Fig 2: SCI induces a similar increase in pain-like behaviors of wild-type, EPAC1−/− and EPAC2−/− mice, as revealed by an operant mechanical conflict test. (A) Sex-specific differences in locomotor recovery post-SCI in mice. Gender significantly affected locomotor recovery post-SCI in mice. The BMS experiments were performed weekly after surgery, and significant differences between males and females were observed as early as 2 weeks post-injury. Mice were monitored during 4 min observation sessions, during which their movements were assessed according to the methodology and BMS scale originally published in (Basso et al., 2006). Statistical comparison was made using 2-way repeated measures ANOVA followed by Sidak tests. (B) Behavioral tests were performed on female mice 3–4 weeks after surgery. SCI significantly decreased the total number of crossings across aversive probes in the mechanical conflict device, and (C) increased the latency to the second complete crossing. Data are shown as mean ± SEM. 2-way ANOVA was used for comparisons of total number of crossings as data were parametric. For comparisons of latencies of second complete crossing, unpaired t-test was used to compare wild-type sham to wild-type SCI mice, as well as wild-type SCI mice to EPAC1−/− while Mann-Whitney U test was performed to compare wild-type SCI mice to EPAC1−/− SCI mice. ANOVA, analysis of variance; BMS, Basso Mouse Scale; EPAC, exchange protein activated by cAMP; KO, knock-out; n.s., non-significant; SCI, spinal cord injury; SEM, standard error of the mean.

Fig 3: Inhibition of both EPAC1 and 2 is necessary to mitigate SCI-induced OA in presumptive mouse nociceptors. To measure extrinsically driven OA, small DRG neurons were artificially depolarized to −45 mV by current injection for 30–60 s. DRG neurons isolated from EPAC1−/− or EPAC2−/− mice were not protected against SCI-induced OA; additional inhibition of the complementary isoform was required to reverse the effect of the injury. (A) Representative 10-second recordings obtained from neurons artificially held at −45 mV. APs are clipped at 0 mV to allow sufficient magnification for clear display of DSFs. (B) Incidence of OA measured at a holding potential of −45 mV. The ratio above each bar denotes the number of neurons with OA/the number of neurons sampled. Statistical comparisons of OA incidence were made with Bonferroni-corrected Fisher’s exact tests on the indicated pairs. DRG, dorsal root ganglion; EPAC, exchange protein activated by cAMP; KO, knock-out; OA, ongoing activity; MP, membrane potential; SCI, spinal cord injury; WT, wild-type.

Fig 4: Hippocampal α7 nAChR, PKA, Epac, Rap1, and pCREB in mice after nicotine place conditioning There was no difference in hippocampal a7 nAChR in male (A) and female mice (B). There was no difference in PKA in male (C) and female mice (D). There was no difference in Epac1 in male (E) and female mice (F). Epac2 level was significantly increased in male (G) and female (H) mice. Rap1 expression was increase in female mice (J), but not in male mice (I). Consistently, p-CREB/CREB ratio expression was decreased in female mice (L), but not in male mice (K). Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, n = 4 in each group.

Fig 5: Epac1 deficiency impairs pathological neovascularization.(A) Hematoxylin and eosin (H&E) staining of ligated carotid artery sections from WT and Epac1−/− mice. (B) Immunofluorescence-stained ligated carotid artery sections from WT and Epac1−/− mice. Green, BS1 lectin-FITC; red, α-SMA; blue, DAPI. Arrows indicate neovascularization. Scale bars, 50 μm. (C) Quantification of neovascularization in ligated carotid artery sections from WT and Epac1−/− mice (n = 7). (D) Images of subcutaneously implanted Matrigel plugs retrieved from WT and Epac1−/− mice. (E) H&E staining of sectioned Matrigel plugs harvested from WT and Epac1−/− mice. Note the presence of microvessel-like structures and abundant blood cells (arrows) in WT Matrigel plugs, which were mostly absent in Epac1−/− Matrigel plugs. Scale bars, 200 μm. (F) Confocal images of immunofluorescence-stained, sectioned Matrigel plug slides harvested from WT and EPAC1−/− mice. Green, BS1 lectin-FITC; red, α-SMA; blue, DAPI. Arrows, microvessel-like structures. Scale bars, 200 μm. (G) Representative images of microvessel outgrowths in VEGF/FGF-treated aortic rings isolated from WT and Epac1−/− mice. (H) Quantification of main microvessel sprouts observed from WT (n = 24) and Epac1−/− (n = 26) aortic rings.