Fig 2: 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 3: Inhibition of both EPAC1 and 2 isoforms is necessary to mitigate SCI-induced hyperexcitability in dissociated mouse DRG neurons. Small to medium-diameter DRG neurons (=30 µm) harvested from lumbar levels were recorded by whole-cell patch clamp 18–30 h after dissociation. Neurons were pretreated with either vehicle, 10–20 µM CE3F4 or 5 µM ESI-05 for 15–20 min before recording. (A) Representative 10-second recordings obtained from neurons at RMP. Arrows indicate two of the larger DSFs. (B) Nociceptors isolated from EPAC1-/- or EPAC2-/- mice were not protected against increased incidence of SCI-induced SA; additional pharmacological inhibition of the complementary isoform was required to bring SA incidence towards a level comparable to neurons isolated from naïve/sham mice. The ratio above each bar denotes the number of neurons with SA/the number of neurons sampled. Statistical comparisons of SA incidence were made with Bonferroni-corrected Fisher’s exact tests on the indicated pairs. (C) SCI-induced depolarization of the RMP in wild-type and EPAC2-/- mice; additional inhibition of the EPAC1 isoform was required to mitigate SCI-induced depolarization within neurons isolated from EPAC2-/- mice. (D) SCI induced significant hyperpolarization of AP voltage threshold in wild-type and EPAC2-/- mice (trending within EPAC1-/- SCI mice); additional inhibition of the EPAC1 isoform did not cause a further significant change in the SCI-induced AP voltage threshold hyperpolarization within nociceptors from EPAC2-/- mice. (E) SCI decreased rheobase in wild-type and EPAC2-/- mice (trending within EPAC1-/- SCI mice); additional inhibition of the EPAC1 isoform did not cause a further significant change in the SCI-induced decrease in rheobase within nociceptors from EPAC2-/- mice. Comparisons of data (mean ± SEM) were made by t-test (for wild-type data), or Kruskal-Wallis followed by Dunn’s method for pairwise comparisons AP, action potential; DRG, dorsal root ganglion; EPAC, exchange protein activated by cAMP; KO, knock-out; MP, membrane potential; NA, nonaccommodating; n.s., non-significant; SA, spontaneous activity; SCI, spinal cord injury; SEM, standard error of the mean; WT, wild-type.

Fig 4: 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 5: 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.