Cell assemblies are considered to be physiological as well as functional units in the brain. A repetitive and stereotypical sequential activation of many neurons was observed, but the mechanisms underlying it are not well understood. Feedforward networks, such as synfire chains, with the pools of excitatory neurons unidirectionally connected and facilitating signal transmission in a cascade-like fashion were proposed to model such sequential activity. When embedded in a recurrent network, these were shown to destabilise the whole network’s activity, challenging the suitability of the model. Here, we investigate a feedforward chain of excitatory pools enriched by inhibitory pools that provide disynaptic feedforward inhibition. We show that when embedded in a recurrent network of spiking neurons, such an augmented chain is capable of robust signal propagation. We then investigate the influence of overlapping two chains on the signal transmission as well as the stability of the host network. While shared excitatory pools turn out to be detrimental to global stability, inhibitory overlap implicitly realises the motif of lateral inhibition, which, if moderate, maintains the stability but if substantial, it silences the whole network activity including the signal. Addition of a disinhibitory pathway along the chain proves to rescue the signal transmission by transforming a strong inhibitory wave into a disinhibitory one, which specifically guards the excitatory pools from receiving excessive inhibition and thereby allowing them to remain responsive to the forthcoming activation. Disinhibitory circuits not only improve the signal transmission, but can also control it via a gating mechanism. We demonstrate that by manipulating a firing threshold of the disinhibitory neurons, the signal transmission can be enabled or completely blocked. This mechanism corresponds to cholinergic modulation, which was shown to be signalled by volume as well as phasic transmission and variably target classes of neurons. Furthermore, we show that modulation of the feedforward inhibition circuit can promote generating spontaneous replay at the absence of external inputs. This mechanism, however, tends to also cause global instabilities. Overall, these results underscore the importance of inhibitory neuron populations in controlling signal propagation in cell assemblies as well as global stability. Specific inhibitory circuits, when controlled by neuromodulatory systems, can robustly guide or block the signals and invoke replay. This mounts to evidence that the population of interneurons is diverse and can be best categorised by neurons’ specific circuit functions as well as their responsiveness to neuromodulators.

A criptococose é uma infecção fúngica sistêmica, predominantemente oportunista, causada por leveduras encapsuladas do gênero Cryptococcus. As infecções por Cryptococcus neoformans são comuns em nível mundial, e as formas graves são observadas nos pacientes imunocomprometidos. A principal fonte de infecção da criptococose são fezes de pássaros (principalmente pombos) contaminadas com o fungo, sendo a inalação de basidiósporos a principal via de infecção. O estabelecimento e a propagação da infecção são altamente dependentes da imunidade do hospedeiro, sendo o sistema imune celular o mecanismo primário de defesa do organismo contra C. neoformans. Nos últimos anos têm-se descrito outros elementos como ativadores e moduladores da resposta imune, destacando o sistema purinérgico e o sistema colinérgico. Desta maneira, este estudo buscou avaliar a influência da criptococose experimental sobre a atividade do sistema purinérgico e colinérgico, através de três objetivos: (1) avaliar a atividade da ecto-difosfoidrolases (E-NTPDase) e ecto-adenosina deaminase (E-ADA) em linfócitos e soro; (2) atividade da acetilcolinesterase (AChE) no cérebro e linfócitos, e butirilcolinesterase (BChE) no soro; e (3) avaliar os níveis de purinas no soro. Os resultados da avaliação do sistema purinérgico demonstraram que a hidrólise do trifosfato de adenosina (ATP) e difosfato de adenosina (ADP) foram diminuídas, bem como a atividade da E-ADA também esteva diminuída. Em relação a dosagem das colinesterases, observou-se um aumento na atividade da AChE nos linfócitos e no cérebro, e diminuição da BChE. Na dosagem do nível de purinas no soro, verificou-se um aumento nos níveis de ATP e adenosina (ADO) no dia 20 pós-infecção (PI), aumento de ATP e diminuição da ADO, inosina e ácido úrico no dia 50 PI. A avaliação da atividade da E-NTPDase e E-ADA levou a conclusão de que seus comportamentos hidrolíticos seriam compensatórios enquanto a E-NTPDase teria uma ação pró-inflamatória a E-ADA teria uma ação anti-inflamatória, gerando mecanismo de proteção contra danos teciduais secundários, possivelmente gerados respostas exacerbadas à infecção por C. neoformans. Adicionalmente, os dados da atividade da AChE, em amostras correspondentes, comprovaram o estabelecimento de uma resposta pró-inflamatória, corroborando com a hipótese da necessidade de um mecanismo de modulação. Por fim, observou-se um aumento nos níveis extracelulares de ATP caracterizando uma resposta pró-inflamatória. Desta forma, foi possível observar que existe uma participação direta dos sistemas purinérgico e colinérgico na imunomodulação da criptococose experimental, contribuindo para a instalação de uma resposta imune celular adequada para combater a proliferação da levedura, e um mecanismo de redução de danos teciduais associados à resposta imune exacerbada. / Cryptococcosis is a systemic fungal infection predominantly opportunistic, caused by encapsulated yeast from Cryptococcus genus. Cryptococcus neoformans infections are common worldwide, and the severe forms are observed in immunocompromised patients. The main source of cryptococcosis infection are bird droppings (especially pigeons) contaminated with the fungus, and the inhalation of basidiospore is the main route of infection. The establishment and spread of infection are highly dependent of the host immunity, and the cellular immune system is the primary mechanism for defense against C. neoformans. In recent years it has been described other elements as activators and modulators of the immune response, highlighting the purinergic and the cholinergic system. Thus, this study aimed to evaluate the influence of experimental cryptococcosis on the activity of purinergic and cholinergic systems through three objectives: (1) to evaluate the activity of the ecto-diphosphohydrolases (E-NTPDase) and ecto-adenosine deaminase (E-ADA ) in lymphocytes and serum; (2) the activity of acetylcholinesterase (AchE) in the brain and lymphocytes, and butyrylcholinesterase (BChE) in serum; and (3) evaluate the serum levels of purines. The results of the evaluation in the purinergic system demonstrated that the hydrolysis of adenosine triphosphate (ATP) and adenosine diphosphate (ADP) was decreased as well as the E-ADA activity. For the dosage of the cholinesterase, there was an increase in AChE activity in lymphocytes and in the brain, and a decreased in BChE. The measurement of serum purine level demonstrate an increase in the levels of ATP and adenosine) on day 20 post-infection (PI), an increased in ATP and decreased in ADO, inosine and uric acid on day 50 PI. The assessment of the E-NTPDase and E-ADA activity led the conclusion that their hydrolytic behavior would be compensatory while the E-NTPDase would have a pro-inflammatory action, E-ADA would have an anti-inflammatory action, generating protective mechanism against secondary damage tissue, producing possibly exacerbated responses to C. neoformans infection. In addition, data of AChE activity in corresponding samples confirmed the establishment of a pro-inflammatory response, corroborating the hypothesis of the need for a modulation mechanism. Finally, there was an increase in extracellular levels of ATP featuring a pro-inflammatory response. In this way, it was observed a direct involvement of the purinergic and cholinergic systems in immunomodulation of experimental cryptococcosis, contributing to the installation of an immune cell response suitable to combat the proliferation of yeast, and a reduction mechanism of tissue damage associated with response immune exacerbated.

A criptococose é uma infecção fúngica sistêmica, predominantemente oportunista, causada por leveduras encapsuladas do gênero Cryptococcus. As infecções por Cryptococcus neoformans são comuns em nível mundial, e as formas graves são observadas nos pacientes imunocomprometidos. A principal fonte de infecção da criptococose são fezes de pássaros (principalmente pombos) contaminadas com o fungo, sendo a inalação de basidiósporos a principal via de infecção. O estabelecimento e a propagação da infecção são altamente dependentes da imunidade do hospedeiro, sendo o sistema imune celular o mecanismo primário de defesa do organismo contra C. neoformans. Nos últimos anos têm-se descrito outros elementos como ativadores e moduladores da resposta imune, destacando o sistema purinérgico e o sistema colinérgico. Desta maneira, este estudo buscou avaliar a influência da criptococose experimental sobre a atividade do sistema purinérgico e colinérgico, através de três objetivos: (1) avaliar a atividade da ecto-difosfoidrolases (E-NTPDase) e ecto-adenosina deaminase (E-ADA) em linfócitos e soro; (2) atividade da acetilcolinesterase (AChE) no cérebro e linfócitos, e butirilcolinesterase (BChE) no soro; e (3) avaliar os níveis de purinas no soro. Os resultados da avaliação do sistema purinérgico demonstraram que a hidrólise do trifosfato de adenosina (ATP) e difosfato de adenosina (ADP) foram diminuídas, bem como a atividade da E-ADA também esteva diminuída. Em relação a dosagem das colinesterases, observou-se um aumento na atividade da AChE nos linfócitos e no cérebro, e diminuição da BChE. Na dosagem do nível de purinas no soro, verificou-se um aumento nos níveis de ATP e adenosina (ADO) no dia 20 pós-infecção (PI), aumento de ATP e diminuição da ADO, inosina e ácido úrico no dia 50 PI. A avaliação da atividade da E-NTPDase e E-ADA levou a conclusão de que seus comportamentos hidrolíticos seriam compensatórios enquanto a E-NTPDase teria uma ação pró-inflamatória a E-ADA teria uma ação anti-inflamatória, gerando mecanismo de proteção contra danos teciduais secundários, possivelmente gerados respostas exacerbadas à infecção por C. neoformans. Adicionalmente, os dados da atividade da AChE, em amostras correspondentes, comprovaram o estabelecimento de uma resposta pró-inflamatória, corroborando com a hipótese da necessidade de um mecanismo de modulação. Por fim, observou-se um aumento nos níveis extracelulares de ATP caracterizando uma resposta pró-inflamatória. Desta forma, foi possível observar que existe uma participação direta dos sistemas purinérgico e colinérgico na imunomodulação da criptococose experimental, contribuindo para a instalação de uma resposta imune celular adequada para combater a proliferação da levedura, e um mecanismo de redução de danos teciduais associados à resposta imune exacerbada. / Cryptococcosis is a systemic fungal infection predominantly opportunistic, caused by encapsulated yeast from Cryptococcus genus. Cryptococcus neoformans infections are common worldwide, and the severe forms are observed in immunocompromised patients. The main source of cryptococcosis infection are bird droppings (especially pigeons) contaminated with the fungus, and the inhalation of basidiospore is the main route of infection. The establishment and spread of infection are highly dependent of the host immunity, and the cellular immune system is the primary mechanism for defense against C. neoformans. In recent years it has been described other elements as activators and modulators of the immune response, highlighting the purinergic and the cholinergic system. Thus, this study aimed to evaluate the influence of experimental cryptococcosis on the activity of purinergic and cholinergic systems through three objectives: (1) to evaluate the activity of the ecto-diphosphohydrolases (E-NTPDase) and ecto-adenosine deaminase (E-ADA ) in lymphocytes and serum; (2) the activity of acetylcholinesterase (AchE) in the brain and lymphocytes, and butyrylcholinesterase (BChE) in serum; and (3) evaluate the serum levels of purines. The results of the evaluation in the purinergic system demonstrated that the hydrolysis of adenosine triphosphate (ATP) and adenosine diphosphate (ADP) was decreased as well as the E-ADA activity. For the dosage of the cholinesterase, there was an increase in AChE activity in lymphocytes and in the brain, and a decreased in BChE. The measurement of serum purine level demonstrate an increase in the levels of ATP and adenosine) on day 20 post-infection (PI), an increased in ATP and decreased in ADO, inosine and uric acid on day 50 PI. The assessment of the E-NTPDase and E-ADA activity led the conclusion that their hydrolytic behavior would be compensatory while the E-NTPDase would have a pro-inflammatory action, E-ADA would have an anti-inflammatory action, generating protective mechanism against secondary damage tissue, producing possibly exacerbated responses to C. neoformans infection. In addition, data of AChE activity in corresponding samples confirmed the establishment of a pro-inflammatory response, corroborating the hypothesis of the need for a modulation mechanism. Finally, there was an increase in extracellular levels of ATP featuring a pro-inflammatory response. In this way, it was observed a direct involvement of the purinergic and cholinergic systems in immunomodulation of experimental cryptococcosis, contributing to the installation of an immune cell response suitable to combat the proliferation of yeast, and a reduction mechanism of tissue damage associated with response immune exacerbated.

O sistema colinérgico está sabidamente envolvido com processos cognitivos. Em trabalho recente mostramos que a infusão do peptídeo A promoveu neurodegeneração e redução da memória de ratos. O treino semanal dos animais em equipamento de esquiva ativa recuperou o desempenho na tarefa e aumentou a densidade de receptores nicotínicos α7 em áreas relacionadas à memória. No presente trabalho, o antagonismo de α7 com metilicaconitina (MLA), em camundongos, promoveu perda cognitiva, porém a recuperação com o treino foi parcial. A infusão conjunta de βA e MLA causou perda da memória, mas essa não foi revertida com o treino semanal. Os animais com MLA apresentaram aumento da atividade da acetilcolinesterase (AChE) e aumento de BDNF, que poderia ser relacionado à resiliência a injúrias. Porém, animais com A e MLA apresentaram aumento da atividade da AChE e redução de BDNF, sugerindo perda dos mecanismos de neuroproteção deflagrados por α7. Com isso, sugere-se que α7 tenha um papel determinante na recuperação da memória e resiliência tecidual, frente à neurodegeneração. / Cholinergic system plays an important role in cognitive processes. In a recent work we showed that infusion of Aβ promoted neurodegeneration and reduction of memory of rats. Week training of animals in active avoidance shuttle box recovered their performance and increased the density of α7 nicotinic receptors in brain areas related to memory. In the present work, infusion of the α7 antagonist methyllycaconitine (MLA), in mice, caused cognitive impairment, but memory recover with week training was partial. Infusion of βA together with MLA promoted memory loss, but this was not recovered with the week training. MLA infused mice presented increase in acetylcholinesterase (AChE) activity and increase in BDNF, which could be related to resilience to tissue injuries. However, animals infused with βA and MLA showed increase in AChE activity and reduction of BDNF, suggesting loss of neuroprotection mechanisms triggered by α7. It is suggested that α7 has a determinant role in memory recover and brain resilience, in neurodegenerative processes.

A formação da memória envolve aprendizagem, consolidação e recuperação de informações e seus processos envolvem circuitos neuronais do córtex, hipocampo e amígdala. A formação da memória de longa duração ocorre a partir da formação da LTP \'\'long term potentiation\'\' que é modulada por diversos sistemas de neurotransmissores, entre eles o sistema colinérgico. Sabe-se que a atividade física aumenta a produção de neurotrofinas levando à melhora de estados patológicos, aumenta a densidade de receptores nicotínicos alfa7 em células do hipocampo. Já foi relatado que o destreinamento por duas semanas leva à reversão dos efeitos benéficos cardiovasculares. Esse projeto avalia os efeitos do treinamento e do destreinamento físico em piscina, sobre a manutenção da memória de longa duração de camundongos fêmeas por determinação da densidade de receptores nicotínicos alfa7 e da neurotrofina BDNF,na região do hipocampo, que não demonstrou diferença significativa entre os grupos avaliados no trabalho. / The processes of memory formation involves learning, consolidation and retrieval of information and processes involving neuronal circuits in the cortex, hippocampus and amygdala. The formation of long term memory occurs through the formation of LTP - \"long term potentiation\" that is modulated by multiple neurotransmitter systems, including the cholinergic system. It is known that physical activity increases the production of neurotrophins leading to an improvement of pathological conditions, increases the density of nicotinic receptors on hippocampal cells alfa7. It has been reported that for two weeks detraining leads to the reversion of beneficial cardiovascular effects. This project evaluates the effects of physical training and detraining on swimming on the maintenance of long-term memory of female mice by determining the density of nicotinic receptors alfa7 and neurotrophin BDNF in hippocampus, than showed no significant differences among the groups at work.

A resposta imune pode ser regulada tanto pelo SNS quanto pelo SNP. Estudos recentes têm identificado uma via colinérgica anti-inflamatória entre as fibras eferentes do nervo vago e direcionadas ao sistema imune. Desta forma, tem-se postulado que esta conexão provê um controle neural da inflamação aguda de uma forma direta e reflexa, sendo então chamada de reflexo inflamatório. Assim, pareceu-nos relevante estudar as influências do SNP na função das DCs, o que foi feito na vigência de um processo inflamatório do tipo antígeno específico produzido por OVA. Para tanto utilizamos: o Betanecol (agonista muscarínico), a Atropina (antagonista muscarínico), a Anabasina (agonista nicotínico) e a Mecamilamina (antagonista nicotínico). No presente trabalho observou-se que: A Anabasina aumentou a porcentagem de células que expressam as moléculas co-estimulatórias B7.1 e B7.2 no baço de camundongos e diminuiu a produção de IL-12 no sobrenadante de co-cultura de células de baço, enquanto que o Betanecol não produziu qualquer efeito no fenótipo das DCs e na produção de citocinas; A Mecamilamina e a Atropina não foram capazes de alterar o fenótipo de DCs de baço e nem a produção de citocinas numa co-cultura de células de baço. A Anabasina, por sua vez: diminuiu a expressão das moléculas co-estimulatórias B7.1 e B7.2 nas DCs de linfonodo; diminuiu a expressão de MHC-II e aumentou a expressão das moléculas co-estimulatórias B7.1 e B7.2 em DCs de baço; diminuiu a expressão de IL-12 intracelular e aumentou a expressão de NF-B de DCs de cultura de hepatócitos; diminuiu os níveis séricos de TNF e MCP-1 e aumentou os níveis séricos de IL-6; diminuiu a resposta de 10 hipersensibilidade do tipo tardia; aumentou a expressão de MHC-II, diminuiu a expressão das moléculas co-estimulatórias B7.1 e B7.2; diminuiu a produção de IL-12 e aumentou a produção de IL-10 nas DCs de medula óssea; aumentou a expressão de mNAChRa7 de DCs maduras provenientes de medula óssea. Os dados obtidos deste trabalho sugerem que o sistema colinérgico diminua a apresentação de antígenos específicos por DCs, atuando de forma anti-inflamatória e produzindo um shift da resposta Th1 para Th2; sugerem, ainda, que estes achados relacionam-se à estimulação da subunidade 7 do receptor nicotínico, com consequente aumento de atividade das DCs e subsequente aumento da expressão de mNAChRa7 / Immune responses might be regulated by the SNS and by PNS. Recently, it was shown the existence of a cholinergic anti-inflammatory pathway that connects vagus nerve afferent/efferent fibers to immune system cells within some organs. These connections make possible a neural control of the inflammatory response both throught a direct and reflex mechanism; the so called inflammatory reflex. Therefore, we thougth that it would be relevant to study the influences of PNS on DCs function in an antigen specific inflammatory process induced by OVA. As pharmacological tools: Bethanechol (muscarinic agonist), Atropine (muscarinic antagonist), Anabasine (nicotinic agonist) and Mecamylamine (nicotinic antagonist) were used. We showed that anabasine increased the percentage of splenocytes expressing co-stimulatory molecules (B7.1 and B7.2) and decreased IL-12p40 production in co-cultured (adherent:non-adherent) splenocytes supernatant. Bethanechol had no effects on DCs phenotype and cytokines production whatsoever. Mecamylamine and atropine also had no effects on DCs phenotype and on cytokines production, as well. Anabasine: decreased co-stimulatory molecules (B7.1 and B7.2) expression on DCs present in lymph nodes.Anabasine also decreased MHC-II expression, while increased the co-stimulatory molecules (B7.1 and B7.2) expression on DCs present in the spleen.Additionally, anabasine decreased intracellular IL-12 expression, while increased NF-B expression in splenocytes culture. Interestingly, anabasine decreased both TNF and MCP-1 and increased IL-6 serum levels. Anabasine also decreased a delayed type hypersensitivity (DTH) response in OVA-sensitized mice. Moreover, Anabasine increased both MHC-II expression and 12 IL-10 productions, while decreased both co-stimulatory molecules (B7.1 and B7.2) expression and IL-12 production in bone marrow derived DCs. Finnally, anabasine increased mNAChRa7 expression in mature bone marrow derived DCs. Taken together, these data suggest that the cholinergic system decreases antigen-specific presentation by DCs, leading to an anti-inflammatory effect, which in turn induces to a shift from Th1 to Th2 responses. Moreover, our data strongly suggest that nicotinic receptor 7 subunit is involved with PNS activity

Submitted by Biblioteca da Faculdade de Farmácia (bff@ndc.uff.br) on 2017-03-24T17:09:38Z No. of bitstreams: 1 Cabo, Carolina Serra Jogaib [Dissertação, 2014].pdf: 1585469 bytes, checksum: c26025e0cb73856aeb3ee836ea718d62 (MD5) / Made available in DSpace on 2017-03-24T17:09:38Z (GMT). No. of bitstreams: 1 Cabo, Carolina Serra Jogaib [Dissertação, 2014].pdf: 1585469 bytes, checksum: c26025e0cb73856aeb3ee836ea718d62 (MD5) / Estudos sobre os efeitos do extrato aquoso da Eugenia punicifolia (EP) demonstram sua ação na neurotransmissão da junção neuromuscular. Entretanto, apesar de saber que o aumento da neurotransmissão colinérgica pode apresentar atividade neuroprotetora e atuar na plasticidade neuronal, não existem estudos sobre o efeito do extrato de EP em células do Sistema Nervoso Central. O objetivo deste trabalho foi estudar o efeito do extrato diclorometano de EP sobre células da retina de ratos neonatos in vitro no que tange à proliferação celular, modulação do fenótipo colinérgico e aos níveis de fator de crescimento do nervo (NGF), fator neurotrófico derivado do cérebro (BDNF) e da interleucina IL-4. Foram realizadas culturas primárias de células da retina de ratos neonatos da linhagem Lister Hooded de ambos os sexos, dia pós-natal 0-2. As culturas foram plaqueadas em placas de Petri pré-tratadas com poli-L-ornitina, na densidade de 1,0x105 cel/cm2, receberam meio 199 ou 1μg/mL do extrato diclorometano de Eugenia punicifolia (EP 1μg/mL) e foram mantidas por 48 horas a 37°C, em atmosfera de 95% de ar e 5% de CO2. O método bioquímico utilizado para análise da proliferação celular foi a incorporação de [3H]-timidina. Os níveis dos receptores muscarínicos, de neurotrofinas e citocinas foram determinados por Western Blot. Todos os dados são apresentados em relação à porcentagem do controle (100%). Os procedimentos experimentais foram aprovados pelo Comitê de Ética no Uso de Animais da UFF (Projeto nº 186/2012). Os resultados mostram que o tratamento das culturas com diferentes concentrações do extrato diclorometano de EP por 48h induziu aumento dependente da concentração na proliferação celular, sendo o aumento mais significativo observado na concentração de 1μg/mL, concentração que foi utilizada em todos os experimentos. Foi observada a redução nos níveis dos receptores muscarínicos M1 e M4, e nos níveis de transportador de acetilcolina associado à vesícula, aumento na expressão do receptor M3 e nenhuma alteração nos níveis do receptor M5. Observou-se, também, que o extrato diclorometano de EP aumenta os níveis de NGF e da interleucina-4, e diminui dos níveis de BDNF. Os resultados sugerem que o extrato diclorometano de EP exerce efeito proliferativo e de diferenciação através da alteração do fenótipo colinérgico da retina e da participação de fatores neurotróficos / Studies on the effects of the Eugenia punicifolia (EP) aqueous extract demonstrate its action on neurotransmission in the neuromuscular junction. However, despite knowing that increased cholinergic neurotransmission may have neuroprotective activity and act in neuronal plasticity, there are no studies on the effect of the extract of EP in cells of the Central Nervous System. The aim of this work was to study the effect of the dichloromethane extract of EP on retinal cells of neonatal rats in vitro with respect to cell proliferation, modulation of the cholinergic phenotype and levels of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and interleukin IL-4. Primary cell cultures of neonatal rat retina from Hooded Lister strain of both sexes, postnatal day 0-2 were performed. Cultures were plated on pre-treated Petri plates with poly-L- ornithine at a density of 1.0 x105 cel/cm2 received medium 199 or 1μg/mL of dichloromethane extract of E. punicifolia (EP 1μg/mL) and kept for 48 hours at 37°C in an atmosphere of 95% air and 5% CO2. The method used for biochemical analysis of cellular proliferation was the incorporation of [3H]-thymidine. The levels of muscarinic receptors, neurotrophins and cytokines were determined by Western blot. All data are presented in relation to the percentage of control (100%). The experimental procedures were approved by the Ethics Committee on Animal Use of UFF (Project nº 186/2012). The results show that treatment of cultures with different concentrations of the dichloromethane extract of EP for 48h induces an increase in cell proliferation, with the most significant increase observed with the concentration of 1μg/mL, concentration, which was used in all experiments. A reduction in levels of muscarinic receptors M1 and M4, and VAChT was observed, an increase in expression of the M3 receptor and no change in the levels of the M5 receptor were observed. Also was observed that the dichloromethane extract of EP increased NGF levels and interleukin-4, and decreases levels of BDNF. The results suggest that the dichloromethane extract of EP exerts a proliferative effect and differentiation by altering the cholinergic phenotype of the retina and the involvement of neurotrophic factors

Current smoking cessation guidelines recommend nicotine replacement therapy (NRT) to assist pregnant smokers to quit, but this is without strong evidence for effectiveness and safety. Nicotine, the main addictive component of tobacco, is known to exert physiological effects by binding to its receptor, the nicotinic acetylcholine receptor (nAChR). Recent studies have identified the presence of nAChRs in non-neuronal cells, and in macrophages, functional alteration upon stimulation with nicotine has been documented. To understand the impact of in utero nicotine exposure on various immune cell disposition and function, we designed preliminary studies using an in vivo model of P. aeruginosa infection. In this model, pregnant mice were exposed to nicotine and after weaning, offspring were infected intra-tracheally and humanely killed 5 days later. Nicotine-exposed mice had a greater weight reduction post-infection. This was accompanied by a decreased number of neutrophil, resident macrophages, and B lymphocytes in the lungs, while the number of B lymphocytes in the lymph nodes were greater than that of the control group. In the lung lavage fluids, IL-6, MCP-1, and TNFα concentrations were elevated in nicotine-exposed mice. In an in vitro system using bone marrow-derived macrophages, a significantly reduced production of IFNγ was observed in nicotine-exposed mice when cells were stimulated with LPS. To characterize and compare gene expression in macrophages isolated from neonates developmentally exposed to nicotine, we designed a clinical study to recruit pregnant mothers who 1) did not smoke during pregnancy, 2) smoked throughout pregnancy, or 3) used NRT during pregnancy. We found that successful RNA isolation can be achieved from neonatal tracheal aspirate samples and cell number and reagent volumes were important determinants of acceptable RNA quality and quantity. Together, these preliminary findings demonstrate a possible alteration in immune response as a result of in utero nicotine exposure and sets a groundwork for future studies in identifying mechanisms underlying the impact of developmental nicotine exposure.

Up to now, solid organ transplantation remains the ultimate life-saving treatment for end-stage organ failure. However, transplantation could be complicated by allograft rejection wherein inflammation plays a pivotal role. In this process, inflammation secondary to ischemia/reperfusion and cell necrosis plays the role of adjuvant and enhances the antigen-specific adaptive response ultimately leading to allograft rejection. Therefore, anti-inflammatory strategies have to be developed to dampen inflammation and secondary alloreactivity. Recently, neuroimmune pathways and particularly the cholinergic anti-inflammatory pathway have been described to modulate inflammation in several experimental models as sepsis. The Vagus Nerve, the α7 nicotinic receptor (α7nAChR) and its agonists are specific targets to regulate the inflammatory response in several pathologies.The aim of this work is to investigate the potential protective effect of the cholinergic pathway in solid organ transplantation. In a model of renal ischemia/reperfusion injury induced by bilateral clamping of renal arteries, nicotine protects from renal dysfunction and tubular damages. This protection is associated to a reduction of inflammatory cytokines and neutrophils and is α7nAChR dependent. In a second part, we test the effect of the α7nACh receptor in a model of minor antigen mismatched skin allograft. Mice deficient for the α7nAChR reject earlier the skin allograft compared to α7nAChR +/+ mice and this is associated to higher Th1 and Th17 T cell responses. α7nAChR expressed on T cells is involved in skin allograft rejection as attested by adoptive transfer experiments in Rag H/H mice with either α7nAChR +/+ or α7nAChR H/H alloreactive T cells. The cholinergic pathway by itself or boosted by nicotinic agonists is able to modulate innate as well as acquired immune components involved in allograft rejection. Other agonists or devices used to stimulate the cholinergic pathway are actually developed in order to be more specific and to reduce toxicity. Our results are particularly relevant in human medicine as grafted organs lose their Vagus Nerve endings and their cholinergic regulatory innervation. / Doctorat en Sciences médicales (Médecine) / info:eu-repo/semantics/nonPublished

Midbrain dopamine (DA) neurons play a key role in a wide range of behaviours, from motor control, motivation, reward and reinforcement learning. Disorders of midbrain dopaminergic signaling is involved in a variety of nervous system disorders including Parkinson’s disease, schizophrenia and drug addiction. Understanding the basis of how dopaminergic neuronal activity in the substantia nigra pars compacta (SNc) governs movements, requires a deep appreciation of how afferent inputs of various neurotransmitter systems create a neuronal circuit that precisely modulates DA neuronal excitability. Two brainstem cholinergic neuclei, the laterodorsal tegmental nucleus (LDT) and the pedunculopontine tegmental nucleus (PPT), have major cholinergic projections to the SNc, despite the fact that the precise mechanisms of cholinergic modulation of DA neuronal activity mediated by nAChRs remain unclear. To dissect out the modulatory roles of the cholinergic system in regulating DAergic neuronal activity in the SNc and locomotion, we employed optogenetics along with electrophysiological and behavioural approaches. My results from whole-cell recordings from lateral and medial SNc DA neurons revealed that lateral DA neurons received predominantly excitatory nAChR mediated cholinergic neurotransmission (monosynaptic nicotinic or disynaptic glutamatergic responses) resulting in greater excitability of DA neurons both at 5 and 15 Hz blue LED light stimulation of cholinergic terminals. However, medial SNc DA neurons received predominantly biphasic current responses that were both inhibitory GABAergic and excitatory nAChR mediated cholinergic neurotransmission. This led to a net inhibition of action potential firing of DA neurons at 5 Hz blue LED light stimulation of cholinergic terminals, while at 15 Hz stimulation there was an initial inhibition followed by a significant increase of the baseline action potential firing frequency. Furthermore, in vivo optogenetic experiments showed that activation of the cholinergic system in the medial SNc resulted in decreased locomotion, while for the lateral SNc led to increased locomotion. Together our findings provide new insights into the role of the cholinergic system in modulating DA neurons in the SNc. The cholinergic inputs to different subregions of the SNc may regulate the excitability of the DA neurons differentially within a tight range from excitation to inhibition which may translate into different kinds of locomotor behaviour. / Graduate