Cocaine reinforcement is transgenerationally inherited

To explore the potential transgenerational effects of cocaine-induced reinforcement, naive male Sprague-Dawley rats were allowed to self-administer cocaine (0.75 mg kg−1 per injection, Cocaine-SA group) or saline (Saline-SA group) by pressing the active lever in 4-h daily sessions. Rats were first subjected to self-administration at a fixed ratio (FR) of 1 (that is, one lever press for an injection), then switched to FR3 and FR5 (three and five lever presses for one injection, respectively) on days 6 and 8, and finally tested on a progressive ratio (PR) schedule on day 13 to assess their motivation for drug reinforcement. Compared with Saline-SA group, Cocaine-SA rats showed significantly higher lever presses during the FR schedules (Fig. 1a left) and higher break point (the number of lever presses to earn the last cocaine injection) in the PR test (Fig. 1a right).

Figure 1: Performance of Addict and Non-addict F0 rats in cocaine self-administration. (a) Naive rats were trained to self-administer saline (Saline SA) or cocaine (Cocaine SA) under a 5-day FR1, 2-day FR3, 5-day FR5, 1-day PR schedule. Cocaine-SA group showed higher lever presses at FR (left) and break point (right) at PR schedules, compared with Saline-SA group. Lever press, Group × FR, mixed model repeated measures (MMRM), χ2 (2)=101.91, P<0.001; Group, ***P<0.001, Saline-SA versus Cocaine-SA. Break point, Mann–Whitney Rank-Sum Test, Z=4.688, ***P<0.001. Saline-SA, n=23; Cocaine-SA, n=134. (b) Scores for cocaine intake and motivation of each Cocaine-SA rat were calculated according to SA performance at FR5 and PR schedules, respectively, and plotted. Cocaine SA rats with a combined drug-seeking behavioural score within the top 25% and the bottom 40% were designated as Addict F0 (red circle) and Non-addict F0 (blue circle), respectively. (c) Addict F0 and Non-addict F0 rats were crossed with naive female rats to generate Addict F1 and Non-addict F1, which were then randomly designated for mating to generate F2 or for behavioural testing. (d) Addict F1 rats displayed higher cocaine consumption and motivation than Non-addict F1. Lever press, Group × FR, MMRM, χ2 (2)=23.33, P<0.001; Group, P=0.041; FR5, ***P<0.001, Non-addict F1 versus Addict F1. Break point, Mann–Whitney Rank-Sum Test, Z=3.272, ***P<0.001. Non-addict F1, n=44; Addict F1, n=45. (e) There was no difference in intake and motivation for sucrose reward in F1. Lever press, Group × FR, MMRM, χ2 (1)=10.1, P=0.32. Break point, Mann–Whitney Rank-Sum Test, Z=1.772, P=0.076. Non-addict F1, n=31; Addict F1, n=32. Data are expressed as mean±s.e.m. Full size image

The individual performance of each of the 134 Cocaine-SA rats in cocaine self-administration was analysed. Uncontrollable urges to seek drugs and excessive drug consumption are important characteristics of addiction. Thus, to reflect the incentive motivational responding to cocaine reward, a combined behavioural score was calculated for each Cocaine-SA rat, by addition of normalized score derived from average lever pressing during FR5 program, and break point during PR schedule. Cocaine-SA rats with the top 25% and the bottom 40% of the behavioural score were designated as Addict F0 (red circle) and Non-addict F0 (blue circle), respectively (Fig. 1b).

The Addict and Non-addict (F0) rats were then mated with naive female rats (Fig. 1c). Addict and Non-addict F1 rats (17 and 16 litters respectively) were obtained, and 2–6 adult male rats from each litter were subjected to the cocaine self-administration test. The F1 offspring of Addict rats, as compared with Non-addict F1, exhibited higher lever presses for cocaine injections during the FR program, and higher motivation for cocaine reinforcement as indicated by break point (Fig. 1d). Interestingly, the third litter of Addict F0, conceived after 6–7 weeks of abstinence, still showed higher voluntary cocaine intake in FR5 sessions compared with the third litter from Non-addict rats (Supplementary Fig. 1). Hyperactivity, reduced cocaine sensitivity and enhanced reward learning ability may increase lever presses in self-administration. Therefore, we conducted three series of experiments to exclude the possibility that any of the above-mentioned factors might have influenced our behavioural readout. In the open field test, no difference in basal locomotor activity and anxiety level between Addict and Non-addict F1 rats was observed (Fig. 2a). Moreover, Addict F1 and Non-addict F1 showed similar dose-dependent responses to cocaine in the sensitivity test (Fig. 2b). Finally, in sucrose and food (natural rewards) self-administration tests, the two groups exhibited equivalent self-administration learning curves and break points (Figs 1e and 2c).

Figure 2: Comparable performance of Addict F1 and Non-addict F1 rats in basal and cocaine-induced locomotion activity and food self-administration. (a) Open field test. Total distance, Student’s t-test, t (64)=0.872, P=0.386. Time in centre, Student’s t-test, t (64)=1.197, P=0.236. Entries to centre, Student’s t-test, t (64)=0.142, P=0.887. Non-addict F1, n=37; Addict F1, n=29. (b) Sensitivity to cocaine-induced locomotor activity. Two-way RM ANOVA, F Group × Dose (4, 80)=0.734, P=0.571. Non-addict F1, n=11; Addict F1, n=11. (c) Food self-administration test. Lever press, Group × FR, MMRM, χ2 (1)=0.19, P=0.663. Break point, Mann–Whitney Rank-Sum Test, Z=1.039, P=0.299. Non-addict F1, n=12; Addict F1, n=17. Data are expressed as mean±s.e.m. Full size image

The phenotype observed in F1 generation could arise from direct exposure of the sperm of F0 rats to cocaine rather than from transgenerational transmission mechanisms. To exclude this possibility, Addict F2 and Non-addict F2 rats, sired by naive F1, were subjected to evaluations in cocaine self-administration. Similar to our observations in their F0 and F1 ascendants, Addict F2 rats displayed higher lever presses under FR5 schedule, and higher break point than Non-addict F2 (Fig. 3a), while their performance in sucrose self-administration (Fig. 3b), open field (Fig. 3c), and their response to acute cocaine doses was comparable to Non-addict F2 group (Fig. 3d). These data suggest that paternal propensity to develop high cocaine-seeking behaviour could be transgenerationally maintained in subsequent generations.

Figure 3: Addict F2 displayed higher responding to cocaine-induced reinforcement than Non-addict F2. (a) Addict F2 rats displayed higher cocaine consumption and motivation than Non-addict F2. Lever press, Group × FR, MMRM, χ2 (2)=28.63, P<0.001; Group, P=0.052; FR5, ***P<0.001, Non-addict F2 versus Addict F2. Break point, Mann–Whitney Rank-Sum Test, Z=2.513, *P=0.013. Non-addict F2, n=14; Addict F2, n=28. (b) Sucrose self-administration. Lever press, Group × FR, MMRM, χ2 (1)=0.29, P=0.588. Break point, Mann–Whitney Rank-Sum Test, Z=0.783, P=0.433. Non-addict F2, n=27; Addict F2, n=21. (c) Open field test. Total distance, Student’s t-test, t (54)=0.0964, P=0.924. Time in centre, Student’s t-test, t (54)=0.729, P=0.469. Entries to centre, Student’s t-test, t (54) =−1.796, P=0.0781. Non-addict F2, n=23; Addict F2, n=33. (d) Cocaine-induced locomotor activity. Two-way RM ANOVA, F group × day (4, 68)=1.170, P=0.295. Non-addict F2, n=10; Addict F2, n=9. Data are expressed as mean±s.e.m. Full size image

Motivation determines cocaine response in offspring

An alternative interpretation for the transgenerational effect is that Addict F0 self-administered more cocaine than Non-addict F0. Thus, inheritance could stem from the exposure to cocaine per se and may not represent incentive motivational effect of cocaine. To examine the effect of cocaine exposure, another cohort of naive rats was given twice-daily intraperitoneal injections of cocaine (15 mg kg−1, Cocaine-IP) or saline (Saline-IP) for 7 days and the response of their F1, F2 offspring in cocaine self-administration was tested (Fig. 4a). Interestingly, Cocaine-IP F1 sired by rats receiving non-contingent cocaine injections showed significantly lower responses than Saline-IP F1 rats (Fig. 4b), while the F2 offspring of Cocaine-IP and Saline-IP exhibited no difference in cocaine self-administration (Fig. 4c). Moreover, as shown in Supplementary Fig. 2, cocaine response of Addict F1 group was significantly higher than Saline F1 under FR5 schedule, and Non-addict F1 exhibited lower breakpoint than Saline F1. These data suggest that chronic cocaine intake results in lower drug responding in F1 generation, that is, a protective effect, and that the high response for cocaine-induced reinforcement observed in Addict offspring is unlikely caused solely by excessive exposure to high doses of cocaine. Previous studies have shown that only a portion of drug users or animals subjected to self-administration of addictive drugs become addicted17,18. Combined with the result of Vassoler et al.19 showing that F1 offspring of rats that self-administered cocaine exhibit lower responding than F1 from saline exposed animals, we speculate that the transgenerational transmission of high cocaine incentive response observed may be dependent on the addiction-like state in the F0 generation, and the incentive motivational effect causes increased susceptibility to cocaine-seeking behaviour or decreased protective effect in offspring.

Figure 4: Non-contingent chronic cocaine injections induce inter-generational cocaine resistance. (a) Rats received 15 mg kg−1 cocaine (Cocaine-IP F0) or equal volume of saline (Saline-IP F0) intraperitoneal injections twice daily for 7 days and were mated with naive females one day after the last injection. Four litters from each of group were randomly designated for behavioural experiment or for mating to generate F2. (b) Cocaine-IP F1 did not show an increase but displayed a decrease tendency in lever press and break point for cocaine in self-administration test, compared with Saline-IP F1 rats. Lever press, Group × FR, MMRM, χ2 (2)=13.00, P=0.0015; FR5, **P=0.009, Saline-IP F1 versus Cocaine-IP F1. Break point, Mann–Whitney Rank-Sum Test, Z=1.060, P=0.289. Saline-IP F1, n=8; Cocaine-IP F1, n=8. (c) There was no statistical difference in lever press or break point between Saline-IP F2 and Cocaine-IP F2. Lever press, Group × FR, MMRM, χ2 (2)=2.60, P=0.272. Break point, Mann–Whitney Rank-Sum Test, Z=0.638, P=0.526. Saline-IP F2, n=8; Cocaine-IP F2, n=8. Full size image

To further discriminate the effects of non-contingent and motivational intake of cocaine, yoked cocaine infusion experiment was carried out. In yoked cocaine delivery procedure, one rat self-administered cocaine by actively pressing the lever (SA), while the paired rat (Yoke) passively received the same dose of cocaine at the same time (Fig. 5a). Addict and Non-addict F0 in SA, as determined by the combined score, and their paired Yoke groups, were mated to generate F1. In contrast to the significantly higher drug intake and motivation for drug reinforcement observed in F1 of Addict-SA group compared with Non-addict-SA group, no difference in lever presses or break point for cocaine was observed between Addict-Yoke F1 and Non-addict-Yoke F1 rats (Fig. 5b). Non-addict-SA F1, but not Addict-SA F1, exhibited lower FR5 responding and break point than Saline-SA F1 rats (Fig. 5b), suggesting a loss of a protective effect in the Addict-SA F1 group. Each pair of Addict-SA and Addict-Yoke F0 rats received the same amount of cocaine simultaneously; however, the offspring of the two groups exhibited significant difference in lever press for cocaine. There was no difference in cocaine self-administration found between offspring of Saline-SA and Saline-Yoke groups. These results support the notion that high incentive motivation for cocaine, but not the intake of an addictive drug itself, renders transgenerational inheritance of vulnerability and/or reduce resistance to drug addiction in descendants.

Figure 5: The inheritance of vulnerability to cocaine reinforcement is contingent on highly motivated cocaine self-administration. (a) Naive male rats were randomly paired. In each pair, one rat was allowed to actively self-administer cocaine or saline by pressing the lever (SA group), and with each drug infusion by SA group rat, a yoked infusion of exactly the same dose of cocaine or saline (Saline-Yoke) was delivered at the same time passively (without lever press) to the paired rat (Yoke group). The yoked rats corresponding to Saline-SA, Addict-SA and Non-addict-SA groups were designated as Saline-Yoke, Addict-Yoke and Non-addict-Yoke, respectively. (b) Addict-SA F1 rats showed significantly higher lever press and break point for cocaine than Non-addict-SA F1, but there was no difference between Addict-Yoke F1 and Non-addict-Yoke F1. Lever press, Group × Yoke × FR, MMRM, χ2 (4)=35.55, P<0.001; P<0.001, Saline F1 versus Non-addict F1, P<0.001, Non-addict F1 versus Addict F1; FR5, ***P<0.001, Addict-SA F1 versus Non-addict-SA F1, *P=0.013, Addict-SA F1 versus Saline-SA F1, ***P<0.001, Addict-SA F1 versus Addict-Yoke F1; *P=0.038, Addict-Yoke F1 versus Saline-Yoke F1. Break point, two-way ANOVA by Ranks, F Group × Yoke (2, 91)=3.604, P=0.090; ***P<0.001, Addict-SA F1 versus Non-addict-SA F1, ***P<0.001, Non-addict-SA F1 versus Saline-SA F1, *P=0.041, Addict-SA F1 versus Addict-Yoke F1. Non-addict-SA F1, n=17; Non-addict-Yoke F1, n=9; Addict-SA F1, n=24; Addict-Yoke F1, n=14; Saline-SA F1, n=20; Saline-Yoke F1, n=12. Data are expressed as mean±s.e.m. Full size image

As Vassoler et al.19 found that the cocaine-resistant phenotype was attributed to increase in BDNF level of F1 male cocaine sired rats, we also measured BDNF level in mPFC of drug- and experiment- naive F1 rats from Saline, Non-addict-SA, Non-addict-Yoke, Addict-SA and Addict-Yoke groups (Fig. 6). The results showed that, F1 descendants of cocaine-experienced fathers (Non-addict-SA, Non-addict-Yoke, Addict-SA, and Addict-Yoke rats) showed cocaine dose-dependent elevation of BDNF level, compared with F1 offspring of Saline-SA group. However, BDNF level between SA and yoked rats from Addict or Non-addict groups was not different. These data indicate that paternal cocaine experience induces BDNF elevation in F1 offspring, and this is correlated with the amount of cocaine taken by F0 fathers, not motivation for reinforcement.

Figure 6: Elevated BDNF level in F1 correlates with the amount of cocaine taken by F0 fathers. BDNF level in mPFC of cocaine naive F1 rats of Saline-SA, Non-addict-SA, Non-addict-Yoke, Addict-SA and Addict-Yoke groups. (a) Representative blotting of two samples from each group was shown. (b) Statistical analysis of BDNF level in each group. BDNF level normalized by β-actin was acquired, and then relative protein level of each sample was divided by average of BDNF/β-actin in Saline F1 group. One-way ANOVA, F (4, 20)=7.251, P<0.001; Bonferroni post hoc, ***P<0.001, compared with Saline F1. n=5 each. Data are expressed as mean±s.e.m. Full size image

Inheritance of drug seeking behaviour is an acquired trait

The transgenerational transmission of high incentive motivation for cocaine could be attributed to pre-existing genetic variations or to acquired heritable epigenetic modifications. To dissociate the two possible mechanisms, we mated naive rats first to generate F1 offspring, and one week after mating, subjected the mated males to cocaine self-administration to identify Addict and Non-addict rats according to their performance (Fig. 7a). The offspring sired by Addict and Non-addict F0 rats before their exposure to cocaine were designated as Pre-Addict F1 and Pre-Non-addict F1 rats. In contrast to what we have observed in descendants of F0 rats mated after drug exposure (Fig. 1d), Pre-Addict F1 and Pre-Non-addict F1 rats exhibited no difference in cocaine self-administered or motivation for cocaine in PR test (Fig. 7b). No differences were observed in the animals’ performance in the open field or the sucrose acquisition and motivation between these two groups (Supplementary Figs. 3a,b). Moreover, cocaine intake and motivation were similar in the mated and non-mated rats (Supplementary Fig. 3c). These data, combined with the results of yoke and intraperitoneal administration experiments, suggest that transgenerational inheritance of high incentive motivation for cocaine is an acquired trait that involves non-genetic mechanisms.

Figure 7: Transgenerational inheritance of cocaine reinforcement is an acquired trait. (a) Naive male rats were crossed with naive females first, and one week after mating were subjected to cocaine self-administration procedures to identify Addict and Non-addict rats according to their cocaine intake and motivation scores. The offspring sired by Addict and Non-addict rats (before their exposure to cocaine) were designated as Pre-Addict F1 and Pre- Non-addict F1. (b) Pre- Non-addict F1 and Pre-Addict F1 rats showed no difference in lever press and break point. Lever press, Group × FR, MMRM, χ2 (2)=0.79, P=0.68. Break point, Mann–Whitney Rank-Sum Test, Z=1.273, P=0.203. Pre- Non-addict F1, n=14; Pre-Addict F1, n=12. Data are expressed as mean±s.e.m. Full size image

Sperm methylome difference is maintained across generations

DNA methylation, a stable epigenetic modification that regulates gene transcription and splicing, may be one of possible mechanisms for transgenerational inheritance of acquired traits20. We therefore performed reduced-representation bisulfite sequencing (RRBS) to assess DNA methylation-enriched sites in sperm samples from Saline F0, Addict F0, Non-addict F0, Addict F1, and Non-addict F1. Sperm samples of 3–4 rats per group were individually sequenced at 20M-read depth. In the 16 samples sequenced, total 1,570,110 CpGs were detected in more than 6 samples and with ≥5 × coverage. We first calculated the associations of generation (F0,F1) and phenotype (Non-addict, Addict, Saline) traits with the global methylation profiles. Moderate general demethylation across all genetic elements analysed was detected in both Addict F0 and Non-addict F0 sperms, compared with Saline F0, likely due to the effect of cocaine intake; and there was also difference between Addict and Non-addict groups, indicating possible association of DNA methylation with incentive motivation for cocaine (Fig. 8a).

Figure 8: Identification of maintained DNA methylation alterations in the sperm of Addict F0 and F1 rats. (a) The average methylation levels of CpG sites upstream of the transcription start site (TSS), within the gene body, and the downstream of transcription termination sites (TES) in all genes detected. Addict F0 and Non-addict F0 showed lower global methylation level than Saline F0. (b) Euclidean distance-based multidimensional scaling indicated segregation of generation and addiction state. Number in circle refers to subject number in each group, corresponding to the subject number in c. (c) Methylation in 1,244 CpGs was significantly different between Addict F0 and Non-addict F0, and 522 differentially methylated CpGs were detected in Addict F1 versus Non-addict F1. The differential methylation was maintained in 475 CpGs across F0 to F1 generation. FDR≤0.05 was considered significant. (d) CpG distributions in TSS±2,000 bp area, exons, introns, and other intergenic elements. CpG methylation differences maintained from F0 to F1 were enriched in TSS±2,000 bp area. Differences maintained from F0 to F1/Global, odds ratio (95% confidence intervals), 273.95 (55.82-1340.73). (e) Ontology analysis of CpGs (in TSS±2,000 bp region) with differential methylation maintained from F0 to F1. Z scores from Enrichr were acquired and ranked. (f) Motif enrichment analysis of TSS±2,000 bp region by rGADEM and MotIV. The results revealed specific enrichment of KLF4 binding sites (e=5.67 × 10−10). (g) Methylation profiles of representative CpGs within differential methylation region (DMR) of Klf4-regulated genes. DMRs were calculated by BiSeq. Group averages are plotted. Btg2: DMR, Chr13: 55970169–55970230, three-way RM ANOVA, F addict × generation × site (9, 81)=7.863, P=3.03 × 10−8; Bonferroni post hoc, P=3.5 × 10−14, Non-addict versus Addict. Nr4a1: DMR, Chr7: 140712984–140713087; three-way RM ANOVA, F addict × generation × site (8, 72)=0.832, P=0.58; Bonferroni post hoc, P=1.1 × 10−11, Non-addict versus Addict. Non-addict F0, n=3; Addict F0, n=3; Non-addict F1, n=4; Addict F1, n=3. Full size image

Multidimensional scaling revealed clear segregation of F0 with F1 generations in Dimension 1, correlating with the global demethylation caused by direct drug exposure, and separation of Addict and Non-addict groups in Dimension 2 (Fig. 8b), Notably, Fisher's exact test also indicate associations of generation and phenotype traits (generation, P=4.0 × 10−5; phenotype, P=4.0 × 10−5; generation × phenotype, P=0.015).

Next, we sought to find the methylation differences, if any, that might be relevant to the transgenerational inheritance. Therefore, we identified CpG sites that were differentially methylated between Addict versus Non-addict F0 and maintained across generations. We found that methylation at 1,244 CpG sites was significantly different between Addict and Non-addict F0 (Fig. 4c, upper panel, P<0.05) and 522 CpG sites were differentially methylated in their F1 offspring (Fig. 8c, middle panel). Importantly, 475 differentially methylated CpG sites were maintained in F0 and F1 generation (hereafter referred to as Maintained Differences, Fig. 8c, lower panel). Maintained methylation differences were enriched in hypo-methylated regions (maximum methylation level ≤0.3, 93.7% in CpGs with Maintained Differences versus 34.2% in CpGs commonly covered) and also featured in TSS±2,000 bp distribution (Fig. 8d; 66.5% in CpGs with Maintained Differences versus 34.8% in CpGs commonly covered), indicating their potential involvement in transcriptional modulation of promoter region. Ontology analysis of genes with Maintained Differences in TSS±2,000 bp region also indicate that GO terms associated with development and morphology are overrepresented (Fig. 8e and Supplementary Table 1). Interestingly, binding motifs for transcription factors, including KLF4, SP1, EGR1 and ZFX, were enriched in these regions (Fig. 8f). Transcription activity of KLF4, which has the most significant e-value, has been demonstrated to be regulated by methylation level21. KLF4 binding sites were detected in promoters of Btg2 and Nr4a1, and they exhibited preserved higher methylation in Addict groups than Non-addict groups (Fig. 8g). This result was consistent with previous report that both Btg2 and Nr4a1 regulate neurogenesis and other brain functions via transcriptional mechanisms22,23.

To provide insight into potential mechanistic link of sperm DNA methylation changes to descendant brain functions, we performed transcriptome sequencing of Non-addict F1 and Addict F1 in the nucleus accumbens (NAc). 292 genes exhibit higher expression level in Non-addict F1 than Addict F1 (fold change ≥2, P<0.05), around 3.5-fold of the number downregulated, coincides with lower DNA methylation level in sperms of Non-addict groups than Addict groups (Supplementary Fig. 4a). Although it is well known that DNA methylation profiles change dynamically during development, and differs from sperm to brain, we found that differential promoter methylation in F1 sperm significantly collapsed with alterations of gene expression in adult NAc (Supplementary Fig. 4b). Forty-nine of the 292 genes, namely Gabrd,Chat,Kcnn3 showed transcriptional differences in F1 of Addict versus Non-addict groups, consistent with DNA methylation differences in sperm maintained from F0 to F1 (Supplementary Figs. 4c, d). The differentially methylated and expressed genes from RRBS and RNA-sequencing (RNA-seq) data sets were compared, and a functionally grouped annotation network was deduced. As shown in Supplementary Fig. 4e, genes that differential methylated between Addict and Non-addict groups were enriched in functions that regulate developmental process, consistent with the result shown in Fig. 8e. Several pathways, such as EGFR regulation, gap junction, and axonal guidance pathways, were co-enriched in methylation and transcription data, but in a co-clustering pathway, often distinct genes from methylation and transcription datasets were involved. Network analysis of genes from these clusters reveals possible signalling network connections between genes enriched in methylation and expression. As shown in Supplementary Fig. 4f, ephrins and EPH-related receptors (EPHA5, EFNA1), SHH, ISL1 and TBX5, which strictly controls embryonic development and were differentially methylated in sperm, regulate key plasticity-regulators, such as BDNF, JUN, and Tubulin family members. These downstream molecules were enriched in transcriptome data.