Objetivou-se comparar os parâmetros comportamentais indicativos de estresse, a qualidade da carne e a influência do enriquecimento ambiental em leitões criados no sistema intensivo confinado, de acordo com a qualidade dos tratamentos recebidos. Para isso, foram utilizados 144 leitões divididos em três grupos que receberam tratamentos distintos: 1) Tratamento Aversivo (TA): pessoa se dirigia aos leitões de forma rude, utilizando tom de voz agressivo e fazendo ameaças posturais; 2) Tratamento Controle (TC): pessoa foi cuidadosa ao se movimentar e usou tom de voz suave; 3) Tratamento Misto (TM): os animais receberam TA durante as fases de maternidade e creche e TC durante as fases de crescimento e terminação. Após o desmame, os leitões foram submetidos a testes para aferir medo e verificar se conseguiam reconhecer seu tratador habitual. Foram observados os comportamentos nas mudanças de ambiente, através de instantâneos de 5 min. Na fase de crescimento/terminação, metade dos animais recebeu enriquecimento ambiental. Após o abate foi realizada a análise da qualidade da carne com relação ao seu pH, temperatura, coloração, perda de água por cocção e exsudação. Variáveis como cortisol, lactato, creatina-fosfoquinase, ganho de peso, conversão alimentar e consumo de ração também foram aferidas. O delineamento experimental foi inteiramente casualizado e para a análise dos dados foi utilizado o procedimento PROC MIXED e GLM do programa estatístico SAS. Leitões que receberam tratamento aversivo apresentaram maior frequência de interações agonísticas na maternidade (P=0,01), creche (P=0,04), crescimento/terminação (P=0,0002) e no pré-abate (P=0,06); além de apresentarem maior frequência de belly-nosing no pós-desmame (P=0,001), maior atividade nos testes de medo (P=0,01), menor tempo (P=0,03) e menor frequência (P=0,005) de contato com o novo objeto. Animais que receberam enriquecimento ambiental apresentaram maior frequência do comportamento exploratório (P=0,01). Não houve diferença no ganho de peso, nas análises de qualidade de carne e de sangue (P>0,05), exceto para lactato que apresentou concentrações mais elevadas para animais tratados de forma aversiva (P<0,05). Leitões tratados de forma aversiva demonstraram um padrão comportamental indicando o elevado estresse. A simples cessação deste tratamento não parece ser suficiente para que os animais retomem o padrão comportamental comum à espécie, indicando um efeito residual do tratamento recebido anteriormente. Além disso, estes animais são mais medrosos à novidade, ao isolamento social e ao tratador aversivo. Mesmo não havendo diferença de desempenho e qualidade de carne, estes resultados indicam um empobrecimento do bem-estar desses animais. Por outro lado, quando cessado o tratamento aversivo e adicionado o enriquecimento ambiental, os efeitos residuais do estresse sofrido nas primeiras fases de vida foram suprimidos e os animais apresentaram uma melhoria na resposta ao estresse e no seu bem-estar. / The aim of this work was to compare the behavioral parameters indicative of stress, meat quality and the influence of enrichment in pigs housed in intensive confined system, according to the quality of the treatment received. For this, 144 piglets were separated in three groups with different treatment: 1) Aversive (TA): the person responsible for the treatment was rude with the animals, used an aggressive tone of voice and moved harshly and unpredictably; 2) Control (TC): the person responsible for this treatment was careful to move around as was using a soft tone of voice; 3) Mixed (TM): the animals received TA during farrowing room and nursery, and with TC during growing and finishing phase. After weaning, piglets were tested to assess fear and if they could recognize its usual handler. Behavioral time budgets at each change of phase, was registered by scan sampling every 5-min. In the growing-finishing phase half of the animals received environmental enrichment. After slaughter, meat quality was analysed in relation to their pH, temperature, colour, drip loss, water holding capacity e tenderness. Variables such as cortisol, creatine phosphokinase, weight gain, feed conversion and consumption of solid food were also assessed. The experimental design was randomized and to analyse the data, we used PROC MIXED and GLM procedure of SAS statistical program. Animals TA had more frequency of agonistics interactions at farrowing room (P=0.01), nursery (P=0.04), growing/finishing phase (P=0.0002) and at the day before the slaughter (P=0.06); in addition they had higher frequency of belly-nosing after weaning (P=0.001), higher activity on fear tests (P=0.01), lower time (P=0.03) and lower frequency (P=0.005) of a contact with the novel object. Animal that received environmental enrichment showed more frequency of exploratory behaviour (P=0.01). No difference was found on weight gain, meat quality and blood analysis (P>0.05), but to lactate that was higher to aversive treatment animals (P<0.05). Piglets that received an aversive treatment showed behavioral patterns indicative of higher stress. Finishing the aversive treatment was not enough to this animal restoring their behavioral pattern, indicating a residual effect of the aversive treatment. In addition, these animals were more fearful to novelty, social isolation and the aversive handling. Although there is no difference in performance and meat quality, the results show a poor animal welfare. In other hand, when the treatment was finished and an environmental enrichment was introduced, the residual effect of stress of the farrowing room and nursery were supressed and piglets showed an improvement of behavioral pattern and animal welfare.

En permettant aux animaux de faire face à des environnements variables, l'apprentissage et la mémoire contribuent à l'optimisation de leur fitness, en leur permettant d'extraire et d'utiliser des informations, de façon à réduire l'incertitude associée à des environnements imprévisibles. Parmi les insectes, la drosophile et l'abeille domestique sont considérés comme des modèles classiques pour l'étude de l'apprentissage et de la mémoire. Les travaux réalisés sur ces derniers ont apporté une quantité considérable d'informations concernant les bases génétiques, neurobiologiques et moléculaires de ces processus, et ont permis de rendre compte du niveau de complexité des capacités cognitives des insectes. Cette somme de connaissances fondamentales acquises chez ces insectes contraste étonnamment avec le faible niveau de connaissance concernant la cognition des espèces impliquées dans des problématiques qui touchent la santé humaine et animale. Pourtant, il est largement admis que l'étude détaillée des capacités cognitives des insectes vecteurs de maladies constitue un aspect prioritaire pour la compréhension de leurs adaptations à la vie hématophage, de leur importance vectorielle, ainsi que pour le développement de nouveaux outils pour leur contrôle. Les travaux réalisés à ce jour chez les vecteurs, principalement chez les moustiques, ont été menés dans des contextes naturels ou peu contrôlés et ne proposent donc pas de démonstration formelle d'apprentissage. Le principal objectif de ce travail de thèse est de proposer un cadre expérimental contrôlé permettant de mettre en évidence et caractériser les capacités d'apprentissage chez la punaise hématophage \textit{Rhodnius prolixus}. \`A la différence des moustiques, les caractéristiques biologiques de cette punaise hématophage, responsable de la transmission de la Maladie de Chagas en Amérique Latine, permettent l'adaptation de protocoles expérimentaux largement validés chez les drosophiles et l'abeille domestique. Nos résultats montrent dans un premier temps que ces insectes sont capables d'apprendre et d'associer la présentation d'une même odeur dite neutre (l'acide lactique), c'est-à-dire qui ne provoque ni attraction ni répulsion lorsqu'elle est présentée seule, avec soit la possibilité d'obtenir une récompense (un repas sanguin, conditionnement appétitif), soit avec la possibilité de recevoir une punition (un choc mécanique, conditionnement aversif). Nous avons également montré que l'apprentissage et la mémoire sont également impliqués dans le choix des hôtes. Les insectes ont en effet associé la présentation d'un choc mécanique avec le complexe d'odeur d'hôtes naturels, biaisant leur préférence lors d'un test de choix réalisé après l'entraînement. Dans un second temps, nous avons adapté à notre modèle d'étude le paradigme de conditionnement de la réponse d'extension du proboscis, développé chez les modèles classiques, ce qui a permis la caractérisation des capacités d'apprentissage, de la durée de rétention à la régulation par des horloges circadiennes. Ces travaux proposent également un paradigme expérimental, reproductible et efficace permettant d'analyser les mécanismes fins qui sous-tendent les processus d'apprentissage et de mémorisation. Dans son ensemble, cette étude apporte la première preuve expérimentale de la capacité d'apprentissage d'insectes vecteurs de la maladie de Chagas et propose des outils expérimentaux et méthodologiques permettant d'améliorer la compréhension des processus associés chez les insectes hématophages en général. Les résultats sont également discutés dans le contexte de la sélection d'hôte et de la transmission des parasites. / Learning and memory contribute to animals' fitness by allowing them adapting to variable environments. Thses two processes make them able to extract and use information from their environment in order to reduce the uncertainty associated with unpredictible environments. Among insects, fruit flies and honeybees are considered as classical models for the study of learning and memory. The amount of work that has been done on these models provide a considerable amount of information regarding the genetic, neurobiological and molecular basis of these processes and revealed the complexity of insects' cognitive abilities. All this knowledge acquired in model species, contrasts surprisingly with the lack of knowledge available regarding insect species that are involved in animal and human diseases transmission. Yet, it has been aknowledge that the detailed study of vectors cognitive abilities would allow the understanding of their adaptation to haematophagy, of their vectorial importance and provide new tools for diseases control. Up to date, studies focusing on disaese vectors, mainly in mosquitoes, were conducted in natural or not completely controled contexts and thus no clear demonstration of learning and memory is availaible.The main goal of this work was to provide a controled experimental context allowing the strudy of learning abilities in the haematophagous bug \textit{Rhodnius prolixus}. Our results show that these insectes are able to learn to associate the delivery of a same neutral odour either with the possibility to obtain a reward (blood-meal, appetitive conditioning) or with the possibility to receive a punishment (mechanical shock, aversive conditioning). We also showed that learning ans memory are involved in host selection processes. In a second part, we adapted to our biological model the paradigm of proboscis extension response conditioning, which allowed us to analyse and characterize its learning abilities. The maximal retention duration as well as the modulation of learning abilities by circadian clocks were evinced. Taken as a whole, this work provides the first experimental demonstration of learning abilities in Chagas disease vectors and provides experimental and methodological tools; These latters should allow improving the understanding of the mechanisms that are underlying cearning abilities of haematophagous insects in general. Results are also discussed in the context of host selection and parasite transmission.

A seleção genética de comportamentos relacionados a um componente emocional do tipo ansiedade, associada ao estudo das possíveis influências desse traço sobre outros componentes emocionais como o medo, impulsividade e tarefas de aprendizagem aversiva, podem contribuir para o entendimento da ansiedade, bem como, fornecer uma base sólida para investigações neurofisiológicas, bioquímicas e moleculares da mesma. Assim, o presente trabalho selecionou duas linhagens de ratos da linhagem Wistar: uma com característica de alta exploração dos braços abertos do labirinto em cruz-elevado (Filô-HE) e outra com baixa exploração desses braços (Filô-LE), e comparou os efeitos da seleção dessas duas linhagens sobre comportamentos associados à ansiedade, medo, impulsividade e aprendizagem aversiva. Para isso, um lote de animais fornecidos pelo Biotério Central da USP-RP foram distribuídos em dois grupos distintos: (1) os que apresentavam maior exploração dos braços abertos do Labirinto em Cruz Elevado (LCE) - (Filô-HE) e (2) os com menor exploração desses braços (Filô-LE) ao longo de doze gerações (de S1 a S12, sem contar o grupo inicial do qual derivam as gerações). Na sexta, nona e décima segunda gerações, os animais foram submetidos aos seguintes testes comportamentais: LCE, Campo Aberto (somente na décima segunda), esquiva passiva, labirinto aquático de Morris (somente na décima segunda), sobressalto (que avalia a resposta de medo), e gradiente de aversão elevado (desenvolvido em nosso laboratório para medir impulsividade). Os dados indicaram a seleção de duas linhagens de ratos Wistar com características distintas para a exploração dos braços abertos do LCE. De um lado, foi selecionada a linhagem Filô-HE, que possui como principal característica, altos índices de exploração de ambientes que geralmente são evitados por outros animais. De outro, foi selecionada (até a oitava geração), a linhagem Filô-LE, que explorava muito pouco ambientes potencialmente aversivos. Tais resultados podem ser interpretados, respectivamente, como (a) baixa e alta frequência de comportamentos associados à ansiedade e medo, (b) baixa e alta frequência de comportamentos associados à impulsividade, (c) altos e baixos níveis de dificuldade na aprendizagem relacionada a memórias emocionais, ou (d) até mesmo o efeito conjunto de todas essas características comportamentais. Porém, para um conhecimento mais confiável, é necessário que os estudos supracitados passem a explorar aspectos neurofisiológicos, psicofarmacológicos, bioquímicos e genéticos, ampliando a investigação dos componentes emocionais selecionados. Bem como, promovendo um controle mais rígido dos traços selecionados e de suas relações com outros componentes que possam interferir na seleção / The genetic selection of behaviors related to an anxiety-like emotional component, associated to the study of the possible influences of this trait over other emotional components like fear, impulsivity and aversive learning, can contribute to the understanding of anxiety as well as supply a solid basis for its neurophysiological, biochemical and molecular investigation. Thus, the present work selected two strains of Wistar rats: one characterized by a high exploration of the open arms of an elevated plus-maze (Filô-HE) and another with a low exploration of these arms (Filô-LE), and compared the effects of such a selection on behaviors associated with anxiety, fear, impulsivity and aversive learning. For this, a batch of animals supplied by the animal house of the USP at Ribeirão Preto were distributed into two distinct groups: (1) one presenting more exploration of the open arms of an elevated plus-maze (EPM) Filô-HE and (2) one with less open arm exploration Filô-LE along twelve generations (from S1 to S12, not counting the initial group from which all generations derived). On the sixth, ninth and twelfth generations the animals were submitted to the following behavioral tests: LCE, open-field (only in the twelfth generation), passive avoidance, Morris aquatic maze (only in the twelfth generation), startle (which evaluates a fear response), and elevated aversion gradient (developed in our laboratory to measure impulsivity). The data indicated a selection of two Wistar rats strains with distinct characteristics concerning the exploration of the open arms of and EPM. On the one hand, the strain Filô-HE was selected exhibiting as the main characteristic high measures of exploration of environments which are usually avoided by other animals. On the other hand, the strain Filô-LE was selected (up to the eighth generation) exhibiting very little exploration of potentially dangerous environments. These results may be interpreted as, respectively, (a) low and high frequency of behaviors associated to anxiety and fear, (b) low and high frequency of behaviors associated to impulsivity, (c) high and low levels of difficulty in learning related to emotional memories, or (d) even the joint effect of all these behavioral characteristics. However, for a more reliable knowledge it is necessary that the studies above begin to explore neurophysiological, psychopharmacological, biochemical and genetic aspects, broadening the investigation ot hte selected emotional components, as well as promoting a more strict control of the selected traits and of its relations with other components that may interfere in the selection

Submitted by ANA KARLA PEREIRA RODRIGUES (anakarla_@hotmail.com) on 2017-10-03T16:47:19Z No. of bitstreams: 1 arquivototal.pdf: 2613082 bytes, checksum: c11de66e961fb0750071b4fd9279d082 (MD5) / Made available in DSpace on 2017-10-03T16:47:19Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 2613082 bytes, checksum: c11de66e961fb0750071b4fd9279d082 (MD5) Previous issue date: 2017-03-31 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Introduction: Adolescence is a crucial period of development, in which the body presents a high level of plasticity. At the present time, adolescents spend more time active and exposed to light at night reducing the amount of daily sleep. To investigate the possible effects of this condition on anxiety and aversive memory, adolescent rats underwent the Open Field (OF) and Passive Avoidance (PA) tasks under a 22h EC cycle. Objective: To evaluate the effects of a 22h light dark (LD) cycle on the locomotor activity of adolescent rats anxiety and aversive memory. Methods: 44 male Wistar rats were divided into two groups: T24, n = 13 (EC 12:12 h) and T22, n = 31 (EC 11:11 h), which were kept under controlled conditions of temperature and humidity, with water and food at will. The tests were performed in the middle of the dark phase for all groups, but the T22 group was subdivided into two groups that performed the tests, respectively: in the middle of the night coincidence between the environment and the biological night of each T22 mouse (n = 16) and in the middle of the night of non-coincidence (n= 15). The OF and PA tasks were performed when the animals had between 40 and 60 days. All sessions were recorded for total distance travelled, total time, velocity and minimum and maximum anxiety indexes (OP), and latency to step down from a platform in PA after 30 min for both groups and after 24 h (For the T24 group) and 22 h (for the T22 group) for evaluation of the short and long term memories, respectively. The locomotor activity was recorded continuously and totalized at 5 min intervals. To compare the means between the groups in the OF test, a 1-way ANOVA was performed for independent samples and the Kruskal Wallis test for PA. Results: T22 animals presented two simultaneous circadian rhythms in locomotor activity: one with the same period of the LD cycle and another that was expressed in free-stroke. All animals in the control group synchronized to LD 24h, showing a stable rhythm synchronized to LD. No differences were found in the performance of the groups in the OF and PA. Conclusions: The T22h promotes dissociation in the circadian rhythm of locomotor activity without altering the levels of anxiety or impairing the aversive memory of adolescent rats. / A adolescência é um período crucial do desenvolvimento, no qual o organismo apresenta um alto nível de plasticidade. Na atualidade, adolescentes passam mais tempo ativos e expostos à luz durante a noite reduzindo a quantidade diária de sono. Para investigar os possíveis efeitos dessa condição na ansiedade e memória aversiva, ratos adolescentes passaram pelas tarefas de Campo Aberto (CA) e Esquiva Passiva (EP) estando sob um ciclo claro-escuro (CE) de 22h. Objetivo: Avaliar os efeitos de um ciclo CE de 22 h sobre a atividade locomotora de ratos adolescentes e ansiedade e memória aversiva. Metodologia: Foram utilizados 44 ratos Wistar machos, divididos em 2 grupos: T24, n=13 (CE 12:12 h) e T22, n=31(CE 11:11 h), que ficaram sob condições controladas de temperatura e umidade, com água e ração à vontade. Os testes aconteceram no meio da fase escura para todos os grupos, porém o grupo T22 foi subdividido em dois grupos que realizaram os testes, respectivamente: no meio da noite de coincidência entre o ambiente e a noite biológica de cada rato do T22 (n=16) e no meio da noite de não-coincidência (n=15). As tarefas de CA e EP foram realizadas quando os animais tinham entre 40 e 60 dias. Todas as sessões foram filmadas para contabilização da distância total percorrida, tempo total, velocidade e índices de ansiedade mínimo e máximo (thigmotaxis) no CA, e latência do tempo de descida da plataforma na EP após 30 min para ambos os grupos e após 24 h (para o grupo T24) e 22 h (para o grupo T22) para avaliação das memórias de curto e longo prazo, respectivamente. A atividade locomotora foi registrada continuamente e totalizada em intervalos de 5 min. Para comparação das médias entre os grupos no teste de CA foi aplicado o teste ANOVA de uma via para amostras independentes e o teste de Kruskal Wallis para a EP. Resultados: Os animais do T22 apresentaram dois ritmos circadianos simultâneos na atividade locomotora: um com o mesmo período do ciclo CE e outro que se expressou em livre-curso. Todos os animais do grupo controle sincronizaram ao CE 24h, demonstrando um ritmo estável sincronizado ao CE. Não foram encontradas diferenças no desempenho dos grupos no CA e na EP. Conclusões: O ciclo CE de 22 h promove dissociação no ritmo circadiano de atividade locomotora sem alterar os níveis de ansiedade, nem prejudicar a memória aversiva de ratos adolescentes.

Orientador: Elenice Aparecida de Moraes Ferrari / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-18T00:49:19Z (GMT). No. of bitstreams: 1 Machado_AlineVilardaSilva_M.pdf: 2487506 bytes, checksum: ec15fc1b78bef814d6d459499276cdf5 (MD5) Previous issue date: 2011 / Resumo: A ritmicidade circadiana, expressa na alteração do comportamento e em aspectos morfofisiológicos e moleculares ao longo das 24 horas do dia, é uma das funções básicas dos organismos vivos. Os processos comportamentais e os mecanismos moleculares no hipocampo, que estão envolvidos na aprendizagem e memória, demonstram oscilação circadiana. Vários estudos sugeriram que o condicionamento clássico aversivo é afetado pelo sistema de temporização circadiana e que a oscilação circadiana de vias moleculares específicas é requerida para a consolidação da memória aversiva. O presente trabalho investigou a oscilação circadiana da expressão da nNOS e da atividade da proteína NOS no hipocampo de pombos e as suas relações com a modulação temporal do condicionamento contextual aversivo. Na Parte I, caracterizou-se o padrão temporal da expressão da nNOS, que foi detectada por Western Blotting e o padrão temporal da atividade enzimática da NOS, determinada pela quantidade de L-citrulina produzida por minuto e por micrograma de proteína na reação. Na Parte II, nos horários de mínima e máxima atividade enzimática da proteína, pombos foram treinados e testados em condicionamento aversivo ao contexto. As sessões foram gravadas para posterior análise comportamental. Após o teste foi realizada a imunoistoquímica para marcação da nNOS em neurônios do hipocampo. Foi evidenciada ritmicidade circadiana significativa (p < 0,05) na expressão protéica da nNOS e na atividade enzimática da NOS, segundo os valores fornecidos pelo método Cosinor para caracterização do padrão temporal. As médias da densitometria óptica dos grupos com horários mais próximos da acrofase - ZT04 (10hs; 0,944 ± 0,12) e a batifase - ZT16-(22hs; 0,572 ± 0,16) foram significativamente diferentes (F5,18 p < 0,0001). Os grupos condicionados, em ambos os horários, mostraram maior duração e maior ocorrência do comportamento de congelamento do que os controles (p < 0,05). Houve uma variação dia-noite para o comportamento de congelamento nos grupos controles (p < 0,05). A marcação de células nNOS-positivas foi maior no hipocampo dos grupos condicionados sendo que o total de células nNOS-positivas na área dorsal do grupo experimental testado à noite foi maior do que aquele observado nos grupos controles e no experimental da manhã (p < 0,05). Os dados mostraram que a expressão protéica da nNOS e da atividade enzimática da NOS no hipocampo de pombos mostram uma oscilação que caracteriza um padrão temporal circadiano. Tanto no horário de máxima como no de mínima atividade da nNOS, o condicionamento contextual aversivo resultou em medo condicionado ao contexto e em expressão de células nNOS-positivas no hipocampo que foi maior nos pombos condicionados do que nos controles. Contudo, no hipocampo do grupo testado à noite houve um maior número de células nNOS-positivas. Esse dado estimula questionamento sobre se ocorreria a ativação de mecanismos compensatórios para o aumento da expressão da proteína nNOS, quanto essa é requisitada em situações de baixa disponibilidade / Abstract: The circadian rhythm, expressed in changing behavior and the morphophysiologic and molecular aspects over 24 hours of the day is one of the basic functions of living organisms. The behavioral processes and molecular mechanisms in the hippocampus, which are involved in learning and memory, show circadian oscillation. Several studies have suggested that classical fear conditioning is affected by the circadian timing system and the circadian oscillation of specific molecular pathways is required for the consolidation of aversive memory. This study investigated the circadian oscillation of nNOS expression and activity of NOS protein in the hippocampus of pigeons and their relationship with the temporal modulation of aversive contextual conditioning. In Part I, we have characterized the temporal pattern of nNOS expression, which was detected by Western blotting and temporal pattern of NOS enzyme activity, determined by the amount of L-citrulline produced per minute and per microgram of protein in the reaction. In Part II, at the times of minimum and maximum activity of the protein, pigeons were trained and tested in aversive conditioning to context. The sessions were taped for later behavioral analysis. After the test was performed immunohistochemical for labeling of nNOS in neurons in the hippocampus. Circadian rhythm was evident (p <0.05) in nNOS protein expression and enzyme activity of NOS, according to figures provided by Cosinor method to characterize the temporal pattern. The mean optical density of groups with times closer to the acrophase - ZT04 (10hrs; 0.944 ± 0.12) and nadir - ZT16-(22hs; 0.572 ± 0.16) were significantly different (F5, 18 p <0.0001 ). The groups conditioned in both schedules, showed more frequent and longer duration of freezing behavior than controls (p <0.05). There was a day-night variation for freezing behavior in control groups (p <0.05). Labeling of nNOS-positive cells was higher in the hippocampus of the groups conditioned with total nNOS-positive cells in the dorsal area of the experimental group tested at night was higher than that observed in control groups and experimental group in the morning (p <0.05). The data showed that nNOS protein expression and enzymatic activity of NOS in the hippocampus of pigeons show an oscillation that characterizes a circadian temporal pattern. Both at the time of maximum as the low activity of nNOS, the aversive contextual conditioning resulted in fear conditioning to context and expression of nNOS-positive cells in the hippocampus was higher in pigeons conditioned than in controls. However, in the hippocampus of the group tested in the evening there was a greater number of nNOS-positive cells. This fact encourages questioning of whether there would be activation of compensatory mechanisms for the increased expression of nNOS protein, as this is required in situations of low availability / Mestrado / Fisiologia / Mestre em Biologia Funcional e Molecular

Orientador: Elenice Aparecida de Moraes Ferrari / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-14T04:52:12Z (GMT). No. of bitstreams: 1 Canova_Fernando.pdf: 32640695 bytes, checksum: 6c06f9d94ba0675ea059bb7d42685788 (MD5) Previous issue date: 2009 / Resumo: Uma grande parte do conhecimento sobre as bases e mecanismos neurais dos processos de aprendizagem, memória e amnésia fundamenta-se na investigação dos correlatos neurais do comportamento de animais não humanos em situações aversivas. Vários estudos têm resultados sugestivos de que o condicionamento clássico aversivo é afetado pelo sistema de temporização circadiana. As análises do condicionamento clássico aversivo e da resposta condicionada de congelamento (FRZ) são úteis para as análises do comportamento e dos processos mecanismos neurais subjacentes. Estudos prévios mostraram a indução da expressão de Zenk no hipocampo de pombos pelo treino em condicionamento clássico aversivo e pelo teste no contexto aversivo. O presente estudo investigou as variações dia - noite na aquisição e na evocação do condicionamento clássico aversivo e na expressão da proteína Zenk no hipocampo. O Experimento I avaliou o condicionamento ao contexto em pombos divididos em grupos condicionados (EC), controle (CC) e manipulação (CM) e o Experimento II investigou o condicionamento aversivo ao som previamente associado ao choque utilizando de animais que receberam som-choque pareados (EP), som-choque não pareados (NP) ou som(GS). Nos dois experimentos foi utilizado o fotoperíodo com pulsos de luz (15 min) às 6h (ZT00) e às 18h (ZT12), sendo as sessões realizadas nos horários ZT02 e ZT14. Os resultados do Experimento I indicaram diferença significativa na ocorrência de congelamento entre os grupos (p<0,05), mas não entre os horários (p>0,05). Comparações entre bloco final do treino e bloco inicial do teste mostraram queda significativa na ocorrência de congelamento no bloco inicial do teste no grupo EC ZT02 (p<0,05, mas não no EC ZT14 (p>0,05). A análise de células Zenk positivas no hipocampo mostrou aumento significativo na região HpV em relação a HpD do grupo EC ZT02 em comparação aos demais grupos (p<0,05), demonstrando uma diferença significativa de horário. Houve aumento significativo na marcação de núcleos Zenk-positivos em HpVM do grupo EC ZT02 em comparação aos demais grupos (p<0,05) e em relação a HpVL (p<0,05). No Experimento II houve maior ocorrência de exploração cautelosa nos grupos EP e NP (p>0,05) em comparação aos grupos GS (p<0,05) e diferença significativa na ocorrência de congelamento entre os grupos EP e GS (p<0,05), mas não entre os horários (p>0,05). A marcação de núcleos Zenk-positivos em HpV foi maior do que em HpD nos animais que receberam som e choque pareados ou não-pareados (p<0,05). Não houve diferenças significativas na marcação de Zenk em HpVL e HpVM nos diferentes grupos e nos dois horários (p>0,05). Os dois experimentos indicaram a expressão de diferentes padrões comportamentais frente ao contexto aversivo condicionado e ao som aversivo condicionado. As variações na expressão de Zenk são indicativas de ativação diferencial de HpD, HpVM e HpVL durante a evocação da memória do contexto e do som. A existência de variações dia - noite na ocorrência de condicionamento ao contexto e na expressão de Zenk no hipocampo sugere uma modulação do sistema temporizador circadiano sobre esses processos. Palavras-Chave: Condicionamento clássico aversivo; Hipocampo; Proteína Zenk; Fotoperiodo esqueleto / Abstract: Part of the knowledge about the mechanisms and neural basis of learning, memory and amnesia is based on the investigation of neural correlates of the behavior of non human animals in aversive situations. Moreover, many studies suggest that these behavioral processes are affected by the circadian timing system. The procedures of classical aversive conditioning and analysis of the conditioned freezing response are useful for the study of behavior and the underlying neural mechanisms. Previous studies showed the induction of Zenk expression in the hippocampus of pigeons after training in classical aversive conditioning. This study investigated day - night variations in the acquisition and retrieval of aversive classical conditioning and in the expression of Zenk protein in the hippocampus of pigeons. The Experiment I evaluated the conditioning to the context in pigeons attributed to conditioned (COND), control (CC) or naive groups (N). The Experiment II investigated the aversive conditioning to the tone in groups of pigeons that received toneshock pairing (PS), unpaired tone and shock (NPS) or tone alone (TS). In both experiments a photoperiod was used with pulses of light (15 min) at 6h (ZT00) and 18h (ZT12) and the sessions were conducted at ZT02 and ZT14. The results of the Experiment I indicated between group differences in the occurrence of freezing (p<0.05). A significant decrease in freezing was observed in the initial block of the test as compared to the final block of the training session of the COND ZT02 (p<0.05), but not in the COND ZT14 group (p>0.05). The analysis of the Zenk-positive nuclei in the hippocampus showed a significant increase in the HpV of the COND ZT02 group compared with the other groups (p<0.05), indicating a significant time difference when compared to COND ZT14 (p<0.05). Higher density of Zenk-positive nuclei was found in HpVM of COND ZT02 group compared to HpVL (p<0.001) and to HpVM of other groups (p<0.05). Experiment II data showed higher occurrence of exploratory risk assessment in PS and NPS groups (p>0.05) as compared to TS (P<0.5). The occurrence of freezing in the PS groups was different from TS groups (p<0,05), but not from NPS groups (p>0.05). No significant time of the day variation of freezing was detected (p> 0.05). Zenk- positive nuclei densities were higher in HpV than in HpD in the animals trained with paired or unpaired tone and shock stimulation (p <0.05). These PS and NPS groups showed no significant differences in the density of labeling of Zenk-positive nuclei in HpVL as compared to HpVM (p>0.05). Data from both experiments showed different behavioral patterns during the exposure to the conditioned aversive context and to the conditioned aversive tone. The variations in Zenk expression indicate differential activation of the HpD, HpVL and HpVM regions during retrieval of aversive memory of the context and of the tone. The day-night variation in the conditioned freezing to the context as well as in Zenk expression in the hippocampus suggests a modulation of these processes by the circadian timing system. Keywords: Classical aversive conditioning; Hippocampus; Zenk protein. Skeleton photoperiod / Universidade Estadual de Campi / Fisiologia / Mestre em Biologia Funcional e Molecular

Orientador: Elenice Aparecida de Moraes Ferrari / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-25T18:07:58Z (GMT). No. of bitstreams: 1 Faria_RodolfoSouza_D.pdf: 4131498 bytes, checksum: 19063a2cb7dfffa9e3e542eeb3359cfc (MD5) Previous issue date: 2014 / Resumo: O Fator Neurotrófico Derivado do Cérebro (BDNF) participa de processos de plasticidade sináptica subjacentes à aprendizagem e memória. A ação do BDNF no hipocampo e na amígdala é necessária para à formação de memória emocionais, incluindo a memória de medo. Além disso, foi demonstrado em roedores que a persistência da memória aversiva depende um pico tardio na expressão de BDNF-maduro que ocorre no hipocampo. Contudo, ainda são necessárias investigações sobre o papel do BDNF-maduro na plasticidade sináptica e processos de memória em aves. Neste estudo o Experimento 1 investigou se o condicionamento som-choque induz um ciclo tardio de expressão do BDNF-maduro no hipocampo e amígdala de pombos. Pombos adultos receberam implante bilateral de microcânula intra-hipocampal e, sete dias após foram treinados com três pareamentos som-choque e designados para um dos três grupos: condicionamento e anisomicina (CondANI) , condicionado e salina (CondSAL) e condicionado não tratado (Cond). O grupo NAIVE não teve tratamento ou condicionamento. O Experimento 2 investigou se o bloqueio da ação do BDNF-maduro por infusão intra-hipocampal de anisomicina, e do antagonista do receptor TrkB, K252a, interferem na persistência da memória de medo condicionado ao contexto e ao som. Foram usados grupos de pombos que receberam infusão intra-hipocampal 11 h após o treino de salina (CondSAL2 e CondSAL7), anisomicina (CondANI2 e CondANI7), ou K252a (CondK2 e CondK7), e também controles não tratados (Cond2 e Cond7). Os animais foram testados ao contexto e a som 2 ou 7 dias após treino. No Experimento 1 a análise por Western blotting indicou que os valores de BDNF-maduro no hipocampo dos pombos Cond e CondSAL foram maiores do que nos pombos CondANI e NAIVE (p < 0,05). Os valores de BDNF-maduro na amígdala não diferiram entre os grupos condicionados (p > 0,05). No Experimento II não houve diferenças significativas entre grupos no teste ao contexto realizado 2 dias após o treino (p > 0,05), mas no quando testados 7 dias após o treino os grupos CondANI7 e CondK7 apresentaram menor ocorrência de congelamento do que Cond7 e CondSAL7 (p < 0,05). Não houve diferenças significativas entre grupos nos testes ao som realizados 2 e 7 dias após o condicionamento (p > 0,05). Os dados do Experimento I indicaram que o treino em condicionamento clássico aversivo induziu o aumento tardio da expressão BDNF-maduro no hipocampo e na amígdala, enquanto que os dados do Experimento 2 demostraram que a infusão intra-hipocampal de anisomicina e de K252a prejudicaram a persistência da memória de medo condicionado ao contexto, mas não a memória de medo condicionado ao som. Esses resultados estariam relacionados com uma dissociação entre o hipocampo e amígdala no processamento das informações contextuais e sonoras no medo condicionado. Em conjunto nossos dados evidenciam que a experiência do condicionamento ativa uma série de eventos moleculares que levam ao aumento da expressão do BDNF-maduro no hipocampo e na amígdala, garantindo a regulação fina da plasticidade neuronal necessária para a memória / Abstract: The Brain Derived Neurotrophic Factor (BDNF) participates in processes of synaptic plasticity underlying learning and memory. The action of BDNF in the hippocampus and amygdala is necessary for the formation of emotional memory, including fear memory. Furthermore, it was demonstrated that rodent aversive memory depends on persistence of a late peak in the expression of mature BDNF, which occurs in the hippocampus. However, we still need further investigations on the role of mature BDNF in synaptic plasticity and memory processes in birds. In this study, Experiment 1 investigated whether the tone-shock conditioning induces a late cycle-expression of mature - BDNF in the hippocampus and amygdala of pigeons. Adult pigeons underwent bilateral implantation of intra-hippocampal microcanullae and seven days later were trained with three tone-shock pairings and assigned to one of three groups: conditioning and anisomycin (CondANI), conditioning and saline (CondSAL) and untreated conditioning (Cond). The NAIVE group had no treatment or conditioning. Experiment 2 investigated whether blockade of the action of mature-BDNF with intra-hippocampal infusion of anisomycin or of TrkB receptor antagonist, K252a, interferes with the persistence of memory of conditioned contextual fear and conditioned tone fear. Groups of birds that at 11 h after the training received intra-hippocampal infusion of saline (CondSAL2 and CondSAL7) anisomycin (CondANI2 and CondANI7) or K252a (CondK2 and CondK7) as well as untreated controls (COND2 and Cond7) were used. Tests to the context and to the tone were conducted 2 days or 7 days after training. In Experiment 1 analysis by Western blotting indicated values of mature-BDNF in the hippocampus of pigeons and Cond CondSAL that were higher than in CondANI and NAIVE pigeons (p < 0.05). The values of mature-BDNF in the amygdala did not differ between the conditioned groups (p > 0.05). In Experiment II there were no significant differences between groups in the context test conducted 2 days after training (p > 0.05), but when tested 7 days after training both the CondANI7 and CondK7 groups showed lower occurrence of freezing than Cond7 and CondSAL7 (p < 0.05). There were no significant differences between groups in the tests to the tone conducted 2 or 7 days after conditioning (p > 0.05). The data of Experiment I indicated that training in classical aversive conditioning induced increase in late expression of mature-BDNF in the hippocampus and amygdala, whereas the data from Experiment 2 showed that intra-hippocampal infusion of anisomycin and K252a disrupted persistence of the memory of contextual fear conditioning, but not of fear memory to the tone. These results could be related to a dissociation between the hippocampus and amygdala in processing auditory and contextual information in fear conditioning. Taken together our data show that the conditioning experience activates a series of molecular events that lead to increased expression of mature-BDNF in hippocampus, and amygdala, which guarantee regulation of neuronal plasticity required for memory / Doutorado / Fisiologia / Doutor em Biologia Funcional e Molecular

The zebrafish (Danio rerio) promises to meet the growing needs of gerontological and neurobehavioral research by possessing highly conserved anatomy and physiology with all other vertebrates, while having low maintenance costs and requiring only simple care. The neurological and physiological bases of learning, memory formation, and memory retention have been studied in a variety of model organisms, such as the rat, mouse, sheep, and several teleost fishes, the notable example being the zebrafish. Unfortunately, most of these animals are poorly suited to senescence research due to costs, care requirements, or long life spans. My research expands upon our rapidly growing understanding of zebrafish neurobiology, learning processes, sensory modalities, and memory retention. Two pairs of distinct aversive conditioning experiments using classic shuttlebox designs compared the effects of sensory modality and conditioned-unconditioned stimulus (CS-US) intervals, the time delay between application of conditioned sensory stimulus and delivery of the stressful unconditioned stimulus in the event of failure to avoid it, upon memory formation and retention. These studies yielded a general spectrum of results against which future conditioning studies may be compared. Both visual and olfactory stimuli were tested, as were 10 second and 15 second CS-US intervals. Successes were scored when the fish crossed the shuttlebox hurtle within the CS-US interval, thereby avoiding the negative unconditioned stimulus. After a three-month delay, ten additional trials were conducted to compare the long-term memory retention resulting from each protocol. When testing a 15s CS-US interval, olfactory conditioning was significantly more likely (39%) to produce a successful outcome (memory formation) than visual conditioning. Grouped results reveal that the second pair of experiments, each with a 10s CS-US interval, yielded significantly more successful memory formation than a 15s CS-US interval. A significant difference was found when comparing the results of any two experiments, except between the results of the visual and olfactory 10s interval experiments). Only the olfactory experiment using a 15s CS-US interval yielded memory retention results significantly higher than the mean of memory retention results from the four experiments. These findings offer inconclusive evidence supporting olfaction’s strong role in memory formation and retention in zebrafish. The results expand our understanding of the relationship between the olfactory and visual senses and memory in the zebrafish and indicate the olfactory sense’s key role in vertebrate neurobiology, warranting further research into the effects of aging on the olfactory-memory modality.

Les ganglions de la base, auxquels appartiennent le striatum et le globus pallidus, sont un ensemble de structures sous-corticales impliquées dans des fonctions motrices, cognitives et motivationnelles. Il a également été montré qu’ils font partie du système de récompense, système assurant la motivation d’un organisme à initier des comportements d’approche, de façon à obtenir une récompense, puis à renforcer les activités ayant produit ces comportements pour pouvoir les reproduire par la suite, ce qui constitue la base de l’apprentissage instrumental. Or, dans certaines situations, le comportement à initier va dépendre d’une motivation à fuir ou à éviter un événement ou une situation aversive. Longtemps, on a pensé que le système du traitement des informations aversives était distinct de celui de la récompense, en termes de réseau et de régions cérébrales. Pourtant, de plus en plus d’études montrent à présent que les ganglions de la base ont certainement un rôle à jouer, non négligeable, dans le traitement des situations aversives. Dans ce travail de thèse, nous avons réalisé chez le primate non-humain des enregistrements d’activité neuronale et des perturbations pharmacologiques locales dans deux régions du système de récompense, le striatum antérieur et le pallidum ventral, au cours d’une tâche comportementale nécessitant tour à tour l’initiation de comportements d’approche vers une récompense et de comportements d’évitement d’un événement aversif. Nous avons montré l’existence d’un codage de l’information aversive dans ces régions tant pour un stimulus prédicteur d’un événement aversif, la préparation ou l’initiation d’un comportement d’évitement de cet événement, que pour l’anticipation et la réception de cet événement. Par ailleurs, les expériences de perturbations locales ont démontré qu’une atteinte du fonctionnement normal du striatum antérieur et du pallidum ventral affectait les comportements initiés normalement en contexte aversif. L’ensemble de ces résultats indique clairement que ces deux structures cérébrales, si elles sont impliquées dans la motivation à initier des comportements dirigés vers un but récompensant et l’apprentissage appétitif, le sont aussi dans la motivation aversive / Striatum and globus pallidus belong to the basal ganglia, which are a group of subcortical structures involved in motor, cognitive and motivational functions. They are also involved in the reward system which enables one’s motivation to initiate approach behaviors in order to get a reward and then consolidate activities that have produced these behaviors. This represents instrumental learning basis. However, in some situations, the behavior to be initiated depends on a motivation to escape or avoid an aversive situation. For a long time, it was thought that aversive information processing system and reward system depended on different networks and cerebral regions. Yet, a growing number of studies tend to show that basal ganglia certainly play an important role in aversive information processing. In this thesis, we recorded neuronal activity and performed local pharmacological perturbations in non-human primates, in two structures of the reward system, anterior striatum and ventral pallidum, while performing a behavioral task requiring them to initiate alternatively approach behaviors toward a reward and avoidance behaviors from an aversive event. We showed aversive information coding for the predictive stimulus, preparation and initiation of the avoidance behavior and anticipation and response to the aversive events. Furthermore, local perturbation experiments demonstrated that a functional impairment of the anterior striatum and the ventral pallidum affects the behaviors usually initiated by the animals in aversive context. Put together, these results clearly show that these two cerebral structures are involved in both appetitive and aversive motivations

Le temps est une dimension essentielle de la vie. Il est nécessaire, entre autres, pour réaliser des mouvements coordonnés, pour communiquer, mais aussi dans la prise de décisions. L’objectif principal de cette thèse était de caractériser le rôle d’un réseau amygdalo-préfronto-dorsostriatal dans la mémorisation et l’encodage du temps chez le rat. Dans un premier temps, nous avons décrit le comportement temporel du rat lors d’une tâche de suppression conditionnée (i.e. la suppression d’une réponse instrumentale d’appui sur levier par la présentation d’un son associé à un stimulus aversif), démontrant ainsi un contrôle temporel fin du comportement dans une situation Pavlovienne aversive. Dans un deuxième temps, nous avons analysé les potentiels de champs locaux (analyse fréquentielle des activités oscillatoires) de notre réseau d’intérêt au début d’un apprentissage associatif et après surentraînement dans la tâche de suppression conditionnée. En effet, le comportement temporel moteur nécessite un grand nombre de séances d’apprentissage pour devenir optimal, alors que l’apprentissage temporel est, lui, très rapide. Cette étude nous a permis de caractériser des corrélats neuronaux temporels au sein de ce réseau, que ce soit au niveau des structures individuelles ou au niveau de l’interaction entre ces structures. De plus, ces corrélats neuronaux sont modifiés selon le niveau d’entraînement des animaux. Enfin, dans une troisième étude, nous avons démontré que des ratons juvéniles (pré-sevrage), qui présentent un cortex préfrontal ainsi qu’un striatum dorsal immatures, peuvent mémoriser et différencier des intervalles de temps, ouvrant donc la question sur le rôle de ce réseau dans l’apprentissage temporel au cours du développement. / Time is an essential dimension of life. It is necessary for coordinating movement, for communication, but also for decision-making. The principal goal of this work was to characterize the role of an amygdalo-prefronto-dorsostriatal network in the memorization and encoding of time in a rat model. Firstly, we described temporal behavior in a conditioned suppression task (i.e. the suppression of an instrumental lever-pressing response for food by the presentation of a cue associated with an aversive event), therefore showing a precise temporal control in Pavlovian aversive conditioning. Secondly, we measured local field potentials in our network of interest at the beginning of associative learning and after overtraining in the conditioned suppression task. In effect, motor temporal behavior requires a large number of training sessions to become optimum, but temporal learning happens very early in training. This study allowed us to characterize, using frequency analysis of oscillatory activities, neuronal correlates of time in this network both at the level of individual structures, but also in their interactions. Interestingly, these neural correlates were modified by the level of training. Finally, we demonstrated that juvenile rats (pre-weaning), with an immature prefrontal cortex and dorsal striatum, can memorize and discriminate temporal intervals, raising questions on the role of this amygdalo-prefronto-dorsostriatal network in temporal learning during development.