Layer 3 (L3) pyramidal neurons are involved in intrinsic and extrinsic corticocortical communications that are integral to area specific cortical functions. The functional and morphological properties of these neurons are altered in the lateral prefrontal cortex (LPFC) of aged rhesus monkeys, changes which parallel the decline of working memory (WM) function. What is not yet understood is the time course of these neuronal alternations during the aging process, or the impact of neuronal changes on the function of local networks that underlie WM. By comparing the properties of L3 pyramidal neurons from the LPFC of behaviorally characterized rhesus monkeys over the adult lifespan using whole cell patch clamp recordings and neuronal reconstructions, the present dissertation demonstrates that WM impairment, neuronal hyperexcitabilty and spine loss begin in middle age. We use bump attractor models to predict how empirically observed changes affect performance on the Delayed Response Task and Delayed Recognition Span Task (spatial). The performance of both models is affected much more by neuronal hyperexcitability than by synapse loss. In a separate study, we examine pathological changes of L3 pyramidal neurons in the perilesional ventral premotor cortex following acute ischemic injury to the primary motor cortex. Neurons from lesioned monkeys exhibit hyperexcitability and changes the excitatory:inhibitory synaptic balance in favor of inhibition. As oxidative stress and inflammation are known to exacerbate both age-related and injury-induced neuronal pathology, we characterize neuronal properties in both conditions after administering therapeutic interventions which target inflammatory pathways and which have previously been shown to ameliorate behavioral deficits. Chronic dietary curcumin treatment dampens neuronal hyperexcitability in middle-aged subjects, but the neuronal changes are not correlated with WM improvements. Treatment with mesenchymal-derived extracellular vesicles lowers firing rates and restores excitatory:inhibitory synaptic balance, and importantly, these changes correlate significantly with motor function.

No description available.

Le Syndrome de l’X Fragile (SXF) est la forme héréditaire de retard mental la plus fréquente et la cause la mieux caractérisée de troubles du spectre autistique (TSA). Elle est causée par une mutation causant l’inactivation du gène Fmr1 (codant pour la protéine FMRP). La sensibilité accrue aux stimuli sensoriels est une caractéristique importante du SXF et des TSA, mais les mécanismes sous-jacents sont encore mal compris. Nous avons constaté que la suppression du gène Fmr1 entrainait une hyperexcitabilité sensorielle dans le modèle murin du SXF. Les souris Fmr1KO nécessitaient significativement moins d'informations tactiles pour l'exploration haptique, et les représentations évoquées par les informations tactiles provenant des vibrisses dans le cortex somatosensoriel primaire (S1) se propageaient à une vitesse plus élevée chez les souris Fmr1KO par rapport aux souris témoins sauvages.Au niveau cellulaire, il a été montré que les ARNm de plusieurs sous-unités de canaux ioniques (par exemple HCN1, KCNMA1) jouant un rôle clé dans le traitement de l'information dendritique / neuronale étaient des cibles de la protéine FMRP (Liao et al, 2008; Darnell et al, 2011). Sur la base de ces observations, nous avons étudié les canalopathies comme une caractéristique importante du SXF. Nous avons testé de possibles dysfonctionnement des canaux ioniques, et leurs conséquences sur le traitement de l'information dendritique dans les neurones pyramidaux du néocortex de la couche 5 chez les souris Fmr1KO, en utilisant une combinaison d’approches électrophysiologiques et d’imagerie calcique bi-photonique. Nos résultats ont montré que les dendrites des neurones pyramidaux du S1 étaient hyperexcitables, facilitant ainsi le couplage des entrées d’information synaptique à la génération de potentiel d'action en sortie dans les neurones. Cette altération était, au moins en partie, attribuable à un dysfonctionnement des canaux Ih et BKCa et a été partiellement restaurée par l'activation pharmacologique des canaux BKCa. Ces résultats plaident en faveur d'un rôle nouveau et crucial des canalopathies dans l'expression de l'hyperexcitabilité sensorielle dans le SXF. / Fragile X Syndrome (FXS) is the most common form of inherited mental retardation syndrome and most well characterized cause of Autism Spectrum Disorders (ASD), and it is caused by a silencing mutation of the gene Fmr1 (encoding the protein FMRP). Increased sensitivity to sensory stimuli is a prominent feature of FXS and ASD, but its underlying mechanisms are poorly understood. We found that deletion of the Fmr1 gene results in somatosensory hyper-excitability in a mouse model for FXS. Fmr1 knockout (Fmr1KO) mice required significantly less tactile information for haptic exploration, and touch-evoked whisker representations in the primary somatosensory cortex (S1) spread with increased velocity in Fmr1KO mice compared to wild-type control. At the cellular level, it has been shown that the mRNAs of several ion channel subunits (e.g. HCN1, KCNMA1) playing key roles in dendritic/neuronal information processing are regulated by FMRP (Liao et al., 2008; Darnell et al., 2011). Based on these observations, we investigated channelopathies as a prominent feature of FXS. We probed ion channel dysfunction, and its consequence for dendritic information processing in neocortical pyramidal neurons of layer 5 in Fmr1KO mice, using a combination of electrophysiological and 2-photon calcium imaging approaches. Our results showed that dendrites of S1 pyramidal neurons were hyper-excitable, facilitating the coupling of synaptic input to the generation of action potential output in these neurons. This defect was, at least in part, attributable to a dysfunction of Ih channels and BKCa channels and was partially rescued by pharmacological activation of BKCa channels. These findings argue for a novel and critical role for channelopathies in the expression of sensory hyper-excitability in FXS.

Epilepsy is characterized by the hyperexcitability of individual neurons and hyper synchronization of groups of neurons (networks). The acquired changes that take place at molecular, cellular and network levels are important for the induction and maintenance of epileptic activity in the brain. Epileptic activity is known to alter the intrinsic properties and signaling of neurons. Understanding acquired changes that cause epilepsy may lead to innovative strategies to prevent or cure this neurological disorder. Advances in in vitro electrophysiological techniques together with experimental models of epilepsy are indispensible tools to understand molecular, cellular and network mechanisms that underlie epileptiform activity. The aim of the study was to investigate the epileptiform activity induced alterations in Ca2+ dynamics in apical dendrites of hippocampal subicular pyramidal neurons in slices and changes in network properties of cultured hippocampal neurons. We have also made attempts to develop an in vitro model of epilepsy using organotypic hippocampal slice cultures. In the first part of the present study, investigations on the basic properties of dendritic Ca2+ signaling in subicular pyramidal neurons during epileptiform activity are described. Subiculum, a part of the hippocampal formation is present, adjacent to the CA1 subfield. It acts as a transition zone between the hippocampus and entorhinal cortex. It receives inputs directly from the CA1 region, the entorhinal cortex, subcortical and other cortical areas. Several forms of evidences support the role of subiculum in temporal lobe epilepsy. Pronounced neuronal loss has been reported in various regions of the hippocampal formation (CA1 and CA3) leaving the subiculum generally intact in human epileptic tissue. It has been observed that epileptic activity is generated in subiculum in cases where the CA3 and CA1 regions are damaged or even absent. However, it is not clear how subicular neurons protect themselves from epileptic activity induced neuronal death. It is widely accepted that epileptiform activity induced neuronal damage is a result of an abnormally large influx of Ca2+ into neuronal compartments. In the present study, combined hippocampus / entorhinal cortical brain slices were exposed to zero Mg2+ + 4-amino pyridine artificial cerebrospinal fluid (ACSF) to generate spontaneous epileptiform discharges. Whole cell current-clamp recordings combined with Ca2+ imaging experiments (by incorporating Oregon green BAPTA-1 in the recording pipette) were performed on subicular pyramidal neurons to understand the changes in [Ca2+]i transients elicited in apical dendrites, in response to spontaneous epileptic discharges. To understand the changes occurring with respect to control, experiments were performed (in both control and in vitro epileptic conditions) where [Ca2+]i transients in dendrites were elicited by back propagating action potentials following somatic current injections. The results show clear distance-dependent changes in decay kinetics of [Ca2+]i transients (τdecay), without change in the amplitude of the [Ca2+]i transients, in distal parts (95–110 µm) compared to proximal segments (30–45 µm) of apical dendrites of subicular pyramidal neurons under in vitro epileptic condition, but not in control conditions. Pharmacological agents that block Ca2+ transporters viz. Na+/Ca2+ exchangers (Benzamil), plasma membrane Ca2+-ATPase pumps (Calmidazolium) and smooth endoplasmic reticulum Ca2+-ATPase pumps (Thapsigargin) were applied locally to the proximal and distal part of the apical dendrites in both experimental conditions to understand the molecular aspects of the Ca2+ extrusion mechanisms. The relative contribution of Na+/Ca2+ exchangers in Ca2+ extrusion was higher in the distal apical dendrite in in vitro epileptic condition. Using computer simulations with NEURON, biophysically realistic models were built to understand how faster decay of [Ca2+]i transients in the distal part of apical dendrite associated with [Ca2+]i extrusion mechanisms affect excitability of the neurons. With a linear increase in the density of Na+/Ca2+ exchangers along the apical dendrite, the decrease in τ decay values of [Ca2+]i transients in distal regions seen in experimental epileptic condition was reproduced in simulation. This linear increase in Na+/Ca2+ exchangers lowered the threshold for firing in response to consecutive synaptic inputs to the distal apical dendrite. Our results thus, show the existence of a novel neuroprotective mechanism in distal parts of the apical dendrite of subicular pyramidal neurons under in vitro epileptic condition with the Na+/Ca2+ exchangers being the major contributors to this mechanism. Although the enhanced contribution of Na+/Ca2+ exchangers helps the neuron in removing excess [Ca2+]i loads, it paradoxically makes the neuron hyperexcitable to synaptic inputs in the distal parts of the apical dendrites. Thus, the Na+/Ca2+ exchangers may actually protect subicular pyramidal neurons and at the same time contribute to the maintenance of epileptiform activity. In the second part of the study, neuronal network topologies and connectivity patterns were explored in control and glutamate injury induced epileptogenic hippocampal neuronal networks, cultured on planar multielectrode array (8×8) probes. Hyper synchronization of neuronal networks is the hallmark of epilepsy. To understand hyper synchronization and connectivity patterns of neuronal networks, electrical activity from multiple neurons were monitored simultaneously. The electrical activity recorded from a single electrode mainly consisted of randomly fired single spikes and bursts of spikes. Simultaneous measurement of electrical activity from all the 64 electrodes revealed network bursts. A network burst represents the period (lasting for 0.1–0.2 s) of synchronized activity in the network and, during this transient period, maximum numbers of neurons interact with each other. The network bursts were observed in both control and in vitro epileptic networks, but the frequency of network bursts was more in the latter, compared to former condition. Time stamps of individual spikes (from all 64 electrodes) during such time-aligned network burst were collected and stored in a matrix and used to construct the network topology. Connectivity maps were obtained by analyzing the spike trains using cross-covariance analysis and graph theory methods. Analysis of degree distribution, which is a measure of direct connections between electrodes in a neuronal network, showed exponential and Gaussian distributions in control and in vitro epileptic networks, respectively. Quantification of number of direct connections per electrode revealed that the in vitro epileptic networks showed much higher number of direct connections per electrode compared to control networks. Our results suggest that functional two-dimensional neuronal networks in vitro are not scale-free (not a power law degree distribution). After brief exposure to glutamate, normal hippocampal neuronal networks became hyperexcitable and fired a larger number of network bursts with altered network topology. Quantification of clustering coefficient and path length in these two types of networks revealed that the small-world network property was lost once the networks become epileptic and this was accompanied by a change from an exponential to a Gaussian network. In the last part of the study, we have explored if an excitotoxic glutamate injury (20 µM for 10 min) that produces spontaneous, recurrent, epileptiform discharges in cultured hippocampal neurons can induce epileptogenesis in hippocampal neurons of organotypic brain slice cultures. In vitro models of epilepsy are necessary to understand the mechanisms underlying seizures, the changes in brain structure and function that underlie epilepsy and are the best methods for developing new antiseizure and antiepileptogenic strategies. Glutamate receptor over-activation has been strongly associated with epileptogenesis. Recent studies have shown that brief exposure of dissociated hippocampal neurons in culture to glutamate (20 µM for 10 min) induces epileptogenesis in surviving neurons. Our aim was to extend the in vitro model of glutamate injury induced epilepsy to the slice preparations with intact brain circuits. Patch clamp technique in current-clamp mode was employed to monitor the expression of spontaneous epileptiform discharges from CA1 and CA3 neurons using several combinations of glutamate injury protocols. The results presented here represent preliminary efforts to standardize the glutamate injury protocol for inducing epileptogenesis in organotypic slice preparations. Our results indicate that glutamate injury protocols that induced epileptogenesis in dissociated hippocampal neurons in culture failed to turn CA1 and CA3 neurons of organotypic brain slice cultures epileptic. We also found that the CA1 and CA3 neurons of organotypic brain slice cultures are resilient to induction of epileptogenesis by glutamate injury protocols with 10 times higher concentrations of glutamate (200µM) than that used for neuronal cultures and long exposure periods (upto 30 min). These results clearly show that the factors involved in induction of epileptiform activity after glutamate injury in neuronal cultures and those involved in making the neurons in organotypic slices resilient to such insults are different, and understanding them could give vital clues about epileptogenesis and its control. The resilience of CA1 and CA3 neurons seen could be due to differences in homeostatic plasticity that operate in both these experimental systems. However, further studies are required to corroborate this hypothesis.

GABA is the major inhibitory neurotransmitter in the central nervous system. It binds to two types of receptors –ionotropic GABAA and metabotropic GABAB. The GABAA receptor directly gates a Clionophore that causes hyperpolarization in mature excitatory neurons while GABAB receptor mediates a slower hyperpolarizing response via G-protein coupled receptor (GPCR) activated potassium channels. This signaling mechanism gets further complicated by the heterogeneous GABA receptor subunit composition that influences the response kinetics in the postsynaptic membrane. In this thesis, the focus has been to decipher the role of GABAA receptors in relation to cellular excitability in the subiculum under physiological and pathophysiological conditions. The subiculum, considered as the output structure of hippocampus, modulates information flow from hippocampus to various cortical and sub-cortical areas and has been implicated in learning and memory, rhythm generation and various neurological disorders. It gates hippocampal activity with its well orchestrated and fine tuned intrinsic and local network properties. Over the years many studies have shown the involvement of subiculum in temporal lobe epilepsy where it forms the focal point of epileptiform activities with altered cellular and network properties. The subiculum is characterized by the presence of a significant population of burst firing neurons that lead local epileptiform activity. By virtue of its bursting nature and recurrent connections, it is a potential site for seizure generation and maintenance. Epileptiform activities are dynamic in nature and change temporally and spatially according to the alterations in electrophysiological properties of neurons. Transitions to different electrical activities in neurons following a prolonged challenge with epileptogenic stimulus have been shown in other brain structures, but not in the subiculum. Considering the importance of the subicular burst firing neurons in the propagation of epileptiform activity to the entorhinal cortex, we have explored the phenomenon of electrophysiological phase transitions in the burst firing neurons of the subiculum in an in vitro brain slice model of epileptogenesis. Whole-cell patch clamp and extracellular field recordings revealed a distinct phenomenon in the subiculum wherein an early hyperexcitable phase was followed by a late suppressed phase upon continuous perfusion with epileptogenic 4-amino pyridine and magnesium-free medium. The late suppressed phase was characterized by inhibitory post-synaptic potentials (IPSPs) in pyramidal excitatory neurons and bursting activity in local fast spiking interneurons at a frequency of 0.1-0.8 Hz. The IPSPs were mediated by GABAA receptors that coincided with excitatory synaptic inputs to attenuate action potential discharge. These IPSPs ceased following a cut between the CA1 and subiculum. Our results suggest the importance of feedforward inhibition in the suppression of epileptiform activity in subiculum to mediate a homeostatic response towards the induced hyper-excitability. GABA release from presynaptic nerve endings activates postsynaptic GABAA receptors, which evoke faster phasic inhibitory postsynaptic currents (IPSCs) and non-inactivating inhibitory tonic current, mediated through extrasynaptic GABAA receptors. These receptors are heteropentameric GABA-gated channels assembled from 19 possible subunits (α1-6, β1-3, γ1-3, δ, π, ρ1-3, θ, and ε). The 2 major subunits involved in tonic GABAA currents in the hippocampus are α5 and δ subunits. Tonic GABAA receptor mediated inhibitory current plays an important role in neuronal physiology as well as pathophysiology such as mood disorders, insomnia, epilepsy, autism spectrum disorders and schizophrenia. While the alterations of various electrical properties due to tonic inhibition have been studied in neurons from different regions, its influence on intrinsic subthreshold resonance in pyramidal excitatory neurons having hyperpolarization-activated cyclic nucleotide-gated (HCN) channels is not known. In the present study, we show the involvement of α5βγ GABAA receptors in mediating picrotoxin sensitive tonic current in subicular pyramidal neurons using known pharmacological agents that target specific GABAA receptor subunits. We further investigated the contribution of tonic conductance in regulating subthreshold electrophysiological properties using current clamp and dynamic clamp experiments. Our experiments suggest that tonic GABAergic inhibition can actively modulate subthreshold properties of subicular pyramidal neurons including resonance due to HCNchannels that may potentially alter the response dynamics in an oscillating neuronal network.

Dendritische Defekte vermitteln Störungen der Erregbarkeit, Modulation und Plastizität von Neuronen, die zur Entwicklung neurodegenerativer Krankheiten führen können. Eine Mutation des Transkriptionsregulators Zeb2 führt zur Entwicklung des Mowat-Wilson-Syndroms, einer Erkrankung, die mit kognitiven Störungen und einem erkennbaren Gesichtsphänotyp einhergeht. Obwohl kognitive Defekte häufig mit Defekten bei der Bildung des dendritischen Baums in Verbindung gebracht werden, wurde die Rolle von Zeb2 bei der dendritischen Entwicklung bisher nicht untersucht. Hier zeige ich, dass Zeb2-defiziente Neuronen in den oberen neokortikalen Schichten einen abnormalen dendritische Baum aufweisen. Außerdem führt der Verlust von Zeb2 zu einer Fehlorientierung des apikalen Dendriten in einer nicht senkrechten Ausrichtung zur Pia. Darüber hinaus habe ich die Signalwege analysiert, die an der Regulierung der Morphologie des Dendritenbaum stromabwärts von Zeb2 beteiligt sind, und zwar durch Deep Sequencing des Transkriptoms von Zeb2-defizienten und Wildtyp-Neokortices sowie durch Massenspektrometrie-Screens auf Veränderungen in der Expression von Zelloberflächenproteinen nach dem Verlust von Zeb2. Für die vielversprechenden Kandidaten habe ich ein in situ-Hybridisierungs-Screening bei E15,5 durchgeführt. Eine Reihe von Genen, die an der neuronalen Morphologie und an Membranproteinen beteiligt sind, darunter Neuropilin1 und Cadherin6, werden in Gehirnen mit Zeb2-Mangel überexprimiert. Insbesondere habe ich die Rolle der neuen nachgeschalteten Zielgene Nrp1, Cdh6 und Wnt5a analysiert. Ich verwendete shRNA von Nrp1, Cdh6 und Wnt5a in neuronale Zellkultur, um zu zeigen, dass Nrp1 und Wnt5a eine erhöhte dendritische Komplexität in den Zeb2-defizienten neuronalen Zellen fördern. Die Überexpression von Nrp1 in Neuronen der oberen Schicht in vivo mittels in utero Elektroporation ist ausreichend, um die dendritische Komplexität zu fördern. Darüber hinaus zeige ich durch in utero Elektroporation einer shRNA gegen Nrp1 in Zeb2-defiziente Tiere, dass die Unterdrückung von Nrp1 durch Zeb2 für die Unterdrückung exzessiver Verzweigungen während der Entwicklung erforderlich ist. Für die Ausrichtung des Dendritenbaum ist sie jedoch nicht erforderlich. Zusammengenommen zeigen diese Daten die wichtige Rolle des Zeb2-Gens bei der Entwicklung des korrekten Dendritenbaum von Neuronen und der Ausrichtung des apikalen Dendriten. / Dendritic defects mediate disturbances in the excitability, modulation and plasticity of neurons, which can lie at the cause of neurodegenerative diseases. Mutation of the transcriptional regulator, Zeb2, causes the development of Mowat-Wilson syndrome, a condition associated with cognitive defects and a recognizable facial phenotype. Although cognitive defects are often associated with defects in the formation of the dendritic tree, the role of Zeb2 in dendritic development had not been studied previously. Here, I show that Zeb2- deficient neurons in the upper neocortical layers have abnormal dendritic trees. Also, loss of Zeb2 results in the mis-orientation of the apical dendrite to a non-perpendicular orientation to the pia. Furthermore, I have analysed the signalling pathways involved in regulation of dendritic tree morphology downstream of Zeb2 by deep sequencing of the transcriptome of Zeb2-deficient and wildtype neocortices and mass spectrometry screens for changes in the expression of cell surface proteins upon loss of Zeb2. For the promising candidates, I have performed in situ hybridization screening at E15.5. A number of genes involved in neuronal morphology and membrane proteins, including Neuropilin1 and Cadherin6, become overexpressed in Zeb2- deficient brains. In particular, I analysed the role of the novel downstream target genes Nrp1, Cdh6 and Wnt5a. I used shRNA of Nrp1, Cdh6 and Wnt5a in cortical neuron cultures to demonstrate that Nrp1 and Wnt5a promote increased dendritic complexity in Zeb2-deficient neuronal cells. Overexpression of Nrp1 in upper layer neurons in vivo, using in utero electroporation, is sufficient to promote dendritic complexity. In addition, I show using in utero electroporation of an shRNA against Nrp1 into Zeb2-deficient animals, that repression of Nrp1 by Zeb2 is required for suppressing excessive branching during development. It is not needed however for the orientation of the dendritic tree. Taken together, these data show the important role of the Zeb2 gene in the development of the correct dendritic tree of neurons and the orientation of the apical dendrite.

Le neurone est une cellule polarisée qui se divise en deux compartiments spécialisés : le compartiment somato-dendritique et le compartiment axonal. Généralement, le premier reçoit l'information en provenance d'autres neurones et le second génère un message en sortie lorsque la somme des entrées dépasse une valeur seuil au segment initial de l'axone. Ce signal de nature discrète appelé potentiel d'action (PA) est propagé activement jusqu'à la terminaison synaptique où il déclenche la transmission chimique de l'information. Cependant, la fonction axonale ne se résume pas à la simple transmission des séquences de PA à l'image d'un câble de télégraphe. L'axone est également capable de transmettre des variations continues de signaux électriques infraliminaires dit analogues et les combiner avec l'information digitale véhiculée par le PA. J'ai consacré la majorité de mon travail de thèse à l'étude de ce nouvel aspect de la fonction axonale dans le cadre de la transmission synaptique entre les neurones pyramidaux au sein du réseau excitateur CA3 de l'hippocampe de rat. Les résultats obtenus à partir d'enregistrements de paires de neurones pendant ma thèse mettent en évidence deux sortes de signalisation analogue et digitale qui aboutissent à la facilitation de la transmission synaptique. La facilitation analogue-digitale (FAD) a été observée lors d'une dépolarisation prolongée, mais également à la suite d'une hyperpolarisation transitoire au niveau du corps cellulaire. Ce sont deux versants d'une même plasticité à court-terme qui découle de l'état biophysique des canaux ioniques sensibles au voltage étant à l'origine du PA. / The neuron is a polarised cell divided into two specialized compartments: the somato-dendritic and the axonal compartment. Generally, the first one receives information arriving from other neurones and the second generates an output message, when the sum of inputs exceeds a threshold value at the axon initial segment. This all-or-none signal, called the action potential (AP) is propagated actively to the synaptic terminal where it triggers chemical transmission of information. However, axonal function is not limited to transmission of AP sequences like a telegraph cable. The axon is also capable of transmitting continuously changing sub-threshold electric signals called analogue signals and to combine them with the digital information carried by the AP. I devoted the majority of my thesis work to the study of these novel aspects of axonal function in the framework of synaptic transmission between pyramidal neurons in the CA3 excitatory network of the rat hippocampus. The results obtained through paired recordings brought to light two kinds of analogue and digital signalling that lead to a facilitation of synaptic transmission. Analogue-digital facilitation (ADF) was observed during prolonged presynaptic depolarization and also after a transient hyperpolarization of the neuronal cell body. These are two sides of the same form of short-term synaptic plasticity depending on the biophysical state of voltage gated ion channels responsible for AP generation. The first variant of ADF induced by depolarization (ADFD) is due to AP broadening and involves Kv1 potassium channels.