Article Figures & Data
Figures
- Figure 1.
Repeated heroin injections caused mechanical hyperalgesia during withdrawal in male and female mice. A, B, Timeline and experimental procedures. Adult male and female mice were habituated for 1 h to the testing apparatus on day 1 (D1). We measured baseline paw withdrawal thresholds on D2. We gave mice twice daily subcutaneous injections of saline (10 ml/kg; nondependent) or increasing doses of heroin (5–40 mg/kg; dependent) from D3 to D5. Bodyweight loss during treatment is shown in Extended Data Figure 1-1. We tested paw withdrawal thresholds 16 h after the last heroin injection on D7 and perfused the mice 1 h later. C, We did not detect significant differences between male and female mice in paw withdrawal thresholds at baseline and 16 h into withdrawal. We found significant effects of treatment (p < 0.0001) and time (p < 0.0001) and a significant treatment × time interaction (p < 0.0001), indicating hyperalgesia in both sexes. The data are expressed as mean ± SEM n = 13–14/group. D, At 16 h into withdrawal, heroin-treated mice (males and females combined) had significant lower paw withdrawal thresholds compared with saline-treated mice (****p < 0.0001) and compared with paw withdrawal thresholds during baseline (####p < 0.0001). E, Heroin-treated mice (males and females combined) exhibited significantly higher hyperalgesia during spontaneous opioid withdrawal than saline-treated mice (****p < 0.0001). We calculated the Δ paw withdrawal threshold (paw withdrawal thresholds at baseline minus paw withdrawal thresholds at testing) to use as a single number for subsequent data analyses. The data are expressed as mean ± SEM n = 27–28/group.
- Figure 2.
Repeated heroin injections produced naloxone-precipitated somatic signs of opioid withdrawal in male and female mice. A, B, Timeline and experimental procedures. We gave mice twice daily injections of saline (10 ml/kg, s.c.) or increasing doses of heroin (10–50 mg/kg, s.c.) from D1 to D5. On D6, we gave mice a single injection of saline or heroin (50 mg/kg, s.c.). Bodyweight loss during treatment is shown in Extended Data Figure 2-1. Two hours after the saline or heroin injection, we precipitated withdrawal with a single intraperitoneal injection of the preferential μ-opioid receptor antagonist naloxone (1 mg/kg) and recorded somatic signs of withdrawal for 20 min. We perfused mice 1 h after the end of behavior scoring (i.e., 80 min after the naloxone injection). C, We did not detect significant differences between male and female mice in paw tremors or “wet-dog” shakes, but heroin-treated mice exhibited a significantly higher number of paw tremors (****p < 0.0001) and “wet-dog” shakes (****p < 0.0001) than saline-treated mice. Heroin-treated female mice exhibited a higher number of jumps compared with heroin-treated male mice (++++p < 0.0001), and heroin-treated mice of both sexes exhibited a significantly higher number of jumps compared with saline-treated mice (****p < 0.0001). D, We found an effect of treatment on opioid withdrawal scores (****p < 0.0001), with no significant differences between male and female mice. We calculated the opioid withdrawal score (sum of the number of occurrences of individual somatic signs) to use as a single number for subsequent data analyses. The data are expressed as mean ± SEM n = 11/group. E, Heroin-dependent mice (male and female data combined) exhibited a higher opioid withdrawal score than saline-treated mice (****p < 0.0001). The data are expressed as mean ± SEM n = 22/group. gf, grams of force.
- Figure 3.
Regional distribution of c-Fos-expressing neurons following the assessment of hyperalgesia during spontaneous heroin withdrawal. Significant effects of treatment on c-Fos expression were found in the PVN (****p < 0.0001), LH (***p < 0.001), CeA (****p < 0.0001), LHb (****p < 0.0001), VTA (****p < 0.0001), DR (**p < 0.01), PBN (****p < 0.0001), and LC (***p < 0.001). The data are expressed as mean ± SEM n = 11–12/group (male and female data combined). A comparison of c-Fos expression levels between heroin-treated and saline-treated male and female mice is shown in Extended Data Figure 3-1.
- Figure 4.
Regional distribution of c-Fos-expressing neurons following the assessment of naloxone-precipitated somatic signs of heroin withdrawal. Significant effects of treatment on c-Fos expression were found in the NAc shell (***p < 0.001), BNST (****p < 0.0001), PVN (****p < 0.0001), PVT (*p < 0.05), LH (****p < 0.0001), CeA (***p < 0.001), LHb (***p < 0.001), SUM (****p < 0.0001), VTA (***p < 0.001), DR (**p < 0.01), PBN (****p < 0.0001), and LC (****p < 0.0001). The data are expressed as mean ± SEM n = 11/group (male and female data combined). A comparison of c-Fos expression levels between heroin-treated and saline-treated male and female mice is shown in Extended Data Figure 4-1.
- Figure 5.
Hyperalgesia during spontaneous heroin withdrawal and naloxone-precipitated somatic signs of heroin withdrawal were associated with c-Fos expression in specific brain areas. A, PCA of hyperalgesia and c-Fos expression in different brain regions in heroin-dependent mice. Hyperalgesia (red circle) and the DR, LC, CeA, VTA LH, and PBN (purple circles) loaded onto the PC1. The BNST, PVT, NAc shell, SUM, and PAG loaded onto the PC2 (green circles). Bodyweight loss (BW) and the PVN (black circles) loaded independently from PC1 and PC2. The distributions of variables are shown along the two factors (independent axes) with factor loadings ≥0.7 (indicated by dotted lines), produced by an orthogonal normalized varimax rotation. n = 12/group (male and female data combined). B, PCA of somatic withdrawal and Fos expression in different brain regions in heroin-dependent mice. The LH, SUM, PBN, BNST, NAc shell, and CeA (green circles) loaded onto PC1. Somatic withdrawal (red circle) and the DR, LC, LHb, and PVT (blue circles) loaded onto PC2. The BW, PVN, VTA, and PAG (black circles) loaded independently from PC1 and PC2. The distributions of variables are shown along the two factors with factor loadings ≥0.7, produced by an orthogonal normalized varimax rotation. n = 11/group (male and female data combined). C, Correlation of c-Fos expression in specific brain regions in heroin-dependent mice following the assessment of hyperalgesia during spontaneous withdrawal. Pearson correlation coefficients (r) inside the boxes correspond to regions that showed significant correlations (p < 0.05). Darker purple shades represent stronger correlations. n = 12/group (male and female data combined). D, Correlations of c-Fos expression in specific brain regions in heroin-dependent mice following the assessment of naloxone-precipitated somatic signs of heroin withdrawal. Pearson correlation coefficients (r) inside the boxes correspond to regions that showed significant correlations (p < 0.05). Darker green shades represent stronger correlations. n = 11/group (male and female data combined).
- Figure 6.
Summary of brain regions that are associated with neuronal c-Fos expression following the assessment of hyperalgesia during spontaneous withdrawal or naloxone-precipitated somatic signs of withdrawal in heroin-dependent mice. The colored brain regions were associated with PCA factors that loaded with hyperalgesia (purple), somatic signs of withdrawal (green), or both (purple/green) in heroin-dependent mice. The PBN, VTA, LH, and CeA are potentially involved in the expression of hyperalgesia during spontaneous withdrawal from heroin. The PVT and LHb are potentially involved in somatic signs of naloxone-precipitated heroin withdrawal. The DR and LC may play a role in both hyperalgesia and somatic signs of heroin withdrawal. The connecting lines represent significant correlations of c-Fos expression in brain regions that were associated with PCA factors that loaded with the behavior. The thickness of the lines represents the strength of correlations (thin = low correlation, r < 0.84; thick = high correlation, r > 0.84).
Tables
- Table 1
Two-way ANOVA results of Fos quantification in analyzed brain regions following hyperalgesia during spontaneous withdrawal
Brain region Treatment effect Sex effect Sex × treatment interaction NAc shell F(1,19) = 0.8349, p = 0.3723 F(1,19) = 10.41, p = 0.0044; F > M F(1,19) = 0.05892, p = 0.8108 BNST F(1,19) = 1.926, p = 0.1813 F(1,19) = 4.275, p = 0.0526 F(1,19) = 0.4395, p = 0.5153 PVN F(1,19) = 30.16, p < 0.0001 F(1,19) = 8.964, p = 0.0075; F > M F(1,19) = 0.2445, p = 0.1344 PVT F(1,19) = 0.1164, p = 0.7367 F(1,19) = 2.544, p = 0.1272 F(1,19) = 0.2627, p = 0.6142 LH F(1,19) = 16.93, p < 0.001 F(1,19) = 5.546, p = 0.0294; F > M F(1,19) = 0.1327, p = 0.7197 CeA F(1,19) = 26.37, p < 0.0001 F(1,19) = 1.876, p = 0.1867 F(1,19) = 0.09373, p = 0.7628 LHb F(1,19) = 40.80, p < 0.0001 F(1,19) = 8.195, p = 0.0100; F > M F(1,19) = 0.9071, p = 0.3528 PAG F(1,19) = 1.136, p = 0.2998 F(1,19) = 8.589, p = 0.0086; F > M F(1,19) = 0.005, p = 0.9441 SUM F(1,19) = 4.195, p = 0.0546 F(1,19) = 14.44, p = 0.0012; F > M F(1,19) = 0.007174, p = 0.9334 VTA F(1,19) = 27.39, p < 0.0001 F(1,19) = 5.773, p = 0.0267; F > M F(1,19) = 0.1231, p = 0.2810 DR F(1,19) = 11.70, p < 0.01 F(1,19) = 1.803, p = 0.1952 F(1,19) = 0.08641, p = 0.3642 PBN F(1,19) = 26.15, p < 0.0001 F(1,19) = 0.2822, p = 0.6014 F(1,19) = 0.1759, p = 0.6796 LC F(1,19) = 17.91, p < 0.001 F(1,19) = 0.5460, p = 0.4695 F(1,19) = 0.5909, p = 0.4520 Males (M), Females (F).
- Table 2
Two-way ANOVA results of Fos quantification in analyzed brain regions following naloxone-precipitated somatic signs of opioid withdrawal
Brain region Treatment effect Sex effect Sex × treatment interaction NAc shell F(1,18) = 15.91, p < 0.001 F(1,18) = 40.50, p < 0.0001; M > F F(1,18) = 1.781, p = 0.1986 BNST F(1,18) = 29.53, p < 0.0001 F(1,18) = 14.97, p = 0.0011; M > F F(1,18) = 1.470, p = 0.2411 PVN F(1,18) = 43.11, p < 0.0001 F(1,18) = 1.915, p = 0.1833 F(1,18) = 0.1469, p = 0.7060 PVT F(1,18) = 5.069, p < 0.05 F(1,18) = 4.738, p = 0.0431; F > M F(1,18) = 1.023, p = 0.3253 LH F(1,18) = 30.28, p < 0.001 F(1,18) = 0.3534, p = 0.5436 F(1,18) = 0.04941, p = 0.8266 CeA F(1,18) = 17.40, p < 0.001 F(1,18) = 23.89, p = 0.0001; M > F F(1,18) = 3.703, p = 0.0703 LHb F(1,18) = 18.28, p < 0.001 F(1,18) = 8.314, p = 0.0099; F > M F(1,18) = 1.297, p = 0.2697 PAG F(1,18) = 3.855, p = 0.0652 F(1,18) = 0.02565, p = 0.8746 F(1,18) = 0.3041, p = 0.5881 SUM F(1,18) = 33.96, p < 0.0001 F(1,18) = 1.128, p = 0.3023 F(1,18) = 6.029, p = 0.0245 VTA F(1,18) = 24.40, p < 0.0001 F(1,18) = 2.406, p = 0.1383 F(1,18) = 1.791, p = 0.1975 DR F(1,18) = 14.54, p < 0.01 F(1,18) = 12.91, p = 0.0021; F > M F(1,18) = 0.116, p = 0.7422 PBN F(1,18) = 27.17, p < 0.0001 F(1,18) = 18.16, p = 0.0005; M > F F(1,18) = 0.1257, p = 0.7271 LC F(1,18) = 88.91, p < 0.0001 F(1,18) = 29.03, p < 0.0001; F > M F(1,18) = 2.218, p = 0.1537 Males (M), Females (F).
Extended Data Figure 1-1
Repeated heroin injections caused bodyweight loss in male and female mice. Percent of bodyweight change in mice that were used for the c-Fos expression experiment following the assessment of hyperalgesia during spontaneous heroin withdrawal. The Student’s t test showed that heroin-treated mice (male and female data combined) weighed significantly less than saline-treated mice (****p < 0.0001). The data are expressed as mean ± SEM. N = 12/group. Download Figure 1-1, TIF file.
Extended Data Figure 2-1
Repeated heroin injections caused bodyweight loss in male and female mice. Percent of bodyweight change in mice used for the c-Fos expression experiment following the assessment of naloxone-precipitated somatic signs of opioid withdrawal. The Student’s t test showed that heroin-treated mice (male and female data combined) weighed significantly less than saline mice (****p < 0.0001). The data are expressed as mean ± SEM. N = 12/group. Download Figure 2-1, TIF file.
Extended Data Figure 3-1
C-Fos expression in neurons distributed in distinct brain regions in male and female mice following the assessment of hyperalgesia during spontaneous heroin withdrawal. Two-way ANOVAs showed no sex × treatment interactions for any of the analyzed brain regions. We found main treatment effect and main sex effect on the number c-Fos-expressing neurons in several brain regions: LH (treatment effect: F(1,19) = 16.93, p < 0.001; sex effect: F(1,19) = 5.546, p = 0.0294; sex × treatment interaction: F(1,19) = 0.1327, p = 0.7197), LHb (treatment effect: F(1,19) = 40.80, p < 0.0001; sex effect: F(1,19) = 8.195, p = 0.0100; sex × treatment interaction: F(1,19) = 0.9071, p = 0.3528), VTA (treatment effect: F(1,19) = 27.39, p < 0.0001; sex effect: F(1,19) = 5.773, p = 0.0267; sex × treatment interaction: F(1,19) = 0.1231, p = 0.2810), PVN (treatment effect: F(1,19) = 30.16, p < 0.0001; sex effect: F(1,19) = 8.964, p = 0.0075; sex × treatment interaction: F(1,19) = 0.2445, p = 0.1344), and SUM (treatment effect: F(1,19) = 4.195, p = 0.0546; sex effect: F(1,19) = 14.44, p = 0.0012; sex × treatment interaction: F(1,19) = 0.007174, p = 0.9334). We found significant treatment effect but no sex effect in the following brain regions: CeA (treatment effect: F(1,19) = 26.37, p < 0.0001; sex effect: F(1,19) = 1.876, p = 0.1867; sex × treatment interaction: F(1,19) = 0.09373, p = 0.7628), DR (treatment effect: F(1,19) = 11.70, p < 0.01; sex effect: F(1,19) = 1.803, p = 0.1952; sex × treatment interaction: F(1,19) = 0.08641, p = 0.3642), PBN (treatment effect: F(1,19) = 26.15, p < 0.0001; sex effect: F(1,19) = 0.2822, p = 0.6014; sex × treatment interaction: F(1,19) = 0.1759, p = 0.6796), and LC (treatment effect: F(1,19) = 17.91, p < 0.001; sex effect: F(1,19) = 0.5460, p = 0.4695; sex × treatment interaction: F(1,19) = 0.5909, p = 0.4520). We did not find significant treatment effects, but we found main sex effects on the number of c-Fos-expressing neurons in three brain regions: NAc shell (treatment effect: F(1,19) = 0.8349, p = 0.3723; sex effect: F(1,19) = 10.41, p = 0.0044; sex × treatment interaction: F(1,19) = 0.05892, p = 0.8108), BNST (treatment effect: F(1,19) = 1.926, p = 0.1813; sex effect: F(1,19) = 4.275, p = 0.0526; sex × treatment interaction: F(1,19) = 0.4395, p = 0.5153), and PAG (treatment effect: F(1,19) = 1.136, p = 0.2998; sex effect: F(1,19) = 8.589, p = 0.0086; sex × treatment interaction: F(1,19) = 0.005, p = 0.9441). We did not find a main treatment effect nor sex effect in the PVT (treatment effect: F(1,19) = 0.1164, p = 0.7367; sex effect: F(1,19) = 2.544, p = 0.1272; sex × treatment interaction: F(1,19) = 0.2627, p = 0.6142). Download Figure 3-1, TIF file.
Extended Data Figure 4-1
C-Fos expression in neurons distributed in distinct brain regions in male and female mice following the assessment of naloxone-precipitated somatic signs of heroin withdrawal. B. Two-way ANOVAs for each brain region showed a significant sex × treatment interaction only for the SUM (treatment effect: F(1,18) = 33.96, p < 0.0001; sex effect: F(1,18) = 1.128, p = 0.0245; sex × treatment interaction: F(1,18) = 6.029, p = 0.0245), but the post hoc comparisons did not detect meaningful differences (heroin females > saline males; heroin males > saline females). We found main treatment effect and main sex effect on the number of c-Fos-expressing neurons in several brain regions: NAc shell (treatment effect: F(1,18) = 15.91, p < 0.001; sex effect: F(1,18) = 40.50, p < 0.0001; sex × treatment interaction: F(1,18) = 1.781, p = 0.1986), BNST (treatment effect: F(1,18) = 29.53, p < 0.0001; sex effect: F(1,18) = 14.97, p = 0.0011; sex × treatment interaction: F(1,18) = 1.470, p = 0.2411), PVT (treatment effect: F(1,18) = 5.069, p < 0.05; sex effect: F(1,18) = 4.738, p = 0.0431; sex × treatment interaction: F(1,18) = 1.023, p = 0.3253), CeA (treatment effect: F(1,18) = 17.40, p < 0.001; sex effect: F(1,18) = 23.89, p = 0.0001; sex × treatment interaction: F(1,18) = 3.703, p = 0.0703), LHb (treatment effect: F(1,18) = 18.28, p < 0.001; sex effect: F(1,18) = 8.314, p = 0.0099; sex × treatment interaction: F(1,18) = 1.297, p = 0.2697), DR (treatment effect: F(1,18) = 14.54, p < 0.01; sex effect: F(1,18) = 12.91, p = 0.0021; sex × treatment interaction: F(1,18) = 0.116, p = 0.7422), PBN (treatment effect: F(1,18) = 27.17, p < 0.0001; sex effect: F(1,18) = 18.16, p = 0.0005; sex × treatment interaction: F(1,18) = 0.1257, p = 0.7271), and LC (treatment effect: F(1,18) = 88.91, p < 0.0001; sex effect: F(1,18) = 29.03, p < 0.0001; sex × treatment interaction: F(1,18) = 2.218, p = 0.1537). We found significant treatment effect but no sex effect in the following brain regions: PVN (treatment effect: F(1,18) = 43.11, p < 0.0001; sex effect: F(1,18) = 1.915, p = 0.1833; sex × treatment interaction: F(1,18) = 0.1469, p = 0.7060), LH (treatment effect: F(1,18) = 30.28, p < 0.001; sex effect: F(1,18) = 0.3534, p = 0.5436; sex × treatment interaction: F(1,18) = 0.04941, p = 0.8266), SUM (treatment effect: F(1,18) = 33.96, p < 0.0001; sex effect: F(1,18) = 1.128, p = 0.3023; sex × treatment interaction: F(1,18) = 6.029, p = 0.0245), and VTA (treatment effect: F(1,18) = 24.40, p < 0.0001; sex effect: F(1,18) = 2.406, p = 0.1383; sex × treatment interaction: F(1,18) = 1.791, p = 0.1975), We did not find a main treatment effect nor sex effect in the in the PAG (treatment effect: F(1,18) = 3.855, p = 0.0652; sex effect: F(1,18) = 0.02565, p = 0.8746; sex × treatment interaction: F(1,18) = 0.3041, p = 0.5881). Download Figure 4-1, TIF file.
Extended Data
Macros used for preprocessing and for morphological segmentation of images using Image-Pro 10.0 software. Download Extended Data 1, DOCX file.
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