Sex differences in the effects of N-ethylpentylone in young CD1 mice: Insights on behaviour, thermoregulation and early gene expression.

BACKGROUND AND PURPOSE: New psychoactive substances such as N-ethylpentylone (NEP) are continuously emerging in the illicit drug market, and knowledge of their effects and risks, which may vary between sexes, is scarce. Our present study compares some key effects of NEP in male and female mice. EXPERIMENTAL APPROACH: Psychostimulant, rewarding and reinforcing effects were investigated by tracking locomotor activity, conditioned place preference (CPP) paradigm and through a self-administration (SA) procedure, respectively, in CD1 mice. Moreover, the expression of early genes (C-fos, Arc, Csnk1e, Pdyn, Pp1r1b and Bdnf in addiction-related brain areas) was assessed by qPCR. Finally, serum and brain levels of NEP were determined by UHPLC-MS/MS. KEY RESULTS: NEP-treated males experimented locomotor sensitisation and showed higher and longer increases in locomotion as well as higher hyperthermia after repeated administration than females. Moreover, while preference score in the CPP was similar in both sexes, extinction occurred later, and reinstatement was more easily established for males. Female mice self-administered more NEP than males at a higher dose. Differences in early gene expression (Arc, Bdnf, Csnk1e and Ppp1r1b) were found, but the serum and brain NEP levels did not differ between sexes. CONCLUSION AND IMPLICATIONS: Our results suggest that male mice are more sensitive to NEP psychostimulant and rewarding effects. These differences may be attributed to different early gene expression but not to pharmacokinetic factors. Moreover, males appear to be more vulnerable to the hyperthermic effects of NEP, while females might be more prone to NEP abuse.

Structure-Activity Relationship of N-Ethyl-Hexedrone Analogues: Role of the α-Carbon Side-Chain Length in the Mechanism of Action, Cytotoxicity, and Behavioral Effects in Mice.

Synthetic cathinones are ß-keto amphetamine derivatives whose appearance has increased dramatically in the past decades. N-Ethyl substituted cathinones have been proven to potently inhibit dopamine (DA) uptake and induce psychostimulant and rewarding effects in mice. However, little is known about the influence of the alpha-carbon side-chain length of N-ethyl cathinones on their pharmacological and toxicological effects. Thus, the aim of this study was to synthesize and investigate the in vitro and in vivo effects of five N-ethyl substituted cathinones: N-ethyl-cathinone (NEC), N-ethyl-buphedrone (NEB), N-ethyl-pentedrone, N-ethyl-hexedrone (NEH), and N-ethyl-heptedrone. HEK293 cells expressing the human DA or serotonin transporter (hDAT and hSERT) were used for uptake inhibition and binding assays. PC12 cells were used for the cytotoxicity assays. Swiss CD-1 mice were used to study the in vivo psychostimulant, anxiogenic, and rewarding properties. Our results show that all tested cathinones are able to inhibit DA uptake and are DAT-selective. The potency of DA uptake inhibitors increases with the elongation of the aliphatic side chain from methyl to propyl and decreases when increasing from butyl to pentyl, which correlates with an inverted U-shape psychostimulant response in mice at the medium dose tested. On the other hand, an increase in the α-carbon side-chain length correlates with an increase in the cytotoxic properties in PC12 cells, probably due to better membrane penetration. Moreover, all the cathinones tested have shown higher cytotoxicity than methamphetamine. Finally, our study not only demonstrated the rewarding properties of NEC and NEB but also the anxiety-like behavior induced at high doses by all the cathinones tested.

Methamphetamine Blocks Adenosine A2A Receptor Activation via Sigma 1 and Cannabinoid CB1 Receptors.

Methamphetamine is, worldwide, one of the most consumed drugs of abuse. One important side effect is neurodegeneration leading to a decrease in life expectancy. The aim of this paper was to check whether the drug affects one of the receptors involved in neurodegeneration/neuroprotection events, namely the adenosine A2A receptor (A2AR). First, we noticed that methamphetamine does not affect A2A functionality if the receptor is expressed in a heterologous system. However, A2AR becomes sensitive to the drug upon complexes formation with the cannabinoid CB1 receptor (CB1R) and the sigma 1 receptor (σ1R). Signaling via both adenosine A2AR and cannabinoid CB1R was affected by methamphetamine in cells co-expressing the two receptors. In striatal primary cultures, the A2AR-CB1R heteromer complex was detected and methamphetamine not only altered its expression but completely blocked the A2AR- and the CB1R-mediated activation of the mitogen activated protein kinase (MAPK) pathway. In conclusion, methamphetamine, with the participation of σ1R, alters the expression and function of two interacting receptors, A2AR, which is a therapeutic target for neuroprotection, and CB1R, which is the most abundant G protein-coupled receptor (GPCR) in the brain.

Effects of High-Fat Diet and Maternal Binge-Like Alcohol Consumption and Their Influence on Cocaine Response in Female Mice Offspring.

BACKGROUND: Prenatal alcohol exposure is a leading cause of neurobehavioral and neurocognitive deficits collectively known as fetal alcohol spectrum disorders, including eating disorders and increased risk for substance abuse as very common issues. In this context, the present study aimed to assess the interaction between prenatal and lactation alcohol exposure (PLAE) and a high-fat diet (HFD) during childhood and adolescence. METHODS: Pregnant C57BL/6 mice underwent a procedure for alcohol binge drinking during gestation and lactation periods. Subsequently, PLAE female offspring were fed with an HFD for 8 weeks, and thereafter, nutrition-related parameters as well as their response to cocaine were assessed. RESULTS: In our model, feeding young females with an HFD increased their triglyceride blood levels but did not induce overweight compared with those fed with a standard diet. Moreover, PLAE affected how females responded to the fatty diet as they consumed less food than water-exposed offspring, consistent with a lower gain of body weight. HFD increased the psychostimulant effects of cocaine. Surprisingly, PLAE reduced the locomotor responses to cocaine without modifying cocaine-induced reward. Moreover, PLAE prevented the striatal overexpression of cannabinoid 1 receptors induced by an HFD and induced an alteration of myelin damage biomarker in the prefrontal cortex, an effect that was mitigated by an HFD-based feeding. CONCLUSION: Therefore, in female offspring, some effects triggered by one of these factors, PLAE or an HFD, were blunted by the other, suggesting a close interaction between the involved mechanisms.

Effects of MDPV on dopamine transporter regulation in male rats. Comparison with cocaine.

RATIONALE: MDPV (3,4-methylenedioxypyrovalerone) is a synthetic cathinone present in bath salts. It is a powerful psychostimulant and blocker of the dopamine transporter (DAT), like cocaine. It is known that acute exposure to psychostimulants induces rapid changes in DAT function. OBJECTIVES: To investigate the effects of MDPV on DAT function comparing with cocaine. METHODS: Binding of [3H]WIN 35428 was performed on PC 12 cells treated with MDPV and washed. Rat striatal synaptosomes were incubated with MDPV or cocaine (1 µM) for 1 h and [3H]dopamine (DA) uptake was performed. Also, different treatments with MDPV or cocaine were performed in Sprague-Dawley rats to assess locomotor activity and ex vivo [3H]DA uptake. RESULTS: MDPV increased surface [3H]WIN 35428 binding on PC 12 cells. In vitro incubation of synaptosomes with MDPV produced significant increases in Vmax and KM for [3H]DA uptake. In synaptosomes from MDPV- (1.5 mg/kg, s.c.) and cocaine- (30 mg/kg, i.p.) treated rats, there was a significantly higher and more persistent increase in [3H]DA uptake in the case of MDPV than cocaine. Repeated doses of MDPV developed tolerance to this DAT upregulation and 24 h after the 5-day treatment with MDPV, [3H]DA uptake was reduced. However, a challenge with the same drugs after withdrawal recovered the DAT upregulation by both drugs and showed an increased response to MDPV vs the first dose. At the same time, animals were sensitized to the stereotypies induced by both psychostimulants. CONCLUSIONS: MDPV induces a rapid and reversible functional upregulation of DAT more powerfully and lasting than cocaine.

Effect of the combination of mephedrone plus ethanol on serotonin and dopamine release in the nucleus accumbens and medial prefrontal cortex of awake rats.

Cathinones, such as mephedrone (Meph), are often co-abused with alcoholic drinks. In the present study, we investigated the combined effects of Meph plus ethanol (EtOH) on neurotransmitter release in the nucleus accumbens (NAc) and the medial prefrontal cortex (mPFC). A guide canula was stereotaxically implanted into either the NAc or the mPFC of male Sprague-Dawley rats. Seven days after surgery, a microdialysis probe was inserted and rats were administered saline, EtOH (1 g/kg, i.p.), Meph (25 mg/kg, s.c.), or their combination, and dialysates were collected. Serotonin (5-HT), dopamine (DA), and their metabolites (5-HIAA, DOPAC and HVA) were determined through high-pressure liquid chromatography coupled to mass spectrometry. 5-HT and DA peaked 40 min after Meph administration (with or without EtOH co-treatment) in both areas. EtOH combined with Meph increased the 5-HT release compared with the rats receiving Meph alone (85% in NAc, 65% in mPFC), although the overall change in the area under the curve only reached statistical significance in the NAc. In mPFC, the increased release of 5-HT lasted longer in the combination than that in the Meph group. Moreover, EtOH potentiated the psychostimulant effect of Meph measured as a locomotor activity. Given that both 5-HT and DA are also related with reward and impulsivity, the observed effects point to an increased risk of abuse liability when combining Meph with EtOH compared with consuming these drugs alone.

Adolescent exposure to MDMA induces dopaminergic toxicity in substantia nigra and potentiates the amyloid plaque deposition in the striatum of APPswe/PS1dE9 mice.

MDMA is one of the most used drugs by adolescents and its consumption has been associated with many psychobiological problems, among them psychomotor problems. Moreover, some authors described that early exposure to MDMA may render the dopaminergic neurons more vulnerable to the effects of future neurotoxic insults. Alzheimer disease (AD) is the main cause of dementia in the elderly and a percentage of the patients have predisposition to suffer nigrostriatal alterations, developing extrapyramidal signs. Nigrostriatal dysfunction in the brain of aged APPswe/PS1dE9 (APP/PS1), a mouse model of familiar AD (FAD), has also been described. The aim of the present study was to investigate the consequences of adolescent exposure to MDMA in APP/PS1 mice, on nigrostriatal function on early adulthood. We used a MDMA schedule simulating weekend binge abuse of this substance. Our MDMA schedule produced a genotype-independent decrease in dopaminergic neurons in the substantia nigra that remained at least 3months. Shortly after the injury, wild-type animals showed a decrease in the locomotor activity and apparent DA depletion in striatum, however in the APP/PS1 mice neither the locomotor activity nor the DA levels were modified, but a reduction in dopamine transporter (DAT) expression and a higher levels of oxidative stress were observed. We found that these disturbances are age-related characteristics that this APP/PS1 mice develops spontaneously much later. Therefore, MDMA administration seems to anticipate the striatal dopaminergic dysfunction in this FAD model. The most important outcome lies in a potentiation, by MDMA, of the amyloid beta deposition in the striatum.

Changes in CREB and deltaFosB are associated with the behavioural sensitization induced by methylenedioxypyrovalerone.

Methylenedioxypyrovalerone (MDPV) is a synthetic cathinone which has recently emerged as a designer drug of abuse. The objective of this study was to investigate the locomotor sensitization induced by MDPV in adolescent mice, and associated neuroplastic changes in the nucleus accumbens and striatum through deltaFosB and CREB expression. Behavioural testing consisted of three phases: Phase I: conditioning regimen with MDPV (0.3 mg/kg/day for five days) or saline; Phase II: resting (11 days); Phase III: challenged with MDPV (0.3 mg/kg), cocaine (10 mg/kg) or saline on day 16 for both groups. Mice repeatedly exposed to MDPV increased locomotor activity by 165-200% following acute MDPV or cocaine administration after an 11-day resting period, showing a MDPV-induced sensitization to itself and to cocaine. An explanation for this phenomenon could be the common mechanism of action between these two psychostimulants. Furthermore, the MDPV challenge resulted in higher levels of phospho-CREB in MDPV-conditioned mice compared with MDPV-naive mice, probably due to an up-regulation of the cAMP pathway. Likewise, MDPV exposure induced a persistent increase in the striatal expression of deltaFosB; the priming dose of MDPV also produced a significant increase in the accumbal expression of this transcription factor. This study constitutes the first evidence that an exposure to a low dose of MDPV during adolescence induces behavioural sensitization and provides a neurobiological basis for a relationship between MDPV and cocaine. We hypothesize that, similar to cocaine, both CREB and deltaFosB play a role in the induction of this behavioural sensitization.

The combination of ethanol with mephedrone increases the signs of neurotoxicity and impairs neurogenesis and learning in adolescent CD-1 mice.

A new family of psychostimulants, under the name of cathinones, has broken into the market in the last decade. In light of the fact that around 95% of cathinone consumers have been reported to combine them with alcoholic drinks, we sought to study the consequences of the concomitant administration of ethanol on mephedrone -induced neurotoxicity. Adolescent male Swiss-CD1 mice were administered four times in one day, every 2h, with saline, mephedrone (25mg/kg), ethanol (2; 1.5; 1.5; 1g/kg) and their combination at a room temperature of 26±2°C. The combination with ethanol impaired mephedrone-induced decreases in dopamine transporter and tyrosine hydroxylase in the frontal cortex; and in serotonin transporter and tryptophan hydroxylase in the hippocampus by approximately 2-fold, 7days post-treatment. Furthermore, these decreases correlated with a 2-fold increase in lipid peroxidation, measured as concentration of malondialdehyde (MDA), 24h post-treatment, and were accompanied by changes in oxidative stress-related enzymes. Ethanol also notably potentiated mephedrone-induced negative effects on learning and memory, as well as hippocampal neurogenesis, measured through the Morris water maze (MWM) and 5-bromo-2'-deoxyuridine staining, respectively. These results are of special significance, since alcohol is widely co-abused with amphetamine derivatives such as mephedrone, especially during adolescence, a crucial stage in brain maturation. Given that the hippocampus is greatly involved in learning and memory processes, normal brain development in young adults could be affected with permanent behavioral consequences after this type of drug co-abuse.

Alcohol enhances the psychostimulant and conditioning effects of mephedrone in adolescent mice; postulation of unique roles of D3 receptors and BDNF in place preference acquisition.

BACKGROUND AND PURPOSE: The psychostimulant mephedrone is often consumed in combination with alcohol (EtOH). This kind of drug consumption during adolescence is a matter of concern. EXPERIMENTAL APPROACH: We studied, in adolescent CD-1 mice, whether EtOH could enhance the psychostimulant (locomotor acivity) and rewarding [conditioned place preference (CPP)] effects of mephedrone. We also determined the transcriptional changes associated with a conditioning treatment with these drugs. KEY RESULTS: Mephedrone (10 mg·kg(-1)) increased locomotor activity, which was further enhanced by 40% when combined with EtOH (1 g·kg(-1)). This enhancement was blocked by haloperidol. Furthermore, mephedrone (25 mg·kg(-1)) induced CPP, which increased by 70% when administered with a dose of EtOH that was not conditioning by itself (0.75 g·kg(-1)). There was enhanced expression of the D3 dopamine receptor mRNA (Drd3) and Arpc5 in all drug-treated groups. The D3 receptor antagonist SB-277011A and the BDNF receptor antagonist ANA-12 completely prevented CPP as well as the increases in Drd3 in all groups. Accordingly, increased expression of BDNF mRNA in medial prefrontal cortex was detected at 2 and 4 h after mephedrone administration. CONCLUSIONS AND IMPLICATIONS: If translated to humans, the enhancement of mephedrone effects by ethanol could result in increased abuse liability. D3 receptors and BDNF play a key role in the establishment of CPP by mephedrone, although an accompanying increase in other synaptic plasticity-related genes may also be necessary.

Concentrations of MDPV in rat striatum correlate with the psychostimulant effect.

3,4-methylenedioxypyrovalerone or MDPV is a synthetic cathinone with psychostimulant properties more potent than cocaine. We quantified this drug in the striatum after subcutaneous administration to rats. MDPV reached the brain around 5 min after its administration and peaked at 20-25 min later. The elimination half-life in the striatum (61 min) correlates with the decrease in the psychostimulant effect after 60 min. Around 11% of the administered dose reached the striatum and, considering a homogeneous brain distribution, we determined that around 86% of the plasma MDPV is distributed to the brain. MDPV induced a dose-dependent increase in locomotor activity, rearing behaviour and stereotypies, all prevented by haloperidol. A plot of locomotor activity or stereotypies versus MDPV striatal concentrations over time showed a direct relationship between factors. No free MDPV metabolites were detected in plasma, at any time, but hydrolysis with glucuronidase allowed us to identify mainly three metabolites, one of them for the first time in rat plasma. The present results contribute to evidence that MDPV induces hyperlocomotion mainly through a dopamine-dependent mechanism. Good correlation between behavioural effects and striatal levels of MDPV leads us to conclude that its psychostimulant effect is mainly due to a striatal distribution of the substance. The present research provides useful information on the pharmacokinetics of MDPV, and can help design new experiments with kinetics data as well as provide a better understanding of the effects of MDPV in humans and its potential interactions.

Neuronal changes and oxidative stress in adolescent rats after repeated exposure to mephedrone.

Mephedrone is a new designer drug of abuse. We have investigated the neurochemical/enzymatic changes after mephedrone administration to adolescent rats (3×25 mg/kg, s.c. in a day, with a 2 h interval between doses, for two days) at high ambient temperature (26±2 °C), a schedule that intends to model human recreational abuse. In addition, we have studied the effect of mephedrone in spatial learning and memory. The drug caused a transient decrease in weight gain. After the first dose, animals showed hypothermia but, after the subsequent doses, temperature raised over the values of saline-treated group. We observed the development of tolerance to these thermoregulatory effects of mephedrone. Mephedrone induced a reduction of the densities of dopamine (30% in the frontal cortex) and serotonin (40% in the frontal cortex and the hippocampus and 48% in the striatum) transporters without microgliosis. These deficits were also accompanied by a parallel decrease in the expression of tyrosine hydroxylase and tryptophan hydroxylase 2. These changes matched with a down-regulation of D2 dopamine receptors in the striatum. Mephedrone also induced an oxidative stress evidenced by an increase of lipid peroxidation in the frontal cortex, and accompanied by a rise in glutathione peroxidase levels in all studied brain areas. Drug-treated animals displayed an impairment of the reference memory in the Morris water maze one week beyond the cessation of drug exposure, while the spatial learning process seems to be preserved. These findings raise concerns about the neuronal long-term effects of mephedrone.

Serotonin is involved in the psychostimulant and hypothermic effect of 4-methylamphetamine in rats.

4-Methylamphetamine (4-MA) has recently emerged as a designer drug of abuse in Europe and it is consumed always with amphetamine. There have been reported some deaths and non-fatal intoxications related to 4-MA. We investigated the changes in locomotor activity and body temperature after 4-MA administration to male Sprague-Dawley rats. Our experiments were carried out at a normal or high ambient temperature. 4-MA (2.5-10mg/Kg, given subcutaneously) increased, in a dose-dependent manner, the horizontal locomotor activity that was significantly reduced by ketanserin, p-cholorophenylalanine (pCPA) or haloperidol, but not by pindolol. In addition, we have studied the effect of 4-MA on core body temperature by means of an implanted electronic thermograph, enabling continuous measurement of body temperature. We observed a dose-dependent hypothermic response to 4-MA that reached a maximum 45 min after a single injection. We also evidenced slight tachyphylaxis to the hypothermic effect when 4-MA was administered four times in a 2h interval. The pre-treatment of animals with pCPA or pindolol, but not with ketanserin, fully abolished the hypothermic effect of 4-MA. With all that, we conclude that hypothermia induced by 4-MA is due to the release of 5-HT which activates postsynaptic 5-HT1A receptors.

Serotonergic impairment and memory deficits in adolescent rats after binge exposure of methylone.

Methylone is a cathinone derivative that has recently emerged as a designer drug of abuse in Europe and the USA. Studies on the acute and long-term neurotoxicity of cathinones are starting to be conducted. We investigated the neurochemical/enzymatic changes indicative of neurotoxicity after methylone administration (4 × 20 mg/kg, subcutaneously, per day with 3 h intervals) to adolescent rats, to model human recreational use. In addition, we studied the effect of methylone on spatial learning ad memory using the Morris water maze paradigm. Our experiments were carried out at a high ambient temperature to simulate the hot conditions found in dance clubs where the drug is consumed. We observed a hyperthermic response to methylone that reached a peak 30 min after each dose. We determined a serotonergic impairment in methylone-treated rats, especially in the frontal cortex, where it was accompanied by astrogliosis. Some serotonergic alterations were also present in the hippocampus and striatum. No significant neurotoxic effect on the dopaminergic system was identified. Methylone-treated animals only displayed impairments in the probe trial of the Morris water maze, which concerns reference memory, while the spatial learning process seemed to be preserved.

3,4-Methylenedioxymethamphetamine enhances kainic acid convulsive susceptibility.

Kainic acid (KA) causes seizures and neuronal loss in the hippocampus. The present study investigated whether a recreational schedule of 3,4-methylenedioxymethamphetamine (MDMA) favours the development of a seizure state in a model of KA-induced epilepsy and potentiates the toxicity profile of KA (20 or 30mg/kg). Adolescent male C57BL/6 mice received saline or MDMA t.i.d. (s.c. every 3h), on 1day a week, for 4 consecutive weeks. Twenty-four hours after the last MDMA exposure, the animals were injected with saline or KA (20 or 30mg/kg). After this injection, we evaluated seizures, hippocampal neuronal cell death, microgliosis, astrogliosis, and calcium binding proteins. MDMA pretreatment, by itself, did not induce neuronal damage but increased seizure susceptibility in all KA treatments and potentiated the presence of Fluoro-Jade-positive cells in CA1. Furthermore, MDMA, like KA, significantly decreased parvalbumin levels in CA1 and dentate gyrus, where it potentiated the effects of KA. The amphetamine derivative also promoted a transient decrease in calbindin and calretinin levels, indicative of an abnormal neuronal discharge. In addition, treatment of cortical neurons with MDMA (10-50µM) for 6 or 48h significantly increased basal Ca(2+), reduced basal Na(+) levels and potentiated kainate response. These results indicate that MDMA potentiates KA-induced neurodegeneration and also increases KA seizure susceptibility. The mechanism proposed includes changes in Calcium Binding Proteins expression, probably due to the disruption of intracellular ionic homeostasis, or/and an indirect effect through glutamate release.

Dose and time-dependent selective neurotoxicity induced by mephedrone in mice.

Mephedrone is a drug of abuse marketed as 'bath salts". There are discrepancies concerning its long-term effects. We have investigated the neurotoxicity of mephedrone in mice following different exposition schedules. Schedule 1: four doses of 50 mg/kg. Schedule 2: four doses of 25 mg/kg. Schedule 3: three daily doses of 25 mg/kg, for two consecutive days. All schedules induced, in some animals, an aggressive behavior and hyperthermia as well as a decrease in weight gain. Mephedrone (schedule 1) induced dopaminergic and serotoninergic neurotoxicity that persisted 7 days after exposition. At a lower dose (schedule 2) only a transient dopaminergic injury was found. In the weekend consumption pattern (schedule 3), mephedrone induced dopamine and serotonin transporter loss that was accompanied by a decrease in tyrosine hydroxylase and tryptophan hydroxylase 2 expression one week after exposition. Also, mephedrone induced a depressive-like behavior, as well as a reduction in striatal D2 density, suggesting higher susceptibility to addictive drugs. In cultured cortical neurons, mephedrone induced a concentration-dependent cytotoxic effect. Using repeated doses for 2 days in an elevated ambient temperature we evidenced a loss of frontal cortex dopaminergic and hippocampal serotoninergic neuronal markers that suggest injuries at nerve endings.

Molecular basis of the selective binding of MDMA enantiomers to the alpha4beta2 nicotinic receptor subtype: synthesis, pharmacological evaluation and mechanistic studies.

The α4ß2 nicotinic acetylcholine receptor (nAChR) is a molecular target of 3,4-methylenedioxymethamphetamine (MDMA), a synthetic drug also known as ecstasy, and it modulates the MDMA-mediated reinforcing properties. However, the enantioselective preference of the α4ß2 nAChR subtype still remains unknown. Since the two enantiomers exhibit different pharmacological profiles and stereoselective metabolism, the aim of this study is to assess a possible difference in the interaction of the MDMA enantiomers with this nAChR subtype. To this end, we report a novel simple, yet highly efficient enantioselective synthesis of the MDMA enantiomers, in which the key step is the diastereoselective reduction of imides derived from optically pure tert-butylsulfinamide. The enantioselective binding to the receptor is examined using [(3)H]epibatidine in a radioligand assay. Even though the two enantiomers induced a concentration-dependent binding displacement, (S)-MDMA has an inhibition constant 13-fold higher than (R)-MDMA, which shows a Hill's coefficient not significantly different from unity, implying a competitive interaction. Furthermore, when NGF-differentiated PC12 cells were pretreated with the compounds, a significant increase in binding of [(3)H]epibatidine was found for (R)-MDMA, indicating up-regulation of heteromeric nAChR in the cell surface. Finally, docking and molecular dynamics studies have been used to identify the binding mode of the two enantiomers, which provides a structural basis to justify the differences in affinity from the differential interactions played by the substituents at the stereogenic centre of MDMA. The results provide a basis to explore the distinct psychostimulant profiles of the MDMA enantiomers mediated by the α4ß2 nAChR subtype.

Repeated doses of methylone, a new drug of abuse, induce changes in serotonin and dopamine systems in the mouse.

RATIONALE: Methylone, a new drug of abuse sold as "bath salts," has similar effects to ecstasy or cocaine. OBJECTIVE: We have investigated changes in dopaminergic and serotoninergic markers, indicative of neuronal damage induced by methylone in the frontal cortex, hippocampus, and striatum of mice, according to two different treatment schedules. METHODS: Methylone was given subcutaneously to male Swiss CD1 mice at an ambient temperature of 26 °C. Treatment A consisted of three doses of 25 mg/kg at 3.5-h intervals between doses for two consecutive days, and treatment B consisted of four doses of 25 mg/kg at 3-h intervals in 1 day. RESULTS: Repeated methylone administration induced hyperthermia and a significant loss in body weight. Following treatment A, methylone induced transient dopaminergic (frontal cortex) and serotoninergic (hippocampus) impairment. Following treatment B, transient dopaminergic (frontal cortex) and serotonergic (frontal cortex and hippocampus) changes 7 days after treatment were found. We found evidence of astrogliosis in the CA1 and the dentate gyrus of the hippocampus following treatment B. The animals also showed an increase in immobility time in the forced swim test, pointing to a depressive-like behavior. In cultured cortical neurons, methylone (for 24 and 48 h) did not induce a remarkable cytotoxic effect. CONCLUSIONS: The neural effects of methylone differ depending upon the treatment schedule. Neurochemical changes elicited by methylone are apparent when administered at an elevated ambient temperature, four times per day at 3-h intervals, which is in accordance with its short half-life.

Protracted treatment with MDMA induces heteromeric nicotinic receptor up-regulation in the rat brain: an autoradiography study.

Previous studies indicate that 3,4-methylenedioxy-methamphetamine (MDMA, ecstasy) can induce a heteromeric nicotinic acetylcholine receptor (nAChR, mainly of α4ß2 subtype) up-regulation. In this study we treated male Sprague-Dawley rats twice-daily for 10 days with either saline or MDMA (7 mg/kg) and sacrificed them the day after to perform [(125)I]Epibatidine binding autoradiograms on serial coronal slices. MDMA induced significant increases in nAChR density in the substantia nigra, ventral tegmental area, nucleus accumbens, olfactory tubercle, anterior caudate-putamen, somatosensory, motor, auditory and retrosplenial cortex, laterodorsal thalamus nuclei, amygdala, postsubiculum and pontine nuclei. These increases ranged from 3% (retrosplenial cortex) to 30 and 34% (amygdala and substantia nigra). No increased α4 subunit immunoreactivity was found in up-regulated areas compared with saline-treated rats, suggesting a post-translational mechanism as occurs with nicotine. The heteromeric nAChR up-regulation in certain areas could account, at least in part, for the reinforcing, sensitizing and psychiatric disorders observed after long-term consumption of MDMA.

MDMA enhances hippocampal-dependent learning and memory under restrictive conditions, and modifies hippocampal spine density.

OBJECTIVES: Addictive drugs produce forms of structural plasticity in the nucleus accumbens and prefrontal cortex. The aim of this study was to investigate the impact of chronic MDMA exposure on pyramidal neurons in the CA1 region of hippocampus and drug-related spatial learning and memory changes. METHODS AND RESULTS: Adolescent rats were exposed to saline or MDMA in a regime that mimicked chronic administration. One week later, when acquisition or reference memory was evaluated in a standard Morris water maze (MWM), no differences were obtained between groups. However, MDMA-exposed animals performed better when the MWM was implemented under more difficult conditions. Animals of MDMA group were less anxious and were more prepared to take risks, as in the open field test they ventured more frequently into the central area. We have demonstrated that MDMA caused an increase in brain-derived neurotrophic factor (BDNF) expression. When spine density was evaluated, MDMA-treated rats presented a reduced density when compared with saline, but overall, training increased the total number of spines, concluding that in MDMA-group, training prevented a reduction in spine density or induced its recovery. CONCLUSIONS: This study provides support for the conclusion that binge administration of MDMA, known to be associated to neurotoxic damage of hippocampal serotonergic terminals, increases BDNF expression and stimulates synaptic plasticity when associated with training. In these conditions, adolescent rats perform better in a more difficult water maze task under restricted conditions of learning and memory. The effect on this task could be modulated by other behavioural changes provoked by MDMA.