Jorge Camarero

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

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. 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. 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−1×3) failed to provide neuroprotection. Pretreatment with clomethiazole (50 mg kg−1×3) was similarly ineffective in protecting against MDMA‐induced dopamine loss. The free radical trapping compound PBN (150 mg kg−1×3) was neuroprotective, but it proved impossible to separate neuroprotection from a hypothermic effect on body temperature. Pretreatment with the nitric oxide synthase (NOS) inhibitor 7‐NI (50 mg kg−1×3) produced neuroprotection, but also significant hypothermia. Two other NOS inhibitors, S‐methyl‐L‐thiocitrulline (10 mg kg−1×3) and AR‐R17477AR (5 mg kg−1×3), provided significant neuroprotection and had little effect on MDMA‐induced hyperthermia. 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.

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

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. 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. Fluoxetine (10 mg kg−1, i.p., ×2, 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., ×2, 60 min apart) only produced neuroprotection when administered concurrently. Fluoxetine (10 mg kg−1, ×2) markedly increased the KD and reduced the Bmax of cortical [3H]‐paroxetine binding 2 and 4 days later. The Bmax was still decreased 7 days later, but the KD was unchanged. [3H]‐Paroxetine binding characteristics were unchanged 24 h after fluvoxamine (15 mg kg−1, ×2). 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. Pretreatment with fluoxetine (10 mg kg−1, ×2) failed to alter cerebral MDMA accumulation compared to saline pretreated controls. Neither fluoxetine or fluvoxamine altered MDMA‐induced acute hyperthermia. 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.

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.

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, using in vivo microdialysis, on the effect of the dopamine uptake inhibitor GBR 12909 on 3,4-methylenedioxymethamphetamine ('ecstasy')-induced dopamine release and free radical formation in the mouse striatum

The present study examined the mechanisms by which 3,4‐methylenedioxymethamphetamine (MDMA) produces long‐term neurotoxicity of striatal dopamine neurones in mice and the protective action of the dopamine uptake inhibitor GBR 12909. MDMA (30 mg/kg, i.p.), given three times at 3‐h intervals, produced a rapid increase in striatal dopamine release measured by in vivo microdialysis (maximum increase to 380 ± 64% of baseline). This increase was enhanced to 576 ± 109% of baseline by GBR 12909 (10 mg/kg, i.p.) administered 30 min before each dose of MDMA, supporting the contention that MDMA enters the terminal by diffusion and not via the dopamine uptake site. This, in addition to the fact that perfusion of the probe with a low Ca2+ medium inhibited the MDMA‐induced increase in extracellular dopamine, indicates that the neurotransmitter may be released by a Ca2+‐dependent mechanism not related to the dopamine transporter. MDMA (30 mg/kg × 3) increased the formation of 2,3‐dihydroxybenzoic acid (2,3‐DHBA) from salicylic acid perfused through a probe implanted in the striatum, indicating that MDMA increased free radical formation. GBR 12909 pre‐treatment attenuated the MDMA‐induced increase in 2,3‐DHBA formation by approximately 50%, but had no significant intrinsic radical trapping activity. MDMA administration increased lipid peroxidation in striatal synaptosomes, an effect reduced by approximately 60% by GBR 12909 pre‐treatment. GBR 12909 did not modify the MDMA‐induced changes in body temperature. These data suggest that MDMA‐induced toxicity of dopamine neurones in mice results from free radical formation which in turn induces an oxidative stress process. The data also indicate that the free radical formation is probably not associated with the MDMA‐induced dopamine release and that MDMA does not induce dopamine release via an action at the dopamine transporter.

3,4‐Methylenedioxymethamphetamine increases interleukin‐1β levels and activates microglia in rat brain: studies on the relationship with acute hyperthermia and 5‐HT depletion

3,4‐Methylenedioxymethamphetamine (MDMA) administration to rats produces acute hyperthermia and 5‐HT release. Interleukin‐1β (IL‐1β) is a pro‐inflammatory pyrogen produced by activated microglia in the brain. We examined the effect of a neurotoxic dose of MDMA on IL‐1β concentration and glial activation and their relationship with acute hyperthermia and 5‐HT depletion. MDMA, given to rats housed at 22°C, increased IL‐1β levels in hypothalamus and cortex from 1 to 6 h and [3H]‐(1‐(2‐chlorophenyl)‐N‐methyl‐N‐(1‐methylpropyl)3‐isoquinolinecarboxamide) binding between 3 and 48 h. Increased immunoreactivity to OX‐42 was also detected. Rats became hyperthermic immediately after MDMA and up to at least 12 h later. The IL‐1 receptor antagonist did not modify MDMA‐induced hyperthermia indicating that IL‐1β release is a consequence, not the cause, of the rise in body temperature. When MDMA was given to rats housed at 4°C, hyperthermia was abolished and the IL‐1β increase significantly reduced. The MDMA‐induced acute 5‐HT depletion was prevented by fluoxetine coadministration but the IL‐1β increase and hyperthermia were unaffected. Therefore, the rise in IL‐1β is not related to the acute 5‐HT release but is linked to the hyperthermia. Contrary to IL‐1β levels, microglial activation is not significantly modified when hyperthermia is prevented, suggesting that it might be a process not dependent on the hyperthermic response induced by MDMA.

MDMA-induced neurotoxicity: long-term effects on 5-HT biosynthesis and the influence of ambient temperature

1 3,4‐Methylenedioxymethamphetamine (MDMA or ‘ecstasy’) decreases the 5‐HT concentration, [3H]‐paroxetine binding and tryptophan hydroxylase activity in rat forebrain, which has been interpreted as indicating 5‐HT neurodegeneration. This has been questioned, particularly the 5‐HT loss, as MDMA can also inhibit tryptophan hydroxylase. We have now evaluated the validity of these parameters as a reflection of neurotoxicity. 2 Male DA rats were administered MDMA (12.5 mg kg−1, i.p.) and killed up to 32 weeks later. 5‐HT content and [3H]‐paroxetine binding were measured in the cortex, hippocampus and striatum. Parallel groups of treated animals were administered NSD‐1015 for determination of in vivo tryptophan hydroxylase activity and 5‐HT turnover rate constant. 3 Tissue 5‐HT content and [3H]‐paroxetine binding were reduced in the cortex (26–53%) and hippocampus (25–74%) at all time points (1, 2, 4, 8 and 32 weeks). Hydroxylase activity was similarly reduced up to 8 weeks, but had recovered at 32 weeks. The striatal 5‐HT concentration and [3H]‐paroxetine binding recovered by week 4 and hydroxylase activity after week 1. In all regions, the reduction in 5‐HT concentration did not result in an altered 5‐HT synthesis rate constant. 4 Administering MDMA to animals when housed at 4°C prevented the reduction in [3H]‐paroxetine binding and hydroxylase activity observed in rats housed at 22°C, but not the reduction in 5‐HT concentration. 5 These data indicate that MDMA produces long‐term damage to serotoninergic neurones, but this does not produce a compensatory increase in 5‐HT synthesis in remaining terminals. It also highlights the fact that measurement of tissue 5‐HT concentration may overestimate neurotoxic damage.

The nNOS inhibitor, AR-R17477AR, prevents the loss of NF68 immunoreactivity induced by methamphetamine in the mouse striatum

The present study examined the time‐course and regionally‐selective changes in the levels of the neurofilament protein NF68 in the mouse brain induced by methamphetamine (METH). The ability of low ambient temperature, or of the specific neuronal nitric oxide synthase (nNOS) inhibitor AR‐R17477AR, to protect against both long‐term striatal NF68 and dopamine loss induced by METH (3 mg/kg, i.p.) was also studied. Seven days after METH administration (3, 6 and 9 mg/kg, i.p., three times at 3 h intervals), mice showed a reduction of about 40% in immunoreactivity for NF68 in the striatum. This effect was not produced in cortex after METH administration at the dose of 3 mg/kg. No difference from controls was observed when measurements were carried out 1 h and 24 h after the last METH injection at the dose of 3 mg/kg. The loss of NF68 immunoreactivity seems to be associated with the long‐term dopamine depletion induced by METH, since no change in serotonin concentration is observed in either the striatum or cortex 7 days after dosing. Animals kept at a room temperature of 4°C showed a loss of NF68 similar to those treated at 22°C but an attenuation of dopamine depletion in the striatum. Pre‐treatment with AR‐R17477AR (5 mg/kg, s.c.) 30 min before each of the three METH (3 mg/kg, i.p.) injections provided complete protection against METH‐induced loss of NF68 immunoreactivity and attenuated the decrease in striatal dopamine and HVA concentrations by about 50%. These data indicate that both the reduction of NF68 immunoreactivity and the loss of dopamine concentration are due to an oxidative stress process mediated by reactive nitrogen species, and are not due to changes in body temperature.

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.

On the protection against methamphetamine-induced neurotoxicity by benzamide, a PARP inhibitor

Repeated administration of methamphetamine (METH) to mice produces neurodegenerative damage to dopamine axons and axon terminals in the striatum and cell body loss in the substantia nigra (Sonsalla et al. 1996; Hirata and Cadet 1997). Damage is reflected in a long-term decrease in the concentration of dopamine and its metabolites, a loss in the density of plasmalemmal and vesicular dopamine transporters and a decrease in tyrosine hydroxylase activity (Sonsalla et al. 1989; Itzhak et al. 2000). The mechanisms underlying this neurodegeneration are not fully understood, but there is considerable evidence that nitrogen reactive species and temperature play essential roles. Damage is prevented by co-administration of neuronal nitric oxide synthase (nNOS) inhibitors by a mechanism not involving changes in rectal temperature (Itzhak et al. 2000), and METH administration causes overexpression of nNOS in the mouse striatum (Deng and Cadet 1999). Furthermore, nNOS deficient mice are resistant to METH neurotoxicity and to 3-nitrotyrosine production (Imam et al. 2001). Benzamide, a poly(ADP-ribose) polymerase (PARP) inhibitor, has been reported to attenuate the long-lasting dopamine depletion induced by METH (Cosi et al. 1996), suggesting that reactive nitrogen species might elicit neurotoxicity by producing DNA strand breaks leading to activation of PARP and, in so doing, provoking a depletion in cellular energy stores (Gaal et al. 1987). However, the effect of benzamide on the hyperthermic response induced by METH was not investigated in that study and there is substantial evidence that preventing METH-induced hyperthermia, or production of frank hypothermia, is neuroprotective (e.g. Miller and O’Callaghan 1994; Albers and Sonsalla 1995). Therefore, we have now examined the effect of benzamide on both body temperature and METH-induced neurotoxicity. All experimental procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85–23, revised 1985). Adult male C57BL/6J mice (Harlan Iberica) weighing 20–25 g were housed in groups of ten, in conditions of constant temperature (21€2 C) and lighting (lights on: 0700– 1900 hours) and given free access to food and water. METH (3 mg/kg, IP) or saline was administered 3 times at 3-h intervals and benzamide (160 or 320 mg/kg, IP) or vehicle was given 30 min before the first and last METH injections. Benzamide was dissolved in saline containing 2% Tween 20 and 1% carboxymethylcellulose (Cosi et al. 1996). Rectal temperature was measured with a digital readout thermocouple probe attached to a CAC-005 Rodent Sensor. In two experiments, the temperature of the mice given METH plus benzamide was kept elevated to match that of mice given only METH, using a Harvard Homeothermic Blanket system placed in the cage. Seven days after treatment, mice were killed by cervical dislocation, the brains rapidly removed and striatal dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) measured by HPLC. Neurochemical data were analysed by one-way ANOVA followed by Newman-Keuls test. Temperature data was analysed by two-way ANOVA with the statistical computer package BMDP/386 Dynamic. Injection of METH (3 mg/kg, IP) produced a rise in rectal temperature (1.5–2.5 C above the saline group) lasting at least 8 h and peaking between 30 and 60 min after each METH injection (Fig. 1a, b). Administration of benzamide (160 mg/kg, IP) 30 min before the first and the third METH injections prevented the hyperthermic response and even produced a marked hypothermia (Fig. 1a). Seven days later, mice injected with METH had a significant loss of striatal dopamine and DOPAC (Fig. 1c, d, e, f), the dopamine loss being partially prevented (63%) by benzamide (160 mg/kg) (Fig. 1c). E. O’Shea · V. Sanchez · J. Camarero · M. I. Colado ()) Departamento de Farmacologia, Facultad de Medicina, Universidad Complutense, 28040 MadridSpain e-mail: colado@med.ucm.es