O hipotálamo modula comportamentos relacionados à motivação, recompensa e punição através de projeções para a área tegmental ventral (VTA), o núcleo dorsal da rafe (DR) e o núcleo tegmental rostromedial (RMTg). Nesse estudo, investigamos através de métodos de rastreamento retrógrado as entradas hipotalâmicas da VTA, do DR e do RMTg e, se neurônios hipotalâmicos individuais inervam mais do que uma dessas regiões. Também determinamos uma possível assinatura GABAérgica ou glutamatérgica das aferências hipotalâmicas, através de rastreamento retrógrado combinado com métodos de hibridação in situ. Observamos que VTA, DR e RMTg recebem um padrão bastante semelhante de entradas hipotalâmicas originando de neurônios de projeção glutamatérgicas e GABAérgicas, a maioria deles (> 90%) inervando somente um desses três alvos. Nossos achados indicam que entradas hipotalâmicas são importantes fontes de sinais homeostáticos para a VTA, o DR e o RMTg. Eles exibem um alto grau de heterogeneidade que permite de excitar ou inibir as três estruturas de forma independente ou em conjunto. / The hypothalamus modulates behaviors related to motivation, reward and punishment via projections to the ventral tegmental area (VTA), dorsal raphe nucleus (DR), and rostromedial tegmental nucleus (RMTg). In this study we investigated by retrograde tracing methods hypothalamic inputs to the VTA, DR, and RMTg, and whether individual hypothalamic neurons project to more than one of these structures. We also determined a possible GABAergic or glutamatergic phenotype of hypothalamic afferents, by combining retrograde tracing with in situ hybridization methods. We found that VTA, DR, and RMTg receive a very similar set of hypothalamic afferents originating from glutamatergic and GABAergic hypothalamic projection neurons, the majority of them (> 90%) only innervating one of these structures. Our findings indicate that hypothalamic inputs are important sources of homeostatic signals for the VTA, DR, and RMTg. They exhibit a high degree of heterogeneity which permits to activate or inhibit the three structures either independently or jointly.

Midbrain dopamine neurons play a critical role in motor function, motivation, reward, and cognition by providing modulatory input to cortical and basal ganglia circuits. Given the importance of dopamine neurotransmission and its dysregulation in disease, mechanistic insight into the molecular underpinnings of dopaminergic neuronal function is needed. This thesis seeks to advance our understanding of dopamine neuronal cell biology by developing and applying cutting edge molecular profiling methods to study the subcellular translatome and proteome of dopamine neurons in mice. Chapter 1 provides an overview of the anatomy and cell biology of midbrain dopamine systems, with a particular emphasis on dopamine neurotransmission, neuronal heterogeneity, and selective vulnerability in Parkinson’s disease. Chapter 2 focuses on methods for studying local translation in neurons and describes newly discovered artifacts associated with two of these methods. Chapter 3 describes a global analysis of ribosome and mRNA localization in dopamine neurons; the results suggest that local translation in dopaminergic dendrites, but not axons, regulates dopamine release. Chapter 4 presents a method for subcellular proteomic profiling of dopamine neurons in the mouse brain, revealing the somatodendritic and axonal polarization of proteins encoded by Parkinson’s disease-linked genes. Emerging data are presented on Synaptotagmin 17, a novel axonal protein identified in midbrain dopamine neurons. Finally, I synthesize key findings regarding the molecular organization underlying dopamine neuronal cell biology and highlight promising areas for future investigation.

Angiotensin II (AII) immunoreactivity in the mesopontine area of the rat brain is distributed through several areas where co-localization of AII receptors has not been established. The current in vitro receptor autoradiography study re-examined the distribution of AII binding using 125I-Sar1, Ile8-AII ([125I]SIAII). When incubations were conducted without sulfhydryl reducing agents, [125I]SIAII binding was observed in the locus coeruleus, inferior colliculus, superior colliculus and the central gray in agreement with previous reports. Novel [125I]SIAII binding sites were detected in the parabrachial nucleus, pedunculopontine tegmental nucleus and the caudal linear raphe nucleus, corresponding with previously reported localization of AII immunoreactivity in these nuclei. [125I]SIAII binding was also found in the paragenual nucleus where the peptide has not been detected. Thus, the observation of novel AII receptors which are sensitive to sulfhydryl reducing agents, resolves several AII-AII receptor mismatches.

The reinforcing properties of nicotine depend partly on cholinergic projections from the pedunculopontine tegmental (PPTg) and laterodorsal tegmental (LDTg) nuclei to midbrain dopamine neurons in the ventral tegmental area (VTA) and substantia nigra pars compacta (SNc). Neuronal activation was investigated using Fos expression in these areas following acute (0, 0.1, 0.4, 0.8mg/kg) or chronic systemic nicotine (0, 0.1, 0.4, 0.8, 1.0mg/kg given once per day for 5 days). We also examined co-localization of Fos expression in bNOS and TH positive neurons to determine what populations of neurons were activated by nicotine. Acute nicotine resulted in dose related Fos expression, with the biggest increase seen after 0.4mg/kg nicotine, but no co-localization occurred with bNOS in the PPTg/LDTg. Surprisingly, nicotine also failed to activate midbrain dopamine neurons. After animals were sensitized to nicotine there was a similar dose response curve in Fos expression, but the biggest increase was seen after 0.8mg/kg nicotine. Chronic nicotine, like acute, also preferentially activated non-cholinergic neurons in the LDTg and PPTg and non-dopamine neurons in the SNc and VTA. Further experiments looked at the mechanisms of Fos expression after nicotine administration. Fos expression in the LDTg/PPTg and SNc/VTA was suppressed after d-amphetamine, despite an increase in locomotor activity, suggesting that the increased Fos expression after chronic nicotine was not simply due to the locomotor activating effects of sensitized nicotine. Blocking autoreceptors in the dopaminergic midbrain by haloperidol pre-treatment did not increase Fos expression in dopamine neurons indicating that the inhibitory mechanism was not dependent on local autoreceptors. Novel methods of visualising and lesioning GABA neurons in the mesopontine tegmentum and midbrain were also examined. The data suggest that the mechanisms by which dopamine is involved in the pharmacological actions of passively administered nicotine are more complex than was first thought and that the role of non-dopamine neurons in the VTA (possibly GABA or glutamate containing) are also important.

Os macacos Cebus apella são hábeis com suas mãos e capazes de utilizar ferrementas (paus e pedras) para resolver problemas, podendo ser treinados como assistentes de pessoas quadriplégicas. São escassas ainda as informações em neuroanatomia desses primatas, o que motiva a presente pesquisa, cujo propósito é o de enfocar a morfologia externa e interna do tronco encefálico. Foram estudados 20 espécimes do macaco Cebus apella que já estavam fixados com formol a 10%, pertencentes ao acervo de pesquisa do laboratório de Anatomia da Universidade Federal de Uberlândia. Após isolamento do encéfalo e remoção das meninges procedeu ? se ao estudo da superfície externa em 14 peças. A dissecção por técnica usual preservou características morfológicas da superfície e origem aparente de nervos cranianos, que foram identificados. Em análise macroscópica, o tronco encefálico mostra ? se bem desenvolvido destacando ? se ainda em sua face lateral a origem aparente do nervo trigêmeo e os pedúnculos cerebelares médios, bem evidentes. Na face dorsal, em relação ao IV ventrículo identificam ? se os véus medulares superior e inferior, bem como o sulco mediano e a eminência medial. Os pedúnculos cerebelares superior e inferior também são bem destacados. Na medula oblonga são identificados, entre outros, os relevos correspondentes às olivas e às pirâmides, além da fissura mediana anterior e a região da decussação das pirâmides, Nem sempre são nítidos o sulco bulbo ? pontino e o forame cego. Os fascículos grácil e cuneiforme são bastante desenvolvidos bem como os tubérculos correspondentes. Para os estudos das estruturas internas do tronco encefálico, 6 peças, já fixadas, foram congeladas com nitrogênio líquido e cortadas no criostato entre ?27º C e ? 30º C, seguindo ? se a feitura de cortes transversais, em seis níveis (colículos superior e inferior, origem aparente do nervo trigêmeo, porções superior, média e inferior da oliva, e decussação das pirâmides), com espessura de 60 µc, corados segundo o método de Mulligan. As estruturas evidenciáveis são descritas com detalhes comparando ? se os dados obtidos com os da literatura consultada. Os termos anatômicos foram referidos segundo a Terminologia Anatômica Internacional, 1ª edição brasileira ? 2001, Ed. Manole Ltda. / The Cebus apella monkey are able to use their nands and to use tools like woods and stones to solve problems. They can be trained to act as quadriplégicas people?s assistants. The Neuroanatomy information about these primates are little, that?s the reason for this research and its purpose is to accomplish a macroscophial anatomic study of encephalic trunk external and internal surface structure. It was studied Cebus apella monkeu 20 especimens that was already fixed on 10% formaldehyde. These monkeys are from research?s heap of Universidade Federal de Uberlândia. The monkey encephalon were removed using an osteótomo. After removing the meninges, theirs external surface study was started on 14 of them. The dissection using usual technique could preserve the morphological characteristics of the surface and cranial nerve apparent origim, that could be identified too. Under a macroscophical analysis, the encephalic trun looks very elaborated and the trigemini nerve and pedunculus cerebellaris aparent origin, very evident, was stood out in its lateral face. In the dorsal face, in relation to fourth ventricle, the inferior and superior velum medullare could be identified as well as the sulcus medianus and the eminentia medialis. The superior pedunculus cerebellaris, as well as the inferior ones, were very stood out too. On the medulla oblongata were identified, beyond others, the relieves corresponding to the oliva and the decussatio pyramidum region. The sulcus bulbus ? pons and the foramen caecum not always are bright. The fasciculus gracilis, as well as the cuneatus ones, are very elaborated as well as the corresponding tuberculuns.The brain stem internal structure study 6 already fixed pieces were froze with liquid nitrong and cut in criostato, under the temperature between ?27ºC and ? 30º C. After this, transversal cuts were made on 6 levels (superior and inferior nerve trigemini aparent origin, olivas superior, medium and inferior portion and decussatio pyramidum), with the thickness of 60µc, roddy according to the Mulligan method. The evidentiable structures are described in details comparing data obtained from the consulted literature. The anatomic terms were aforesaid according to Terminologia Anatômica Internacional, 1ª edição brasileira ? 2001, Ed. Manole Ltda.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Os macacos Cebus apella são hábeis com suas mãos e capazes de utilizar ferrementas (paus e pedras) para resolver problemas, podendo ser treinados como assistentes de pessoas quadriplégicas. São escassas ainda as informações em neuroanatomia desses primatas, o que motiva a presente pesquisa, cujo propósito é o de enfocar a morfologia externa e interna do tronco encefálico. Foram estudados 20 espécimes do macaco Cebus apella que já estavam fixados com formol a 10%, pertencentes ao acervo de pesquisa do laboratório de Anatomia da Universidade Federal de Uberlândia. Após isolamento do encéfalo e remoção das meninges procedeu ? se ao estudo da superfície externa em 14 peças. A dissecção por técnica usual preservou características morfológicas da superfície e origem aparente de nervos cranianos, que foram identificados. Em análise macroscópica, o tronco encefálico mostra ? se bem desenvolvido destacando ? se ainda em sua face lateral a origem aparente do nervo trigêmeo e os pedúnculos cerebelares médios, bem evidentes. Na face dorsal, em relação ao IV ventrículo identificam ? se os véus medulares superior e inferior, bem como o sulco mediano e a eminência medial. Os pedúnculos cerebelares superior e inferior também são bem destacados. Na medula oblonga são identificados, entre outros, os relevos correspondentes às olivas e às pirâmides, além da fissura mediana anterior e a região da decussação das pirâmides, Nem sempre são nítidos o sulco bulbo ? pontino e o forame cego. Os fascículos grácil e cuneiforme são bastante desenvolvidos bem como os tubérculos correspondentes. Para os estudos das estruturas internas do tronco encefálico, 6 peças, já fixadas, foram congeladas com nitrogênio líquido e cortadas no criostato entre ?27º C e ? 30º C, seguindo ? se a feitura de cortes transversais, em seis níveis (colículos superior e inferior, origem aparente do nervo trigêmeo, porções superior, média e inferior da oliva, e decussação das pirâmides), com espessura de 60 µc, corados segundo o método de Mulligan. As estruturas evidenciáveis são descritas com detalhes comparando ? se os dados obtidos com os da literatura consultada. Os termos anatômicos foram referidos segundo a Terminologia Anatômica Internacional, 1ª edição brasileira ? 2001, Ed. Manole Ltda. / The Cebus apella monkey are able to use their nands and to use tools like woods and stones to solve problems. They can be trained to act as quadriplégicas people?s assistants. The Neuroanatomy information about these primates are little, that?s the reason for this research and its purpose is to accomplish a macroscophial anatomic study of encephalic trunk external and internal surface structure. It was studied Cebus apella monkeu 20 especimens that was already fixed on 10% formaldehyde. These monkeys are from research?s heap of Universidade Federal de Uberlândia. The monkey encephalon were removed using an osteótomo. After removing the meninges, theirs external surface study was started on 14 of them. The dissection using usual technique could preserve the morphological characteristics of the surface and cranial nerve apparent origim, that could be identified too. Under a macroscophical analysis, the encephalic trun looks very elaborated and the trigemini nerve and pedunculus cerebellaris aparent origin, very evident, was stood out in its lateral face. In the dorsal face, in relation to fourth ventricle, the inferior and superior velum medullare could be identified as well as the sulcus medianus and the eminentia medialis. The superior pedunculus cerebellaris, as well as the inferior ones, were very stood out too. On the medulla oblongata were identified, beyond others, the relieves corresponding to the oliva and the decussatio pyramidum region. The sulcus bulbus ? pons and the foramen caecum not always are bright. The fasciculus gracilis, as well as the cuneatus ones, are very elaborated as well as the corresponding tuberculuns.The brain stem internal structure study 6 already fixed pieces were froze with liquid nitrong and cut in criostato, under the temperature between ?27ºC and ? 30º C. After this, transversal cuts were made on 6 levels (superior and inferior nerve trigemini aparent origin, olivas superior, medium and inferior portion and decussatio pyramidum), with the thickness of 60µc, roddy according to the Mulligan method. The evidentiable structures are described in details comparing data obtained from the consulted literature. The anatomic terms were aforesaid according to Terminologia Anatômica Internacional, 1ª edição brasileira ? 2001, Ed. Manole Ltda.

Les mécanismes neuronaux contrôlant la respiration sont présentement explorés à l’aide de plusieurs modèles animaux incluant le rat et la grenouille. Nous avons utilisé la lamproie comme modèle animal nous permettant de caractériser les réseaux de neurones du tronc cérébral qui génèrent et modulent le rythme respiratoire. Nous avons d’abord caractérisé une nouvelle population de neurones, dans le groupe respiratoire paratrigéminal (pTRG), une région du tronc cérébral essentielle à la genèse du rythme respiratoire chez la lamproie. Les neurones de cette région sont actifs en phase avec le rythme respiratoire. Nous avons montré que ces neurones possèdent une arborisation axonale complexe, incluant des projections bilatérales vers les groupes de motoneurones du tronc cérébral qui activent les branchies ainsi que des connexions reliant les pTRG de chaque côté du tronc cérébral. Ces résultats montrent que le pTRG contient un groupe de cellules qui active les motoneurones respiratoires des deux côtés et qui pourrait être impliqué dans la synchronisation bilatérale du rythme respiratoire. Nous avons ensuite étudié les mécanismes neuronaux par lesquels le rythme respiratoire est augmenté en lien avec l’effort physique. Nous avons montré que la région locomotrice du mésencéphale (MLR), en plus de son rôle dans la locomotion, active les centres respiratoires pendant la nage, et même en anticipation. Les neurones de la MLR projetant vers les centres locomoteurs et respiratoires sont ségrégés anatomiquement, les neurones localisés plus dorsalement étant ceux qui possèdent des projections vers les centres respiratoires. Nous avons aboli la contribution de la partie dorsale de la MLR aux changements respiratoires en injectant des bloqueurs des récepteurs glutamatergiques localement, sur des préparations semi-intactes. Nous avons montré que lors d’épisodes de nage, une majeure partie de l’effet respiratoire est abolie par ces injections, suggérant un rôle prépondérant des neurones de cette région dans l’augmentation respiratoire pendant la locomotion. Nos résultats confirment que le rythme respiratoire est généré par une région rostrolatérale du pons de la lamproie et montrent que des connexions des centres locomoteurs arrivent directement à cette région et pourraient être impliquées dans l’augmentation respiratoire reliée à l’effort physique. / The neural control of breathing is currently investigated on multiple animal models such as frogs and rats. We have used the lamprey as an experimental model to characterize the brainstem neural networks involved in the genesis and modulation of the respiratory rhythm. We have first characterized a new population of respiratory neurons in the paratrigeminal respiratory group (pTRG). The pTRG is a region that was shown to be essential to respiratory rhythmogenesis in lampreys. We have shown that the pTRG contains a group of neurons with complex axonal arborisations, including bilateral projections to the motoneuron pools of the brainstem that activate gills, as well as bilateral projections connecting the pTRGs on the two sides of the brainstem. These results suggest that pTRG neurons could participate in the descending control of respiratory motoneurons as well as the bilateral synchrony of the respiratory rhythm. We have then studied the neural mechanisms by which respiration is increased during locomotion. We have shown that the mesencephalic locomotor region (MLR), in addition to its role in controlling locomotion, also increases breathing during locomotion. Neurons in the MLR are anatomically segregated, those projecting to the respiratory centers being located more dorsally. We have abolished the contribution of the dorsal part of the MLR to respiratory changes by injecting glutamate receptor blockers locally in semi-intact preparations. We have shown that during swimming episodes, a major part of the respiratory effect is dependent on the dorsal part of the MLR. Our results confirm that the respiratory rhythm is generated by a rostrolateral region in the pons of lampreys and show that connections from locomotor centers can directly activate this region. These connections could be implicated in the increase of breathing activity related to locomotion.

Diss. (sammanfattning) Umeå : Univ., 2008. / Härtill 3 uppsatser.

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