Les cellules ciliées sont les cellules sensorielles de la cochlée, l’organe de l’audition, et assurent la transduction des stimulations acoustiques en message nerveux compréhensible par le système nerveux central. Nous avons étudié le rôle du cytosquelette dans le transfert synaptique et dans le cadre d’une neuropathie auditive.La première partie de cette thèse a consisté à déterminer le rôle des filaments d’actine dans l’exocytose des cellules ciliées. Pour ce faire, nous avons infusé des toxines connues pour dépolymériser les filaments d’actine directement dans les cellules ciliées internes. Après 10 minutes d’infusion, nous avons mesuré l’exocytose des cellules ciliées à partir de l’enregistrement de la capacité membranaire, indice de la fusion vésiculaire. Dans nos expériences, la dépolymérisation de l’actine provoquait une augmentation de l’exocytose. De plus nos résultats suggèrent qu’une fraction des vésicules éloignées des canaux calciques se rapproche des sites de libération après dépolymérisation du réseau d’actine. Ces travaux ont donc permis d’identifier une sous-population de vésicules synaptiques dont la disponibilité aux sites de fusion est dépendante des filaments d’actine. La seconde partie de cette thèse porte sur les mécanismes à l’origine d’une neuropathie auditive, la surdité AUNA1. Cette dernière se caractérise par la surexpression cytoplasmique de la protéine Diaph3, dont la fonction est de réguler la nucléation de l'actine et des microtubules. Pour étudier AUNA1, nous avons montré que les souris transgéniques qui surexpriment la protéine Diap3 (protéine murine) développent une surdité progressive, similaire à la neuropathie auditive AUNA1 : soit une élévation des seuils auditifs et des otoémissions normales, témoins de l’activité des cellules ciliées externes (qui ont pour rôle d’amplifier les stimulations sonores). En outre, le potentiel de sommation, qui reflète l’activité in vivo des cellules ciliées internes est altéré chez les souris transgéniques. L’observation des cellules ciliées internes en microscopie électronique à balayage montre un gonflement de la plaque cuticulaire, qui est une plateforme dense en actine servant à ancrer les stétéocils. L’observation en microscopie confocale du réseau de microtubules montre que ce dernier entoure la plaque cuticulaire chez les souris sauvages. A l’inverse, les microtubules envahissent la plaque cuticulaire chez les souris transgéniques. L’ensemble de ces résultats a donc permis de montrer un défaut d’adressage des microtubules à l’origine de la surdité AUNA1. / Inner hair cells transduce sound stimulation into neurotransmitter release onto the afferent auditory nerve fibers. Here, we studied how cytoskeleton modulates the transduction capabilities of the inner hair cells. Exocytosis at the inner hair cell ribbon synapse is achieved through the coupling between calcium channels and glutamate-filled synaptic vesicles. Using membrane capacitance measurements, we probed whether the actin filament network regulates the exocytosis of synaptic vesicles at the auditory hair cell. Our results suggest that actin network disruption increases exocytosis and that actin filaments may spatially organize a sub-fraction of synaptic vesicles with respect to the calcium channel.The auditory neuropathy 1 (AUNA1) is a form of human deafness, which results from a point mutation in the 5’untranslated region of the Diaphanous homolog 3 (DIAPH3) gene. Strikingly, the DIAPH3 mutation leads to the overexpression of the Diaph3 protein, a formin family member involved in the cytoskeleton nucleation and stabilization. Here, we examined in further details the anatomical, functional and molecular mechanisms that account for AUNA1. We found out that the Diap3-overexpressing transgenic mice show a progressive threshold shift associated to a defect in the inner hair cells. While synaptic function was not affected, Diap3-overexpression results into a selective and early-onset alteration of the inner hair cells cuticular plate, a dense plateform anchoring the stereocilia bundle. Molecular dissection of the apical components revealed that the microtubule meshwork undergoes an aberrant targeting into the cuticular plate of the transgenics’ inner hair cells at early onset, leading to the inabilities of these sensory cells to transduce incoming sound stimulation at later stages.

Alzheimer’s disease is a debilitating, progressive neurodegenerative disorder in the elderly, characterized by severe loss of memory and higher cognitive functions. In the hundred years since its discovery, Alzheimer’s disease (AD) has traversed from the status of a ‘rare neurological oddity’ to one of the greatest challenges faced by healthcare and medicine in this millennium. A reported 44 million people currently suffer from AD but only 1 in 4 people have been diagnosed. Although AD has been an area of intense research for almost 50 years now, most studies have focused on the end stage disease. Years of study on the pathological cause underlying AD; have conclusively shown that the accumulation of the sticky peptide, Aβ, is one of the major triggers of AD pathogenesis. However, after the initial Aβ trigger, multiple processes contribute to disease progression, so that by the time a patient is diagnosed on the basis of overt behavioral phenotypes, it is difficult to understand and differentiate between the causative mechanisms and the consequential effects of the disease. It is, perhaps, because of this, that we are still struggling to find therapies for AD which will stop or at the very least slow the course of the disease. In the 2015 report on AD, issued by the Alzheimer’s association, much emphasis has been placed on the early diagnosis of AD and the revision of the diagnostic criteria for AD. According to the new guidelines proposed in 2011, AD has been divided into three stages where the first stage occurs before the appearance of overt behavioral symptoms such as memory loss, whereas by the 1984 guidelines, cognitive disabilities must have already occurred for diagnoses of AD. This proposed preclinical stage of AD has been defined, reflecting the current belief that AD pathogenesis begins almost 20 years before the occurrence of behavioral dysfunction. However, no diagnostic criteria are currently available to establish this stage. Hence, there is a need to understand the early pathogenic mechanisms of AD, which will yield early therapeutic targets as well as early diagnostic markers of AD. One of the earliest documented events in AD pathogenesis is synaptic dysfunction, which is later manifested as loss of dendritic spines. Deficits in long term potentiation (LTP) has been demonstrated in Aβ exposed hippocampal slices as well as in mouse models of AD, much before the appearance of pathological hallmarks such as plaques and tangles as well as overt behavioral phenotypes. While these and other studies indicate clearly that elevated levels of soluble Aβ peptide leads to impairment of synaptic function, the underlying molecular mechanisms are yet to be elucidated. One of the purported mediators of Aβ induced dysfunction is oxidative stress. The Aβ peptide, especially the Aβ42, is a self aggregating peptide with a propensity to form peptidyl radicals. Interaction of the peptidyl radicals with biomolecules leads to the generation of more free radical species via cascading chain reactions. Additionally, Aβ peptide has also been demonstrated to have synaptotoxic effects via its effect on NMDA receptors and calcium influx leading to deregulated reactive oxygen species (ROS) production as well as excitotoxicity. Hence, with a view to understanding Aβ mediated early synaptic dysfunction in AD, we studied early signaling changes in the synaptosomes derived from the cortex of APP/PS1 mice model of AD at various ages. The APP/PS1 model contains a mouse/human chimeric APP gene bearing the KM670/671NL Swedish mutation and the human PS1 gene with an exon 9 deletion. These mice exhibit behavioral deficits from 7 months of age while plaque deposition and gliosis become apparent by 9 months of age. We chose to study both pre-symptomatic ages (1 and 3 months old) as well as post symptomatic (9 months old) mice. Post nuclear supernatant (PNS) as well as synaptosomes were isolated from the cortex of APP/PS1 and age matched control mice. We assayed the levels of reactive oxygen species (ROS) in the PNS and the synaptosomes of post symptomatic 9 months old APP/PS1 mice and age matched controls. In contrast to reports of enhanced oxidative stress markers in the brains of AD patients, we did not find any increase in the levels of ROS in the PNS of post symptomatic APP/PS1 mice compared to age matched controls. However, synaptosomes from the cortex of these animals exhibited a significant increase in ROS levels in APP/PS1 mice compared to controls. We further found that there was significant increase in the ROS levels in synaptosomes, but not PNS, of very young asymptomatic 1 and 3 months old APP/PS1 mice. This is a first demonstration of synapse specific increase in oxidative stress in AD mice, as young as 1 month of age, indicating that disease specific mechanisms operate at the synapse much before the appearance of any overt cellular or behavioral symptoms. The increase in synaptic ROS levels correlated with a small but significant increase in the levels of Aβ42 in the brains of APP/PS1 mice compared to controls. We also found a concurrent change in the redox status of the cytoskeletal protein, actin, at the synapse. As early as 1 month of age, there was a significant decrease in the protein level of reduced actin indicating that there is an increase in the level of oxidized actin at the synapse. This loss of reduced actin was specific to the fibrillar pool of actin while no significant change was observed in the redox status of the monomeric globular pool of actin. Oxidation of actin has been demonstrated to lead to its depolymerization. Concurrently, we found a significant loss of fibrillar actin in the synaptosomes of APP/PS1 mice. Actin is the major cytoskeletal protein at the synapse. Changes in the globular to fibrillar actin ratio at the synapse at early pre-symptomatic ages in APP/PS1 mice will likely lead to structural and consequent functional changes at the synapse. This could potentially be one of the triggers of synaptic dysfunction in AD. Furthermore, changes in the Akt-mTOR signaling pathway was also observed in the synaptosomes of 1 month old APP/PS1 mice, which is sustained at 9 months. There was a significant loss of the mTOR-pS6K-4EBP1 axis in the synaptosomes, but not PNS, of APP/PS1 mice. We found that loss of Akt signaling, as evinced by loss of Akt phosphorylation, Akt kinase activity as well as loss of phosphorylation of downstream effector GSK3β, potentially underlies the loss of mTOR signaling. Further, the loss of Akt signaling is mediated by synapse specific redox modification of Akt and consequent interaction with the protein phosphatase PP2a. Loss of the Akt-mTOR signaling at the synapse is indicative of deficits in local protein translation. Loss of this essential synaptic function, which plays critical roles in synapse maintenance as well as synaptic plasticity during learning and memory, at an early age, will have long ranging impact on synaptic function such as long term potentiation (LTP) in APP/PS1 mice. Our study is the first demonstration of oxidative stress and consequent signaling changes which occur specifically at the synapse of very young 1 month old APP/PS1 mice. These changes occur much before the appearance of overt phenotype such as plaque deposition and behavioral dysfunction but sustain till the appearance of classical pathological hallmarks. Hence, the study demonstrates that disease progression starts much before previously thought and provides us a critical time window during which therapeutic strategies designed to delay or stop these changes might change the course of AD.

Calpains, a highly conserved family of calcium-dependent cysteine proteases, are divided in two groups; classical and non-conventional calpains. Calpain-1 and calpain-2, the classical ones, are ubiquitously expressed and abundant in the CNS. Findings through different experimental approaches, predominantly pharmacological calpain inhibitors, proposed the necessity of the proteases for the modulation of various biological events particularly in the CNS, or a functional link between calpain and neurodegeneration. Significant functions associated with calpain activity are neuronal proliferation/differentiation, signal transduction, apoptosis, and synaptic plasticity; or neuronal death in Alzheimer’s disease, Huntington’s disease, Parkinson’s disease, and ischemic stroke. However, due to limited insights of the approaches taken, such as non-specificity of the inhibitors, the exact roles of calpains in the CNS and the key mechanisms underlying them remain controversial. Calpain-1/calpain-2 germline knock-out are embryonic lethal at a very early stage hindering the use of these lines as mouse models for CNS studies. Accordingly, this thesis research introduced a unique brain-specific calpain-1/calpain-2 knock-out and explored the role of the proteases in brain development/function and in neuronal death. The first set of analyses examined how the elimination of calpain-1/calpain-2 activities in mouse brain impacts CNS development in general and synaptic plasticity in CA1 neurons of hippocampus. CNS-specific elimination of CAPNS1, the common small subunit, abolished calpain-1/calpain-2 activities in mouse brain. In contrast to Calpain-1/calpain-2 germ line knock-outs, the brain-specific knock-outs are viable and the general development of mouse brain is normal. However, morphology of dendrites in pyramidal neurons of the hippocampal CA1 region showed significantly decreased dendritic branching complexity and spine density. Consistent with dendrite morphological abnormalities, electrophysiological analyses revealed a significant decrease in field excitatory postsynaptic potentials, long term potentiation, and learning and memory in the hippocampal CA1 neurons of the mutants. In the second part of this research we investigated the direct role of the calpains in neuronal death and their potential downstream targets in in vitro models of PD and ischemic stroke. Our findings indicated that ablation of calpains activity improves survival of different types of neurons against mitochondrial toxin 1-methyl-4-phenylpyridinium (MPP+), glutamate, and hypoxia. Importantly, we demonstrated an increase in p35-cleavage to p25, a cyclin dependent kinase 5 (Cdk5) activator, and that restoration of p25 significantly suppresses the neuronal survival associated with calpain deficiency. Taken together, this work unequivocally establishes two central roles of calpain-1/calpain-2 in CNS function in plasticity and neuronal death.

Homeostatic synaptic plasticity (HSP) regulates synaptic strength in response to changing neuronal firing patterns. This form of plasticity is defined by neurons’ ability to sense and over time integrate their level of firing activity, and to actively maintain it within a defined range. For instance, a compensatory increase in synaptic strength occurs when neuronal activity is chronically attenuated. However, the underpinning cellular mechanisms of this fundamental neural process remain poorly understood. We previously found that during activity deprivation, HSP leads to an increase in α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic (AMPA) receptor function as well as a shift in subunit composition from Ca2+-impermeable GluA2-containing AMPA receptors to Ca2+-permeable GluA2-lacking AMPA receptors not only at synapses, but also at extrasynaptic sites. Neurons therefore appear to be actively enhancing Ca2+ entry, possibly as a compensatory mechanism in response to a prolonged Ca2+ deficit. To test whether the homeostatic response may, at least in part, be mediated by internal Ca2+ stores, we depleted endoplasmic reticulum (ER) Ca2+ stores by using the Sarco/endoplasmic reticulum Ca2+ ATPases (SERCA) pump blocker cyclopiazonic acid (CPA) for a prolonged period. Interestingly, we have found that prolonged Ca2+-store depletion leads not only to an increase in synaptic strength per se, but also a cell-wide increase in synaptic Ca2+-permeable GluA2-lacking AMPARs. This increase in Ca2+ influx following periods of inactivity is conceptually highly reminiscent of a store-operated response, whereby cells re-establish their calcium levels following Ca2+ store depletion using cell surface Ca2+ channels. Our results suggest that neurons use synaptic receptors as means to regulate store Ca2+ levels, thus significantly expanding our understanding of the repertoire used by neurons to modulate cellular excitability.

[résumé trop long] / The important relationship between structure and function has always been a fundamental question in neuroscience research. In particular in the case of movement, brain controls large groups of muscles and combines it with sensory informations from the environment to execute purposeful motor behavior. Mapping dynamics encoded in a high dimensional neural space onto low-dimensional behavioral space has always been a difficult challenge as far as theory is concerned. Here, we develope a framework to study spike/burst dynamics having low dimensional phase description, which can readily be extended under certain biological constraints on the coupling to low dimensional functional descriptions. In general, phase models are amongst the simplest of neuron models reproducing spike-burst behavior, excitability and bifurcations towards periodic firing. However, the coupling among neurons has only been considered using generic arguments valid close to the bifurcation point, and the distinction between electric and synaptic coupling remains an open question. In this thesis we aim to address this question and derive a mathematical formulation for the various forms of biologically realistic coupling. We begin by constructing a mathematical model based on a planar simplification of the Morris-Lecar model. Using geometric arguments we then derive a phase description of a network of neurons with biologically realistic electric coupling and subsequently with chemical coupling under the fast synapse approximation. We then demonstrate that electric and synaptic coupling are expressed differently on the level of the network’s phase description, exhibiting qualitatively different dynamics. Our numerical investigations confirm these findings and show excellent correspondence between the dynamics of the full network and the network’s phase description. Following the success of the phase description of the spiking neural network, we extend this approach in order to propose a generating mechanism for parabolic bursting captured by only a single phase variable. This is the first model in the literature which captures bursting dynamics in one dimension. In order to study the emergent behavior we extend this to a network of bursters with global coupling and analytically reduce a high dimensional system to only two dimensions. Further, we investigate the bifurcation properties numerically as well as analytically. One of the key conclusion is that the stability states remain invariant to the increasing number of spikes per burst. Finally we investigate a spikeburst neuron network coupled via mean field type of fast synapses developed in this thesis and systematically carry out a detailed bifurcation analysis of the model, for a tractable special case. Numerical simulations investigate this mean field model beyond special case and clearly reveals qualitative correspondence with the full network model. Moreover, these network displays rich collective dynamics as a function of two parameters, mainly the synaptic coupling strength and the width of the distribution in applied stimulus. Besides incoherence, frequency locking, and oscillator death (a total cessation of firing caused by excessively strong coupling), there exist multistable solutions in the full and the phase network of neurons.

The effects of a range of neuronal excitants were examined on the firing of central neurones of the cerebral cortex, ventrobasal thalamus, dentate gyrus and dorsal and ventral horns of the spinal cords of urethane anaesthetized rats. These responses were pharmacologically characterized on the basis of their susceptibilities to a number of antagonists and from these results, inferences were made concerning probable receptor mechanisms employed by the agonists. Throughout these experiments the technique of iontophoresis was found to be an ideal one for evaluating the effects of agonists and antagonists on single neurones. Neurones in the cortex, thalamus and Renshaw cells of the spinal cord were readily excited by acetylcholine. These responses were elicited also by both nicotinic and muscarinic cholinomimetics. Excitations produced by acetylcholine and acetyl-β-methylcholine were antagonized by atropine and those of acetylcholine and nicotinic agonists were blocked by nicotinic antagonists. The results may be interpreted as revealing a difference between excitatory cholinergic receptors in the rat and in the cat; the nature of these receptors is discussed. to The excitatory responses of ventrobasal thalamic neurones iontophoretically applied amino acids related to glutamate and aspartate could be blocked both by glutamate diethylester and α-aminoadipate. These two antagonists were found to possess different mechanisms of action however, as the ranking orders of susceptibility of the agonists differed for each antagonist. An analysis of these orders led to the proposal that more than one and possibly as many as three different receptors for the excitatory amino acids exist on central neurones. A number of additional compounds were tested for an evaluation of their antagonistic properties against the amino acid induced responses, and these results are discussed in light of possible steric requirements of the receptors. Granule cells of the dentate gyrus were excited by the amino acids and by their synaptic responses to stimulation of perforant path and commissural inputs. A differential effectiveness of glutamate diethylester and α-aminoadipate was suggestive that two distinct excitatory amino acid receptors, both of which appear to be of synaptic significance, coexist on the same neurones. The effects of octopamine were compared with those of catecholamines on neurones of the cortex and dorsal horn of the spinal cord. Both excitation and depression of neuronal firing was observed with octopamine and these responses appeared not to be correlated with those effected by the catecholamines. A further separation of the actions of octopamine and the catecholamines was evident when the amine induced responses were compared in the presence of the antagonists, propranolol and α-flupenthixol. These blocking compounds were effective in attenuating the effects of the catecholamines, but had no effect upon the octopamine induced changes in firing rate. The results suggest that receptors sensitive to octopamine and which appear to be pharmacologically distinct from those previously categorized as catecholamine receptors, may exist on central neurones of the rat. On the basis of the present findings, it was evident that when the technique of iontophoresis is combined with standard neurophysiological methods of identifying central neurones by their responses to synaptic stimulation, valuable information can be obtained concerning the nature of the synaptic transmitters employed by these cells. / Medicine, Faculty of / Cellular and Physiological Sciences, Department of / Graduate

Contrairement à l'opinion populaire l'oubli pourrait être utile: il permettrai le filtrage des informations non-essentielles. Le travail de cette thèse vise à déterminer les bases biologiques de cet oubli adaptatif, en particulier dans le contexte de la mémoire de travail (MT). Nous avons adopté une approche comparative grâce à l'entrainement de rats dans un trois taches comportementales dans un labyrinthe radial visant à tester trois processus cognitifs distincts: la mémoire à long terme, la MT et le traitement des interférences proactive (IP). Nous avons montré que l'information supposée être stockée en MT pouvait perdurer plus longtemps que nécessaire et interférer, plus tard, avec le stockage de nouvelles informations. L'oubli des premiers essais est donc nécessaire pour éviter les IP. Pour comprendre les bases biologiques de cet oubli, nous avons utilisé trois approches. Nous avons effectué une étude immunohistochimique visant à comprendre dans quelle région du cerveau le traitement des IP se produit. Cette étude a montré que ce traitement requiert l'inactivation du gyrus denté de l'hippocampe. Nous avons ensuite effectué une analyse en western-blot pour identifier les processus moléculaires à la base de cette inhibition. Cette étude montre que, dans l'hippocampe, différents processus de plasticité synaptique pourraient se produire pendant le traitement des IP. La troisième approche, vise à comprendre à quel moment ce traitement se produit. Cette étude montre un rôle du sommeil lent dans le traitement des IP. Ces travaux nous aident donc à identifier les mécanismes responsables de l'oubli utile d'informations et donc à mieux comprendre comment le cerveau gère les IP / Contrary to popular opinion forgetting can be useful: it will allow the filtering of non-essential information. The work of this thesis is to determine the biological basis of such adaptive forgetting, especially in the context of working memory (WM). We have adopted a comparative approach through the training of rats in a three behavioral tasks in a radial maze designed to test three distinct cognitive processes: the long-term memory, WM and treatment of proactive interference (PI). We have shown that information supposed to be stored in WM could last longer than necessary and interfere later with the storage of new information. Forgetting the first tests is therefore necessary to avoid PI. To understand the biological basis for this forgetting, we used three methodological approaches. We performed an immunohistochemical study aiming to understand what the brain region underlies the PI processing. This study showed that this processing requires inactivation of the dentate gyrus of the hippocampus. We then performed Western blot analysis in order to identify the molecular processes underlying this inhibition. This study shows that, in the hippocampus, different synaptic plasticity processes may occur during treatment of PI. The third approach is to understand when this processing occurs. This study shows a slow sleep role in the treatment of PI

No description available.

L’élément constitutif des synapses centrales est le site synaptique individuel, comprenant une zone active du côté présynaptique et une densité postsynaptique associée. Du fait de limitations techniques nos connaissances sur le mode de fonctionnement d’un site synaptique restent insuffisantes. Pour faire progresser cette question nous projetons d’effectuer des enregistrements en paires entre interneurones de la couche moléculaire du cervelet. Ces neurones forment des synapses qui ont des signaux élémentaires quantiques de grande taille, et les synapses comprennent parfois un seul site synaptique, ce qui fait qu’ils offrent des avantages décisifs pour ce projet. Les réponses postsynaptiques à des trains de potentiels d’action seront étudiées dans différentes conditions expérimentales. Les résultats seront interprétés par un modèle supposant que les vésicules synaptiques doivent se lier à un petit groupe de sites d’arrimage avant l’exocytose. / The unitary element of central synaptic transmission is a single synaptic site, with one active zone as presynaptic component and the postsynaptic density as postsynaptic partner. Due to technical limitations there is much uncertainty on the mode of functioning of a single synaptic site. To address this issue it is planned to perform paired recordings between interneurons of the molecular layers of the cerebellum. These neurons form synapses with a large quantal size, and occasionally displaying a single release site, and are thus favorable for this study. Postsynaptic responses will be studied in response to trains of presynaptic action potentials under various conditions. The results will be compared to a model supposing the obligatory binding of vesicles to a small complement of docking sites prior to exocytosis.

No description available.