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.

Adenosine Release during Seizures Attenuates GABAAReceptor-Mediated Depolarization

“…3). In agreement with previous studies of chloride homeostasis, we found that the recovery of depolarizing GABA A R responses could be estimated using an exponential fit (Brumback and Staley, 2008;Nardou et al, 2011). Under control conditions we found that the recovery rate had a time constant of 34.7 Ϯ 2.8 s and that this was not affected by AR blockade with CGS 15943, when the mean time constant was 31.6 Ϯ 3.0 (p ϭ 0.22, n ϭ 8, paired t test).Together, these results show that changes in Cl Ϫ homeostasis mechanisms are unlikely to contribute to the attenuation of the GABA A R-mediated potentials.…”

Paradoxical Benzodiazepine Response: A Rationale for Bumetanide in Neurodevelopmental Disorders?

“…6 The present article provides the first case example in which a behavioral disinhibition to a benzodiazepine was noted and used as a rationale to initiate bumetanide treatment. Indeed, adverse effects to GABA-enforcing agents have been experimentally linked to disturbed chloride homeostasis, 18,19 and behavioral disinhibition after benzodiazepine treatment is a common phenomenon 2,3,20-24 also noted in patients with ASD. 4 Bumetanide led to a remarkable improvement in sensory behaviors, rigidity, memory, and learning in our patient, which suggested that the diuretic had improved neural functioning.…”

Cation-chloride cotransporters in neuronal development, plasticity and disease

“…NKCC1 is the principal transport mechanism responsible for Cl − uptake and for the depolarizing GABA A R responses of immature neurons 10,94,97 (TABLE 1). By contrast, in some other neuronal populations, such as the brainstem superior olivary complex, other — probably HCO 3 − -dependent — mechanisms of Cl − uptake are important 69 .…”