Fig 2: Schematic model of GEMS molecular cascade mediating the enhancement of stress-related memories. During stress, the increase in glucocorticoid secretion (1) activates the glucocorticoid receptors (GR) inducing its nuclear translocation (2). GR trigger the synthesis of the transcription factor Egr-1 (3) and then the activation of the MAPK pathway (4), which further increases Egr-1 production (5). Egr-1 stimulates the transcription of synapsin-Ia/Ib (6), which increases the number of SVs bound to actin and in this way the pool of readily releasable neurotransmitters. Activated MAPK do not modify synapsin-I expression, but induce their phosphorylation (7). Phosphorylated synapsin-Ia/Ib releases SVs bound to actin (8) allowing their transport to the pre-synaptic membrane (9) and neurotransmitter release (10). The double action of GR-induced Egr-1 expression and MAPK activation on both synapsin-I synthesis and phosphorylation can guarantee a sustained increase in neurotransmitter release. Specifically, an increase in glutamate release is likely to mediate the enhancement of stress-related memories that can be blocked both by inhibiting MAPK phosphorylation and synapsin-I expression. This model is based on several studies, including ours, which are discussed in the main text.

Fig 3: Corticosterone stimulation of synapsin-I expression depends on Egr-1. (a) Schematic illustration of the protocol used to silence Egr-1 expression by using a small-interfering RNA strategy. PC12 cells and hippocampal neurons were transfected with siRNA (-Egr-1, -ctrl, -Alexa Fluor 546, or EGFP); 48 to 65 h later, the cells were exposed to corticosterone (Cort) for 1 h and then proteins were analyzed by western blot (PC12) or immunostaining (neurons). (b) The transfection efficiency in PC12 cells was evaluated by flow cytometry after transfecting siRNA-Alexa Fluor 546. (c) Western blot analyses of the effects of corticosterone administration (10 n) on synapsin-I and Egr-1 expressions after silencing Egr-1 expression using an siRNA strategy. ßIII-tubulin was used as a loading control. (d) Quantification by densitometry (optical density OD, mean±s.e.m., n=3) of the western blot experiments. *P<0.05, **P<0.005, in comparison with untreated cells (ANOVA). (e) Immunostaining of hippocampal neurons cultured for 9 days in vitro showing the sublocalization of the GR proteins (green) in the absence or presence of 100 n of corticosterone for 1 h. Nuclei were counterstained with DAPI (blue). Merge of the two signals is shown. Scale bar=25 µm. (f) Co-immunolocalization of Egr-1 (green) and synapsin-I (red) proteins on hippocampal neurons cultured for 9 days in vitro in the absence or presence of 100 n of corticosterone during 1 h. Scale bar=25 µm. (g, h) Quantification of the fluorescence intensity (AU, arbitrary unit) of Egr-1 (g) and synapsin-I (h) -immunoreactive hippocampal neurons in the absence or presence of 100 n of corticosterone (Cort) during 1 h. (i) After corticosterone treatment, the expression of Egr-1 and synapsin-I proteins in Egr-1-expressing hippocampal neurons increased proportionally. Values shown are mean±s.e.m. **P<0.005, ***P<0.001, in comparison with untreated cells. (j) Immunofluorescence analysis of hippocampal neurons treated with corticosterone (100 n) for 1 h and earlier co-transfected with EGFP cDNA and siRNA control or siRNA silencing Egr-1 protein. Nuclei were counterstained with DAPI (blue). The white arrows show the mapping of the synapsin-I protein in EGFP-positive hippocampal neurons transfected either with siRNA-ctrl or siRNA-Egr-1. Scale bar=25 µm. (k, l) Quantification of the fluorescence intensity (AU, arbitrary unit) of Egr-1 (k) and synapsin-I (l) -immunoreactive hippocampal neurons transfected with siRNA-ctrl or siRNA-Egr-1 in the presence of 100 n of corticosterone (Cort) during 1 h. (m) Proportional decrease between Egr-1 and synapsin-I proteins in neurons in which Egr-1 expression was silenced compared with siRNA-ctrl transfection. Values shown are mean±s.e.m. **P<0.005, ***P<0.001, in comparison with siRNA-ctrl transfected cells.