Table 1. P values of the graphs presented.
Figure 1. Anhedonia- and anxiety-like behaviour induced by inflammatory pain in male and female rats. A) Timeline of the experimental design B) Sucrose consumption during 48 hours in male, expressed as mean ± SEM of mg/kg/24 hours C) Sucrose consumption during 48 hours in female, expressed as mean ± SEM of mg/kg/24hours D) Sucrose consumption during 2 hours in male, expressed as mean ± SEM of mg/kg/2 hours E) Sucrose consumption during 2 hours in female, expressed as mean ± SEM of mg/kg/2 hours F) Time (seconds) in light chamber in the light dark box test (LDB) in male, expressed as mean ± SEM G) Time (seconds) in light chamber in the LDB in male, expressed as mean ± SEM H) Number of transitions between chambers in the LDB in male, expressed as mean ± SEM I) Number of transitions between chambers in the LDB in female, expressed as mean ± SEM J) Mechanical sensitivity threshold in male, expressed as mean ± SEM of paw withdrawal in grams K) Mechanical sensitivity threshold in female, expressed as mean ± SEM of paw withdrawal in grams. Black and red bars and symbols represent saline- and CFA-treated male respectively, and grey and orange bars and symbols represent saline- and CFA-treated female respectively. *, ** and *** denotes significant differences between groups (two-way ANOVA for repeated measures followed by Bonferroni multiple comparisons test, p< 0.05, p< 0.01 and p< 0.001, respectively), and #, ## and ### denotes significant differences within groups compared with the basal measures (two-way ANOVA for repeated measures followed by Bonferroni multiple comparisons test, p< 0.05, p< 0.01 and p< 0.001, respectively). Abbreviations: SPT, sucrose preference test; VFT, Von Frey test; CFA, Complete Freund’s Adjuvant; SAL, saline; LDB, light-dark box test.
Figure 2. Corticotropin-releasing factor system alterations induced by inflammatory pain. A) Mean ± SEM of CRF mRNA relative gene expression in NAc of male rats. B) Mean ± SEM of CRF mRNA relative gene expression in Amy of male rats. C) Mean ± SEM of CRF1R mRNA relative gene expression in NAc of male rats. D) Mean ± SEM of CRF1R mRNA relative gene expression in Amy of male rats. E) Mean ± SEM of CRF1R protein expression in NAc of male rats (CRF1R/GAPDH arbitrary units). F) Mean ± SEM of CRF1R protein expression in Amy of male rats (CRF1R/GAPDH arbitrary units). G) Plasma cortisol levels in male rats, expressed as mean ± SEM of ng/mL. H) Mean ± SEM of CRF mRNA relative gene expression in NAc of female rats I) Mean ± SEM of CRF mRNA relative gene expression in Amy of male rats. C) Mean ± SEM of CRF1R mRNA relative gene expression in NAc of female rats. J) CRF1R expression in NAc in female, expressing as mean ± SEM of relative gene expression. K) Mean ± SEM of CRF1R mRNA relative gene expression in Amy of female rats. L) Mean ± SEM of CRF1R protein expression in NAc of female rats (CRF1R/GAPDH arbitrary units). M) Mean ± SEM of CRF1R protein expression in Amy of female rats (CRF1R/GAPDH arbitrary units). O) Plasma cortisol levels in female rats, expressed as mean ± SEM of ng/mL. Black and red bars represent saline- and CFA-treated male respectively, and grey and orange bars represent saline- and CFA-treated female respectively. * Denotes significant differences between groups (t-student for independent samples or Welch’s t test p < 0.05).
Figure 3. Kappa opioid receptor/Dynorphinergic system alterations induced by inflammatory pain in male and female rats. A) Mean ± SEM of pDYN mRNA relative gene expression in NAc of male rats. B) Mean ± SEM of pDYN mRNA relative gene expression in Amy of male rats. C) Mean ± SEM of KOR mRNA relative gene expression in NAc of male rats. D) Mean ± SEM of KOR mRNA relative gene expression in Amy of male rats. E) Mean ± SEM of DYN protein expression (ug/tissue) in NAc of male rats. F) Mean ± SEM of DYN protein expression (ug/tissue) in Amy of male rats. G) Mean ± SEM of KOR protein expression in NAc of male rats (KOR/GAPDH arbitrary units). H) Mean ± SEM of KOR protein expression in Amy of male rats (KOR/GAPDH arbitrary units). I) Mean ± SEM of pDYN mRNA relative gene expression in NAc of female rats. J) Mean ± SEM of pDYN mRNA relative gene expression in Amy of female rats. K) Mean ± SEM of KOR mRNA relative gene expression in NAc of female rats. L) Mean ± SEM of KOR mRNA relative gene expression in Amy of female rats. M) Mean ± SEM of DYN protein expression (ug/tissue) in NAc of female rats. N) Mean ± SEM of DYN protein expression (ug/tissue) in Amy of female rats. O) Mean ± SEM of KOR protein expression in NAc of female rats (KOR/GAPDH arbitrary units). P) Mean ± SEM of KOR protein expression in Amy of female rats (KOR/GAPDH arbitrary units). * Denotes significant differences between groups (t-student for independent samples or Welch’s t test p < 0.05).
Figure 4. Anhedonia- and anxiety-like behaviour induced by inflammatory pain in female rats is blocked by NorBNI intra-NAc administration. A) Timeline of the experimental design. B) Sucrose consumption during 48 hours in female rats, expressed as mean ± SEM of mg/kg/24 hours mean ± SEM. C) Sucrose consumption during 2 hours in female rats, expressed as mean ± SEM of mg/kg/2 hours. D) Time in light chamber (seconds) in the light-dark box test (LDB). E) Number of transitions between chambers in the LDB, expressed as mean ± SEM. F) Mechanical sensitivity threshold (g) in female rats, expressed as mean ± SEM of paw withdrawal threshol. Grey and orange bars represent saline- and CFA-treated female rats respectively, and empty and full bars represents the intracranial treatments of aCSF and NorBNI respectively. *, ** and *** denotes significant differences between groups (three-way ANOVA for repeated measures followed by Bonferroni multiple comparisons test, p < 0.05, p < 0.01 and P < 0.001, respectively), and #, ## and ### denotes significant differences within groups compared with the basal measures (two-way ANOVA for repeated measures followed by Bonferroni multiple comparisons test, p < 0.05, p < 0.01 and P < 0.001, respectively). Abbreviation: SPT, sucrose preference test; VFT, Von Frey test; CFA, Complete Freund’s Adjuvant; SAL, saline; LDB, light-dark box test; NorBNI, Norbinaltorphimine.
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Figure S1. Daily calendar of the behavioural protocol. CFA, complete Freund adjuvant; SAL, saline; LDB, light–dark box test; SPT, sucrose preference test; VFT, von Frey test.
Figure S2. Schematic of the brain areas dissection. A) Area of the NAc dissection B) Area of the amygdala dissection.
Figure S3. Daily calendar of the behavioural protocol of the Experiment 2 and intracranial administration verification. A) Daily calendar of the behavioural protocol. B) Dots represent the site where aCSF and NorBNI were injected: Black represent saline/aCSF treated female rats, grey represent saline/NorBNI treated female rats, red represents CFA/aCSF treated female rats, and orange represents CFA/NorBNI treated female rats. Abbreviatures: CFA, complete Freund adjuvant; SAL, saline; LDB, light–dark box test; SPT, sucrose preference test; VFT, von Frey test.