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alproate Corrects the Schizophrenia-like Epigeneticehavioral Modifications Induced by Methionine

n Miceucio Tremolizzo, Mohemed-Salim Doueiri, Erbo Dong, Dennis R. Grayson, John Davis, Graziano Pinna,atricia Tueting, Virginia Rodriguez-Menendez, Erminio Costa, and Alessandro Guidotti

ackground: Reelin and GAD67 expression is downregulated in cortical interneurons of schizophrenia (SZ) patients. Thisownregulation is probably mediated by epigenetic hypermethylation of the respective promoters caused by the selective increase ofNA-methyltransferase 1 in GABAergic neurons. Mice receiving methionine (MET) provide an epigenetic model for neuropathologies

elated to SZ. We studied whether MET-induced epigenetic reelin promoter hypermethylation and the associated behavioral alterationsan be reduced by valproate in doses that inhibit histone deacetylases (HDACs).ethods: Mice treated with either methionine (MET) (5.2 mmol/kg/SC/twice daily) or valproate (1.5 mmol/kg/SC/twice daily) or MET�

alproate combination were tested for prepulse inhibition of startle (PPI) and social interaction (SI). S-adenosylmethionine, acetylatedistone 3, reelin promoter methylation, and reelin mRNA were assayed in the frontal cortex.esults: Valproate enhances acetylated histone 3 content, and prevents MET-induced reelin promoter hypermethylation, reelin mRNAownregulation, and PPI and SI deficits. Imidazenil, a positive allosteric modulator at GABAA receptors containing �5 subunits butnactive at receptors including �1 subunits, normalizes MET-induced behavioral changes.onclusion: This MET-induced epigenetic mouse models the neurochemical and behavioral aspects of SZ that can be corrected byositively modulating the action of GABA at �5-containing GABAA receptors with imidazenil or by inhibiting HDACs with valproate,

hus opening exciting new avenues for treatment of epigenetically modified chromatin in SZ morbidity.

ey Words: Behavioral deficits, epigenetic, GAD67, methionine,eelin, schizophrenia

he availability of the complete sequence of human andmouse genomes has allowed the study of epigeneticfactors that induce complex behavioral modifications

Francis et al 2003) via gene promoter hypermethylation inpecific neuronal populations (Costa et al 2002b, 2003a, 2003b;eaver et al 2002). Genetic factors play an important role in

culpting brain cortical architecture and thereby contribute tohape the expression of complex behaviors; however, epigeneticypermethylation of neuron-specific gene promoters and hyper-cetylation of nucleosomal chromatin triggered by drugs (Trem-lizzo et al 2002) or environmental stimuli (Francis et al 2003;eaver et al 2002) may also be operative in regulating the

rchitecture of telencephalic neuronal circuits underlying thexpression of specific behavioral patterns. For example, in theeveloping cerebral cortex of mammals, the tone of the GABAer-ic interneurons can be affected by drugs or other environmentaltimuli, thereby influencing the correct expression of ocularominance patterns (Fagiolini et al 2004; Hensch et al 2004). Aeficit of prefrontal cortex (PFC) GABAergic transmission includ-ng a downregulation of GABA and reelin expression (Akbariant al 1995; Benes and Berretta 2001; Costa et al 2002b, 2003a,003b; Eastwood and Harrison 2003; Fatemi et al 2000; Guidottit al 2000; Knable et al 2004; Lewis et al 2000) has beenssociated with the cortical neuron hypoplasticity (Black et al004; Dong et al 2003; Glantz and Lewis 2000; Liu et al 2001;

rom the Department of Psychiatry, Psychiatric Institute, College of Medi-cine, University of Illinois at Chicago, Chicago, Illinois.

ddress reprint requests to Erminio Costa, University of Illinois at Chicago,1601 Taylor Street Chicago, IL 60612; E-mail: Costa@psych.uic.edu.

eceived July 23, 2004; revised October 14, 2004; accepted November 23,

2004.

006-3223/05/$30.00oi:10.1016/j.biopsych.2004.11.046

Selemon and Goldman-Rakic 1999; Volk et al 2000; Weeber et al2002) operative in patients with schizophrenia.

Several lines of independent investigation reviewed by Costaet al (2002b, 2003a, 2003b) suggest that epigenetic mechanisms,likely mediated by a DNA-methyltransferase (DNMT) overex-pression detected in GABAergic neurons of various cortical areasof schizophrenia patients, are responsible for the downregula-tion of GAD67 and reelin expression observed in these corticalareas. The overexpression of DNMT1 in schizophrenia cortex(Veldic et al 2004), presumably by hypermethylating the promot-ers of reelin, GAD67, and probably of other genes enriched incytosine/guanine dinucleotides (CpG) islands contributes to thedownregulation of reelin and GAD67 expression observed inprefrontal cortex of schizophrenia patients (Guidotti et al 2000).Although the cause of this DNMT1 overexpression is unclear andstill under investigation, it is known that schizophrenia symp-tomatology is exacerbated by administering large doses ofmethionine (MET; 20–40 g/day, up to 4 weeks), whereasidentical treatment with MET changes neither thought processesnor behavior of normal subjects (Wyatt et al 1971).

We recently reported (Tremolizzo et al 2002) that micereceiving a protracted treatment with MET (6.6 mmol/kg subcu-taneously [SC] twice a day for 15 days) express an interestingendophenotype that may help to unveil the primary epigeneticmechanism that causes the downregulation of reelin and GAD67

expression in the brain of schizophrenia patients. In mice treatedwith MET, there is an increase of brain S-adenosylmethionine(SAM), a cofactor operative in the hypermethylation of CpGislands expressed in several promoters including promoters ofreelin and GAD67, which are reduced in various cortical andhippocampal areas (Tremolizzo et al 2002). This downregulationcan be prevented by valproate (VPA), which, when administeredin doses that inhibit histone deacetylases (HDACs; Phiel et al2001), hyperacetylates the N-terminal tails of brain nucleosomalhistones (Tremolizzo et al 2002). Such hyperacetylation favorsthe stabilization of an open chromatin state (euchromatin) and

an associated induction of DNA demethylase activity in chroma-

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in (Detich et al 2003). We have also shown that in the reelinromoter of MET-treated mice, the number of methylated CpGinucleotides is inversely correlated with the decrease of reelinxpression in the brain (Tremolizzo et al 2002).

Stimulated by recent clinical reports that VPA administered inonjunction with antipsychotics accelerates the onset of antipsy-hotic actions on schizophrenia symptomatology (Casey et al003; Wassef et al 2003), we studied whether hypermethylationf reelin promoter and schizophrenia-like behavioral traits in-uced by MET in mice can be prevented by VPA-inducedyperacetylation of chromatin histone tails.

To further explore whether MET induces the earlier-men-ioned behavioral modifications, at least in part by downregulat-ng GABAergic neurotransmission, we administered imidazenilo MET-treated mice. Imidazenil is a potent positive allostericodulator of GABA action on �5 expressing GABAA receptors,ut it does not enhance GABA action at GABAA receptors thatnclude the �1 subunit (Costa et al 2002a). In fact, imidazenil is andeal drug to study the role of specific GABAA receptors,ncluding �5 subunits, on behavior because unlike diazepam,midazenil fails to induce tolerance or sedation, reduce attention,odify cognition, or alter muscular tone and locomotor activity

t doses that inhibit anxiety and seizures (Costa et al 2002a).

ethods and Materials

nimals and Drug Administration ScheduleB6C3Fe male mice (60–80 days old) received SC vehicle

VEH; 10 mL/kg saline), L-methionine (MET; 5.2 mmol/kg), oralproate ([VPA] 1.5 mmol/kg), or a combination of the tworugs [MET�VPA] twice daily for 15 days. Unless otherwisendicated, mice were left untreated for 16–20 hours beforeeurochemical or behavioral measurements. Imidazenil, 3.3mol/kg was administered SC 60 min before the PPI test and 120in before the social interaction test. Doses and schedule of

dministration were selected on the basis of previously reportedose and time related pharmacodynamic experiments (Costa etl 2002c; Tremolizzo et al 2002). The number of animals used inhe various experiments is specified in the figure and tableegends. All animal procedures were approved by the Universityf Illinois at Chicago Animal Care Committee.

issue Levels of S-Adenosyl-Methionine (SAM) and-Adenosyl-Homocysteine (SAH)

Frontal cortex (FC) samples (50 mg, 2 mm anterior to theregma) were homogenized (100 �L/10 mg tissue) in .4 M HClO4

ontaining traces of 3H-SAM (� 2000 CPM, � .02 pmol eachample) to monitor recovery. After centrifugation, an aliquot ofhe supernatant (.4 mL) was applied to a reverse-phase high-erformance liquid chromatography (HPLC) column Symmetry

18 4.6 � 250 mm (Waters, Milford, Massachusetts) developed asescribed earlier (Tremolizzo et al 2002). Standard solutions ofAM and SAH containing 50–10,000 pmol of each compoundnd 3H-SAM (as described earlier) were injected in triplicate intohe HPLC and used for calibration and quantitation.

estern BlottingThe FC samples were homogenized in Laemmli buffer (100

L/10 mg of tissue) and 25 �L were separated by a 10%–20%radient tris-glycine gel (Invitrogen, Carlsbad, California). Aftervernight blotting at 4°C onto Hybond ECL nitrocellulose mem-rane (Amersham Pharmacia, Piscataway, New Jersey), the blotas incubated overnight at 4oC with one of the following

rimary antibodies (Ab): polyclonal anti-dimethyl (K9) histone3

(H3) (1:1,000, Upstate Biotechnology, Waltham, Massachusetts),polyclonal anti-total acetyl H3 (1:1,000, Upstate Biotechnology),or anti-�-actin (1:10,000, Clone AC-15, Sigma, Milwaukee, Wis-consin). After incubation for 1 hour at room temperature with thecorrespondent secondary Ab: HRP-conjugated anti-rabbit Ab(1:1,000, Amersham Pharmacia) for acetyl H3 and dimethyl H3,and HRP-conjugated anti-mouse Ab (1:5,000, Sigma) for �-actin,the ECL Plus chemiluminescence Western blotting kit (Amer-sham Pharmacia) was applied to reveal the immunoreactivebands. The analysis was performed by using the Blue Fluores-cence/Chemifluorescence Storm System (Molecular Dynamics,Sunnyvale, California) with IMAGEQUANT analysis software,and the values were expressed as an optical density (OD) ratiowith respect to �-actin. We previously reported details of analy-ses and examples of blots (Tremolizzo et al 2002).

ImmunohistochemistryTissues were fixed with 4% paraformaldehyde containing .1%

glutaraldehyde in phosphate-buffered saline. Acetyl H3 immu-nohistochemical quantification was carried out in FC floatingsections (15 �m) incubated for 24 hours with total anti-acetyl-H3Ab (Upstate), diluted 1:1000 according to the procedure de-scribed by Rodriguez et al (2002).

Bisulfite-Modified Reelin Promoter SequencingGenomic DNA (� 2 �g) isolated from FC specimens of mice

receiving VEH, MET (5.2 mmol/kg), or VPA (1.5mmol/kg), or acombination of these drugs, SC for 15 days (twice daily) wasbisulfite modified (Grunau et al 2001; Tremolizzo et al 2002), andthe reelin promoter region between �340 and �160 bp, waspolymerase chain reaction (PCR)-amplified; the resulting ampli-cons were gel purified and then subcloned into the pGEM T-easyvector. For this experiment, we used four mice per group. Fourclones from each animal were sequenced at the University ofChicago Cancer Research Center DNA Sequencing Facility usingT7-long and SP6 primers.

Quantitative Reelin mRNA Reverse Transcriptase PCR(RT-PCR) Analysis

Total RNA was extracted as previously described (Impagna-tiello et al 1998). Reelin mRNA content was measured by RT-PCRanalysis with internal standards (IS) and the following amplifica-tion primers: forward, bp 9211–9234; reverse, bp 9549–9572(GenBank accession No. HSU79716; Guidotti et al 2000; Tuetinget al 1999).

Behavioral TestsSocial Interaction in the Home Cage. We used the experi-

mental paradigm described by Ferguson et al (2000) to measuresocial recognition. In a first trial (5 min duration), a male residentmouse housed alone in a home cage (29 � 18 � 12 cm) for 2days before trial, investigated a novel mouse introduced into hiscage by close following, inspection, anogenital sniffing, andother social body contacts. Four trials with an intertrial interval of30 min were used to generate the habituation curve (Ferguson etal 2000). To test individual olfactory acuity, we used the recog-nition of a lemon-juice-soaked cotton ball scent.

Social Interaction in a Novel Environment. Briefly, two malemice, group-housed in separate cages, were placed together in anovel cage (29 � 18 � 12 cm), and the interaction between thetwo mice was recorded for 10 min with an infrared digital videocamera (Samsung, Korea). The output of the video camera wasfed into a personal computer, and latency in sec to initiate the

interaction and the duration and number of interactions were

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cored by two blind raters. An interaction was defined by bodyontact including inspection and anogenital sniffing. Reliabilityf measurements was assessed by correlating the scores of thewo raters.

Social Isolation Induced-Aggression (Resident-Intruderest). Male mice were socially isolated for several weeks. Theome cage of the resident mouse was not changed during theast week before the test. At the time of the test a young adultale mouse (intruder) was introduced into the home cage (27 �

7 � 12 cm) of an isolated (resident) mouse. In control condi-ions, the resident mouse typically initiates aggressive contacts aew seconds after the introduction of the intruder. These areharacterized by a pattern of exploratory activity toward thentruder, followed by rearing and tail rattles, accompanied in aew seconds by wrestling, violent biting attacks, or both. Attacksere defined as a fierce lunging of the resident at the intruderouse, often associated with biting. Wrestling was defined as a

ideways or an upright stance with head and forebody move-ents toward the intruder and trying to bite the intruder. Theehavior of the animals during the interaction was videotapednd the total duration of the attacks or wrestling (lasting 3–10ec) during a 10-min observation period was measured by twoighly experienced observers who were blind to the type ofreatment (Pinna et al 2003).

Prepulse Inhibition of Startle (PPI). The SR-Lab Startle Re-ponse System (San Diego, California) and PPI procedures haveeen described previously (Tremolizzo et al 2002; Tueting et al999). Startle-only trails (115 dB, 30 msec) or prepulse � startlerials, consisting of an 85-dB, 20-msec prepulse stimulus fol-owed after a silent interval (40, 70, 100, 140, 280, or 420 msec)y the startle stimulus, were randomly presented. The amount ofnhibition was calculated as a ratio (mean startle for startle onlyrials minus mean startle for prepulse trials divided by meantartle for startle only trials).

Locomotor Activity and Movement Tracking. A computer-zed 6 Animal Activity Monitoring System with VersaMax softwareAccuScan Instruments, Columbus, Ohio) was used to quantify andrack locomotor activity in mice, as previously described (Carboni etl 2004). Each activity cage consisted of a Perspex box (20 � 20 �0 cm) surrounded by horizontal and vertical infrared sensor beams.he total number of interruptions of the horizontal sensors wasaken as a measure of horizontal activity, whereas that of verticalensors was used as a measure of vertical activity. Activity wasecorded for 15 min between 1 and 3 PM.

Statistical Analysis. Results are expressed as mean � SE.tudent t test, one-way analysis of variance (ANOVA) followed byultiple comparisons with the Bonferroni test or two-way repeated-easures ANOVA followed by multiple comparisons with Student–ewman–Keuls test were used as indicated in the figure and table

egends. The criterion for significance was p .05, two-tailed.

esults

PA-Induced Acetylation of Histone TailsHistone 3 (H3)-Acetylation. Valproate (1.5 mmol/kg SC)

dministered to mice treated for 15 days with VEH inducedithin 2 hours a three-fold increase of acetylated H3 in FC

Figure 1). In mice that received protracted treatment with VPA,he injection of VPA (1.5 mmol/kg) induced within 2 hours anncrease of hyperacetylated H3-like immunoreactivity in the FChat was similar to that of VEH-treated mice (Figure 1). Thisncrease of acetylated H3 was completely gone 12 hours after the

PA injection (Figure 1).

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We also found that the duration of the increased acety-lated-H3 in FC was similar to that shown in Figure 1 when VPAwas administered to mice receiving MET twice daily for 15 days(data not shown).

As shown in Figure 2, 2 hours after SC injection of VPA, theintensity and number of acetyl H3 immunoreactive neurons wasincreased compared with VEH-treated mice. The increase in theintensity and number of neurons with acetylated H3 appeared tobe greater in cortical layers I and II than in layers IV and V. Nisslcounterstaining (Figure 2) suggests that this increase occurspreferentially in small round cell bodies, which are likelyGABAergic interneurons.

H3-Methylation. Protracted injections of MET (5.2 mmol/kgSC, twice daily for 15 consecutive days) more than doubled theFC content of dimethyl K9-H3 (Figure 3). Repeated doses of VPAfor 15 days failed to change the basal expression of dimethylK9-H3 (Figure 3). When VPA was coadministered with MET,however, VPA inhibited the hypermethylation of dimethyl K9-H3induced by MET (Figure 3). This VPA inhibition was not due toan inhibition of SAM biosynthesis or metabolism. In fact, theincrease in SAM and of its metabolite SAH in the FC of micereceiving MET was similar to that found in mice treated on asimilar schedule with either VEH or VPA (Table 1).

VPA Prevents MET-Induced Reelin PromoterHypermethylation

In the CpG-rich reelin promoter region included between

Figure 1. Western blot analyses in mice treated for 15 days twice a day witheither vehicle (VEH) or VPA (same dose as the challenge). Valproate (VPA)increased acetyl H3-like immunoreactivity in mouse frontal cortex. A chal-lenge dose of VPA (1.5 mmol/kg) was given subcutaneously (s.c.) 2 or 12hours before. The last injection of protracted treatment was administered16 –20 hours before challenge with VPA. Each bar represents the mean � SE;n 3; *p .05 versus VEH, Student t test. O.D., optical density.

bases �340 and �160 (Figure 4), we detected two cytosine

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esidues (�319 and �145) that were consistently methylated inll clones (so called hot spots). Because these two hot spotsailed to change in mice receiving different treatments, they werexcluded from the final analyses.

igure 2. Valproate (VPA) enhanced acetyl-H3 immunoreactivity expressioours before analyses. Coronal sections 2 mm anterior to the bregma (15-�mith thionine (violet). In the inset, large magnification of cells is indicated b

igure 3. Results of Western blot analyses of mice FC extracts carried out 12–16ours after the last methionine (MET), valproate (VPA), or MET�VPA treatment.he combination of VPA and MET administration reduced the dimethyl (K9) H3

mmunoreactivity increase elicited by protracted MET treatment. MET 5.2mol/kg SC, twice daily for 15 days. VPA 1.5 mmol/kg SC twice daily for 15

ays. Each bar is the mean � SE of four mice. Analysis of variance: p .01,ollowed by Student–Newman–Keuls comparisons, *p .05 MET versus VEH,

ersus VPA, or versus MET�VPA. O.D., optical density.

The reelin promoter region included between the two hotspots (�319 and �145 bp) contains a total of 71 CpG dinucle-otide sites (Figure 4) that were hypomethylated or virtuallyunmethylated in VEH- and VPA-treated mice (Table 2). Incontrast, the protracted administration of MET methylates 10CpGs expressed between CpGs 14 and 47 (Figure 4 and Table 2).

ouse FC neurons. Mice received vehicle (VEH) or VPA (1.5 mmol/kg SC) 2s) were stained with anti-acetyl-H3 antibodies (brown) and counterstainedws.

Table 1. Increase of SAM and SAH in FC of Mice Receiving MET orValproate Coadministered with MET

Repeated TreatmentaSingle Treatment

Challengeb SAMc SAHc

VEH VEH 320 � 6.2 134 � 12VEH METd 480 � 45g 177 � 18METe VEH 420 � 36g 151 � 2.4METe METd 470 � 24g 192 � 4.2g

METe � VPAf METd 500 � 31h 195 � 8.8h

Each value is the mean � SE of 4 animals. SAH, S-adenosyl-homocys-teine; SAM, S-adenosylmethionine; VEH, vehicle; MET, methionine.

aTwice daily for 15 days.b2 hours before measurement.cpmol/mg protein.dMET 5.2 mmol/kg, injected subcutaneously.eMET 5.2 mmol/kg, injected subcutaneously. Animals were kept drug

free for 16 –20 hours before administration of a single challenge dose of VEHor MET.

fVPA 1.5 mmol/kg. Mice were without valproate for 16 hours beforeadministration of a single challenge dose of MET.

gp .05. versus VEH�VEH-treated group.hp .01. versus VEH�VEH-treated group.Analysis of variance followed by Student–Newman–Keuls comparison

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t is interesting that within the reelin promoter the transcriptiontart site region, included between �54 and �150 bp, there are2 CpGs that remained virtually unmethylated in MET-treatedice (Figure 4, Table 2).Mice treated twice daily with 5.2 mmol/kg of MET for 15 days

howed a decrease of reelin mRNA expression from 9.5 � .1 to.2 � .05 pmol/ug RNA (p .05; n 5). Valproate coadminis-ered with MET virtually caused the disappearance of MET-nduced hypermethylation from the reelin promoter region in-luding CpGs 14–47 (Table 2).

PA Attenuates the Expression of MET-Induced Behavioralhanges

Social Interaction and Social Recognition. Home cage test:EH-treated male mice isolated for 2 days in their home cagend then exposed to an unfamiliar VEH-treated male intrudernitiated social contacts toward the intruder with a latency ofpproximately 2 sec. The total duration of the test was 5 min,nd in this period of time the resident spent a considerablemount of time inspecting the unfamiliar individual (FigureA). If after 5 min the intruder was removed for 30 min fromhe test cage and was then returned to the test cage, theuration of social interaction was shortened in the secondrial. If social interaction time was measured during the thirdr fourth trial the duration of interaction progressively short-ned (Figure 5A). This shortening during repeated trials wasnterpreted as development of social recognition. In fact, if in

igure 4. Map of CpG dinucleotide location in the mouse reelin promoterlignment tool (BLAT) search of the University of California Santa Cruz (UCShe numbering is related to the 5’ RNA start site reported (Royaux et al 1997egion are represented by the vertical bars and numbered sequentially fromhot spots” that are methylated in all mice irrespective of the treatment. Aste

ice (5.2 mmol/kg, twice daily for 15 days). Results are from four clones obt6 clones per treatment).

able 2. Valproate Corrects methionine-Induced Reelin Promoter Hyperm

Cp

4–13 14–16 17–19 20–22 23–25 26–

reatment NumVEH 1MET 2 1 1 2VPAVPA�MET 1

Total methylated 5-cytosines in four clones obtained from the frontal coET (5.2 mmol/kg, twice daily for 15 days), valproate (1.5 mmol/kg, twice d

ites 3 and 79 (see Figure 4) were methylated in nearly every sequenced clonhot spots; for more details see Tremolizzo et al 2002). For CpG-location of melone was .68 � .24 in MET-treated mice, .12 � .006 in VPA � MET-treated mPA-treated mice. Differences between MET and the other conditions were aultiple comparisons. The number of methylated cytosine per clone in

alproate; VEH, vehicle.

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the fifth trial an unfamiliar intruder was presented, an imme-diate restoration of a lasting investigative social interactiontook place (Figure 5A).

Resident mice receiving protracted MET treatment (scheduledescribed earlier), similar to that of VEH-treated mice, initiatedinteractions with a MET-treated intruder after a latency of 2 sec;however, MET-treated resident mice displayed 35%–40% lesssocial interaction time when presented with a MET-treatedintruder (Figure 5A). Moreover, MET-treated mice failed tohabituate to an unfamiliar intruder, suggesting they had lost thecapability to recognize the intruder. When the same mice weretested for their ability to recognize a lemon scent, both MET andVEH groups rapidly habituated to this stimulus (Figure 5B) anddishabituated when a vanilla scent was substituted. Although thenumber of lemon-scented cotton investigations was initiallyslightly smaller in MET- than in VEH-treated mice, this differencewas not statistically significant (repeated-measures ANOVA, us-ing treatment and trials as variables), suggesting that MET doesnot change olfactory acuity.

Novel environment test: Mice receiving protracted VEH treat-ment placed in a novel environment initiated reciprocal contactsin approximately 8 � 2 sec. In contrast, MET-treated miceinteracted in approximately 2 � .25 sec (p .05; Student t test).During the 10-min social interaction test, the duration of interac-tions between two mice treated for 15 consecutive days withMET was significantly less than between two VEH-treated mice(Table 3).

island. The reference sequence was obtained by performing a basic localuse Genome Browser using the sequences reported in Royaux et al (1997).

h the first base corresponding to �1. The CpG dinucleotides present in this’. The RNA start site is indicated. Arrows indicate the location of methylation) denotes methylated 5-cytosines present only in methionine (MET)-treatedfrom the frontal cortex DNA extracted from each of the four animals (total

tion in Mouse Frontal Cortex

ucleotide Number

29–31 32–34 35–37 38–40 41–43 44–47 48–78

ethylated Cytosines

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NA of each of four animals per group (total 16 clones per treatment): VEH,r 15 days) coadministration of MET � VPA twice daily for 15 days. The CpG

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MET versus VEH was not significantly different. MET, methionine; VPA,

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Because in both the first trial of the home cage test and theovel environment test MET-treated mice showed comparableeduction of social interaction, we selected the novel environ-ent test to study whether VPA, in doses that reduce reelinromoter methylation (Figure 4 and Table 2), also reverses theocial interaction deficit that MET elicits.

When VPA was coadministered with MET for 15 days, itrevented the social interaction shortening elicited by METTable 3). If after a protracted treatment MET administration isbruptly discontinued, the abnormal social behavior persistedonger than 72 hours (data not shown). Importantly, VPA inoses that failed to change the duration of social interactionrevented the social interaction shortening elicited by METTable 3). As a further control, the duration of social interactionuration failed to change after a glycine dose equimolar to theET dose. No differences among the different groups werebserved in vertical or horizontal locomotor activity (i.e., valuesor horizontal are VEH treatment 1699 � 72; MET treatment 524 � 125; VPA�MET 1390 � 92 beam breaks; n 10)

Decreased Aggression in Socially Isolated Mice. As shownn Figure 6A, VEH-treated male mice socially isolated for 2–3eeks were very aggressive toward an untreated intruder of the

ame sex. Methionine administration to resident mice during thesolation period significantly prolonged the latency of attacks atigher doses (from 100 � 13 sec in VEH-treated mice, to 89 � 10n mice treated with .66 mmol/kg MET, 131 � 18 in mice treatedith 3.3 mmol/kg, 217 � 38 in mice treated with 5.2 mmol/kg,nd 344 � 49 in mice treated with 6.6 mmol/kg; n 5) andeduced the duration of attacks against the intruder mouseFigure 6A). This reduction of aggression was prevented by VPAoadministered with MET (Figure 6C). A VPA�MET treatedesident attacked the intruder with a similar latency and durations a VEH-treated resident, whereas VPA alone failed to modifyggressive behavior. The MET-induced modification of aggres-ive behavior was dose related (Figure 6B) and persisted longerhan 1 week following MET withdrawal (Figure 6A).

Impaired PPI. In male mice, the PPI ratio reached a maxi-

igure 5. Protracted methionine (MET) treatment reduced social interactionime occurring in the home cage. Groups of 10 male mice were treated twiceaily for 15 days with either vehicle (VEH; black circles) or with MET (5.2mol/kg SC twice daily for 15 days; gray squares). Each value (n 5) is the

roup mean � SE. (A) During the first social interaction trial (5-min durationor each trial), MET treatment reduced social interaction time compared

ith VEH treatment (*p .01 Student t test). If mice were subjected toepeated 5-min trials separated by a 30-min intertrial interval, the investiga-ive behavior of VEH-treated mice habituated. If after five trials, a newntruder mouse was substituted, the resident mouse investigated the novelntruder mouse with an interest similar to that on the first trial. In contrast, in

ET-treated mice habituation of investigative behavior failed to occur, sug-esting that these mice have a social recognition deficit. (B) The number ofontacts per min in investigating a lemon-scented cotton ball. A novel scentvanilla) was introduced in trial 4.

um when a soft auditory prepulse stimulus preceded a loud

startle stimulus by a 70-msec interval. By prolonging this intervalup to 420 msec, we observed a gradual decline of inhibition(Figure 7). In mice receiving our standard 15-day MET treatmentand tested 16–20 hours after the last injection of MET, the PPIratio was significantly lower than in VEH-treated mice at 70 msec,and PPI declined at a faster rate as the prepulse startle intervalwas prolonged (Figure 7A).

A single MET injection given 16–20 hours before PPI testingwas without effect. Body weight and locomotor activity wereapproximately equal in mice receiving VEH and protracted METtreatment.

When VPA (1.5 mmol/kg, twice daily for 15 days) wascoadministered with MET, VPA antagonized the decrease in PPIratio induced by MET (Figure 7B), whereas in mice receivingVPA alone, there were no significant changes in PPI decay rates(Figure 7B). A single injection of VPA administered after 15 daysof MET treatment also failed to change the characteristic MET-induced decay of inhibition.

Imidazenil Normalizes Behavioral Deficits Induced by METFigure 7C shows that imidazenil reduced the loss of PPI in

mice receiving protracted MET treatment. Table 3 shows thatimidazenil administered in a single dose 120 min before thesocial interaction test also normalized the social deficit inducedby protracted MET treatment. At this dose, imidazenil failed tomodify locomotor activity (VEH 1560 � 103 unit/15 min; imida-zenil 1320 � 117 beam breaks/15 min; n 8, ns).

Discussion

MET as a Tool to Investigate Epigenetic Regulation of ReelinPromoter Hypermethylation

Methionine, administered to mice in doses that increase FCcontent of the methyl donor SAM, elicits reelin promoter hyper-methylation and the subsequent downregulation of the expres-sion of the reelin gene by an extent (� 50%) that is comparableto the reelin defect detected in the prefrontal cortex (PFC) ofschizophrenia patients (Guidotti et al 2000). The analysis ofsingle CpG dinucleotide methylation in the mouse reelin pro-moter region comprising �340 and �160 bp indicates that afterMET treatment, the hypermethylation is primarily restricted toCpGs 14–47, a region just upstream to the transcriptional startsite (Figure 4). Computer analyses (Transfac Professional 7.4.1,Biobase, Wolferbuttel, Germany) suggests that this reelin pro-

Table 3. Valproate Prevents the Methionine-Induced Decrease in SocialInteraction Timea in a Novel Environment

ProtractedTreatment

Protracted Cotreatment Single Treatment

VEH VPAd IMDe

VEHb 215 � 6 189 � 19 260 � 20METc 110 � 11f 161 � 12 200 � 15

Each value is the mean � SE of six pairs of mice. Analysis of variance p .001 followed by Student–Newman–Keuls multiple comparison tests. MET,methionine; VPA, valproate; VEH, vehicle; IMD, imidazenil.

aSocial interaction, sec/10 min.bVEH .1 mL saline/10 g body weight injected subcutaneously twice a

day for 15 days.cMET 5.2 mmol/kg, injected subcutaneously twice a day for 15 days.dVPA 1.5 mmol/kg, injected subcutaneously twice a day for 15 days.eIMD 3.3 �mol/kg, injected subcutaneously 2 hours before test.fp .05, comparing (METc � VEH) versus (VEHb � VEH) or versus (METc �

VPAd), or versus (METc � imidazenil). Social interaction was determined

16 –20 hours after the last protracted injections.

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oter region expresses sequences that are recognized by severalranscription factor binding sites (Pax6, Sp1, Trb1) and that thesere important for reelin transcriptional regulation. The binding ofhese transcription factors to putative reelin promoter regulatoryites may be affected by methylation at CpGs that control reelinxpression.

The mechanisms by which MET induces reelin promoter CpGinucleotide hypermethylation depend on an increase of brainAM content that alters high-order chromatin remodeling inABAergic neurons by inducing nucleosomal histone tail hyper-ethylation. This change may recruit multifunctional repressor

omplexes comprising DNMTs and methyl binding domainroteins (MBD; Burgers et al 2002; Jenuwein 2002; Johnstone002).

PA Activation of Histone Acetylation and Methylationynamics and the Epigenetic Hypermethylation of the Reelinromoter

Valproate, a drug used for the treatment of epilepsy and moodisorders (Loscher 1999), is now frequently prescribed with anti-sychotics as adjunctive medication for the treatment of schizophre-ia (Casey et al 2003; Wassef et al 2003). At the doses used in theseouse experiments, VPA is an effective HDAC inhibitor (Tremol-

zzo et al 2002) that maintains unabated its HDAC inhibitory actionven after protracted treatment (Figure 1).

We show here that VPA coadministered with MET preventshe histone N-terminal tail hypermethylation (Figure 3) and theeelin promoter hypermethylation (Figure 4, Table 2) induced byET while favoring neuronal histone tail hyperacetylation by

nhibiting neuronal HDACs (Figures 1 and 2). This VPA actionails to change the brain increase of SAM or SAM metabolicroducts induced by MET (see Table 1).

In preliminary experiments using ChIP assay, we have studiedhether VPA induces hyperacetylated H3 associated with reelinromoter and found a twofold to threefold increase of acetylatedistones associated with reelin promoter after VPA administra-ion. Thus, the results suggest that VPA-induced histone hyper-cetylation associated with reelin promoter, stabilizing an openhromatin state (euchromatin) and favoring the associated induc-ion of DNA demethylase activity (Detich et al 2003), is respon-ible for the prevention of MET-induced cytosine hypermethyl-tion of reelin promoter.

igure 6. Protracted methionine (MET) treatment prevented developmenesponse, and (C) prevention by valproate (VPA). The level of aggression ofttacks in 10 min. Each value is the mean � SE of five animals. Results in A,mol/kg twice a day for the weeks of isolation indicated. *p .01 or **p .0

analysis of variance [ANOVA] followed by Bonferroni multiple comparisoggressive behavior was assessed 16 –20 hours after the last protracted treith VEH-treated mice (ANOVA followed by Bonferroni multiple comparis

dministered twice a day for 3 weeks. Aggressive behavior was measured 16y Bonferroni multiple comparison test between VEH- and MET-treated mic

It is worth noting that the dose of VPA administered to mice

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(1.5 mmol/kg) appears disproportionately larger than the dosesof VPA administered to psychiatric patients (.15–.30 mmol/kg).Thus, the question is whether VPA, at therapeutic doses, willhyperacetylate histones in human brain. Pharmacokinetic con-siderations suggest that VPA half-life of 9–18 hours in humansversus .8 hours in mice and plasma binding proteins of 80%–95%in humans versus 12% in mice may affect plasma and brain levelsof VPA differently in mice and humans (Loscher 1999). Forexample, it has been established that the brain concentration ofVPA in mice treated with 1 mmol/kg of the drug (the EC50 forantimetrazol convulsions) are similar to those measured in brainbiopsies of epileptic patients treated with .1–.15 mmol/kg of VPA(Loscher 1999).

Based on these data, we suggest that the concentrations ofVPA in the brain of our animal model are comparable to the

ggressive behavior in socially isolated (SI) mice. (A) Time course, (B) doseent male mouse against an intruder was measured as the total duration of

d C are from three experiments. (A) MET was administered in a dose of 5.2en MET-treated animals were compared with vehicle (VEH)-treated animals) MET was administered twice a day for 3 weeks at the doses indicated.t injection. *p .01 or **p .001 when MET-treated mice were compared

(C) VEH, MET (5.2 mmol/kg/sc), or VPA (1.5 mol/kg/sc) or VPA�MET wereours after the last protracted treatment injection. *p .01 (ANOVA followed

Figure 7. Mice receiving protracted treatment with methionine (MET)showed an acceleration of the decline of prepulse inhibition elicited byincreasing the delay in the prepulse–startle intervals: modifications wereinduced by pharmacologic interventions with valproate (VPA) and imidaze-nil (IMD). Six groups of mice (vehicle [VEH], MET, VPA, MET�VPA, IMD, andIMD�MET) were run simultaneously in the experiment. For presentationclarity, the results are presented in separate panels. Statistical analyses wererun by two sets of 2-by-2 design. One set with MET as one factor and VPA asa second factor. A second set with MET as one factor and VPA as secondfactor. (A) Comparison between VEH- and MET-treated mice: p .01; re-peated-measures analysis of variance (ANOVA). (B) Comparison betweenVPA and VPA�MET coadministration: no significant differences; repeated-measures ANOVA. (C) Comparison between IMD and IMD�MET: no signifi-cant differences; repeated-measures ANOVA. The VPA- and IMD-treatedmice did not differ from VEH-treated mice. MET 5.2 mmol/kg SC twicedaily for 15 days. VPA 1.5 mmol/kg SC twice daily for 15 days. IMD

t of aa residB, an01 whns). (Batmenons).

3.3umol/kg, 1 hour before test. Each value is the mean � SE of six mice.

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ffective concentrations of VPA in the brain of schizophreniaatients treated with .15–.30 mmol/kg of VPA. Thus, the ability ofPA to hyperacetylate histones in mice can be extrapolated touman brain following effective therapeutic doses.

To clarify whether the molecular mechanisms underlying thection of VPA on reelin expression in mice are related to thelockade of HDACs or to some other action of VPA (as discussedy Tremolizzo et al 2002), we (Chen et al 2002) studied in neuralrogenitor cells in vitro the effect of VPA in comparison to that ofther chemically unrelated HDAC inhibitors and found thatDAC inhibitors such as trichostatin A elicit the same effects asPA on the regulation of reelin expression. Collectively, thesexperiments suggest that the molecular mechanism by whichPA modulates reelin expression in brain may be mediated by alockade of HDACs.

It remains to be studied in future experiments whether thepregulation of GAD67 expression induced by VPA (Losher et al999; Tremolizzo et al 2002) is also mediated via inhibition ofrain HDAC activity.

elationship of Mouse Schizophrenia-like Behavior topigenetic Mechanisms Involving Promoterypermethylation

Mice receiving protracted MET treatment express a number ofharacteristic behavioral patterns that resemble certain endophe-otypic symptoms that have been considered in other rodentodels of schizophrenia morbidity (e.g., NMDA KO [Mohn et al

999]; calcinurin KO [Miyakawa et al 2003]; dopamine transporterO [Gainetdinov et al 2001]; enhanced nucleus accumbens andtriatal dopaminergic system activity [Lipska and Weinberger000]; and reelin heterozygous mice [Carboni et al 2004; Larsont al 2003]).

We are aware that any animal model for studying andnderstanding schizophrenia has limitations. For example, theres concern that neuronal circuits in mouse neocortex are noteveloped to the level necessary to model a human disorderuch as schizophrenia, and there are also criticisms related to thealidity of rodent behavior simulation of positive or negativeymptoms of schizophrenia. There are behaviors (e.g., auditoryensorimotor gating, social interaction) and neurobiologicalhanges in genetic or epigenetic altered mice, however, that doppear to mimic specific phenotypic aspects of schizophreniand have been shown to be modified by antipsychotic drugreatment, thus supporting a model of schizophrenia in rodentsGainetdinov et al 2001; Lipska and Weinberger 1999).

Social withdrawal, attention deficit, and cognitive dysfunctionre characteristic signs associated with schizophrenia vulnerabil-ty. Thus, a schizophrenia animal model is expected to induce aecrease in social interaction (social withdrawal), a decrease inttention, and cognitive impairment (Miyakawa et al 2003; Mohnt al 1999).

In this study, we have shown that mice receiving protractedET treatment display a suppression of social interaction (Figure, Table 3) as well as impaired attention and cognition, asnferred by the faster rate of PPI decline induced by prolonginghe prepulse and startle pulse interval (Figure 7). These mice alsoxpress decreased aggression after social isolation (Figure 6). Weelieve that these behavioral modifications model phenotypicspects associated with schizophrenia vulnerability (Miyakawa etl 2003; Mohn et al 1999).

To support the hypothesis that epigenetic mechanisms maye responsible for the MET-induced behavioral alterations re-

embling schizophrenia, we coadministered VPA with MET for

15 days. In doses that maximally hyperacetylate lysines ex-pressed in histone N-terminal tails and that virtually abolishMET-induced reelin promoter hypermethylation (Table 2), VPAreverses or abolishes MET induced changes in PPI, social inter-action, and aggression.

The lack of a direct action of VPA on aggressive behaviorobserved in our experiments and the reversal by VPA of aMET-induced decrease in aggressive behavior in socially isolatedmice might appear contradictory to a number of studies (Beloz-ertseva and Andreev 1997; Molina et al 1986; Oehler et al 1985;Sulkova et al 1981) reporting that VPA administration reducesaggressive behavior in mice and rats. We believe that thisapparent discrepancy can be reconciled by considering thepharmacokinetic and pharmacodynamic properties of VPA. Inthe experiments of Molina et al (1986), Sulkova et al (1981), andBelozertseva and Andreev (1997), the antiaggressive effect ofVPA in mice and rats was observed only for a short period of timeafter a single VPA injection (� 1 hour, which is the half-life ofVPA in rodents; Loscher 1999). In the experiments of Oehler et al(1985), however, chronic administration of VPA (4 weeks) indoses up to 200 mg/kg/day did not lead to prevention ofisolation induced aggression. Our results agree with the Oehleret al’s (1985) observations, because protracted administration ofVPA in relevant pharmacologic doses failed to decrease aggres-sive behavior when animals were tested 16–20 hours after thelast VPA injection. Several lines of evidence show that VPA exertsshort-lasting direct effects on neuronal membrane excitability,reducing sodium current (Loscher 1999). These effects are re-lated to relatively high concentrations of VPA in plasma andbrain, but as the concentration rapidly declines, these earlyeffects rapidly disappear. Late effects of VPA are not due tomembrane effects on receptors or channels but may rather bedue to intracellular signaling mechanisms (Hao et al 2004) orgenomic actions (i.e., HDAC inhibition; Tremolizzo 2002); thus,they are long-lasting (hours and days) and may produce effectscompletely different from the early short-lasting effects that areobserved in the first hours after single administration.

Relationship Between GABAergic Hypofunction andBehavioral Alterations

It is well established that protracted VPA treatment increasesGAD67 mRNA expression in brain and thereby facilitatesGABAergic neurotransmission (Loscher 1999; Tremolizzo et al2002). Because VPA/MET coadministration prevents MET-in-duced reelin and GAD67 downregulation (Tremolizzo et al 2002)and associated MET-induced behavioral alterations, we haveconsidered that GABAergic transmission downregulation maycontribute to the onset of schizophrenia-like behavior in MET-treated mice. To further investigate this hypothesis, we studiedwhether MET-induced behavioral alterations could be attenuatedby imidazenil, a benzodiazepine that selectively and in a positiveallosteric manner modulates the action of GABA at GABAA

receptors expressing the �5 subunit but is virtually devoid ofsedation and intrinsic activity at the GABAA receptors expressing�1 subunits (Costa et al 2002a). In Table 3 and Figure 7, we showthat single doses of 3.3 �mol/kg of imidazenil administered tomice 1–2 hours before either a social interaction test or anauditory sensorimotor gating session reduces the effects ofprotracted MET treatment.

These data allow us to postulate that imidazenil may be theprototype of a new family of antipsychotics with a unique actionon cortical GABAergic dysfunction operative in schizophrenia.

To this end, it is important to stress that imidazenil does not have

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olerance and dependence liabilities or liver and other organoxicity even at doses 100- to 200-fold higher than the doses thatreferentially and positively modulate GABA action at �5 con-aining recombinant GABAA receptors (Costa et al 2002a).

Taken collectively, these data suggest that a dysfunction ofABAergic transmission with a reduction of GABA and reeliniosynthesis and secretion may be operative in the developmentf the behavioral syndrome expressed in MET-treated mice.hese results are in agreement with previous studies in heterozy-ous reeler mice (HRM), in which the reelin haploinsufficiencynd a 25%–30% reduction of GAD67 and GABA turnover in FCnd hippocampus results in deficits in PPI (Tueting et al 1999),lfactory memory (Larson et al 2003), and spatial memory testsfter treatment with N-methyl-D-aspartate (NMDA) receptor an-agonists (Carboni et al 2004). The results of these studies,owever, do not exclude the possibility that, in addition to aecrease of reelin and GAD67 mRNAs, the decrease of other generoducts considered important in causing schizophrenia symp-oms (e.g., receptors for dopamine, glutamate, 5HT, acetylcho-ine, or their respective transmitter synthesizing or metabolizingnzymes) might contribute to the MET-induced syndrome.

In fact, it is possible that MET, by increasing SAM in glutama-ergic, serotonergic, catecholaminergic, and cholinergic neurons,ay facilitate methylation of gene promoters related to the

unction of these specific transmitters or influence hormoneeceptors with functions implicated in the pathophysiology ofchizophrenia.

onclusionsFurther studies with the MET mouse model described in this

eport may allow us to evaluate whether the PPI deficit, socialnteraction deficits, or the failure to develop aggressive behaviorsfter social isolation will become more severe after administra-ion of NMDA receptor blockers or of dopamine-mimetic drugsi.e., amphetamines). It will be even more relevant to schizo-hrenia morbidity to determine whether these behaviors im-rove with long-term treatment involving typical or atypicaleuroleptics administered alone or with VPA.

Treatment with MET, which aggravates the symptoms ofchizophrenia in humans and can elicit the onset of psychoticpisodes in asymptomatic patients, is becoming an importantnd useful tool in rodents to model schizophrenia symptomatol-gy and to study mechanisms whereby antipsychotics and VPAmeliorate the consequences of schizophrenia morbidity.

This work was supported by NIMH grants MH06020 to EC,H62682 to DRG, and MH062188 to AG.

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