Abstracts

RATIONALE: Cortical dysplasia refers to a spectrum of developmental disorders that are highly associated with epilepsy. Although 20% of epilepsy patients appear to have some form of dysplasia, the relationship of specific malformations to epileptogenesis remains unclear. By studying animal models of cortical dysplasia, we hope to elucidate these relationships. The p35 knockout mouse exhibits spontaneous seizures, and displays both neocortical and hippocampal dysplasia. The dispersed granule cell pattern is particularly intriguing since it closely resembles a clinical picture seen in temporal lobe epilepsy. The aim of the current research is to characterize the heterotopically organized dentate during development. By elucidating developmental changes, we hope to gain a better understanding of the relationship between structural abnormalities and seizure generation.
METHODS: Animals were deeply anesthetized with Nembutal and transcardially perfused with paraformaldehyde. Brains were extracted, post-fixed, and cryoprotected. Forty mm sections were cut on a vibratome. Histology using cresyl violet, and immunocytochemistry using an antibody against parvalbumin were used to characterize dispersed granule cells (GCs) and interneurons in the dentate gyrus of p35 knockout (KO) and age-matched wildtype (WT) mice at postnatal ages P3, P7, P14 and P30 (n=2 from each group).
RESULTS: Cresyl violet analysis indicates ongoing proliferation and migration of dentate GCs in both KO and WT mice at P3. At P7, in both WT and KO, the granule cell layer (GCL) and molecular layers (MLs) are differentiated, but the border between the superior blade of the GCL and the ML is indistinct. The P14 WT dentate shows an adult-like pattern in which the GCL, ML and hilus (H) are fully developed; the ML and H are devoid of displaced GCs. P14 KOs show similar development, but the superior blade of GCL continues to lack definition; GCs blend into both the ML and the H. At P30, KOs exhibit clear dispersion of GCs into the ML and H. immunocytochemistry first reveals parvalbumin-positive (PV+) cells, along the inner border of the GCL and in the H, in P7 WT animals; a PV+ axonal plexus is also evident in the superficial layer of GCs. KO animals show no immunoreactivity in the hippocampus at P7. In WT mice, the PV+ pattern intensifies at P14 and P30, with additional PV+ cells appearing in the inner and outer MLs. In the KO, PV+ cells and axonal plexus are first apparent at P14, with cells appearing randomly through H, GCL, and MLs. The PV+ axonal plexus is less pronounced at P30 in the KO than in the WT.
CONCLUSIONS: Granule cell dispersion in the p35 knockout dentate gyrus is first recognized at P14; before that time, the GCL - even in WT animals - had not yet coalesced. In addition, there is a delayed appearance of PV+ cells in p35 KO mice, suggesting a slowed (and incomplete) development of inhibitory circuitry. These developmental changes in p35 KO animals may correlate with the development of spontaneous seizures in this model of cortical dysplasia.
[Supported by: NIH NS18895]