Im Subikulum der Ratte finden sich zwei unterschiedliche Typen von Pyramidalzellen, die sich auf Grund ihres intrinsischen Entladungsverhaltens unterscheiden. Die Funktion dieser beiden Zelltypen hinsichtlich der synaptischer Neurotransmission ist unklar. Bursterzellen und regulär feuernde Zellen zeigten nach tetanischer Reizung ein unterschiedliches Ausmaß der LTP. Neben der zellspezifischen Ausprägung der LTP fanden sich mehrere Hinweise auf eine zielspezifische Projektion der Efferenzen der vorgeschalteten Area CA1. Die durchgeführten Experimente legen den Schluss nahe, dass Axone von Pyramidalzellen der Area CA1 selektiv auf subikuläre Pyramidenzellen projizieren und so den hippokampalen Informationsfluss steuern und regulieren können. NMDA-Rezeptoren auf beiden Seiten des synaptischen Spaltes spielen hier eine besondere Rolle. Präsynaptische NMDAR der Untereinheit NR2B scheinen an der LTP in Bursterzellen beteiligt zu sein und über einen vermehrten Kalziumeinstrom in die Präsynapse eine langanhaltende Erhöhung der Transmitterausschüttung herbeizuführen. Ebenso zeigten sich abhängig von der Zielzelle Hinweise auf eine unterschiedliche Aktivierung der präsynaptischen Adenylylcyclase-cAMP Kaskade. In Pilokarpin-behandelten Tieren ließ sich nach hochfrequenter Reizung keine langanhaltende Potenzierung der synaptischen Antworten nachweisen. Stattdessen scheinen polysynaptisch latente Verbindungen mittels tetanischer Stimulation aktivierbar zu sein. In einigen Fällen waren diese polysynaptisch latenten Verbindungen per se, in anderen Fällen nach Blockade der GABAergen Neurotransmission aktiv. In Hirnschnittpräparaten von Patienten mit pharmakoresistenter Temporallappenepilepsie konnte im Subikulum spontane rhythmische Aktivität mit einer Frequenz von 0,75 bis 3 Hz aufgezeichnet werden. Diese Aktivität, bestehend aus EPSP/IPSP Sequenzen, wurde sowohl in sklerotischem als auch in nicht sklerotischem Gewebe gefunden. In beiden Gruppen korrelierte die in vitro Aktivität sehr gut mit dem präoperativen Auftreten elektroenzephalografisch detektierter interiktaler Aktivität. Die Blockade GABAerger oder glutamaterger Neurotransmission hob die inhibitorische bzw. exzitatorische Aktivität auf. Dies legt den Schluss nahe, dass sowohl Interneurone wie Pyramidalzellen an der spontanen rhythmischen Aktivität beteiligt sind. / The subiculum plays a key role in processing memory information from the hippocampus to different cortical and subcortical brain regions. Subicular pyramidal cells are classified as regular firing or bursting cells according to their responses to supra-threshold depolarizing current pulses. Synaptic terminals arising from CA1 pyramidal cells do not function as a single compartment but show a specialized synaptic plasticity onto subicular pyramidal cells depending on the discharge properties of the synaptic target. Tetanic stimulation of CA1 axons caused a significantly stronger long-term potentiation (LTP) in bursting cells than in regular firing cells. Postsynaptic bursting was not necessary for the enhanced synaptic potentiation in bursting cells. The LTP in bursting neurons was independent of postsynaptic calcium, induced by presynaptic NR2B-containing autoreceptors and mediated via a adenylyl cylcase-cAMP-dependent signaling cascade. In pilocarpine-treated animals subicular LTP was impaired. A long-lasting increase in synaptic transmission could not be observed after titanic stimulation neither in regular firing cells nor in bursting cells. In human brain slices resected from patients from with drug-resistant temporal lobe epilepsy the subiculum displayed spontaneous rhythmic activity. In sclerotic but also in non-sclerotic hippocampal tissue the subiculum showed cellular and synaptic changes which suffice to generate spontaneous rhythmic activity that is correlated with the occurrence and frequency of interictal discharges recorded in the electroencephalograms of the corresponding patients.

No description available.

DYRK1A es uno de los principales genes candidatos que podrían explicar algunos de los defectos neurológicos asociados al fenotipo Síndrome de Down (SD); desde el retraso mental, rasgo común a todos los individuos con SD hasta los déficits motores, también muy frecuentes entre la población con SD. Con el fin de validar la implicación de DYRK1A en el fenotipo SD se ha desarrollado una estrategia de terapia génica basada en la reducción de la expresión del gen mediante interferencia del RNA, en el modelo transgénico TgDyrk1A, y se han evaluado los efectos en el fenotipo motor de estos animales. Además se ha estudiado la implicación de los receptores glutamatérgicos de tipo NMDA en las alteraciones motoras descritas en el modelo. Los resultados obtenidos en este trabajo ponen de manifiesto la validez de la estrategia desarrollada y apuntan a una desregulación de los receptores de NMDA como uno de los mecanismos moleculares subyacentes de las disfunción motora presente en el modelo TgDyrk1A. / The are growing evidences to consider DYRK1A as a candidate gene for some of the neurological alterations present in DS phenotype such as mental retardation which is a common feature in the syndrome, or motor deficits which show a high prevalence among DS individuals. With the aim to validate the contribution of Dyrk1A to DS phenothype, we have developped a gene therapy strategy based on RNA interference to reduce gene expression in the transgenic model TgDyrk1A, and we have evaluated the effects in the motor phenotype of these animals. Moreover, we have studied the implication of the NMDA glutamate receptor in the motor alterations present in the model. The results obtained validate the strategy developped and suggest the deregulation of the NMDA receptor as one of the main causes underlying motor dysfunction in TgDyrk1A mice.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

Dopamine (DA) neurons of the substantia nigra pars compacta (SNc) have a key role in regulation of voluntary movement control. Their death is a hallmark of Parkinson’s disease, characterised by inhibited motor control, including muscle rigidity and tremor. Excitatory input to SNc-DA neurons is primarily from the subthalamic nucleus, and in PD these afferents display a higher frequency firing, as well as increased burst firing, which could cause increased excitatory activity in SNc-DA neurons. NMDA receptors (NMDARs) bind the excitatory neurotransmitter glutamate, and are essential for learning and memory. In SNc-DA neurons, NMDARs have a putative triheteromeric subunit arrangement of GluN1 plus GluN2B and/or GluN2D. Wild type (WT) mice, and those lacking the gene for GluN2D (Grin2D-null), were used to explore its role in various aspects of DA neuronal function and dysfunction using patch-clamp electrophysiology, viability assaying, and immunofluorescence. Pharmacological intervention using subunit-specific inhibitors ifenprodil and DQP-1105 on elicited NMDAR-EPSCs suggested a developmental shift from primarily GluN2B to GluN2B/D. Activity dependent regulation was assessed by high frequency burst stimulation of glutamatergic afferents: in comparison to controls, significant downregulation of NMDARs was observed in SNc-DA neurons, though no differences were observed based on genotype. This regulatory function may be a neuroprotective or homeostatic response. Ambient extracellular glutamate elicits tonic NMDAR activity in SNc-DA neurons, which may be important for maintaining basal levels of excitability: the role of GluN2D was assessed by recording the deflection in baseline current caused by application of competitive NMDAR antagonist D-AP5. There was a significantly larger NMDAR-mediated current in WT vs Grin2D-null mice, indicating that GluN2D has a role in binding ambient glutamate. Dysfunction of glutamate uptake could be a secondary pathophysiological occurrence in the SNc, leading to increased ambient glutamate: the effect of this was explored by application of the competitive glutamate transporter blocker TBOA. Here, the NMDAR-mediated portion of this current was significantly higher in WT mice in comparison to Grin2D-null. Interestingly, dose-response data obtained from bath application of NMDA showed significantly larger currents in Grin2D-null animals vs WT, but only at the top of the response curve (~1-10 mM), which may indicate a capability for larger conductance in Grin2D-null animals at high NMDAR saturation due to replacement of GluN2D with GluN2B. GluN2D may therefore be neuroprotective, by attenuating peak current flow in response to very high agonist concentrations. Lastly, GluN2D has been found to decrease NMDAR open probability under hypoxic conditions, potentially conferring resistance to hypoxia / ischemia related excitotoxicity. Therefore, low (15% O2 / 80% N2 / 5% CO2) vs high (95% O2 / 5% CO2) oxygen conditions were used along with immunofluorescent propidium iodide cell death assaying and immunofluorescent labeling for DA neurons in order to compare levels of DA neuronal death in the SNc based on oxygen status and genotype. Whilst there was a significant submaximal effect based on O2 status, genotype did not confer a practical resistance under these conditions. In summary, NMDARs have diverse roles in SNc-DA neurons which may both serve to maintain normal function and protect the cell against potentially pathological conditions.

No description available.

Initialement encodés dans l’hippocampe, les nouveaux souvenirs déclaratifs deviennent progressivement dépendants d’un réseau distribué de neurones corticaux au cours de leur maturation dans le temps. Cependant, les mécanismes cellulaires et moléculaires sous-­‐tendant la consolidation et le stockage à long terme de ces nouveaux souvenirs au sein des réseaux corticaux restent à élucider. Les récepteurs N-­‐méthyl-­‐D-­‐aspartate (RNMDA) jouent un rôle essentiel dans l’induction et la régulation des changements synaptiques sous-­‐tendant les processus mnésiques de type associatifs. Sur la base de leurs propriétés biophysiques respectives, nous avons formulé l’hypothèse que la redistribution synaptique des deux formes principales de sous-­‐unités GluN2 exprimées dans le néocortex adulte (GluN2A and GluN2B), pourrait constituer un mécanisme de régulation de la plasticité synaptique supportant l’intégration et la stabilisation progressive des souvenirs au niveau cortical au cours du processus de consolidation mnésique. En combinant, chez le rat adulte, une approche comportementale, biochimique, pharmacologique et des stratégies innovantes consistant à manipuler le trafic de sous-­‐unités des RNMDA à la surface synaptique, nos résultats mettent en évidence un changement cortical dans la composition synaptique en sous unités GluN2, lequel régule la stabilisation progressive de la mémoire à long terme au sein des réseaux corticaux. Nous avons d'abord établi que les RNMDA contenant la sous-­‐unité GluN2B, via leur interaction spécifique avec une protéine clé de la signalisation synaptique, la CaMKII, sont préférentiellement recrutés lors de la phase d’encodage pour permettre l’allocation des nouveaux souvenirs olfactifs associatifs dans un réseau de neurones corticaux spécifique. Au cours du processus de consolidation, nous avons révélé que la redistribution des RNMDA corticaux contenant les sous-­‐unités GluN2B vers l’extérieur ou l’intérieur de l’espace synaptique suite à l’apprentissage, contrôle respectivement la stabilisation de la mémoire à long terme et son oubli au cours du temps. Enfin, renforcer l’acquisition initiale conduit à une augmentation plus rapide du ratio post-­‐synaptique GluN2A/GluN2B et accélère la cinétique du dialogue hippocampo-­‐cortical, ce qui se traduit par une stabilisation accélérée des souvenirs au sein des réseaux corticaux. Pris dans leur ensemble, nos travaux montrent que le trafic des GluN2B-­‐RNMDA corticaux représente un mécanisme cellulaire majeur conditionnant le devenir des traces mnésiques (i.e. stabilisation versus oubli) et apporte un éclairage nouveau sur la façon dont le cerveau organise les souvenirs récents et anciens. / Initially encoded in the hippocampus, new declarative memories are thought to become progressively dependent on a broadly distributed cortical network as they mature and consolidate over time. Although we have a good understanding of the mechanisms underlying the formation of new memories in the hippocampus, little is known about the cellular and molecular mechanisms by which recently acquired information is transformed into remote memories at the cortical level. The N-­‐methyl-­‐D-­‐aspartate receptor (NMDAR) is widely known to be a key player in many aspects of long-­‐term experience-­‐dependent synaptic changes underlying associative memory processes. Based on their distinct biophysical properties, we postulated that the activity-­‐dependent surface dynamics of the two predominant GluN2 subunits (GluN2A and GluN2B) of NMDARs present in the adult neocortex could provide a metaplastic control of synaptic plasticity supporting the progressive embedding and stabilization of long-­‐lasting associative memories within cortical networks during memory consolidation. By combining, in adult rats, behavioral, biochemical, pharmacological and innovative strategies consisting in manipulating trafficking of NMDAR subunits at the cell membrane, our results identify a cortical switch in the synaptic GluN2-­‐containing NMDAR composition which drives the progressive embedding and stabilization of long-­‐lasting memories within cortical networks. We first established that cortical GluN2B-­‐containing NMDARs and their specific interactions with the synaptic signaling CaMKII protein are preferentially recruited upon encoding of associative olfactory memories to enable neuronal allocation, the process via which a new memory trace is thought to be allocated to a given neuronal network. As these memories are progressively processed and embedded into cortical networks, we observed a learning-­‐induced surface redistribution of cortical GluN2B-­‐containing NMDARs outwards or inwards synapses which respectively drives the progressive stabilization and subsequent forgetting of remote memories over time. Finally, increasing the strength, upon encoding, of the initial memory leads to a faster increase of the cortical GluN2A/GluN2B synaptic ratio and accelerates the kinetics of hippocampal-­‐cortical interactions, which translated into a faster stabilization of memories within cortical networks. Taken together, our results provide evidence that GluN2B-­‐NMDAR surface trafficking controls the fate of remote memories (i.e. stabilization versus forgetting), shedding light on a novel mechanism used by the brain to organize recent and remote memories.

Le récepteur du glutamate de type N-méthyl-D-aspartate (NMDAR) est un transducteur clef dans la physiologie du système nerveux et dans nombre de ses pathologies, selon qu’il est localisé à la synapse ou en position extra-synaptique respectivement. Son activité est sous le contrôle étroit du ‘site-glycine’, dont l’activation est gouvernée par la disponibilité en coagoniste. Pourtant, on ignore encore largement les règles qui régissent cette étape limitante de l’activation des NMDARs in situ. Par ailleurs, l’ensemble des onnaissances actuelles suggère que les astrocytes pourraient contrôler les NMDARs dans le contexte des interactions entre cellules gliales et neurones, en particulier via la libération du gliotransmetteur D-sérine. Le principal objectif de ce travail de thèse a été de comprendre les modalités du contrôle endogène des NMDARs par leur site co-agoniste, dans la région CA1 de l’hippocampe. Nous avons porté notre attention, avant tout, sur les acteurs de ce contrôle : la glycine et la D-sérine, qui sont les ligands endogènes du site-co-agoniste. Nous nous sommes intéressés à leur contribution respective dans le contrôle des NMDARs, aux dynamiques de ce contrôle en fonction de l’activité neuronale, à ses variations en fonction de la localisation des NMDARs, ainsi qu’à ses modifications développementales. Nous montrons par des approches d’électrophysiologie que la D-sérine, et non la glycine, est le co-agoniste endogène des NMDARs à la synapse CA3-CA1 chez l’adulte. Elle est délivrée par les prolongements astrocytaires environnants, d’une manière qui est influencée par l’activité synaptique. Sa libération répond à un mécanisme vésiculaire et est dépendante de la signalisation calcique intra-astrocytaire. De cette manière, les astrocytes exercent un contrôle étroit et dynamique des NMDARs à l’état basal et au cours de phénomènes de plasticité synaptique. En contre partie, à l’inverse de leurs homologues localisés à la synapse, les NMDARs extrasynaptiques sont contrôlés par la glycine à l’âge adulte. Cette compartimentation spatiale est dictée par une disponibilité différentielle des deux co-agonistes aux différents sites. Elle est également favorisée par une composition en sous-unités des NMDARs synaptiques et extra-synaptiques différente qui leur confère une affinité distincte pour la glycine et la D-sérine. Enfin, le contrôle des NMDARs par la D-sérine astrocytaire observé à l’âge adulte n’est pas opérationnel à la naissance. En effet, il ne se met en place qu’au cours du premier mois post-natal, de façon concomitante au changement de composition en sous-unités des NMDARs. / N-methyl D-aspartate receptors (NMDARs) are central to many aspects of brain physiology and pathology, which they impact differently depending on their synaptic or extrasynaptic location, respectively. In addition to glutamate, they are gated by the necessary binding of a co-agonist on the so-called ‘glycine-binding site’. However, very little is known about the rules that govern the control of NMDARs through this site, in situ. Evidence now suggests that astrocytes could play a critical role in controlling NMDARs activity, in particular through the release of the gliotransmitter D-serine. In the present work, we aimed at understanding how NMDARs are endogenously controlled through their co-agonist binding site, in the CA1 region of rat hippocampus. We primarily focused on the role of two endogenous ligands of this site: glycine and D-serine. We investigated their relative contribution in the control of NMDARs at the different subcellular locations, the dynamics of such control according to synaptic activity, as well as possible changes during post-natal development. Using elecrophysiological approaches, we demonstrate that NMDARs are gated by Dserine, but not glycine, at CA3-CA1 synapses in adults. D-serine is supplied at least in part by surrounding astrocytes in an activity-dependant manner. Its release occurs in response to calcium signalling within the astrocyte and in a vesicular way. Correspondingly, we found astrocytic supply of D-serine to be essential for NMDARs-dependant functions such as synaptic plasticity. In contrast with their synaptic counterparts, extrasynaptic NMDARs are gated by endogenous glycine and not by D-serine. We provide evidence that this compartmentation relies on the differential availability of the two co-agonists at synaptic and extrasynaptic sites. Besides, due to differences in their subunit composition, synaptic and extrasynaptic NMDARs may have preferential affinity for D-serine and glycine respectively. Finally, we show that the control of the NMDAR co-agonist site is developmentally regulated. Early after birth, glycine is the endogenous co-agonist of synaptic NMDARs. The control exerted by D-serine only progressively appears during the first post-natal month, as the switch in NMDARs subunit composition occurs, suggesting a maturation of cellular interactions at the tripartite synapse.

In preclinical research on Alzheimer's disease pharmacotherapy, attention is paid to multipotent compounds, enabling intensification of the effect by targeting multiple pathophysiological mechanisms. The aim of the thesis was to assess the effect of multipotent compounds and combination therapy in models of cognitive deficit in the rat. The mechanism of action of the tested compounds was modulation of neurotransmitter systems. The aim of the first part of the study was to compare the effect of experimental monotherapy and combination therapy with an N-methyl-D-aspartate (NMDA) receptor antagonist and a γ-aminobutyric acid type A (GABAA) receptor positive modulator in the trimethyltin-induced model. Superiority of the combination therapy was proven by histological analysis of hippocampal neurodegeneration; however, it did not reach statistical significance in the cognitive test. The other part of the thesis focused on multipotent tacrine derivatives. We demonstrated a positive effect of 6- chlorotacrine-6-nitrobenzothiazole hybrid, as well as 6-chlorotacrine-L-tryptophan hybrid, acting as acetylcholinesterase inhibitors, in the scopolamine-induced model of cognitive deficit. Besides, we demonstrated a low risk of serious side effects of other tacrine derivatives acting as NMDA receptor antagonists....

認知功能的提升是當今神經科學領域中的研究重點之一，但其神經機制尚有待釐清。本研究利用一種用於改善阿茲海默症臨床的非競爭型N-甲基-D-天門冬胺酸受體拮抗劑memantine，檢測其對於大白鼠在不同時距相關操作式制約行為及空間工作記憶行為之影響效果。實驗一為針對時間屬性的操作式制約行為實驗，運用大白鼠的區辯性增強低頻反應作業（DRL 10秒行為）與固定時距作業（FI 30秒行為）之行為作業，並操弄連續訓練與間歇訓練的兩種不同模式，測試memantine對前述四組受試的操作式制約行為在表現、消除與自發恢復等三階段之劑量反應。實驗二利用配對性延遲T迷津作業區分出不等基準線(表現好與表現差)之受試，再加以藥理實驗，測試memantine對於前述兩組受試之劑量反應。實驗一結果顯示，受試在兩種不同訓練模式下經十五次習得訓練後，在兩種操作式壓桿行為的壓桿反應相關指標中都有明顯的差異，這證實不同的行為訓練模式會導致學習後的表現有差異之別。memantine藥理實驗結果顯示，此藥對於上述四組受試的操作式行為之三階段的影響效果，會因為不同訓練模式與不同作業而異。實驗二結果顯示，memantine提高空間工作記憶的正確率在表現不好的組別有很顯著的藥效，這證實memantine對於空間式工作記憶行為的影響，也會因學習基準線的不同水平而異。在行為實驗後所進行的蛋白質表現量檢測中，memantine（5 mg/kg）只對五個測試腦區中的背側紋狀體中ERK1磷酸化程度有明顯上升的影響，而其對ERK2及CREB的磷酸化在所有腦組織中皆沒有顯著的影響。綜合以上結果，memantine影響時間與空間屬性的相關行為之藥理效果，會依行為的不同習得歷程(或行為背景經驗)及基準線表現程度而異，而此項行為藥理效果，可能與紋狀體中ERK1的磷酸化有關。 / The neural basis of cognitive enhancement is one of the intriguing topics in neuroscience research; however, the underlying neural mechanisms remain to be elucidated. This study examined the effects of memantine, a non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist which is used to treat Alzheimer’s disease in clinic, on operant behaviors and spatial working memory. In Experiment 1, using the differential reinforcement for low-rate-response 10 sec (DRL 10s) and the fixed-interval 30 sec (FI 30s) operant tasks, and with the manipulation of two different training regimens (continuous vs. intermittent) in the acquisition phase, the effects of memantine were evaluated in three stages of behavioral tests including the performance (right after the end of 15-day acquisition), the extinction, and the spontaneous recovery (after the extinction). In Experiment 2, memantine were tested in the subjects with different level of baseline performance (good vs. bad) on the distinctive patterns of operant responding in four different groups which received DRL 10s and FI 30s with different training regimens; indicating that behavioral task and training background are critical to the operant performance of temporal operant behaviors. Such behavioral outcomes led the dissociable effects of memantine appeared in between the four groups as tested in all three different stages. The results of Experiment 2 showed a profound improvement of the correct responses rate on spatial working memory in the low-baseline group as compared to the higher-baseline group. With a pretreatment of memantine (5 mg/kg), brain tissues in five selected areas were collected for western blot assays of ERK 1, ERK 2, and CREB. The results only revealed a significant increase of ERK 1 phosphorylation in the dorsal striatum. Together, the effects of memantine to improve cognition-associated processes in the temporal operant behaviors and the baseline of performance, and the present observation of cognition-enhancing effects of memantine may be resulted by the ERK 1 phosphorylation in the dorsal striatum.