Neuronal alterations resulting from exposure to Parkinsonian-inducing 1-methyl-4- phenyl-1,2,3,6-tetrahydropyridine (MPTP) in an in vitro model SH-SYSY human neuroblastoma cells were explored using cytotoxic effects, neurochemical changes and pathological injury as endpoints. The results suggested that: MPTP entered the SH-SYS5Y human neuroblastoma cells through a non-dopamine transport mechanism, and was metabolized to 1-methyl-4-phenyl-2,3-dihydropyridium (MPDP⁺) and 1-methyl-4-phenylpyridium (MPP⁺) by monoamine oxidase (MAO). MPP⁺, the neurotoxic analog of MPTP, was taken up into cells through a dopamine (DA) uptake mechanism. MPTP, via its metabolite MPP⁺, inhibited NADH dehydrogenase activity. The MPTP-induced alterations of morphology included formation of blebs, attenuated neutrites, abnormal mitochondria with electron-density of matrix and disorganization of cristae, and abnormal aggregation of filamentous material of the cytoskeleton. MPTP was neurotoxic to the dopaminergic system, inhibiting monoamine oxidase (MAO) activity, and decreasing levels of dopamine (DA) and other catecholamines. In addition, MPTP enhanced ³H-DA release from cells, and its metabolite MPP⁺ inhibited ³H-DA uptake. MPTP was found to directly act on the cholinergic system in SH-SY5Y cells, causing dose-related decreases in the binding at muscarinic and nicotinic receptors. MPTP also inhibited acetylcholinesterase (AChE) activity and increased choline levels. The MPTP-induced increase in DA release and the decreases in catecholamines in SH-SYSY cells were blocked by pretreatment with acetylcholine receptor antagonists atropine and d-tubocurarine. MPTP caused increases in tau proteins, and also caused an increased expression of the reverse transcriptase polymerase chain reaction (RT-PCR) product after treatment for 2 to 5 days at 10⁻³ to 10⁻⁴ M. The results, for the first time, demonstrated that MPTP affected cytoskeletal associated tau protein and altered its mRNA. These results demonstrated that the human neuroblastoma cell line, SH-SYSY, can be used as an in vitro model for the study of the neurotoxicity of MPTP, including the mechanisms associated with exposure to this neurotoxicitant. / Ph. D.

o óxido nítrico tem sido apontado como um importante fator de desenvolvimento neural, participando ativamente como sinalizador espacial durante o processo de refinamento sináptico. Avaliamos nesse estudo a participação do óxido nítrico durante esse processo em tectos ópticos de embriões de pintos (Gallus gallus). Para tal avaliamos a imunorreatividade dos neurônios para as proteínas que formam os receptores colinérgicos e glutamatérgicos em diferentes fases do desenvolvimento embrionário, com e sem o bloqueio farmacológico da enzima de síntese do óxido nítrico (NOS). Durante o desenvolvimento embrionário, as subunidades GluR2/3 dos receptores glutamatérgicos são as que aparecem mais precocemente, no 6°dia de desenvolvimento embrionário (E6), juntamente com os neurônios imunorreativos para NOS e somente a partir de E8 é possível identificar a arquitetura laminar típica do tecto óptico.O bloqueio farmacológico da NOS mostrou diferentes padrões de marcação dependendo do tempo de sobrevida do animal. Em animais eutanasiados 24 h após o bloqueio farmacológico observou-se uma alteração na organização das camadas e no padrão radial de organização dos neurônios imunorreativos. Esse dado sugere que haja um atraso no refinamento sináptico, que se reflete em desorganização das camadas do tecto e alterações morfológicas nos neurônios. / O óxido nítrico tem sido apontado como um importante fator de desenvolvimento neural, participando ativamente como sinalizador espacial durante o processo de refinamento sináptico. Avaliamos nesse estudo a participação do óxido nítrico durante esse processo em tectos ópticos de embriões de pintos (Gallus gallus). Para tal avaliamos a imunorreatividade dos neurônios para as proteínas que formam os receptores colinérgicos e glutamatérgicos em diferentes fases do desenvolvimento embrionário, com e sem o bloqueio farmacológico da enzima de síntese do óxido nítrico (NOS). Durante o desenvolvimento embrionário, as subunidades GluR2/3 dos receptores glutamatérgicos são as que aparecem mais precocemente, no 6°dia de desenvolvimento embrionário (E6), juntamente com os neurônios imunorreativos para NOS e somente a partir de E8 é possível identificar a arquitetura laminar típica do tecto óptico.O bloqueio farmacológico da NOS mostrou diferentes padrões de marcação dependendo do tempo de sobrevida do animal. Em animais eutanasiados 24 h após o bloqueio farmacológico observou-se uma alteração na organização das camadas e no padrão radial de organização dos neurônios imunorreativos. Esse dado sugere que haja um atraso no refinamento sináptico, que se reflete em desorganização das camadas do tecto e alteração morfológicas nos neurônios.

Palmoplantar pustulosis (PPP) is a chronic disease of unknown pathogenesis. Most of the patients were smokers. High prevalence of a number of autoimmune diseases was observed among the patients (thyroid disease 14%, gluten intolerance 8%, diabetes type 1 3%). Eosinophils and neutrophils were found in large numbers in the pustules. Massive infiltrates of lymphocytes and mast cells in the dermis below the pustule and an abnormal acrosyringial pattern indicate that the acrosyringium is the target for the inflammation. Immunofluorescence (IF) revealed decreased innervation of the sweat gland, outward migration of substance P-positive granulocytes in the acrosyringium and an increased number of contacts between mast cells and nerve fibres in the dermis. Distributions of choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) were studied, since they regulate the level of acetylcholine, the main inducer of sweating. The most intense AChE-like immunoreactivity (LI) was observed in the acrosyringium in the lowest part of the stratum corneum, corresponding to the site of the pustule in PPP. ChAT-LI in granulocytes and AChE-LI in mast cells were demonstrated, which may have implications for inflammatory processes in general. Nicotinic acetylcholine receptors (nAChR) are activated by acetylcholine but also by nicotine. Immunohistochemstry of α-3 and α-7 subtypes of the nAChRs showed that the nAChR expression in healthy skin was influenced by smoking. A highly abnormal α-7 nAChR distribution in PPP skin was observed. The levels of nAChR antibodies were elevated in 42% of the PPP sera, and 68% of these sera gave specific endothelial IF in the papillary dermis in skin from non-smokers. Positive IF in the acrosyringium was also noted in skin from smokers. Conclusions: Smoking seems to induce up-regulation of an antigen in palmar skin. The results indicate that PPP is an autoimmune disease and that nicotine might have a role in the onset of the inflammation.

o óxido nítrico tem sido apontado como um importante fator de desenvolvimento neural, participando ativamente como sinalizador espacial durante o processo de refinamento sináptico. Avaliamos nesse estudo a participação do óxido nítrico durante esse processo em tectos ópticos de embriões de pintos (Gallus gallus). Para tal avaliamos a imunorreatividade dos neurônios para as proteínas que formam os receptores colinérgicos e glutamatérgicos em diferentes fases do desenvolvimento embrionário, com e sem o bloqueio farmacológico da enzima de síntese do óxido nítrico (NOS). Durante o desenvolvimento embrionário, as subunidades GluR2/3 dos receptores glutamatérgicos são as que aparecem mais precocemente, no 6°dia de desenvolvimento embrionário (E6), juntamente com os neurônios imunorreativos para NOS e somente a partir de E8 é possível identificar a arquitetura laminar típica do tecto óptico.O bloqueio farmacológico da NOS mostrou diferentes padrões de marcação dependendo do tempo de sobrevida do animal. Em animais eutanasiados 24 h após o bloqueio farmacológico observou-se uma alteração na organização das camadas e no padrão radial de organização dos neurônios imunorreativos. Esse dado sugere que haja um atraso no refinamento sináptico, que se reflete em desorganização das camadas do tecto e alterações morfológicas nos neurônios. / O óxido nítrico tem sido apontado como um importante fator de desenvolvimento neural, participando ativamente como sinalizador espacial durante o processo de refinamento sináptico. Avaliamos nesse estudo a participação do óxido nítrico durante esse processo em tectos ópticos de embriões de pintos (Gallus gallus). Para tal avaliamos a imunorreatividade dos neurônios para as proteínas que formam os receptores colinérgicos e glutamatérgicos em diferentes fases do desenvolvimento embrionário, com e sem o bloqueio farmacológico da enzima de síntese do óxido nítrico (NOS). Durante o desenvolvimento embrionário, as subunidades GluR2/3 dos receptores glutamatérgicos são as que aparecem mais precocemente, no 6°dia de desenvolvimento embrionário (E6), juntamente com os neurônios imunorreativos para NOS e somente a partir de E8 é possível identificar a arquitetura laminar típica do tecto óptico.O bloqueio farmacológico da NOS mostrou diferentes padrões de marcação dependendo do tempo de sobrevida do animal. Em animais eutanasiados 24 h após o bloqueio farmacológico observou-se uma alteração na organização das camadas e no padrão radial de organização dos neurônios imunorreativos. Esse dado sugere que haja um atraso no refinamento sináptico, que se reflete em desorganização das camadas do tecto e alteração morfológicas nos neurônios.

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No. of bitstreams: 1 DissCAPS.pdf: 2325968 bytes, checksum: 1ef9c551b609d3ee485174289e0d2d65 (MD5) Previous issue date: 2016-02-25 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / In the last 40 years, the use of pesticides increased 700% in Brazil, setting this country into the very consumers of this kind of chemicals in the world. There among the most used pesticides in Brazil are the pyrethroids, a class of insecticides largely used for the stability to light rays and being not accumulated in the trophic chain. Deltamethrin is a pyrethroid classified as moderately toxic and it may work as endocrine disruptor. It is widely used in the farms and preserving stocked foods. Pyrethroids are low toxic to mammals and birds. Although, new studies pointed out their noxiousness to aquatic organisms such as fishes. The high toxicity of pyrethroids to fishes may be related to damages caused to nervous system and to their mechanisms of biochemical degradation. Biomarkers are a way of monitoring the pesticides effects on the environments. Among them, cholinesterases and metabolites such as glucose, cortisol and lactate can be used. In the present monography is reported a work carried out in vivo with matrinxa exposed to deltamethrin (Keshet®) at 20, 40 and 60% of CL50:96h for 96 hours, and other in vitro with matrinxa exposed to the analytical formulation of deltamethrin (Pestanal®). After the pesticide exposure, fish were anesthetized, blood was withdrawn and then the fish were killed for brain, liver, gut, gills and white muscle excision. From the observations we can say that glucose, protein and ammonia were increased in liver, and amino acids and ammonia were increased in plasma. The glycogen bulks of liver and plasma lactate were decreased. In white muscle, protein and amino acids levels were decreased, and ammonia, glucose and lactate increased. Concerning the studied enzymes, it was observed inhibition of brain AChE in vivo and in vitro activities while CbE was inhibited in the gills and gut but increased in white muscle and plasma. The enzyme activities of ALAT and ASAT increased in liver and LDH activity was reduced. Therefore, we may conclude that matrinxa presented metabolic changes in order to supply the energetic demand caused by deltamethrin poisoning. The inhibition of AChE was observed, as expected, in addition to CbE increase, showing the activation of mechanisms to degrade deltamethrin. It is possible to assert that Brycon amazonicus exposed to deltamethrin is biochemically responsive to the poisoning using adaptive strategies which enable it to escape from toxic effects of a contaminated environment by such xenobiotic. / Nos últimos 40 anos, o consumo nacional de agrotóxicos aumentou 700%, fazendo do Brasil um dos maiores consumidores desse tipo de compostos no mundo. Dentre os agrotóxicos mais utilizados no Brasil estão os piretroides, um grupo de inseticidas muito utilizados por ter alta estabilidade à luz e não sofrer bioacumulação na cadeia trófica. A deltametrina é um piretróide, classificado como medianamente tóxico e pode funcionar como um interruptor endócrino sendo amplamente utilizado nas lavouras e na conservação de produtos estocados. Os piretroides possuem baixa toxicidade aos mamíferos e aves. Contudo, estudos recentes apontam sua nocividade a organismos aquáticos, tais como os peixes. A alta toxicidade dos piretroides aos peixes pode estar relacionada aos danos causados ao sistema nervoso e aos mecanismos de metabolização desse xenobiótico. Uma forma de monitorar os efeitos dos agrotóxicos no meio ambiente é através de biomarcadores, como as colinesterases e os intermediários metabólicos, como glicose, cortisol e lactato. Assim, foi feito nesse trabalho um experimento in vivo com B. amazonicus exposto por 96h a 20%, 40% e 60% da CL/50 96h de deltametrina (Keshet®), e um experimento in vitro com o B. amazonicus exposto por uma hora a deltametrina na formulação analítica (Pestanal®). Após a exposição os peixes foram anestesiados para retirada de sangue e posteriormente abatidos para retirada de: cérebro, fígado, intestino, brânquias e músculo branco. De acordo com os resultados observados, no fígado houve aumento dos níveis de glicose, proteína e amônia, enquanto no plasma houve aumento das concentrações de aminoácidos e amônia. Em contrapartida, os níveis de glicogênio caíram no fígado, assim como os níveis de lactato do plasma. No músculo observou-se redução nos níveis de proteína e aminoácidos e aumento de amônia, glicose e lactato. Com relação aos parâmetros enzimáticos observou-se inibição da colinesterase cerebral “in vivo” e “in vitro”, enquanto a CbE apresentou inibição nas brânquias e intestino e aumento da atividade no músculo e plasma. As atividades enzimáticas de ALAT E ASAT aumentaram no fígado enquanto a LDH apresentou inibição. Sendo assim, podemos concluir que o B. amazonicus apresentou alterações do metabolismo intermediário no sentido de suprir a demanda energética causada pelos processos de intoxicação causada pela deltametrina. E apresentou inibição da colinesterase, tal como esperado, além de apresentar atividade da CbE evidenciando a tentativa de degradar a deltametrina. Podemos assim afirmar que Brycon amazonicus exposto à deltametrina responde bioquimicamente à intoxicação através de respostas adaptativas que o permitem inicialmente escapar aos efeitos tóxicos do meio contaminado por esse xenobiótico.

The high intensity exercise produces physiological, neural and cognitive benefits. Caffeine has been considered an important ergogenic resource in exercise, due to its ability to block adenosine receptors. Due to fact the mechanisms related to effect of caffeine on physical exercise has not yet been fully elucidated, in this study we evaluated the effects of caffeine treatment alone or in association with high intensity exercise on the enzymes NTPDase, 5´- nucleotidase, adenosine deaminase (ADA) and acetylcholinesterase (AChE) in brain of rats. Animals were divided into six groups: I (Control untrained); II (untrained plus caffeine 4 mg/kg); III (untrained plus caffeine 8 mg/kg); IV- (only trained) V- (trained plus caffeine 4 mg/Kg) and VI (trained plus caffeine 8 mg/Kg). Swimming was used as a model of intermittent exercise. Animals were trained three times a week, gradually increasing the workload up to 2.5% of the animal's body weight per week. Caffeine was administered by gavage one hour before the training for 5 days a week. Animals were submitted to a period of six weeks to protocol exercise and caffeine treatment. Results showed no alterations in the ATP (10,14%), ADP (23,2%), AMP (45,89%) hydrolysis and ADA (27,33) activity in synaptossomes of cerebral cortex of trained rats (Grupo IV). However, when trained rats were treated with caffeine we observed a significant increase in the ADP, AMP hydrolysis and ADA activity in synaptosomes of the cerebral cortex (V and VI) when compared to other groups (P<0.05). In relation to the AChE activity, we observed a decrease in the activity of this enzyme in synaptosomes from cerebral cortex in the trained group (IV) and in the animals from the groups V and VI when compared to the control group (P <0.05). Exercise alone did not alter the AChE activity in homogenates from cerebral cortex, striatum, cerebellum or hippocampus (group IV). Group V showed a decrease in the AChE activity in hippocampus and an increase in the activity of this enzyme in striatum and cerebral cortex in relation to control (P<0.05). In the animals from group VI, caffeine increased the AChE activity in cerebral cortex and in cerebellum in relation to other groups (P<0.05). These findings suggest that caffeine modulates the purinergic and cholinergic signaling associated with high intensity exercise. / O exercício físico de alta intensidade produz benefícios fisiológicos, cognitivos e neurais. A cafeína tem sido considerada um recurso ergogênico associada ao exercício, devido a sua capacidade de bloquear os receptores de adenosina. Devido ao fato dos mecanismos relacionados ao efeito da cafeína no exercício físico ainda não serem totalmente elucidados, o objetivo desse estudo foi avaliar o efeito da cafeína per se ou em associação ao exercício de alta intensidade na atividade das enzimas NTPDase, 5´-nucleotidase, adenosina desaminase (ADA) e acetilcolinesterase (AChE) em encéfalo de ratos. Os animais foram divididos em seis grupos: I (controle/sedentário); II (sedentário tratado com cafeína 4mg/kg); III (sedentário tratado com cafeína 8mg/kg); IV (treinado); V(treinado e tratado com cafeína 4mg/kg) e VI (treinado e tratado com cafeína 8mg/kg). A natação com adição de sobrecarga ao dorso de ratos foi utilizada como modelo de exercício sendo que os ratos foram treinados 3 vezes por semana com aumentos progressivos (2,5% do peso corporal) a cada semana até 23% do peso corporal. Cafeína foi administrada durante cinco dias na semana por gavagem uma hora antes do exercício. Os ratos foram submetidos ao tratamento com cafeína associado ao protocolo de exercício durante seis semanas. Os resultados demonstraram que não houve alterações na hidrólise do ATP (10,14%), ADP (23,2%) e AMP (45,89%) bem como na atividade da ADA (27,33%) em sinaptossomas de córtex cerebral do grupo treinado (IV). Quando ratos treinados foram tratados com cafeína ocorreu um aumento na hidrólise do ADP, AMP e na atividade da ADA em sinaptossomas de córtex cerebral (grupos V e VI) (P<0.05). Em relação a AChE, uma diminuição aproximada de 40% na atividade dessa enzima ocorreu em sinaptossomas de córtex cerebral de ratos treinados e/ou tratados com cafeína (grupo IV, V e Vl) (P<0.05). O exercício per se não alterou a atividade da AChE em homogeneizado de córtex cerebral, estriado, cerebelo e hipocampo (grupo IV). Nos ratos do grupo V foi observado uma diminuição na atividade da AChE em hipocampo e um aumento em estriado e córtex cerebral (P<0.05) enquanto que nos animais do grupo VI ocorreu um aumento na atividade da AChE em córtex cerebral e cerebelo quando comparado com outros grupos (P<0.05). Esses resultados sugerem que a cafeína per se ou associada ao exercício de alta intensidade modula a sinalização purinérgica e colinérgica em ratos.

Nos travaux visaient à réaliser un « modèle rongeur » (souris C57Bl/6) de l’altération de la mémoire de travail (MDT) lors du vieillissement, et comparer le potentiel promnésiant de divers composés pharmacologiques. Nous montrons que des souris âgées (18-19 mois) présentent une sensibilité accrue aux interférences proactives délai-dépendantes dans une épreuve d’alternance spontanée par rapport à des souris jeunes (4-5 mois). Une étude immunohistochimique centrée sur l’activité CREB révèle que les souris âgées présentent une suractivation du cortex préfrontal (CPF) inversement corrélée aux performances. La relation entre le déficit cognitif et la suractivation du CPF est confortée par la restauration des performances suite à l’injection dans le CPF d’un inhibiteur de la kinase activatrice de CREB.L’administration de composés cholinergiques permet de restaurer les capacités mnésiques des souris âgées par des mécanismes bien dissociés. Le donepezil, inhibiteur de l’acétylcholinestérase, augmente l’activation de l’hippocampe, alors que le S 38232, un agoniste des récepteurs nicotiniques α4β2, agit en supprimant la suractivation préfrontale. Finalement, l’utilisation de souris invalidées génétiquement pour la sous-unité β2 révèle des mécanismes de régulation complexes au sein du système cholinergique.Nos travaux démontrent la validité de notre modèle pour l’étude des troubles de la MDT induits par le vieillissement. De plus, ils confortent des études récentes chez l’Homme indiquant un lien important entre la suractivation du CPF et le déclin cognitif lié à l’âge. Au regard de l’effet du S 38232 qui, en diminuant la suractivation préfrontale, permet de restaurer les performances de MDT, le récepteur nicotinique α4β2 apparaît comme une cible potentielle pour le développement de nouvelles stratégies thérapeutiques. / The aim of this study was to realise a “rodent model” (C57Bl/6 mice) of age-related working memory (WM) impairments, and to evaluate the procognitive impact of several pharmacological compounds. We show that compared to young adult mice (4-5 months), aged mice (18-19 months) exhibit an exaggerated vulnerability to delay-dependant interference in a sequential spontaneous alternation task. CREB activity assessed by immunohistochemistry demonstrates that aged mice show an over-activation of the prefrontal cortex (PFC) negatively correlated with behavioural performance. Infusion of an inhibitor of CREB activation into the PFC restores WM performance in aged mice. Both results highlight the link between the PFC over-activation and the age-related cognitive deficit.Pharmacological study reveals that cholinergic compounds restore cognitive performance of aged mice and act differently on brain structures sustaining WM. On one hand, donepezil – an inhibitor of acetylcholinesterase – increases CREB activation of the hippocampus. On the other hand, S 38232 – an agonist of α4β2 nicotinic receptors – decreases the CREB age-induced over-activation of the PFC. Finally, using β2 knock-out mice, we show that the regulation of the cholinergic system is submitted to complex mechanisms.Overall, our experimental set up demonstrates that spontaneous alternation is a valuable model for studying age-related WM impairments. In accordance with human’s findings, our results highlight the link between prefrontal over-activation and the cognitive decline occurring during ageing. Considering that S 38232, by decreasing the CPF over-activation, alleviates the WM deficit observed in aged mice, the α4β2 nicotinic receptor appears to be an efficient target for new therapeutic strategies.

Bien que la maladie d’Alzheimer (MA) soit la cause de démence la plus fréquente, lesmécanismes qui sous-tendent les déficits cognitifs chez les patients restent mal connus.Cependant, les peptides amyloïdes (Aβ) semblent être un acteur majeur impliqué dansl’apparition des troubles mnésiques au cours de l’évolution de la maladie, notamment de parleur capacité à induire un hypofonctionnement du système cholinergique associé au déclinmnésique. Sur la base de ces observations, le rôle joué par les récepteurs cholinergiquesnicotiniques α7 (α7-nAChRs) a été largement étudié, au vue de leur capacité à interagir avecles Aβ, sans toutefois dégager un consensus quant à l’implication de ces récepteurs dans lesdéficits mnésiques induits par les Aβ.Afin d’améliorer notre compréhension quant aux mécanismes sous-tendant les effetsdélétères induits par les Aβ dans les déficits mnésiques, notre travail visait à identifier le rôlejoué par les récepteurs α7-AChRs via une approche comportementale, pharmacologique etmoléculaire. Ainsi, nous avons utilisé un modèle « souris » basé sur des injections de formesoligomériques d’Aβ(1-42) (Aβo(1-42)) dans la région CA1 de l’hippocampe dorsal (dCA1),structure cérébrale impliquée dans les processus mnésiques, atteinte de manière précoce dansla MA et exprimant fortement les récepteurs α7-nAChRs.La première partie de cette étude a consisté à mettre au point et à valider notre modèleanimal d’étude des effets induits par les Aβo(1-42) dans le dCA1 par une approchecomportementale et moléculaire. Nous montrons que les injections répétées d’Aβo(1-42) dans ledCA1 induisent une perturbation spécifique de la mémoire de travail alors que la mémoirespatiale est préservée lorsque les performances mnésiques sont évaluées 7 jours après ladernière injection. Nous avons également montré que cette perturbation de la mémoire detravail est associée à une absence d’activation/phosphorylation de ERK1/2 au sein du réseauhippocampo-frontal et septo-hippocampique. Ces données nous ont permis de valider notremodèle expérimental permettant d’étudier spécifiquement l’impact des Aβo(1-42) dansl’hippocampe dorsal.Dans une seconde partie, nous nous sommes focalisés sur le rôle joué par lesrécepteurs α7-nAChRs dans les perturbations mnésiques induites par les Aβo(1-42). Nosrésultats montrent que (1) les souris KOα7 ne présentent pas de déficits de mémoire de travailconsécutivement aux injections intra-dCA1 d’Aβo(1-42), (2) les déficits mnésiques ainsi que lala perturbation de l’activation de ERK1/2 induits par les Aβo(1-42) sont compensés par destraitements pharmacologiques agoniste partiel et antagoniste des récepteurs α7-nAChRs, (3)le traitement par un agoniste complet des récepteurs α7-nAChRs ne permet pas de prévenir lesdéficits mnésiques. Au regard de ces résultats, le récepteur α7-nAChRs semble être essentielau développement des déficits mnésiques induits par les Aβo(1-42), et l’utilisationd’antagonistes de ces récepteurs pourraient être une cible potentielle pour le développementde nouvelles stratégies thérapeutiques. / Although Alzheimer’s disease (AD) has been considered as one of the major causesfor dementia, the mechanisms by which cognitive decline appear still remain unclear.However, amyloid-β peptides (Aβ) seem to play a central role in the appearance of memoryimpairments in the time course of the disease, inducing down-regulation of the cholinergicsystem which is associated with cognitive decline. Based on these observations, the role of α7nicotinic receptors (α7-nAChRs) which can interact with Aβ was widely studied withoutconsensus about the involvement of these receptors in memory deficits induced by Aβ.In order to improve our knowledge about the mechanisms involved in Aβ side effects,our work aims at identify the role of α7-nAChRs via behavioral and molecular approaches.Thus, we used a mice model based on injections of oligomeric assemblies of Aβo(1-42) (Aβo(1-42)) in the CA1 field of the dorsal hippocampus (dCA1) which is a brain structure stronglyinvolved in memory processes, precociously affected in the AD and with a high density of α7-nAChRs.The first part of this study was to develop and validate this animal model to studythe effects induced by Aβo(1-42) in the dCA1 by behavioral and molecular approaches. Weshow that repeated injections of Aβo(1-42) in the dCA1 induce a specific disruption of workingmemory 7 days after the last injection whereas spatial memory is spared. We also showed thatworking memory disturbance is associated with decreased activation / phosphorylation ofERK1 / 2 in the hippocampo-frontal and septo-hippocampal networks. These data allowed usto validate our experimental model to specifically study the impact of Aβo(1-42) into the dorsalhippocampus.In the second part, we focused on the role played by the α7- nAChRs receptors inmemory disturbances induced by Aβo(1-42). Our results show that (1) KOα7 mice do notexhibit working memory deficits consecutively to intra-dCA1 Aβo(1-42) injections, (2) thememory deficits and decreasing activation of ERK1/2 induced by Aβo(1-42) are offset bypharmacological treatments partial agonist and antagonist of α7-nAChRs receptors, (3)treatment with a full agonist of α7-nAChRs receptors does not prevent memory deficits .Given these results, the α7-nAChRs receptor appears to be essential to the development ofmemory deficits induced by Aβo(1-42), and the use of antagonists of these receptors might be apotential target for developing new therapeutic strategies for AD.

Due to the persisting lack of reliable animal models of cognitive impairment with good translational validity, researches strive to discover new ways and tools to replicate symptoms of human neurodegenerative diseases in rodents. Recently, biperiden, an M1- selective muscarinic antagonist, has been proposed as a potential tool for generating fast screening models of mnemonic deficits such as seen in patients with Alzheimer's disease. Being highly selective for the M1 receptor, a predominant type of muscarinic acetylcholine receptors in the brain involved in cognitive processes, it has been speculated to possibly only influence cognition without causing sensorimotor side effects. Studies assessing the usability of this drug reported conflicting results. We have decided to expand the experimental data and evaluate biperiden's validity in several variants of the Morris water maze. The results of this study showed no significant effect of biperiden on cognitive flexibility, tested by reversal learning. In delayed-matching-to-position paradigm, which tests assesses working memory, we found a difference in performance between the two experimental groups; however, it cannot be unequivocally attributed to a memory impairment. No effects were observed in visible platform task, confirming a lack of...

Le système cholinergique basalo-cortical joue un rôle crucial dans la régulation de la fonction visuelle grâce à son contrôle sur le cortex visuel primaire (V1). Ce système influence particulièrement la plasticité corticale, les processus d'attention et les mécanismes d'apprentissage. Les neurones cholinergiques, en particulier, jouent un rôle fondamental dans les processus d'attention et les mécanismes d'apprentissage, deux aspects clés de la cognition. Une caractéristique remarquable de ce système est sa capacité à moduler la fonction des neurones visuels. La stimulation des neurones cholinergiques, par exemple, peut entraîner une augmentation du fonctionnement de ces neurones, ce qui se traduit par une amélioration de leur sélectivité pour des tâches visuelles spécifiques. Un exemple frappant de cet effet est observé dans la sensibilité au contraste, une fonction cruciale pour la perception visuelle. Dans ce contexte, notre étude cherche à explorer et à comparer les caractéristiques distinctes de la libération d'acétylcholine (ACh) et de l'activité neuronale au sein du cortex visuel. Nous nous concentrons particulièrement sur les variations de contraste et de mouvement, deux éléments essentiels de l'environnement visuel, pour mieux comprendre comment le système cholinergique influence ces aspects de la perception visuelle. Pour ce faire, nous avons recours à l'imagerie mésoscopique, une technique avancée permettant d'observer l'activité calcique et cholinergique au niveau neuronal. L'imagerie mésoscopique de l'activité calcique et cholinergique a été réalisée chez des souris transgéniques de Thy1-gCAMP6s et des souris gACh-3.0 (senseur d’ACh transfecté par un virus adeno-associé). Dans cette étude, nous avons utilisé un réseau sinusoïdal horizontal de fréquence spatiale de 0,3 cycles par degré et de contraste variable de 30%, 50%, 75%, et 100%. La stimulation sur des moniteurs a inclus 10 répétitions de 2 secondes, avec des intervalles de 8 secondes. L’amplitude maximale des signaux calcique et cholinergiques a été calculée à l'aide d'un système d'imagerie optique modulaire et d'une caméra scientifique complémentaire métal-oxyde-semi-conducteur, CMOS. Ces mesures ont été effectuées au niveau du V1 ainsi que des zones extrastriées, y compris le cortex occipital latéral (LM), le cortex temporal intermédiaire postérieur (PM) et lateral (AL). L'examen des variationsde l'ACh et des signaux de calcium a été effectué en utilisant l'outil universal mesoscale Imaging dans le logiciel MATLAB. Des changements significatifs dépendant du contraste des signaux provenant de l'indicateur cholinergique (ACh) et calcium (Ca)ont été observés dans toutes les zones visuelles étudiées, à savoir V1, AL et PM, à l'exception de LM. Par exemple, l'amplitude moyenne pour groupe de l'expérience gACh 3.0 a été multipliée par trois lorsque l'on compare la condition de 30 % à la condition de 100 % et pour le groupe gCAMP6s plus de trois fois dans le cortex visuel primaire. En outre, la latence pour la zone V1 a été mesurée, révélant une diminution du temps de réaction à mesure que l'intensité du stimulus augmentait en fonction du contraste, statistiquement significatif pour le groupe gCAMP6s mais non statistiquement significatif pour gACh3.0. La sensibilité au mouvement a été étudiée quant à elle grâce à la projection d’un kinématogramme de points aléatoires (RDK) dont la cohérence de direction variait (de 30%, 50%, 75%, à 100%). Ni le signal calcique si celui d’ACh était sensible à la variation de la cohérence de mouvement. L'efficacité du donepezil (0.1 et 1mg/kg), qui potentialise la transmission cholinergique, était dépendante de la dose et augmentait la libération d’ACh signal mais pas le signal calcique. L’antagonisme des récepteurs muscarinique à l’ACh par la scopolamine (1mg/kg), diminuait le signal calcique. L'activité à l'état de repos présentait une corrélation modeste entre les différentes aires corticales et n’a pas été affectée par le DPZ dans le groupe gACh3.0. Cependant, dans le groupe de la gCAMP6s, les corrélations ont été renforcées après l'administration des injections. En conclusion, les résultats ont révélé une sensibilité accrue au contraste pour la signalisation du calcium et de l'ACh, où les signaux de calcium ont montré une plus grande activation par rapport aux signaux cholinergiques. Cependant les signaux n’étaient pas sensibles à la cohérence des points en mouvement. Conclusion : La libération d’ACh varie en fonction du stimulus visuel et semble avoir un impact sur l’intensité de la réponse neuronale au stimulus. Les médicaments cholinergiques et anticholinergiques, en particulier lorsqu'ils sont administrés à des doses élevées, peuvent induire des altérations de l'amplitude de l’activité corticale. / The basalo-cortical cholinergic system plays a crucial role in the regulation of visual function through its control over the precise adjustment of cortical processing. This system particularly influences cortical plasticity, attentional processes, and learning mechanisms. Cholinergic neurons, in particular, play a critical role in attention processes and learning mechanisms, which are key aspects of cognition. A notable feature of this system is its ability to modulate the function of visual neurons. For instance, stimulation of cholinergic neurons can lead to an enhanced operation of these neurons, resulting in improved selectivity for specific visual tasks. This effect is prominently observed in contrast sensitivity, a crucial function for visual perception. In this context, our study aims to explore and compare the distinct characteristics of acetylcholine (ACh) release and neuronal activity within the visual cortex. We are especially focused on variations in contrast and motion, two essential components of the visual environment, to better understand how the cholinergic system influences these aspects of visual perception. To achieve this, we employ mesoscopic imaging, an advanced technique for observing calcium and cholinergic activity at the neuronal level. Mesoscopic imaging of calcium and cholinergic activity was conducted in Thy1-gCAMP6s transgenic mice and gACh-3.0 mice (ACh sensor transduced by adeno-associated virus). In this study, we used a horizontal sinusoidal grating of 0.3 cycles per degree spatial frequency with varying contrast levels of 30%, 50%, 75%, and 100%. Stimulation on BenQ monitors included 10 repetitions of 2 seconds, with 8-second intervals. The maximum amplitude of calcium and cholinergic signals was calculated using a modular optical imaging system and a complementary metal-oxide-semiconductor, CMOS, scientific camera. These measurements were taken at V1 and extrastriate areas, including the lateral occipital cortex (LM), posterior intermediate temporal cortex (PM), and lateral (AL). Examination of ACh and calcium signal variations was performed using the universal mesoscale Imaging tool in MATLAB software. Significant contrast-dependent changes in cholinergic (ACh) and calcium (Ca) indicator signals were observed in all visual areas studied, namely V1, AL, and PM, except for LM. For instance, the mean amplitude for the gACh 3.0 experimental group was tripled when comparing the 30% to the 100% condition, and for the gcamp6s group, it was more than tripled in the primary visual cortex. Moreover, the latency for the V1 area was measured, revealing a decrease in reaction time as stimulus intensity increased according to contrast statistically significant for gCAMP6s group but not significant for gACh3.0. Motion sensitivity was studied by projecting a random dot kinematogram with varying directional coherence (from 30%, 50%, 75%, to 100%). Neither the CaS nor the ACh signal was sensitive to variation in motion coherence. The efficacy of DPZ (0.1 and 1mg/kg), which potentiates cholinergic transmission, was dose-dependent and increased ACh release but not calcium signal. Muscarinic ACh receptor antagonism by scopolamine (1mg/kg) decreased calcium signaling. Resting-state activity correlated modestly between the different cortical areas and was not affected by DPZ in the gACh3.0 group. The resting state activity exhibited a modest correlation and was infrequently impacted by treatments in the gACh3.0 group. However, in the gCAMP6s group, both positive and negative correlations were enhanced subsequent to the administration of injections. As a conclusion, the research findings revealed a strong contrast sensitivity of both calcium and ACh signalling, wherein calcium signals exhibited greater activation compared to ACh signals. The influence of ACh on visual processing is thus shown at a very low cognitive level. The signals were not changed by the coherence of moving dots. Cholinergic and anticholinergic drugs, particularly when administered in high dosages, influence the visual processing.