Article Figures & Data
Figures
- Figure 1.
Experimental timeline. Perinatal morphine treatment to mouse dams (MO, 10 mg/kg, s.c.) versus SAL began 7 d before breeding [gestational day 7 (GD-7), start of the study], continued through birth and lactation until offspring were weaned on P21. Maternal plasma was collected on P22 for LC-MS/MS, 20 h after the final MO injection. One cohort of mouse offspring was used for P21 gene expression. A second behaviorally naive cohort was used for immunohistochemistry at 23 weeks (w) of age. The third cohort underwent testing for three-chambered social interaction, sucrose/high-fat diet preference, and operant testing (FR1; PR, progressive ratio), and brains were used for gene expression at 22 weeks. Finally, a smaller separate cohort of dams were injected at GD14–GD18, and maternal plasma, placentas, and fetal brains were collected at 1 and 4 h after MO injection for LC-MS/MS. Refer to Extended Data Figure 1-1 for sample sizes, and Extended Data Figures 1-2, 1-3, 1-4 for gene expression targets. Refer to Extended Data Figures 1-5, 1-6, 1-7 for additional birthing and offspring outcomes.
- Figure 2.
Social behavior and basic operant performance. A, B, Perinatal MO increased social preference (MO: *p < 0.05 main effect of MO vs SAL via two-way ANOVA; A) but did not affect social novelty (B). C, D, Male MO offspring had delayed learning of FR1 (MO: *p < 0.05 vs SAL via Log rank survival analysis; C), but not females (D). E, F, Male MO offspring had reduced break point in progressive ratio (PR; *p < 0.05 vs SAL via t test; E), but not females (F). Refer to Extended Data Figure 2-1 for high-fat and sucrose preference test results.
- Figure 3.
5CSRTT. A–D, Male MO offspring had reduced accuracy (A), increased percentage of omissions (B), reduced number of premature responses (C), and no change in the total number of trials (D) in the initial training phase with a 16 s stimulus before reaching criterion (MO: *p < 0.05 main effect of MO vs SAL via two-way repeated-measures ANOVA). E–H, Female MO offspring had no change in accuracy (E) or omissions (F), but had an increase in premature responses (G) and total number of trials (H) during 16 s stimulus training (MO: *p < 0.05 main effect of MO vs SAL via two-way repeated-measures ANOVA). I–K, Upon successfully passing criterion at the 16, 8, and 4 s stimulus lengths, male MO offspring had reduced accuracy (I), no change in omissions (J), and reduced premature responses (K; MO: *p < 0.05 main effect of MO vs SAL via two-way repeated-measures ANOVA). L, In the final titrated version of the 5CSRTT, male MO offspring had reduced premature responses (*p < 0.05 vs SAL, via t test).
- Figure 4.
Behavioral correlations. A, B, Male (A) and female (B) correlation plots with circles denoting significant p-values (larger circle size showing smaller p-value) and the color of the circle showing the Pearson r value. C, D, Male (C) and female (D) linear regression plots depicting the correlation between premature and correct responses, further divided by MO versus SAL (*p < 0.05 via Pearson correlation). FR1 crit, Days to reach FR1 criterion; PRbp, break point in progressive ratio; ACC16s, average percentage accuracy (ACC) across initial 5CSRTT training with 16 s stimulus (16 s); PREMAT16s, average premature responses (16 s); OMIT16s, average percentage omissions (16 s); TRIALS16s, average trials (16s); CORRECT16s, average number of correct trials (16 s); SocPref, percentage preference for mouse versus empty cup in three-chamber social interaction test; SocNovel, percentage preference for novel versus familiar mouse in three-chamber social interaction test.
- Figure 5.
Postnatal day 21 and adult offspring gene expression (qPCR). A–F, In the mPFC at P21, MO offspring had increased expression of ITGAM (CR3/CD11b; A), MYD88 (B), DLG4 (PSD-95; C), COMT (D), PNOC (E), and DNMT3A (F) (A-F, MO: *p < 0.05 drug main effect, two-way ANOVA). G, In the AMG, MO offspring had reduced SLC17A7 (VGluT1) expression (MO: *p < 0.05 drug main effect, two-way ANOVA). H, In the VTA, male MO had reduced TLR4 expression compares to male SAL (#p < 0.05 drug × sex interaction, *p < 0.05 vs male SAL, two-way ANOVA). I, J, In the adult AMG, male MO offspring had increased expression of IL-1β (I) and NTS (J). *p < 0.05 versus male SAL, t test. Refer to Extended Data Figure 5-1 for effects of sex at P21.
- Figure 6.
Adult Iba1 and CD68 immunohistochemistry. A–C, Anatomical description of the m-mPFC (A), c-mPFC (B), and MeA (C). D, E, In 20× m-mPFC images, female MO offspring had increased Iba1 (D) and CD68 (E) integrated density normalized to cell count. F, In 20× MeA images, male MO offspring had reduced CD68 integrated density normalized to cell count. G, In 40× m-mPFC images, female MO offspring had increased Iba1 integrated density within the cell body. H, I, In 40× PFC images, male MO offspring had reduced Iba1 (H) and reduced CD68 integrated densities (I) within the cell body in the m-mPFC and c-mPFC, respectively. *p < 0.05 versus SAL, t test. Extended Data Figure 6-1 shows male m-mPFC values, and Extended Data Figure 6-2 shows representative immunohistochemical images.
- Figure 7.
Postnatal day 21 mPFC exploratory RNA-seq. A, B, Male (A) and female (B) differentially expressed gene lists based on MO versus SAL comparison yielding unadjusted p < 0.05 and log2FoldChange >1.2 or less than −1.2. a–f, Genes highlighted in yellow depict contributions to significantly enriched pathways, annotated by functions. Hierarchical clustering gene × drug group dendrograms were computed with the Spearman rank-based similarity measure.
Tables
Result Data structure Type of test Power: 95% CI, MO vs SAL a Normal distribution Two-way ANOVA −0.9729 to 1.565 b Normal distribution Two-way ANOVA 12.37–57.91 c Normal distribution t test −42.15 to −2.805 d Normal distribution Two-way ANOVA −13.95 to −1.238 e Survival analysis Log-rank (Mantel–Cox) 0.8219–4.835 f Normal distribution t test −16.25 to −3.751 g Normal distribution One-way ANOVA 0.8553–20.70 h Normal distribution One-way ANOVA −8.720 to −2.726 i Normal distribution One-way ANOVA 2.081–8.610 j Normal distribution One-way ANOVA −11.40 to −1.271 k Normal distribution One-way ANOVA −22.71 to −3.676 l Normal distribution One-way ANOVA 0.1384–8.060 m Normal distribution One-way ANOVA 2.606–7.030 n Normal distribution t test −5.397 to −0.7243 o Normal distribution Linear Regression Slope 95% CI: 0.2513–2.790 p Normal distribution Linear Regression Slope 95% CI: 0.3514–1.937 q Normal distribution Linear Regression Slope 95% CI: −0.189 to 0.891 r Normal distribution Linear Regression Slope 95% CI: 0.0744–1.204 s Normal distribution Two-way ANOVA −0.3064 to −0.07172 t Normal distribution Two-way ANOVA −0.1778 to −0.002775 u Normal distribution Two-way ANOVA −0.2946 to −0.01059 v Normal distribution Two-way ANOVA −0.1942 to −0.03028 w Normal distribution Two-way ANOVA −0.3187 to −0.05020 x Normal distribution Two-way ANOVA −0.3454 to −0.04435 y Normal distribution Two-way ANOVA 0.002719–0.2159 z Normal distribution Two-way ANOVA 0.06831–0.3622 aa Non-normal Mann Whitney U test 0.04579–1.417 ab Non-normal Mann Whitney U test 0.06866–0.5624 ac Normal distribution t test 10,716–8,233,887 ad Normal distribution t test 478,164–3,712,384 ae Normal distribution t test 0.6423–9.446 af Normal distribution t test −17.07 to −2.215 ag Normal distribution t test −14.15 to −4.105 ah Normal distribution t test −4,454,906 to −331,789 The Result column indicates superscript letters for each statistical result presented in this section.
Tissue Time (h) Total morphine ± SE Maternal plasma (n = 2) 1 2825.5 ± 35.0 (ng/ml) Placenta (n = 7) 1 2704.6 ± 99.4 (ng/g) Fetal brain (n = 7) 1 1267.2 ± 45.4 (ng/g) Maternal plasma (n = 3) 4 521.7 ± 73.6 (ng/ml) Placenta (n = 11) 4 415.4 ± 10.4 (ng/g) Fetal brain (n = 11) 4 434.0 ± 11.2 (ng/g) Maternal plasma (n = 2) 20 <2 ng/ml Total morphine detected at 1, 4, and 20 h after maternal injection in maternal plasma. Tissue levels in placenta and fetal brain samples were measured at 1 and 4 h only.
Figure 1-1
End point, variables measured, offspring sample size, and litter representation for each outcome assessed. Download Figure 1-1, DOCX file.
Figure 1-2
Gene expression targets for P21 mPFC, AMG, VTA, and NAc. The + and – symbols indicate included (+) or omitted (–) targets for adult operant PFC. The ^ and ᵒ symbols indicate included (^) or omitted (ᵒ) targets for adult operant AMG. Download Figure 1-2, DOCX file.
Figure 1-3
Targets added for the adult operant mPFC gene expression analysis. The ^ and ᵒ symbols indicate included (^) or omitted (ᵒ) targets for adult operant AMG. Download Figure 1-3, DOCX file.
Figure 1-4
Targets added for the adult operant AMG gene expression analysis. Download Figure 1-4, DOCX file.
Figure 1-5
Maternal and birth characteristics. A, B, Maternal MO treatment did not alter gestational weight (A) or pup birth weight (B). C, Assessed during the first postnatal week, MO injection acutely reduced time spent in the nest at 1 h, but this was recovered by 4 h (#p < 0.05, drug × time interaction; MO: *p < 0.05 main effect of drug; *p < 0.05 MO vs SAL at 1 h, two-way repeated-measures ANOVA). D, On postnatal day 2, MO dams returned their pups faster on average than SAL dams (*p < 0.05 vs SAL, t test). Download Figure 1-5, TIF file.
Figure 1-6
Values for maternal and birthing outcomes that did not reach statistical significance (mean ± SEM) or median (range). Download Figure 1-6, DOCX file.
Figure 1-7
Values for offspring body weight (mean ± SEM). Download Figure 1-7, DOCX file.
Figure 2-1
High-fat and sucrose preference tests. A, B, There were no group differences in high-fat (A) or sucrose preference (B) tests. Download Figure 2-1, TIF file.
Figure 5-1
P21 fold change values by group (mean ± SEM) for observed sex differences in gene expression. Download Figure 5-1, DOCX file.
Figure 6-1
Male m-mPFC values for Iba1 and CD68 reported as the mean ± SEM. Download Figure 6-1, DOCX file.
Figure 6-2
Representative images for CD68/Iba1 immunohistochemistry. A–D, Representative m-mPFC Iba1/CD68 40× images of female SAL (A) and female MO (B) depict increased Iba1 in female MO offspring; male SAL (C) and male MO (D) depict reduced Iba1 in male MO offspring. Representative c-mPFC CD68 40× images of male SAL (L) and male MO (O) depict reduced CD68 in male MO offspring. Download Figure 6-2, TIF file.
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