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Neuroscience 168 (2010) 359–370

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ARLY LIFE PROTEIN RESTRICTION ALTERS DOPAMINE CIRCUITRY

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. VUCETIC,a K. TOTOKI,a H. SCHOCH,a

. W. WHITAKER,b1 T. HILL-SMITH,c I. LUCKIc AND. M. REYESa*

Department of Pharmacology, Institute for Translational Medicine andherapeutics, University of Pennsylvania School of Medicine, Phila-elphia, PA 19104, USA

Department of Biochemistry, Scripps Florida, Jupiter, FL 33458, USA

Department of Psychiatry, Institute for Translational Medicine andherapeutics, University of Pennsylvania School of Medicine, Phila-elphia, PA 19104, USA

bstract—Adverse prenatal environment, such as intrauter-ne growth retardation (IUGR), increases the risk for negativeeurobehavioral outcomes. IUGR, affecting approximately0% of all US infants, is a known risk factor for attentioneficit hyperactivity disorder (ADHD), schizophrenia spec-rum disorders and addiction. Mouse dams were fed a proteineficient (8.5% protein) or isocaloric control (18% protein)iet through pregnancy and lactation (a well validated rodentodel of IUGR). Dopamine-related gene expression, dopa-ine content and behavior were examined in adult offspring.

UGR offspring have six to eightfold over-expression of do-amine (DA)-related genes (tyrosine hydroxylase (TH) andopamine transporter) in brain regions related to reward pro-essing (ventral tegmental area (VTA), nucleus accumbens,refrontal cortex (PFC)) and homeostatic control (hypothala-us), as well as increased number of TH-ir neurons in theTA and increased dopamine in the PFC. Cyclin-dependentinase inhibitor 1C (Cdkn1c) is critical for dopaminergic neu-on development. Methylation of the promoter region ofdkn1c was decreased by half and there was a resultant two

o sevenfold increase in Cdkn1c mRNA expression acrossrain regions. IUGR animals demonstrated alterations in do-amine-dependent behaviors, including altered reward-pro-essing, hyperactivity and exaggerated locomotor responseo cocaine. These data describe significant dopamine-related

olecular and behavioral abnormalities in a mouse model ofUGR. This animal model, with both face validity (behavior)nd construct validity (link to IUGR and dopamine dysfunc-ion) may prove useful in identifying underlying mechanismsinking IUGR and adverse neurobehavioral outcomes such asDHD. © 2010 IBRO. Published by Elsevier Ltd. All rights

eserved.

ey words: dopamine, neurodevelopmental programming,pigenetics, addiction, perinatal nutrition.

Present address: K. W. Whitaker, Institute for Neuroscience, Univer-ity of Texas at Austin.Corresponding author. Tel: �1-215-573-2991; fax: �1-215-573-9004.-mail address: reyestm@mail.med.upenn.edu (T. M. Reyes).bbreviations: ADHD, attention deficit hyperactivity disorder; CDKN1C,yclin-dependent kinase inhibitor 1C; CLAMS, Comprehensive Labnimal Monitoring System; CNS, central nervous system; DA, dopa-ine; DAT, dopamine reuptake transporter; DOPAC, 3,4-dihydroxy-henylacetic acid; HVA, homovanillic acid; Hyp, hypothalamus; IUGR,

ntrauterine growth retardation; LP, low protein; MeDIP, methylated

iNA immunoprecipitation; NAc, nucleus accumbens; PFC, prefrontalortex; TH, tyrosine hydroxylase; VTA, ventral tegmental area.

306-4522/10 $ - see front matter © 2010 IBRO. Published by Elsevier Ltd. All rightoi:10.1016/j.neuroscience.2010.04.010

359

suboptimal prenatal environment, typically indicated byow birth weight or being small for gestational age (SGA),an increase the risk for adverse neurobehavioral out-omes, including ADHD (Hultman et al., 2007; Lahti et al.,006), schizophrenia spectrum disorders (Susser et al.,008; St Clair et al., 2005), major affective disorder/de-ression (Brown et al., 2000, 1995), antisocial personalityisorder (Neugebauer et al., 1999) and addiction (Franzekt al., 2008). Intrauterine growth retardation (IUGR), whichesults in SGA infants, affects roughly 10% of all US birthsnd is associated with adverse pregnancy conditions, suchs maternal malnutrition, hypertension, uterine or placentalysfunction, smoking or drug use, and multiple births.hile adverse metabolic and cardiovascular outcomes

hypertension, cardiovascular disease, insulin resistance;luckman and Hanson, 2008; de Rooij et al., 2007) haveeen well characterized in IUGR animal models, the coin-ident neurobehavioral disabilities and specific central ner-ous system (CNS) abnormalities have received signifi-antly less attention. Early life protein restriction, an estab-

ished rodent model of IUGR used extensively in rats, hasnly recently been shown to result in similar metabolichenotypes in the mouse (Goyal et al., 2010; van Stratent al., 2010; Bol et al., 2009; Chen et al., 2009). Neurobe-avioral outcomes in a mouse model of IUGR have noteen reported.

Dopamine (DA) participates in locomotor activity, re-ard, motivation and feeding behavior and its dysfunction

s implicated in several neurobehavioral disorders includ-ng ADHD and addiction. The central DA system consistsf neurons that originate in substantia nigra pars compactaSNpc), ventral tegmental area (VTA), and project throughigrostriatal (locomotor activity), mesolimbic (reward, feed-

ng), and mesocortical (motivation, emotional) pathways. Do-amine-related gene expression is also prominent withinhe hypothalamus. A broad range of prenatal insults, in-luding malnutrition, maternal stress or infection and envi-onmental toxins, have been found to alter dopamine func-ion (Wang et al., 2009; Zhou et al., 2009; Palmer et al.,008; McArthur et al., 2007; Son et al., 2007; Stanwoodnd Levitt, 2007; Valdomero et al., 2005).

The molecular link between adverse neurobehav-oral outcomes and a suboptimal prenatal environment

ay involve epigenetic changes (DNA methylation, his-one methylation or acetylation; Jirtle and Skinner, 2007;

eaver et al., 2004; Szyf, 2009). Imprinted genes, dueo their monoallelic and epigenetically regulated expres-ion, are particularly susceptible to dysregulation as aesult of suboptimal prenatal conditions (Jirtle and Skin-er, 2007; Kwong et al., 2006). Similarly, disruption of the

mprinting status of several imprinted genes in the CNSs reserved.

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Z. Vucetic et al. / Neuroscience 168 (2010) 359–370360

eads to neurodevelopmental disorders (e.g, Prader–Williyndrome, Angelman syndrome) (Davies et al., 2005).yclin-dependent kinase inhibitor 1C (Cdkn1c/p57) is an

mprinted gene located in an imprinted region on mousehromosome 7 encoding a cyclin-dependant kinase in-ibitor that acts to negatively regulate cell proliferationnd, in some tissues, to actively direct differentiationSmith et al., 2007; Ye et al., 2009). In the brain, Cdkn1cs expressed in dopaminergic neurons where it exerts arucial role in dopamine neuron differentiation (Josepht al., 2003; Freed et al., 2008). Furthermore, overex-ression of Cdkn1c in a transgenic mouse model leadso embryonic growth retardation and low birth weight, aharacteristic IUGR phenotype (Andrews et al., 2007).gf2, another imprinted gene in this locus, has also beeninked to fetal growth (Zeisel, 2009). Therefore, genexpression and differential methylation was determinedor genes within the Cdkn1c locus.

IUGR infants are at significant risk for long term neurobe-avioral disabilities, including ADHD, depression, and ad-iction. In utero malnutrition does not lead to major struc-ural or anatomical deficits within the CNS, but rather per-anent suboptimal development (Ranade et al., 2008).xperiments in the present manuscript were designed toxamine dopaminergic dysregulation at the molecular andehavioral level in a mouse model of IUGR. Vulnerability topigenetic modifications was determined for dopamine-elated genes as well as potentially more vulnerable im-rinted genes within the Cdkn1c locus.

EXPERIMENTAL PROCEDURES

nimals and experimental model

57BL/6J females were mated with DBA/2J males and fed eithercontrol (18% protein) or isocaloric 8.5% low protein (LP) diet

uring breeding, pregnancy and lactation (diet details below). Atirth, litters were culled to six to eight pups, and at weaning, allffspring were maintained on the control diet. A subset of bothontrol and IUGR animals was placed on a high-fat diet (60%alories from fat) from weaning and tested to determine whetherocomotor activity was affected by postnatal diet, as consumptionf a Western diet has been shown to affect locomotor activityBjursell et al., 2008). One animal per litter was randomly chosenor use in individual experiments, to control for any litter effect.here was no difference in litter size between LP and controlregnancies. Body weights were recorded weekly, and male miceged 18–20 weeks of age (adulthood) were used in all experi-ents.

iet composition

otal energy content of control diet (Test Diet 5755, Richmond, IN,SA) was 4.09 kcal/g with 18% of total energy calories fromrotein, 22% from fat, and 60% from carbohydrate. Low proteiniet (Test Diet 5769) was 8.5% protein purified diet with totalnergy content of 4.13 kcal/g with 8.5% of total energy calories

rom protein, 22% from fat, and 69.5% from carbohydrate. High fatiet (Test Diet 58G9) has a total energy content of 5.21 kcal/g with8% of total energy calories from protein, 60% from fat, and 22%

rom carbohydrate. d

ucrose preference and locomotor activity

ucrose preference was determined in standard cages using awo bottle choice test with 4% sucrose (details in Supplementalethods.) Locomotor activity was measured using the Compre-ensive Lab Animal Monitoring System (CLAMS; Columbus In-truments, Columbus, OH, USA), which measures animal move-ent in the x- and z-axes. While in the CLAMS cages, animalsad ad libitum access to powdered diet and water. Food intakeas normalized to body weight.

ocomotor response to cocaine

nimals were tested for their locomotor response to cocaine usinghe CLAMS cages. Animals previously acclimated to the cagesere housed in the cages for 3 days. On day 1 animals were leftndisturbed, on day 2, they were injected with saline (11 AM) andn day 3, they were injected with cocaine (11 AM, 20 mg/kg, IP).ood and water was available ad libitum.

enomic DNA and total RNA isolation from brain

nimals were euthanized with an overdose of carbon dioxide,ollowed by cervical dislocation. This method is consistent with theecommendations of the Panel on Euthanasia of the Americaneterinary Medical Association and was approved by the Institu-

ional Animal Care and Use Committee (IACUC) of the Universityf Pennsylvania. After the animals were killed, the brains wereapidly removed and placed in RNAlater (Ambion, Austin, TX,SA) for 4 h before dissections. Specific regions were identifiednd macrodissected using their approximate mouse stereotaxicoordinates according to Mouse Brain Atlas (NAc, bregma�1.10m; prefrontal cortex (PFC) ��2 to bregma at posterior surface;TA, bregma �3.64 mm). Briefly, brains were positioned in a 1m mouse brain matrix (ASI Instruments, Warren, MI, USA)butting a razor blade placed in the first slot. The slot nearest theoundary of medulla was the first landmark. Next, double-edgedazor blades were placed in slots one and two mm anterior to thatoundary. Next, razor blades were placed in slots one, two andhree mm posterior to blade at anterior surface. The anterior set ofhree blades was removed from the block. Proceeding anterior toosterior, the first section was discarded. From second section��2 to bregma at posterior surface), cortex was cut from dorsalalf of section. From third section (��1 to bregma at posteriorurface), the nucleus accumbens was removed. Hypothalamusas dissected from the section in between blade 3 and 4. The slabas placed flat and the cuts were placed on either side of the optichiasm and dorsal to the third ventricle. The posterior set of bladesas removed from the block and VTA/SN region was dissected.enomic DNA and total RNA were isolated simultaneously usingllPrep DNA/RNA Mini Kit (Qiagen).

ene expression analysis by quantitativeeal-time PCR

or each individual sample, 500 ng of total RNA was used ineverse transcription using High Capacity Reverse Transcriptionit (ABI). Expression of target genes was determined by quanti-

ative RT-PCR using gene-specific TaqMan probes (ABI, Fosterity, CA, USA) with TaqMan Gene Expression Master Mix (ABI,oster City, CA, USA) on the ABI7900HT Real-Time PCR Cycler.robes used for RT-PCR are listed in supplemental material. The

elative amount of each transcript was determined using delta Ctalues as previously described (Pfaffl, 2001). Changes in genexpression were calculated using relative quantitation of a targetene against endogenous, unchanged GAPDH and B-ACTN stan-

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Z. Vucetic et al. / Neuroscience 168 (2010) 359–370 361

ethylated DNA immunoprecipitation (MeDIP) assay

eDIP assay was performed as described (Weber et al., 2005).ethylated DNA was immunoprecipitated using 10 �g of mouseonoclonal 5-methylcytidine antibody (Eurogentec) or mousere-immune serum. Enrichment in the MeDIP fraction was deter-ined by quantitative RT-PCR using ChIP-qPCR Assay Masterix (SuperArray) on the ABI7900HT Real-Time PCR Cycler. Forll genes examined, primers were obtained from SuperarrayChIP-qPCR Assays (�01) kb tile, SuperArray) for the amplifica-ion of genomic regions spanning the CpG sites located approxi-ately 300–500 bp upstream of the transcription start sites (seeupplemental Material for primer sequences). MeDIP results werexpressed as fold enrichment of immunoprecipitated DNA forach specific site. To calculate differential occupancy fold change% enrichment), MeDIP DNA fractions’ Ct value were normalizedo the Input DNA fraction Ct value (see Supplemental Material).inally, the normalized level of DNA methylation at a particular site

s expressed as relative to control group set to 1.

mmunohistochemistry

etails for tissue processing and immunohistochemistry haveeen described (Reyes et al., 2003) and are included in theupplemental methods. The primary antibodies were (1) a THabbit polyclonal directed against rat tyrosine hydroxylase (TH)Pel Freeze Biologicals, Rogers, AR, USA used at 1:2K) and (2) a-Fos rabbit polyclonal antiserum directed against a syntheticeptide corresponding to the n-terminal portion (amino acids 5-16)f human Fos protein (Santa Cruz Biotechnologies, Santa Cruz,A, USA, used at 1:10K). Localization was performed using aonventional avidin–biotin immunoperoxidase method. Differ-nces in the relative abundance of positive cells were estimatedy simple cell counting. Counts of stained neurons were made iningle sections from multiple animals (n�3–4/group) in either theTA (TH-ir) or NAc (Fos-ir) defined on the basis of adjoining Nissltained sections.

eurochemical procedure

opamine (DA) and its metabolites homovanillic acid (HVA), and,4-dihydroxyphenylacetic acid (DOPAC) were measured by high-erformance liquid chromatography analysis. Brains were re-oved from the skull and rinsed in ice cold water to remove any

urface blood. First, the brain was partially cut in half into right andeft hemisphere. Next, a stiff brush was placed into a hippocampalrove and rotated to displace and remove the hippocampus. After

he cortex was peeled apart and collected, the exposed striatumas dissected and harvested. The right and left striata and theortex were quickly frozen on dry ice, and then stored at �80 °Cntil analysis. Tissue samples were homogenized in 0.1 N per-hloric acid with 100 �M EDTA (15 �l/mg of tissue) using aissuemizer (Tekmar, Cleveland, OH, USA). Samples were cen-

rifuged at 15,000 rpm for 15 min at 2–8 °C. The supernatant wasxtracted and filtered through 0.45-�m nylon acrodisk syringelters and analyzed immediately. The Bioanalytical SystemsPLC (West Lafayette, IN, USA) consisted of a PM-80 pump, aample Sentinel autosampler, and an LC-4C electrochemical de-

ector. The samples (12 �l) were injected and pumped through aeverse phase microbore column (ODS 3 �m, 1�100 mm), at aow rate of 0.6 ml/min with electrodetection at �0.6 V. Separationor DA and the metabolites was accomplished by using a mobilehase consisting of 90-mM sodium acetate, 35-mM citric acid,.34-mM ethylenediamine tetraacetic acid, 1.2-mM sodium octylulfate, and 15% methanol v/v at a pH of 4.2 (Mayorga et al.,

001). c

tatistical analyses

ata, presented as means�SEM, were analyzed using SPSStatistical package and Excel tools for statistical analysis. Stu-ent’s t-test or Mann–Whitney U was used to analyze differencesetween IUGR and control animals, with Bonferroni correction forultiple t-tests (multiple genes within a brain region) applied asarranted. Repeated-measures ANOVA followed by Newman–euls posthoc analysis was used to calculate statistically signifi-ant difference between groups in Figs. 5 and 6. A P-value of 0.05r lower was considered significant.

RESULTS

xperimental animal model of IUGR

n order to eliminate any potential adverse effect ofandling on the development of offspring behavior andNS gene expression (Murgatroyd et al., 2009), a sep-rate group of dams and offspring were used to deter-ine the maternal response to the LP diet (Suppl. Table). Consumption of the low protein diet did not affectaternal weight gain through pregnancy, litter size, orregnancy duration. There was a nonsignificant trend forlower birthweight in the IUGR animals, however, byeaning IUGR animals weighed significantly less. Com-arable to IUGR infants, animals from maternal LP preg-ancies, termed IUGR mice, weighed 37% less at wean-

ng than animals from control pregnancies, (n�5, t-test,�0.002) and maintained 10%–15% lower weight than

he controls until postnatal week 9 (Suppl. Fig. 1). Bodyeight of IUGR mice normalized by 10 weeks of age.herefore, when tested at 18 –20 week of age, animals

n the IUGR group weighed on average the same asontrols.

he mesocorticolimbic and hypothalamic dopamineDA) system is altered in IUGR adult animals

xperiments were initiated to determine the extent tohich dopamine-related genes were altered within specificNS regions in IUGR animals. Quantitative real-time PCR

qRT-PCR) was used to measure the relative expressionf genes involved in the synthesis (TH, tyrosine hydroxy-

ase), reuptake (DAT, dopamine reuptake transporter), andegradation of dopamine (COMT, catechol-O-methyltrans-erase) and modulation and transmission of dopamine sig-al (dopamine receptor D1, dopamine receptor D2, andopamine- and cyclic AMP-regulated phosphoproteinARPP-32) in brain regions associated with reward pro-essing (VTA, PFC, NAc) and homeostasis (hypothala-us) of adult IUGR and control animals (Fig. 1). Thexpression of TH was upregulated six to eightfold inTA, PFC, NAc and hypothalamus of IUGR animals

n�5, t-test, P�0.019, 0.012, 0.008, 0.012, respec-ively). Additionally, IUGR animals showed overexpres-ion of DAT, with a three to fourfold increase in the VTAn�5, t-test, P�0.017), a nearly eightfold (n�5, t-test,�0.004) increase in the NAc, and a nonsignificant

P�.042; Bonferroni corrected �-level�.02) increase inhe hypothalamus. DARPP-32 expression was signifi-

antly increased only in the hypothalamus of IUGR an-

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Z. Vucetic et al. / Neuroscience 168 (2010) 359–370362

mals (n�5, t-test, P�0.01). On the other hand, expres-ion levels of dopamine receptors, D1 and D2, or COMTid not differ (data not shown) regardless of the brainegion. Taken together, these data demonstrate thatUGR due to exposure to prenatal and early postnatal LPiet results in altered expression of key dopaminergicignaling molecules within mesocorticolimbic circuitrynd in the hypothalamus.

Experiments were then conducted to examine theumber of TH-ir neurons within the VTA, the primaryource of dopaminergic innervation to the PFC and VTA.s predicted based on the increased TH mRNA in theTA, there were nearly 30% more TH� neurons in the

ig. 1. Dopamine-related gene expression within mesocorticolim-ic circuitry and hypothalamus of IUGR and control mice. mRNAxpression levels of dopamine pathway related genes TH, DAT,nd DARPP-32 in the ventral tegmental area (VTA), nucleus ac-umbens (NAc), prefrontal cortex (PFC) and hypothalamus of controlgrey bars) and IUGR (black bars) mice was determined by qRT-PCRn�5/group). Expression level of each gene examined was normalizedo the GAPDH and �-Actin levels and expressed relative to the controlroup set to 1. n�5/group. Values are mean�SEM. * P�0.05,* P�.01, # P�.005.

TA of IUGR animals (Fig. 2A, P�.05, t-test, representa- n

ive images are shown in 2C (control) and 2D (IUGR)).opamine and dopamine metabolites (DOPAC and HVA)ere measured in PFC and striatum, projection fields of

he VTA. A significant increase in DA was detected in theFC, as well as a significant decrease in the ratio ofOPAC:DA, an indication of dopamine turnover (Fig. 2B,�.05, Mann–Whitney U). No changes were evident inOPAC or HVA concentrations in PFC, and there were noignificant differences in DA or DA metabolites within thetriatum.

erinatal protein restriction affects promoterethylation of Cdkn1c

e then examined gene-specific promoter methylationithin the CpG islands of TH and DAT, genes that wereignificantly overexpressed in IUGR animals. We usedffinity purification of methylated DNA (methyl DNA immu-oprecipitation (MeDIP) assay) followed by qRT-PCR toetermine methylation levels of proximal promoter regionsf TH and DAT (Fig. 3). The amount of 5-meC-DNA en-iched fraction in the promoters of TH and DAT of IUGRnimals did not differ significantly from the controls in anyf the brain regions examined.

We next examined methylation status of several im-rinted genes in the Cdkn1c-Igf2 locus on mouse Ch7 (Fig.a). Expression of genes located in Cdkn1c-Igf2 locus cane regulated by environmental inputs during developmentKwong et al., 2006; Waterland et al., 2006), are importantor differentiation of neuronal precursor cells into mature DAeurons (Joseph et al., 2003; Freed et al., 2008), and neu-onal-specific overexpression of Cdkn1c leads to embryonicrowth retardation (Andrews et al., 2007). We performedeDIP analysis of proximal promoter regions (�350 bppstream of transcription start site) of the Cdkn1c and Igf2enes. Adult IUGR animals showed a 50%–75% reduction

n methylation of Cdkn1c promoter in PFC, NAc, and hy-othalamus compared to controls (Fig. 3; n�3, t-test,�.01, P�.008, P�.013, respectively), while Igf2 pro-oter methylation did not differ in any brain region tested.he primarily unmethylated, control Gapdh promoter wasot affected.

rain-region specific overexpression of genes inmprinted Cdkn1c - Igf2 locus that regulate dopamineeuron differentiation

e next asked whether hypomethylation of the Cdkn1cromoter in IUGR animals was accompanied by alteredxpression of genes in the Cdkn1c-Igf2 gene cluster (Fig.b). We observed a statistically significant increase in thexpression of Cdkn1c in PFC, NAc and hypothalamus ofUGR animals (n�5, t-test, P�0.0001, P�0.012, P�.013, respectively), all regions in which promoter hypom-thylation was observed. Igf2 was overexpressed in PFCnd hypothalamus (n�5, t-test, P�0.004, and P�0.03),hile Tssc4 (tumor-suppressing subchromosomal trans-

erable fragment 4) showed a significant increase in hypo-halamus only (n�5, t-test, P�0.037). On the other hand,he relative expression of potassium voltage-gated chan-

el Q1(Kcnq1), as well as pleckstrin homology-like domain

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Z. Vucetic et al. / Neuroscience 168 (2010) 359–370 363

2 (Phlda2) and solute carrier family 22 (Slc22a1) geneere not affected by IUGR in any of the brain regions

ested (data not shown). Interestingly, out of seven geneshat were tested in the Cdkn1c-Igf2 locus, only the geneshown to be important in differentiation and specification ofA neurons (TH, Cdkn1c, Igf2; Freed et al., 2008) wereverexpressed in IUGR offspring.

erinatal exposure to low protein diet altersopamine-associated behaviors

Altered sucrose preference and locomotor behavior inUGR offspring. We evaluated the response to a natu-al reward, sucrose, using a two-bottle, 4% sucroseolution preference test. All mice preferred sucrose overater, but that preference was significantly decreased in

UGR mice (Fig. 5a, P�.02). Locomotor activity duringhe dark period (active phase for mice, n�4 mice/con-ition) was also assessed every 3 weeks beginning at 14eeks of age in a cohort of mice. When animals were

ed the control diet, there was no difference in locomotorctivity (Fig. 5b). However, when animals (both controlsnd IUGR animals) were placed on an HF diet at wean-

ng, the IUGR animals demonstrated a significant hyper-ctivity (Fig. 5c). One-way repeated measures ANOVAevealed a significant interaction between group andime (F(4,20)�24.6, P�.0008), with significant main ef-ects for both group and time (F(1,20)�38.0, P�.003,(4,20)�19.4, P�.003, respectively).

Augmentation of locomotor response and c-fos activa-ion after acute cocaine administration in IUGR offspring.

ig. 2. Increased TH-ir cells in the VTA and dopamine in the PFC.P�.05). Representative images are shown in the lower panels (C: c

he PFC of control (grey) and IUGR animals (black, n�6/group). Thereurnover (DOPAC:DA ratio), * P�.05.

UGR and control animals (n�10 –12/ group) were t

ested for their locomotor response to acute cocainedministration (20 mg/kg) (Fig. 6). There were no differ-nces in locomotor activity in response to the saline

njection on d2 or in the 30 min prior to cocaine injectionn d3 (Fig. 6A). In response to cocaine, both groups ofnimals had an increase in locomotor activity (significantain effect for time (F(3,60)�65.6, P�.0001)), however,

UGR offspring had a significantly greater response tohe cocaine (significant main effect for group; F(1,60)�.36, P�.05)).

Additionally, expression of the immediate early generoduct, Fos (a generic marker of neuronal activation), wasuantified in the NAc 2 h following saline or cocaine injec-ion. As expected, there were few Fos-ir cells in saline-njected animals (Fig. 6C, D, insets) and both control andUGR animals showed an increase in Fos-ir cells in theAc after cocaine injection (20 mg/kg), a response shownreviously to be associated with the locomotor response toocaine (Hooks et al., 1991). Consistent with the exagger-ted locomotor response, there was a significant increase

n the number of activated cells present in the NAc of IUGRersus control animals (Fig. 6B; 176 � 27 cells 112�14ells, respectively, P�.03).

DISCUSSION

dverse prenatal environment has been linked to in-reased risk of adult neurobehavioral disorders andhronic metabolic diseases. These data highlight signif-

cant vulnerability of the dopamine system to early liferotein restriction. We utilized maternal protein restric-

nificant increase in TH-ir neurons was seen in the VTA (n�4/group,: IUGR). (B) Dopamine and dopamine metabolites were measured innificant increase in dopamine, and a significant decrease in dopamine

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ion during pregnancy and lactation, a well characterized

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Z. Vucetic et al. / Neuroscience 168 (2010) 359–370364

at model for IUGR, which has been used extensively toxamine both metabolic and CNS outcomes. Recenttudies (Goyal et al., 2010; van Straten et al., 2010; Bolt al., 2009; Chen et al., 2009) combined with our dataonfirm this as a valid mouse model of IUGR. As therst report of adverse neurobehavioral outcomes in a

ig. 3. DNA methylation changes at regulatory regions of dopamineelated genes in IUGR mice. The enrichment of DNA methylationelative to input genomic DNA at proximal promoter regions of TH,AT, Cdkn1c, Igf2 and Gapdh was quantified in ventral tegmental area

VTA), prefrontal cortex (PFC), nucleus accumbens (NAc), and hypo-halamus (Hyp) by qRT-PCR after MeDIP. No differences were ob-erved in TH, DAT or IGF2, however, the Cdkn1c promoter region wasignificantly hypomethylated in PFC, NAc, and hypothalamus. n�3/roup. Values are mean�SEM. * P�0.05, ** P�.01.

ouse, we report large increases in TH and DAT mRNA a

xpression, increased number of TH-ir cells in the VTAnd increased dopamine in the PFC. These changes inopamine expression are paralleled by disruptions inA-dependent behaviors, including hyperactivity and

ncreased locomotor response to cocaine. Additionally,e identified hypomethylation and overexpression ofdkn1c, which is critical for driving dopamine neuronevelopment and differentiation and which has been

inked to IUGR.Early life exposure to LP diet increased expression

f TH in all regions examined, while upregulation of DATnd DARPP-32 was evident in only select brain regions.

mportantly, we also observed an increase in TH-ir neu-ons within the VTA, and an increase in DA within theFC. Dopamine levels within the striatum were not al-

ered, suggesting that VTA to PFC dopaminergic projec-ions may be uniquely sensitive to this perinatal chal-enge or that the increased expression of DAT in theAc (which was not elevated in the PFC) participated inormalizing dopamine levels. These data complementeports of increased whole brain DA levels (Chen et al.,997; Marichich et al., 1979), TH activity (Marichich etl., 1979), and altered dopamine receptor binding in thetriatum in rats from protein-restricted pregnanciesPalmer et al., 2008; Marichich et al., 1979). TH-produc-ng neurons within the PFC are likely non-classical TH-roducing interneurons (Asmus et al., 2008) although

ittle is known about these cells, including their finalransmitter phenotype. These neurons lack additionalownstream enzymes necessary for catecholamine syn-

hesis and these neurons have been termed “dopaergic”Ugrumov et al., 2004). Interestingly, both classical andon-classical catecholaminergic cell groups respondedimilarly to the early life protein restriction.

Altered DA tone has significant implications for humanealth, given the broad range of normal and pathologicalehaviors that rely on dopamine. Studies in infants havehown that reduced fetal growth represents a consistentisk for ADHD (Hultman et al., 2007; Lahti et al., 2006; Mickt al., 2002). Although the relative importance of dopamine

n ADHD pathology has been debated (Gonon, 2009),here is a wealth of data that support a role for dopamineysfunction in ADHD pathology (van der Kooij and Glen-on, 2007; Madras et al., 2005; DiMaio et al., 2003). Over-xpression of DAT in the NAc (similar to the increasedccumbens DAT expression observed in the IUGR mice)as shown to increase impulsivity and risk taking behavior

Adriani et al., 2009), prominent behavioral components ofDHD, and methylphenidate administration was shown toecrease DAT expression in the striatum (Moll et al.,001), which may contribute to its therapeutic effective-ess. However, decreases in DAT (and D2/D3) in the leftidbrain and accumbens have been linked to measures of

nattention in ADHD patients (Volkow et al., 2009), high-ighting the difficulty of replicating the totality and hetero-eneity of ADHD symptoms in any one animal model.yperactivity (Wilson, 2000), a component of ADHD, waslso present in the IUGR mice, but was only evident in

nimals fed a high fat diet. While the explanation for this is

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Z. Vucetic et al. / Neuroscience 168 (2010) 359–370 365

urrently unknown, consumption of a high fat diet is knowno alter dopaminergic activity (Lee et al., 2010). It is pos-ible that the dopaminergic alterations due to IUGR estab-ish an underlying vulnerability to hyperactivity and whenoupled with altered dopamine signaling in response toonsumption of high fat diet, hyperactivity emerged. Theresent data suggest that altered DA in response to auboptimal perinatal environment may contribute to somef the behavioral components of ADHD observed in IUGR

nfants.Altered reward processing (Haenlein and Caul, 1987)

s also a component of ADHD. IUGR mice demonstrated aecrease in sucrose preference, reflecting a decreasedesponsiveness to rewarding stimuli. Individual differencesn sucrose consumption (high preference vs. low prefer-nce) positively predict amphetamine self-administrationDeSousa et al., 2000) and correlate with D2 binding in thetriatum (Tonissaar et al., 2006). Decreased sucrose pref-rence in response to stress has also been associated with

ncreased dopamine in the hypothalamus, striatum and

ig. 4. (a). Cdkn1c-Igf2 locus. Schematic representation of Cdkn1c-Igenes in this locus. Black and gray boxes indicate maternally andxpressed genes. Arrows are the direction of transcription. (b) CNS exdkn1c, Tssc4, and Igf2 in VTA, PFC, NAc and hypothalamus of contata are means�SEM. * P�0.05, ** P�.01, # P�.005, ## P�.001.

FC (Bekris et al., 2005), which is similar to our observa- s

ion of increased DA in the PFC and increased TH in theypothalamus. It is important to note, however that dopa-ine deficient mice have been shown to have an intact

ucrose preference (Cannon and Palmiter, 2003), and ad-itional neurotransmitter systems are clearly involved. Anxtensive literature has documented decreased sucrosereference in response to chronic mild stress, commonly

nterpreted as an animal model of “anhedonia”. Accord-ngly, this decrease can be ameliorated by antidepressantreatment (i.e., broad acting tricyclics such as imiprimine;ekris et al., 2005, as well as selective serotonin reuptake

nhibitors like citalopram; Rygula et al., 2006). In addition toopamine, decreased activity of serotonin in the PFC haslso been linked to reductions in sucrose preferenceBekris et al., 2005) and citalopram can reverse both theehavior and the neurochemical changes (Rygula et al.,006). Of note, however, citalopram also decreases stria-al dopamine and increases D2 binding (Dewey et al.,995), indicating that an interaction between serotonin andopamine systems is likely to be important in mediating

with two imprinted centers (IC1 and IC2) regulating the expression ofy imprinted genes, respectively. White boxes represent biallelicallyof Cdkn1c-Igf2 genes. Expression of Cdkn1c-Igf2 locus genes Kcnq1,bars), IUGR (black bars) mice was assayed by RT-PCR. n�5/group.

f2 genespaternallpression

ucrose preference. Activation of the reward system by

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22

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Z. Vucetic et al. / Neuroscience 168 (2010) 359–370366

rugs of abuse (Martinez et al., 2007; Ron and Jurd, 2005),oupled with underactivation of reward circuitry in re-ponse to natural rewards (i.e., sucrose) may act syner-istically in the development of addiction (Koob and Leoal, 2008). These two behaviors were noted in IUGRnimals (exaggerated response to cocaine and decreaseducrose preference), suggesting that the IUGR animalsay represent an animal model of increased addiction

isk as a result of suboptimal prenatal environment.hese findings are consistent with other observations in

he literature linking malnutrition and addictive behavior

ig. 5. Sucrose intake and locomotor activity. Sucrose preference (a)as evaluated in IUGR (gray bars) and control (black bars) mice

n�4–5/group). IUGR mice show reduced preference for sucrose.P�0.05. Locomotor activity was evaluated every 3 weeks in theLAMS cages for 12 h, beginning at the onset of lights off. Animals

ed the control diet (b) did not differ in activity levels, however, whennimals were fed a high-fat diet (c) IUGR animals demonstratedignificant hyperactivity (main effect for group, * P�0.003). n�4/roup.

n rodents (Palmer et al., 2008; Valdomero et al., 2007, r

006; Shultz et al., 1999) and humans (Franzek et al.,008).

In an effort to define potential mechanisms driving thebserved gene expression changes, we investigated theethylation status of specific target genes. A recent reportxamined genome-scale methylation status of CpG is-

ands in the liver in response to maternal malnutrition (vantraten et al., 2010), and found that less than 0.5% of CpG

slands were differentially methylated. Further, they ob-erved equal amounts of hyper- and hypo-methylation,uggesting that limited substrate (e.g., methyl donors) dueo the low protein diet was not likely to be responsible forhe differential methylation patterns. Further, CpG islandsithin promoter regions are known to demonstrate varyingegrees of sensitivity to aberrant methylation (Feltus et al.,003), and here we report that TH, DAT and Igf2 are notusceptible to differential methylation, but Cdkn1c is vul-erable to differential methylation in response to early liferotein restriction. Hypomethylation and subsequent over-xpression of Cdkn1c in IUGR animals is notable for threerimary reasons: (1) Cdkn1c is involved in DA neuronifferentiation, (2) neural overexpression of Cdkn1c results

n growth retardation (Andrews et al., 2007) and (3) as anmprinted locus, this region may be more susceptible topigenetic dysregulation as a result of suboptimal prenatalonditions. Cdkn1c is important for neuronal migration andoordination of the timing of progenitor cell cycle exit ineveloping mouse neocortex (Ye et al., 2009; Itoh et al.,007) and most likely partners with Nurr1 to promote ter-inal differentiation of DA neurons (Joseph et al., 2003;reed et al., 2008). Interestingly, Igf2, overexpressed in

he PFC and hypothalamus of IUGR mice, has also beenhown to play a role in DA neuron development (Vazin etl., 2009). Counts of TH-ir neurons were made within theTA, as this is the primary source of dopaminergic inner-ation of the PFC where an increase in DA was detected,ven though we did not detect an increase in Cdkn1c inhis region. It is possible that elevations in Cdkn1c were notetected using qRT-PCR in a mixed (VTA/SN) dissectionr it is possible that Cdkn1c levels were elevated at anarlier timepoint in development. At this time, it is prema-ure to define Cdkn1c overexpression as causative in theopaminergic changes observed in the IUGR animals.owever, given its dysregulation in this model of IUGR,

he importance of Cdkn1c to dopamine neuron develop-ent, and the link between neuronal overexpression ofdkn1c and embryonic growth retardation, our dataighlight Cdkn1c as an important molecule for futureesearch within this context. Only a subset of genes indkn1c-Kcnq1 locus was differentially expressed in

UGR animals, which implies that the methylation statusf IC1 (see Fig. 4a), normally inherited from gametes, isot responsible for observed gene expression changes.

nstead the Cdkn1c promoter is hypomethylated in IUGRffspring, which is established during the post-implanta-

ion period (Bhogal et al., 2004), identifying this as aulnerable timeframe for long-lasting epigenetic dys-

egulation of Cdkn1c gene.

tnmd3pdgtportrns2aptprMfre

ie(repeoicsan

ImmtprdIwd

Fc(aC os-ir cellsi

Z. Vucetic et al. / Neuroscience 168 (2010) 359–370 367

In these studies, maternal exposure to the low pro-ein diet extended through the entirety of breeding, preg-ancy and lactation. At birth, rodents are consideredore “immature” than humans, with significant brainevelopment occurring postnatally (e.g., postnatal days–10 are thought to mirror the third trimester of a humanregnancy (Livy et al., 2003)) with precise pairing ofevelopmental time periods dependent upon brain re-ion (Clancy et al., 2007). Therefore, to model the en-irety of human gestation, we chose to maintain the lowrotein diet through pregnancy and lactation. A numberf factors may participate in driving the observed neu-odevelopmental phenotype, including the prenatal pro-ein restriction of the pup or the maternal endocrineesponse (Langley-Evans et al., 1996). Postnatal mater-al behavior has been shown to modify the methylationtatus of the glucocorticoid receptor (Weaver et al.,004) and estrogen receptor (Champagne et al., 2006),nd differences in maternal care between the groups areossible (i.e., while the direct effect of protein malnutri-ion on the maternal behavior has not been reported,rotein-deprived rat pups nursed by control dams expe-ienced an increase in active nursing and grooming;assaro et al., 1974). Defining the causative factor(s)

or the observed phenotypes remains an area of activeesearch. As these experiments represented an initial

ig. 6. Locomotor and neuronal response to cocaine. (A) Locomotorircles) was measured. IUGR animals had a significantly greater resB–D) Immunohistochemistry was used to localize the expression of tctivation, within the NAc. As expected, there were few activated (Fos, D). In response to cocaine (20 mg/kg, IP), a significant increase in F

n IUGR animals (* P�.05, n�3 mice/group).

valuation of maternal low protein diet on neurobehav- v

oral outcomes in the offspring, experiments were nec-ssarily limited with regard to age of assessmentadults) and sex (males). Future work can now be di-ected at defining other key variables. For example, anxamination of the critical period for exposure to lowrotein diet (e.g., early vs. late gestation, inclusion orxclusion of lactation) will provide important informationn specific windows of vulnerability. Further, future stud-

es may extend the analyses to female offspring, asertain developmental programming effects have beenhown to be sex-specific (Skinner et al., 2008) or otherssessment timepoints that represent particularly vul-erable ages (e.g., adolescence).

CONCLUSION

n conclusion, early life protein deficiency results in aouse model of IUGR with persistent alterations in dopa-ine circuitry and dopamine-dependent behaviors. Addi-

ionally, we have identified hypomethylation and overex-ression of Cdkn1c, a molecule known to have a causativeole in growth retardation and to play a critical role inopamine neuron development. This mouse model ofUGR may represent a novel animal model for ADHD, asell as other disorders linked to both IUGR and dopamineysfunction (schizophrenia and addiction), with both face

to cocaine (20 mg/kg, IP) in IUGR (black square) and control (greycocaine, particularly at 30–60 min post-injection (n�10–12/group).iate early gene protein product, c-Fos, a marker of general neuronal

n saline-injected animals, which did not differ between groups (insets,was observed in the NAc, and this response was significantly greater

responseponse tohe immed-ir) cells i

alidity (behaviors) and construct validity (IUGR-induced

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opaminergic dysregulation), which would provide an ad-itional tool to explore questions of mechanism and poten-

ial therapeutics.

cknowledgments—The current work was supported by NIHK064086 (Reyes) and MH087978 (Reyes). Irwin Lucki has beenn the Scientific Advisory Board for Wyeth and has receivedesearch support from AstraZeneca, Wyeth, Forest and Epix phar-aceutical companies during the past 3 years.

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APPENDIX

upplementary data

upplementary data associated with this article can be found, in

he online version, at doi:10.1016/j.neuroscience.2010.04.010.

(Accepted 5 April 2010)(Available online 13 April 2010)