title: Imaging cytoplasmic cAMP in mouse brainstem neurons.
abstract: BACKGROUND
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: DFG-Center of Molecular Physiology of the Brain, Department of Neuro- and Sensory Physiology, Humboldtallee 23, Georg-August-University, 37073 Göttingen, Germany. smirono@gwdg.de
authors.forename: S L
authors.initials: SL
authors.lastname: Mironov
authors.forename: E
authors.initials: E
authors.lastname: Skorova
authors.forename: G
authors.initials: G
authors.lastname: Taschenberger
authors.forename: N
authors.initials: N
authors.lastname: Hartelt
authors.forename: V O
authors.initials: VO
authors.lastname: Nikolaev
authors.forename: M J
authors.initials: MJ
authors.lastname: Lohse
authors.forename: S
authors.initials: S
authors.lastname: Kügler