In rats that had been treated neonatally with 6-hydroxydopamine (6OHDA) to deplete striatal dopamine more than 95%, a single injection of p-chloroamphetamine (pCA) (5 or 10 mg/kg, i.p.) resulted in depletion of striatal and hippocampal serotonin at 1 week to a similar extent as in control rats. These findings suggest that striatal dopamine is not essential to the long-lasting depletion of brain serotonin by pCA in rats.

Because chronic haloperidol-treated rats demonstrate an increased incidence of spontaneous oral activity, while neonatal 6-hydroxydopamine-lesioned rats demonstrate an increased incidence of dopamine agonist-induced oral activity, we studied the influence of haloperidol in 6-hydroxydopamine-lesioned rats. At 3 days after birth rats received 6-hydroxydopamine hydrobromide (200 μg intracerebroventricularly; desipramine pretreatment, 20 mg/kg i.p., 1 h) or vehicle. Two months later haloperidol (1.5 mg/kg per day × 2 days per week, for 4 weeks; then 1.5 mg/kg per day, every day for 10 months) was added to the drinking water. After 15 weeks the level of spontaneous oral activity was stable. At 11 months there were 35.8 ± 4.9 vs. 18.4 ± 2.1 oral movements in 6-hydroxydopamine-lesioned vs. intact rats receiving haloperidol. This effect persisted unabated in lesioned rats for 4 months after haloperidol withdrawal. This stable high frequency of oral dyskinesias is an advantage for studying putative therapeutic drugs for tardive dyskinesia.

In rat, the neonatal destruction of nigrostriatal dopamine (DA) neurons by intracerebral administration of 6-hydroxydopamine entails dramatic changes in serotonin (5-hydroxytryptamine, 5-HT) as well as DA function. Most striking is the 5-HT hyperinnervation of the adult neostriatum, associated with increases in density of various 5-HT receptor subtypes and enhanced neuronal responsiveness to the iontophoretic application of 5-HT and its 5- HT(1B/2C) and 5-HT(2A/2C) receptor agonists, m-chlorophenylpiperazine and iododimethoxyphenylaminopropane. The topographical distribution of these changes is consistent with up-regulation and/or increased production and transport of 5-HT(1B) and 5-HT(2A) receptors by the neostriatal projection neurons, as confirmed for the 5-HT(2A) receptor in a recent in situ hybridization study. It is interesting that this study has also shown that increases in both 5-HT(2A) binding and mRNA level were abolished by chronic pretreatment with the DA agonists, apomorphine and SKF 38393, suggesting a regulatory influence of DA in the expression of this 5-HT receptor. D1 receptor binding is known to be slightly reduced in the rostral neostriatum of these rats, a down-regulation apparently imputable to a reduced rate of synthesis of the receptor. In contrast, D2 receptor binding is increased throughout the DA-denervated and 5-HT-hyperinnervated neostriatum, perhaps due to some post-transcriptional modifications. Stereotyped and motor behaviors induced by systemic treatment with D1 and D2 agonists are markedly enhanced in these rats (behavioral supersensitivity), although priming is commonly required to unmask a latent D1 supersensitivity. In the case of oral activity, however, overt behavioral supersensitivity is induced by D1 as well as D2 agonists. Moreover, there is overt supersensitivity of oral activity in response to the 5-HT receptor agonist m- chlorophenylpiperazine, which is presumably imputable to 5-HT(2C) receptors and may be demonstrated even in the absence of supersensitivity to D1 receptor agonist. 5-HT adaptations, therefore, seem to play a role not only in the abnormal spontaneous behavior, but also in the behavioral supersensitivity to 5-HT as well as DA receptor agonists in these rats.

Les stratégies thérapeutiques mises en place dans la maladie de Parkinson sont symptomatiques et ne permettent pas de ralentir la progression de la maladie, nécessitant le développement de nouvelles approches neuroprotectrices. La neuroinflammation joue un rôle majeur dans le processus neurodégénératif où elle se manifeste précocement par l’activation de cellules gliales (microglie et astrocytes). En s’appuyant sur l’utilisation de modèles animaux mimant les stades précoces de la maladie, l’élaboration de stratégies thérapeutiques à visée anti-inflammatoire constitue donc une approche thérapeutique prometteuse. Ce travail de thèse a consisté à mettre au point et à caractériser un modèle de lésion partielle à la 6-hydroxydopamine chez le rat afin d’évaluer les effets d’une stratégie thérapeutique originale basée sur l’utilisation en combinaison d’un agoniste des récepteurs nicotiniques α7 et d’un agoniste des récepteurs σ1. En utilisant différentes approches expérimentales, nous avons tout d’abord évalué le processus neurodégénératif et la neuroinflammation dans le modèle que nous avons mis en place. Nos résultats ont montré une dégénérescence partielle et reproductible des neurones dopaminergiques nigro-striataux associée à une importante neuroinflammation. Nos analyses métabolomiques ont également révélé plusieurs altérations spécifiques, apportant ainsi de nouvelles informations sur les mécanismes intervenant dans le processus neurodégénératif. En s’appuyant sur l’utilisation de la tomographie par émission de positrons, nous avons ensuite évalué longitudinalement le profil d’expression des récepteurs nicotiniques α7 dans les structures clés de la voie nigrostriée. Nos résultats ont montré des modifications transitoires de la densité de ces récepteurs pouvant être liées à des réponses microgliales biphasiques en association avec la cinétique de la dégénérescence neuronale. Ainsi, ces résultats renforcent l’idée de cibler spécifiquement les récepteurs nicotiniques α7 dans l’atténuation des processus neuroinflammatoires. Nous avons enfin évalué les effets de notre stratégie thérapeutique dans le modèle et nos résultats ont permis de montrer que ce type de combinaison préserve partiellement l’intégrité des neurones dopaminergiques nigro-striataux et réduit les réactions gliales chez les animaux lésés. Bien qu’il sera nécessaire de confirmer et de compléter ces résultats avec d’autres analyses, ce type de combinaison pourrait constituer une nouvelle entité biochimique prometteuse dans le traitement de la maladie de Parkinson. / Currently, therapeutic strategies in Parkinson’s disease are symptomatic and the progression of the disease is uncontrolled, requiring the development of new neuroprotective approaches. Neuroinflammation plays a major role in the neurodegenerative process where it occurs early through the activation of glial cells (microglia and astrocytes). Based on the use of animal models mimicking the early stages of the disease, the development of anti-inflammatory strategies is therefore a promising therapeutic approach. This thesis work consisted in the development and the characterisation of a partial 6-hydroxydopamine lesion model in rats in order to evaluate the effects of an original therapeutic strategy based on the combined use of a α7 nicotinic receptors agonist and a σ1 receptors agonist. Using different experimental approaches, we first evaluated the neurodegenerative and neuroinflammation processes in the model that we developped. Our results showed a partial and reproductible degeneration of nigro-striatal dopaminergic neurons associated with a marked neuroinflammation. Our metabolic analyses have also revealed several specific alterations, providing new insight on the mechanisms involved in the neurodegenerative process. Using positron emission tomography imaging, we then evaluated longitudinally the expression profile of α7 nicotinic receptors in the key structures of the nigro-striatal pathway. Our results showed transient changes in the density of these receptors that may be linked to biphasic microglial responses in association with the kinetics of neuronal degeneration. Thus, these results reinforce the hypothesis of specifically targeting α7 nicotinic receptors in order to reduce the neuroinflammatory processes. Finally, we evaluated the effects of our therapeutic strategy in the model and our results showed that this type of combination partially preserves the integrity of nigro-striatal dopaminergic neurons and reduces glial reactions in lesioned animals. Although it is necessary to confirm and extend these results, this type of combination could represent a promising new pharmacological approach in the treatment of Parkinson’s disease.

Au delà des symptômes moteurs classiques de la maladie de Parkinson (MP), d'autres troubles comportementaux, émotionnels, ou cognitifs sont fréquemment observés chez le patient parkinsonien. L'apathie, définie comme une réduction des comportements motivés, est l'un des troubles neuropsychiatriques les plus souvent rapportés en clinique, en particulier chez les patients traités par stimulation haute fréquence du noyau sous-thalamique. De nombreuses données récentes ont permis de suggérer que la résurgence de l'état apathique chez ces patients stimulés pourrait être liée à la diminution du traitement dopaminergique. Plus précisément, il semble que ce déficit motivationnel puisse s'expliquer, au moins en partie, par un hypofonctionnement affectant le système dopaminergique. En se basant sur des approches de lésions sélectives, partielles et bilatérales des neurones dopaminergiques du mésencéphale, notre laboratoire a récemment développé un modèle animal chez le rat reproduisant un déficit motivationnel pouvant s'apparenter à l'apathie parkinsonienne. L'objectif de ce travail doctoral a été d'élucider les mécanismes neurobiologiques qui sont à l'origine de l'apparition du déficit motivationnel observé dans ce modèle animal (rat 6-OHDA SNc). Dans une première partie, en utilisant la microdialyse intracérébrale, nous avons analysé les modifications neurochimiques induites par les lésions mésencéphaliques réalisées, au sein de différents territoires cibles (le noyau accumbens ou N.Acc et le striatum dorsolatéral ou DLS) des projections dopaminergiques ascendantes. Nous avons ainsi pu montrer que le DLS est affecté par une situation d'hypodopaminergie chez les rats 6-OHDA SNc. Dans une deuxième partie expérimentale, nous avons étudié les modifications d'expression du transporteur de la dopamine et des récepteurs D1, D2 et D3 (RD3), au niveau du mésencéphale et des territoires de projections dopaminergiques. Cette étude autoradiographique nous a permis de mettre en évidence une diminution d'expression des RD3 qui concerne spécifiquement le DLS chez les rats 6-OHDA SNc. Enfin, dans une troisième partie, nous avons utilisé des approches de micro-injections in situ avec des antagonistes dopaminergiques sélectifs, qui nous ont permis de caractériser le rôle fonctionnel des RD3 au sein du striatum dorsal (DLS et striatum dorsomédian) et du N.Acc dans les comportements motivés. Les données pharmaco-comportementales que nous avons obtenues suggèrent une implication centrale de la neurotransmission médiée par les RD3 dorsostriataux dans les processus motivationnels qui sous-tendent le phénotype apathique observé dans notre modèle animal. Au total, les données obtenues au cours de ce travail doctoral confirment l'implication de la voie dopaminergique nigro-striée, et en particulier du DLS, dans la physiopathologie des troubles apathiques. De plus, cette étude a permis d'identifier les RD3 comme une cible thérapeutique intéressante pour améliorer les troubles motivationnels de la MP. / Beyond the classical motor symptoms of Parkinson's disease (PD), behavioural, emotional or cognitive impairments are also commonly observed in PD patients. Apathy, which is defined as a decrease in motivated behaviours, is one of the most frequently reported neuropsychiatric symptom in PD, especially in patients with high frequency stimulation of the subthalamic nucleus. A growing body of data suggests that the resurgence of an apathetic state in these stimulated patients may be linked, at least in part, to a dopaminergic hypofunction. Through neurotoxic selective, partial and bilateral lesion-based approaches of mesencephalic dopaminergic neurons, we recently developed in the laboratory an experimental model in the rat of motivational deficits that are reminiscent of the Parkinsonian apathy. The aim of this thesis was to better understand the neurobiological mechanisms underlying the motivational deficits observed in this animal model (6-OHDA-SNc rats). In a first part, we have investigated, with intracerebral microdialysis, the neurochemical alterations induced by our lesional approach of the main projection territories of the mesencephalic dopaminergic neurons, namely the nucleus accumbens (N.Acc) and the dorsolateral striatum (DLS). Hence, we have confirmed the presence of a hypodopaminergic state within the DLS, in our 6-OHDA-SNc experimental model. Next, we have studied the modifications of dopamine transporter and D1, D2 and D3 receptors (D3R) expression in different mesencephalic and striatal sub-regions. In this autoradiographic study, we have found a specific decrease in D3R levels within the DLS of 6-OHDA-SNc rats. Lastly, in a third part, we set out to determine the functional implication of D3R in motivated behaviors, with in situ microinjections of selective dopaminergic receptor subtype antagonists within the DLS, the dorsomedial striatum and the N.Acc. The psychopharmacological data obtained, suggest a central role of dorsostriatal D3R-mediated neurotransmission in the motivational processes that underlie the apathetic-like phenotype observed in our model. Finally, the data obtained during this doctoral work confirm the implication of the nigrostriatal dopaminergic pathway, and particularly of the DLS, in the pathophysiology of apathy. Moreover, we identified the D3R as a potential interesting target for treating motivational impairments in PD.

Submitted by Angela Maria de Oliveira (amolivei@uepg.br) on 2018-12-11T17:15:35Z No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) ANA PAULA CHUPROSKI.pdf: 2454041 bytes, checksum: 5a8faf5a6715ca365008970cde8b1ed7 (MD5) / Made available in DSpace on 2018-12-11T17:15:35Z (GMT). No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) ANA PAULA CHUPROSKI.pdf: 2454041 bytes, checksum: 5a8faf5a6715ca365008970cde8b1ed7 (MD5) Previous issue date: 2018-02-05 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A doença de Parkinson (DP) é uma desordem crônica, degenerativa e progressiva causada pela perda dos neurônios dopaminérgicos da substância negra pars compacta (SNpc) do mesencéfalo. Acompanhando ou antecedendo os sinais motores, são observadas alterações sensoriais, autonômicas e distúrbios neuropsiquiátricos como depressão, ansiedade e demência, chamados de sintomas não-motores da DP. Como o tratamento atual para a DP permanece sintomático e paliativo, busca-se terapias neuroprotetoras capazes de retardar a degeneração neuronal em fases iniciais. Entre estas substâncias estão os ácidos graxos ômega-3 (ω-3), componentes alimentares com funções estruturais, metabólicas e funcionais, cuja importância vem sendo demonstrada para o bom funcionamento do sistema nervoso, no neurodesenvolvimento e na prevenção de doenças neurodegenerativas, entre elas a DP. Apesar disso, em decorrência de mudanças do comportamento alimentar da sociedade atual, a ingestão de alimentos ricos em ω-3 vem reduzindo significativamente. O objetivo deste projeto é avaliar os efeitos da suplementação subcrônica com ω-3 em relação a um possível papel neuroprotetor para os comportamentos tipo depressivo, ansioso e déficit de memória em ratos adultos lesados pela 6-OHDA e se este efeito neuroprotetor está relacionado ao aumento da capacidade antioxidante neuronal pelos níveis de glutationa (GSH). Para realização dos experimentos, foram utilizados 49 ratos machos linhagem Wistar com peso entre 280 a 320 g, divididos em 4 grupos: SHAM/salina (n=12), SHAM/OP (n=12), 6-OHDA/salina (n=14), 6-OHDA/OP (n=11). Nos grupos OP, foi administrado via gavagem óleo de peixe (OP) contendo ácidos graxos ω-3 (50%DHA e 20% EPA) durante o período de 21 dias (2 g/kg/dia), sendo 14 dias antes e 7 dias após a lesão nigroestriatal. Os animais foram submetidos à cirurgia estereotáxica para infusão da neurotoxina 6-OHDA ou solução salina (grupos SHAM) na SNpc. Após o intervalo de três semanas, realizaram-se os testes comportamentais: campo aberto, labirinto em cruz elevado, reconhecimento de objetos, preferência à sacarose e natação forçada. Ao término dos testes, os animais foram sacrificados, sendo retiradas amostras de córtex cerebral, estriado e mesencéfalo para análise bioquímica e histológica, respectivamente. Foi realizada análise bioquímica da concentração de GSH no córtex frontal e estriado. Verificou-se que a lesão bilateral gerada pela 6-OHDA na SNpc foi capaz de provocar um comportamento tipo depressivo, sendo atenuado nos animais suplementados com ω-3. Da mesma forma, a lesão causou déficit na memória de reconhecimento, que se apresentou menos intenso nos animais suplementados com ω-3. Entretanto, não houve alteração nos níveis de GSH em decorrência da lesão, não sendo possível estabelecer uma relação entre os níveis desta enzima e a neuroproteção. Portanto, pode-se inferir que a suplementação com ácidos graxos ω-3 presentes no óleo de peixe ofereceu uma resposta neuroprotetora frente a este modelo animal de DP. / Parkinson's disease (PD) is a chronic, degenerative and progressive disorder caused by the loss of the dopaminergic neurons from the substantia nigra pars compacta (SNpc) on the midbrain. Accompanying or predating motor signals, sensorial, autonomic and neuropsychiatric disorders such as depression, anxiety and dementia, are observed, being called non-motor symptoms of PD. As the current treatment for PD remains symptomatic and palliative, there is a need for neuroprotective therapies capable of slowing neuronal degeneration in the early stages. Among these substances omega-3 fatty acids (ω-3) are found, food components with structural, metabolic and functional roles whose importance has been demonstrated for the proper functioning of the nervous system, neurodevelopment and prevention of neurodegenerative diseases, including PD. Despite this, due to last decades changes in the eating behavior, the ingestion of foods rich in ω-3 has been reducing significantly. The aim of this work was to evaluate the possible neuroprotective effects of subchronic ω-3 supplementation on depressive-like, anxiety-like and memory deficit of 6-OHDA-injured adult rats and if this neuroprotective effect is related to the improvement of the neuronal antioxidant capacity by glutathione levels (GSH). For the experiments, 49 male Wistar rats weighing between 280 and 320 g were divided into 4 groups: SHAM/saline (n=12), SHAM/OP (n=12), 6-OHDA/saline (n=14), 6-OHDA/OP (n=11). In the OP groups, fish oil containing ω-3 fatty acids (50% DHA and 20% EPA) was administered via gavage during the 21-day period (2 g/kg/day), 14 days before and 7 days after the injury. The animals were submitted to stereotaxic surgery to infuse neurotoxin 6-OHDA or saline (SHAM groups) into the SNpc. After the three-week interval, the behavioral tests were performed: open field, elevated plus maze, object recognition task, sucrose preference and forced swimming. At the end of the tests, the animals were sacrificed, and samples of cerebral cortex, striatum and midbrain were removed for biochemical and histological analysis. Biochemical analysis of the concentration of GSH in the frontal cortex and striatum was performed. It was verified that the bilateral lesion generated by 6-OHDA in SNpc was able to induce a depressive-like behavior, which was attenuated in the animals supplemented with ω-3. Similarly, the lesion caused recognition memory deficit, which was less intense in those supplemented with ω-3. There was no change in GSH levels after the lesion, and it was not possible to establish a relationship between the levels of this enzyme and neuroprotection. Therefore, it can be deduced that the supplementation with ω-3 fatty acids present in the fish oil demonstrated a possible neuroprotective response in this animal model of PD.

In zebrafish, the ventilatory response to hypoxia first develops at 3 days post-fertilization (d.p.f.) before O2-chemoreceptive neuroepithelial cells (NECs) of the gill appear at 7 d.p.f. This indicates the presence of extrabranchial chemoreceptors in embryos and a developmental transition to primarily gill O2 sensing. This thesis examined the skin NECs, which reach peak density in embryos but decline as gill NECs appear. Exposure of embryos and larvae to chronic hypoxia prevented the loss of skin NECs, shifted peak basal ventilation to a later developmental stage, and induced a hypoventilatory response to acute hypoxia. Chronic exposure to hyperoxia rapidly diminished skin NECs, shifted peak ventilation to earlier stages and eliminated the response to acute hypoxia. Administration of the neurotoxin 6-hydroxydopamine degraded nerve terminals that contact skin NECs and reduced both basal ventilation frequency and the hypoxic ventilatory response. Thus, skin NECs are candidates for extrabranchial O2 chemoreceptors in developing zebrafish.

In zebrafish, the ventilatory response to hypoxia first develops at 3 days post-fertilization (d.p.f.) before O2-chemoreceptive neuroepithelial cells (NECs) of the gill appear at 7 d.p.f. This indicates the presence of extrabranchial chemoreceptors in embryos and a developmental transition to primarily gill O2 sensing. This thesis examined the skin NECs, which reach peak density in embryos but decline as gill NECs appear. Exposure of embryos and larvae to chronic hypoxia prevented the loss of skin NECs, shifted peak basal ventilation to a later developmental stage, and induced a hypoventilatory response to acute hypoxia. Chronic exposure to hyperoxia rapidly diminished skin NECs, shifted peak ventilation to earlier stages and eliminated the response to acute hypoxia. Administration of the neurotoxin 6-hydroxydopamine degraded nerve terminals that contact skin NECs and reduced both basal ventilation frequency and the hypoxic ventilatory response. Thus, skin NECs are candidates for extrabranchial O2 chemoreceptors in developing zebrafish.

In zebrafish, the ventilatory response to hypoxia first develops at 3 days post-fertilization (d.p.f.) before O2-chemoreceptive neuroepithelial cells (NECs) of the gill appear at 7 d.p.f. This indicates the presence of extrabranchial chemoreceptors in embryos and a developmental transition to primarily gill O2 sensing. This thesis examined the skin NECs, which reach peak density in embryos but decline as gill NECs appear. Exposure of embryos and larvae to chronic hypoxia prevented the loss of skin NECs, shifted peak basal ventilation to a later developmental stage, and induced a hypoventilatory response to acute hypoxia. Chronic exposure to hyperoxia rapidly diminished skin NECs, shifted peak ventilation to earlier stages and eliminated the response to acute hypoxia. Administration of the neurotoxin 6-hydroxydopamine degraded nerve terminals that contact skin NECs and reduced both basal ventilation frequency and the hypoxic ventilatory response. Thus, skin NECs are candidates for extrabranchial O2 chemoreceptors in developing zebrafish.

In zebrafish, the ventilatory response to hypoxia first develops at 3 days post-fertilization (d.p.f.) before O2-chemoreceptive neuroepithelial cells (NECs) of the gill appear at 7 d.p.f. This indicates the presence of extrabranchial chemoreceptors in embryos and a developmental transition to primarily gill O2 sensing. This thesis examined the skin NECs, which reach peak density in embryos but decline as gill NECs appear. Exposure of embryos and larvae to chronic hypoxia prevented the loss of skin NECs, shifted peak basal ventilation to a later developmental stage, and induced a hypoventilatory response to acute hypoxia. Chronic exposure to hyperoxia rapidly diminished skin NECs, shifted peak ventilation to earlier stages and eliminated the response to acute hypoxia. Administration of the neurotoxin 6-hydroxydopamine degraded nerve terminals that contact skin NECs and reduced both basal ventilation frequency and the hypoxic ventilatory response. Thus, skin NECs are candidates for extrabranchial O2 chemoreceptors in developing zebrafish.