Journal: bioRxiv

Article Title: Hippocampal cAMP-dependent synaptic potentiation, ERK-dependent immediate-early gene activation, and context-dependent fear conditioning in mice are linked through dependence on the guanine nucleotide exchange factor RapGEF2

doi: 10.1101/2022.04.30.490162

Figure Lengend Snippet: (A) Representative immunohistochemical images with RapGEF2 antibody (NNLE-2) for hippocampal subregions (CA1, DG and CA3) from flox and cKO mice at 5-weeks old (left panel) and 10~20 weeks old (right panel). Scale bar: 100μm. (B) RapGEF2 immunoreactive (IR) signals in hippocampal CA1, DG and CA1 from cKO mice (10~20 weeks old) were quantified with NIH Image J and compared to RapGEF2 IR signals from flox mice. The result indicated a significant reduction in RapGEF2 levels in the CA1 and DG hippocampal subregions, but not in the CA3 subregion. N=4~5 for each group. Student’s t-test for each region, ** p <0.001. (C) Western blots using protein lysates from hippocampal subregions of flox and cKO mice showed similar results that RapGEF2 was downregulated in CA1 and DG. N=3~7 for each group, Student’s t-test for each region, ** p <0.001.

Article Snippet: The primary antibodies used were rabbit anti-pERK (1:1500, Cell Signaling Technology, Danvers, MA), anti-c-Fos (1:5000, EnCor Biotechnology Inc., Gainesville, FL), anti-Egr-1 (15F7) (1:1000, Cell Signaling Technology, Danvers, MA) and rabbit anti-Rapgef2 (NNLE-2, custom-made by Anaspec, see ref. ( )).

Techniques: Immunohistochemical staining, Western Blot

Journal: bioRxiv

Article Title: Hippocampal cAMP-dependent synaptic potentiation, ERK-dependent immediate-early gene activation, and context-dependent fear conditioning in mice are linked through dependence on the guanine nucleotide exchange factor RapGEF2

doi: 10.1101/2022.04.30.490162

Figure Lengend Snippet: (A) & (B) Representative images of phospho-ERK staining (in red) in hippocampal CA1 pyramidal cell layer (A) or hippocampal DG granule cell layer (B) of flox and cKO mice 10 min (FC10 min) or 30 min (FC 30 min) or 60 min (FC 60 min) after fear conditioning or without fear conditioning (NFC). Scale bar: 50 μm. (C) & (D) Quantification of phospho-pERK IR in the CA1 (C) or DG (D) of flox and cKO mice at different time points after fear conditioning, showed RapGEF2-dependent activation of ERK 10 and 30 min after training. N=3~5 for each group. Two-way ANOVA following by post hoc Bonferroni t-test, *p<0.05; **p<0.001.

Article Snippet: The primary antibodies used were rabbit anti-pERK (1:1500, Cell Signaling Technology, Danvers, MA), anti-c-Fos (1:5000, EnCor Biotechnology Inc., Gainesville, FL), anti-Egr-1 (15F7) (1:1000, Cell Signaling Technology, Danvers, MA) and rabbit anti-Rapgef2 (NNLE-2, custom-made by Anaspec, see ref. ( )).

Techniques: Staining, Activation Assay

Journal: bioRxiv

Article Title: Hippocampal cAMP-dependent synaptic potentiation, ERK-dependent immediate-early gene activation, and context-dependent fear conditioning in mice are linked through dependence on the guanine nucleotide exchange factor RapGEF2

doi: 10.1101/2022.04.30.490162

Figure Lengend Snippet: (A) Representative images of cFos immunostaining in hippocampal CA1, DG and CA3 and basolateral amygdala of flox and cKO mice that were sacrificed 1hr after fear conditioning (FC) or stayed in the home cage (NFC). Scale bar: 200 μm (B) Quantification of cFos IR in hippocampal subregions and basolateral amygdala of flox and cKO mice indicated that fear-conditioning induced cFos increase in all these regions. No significant difference was observed between flox and cKO mice. N=3~6 for each group. Two-way ANOVA following by post hoc Bonferroni t-test, *p<0.05, **p<0.001. (C) Representative images of Egr-1 immunostaining in hippocampal CA1, DG and CA3 and basolateral amygdala of flox and cKO mice after fear conditioning. Scale bar: 200 μm (D) Quantification of Egr-1 IR indicated that fear-conditioning induced Egr-1 increase in CA1 and DG is RapGEF2-dependent. cKO mice with RapGEF2 ablation in CA1 and DG showed attenuated Egr-1 increase in CA1 and DG 1hr after fear conditioning, compared to flox mice. N=3~6 for each group. Two-way ANOVA following by post hoc Bonferroni t-test, *p<0.05, **p<0.001. (E) RNAscope with egr-1 (in red) and c-fos (in green) probes indicated that upregulation of c-fos mRNA in hippocampal CA1 and DG following fear conditioning occurred exclusively in the neurons with upregulation of egr-1 mRNA. In CA1, 67.77% ± 10.92% of Egr-1 positive neurons are c-fos positive. In DG, 87.89% ± 7.67% of egr-1 positive neurons are cfos positive. Scale bar: 100 μm (left panels), 20 μm (right panels).

Article Snippet: The primary antibodies used were rabbit anti-pERK (1:1500, Cell Signaling Technology, Danvers, MA), anti-c-Fos (1:5000, EnCor Biotechnology Inc., Gainesville, FL), anti-Egr-1 (15F7) (1:1000, Cell Signaling Technology, Danvers, MA) and rabbit anti-Rapgef2 (NNLE-2, custom-made by Anaspec, see ref. ( )).

Techniques: Immunostaining