Email updates

Keep up to date with the latest news and content from BMC Neuroscience and BioMed Central.

Open Access Highly Accessed Research article

Imaging cytoplasmic cAMP in mouse brainstem neurons

SL Mironov1*, E Skorova1, G Taschenberger2, N Hartelt1, VO Nikolaev3, MJ Lohse3 and S Kügler2

Author Affiliations

1 DFG-Center of Molecular Physiology of the Brain, Department of Neuro- and Sensory Physiology, Humboldtallee 23, Georg-August-University, 37073 Göttingen, Germany

2 University Medical Center Göttingen, Department of Neurology, Waldweg 33, 37073 Göttingen, Germany

3 Institute of Pharmacology and Toxicology, University of Würzburg, Versbacher Str. 9, 97078 Würzburg, Germany

For all author emails, please log on.

BMC Neuroscience 2009, 10:29  doi:10.1186/1471-2202-10-29

Published: 27 March 2009



cAMP is an ubiquitous second messenger mediating various neuronal functions, often as a consequence of increased intracellular Ca2+ 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.


Using a strictly neuron-restricted promoter we virally transduced neurons in the organotypic brainstem slices which contained pre-Bötzinger 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 Ca2+ from internal stores. The effects developed slowly and reached their maximum after transient [Ca2+]i elevations subsided. Ca2+-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 Ca2+ 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.


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 Ca2+ and cAMP signalling and revealed a synergism of actions of these two second messengers.