A computational study of astrocytic glutamate influence on post-synaptic neuronal excitability

<p><b>Postsynaptic activity due to synaptic and intrinsic currents</b>, triggered by (a) synaptic glutamate [Glu]_syn (b-d) simulation with [Glu]_ast,eq = 1.5mM, 5mM, and 10mM respectively, synaptic currents (I_syn) combined AMPA- and NMDA-mediated currents in response to synaptic glutamate, membrane potential (V_m) of postsynaptic neuron resulting from combination of I_syn and voltage-gated currents (Na⁺, K⁺ and leak). Prolonged time course of synaptic glutamate leads to enhanced synaptic currents (I_syn) and higher frequency postsynaptic firing response (V_m depolarisations) as [Glu]_ast,eq increases.</p

Ion homeostasis in rhythmogenesis : the interplay between neurons and astroglia

Proper function of all excitable cells depends on ion homeostasis. Nowhere is this more critical than in the brain where the extracellular concentration of some ions determines neurons' firing pattern and ability to encode information. Several neuronal functions depend on the ability of neurons to change their firing pattern to a rhythmic bursting pattern, whereas, in some circuits, rhythmic firing is, on the contrary, associated to pathologies like epilepsy or Parkinson's disease. In this review, we focus on the four main ions known to fluctuate during rhythmic firing: calcium, potassium, sodium, and chloride. We discuss the synergistic interactions between these elements to promote an oscillatory activity. We also review evidence supporting an important role for astrocytes in the homeostasis of each of these ions and describe mechanisms by which astrocytes may regulate neuronal firing by altering their extracellular concentrations. A particular emphasis is put on the mechanisms underlying rhythmogenesis in the circuit forming the central pattern generator (CPG) for mastication and other CPG systems. Finally, we discuss how an impairment in the ability of glial cells to maintain such homeostasis may result in pathologies like epilepsy and Parkinson's disease

A Mathematical Model of CA1 Hippocampal Neurons with Astrocytic Input

Over time astrocytes have been thought to function in an auxiliary manner, providing neurons with metabolic and structural support. However, recent research suggests they may play a fundamental role in the generation and propagation of focal epileptic seizures by causing synchronized electrical bursts in neurons. It would be helpful to have a simple mathematical model that represents this dynamic and incorporates these updated experimental results. We have created a two-compartment model of a typical neuron found in the hippocampal CA1 region, an area often thought to be the origin of these seizures. The focus is on properly modeling the astrocytic input to examine the pathological excitation of these neurons and subsequent transmission of the signals. In particular, we consider the intracellular astrocytic calcium fluctuations which are associated with slow inward currents in neighbouring neurons. Using our model, a variety of experimental results are reproduced, and comments are made about the potential differences between graded and “all-or-none” astrocytes

Incessant transitions between active and silent states in cortico-thalamic circuits and altered neuronal excitability lead to epilepsy

La ligne directrice de nos expériences a été l'hypothèse que l'apparition et/ou la persistance des fluctuations de longue durée entre les états silencieux et actifs dans les réseaux néocorticaux et une excitabilité neuronale modifiée sont les facteurs principaux de l'épileptogenèse, menant aux crises d’épilepsie avec expression comportementale. Nous avons testé cette hypothèse dans deux modèles expérimentaux différents. La déafférentation corticale chronique a essayé de répliquer la déafférentation physiologique du neocortex observée pendant le sommeil à ondes lentes. Dans ces conditions, caractérisées par une diminution de la pression synaptique et par une incidence augmentée de périodes silencieuses dans le système cortico-thalamique, le processus de plasticité homéostatique augmente l’excitabilité neuronale. Par conséquent, le cortex a oscillé entre des périodes actives et silencieuses et, également, a développé des activités hyper-synchrones, s'étendant de l’hyperexcitabilité cellulaire à l'épileptogenèse focale et à des crises épileptiques généralisées. Le modèle de stimulation sous-liminale chronique (« kindling ») du cortex cérébral a été employé afin d'imposer au réseau cortical une charge synaptique supérieure à celle existante pendant les états actifs naturels - état de veille ou sommeil paradoxal (REM). Dans ces conditions un mécanisme différent de plasticité qui s’est exprimé dans le système thalamo-corticale a imposé pour des longues périodes de temps des oscillations continuelles entre les époques actives et silencieuses, que nous avons appelées des activités paroxysmiques persistantes. Indépendamment du mécanisme sous-jacent de l'épileptogenèse les crises d’épilepsie ont montré certaines caractéristiques similaires : une altération dans l’excitabilité neuronale mise en évidence par une incidence accrue des décharges neuronales de type bouffée, une tendance constante vers la généralisation, une propagation de plus en plus rapide, une synchronie augmentée au cours du temps, et une modulation par les états de vigilance (facilitation pendant le sommeil à ondes lentes et barrage pendant le sommeil REM). Les états silencieux, hyper-polarisés, de neurones corticaux favorisent l'apparition des bouffées de potentiels d’action en réponse aux événements synaptiques, et l'influence post-synaptique d'une bouffée de potentiels d’action est beaucoup plus importante par rapport à l’impacte d’un seul potentiel d’action. Nous avons également apporté des évidences que les neurones néocorticaux de type FRB sont capables à répondre avec des bouffées de potentiels d’action pendant les phases hyper-polarisées de l'oscillation lente, propriété qui peut jouer un rôle très important dans l’analyse de l’information dans le cerveau normal et dans l'épileptogenèse. Finalement, nous avons rapporté un troisième mécanisme de plasticité dans les réseaux corticaux après les crises d’épilepsie - une diminution d’amplitude des potentiels post-synaptiques excitatrices évoquées par la stimulation corticale après les crises - qui peut être un des facteurs responsables des déficits comportementaux observés chez les patients épileptiques. Nous concluons que la transition incessante entre des états actifs et silencieux dans les circuits cortico-thalamiques induits par disfacilitation (sommeil à ondes lentes), déafférentation corticale (épisodes ictales à 4-Hz) ou par une stimulation sous-liminale chronique (activités paroxysmiques persistantes) crée des circonstances favorables pour le développement de l'épileptogenèse. En plus, l'augmentation de l’incidence des bouffées de potentiels d’actions induisant une excitation post-synaptique anormalement forte, change l'équilibre entre l'excitation et l'inhibition vers une supra-excitation menant a l’apparition des crises d’épilepsie.The guiding line in our experiments was the hypothesis that the occurrence and / or the persistence of long-lasting fluctuations between silent and active states in the neocortical networks, together with a modified neuronal excitability are the key factors of epileptogenesis, leading to behavioral seizures. We addressed this hypothesis in two different experimental models. The chronic cortical deafferentation replicated the physiological deafferentation of the neocortex observed during slow-wave sleep (SWS). Under these conditions of decreased synaptic input and increased incidence of silent periods in the corticothalamic system the process of homeostatic plasticity up-regulated cortical cellular and network mechanisms and leaded to an increased excitability. Therefore, the deafferented cortex was able to oscillate between active and silent epochs for long periods of time and, furthermore, to develop highly synchronized activities, ranging from cellular hyperexcitability to focal epileptogenesis and generalized seizures. The kindling model was used in order to impose to the cortical network a synaptic drive superior to the one naturally occurring during the active states - wake or rapid eye movements (REM) sleep. Under these conditions a different plasticity mechanism occurring in the thalamo-cortical system imposed long-lasting oscillatory pattern between active and silent epochs, which we called outlasting activities. Independently of the mechanism of epileptogenesis seizures showed some analogous characteristics: alteration of the neuronal firing pattern with increased bursts probability, a constant tendency toward generalization, faster propagation and increased synchrony over the time, and modulation by the state of vigilance (overt during SWS and completely abolished during REM sleep). Silent, hyperpolarized, states of cortical neurons favor the induction of burst firing in response to depolarizing inputs, and the postsynaptic influence of a burst is much stronger as compared to a single spike. Furthermore, we brought evidences that a particular type of neocortical neurons - fast rhythmic bursting (FRB) class - is capable to consistently respond with bursts during the hyperpolarized phase of the slow oscillation, fact that may play a very important role in both normal brain processing and in epileptogenesis. Finally, we reported a third plastic mechanism in the cortical network following seizures - a decreasing amplitude of cortically evoked excitatory post-synaptic potentials (EPSP) following seizures - which may be one of the factors responsible for the behavioral deficits observed in patients with epilepsy. We conclude that incessant transitions between active and silent states in cortico-thalamic circuits induced either by disfacilitation (sleep), cortical deafferentation (4-Hz ictal episodes) and by kindling (outlasting activities) create favorable circumstances for epileptogenesis. The increase in burst-firing, which further induce abnormally strong postsynaptic excitation, shifts the balance of excitation and inhibition toward overexcitation leading to the onset of seizures

Astrocytic modulation of neuronal network oscillations

The synchronization of the neuron’s membrane potential results in the emergence of neuronal oscillations at multiple frequencies that serve distinct physiological functions (e.g. facilitation of synaptic plasticity) and correlate with different behavioural states (e.g. sleep, wakefulness, attention). It has been postulated that at least ten distinct mechanisms are required to cover the large frequency range of neuronal oscillations in the cortex, including variations in the concentration of extracellular neurotransmitters and ions, as well as changes in cellular excitability. However, the mechanism that gears the transition between different oscillatory frequencies is still unknown. Over the past decade, astrocytes have been the focus of much research, mainly due to (1) their close association with synapses forming what is known today as the “tripartite synapse”, which allows them to bidirectionally interact with neurons and modulate synaptic transmission; (2) their syncytium-like activity, as they are electrically coupled via gap junctions and actively communicate through Ca2+ waves; and (3) their ability to regulate neuronal excitability via glutamate uptake and tight control of the extracellular K+ levels via a process termed K+ clearance. In this thesis we hypothesized that astrocytes, in addition to their role as modulators of neuronal excitability, also act as “network managers” that can modulate the overall network oscillatory activity within their spatial domain. To do so, it is proposed that astrocytes fine-tune their K+ clearance capabilities to affect neuronal intrinsic excitability properties and synchronization with other neurons, thus mediating the transitions between neuronal network oscillations at different frequencies. To validate or reject this hypothesis I have investigated the potential role of astrocytes in modulating cortical oscillations at both cellular and network levels, aiming at answering three main research questions: a) what is the impact of alterations in astrocytic K+ clearance mechanisms on cortical networks oscillatory dynamics? b) what specific neuronal properties underlying the generation of neuronal oscillations are affected as a result of impairments in the astrocytic K+ clearance process? and c) what are the bidirectional mechanisms between neurons and astrocytes (i.e. neuromodulators) that specifically affect the K+ clearance process to modulate the network activity output? In the first experimental chapter I used electrophysiological recordings and pharmacological manipulations to dissect the contribution of the different astrocytic K+ clearance mechanisms to the modulation of neuronal network oscillations at multiple frequencies. A key finding was that alterations in membrane properties of layer V pyramidal neurons strongly correlated with the network behaviour following impairments in astrocytic K+ clearance capabilities, depicted as enhanced excitability underlying the amplification of high-frequency oscillations, especially within the beta and gamma range. The second experimental chapter describes a combinatorial approach based on K+-selective microelectrode recordings and optical imaging of K+ ions used to quantitatively determine extracellular K+ changes and to follow the spatiotemporal distribution of K+ ions under both physiological and altered K+ clearance conditions, which affected the K+ clearance rate. The impact of different neuromodulators on astrocytic function is discussed in the third experimental chapter. Using extracellular K+ recordings and Ca2+ imaging I found that some neuromodulators act specifically on astrocytic receptors to affect both K+ clearance mechanisms and Ca2+ signalling, as evidenced by reduced K+ clearance rates and altered evoked Ca2+ signals. Overall, this thesis provides new insights regarding the impact of astrocytic K+ clearance mechanisms on modulating neuronal properties at both cellular and network levels, which in turn imposes alterations on neuronal oscillations that are associated with different behavioural states

PRRT2-Na+ CHANNELS INTERACTION: PATHOGENETIC BASIS OF PRRT2-ASSOCIATED PAROXYSMAL DISORDERS AND NEW THERAPEUTIC STRATEGIES

Proline-Rich Transmembrane protein 2 (PRRT2) is a neuron-specific protein whose mutations are involved in pleiotropic paroxysmal syndromes including epilepsy, kinesigenic dyskinesia, episodic ataxia and migraine. PRRT2 is a type-2 membrane protein with a transmembrane domain and a long proline-rich N-terminal cytoplasmic region. According to several data, PRRT2 regulates membrane exposure and the biophysical properties of voltage-dependent Na+ channels (Nav) 1.2 and 1.6 that negatively modulate intrinsic excitability. Nav channels form complexes with β-subunits that facilitate the membrane targeting and the activation of the α-subunits. The objective of this thesis is to characterize the molecular and functional PRRT2-Nav interaction clarify: (i) whether PRRT2 and β-subunits interact or compete for common binding sites on the α-subunit, generating Nav complexes with distinct functional properties, (ii) based on its membrane topology, study the structure-function PRRT2 relationships regarding the interaction with Nav, (iii) focus on some point PRRT2 mutations involved in the binding to the Nav directly implicated in PRRT2-related pathologies. Since PRRT2 and β-subunits have opposite effects on Nav channels, it is unclear whether PRRT2 and β-subunits interact or compete for common binding sites on the α-subunit, leading to Nav complexes with different functional features. Using a heterologous expression system, we observed that β-subunits and PRRT2 do not interact with each other acting as independent non-competitive modulators of Nav1.2 channel trafficking and biophysical properties. The data indicate that β4-subunit and PRRT2 form a push-pull system that finely tunes the membrane expression and function of Nav channels and the intrinsic neuronal excitability. In addition, we observed that the unstructured N-terminal cytoplasmic region mimicked full-length PRRT2 by binding to the Nav1.2 more efficiently than the isolated transmembrane domain. Only the C-terminal intramembrane domain was able to modulate Nav properties, maintaining the striking specificity for Nav1.2 vs Nav1.1 channels. These results identify PRRT2 as a multi-domain protein in which the N-terminal cytoplasmic region acts as a binding antenna for Na+ channels, while the transmembrane domain mechanistically regulates channel exposure on the membrane and its biophysical properties. Since the majority of the PRRT2 pathogenic mutations cause the loss of protein expression making in vitro studies difficult or impossible, a restricted number of missense mutations maintains the protein expression and the trafficking to the membrane allowing their characterization. Hence, their expression and function were studied in the same system used for the previous points. Two residues were identified, V286M and A320, that, if mutated, cause Nav binding alteration, and therefore that can be involved in the direct modulation of PRRT2 functions

Development and Analysis of Engineered Brain Cell Microenvironments Mimicking Healthy and Diseased Neuronal Circuits

Astrocytes and microglia (glial cells) are active elements of the brain maintaining numerous homeostatic functions. Disturbances result in worsening of neuro-inflammation, traumatic brain injury, and various stages of brain tumors. Glial cells contribute to homeostasis for dynamic second messengers in the CNS, including intracellular calcium concentration ([Ca2+] i). Calcium is a central secondary messenger which signals for example, through the N-methyl-D-aspartate (NMDA) glutamate receptor on the neuronal membrane. A large, dynamic Ca2+ influx ensues after glutamate binds to the NMDA receptor. This influx initiates several molecular mechanisms within the cell. Disturbances in calcium homeostasis can lead to neurological diseases such as epilepsy and major depressive disorder. In this project, we set out to gain a better understanding of the effect of glial density on neural signalling. This was done by accomplishing four main objectives: 1. Develop neural micro-environments with quantifiable variations in glial cell densities. 2. Use calcium imaging methods to analyze the calcium information processing capacity of the various neural micro-environment developed. 3. Develop mathematical tools for testing calcium dynamics iv 4. Study short term and interactions of novel biomaterial (CuHARS) used for tissue engineering in brain cell micro-environments (Ca+2 signaling as an indicator of cell “health”) To do so, tissue engineered microenvironments were constructed to test the effects of the glial cell density have on calcium information processing. We investigated the response of glia rich, mildly glia depleted, partially depleted, and severely depleted neuronal cultures to sub-maximal (nM to µM) glutamate concentrations using calcium imaging. This was used to assist in predicting and interpreting chaotic neural networks experimentally. Anti-mitotic agents, cytosine arabinoside (AraC), or 2-deoxy-5- fluorouridine (FdU) were used to inhibit proliferating glia and develop the three classes of glia density. Imaging was done with Fluo 3/AM, nine to fourteen days after plating. Neuronal cultures severely depleted (greater than sixty percent depletion) of glia responded to increasing glutamate additions with large, slightly unsynchronized responses with the greatest area under the curve (AUC) observed which returned to baseline the slowest of the three micro-environments developed. Cultures partially depleted (thirty to sixty percent depletion) of glia, responded to increasing glutamate addition with mid-sized, synchronized responses with lower AUC than cultures with severely depleted glial cells. Mildly depleted cultures behaved similarly to glia rich cultures. The difference between their AUC was not statistically significant. Studying how the brain behaves in altered systems, such as in glia depleted micro-environments will help us explore cell loss in the brain and develop more targeted protective strategies

Underlying Mechanisms of Epilepsy

This book is a very provocative and interesting addition to the literature on Epilepsy. It offers a lot of appealing and stimulating work to offer food of thought to the readers from different disciplines. Around 5% of the total world population have seizures but only 0.9% is diagnosed with epilepsy, so it is very important to understand the differences between seizures and epilepsy, and also to identify the factors responsible for its etiology so as to have more effective therapeutic regime. In this book we have twenty chapters ranging from causes and underlying mechanisms to the treatment and side effects of epilepsy. This book contains a variety of chapters which will stimulate the readers to think about the complex interplay of epigenetics and epilepsy