SUMMARY Purpose Expression of the protein subunits that make up the GABAA receptor pentamer is known to change during postnatal brain development in animal models. In the present study, analysis of cortical GABAA subunit expression was performed in control human tissue obtained from infancy through adolescence, and was compared to that from similarly aged children with intractable focal epilepsy. Methods Twenty frozen pediatric control and 25 epileptic neocortical specimens were collected. The membrane fractions were isolated and subjected to quantitative Western blot analysis. Subunit expression was correlated with clinical factors including age, pathology, and medication exposure. Results In control cortical samples, α1 and γ2 GABAA receptor subunits exhibited low expression in infancy which increased over the first several years of life and then stabilized through adolescence. In contrast, α4 subunit expression was higher in infants than in older children. The level of the chloride transporter KCC2 increased markedly with age, while that of NKCC1 decreased. These patterns were absent in the epileptic children, both in those with focal cortical dysplasia and in those with cortical gliosis. While there was marked variability in GABAA receptor subunit expression amongst the epileptic children, identifiable patterns of subunit expression were found in each individual child. Discussion Maturation of cortical GABAA receptor subunit expression continues over the first several years of postnatal human development. Intractable focal epilepsy in children is associated with disruption of this normal developmental pattern. These findings have significant implications for the treatment of children with medications that modulate GABAA receptor function.

The neurodevelopmental disorder Angelman syndrome is most frequently caused by deletion of the maternally-derived chromosome 15q11-q13 region, which includes not only the causative UBE3A gene, but also the β3-α5-γ3 GABAA receptor subunit gene cluster. GABAergic dysfunction has been hypothesized to contribute to the occurrence of epilepsy and cognitive and behavioral impairments in this condition. In the present study, analysis of GABAA receptor subunit expression and pharmacology was performed in cerebral cortex from four subjects with Angelman syndrome and compared to that from control tissue. The membrane fraction of frozen postmortem neocortical tissue was isolated and subjected to quantitative Western blot analysis. The ratios of β3/β2 and α5/α1 subunit protein expression in Angelman syndrome cortex were significantly decreased when compared with controls. An additional membrane fraction was injected into Xenopus oocytes, resulting in incorporation of the brain membrane vesicles with their associated receptors into the oocyte cellular membrane. Two-electrode voltage clamp analysis of GABAA receptor currents was then performed. Studies of GABAA receptor pharmacology in Angelman syndrome cortex revealed increased current enhancement by the α1-selective benzodiazepine site agonist zolpidem and by the barbiturate phenobarbital, while sensitivity to current inhibition by zinc was decreased. GABAA receptor affinity and modulation by neurosteroids were unchanged. This shift in GABAA receptor subunit expression and pharmacology in Angelman syndrome is consistent with impaired extrasynaptic but intact to augmented synaptic cortical GABAergic inhibition, which could contribute to the epileptic, behavioral, and cognitive phenotypes of the disorder.

Catastrophic epilepsy due to cortical dysplasia is often intractable to anticonvulsant treatment. Many of the medications used unsuccessfully in treating this disorder are thought to exert at least a portion of their action through enhancement of inhibitory GABA A neurotransmission. In the present study, GABA A receptor properties in resected brain tissue from four infants with infantile spasms and intractable epilepsy due to cortical dysplasia were measured to determine if this clinical resistance to pharmacologic treatment correlates with alterations in receptor function. Results from epileptic cortex were compared with those from autopsy control samples. To perform these studies, we utilized the technique of injection of brain cellular membrane preparations into the Xenopus oocyte, which results in the incorporation of human GABA A receptors in their native configuration into the oocyte plasma membrane. Two-electrode voltage clamp electrophysiology analysis was then performed to assess GABA A receptor pharmacologic properties. The intrinsic properties of affinity, reversal potential, current decay, and current rundown were unchanged in the epileptic infants. Current enhancement by benzodiazepines was also unaltered, as was the response to barbiturates. However, a significant decrease was found in the degree of GABA A current enhancement by neurosteroids in the epileptic infants, along with an increase in current inhibition by zinc. These findings may contribute to the mechanisms of intractability in catastrophic infantile epilepsy due to cortical dysplasia, and suggest alternative therapeutic approaches.