Fig. 3.

Time-course of ERK activation in presynaptic terminals. (A–C) Laser confocal images show double-immunofluorescence labeling for pERK and Syn I in CA3 stratum lucidum of representative slices under baseline stimulation (A) or following HFS (B, 2 min after 50-Hz tetanus; C, 5 min after 100-Hz tetanus). Note that both HFS protocols resulted in a noticeable increase in the density of pERK-positive puncta colocalized with Syn I (arrows). (D and E) Bar graphs illustrate the time course of synaptic ERK activation in CA3 stratum lucidum after delivery of either 50-Hz (D) or 100-Hz (E) HFS to the MF-CA3 pathway. Data are expressed as mean number of pERK/Syn I-positive puncta in confocal fields of 3772.42 μm². With both stimulation frequencies, the densities of pERK-positive terminals was significantly increased in the 0.5–15 min interval with respect to control values (2-way ANOVA, P < 0.0001). However, the pattern of ERK activation was indistinguishable between the 50 Hz and 100 Hz stimulation protocols (2-way ANOVA, P = 0.39). For 50-Hz HFS, data are from 8 (control), 5 (0.5 min), 6 (2 min), 6 (5 min), 5 (10 min), 5 (15 min), and 5 (30 min) slices. For 100-Hz HFS, data are from 9 (control), 4 (0.5 min), 4 (2 min), 6 (5 min), 4 (10 min), 4 (15 min), and 4 (30 min) slices. (F and G) Bar graphs show the mean number of Syn-I positive puncta per field after 50-Hz (F) or 100-Hz (G) HFS (quantified in the same images used for D and E). No statistically significant variations were observed along the time course. (H–J) 20-Hz tetanization of the MF-CA3 pathway does not induce ERK activation in MF terminals. No variation in presynaptic pERK expression (arrows) could be seen in MF terminals of slices that were potentiated and harvested 2 min (I) or 5 min (J) after the onset of HFS (1 sec at 20 Hz) delivered at MF-CA3 synapse, compared with slices that received baseline stimulation (H). **, P < 0.01; #, P > 0.12. (Scale bars, 10 μm.)