The Journal of Neuroscience, November 8, 2006, ():
Protein Kinase A-Mediated Synapsin I Phosphorylation Is a Central Modulator of Ca2+-Dependent Synaptic Activity
J. Neurosci. Menegon et al.
26: 11670
Supplemental Data
Files in this Data Supplement:
- supplemental material
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Supplementary Figure 1. Effects of the expression of wild-type and S9A synapsin in hippocampal neurons.
(A) Hippocampal neurons from synapsin I knock-out mice infected to express either ECFP-SynI or ECFP-SynI S9A under the PGK promoter (blue) were depolarized for 1 min and processed for double immunofluorescence with anti-synapsin antibodies specific for either phosphosite-1 (green) or phosphosite-3 (red). In ECFP-SynI-expressing neurons note the presence of synapses positive for both phosphosite-1 and 3 (arrowhead) as well as of synapses positive exclusively for phosphosite-1 (arrow).
(B) Rat hippocampal neurons double infected to express SypI-EYFP and either ECFP-SynI or ECFP-SynI S9A were imaged after a 10 min-incubation with forskolin. Forkolin promotes the translocation out of synapses of ECFP-SynI but not of ECFP-SynI S9A. SypI-EYFP remains confined to the terminals in all cases. Bar: A, 7 ?m; B, 15 ?m.
(C) Hippocampal neurons in culture from synapsin I knock-out mice infected to express either ECFP-SynI, ECFP-SynI S9A, or ECFP-VAMP2 under the PGK promoter were fixed and stained for synaptophysin immunoreactivity. Left panel: mean levels (± S.E.) of endogenous SypI immunoreactivity. p > 0.05, Bonferroni’s multiple comparison test (n= 20 fields of view for each condition). Right panel: mean density of synaptic boutons. p > 0.05, Bonferroni’s multiple comparison test (n= 20 fields of view for each condition).
- supplemental material
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Supplementary Figure 2. mEPSCs recorded at the soma of whole-cell clamped hippocampal neurons.
A: Representative traces of miniature EPSCs (mEPSCs) in Syn I KO neurons expressing either ECFP-SynI (upper traces) or ECFP-SynI S9A (lower traces). B and C: Cumulative distribution of the interevent intervals and cumulative amplitude distribution curves calculated for the two representative neurons of panel A. D: Time course of mEPSC frequency calculated every 10 s. Application of forskolin increases the frequency of mEPSCs in a representative Syn I knock-out neuron expressing ECFP-Syn I. E: Cumulative distribution of the interevent intervals calculated for the representative neuron of panel D. F: Cumulative amplitude distribution curves for two representative neurons expressing either ECFP-Syn I or ECFP-SynI S9A and exposed to forskolin. G and H: Mean amplitude (G) and frequency (H) of mEPSCs calculated from groups of Syn I knock-out neurons expressing either ECFP-Syn I (n=7) or ECFP-SynIS9A (n=6) in the absence or presence of forskolin. Forskolin induces a significant increase in the frequency of mEPSCs in both types of neurons (p < 0.01, Student’s t test).
Methods: mEPSCs were acquired with sample frequency ranging between 10 and 20 kHz and filtered at half the acquisition rate with an 8-pole lowpass Bessel filter. Tetrodotoxin (0.3 ?M) was added to block spontaneous action potential propagation. Neurons were voltage-clamped at an holding potential of -70mV and at least 4 min of continuous stationary activity was recorded,. The mEPSC data analysis was performed with the MINIANALYSIS program (Synaptosoft, Leonia, NJ). The amplitude and frequency of mEPSCs were calculated using a peak detector function with the threshold amplitude set at 3 pA and threshold area at 50 pA*ms for all the experiments. Data were expressed as mean ± S.E. for 6
