Sigma receptor ligands haloperidol and ifenprodil attenuate hypoxia induced dopamine release in rat striatum.

OBJECTIVE: We aimed to investigate the hypothesis that sigma receptor ligands, haloperidol and ifenprodil, attenuate hypoxia-induced striatal dopamine release in vitro and determine the possible mechanisms. METHODS: Extracellular concentrations of dopamine were measured using acute brain slices method under hypoxic, aglycemic and ischemic conditions. Sigma receptor ligands haloperidol and ifenprodil attenuate striatal dopamine release induced by hypoxia in contrast to aglycemia and ischemia. To determine the possible contribution of glutamatergic system on this effect, we compared the effect of NMDA receptor antagonist MK-801 and haloperidol in hypoxia induced by Na-K-ATPaz enzyme inhibitor ouabain. Also, we compared the effect of dopamine uptake blocker nomifensine and haloperidol to determine the role of dopamine transporter on this effect. RESULTS: Haloperidol and nomifensine almost completely abolish ouabain-induced dopamine release unlike MK-801. Different effects of sigma ligands and glutamate receptor antagonists on the hypoxia and ouabain induced dopamine release show that glutamate receptor blockade is partial involved in inhibitory effect of sigma ligand on dopamine release under hypoxic conditions. Similar effect of dopamine uptake blocker nomifensine and sigma receptor ligand haloperidol on ouabain induced dopamine release supports the possibility that inhibition of reverse dopamine transport by sigma ligands might be involved in their protective effect. CONCLUSIONS: Data in this study suggest that sigma ligands may be a new therapeutic intervention for the management of hypoxic conditions.

Glutamate-induced modulation in energy metabolism contributes to protection of rat cortical slices against ischemia-induced damage.

OBJECTIVES: Glutamate excitotoxicity contributes to neurodegeneration during cerebral ischemia. Recent studies in the protective effect of glutamate against ischemia and hypoxia have shown the need for questioning the role of glutamate in energy metabolism during ischemia. Current study investigates the effect of glutamate on energy substrate metabolites such as alpha-ketoglutarate, lactate, and pyruvate release during control, oxygen-glucose deprivation (OGD), and reoxygenation (REO) conditions. METHODS: The effects of 0.5 and 2 mM glutamate on spontaneous alpha-ketoglutarate, lactate, and pyruvate release were tested in vitro, on acute rat cortical slices. Alpha-ketoglutarate, lactate, and pyruvate levels were determined by HPLC with UV detector. RESULTS: We observed that glutamate added into medium significantly increased alpha-ketogluarate release under control conditions. Although OGD and REO also had a glutamate-like effect, only REO-induced rise further enhanced by glutamate. In contrast to alpha-ketoglutarate, both OGD and REO conditions caused significant declines in pyruvate and lactate outputs. While OGD and REO-induced declines in pyruvate outputs were further potentiated, lactate output was not altered by glutamate added into the medium. Glutamate and alpha-ketoglutarate, moreover, also ameliorated OGD- and REO-induced losses in 2,3,5-triphenyltetrazolium chloride staining with a similar degree. CONCLUSION: These results indicate that glutamate probably increases alpha-ketoglutarate production as an alternative energy source for use in the TCA cycle under energy-depleted conditions. Thus, increasing the alpha-ketoglutarate production may represent a new therapeutic intervention for neurodegenerative disorders, including cerebral ischemia.

K channel blockage with 3,4-diaminopyridine potentiates the effect of L-DOPA on dopamine release in striatal slices prepared from 6-OHDA pre-treated rats.

Although L-DOPA revolutionized in the treatment of Parkinson's disease, most patients developed motor complications after several years of treatment. Adjunctive therapy to L-DOPA with drugs related to dopaminergic signaling may reduce its dose without decreasing the therapeutic efficiency and thus ameliorates its adverse effects. It has been shown that 3,4-diaminopyridine (3,4-DAP), a K channel blocker, increased dopamine release from striatal slices by increasing neuronal firing in striatal dopaminergic terminals. The current study investigates whether 3,4-DAP may enhance L-DOPA-induced dopamine (DA) release from striatal slices by increasing neuronal firing in striatal dopaminergic terminals. The effects of L-DOPA and 3,4-DAP on spontaneous DA and DOPAC release were tested in vitro, on acute rat striatal slices prepared from non-treated and 6-hydroxydopamine-pre-treated rats. DA and DOPAC levels were determined by HPLC methods. When 3,4-diaminopyridine was combined with L-DOPA, the observed effect was considerably greater than the increases induced by L-DOPA or 3,4-DAP alone in normoxic and neurodegenerative conditions produced by FeSO4 and 6-hydroxydopamine. Furthermore, L-DOPA plus 3,4-DAP also ameliorated DOPAC levels in neurodegenerative conditions. These data indicate that 3,4 DAP plus L-DOPA activates striatal dopaminergic terminals by increasing the DA release and, thus, could be considered as a promising finding in treatment of acute and chronic injury in dopaminergic neurons.

Brain slice viability determined under normoxic and oxidative stress conditions: involvement of slice quantity in the medium.

Objective: In vitro acute adult brain slice methods are instruments in developing our knowledge of the nervous system. Optimization of this method for obtaining high-quality brain slices is extremely important in terms of consistency and reliability of the experimental results. Although some important topics such as slice thickness, temperature, and composition of the physiological medium have been studied for optimization, involvement of slice quantity in medium on tissue viability has not been investigated yet.Methods: Different number of slices (1, 3, or 6 slices) were incubated under normoxic or some prooxidant stress conditions induced by oxygen-glucose deprivation (OGD), H2O2, FeSO4+ ascorbic acid, or menadione to evaluate the effect of slice density on tissue viability.Results:Slice quantity in the normoxic incubation medium caused a significant increase in 2,3,5-triphenyltetrazolium chloride (TTC) staining intensity of the slices. Similarly, increase in the slice quantity in the medium also protected the slices against either OGD, H2O2, FeSO4, or menadione-induced decrease in TTC staining. In addition to TTC staining, lactate dehydrogenase leakage or malondialdehyde and reactive oxygen species production under normoxic or ischemia-like conditions were also attenuated by increasing slice quantity in the medium.Conclusion: These results show that when using brain slices method for investigating the structural and functional features of brain at the molecular and cellular levels, both slice quantity in the medium and incubation volume should be considered first. Increasing slice quantity or decreasing incubation volume probably causes an increase in the concentration of endogenous substance(s) involved in neuroprotection.

The effect of Gly-Gln [ß-endorphin30-31] on morphine-evoked serotonin and GABA efflux in the nucleus accumbens of conscious rats.

Glycyl-L-glutamine (Gly-Gln; ß-endorphin30-31) is an endogenous dipeptide synthesized through the post-translational processing of ß-endorphin1-31. Central Gly-Gln administration inhibits the rewarding properties of morphine and attenuates morphine tolerance, dependence and withdrawal although it does not interfere with morphine analgesia. In an earlier study, we found that Gly-Gln inhibits morphine-induced dopamine efflux in the nucleus accumbens (NAc), consistent with its ability to inhibit morphine reward. To further investigate the mechanism responsible for its central effects we tested whether i.c.v. Gly-Gln administration influences the rise in extracellular serotonin and GABA concentrations evoked by morphine in the NAc. Conscious rats were treated with Gly-Gln (100nmol/5µl) or saline i.c.v. followed, 2min later, by morphine (2.5mg/kg) or saline i.p. and extracellular serotonin and GABA concentrations were analyzed by microdialysis and HPLC. Morphine administration increased extracellular serotonin and GABA concentrations significantly within 20min, as shown previously. Unexpectedly, Gly-Gln also increased extracellular serotonin concentrations significantly in control animals. Combined treatment with Gly-Gln+morphine also elevated extracellular serotonin concentrations although the magnitude of the response did not differ significantly from the effect of Gly-Gln or morphine, given alone suggesting that Gly-Gln suppressed morphine induced serotonin efflux. Gly-Gln abolished the morphine-induced rise in extracellular GABA concentrations but had no effect on extracellular GABA when given alone to otherwise untreated animals. These data show that Gly-Gln stimulates NAc serotonin efflux and, together with earlier studies, support the hypothesis that Gly-Gln inhibits the rewarding effects of morphine by modulating morphine induced dopamine, GABA and serotonin efflux in the NAc.

High glutamate attenuates S100B and LDH outputs from rat cortical slices enhanced by either oxygen-glucose deprivation or menadione.

One hour incubation of rat cortical slices in a medium without oxygen and glucose (oxygen-glucose deprivation, OGD) increased S100B release to 6.53 ± 0.3 ng/ml/mg protein from its control value of 3.61 ± 0.2 ng/ml/mg protein. When these slices were then transferred to a medium containing oxygen and glucose (reoxygenation, REO), S100B release rose to 344 % of its control value. REO also caused 192 % increase in lactate dehydrogenase (LDH) leakage. Glutamate added at millimolar concentration into the medium decreased OGD or REO-induced S100B release and REO-induced LDH leakage. Alpha-ketoglutarate, a metabolic product of glutamate, was found to be as effective as glutamate in decreasing the S100B and LDH outputs. Similarly lactate, 2-ketobutyrate and ethyl pyruvate, a lipophilic derivative of pyruvate, also exerted a glutamate-like effect on S100B and LDH outputs. Preincubation with menadione, which produces H2O2 intracellularly, significantly increased S100B and LDH levels in normoxic medium. All drugs tested in the present study, with the exception of pyruvate, showed a complete protection against menadione preincubation. Additionally, each OGD-REO, menadione or H2O2-induced mitochondrial energy impairments determined by 2,3,5-triphenyltetrazolium chloride (TTC) staining and OGD-REO or menadione-induced increases in reactive oxygen substances (ROS) determined by 2,7-dichlorofluorescin diacetate (DCFH-DA) were also recovered by glutamate. Interestingly, H2O2-induced increase in fluorescence intensity derived from DCFH-DA in a slice-free physiological medium was attenuated significantly by glutamate and alpha-keto acids. All these drug actions support the conclusion that high glutamate, such as alpha-ketoglutarate and other keto acids, protects the slices against OGD- and REO-induced S100B and LDH outputs probably by scavenging ROS in addition to its energy substrate metabolite property.

Glycyl-glutamine (beta-endorphin(30-31)) inhibits morphine-induced dopamine efflux in the nucleus accumbens.

Glycyl-glutamine (Gly-Gln) is an endogenous dipeptide that is synthesized from beta-endorphin post-translationally. Previously, we showed that Gly-Gln prevents acquisition of morphine-conditioned place preference, a behavioral test of morphine reward, but does not interfere with morphine analgesia. In this study, we tested the hypothesis that Gly-Gln inhibits morphine reward by blocking morphine-induced dopamine efflux in the nucleus accumbens (NAc). Extracellular dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC) were sampled by microdialysis and analyzed by high-performance liquid chromatography with electrochemical detection. Guide cannulas were implanted in the right NAc and left lateral ventricle of male Sprague-Dawley rats stereotaxically. Approximately 24 h later, a microdialysis probe was inserted into the NAc and perfused at 1 microl/min. Gly-Gln (1, 3, 30, or 100 nmol/5 microl) or saline was administered intracerebroventricularly, morphine (2.5 mg/kg) was injected intraperitoneally (i.p.) 2 min later, and extracellular dopamine and DOPAC were sampled at 20-min intervals. Morphine administration increased extracellular dopamine concentrations by approximately 600% within 40 min. Gly-Gln pretreatment inhibited the rise in extracellular dopamine in a dose-related manner; the lowest significantly inhibitory dose was 1 nmol. Gly-Gln also inhibited the morphine-induced rise in extracellular DOPAC concentrations but did not affect extracellular dopamine or DOPAC in control animals. Gly-Gln (100 nmol/5 microl) prevented morphine-induced dopamine efflux in rats treated with morphine chronically (10 mg/kg, i.p. twice daily for 6 days), although it did not affect DOPAC concentrations significantly. These data support the hypothesis that Gly-Gln abolishes the rewarding effect of morphine by inhibiting the ability of morphine to stimulate dopamine release in the NAc.

Mechanism of S100b release from rat cortical slices determined under basal and stimulated conditions.

Incubation of rat cortical slices in a medium that was not containing oxygen and glucose (oxygen-glucose deprivation, OGD) caused a 200% increase in the release of S100B. However, when slices were transferred to a medium containing oxygen and glucose (reoxygenation conditions, or REO), S100B release reached 500% of its control value. Neither inhibition of nitric oxide (NO) synthase by L-NAME nor addition of the NO donors sodium nitroprussid (SNP) or hydroxylamine (HA) to the medium altered basal S100B release. Similarly, the presence of SNP, HA or NO precursor L: -arginine in the medium, or inhibition of NO synthase by L-NAME also failed to alter OGD- and REO-induced S100B outputs. Moreover, individual inhibition of PKC, PLA(2) or PLC all failed to attenuate the S100B release determined under control condition or enhanced by either OGD or REO. Blockade of calcium channels with verapamil, chelating the Ca(+2) ions with BAPTA or blockade of sodium channels with tetrodotoxin (TTX) did not alter OGD- and REO-induced S100B release. In contrast to the pharmacologic manipulations mentioned above, glutamate and alpha-ketoglutarate added at high concentrations to the medium prevented both OGD- and REO-induced S100B outputs. These results indicate that neither NO nor the activation of PKC, PLA(2) or PLC seem to be involved in basal or OGD- and REO-induced S100B outputs. Additionally, calcium and sodium currents that are sensitive to verapamil and TTX, respectively, are unlikely to contribute to the enhanced S100B release observed under these conditions.

Resveratrol protects rat striatal slices against anoxia-induced dopamine release.

Incubation of rat striatal slices in anoxic medium caused significant alterations in dopamine (DA) and 3,4-dihydroxyphenylacetic acid (DOPAC) outputs; while DA release increased several times, 50% decline in DOPAC output was observed under this condition. Tissue ATP level, on the other hand, was decreased 40% by anoxia. Presence of resveratrol in the medium decreased anoxia-induced DA release in a concentration-dependent manner. Enhanced DA output, however, was declined slightly by epicatechine and catechine, and not altered significantly by morin hydrate and quercetin dehydrate which are other penolic compounds present in the red wine. In contrary to DA output, anoxia-induced decline in tissue ATP level was not ameliorated by resveratrol. In addition to anoxia, resveratrol, as observed with DA uptake blocker nomifensine, also reduced DA release stimulated by ouabain. Efficiencies of both resveratrol and nomifensine to attenuate ouabain-induced DA output, however, were closely dependent on ouabain concentration in the medium. These results indicate that some phenolic compounds, particularly resveratrol decrease anoxia-induced DA output and appear promising agents to improve the alterations occurred under anoxic-ischemic conditions.