Microbeam laser-injured neurons increase in vitro astrocytic gap junctional communication as measured by fluorescence recovery after laser photobleaching

Abstract

An important aspect of the neuronal-astrocyte relationship is the interaction of reactive astrocytes with injured and/or dying neurons. Few studies have focused on the signaling of astrocytes by injured neurons or on the possibility that neurons can alter astrocytic gap junctional communication. The purpose of this study was to determine whether the presence of injured neurons could alter astrocytic gap junctional coupling by establishing an in vitro method of microbeam laser neuronal injury and coculturing these neurons with astrocytes. Neurons from two rat neuronal clonal cell lines were injured using a 20-W argon laser operating on the ultraviolet (UV) multiline (351-361 nm) directed through a X40 objective of an inverted microscope. After laser injury, the glass slide with the injured neurons was sandwiched with a slide on which primary rat astrocytes were grown. Although the neurons and astrocytes were bathed in the same medium, they were not in direct contact during the coculture period (24, 48, or 72 hr). Astrocytic gap junctional dye coupling was examined using the fluorescence recovery after laser photobleaching (gap-FRAP) analysis technique. Astrocytes cocultured with the injured neurons for 24 to 48 hr did not show a significant difference in fluorescence recovery when compared to control values. After 72 hr of coculture, there was a significant increase in the gap junctional dye coupling. These results indicate that injured neurons influence in vitro astrocytic gap junctional conductance after 72 hr of coculture as measured by dye coupling.