Increased kynurenine concentration attenuates serotonergic neurotoxicity induced by 3,4-methylenedioxymethamphetamine (MDMA) in rats through activation of aryl hydrocarbon receptor.

3,4-Methylenedioxymethamphetamine (MDMA) is an amphetamine derivative that has been shown to produce serotonergic damage in the brains of primates, including humans, and of rats. Tryptophan, the precursor of serotonin, is primarily degraded through the kynurenine (KYN) pathway, producing among others KYN, the main metabolite of this route. KYN has been reported as an endogenous agonist of the aryl hydrocarbon receptor (AhR), a transcription factor involved in several neurological functions. This study aims to determine the effect of MDMA on the KYN pathway and on AhR activity and to establish their role in the long-term serotonergic neurotoxicity induced by the drug in rats. Our results show that MDMA induces the activation of the KYN pathway, mediated by hepatic tryptophan 2,3-dioxygenase (TDO). MDMA also activated AhR as evidenced by increased AhR nuclear translocation and CYP1B1 mRNA expression. Autoradiographic quantification of serotonin transporters showed that both the TDO inhibitor 680C91 and the AhR antagonist CH-223191 potentiated the neurotoxicity induced by MDMA, while administration of exogenous l-kynurenine or of the AhR positive modulator 3,3'-diindolylmethane (DIM) partially prevented the serotonergic damage induced by the drug. The results demonstrate for the first time that MDMA increases KYN levels and AhR activity, and these changes appear to play a role in limiting the neurotoxicity induced by the drug. This work provides a better understanding of the physiological mechanisms that attenuate the brain damage induced by MDMA and identify modulation of the KYN pathway and of AhR as potential therapeutic strategies to limit the negative effects of MDMA.

Implicación de la molécula de adhesión ALCAM en el daño cerebral. Modelo de daño neuronal mediante la administración de MDMA/éxtasis en rata / Implication of the adhesion molecule ALCAM in the experimental rat model of brain damage caused by MDMA/ecstasy

Objetivo: Caracterizar la expresión de ALCAM en vasos de corteza cerebral de ratas tratadas con MDMA. 2) Estudiar el efecto que sobre su expresión y sobre la neurotoxicidad producida por MDMA tiene ibuprofeno. Materiales y métodos: Se administró una dosis neurotóxica de MDMA a ratas Dark Agouti e buprofeno a diferentes tiempos. Se midió la temperatura de los animales durante los tratamientos y se estudió la expresión de ALCAM en los vasos de corteza. El daño cerebral se estudió midiendo los niveles de ácido 5-indolacético, serotonina y la densidad de su transportador. Resultados: MDMA produce un aumento de ALCAM a las 24 horas (p<0.01). El co-tratamiento con ibuprofeno lo disminuye (p<0.01) y atenúa el daño cerebral disminuyendo los efectos neurotóxicos de MDMA sobre los niveles de serotonina cortical (p<0.0001) y la densidad del transportador (p<0.0001). Ibuprofeno disminuye ligeramente la hipertermia producida por MDMA. Conclusiones: MDMA aumenta la expresión de ALCAM. Los datos sugieren la posibilidad de utilizar compuestos anti-inflamatorios como ibuprofeno que reducen este efecto sobre ALCAM y que disminuyen parcialmente el daño cerebral, si bien es necesario analizar la participación de la disminución de la temperatura en dicha protección (AU)

Objective: 1) Characterization of ALCAM adhesion molecule expression in cortical vessels of MDMA-treated rats. 2) Study of the effect of the anti-inflammatory compound ibuprofen on ALCAM expression and on the neurotoxicity produced by MDMA. Material and methods: Male Dark Agouti rats were given a neurotoxic dose of MDMA. Ibuprofen was given before and at various times after MDMA. Rectal temperature was monitored during the treatment and ALCAM expression in vessels from cerebral cortex was determined at 24 h. In neurotoxicity studies, cortical 5-HT tissue levels and 5-HT transporter density were measured. Results: ALCAM expression was increased 24 h after MDMA treatment (p<0.01). Co-treatment with ibuprofen attenuated the increase in ALCAM levels (p<0.01) and partially prevented cerebral injury, reducing MDMA-induced 5-HT (p<0.0001) and 5-HT transporter (p<0.0001) loss. Ibuprofen produced a minor modification in the MDMA-induced hyperthermia. Conclusions: Our study demonstrates an effect of MDMA on ALCAM expression. Thus, anti-inflammatory compounds such as ibuprofen may result useful in brain protection by inhibiting the effects of ALCAM and reducing brain damage although the potential contribution of the attenuation of MDMA-induced hyperthermia must also be considered (AU)

Changes in the serotonergic system and in brain-derived neurotrophic factor distribution in the main olfactory bulb of pcd mice before and after mitral cell loss.

The serotonergic centrifugal system innervating the main olfactory bulb (MOB) plays a key role in the modulation of olfactory processing. We have previously demonstrated that this system suffers adaptive changes under conditions of a lack of olfactory input. The present work examines the response of this centrifugal system after mitral cell loss in the Purkinje cell degeneration (pcd) mutant mice. The distribution and density of serotonergic centrifugal axons were studied in the MOB of control and pcd mice, both before and after the loss of mitral cells, using serotonin (5-HT) and 5-HT transporter immunohistochemistry. Studies of the amount of 5-HT and its metabolite, 5-hydroxyindole acetic acid (5-HIAA), were performed by means of high-performance liquid chromatography (HPLC), and the relative amounts of brain-derived neurotrophin factor, BDNF, and its major receptor, tropomyosin-related kinase B (TrkB), were measured by Western blot. Our study revealed that the serotonergic system develops adaptive changes after, but not before, mitral cell loss. The lack of the main bulbar projection cells causes a decrease in the serotonergic input received by the MOB, whereas the number of serotonergic cells in the raphe nuclei remains constant. In addition, one of the molecules directly involved in serotonergic sprouting, the neurotrophin BDNF and its main receptor TrkB, underwent alterations in the MOBs of the pcd animals even before the loss of mitral cells. These data indicate that serotonergic function in the MOB is closely related to olfactory activity and that mitral cell loss induces serotonergic plastic responses.

Dopamine D(1) receptor deletion strongly reduces neurotoxic effects of methamphetamine.

Methamphetamine (METH) is a potent, highly addictive psychostimulant consumed worldwide. In humans and experimental animals, repeated exposure to this drug induces persistent neurodegenerative changes. Damage occurs primarily to dopaminergic neurons, accompanied by gliosis. The toxic effects of METH involve excessive dopamine (DA) release, thus DA receptors are highly likely to play a role in this process. To define the role of D(1) receptors in the neurotoxic effects of METH we used D(1) receptor knock-out mice (D(1)R(-/-)) and their WT littermates. Inactivation of D(1)R prevented METH-induced dopamine fibre loss and hyperthermia, and increases in gliosis and pro-inflammatory molecules such as iNOS in the striatum. In addition, D(1)R inactivation prevented METH-induced loss of dopaminergic neurons in the substantia nigra. To explore the relationship between hyperthermia and neurotoxicity, METH was given at high ambient temperature (29 °C). In this condition, D(1)R(-/-) mice developed hyperthermia following drug delivery and the neuroprotection provided by D(1)R inactivation at 23 °C was no longer observed. However, reserpine, which empties vesicular dopamine stores, blocked hyperthermia and strongly potentiated dopamine toxicity in D(1)R(-/-) mice, suggesting that the protection afforded by D(1)R inactivation is due to both hypothermia and higher stored vesicular dopamine. Moreover, electrical stimulation evoked higher DA overflow in D(1)R(-/-) mice as demonstrated by fast scan cyclic voltammetry despite their lower basal DA content, suggesting higher vesicular DA content in D(1)R(-/-) than in WT mice. Altogether, these results indicate that the D(1)R plays a significant role in METH-induced neurotoxicity by mediating drug-induced hyperthermia and increasing the releasable cytosolic DA pool.

Involvement of 2-arachidonoyl glycerol in the increased consumption of and preference for ethanol of mice treated with neurotoxic doses of methamphetamine.

BACKGROUND AND PURPOSE: Methamphetamine (METH) is a psychostimulant amphetamine that causes long-term dopaminergic neurotoxicity in mice. Hypodopaminergic states have been demonstrated to increase voluntary ethanol (EtOH) consumption and preference. In addition, the endocannabinoid system has been demonstrated to modulate EtOH drinking behaviour. Thus, we investigated EtOH consumption in METH-lesioned animals and the role of cannabinoid (CB) signalling in this EtOH drinking. EXPERIMENTAL APPROACH: Mice were treated with a neurotoxic regimen of METH, and 7 days later exposed to increasing concentrations of drinking solutions of EtOH (3, 6, 10 and 20%). Seven days after neurotoxic METH, the following biochemical determinations were carried out in limbic forebrain: CB(1) receptor density and stimulated activity, 2-arachidonoyl glycerol (2-AG) and monoacylglycerol lipase (MAGL) activity, dopamine levels and dopamine transporter density. KEY RESULTS: EtOH consumption and preference were increased in METH-treated mice. Seven days after METH, a time at which both dopamine levels and density of dopamine transporters in limbic forebrain were decreased, CB(1) receptor density and activity were unaltered, but 2-AG levels were increased. At this same time-point, MAGL activity was reduced. The CB(1) receptor antagonist AM251 prevented the METH-induced increase in EtOH consumption and preference, while N-arachidonoyl maleimide, an inhibitor of MAGL, increased EtOH consumption and preference in both saline- and METH-treated mice. CONCLUSIONS AND IMPLICATIONS: An increase in endocannabinoid tone may be involved in the increased consumption of and preference for EtOH displayed by METH-lesioned mice as blockade of the CB(1) receptor decreased EtOH-seeking behaviours, whereas the MAGL inhibitor increased EtOH consumption.

Dopamine transporter down-regulation following repeated cocaine: implications for 3,4-methylenedioxymethamphetamine-induced acute effects and long-term neurotoxicity in mice.

BACKGROUND AND PURPOSE: 3,4-Methylenedioxymethamphetamine (MDMA) and cocaine are two widely abused psychostimulant drugs targeting the dopamine transporter (DAT). DAT availability regulates dopamine neurotransmission and uptake of MDMA-derived neurotoxic metabolites. We aimed to determine the effect of cocaine pre-exposure on the acute and long-term effects of MDMA in mice. EXPERIMENTAL APPROACH: Mice received a course of cocaine (20 mg*kg(-1), x2 for 3 days) followed by MDMA (20 mg*kg(-1), x2, 3 h apart). Locomotor activity, extracellular dopamine levels and dopaminergic neurotoxicity were determined. Furthermore, following the course of cocaine, DAT density in striatal plasma membrane and endosome fractions was measured. KEY RESULTS: Four days after the course of cocaine, challenge with MDMA attenuated the MDMA-induced striatal dopaminergic neurotoxicity. Co-administration of the protein kinase C (PKC) inhibitor NPC 15437 prevented cocaine protection. At the same time, after the course of cocaine, DAT density was reduced in the plasma membrane and increased in the endosome fraction, and this effect was prevented by NPC 15437. The course of cocaine potentiated the MDMA-induced increase in extracellular dopamine and locomotor activity, following challenge 4 days later, compared with those pretreated with saline. CONCLUSIONS AND IMPLICATIONS: Repeated cocaine treatment followed by withdrawal protected against MDMA-induced dopaminergic neurotoxicity by internalizing DAT via a mechanism which may involve PKC. Furthermore, repeated cocaine followed by withdrawal induced behavioural and neurochemical sensitization to MDMA, measures which could be indicative of increased rewarding effects of MDMA.

Éxtasis (MDMA) y drogas de diseño: estructura, farmacología, mecanismos de acción y efectos en el ser humano / Ecstasy (MDMA) and designer drugs: structure, pharmacology, mechanism of action and effects in human beings

Objetivo. El derivado anfetamínico 3,4-metilenodioximetanfetamina (MDMA, éxtasis) es una droga recreativa muy popular entre los jóvenes, particularmente entre aquéllos implicados en la cultura de la música electrónica. El consumo es preocupante porque estudios realizados en diversos laboratorios han demostrado que la MDMA es una potente neurotoxina en el cerebro de diversas especies animales. El objetivo de esta revisión ha sido examinar los efectos inmediatos y a largo plazo producidos por la MDMA en consumidores y analizar las características y tendencias de consumo en la población escolar. Material y métodos. Se revisan las acciones farmacológicas y la toxicidad del éxtasis, así como las pautas de consumo del éxtasis en la población escolar. Resultados. Numerosos estudios realizados con animales de experimentación demuestran que la MDMA origina a largo plazo una pérdida de terminales serotonérgicos en el cerebro de la rata, cobayo y mono. Existen datos funcionales y bioquímicos que sugieren la existencia de daño neuronal en el cerebro de los consumidores habituales de esta droga. En estos individuos aparecen también cambios fisiológicos y psicológicos compatibles con una disminución de la función serotonérgica. Conclusiones. Cada vez es más numerosa la evidencia científica que sugiere que los consumidores habituales de MDMA son susceptibles a los efectos que la droga produce sobre las neuronas serotonérgicas cerebrales de los animales de experimentación. No obstante, hay que considerar que la mayor parte de los sujetos estudiados son o han sido policonsumidores y que se desconocen completamente las dosis ingeridas

Abstract. Objective. The amphetamine derivative3,4-methylenedioxymethamphetamine (MDMA,ecstasy) is a popular recreational drug amongyoung people, particularly those involved in thedance culture. The consumption of this drug isworrying because studies carried out in several laboratories have shown that MDMA is a potent neurotoxinin the brain of experimental animals. Theobjective of this review is to examine the acute andlong-term effects induced by MDMA in humanbeings and analyse the properties and tendencies ofconsumption in the school population.Material and methods. The pharmacological andtoxicological actions of ecstasy, as well as the patternsof ecstasy use in the school aged populationare reviewed. Results. Several studies performed in experimental animals have shown that MDMA produces along-term loss of serotonergic nerve terminals in thebrain of rats, guinea-pigs and monkeys. There arefunctional and biochemical data suggesting the existenceof neuronal damage in the brain of humanbeings. In these subjects physiological and psychologicalchanges also appear which are consistentwith decreased serotonergic function.Conclusion. Evidence for the occurrence of MDMAinducedneurotoxic damage in human users remainsequivocal, although there is a significantamount of data suggesting that damage may occurin the brains of heavy users. However, such evidenceis complicated by the lack of knowledge of dosesingested and the fact that many subjects studied areor have been poly-drug users

Éxtasis (MDMA): estudios neurobiológicos en el laboratorio / Ecstasy (MDMA): neurobiological studies in the laboratory

Objetivo. Describir los efectos agudos y a largo plazo producidos por la 3,4-metilenodioximetanfetamina (MDMA) en animales de experimentación y establecer las diferencias más importantes que existen entre especies. Material y métodos. Se revisa la neurobiología del MDMA. Resultados. Inmediatamente después de la administración de MDMA se observa un incremento en la liberación de serotonina y dopamina de los terminales nerviosos cerebrales. Aparece también una respuesta hipertérmica y un complejo conjunto de síntomas que constituyen el síndrome serotonérgico. Los efectos a largo plazo son especie-específicos, de tal manera que en la rata se observa una degeneración selectiva de los terminales serotonérgicos mientras que en el ratón MDMA produce una lesión en los terminales dopaminérgicos estriatales. El efecto neurodegenerativo se mantiene durante meses en la rata y durante años en el ratón. Un incremento en la formación de radicales libres en diversas estructuras cerebrales y un aumento de la temperatura corporal son algunos de los factores implicados en el mecanismo de neurotoxicidad. Estas especies reactivas de oxígeno podrían formarse en el transcurso del metabolismo de la MDMA. Conclusión. MDMA es una neurotoxina seronérgica selectiva en la mayoría de las especies animales examinadas. Sin embargo, en el ratón lesiona selectivamente las neuronas dopaminérgicas. Esta selectividad puede ser una consecuencia de la capacidad atrapadora de radicales libres de los terminales nerviosos específicos

ABSTRACT. Objective. To describe the acute- andlong-term effects induced by MDMA in experimentalanimals and establish differences among species.Material and methods. The neurobiology of MDMAwas reviewed. Results. Immediately after MDMA administration there is an increase in serotonin and dopamine release from brain nerve terminals. An hyperthermic response and complex set of symptoms knowas the serotonergic syndrome are also evident. Thelong-term effects are species-specific. In the rat,there is a selective degeneration of serotonin nerveterminals. In contrast, in mice MDMA damagesstriatal dopamine terminals. The neurodegenerativeeffects remain evident for several months in ratsand for several years in monkeys. An increased freeradical formation in several brain areas and an increasedbody temperature are some of the factorsinvolved in the mechanism of neurotoxicity. Thesereactive oxygen species could be formed throughMDMAmetabolism. Conclusion. While MDMA appears to be a selective serotonin neurotoxin in most species, it is a selective dopamine neurotoxin in mice. This selectivitymay be a consequence of the endogenous freeradical trapping ability of specific nerve endings

Sex differences in catechol contents in the olfactory bulb of control and unilaterally deprived rats.

The dopaminergic system plays important roles in the modulation of olfactory transmission. The present study examines the distribution of dopaminergic cells and the content of dopamine (DA) and its metabolites in control and deprived olfactory bulbs (OB), focusing on the differences between sexes. The content of DA and of its metabolites, dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), were measured by HPLC. The morphology and distribution of dopaminergic neurons were studied using tyrosine hydroxylase (TH) immunohistochemistry. Cells were typified with TH-parvalbumin, TH-cholecystokinin or TH-neurocalcin double-immunofluorescence assays. Biochemical analyses revealed sex differences in the content of DA and of its metabolites. In normal conditions, the OBs of male rats had higher concentrations of DA, DOPAC and HVA than the OBs of females. The immunohistochemical data pointed to sex differences in the number of TH-immunopositive cells (higher in male than in female rats). Colocalization analyses revealed that dopaminergic cells constitute a different cell subpopulation from those labelled after parvalbumin, cholecystokinin or neurocalcin immunostaining. Unilateral olfactory deprivation caused dramatic alterations in the dopaminergic system. The DA content and the density of dopaminergic cells decreased, the contents of DA and DOPAC as well as TH immunoreactivity were similar in deprived males and females and, finally, the metabolite/neurotransmitter ratio increased. Our results show that the dopaminergic modulation of olfactory transmission seems to differ between males and females and that it is regulated by peripheral olfactory activity. A possible role of the dopaminergic system in the sexually different olfactory sensitivity, discrimination and memory is discussed.

Changes in the serotonergic system in the main olfactory bulb of rats unilaterally deprived from birth to adulthood.

The serotonergic system plays a key role in the modulation of olfactory processing. The present study examined the plastic response of this centrifugal system after unilateral naris occlusion, analysing both serotonergic afferents and receptors in the main olfactory bulb. After 60 days of sensory deprivation, the serotonergic system exhibited adaptive changes. Olfactory deprivation caused a general increase in the number of fibres immunopositive for serotonin but not of those immunopositive for the serotonin transporter. HPLC data revealed an increase in serotonin levels but not in those of its major metabolite, 5-hydroxyindole acetic acid, resulting in a decrease in the 5-hydroxyindole acetic acid/serotonin ratio. These changes were observed not only in the deprived but also in the contralateral olfactory bulb. Double serotonin-tyrosine hydroxylase immunolabelling revealed that the glomerular regions of the deprived olfactory bulb with a high serotonergic fibre density showed a strong reduction in tyrosine hydroxylase. Finally, the serotonin(2A) receptor distribution density and the number of juxtaglomerular cells immunopositive for serotonin(2A) receptor remained unaltered after olfactory deprivation. Environmental stimulation modulated the serotonergic afferents to the olfactory bulb. Our results indicate the presence of a bilateral accumulation of serotonin in the serotonergic axon network, with no changes in serotonin(2A) receptor density after unilateral olfactory deprivation.

Differential effects of unilateral olfactory deprivation on noradrenergic and cholinergic systems in the main olfactory bulb of the rat.

The lack of environmental olfactory stimulation produced by sensory deprivation causes significant changes in the deprived olfactory bulb. Olfactory transmission in the main olfactory bulb (MOB) is strongly modulated by centrifugal systems. The present report examines the effects of unilateral deprivation on the noradrenergic and cholinergic centrifugal systems innervating the MOB. The morphology, distribution, and density of positive axons were studied in the MOBs of control and deprived rats, using dopamine-beta-hydroxylase (DBH)-immunohistochemistry and acetylcholinesterase (AChE) histochemistry in serial sections. Catecholamine content was compared among the different groups of MOBs (control, contralateral, and ipsilateral to the deprivation) using high-performance liquid chromatography analysis. Sensory deprivation revealed that the noradrenergic system developed adaptive plastic changes after olfactory deprivation, including important modifications in its fiber density and distribution, while no differences in cholinergic innervation were observed under the same conditions. The noradrenergic system underwent an important alteration in the glomerular layer, in which some glomeruli showed a dense noradrenergic innervation that was not detected in control animals. The DBH-positive glomeruli with the highest noradrenergic fiber density were compared with AChE-stained sections and it was observed that the strongly noradrenergic-innervated glomeruli were always atypical glomeruli (characterized by their strong degree of cholinergic innervation). In addition to the morphological findings, our biochemical data revealed that olfactory deprivation caused a decrease in the content of dopamine and its metabolite 3,4-dihydroxyphenylacetic acid in the ipsilateral MOB in comparison to the contralateral and control MOBs, together with an increase in noradrenaline levels in both the ipsilateral and contralateral MOBs. Our results show that regulation of the noradrenergic centrifugal system in the MOB depends on environmental olfactory stimulation and that it is highly reactive to sensory deprivation. By contrast, the cholinergic system is fairly stable and does not exhibit clear changes after the loss of sensory inputs.

3,4-Methylenedioxymethamphetamine increases pro-interleukin-1beta production and caspase-1 protease activity in frontal cortex, but not in hypothalamus, of Dark Agouti rats: role of interleukin-1beta in neurotoxicity.

3,4-Methylenedioxymethamphetamine (ecstasy) increases mature interleukin-1beta production in rat brain shortly after injection. This effect is a consequence of the 3,4-methylenedioxymethamphetamine-induced hyperthermia and is reduced when rats are maintained at low ambient room temperature. Since interleukin-1beta is generated as an inactive 31-kDa precursor protein and processed into mature form by caspase-1, we have now examined the effect of 3,4-methylenedioxymethamphetamine on pro-interleukin-1beta production and caspase-1-like protease activity in the hypothalamus and frontal cortex of Dark Agouti rats. 3,4-Methylenedioxymethamphetamine increased the immunoreactivity of pro-interleukin-1beta in frontal cortex, not in hypothalamus, 3 h and 6 h after administration. Caspase-1-like protease activity was increased in frontal cortex 3 h after 3,4-methylenedioxymethamphetamine injection compared with saline-treated animals. 3,4-Methylenedioxymethamphetamine did not modify the expression of pro-caspase-1 but increased the immunoreactivity for the caspase-1 active cleavage product (p20) in frontal cortex 3 h after dosing. No change on caspase-1-like protease activity was observed in hypothalamus. The basal immunoreactivity of pro-interleukin-1beta and caspase-1-like protease activity was higher in the hypothalamus than in frontal cortex of control (saline-treated) animals. These data indicate that 3,4-methylenedioxymethamphetamine alters, in a region-specific manner, the mechanisms which regulate interleukin-1beta production in the brain of Dark Agouti rats and suggest that the release of interleukin-1beta in hypothalamus may be regulated independently of caspase-1 activation. Administration (i.c.v.) of interleukin-1beta enhanced the 3,4-methylenedioxymethamphetamine-induced long-term loss of brain 5-HT parameters and immediate hyperthermia. Neither of these effects was observed when interleukin-1beta was given into hippocampus. These results indicate that exogenous interleukin-1beta potentiates 3,4-methylenedioxymethamphetamine neurotoxicity as a consequence of its effect on body temperature and suggest that the 3,4-methylenedioxymethamphetamine-induced rise in interleukin-1beta levels could in turn contribute to the maintenance of 3,4-methylenedioxymethamphetamine-induced hyperthermia and subsequent neurotoxicity.

Differential effect of dietary selenium on the long-term neurotoxicity induced by MDMA in mice and rats.

We examined the effect of dietary selenium (Se) on the long-term effect of 3,4-methylenedioxymethamphetamine (MDMA) on dopamine (DA) and 5-hydroxytryptamine (5-HT) containing neurons in the brain of mice and rats. Animals were fed either a Se-deficient (<0.02 ppm) or Se-replete (0.2 ppm) diet for 8 weeks. On the seventh week mice received three injections of MDMA (15 mg/kg, i.p. 3 h apart) or saline and rats a single dose of MDMA (12.5 mg/kg i.p.) or saline. All animals were sacrificed 7 days later. MDMA administration to mice depleted striatal DA concentration in both dietary groups, although depletion was considerably larger in the Se-deficient mice (64%) than Se-replete mice (30%). In addition, a decrease in 5-HT (17-32%) occurred in brain regions of Se-deficient but not Se-replete mice. In rats, MDMA decreased cortical [(3)H]-paroxetine binding (62%) and 5-HT content, the depletion being similar in the Se-deficient and Se-replete groups. No DA loss occurred in either group. There was no difference in the hyperthermic response induced by MDMA in Se-deficient or Se-replete animals. The Se-deficient diet decreased glutathione peroxidase (GPx) activity by 30% in mouse striatum and cortex and increased the degree of lipid peroxidation in cortical synaptosomes. Se-deficient rats also showed a decrease in brain GPx activity compared with the Se-replete group, but the degree of lipid peroxidation in synaptosomes was similar in both dietary groups. These results suggest that the antioxidant capacity of rats and mice differ leading to a differential susceptibility to the oxidative stress caused by MDMA in situations of low dietary Se.

A study of the mechanisms involved in the neurotoxic action of 3,4-methylenedioxymethamphetamine (MDMA, 'ecstasy') on dopamine neurones in mouse brain.

1. Administration of 3,4-methylenedioxymethamphetamine (MDMA, 'ecstasy') to mice produces acute hyperthermia and long-term degeneration of striatal dopamine nerve terminals. Attenuation of the hyperthermia decreases the neurodegeneration. We have investigated the mechanisms involved in producing the neurotoxic loss of striatal dopamine. 2. MDMA produced a dose-dependent loss in striatal dopamine concentration 7 days later with 3 doses of 25 mg kg(-1) (3 h apart) producing a 70% loss. 3. Pretreatment 30 min before each MDMA dose with either of the N-methyl-D-aspartate antagonists AR-R15896AR (20, 5, 5 mg kg(-1)) or MK-801 (0.5 mg kg(-1)x3) failed to provide neuroprotection. 4. Pretreatment with clomethiazole (50 mg kg(-1)x3) was similarly ineffective in protecting against MDMA-induced dopamine loss. 5. The free radical trapping compound PBN (150 mg kg(-1)x3) was neuroprotective, but it proved impossible to separate neuroprotection from a hypothermic effect on body temperature. 6. Pretreatment with the nitric oxide synthase (NOS) inhibitor 7-NI (50 mg kg(-1)x3) produced neuroprotection, but also significant hypothermia. Two other NOS inhibitors, S-methyl-L-thiocitrulline (10 mg kg(-1)x3) and AR-R17477AR (5 mg kg(-1)x3), provided significant neuroprotection and had little effect on MDMA-induced hyperthermia. 7. MDMA (20 mg kg(-1)) increased 2,3-dihydroxybenzoic acid formation from salicylic acid perfused through a microdialysis tube implanted in the striatum, indicating increased free radical formation. This increase was prevented by AR-R17477AR administration. Since AR-R17477AR was also found to have no radical trapping activity this result suggests that MDMA-induced neurotoxicity results from MDMA or dopamine metabolites producing radicals that combine with NO to form tissue-damaging peroxynitrites.

Neuroprotective effect of aspirin by inhibition of glutamate release after permanent focal cerebral ischaemia in rats.

Aspirin reduces the size of infarcts after ischaemic stroke. Although this fact has been attributed to its anti-platelet actions, direct neuroprotective effects have also been reported. We have recently demonstrated that aspirin is neuroprotective by inhibiting glutamate release in 'in vitro' models of brain ischaemia, via an increase in ATP production. The present study was designed to determine whether the inhibition of glutamate release induced by aspirin might be protective in a whole-animal model of permanent focal brain ischaemia. Focal brain ischaemia was produced in male adult Fischer rats by occluding both the common carotid and middle cerebral arteries. Central and serum glutamate levels were determined at fixed intervals after occlusion. The animals were then killed and infarct volume was measured. Aspirin (30 mg/kg i.p. administered 2 h before the occlusion) produced a significant reduction in infarct volume, an effect that correlated with the inhibition caused by aspirin on ischaemia-induced increase in brain and serum glutamate concentrations after the onset of the ischaemia. Aspirin also inhibited ischaemia-induced decrease in brain ATP levels. Our present findings show a novel mechanism for the neuroprotective effects of aspirin, which takes place at concentrations in the anti-aggregant-analgesic range, useful in the management of patients with risk of ischaemic events.

Studies on the neuroprotective effect of the enantiomers of AR-A008055, a compound structurally related to clomethiazole, on MDMA ("ecstasy")-induced neurodegeneration in rat brain.

RATIONALE: 3,4-Methylenedioxymethamphetamine (MDMA, "ecstasy") administration produces neurotoxic degeneration of 5-HT nerve endings in several regions of rat brain. Administration of the GABAmimetic drug clomethiazole protects against this damage. OBJECTIVE: We wished to see whether the enantiomers of AR-A008055 (1-4-methyl-5-thiazolyl-1-phenyl-methylamine), a compound structurally related to clomethiazole, were also neuroprotective against MDMA-induced degeneration. METHODS: (R)-(+)-AR-A008055 or (S)-(-)-AR-A008055 (100 mg/kg IP) was injected 5 min prior to and 55 min after MDMA (15 mg/kg IP) administration to Dark Agouti rats. Rectal temperature was measured during this time and the concentration of 5-HT and 5-HIAA measured in hippocampus, cortex and striatum 7 days later. [3H]-Paroxetine binding was also measured in cortex. RESULTS: Both enantiomers abolished the acute MDMA-induced hyperthermia and attenuated the subsequent neurotoxic loss of 5-HT, 5-HIAA and [3H]-paroxetine binding. When rats given the enantiomer plus MDMA were warmed to keep their rectal temperature elevated to near that of animals given only MDMA, the neuroprotective effect of (S)-(-)-AR-A008055 was still seen, while the effect of (R)-(+)-AR-A008055 was abolished. Protection was also seen when (S)-(-)-AR-A008055 (50 mg/kg) was given, a dose which produced only a modest attenuation of MDMA-induced hyperthermia. CONCLUSIONS: The current data suggest that a major proportion of the neuroprotective action of (S)-(-)-AR-A008055 did not involve an attenuating effect on MDMA-induced hyperthermia. The protection afforded by (R)-(+)-AR-A008055, which is not a GABA agonist, appears to be solely due to its action on body temperature, strengthening the contention that abolishing the acute MDMA-induced hypothermia can produce neuroprotection. Since (S)-(-)-AR-A008055 has a similar pharmacology to clomethiazole, these data suggest that drugs which increase GABAA receptor channel opening are neuroprotective against MDMA-induced damage.

The mechanisms involved in the long-lasting neuroprotective effect of fluoxetine against MDMA ('ecstasy')-induced degeneration of 5-HT nerve endings in rat brain.

1. It has been reported that co-administration of fluoxetine with 3,4-methylenedioxymethamphetamine (MDMA, 'ecstasy') prevents MDMA-induced degeneration of 5-HT nerve endings in rat brain. The mechanisms involved have now been investigated. 2. MDMA (15 mg kg(-1), i.p.) administration produced a neurotoxic loss of 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) in cortex, hippocampus and striatum and a reduction in cortical [3H]-paroxetine binding 7 days later. 3. Fluoxetine (10 mg kg(-1), i.p., x2, 60 min apart) administered concurrently with MDMA or given 2 and 4 days earlier provided complete protection, and significant protection when given 7 days earlier. Fluvoxamine (15 mg kg(-1), i.p., x2, 60 min apart) only produced neuroprotection when administered concurrently. Fluoxetine (10 mg kg(-1), x2) markedly increased the K(D) and reduced the B(max) of cortical [3H]-paroxetine binding 2 and 4 days later. The B(max) was still decreased 7 days later, but the K(D) was unchanged. [3H]-Paroxetine binding characteristics were unchanged 24 h after fluvoxamine (15 mg kg(-1), x2). 4. A significant cerebral concentration of fluoxetine plus norfluoxetine was detected over the 7 days following fluoxetine administration. The fluvoxamine concentration had decreased markedly by 24 h. 5. Pretreatment with fluoxetine (10 mg kg(-1), x2) failed to alter cerebral MDMA accumulation compared to saline pretreated controls. 6. Neither fluoxetine or fluvoxamine altered MDMA-induced acute hyperthermia. 7. These data demonstrate that fluoxetine produces long-lasting protection against MDMA-induced neurodegeneration, an effect apparently related to the presence of the drug and its active metabolite inhibiting the 5-HT transporter. Fluoxetine does not alter the metabolism of MDMA or its rate of cerebral accumulation.

A neurotoxic dose of 3,4-methylenedioxymethamphetamine (MDMA; ecstasy) to rats results in a long-term defect in thermoregulation.

RATIONALE: 3,4-Methylenedioxymethamphetamine (MDMA; "ecstasy") administration to rats produces damage to cerebral 5-HT nerve endings; however, the long-term functional consequences of this damage are poorly understood. OBJECTIVE: To confirm that MDMA administration produces a long-term effect on thermoregulation and investigate the mechanisms involved. METHODS: Male Dark Agouti rats were injected with a neurotoxic dose of MDMA (12.5 mg/kg i.p.). Five to 6 weeks later, they were exposed to high ambient temp (30 degrees C) for 60 min followed by a return to normal temp (20 degrees C), with rectal temperature being measured under both conditions. Further groups of MDMA-pretreated rats were challenged with 8-OH-DPAT and their temperature response measured. RESULTS: MDMA administration produced acute hyperthermia. Rectal temperature had normalised 24 h later and was similar to saline-injected controls over the following 15 days. MDMA administration produced a 37% loss in hypothalamic 5-HT content 18 days later. When MDMA-pretreated rats were subjected to high ambient temperature 33 days posttreatment, they displayed both a faster rise in rectal temperature and sustained hyperthermia when returned to normal conditions. There was no difference in their hypothermic response to the 5-HT1A agonist 8-OH-DPAT. CONCLUSIONS: A neurotoxic dose of MDMA resulted in impaired thermoregulation when rats were exposed to high ambient temperature. 5-HT1A receptor mechanisms were unaltered. Impaired serotonergic function following MDMA presumably alters the neurotransmitter balance, thereby compromising thermoregulation. Heavy recreational users of MDMA may also have impaired thermoregulation and thus be at greater risk of an acute adverse response to MDMA in a hot crowded dance environment.

3,4-Methylenedioxymethamphetamine induces monoamine release, but not toxicity, when administered centrally at a concentration occurring following a peripherally injected neurotoxic dose.

RATIONALE: There is good evidence that 3,4-methylenedioxymethamphetamine (MDMA)-induced neurotoxicity results from free radical formation. However, it is unclear whether it is the presence of MDMA or a metabolite in the brain that initiates this process. OBJECTIVE: We wished to measure the concentration of MDMA in the brain following peripheral administration of neurotoxic doses and examine the effect on acute monoamine release and the subsequent neurotoxic loss in 5-hydroxytryptamine (5-HT) content when a high concentration of MDMA was infused into cerebral tissue. METHODS: Selectively placed microdialysis probes were used to determine both the concentration of MDMA in the brain following peripheral injection and the degree of 5-HT release. Monoamines in dialysate and tissue were measured with standard HPLC techniques. RESULTS: MDMA, administered intraperitoneally, at doses of 10 and 15 mg/kg, which produce neurodegeneration, resulted in an estimated cerebral extracellular concentration of MDMA of 11 and 20 microM, respectively. When MDMA (100-400 microM) was perfused through a selectively placed microdialysis probe it dose-dependently increased 5-HT release in the hippocampus and dopamine release in the striatum. Seven days after perfusion of MDMA the concentration of 5-HT and its metabolite, 5-hydroxyindoleacetic acid was unchanged in the ipsilateral side of the brain of normothermic rats and also in the brains of animals made hyperthermic to mimic the acute effect of MDMA given peripherally. In contrast, perfusion with 5,7-dihydroxytryptamine (400 microM) markedly decreased the cerebral 5-HT content. A second probe, also placed in the hippocampus at a distance of 1 mm from the main probe, revealed that during the perfusion of MDMA (400 microM) the estimated extracellular concentration of MDMA in the hippocampus was between 10.4 and 19.5 microM, i.e. in the range of concentrations observed after systemic injection of neurotoxic doses of MDMA. CONCLUSIONS: These data demonstrate that MDMA when injected directly into the brain produces 5-HT release but no neurotoxicity, suggesting that it must be metabolised peripherally in order to produce compounds that induce free radical formation and neurotoxicity in the brain.

Effect of GBR 12909 and fluoxetine on the acute and long term changes induced by MDMA ('ecstasy') on the 5-HT and dopamine concentrations in mouse brain.

We examined the long term effect of 3,4 methylenedioxymethamphetamine (MDMA, 10, 20 and 30 mg/kg, i.p.) on the cerebral 5-hydroxytryptamine (5-HT) and dopamine content in Swiss Webster mice. Three injections of MDMA (20 or 30 mg/kg, i.p.) given 3 h apart produced a marked depletion in the striatal content of dopamine and its metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) 7 days later. None of the doses administered altered the concentration of 5-HT or its metabolite 5-hydroxyindoleacetic acid (5-HIAA) in several brain areas. Pre-treatment with the dopamine uptake inhibitor GBR 12909 (10 mg/kg, i.p.), 30 min before each of the three MDMA (30 mg/kg, i.p.) injections, completely prevented the long term loss in the striatal catechol concentrations. However, GBR 12909 (10 mg/kg, i.p.) not only failed to prevent the acute effects induced by MDMA (30 mg/kg x 3, i.p.) on dopamine metabolism 30 min later, but in fact potentiated them. The 5-HT uptake inhibitor, fluoxetine (10 mg/kg, i. p.) failed to prevent both the acute and long term dopaminergic deficits. MDMA (30 mg/kg x 3) altered the body temperature of the mice biphasically, producing a rapid hyperthermia followed by prolonged hypothermia. In contrast, MDMA (20 mg/kg x 3) produced an initial hypothermia followed by hyperthermia. The present experiments therefore appear to rule out any direct relationship between the neurotoxic effects of MDMA and its acute effects on body temperature in mice. Fluoxetine administered 30 min before each MDMA (30 mg/kg) injection prevented these temperature changes, while GBR 12909 was without effect. This suggests that the neuroprotective effect of GBR 12909 against MDMA-induced neurotoxicity is not directly related to its ability to inhibit the MDMA-induced acute effects on dopamine metabolism or alter the MDMA-induced temperature change. The data illustrate major differences in the neurotoxic profile of MDMA in mice and rats.