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Proenkephalin Mediates the Enduring Effects of Adolescent Cannabis Exposure Associated with Adult Opiate Vulnerability

  • Hilarie C. Tomasiewicz
    Affiliations
    Fishburg Department of Neuroscience, Mount Sinai School of Medicine, New York, New York

    Department of Psychiatry, Mount Sinai School of Medicine, New York, New York

    Friedman Brain Institute, Mount Sinai School of Medicine, New York, New York
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  • Michelle M. Jacobs
    Affiliations
    Department of Psychiatry, Mount Sinai School of Medicine, New York, New York

    Department of Pharmacology and Systems Therapeutics, Mount Sinai School of Medicine, New York, New York

    Friedman Brain Institute, Mount Sinai School of Medicine, New York, New York
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  • Matthew B. Wilkinson
    Affiliations
    Fishburg Department of Neuroscience, Mount Sinai School of Medicine, New York, New York

    Friedman Brain Institute, Mount Sinai School of Medicine, New York, New York
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  • Steven P. Wilson
    Affiliations
    Department of Pharmacology, Physiology and Neuroscience, University of South Carolina School of Medicine, Columbia, South Carolina
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  • Eric J. Nestler
    Affiliations
    Fishburg Department of Neuroscience, Mount Sinai School of Medicine, New York, New York

    Department of Psychiatry, Mount Sinai School of Medicine, New York, New York

    Department of Pharmacology and Systems Therapeutics, Mount Sinai School of Medicine, New York, New York

    Friedman Brain Institute, Mount Sinai School of Medicine, New York, New York
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  • Yasmin L. Hurd
    Correspondence
    Address correspondence to Yasmin Hurd, Ph.D., Departments of Psychiatry, Pharmacology and Systems Therapeutics, and Neuroscience, Mount Sinai School of Medicine, New York, NY
    Affiliations
    Fishburg Department of Neuroscience, Mount Sinai School of Medicine, New York, New York

    Department of Psychiatry, Mount Sinai School of Medicine, New York, New York

    Department of Pharmacology and Systems Therapeutics, Mount Sinai School of Medicine, New York, New York

    Friedman Brain Institute, Mount Sinai School of Medicine, New York, New York

    James J. Peters VA Medical Center, New York, New York
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      Background

      Marijuana use by teenagers often predates the use of harder drugs, but the neurobiological underpinnings of such vulnerability are unknown. Animal studies suggest enhanced heroin self-administration (SA) and dysregulation of the endogenous opioid system in the nucleus accumbens shell (NAcsh) of adults following adolescent Δ9-tetrahydrocannabinol (THC) exposure. However, a causal link between proenkephalin (Penk) expression and vulnerability to heroin has yet to be established.

      Methods

      To investigate the functional significance of NAcsh Penk tone, selective viral-mediated knockdown and overexpression of Penk was performed, followed by analysis of subsequent heroin SA behavior. To determine whether adolescent THC exposure was associated with chromatin alteration, we analyzed levels of histone H3 methylation in the NAcsh via chromatin immunoprecipitation at five sites flanking the Penk gene transcription start site.

      Results

      Here we show that regulation of the Penk opioid neuropeptide gene in NAcsh directly regulates heroin SA behavior. Selective viral-mediated knockdown of Penk in striatopallidal neurons attenuates heroin SA in adolescent THC-exposed rats, whereas Penk overexpression potentiates heroin SA in THC-naïve rats. Furthermore, we report that adolescent THC exposure mediates Penk upregulation through reduction of histone H3 lysine 9 (H3K9) methylation in the NAcsh, thereby disrupting the normal developmental pattern of H3K9 methylation.

      Conclusions

      These data establish a direct association between THC-induced NAcsh Penk upregulation and heroin SA and indicate that epigenetic dysregulation of Penk underlies the long-term effects of THC.

      Key Words

      Drug addiction is a chronic and relapsing disease that often begins during adolescence. Epidemiologic evidence documents an association between marijuana use during adolescence and subsequent abuse of drugs such as heroin and cocaine (
      • Kandel D.
      Stages in adolescent involvement in drug use.
      ,
      • Hall W.D.
      • Lynskey M.
      Is cannabis a gateway drug? Testing hypotheses about the relationship between cannabis use and the use of other illicit drugs.
      ). Although many factors including societal pressures, family, culture, and drug availability can contribute to this apparent “gateway” association, little is known about the neurobiological basis underlying such potential vulnerability. Of the neural substrates that have been investigated, the enkephalinergic opioid system is consistently altered by developmental marijuana exposure (
      • Ellgren M.
      • Spano S.M.
      • Hurd Y.L.
      Adolescent cannabis exposure alters opiate intake and opioid limbic neuronal populations in adult rats.
      ,
      • Spano M.S.
      • Ellgren M.
      • Wang X.
      • Hurd Y.L.
      Prenatal cannabis exposure increases heroin seeking with allostatic changes in limbic enkephalin systems in adulthood.
      ,
      • Ellgren M.
      • Artmann A.
      • Tkalych O.
      • Gupta A.
      • Hansen H.S.
      • Hansen S.H.
      • et al.
      Dynamic changes of the endogenous cannabinoid and opioid mesocorticolimbic systems during adolescence: THC effects.
      ), perhaps reflecting neuroanatomic interactions between cannabinoid receptor type 1 and the enkephalinergic system (
      • Rodriguez J.J.
      • Mackie K.
      • Pickel V.M.
      Ultrastructural localization of the CB1 cannabinoid receptor in mu-opioid receptor patches of the rat caudate putamen nucleus.
      ,
      • Pickel V.M.
      • Chan J.
      • Kash T.L.
      • Rodriguez J.J.
      • MacKie K.
      Compartment-specific localization of cannabinoid 1 (CB1) and mu-opioid receptors in rat nucleus accumbens.
      ). Debates exist, however, regarding the relationship between proenkephalin (Penk) dysregulation and opiate susceptibility. We previously reported that adult rats exposed to Δ9-tetrahydrocannabinol (THC; the primary psychoactive component of marijuana) during adolescence exhibit increased heroin self-administration (SA) as well as increased expression of Penk, the gene encoding the opioid neuropeptide enkephalin, in the nucleus accumbens shell (NAcsh), a mesolimbic structure critically involved in reward-related behaviors (
      • Ellgren M.
      • Spano S.M.
      • Hurd Y.L.
      Adolescent cannabis exposure alters opiate intake and opioid limbic neuronal populations in adult rats.
      ). Although these data suggest that increased NAcsh Penk expression and heroin SA behavior are independent consequences of adolescent THC exposure, they do not address a possible causal relationship between THC-induced Penk upregulation in NAcsh and enhanced behavioral susceptibility to opiates. Moreover, insights regarding the neurobiological mechanisms through which adolescent THC exposure maintains upregulation of Penk into adulthood remain unknown.
      Here we take advantage of viral-mediated gene transfer strategies to show that adulthood addiction-like behaviors induced by adolescent THC exposure are dependent on discrete regulation of NAcsh Penk gene expression. A number of recent studies have demonstrated an important role for histone methylation in the regulation of drug-induced behaviors and transcriptional plasticity, particularly alteration of repressive histone H3 lysine 9 (H3K9) methylation at NAc gene promotors (
      • Renthal W.
      • Kumar A.
      • Xiao G.
      • Wilkinson M.
      • Covington 3rd, H.E.
      • Maze I.
      • et al.
      Genome-wide analysis of chromatin regulation by cocaine reveals a role for sirtuins.
      ,
      • Maze I.
      • Covington 3rd, H.E.
      • Dietz D.M.
      • LaPlant Q.
      • Renthal W.
      • Russo S.J.
      • et al.
      Essential role of the histone methyltransferase G9a in cocaine-induced plasticity.
      ). We report here that one mechanism through which adolescent THC exposure may mediate Penk upregulation in adult NAcsh is through reduction of H3K9 di- and trimethylation, a functional consequence of which may be decreased transcriptional repression of Penk.

      Methods and Materials

      Animals and THC Treatment

      Male 21-day-old Long Evans rats (Taconic, Hudson, New York) were used; procedures conducted in accordance with approved protocols. Rats received intraperitoneal injections of THC (1.5 mg/kg; RTI International, Durham, North Carolina) or vehicle (.9% sodium chloride with .3% Tween 80) every third day (eight injections) during adolescence (postnatal days 28–49) (
      • Ellgren M.
      • Spano S.M.
      • Hurd Y.L.
      Adolescent cannabis exposure alters opiate intake and opioid limbic neuronal populations in adult rats.
      ). For SA experiments, Penk- and green fluorescent protein (GFP)-infused rats were treated with vehicle during adolescence and GFP-, microRNA (miR) control (ctrl)-, and miR Penk-infused rats were treated with THC during adolescence.

      Lentiviral Vectors

      Lentiviral vectors were constructed as described (Supplement 1). In all cases, in vivo transgene expression was validated and NAcsh-specific expression confirmed via in situ hybridization histochemistry.

      Surgeries

      Two weeks after final THC treatment, rats were anesthetized with isoflurane/oxygen (2.5%–4.5%) and bilaterally stereotaxically infused with .5 μL virus (Penk or GFP) or 1.0 μL virus (GFP, miR ctrl, miR Penk) into NAcsh (from bregma: anteroposterior +1.7 mm; mediolateral +2.3 mm; dorsoventral −6.8 mm [from dura], 10° from midline) at .1 μL/min. Two weeks subsequently, rats underwent surgery for jugular catheters (Brian Fromant, Cambridge, United Kingdom) for future SA experiments.

      Heroin Self-Administration and Locomotor Activity

      Heroin SA was conducted according to published protocols (
      • Ellgren M.
      • Spano S.M.
      • Hurd Y.L.
      Adolescent cannabis exposure alters opiate intake and opioid limbic neuronal populations in adult rats.
      ,
      • Ellgren M.
      • Artmann A.
      • Tkalych O.
      • Gupta A.
      • Hansen H.S.
      • Hansen S.H.
      • et al.
      Dynamic changes of the endogenous cannabinoid and opioid mesocorticolimbic systems during adolescence: THC effects.
      ) (Supplement 1). Briefly, rats self-administered heroin (30 μg/kg/injection) 3-hour daily under a fixed-ratio 1 reinforcement schedule until stable baseline responding was established. Following stabilization, a between-session dose response was conducted (30/7.5/100/15/60 μg/kg/injection; 1 dose/day). After a 3-week abstinence period, cue-induced drug-seeking behavior was evaluated (depression of the drug-paired lever had no programmed consequence) followed 1 week later by mild stress (24-hour food deprivation)-induced drug seeking. Both tests were 1 hour. Activity was measured by infrared beams during the SA sessions.

      In Situ Hybridization Histochemistry

      In situ hybridization histochemistry was conducted according to published protocols (
      • Ellgren M.
      • Spano S.M.
      • Hurd Y.L.
      Adolescent cannabis exposure alters opiate intake and opioid limbic neuronal populations in adult rats.
      ,
      • Hurd Y.L.
      In situ hybridization with isotopic riboprobes for detection of striatal neuropeptide mRNA expression after dopamine stimulant administration.
      ) (Supplement 1). [35S]-labeled Penk riboprobe (generated from a polymerase chain reaction [PCR]-derived complementary DNA (cDNA) fragment: bases 585–1140; Genbank accession: NM_017139) was applied to duplicate brain sections (2 × 103 cpm/mm2), overnight 55°C hybridization, and sections exposed (imaging plate; Fujifilm, Tokyo, Japan) with 14C standards for 28hr. Disintegrations-per-minute (dpm/mg) autogradiographic measurements were obtained for the NAc and averaged/animal.

      Gene Expression Analysis

      RNA was prepared from fresh-frozen bilateral NAcsh punches. cDNA was obtained using a first-strand synthesis kit (Quanta Biosciences, Gaithersburg, Maryland). Quantitative real-time PCR analysis was performed using Taqman-based probes (Applied Biosystems, Carlsbad, California; Penk, Rn00567566_m1; 18 S, 4319413E; Pdyn, Rn00571351_m1.); reactions run in triplicate, each gene run separately. Data normalized to eukaryotic 18S ribosomal RNA and analyzed via the ΔΔCT method (
      • Tsankova N.M.
      • Berton O.
      • Renthal W.
      • Kumar A.
      • Neve R.L.
      • Nestler E.J.
      Sustained hippocampal chromatin regulation in a mouse model of depression and antidepressant action.
      ).

      Chromatin Immunoprecipitation

      Fresh tissue was prepared for chromatin immunoprecipitation as previously described (
      • Renthal W.
      • Kumar A.
      • Xiao G.
      • Wilkinson M.
      • Covington 3rd, H.E.
      • Maze I.
      • et al.
      Genome-wide analysis of chromatin regulation by cocaine reveals a role for sirtuins.
      ,
      • Kumar A.
      • Choi K.H.
      • Renthal W.
      • Tsankova N.M.
      • Theobald D.E.
      • Truong H.T.
      • et al.
      Chromatin remodeling is a key mechanism underlying cocaine-induced plasticity in striatum.
      ) with minor modifications (Supplement 1). Briefly, two bilateral NAcsh punches/rat (three rats pooled per sample) were collected and processed. Immunoprecipitated samples (antibodies: H3K9me2, ab1220; H3K9me3, ab8898; H3K36me3, ab9050; H3K4me3, ab8580; abcam, Cambridge, Massachusetts) were subject to quantitative PCR (SYBR Green; Roche, Basel, Switzerland) and normalized to their non-immunoprecipitated input controls. Each reaction was run in triplicate and analyzed via the ΔΔCT method (
      • Tsankova N.M.
      • Berton O.
      • Renthal W.
      • Kumar A.
      • Neve R.L.
      • Nestler E.J.
      Sustained hippocampal chromatin regulation in a mouse model of depression and antidepressant action.
      ).

      Statistics

      We used two-tailed unpaired Student's t tests (for comparison of two groups), one-way analyses of variance (ANOVAs) followed by followed by Tukey's honest significant difference test or two-tailed Student's t tests when appropriate (for three groups), and two-way repeated-measures ANOVAs followed by one-way ANOVAs (to examine significant repeated-measure effects). All values represent mean ± SEM. Statistical calculations performed using JMP software (SAS, Cary, North Carolina).

      Results

      Penk Overexpression in NAcsh Increases Heroin Self-Administration

      To investigate the direct role of NAcsh Penk tone in the regulation of heroin SA behavior, we first verified the effects of local overexpression of Penk by infusing a lentiviral vector encoding Penk and GFP into NAcsh of adult rats (Figure 1A, B). Viral spread was 1 mm3 from the needle tip.
      Figure thumbnail gr1
      Figure 1Lentivirus-mediated Penk gene manipulation and heroin self-administration. (A) Green fluorescent protein (GFP) expression is restricted to nucleus accumbens shell (NAcsh; reprinted from Paxinos and Watson [
      • Paxinos G.
      • Watson C.
      The Rat Brain in Stereotaxic Coordinates.
      ] with permission from Elsevier, copyright 2007). (B) Lentiviral vectors. (C) Behavioral research design. Ψ, encapsidation signal including the 5' portion of the gag gene (GA); aca, anterior commissure; DAPI, 4',6-diamidino-2-phenylindole; eGFP, enhanced green fluorescent protein; EmGFP, emerald green fluorescent protein; HIV LTR, human immunodeficiency virus long terminal repeat; IRES, encephalomyocarditis virus internal ribosome entry site; LTR*, LTR with deletion in the U3 region; LTR, long terminal repeat; LV, lentiviral vector; miR NC, microRNA targeting nonvertebrate gene (Invitrogen, Carlsbad, California); miR ctrl, microRNA control; miR Penk, miRNA targeting nucleotides 709-729 of rat Penk coding region; mPGK1p, mouse phosphoglycerate kinase-1 promoter; NAcc, nucleus accumbens core; Penk, 956 nucleotide fragment containing the coding region of the rat Penk cDNA; PND, postnatal day; RRE, Rev-responsive element; sac, sacrifice; WPRE, woodchuck post-transcriptional regulatory element. See also .
      To investigate the functional significance of enhanced NAcsh Penk tone, we tested the involvement of increased NAcsh Penk expression on a fixed-ratio 1 schedule of heroin SA. Two cohorts of animals were treated with vehicle during adolescence and then given bilateral NAcsh infusions of lentivirus vectors expressing Penk or GFP in young adulthood. A third cohort of rats was treated with THC during adolescence (Figure 1C; Table S1 in Supplement 1). Bilateral NAcsh infusions of Penk enhanced responding for heroin [treatment by session interaction, F(22,213) = 2.036, p < .01, Figure 2A] and mean heroin intake [treatment by session interaction, F(22,213) =2.589, p < .001, Figure 2B], without modifying inactive lever pressing or locomotor activity (Figure S1A in Supplement 1), compared with GFP-infused controls. Consistent with previous data (
      • Ellgren M.
      • Spano S.M.
      • Hurd Y.L.
      Adolescent cannabis exposure alters opiate intake and opioid limbic neuronal populations in adult rats.
      ), animals treated with THC during adolescence also exhibited increased heroin SA compared to control conditions (GFP-infusion), an effect similar in magnitude to that resulting from NAcsh Penk overexpression (Figure 2A, B). Animals treated with THC during adolescence also exhibited increased heroin SA compared with vehicle-exposed “sham” animals that had undergone bilateral NAcsh infusion with saline (Figure S2 in Supplement 1). Following stabilization of heroin SA behavior, we next examined whether NAcsh Penk overexpression affected dose-dependent responding for heroin in a between-session dose response test. Penk overexpression and adolescent THC treatment led to upward vertical shifts in the heroin dose-response function, including higher peak SA rates on the descending limb of the dose-response curve [treatment by dose interaction, F(8,74) = 3.859, p < .001, Figure 2C] and higher drug intake across the range of doses studied (Figure 2D).
      Figure thumbnail gr2
      Figure 2Penk overexpression in nucleus accumbens shell (NAcsh) potentiates heroin self-administration. (A) Acquisition of heroin self-administration (SA; fixed-ratio 1, 30 μg/kg/injection). (B) Mean heroin intake. (C) Between-session dose-response (7.5, 15, 30, 60, 100 μg/kg/infusion; randomized order). (D) Mean heroin intake. (E) Heroin-seeking behavior (cue- and stress-induced) in Penk-infused, green fluorescent protein (GFP)-infused, or Δ9-tetrahydrocannabinol (THC)-exposed rats. (F) Penk mRNA levels in NAcsh, nucleus accumbens (NAc) core, and caudate-putamen (CPu) following heroin SA. (G) Representative in situ hybridization autoradiograms demonstrating striatal Penk mRNA expression following heroin SA. For all figures, n = 5–9 per group. Data shown as mean ± SEM. *p < .05; **p < .01 compared with GFP-expressing controls for each session. AL, active lever; IL, inactive lever, dpm, disintegrations per minute. See also .

      Penk Overexpression in NAcsh Promotes Enhanced Heroin Seeking

      To determine whether NAcsh Penk infusion facilitated subsequent behavioral susceptibility to relapse, we measured heroin-seeking behavior following abstinence. In light of self-reports by drug-dependent individuals indicating that exposure to drug-associated stimuli and stress precipitate drug craving and relapse (
      • Jaffe J.H.
      • Cascella N.G.
      • Kumor K.M.
      • Sherer M.A.
      Cocaine-induced cocaine craving.
      ,
      • O'Brien C.P.
      • Childress A.R.
      • McLellan A.T.
      • Ehrman R.
      Classical conditioning in drug-dependent humans.
      ,
      • Kreek M.J.
      • Koob G.F.
      Drug dependence: Stress and dysregulation of brain reward pathways.
      ), we first assessed heroin-seeking behavior under cue-induced reinstatement conditions. The magnitude of heroin-seeking behavior in the absence of reinforcement was assessed by the amount of responding at the previously drug-paired lever. Adolescent THC exposure and NAcsh Penk overexpression led to an increase in drug-paired lever responding [F(2,20) = 3.859, p < .05, Figure 2E] compared with GFP-infused controls, indicating that prior NAcsh Penk infusion enhanced the ability of the drug-associated environment to elicit drug-seeking behavior. We next examined whether NAcsh Penk infusion enhanced drug-seeking behavior triggered by exposure to a stressor previously shown to increase heroin seeking (
      • Spano M.S.
      • Ellgren M.
      • Wang X.
      • Hurd Y.L.
      Prenatal cannabis exposure increases heroin seeking with allostatic changes in limbic enkephalin systems in adulthood.
      ). One week after the first drug-seeking test, we evaluated drug-seeking behavior following 24-hour food deprivation. NAcsh Penk overexpression, but not adolescent THC-exposure, potentiated stress-induced heroin-seeking compared with GFP-infused controls [F(2,19) = 4.829, p < .05; Figure 2E]. Thus, increased Penk tone in NAcsh increased heroin-seeking behavior triggered by a mildly stressful event, indicating an enhanced propensity for relapse in these animals. Importantly, autoradiographic in situ hybridization histochemistry image analysis revealed significant upregulation of Penk messenger (m)RNA expression in NAcsh of Penk-infused and adolescent THC-exposed animals compared to GFP-infused control animals [F(2,23) = 11.026, p < .05; Figure 2F, G) following the completion of behavioral experiments. In contrast, Pdyn mRNA levels in NAcsh were unchanged in vehicle-exposed, GFP-infused, and Penk-infused animals (Figure S3 in Supplement 1). These data demonstrate that specific Penk upregulation in NAcsh promotes drug seeking after prolonged abstinence from heroin, indicating an important role for increased NAcsh Penk tone in the propensity for both cue- and stress- induced heroin-seeking behavior.

      NAcsh Penk Knockdown Attenuates Adolescent THC-Induced Heroin Self-Administration

      The pronounced increase in heroin-taking and heroin-seeking behavior resulting from NAcsh Penk overexpression prompted us to assess whether Penk-mediated regulation of these behaviors were selective to striatopallidal neurons that preferentially express the Penk gene. To this end, we used a microRNA (miR) targeting the Penk mRNA, allowing for miR-mediated mRNA cleavage specific to Penk-expressing neurons. After confirming the specific activity of the Penk miR in vitro (Figure S4 in Supplement 1), we verified the effects of local overexpression of a lentiviral vector containing the Penk miR tagged with GFP (miR Penk) into NAcsh (Figure 1B).
      To investigate whether miR-mediated Penk knockdown could reverse the behavioral phenotype that was induced by adolescent THC exposure and NAcsh Penk infusion, three cohorts of animals were treated with THC during adolescence and then given bilateral NAcsh infusions of the miR Penk lentivirus or one of two lentiviral control vectors, one of which contained no targeting miR sequence (GFP) and the other a miR known to target a sequence not found in vertebrate DNA (miR ctrl), in young adulthood (Figure 1C; Table S1 in Supplement 1). Despite the marked downregulation of Penk mRNA expression that resulted from miR Penk infusion, animals readily learned the SA paradigm. miR Penk in NAcsh reduced both overall responding for heroin [treatment by session interaction, F(22,213) = 1.759, p < .05, Figure 3A] and mean heroin intake [treatment by session interaction, F(22,211) = 2.136, p < .01, Figure 3B] compared with GFP- and miR ctrl-infused control animals. Interestingly, the heroin-taking behavior exhibited by miR Penk animals was similar to the behavior displayed by GFP animals unexposed to THC during adolescence (Figure 2A and 2B). NAcsh miR Penk also increased locomotor activity [F(2,15) = 11.441, p < .001; Figure S1B in Supplement 1], in line with the inhibitory role of the striatopallidal pathway in regulating motor behavior. Overall, these data provide evidence that NAcsh miR Penk blocks the behavioral phenotype induced by adolescent THC and further implicate a role for NAcsh Penk as a key mediator of heroin susceptibility.
      Figure thumbnail gr3
      Figure 3Penk knockdown in nucleus accumbens shell (NAcsh) attenuates heroin self-administration. (A) Acquisition of heroin self-administration (SA; fixed-ratio 1, 30 μg/kg/injection). (B) Mean heroin intake. (C) Between-session dose-response (7.5, 15, 30, 60, 100 μg/kg/infusion; randomized order). (D) Mean heroin intake. (E) Heroin-seeking behavior (cue- and stress-induced) in microRNA (miR) Penk-infused, miR c-infused, or green fluorescent protein (GFP)-infused rats exposed to adolescent Δ9-tetrahydrocannabinol. (F) Penk mRNA levels in NAcsh, nucleus accumbens (NAc) core, and caudate-putamen (CPu) following heroin SA. (G) Representative in situ hybridization autoradiograms demonstrating striatal Penk mRNA expression following heroin SA. For all figures, n = 5–9 per group. Data shown as mean ± SEM. *p < .05; **p < .01 compared with GFP-expressing controls for each session; ***p < .001 compared with GFP and miR controls. AL, active lever; IL, inactive lever, dpm, disintegrations per minute. See also .
      In contrast to Penk overexpression, miR Penk in NAcsh led to a downward shift in the dose–response function, including both lower maximal SA rates [treatment by dose interaction, F(8,77) = 4.120, p < .001, Figure 3C] and heroin intake (Figure 3D) on the lower end of the dose–response curve compared with GFP and miR ctrl-infused control animals. Because a downward shift in the dose–response curve opposes alterations thought to be associated with the transition to more addicted states, these data indicate that reduced shell Penk tone decreases apparent behavioral susceptibility to heroin reinforcement. Given that NAcsh miR Penk attenuated the behavioral phenotype induced by adolescent THC exposure, we next investigated whether NAcsh miR Penk affected behavioral susceptibility to drug seeking. Mir Penk did not affect cue- or stress-induced heroin seeking when compared with GFP and miR ctrl controls (Figure 3E). There was a significant downregulation of NAcsh Penk mRNA expression in miR Penk animals [F(2,24) = 222.929, p < .001; Figure 3F, G] compared with GFP and miR ctrl control animals following the completion of behavioral experiments. The viral manipulation was specific to the Penk gene as Pdyn mRNA levels in NAcsh were unchanged as a result of miR Penk infusion (Figure S3 in Supplement 1). Taken together, these data establish a causal link between adolescent THC-mediated Penk dysregulation and the subsequent expression of behavioral susceptibility to heroin.

      Adolescent THC Regulates Repressive Histone H3 Methylation at the Penk Gene in NAcsh

      Given the protracted behavioral consequences of THC exposure during adolescence (
      • Ellgren M.
      • Spano S.M.
      • Hurd Y.L.
      Adolescent cannabis exposure alters opiate intake and opioid limbic neuronal populations in adult rats.
      ), we aimed to identify whether alterations at the level of chromatin regulation were associated with the transcriptional dysregulation of Penk that follows adolescent THC exposure. One day after the last THC treatment, NAcsh Penk mRNA levels were not significantly altered (Figure 4A); however, consistent with previous data, Penk mRNA expression was significantly increased in NAcsh 30 days after cessation of adolescent THC administration [t(15) = 2.78, p < .05; Figure 4A] compared with control animals. As a first step toward characterizing the potential epigenetic regulation of Penk, we investigated whether adolescent THC exposure was associated with altered levels of histone H3 methylation. In light of recent reports describing cocaine-induced alteration of repressive histone H3 lysine 9 (H3K9) methylation at gene promotors in the NAc (
      • Renthal W.
      • Kumar A.
      • Xiao G.
      • Wilkinson M.
      • Covington 3rd, H.E.
      • Maze I.
      • et al.
      Genome-wide analysis of chromatin regulation by cocaine reveals a role for sirtuins.
      ,
      • Maze I.
      • Covington 3rd, H.E.
      • Dietz D.M.
      • LaPlant Q.
      • Renthal W.
      • Russo S.J.
      • et al.
      Essential role of the histone methyltransferase G9a in cocaine-induced plasticity.
      ), we studied di- and trimethylation of H3K9 (H3K9me2, H3K9me3) at the Penk gene, as well as trimethylation of histone H3 lysine 36 (H3K36me3) and lysine 4 (H3K4me3), marks that have been associated with transcriptional activation (
      • Li B.
      • Carey M.
      • Workman J.L.
      The role of chromatin during transcription.
      ,
      • Shilatifard A.
      Molecular implementation and physiological roles for histone H3 lysine 4 (H3K4) methylation.
      ). Animals were treated with THC during adolescence and levels of H3K9me2, H3K9me3, H3K4me3, and H3K36me3 were analyzed in NAcsh via chromatin immunoprecipitation followed by quantitative PCR analysis of five sites flanking the Penk gene transcription start site, three spanning regulatory elements in the 5′ untranslated region and two in the coding region (Figure S5 and Table S2 in Supplement 1). One day following the final THC treatment, H3K9me2 was decreased at two sites in the Penk promoter region in the most upstream regions evaluated [−1.5−t(11) = −2.417, p < .05; −.9−t(11) = −2.738, p < .05; point-wise comparison Figure 4B] compared with vehicle-treated adolescent control animals. H3K9me3 did not differ statistically between the groups but did tend to be increased at the same promoter regions where H3K9me2 was decreased (Figure 4C). No change was observed in H3K36me3 (Figure 4D). H3K4me3 levels were increased at each region evaluated [−1.5−t(10) = 2.545, p < .05; −.9−t(9) = 7.109, p < .0001; −.6−t(10) = 5.621, p < .001; +.2−t(10) = 3.550, p < .01; +.4−t(10) = 8.144, p < .0001; Figure 4E].
      Figure thumbnail gr4
      Figure 4Adolescent Δ9-tetrahydrocannabinol (THC) regulates Penk gene expression and histone H3 methylation in nucleus accumbens shell (NAcsh). (A) NAcsh Penk messenger RNA (mRNA) levels 1 day (adolescent) and 30 days (adult) after adolescent THC or vehicle (n = 9–10/group). (B–E) NAcsh histone H3 methylation fold changes at the Penk gene 1 day and 30 days after the last adolescent exposure to THC relative to vehicle treated animals (n = 6–8/group; 3 animals pooled/n). (B) H3K9me2. (C) H3K9me3. (D) H3K36me3. (E) H3K4me3. Data shown as mean ± SEM. *p < .05; **p < .01; ***p < .001 compared with vehicle-exposed animals at the same time point. kb, kilobases; TSS, transcription start site. See also .
      One month following cessation of adolescent THC treatment, H3K9me2 remained decreased at Penk in adult NAcsh, but significant effects were observed at promoter sites .9 kb and .6 kb upstream of the Penk transcription start site [−.9−t(9) = −2.260, p < .05; −.6−t(8) = −2.480, p < .05; Figure 4B]. In contrast to the pattern of H3K9me3 observed in adolescent NAcsh, however, H3K9me3 was decreased at all regions of the Penk gene in adult NAcsh [−1.5−t(10) = −4.698, p < .001; −.9−t(10) = −7.172, p < .0001; −.6−t(10) = −6.959, p < .0001; +.2−t(10) = −5.681, p < .001; +.4−t(10) = −6.451, p < .0001; Figure 4C], a finding consistent with the increased Penk gene expression (Figure 4A) in these animals. No alterations were observed in H3K36me3 or H3K4me3 in adult animals (Figure 4D and 4E). Taken together, these data suggest that decreases in H3K9me2 and H3K9me3 binding at the Penk promoter in adult NAcsh may mediate the upregulation of Penk transcription characteristic of adult animals with adolescent THC exposure.

      Developmental Regulation of Histone H3 Methylation at the Penk Gene in NAcsh

      Given that few studies have investigated the ontogeny of the enkephalinergic system, we were interested to study potential developmental differences in the regulation of Penk gene expression and histone H3 methylation at the Penk gene. Evaluation of NAcsh Penk mRNA levels in THC-naïve adolescent and adult animals revealed no significant difference between developmental periods (Figure 5A). We next examined H3K9me2, H3K9me3, H3K36me3, and H3K4me3 levels at the Penk gene of THC-naïve adolescent and adult animals. While H3K9me2 and H3K36me3 levels were similar between adolescent and adult animals, levels of H3K9me3 were increased in adult NAcsh at all regions studied [−1.5−t(9) = 4.941, p < .0001; −.9−t(9) = 8.589, p < .0001; −.6−t(10) = 7.160, p < .0001; +0.2−t(10) = 5.473, p < .0001; +0.4−t(10) = 6.249, p < .0001; Figure 5B]. H3K4me3 was also elevated in adulthood compared with the adolescent period at all regions [−1.5−t(9) = 4.211, p < .01; −.9−t(9) = 5.172, p < .001; −.6−t(9) = 5.586, p < .001; +.2−t(9) = 4.179, p < .01; +.4−t(9) = 5.434, p < .001; Figure 5B]. The concomitant enrichment of both H3K9me3 and H3K4me3 at the Penk gene could account for the lack of significant difference observed in Penk mRNA levels between adolescence and adulthood. These data provide evidence that specific histone H3 methyl marks in NAcsh are developmentally regulated.
      Figure thumbnail gr5
      Figure 5Histone H3 methylation at the Penk gene in nucleus accumbens shell (NAcsh) is dynamic during normal development. (A) Adolescent and adult NAcsh Penk messenger RNA (mRNA) levels (n = 9–10/group). (B) NAcsh histone H3 methylation fold changes at the Penk gene (n = 6–8/group; 3 animals pooled/n). Data shown as mean ± SEM. *p < .05; **p < .01; ***p < .001 compared with adolescent animals. kb, kilobases.

      Discussion

      This studies reveal a direct link between NAcsh Penk gene expression and enhanced behavioral susceptibility to heroin SA that mimics that seen in adult animals exposed to THC during adolescence. Such findings lend strong support to the hypothesis that adolescent THC exposure contributes to an opiate-vulnerable phenotype in adulthood. Here, we show that overexpression of NAcsh Penk in THC-naïve animals potentiates heroin SA, a behavioral effect that is attenuated by striatopallidal Penk knockdown in THC-exposed animals. Together, these data indicate a direct relationship between adolescent THC-induced Penk upregulation and heightened heroin taking in adulthood. Furthermore, we suggest that adolescent THC exposure may mediate adult NAcsh Penk upregulation through regulation of repressive histone H3K9 methylation, an epigenetic effect that represents a profound pathologic departure from the distinct developmental pattern of histone H3 methylation that normally occurs at Penk in NAcsh across the transition from adolescence to adulthood.
      Of the opioid neuropeptides, enkephalin is consistently associated with regulating hedonic state (
      • Kelley A.E.
      • Bakshi V.P.
      • Haber S.N.
      • Steininger T.L.
      • Will M.J.
      • Zhang M.
      Opioid modulation of taste hedonics within the ventral striatum.
      ,
      • Skoubis P.D.
      • Lam H.A.
      • Shoblock J.
      • Narayanan S.
      • Maidment N.T.
      Endogenous enkephalins, not endorphins, modulate basal hedonic state in mice.
      ). Although our SA paradigm was not designed to dissociate between reward and incentive motivational state, heroin SA behavior did not differ between groups during the early stages of acquisition, arguing against a Penk-mediated generalized impairment of basal hedonic tone. Moreover, although selective knockdown of Penk expression reduced overall heroin intake over time, it did not affect acquisition of SA behavior. Instead, Penk-overexpressing and THC-exposed animals continued to increase their heroin intake, ultimately stabilizing at a higher drug intake level during the maintenance phase, suggesting that these animals have different hedonic set points compared to controls (
      • Ellgren M.
      • Spano S.M.
      • Hurd Y.L.
      Adolescent cannabis exposure alters opiate intake and opioid limbic neuronal populations in adult rats.
      ). Additionally, the present data demonstrate that animals with elevated NAcsh Penk expression exhibit potentiated drug-seeking behavior induced by drug-associated environmental cues and mild stress. Interestingly, stress-induced sensitivity to heroin drug seeking was also apparent in adults following prenatal THC exposure (
      • Spano M.S.
      • Ellgren M.
      • Wang X.
      • Hurd Y.L.
      Prenatal cannabis exposure increases heroin seeking with allostatic changes in limbic enkephalin systems in adulthood.
      ). Although the animals' affective state underlying sensitivity to heroin is not yet fully understood, the present experiments implicate a direct role for NAcsh Penk in the opiate-susceptible behavioral phenotype similar to the consequence of adolescent THC exposure.
      In the NAc, Penk is predominantly expressed in striatopallidal medium spiny neurons that project to ventral pallidum (
      • Zhou L.
      • Furuta T.
      • Kaneko T.
      Chemical organization of projection neurons in the rat accumbens nucleus and olfactory tubercle.
      ). Viral overexpression of Penk was not localized to a specific striatal subpopulation in our study, but it nevertheless resulted in the same behavioral pattern of heroin SA demonstrated by rats exposed to adolescent THC, suggesting that an increase in NAcsh enkephalinergic tone may be sufficient to affect opioid susceptibility. In contrast, miR knockdown of Penk is inherently specific to striatopallidal cells, and such manipulations attenuated the enhancement of heroin SA induced by adolescent THC exposure. Importantly, NAcsh Pdyn levels were unaffected by any of the manipulations, indicating specificity of the behavioral alterations to selective NAcsh Penk alteration. Together, these findings emphasize the important role of Penk in mediating long-term effects of THC that contribute to opiate susceptibility. How regulation of Penk striatopallidal regulation contributes to specific components of addiction-related behavior in the nondrug state remains to be established.
      Given the protracted behavioral effects of adolescent THC exposure, alterations at the level of chromatin regulation are prime candidates for investigation. Although several studies have suggested an important role for transient histone modifications in the regulation of drug-induced behaviors, only recently has histone methylation, a more stable modification, been demonstrated as a potential mediator of drug-induced transcriptional plasticity (
      • Renthal W.
      • Kumar A.
      • Xiao G.
      • Wilkinson M.
      • Covington 3rd, H.E.
      • Maze I.
      • et al.
      Genome-wide analysis of chromatin regulation by cocaine reveals a role for sirtuins.
      ,
      • Maze I.
      • Covington 3rd, H.E.
      • Dietz D.M.
      • LaPlant Q.
      • Renthal W.
      • Russo S.J.
      • et al.
      Essential role of the histone methyltransferase G9a in cocaine-induced plasticity.
      ). Histone methylation is highly complex; N-terminal histone lysine residues can be mono, di, or trimethylated, with each valence state differentially regulating the recruitment of proteins that activate or repress transcription (
      • Rice J.C.
      • Allis C.D.
      Histone methylation versus histone acetylation: New insights into epigenetic regulation.
      ,
      • Martin C.
      • Zhang Y.
      The diverse functions of histone lysine methylation.
      ). Although increased H3K9me2 binding has been demonstrated at promoters of repressed eukaryotic genes, our findings confirm that reduced H3K9me2 binding plays a role at promoters of activated eukaryotic genes. Of the histone marks quantified in this study, dimethylation of H3K9 at upstream regions of the Penk gene in NAcsh was reduced both 1 day and 30 days after THC administration. In contrast, the pronounced enrichment across the Penk promoter of the activating mark H3K4me3 seen 1 day after THC exposure was normalized by adulthood.
      In addition to modulation of H3K9me2, adolescent THC exposure also had significant effects on H3K9me3, an unexpected finding given that H3K9me3 is typically enriched at pericentromeric heterochromatin and sites of repressed chromatin (
      • Cao R.
      • Wang L.
      • Wang H.
      • Xia L.
      • Erdjument-Bromage H.
      • Tempst P.
      • et al.
      Role of histone H3 lysine 27 methylation in Polycomb-group silencing.
      ,
      • Rice J.C.
      • Briggs S.D.
      • Ueberheide B.
      • Barber C.M.
      • Shabanowitz J.
      • Hunt D.F.
      • et al.
      Histone methyltransferases direct different degrees of methylation to define distinct chromatin domains.
      ,
      • Schotta G.
      • Lachner M.
      • Sarma K.
      • Ebert A.
      • Sengupta R.
      • Reuter G.
      • et al.
      A silencing pathway to induce H3-K9 and H4-K20 trimethylation at constitutive heterochromatin.
      ,
      • Maze I.
      • Feng J.
      • Wilkinson M.B.
      • Sun H.
      • Shen L.
      • Nestler E.J.
      Cocaine dynamically regulates heterochromatin and repetitive element unsilencing in nucleus accumbens.
      ). However, several groups have reported the presence of H3K9me3 in transcribed regions of active mammalian genes (
      • Brinkman A.B.
      • Roelofsen T.
      • Pennings S.W.
      • Martens J.H.
      • Jenuwein T.
      • Stunnenberg H.G.
      Histone modification patterns associated with the human X chromosome.
      ,
      • Rougeulle C.
      • Chaumeil J.
      • Sarma K.
      • Allis C.D.
      • Reinberg D.
      • Avner P.
      • et al.
      Differential histone H3 Lys-9 and Lys-27 methylation profiles on the X chromosome.
      ,
      • Squazzo S.L.
      • O'Geen H.
      • Komashko V.M.
      • Krig S.R.
      • Jin V.X.
      • Jang S.W.
      • et al.
      Suz12 binds to silenced regions of the genome in a cell-type-specific manner.
      ,
      • Vakoc C.R.
      • Mandat S.A.
      • Olenchock B.A.
      • Blobel G.A.
      Histone H3 lysine 9 methylation and HP1gamma are associated with transcription elongation through mammalian chromatin.
      ). The current finding that H3K9me3 was decreased long term (30 days) in the transcribed regions of Penk in the adult NAcsh as a consequence of adolescent THC exposure raises the possibility that reduced H3K9me3 in the coding regions of active genes may also contribute to transcriptional plasticity. Current technologies cannot establish causal regulation of histone methylation at a single gene level, but accumulating evidence suggests that H3K9me3 may play a significant role in regulating active genes (
      • Vakoc C.R.
      • Mandat S.A.
      • Olenchock B.A.
      • Blobel G.A.
      Histone H3 lysine 9 methylation and HP1gamma are associated with transcription elongation through mammalian chromatin.
      ,
      • Vakoc C.R.
      • Sachdeva M.M.
      • Wang H.
      • Blobel G.A.
      Profile of histone lysine methylation across transcribed mammalian chromatin.
      ). Given the low levels of H3K9me3 at most expressed genes (
      • Maze I.
      • Feng J.
      • Wilkinson M.B.
      • Sun H.
      • Shen L.
      • Nestler E.J.
      Cocaine dynamically regulates heterochromatin and repetitive element unsilencing in nucleus accumbens.
      ), however, the magnitude of the fold changes seen with adolescent THC in the adult may be artificially enhanced. Although it is currently impossible to know the absolute neurobiological consequences of small relative changes in histone marks, the differential profile of H3K9me3 at the Penk gene 1 day compared with 30 days after THC exposure, coupled with the potentiated SA behavior evident in adult animals, support a functional role for even small perturbations in H3K9me3 at the Penk gene and thus requires further investigation.
      To date, no studies have examined histone methylation during normal development. Adolescence is a critical phase of brain maturation, and the current results demonstrate distinct development-specific patterns of histone H3 modifications at the NAcsh Penk gene. Although stable levels of H3K9me2 and H3K36me3 were observed in NAcsh of adolescent and adult animals, the profiles of H3K9 and H3K4 trimethylation varied across this developmental period. The chromatin landscape is highly complex, but trimethylation of H3K4 (transcriptional activation) concomitant with trimethylation of H3K9 (transcriptional repression) may account for the developmental transcriptional stability of NAcsh Penk because there was no difference in Penk mRNA levels in adolescent versus adult. Furthermore, that H3K9me3 and H3K4me3 displayed similar magnitudes of induction and distribution across the Penk gene in adolescent NAcsh suggests that trimethylation of these marks may be coordinated (
      • Vakoc C.R.
      • Mandat S.A.
      • Olenchock B.A.
      • Blobel G.A.
      Histone H3 lysine 9 methylation and HP1gamma are associated with transcription elongation through mammalian chromatin.
      ) during normal development. Although the functional consequences on NAcsh Penk gene expression did not differ between adolescence and adulthood, the distinct epigenetic profiles during these ontogenetically disparate periods may allow the Penk gene to be “primed” to respond differentially to similar environmental cues. Limited studies have investigated the differential neurobiological effects of THC exposure in adolescence versus adulthood, but mounting evidence documents differential responsivity to drugs of abuse in the adolescent compared with the adult brain (
      • Schramm-Sapyta N.L.
      • Cha Y.M.
      • Chaudhry S.
      • Wilson W.A.
      • Swartzwelder H.S.
      • Kuhn C.M.
      Differential anxiogenic, aversive, and locomotor effects of THC in adolescent and adult rats.
      ,
      • Izenwasser S.
      Differential effects of psychoactive drugs in adolescents and adults.
      ). Overall, our study emphasizes that adolescent THC exposure leads to a departure of the normal trajectory of the transcriptional and epigenetic state of the Penk gene, a disruption that may mediate the expression of enhanced behavioral vulnerability to opiates in adulthood.
      In conclusion, our findings indicate that marijuana exposure in and of itself can serve as a risk factor that acts “above the genome” and can imprint on the existing epigenetic landscape of adolescent neurodevelopment. Thus, the epigenetic effects of adolescent THC exposure may act in concert to augment future behavioral responses to drugs of abuse via stable and long-term regulation of genes at the transcriptional level. The results also support a novel role for the Penk gene as an emergent endogenous risk factor resulting from adolescent THC exposure, the dysregulation of repressive histone H3 methylation of which may underlie the long-term behavioral consequences of adolescent THC.
      This work was funded by the National Institute on Drug Abuse , Grant Nos. DA024929 (HCT), T32 DA007135 (MMJ), DA08227 (EJN), DA030359 (YLH), and DA19350 (YLH).
      We thank Dr. Ian Maze for comments on the manuscript and Dr. Yanhua Ren for technical advice.
      The authors report no biomedical financial interest or potential conflicts of interest.

      Supplementary data

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