Paternal heroin self-administration increases heroin seeking in male F1 offspring compared with saline- and yoked-sired controls

Naïve male SD rats were allowed to self-administer heroin (HSA group) or saline (SSA group) on a FR schedule for 30 days. On day 31, they were subjected to a PR test (Fig. S1a–c), and mated with naïve females to produce offspring (Fig. 1a). Male F1 offspring sired by heroin-SA-experienced fathers showed higher lever presses for heroin (Fgroup(1, 39.9) = 5.199, P = 0.028), higher infusions in FR (Fgroup(1, 38) = 9.18, P = 0.00439) and increase of infusions under PR schedule (Fgroup(1, 38) = 4.87, P = 0.0335), compared with saline-sired F1 (Fig. 1b). In female offspring, no significant difference was found under FR (lever, Fgroup(1, 34.8) = 0.718, P = 0.403; infusions, Fgroup(1, 34) = 1.273, P = 0.267), but there was an increase of infusions under PR schedule (Fgroup(1, 34) = 4.19, P = 0.0485), compared with saline-sired progeny (Fig. 1c).

Fig. 1: Effects of paternal heroin self-administration on heroin and sucrose self-administration behavior in offspring.

a Mating and behavioral assessment schedule. Rats trained to self-administer heroin (45 μg/kg/inf, HSA) or saline (SSA) were mated with naïve female rats, and offspring generated were subjected to behavioral tests. F2 was obtained by mating random F1 male rats with naïve female rats. b Lever pressed in FR sessions, infusions in FR sessions, and infusions in PR session in heroin SA tests of F1 male offspring. c Lever pressed in FR sessions, infusions in FR sessions, and infusions in PR session in heroin SA tests of F1 female offspring. d Lever pressed in FR sessions, pellets gained in FR sessions, and infusions in PR session in sucrose SA tests of male F1 offspring. e Lever pressed in FR sessions, pellets gained in FR sessions, and infusions in PR session in sucrose SA tests of female F1 offspring. f Lever pressed in FR sessions, infusions in FR sessions, and infusions under PR schedule in PR session in heroin SA tests of F2 male offspring. g Lever pressed in FR sessions, infusions in FR sessions, and infusions in PR session in heroin SA tests of F2 female offspring. For FR tests, results are presented as mean ± s.e.m in the line plot. For PR tests, polygon represents density estimates of data, the box represents the 25th and 75th percentiles, the whiskers show the minimum and maximum of the data, and the line inside the box denotes the median. *P < 0.05, ***P < 0.001.

As significant individual differences in abuse potential have been reported in both human [32] and rodent models [33], we examined whether the pattern of paternal drug-seeking behavior would affect their offspring (Fig. S1d). For a random cohort of 16 SD rats and their offspring, the lever-pressing performance was compared. On day 9 of SA schedule, the lever presses of heroin-experienced F1 generation were significantly higher than that of F0, which restated that paternal heroin-seeking increased heroin seeking in F1 generation (Fig. S1e). Using principal component analysis (PCA), we roughly divided F0 into three clusters: Hi-SA, Med-SA, and Lo-SA (Fig. S1f). Although the three groups showed significant differences in lever pressing during FR (Fig. S1f), no-drug periods (Fig. S1i), and PR tests (Fig. S1l), their offspring performed similarly (Fig. S1g, j, m). No significant correlation was observed in the standardized scores for FR, no-drug period lever pressed, or PR between F0 and F1 (Fig. S1h, k, n). Furthermore, we observed comparable performance in sucrose SA (Fig. 1e–f. Male: FR, Fgroup(1, 21) = 0.171; pellets, Fgroup(1, 21) = 0.801, P = 0.381; infusions under PR, Fgroup(1, 44) = 2.34, P = 0.133. Female: FR, Fgroup(1, 17.2) = 0.016, P = 0.9; pellets, Fgroup(1, 17.212) = 0.219, P = 0.646; infusions under PR, Fgroup(1, 16.9) = 0.005, P = 0.946), distance traveled (male, t(14.5) = -0.174, P = 0.864; female, t(16.5) = 0.937, P = 0.326), time spent in the center area (male, t(14.5) = −0.174, P = 0.864; female, t(16.5) = 0.937, P = 0.362) and entries to the center (male, t(14.5) = −0.174, P = 0.864; female, t(16.5) = 0.937, P = 0.362) of OFT test, time in the light box of LDB test (male, t(36.0) = −0.757, P = 0.454; female, t(15.7) = −0.483, P = 0.636), time in the open arm of EPM test (male, t(13.4) = −1.139, P = 0.275; female, t(15.7) = −0.483, P = 0.636), ratio in novel arm of Y-maze (male, t(36.0) = 1.059, P = 0.297; female, t(15.8) = −0.587, P = 0.565), sociability tests (male, Fgroup×test(1, 72) = 2.73, P = 0.103; female, Fgroup×test(1, 70) = 0.128, P = 0.722) or social recognition tests (male, Fgroup×test(1, 72) = 0.746, P = 0.39; female, Fgroup×test(1, 70) = 1.126, P = 0.292), between the two groups in both sexes (Fig. S2).

Naïve male F1 rats were co-housed with naïve females to produce F2 offspring (Fig. 1a). Both male and female F2 offspring from these two groups exhibited similar lever pressing for heroin under the FR schedule (Fig. 1f-g. FR, male, Fgroup(1, 69.6) = 0.517, P = 0.475; female, Fgroup(1, 21.8) = 0.269, P = 0.609. Infusions, male, Fgroup(1, 67) = 1.14, P = 0.29; female, Fgroup(1, 28.8) = 0.036, P = 0.852), but female F2 offspring sired by heroin-SA experienced grandfathers had a higher infusions under PR schedule (male, Fgroup(1, 24.2) = 0.918, P = 0.347; female Fgroup(1, 47) = 4.14, P = 0.0476). This suggests paternal heroin reinforcement may lead to transgenerational epigenetic inheritance, and significant sex difference in behavior.

Additionally, we conducted a yoked heroin infusion experiment, pairing each SA rat with a yoked control receiving the same dose of drug at the same time (Fig. 2a). Male F1 offspring sired by heroin-SA-experienced fathers showed significantly higher FR5 lever presses than saline-sired F1 rats (lever, Fdrug×deliveryMethod(1, 29.3) = 2.37, P = 0.135; FR5, ***P = 0.00018; infusions, Fdrug×deliveryMethod(1, 29.7) = 3.16, P = 0.0857; FR5 **P = 0.0081), while under yoked infusions, paternal heroin infusions did not induce significant changes in F1 offspring (lever, FR5, P = 0.91019; infusions, P = 0.7521, Fig. 2b). We failed to grasp a significant difference in infusions under PR schedule between the groups (Fig. 2b, Fdrug×deliveryMethod(1, 32.3) = 3.45, P = 0.0724). Male F1 offspring from heroin SA-experienced fathers had higher lever presses than other F1 offspring at various doses (Fig. 2c, lever, Fdrug×deliveryMethod(1, 35.0) = 1.63, P = 0.210; 4.5 μg*kg/inf, HSA-sired F1 vs. HYoke-sired F1, *P = 0.0192; 6.8 μg*kg/inf, HSA-sired F1 vs. SSA-sired F1, *P = 0.0253, HSA-sired F1 vs. HYoke-sired F1, **P = 0.0084; 20 μg*kg/inf, HSA-sired F1 vs. SSA-sired F1, *P = 0.0494). Positive correlations of lever presses at 4.5 μg*kg/inf and 20 μg*kg/inf doses were observed in all groups (Fig. 2d), with similar coefficients (HSA-sired F1 vs. SSA-sired F1, Z = −0.212, P = 0.832; HSA-sired F1 vs. HYoke-sired F1, Z = 1.512, P = 0.131). This suggests an upward shift in the dose-response curve for male F1 offspring sired by heroin self-administration-experienced fathers, likely due to increased motivation rather than enhanced sensitivity to heroin. Overall, these findings indicate that paternal drug-seeking behavior for heroin reinforcement, rather than heroin exposure itself, is crucial for increased heroin self-administration in offspring.

Fig. 2: The inheritance of increased heroin self-administration behavior is contingent on paternally motivated voluntary administration of heroin.

a Naive male rats were randomly paired. In each pair, one rat was allowed to self-administer heroin or saline by pressing the lever (SA rat), while the other passively received the same dose of infusion at the same time when the SA rat pressed the lever (yoked rat). Pairs of Her and Sal groups were used to generate F1 offspring. b Lever pressed in FR sessions, infusions in FR sessions, and infusions in PR session in heroin SA tests of male F1 offspring. c Lever pressed and dosage of drug obtained in heroin dose-response teste of F1 offspring. *, HSA-sired F1 vs. SSA-sired F1. #, ##, HYoke-sired F1 vs. HSA-sired F1. d Correlation of lever press at heroin dose of 4.5 μg/kg/inf and 20 μg/kg/inf of F1 offspring. For FR tests, results are presented as mean ± s.e.m in the line plot. For PR tests, polygon represents density estimates of data, the box represents the 25th and 75th percentiles, the whiskers show the minimum and maximum of the data, and the line inside the box denotes the median. * or # P < 0.05, ** or ## P < 0.01, ***P < 0.001.

Alterations of sperm RNA are causal to the increased heroin self-administration phenotype in offspring

Increasing evidence suggests that alterations in sperm epigenetic signatures, including non-coding RNAs, correlate with phenotypic changes in subsequent generations. We isolated total RNA from sperm of HSA, HYoke, and SSA groups. This RNA was then microinjected into naïve fertilized oocytes (Fig. 3a). The resulting HSA-RNA F1 male exhibited significantly higher lever presses (Fgroup(2, 38.0) = 4.6, P = 0.0163; FR5, HSA-RNA F1 vs. SSA-RNA F1, ***P = 4.00e-4; HSA-RNA F1 vs. HYoke-RNA F1, ***P = 0.000406), and received more infusions (Fgroup(2, 37.0) = 5.97, P = 0.00566; HSA-RNA F1 vs. SSA-RNA F1, *P = 0.0178; HSA-RNA F1 vs. HYoke-RNA F1, *P = 0.0146) for heroin in FR, and higher infusions under PR schedule compared with the F1 offspring from saline-SA-experienced or yoke heroin-infused fathers (Fig. 3b, F(2, 37) = 7.936, P = 0.00136; HYoke-RNA F1 vs.HSA-RNA F1,**P = 0.00556; SSA-RNA F1 vs.HSA-RNA F1,**P = 0.00532), but in female, no significant difference was observed (Fig. 3c, lever, Fgroup(2, 32.1) = 1.40, P = 0.262; infusions, Fgroup(2, 31) = 3.31, P = 0.0499; infusions under PR, F(2, 31) = 0.5, P = 0.612). Furthermore, there were no notable differences in sucrose self-administration behavior among the groups across both sexes (Fig. 3d-e. Male, lever, Fgroup(2, 29.0) = 0.004, P = 0.996; pellets, Fgroup(2, 29) = 0.003, P = 0.997; infusions under PR, Fgroup (2, 29) = 0.995, P = 0.382. Female, lever, Fgroup(2, 32) = 0.493, P = 0.616; pellets, Fgroup(2, 32) = 0.277, P = 0.76; infusions under PR, Fgroup (2, 32) = 0.284, P = 0.755). These data support a causal role for alterations in sperm RNA in the development of increased heroin self-administration behavior in male offspring.

Fig. 3: Alterations in sperm RNA are causally linked to increased heroin self-administration in male F1 offspring.

a Experimental scheme. Total RNAs purified from sperm of HSA, HYoke, or SSA F0 generation were microinjected into naïve fertilized oocytes and then transferred into the oviduct of surrogate rats. b Lever pressed in FR sessions, infusions in FR sessions, and infusions in PR session in heroin SA tests of RNA male offspring. c Lever pressed in FR sessions, infusions in FR sessions, and infusion in PR session in heroin SA tests of RNA female offspring. d Lever pressed in FR sessions, pellets gained in FR sessions, and infusions in PR session in sucrose SA tests of RNA male offspring. e Lever pressed in FR sessions, pellets gained in FR sessions, and infusions in PR session in sucrose SA tests of RNA female offspring. For FR tests, results are presented as mean ± s.e.m in the line plot. For PR tests, polygon represents density estimates of data, the box represents the 25th and 75th percentiles, the whiskers show the minimum and maximum of the data, and the line inside the box denotes the median. **P < 0.01.

Alterations of miRNA expression profiles in sperm and brain after heroin SA are correlated

We performed small RNA sequencing on sperm of F0 generation. PCA revealed key contributions to the differences of miRNAs, tRNAs, and rRNAs (Fig. 4a). Furthermore, analysis of RNA content revealed a notable depletion of sperm miRNAs in heroin self-administration experienced rats compared with the saline control group (Fig. S3a–c, log2(fold change) = −3.54, P = 2.62e-9). Consequently, we focused our further investigations on the differences in miRNA expression.

Fig. 4: Correlation of miRNA expression patterns in sperm and the nucleus accumbens of the F0 generation.

a Principal component analysis of F0 sperm non-coding RNA profiles. Arrows indicate the contribution of each non-coding RNA type to each dimension. b Heatmap and hierarchical clustering of differentially expressed miRNAs between SSA, HSA and HYoke of F0 sperm. c Cellular component enrichment of the four clusters of differentially expressed sperm miRNAs. d Rank Rank Hypergeometric Overlap (RRHO) analysis of differentially expressed miRNAs and tsRNAs in the NAc vs. sperm of the F0 generation. The upper-right and lower-left region of each plot represents co- up/down regulation, while the upper-left and lower-right regions shows inconsistent changes. Color indicates significance. e Overlaps of HYoke vs. SSA, HSA vs. SSA, HSA vs. HYoke. Venn diagram (top) and heatmap (bottom) of candidate miRNAs. The heatmap shows the log10(Counts) of each miRNA in F0 sperm (bars) and the relative fold change of each miRNA in both the sperm and the nucleus accumbens of F0 generation was shown. Colors of miRNA names correspond to the clustering of the miRNA in (b). f Correlation plot. Significance of correlation between sperm and NAc of F0 HSA group (−log10(P-value)) vs. the enrichment score of addiction-related miRNA targets (NPES score). Size represents Pearson correlation coefficient of miRNA expression level between sperm and NAc of F0 HSA group. Colors correspond to the clustering of the miRNA in (b). g Relative expression of candidate miRNAs in sperm and NAc of the F0 generation. Results are shown as mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001.

Hierarchical clustering of differentially expressed miRNAs revealed four distinct clusters of expression patterns (Fig. 4b). Clusters 1 and 3 enriched miRNAs significantly upregulated/downregulated in the sperm of heroin-SA experienced rats, respectively. Cluster 2 contained miRNAs downregulated in response to heroin exposure. In contrast, Cluster 4 was enriched with miRNAs that were inversely expressed in HSA and HYoke groups compared to SSA. Using miRNA set enrichment analysis tool (TAM 2.0) [34], we discovered that altered sperm miRNAs were enriched particularly in the brain, in Clusters 2, 3, and 4 (Fig. 4c). Similarly, miEAA indicated Cluster 3 originated from the cerebrum-thalamus and pituitary (Fig. S3d), and unveiled distinct overrepresented pathways (Fig. S3e). Then we included NAc and mPFC for small RNA-seq, and surprisingly found, using RRHO analysis, a positive correlation between changes of sperm and NAc miRNA profiles (Fig. 4d, upper), but not in tsRNAs (Fig. 4d, lower). Furthermore, the correlation was not found in the mPFC vs. sperm (Fig. S4g). These observations implicate a potential association between heroin-induced epigenetic remodeling of miRNA profiles in NAc brain region and changes in sperm.

By comparing differentially expressed sperm miRNAs among the three groups, we identified 28 core miRNAs with consistent significant alterations in sperm and the NAc (Fig. 4e) from the sequencing data. We also performed transcriptome sequencing in the NAc of F1 (Fig. S4a–b), and confirmed that these core miRNAs indeed significantly modulate gene expression differences in NAc (Fig. S4c). We ranked these core miRNAs for their potential to target addiction-regulating genes using a network propagation strategy [35]. The top-ranked miRNAs-miR-144, let-7 family, miR-19b (Cluster 2), miR-19a (Cluster 3), and miR-181a/b, miR-29a/c (Cluster 4) were selected for further validation in a separate batch of samples of F0 and their F1 offspring. Although we observed some considerable inconsistencies in the HYoke group, compared with sequencing results, the high correlation coefficients between sperm and NAc persisted (Fig. 4f, Fig. S5). Among these, miR-19b exhibited the strongest significance (Fig. 4g) (F(2, 31) = 6.041, P = 0.006, SSA vs. HSA, **P = 0.007; SSA vs HYoke, *P = 0.041) and was altered in the NAc (Fig. S4d, F(2, 9) = 10.01, P = 0.00515, male, HSA-RNA F1 vs. SSA-RNA F1, **P = 0.00215, HYoke-sired-F1 vs. SSA-sired-F1, **P = 0.00381; female, HSA-RNA F1 vs. SSA-RNA F1, *P = 0.0394) and sperm of F1 offspring sired by heroin-SA experienced fathers (Fig. S4e, F(2, 24) = 5.073, P = 0.015; SSA-sired-F1 vs. HSA-sired-F1, *P = 0.012). The results of the PCR study indicated the necessity of further investigation into the role of miR-19b in the mediation of trait transmission and the regulation of heroin SA behavior.

MiR-19b is critically involved in the epigenetic transmission and the increased heroin SA in Heroin F1 offspring

To establish a causal relationship between miR-19b downregulation in sperm of heroin-SA experienced rats and increased heroin self-administration in their offspring, we supplemented sperm RNA of heroin-SA experienced rats with synthetic miR-19b to match levels of saline-SA rats (Fig. S6) and microinjected these into naive fertilized oocytes (Fig. 5a). Normalizing miR-19b levels reduced heroin self-administration behavior of the male HSA-RNA F1 group (Fig. 5b, lever, Fgroup(2, 38.3) = 8.64, P = 0.000798; FR5, HSA-RNA+19b F1 vs. HSA-RNA F1, **P = 0.00327; infusions, Fgroup(2, 37) = 9.561, P = 0.000449; HSA-RNA+19b F1 vs. HSA-RNA F1, P = 0.194; infusions under PR schedule, FGroup(2, 37) = 7.867, P = 0.00142; HSA-RNA+19b F1 vs. HSA-RNA F1,**P = 0.00135), suggesting the critical role of miR-19b in the epigenetic transmission of heroin-induced changes.

Fig. 5: MiR-19b is critical for the epigenetic inheritance and the increased heroin self-administration phenotype in F1 offspring.

Schematics (a) and heroin self-administration tests (b) on RNA offspring obtained by microinjection of RNA from SSA-RNA (SSA-RNA-F1), HSA-RNA + Scr-RNA (HSA-RNA-Scr-F1), miR-19b-normalized HSA-RNA (HSA-RNA-19b-F1) into naïve fertilized oocytes were tested. c In vivo transfection of candidate miRNAs and RNA-seq of the transfected NAc. Upper, Agomir-Scr, or Agomir-19b were in vivo transfected in the NAc of HSA-F1, validated for target miRNA overexpression, and then subjected to RNA-seq. Lower, heat map of differentially expressed genes between groups. d Differential overrepresentation of pathways in agomir-transfected F1. e Performance of heroin SA behavior before and after miR-19b over-expression in the NAc of male HSA-sired F1 generation. f Performance of heroin SA behavior before and after miR-19b knockdown with TuD-miR-19b in the NAc. For FR tests, results are presented as mean ± s.e.m in the line plot. For PR tests, polygon represents density estimates of data, the box represents the 25th and 75th percentiles, the whiskers show the minimum and maximum of the data, and the line inside the box denotes the median. *P < 0.05, **P < 0.01, ***P < 0.001.

Next, we transfected miR-19b in vivo into the NAc of F1 offspring sired by heroin self-administration-experienced fathers and extracted RNA samples for expression analysis and sequencing (Fig. 5c). Both heatmap and pathway overrepresentation analyses confirmed that miR-19b overexpression shifted its transcriptome profiles toward those of F1 sired by yoke heroin-infused fathers, indicating miR-19b’s crucial role in mediating changes induced by paternal heroin-seeking behavior (Fig. 5c, d).

Then, male F1 rats sired by heroin self-administration-experienced fathers were split into two groups based on equal lever pressure after initial heroin-SA training, and received viral injection into the NAc to overexpress miR-19b or miR-Scramble (Fig. 5e, S7). We found that viral expression of miR-19b, unlike the scramble control, significantly attenuated the escalation of lever presses for heroin in the FR5 schedule (Fgroup(2, 28.9) = 4.28, P = 0.0236; FR5-2, **P = 0.00867), down-regulated drug infusions (Fgroup(2, 29.0) = 4.08, P = 0.0276; FR5-2, **P = 0.0178), and the infusions under PR schedule was indifferent compared with SSA-sired F1 (Fgroup (2, 28) = 4.46, P = 0.0209; HSA-F1-AAV-19b vs. SSA-sired F1, P = 1, Fig. 5e). Conversely, we constructed a miRNA sponge (TuD) to antagonize miR-19b (Fig. S8a, b). Naive rats were trained to self-administer heroin and randomly assigned to express TuD-miR-19b or TuD-Scramble in the NAc (Fig. 5f). Inhibition of miR-19b expression decreased lever presses for heroin (Fgroup(1, 16.7) = 1.38, P = 0.257; FR5-2, **P = 0.00229), and exhibited a trend of decrease in number of daily drug infusions (Fgroup(1, 16) = 3.61, P = 0.0759; FR5-2, P = 0.0759) after viral delivery, and down-regulated the infusions under PR schedule in the PR schedule, in contrast to the injection of scramble-TuD (t(15.9) = −2.643, *P = 0.0178). In summary, these results establish a causal link between deregulation of miR-19b and increased heroin self-administration behavior in F1 offspring, supporting the role of miR-19b in mediating increased heroin-seeking behavior and participating in cross-generational epigenetic transmission.