Introdução: A inflamação constitui um dos fatores mais importantes da fisiopatologia da asma brônquica, caracterizada por uma resposta eosinofílica com produção de citocinas de perfil Th2. A persistência desta inflamação induz no pulmão um processo de reparo associado à redução progressiva da função pulmonar, que nem sempre é revertida pelos tratamentos disponíveis. O sistema colinérgico anti-inflamatório é descrito como um mecanismo neural que suprime a resposta imune e controla a inflamação principalmente pelo efeito da acetilcolina em receptores nicotínicos do tipo alfa7 (alfa7nAChR) encontrados em células do sistema imune. A acetilcolina (ACh) exerce um importante efeito na asma e recentemente demonstramos que a redução parcial da liberação da acetilcolina induz per se a inflamação pulmonar. Embora se saiba que os receptores muscarínicos (mAChRs) exercem um efeito pró-inflamatório e broncoconstritor na asma, a ativação de receptores nicotínicos (nAChRs) poderia ter um efeito benéfico reduzindo a inflamação pulmonar, fato demonstrado em modelos de inflamação sistêmica e aguda. O efeito da ativação do alfa7nAChR na fisiopatologia da asma ainda não está claramente elucidado. Objetivo: Investigar o efeito do tratamento com PNU-282987 (PNU), um agonista específico do alfa7nAChR, em um modelo murino de inflamação alérgica crônica das vias aéreas. Metodologia: Camundongos BALB/c foram submetidos ao protocolo de indução alérgica crônica das vias aéreas com ovoalbumina (OVA) ou salina intraperitoneal (i.p.) e posterior desafios inalatórios. A partir do 22° dia, os animais receberam diariamente tratamento com PNU ou veículo (Ve) até o 28° dia. Foram testadas três doses de PNU (0,5, 1,0 e 2,0 mg/Kg). A fim de evidenciar se o efeito obtido no tratamento com PNU era dependente do receptor alfa7nAChR, um grupo de animais foi tratado com MLA (antagonista do alfa7nAChR), previamente ao tratamento com PNU. No 29° dia do protocolo, os animais foram eutanasiados e foram avaliados o número de células inflamatórias no lavado broncoalveolar (LBA) e no sangue, os níveis de citocinas no LBA, a expressão do alfa7nAChR e mAchRs do tipo 3 (M3) e a ativação do fator de transcrição nuclear kB (NF-kB) no pulmão. O remodelamento brônquico foi avaliado por morfometria. As análises estatísticas foram realizadas por meio do programa SigmaStat (Jandel Scientific, San Rafael, CA), onde um P < 0,05 foi considerado estatisticamente significativo. Resultados: Houve expressão do alfa7nAChR e M3 no homogenato de pulmão de animais controle e sensibilizados. Determinamos por meio da redução de eosinófilos que a dose de 0,5 mg/Kg do tratamento com PNU foi a mais efetiva. Assim, observamos que o tratamento com PNU0,5 nos animais sensibilizados reduziu o número de células totais, eosinófilos, neutrófilos, macrófagos e linfócitos no LBA, assim como número de eosinófilos no sangue periférico e ao redor das vias aéreas. O tratamento com PNU reduziu os níveis de IgE no sangue e as citocinas IL-4, IL-13 e IL-17 no LBA. Todos estes efeitos foram revertido com o pré-tratamento com MLA, exceto para a citocina IL-17. Alem disso, o tratamento com PNU reduziu o remodelamento brônquico (área de edema, de epitélio e de músculo liso e deposição de fibras colágenas) assim como o número de células positivas para MMP-9 e TIMP-1 ao redor das vias aéreas. No pulmão a expressão do p-65-NF-kB, STAT3 fosforilado e o SOCS3 foram inibidas pelo PNU. Conclusão: Estes dados claramente demonstram que o alfa7nAChR está envolvido no controle da resposta inflamatória pulmonar alérgica e de remodelamento brônquico em modelo experimental de asma alérgica e portanto é um novo alvo com potencial terapêutico a ser explorado na fisiopatologia da asma brônquica / Background: Inflammation is one of the most important features in asthma pathophysiology, characterized by eosinophilic response with production of Th2 cytokine profile. The persistence of this inflammation can induce a lung repair process associated with a progressive reduction in lung function, which is not always reversed by available treatments. The anti-inflammatory cholinergic system was described as a neural mechanism that suppresses the immune response and controls inflammation mainly by the activaction of acetylcholine alfa7 nicotinic receptors (alfa7nAChR) found on immune cells. Acetylcholine (ACh) is an important mediator in asthma and we recently demonstrated that partial reduction on ACh release induced lung inflammation per se. Although it is known that muscarinic receptors (mAChRs) has a pro-inflammatory action and causes bronchoconstriction in asthma, the activation of nicotinic receptors (nAChRs) could have a beneficial effect reducing pulmonary inflammation as demonstrated in models of acute and systemic inflammation. The effects of alfa7nAChR activation in the pathophysiology of asthma have not been clearly elucidated. Aim: To investigate the effects of PNU- 282987 (PNU) treatment, a specific alfa7nAChR agonist, in a murine model of chronic allergic airway inflammation. Methods: BALB/c mice were subjected to a protocol of chronic allergic inflammation induced by intraperitoneal ovalbumin (OVA) or saline and subsequent challenges with inhalation. From the 22th day, the animals daily received PNU or vehicle (Ve) until the 28th day. PNU were tested in three differents doses (0.5, 1.0 and 2.0 mg/kg). In order to demonstrate that the effects obtained by PNU treatment was dependent on alfa7nAChR, a group of animals was treated with MLA (antagonist of alfa7nAChR) prior to the PNU treatment. On the 29th day of the protocol, the animals were euthanised and the number of inflammatory cells in the bronchoalveolar lavage fluid(BALF) fluid and blood, cytokine levels in BALF, the expression of alfa7nAChR and mAChRs type 3 (M3), and activation of nuclear transcription factor kB (NF-kB) in the lung were evaluated. Bronchial remodeling was assessed by morfometric methods. Statistical analyses were performed using the SigmaStat (Jandel Scientific, San Rafael, CA) and P < 0.05 is considered statistically significant. Results: ?7nAChR and M3 expression was detected in control and sensitized lung homogenate. The most effective dose of PNU treatment was 0.5 mg/kg evaluated by the effects on eosinophil reduction. Thus, we observed that treatment with PNU0,5 reduced the number of total cells, eosinophils, neutrophils, macrophages and lymphocytes in BALF, as well as number of eosinophils in peripheral blood and around the airways of sensitized animals. The treatment with PNU also reduced IgE levels in the blood, and cytokines IL-4, IL-13 and IL-17 in BALF. All these effects were reversed by pretreatment with MLA, except for IL-17 cytokine. Furthermore, treatment with PNU reduced bronchial remodeling (edema, epithelium and smooth muscle area and airway collagen deposition) as well as the number of positive cells for MMP-9 and TIMP-1 around the airways. The lung p-65-NF-kB, phosphorylated STAT3 and the SOCS3 expression were inhibited by PNU-282987. Conclusion: These data clearly demonstrate that the alfa7nAChR is involved in the control of allergic pulmonary inflammatory response and in bronchial remodeling in an experimental model of allergic asthma and it can be a new target with therapeutic potential to be explored in the pathophysiology of asthma

Introdução: A inflamação constitui um dos fatores mais importantes da fisiopatologia da asma brônquica, caracterizada por uma resposta eosinofílica com produção de citocinas de perfil Th2. A persistência desta inflamação induz no pulmão um processo de reparo associado à redução progressiva da função pulmonar, que nem sempre é revertida pelos tratamentos disponíveis. O sistema colinérgico anti-inflamatório é descrito como um mecanismo neural que suprime a resposta imune e controla a inflamação principalmente pelo efeito da acetilcolina em receptores nicotínicos do tipo alfa7 (alfa7nAChR) encontrados em células do sistema imune. A acetilcolina (ACh) exerce um importante efeito na asma e recentemente demonstramos que a redução parcial da liberação da acetilcolina induz per se a inflamação pulmonar. Embora se saiba que os receptores muscarínicos (mAChRs) exercem um efeito pró-inflamatório e broncoconstritor na asma, a ativação de receptores nicotínicos (nAChRs) poderia ter um efeito benéfico reduzindo a inflamação pulmonar, fato demonstrado em modelos de inflamação sistêmica e aguda. O efeito da ativação do alfa7nAChR na fisiopatologia da asma ainda não está claramente elucidado. Objetivo: Investigar o efeito do tratamento com PNU-282987 (PNU), um agonista específico do alfa7nAChR, em um modelo murino de inflamação alérgica crônica das vias aéreas. Metodologia: Camundongos BALB/c foram submetidos ao protocolo de indução alérgica crônica das vias aéreas com ovoalbumina (OVA) ou salina intraperitoneal (i.p.) e posterior desafios inalatórios. A partir do 22° dia, os animais receberam diariamente tratamento com PNU ou veículo (Ve) até o 28° dia. Foram testadas três doses de PNU (0,5, 1,0 e 2,0 mg/Kg). A fim de evidenciar se o efeito obtido no tratamento com PNU era dependente do receptor alfa7nAChR, um grupo de animais foi tratado com MLA (antagonista do alfa7nAChR), previamente ao tratamento com PNU. No 29° dia do protocolo, os animais foram eutanasiados e foram avaliados o número de células inflamatórias no lavado broncoalveolar (LBA) e no sangue, os níveis de citocinas no LBA, a expressão do alfa7nAChR e mAchRs do tipo 3 (M3) e a ativação do fator de transcrição nuclear kB (NF-kB) no pulmão. O remodelamento brônquico foi avaliado por morfometria. As análises estatísticas foram realizadas por meio do programa SigmaStat (Jandel Scientific, San Rafael, CA), onde um P < 0,05 foi considerado estatisticamente significativo. Resultados: Houve expressão do alfa7nAChR e M3 no homogenato de pulmão de animais controle e sensibilizados. Determinamos por meio da redução de eosinófilos que a dose de 0,5 mg/Kg do tratamento com PNU foi a mais efetiva. Assim, observamos que o tratamento com PNU0,5 nos animais sensibilizados reduziu o número de células totais, eosinófilos, neutrófilos, macrófagos e linfócitos no LBA, assim como número de eosinófilos no sangue periférico e ao redor das vias aéreas. O tratamento com PNU reduziu os níveis de IgE no sangue e as citocinas IL-4, IL-13 e IL-17 no LBA. Todos estes efeitos foram revertido com o pré-tratamento com MLA, exceto para a citocina IL-17. Alem disso, o tratamento com PNU reduziu o remodelamento brônquico (área de edema, de epitélio e de músculo liso e deposição de fibras colágenas) assim como o número de células positivas para MMP-9 e TIMP-1 ao redor das vias aéreas. No pulmão a expressão do p-65-NF-kB, STAT3 fosforilado e o SOCS3 foram inibidas pelo PNU. Conclusão: Estes dados claramente demonstram que o alfa7nAChR está envolvido no controle da resposta inflamatória pulmonar alérgica e de remodelamento brônquico em modelo experimental de asma alérgica e portanto é um novo alvo com potencial terapêutico a ser explorado na fisiopatologia da asma brônquica / Background: Inflammation is one of the most important features in asthma pathophysiology, characterized by eosinophilic response with production of Th2 cytokine profile. The persistence of this inflammation can induce a lung repair process associated with a progressive reduction in lung function, which is not always reversed by available treatments. The anti-inflammatory cholinergic system was described as a neural mechanism that suppresses the immune response and controls inflammation mainly by the activaction of acetylcholine alfa7 nicotinic receptors (alfa7nAChR) found on immune cells. Acetylcholine (ACh) is an important mediator in asthma and we recently demonstrated that partial reduction on ACh release induced lung inflammation per se. Although it is known that muscarinic receptors (mAChRs) has a pro-inflammatory action and causes bronchoconstriction in asthma, the activation of nicotinic receptors (nAChRs) could have a beneficial effect reducing pulmonary inflammation as demonstrated in models of acute and systemic inflammation. The effects of alfa7nAChR activation in the pathophysiology of asthma have not been clearly elucidated. Aim: To investigate the effects of PNU- 282987 (PNU) treatment, a specific alfa7nAChR agonist, in a murine model of chronic allergic airway inflammation. Methods: BALB/c mice were subjected to a protocol of chronic allergic inflammation induced by intraperitoneal ovalbumin (OVA) or saline and subsequent challenges with inhalation. From the 22th day, the animals daily received PNU or vehicle (Ve) until the 28th day. PNU were tested in three differents doses (0.5, 1.0 and 2.0 mg/kg). In order to demonstrate that the effects obtained by PNU treatment was dependent on alfa7nAChR, a group of animals was treated with MLA (antagonist of alfa7nAChR) prior to the PNU treatment. On the 29th day of the protocol, the animals were euthanised and the number of inflammatory cells in the bronchoalveolar lavage fluid(BALF) fluid and blood, cytokine levels in BALF, the expression of alfa7nAChR and mAChRs type 3 (M3), and activation of nuclear transcription factor kB (NF-kB) in the lung were evaluated. Bronchial remodeling was assessed by morfometric methods. Statistical analyses were performed using the SigmaStat (Jandel Scientific, San Rafael, CA) and P < 0.05 is considered statistically significant. Results: ?7nAChR and M3 expression was detected in control and sensitized lung homogenate. The most effective dose of PNU treatment was 0.5 mg/kg evaluated by the effects on eosinophil reduction. Thus, we observed that treatment with PNU0,5 reduced the number of total cells, eosinophils, neutrophils, macrophages and lymphocytes in BALF, as well as number of eosinophils in peripheral blood and around the airways of sensitized animals. The treatment with PNU also reduced IgE levels in the blood, and cytokines IL-4, IL-13 and IL-17 in BALF. All these effects were reversed by pretreatment with MLA, except for IL-17 cytokine. Furthermore, treatment with PNU reduced bronchial remodeling (edema, epithelium and smooth muscle area and airway collagen deposition) as well as the number of positive cells for MMP-9 and TIMP-1 around the airways. The lung p-65-NF-kB, phosphorylated STAT3 and the SOCS3 expression were inhibited by PNU-282987. Conclusion: These data clearly demonstrate that the alfa7nAChR is involved in the control of allergic pulmonary inflammatory response and in bronchial remodeling in an experimental model of allergic asthma and it can be a new target with therapeutic potential to be explored in the pathophysiology of asthma

Substance use disorders are serious health concerns in the United States. Furthermore, the National Survey on Drug Use and Health reports a continuous increase in substance use disorders in the United States during the last 10 years. However, there are not many effective pharmacotherapeutics available for substance use disorders. The current dissertation is focused on research aimed at discovering pharmacotherapeutics for substance use disorders. First part of dissertation focused on discovering methamphetamine (METH) use disorder therapeutics targeting specific mechanism of METH action on dopaminergic neurons. The second part of dissertation focused on opioids and cocaine use disorder therapeutics targeting rewarding pathway commonly activated by opioids and cocaine. With respect to METH, it induces release of dopamine (DA) in neuronal terminals by interacting with the vesicular monoamine transporter-2 (VMAT2) and DA transporter (DAT). VMAT2 inhibitors have been found by our research group to decrease METH-evoked DA release, METH-induced hyperlocomotion, and METH self-administration in rats. However, these VMAT2 inhibitors lacked selectivity and tolerance developed to these pharmacologic effects after repeated administration, thereby limiting their potential as pharmacotherapeutics for METH use disorders. In the current study, analogs from a novel scaffold were found to selectively inhibit VMAT2 and were evaluated using neurochemical and behavioral pharmacological approaches. R- and S-3-(4-methoxyphenyl)-N-(1-phenylpropan-2-yl)propan-1-amine (GZ-11610 and GZ-11608, respectively) exhibited 94- to 3450-fold selectivity for VMAT2 over human-ether-a-go-go (hERG) channel, DAT, serotonin transporter, and nicotinic acetylcholine receptors. GZ-11608 competitively and concentration-dependently inhibited METH-evoked DA release via VMAT2. Also, GZ-11610 (56-300 mg/kg, oral) and GZ-11608 (300 mg/kg, oral; 10-30 mg/kg, s.c.) reduced METH-induced hyperlocomotor activity in METH-sensitized rats. Furthermore, GZ-11608 (1-30 mg/kg, s.c.) inhibited METH self-administration, cue- and METH-induced reinstatement in a dose-dependent manner, and 30 mg/kg (s.c.), 10 mg/kg (s.c.), and 17 mg/kg (s.c.) produced significant effect, respectively. Importantly, the GZ-11608-induced decrease in METH self-administration was not surmounted by increasing the amount of METH available. GZ-11608 did not substitute for METH and did not serve as a reinforcer in rats self-administering METH and drug naïve rats, respectively. Thus, these VMAT2 inhibitors incorporating a new scaffold are novel leads for new pharmacotherapeutics to treat METH use disorders. Substances with high abuse potential including opioids and cocaine elevate extracellular DA concentration in the nucleus accumbens, and this mechanism has long been considered to underly substance-induced reward. DA in the nucleus accumbens originates from DA neuron cell bodies located in the ventral tegmental area in the midbrain. Interestingly, M5 muscarinic acetylcholine receptors (mAChRs) are proteins that are highly expressed on ventral tegmental area DA neurons. Also, studies investigating M5 mAChRs knockout mice showed reduced responding for cocaine in cocaine self-administration and decreased time spent in cocaine-paired and morphine-paired place preference studies. Pharmacological inhibition of M5 mAChRs function via microinfusing mAChR antagonists exhibiting no selectivity among M1-M5 mAChRs subtypes into the ventral tegmental area where expression of M5 mAChRs are dominant, reduced morphine-induced hyperlocomotion and cocaine seeking behaviors in rats. These studies support therapeutic potential of M5 mAChRs selectivity antagonists in opioids and cocaine use disorders. Thus, in the current study, affinity of a series of pethidine and quinuclidinyl N-phenylcarbamate analogs for M5 mAChRs was evaluated using in vitro and ex vivo neuropharmacological assays. Among the pethidine analogs, compound 6a showed the highest binding affinity at M5 (Ki = 0.38 µM), but also high affinity at M1 and M3 mAChRs (0.67 and 0.37 µM, respectively). Among the quinuclidinyl N-phenylcarbamate analogs, compound 13c exhibited the highest affinity at M5 (Ki = 1.8 nM), but also high affinity at M1, M2, M3 and M4 mAChRs (Ki = 1.6, 13, 2.6, 2.2 nM, respectively). Also, 13c acted as an agonist of mAChRs on oxotremorine-induced DA release from rat striatal slices. In addition, compound 13b was found exhibiting the highest selectivity (17-fold) at M3 over M2 mAChRs, suggesting potential of 13b as a chronic obstructive pulmonary disease therapeutics. Taken together, these novel analogs serve as leads for further discovery of subtype-selective M5 mAChR antagonists that may have potential as therapeutics for substance use disorders, as well as for chronic obstructive pulmonary disease.

<p>The use of positron-emitting tracers has been extended beyond tomographic facilities in the last few years, giving rise to a general positron-emitting tracing technique. The methodological part of the present thesis involved the evaluation of the performance of storage phosphor (SP) plates, with tracers labeled with high-energy, short-lived, positron-emitting radionuclides, using homogenized tissue specimens and autoradiography with frozen brain sections. The SP plates showed superior sensitivity and a linear response over a wide radioactivity range. Autoradioradiography provided reliable results due to (a) adequate sensitivity for low radioactivity concentration, b) an excellent linear range, and (c) satisfactory resolution. Though equilibration time of receptor-ligand interaction was dependent upon section thickness, quantification was possib with thinner sections.</p><p>An initial finding using frozen section autoradiography of rat brain and spinal cord showed preferential binding of [¹¹C]4-NMPB, a muscarinic acetylcholine (mACh) receptor antagonist, to the M4 subtype of mACh receptors. Further work to ascertain this specificity, by use of binding studies on cell membranes from CHO-K1 cells expressing individual subtypes of human mACh receptors, suggested lack of subtype selectivity. With respect to the possible cliinical use in glutamatergic neuropathology, [¹¹C]cyano-dizocilpine, as a potential PET tracer for the N-methyl-D-aspartate (NMDA) subtype of glutamate receptors, was studied. The <i>in vivo</i> visualization of specific binding could not be achieved, though <i>in vitro</i> binding demonstrated good specificity and preferential binding to the activated for of the NMDA receptors.</p><p>The use of the glucose analogue [¹⁸F]fluorodeoxyglucose (FDG) to study glucose utilization was evaluated in experimental traumatic brain injury (TBI). A trauma-induced increased uptake of FDG was seen, whereas the uptake of [1-¹⁴C]glucose remained unchanged. This discrepancy might be due to the increased postraumatic affinity of FDG for the endothelial glucose transporter proteins and/or to the hexokinase enzyme. [¹¹C]Cyano-dizocilpine, [¹¹C]4-NMPB, and [¹¹C]flumazenil were utilized in autoradiography to evaluate changes in NMDA, mACh, and GABA_A receptors, espectively, in experimental TBI. Observations showed a global decrease in the binding potential BP) of (i) [¹¹C]cyano-dizocilpine acutely and 12 hrs after TBI, and (ii) of [¹¹C]4-NMPB at 12 hrs after TBI, and (iii) a decrease in the BP of [¹¹C]flumazenil in the cortex and hippocampus ipsilateral to the site of injury. The demonstrated changes in receptor binding after TBI are indicative of a widely dissipated effect of TBI on the particular neurotransmitter receptor systems as compared with what would be expected from FDG studies after TBI, i.e., a local disturbed neurotransmission.</p>

The use of positron-emitting tracers has been extended beyond tomographic facilities in the last few years, giving rise to a general positron-emitting tracing technique. The methodological part of the present thesis involved the evaluation of the performance of storage phosphor (SP) plates, with tracers labeled with high-energy, short-lived, positron-emitting radionuclides, using homogenized tissue specimens and autoradiography with frozen brain sections. The SP plates showed superior sensitivity and a linear response over a wide radioactivity range. Autoradioradiography provided reliable results due to (a) adequate sensitivity for low radioactivity concentration, b) an excellent linear range, and (c) satisfactory resolution. Though equilibration time of receptor-ligand interaction was dependent upon section thickness, quantification was possib with thinner sections. An initial finding using frozen section autoradiography of rat brain and spinal cord showed preferential binding of [11C]4-NMPB, a muscarinic acetylcholine (mACh) receptor antagonist, to the M4 subtype of mACh receptors. Further work to ascertain this specificity, by use of binding studies on cell membranes from CHO-K1 cells expressing individual subtypes of human mACh receptors, suggested lack of subtype selectivity. With respect to the possible cliinical use in glutamatergic neuropathology, [11C]cyano-dizocilpine, as a potential PET tracer for the N-methyl-D-aspartate (NMDA) subtype of glutamate receptors, was studied. The in vivo visualization of specific binding could not be achieved, though in vitro binding demonstrated good specificity and preferential binding to the activated for of the NMDA receptors. The use of the glucose analogue [18F]fluorodeoxyglucose (FDG) to study glucose utilization was evaluated in experimental traumatic brain injury (TBI). A trauma-induced increased uptake of FDG was seen, whereas the uptake of [1-14C]glucose remained unchanged. This discrepancy might be due to the increased postraumatic affinity of FDG for the endothelial glucose transporter proteins and/or to the hexokinase enzyme. [11C]Cyano-dizocilpine, [11C]4-NMPB, and [11C]flumazenil were utilized in autoradiography to evaluate changes in NMDA, mACh, and GABAA receptors, espectively, in experimental TBI. Observations showed a global decrease in the binding potential BP) of (i) [11C]cyano-dizocilpine acutely and 12 hrs after TBI, and (ii) of [11C]4-NMPB at 12 hrs after TBI, and (iii) a decrease in the BP of [11C]flumazenil in the cortex and hippocampus ipsilateral to the site of injury. The demonstrated changes in receptor binding after TBI are indicative of a widely dissipated effect of TBI on the particular neurotransmitter receptor systems as compared with what would be expected from FDG studies after TBI, i.e., a local disturbed neurotransmission.

La distractibilité est un syndrome qui se caractérise par une réactivité exagérée d'un individu vis-à-vis de son environnement. Il a été démontré que des atteintes limitées au cortex préfrontal dorsolatéral (CPFDL) sont responsables d'un tel syndrome chez l'homme. Ce trouble peut être quantifié par un test spécifique et sensible appelé " test des antisaccades ". Ce syndrome se rencontre dans de nombreuses pathologies neurodégénératives telles que la Paralysie supranucléaire progressive (PSP) ou encore psychiatriques telles que la schizophrénie. L'objectif de cette recherche est de tenter de mieux comprendre ce syndrome de distractibilité à la fois sur le plan anatomique et pharmacologique. Le projet anatomique débute par une étude chez l'homme de lésions au niveau sous cortical pour déterminer l'implication réelle du sous cortex dans ce syndrome. Afin d'apporter des données indiscutables sur le rôle du CPFDL dans la distractibilité, nous avons réalisé une étude chez le primate de microinjections intracérébrales de muscimol au niveau de cette aire cérébrale. L'approche pharmacologique a consisté à réaliser un modèle animal de distractibilité en reproduisant chez le primate ce syndrome par administration de kétamine à dose subanesthésique. Celle-ci est connue pour induire des symptômes " shizophréniques like " avec des troubles liés à un dysfonctionnement du cortex préfrontal. La seconde étude a pour but d'évaluer l'effet d'un inhibiteur de l'acétylcholine estérase sur le syndrome de distractibilité chez des patients atteints de PSP. Cette recherche, en particulier les études chez le primate devraient permettre à terme de développer une thérapeutique pour enrayer ce trouble.

Cholinergic inputs from mesopontine tegmentum activate midbrain dopamine (DA) neurons via M5 muscarinic receptors. The M5 receptor is important for mesopontine stimulation-induced accumbal or striatal DA efflux, brain stimulation reward or morphine-induced conditioned place preference (CPP). M5 receptor knockout (KO) mice show 40-50% less morphine-induced locomotion. Pedunculopontine tegmental nucleus (PPT) lesions in rodents block morphine CPP, but are ineffective after 18 hours food deprivation, opiate dependence, or intra-VTA BDNF. Based on these findings, we investigated whether acute food deprivation or intra-VTA BDNF alters morphine-induced locomotion (3 and 10 mg/kg, i.p.) in C57BL/6 M5 KO mice. Non-deprived M5 KOs showed reduced morphine-induced locomotion, suggesting M5 receptors partly mediate morphine-induced locomotion. Morphine-induced locomotion was reversed in food-deprived mice, suggesting the stimulant effects of morphine were altered to bypass the PPT. Unexpectedly, intra-VTA BDNF infusions were ineffective in altering morphine-induced locomotion. Additionally, M5 KOs receiving intra-VTA saline showed no deficits in morphine-induced locomotion.

Cholinergic inputs from mesopontine tegmentum activate midbrain dopamine (DA) neurons via M5 muscarinic receptors. The M5 receptor is important for mesopontine stimulation-induced accumbal or striatal DA efflux, brain stimulation reward or morphine-induced conditioned place preference (CPP). M5 receptor knockout (KO) mice show 40-50% less morphine-induced locomotion. Pedunculopontine tegmental nucleus (PPT) lesions in rodents block morphine CPP, but are ineffective after 18 hours food deprivation, opiate dependence, or intra-VTA BDNF. Based on these findings, we investigated whether acute food deprivation or intra-VTA BDNF alters morphine-induced locomotion (3 and 10 mg/kg, i.p.) in C57BL/6 M5 KO mice. Non-deprived M5 KOs showed reduced morphine-induced locomotion, suggesting M5 receptors partly mediate morphine-induced locomotion. Morphine-induced locomotion was reversed in food-deprived mice, suggesting the stimulant effects of morphine were altered to bypass the PPT. Unexpectedly, intra-VTA BDNF infusions were ineffective in altering morphine-induced locomotion. Additionally, M5 KOs receiving intra-VTA saline showed no deficits in morphine-induced locomotion.

Myopia is a refractive defect of the eye in which collimated light produces images focused in front of the retina. Myopia can be artificially induced in animal models by form deprivation (form deprivation myopia, FDM) or by application of negative lenses (lens induced myopia, LIM). In this study myopia was induced using diffusers. The project had two main aims: 1. To determine if there is an interaction between the GABAergic system and dopaminergic system in the retina in terms of myopia? 2. To determine if there is an interaction between the GABAergic system and cholinergic system in the retina in terms of myopia? Firstly, an experiment focusing on the interaction between dopaminergic receptors antagonists and GABAC receptor antagonist was developed. Comparison of the different drug treated eye with the control was found and the effects of combination injections were compared to individual drug injections. Use of different blockers for various subtype of receptors simplified the understandings the underlying pharmacological interventions for GABAC receptor antagonist TPMPA. The D1 subtype of receptors was found to be involved in transmission of signals from GABAC receptors. Our results showed that D1 receptor antagonist SCH-23390 antagonizes the actions of TPMPA. In addition to this it was also found that possibly 5HT receptor may also play an important role in modulation of signaling from GABA receptor to dopaminergic receptors in the retina. These results were consistent with the drug combination effects for agonists. GABA A/C receptor agonist muscimol negativate the efficacy of D1 receptor agonist SKF-38393 but the activity of D2/4 receptor agonist quinpirole was not affected by muscimol. Although dopaminergic receptors are found to interact with GABAergic signaling, but an alternative interaction with anticholinergic (most widely studied antimyopic agents) could not be ruled out. This problem led to a follow-up experiment, in which GABA receptors intervention in anticholinergic agents was studied. The GABAergic receptor agonist muscimol when injected with anticholinergics (atropine and pirenzepine) showed a moderate interaction. As muscimol interacted with atropine to a lesser extent a more specific M1/5 receptor antagonist pirenzepine (earlier found to inhibit myopia) was used under these circumstances. The second aim to study the interaction between muscimol and pirenzepine showed more interaction with GABAA/C receptor agonist. There were data suggesting that there is a muscarinic and GABAergic interaction in retina, such that each modulation of each receptor had an effect on FDM. However, a drug combination treatment helped in understanding the underlying mechanism. Several previous studies have indicated that there exist a strong interaction between excitatory neurotransmitter acetylcholine and inhibitory transmitter GABA in retina. The results of this study indicate a similar finding. Thus results of this study may be summarized as: 1. D1 antagonists and not D2 antagonists blocks the antimyopic effects of GABAC antagonist TPMPA 2. GABA A/C agonist muscimol partially blocks the antimyopic activity of anticholinergics (e.g. atropine and pirenzepine).

Submitted by Ronildo Prado (ronisp@ufscar.br) on 2017-08-10T14:10:10Z No. of bitstreams: 1 DissWIF.pdf: 2975197 bytes, checksum: 0039c1cb759b3d42c9bd5b659146af6a (MD5) / Approved for entry into archive by Ronildo Prado (ronisp@ufscar.br) on 2017-08-10T14:10:20Z (GMT) No. of bitstreams: 1 DissWIF.pdf: 2975197 bytes, checksum: 0039c1cb759b3d42c9bd5b659146af6a (MD5) / Approved for entry into archive by Ronildo Prado (ronisp@ufscar.br) on 2017-08-10T14:10:28Z (GMT) No. of bitstreams: 1 DissWIF.pdf: 2975197 bytes, checksum: 0039c1cb759b3d42c9bd5b659146af6a (MD5) / Made available in DSpace on 2017-08-10T14:10:35Z (GMT). No. of bitstreams: 1 DissWIF.pdf: 2975197 bytes, checksum: 0039c1cb759b3d42c9bd5b659146af6a (MD5) Previous issue date: 2013-08-26 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / The nucleus of the solitary tract (NTS) is the primary synaptic site of the peripheral baroreceptors and chemoreceptors. It has been shown that acetylcholine (ACh) microinjected into the NTS of rats induces hypotension and bradycardia. However, the contribution of cholinergic mechanisms at different NTS subnuclei (intermediate and commissural) as well as the cholinergic receptors blockade on the control of sympathetic (SNA) and phrenic (PNA) nerve activities have not been studied yet. In this study we assessed the role of ACh and its cholinergic receptors at the intermediate NTS (iNTS) and commissural NTS (cNTS) on the control of SNA, PNA and electrophysiological properties of these subnuclei neurons, as well as on baro and chemoreflex responses. Decorticated arterially-perfused in situ preparations of male juvenile rats were used to record SNA and PNA. Microinjections of ACh and cholinergic antagonists were performed into the iNTS or cNTS. Coronal slices of the brainstem containing either cNTS or iNTS subnuclei were obtained from male juvenile rats and used in whole cell patch clamp – current clamp recordings. It was observed that ACh microinjected into the iNTS inhibited both SNA and PNA. These effects were reduced by the pre-treatment with atropine (muscarinic antagonist) or mecamylamine (nicotinic antagonist). The cholinergic antagonists into the iNTS did not change the effects on SNA and PNA induced by baro and chemoreflex activation. In contrast, microinjections of ACh into the cNTS did not induce changes in SNA, but increased PNA. Despite the absence of changes in SNA, ACh into the cNTS changed the pattern of respiratory-sympathetic coupling. Both atropine and mecamylamine into the cNTS inhibited the ACh-induced tachypnea, but only mecamylamine inhibited the chemoreflex-induced tachypnea and the ACh-induced change in respiratory-sympathetic coupling. In vitro studies demonstrated that ACh promotes depolarization in both iNTS and cNTS neurons. Both muscarinic and nicotinic antagonism in the iNTS inhibited the ACh-induced depolarization. However, only nicotinic antagonist was effective in diminishing this response in the cNTS. The results suggest that ACh plays an important role in the control of cardiovascular and respiratory activities, with distinct functions between iNTS and cNTS. This cholinergic control involves activation of both muscarinic and nicotinic receptors within NTS, but only nicotinic receptors are involved in the chemoreflex tachypneic response. / O núcleo do trato solitário (NTS) é o sítio primário de aferências dos barorreceptores arteriais e quimiorreceptores. Sabe-se que a acetilcolina (ACh) injetada no NTS de ratos provoca hipotensão e bradicardia. Entretanto, não se sabe até o momento qual o papel do sistema colinérgico nos diferentes subnúcleos do NTS (intermediário ou comissural) ou o bloqueio de seus receptores na atividade simpática (SNA) ou na atividade do frênico (PNA). No presente estudo avaliamos os efeitos da ACh e seus receptores no NTS intermediário (NTSi) e comissural (NTSc) sobre a SNA, PNA e sobre as propriedades eletrofisiológicas dos neurônios desses subnúcleos, bem como nas respostas do baro e quimiorreflexos. Preparações in situ decorticadas de ratos jovens foram utilizadas para registro da SNA e PNA, e ACh e antagonistas colinérgicos foram microinjetados no NTSi ou NTSc. Cortes coronais bulbares contendo o NTSi ou NTSc foram obtidos de ratos jovens e utilizados para registro de neurônios através da técnica whole cell patch clamp – current clamp. Foi observado que a microinjeção de ACh no NTSi inibe tanto a SNA quanto a PNA, sendo que tais efeitos são inibidos pelo tratamento com atropina (antagonista muscarínico) ou mecamilamina (antagonista nicotínico). Os antagonistas colinérgicos no NTSi não alteraram os efeitos na SNA e PNA induzidos pela ativação do baro e quimiorreflexos. Por outro lado, microinjeções de ACh no NTSc não altera a SNA mas promove aumento da PNA. Apesar de não alterar a SNA, a ACh no NTSc promove alterações no acoplamento simpato-respiratório. Tanto a atropina quanto a mecamilamina microinjetadas no NTSc inibiram a taquipnéia induzida pela ACh, mas apenas a mecamilamina inibiu a taquipnéia resultante do quimiorreflexo e a alteração no acoplamento simpato-respiratório induzida pela ACh no NTSc. Nos estudos in vitro, observamos que a ACh promove respostas de despolarização em neurônios tanto do NTSi quanto do NTSc. O bloqueio de receptores tanto nicotínicos quanto muscarínicos no NTSi diminui a despolarização evocada pela ACh. No entanto, apenas o antagonista nicotínico foi capaz de diminuir essa resposta no NTSc. Os resultados sugerem que a ACh desempenha um importante papel no controle cardiorrespiratório, com funções distintas entre NTSi e NTSc. Esse controle colinérgico envolve a ativação de receptores tanto nicotínicos quanto muscarínicos no NTS, mas apenas receptores nicotínicos estão envolvidos na resposta de taquipnéia do quimiorreflexo. / FAPESP: 2010/17218-0