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Figure 5 | BMC Neuroscience

Figure 5

From: Imaging cytoplasmic cAMP in mouse brainstem neurons

Figure 5

Calibration of cAMP levels in neurons. A– Increases in 470/535 nm emission ratio during sequential additions of specific membrane-permeable Epac agonist 8-Bromo-2'-OMe-cAMP. B – Calibration of Epac-1-camps. Mean ratios ± S. E. M were obtained for 8-Bromo-2'-OMe-cAMP in vivo and in vitro, and for cAMP in vitro. The data are plotted against nucleotide concentration and the smooth curve was drawn according to the Michaelis – Menten-like equation with K d = 1.5 μM. C – Typical Ca2+-dependent changes in [cAMP] after membrane depolarisation. Shown are the raw traces of Epac-1-camps emission at 470 nm (CFP) and 535 nm (FRET) excited at 430 nm and of fura-2 emission at 535 nm excited at 350 and 380 nm as indicated. The lower two traces demonstrate changes in the fura-2 ratio at excitation wavelengths 350 and 380 nm and those obtained after subtraction of 15% of Epac1-camps signal (Ex 430 nm, Em 535 nm) from fura-2 signal (Ex 380, Em 535 nm).

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