Studying neuronal peptide release and secretory granule dynamics with green fluorescent protein

Neurons and endocrine cells release peptides stored in a limited pool of secretory granules. Although it has been possible to measure secretion or exocytosis, studying events within cells that influence the size and speed of secretory responses has been difficult. Here we describe how green fluorescent protein (GFP)-tagged hormones can be used to measure release and granule mobility. Epifluorescence allows one to measure total peptide content and granule trajectories within a plane of focus. Confocal microscopy facilitates measurements within a better defined optical section. Granule mobility can also be measured with fluorescence recovery after photobleaching (FRAP). Application of these optical approaches to the study of neuronal peptide secretion has revealed that the releasable pool is not spatially defined and that sustained peptide release is limited by the availability of mobile secretory granules.