Zugl.: Berlin, Univ. der Künste, Diss., 2008

Um acúmulo de evidências sugere que animais vertebrados são capazes de detectar informações geomagnéticas, dentre eles, podemos citar os mais estudados, o caso clássico do pombo-correio (Columba livia). A descoberta de material biomagnético no epitélio do bico superior de pombos sugere a possibilidade de um mecanismo transdutor de campo magnético situado nessa região. Pelos resultados obtidos em outros contextos, a utilização de genes de resposta imediata (IEGs) combinada com tratamentos disruptivos e tarefas clássicas de orientação é tida como metodologia promissora para se conseguir uma descrição mais compreensiva dos canais sensoriais e dos mecanismos de processamento nervoso envolvidos no comportamento de orientação, entre os quais a putativa magnetocepção. Pela atividade de IEGs, especificamente a expressão da proteína ZENK, avaliamos diferencialmente áreas cerebrais ativadas em pombos expostos ou não a uma estimulação magnética, 1h e 3h após a estimulação. A análise quantitativa (teste T) mostrou um aumento no número de neurônios ZENK positivos na região do córtex pré-piriforme de pombos estimulados (p=0,051) em relação aos controles, não havendo diferença entre os grupos de 1h e 3h (ANOVA, uma via). Esses neurônios estão relacionados ao sistema olfativo, o que reforça a idéia de que esse sistema seja importante no comportamento de orientação mas ao mesmo tempo apresenta a primeira evidência experimental de um possível envolvimento da via olfatória na magnetocepção. Esse resultado indica que é necessário tomar o maior cuidado na interpretação de experimentos comportamentais inibindo a via olfativa, pois, além de causar anosmia, essas manipulações poderiam também lesar mecanismos magnetoceptivos. / Much evidence suggests that vertebrate animals are capable of detecting geomagnetic information, among them, we can cite the best studied classic example of homing pigeon (Columba livia). The discovery of biomagnetic material in the pigeon upper beak suggest the possibility of a magnetoceptor transduction mechanism situated in this area. Because of the results obtained in other contexts, the use of immediate early genes (IEGs), combined with disruptive treatments and classic orientation tasks is a promising tool towards a more accurate description of sensory channels and of neural processing mechanisms involved in orientation behaviour, particularly the putative magnetoception mechanism. Using IEG activity, specifically, the ZENK-immunoreactivity protein, we studied different pigeon brain areas activated after magnetic stimulation, compared to no stimulation, at two periods of latency after the end of the experimental session (1h and 3h). Quantitative analysis (T test) showed an increase in labeling in the prepiriform cortex (CPP) of stimulated pigeons in relatioin to controls (p=0,051), but no difference between 1h and 3h groups (one-way ANOVA). These neurons are related to the olfactory system, reforcing the idea that this system is important for orientation, but at the same time presents the first experimental evidence of a possible participation of olfactory pathways in magnetoception. This result indicates that it is important to take utmost care in interpreting results of behavioural experiments in which olfaction is inhibited, since such manipulations may not only cause anosmia, but also loss of magnetic sensitivity.

The aim of this study was to provide deeper insights in passerine phylogenetic relationships using new molecular markers. The monophyly of the largest avian order Passeriformes (~59% of all living birds) and the division into its suborders suboscines and oscines are well established. Phylogenetic relationships within the group have been extremely puzzling, as most of the evolutionary lineages originated through rapid radiation. Numerous studies have hypothesised conflicting passerine phylogenies and have repeatedly stimulated further research with new markers. In the present study, I used three different approaches to contribute to the ongoing phylogenetic debate in Passeriformes. I investigated the recently introduced gene ZENK for its phylogenetic utility for passerine systematics in combination and comparison to three already established nuclear markers. My phylogenetic analyses of a comprehensive data set yielded highly resolved, consistent and strongly supported trees. I was able to show the high utility of ZENK for elucidating phylogenetic relationships within Passeriformes. For the second and third approach, I used chicken repeat 1 (CR1) retrotransposons as phylogenetic markers. I presented two specific CR1 insertions as apomorphic characters, whose presence/absence pattern significantly contributed to the resolution of a particular phylogenetic uncertainty, namely the position of the rockfowl species Picathartes spp. in the passerine tree. Based on my results, I suggest a closer relationship of these birds to crows, ravens, jays, and allies. For the third approach, I showed that CR1 sequences contain phylogenetic signal and investigated their applicability in more detail. In this context, I screened for CR1 elements in different passerine birds, used sequences of several loci to construct phylogenetic trees, and evaluated their reliability. I was able to corroborate existing hypotheses and provide strong evidence for some new hypotheses, e.g. I suggest a revision of the taxa Corvidae and Corvinae as vireos are closer related to crows, ravens, and allies. The subdivision of the Passerida into three superfamilies, Sylvioidea, Passeroidea, and Muscicapoidea was strongly supported. I found evidence for a split within Sylvioidea into two clades, one consisting of tits and the other comprising warblers, bulbuls, laughingthrushes, whitethroats, and allies. Whereas Passeridae appear to be paraphyletic, monophyly of weavers and estrild finches as a separate clade was strongly supported. The sister taxon relationships of dippers and the thrushes/flycatcher/chat assemblage was corroborated and I suggest a closer relationship of waxwings and kinglets to wrens, tree-creepers, and nuthatches. / Das Ziel dieser Arbeit war es, mittels neuer molekularer Marker zusätzliche Informationen über die phylogenetischen Verwandtschaftsverhältnisse der Sperlingsvögel (Passeriformes) zu erhalten. Die Monophylie der Passeriformes, der größten Vogelgruppe (~59% aller lebenden Arten), sowie ihrer Unterteilung in Suboscines und Oscines sind gut belegt. Die phylogenetischen Verwandtschaftsverhältnisse innerhalb dieser Gruppen sind jedoch seit jeher sehr schwer zu entschlüsseln, da sich die meisten Linien durch eine schnelle Radiation entwickelten. Zahlreiche Studien haben verschiedene Hypothesen zur Phylogenie der Sperlingsvögel aufgestellt und damit die Suche nach neuen Markern initiiert. In meiner Untersuchung habe ich drei verschiedene Ansätze benutzt, um zur Klärung der Phylogenie beizutragen. Ich untersuchte das kürzlich als Marker eingeführte ZENK-Gen im Hinblick auf seinen Nutzen in der Systematik der Sperlingsvögel in Kombination und im Vergleich zu drei bereits etablierten nukleären Markern. Meine phylogenetischen Analysen eines umfassenden Datensatzes ergaben hoch aufgelöste, konsistente und stark unterstütze Stammbäume, so dass ich den hohen Nutzwert des ZENK-Gens für die Klärung phylogenetischer Verwandtschaftsverhältnisse der Passeriformes zeigen konnte. Für den zweiten und dritten Ansatz habe ich Chicken Repeat 1 (CR1) Retrotransposons als phylogenetische Marker benutzt. Anhand zweier spezifischer CR1 Insertionen als apomorphe Merkmale und deren Insertionsmuster in verschiedenen Sperlingsvögeln konnte ich die phylogenetische Position der afrikanischen Felshüpfer, Picathartes spp., klären. Aufgrund meiner Ergebnisse schließe ich auf eine engere Verwandtschaft der Felshüpfer zu den Rabenvögeln. Durch meinen dritten Ansatz konnte ich nachweisen, dass CR1-Sequenzen phylogenetische Informationen enthalten, und untersuchte detailliert deren Anwendung als Marker. Dafür habe ich in verschiedenen Sperlingsvögeln nach CR1 Elementen gesucht und mit einigen dieser Sequenzen Stammbäume berechnet, um die Verlässlichkeit der Marker zu überprüfen. Durch meine Untersuchungen konnte ich existierende Hypothesen stützen und zusätzlich starke Hinweise auf neue Hypothesen finden. Beispielsweise schlage ich eine Revision der Taxa Corvidae und Corvinae vor, da Vireos eng mit den Rabenvögeln verwandt sind. Die Unterteilung der Passerida in die drei Unterfamilien Sylvioidea, Passeroidea und Muscicapoidea konnte deutlich bestätigt werden. Ich habe Hinweise auf eine Trennung der Sylvioidea in zwei taxonomische Gruppen erhalten, einer bestehend aus Meisen und Verwandten und der andere aus Grasmücken, Bülbüls, Häherlingen, Brillenvögeln und Verwandten. Während die Passeridae paraphyletisch sind, wurde die Monophylie der Weber und Astrilden als ein eigenes Taxon unterstützt. Das Schwestergruppenverhältnis zwischen Wasseramseln und dem Drossel/Fliegenschnäpper/Schmätzer-Taxon wurde ebenfalls bestätigt. Außerdem habe ich Hinweise auf eine nähere Verwandtschaft zwischen Seidenschwänzen und Goldhähnchen zu Zaunkönigen, Baumläufern und Kleibern gefunden.

Um acúmulo de evidências sugere que animais vertebrados são capazes de detectar informações geomagnéticas, dentre eles, podemos citar os mais estudados, o caso clássico do pombo-correio (Columba livia). A descoberta de material biomagnético no epitélio do bico superior de pombos sugere a possibilidade de um mecanismo transdutor de campo magnético situado nessa região. Pelos resultados obtidos em outros contextos, a utilização de genes de resposta imediata (IEGs) combinada com tratamentos disruptivos e tarefas clássicas de orientação é tida como metodologia promissora para se conseguir uma descrição mais compreensiva dos canais sensoriais e dos mecanismos de processamento nervoso envolvidos no comportamento de orientação, entre os quais a putativa magnetocepção. Pela atividade de IEGs, especificamente a expressão da proteína ZENK, avaliamos diferencialmente áreas cerebrais ativadas em pombos expostos ou não a uma estimulação magnética, 1h e 3h após a estimulação. A análise quantitativa (teste T) mostrou um aumento no número de neurônios ZENK positivos na região do córtex pré-piriforme de pombos estimulados (p=0,051) em relação aos controles, não havendo diferença entre os grupos de 1h e 3h (ANOVA, uma via). Esses neurônios estão relacionados ao sistema olfativo, o que reforça a idéia de que esse sistema seja importante no comportamento de orientação mas ao mesmo tempo apresenta a primeira evidência experimental de um possível envolvimento da via olfatória na magnetocepção. Esse resultado indica que é necessário tomar o maior cuidado na interpretação de experimentos comportamentais inibindo a via olfativa, pois, além de causar anosmia, essas manipulações poderiam também lesar mecanismos magnetoceptivos. / Much evidence suggests that vertebrate animals are capable of detecting geomagnetic information, among them, we can cite the best studied classic example of homing pigeon (Columba livia). The discovery of biomagnetic material in the pigeon upper beak suggest the possibility of a magnetoceptor transduction mechanism situated in this area. Because of the results obtained in other contexts, the use of immediate early genes (IEGs), combined with disruptive treatments and classic orientation tasks is a promising tool towards a more accurate description of sensory channels and of neural processing mechanisms involved in orientation behaviour, particularly the putative magnetoception mechanism. Using IEG activity, specifically, the ZENK-immunoreactivity protein, we studied different pigeon brain areas activated after magnetic stimulation, compared to no stimulation, at two periods of latency after the end of the experimental session (1h and 3h). Quantitative analysis (T test) showed an increase in labeling in the prepiriform cortex (CPP) of stimulated pigeons in relatioin to controls (p=0,051), but no difference between 1h and 3h groups (one-way ANOVA). These neurons are related to the olfactory system, reforcing the idea that this system is important for orientation, but at the same time presents the first experimental evidence of a possible participation of olfactory pathways in magnetoception. This result indicates that it is important to take utmost care in interpreting results of behavioural experiments in which olfaction is inhibited, since such manipulations may not only cause anosmia, but also loss of magnetic sensitivity.

Relatively little is known about the existence and traits of a possible nucleus accumbens (Acc) region in non-mammals. The current project investigated a likely candidate for such a structure in pigeons, the medioventral (mvMSt) and mediodorsal (mdMSt) parts of avian medial striatum (MSt). The methods employed were threefold: 1) tract-tracing to determine anatomical connections of the MSt; 2) lesion studies to assess MSt's role in a cognitive task (reversal learning); and 3) measuring an immediate-early gene induced protein, ZENK, in striatal regions during courtship behavior in male pigeons. The MSt was found to have many forebrain (amygdala, hippocampus, dorsal thalamus) and midbrain (ventral tegmental area, substantia nigra) connections similar to those of Acc. In addition, differences in connection patterns between mvMSt and mdMSt indicated that mvMSt was comparable to the shell of Acc, while the mdMSt showed characteristics of Acc core. Effects of MSt lesions on pattern discrimination and reversal learning were assessed. Both lesion subjects and controls performed similarly on original discrimination. Furthermore, there were no significant differences in MSt lesioned birds compared to controls. However, there was a tendency for the two groups to make different types of errors. Error patterns indicated that sham-lesioned birds had deficits due to key preference, whereas lesioned birds had fixation on previous reward contingencies (perseverative errors). The performance of the lesioned birds was consistent with Acc lesion effects on reversal learning in mammals. The expression of ZENK in the mvMSt, mdMSt, lateral MSt, and lateral striatum of male birds exposed to either an empty cage or a live female pigeon was quantified. Higher ZENK expression was found in the live pigeon condition for all the striatal structures. However, the degree of difference between live and empty was much higher in the mvMSt and mdMSt than in the other areas. Therefore, mvMSt and mdMSt appear to play a role in anticipatory sexual behaviors, as has been shown in Acc. The anatomical and functional data from the current study indicate that avian mMSt has numerous similarities with mammalian Acc. These findings will contribute to understanding the evolution of mammalian Acc and identifying the functional significance of avian MSt.

The developmental stress hypothesis predicts that an aversive condition, such as decreased food intake, predation, and social isolation, in the early developmental stage could have long term effects on behaviors and brain development of an animal. In nature, bird nestlings are susceptible to various factors, such as malnutrition, infections, and parasites. Effects of early life stress on adulthood have been extensively studied with some stressors including malnutrition. However, immune challenges as an early life stressor and their long-term programming effects on adult behaviors are yet to be studied in detail. The goal of the current study was to investigate changes in growth rate, personality, mate selection behaviors and brain development in zebra finch nestlings after injection with a viral infection mimicking agent, Polyinosinic: polycytidylic acid (Poly I:C). By using Poly I:C, it was possible to isolate long-term effects to the immune response of the bird. After Poly I:C injection on post-hatch day (PD) 14, morphological measures were conducted to detect changes in body growth rate. When birds became sexually mature (> ~PD 200), behaviors of birds were observed in different conditions to detect changes associated with the personality traits of animals. In mate choice trials, both attractiveness of males and mate selection behaviors of males and females were investigated. Finally, the development and neuronal activity of specific brain nuclei involved in courtship (i.e., HVC and RA) and social/sexual behaviors (nucleus taeniae of the amygdala, TnA) were investigated. The results showed that nestlings’ growth rate was not affected. However, Poly I:C injection had some effects on certain, but not all, personality traits observed in the study. Such effects were found only in female zebra finches, suggesting that there was a limited sex-specific influence of an early life immune challenge on personalities of adults. The results also showed that Control females tended to choose untreated males over Poly I:C injected males in mate choice trials. Finally, Poly I:C injection negatively affected the overall development of targeted brain nuclei. In addition, neuronal activity in TnA was higher in Poly I:C injected birds. Results of the present study suggest that one time injection with Poly I:C early in the life causes long term effects on adulthood. These findings are further discussed regarding their relevance to the developmental stress hypothesis.

Early visual processing in the avian brain has been studied extensively, the retina and midbrain in particular. However, a clear understanding of the higher visual centers in the forebrain (the telencephalon and thalamus) remains poor. Two structures located within the avian visual telencephalon, the entopallium (E) and the lateral portion of the intermediate nidopallium (NIL), merit extensive investigation based on their critical role in visual processing. The goal of the current study was to further clarify the anatomical characteristics of E and NIL. Visual information that reaches these telencephalic structures is mostly from the contralateral retina. Thus, blocking visual input on one side affects the opposite hemisphere, but leaves the hemisphere on the same side largely unaffected. This unique property of the avian visual system was used in order to emphasize neurochemical expression in the higher visual structures. After blocking visual input to one hemisphere either by monocular occlusion or unilateral lesion of the nucleus rotundus we examined the expression of specific neuroanatomical markers; namely cytochrome oxidase (CO) and the calcium binding protein, parvalbumin (PV) in E and ZENK protein expression in NIL. . Our results showed there were significantly more PV-ir cell bodies in the inner region of E compared to the outer region. In terms of PV-ir neuropil, regional differences within E were significant. In particular, the ventrolateral E tended to have a higher density of PV-ir neuropil than other regions except the most ventromedial area which was also high in PV-ir. Differential CO staining patterns were observed as well. However, unlike PV-ir neuropil the ventro-intermedial area showed a low level of CO staining compared to the other areas. Finally, there was a significant reduction of ZENK-ir cell bodies on NIL in the experimental hemisphere compared to control side. Based on this differential expression of ZENK, it was possible for the first time to visualize the location of NIL. The findings presented here are discussed in terms of their relevance to the identification of the size and extent of NIL and the heterogeneous nature of E.

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

No description available.