Figure 1.

PKA modulates SV recycling. A, Rat hippocampal neurons expressing SypI–EYFP (left) were loaded with FM4-64 by exposure for 1 min to KRH–55 mm KCl (depolarizing solution) (middle). Neurons were subsequently exposed to a second depolarizing stimulus to unload the dye (right). Scale bar, 20 μm. B–D, Quantitative analysis of FM4-64 uptake in SypI–EYFP-positive synapses. The intensity of FM4-64 uptake was measured at single synaptic boutons. The distributions of classes of FM4-64 fluorescence are shown as smoothened histograms, and the mean ± SE fluorescence values (n = 3 independent experiments) expressed in arbitrary units (A.U.) are shown in the inset with the corresponding color coding. B, Neurons were incubated for 30 min in KRH in the presence or absence of H89, KN93, or W7 and were then loaded with FM4-64 during a 1 min incubation in depolarizing solution; a 1 min incubation in KRH was used as control (black). p < 0.001 for KCl versus KCl/W7, KCl/H89, or KCl/KN93, Bonferroni's multiple comparison test (n = 250 synapses from 3 independent experiments). C, Quantitative analysis of FM4-64 uptake at SypI-EYFP-positive synapses elicited by a depolarizing stimulus applied for 30 s after a 10 min incubation in KRH in either the absence (black) or presence (red) of forskolin. p < 0.001, Bonferroni's multiple comparison test (n = 250 synapses from 3 independent experiments). D, Quantitative analysis of FM4-64 uptake at SypI–EYFP-positive synapses elicited by a depolarizing stimulus applied for 1 min after a 30 min incubation in the absence (black) or presence of either W7 (blue) or W7/dibutyryl-cAMP (red). p < 0.001, Bonferroni's multiple comparison test (n = 350 synapses from 3 independent experiments).