Os endocanabinóides (ECs) são neuromoduladores lipídicos que são produzidos por demanda tendo ação retrógrada. Existem duas moléculas que são atualmente reconhecidos como os principais ECs, a anandamida (AEA) e 2-araquidonoilglicerol (2-AG). O hipocampo sintetiza endocanabinóides e expressa seus receptores (CB1, CB2, TRPV1).Existe uma discrepância de efeitos dos 2 endocanabinóides na potenciação á longo prazo (LTP) hipocampal que pode ser um resultado da AEA agir tanto em receptores CB1 quanto em receptores TRPV1.Tendo em vista que a ativação dos receptores TRPV1 potenciam a LTP hipocampal, e não a inibem como é observado com a AEA, então qual seria o mecanismo de ação da AEA em inibir a LTP? Seria possível que a AEA estivesse preferencialmente inibindo a produção de 2-AG e assim inibindo a LTP? Para testar essa hipótese usamos inibidores farmacológicos da degradação hidrolítica do 2-AG e da AEA e também usamos antagonistas dos receptores TRPV1 e um animal knock-out para o receptor TRPV1. Camundongos machos BlackC57 e knockout(KO) para TRPV1 com idade entre 35 a 49 dias foram utilizados para obtenção de fatias do hipocampo (400µm), e a potenciação a longo prazo na via Schaffer-CA1 estudada. Não observamos efeito dos inibidores da degradação hidrolítica e oxidative dos endocanabinóides na LTP. A LTP do camundongo knock-out era inibida, porem o antagonismo farmacológico dos receptores TRPV1 não minetizou esse efeito. Já o agonista dos receptors canabinóides WIN55212-2 inibiu a indução da LTP. Concluimos que o aumento dos endocanabinóides pela inibição da sua degradação não foi eficiente em alterar a LTP hippocampal em nosso modelo experimental. Aprovação do CONCEA-FMRP- nº008/2017 / Endocannabinoids (ECs) are lipid neuromodulators that are produced on demand having retrograde action. There are two molecules that are currently recognized as the major ECs, anandamide (AEA) and 2-arachidonoylglycerol (2-AG). The hippocampus synthesizes endocannabinoids and expresses their receptors (CB1, CB2, TRPV1). There is a discrepancy of endocannabinoid effects on hippocampal long term potentiation (LTP) which may be a result of AEA acting on both CB1 and TRPV1 receptors. Given that the activation of TRPV1 receptors potentiate hippocampal LTP, and do not inhibit it as observed with AEA, then what would be the mechanism of action of AEA in inhibiting LTP? Was it possible that AEA was preferentially inhibiting 2-AG production and thus inhibiting LTP? To test this hypothesis, we used pharmacological inhibitors of the hydrolytic degradation of 2-AG and AEA and also used TRPV1 receptor antagonists and a knock-out animal for the TRPV1 receptor. Male BlackC57 mice and TRPV1 knockout (KO) aged 35 to 49 days were used to obtain hippocampal slices (400 ?m), and the long-term potentiation in the Schaffer-CA1 pathway studied). The LTP of the knock-out mouse was inhibited, but the pharmacological antagonism of TRPV1 receptors did not mimic this effect, whereas the WIN55212-2 cannabinoid receptor agonist inhibited the induction of LTP. We conclude that increasing the levels of endocannabinoids by inhibiting their degradation was not efficient in altering hippocampal LTP in our experimental model Approval of CONCEA-FMRP- nº 008/2017

O início do desenvolvimento do sistema nervoso central (SNC) é caracterizado por diversos processos, com períodos críticos, que podem ser influenciados por xenobióticos. Este estudo teve como objetivo comparar os efeitos das diferentes concentrações de nicotina em elementos da transmissão sináptica durante o desenvolvimento de camundongos C57Bl/6. Para tanto, foram utilizados cigarros com duas diferentes concentrações de nicotina, o 3R4F (0,73 mg de nicotina/cigarro) e o 4A1 (0,16 mg de nicotina/cigarro). As exposições ocorreram diariamente a partir do 3° dia de vida (P3) até o 14° dia (P14) por duas horas, e os animais controle foram expostos somente ao ar sintético. Os animais foram eutanasiados em P15, P35 e P65 e avaliamos as seguintes proteínas: sinapsina (SYN1), sinaptotagmina (SYT), sinaptofisina (SYP), sinaptobrevina (SYB), PSD-95 e EGR-1, por Western blotting; o gene Egr-1 por PCR Real Time; MAP-2 e neurofilamentos (Neu) por imunofluorescência no hipocampo, tronco encefálico e cerebelo. Nossos resultados mostraram que na infância (P15) a fumaça do cigarro 3R4F diminuiu SYN1, assim como EGR-1, MAP-2 e Neu no cerebelo. No hipocampo houve aumento de SYN1 e MAP-2, diminuição de PSD-95, Egr-1 e Neu. No tronco encefálico esse cigarro não modificou nenhum dos parâmetros avaliados. Nessa mesma idade, a fumaça do cigarro 4A1 diminuiu MAP-2, Neu e a expressão gênica de Egr-1 no cerebelo; diminuiu MAP-2 e Neu no hipocampo e não promoveu alterações no tronco encefálico. Em P35 (adolescência) o 3R4F manteve reduzido SYN1, EGR-1, MPA-2 and Neu, e também reduziu Egr-1 no cerebelo. A diminuição de SYN1 também foi observada no hipocampo, mas nessa estrutura o 3R4F também aumentou SYT, SYP, MAP-2 and Neu; diminuiu EGR-1 e a expressão de Egr-1. No entanto, no tronco encefálico, foram observados aumento de SYN1 e MAP-2, e uma diminuição de Neu. Em relação à fumaça do 4A1, em P35, nossos resultados mostraram diminuição de SYB, Egr-1, MAP-2 and Neu no cerebelo; aumento de Egr-1, MAP-2 e diminuição de Neu no hipocampo; no tronco encefálico houve aumento de SYB, Egr-1, Neu e diminuição de MAP-2. Na fase adulta (P65) as únicas diferenças estatísticas encontradas foram: no cerebelo: diminuição de SYB pelos cigarros 4A1, aumento de EGR-1 pelo 3R4F e aumento de Egr-1 e MAP-2 por ambos os cigarros; no hipocampo: aumento de Neu por ambos os cigarros; tronco encefálico: aumento de SYB e EGR-1 e diminuição de SYT pelo 4A1, diminuição de SYN1, aumento de EGR-1, Egr-1 e MAP-2 pelo 3R4F. Nossos resultados indicam que a exposição à fumaça do cigarro nos primeiros dias de vida de camundongos, mesmo que em baixas concentrações de nicotina, podem acarretar em alterações em elementos da transmissão sináptica em várias regiões encefálicas durante a infância, adolescência e na fase adulta. / The initial steps of the development of the central nervous system are characterized by several critical processes, which can be affected by xenobiotics. The present study aimed to compare the effect of two different nicotine concentrations in cigarettes on synaptic transmission elements during the development of C57/Bl6 mice. To reach this aim we exposed C57Bl/6 mice to smoke from two different cigarettes - 3R4F (0.73mg of nicotine/cigarette) or 4A1 (0.16mg of nicotine/cigarette), from the third day of life (P3) until the fourteenth (P14) for a period of 1h, twice a day (at 8am and at 3pm). The control animals were exposed only to synthetic air. At P15, P35 and P65, the animals were submitted to euthanasia and different encephalic areas were collected (cerebellum, hippocampus and brainstem); quantification of synapsin (SYN1), synaptotagmin (SYT), synaptophysin (SYP), synaptobrevin (SYB), PSD-95 and EGR1 protein expression was assessed by Western blotting, gene expression of Egr-1 by was assessed by RT-PCR and MAP-2 and neurofilaments (Neu) were assessed by immunofluorescence. Our results showed that exposure to 3R4F smoke decreased the quantification of SYN1 at infancy (P15), as well as EGR-1, MAP-2 and Neu at cerebellum. At hippocampus, there was an increase of SYN1 and MAP-2, decrease of PSD-95, Egr-1 and Neu. At brainstem, 3R4F smoke did not modify any parameter. At the same age, 4A1 smoke decreased the quantification of MAP-2, Neu and the expression of Egr-1, at cerebellum; decreased MAP-2 and Neu at hippocampus and did not alter any parameter at brainstem. At P35 (adolescence) 3R4F smoke still reduced SYN1, EGR-1, MAP-2 and Neu, and reduced Egr-1 at cerebellum. The reduction of SYN1 quantification was also observed at hippocampus, but at this area, 3R4F smoke also increased the quantification of SYT, SYP, MAP-2 and Neu and decreased EGR-1 and the expression of Egr-1. However, at the brainstem, an increased quantification of SYN1 and MAP-2 and a decrease of Neu were observed. Regarding 4A1 smoke, at the same age, our results showed a decreased quantification of SYB, Egr-1, MAP-2 and Neu at cerebellum; increase of Egr-1, MAP-2 and decrease of Neu at hippocampus; and at brainstem, increase of SYB, Egr-1, Neu and decrease of MAP-2. At adulthood (P65) the only statistical differences were: at cerebellum - decreased quantification of SYB by 4A1 cigarette smoke, increase of EGR-1 by 3R4F and increase of Egr-1 and MAP-2 by smoke of both cigarettes; at hippocampus - increase of Neu by smoke of both cigarettes; at brainstem - increase of SYB and EGR-1 and decrease of SYT by 4A1 smoke, decrease of SYN1, increase of EGR-1, Egr-1 and MAP-2 by 3R4F smoke. Thus, we can conclude that exposure to cigarette smoke early in life, even at low nicotine concentrations can modify elements of synaptic transmission, compromising such transmission at several encephalic areas.

A hipóxia mantida de curta duração (HM) está associada a alterações cardiorrespiratórias e ao desencadeamento de processo inflamatório em humanos e modelos experimentais. Ademais, há evidências de que a HM pode alterar a transmissão sináptica na região do Núcleo do Trato Solitários (NTS). No presente estudo, utilizamos a minociclina, um inibidor da ativação microglial e antiinflamatório, para avaliar a influência da inflamação desencadeada pela HM sobre a neurotransmissão glutamatérgica nos neurônios do NTS que enviam projeções para a região ventrolateral da medula (NTS-VLM). A hipótese geral do nosso estudo foi a seguinte: a HM induz processo inflamatório no tronco encefálico, o qual contribui para o aumento da neurotransmissão glutamatérgica em neurônios NTS-VLM, colaborando para a elevação da pressão arterial média (PAM) observada nestes ratos. Embora tenhamos observado aumento da pressão arterial média em ambos os grupos de ratos tratados com veículo (solução salina + água destilada, ip) ou minociclina [(30mg/Kg ip por 3 dias) submetidos a 24h de HM (FiO2 0.1) em relação aos seus respectivos grupos controle (FiO2 0,28), o aumento da MAP foi menor nos ratos previamente tratados com minociclina. Os registros eletrofisiológicos utilizando a técnica de whole cell patch-clamp mostraram que a HM não produziu alterações nas propriedades ativas e passivas dos neurônios NTS-VLM. No entanto, os neurônios de ratos submetidos a HM apresentaram aumento nas correntes glutamatérgicas espontâneas e evocadas pelo estímulo do trato solitário. Esse grupo de animais também apresentou aumento no número de microgliais na região do NTS. As alterações mencionadas foram atenuadas pelo tratamento prévio com minociclina. Concluímos que a inflamação induzida pela HM contribui para o aumento da neurotransmissão glutamatérgica nos neurônios NTS-VLM o qual poderia estar relacionado com a hipertensão arterial observada nestes ratos. / Short-term Sustained hypoxia (SH) is associated with cardiorespiratory changes and inflammatory process in humans and experimental models. There is also evidence that SH can change the synaptic transmission in the nucleus tractus solitarius (NTS) region. Here we use the minocycline, an anti-inflammatory and microglial inhibitor, to evaluate the role of inflammation triggered by SH on the excitatory neurotransmission in the NTS neurons sending projections to the ventrolateral medulla (NTS-VLM). We hypothesized that SH induces brainstem inflammatory process, which may contribute to increase in excitatory neurotransmission and excitability of the NTS-VLM neurons, collaborating to the high blood pressure observed on these rats. Although we have observed increased MAP in both groups of rats treated with vehicle (saline + distilled water, i.p) or minociclina [(30mg/Kg i.p for 3 days) submitted to 24h of SH (FiO2 0.1) in relation to their respective control groups (FiO2 0.28), the MAP increase was lower in rats treated with minociclina. The whole cell patch-clamp recordings showed that SH produced no changes in active properties of NTS neurons. However, neurons of rats submitted SH presented an increase in the glutamatergic neurotransmission and the number of microglial at the NTS region. These increases were prevented in the groups previously treated with minociclina. We conclude that inflammation induced by SH contributes to the increased excitatory neurotransmission in NTS-VLM neurons that could be associated to high blood pressure observed in these rats.

Cooperative behaviour among unrelated individuals is an evolutionary paradox. Research suggests that an individual’s propensity to cooperate and its response to experiencing cooperation or defection from its social environment consistently varies among individuals and as a function of external factors. The biological and psychological underpinnings of such behavioural variation remain unknown; they can, however, provide more insight into the evolution and maintenance of cooperation among non-kin. This thesis explores the proximate effects of experiences of cooperation or defection from the social environment, as well as possible proximate drivers of cooperative behaviour, using the Trinidadian guppy (Poecilia reticulata) as a study system. Firstly, the behavioural rules underpinning an individual’s decision to cooperate or not with unfamiliar individuals in the presence of specific or non-specific information were explored. When fish had information about their social partner’s cooperativeness, they behaved in a manner consistent with direct reciprocity, copying their partner’s last move. When paired with an ostensibly novel partner, a different, or at least additional, behavioural rule seemed to be employed. In order to help understand the drivers of individual variation in cooperative behaviour, phenotypic selection on cooperativeness was carried out over three filial generations, resulting in fish of high cooperativeness (HC) and low cooperativeness (LC). The divergence of individual cooperativeness observed between the two phenotypic selection lines suggests that cooperative behaviour in the context of predator inspection is at least in part heritable. Cooperative behaviour of F3 fish was found not to correlate with boldness or exploratory behaviour; HC and LC fish did, however, differ in some aspects of sociability and agonistic behaviour. Possible proximate neuromodulatory mechanisms underlying these differences in cooperativeness were also explored, focusing on brain expression patterns for the isotocin receptor (itr) gene in F3 females. HC females were found to have higher mid-section itr expression levels than LC females. Finally, I explored the effects of experiencing cooperation or defection on monoaminergic neurotransmission, which is thought to instantiate the effects of such experiences on the individual’s internal state. My findings suggest that experiencing cooperation or defection from the social environment affects internal state; this phenomenon may be crucial for the appropriate adjustment of the behavioural response to such experiences, and for the emergence of behavioural rules such as generalised reciprocity. Taken together these results suggest that neuromodulatory mechanisms are pivotal for the perception of stimuli from the social environment in the tested cooperative context and that variation in cooperative behaviour may be underpinned by individual differences in the structural properties of such systems. They also provide insight into how behavioural input may affect the behavioural response to such experiences, and ultimately how such mechanisms may lead to the evolution and maintenance of cooperation.

Fipronil é um inseticida fenilpirazol de amplo espectro, desenvolvido para inibir seletivamente receptores GABA associados a canais de cloreto de insetos; tanto em Medicina Veterinária, como em Agricultura tem sido utilizado para o controle de pragas. Embora vários estudos procurem compreender os mecanismos da toxicidade neuronal dos praguicidas em mamíferos, há poucos relacionados aos efeitos neurocomportamentais. Assim, o presente estudo investigou os efeitos da exposição prolongada de ratos ao fipronil, observando-se alguns comportamentos ligados ao sistema GABAérgico, como atividade no campo aberto, no labirinto em cruz elevado (LCE), bem como na convulsão induzida por picrotoxina e pentilenotetrazol; avaliou-se ainda os níveis cerebrais de alguns neurotransmissores, como GABA, dopamina, noradrenalina, serotonina e seus respectivos metabólitos. Além disso, foram realizadas análises anátomo-histopatológicas em fígado, cérebro e rins dos animais. Ratos Wistar receberam, por via intragástrica (gavage), durante 28 dias um dos seguintes tratamentos: fipronil (0,1; 1,0 ou 10,0 mg/Kg/dia) ou água destilada 1 mL/Kg/dia. Após uma hora da última administração do fipronil ou água destilada, os ratos tiveram seu comportamento avaliado no campo aberto e no LCE com auxílio do sistema computadorizado EthoVision®. Observou-se no campo aberto apenas redução significante do nº de levantamentos nos animais tratados com 10,0 mg/Kg de fipronil, quando comparados com os ratos do grupo controle. No LCE, a exposição prolongada de ratos ao fipronil nas doses de 1,0 e 10,0 mg/Kg provocou redução na distância percorrida pelos animais e o tempo de permanência dos animais nos braços abertos, além de aumentar o tempo de permanência nos braços fechados. Para o cálculo da dose convulsivante mínima, seguiu-se o mesmo protocolo de tratamento, sendo observada diminuição significante na dose de picrotoxina e de pentilenotetrazol necessária para a indução da convulsão em animais tratados com 10,0 mg/Kg fipronil. A determinação dos níveis de neurotransmissores e seus respectivos metabólitos mostrou aumento dos níveis de dopamina no córtex frontal de ratos tratados com 10,0 mg/Kg de fipronil, redução nos níveis de GABA no striatum de ratos tratados com 1,0 mg/Kg de fipronil. Em relação à serotonina houve aumento de seus níveis no córtex frontal de ratos tratados com 1,0 mg/Kg e seu metabólito, ácido 5 (5HIAA), foi observado redução de seus níveis no córtex frontal de ratos tratados com 0,1 e 1,0 mg/kg p.c. de fipronil. No striatum também houve redução de seus níveis em ratos tratados com 0,1 e 1,0 mg/Kg de fipronil. O estudo anátomo-histopatológico revelou que a exposição prolongada ao fipronil pode causar hepatotoxicidade, caracterizada por aumento dos pesos absoluto e relativo do fígado e tumefação celular de hepatócitos. Em conclusão, os resultados mostraram que a exposição de ratos por 28 dias, via gavage, ao fipronil pode causar efeitos comportamentais relacionados a alterações em sistemas centrais de neurotransmissão e também hepatotoxicidade. / Fipronil is a phenylpyrazole insecticide with broad spectrum, developed to selectively inhibit insect GABA-gated chloride channels; both in Veterinary Medicine, and in Agriculture it has been used for the control of nuisances. 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 long term exposure in rats, being observed some behaviors connected to GABAergic system, as activity in open field, in elevated plus maze (EPM), as well as in the seizures induced by picrotoxinin and pentylenotetrazole. In addition, this study determinated the brain levels of some neurotransmitter as GABA, dopamine, noradrenaline, serotonin and metabolic levels and investigated liver, brain and kidney damage. Wistar rats received by intragastric (gavage) way during 28 days one of this treatment: fipronil (0,1, 1,0 or 10,0 mg/Kg) or distilled water; the rats behavior were evaluated in open field and elevated plus maze apparatus with system computer EthoVision®. There was a significant reduction of rearing in open field in rats treated with 10,0 mg/Kg of fipronil when compared with control rats. In the EPM, there was a decrease of distance moved in open arms and increase of time spent in closed arms to rats treated with 1,0 and 10,0 mg/Kg of fipronil when compared to control. It was still noticed the reduction in the time spent in open arms EPM in rats treated with 1,0 and 10,0 mg/Kg of fipronil when compared to control group. To calculate the minimum convulsant dose, was used the same treatment protocol and it was observed a significant reduction in picrotoxinin and pentylenotetrazole necessary dose to induction of seizure in animals treated with fipronil 10,0 mg/Kg. The level determination of neurotransmitters and their metabolites, it was observed the dopamine increasing in forebrain from fipronil treated rats by 10,0 mg/Kg. GABA levels decreased in fipronil treated rats striatum by 1,0 mg/Kg. Serotonin neurotransmitter was increased in fipronil treated rats forebrain by 1,0 mg/Kg to and its metabolite, 5HIAA, it was observed a levels decrease in fipronil forebrain treated rats by 0,1 e 1,0 mg/kg. There was decrease of 5HIAA levels in fipronil treated rats striatum by 0,1 e 1,0 mg/Kg. According to organ analyses, it was observed dose-response liver damage such as swelling liver cells. In conclusion, the data showed that fipronil administered in rats by 28 days caused behavioral effects related to central neurotransmitter alterations and hepatotoxicity.

O início do desenvolvimento do sistema nervoso central (SNC) é caracterizado por diversos processos, com períodos críticos, que podem ser influenciados por xenobióticos. Este estudo teve como objetivo comparar os efeitos das diferentes concentrações de nicotina em elementos da transmissão sináptica durante o desenvolvimento de camundongos C57Bl/6. Para tanto, foram utilizados cigarros com duas diferentes concentrações de nicotina, o 3R4F (0,73 mg de nicotina/cigarro) e o 4A1 (0,16 mg de nicotina/cigarro). As exposições ocorreram diariamente a partir do 3° dia de vida (P3) até o 14° dia (P14) por duas horas, e os animais controle foram expostos somente ao ar sintético. Os animais foram eutanasiados em P15, P35 e P65 e avaliamos as seguintes proteínas: sinapsina (SYN1), sinaptotagmina (SYT), sinaptofisina (SYP), sinaptobrevina (SYB), PSD-95 e EGR-1, por Western blotting; o gene Egr-1 por PCR Real Time; MAP-2 e neurofilamentos (Neu) por imunofluorescência no hipocampo, tronco encefálico e cerebelo. Nossos resultados mostraram que na infância (P15) a fumaça do cigarro 3R4F diminuiu SYN1, assim como EGR-1, MAP-2 e Neu no cerebelo. No hipocampo houve aumento de SYN1 e MAP-2, diminuição de PSD-95, Egr-1 e Neu. No tronco encefálico esse cigarro não modificou nenhum dos parâmetros avaliados. Nessa mesma idade, a fumaça do cigarro 4A1 diminuiu MAP-2, Neu e a expressão gênica de Egr-1 no cerebelo; diminuiu MAP-2 e Neu no hipocampo e não promoveu alterações no tronco encefálico. Em P35 (adolescência) o 3R4F manteve reduzido SYN1, EGR-1, MPA-2 and Neu, e também reduziu Egr-1 no cerebelo. A diminuição de SYN1 também foi observada no hipocampo, mas nessa estrutura o 3R4F também aumentou SYT, SYP, MAP-2 and Neu; diminuiu EGR-1 e a expressão de Egr-1. No entanto, no tronco encefálico, foram observados aumento de SYN1 e MAP-2, e uma diminuição de Neu. Em relação à fumaça do 4A1, em P35, nossos resultados mostraram diminuição de SYB, Egr-1, MAP-2 and Neu no cerebelo; aumento de Egr-1, MAP-2 e diminuição de Neu no hipocampo; no tronco encefálico houve aumento de SYB, Egr-1, Neu e diminuição de MAP-2. Na fase adulta (P65) as únicas diferenças estatísticas encontradas foram: no cerebelo: diminuição de SYB pelos cigarros 4A1, aumento de EGR-1 pelo 3R4F e aumento de Egr-1 e MAP-2 por ambos os cigarros; no hipocampo: aumento de Neu por ambos os cigarros; tronco encefálico: aumento de SYB e EGR-1 e diminuição de SYT pelo 4A1, diminuição de SYN1, aumento de EGR-1, Egr-1 e MAP-2 pelo 3R4F. Nossos resultados indicam que a exposição à fumaça do cigarro nos primeiros dias de vida de camundongos, mesmo que em baixas concentrações de nicotina, podem acarretar em alterações em elementos da transmissão sináptica em várias regiões encefálicas durante a infância, adolescência e na fase adulta. / The initial steps of the development of the central nervous system are characterized by several critical processes, which can be affected by xenobiotics. The present study aimed to compare the effect of two different nicotine concentrations in cigarettes on synaptic transmission elements during the development of C57/Bl6 mice. To reach this aim we exposed C57Bl/6 mice to smoke from two different cigarettes - 3R4F (0.73mg of nicotine/cigarette) or 4A1 (0.16mg of nicotine/cigarette), from the third day of life (P3) until the fourteenth (P14) for a period of 1h, twice a day (at 8am and at 3pm). The control animals were exposed only to synthetic air. At P15, P35 and P65, the animals were submitted to euthanasia and different encephalic areas were collected (cerebellum, hippocampus and brainstem); quantification of synapsin (SYN1), synaptotagmin (SYT), synaptophysin (SYP), synaptobrevin (SYB), PSD-95 and EGR1 protein expression was assessed by Western blotting, gene expression of Egr-1 by was assessed by RT-PCR and MAP-2 and neurofilaments (Neu) were assessed by immunofluorescence. Our results showed that exposure to 3R4F smoke decreased the quantification of SYN1 at infancy (P15), as well as EGR-1, MAP-2 and Neu at cerebellum. At hippocampus, there was an increase of SYN1 and MAP-2, decrease of PSD-95, Egr-1 and Neu. At brainstem, 3R4F smoke did not modify any parameter. At the same age, 4A1 smoke decreased the quantification of MAP-2, Neu and the expression of Egr-1, at cerebellum; decreased MAP-2 and Neu at hippocampus and did not alter any parameter at brainstem. At P35 (adolescence) 3R4F smoke still reduced SYN1, EGR-1, MAP-2 and Neu, and reduced Egr-1 at cerebellum. The reduction of SYN1 quantification was also observed at hippocampus, but at this area, 3R4F smoke also increased the quantification of SYT, SYP, MAP-2 and Neu and decreased EGR-1 and the expression of Egr-1. However, at the brainstem, an increased quantification of SYN1 and MAP-2 and a decrease of Neu were observed. Regarding 4A1 smoke, at the same age, our results showed a decreased quantification of SYB, Egr-1, MAP-2 and Neu at cerebellum; increase of Egr-1, MAP-2 and decrease of Neu at hippocampus; and at brainstem, increase of SYB, Egr-1, Neu and decrease of MAP-2. At adulthood (P65) the only statistical differences were: at cerebellum - decreased quantification of SYB by 4A1 cigarette smoke, increase of EGR-1 by 3R4F and increase of Egr-1 and MAP-2 by smoke of both cigarettes; at hippocampus - increase of Neu by smoke of both cigarettes; at brainstem - increase of SYB and EGR-1 and decrease of SYT by 4A1 smoke, decrease of SYN1, increase of EGR-1, Egr-1 and MAP-2 by 3R4F smoke. Thus, we can conclude that exposure to cigarette smoke early in life, even at low nicotine concentrations can modify elements of synaptic transmission, compromising such transmission at several encephalic areas.

Placebo treatments are inert but are known to alleviate symptoms across numerous clinical conditions. One of the most studied placebo effects is placebo analgesia, which is a placebo effect limited to pain relief. This thesis aims to introduce the current state of research regarding the neuroscience of placebo analgesia and specifically to present research findings regarding the neurotransmission. Studies have demonstrated that placebo analgesia can be elicited through two separate processes interacting with each other; manipulation of expectations and through conditioning. These processes seem to affect neurotransmission in different ways. Many brain areas have been found to be correlated to placebo analgesia. Besides the pain-processing brain areas, studies point to that the prefrontal cortex can have a vital role in the placebo analgesic effect. Known neurotransmitters that have shown to be involved in placebo analgesia are endogenous opioids, cholecystokinin (CCK), and endocannabinoids. Studies point to that endogenous opioids are involved in the placebo analgesic effect when elicited by expectation or conditioned by an opioid drug. CCK act on placebo analgesia by affecting the release of endogenous opioids and endocannabinoids seem to be involved in placebo analgesia while it occurs due to conditioning with non-opioid drugs. Getting a better understanding of placebo analgesia and find ways to apply this knowledge in the clinical context could powerfully develop the whole medical society.

Glycine receptors (GlyRs) are ligand-gated ion channels (LGICs) that, along with other members of the cys-loop superfamily of receptors, mediate a considerable portion of fast neurotransmission in the central nervous system (CNS). GlyRs are pentameric channels, organized quasi-symmetrically around an ion-conducting pore. Opening of the integral ion pore depends on ligand binding and transduction of this binding signal to the channel gate. Research presented in this dissertation describes a number of critical electrostatic interactions that play a role in conserving the closed-state stability of the receptor in the absence of ligand, ensuring that receptor activation occurs only upon neurotransmitter binding. These amino acids, aspartic acid at position 97 (D97), lysine 116 (K116), arginine 119 (R119) and arginine R131 (R131) are charged residues that interact with one another through electrostatic attraction. When D97 is replaced with any other amino acid this destabilizes the closed state of the channel and causes spontaneous GlyR channel opening. I show that restoration of this electrostatic interaction in GlyR bearing double mutations in which the charges are swapped (D97R/R119E and D97R/R131D) markedly decreases this spontaneous current. In addition, I investigate how these residues that interact at the interfaces between receptor subunits affect the efficacies of GlyR partial agonists. My work shows that the partial agonist taurine is converted into a full agonist at both D97R and R131D receptors. Furthermore, I analyzed the structure of the more extracellular part of the transmembrane (TM) 2 segment that lines the ion channel pore, showing that it is unlikely that this fragment (stretching from T13’ to S18’) is constrained in a true alpha helical conformation. From this work, using disulfide trapping and whole cell electrophysiology, I conclude that a significant level of flexibility characterizes this part of the TM2 domain. This segment includes residue S267, previously shown to be significant for alcohol and anesthetic actions, as well as residue Q266 that, when mutated, produces a hyperekplexia-like phenotype. The range of movement of residues in this region may therefore play an important role not only in channel gating but also in how modulators of GlyR function exert their actions. / text

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Fipronil é um inseticida fenilpirazol de amplo espectro, desenvolvido para inibir seletivamente receptores GABA associados a canais de cloreto de insetos; tanto em Medicina Veterinária, como em Agricultura tem sido utilizado para o controle de pragas. Embora vários estudos procurem compreender os mecanismos da toxicidade neuronal dos praguicidas em mamíferos, há poucos relacionados aos efeitos neurocomportamentais. Assim, o presente estudo investigou os efeitos da exposição prolongada de ratos ao fipronil, observando-se alguns comportamentos ligados ao sistema GABAérgico, como atividade no campo aberto, no labirinto em cruz elevado (LCE), bem como na convulsão induzida por picrotoxina e pentilenotetrazol; avaliou-se ainda os níveis cerebrais de alguns neurotransmissores, como GABA, dopamina, noradrenalina, serotonina e seus respectivos metabólitos. Além disso, foram realizadas análises anátomo-histopatológicas em fígado, cérebro e rins dos animais. Ratos Wistar receberam, por via intragástrica (gavage), durante 28 dias um dos seguintes tratamentos: fipronil (0,1; 1,0 ou 10,0 mg/Kg/dia) ou água destilada 1 mL/Kg/dia. Após uma hora da última administração do fipronil ou água destilada, os ratos tiveram seu comportamento avaliado no campo aberto e no LCE com auxílio do sistema computadorizado EthoVision®. Observou-se no campo aberto apenas redução significante do nº de levantamentos nos animais tratados com 10,0 mg/Kg de fipronil, quando comparados com os ratos do grupo controle. No LCE, a exposição prolongada de ratos ao fipronil nas doses de 1,0 e 10,0 mg/Kg provocou redução na distância percorrida pelos animais e o tempo de permanência dos animais nos braços abertos, além de aumentar o tempo de permanência nos braços fechados. Para o cálculo da dose convulsivante mínima, seguiu-se o mesmo protocolo de tratamento, sendo observada diminuição significante na dose de picrotoxina e de pentilenotetrazol necessária para a indução da convulsão em animais tratados com 10,0 mg/Kg fipronil. A determinação dos níveis de neurotransmissores e seus respectivos metabólitos mostrou aumento dos níveis de dopamina no córtex frontal de ratos tratados com 10,0 mg/Kg de fipronil, redução nos níveis de GABA no striatum de ratos tratados com 1,0 mg/Kg de fipronil. Em relação à serotonina houve aumento de seus níveis no córtex frontal de ratos tratados com 1,0 mg/Kg e seu metabólito, ácido 5 (5HIAA), foi observado redução de seus níveis no córtex frontal de ratos tratados com 0,1 e 1,0 mg/kg p.c. de fipronil. No striatum também houve redução de seus níveis em ratos tratados com 0,1 e 1,0 mg/Kg de fipronil. O estudo anátomo-histopatológico revelou que a exposição prolongada ao fipronil pode causar hepatotoxicidade, caracterizada por aumento dos pesos absoluto e relativo do fígado e tumefação celular de hepatócitos. Em conclusão, os resultados mostraram que a exposição de ratos por 28 dias, via gavage, ao fipronil pode causar efeitos comportamentais relacionados a alterações em sistemas centrais de neurotransmissão e também hepatotoxicidade. / Fipronil is a phenylpyrazole insecticide with broad spectrum, developed to selectively inhibit insect GABA-gated chloride channels; both in Veterinary Medicine, and in Agriculture it has been used for the control of nuisances. 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 long term exposure in rats, being observed some behaviors connected to GABAergic system, as activity in open field, in elevated plus maze (EPM), as well as in the seizures induced by picrotoxinin and pentylenotetrazole. In addition, this study determinated the brain levels of some neurotransmitter as GABA, dopamine, noradrenaline, serotonin and metabolic levels and investigated liver, brain and kidney damage. Wistar rats received by intragastric (gavage) way during 28 days one of this treatment: fipronil (0,1, 1,0 or 10,0 mg/Kg) or distilled water; the rats behavior were evaluated in open field and elevated plus maze apparatus with system computer EthoVision®. There was a significant reduction of rearing in open field in rats treated with 10,0 mg/Kg of fipronil when compared with control rats. In the EPM, there was a decrease of distance moved in open arms and increase of time spent in closed arms to rats treated with 1,0 and 10,0 mg/Kg of fipronil when compared to control. It was still noticed the reduction in the time spent in open arms EPM in rats treated with 1,0 and 10,0 mg/Kg of fipronil when compared to control group. To calculate the minimum convulsant dose, was used the same treatment protocol and it was observed a significant reduction in picrotoxinin and pentylenotetrazole necessary dose to induction of seizure in animals treated with fipronil 10,0 mg/Kg. The level determination of neurotransmitters and their metabolites, it was observed the dopamine increasing in forebrain from fipronil treated rats by 10,0 mg/Kg. GABA levels decreased in fipronil treated rats striatum by 1,0 mg/Kg. Serotonin neurotransmitter was increased in fipronil treated rats forebrain by 1,0 mg/Kg to and its metabolite, 5HIAA, it was observed a levels decrease in fipronil forebrain treated rats by 0,1 e 1,0 mg/kg. There was decrease of 5HIAA levels in fipronil treated rats striatum by 0,1 e 1,0 mg/Kg. According to organ analyses, it was observed dose-response liver damage such as swelling liver cells. In conclusion, the data showed that fipronil administered in rats by 28 days caused behavioral effects related to central neurotransmitter alterations and hepatotoxicity.