Abstracts

Cortical malformations are a frequent cause of pediatric epilepsy and are best understood in terms of brain development. In rats, prenatal exposure to methyazoxymethanol (MAM) consistently yields offspring with cortical malformations mimicking those seen clinically. Here we designed studies to establish a timeline of the two hallmark abnormalities in the MAM brain: (i) widespread neocortical dysplasia and (ii) nodular heterotopia in hippocampus. Results of antibody staining in tissue sections obtained from MAM-exposed offspring and analysis of organotypic slice cultures are presented. [italic]MAM exposure: [/italic]Pregnant S-D rats were injected with 25 mg/kg (i.p.)methylazoxymethanol (MAM) on day 15 of gestation (E15). [italic]Immunohistochemistry: [/italic]Tissue sections (30 [mu]m-thick coronal slices) were cut on a cryostat, and sections were treated with an various antibodies. Staining was visualized using the Vectastain ABC kit. [italic]Slice Culture: [/italic]E16 embryos and P0 rat brains were embedded in 5% low melting point agarose and cut into 250 [mu]m-thick sections. Slices were cultured on serum free Neurobasal media. Slices were fixed in 4% PFA, cryoprotected, and stained with the DNA dye, TO-PRO 3. In the MAM model, abnormal cortical development is evident as early as E17 and increases with time. Invasion of the hippocampal heterotopia begins with the postnatal erosion of ventricular zone and subsequent interruption of CA1 region. Cortical markers, such as Lis1, show that there is severe disorganization soon after MAM exposure and complete disruption of the cortical plate. Nestin staining, a radial glial marker, shows the breakdown of the radial glia scaffold in neocortex; the hippocampal scaffold remains intact. We also observed an expansion of neocortex marginal zone with an increase in reelin-positive cells; this change is first evident at E18. There is no abnormality seen with the reelin-positive cells in the hippocampus. Immunostaining for calretinin and reelin identifies the departing cells of Layer I as Cajal-Retzius cells. P75 expression shows that the subplate is disorganized and has lost its integrity. Interneuronal migration is also disturbed in the MAM brain; the expression pattern for GABA, an interneuronal marker, shows their migratory path is distorted. This study uses a well-established rat model of cortical malformation (prenatal MAM exposure) and shows that the emergence of a hippocampal heterotopia is a postnatal event, with the cluster first appearing at P2. Dramatic changes in the expression of molecular markers such as p75, nestin, and reelin demonstrate that the abnormal developmental changes in the MAM-treated rat begin prenatally. Knowledge of how the abnormal brain is formed will provide insight into the cause(s) of epileptogenesis and can contribute to advances in therapies for patients with cortical malformations. (Supported by EFA Predoctoral Fellowship (MFP) and NIH NS40272 (SCB))