La plasticité cérébrale est une capacité remarquable des cellules du cerveau à adapter leur structure et fonction en réponse à l’expérience et l’environnement. Cette plasticité cérébrale est favorisée par des conditions de vie favorables qui peuvent être modélisées chez le rongeur par le modèle de l’Environnement Enrichi (EE). L’EE consiste à mettre un grand nombre de souris dans de grandes cages comprenant de nombreux objets (nids, tunnels, roues…) qui sont changés régulièrement. L’EE induit une activité physique volontaire accrue, des conditions optimales pour la stimulation des interactions sociales, du comportement exploratoire et des fonctions cognitives. L’EE exerce des effets bénéfiques sur les processus physiologiques au niveau de nombreux systèmes (hormonal, cardiovasculaire, immunitaire…). L’EE réduit les comportements anxio-dépressifs, améliore l'apprentissage et la mémorisation. Ces effets sont sous-tendus par des changements au niveau du cerveau et en particulier de l’hippocampe, où l’on observe en EE plus de neurogenèse et synaptogenèse. De manière intéressante, chez les souris immunodéficientes, les performances mnésiques et la neurogenèse sont très altérées, suggérant une interaction bidirectionnelle entre le système immunitaire et le cerveau. Parmi les cellules du système immunitaire, les lymphocytes T (LT) semblent jouer un rôle particulièrement important dans les mécanismes de plasticité neuronale. Notre objectif a été de caractériser le rôle des LT dans les effets de l’EE sur la plasticité cérébrale et de chercher si ces effets impliquent une modification des LT par l’EE. / Cerebral plasticity is a remarkable ability of brain cells to adapt their structure and function in response to experience and the environment. This cerebral plasticity is enhanced by favorable living conditions that can be modeled in the rodent by the Enriched Environment (EE) model. The EE consists in large number of mice in large cages including numerous objects (nests, tunnels, wheels ...) which are changed frequently. EE induces increased voluntary physical activity, optimal conditions for stimulation of social interactions, exploratory behavior and cognitive functions. EE has beneficial effects on physiological processes in many systems (hormonal, cardiovascular, immune system...). EE reduces anxio-depressive behavior, improves learning and memory. These effects are underpinned by changes in the brain and particularly in the hippocampus, where EE induce more neurogenesis and synaptogenesis. Interestingly, in immunodeficient mice, memory performance and neurogenesis are highly impaired, suggesting a bidirectional interaction between the immune system and the brain. Among the cells of the immune system, T cells appear to play a major role in neuronal plasticity mechanisms. Our objective was to characterize the role of T cells in EE’s effects on cerebral plasticity and to investigate whether these effects imply a modification of T cells by EE.

of Ph.D. thesis Molecular mechanisms of regulation of trafficking and function of different subtypes of NMDA receptors in hippocampal neurons Mgr. Kristýna Skřenková N-methyl-D-aspartate (NMDA) receptors are ionotropic glutamate receptors that play a key role in the mammalian central nervous system. Under physiological conditions, these receptors are important for excitatory synaptic transmission and memory formation. However, under pathological conditions, their abnormal regulation or activation may lead to many neurological and psychiatric disorders, such as Alzheimer's disease, Parkinson's disease, Huntington's disease, epilepsy or schizophrenia. Previous studies have shown that the number and type of NMDA receptors on the cell surface are regulated at multiple levels, including their synthesis, folding, internalization or degradation. During the trafficking of NMDA receptors to the cell surface membrane, both the agonist binding and receptor activation are examined. Moreover, NMDA receptors undergo many posttranslational modifications such as palmitoylation, phosphorylation or N-glycosylation. In this thesis, we studied the molecular mechanisms that may affect the trafficking and functional properties of NMDA receptors in mammalian cells and rat hippocampal neurons. Specifically, we studied i)...

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This study investigated memory in children with temporal lobe epilepsy and the ability to discern hippocampal dysfunction with conventional memory tests that are typically used to detect more global memory impairment. All data was obtained retrospectively from the epilepsy surgery program at a local children’s hospital. The research population consisted of 54 children with intractable epilepsy of temporal onset, balanced across pathology types (with and without hippocampal disease) and other demographics. Each was given a clinical battery prior to surgical intervention, which included the WRAML/WRAML2 Verbal Learning subtest from which the dependent variables for this study were extracted. The research hypothesis had predicted that memory retention between verbal learning and recall would be worse for participants with pathology that included hippocampal sclerosis than for those with non-hippocampal temporal lobe pathology. A two-way mixed-design ANOVA was used to test the hypothesis, which allowed incorporation of variables of interest related to memory factors, pathology type, and hemispheric laterality, as well as their various interactions. There was a significant main effect for change in the number of words retained from the final learning trial to the delayed recall. Although the interaction between memory retention and pathology type was not statistically significant, the average of the memory scores as it related to pathology by side did show significance. Thus, results did not support the hypothetical relationship between retention and hippocampal function. However, additional exploratory analyses revealed that the final learning trial by itself was associated with hippocampal pathology, which applied only to those participants with left-hemisphere lesions. Logistic regression with the final learning trial correctly classified 74 percent of participants into the appropriate pathology category, with 81 percent sensitivity to hippocampal dysfunction. Mean participant memory scores were nearly one standard deviation below the normative mean for both delayed recall and total learning scaled scores, regardless of pathology type or lesion hemisphericity. Thus, while the conventionally used indices of the WRAML Verbal Learning test are useful for determining overall memory status, they are not specific to pathological substrate. The within-subject main effect showed an expected loss of information across the time of the delay, but overall the recall score showed no association with hippocampal functioning. This study revealed the possibility of measuring hippocampal function at statistically significant group levels using learning scores from a widely used measure of verbal memory, even in participants with intact contralateral mesial temporal structures. It also indicated that hippocampal structures do not play a role during recall measures given after a standard time delay. Data further demonstrated a role of the hippocampus for encoding and transferring information beyond short term/working memory into long term. During the learning process, the hippocampus appears to work in concert with short-term memory systems, but does not take over the encoding process until enough repetitions have occurred to saturate the working memory buffer. This research represents a small, yet important step forward in our understanding of the hippocampus, with potentially important implications for the future study of memory constructs and mensuration.

We have previously devised a semiquantitative grading system for hippocampal sclerosis (HS) in specimens resected for intractable temporal lobe epilepsy. The grades range from zero to four based on the amount and distribution of neuronal loss and gliosis. In the present study hippocampal sections from 25 patients who had temporal lobe epilepsy and had previously been assigned a grade were examined with synaptophysin immunohistochemistry, and the synaptic content in specific hippocampal fields was correlated with the results of the HS grading system. There was evidence of both significant synaptic loss and increased synaptic density in different fields of the hippocampus with increasing HS. A marked decrement of synaptic immunostaining was present in fields CA1 and CA4 that were highly correlated with HS grade. Sector CA4 seemed to respond in a more graded or continuous way to the pathological insults occurring in temporal lobe epilepsy than did CA1, which appeared to exhibit an all or nothing response. Also, while the width of the outer part of the molecular layer of the dentate (mld) gyrus decreased with increasing HS grade, the inner part of the mld became wider and showed an increased synaptic density so that the overall width of the mld was increased in the high-grade group. We conclude that quantitative measurement of synaptic loss in CA1 and CA4 using synaptophysin immunohistochemistry is a sensitive method for detecting HS and correlates well with the empirically derived HS grading scale, with CA4 exhibiting a more graded response than CA1. In addition, a plasticity response in the inner part of the mld in patients with high-grade HS has been confirmed and quantitated.

Neuronal nicotinic acetylcholine receptors (nAChRs) are expressed in both the periperhal and central nervous systems, and are involved in pre-, post-, and non-synaptic control of neuronal activation. In the brain, these receptors play an important role in a variety of physiological processes such as cognition, development, learning, and memory formation. Malfunction of these receptors have been implicated in neurodegenerative diseases like Alzheimer's disease (AD), schizophrenia, and Parkinson's disease. To date, 17 different nAChR subunits, including α2-α7 and β2-β4, have been cloned that can form homo- and/or hetero-pentameric ionotropic receptors. The unique combinations of subunit pentamers manifest in distinct functional receptors. Using single-cell real-time quantitative RT-PCR, we identified the individual expression rates and co-expression rates of the different nAChR subunits in rat CA1 hippocampal interneurons in efforts to characterize functional receptors involved in learning and memory. The two-way combination of subunits with highest expression in hippocampal interneurons was α3β2. Moreover, this combination was expressed in ratios near 1:3 or 3:1 α3 to β2 respectively. To investigate the functionality of α3β2 receptors in different stoichiometries, we injected human α3 and rat β2 subunit mRNA in 1:3, 1:1, and 3:1 ratios into Xenopus laevis oocytes for expression. Two-electrode voltage clamp was then performed with the application of different concentrations of ACh to produce full dose-response curves and channel kinetics data. Distinct α3β2 functional channels were identified from the different expression ratios based on significant differences in channel kinetics (i.e.- peak current rise times, peak current decay times, steady state current in forced desensitization) Dose-response curves produced no significant difference in EC50 values in the different expression groups. However, there was a trend to greater agonist sensitivity with increased α3 expression relative to β2. α3β2 receptors were further characterized through forced desensitization of the receptors and generation of IV plots. The findings from this study elucidate the neuronal nAChR subunit combinations that form functional channels in hippocampal interneurons.

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Yes / Evidence suggests that flavonoid-rich foods are capable of inducing improvements in memory and cognition in animals and humans. However, there is a lack of clarity concerning whether flavonoids are the causal agents in inducing such behavioral responses. Here we show that supplementation with pure anthocyanins or pure flavanols for 6 weeks, at levels similar to that found in blueberry (2% w/w), results in an enhancement of spatial memory in 18 month old rats. Pure flavanols and pure anthocyanins were observed to induce significant improvements in spatial working memory (p = 0.002 and p = 0.006 respectively), to a similar extent to that following blueberry supplementation (p = 0.002). These behavioral changes were paralleled by increases in hippocampal brain-derived neurotrophic factor (R = 0.46, p<0.01), suggesting a common mechanism for the enhancement of memory. However, unlike protein levels of BDNF, the regional enhancement of BDNF mRNA expression in the hippocampus appeared to be predominantly enhanced by anthocyanins. Our data support the claim that flavonoids are likely causal agents in mediating the cognitive effects of flavonoid-rich foods.

Les travaux de cette thèse ont pour but de mettre en œuvre des outils de modélisation et de simulation numériques de mécanismes sous-tendant l’activité neuronale, afin de promouvoir la découverte de médicaments pour le traitement des maladies du système nerveux. Les modèles développés s’inscrivent à différentes échelles : 1) les modèles dits « élémentaires » permettent de simuler la dynamique des récepteurs, des canaux ioniques, et les réactions biochimiques des voies de signalisation intracellulaires ; 2) les modèles de neurones permettent d’étudier l’activité électrophysiologique de ces cellules ; et 3) les modèles de microcircuits permettent de comprendre les propriétés émergentes de ces systèmes complexes, tout en conservant les mécanismes élémentaires qui sont les cibles des molécules pharmaceutiques. À partir d’une synthèse bibliographique des éléments de neurobiologie nécessaires, et d’une présentation des outils mathématiques et informatiques mis en œuvre, le manuscrit décrit les différents modèles développés ainsi que leur processus de validation, allant du récepteur de neurotransmetteur au microcircuit. D’autre part, ces développements ont été appliqués à trois études visant à comprendre : 1) la modulation pharmacologique de la potentialisation à long terme (LTP) dans les synapses glutamatergiques de l’hippocampe, 2) les mécanismes de l'hyperexcitabilité neuronale dans l'épilepsie mésio-temporale (MTLE) à partir de résultats expérimentaux in vitro et in vivo, et 3) la modulation cholinergique de l'activité hippocampique, en particulier du rythme thêta associé à la voie septo-hippocampique. / The work of this thesis aims to apply modeling and simulation techniques to mechanisms underlying neuronal activity, in order to promote drug discovery for the treatment of nervous system diseases. The models are developed and integrated at different scales: 1) the so-called "elementary models" permit to simulate dynamics of receptors, ion channels and biochemical reactions in intracellular signaling pathways; 2) models at the neuronal level allow to study the electrophysiological activity of these cells; and 3) microcircuits models help to understand the emergent properties of these complex systems, while maintaining the basic mechanisms that are the targets of pharmaceutical molecules. After a bibliographic synthesis of necessary elements of neurobiology, and an outline of the implemented mathematical and computational tools, the manuscript describes the developed models, as well as their validation process, ranging from the neurotransmitter receptor to the microcircuit. Moreover, these developments have been applied to three studies aiming to understand: 1) pharmacological modulation of the long-term potentiation (LTP) of glutamatergic synapses in the hippocampus, 2) mechanisms of neuronal hyperexcitability in the mesial temporal lobe epilepsy (MTLE), based on in vitro and in vivo experimental results, and 3) cholinergic modulation of hippocampal activity, particularly the theta rhythm associated with septo-hippocampal pathway.

One of the defining characteristics of the brain is its plasticity, which is the ability to alter and reorganize neuronal circuits. The brain is constantly being shaped and moulded by the external world through endogenous factors like neurotransmitters, growth factors and circulating hormones. 17β-estradiol, which is the most potent estrogen among the group of ovarian steroid hormones, has widespread effects throughout the central nervous system. Apart from its actions on regions of the brain concerned with reproduction, estradiol has profound effects on brain areas not classically associated with reproductive function like cerebral cortex, midbrain, brainstem, hippocampus and spinal cord. This enables the hormone to influence learning and memory, emotions, affective state, cognition, motor coordination and pain sensitivity. Estradiol exerts these effects by regulating gene expression via intracellular estrogen receptors. In addition to this, the hormone interacts with receptors at the cell membrane to rapidly alter the electrical activity of neurons and astrocytes, and regulate second messenger systems. The aim of this study was to investigate the cellular and functional effects of estradiol on neuronal networks and on signaling between neurons and astrocytes in primary mixed hippocampal cultures. Estradiol is proconvulsant; it increases neuronal excitability and decreases the threshold for seizures. This property of estradiol is instrumental in precipitating catamenial seizures in women with epilepsy. These are epileptic seizures influenced by cyclical hormone changes and occur in over one-third to half of women with epilepsy. In the first part of the work, the effects of 24-hour estradiol treatment on hippocampal neurons were investigated using fluorescence imaging and electrophysiological techniques. Further, the ability of gabapentin, an antiepileptic drug sometimes used to treat hormone sensitive seizures, to counteract the effects of estradiol was studied. Synaptic vesicles were labeled by uptake of FM 1-43, and high K+- triggered exocytotic release was monitored by fluorescence imaging. The reduction in intensity of FM 1-43 fluorescence, which is a measure of vesicular release, was enhanced by estradiol, suggesting that estradiol upregulates the exocytotic machinery. The high K+-evoked intracellular Ca2+ rise in neurons, studied by loading the neurons with the Ca2+ indicator dye fluo-3 AM, was potentiated following estradiol treatment. Electrophysiological recordings from neurons following estradiol treatment showed an increase in the frequency of miniature excitatory postsynaptic currents (mEPSCs) and a larger number of mEPSC events with a predominant NMDA component. Many of the estradiol-induced excitatory effects on the neuronal network were abolished by incubating the cultures with a combination of estradiol and gabapentin suggesting a mechanism of action for the drug in the treatment of hormone sensitive seizures. Glial cells were once regarded as passive, supportive elements in the nervous system. This view of glial cells has drastically changed over the past decade and it is now known that glial cells are dynamic signaling elements in the brain. In view of the emerging importance of glia in the physiology of the nervous system and accumulating evidence of direct effects of steroid hormones on these cells, the subsequent part of the work delves into the consequences of 24-hour estradiol treatment on astrocytes and neuron-to-astrocyte signaling. Estrogen receptors have been described on both neurons and astrocytes in the hippocampus suggesting a complex interplay between the two cell types in mediating the effects of the hormone. Astrocytes sense and respond to neuronal activity with a rise in intracellular calcium concentration, ([Ca2+]i). Astrocyte ([Ca2+]i) transients can modulate neuronal activity, indicating a bi-directional form of communication between neurons and astrocytes. Using simultaneous electrophysiology and calcium imaging techniques, neuronal activity-evoked ([Ca2+]i) changes in fluo-3 AM loaded astrocytes were monitored. Action potential firing in neurons, elicited by injecting depolarizing current pulses, was associated with ([Ca2+]i) elevations in adjacent astrocytes which could be blocked by 200 µM MCPG and also 1 µM TTX. Comparison of astrocytic ([Ca2+]i) transients in control and estradiol treated cultures revealed that the amplitude of the ([Ca2+]i) transient, the number of responsive astrocytes and the ([Ca2+]i) wave velocity were all significantly reduced in estradiol treated cultures. ([Ca2+]i) rise in astrocytes in response to local application of the metabotropic glutamate receptor agonist t-ACPD was attenuated in estradiol treated cultures suggesting functional changes in the astrocyte metabotropic glutamate receptor following 24-hour treatment with estradiol. Since astrocytes can modulate synaptic transmission by release of glutamate, the attenuated ([Ca2+]i) response seen following estradiol treatment could have functional consequences on astrocyte-neuron signaling. The acute effects of estradiol on astrocyte-to-astrocyte and astrocyte-to-neuron signaling have been addressed in the next part of the study. Bidirectional communication between neurons and astrocytes involves integration of neuronal inputs by astrocytes, and release of gliotransmitters that modulate neuronal excitability and synaptic transmission. In addition to its rapid actions on neuronal electrical activity, estradiol can rapidly alter astrocyte ([Ca2+]i) levels through a plasma membrane-associated estrogen receptor. The functional consequences of acute estradiol treatment (5 min) on astrocyte-astrocyte and astrocyte-neuron communication were investigated using calcium imaging and electrophysiological techniques. Mechanical stimulation of an astrocyte evoked a ([Ca2+]i) rise in the stimulated astrocyte, which propagated to the surrounding astrocytes as a ([Ca2+]i) wave. Following acute treatment with estradiol, the amplitude of the ([Ca2+]i) elevation in astrocytes around the stimulated astrocyte was attenuated. Further, estradiol inhibited the ([Ca2+]i) rise in individual astrocytes in response to the metabotropic glutamate receptor agonist, t-ACPD. Mechanical stimulation of astrocytes induced ([Ca2+]i) elevations and electrophysiological responses in adjacent neurons. Estradiol rapidly attenuated the astrocyte-evoked glutamate-mediated ([Ca2+]i) rise and slow inward current in neurons. Also, the incidence of astrocyte-induced increase in spontaneous postsynaptic current frequency was reduced in presence of estradiol. The effects of estradiol were stereo-specific and reversible following washout. These findings indicate that the regulation of neuronal excitability and synaptic transmission by astrocytes is sensitive to rapid estradiol mediated hormonal control.