Evidence for a role of Hsp70 in the neuroprotection induced by heat shock pre-treatment against 3,4-methylenedioxymethamphetamine toxicity in rat brain

3,4-Methylenedioxymethamphetamine (MDMA, 'ecstasy') produces acute hyperthermia which increases the severity of the selective serotoninergic neurotoxicity produced by the drug in rats. Heat shock protein 70 (Hsp70) is a major inducible cellular protein expressed in stress conditions and which is thought to exert protective functions. MDMA (12.5 mg/kg, i.p.), given to rats housed at 22 degrees C, produced an immediate hyperthermia and increased Hsp70 in frontal cortex between 3 h and 7 days after administration. MDMA, given to rats housed at low ambient temperature (4 degrees C) produced transient hypothermia followed by mild hyperthermia but no increase in Hsp70 expression, while rats treated at elevated room temperature (30 degrees C) showed enhanced hyperthermia and similar expression of Hsp70 to that seen in rats housed at 22 degrees C. Fluoxetine-induced inhibition of 5-HT release and hydroxyl radical formation did not modify MDMA-induced Hsp70 expression 3 h later. Four- or 8-day heat shock (elevation of basal rectal temperature by 1.5 degrees C for 1 h) or geldanamycin pre-treatment induced Hsp70 expression and protected against MDMA-induced serotoninergic neurotoxicity without affecting drug-induced hyperthermia. Thus, MDMA-induced Hsp70 expression depends on the drug-induced hyperthermic response and not on 5-HT release or hydroxyl radical formation and pre-induction of Hsp70 protects against the long-term serotoninergic damage produced by MDMA.     

Changes in brain monoamine oxidase activity in conditioned passive avoidance reaction in rats]

Activity of the enzyme monoamine oxidase (MAO) and kinetic parameters (Km, Vmax) for the 5-hydroxytryptamine (5-HT) and dopamine deamination were examined in the brain of rats with conditioned passive avoidance recall. Changes of the 5-HT and dopamine deamination were found in amygdala, striatum and frontal cortex. MAO activity was not changed in hippocampus. In amygdala the rate of 5-HT deamination was significantly increased and kinetic studies revealed increased affinity of the enzyme for 5-HT. The metabolism of dopamine in amygdala was unchanged. In frontal cortex the deamination of 5-HT was not changed, but the dopamine deamination significantly decreased. This decrease was due to lowering of MAO affinity for dopamine. In striatum the metabolism of both 5-HT and dopamine was reduced, and kinetic studies showed the lowering of Vmax for 5-HT and dopamine deamination.     

Preferential Potentiation of the Effects of Serotonin Uptake Inhibitors by 5-HT1A Receptor Antagonists in the Dorsal Raphe Pathway: Role of Somatodendritic Autoreceptors

Abstract: 5-HT_1A autoreceptor antagonists enhance the effects of antidepressants by preventing a negative feedback of serotonin (5-HT) at somatodendritic level. The maximal elevations of extracellular concentration of 5-HT (5-HT_ext) induced by the 5-HT uptake inhibitor paroxetine in forebrain were potentiated by the 5-HT_1A antagonist WAY-100635 (1 mg/kg s.c.) in a regionally dependent manner (striatum > frontal cortex > dorsal hippocampus). Paroxetine (3 mg/kg s.c.) decreased forebrain 5-HT_ext during local blockade of uptake. This reduction was greater in striatum and frontal cortex than in dorsal hippocampus and was counteracted by the local and systemic administration of WAY-100635. The perfusion of 50 µmol/L citalopram in the dorsal or median raphe nucleus reduced 5-HT_ext in frontal cortex or dorsal hippocampus to 40 and 65% of baseline, respectively. The reduction of cortical 5-HT_ext induced by perfusion of citalopram in midbrain raphe was fully reversed by WAY-100635 (1 mg/kg s.c.). Together, these data suggest that dorsal raphe neurons projecting to striatum and frontal cortex are more sensitive to self-inhibition mediated by 5-HT_1A autoreceptors than median raphe neurons projecting to the hippocampus. Therefore, potentiation by 5-HT_1A antagonists occurs preferentially in forebrain areas innervated by serotonergic neurons of the dorsal raphe nucleus.     

3,4-Methylenedioxymethamphetamine increases interleukin-1beta 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-1beta (IL-1beta) is a pro-inflammatory pyrogen produced by activated microglia in the brain. We examined the effect of a neurotoxic dose of MDMA on IL-1beta concentration and glial activation and their relationship with acute hyperthermia and 5-HT depletion. MDMA, given to rats housed at 22 degrees C, increased IL-1beta levels in hypothalamus and cortex from 1 to 6 h and [(3)H]-(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-1beta release is a consequence, not the cause, of the rise in body temperature. When MDMA was given to rats housed at 4 degrees C, hyperthermia was abolished and the IL-1beta increase significantly reduced. The MDMA-induced acute 5-HT depletion was prevented by fluoxetine coadministration but the IL-1beta increase and hyperthermia were unaffected. Therefore, the rise in IL-1beta is not related to the acute 5-HT release but is linked to the hyperthermia. Contrary to IL-1beta 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.     

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.     

Monoaminoxidase activity and serotonin metabolism in the brain of rats during latent inhibition]

The monoamine oxidase (MAO) activities and the concentrations of 5-HT and 5-hydroxyindoleacetic acid were investigated in four brain regions in rats during the acquisition of latent inhibition in one-trial passive avoidance task. 5-HT metabolism was not altered in the hippocampus. Changes of 5-HT metabolism were found in the frontal cortex during testing of latent inhibition and were accompanied by lowering of MAO activity. No change of 5-HT metabolism was observed in this structure at the stage of pre-exposition to conditioned stimulus. 5-HT metabolism was activated at the stage of pre-exposition to conditioned stimulus in the amygdala and striatum and was maintained on high level, in these structures during testing of latent inhibition. The data presented here indicate that serotoninergic system in various brains regions is specifically involved in the formation of different stages of latent inhibition.     

Ketanserin pretreatment attenuates MDMA-induced dopamine release in the striatum as measured by in vivo microdialysis

Systemic administration of the amphetamine analogue, 3,4-methylenedioxymethamphetamine (MDMA) produced a dose-dependent increase in the extracellular concentration of dopamine (DA) in the striatum as measured by in vivo microdialysis in awake, freely-moving rats. The extracellular concentration of the DA metabolite, 3,4-dihydroxyphenylacetic acid (DOPAC), was significantly decreased in dialysate samples following the administration of MDMA (10 and 20 mg/kg, i.p.). The serotonin-2 (5-HT2) antagonist ketanserin (3 mg/kg, i.p.) had no effect on the extracellular concentration of DA or DOPAC in the striatum of vehicle- treated rats. The administration of ketanserin (3 mg/kg) 1 hr prior to MDMA (20 mg/kg) significantly attenuated the MDMA- induced increase in the extracellular concentration of DA without affecting the decrease in DOPAC concentrations. These data are suggestive that MDMA administration increases DA release in the striatum of awake, freely-moving rats. In addition, MDMA-induced increase in the extracellular concentration of DA in the striatum is mediated, in part, via 5-HT2 receptor mechanisms.     

Distinctions in metabolism of serotonin and dopamine in rat brain during latent inhibition

Latent inhibition (LI) is a behavioral phenomenon, in which repeated presenting of a non-reinforced stimulus retards conditioning to this stimulus when it is coupled with a reinforcer. In order to find specific serotonin (5-HT- and dopamine (DA) changes mediating the LI, the 5-HT and DA metabolism was investigated in certain brain regions. Oxidative deamination of 5-HT and DA by monoamine oxidase (MAO) was determined in the prefrontal cortex, striatim, amygdala, and hippocampus at preexposure and testing stages of the LI using the passive avoidance procedure in rats. Preexposed animals demonstrated high MAO activity for 5-HT deamination in the amygdala and striatum and lower MAO activity for DA deamination in the amygdala and hippocampus. After testing the LI, a high level of 5-HT deamination by MAO was revealed in the amygdala, white the lower level of 5-HT deamination by MAO was shown in the prefrontal cortex. At the same time, no changes in DA metabolism were found in all the brain regions studied. Thus, the role of dopaminergic system in the LI effect may be limited by the preexposure stage. The obtained evidence suggests that the enhanced 5-HT activity in the amygdala and striatum induced by the preexposed stimulus is a principal biochemical mechanism underlying the LI.     

Effects of sex factors and hemispheric localization on the involvement of serotonin from the frontal cortex, striatum, and nucleus accumbens into the processing of novel and repeatedly presented information in rats

The effects of novel or relevant (a single exposure to experimental chamber) and irrelevant (20 exposures to experimental chamber) stimuli on the levels of serotonin (5-HT) and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) in the frontal cortex, striatum, and nucleus accumbens in the left and right hemispheres were studied in male and female rats. It was found that 5-HT and 5-HIAA contents in the frontal cortex changed in response to neither relevant nor irrelevant stimuli. However, there were hemispheric difference in 5-HT and 5-HIAA in the frontal cortex of intact animals. The level of 5-HT in males and the level of 5-HIAA in females were higher in the left frontal cortex. In females, the level of 5-HIAA in the left striatum decreased in response to the novel stimulus. Sex differences in: a) 5-HT metabolism (increase in the level of 5-HIAA in males and increase in 5-HT in females) and b) lateralization (the striatal 5-HT metabolism in males changed bilaterally and only in the left hemisphere in females) were observed in reactions to irrelevant stimuli. Both in male and female rats, serotonin content in the nucleus accumbens changed only in response to the irrelevant stimuli. The 5-HT level increased in the left and right hemispheres independently of sex, but hemispheric difference was revealed only in females, in which the serotonin level was higher in the left nucleus accumbens. It is concluded that serotonergic neurotransmitter mechanisms are involved in hemispheric and sex differences in selective attention.     

Carrier-Mediated Release of Serotonin by 3,4-Methylenedioxymethamphetamine: Implications for Serotonin-Dopamine Interactions

Abstract: In vivo microdialysis was used to determine whether the 3,4-methylenedioxymethamphetamine (MDMA)-induced release of serotonin (5-HT) in vivo involves a carrier-mediated process and to investigate further the state-dependent interaction between 5-HT and dopamine. MDMA produced a dose-dependent increase in the extracellular concentration of 5-HT in the striatum and prefrontal cortex that was attenuated by treatment with fluoxetine but not by tetrodotoxin. Suppression by fluoxetine of the MDMA-induced release of 5-HT was accompanied by a suppression of the MDMA-induced release of dopamine. Administration of MDMA to rats treated with carbidopa and l -5-hydroxytryptophan resulted in a synergistic elevation of the extracellular concentration of 5-HT that was much greater than that produced by either treatment alone. The MDMA-induced release of dopamine by MDMA also was potentiated in 5-hydroxytryptophan-treated rats. These data are consistent with the view that MDMA increases the extracellular concentration of 5-HT by facilitating carrier-mediated 5-HT release, which can be enhanced greatly under conditions in which 5-HT synthesis is stimulated. Moreover, these data are supportive of a state-dependent, stimulatory role of 5-HT in the regulation of dopamine release.     

Verbal Memory Deficits Are Correlated with Prefrontal Hypometabolism in 18FDG PET of Recreational MDMA Users

Introduction

3,4-Methylenedioxymethamphetamine (MDMA, “ecstasy”) is a recreational club drug with supposed neurotoxic effects selectively on the serotonin system. MDMA users consistently exhibit memory dysfunction but there is an ongoing debate if these deficits are induced mainly by alterations in the prefrontal or mediotemporal cortex, especially the hippocampus. Thus, we investigated the relation of verbal memory deficits with alterations of regional cerebral brain glucose metabolism (rMRGlu) in recreational MDMA users.

Methods

Brain glucose metabolism in rest was assessed using 2-deoxy-2-(¹⁸F)fluoro-D-glucose positron emission tomography (¹⁸FDG PET) in 19 male recreational users of MDMA and 19 male drug-naïve controls. ¹⁸FDG PET data were correlated with memory performance assessed with a German version of the Rey Auditory Verbal Learning Test.

Results

As previously shown, MDMA users showed significant impairment in verbal declarative memory performance. PET scans revealed significantly decreased rMRGlu in the bilateral dorsolateral prefrontal and inferior parietal cortex, bilateral thalamus, right hippocampus, right precuneus, right cerebellum, and pons (at the level of raphe nuclei) of MDMA users. Among MDMA users, learning and recall were positively correlated with rMRGlu predominantly in bilateral frontal and parietal brain regions, while recognition was additionally related to rMRGlu in the right mediotemporal and bihemispheric lateral temporal cortex. Moreover, cumulative lifetime dose of MDMA was negatively correlated with rMRGlu in the left dorsolateral and bilateral orbital and medial PFC, left inferior parietal and right lateral temporal cortex.

Conclusions

Verbal learning and recall deficits of recreational MDMA users are correlated with glucose hypometabolism in prefrontal and parietal cortex, while word recognition was additionally correlated with mediotemporal hypometabolism. We conclude that memory deficits of MDMA users arise from combined fronto-parieto-mediotemporal dysfunction.     

Short- and Long-Term Effects of p-Ethynylphenylalanine on Brain Serotonin Levels

Changes in tissue and extracellular serotonin (5-HT) in raphe dorsalis, raphe medialis and in their main projections areas (hippocampus, striatum and frontal cortex) were investigated at short and long-term times after single injection (5 mg/kg ip) of a novel tryptophan hydroxylase inhibitor, p-ethynylphenylalanine (p-EPA). The 5-HT tissue concentration decreased significantly in raphe nuclei, 30 min post-injection and for 4 days, whereas it decreased from 24 hours post-injection in the 5-HT projections. Normal 5-HT levels reappeared after 12 days post-injection in all areas. Moreover, in the projection areas, the extracellular 5-HT levels decreased rapidly, 90, 40 and 30 min after p-EPA injection, in hippocampus, striatum and frontal cortex, respectively. Decreased accumulation of 5-hydroxytryptophan (5-HTP) under NSD-101 perfusion in the serotoninergic projections after p-EPA injection, confirmed the direct inhibitory effect of the drug on the tryptophan hydroxylase activity. These results demonstrated that p-EPA is a useful pharmacological tool which powerfully, acutely and irreversibly reduces the 5-HT levels.     

n-3 polyunsaturated fatty acid supplementation reverses stress-induced modifications on brain monoamine levels in mice

The aim of this study was to examine the effects of supplementation with n-3 polyunsaturated fatty acids (PUFAs) on stress responses in mice subjected to an unpredictable chronic mild stress (UCMS) procedure. Stress-induced modifications in coat and aggressiveness were evaluated, and phospholipid PUFA profiles and monoamine levels were analyzed in the frontal cortex, hippocampus, and striatum. The results showed that repeated exposure to mild stressors induced degradation in the physical state of the coat, lowered body weight gain, and increased aggressiveness, without any effect of n-3 PUFA supplementation. The UCMS induced a significant decrease in the levels of norepinephrine in the frontal cortex and striatum, and a nonsignificant decrease in the hippocampus. The tissue levels of serotonin (5-HT) were 40% to 65% decreased in the three brain regions studied. Interestingly, the n-3 PUFA supplementation reversed this stress-induced reduction in 5-HT levels. These findings showed that supplementation in n-3 long-chain PUFAs might reverse certain effects of UCMS in cerebral structures involved in stress-related behaviors.     

Regional Distribution of Extracellular 5-Hydroxytryptamine and 5-Hydroxyindoleacetic Acid in the Brain of Freely Moving Rats

The extracellular concentrations of 5-hydroxytryptamine (5-HT) and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) have been determined in six brain areas of awake rats (frontal cortex, striatum, hypothalamus, hippocampus, inferior colliculus, and raphe nuclei) using intracerebral microdialysis. The extracellular levels of 5-HT showed no significant differences among the brain regions studied. The tissue levels of 5-HT and 5-HIAA as well as the extracellular concentration of 5-HIAA were significantly higher in raphe nuclei. The regional distribution of tissue and extracellular 5-HIAA were very similar, suggesting that extracellular 5-HIAA depends mainly on the output from the intracellular compartment. On the other hand, extracellular 5-HT and tissue 5-HT showed a different distribution pattern. The tissue/extracellular ratio for 5-HT ranged from 739 in frontal cortex to 2,882 in raphe, whereas it only amounted to 1.8-3.6 for 5-HIAA. The relationship between the present results and the density of 5-HT uptake sites in these areas is discussed.     

The effects of acute ethanol exposure and ageing on rat brain glutathione metabolism

Binge alcohol consumption in adolescents is increasing, and it has been proposed that immature brain deals poorly with oxidative stress. The aim of our work was to study the effect of an acute dose of ethanol on glutathione (GSH) metabolism in frontal cortex, hippocampus and striatum of juvenile and adult rats. We have observed no change in levels of glutathione produced by acute alcohol in the three brain areas studied of juvenile and adult rats. Only in the frontal cortex the ratio of GSH/GSSG was increased in the ethanol-treated adult rats. GSH levels in the hippocampus and striatum were significantly higher in adult animals compared to young ones. Higher glutathione peroxidase (GPx) activity in adult rats was observed in frontal cortex and in striatum. Our data show an increased GSH concentration and GPx activity in different cerebral regions of the adult rat, compared to the young ones, suggesting that age-related variations of total antioxidant defences in brain may predispose young brain structures to ethanol-induced, oxidative stress-mediated tissue damage.     

The effects of acute ethanol exposure and ageing on rat brain glutathione metabolism

Abstract

Binge alcohol consumption in adolescents is increasing, and it has been proposed that immature brain deals poorly with oxidative stress. The aim of our work was to study the effect of an acute dose of ethanol on glutathione (GSH) metabolism in frontal cortex, hippocampus and striatum of juvenile and adult rats. We have observed no change in levels of glutathione produced by acute alcohol in the three brain areas studied of juvenile and adult rats. Only in the frontal cortex the ratio of GSH/GSSG was increased in the ethanol-treated adult rats. GSH levels in the hippocampus and striatum were significantly higher in adult animals compared to young ones. Higher glutathione peroxidase (GPx) activity in adult rats was observed in frontal cortex and in striatum. Our data show an increased GSH concentration and GPx activity in different cerebral regions of the adult rat, compared to the young ones, suggesting that age-related variations of total antioxidant defences in brain may predispose young brain structures to ethanol-induced, oxidative stress-mediated tissue damage.     

Age dependence of somatostatin levels and somatostatin binding in the rat brain

1. Somatostatin-like immunoreactivity (SLI) and 125I-Tyrl-somatostatin binding were measured from the brains of rats aged 1, 8 and 18 months. 2. Somatostatin binding was reduced in the striatum, frontal cortex, hypothalamus and hippocampus of the 8-month-old rats compared to the 1-month-old group. 3. Somatostatin binding was reduced in the striatum, frontal cortex and hippocampus of the 18-month-old rats compared to the 1-month-old group. 4. The reduction (40%) was most striking in the frontal cortex. 5. In no area of the brain did changes in SLI differ significantly between the different age groups.     

Molecular and Cellular Mechanisms of Ecstasy-Induced Neurotoxicity: An Overview

“Ecstasy” [(±)-3,4-methylenedioxymethamphetamine, MDMA, XTC, X, E] is a psychoactive recreational hallucinogenic substance and a major worldwide drug of abuse. Several reports raised the concern that MDMA has the ability to induce neurotoxic effects both in laboratory animals and humans. Despite more than two decades of research, the mechanisms by which MDMA is neurotoxic are still to be fully elucidated. MDMA induces serotonergic terminal loss in rats and also in some mice strains, but also a broader neuronal degeneration throughout several brain areas such as the cortex, hippocampus, and striatum. Meanwhile, in human “ecstasy” abusers, there are evidences for deficits in seronergic biochemical markers, which correlate with long-term impairments in memory and learning. There are several factors that contribute to MDMA-induced neurotoxicity, namely, hyperthermia, monoamine oxidase metabolism of dopamine and serotonin, dopamine oxidation, the serotonin transporter action, nitric oxide, and the formation of peroxinitrite, glutamate excitotoxicity, serotonin 2A receptor agonism, and, importantly, the formation of MDMA neurotoxic metabolites. The present review covered the following topics: history and epidemiology, pharmacological mechanisms, metabolic pathways and the influence of isoenzyme genetic polymorphisms, as well as the acute effects of MDMA in laboratory animals and humans, with a special focus on MDMA-induced neurotoxic effects at the cellular and molecular level. The main aim of this review was to contribute to the understanding of the cellular and molecular mechanisms involved in MDMA neurotoxicity, which can help in the development of therapeutic approaches to prevent or treat the long-term neuropsychiatric complications of MDMA abuse in humans.     

Berberine produces antidepressant-like effects in the forced swim test and in the tail suspension test in mice

This study investigated the effect of berberine (BER) in the mouse forced swim test (FST) and in the tail suspension test (TST), two models predictive of antidepressant activity. We also investigated the antidepressant-like mechanism of BER by the combination of the desipramine [DES, an inhibitor of reuptake of noradrenaline (NA) and serotonin (5-HT)], maprotiline (MAP, selective NA reuptake inhibitor), fluoxetine (FLU, selective 5-HT reuptake inhibitor) and moclobemide [MOC, monoamine oxidase (MAO) A inhibitor). Then we further measured the levels of monoamines [NA, dopamine (DA) and 5-HT) in mice striatum, hippocampus and frontal cortex. The results show that BER (10, 20 mg/kg, p.o.), significantly reduced the immobility time during the FST and the TST. The immobility time after treatment with BER (20 mg/kg, p.o.) in FST was augmented by DES, FLU and MOC, and not affected by MAP. Furthermore, BER (20 mg/kg, p.o.) increased NA and 5-HT levels in the hippocampus and frontal cortex. Our findings support the view that BER exerts antidepressant-like effect. The antidepressant-like mechanism of BER may be related to the increase in NA and 5-HT levels in the hippocampus and frontal cortex.