Abstracts

Rationale: Around 30-50% of neonatal seizures are resistant to currently available treatments, such as phenobarbital. In addition to poor efficacy, phenobarbital also induces neuronal apoptosis independent of seizures in mouse models. Neonatal seizures become more challenging to handle when there is delayed treatment, but detection is not always immediate, especially in medically underserved areas. Traditional GABAergic antiseizure medications target synaptic GABA_A receptors, which mediate around 25% of the total inhibitory tone of a given neuron (phasic inhibition). In comparison, extrasynaptic GABA_ARs mediate 75% of the inhibitory current (tonic inhibition). Our prior work demonstrated that enhancing tonic inhibition significantly reduces seizure-like activity in acute neonatal brain slices. However, it is unknown how prolonged seizure-like activity alters tonic conductances in neonatal mice, if enhancing tonic inhibition effectively reduces prolonged seizure-like activity, and if it alters neuronal death.

Methods: Experiments were performed using C57BL/6J neonatal acute brain slices (P5-P9). We induced seizure-like activity in the neocortex and hippocampus of acute brain slices using the Low-Mg²⁺ model. Whole-cell patch recordings of isolated GABAergic tonic currents were measured before and after 8 hours of Low-Mg²⁺ incubation in the neocortex L4/5 pyramidal neurons. Additionally, the magnitude of seizure-like activity was recorded before and after enhancing tonic inhibition with 4,5,6,7-tetrahydroisoxazolo(5,4-c)pyridin-3-ol (THIP) at different time points up to 8 hours using field potentials. Brain slice immunofluorescent labeling of cleaved-caspase 3 (CC3) and Fluoro-jade C (FJC) was used to assess the effect of enhancing tonic inhibition on neuronal apoptosis and degeneration, respectively.

Results: Tonic conductances in neocortical layer IV/V significantly increased after 8 hours of prolonged neonatal seizure-like activity. Enhancing tonic inhibition in the neocortex and the CA1 pyramidal layer, which experienced protracted seizure-like activity, showed increased treatment efficacy over time. Immunofluorescent labeling showed that enhancing tonic inhibition did not increase neuronal neurodegeneration or apoptosis above controls.

Conclusions: Enhancing tonic inhibition may serve as a viable treatment alternative for neonates experiencing prolonged, pharmacoresistant seizures.

Funding: NIH/NINDS R01NS115800, Iowa Neuroscience Institute.