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No / A neurological disorder is any disorder or abnormality in the nervous system. Among different neurological disorders, Alzheimer’s disease (AD) is recognized as the sixth leading cause of death globally. Considerable research has been conducted to find pioneer treatments for this devastating disorder among which cell therapy has attracted remarkable attentions over the last decade. Up to now, targeted differentiation into specific desirable cell types has remained a major obstacle to clinical application of cell therapy. Also, potential risks including uncontrolled growth of stem cells could be disastrous. In our novel protocol, we used basal forebrain cholinergic progenitor cells (BFCN) derived from human chorion-derived mesenchymal stem cells (hC-MSCs) which made it possible to obtain high-quality population of cholinergic neurons and in vivo in much shorter time period than previous established methods. Remarkably, the transplanted progenitors fully differentiated to cholinergic neurons which in turn integrated in higher cortical networks of host brains, resulting in significant improvement in cognitive assessments. This method may have profound implications in cell therapies for any other neurodegenerative disorders. / This work was carried outwithin the framework of a collaborative project (Project Grant No. 94-02-30-25922) by the School of Medicine, Iran University of Medical Sciences, (Project Grant No. REP209) council for stem cell sciences and technologies (Presidency of the Islamic Republic of Iran, vice-presidency for science and technology), and Iran National Science Foundation (INSF).

Investigations of cholinergic and adrenergic tone on heart rate (fH) and mean arterial pressure (Pm) during embryonic development have been conducted on numerous avian species. While these investigations have documented that adrenergic tone, a continuous stimulation, on fH and Pm is vital to embryonic development in the birds studied to date, development of cholinergic tone on fH has been shown to vary even within species. Further, past studies have been bias to focus primarily on precocial species while altricial species remain poorly understood in this context. The goal of this investigation was to investigate the role of cholinergic and adrenergic tone on fH and Pm of an altricial species, the neotropic cormorant (P. brasilianus) to address this bias. The embryonic neotropic cormorant possesses B-and-a adrenergic tone on fH and Pm at 70% and 90% incubation while cholinergic tone on fH occurs at 90% incubation. This pattern of control is similar to that previously reported for several species of precocial birds suggesting the development of tonic cardiovascular regulation may be conserved across avian taxa.

The reinforcing properties of nicotine depend partly on cholinergic projections from the pedunculopontine tegmental (PPTg) and laterodorsal tegmental (LDTg) nuclei to midbrain dopamine neurons in the ventral tegmental area (VTA) and substantia nigra pars compacta (SNc). Neuronal activation was investigated using Fos expression in these areas following acute (0, 0.1, 0.4, 0.8mg/kg) or chronic systemic nicotine (0, 0.1, 0.4, 0.8, 1.0mg/kg given once per day for 5 days). We also examined co-localization of Fos expression in bNOS and TH positive neurons to determine what populations of neurons were activated by nicotine. Acute nicotine resulted in dose related Fos expression, with the biggest increase seen after 0.4mg/kg nicotine, but no co-localization occurred with bNOS in the PPTg/LDTg. Surprisingly, nicotine also failed to activate midbrain dopamine neurons. After animals were sensitized to nicotine there was a similar dose response curve in Fos expression, but the biggest increase was seen after 0.8mg/kg nicotine. Chronic nicotine, like acute, also preferentially activated non-cholinergic neurons in the LDTg and PPTg and non-dopamine neurons in the SNc and VTA. Further experiments looked at the mechanisms of Fos expression after nicotine administration. Fos expression in the LDTg/PPTg and SNc/VTA was suppressed after d-amphetamine, despite an increase in locomotor activity, suggesting that the increased Fos expression after chronic nicotine was not simply due to the locomotor activating effects of sensitized nicotine. Blocking autoreceptors in the dopaminergic midbrain by haloperidol pre-treatment did not increase Fos expression in dopamine neurons indicating that the inhibitory mechanism was not dependent on local autoreceptors. Novel methods of visualising and lesioning GABA neurons in the mesopontine tegmentum and midbrain were also examined. The data suggest that the mechanisms by which dopamine is involved in the pharmacological actions of passively administered nicotine are more complex than was first thought and that the role of non-dopamine neurons in the VTA (possibly GABA or glutamate containing) are also important.

This thesis deals with effects of some chosen 7-methoxitacrine (7-MEOTA) analogues on enzymatic activity of acetylcholinesterase (AChE). 7-MEOTA is a derivative of tacrine, which had been used for symptomatic treatment of Alzheimer's disease (AD), until drugs with better therapeutic index were developed. 7-MEOTA the same way as tacrine therapeutically acts by inhibition of acetylcholinesterase and a neurotransmitter acetylcholine rise in the organism. It shows similar strength and type of inhibition, but it's less toxic contrary to tacrine. Some of the previously examined analogues of 7-MEOTA were as strong or even stronger AChE inhibitors than 7-MEOTA and so promising future medicaments. However, all the compounds analyzed in this thesis showed weaker enzymatic reaction inhibition and AChE affinity. For each of the examined compounds IC50, Ki and Ki' were calculated and AChE inhibition type was determined. All the 7-MEOTA analogues showed a mixed type of the inhibition. The theoretical part of this thesis deals with manifestations and origins of AD, its genetic factors etc. and tries to show some of the anthropological findings a theories connected with the theme.

This thesis deals with effects of some chosen 7-methoxitacrine (7 MEOTA) analogues on enzymatic activity of acetylcholinesterase (AChE). 7-MEOTA is a derivative of tacrine, which had been used for symptomatic treatment of Alzheimer's disease (AD), until drugs with better therapeutic index were developed. 7-MEOTA the same way as tacrine therapeutically acts by inhibition of acetylcholinesterase and a neurotransmitter acetylcholine rise in the organism. It shows similar strength and type of inhibition, but it's less toxic contrary to tacrine. Some of the previously examined analogues of 7-MEOTA were as strong or even stronger AChE inhibitors than 7-MEOTA and so promising future medicaments. However, all the compounds analyzed in this thesis showed weaker enzymatic reaction inhibition and AChE affinity. For each of the examined compounds IC50, Ki and Ki' were calculated and AChE inhibition type was determined. All the 7 MEOTA analogues showed a mixed type of the inhibition. The theoretical part of this thesis deals with manifestations and origins of AD, its genetic factors etc. and tries to show some of the anthropological findings a theories connected with the theme. Powered by TCPDF (www.tcpdf.org)

L'addiction est un comportement compulsif de recherche et de prise de drogues alternant des phases d'abstinence et de rechute malgré les conséquences négatives sur la vie de l'individu. Les êtres humains ne sont pas égaux devant l'addiction et les mécanismes moléculaires sous jacents sont encore mal compris. De nombreuses structures cérébrales, telles que l'aire tegmentale ventrale (VTA), le cortex préfrontal ou l'amygdale convergent sur le noyaux accumbens (NAc) pour réguler les circuits de la " récompense ". Les neurones GABAergiques épineux de taille moyenne (MSN) sont à la fois la voie d'entrée et de sortie majeure du NAc. Les MSNs sont régulés de façon dynamique par les fibres dopaminergiques provenant de la VTA ainsi que par les interneurones cholinergiques locaux (TANs). La destruction sélective des TANs entraine une importante modification des propriétés renforçantes des psychostimulants tel que la cocaïne. En 2002 nous avons découvert que, de façon surprenante, ces neurones expriment à la fois le transporteur vésiculaire de l'acétylcholine (VAChT) et le transporteur vésiculaire du glutamate de type 3 (VGLUT3). Plus récemment nous avons établi que VGLUT3 augmentait le stockage vésiculaire ainsi que la libération d'acétylcholine (ACh) par un mécanisme que nous avons appelé " synergie vésiculaire ". De plus, il a été observé que VGLUT3 confère aux TANs la capacité d'utiliser le glutamate aussi bien qu'avec l'ACh pour communiquer. De façon surprenante, des souris ayant perdu la capacité de libérer l'ACh dans le NAc ne présente que très peu d'altération de leurs réponses comportementales à la cocaïne. Ce résultat suggère que l'ACh n'est pas indispensable à la régulation des comportements de « récompense ». 1.2 Afin de déterminer le rôle de la signalisation VGLUT3-dépendante par les TANs nous avons utilisé une souris n’exprimant plus VGLUT3. Au cours de cette thèse j’ai pu établir que l’absence de VGLUT3 exacerbe les effets comportementaux induit par la cocaïne. Il semble donc que les TANs utilisent l’ACh ou le glutamate pour réguler différentiellement la libération de DA. Nous avons des résultats préliminaires suggérant que le glutamate libéré par les TANs va activer des mGluR qui exercent un contrôle inhibiteur sur la libération de DA. De plus j’ai observé que l’augmentation de libération de DA chez les souris VGLUT3-KO entraine une activation des cascade de signalisation DR1-dépendantes. De plus les MSNs du NAc des souris VGLUT3-KO présentent des augmentations morphologiques et synaptiques de l’activité glutamatergique du NAc. Finalement une augmentation de la fréquence des mutations du gène codant pour VGLUT3 a été trouvée dans une cohorte de sujets souffrants de formes sévères d’addictions. L’ensemble de ces résultats suggère que la régulation concomitante de la signalisation DAergique et glutamatergique dans le NAc agit comme un filtre protecteur contre les effets renforçant de la cocaïne. / Drug addiction is a compulsive pattern of drug-taking/drug-seeking behavior with alternate phases of abstinence and relapse despite adverse consequences. Human beings are not equally susceptible to addictions and molecular mechanisms underlying addiction are still poorly understood. Numerous brain structures such as the ventral tegmental area (VTA), the prefrontal cortex, the amygdala or the hippocampus converge onto the nucleus accumbens (NAc) to regulate reward. GABAergic medium spiny neurons (MSN) are the major input target as well as output pathway of the NAc. MSNs are dynamically regulated by dopaminergic fibers originating from the VTA and by local tonically active cholinergic interneurons (TANs). The selective destruction of TANs modulates rewarding properties of psychostimulant such as cocaine. Twelve years ago we made the surprising discovery that these neurons express both the vesicular acetylcholine transporter (VAChT) and the vesicular glutamate transporter type 3 (VGLUT3). We recently established that VGLUT3 increases the acetylcholine (ACh) vesicular accumulation (and release) by a mechanism named vesicular synergy. Furthermore, the presence of VGLUT3 confers to TANs the ability to release glutamate in addition to ACh. Unexpectedly, mice that have lost the ability to secrete ACh in the NAc show minimal alteration of their behavioral response to cocaine. This result suggests that ACh is not sufficient to modulate reward.To investigate the role of VGLUT3-mediated signaling by TANs we used a mouse line that no longer expressed VGLUT3. During this PhD I established that silencing VGLUT3 in mice dramatically exacerbated cocaine-induced behaviors. Furthermore, we found that VAChT-KO and VGLUT3-KO mice showed a decreased and increased DA release (respectively) in the NAc. Therefore, TANs use ACh and glutamate to differentially regulate DA release. We have preliminary data suggesting the glutamate released by TANs activate mGluR that negatively control DA release. I further observed that in VGLUT3-KO mice the increased DA release enhanced DR1-signaling cascades. In addition, MSNs from the NAc of VGLUT3-KO mice had increased morphologic and synaptic glutamatergic activity in the NAc. Finally, we report non-synonymous mutations in the gene encoding VGLUT3 in patients with severe addictions. Our results suggested that the concomitant regulation of the dopaminergic and glutamatergic tone by VGLUT3 in the NAc acted as a protective filter against reinforcing properties of cocaine.

Introduction : La dystonie est définie comme un syndrome de cocontractions musculaires soutenues aboutissant à des mouvements répétitifs et des postures anormales. Cependant la physiopathologie des dystonies reste mal comprise. Les études menées chez l’homme soulignent le rôle crucial des ganglions de la base dans la physiopathologie des dystonies. Des données récentes obtenues chez le rongeur suggèrent l’implication d’un désordre de la transmission cholinergique striatale mais es modèles qu’ils soient génétiques ou pharmacologiques n’aboutissent pas toujours à un phénotype de dystonie. C’est pourquoi il était important de proposer une étude chez le primate non humain, visant à vérifier notre hypothèse de travail, à savoir : est-ce qu’une augmentation de la transmission cholinergique dans le putamen est capable d’induire un phénotype clinique de dystonie similaire à celui rencontré chez l’homme.Méthodes : Nous avons réalisé des infusions chroniques d’un agoniste muscarinique non sélectif (Oxotremorine) au sein du territoire sensori-moteur du striatum chez le primate non-humain. Les symptômes cliniques induits par ce produit ont été évalués à l’aide de l’échelle de Burke-Fahn-Marsden (BFM) adaptée à l’animal. Nous avons également utilisé une approche électromyographique pour caractériser l’activité musculaire en lien avec la clinique ainsi que des enregistrements de l’activité Multi-Unitaire et Unitaire au sein des ganglions de la base afin d’établir des corrélations électro-cliniques.Résultats : Les infusions d’Oxotremorine nous ont permis d’observer : (i) des postures et des mouvements anormaux similaires aux mouvements dystoniques rencontrés en pathologie humaine ; (ii) une fréquence de décharge neuronale anormalement basse dans le GPi (13,5Hz) et un pattern de décharge de type « bursty » principalement lorsque les symptômes sont sévères ; (iii) une activité oscillatoire (28-30Hz) au sein du putamen, du GPe et du GPi; (iv) l’absence de cohérence de l’activité oscillatoire entre ces structures ; (v) que le GPi est la seule structure à présenter une cohérence de l’activité oscillatoire.Conclusion : Nos travaux démontrent pour la première fois qu’un modèle de dystonie chronique peut être obtenu chez le primate non humain par augmentation du tonus cholinergique dans le putamen. Ce travail valide l’hypothèse de l’implication des interneurones cholinergiques dans la physiopathologie des dystonies. Ils confortent l’idée qu’une augmentation du tonus cholinergique peu à elle seule induire un phénotype de dystonie. / Introduction: Dystonia is defined as a syndrome of sustained muscular cocontractions leading to repetitive movements and abnormal postures. However, the pathophysiology of dystonia remains poorly understood. Studies in humans emphasize the crucial role of basal ganglia in the pathophysiology of dystonia. Recent data in rodents suggest the involvement of a disorder in the striatal cholinergic transmission. But these genetic or pharmacological rodent models do not always express the phenotype of dystonia. Therefore, it was important to propose a primate study to test whether an increase of cholinergic transmission within the putamen is able to induce a clinical phenotype of dystonia similar to that seen in humans.Methods: To verify our hypothesis, we chronically infused non-selective muscarinic agonist (Oxotremorine) in the sensory-motor striatum in non-human primates. Dystonic clinical symptoms induced by this drug were assessed using the Burke-Fahn-Marsden (BFM) scale adapted to animals. We used electromyographic approach to characterize muscular activity linked to clinical symptoms, and we recorded Multi-Unit and Single-Unit neuronal activity in basal ganglia to establish electro-clinical correlations.Results: The infusions of Oxotremorine allowed us to observe: (i) abnormal postures and movements similar to the dystonic movements encountered in human pathology; (ii) an abnormally low neuronal firing frequency in the GPi (13.5Hz) and a bursty firing pattern mainly when the symptoms where severe; (iii) oscillatory activity (28-30Hz) within the putamen, GPe and GPi; (iv) the lack of coherence of the oscillatory activity between these structures; (v) that the GPi is the only structure to present a coherence of the oscillatory activity.Conclusion: We have demonstrated for the first time that a model of chronic dystonia can be obtained in non-human primates by increasing cholinergic tone in the putamen. This work validates the hypothesis of an involvement of cholinergic interneurons and striatal acetylcholine levels in the pathophysiology of dystonia.

Durante situações aversivas, como choque nas patas, imobilidade ou restrição dos movimentos, há um aumento nos níveis de acetilcolina no hipocampo de ratos. Além disso, o aumento desse neurotransmissor está envolvido com a modulação comportamental do MCC, principalmente em sua porção dorsal, HD. Diante disso, é proposto neste estudo que os receptores muscarínicos presentes no HD de ratos, modulam a expressão da REC, no modelo do MCC. Ratos wistar foram submetidos às sessões de condicionamento aversivo contextual, sendo divididos nos grupos: condicionado (6 choques de 3s; 1,5 mA) e não condicionado (sem choques) . Quarenta e oito horas após esta sessão foram avaliadas a comportamental (congelamento) e respostas autonômicas (PAM, FC e queda da TC). Além disso, foi verificada a distribuição de receptores M1 e M3 nas subáreas do HD e a quantificação dos mesmos em animais condicionados, não condicionados e naive, 48 após a sessão de condicionamento. A microinjeção bilateral de hemicolínio, inibidor da captação de colina, no HD promoveu uma redução do tempo de congelamento durante a reexposição ao contexto aversivo, caracterizando um efeito do tipo ansiolítico. Além disso, este tratamento inibiu o aumento da PAM, FC e a queda da TC induzidos pelo MCC. O mesmo ocorreu com a administração de atropina, antagonista não seletivo de receptores muscarínicos, de forma dose-dependente em todas as respostas observadas. Adicionalmente, a microinjeção de diferentes doses de J104129 fumarate bloquearam o tempo de congelamento (de forma dose-dependente, semelhante a atropina), a elevação da PAM, FC e a queda da TC durante a re-exposição ao contexto aversivo. Como o J104129 fumarate bloqueia receptores muscarínicos tanto M1 quanto M3, foi utilizado um antagonista de maior afinidade para receptores do tipo M1, pirenzepina. Todas as doses utilizadas de pirenzepina inibiram as respostas autonômicas, sem afetar o tempo de congelamento induzidos pelo MCC. A análise de imunofluorescência, por duplamarcação, mostrou que receptores M1 e M3 estão distribuídos nos mesmos compartimentos celulares nas subáreas do HD. A quantificação dos receptores 48 horas após o condicionamento evidencia apenas o aumento de receptores M3 no hipocampo de ratos, sem alteração na população de receptores M1. Com este conjunto de resultados podemos concluir que a ACh no HD é essencial para a expressão da REC. Especificamente, eles sugerem que os receptores muscarínicos do tipo M1 presentes nesta estrutura estão envolvidos com as respostas autonômicas e somente os receptores M3 participam das respostas comportamentais. / During aversive and stressful situations, such as footshock, stillness and restriction of movements, there is an increase in the levels of acetylcholine in rat hippocampus. Moreover, the increasing in the synaptic level of this neurotransmitter is involved with behavioral modulation of contextual fear conditioning (CFC), especially in the dorsal portion, DH. Therefore, this study investigated the involvement of muscarinic receptors in DH of rats in the expression of conditioned emotional response (CER) in the CFC. Moreover, we verified the expression of these receptors in the DH by double labeling immunofluorescence. Male Wistar rats were subjected to aversive contextual conditioning sessions and were divided into two groups: conditioned and unconditioned. Forty-eight hours after this session were evaluated autonomic (mean arterial pressure, MAP, heart rate, HR and tail temperature, TT) and behavioral (freezing) responses. Moreover, the distribution of M1/M3 receptors in subareas of DH was observed and the quantification of these receptors were performed 48 hours after the conditioning session in conditioned, unconditioned and naive animals. The bilateral microinjection of hemicholinium, inhibitor of choline reuptake, in DH, caused a decrease of freezing during re-exposure to the aversive context, featuring an anxiolytic-like effect. Furthermore, this treatment inhibited the increase in MAP, HR and TT drop by CFC. DH muscarinic receptors antagonism evoked by atropine, a non-selective muscarinic antagonist, reduced the freezing, in a dose-dependent manner. Similar reduction was observed in autonomic responses. The selective antagonism of M1/M3 receptors evoked by J104129 fumarate also reduced freezing, in a dose-dependent manner, when compared with control animals. The same effect was observed with autonomic responses in all the tested doses. As J104129 fumarate blocks both M1 muscarinic as M3 muscarinic, was used a higher-affinity M1 antagonist, pirenzepine. All doses inhibited cardiovascular responses and decrease the TT drop, without affecting the freezing induced by CFC. The immunofluorescence analysis revealed that M1 and M3 receptors are distributed in the same cellular compartments in DH. The quantification of receptors showed an increase of M3 receptors in rat hippocampus, while no change in the density of receptors M1 was detected. These findings support that cholinergic neurotransmission present in DH is involved with the expression of responses evoked by fear contextual conditioning, through muscarinic receptors activation. In particular, M3 muscarinic receptors modulate behavioral responses, M1 and M3 muscarinic receptors modulate the autonomic responses.