title: Neuronal chloride accumulation and excitatory GABA underlie aggravation of neonatal epileptiform activities by phenobarbital.
abstract: Phenobarbital produces its anti-epileptic actions by increasing the inhibitory drive of γ-aminobutyric acid. However, following recurrent seizures, γ-aminobutyric acid excites neurons because of a persistent increase of chloride raising the important issue of whether phenobarbital could aggravate persistent seizures. Here we compared the actions of phenobarbital on initial and established ictal-like events in an in vitro model of mirror focus. Using the in vitro three-compartment chamber preparation with the two hippocampi and their commissural fibres placed in three different chambers, kainate was applied to one hippocampus and phenobarbital contralaterally, either after one ictal-like event or after many recurrent ictal-like events that produce an epileptogenic mirror focus. Field, perforated patch and single-channel recordings were used to determine the effects of γ-aminobutyric acid and their modulation by phenobarbital, and alterations of the chloride cotransporters were investigated using sodium-potassium-chloride cotransporter 1 and potassium chloride cotransporter 2 antagonists, potassium chloride cotransporter 2 immunocytochemistry and sodium-potassium-chloride cotransporter 1 knockouts. Phenobarbital reduced initial ictal-like events and prevented the formation of a mirror focus when applied from the start. In contrast, phenobarbital aggravated epileptiform activities when applied after many ictal-like events by enhancing the excitatory actions of γ-aminobutyric acid due to increased chloride. The accumulation of chloride and the excitatory actions of γ-aminobutyric acid in mirror foci neurons are mediated by the sodium-potassium-chloride cotransporter 1 chloride importer and by downregulation and internalization of the chloride-exporter potassium-chloride cotransporter 2. Finally, concomitant applications of the sodium-potassium-chloride cotransporter 1 antagonist bumetanide and phenobarbital decreased excitatory actions of γ-aminobutyric acid and prevented its paradoxical actions on mirror focus. Therefore, the history of seizures prior to phenobarbital applications determines its effects and rapid treatment of severe potentially epileptogenic-neonatal seizures is recommended to prevent secondary epileptogenesis associated with potassium chloride cotransporter 2 downregulation and acquisition of the excitatory γ-aminobutyric acid phenotype.
authors: INSERM U-901, Marseille, 13009, France.
authors: Romain
authors: R
authors: Nardou
authors: Sumii
authors: S
authors: Yamamoto
authors: Geneviève
authors: G
authors: Chazal
authors: Asma
authors: A
authors: Bhar
authors: Nadine
authors: N
authors: Ferrand
authors: Olivier
authors: O
authors: Dulac
authors: Yehezkel
authors: Y
authors: Ben-Ari
authors: Ilgam
authors: I
authors: Khalilov
mesh_terms: D000200:Action Potentials / Q000187:drug effects / Q000502:physiology; D000818:Animals; D000831:Animals, Newborn; D004594:Electrophysiology; D006624:Hippocampus / Q000187:drug effects / Q000502:physiology*; D007150:Immunohistochemistry; D051379:Mice; D018345:Mice, Knockout; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D010634:Phenobarbital / Q000494:pharmacology*; D051381:Rats; D017208:Rats, Wistar; D028021:Sodium-Potassium-Chloride Symporters / Q000235:genetics / Q000378:metabolism; D064506:Solute Carrier Family 12, Member 2; D027981:Symporters / Q000235:genetics / Q000378:metabolism; D005680:gamma-Aminobutyric Acid / Q000378:metabolism*; D000096922:K Cl- Cotransporters
chemical_list: D010634:Phenobarbital; D028021:Sodium-Potassium-Chloride Symporters; D064506:Solute Carrier Family 12, Member 2; D027981:Symporters; D005680:gamma-Aminobutyric Acid; D000096922:K Cl- Cotransporters; C578878:Slc12a2 protein, mouse; C578879:Slc12a2 protein, rat