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Acta Physiologica 2007; Volume 190, Supplement 655
XXXIV Congress of The Spanish Society for Physiological Sciences
7/3/2007-7/7/2007
Valladolid, Spain
FRET PROBES TO MONITOR THE SUBCELLULAR DYNAMICS OF SECOND MESSENGERS IN LIVING NEURONES.
Abstract number: W06
Vincent1 P, Hepp1 R, Gervasi1 N, Hu1 E, Lambolez1 B, Paupardin-Tritsch1 D
1CNRS Universit Pierre et Marie Curie Paris6 UMR7102 Neurobiologie des Processus Adaptatifs 75005 Paris
Cyclic nucleotides (cyclic AMP and cyclic GMP) activate protein kinase A and G respectively, thereby exerting pleiotropic effects on neuronal properties. Nevertheless, the spatial and temporal properties of these signaling cascades remain poorly understood in neurons, because of a lack of suitable methodological approach allowing a direct measurement.
We used genetically-encoded FRET probes to monitor in real time these second messenger cascades in individual neurons in brain slice preparations. These probes are expressed in neurons using a viral vector. We verified the specificity of expression as well as lack of deleterious effects of probe expression. For these recordings, we used wide-field low-noise fluorescence ratiometric imaging.
Using the Cygnet probe, we observed that cGMP level results from an equilibrium between NO-dependent tonic synthesis, and degradation by type II phosphodiesterases.
We also determined the kinetics of PKA activation by measuring the FRET signal emitted by the AKAR2 probe while simultaneously measuring the downstream effect of PKA on membrane potassium channels. Increases in PKA activity in response to 5-HT7 receptor activation or direct stimulation of adenylyl cyclase with forskolin reduced the potassium current within 30 s. However, activation of 5-HT7 receptors had a slower effect on AKAR2 phosphorylation level and nuclear effects were slower still, reaching a maximum after 17 min. These results are consistent with the current view of signaling microdomains where the cAMP/PKA signal is restricted to specific compartments of cells.
To cite this abstract, please use the following information:
Acta Physiologica 2007; Volume 190, Supplement 655 :W06