Imaging cytoplasmic cAMP in mouse brainstem neurons
BMC Neurosci, (2009)Mironov, S L Skorova, E Taschenberger, G Hartelt, N Nikolaev, V O
Lohse, M J Kugler, S England BMC neuroscience BMC Neurosci. 2009
Mar 27;10:29..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-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 cAMPi changes in the micromolar
range after modulation of adenylate cyclase, inhibition of phosphodiesterase
and activation of G-protein-coupled metabotropic receptors. cAMPi
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
cAMPi 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 cAMPi 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 Ca2+ and cAMP signalling and revealed
a synergism of actions of these two second messengers.