Phencyclidine Rapidly Decreases NeuronalmRNA of Brain-Derived Neurotrophic

FactorYUSUKE KATANUMA,1,2 TADAHIRO NUMAKAWA,1,3* NAOKI ADACHI,1,3 NORIKO YAMAMOTO,1

YOSHIKO OOSHIMA,1 HARUKI ODAKA,1,2 TAKAFUMI INOUE,2 AND HIROSHI KUNUGI1,3

1Department of Mental Disorder Research, National Institute of Neuroscience, National Center of Neurology andPsychiatry, Tokyo, Japan

2Department of Life Science and Medical Bioscience, School of Advanced Science and Engineering,Waseda University, Tokyo, Japan

3Core Research for Evolution Science and Technology Program (CREST), Japan Science and Technology Agency(JST), Tokyo, Japan

KEY WORDS phencyclidine; BDNF; NMDA receptor; schizophrenia

ABSTRACT Downregulation of brain-derived neurotrophic factor (BDNF), a mem-ber of neurotrophin family, has been implicated in psychiatric diseases including schiz-ophrenia. However, detailed mechanisms of its reduction in patients withschizophrenia remain unclear. Here, using cultured cortical neurons, we monitoredBDNF mRNA levels following acute application of phencyclidine [PCP; an N-methyl-D-aspartate (NMDA) receptor blocker], which is known to produce schizophrenia-likesymptoms. We found that PCP rapidly caused a reduction in total amount of BDNFtranscripts without effect on cell viability, while mRNA levels of nerve growth factorwas intact. Actinomycin-D (ActD), an RNA synthesis inhibitor, decreased total BDNFmRNA levels similar to PCP, and coapplication of ActD with PCP did not show furtherreduction in BDNF mRNA compared with solo application of each drug. Among BDNFexons I, IV, and VI, the exon IV, which is positively regulated by neuronal activity, washighly sensitive to PCP. Furthermore, PCP inactivated cAMP response element-binding protein (CREB; a regulator of transcriptional activity of exon IV). The inacti-vation of CREB was also achieved by an inhibitor for Ca21/calmodulin kinase II (CaM-KII), although coapplication with PCP induced no further inhibition on the CREBactivity. It is possible that PCP decreases BDNF transcription via blocking the NMDAreceptor/CaMKII/CREB signaling. Synapse 68:257–265, 2014. VC 2014 Wiley Periodicals, Inc.

INTRODUCTION

Schizophrenia affects about 1% of the populationand is characterized by hallucinations, delusions (pos-itive-symptoms), emotional dullness (negative-symp-toms), and cognitive deficits (Favalli et al., 2012).Importantly, it has been reported that dysfunction ofthe prefrontal cortex (PFC) is closely related to theschizophrenia-like behaviors. Cognitive performancedeficits in schizophrenia patients result, at least inpart, from impaired function of the PFC (Weinbergeret al., 1986). Abnormal glutamatergic neurotransmis-sion in the PFC has been suggested to be involved inthe pathology of schizophrenia (Frankle et al., 2003).

Phencyclidine (PCP), a noncompetitive antagonistfor N-methyl-D-aspartate (NMDA) receptor, caninduce positive and negative symptoms in humans(Domino and Luby, 2012). Because PCP-treatedrodents also show schizophrenia-like behaviors

(Domino and Luby, 2012; Noda et al., 1995), they arewidely used as an animal model of schizophrenia.Notably, adverse effects of PCP on neuronal functionas well as behavior are evident (Domino and Luby,

Contract grant sponsor: Health and Labor Sciences Research Grants (Com-prehensive Research on Disability, Health, and Welfare H21-kokoro-002, H.K.); Contract grant sponsor: Core Research for Evolutional Science and Tech-nology Program and CREST, Japan Science and Technology Agency (JST, T.N.,N.A. and H.K.); Contract grant sponsor: Takeda Science Foundation (T. N.);Contract grant sponsor: Grant-in-Aid for Scientific Research (B); Contractgrant number: JSPS KAKENHI 24300139 (T. N.); Contract grant sponsor:Grant-in-Aid for Challenging Exploratory Research; Contract grant number:JSPS KAKENHI 25640019 (T. N.); Contract grant sponsor: Ministry of Educa-tion, Culture, Sports, Science, and Technology of Japan.

*Correspondence to: Tadahiro Numakawa, Ph.D., Department of MentalDisorder Research, National Institute of Neuroscience, National Center ofNeurology and Psychiatry, 4-1-1 Ogawa-Higashi, Kodaira, Tokyo 187–8502,Japan. E-mail: numakawa@ncnp.go.jp

Received 26 September 2013; Accepted 7 February 2014

DOI: 10.1002/syn.21735

Published online 12 February 2014 in Wiley Online Library(wileyonlinelibrary.com).

� 2014 WILEY PERIODICALS, INC.

SYNAPSE 68:257–265 (2014)

2012; Wang et al., 2001). Continuous administrationof PCP caused neurodegeneration in the posteriorentorhinal cortex, ventral dentate gyrus, and cingu-late cortex (Ellison and Switzer, 1993). The numberof dendritic spines in the rat PFC was decreasedafter subchronic PCP administration (Hajszan et al.,2006). Consistently, a significant decrease in immuno-reactivity of synaptophysin, one of synaptic vesicleproteins, in the PFC of patients with schizophreniawas confirmed (Glantz and Lewis, 1997).

Brain-derived neurotrophic factor (BDNF) isbroadly expressed in the central nervous system(CNS) and has critical roles in neuronal survival andmaturation, neurotransmitter release, and synapticplasticity via stimulating several intracellular signal-ing pathways including phosphoinositide 3-kinase/Akt (PI3K/Akt), extracellular signal-regulated kinase(ERK), and phospholipase C-g pathways (Huang andReichardt, 2003; Minichiello et al., 2009; Numakawaet al., 2013; Russo et al., 2009). Reduced BDNF func-tion and/or expression are suggested to be involved inthe pathogenesis of a variety of brain diseases suchas major depression and schizophrenia (Angelucciet al., 2005; Favalli et al., 2012; Javitt and Zukin,1991; Karege et al., 2002). Functional magnetic reso-nance imaging and postmortem studies have reporteddecreased expression levels of BDNF mRNA and pro-tein in the PFC of schizophrenia patients (Callicottet al., 2000; Weickert et al., 2003), suggesting thatdecreased cortical BDNF function may contribute tothe development of schizophrenia, although molecu-lar mechanisms underlying such a reduction inBDNF expression have not been fully elucidated. Onestudy has shown that BDNF mRNA expression invarious brain regions including the PFC was reducedby subchronic PCP administration in female rats(Snigdha et al., 2011).

As both PCP-mediated neuronal damage andBDNF dysfunction are key concerns to investigateschizophrenia-like behaviors, it is intriguing to clarifythe relationship between the two molecules. Werecently found that a marked impairment of synapticfunction occurs due to reduced secretion of BDNFprotein after subchronic PCP treatment in culturedcortical neurons (Adachi et al., 2013). In the presentstudy, we investigated an effect of PCP on BDNFtranscription and found a rapid decrease of BDNFmRNA via suppression of neural activity-mediatedmechanisms.

MATERIALS AND METHODSPreparation of primary cortical neurons

The cerebral cortex was removed from postnatal 1-to 2-day old Wistar rats (SLC, Shizuoka, Japan), anddissociated cultures were prepared as described inour previous report (Numakawa et al., 2009). Briefly,cortical tissues were digested with papain solution,

and then the dissociated cortical neurons in culturemedia, which consists of 1:1 mixture of Dulbecco’smodified Eagle’s medium and Ham’s F-12 medium,5% fetal bovine serum (FBS), 5% heated-inactivatedhorse serum, and penicillin–streptomycin (penicillin:18 units/mL, streptomycin: 18 mg/mL) were plated onpolyethylenimine-coated 3.5 cm dishes or plates (BDFalcon, CA). All experiments were conducted accord-ing to the laboratory animals ethical guidelines of theNational Institute of Neuroscience, National Centerof Neurology and Psychiatry, Japan.

For astrocyte culture, cortical cells were plated ona noncoated 75 cm2 flask (Corning, NY). Cell mainte-nance was performed with minimum essentialmedium-based growth medium (containing 100 mg/Lepidermal growth factor, 5% FBS, and 0.5 mM gluta-mine), and astroglial cells were replated on 3.5 cmdishes before experiments.

Drug treatment

Cultured cortical neurons at 11–12 days in vitro or60–80% confluent astrocytes were treated with PCP(Sigma-Aldrich, MO), actinomycin D (ActD, Sigma-Aldrich, MO), D-(2)-amino-5-phosphonopentanoic acid(D-APV, Tocris Bioscience, UK), MK801 (Sigma-Aldrich, MO), KN-93 (5 mM, Calbiochem, CA),KT5720 (200 nM, Calbiochem), and K252a (200 nM,Sigma-Aldrich, MO). Except for the examination ofdose- or time-dependent effect, PCP, ActD, and D-APVwere applied for 3 h at 1, 1, and 50 mM, respectively.All drugs were applied to cultured cells after dilutionwith water or dimethyl sulfoxide and correspondingvehicle was treated as control.

Total RNA extraction, reverse transcription,and quantitative PCR

Total RNA extraction was conducted using mirVa-naTM miRNA Isolation Kit (Life Technologies, CA)according to the manufacture’s protocol. Reverse tran-scription reaction (25�C, 10 min, 42�C, 60 min, and85�C, 5 min) with the same amount of RNA was per-formed using GeneAmp polymerase chain reaction(PCR) System 9700 (Applied Biosystems, CA). Theobtained complementary DNA was determined by thequantitative PCR with ABI Prism 7000 (Applied Bio-systems). After an initial denaturation step at 95�C for10 min, 40 PCR cycles of denaturation at 95�C for 15 sand annealing/extension at 60�C for 1 min were done.Amount of each mRNA was determined by calculatingfrom the obtained threshold cycle. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was usedfor normalization. The primer and probe sets (TaqManGene Expression Assays from Applied Biosystems)were as follows: BDNF exon I: Rn01484924_m1,BDNF exon IV: Rn01484927_m1, BDNF exon VI:Rn01484928_m1, total BDNF: Rn02531967_s1,Microtubule-associated protein 2 (MAP2):

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Rn00565046_m1, nerve growth factor (NGF):Rn01533872_m1, GAPDH: 4352338E.

MTT assay

After aspiration of culture medium, 3-(4,5-di-meth-ylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, tetra-zole (MTT) diluted in fresh medium was applied tocortical neurons and incubated for 1.5–2.5 h at 37�C.Then lysis buffer including sodium dodecyl sulfate(SDS) (0.1 g/mL) was added for 1 h. The absorbanceat 550 nm was measured by a microplate reader (Bio-Rad Laboratories, CA).

Western blotting

Immunoblotting was performed as previouslyreported (Numakawa et al., 2009). The same amountof protein was electrophoresed in the SDS-polyacrylamide gel and transferred to a membrane. After block-ing with 5% skim milk for 1–2 h, anti-CREB (1:500,Cell Signaling Technology, MA), anti-pCREB (1:500,Cell Signaling Technology, MA), anti-NR2A (1:500,Sigma-Aldrich, MO), anti-NR2B (1:500, Sigma-Aldrich, MO), anti-GluR1 (1:500, Chemicon Interna-tional, CA), or anti-bactin antibody (as a control pro-tein, 1:5000, Sigma-Aldrich, MO) was incubatedovernight at 4�C. Observation of immunoreactivityand data analysis was performed with Ez-capture con-trolled by ImageSaver5 software and CS Analyzer 3.0software, respectively (ATTO, Tokyo, Japan).

Statistical analysis

The experimental data were expressed as mean-6 standard deviation (SD). The statistical significancewas evaluated with Student’s t-test, or one- or two-way analysis of variance (ANOVA) followed by Bon-ferroni post hoc test in SPSS ver.18 (IBM, Japan) andconsidered to be significant when the probabilityvalue was less than 5%.

RESULTSPCP rapidly decreased total BDNF mRNA in

cultured cortical neurons

To examine the influence of PCP on BDNF mRNAlevels, matured cortical neurons at DIV11–12 wereexposed to PCP at final concentrations of 0.1–10 mMfor 3 h. As shown in Figure 1A, 1 and 10 mM of PCPsignificantly reduced total BDNF mRNA levels. Atime-course analysis of the PCP effect (1 mM) alsorevealed significant reductions in the total BDNFmRNA after 1, 3, and 6 h PCP treatment (Fig. 1B).When an influence of PCP on BDNF mRNA expres-sion in pure astroglial culture was examined, no sig-nificant change was observed (Fig. 1C), indicatingthat the observed BDNF mRNA reduction by PCP isattributable to neuronal response. To determinewhether the PCP-induced reduction in total BDNFmRNA immediately affects neuronal viability or not,

the MTT assay was conducted. PCP did not alter cellviability at any dose examined (0.1–10 mM, 3 h; Fig.1D). To see specificity of the PCP effect in corticalneurons, MAP2 and NGF mRNA levels after PCPtreatment (1 mM, 3 h) were determined. Both MAP2and NGF mRNA expressions were unchanged byPCP while BDNF mRNA was affected (Fig. 1E).

PCP inhibited transcriptional activityin BDNF exon IV

The decreased total BDNF mRNA caused by PCPis attributable to suppression of transcriptional activ-ity and/or acceleration of degradation. Therefore,next we examined the possible effect of ActD, a tran-scription inhibitor. Importantly, ActD also decreasedBDNF mRNA expression in a similar time course asPCP (see Figs. 1B and 2A). The degree of change intotal BDNF mRNA levels after coapplication of ActDwith PCP (1 mM, 3 h) was not significantly differentfrom that after solo application of each drug(Fig. 2B). There was a significant interaction betweenPCP and ActD treatments (two-way ANOVAF(1,20) 5 19.3, P< 0.001). If PCP promoted degrada-tion of mRNA, an additional reduction in levels ofBDNF mRNA would have been achieved by the coap-plication. These data, therefore, suggested that PCPdiminishes the amount of BDNF mRNA mainly viarepressing transcriptional activity. To confirm contri-bution of NMDA receptor activity to the reducedBDNF mRNA levels, other inhibitors for NMDAreceptors were tested. Three-hour treatment with D-APV (a competitive antagonist) at the concentrationof 50 mM, the dose of which abolishes NMDAreceptor-mediated current (Liu et al., 2004), caused acomparable reduction in total BDNF mRNA to that ofPCP (1 mM, 3 h) or ActD (1 mM, 3 h; Fig. 2C), imply-ing that impairment in the NMDA receptor-mediatedtranscription is a major contributor to the decrease intotal BDNF mRNA. MK-801, a noncompetitiveNMDA receptor antagonist, also showed a significantinhibitory effect on BDNF mRNA levels (Fig. 2D).

The rodent BDNF gene has at least nine promoters(BDNF pI–IX) and nine exons (BDNF exon I–IX, Aidet al., 2007). As exon I and IV are positively regu-lated by neuronal activity (Pruunsild et al., 2011), weexamined BDNF transcripts containing these exons.In addition, we measured the exon VI transcriptwhich was assumed to be activity independent. Wefound that the basal levels of exon IV transcript weremuch higher than that of exon I or VI in cortical cul-tures (Fig. 2E). Conversely, exon VI transcript wasthe major population in astroglial cell cultures (Fig.2F). We then investigated effects of PCP, ActD, andD-APV on expression levels of exon I, IV, and VI tran-scripts. In contrast to the total BDNF mRNA, eachexon-containing transcript showed various degrees ofreduction in response to these drugs. Regarding exon

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I and VI, ActD strongly reduced their transcript lev-els while weaker suppression was observed in PCP orD-APV application (Fig. 2G). For exon IV, by contrast,PCP, D-APV, and ActD similarly and dramaticallydecreased its transcript (Fig. 2G). Effect of PCP oneach exon-containing transcript was also determinedin astrocytes. As shown in Figure 2H, the amount ofexon I, IV, or VI transcript in pure astroglial culturesshowed no significant change after PCP application.

PCP suppressed CaMKII-CREB system

As PCP putatively acts on NMDA receptors, weexamined endogenous expressions of NR2A, NR2B(NMDA receptor subunits), and GluR1 (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptorsubunit) in our cortical neurons. We found thatexpressions of these glutamate receptors graduallyincreased during in vitro maturation (Fig. 3A–D),indicating an adequate amount of glutamate

Fig. 1. Effect of PCP on total mRNA expression of BDNF. (A)PCP (0.1–10 mM) was added to cortical neurons at DIV11–12. TotalBDNF mRNA levels were determined 3 h after PCP treatment.(n 5 5–6, n indicates the number of culture dishes in each experi-mental condition). *P< 0.05, **P< 0.01 by one-way ANOVA followedby Bonferroni post-hoc test compared with con (vehicle treated). (B)Time dependency of PCP effect (1 mM) was examined. The amountof total BDNF mRNA in cortical neurons was gradually decreasedduring 6-h treatment. (n 5 3–4) *P< 0.05, **P< 0.01 by one-way

ANOVA followed by Bonferroni post-hoc test compared with con. (C)PCP (1 mM, 3 h) did not change BDNF mRNA expression in astro-glial cells. (n 5 6). (D) Cell survival was not affected by PCP (0.1–10mM, 3 h). The MTT assay was conducted to determine cell viabilityof cultured cortical neurons. (n 5 6). (E) Expression levels of BDNF,MAP2, and NGF mRNAs after PCP (1 mM, 3 h) treatment. No alter-ation in mRNAs of both MAP2 and NGF was observed. (n 5 5–6).***P< 0.001 by Student’s t-test. All data represent mean 6 SD.

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Fig. 2. PCP decreased BDNF mRNA containing exon IV viainhibiting transcriptional activity. (A) Time course analysis of totalBDNF mRNA expression after ActD (a transcription inhibitor)treatment. ActD (1 mM) was applied to cultured cortical neurons(n 5 4). Note that ActD decreased BDNF mRNA levels in a similartime course as PCP application (see Fig. 1B). *P< 0.05, ***P< 0.001by one-way ANOVA followed by Bonferroni post-hoc test comparedwith con. (B) Cotreatment of ActD (1 mM) with PCP (1 mM) for 3 hwas performed. Additional or synergistic decrease in total BDNFmRNA levels was not detected. (n 5 6). ***P< 0.001 by two-wayANOVA followed by Bonferroni post hoc test compared with con.n.s. means not significant difference between the PCP (or ActD)solo application and coapplication with both drugs. (C) Exposure toActD or D-APV (NMDA receptor antagonist, 50 mM, 3 h) caused thesimilar reduction in total BDNF mRNA as PCP. (n 5 5–6). **P< 0.01

by one-way ANOVA followed by Bonferroni post hoc test comparedwith con. (D) Effect of MK-801, a noncompetitive NMDA receptorblocker, on total BDNF mRNA levels. (n 5 5–6). *P< 0.05 by one-way ANOVA followed by Bonferroni post hoc test compared withcon. (E) The basal levels of BDNF exon IV transcript were higher incortical neurons than that of exon I or VI. (n 5 6). (F) Astroglialexon I and IV transcripts levels were very low in comparison withexon VI. (n 5 6). (G) The amount of BDNF exon I, IV, and VI tran-scripts in cortical neurons were decreased by PCP, ActD, or D-APVapplication. Negative effect on exon IV transcript was similarlyexerted by solo application of PCP, ActD, or D-APV. (n 5 3–6).*P< 0.05, ***P< 0.001 by one-way ANOVA followed by Bonferronipost hoc test compared with con. (H) Glial BDNF exon I, IV, and VItranscripts were unchanged by PCP. (n 5 6). All data representmean 6 SD.

Fig. 3. PCP reduced phosphorylation of CREB. (A) Expressionlevels of GluR1, NR2A, and NR2B were gradually increased duringin vitro development. Quantification of developmental changes inthe expression of GluR1 (B), NR2A (C), and NR2B (D), were con-ducted. (n 5 5). (E) Levels of pCREB (phosphorylation form) andtotal CREB after PCP exposure (1 mM, 3 h) were analyzed by west-ern blotting. Reduced pCREB was observed after PCP stimulationwhile total CREB expression was not altered. (F) Quantification ofthe ratio of pCREB to total CREB levels was shown. (n 5 4).**P< 0.01 by Student’s t-test. (G and H) Cotreatment of KN-93

(a CaMKII inhibitor, 5 mM) with PCP (1 mM) for 3 h was deter-mined, n 5 6. **P< 0.01, ***P< 0.001 by two-way ANOVA followedby Bonferroni post hoc test compared with con. n.s. means not sig-nificant difference between PCP (or KN-93) solo application and thecoapplication with both drugs. (I and J) Coapplication of KT5720 (aPKA inhibitor, 200 nM) with 1 mM PCP for 3 h. ***P< 0.001 by two-way ANOVA followed by Bonferroni post hoc test compared withcon. n.s. means not significant difference between con (vehicletreated) and KT5720 solo application, and PCP solo application andthe coapplication with both drugs. All data represent mean 6 SD.

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receptors in neuronal cultures (at DIV 11) to beblocked by PCP. Since the promoter region of BDNFexon IV has a cAMP-response element (CRE) sitewhere phosphorylated CREB (pCREB, an activatedCREB) binds to stimulate transcription from the exon(Shieh et al., 1998; Tao et al., 1998), we examined thelevels of pCREB with or without PCP application. Asexpected, pCREB was reduced by 3-h PCP treatmentwhile total CREB was unchanged (Fig. 3E). The ratioof pCREB to total CREB displayed a markeddecrease by PCP (Fig. 3F). Some reports indicate thatCaMKII and/or protein kinase A (PKA) function asupstream regulators for CREB activation (Ebertet al., 2013; Zheng et al., 2011). Indeed, KN-93 (aninhibitor for CaMKII, 5 mM) significantly decreasedlevels of pCREB, although additional reduction ofpCREB was not caused by its coapplication with PCP(Fig. 3G and 3H). There was a significant interactionbetween PCP and KN-93 treatments (two-wayANOVA F(1,20) 5 4.7, P< 0.05). In contrast, KT5720(200 nM), a PKA inhibitor, has no effect on pCREBlevels in the presence or absence of PCP (Fig. 3I and3J). There was no significant interaction betweenPCP and KT5720 treatments (two-way ANOVAF(1,16) 5 0.006, P> 0.05). These data suggest thatPCP decreased CREB activation via repressing activ-ity of NMDA receptor/CaMKII signaling.

DISCUSSION

We found that PCP rapidly decreased total BDNFmRNA levels within 1 h via inhibiting transcriptionactivity in cultured cortical neurons. To our knowl-edge, such an acute effect of PCP on BDNF mRNA hasnot been reported. Among BDNF exon transcripts ana-lyzed in this study, activity-dependent exon IV tran-script was dominant in cortical neurons and wasstrongly suppressed by PCP as well as other NMDAreceptor blockers (D-APV and MK801). Furthermore,cotreatment with PCP and CaMKII inhibitor similarlycaused suppression of pCREB levels compared withsolo treatment with each drug, suggesting that PCPinfluenced total BDNF mRNA expression mainly byrepression of the exon IV-derived transcriptionthrough blockade of NMDA receptor/CREB function.

It is suggested that expression/secretion of BDNFis regulated by neuronal activity (Lessmann andBrigadski, 2009). Because CREB can be phosphoryl-ated in the downstream of BDNF/TrkB (receptor forBDNF) signaling pathway (Finkbeiner et al., 1997),the downregulation of pCREB by PCP might be indi-rect phenomenon due to decreased BDNF productionand/or secretion. However, K252a (an inhibitor of Trkkinases, 200 nM, 3 h,) had no influence on pCREBlevels (94.7 6 18.0 % of control, P> 0.05), suggestingthat secreted BDNF in the basal condition would notaffect CREB activity in our system. Therefore, it is

possible that PCP inhibits CREB system directly viarepressing intracellular signaling.

Ca21 influx through NMDA receptors and L-typevoltage-sensitive calcium channels stimulate multiplecalcium-dependent signaling molecules includingCaMKs, PKA, and mitogen-activated protein kinase(Ebert et al., 2013). In the present study, a CaMKIIinhibitor KN-93 suppressed basal CREB phosphoryla-tion although the coapplication of the inhibitor withPCP had no additional inhibitory effect. Conversely,KT5720, a PKA inhibitor, had no significant effect onthe CREB phosphorylation. Zheng et al. (2011)showed that overexpression of dominant negativeform of PKA in cultured cortical neurons slightlyreduced the NMDA-mediated activity of BDNF pro-moter IV. Therefore, PKA activity would not contrib-ute to the BDNF exon IV transcription under thebasal neuronal activity level.

In our cultures, 1 mM PCP significantly suppressedactivity of BDNF transcription. Based on pharmaco-kinetic analysis, Proksch et al. estimated that humanserum PCP concentrations in emergency roompatients after PCP overdose (Walberg et al., 1983)are approximately 0.2–3.5 mg/kg (Proksch et al.,1998). Generally, to make animal models of schizo-phrenia, 1–10 mg/kg PCP injection are performed tocause schizophrenia-like behavioral changes inrodents (see review Mouri et al., 2007). Furthermore,intravenous (i.v.) injection of 1 mg/kg PCP producedtransient and high concentration of PCP (�10 mM) inthe rat brain, which was about 10 times higher thanits serum concentration (Proksch et al., 2000). Takentogether, 1 mM PCP-induced suppression of BDNFtranscription possibly occurs in both rodent andhuman brain after PCP administration.

A previous study using MK-801 also showeddecreased BDNF mRNA levels in cultured neurons(Zafra et al., 1991). However, both increased anddecreased BDNF levels in the rat brain after PCPinjection have been reported. Increased BDNF pro-tein levels were observed in the adult rat cortex afteracute and chronic PCP administration (10 mg/kg, sin-gle or repeated injection for 14 days, respectively;Takahashi et al., 2006) and in the hippocampus (5mg/kg, bidaily for 7 days; Harte et al., 2007). In con-trast, reduced levels of BDNF mRNA in hippocampaland cortex of adult female rats were observed even 6weeks after chronic PCP treatment (twice a day for 7days, 2 mg/kg; Snigdha et al., 2011). Semba et al.showed that acute PCP (5, 10 mg/kg, 24 h) adminis-tration decreased the amount of hippocampal BDNFprotein only in the neonatal (postnatal day 15) butnot in the adult (postnatal day 48) rats (Semba et al.,2006). Considering these results, our system usingprimary neuronal cultures could be beneficial to pre-dict what occurs in the developing brain tissue afteracute PCP administration.

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In contrast to BDNF mRNA, NGF, and MAP2mRNAs were not affected by PCP, suggesting thatregulation systems of these NGF and MAP2 genesare different. A transcriptional factor CCAAT/enhancer-binding protein d (C/EBPd) binds to a pro-moter region of NGF and stimulates transcriptionafter b2-adrenergic receptor activation (McCauslinet al., 2006). A putative CRE site within the NGFpromoter region and a cooperative transcriptionalregulation between C/EBPd and CREB were alsodemonstrated. Given that NGF mRNA levels showedno change after PCP exposure, such C/EBPd=CREBcooperative system may not be active in the basalstate of cortical neurons. Although MAP2 mRNA con-tains the cytoplasmic polyadenylation element (CPE)site in the 30-UTR by which it is targeted to dendritesby CPE-binding protein after NMDA receptor activa-tion (Huang et al., 2003), its transcriptional activitymight not be mediated by NMDA receptor function.

CREB has crucial roles in the CNS including synap-tic regulation and consolidation of memory (Benito andBarco, 2010; Silva et al., 1998). pCREB levels werereduced in the PFC of PCP-treated rats (20 mg/kg, 14days; Molteni et al., 2008) and in cultured organotypiccorticostriatal slices (3 mM, 2–8 h; Xia et al., 2010). Con-sistent with these data, our results showed decreasedpCREB by PCP (1 mM, 3 h) in cortical neurons. Interest-ingly, in our system, PCP decreased BDNF exon IVtranscript by 80%, while exon I, VI transcripts byaround 40%. It has been reported that the promoter IVcontains CRE site and its regulation depends on theneuronal activity via CREB function. Using BDNF-BAC (bacterial artificial chromosome) mice, Timmuskand colleagues have reported that depolarization of pri-mary mouse cortical neurons induced transcriptionfrom BDNF exon I and IV of both human and mousegenes, though exon VI transcript was not stimulated(Pruunsild et al., 2011). Of note, a mutation of a puta-tive CRE site in the BDNF promoter I failed to cancelthe depolarization-dependent increase in exon I tran-script (Pruunsild et al., 2011). These data includingours suggest that the CREB-dependent transcriptionusing exon IV is constitutively activated in cortical sys-tem, and that an acute suppression of basal NMDAfunction (for example, by PCP administration) isenough to cause a downregulation of BDNF transcript,although CREB-independent mechanism may also beinvolved in the negative regulation by PCP.

Recently, it has been shown that mutant mice lack-ing BDNF promoter IV exhibit an impairment ofinhibitory synaptic transmission in the PFC (Sakataet al., 2009). Considering that impairment of theactivity-dependent transcription of BDNF causes dys-function of synaptic plasticity, unveiling the mecha-nisms underlying the transcriptional downregulationof BDNF using our in vitro system might be useful tounderstand the molecular basis of schizophrenia.

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