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] 0006-8993/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2005.01.093 A. Freezer et al. / Brain Research 1041 (2005) 48–55 49 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.