No / The human epidermis holds the full machinery for cholinergic signal transduction. However, the presence of the vesicular transporter (vesicular acetylcholine (ACh) transporter (VAChT)) for both choline and ACh has never been shown in this compartment. The results of this study confirm the presence of VAChT in cutaneous nerves and in both epidermal melanocytes and keratinocytes as well as in their nuclei using immunofluorescence labelling in situ and in vitro, Western blot analysis of cellular and nuclear extracts and reverse transcription-PCR. These results underline that ACh/choline transport in the non-neuronal epidermis is no different from the neuronal pathway. However, the function of VAChT in the nucleus remains to be shown.

Doctor of Philosophy(PhD) / The aims of this project were to contribute to the elucidation of the role of the cholinergic system in attention and memory, two cognitive processes severely compromised in Alzheimer’s disease (AD), and to evaluate and develop tools for the functional molecular imaging of this system with a view to improving knowledge of AD and other neurological disorders. Towards the first aim, the specific anti-cholinergic toxin 192 IgG-saporin (SAP) was administered to female Sprague-Dawley rats via either an intracerebroventricular (icv) or an intracortical route and animals were tested with a vibrissal-stimulation reaction-time task and an object recognition task to evaluate their attentional and mnemonic function, respectively. The second aim was approached in two ways. Firstly, relative neuronal densities from animals with icv lesions were assessed with both ex vivo and in vitro autoradiography with the specific cholinergic radiopharmaceuticals [123I]iodobenzovesamicol (123IBVM) and 125I-A-85380, ligands for the vesicular acetylcholine transporter and the nicotinic acetylcholine receptor, respectively. Secondly, a number of in vivo and in vitro studies were performed on a novel and unique molecular imaging system (TOHR), with which it had been hoped initially to image eventually SAP-lesioned animals, with a view to measuring and ameliorating its performance characteristics and assessing its in-principle suitability for small-animal molecular imaging. The behavioural studies support a critical role for the cholinergic system in normal attentional function. Additionally, in accord with literature evidence, no significant impairment was observed in mnemonic function. It is postulated however that the results observed in the intracortically-lesioned animals support the published hypothesis that cholinergic projections to the perirhinal cortex are critical for object-recognition memory. In autoradiographic studies, SAP-lesioned animals demonstrated reduced uptake of 123IBVM in multiple regions. A reduction of nicotinic receptors was also seen in SAP-lesioned animals, a novel finding supportive of the excellent characteristics of radioiodinated I-A-85380. Examination of the performance characteristics of the TOHR support in principle its utility for targeted small-animal molecular imaging studies.

Doctor of Philosophy(PhD) / The aims of this project were to contribute to the elucidation of the role of the cholinergic system in attention and memory, two cognitive processes severely compromised in Alzheimer’s disease (AD), and to evaluate and develop tools for the functional molecular imaging of this system with a view to improving knowledge of AD and other neurological disorders. Towards the first aim, the specific anti-cholinergic toxin 192 IgG-saporin (SAP) was administered to female Sprague-Dawley rats via either an intracerebroventricular (icv) or an intracortical route and animals were tested with a vibrissal-stimulation reaction-time task and an object recognition task to evaluate their attentional and mnemonic function, respectively. The second aim was approached in two ways. Firstly, relative neuronal densities from animals with icv lesions were assessed with both ex vivo and in vitro autoradiography with the specific cholinergic radiopharmaceuticals [123I]iodobenzovesamicol (123IBVM) and 125I-A-85380, ligands for the vesicular acetylcholine transporter and the nicotinic acetylcholine receptor, respectively. Secondly, a number of in vivo and in vitro studies were performed on a novel and unique molecular imaging system (TOHR), with which it had been hoped initially to image eventually SAP-lesioned animals, with a view to measuring and ameliorating its performance characteristics and assessing its in-principle suitability for small-animal molecular imaging. The behavioural studies support a critical role for the cholinergic system in normal attentional function. Additionally, in accord with literature evidence, no significant impairment was observed in mnemonic function. It is postulated however that the results observed in the intracortically-lesioned animals support the published hypothesis that cholinergic projections to the perirhinal cortex are critical for object-recognition memory. In autoradiographic studies, SAP-lesioned animals demonstrated reduced uptake of 123IBVM in multiple regions. A reduction of nicotinic receptors was also seen in SAP-lesioned animals, a novel finding supportive of the excellent characteristics of radioiodinated I-A-85380. Examination of the performance characteristics of the TOHR support in principle its utility for targeted small-animal molecular imaging studies.

Half of the cholinergic neurons of human and primate intrinsic cardiac ganglia (ICG) have a dual cholinergic/noradrenergic phenotype. Likewise, a large subpopulation of cholinergic neurons of the mouse heart expresses enzymes needed for synthesis of norepinephrine (NE), but they lack the vesicular monoamine transporter type 2 (VMAT2) required for catecholamine storage. In the present study, we determined the full scope of noradrenergic properties (i.e. synthetic enzymes and transporters) expressed by cholinergic neurons of mouse ICG, estimated the relative abundance of neurons expressing different elements of the noradrenergic phenotype, and evaluated the colocalization of cholinergic and noradrenergic markers in atrial nerve fibers. Stellate ganglia were used as a positive control for noradrenergic markers. Using fluorescence immunohistochemistry and confocal microscopy, we found that about 30% of cholinergic cell bodies contained tyrosine hydroxylase (TH), including the activated form that is phosphorylated at Ser-40 (pSer40 TH). Dopamine β-hydroxylase (DBH) and norepinephrine transporter (NET) were present in all cholinergic somata, indicating a wider capability for dopamine metabolism and catecholamine uptake. Yet, cholinergic somata lacked VMAT2, precluding the potential for NE storage and vesicular release. In contrast to cholinergic somata, cardiac nerve fibers rarely showed colocalization of cholinergic and noradrenergic markers. Instead, these labels were closely apposed but clearly distinct from each other. Since cholinergic somata expressed several noradrenergic proteins, we questioned whether these neurons might also contain trophic factor receptors typical of noradrenergic neurons. Indeed, we found that all cholinergic cell bodies of mouse ICG, like noradrenergic cell bodies of the stellate ganglia, contained both tropomyosin-related kinase A (TrkA) and p75 neurotrophin receptors. Collectively, these findings demonstrate that mouse intrinsic cardiac neurons (ICNs), like those of humans, have a complex neurochemical phenotype that goes beyond the classical view of cardiac parasympathetic neurons. They also suggest that neurotrophins and local NE synthesis might have important effects on neurons of the mouse ICG.

No description available.

INTRODUÇÃO: A Asma Brônquica é caracterizada por obstrução ao fluxo aéreo, reversível ou não, e processo inflamatório pulmonar, caracterizado principalmente por eosinofilia. A persistência da inflamação pode induzir processo de reparo pulmonar associado à redução progressiva da função pulmonar. A recente descrição do sistema colinérgico anti-inflamatório, um mecanismo neural que suprime a resposta imune inata e controla a inflamação por inibição de citocinas proinflammatórias, e a detecção de alguns de seus componentes em células de vias aéreas sugerem uma importante participação deste sistema na fisiopatologia de doenças pulmonares. O principal mediador deste sistema é a acetilcolina (ACh), que é estocada em vesículas sinápticas pelo transportador vesicular de ACh (VAChT), proteína essencial para sua liberação. OBJETIVOS: Avaliar os efeitos da deficiência colinérgica por redução da VAChT nas alterações pulmonares observadas em modelo experimental de inflamação pulmonar induzida pela exposição crônica a ovoalbumina. METODOLOGIA: A redução colinérgica foi induzida pela modificação genética nos níveis de VAChT. Camundongos machos selvagens e mutantes foram submetidos ao protocolo de sensibilização subcutânea com ovoalbumina ou salina nos dias 0, 7 e 14. Após, foram submetidos a desafios inalatórios com ovalbumina a 1% ou inalações de salina por 20 minutos nos dias 26, 27 e 28. No dia 29, foi realizada a avaliação da mecânica pulmonar, da inflamação no lavado broncoalveolar e no tecido e análise histológica de remodelamento e expressão de MMP-9 e TIMP-1 por imunohistoquímica. Também foi quantificado por ELISA níveis de IL-4, IL-10 e de TNF-a no homogenato pulmonar. A análise estatística considerou um p<0,05 como significativo. RESULTADOS: Animais sensibilizados apresentaram hyperresponsividade brônquica, inflamação e edema peribrônquico e deposição de fibras colágenas e elásticas ao redor das vias aéreas comparado ao grupo salina (p<0,05). Além disso houve aumento de IL-4 no homogenato pulmonar e da expressão de MMP-9 e TIMP-1 nas células inflamatórias. Os animais mutantes, independente de serem ou não sensibilizados, apresentaram aumento de TNF-a no pulmão. Os animais mutantes que foram submetidos ao protocolo de sensibilização mostraram aumento da hiperresponsividade brônquica, do eosinófilos, do edema e da deposição de colágeno comparado aos animais selvagens que também foram sensibilizados com ovoalbumina. Estas alterações podem ser atribuídas ao aumento de IL-4 e de MMP-9/TIMP-1 que foi observado nos animais mutantes e sensibilizada em comparação com o os selvagens sensibilizados. Não houve diferença nos níveis de IL-10 nos grupos experimentais. Conclusão: A deficiência colinérgica piora a hiperresponsividade brônquica, a inflamação eosinofílica e o remodelamento, principalmente por interferir com a citocina pró-inflamatória IL-4 e na proporção de MMP-9 e TIMP-1. Estes dados sugerem que a via colinérgica antiinflamatória está envolvida na fisiopatogenia da asma e necessita ser mais investigada / BACKGROUND: Bronchial asthma is characterized by reversible or not airflow obstruction and pulmonary inflammation, mainly characterized by eosinophilia. The persistence of inflammation can induce lung repair process associated with progressive reduction in lung function. Recent evidence of the cholinergic anti-inflammatory system, a neural mechanism that suppresses the innate immune response and control inflammation by proinflammatory cytokines inhibition, and the detection of some of its components in airway cells suggest an important role of this system in pulmonary physiopathology. The main mediator of this system is acetylcholine (ACh), which is stored in synaptic vesicles by vesicular acetylcholine transporter (VAChT), an essential protein for ACh release. AIMS: To evaluate the effects of cholinergic deficiency by VAChT reduction on pulmonary alterations observed in an experimental model of pulmonary inflammation induced by chronic exposure to ovalbumin. METHODS: The cholinergic deficiency was induced by genetic modification on VAChT levels. Wild-type and mutant male mice were submitted to subcutaneous ovalbumin sensitization or saline protocol on days 0, 7 and 14. After, animals were submitted to inhalation challenge with ovalbumin 1% or saline for 20 minutes on days 26, 27 and 28. On day 29, we evaluated the pulmonary mechanics, inflammation in bronchoalveolar lavage and in airways, histological analysis of airway remodeling and the expression of MMP-9 and TIMP-1 by immunohistochemistry. It was also quantified by the levels of IL-4, IL-10 and TNF-a in lung homogenate. The statistical analysis were performed and a p<0.05 was considered significant. RESULTS: Sensitized animals presented bronchial hyperresponsividade, airway inflammation and edema and collagen and elastic fibers deposition of collagen and elastic fibers around the airways compared to saline group (p <0.05). Furthermore, there was an increase of IL-4 in lung homogenate and the expression of MMP-9 and TIMP-1 in inflammatory cells. The mutant animals, regardless the sensitization, showed an increase in lung content of TNF-a. The mutant and sensitized animals showed an increase in bronchial hyperresponsiveness, in eosinophils, edema and collagen deposition in airways compared to the wild type and sensitized animals. These changes can be attributed to increased IL-4 and MMP-9/TIMP-1 that were observed in mutant and sensitized animals. There was no difference in levels of IL-10 in the experimental groups. Conclusion: The cholinergic deficiency worsens bronchial hyperresponsiveness, eosinophilic inflammation, and airway remodeling mainly by interfering with the pro-inflammatory cytokine IL-4 and in MMP-9/TIMP-1 ratio. These data suggest that anti-inflammatory cholinergic pathway is involved in the asthma pathogenesis deserves further investigation

INTRODUÇÃO: A Asma Brônquica é caracterizada por obstrução ao fluxo aéreo, reversível ou não, e processo inflamatório pulmonar, caracterizado principalmente por eosinofilia. A persistência da inflamação pode induzir processo de reparo pulmonar associado à redução progressiva da função pulmonar. A recente descrição do sistema colinérgico anti-inflamatório, um mecanismo neural que suprime a resposta imune inata e controla a inflamação por inibição de citocinas proinflammatórias, e a detecção de alguns de seus componentes em células de vias aéreas sugerem uma importante participação deste sistema na fisiopatologia de doenças pulmonares. O principal mediador deste sistema é a acetilcolina (ACh), que é estocada em vesículas sinápticas pelo transportador vesicular de ACh (VAChT), proteína essencial para sua liberação. OBJETIVOS: Avaliar os efeitos da deficiência colinérgica por redução da VAChT nas alterações pulmonares observadas em modelo experimental de inflamação pulmonar induzida pela exposição crônica a ovoalbumina. METODOLOGIA: A redução colinérgica foi induzida pela modificação genética nos níveis de VAChT. Camundongos machos selvagens e mutantes foram submetidos ao protocolo de sensibilização subcutânea com ovoalbumina ou salina nos dias 0, 7 e 14. Após, foram submetidos a desafios inalatórios com ovalbumina a 1% ou inalações de salina por 20 minutos nos dias 26, 27 e 28. No dia 29, foi realizada a avaliação da mecânica pulmonar, da inflamação no lavado broncoalveolar e no tecido e análise histológica de remodelamento e expressão de MMP-9 e TIMP-1 por imunohistoquímica. Também foi quantificado por ELISA níveis de IL-4, IL-10 e de TNF-a no homogenato pulmonar. A análise estatística considerou um p<0,05 como significativo. RESULTADOS: Animais sensibilizados apresentaram hyperresponsividade brônquica, inflamação e edema peribrônquico e deposição de fibras colágenas e elásticas ao redor das vias aéreas comparado ao grupo salina (p<0,05). Além disso houve aumento de IL-4 no homogenato pulmonar e da expressão de MMP-9 e TIMP-1 nas células inflamatórias. Os animais mutantes, independente de serem ou não sensibilizados, apresentaram aumento de TNF-a no pulmão. Os animais mutantes que foram submetidos ao protocolo de sensibilização mostraram aumento da hiperresponsividade brônquica, do eosinófilos, do edema e da deposição de colágeno comparado aos animais selvagens que também foram sensibilizados com ovoalbumina. Estas alterações podem ser atribuídas ao aumento de IL-4 e de MMP-9/TIMP-1 que foi observado nos animais mutantes e sensibilizada em comparação com o os selvagens sensibilizados. Não houve diferença nos níveis de IL-10 nos grupos experimentais. Conclusão: A deficiência colinérgica piora a hiperresponsividade brônquica, a inflamação eosinofílica e o remodelamento, principalmente por interferir com a citocina pró-inflamatória IL-4 e na proporção de MMP-9 e TIMP-1. Estes dados sugerem que a via colinérgica antiinflamatória está envolvida na fisiopatogenia da asma e necessita ser mais investigada / BACKGROUND: Bronchial asthma is characterized by reversible or not airflow obstruction and pulmonary inflammation, mainly characterized by eosinophilia. The persistence of inflammation can induce lung repair process associated with progressive reduction in lung function. Recent evidence of the cholinergic anti-inflammatory system, a neural mechanism that suppresses the innate immune response and control inflammation by proinflammatory cytokines inhibition, and the detection of some of its components in airway cells suggest an important role of this system in pulmonary physiopathology. The main mediator of this system is acetylcholine (ACh), which is stored in synaptic vesicles by vesicular acetylcholine transporter (VAChT), an essential protein for ACh release. AIMS: To evaluate the effects of cholinergic deficiency by VAChT reduction on pulmonary alterations observed in an experimental model of pulmonary inflammation induced by chronic exposure to ovalbumin. METHODS: The cholinergic deficiency was induced by genetic modification on VAChT levels. Wild-type and mutant male mice were submitted to subcutaneous ovalbumin sensitization or saline protocol on days 0, 7 and 14. After, animals were submitted to inhalation challenge with ovalbumin 1% or saline for 20 minutes on days 26, 27 and 28. On day 29, we evaluated the pulmonary mechanics, inflammation in bronchoalveolar lavage and in airways, histological analysis of airway remodeling and the expression of MMP-9 and TIMP-1 by immunohistochemistry. It was also quantified by the levels of IL-4, IL-10 and TNF-a in lung homogenate. The statistical analysis were performed and a p<0.05 was considered significant. RESULTS: Sensitized animals presented bronchial hyperresponsividade, airway inflammation and edema and collagen and elastic fibers deposition of collagen and elastic fibers around the airways compared to saline group (p <0.05). Furthermore, there was an increase of IL-4 in lung homogenate and the expression of MMP-9 and TIMP-1 in inflammatory cells. The mutant animals, regardless the sensitization, showed an increase in lung content of TNF-a. The mutant and sensitized animals showed an increase in bronchial hyperresponsiveness, in eosinophils, edema and collagen deposition in airways compared to the wild type and sensitized animals. These changes can be attributed to increased IL-4 and MMP-9/TIMP-1 that were observed in mutant and sensitized animals. There was no difference in levels of IL-10 in the experimental groups. Conclusion: The cholinergic deficiency worsens bronchial hyperresponsiveness, eosinophilic inflammation, and airway remodeling mainly by interfering with the pro-inflammatory cytokine IL-4 and in MMP-9/TIMP-1 ratio. These data suggest that anti-inflammatory cholinergic pathway is involved in the asthma pathogenesis deserves further investigation

Introduction. Plusieurs maladies neurodégénératives bénéficieraient de meilleures ap-proches diagnostiques, dont la maladie d’Alzheimer. Celle-ci affecte en particulier les systèmes cholinergiques du SNC, et de nombreuses études d’imagerie ont tenté d’évaluer la dégénérescence de ce système à des fins diagnostiques, à l’aide de ligands radioactifs de diverses composantes du système ACh. En définitive, la plupart de ces études ne se sont pas montrées satisfaisantes. À la recherche de meilleures approches dans ce domaine, nous avons décidé d’évaluer les possibilités offertes par le (-)-[18F]Fluoroethoxy-benzovesamicol ((-)-[18F]FEOBV), un agent émetteur de positons se liant au VAChT de façon spécifique et réversible. Avant d’en arriver à une utilisation humaine cependant, une validation animale en plusieurs étapes s’avère nécessaire, mais celle-ci nous est apparue justifiée à la lumière de résultats d’études préliminaires en TEP chez le rat, qui se sont montrées très prometteuses. Nous nous sommes donc attaqués à la caractérisation du métabolisme de cet agent. Ceci a exigé, dans un premier temps, la mise au point d’une méthode chromatographique d’analyse des métabolites sanguins et, dans un deuxième temps, l’évaluation de ces métabolites et de leur cinétique chez le rat. Ces données permettront ultérieurement, chez l’humain, de procéder à des études quantitatives en TEP. Étude #1: Une fois les paramètres chromatographiques optimisés, le TR du (–)-FEOBV fut établi à 7.92 ± 0.18 minutes. Étude #2 : Le métabolisme in vivo s’est montré très rapide et temporellement variable, mais un seul métabolite hydrophile a été identifié. La fonction d’apport au cerveau du (–)-[18F]FEOBV a pu être établie après correction pour la présence du métabolite détecté. Conclusion. Dans l’ensemble, le (–)-[18F]FEOBV semble très prometteur en tant que marqueur biologique du système cholinergique pré-synaptique. / Background. Several neurodegenerative diseases would benefit from better diagnostic tools, and Alzheimer’s disease is an obvious point in case. Of interest, that disease par-ticularly affects CNS cholinergic systems. Many studies have evaluated neurodegenera-tion in that system during the course of Alzheimer’s disease, some using imaging tech-niques with radioactive ligands targeting the cholinergic system. However, most of those studies have shown rather unsatisfying results. Therefore, our team has decided to evaluate a so far never used in primates positron emitting ligand of the VAChT which reversibly binds to its target, (-)-[18F]Fluoroethoxy-benzovesamicol ((-)-[18F]FEOBV). Of course, before being able to use this ligand in a clinical setting, a multi-step animal validation needs to be performed. As initial experiments with PET imaging yielded encouraging results, assessing the metabolism of (-)-[18F]FEOBV was the next logical step. First, an HPLC methodology had to be developed to analyse blood metabolites. Then, we were able to use that methodology to analyse metabolites and their kinetics in the rat. That data will allow quantitative studies in humans with PET. Study #1: After the chromatographic parameters had been optimised, the TR of (–)-FEOBV was established at 7.92 ± 0.18 minutes. Study#2 In vivo metabolism was found to be fairly rapid and somewhat temporally variable, but a lone hydrophilic metabolite was identified. The plasmatic input function was obtained and corrected for metabolism. Conclusion. Overall, (–)-[18F]FEOBV holds promise as a potential ACh system pre-synaptic marker.

No description available.

Grundlage der vorgelegten Arbeit sind die bei der Alzheimerschen Erkrankung beobachtbaren pathologischen Merkmale, wie die progressive Akkumulation von β-Amyloid-Plaques, cholinerger Dysfunktion und zerebrovaskuläre Abnormalitäten. Die in englischer Sprache verfasste Dissertation ist eine tierexperimentelle Studie, die versucht, den Zusammenhang von β-Amyloid, cholinerger Neurotransmission und zerebralem Gefäßsystem bei der Alzheimerschen Erkrankung näher zu charakterisieren. An Hirnmaterial aus der transgenen Maus Tg2576, die die schwedische Mutation des humanen Amyloidpräkursorproteins als Transgen trägt und ab dem 10. Lebensmonat durch humane β-Amyloid-Plaqueablagerungen in der Hirnrinde imponiert, wurden im Altersverlauf (4 bis 18 Monate) immunhistochemische Untersuchungen zur morphologischen Integrität der zerebralen Mikrogefäße, der kortikalen cholinergen Nervterminalen und der intrazerebralen cholinergen neurovaskulären Innervation durchgeführt. Am somatosensorischen Kortex werden beispielhaft die Expression des Glukosetransporters 1 oder Solanum tuberosum Lektin als Kapillarmarker und des vesikulären Acetylcholintransporters als Marker für cholinerge Fasern mittels Immunfluoreszenz und Laser-Scanning Mikroskopie erfasst, einer semiquantitativen Computer-gestützten Bildanalytischen Auswertung unterzogen und mit dem Ausmaß der kortikalen Plaquebeladung korreliert. So konnte gezeigt werden, dass die Dichte der Blutgefäße und cholinergen Fasern im somatosensorischen Kortex von transgenen Tieren mit dem Alter im Vergleich zu nichttransgenen Kontrolltieren abnimmt, was mit einer Reduktion der perivaskulären cholinergen Innervation einhergeht. Die erhobenen Befunde stützen die von J.C. de la Torre und T. Mussivand schon im Jahre 1993 formulierte „vaskuläre Hypothese“, wonach bei der sporadischen Form der Alzheimerschen Erkrankung alters- und Lebensstil-bedingte Schädigungen des zerebralen Gefäßsystems eine zentrale Rolle bei der Manifestierung der Erkrankung spielen.