Abstracts

p35 is a highly expressed protein in the brain and a specific activator of cyclin-dependent kinase 5 (Cdk5). The p35/Cdk5 complex, in turn, is part of a signaling mechanism that regulates neuronal migration during development. The p35 knockout mouse exhibits hippocampal aberrations in neuronal development and migration, as well as spontaneous seizure activity. To better understand the correlation between morphological abnormalities and seizures, we have begun to study dentate development in hippocampal organotypic cultures from p35 knockout (-/-) mice. We hypothesize that granule cells (GC) in the p35 -/- dentate gyrus (DG) in vitro will show migrational and morphological abnormalities similar to those observed in vivo. Organotypic slice cultures were prepared based on the method of Stoppini et al. (1991). Seven- to eight-day old p35 wildtype and knockout mouse pups were used to generate cultures. Under sterile conditions, 400-[mu]m transverse sections were cut from the hippocampus (with the attached entorhinal cortex). Slice cultures were maintained for up to 21 days in vitro (DIV); they were prepared for histological analyses (fixed in 4% paraformaldehyde) at three developmental timepoints - 7, 14, or 21 DIV. Cell staining (cresyl violet, biocytin) and immunocytochemistry (NeuN, GFAP, GABA, parvalbumin) were used to follow and characterize cell features. The dentate granule cells in slice cultures from wildtype mice developed organotypically, showing a normal morphological progression similar to GCs in vivo. Typical neuroanatomical features of the dentate included: a clear boundary between the GCs and the hilus and molecular layer; GC dendrites with regular apical orientation and branching, dendritic spines, GC axonal arborization within the hilus, and GABA and parvalbumin expression in DG interneurons. GC development in slice cultures from knockout mice recapitulated many of the abnormal characteristics described for this model in vivo, including: aberrant GC migration into the hilar and molecular regions; and presence of abnormal dendritic trees (e.g., basal dendrites, misoriented branches). The present study demonstrates the preservation of typical GC development in organotypic hippocampal slice cultures. Moreover, dentate granule cells in cultures from p35 mutant mice exhibit abnormal migration patterns and histopathological features comparable to those observed in the in vivo p35 -/- preparation. These baseline data will allow us to use in vitro methods to elucidate the regulation of granule cell migration and development as they relate to developing epileptogenicity. (Supported by U.C. Davis Health Sciences Research Award (HJW))