Abstracts

Rationale: Seizures may cause brain injury via a variety of mechanisms, potentially contributing to cognitive deficits in epilepsy patients. Although seizures induce neuronal death in some situations, they may also have “non-lethal” pathophysiological effects on neuronal structure and function, such as modifying dendritic morphology. Previous studies involving conventional fixed tissue analysis have demonstrated a chronic loss of dendritic spines following seizures in animal models and human tissue. More recently, in vivo time-lapse imaging methods have been used to monitor acute changes in spines directly during seizures, but documented spine loss only under severe conditions. Here, we examined effects of secondary generalized seizures induced by kainate, on dendritic structure of neocortical neurons utilizing multiphoton imaging in live mice in vivo and investigated molecular mechanisms mediating these structural changes.Methods: Multiphoton imaging was performed through a craniotomy window to visualize dendrites and associated spines of neocortical neurons in GFP-expressing transgenic mice before and after kainate-induced seizures of varying severity. The same dendrities were followed sequentially by time-lapse imaging, monitoring for overt morphological changes (beading) and changes in spine number over a several hour period. Phalloidin-rhodamine labeling and Western blotting were used to analyze changes in actin polymerization and actin-regulatory factors.Results: Higher stage kainate-induced seizures caused dramatic dendritic beading and loss of spines within minutes, in the absence of neuronal death or changes in systemic oxygenation. Although the dendritic beading improved rapidly following the seizures, the spine loss recovered only partially over a several hour period. Kainate seizures also resulted in activation of the actin-depolymerizing factor, cofilin, and a corresponding decrease in filamentous actin, indicating that depolymerization of actin may mediate the morphological dendritic changes. Finally, an inhibitor of the calcium-dependent phosphatase, calcineurin, antagonized the effects of seizures on cofilin activation and spine morphology.Conclusions: These dramatic in vivo findings demonstrate that seizures produce acute dendritic injury in neocortical neurons via calcineurin-dependent regulation of the actin cytoskeleton and suggest novel therapeutic targets for preventing seizure-induced brain injury.