State of actin filaments is changed by anoxia in cultured rat neocortical neurons

Abstract

Cultured necortical neurons respond rapidly to oxygen deprivation. Within minutes they demonstrate an increase in intracellular calcium and pronounced changes in their morphology. These changes include swelling, bleb formations, process retraction and a change in shape of the soma from pyramidal or ellipsoidal to round. Since the cytoskeleton is responsible for the maintenance of cell shape, we investigated the changes in state of a major component of the neuronal cytoskeleton, the actin filaments. Actin exists in a dynamic equilibrium between the monomeric and filamentous states. This equilibrium is dependent, in part, upon intracellular ATP, which is reduced during anoxia. We differentially labelled monomeric actin with Texas Red-tagged DNase-1 and the polymeric form with Bodipy–phallicidin. Using confocal microscopy and image reconstruction, we have found that the ratio of filamentous to monomeric actin increases on average three-fold following 10 min of anoxia. In addition, filamentous actin redistributes within the soma and appears to have a more homogeneous distribution than in normoxic neurons. Our results show that, in cultured neocortical neurons, actin filaments are modulated by anoxia. We believe that, although the filamentous/monomeric ratio is modulated, at least in part, by the levels of ATP and ADP, the relative distribution and amount of the filamentous to monomeric form within neurons are likely regulated by other factors such as calcium-sensitive actin-binding and severing proteins.