Objective: Dysregulation of γ-aminobutyric acidergic (GABAergic) transmission has been reported in lesional acquired epilepsies (gliomas, hippocampal sclerosis). We investigated its involvement in a developmental disorder, human focal cortical dysplasia (FCD), focusing on chloride regulation driving GABAergic signals. Methods: In vitro recordings of 47 human cortical acute slices from 11 pediatric patients who received operations for FCD were performed on multielectrode arrays. GABAergic receptors and chloride regulators were pharmacologically modulated. Immunostaining for chloride cotransporter KCC2 and interneurons were performed on recorded slices to correlate electrophysiology and expression patterns. Results: FCD slices retain intrinsic epileptogenicity. Thirty-six of 47 slices displayed spontaneous interictal discharges, along with a pattern specific to the histological subtypes. Ictal discharges were induced in proepileptic conditions in 6 of 8 slices in the areas generating spontaneous interictal discharges, with a transition to seizure involving the emergence of preictal discharges. Interictal discharges were sustained by GABAergic signaling, as a GABA A receptor blocker stopped them in 2 of 3 slices. Blockade of NKCC1 Cl − cotransporters further controlled interictal discharges in 9 of 12 cases, revealing a Cl − dysregulation affecting actions of GABA. Immunohistochemistry highlighted decreased expression and changes in KCC2 subcellular localization and a decrease in the number of GAD67-positive interneurons in regions generating interictal discharges. Interpretation: Altered chloride cotransporter expression and changes in interneuron density in FCD may lead to paradoxical depolarization of pyramidal cells. Spontaneous interictal discharges are consequently mediated by GABAergic signals, and targeting chloride regulation in neurons may be considered for the development of new antiepileptic drugs. ANN NEUROL 2019;85:204-217 M alformations of cortical development are an emblematic etiology of drug-resistant lesional epilepsy in children. Since the pioneer description of focal cortical dysplasia (FCD) by Taylor et al, 1 the classification has evolved into 3 main types of FCD according to the pattern of dyslamination, the presence of abnormal cells (cytomegalic neurons and balloon cells), and the association with another brain lesion. 2,3 This histological classification is crucial to categorize the various features of FCDs, but it does not describe the mechanisms of the View this article online at wileyonlinelibrary.com.

The solute carrier family 25 (SLC25) drives the import of a large diversity of metabolites into mitochondria, a key cellular structure involved in many metabolic functions. Mutations of the mitochondrial glutamate carrier SLC25A22 (also named GC1) have been identified in early epileptic encephalopathy (EEE) and migrating partial seizures in infancy (MPSI) but the pathophysiological mechanism of GC1 deficiency is still unknown, hampered by the absence of an in vivo model. This carrier is mainly expressed in astrocytes and is the principal gate for glutamate entry into mitochondria. A sufficient supply of energy is essential for the proper function of the brain and mitochondria have a pivotal role in maintaining energy homeostasis. In this work, we wanted to study the consequences of GC1 absence in an in vitro model in order to understand if glutamate catabolism and/or mitochondrial function could be affected. First, short hairpin RNA (shRNA) designed to specifically silence GC1 were validated in rat C6 glioma cells. Silencing GC1 in C6 resulted in a reduction of the GC1 mRNA combined with a decrease of the mitochondrial glutamate carrier activity. Then, primary astrocyte cultures were prepared and transfected with shRNA-GC1 or mismatch-RNA (mmRNA) constructs using the Neon® Transfection System in order to target a high number of primary astrocytes, more than 64%. Silencing GC1 in primary astrocytes resulted in a reduced nicotinamide adenine dinucleotide (Phosphate) (NAD(P)H) formation upon glutamate stimulation. We also observed that the mitochondrial respiratory chain (MRC) was functional after glucose stimulation but not activated by glutamate, resulting in a lower level of cellular adenosine triphosphate (ATP) in silenced astrocytes compared to control cells. Moreover, GC1 inactivation resulted in an intracellular glutamate accumulation. Our results show that mitochondrial glutamate transport via GC1 is important in sustaining glutamate homeostasis in astrocytes.Main Points: The mitochondrial respiratory chain is functional in absence of GC1Lack of glutamate oxidation results in a lower global ATP levelLack of mitochondrial glutamate transport results in intracellular glutamate accumulation