Abstracts

Rationale: We have demonstrated that maturation of GABA-A receptor subunit expression patterns and pharmacologic properties occurs during normal postnatal development in human cortex, and that this maturation is absent from specimens resected from children with intractable epilepsy due to cortical dysplasia. In the present study, we investigated the hypothesis that GABA-A receptors in epileptogenic tissue may instead demonstrate dysmaturity , with acquisition of unique structural and pharmacologic properties.Methods: The membrane fractions of surgically resected and autoptic cortical tissues were isolated and injected into Xenopus oocytes, resulting in incorporation of the brain membrane vesicles with their associated receptors into the oocyte cellular membrane, allowing two-electrode voltage clamp analysis of GABA-A receptor currents. Formalin-fixed, paraffin-embedded sections were used for fluorescence immunohistochemistry studies, which were performed utilizing standard techniques.Results: In control pediatric cortex, type I benzodiazepine responses were positively correlated with both age and GABA-A ?₁ subunit expression levels. Unexpectedly, in epileptogenic focal cortical dysplasia (FCD) type 2A specimens, a negative correlation was observed between these parameters. This finding suggested that in the older FCD specimens, ?₁ subunits were no longer predominantly associated with benzodiazepine-sensitive ?₁?_x?₂ receptors, and were instead associated with benzodiazepine-insensitive ?₁?_x? or ?₁?_x receptors. This also suggested that ?₂ subunits could be associated with alternative ? subunits, such as ?₄, which is expressed at high levels in FCD 2A specimens. Indeed, GABA-A currents from older FCD 2A tissues exhibited increased enhancement by the ? subunit-selective agonist DS2 and by the ?₄?_x?₂ agonist Ro15-4513, as well as increased inhibition by zinc, which has higher efficacy on receptors that do not contain a ?₂ subunit. In order to assess whether this change in pharmacology was associated with a change in GABA-A receptor subunit cellular distribution, we performed double-label fluorescence immunohistochemistry utilizing ?₁ or ? subunit antibodies along with antibodies against CAMKII? (pyramidal cell marker) or GAD 65/67 (interneuron marker). In control cortex, ?₁ immunofluorescence intensity was nearly equivalent in pyramidal cells, interneurons, and the cortical neuropil, while in FCD 2A specimens, ?₁ immunofluorescence was much more prominent in interneurons than elsewhere. In contrast, in both the control and FCD 2A samples, ? subunit immunofluorescence was equivalent in pyramidal cells and interneurons, but relatively low in the neuropil. These results provide structural evidence of altered distribution of the ?₁ subunit in epileptogenic FCD, and indicate that association with the ? subunit may occur in cortical interneurons.Conclusions: GABA-A receptor subunit redistribution in epileptogenic pediatric cortical dysplasia produces unique pharmacological properties that may serve as targets for novel anticonvulsant agents.