Substantial evidence in the literature shows that tobacco smoking has complex and divergent effects on inflammatory bowel diseases (IBD). It ameliorates ulcerative colitis (UC); whereas it aggravates the risk of Crohn’s disease (CD) and affects the disease course and severity. Studies have shown that nicotine has a positive influence on symptoms of UC. Also, it is demonstrated that nicotinic acetylcholine receptor, especially α7 subunit plays an essential component in the vagus nerve-based cholinergic anti-inflammatory effects. In the present study, we explored the effect of nicotine and α7 nicotinic agonists treatment in the DSS colitis mouse model. We also investigated the effects of cotinine, a major metabolite of nicotine, in the model. Methods: Different groups of C57BL6 mice, as well as α7, α5, and β2 nicotinic receptor knock out mice, and their littermates wild-type nicotinic receptor male adult mice were given DSS solution freely in the drinking water for 7 consecutive days after which tap water was given on the 8th day. We measured a Disease Activity Index (DAI) that includes body weight loss, blood presence in stools, stool consistency, local rectal irritation and length of the colon. The mice were then sacrificed on day 8 to allow examination of the entire colon. Disease severity and colon tissue histology and inflammatory markers including colonic myeloperoxidase (MPO) and colonic tumor necrosis factor-α (TNF-α) were evaluated. Levels of MPO and TNF-α were determined by enzyme-linked immunosorbent assay analysis of the homogenized colon samples. The effect of oral, subcutaneous, mini pump nicotine, and oral cotinine treatments were examined on experimental colitis induced by 2.5% DSS in mice. In addition, we measured the plasma levels of the nicotine and cotinine in our treatment protocols. Results: The DSS 2.5% model of colitis is easily induced in mice. Administration of low doses of oral nicotine (12.5 and 25 μg/ml), but not high doses in DSS-treated mice displayed a significant decrease in disease activity index value, total histological damage scores, as well as colonic level of TNF-α compared to the control group. However, the anti-inflammatory effect of nicotine was not seen with chronic s.c., mini pump nicotine or oral cotinine administration. Differences in plasma levels of nicotine and cotinine do not seem to account for this lack of effect. Moreover, neither nicotine nor cotinine reversed colon length shortening in DSS-treated mice, except with the 0.5 mg/kg s.c. dose of nicotine. There was no change in MPO activity among the groups treated with oral or s.c. nicotine. Cotinine oral administration on its own failed to show a significant effect in the DSS model of colitis. α7 KO mice displayed a significantly increased in DAI value starting from day 4 till day 8, histological damage scores and TNF-α levels of were increased significantly compared to their littermate WT mice. Moreover, pretreatments with PHA-543613 (8 mg/kg), a selective α7 agonist, and choline chloride (40 ug/ml), an α7 nAChR natural agonist, significantly reduced clinical parameters in DSS-treated mice; however, they slightly inhibited the increase in the colonic TNF-α levels compare with vehicle DSS-treated mice. Moreover, PNU-120596 (3 mg/kg), a positive allosteric modulator for α7 nAChRs, significantly reduced DAI value and total histological damage score in DSS-treated mice. Conclusion: Results obtained from this study highlight that dose and route of administration play a critical role in the protective effect of nicotine in the DSS mouse colitis model. Also, these data suggest that α7 nAChR has a protective role in colitis with narrower therapeutic index. Data obtained from this study further understanding of the effect of nicotine in UC and may contribute in the development of new pharmaceutical designs for targeting nAChRs for the treatment of ulcerative colitis.

des-Formylflustrabromine (dFBr), isolated from the marine organism Flustra foliacea, is the first selective, positive allosteric modulator (PAM) of α4β2 nicotinic acetylcholine receptors that potentiates the action of the neurotransmitter acetylcholine (ACh). Most agonists for this receptor population are not selective and can activate other nACh receptors. A selective PAM, which activates α4β2 nACh receptors only in the presence of ACh, might find application in the treatment of of various neurological diseases such as Alzheimer’s disease or autism. dFBr was examined and found to produce a biphasic dose-response curve over a wide concentration range (i.e., potentiation at low concentration, but inhibition of the ACh-induced response at high concentrations). Our goal was to examine various structural features of dFBr required for potentiation; a secondary goal was to examine the same for inhibition. To understand the structural requirements of dFBr, a systematic ‘deconstruction reconstruction and elaboration’ approach (see p. 48) was employed to determine the contribution of various structural components of dFBr to its activity at α4β2 nACh receptors. Novel compounds were synthesized and characterized. Human α4β2 nACh receptors were expressed in Xenopus oocytes and the actions of dFBr and its analogs were measured using a two-electrode voltage clamp technique. Dose-response curves were obtained for the compounds in the absence and presence of 100 μM ACh. Structural features of dFBr optimal and/or required for PAM action at 42 nACh receptors were identified. A novel reconstructed analog with all the essential features for PAM action was synthesized and submitted for biological testing. Elaborated analogs of dFBr further helped in identification of various structural features important for PAM action and the inhibition of action of ACh. The ‘deconstruction reconstruction and elaboration’ approach (see p.48) identified important structural features of dFBr that modify its actions as a PAM or an antagonist (NAM? or channel blocker?) at α4β2 nACh receptors. This information should be useful for the subsequent design of novel analogs to evaluate their potential for the treatment of neurological disorders associated with ACh.

A glândula pineal sintetiza o hormônio melatonina no período escuro. No rato, a ativação dos receptores a e b-adrenérgicos aumenta os níveis de AMPc, levando à síntese e ativação da enzima arilalquilamina-N-acetiltransferase (AANAT). A glândula recebe também inervação parassimpática, sendo inibitório o efeito da acetilcolina. A metilecgonidina (AEME) é o produto da pirólise da cocaína, quando esta é usada sob a forma de \"crack\". Neste trabalho estudamos os efeitos e os mecanismos de ação da AEME sobre a síntese da melatonina. Foram investigados a atividade da AANAT, o Ca2+, o AMPc e a viabilidade celular e a fragmentação do DNA. A AEME reduziu a síntese da melatonina in vivo e in vitro, sendo este efeito revertido pela atropina. A AEME induziu um aumento do Ca2+, não alterando o AMPc e a atividade da AANAT. A viabilidade celular e fragmentação do DNA não foram modificadas pela AEME. Em conclusão, a AEME reduz a síntese da melatonina, in vitro e in vivo, e a sua ação se dá por interferir com o sistema colinérgico muscarínico. / The pineal gland synthesizes the hormone melatonin in the dark. In rats, the activation of a and b-adrenergic receptors increases cAMP levels and the synthesis and activity of arylalkylamine-N-acetyltransferase enzyme (AANAT). The pineal gland is also innervated by parasympathetic fibers, being inhibitory the effect of acetylcholine. Methylecgonidine (AEME) is the pyrolysis product of cocaine when it is used as \"crack.\" In this work we studied the effects of AEME on the melatonin synthesis, in vitro and in vivo. We investigated AANAT activity, iCa2+, cAMP, cell viability and DNA fragmentation. AEME reduced melatonin synthesis in vivo and in vitro, and this effect was reversed by atropine. There was an increase in Ca2+, but not in cAMP or AANAT activity induced by AEME. Cell viability and DNA fragmentation were not affected by AEME. In conclusion, AEME reduced melatonin synthesis in vitro and in vivo, being this effect mediated by the muscarinic cholinergic system.

A farmacodependência é considerada uma doença crônica e sujeita à recaídas, na qual o indivíduo perde o controle sob a utilização de determinada droga de abuso. Conforme o usuário persiste com o uso da droga, ocorrem alterações anatômicas, fisiológicas e neuroquímicas no sistema nervoso central (SNC), as quais podem culminar no desenvolvimento de um comportamento compulsivo. A neurobiologia deste processo é complexa e envolve mecanismos de plasticidade em diferentes sistemas neurotransmissores. O principal deles é o sistema mesocorticolímbico dopaminérgico, constituído por neurônios da área ventral do tegmento mesencefálico (VTA) que se projetam para o núcleo accumbens (NAc) e ao córtex pré-frontal (CPF), diretamente relacionado aos processos motivação e recompensa. Contudo, o mesmo não é suficiente para elucidar a complexidade da doença, o que levou ao entendimento da presença de outros sistemas neurotransmissores neste processo. Sabe-se que o sistema colinérgico muscarínico está diretamente envolvido em diferentes doenças neuropsiquiátricas, incluindo a farmacodependência. Além disso, os receptores colinérgicos muscarínicos (mAChRs) estão densamente presentes em regiões límbicas, onde acetilcolina e dopamina interagem por neuromodulação. Diante disto, o objetivo deste trabalho foi investigar as possíveis alterações plásticas no sistema colinérgico muscarínico resultantes de tratamentos com cocaína que mimetizaram o consumo compulsivo humano. Para tanto, foram realizados ensaios comportamentais com camundongos Swiss machos adultos em campo aberto, tratados durante um (acute binge paradigm, 30 mg/kg) ou 14 dias (escalating dose binge paradigm, 15 - 30 mg/kg) com cocaína. Os animais receberam 3 injeções intraperitoneais (i.p.) de cocaína com intervalos de 60 minutos, durante os quais a atividade locomotora foi avaliada. Após a análise comportamental, os animais foram eutanasiados por decapitação para a remoção do encéfalo e dissecação do estriado, CPF e hipocampo, regiões cerebrais cruciais para o processo fisiopatológico da farmacodependência. Componentes do sistema dopaminérgico (receptores D1 e D2) e colinérgico muscarínico (M1-M5 mAChRs, ChAT, VAChT e AChE) foram avaliados por Immunoblotting. O sangue dos animais foi coletado para a realização das dosagens de cocaína e benzoeilecgonidina por UPLC-MS/MS. O desempenho locomotor total dos animais tratados com cocaína foi superior ao dos animais controle. O grupo tratado com escalonamento de dose desenvolveu sensibilização comportamental aos efeitos psicoestimulantes da cocaína no segundo dia de tratamento e, a partir dele, a atividade locomotora total manteve a mesma magnitude. Além disso, conforme o aumento da dose, os animais mantiveram um nível de atividade superior ao basal, mesmo após o término do experimento. As análises de Immunoblotting mostraram alterações dopaminérgicas e colinérgicas. No estriado observou-se redução da densidade de D2R após o tratamento de 14 dias e aumento na densidade de M3 mAChR após o tratamento agudo. Já no hipocampo observou-se redução de D1R e aumento de D2R, M1 e M5 mAChR após o tratamento crônico; e um aumento na densidade de M3 mAChR após o tratamento agudo. No CPF, foi evidenciada redução de M3 e de M5 mAChR após o tratamento cônico de 14 dias. Em relação às moléculas colinérgicas, observou-se, após o tratamento crônico, aumento da quantidade de ChAT em todas as estruturas estudadas. Além disso, VAChT mostrou-se aumentado no hipocampo após ambos os tratamentos. As dosagens plasmáticas revelaram a presença de 20,38 ± 3,4 ng/mL de cocaína e 224,6 ± 24,02 ng/mL de benzoilcgonina (BZE) nos animais do grupo agudo e, nos do grupo crônico, 62,26 ± 10,56 ng/mL e 375,1 ± 25,62 ng/mL de cocaína e BZE respectivamente. / Drug addiction is a chronic releapsing disorder characterized by the loss of control in limiting drug intake. As the drug use persists, anatomical, physiological and neurochemical changes occur in the central nervous system (CNS), which may lead to the development of compulsive behaviors. The neurobiology of this process is complex and involves mechanisms of plasticity in different neurotransmitter systems. The main one is the mesocorticolimbic dopaminergic system, composed by neurons from the ventral tegmental area (VTA) that projects to the nucleus accumbens (NAc), which is directly related to motivation and reward processes. However, just dopamine is not enough to elucidate the complexity of the disease, leading to the comprehension of another neurotransmitters system involved. It is known that the cholinergic system is involved in different neuropsychiatric disorders, including drug addiction. Furthermore, cholinergic muscarinic receptors (mAChRs) are densely present in limbic regions, where acetylcholine and dopamine interact by neuromodulation. Considering that, the aim of this study was to evaluate the existence of neuroadaptative changes in the cholinergic muscarinic system induced by cocaine in a compulsive-like behavior model in mice. Swiss-Webster adult male mice received 3 daily injections (i.p) of cocaine or saline, with a 60-min interval among them, either acutely (acute binge paradigm) or for 14 consecutive days (escalating dose binge paradigm). The locomotor activity was monitored in the open field during 60 min, in 5 min bins, after each injection. After behavioral analysis animals were euthanized by decapitation and the brain regions of striatum, hippocampus and prefrontal cortex, involved in the pathophysiology of addiction were dissected. Dopaminergic receptors (D1R and D2R), cholinergic muscarinic receptors (M1-M5 mAChRs), choline acetylytransferase (ChAT), acetylcholine vesicular transporter and acetylcholinesterase (AChE) were quantified by Immunoblotting. Blood samples were collected with heparin and plasma was separated and stored with 2% sodium fluorite at -80ºC for cocaine and benzoilecgonine quantification by UPLC-MS/MS. In the open field, animals treated with cocaine showed an increase in locomotor activity compared to control. Cocaine induced behavioral sensitization, in the escalating dose group on day 2, and after that the locomotor activity had the same magnitude until day 14th. These animals also kept the locomotor activity elevated even after the last injection. Immunobltting shows dopaminergic and cholinergic changes. An increase in M3 was observed in both hippocampus and striatum of animals acutely treated. After 14 days, there was an increase in M1, M5 and D2 and a decrease in D1 in hippocampus. There was also a decrease in D2 in the striatum; and finally, there was a decrease in M5 and M3 in the prefrontal cortex. ChAT densities were higher in all regions after the chronic treatment. Besides that, VAChT were higher in the hippocampus after both acute and chronic treatments. UPLC-MS/MS for cocaine and benzoilecgonine demonstrated the presence of 20,38 ± 3,4 ng/mL of cocaine and 224,6 ± 24,02 ng/mL of BZE in the acute binge group; and, 62,26 ± 10,56 ng/mL and 375,1 ± 25,62 ng/mL of cocaine and BZE, respectively in the escalating dose animals.

A sepse é caracterizada por um desequilíbrio entre a resposta pró- e anti-inflamatória às infecções. Um dos principais componentes da resposta do hospedeiro no choque séptico são as interações recíprocas entre o sistema imune e o sistema nervoso central, desta forma o objetivo deste estudo foi investigar o desenvolvimento de alterações neurológicas e sua associação com alterações imunológicas em fases iniciais e tardias após a sepse por ligação e perfuração cecal (CLP). Dividimos em três experimentos: agudo, crônico e efeito da ACh na evolução tardia da sepse. No experimento agudo utilizamos camundongos Balb/c, induzimos sepse por CLP em diferentes gravidades (leve, moderado e grave), 6 horas após o CLP foi realizado teste comportamental SHIRPA e logo após os animais foram sacrificados. No experimento crônico os camundongos Balb/c foram submetidos ao CLP leve, o SHIRPA foi realizado 6 horas e 15 dias após o CLP e os animais foram sacrificados 15 dias após o CLP. No experimento dos efeitos da ACh utilizamos camundongos Balb/c que receberam a droga donepezila (5 mg/kg/dia, oralmente) sete dias antes do CLP leve até o dia do sacrifício e os camundongos homozigotos mutantes VAChT KD também submetidos ao CLP leve. O teste comportamental SHIRPA foi realizado 6 horas após o CLP e os animais sacríficos 15 dias após o CLP. O plasma, o baço e o hipocampo foram removidos em todos os experimentos. Os níveis do S100? foram medidos no plasma. Os baços foram pesados, e por citometria de fluxo foi caracterizado os linfócitos (linfócitos T citotóxicos, linfócitos T auxiliares, linfócitos B, células T reguladoras e células Th17) e morte celular (Apoptose inicial, necrose e apoptose tardia). Os níveis de citocinas no baço, hipocampo e plasma foram determinados por ELISA. Nossos resultados mostram que no experimento agudo, 6 horas após o CLP a encefalopatia é diferente dependendo da gravidade da sepse, e o perfil de linfócitos no baço não é alterado por nenhuma gravidade da sepse. No entanto, a ativação de células do baço foi indicada no nosso estudo por variações na quantidade de citocinas no baço. No experimento crônico observamos que 15 dias após o CLP os animais apresentam encefalopatia séptica, e esta está correlacionada com a diferenciação e morte celular de linfócitos do baço, o que leva a um alto perfil imunossupressor. No experimento da ACh mostramos que a estimulação da transmissão colinérgica, utilizando donepezila, diminui a inflamação, por aumentar linfócitos, morte linfocitária e diminuir citocinas pró-inflamatória. E, ao contrário, a diminuição da transmissão colinérgica, experimento VAChT KD, observouse uma diminuição de linfócitos, sem morte celular e aumento da inflamação. Desta forma, concluímos que a alteração neurológica nos animais com sepse está associada com as alterações imunológicas tardias e que a ACh tem um importante papel no perfil imunológico 15 dias após o CLP / Sepsis is characterized by an imbalance between pro- and anti-inflammatory responses to infection. One of the main components of the host response in septic shock are the reciprocal interactions between the immune system and the central nervous system, so the aim of this study was to investigate the development of neurological disorders and their association with immunological changes in early and late stages after sepsis by cecal ligation and puncture (CLP). We divided in three experiments: acute, chronic and chronic ACh. In acute experiment we use Balb/c mice, induce sepsis by CLP in different severities (mild, moderate and severe), 6 hours after CLP was conducted behavioral test SHIRPA and after the animals were sacrificed. In the chronic experiment Balb/c mice were subjected to CLP mild, the SHIRPA was performed 6 hours and 15 days after CLP, and animals were sacrificed 15 days after CLP. In chronic ACh experiment use Balb/c mice that received the drug Donepezil (5 mg/kg/day, orally) seven days before the CLP mild until the day of sacrifice and use too mice homozygous mutants KD VAChT also submitted to CLP mild. The SHIRPA behavioral test was performed 6 hours after CLP and the animals were sacrificed 15 days after CLP. The plasma, spleen and hippocampus were removed in all experiments. The levels of S100? were measured in plasma. The spleens were weighed, and flow cytometry was characterized lymphocytes (cytotoxic T lymphocytes, helper T lymphocytes, B lymphocytes, regulatory T cells and Th17 cells) and cell death (apoptosis initial, necrosis and DNA fragmentation). Cytokine levels in the spleen, hippocampus and plasma were determined by ELISA. Our results show that in the acute experiment, 6 hours after CLP encephalopathy is different depending on the severity of sepsis, since the profile of the spleen lymphocytes is not changed by any severity of sepsis. However, the spleen cell activation was shown in this study by variations in the quantity of cytokines in the spleen. In the chronic experiment we observed that 15 days after CLP animals have septic encephalopathy, and this correlates with cell differentiation and the death of spleen lymphocytes, which leads to a high immunosuppressive profile. Since in the chronic ACh experiment have shown that stimulation of cholinergic transmission, using donepezil, reduces inflammation by increasing lymphocytes, lymphocyte death and decreasing proinflammatory cytokine. And, conversely, the reduction in cholinergic transmission, KD VAChT experiment, we observed a decrease of lymphocytes, and increase cell death without inflammation. Thus, we conclude that the neurological deficits in animals with sepsis is associated with immunological late changes and ACh plays an important role in the immune profile 15 days after CLP

O infarto do miocárdio (IM), uma das principais causas de morte nas sociedades industrializadas, é sempre acompanhado por uma notável alteração da modulação autonômica, caracterizada por hiperatividade simpática e diminuição do tono parassimpático ao coração. O bloqueio da atuação do simpático cardíaco tem sido amplamente utilizado como estratégia terapêutica eficaz para redução da morbi-mortalidade em pacientes com IM. Entretanto, há evidências de que o restabelecimento da função parassimpática ao coração pode ser igualmente benéfica, uma vez que a diminuição do parassimpático cardíaco é um fator de risco independente de morte súbita. Assim, o objetivo do presente estudo foi avaliar a influência da inibição da acetilconinesterase plasmática (AChE), por meio da administração endovenosa do brometo de piridostigmina (PIR), sobre o eletrocardiograma (ECG), hemodinâmica e modulação autonômica apos o IM agudo em ratos. Ratos foram anestesiados com uretana e mantidos a uma temperatura de 36-37 ºC. Tiveram eletrodos subcutâneos para registro do ECG implantados, e a artéria e veia femoral cateterizadas para medida direta de PA e administração de drogas, respectivamente. Experimentos preliminares foram realizados para determinação de uma dose de PIR que não causasse grande repercussão hemodinâmica. A atividade da acetilcolinesterase plasmática, bem como o tono autonômico cardíaco também foram avaliados em ratos normais. Em outro protocolo, ratos anestesiados, sob registro contínuo do ECG e PA, tiveram o ramo descendente anterior da artéria coronária esquerda ligado para provocar um extenso IM e, após 10 min, foram tratados com PIR (0,25 mg/kg, i.v) ou salina (solução fisiológica 0,9%), e os registros foram acompanhados por 4 h. Ratos controles tiveram o tórax aberto, mas a artéria coronária foi mantida intacta. Ao final, os ratos tiveram o coração retirado para avaliação da extensão da isquemia miocárdica e para o estudo da conexina 43. A administração endovenosa de PIR foi efetiva em reduzir a atividade da AChE e provocou uma discreta redução da FC (438±8 para 387±10 bpm), sem alteração da PA. Ratos tratados com PIR tiveram menor tono simpático e maior tono vagal cardíaco que os ratos que receberam salina. O tratamento com PIR diminuiu a incidência de arritmias nos animais com IM e aumentou a porcentagem de ratos que sobreviveram até a 4ª hora após o infarto (72 vs 58% nos não tratados). A PIR também preveniu o aumento do intervalo QTc, observado após o IM em ratos não tratados (=-2±4, vs 33±13 ms). A quantidade de conexina 43 foi marcadamente reduzida pelo IM em ratos não tratados (0,7±0,1 vs 2,2±0,4 ua), redução esta que não ocorreu nos ratos com IM tratados com PIR (1,3±0,3 ua). Por fim, foi realizado um ensaio, in vitro, em cardiomiócitos da linhagem H9c2 em cultura, e foi observado que a PIR preveniu a degradação da Cx43 induzida por meio isquêmico durante 4 horas. Portanto, a administração aguda de PIR provocou uma bradicardia pouco intensa, sem repercussões hemodinâmicas importantes, aumentou o tono vagal cardíaco, preveniu o prolongamento do intervalo QTc, diminuiu a incidência de arritmias, e preveniu a degradação da Cx43 nos corações dos ratos infartados. / Myocardial infarction (MI), a leading cause of death in industrialized societies, is always accompanied by a remarkable change in cardiovascular autonomic modulation, characterized by sympathetic overactivity and decreased vagal tone to the heart. The blockade of the cardiac sympathetic activity has been widely used as an effective therapeutic strategy to reduce morbidity and mortality in patients with MI. However, there is evidence that improvement of parasympathetic function can also be beneficial since reduction of cardiac vagal function is an independent risk factor for sudden death. The aim of this study was to evaluate the influence of inhibition of plasma acetilconinesterase (AChE), by intravenous administration of pyridostigmine bromide (PYR) on the electrocardiogram (ECG), hemodynamics and cardiovascular autonomic modulation shortly after MI in rats. Rats were anesthetized with urethane and maintained on a heating pad. Subcutaneous electrodes to record the ECG were installed and catheters were inserted into femoral artery and vein for measurement of blood pressure and drug administration, respectively. Preliminary experiments were performed to determine a dose of PYR that would not cause major hemodynamic consequences. Plasma AChE activity, and cardiac autonomic tone were also evaluated in normal rats. Then, anesthetized rats under continuous recording of ECG and BP, had the anterior descending branch of the left coronary artery ligated to elicit extensive MI and, after 10 min, were treated with PYR (0.25 mg/kg, iv) or saline (0.9% NaCl) and monitored for the next 4 h. Control rats had the chest open, but coronary artery was kept intact. At the end, the rats had the heart removed to determine the size of myocardial isquemia and to study connexin 43. Intravenous administration of PYR was effective in reducing AChE activity of and caused a mild reduction in HR (438±8 to 387±10 bpm) with no change in BP. Also, rats treated with PYR had lower sympathetic and higher cardiac vagal tone as compared to untreated rats. The treatment with PYR decreased the incidence of arrhythmias after MI and increased the percentage of rats that survived until the 4th hour after infarction (72 vs 58 % in untreated). PYR also prevented the increase in QTc interval observed after MI in untreated rats (=-2±4 vs 33±13 ms). The amount of connexin 43 was markedly reduced by MI in untreated rats (2.24±0.46 vs 0.72±0.14 au), nevertheless, this reduction was not observed in infarcted rats that received PYR (1.31±0.29 au). Finally, an in vitro assay was performed on the line H9c2 cardiomyocytes in culture, and it was observed that PYR prevented the degradation of Cx43 induced by ischemic medium for 4 hours. Therefore, the acute administration of PYR caused mild bradycardia without hemodynamic repercussions, increased vagal tone, prevented the prolongation of the QTc interval and decreased the incidence of arrhythmias and prevented the degradation of Cx43 in infarcted rat hearts.

La locomotion, comme toute autre forme d'activité physique, mobilise le système nerveux autonome pour faire face à la demande physiologique croissante. Ces réponses autonomes impliquent un couplage entre les activités motrices sympathiques et somatiques. De manière intéressante, à la fois les réseaux locomoteurs spinaux, ainsi que les neurones préganglionnaires sympathiques intermédiolatéraux (IMLs) sont les cibles d’une modulation par le système cholinergique propriospinal. Dans ce contexte, le but de mon travail doctoral a été d'étudier le rôle du système cholinergique propriospinal dans la coordination entre ces deux systèmes. En utilisant une préparation de moelle épinière de rat nouveau-né isolée in vitro, nous avons montré que l’acétylcholine pourrait permettre un couplage entre les réseaux locomoteurs et sympathiques via l’activation de récepteurs muscariniques. En effet, l'oxotrémorine, un agoniste non-sélectif de ces récepteurs, induit une activité rythmique lente bloquée par des antagonistes des récepteurs muscariniques M1, M2, M3 et M4. De plus, l’oxotrémorine permet de révéler des capacités rythmogènes endogènes de la moelle épinière thoracique. Nous avons observé que les motoneurones thoraciques étaient rythmiquement actifs à la fois durant des épisodes de locomotion fictive et lors de l’application d'oxotrémorine. A l’inverse, les IMLs présentaient une activité rythmique uniquement en présence d'oxotrémorine. Cette étude fournit ainsi de nouveaux éléments concernant les processus neuronaux à l'origine du couplage entre les systèmes somatiques et sympathiques. Nous proposons ici que ces mécanismes de synchronisation sont réalisés en partie via un réseau intraspinal pouvant être activé conditionnellement par le système cholinergique propriospinal. / Locomotion, as any other forms of physical activity, mobilizes the autonomic nervous system to match the increasing physiological demand. These autonomic responses mostly rely on the coupling between sympathetic and somatic motor activities. The propriospinal cholinergic system plays an important role in the control of locomotor networks, and several lines of evidences suggest that it may also activate sympathetic preganglionic neurons from the intermediolateral nucleus (IMLs). The aim of my doctoral thesis was to investigate the role of the cholinergic propriospinal system in the coordination between these two systems. Using the in vitro isolated spinal cord from new born rat, we showed that application of acetylcholine synchronized the locomotor and sympathetic networks, via the activation of muscarinic receptors. Indeed, the non-selective agonist oxotremorine induced slow rhythmic activity blocked by M1, M2, M3 and M4 muscarinic receptor antagonists. In addition, oxotremorine revealed endogenous rhythmogenic capabilities of the thoracic segments. This slow oscillatory activity propagated from thoracic ventral roots to lumbar ones, but not the reverse. We observed that thoracic MNs were rhythmically activated during both locomotorlike activity and oxotremorine-induced rhythm. In contrast, IMLs were rhythmically activated solely in the presence of oxotremorine. This study provides new light on the origin of the coupling between the somatic and the sympathetic systems. We propose that synchronizing mechanisms are achieved in part by an intraspinal network which may be activated under the control of the cholinergic propriospinal system.

Nematodes of the genus Ascaris are large gastrointestinal parasites. Ascaris lumbricoides infects ~1 billion people globally; causing malnutrition and general morbidity, and can block the gut or bile duct causing fatal complications. Ascaris suum is a parasite of pigs; in addition to its veterinary significance, it can occasionally be zoonotic, and is a good model of the human parasite. One of the main classes of drugs used to treat parasitic nematode infections are the cholinergic anthelmintics, such as levamisole and pyrantel, which act as agonists of nicotinic acetylcholine receptors at the nematode neuromuscular junction.

α7 nicotinic acetylcholine receptors (nAChR) are highly permeable to Ca2+ and are clinical targets for Alzheimer’s disease and schizophrenia. The aim of this work was to examine α7 nAChR-mediated Ca2+ signalling in neuronal cells using three different methods, and to evaluate the effects of the desensitizing agonist and prototypical smoking-cessation drug sazetidine-A on α7 nAChRs. Initial studies used 96-well plate assays with SH-SY5Y cells to characterize responses evoked by the α7 nAChR-selective agonist PNU-282987 and positive allosteric modulator PNU-120596. This was complemented by live-imaging of cortical cultures, where the compounds evoked robust Ca2+ responses from 12 % of cells. Co- application with Cd2+, ryanodine and xestospongin-C significantly inhibited these responses, suggesting the involvement of voltage-gated Ca2+ channels and Ca2+- induced Ca2+-release. CNQX and MK801 also significantly inhibited α7 nAChR mediated Ca2+ elevations, indicating a role for glutamate release. A high-content screening assay was developed to further examine these phenomena. Exploratory experiments using KCl, AMPA and NMDA validated a protocol that could be used to image Ca2+ elevations in large cell populations. Inconsistent responses to PNU-120596 and PNU2-282987 were also observed, reflecting the scarcity of α7 nAChRs in cortical cultures and the need for assay optimization. Combination with immunofluorescent labelling revealed α7 nAChR mediated Ca2+ elevations in a subpopulation of astrocytes and neurons, some of which were GABAergic. PNU-120596 potentiated the effects of sazetidine-A in SH-SY5Y cells (EC50 0.4 μM) eliciting responses in 14 % of cells in cortical cultures in a methyllycaconitine- sensitive manner, consistent with α7 nAChR activation. Pre-incubation with sazetidine-A concentration-dependently attenuated subsequent α7 nAChR-mediated responses in SH-SY5Y cells (IC50 476 nM) and cortical cultures, suggesting that α7 nAChRs could play a role in the behavioural effects of sazetidine-A. These comparative experiments enhance our understanding of α7 nAChR signalling and provide a new method to study them further.

Pneumonia is the second leading cause of disability-adjusted life-years lost worldwide and the eighth leading cause of death in the United States. Traumatic brain injury (TBI) patients have classically been considered immunosuppressed, but recent research reported that mild head trauma patients have reduced incidence of pneumonia compared to blunt trauma patients. Using our mild TBI model followed by bacterial pneumonia, we investigated the effect of neuronal signaling on innate immune function. To test whether any mild injury primes host immune responses to pneumonia, we generated a mild tail trauma (TT) model. mTBI mice showed protection from bacterial pneumonia while TT mice did not. Using an FDA-approved neurokinin-1 receptor (NK1R) antagonist, aprepitant, we confirmed our previous findings that substance P (SP) is a key mediator of enhanced resistance to pneumonia. Blocking NK1R showed that mTBI-induced release of SP augments pulmonary neutrophil recruitment and microbicidal activity to pulmonary bacterial pathogens. In TT mice, NK1R agonism enhanced the same neutrophil functions, further supporting the hypothesis. No differences were found between mTBI and TT neutrophils’ ability to phagocytose, generate oxidative burst, or acidify phagosomes. However, neutrophils from mTBI mice produced more neutrophil extracellular traps in response to bacterial challenge. These studies show that neurokinin signaling in our model contributes to enhanced bacterial clearance. Cholinergic anti-inflammatory pathway signaling though the α7 nicotinic acetylcholine receptor (α7 nAChR) is also a critical component of improved survival. Blockade of α7 nAChR abrogated the mTBI survival benefit. Mimicking cholinergic signaling using α7 nAChR agonist recapitulated the mTBI reduced pro-inflammatory cytokine production and improved survival. No physiologic differences emerged within 24h following pneumonia, but mTBI and α7 agonist treated mice had significantly lower TNFα in bronchoalveolar fluid, suggesting reduced injurious pulmonary inflammation. However, replacing early TNFα during pneumonia did not increase mortality. Western blot analysis showed downregulation of HMGB1 release in mTBI mice, suggesting that vagal cholinergic signaling reduces late mediators of organ damage. Our experiments show that mTBI enhances resistance to pneumonia by activating the vagus nerve signaling through neurokinin and cholinergic pathways. Translation of these findings could be innovative solutions to fighting or preventing infections.