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

Ion channels as drug targets in central nervous system disorders

Ion channel targeted drugs have always been related with either the central nervous system (CNS), the peripheral nervous system, or the cardiovascular system. Within the CNS, basic indications of drugs are: sleep disorders, anxiety, epilepsy, pain, etc. However, traditional channel blockers have multiple adverse events, mainly due to low specificity of mechanism of action. Lately, novel ion channel subtypes have been discovered, which gives premises to drug discovery process led towards specific channel subtypes. An example is Na+ channels, whose subtypes 1.3 and 1.7-1.9 are responsible for pain, and 1.1 and 1.2 - for epilepsy. Moreover, new drug candidates have been recognized. This review is focusing on ion channels subtypes, which play a significant role in current drug discovery and development process. The knowledge on channel subtypes has developed rapidly, giving new nomenclatures of ion channels. For example, Ca2+ channels are not any more divided to T, L, N, P/Q, and R, but they are described as Cav1.1-Cav3.3, with even newer nomenclature α1A-α1I and α1S. Moreover, new channels such as P2X1-P2X7, as well as TRPA1-TRPV1 have been discovered, giving premises for new types of analgesic drugs

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

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

Optogenetic investigation of cortical network dynamics in epilepsy

Ph. D. ThesisUnderstanding the cortical network properties which determine the susceptibility of cortex to the onset of seizures remains a major goal of epilepsy research. The determinants of seizure risk in cortical networks are dynamic, showing dependency on intrinsic cortical activity and environmental influences. The failure to identify reliable electrographic indicators of imminent seizure onset suggests that the contributory factors may not be electrographically obvious. A strong candidate for such a property is the activity dependent disinhibition of the excitatory network which results from increases in intracellular chloride concentration. Chloride loading has been shown previously to occur during periods of intense neuronal activity, resulting from concomitant excitatory and inhibitory synaptic transmission. To explore how network dynamics evolve from a stable healthy state to one permissive for the onset and propagation of seizures, I used an optogenetic approach to selectively interrogate dynamic changes to excitatory transmission between the principal cells of the cortical circuit following an acute ictogenic challenge, both in vitro and in vivo. Using ultra-low frequency optogenetic stimulation genetically targeted to the pyramidal cells of neocortex, I demonstrate that epileptiform activity, which develops spontaneously following an acute chemoconvulsant challenge, can both be reduced and monitored, using an active probing strategy. Delivering continuous and focal optogenetic stimulations to superficial neocortex and regions of the hippocampal formation evokes glutamatergic responses in the LFP which can be used to assay dendritic excitability in the network. At ultralow frequencies, between 0.1-0.033 Hz, optogenetic stimulation markedly reduced the rate of evolution of epileptiform activity, when delivered to neocortex or hippocampal structures, in acutely prepared adult mouse brain slices bathed in 0Mg2+ perfusate. The response evoked by these test pulses undergoes an all-or-nothing transformation observable in the LFP which reliably telegraphed the onset of ictal activity in two models of epilepsy. Using electrophysiological tools and 2-photon calcium imaging of individual dendrites, I demonstrate that this phenomenon likely reflects a reduction in the threshold for dendritic spikes. Using an anatomically realistic computational model pyramidal cell I show that this effect is reproduced by modest positive shifts in the GABAergic reversal potential in distal pyramidal cell dendrites. Finally, I report preliminary data demonstrating a potential mechanism for the diurnal modulation of seizure risk. Diurnal periodicity in seizure susceptibility have been observed longitudinal recordings from both patients and chronically epileptic experimental animals. Using the optical chloride sensor ClopHensor I examine steady-state pyramidal cell chloride concentration over the diurnal period and show that periodicity in chloride homeostasis is consistent with the phase of diurnally modulated seizure risk. In this thesis I use a range of optical and electrophysiological tools to explore the contribution of dynamic chloride concentration in pyramidal cells in determining cortical susceptibility to seizures onset. Using two acute epilepsy models I demonstrate that an assayable increase in dendritic excitability precedes ictogenesis, and demonstrate a potential mechanism by which variation in [Cl-]i can give rise to this effect. I go on to show diurnal variation in [Cl-]i in cortical pyramidal cells, and link this to circadian modulation of susceptibility to chemoconvulsants, suggesting a functional mechanism for the dynamic seizure risk observed in epileptic patients

A study of modulation of P2X3 and TRPV1 receptors by the B-type natriuretic peptide and novel synthetic compounds in trigeminal sensory neurons of wild type and migraine-model mice

Background Trigeminal ganglion (TG) is a key player in processing noxious stimuli. Among many ligand-gated ion channels, trigeminal sensory neurons express on their membranes purinergic P2X3 receptors and capsaicin-sensitive transient receptor potential vanilloid 1 channels (TRPV1). These receptors are thought to be involved in pain transduction and pathophysiology of different pain syndromes, including migraine disorders. P2X3 and TRPV1 channels are continuously regulated by a variety of endogenous modulators, which, upregulating these receptors, can cause sensitization and promote development of pathological pain conditions. Although positive P2X3 and TRPV1 regulators are well studied, not much is known about those which might restrain the activity of these receptors. One candidate for the role of endogenous negative regulator of sensory ganglion activity is the brain natriuretic peptide (BNP). In fact, BNP was recently reported to downregulate inflammatory pain and firing frequency of small neurons in dorsal root ganglia via its receptor NPR-A. Aims In order to investigate the role of BNP/NPR-A system in trigeminal ganglion in control conditions and in migraine pathology we used wild-type (WT) mice and transgenic R192Q KI mice of the familial hemiplegic migraine type 1 (FHM1) model. First we characterized BNP and NPR-A expression and functional properties of the BNP/NPR-A pathway in trigeminal ganglions of WT and KI mice. To understand if this pathway can affect the properties of sensory neurons in TG we studied the effects of endogenous and exogenous BNP on P2X3 and TRPV1 receptors responses in vitro. Investigating molecular mechanisms underneath P2X3 receptor modulation we carefully examined changes in P2X3 phosphorylation and membrane distribution and considered involvement of particular kinases and phosphatases in this process. Firing activity of the WT and KI trigeminal neurons were also evaluated to find out if the modulatory effects of BNP/NPR-A system on the P2X3 channels are reflected in neuronal excitability. Additionally, in search for new potent P2X3 antagonists a variety of diaminopurine derivatives as well as several adenosine nucleotide analogues were evaluated on recombinant P2X3 receptors in HEK cells and on native P2X3 receptors of TG sensory neurons. Results We found abundant expression of NPR-A in trigeminal ganglion along with low levels of BNP itself; the BNP/NPR-A pathway in both WT and KI neurons proved to be functional. Exogenously applied BNP inhibited TRPV1-mediated responses in WT and KI trigeminal neurons without any changes in the receptor\u2019s expression level. On the other hand, P2X3 receptors were not sensitive to additional exogenous BNP, but appeared to be downregulated by the low amount of endogenous BNP already present in WT TG cultures. This negative modulation included P2X3 serine phosphorylation and receptor redistribution to the non-lipid raft membrane compartments. Both mechanisms were dependent on the activity of protein kinase G. Interestingly, in KI mice NPR-A-mediated P2X3 inhibition could not be seen and receptors remained upregulated, most probably due to the increased activity of P/Q calcium channels and high concentration of calcitonin gene related peptide (CGRP). Considering firing properties of trigeminal neurons, inactivation of BNP/NPR-A system with NPR-A antagonist anantin caused a hyperexcitability phenotype of WT cultures, which was very similar to what is typical for KI neurons. KI cultures remained unaltered, consistent with lack of BNP/NPR-A regulation over P2X3 activity. Experiments with new diaminopurine compounds and adenosine nucleotide derivatives resulted in molecules which showed antagonistic behavior towards P2X3 receptors with IC50 values in low micromolar and nanomolar range, respectively. Conclusion The main result of the present study is the identification of BNP/NPR-A pathway as an intrinsic negative modulatory system for P2X3 and TRPV1 receptors activity in sensory neurons of mouse trigeminal ganglion and related neuronal excitability. However, in a mouse FHM1 migraine model BNP/NPR-A lacked the inhibitory effect on P2X3 receptors due to the overall amount of activation these receptors undergo in KI neurons. Modifications of diaminopurine and adenosine scaffold could serve as a promising strategy in search for new potent antagonists of P2X3 receptors