Anna-Lucia Koerling

Tanja Fuchsberger

Ole Paulsen

Y Audrey Hay

Audrey Hay

Partial restoration of

INTRODUCTION

Epileptic seizures are defined by paroxysmal hyper-synchronizations of neuronal activity and most therapeutic strategies aim at preventing this neuronal network synchronization by reducing neuronal or network excitability. However, synchronization of neuronal activity is also a physiological process necessary to perform cognitive tasks, from sensory perception to memory acquisition and consolidation [START_REF] Major | Persistent neural activity: prevalence and mechanisms[END_REF][START_REF] Compte | Computational and in vitro studies of persistent activity: edging towards cellular and synaptic mechanisms of working memory[END_REF]. The distinction between physiological and pathological rhythms is often subtle and it is hypothesized that the specific neuronal circuits that are involved in physiological rhythms are highjacked by pathological events [START_REF] Beenhakker | Neurons that fire together also conspire together: is normal sleep circuitry hijacked to generate epilepsy?[END_REF][START_REF] Bragin | Local generation of fast ripples in epileptic brain[END_REF][START_REF] Huguenard | Intrathalamic rhythmicity studied in vitro: nominal Tcurrent modulation causes robust antioscillatory effects[END_REF]. Most anti-convulsant drugs affect both physiological and pathological events and thus induce significant cognitive impairments. Models to compare the effects of drugs on physiological and pathological activity are needed to especially address whether drugs allow for the return from a pathological to a physiological network state.

The link between sleep and epilepsy has been highlighted since the beginning of electroencephalographic (EEG) recordings [START_REF] Lennox | Epilepsy and related disorders[END_REF]. Seizures during sleep are associated with multiple forms of epilepsy and 12 % of epileptic patients suffer from nocturnal epilepsy syndrome, which is defined by a frequency of 90 % or more of the seizures at night [START_REF] Bazil | Effects of sleep and sleep stage on epileptic and nonepileptic seizures[END_REF][START_REF] Crespel | The relationship between sleep and epilepsy in frontal and temporal lobe epilepsies: practical and physiopathologic considerations[END_REF]. In particular, epileptiform activity is increased during slow wave sleep (SWS), while seizures are virtually absent during rapid eye movement (REM) sleep [START_REF] Bazil | Effects of sleep and sleep stage on epileptic and nonepileptic seizures[END_REF]. During SWS, active UP-states that are characterized by depolarized neurons and a high level of synaptic activity, alternate with quiescent DOWN-states, during which neurons are hyperpolarized and mostly silent [START_REF] Steriade | Thalamocortical oscillations in the sleeping and aroused brain[END_REF]. In contrast, epileptic seizures are associated with hypersynchronous neuronal activity, generally initiated locally, which can spread throughout the brain, sometimes resulting in long-lasting generalized seizure states called status epilepticus, and often associated with slow wave sleep [START_REF] Berg | Revised terminology and concepts for organization of seizures and epilepsies: Report of the ILAE Commission on Classification and Terminology 2005-2009[END_REF][START_REF] Singhal | Continuous spike-wave during slow wave sleep and related conditions[END_REF][START_REF] Trinka | A definition and classification of status epilepticus -Report of the ILAE Task Force on Classification of Status Epilepticus[END_REF]. At the cellular level, they are characterized by paroxysmal depolarizing shifts and abnormally elevated firing rate [START_REF] Berg | Revised terminology and concepts for organization of seizures and epilepsies: Report of the ILAE Commission on Classification and Terminology 2005-2009[END_REF]. Because spontaneous UP-states during SWS are due to the synchronous but balanced activation of excitatory and inhibitory neuronal populations, it is crucial to understand how a balanced ratio between excitation and inhibition may become dysregulated and lead to epileptic seizures.

Gamma-aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the cortex and plays a fundamental role in preventing paroxysmal excitation. GABA acts pre-and postsynaptically via ionotropic GABA A receptors (GABA A Rs) and metabotropic GABA B receptors (GABA B Rs). During SWS, balanced activation of GABA A Rs has been suggested to contribute to the stability of UP-states while activation of GABA B Rs promotes the transition from UP-to DOWN-state. Indeed, blocking even partially GABA A Rs leads to epileptiform bursts while blocking GABA B Rs prolongs UP-state duration [START_REF] Mann | Distinct roles of GABA A and GABA B receptors in balancing and terminating persistent cortical activity[END_REF]. The GABA signalling pathway is one of the main therapeutic targets of anti-epileptic drugs, and tiagabine, an inhibitor of GABA uptake, is used clinically to treat certain forms of epilepsy (Schachter, 1999). However, it is unclear whether GABA uptake inhibitors have their effect primarily via GABA A Rs or GABA B Rs or both, and their effects on SWS oscillations have not been thoroughly investigated. We therefore decided to compare the effects of classic GABA uptake-blocking anticonvulsant drugs on both SWS-like and epileptiform activity using a classic ex vivo preparation, in which neurons spontaneously fluctuate between long quiescent DOWN-states and short active UP-states [START_REF] Sanchez-Vives | Cellular and network mechanisms of rhythmic recurrent activity in neocortex[END_REF][START_REF] Digby | Distinct mechanisms of Up state maintenance in the medial entorhinal cortex and neocortex[END_REF]. These brain slices are used to investigate the mechanisms un-derlying this bistable mode of cortical activity, which gives insight into the mechanisms of deep sleep and the dynamics of cortical networks.

In this study, we addressed the role of GABAergic pathways in the modulation of spontaneous physiological and pathological epileptiform events, and whether anti-convulsant drugs can restore physiological activity in a pathological context. To that end, we combined whole-cell patch-clamp recordings of neocortical neurons from juvenile thalamocortical slices of mouse brain with pharmacology to study the transition from spontaneous physiological activity to status epilepticus-like state. We used two classic models of epilepsy: low inhibition, using synaptic GABA A Rs blockers (gabazine) to isolate GABA B Rs and extrasynaptic GABA A Rs component, and enhanced excitation, using low Mg 2+ extracellular concentration that is known to more realistically preserve the properties of epileptiform burst propagation in the neocortex [START_REF] Walther | Epileptiform activity in combined slices of the hippocampus, subiculum and entorhinal cortex during perfusion with low magnesium medium[END_REF]Trevalyan et al., 2006). We investigated the modulatory roles of GABA A Rs and GABA B Rs on UP-states and epileptiform bursts. We show that GABA B R antagonists prolong both UP-states and epileptiform bursts, while GABA B R agonists reduce the incidence of epileptiform bursts. Furthermore, blocking GABA uptake using classical anti-convulsant drugs shortens and decreases the incidence of epileptiform bursts. Finally, we show that GABA uptake blockers can partially restore physiological UP-states in a pharmacologically induced model of epilepsy.

MATERIALS AND METHODS

Mice

All experiments were performed in accordance with United Kingdom Home Office regulations (Animals (Scientific Procedures) Act 1986 Amendment Regulations 2012) following ethical review by the University of Cambridge Animal Welfare and Ethical Review Body (AWERB). All animal procedures were authorized under Personal and Project licences held by the authors. Mice were provided ad libitum access to food and water and housed in a room maintained on a 12h-12h light-dark cycle, with temperature maintained at 22-24 °C and relative humidity kept at 50-55%.

Experiments were carried out on wild-type C57/BL6J mice (Harlan, Bicester, UK), bred in the facility.

Slice preparation and Electrophysiology

Thalamocortical slices of 400 μm thickness were prepared from postnatal mice (P12-21).

Mice were deeply anaesthetized with isofluorane (4%) in 100% oxygen and decapitated.

The brain was then excised and immersed in oxygenated ice-cold artificial cerebrospinal fluid (aCSF) containing: 126 mM NaCl, 3.5 mM KCl, 1.25 mM NaH 2 PO4, 1 mM MgSO 4 , 1.2 mM CaCl 2 , 26.4 mM NaHCO 3 , and 10 mM glucose (pH 7.2). Thalamocortical slices were prepared as described in [START_REF] Agmon | Thalamocortical responses of mouse somatosensory (barrel) cortex in vitro[END_REF] to preserve corticocortical connections in the barrel cortex and as many of thalamocortical connections as possible.

Slices were then incubated for at least 60 minutes at room temperature (22-24 °C) in an interface chamber between aCSF and humidified carbogen gas (95% O 2 , 5% CO 2 ).

For recording, slices were mounted on coverslips coated in 0.1% poly-L-lysine and transferred to the recording chamber perfused at 5-6 mL/min with aCSF maintained at 30-32 °C. Slices were used for up to 10 hours following sectioning.

Neurons were visualized and selected via infrared videomicroscopy using a waterimmersion objective. Whole-cell patch-clamp recordings were performed using glass pipettes (3-6 MOhm), pulled from standard borosilicate glass. Pipettes were filled with a solution containing: 110 mM K-gluconate, 40 mM HEPES, 2 mM ATP-Mg, 0.3 mM GTP, 4 mM NaCl, 2 mg/mL biocytin (270-285 mOsm/L, pH 7.2-7.3). All experiments were performed in current-clamp mode and the cells were maintained at their resting membrane potential without current injection. No correction for liquid junction potentials was applied.

Cells with a membrane potential positive to -55 mV during Down states and cells showing a change in input resistance greater than 20% in the first 10-minute control recording were excluded from the study. Data were collected using an Axon Multiclamp 700B amplifier (Molecular Devices, Sunnyvale, CA, USA), acquired and digitized at 20 kHz using an Instrutech ITC-18 A/D board and custom-made acquisition procedures in Igor Pro (Wavemetrics). Offline analyses were done using custom-made procedures in Igor Pro version 6. Automated detection of UP-states and epileptiform bursts were performed as previously described [START_REF] Craig | Distinct roles of GABA B1a -and GABA B1b -containing GABAB receptors in spontaneous and evoked termination of persistent cortical activity[END_REF]. Briefly, membrane potential noise (RMS) was computed on 4 successive nonoverlapping windows (noise_win_a, noise_win_b, noise_win_c, noise_win_d, 6 ms each), noise_win_a/noise_win_b ratio was compared to noise_win_c/noise_win_d and changes of 10 % (this value was adjusted between 6 % for epileptiform events and 15 % for UPstates in some recordings) was considered as a state transition. Each event was then manually confirmed.

Drugs and Chemicals

SR95531 (gabazine), 1-[2-[tris(4-methoxyphenyl)methoxy]ethyl]-(S)-3-piperidinecarboxylic acid (SNAP-5114), and (-)-(R)-1-[4,4-bis(3-methyl-2-thienyl)-3-butenyl]nipecotic acid hydrochloride (tiagabine) were obtained from Sigma (Saint Louis, MO, USA) and Fisher Scientific UK Ltd (Loughborough, UK). CGP55845 hydrochloride and baclofen were purchased from Bio-techne (Abingdon, UK).

Statistics

All statistics and graphical representations were performed using R (http://www.cran.rproject.org/). Results are given as mean ± standard error of the mean (SEM). Paired and unpaired comparisons were performed using non-parametric Wilcoxon-Signed Rank test.

For the initial characterization of UP-state parameters, one-way ANOVA was used. Homogeneity of variances was tested prior to ANOVA using a Bartlett test; if the homogeneity of variance was not satisfied, Welsh corrections were applied giving rise to decimal number for the degrees of freedom (ONEWAY test with non equal variance in R). A p-value below 0.05 was considered statistically significant.

RESULTS

Characterization of spontaneous UP-states

UP-states can occur spontaneously in slices from the barrel cortex of juvenile mice maintained at physiological ion concentrations and temperature [START_REF] Digby | Distinct mechanisms of Up state maintenance in the medial entorhinal cortex and neocortex[END_REF]. These spontaneous events reflect the preservation of the cortical network architecture and circuit dynamics in the slice and, thus, spontaneously active slices can serve as a model to understand better the mechanisms of transition from physiological to abnormal, epileptiform neocortical activity. In order to investigate the mechanism of transition to epileptiform burst generation, we first characterized the UP-states occurring spontaneously in thalamocortical slices from juvenile (P12-P18) wild-type mice. We performed whole-cell recording during spontaneous UP-states from 85 neurons located in layers 2-5 (41 mice).

Under our conditions, UP-states occurred at a rate of 1.11 ± 0.08 min -1 (n = 85), lasted on average 1.14 ± 0.09 s, and were crowned by 2.25 ± 0.38 action potentials (Figure 1). A similar incidence of UP-states was observed in coronal slices (0.87 ± 0.21 min -1 , n = 7, p = 0.59), suggesting that under our experimental conditions, UP-states are mostly generated within the local cortical network. To verify that UP-states are network driven events, we recorded UP-states simultaneously extracellularly and from one neuron in whole-cell configuration. Patch-clamp and field electrodes were placed approximately 200 µm apart.

Extracellularly recorded and whole-cell recorded UP-states occurred simultaneously in all recordings (n = 50 events from 5 recordings, Figure 1A), confirming that UP-states are network-driven events. To optimize the read-out of UP-state parameters, whole-cell recordings were performed with an E Cl -set at -87 mV. To verify the effect of the recorded neurons E Cl -on the event parameters, we recorded neurons (n = 5) with a modified pipette solution containing 8.5 mM Cl -. In those conditions, the Down state membrane potential measured -68.6 ± 1.7 mV but UP-state incidence was similar to those recorded in control conditions (0.88 ± 0.31 min -1 , n = 5, p = 0.64).

We next tested whether UP-state properties vary with the age of the animal. Recordings were separated into 3 age categories, but no significant differences were observed in UPstate incidence (P12-P13: 1.35 ± 0.23 min -1 , n = 14; P14-16, n = 58: 1.15 ± 0.10 min -1 ; P17-18: 0.69 ± 0.10 min -1 , n = 13; one-way ANOVA: F(2,82) = 2.957, p = 0.0575, Figure 1B), the duration of individual events (P12-13: 1. F(2,30.393) = 0.419, p = 0.66). However, to reduce variability in our results, throughout the study we performed recordings primarily on slices from animals aged P14-P16.

UP-state parameters may also depend on which layer the neurons are recorded from. We separated our sample into 3 groups: neurons from the supragranular layers (L2/3, n = 52), granular layer (L4, n = 6) and infragranular layers (L5, n = 34). Our results showed no significant difference in UP-state incidence between layers (L2/3: 1.11 ± 0.10 min -1 ; L4: 1.55 ± 0.42 min -1 ; L5: 1.02 ± 0.12 min -1 ; one-way ANOVA: F(2,89) = 1.281, p = 0.28; Figure 1C). However, UP-states were significantly shorter in L2/3 compared to L5 (L2/3: 0.80 ± 0.08 s; L4: 1.40 ± 0.46 s; L5: 1.48 ± 0.16s; one-way ANOVA: F(2,12.635) = 7.073, p = 0.0087; Figure 1C) consistent with the initiation and longer persistence of UP-states in layer 5 in ferret neocortical slices [START_REF] Sanchez-Vives | Cellular and network mechanisms of rhythmic recurrent activity in neocortex[END_REF]. In further agreement with this, DOWN-state membrane potential was more depolarized in L5 compared to L2/3 (L2/3: -68.0 ± 1.5 mV; L5: -63.0 ± 0.5 mV; Mann-Whitney: p = 1.8 . 10 -5 ), but action potential discharge during the UP-state was not significantly different between layers (L2/3: 2.58 ± 0.60 APs/UP-state; L4: 1.44 ± 0.40 APs/UP-state; L5: 2.39 ± 0.33 APs/UPstate; one-way ANOVA: F(2,25) = 2.040, p = 0.15). Taken together, these results suggest that the layer of recording did not influence the UP-state incidence but might affect the duration of UP states. Since we were primarily interested in the incidence of events, we collated results from each layer, but ensured that we recorded a similar number of neurons from infra-and supragranular layers to counteract any potential layer-dependent differences.

Induction of epileptiform bursts in spontaneously active slices

We asked whether pharmacological protocols used to facilitate the emission of an epileptiform burst in response to an electrical stimulation in slices are sufficient to induce the transition from spontaneous UP-states to spontaneous epileptiform bursts. We addressed this question in two classic models of epilepsy in slice: the reduced inhibition model, which entails blocking GABA A Rs with gabazine (GBZ), and the elevated excitation model, which involves reducing extracellular concentration of Mg 2+ . Increasing gabazine concentration above 200 nM resulted in spontaneous epileptiform activity. The frequency of epileptiform burst events was significantly more frequent than the frequency of spontaneous UP-state events in control condition (Control: 0.7 ± 0.1 min -1 , GBZ, 3 μM: 2.4 ± 0.3 min -1 , n = 20, p = 1.7 . 10 -4 ; Figure 2A). Even though the mean duration of the events was not significantly altered by the addition of gabazine (Control: 1.10 ± 0.24 s, GBZ: 0.93 ± 0.19 s, n = 20, p = 0.55), their morphology was changed. Blocking GABA A Rs induced an increase of AP discharge that clustered at the beginning of the burst (Control: 1.80 ± 0.35

APs/UP-state, GBZ: 15.64 ± 3.68 APs/burst, n = 20, p = 1.4 . 10 -4 ). During the events, the cell membrane potential was more depolarized in the presence of gabazine than in control condition (Control UP-state membrane potential: -56.3 ± 1.0 mV, GBZ burst membrane potential: -33.7 ± 1.7 mV, n = 20, p = 9.5 . 10 -5 , Figure 2A).

Similarly, slices immersed in low-magnesium aCSF (LowMg; nominally 0 Mg 2+ ), a condition known to induce epileptiform activity [START_REF] Walther | Epileptiform activity in combined slices of the hippocampus, subiculum and entorhinal cortex during perfusion with low magnesium medium[END_REF], robustly showed spontaneous epileptiform activity. The rate of epileptiform burst events was significantly higher than the rate of spontaneous UP-state events in control condition (Control: 0.8 ± 0.1 min -1 , LowMg: 7.5 ± 1.1, n = 15, p = 6.1 . 10 -5 ; Figure 2B). Similarly to gabazine-induced events, epileptiform events in the presence of low Mg 2+ (LowMg) were of similar duration to the spontaneous UP-states (Control: 1.53 ± 0.28 s, LowMg: 1.64 ± 0.22 s, n = 15, p = 0.64).

Other parameters were qualitatively similar to those observed upon application of gabazine, with an increase of action potential discharge during the events sustained by a higher level of depolarization of the cell compared to spontaneous UP-states (Control UPstate membrane potential: -56.7 ± 1.1 mV and 2.5 ± 0.6 APs/event; LowMg burst membrane potentials: -41.1 ± 1.5 mV and 12.2 ± 2.5 APs/event, n = 15; p = 7.2 . 10 -4 ; Figure 2B).

These results show that in spontaneously active slices, the application of gabazine or the lowering of Mg 2+ concentration is sufficient to induce a transition from spontaneous UPstates to pathological epileptiform bursts.

GABA B R antagonists prolong spontaneous epileptiform bursts in a dosedependent manner

GABA B R antagonists have been shown to prolong UP-state duration in the entorhinal cortex [START_REF] Mann | Distinct roles of GABA A and GABA B receptors in balancing and terminating persistent cortical activity[END_REF] and increase the frequency of epileptiform bursts in the hippocampus [START_REF] Lang | Regulating hippocampal hyperexcitability through GABA B Receptors[END_REF]. We thus aimed to characterize their influence on spontaneous epileptiform activity in the barrel cortex. As reported in the entorhinal cortex, the application of the GABA B R antagonist CGP55845 (CGP, 1µM) prolonged UP-state duration in the barrel cortex (Control: 0.89 ± 0.16 s, CGP: 1.66 ± 0.38 s, n = 9, p = 0.008; Figure 3A).

However, blocking GABA B Rs did not significantly affect UP-state incidence (Control: 0.9 ± 0.1 min -1 , CGP: 0.7 ± 0.1 min -1 , n = 9, p = 0.06), indicating an effect primarily on individual UP-states rather than on the quiescent period between the UP-states.

We then investigated how CGP affects spontaneous epileptiform activity. For each recording, spontaneous UP-states were first monitored to ensure that the slices were spontaneously active before applying gabazine. After 10 minutes of baseline recording in the presence of gabazine, different doses of CGP were applied and the parameters of the epileptiform bursts were quantified for the maximal dose of CGP (5 μM, Figure 3B). At this saturating concentration, CGP had no significant effect on the rate of burst frequency (GBZ: 2.8 ± 0.5 min -1 , GBZ+CGP5µM: 2.1 ± 0.5 min -1 , n = 6, p = 0.14), but prolonged the duration of individual bursts and promoted action potential discharge during the burst (GBZ: burst duration: 1.45 ± 0.36 s, AP frequency: 24.6 ± 5.0 APs/burst; GBZ+CGP5µM: burst duration: 3.68 ± 0.55 s, AP frequency: 62.7 ± 15.7 APs/burst; n = 6, p = 0.03 for both). We then determined how burst duration is modulated by different concentrations of CGP. The dose-response curve showed that already from 10 nM of CGP, burst duration was doubled compared to baseline gabazine-induced bursts (202% of GBZ condition, n = 5) and the increase in burst duration plateaued for 1 uM of CGP (345% of GBZ condition, n = 3). This underlines the importance of GABA B R activation in modulating individual epileptiform burst morphology.

Because the gabazine condition blocks the activation of synaptic GABA A Rs and thus leaves extrasynaptic GABA A and GABA B receptors as the only sources of local inhibition in the preparation, we also investigated how GABA B R blockade affects epileptiform activity with intact synaptic GABA A R function in the LowMg condition. Application of CGP (5 µM) increased burst frequency (LowMg: 9.3 ± 1.6 min -1 , LowMg+CGP5μM: 16.0 ± 2.9 min -1 , n = 10, p = 0.027), which was not observed in the gabazine condition. However, CGP had no significant influence on burst duration nor on action potential discharge frequency during bursts (LowMg: burst duration: 1.08 ± 0.11 s, AP frequency: 9.9 ± 2.5 APs/burst; LowMg+CGP5µM: burst duration: 1.11 ± 0.08 s, AP frequency: 7.8 ± 1.7 APs/burst; n = 10, p = 0.85 and p = 0.38, respectively). The apparent absence of effect of CGP on burst duration in low Mg 2+ condition might be explained by the higher incidence of bursts before the application of CGP, which limits the possible burst duration.

Altogether, these results suggest that GABA B Rs have two major effects on the epileptiform slice: in the reduced inhibition model (gabazine condition), GABA B R activation during the burst decreases the burst duration, while in the elevated excitation model (LowMg condition), GABA B R activation contributes to a refractory period that decreases the incidence of epileptiform bursts. Thus, GABA B R activation may affect the epileptic brain in two ways, by decreasing the frequency of seizures as well as their severity.

GABA B R agonists reduce the incidence of both UP-states and epileptiform bursts

To further test this hypothesis, we next applied the GABA B R agonist baclofen (1-10 μM).

We first investigated the effect of baclofen on spontaneously occurring UP-states. Neither the resting membrane potential (Control: -68.7 ± 1.3 mV, Baclofen: -67. 4A). However, baclofen reduced the UP-state incidence (Control: 0.17 ± 0.03 min -1 , Baclofen: 0.10 ± 0.02 min -1 ; n = 6; p = 0.04) indicating that the tonic activation of GABA B Rs reduces overall excitability. Furthermore, we observed a clustering of UPstates characterized by the occurrence of 2-3 UP-states in a short interval followed by a longer silent period. This clustering was never observed in baseline conditions and might reflect the tonic activation of GABA B Rs, as opposed to a phasic activation at the end of UP-states, occluding the refractory period between UP-states (Figure 4A).

We then tested how baclofen affects the spontaneous epileptiform activity. In the presence of baclofen, epileptiform burst incidence dropped to 41.0 ± 6.7 % of the baseline incidence in the gabazine model (GBZ: 2.84 ± 0.30 min -1 ; GBZ+Baclofen: 1.23 ± 0.35 min -1 ; n = 11; p = 0.002; Figure 4B) and to 22.8 ± 4.1 % of the baseline incidence in the low Mg 2+ model (LowMg: 8.61 ± 0.78 min -1 ; LowMg+Baclofen: 1.72 ± 0.24 min -1 ; n = 14; p = 0.001; Figure 4C). Interestingly, the baclofen-induced clustering of events observed for spontaneous UP-states was seen also in 11 out of 14 recordings in the presence of reduced extracellular Mg 2+ concentration (see examples in Figure 4B 1 -B 2 ) but not in the gabazine model. Individual burst duration and frequency of action potential discharge were not significantly affected by baclofen superfusion in the gabazine model (GBZ: burst duration: 0.45 ± 0.076 s, AP frequency: 8.1 ± 1.3 APs/event; GBZ+Baclofen: burst duration: 0.54 ± 0.09 s, AP frequency: 7.8 ± 1.1 APs/event; n = 11; p = 0.10 and 0.90, respectively; Figure 4B) but event duration was prolonged in the Low Mg 2+ model (LowMg: burst duration: 1.38 ± 0.21 s, AP frequency: 5.3 ± 1.1 APs/event; LowMg+Baclofen: burst duration: 2.09 ± 0.33 s, AP frequency: 6.1 ± 2.1 APs/event; n = 14; p = 0.03 and 0.72, respectively; Figure 4C).

Altogether, these results indicate that GABA B R agonists significantly decrease the event frequency of epileptiform bursts and also the event frequency of spontaneous UP-states in control conditions. Moreover, burst duration was significantly increased in the low Mg 2+ model and a trend towards an increased duration was observed for UP-states and bursts in the gabazine model, maybe due to the tonic activation of GABA B Rs by baclofen which might occlude phasic GABA B R activation during events. Baclofen also affects the regularity of the spontaneous physiological UP-states and clusters events in the low Mg 2+ model, indicating that, although the overall epileptiform burst frequency decreases, clusters of epileptiform events still occur and could have undesirable effects on the network properties.

Blocking GABA uptake weakens UP-states and strongly reduces epileptiform activity

Inhibitors of GABA transporters have anti-convulsive properties and tiagabine, a specific inhibitor of GABA transporter 1 (GAT1), is approved as an anti-epileptic drug (Schachter, 1999). Tiagabine is known to promote SWS [START_REF] Mathias | The GABA uptake inhibitor tiagabine promotes slow wave sleep in normal elderly subjects[END_REF], however, it is unclear whether this anti-convulsive drug affects spontaneous UP-states and hence how SWS patterns are affected. We investigated how GAT inhibitors affect spontaneous UP-states and epileptiform activity in the spontaneously active cortical slice preparation. Co-application of GAT1 inhibitor tiagabine (100 nM) and GAT2-3 inhibitor SNAP-5114 (50 nM) had no significant effect on the incidence of UP-states (Control: 0.69 ± 0.2 min -1 ; Tiagabine + SNAP: 0.86 ± 0.2 min -1 ; n = 10; p = 0.68; Figure 5A-B) and did not affect the DOWN-state membrane potential (Control: -67.8 ± 1.4 mV; Tiagabine + SNAP: -67.5 ± 1.8 mV; n = 10; p = 0.77), suggesting that the drugs do not prevent SWS-like activity in non-pathological conditions. However, individual events were weakened (Figure 5A): UP-states were shorter, the mean depolarization during UP-states was reduced and fewer spikes were emitted during each UP-state (Control: Vm during UP-states: -55.5 ± 1.7 mV, Up-state duration: 0.53 ± 0.06 s, AP frequency: 3.08 ± 0.82 APs/UP-state; Tiagabine + SNAP: Vm during UPstates: -58.0 ± 1.8 mV, UP-state duration: 0.43 ± 0.07 s, AP frequency: 1.92 ± 0.58 APs/UP-state; n = 10; p = 0.04 for the three parameters), indicating that during UP-states the excitability is reduced by the excess extracellular GABA.

While inhibiting GABA uptake has been well-established as efficient treatment for some types of epilepsies, whether the anti-convulsive effect is mediated by GABA A or GABA B receptor activation has not been investigated. Hence, we next investigated the simultane-ous inhibition of GAT1 and GAT2-3 on gabazine-and LowMg-induced epileptiform activity.

In both gabazine and LowMg conditions, application of GAT1 inhibitor tiagabine and GAT2-3 inhibitor SNAP-5114 induced a reduction of the event incidence (GBZ: 1.6 ± 0.3 min -1 ; GBZ+SNAP+Tiagabine: 0.9 ± 0.1 min -1 ; n = 8; p = 0.042; LowMg: 8.5 ± 1.3 min -1 ;

LowMg+SNAP+Tiagabine: 6.8 ± 1.5 min -1 ; n = 13; p = 0.036; Figure 5B-C). These results confirm the anti-convulsive effects of GAT inhibitors and validate our experimental model for investigation of possible pharmaceutical targets for epilepsy treatment. Furthermore, in gabazine-treated slices, application of SNAP and tiagabine reduced significantly the mean duration of the bursts (GBZ: 0.45 ± 0.4 s; GBZ+SNAP+Tiagabine: 0.38 ± 0.4 s; n = 8; p = 0.039; Figure 5B 1-2 ), which were terminated by a marked hyperpolarization (Figure 5B 3-4 ) most likely due to postsynaptic GABA B R or extra-synaptic GABA A R conductance as synaptic GABA A R conductance is blocked by gabazine (Bai et al., 2000). However, in LowMg condition, burst duration was not significantly reduced by SNAP + tiagabine (LowMg: 1.60 ± 0.23 s; LowMg+SNAP+Tiagabine: 1.35 ± 0.30 s; n = 13; p = 0.27), maybe because the burst duration is extended by the NMDAR conductances and less sensitive to GABA B R conductance. However, the spike frequency was significantly reduced (LowMg: 13.14 ± 2.20 APs/burst; LowMg+SNAP+Tiagabine: 6.92 ± 1.80 APs/burst; n = 13; p = 0.027), suggesting that GAT inhibitors could reduce the severity of the events without affecting their mean duration.

Blocking GABA uptake allows for a partial return of physiological UP-states in the low Mg 2+ model

The ultimate aim in anti-convulsive drugs development is to restore physiological activity.

We asked whether GABA B R agonists or GABA uptake blockers achieve at least partially this goal. We quantified the UP-state like events interleaved between epileptiform bursts in four recording conditions: gabazine + baclofen, gabazine + SNAP + tiagabine, low Mg 2+ + baclofen and low Mg 2+ + SNAP + tiagabine (Figure 6). UP-state-like events were discriminated from epileptiform bursts by their slow rising phases due to the summation of postsynaptic potentials and non-saturating action potential discharge (see examples in Figure 6B 1-C 1 ). We observed that during reduced inhibition (gabazine model), GABA B R agonist baclofen and GABA uptake blockers allow for the partial return of UP-state-like events in only very few recordings (2 out of 11 neurons for baclofen and 1 out of 8 neurons for SNAP + tiagabine, Figure 6A). Partial return of physiological activity was observed only in recordings showing also a reduction of epilepsy severity, defined by the reduced frequency of epileptiform bursts upon drug application (9 out of 11 neurons for baclofen and 8 out of 8 neurons for SNAP + tiagabine, Figure 6A). In the elevated excitation model (LowMg condition), epileptiform activity was reduced in 10 out of 13 recordings after application of GABA uptake blockers and in all recordings in the presence of baclofen (n = 14). Within these samples, a partial restoration of physiological activity was observed in 7 neurons in the presence of GABA uptake blockers and in 6 neurons after application of baclofen (Figure 6). In both conditions, although physiological activity was partially restored, we observed shorter UP-states than those recorded in control conditions (Ctrl: 1.32 ± 0.30 s;

LowMg+SNAP+Tiagabine: 0.37 ± 0.10 s; n = 7; p = 0.015; Ctrl: 2.13 ± 0.51 s;

LowMg+Baclofen: 0.76 ± 0.14 s; n = 6; p = 0.031) and a trend towards a reduction of the number of action potentials fired during the UP-states (Ctrl: 6.00 ± 3.74 APs/UP-state;

LowMg+SNAP+Tiagabine: 1.67 ± 0.31 APs/UP-state; n = 7; p = 0.29; Figure 6C 2 ; Ctrl:

3.8 ± 1.9 APs/UP-state; LowMg+Baclofen: 0.16 ± 0.16 APs/UP-state; n = 6; p = 0.28;

Figure 6B 2 ). These results show that the gabazine model might be too powerful to address the restoration of physiological activity. More importantly, these results demonstrate that both GABA B R agonist and GABA uptake blockers allow for a partial restoration of physiological UP-states in the low Mg 2+ model, but their characteristics are altered, reflecting the need to develop more subtle treatment to block epileptiform bursts whilst preserving physiological activity.

DISCUSSION

The present study compared GABA pharmacology of spontaneous epileptiform events and physiological UP-and DOWN-states in spontaneously active neocortical slices. We showed that spontaneous epileptiform events can be induced by reducing inhibition (gabazine) or increasing excitation (low Mg 2+ ) in a spontaneously active slice preparation, thus mimicking the status epilepticus. Our results demonstrate that tonic activation of GABA B Rs decreases the frequency of both UP-states and epileptiform bursts. Conversely, inhibition of GABA B Rs increases the duration of UP-states and of epileptiform events in the reduced inhibition epilepsy model, while it increases the incidence of epileptiform events in the elevated excitability epilepsy model. Furthermore, we show that blocking GABA uptake decreases the incidence of epileptiform bursts in both epilepsy models and also disrupts the morphology of spontaneous UP-states. Finally, we observe that GABA B R agonist and GABA uptake blockers allow for a partial restoration of physiological UP-state activity in the low Mg 2+ model.

Spontaneously active neocortical slices as a model to compare the effects of anti-convulsant drugs on physiological and pathological network events

Several approaches have been developed to induce epileptiform events in neocortical acute brain slices. Most commonly used are pharmacological strategies that result in biasing the excitation/inhibition balance toward excitation [START_REF] Walther | Epileptiform activity in combined slices of the hippocampus, subiculum and entorhinal cortex during perfusion with low magnesium medium[END_REF][START_REF] Pinto | Initiation, propagation, and termination of epileptiform activity in rodent neocortex in vitro involve distinct mechanisms[END_REF][START_REF] Trevelyan | Modular propagation of epileptiform activity: evidence for an inhibitory veto in neocortex[END_REF]. Alternatively, chemoconvulsants such as kainic acid, NMDA or pilocarpine can be injected in vivo, which will after a few days generate recurrent seizures in the animals. These can then be used for the preparation of acute slices to stimulate epileptiform bursts. However, in all these cases slices are silent and an electrical stimulation is required to induce a short train of epileptiform bursts, preventing the study of ini-tiation and spreading of spontaneous events and the restoration of physiological events.

Using electrical stimulation to induce epileptiform bursts shows analogy with seizures triggered by external stimuli, called reflex epilepsies that have a prevalence of 4-7 % (Panayiotopoulos [START_REF] Panayiotopoulos | Chapter 13: Reflex Seizures and Reflex Epilepsies, in: The Epilepsies: Seizures, Syndromes and Management[END_REF][START_REF] Pinto | Initiation, propagation, and termination of epileptiform activity in rodent neocortex in vitro involve distinct mechanisms[END_REF][START_REF] Trevelyan | Modular propagation of epileptiform activity: evidence for an inhibitory veto in neocortex[END_REF]. However, epilepsy is often associated with sleep [START_REF] Bazil | Effects of sleep and sleep stage on epileptic and nonepileptic seizures[END_REF][START_REF] Crespel | The relationship between sleep and epilepsy in frontal and temporal lobe epilepsies: practical and physiopathologic considerations[END_REF] and in particular with SWS, which is characterized in the neocortex by intrinsic slow oscillations and the relative absence of external sensory inputs. It is noteworthy that epilepsies associated with a neocortical focus, such as frontal lobe epilepsy, present a higher frequency of seizures during SWS than epilepsies with a focus located in the hippocampus or the amygdala [START_REF] Bazil | Effects of sleep and sleep stage on epileptic and nonepileptic seizures[END_REF][START_REF] Crespel | The relationship between sleep and epilepsy in frontal and temporal lobe epilepsies: practical and physiopathologic considerations[END_REF]. This suggests that there might be a causal link between neocortical slow oscillations and cortical epileptic seizures.

SWS is characterized by the alternation of two stable states, a quiescent state called DOWN-state, during which neurons are relatively silent and the synaptic activity weak, and an active state or UP-state, in which neurons fire action potentials and the synaptic activity is high. This bistable mode, especially the transitions between DOWN-states and UPstates, requires a fine-tuned balance of excitation and inhibition [START_REF] Sanchez-Vives | Cellular and network mechanisms of rhythmic recurrent activity in neocortex[END_REF][START_REF] Shu | Turning on and off recurrent balanced cortical activity[END_REF]. A certain level of excitability is required to generate and maintain active states, while a powerful inhibitory network is necessary to prevent excitation to build up too much [START_REF] Shu | Turning on and off recurrent balanced cortical activity[END_REF][START_REF] Haider | Neocortical network activity in vivo is generated through a dynamic balance of excitation and inhibition[END_REF]. This fine-tuned balance is fragile and, if the balance breaks down, epileptiform bursts may occur [START_REF] Epsztein | Ongoing epileptiform activity in the post-ischemic hippocampus is associated with a permanent shift of the excitatory-inhibitory synaptic balance in CA3 pyramidal neurons[END_REF][START_REF] Bragin | Patterns of the UP-Down state in normal and epileptic mice[END_REF][START_REF] Dehghani | Dynamic balance of excitation and inhibition in human and monkey neocortex[END_REF]. Indeed, a gradual inhibition of GABA A Rs progressively reduces UP-state duration, and increases AP discharge during UP-states, leading eventually to a complete transition from UP-state to epileptiform burst [START_REF] Mann | Distinct roles of GABA A and GABA B receptors in balancing and terminating persistent cortical activity[END_REF]. Similar associations between epilepsies and physiological rhythms occurring during sleep have been reported in other brain areas such as thalamic sleep spindles or hippocampal sleep ripples [START_REF] Beenhakker | Neurons that fire together also conspire together: is normal sleep circuitry hijacked to generate epilepsy?[END_REF][START_REF] Bragin | Local generation of fast ripples in epileptic brain[END_REF][START_REF] Huguenard | Intrathalamic rhythmicity studied in vitro: nominal Tcurrent modulation causes robust antioscillatory effects[END_REF]. Therefore, a better understanding of the nature of the relation between physiological sleep rhythms and spontaneous epileptiform bursts is needed.

Our slice model combines physiological recording conditions known to allow for spontaneous generation of UP-states [START_REF] Sanchez-Vives | Cellular and network mechanisms of rhythmic recurrent activity in neocortex[END_REF] and classic pharmacological conditions known to destabilise E/I balance and promote epileptiform activity [START_REF] Walther | Epileptiform activity in combined slices of the hippocampus, subiculum and entorhinal cortex during perfusion with low magnesium medium[END_REF][START_REF] Pinto | Initiation, propagation, and termination of epileptiform activity in rodent neocortex in vitro involve distinct mechanisms[END_REF][START_REF] Trevelyan | Modular propagation of epileptiform activity: evidence for an inhibitory veto in neocortex[END_REF]. Besides allowing us to record spontaneous epileptiform bursts, we were able to compare them with physiological UP-states thus evaluating physiological and pathological conditions in the same preparation. Moreover, importantly, our preparation allowed us to investigate whether anti-epileptic drugs permit the restoration of physiological rhythms. We observed only a partial restoration of physiological UP-states, which may be due to the fact that the GABA B R agonists and GABA uptake blockers interfere with the initiation, maintenance and termination of spontaneous UP-states in physiological contexts (as shown in Figures 3A, 4A and5A).

Nevertheless, this study addresses in slice the consideration of physiological activity in the evaluation of antiepileptic drugs. Therefore, we suggest that this model could provide an efficient pipeline to evaluate the ability of anti-convulsant drugs to both suppress pathological events and restore physiological activity.

GABA B Rs as targets to reduce epileptiform bursts

GABA neurotransmission is an obvious target for anti-epileptic treatment as the activation of this mechanism leads to an increase of the inhibitory tone that counterbalances paroxysmal excitation during epileptiform bursts. A first approach to modulate GABAergic pathways is to pharmacologically target the GABA receptors. GABA A R modulators, such as benzodiazepines, are a first line treatment in status epilepticus as they powerfully depress neuronal activity (Grover et al., 2015). However, they also strongly affect intrinsic neuronal rhythms, especially SWS oscillations, and impair learning and memory [START_REF] Roth | Benzodiazepines and memory[END_REF].

In our preparation, application of muscimol, a GABA A R agonist, abolished spontaneous UP-states (data not shown). Therefore GABA A R receptors are a pharmaceutical target for a rapid treatment of ictal episodes but may be less suitable for long-term anti-epileptic treatment.

GABA B R agonists mediate a slow inhibition through G protein-mediated activation of K + channels and thus can reduce network excitability. Here, we investigated GABA B R pharmacology in a neocortical slice model that mimics the epileptiform events observed during SWS, which often occur in frontal lobe epilepsy. GABA B R agonists have pro and antiepileptic effects depending on the syndrome studied but they have been mostly tested for their pro-epileptic properties on absence epilepsy models [START_REF] Hosford | The role of GABA B receptor activation in absence seizures of lethargic (lh/lh) mice[END_REF][START_REF] Han | GABA B receptor and absence epilepsy[END_REF][START_REF] Mccafferty | Cortical drive and thalamic feed-forward inhibition control thalamic output synchrony during absence seizures[END_REF] and temporal lobe epilepsy syndromes (Burgard and Sarvey, 1991). Therefore, how baclofen acts on frontal lobe epilepsy syndrome is poorly documented. We found that baclofen, a potent GABA B R agonist, reduces the frequency of cortical epileptiform bursts, which is consistent with reports on subcortical convulsive epileptic syndromes induced with focal GABA A R antagonist applications [START_REF] Wurpel | Baclofen inhibits amygdala kindling in immature rats[END_REF][START_REF] Chen | GABA-B receptor activation in the rat globus pallidus potently suppresses pentylenetetrazol-induced tonic seizures[END_REF]. In contrast, CGP55489, a potent GABA B R antagonist, caused an increase in epileptic burst duration in the reduced inhibition model and an increase of burst frequency in the enhanced excitation model. This is consistent with the increased excitability reported after GABA B R antagonist application in the epileptic hippocampus [START_REF] Lang | Regulating hippocampal hyperexcitability through GABA B Receptors[END_REF] and with its anticonvulsant effect in tonic-clonic and clonic epilepsy models (Velíšková et al., 1993). Thus GABA B Rs are spontaneously activated during epileptiform bursts in convulsive epilepsy models, limiting the extent of paroxysmal events, and therefore are a potentially good target for anticonvulsant drug development. Refinement of GABA B R pharmacology could involve targeting specific GABA B R isoforms. GABA B Rs are heterodimers composed of one GABA B1 subunit, which exists in two isoforms, and one GABA B2 subunit. GABA B Rs containing the GABA B1a isoform are located primarily presynaptically while GABA B1b -containing receptors are located primarily post-synaptically [START_REF] Vigot | Differential compartmentalization and distinct functions of GABA B receptor variants[END_REF][START_REF] Benke | Molecular organization, trafficking, and degradation of the GABA B receptor[END_REF]. Activation of postsynaptic GABA B Rs has a straightforward hyperpolarizing effect on its targets while activation of presynaptic GABA B Rs reduces the transmitter release probability at both glutamatergic and GABAergic synapses.

Moreover, most studies have investigated the effect of GABA B R pharmacology using systemic injections and without precise temporal control, thus more refined experiments could be carried out to test the effect of GABA B R pharmacology on frontal lobe epilepsy.

Recent optogenetic experiments showed that specific activation of GABAergic interneurons can powerfully terminate epileptiform bursts in the hippocampus [START_REF] Krook-Magnuson | On-demand optogenetic control of spontaneous seizures in temporal lobe epilepsy[END_REF][START_REF] Ledri | Global optogenetic activation of inhibitory interneurons during epileptiform activity[END_REF]). However, cortical interneurons are diverse, each subtype showing specific projections pattern and activating preferentially GABA A Rs or GABA B Rs (De Felipe et al., 2013). Parvalbumin-positive interneurons powerfully inhibit pyramidal neurons via GABA A Rs activation [START_REF] Kawaguchi | GABAergic cell subtypes and their synaptic connections in rat frontal cortex[END_REF]. Selective optogenetic activation of these interneurons during neocortical epileptiform bursts only terminates 30% of the events in vivo and can also induce epileptiform bursts when stimulated between events (Assaf and Schiller, 2016), suggesting limited promise of parvalbumin-positive interneurons as an anti-epileptic target. However, optogenetic stimulation of somatostatinpositive interneurons, which activate both GABA A and GABA B receptors, reliably reduces the frequency of epileptiform bursts [START_REF] Ledri | Global optogenetic activation of inhibitory interneurons during epileptiform activity[END_REF].

GABA uptake blockers in epilepsy treatment

GABAergic pathways can be pharmacologically manipulated upstream of GABA receptors by preventing the uptake of GABA from the extracellular space and thus prolong the activation of GABA receptors [START_REF] Frahm | Efficacy of background GABA uptake in rat hippocampal slices[END_REF][START_REF] Semyanov | GABA uptake regulates cortical excitability via cell type-specific tonic inhibition[END_REF]. This strategy has been successfully developed to treat certain forms of epilepsy and tiagabine is a commonly used anti-epileptic drug since the mid 1990s (Schachter, 1999). Blocking GABA uptake affects synaptic and extrasynaptic GABA receptors [START_REF] Semyanov | GABA uptake regulates cortical excitability via cell type-specific tonic inhibition[END_REF] but its mechanism of action on spontaneous epileptiform bursts is unknown. Both epilepsy models used in this study demonstrate the potent effect of GABA uptake blockers in decreasing the event frequency and event strength, which is consistent with previous reports [START_REF] Nielsen | Characterization of tiagabine (NO-328), a new potent and selective GABA uptake inhibitor[END_REF][START_REF] Lin | Effects of anti-epileptic drugs on spreading depolarization-induced epileptiform activity in mouse hippocampal slices[END_REF]. By using gabazine to reduce the inhibitory drive in the slice, we selectively blocked synaptic GABA A Rs [START_REF] Bai | Distinct functional and pharmacological properties of tonic and quantal inhibitory postsynaptic currents mediated by gamma-aminobutyric acid(A) receptors in hippocampal neurons[END_REF]. Therefore, the pronounced hyperpolarization following a burst and the reduction of burst frequency in the presence of GAT blockers is likely due to activation of extra-synaptic GABA A Rs and/or GABA B Rs. Extra-synaptic GABA A Rs are present on both pyramidal cells and inhibitory interneurons, but in the hippocampus only interneurons showed a tonic inhibitory current due to basal activation of extrasynaptic GABA A Rs [START_REF] Semyanov | GABA uptake regulates cortical excitability via cell type-specific tonic inhibition[END_REF]. Blocking GABA uptake allows for the expression of such tonic inhibition also of pyramidal neurons, thus reducing the excitability of pyramidal cells [START_REF] Semyanov | GABA uptake regulates cortical excitability via cell type-specific tonic inhibition[END_REF]. Although we did not show direct evidence for a similar mechanism in the neocortex, our results are consistent with such a mechanism.

We also tested the effect of GAT blockers in our preparation to assess the relevance of our spontaneously active slice model to study spontaneous epileptic bursts and to compare the effects between physiological and pathological network events. Tiagabine has been reported to promote deep sleep stages [START_REF] Mathias | The GABA uptake inhibitor tiagabine promotes slow wave sleep in normal elderly subjects[END_REF][START_REF] Walsh | Tiagabine enhances slow wave sleep and sleep maintenance in primary insomnia[END_REF].

Accordingly, we found no reduction of UP-state frequency, however the weakening of individual events indicates that if the overall structure of the SWS is preserved, the firing rate of individual neurons might be reduced during SWS in the presence of GAT inhibitors.

SWS is associated with memory consolidation and the time spent in SWS is positively correlated to memory consolidation while SWS deprivation prevents optimal memory acquisition and consolidation [START_REF] Fowler | Sleep and memory[END_REF][START_REF] Major | Persistent neural activity: prevalence and mechanisms[END_REF][START_REF] Compte | Computational and in vitro studies of persistent activity: edging towards cellular and synaptic mechanisms of working memory[END_REF]. Precise spike timing during UP-states is necessary to consolidate synaptic connections (González-could have adverse consequences for memory consolidation. Therefore we suggest that the reduction of AP discharge we observe might explain why tiagabine fails at enhancing memory consolidation despite prolonging the time spent in SWS [START_REF] Feld | Slow wave sleep induced by GABA agonist tiagabine fails to benefit memory consolidation[END_REF].

Conclusion

In conclusion, our results show that the GABAergic pathway is an interesting therapeutic target to treat sleep-associated epilepsy as GABA B R agonists and GABA uptake inhibitors both decrease the incidence of the epileptiform events and the strength of individual events while partially restoring physiological events. However, we also show that GABA B R agonists and GABA uptake blockers affect the characteristics of physiological UP-states in control condition and only allow for a partial restoration of altered UP-states in pathological contexts. Therefore, it is likely that cognitive performance would only be partially restored by these drugs and more investigation is needed to identify anti-epileptic targets that leave physiological processes less affected.

Figure 1 :

 1 Figure 1: Spontaneous UP-states in the barrel cortex.

Figure 2 :

 2 Figure 2: Induction of epileptiform activity in spontaneously active slices.

Figure 3 :

 3 Figure 3: GABA B R antagonist prolongs UP-states and epileptiform bursts.

Figure 4 :

 4 Figure 4: GABA B R agonist reduces the incidence of UP-states and epileptiform bursts.

Figure 5 :

 5 Figure 5: GABA uptake blockers differentially impact spontaneous UP-states and

Figure 6 :

 6 Figure 6: GABA uptake blockers partially restore physiological UP-state in the low Mg 2+

Acknowledgements

We would like to thank Drs Vincent Magloire and Susanna Mierau for their helpful inputs on the manuscript.

This work was supported by an Herchel Smith Fellowship for YAH. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.