The NOX1/4 inhibitor GKT136901 as selective and direct scavenger of peroxynitrite.

NADPH oxidases (NOX), catalyzing the reduction of molecular oxygen to form the superoxide radical anion (•O2⁻) and hydrogen peroxide (H2O2), are involved in several pathological conditions, such as stroke, diabetes, atherosclerosis, but also in chronic neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, or multiple sclerosis. GKT136901 is a novel NOX-1/4 inhibitor with potential application in the areas of diabetic nephropathy, stroke, or neurodegeneration. In the present study, we investigated additional pharmacological activities of the compound with respect to direct free radical scavenging. GKT136901 did not interact with nitric oxide (•NO), •O2⁻, or hydroxyl radicals (•OH), but it acted as selective scavenger of peroxynitrite (PON) already in the submicromolar concentration range. Alpha synuclein (ASYN) is a protein involved in the pathogenesis of Parkinson's disease and a known target for PON-dependent tyrosine nitration. Submicromolar concentrations of GKT136901 prevented tyrosine nitration and di-tyrosine-dependent dimer formation of ASYN by PON as indicated by Western blot and mass spectrometric analysis. GKT136901 itself was degraded when exposed to PON. In a human neuronal cell model, GKT136901 prevented both the depletion of reduced intracellular glutathione, and the degeneration of neurites when present during PON treatment of the cells. When GKT136901 was applied after PON treatment, no protective effect was observed, thus excluding an impact of GKT136901 on cellular death/survival pathways. In summary, selective scavenging of PON is an additional pharmacological property of the NOX-1/4 inhibitor GKT136901, and this may add to the efficiency of the drug in several disease models.

Site-specific action of L-3,4-dihydroxyphenylalanine in the striatum but not globus pallidus and substantia nigra pars reticulata evokes dyskinetic movements in chronic L-3,4-dihydroxyphenylalanine-treated 6-hydroxydopamine-lesioned rats.

Dyskinesia eventually develops in the majority of Parkinson's disease patients treated with l-3,4-dihydroxyphenylalanine (l-DOPA). We have investigated the effect of an acute and local administration of L-DOPA, GABA and glutamate to provoke dyskinetic movements in three basal ganglia structures (striatum, globus pallidus (GP) and substantia nigra pars reticulata (SNr)) of chronically L-DOPA-treated, unilaterally 6-hydroxydopamine-lesioned rats. We demonstrated that L-DOPA administration into the lesioned striatum using the technique of reverse in vivo microdialysis was an effective trigger to switch on dyskinesia. Notably, local L-DOPA perfusion at the same concentration in the ipsilateral GP and SNr did not provoke significant dyskinetic behaviour. Neither GABA nor glutamate triggered dyskinetic movements in the striatum, GP or SNr. We postulate a site-specific action of L-DOPA for the evocation of already established dyskinesia since L-DOPA in the striatum but not in the GP or SNr switched on dyskinetic behaviour.

Comparison of intrastriatal administration of noradrenaline and l-DOPA on dyskinetic movements: a bilateral reverse in vivo microdialysis study in 6-hydroxydopamine-lesioned rats.

l-DOPA-induced dyskinesia is known as involuntary debilitating movement, which limits quality of life in patients suffering from Parkinson's disease. The present study focuses on the role of the neurotransmitter noradrenaline (NA) on dyskinetic movements in comparison to the effect of l-DOPA. Rats were unilaterally lesioned with 6-hydroxydopamine and treated with l-DOPA/benserazide (6/15 mg/kg, p.o.) to induce stable dyskinetic movements. On the day of the experiment, NA (0.04 nmol/min, 0.4 nmol/min) and l-DOPA (0.04 nmol/min, 0.4 nmol/min) were perfused into the lesioned and non-lesioned striatum of dyskinetic rats using the reverse in vivo microdialysis technique. Neither NA nor l-DOPA treatment of the non-lesioned striatum produced any dyskinetic behavior. In contrast, administration of l-DOPA 0.4 nmol/min into the lesioned striatum led to a significant increase in dyskinesia indicated by abnormal axial, limb and orolingual movements. Notably, perfusion with NA 0.4 nmol/min into the lesioned striatum revealed a highly significant induction of dyskinetic movements, which are similar to the dyskinesia subtype profile of l-DOPA. In conclusion, NA is as potent as l-DOPA to express dyskinetic movements in l-DOPA-primed rats.

Activation of dopaminergic neurotransmission in the medial prefrontal cortex by N-methyl-d-aspartate stimulation of the ventral hippocampus in rats.

Many behavioral functions-including sensorimotor, attentional, memory, and emotional processes-have been associated with hippocampal processes and with dopamine transmission in the medial prefrontal cortex (mPFC). This suggests a functional interaction between hippocampus and prefrontal dopamine. The anatomical substrate for such an interaction is the intimate interconnection between the ventral hippocampus and the dopamine innervation of the mPFC. The present study yielded direct neurochemical evidence for an interaction between ventral hippocampus and prefrontal dopamine transmission in rats by demonstrating that subconvulsive stimulation of the ventral hippocampus with N-methyl-d-aspartate (NMDA; 0.5 mug/side) activates dopamine transmission in the mPFC. Postmortem measurements revealed that bilateral NMDA stimulation of the ventral hippocampus, resulting in locomotor hyperactivity, increased the homovanillic acid/dopamine ratio, an index of dopamine transmission, in the mPFC; indices of dopamine transmission in any of five additionally examined forebrain regions (amygdala, nucleus accumbens shell/core, lateral prefrontal cortex, caudate putamen) were unaltered. In vivo microdialysis measurements in freely moving rats corroborated the suggested activation of prefrontal dopamine transmission by demonstrating that unilateral NMDA stimulation of the ventral hippocampus increased extracellular dopamine in the ipsilateral mPFC. The suggested influence of the ventral hippocampus on prefrontal dopamine may be an important mechanism for hippocampo-prefrontal interactions in normal behavioral processes. Moreover, it indicates that aberrant hippocampal activity, as found in neuropsychiatric diseases, such as schizophrenia and mood disorders, may contribute to disruption of certain cognitive and emotional functions which are extremely sensitive to imbalanced prefrontal dopamine transmission.

Effect of repeated treatment with high doses of selegiline on behaviour, striatal dopaminergic transmission and tyrosine hydroxylase mRNA levels.

The anti-parkinsonian drug selegiline is a monoamine oxidase B (MAO-B) inhibitor and a potential neuroprotective agent which facilitates dopaminergic transmission. Its metabolites (-)-amphetamine and (-)-metamphetamine might contribute to the pharmacological effects as they are also able to increase dopaminergic transmission and in addition might lead to behavioural sensitization after repeated administration. We investigated the effects of acute and repeated treatment with a high dose of selegiline on dopamine overflow in the striatum as well as on behaviour and on tyrosine hydroxylase (TH) mRNA levels in midbrain. Two experiments were performed. In the first one, rats were implanted with microdialysis probes into the striatum and received daily injections of selegiline (10 mg/kg, i.p.) for 1 or 8 days or a single dose of saline. In vivo microdialysis was carried out on days 1, 8 or 17 (after withdrawal of 9 days) to measure dopamine overflow. Motility was measured at the same time. In the second experiment, rats were injected daily with selegiline (10 mg/kg, i.p.) or saline over a time period of 6 weeks or only once before the brains were processed for in situ hybridization with a (35)S-radiolabelled probe for TH. Repeated treatment led to higher levels in motility scores than acute administration after administration of the same dose, indicating behavioural sensitization, which was still manifest after an interruption of 9 days in the supply of selegiline. In contrast, acute administration of selegiline increased dopamine levels to a similar degree as the same dose after subchronic treatment, with or without interruption of 9 days. The dopamine metabolite DOPAC was reduced by more than 50% after acute administration of selegiline and even more so on day 8 by the same dose, after repeated administration. The basal concentrations of dopamine (before challenge with selegiline) were not altered by the repeated administration, whereas the basal concentrations of DOPAC were decreased by more than 80% by the repeated administration of selegiline, suggesting a decrease in dopamine turnover. Acute administration did not have any influence on TH mRNA levels, whereas chronic treatment significantly reduced TH mRNA levels in substantia nigra and ventral tegmental area. In conclusion, repeated administration of selegiline leads to behavioural sensitization independent of altered dopamine levels. In addition, it leads to a decrease, probably due to a down-regulation, of dopamine turnover and tyrosine hydroxylase.

Dopamine mediates striatal malonate toxicity via dopamine transporter-dependent generation of reactive oxygen species and D2 but not D1 receptor activation.

Intrastriatal injection of the reversible succinate dehydrogenase inhibitor malonate results in both chemically induced hypoxia and striatal lesions that are similar to those seen in Huntington's disease and cerebral ischaemia. The mechanisms leading to neuronal death involve secondary excitotoxicity, the release of dopamine from nigrostriatal fibres and the generation of reactive oxygen species (ROS) including nitric oxide (NO) and hydroxyl radicals. Here, we further investigated the contribution and mechanism of dopamine on malonate-induced striatal lesions. Prior lesions of the nigrostriatal pathway with 6-OHDA or the depletion of striatal dopamine stores by pretreatment with reserpine, an inhibitor or the vesicular monoamine transporter type-2 (VMAT2), in combination with alpha-methyl-p-tyrosine resulted in a significant reduction of malonate-induced striatal lesion volumes. This was paralleled by block or reduction of the malonate-induced generation of ROS, as measured by the conversions of salicylate to 2,3-dihydroxybenzoic acid (2,3-DHBA) using microdialysis. Systemic or intrastriatal application of L-DOPA or dopamine, respectively, reconstituted malonate toxicity and the generation of ROS in 6-OHDA-lesioned rats. Block of the dopamine transporter by GBR12909 did not result in a reduction of malonate-induced dopamine release, but significantly reduced the generation of hydroxyl radicals. The D2 receptor agonist lisuride and the mixed D1 and D2 receptor agonist apomorphine, but not the D1 receptor agonist SKF38393, partially restored malonate toxicity in 6-OHDA-lesioned rats without increasing the generation of ROS. In line with these results sulpiride, an inhibitor of D2 receptors, reduced the malonate-induced lesion volume, whereas SCH23390, an inhbitor of D1 receptors, was ineffective. Our data suggest that malonate-induced dopamine toxicity to energetically impaired neurons is mediated by two independent pathways: (i) dopamine transporter uptake-dependent, dopamine receptor-independent generation of ROS, and (ii) excessive stimulation of D2 receptors.

6-hydroxydopamine increases the hydroxylation and nitration of phenylalanine in vivo: implication of peroxynitrite formation.

In the present study, we investigated the effect of the dopaminergic neurotoxin 6-hydroxydopamine (6-OHDA) on hydroxyl free radical and peroxynitrite formation in vivo using D-phenylalanine as a novel mechanistic probe. In vivo microdialysis was carried out in the striatum of freely moving male Wistar rats. The microdialysis probes were perfused with artificial cerebrospinal fluid containing 5 mM D-phenylalanine (flow rate 2 microL/min). After obtaining a stable baseline 6-OHDA was delivered into the striatum via reverse microdialysis for 60 min. HPLC measurements of the effluent were performed using photodiode array detection for determination of phenylalanine derived o-tyrosine and m-tyrosine (as hydroxylation markers) as well as of nitrotyrosine and nitrophenylalanine (as nitration markers). The basal levels of the hydroxylation derived products of phenylalanine were approximately 100-fold higher than those of the nitration derived products. 6-OHDA (0.1, 1, 10 mM) significantly increased o- and m-tyrosine up to nine- and 13-fold, respectively, whereas levels of 3-nitrotyrosine and 4-nitrophenylalanine were significantly increased up to 422- and 358-fold, respectively. The results demonstrate that phenylalanine is a sensitive in vivo marker for 6-OHDA-induced hydroxylation and nitration reactions which are clearly concentration dependent. We conclude that peroxynitrite formation is involved in 6-OHDA-induced neurochemical effects.

Comparison of two independent aromatic hydroxylation assays in combination with intracerebral microdialysis to determine hydroxyl free radicals.

The phenylalanine- and salicylate assay were compared to investigate the production of hydroxyl free radicals. In vitro experiment: Phenylalanine (100 micromol/l) or salicylic acid (100 micropmol/l) were incubated in a hydroxyl radical generating in vitro Fenton system with increasing concentrations (1.25--40 micromol/l) of equimolar hydrogen peroxide and ferrous ions. Both, phenylalanine and salicylic acid were able to trap hydroxyl radicals in a reliable way indicated by the linear relationship between the concentration of the Fenton reagents and either the phenylalanine derived products (ortho-, meta-, para-tyrosine) or the salicylic acid-derived products (2,3- and 2,5-dihydroxybenzoic acid (DHBA)). In vivo experiment: Wistar rats were implanted with microdialysis probes and striatal perfusion with either 5 mmol/l phenylalanine or 5 mmol/l salicylic acid was performed. Addition of the dopaminergic neurotoxin 6-hydroxydopamine (100 micromol/l, flow rate 2 microl/min, 60 min) to the perfusion fluid significantly increased the concentrations of ortho- and meta-tyrosine or 2,3-DHBA in comparison to control animals. All increases determined were rapidly reversible after changing back to pre-stimulation conditions. The results demonstrate that aromatic hydroxylation of phenylalanine or salicylic acid is a useful technique to investigate hydroxyl free radical formation in vitro and in vivo. Advantages and disadvantages of both methods are discussed.

6-hydroxydopamine increases hydroxyl free radical production and DNA damage in rat striatum.

Oxidative damage is considered to be an important factor of 6-hydroxydopamine (6-OHDA) toxicity. To address this issue, microdialysis probes were implanted into the striatum of Wistar rats and perfused with 6-OHDA. Salicylate was included in the perfusion fluid to measure 2,3-dihydroxybenzoic acid (2,3-DHBA) as a marker of hydroxyl radical formation using HPLC with electrochemical detection. Additionally, striatal tissue was analysed for DNA base alterations using gas chromatography-mass spectrometry. 6-OHDA administration resulted in a rapid and substantial 6.6-fold increase in 2,3-DHBA formation and also increased levels of the modified DNA bases 5-hydroxycytosine, hypoxanthine and 2,6-diamino-4-hydroxy-5-formamidopyrimidine. Hydroxyl radical formation and DNA base alterations are early phenomena of 6-OHDA toxicity and provide clues to the processes that may be involved in the initiation of cell death in Parkinson's disease.

Sensitization to the behavioural effects of cocaine: alterations in tyrosine hydroxylase or endogenous opioid mRNAs are not necessarily involved.

After repeated administration of cocaine at intervals, sensitization phenomena can be observed, so that its behavioural effects are enhanced. Since this phenomenon is long-lasting, it was of interest to study which persistent alterations in the activity of dopaminergic neurones or of endogenous opioid systems downstream of dopaminergic synapses in the basal ganglia are involved in the sensitization. Cocaine (10 mg/kg i.p.) was administered to rats on days 1, 3, 5 and 7 and saline on days 2, 4 and 6 ("repeated cocaine"), or saline was injected on days 1-6 and cocaine on day 7 ("acute cocaine"), or saline was injected on days 1-7 ("saline group"). The "repeated cocaine" schedule led to a significant sensitization to the locomotor activation produced by cocaine on day 7 or on day 17, 10 days after the end of sensitization protocol. Microdialysis in the nucleus accumbens which was performed after administration of cocaine (10 mg/kg i.p.) on day 7, or after an administration of the same dose 10 days after the last administration of cocaine, respectively, revealed significant acute increases of extracellular dopamine to about 200% of basal values. These increases were similar in "acute cocaine" and in "repeated cocaine" animals both after 7 days and after 17 days. For in situ hybridization studies, rats were sacrificed on day 7, 4.5 h after the last cocaine or saline administration. The mRNA for tyrosine hydroxylase (TH) in substantia nigra + ventral tegmental area was significantly elevated to about 140% of saline controls both in the "repeated cocaine" and the "acute cocaine" group as compared with the "saline group". In contrast, there were no differences between the three groups in the mRNAs of preprodynorphin or preproenkephalin levels measured in the nucleus accumbens (core and shell). These results suggest that sensitization phenomena to cocaine are not necessarily connected with alterations in the dopaminergic activity in the mesolimbic system or in the transcription of precursors of endogenous opioid peptides which are located downstream of the dopaminergic synapses.

Nuclear factor-kappaB activation is not involved in a MPTP model of Parkinson's disease.

In the present study the involvement of hydroxyl free radicals and nuclear factor-kappaB (NF-kappaB) activation was investigated in the MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) model of Parkinson's disease. MPTP (30 mg/kg, s.c.) produced a significant 2-fold increase in hydroxyl free radicals in the striatum of C57BL/6 mice determined by microdialysis in combination with the salicylate hydroxylation assay. Electrophoretic mobility shift assays did not detect NF-kappaB activation after MPTP treatment. Furthermore, p50-deficient mice showed only minor differences in striatal dopamine and metabolite levels as well as tyrosine hydroxylase immunoreactivity after MPTP administration in comparison to wildtype mice. We postulate that, although hydroxyl radical production was enhanced, NF-kappaB plays only a minor role in the MPTP model because neither neurochemical nor immunocytochemical parameters were altered in p50-deficient mice in comparison to controls.

Inhibition of the cyclooxygenase isoenzymes COX-1 and COX-2 provide neuroprotection in the MPTP-mouse model of Parkinson's disease.

To study the possible role of the isoenzymes of cyclooxygenase COX-1 and COX-2 in the MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) mouse model of Parkinson's disease we used acetylsalicylic acid, a COX-1/COX-2 inhibitor, in comparison with meloxicam, a preferential COX-2 inhibitor. As markers of protection we determined the effects on MPTP-induced striatal dopamine depletion, locomotor activity, cell loss, and tyrosine hydroxylase immunoreactivity (TH-IR) in the substantia nigra pars compacta. Male C57BL/6 mice (n = 82) were treated with a single dose of acetylsalicylic acid (10, 50, 100 mg/kg i.p.) or meloxicam (2, 7.5, 50 mg/kg i.p.) immediately prior to administration of MPTP (30 mg/kg s.c.) or saline. After 7 days the mice were sacrificed to analyze striatal dopamine and metabolite levels. Nigral sections were processed for Nissl-staining and TH-IR. In the saline-treated MPTP control group striatal dopamine levels were reduced to 15.9% of control values. Dopamine depletion was significantly attenuated to values of 37.1 and 38.6% of saline control values by acetylsalicylic acid (50 and 100 mg/kg) and to values of 36 and 40% by meloxicam (7.5 and 50 mg/kg), respectively. MPTP-induced decrease of locomotor activity was significantly attenuated by acetylsalicylic acid and meloxicam. Remarkably, the MPTP-induced decrease of TH-IR as well as the loss of nigral neurons was nearly completely prevented by acetylsalicylic acid (100 mg/kg) and meloxicam (7.5 and 50 mg/kg). In conclusion, the inhibition of either COX-1/COX-2 by acetylsalicylic acid or preferentially COX-2 by meloxicam provided a clear neuroprotection against MPTP-toxicity on the striatal and nigral levels.

Neuroprotective effects of (+/-)-kavain in the MPTP mouse model of Parkinson's disease.

This is the first study to investigate the potential protective effects of the lipophilic kavapyrone (+/-)-kavain in the experimental MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) model of Parkinson's disease (PD). Male C57BL/6 mice were treated with (+/-)-kavain (50, 100, or 200 mg/kg i.p.) or vehicle 60 min before and 60 min after a single administration of MPTP (30 mg/kg s.c.) or saline, respectively. Mice were sacrificed after 7 days and the neostriatum was analyzed for dopamine and its metabolites using HPLC with electrochemical detection. Furthermore, nigral sections were processed for tyrosine hydroxylase (TH) immunocytochemistry. To determine the effects of (+/-)-kavain (200 mg/kg) on MPTP metabolism, HPLC analysis of striatal MPP(+) (1-methyl-4-phenylpyridinium) levels was performed. MPTP treatment alone led to a significant depletion of striatal dopamine levels to 12.61% of saline controls. The lower dosages of (+/-)-kavain (50 and 100 mg/kg) showed only a nonsignificant attenuation of MPTP-induced dopamine depletion, but a high dosage of (+/-)-kavain (200 mg/kg) significantly antagonized the dopamine depletion to 58.93% of saline control values. Remarkably, the MPTP-induced decrease of TH-immunoreactivity as well as the loss of nigral neurons was completely prevented by (+/-)-kavain (200 mg/kg). Striatal MPP(+) levels were not altered by (+/-)-kavain treatment. In conclusion, we found that MPTP metabolism was not influenced by (+/-)-kavain and postulate the antiglutamatergic effects of (+/-)-kavain for its protective effects against MPTP toxicity. (+/-)-Kavain may be a novel candidate for further preclinical studies in animal models of PD and other disorders with glutamatergic overactivity.

Neurochemical findings in the MPTP model of Parkinson's disease.

Animal models are a very important approach to study the pathogenesis and therapeutic intervention strategies of human diseases. Since many human disorders do not arise spontaneously in animals, characteristic functional changes have to be mimicked by neurotoxic agents. For instance, the application of the dopaminergic neurotoxin MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) is able to produce striking similarities to Parkinson's disease (PD) diagnosed in humans. MPTP is thought to selectively damage dopaminergic neurons predominantly those originating in the substantia nigra pars compacta (SNc) which leads to impaired dopaminergic neurotransmission accompanied by a loss of dopaminergic nerve terminals in the striatum. MPTP-induced neurochemical, behavioral, and histopathological alterations replicate very closely the clinical symptoms of PD patients, which will be discussed in this paper and render the MPTP model currently the most favored PD model to study therapeutic intervention strategies in an easy and reliable way in preclinical studies. We and many other research groups propose that the knowledge about the neurotoxic mechanisms of MPTP such as mitochondrial dysfunction with breakdown of energy metabolism and free radical production will help us to understand the underlying mechanisms of PD, which are not fully understood yet. In particular, the novel aspects of inflammatory processes and the involvement of reactive nitrogen species in addition to reactive oxygen species seem to be important milestones for a better understanding of the neurodegenerative effects of MPTP. In this review we focus on the MPTP mouse model which is easy practicable and widely used in neuroscience research and draw comparisons to the human pathology in PD.

The dopamine agonist pramipexole scavenges hydroxyl free radicals induced by striatal application of 6-hydroxydopamine in rats: an in vivo microdialysis study.

Hydroxyl free radical production seems to play an important role in the pathogenesis of Parkinson's disease. In the present study, we investigated the dopamine agonists pramipexole and pergolide as well as the nitrone compound S-PBN (N-tert-butyl-alpha-(2-sulfophenyl)nitrone) to reduce hydroxyl radical formation. Microdialysis experiments were carried out in non-anaesthetized Wistar rats. Salicylate was incorporated into the perfusion fluid to measure indirectly hydroxyl radicals indicated by 2,3-dihydroxybenzoic acid (2,3-DHBA). Local perfusion with 0.2 or 2 nmol/2 microl/min 6-hydroxydopamine (6-OHDA) via the microdialysis probe significantly increased 2,3-DHBA levels 14-fold and 47-fold, respectively. Systemic application of either pergolide (0.05 mg/kg) or pramipexole (1 mg/kg) failed to significantly reduce 6-OHDA-induced hydroxyl radical production. In contrast, a 40 min pretreatment with pramipexole (2 and 10 nmol/2 microl/min via the probe) before onset of 6-OHDA perfusion, significantly attenuated 2, 3-DHBA levels compared with vehicle controls. S-PBN pretreatment (2 nmol/2 microl/min) was not effective to reduce 2,3-DHBA levels. In conclusion, pramipexole was able to reduce hydroxyl radical levels induced by 6-OHDA in vivo after local application. This property of pramipexole may be beneficial under conditions of enhanced hydroxyl radical formation in parkinsonian brains and may add to its well known dopamine D(2)-like receptor agonistic effects.

The associative type of sensitization to d-amphetamine is expressed as an NO-dependent dramatic increase in extracellular dopamine in the nucleus accumbens.

After repeated administration of psychomotor-stimulant drugs such as d-amphetamine, a sensitization to its stimulant effects such as locomotor activity and stereotyped behaviour can be observed depending on the treatment schedule. It was the aim of the present study to test whether (1) associative (conditioning) and non-associative (pseudoconditioning) types of sensitization differ in the corresponding alterations in extracellular dopamine in the nucleus accumbens, and (2) the inhibitor of nitric oxide synthase (NOS), N(G)-nitro-L-arginine methyl ester (L-NAME), influences the two types of sensitization in a different way. Rats were treated with d-amphetamine (1 mg/kg i.p.) on days 1, 3, 5 and 7 and alternatively with saline on days 2, 4 and 6 under associative or non-associative conditions, respectively. A third (saline control) group was treated with saline daily for 7 days. Subsequently, probes for microdialysis were implanted into the nucleus accumbens. After a drug-free period of 10 days, on day 17, the increase in extracellular dopamine produced by 1 mg/kg d-amphetamine was much more pronounced in associative (conditioned) than in non-associative (pseudoconditioned) or naive rats. Pretreatment with L-NAME (100 mg/kg i.p.) on day 17 did not significantly alter the baseline concentrations of dopamine, but it inhibited the dopamine increase much more in associative than in naive rats and did not significantly affect it in non-associative rats. The results suggest that (1) associative sensitization to d-amphetamine leads to the most pronounced increase in extracellular dopamine in the nucleus accumbens, and (2) NO is very much involved in the expression of the associative increase in extracellular dopamine in the nucleus accumbens.

Effects of ensaculin on dopamine metabolite levels and K(+)-induced glutamate release.

In vivo microdialysis with the new antidementia compound ensaculin was performed in freely moving rats to study the alterations in dopaminergic and glutamatergic neurotransmission. Ensaculin (0.1 and 1 mg/kg i.p.) significantly increased extracellular levels of the dopamine metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA). Furthermore, ensaculin (1 mg/kg i.p.) showed a non-significant tendency to reduce the K(+)-induced glutamate release. The data suggest that ensaculin may have moderate D(2) antagonistic properties. Thus, besides its possible role as a cognitive enhancer, ensaculin may also have moderate antipsychotic properties.

In vivo effects of the putative cognitive enhancer KA-672.HCl in comparison with 8-hydroxy-2-(di-N-propylamino) tetralin and haloperidol on dopamine, 3,4-dihydroxyphenylacetic acid, serotonin and 5-hydroxyindoleacetic acid levels in striatal and cortical brain regions.

1. KA-672.HCl (7-methoxy-6-[3-[4-(2-methoxyphenyl)piperazin-1-yl]propoxy]-3,4-di methyl-2H-1-benzopyran-2-one hydrochloride), designed as a cognitive enhancer, has been investigated through behavioural and binding studies. However, little is known about its biochemical effects on the dopaminergic and serotoninergic system in vivo. 2. In the present study the authors investigated the effects of KA-672.HCl (0.1 mg/kg and 1 mg/kg), 8-hydroxy-2-(di-N-propylamino)tetralin (8-OH-DPAT) (1 mg/kg), haloperidol (0.1 mg/kg) and a mixture of haloperidol and 8-OH-DPAT on dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), serotonin (5-HT) and 5-hydroxyindolacetic acid (5-HIAA) levels, in striatum and cerebral cortex of rats. 3. Male Wistar rats received an intraperitoneal injection of the drugs or vehicle 1 hour before striatal and cortical brain tissues were dissected out for neurochemical analysis. 4. KA-672.HCl, 8-OH-DPAT and haloperidol significantly reduced striatal DA levels, whereas only KA-672.HCl significantly reduced cortical DA levels. 8-OH-DPAT and haloperidol induced a significant increase in cortical DOPAC levels but only haloperidol significantly elevated the striatal DOPAC content. In contrast, only the higher dose of KA-672.HCl elevated striatal DOPAC levels. Furthermore, KA-672.HCl significantly reduced striatal 5-HT levels and slightly elevated striatal 5-HIAA concentrations. 8-OH-DPAT significantly decreased striatal 5-HIAA levels. All substances were able to enhance the cortical and striatal DA turnover. 5. The cortical and striatal 5-HT turnover was significantly decreased following 8-OH-DPAT treatment and significantly increased in the striatum after haloperidol and KA-672.HCl treatment. 6. The data suggest that KA-672.HCl possesses D2 antagonistic as well as 5-HT1A agonistic properties. However, additional mechanisms of actions by interaction with other neurotransmitter systems such as acetylcholine, excitatory or inhibitory amino acids need to be determined.

The salicylate hydroxylation assay to measure hydroxyl free radicals induced by local application of glutamate in vivo or induced by the Fenton reaction in vitro.

The direct measurement of hydroxyl radicals in vivo is extremely difficult. Therefore, the indirect determination of hydroxyl radicals using salicylate (2-hydroxybenzoate) is widely accepted. Reverse microdialysis with glutamate led to a dose-dependent production of hydroxyl free radicals indicated by the hydroxylation adduct of salicylate, namely 2,3-dihydroxybenzoic acid. The local stimulation of hydroxyl free radical formation seems to be suitable to investigate a radical-scavenging property of potential neuroprotective drugs. In vitro experiments using the Fenton reaction may be a helpful tool to assess whether or not a substance is able to act as a radical scavenger in a cell free environment, which is easy to handle and a simple screening method before in vivo experiments were performed. In the present study we present an in vivo approach using local application of glutamate into the striatum and an in vitro screening using the Fenton reaction to induce hydroxyl radical formation. The main goal is to reliable measure hydroxyl free radicals, which are the most reactive oxygen radicals in biology and medicine.

Comparison of the novel drug Ensaculin with MK-801 on the reduction of hydroxyl radical production in rat striatum after local application of glutamate.

Ensaculin interacts with various neurotransmitter systems (e.g., dopaminergic, serotoninergic, glutamatergic) and was originally designed for the treatment of dementia. In the present study Ensaculin was tested for its possible reduction of glutamate-induced hydroxyl free radical formation in vivo. The microdialysis experiment was carried out in non-anaesthetized Wistar rats, which were implanted with a microdialysis probe into the striatum. Salicylate (10 nmol/2 microl/min) was incorporated into the perfusion fluid to measure indirectly hydroxyl radicals indicated by 2,3-dihydroxybenzoic acid (DHBA) formation. After baseline recording, glutamate (100 or 500 nmol/2 microl/min) was perfused through the microdialysis probe (CMA 12, 4 mm, flow rate 2 microl/min). Ensaculin (0.1, 1 and 10 mg/kg), MK-801 (1 mg/kg) or saline was injected i.p. 20 min after the onset of glutamate perfusion (500 nmol/2 microl/min). Glutamate (100 nmol/2 microl/min) and (500 nmol/2 microl/min) perfusion produced a 2.6- and 17-fold increase of 2,3-DHBA, respectively. Treatment with Ensaculin (1 and 10 mg/kg i.p. ) significantly antagonized the formation of 2,3-DHBA, to values of 60.5% and 56.7% of control levels, respectively. In comparison, MK-801 attenuated 2,3-DHBA levels, to values of 65.8% compared to control values. Ensaculin may be useful in the treatment of neurodegenerative disorders associated with elevated hydroxyl free radicals and excitotoxicity.