; O'Shea et al., 1998). The on cortical 5-HT and 5-HIAA. A 20 mg/kg MDMA dose using 10 mg/kg regimen of MDMA provided an intermediate dose this regimen has been shown to produce maximal depletions of where significant reductions in cortical [3H]-paroxetine binding 5-HT in cortical regions (Battaglia et al., 1988). As no effect of were seen, yet were still significantly less than the 20 mg/kg increased ambient temperature 6 h post treatment was seen, regimen, as previously shown (Battaglia et al., 1988; O'Shea together with the complicating factor of substantial difference in et al., 1998). SERT binding has been shown previously to be a metabolism using multiple daily dosing, animals were housed at more sensitive measure of MDMA induced degeneration room temperature for all drug treatments. It is intriguing that in (Battaglia et al., 1987; Green et al., 2003), which lead us to spite of increasing the frequency of dosage of drug per day, use of this marker with the once daily treatment regimen. cortical 5-HT content was unaltered by PMA treatment. As In contrast, the long term effects of PMA administration on previously mentioned, the effects of MDMA on cortical 5-HT/5- [3H]-paroxetine binding are less clear. Repeated PMA treatment HIAA were comparable to the reductions seen in the literature was shown to reduce cortical [3H]-paroxetine binding, indi- (Battaglia et al., 1988; Green et al., 2003; McKenna and cating it may indeed induce 5-HT fibre degeneration like Peroutka, 1990). However PMA treatment did reduce cortical 5- MDMA. Both doses (10 and 20 mg/kg) produced maximal HIAA content, which is difficult to reconcile. PMA is known to effects, but were significantly less than the 20 mg/kg MDMA be a potent reversible inhibitor of monoamine oxidase A, a key treatment (at ambient temperature). The use of higher doses of enzyme in 5-HT metabolism (Green and El Hait, 1980). How- PMA would not be advisable, as greater doses than 20 mg/kg of ever, within 24 h approximately 70% of PMA dose is meta- PMA produce significantly greater hyperthermic effects (com- bolised to its primary metabolite and excreted, indicating PMA pared to the same dose of MDMA) which often lead to signi- would not be present in vivo after 1 week (Kitchen et al., 1979). ficant acute morbidity (Daws et al., 2000). Evidence for a potential neurodegenerative action of PMA has P.D. Callaghan et al. / European Journal of Pharmacology 546 (2006) 74–81 79 been investigated in only two prior studies. Steele and co- to be due to degeneration of 5-HT fibres when cortical 5-HT workers found a 30–46% reduction in hippocampal, hypotha- concentrations are not reduced. lamic and frontal cortical 5-HT and 5-HIAA content in rats one In this study, the two markers chosen indicate repeated PMA week after PMA (80 mg/kg, twice daily for 4 days) (Steele et al., treatment results in alteration of cortical 5-HT neurotransmis- 1992). The dose used, however, was far in excess of the LD50 sion, which could be explained by mechanisms such as down- (40–70 mg/kg) of PMA (Nichols et al., 1975), and as such, regulation of SERT (consistent with reduced cortical [3H]- complications exist with the interpretation of the data if only the paroxetine binding) and/or degeneration of 5-HT fibres. The surviving animals were represented. Concurrent studies from reduction in 5-HIAA content with no effect on cortical 5-HT our laboratory have also shown repeated PMA treatment (15 mg/ content after PMA administration could also be as a result of kg for 4 days, twice daily) also resulted in reduced hippocampal reduced 5-HT synthesis and monoamine oxidase function. In SERT binding but not 5-HT content, 2 weeks after final dosing contrast, MDMA treatment was shown to produce significant (Callaghan et al., in press). Again, as seen in the current study, reductions in all the aforementioned indices of 5-HT function, repeated MDMA treatment (15 mg/kg for 4 days, twice daily) yet a 6 h period of increased ambient temperature did not appear reduced hippocampal SERT binding and 5-HT content. Inter- to be long enough to potentiate the effects of either PMA or estingly, the reduction in SERT binding after PMA administra- MDMA on reductions in SERT binding. In summary, a more tion in that study resulted in a reduction in 5-HT clearance in comprehensive study including other measures of 5-HT neu- vivo, again consistent with serotonergic degeneration. Intrigu- ronal localisation and function is required to establish the exact ingly, repeated MDMA administration did not alter in vivo 5-HT nature of the long term effects of PMA on 5-HT neurotrans- clearance, which was an unexpected finding. The consistency in mission, and to determine whether these effects generalise to the findings of this and the current study, in two brain regions, other 5-HT innervated regions of the brain. indicates the differences in alteration of 5-HT neurotransmission between PMA and MDMA may be also seen in other brain Acknowledgements nuclei with serotonergic innervation. The mechanisms underly- ing this difference warrant further investigation. This study was supported by funding from the NHMRC to Interestingly, some preliminary evidence exists indicating RJI and an Australian Postgraduate Award to PDC. PMA produces peroxynitrite radicals in vivo to a significantly greater extent than MDMA, indicating a potential mechanism References for degeneration of nerve terminals in common between these drugs (Imam et al., 2001a). Peroxynitrite radicals have been Battaglia, G., Yeh, S.Y., O'Hearn, E., Molliver, M.E., Kuhar, M.J., De Souza, E.B., 1987. 3,4-Methylenedioxymethamphetamine and 3,4-methylenedioxyampheta- implicated in MDMA and methamphetamine induced neuro- mine destroy serotonin terminals in rat brain: quantification of neurodegeneration degeneration (Darvesh et al., 2005; Imam et al., 2001b). by measurement of [3H]paroxetine-labeled serotonin uptake sites. J. Pharmacol. Recent studies indicate that data providing evidence for Exp. Ther. 242, 911–916. 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