The use of fluorescence resonance energy transfer (Fret) to measure axon growth and guidance-related intracellular signalling in live dorsal root ganglia neuronal growth cones

Abstract

The measurement of signalling by traditional methods in primary neuronal cultures is often limited by cell numbers within the culture and restricted division among these cells. Further limitations are seen with modern fluorescent imaging techniques on account of difficulties with transfection of these cell types. Here, we describe successful transfection of dorsal root ganglion (DRG) primary neuronal cultures with cDNA encoded fluorescence resonance energy transfer (FRET) probes for various signalling moieties, and subsequent measurement of FRET as an index of signalling within these cells. Furthermore, these measurements were made within live neuronal growth cones, which are thin, fragile, and dynamic structures central to axonal growth, repair, and regeneration. This provides novel, physiological insight into the signaling processes driving these axonal behaviors.