A Doença de Parkinson, uma condição neurodegenerativa e progressiva, está relacionada à morte de neurônios localizados na Substância Negra compacta, um dos componentes dos Núcleos da Base. Quando há a morte de neurônios dopaminérgicos nigrais, esta via modulatória é perdida, levando ao desequilíbrio entre as vias direta e indireta, esta última tendo sua atividade aumentada em detrimento da outra. O estriado tem um papel importante no recebimento e filtração de sinais motores corticais e talâmicos e suas maiores populações neuronais são GABAérgicas, demonstrando a importância do neurotransmissor GABA nesta modulação. O estriado recebe projeções dopaminérgicas vindas da Substância Negra compacta e, na falta desta aferentação, surgem os sintomas e sinais da Doença de Parkinson. Nosso objetivo é caracterizar a liberação de GABA nesta estrutura, avaliando os efeitos de outros transmissores e também o papel de alguns sinalizadores intracelulares neste processo. Para isto, empregamos o método de superfusão e liberação de GABA radiomarcado, previamente carregado, em tecido picado in vitro. A lesão nigral é produzida por cirurgia estereotáxica e microinjeção de 6-OHDA no feixe medial prosencefálico (mfb). Diversas drogas foram utilizadas para avaliarmos diferentes passos na liberação do transmissor. Concluímos que a liberação é fortemente dependente de cálcio e segue o modelo de exocitose vesicular, além de a subpopulação neuronal GABAérgica estrital estudada sofrer pouca influência de aferências glutamatérgicas e colinérgicas. No entanto, drogas dopaminérgicas regulam complexamente a liberação de GABA no estriado e ela também é bastante dependente de calmodulina. Conjecturamos se algumas drogas antipsicóticas que agem sobre calmodulina devem seu efeito terapêutico, ou parte dele, a esta ação e se, no modelo de DP de lesão unilateral por 6-OHDA, há comunicação entre os hemisférios lesado e não lesado após o estabelecimento da lesão e processo de rearranjo neuronal / Parkinsons disease, a progressive and neurodegenerative condition, is related to the death of neurons located in Substantia Nigra compacta, a component of Basal Ganglia. When nigral dopaminergic neurons die, this modulatory pathway is lost leading to imbalance between direct and indirect pathways, the latter having its activity increased over the former. Striatum has an essential role in receiving and filtering motor signals from cortex and thalamus and its major neuronal populations are composed by GABAergic neurons, showing how important is GABA in this modulation. Striatum receives dopaminergic projections from Substantia Nigra compacta and in its absence the typical signals and symptoms of the disease arise. We aimed to characterize GABA relase at this structure, assessing the effect of other transmitters as well the role of some intracellular signaling molecules in this process. For that, we employed the superfusion method and release of preloaded radiolabeled GABA from chopped striatal tissue. Nigral injury was produced by stereotaxic surgery and 6-OHDA microinjection at medial forebrain bundle (mfb). Several drugs were used to evaluate different steps in transmitter release. We concluded that the release is strongly calcium-dependent and follows vesicular exocytosis model; in addition the striatal GABAergic subpopulation of neurons studied here undergo little influence of glutamatergic and cholinergic afferents. However, dopaminergic drugs complexly regulate striatal GABA release and it also shows high involvement of calmodulin. We wonder if some antipsychotic drugs that act over calmodulin owe their therapeutical effects, or at least part of it, to this activity and if in 6-OHDA unilateral lesion parkinsonism model there is communication between injuried and healthy hemispheres after the establishment of the injury and neuronal rearrangement process

O fipronil é um inseticida fenilpirazol de amplo espectro, empregado na Medicina Veterinária e na Agricultura para o controle de pragas; é um potente inibidor do canal de cloreto ligado ao ácido gama aminobutírico (GABA), um dos neurotransmissores responsáveis por efeitos inibitórios no sistema nervoso central (SNC) de mamíferos. Embora vários estudos procurem compreender os mecanismos da toxicidade neuronal dos praguicidas em mamíferos, há poucos relacionados aos efeitos neurocomportamentais. Neste trabalho estudou-se os efeitos da exposição aguda a ao fipronil, utilizando-se modelos comportamentais ligados ao sistema GABAérgico: campo-aberto (CA), labirinto em cruz elevado (LCE), dose convulsivante mínima (DCM) com picrotoxina e pentilenotetrazol, e avaliação dos níveis cerebrais de alguns neurotransmissores e metabólitos. Ratos Wistar machos receberam, por via intragástrica (gavage), dose única de fipronil (1,0; 10,0; 30,0 ou 100,0 mg/Kg) ou água destilada 1 mL/Kg. No CA, uma hora após o tratamento, houve redução significante da distância percorrida, da velocidade média, do tempo em movimento e do número de levantamentos nos animais tratados. No LCE, observou-se diminuição da distância percorrida e número de entradas nos braços fechados e diminuição do número de movimentos iniciados na arena, nos animais tratados. Quanto às convulsões, foi observada redução significante apenas na DCM de pentilenotetrazol em animais expostos a maior dose de fipronil. Uma hora após o tratamento não foram observadas alterações relevantes nos níveis dos neurotransmissores (noradrenalina; dopamina, serotonina e GABA) e seus metabólitos no córtex, no hipotálamo e no striatum. Finalizando, os resultados do presente estudo mostraram que ratos expostos a dose única de fipronil apresentaram alterações comportamentais, caracterizadas por redução da atividade motora no campo aberto, no labirinto em cruz elevado e na dose convulsivante mínima do pentilenotetrazol. Além disso, a maior dose de fipronil (100,0 mg/Kg) causou a morte de 60% dos ratos, num período entre 24 horas a 7 dias após a exposição oral. Esses efeitos comportamentais do fipronil não puderam ser atribuídos a um único sistema de neurotransmissão central. / The Fipronil is a phenylpyrazole insecticide with broad spectrum, both in Veterinary Medicine, and in Agriculture it has been used for the control of nuisances, is a potent inhibitor of chloride channel connected to the gamma aminobutyric acid (GABA), one of the neurotransmitters responsible for inhibitory effects on the central nervous system (CNS) of mammals. Although several studies try to understand the mechanism of neuronal toxicity of pesticide in mammalian, there are few studies related to neurobehavioral effects. Thus, the present study investigated the effects of fipronil acute exposure, being observed some behaviors models connected to GABAergic system, as open field, elevated plus maze (EPM), as well as in the seizures induced by picrotoxinin and pentylenotetrazole and determinate the brain levels of some neurotransmitter and metabolic levels. Male Wistar rats received by intragastric (gavage), single dose of fipronil (1.0; 10.0; 30.0 or 100.0 mg/Kg) or distilled water. In open field, one hour after treatment, there was a significant reduction of distance moved, average speed, time moving and the number of surveys in treated animals. In the EPM, it was observed decrease in the distance moved, in the number of entries in the closed arms and decrease the number of started movements in the arena, in treated animals. As for seizures, a significant decrease was observed only in minimum convulsant dose of pentylenotetrazole in animals exposed to higher dose of fipronil. One hour after treatment were not observed any relevant changes in the levels of neurotransmitters (norepinephrine, dopamine, serotonin and GABA) and its metabolites in the cortex, the hypothalamus and the striatum. Finally, the results of this study showed that rats exposed to single dose of fipronil showed behavioral changes, characterized by reduction of motor activity in the open field, in the elevated plus-maze and the minimum convulsant dose of pentyletetrazole. Moreover, the higher dose of fipronil (100.0 mg/Kg) caused the death of 60% of rats, over a period from 24 hours to 7 days after oral exposure. These behavioral effects of fipronil could not be attributed to a single system of central neurotransmission.

GABA and GABA type A receptor (GABAAR) are expressed in pancreatic β-cells and comprise an autocrine signaling system. How the GABA-GABAAR system is regulated is unknown. In this study, I investigated insulin’s effect on this system in the INS-1 β-cell line. I found that GABA evoked current (IGABA) in INS-1 cells, resulting in membrane depolarization. Perforated-patch recordings showed that pre-treatment of insulin or zinc-free insulin suppressed IGABA in INS-1 cells (p < 0.01). Radioimmunossay showed that GABA (30 μM) increased C-peptide secretion from INS-1 cells, which was blocked by GABAAR antagonist picrotoxin, indicating that GABA increased insulin secretion through activation of GABAAR. However, insulin significantly reduced the stimulatory effect of GABA on C-peptide secretion (p < 0.05). These data suggest that GABA released from β-cells positively regulates insulin secretion via GABAAR activation, and that insulin negatively regulates the β-cell secretory pathway likely via inhibiting the GABA-GABAAR system in β-cells.

GABA and GABA type A receptor (GABAAR) are expressed in pancreatic β-cells and comprise an autocrine signaling system. How the GABA-GABAAR system is regulated is unknown. In this study, I investigated insulin’s effect on this system in the INS-1 β-cell line. I found that GABA evoked current (IGABA) in INS-1 cells, resulting in membrane depolarization. Perforated-patch recordings showed that pre-treatment of insulin or zinc-free insulin suppressed IGABA in INS-1 cells (p < 0.01). Radioimmunossay showed that GABA (30 μM) increased C-peptide secretion from INS-1 cells, which was blocked by GABAAR antagonist picrotoxin, indicating that GABA increased insulin secretion through activation of GABAAR. However, insulin significantly reduced the stimulatory effect of GABA on C-peptide secretion (p < 0.05). These data suggest that GABA released from β-cells positively regulates insulin secretion via GABAAR activation, and that insulin negatively regulates the β-cell secretory pathway likely via inhibiting the GABA-GABAAR system in β-cells.

Not available / text

Diss. (sammanfattning) Umeå : Univ., 2007. / Härtill 4 uppsatser.

Made available in DSpace on 2014-06-11T19:23:33Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-06-22Bitstream added on 2014-06-13T20:11:01Z : No. of bitstreams: 1 silva_tm_me_araca.pdf: 1258731 bytes, checksum: 9d2da0efd667a88f8de0c4364d2a3ab8 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Injeções no núcleo parabraquial lateral (NPBL) de muscimol, agonista de receptores GABAA, promove aumento de pressão arterial e induz ingestão de NaCl 0.3 M e água em ratos normovolêmicos, saciados e depletados de sódio. A doença periodontal é uma condição inflamatória que promove a liberação de citocinas próinflamatórias, tais como IL-6 e TNF-α e a destruição das estruturas de suporte do dente. As citocinas pró-inflamatórias podem modular a neurotransmissão GABAérgica e ativar neurônios do NPBL. Neste estudo, investigamos o efeito da ativação GABAérgica no NPBL com muscimol na ingestão de NaCl hipertônico e água e parâmetros cardiovasculares em ratos normovolêmicos, saciados e depletados pelo modelo FURO+CAP com doença periodontal. Foram utilizados ratos Wistar divididos em dois grupos: com doença periodontal induzida por ligadura (DP) e sem doença periodontal (grupo controle). Quinze (15) dias após a indução da doença periodontal em ambos os grupos foram implantadas cânulas bilaterais no NPBL. Ratos saciados controles que receberam injeções bilaterais no NPBL de muscimol tiveram aumento na ingestão... / GABAA receptor activation with muscimol in the lateral parabrachial nucleus (LPBN) induces water and hypertonic sodium chloride (NaCl) intake in rats. The purpose of this study was to investigate whether local inflammatory event, such as periodontal disease, is able to alter the effects of injections of muscimol (GABAA receptor agonist) into the LPBN on water and 0.3 M NaCl intake in fluid replete rats and in rats treated with diuretic furosemide (FURO) combined with a low dose of the angiotensin-converting enzyme inhibitor captopril (CAP) injected subcutaneously. Male Wistar rats were divided into two groups: with experimental ligature-induced periodontal disease (PD) and those without PD (control conditions). Fifteen days after application of the ligature, both groups had cannulas implanted bilaterally into the LPBN, and were given simultaneous access to water and 0.3 M NaCl intake. In fluid replete rats without PD, injections of muscimol (0.5 nmol/0.2 μl) into the LPBN induced 0.3 M NaCl intake (15.8 ± 2.4 vs. saline: 0.2 ± 0.05 ml/210 min), water intake (14.2 ± 1.2 vs. saline: 0.6 ± 0.3 ml/210 min) and pressor response (15 ± 3.3 mmHg, vs. saline: 0.6 ± 1.3 mmHg). In fluid replete rats (PD group), a decrease was observed in water intake (6.0 ± 1.4 ml/210 min), pressor response (7.5 ± 3.1 mmHg), but not in 0.3 M NaCl intake induced by muscimol. In rats with FURO + CAP-treatment (control group), injections of muscimol into the LPBN increased 0.3 M NaCl (25.9 ± 5.8 vs. saline: 5.7  1.0 ml/210 min) and water intake (19.9  1.2 vs. saline: 11.2  1.0 ml/210 min). In rats with FURO + CAP-treatment (PD group), a decrease was observed in 0.3 M NaCl intake (10.9 ± 2.9 ml/210 min) and water intake (13.4  2.3 ml/210 min) after... (Complete abstract click electronic access below)

Le diabète de type 1 (DT1) résulte de la destruction des cellules β productrices d’insuline par le système immunitaire. Cette condition représente un enjeu de santé publique majeur car, malgré les thérapies actuelles, les patients atteints développent trop souvent des complications cardio-vasculaires. Des thérapies alternatives se doivent donc d’être mises au point. Ainsi, diverses approches visent à reprogrammer/différencier (in vitro ou in vivo) différents types cellulaires pancréatiques afin de générer des cellules β (productrices d’insuline) fonctionnelles. Dans ce but, notre laboratoire a notamment montré que les cellules α (productrices de glucagon) embryonnaires peuvent être régénérées et converties en cellules β fonctionnelles par l’expression ectopique du seul gène Pax4 (un gène normalement impliqué dans la spécification embryonnaire du lignage β - (Collombat and Mansouri, 2009)). Dans la première partie de ce travail, nous démontrons que les cellules α à l’âge adulte (Al-Hasani et al., 2013) retiennent leur capacité de régénération et de conversion en cellules β, celles-ci étant fonctionnelles et capable de remplacer plusieurs fois l’ensemble des cellules β du pancréas. Cependant, cette approche transgénique serait difficile à mettre en œuvre chez l’homme. De nombreux cribles furent donc initiées dans le but de trouver des petites molécules/composés chimiques mimant les effets de Pax4. Un composé potentiel, GABA, fut ainsi identifié et caractérisé. / Type 1 diabetes (T1D) results from the destruction of insulin-producing β-cells by the immune system. This condition is a major public health issue because, despite current therapies, patients often develop cardiovascular complications. Therefore alternative therapies need to be developed. Thus, various approaches are designed to reprogram / differentiate (in vitro or in vivo) different pancreatic cell types to generate functional (insulin-producing) β-cells. To this end, our laboratory has shown that especially the embryonic α-cells (producing glucagon) can be regenerated and converted into functional β-cells by the ectopic expression of the Pax4 gene (usually a gene involved in the specification of embryonic lineage β - (Collombat and Mansouri, 2009)). In the first part of this work, we show that α-cells in adulthood (Al-Hasani et al., 2013) retain their capacity for regeneration and conversion into β-cells, the latter being functional and able to replace repeatedly all the β-cells of the pancreas. However, this transgenic approach would be difficult to implement in humans. Many screens were therefore initiated in order to find small molecules / chemical compounds that mimic the effects of Pax4. A potential compound, GABA, was identified and characterized. Our results demonstrate that treatment of WT mice with GABA results in a significant increase in the number and size of the islets (caused by insulin+ cell hyperplasia). By using lineage tracing tools, our results indicate that these "β-like" neo-generated cells are coming from glucagon+ cells.

Chapter 1 provides a general introduction on the role of bioisosterism of fluorine aiming to improve the pharmacokinetics properties of lead compounds. GABAA receptors specifically, synaptic GABAA receptors, extrasynaptic GABAA receptors and GABAA rho receptors are then presented. Compounds that exhibit agonist and partial agonist effects at these receptors are also discussed. The applications of some compounds as GABAA receptor PET radiotracers are also described. Chapter 2 details the synthesis of two fluorinated analogues of muscimol, fluoromuscimol and trifluoromethylmuscimol. Fluoromuscimol was obtained from the lithiation of a Boc-protected isoxazole followed by in-situ fluorination using NFSI, whereas trilfuoromethylmuscimol was obtained from the coupling of a heteroaryl iodide with trifluoromethylcopper species, which was generated in-situ from MFSDA in the presence of CuI. Fluoromuscimol and trifluoromethylmuscimol were assessed on human synaptic, (α₁β₂γ₂), extrasynaptic, (α₄β₂δ) and ρ₁ subunits of the GABAA receptor. The biological results show that fluoromuscimol exhibits greater maximum response in comparison to GABA at the extrasynaptic GABAA receptors (α₄β₂δ), but lower overall potency, whereas trifluoromethylmuscimol was inactive at all the tested GABAA receptors. Chapter 3 discusses the synthesis and late stage fluorination of diaryliodonium salts as precursors to fluoromuscimol. Application of iodonium salts as precursors for nucleophilic fluorination in PET studies are also highlighted. The last part of this chapter focuses on the synthesis of iodomuscimol as a potential alternative SPECT radiotracer to fluoromuscimols in probing GABA binding sites on GABAA receptors.

Orientador: João Carlos Callera / Banca: José Vanderlei Menani / Banca: Juliana Irani Fratucci De Gobbi / Resumo: Injeções no núcleo parabraquial lateral (NPBL) de muscimol, agonista de receptores GABAA, promove aumento de pressão arterial e induz ingestão de NaCl 0.3 M e água em ratos normovolêmicos, saciados e depletados de sódio. A doença periodontal é uma condição inflamatória que promove a liberação de citocinas próinflamatórias, tais como IL-6 e TNF-α e a destruição das estruturas de suporte do dente. As citocinas pró-inflamatórias podem modular a neurotransmissão GABAérgica e ativar neurônios do NPBL. Neste estudo, investigamos o efeito da ativação GABAérgica no NPBL com muscimol na ingestão de NaCl hipertônico e água e parâmetros cardiovasculares em ratos normovolêmicos, saciados e depletados pelo modelo FURO+CAP com doença periodontal. Foram utilizados ratos Wistar divididos em dois grupos: com doença periodontal induzida por ligadura (DP) e sem doença periodontal (grupo controle). Quinze (15) dias após a indução da doença periodontal em ambos os grupos foram implantadas cânulas bilaterais no NPBL. Ratos saciados controles que receberam injeções bilaterais no NPBL de muscimol tiveram aumento na ingestão... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: GABAA receptor activation with muscimol in the lateral parabrachial nucleus (LPBN) induces water and hypertonic sodium chloride (NaCl) intake in rats. The purpose of this study was to investigate whether local inflammatory event, such as periodontal disease, is able to alter the effects of injections of muscimol (GABAA receptor agonist) into the LPBN on water and 0.3 M NaCl intake in fluid replete rats and in rats treated with diuretic furosemide (FURO) combined with a low dose of the angiotensin-converting enzyme inhibitor captopril (CAP) injected subcutaneously. Male Wistar rats were divided into two groups: with experimental ligature-induced periodontal disease (PD) and those without PD (control conditions). Fifteen days after application of the ligature, both groups had cannulas implanted bilaterally into the LPBN, and were given simultaneous access to water and 0.3 M NaCl intake. In fluid replete rats without PD, injections of muscimol (0.5 nmol/0.2 μl) into the LPBN induced 0.3 M NaCl intake (15.8 ± 2.4 vs. saline: 0.2 ± 0.05 ml/210 min), water intake (14.2 ± 1.2 vs. saline: 0.6 ± 0.3 ml/210 min) and pressor response (15 ± 3.3 mmHg, vs. saline: 0.6 ± 1.3 mmHg). In fluid replete rats (PD group), a decrease was observed in water intake (6.0 ± 1.4 ml/210 min), pressor response (7.5 ± 3.1 mmHg), but not in 0.3 M NaCl intake induced by muscimol. In rats with FURO + CAP-treatment (control group), injections of muscimol into the LPBN increased 0.3 M NaCl (25.9 ± 5.8 vs. saline: 5.7  1.0 ml/210 min) and water intake (19.9  1.2 vs. saline: 11.2  1.0 ml/210 min). In rats with FURO + CAP-treatment (PD group), a decrease was observed in 0.3 M NaCl intake (10.9 ± 2.9 ml/210 min) and water intake (13.4  2.3 ml/210 min) after... (Complete abstract click electronic access below) / Mestre