Abstracts

Rationale: Mutations in the Aristaless-related homeobox protein, ARX, have been linked to a number epilepsy and mental retardation (MR) phenotypes in humans. ARX is known to function in early cortical development with an established role in neuronal migration. How mutations in ARX result in epilepsy and MR is unknown. Unfortunately deletion of Arx is lethal in mice by postnatal (P) day 2, precluding examination of any developmental or epilepsy phenotypes. To test the role of ARX in epilepsy and MR, we generated a conditional mutant mouse line, Arx^flox and crossed this line to several Cre driver lines in an effort to model and further study the pathogenesis of epilepsy and MR related to ARX mutations.Methods: Arx^flox mice crossed with Dlx5/6^cre-IRES-GFP generated Arx mutant male mice, Arx^+/- female mice, males and females carrying the Arx^flox allele, and Dlx5/6^cre-IRES-GFP mice of both gender. All of these mice survived and video-EEG recordings performed at two time points, P12-P14 and between 3-12 months. Digital EEG recorded at 200Hz was first visually reviewed to monitor for the presence of seizures. Mean and instantaneous RMS energy and power spectrums were computed to measure background differences amongst the genotypes. After recordings, the brains were prepared for histological and immunohistochemical analysis. Cell counts of interneuron subtypes and region and layer areas were measured. Groups were compared using ANOVA and students t-tests.Results: All genotypes were successfully generated, but Arx^-/y mice were present at a reduce frequency. At P12-14, the equivalent of early childhood in humans, seizures were present in 50% (2/4) of the Arx^+/- female mice (Arx^-/y males not yet recorded) but none of the control genotypes. By adult ages, 44.4% (4/9) of Arx^+/- female mice developed seizures as did the single surviving Arx^-/y male. Quantitative analysis of background EEG characteristics revealed no clear differences in RMS energy between groups for cortex or hippocampal signals. Power spectral EEG differences existed between hippocampal signals, with the Arx^+/- female mice having an increase in faster frequency activity compared to all control genotypes. There were no significant differences between control genotypes background EEG characteristics. The immunohistological analysis revealed that brain weights and gross anatomy appeared identical between all genotypes in the adults, as did all histological parameters. However, the calretinin interneurons appeared misplaced in the adult Arx^+/- females without changes in the absolute number this subtype.Conclusions: In conclusion, targeted deletion of Arx in interneurons during development appears to result in subtle alteration in interneuron location and function resulting in measurable electrophysiological alterations that lead to epilepsy beginning in the post-natal period and extending into mature animals. Further time course, anatomic and histologic studies of these mice should shed light on the underlying pathophysiology of epilepsy in patients with ARX mutations.