Imaging cytoplasmic cAMP in mouse brainstem neurons

Background: cAMP is an ubiquitous second messenger mediating various neuronal functions, often as a consequence of increased intracellular Ca(2+) levels. While imaging of calcium is commonly used in neuroscience applications, probing for cAMP levels has not yet been performed in living vertebrate neuronal tissue before. Results: Using a strictly neuron-restricted promoter we virally transduced neurons in the organotypic brainstem slices which contained pre-Botzinger complex, constituting the rhythm-generating part of the respiratory network. Fluorescent cAMP sensor Epac1-camps was expressed both in neuronal cell bodies and neurites, allowing us to measure intracellular distribution of cAMP, its absolute levels and time-dependent changes in response to physiological stimuli. We recorded [cAMP](i) changes in the micromolar range after modulation of adenylate cyclase, inhibition of phosphodiesterase and activation of G-protein-coupled metabotropic receptors. [cAMP](i) levels increased after membrane depolarisation and release of Ca(2+) from internal stores. The effects developed slowly and reached their maximum after transient [Ca(2+)](i) elevations subsided. Ca(2+)-dependent [cAMP](i) transients were suppressed after blockade of adenylate cyclase with 0.1 mM adenylate cyclase inhibitor 2'5'-dideoxyadenosine and potentiated after inhibiting phosphodiesterase with isobutylmethylxanthine and rolipram. During paired stimulations, the second depolarisation and Ca(2+) release evoked bigger cAMP responses. These effects were abolished after inhibition of protein kinase A with H-89 pointing to the important role of phosphorylation of calcium channels in the potentiation of [cAMP](i) transients. Conclusion: We constructed and characterized a neuron-specific cAMP probe based on Epac1-camps. Using viral gene transfer we showed its efficient expression in organotypic brainstem preparations. Strong fluorescence, resistance to photobleaching and possibility of direct estimation of [cAMP] levels using dual wavelength measurements make the probe useful in studies of neurons and the mechanisms of their plasticity. Epac1-camps was applied to examine the crosstalk between Ca(2+) and cAMP signalling and revealed a synergism of actions of these two second messengers.