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Green Fluorescent Protein in the Study of Neuronal Signaling Pathways

In recent years, techniques have been established for transiently co‐transfecting cells with cDNA of the jellyfish green fluoresent protein (GFP), a reporter gene that encodes a non‐toxic marker. This approach can be applied to primary neurons where it has become especially useful for the study of neuronal second messenger pathways. This unit describes procedures for transfecting neurons in primary culture: transfection with GFP DNA, including co‐transfecting with separate GFP and gene‐of‐interest constructs, transfecting with a single construct containing the gene of interest fused to a GFP gene, and transfecting with a single construct containing separate gene‐of‐interest and GFP cassettes. Also included is a method for the rapid, large‐scale preparation of a nearly homogeneous population of neurons from rat cerebellum. The Commentary provides several examples of how this approach can be applied to specific biological questions on neuronal signaling pathways.