Effects of 3,4-Methylenedioxymethamphetamine (MDMA, Decstasyt) and Para-Methoxyamphetamine on Striatal 5-HT When Co-Administered with Moclobemide

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Brain Research 1041 (2005) 48–55 www.elsevier.com/locate/brainres Research report Effects of 3,4-methylenedioxymethamphetamine (MDMA, dEcstasyT) and para-methoxyamphetamine on striatal 5-HT when co-administered with moclobemide

Alexander Freezer, Abdallah SalemT, Rodney J Irvine

Department of Clinical and Experimental Pharmacology, Medical School North, University of Adelaide, Adelaide, South Australia 5005, Australia

Accepted 27 January 2005 Available online 5 March 2005

Abstract

3,4-Methylenedioxymethamphetamine (MDMA, becstasyQ) and para-methoxyamphetamine (PMA) are commonly used recreational drugs. PMA, often mistaken for MDMA, is reported to be more toxic in human use than MDMA. Both of these drugs have been shown to facilitate the release and prevent the reuptake of 5-hydroxytryptamine (5-HT, serotonin). PMA is also a potent inhibitor of monoamine oxidase type A (MAO-A), an enzyme responsible for the catabolism of 5-HT, and this characteristic may contribute to its increased toxicity. In humans, co-administration of MDMA with the reversible MAO-A inhibitor moclobemide has led to increased apparent toxicity with ensuing fatalities. In the present study, using microdialysis, we examined the effects of co-administration of MDMA and PMA with moclobemide on extracellular concentrations of 5-HT and 5-hydroxy indol acetic acid (5-HIAA) in the striatum of the rat. 5-HT-mediated effects on body temperature and behavior were also recorded. Rats were pretreated with saline or 20 mg/kg (i.p.) moclobemide and 60 min later injected with 10 mg/kg MDMA, PMA, or saline. Dialysate samples were collected every 30 min for 5 h and analyzed by HPLC-ED. Both MDMA and PMA produced significant increases in extracellular 5-HT concentrations (590% and 360%, respectively, P b 0.05). Rats treated with PMA and MDMA displayed significantly increased 5-HT-related behaviors (P b 0.05). Furthermore, only MDMA was capable of producing additional significant increases in 5-HT concentrations (980%, P b 0.05) when co-administered with moclobemide. These data suggest that co-administration of MDMA with moclobemide increases extracellular 5-HT and 5-HT-mediated behaviors and may cause increased 5-HT related toxicity similar to that reported with PMA. D 2005 Elsevier B.V. All rights reserved.

Theme: Neural basis of behavior Topic: Drugs of abuse: amphetamine and other stimulants

Keywords: 3,4-Methylenedioxymethamphetamine; para-methoxyamphetamine; Ecstasy; Monoamine oxidase-A inhibitor; Moclobemide

1. Introduction aged 20–29 in Australia are reported to have used it recently [8]. The effects of both drugs are characterized by sensory 3,4-Methylenedioxymethamphetamine (MDMA; euphoria, a sense of well-being and increased energy, though becstasyQ)andpara-methoxyamphetamine (PMA) are illicit the effects of PMA have a more hallucinogenic profile. substituted amphetamine drugs used recreationally. MDMA There have been a number of studies demonstrating that has become increasingly popular in Australia and worldwide both MDMA and PMA elevate the concentrations of over the last 15 years as a party drug, and is currently the monoamine neurotransmitters dopamine and 5-HT via the third most popular illicit drug in Australia. 10% of people release of vesicle stores and the inhibition of their reuptake [10,12,31]. The acute toxicity of MDMA and PMA is T Corresponding author. Fax: +61 8 8224 0685. attributed to 5-HT, since their effects are similar to the E-mail address: [email protected] (A. Salem). condition known as the serotonin syndrome [27]. The URL: http://www.adelaide.edu.au/health/pharm. serotonin syndrome has been reported both in animals [11]

and clinically [9] and is due to increased activation of 5-HT1 2.2. Brain microdialysis and 5-HT2 receptors in the central nervous system and periphery. The serotonin syndrome produces hyperthermia, Rats anesthetized with 400 mg/kg chloral hydrate were rhabdomyolysis, and hyponatremia which in extreme cases placed on heat pad to maintain body temperature at 37 8C can lead to the onset of a coma state, cerebral edema or and the head was fixed in a stereotaxic frame (KOPF death [12]. Although PMA is a structurally and pharmaco- instruments). The skull was exposed, bregma located and an logically similar to MDMA, it is regarded as having higher intracerebral cannula and guide stylet (BAS MD-2251) was toxicity in human use [2]. implanted into the striatum. The coordinates used to situate A possible reason for PMA’s increased toxicity may be the cannula were A, +1.2 mm; L, 2.2 mm relative to bregma due to its greater potency as an inhibitor of MAO-A and V, À5.5 mm below the dura according to Paxinos and compared to MDMA. Although in vitro studies have revealed Watson [28]. The cannula was held in place with dental that MDMA is able to inhibit MAO-A activity [20], PMA is cement. Animals were kept on the heating pad until they one hundred times more potent than MDMA [29] and this regained full consciousness. The animals were allowed may underlie its apparent greater toxicity when compared to recover for 24 h before the microdialysis experiment with MDMA. Mitochondrial MAO-A is an enzyme involved started. in the catabolism of monoamine neurotransmitters thereby During the recovery period and throughout the micro- contributing to the regulation of their synaptic concentrations dialysis experiment, animals were kept in a clear Perspex [4]. MAO-A is specific for 5-HTas a substrate, deaminating it observation bowl (BAS bBee KeeperQ rodent residence) and into the neurochemically inactive 5-HIAA [14,15]. given ad libitum access to food and water. All experiments Reversible MAO-A inhibitors such as moclobemide are were conducted at normal room temperature (22 8C) and the currently prescribed clinically to treat depression and social relative humidity was 50–52%. Following the 24-h recovery anxiety [33]. There is evidence that the presence of a MAO- period, microdialysis probes (BAS MD-2004, RR-4) were A inhibitor increases MDMA toxicity. Vuori et al. [32] inserted and perfused at a flow rate of 1.5 Al/min using a reported four deaths in Finland attributed to the co- BAS pump with artificial cerebrospinal fluid (aCSF) (in administration of MDMA with moclobemide. None of the mM): NaCl 125, KCl 2.5, MgCl2.6H2O 1.18, Na2HPO4 2, victims reported by Vuori et al. [32] had a valid prescription adjusted to pH 7.4 with phosphate buffer. Samples were for the moclobemide they ingested. It is possible that collected every 30 min into 1.5-ml Eppendorf tubes MDMA users purposefully co-administer MAO-A inhibi- containing 10 Al of 2% acetic acid for 6 h. To determine tors to increase and prolong the effects of MDMA. in vitro recovery, microdialysis probes were immersed in a It is hypothesized that co-administration of MDMA with 10 ng/ml solution of 5-HT and 5-HIAA and perfused with a potent MAO-A inhibitor moclobemide will potentiate the aCSF at 1.5 Al/min. Dialysate samples were immediately brain 5-HT levels and 5-HT-mediated behavior and body analyzed for 5-HT and 5-HIAA by high-performance liquid temperature. Conversely, co-administration of PMA with chromatography coupled with electrochemical detection moclobemide will not lead to potentiation of the 5-HT effects (HPLC-ED) as described below. as PMA is already a potent MAO-A inhibitor. The aim of this study was to determine the neurochemical and 5-HT- 2.3. HPLC-ED mediated behavioral effects of MDMA and PMA when co- administered with moclobemide. We used in vivo micro- The HPLC-ED system consisted of a controller (Shi- dialysis in fully conscious rats to monitor the extracellular 5- madzu LC-10AD), degasser (Shimadzu DGU-14AD) and HT and 5-HIAA concentrations in the striatum. Doses of BAS LC-4B fitted with a working electrode potential set at MDMA and PMA used in our study are based on previous 0.7 V with a range of 0.1 nA. The mobile phase was microdialysis work [10] demonstrating moderate changes in composed of (in mM): NaH2PO4 100, octanesulphonic acid extracellular 5-HT and 5-HIAA concentrations. Animal 1, EDTA 0.1, and 16% methanol, and was adjusted to pH behavior was simultaneously observed and core body 2.9 with phosphoric acid. The mobile phase was filtered, temperature was measured aurally at 30-min intervals. degassed, and delivered at a flow rate of 0.07 ml/min. Compounds of interest were separated using 100 Â 1.0 AD Luna 3A c18 column (Phenomenex) and sampling was 2. Materials and methods recorded using an ICI DP800 Chromatograph Data station (Version 2.5). 2.1. Animals 2.4. Drug treatments Male Sprague–Dawley rats weighing 250 g to 310 g were purchased from the Adelaide University Animal House MDMA, PMA, and moclobemide were dissolved in and kept on a 12/12-h light/dark cycle. Animals were given 0.9% saline solution and administered via the intraperitoneal ad libitum access to food and water. Ethics approval was route (i.p). 60 min after microdialysis probe insertion, obtained from the University of Adelaide ethics committee. animals were injected with 20 mg/kg moclobemide or saline 50 A. Freezer et al. / Brain Research 1041 (2005) 48–55 and challenged with 10 mg/kg MDMA, 10 mg/kg PMA, or 2.8. Chemicals and reagents saline 120 min post microdialysis probe insertion. (F)-MDMA and (F)-PMA were provided by the 2.5. Behavior and temperature Australian Government Analytical Laboratories (Sydney, Australia). 4-Chloro-N(2-morpholinoethyl)benzamide, Behavior was scored every 30 min for 5 h using a (C13H17O2Cl, moclobemide) was generously supplied by behavioral scale previously used by Molloy and Wadding- Hoffman La Roche, Switzerland. Chloral hydrate ton [21]. The scale was used to measure the effects of (C2H3Cl3O2), sodium chloride, potassium chloride, di- stimulant drugs on the behavioral response of male sodium hydrogen orthophosphate, sodium dihydrogen Sprague–Dawley rats and they are similar to behavioral orthophosphate, acetic acid, hydrochloric acid, and meth- features of the bserotonin syndromeQ observed in rats. In anol were provided by BHD Laboratory Supplies Pty. addition to using the behavioral scale described by Molloy Limited (Victoria, Australia). Ethylenediaminetetraacetic and Waddington [21], we have also recorded additional acid (EDTA) and 1-Octanesulphonic acid (OSA) were classic symptoms of the bserotonin syndromeQ in rats such sourced from Sigma-Aldrich, St. Louis, USA. as forepaw treading, penile erection, pilorection and proptosis of the eyes. At each measurement, the rat’s behavior was rated and the following scores were allocated: 3. Results 0, asleep, inactive; 1, episodes of normal activity; 2, discontinuous activity with bursts of prominent sniffing or 3.1. Experimental validation rearing; 3, continuous stereotyped behavior along a fixed path; 4, continuous stereotyped behavior fixated in one Precision and accuracy for 5-HT and 5-HIAA were location; 5, stereotyped behavior with bursts of licking or within acceptable limits (range 1.9–10%). Coefficient gnawing; and 6, continuous licking or gnawing. Core body values generated from the calibration curve were greater temperature was recorded using infrared aural thermometer than 0.99 for both 5-HT and 5-HIAA: 0.9981 for 5-HT, (Braun ThermoScan) as described by O’Loinsigh et al. 0.9942 for 5-HIAA (n = 4). In vitro microdialysis probe [25]. recovery recorded for 5-HT was 10.89 F 0.74% and 13.93 F 1.12% for 5-HIAA (n = 4). Histological sections performed 2.6. Histology on each rat confirmed correct probe placement in the striatum and schematic representation of microdialysis At the end of each experiment, animals were sacrificed probe placement is shown in Fig. 1. via anesthetic overdose with chloral hydrate. Brains were stored on formalin until further histology was performed. 3.2. Effects of MDMA and moclobemide on extracellular 50- to 100-Am histological slices were cut along the coronal 5-HT and 5-HIAA concentrations plane using a Vibratome 1000 (Lancer). Slices were mounted onto gelatin-coated slides, stained with cresylecht Basal 5-HT values of the different treatment groups did violet and used in the verification of probe placement under not differ significantly. The means (F SEM; n = 3–4) of the a light microscope. basal concentration in the striatum dialysate were as follows: 5-HT, 18 F 0.34 pg/40 Al; and 5-HIAA, 192 F 4.88 pg/40 2.7. Data analysis Al. Fig. 2 displays the effects of 10 mg/kg MDMA alone and when co-administered with 20 mg/kg moclobemide (Moc) 5-HT and 5-HIAA concentrations for each treatment on extracellular 5-HT levels in the striatum. Data are group were calculated by linear regression of peak area from expressed as dialysate 5-HT (% of Baseline) F SEM and the standard curve. Increases in concentrations of 5-HT and as area under curve (AUC) 0 to 240 min after saline (Sal), 5-HIAA are expressed as percentage change from the mean pre-drug baseline values. Changes in extracellular neurotransmitters for each treatment group were assessed using one-way ANOVA with Tukey post hoc test. Area under the curve (AUC) for each treatment group was calculated from 0 to 240 min and compared with one-way ANOVA with Tukey post hoc test. Due to its non-parametric nature, behavioral data were analyzed using a Kruskal–Wallis test followed by Dunn’s post test. P b 0.05 was considered statistically significant. Temperature data were analyzed in two ways: (1) to identify within group changes from 0 to Fig. 1. Schematic representation of microdialysis probe placement. Arrow 240 time point and (2) to identify changes between groups on the left side of the picture represents the probe track in the striatum using using one-way ANOVA with Tukey post Hoc test. coordinates according to Paxinos and Watson [28]. A. Freezer et al. / Brain Research 1041 (2005) 48–55 51

shown in Fig. 3. 5-HIAA concentrations decreased significantly in Moc/Sal (66.6 F 3.2%, P b 0.05) and Moc/MDMA (69.2 F 3.4%, P b 0.05)-treated rats.

Fig. 2. The effects of MDMA (A) or PMA (B) when co-administered with moclobemide (Moc) or saline (Sal) on extracellular 5-HT concentrations in the rat striatum. Sal or Moc (20 mg/kg, i.p.) was administered at À60 min as indicated by the first arrow; Sal, MDMA (10 mg/kg, i.p.) or PMA (10 mg/ kg, i.p.) was administered at 0 min as indicated by the second arrow. Data expressed as dialysate 5-HT (% of Baseline) F SEM (n = 3–4). Histogram (C) represents area under curve (AUC) 0 to 240 min after MDMA or PMA injection. *P b 0.05 compared with Sal/Sal, one-way ANOVA, Tukey post test.

Fig. 3. The effects of MDMA (A) or PMA (B) when co-administered with MDMA or PMA injection (n = 3–4). Significant increases in moclobemide (Moc) or saline (Sal) on extracellular 5-HIAA concentrations in 5-HT concentrations were seen in Sal/MDMA-treated rats the rat striatum. Sal or Moc (20 mg/kg, i.p.) was administered at À60 min as (590 F 180% P b 0.05) and Moc/MDMA treatment indicated by the first arrow; Sal, MDMA (10 mg/kg, i.p), or PMA (10 mg/kg, i.p) was administered at 0 min as indicated by the second arrow. Data ex- produced additional significant increase in 5-HT concen- pressed as dialysate 5-HIAA (% of Baseline) F SEM (n = 3–4). Histogram (C) trations (988.9 F 172.4%, P b 0.05). The effects of MDMA represents area under curve (AUC) 0 to 240 min after MDMA or PMA in- and moclobemide treatment on 5-HIAA concentrations are jection. *P b 0.05 compared with Sal/Sal, one-way ANOVA,Tukey post test. 52 A. Freezer et al. / Brain Research 1041 (2005) 48–55

3.3. Effects of PMA and moclobemide on extracellular 5-HT and 5-HIAA concentrations

Significant increases in 5-HT concentrations were seen in Sal/PMA-treated (360.5 F 140.5%, P b 0.05) and Moc/ PMA-treated (418 F 166.3%, P b 0.05) rats (Fig. 2). No further significant increases were observed when PMA was co-administered with moclobemide. The effects of PMA on 5-HIAA concentrations are shown in Fig. 3. 5-HIAA concentrations decreased significantly in Sal/PMA-treated (63.2 F 9.8%, P b 0.05) and Moc/PMA-treated (47.9 F 6.6%, P b 0.05) rats. After reaching minimum levels, 5- HIAA concentrations began to either increase again as seen in with Moc/Sal or remained depressed as seen with Moc/PMA and Sal/PMA.

3.4. Effects of MDMA and PMA on behavior

Fig. 4 shows substituted amphetamine-treated animals had significantly increased ( P b 0.05) behavior scores over saline-only treated controls. Moc/MDMA treatment produced additional significantly increased behavior ( P b 0.05, AUC) compared to MDMA-only treated rats. In addition to the onset of increased stereotyped scores observed in MDMA-treated rats, all rats treated with MDMA or PMA exhibited the following behaviors: forepaw treading, penile erection, pilorection and proptosis of the eyes.

3.5. The effects of MDMA and PMA co-administration with moclobemide on core body temperature

Fig. 5 shows the temporal changes of core body temperature caused by MDMA and PMA. Core body temperature in Sal/PMA-treated rats was significantly increased at 60 min compared to Sal/Sal (P b 0.05) and Moc/Sal ( P b 0.05). Within-group statistical analysis (comparison of initial to peak temperatures) revealed that the following treatment groups had significant increases: Moc/MDMA (2.3 F 0.4 8C, P b 0.05), Moc/PMA (2.7 F 0.4 8C, P b 0.05), and Sal/PMA (2.9 F 0.3 8C, P b 0.05) (n = 4 for all). Sal/Sal-, Moc/Sal-, and Sal/MDMA-treated rats did not have any significant increases.

Fig. 4. Behavioral effects of MDMA (A) and PMA (B) at doses of 10 mg/kg, 4. Discussion i.p. when co-administered with moclobemide (Moc) or saline (Sal). Sal or Moc was administered at À60 min as shown by the first arrow; Sal, MDMA Consistent with earlier in vivo microdialysis studies (10 mg/kg, i.p.), or PMA (10 mg/kg, i.p.) was administered at 0 min as shown F [10,13,34], our results have shown that both MDMA and by the second arrow. Data are given as mean SEM (n = 4). Histogram (C) represents area under curve (AUC) 0 to 240 min after MDMA or PMA PMA produced significant increase in extracellular 5-HT injection. *P b 0.05, compared with Sal/Sal, Kruskal–Wallis non-parametric concentrations in the striatum of rats. Furthermore, the data test with Dunn’s post test. described here have also shown that MDMA produced a significantly greater increase in 5-HT concentrations when co-administered with the MAO-A inhibitor moclobemide ramifications relating to increased serotonin toxicity in while PMA-treated rats showed no additional significant humanscausedbyco-administrationofMDMAwith increases in 5-HT concentrations. These findings may have moclobemide. A. Freezer et al. / Brain Research 1041 (2005) 48–55 53

trations by 62%, and showed a decrease throughout the 160- min sampling period. The results of the current study showed a statistically significant decrease in 5-HIAA levels after 120 min and returned to basal levels 4 h after moclobemide administration. Effects of moclobemide on 5-HIAA concentrations is in agreement with its reported half life in the rat [24]. Although increases in 5-HT concentrations were seen following moclobemide administration at 60 and 180 min, due to small group numbers, it did not achieve statistical significance. It should also be noted that Da Prada et al. [6] reported orally administered moclobemide (10 mg/kg) in rats increased brain levels of 5- HT by only 150% which is comparable to values reported in this study. Animals treated with moclobemide and MDMA showed a 2-fold increase in extracellular 5-HT concentrations compared to MDMA-only treated rats. Moclobemide pretreatment of MDMA also reduced 5-HIAA levels significantly, suggesting effective MAO-A inhibition. In addition, our results demonstrated that when moclobomide is co-administered with MDMA, 5-HT concentrations peaked at 60 min and this significant increase was sustained up to 180 min and it appears to parallel the time course of MAO-A inhibition. This is in contrast to MDMA effect on 5-HT concentrations where it peaked at 60 min and then started to declined at 90 min. In fact, based on the data presented in this paper, it could be argued that moclobemide pretreatment increases MDMA induced 5-HT effects. Rats treated with PMA did not show any statistically significant increase in 5-HT levels when pretreated with moclobemide. In contrast to MDMA, PMA almost exclu- sively affects the release and inhibits the reuptake of 5-HT with limited effect on other monoamine neurotransmitters [7,30]. Daws et al. [7] have postulated that PMA is more potent than MDMA in increasing 5-HT levels in vivo due to PMA’s potent MAO-A inhibition in vitro [29]. However, to date, there has been little animal data to support this notion of PMA’s increased 5-HT effect. Microdialysis studies using PMA (10 mg/kg) have shown statistically significant increases in 5-HT levels (350%), yet the increases were not statistically significant when compared to MDMA [10]. The present study also found no significant differences between PMA- and MDMA-mediated increases in 5-HT Fig. 5. Effects of MDMA (A) and PMA (B) co-administered with concentrations. Since PMA inhibits MAO-A and subse- moclobemide (Moc) on core body temperature. Saline (Sal) or Moc was administered at À60 min as shown by the first arrow; Sal, MDMA (10 mg/ quent deamination of 5-HT to 5-HIAA, decreases in 5- kg, i.p.) or PMA (10 mg/kg, i.p.) was administered at 0 min as shown by the HIAA concentrations in the current study could be second arrow. Data are given as mean F SEM (n = 4). Histogram expected. In accordance with this hypothesis, and consistent (C) represents area under curve (AUC) 0 to 240 min after MDMA or PMA with previous microdialysis studies [10], PMA-mediated b b injection. *P 0.05, **P 0.01, compared with Sal/Sal one-way ANOVA decreases in concentrations of 5-HIAA were statistically with Tukey post test. ##P b 0.01 for comparison to Moc/Sal using one-way ANOVA, Tukey post test. significant. The serotonin syndrome was first described by Grahame- Smith [11], and similar effects such as hyperactivity, head Moclobemide treatment has been shown to result in weaving, and forepaw treading have been observed in significant decreases in 5-HIIA concentrations in rats [17]. MDMA-treated rats in a dose-dependent fashion [15,30]. A microdialysis study by Iurlo et al. [17] reported The current study found that the onset of the stereotyped moclobemide (20 mg/kg, i.p.) reduced 5-HIAA concen- behavior was increased in rats treated with MDMA when 54 A. Freezer et al. / Brain Research 1041 (2005) 48–55 compared to PMA-treated rats. In addition, moclobemide MDMA have been previously reported [32]. The increases pretreatment produced higher behavior scores for MDMA- seen in 5-HT and related effects in our animal data strongly treated rats. Peaks in behavioral activity coincided with suggest that the underlying mechanisms involve increased peaks in extracellular 5-HT concentrations measured in the brain extracellular concentrations of 5-HT. The data striatum, giving correlation to their effect. Since other presented here also support the hypothesis that the greater workers have observed that MDMA has more locomotor toxicity reported with the human use of PMA may be effects compared to PMA [7,16], the pronounced behavioral partially due to its potent effects on the inhibition of MAO. effect caused by MDMA is not surprising. This is thought to It would be desirable to gain further knowledge of this be due to the lack of dopamine activity in PMA-treated interaction at various MDMA and PMA doses and at animals [16]. Moclobemide pretreated rats experienced increased ambient temperatures to simulate the conditions increased onset of penile erection, pilorection and proptosis where these drugs are used in the community. Examination of the eyes, which are classic symptoms of the serotonin of the role of other neurotransmitters, such as dopamine, and syndrome [11]. Low behavior scores for moclobemide- other brain regions would also be desirable. treated rats confirmed reports by Burkard et al. [1] that it has minimal effect on rat behavior. Lin et al. [18] demonstrated that increases in 5-HT levels Acknowledgments in the median preoptic hypothalamus may play a role in causing hyperthermic effects. Ling et al. [19] considered the Gratitude is expressed to Paul Callaghan and Robert hyperthermic effects of MDMA and PMA contribute Moyer for their technical assistance in preparing brain slices significantly to their acute toxicity. Earlier studies have used in the verification of probe placement. They also thank recorded MDMA-induced increases in core body temper- the National Health and Medical Research Council of ature in rats at normal room temperature [3,21,22,25,26]. Australia for their financial support. However, Dafters and Lynch [5] have shown that temperature responses to MDMA vary, with hyperthermia and hypothermia experienced at ambient temperatures of 24 8C and 17 8C, respectively. Although our data did not References demonstrate that the combination of Moc/MDMA produced a statistically significant increase in body temperature [1] W.P. Burkard, M. Da Prada, H.H. Keller, R. Kettler, W. Haefely, Pre- clinical pharmacology of moclobemide: a review of published studies, compared with Sal/MDMA, it was apparent that moclobe- Br. J. Psychiatry 155 (1989) 84–88. mide pretreatment significantly enhanced the hyperthermic [2] D.G. Caldicott, N.A. Edwards, A. Kruys, K.P. Kirkbride, D.N. Sims, responses elicited by MDMA alone. Within-group statistical R.W. Byard, M. Prior, R.J. Irvine, Dancing with bdeathQ: p- analysis revealed that MDMA alone was unable to produce methoxyamphetamine overdose and its acute management, J. Toxicol., significant increase in body temperature; however, moclo- Clin. Toxicol. 41 (2003) 143–154. [3] M.I. Colado, T.K. Murray, A.R. Green, 5-HT lose in rat brain following bemide pretreatment caused significant increase in the 3,4-methylenedioxymethamphetamine (MDMA), p-chloroamphet- maximum core body temperature. The present data suggest amine and fenfluramine administration and effects of chlormethiazole a possible link between increases in striatal 5-HT concen- and dizocilpine, Br. J. Pharmcol. 108 (1993) 582–583. tration and hyperthermic responses elicited by MDMA. [4] A. Colzi, F. D’Agostini, A.M. Cesura, E. Borroni, M. Da Prada, PMA is also reported to significantly increase core body Monoamine oxidase-A inhibitors and dopamine metabolism in rat caudatus: evidence that an increased cytosolic level of dopamine temperature in the animal model [23] and it is more potent displaces reversible monoamine oxidase-A inhibitors in vivo, than MDMA at increasing body temperature [2].The J. Pharmacol. Exp. Ther. 265 (1992) 103–111. present study partially supports this findings, with PMA [5] R. Dafters, E. Lynch, Persistent loss of thermoregulation in rat induced producing statistically significantly higher peak temper- by 3,4 methyl-enedioxymethamphetamine (MDMA or becstasyQ ) but atures compared to saline controls. Other studies [7] have not by fenfluramine, Psychopharmacology 138 (1998) 207–212. [6] M. Da Prada, R. Kettler, H.H. Keller, W.P. Burkard, D. Muggli- reported attenuated increases in body temperature compared Maniglio, W.E. Haefely, Neurochemical profile of moclobemide, a to MDMA. Pretreatment with moclobemide in PMA-treated short acting and reversible inhibitor of monoamine oxidase type A, rats did not change the intensity of the temperature response J. Pharmacol. Exp. Ther. 248 (1989) 400–414. but did increase the time taken for the peak in core body [7] L.C. Daws, R.J. Irvine, P.D. Callaghan, N.P. Toop, J.M. White, F. temperature to occur. Previous rat fatalities have been Bochner, Differential behavioural and neurochemical effects of para-methoxyamphetamine and 3,4-methylenemethamphetamine in recorded using PMA [7] where maximum core body the rat, Prog. Neuro-Psychopharmacol. Biol. Psychiatry 24 (2000) temperatures of 42 8C were exceeded. No animals in the 955–977. present study attained body temperatures above 42 8C, nor [8] L. Degenhardt, B. Barker, L. Topp, Patterns of ecstasy use in were any drug-related fatalities recorded. The fatalities Australia: findings from a national household survey, Addiction 99 observed by Daws et al. [7] were recorded at ambient (2004) 187–195. [9] R.A. Ener, S.B. Meglathery, W.A. Van Decker, R.M. Gallagher, temperatures of 30 8C. Serotonin syndrome and other serotonergic disorders, Pain Med. 4 Human deaths attributed to the serotonin syndrome (2003) 63–74. caused by the co-administration of moclobemide and [10] B. Gough, S.Z. Imam, B. Blough, W. Slikker, S.F. Ali, Comparative A. Freezer et al. / Brain Research 1041 (2005) 48–55 55

effects of substituted amphetamines (PMA, MDMA and METH) on and corticosterone concentrations in the rat after the administration of monoamines in rat caudate: a microdialysis study, Ann. N. Y. Acad. 3,4-methylenedioxymethamphetamine, J. Pharmcol. Exp. Ther. 245 Sci. 965 (2002) 410–420. (1988) 873–879. [11] D.G. Grahame-Smith, Studies in vivo on the relationship between [23] D.E. Nichols, M. Ilhan, J.P. Long, Comparison of cardiovascular, brain tryptophan, brain 5-HT synthesis and hyperactivity in rats treated hyperthermic and toxic effects of para-methoxyamphetamine (PMA) with a monoamine oxidase inhibitor and l-tryptophan, J. Neurochem. and 3,4-methylene dioxyamphetamine (MDA), Arch. Int. Pharmaco- 18 (1971) 1053–1066. dyn. Ther. 214 (1975) 133–140. [12] A.R. Green, A.O. Mechan, J.M. Elliott, E. O’Shea, M.I. Colado, The [24] S. Oie, T.W. Guentert, L. Tolentino, G. Hermodsson, Pharmacoki- pharmacology and clinical pharmacology of 3,4-methylenedioxyme- netics of moclobemide in male, virgin female, pregnant and nursing thamphetamine (MDMA, bEcstasyQ), Pharmacol. Rev. 55 (2003) rats, J. Pharm. Pharmacol. 44 (1992) 413–418. 463–508. [25] E.D. O’Loinsigh, G. Boland, J.P. Kelly, K.M. O’Boyle, Behavioural, [13] G.A. Gudelsky, J.F. Nash, Carrier-mediated release of serotonin by hyperthermic and neurotoxic effects of 3,4-methylenedioxymetham- 3,4-methylenedioxymethamphetamine—implications for serotonin– phetamine analogues in the Wistar rat, Prog. Neuro-Psychopharmacol. dopamine interactions, J. Neurochem. 66 (1996) 243–249. Biol. Psychiatry 25 (2001) 621–638. [14] W. Haefely, W.P. Burkard, A.M. Cesura, R. Kettler, H.P. Lorez, J.R. [26] E. O’Shea, R. Granados, B. Esteban, M.I. Colado, A.R. Green, The Martin, J.G. Richards, R. Scherschlicht, M. Da Prada, Biochemistry relationship between the degree of neurodegeneration of rat brain 5- and pharmacology of moclobemide, a prototype RIMA, Psychophar- HT nerve terminals and the dose frequency of administration of macology 106 (1992) S6–S14. MDMA (becstasyQ), Neuropharmacology 37 (1998) 919–926. [15] W. Haefely, W.P. Burkard, A.M. Cesura, R. Kettler, H.P. Lorez, J.R. [27] A.C. Parrott, Recreational ecstasy/MDMA, the serotonin syndrome Martin, J.G. Richards, R. Scherschlicht, M. Da Prada, Pharmacology and serotonergic neurotoxicity, Pharmacol. Biochem. Behav. 71 of moclobemide, Clin. Neuropharmacol. 16 (1993) S8–S18. (2002) 837–844. [16] K.M. Hegadoren, M.T. Martin-Iverson, G.B. Baker, Comparative [28] G. Paxinos, C. Watson, The Rat Brain in Stereotaxic Coordinates, behavioural and neurochemical studies with a psychomotor stimulant, 2nd ed., Academic Press, Sydney, 1986. an hallucinogen and 3,4-methlenedioxy analogues of amphetamine, [29] M.C. Scorza, C. Carrau, R. Silveira, G. Zapata-Torres, B.K. Cassels, Psychopharmacology 118 (1995) 295–304. M. Reyes-Parada, Monoamine oxidase inhibitory properties of some [17] M. Iurlo, G. Leone, B. Schilstrfm, L. Linne´r, G. Nomikos, P. Hertel, methoxylated and alkylthio amphetamine derivatives, Biochem. B. Silvestrini, H. Svensson, Effects of harmine on dopamine output Pharmacol. 54 (1997) 1361–1369. and metabolism in rat striatum: role of monoamine oxidase-A [30] L.J. Spanos, B.K. Yamamoto, Acute and subchronic effects of inhibition, Psychopharmacology 159 (2001) 98–104. methylenedioxymethamphetamine[FMDMA] on locomotion and the [18] M.T. Lin, H.J. Tsay, W.H. Su, F.Y. Chueh, Changes in extracellular serotonin syndrome behaviour in the rat, Pharmacol. Biochem. Behav. serotonin in rat hypothalamus affect thermoregulatory function, Am. 32 (1989) 835–840. J. Physiol. 274 (1998) R1260–R1267. [31] L.F. Tseng, R.J. Hitzemen, H.H. Loh, Comparative effects of dl-p- [19] L.H. Ling, C. Marchant, N.A. Buckley, M. Prior, R.J. Irvine, methoxyamphetamine and d-amphetamine on catecholamine release Poisoning with the recreational drug paramethoxyamphetamine, and reuptake in vitro, J. Pharmacol. Exp. Ther. 189 (1974) 708–715. Med. J. Aust. 174 (2001) 453–455. [32] E. Vuori, J.A. Henry, I. Ojanper7, R. Nieminen, T. Savolainen, P. [20] E.T. Leonardi, E.C. Azmitia, MDMA (ecstasy) inhibition of MAO Wahlsten, M. J7ntti, Death following ingestion of MDMA (ecstasy) type A and type B: comparisons with fenfluramine and fluoxetine and moclobemide, Addiction 98 (2003) 365–368. (Prozac), Neuropsychopharmacology 10 (1994) 231–238. [33] M. Yamada, H. Yasuhara, Clinical pharmacology of MAO inhibitors: [21] A.G. Molloy, J.L. Waddington, Assessment of grooming and other safety and future, Neurotoxicology 25 (2004) 215–221. behavioural responses to the D-1 dopamine receptor agonist SK and F [34] B.K. Yamamoto, J.F. Nash, G.A. Gudelsky, Modulation of methyl- 38393 and its R-andS-enantiomers in the intact adult rat, enedioxymethamphetamine-induced striatal dopamine release by the Psychopharmacology 92 (1987) 164–168. interaction between serotonin and g-aminobutyric acid in the [22] J.F. Nash, H.Y. Meltzer, G.A. Gudelsky, Elevation of serum prolactin substantia nigra, J. Pharmacol. Exp. Ther. 273 (1995) 1063–1070.