Towards finding a population biomarker for wastewater epidemiology studies.

Wastewater analysis has the potential to provide objective information on community drug use. Introducing a population biomarker (PB) in the sample analysis may significantly reduce errors in the back-calculation associated with population estimation and wastewater volume measurement. A number of potential PBs have been suggested but no systematic evaluation has been conducted so far. This study evaluated the eligibility of the previously suggested PB candidates (creatinine, cholesterol, coprostanol and cotinine) as well as three new ones (cortisol, androstenedione and 5-hydroxyindoleacetic acid (5-HIAA)) using five criteria. We assessed the quantification method, affinity to particulate matter and stability of candidates in wastewater, as well as the constancy of inter-day excretion and correlation between excretion and census population. All PB candidates were quantifiable in wastewater. Cholesterol and coprostanol were eliminated from further consideration due to affinity to particulate matters in the wastewater. Creatinine, cortisol and androstenedione were disqualified for stability reasons. On a population scale, both cotinine and 5-HIAA were excreted (RSD=8.01 ± 1.13% and 10.20 ± 0.89%, respectively) at a constant rate and concentrations of each correlated well with the census population (r=0.9809 and 0.9442, respectively). Overall, both cotinine and 5-HIAA are eligible PBs, but the neurotransmitter metabolite 5-HIAA may be more suitable for international comparisons.

Increases in use of novel synthetic stimulant are not directly linked to decreased use of 3,4-methylenedioxy-N-methylamphetamine (MDMA).

A decline in 3,4-methylenedioxy-N-methylamphetamine (MDMA) use in Adelaide, Australia from 2009 to 2010 was confirmed by us previously. Reports suggested that the shortage in MDMA supply was associated with an increased prevalence of other synthetic stimulants, but quantitative measurements were unavailable. To obtain objective data on the community use of synthetic stimulants, we collected wastewater samples from multiple treatment plants in Adelaide, Australia from 2009 to 2011 and analysed them using solid-phase extraction/liquid chromatography/tandem mass spectrometry (SPE-LC-MS/MS), targeting MDMA and some of the most reported synthetic cathinones and piperazines. Data were temporally compared. MDMA and six other synthetic stimulants were detected and quantified in wastewater samples. While MDMA level decreased markedly from 2009 to 2010 and remained low in 2011, localized increased use of mephedrone, methylone, methylenedioxypyrovalerone (MDPV), benzylpiperazine (BZP), 3-trifluoromethylphenylpiperazine (TFMPP), but not methcathinone, was observed in 2010 and 2011. This suggested that the decline in MDMA use was associated with an increase in the use of a number of other synthetic stimulants. However, the lag time from the decrease in MDMA to the increase in use of a number of these stimulants, together with the highly regionalized use of all synthetic stimulants except methcathinone indicates that there was no direct population wide substitution in response to the reduction in MDMA.

Evaluation of pre-analysis loss of dependent drugs in wastewater: stability and binding assessments.

Wastewater analysis has the potential to provide objective and timely data on population drug consumption, but some crucial factors such as pre-analysis drug loss during sample storage and filtration could affect the accuracy and reliability of the method, and these uncertainties have yet to be fully assessed. This study was designed to evaluate analyte stability in wastewater stored under different conditions with the aim of optimizing the sample storage procedures for future studies. It also investigated whether there is significant analyte loss during filtration before sample extraction and storage after that. The studied substances and metabolites were: cotinine, cocaine and its metabolite benzoylecgonine, phenethylamines amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA), opioids including codeine, methadone, 6-monoacetylmorphine (MAM) and morphine. In most situations, storing samples at 4 °C is sufficient to stabilize analytes for at least 2 weeks, and refrigeration is unnecessary during sample transportation within 3 days. However, additional measures need to be taken if unstable analytes such as cocaine and MAM are to be analyzed. No significant analyte loss was observed in the filtration process or in reconstituted extract stored at 4 °C or -20 °C for 2 weeks. By choosing stable analytes and proper storage conditions, wastewater analysis has the potential to provide accurate data for estimation of community drug use.

Antidepressant-like effects of ecstasy in subjects with a predisposition to depression.

INTRODUCTION: Positive effects of ecstasy on mood and self-esteem due to increased synaptic serotonin levels may indicate a potential antidepressant-like action. This effect may be more prominent in subjects with a pre-existing mood disturbance who may use ecstasy more frequently as a 'self-medication'. This study compared depressive symptoms and the immediate effects of ecstasy on mood in subjects with (WP) and without (NP) a predisposition to depression. METHODS: Current ecstasy users were assessed using the profile of mood states (POMS) and beck depression inventory (BDI) when drug-free, and during social gathering, when 20 subjects voluntarily consumed ecstasy (ecstasy group) and 20 abstained from ecstasy (control group). Predisposition to depression was determined using the Brief Symptom Inventory. During social gathering, POMS and BDI were administered 60 min after ecstasy consumption, or at matched time for controls. 3,4-Methylenedioxymethamphetamine (MDMA) exposure was confirmed using saliva samples collected 60 min after pill ingestion. RESULTS: There was no difference in ecstasy use patterns between the groups. When drug-free, the WP subjects had greater mood disturbance and depressive symptoms than the NP group (POMS: NP 5.85±1.63, WP 14.5±2.81, p<0.05, BDI: NP 4.9±0.86, WP 11.2±1.65, p<0.01). During social gathering, WP subjects who consumed ecstasy reported a significant decrease in depressive symptoms (F(1,35)=5.47, p<0.05). CONCLUSIONS: A decrease in depressive symptoms was observed in subjects predisposed to depression. This antidepressant-like action of MDMA may contribute to its use, particularly among people with an existing or latent depressive disorder.

Antidepressant-like effects of 3,4-methylenedioxymethamphetamine in an animal model of depression.

Increased synaptic serotonin (5-hydroxytryptamine) levels may underlie antidepressant-like effects of 3,4-methylenedioxymethamphetamine (MDMA) that may be more prominent in subjects with mood disturbance. The Flinders Sensitive Line (FSL) strain is an important animal model of depression. These rats are more immobile in the forced swimming test (FST), and their immobility is reversed by known antidepressants after prolonged administration. The objective of this study was to determine whether MDMA administration has a dose-dependent antidepressant-like effect in this animal model of depression. The effects of MDMA at 5 and 10 mg/kg following single and repeated administration were assessed in FSL rats using the FST. Sprague-Dawley rats were used as a control. During both FST sessions, saline-treated FSL rats were significantly more immobile than Sprague-Dawley rats (P<0.001). Acute MDMA administration had a dose-dependent antidepressant-like effect in FSL rats, which was most evident after 10 mg/kg. This effect was diminished after repeated administration. Methamphetamine 2 mg/kg, which was used as a positive control for locomotor activity induction, did not affect the depressive-like state in FSL rats. There were no changes in the cortical levels of 5-hydroxytryptamine and 5-hydroxyindoleacetic acid after treatments. It is concluded that MDMA exhibited an antidepressant-like effect in FSL rats, which was most evident following acute administration.

Marked decline in 3,4-methylenedioxymethamphetamine (MDMA) based on wastewater analysis.

OBJECTIVE: Recent reports in Europe suggest a decline in 3,4-methylenedioxymethamphetamine (MDMA; Ecstasy) use, but quantifiable and objective measurement is unavailable. The global extent of changes in MDMA and related stimulant use is also unclear. This study aims to quantify changes in MDMA use in Australia and determine whether these changes have been accompanied by differing amounts of other stimulant use. METHOD: We acquired information on recent use of MDMA and related illicit stimulants in Australia using the method of wastewater analysis. Untreated wastewater samples collected from three metropolitan treatment plants in Adelaide from May to July 2009 and the same months in 2010 were analyzed. Concentrations of MDMA, methamphetamine, and benzoylecgonine (a metabolite of cocaine) were determined using solid phase extraction-liquid chromatography- tandem mass spectrometry. Weekly consumed doses of MDMA, methamphetamine, and cocaine per 1,000 people were estimated. RESULTS: From 2009 to 2010, weekly consumption of MDMA decreased from mean of 4.52 (SEM = 0.74) doses/week per 1,000 people to 0.08 (0.01) doses/week per 1,000 people (p < .001); weekly consumption of methamphetamine increased from a mean of 48.35 (6.13) doses/week per 1,000 people to 68.13 (5.33) doses/week per 1,000 people (p < .05); and weekly consumed doses of cocaine did not significantly change. Local roadside saliva testing data also showed that the MDMA-positive test rate decreased from 0.30% to 0.05% and the methamphetamine-positive test rate increased from 1.43% to 1.52% during the past 2 years. CONCLUSIONS: This study shows a 50-fold decrease in consumed doses of MDMA with a rise in methamphetamine use in Australia over a 1-year period.

Population drug use in Australia: a wastewater analysis.

Accurate information on drug use in communities is essential if health, social and economic harms associated with illicit drug use are to be addressed efficiently. In most countries population drug use is estimated indirectly via surveys, medical presentations and police and custom seizures. All of these methods have at least some problems due to bias, small samples and/or long time delays between collecting the information and analysing the results. Recently the direct quantification of drug residues in wastewater has shown promise as a means of monitoring drug use in defined geographical areas. In this study we measured 3,4-methylenedioxymethamphetamine (MDMA), methamphetamine and benzoylecgonine in sewage inflows in metropolitan and regional areas of Australia and compared these data with published European data. Cocaine use was small compared to European cities (p<0.001) but was compensated for by much greater consumption of methamphetamine (p<0.001) and MDMA (p<0.05). MDMA was more popular in regional areas (p<0.05) whereas methamphetamine and cocaine were mainly consumed in the city (p<0.05). Greater than 5-fold increases in MDMA use were detected on weekends (p<0.001). This approach has the potential to improve our understanding of drug use in populations and should be further developed to improve prevention and treatment programs.

Pill content, dose and resulting plasma concentrations of 3,4-methylendioxymethamphetamine (MDMA) in recreational 'ecstasy' users.

AIMS: To improve our understanding of the pharmacology of 'ecstasy' in recreational environments; in particular, to describe the composition of ecstasy pills, patterns of ecstasy use and the relationship between dose of 3,4-methylendioxymethamphetamine (MDMA) and resulting plasma concentrations. DESIGN, SETTING AND PARTICIPANTS: A naturalistic observational study of 56 experienced 'ecstasy' users in recreational settings in Australia. MEASUREMENTS: Drug use patterns (number of pills consumed, other drugs consumed). drug content of pills and resultant plasma concentrations of MDMA and related drugs were assessed by gas chromatography/mass spectrometry (GC/MS). FINDINGS: Ecstasy pills generally contained MDMA, but this was often combined with other drugs such as 3,4-ethylendioxyethylamphetamine (MDEA) and methamphetamine. The dose of MDMA per pill ranged from 0 to 245 mg and users consumed from one-half to five pills, with the total dose consumed ranging up to 280 mg. Plasma concentrations of MDMA increased with number of pills consumed and cumulative MDMA dose. Use of larger numbers of pills was associated with extended exposure to the drug. CONCLUSIONS: MDMA is the major active drug in ecstasy pills, but there is a high degree of variation in doses. Use of multiple pills over the course of one session is common and results in a sustained increase in MDMA plasma concentrations over a number of hours. This is likely to lead to a much greater exposure of the brain to MDMA than would be predicted from controlled single-dose pharmacokinetic studies.

Increased effects of 3,4-methylenedioxymethamphetamine (ecstasy) in a rat model of depression.

3,4-Methylenedioxymethamphetamine (MDMA, ecstasy) is associated with increases in core body temperature (T(C)) and depressive mood states in users. Flinders Sensitive Line (FSL) rats represent a rat model of depression originally bred from Sprague-Dawley (SD) rats. They are more sensitive to both muscarinic and serotonergic agonists and have altered thermoregulatory responses to various drugs. To examine the link between MDMA and depression, eight FSL and eight SD rats were administered saline and 5 and 7.5 mg/kg MDMA. Immediately following administration, rats were confined to an area with an ambient temperature (T(A)) of 30 ± 1°C for 30 minutes before being allowed access to a thermal gradient for four hours. The brains were removed one week after final dose of MDMA and concentrations of serotonin and dopamine were measured. Treatment with MDMA at both doses led to a higher T(C) in the FSL rats than the SD rats at high T(A) (P < 0.01). Fatalities due to hyperthermia occurred in the FSL rats after both doses, whereas all but one of the SD rats recovered well. Heart rate was also much higher after MDMA in the FSL rats throughout the experiments. The FSL rats showed significant decreases in all transmitters measured (P < 0.05). These differences between strains were not accounted for by altered blood or brain concentrations of MDMA. The results indicate that the FSL rats may be more susceptible to developing MDMA-induced hyperthermia and possible damage to the brain. These findings may be of importance to human users of MDMA who also have depression.

A new metabotropic glutamate receptor agonist with in vivo anti-allodynic activity.

As part of the vital search towards improved therapeutic agents for the treatment of neuropathic pain, the central nervous system glutamate receptors have become a major focus of research. Outlined herein are the syntheses of two new biologically active 3'-cycloalkyl-substituted carboxycyclopropylglycines, utilizing novel synthetic chemistry. The reaction between substituted 1,2-dioxines and an aminophosphonate furnished the cyclopropane core in a single step with all required stereochemistry of pendant groups. In vitro binding assays at metabotropic glutamate receptors revealed selective activity. In vivo testing in a rodent model of neuropathic pain indicated one amino acid significantly and dose-dependently decreased mechanical allodynia.

Potentiation of 3,4-methylenedioxymethamphetamine-induced 5-HT release in the rat substantia nigra by clorgyline, a monoamine oxidase A inhibitor.

1. It is well established that the commonly used recreational drugs 3,4-methylenedioxymethamphetamine (MDMA, 'ecstasy') and para-methoxyamphetamine (PMA) facilitate the release and prevent the reuptake of 5-hydroxytryptamine (5-HT, serotonin). Although these drugs have similar potencies for their abilities to increase the release and inhibit the re-uptake of 5-HT, PMA has greater potency as an inhibitor of monoamine oxidase (MAO)-A. 2. The present study compared the abilities of PMA and MDMA to increase extracellular 5-HT concentrations in animals with functional MAO-A and when MAO-A activity was inhibited by clorgyline. 3. Samples of extracellular fluid from rat substantia nigra were collected using microdialysis and then analysed for 5-HT and 5-hydroxyindol acetic acid (5-HIAA) by high-performance liquid chromatography coupled with electrochemical detection. The 5-HT-mediated effects on body temperature and behaviour were also recorded. Rats were pretreated with saline or 10 mg/kg, i.p., clorgyline and, 24 h later, injected with 10 mg/kg MDMA, PMA or saline. 4. Both MDMA and PMA produced significant increases in extracellular 5-HT concentrations (482 +/- 83 and 726 +/- 287%, respectively; P < 0.05). Rats treated with PMA and MDMA displayed significantly increased 5-HT-related behavours (P < 0.05). Furthermore, only MDMA was capable of producing additional significant increases in 5-HT concentrations (1033 +/- 131%; P < 0.01) when coadministered with clorgyline. 5. The results of the present study suggest that PMA and MDMA are similar in their abilities to increase extracellular 5-HT levels in animals with functional MAO-A activity. However, coadministration of these substituted amphetamines with an MAO-A inhibitor causes significant potentiation in the ability to increase extracellular levels of 5-HT for MDMA, but not PMA.

Nandrolone potentiates arrhythmogenic effects of cardiac ischemia in the rat.

Anabolic steroid abuse has been associated with thrombosis and arteriosclerosis, both of which predispose to myocardial ischemia and infarction. However, there are reports of sudden cardiac death in the absence of thrombus and atheroma following anabolic steroid use. Although treatment with the commonly abused steroid, nandrolone, has been shown to decrease recovery of systolic function following ischemia in isolated rat hearts, it is unknown whether anabolic steroids can increase the incidence of fatal arrhythmia associated with cardiac ischemia. Anesthetized male Sprague-Dawley rats were administered vehicle or nandrolone (10-160 microg/kg/min iv) 10 min prior to 15-min occlusion of the left anterior descending coronary artery followed by 10-min reperfusion. Nandrolone, in this dose range, did not significantly change heart rate, blood pressure, or cardiac rhythm in the absence of ischemia. However, the fraction of rats surviving ischemia was significantly (p < 0.05) decreased by nandrolone at both 40 and 160 microg/kg/min, while survival time during ischemia was decreased significantly (p < 0.001) by nandrolone 160 microg/kg/min. An increase (p < 0.05) in the duration of ventricular fibrillation was noted at the highest compared to the lowest dose of nandrolone, corresponding to a significant increase in the fraction of rats experiencing ventricular fibrillation (p < 0.01). Nandrolone had no effect on the frequency or duration of ventricular fibrillation or survival time during reperfusion. Although the mechanisms underlying these effects are currently unclear, they indicate that exposure to anabolic steroids in combination with transient reductions in coronary blood flow may explain some reports of sudden cardiac death in anabolic steroid users.

In vivo analysis of serotonin clearance in rat hippocampus reveals that repeated administration of p-methoxyamphetamine (PMA), but not 3,4-methylenedioxymethamphetamine (MDMA), leads to long-lasting deficits in serotonin transporter function.

p-Methoxyamphetamine (PMA) has been implicated in fatalities as a result of 'ecstasy' (MDMA) overdose worldwide. Like MDMA, acute effects are associated with marked changes in serotonergic neurotransmission, but the long-term effects of PMA are poorly understood. The aim of this study was to determine the effect of repeated PMA administration on in vitro measures of neurodegeneration: serotonin (5-HT) uptake, 5-HT transporter (SERT) density and 5-HT content in the hippocampus, and compare with effects on in vivo 5-HT clearance. Male rats received PMA, MDMA (4 or 15 mg/kg s.c., twice daily) or vehicle for 4 days and 2 weeks later indices of SERT function were measured. [(3)H]5-HT uptake into synaptosomes and [(3)H]cyanoimipramine binding to the SERT were significantly reduced by both PMA and MDMA treatments. 5-HT content was reduced in MDMA-, but not PMA-treatment. In contrast, clearance of locally applied 5-HT measured in vivo by chronoamperometry was only reduced in rats treated with 15 mg/kg PMA. The finding that 5-HT clearance in vivo was unaltered by MDMA treatment suggests that in vitro measures of 5-HT axonal degeneration do not necessarily predict potential compensatory mechanisms that maintain SERT function under basal conditions.

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.

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 [(3)H]-paroxetine binding (SERT density) one week after final drug administration. When rats were housed at a higher ambient temperature (28 degrees C vs. 22 degrees 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.

Reversal of morphine, methadone and heroin induced effects in mice by naloxone methiodide.

Opioid overdose, which is commonly associated with opioid induced respiratory depression, is a problem with both therapeutic and illicit opioid use. While the central mechanisms involved in the effects of opioids are well described, it has also been suggested that a peripheral component may contribute to the effects observed. This study aimed to further characterise the effects of the peripherally acting naloxone methiodide on the respiratory, analgesic and withdrawal effects produced by various opioid agonists. A comparison of the respiratory and analgesic effects of morphine, methadone and heroin in male Swiss-Albino mice was conducted and respiratory depressive ED(80) doses of each opioid determined. These doses (morphine 9 mg/kg i.p., methadone 7 mg/kg i.p., and heroin 17 mg/kg i.p.) were then used to show that both naloxone (3 mg/kg i.p.) and naloxone methiodide (30-100 mg/kg i.p.) could reverse the respiratory and analgesic effects of these opioid agonists, but only naloxone precipitated withdrawal. Further investigation in female C57BL/6J mice using barometric plethysmography found that both opioid antagonists could reverse methadone induced decreases in respiratory rate and increases in tidal volume. Its effects do not appear to be strain or sex dependent. It was concluded that naloxone methiodide can reverse the respiratory and analgesic actions of a variety of opioid agonists, without inducing opioid withdrawal.

Pharmacokinetic profile of single and repeated oral doses of MDMA in squirrel monkeys: relationship to lasting effects on brain serotonin neurons.

A large body of data indicates that (+/-)3,4-methylenedioxymethamphetamine (MDMA, 'ecstasy') can damage brain serotonin neurons in animals. However, the relevance of these preclinical data to humans is uncertain, because doses and routes of administration used in animals have generally differed from those used by humans. Here, we examined the pharmacokinetic profile of MDMA in squirrel monkeys after different routes of administration, and explored the relationship between acute plasma MDMA concentrations after repeated oral dosing and subsequent brain serotonin deficits. Oral MDMA administration engendered a plasma profile of MDMA in squirrel monkeys resembling that seen in humans, although the half-life of MDMA in monkeys is shorter (3 vs 6-9 h). MDMA was biotransformed into MDA, and the plasma ratio of MDA to MDMA was 3-5 / 100, similar to that in humans. MDMA accumulation in squirrel monkeys was nonlinear, and plasma levels were highly correlated with regional brain serotonin deficits observed 2 weeks later. The present results indicate that plasma concentrations of MDMA shown here to produce lasting serotonergic deficits in squirrel monkeys overlap those reported by other laboratories in some recreational 'ecstasy' consumers, and are two to three times higher than those found in humans administered a single 100-150 mg dose of MDMA in a controlled setting. Additional studies are needed on the relative sensitivity of brain serotonin neurons to MDMA toxicity in humans and non-human primates, the pharmacokinetic parameter(s) of MDMA most closely linked to the neurotoxic process, and metabolites other than MDA that may play a role.

Plasma drug concentrations and physiological measures in 'dance party' participants.

The increasing use of (+/-) 3,4-methylenedioxymethamphetamine (MDMA) in the setting of large dance parties ('raves') and clubs has been the source of some concern, because of potential acute adverse events, and because animal studies suggest that MDMA has the potential to damage brain serotonin (5-HT) neurons. However, it is not yet known whether MDMA, as used in the setting of dance parties, leads to plasma levels of MDMA that are associated with toxicity to 5-HT neurons in animals. The present study sought to address this question. Plasma MDMA concentrations, vital signs, and a variety of blood and urine measures were obtained prior to, and hours after, individuals attended a dance party. After the dance party, subjects were without clinical complaints, had measurable amounts of residual MDMA in plasma, and nearly half of the subjects also tested positive for methamphetamine, another amphetamine analog that has been shown to have 5-HT neurotoxic potential in animals. Plasma concentrations of MDMA did not correlate with self-reported use of 'ecstasy' and, in some subjects, overlapped with those that have been associated with 5-HT neurotoxicity in non-human primates. Additional subjects were likely to have had similar concentrations while at the dance party, when one considers the reported time of drug ingestion and the plasma half-life of MDMA in humans. Hematological and biochemical analyses were generally unremarkable. Moderate increases in blood pressure, heart rate and body temperature were observed in the subjects with the highest MDMA plasma concentrations. These findings are consistent with epidemiological findings that most people who use MDMA at dance parties do not develop serious clinical complications, and suggest that some of these individuals may be at risk for developing MDMA-induced toxicity to brain serotonin neurons.

Differences in the in vivo dynamics of neurotransmitter release and serotonin uptake after acute para-methoxyamphetamine and 3,4-methylenedioxymethamphetamine revealed by chronoamperometry.

Illicit use of p-methoxyamphetamine (PMA) is rapidly increasing. However, little is known about the acute effects of PMA on neurotransmission in vivo. High-speed chronoamperometry was used to monitor neurotransmitter release and clearance in anesthetized rats after local application of PMA or 3,4-methylenedioxymethamphetamine (MDMA). In striatum, PMA caused less neurotransmitter release than MDMA. PMA-evoked release could be partially blocked by pre-treatment with a serotonin (5-HT) reuptake inhibitor, suggesting that evoked 5-HT release contributed to the electrochemical signal and was mediated by the 5-HT transporter (SERT). MDMA-evoked release was not blocked by a SERT inhibitor, suggesting that primarily DA was released. To study the effect of these amphetamines on clearance of 5-HT mediated specifically by the SERT, clearance of exogenously applied 5-HT was measured in the CA3 region of the hippocampus. In contrast to the striatum where 5-HT is cleared by both the SERT and the dopamine transporter (DAT), 5-HT is cleared primarily by the SERT in the CA3 region. This is also a region where neither PMA nor MDMA evoked release of neurotransmitter. The maximal inhibition of 5-HT clearance was greater after PMA than MDMA. These data demonstrate in vivo (1) brain region variability in the ability of PMA and MDMA to evoke release of neurotransmitter; (2) that clearance of 5-HT in the striatum is mediated by both the SERT and the DAT; (3) distinct differences in the amount and nature of neurotransmitter released in the striatum after local application of PMA and MDMA and (4) that PMA is a more efficacious inhibitor of 5-HT clearance in the hippocampus than MDMA. These fundamental differences may account for the more severe adverse reactions seen clinically after PMA, compared to MDMA.

Effects of 3,4-methylenedioxymethamphetamine and related amphetamines on autonomic and behavioral thermoregulation.

3,4-Methylenedioxymethamphetamine (MDMA, 'ecstasy') and related amphetamines such as para-methoxyamphetamine (PMA) disrupt normal thermoregulation in humans and rats. Behavior, an important component of thermoregulation in mammals, has not been investigated with respect to these drugs. This is surprising as harm minimization depends on appropriate thermoregulatory behavior by drug users. The effects of MDMA (10 mg/kg), PMA (10 mg/kg) and d-amphetamine (2 mg/kg) were therefore studied in Sprague-Dawley rats, with telemetry implants measuring core body temperature (T(C)), locomotor activity and heart rate. Rats were administered an amphetamine or saline and confined to an ambient temperature of 21, 30 or 15 degrees C for 30 min, before being able to choose their preferred temperature (T(P)) on a thermally graded runway (11-41 degrees C). Confinement at 21 degrees C had little effect on T(C) in any group. At 30 degrees C MDMA and PMA increased T(C) compared to saline (p<0.001). MDMA treated animals behaviorally overcompensated for this effect (p<0.01). Locomotor activity after MDMA treatment was significantly elevated compared with saline (p<0.01). In contrast, at 15 degrees C MDMA administration resulted in a lower T(C) than saline (p<0.001). MDMA and PMA disrupt autonomic components of thermoregulation, while behavioral components are disrupted to a lesser extent. These results highlight differences in thermoregulatory responses to individual drugs, which were only evident when behavior was measured, and this may be important in assessing their risk.

Effects of 3,4-methylenedioxymethamphetamine (MDMA, 'Ecstasy') and para-methoxyamphetamine on striatal 5-HT when co-administered with moclobemide.

3,4-Methylenedioxymethamphetamine (MDMA, "ecstasy") 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 < 0.05). Rats treated with PMA and MDMA displayed significantly increased 5-HT-related behaviors (P < 0.05). Furthermore, only MDMA was capable of producing additional significant increases in 5-HT concentrations (980%, P < 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.