European Journal of Neuroscience, Vol. 39, pp. 788–799, 2014

doi:10.1111/ejn.12441

NEUROSYSTEMS

Acute effect of carbamazepine on corticothalamic 5–9-Hz and thalamocortical spindle (10–16-Hz) oscillations in the rat Thomas W. Zheng,1,2,3 Terence J. O’Brien,3 Sofya P. Kulikova,1,2 Christopher A. Reid,4 Margaret J. Morris5 and Didier Pinault1,2


nie, INSERM U1114, Strasbourg, France Neuropsychologie cognitive et physiopathologie de la schizophre
de
ration de Me
decine Translationnelle de Strasbourg (FMTS), NeuroPole de Strasbourg, Faculte
de me
decine, Universite
de Fe Strasbourg, INSERM U1114, 11 rue Humann, Strasbourg, 67085, France 3 Department of Medicine, The Royal Melbourne Hospital, The University of Melbourne, Parkville, Vic., Australia 4 Florey Institute of Neuroscience and Mental Health, The University of Melbourne, Parkville, Vic., Australia 5 Department of Pharmacology, University of New South Wales, Kensington, NSW, Australia 1 2

Keywords: 5–9-Hz oscillations, drowsiness, EEG, intracellular, sleep spindles, somatosensory system

Abstract A major side effect of carbamazepine (CBZ), a drug used to treat neurological and neuropsychiatric disorders, is drowsiness, a state characterized by increased slow-wave oscillations with the emergence of sleep spindles in the electroencephalogram (EEG). We conducted cortical EEG and thalamic cellular recordings in freely moving or lightly anesthetized rats to explore the impact of CBZ within the intact corticothalamic (CT)–thalamocortical (TC) network, more specifically on CT 5–9-Hz and TC spindle (10–16-Hz) oscillations. Two to three successive 5–9-Hz waves were followed by a spindle in the cortical EEG. A single systemic injection of CBZ (20 mg/kg) induced a significant increase in the power of EEG 5–9-Hz oscillations and spindles. Intracellular recordings of glutamatergic TC neurons revealed 5–9-Hz depolarizing wave–hyperpolarizing wave sequences prolonged by robust, rhythmic spindle-frequency hyperpolarizing waves. This hybrid sequence occurred during a slow hyperpolarizing trough, and was at least 10 times more frequent under the CBZ condition than under the control condition. The hyperpolarizing waves reversed at approximately 70 mV, and became depolarizing when recorded with KCl-filled intracellular micropipettes, indicating that they were GABAA receptor-mediated potentials. In neurons of the GABAergic thalamic reticular nucleus, the principal source of TC GABAergic inputs, CBZ augmented both the number and the duration of sequences of rhythmic spindle-frequency bursts of action potentials. This indicates that these GABAergic neurons are responsible for the generation of at least the spindle-frequency hyperpolarizing waves in TC neurons. In conclusion, CBZ potentiates GABAA receptor-mediated TC spindle oscillations. Furthermore, we propose that CT 5–9-Hz waves can trigger TC spindles.

Introduction Carbamazepine (CBZ) is a widely prescribed drug for the treatment of focal epileptic seizures, as well as several other neurobiological diseases, such as schizophrenia, bipolar disorders, and neuropathic pain (Post, 1982; Simhandl & Meszaros, 1992; Leucht et al., 2007). CBZ is ineffective in the treatment of typical absence epilepsy, aggravating the seizures in some patients (Genton, 2000) and in animal models (Liu et al., 2006), and has even been reported to trigger the de novo generation of epileptic discharges in humans (Monji et al., 2004). Owing to the tricyclic structural features of CBZ, it is also has a broad spectrum of action, resulting in significant side

Correspondence: Didier Pinault, 2F
ed
eration de M
edecine Translationnelle de Strasbourg (FMTS), INSERM U1114, Universit
e de Strasbourg, as above. E-mail: [email protected] Received 19 June 2013, revised 21 October 2013, accepted 4 November 2013

effects, including drowsiness, weight gain, and seizure aggravation (Levy et al., 1985; Brady, 1989; Liu et al., 2006). Apart from its primary therapeutic mechanism of use-dependent Na+ channel blockage, CBZ interacts directly with several molecular targets, including GABAA receptors (Granger et al., 1995; Liu et al., 2006), adenosine A1 receptors (Biber et al., 1999), and Ca2+ currents (Walden et al., 1993). Importantly, CBZ has been shown to directly potentiate GABAA currents in recombinant receptors expressed in human embryonic kidney cells (Granger et al., 1995) and Xenopus oocytes (Liu et al., 2006). Deciphering the mechanisms of CBZ action in living intact neural networks is likely to help increase our understanding of its efficacy and side-effect profiles. The thalamocortical (TC) system is an appropriate model with which to study the effects of neuroactive substances on GABAA receptor-mediated oscillations (von Krosigk et al., 1993; Ulrich & Huguenard, 1997; Crunelli et al., 2011). The TC neurons are glutamatergic

© 2013 Federation of European Neuroscience Societies and John Wiley & Sons Ltd

CBZ potentiates thalamocortical spindles 789 and are the principal cells of the dorsal thalamus. They are reciprocally connected with both the neocortex and the GABAergic thalamic reticular nucleus (TRN), this latter structure being an important source of GABA receptor-mediated inhibition in TC neurons (Pinault, 2004). In the rodent, at least two types of GABAA receptor-mediated oscillation have been investigated: wake-related corticothalamic (CT) 5–9-Hz oscillations and sleep-related TC spindles. Sleep spindles are a unique EEG signature of the early stages of sleep, and start to emerge during drowsiness (De Gennaro & Ferrara, 2003). They may be involved in processes involved in memory consolidation (Tamminen et al., 2010). They are characterized by short-lasting (< 2 s) bouts of medium-voltage (< 100 lV) 12–14-Hz oscillations in humans, 7–14-Hz oscillations in cats (Contreras & Steriade, 1996), and 10–16-Hz oscillations in rodents (Pinault et al., 2001). They generally have a singular sinusoidal waveform that progressively increases and then gradually decreases in amplitude. Sleep spindles are generated in TC neurons, via GABAA receptor-mediated mechanisms driven by TRN neurons (von Krosigk et al., 1993; Steriade, 1995; Ulrich & Huguenard, 1997). Wake-related CT 5–9-Hz oscillations have been recorded in rodent sensory cortices, especially during quiet immobile wakefulness (Pinault et al., 2001) and sensory cortical responses (Tort & Katz, 2010). The function of this type of rhythmic activity is still unknown. The thalamic intracellular events associated with 5–9-Hz and spindle oscillations are distinguishable (Pinault et al., 2006). In TC neurons, 5–9-Hz oscillations are characterized by a rhythmic excitatory postsynaptic potential (EPSP) wave–inhibitory postsynaptic potential (IPSP) wave sequence that occurs during a slow hyperpolarizing trough. The EPSP wave (summation of unitary EPSPs) can trigger a low-threshold Ca2+ potential crowned by a high-frequency burst of action potentials (Pinault, 2003). Oscillations of 5–9 Hz usually emerge from a membrane potential that is more depolarized, with more fast rhythmic activities than the membrane potential from which spindle oscillations emerge (Pinault et al., 2006). In TC neurons, the rhythmic 5–9-Hz and spindle IPSPs are mediated by activation of GABAA receptors. Therefore, our aim was to test the hypothesis that CBZ-induced drowsiness is associated with CT–TC oscillations involving GABAA receptor-mediated mechanisms. The hypothesis was tested in the rat somatosensory CT–TC system, by the use of intracellular recording of glutamatergic TC neurons and extracellular recordings of GABAergic TRN neurons under light anesthesia. The present results indicate that CBZ potentiates GABAA receptor-mediated TC spindles.

Materials and methods Animals Experiments were conducted in inbred adult male rats (n = 39), which originate from a colony of Wistar-derived non-epileptic control rats that are selectively bred in our laboratory so that they do not show absence-related spike-and-wave discharges (Vergnes et al., 1982). All procedures were approved by the Comit
e r
egional d’
ethique en matiere d’exp
erimentation animale (CREMEAS), the Universit
e de Strasbourg (AL/01/23/11/07), and the University of Melbourne Animal Ethics Committee (AEC #0810999), and were performed in accordance with the guidelines published by the Australian National Health and Medical Research Council (7th Edn, 2004) and the European Union (directive 2010/63/EU) for the use of animals in research. All experimental procedures also complied with the ARRIVE guidelines (Kilkenny et al., 2010). Rats were at least 13 weeks of age, and weighed 250–350 g at the time of the experiments. Rats were housed and kept under controlled

environmental conditions (12-h light/dark cycle; lights on at 07:00 h), with food and water available ad libitum. Recordings were performed in the middle of the light phase (10:00 to 16:00 h). Anesthesia and surgery Adult male rats (n = 8) were chronically implanted with extradural EEG electrodes (stainless steel screws; bilateral electrode placements – frontal parietal bone, 0.5 mm anterior to the coronal suture; ground and reference – parietal bone, 2 mm anterior to the lambdoid suture) under general anesthesia (isoflurane, 5% induction, 1.5–2.5% maintenance). Rats were given a 7-day recovery period. The acute experiments (cortical EEG, juxtacellular and intracellular recordings) were conducted under light anesthesia (neuroleptic analgesia; 31 rats). Details of all procedures are available in previous reports (Pinault, 2003; Kulikova et al., 2012). The light anesthesia was induced by continuous intravenous injection of the following mixture (quantity given per hour for a 300-g rat): fentanyl (2 lg) and glucose (25 mg). Muscle rigidity and tremors were blocked with intravenous administration of D-tubocurarine chloride (0.4 mg/h). The rats were artificially ventilated in the pressure mode (8–12 cm H2O; 60 beats/min) with an O2-enriched gas mixture (50% air/50% O2). The rat’s rectal temperature was maintained at its physiological level (37–38.3 °C) with a thermoregulated blanket. The cortical EEG and the heart rate were also continuously monitored to maintain a steady depth of light anesthesia, either by giving a bolus or adjusting the injection rate of the sedating mixture. The depth of anesthesia was ascertained by the occurrence of slow waves in the EEG. Recording sessions started 3 h after state induction. Local anesthetic (lidocaine, 2%) was infiltrated into all surgical wounds every 2 h. Drugs Carbamazepine (Sigma-Aldrich) was dissolved in vehicle containing 10% dimethylsulfoxide, 40% propylene glycol, and 50% saline. CBZ was injected intraperitoneally for EEG recordings in freely moving rats (n = 8), and subcutaneously for rats under neuroleptic analgesia (n = 31). The administered doses (10–20 mg/kg) were those that aggravate absence seizures (Liu et al., 2006). Electrophysiology Freely moving condition For four rats, recordings were made with a Compumedics EEG acquisition system (Compumedics, Melbourne, Australia) with a sampling rate of 256 Hz, and for another four rats, recordings were made with ultralow-noise amplifiers (AI 402, 950; Axon Instruments) with a sampling rate of 10 kHz (bandpass: 0.1–800 Hz). EEG recordings were performed in a well-lit, quiet room with rats remaining in their home cages. The recording session started after 15 min of habituation to the experimental conditions; this was followed by a 30-min baseline period and a 90-min post-injection period. The behavior was rated every minute as immobile, active (exploration, whisking, eating, chewing, motion, grooming, etc.), sleepy, eyes closed or open, etc. Light-anesthesia condition Glass micropipettes were filled with a solution containing 1.5% N-(2-amino ethyl)-biotin amide hydrochloride (Neurobiotin; Vector

© 2013 Federation of European Neuroscience Societies and John Wiley & Sons Ltd European Journal of Neuroscience, 39, 788–799

790 T. W. Zheng et al. Laboratories, Burlingame, CA, USA) dissolved in 0.5–1 M CH3COOK. The micropipette (25–70 MO) was stereotaxically (Paxinos & Watson, 1998) lowered with a stepping micro-driver into the medial part of the ventral posterior thalamic nucleus and into the related TRN (intermediate part) to record, extracellularly or intracellularly, a single TC or TRN neuron along with the surface EEG of the related somatosensory cortex. Additional recordings were performed in three rats with intracellular glass micropipettes filled with KCl (3 M). The EEG and cellular signals were digitized at a sampling rate of 20 kHz, with bandpasses of 0.1–800 Hz and 0.1– 6 kHz, respectively. During the intracellular recording session, a current pulse in the range of 0.2 to 0.5 nA was applied every 2 s to keep the Wheatstone bridge well balanced. Histology At the end of the recording session, the neurons were individually labeled by use of the juxtacellular (Pinault, 1996) or intracellular tracer micro-iontophoresis technique for standard histological identification and localization. After a survival period of 15–30 min, the rat was killed with an intravenous overdose of pentobarbital, followed by transcardial perfusion with a fixative containing 4% paraformaldehyde and 0.25% glutaraldehyde in 10 mM phosphate-buffered saline. Brains were kept in 4% paraformaldehyde overnight. Serial slices of 100 lm of the required region were cut with a vibratome, and processed with standard histological techniques for retrieval of the tracer-stained neurons or regions (Pinault, 1996).

puted, one with a 5–9-Hz burst, and the other with a spindle burst (Fig. 6A). The corresponding extracted EEG segments were subjected to fast Fourier transform analysis. Pearson’s correlation coefficient (implemented with Bonferroni probability) was used to provide an index of the power coherence of 5–9-Hz and spindle (10–16-Hz) oscillations between the membrane potential of thalamic neurons and the EEG recording of the related frontoparietal cortex. We used the intracellular membrane potential oscillations because they are composed of intrinsic and postsynaptic potentials, which contribute more than action potentials to extracellular local field potential oscillations (Buzs
aki, 2004). The linear correlation was estimated from paired values of the total power from 500-ms recording epochs that included a hybrid 5–9-Hz spindle pattern in the intracellularly recorded TC neurons. From every hybrid pattern, two epochs were extracted for analysis: the first epoch targeted the first component that included at least two 5–9-Hz waves, and the second epoch targeted the spindle component. For each set of data, normality was checked with the D’Agostino and Pearsons omnibus normality test (a = 0.05; GRAPHPAD PRISM, V5.04). For group analysis of non-parametric data, the Kruskal– Wallis test was used followed by Dunn’s post hoc test. t-Tests were used for parametric data, for comparison between groups. Data (mean  standard error of the mean) were considered to be significant at P < 0.05.

Results

Data analysis

CBZ potentiates cortical EEG 5–9-Hz and 10–16-Hz (spindle) oscillations

The tracer-filled neurons were examined with a light microscope. Their location was ascertained by reference to the stereotaxic atlas (Paxinos & Watson, 1998). Electrophysiological recordings were analyzed with Axon software (CLAMPEX, v7; Axon Instruments), and DataWave software (SCIWORKS, v8; DataWave Technologies, Berthoud, CO, USA). The average membrane potential of the recorded neurons was estimated from at least 30 values collected during periods free of slow oscillations (< 17 Hz) at rest (no holding current). Fast Fourier transform analysis (hamming windowing) of EEG and intracellular signals was based on epochs of ≥ 500 ms, with a resolution of 0.6–2.4 Hz. The total power of a given frequency band was computed as the sum of all of the corresponding fast Fourier transformation values. The thalamic high-frequency bursts of action potentials recorded in the intact brain were identified according to standard criteria (Domich et al., 1986; Pinault et al., 2006). In short, a typical burst contains at least two action potentials with an interval of < 3 ms. The action potential discharge in the 5–9-Hz bursts shows a less marked acceleration–deceleration pattern than that in spindle bursts (Pinault et al., 2006). Autocorrelation histograms of EEG and intracellular oscillations and inter-action potential interval histograms of TRN neuron firing were computed over periods of time of > 10 s. Burst-triggered averages (BTAs) of the cortical EEG recording and of the corresponding fast Fourier transformation were computed over a period of time of  250 ms. The first action potential of a 5–9-Hz or spindle TRN extracellular burst was the triggering event. As it is known that the rhythmic 5–9-Hz and spindle firing of TRN neurons is more reliable than that of TC neurons (Pinault et al., 2006), the BTAs were computed from the recordings of TRN neurons along with the related cortical EEG. Two BTAs were com-

Under the drug-free condition, the behavior of the rats fluctuated from active and quiet immobile wakefulness to drowsiness with an inclination to sleep. Rats were left undisturbed as much as possible (at times, rats were gently handled to untwist recording cables) throughout the recording sessions. During quiet immobile wakefulness, the frontoparietal sensorimotor cortex spontaneously showed desynchronized low-voltage (< 0.1-mV) fast (17–160-Hz) EEG oscillations, which were at times (0.5–4/min) interrupted by the occurrence of short-lasting (< 3-s) bouts of medium-voltage (< 0.2mV) slower waves, the most prominent being 5–9-Hz oscillations (Pinault et al., 2001). The rats entered the first stages of sleep (curled-up posture) 20–40 min after the beginning of the recording sessions. The sleep-related cortical EEG was characterized by ongoing slow-frequency (< 16-Hz) oscillations, including a large amount of delta (1–4-Hz) waves (Fig. 1). The CBZ-treated rats were behaviorally much less reactive to gentle handling than the control rats. Indeed, under the CBZ condition, the rats often fell asleep < 2 min after handling, whereas the control rats could stay awake and active for > 2 min (Fig. S1). 5–9 Hz and spindle oscillations were more prominent during CBZ-induced drowsiness than during natural drowsiness (Fig. 1A, B2, and B3). There was a significant overall difference in the 5–9-Hz and 10–16-Hz frequency bands after CBZ (20 mg/kg) injection (Kruskal–Wallis test, n = 4; Fig. 1B2, P = 0.018; Fig. 1B3, P = 0.013). Dunn’s post hoc test showed significant increases in both frequencies following the administration of 20 mg/kg CBZ (n = 4; Fig. 1B2, P = 0.03; Fig. 1B3, P = 0.007). A significant effect was measured with 20 mg/kg CBZ. On the basis of the internal frequency and waveform, 5–9-Hz oscillations could be distinguished from spindles (Fig. 2A, C, and D) [see also Pinault et al. (2006)]. Following the administration of CBZ, spindles occurred either alone or after a few 5–9-Hz waves or a K complex (Fig. 2B), this latter event

© 2013 Federation of European Neuroscience Societies and John Wiley & Sons Ltd European Journal of Neuroscience, 39, 788–799

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Fig. 1. CBZ increases 5–9-Hz and spindle oscillations in the frontoparietal cortex. (A) Top: 20-min records (15-s bouts extended just below) of the surface EEG under control (left records: drowsiness; +60–80 min after vehicle injection) and CBZ (right records: drowsiness; +60–80 min after 20 mg/kg injection) conditions (from the same rat, 3-day interval). Below: time–frequency spectra from 30-s epochs (fast Fourier transformation, resolution of 2.4 Hz, hamming windowing) of the surface cortical EEG recording under control and CBZ conditions. The white dotted lines indicate the putative limits between 5–9-Hz and spindle (10–16-Hz) oscillations, although they share a common frequency band. (B1–B3): Spectral power of the 1–4-Hz (B1), 5–9-Hz (B2) and 10–16-Hz (B3) oscillations under three experimental conditions (veh, vehicle; CBZ10, CBZ 10 mg/kg; and CBZ20, CBZ 20 mg/kg). Dunn’s post hoc test shows significant increases in both frequencies following administration of 20 mg/kg CBZ (n = 4; B2, *P = 0.03; B3, *P = 0.007).

representing an isolated cortical down-state (Cash et al., 2009). Curiously, we never observed a 5–9-Hz oscillation at the end of a typical spindle, i.e. a frank deceleration of the spindle (10–16-Hz) oscillations. During quiet immobile wakefulness, in contrast to the CBZ condition, following the administration of vehicle spindle oscillations were not visible in EEG recordings even though a certain amount of 10–16-Hz oscillations could be measured with mathematical tools (not shown).

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CBZ increases GABAA receptor-mediated potentials in TC neurons To determine whether the CBZ-induced 5–9-Hz and spindle oscillations involved a GABAA receptor-mediated component, we performed intracellular recordings of TC neurons under neuroleptic analgesia, a condition that allows recording of spontaneously occurring EEG 5–9-Hz and spindle oscillations (Pinault et al., 2006; Zheng et al., 2012). It is worth noting that this light anesthesia itself maintains the animals in a relatively stable state that is electrophysiologically equivalent to quiet immobile wakefulness (Pinault et al., 2001). After CBZ administration, the rats’ state consistently evolved towards a sleepy state accompanied by more low-frequency

Fig. 2. Characteristics of 5–9-Hz, spindle and ‘hybrid’ EEG oscillations. (A) Typical traces of cortical 5–9-Hz and spindle oscillations recorded in freely behaving rats. (B) A spindle (sp) can occur after either a few 5–9-Hz waves (hybrid) or after a K complex (k). (C and D) Autocorrelation histograms computed from three successive bouts of 5–9-Hz and spindle oscillations, respectively.

© 2013 Federation of European Neuroscience Societies and John Wiley & Sons Ltd European Journal of Neuroscience, 39, 788–799

792 T. W. Zheng et al. (< 20-Hz) waves, including spindle oscillations (Fig. S2), which were similar to those recorded in freely behaving rats (Fig. S3). Intracellular recordings were made from 10 TC neurons under the control condition and from eight TC neurons 20–60 min after a single CBZ injection. Under the control condition, TC neurons had an estimated average resting membrane potential of 56  5 mV, an average input resistance of 26  9 MΩ, and action potential amplitude above 50 mV. After a single injection of CBZ, the membrane potential was more hyperpolarized [ 61  5 mV; Student’s nondirectional t-test (equal variances), P = 0.007], revealing subthreshold oscillations, including rhythmic 5–9-Hz and spindle waves (Fig. 3), the latter waves being more prominent in power under the CBZ condition (Fig. 3A2, B2, and C). Cortical EEG and thalamic cellular 5–9-Hz oscillations were spatiotemporally more coherent in power than spindles (Fig. S4). Indeed, a thalamic cellular spindle was not systematically time-locked with a cortical EEG spindle, although spindles were increased in both the cortex and the thalamus under the CBZ condition relative to the control condition. All recorded neurons showed a putative low-threshold Ca2+ potential topped by a high-frequency burst of action potentials (Fig. 3D).

Following the administration of CBZ, the membrane potential of TC neurons often showed 5–9-Hz oscillations that were suddenly prolonged by pronounced spindle oscillations, forming a 5–9-Hz– spindle hybrid sequence. This was characterized by two to four cycles of a rhythmic 5–9-Hz depolarizing wave–hyperpolarizing wave sequence extended by a typical series of 3–10 rhythmic (10–16-Hz) short-lasting (66.5  1.1-ms, n = 35, from five rats) hyperpolarizations (Fig. 3A2 and B2). Both 5–9-Hz and spindle oscillations always occurred during a slow hyperpolarizing trough (Fig. 3B1 and B2). The first spindle IPSP abruptly appeared immediately after either a 5–9-Hz EPSP wave–IPSP wave sequence or a 5–9-Hz EPSP wave (Fig. 3B2). On the other hand, intracellular spindles were never followed by 5–9-Hz oscillations. It is important to emphasize that, like the oscillations observed in the frontoparietal EEG, intracellular TC spindles were extremely rare in control rats (one or two cases during a recording session of several tens of minutes in two of 10 TC neurons). Such a rare case is shown in Fig. 3A2. In contrast, under the CBZ condition, intracellular spindles frequently occurred (up to 12/min), and lasted for no more than 1.5 s (average of 0.82  0.05 s, n = 40, in five of eight recorded

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Fig. 3. Carbamazepine (CBZ) increases intracellular voltage spindle oscillations in TC neurons. (A and B) Simultaneous intracellular TC recordings and frontoparietal cortical (FPcx) EEG obtained under control (A1 and A2) and CBZ (10 mg/kg, subcutaneous; B1 and B2) conditions. Sections highlighted in gray in A1 and B1 are expanded in A2 and B2, respectively. In A2 and B2, stars indicate depolarizing waves, vertical arrows hyperpolarizing waves, and curved arrows action potentials (truncated). The 5–9-Hz depolarizing wave–hyperpolarizing wave sequences are indicated by a horizontal curved bar. The inset in A2 is an autocorrelation histogram of the 1-s firing period that just precedes the subthreshold spindle hyperpolarizing waves (indicated by the arrows). The photomicrograph in B2 is from a 100-lm-thick section of the recorded neuron, revealing part of its soma and dendrites. (C) Spectral analysis (average of eight 2.5-s epochs) of fast Fourier transform data with a resolution of 0.76 Hz. The histogram shows the average (eight values from four rats) total power of 10–16-Hz oscillations (t-test, *P < 0.001). (D) A typical low-threshold Ca2+ potential topped by a high-frequency burst of action potentials triggered by a depolarizing pulse from a hyperpolarizing holding current ( 79 mV). (E and F) Superimposition of three successive rhythmic depolarizations, which occur during 5–9-Hz oscillations (E) and during spindle oscillations (F) in the TC neuron. In E, one of the three 5–9-Hz depolarizations triggers a putative low-threshold Ca2+ spike topped by a high-frequency burst of APs, whereas in F only a single action potential was evoked during one of the spindle events. © 2013 Federation of European Neuroscience Societies and John Wiley & Sons Ltd European Journal of Neuroscience, 39, 788–799

CBZ potentiates thalamocortical spindles 793 TC neurons). In contrast to spindle waves, a 5–9-Hz depolarizing wave could contribute to the generation of a putative low-threshold Ca2+ potential crowned by a high-frequency burst of action potentials (Fig. 3E and F). In an attempt to determine whether the hyperpolarizing waves of the spindle oscillations were GABAA receptor-mediated IPSPs, we applied a sustained hyperpolarizing current of increasing intensity to move the membrane potential from rest (with a holding current of 0 nA) to more hyperpolarized levels (Fig. 4). In all TC neurons that showed spindle oscillations (five of five rats) under the CBZ condition, the polarity of the 5–9-Hz and spindle hyperpolarizations reversed at a membrane potential of approximately 70 mV (range between 67 and 73 mV; Fig. 4B1 and B2). Moreover, when the membrane potential was set below 80 mV, all of the components (EPSPs and IPSPs) of the hybrid 5–9-Hz–spindle sequence were depolarizing (Fig. 4C2). The slow hyperpolarizing trough was, in great part, reversed. These observations are consistent with previous

data (Pinault et al., 1998), suggesting the reversal potential of chloride ions. Indeed, in three TC neurons that were recorded for at least 10 min with an intracellular KCl-filled micropipette, putative rhythmic 5–9-Hz and spindle IPSPs became depolarizing and triggered high-frequency bursts of action potentials (Fig. S5). These observations substantiate the hypothesis of the involvement of activation of GABAA receptors in the generation of 5–9-Hz and spindle hyperpolarizations, which are probably IPSPs. CBZ increases spindle burst firing in TRN neurons The TRN is the principal source of GABA receptor-mediated inhibition of the TC neurons (Bal & McCormick, 1993; Pinault, 2004; Astori et al., 2011). If the TC rhythmic hyperpolarizing waves at the spindle frequency were indeed GABAA receptor-mediated IPSPs, we would record rhythmic burst firing in TRN neurons at the same frequency (10–16 Hz). We performed, under light anesthesia,

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Fig. 4. Reversal of intracellular spindle hyperpolarizing waves in TC neurons under the CBZ condition. All voltage recordings are from the same TC neuron along with the frontoparietal cortical (FPcx) EEG. (A1 and A2) In the absence of holding current (0 nA), the spindle hyperpolarizing waves are obvious at the resting membrane potential ( 59 mV). (B1 and B2) At a membrane potential in the range of 65 to 72 mV (holding hyperpolarizing current of 0.8 nA), the hyperpolarizing waves are not identifiable. The curved arrow indicates a putative low-threshold Ca2+ potential topped by a high-frequency burst of action potentials. (C1 and C2) At a more hyperpolarized level (lower than 80 mV with a holding current of 4.6 nA), the hyperpolarizing waves are fully reversed (the last six depolarizing events in C2). In C2, the first two depolarizing events presumably include increased EPSPs plus reversed IPSPs (two cycles of 5–9-Hz oscillations extended by a series of six reversed spindle IPSPs). B2 and C2 also include autocorrelation histograms of an intracellular spindle oscillation occurring at 59 mV (rhythmic hyperpolarizing waves) and an intracellular spindle oscillation occurring at 88 mV (rhythmic depolarizing waves), respectively.

© 2013 Federation of European Neuroscience Societies and John Wiley & Sons Ltd European Journal of Neuroscience, 39, 788–799

794 T. W. Zheng et al. A2; vertical arrows for spindle bursts and asterisks for 5–9-Hz bursts). This hybrid 5–9-Hz–spindle sequence was accompanied by a larger amount of 5–9-Hz than of spindle waves recorded in the related cortex (Fig. 6), supporting the hypothesis that the cortex plays a key role in the generation of CT 5–9-Hz oscillations (Pinault, 2003).

extracellular single-unit recordings of GABAergic TRN neuronss (five units from five rats) along with the related frontoparietal cortical EEG. The state of the EEG was more synchronized under the CBZ condition than under the control condition (Fig. 5A1 and A2). In all five recorded TRN neurons, inter-action potential interval histograms did not show any significant difference in the principal intrinsic firing features recorded between the vehicle and CBZ conditions (Fig. 5B), with averaged instantaneous frequencies of 270  12 Hz (maximum, 646 Hz; 316 action potentials) and 251  12 Hz (maximum, 667 Hz; 366 action potentials), respectively. The related TRN spindle bursts are, in great part, underpinned by low-threshold T-type Ca2+ channels of the Cav3.3 type (Astori et al., 2011). Under the CBZ condition, a rhythmic spindlefrequency burst firing pattern was significantly more frequent and longer in duration (Fig. 5A1 and A2, vertical arrows; Fig. 5C and D; Fig. 5E, spindle frequency, Wilcoxon matched-paired signed rank test, P = 0.03; Fig. 5F, spindle duration, Mann–Whitney test, U = 1056, P < 0.0001). It was also striking that the robust spindle burst firing often occurred immediately after two or three cycles of 5–9-Hz high-frequency bursts of action potentials (Fig. 5A1 and

Discussion The present study, carried out in vivo in the rat TC system, demonstrates for the first time that drowsiness induced by a single injection of CBZ is principally associated with an increase in sleep spindle power, number, and duration. Indeed, in somatosensory TC neurons, spindles constituted a manifestation of GABAA receptor-mediated rhythmic IPSPs, which were triggered by high-frequency bursts of action potentials in TRN neurons. In TC neurons, spindle oscillations often occurred after two or three CT 5–9-Hz EPSP–IPSP waves. In TRN neurons, spindle bursts often occurred after a few (two or three) 5–9-Hz bursts. This is the first time that this hybrid 5–9-Hz–spindle pattern has been reported. These findings supports

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Fig. 5. CBZ increases spindle burst firing in TRN neurons. (A1 and A2) Simultaneous cortical EEG recording from the frontoparietal cortex (FPcx) and extracellullar recording of a TRN neuron under the control (vehicle, A1) condition and approximately 40 min after CBZ (A2) injection. Stars indicate typical 5–9Hz burst firing, and arrows indicate typical spindle burst (10–16-Hz) firing. Below (A2) is shown a typical hybrid 5–9-Hz–spindle sequence (high-pass filter cut-off at 200 Hz) and expanded 5–9-Hz (left) and spindle (right) high-frequency bursts of action potentials. (B) Inter-action potential interval histograms under vehicle and CBZ conditions (resolution, 1 ms; 10-s epoch during synchronized EEG; CBZ, 366 action potentials; vehicle, 316 action potentials). (C and D) Autocorrelation histograms of typical 5–9-Hz burst and spindle-burst firing, respectively. (E and F) Both the frequency (E; P = 0.03, n = 5) and the duration (F; CBZ, 87 spindles from five rats; vehicle, 54 spindles from five rats) of spindle firing are significantly increased after systemic injection of CBZ (10 mg/kg, subcutaneous). *P < 0.05 (see text for detail). © 2013 Federation of European Neuroscience Societies and John Wiley & Sons Ltd European Journal of Neuroscience, 39, 788–799

CBZ potentiates thalamocortical spindles 795 A

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Fig. 6. Relationship of the TRN hybrid 5–9-Hz–spindle sequence with the simultaneous EEG from the frontoparietal cortex (FPcx) under the CBZ condition. A typical example of a TRN hybrid sequence with the related cortical activity filtered at 2–20 Hz is shown in A. The two vertical arrows indicate a 5–9-Hz and spindle burst used to compute the BTA of the related EEG (B1 and B2) and corresponding fast Fourier transformation (C1 and C2). Averaging is computed from 10 hybrid sequences, like the one shown in A. The inserted histogram in C2 shows the total power of 5–9-Hz (black) and spindle (10–16-Hz, white) oscillations computed from 22 TRN hybrid 5–9-Hz–spindle sequences from four TRN cells (four rats). The histogram includes the values of the spectra in C1 and C2. Note that the power of 5–9-Hz oscillations is much higher than that of spindle oscillations during both the 5–9-Hz and the spindle TRN bursts in the hybrid pattern. t-test: ns, not significant.

the hypothesis that CBZ potentiates GABAA currents (Liu et al., 2006). Furthermore, we propose that the cortex can play a leading role in the triggering of TC spindles. EEG features associated with CBZ-induced drowsiness Consistently, increased spindle power in the cortical EEG was the most important electrophysiological feature recorded here in freely moving rats treated with a relatively high dose of CBZ (20 mg/kg), which induced drowsiness. At a lower dose (10 mg/kg), the amount of spindles remained in the control range. It is worth remembering that the spindle power computed with the fast Fourier transform does not differentiate between baseline EEG sigma (10–16-Hz) oscillations and transient spindle oscillations (De Gennaro & Ferrara, 2003). The increase in EEG spindle power was confirmed by the cellular correlates recorded in TC (spindle IPSPs) and TRN (spindle bursts) neurons after a single CBZ injection (see below). Our results are consistent with a recent human study showing that CBZ enhanced frontal lobe spindle activity and slow oscillations (Ayoub et al., 2013). The power of EEG 5–9-Hz oscillations was also significantly increased after a single CBZ injection in freely moving (CBZ, 20 mg/ kg) and in lightly anesthetized (CBZ, 10 or 20 mg/kg) rats. The increase in 5–9-Hz waves was not obvious in cellular recordings,

suggesting that the spectral analysis included a frequency band common to both 5–9-Hz and spindle oscillations. Oscillations of 5–9 Hz are recorded in the rodent frontoparietal cortex during quiet immobile wakefulness, but not during active wakefulness (present study) [see also Pinault et al. (2001)]. In genetic models of absence epilepsy, 5–9-Hz oscillations suddenly become hypersynchronized, giving rise to generalized epileptic discharges in CT–TC systems (Pinault et al., 2001; Polack et al., 2007; Zheng et al., 2012). The physiological elements that underlie these 5–9-Hz oscillations remain to be determined. They belong to a puzzling continuum of theta-frequency, alpha-frequency, mu-frequency and sigma-frequency oscillations (Tiihonen et al., 1989; Pfurtscheller et al., 1997; M€ olle et al., 2002). Their function is still under debate and investigation (Tort & Katz, 2010). Their frequency band is lower but overlaps with that of spindles, reaching a maximum frequency of 12 Hz (Pinault et al., 2001). There is also an overlap with lower-frequency delta oscillations, and both can coexist. As 5–9-Hz oscillations are recorded in the somatosensory system, it is tempting to suggest that they could be the equivalent of either the alpha rhythm (8–12 Hz) recorded in the visual CT– TC systems, or the theta rhythm, which is known to increase during the first stages of sleep (Silber et al., 2007). Further investigation is required to clarify this intriguing point. Another remarkable EEG feature consistently recorded under the control condition (freely moving or light anesthesia) but more

© 2013 Federation of European Neuroscience Societies and John Wiley & Sons Ltd European Journal of Neuroscience, 39, 788–799

796 T. W. Zheng et al. frequently after CBZ administration was 5–9-Hz waves followed by a robust spindle (Fig. 7). Intriguingly, the order of this stereotyped hybrid pattern seems to be fixed. Indeed, in EEG and intracellular recordings, spindles were never followed by 5–9-Hz oscillations. Furthermore, both the autocorrelogram of the cortical EEG recording and the intracellular recording of thalamic neurons revealed that spindle oscillations were more stereotyped than 5–9-Hz oscillations. As 5–9-Hz oscillations are generated in the cortex (Pinault, 2003; Pinault et al., 2006) and spindles in the thalamus (von Krosigk et al., 1993), we propose that CT 5–9-Hz oscillations can trigger TC spindles, sometimes under the physiological condition, and more frequently after the administration of CBZ. This proposal is in agreement with previous in vitro and in vivo studies (Bal et al., 2000; Blumenfeld & McCormick, 2000; Bonjean et al., 2011). In other words, CT 5–9-Hz oscillations would make the TC system resonate at the spindle frequency. The electrophysiological hybrid 5–9-Hz–spindle sequence might be a signature of CBZ effects, as it was at least 10 times more frequent during the CBZ condition than during the control condition. Moreover, what is intriguing is that the 5–9-Hz rhythm, which is more related to wakefulness than to sleep (Pinault et al., 2001), can immediately be prolonged by sleep spindles, suggesting that a certain amount of residual wake-related

Fig. 7. Prediction that CT 5–9-Hz oscillations can trigger TC spindles. This sketch predicts cortical and thalamic events that can be recorded in rats with CBZ-induced drowsiness. On the left, the three principal elements that make up the somatosensory TC system are shown. Note that the CT neuron of layer VI innervates both the GABAergic TRN neuron and the glutamatergic TC neuron. The surface cortical EEG recording shows a series of 5–9-Hz waves followed by a typical spindle (sinusoidal waves at 10–16 Hz). This sequence was recorded in a drowsy rat (freely moving condition). The TRN neuron shows a series of four 5–9-Hz high-frequency (300–650-Hz) bursts of action potentials (n = 3–15). The membrane potential of the TC neuron shows a few waves at 5–9 Hz followed by a spindle oscillation during a steady envelope-like hyperpolarization. A depolarizing 5–9-Hz wave can trigger a low-threshold Ca2+ potential crowned by a high-frequency burst of action potentials (left curved arrow). The following spindle oscillations are subthreshold. Such a 5–9-Hz–spindle sequence can be terminated by a rebound firing (right curved arrow), which usually appears at the termination of the steady envelope-like hyperpolarization. The TRN and TC neurons were recorded under light anesthesia. On the basis of the current literature, the origin of 5–9-Hz oscillations is led by CT neurons of layer VI (green bar), whereas the generation of spindles involves reciprocal TC–TRN interactions (red and purple bars).

5–9-Hz oscillations persist during CBZ-induced drowsiness. Thus, the question of whether this singular hybrid pattern has a physiological or pathophysiological function remains open. The thalamic cellular correlates of 5–9-Hz and spindle oscillations The present cellular data are in agreement with those from previous studies (Pinault, 2003; Pinault et al., 2006). Here, it is further shown that, through unknown prethalamic and/or intrathalamic mechanisms, CBZ significantly hyperpolarizes the membrane potential of thalamic neurons and increases spindle oscillations. We predict that CBZ likewise hyperpolarizes other types of cortical and subcortical neurons. CT neurons of layer VI, which are massive in number (Jones, 1985), exert a strong facilitatory effect on thalamic neurons (Yuan et al., 1986). Therefore, it is reasonable to propose that the hyperpolarizations recorded in thalamic neurons under the CBZ condition might be the result of disfacilitation of CT inputs. The CT feedback on TC and TRN neurons is thought to play a central role in attentional processes (Sillito et al., 1994; Pinault, 2004). Concerning the hyperpolarizing trough that is associated with the rhythmic events, which include GABAA receptor-mediated IPSPs, the present results (partial reversal at approximately 70 mV) suggest the involvement of a cumulative effect of the repetitive activation of GABAA receptors. However, we cannot exclude the possibility of a contribution of the cation currents IK-leak and/or Ih to the slow hyperpolarizing trough that is associated with rhythmic 5–9-Hz and spindle activity in TC neurons (Meuth et al., 2006). Further investigation is required to better understand the relative contributions of synaptically mediated (GABAA) and intrinsically mediated (IK-leak and Ih) currents during 5–9-Hz and spindle oscillations. The thalamic intracellular rhythmic events associated with 5–9-Hz and spindle oscillations are distinguishable (Pinault et al., 2006). In TC neurons, the rhythmic 5–9-Hz and spindle IPSPs are mediated by activation of GABAA receptors. The present study demonstrates that the GABAA receptor-mediated rhythmic 5–9-Hz and spindle IPSPs are caused by rhythmic burst discharges in related GABAergic TRN neurons. The 5–9-Hz oscillations recorded in TC neurons are very probably initiated by the glutamatergic layer VI CT neurons (Pinault, 2003), whereas the electrogenesis of spindles principally involves the functional reciprocal interaction in the glutamatergic TC–GABAergic TRN circuit (von Krosigk et al., 1993; Steriade, 1995). Importantly, both the initiation and the termination of sleep spindles are under the control of CT activities (Bonjean et al., 2011). Figure 7 is a sketch presenting the likely scenario of the anatomofunctional events underlying the hybrid 5–9-Hz–spindle pattern present in the surface cortical EEG during drowsiness or the first stages of non-rapid eye movement sleep. On the basis of the present findings, we conclude that CBZ significantly and steadily hyperpolarizes both TC and TRN neurons. The glutamatergic layer VI neurons, which innervate TRN and TC neurons, sometimes rhythmically discharge at 5–9 Hz, imposing at that frequency a series of high-frequency bursts of action potentials in TRN neurons, which have a higher propensity to burst than TC neurons, because of their reliable electro-responsive properties (Llin
as, 1988; Huguenard & Prince, 1992). The number of excitatory CT neurons is large; they are approximately 10-fold more abundant than TC neurons (Jones, 1985). In response to the CT discharges, TC neurons generate subthreshold EPSP waves, because they become more hyperpolarized during the first stages of sleep (natural or CBZ-induced). However, such a TC EPSP wave can trigger a low-threshold Ca2+ potential

© 2013 Federation of European Neuroscience Societies and John Wiley & Sons Ltd European Journal of Neuroscience, 39, 788–799

CBZ potentiates thalamocortical spindles 797 (McCormick & Pape, 1990). In turn, the TRN 5–9-Hz bursts directly trigger a GABAA receptor-mediated IPSP in TC neurons immediately after the subthreshold EPSP wave. Intriguingly, the few TC 5–9-Hz EPSP wave–IPSP wave sequences are suddenly prolonged by a spindle, which corresponds to a resonant train of rhythmic 10–16-Hz bursts in TRN neurons and to a subsequent typical series of rhythmic 10–16-Hz IPSPs in related TC neurons. This TC spindle usually terminates at the end of the steady hyperpolarization, and occasionally shows rebound firing, very likely under the depolarizing influences of a hyperpolarization-activated cation current (Ih) and of CT inputs (Bal & McCormick, 1996; Budde et al., 1997; Bonjean et al., 2011). The hybrid 5–9-Hz–spindle pattern is, to all appearances, a hallmark of drowsiness or of the first stages of nonrapid eye movement sleep. It appears as an index of functional interactions between the cortex and thalamus. Moreover, it supports the hypothesis of an active contribution of the cortex to the initiation of sleep spindles (Bonjean et al., 2011). Whether the hybrid pattern is an epiphenomenon or is implicated in a given function or dysfunction remains an open question. As no intermediate event was apparent in between the 5–9-Hz oscillations and the 10–16-Hz spindle oscillations, one may wonder what is (are) the factor(s) responsible for the sudden switch from the former to the latter oscillation type. First, one important factor is the state of the system, which becomes increasingly hyperpolarized (at least in TC and TRN neurons) during 5–9-Hz oscillations (Fig. 3). Such a hyperpolarized state is a prerequisite for triggering the spindle pacemaker properties of TRN neurons, which synaptically and mutually work with the related TC neurons (von Krosigk et al., 1993). This likely scenario is supported by previous studies showing that cortical electrical stimulation generates a long-lasting hyperpolarization in TRN neurons, immediately followed by a spindle burst sequence (Contreras et al., 1996), and that activation of the CT pathway triggers intra-TRN inhibition (Zhang & Jones, 2004). Therefore, it is tempting to put forward the concept that, after CT 5–9-Hz oscillations, a spindle is a post-inhibitory all-or-none resonant rebound activity in TRN (series of spindle bursts) and TC (subsequent series of spindle GABAA IPSPs) neurons. The present findings highlight the persuasive notion that the CT–TC system is characterized by plastic mechanisms during the wake–sleep cycle (Steriade, 2006; Timofeev, 2011), in which the TRN, with its powerful intrinsic properties, plays a pivotal role, at least in the generation of sleep spindles (Steriade et al., 1987). In agreement with a previous study (Pinault et al., 2006), the present investigation shows that, in rat TC neurons, the series of rhythmic spindle-frequency IPSPs does not trigger rebound burst firing, as would have been expected from previous in vivo and in vitro studies (Desch^enes et al., 1984; von Krosigk et al., 1993). This may be the reason why TC spindles never triggered 5–9-Hz waves in the somatosensory CT–TC system. The lack of spindle-frequency burst firing in the recorded TC neurons is discussed in a previous report (Pinault et al., 2006). The cellular and ionic mechanisms underlying sleep spindles have been well described (Beenhakker & Huguenard, 2009). Whether the CT–TC systems are the primary sites of action of CBZ, and, similarly, whether GABAergic transmission is the primary target of CBZ, are questions that deserve further investigation. Although the CBZ-binding site on GABAA receptors is not known, it has been demonstrated to be a positive allosteric modulator on GABAA receptor subtypes that are naturally expressed in the thalamus (Granger et al., 1995; Liu et al., 2006; Zheng et al., 2009). We should not exclude the possibility that CBZ may have other molecular targets. Spindle bursting in TRN neurons involves an interplay

between Cav3.3-type Ca2+ channels and Ca2+-dependent smallconductance type 2 (SK2) K+ channels (Astori et al., 2011). As enhanced SK2 channel activity sustains sleep spindles (Wimmer et al., 2012), CBZ might directly or indirectly (e.g. via GABA receptors) potentiate the activity of SK2 channels. Functional implications The present study demonstrates that CBZ is a sleep spindle-promoting agent, and this property is probably responsible for provoking persistent somnolence and attention deficit, some of the drug’s major side effects (Levy et al., 1985). These findings raise a number of questions requiring further investigation. Are the behavioral and neurophysiological effects of chronic CBZ treatment similar to those induced by a single CBZ injection? Do the neurocognitive side effects of CBZ (Dias et al., 2012) result from sleep disorders, or more specifically from an increase in sleep spindles? Could the CBZ-induced spindle increase disrupt the beneficial effects of sleep, e.g. by disturbing synaptic homeostasis (Tononi & Cirelli, 2012)? In patients with schizophrenia, sleep spindles are reduced (Ferrarelli et al., 2007, 2010). Therefore, another question arising from the current study is whether CBZ-induced renormalization of the amount of spindles could be associated with beneficial cognitive effects in patients with schizophrenia. Despite the effective use of CBZ in the treatment of focal seizures, the mechanisms underlying significant side effects of the drug, such as drowsiness and severe alteration of sleep architecture (Levy et al., 1985; Yang et al., 1989), remain unclear. Pharmacotherapies that induce drowsiness or sedation, whether as the primary therapeutic mechanism of action or as an adverse effect, commonly involve GABAA receptor-mediated neurotransmission. This diverse class of drugs includes benzodiazepines, barbiturates, ethanol, and several anesthetics that have been used either therapeutically or as experimental tools to study sleep physiology. GABAA agonists such as muscimol are known to induce sleep that is associated with an increase in slow waves (0.5–4 Hz) and the generation of spindles (11–16 Hz) in rats (Lancel et al., 1996). Treatments for insomnia have also been dominated by positive modulators of GABAA receptors, such as barbiturates and benzodiazepines, which act at different binding sites on GABAA receptors to increase spindle-frequency activity (Winsky-Sommerer, 2009). In short, the direct and/or indirect mechanisms by which CBZ modulates GABA-related oscillations in CT–TC systems remain to be further investigated.

Conclusion The present study shows that a single systemic administration of CBZ induces a dysfunctional state in CT–TC systems, characterized by an excessive increase in GABAA receptor-mediated TC sleep spindles. They often appeared after a few CT 5–9-Hz waves, forming a unique hybrid CT 5–9-Hz–TC spindle pattern. We propose that, at least in drowsy rodents, the rhythmic CT 5–9-Hz activity can make the TC system resonate at the spindle frequency.

Supporting Information Additional supporting information can be found in the online version of this article: Fig. S1. Electrophysiological indication of CBZ-induced sedation. Fig. S2. Under the light anesthesia condition, CBZ increases the amount of cortical low-frequency (< 20-Hz) oscillations.

© 2013 Federation of European Neuroscience Societies and John Wiley & Sons Ltd European Journal of Neuroscience, 39, 788–799

798 T. W. Zheng et al. Fig. S3. Cortical 5–9-Hz and spindle (10–16-Hz) oscillations recorded under light anesthesia are similar to those recorded in freely behaving rats. Fig. S4. Oscillations at 5–9 Hz are more coherent than spindle oscillations in power between the membrane potential of TC neurons and the EEG of the related frontoparietal cortex (FPcx). Fig. S5. Thalamocortical hybrid 5–9-Hz spindle pattern recorded intracellularly with a KCl (3 M)-filled micropipette.

Conflict of interest All of the authors report no biomedical financial interests or potential conflicts of interest.

Author contributions T. W. Zheng, S. P. Kulikova, and D. Pinault: designed the study, performed experiments and data analysis, and wrote the manuscript. T. J. O’Brien, C. A. Reid, and M. J. Morris: designed the study and wrote the manuscript.

Acknowledgements We thank Anne-Sophie Bouillot (INSERM Apprenticeship, Professional Licence, University Joseph Fourier, Grenoble, France) for her excellent technical assistance. T. W. Zheng is the recipient of an Eiffel Doctorate Fellowship of Excellence. S. P. Kulikova is a fellow of the Trinational Neurex Joint Master in Neuroscience (Basel-Strasbourg-Freiburg) and of INSERM. This work was supported by grants from the French Institute of Health and Medical Research (INSERM), the Universit
e de Strasbourg, FMTS, Strasbourg and Neurex to D. Pinault. It was also supported by a Project Grant from the NH&MRC (Australia) (#568729) to T. J. O’Brien and M. J. Morris.

Abbreviations BTA, burst-triggered average; CBZ, carbamazepine; CT, corticothalamic; EEG, electroencephalogram; EPSP, excitatory postsynaptic potential; IPSP, inhibitory postsynaptic potential; SK2, Ca2+-dependent small-conductance type 2; TC, thalamocortical; TRN, thalamic reticular nucleus.

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