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What causes absence seizures and are they really benign? Crunelli et al. review the pathophysiology, pharmacotherapy and neuropsychiatric comorbidities of absence seizures, and highlight the key role of cortical, thalamic and basal ganglia mechanisms in driving absence seizure ictogenesis., Absence seizures in children and teenagers are generally considered relatively benign because of their non-convulsive nature and the large incidence of remittance in early adulthood. Recent studies, however, show that 30% of children with absence seizures are pharmaco-resistant and 60% are affected by severe neuropsychiatric comorbid conditions, including impairments in attention, cognition, memory and mood. In particular, attention deficits can be detected before the epilepsy diagnosis, may persist even when seizures are pharmacologically controlled and are aggravated by valproic acid monotherapy. New functional MRI-magnetoencephalography and functional MRI-EEG studies provide conclusive evidence that changes in blood oxygenation level-dependent signal amplitude and frequency in children with absence seizures can be detected in specific cortical networks at least 1 min before the start of a seizure, spike-wave discharges are not generalized at seizure onset and abnormal cortical network states remain during interictal periods. From a neurobiological perspective, recent electrical recordings and imaging of large neuronal ensembles with single-cell resolution in non-anaesthetized models show that, in contrast to the predominant opinion, cortical mechanisms, rather than an exclusively thalamic rhythmogenesis, are key in driving seizure ictogenesis and determining spike-wave frequency. Though synchronous ictal firing characterizes cortical and thalamic activity at the population level, individual cortico-thalamic and thalamocortical neurons are sparsely recruited to successive seizures and consecutive paroxysmal cycles within a seizure. New evidence strengthens previous findings on the essential role for basal ganglia networks in absence seizures, in particular the ictal increase in firing of substantia nigra GABAergic neurons. Thus, a key feature of thalamic ictogenesis is the powerful increase in the inhibition of thalamocortical neurons that originates at least from two sources, substantia nigra and thalamic reticular nucleus. This undoubtedly provides a major contribution to the ictal decrease in total firing and the ictal increase of T-type calcium channel-mediated burst firing of thalamocortical neurons, though the latter is not essential for seizure expression. Moreover, in some children and animal models with absence seizures, the ictal increase in thalamic inhibition is enhanced by the loss-of-function of the astrocytic GABA transporter GAT-1 that does not necessarily derive from a mutation in its gene. Together, these novel clinical and experimental findings bring about paradigm-shifting views of our understanding of absence seizures and demand careful choice of initial monotherapy and continuous neuropsychiatric evaluation of affected children. These issues are discussed here to focus future clinical and experimental research and help to identify novel therapeutic targets for treating both absence seizures and their comorbidities.

International audience; Neuroscience Methods Vincenzo stepped down on 31st December 2017. His experience and dedication have been invaluable to set the Journal as a leading academic journal in the field of neuroscience methods and techniques. One of his initiatives was the regular publication of journal issues dedicated to the review of emerging thecnological advances and questions on a specific topic. Some of these Special Issues gathered contributions presented at international workshops dealing for example with computational neuroscience (volumes 169 (2008); 210 (2012); 220 (2013)) or behavioral measurements (volumes 268 (2016) and 300 (2018)). Others focused on methods and models for the study of neu-rological diseases (volumes 252 (2015); 260 (2016); 265 (2016); 272 (2016); 292 (2017)) or reported the outcome of a challenge for machine learning algorithms and analysis of Alzheimer's disease neuroimaging data (volume 300 (2018)). In honor of Vincenzo's own research interests , we decided to launch a Special Issue on sleep-a complex beha-vioral state shared by all animals. Vincenzo's interest in sleep mechanisms spans decades, from his initial work on thalamic excitability. His research during this time tackled the issue of thalamocortical network dynamics during non-REM sleep at multiple levels, including in vitro studies of neuronal excit-ability, recordings of cellular activities during natural sleep in rodents, and the development of computational models to predict and explain in vivo experimental observations. Vincenzo's laboratory contributed to our current understanding of the cellular and network mechanisms underlying non-REM sleep delta waves and established that the tha-lamus is essential to the full expression of non-REM sleep. His results emphasized the essential role of thalamic T-type calcium channels to the expression of slow waves of natural sleep and the key role played by calcium entry through these channels in the modulation of other voltage dependent channels critical for the generation of both non-REM slow waves and sleep spindles. A major focus of contributions to this Special Issue is the analysis of electroencephalographic (EEG) activities. Although EEG signals have been recorded for almost a century, the detection and analysis of specific waves characterizing different sleep stages is still a challenge. Two papers, Lacourse et al. and Sampson et al., propose new methods to detect sleep spindles. Sakellariou et al. describe a novel approach to estimate brain functional connectivity across subjects from the scalp EEG signal and illustrate it by analyzing the connectivity underlying fast sleep spindles. Lazar et al. report that infraslow oscillations in the EEG are a primarily a non-REM Stage 2, sigma band, and sleep spindle-specific phenomenon. Bukhiyarova et al. compare four different methods to detect slow waves, concluding that a neural network pattern recognition method provides the lowest false-positive rate. Rose et al. propose an approach based on an integrated multiscale model framework to decipher the relationships between EEG and transmembrane ion flows. Several papers of this Special Issue deal with development or improvement of recording techniques for recording from neuronal circuits , using electrophysiological or imaging approaches. Fiath et al. report the development of a novel high-density silicon probe to record neuronal activities from multiple cortical layers simultaneously with a very high spatial resolution during non-REM oscillations. Sigt-Glöcknet and Seibt review how recent advances in in vivo brain imaging, from single synapse to large-scale network activity, can be used in rodents to investigate sleep's influence on cognition. Moehlen et al. tackle the issue of the sleep-adverse conditions inside an fMRI scanner and provide a methodological validation of procedures for obtaining all-night fMRI data from sleeping subjects. Recent work has aimed at deciphering the mechanisms regulating sleep and arousal at the molecular level. Yamaguchi and de Lecea review how the newly developed CRISP/Cas9 toolbox can be used to investigate gene functions within genetically-defined brain cell populations in the regulation of sleep behavior. Sleep behavior is associated with changes in numerous physiological parameters in addition to alterations in patterns of brain activity. Massot et al. report on the development of a novel tool designed to record electrophysiological and metabolic signals, together with beha-vioral information, in small freely moving animals for extended periods of time. Finally, two papers represent the very active field of research dealing with the cognitive functions of sleep. Ngo et al. tested the possibility that memory consolidation can be promoted by enhancing non-REM spindle activity using auditory close-loop stimulation in the context of ongoing slow oscillations. Cellini and Mednick provide an overview of multiple available techniques to non-invasively modulate sleep physiology, as a mechanism to promote optimal memory consolidation. We hope that readers will find this Special Issue stimulating. We would like to thank all the contributors for making it a success.

International audience

Behaviorally and pathologically relevant cortico-thalamo-cortical oscillations are driven by diverse interacting cell-intrinsic and synaptic processes. However, the mechanism that gives rise to the paroxysmal oscillations of absence seizures (ASs) remains unknown. Here we report that, during ASs in behaving animals, cortico-thalamic excitation drives thalamic firing by preferentially eliciting tonic rather than T-type Ca2+ channel (T-channel)-dependent burst firing in thalamocortical (TC) neurons and by temporally framing thalamic output via feedforward reticular thalamic (NRT)-to-TC neuron inhibition. In TC neurons, overall ictal firing was markedly reduced and bursts rarely occurred. Moreover, blockade of T-channels in cortical and NRT neurons suppressed ASs, but such blockade in TC neurons had no effect on seizures or on ictal thalamic output synchrony. These results demonstrate ictal bidirectional cortico-thalamic communications and provide the first mechanistic understanding of cortico-thalamo-cortical network firing dynamics during ASs in behaving animals.

International audience; During inattentive wakefulness and non-rapid eye movement (NREM) sleep, the neocortex and thalamus cooperatively engage in rhythmic activities that are exquisitely reflected in the electroencephalogram as distinctive rhythms spanning a range of frequencies from

Statistical models that predict neuron spike occurrence from the earlier spiking activity of the whole recorded network are promising tools to reconstruct functional connectivity graphs. Some of the previously used methods are in the general statistical framework of the multivariate Hawkes processes. However, they usually require a huge amount of data, some prior knowledge about the recorded network, and/or may produce an increasing number of spikes along time during simulation.Here, we present a method, based on least-square estimators and LASSO penalty criteria, for a particular class of Hawkes processes that can be used for simulation.Testing our method on small networks modeled with Leaky Integrate and Fire demonstrated that it efficiently detects both excitatory and inhibitory connections. The few errors that occasionally occur with complex networks including common inputs, weak and chained connections, can be discarded based on objective criteria.With respect to other existing methods, the present one allows to reconstruct functional connectivity of small networks without prior knowledge of their properties or architecture, using an experimentally realistic amount of data.The present method is robust, stable, and can be used on a personal computer as a routine procedure to infer connectivity graphs and generate simulation models from simultaneous spike train recordings.

Although the thalamus presents a rather limited repertoire of GABAergic cell types compare to other CNS area, this structure is a privileged system to study how GABA impacts neuronal network excitability. Indeed both glutamatergic thalamocortical (TC) and GABAergic nucleus reticularis thalami (NRT) neurons present a high expression of T-type voltage-dependent Ca

Although the thalamus presents a rather limited repertoire of GABAergic cell types compare to other CNS area, this structure is a privileged system to study how GABA impacts neuronal network excitability. Indeed both glutamatergic thalamocortical (TC) and GABAergic nucleus reticularis thalami (NRT) neurons present a high expression of T-type voltage-dependent Ca2+ channels whose activation that shapes the output of the thalamus critically depends upon a preceding hyperpolarisation. Because of this strict dependence, a tight functional link between GABA mediated hyperpolarization and T-currents characterizes the thalamic network excitability. In this review we summarize a number of studies showing that the relationships between the various thalamic GABAA/B receptors and T-channels are complex and bidirectional. We discuss how this dynamic interaction sets the global intrathalamic network activity and its long-term plasticity and highlight how the functional relationship between GABA release and T-channel-dependent excitability is finely tuned by the T-channel activation itself. Finally, we illustrate how an impaired balance between T-channels and GABA receptors can lead to pathologically abnormal cellular and network behaviours. This article is part of the "Special Issue Dedicated to Norman G. Bowery".

Non-technical summary Voltage-dependant calcium channels constitute a heterogeneous group playing ubiquitous roles in excitable cells. Among them the low-voltage activated T-type channels generate a family of currents that differ in their biophysical properties reflecting structural or neuromodulatory diversity. These T-type calcium channels are highly expressed in neurons located in the thalamus, a brain structure considered as the gateway to the cortex. Thalamic T-type calcium channels are critically involved in oscillatory neuronal activities associated with sleep or epilepsy and may contribute to sensory processing. Using injections of computer-simulated T-type conductances (a real time mimicry of ionic currents) in biological thalamic neurons, we dissect how the diversity in T-type currents impact on the output of thalamic neurons. We show that very subtle modifications in the properties of the T current that were overlooked so far affect drastically the physiological output of the thalamic neurons and therefore condition the dynamics of thalamo-cortical information integration.

Childhood absence epilepsy is an idiopathic, generalized, nonconvulsive epilepsy with a multifactorial genetic etiology. The KCNK9 gene coding for the TASK3 (Twik-like acid-sensitive K +) channel is present on chromosome 8 at position 8q24, a locus that has shown positive linkage to the human absence epilepsy phenotype. Sequencing of the KCNK9 gene in the genetic absence epilepsy rats from Strasbourg (GAERS), a well established genetic model of this disease, reveals an additional alanine residue in a polyalanine tract within the C-terminal intracellular domain. This additional alanine is absent in the inbred nonepileptic control (NEC) strain, Wistar, and Wistar albino Glaxo strain bred in Rijswijk, another inbred rat model of absence epilepsy. Expression of the mutant channel in CHO cells produces a K+ current that is blocked by acidic pH and millimolar concentrations of barium or ruthenium red and is not different from the wild-type channel. In brain slices, thalamic neurons display a prominent pH-sensitive tonic K+ current, but no difference was observed between GAERS and NEC or Wistar rats. Ruthenium red had no effect in cortical, reticular thalamic, or sensory thalamic neurons in either GAERS or NEC, indicating that the TASK3 homodimer is not present in these structures. Twik-like acid-sensitive K+ (TASK3) channels, therefore, are probably associated with TASK1 to form ruthenium red-insensitive heterodimers in these neurons. Finally, no difference was found between GAERS and NEC rats in the modulation of the leak K+ current following activation of muscarinic receptors. These studies describe the first mutation found in a genetic model of absence epilepsy. Although our experiments showed no difference in the leak K+ current between GAERS and NEC rats, further work is needed to ascertain whether this mutation contributes to the generation of absence seizures, possibly by mechanisms related to the expansion of the polyalanine run.

Inhibitory neurotransmission in the mammalian brain is principally mediated by gamma-aminobutyric acid (GABA) acting through different subtypes of cell membrane GABA receptor (GABAR). The expression of one GABAR gene, GABABR1, is distinguished by the expression of multiple splice variants that encode different isoforms of the receptor. In the present study, we have identified two novel GABABR1 variants, GABABR1h (R1h) and GABABR1i (R1i), which appear to arise from alternative splicing of the GABABR1 gene. The expression of R1h and R1i is differentially regulated in brain and peripheral tissues, but expression is not altered in the brain of a genetic model of absence epilepsy (GAERS rat [genetic absence epilepsy rat from Strasbourg]). Both the R1h and R1i variants exhibit a novel 80-bp insert downstream of exon 4 that is flanked by consensus splice sites, and both encode C-terminal-truncated proteins. The new insight into the family of GABABR1 variants gained from this study identifies exon 4 as a preferred locus, or hot spot for regulated splicing in the GABABR1 gene. This finding correlates with the micro-exonic nature of exon 4 (21 bp). Bioinformatic analysis of micro-exon 4 and its flanking pre-mRNA sequences has revealed multiple, potentially competitive, exonic splicing enhancers that provide a mechanistic basis for the preponderance of alternative splicing events at this locus. Conservation of GABABR1 micro-exon 4 across species suggests a conserved functional role, facilitating either N-terminal protein production or post-transcriptional gene regulation through regulated splicing coupled to transcript decay.

Absence epilepsy is characterised by recurrent periods of physical and mental inactivity coupled to bilateral, synchronous spike and wave discharges (SWDs) on the electroencephalogram. The mechanism of action of ethosuximide (ETX), a drug specific for absence seizures, is believed to involve a reduction in the low threshold T-type Ca(2+) current in thalamocortical and nucleus reticularis thalami (NRT) neurones, although other electrophysiological data have questioned this. Here, we employed a genetic rat model of absence seizures to investigate the effects of directly administering ETX to the thalamus.SWDs were immediately and substantially reduced (approximately 90%) by systemic administration of ETX (177-709 micromol/kg), or by bilateral microinfusion into the thalamus of the GABA(B) antagonist, CGP 36742 (5-27 nmol per side). However, infusion of ETX (1-200 nmol per side) into the ventrobasal complex or the NRT resulted in a reduction of SWDs that was delayed (30-60 min) and less marked (approximately 50%). Administration of ETX (0.2 mM to 1M) to a greater volume of thalamus by reverse microdialysis also produced significant but delayed reduction of SWDs at concentrations >1mM. Only at 5mM were seizures significantly reduced (approximately 70%) within 30 min of administration. These results suggest that targeting of the thalamus alone may be insufficient for an immediate and full anti-absence action for ETX. Concomitant or exclusive actions in the cortex remain a possibility.

Slow waves of non-REM sleep are suggested to play a role in shaping synaptic connectivity to consolidate recently acquired memories and/or restore synaptic homeostasis. During sleep slow waves, both GABAergic neurons of the nucleus reticularis thalami (NRT) and thalamocortical (TC) neurons discharge high-frequency bursts of action potentials mediated by low-threshold calcium spikes due to T-type Ca2+channel activation. Although such activity of the intrathalamic network characterized by high-frequency firing and calcium influx is highly suited to modify synaptic efficacy, very little is still known about its consequences on intrathalamic synapse strength. Combiningin vitroelectrophysiological recordings and calcium imaging, here we show that the inhibitory GABAergic synapses between NRT and TC neurons of the rat somatosensory nucleus develop a long-term depression (I-LTD) when challenged by a stimulation paradigm that mimics the thalamic network activity occurring during sleep slow waves. The mechanism underlying this plasticity presents unique features as it is both heterosynaptic and homosynaptic in nature and requires Ca2+entry selectively through T-type Ca2+channels and activation of the Ca2+-activated phosphatase, calcineurin. We propose that during slow-wave sleep the tight functional coupling between GABAAreceptors, calcineurin, and T-type Ca2+channels will elicit LTD of the activated GABAergic synapses when coupled with concomitant activation of metabotropic glutamate receptors postsynaptic to cortical afferences. This I-LTD may be a key element involved in the reshaping of the somatosensory information pathway during sleep.

Background: The advent of optogenetics has given neuroscientists the opportunity to excite or inhibit neuronal population activity with high temporal resolution and cellular selectivity. Thus, when combined with recordings of neuronal ensemble activity in freely moving animals optogenetics can provide an unprecedented snapshot of the contribution of neuronal assemblies to (patho)physiological conditions in vivo. Still, the combination of optogenetic and silicone probe (or tetrode) recordings does not allow investigation of the role played by voltage- and transmitter-gated channels of the opsin-transfected neurons and/or other adjacent neurons in controlling neuronal activity. New method and results: We demonstrate that optogenetics and silicone probe recordings can be combined with intracerebral reverse microdialysis for the long-term delivery of neuroactive drugs around the optic fiber and silicone probe. In particular, we show the effect of antagonists of T-type Ca2+ channels, hyperpolarization-activated cyclic nucleotide-gated channels and metabotropic glutamate receptors on silicone probe-recorded activity of the local opsin-transfected neurons in the ventrobasal thalamus, and demonstrate the changes that the block of these thalamic channels/receptors brings about in the network dynamics of distant somatotopic cortical neuronal ensembles. Comparison with existing methods: This is the first demonstration of successfully combining optogenetics and neuronal ensemble recordings with reverse microdialysis. This combination of techniques overcomes some of the disadvantages that are associated with the use of intracerebral injection of a drug-containing solution at the site of laser activation. Conclusions: The combination of reverse microdialysis, silicone probe recordings and optogenetics can unravel the short and long-term effects of specific transmitter- and voltage-gated channels on laser-modulated firing at the site of optogenetic stimulation and the actions that these manipulations exert on distant neuronal populations., peer-reviewed

The action of ethosuximide (ETX) on Na+, K+, and Ca2+currents and on tonic and burst-firing patterns was investigated in rat and cat thalamic neuronsin vitroby using patch and sharp microelectrode recordings. In thalamocortical (TC) neurons of the rat dorsal lateral geniculate nucleus (LGN), ETX (0.75–1 mm) decreased the noninactivating Na+current,INaP, by 60% but had no effect on the transient Na+current. In TC neurons of the rat and cat LGN, the whole-cell transient outward current was not affected by ETX (up to 1 mm), but the sustained outward current was decreased by 39% at 20 mV in the presence of ETX (0.25–0.5 mm): this reduction was not observed in a low Ca2+(0.5 mm) and high Mg2+(8 mm) medium or in the presence of Ni2+(1 mm) and Cd2+(100 μm). In addition, ETX (up to 1 mm) had no effect on the low-threshold Ca2+current,IT, of TC neurons of the rat ventrobasal (VB) thalamus and LGN and in neurons of the rat nucleus reticularis thalami nor on the high-threshold Ca2+current in TC neurons of the rat LGN.Sharp microelectrode recordings in TC neurons of the rat and cat LGN and VB showed that ETX did not change the resting membrane potential but increased the apparent input resistance at potentials greater than −60 mV, resulting in an increase in tonic firing. In contrast, ETX decreased the number of action potentials in the burst evoked by a low-threshold Ca2+potential. The frequency of the remaining action potentials in a burst also was decreased, whereas the latency of the first action potential was increased. Similar effects were observed on the burst firing evoked during intrinsic δ oscillations.These results indicate an action of ETX onINaPand on the Ca2+-activated K+current, which explains the decrease in burst firing and the increase in tonic firing, and, together with the lack of action on low- and high-threshold Ca2+currents, the results cast doubts on the hypothesis that a reduction ofITin thalamic neurons underlies the therapeutic action of this anti-absence medicine.

International audience; Since their discovery more than 30 years ago, low-threshold T-type $Ca 2+$ channels (T channels) have been suggested to play a key role in many EEG waves of non-REM sleep, which has remained exclusively linked to the ability of these channels to generate low-threshold $Ca 2+$ potentials and associated high-frequency bursts of action potentials. Our present understanding of the biophysics and physiology of T channels, however, highlights a much more diverse and complex picture of the pivotal contributions that they make to different sleep rhythms. In particular, recent experimental evidence has conclusively demonstrated the essential contribution of thalamic T channels to the expression of slow waves of natural sleep and the key role played by $Ca 2+$ entry through these channels in the activation or modulation of other voltage-dependent channels that are important for the generation of both slow waves and sleep spindles. However, the precise contribution to sleep rhythms of T channels in cortical neurons and other sleep-controlling neuronal networks remains unknown, and a full understanding of the cellular and network mechanisms of sleep delta waves is still lacking.

International audience; Since the discovery of low-voltage activated T-type calcium channels in sensory neurons and the initial characterization of their physiological function mainly in inferior olive and thalamic neurons, studies on neuronal T-type currents have predominantly focused on the generation of low-threshold spike (and associated action potential burst firing) which is strictly conditioned by a preceding hyperpolarization. This T-type current mediated activity has become an archetype of the function of these channels, constraining our view of the potential physiological and pathological roles that they may play in controlling the excitability of single cells and neural networks. However, greatly helped by the recent availability of the first potent and selective antagonists for this class of calcium channels, novel T-type current functions are rapidly being uncovered, including their surprising involvement in neuronal excitability at depolarized membrane potentials and their complex control of dendritic integration and neurotransmitter release. These and other data summarized in this short review clearly indicate a much wider physiological involvement of T-type channels in neuronal activity than previously expected.

International audience; The temporal coincidence of sleep spindles and spike-and-wave discharges (SWDs) in patients with idiopathic generalized epilepsies, together with the transformation of spindles into SWDs following intramuscular injection of the weak GABAA receptor (GABAAR) antagonist, penicillin, in an experimental model, brought about the view that SWDs may represent 'perverted' sleep spindles. Over the last 20 years, this hypothesis has received considerable support, in particular by in vitro studies of thalamic oscillations following pharmacological/genetic manipulations of GABAARs. However, from a critical appraisal of the evidence in absence epilepsy patients and well-established models of absence epilepsy it emerges that SWDs can occur as frequently during wakefulness as during sleep, with their preferential occurrence in either one of these behavioural states often being patient dependent. Moreover, whereas the EEG expression of both SWDs and sleep spindles requires the integrity of the entire cortico-thalamo-cortical network, SWDs initiates in cortex while sleep spindles in thalamus. Furthermore , the hypothesis of a reduction in GABAAR function across the entire cortico-thalamo-cortical network as the basis for the transformation of sleep spindles into SWDs is no longer tenable. In fact, while a decreased GABAAR function may be present in some cortical layers and in the reticular thalamic nucleus, both phasic and tonic GABAAR inhibitions of thalamo-cortical neurons are either unchanged or increased in this epileptic phenotype. In summary, these differences between SWDs and sleep spindles question the view that the EEG hallmark of absence seizures results from a transformation of this EEG oscillation of natural sleep.

Although it is well established that low-voltage-activated T-type Ca2+channels play a key role in many neurophysiological functions and pathological states, the lack of selective and potent antagonists has so far hampered a detailed analysis of the full impact these channels might have on single-cell and neuronal network excitability as well as on Ca2+homeostasis. Recently, a novel series of piperidine-based molecules has been shown to selectively block recombinant T-type but not high-voltage-activated (HVA) Ca2+channels and to affect a number of physiological and pathological T-type channel-dependent behaviors. Here we directly show that one of these compounds, 3,5-dichloro-N-[1-(2,2-dimethyl-tetrahydro-pyran-4-ylmethyl)-4-fluoro-piperidin-4-ylmethyl]-benzamide (TTA-P2), exerts a specific, potent (IC50= 22 nm), and reversible inhibition of T-type Ca2+currents of thalamocortical and reticular thalamic neurons, without any action on HVA Ca2+currents, Na+currents, action potentials, and glutamatergic and GABAergic synaptic currents. Thus, under current-clamp conditions, the low-threshold Ca2+potential (LTCP)-dependent high-frequency burst firing of thalamic neurons is abolished by TTA-P2, whereas tonic firing remains unaltered. Using TTA-P2, we provide the first direct demonstration of the presence of a window component of Ca2+channels in neurons and its contribution to the resting membrane potential of thalamic neurons and to the Up state of their intrinsically generated slow (

International audience; Gamma-hydroxybutyric acid (GHB) is an endogenous brain substance that has diverse neuropharmacological actions, including rewarding properties in different animal species and in humans. As other drugs of abuse, GHB affects the firing of ventral tegmental neurons (VTA) in anaesthetized animals and hyperpolarizes dopaminergic neurons in VTA slices. However, no direct behavioural data on the effects of GHB applied in the VTA or in the target regions of its dopaminergic neurons, e.g. the nucleus accumbens (NAc), are available. Here, we investigated the effects of various doses of intravenous GHB in maintaining self-administration (from 0.001 to 10 mg/kg per infusion), and its ability to induce conditioned place preference (CPP) in rats when given orally (175-350 mg/kg) or injected directly either in the VTA or NAc (from 10 to 300 mug/0.5 mul per side). Our results indicate that while only 0.01 mg/kg per infusion GHB maintained self-administration, although not on every test day, 350 mg/kg GHB given orally induced CPP. CPP was also observed when GHB was injected in the VTA (30-100 mug/0.5 mul per side) but not in the NAc. Together with recent in-vitro findings, these results suggest that the rewarding properties of GHB mainly occur via disinhibition of VTA dopaminergic neurons.

A growing number of in vivo experiments shows that high frequency bursts of action potentials can be recorded in thalamocortical neurons of awake animals. The mechanism underlying these bursts, however, remains controversial, because they have been proposed to depend on T-type Ca 2+ channels that are inactivated at the depolarized membrane potentials usually associated with the awake state. Here, we show that the transient potentiation of the T current amplitude, which is induced by neuronal depolarization, drastically increases the probability of occurrence and the temporal precision of T-channel-dependent high frequency bursts. The data, therefore, provides the first biophysical mechanism that might account for the generation of these high frequency bursts of action potentials in the awake state. Remarkably, this regulation finely tunes the response of thalamocortical neurons to the corticofugal excitatory and intrathalamic inhibitory afferents but not to sensory inputs.

Human and experimental studies indicate that molecular genetic changes in GABAA receptors may underlie the expression of spike-and-waves discharges (SWDs) occurring during absence seizures. However, the full spectrum of the genetic defects underlying these seizures has only been partially elucidated, the expression and functional profiles of putative abnormal protein(s) within the thalamocortical network are undefined, and the pathophysiological mechanism(s) by which these proteins would lead to absence paroxysms are poorly understood. Here we investigated GABAA inhibitory postsynaptic currents (IPSCs) in key thalamocortical areas, i.e., the somatosensory cortex, ventrobasal thalamus (VB) and nucleus reticularis thalami (NRT), in preseizure genetic absence epilepsy rats from Strasbourg (GAERS), a well-established genetic model of typical absence seizures that shows no additional neurological abnormalities, and compared their properties to age-matched non-epileptic controls (NECs). Miniature GABAA IPSCs of VB and cortical layers II/III neurons were similar in GAERS and NEC, whereas in GAERS NRT neurons they had 25% larger amplitude, 40% faster decay. In addition, baclofen was significantly less effective in decreasing the frequency of NRT mIPSCs in GAERS than in NEC, whereas no difference was observed for cortical and VB mIPSCS between the two strains. Paired-pulse depression was 45% smaller in GAERS NRT, but not in VB, and was insensitive to GABAB antagonists. These results point to subtle, nucleus-specific, GABAA receptor abnormalities underlying SWDs of typical absence seizures rather than a full block of these receptors across the whole thalamocortical network, and their occurrence prior to seizure onset suggests that they might be of epileptogenic significance.

Despite the marked influence on neuronal physiology of the low-voltage activated T-type Ca2+currents, little is known about the intracellular pathways and neurotransmitters involved in their regulations. Here, we report that in thalamocortical neurons a phosphorylation mechanism induces an increase both in the current amplitude (1.5 ± 0.27-fold in the ventrobasal nucleus) and its inactivation kinetics. Dialysis of the neuron with an ATP-free solution suppresses the T-current potentiation, whereas it becomes irreversible in the presence of ATPγS. Phosphorylation occurs when the channels are inactivated and is slowly removed when they recover from inactivation and remain in closed states (time constants of the induction and removal of the potentiation: 579 ± 143 msec and 4.9 ± 1.1 sec, respectively, at 25°C). The resulting apparent voltage sensitivity of this regulation follows the voltage dependence of the current steady-state inactivation. Thus, the current is paradoxically inhibited when the preceding hyperpolarization is lengthened, and maximal currents are generated after transient hyperpolarizations with a duration (0.7-1.5 sec) that is defined by the balance between the kinetics of the dephosphorylation and deinactivation. In addition, the phosphorylation will facilitate the generation of T current at resting membrane potential. This potentiation, which is specific to sensory thalamocortical neurons, would markedly influence the electroresponsiveness of these neurons and represent the first evidence of a regulation of native Cav3.1 channels.

Childhood absence epilepsy is an idiopathic, generalized, nonconvulsive epilepsy with a multifactorial genetic etiology. The KCNK9 gene coding for the TASK3 (Twik-like acid-sensitive K/U)+) channel is present on chromosome 8 at position 8q24, a locus that has shown positive linkage to the human absence epilepsy phenotype. Sequencing of the KCNK9 gene in the genetic absence epilepsy rats from Strasbourg (GAERS), a well established genetic model of this disease, reveals an additional alanine residue in a polyalanine tract within the C-terminal intracellular domain. This additional alanine is absent in the inbred nonepileptic control (NEC) strain, Wistar, and Wistar albino Glaxo strain bred in Rijswijk, another inbred rat model of absence epilepsy. Expression of the mutant channel in CHO cells produces a K+ current that is blocked by acidic pH and millimolar concentrations of barium or ruthenium red and is not different from the wild-type channel. In brain slices, thalamic neurons display a prominent pH-sensitive tonic K+ current, but no difference was observed between GAERS and NEC or Wistar rats. Ruthenium red had no effect in cortical, reticular thalamic, or sensory thalamic neurons in either GAERS or NEC, indicating that the TASK3 homodimer is not present in these structures. Twik-like acid-sensitive K+(TASK3) channels, therefore, are probably associated with TASK1 to form ruthenium red-insensitive heterodimers in these neurons. Finally, no difference was found between GAERS and NEC rats in the modulation of the leak K+ current following activation of muscarinic receptors. These studies describe the first mutation found in a genetic model of absence epilepsy. Although our experiments showed no difference in the leak K+ current between GAERS and NEC rats, further work is needed to ascertain whether this mutation contributes to the generation of absence seizures, possibly by mechanisms related to the expansion of the polyalanine run.

Absence epilepsy is characterised by a paroxysmal loss of consciousness, of abrupt onset and termination, and is associated with a bilateral synchronous spike and wave discharge (SWD) on the electroencephalogram. Absence seizures involve an interplay between thalamic and cortical structures, although most research has so far focussed on sensory thalamic nuclei and the reticular thalamic nucleus (RTN). Thus, microinfusion of ethosuximide (ETX), a first choice anti-absence drug, into either the ventrobasal thalamus or RTN of the genetic absence epilepsy rat from Strasbourg (GAERS), a validated rat model of absence epilepsy, does not produce immediate cessation of seizure activity, as is seen following systemic administration. As recent evidence indicates a seizure initiation site within the peri-oral region of the primary somatosensory cortex (S1po), we have now applied ETX into S1po as well as the somatosensory cortex forelimb region (S1FL) and the motor cortex (M1) of freely moving GAERS. Microinfusion of 10 or 20 nmol/side of ETX into S1po produced an immediate cessation of seizure activity. A less marked response was produced when even a higher dose (200 nmol/side) was infused into S1FL. No reduction of SWD was seen when ETX was infused into M1. Microinfusion of CGP 36742 (5 nmol/side), a GABABantagonist, produced immediate cessation of seizure activity in both S1po and M1 and a delayed effect in S1FL. These data suggest that the ability of ETX to abolish genetically determined absence seizures is cortical-area specific and support the involvement of S1po in the initiation of SWDs.

The systemic injection of γ-hydroxybutyric acid (GHB) elicits spike and wave discharges (SWDs), the EEG hallmark of absence seizures, and represents a well established, widely used pharmacological model of this nonconvulsive epilepsy. Despite this experimental use of GHB, as well as its therapeutic use in narcolepsy and its increasing abuse, however, the precise cellular mechanisms underlying the different pharmacological actions of this drug are still unclear.Because sensory thalamic nuclei play a key role in the generation of SWDs and sleep rhythms, and because direct injection of GHB in the ventrobasal (VB) thalamus elicits SWDs, we investigated GHB effects on corticothalamic EPSCs and GABAergic IPSCs in VB thalamocortical (TC) neurons. GHB (250 μm-10 mm) reversibly decreased the amplitude of electrically evoked EPSCs and GABAAIPSCs via activation of GABABreceptors; however, ∼60% of the IPSCs were insensitive to low (250 μm-1.0 mm) GHB concentrations. The putative GHB receptor antagonist NSC 382 applied alone had a number of unspecific effects, whereas it either had no action on, or further increased, the GHB-elicited effects on synaptic currents. Low GHB concentrations (250 μm) were also effective in increasing absence-like intrathalamic oscillations evoked by cortical afferent stimulation.These results indicate that low concentrations of GHB, similar to the brain concentrations that evoke SWDsin vivo, differentially affect excitatory and inhibitory synaptic currents in TC neurons and promote absence-like intrathalamic oscillations. Furthermore, the present data strengthen previous suggestions on the GHB mechanism of sleep promotion and will help focus future studies on the cellular mechanisms underlying its abuse.

De nombreux processus physiologiques sous le controle du Ca 2+ intracellulaire surviennent a des potentiels membranaires proches du potentiel de repos. A ces potentiels, les canaux calcium de type T sont des acteurs privilegies du controle calcique, capables d'engendrer soit un courant depolarisant de grande amplitude mais transitoire, soit un courant de faible amplitude mais soutenu. Dans les deux cas, ces courants participent etroitement a l'homeostasie calcique intracellulaire et conditionnent l'integration des signaux cellulaires. Le clonage en 1998-1999 par l'equipe de Perez-Reyes - et depuis par d'autres equipes - de trois canaux T permet d'esperer un progres rapide des etudes au niveau cellulaire, de mieux comprendre les fonctions physiologiques et d'ouvrir un nouveau champ d'investigations a visee therapeutique.

THE low threshold calcium current (IT) in thalamo-cortical neurones contributes to the generation of spike and wave discharges (SWDs) characteristic of generalized, non-convulsive absence epilepsy. The biophysical properties of this current were analysed in dorsal lateral geniculate neurones from the Genetic Absence Epilepsy Rats from Strasbourg (GAERS + ). No difference was found in the voltage dependence and kinetics of IT between GAERS + and rats of the non epileptic control strain (GAERS — ). Thus, a dysfunction of IT does not appear to underlie the occurrence of SWDs in absence epilepsy.

NMDA receptors (NMDARs) are glutamate-gated ion channels of the CNS. The best characterized and most common forms of NMDARs are composed by two GluN1 subunits binding glycine and two GluN2 subunits binding glutamate. Our attention focused on the less known glycine-binding GluN3A subunit of the third NMDAR group. GluN3A can form two distinct NMDAR flavors. First, GluN3A can constitute diheteromeric GluN1/GluN3ARs activated by glycine and insensitive to glutamate. GluN3A can also generate with GluN1 and GluN2 glutamate-sensitive triheteromeric GluN1/GluN2/GluN3ARs with different properties from classic NMDARs. GluN3A expression is assumed to be limited to the first postnatal weeks. Therefore, its identification in neurons has so far been limited to the juvenile brain, where triheteromers were shown to play a role in synaptic maturation. GluN1/GluN3ARs had however never been identified in native tissues. During my thesis, on the basis of novel immunohistological evidence on GluN3A expression in the adult brain, and using a unique compound, CGP78608, I showed that GluN1/GluN3ARs are functional in several nuclei, comprising the adult Medial Habenula and juvenile hippocampus. Concerning GluN1/GluN2/GluN3ARs, I could demonstrate that they are functional in the paraventricular nucleus of the adult thalamus. Among several cell subgroups examined, I could show that neurons expressing calbindin 2 and melanocortin receptors 4 from the parabrachial nucleus are physiological activators of these receptors. In conclusion, my work led to a shift in our perception of how GluN3A subunits are expressed and opens new hypotheses on how they can contribute to the activity of neural circuits., GluN3A est une sous-unité particulière des récepteurs NMDA (NMDAR), jusqu'a présent très peu étudiée. Les NMDAR classiques sont constitués de 2 GluN1 qui lient la glycine et 2 GluN2 qui lient le glutamate. Liant la glycine, GluN3A peut s’associer à GluN1 et former des récepteurs GluN1/GluN3Rs activés par la glycine seule. GluN3A peut également s’associer à GluN1 et GluN2 et générer des récepteurs GluN1/GluN2/GluN3Rs activés à la fois par la glycine et le glutamate, avec des propriétés altérées par rapport aux récepteurs classiques. Son expression supposée limitée à la vie postnatale précoce, GluN3A a été uniquement identifiée dans des neurones du cerveau juvénile, où les GluN1/GluN2/GluN3ARs semblent jouer un rôle dans la maturation synaptique. Les GluN1/GluN3ARs par contre n’avaient jamais été observés dans des neurones natifs; Durant mon doctorat, de nouvelles données immunohistochimiques ont montré une forte expression de GluN3A dans le cerveau adulte. En me basant sur ces données, et grâce à une nouvelle drogue, la CGP78608, j’ai pu révéler la présence de GluN1/GluN3ARs fonctionnels dans l’hippocampe juvénile et dans des structures du cerveau adulte, à noter l’Habenula Médiale. Quant aux GluN1/GluN2/GluN3ARs, j’ai pu montré qu'ils sont présents et fonctionnels dans le noyau paraventriculaire du thalamus (PVT) adulte, et qu'uniquement certaines sous-populations cellulaires du noyau parabrachial (PB) activent physiologiquement ces récepteurs à la synapse avec le PVT. En conclusion, mes données ont mené à un changement dans notre perception concernant l’expression de GluN3A dans le cerveau, ouvrant de nouvelles hypothèses sur leur contribution à l’activité neuronale.

Thalamocortical (TC) glutamatergic neurons from the thalamus are the last relay in the flow of information from the periphery to the cortex. Intrathalamic network is based on the interactions between these TC neurons and the GABAergic neurons located in the Thalamic Reticularis Nucleus (TRN). These two types of neurons contribute to the processing of information arising from the periphery and to the generation of oscillatory activities during sleep. During slow-waves-sleep, NRT and TC neurons discharge rhythmically and synchronously high frequency bursts of action potentials associated with calcium influx occurring through T-type calcium channels. During my thesis, I sought to determine whether these activities were capable of changing synaptic strength. Using an induction protocol mimicking the excitability of thalamic neurons during slow-wave sleep, I highlighted a long-term depression (LTD) of the synapse between NRT GABAergic neurons and TC neurons. I showed that LTD induction had a postsynaptic origin and required synergistically 1) the synaptic activation of GABAA receptors, 2) a high calcium entry in TC neurons specifically through T-type calcium channels 3) the activation of metabotropic glutamate receptors. The requirement of calcium influx through the T-type calcium channels suggests that only thalamocortical activities associated with slow wave sleep may trigger this LTD., Le thalamus, via les neurones glutamatergiques thalamocorticaux (TC), est le dernier relai dans le cheminement des informations de la périphérie vers le cortex. Le réseau intrathalamique repose sur les interactions entre ces neurones TC et des neurones GABAergiques regroupés dans le Noyau Réticulé Thalamique (NRT). Ces deux types de neurones contribuent au traitement des informations issues de la périphérie et à la genèse des activités oscillantes pendant le sommeil. Pendant les phases de sommeil à ondes lentes les neurones TC et NRT émettent de manière périodique et synchrone des bouffées de potentiels d'action à haute fréquence associées à de forts influx de calcium dus à l'activation des canaux calciques de type T. Au cours de ma thèse, j'ai cherché à déterminer si ces activités étaient susceptibles de modifier la force synaptique. En utilisant un protocole d'induction mimant l'excitabilité des neurones thalamiques durant le sommeil à ondes lentes, j'ai mis en évidence une dépression à long terme (LTD) de la synapse GABAergique entre les neurones NRT et TC. J'ai montré que cette LTD était d'origine post-synaptique et nécessitait en synergie 1) l'activation des récepteurs GABAA présents à la synapse, 2) une forte entrée de calcium dans les neurones TC spécifiquement par les canaux calciques de type T, 3) l'activation des récepteurs métabotropiques du glutamate. L'exigence d'une entrée massive de calcium par les canaux calciques de type T suggère que seules les activités thalamocorticales associées au sommeil à ondes lentes sont susceptibles de déclencher cette LTD.