Figure 7.

PKA-mediated synapsin phosphorylation at site 1 modulates SV recycling. A, Rat hippocampal neurons transfected to express either ECFP–SynI or ECFP–SynI S9A under the control of the CMV promoter were loaded with FM4-64 by exposure to 55 mm KCl for 1 min. The distribution of classes of FM4-64 fluorescence in ECFP fluorescent synapses is shown in the histograms with the corresponding color coding. p < 0.001 for ECFP–SynI versus ECFP–SynIS9A, Bonferroni's multiple comparison test (n = 300 synapses from 3 independent experiments). Mean ± SE values of FM4-64 uptake (n = 3 independent experiments) are shown in the insets. A.U., Arbitrary units. B, Rat hippocampal neurons transfected to express either ECFP–SynI or ECFP–SynI S9A under the CMV promoter were loaded with FM4-64 by exposure to 55 mm KCl for 1 min after a 30 min incubation with H89. A second epoch of FM4-64 uptake was induced by applying the same depolarizing stimulus 30 min after the washing out of H89 and bleaching of the residual fluorescence. FM4-64 was visualized exclusively in the synaptic boutons of the transfected neurons, selected using a digital masking system. The increase in FM4-64 uptake after removal of the PKA inhibition is prominent in synapses expressing ECFP–SynI (arrowheads), whereas it is partially inhibited in synapses expressing ECFP–SynI S9A (arrows). Scale bar, 4 μm. C, Quantitative analysis of FM4-64 uptake. p < 0.001 for KCl/H89 versus KCl for both chimeras and for ECFP–Syn I KCl versus ECFP–Syn I S9A KCl, Bonferroni's multiple comparison test (n = 200 synapses from 3 independent experiments).