Search

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.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.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.

The pro-oxidative effect of methamphetamine (METH) in dopamine terminals was studied in rat striatal synaptosomes. Flow cytometry analysis showed increased production of reactive oxygen species (ROS) in METH-treated synaptosomes, without reduction in the density of dopamine transporters. In synaptosomes from dopamine (DA)-depleted animals, METH did not induce ROS production. Reserpine, in vitro, completely inhibited METH-induced ROS production. These results point to endogenous DA as the main source of ROS induced by METH. Antioxidants and inhibitors of neuronal nitric oxide synthase and protein kinase C (PKC) prevented the METH-induced oxidative effect. EGTA and the specific antagonist methyllycaconitine (MLA, 50 microM) prevented METH-induced ROS production, thus implicating calcium and alpha7 nicotinic receptors in such effect. Higher concentrations of MLA (>100 microM) showed nonspecific antioxidant effect. Preincubation of synaptosomes with METH (1 microM) for 30 min reduced [(3)H]DA uptake by 0%. The METH effect was attenuated by MLA and EGTA and potentiated by nicotine, indicating that activation of alpha(7) nicotinic receptors and Ca(2+) entry are necessary and take place before DAT inhibition. From these findings, it can be postulated that, in our model, METH induces DA release from synaptic vesicles to the cytosol. Simultaneously, METH activates alpha(7) nicotinic receptors, probably inducing depolarization and an increase in intrasynaptosomal Ca(2+). This would lead to DAT inhibition and NOS and PKC activation, initiating oxidation of cytosolic DA.

The consequences of the neurotoxic insult induced by 3,4-methylenedioxymethamphetamine (MDMA, an amphetamine derivative with specific action on the serotonergic system) were compared with those of methamphetamine (a derivative with specific action on dopaminergic system) in rats. Both drugs induced a very similar loss of body weight, especially evident 24 h after treatment. Their hyperthermic profile was also very similar and was dependent on ambient temperature, corroborating the thermo-dysregulatory effect of both substances. Methamphetamine (four injections of 10 mg kg(-1) s.c. at 2-h intervals) induced the loss of dopaminergic (35%) but not of serotonergic, terminals in the rat striatum and, simultaneously, a significant increase in striatal peripheral-type benzodiazepine receptor density, pointing to a glial reaction. Evidence for this drug-induced astrogliosis was the increased heat shock protein 27 (HSP27) expression in striatum, cortex and hippocampus. MDMA (20 mg kg(-1) s.c. b.i.d. for 4 days) induced a similar dopaminergic lesion in the striatum 3 days post-treatment, which reversed 4 days later. An important neurotoxic effect on serotonergic terminals was also observed in the cortex, striatum and hippocampus 3 days post-treatment, which partially reversed 4 days later in the striatum and hippocampus. No microglial activation was noticeable at either 3 or 7 days after MDMA treatment. This lack of effect on microglial cells was assessed by [(3)H]PK 11195 binding and OX-6 immunostaining, which were unchanged in the striatum and cortex after MDMA treatment. A non-significant tendency to increase was noted in the hippocampus 3 days after MDMA treatment. Furthermore, in MDMA-treated rats, neither HSP27 expression nor an increase in HSP27 immunoreactivity were detected. This result, together with the lack of increase in glial fibrilliary acidic protein (GFAP) immunoreactivity, indicate no astroglial activation at either 3 or 7 days post-treatment. Without microglial activation, an inflammatory process would not accompany the lesion induced by MDMA. The differences in glial activation between methamphetamine and MDMA observed in the present study could have implications for the prognosis of the injury induced by these drugs.

The overexcitation of glutamate receptors is believed to be the cause of several neurodegenerative disorders. The determination of calcium fluxes, mitochondrial membrane potential (MMP) variations or the production of reactive oxygen species (ROS) in mammalian cells are usually measured during the development of potentially useful drugs that might interfere in the events induced by glutamate receptor activation. By using flow cytometry with dissociated cerebellar granule cells, we have developed a rapid and economical method to measure changes in biochemical parameters that are involved in neuronal cell death. The formation of intracellular ROS is measured using 2',7'-dichlorofluorescin diacetate (DCFH-DA). The mitochondrial membrane potential is assessed by the retention of rhodamine 123 (Rh123), a specific fluorescent cationic dye that is readily sequestered by active mitochondria, depending on their transmembrane potential. Finally, intracellular calcium increases are detected by using the calcium-selective indicator Indo-1. Cell viability is also assessed by using propidium iodide (PI) which stains DNA strands of permeabilized cells. This method might be useful for the screening of new drugs with potential neuroprotective activity, with improved cost/effectiveness ratio compared to other techniques.

The anticholinergic drug orphenadrine is used in the treatment of Parkinson's disease. In this study we evaluate the neuroprotective effects of orphenadrine on excitotoxicity in vivo and in vitro. Orphenadrine prevented the mitochondrial and the cytoplasmic membrane potential decrease evoked by NMDA (100 microM) in rat dissociated cerebellar granule cells showing an IC50 value of 11.6 +/- 4.7 microM (mean +/- SEM, n = 5) and 13.5 +/- 2.3 microM (n = 3), respectively. Orphenadrine was able to protect cerebellar granule cell cultures from glutamate-induced neurotoxicity. Kainic acid (KA, 10 mg/kg)-induced excitotoxicity was evaluated in vivo using the microglial marker peripheral-type benzodiazepine receptor (PBR) and heat shock protein 72 (HSP72) expression in the hippocampus. The Bmax of PBR for control tissues was 589.1 +/- 40.0 fmol/mg protein (n = 4), increasing to 1692.5 +/- 51.6 fmol/mg protein (n = 5) after the KA treatment. Pretreatment with orphenadrine (10 mg/kg) blocked the KA-induced increase in PBR density. As expected, KA-administration induced the expression of HSP72 that was blocked in the orphenadrine + KA-treated rats. We demonstrate that orphenadrine, interacting at the NMDA receptor, is able to prevent the neurotoxicity mediated by activation at glutamate ionotropic receptors.

Activation of NMDA receptors in dissociated cerebellar granule cells reduced mitochondrial membrane potential (MMP), as measured by rhodamine 123 fluorescence in a flow cytometer. This effect was inhibited by several NMDA-receptor antagonists with the following rank order of potency: MK-801 > PCP > TCP > dextrorphan > dichlorokynurenic acid > D-AP5 > dextromethorphan. Neither spermine nor arcaine modified the NMDA-induced reduction in MMP, whereas ifenprodil and eliprodil inhibited this response in the micromolar range. The mechanism responsible for the alteration of MMP mediated by NMDA was studied. Mepacrine and dibucaine prevented the MMP reduction induced by NMDA, as did W13 (calmodulin antagonist). In contrast, this effect was not blocked by cyclooxygenase or lipooxygenase inhibitors, H7 (a protein kinase C inhibitor) or nitroarginine (nitric oxide synthase inhibitor). These data suggest a direct interaction between NMDA-receptor activation and arachidonic acid formation, and indicate that NMDA receptor-mediated effect on MMP could involve arachidonic acid.

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.

Previous work from our laboratory has demonstrated the presence of high-affinity binding sites for [3H]nitrobenzylthioinosine ([3H]NBTI), a marker of adenosine uptake systems, in the mitochondrial fraction of rat testis. Here, we characterize this system functionally through [3H]adenosine uptake assays. This system (K(m)=2+/-1.3 microM; V(max)=86.2+/-15.5 pmol/mg protein/min) was found to be saturable, non sodium-dependent and sensitive to temperature, pH and osmolarity. [3H]Adenosine incorporation was potently inhibited by hydroxynitrobenzylthioguanosine (HNBTG, IC(50)=3 nM) although NBTI inhibited this uptake weakly (IC(50)=72. 7+/-37.1 microM). Dilazep>dipyridamole>/=hexobendine inhibited [3H]adenosine incorporation at low micromolar concentrations. The nucleosides inosine and uridine were weak inhibitors of this system. The adenosine receptor ligands N(6)-phenylisopropyladenosine (PIA) and 2-chloroadenosine inhibited the uptake only at micromolar concentrations. Neither 5'-(N-ethylcarboxamido)-adenosine (NECA) nor theophylline inhibited adenosine uptake by more than 60% but the mitochodrial benzodiazepine receptor ligands 4'-chloro-diazepam (Ro 5-4864) and 1-(2-chlorophenyl)-N-methyl-N-(1-methyl-propyl) isoquinoline carboxamide (PK 11195) were able to inhibit it. The lack of inhibition by the blockers of the mitochondrial adenine-nucleotide carrier, atractyloside and alpha, beta-methylene-ATP, indicates that [3H]adenosine uptake occurs via a transporter other than this carrier. All these results support the existence of an equilibrative adenosine transport system, which might mediate the passage of adenosine formed in the mitochondria to the cytoplasm.

The effects of the lazaroid compound U-83836E on the glutamate-induced production of reactive oxygen species (ROS) were studied in dissociated rat cerebellar granule cells by flow cytometry. U-83836E completely inhibited ROS production with an estimated IC50 value of 21.7 +/- 9.1 nM. However, U-83836E did not inhibit the glutamate-evoked decrease in mitochondrial membrane potential (MMP). Nevertheless, U-83836E (10 nM to 10 microM) prevented cell death induced by 10 mM of glutamate. At concentrations above 10 microM, U-83836E by itself showed slight cytotoxicity, which was significant at a 100 microM concentration. U-83836E (25 to 200 microM) also increased the cytosolic calcium levels in a concentration-dependent manner. Our results indicate that the cytotoxic effects found at micromolar concentrations of U-83836E could be explained by an increase in [Ca2+]i. Finally, since U-83836E did not prevent the MMP decrease evoked by glutamate, it is suggested that antioxidant pharmacotherapy would not be sufficient to block the neurotoxic effects of glutamate.

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.

The effect of kainic acid (KA) on mitochondrial membrane potential (MMP) and reactive-oxygen species (ROS) production was studied in dissociated cerebellar granule cells from rat pups. KA induced a maximum increase of 361%+/-35% in ROS production. The lazaroid compound U-83836E (at concentrations ranging from 10(-9) to 5x10(-6) M) completely inhibited this increase, with an IC50 value of 3.02+/-1.08x10(-7) M. KA also decreased the mitochondrial membrane potential (MMP), with a maximum decrease of about 30%. Absence of Na+ in the incubation medium did not significantly alter the effect of KA on MMP. As expected, the AMPA/kainate receptor antagonist NBQX inhibited the effects of KA on MMP with an IC50 value of 1.1+/-0.8 microM. However, the lazaroid U-83836E, indomethacin, nor-dihydroguaiaretic acid and L-nitroarginine all failed to inhibit the KA-induced decrease in the MMP. Finally, to assess the neuroprotective effect of U-83836E on KA-induced neurotoxicity in vivo, the increase in the peripheral-type benzodiazepine receptor density in rat hippocampus was measured. Treatment with KA increased the Bmax to 1341+/-192 fmol mg(-1). When U-83836E was coadministered with KA, the Bmax was reduced to 765+/-122 fmol mg(-1), which was not significantly different from the Bmax obtained from untreated rats (Bmax: 518+/-33 fmol mg(-1)). We conclude that treatment with the lazaroid U-83836E might be a suitable therapeutic strategy in neurodegenerative disorders.

Sigma (sigma) and phencyclidine (PCP) receptor ligands, apart from their main effects on sigma receptors and NMDA receptor-mediated neurotransmission, have been found to interact with catecholamine systems in several central and peripheral tissues. In the present study the binding profile of [3H]nisoxetine ([3H]NIS), a selective marker of the noradrenaline transporter, has been characterized in rat vas deferens membranes to further study its modulation by a number of characteristic sigma and PCP ligands. The binding of [3H]NIS was found to be of high affinity (Kd = 1.63 +/- 0.36 nM), saturable, sodium-dependent and to a single population of binding sites (nH = 1.003 +/- 0.017). The maximal binding capacity was 1,625 +/- 500 fmol/mg of protein. Kinetic experiments gave a k(+1) of 3.9 x 10(7) min(-1)M(-1) and a k(-1) of 0.005 min(-1). The [3H]NIS binding was totally inhibited, with IC50 values in the micromolar range, by all the sigma and PCP ligands tested, with the following order of potency: haloperidoldextromethorphandizocilpinedextrorphan(+)-3-PPPPCPtenocyclidine. This order correlates well with that described in other tissues using [3H]desmethylimipramine. The inhibition by all these compounds, except that of (+)-3-PPP, was competitive. These results suggest that sigma and PCP ligands bind, at low micromolar concentrations, to a site in the noradrenaline transporter that is labelled by [3H]NIS.

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.

The effect of three different glutamate receptor ligands on mitochondrial membrane potential has been studied in rat pup dissociated cerebellar cells by measuring rhodamine 123 fluorescence. L-glutamate, NMDA (N-methyl-D-aspartate) and kainate (from 10(-8) to 10(-3) M) decreased in a concentration-dependent manner the mitochondrial membrane potential with EC50 values of 6.7 +/- 1.7, 3.8 +/- 0.5, and 37.4 +/- 14 microM, respectively. Dizocilpine ((+)MK 801) was able to inhibit the NMDA- and L-glutamate-induced decrease in rhodamine 123 fluorescence, while kainate-induced fluorescence-decreases were unaffected. However, 6-cyano-7-nitroquinoxaline-2, 3-dione (CNQX) totally prevented the effect of kainate on mitochondrial membrane potential, but failed to block the L-glutamate effect. It is concluded that, in our cell preparation, L-glutamate exerts its action mainly through NMDA-subtype receptors, and that Ca2+ and Na+ entry through ionotropic glutamate receptors could be responsible for an impairment of mitochondrial membrane potential.

1. 1. The peripheral-type benzodiazepine receptor was characterized in rat prostate and seminal vesicle using [ 3 H]Ro 5-4864 as radioligand. 2. 2. The affinity of this radioligand for this receptor was higher in rat prostate ( K D = 4.36 ± 1.02 nM) than in seminal vesicle ( K D = 8.45 ± 1.34 nM). 3. 3. The density of binding sites obtained in these two tissues was B max = 4164 ± 873 fmol/mg in prostate and 5978 ± 1022 fmol/mg in seminal vesicle. 4. 4. The [ 3 H]Ro 5-4864 binding was inhibited non-competitively by atractyloside and α,β-methyleneATP, suggesting a modulation by the ADP/ATP mitochondrial carrier. 5. 5. Flutamide was able to displace bound [ 3 H]Ro 5-4864 with an IC 50 similar to protoporphyrin IX.