Esse trabalho consiste no estudo de alguns mecanismos responsáveis pela geração das oscilações elétricas observadas no sistema olfativo de vertebrados e das possíveis funções que essas oscilações possam ter no processamento da informação olfativa. Da-se especial atenção ao papel desempenhado pelo ritmo respiratório e pelas sinapses químicas e elétricas nesse processo. Para realizar essa investigação, foram utilizados modelos computacionais que reproduzem aspectos da anatomia e da fisiologia do epitélio olfativo, do bulbo olfativo e do córtex piriforme. Os modelos foram desenvolvidos e simulados no neurossimulador GENESIS, funcionando no sistema operacional LINUX. A análise dos resultados foi feita no programa MATLAB (Mathworks™). Inicialmente, a tese faz uma descrição do substrato neurobiológico que compõe as camadas iniciais do sistema olfativo, incluindo o epitélio, bulbo e córtex olfativo, e de como a informação olfativa é processada por cada camada, discutindo a importância do sentido olfativo e a relevância da neurociência computacional no estudo da origem e do papel das oscilações elétricas existentes nesse sistema (Capítulo 1). O capítulo 2 descreve os materiais e métodos utilizados para a construção dos modelos computacionais e para análise dos resultados. O capítulo 3 faz uma descrição detalhada do modelo computacional utilizado e dos experimentos realizados com o modelo. Finalmente, o capítulo 4 apresenta e discute os resultados das simulações realizadas e o capítulo 5 estende essa discussão, concluindo a tese. O capítulo 6 contém as referências bibliográficas utilizadas no trabalho. Os resultados do trabalho sugerem que as oscilações elétricas no sistema olfativo poderiam ser geradas em várias estruturas e níveis de organização, abrangendo os níveis moleculares, celulares e de sistemas neurais. E que as sinapses químicas e elétricas, assim como os ritmos respiratórios, podem ter um papel fundamental na geração dessas oscilações. Assim, o modelo construído propõe uma explicação plausível para a origem das oscilações elétricas no sistema olfativo de vertebrados e discute as possíveis funções que essas oscilações teriam no contexto do processamento da informação sensorial. / This work is a study of some mechanisms associated with the generation of electric oscillations in the vertebrate olfactory system. Special attention is given for the role of the respiratory rhythm, chemical synapses and electrical synapses in this process. The possible functions of the electric oscillations in olfactory information processing are explored. A computational model that reproduces aspects of the anatomy and physiology of the olfactory epithelium, bulb and piriform cortex was utilized to realize this investigation. The models were developed and simulated in the GENESIS neurosimulator, running under the LINUX operational system. The analysis of the results was made in the software MATLAB (Mathworks™). In the beginning, the thesis describe the neurobiological substracts of the initial layers of the olfactory system, including the olfactory epithelium, bulb and piriform cortex, and explore how the olfactory information is processed by each layer. The chapter 1 presents the importance of the olfactory sense and the use of computational neuroscience to study the role of the electric oscillations in this system. The chapter 2 explains the material and methods utilized to develop the computational model and to analyse the data generated by the model. The chapter 3 describes the used computational model and the experiments realized with the model. Finally, the chapter 4 presents and discusses the results of the simulations. The chapter 5 extends the discussion and concludes the thesis. The chapter 6 contains the bibliographic references. The results of the work suggest that electric oscillations in the olfactory system could be generated in several structures and organizational levels, including the molecular level, the cellular and neural systems level. In particular, the results shown that chemical and electric synapses, as well as the respiratory rhythm, may have a fundamental role in the generation of these oscillations. Indeed, the constructed model proposes a plausible explanation for the origin of the electrical oscillations in the vertebrate olfactory system and discusses the possible function of these oscillations in the context of sensorial information processing.

Orientador: Maria Aparecida Azevedo Pereira da Silva / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-19T19:30:51Z (GMT). No. of bitstreams: 1 Natalicio_MariaAngelica_D.pdf: 1137447 bytes, checksum: bf1a6d401d0100649213c09ece37f7f9 (MD5) Previous issue date: 2012 / Resumo: Testes sensoriais para a avaliação da função olfatória de indivíduos têm sido validados e utilizados em diagnósticos da população em geral, e em portadores de desordens cerebrais e pesquisas em neurociência em particular. No Brasil, testes para serem utilizados com segurança e eficiência para a avaliação da capacidade de identificação e discriminação de odores de indivíduos ainda não foram adequadamente desenvolvidos e validados. Assim, os objetivos da presente pesquisa foram: i) desenvolver, testar e validar testes de identificação e discriminação de odores para avaliar a função olfatória de indivíduos brasileiros, ii) avaliar a potencialidade das metodologias desenvolvidas para uso em pré-diagnóstico clínico de indivíduos da terceira idade e pacientes portadores de epilepsia, iii) verificar o desempenho, em portadores de epilepsia, de uma metodologia já validada e utilizada mundialmente para avaliar a capacidade de identificação de odores de indivíduos e, iv) avaliar em portadores de epilepsia, funções que são processadas por substratos neurais comuns à função olfatória, neste caso, a capacidade de reconhecimento de emoção facial e vocal. Para o teste de identificação de odores, foi desenvolvido um instrumento intitulado &quot;Pastilhas de Odor¿ contendo em pastilhas individuais, 36 odores familiares aos brasileiros. Os odores foram caracterizados quanto à intensidade, aceitação, pungência, refrescância e familiaridade, sendo considerados adequados para comporem um teste de avaliação da função olfatória. A identificação dos odores de &quot;Pastilhas de Odor¿ foi realizada através de um teste de múltipla escolha contendo quatro alternativas, das quais apenas uma é a correta. Para o desenvolvimento do teste de discriminação de odores, 24 voláteis odoríferos puros (P.A.), associados a 6 diferentes categorias de odor - doce, verde, frutal, cítrico, floral e desagradável - foram selecionados e diluídos em propilenoglicol. Com essas amostras, 36 testes de comparação pareada foram construídos, onde em cada categoria de odor, uma amostra alvo foi selecionada para ser comparada com as demais da mesma categoria. O desempenho de cada indivíduo neste teste é analisado utilizando-se a teoria &quot;signaldetection¿, através dos seguintes parâmetros: taxa de acertos (HR), taxa de falsos alarmes (FR), poder discriminativo (d¿L) e vício de resposta (CL). Os dois testes desenvolvidos - &quot;Pastilhas de Odor¿ e teste de discriminação - foram validados com a participação de três grupos de indivíduos: grupo controle, grupo da terceira idade e grupo de indivíduos portadores de epilepsia do lobo temporal (ELT). A capacidade de identificação de odores, de pacientes norte-americanos portadores de ELT foi também avaliada, utilizando-se com esse fim, o teste já validado, denominado &quot;University of Pennsylvania Smell Identification Test¿ (UPSIT), o qual consiste em um teste de múltipla escolha, composto por 40 estímulos odoríferos microencapsulados. Adicionalmente, avaliou-se nesses pacientes, a capacidade de reconhecimento de emoção facial e vocal, utilizando-se uma nova ferramenta intitulada &quot;Comprehensive Affect Testing System¿ (CATS). Com relação à capacidade de identificação de odores dos indivíduos brasileiros, os resultados obtidos através do teste &quot;Pastilhas de Odor¿ revelaram significância estatística tanto para o efeito &quot;sexo¿ (p=0,0003), como para o efeito &quot;idade¿ (p<0,001). O teste também permitiu identificar que os portadores de ELT, sem cirurgia e após ressecção do lobo temporal, apresentavam menor capacidade de identificação de odores comparativamente ao grupo controle (p= 0,05); este mesmo resultado foi observado ao aplicar o UPSIT em portadores de ELT norte-americanos, antes e após os mesmos terem sido submetidos à mencionada cirurgia. O teste &quot;Pastilhas de Odor¿ mostrou ser de fácil manuseio e aplicação em indivíduos adultos, apresentou alto coeficiente de confiabilidade no teste-reteste (r=0,87, p<0,001) e suas pastilhas apresentaram boa estabilidade ao armazenamento durante 3 meses. Por sua vez, os dados obtidos através do teste de discriminação de odores identificaram que tanto o grupo da terceira idade como o dos portadores de ELT apresentaram poder discriminativo e taxa de acertos inferiores (p=0.05) aos indivíduos do grupo controle. Os resultados obtidos pela aplicação do teste CATS em indivíduos norte-americanos, revelaram que pacientes ELT tanto pré- como póscirúrgicos apresentaram menor reconhecimento de emoção facial e vocal quando comparados com o grupo controle, principalmente para as emoções negativas. O fato dos testes &quot;Pastilhas de Odor¿ e de discriminação de odores desenvolvidos na presente pesquisa terem detectado deficiência olfatória nos indivíduos brasileiros da terceira idade e portadores de epilepsia, constrói validade para a utilização dos mesmos em diagnósticos clínicos associados a essas populações / Abstract: Sensory tests to evaluate olfactory function have been validated and used in the diagnosis of subjects, mainly in brain disorders patients, and in the neuroscience research. In Brazil, reliable tests of odor discrimination and identification have not been appropriately developed and validated. The aims of the present study were: i) to develop, test and validate odor discrimination and identification tests to assess olfactory function of Brazilian population; ii) to evaluate the methodologies performance in the diagnosis of elderly subjects and epilepsy patients. For odor identification test, the developed instrument was entitled &quot;Odor Tablets¿, with 36 different odors familiar to Brazilians; to verify the performance in patients with epilepsy, a methodology previously validated and used worldwide to evaluate the ability to identify odors of individuals, and iv) to evaluate in patients with epilepsy, functions that are processed by common neural substrates for olfactory function, in this case, the ability of recognition of voice and facial emotion. Odors were rated as to their intensity, pleasantness, pungency, coolness and familiarity and they were considered suitable for composing a test to assess olfactory function. &quot;Odor Tablets¿ proceeded through multiple-choice test with four alternatives which only one was correct. For the development of odor discrimination test, 24 pure odorants, associated with six different odor categories ¿ sweet, green, fruity, citric, floral and unpleasant - were selected and diluted in propylene glycol. With these samples, 36 paired comparison tests were constructed, where in each odor category, a target sample was selected to be compared with the others in the same category. The participant¿s performance in the odor discrimination test was analyzed using the &quot;signal-detection" theory through the following parameters: hit rate (HR), false-alarm rate (FR), discrimination measurement (d 'L) and bias response (CL). Both tests, &quot;Odor Tablets¿ and odor discrimination test, were validated with three groups of subjects: control group, elderly group and temporal lobe epilepsy patients group (TLE). The ability to identify odors from North American TLE patients was also measured, using for this purpose, the validated test "University of Pennsylvania Smell Identification Test" (UPSIT), a multiple choice test consisting of 40 microencapsulated odor stimuli. In addition, the ability of recognition of voice and facial emotion of TLE patients were evaluated using a new tool entitled "Comprehensive Affect Testing System" (CATS). For the subject¿s ability to identify odors, there were significant differences for gender (p=0.0003) and age (p<0.001). Also, for this test, the results showed that pre- and postsurgery epilepsy patients presented lower performance than the control group (p= 0.05); This same result was observed when applying the UPSIT in American TLE patients before and after surgery. &quot;Odor Tablets¿ proved to be easy to administer in adult subjects, showed a high coefficient of the test-retest reliability (r = 0.87, p<0.001), and the tablets presented a storage stability for 3 months. The discrimination test results showed that elderly and epilepsy patient groups presented lower performance in the discrimination measurement and hit rate parameters than the control group. The results obtained using the CATS test in American TLE patients revealed that both pre-and post-surgery showed deficits in the facial and vocal emotions when compared with the control group, especially for negative emotions. The fact that the &quot;Odor Tablets¿ and the discrimination odor test developed in the present study had detected olfactory dysfunction in elderly subjects and epilepsy patients, provides their validity for use in the diagnoses of these population / Doutorado / Consumo e Qualidade de Alimentos / Doutor em Alimentos e Nutrição

Precursores neurais originados na zona subventricular (ZSV) de algumas espécies animais possuem uma rota de migração neuronal destinada ao bulbo olfatório principal (BOP), onde os neuroblastos migrantes se diferenciam em interneurônios. Esta corrente migratória é mantida na idade adulta. A compreensão de como se organiza na idade fetal é essencial para a compreensão geral e estabelecimento de novas terapias celulares. O objetivo deste estudo é caracterizar a composição celular e organização morfológica da ZSV e da corrente rostral migratória (CRM) em encéfalos de fetos caninos. A ZSV, CRM e BOP foram obtidos de fetos caninos de aproximadamente 57 dias de idade gestacional. O tecido foi analisado através de coloração de Nissl, método de imunohistoquímica de dupla marcação com duplacortina (DCX), fator de transcrição SOX2, proteína glial fibrilar ácida (GFAP), calbindina (CALB), calretinina (CALR) e tirosina-hidroxilase (TH). Foram feitas a análise relativa da expressão da imunorreatividade e análise quantitativa de colocalização celular, além do método de microscopia eletrônica de transmissão. Os resultados mostram que a ZSV dorsal possui células imunorreativas (ir) para o DCX ao longo da parede ventricular, dispostas tangencialmente e fileiras de células SOX2-ir foram encontradas na mesma orientação. A imunorreatividade de GFAP foi mais forte na ZSV dorsal e as células possuem fibras dirigidas tangencialmente adjacentes ao ventrículo lateral e fibras orientadas radialmente em direção ao córtex. A CRM de feto de cão tem início na ZSV anterior e segue caudalmente ao redor da cabeça do núcleo caudado e desce na vertical até se curvar rostralmente em direção ao BOP onde termina na camada de células granulares (CCG). A CRM tem aparência homogênea e densa e possui células positivas para o DCX nas porções iniciais e para SOX2 e GFAP por toda a extensão. Não houve células positivas para CALB, CALR e TH em nenhuma região da ZSV e CRM. No BOP, os resultados mostraram que a camada glomerular (CG) possui células imunorreativas a CALR, TH, SOX2 e GFAP. Na camada plexiforme externa (CPE) houve células imunorreativas a CALB, CALR, SOX2 e GFAP e na CCG, houve células imunorreativas a CALR, SOX2 e GFAP. Na análise de colocalização, foram encontrados na CG neurônios CALR que colocalizam com células SOX2 e uma baixa colocalização de neurônios TH e células SOX2. Na CPE, foi observado um baixo número de colocalização de neurônios CALR e CALB e na CCG, as células SOX2 colocalizam com os neurônios CALR. As conclusões mostram que o feto de cão possui uma CRM em direção BOP, com imunorreatividade celular para DCX, SOX2 e GFAP na ZSV e CRM e para CALB, CALR, TH, SOX2 e GFAP nas principais camadas do BOP / Neural precursors originated in the subventricular zone (SVZ) of some animal species have a migration route destined for main olfactory bulb (MOB), where migrants neuroblasts differentiate into olfactory interneurons. This migratory stream is maintained in adulthood. Understanding how it is organized in fetal age is essential for general understanding and establishment of new cell therapies. The aim of this study is characterize the cellular composition and morphological organization of the SVZ and rostral migratory stream (RMS) of brains of canine fetuses. The SVZ, RMS and MOB was obtained from canine fetuses of the approximately 57 gestacional days-old. The tissue was analyzed by Nissl staining and by immunohistochemical methods for double labelling with doublecortin (DCX), transcription factor SOX2, glial fibrillary acid protein (GFAP), calbindin (CALB), calretinin (CALR) and tyrosinehydroxylase (TH). Semiquantitative analysis of immunoreactivity and quantitative analysis of colocalization were realized, besides ultrastructural analysis by electron microscopy. The results show that in dorsal SVZ, DCX immunoreactive cells were found along the ventricular wall, arranged tangentially and lines of SOX2 cells were also found in the same orientation. The GFAP immunostaining is stronger in dorsal SVZ with tangentially directed fibers near the lateral ventricle and radially oriented fibers toward the cortex. The RMS of dog fetus begins at anterior SVZ and follows caudally around the head of the caudate nucleus and vertically descends to bend rostrally into the MOB, where it ends in the granular cell layer (GCL).The RMS have SOX2 positive cells on entire length, showing a homogeneous appearance and high cell density. There is no positive CALB cells or CALR in any region of the SVZ and RMS. The results of the MOB show that the glomerular layer (GL) there were cells immunoreactive to CALR, TH, SOX2 and GFAP. In the external plexiforme layer (EPL) there were immunoreactive cells for CALR, CALB, SOX2 and GFAP and, the GCL, the prevalence is higher for CALR neurons, SOX2-ir and GFAP-ir cells. In colocalization analysis, they were found a some CALR positive neurons in GL that colabeled with SOX2 cells and a low colocalization of TH neurons and SOX2 cells. In EPL, was observed a low colocalization number of CALR and CALB neurons and in GCL, SOX2 cells colabeled with CALR neurons. The conclusions show that the dog fetus has a RMS directed to the MOB, with cellular immunoreactivity for DCX, SOX2 and GFAP in the ZSV and RMS and cellular immunoreactivity for SOX2 CALB, CALR, TH and GFAP in main olfactory bulb layers

Les cellules periglomerulaires du bulbe olfactif conforment une population hétérogène avec des propriétés moléculaires, synaptiques, morphologiques et biophysiques diverses toujours étudiés de façon indépendante. Toutefois, cette diversité suggère que des groupes différents des cellules periglomerulaires pourraient avoir des rôles différents. Dans la première partie de ma thèse je cherche à associer, pour la première fois, différents marqueurs de la diversité des neurones periglomerulaires de façon à aider à comprendre les potentielles implications fonctionnelles que les cellules periglomerulaires pourraient avoir dans le traitement de l’information olfactive. Les cellules periglomerulaires reçoivent des courants inhibiteurs postsynaptiques mais les circuits responsables de cette inhibition restent méconnus. À l’aide des enregistrements électrophysiologiques dans des tranches aiguës horizontales de bulbe olfactif de souris et des techniques d’optogénétique je montre que des projections centrifuges GABAergiques en provenance du télencéphale basal modulent fortement l’inhibition des cellules periglomerulaires de type 2 ainsi que des cellules granulaires et des cellules à axone courtes. / In the olfactory bulb periglomerular cells form a heterogeneous population with diverse molecular, synaptic, morphological and biophysical properties that have always been considered independently and never explored together. However, such diversity suggests different functional implications. On the first part of this thesis, I aim to associate, for the first time, different markers of periglomerular diversity together to put in perspective the functional implications that differebt subgroups of these cells could exert in odor processing. Periglomerular cells receive inhibitory postsynaptic currents but the circuits mediating this inhibition remain poorly understood. Using a combination of patch-clamp recordings in mouse horizontal olfactory bulb slices and optogenetics I demonstrate that centrifugal GABAergic projections from the basal forebrain strongly mediate inhibition of type 2 periglomerular cells but also granule cells and deep short axon cells.

Les projections corticales de retour conduisent l'information vers des relais de traitement de l'information plus précoces. Elles sont essentielles pour la perception sensorielle. En ce qui concerne l'olfaction, l'information sensorielle est constituée d'une multitude de molécules odorantes, et c'est ce mélange complexe qui pénètre dans la cavité nasale. En fonction du contexte, c'est une partie ou une autre de cet ensemble de molécules qui va être importante d'un point de vue comportemental. Les signaux corticaux de retour permettraient de focaliser son attention sur les odeurs pertinentes de l'environnement. Au cours de mon doctorat, j'ai étudié le rôle de la signalisation inhibitrice GABAergique dans ces retours corticaux vers le bulbe olfactif, le premier relais de l'information olfactive. La première partie de mon travail a mis en évidence une modulation métabotropique GABAergique du retour cortical excitateur. Nos expériences caractérisent ensuite l'effet produit par cette modulation sur le bulbe olfactif. Nous avons ainsi démontré que la signalisation GABAergique au niveau de retours corticaux change de manière profonde la réponse du bulbe olfactif aux stimuli olfactifs. Dans un deuxième temps, j'ai trouvé que le cortex olfactif envoie non seulement des projections de retour excitatrices, mais aussi des retours inhibiteurs. Des expériences précisent ensuite la localisation de ce retour GABAergique, ainsi que son impact sur le bulbe olfactif. Nous avons notamment observé qu'en manipulant l'activité de ces fibres GABAergiques, nous pouvions modifier le comportement olfactif. / Cortical feedback conducts information towards earlier relays of information processing. It is instrumental for sensory perception. In the olfactory system, odorants are never experienced in isolation by the nose, and they might be meaningful to the animal or not depending on the context. Feedback inputs onto early processing stages are poised to permit selective attention to the relevant odorants in the olfactory scene. During my thesis work, I focused on understanding the key role that inhibitory GABAergic signaling plays in the cortical feedback to the olfactory bulb in mice.The first part of my work started with the discovery of excitatory transmission between cortical feedback inputs and the olfactory bulb is modulated by metabotropic receptors for GABA. Next, the impact of this regulation on the olfactory bulb network was investigated. We found that GABAergic signaling at cortical feedback axons profoundly changes the response of the olfactory bulb output cells to odor stimulation. In the second part of my thesis, I found that the cortical projections to the olfactory bulb not only comprises of excitatory components, but also inhibitory components. The precise origin of this GABAergic feedback was then determined and its impact on the olfactory bulb network is currently assessed. In particular, we observed that manipulating the activity of this GABAergic feedback perturbs olfactory behavior.

The aim of the studies included in the thesis is to better understand the interplay between neuronal activity-dependent gene regulation and the bioactive vitamin A metabolite all-trans-retinoic acid (RA) during postnatal development, refinement and maintenance of precise neuronal connectivity using the olfactory sensory neuron (OSN) in the olfactory epithelium (OE) of genetically modified mice as a model. We show that: Inhibition of RA receptor (RAR)-mediated transcription in OSNs reduces expression of the olfactory cyclic nucleotide-gated (CNG) ion channel, which is required for odorant receptor (OR)-mediated stimulus transduction. This, results in increased OSN death and errors in precise connectivity. The increased cell death may be a consequence of reduced intrinsic excitability and/or reduced influx of Ca2+ ions while the errors in connectivity may be due to altered OR-dependent expression of axonal guidance proteins, such as Kirrel-2 and Neuropilin-1. Expression of the RA catabolic enzyme Cyp26B1 in OSNs is positively regulated by RAR-mediated transcription as well as sensory stimulation in a CNG channel-dependent manner. This shows that neuronal activity and local vitamin A metabolism are parts of novel regulatory feedback loop controlling precise connectivity and neuronal survival. The feedback loop may be a form of homeostatic plasticity in response to global changes in neuronal activity. BACE1, an enzyme is implicated in Alzheimer´s disease, and Cyp26B1 are inversely regulated by CNG channel-dependent sensory stimulation. Cyp26B1 expression is switched on at birth, forms a topographic expression gradient in OE and inhibits BACE1 expression into an inverse counter gradient. Taken together these results reveal a novel neuronal activity-dependent mechanism by which sensory stimuli can shape spatial gene expression via altered RA bioavailability. Increased Cyp26B1 expression stimulates turnover of OSNs during adult neurogenesis by a non-cell-autonomous mechanism. The gradient of Cyp26B1 expression correlates with spatially-regulated diversification of OSNs into subpopulations that express different subsets of OR genes. Cyp26B1 expression influences spatial OR diversification of OSNs by two different mechanisms. In the ventrolateral OE, Cyp26B1 inhibits OR expression by blocking OSN differentiation at a stage that may be associated with the cell intrinsic mechanism regulating OR gene choice. In the dorsomedial OE the expression frequency of some ORs is unaltered while other increases, presumably as a consequence of neuronal activity-dependent competition. A probable function of graded and activity-dependent Cyp26B1 expression is to form a topographic partitioning of the olfactory sensory map into functional domains, which gradually differ from each other with regard to experience-driven plasticity and neurogenic potential along the dorsomedial-ventrolateral axis of OE.

La neuromodulation de circuits olfactifs par l'acétylcholine (ACh) joue un rôle important dans la discrimination et l'apprentissage d’odeur. Le traitement précoce des signaux chimiosensoriels se produit dans deux régions fonctionnellement et anatomiquement distinctes, les principaux et accessoires bulbes olfactifs (MOB et AOB), qui reçoivent entrée cholinergique significative du cerveau antérieur basal. Ici, nous explorons la régulation des circuits de l’AOB et la MOB par ACh, et comment cette modulation influence le comportement à médiation olfactifs. De manière surprenante, malgré la présence d'un circuit conservé, l'activation des récepteurs muscariniques de l'ACh révèle des différences marquées dans la modulation cholinergique des neurones de sortie: l’excitation de l’AOB et l'inhibition de la MOB. Les cellules granulaires (GCs), le neurone intrinsèque le plus abondant dans l'OB, présentaient également une réponse muscarinique complexe. Alors que les GCs de l’AOB ont été excitées, les GCs de la MOB présentaient une action muscarinique double, une hyperpolarisation et une augmentation de l'excitabilité non couvert par la dépolarisation cellulaire. Par ailleurs, l’ACh a eu un effet différent sur la relation d'entrée / sortie des MCs dans l’AOB et la MOB, montrant un effet net sur le gain en les MCs de la MOB, mais pas dans l'AOB. Fait intéressant, malgré les différences frappantes dans les actions neuromodulateurs sur les neurones de sortie, l'inhibition de la libération d'ACh chemogenetic produit des perturbations similaires dans les comportements olfactifs médiés par ces deux régions. La diminution de l’ACh dans l'OB a perturbé la discrimination naturelle des odeurs liées moléculairement et l'enquête naturelle des odeurs associées à des comportements sociaux. Ainsi, la neuromodulation distincte par l’ACh dans ces circuits pourrait déclencher des solutions différentes générales pour le traitement des odeurs et les médiateurs chimiques, ainsi que les comportements olfactifs diverses qu'ils déclenchent. / Neuromodulation of olfactory circuits by acetylcholine (ACh) plays an important role in odor discrimination and learning. Early processing of chemosensory signals occurs in two functionally and anatomically distinct regions, the main and accessory olfactory bulbs (MOB and AOB), which receive significant cholinergic input from the basal forebrain. Here we explore the regulation of AOB and MOB circuits by ACh, and how this modulation influences olfactory mediated behaviors. Surprisingly, despite the presence of a conserved circuit, activation of muscarinic ACh receptors revealed marked differences in cholinergic modulation of output neurons: excitation in the AOB and inhibition in the MOB. Granule cells (GCs), the most abundant intrinsic neuron in the OB, also exhibited a complex muscarinic response. While GCs in the AOB were excited, MOB GCs exhibited a dual muscarinic action, a hyperpolarization and an increase in excitability uncovered by cell depolarization. Furthermore, ACh had a different effect on the input/output relationship of MCs in the AOB and MOB, showing a net effect on gain in MCs of the MOB, but not in the AOB. Interestingly, despite the striking differences in neuromodulatory actions on output neurons, chemogenetic inhibition of ACh release produced similar perturbations in olfactory behaviors mediated by these two regions. Decreasing ACh in the OB disrupted the natural discrimination of molecularly related odors and the natural investigation of odors associated with social behaviors. Thus, the distinct neuromodulation by ACh in these circuits could underlie different solutions to the processing of general odors and semiochemicals, and the diverse olfactory behaviors they trigger.

Le cerveau de la drosophile est le siège de processus neuronaux complexes, et la drosophile se révèle être un organisme de choix pour leur étude grâce en particulier aux puissants outils de génétique moléculaire. Une drosophile peut former une mémoire olfactive associative aversive ou appétitive, selon qu’une odeur est associée à une punition ou à une récompense. En aversif, si le conditionnement est répété au moins cinq fois avec intervalles de repos, la mémoire se consolide en impliquant une synthèse protéique de novo et peut alors durer plus d’une semaine : on parle de mémoire à Long Terme (MLT). Dans le cadre du paradigme appétitif, il existe également une MLT dépendant de la synthèse protéique de novo, mais sa formation est engagée dès le premier cycle d’apprentissage. Les Corps Pédonculés sont le centre cérébral où est encodée la mémoire olfactive et comprennent 4000 neurones, les Cellules de Kenyon (KC). Ils sont contactés par environ 150 neurones de projection cholinergiques leur apportant l’information olfactive, mais également par environ 130 neurones dopaminergiques afférents et seulement 34 neurones efférents. Beaucoup des neurones impliqués dans la formation et le stockage des mémoires olfactives ont été identifiés au cours des 15 dernières années. Le premier objectif de mes travaux de thèse a été d’identifier précisément quels neurones encodent la MLT au sein des Corps Pédonculés et quels neurones convoient l’information mnésique hors des Corps Pédonculés. Pour ce faire, nous avons mis à profit des outils thermogénétiques permettant de bloquer la transmission synaptique de neurones choisis, et ce à un moment donné des processus mnésiques. Après avoir induit la formation de MLT aversive ou appétitive, il nous est possible d’inhiber les KC ou les neurones efférents aux Corps Pédonculés lors de la remobilisation, 24 heures après le conditionnement, des informations enregistrées. Ensuite, pour comprendre la physiologie des neurones identifiés, c’est à dire comment leur activité leur permet d’assurer leur fonction dans la mémoire, nous révélons leur activité grâce à une sonde calcique fluorescente exprimée génétiquement et nous enregistrons cette activité in vivo par microscopie confocale. Pour mimer les conditions de rappel de la MLT, nous représentons l'odeur ayant servi au conditionnement à des drosophiles ayant formé une MLT... / Drosophila brain is subject to complex neuronal processes, and their study is very convenient in drosophila due to powerful genetic tools. Drosophila can form aversive or appetitive olfactory associative memory, if an odor is associated to a punishment or a reward. When an aversive conditioning is repeated more than five times with rest intervals, the memory is strengthened, implying de novo protein synthesis and lasting over one week, in what we call Long Term Memory (LTM). With appetitive paradigm, a protein synthesis dependent LTM can also be formed, but from only one conditioning cycle.Mushroom Bodies (MB) are the brain memory center where olfactory memory is encoded, and they comprise 4000 neurons, the Kenyon Cells (KC). They are targeted by around 150 cholinergic projection neurons, bringing olfactory information, but also by 130 afferent dopaminergic neurons and only 34 efferent neurons. Over the past 15 years, one have identified many neurons involved in olfactory memory formation and storage. The first goal of my PhD work was to precisely identify which neurons encode LTM within MB and which neurons carry mnesic information out of MB. To this purpose, we used thermogenetic tools to block synaptic transmission, in precise neuronal populations and at precise time windows. After the induction of aversive or appetitive LTM formation, we can inhibit KC or MB output neurons during the retrieval of recorded informations, 24H later. Then, aiming at understanding the physiology of the unravelled neurons, i.e. how their activity support their role in memory, we record their calcic activity with a genetically encoded fluorescent probe, in vivo, with a confocal microscopy device. To mimick LTM retrieval conditions, we present the conditioned odor to LTM trained flies...

Neurogenesis occurs in exclusive regions in the adult nervous system, the subventricular zone and dentate gyrus in the brain, and olfactory epithelium (OE) in the periphery. Cell replacement after death or injury, occurs to varying degrees in neural tissue, and is thought to be dependent upon the biological responses of stem and/or progenitor cells. Despite the progress made to identify adult OE and central nervous system (CNS) progenitors and lineage trace their progeny, our spatial and temporal understanding of embryonic OE neuroglial progenitors has been stalled by the paucity of identifiable genes able to distinguish individual candidate progenitors. In the developing CNS, radial glia serve as both neural progenitors and scaffolding for migrating neuroblasts and are identified by the expression of a select group of antigens, including nestin. Here, I show that the embryonic OE contains a novel radial glial-like progenitor (RGLP) that is not detected in adult OE. RGLPs express the radial glial antigens nestin, GLAST and RC2, but not brain lipid binding protein (BLBP), which, distinct from CNS radial glia, is instead found in olfactory ensheathing cells, a result confirmed using lineage tracing with BLBP-cre mice. Nestin-cre-mediated lineage tracing with three different reporters reveals that only a subpopulation of nestin-expressing RGLPs activate the “CNS-specific” nestin regulatory elements, and produce spatially restricted neurons in the OE and vomeronasal organ. The dorsal-medial restriction of transgene-activating cells is also seen in the embryonic OE of Nestin-GFP transgenic mice, where GFP is found in a subpopulation of GFP+ Mash1+ neuronal progenitors, despite the fact that endogenous nestin expression is found in RGLPs throughout the OE. In vitro, embryonic OE progenitors produce three biologically distinct colony subtypes, that when generated from Nestin-cre/ZEG mice, produce GFP+ neurons, recapitulating their in vivo phenotype, and are enriched for the most neurogenic colony subtype. Neurogenesis in vitro is driven by the proliferation of nestin+ progenitors in response to FGF2. I thus provide evidence for a novel neurogenic precursor, the RGLP of the OE, that can be regulated by FGF2, and provide the first evidence for intrinsic differences in the origin and spatiotemporal potential of distinct progenitors during OE development. / Medicine, Faculty of / Medicine, Department of / Experimental Medicine, Division of / Graduate

Background: Roux-en-y gastric bypass (RYGB) surgery produces significant weight loss, however a number of patients experience some and/or complete recidivism of weight years after surgery. Limited research has investigated why patients are experiencing weight regain after surgical interventions. Our objective was to identify appetite-related measures associated with weight regain after RYGB surgery. Methods: Using a cross-sectional design, 29 participants (49.6 ± 9.1 years of age; 29-62 months post-RYGB) were divided into three weight categories; (weight maintainers, n = 9; low weight regainers, n = 10; and high weight regainers, n = 10). Appetite, smell function, eating behaviours and food reward were measured in response to a standardized meal. Results: Weight regain increased significantly in association with time after surgery (rs = 0.768, p = 0.016). High regainers gained on average 8.6 kg/year, compared to low regainers and maintainers, 3.8 ± 0.9 kg/year and 0.9 ± 0.9 kg/year, respectively (p < 0.001). Dietary restraint (using the Three Factor Eating Questionnaire) was significantly higher in weight maintainers and low regainers compared to high regainers using clinical subscales (p < 0.05). Weight regain was associated with higher “liking” of high-fat sweet foods (measured with the Leeds Food Preference Questionnaire) among high weight regainers. Conclusion: Weight regain after RYGB may be associated with higher preferences for high-fat sweet foods, whereas, higher dietary restraint may be associated with lower wanting of high-fat sweet foods among weight maintainers. Findings provide insight into why some patients after RYGB regain weight, while others maintain their weight. Future research is needed to further explore the relationships between appetite-related factors and weight regain after RYGB employing a longitudinal study design.