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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.

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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".

During non-REM sleep the EEG shows characteristics waves that are generated by the dynamic interactions between cortical and thalamic oscillators. In thalamic neurons, low-threshold T-type Ca2+ channels play a pivotal role in almost every type of neuronal oscillations, including slow (

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.

International audience; Voltage-dependent calcium channels (Cav) of the T-type family (Cav3.1, Cav3.2, and Cav3.3) are activated by low threshold membrane depolarization and contribute greatly to neuronal network excitability. Enhanced T-type channel activity, especially Cav3.2, contributes to disease states, including absence epilepsy. Interestingly, the intracellular loop connecting domains I and II (I-II loop) of Cav3.2 channels is implicated in the control of both surface expression and channel gating, indicating that this I-II loop plays an important regulatory role in T-type current. Here we describe that co-expression of this I-II loop or its proximal region (⌬1-Cav3.2; Ser 423 –Pro 542) together with recombinant full-length Cav3.2 channel inhibited T-type current without affecting channel expression and membrane incorporation. Similar T-type current inhibition was obtained in NG 108-15 neuroblastoma cells that constitutively express Cav3.2 channels. Of interest, ⌬1-Cav3.2 inhibited both Cav3.2 and Cav3.1 but not Cav3.3 currents. Efficacy of ⌬1-Cav3.2 to inhibit native T-type channels was assessed in thalamic neurons using viral transduction. We describe that T-type current was significantly inhibited in the ventrobasal neurons that express Cav3.1, whereas in nucleus reticularis thalami neurons that express Cav3.2 and Cav3.3 channels, only the fast inactivating T-type current (Cav3.2 component) was significantly inhibited. Altogether, these data describe a new strategy to differentially inhibit Cav3 isoforms of the T-type calcium channels.

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.

As T-type calcium channels open near resting membrane potential and markedly influence neuronal excitability their activity needs to be tightly regulated. Few neuronal T-current regulations have been described so far, but interestingly some of them involve unusual mechanisms like G protein-independent but receptor-coupled modulation, while the use of recombinant channels has established both a direct action of Gbetagamma subunits, anandamide, arachidonic acid and a phosphorylation process by CaMKII. Nearly all reported types of modulation involve Cav3.2 channels while no regulation of Cav3.1 has been reported, a difference that may originate from diversities in the intracellular loop connecting the II and III domains of the two isotypes. The search for T-current regulators requires taking into account their peculiar activation properties, since a close link may exist between the channel conformation and its modulation. Indeed, in thalamocortical neurons a phosphorylation-mediated regulation of the amplitude of the T-current has been shown to be highly dependent upon the state of the channel and only to become apparent when the channels are in the voltage range close to neuronal resting membrane potential.

T-type calcium channels (the Ca(V)3 channel family) are involved in defining the resting membrane potential and in neuronal activities such as oscillations and rebound depolarization. Their physiological roles depend upon the channel activation and inactivation kinetics. A fast inactivation that stops the ionic flux of calcium in tens of milliseconds has already been described in both native and heterologously expressed channels. Here, using HEK 293 cells expressing the rat Ca(V)3.1 channel and whole-cell voltage clamp, we investigate an additional inactivation process, which can be distinguished from the previously described fast inactivation by its slow time course of recovery from inactivation (tau= 1 s) and by its sensitivity to external calcium. Steady-state slow inactivation is voltage dependent around the resting membrane potential (the potential of half-inactivation (V(0.5)) =-70 mV, slope factor = 7.4 mV) and can reduce the calcium current by up to 50%. Near resting potential, the slow inactivation displays a half-time of induction of tens of seconds. The slow inactivation therefore modulates the availability of T-type calcium channels depending upon recent cell history, providing a mechanism to store information in a time scale of seconds.

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

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.

Voltage-dependent activity around the resting potential is determinant in neuronal physiology and participates in the definition of the firing pattern. Low-voltage-activated T-type Ca2 + channels directly affect the membrane potential and control a number of secondary Ca2 + -dependent permeabilities. We have studied the ability of the cloned T-type channels (alpha1G,H,I) to carry Ca2 + currents in response to mock action potentials. The relationship between the spike duration and the current amplitude is specific for each of the T-type channels, reflecting their individual kinetic properties. Typically the charge transfer increases with spike broadening, but the total Ca2 + entry saturates at different spike durations according to the channel type: 4 ms for alpha1G; 7 ms for alpha1H; and > 10 ms for alpha1I channels. During bursts, currents are inhibited and/or transiently potentiated according to the alpha1 channel type, with larger effects at higher frequency. The inhibition may be induced by voltage-independent transitions toward inactivated states and/or channel inactivation through intermediate closed states. The potentiation is explained by an acceleration in the channel activation kinetics. Relatively fast inactivation and slow recovery limit the ability of alpha1G and alpha1H channels to respond to high frequency stimulation ( > 20 Hz). In contrast, the slow inactivation of alpha1I subunits allows these channels to continue participating in high frequency bursts (100 Hz). The biophysical properties of alpha1G, H and I channels will therefore dramatically modulate the effect of neuronal activities on Ca2 + signalling.

ClC-K channels are Cl- channels specifically expressed in vertebrate kidneys. Although their heterologous functional expression is still controversial, indirect evidence points to them as major factors involved in Cl- reabsorption in the nephron. We cloned xClC-K, an amphibian (Xenopus) homologue of mammalian ClC-K. The cDNA encodes a 77 kDa protein presenting 62% similarity with human ClC-Kb. The protein is monoglycosylated and is expressed primarily in the Xenopus kidney. It is localized in the basolateral membranes of proximal convoluted tubules of the nephron and in the apical region of the diluting segments. Heterologous expression of xClC-K in HEK-293 cells showed that the full-length protein is glycosylated and targeted to the cell membrane, but no associated Cl- current could be observed with the patch-clamp recording technique. N-glycosylation of both the native kidney channel and the recombinant protein expressed in HEK-293 conferred on them anomalous behaviour in denaturing PAGE, which is indicative of strong interactions at the extracellular side of the plasma membrane. The expression of ClC-K channels in both mesonephric and metanephric kidneys will permit further comparative physiological studies of Cl- permeabilities at the molecular level.

At the molecular level, our knowledge of the low voltage-activated Ca2+channel (T-type) has made little progress. Using an antisense strategy, we investigated the possibility that the T-type channels have a structure similar to high voltage-activated Ca2+channels. It is assumed that high voltage-activated channels are made of at least three components: a pore forming α1subunit combined with a cytoplasmic modulatory β subunit and a primarily extracellular α2δ subunit. We have examined the effect of transfecting cranial primary sensory neurons with generic anti-β antisense oligonucleotides. We show that in this cell type, blocking expression of all known β gene products does not affect T-type current, although it greatly decreases the current amplitude of high voltage-activated channels and modifies their voltage dependence. This suggests that β subunits are likely not constitutive of T-type Ca2+channels in this cell type.

Slow waves represent one of the prominent EEG signatures of non-rapid eye movement (non-REM) sleep and are thought to play an important role in the cellular and network plasticity that occurs during this behavioral state. These slow waves of natural sleep are currently considered to be exclusively generated by intrinsic and synaptic mechanisms within neocortical territories, although a role for the thalamus in this key physiological rhythm has been suggested but never demonstrated. Combining neuronal ensemble recordings, microdialysis, and optogenetics, here we show that the block of the thalamic output to the neocortex markedly (up to 50%) decreases the frequency of slow waves recorded during non-REM sleep in freely moving, naturally sleeping-waking rats. A smaller volume of thalamic inactivation than during sleep is required for observing similar effects on EEG slow waves recorded during anesthesia, a condition in which both bursts and single action potentials of thalamocortical neurons are almost exclusively dependent on T-type calcium channels. Thalamic inactivation more strongly reduces spindles than slow waves during both anesthesia and natural leep. Moreover, selective excitation of thalamocortical neurons strongly entrains EEG slow waves in a narrow frequency band (0.75–1.5 Hz) only when thalamic T-type calcium channels are functionally active. These results demonstrate that the thalamus finely tunes the frequency of slow waves during non-REM sleep and anesthesia, and thus provide the first conclusive evidence that a dynamic interplay of the neocortical and thalamic oscillators of slow waves is required for the full expression of this key physiological EEG rhythm., peer-reviewed

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.

To study neuronal ion channel function with antisense oligonucleotides, a reliable method is needed which allows different neuronal cell types to be transfected without artifactual disruptive effects on their electrical properties. Here we report that use of the recently introduced transfecting agent, polyethylenimine, fulfills this requirement. Four days after transfection, in both central and peripheral neurons, an antisense designed to block the synthesis of the Ca 2+ channel β subunits induced a maximal decrease of the Ca 2+ current amplitude and modification of their kinetics and voltage-dependence. Controls with scrambled oligonucleotides, as well as Na + current recordings of antisense transfected neurons, confirm both that the transfecting agent does not modify the electrophysiological properties of the neurons and that the effect of the antisense is sequence specific.

Activity of high-threshold voltage activated neuronal Ca2+ channels, including dihydropyridine-sensitive (L-type) channels, rapidly disappears during cell dialysis in whole-cell recording conditions or after excision of a patch. To date, this phenomenom has been mainly related to phosphatase or protease activity. On the other hand, it has been suggested that Ca2+ channels may be regulated by G-proteins. Therefore, disruption of this regulatory pathway may also be involved directly or indirectly in the rundown process. Here, we show that treatment of cultured cerebellar granule cells with pertussis toxin (PTX) increases to 70% the probability for excising patches that display L-type Ca2+ channels activity in the inside-out recording configuration. Quantitative study indicates that, except a half decrease in the open probability, most features of the channel activity are retained after patch excision with minor modifications. The characteristics of the channel activity did not change with time during at least the first 9 min of the inside-out configuration. In addition, comparison of unitary currents recorded in the cell-attached, configuration on treated and nontreated cells demonstrates that the PTX treatment slows the activation kinetics of the current and increases the duration of channel openings evoked at -20 mV but not at 0 mV depolarizing potential. These data suggest that L-type Ca2+ channel activity are under a tonic regulation of a PTX-sensitive mechanism, which is implied in the run-down process.

International audience; The thalamic output during different behavioral states is strictly controlled by the firing modes of thalamocortical neurons. During sleep, their hyperpolarized membrane potential allows activation of the T-type calcium channels, promoting rhythmic high-frequency burst firing that reduces sensory information transfer. In contrast, in the waking state thalamic neurons mostly exhibit action potentials at low frequency (i.e., tonic firing), enabling the reliable transfer of incoming sensory inputs to cortex. Because of their nearly complete inactivation at the depolarized potentials that are experienced during the wake state, T-channels are not believed to modulate tonic action potential discharges. Here, we demonstrate using mice brain slices that activation of T-channels in thalamocortical neurons maintained in the depolarized/wake-like state is critical for the reliable expression of tonic firing, securing their excitability over changes in membrane potential that occur in the depolarized state. Our results establish a novel mechanism for the integration of sensory information by thalamocortical neurons and point to an unexpected role for T-channels in the early stage of information processing.

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 (

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.

Genomic mining, the process of sifting through billions of genomic and EST sequences of several different species has led to the molecular identification of a family of low voltage activating channels, more commonly referred to as T-type channels. Historically, these channels were initially identified through the use of the patch-damp technique on various neuronal preparations. They were characterized by their small conductance, rapid voltage-dependent inactivation, a small window current and slow deactivation kinetics and their remarkable property of being able to open at membrane potentials just above the resting membrane potential of neurons. This property would allow for the entry of Ca2+ without the initiation of an action potential triggered by the opening of sodium channels. Thus the activity of these channels would contribute to modifying membrane excitability, allowing Ca2+ signaling events to occur at subthreshold potentials, and potentially modulate waveform patterns.

International audience; Only a few methods permit researchers to study selected regions of the central and peripheral nervous systems with a spatial and time resolution sufficient to image the function of neural structures. Usually, these methods cannot analyse deep-brain regions and a high-resolution method, which could repeatedly probe dynamic processes in any region of the central and peripheral nervous systems, is much needed. Here, we show that fibred fluorescence microscopy-which uses a small-diameter fibre-optic probe to provide real-time images-has the spatial resolution to image various neural structures in the living animal, the consistency needed for a sequential, quantitative evaluation of axonal degeneration/regeneration of a peripheral nerve, and the sensitivity to detect calcium transients on a sub-second timescale. These unique features should prove useful in many physiological studies requiring the in situ functional imaging of tissues in a living animal.

International audience; In thalamocortical neurons relaying sensory information, we recently described a phosphorylation mechanism that induces a marked increase in the amplitude of the low-voltage activated Ca 2þ current (T-type). Surprisingly, potentiation of the T-current closely depends on the state of the channel and is, therefore, both voltage-and ATP-dependent. Further analysis of the modification of channel activity induced by this regulation, and underlying the increase in the macroscopic current amplitude, requires a detailed study of the T-current biophysical properties that, unfortunately, might be constrained by the technical limitations of whole-cell recordings. Therefore, in the present study we have developed an alternative approach that is based on computational models of T-channel activity using Markov gating schemes. We show that both modifications in the activation kinetics of the channels and/or the existence of a second channel population with a conducting state conditioned by a phosphorylation step can explain the specific properties of T-currents that have been observed in thalamocortical neurons as a result of their ATP/voltage-dependent regulation. The flexibility in the T-type current behavior that is incorporated in these models might also help to unravel new roles for T-channels in shaping the different firing properties of thalamocortical neurons.

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.

Publisher Summary Current gene cloning and genomic initiatives provide neuroscientists with an exponentially increasing bank of genetic sequence data that details the molecular identity of novel brain proteins. Often in the initial absence of selective ligands, the functional properties of the more recently cloned brain proteins remain unclear. The antisense approach conceptually offers a solution to this problem. Multiple mechanisms have been proposed that describe the molecular events that precipitate gene-specific knockdown by antisense oligonucleotides. These include inhibition of transcription, translational arrest, disruption of ribonucleic acid (RNA) processing, and RNase H–mediated transcript degradation. Despite the many questions that remain unanswered, antisense oligonucleotides have been applied successfully in the central nervous system (CNS) research. This chapter discusses their in vitro applications and describes both the strategies and methods that have been developed and applied successfully to the studies of neuronal voltage-dependent ion channels. Wherever possible, the effects of antisense treatment have been examined at multiple levels—that is, transcription transduction, messenger RNA (mRNA), protein and/or functionally by electrophysiological measurements. Each level of analysis affords complementary data that when taken together greatly facilitate the final interpretation of results.

Recently, two members of a new family of Ca2+channel α1 subunits, α1G (or CavT.1) and α1H (or CavT.2), have been cloned and expressed. These α1 subunits generate Ba2+currents similar to the T-type Ca2+currents present in sensory neurons. Here, we use three methods to investigate whether the T currents of nodosus ganglion neurons are encoded by members of the CavT family. PCR detected the presence of mRNA encoding both α1G and α1H, as well as a third highly related sequence, α1I.In situhybridizations performed on nodosus ganglia demonstrate a high expression of α1H subunit RNAs. Transfection of nodosus ganglion neurons with a generic antisense oligonucleotide against this new α1 subunit family selectively suppresses the low-voltage-activated Ca2+current. The antisense oligonucleotide effect increased with time after transfection and reached a maximum 3 d after treatment, indicating a 2–3 d turnover for the α1 proteins. Taken together, these results suggest that the T-type current present in the sensory neurons is mainly attributable to α1H channels. In addition, taking advantage of the high specificity of the antisense ON to the cloned channels, we showed that T-type currents greatly slowed the repolarization occurring during an action potential and were responsible for up to 51% of the Ca2+entry during spikes. Therefore, the antisense strategy clearly demonstrates the role of low-voltage-activated Ca2+current in affecting the afterpotential properties and influencing the cell excitability. Such tools should be beneficial to further studies investigating physiological roles of T-type Ca2+currents.

The pharmacology of the majority of Ca 2+ channels in the nervous system is very limited. Although attempts have been made to constrain native Ca 2+ channels into the framework provided by the six pore-forming molecules cloned to date, refined biophysical analysis of Ca 2+ currents, expression techniques and the use of selective toxins have helped to identify unambiguously only a limited number of Ca 2+ channels. In fact, many native Ca 2+ channel activities remain as `orphans', waiting for their molecular counterparts to be defined. In this article, Janet Nooney, Regis Lambert and Anne Feltz systematically delineate the well characterized non-L Ca 2+ channel activities and the missing elements in our knowledge of the Ca 2+ channel family.

Intracellular pH (pH i ) was measured at 37°C in mature rat cerebellar oligodendrocytes dissociated in culture by using the pH-sensitive probe BCECF. Cells were identified by anti-galactocerebroside antibody. The mean steady-state pH i was 7.02 in the absence of CO 2 /bicarbonate (Hepes-buffered solution) at an external pH of 7.40 and 7.04 in 5% CO 2 /25 mM bicarbonate-buffered solution at the same external pH; this value was modified neither by the removal of external chloride nor by the addition of the chloride-coupled transport blocker DIDS. In both external solutions steady-state pH i values were strongly dependent on external pH. In Hepes-buffered solution pH i recovery following an acid load required external Na + and was completely inhibited by amiloride, indicating the presence of a Na + /H + exchanger. In CO 2 /bicarbonate-buffered solution amiloride partially reduced the pHi recovery rate, indicating the presence ofa bicarbonate-dependent pH i regulating mechanism. Membrane depolarization induced by increasing external K + concentration elicited an alkalinization only in the presence of external Na + and bicarbonate. Analysis of the calculated HCO 3 fluxes with respect to membrane potential indicated that these fluxes were mediated by a Na + -HCO 3 - cotransport with a stoichiometry of 1:3. These results demonstrate that a Na + /H + exchanger and a Na + -HCO 3 - cotransporter are involved in pH i regulation of mature oligodendrocytes.

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.