Activity of the NMDA receptor is crucial for CA1 plasticity. Functional modification of the receptor is one way to modulate synaptic plasticity and affect hippocampus dependent behaviours. Two GPCRs, the dopamine receptor D1 and the PACAP38 receptor PAC1, have been shown to enhance NMDA activity via Gq and Gs signaling pathways respectively. Enhancement of NMDAR activity by the D1/Gs pathway depends on phosphorylation of the NR2B subunit by Fyn kinase. Conversely, enhancement by the PAC1/Gq pathway depends on phosphorylation of the NR2A subunit by Src kinase. SKF81297, a D1 agonist, was shown to enhance LTD whereas PACAP38, through the PAC1 pathway, was shown to lower the threshold for LTP. Both effects were blocked by specific antagonists and shown to be dependent on NR2 subunit phosphorylation. Ultimately, physiological metaplasticity at the CA1 synapse may be mediated by the relative activation of many GPCR signaling pathways via modification of the NR2 subunit.

Activity of the NMDA receptor is crucial for CA1 plasticity. Functional modification of the receptor is one way to modulate synaptic plasticity and affect hippocampus dependent behaviours. Two GPCRs, the dopamine receptor D1 and the PACAP38 receptor PAC1, have been shown to enhance NMDA activity via Gq and Gs signaling pathways respectively. Enhancement of NMDAR activity by the D1/Gs pathway depends on phosphorylation of the NR2B subunit by Fyn kinase. Conversely, enhancement by the PAC1/Gq pathway depends on phosphorylation of the NR2A subunit by Src kinase. SKF81297, a D1 agonist, was shown to enhance LTD whereas PACAP38, through the PAC1 pathway, was shown to lower the threshold for LTP. Both effects were blocked by specific antagonists and shown to be dependent on NR2 subunit phosphorylation. Ultimately, physiological metaplasticity at the CA1 synapse may be mediated by the relative activation of many GPCR signaling pathways via modification of the NR2 subunit.

Theta-burst stimulation (TBS) is a form of repetitive transcranial magnetic stimulation (TMS) developed to induce neuroplasticity. TBS usually consists of 50 Hz bursts at 5 Hz intervals. When applied intermittently, it can lead to facilitation of motor evoked potentials (MEPs), although these effects can be variable between individuals. Here, we aimed to determine whether a version of intermittent TBS (iTBS) consisting of 30 Hz bursts at 6 Hz intervals would produce less variable modulation. Nineteen healthy adults underwent single-pulse TMS to assess corticomotor excitability at baseline as reflected in MEP amplitude. 30 Hz iTBS was then administered and MEP amplitude was reassessed at 5-, 20- and 45-mins after the iTBS protocol. Compared to baseline, MEPs were significantly facilitated up to 45-min post-iTBS and most participants exhibited the expected facilitation. These observations suggest that 30 Hz/6 Hz iTBS may provide a sound alternative to induce consistent neuromodulatory effects over the commonly used 50 Hz/5 Hz protocol.

This thesis concerns with change detection problematics in digital images captured under indoor conditions with an ordinary integrated camera in two consecutive moments. All challenges that accompany this problem will be discussed, starting with preprocessing and arriving to evaluation of the results. Currently used methods from this field are described and compared with each other such as differencing and LCP (Local Correlation Peak). A novel method, based on LTP descriptors, effectively solving this problem is proposed in this work. The proposed method is then tested on real data. The results of this tests are discussed subsequently. Besides the change detection method a method for parallax error minimization is proposed here.

Le système endocannabinoïde est un important modulateur des fonctions physiologiques. Il est composé des récepteurs aux cannabinoïdes, de ses ligands lipides endogènes (les endocannabinoïdes) et de la machinerie enzymatique pour leur synthèse et leur dégradation. Les récepteurs aux cannabinoïdes de type 1 (CB1) sont exprimés dans différents types cellulaires dans le cerveau et sont connus pour être impliqués dans les processus mnésiques. Les endocannabinoïdes sont mobilisés dépendamment de l’activité notamment dans les régions cérébrales impliquées dans la mémoire telle que l’hippocampe. Dans cette région, les récepteurs CB1 sont exprimés au niveau des terminaisons neuronales présynaptiques où leur stimulation inhibe la libération de neurotransmetteurs, modulant ainsi différentes formes d’activité synaptique. Outre leur expression sur les neurones, les récepteurs CB1 sont également exprimés par les astrocytes. Avec l’élément pré- et post-synaptique, les astrocytes font partis de la « synapse tripartite » où ils participent à la plasticité synaptique et les processus mnésiques associés. De manière intéressante, la stimulation des récepteurs CB1 astrocytaires facilite la transmission glutamatergique dans l’hippocampe. Dans cette région, les astrocytes régulent l’activité des N-methyl-Daspartate receptors (NMDARs) à travers le contrôle des niveaux synaptiques de leur co-agoniste, la D-serine, modulant ainsi la plasticité synaptique à long terme. Cependant, le mécanisme entrainant la libération de D-serine par les astrocytes n’est pas identifié. De manière intéressante, notre laboratoire a montré que les effets délétères des cannabinoïdes exogènes sur la mémoire de travail spatial sont médiés par les récepteurs CB1 astrocytaires à travers un mécanisme dépendant des NMDARs dans l’hippocampe. Cependant, le rôle physiologique des récepteurs CB1 astrocytaires restent méconnus. Une des formes de mémoire impliquant le récepteurs CB1 est la mémoire de reconnaissance d’objet (NOR). La stimulation exogène des récepteurs CB1 hippocampique inhibe la consolidation de la NOR mais la délétion constitutive des récepteurs CB1 n’affecte pas la NOR, suggérant que la signalisation des récepteurs CB1 endogènes n’est pas nécessaire. Cependant, de récentes études soulignent que la délétion globale du gène CB1 pourrait masquer le rôle des récepteurs CB1 des différents types cellulaires. Ceci indique la nécessité de nouveaux outils plus sophistiqués afin de totalement comprendre le rôle physiologique du système endocannabinoïde dans des comportements complexes. Dans cette étude, nous avons étudié le rôle physiologique des récepteurs CB1 astrocytaires dans la formation de la NOR et la plasticité synaptique. En utilisant une combinaison d’approches génétiques, comportementales, électro-physiologiques, d’imagerie et de biochimie, nous avons montré que l’activation endogène des récepteurs CB1 astrocytaires est nécessaire pour la consolidation de la NOR à long terme, et ceci à travers un mécanisme impliquant l’apport en D-sérine, afin de stimuler l’activité des NMDARs synaptiques de l’hippocampe dorsal. Cette étude révèle un mécanisme inattendu à la base de la libération de D-sérine, entrainant l’activité des NMDARs et la formation de la mémoire à long terme. / The endocannabinoid system is an important modulator of physiological functions. It is composed of cannabinoid receptors, their endogenous lipid ligands (the endocannabinoids) and the enzymatic machinery for endocannabinoid synthesis and degradation. The type-1 cannabinoid receptors (CB1) are expressed in different cell types of the brain and are known to be involved in memory processes. Endocannabinoids are mobilized in an activity-dependent manner in brain areas involved in the modulation of memory such as the hippocampus. In this brain region, CB1 receptors are mainly expressed at neuronal pre-synaptic terminals where their stimulation inhibits the release of neurotransmitters, thereby modulating several forms of synaptic activity. Besides their expression in neurons, CB1 receptors are also expressed in astrocytes. Along with the pre- and post-synaptic neurons, astrocytes are part of the “tripartite synapse”, where they participate in synaptic plasticity and associated memory processes. Interestingly, modulation of astroglial CB1 receptors has been proposed to facilitate glutamatergic transmission in the hippocampus. In this brain area, astrocytes regulate the activity of N-methyl-D-aspartate receptors (NMDARs) through the control of the synaptic levels of their co-agonist D-serine, thereby mediating long-term synaptic plasticity. However, the mechanisms inducing D-serine release by astrocytes are still not identified. Interestingly, our laboratory showed that the negative effect of exogenous cannabinoids on spatial working memory is mediated by astroglial CB1 receptors through a NMDAR-dependent mechanism in the hippocampus, but the physiological role of astroglial CB1 remains unknown. One of the forms of memory involving CB1 receptors is novel object recognition (NOR) memory. The exogenous stimulation of hippocampal CB1 receptors inhibits the consolidation of long-term NOR formation. Constitutive global deletion of CB1 receptors in mice leaves NOR memory intact, suggesting that endogenous CB1 receptor signaling is not necessary for long-term NOR. However, recent studies pointed-out that, likely due to compensatory mechanisms, the global deletion of the CB1 gene might mask cell type-specific roles of CB1 receptors, indicating that more sophisticated tools are required to fully understand the physiological roles of the endocannabinoid system in complex behavioral functions. In this work, we investigated the physiological role of the astroglial CB1 receptors on NOR memory formation and synaptic plasticity. By using a combination of genetic, behavioral, electrophysiological, imaging and biochemical techniques, we showed that endogenous activation of astroglial CB1 receptors is necessary for the consolidation of long-term NOR memory, through a mechanism involving the supply of D-serine to enhance synaptic NMDARs-dependent plasticity in the dorsal hippocampus. This study uncovers an unforeseen mechanism underlying D-serine release, triggering NMDARs activity and long-term memory formation.ory.

This thesis deals with a newly-developed model for the spatiotemporal calcium dynamics within presynaptic terminals. The model is based on single-protein kinetics and has been used to successfully describe different neuron types such as pyramidal neurons in the rat neocortex and the Calyx of Held of neurons from the rat brainstem. A limited number of parameters had to be adjusted to fluorescence measurements of the calcium concentration. These values can be interpreted as a prediction of the model, and in particular the protein densities can be compared to independent experiments. The contribution of single proteins to the total calcium dynamics has been analysed in detail for voltage-dependent calcium channel, plasma-membrane calcium ATPase, sodium-calcium exchanger, and endogenous as well as exogenous buffer proteins. The model can be used to reconstruct the unperturbed calcium dynamics from measurements using fluorescence indicators. The calcium response to different stimuli has been investigated in view of its relevance for synaptic plasticity. This work provides a first step towards a description of the complete synaptic transmission using single-protein data.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Many control techniques applied to converters with power factor correction (PFC) use cascaded control to compensate the output voltage and the input current. These techniques usually employ linear time-invariant (LTI) models and ignore the periodic output voltage oscillation by using compensator with low-pass characteristic. To ensure correlation between the rectifier and its LTI model, as well as power factor correction, these techniques reduce the crossing frequency, which result in a closed-loop voltage with poor dynamic response. Aiming to reduce the time-response designers have associated notch filters into the controller to increase the bandwidth. These filters increase the attenuation in ripple frequencies making possible to enlarge system bandwidth without compromising power factor correction. However, increasing the crossing frequency enhances the interaction between ac output components and the feedback system. In these cases, linear time-invariant (LTI) approach can not adequately represent the closed loop dynamics. As a result, LTI stability analysis based on LTI approach is no longer valid. On the other hand, a better dynamic representation can be achieved by using linear time-periodic models (LTP). These models include the modulation effects inherent in the output voltage dynamics. This work presents an alternative systematic methodology to perform the stability analysis for continuous-time closed-loop control systems using linear time-periodic (LTP) models. This methodology use an LTP representation in frequency domain called harmonic transfer function (HTF). This method extends the multi-variable LTI techniques for LTP approach. Finally is detailed the stability analysis of full-bridge and half-bridge single-phase PFC rectifiers. Theoretical, simulation and experimental results are presented to validate the proposed technique. / Muitas técnicas de controle aplicadas a retificadores monofásicos com correção do fator de potência (PFC) utilizam controladores em cascata para compensação da tensão de saída e da corrente de entrada. Essas técnicas, em geral, empregam modelos lineares e invariantes no tempo (LTI) e desconsideram as oscilações periódicas presentes nas tensões de saída, fazendo uso de leis de controle com características passa-baixa. Para garantir a correlação entre o modelo LTI e o retificador, bem como a correção do fator de potência, estas estratégias utilizam banda-passante reduzida, o que limita consideravelmente a resposta dinâmica da compensação da tensão. No intuito de reduzir o tempo de resposta da malha de tensão, sem comprometer a correção do fator de potência, projetistas têm associado filtros rejeita-banda à malha de controle. Esses filtros acentuam a atenuação nas frequências de interesse e, assim, possibilitam elevar a banda-passante sem comprometer a correção do fator de potência na entrada. Porém, a elevação da banda-passante aumenta a interação das componentes alternadas da tensão do lado cc com o sistema de compensação. Nestes casos, a abordagem LTI pode não representar adequadamente as dinâmicas de malha fechada e, consequentemente, não é válida para a análise da estabilidade. Por outro lado, uma melhor representação dinâmica da malha de compensação da tensão de retificadores PFC monofásicos pode ser obtida com a utilização de modelos lineares periódicos no tempo (LTP). Esses modelos dinâmicos incluem os efeitos de modulação inerentes da dinâmica da tensão de saída. Este trabalho apresenta uma metodologia sistemática alternativa para a análise da estabilidade de malha fechada de retificadores PFC monofásicos empregando-se modelos LTP em tempo contínuo. A metodologia de análise da estabilidade utiliza uma representação dos modelos LTP no domínio da frequência, denominado função de transferência harmônica (HTF). Esta representação estende as técnicas clássicas de análise da estabilidade de sistemas LTI multivariáveis para a abordagem LTP. Por fim, são detalhadas as análise de estabilidade dos retificadores monofásicos em ponte completa e meia ponte, considerando-se a operação com correção do fator de potência de entrada. Resultados teóricos, de simulação e experimentais são apresentados para validar a técnica de análise da estabilidade.

Milk is one of the most consumed fresh food, considered a nutritionally complete product. Veterinary drugs are used in order to increase productivity. However, improper use of these products may result in the presence of residues in milk. This study aims to develop a method for determination of antiparasitic residues in bovine milk using fast liquid-liquid low temperature partitioning (LLE-LTP) method and liquid chromatographic coupled to tandem mass spectrometry (LC-MS/MS). Different classes were studied for antiparasitic, such as macrocyclic lactones, pyrethroids, benzimidazoles, organophosphates, carbamates, among others. The spikes were held comprising the Maximum Residue Limits (MRLs) of each compound at levels 1- 200 ug L-1. For validation step was adopted the following parameters: selectivity, linear range, matrix effect, limits of detection (LOD) and quantitation (LOQ), decision limit (CCα) and detection capability (CCβ), precision, accuracy. The method was considered valid to 55 antiparasitic and presented LOD from 0.03 to 6.0 μg L-1 and LOQ from 1 to 20 ug L-1. Recoveries were between 70 and 120% with RSD ≤ 20%, that were considered acceptable. The proposed method was applied to 15 real samples where all samples presented residues of at least one antiparasitic. Of these, two samples contained cyhalothrin and nicarbazin residues above the MRL. However, the method was efficient in determining antiparasitic residues in bovine milk using LLE-LTP and LCMS/ MS, showing good sensitivity and selectivity. / O leite é um dos alimentos in natura mais consumidos, por ser considerado um produto completo nutricionalmente. A fim de tratar e assegurar a sanidade do rebanho, faz-se uso de medicamentos veterinários. No entanto, a utilização inadequada destes produtos pode acarretar na presença de resíduos destes no leite. Este estudo tem como objetivo desenvolver um método para determinação de resíduos de antiparasitários em leite bovino, empregando método extração líquido-líquido com rápida partição a baixa temperatura (LLELTP) e cromatografia líquida acoplada à espectrometria de massas em série (LC–MS/MS). Avaliou-se diferentes classes de antiparasitários, como lactonas macrocíclicas, piretróides, benzimidazóis, organofosforados, carbamatos, entre outros. As fortificações foram realizadas abrangendo os Limites Máximos de Resíduos (LMRs) de cada composto, em níveis de 1 a 200 μg L-1. Para a etapa de validação do método avaliou-se os seguintes parâmetros: seletividade, faixa linear, efeito matriz, limites de detecção (LOD) e quantificação (LOQ), limite de decisão (CCα), capacidade de detecção (CCβ), precisão e exatidão. O método foi considerado validado para 55 antiparasitários e apresentou valores de LOD de 0,03 a 6,0 μg L-1 e de LOQ de 1 a 20 μg L-1. As recuperações ficaram entre 70 e 120%, e RSD ≤ 20%, sendo considerados aceitáveis. O método proposto foi aplicado em 14 amostras reais, onde todas apresentaram resíduos de pelo menos um antiparasitário. Destas, duas amostras continham resíduos de cialotrina e nicarbazin acima do LMR. O método mostrou-se eficiente na determinação de resíduos de antiparasitários em leite bovino empregando LLELTP e LC–MS/MS, demonstrando boa sensibilidade e seletividade.

Les champs récepteurs corticaux se réorganisent en réponse aux changements de l'environnement. Par exemple, suite à une lésion périphérique, les modalités sensorielles préservées gagnent de l'espace cortical au détriment de celles lésées. L'étude du cortex somatosensoriel en tonneau des rongeurs a fourni des données importantes pour la compréhension des mécanismes synaptiques à l'origine de cette réorganisation corticale. En condition normale, les neurones de chaque colonne corticale répondent préférentiellement à la stimulation d'une seule vibrisse principale ("Principal Whisker, PW"). Au contraire, suite à l'amputation de l'ensemble des vibrisses sauf une ("Single Whisker Experience, SWE"), les neurones des colonnes associées aux vibrisses amputées répondent à la stimulation de la vibrisse conservée, à l'origine du renforcement et de l'expansion des représentations corticales des vibrisses conservées. Bien que des preuves indirectes aient révélées un rôle de la potentialisation à long terme ("Long-Term Potentiation, LTP") de synapses préexistantes dans la modification des cartes corticales, probablement via une augmentation du nombre des récepteurs AMPA (AMPARs) aux synapses, un lien direct entre la LTP, la réorganisation des cartes corticales, et l'adaptation des comportements sensori-moteurs suite à une altération des entrées sensorielles n'a pas encore été démontré. L'objectif de cette thèse a donc été de mettre en évidence cette relation de façon expérimentale et en condition physiologique. Pour cela, nous avons mis au point une stratégie in vivo combinant des enregistrements électrophysiologiques, de l'imagerie biphotonique et l'analyse du comportement d'exploration chez la souris contrôle ("Full Whisker Experience, FWE) et amputée de certaines vibrisses (SWE). Nous avons d'abord confirmé que la stimulation rythmique de la PW ("Rhytmic Whisker Swtimulation, RWS") renforce les synapses excitatrices (RWS-LTP) in vivo des souris anesthésiées FWE. Au contraire des souris FWE, les neurones pyramidaux des souris SWE présentent une augmentation de l'excitabilité neuronale et une absence de RWS-LTP, indiquant ainsi que les synapses corticales associées à la vibrisse intacte ont été potentialisées en réponse au protocole SWE. Pour mieux comprendre l'implication de la RWS-LTP dans la réorganisation des cartes corticales et l'adaptation des comportements sensori-moteurs, nous avons développé une nouvelle approche pour manipuler la LTP in vivo grâce à l'immobilisation des AMPARs par des anticorps extracellulaires ("cross-linking"). En effet, notre équipe a montré précédemment que le cross-linking des AMPARs empêche la LTP in vitro. Par ailleurs, une accumulation des AMPARs au niveau post-synaptique a été démontrée in vivo par imagerie biphotonique au cours d'une stimulation RWS, suggérant un rôle de la mobilité de ces récepteurs dans cette RWS-LTP. Au cours de cette thèse, nous avons démontré que le cross-linking des AMPARs in vivo bloque également l'expression de la RWS-LTP, mais sans affecter la transmission synaptique basale, ni l'induction de la RWS-LTP, indiquant ainsi que la mobilité des AMPARs est également fondamental pour l'expression de la LTP in vivo. De façon importante, le cross-linking des AMPARs de façon chronique, au cours du SWE, permet non seulement de rétablir la RWS-LTP et l'excitabilité neuronale, et donc de bloquer la réorganisation corticale, mais aussi de modifier les capacités de récupération sensori-motrices des souris amputées. Dans l'ensemble, nos données démontrent pour la première fois un rôle critique et direct de la RWS-LTP dans le réarrangement des circuits en réponse à l'amputation de certaines vibrisses. La réorganisation des cartes corticales serait ainsi assurée par le renforcement de la transmission synaptique, et constituerait alors un mécanisme compensatoire pour optimiser le comportement sensorimoteur de l'animal lors de l'altération des entrées sensorielles. / Neuronal receptive fields in the cerebral cortex change in response to peripheral injury, with active modalities gaining cortical space at the expense of less active ones. Experiments on the mouse whisker-to-barrel cortex system provided important evidences about the synaptic mechanisms driving this cortical remapping. Under normal conditions, neurons in each barrel-column have receptive fields that are strongly tuned towards one principal whisker (PW). However, trimming all the whiskers except one (single-whisker experience, SWE) causes layer (L) 2/3 pyramidal neurons located in the deprived and spared-related columns to increase their response towards the spared input. This results in a strengthening and expansion of the spared whisker representation within the barrel sensory map. Indirect evidences suggest that these cortical alterations might depend on the activity-dependent potentiation of pre-existing excitatory synapses (LTP), likely through increased levels of postsynaptic AMPA receptors (AMPARs). However, a clear link between LTP, cortical remapping, and the adaptation of sensorimotor skills following altered sensory experience has not yet convincingly been demonstrated. Here, we combined in vivo whole-cell recordings, 2-Photon calcium imaging and a whisker-dependent behavior protocol to directly demonstrate this relationship. It has been described that rhythmic whisker stimulation potentiates cortical synapses (RWS-LTP) in vivo. An accumulation of postsynaptic AMPARs during similar sensory stimulation was also reported by imaging evidences. Our data demonstrates that this potentiation is occluded by SWE, suggesting that cortical synapses are already potentiated by this trimming protocol. This is translated into an increased neuronal excitability in the spared column and sensorimotor recovery by the spared whisker. To better understand the implication of LTP in cortical remapping, we developed a novel approach to manipulate LTP in vivo without affecting overall circuit properties. Our team showed previously that the blockage of AMPARs synaptic recruitment by extracellular antibody cross-linking prevents LTP in vitro. Here, we report that in vivo cross-linking of AMPARs blocks the expression but not the induction of RWS-LTP, suggesting that the synaptic recruitment of AMPARs is fundamental for in vivo LTP as well. Moreover, chronic AMPAR cross-linking during SWE reverts RWS-LTP occlusion and the increased neuronal excitability caused by whisker trimming. As consequence, the sensorimotor performance by the spared whisker is permanently impaired by the blockage of cortical remapping. Altogether, these evidences led us to define a critical role for synaptic LTP on circuit re-arrangement after whisker trimming. Our data shows that LTP-driven cortical remapping is a compensatory mechanism to optimize animal’s sensorimotor behavior upon altered sensory experience.

This thesis deals with a newly-developed model for the spatiotemporal calcium dynamics within presynaptic terminals. The model is based on single-protein kinetics and has been used to successfully describe different neuron types such as pyramidal neurons in the rat neocortex and the Calyx of Held of neurons from the rat brainstem. A limited number of parameters had to be adjusted to fluorescence measurements of the calcium concentration. These values can be interpreted as a prediction of the model, and in particular the protein densities can be compared to independent experiments. The contribution of single proteins to the total calcium dynamics has been analysed in detail for voltage-dependent calcium channel, plasma-membrane calcium ATPase, sodium-calcium exchanger, and endogenous as well as exogenous buffer proteins. The model can be used to reconstruct the unperturbed calcium dynamics from measurements using fluorescence indicators. The calcium response to different stimuli has been investigated in view of its relevance for synaptic plasticity. This work provides a first step towards a description of the complete synaptic transmission using single-protein data.