Measurement of spontaneous neuronal membrane activity by time lapse confocal microscopy

Abstract

Cellular electrophysiological properties are normally monitored by standard patch clamp techniques . The combination of membrane potential dyes with time-lapse laser confocal microscopy provides a more direct, least destructive rapid method for monitoring changes in neuronal electrical activity. Using membrane potential dyes we found that spontaneous action potential firing can be detected using time-lapse confocal microscopy. Initially, patch clamp recording techniques were used to verify spontaneous electrical activity in GH4\C1 pituitary cells. It was found that serum depleted cells had reduced spontaneous electrical activity. Brief exposure to the serum derived growth factor, IGF-1, reconstituted electrical activity. We have examined the possibility of developing a rapid fluorescent assay to measure neuronal activity using membrane potential dyes. This neuronal regeneration assay has been adapted to run on a confocal microscope. Quantitative fluorescence is then used to measure a compounds ability to regenerate neuronal firing.The membrane potential dye di-8-ANEPPS was selected for these experiments. Di-8- ANEPPS is internalized slowly, has a high signal to noise ratio (40:1), has a linear fluorescent response to change in voltage.