Progress in Neuro-Psychopharmacology & Biological Psychiatry 56 (2015) 149–154
Contents lists available at ScienceDirect
Progress in Neuro-Psychopharmacology & BiologicalPsychiatry
Perinatal asphyxia alters neuregulin-1 and COMT gene expression in themedial prefrontal cortex in rats
Tomoyasu Wakuda a, Keiko Iwata b, Yasuhide Iwata a, Ayyappan Anitha c, Taro Takahashi a, Kohei Yamada c,Mahesh Mundalil Vasu a, Hideo Matsuzaki b, Katsuaki Suzuki a,⁎, Norio Mori a,c
a Department of Psychiatry, Hamamatsu University School of Medicine, Hamamatsu, Shizuoka 431-3192, Japanb Research Center for Child Mental Development, University of Fukui, Eiheiji-cho, Japanc Research Center for Child Mental Development, Hamamatsu University School of Medicine, Hamamatsu, Japan
Abbreviations:AKT1, V-aktmurine thymoma viral oncysis of variance; BDNF, brain-derived neurotrophic factoCOMT, catechol-O-methyltransferase; ErbB4, v-erb-a erytgenehomolog4;GAPDH,glyceraldehydes-3-phosphatedeNRG1,neuregulin-1;PBS,phosphatebufferedsaline;PFC,ptive polymerase chain reaction.⁎ Corresponding author at: Department of Psychiatry, H
Epidemiological studies suggest that perinatal complications, particularly hypoxia-related ones, increase the riskof schizophrenia. Recent genetic studies of the disorder have identified several putative susceptibility genes,some of which are known to be regulated by hypoxia. It can be postulated therefore that birth complicationsthat cause hypoxia in the fetal brain may be associated with a dysregulation in the expression of some of theschizophrenia candidate genes. To test this, we used an animal model of perinatal asphyxia, in which rat pupswere exposed to 15 min of intrauterine anoxia during Caesarean section birth, and examined the expression ofmRNAoffive of the putative susceptibility genes (NRG1, ErbB4, AKT1, COMT and BDNF) by real-time quantitativePCR in themedial prefrontal cortex (mPFC) and the hippocampus at 6 and 12 weeks after birth. The expression ofNRG1mRNAwas significantly decreased in themPFC, but not in the hippocampus, at 6 and 12 weeks after birth.In addition, a significant increase in COMT mRNA expression was observed in the mPFC at 12 weeks. The alter-ation in mRNA levels of NRG1 and COMT was not associated with a change in their protein levels. These resultssuggest that perinatal asphyxia may lead to disturbances in the PFC, which in turnmay exert a long-lasting influ-ence on the expression of specific genes, such as NRG1 and COMT. Our results also suggest that translational in-terruption may occur in this model of perinatal asphyxia.
Schizophrenia is a chronic, severe, and disabling brain disorder thathas affected people throughout history. Although the etiology of schizo-phrenia is not well understood, findings from epidemiological and neu-ropathological studies indicate that pathogenic processes thatculminate in the development of schizophrenia are initiated early inlife (Murray, 1994). The neurodevelopmental hypothesis that schizo-phrenia has its origin in aberrant brain development is supported byevidence that obstetric complications at or close to the time of birthcontribute to the risk for the development of schizophrenia later in life(Byrne et al., 2007; Cannon et al., 2002).
amamatsu University School ofan. Tel.: +81 53 435 2295; fax:
.
The issue of perinatal asphyxia has been studied in a rodentmodel ofglobal hypoxia during Caesarean section (C-section) birth (Bjelke et al.,1991). In thismodel, the intact uterus containing rodent pups is isolatedfrom an anesthetized dam by a C-section on the expected day of birthand immersed in a water bath kept at 37 °C for 14–17 min to induceintra-uterine global hypoxia. Such global hypoxia can lead to alterationsin central dopamine function during adulthood that are consistent withthe postulated pathophysiology of schizophrenia (Boksa and El-Khodor,2003). For instance, we have recently produced perinatal asphyxia in ratpups by exposing them to 15min of intrauterine anoxia during C-sectionbirth (Wakuda et al., 2008). Whenmethamphetamine-induced locomo-tor activity was tested at adulthood (12 weeks after birth), it was greatlyincreased, accompanied by an increase of dopamine release in the nucle-us accumbens. Suchfindingswere not observed at adolescence (6 weeksafter birth).
Accumulating evidence from recent studies has advanced our un-derstanding of the neurobiology of schizophrenia. One of the majorareas of progress has been the identification of putative susceptibilitygenes for schizophrenia, which has been made by family studies(Aoki-Suzuki et al., 2005; Chen et al., 2004; Lichtenstein et al., 2009;Yamada et al., 2004), national record linkage studies (Eaton et al.,2006; Ekelund et al., 2004; Numakawa et al., 2004), and emerging
150 T. Wakuda et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 56 (2015) 149–154
evidence from a genome-wide association study (SchizophreniaPsychiatric Genome-Wide Association Study [GWAS] Consortium,2011). Some of these candidate genes are responsive to hypoxia(Schmidt-Kastner et al., 2006). Neuregulin-1 (NRG1) has been one ofthe candidate genes most associated with an increased risk of schizo-phrenia (Gong et al., 2009;Munafò et al., 2008). NRG1 is a trophic factorthat contains an epidermal growth factor (EGF)-like domain that signalsby stimulating ErbB receptor tyrosine kinases. The functional NRG1 re-ceptor v-erb-a erythroblastic leukemia viral oncogene homolog 4(ErbB4) is expressed in the midbrain dopaminergic neurons in miceand primates (Abe et al., 2009; Zheng et al., 2009). Furthermore, the sig-naling of NRG1 and its receptor ErbB4 has been implicated in importantneurodevelopmental processes of putative relevance to theaetiopathology of schizophrenia (Corfas et al., 2004; Fazzari et al., 2010;Hahn et al., 2006; Jaaro-Peled et al., 2009; Mei and Xiong, 2008). Boththe catechol-O-methyltransferase (COMT) and brain-derived neuro-trophic factor (BDNF) genes are also regulated by hypoxia (Schmidt-Kastner et al., 2006), and are associated with the dopamine system inthe brain; COMT is an enzyme involved in the degradation of dopamineand BDNF is one of the trophic factors involved in the development of do-paminergic neurons (Baquet et al., 2005). V-akt murine thymoma viraloncogene homolog 1 (AKT1), a component of the downstream signalingpathways of bothNRG1/ErbB4 and BDNF, is also one of the schizophreniacandidate genes that are regulated by hypoxia (Emamian et al., 2004;Schmidt-Kastner et al., 2006; Thiselton et al., 2008). Moreover, AKT1me-diates dopaminergic neurotransmission (Beaulieu et al., 2005).Nicodemus et al. (2010) have suggested that NRG1/ErbB4 and AKT1 sig-naling is implicated in the pathogenesis of schizophrenia.
To obtain insight into the delayed alterations in central dopaminefunction during adulthood that are consistentwith the postulated path-ophysiology of schizophrenia, we used an animal model of rats born byC-section and exposed to additional global hypoxia (Wakuda et al.,2008). Subsequently, we examined the mRNA expression levels of thefive schizophrenia candidate genes described above (NRG1, ErbB4,AKT1, COMT and BDNF) in the prefrontal cortex (PFC) and the hippo-campus (HP), in addition to their protein levels in the PFC, at two devel-opmental periods, adolescence (6 weeks of age) and adulthood(12 weeks of age).
Fig. 1. Schematic representation of the rat brain regions analyzed for real-time qPCR. Thebrain regions of mPFC sampled for analysis are indicated by the filled rectangles. Thedrawings were made according to the atlas of Paxinos and Watson (1997), in a caudalto rostral distribution using the bregma (mm) as a reference point.
2. Methods
2.1. Animals and induction of perinatal asphyxia
All experiments were performed in accordance with the Guide forAnimal Experimentation of the Hamamatsu University Schoolof Medicine. Intrauterine anoxia was induced in rats delivered byC-section according to the method described originally by Bjelkeet al. (1991). Pregnant female Sprague–Dawley rats (Japan SLC,Hamamatsu, Japan) within the last day of gestation were anesthe-tized by diethyl ether and hysterectomized. The uterus, including fe-tuses, was placed in a water bath at 37 °C for induction of 15 min ofasphyxia. Rats that had delivered normally on the day of the experi-ment were used as surrogate mothers. Each surrogate mother re-ceived four pups from another surrogate mother, four C-section-delivered and four asphyxia-exposed pups. At 3 weeks after birth,male rats were selected for the experiments described below andwere housed three per cage in a temperature- and humidity-controlled colony room, which was maintained on a 12-h light/darkcycle (07:00 to 19:00 h lights on) and with food and water providedad libitum. The animals were divided into 2 groups in terms of deliv-ery: birth by C-section alone (C group, n = 28) or by C-section plus15 min of global perinatal asphyxia (A group, n = 28).
2.2. RNA preparation
To determine the target gene expression levels, 12 animals (6-week-old, n=6; 12-week-old, n=6) from each of the two groups were used.All the animalswere deeply anesthetizedwithpentobarbital (100 mg/kg,intraperitoneally) and rapidly decapitated. The bilateral medial PFC(mPFC) and the whole HP were dissected on ice and used for the poly-merase chain reaction (PCR) quantification analysis. The region of mPFCwas defined as the cingulate (Cg), prelimbic (PrL) and infralimbic (IL)cortices according to the atlas of Paxinos and Watson (1997) (Fig. 1).
Total RNAs from ratmPFC andHP tissuewere isolated frombrain tis-sues using TRIZOL reagent (Invitrogen Life Technologies, Carlsbad, CA).The RNAs were cleaned up using RNeasy Mini Kit and DNase set(Qiagen, Hilden, Germany). The quality and quantity of RNA were mea-sured using a NanoDrop 1000 spectrophotometer (Thermo Scientific,Yokohama, Japan). RNA with a 260/280 nm ratio in the range of 1.8–2.0 was considered high quality. The complementary DNAs (cDNAs)from the RNA preparations were synthesized using a SuperScript IIIFirst-Strand Synthesis System (Invitrogen Life Technologies).
151T. Wakuda et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 56 (2015) 149–154
2.3. Real-time quantitative PCR conditions
Real-time quantitative PCR (qPCR) was performed using an ABIPRISM 7700 Sequence Detection System in combination with continu-ous SYBR Green detection (Applied Biosystems, Foster City, CA). Real-time qPCR was performed in a 25 μl reaction volume containing 2.5 μlcDNA, 12.5 μl SYBR Green PCR Master Mix (Qiagen), 2.5 μl each of thesense and antisense primers (10 μM), and 5 μl H2O. The general PCR con-dition profile was as follows: polymerase activation at 95 °C for 15 min,followed by 50 cycles of denaturing at 95 °C for 15 s, annealing at 60 °Cfor 30 s, and extension at 72 °C for 60 s. After amplification, a meltingcurve was acquired to determine the optimal PCR conditions by heatingthe PCR products at 20 °C/s to 95 °C, then cooling at 20 °C/s to 60 °C.Primer sequences for qPCR amplification were designed using onlinePrimer3 software (http://biocore.unl.edu/cgi-bin/primer3/primer3_www.cgi). The sequences of primers and sizes of PCR amplicons arelisted in Table 1.
The ΔΔCt method (comparative Ct method) (Livak and Schmittgen,2001)was employed for the diagnostic assays. The Ct valueswere calcu-lated using three kinds of housekeeping genes (glyceraldehydes-3-phosphate dehydrogenase [GAPDH], cyclophilin A and β-actin). Thehousekeeping genes fulfilled the criterion that the absolute value ofthe slope of the log input amount vs. ΔCt should be b0.1, which was se-lected as the internal reference value.
2.4. Western blot analysis
To determine the protein quantity, we used another set of 16 ani-mals (6-week-old, n = 8; 12-week-old, n = 8) from each of the twogroups. Total protein from the rat mPFC tissue was quantified with aPierce BCA Protein Assay kit (Thermo Scientific, Barrington, IL).GAPDH was used as the internal control to normalize the expressionof proteins. We used the following primary antibodies for westernblot detection: ErbB4 (ab32375), COMT (ab126618), GAPDH (ab8245)(all from Abcam, Tokyo, Japan), NRG1 (sc-348), BDNF (sc-546) (bothfrom Santa Cruz Biotechnology, Santa Cruz, CA), and AKT1 (#2938)(Cell Signaling Technology, Danvers, MA). Fluorescent-labeled anti-rabbit and anti-mouse secondary antibodies (Rockland, Gilbertsville,PA) were used for the detection of proteins with an Odyssey InfraredImaging System (Li-cor Bioscience, Lincoln, NE). Representative imagesof the western blots are shown in Fig. 2.
2.5. Statistical analysis
The data are expressed as the mean ± standard error of the mean(SEM). In the RT-PCR analysis, the values for the A group are relativeto the mean of the corresponding values of the C group, which was a
Table 1Primer sequences and length of PCR products for each gene used in the real-time quantitative
reference set as 100. In the Western blot analysis, the values indicatethe relative protein levels normalized to GAPDH expression. Differencesin the gene and protein expression levels between the A and C groupswere determined by Student's t-test as well as two-way analysis of var-iance (ANOVA), in which treatment (C group and A group) and age (i.e.6-week-old and 12-week-old) were the independent variables. In thetwo-way ANOVA, the interaction between treatment and age was alsotested. We used SPSS version 22J (IBM Corp., Tokyo) for the statisticalanalyses. The level of statistical significance was set at p b 0.05.
3. Results
Significant changes in the mRNA levels of NRG1 and COMT were ob-served in themPFC. ThemRNA levels of NRG1 in the A groupwere signif-icantly decreased at 6 (16% decrement) (t=−2.24, df=10, p= 0.049)and 12 weeks of age (24% decrement) (t=−2.61, df=10, p= 0.026),compared with those in the C group (Table 2). There were significantmain effects of treatment [F(1,20) = 11.771; p = 0.003] and age[F(1,20) = 30.235; p b 0.001], but no significant interaction betweentreatment and age [F(1,20) = 0.510; p = 0.483]. Compared with thoseof the C group, the A group rats showed a significant increase in themRNA expression levels of COMT at 12 weeks, but not 6 weeks (48% in-crement) (t= 2.80, df=10, p= 0.019) (Table 2). Therewere significantmain effects of treatment [F(1,20) = 8.313; p= 0.009] and age [F(1,20) =49.079; p b 0.001], but no significant interaction between treatment andage [F(1,20) = 0.412; p = 0.528]. There was no significant difference inthe mRNA expression levels for the remaining three genes (ErbB4,AKT1, and BDNF) at 6 or 12 weeks. In the HP, there was no overt changein the mRNA expression for the five genes tested between the A and Cgroups, at either 6 or 12 weeks after birth.
There was no apparent change in the protein quantity for the fivegenes tested between the A and C groups, at either 6 or 12 weeksafter birth (Table 3).
4. Discussion
We evaluated the mRNA levels of five genes that are related to theregulation of the dopaminergic system (Abe et al., 2009; Baquet et al.,2005; Beaulieu et al., 2005; Zheng et al., 2009), NRG1, ErbB4, AKT1,COMT and BDNF, in the mPFC and HP in rats born by C-section withadded global hypoxia at two developmental periods, adolescence(6 weeks of age) and adulthood (12 weeks of age). Among the fivegenes, apparent changes were observed in two genes, NRG1 andCOMT: expression of the mRNA of NRG1 was decreased at 6 and12 weeks, while expression of the mRNA of COMT was increased at12 weeks after delivery. These changes were observed in the mPFC,but not the HP. Since both NRG1 and COMT are strongly implicated in
Fig. 2. A representative gel showing the protein levels of ErbB4, AKT1, NRG1, COMT, and BDNF in the mPFC brain region. GAPDH was used as the housekeeping protein. The molecularweight of the protein marker is shown in kDa. (A) 7.5% gel; ErbB4, 147 kDa; AKT1, 60 kDa. (B) 10% gel; NRG1, 50 kDa; COMT, 24–30 kDa. (C) 15% gel; BDNF, 32 kDa. M,molecular weightmarker; Lane C, mPFC of the rats delivered by cesarean section (C group); Lane A, mPFC of the rats delivered by cesarean section with 15 min anoxia (A group).
152 T. Wakuda et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 56 (2015) 149–154
schizophrenia at both the gene and transcript levels, our results furthersupport the general concept that perinatal asphyxia interacts with thegenes for schizophrenia (Schmidt-Kastner et al., 2006).
It is not known when the levels of the expression of NRG1 mRNAbegin to decrease. Although there are no available reports in this regard,in the present study the reduction of the gene mRNA levels was ob-served at two developmental periods, i.e., 12 and 6 weeks after birth.Therefore, alterations of the expression of NRG1 mRNA may be a long-lasting phenomenon. Our data may not be comparable with those of aprevious study in which 6-day-old rat pups were exposed to a hypoxiacondition (an air mixture of 8% O2, 0.03% CO2, and balance N2 for120 min), and then protein levels of the NRG1α isoform in the frontalcortex were measured in 56-day-olds, and found to be elevated (Nadriet al., 2007). A similar inconsistency is found among the postmortem
Table 2Normalized relativemRNA expression in rats born by the C-sectionwith orwithout globalperinatal asphyxia.
Values are expressed as the means ± SEM. N = 6 animals in each group. Note a statisti-cally significant difference, ⁎p b 0.05.
studies of schizophrenic patients. Bertram et al. (2007) reportedNRG1α isoform protein reductions in the PFC of schizophrenic patients.However, isoform-specific increases in NRG1 mRNA expression in thePFC have also been observed in schizophrenic patients (Hashimotoet al., 2004; Law et al., 2006, 2007; Silberberg et al., 2006). The inconsis-tent results may be due to the use of different samples, i.e., patients orvarious animal models. Nonetheless, all the results from animal and pa-tient studies seem to show that aberrant signals of NRG1may play rolesin schizophrenia, because both mutant mice deficient in NRG1 andtransgenic mice overexpressing NRG1 are known to exhibitschizophrenia-like phenotypes (Ehrlichman et al., 2009; Kato et al.,2010; O'Tuathaigh et al., 2007, 2010).
In contrast to NRG1mRNA levels in themPFC, whichwere altered at6 and 12 weeks after delivery, COMT mRNA levels in the mPFC
Table 3Normalized protein levels of themPFC of rats delivered by C-sectionwith or without glob-al perinatal asphyxia.
153T. Wakuda et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 56 (2015) 149–154
increased at 12 weekswith no change at 6 weeks. These results may becompatible with our previous study employing the same perinatalmodel, in which we showed that methamphetamine-induced locomo-tor activity can be greatly increased at 12 weeks, with no change at6 weeks after birth (Wakuda et al., 2008). Dopaminergic projectionsfrom the ventral tegmental area to the cortex exhibit marked postnatalmaturation (Rosenberg and Lewis, 1995; Tseng et al., 2007). Until youngadulthood, the concentration of dopamine continues to increase in thePFC (Lambe et al., 2000; Rosenberg and Lewis, 1995). The delayed oc-currence of the increase in COMT mRNA levels may reflect functionalimpairment of thematuration of themesocortical dopaminergic projec-tions. However, the degree towhich the disturbance of themesocorticaldopaminergic system affects the methamphetamine-induced locomo-tor activity (Wakuda et al., 2008) is unclear from the current study. Ina previous postmortem study, COMTmRNA overexpression due to pro-moter hypomethylation was observed in the frontal lobes of patientswith schizophrenia (Abdolmaleky et al., 2006). Since perinatal asphyxiamay change the methylation patterns of gene promoters(Herrera-Marschitz et al., 2011; Kumral et al., 2013), epigenetic modu-lation may be involved in the increase in COMT mRNA observed in themPFC of animals experiencing perinatal asphyxia.
In a previous study, we used the same animal model of perinatal as-phyxia as used in the present study, and assessed behaviors using amethamphetamine-induced locomotion test at 6 and 12 weeks afterbirth. At 6 weeks, there was no change in the methamphetamine-induced locomotion. However, at 12 weeks, we found an elevation inmethamphetamine-induced locomotor activity (Wakuda et al., 2008).This previous observation suggests that perinatal asphyxia can causedelayed alterations in central dopamine function. The present resultssuggest a possible explanation for this phenomenon. In this study, de-creased NRG1 mRNA levels in the mPFC were observed at 6 and12 weeks after birth. Since the NRG1-ErbB signaling cascade is involvedin the regulation of neurodevelopment and neurotransmission, includ-ing via dopamine pathways (Roy et al., 2007), an interruption in the de-velopment of dopamine systems in the mPFC may begin at least fromadolescence (at 6 weeks) and persist during adulthood (at 12 weeks).The present study also showed increased COMT mRNA levels in themPFC at 12, but not 6, weeks after birth. Since COMT is critically in-volved in the degradation of synaptic dopamine (Gogos et al., 1998),an increase of dopamine turnover in the mPFC may emerge duringadulthood (at 12 weeks). Taken together, these results suggest thatthe immaturity of the mesocortical dopamine system in the peripheralasphyxia model might arise through a disturbance of the NRG1-ErbBsignaling cascade, in addition to enhancement of the COMT functionduring adulthood. Such a mechanism might be responsible for the de-layed appearance of dopamine dysfunction, as represented by the exhi-bition of aberrant behavior after methamphetamine during adulthood,but not in adolescence (Wakuda et al., 2008).
The HP is one of the brain regionswhich have been shown to be vul-nerable to hypoxia. Indeed, in our previous study using the same animalmodel, we observed a decrease in the number of dentate granule cells inthe hippocampus at 12 weeks (Wakuda et al., 2008). New neurons arecontinuously generated in the dentate gyrus throughout the adult lifeof a variety of mammals (Eriksson et al., 1998), and all of the 5 genestested here, NRG1, ErbB4, AKT1, COMT and BDNF, appear to increasethe growth and development of newly born dentate granule cells.NRG1 induces an increase in cell proliferation and migration in theadult dentate gyrus (Mahar et al., 2011), while ErbB4 plays a fundamen-tal role in controlling NGR1-induced migration (Gambarotta et al.,2004). AKT1-knockout mice have lower levels of cell proliferation inthe dentate gyrus (Balu et al., 2012), indicating that AKT1 can activateneurogenesis in dentate granule cells. It has been proposed that dopa-mine modulates the maturation of newly born dentate granule cells(Mu et al., 2011). BDNF is strongly expressed in the dentate granulecells, and increases neurogenesis in the adult dentate gyrus(Scharfman et al., 2005). In spite of evidence that the 5 genes are
particularly well-documented pro-proliferative factors, there was noapparent change inmRNA levels of any of the 5 genes. Therefore, the de-crease in the number of dentate granule cells observed in the previousstudy may have been caused by factors other than the 5 genes.
Although proteins, as the end products of gene expression, are themajor executors of biological processes, we observed no change in theprotein levels of NRG1 and COMT in the mPFC at 6 and 12 weeks. Thatthe mRNA levels of NRG1 and COMT were altered without any changein the protein levels could suggest that the protein translation isinterrupted during steady-state measurement in perinatal asphyxiamodel. A similar discordance between mRNA and protein levels hasbeen reported in schizophrenic patients (Baracskay et al., 2006). Alter-natively, the protein may be less abundant by steady-state measure-ment in a perinatal asphyxia model. Whatever the reason, themismatch in the mRNA and protein levels may still provide importantinformation in regard to the pathophysiology of the perinatal asphyxiamodel. The decreased NRG1 mRNA levels without a correspondingchange in the protein levels may have a lower rate of degradation, lead-ing to down-regulation of the NRG1 signal, while the increased COMTmRNA levels without a change in the protein levels may have a higherrate of degradation, causing the up-regulation of the COMT signal.Such altered metabolic mechanisms in the perinatal asphyxia modelmight partially contribute to the potentiated responsiveness to meth-amphetamine (Wakuda et al., 2008). Clearly, further work on the rela-tionship between mRNA levels and the corresponding proteinexpressions will be needed to gain additional insights into the detailedpathophysiology of schizophrenia.
There were limitations in the present study. The multiple compari-sons of five genes in two regions and the relatively small sample size,which might have caused type I and type II errors, respectively, renderthe data presented here preliminary. In addition,we only tested two de-velopmental periods, i.e., 6 and 12 weeks after birth. Given theneurodevelopmental hypothesis for schizophrenia and the fact thatthe genes examined here have been shown to be regulated by hypoxia(Schmidt-Kastner et al., 2006), further studies of the earlier periods ofdevelopment (from birth to 6 weeks of life) would be informative.
5. Conclusion
The findings from the current study suggest that perinatal asphyxiamay induce the alteration of both NRG1 and COMTmRNA expression inthe mPFC, which may account for the dysfunction in the mesocorticaldopaminergic system in schizophrenia. Our findings of altered gene ex-pressions associated with exposure to hypoxia during the perinatal pe-riodmay provide an explanation for the epidemiological evidence that ahistory of perinatal hypoxia is a risk factor for the development ofschizophrenia. This animal model was thus found to be useful for re-vealing the pathogenesis of schizophrenia, especially at the cellularand molecular levels.
Acknowledgments
This study was supported in part by a Grant-in-Aid for ScientificResearch (C) from the Ministry of Education, Culture, Sports, Science,and Technology of Japan (#24591702) and grants from the SENSHINMedical Research Foundation (to T.W.). The authors thank Mses. TaeTakahashi and Mika Oyaizu for their excellent technical assistance.
References
Abdolmaleky HM, Cheng KH, Faraone SV, Wilcox M, Glatt SJ, Gao F, et al. Hypomethyla-tion of MB-COMT promoter is a major risk factor for schizophrenia and bipolar disor-der. Hum Mol Genet 2006;15:3132–45.
Abe Y, Namba H, Zheng Y, Nawa H. In situ hybridization reveals developmental regulationof ErbB1-4 mRNA expression in mouse midbrain: implication of ErbB receptors fordopaminergic neurons. Neuroscience 2009;161:95–110.
154 T. Wakuda et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 56 (2015) 149–154
Aoki-Suzuki M, Yamada K, Meerabux J, Iwayama-Shigeno Y, Ohba H, Iwamoto K, et al. Afamily-based association study and gene expression analyses of netrin-G1 and -G2genes in schizophrenia. Biol Psychiatry 2005;57:382–93.
Balu DT, Carlson GC, Talbot K, Kazi H, Hill-Smith TE, Easton RM, et al. Akt1 deficiency inschizophrenia and impairment of hippocampal plasticity and function. Hippocampus2012;22:230–40.
Baquet ZC, Bickford PC, Jones KR. Brain-derived neurotrophic factor is required for the es-tablishment of the proper number of dopaminergic neurons in the substantia nigrapars compacta. J Neurosci 2005;25:6251–9.
Baracskay KL, Haroutunian V, Meador-Woodruff JH. Dopamine receptor signaling mole-cules are altered in elderly schizophrenic cortex. Synapse 2006;60:271–9.
Bertram I, Bernstein HG, Lendeckel U, Bukowska A, Dobrowolny H, Keilhoff G, et al. Im-munohistochemical evidence for impaired neuregulin-1 signaling in the prefrontalcortex in schizophrenia and in unipolar depression. Ann N Y Acad Sci 2007;1096:147–56.
Bjelke B, Andersson K, Ogren SO, Bolme P. Asphyctic lesion: proliferation of tyrosinehydroxylase-immunoreactive nerve cell bodies in the rat substantia nigra andfunctional changes in dopamine neurotransmission. Brain Res 1991;543:1–9.
Boksa P, El-Khodor BF. Birth insult interacts with stress at adulthood to alter dopaminer-gic function in animal models: possible implications for schizophrenia and otherdisorders. Neurosci Biobehav Rev 2003;27:91–101.
Byrne M, Agerbo E, Bennedsen B, EatonWW,Mortensen PB. Obstetric conditions and riskof first admission with schizophrenia: a Danish national register based study.Schizophr Res 2007;97:51–9.
Cannon M, Jones PB, Murray RM. Obstetric complications and schizophrenia: historicaland meta-analytic review. Am J Psychiatry 2002;159:1080–92.
Chen WY, Shi YY, Zheng YL, Zhao XZ, Zhang GJ, Chen SQ, et al. Case–control study andtransmission disequilibrium test provide consistent evidence for association betweenschizophrenia and genetic variation in the 22q11 gene ZDHHC8. Hum Mol Genet2004;13:2991–5.
Corfas G, Roy K, Buxbaum JD. Neuregulin 1-erbB signaling and the molecular/cellularbasis of schizophrenia. Nat Neurosci 2004;7:575–80.
Eaton WW, Byrne M, Ewald H, Mors O, Chen CY, Agerbo E, et al. Association of schizo-phrenia and autoimmune diseases: linkage of Danish national registers. Am J Psychi-atry 2006;163:521–8.
Ehrlichman RS, Luminais SN, White SL, Rudnick ND, Ma N, Dow HC, et al. Neuregulin 1transgenic mice display reduced mismatch negativity, contextual fear conditioningand social interactions. Brain Res 2009;1294:116–27.
Ekelund J, Hennah W, Hiekkalinna T, Parker A, Meyer J, Lönnqvist J, et al. Replication of1q42 linkage in Finnish schizophrenia pedigrees. Mol Psychiatry 2004;9:1037–41.
Emamian ES, Hall D, BirnbaumMJ, KarayiorgouM, Gogos JA. Convergent evidence for im-paired AKT1-GSK3beta signaling in schizophrenia. Nat Genet 2004;36:131–7.
Eriksson PS, Perfilieva E, Björk-Eriksson T, Alborn AM, Nordborg C, Peterson DA, et al.Neurogenesis in the adult human hippocampus. Nat Med 1998;4:1313–7.
Fazzari P, Paternain AV, Valiente M, Pla R, Luján R, Lloyd K, et al. Control of cortical GABAcircuitry development by Nrg1 and ErbB4 signalling. Nature 2010;464:1376–80.
Gambarotta G, Garzotto D, Destro E, Mautino B, Giampietro C, Cutrupi S, et al. ErbB4 ex-pression in neural progenitor cells (ST14A) is necessary to mediate neuregulin-1beta1-induced migration. J Biol Chem 2004;279:48808–16.
Gogos JA, Morgan M, Luine V, Santha M, Ogawa S, Pfaff D, et al. Catechol-O-methyltrans-ferase-deficient mice exhibit sexually dimorphic changes in catecholamine levels andbehavior. Proc Natl Acad Sci U S A 1998;95:9991–6.
Gong YG, Wu CN, Xing QH, Zhao XZ, Zhu J, He L. A two-method meta-analysis ofNeuregulin 1 (NRG1) association and heterogeneity in schizophrenia. SchizophrRes 2009;111:109–14.
Hahn CG, Wang HY, Cho DS, Talbot K, Gur RE, Berrettini WH, et al. Altered neuregulin1-erbB4 signaling contributes to NMDA receptor hypofunction in schizophrenia.Nat Med 2006;12:824–8.
Hashimoto R, Straub RE, Weickert CS, Hyde TM, Kleinman JE, Weinberger DR. Expressionanalysis of neuregulin-1 in the dorsolateral prefrontal cortex in schizophrenia. MolPsychiatry 2004;9:299–307.
Herrera-Marschitz M,Morales P, Leyton L, Bustamante D, Klawitter V, Espina-Marchant P,et al. Perinatal asphyxia: current status and approaches towards neuroprotectivestrategies, with focus on sentinel proteins. Neurotox Res 2011;19:603–27.
Jaaro-Peled H, Hayashi-Takagi A, Seshadri S, Kamiya A, Brandon NJ, Sawa A.Neurodevelopmental mechanisms of schizophrenia: understanding disturbed post-natal brain maturation through neuregulin-1-ErbB4 and DISC1. Trends Neurosci2009;32:485–95.
Kato T, Kasai A, Mizuno M, Fengyi L, Shintani N, Maeda S, et al. Phenotypic characteriza-tion of transgenic mice overexpressing neuregulin-1. PLoS One 2010;5:e14185.
Kumral A, Tuzun F, Tugyan K, Ozbal S, Yılmaz O, Yesilirmak CD, et al. Role of epigeneticregulatory mechanisms in neonatal hypoxic-ischemic brain injury. Early Hum Dev2013;89:165–73.
Lambe EK, Krimer LS, Goldman-Rakic PS. Differential postnatal development of catechol-amine and serotonin inputs to identified neurons in prefrontal cortex of rhesusmonkey. J Neurosci 2000;20:8780–7.
Law AJ, Lipska BK, Weickert CS, Hyde TM, Straub RE, Hashimoto R, et al. Neuregulin 1transcripts are differentially expressed in schizophrenia and regulated by 5′ SNPs as-sociated with the disease. Proc Natl Acad Sci U S A 2006;103:6747–52.
Law AJ, Kleinman JE, Weinberger DR, Weickert CS. Disease-associated intronic variants inthe ErbB4 gene are related to altered ErbB4 splice-variant expression in the brain inschizophrenia. Hum Mol Genet 2007;16:129–41.
Lichtenstein P, Yip BH, Bjork C, Pawitan Y, Cannon TD, Sullivan PF, et al. Common geneticdeterminants of schizophrenia and bipolar disorder in Swedish families: apopulation-based study. Lancet 2009;373:234–9.
Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quanti-tative PCR and the 2−ΔΔCT method. Methods 2001;25:402–8.
Mahar I, Tan S, Davoli MA, Dominguez-Lopez S, Qiang C, Rachalski A, et al. Subchronic pe-ripheral neuregulin-1 increases ventral hippocampal neurogenesis and inducesantidepressant-like effects. PLoS One 2011:e26610.
Mei L, XiongWC. Neuregulin 1 in neural development, synaptic plasticity and schizophre-nia. Nat Rev Neurosci 2008;9:437–52.
Mu Y, Zhao C, Gage FH. Dopaminergic modulation of cortical inputs during maturation ofadult-born dentate granule cells. J Neurosci 2011;31:4113–23.
MunafòMR, Attwood AS, Flint J. Neuregulin 1 genotype and schizophrenia. Schizophr Bull2008;34:9–12.
Murray RM. Neurodevelopmental schizophrenia: the rediscovery of dementia praecox. BrJ Psychiatry Suppl 1994:6–12.
Nadri C, Belmaker RH, Agam G. Oxygen restriction of neonate rats elevates neuregulin-1alpha isoform levels: possible relationship to schizophrenia. Neurochem Int 2007;51:447–50.
Nicodemus KK, Law AJ, Radulescu E, Luna A, Kolachana B, Vakkalanka R, et al. Biologicalvalidation of increased schizophrenia risk with NRG1, ERBB4, and AKT1 epistasisvia functional neuroimaging in healthy controls. Arch Gen Psychiatry 2010;67:991–1001.
Numakawa T, Yagasaki Y, Ishimoto T, Okada T, Suzuki T, Iwata N, et al. Evidence of novelneuronal functions of dysbindin, a susceptibility gene for schizophrenia. Hum MolGenet 2004;13:2699–708.
O'Tuathaigh CM, Babovic D, O'Sullivan GJ, Clifford JJ, Tighe O, Croke DT, et al. Phenotypiccharacterization of spatial cognition and social behavior in mice with ‘knockout’ ofthe schizophrenia risk gene neuregulin 1. Neuroscience 2007;147:18–27.
O'Tuathaigh CM, Harte M, O'Leary C, O'Sullivan GJ, Blau C, Lai D, et al. Schizophrenia-related endophenotypes in heterozygous neuregulin-1 ‘knockout’ mice. Eur JNeurosci 2010;31:349–58.
Paxinos G, Watson C. The rat brain in stereotaxic coordinates. 3rd ed. New York: Academ-ic Press; 1997.
Rosenberg DR, Lewis DA. Postnatal maturation of the dopaminergic innervation of mon-key prefrontal and motor cortices: a tyrosine hydroxylase immunohistochemicalanalysis. J Comp Neurol 1995;358:383–400.
Roy K, Murtie JC, El-Khodor BF, Edgar N, Sardi SP, Hooks BM, et al. Loss of erbB signaling inoligodendrocytes alters myelin and dopaminergic function, a potential mechanismfor neuropsychiatric disorders. Proc Natl Acad Sci U S A 2007;104:8131–6.
Scharfman H, Goodman J, Macleod A, Phani S, Antonelli C, Croll S. Increased neurogenesisand the ectopic granule cells after intrahippocampal BDNF infusion in adult rats. ExpNeurol 2005;192:348–56.
Schizophrenia Psychiatric Genome-Wide Association Study (GWAS) Consortium.Genome-wide association study identifies five new schizophrenia loci. Nat Genet2011;43:969–76.
Schmidt-Kastner R, van Os J, Steinbusch HWM, Schmitz C. Gene regulation by hypoxiaand the neurodevelopmental origin of schizophrenia. Schizophr Res 2006;84:253–71.
Silberberg G, Darvasi A, Pinkas-Kramarski R, Navon R. The involvement of ErbB4 withschizophrenia: association and expression studies. Am J Med Genet B NeuropsychiatrGenet 2006;141B:142–8.
Thiselton DL, Vladimirov VI, Kuo PH, McClay J, Wormley B, Fanous A, et al. AKT1 is asso-ciated with schizophrenia across multiple symptom dimensions in the Irish study ofhigh density schizophrenia families. Biol Psychiatry 2008;63:449–57.
Tseng KY, Lewis BL, Lipska BK, O'Donnell P. Post-pubertal disruption of medial prefrontalcortical dopamine–glutamate interactions in a developmental animal model ofschizophrenia. Biol Psychiatry 2007;62:730–8.
Wakuda T, Matsuzaki H, Suzuki K, Iwata Y, Shinmura C, Suda S, et al. Perinatal asphyxiareduces dentate granule cells and exacerbates methamphetamine-inducedhyperlocomotion in adulthood. PLoS One 2008;3:e3648.
Yamada K, Nakamura K, Minabe Y, Iwayama-Shigeno Y, Takao H, Toyota T, et al. Associa-tion analysis of FEZ1 variants with schizophrenia in Japanese cohorts. Biol Psychiatry2004;56:683–90.
Zheng Y,Watakabe A, TakadaM, Kakita A, Namba H, Takahashi H. Expression of ErbB4in substantia nigra dopamine neurons of monkeys and humans. ProgNeuropsychopharmacol Biol Psychiatry 2009;33:701–6.
