ERK activation in axonal varicosities modulates presynaptic plasticity in the CA3 region of the hippocampus through synapsin I

Abstract

Activity-dependent changes in the strength of synaptic connections in the hippocampus are central for cognitive processes such as learning and memory storage. In this study, we reveal an activity-dependent presynaptic mechanism that is related to the modulation of synaptic plasticity. In acute mouse hippocampal slices, high-frequency stimulation (HFS) of the mossy fiber (MF)-CA3 pathway induced a strong and transient activation of extracellular-regulated kinase (ERK) in MF giant presynaptic terminals. Remarkably, pharmacological blockade of ERK disclosed a negative role of this kinase in the regulation of a presynaptic form of plasticity at MF-CA3 contacts. This ERK-mediated inhibition of post-tetanic enhancement (PTE) of MF-CA3 synapses was both frequency- and pathway-specific and was observed only with HFS at 50 Hz. Importantly, blockade of ERK was virtually ineffective on PTE of MF-CA3 synapses in mice lacking synapsin I, 1 of the major presynaptic ERK substrates, and triple knockout mice lacking all synapsin isoforms displayed PTE kinetics resembling that of wild-type mice under ERK inhibition. These findings reveal a form of short-term synaptic plasticity that depends on ERK and is finely tuned by the firing frequency of presynaptic neurons. Our results also demonstrate that presynaptic activation of the ERK signaling pathway plays part in the activity-dependent modulation of synaptic vesicle mobilization and transmitter release.

Fig. 1.

ERK activation occurs in excitatory giant terminals after tetanization of the CA3-MF pathway. (A and B) Confocal images show the distribution of immunolabeling for pERK in CA3 fields of a representative control slice that received baseline stimulation (A) and a potentiated slice (B) that was harvested 2 min after tetanization (1 × 50 Hz) of MF-CA3 synapses (s.l., stratum lucidum; s.r., stratum radiatum; s.p., stratum piramidale). (C and D) Representative confocal micrographs showing pERK-positive MF terminals in CA3 stratum lucidum after 50-Hz stimulation. pERK-IR (red) is clearly colocalized (arrows) with VGluT1 (green, C) but not with VGAT (green, D) immunosignal (arrows). (E and F) Confocal micrographs show double labeling for pERK (red) and Syn I (green) in CA3 stratum lucidum of representative tetanized (1 × 50 Hz) slices in the presence of either vehicle (E) or U0126 20 μM (F). Note the colabeled profiles representing ERK activation in Syn I-positive mossy fiber terminals (arrows in E) and the complete absence of pERK labeling after application of U0126 (F). White box in B indicates the location where images C–F were acquired. [Scale bars: 100 μm (A and B); 10 μm (C–F).]

Fig. 2.

ERK modulates short-term presynaptic plasticity in the MF-CA3 pathway at a specific stimulation frequency. (A and B) Basal transmission in MF was unaltered by U0126 application. Input–output curves show that both fiber volley (A) and fEPSP (B) of synaptic responses were intact in the presence of the inhibitor [n = 6 both for DMSO (●) and U0126 (○)]. (C) Paired-pulse facilitation (PPF) was not changed by U0126 (DMSO, n = 8; U0126, n = 7). (D) The magnitude of PTE induced by one 50-Hz burst of 1 s was enhanced in slices treated with U0126. Mean fEPSP slope, during mins 2–3 post-tetanus, and time constant of decay of PTE were significantly increased after the delivery of the tetanus in U0126-treated slices (○, n = 8) compared with slices perfused with vehicle (●, n = 7). (E) PTE induced by a 20-Hz tetanus for 1 s was unmodified by U0126 (vehicle, n = 8; U0126, n = 5). (F) PTE induced by 3 trains of 100 Hz for 1 s did not differ between vehicle treated slices (n = 6) and U0126 treated slices (n = 6). (G) The magnitude of LTP after 100-Hz tetanization was also unchanged in the presence of U0126 (n = 6 for both vehicle and U0126-treated groups). (H) PTP of the Schaffer collateral-CA1 pathway was similar in slices treated with U0126 (n = 8) or with vehicle (n = 9).

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.)

Fig. 4.

Effects of PTE induction in the CA3 area of the hippocampus on the endogenous phosphorylation of ERK 2 and Syn I. Hippocampal slices incubated under basal conditions (Ctrl) or subjected to HFS (50 Hz) to induce PTE were harvested and homogenized. After homogenization, equal amounts of protein (10 μg protein/sample) were subjected to immunoblotting for the total and phosphorylated forms of ERK2 and Syn I, respectively. A representative immunoblot is reported in A. (B) For each slice (n = 5 per experimental group), the amounts of total and phosphorylated ERK 2 and Syn I were quantified and normalized by an internal standard of brain homogenate. Immunoreactivity levels for both total and phosphorylated proteins detected in HFS-treated slices are expressed in percent of the respective levels in control slices (means ± SEM). *, P < 0.05; **, P < 0.01; 1-way ANOVA vs. respective control.

Fig. 5.

ERK modulation of 50 Hz-induced PTE requires Syn I. (A and B) ERK activation in MF terminals in response to a 50-Hz tetanus was normal in Syn I KO mice. ERK activation is visible in MF terminals of Syn I KO mice in response to a 50-Hz tetanus (B). Note that the immunolabeling pattern for pERK in large MF terminals is similar in both genotypes, despite the absence of Syn I immunoreactivity in mutant slices. (Scale bar, 10 μm.) (C) PTE of MF-CA3 synapses induced with a single 50-Hz burst is similar in hippocampal slices from Syn-I KO mice (●, n = 12) and WT mice (gray triangles, data partially replotted from Fig. 2D). The magnitude and kinetics of this PTE were not affected by the application of ERK inhibitor U0126 (○, n = 11). (D) The duration of PTE induced by 50 Hz in synapsin TKO mice (○, n = 10) was found to be significantly increased with respect to WT animals (●, n = 5).