Changes in the striatal extracellular levels of dopamine and dihydroxyphenylacetic acid evoked by ammonia and N-methyl-D-aspartate: modulation by taurine

Acute hyperammonemia is associated with motor disturbances that are thought to involve striatal dopaminergic dysfunction. Discharge of striatal dopaminergic neurons is controlled by N-methyl-D-aspartate (NMDA) receptors, the excessive activation of which contributes to ammonia neurotoxicity. Here we show that ammonium chloride ("ammonia", extracellular concentration 5 mM) or NMDA (1 mM), when directly administered to the rat striatum via a microdialysis probe, evoke a prompt accumulation of dopamine (DA) in the microdialysates. However, while ammonia increases, NMDA decreases, the extracellular dihydroxyphenylacetate (DOPAC) level. The results point to the NMDA receptor-mediated enhancement of DA release and increased DA metabolism as two independent ways by which ammonia affects the striatal dopaminergic system. Taurine (extracellular concentration 10 mM) attenuated the NMDA- and ammonia-evoked DA release and ammonia-induced accumulation of DOPAC, reflecting two different neuroprotective mechanisms of this amino acid.     

Nigrostriatal dopaminergic neurotoxicity of L-cysteine after stereotaxic administration into the substantia nigra of rats: Potential implications for MPTP-induced neurotoxicity and parkinson’s disease

Stereotaxic administration of L-cysteine (CySH) into the rat substantia nigra pars compacta (SN_C) evokes a dose-dependent fall of striatal levels of dopamine. This, together with decreased tyrosine hydroxylase immunoreactivity in the striatum and SN_cand decreased nigral staining for Niss1 substance indicate that CySH is a dopaminergic neurotoxin. The neurotoxic effects of CySH infusion into the rat SN_C were blocked by prior administration of the non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist MK-801. Previous studies have demonstrated that administration of the neurotoxin l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP) not only evokes the degeneration of nigrostriatal dopamine neurons but also causes a significant fall of glutathione (GSH) without corresponding increases of glutathione disulfide (GSSG). Furthermore, microdialysis studies have demonstrated that when perfusions of l-methyl-4-phenylpyridinium (MPP⁺), the active metabolite of MPTP, into the rat SN_C are discontinued extracellular levels of GSH massively but transiently increase followed by a prolonged elevation of extracellular CySH, the latter effect being blocked by inhibition of γ-glutamyl transpeptidase (γ-GT). These observations, together with the present results, suggest that the delayed but prolonged elevation of extracellular CySH that occurs as a MPP⁺-induced dopaminergic SN_ccell energy impairment begins to subside might evoke NMDA receptor mediated excitotoxicity. The potential roles of elevated extracellular CySH in MPTP/MPP⁺-induced dopaminergic neurotoxicity and in the pathogenesis of Parkinson’s disease are discussed.     

Acute effects of intranigral application of MPP+ on nigral and bilateral striatal release of dopamine simultaneously recorded by microdialysis.

Intracerebral microdialysis in freely moving rats was used to investigate the effects of perfusions with the 1-methyl-4-phenylpyridinium ion (MPP+) in the substantia nigra (SN) on the extracellular levels of dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA) in the perfused SN and in the ipsi- and contralateral striata. Following MPP+ perfusion, the release of DA in the SN increased markedly from nondetectable basal levels to about 105 fmoles/min, whereas the output of DOPAC, HVA and 5-HIAA decreased below 25% of basal levels. The intranigral MPP+ application induced, at the same time, an almost immediate, long-lasting decrease in the release of DA in the ipsilateral striatum to less than 20% of basal levels and a moderate increase in the DOPAC and HVA levels, without affecting 5-HIAA output. In the contralateral striatum, the extracellular levels of DA, DOPAC, HVA and 5-HIAA remained unchanged during the entire perfusion experiment. These results suggest that infusion of 10 mM MPP+ into the SN produces an almost immediate blockade of neuronal impulse flow, as shown by the rapid decline in DA release from the ipsilateral striatal nerve terminals. The simultaneously occurring massive increase of the extracellular DA in the SN is, therefore, probably the result of destruction of the nigral cell bodies and/or dendrites following locally applied MPP+. This study clearly illustrates the possibilities of simultaneous microdialysis in various brain areas, allowing pharmacological manipulations on the levels of the cell bodies, while monitoring events in the terminal areas.     

Release of dopamine by perfusion with 1-methyl-4-phenylpyridinium ion (MPP) into the striatum is associated with hydroxyl free radical generation

In Parkinson's disease (PD), the dopamine (DA) neuronal cell death in the nigrostriatal system has been proposed to be mediated by reactive oxygen radicals such as hydroxyl radicals (.OH). This.OH production may cause lipid peroxidation of cell membranes leading to neuronal cell death. This paper report that the DA-selective neurotoxin, 1-methyl-4-phenylpyridinium ion (MPP(+)), (1 nmol/microl per min for 1 h) infusion into the striatum of rats induces elevation of extracellular DA and.OH formation. These elevations seem to induce lipid peroxidation of striatum membranes, as detected by increases in non-enzymatic formation of 2,3-dihydroxybenzoic acid (DHBA) levels. To test the involvement of DA release in the.OH generation and lipid peroxidation, the rats were pretreated with reserpine (5 mg/kg, i.v., 24 h before MPP(+) or without MPP(+)) to deplete presynaptic DA. Reserpine treatment alone did not change the levels of DA or 2,3-DHBA, while the combined treatment with both MPP(+) and reserpine clearly decreased 2,3-DHBA, as well as DA levels, compared to those in the group treated with MPP(+) alone. After injection into reserpinized rats, DA at various doses (2, 5 and 10 microM) small increased 2,3-DHBA levels dose-dependently, as compared to the MPP(+) alone-treated group. These results clearly indicate that MPP(+) perfusion into the striatum increases extracellular DA levels and this increase may concomitantly induce the formation of reactive free oxygen radicals, such as.OH free radicals. These events may contribute, at least in part, to the nigrostriatal neurons cell death after MPP(+).     

Subtoxic concentration of manganese synergistically potentiates 1-methyl-4-phenylpyridinium-induced neurotoxicity in PC12 cells

Endogenous or exogenous substances that are toxic to dopaminergic cells have been proposed as possible cause of idiopathic Parkinson's disease (PD). 1-Methyl-4-phenylpyridinium (MPP(+)) and manganese are dopaminergic neurotoxins causing a parkinsonism-like syndrome. Here, we studied the possible synergistic reaction between these two neurotoxins using rat PC12 pheochromocytoma cells. MPP(+) induced a delayed neurotoxicity in PC12 cells. Although low concentration of manganese did not cause cell damage, it markedly enhanced MPP(+)-induced neurotoxicity with characteristics of apoptosis, such as DNA laddering and activation of caspase-3. To understand the mechanism of enhancement of subtoxic concentration of manganese on MPP(+)-induced neurotoxicity, we investigated the reactive oxygen species (ROS) generation using a molecular probe, 2',7'-dichlorofluorescein diacetate. Although subtoxic concentration of manganese alone did not induce ROS increase, it significantly enhanced the ROS generation induced by MPP(+). We also determined the intracellular MPP(+) content. A time- and concentration-dependent increase of MPP(+) levels was found in PC12 cells treated with MPP(+). The accumulation of MPP(+) by PC12 cells was not affected by manganese. Taken together, these studies suggest that co-treatment with MPP(+) and manganese may induce synergistic neurotoxicity in PC12 cells and that subtoxic concentration of manganese may potentiate the effect of MPP(+) by an ROS-dependent pathway.     

The role of phospholipid methylation in 1-methyl-4-phenyl-pyridinium ion (MPP+)-induced neurotoxicity in PC12 cells

Excessive methylation has been proposed to be involved in the pathogenesis of Parkinson's disease (PD), via mechanisms that involve phospholipid methylation. Meanwhile, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was found to stimulate phospholipid methylation via the oxidized metabolite, 1-methyl-4-phenyl-pyridinium (MPP+), in the rat brain and liver tissues. In the present study, we investigated the effect of MPP+ on phosphatidylethanolamine N-methyltransferases (PENMT) and the potential role of this pathway in MPP(+)-induced neurotoxicity using PC12 cells. The results obtained indicate that MPP+ stimulated phosphatidylethanolamine (PTE) methylation to phosphatidylcholine (PTC) and correspondingly increased the formation of lysophosphatidylcholine (lyso-PTC). Moreover, the addition of S-adenosylmethionine (SAM) to the cell culture medium increases MPP(+)-induced cytotoxicity. The incubation of 1mM MPP+ and various concentrations of SAM (0-4 mM) decreased the viability of PC12 cells from 80% with MPP+ alone to 38% viability with 4 mM SAM for 4 days incubation. The data also revealed that the addition of S-adenosylhomocysteine (SAH), a methylation inhibitor, offered significant protection against MPP(+)-induced cytotoxicity, indicating that methylation plays a role in MPP(+)-induced cytotoxicity. Interestingly, lyso-PTC showed similar actions to MPP+ in causing many cytotoxic changes with at least 10 times higher potency. Lyso-PTC induced dopamine release and inhibited dopamine uptake in PC12 cells. Lyso-PTC also caused the inhibition of mitochondrial potential and increased the formation of reactive oxygen species in PC12 cells. These results indicate that phospholipid methylation pathway might be involved in MPP+ neurotoxicity and lyso-PTC might play a role in MPP(+)-induced neurotoxicity.     

Alpha 2-adrenoceptors are not involved in the regulation of striatal glutamate release: comparison to dopaminergic inhibition.

Striatal slices from the rat were loaded with [3H]glutamate ([3H]Glu) and superfused in order to measure release of radioactivity at rest and during potassium-evoked depolarization. Addition of KCl (22-40 mmol/liter) to the perfusion fluid enhanced the release of tritium in a concentration-dependent manner, and this release was abolished by omission of CaCl2. High-performance liquid chromatography (HPLC) separation coupled with radiochemical detection revealed that 23% and 41% of the tritium efflux detected in the perfusion fluid under resting conditions and during potassium stimulation, respectively, was due to [3H]Glu. At the end of the superfusion about 63% of residual tritium content in the tissue was [3H]Glu. Tritium efflux in response to KCl excess was significantly higher from striatum dissected from 6-hydroxydopamine-pretreated rats. Apomorphine decreased the KCl-evoked release of [3H]Glu, and haloperidol exerted the opposite effect. Yohimbine, which antagonized the decrease of dopa accumulation elicited by apomorphine in NSD-1015 and gamma-butyrolactone-pretreated rat caudate nucleus, also reversed the apomorphine inhibition of the release of [3H]Glu evoked by depolarization. The selective alpha 2-adrenoceptor antagonist CH-38083, however, did not modify the apomorphine inhibition of [3H]Glu release or dopa accumulation, and the alpha 2-adrenoceptor agonist xylazine did not alter tritium efflux from striatum preloaded with [3H]Glu. These findings suggest that release of glutamate (Glu) from the corticostriatal pathway is under tonic control of dopamine released from nigrostriatal neurons, and alpha 2-adrenoceptors are not involved in the regulation of glutamatergic transmission in the rat striatum.(ABSTRACT TRUNCATED AT 250 WORDS)     

Estrogen modulates responses of striatal dopamine neurons to MPP(+): evaluations using in vitro and in vivo techniques

In vitro superfusion and in vivo electrochemistry were used to investigate the role of estrogen in modulating MPP(+)-induced dopamine output in the corpus striatum and nucleus accumbens of ovariectomized female rats. For in vitro superfusion experiments, dopamine and dihydroxyphenylacetic acid release were determined using HPLC with electrochemical detection from superfusion of corpus striatum fragments with Kreb's ringer phosphate buffer pulsed with MPP(+) alone or MPP(+) with estrogen. The in vivo electrochemistry experiments recorded the dopamine signal from carbon fiber microelectrodes stereotaxically passed through the corpus striatum and nucleus accumbens. Dopamine release was stimulated by pressure ejection of MPP(+) alone or in combination with estrogen through glass micropipettes fastened to the electrodes. Dopamine output from superfusion chambers which received infusion of MPP(+) with estrogen showed significantly lower output of dopamine compared with chambers which received MPP(+) alone. Outputs of dihydroxyphenylacetic acid did not increase following MPP(+) infusions. Data from the electrochemistry experiments demonstrated that estrogen significantly reduced both the amplitude and clearance rates of the MPP(+)-evoked dopamine signal in both the corpus striatum and nucleus accumbens. Results of this study demonstrate that: (1) MPP(+) evokes striatal dopamine release and this effect is significantly reduced in the presence of estrogen as determined by both in vivo electrochemistry and in vitro superfusion: (2) similar, albeit attenuated effects are observed in the nucleus accumbens as determined with in vivo electrochemistry; (3) estrogen acts to inhibit the clearance of dopamine in both the striatum and nucleus accumbens; and (4) estrogen may function as a neuroprotectant by reducing the uptake of neurotoxin into dopaminergic neurons.     

High potassium induces taurine release by osmosensitive and osmoresistant mechanisms in the rat hippocampus in vivo

The high potassium-evoked taurine efflux in the nervous tissue has been entirely considered to be the result of the cell swelling produced by KCl influx via passive Donnan forces. However, the extracellular taurine increase evoked in the hippocampus by applying 6-100 mM KCl through microdialysis probes, which saturates at a concentration of 25 mM KCl, is not congruent with the mentioned osmosensitive release of taurine stimulated by high potassium. Therefore, we studied whether the taurine release elicited by different high KCl concentrations (25, 50, 75, or 100 mM) was blocked under hypertonic conditions (+100 mM sucrose). Taurine release stimulated by 25 mM KCl was totally osmosensitive, but that released by higher KCl concentrations became progressively osmoresistant, achieving more than the 60% of the extracellular taurine enhancement during 100 mM KCl perfusion. The osmoresistant taurine release evoked by 100 mM KCl perfusion was partially reduced by a solution without Ca(2+) and with high Mg(2+), or by D,L-2-amino-5-phosphopentanoic acid, an N-methyl-D-aspartic acid (NMDA) receptor antagonist. Moreover, the release of taurine induced by a hypoosmotic solution was reduced by the presence of either high K(+) (75 mM) or NMDA (100 microM). These results indicate that although moderately high [K(+)] evoke the osmosensitive release of taurine, higher [K(+)] inhibit it and trigger the release of taurine by an osmoresistant mechanism. This last component is partially mediated by NMDA receptors activated by the glutamate released during potassium-induced depolarization.     

Imidaprilat, an angiotensin-converting enzyme inhibitor exerts neuroprotective effect via decreasing dopamine efflux and hydroxyl radical generation induced by bisphenol A and MPP+ in rat striatum

The present study examined the ability of antioxidant effects of angiotensin-converting enzyme (ACE) inhibitor, imidaprilat, on the synergistic effect of bisphenol A and 1-methyl-4-phenylpyridinium ion (MPP(+))-induced hydroxyl radical (*OH) formation and dopamine (DA) efflux in extracellular fluid of rat striatum. Bisphenol A clearly enhanced OH formation and DA efflux induced by MPP(+). When imidaprilat was infused in bisphenol A and MPP(+)-treated rats, DA efflux and OH formation significantly decreased, as compared with that in the bisphenol A and MPP(+) treated control. These results suggest that ACE inhibitors may protect against the synergistic effect of bisphenol A and MPP(+)-induced OH formation via suppressing DA efflux in the rat striatum.     

MPP(+) injection into rat substantia nigra causes secondary glial activation but not cell death in the ipsilateral striatum

Injection of MPP(+) into the substantia nigra causes extensive necrosis and anterograde degeneration of pars compacta dopaminergic neurons. We studied secondary effects in the ipsilateral striatum by examining dopaminergic terminals, signs of neuronal damage, and glial reactivity at 1, 2, 3, and 7 days after injection of MPP(+) into the substantia nigra. Dopaminergic terminals and uptake sites were evaluated with [(3)H]GBR-12935 binding and tyrosine hydroxylase immunoreactivity. Glial reaction was examined with markers of astrocytes and microglia. Stereology was used to evaluate any changes in neuronal density. Tyrosine hydroxylase immunoreactivity and [(3)H]GBR-12935 binding markedly decreased (74%) from days 2 to 7. Loss of dopaminergic terminals in the ipsilateral striatum was accompanied by an intense astroglial and, to a lesser extent, microglial reaction. However, no signs of cell damage, neuronal loss, or disruption of the blood-brain barrier were found in the striatum. Resident astroglial and microglial cells showed a morphological shift and notable changes in protein expression typical of glial reactivity, yet the presence of macrophage-like cells was not detected. This study shows that injection of MPP(+) in the substantia nigra causes a secondary reaction within the ipsilateral striatum involving the transformation of quiescent glia to reactive glia. It is suggested that stimuli derived from damaged dopaminergic terminals within the striatum are able to activate resident glia and that this glial transformation may promote repair and regeneration.     

Regulation of excitatory amino acid release by N-methyl-D-aspartate receptors in rat striatum: in vivo microdialysis studies.

The microdialysis technique was utilized to study the effects of N-methyl-D-aspartate (NMDA) receptor ligands on the in vivo release of endogenous glutamate (Glu) and aspartate (Asp) from the rat striatum. Addition of NMDA (250 and 500 microM) to the dialysis perfusion solution resulted in a striking dose-dependent increase in extracellular concentrations of Glu and Asp in the striatum. The NMDA-induced effects were reduced in a dose-related way by prior perfusion with 75 microM dizocilpine (MK-801), a non-competitive NMDA receptor antagonist. MK-801, at 75 microM, produced no changes on basal levels of Glu and Asp. However, 100 microM MK-801 did increase Glu and Asp extracellular concentrations. Local infusion with 500 microM D-serine, an agonist at the glycine site associated to the NMDA receptor, significantly increased basal level of Glu, but not Asp. Such D-serine-induced effects were reduced by 7-Cl-kynurenic acid (200 microM), a selective blocker of the glycine site present in the NMDA receptor. It is proposed that activation of NMDA receptors by endogenous Glu and Asp enhances the subsequent release of these excitatory amino acids in the striatum. Part of these NMDA receptors might be located presynaptically on cortico-striatal nerve endings. In addition, postsynaptic NMDA receptors present in the striatum may also indirectly modulate the release of Glu and Asp, through trans-synaptic mechanism.     

MPP+-induced pathophysiology demonstrates advantages of neurotoxicology studies in brain slices

Since MPTP and its metabolite MPP+ produce nigrostriatal lesions and symptoms similar to Parkinson's disease, recent studies have aimed toward defining their selectivity and neurotoxic mechanisms. In mitochondria in vitro, MPP+ blocked electron transport and decreased oxygen consumption. However, these effects were not selective to striatal mitochondria or even to mitochondria from brain, they required concentrations of MPP+ much greater than those found in vivo, and physiological actions could not be related to intramitochondrial changes. Lower doses of MPP+ did produce highly selective degeneration of dopaminergic (DA) neurons in cell cultures. We report here that MPP+ provoked large (80%) oxidations of cytochrome b and large K+o increments (approximately 30 mM) in rat striatal slices. These effects were slowed by mazindol, which inhibits DA uptake, and were markedly attenuated in rat hippocampal slices which have little DA input. Since DA terminals comprise only 2-4% of the striatal mass, the large MPP+-induced changes suggest that while MPP+ neurotoxicity in brain requires the presence of functioning DA terminals, effects are not confined to these terminals. Such studies illustrate the complexity of MPP+ neurotoxicity and demonstrate the importance of investigations in models such as brain slices with an extracellular space and intracellular relationships as in intact brain.     

Apparent opposite effects of tetrabenazine and reserpine on the toxic effects of 1-methyl-4-phenylpyridinium or 6-hydroxydopamine on nigro-striatal dopaminergic neurons

It is well documented that VMAT2 protects nigrostriatal DA neurons against MPP(+) by sequestering it inside vesicles away from its mitochondrial site of neurotoxic action. However, the implication of the VMAT2 in the mechanism of action exerted by 6-OHDA has received little attention. Therefore, the aim of the present study was to determine whether the vesicular sequestration of 6-OHDA would protect dopaminergic neurons from its toxicity similarly to what is observed with MPP(+). We injected mice with 6-OHDA 90 min after TBZ treatment. Since, unexpectedly, TBZ pretreatment prevented 6-OHDA neurotoxicity, we performed a similar experience replacing 6-OHDA with MPP(+) in order to check our experimental protocol. TBZ pretreatment similarly prevented MPP(+) neurotoxicity. This discrepancy with what is commonly describe in the literature, led us to use reserpine. Indeed, the long lasting VMAT2 inhibition induced by reserpine allowed us to inject neurotoxins while mice no longer presented hypothermia. Contrary to TBZ pretreatment, reserpine pretreatment potentiated both 6-OHDA and MPP(+) toxicity on dopaminergic neurons. Hypothermia elicited by TBZ appeared to be responsible, at least in part, for the neuroprotective effect observed. To verify this hypothesis, we investigated the influence of hypothermia on the toxic activity of both neurotoxins. A hypothermia similar to that induced by TBZ was obtained by a forced swimming test of putting mice into cool water (23 degrees C). The hypothermia prevented both 6-OHDA and MPP(+)-induced neurotoxicity. We finally reported that VMAT2 inhibition potentiates both MPP(+) and 6-OHDA neurotoxicity.     

D-deprenyl protects nigrostriatal neurons against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced dopaminergic neurotoxicity

Selegiline (L-deprenyl) is believed to render protection against l-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-neurotoxicity to a significant extent via a free radical scavenging mechanism, which is independent of its ability to inhibit monoamine oxidase-B (MAO-B) in the brain. We investigated the hydroxyl radical (.OH) scavenging action and neuroprotective effect of D-deprenyl, its less active isomer, in MPTP-induced dopaminergic neurotoxicity in mice to test whether the chemical structure of the molecule or its biological effects contribute to this property. To achieve this goal we studied the effects of D-deprenyl on: (1).OH production in a Fenton reaction; (2) MPTP-induced.OH generation and dopamine (DA) depletion in vivo, employing a sensitive HPLC-electrochemical procedure; and (3) formation of MPP(+) in vivo in the striatum following systemic administration of MPTP, employing an HPLC-photodiode array detection system. D-deprenyl inhibited ferrous citrate-induced.OH in vitro (0.45 microM) and MPTP-induced.OH in vivo in substantia nigra (SN) and in the striatum (1.0 mg/kg, i.p.). D-deprenyl did not, but L-deprenyl (0.5 mg/kg dose) did significantly inhibit formation of MPP(+) in the striatum 90 min following systemic MPTP injection. It failed to affect MAO-B activity at 0.5 mg/kg in the striatum, but effectively blocked MPTP-induced striatal DA depletion. The potency of D-deprenyl to scavenge MPTP-induced.OH in vivo and to render protection against the dopaminergic neurotoxicity without affecting dopamine turnover, MAO-B activity, or formation of MPP(+) in the brain indicates a direct involvement of.OH in the neurotoxic action of MPTP and antioxidant effect in the neuroprotective action of deprenyl.     

Influence of manganese on the release of neurotransmitters in rat striatum

On the basis of the evidence that manganese may be released along with glutamate into the extracellular space in the hippocampus and amygdala, the release of manganese and its influence in the striatum was examined by using the in vivo microdialysis method in the present study. The release of 54Mn previously taken up by the striatum into the extracellular space was enhanced during stimulation with 100 mM KCl. This enhancement of 54Mn release into the striatal extracellular space was inhibited by addition of 1 micro M tetrodotoxin. When the rat striatum was perfused with artificial CSF containing 200 nM MnCl(2), the levels of GABA in the perfusate were remarkably decreased, while the levels of glutamate, aspartate, and glycine in the perfusate were not appreciably decreased. These results suggest that manganese released into the synaptic cleft in a calcium- and impulse-dependent manner may influence GABA release in the striatum.     

Topological and chronological features of the impairment of glucose metabolism induced by 1-methyl-4-phenylpyridinium ion (MPP<Superscript>+</Superscript>) in rat brain slices

Summary 1-Methyl-4-phenylpyridinium (MPP⁺) was added directly to fresh rat brain slices and the dynamic changes in the cerebral glucose metabolic rate (CMRglc) were serially and two-dimensionally measured with [¹⁸F]2-fluoro-2-deoxy-D-glucose as a tracer. MPP⁺ dose-dependently increased CMRglc, reflecting enhanced glycolysis compensating for the decrease in aerobic metabolism. While the CMRglc enhancement induced by MPP⁺ (<10 μM) was restricted to the striatum, MPP⁺ (≥10 μM) induced a significant CMRglc enhancement in all brain regions. MPP⁺ at high concentration (1 mM) eventually initiated rapid metabolic collapse, with failure to sustain anaerobic glycolysis.     

Validity of a quantitative technique to study striatal dopaminergic neurodegeneration by in vivo microdialysis

The development of a technique that allows the direct quantitative study of the damage produced by a toxin on a specific neurotransmitter system is very important. For that, we have used the microdialysis technique to validate a method to study the specific drug's toxicity on dopaminergic (DAergic) striatal terminals. We perfused different MPP(+) and 6-hydroxydopamine (6-OHDA) concentrations, with different toxicity for DAergic terminals, 24 h after the implantation of the microdialysis probe (day 1). One day later (day 2), MPP(+) was perfused through the microdialysis probe and DA extracellular output measured. We hypothesize that the amount of extracellular dopamine (DA) obtained on day 2 is directly proportional to the neurotoxic damage produced on day 1. To corroborate this hypothesis tyrosine hydroxylase (TH) immunohistochemistry was also carried out on day 2. There was a clear correlation index between the amount of DA measured after MPP(+) perfusion and the lack of TH immunoreactivity measured as the radius of the area showing decrease in TH immunoreactivity around the cannula. These results show the possibility to measure DAergic remaining terminals after a toxic drug exposure by in vivo MPP(+) perfusion. The possibility to extend this neurotoxic study to another neurotransmitter systems is suggested.     

Non-steroidal anti-inflammatory drug sodium salicylate,but not diclofenac or celecoxib,protects against 1-methyl-4-phenyl pyridinium-induced dopaminergic neurotoxicity in rats

We evaluated the hydroxyl radical (*OH) scavenging action of nonsteroidal anti-inflammatory drugs (NSAIDs), sodium salicylate (SA), diclofenac and celecoxib in Fenton's reaction and their neuroprotective effects in 1-methyl-4-phenylpyridinium (MPP(+))-induced striatal dopamine (DA) depletion in rats. Salicylate hydroxylation procedure employing HPLC-electrochemistry was used to assay formation of *OH in Fenton's reaction in test tubes. While SA dose- and time-dependently hydroxylated itself and inactivated *OH, celecoxib (up to 10 mM) showed no effect on *OH formation and diclofenac caused a reduction in *OH generation only at high doses (100 microM-10 mM). Administration of the non-selective cyclooxygenase (COX) inhibitor, SA (50, 100 mg/kg, i.p.) significantly attenuated striatal DA depletion caused by intrastriatal infusion of MPP(+) (100 nmol in 4 microl). Treatment with another nonselective, reversible COX inhibitor, diclofenac (5, 10 mg/kg) did not protect against MPP(+)-induced DA depletion. The selective COX-2 inhibitor, celecoxib (2.5-50 mg/kg) treatment exacerbated MPP(+)-induced decrease in DA. Failure of celecoxib or diclofenac to render protection in animals against MPP(+)-induced DA depletion indicates absence of prostaglandin involvement in MPP(+) action. These results also suggest that the neuroprotective ability of SA is independent of prostaglandin mediation. A relationship between inactivation of *OH by SA and its ability to protect DA depletion in the striatum caused by MPP(+) indicates a direct involvement of *OH in the action of this neurotoxin. The present study establishes potent neuroprotective activity of SA and suggests the use of aspirin in adjuvant therapy in Parkinson's disease.     

Zinc neurotoxicity is dependent on intracellular NAD levels and the sirtuin pathway

Zinc neurotoxicity has been demonstrated in ischemic, seizure, hypoglycemic, and trauma-induced neuronal death where Zn(2+) is thought to be synaptically released and taken up in neighbouring neurons, reaching toxic concentrations. We previously demonstrated that toxicity of extracellular Zn(2+) depended on entry, elevation in intracellular free Zn(2+) ([Zn(2+)](i)), a reduction in NAD(+) and ATP levels, and dysfunction of glycolysis and cellular metabolism. We suggested that PARP-1 activation alone can not explain this loss of neuronal NAD(+). NAD(+) was recently demonstrated to permeate neurons and glia, and we have now shown that exogenous NAD(+) can reduce Zn(2+) neurotoxicity, and 3-acetylpyridine, which generates inactive NAD(+), potentiated Zn(2+) neurotoxicity. Sirtinol and 2-hydroxynaphthaldehyde, inhibitors of the sirtuin pathway (SIRT proteins are NAD(+)-catabolic protein deacetylases), attenuated both acute and chronic Zn(2+) neurotoxicity. Resveratrol and fisetin (sirtuin activators) potentiated NAD(+) loss and Zn(2+) neurotoxicities. Furthermore, neuronal cultures derived from the Wld(s) mouse, which overexpress the NAD(+) synthetic enzyme nicotinamide mononucleotide adenyl transferase (NMNAT-1), had reduced sensitivity to Zn(2+) neurotoxicity. Finally, nicotinamide was demonstrated to attenuate CA1 neuronal death after 10 min of global ischemia in rat even if administered 1 h after the insult. Together with previous data, these results further implicate NAD(+) levels in Zn(2+) neurotoxicity.