Actin dynamics play an important role in the developmental outgrowth of neurites, the formation of synapses, and the modulation of synaptic activity at mature synapses. In addition, actin may play a significant role in neurodegenerative diseases, since actin depolymerization can protect against excitotoxicity and the sprouting of mossy fibers induced by epilepsy may be due to the re-engagement of developmental growth mechanisms. Actin-depolymerizing factor (ADF) is an important regulator of actin dynamics whose activity can be regulated by phosphorylation. ADF is localized to synapses and growth cones in neurons. ADF over-expression increases actin turnover and neurite outgrowth. The proposed experiments will test the hypothesis that the activity of ADF is modified by seizure activity, and changes in ADF activity in turn influence synaptic properties. The distribution of ADF and phosphorylated forms of ADF and cofilin in rat brain will first be determined by immunohistochemistry. Then changes in phosphorylation state and localization after kainate-induce seizures will be investigated using western blot and immunohistochemical analyses. To determine how changes in ADF activity affect synaptic properties a cell culture model of spontaneous seizure activity will be used. It will first be determined if seizure-induced changes in ADF phosphorylation and translocation in the cell culture model are similar to those observed in vivo. ADF activity will be altered in the cell culture system by recombinant adenovirus-mediated expression of mutant forms of ADF and LIM kinase, the ADF kinase. Effects of altered ADF activity on synaptic function will then be assessed by effects on F-actin distribution and turnover, calcium signaling and excitotoxicity.
|Meberg, Peter J; Miller, Matthew W (2003) Culturing hippocampal and cortical neurons. Methods Cell Biol 71:111-27|