Jorge Camarasa

Comparative neuropharmacology of three psychostimulant cathinone derivatives: butylone, mephedrone and methylone

BACKGROUND AND PURPOSE Here, we have compared the neurochemical profile of three new cathinones, butylone, mephedrone and methylone, in terms of their potential to inhibit plasmalemmal and vesicular monoamine transporters. Their interaction with 5‐HT and dopamine receptors and their psychostimulant effect was also studied.

Interaction of mephedrone with dopamine and serotonin targets in rats

INTRODUCTION We described a first approach to the pharmacological targets of mephedrone (4-methyl-methcathinone) in rats to establish the basis of the mechanism of action of this drug of abuse. EXPERIMENTAL PROCEDURES We performed in vitro experiments in isolated synaptosomes or tissue membrane preparations from rat cortex or striatum, studying the effect of mephedrone on monoamine uptake and the displacement of several specific radioligands by this drug. RESULTS In isolated synaptosomes from rat cortex or striatum, mephedrone inhibited the uptake of serotonin (5-HT) with an IC ₅₀ value lower than that of dopamine (DA) uptake (IC ₅₀=0.31±0.08 and 0.97±0.0 5μM, respectively). Moreover, mephedrone displaced competitively both [³H]paroxetine and [³H]WIN35428 binding in a concentration-dependent manner (Ki values of 17.55±0.78μM and 1.53±0.47 μM, respectively), indicating a greater affinity for DA than for 5-HT membrane transporters. The affinity profile of mephedrone for the 5-HT₂ and D₂ receptors was assessed by studying [³H]ketanserin and [³H] raclopride binding in rat membranes. Mephedrone showed a greater affinity for the 5-HT₂ than for the D₂ receptors. DISCUSSION These results provide evidence that mephedrone, interacting with 5-HT and DA transporters and receptors must display a similar pattern of other psychoactive drugs such as amphetamine-like compounds.

Mitochondrial membrane potential measurement in rat cerebellar neurons by flow cytometry.

Mitochondrial membrane potential (MMP) in dissociated rat cerebellar neurons was measured using rhodamine 123 (Rh 123) as fluorescent dye, and flow cytometry. Dye distribution was studied by confocal scanning microscopy. Propidium iodide (PI)-marked cells (dead cells) were not stained by Rh 123, while the green fluorescence of living cells was restricted to mitochondria. Incubation of cells with different ionophores resulted in a maximal inhibition of Rh 123 fluorescence of 27.0 +/- 5.9% (valinomycin), 55.6 +/- 7.2% (ionomycin), and 37.3 +/- 5.1% (gramicidin). Ionophores decreased cell viability at high concentrations, measured as the number of propidium iodide-marked cells. Exposure of cell suspensions to the mitochondrial specific uncoupling agent CCCP caused a decrease in Rh 123 fluorescence (40 +/- 6.1%). Conversely, oxidative stress induced by H2O2 did not affect Rh 123 fluorescence. Impairment of glucose bioavailability reduced Rh 123 fluorescence. 2-Deoxy-D-glucose decreased the MMP with a maximal inhibition of 24.0 +/- 4.4%. Lack of glucose in the incubation medium also resulted in a decrease in MMP. Moreover, application of L-glutamate and N-methyl-D-aspartate (NMDA) (the excitatory amino acids) decreased Rh 123 uptake in a dose-dependent manner, which suggests that the measurement of MMP in dissociated cerebellar neurons by flow cytometry is a suitable method to detect the activity of drugs acting on glutamate receptors.

Memantine is a useful drug to prevent the spatial and non-spatial memory deficits induced by methamphetamine in rats

Methamphetamine (METH) is a street drug that is abused by young people. In previous studies, we demonstrated the effectiveness of alpha-7 nicotinic receptor antagonists in preventing the neurotoxicity induced by this amphetamine derivative. The present study seeks to determine whether pre-treatment with memantine (MEM) (an antagonist of both NMDA and alpha-7 nicotinic receptors) counteracts the memory impairment induced by METH administration in male Long Evans rats. Non-spatial memory was tested in the object recognition test and spatial learning memory was tested in the Morris water maze. In our experimental conditions, rats that received the MEM (5 mg/kg, intraperitoneally) pre-treatment recovered the ability to discriminate between a familiar and a novel object. This ability had been abolished by METH (10 mg/kg, subcutaneously) at 72 h and 1 week after treatment. Moreover, MEM pre-treatment also inhibited the thigmotaxis behaviour induced by METH. Rats treated with METH showed impaired learning in the Morris water maze. The results of the probe trial demonstrated that METH-treated rats did not remember the location of the platform, but this memory impairment was also prevented by MEM pre-treatment. Moreover, MEM by itself improved the learning of the task. Finally, MEM significantly improved the learning and memory impairment induced by METH. Therefore, MEM constitutes the first successful approach to prevent the cognitive deficits induced by amphetamine derivatives which are frequently abused in western countries.

DETERMINATION OF NITRIC OXIDE GENERATION IN MAMMALIAN NEURONS USING DICHLOROFLUORESCIN DIACETATE AND FLOW CYTOMETRY

A method for the rapid detection of intracellular nitric oxide (NO) generation in dissociated cerebellar granule cells using dichlorofluorescin (DCFH) and flow cytometry was developed. DCFH can be oxidized specifically by NO and this was assessed by 1) the use of SIN-1 (10 nM-100 microM), an NO donor, that induced a concentration-dependent increase in dichlorofluorescein (DCF) fluorescence and 2) the use of hemoglobin (10 microM), an NO-scavenger, that totally inhibited the increase of fluorescence induced by SIN-1 (10 microM). This assay was used to determine the ability to kainate to stimulate NO production in dissociated cerebellar granule cells. Kainate (1 microM-10 mM) induced an increase in DCF fluorescence that was partially reduced by NG-nitro-L-arginine (1 nM-10 microM), a nitric oxide synthase inhibitor (61.9% +/- 9.1), or hemoglobin (10 microM) (55.0% +/- 4.1). The method described allows evaluation of the oxidation of DCFH to produce DCF as a parameter for measuring intracellular NO generation. The extent of DCFH oxidation by NO and ROS can be determined by using NO scavengers or NO synthase inhibitors.

Memantine prevents the cognitive impairment induced by 3,4-methylenedioxymethamphetamine in rats

Amphetamine abuse is an important risk factor for the development of cognitive impairment involving learning and memory. Since in previous studies we have demonstrated the effectiveness of alpha-7 nicotinic receptor antagonists in preventing the neurotoxicity induced by amphetamine derivatives, the present paper seeks to determine whether pre-treatment with memantine (MEM) (an antagonist of both nicotinic and NMDA receptors) counteracts the memory impairment induced by 3,4-methylenedioxymethamphetamine (MDMA or ecstasy) administration in male Long Evans rats. In mice, MDMA and MEM induced a locomotor stimulant response but with a different profile. Moreover, MEM inhibited the rearing and thygmotaxis behaviour induced by MDMA. Non-spatial memory was tested in the object recognition test and the spatial learning and memory was tested in the Morris water maze. In our experimental conditions, rats receiving MEM pre-treatment recovered the ability to discriminate between the familiar and the novel object that had been abolished by MDMA treatment. Animals treated with MDMA showed impaired learning in the Morris water maze. Results of the probe trial demonstrated that MDMA-treated rats did not remember the location of the platform, but this memory impairment was also prevented by the MEM pre-treatment. Moreover, MEM alone improved the learning task. No differences were observed between the different groups as regards swim speed. In conclusion, MEM significantly improved the learning and memory impairment induced by MDMA and constitutes the first approach to the treatment of the long-term cognitive deficits found in ecstasy users.

A new aspect of the antiproliferative action of peripheral-type benzodiazepine receptor ligands

Ro 5-4864 (4-chlorodiazepam), PK 11195 (1-(2-chlorophenyl)-1,3-dihydro-1-methyl-propyl)isoquinoline carboxamide) and diazepam inhibit, in a concentration-dependent way, the proliferation of V79 Chinese hamster lung cells (IC50 values were: 65.0 +/- 3.73 microM, 57.70 +/- 4.75 microM and 106.80 +/- 8.89 microM, respectively) without being cytotoxic. This antiproliferative effect is mediated by mitosis arrest in the G2 + M stage without affecting DNA synthesis and seems unrelated to a specific interaction of these drugs with the peripheral-type benzodiazepine receptor.

Flavonoids as inhibitors of rat liver cytosolic glutathione S-transferase

The inhibitory potencies of different flavonoids for rat liver cytosolic glutathione S-transferase activity varied over 30-fold, depending on the pattern of hydroxylation, the presence of a C-2, C-3 double bond and the substitution of a hydroxyl group with a sugar moiety. Kinetic inactivation studies of the enzyme with the inhibitor quercetin revealed a non-competitive profile versus both glutathione and 1-chloro-2,4-dinitrobenzene.

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.