Abstracts

RATIONALE: Cortical dysplasia is a common cause of medically-intractable epilepsy. To elucidate the relationship between dysplasia and epilepsy, we have studied the p35 [dsquote]knockout[dsquote] (-/-) mouse. These animals develop severe defects in brain morphology as well as spontaneous seizures. The hippocampal dentate gyrus is particularly interesting in these animals, since it exhibits granule cell (GC) dispersion, mossy fiber (MF) sprouting, and GC basal dendrites [ndash] features typically seen in animal models and human patients with epileptic phenotypes. We hypothesize that these morphological features are part of a recurrent excitatory network that contributes to the epileptic state. To begin to test this hypothesis, we have examined the synaptic connections of dye-labeled GCs in p35 -/- hippocampus.
METHODS: Electron microscopic (EM) analyses were carried out on GCs that were electrophysiologically characterized and biocytin-labeled in hippocampal slices obtained from p35 (-/-) and wild-type mice (see accompanying poster by LS Patel et al.). Immunocytochemistry (ICC), EM, and 3D-reconstruction were carried out on sprouted MF axons in the granule cell and molecular layers, and on basal GC dendrites localized within the hilus. Pre-embedding ICC EM for zinc transporter (ZnT3) was used to identify MF boutons, and post-embedding ICC for glutamate and GABA were used to characterize the post-synaptic targets of sprouted MF axons.
RESULTS: The dentate gyrus of p35 (-/-) mice exhibits GC dispersion into the molecular layer and hilus. Biocytin-labeled GCs in this tissue exhibit abnormal axonal and dendritic arbors. Four recurrent MF axon collaterals localized within granule cell and molecular layers were examined at the EM level, and axon segments reconstructed. The MF axon collaterals form periodic small varicosities (less than 2 [mu]m in diameter), which synapse predominantly with dendritic spines (over 85% of synapses) and dendritic shafts, and occasionally with GC somata. Only a few varicosities form synapses with dendritic shafts of interneurons (as confirmed by their morphological features and/or GABA immunoreactitvity). Two basal dendrites of GCs were examined and 3D-reconstructed; they exhibit typical features of GC dendrites (e.g., complex spines), and form numerous axo-spinous and axo-dendritic synapses with axonal varicosities and with MF boutons.
CONCLUSIONS: Microscopic analyses of electrophysiological-identified GCs reveal a high degree of abnormality of GC morphology and synaptic connectivity, including sprouting of recurrent MF axon collaterals. EM reconstructions show that both abnormal axons and dendrites participate in excitatory synaptic interactions, and may thus contribute to a recurrent excitatory network in the dentate gyrus of p35 (-/-) mice.
[Supported by: NIH NS 18895 and GM 07108]