Long-Term Parental Methamphetamine Exposure of Mice Influences

ORIGINAL ARTICLE Long-term parental methamphetamine exposure of mice inﬂuences behavior and hippocampal DNA methylation of the offspring

Y Itzhak1, I Ergui2 and JI Young2,3

The high rate of methamphetamine (METH) abuse among young adults and women of childbearing age makes it imperative to determine the long-term effects of METH exposure on the offspring. We hypothesized that parental METH exposure modulates offspring behavior by disrupting epigenetic programming of gene expression in the brain. To simulate the human pattern of drug use, male and female C57Bl/6J mice were exposed to escalating doses of METH or saline from adolescence through adulthood; following mating, females continue to receive drug or saline through gestational day 17. F1 METH male offspring showed enhanced response to cocaine-conditioned reward and hyperlocomotion. Both F1 METH male and female offspring had reduced response to conditioned fear. Cross-fostering experiments have shown that certain behavioral phenotypes were modulated by maternal care of either METH or saline dams. Analysis of offspring hippocampal DNA methylation showed differentially methylated regions as a result of both METH in utero exposure and maternal care. Our results suggest that behavioral phenotypes and epigenotypes of offspring that were exposed to METH in utero are vulnerable to (a) METH exposure during embryonic development, a period when wide epigenetic reprogramming occurs, and (b) postnatal maternal care.

Molecular Psychiatry (2015) 20, 252–262; doi:10.1038/mp.2014.7; published online 18 February 2014 Keywords: behavior; cross-fostering; epigenetics; methamphetamine; prenatal exposure

INTRODUCTION on neurobehavioral effects in adolescence and adulthood are Methamphetamine (METH) is a psychostimulant and a major drug currently unknown. A few studies have investigated F1 rat of abuse in many parts of the world. Amphetamines inhibit the progeny of dams exposed to METH during gestation. Male F1 re-uptake of dopamine (DA), leading to reverse transport of DA rat progeny were sensitized to the psychomotor stimulating effect from the cytoplasm to the extracellular space, resulting in a of METH.28,29 METH caused oxidative DNA damage in embryonic massive increase in extracellular DA.1,2 METH users suffer from and fetal brain, leading to long-term postnatal neurodevelop- depression, suicidal behavior and anxiety,3–7 and METH-induced mental deficits unrelated to dopaminergic neurotoxicity.30 How- psychosis has been the focus of several studies.8,9 The conse- ever, aside from these reports, the consequences of prenatal quence of METH on cognitive function is long lasting; both METH exposure are unknown. Moreover, studies on animal models abstinent and current users show significant memory of the effect of in utero psychostimulant exposure on offspring impairments.10 Based on 2004–2005 and 2006–2007 surveys of were routinely carried out by administering the drug from past-month illicit drug use among females aged 15–44 years, gestational day 8 through 19. Although this short exposure of 5.3–7% of these women were pregnant and continued to use rats to drugs of abuse may reflect the first and second trimester in drugs during all three trimesters of pregnancy; 2000–3000 abused human,31 it does not simulate the human drug addict experience METH.11 Although serious concerns were raised about pregnant that starts usually in adolescence and continues through women using METH,12 the literature on METH-exposed children is adulthood, and then through pregnancy. sparse compared with that on cocaine-exposed babies.13 Reported The goal of this study was to simulate the human pattern of effects of METH exposure in children include reduced birth weight parental METH exposure and its effects on the offspring. To this – – and size,14 17 poor growth,18 neuroanatomical changes,19 21 end, male and female mice (F0) were exposed to an intermittent increased stress,22 and learning and memory deficits.23,24 These escalating regimen of METH or saline from adolescence through outcomes may result from direct uterine exposure to METH and adulthood. In adulthood, parental F0 METH produced F1 METH also from maladaptive parenting and caregiving.25,26 Further, poor progeny, and parental F0 saline produced F1 saline progeny. To maternal care by METH-exposed women may arise not only from investigate the effect of maternal care on F1 progeny, half of F1 drug use per se but also from other socioeconomic factors.27 METH progeny was cross-fostered to saline dams and half of the Owing to the scarcity of longitudinal studies on METH-exposed F1 saline progeny was cross-fostered to METH dams. In children, the long-term consequences of in utero METH exposure adolescence, a subset of F1 mice was subjected to several

1Department of Psychiatry and Behavioral Sciences, Cellular and Molecular Pharmacology and Neuroscience Division, University of Miami Miller School of Medicine, Miami, FL, USA; 2John P. Hussman Institute for Human Genomics, University of Miami Miller School of Medicine, Miami, FL, USA and 3Dr John T. Macdonald Foundation Department of Human Genetics, University of Miami Miller School of Medicine, Miami, FL, USA. Correspondence: Professor Y Itzhak or Dr JI Young, Department of Psychiatry and Behavioral Sciences, Cellular and Molecular Pharmacology and Neuroscience Division, University of Miami Miller School of Medicine, 1011 NW 15th Street, Gautier Buillding, Room 503, Miami, FL 33136, USA. E-mail: [email protected] or [email protected] Received 12 September 2013; revised 6 January 2014; accepted 9 January 2014; published online 18 February 2014 Consequences of parental methamphetamine exposure Y Itzhak et al 253 behavioral assessments and others were set aside for analysis of of each offspring group (METH,n= 4 and saline, n = 4) were combined. DNA methylation profile in the hippocampus. We report that both Concentrations of DA and its metabolites, 3,4-dihydoxyphenylacetic acid in utero METH exposure and maternal care influence adolescent and homovanillic acid, in the striatum were quantitated by a modified method of high-performance liquid chromatography combined with offspring response to novelty and aversive and appetitive stimuli. 36 These phenotypes are accompanied by significant changes in electrochemical detection as we previously described. hippocampal DNA methylation. Our findings suggest that long- term parental METH exposure including in utero drug exposure Maternal behavior lead to long-lasting changes in DNA methylation and behavior of Maternal behavior of METH dams (n = 5) and saline dams (n = 5 ) was the offspring, resulting from direct drug exposure and altered monitored by an observer blind to the treatments. On PD6, PD7, PD10, postnatal maternal influence. PD12, PD14 and PD17 three major activities were recorded for 15 min and summarized as percent time dams spent in (1) crouch (arched back) and nursing, (2) self-grooming and (3) away from the pups. MATERIALS AND METHODS Subjects Behavioral tests of F1’s Drug treatments and generation of F1 progeny. Eight-week-old C57Bl/6J Cocaine-induced conditioned place preference and locomotor activity. The mice (Jackson Laboratory; Bar Harbor, Maine, USA) were mated and conditioned place preference (CPP) paradigm is a useful tool for evaluating produced 22 offspring that were born on the same day. Following weaning the motivational effect of drugs of abuse, drug discrimination studies and 37 on postnatal day (PD) 28, mice were housed in same sex groups of 4–5 per the reinstatement of drug-seeking behavior. It was recently reported that cage. On PD32, mice were randomly assigned to two treatment groups: the male progeny of sirs who were subjected to cocaine self- METH (n = 6 males and n = 5 females) and saline (n = 5 males and n =5 administration showed reduced propensity to cocaine self- 38 females). METH-HCl (Sigma-Aldrich, St Louis, MO, USA) was dissolved in administration. Although our experimental design of parental drug saline. Starting on PD33, mice of both sexes received every-other-day exposure was different, we sought to investigate cross-sensitivity of F1 intraperitoneal (i.p.) injections of either saline or an escalating dose of METH progeny to the rewarding effect of cocaine. METH (0.1 ml per 10 g weight) as follows: PD33–39, 0.5 mg kg − 1 (four Custom-designed Plexiglas cages (42(length) × 20(width) × 20(height) cm; injections per week); PD40–46, 1mg kg − 1 (three injections per week); Opto-Max Activity Meter v2.16; Columbus Instruments, Columbus, OH, USA) PD47–53, 2 mg kg − 1 (four injections per week); PD54–60, 4 mg kg − 1 (three were used to monitor place preference. The training context consisted of injections per week). On PD60, which was injection-free day, saline males two compartments separated by a divider. One compartment had black- were mated with saline females and METH males were mated with METH and white-striped (2.0 cm apart) walls and a white floor covered with females. From PD61 and onward, saline and a fixed dose of METH (4 mg stainless-steel grid, whereas the other compartment had black walls and kg − 1) were administered to saline and METH mating pairs every-other-day, smooth black floor, thus providing distinct visual and tactile cues. Each except that females now received the injections subcutaneously (s.c.). The compartment was scanned by seven infrared beams at a rate of 10 Hz (2.54 pharmacokinetics of i.p. and s.c. METH administration are quite similar.32 cm intervals). Time spent in each compartment and locomotor activity were Females were examined for vaginal plug every morning; by PD63–64 recorded and analyzed by the Opto-max interface and software (Columbus vaginal plugs were observed in both saline and METH females. Gestational Instruments, Columbus, OH, USA). Experiments were performed as we 37,39 length was 19 days for both groups; all injections stopped on gestation day previously described. On the first day, mice were habituated (20 min) to 17. Body weight of saline and METH dams during gestation were recorded a two-compartment apparatus. During the next 4 days, conditioning was and subsequently pups’ weights were recorded. The average litter size of performed in an unbiased design (cocaine was paired with either the saline and METH pups was 7.8 and 7.4, respectively. To confirm the results striped or the black compartment). A morning session involved saline of the first set of experiments, a second set included F0 males and females injection (i.p.) and confinement to one compartment (30 min); the − 1 that received the same schedule of METH (n = 3/sex) and saline (n = 3/sex). afternoon session involved cocaine injection (i.p.; 10 mg kg ) and Because results of the two sets of experiments were similar, the final confinement to the other compartment. Time spent in each compartment analysis included offspring of eight METH and eight saline dams. was recorded (20 min) 72 h following the last training session. Half of the subjects from each of the four groups (n =13–14) were – Cross-fostering. Several studies suggest that early rearing conditions, and trained for CPP during late adolescence (PD46 51) and the other half – fl (n =13–14) were trained in mid-adulthood (PD87–94). Routinely, each in particular, the quality of mother infant interactions, may in uence brain – development resulting in long-lasting implications for behavioral and group (n =13 14) contained a similar number of males and females. To physiological responses through epigenetic changes in gene expression counterbalance the order of conditioning experiments, the late-adolescent 33,34 group was tested for fear conditioning in mid-adulthood; mice that were that persist into adulthood. One of our goals was to investigate the fi effect of fostering of METH pups to saline dams and saline pups to METH tested for CPP in mid-adulthood were rst tested for fear conditioning in late adolescence. Data analysis showed neither significant ‘previous- dams. We followed a fostering paradigm that described both partial and ’ fi full replacement of pups.35 Within 24–36 h of parturition (PD0), litters were conditioning effect nor signi cant age effect, and thus results of the two culled into 6–8 pups and half of the pups from each litter were transferred sub-groups were collapsed. to an opposite-treatment dam. Consequently, each dam reared 3–4 of her own biological pups and 3–4 fostered pups. The weight of the pups was Fear conditioning. The goal of this experiment was to investigate whether recorded from PD3 to PD27. in utero METH exposure and maternal care influence formation of long- Subsequently, saline pups (total n = 62) and METH pups (n = 60) yielded term memory of an aversive stimulus. Fear conditioning training and four groups (comprising both sexes) as follows: (1) saline pups reared by testing occurred in Plexiglas chambers (30.5 × 30.5 × 43.5 cm; Noldus biological saline dams (SpSd group; males n = 14 and females n = 16); (2) Information Technology, Leesburg, VA, USA). Each chamber was equipped saline pups fostered by METH dams (SpMd group; males n = 15 and with a stainless-steel rod floor through which an electric shock was females n = 17); (3) METH pups reared by biological METH dams (MpMd delivered, and an upper control panel containing a video camera, a sound group; males n = 15 and females n = 16); (4) METH pups fostered by saline emitter and a white light illuminating one corner of the chamber. Mice dams (MpSd group; males n = 14 and females n = 15). The four F1 groups were fear conditioned to an auditory cue that was followed by a single 40 are described as SpSd, SpMd, MpMd and MpSd; the first letter signifies the footshock (0.75 mA; 2 s) in context A, as we previously described. After pup origin and the second letter signifies the nursing dam. 24 h, mice were placed in context B that differed from context A, and percent freezing during the sound of the auditory cue (2 min) was analyzed. The freezing magnitude in response to the auditory cue 24 h Striatal DA levels after training represents expression of long-term memory. We investigated whether administration of METH to dams and offspring in utero METH exposure resulted in depletion of striatal DA and its Spontaneous locomotor activity. The locomotor activity cages were metabolites. METH dams (n = 3) and saline dams (n = 3) were killed standard transparent rectangular rodent cages (42 × 24 × 20 cm high). following pups’ weaning and the striatum was extracted. F1 METH male Each mouse was placed in the cage for 30 min and locomotion was (n = 2) and female (n = 2) offspring of the MpMd group were killed around recorded by an activity meter (Opto-Varimex-Mini Model B; Columbus PD40–PD45. Saline offsprings were used as controls. Results of both sexes Instruments) that consisted of an array of 15 infrared emitter/detector

© 2015 Macmillan Publishers Limited Molecular Psychiatry (2015), 252 – 262 Consequences of parental methamphetamine exposure Y Itzhak et al 254 pairs, spaced at 2.65 cm intervals, measuring activity along a single axis of For the MeDIP studies, to compare differentially enriched regions motion. Spontaneous locomotor activity was recorded on PD41 and PD42. between samples, the log2 ratio values were averaged and then used to calculate the M′ value (M_ = average(log2 MeDIPE/inputE) − average(log2 fi Preference for black and white compartments. The two-compartment MeDIPC/ inputC)) for each probe. NimbleScan sliding-window peak- nding algorithm was run on these data to find the DEPs. The DEPs, called by the black/dark and white/light box is used to assess anxiety-like behavior in fi rodents.41,42 Usually, mice spend 60–65% of their time in a dark NimbleScan algorithm, were ltered according to the following criteria: (1) 43 at least one of the groups had a median value of log2 MeDIP/Input ⩾0.3 compartment; strong preference for the black or white compartment ′ suggests anxiogenic or anxiolytic response, respectively. This experiment and a median value of M >0 within each peak region; (2) at least half of – the probes in a peak had a median value of coefficient of variability ⩽0.8 in was performed on PD42 PD43. fi The number of F1 subjects tested for spontaneous locomotor activity all groups within each peak region. The bisul te PCR sequencing data was analyzed by the Mann–Whitney analysis (VassarStats) with a significance and preference for black and white compartment was as follows: SpSd, α o males n = 14, females n = 13; SpMd, males n = 15, females n = 14; MpMd, level of = 0.05 (P 0.05, two tailed). males n = 15, females n = 13; MpSd, males n = 14, females n = 12.

RESULTS DNA methylation METH had no effect on body weight of dams and nursing pups Adolescent (PD40–PD45) F1 females from the four groups SpSd, SpMd, − 1 MpMd and MpSd who were not subjected to behavioral tests were killed Body weight of saline and METH (4 mg kg ) dams was recorded by cervical dislocation and the hippocampus was removed, snapped from gestation day 3 through 17. Two-way repeated measure frozen and kept at −80 °C. Genomic DNA was extracted using a DNeasy ANOVA and the Greenhouse–Geisser test resulted in significant Blood and Tissue kit (Qiagen, Valencia, CA, USA) according to manufac- time effect F = 1087.58; Po0.0001 and non-significant group turer's instructions and sonicated to 200–1000 bp with a Bioruptor effect F = 3.09; P = 0.122, suggesting that METH had no significant sonicator (Diagenode, Denville, NJ, USA). One microgram of sheared effect on body weight of dams (Supplementary Figure 1A). DNA was denatured and used for immunoprecipitation using a mouse Therefore, the inclusion of a pair-fed group was unnecessary. The monoclonal anti-5-methylcytosine antibody (Diagenode) overnight at 4 °C. lack of weight loss in the METH dams may be because of the Anti-mouse IgG magnetic beads (Dynabeads, Life Technologies, Gaithers- following factors: (a) the prolonged exposure to the drug from burg, MD, USA) were added and incubated for additional 2 h at 4 °C. After immunoprecipitation, five washes were performed with ice-cold buffer adolescence through gestation; and (b) the relatively low dose of METH. Likewise, it has been reported that 14 days cocaine self- (0.5% bovine serum albumin in PBS) and beads resuspended in Tris-EDTA 45 buffer with 0.25% SDS and 0.25 mg ml − 1 proteinse K and incubated for 2 h administration in adolescent rats had no effect on body weight. at 65 °C. Methylated DNA immunoprecipitation (MeDIP)-enriched DNA was The cross-fostering design yielded four groups of pups such as purified using Qiagene MinElute columns (Qiagen), amplified using a SpSd (n = 30), SpMd (n = 32), MpMd n = 31 and MpSd (n = 29). Each whole-genome Amplification kit (GenomePlex Complete WGA2, Sigma- group contained about similar number of males and females. We Aldrich) and repurified with Qiaquick PCR purification kit (Qiagen). The posit that METH dams who fostered saline pups did not expose NimbleGen Dual-Color DNA labeling kit (Roche NimbleGen) was used to them to METH or amphetamine (METH metabolite) through the label equal amounts of pull down DNA and input samples with Cy5 – ’ milk because (a) the t1/2 of METH is 51 70 min and the t1/2 of (control) or Cy3, respectively, according to manufacturer s protocol. – 32,46 Microarrays (Roche NimbleGen MM8 Meth 385 K CpG plus Promoter; amphetamine is 79 88 min (s.c. and i.p.); and (b) the last METH Roche NimbleGen), which contain ~385 000 probes covering 15 936 injection was given 48 h before parturition. University of California Santa Cruz-annotated CpG islands and all RefSeq Pups’ weights were recorded from PD3 through PD27. Because gene promoter regions, were hybridized at 42 °C for 16–20 h according to there was no significant sex-dependent difference in pups’ weight, the standard procedure by Roche NimbleGen. Sample labeling, hybridiza- results of both sexes were collapsed. Two-way repeated measure tion and processing were performed at Arraystar (Rockville, MD, USA). ANOVA and the Greenhouse–Geisser test resulted in significant Array raw data were extracted as pair files by NimbleScan software (Roche time effect F = 676.89, Po0.0001, but non-significant group NimbleGen). Median-centering quantile normalization and linear smooth- effect F = 3.89, P = 0.156, suggesting that neither in utero METH ing by Bioconductor packages Ringo, limma and MEDME were performed. exposure nor maternal care had an effect on pups’ body weight From the normalized log2 ratio data, a sliding-window peak-finding algorithm provided by NimbleScan v2.5 (Roche NimbleGen) was applied to (Supplementary Figure 1B). find the enriched peaks with specified parameters (sliding-window width: 750 bp; mini probes per peak: 2; P-value minimum cutoff: 2; maximum Striatal DA levels spacing between nearby probes within peak: 500 bp). The identified peaks were mapped to genomic features such as transcripts and CpG Islands. A Levels of striatal DA in saline and METH dams were 1304 ± 123 and list of the differentially enriched peaks (DEPs) that reached statistical 1227 ± 134 ng per 100 mg tissue, respectively. Levels of striatal 3,4- significance is provided in Supplementary Table 1. dihydoxyphenylacetic acid in saline and METH dams were 105 ± 21 and 128 ± 19 ng per 100 mg tissue, respectively, and Bisulfite sequencing. Bisulfite PCR primers were designed with the levels of striatal homovanillic acid in saline and METH dams were BiSearch program44 (Supplementary Table 1). Bisulfite-modified genomic 155 ± 21 and 141 ± 19 ng per 100 mg tissue, respectively. Because DNA from each sample was amplified using AmpliTaq Gold (Applied there were no statistical significant differences between saline and Biosystems, Foster City, CA, USA) at annealing temperatures of 50–62 °C. METH dams, results suggest that the current METH regimen did The obtained PCR products were cloned into pCR2.1 TOPO vector not induce dopaminergic depletion (for example, neurotoxicity). (Invitrogen, Carlsbad, CA, USA). Colonies were screened by colony PCR Results of the levels of DA and its metabolites in METH offspring and eight positive clones were picked from each single bisulfite-treated fi fi – were not signi cantly different from saline offspring, suggesting DNA. A minimum of ve clones per sample (n =15 31 clones per group) that in utero METH exposure did not show evidence of were sequenced and the data were analyzed for statistical significance. dopaminergic neurotoxicity in adulthood.

Statistics Maternal behavior ’ ’ Dams and pups weights were analyzed by two-way repeated measure Maternal behavior was observed on PD6, PD7, PD10, PD12, PD14 analysis of variance (ANOVA) and the Greenhouse–Geisser test. Behavioral tests were usually analyzed by three-way ANOVA (sex × pup: saline/ and PD17. Three major activities were recorded, such as (a) METH × dam: saline/METH) that allowed us to determine differences crouch over the pups including nursing; (b) self-grooming; and between sexes, saline vs METH-exposed offspring and maternal care, (c) away from the pups. Time spent in each of these activities and saline vs METH dams. When applicable, the Bonferroni post hoc during a 15-min period was recorded and expressed as % time analysis was used. Signiﬁcance was set at a P-value o0.05. (Supplementary Figure 2); results were analyzed by two-repeated

Molecular Psychiatry (2015), 252 – 262 © 2015 Macmillan Publishers Limited Consequences of parental methamphetamine exposure Y Itzhak et al 255 measure ANOVA (treatment × time). There was no significant interaction F(1,102) = 29.45, Po0.001; significant pup × dam treatment effect for each of the activities; however, there was a interaction F(1,102) = 15.13, Po0.001; and significant sex × pup × significant time effect (Supplementary Figure 2). Saline (n = 8) and dam interaction F(1,102) = 13.45, Po0.001. The finding that MpSd METH (n = 8) dams spent between 80% (PD6) and 60% (PD17) of and MpMd males had significantly higher CPP than SpSd and the time in crouch position and nursing, respectively. Dams of SpMd males (Figure 1a) suggests that heightened response to both groups spent about 15–25% of the time self-grooming or cocaine-conditioned reward is the result of in utero METH away from the pups. In both groups, the amount of time spent exposure (pup effect) that was not influenced by maternal care. with the pups decreased over time, whereas the amount of time However, the response of female offspring, both saline and METH spent away from the pups increased over time (Supplementary in utero exposed, was influenced by maternal care of both saline Figure 2). Based on the three phenotypes we observed, results and METH dams (Figure 1a). Hence, it appears that maternal care suggest no significant differences between saline and METH influenced female but not male offspring response to cocaine maternal behavior. reward.

Cocaine-induced hyperactivity. Locomotor activity (30 min) was Cocaine-induced CPP and hyperactivity in F1 offspring − recorded during the cocaine (10 mg kg 1) conditioning session, Conditioned place preference. Results were analyzed by three-way daily, for 4 days. A two-way ANOVA (group × time) revealed a ANOVA (sex × pup: saline/METH × dam: saline/METH). There was a significant group effect F(7,407) = 45.97, Po0.0001, but non- fi o signi cant pup effect F(1,102) = 18.5, P 0.001; and sex effect F significant time effect F(3,407) = 3.56, P = 0.086 (for example, lack o fi (1,102) = 21.8, P 0.001. Also there was signi cant sex × dam of sensitization over time). Results of cocaine-induced hyperlocomotion are similar to results of cocaine-induced place preference. Post hoc analysis showed that MpMd and MpSd males developed significantly higher hyperlocomotion compared with SpSd and SpMd males (Figure 1b). This finding suggests again pup effect that is not influenced by maternal care. Sexual dimorphism was also observed because the response of MpMd males was higher than the female counterparts (MpMd; Figure 1b).

Fear conditioning Mice were fear conditioned to an auditory cue by a single footshock (0.75 mA) in context A, and tested for cued-freezing in context B after 24 h. Differences in freezing magnitude among the groups was analyzed by three-way ANOVA (sex × pup: saline/ METH × dam: saline/METH). There was a signiﬁcant (a) pup effect F

Figure 1. Cocaine-induced conditioned place preference (CPP) and hyperlocomotion and fear conditioning. Four groups such as SpSd, SpMd, MpSd and MpMd (n = 26–28 per group) each containing a similar number of males and females were tested. (a) Results of CPP were analyzed by three-way analysis of variance (ANOVA; sex × pup: saline/METH (methamphetamine) × dam: saline/METH). There was a significant pup effect F(1,102) = 18.5, Po0.01, and sex effect F (1,102) = 21.8, Po0.001. Also, there was significant sex × dam interaction F(1,102) = 29.45, Po0.001; significant pup × dam interaction F(1,102) = 15.13, Po0.001; and significant sex × pup × dam interaction F(1,102) = 13.45, Po0.001. MpSd and MpMd males had significantly higher CPP than SpSd and SpMd males (a), which suggests that heightened CPP response was the result of in utero METH exposure (pup effect) that was not influenced by maternal care. Female offspring, both saline and METH in utero exposed, was influenced by maternal care of both saline and METH dams (*Po0.05 between group comparison). (b) Cocaine-induced hyperactivity. Results of cocaine-induced hyperlocomotion are similar to results of cocaine-induced CPP. MpMd and MpSd males developed significantly higher hyperlocomotion compared with SpSd and SpMd males (b;*Po0.05). Sexual dimorphism is shown by the finding that MpDp females had lower cocaine-induced locomotor activity compared with male counterparts (*Po0.05). (c) Fear conditioning. Three-way ANOVA (sex × pup: saline/METH × dam: saline/METH) resulted in significant (1) pup effect F(1,104) = 33.48, Po0.001; (2) dam effect F(1,104) = 54.76, Po0.001; and (3) interaction of pup × dam F(1,104) = 63.39, Po0.001. Post hoc analysis showed significantly low-freezing response in METH pups reared by METH dams (MpMd of both sexes) compared saline counterparts (gray bars *Po0.05). However, METH pups fostered by saline dams showed normal freezing response compared METH pups fostered by METH dams (#Po0.05), suggesting saline maternal effect. However, because saline pups (of both sexes) fostered by METH dams (SpMd) were not significantly different than controls SpSd (c), it appears that METH dams had not effect on offspring fear conditioning.

© 2015 Macmillan Publishers Limited Molecular Psychiatry (2015), 252 – 262 Consequences of parental methamphetamine exposure Y Itzhak et al 256 (1,104) = 33.48, Po0.001; (b) dam effect F(1,104) = 54.76, Po0.001; and (c) interaction of pup × dam F(1,104) = 63.39; Po0.001. Post hoc analysis showed significantly low-freezing response in METH pups reared by METH dams (MpMd of both sexes) compared with all other groups (Figure 1c). The finding that METH pups fostered by saline dams did not show deficit in freezing response (Figure 1c) suggests that saline dams may restore the dampened response of METH pups. However, because saline pups (of both sexes) fostered by METH dams (SpMd) were not significantly different than controls SpSd (Figure 1c), it appears that METH dams had no effect on offspring fear conditioning.

F1 spontaneous locomotor activity and time in black compartment Locomotion. On PD41 and PD42, spontaneous locomotor activity was recorded. Because sex-dependent differences in locomotor activity were not observed, results of both sexes were collapsed and analyzed by two-way ANOVA (pup saline/METH × dam saline/ METH). There was a signiﬁcant dam effect F(1,110) = 35.57, Po0.0001 but not pup effect F(1,101) = 1.51, P = 0.221. Pair-wise comparisons showed that locomotor activity of the MpMd and the SpMd groups was signiﬁcantly lower than SpSd and MpSd groups (Figure 2a). Results suggest that maternal care of METH dams induced lower spontaneous locomotor activity, regardless whether pups were exposed to METH or saline in utero. If we assume that METH pups do not have motor impairment, then it appears that saline dams had no effect on motor behavior because locomotor activity of SpSd and MpSd groups was similar (Figure 2a).

Time in black compartment. Mice usually spend 60–65% of their time in a dark/black compartment.43 On PD45 and PD46, percent time spent in the black compartment (15 min period) was analyzed by three-way ANOVA (sex × pup: saline/METH × dam: saline/METH). There was a significant sex effect F(1,96) = 85.00, o fi P 0.0001; signi cant interactions of sex × dam F(1,96) = 37.80, Figure 2. Maternal care influenced spontaneous locomotor activity Po0.0001; sex × pup F(1,96) = 39.11, Po0.0001; and sex × dam × and time spent in black compartment. Four groups such as SpSd, pup F(1,96) = 34.50, Po0.0001. Results depicted in Figure 2b SpMd, MpSd and MpMd (n = 26–28 per group) each containing a shows that SpMd males spent significant amount of time in the similar number of males and females were tested. (a) Reduced black zone, whereas SpMd females spent the least amount of time locomotor activity was observed in the SpMd and MpMd groups in the black zone. Results suggest that maternal care of METH compared with SpSd and MpSd (*Po0.05) groups, suggesting fi METH maternal effect on spontaneous locomotor activity. Because dams induced signi cant sex-dependent effect on anxiety-like fi behavior of saline offspring. no signi cant sex effect was observed, results of both sexes were collapsed. (b) SpMd males spent significantly more time in the black zone compared with SpSd males (*Po0.05), whereas SpMd females DNA methylation spent less time in the black zone compared with SpSd females o fi One possible mechanism by which prenatal exposures affect long- (*P 0.05), suggesting that METH maternal care induced signi cant term phenotypes later in life involves alterations of DNA sex-dependent effect on anxiety-like behavior of saline offspring. methylation marks in the genome. To determine the role of DNA methylation in regulating behavioral responses following in utero METH exposure, we compared the DNA methylome of the MpMd vs SpSd samples identified 1822 methylation peaks hippocampus of the F1 SpSd, MpMd, SpMd and MpSd mice. aligning to gene promoter regions and 1808 peaks aligning to Although the nucleus accumbens is the major reward substrate in CpG islands as differentially methylated regions (DMR). The brain, we chose to investigate the hippocampus because it has a majority of these DMR showed hypermethylation in MpMd when border role in cognition and emotional behavior, and little is compared with SpSd (Supplementary Table 4). When applying the known about the role of the hippocampus in the motivational extremely stringent criteria of looking at peaks that were present effects of psychostimulants (see Discussion for more details). in all three samples of either MpMd or SpSd and absent in all three DNA methylation patterns were assayed by MeDIP-chip. The samples of the comparison group, we restricted the list of DMR to MeDIP procedure is based on the enrichment of methylated DNA 545 CpG islands and 156 promoter regions hypermethylated in with an antibody that specifically binds to 5-methylcytidine that MpMd and 209 CpG islands and 46 promoters with highest was followed by hybridization to a microarray containing all methylation in SpSd (Table 2); 75% of the DMR identified by these annotated CpG Islands and promoter regions (from about −1.3 to stringent criteria showed methylation differences by bisulfite 0.5 kb of the transcriptional start site). We identified an average of sequencing concordant with the array data (Figure 3). Of note, 5135.5 methylation-enriched peaks in gene promoters and 6279.5 there is more variability in methylation levels in samples from methylation-enriched peaks in CpG islands per sample maternally METH-exposed mice than in SpSd samples, suggesting (Supplementary Table 3). The group that exhibited the highest that the response of hippocampal cells to maternal METH number of identified peaks was MpMd, suggesting that the exposure is heterogeneous. As the physiological relevance of combination of in utero exposure to METH and METH-induced intergenic CpG islands is unknown and the promoter-associated maternal care mostly promotes DNA methylation. A comparison of CpG islands showed a methylation profile similar to the promoter

Molecular Psychiatry (2015), 252 – 262 © 2015 Macmillan Publishers Limited Consequences of parental methamphetamine exposure Y Itzhak et al 257 hippocampal transcriptional landscape of the progeny; promoters that are usually active become methylated, whereas inactive promoters are demethylated. The most enriched gene ontology terms in the DMR were ‘cerebral cortex GABAergic interneuron differentiation’ for the hypermethylated (P = 0.002 after Bonferroni correction for multiple comparisons) and ‘embryonic development’ for the hypomethylated (P = 0.01 after Bonferroni), suggesting that the observed phenotypes could be the result of abnormal brain development. Classiﬁcation according to the Kyoto Encyclopedia of Genes and Genomes pathway placing identiﬁed enrichment in transcriptional regulation (P = 0.0003) and the mitogen-activated protein kinase (Mapk) signaling pathway (P = 0.04) for hypermethylated and hypomethylated DMR in MpMd as compared with SpSd, respectively.

DISCUSSION Current use of METH surpasses the use of cocaine and opiates,49 and ~7% of METH users are pregnant women.50 However, little is known about the impact of prenatal METH exposure on developmental outcomes of the progeny.51,52 This is mostly because of the difficulty of investigating the effects of maternal drug exposure in humans that are often multidrug users, and their response could be influenced by a multitude of factors such as frequency, dose and route of drug exposure, and social and genetic determinants. We sought to conduct preclinical studies in mice to investigate the long-term behavioral consequences induced by parental METH exposure and the molecular alterations associated with the behavioral phenotypes. We used a method of drug exposure that mimics human drug use: escalating doses Figure 3. Methylation analysis by bisulfite sequencing. (a) Repre- starting at adolescence and continuing during pregnancy. sentative alleles for Bcl7c and Col24a1 for bisulfite sequencing of The extrapolation of daily METH dosage from human drug users DNA from a SpSd and MpMd hippocampus sample. Each circle to animals may be a challenge because human drug use varies along a horizontal row depicts a CpG site in a cloned allele with upon the degree of addiction, route of drug intake, availability, white representing unmethylated and black representing methy- cost and so on. Among METH users in the United States, self- lated. (b) Percent methylation (y axis) per clone and loci on the x reported drug use was 13–22 days within a 30-day period,53 axis. An average of 18 alleles were sequenced for each group. 54 − 1 o o averaging 3.1 g a week, which equals about 6.1 mg kg per *P 0.05, **P 0.01. day. In another study, self-reported METH use frequency was 5.3 days per week at a dose of 0.53 g per day (median), which equals about 7.5 mg kg − 1 per hit. 55 Based on these reports and a direct in the vast majority of analyzed DMR, we focused our report on mg per kg dose comparison, the doses of METH given to mice promoter DMR. To differentiate between the effects that arise (0.5–4.0 mg kg − 1 METH every-other-day) do not exceed the from direct METH exposure and maternal care, we compared the quantities of METH abused by humans. methylation profiles from all experimental groups (Supplementary Exposure to drugs of abuse before and during pregnancy could Figure 2). We identified 62 and 35 promoter regions in which DNA potentially affect the two developmental periods in which methylation was elevated and reduced, respectively, in the F1 epigenetic reprogramming occurs genome wide, such as germ mice due to in utero METH exposure that was insensitive to cells and preimplantation embryos. Our results indicate that maternal-fostering effects: DMR found in MpMd vs SpSd that were parental METH exposure, in a manner that resembles human use, also found in MpMd vs SpMd and MpSd vs SpSd but were not produces alterations in offspring’s behavior and DNA methylation observed in the MpMd vs MpSd comparison (Table 1). patterns in the hippocampus. The data demonstrate that both, the We also identified DMR induced by METH-related maternal epigenotype and the behavioral phenotype of the offspring, are effects: 70 hypermethylated and 39 undermethylated loci vulnerable to METH exposure during embryonic development, a identified by their presence in MpMd vs SpSd and also in SpMd period when wide epigenetic reprogramming occurs. In addition, vs SpSd (methylation), and in SpSd vs SpMd and also in SpSd vs the cross-fostering experiments suggest that this vulnerability MpMd (demethylation) (Table 1). extends into the postnatal period as both, the DNA methylome We performed bioinformatic analysis of the SpSd vs MpMd DMR and the behavior of the pups, were modulated by postnatal associated to gene promoters using the software application maternal influences. Thus, we show that METH exposure before Enrichr.47 Notably, enrichment analysis using histone modifica- and during pregnancy results in significant lasting effects on DNA tions chromatin immunoprecipitation-seq gene-set libraries methylation that consequently may influence gene expression showed that the most enriched terms were those associated with and produce abnormal phenotypes across the life course. H3K4me3 for the parental exposure to METH-induced methylation Our results suggest that the aberrant behavioral phenotypes events and H3K27me3 when the input was the demethylated and DNA methylation we observed in F1 offspring are not due to DMR (Table 2). These signature patterns of the unbiased promoter dopaminergic neurotoxicity. Levels of striatal DA and its metabo- lists are rather interesting, as the H3K4me3 and H3K27me3 lites 3,4-dihydoxyphenylacetic acid and homovanillic acid were modifications are usually associated with transcribed and silenced not significantly different between (a) METH and saline dams, and promoters, respectively, whereas promoter methylation is gen- (b) F1 METH and saline offspring. In our previous studies, METH- erally non-permissive for transcription.48 Thus, these data suggest induced dopaminergic neurotoxicity was observed following that parental METH exposure has important consequences on the acute administration of three injections of METH (5 mg kg − 1)in

Table 1. Promoter methylation changes induced by either METH exposure or maternal care

Methylation induced Demethylation induced Methylation induced by Demethylation induced Methylation induced by Demethylation by METH exposure by METH exposure METH-altered maternal by METH-altered interaction of METH induced by interaction care maternal care exposure and METH- of METH exposure and altered maternal care METH-altered maternal care

1110054O05Rik 1110008L16Rik 1700019N12Rik 4930539J05Rik 1110002L01Rik 4833439L19Rik 1700020O03Rik 7420426K07Rik 1700037C18Rik Abca3 1300001I01Rik Acer1 4933433P14Rik Accn4 4921504E06Rik Add3 2610204G22Rik Arhgap23 Adora1 Aldh3b1 5930434B04Rik Atp5l 3010026O09Rik Btbd11 Aimp1 Allc Abhd11 B3galtl 3110001I22Rik Cacna1c Akap5 Ankrd23 Acot13 BC022687 Aagab Col24a1 Atg2b Armc7 Adcy8 C030019I05Rik Aasdhppt Gyltl1b Atp5s Atp2c1 Ahsg Camk2a Anapc7 Hdac5 B4galnt2 BC020402 Aph1b Catsperg1 Ankrd9 Hipk2 BC048679 Bex1 AW209491 Cbfa2t3 Arhgap29 Hras1 Bcl7c C1ql4 Btg2 Celf1 Asxl2 Limch1 Cacna1g Camkk2 Cacng4 Dnajc13 Atad3a Lmna Cdc23 Car9 Cd2ap Fbrsl1 Atl1 Rbp1 Chrm4 Cldn19 Cep57 Fbxl3 Atxn7l1 Rps8 Cnga3 Crhr2 Cib1 Fndc4 Bfar Snord38a Cnst Cyp4f15 Cln8 Hdac11 Cdca4 Snord55 Cpz Ddhd2 Cluap1 Lrrc4b Cenpp Spink2 Dhx16 Dlgap2 Ctnnd2 Lrrc8b Ctdsp2 Spint2 Elk3 Dpp6 Cygb Mapk12 E2f8 Srgap1 Eme2 Fam100a D330012F22Rik Mier2 Edem3 Strn Exosc6 Gas2l3 Daam1 Ndufb4 Ehmt1 Zdhhc2 F11r Lsp1 Dennd2a Nkx2-1 F730043M19Rik Fuca1 Mpped2 Depdc6 Oas1a Fam19a2 Gatad2a Mysm1 Dusp7 Slc46a1 Fastkd3 Gdap1l1 Nell1 Ednra Slc6a1 Gatsl3 Ggct Pdia5 Eef1d Slc9a7 Gsk3b Glyr1 Pnck Fam171b Taf13 Insm2 Gm266 Ppp2r3c Fam76b Timm50 Iqch Gnb1l Rarg Gdf11 Tmem176a Irak1 Gpatch3 Rusc2 Gpd2 Tmem176b Jmy Grinl1a Smurf1 Gpr35 Trhr2 Kbtbd3 Hrk Socs1 H2-Ke6 Uba6 Kcnmb4 Hspb8 Timm13 Hadha Uqcrq Ldoc1l Ilk Trpm4 Hadhb Lemd1 Kcnab2 Wdr12 Irak4 Map4k5 L2hgdh Lhx2 Mdﬁ Mir762 Lhx4 Mga Mrps34 Lox Mir718 Mst1 Mdc1 Mtrr Nphp4 Nat10 Ncapd3 Nudt16l1 Ndrg3 Nol8 Pcdhgc3 Pabpc5 Nrarp Pcf11 Papss1 Nup50 Pgam1 Pax8 Pcx Rrp8 Pcdh8 Pds5b Sep15 Plrg1 Pgls Six6 Pomc Raver1 Snx7 Prss28 Sesn2 Sorbs1 Ptma Sorcs1 Srsf5 Pus7l Srrm1 Tatdn2 Rarres2 Ssr4 Tbck Rtn1 Tgfb3 Tex261 Sec24d Tifab Tfb2m Shfm1 Tmem39b Tia1 Slc38a4 Ubxn4 Tsen34 Stat6 Vamp4 Txnrd3 Stoml2 Vps26b Ube1y1 Tbkbp1 Zfp191 Ubn1 Tbx5 Zfp346 Vbp1 Tdp2 Zc3h7a Tkt Zic3 Tmem147 Tmem57 Tpm4 Trabd Vstm2a Zfp191 Zfp316 Zfp346 Zfp384 Abbreviations: DMR, differentially methylated regions; Mapk, mitogen-activated protein kinase; METH, methamphetamine. Last two columns containtop5% of DMR.

Molecular Psychiatry (2015), 252 – 262 © 2015 Macmillan Publishers Limited Consequences of parental methamphetamine exposure Y Itzhak et al 259 showed significant higher CPP (Figure 1a) and cocaine-induced Table 2. Enriched histone modification signatures of the SpSd vs hyperlocomotion (Figure 1b) compared with SpSd and SpMd MpMd promoter DMR males suggest that prenatal METH exposure of males render them Index Name P-value Z-score Combined highly sensitive to cocaine effect. The enhanced response of F1 score METH males to cocaine-conditioned reward is again suggestive of a lack of METH-induced neurotoxicity, which significantly A diminishes cocaine CPP in mice.56 1 H3K27me3 fetal lung 8.73e–4 −1.52 10.68 Sex-dependent differences were observed as MpMd males were 2 H3K27me3 H1 BMP4 derived 3.51e–3 −1.65 9.34 more sensitive to cocaine than the female counterparts in both trophoblast cultured cells − CPP (Figure 1a) and locomotor activity (Figure 1b) experiments. 3 H3K27me3 H1 0.01 1.87 8.59 fl − Maternal in uence of METH and saline dams on female offspring 4 H3K27me3 colonic mucosa 0.02 1.69 6.08 fi 5 H3K27me3 stomach smooth 0.03 −1.40 4.74 response to cocaine was observed. Together, these ndings muscle suggest that the response of female— but not male— offspring to 6 H3K27me3 CD4 memory 0.04 −1.46 4.60 cocaine reward was modulated by maternal care. The enhanced primary cells sensitivity of F1 METH males to cocaine reward is seemingly 7 H3K27me3 CD4 naive primary 0.03 −1.29 4.48 different than the reduced response to cocaine of male offspring cells of sirs who were exposed to cocaine self-administration.38 8 H3K27me3 CD3 primary cells 0.06 −1.45 4.05 However, because in the latter study offspring were not exposed − 9 H3K27me3 penis foreskin 0.04 1.27 4.00 to the drug in utero it is difficult to compare the results of these keratinocyte primary cells 10 H3K27me3 CD8 memory 0.06 −1.39 3.89 two studies. primary cells DNA methylation is an important epigenetic mechanism regulating cocaine-induced structural plasticity.60 Although the B nucleus accumbens is the major reward substrate in brain, we 1 H3K4me3 H1 3.93e–22 −2.26 98.08 chose to investigate the hippocampus because it has a border role 2 H3K4me3 H1 derived 1.10e–14 −2.60 70.01 in cognition and emotional behavior, and little is known about the mesenchymal stem cells role of the hippocampus in the motivational effects of cocaine.61 3 H3K4me3 H9 1.68e–12 −2.84 64.02 The dorsal hippocampus is primarily involved in spatial learning – − 4 H3K4me3 iPS DF 6.9 4.61e 12 2.79 60.87 and the ventral hippocampus is associated with motivational and 5 H3K4me3 CD8 naive primary 1.59e–13 −2.39 58.81 62,63 cells emotional behavior. Relevant to CPP studies, ablation or – − blockade of dorsal hippocampus precluded formation of cocaine 6 H3K4me3 iPS DF 19.11 3.63e 10 2.81 49.50 64,65 7 H3K4me3 CD19 primary cells 7.63e–10 −2.64 44.87 CPP, and evidence supports the role of hippocampal DA in 66 8 H3K9ac H1 4.42e–8 −3.10 40.60 long-term memory of motivationally significant events. Ventral 9 H3K4me3 H1 BMP4 derived 7.84e–8 −2.84 35.95 CA1 projections to the medial nucleus accumbens shell was trophoblast cultured cells predominately and selectively potentiated after cocaine expo- 10 H3K4me3 IMR90 1.77e–6 −2.95 30.24 sure,67 supporting the role of VHP in motivational effects of Abbreviation: DMR, differentially methylated regions. (A) Top 10 enriched cocaine. Ventral CA1 innervates regions of the prefrontal cortex, histone terms ranked based on the level of significance for the that is, the prelimbic and medial orbital areas that project to the hypermethylated DMR. (B) Top 10 enriched histone terms ranked based hypothalamus, supporting the role of ventral hippocampus in on the level of significance for the demethylated DMR. stress response.68 Therefore, we posit that changes in DNA methylation in the hippocampus are relevant for appetitive and aversive learning and memory. a single day.56 It has also been reported that pretreatment with Acquired fear response, following classical Pavlovian fear intermittent low doses of METH protects against dopaminergic conditioning, is primarily dependent on the integrity of the and serotonergic neurotoxicity caused by higher doses of METH.57 hippocampus and amygdala. We found that both male and female Hence, it appears that our schedule of METH administration did METH offspring fostered by METH dams showed significant lower not produce dopaminergic neurotoxicity. cued-freezing response (Figure 1c). The dampened freezing The behavioral characterization showed several aberrations in response was ‘rescued’ by saline dams. The finding that saline the F1 offspring that could be a direct consequence of germ cell, pups fostered by METH dams (SpMd) were not different than in utero METH exposure, indirect result of altered maternal control pups (SpSd) suggests that the reduced freezing response behavior or the combination of all. Although our investigation of the METH pups is primarily because of in utero METH exposure of maternal behavior did not reveal significant differences that was not influenced by maternal care of METH dams. Thus, between METH and saline dams with reference to nursing, self- cognitive deficits in METH offspring could be the result of in utero grooming and time spent away from the pups, results of the cross- METH exposure. fostering studies suggest that some behavioral phenotypes of F1 Although it is well established that cued fear memory is progenies were influenced by maternal care. primarily amygdala dependent, several studies have shown that The influence of cross-fostering within the same strain or hippocampal lesions may also impair cued fear memory.69–72 between different mouse strains is a matter of debate; some Given the role of hippocampal DNA methylation in learning and suggest no significant differences in pup development,58 whereas memory,73 the deficits in fear conditioning of MpMd offspring others suggest some increased nursing of C57Bl/6 fostered pups could have a relationship with aberrant hippocampal DNA by C57Bl/6 dams.59 In the current study, we did not attempt to methylation we observed. It appears, however, that METH- investigate the effect of cross-fostering per se. It should be noted, induced aberrant DNA methylation may be offset by maternal however, that body weight of all pups from PD3 through care, because METH pups fostered by saline dams did not show adolescence (PD27), regardless of sex, in utero METH exposure the deficits in cued fear memory. Additional studies are needed to and fostering dam, was the same in all four groups investigate the profile of DNA methylation in the amygdala and (Supplementary Figure 1B), suggesting that in utero METH other brain regions involved in acquisition of fear memory (for exposure and maternal care had no effect on body weight. example, prefrontal cortex). The response of offspring to cocaine reward was determined by Although maternal effect of METH dams on offspring response the CPP paradigm. The findings that MpMd and MpSd males to cocaine-conditioned response and hyperlocomotion as well as

© 2015 Macmillan Publishers Limited Molecular Psychiatry (2015), 252 – 262 Consequences of parental methamphetamine exposure Y Itzhak et al 260 fear conditioning was unnoticeable, the influence of METH dams DNA methylation may not be the sole cause for the changes in on spontaneous locomotor activity (Figure 2a) and anxiety-like behavior we observed. Numerous changes in synaptic plasticity behavior (Figure 2b) is evident. Both saline pups and METH pups because of in utero drug exposure may have long-term effect on fostered by METH dams showed significantly lower spontaneous the offspring; additional studies are necessary to investigate the locomotor activity compared with saline and METH pups fostered consequences of parental METH exposure on the offspring. by saline dams (Figure 2a). In the choice between black and white We identified DMR located in promoters of genes already linked compartments of a cage, METH dams had significant effect on to drug response, such as Adora1 (METH and cocaine76), ILK saline pups but not on METH pups. The former suggests that (integrin-linked kinase; cocaine77), Akap5 (A-kinase anchoring METH dams induced anxiogenic-like phenotype in saline males protein-5; cocaine78), Camkk2 (METH79), Hdac5 (cocaine and (preferring black) and anxiolytic-like phenotype in saline females METH80), Pomc (heroin81), Camk2a (METH and cocaine,82 Gsk-3-β (preferring white). It has been shown that early-life experience and (glycogen synthase kinase 3 beta; cocaine83,84) and PCDH8 maternal care influence hippocampal DNA methylation profile in (cocaine85). These findings suggest that in utero METH exposure rats and humans.74 Thus, the detected maternal METH effects on may induce changes in several genes that are modulated by direct saline offspring behavior may be related to the effect of METH exposure to other drugs of abuse. Some of these genes have dams on hippocampal DNA methylation of the offspring we critical role in synaptic plasticity and learning and memory. observed (Supplementary Figure 2). Alteration in ILK–glycogen synthase kinase 3 beta (Gsk-3-β) The literature on METH-exposed children is sparse compared signaling was identified in an animal model associated with with that on cocaine-exposed babies;13 and therefore it is difficult memory loss and Alzheimer's disease.86 A-kinase anchoring to identify specific behavioral phenotypes in our animal model proteins (APAKs) constitute a family of scaffolding proteins that that may correspond to abnormalities in children exposed to bind to protein kinase A and regulate the phosphorylation of METH in utero. However, reported effects of METH exposure in various proteins that have been implicated in synaptic plasticity children include reduced birth weight and size,14–17 poor and memory consolidation.87 Likewise calcium/calmodulin-depen- growth,18 neuroanatomical changes,19–21 increased stress,22 and dent kinases (Camkk2 and Camk2a) and histone deacetylase 5 learning and memory deficits.23,24 The latter may relate to the (Hdac5) have been implicated in synaptic plasticity and learning deficits in fear conditioning we observed (Figure 1c). However, and memory. Therefore, we postulate that DMR located in longitudinal studies on children that were exposed to METH in promoters of these genes may have profound implications on utero and their susceptibility to drug abuse in adolescence are behavioral phenotypes associated with appetitive- and aversive- missing but undoubtedly essential. associative learning we observed. Examination of the methylation status of hippocampal DNA of We observed that the methylation of a large number of gene female subjects identified several interesting candidates as promoters was changed as a result of fostering by METH dams. mediators of the aberrant behaviors. The DMR identified showed This finding suggests that METH maternal behavior significantly a remarkable different profile depending on the methylation contributes to the offspring epigenetic response. Interestingly, we change. METH-induced methylation, which should produce gene identified DMR in gene promoters in which the methylation or silencing, occurs in promoters that have a histone modification expression pattern was already reported to be sensitive to signature of active transcription. Conversely, METH-induced maternal influences, making them good candidates to mediate demethylation was observed in promoters associated with an the maternal METH influences. For instance, the methylation epigenetic signature of transcriptional silencing. The mechanism status of the Pomc (pro-opiomelanocortin) and Cln8 (claudin 8) by which prenatal METH exposure alters the epigenotype genes is altered by intrauterine exposure to alcohol88 and is not known. However, our data suggest that it likely involves maternal diabetes,89 respectively. Maternal effect on expression alterations at the levels of wide-range chromatin conformation, of Rtn1 in the offspring was reported for maternal hypothyroid- rather than modifying the expression of specific methyltrans- ism,90 and exposure to maternal diabetes resulted in dysregula- ferases, demethylases or their regulators. Concurrent with tion of TBX5.91 The data suggest that maternal effects on the this premise is the finding that altered hippocampal DNA offspring induced by a variety of stimuli (exposure to drugs, methylation in F1 offspring following in utero cocaine exposure disease states and nutritional status) may be mediated by a (gestational days 8–19) was observed in the absence of alterations common set of genes. A very likely candidate for this mediation is in expression of DNMTs.75 Pomc that is capable of sensing homeostatic perturbations and Functional categorization of the DMR identified in the SpSd vs participates in the modulation of behavioral responses such as MpMd comparison, regardless of whether the effects are because anxiety, cognition and response to drugs.92,93 of a direct METH exposure or maternal care, revealed enrichment In summary, the current study demonstrates that prenatal of genes involved in embryonic development and pattern METH exposure produces long-lasting changes in the offspring specification. This suggests that the observed phenotype may brain epigenome that could contribute to the initiation and result from abnormal development. The phenotypic response to maintenance of the observed behavioral phenotypes. Moreover, prenatal exposure of METH is likely the result of the compound our experiments indicate a significant influence of maternal activity of a large set of dysregulated genes, rather than the effects on the epigenotype and behavioral phenotype of the F1 product of a handful of genes. Our observation of promoter DMR progeny. Future studies will determine whether sex-dependent enrichment in terms such as transcriptional regulation and MAPK behavioral phenotypes parallel sex-dependent changes in hippo- signaling pathways supports this idea. Hypermethylation of CpG campal DNA methylation, and whether changes in epigenome islands in gene promoter of MAPK1 was also detected in the and behavior due to METH exposure are trans-generationally hippocampus of prenatally cocaine-exposed offspring.75 Also, transmitted to F2 and F3 generations. some of the identified DMR are associated with genes that could be central to the alterations of these pathways. These include histone modifiers such as deacetylases Hdac5 and Hdac11, CONFLICT OF INTEREST methyltransferase Ehmt1, H2A deubiquitinase Mysm1, and hub The authors declare no conflict of interest. kinases of the Mapk pathway such as Hras1 and Mapk12. Currently, it is unclear how exactly these DNA methylation changes correlate with the behavioral observations; further ACKNOWLEDGMENTS studies are necessary to determine the role they have in the This work was supported in part by grant RO1DA026878 and R21DA029404 from the development of the aberrant behavioral phenotypes. Changes in National Institute on Drug Abuse, National Institutes of Health (to YI) and award from

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