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Behavioural Brain Research 256 (2013) 655– 661

Contents lists available at ScienceDirect

Behavioural Brain Research

j ourna l h om epage: www.elsev ier .com/ locate /bbr

esearch report

ersistent gene expression changes in NAc, mPFC, and OFC associatedith previous nicotine or amphetamine exposure

ichelle Mychasiuka,∗, Arif Muhammada, Slava Ilnytskyyb, Bryan Kolba

Canadian Centre for Behavioural Neuroscience, University of Lethbridge, CanadaDepartment of Biology, University of Lethbridge, Canada

i g h l i g h t s

Exposure to amphetamine or nicotine decreases DNA methylation in the reward circuit.Psychomotor stimulants produce persistent epigenetic changes in PFC and NAc.The epigenetic changes are dependent upon the drug (amphetamine/nicotine).The epigenetic changes are region specific; differing in the mPFC, OFC and NAc.Persistent changes differ from epigenetic responses immediately following exposure.

r t i c l e i n f o

rticle history:eceived 27 August 2013ccepted 1 September 2013vailable online xxx

eywords:pigeneticethylation

ddictionsychomotor stimulantong-Evans rat

a b s t r a c t

Highly addictive drugs like nicotine and amphetamine not only change an individual’s behaviour inthe short and long-term, they also induce persistent changes in neuronal excitability and morphology.Although research has started to examine the epigenetic changes that occur immediately after drugexposure, there has been little investigation into the persistent modifications to the epigenome that likelymoderate the stable maintenance of the neurological changes. Male Long-Evans rats were administeredamphetamine, nicotine, or saline for 14 consecutive days, given a 14 day withdrawal period, and thensacrificed. DNA from the mPFC, OFC, and nucleus accumbens (NAc) was used for global DNA methylationanalysis and RNA from the same brain regions was used for gene expression analysis. Following thetwo-week withdrawal period, exposure to amphetamine or nicotine was associated with a decrease inglobal DNA methylation in each brain region examined. Previous exposure to nicotine was associated

with changes in expression of 16 genes (NAc:6, mPFC:5, OFC:5) whereas exposure to amphetamine wasassociated with changes in expression of 25 genes (NAc:13, OFC:8, mPFC:4). The persistent epigeneticchanges associated with exposure to amphetamine and nicotine were region and drug dependent, anddiffer from the latent epigenetic changes that occur immediately after drug exposure. The changes in DNAmethylation are consistent with the gene expression results and provide further support to the notionthat DNA methylation is the key regulatory mechanism for experience dependent changes.

. Introduction

Epigenetic modifications to protein expression likely underliehe plastic nature of synaptic connections and cortical reorgani-ation [1]. A neuron must have mechanisms capable of ‘memory’

or experiences in the past, but also ‘plasticity’ to respond to cur-ent experiences [2]. Certain experiences, such as exposure to drugsf abuse, likely exploit the plastic nature of the epigenome to

∗ Corresponding author at: Canadian Centre for Behavioural Neuroscience, Uni-ersity of Lethbridge, 4401 University Drive, Lethbridge, AB T1K 3M4, Canada.el.: +1 403 329 2405.

E-mail address: r.mychasiuk@uleth.ca (R. Mychasiuk).

166-4328/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.bbr.2013.09.006

© 2013 Elsevier B.V. All rights reserved.

induce long-term changes in brain structure and function. Thepersistent effects induced by drugs of abuse are evident in drugcravings and relapse that occur decades after the last exposure.For changes in gene expression and chromatin organization toaffect complex behaviours like addiction, they must affect func-tionality at a higher level, such as neuronal excitability or synapticconnectivity [3–5]. As almost every drug of abuse has been demon-strated to induce structural plasticity, most specifically alterationsto dendritic spines, it is important to understand the molecu-lar mechanisms that regulate these changes [6]. Although DNA

methylation is a primary contributor to the stable maintenance ofparticular gene expression states [7] and may be critical for imme-diate brain changes associated with drug exposure [8,9], othergenes may be responsible for maintaining the persistent changes

6 l Brain Research 256 (2013) 655– 661

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n functionality. The continual activation of the reward system byrugs of abuse leaves the epigenome vulnerable to transient andtable modification.

Evidence indicates that the epigenetic mechanisms involvedn sensitization and addiction likely require multiple stages of

odification; a latent phase immediately following drug expo-ure or learning that is characterized by changes in expressionf DNMTs, HDACs, and HATs [8,10,11], and a regulatory phase,hanges in expression of genes as a result of the DNMT, HDAC,nd HAT activity that maintain the structural and functionalhanges. Multiple studies have examined the latent phase; forxample, LePlant et al. eloquently demonstrated that DNA methyla-ion, via DNMT3, regulates immediate drug sensitization-inducedhanges in NAc spine density. Furthermore, Guan et al. demon-trated that changes in HDAC expression can regulate the formationnd retrieval of memories from the hippocampus. Although stud-es have nicely examined the early epigenetic response, thereppears to be a limited number of studies in the literaturexamining gene expression changes that persist weeks after lastncountering the drug [12]. In addition, despite the fact thathere are a multitude of brain regions involved in reward-basedearning and addiction, epigenetic examination of drug inducedhanges have been limited to the NAc [8,10,13] and hippocampus14,15].

The purpose of this study was to examine the persistent changesn gene expression two weeks after chronic administration oficotine or amphetamine. This study sought to investigate thepigenetic effects of psychomotor stimulants using global DNAethylation and mRNA biosequencing, on three critical structures

f the brain’s reward circuitry; mPFC, OFC, and the NAc. Identi-cation of the genes exhibiting stable changes in expression willelp researchers understand the mechanisms driving the persistentature of addiction and drug abuse.

. Materials and methods

.1. Subjects and drug administration

All experiments were carried out in accordance with the Cana-ian Council of Animal Care and approved by the Universityf Lethbridge Animal Care Committee. Twenty-seven male Longvans rats were ordered from Charles Rivers Laboratories andrrived at the age of postnatal day 70 (P70). All rats were housedn standard shoebox cages (3 rats/cage) and maintained on a2:12 h light:dark cycle in a temperature controlled room (21 ◦C).he rats were handled for 20 min/day for the first 10 days afterrrival to reduce the stress associated with travel. On P80, the ratsere randomly assigned to 1 of 3 groups, Nicotine injections (9),mphetamine injections (9) or Saline injections (9).

Rats were administered nicotine, amphetamine or saline from80–P93 (14 days), given a 14 day withdrawal period and thenacrificed at P108. Drug doses and treatment regimes were cho-en to replicate procedures previously used in this laboratory totudy persistent changes in dendritic morphology and synapticonnectivity following exposure to psychomotor stimulants [4,16].ocomotor activity, used as an index of behavioural sensitization17], was recorded using the Accuscan activity monitoring systemnd recorded using the VersaMax software program. The rats wereabituated to the activity box for 30 min, administered the drug,nd then immediately returned to the activity box for 90 min. Drugsere administered (1.0 mg/kg body weight, i.p. for amphetamine

nd 0.3 mg/kg for nicotine) at 9:00 am daily. Amphetamine (d-mphetamine sulfate) and nicotine (nicotine hydrogen tartratealt) (Sigma Aldrich, St. Louis, MO, USA) were dissolved in ster-le 0.9% saline. Saline administration (0.9%) was also completed

Fig. 1. Illustrative representation of brain areas used for epigenetic analysis.

based on a 1 ml/kg paradigm. Once recording of activity wascompleted, rats were returned to their home cages. The devel-opment of sensitization was determined by analysing activityrecorded over the 14-day period using a repeated measure ANOVAwith drug (Amphetamine, Nicotine, or Saline) and day (1–14)as factors, followed by Bonfoerroni’s post hoc test for multiplecomparisons.

2.2. Sacrifice, methylation and gene expression

Rats were subjected to isoflourane inhalation, weighed andquickly decapitated. The brains were rapidly dissected using theZilles [18] rat brain atlas, to remove mPFC (Cg3, Cg2, PL & IL) OFC(AID) and nucleus accumbens from each rat. Tissue was immersedin RNAlater RNA stabilization Reagent (Qiagen, Valencia), flashfrozen and stored at −80 ◦C. See Fig. 1 for an illustration of the brainregions extracted.

Genomic DNA and RNA were extracted from the brain tis-sue using the Qiagen Allprep DNA/RNA Mini Kit according to themanufacturer’s recommendations (Qiagen, 2005). DNA and RNAwere stored at −20 ◦C until used for analysis. A spectrophotometer(NanoDrop, 2000: Thermo Fisher Scientific) was used to determinethe quantity and quality of the samples. All sample concentrationswere over 100 ng/�l and produced absorbance ratios (A260/280)between 1.95 and 2.05.

2.2.1. Global DNA methylationWe utilized a well-established radiolabelled [3H]-dCTP exten-

sion assay [19] to determine the extent of global DNA methylation.This assay exploits two similar restriction endonucleases, HpaIIand MspI (New England Biolabs, Beverly, MA, USA) to measure theproportion of CCGG sites that have lost methyl groups on bothstrands. HpaII cleaves unmethylated CCGG sequences on eitherstrand, whereas MspI cleaves CCGG sites regardless of methyla-tion state. The absolute percent of double-stranded unmethylated

CCGG sites can be calculated by relating the data of the HpaIIand MspI digests [20]. Using a radiolabelled tag, [3H]-dCTP that isadded to the cleaved cytosine residues we can measure the amountof radioactive incorporation. The analyst conducted two blinded

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echnical repeats of each experiment to ensure accuracy. Methyla-ion data was analyzed using SPSS 19.0 for Mac. One-way ANOVAsith drug (Nicotine, Amphetamine, or Saline) were run for each

rain region examined (mPFC, OFC & NAc).

.2.2. Bio-sequencing mRNA for gene expressionRNA samples for gene expression analysis were pooled; RNA

rom three rats in the same experimental group were pooled to pro-uce the samples used in the analysis (i.e. 9 rats with mPFC samplesrom the Amphetamine group; this was pooled into 3 groups, eachontaining RNA from 3 rats). Each rat contributed equal RNA to theooled sample.

The Illumina GAIIx sequencing platform was used to iden-ify differentially expressed genes between each of the two drugreatment groups and the control group in the three differentrain regions (i.e. mPFC – Amphetamine compared to Control, andeparately, Nicotine compared to Control). Prior to initiating theequencing platform all RNA samples were run through a qual-ty control analysis and reached criteria. In addition, the qualityf the sequencing reads was assessed before analyzing the out-ut reads; all of the libraries were of high quality with reads inhe range of 6–10 millions. The parameters used for analysis were:llumina GAIIx; Single-End; multiplexed; 36 cycles; 9 samples perane, each sample was run in duplicate across two lanes; sam-les were balanced across the lanes according to treatment groups.asecalling and demultiplexing were performed using the IlluminaASAVA 1.8.1 with default parameters according to the manufac-urer’s manual. The Rat, rn4 UCSC, downloaded from the IlluminaGENOME site was used for referencing. The annotation information

as downloaded from Ensembl.Read alignment was performed using the bwa aligner version

.6.2 [21]. Htseq-count script considers only unique alignments.ntersection-strict mode ensures that only those reads that fallntirely inside the feature get counted, also reads that aligned toultiple features are removed to avoid double counting. Statis-

ical comparisons between the control and drug groups for eachrain region were performed using DESeq Bioconductor packageversion 1.10.1) [22]. Only genes with a p-value adjusted for the

alse discovery rate (Benjamini–Hochberg method) [23] of lesshan 0.25 were considered to be significantly altered; all of theseenes have p values less than .0001. All genes identified are proteinoding.

ig. 2. Mean (±SEM) locomotor activity (beam crossings × 103) recorded for 90 min after irug administration period. Animals that received injections of nicotine or amphetaminep < .01).

Research 256 (2013) 655– 661 657

2.2.3. Quantitative Real Time PCR (qRT-PCR)Expression changes in six of the genes that showed up- or down-

regulation on the microarray were validated with qRT-PCR. Thegenes selected represent both amphetamine and nicotine groupsfor each of the 3 brain regions examined. Descriptive methodologyincluding the primers utilized can be found in the SupplementalFile 1.

3. Results

3.1. Animal characteristics and drug sensitization

Chronic administration of amphetamine and nicotine did notaffect weight gain during drug administration or the withdrawalperiod. Similarly, drug exposure did not alter brain weight atthe time of sacrifice, F(2, 25) = 0.18, p = .84. All rats administeredamphetamine or nicotine exhibited sensitization to the drugs, asdemonstrated via increased locomotor activity over the 14 daytrial period, see Fig. 2. The repeated measures ANOVA with drug(amphetamine, nicotine, or saline) as independent factors and daysas the repeated measure, demonstrated that repeated exposure tonicotine and amphetamine resulted in behavioural sensitization,F(2, 48) = 103.22, p < .01.

3.2. Global DNA methylation

Chronic exposure to nicotine and amphetamine resulted in asignificant increase in global DNA methylation in all three brainregions examined following the two-week withdrawal period. Aone-way ANOVA with drug (amphetamine, nicotine, or saline) asthe factor was run for global methylation in each of the brainregions examined. All three brain regions, the NAc, mPFC, and OFC,demonstrated a main effect of drug, F(2, 16) = 7.89, p < .01; F(2,16) = 5.35, p = .02; F(2, 16) = 9.12, p < .01, respectively. See Fig. 3.

3.3. mRNA sequencing for gene expression

Following the two week withdrawal period, exposure to nico-

tine resulted in significant changes in the expression of 16 distinctgenes, of which 14 were down-regulated, see Table 1. This down-regulation of expression is consistent with the increased globalmethylation levels identified through our DNA analysis. In the NAc

njections of saline, nicotine (0.3 mg/kg) or amphetamine (1.0 mg/kg) for the 14-day were significantly more active than animals that received daily injections of saline

658 R. Mychasiuk et al. / Behavioural Brain Research 256 (2013) 655– 661

Table 1List of genes with significant expression differences between control rats and rats chronically administered nicotine (0.3 mg/kg) following a two-week withdrawal period.

Brain area Gene ID Rat genome database ID Gene name Log2 fold change

NAc

Nefm 3160 Neurofilament medium polypeptide −1.198Pcp4l1 1593809 Purkinje cell protein 4-like 1 −1.305Pcp4 3271 Purkinje cell protein 4 −1.007Hhatl 1311911 Hedgehog acyltransferase-like −2.087Nts 621612 Neurotensin −2.170Sema7a 1305192 Semaphorin 7A, GPI membrane anchor −0.957

OFC

Kitlg 3086 KIT ligand −0.580Cacna1h 68943 Calcium channel, voltage dependent, T type 0.516Pdyn 62054 Prodynorphin −0.759Luzp1 61839 Leucine zipper protein 1 −0.517Prr7 1561898 Proline rich 7 (synaptic) 1.026

mPFC

Rprm 1595512 Reprimo, TP53 dependent G2 arrest mediator −1.011Shisa3 1565710 Shisa Homolog 3 −1.616Slc13a4 1359715 Solute carrier family 13, member 4 −1.196RGD1359529 1359529 Similar to chromosome 1 open reading frame 63 −0.643

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ix genes displayed reduced expression in the nicotine comparedo the control group (Nefm, Pcp4l1, Pcp4, Hhatl, Nts, and Sema7a),hereas there were five genes significantly altered in both of theFC (Kitlg, Cacna1h, Pdyn, Luzp1, and Prr7) and the mPFC (Rprm,hisa3, Slc13a4, RGD1359529, and Arl4d).

Similarly, exposure to amphetamine resulted in significantlteration to 25 distinct genes, with more variability related to theilencing or enhancement of gene expression, see Table 2. This how-ver is also consistent with the global DNA methylation analysis;lobal methylation patterns in the amphetamine group were lessrofound than that of the nicotine group, suggesting genes wereoth up and down-regulated. Gene expression in the NAc was mosttrongly affected by amphetamine, with 13 genes exhibiting alteredevels of expression in the drug compared to the control groupKlf4, Notch3, Htr2a, RGD1359529, Nefl, Hsd11b1, Glrb, Acer2, Gabrg2,lc6a7, Shisa3, Pyroxd2, and Nefm). In the mPFC, four genes were upegulated (Arc, Atf3, Nr4a1, and Dusp1) whereas eight genes wereound to be altered in the OFC (Retsat, Bhlhe40, Dusp6, Lpar1, Synpo,srp1, Dnajb5, and Ahdc1).

Of the 41 genes affected by chronic exposure to the psychomo-or stimulants, one gene Nefm – neurofilament medium polypeptideas down-regulated in the NAc of both amphetamine and nicotine

xposed rats. Two additional genes, RGD1359529 and Shisa3 were

own-regulated in the NAc of amphetamine exposed rats and thePFC of nicotine treated rats. The remaining 35 genes were dis-

inct, failing to demonstrate further overlap between brain regionr drug exposure.

ig. 3. Average global DNA methylation for each brain region two weeks after ani-als received daily injections of saline, nicotine, or amphetamine for a 14-day period

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3.4. qRT-PCR validation

Confirmations of the changes identified with the mRNA geneexpression-sequencing platform were obtained for the six genesvalidated with qRT-PCR. Furthermore, there was significant corre-lation between the Log2 expression change obtained in the qRT-PCRand mRNA analysis, r = .88, p = .01. Figures and statistical analysescan be found in Supplemental File 1.

4. Discussion

Using both global DNA methylation and mRNA bio-sequencing,this study has demonstrated that chronic exposure to nicotine andamphetamine differentially affect gene expression in three dis-tinct regions of the brain’s reward circuit. One novel finding is thatthe epigenetic changes in the reward circuit are primarily area-and drug-dependent. The global DNA methylation results are quitereliable as a general predictor of the directionality (up or down reg-ulation) of the majority of significantly altered genes. Furthermore,the changes in global DNA methylation in this study provide fur-ther support to the widely held belief that experience dependentchanges in gene expression are likely regulated by DNA methyla-tion [7,24,25].

Several brain circuits are known to be involved in the neu-robiology of reward and addiction, with the interaction betweenthe nucleus accumbens (NAc) and the prefrontal cortex, specifi-cally the medial prefrontal cortex (mPFC) and the orbital prefrontalcortex (OFC), playing a particularly vital role [26,27]. Psychomo-tor stimulants such as amphetamine and nicotine, like all drugs ofabuse, activate the reward circuitry far more strongly and persis-tently than natural rewards [28]. Activation of the reward systemstimulates dopamine and other neurotransmitter systems, whichalter intracellular signalling cascades leading to the activation orinhibition of transcription factors and other chromatin regulat-ing proteins [3]. When psychomotor stimulant-induced changes inneuronal structure are measured using Golgi–Cox analysis, thereis a general tendency to see similar changes in NAc and mPFC(increases in dendritic branching and spine density) with the OFCexhibiting the opposite effects (decreased dendritic branching andspine density) (for review see, Robinson and Kolb [4]). Further-more, the morphological changes are consistent across drug type,

with different psychomotor stimulants, amphetamine or nicotine,exhibiting similar responses. Whats puzzling is the contradictoryresponse of the mPFC and OFC to drugs of abuse because bothof these brain regions receive projections from the mediodorsal

R. Mychasiuk et al. / Behavioural Brain Research 256 (2013) 655– 661 659

Table 2List of genes with significant expression differences between control rats and rats chronically administered amphetamine (1.0 mg/kg) following a two-week withdrawalperiod.

Brain area Gene ID Rat genome database ID Gene name Log2 fold change

NAc

Klf4 621445 Kruppel-like factor 4 1.503Notch3 620761 Notch 3 1.742Htr2a 61800 5-Hydroxytryptamine (serotonin) receptor 2A, G-coupled protein −0.965RGD1359529 1359529 Similar to chromosome 1 open reading frame 63 1.734Nefl 621458 Neurofilament, light polypeptide −0.554Hsd11b1 2834 Hydroxysteroid 11-beta dehydrogenase 1 1.599Glrb 2706 Glycine receptor, beta −0.578Acer2 1304629 Alkaline ceramidase 2 0.810Gabrg2 61966 Gamma-aminobutyric acid (GABA) A receptor −0.581Slc6a7 620928 Solute carrier family 6, member 7 −0.611Shisa3 1565710 Shisa Homolog 3 −1.140Pyroxd2 1303232 Pyridine nucleotide-disulphide oxidoreductase domain 2 1.141Nefm 3160 Neurofilament, medium polypeptide −0.474

OFC

Retsat 628802 Retinol saturase 0.672Bhlhe40 68439 Basic helix-loop-helix family, member e40 −0.623Dusp6 70978 Dual specificity phosphatase 6 −0.648Lpar1 620563 Lysophosphatidic acid receptor 0.596Synpo 620668 Synaptopodin −0.420Csrp1 62053 Cystine and glycine-rich protein 0.394Dnajb5 1307453 DNAJ (Hsp40) homolog −0.511Ahdc1 1310031 AT hook, DNA binding motif −0.452

mPFC

Arc 62037 Activity-regulated cytoskeleton-associated protein 1.550Atf3 2165 Activating transcription factor 3 3.430Nr4a1 620029 Nuclear receptor subfamily 4 0.637

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ucleus of the thalamus, are primary cortical targets of mesocorti-al dopamine inputs, both are interconnected to the amygdala andoth send excitatory projections to the NAc [3,29]. As it has beenypothesized that the psychomotor stimulant induced changes inendritic morphology result from activation of signalling cascadeshat alter transcriptional regulation of actin assembly processes30], the OFC and mPFC must encompass underlying differencesn epigenetic susceptibility to drugs of abuse.

In this particular study, the changes in gene expression wereery different for the mPFC and OFC in response to the same drug.lthough there were no obvious changes in gene expression thatould induce the opposing structural changes in spine density andendritic branching generally found in the OFC and mPFC follow-

ng stimulant administration, two genes, Dusp1 and Dusp6, maylay a contributory role. In response to amphetamine treatment,usp1 was up-regulated in the mPFC whereas, Dusp6 was down-

egulated in the OFC. Dusp1 and Dusp6 are both phosphatases thategatively regulate/inactivate MAP kinase. MAP kinases have manyoles and are involved in directing the cellular response to a diverserray of potentially harmful stimuli such as DNA damage and oxida-ive stress, but they also regulate expression of certain genes likerc [31]. Transcription of Arc, an immediate early gene, is entirelyependent upon MAP kinase activity [32]. Similar to Dusp1, Arc wasp-regulated in the mPFC amphetamine treated animals, but notresent in the OFC where Dusp6 was down-regulated. As Arc haseen used as a marker of plastic changes in the brain and is believedo be involved in producing the structural alterations in neuronshat are triggered by drugs of abuse [33,34], the opposing down-egulation of Dusp6 in OFC and up-regulation of Dusp1 and Arc inhe mPFC may contribute to the increased dendritic complexity ofhe mPFC and the decrease in neuronal branching and spine densityresent in the OFC.

Owing to similar drug induced changes in structure and func-

ion, the mPFC and NAc were expected to have greater overlap inheir epigenetic response. Although not in response to the samerug, the NAc (amphetamine group) and mPFC (nicotine group)oth exhibited changes in expression of RGD1359529 and Shisa3.

ty phosphatase 1 0.543

The function of RGD1359529 is not well established so it is difficultto hypothesize how and why drugs of abuse affect its expression.Conversely Shisa3 is known to antagonize Wnt and Fgf signalling[35]. Wnt signalling has many roles including embryonic pattern-ing, cell communication for proliferation and differentiation, andaxon guidance [36], whereas Fgf signalling plays an important rolein neurogenesis, axon growth, and neuronal survival [37]. As Shisa3is an antagonist to these two signalling pathways and was downregulated in both conditions, the brain may have been promotingneuronal survival and axon growth in response to chronic drugadministration. However, this is only speculation as we did notexamine these types of brain changes.

As would be expected, the NAc was a main source of epigeneticmodulation following chronic exposure to drugs of abuse. Althougha likely naïve hypothesis, one could predict the epigenome of theNAc would respond to all rewards in a similar fashion. Only 1 gene(Nefm) was altered in the NAc in response to both psychomotorstimulants. However, Nefm and Nefl exhibited down-regulation inthe NAc of amphetamine treated animals and Nefm was reduced inthe nicotine treated animals. The level of neurofilament expressionseems to directly regulate axon diameter, which is turn controlshow fast electrical signals travel down the axon [38]. Neurofil-aments are required for proper postnatal growth of axons, andthe lack of Nefm renders some axons selectively vulnerable toage-dependent atrophic processes [39]. In adult mammals neuro-filaments are made up of heterpolymers that contain Nefl plus Nefmor Nefh. When examining the effects of drugs of abuse on neuronalstructure, researchers generally analyze dendritic alterations pay-ing little attention to axonal characteristics. Given that the primaryfunction of Nefm is the maintenance of axonal integrity and calibre,future studies may need to focus on axonal dysfunction followingchronic drug exposure. Down regulation of the Nefm gene may indi-cate that connectivity between the NAc and other brain regions is

compromised as axonal integrity is lost.

The Pcp4 gene exhibited decreased expression in the NAcof nicotine treated animals. Pcp4 (a.k.a., brain-specific polypep-tide) is believed to be involved in calcium signalling, neuronal

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poptosis, and the release of dopamine and acetylcholine [40].ver-expressing Pcp4 has been shown to inhibit apoptosis [41]hereas under-expression has been associated with neurodegen-

ration. Research has demonstrated down-regulation of Pcp4 in theerebellum of Alzheimer’s disease patients [42] and in the cau-ate nucleus, putamen, globus pallidum, and substantia nigra ofuntington’s disease patients [43]. Down regulation of Pcp4 andefh (neurofilament, heavy polypeptide) has been demonstrated in

he prefrontal cortex of alcoholics and it is hypothesized that thispigenetic patterning is associated with neurodegeneration [44].nterestingly, the epigenetic profile discovered in alcoholics is sim-lar to our down-regulation of Nefm and Pcp4 in the NAc of nicotinereated animals.

Four genes important for cell-to-cell signalling in the brain werelso altered in the NAc. Htr2a, Gabrg2, and Hsd11b1 were down-egulated in the amphetamine treated group whereas, Nts wasown-regulated in the nicotine treated group. Htr2a (a.k.a. 5-HT2Aeceptor) is the main excitatory receptor subtype among the Grotein-coupled receptors that belong to the serotonin receptoramily. Down-regulation of Htr2a is an adaptive process inducedy chronic administration of SSRIs or antipsychotics [45] (and nowhronic administration of amphetamine). Down-regulation of the-HT2A receptor would reduce the amount of serotonin needed toctivate cellular signalling.

Similarly, amphetamine administration also decreased expres-ion of Gabrg2 which encodes subunit 2 of the GABA(A) receptor.he GABA(A) receptor binds GABA, the major inhibitory neu-otransmitter in the brain. Research has previously identifiedABA(A) receptor modulation as a novel mode of action for drugsuch as MDMA and MDA [46]. Owing to the fact that individ-als with cocaine addictions exhibit decreased levels of GABA,nd administration of GABA(A) receptor agonists can reduce self-dministration of cocaine in rats [47], it is possible that the chronicdministration of amphetamine also decreased GABA in the NAcesulting in a decreased need for GABA(A) receptors.

Amphetamine also down-regulated expression of Hsd11b1encodes the enzyme hydroxysteroid (11-beta) dehydrogenase 1)n the NAc. This enzyme acts bidrectionally, catalyzing the conver-ion of cortisol to the inactive metabolite cortisone or as a reductaseor gluccorticoid synthesis. Up-regulation of this enzyme is likelyn adaptive response to alterations in the stress response. Researchas demonstrated that administration of psychomotor stimulants

ncreases HPA axis activity, in turn increasing cortisol release [48].t could be hypothesized that in order to counteract the damag-ng effects of excess cortisol in the brain, Hsd11b1 expression wasncreased.

Finally, Nts (a.k.a. neurotensin), which was down-regulatedt the NAc of nicotine treated animals, is widely distributedhroughout the CNS and may function as a neurotransmitter or neu-omodulator. Researchers have hypothesized that Nts may functions an endogenous antipsychotic compound; Nts is markedlynhanced after treatment with antipsychotic drugs and is abnor-ally low in the CSF of untreated patients with schizophrenia

49,50]. Nts acts through the dopaminergic pathways and the vastajority of dopamine neurons in the mesocorticolimbic and nigros-

riatal pathways express neurotensin receptors [51]. Binding of Ntso Nts receptors results in a net increase in the number of sponta-eously active dopamine neurons [51]. Consistent with literatureemonstrating that schizophrenic individuals self-medicate withigarettes [52], one could speculate that chronic administration oficotine continuously activated the same dopamine neurons as Nts,ecreasing the need for endogenous Nts.

The last gene to be discussed in this article is Pdyn which wasown-regulated in the OFC in response to nicotine. Pdyn binds to-opioid receptors, regulates dopaminergic tone and is thereforen important factor in the reinforcing and rewarding properties of

Research 256 (2013) 655– 661

drugs of abuse [53]. Pdyn’s are the building blocks to endogenousendorphins and are thought to modulate the brain’s response toseveral psychoactive substances such as cocaine (and in this studynicotine) [54]. Human association studies have found that certainpolymorphisms in the Pdyn gene place individuals at increased riskfor opioid dependence [55]. Pdyn is believed to contribute to thenegative emotional states such as dysphoria, experienced duringwithdrawal, thus contributing to an increased desire to continuedrug use [55]. It is interesting that this gene was altered in the OFC,which is generally believed to control behaviour based upon thevalue of the expected outcome [56].

5. Conclusions

The data presented in this study demonstrate that the epige-netic modifications to the brain’s reward circuit following a twoweek withdrawal period are different than the immediate epi-genetic response. Although this study does not fully explain therelationship between gene expression changes and neuronal mod-ification, it does provide an excellent platform to build from. TheDNA methylation patterns were a good predictor of the direc-tionality of genetic change and the genes identified using thebio-sequencing platform provide further insight into the brainchanges that occur in response to drugs of abuse. The data clearlydemonstrate that the epigenome is differentially susceptible tovarying drug types; although psychomotor stimulants producesimilar changes in dendritic morphology and synaptic connectivity,the mechanistic pathophysiology likely varies dramatically. Fur-thermore, as would have been predicted, the various regions of thebrain’s reward circuit respond to psychomotor stimulants throughvery different pathways. A limitation to the study is in the useof a single time point for analysis. However, because the persis-tent changes in dendritic morphology and spine density seen withGolgi–Cox staining are present 14 days after drug administrationis completed, we felt this time period was an adequate withdrawaltime frame in which to examine persistent changes at the epi-genetic level. It is however, becoming increasingly clear in theliterature that gene expression changes are time dependent andmRNA values may differ depending on the time points that youchose to look. It is also possible that the gene expression changeswe identified are related to the withdrawal process and not main-tenance of the modifications to neuronal structure and function. Itremains to be seen how other classes of psychoactive drugs mightalso affect gene expression in these regions or how mode of drugexposure might differentially alter gene expression. For example,in contrast to stimulants, morphine has the opposite effect of neu-ronal morphology. Similarly, IP injection of amphetamine inducesdifferent neuronal changes than injection of the same drug into theventral tegmental area [57]. Although the current study has pro-vided novel findings to build upon, these questions may lay thefoundation for future experiments.

Acknowledgements

The research group would like to thank the University of Leth-bridge Sequencing Facility for their mRNA biosequencing platformand analysis, as well as the Norlein Foundation and NSERC for theirfinancial support of the experiment.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.bbr.2013.09.006.

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