Abstracts

Neonatal seizures can be refractory to common AEDs such as phenobarbital and lorazepam, which are GABA receptor agonists. GABA inhibits neurons by gating influx of the anion chloride. Experimental data show show that GABA is excitatory in immature rodent brain. The excitation is due to neuronal accumulation of intracellular chloride, which is regulated by the chloride importer NKCC1 and exporter KCC2. In immature rodent neurons, KCC2 expression is at low levels and hence GABA[sub]A[/sub] receptor activation is excitatory due to net anion efflux. It is unknown whether GABA[sub]A[/sub] receptor activation is similarly excitatory in the human, and hence we examined the developmental expression by western blot and immunocytochemical double labeling in human cortex and compared these expression profiles to those in the adult. We hypothesized that KCC2 expression would be low in neonatal human cortex, an age characeterized by seizures refractory to GABA receptor agonists. The developmental regulation of NKCC1 and KCC2 in immature rat and human postmortem brain was examined by western blot. Cortical tissue was prepared from rats (P3-P60, n=14) and 12 normal human parietal lobe autopsy samples (20 postconceptional weeks (PCW) to 57 yrs). Double labeling of sections from 10 human cases was performed with neuronal and transporter antibodies. KCC2 (Upstate), NKCC1 (Chemicon), NeuN(Chemicon), SMI-11(Sternberger) were used for western blot and immunocytochemistry. In rat between P3-14, KCC2 was [lt]10% of adult (p[lt]0.001). NKCC1 transiently increased to 1200% of adult between P3-14 (p[lt]0.001). Human KCC2 expression was [lt]25% of adult between 20-54 PCW (p[lt]0.01), rising to adult levels over the first year. NKCC1 exceeded adult levels by approximately 200% between 31-54 PCW (p[lt]0.001), returning to adult levels by 1 year. NKCC1:KCC2 ratios were also higher during development that adulthood in both species (p[lt]0.002). Immunocytochemical labeling with KCC2 and NeuN/SMI-11 antibodies confirmed that neuronal expression of KCC2 was not abundant before 1 year of life. In both rat and human, there is a relative lack of KCC2 and high NKCC1:KCC2 ratios during development.in both immature rat and human brain, when seizure susceptibility is highest. Furthermore, NKCC1 expression transiently exceeds adult in the perinatal rat and human, which could further increase intracellular chloride concentrations. Low KCC2 and high NKCC1 expression in human neonatal cortex suggests the presence of depolarizing GABARs. Depolarizing GABARs may play a role in increased seizure susceptibility in the neonate and may contribute to the relative refractoriness of neonatal seizures to GABA agonist AEDs. (Supported by NIH NS 31718 (FEJ); Epilepsy Foundation (DMT).)