In agreement with previous work, adult rats given selective lesions to dopamine (DA)-containing neurons as neonates exhibited a greater behavioral sensitization to repeated phencyclidine (PCP) treatment in comparison to sham-lesioned controls. Acute administration of olanzapine (1-5 mg/kg ip) or clozapine (15 mg/kg ip) decreased sensitized PCP-induced activity in both lesioned and control animals. Acute haloperidol (0.5 mg/kg ip) had no impact on PCP responsiveness in lesioned animals, but significantly antagonized PCP effects in sham-lesioned controls. Ketanserin, a selective 5-HT 2A/5-HT2C-receptor antagonist, significantly reduced PCP activation in both lesioned and control rats, suggesting that the efficacy of atypical antipsychotics against PCP-induced sensitized responses may be mediated by one of the 5-HT2-receptor subtypes. A 6-week chronic regimen of orally administered olanzapine, clozapine, or haloperidol failed to block the sensitization induced by repeated PCP exposure. However, a 10-month oral olanzapine treatment significantly blunted the behavioral sensitization to repeated PCP exposure in lesioned animals, even after withdrawal from chronic olanzapine for more than 3 weeks. A 10-month oral haloperidol treatment had no effect on the sensitization induced by repeated PCP dosing. The persistent effect of chronic olanzapine administration on PCP sensitization may be relevant to the chronic therapeutic efficacy of atypical antipsychotics treating schizophrenia - a clinical syndrome linked to enhanced sensitivity to N-methyl-D-aspartate (NMDA)-receptor antagonists.

To explore associations of neostriatal (NST) endogenous levels of dopamine (DA) and serotonin (5-HT) with sensitivity of their receptors, graded doses of 6-hydroxydopamine HBr (0 to 400 μg, ICV; 6-OHDA; desipramine pretreatment, 20 mg/kg IP) were given to rats between birth (P 0) and P 42. Numbers of vacuous chewing movements (VCMs) induced by SKF 38393 or m-chlorophenylpiperazine (m-CPP), respective DA D1 and 5-HT2 agonists, were subsequently determined. Enhanced SKF 38393-induced VCMs occurred when NST DA was reduced 97%-98% by high dose 6-OHDA (100-134 μg) at P 0 or P 3, but not in rats with 95%-97% loss in DA produced by 6-OHDA at P7 (134 μg) or P3 (67 μg). Enhanced m-CPP-induced VCMs occurred even when NST 5-HT content was not elevated after 6-OHDA (134 μg at P 10). Accordingly, D1 and 5-HT receptor sensitivity is not correlated with respective NST DA and 5-HT contents. The stage of ontogeny at the time of DA denervation may be the governing influence on receptor sensitivity.

Since prolyl-leucyl-glycinamide (MIF-1) modifies the behavior of adult rats after treatment with neuroleptics, we examined whether MIF-1 would also modify adult behavior after treatment of neonatal rats with 6-hydroxydopamine (6-OHDA). Rats received 6-OHDA (100 μg i.c.v.) or diluent at 3 days after birth and either MIF-1 (2.0 mg/kg per day s.c. × 10 days) or diluent beginning at 28 or 29 days after birth. At 5 weeks, a low dose (0.1 mg/kg s.c.) of apomorphine increased the distance traveled, time in ambulation, number of stereotypic movements, and number of movements per time in stereotypy, but decreased the time in stereotypy in the 6-OHDA group. MIF-1 (× 7 or 8 days) showed a tendency to attenuate the increased number of movements and significantly (P < 0.05) reduced all of the other effects of neonatal 6-OHDA. Behavior induced by higher doses of apomorphine in the 6-OHDA group (reduced licking and head nodding; increased paw treading, taffy pulling and self-biting) were not attenuated by MIF-1. At 38 or 39 days, total in vitro binding of [3H]SCH-23390 and [3H]spiroperidol to striatal homogenates was not altered in any of the groups. The findings demonstrate that specific early developmental alterations in apomorphine-induced behaviors can be modified by treatment of adult rats with MIF-1, even in the absence of overt changes in the binding of striatal dopamine D-1 and D-2 receptors.

Serotoninergic nerves are known to modulate sensitization of dopamine receptors (DA-R) in a rodent model of Parkinson’s disease (PD). However, serotoninergic nerves are not known to have a prominent role on DA exocytosis in intact rats. The current study was undertaken to explore the possible influence of serotoninergic nerves on DA exocytosis in Parkinsonian rats. Rat pups were treated at 3 days after birth with the neurotoxin 6-hydroxydopamine (6-OHDA; 134 μg icv, half into each lateral ventricle; desipramine, 1 h pretreatment), in order to produce marked long-lasting destruction of neostriatal dopaminergic innervation, as evidenced by the 90–95% depletion of DA (p < 0.001) [HPLC/ED] into adulthood. Controls received vehicle/desipramine in place of 6-OHDA. Other groups received the serotoninergic neurotoxin 5,7-dihydroxytryptamine (5,7-DHT; 25 μg base, icv, half in each lateral ventricle; desipramine, 1 h; 75 mg/kg pargyline HCl, 30 min) at 3 days post-birth; or both 6-OHDA+5,7-DHT treatments. In adulthood, an in vivo microdialysis study was undertaken to ascertain that p-chloroamphetamine (PCA, 1 mM in the microdialysate)-evoked DA release in the neostriatum was reduced approximately 50% in the 6-OHDA group, while PCA-evoked DA release in the 6-OHDA+5,7-DHT group was substantially increased, to a level equivalent to that of the vehicle control. The baseline neostriatal microdialysate level of 3,4-dihydroxyphenylacetic acid (DOPAC) was also higher in the 6-OHDA+5,7-DHT group vs 6-OHDA group; also, during the 2nd hour of PCA infusion. PCA-enhanced DA exocytosis occurred in the absence of changes in hydroxyl radical (HO·) in the microdialysate (i.e., assay of 2,3- and 2,5-dihydroxybenzoic acid, 2,3-DHBA; 2,5-DHBA). The overall findings demonstrate that an adulthood serotoninergic nerve lesion enhanced PCA-evoked DA exocytosis in a rodent model of severe PD, while susceptibility to oxidative stress was unchanged. The implication is that serotoninergic nerves may normally suppress the release of DA and/or act as an uptake site and storage sink for accumulated DA in parkinsonian-like neostriatum. Potentially, serotoninergic agonists or antagonists, targeting subtype-selective serotonin receptors, may be viable therapeutic adjuncts in PD.

No description available.

The neurotoxin 6-hydroxydopamine (6-OHDA), following pretreatment with the norepinephrine transport inhibitor desipramine, selectively destroys dopaminergic neurons. When given to rats, neonatal 6-OHDA (n6-OHDA) crosses the blood-brain barrier to destroy 90-99% of dopaminergic nerves in pars compacta substantia nigra (SNpc). The n6-OHDA-lesioned rat is posed as a reasonable animal model for PD: (a) the magnitude of dopaminergic neuronal destruction is expansive, (b) mapping of dopaminergic denervation has been defined, (c) effects on dopamine (DA) receptor alterations have been elucidated (d) as well as changes in receptor sensitivity status, (e) reactive sprouting of serotoninergic innervation (i.e. hyperinnervation) has been mapped, and (f) interplay between serotoninergic and dopaminergic systems is characterized. (g) A broad range of locomotor and stereotyped behaviors has been assessed and (h) large numbers of neurochemical assessments have been attained. (i) n6-OHDA-lesioned rats survive 6-OHDA lesioning and (j) the rat is behaviorally indistinguishable from controls. Dopaminergic destruction in early ontogeny rather in adulthood is a 'treatment liability' of this model, yet other animal models have liability issues of a serious nature-the initial one being use of a neurotoxin to produce the animal model of PD. The n6-OHDA-lesioned rat is proposed as a PD model for its value in associating the SNpc dopaminergic lesion with behavioral outcomes, also for replicability of dopaminergic destruction, and the accompanying neuronal adaptations and interplay between neuronal phenotypes in brain-which provide a means to better define and understand the range of deficits and neuronal adaptations that are likely to occur in human PD.

Yes / Background: Riluzole is a neuroprotective drug used in the treatment of motor neurone disease. Recent evidence suggests that riluzole can up-regulate the expression and activity of the astrocyte glutamate transporter, GLT-1. Given that regulation of glutamate transport is predicted to be neuroprotective in Parkinson’s disease, we tested the effect of riluzole in parkinsonian rats which had received a unilateral 6-hydroxydopamine injection into the median forebrain bundle. Results: Rats were treated with intraperitoneal riluzole (4 mg/kg or 8 mg/kg), 1 hour before the lesion then once daily for seven days. Riluzole produced a modest but significant attenuation of dopamine neurone degeneration, assessed by suppression of amphetamine-induced rotations, preservation of tyrosine hydroxylase positive neuronal cell bodies in the substantia nigra pars compacta and attenuation of striatal tyrosine hydroxylase protein loss. Seven days after 6-hydroxydopamine lesion, reactive astrocytosis was observed in the striatum, as determined by increases in expression of glial fibrillary acidic protein, however the glutamate transporter, GLT-1, which is also expressed in astrocytes was not regulated by the lesion. Conclusions: The results confirm that riluzole is a neuroprotective agent in a rodent model of parkinson’s disease. Riluzole administration did not regulate GLT-1 levels but significantly reduced GFAP levels, in the lesioned striatum. Riluzole suppression of reactive astrocytosis is an intriguing finding which might contribute to the neuroprotective effects of this drug.

Les neurones dopaminergiques (DAergiques) libèrent non seulement de la DA à partir de leurs terminaisons axonales, mais également dans le mésencéphale au niveau de la substance noire (SN) et l’aire tegmentaire ventrale (ATV). À cet endroit, un mécanisme de libération somatodendritique (STD) de DA a été proposé et impliquerait des senseurs calciques différents de ceux retrouvés du côté axonal. Au niveau axonal, la synaptotagmine 1 (Syt1) est une protéine essentielle à la libération rapide de DA. Toutefois, des études de notre laboratoire sur des knockout conditionnels (cKO) de Syt1 dans les neurones DA démontrent une diminution substantielle de la libération de DA au niveau axonal, mais aussi dans le mésencéphale. Une première hypothèse expliquant cette diminution dans le mésencéphale serait que Syt1 est impliquée dans la libération STD. Cependant, nous observons par microscopie à super-résolution que Syt1 ne se retrouve pas dans le compartiment STD des neurones DAergiques. Une autre possibilité serait la présence de collatérales axonales DAergiques dans le mésencéphale. Par imagerie confocale et électronique, nous observons que le mésencéphale contient plusieurs varicosités axonales asynaptiques et quelques varicosités axonales synaptiques. Enfin, nous avons évalué la plasticité des collatérales axonales DAergiques dans un modèle de lésion partielle des neurones DAergiques induite par la 6-OHDA. Malgré la perte de plus de 40% des neurones DA, la libération de DA dans la SN persiste 14 jours après lésion et s’accompagne d’une augmentation de l’expression axonale de Syt1, suggérant qu’un mécanisme de compensation axonale contribue à la résilience de la libération de DA. / Dopaminergic (DA) neurons not only exhibit a classic vesicular release from their axons in the striatum, but they also release DA in the midbrain in the substantia nigra (SN) and ventral tegmental area (VTA). In this region, somatodendritic (STD) release occurs and it requires different calcium sensors than those found in the axons. Of interest, synaptotagmin 1 (Syt1) has been shown to be implicated in fast DA release in the axons. However, recent research in our lab shows that in mice with conditional knockout (cKO) of Syt1 in DA neurons, there is a substantial decrease of DA release not only in the striatum, but also in the midbrain. Our first hypothesis is that Syt1 is directly involved in STD release. With super-resolution microscopy, we concluded that Syt1 is not localized in the STD compartment of DA neurons. This brings us to our second hypothesis, where local DA axon collaterals contribute to DA release in the midbrain. Through confocal and electron microscopy, we observed that the midbrain contains asynaptic varicosities as well as local DA synapses. In light of these results, we explored the contribution of axonal release to the resilience of SN DA release in a partial 6-OHDA lesion model. We observed that, following a loss of more than 40% of DA neurons, DA release in the SNc persists 14 days after lesion and that this is accompanied by an increase in Syt1 expression in DA axons which suggest that local axonal release is increased to compensate for DA loss.

Os mecanismos bioquímicos envolvidos na maioria das doenças psiquiátricas ainda não estão esclarecidos. Estudos recentes têm sugerido a participação de espécies ativas de oxigênio em diversas patologias. Baseados em informações pre liminares da literatura, propusemo-nos a investigar o possível envolvimento de radicais de oxigênio e enzimas antioxidantes na esquizofrenia e psicose maníaco-depressiva. A análise das atividades eritrocitárias da superóxido dismutase (SOD) e glutationa peroxidase (GSH-Px) nestes pacientes mostrou níveis significativamente elevados de SOD (cerca de 1,5 vezes) em relação aos indivíduos normais. A comparação das atividades enzimáticas, em pacientes esquizofrênicos tratados e não-tratados com neurolépticos, tais como clorpromazina, haloperidol e prometazina, mostrou que o aumento da atividade de SOD não e dependente da farmacoterapia. O efeito da medicação sobre os níveis de atividade da SOD e GSH-Px foi avaliado em estudos-modelo com ratos, constatando-se que o tratamento agudo com clorpromazina e Li2C03 não alterou a atividade destas enzimas em eritrócitos, fígado ou cérebro. O tratamento crônico com clorpromazina resultou em uma diminuição significativa na atividade de SOD eritrocitária e da SOD total dos hemisférios cerebrais e cerebelo Um modelo para a geração de \"stress oxidativo\" cerebral foi proposto administrando-se a neurotoxina 6-hidroxidopamina (6-0HDA) no cérebro de ratos por via intraventricular. Com este modelo estudou-se a indução de SOD pela 6-hidroxidopamina, a nível cerebral e eritrocitário, observando-se aumento significante da atividade de SOD nos dois tecidos. O tratamento prévio com clorpromazina não modificou o efeito da 6-0HDA sobre a atividade da SOD sugerindo que o tratamento com neurolépticos não exclui a ocorrência de um \"stress oxidativo\" no cérebro embora tenha atuado como inibidor de peroxidação lipídica em estudos-modelo. A indução de lipoperoxidação por 6-OHDA em meio aerado tamponado foi demostrada em lipossomos multilamelares de lecitina de ovo, constatando-se inibição do processo pela clorpromazina. Analisando-se o efeito de SOD, catalase e sequestradores de radical hidroxila sobre a velocidade de peroxidase, constatou-se que este radical é o provável agente iniciador do processo. Pela técnica de ressonância paramagnética eletrônica usando captador de spin demonstrou-se a formação de radical hidroxila durante a autoxidação de 6-0HDA em pH fisiológico. Postulou-se que os efeitos lesivos da 6-0HDA possam ser devidos em parte à peroxidação lipídica induzida pelo radical hidroxila, gerado na autoxidação de 6-0HDA no cérebro. A formação de 6-OHDA, \"in vivo\", é um tema controverso na literatura. Tentou-se detectar a formação de cromatografia liquida de alta pressão, em homogenatos do corpo estriado de ratos tratados com inibidores da monoamino oxidase e da catecol-O-metil transferase e com D-anfetamina. Este tratamento foi realizado com o objetivo de simular uma condição onde ocorre uma hiperatividade dopaminérgica, como é proposto na esquizofrenia. Nossos resultados mostraram a formação de uma substância com o mesmo tempo de retenção da 6-OHDA, que, no entanto, não foi identificada como 6-OHDA. Sugere-se que esta substância possa ser outro produto hidroxilado da dopamina, tal como a 5-hidroxidopamina. O modelo experimental sugerido neste trabalho foi adequado para estudar a indução de \"stress oxidativo\" cerebral e deverá ser utilizado em estudos futuros, enfocando a indução de lesões a nível de barreira hemotoencefálica por espécies ativas de oxigênio, geradas por produtos da hidroxilados da dopamina. A formação exacerbada de espécies ativas de oxigênio por substratos endógenos autoxidáveis tais como derivados hidroxilados da dopamina, poderia provocar lesões em estruturas importantes do sistema nervoso central. O aumento da atividade eritrocitária de SOD, em pacientes psiquiátricos, pode ser interpretado como uma resposta da defesa do organismo contra os efeitos deletérios de espécies ativas de oxigênio, geradas no cérebro por estas substâncias endógenas, passiveis de romper a barreira hematoencefálica e atingir eritroblastos, a nível de medula óssea, onde poderiam induzir a síntese de SOD. / The biochemical mechanisms underlying the psychiatric disorders still are to be elucidated. Recent studies have suggested the participation of active oxygen species in a number of normal and pathological processes, including mental diseases. We have then decided to investigate the role of oxyradicals and anti-oxidant enzymes in the cases of schizophrenia and manic-depressive psychosis. The erythrocytic activities of superoxide dismutase (SOD) and of glutathione peroxidase (GSH-Px) in blood obtained from schizophrenic and manic-depressive patients were shown to be 1.5 times higher than in normal individuals. Medication with neuroleptic agents such as chlorpromazine, holoperidol and promethazine did not affect the enzyme activities. In the case of manic-depression carriers, the trends for high anti-oxidant enzyme activities were also shown to be independent of lithium therapy. Model studies carried out with rats revealed that acute treatment with chlorpromazine and Li2C03 does not alter the enzyme activities in either blood, liver or brain. Chronic treatment with chlorpromazine led to significant decrease of erythrocytic SOD and total SOD in cerebral hemispheres and cerebelum. Oxidative stress in the brain of rats triggered by 6-hydroxydopamine (6-0HDA) - a well known neurotoxin is here described as a model aiming to shed some light to the present knowledge of the biochemical basis for those mental disorders. In fact, intraventricular administration of 6-0HDA to the brain of rats resulted in increased SOD levels of blood and brain tissues. Pre-treatment with chlorpromazine did not modify the SOD values, indicating that neuroleptic agents probably do not abolish the cerebral oxydative stress. In vitro studies showed that chlorpromazine does not affect the rate of 6-0HDA aerobic oxidation but inhibits the peroxidation of egg lechitin liposomes. That hydroxyl radicals generated during 6-0HDA autoxidation iniciates the lipoperoxidation is indicated by the observed inhibitory effect of added SOD, catalase and hydroxyl scavengers. Using EPR trapping techniques, hydroxyl radical formation during 6-0HDA autoxidation was confirmed. In vivo occurrence of 6-0HDA still remains a controverse fact in the literature. Using HPLC, we have tried to detect the accummulation of hydroxylated dopamines in homogenates prepared from the brain \"striatum\" of anphetamine-treated rats under concomitant treatment with monoamine oxidase and cathecol-O-methyltransferase inhibitors. Inject a component with approximately the same retention time as that of 6-0HDA was found in the chromatogram, perhaps the 5-0HDA. Based on those events we have tentatively proposed that active oxygen species produced by cerebral endogenous autoxidazable substracts such as 6-0HDA can indeed cause chemical lesions to important structures of the central nervous system, resulting neuro-psychiatria manifestations. In this study, we have interpreted the elevated erythrocytic SOD activities found in mental patients as a response against the deleterious effects of active oxygen species generated in the brain by such autoxidazable substracts which might cross the brain blood barrier, reach the erythroblasts and there induce the SOD biosynthesis.

The dopaminergic neurodegeneration in the substantia nigra pars compacta (SNpc) is responsible for the marked motor impairment observed in Parkinson's disease (PD). However, the molecular mechanisms underlying this are not completely understood. Since by the time of diagnosis, 50-70% of the dopaminergic neurons of the nigrostriatal pathway have already been degenerated, it is difficult to investigate the early-stage events of disease pathogenesis. Due to inaccessibility of the human brain to study initial pathogenic mechanisms of the disease, experimental models have been developed in an attempt to elucidate PD etiology and its progression. Nevertheless, PD models are a controversial issue in neuroscience research since it is challenging to mimic human neuronal complexity. Therefore, the lack of optimal models that recreate disease pathology is one of the causes of failure of clinical trials that have attempted to find new/better PD therapies. Taking this in consideration, the development of more suitable models is necessary to improve our knowledge regarding PD etiological mechanisms. Additionally, the understanding of the advantages and disadvantages of models already established would also be beneficial for PD research, which our group addressed by reviewing this subject. Considering this, we chose SH-SY5Y cells as a PD model for our studies. To investigate the initial stages of PD-induced neurodegeneration, our work focused in the role of cofilin-1, a protein involved in mitochondrial dysfunction caused by oxidant-induced-apoptosis, which are two pathogenic processes strongly related to PD. Hence, in the thesis, we aimed to validate the use of retinoic-acid-(RA)-differentiated SH-SY5Y cells as an in vitro model and use it to investigate the potential role of cofilin-1 in the initial molecular and cellular mechanisms of PD. Although SH-SY5Y cells are widely used in PD research, their major drawback is their lack of important neuronal features, such as low levels of proliferation and stellate morphology. On the other hand, SH-SY5Y cells can acquire a neuronal phenotype when treated with differentiation agents such as RA. Since several protocols have been described, the consequence of which may be the discrepancies observed among studies regarding neuronal and dopaminergic features. In Chapter I, we aimed to validate a RA-differentiation protocol for SH-SY5Y cells previously established by our research group, focusing upon characterization of neuronal features and its subsequent response to 6-hydroxydopamine (6-OHDA), a toxin widely used to induce dopaminergic degeneration. RA-differentiated SH-SY5Y cells have low proliferative rates, a pronounced neuronal morphology and high expression of genes related to synapse vesicle cycle, dopamine synthesis/degradation, and dopamine transporter (DAT). After exploring phenotypic differences between these two models, we verified that RA-differentiated cells were more sensitive to 6-OHDA toxicity than undifferentiated cells, which could be related to an increase of DAT immunocontent. Many lines of evidence have showed that DAT is responsible for 6-OHDA uptake in vivo. Once inside the neuron, 6-OHDA underwent auto-oxidation causing a significant increase in oxidative stress. However, toxin uptake is not an essential step in undifferentiated SH-SY5Y cells, as auto-oxidation occurs extracellularly. We showed here, for the first time, that RA-differentiated SH-SY5Y cells can mimic, at least in part, an important mechanism of the 6-OHDA-induced cell death found in previous in vivo studies. Hence, the cellular model established by our research group presents essential neuronal features, being a suitable model for PD research. In Chapter II, RA-differentiated SH-SY5Y cells were used as cellular model to investigate disease molecular mechanisms, focusing upon cofilin-1. Our previous data have shown that oxidation of non-phosphorylate (activated) cofilin-1 leads to mitochondrial dysfunction and cell death induced by apoptosis in tumour cells. Here we found that cofilin-1 played a role in early stages of neuronal apoptosis induced by 6-OHDA in our cellular model since cofilin-1 mitochondrial translocation precedes organelle dysfunction. Overexpression of wild type CFL1 resulted in increased sensitivity of SH-SY5Y cells to 6-OHDA-induced neuronal cell death. Furthermore, overexpression of non-oxidizable CFL1 containing Cys-to-Ala mutations (positions 39, 80 and 139) increased neuronal resistance to this toxin, suggesting that oxidation is an important step in 6-OHDA toxicity. Follow-up experiments were performed in order to evaluate clinically whether cofilin-1 pathway proteins content is altered in PD post mortem human brain. Our findings showed a significant decrease in p-cofilin-1/cofilin-1 ratio in PD patients, which indicates an increase in the amount of activated cofilin-1 available for oxidation. Moreover, through principal component analysis, the immunodetection of cofilin-1 pathway proteins were able to discriminate controls and PD individuals during the early-stage of neuropathological changings. Hence, we demonstrated, for the first time, a possible role for cofilin-1 in PD pathogenesis and its potential use as biomarker. Taken together, our data showed that RA-differentiated SH-SY5Y cells present terminally-differentiated dopaminergic neuron features, that are essential to mimic dopaminergic neurons. By using this cellular model and post mortem brain tissue, we also demonstrated a possible role for cofilin-1 in early steps of the neurodegeneration process found in PD, which it could impact drug and biomarker discovery researches.