Green fluorescent cAMP indicator of high speed and specificity suitable for neuronal live-cell imaging.

ABSTRACT

Cyclic adenosine monophosphate (cAMP) is a canonical intracellular messenger playing diverse roles in cell functions. In neurons, cAMP promotes axonal growth during early development, and mediates sensory transduction and synaptic plasticity after maturation. The molecular cascades of cAMP are well documented, but its spatiotemporal profiles associated with neuronal functions remain hidden. Hence, we developed a genetically encoded cAMP indicator based on a bacterial cAMP-binding protein. This indicator "gCarvi" monitors [cAMP]i at 0.2 to 20 µM with a subsecond time resolution and a high specificity over cyclic guanosine monophosphate (cGMP). gCarvi can be converted to a ratiometric probe for [cAMP]i quantification and its expression can be specifically targeted to various subcellular compartments. Monomeric gCarvi also enables simultaneous multisignal monitoring in combination with other indicators. As a proof of concept, simultaneous cAMP/Ca2+ imaging in hippocampal neurons revealed a tight linkage of cAMP to Ca2+ signals. In cerebellar presynaptic boutons, forskolin induced nonuniform cAMP elevations among boutons, which positively correlated with subsequent increases in the size of the recycling pool of synaptic vesicles assayed using FM dye. Thus, the cAMP domain in presynaptic boutons is an important determinant of the synaptic strength.