Elena Escubedo

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.

Kainic acid-induced apoptosis in cerebellar granule neurons: an attempt at cell cycle re-entry

This study was undertaken to investigate whether kainic acid (KA) may regulate the expression of several proteins which plays an important role in cell-cycle progression in cerebellar granule neurons (CGNs). KA induced decrease in MTT values in a concentration dependent way. Flow cytometric analysis showed that KA was able to induce 30% apoptosis in CGNs. Apoptotic nuclear condensation were detected 24 h of exposure to KA (200 μM). An associated marked increase in DNA synthesis, measured by BrdU incorporation, was observed. Western blot analysis showed that KA induced an increase in the expression of Cdk2, cyclin E and E2F-1. It is proposed that, in post-mitotic cells like CGNs, re-entry cell cycle could be responsible for the apoptotic effect of KA.

Microgliosis and down-regulation of adenosine transporter induced by methamphetamine in rats

Chronic administration of methamphetamine to rats induces neurotoxicity characterized by a loss of striatal dopaminergic terminals and reactive gliosis. Subcutaneous administration of methamphetamine in a scheduled procedure of four doses (10 mg/kg) at 2 h interval also induces a significant increase in the peripheral-type benzodiazepine receptor (PBR) density. This increase is maximum (76%) at 72 h post-treatment in the striatum and disappears at 7 days, suggesting that microglia may have a predominant role in necrosis-phagocytosis of neuronal debris rather than acting in a restorative manner. Microgliosis is not restricted to the striatum since it is also evident in cerebellum (75.4% of PBR increase) and hippocampus (37.2% of PBR increase). In the areas with high density of adenosine transporter, the microgliosis phenomenon correlates well with a decrease of this nucleoside transporter (about 39%). Although the microgliosis and the decrease in adenosine transporter could be parallel and not related events, we can speculate that when microglia are activated, a down-regulation of adenosine transporter occurs, playing a role in tissue homeostasis. With the same dosing schedule, methamphetamine induces HSP72 expression in both cytoplasmic and nuclear fractions of the striatum, cerebellum and hippocampus. This expression is also evident in the cerebral cortex, where adenosine transporter population did not show any variation.

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 protects against amphetamine derivatives-induced neurotoxic damage in rodents.

We hypothesize that 3,4-methylenedioxymethamphetamine (MDMA) and methamphetamine (METH) interact with alpha-7 nicotinic receptors (nAChR). Here we examine whether memantine (MEM), an antagonist of NMDAR and alpha-7 nAChR, prevents MDMA and METH neurotoxicity. MEM prevented both serotonergic injury induced by MDMA in rat and dopaminergic lesion by METH in mice. MEM has a better protective effect in front of MDMA- and METH-induced neurotoxicity than methyllycaconitine (MLA), a specific alpha-7 nAChR antagonist. The double antagonism that MEM exerts on NMDA receptor and on alpha-7 nAChR, probably contributes to its effectiveness. MEM inhibited reactive oxygen species production induced by MDMA or METH in synaptosomes. This effect was not modified by NMDA receptor antagonists, but reversed by alpha-7 nAChR agonist (PNU 282987), demonstrating a preventive effect of MEM as a result of it blocking alpha-7 nAChR. In synaptosomes, MDMA decreased 5-HT uptake by about 40%. This decrease was prevented by MEM and by MLA but enhanced by PNU 282987. A similar pattern was observed when we measured the dopamine transport inhibited by METH. The inhibition of both transporters by amphetamine derivatives seems to be regulated by the calcium incorporation after activation of alpha-7 nAChR. MDMA competitively displaces [(3)H]MLA from rat brain membranes. MEM and METH also displace [(3)H]MLA with non-competitive displacement profiles that fit a two-site model. We conclude that MEM prevents MDMA and METH effects in rodents. MEM may offer neuroprotection against neurotoxicity induced by MDMA and METH by preventing the deleterious effects of these amphetamine derivatives on their respective transporters.

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.

Protection against MDMA-induced dopaminergic neurotoxicity in mice by methyllycaconitine : Involvement of nicotinic receptors

Methylenedioxymethamphetamine (MDMA) is a relatively selective dopaminergic neurotoxin in mice. Previous studies demonstrated the participation of alpha-7 nicotinic receptors (nAChR) in the neurotoxic effect of methamphetamine. The aim of this paper was to study the role of this receptor type in the acute effects and neurotoxicity of MDMA in mice. In vivo, methyllycaconitine (MLA), a specific alpha-7 nAChR antagonist, significantly prevented MDMA-induced neurotoxicity at dopaminergic but not at serotonergic level, without affecting MDMA-induced hyperthermia. Glial activation was also fully prevented by MLA. In vitro, MDMA induced intrasynaptosomal reactive oxygen species (ROS) generation, which was calcium-, nitric-oxide synthase-, and protein kinase C-dependent. Also, the increase in ROS was prevented by MLA and alpha-bungarotoxin. Experiments with reserpine point to endogenous dopamine (DA) as the main source of MDMA-induced ROS. MLA also brought the MDMA-induced inhibition of [3H]DA uptake down, from 73% to 11%. We demonstrate that a coordinated activation of alpha-7 nAChR, blockade of DA transporter function and displacement of DA from intracellular stores induced by MDMA produces a neurotoxic effect that can be prevented by MLA, suggesting that alpha-7 nAChR have a key role in the MDMA neurotoxicity in mice; however, the involvement of nicotinic receptors containing the beta2 subunit cannot be conclusively ruled out.

Neuroprotective action of flavopiridol, a cyclin-dependent kinase inhibitor, in colchicine-induced apoptosis

Flavopiridol was developed as a drug for cancer therapy due to its ability to inhibit cell cycle progression by targeting cyclin-dependent kinases (CDKs). In this study, we show that flavopiridol may also have a neuroprotective action. We show that at therapeutic dosage (or at micromolar range), flavopiridol almost completely prevents colchicine-induced apoptosis in cerebellar granule neurones. In agreement with this, flavopiridol inhibits both the release of cyt c and the activation of caspase-3 induced in response to colchicine treatment. We demonstrate that in this cellular model for neurotoxicity, neither re-entry in the cell cycle nor activation of stress-activated protein kinases, such as c-Jun N-terminal kinase (JNK) or p38 MAP kinase, is involved. In contrast, we show that colchicine-induced apoptosis correlates with a substantial increase in the expression of cdk5 and Par-4, which is efficiently prevented by flavopiridol. Accordingly, a cdk5 inhibitor such as roscovitine, but not a cdk4 inhibitor such as 3-ATA, was also able to protect neurons from apoptosis as well as prevent accumulation of cdk5 and Par-4 in response to colchicine. Our data suggest a potential therapeutic use of flavopiridol in disorders of the central nervous system in which cytoskeleton alteration mediated by cdk5 activation and Par-4 expression has been demonstrated, such as Alzheimers disease.

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.