Prenatal Phencyclidine Exposure AltersHippocampal Cell Proliferation in

Offspring RatsATSUSHI TANIMURA,1 JUAN LIU,1,2 TAKASHI NAMBA,3 TATSUNORI SEKI,4 YOICHIRO MATSUBARA,1

MASANOBU ITOH,1 TOSHIHITO SUZUKI,1* AND HEII ARAI11Department of Psychiatry, Juntendo University School of Medicine, Bunkyo, Tokyo, Japan2Department of Human Anatomy, Ning Xia Medical College, Yin Chuan, Ning Xia, China3Department of Neurochemistry, National Institute of Neuroscience, Kodaira, Tokyo, Japan

4Division of Developmental Neuroscience, Center for Translational and Advanced Animal Research (CTAAR),Tohoku University Graduate School of Medicine, Sendai, Miyagi, Japan

KEY WORDS phencyclidine; drug abuse; prenatal stress; hippocampal neurogene-sis; neonatal rats; CNS development

ABSTRACT Multiple case reports have described pregnancy in phencyclidinehydrochloride (PCP) abusers. Characteristic clinical symptoms of PCP-exposed infantshave revealed neurobehavioral or physical abnormalities. We designed this study toevaluate whether chronic prenatal exposure to PCP during the last 2 weeks of gesta-tion in rats produces alterations of hippocampal neurogenesis in offspring. Ratsreceived repeated subcutaneous injection of PCP (5 mg/kg) once daily during the last 2weeks of gestation. Control animals received subcutaneous injection of physiologicalsaline during gestation. Dams receiving repeated PCP administrations showed mark-edly increased locomotor activities on days 1, 5, and 10 during the last 2 weeks of ges-tation. At 21 days after birth, 5-bromo-20-deoxyuridine (BrdU)-positive cells of off-spring were counted in the granule cell layer (GCL) and subgranular zone of the den-tate gyrus. The numbers of BrdU-positive cells in the GCL in male and femaleoffspring of the PCP-treated group were significantly increased by �77% comparedwith those from the control group. At 56 days, the number of surviving BrdU-positivecells also remained to be increased by 74% in the GCL in PCP-treated group. At 21days, locomotor activities of offspring in the PCP-treated group were significantlydecreased by �30% compared with those in the control group. However, neuronal dif-ferentiation of newly formed cells and cell survival were not influenced at 5 weeksafter BrdU injections. Some altered biochemical or physiological conditions of offspringfrom dams receiving repeated PCP injections during pregnancy could influencechanges in cell proliferation in the GCL of offspring during early development.Changes to cell proliferation in the hippocampus may affect behavioral abnormalitiesduring infancy in offspring. Synapse 63:729–736, 2009. VVC 2009 Wiley-Liss, Inc.

INTRODUCTION

Phencyclidine hydrochloride (PCP) is a psychosti-mulant that noncompetitively blocks N-methyl-D-aspartate (NMDA) receptors and inhibits uptake ofdopamine and serotonin (Martin et al., 1979). In adultrats, single exposure to PCP results in abnormalbehaviors such as increased locomotor activity orataxia and disruption in several cognitive functions(Castner et al., 2004; Le Pen et al., 2003). In addition,prolonged exposure to PCP, cocaine, or methamphet-amine can cause long-lasting behavioral sensitizationand biochemical alterations in certain regions of the

rat brain, and more persistent psychotic symptoms,flattened affect and amotivation in human (Adamsand Moghaddam, 1998). We have previously demon-

A. T. and J. L. contributed equally to this work.

Contract grant sponsor: Research Support Foundation of Juntendo Instituteof Mental Health.

*Correspondence to: T. Suzuki, Juntendo Koshigaya Hospital, 560 Fukur-oyama, Koshigaya, Saitama, Japan 343-0032. E-mail: psuzukit@juntendo.ac.jp

Received 17 October 2008; Accepted 24 December 2008

DOI 10.1002/syn.20660

Published online in Wiley InterScience (www.interscience.wiley.com).

VVC 2009 WILEY-LISS, INC.

SYNAPSE 63:729–736 (2009)

strated that repeated administration of PCP producestransient disturbances of cell proliferation in the den-tate gyrus that may result from adaptation to newpharmacological or stressful conditions under behav-ioral sensitization (Liu et al., 2006).

PCP is known to be readily cross the blood-placentabarrier and reach the fetus in human (Nicholas et al.,1982). In addition, PCP has been detected in neonatalurine at 7 days postpartum (Marble et al., 1980). Mul-tiple case reports of pregnant PCP abusers havedescribed characteristic clinical symptoms of PCP-exposed infants exposed to PCP before a birthsuggesting neurobehavioral changes including motordysfunction, i.e., hypertonicity or fine dysmovement,cognitive or learning problems, and physical abnor-malities (Golden et al., 1987; Rahbar et al., 1993; VanDyke and Fox, 1990). And controversial result, i.e., noevidence for an increase in low-birth weight, has pre-viously been described (Mvula et al., 1999). Mental orbehavioral abnormalities, typical withdrawal syn-drome with irritability, and hypertonia have beenseen postpartum (Strauss et al., 1981). Sudden out-bursts of agitation, rapid changes in level of con-sciousness, and marked reactivity to auditory stimulihave also been noted (Chasnoff et al., 1983).Increased lability of state has been revealed comparedwith other drugs, i.e., sedative/stimulant drugs(Chasnoff et al., 1986). Investigations on the influen-ces of PCP have been limited because most PCP usersare multidrug users, making interpretation difficult.

Nabeshima et al. (1987, 1988) reported that prena-tal administration of PCP to pregnant rats produces adelay in learning and memory processes in rat off-spring, along with disturbances of viability index, andemotional indicators, i.e., face-washing, at 4–5 weeksold, accompanied by a decrease in brain weight.These behavioral disturbances were noted in damsthat received repeated PCP injection during mid andlate gestation (Fico and Vanderwende, 1988; Nabe-shima et al., 1987, 1988).

Until recently, little attention has been given to theadverse effects of PCP exposure in pregnant rats ondevelopment of the central nervous system and neo-natal behaviors. We designed the present experimentsto evaluate whether chronic prenatal exposure toPCP during the last 2 weeks of gestation in rats pro-duces alterations in development of hippocampalneurogenesis in offspring.

MATERIALS AND METHODSMaterials and drugs

PCP was purchased from Shinnihonyakugyo Indus-tries (Tokyo, Japan). All other chemicals used in thisstudy were obtained from Sigma Chemicals (St.Louis, MO) or Wako Chemicals (Tokyo, Japan).

Animals and experimental design,drug administration

Pregnant female Sprague-Dawley rats (SankyoLabo Service, Tokyo, Japan) weighing 240–290 g at10–11 weeks old were caged individually on a 12/12-hlight-dark cycle at constant temperature, with ad libi-tum access to standard laboratory feed and tap water.All procedures performed on animals were approvedby the Institutional Animal Care and Use Committeeof Juntendo University School of Medicine (approval#180140).

Experimental designs

Dams received repeated dorsal subcutaneous injec-tions of PCP (5 mg/kg) once daily for 14 consecutivedays during the last 2 weeks of gestation (Fig. 1). Wehave previously confirmed the establishment of sensi-tization of dopaminergic abnormal behaviors and tol-erance of serotonergic ataxic behaviors using dailydoses of 7.5 mg/kg (Liu et al., 2006). In this study, thedaily dose of PCP was reduced to reduce risk toabnormal development of the fetus. Control animalsreceived subcutaneous injections of physiological sa-line for 14 days in the same period. The control groupwas divided into two groups as follows: Control I andControl II. Due to a lack of feeding ability in damsreceiving repeated PCP, litters of PCP-treated damswere surrogate fostered to dams without PCP injec-tions in Control I. Since the effect of maternal behav-iors on development of pups needed to be controlledfor in these experiments, dams from the Control Igroup nursed and fostered pups from the PCP-treatedgroup, and dams from the Control II group nursedand fostered pups from the Control I group. The fos-ter rats were from the same strain as the PCP-treated rats and the two Control groups. Pups fromtwo groups were thus fed by surrogate dams untilpostnatal 3 weeks at a decapitation. At birth, thenumber of live births and birth weights wererecorded. On postnatal day (PD) 14, litters wereexamined for sex, and the number of pups per damwas controlled to approximately eight pups, compris-ing four males and four females, to exclude any differ-ences in physical development among litters of eachexperiment.

We focused on the changes of hippocampal neuro-genesis in pups before puberty, since some neurobeha-vioral abnormalities have been reported in PCP-exposed infants (Golden et al., 1987; Rahbar et al.,1993; Van Dyke and Fox, 1990). According to our pre-vious study (Namba et al., 2005) about developmentalchanges in the distribution of Ki67-positive cells inthe dentate gyrus, Ki67-positive cells are localizedmainly in the GCL at PD21 before puberty. Thus, cellproliferation was investigated in pups from both PCP-treated rats and controls at PD21. Each treatment

730 A. TANIMURA ET AL.

Synapse

group (PCP-group, Control I group, and Control IIgroup) in these experiments comprised eight malerats and eight female rats from two dams.

For cell differentiation (as neuronal or glial pheno-type) and cell survival, pups in each group were per-fused with 0.1 M phosphate-buffered saline (PBS) 5weeks after 5-bromo-20-deoxyuridine (BrdU) injectionon PD18-20. At 5 weeks after BrdU injections, newlyformed cells differentiate into mature neurons or gliaand integrate into the existing hippocampal circuitry.

Behavioral assessment of dams

Behavioral assessments were performed on dams toconfirm locomotor activities on days 1, 5, and 10 of re-petitive administration without day 14 to avoid anyobstacle to delivery. Dams were individually placed ina transparent 35 3 35 3 25 cm plastic cage. After 30min of acclimatization, dams received a PCP injec-tion, then behavioral screening of locomotor activitywas automated and assessed for 60 min using aSupermex behavior-analyzing system (MuromachiKikai, Tokyo, Japan), which is an apparatus with aninfrared sensor that detects thermal radiation fromanimals.

Behavioral assessment of pups

We selected a simple method to count a locomotoractivity as assessment for behavioral abnormalitywithin a day on PD21, as learning or memory testsgenerally need much more time to obtain a data.Locomotor activities of pups on PD21 after a birth

were measured in the same plastic cage using theSupermex behavior-analyzing system. After 30 min ofacclimatization, activities were assessed for 1 h justbefore perfusion.

Immunohistochemistry for BrdU-positive cells

All animals received subcutaneous injections ofBrdU (50 mg/kg) dissolved in 0.9% NaCl total threetimes, once on each PD18, PD19, and PD20. At 24 hafter last injection of BrdU, rats were deeply anesthe-tized with sodium pentobarbital and perfused intra-cardially with 0.01 M PBS (pH 7.4), followed by 4%paraformaldehyde in 0.1 M phosphate buffer (pH 7.4)at room temperature. The brain was removed fromthe skull and postfixed overnight in the same solutionat 48C. Fixed brains were washed twice with PBSand embedded in 10% sucrose. Cerebral cortices con-taining the hippocampal formation were dissectedaway from the remaining brain structure, and slices1–2 mm thick were cut in a plane perpendicular tothe septotemporal axis of the dorsal hippocampal for-mation at the approximate midpoint of the axis. Sli-ces were postfixed overnight in paraformaldehyde at48C, and then embedded in 5% agarose in PBS. Serial50-lm sections were cut using a microtome and slide-mounted before immunocytochemical processing toensure objectivity. Every second section was thenused for BrdU immunocytochemistry.

For BrdU immunostaining, sections were subse-quently treated with 2-N HCl at 378C for 30 min todenature DNA, then neutralized with 0.1 M borate

Fig. 1. Experimental design of PCP-treated group (dams withrepeated PCP treatment) and the control groups (I and II). Damsreceived repeated dorsal subcutaneous injections of PCP (5 mg/kg)once daily for 14 consecutive days during the last 2 weeks of gesta-tion. The control group was divided into two groups. Dams from theControl I group nursed and fostered pups from the PCP-treated

group, and dams from the Control II group nursed and fosteredpups from the Control I group. Pups from two groups were thus fedby surrogate dams until postnatal 3 weeks at a decapitation. Cellproliferation was investigated in pups from both PCP-treated ratsand controls at PD21.

731PHENCYCLIDINE ON HIPPOCAMPAL NEUROGENESIS OF PUPS

Synapse

buffer (pH 8.5) at room temperature for 10 min. Next,sections were washed twice with PBS and incubatedwith rat IgG monoclonal anti-BrdU (Immunologicals-Direct.com, UK) at 48C overnight, then washed threetimes with PBS. Sections were incubated with Cy3-conjugated donkey anti-rat IgG (1:200; JacksonImmunoResearch) at room temperature for 1 h, andthen washed three times with PBS.

For triple-label immunohistofluorescence to examinethe differentiation (astrocyte or neuron phenotype) ofmature BrdU-positive cells, sections were further incu-bated with a mixture of rabbit antibody against S-100b (Swant, Bellinzona, Switzerland) and mouse IgGmonoclonal anti-NeuN (1:200; Chemicon Interna-tional, Temecula, CA) at 48C for 24 h to examine neu-rons. Sections were then incubated with a mixture offluorescein isothiocyanate (FITC)-conjugated donkeyanti-rabbit IgG (1:200; Jackson ImmunoResearch) andCy5-conjugated donkey anti-mouse IgG (1:200; Jack-son ImmunoResearch) at room temperature for 1 h.Slices were analyzed under confocal microscopy, asdescribed above. In each animal, �50 BrdU-positivecells were examined to confirm colocalization of bothBrdU and each marker for neurons and glia.

Quantification of BrdU-positive cells

To distinguish each cell within proliferating cellclusters, all counts were performed using an LSM 510confocal laser scanning miscroscope (Zeiss, Germany)with a 403 objective lens. Stacks of optical sections(thickness: 1.8 lm for 403 objective) were obtained at2.1-lm increments in the z-axis for 203 objective, 0.9lm for 403 objective analysis. Images were correctedfor brightness and contrast using LSM Image Browsersoftware (Zeiss) and Photoshop 5.0 software (Adobe,Mountain View, CA). To determine the number ofBrdU-positive cells in the granule cell layer (GCL) andsubgranular zone (SGZ), all BrdU-labeled cells in thedentate gyrus (GCL) were counted regardless of size orshape in each section by an experimenter blinded tothe study code. A cell was counted as being within theSGZ of the dentate gyrus (a two cell body wide zone atthe border between granule cell layer and hilus) andthe GCL. A total of five sections per rat were counted.The total number of BrdU-positive cells in the dentategyrus was multiplied by two and reported as totalnumber of cells per region.

Quantitative comparisons of counts of BrdU-positivecells in the dentate gyrus were performed at PD21 (cellproliferation) and PD56 (survival) between two groupsof pups from PCP-treated and the control groups.

Statistical analysis

Values of BrdU-positive cell counts were deter-mined based on eight rats for cell proliferation, fourrats for cell differentiation, and 12 rats for locomotor

activity of pups in each group. Values are expressedas mean 6 standard error of the mean (SEM). BrdU-positive cell counts in the hippocampus and locomotoractivity of pups on PD21 were determined usingunpaired Student’s t-test. Values of P < 0.05 wereconsidered statistically significant.

RESULTSInfluences of PCP treatment on physical

conditions of dams during the last 2 weeksof gestation

No maternal deaths occurred among any PCP-treated or control dams under a daily PCP dose of 5mg/kg. Mean body weight gain of dams for the last 2weeks at gestational day 21s was 63.0 6 8.5 g forPCP-treated dams and 105.3 6 7.7 g for controldams. PCP-treated dams displayed significantly lowermean body weight gain compared with controls (P 50.021, 240.2%), but no significant differences in num-ber of pups born from a dam were seen groups (PCP-treated group, 9.7 6 1.2; controls, 12.7 6 1.5).

Number and body weight gain of pups fromdams with and without repeated PCP

The number of pups born on to a mother was 8–15pups. At delivery, no gross physical abnormalities wereobserved in offspring. No significant difference in bodyweight of pups at 3 weeks after birth was apparentbetween the PCP-treated and control groups (males:32.5 6 1.2 g in PCP-treated group, 35.1 6 0.6 g in con-trols, P 5 0.073; females: 32.4 6 0.96 g in PCP-treatedgroup, 32.06 0.84 g in controls, P5 0.762)

Cell proliferation of BrdU-positive cells in theGCL of offspring at PD21 between PCP-treated

and the control groups

Before cell counting, BrdU-positive cells were typi-cally observed in the SGZ and GCL of the dentategyrus of pups. Cells in PCP-treated and the controlgroups did not differ in terms of gross morphology orlocation of labeled cells with in the GCL. At PD21,numbers of BrdU-positive cells in the GCL in maleand female offspring of the PCP-treated group weresignificantly increased by �77.5% in males and 77.7%in females compared with those in the control group(n 5 8). This increase in BrdU-positive cells was sig-nificantly found in both males (P 5 0.00012) andfemales (P 5 0.00043) (Fig. 2).

Behavioral changes in offspring at PD21between PCP-treated and the control groups

Locomotor activities of pups on PD21 after a birthwere measured for 1 h using the Supermex behavior-analyzing system after 30 min of acclimatization. Asshown in Figure 3, counts of locomotor activities in

732 A. TANIMURA ET AL.

Synapse

the PCP-treated group at PD21 were significantlydecreased by 28.9% (P 5 0.0066; n 5 12) comparedwith the control group (Fig. 3).

Phenotype of BrdU-positive cells in theGCL of offspring between PCP-treated

and the control groups

Triple-labeling was performed in the dentate gyrusof the contol rats using confocal microscopy. (Fig. 4)Cells were labeled for BrdU (red), neuronal markerNeuN (blue), and glial marker S-100b (green). No dif-ferences in morphology, distribution, or phenotype ofnew cells were noted between PCP-treated and con-trol groups. Concerning cell differentiation of BrdU-positive cells in the GCL at PD56, no difference in ra-tio of neurons to glia was seen between PCP-treatedand Control groups. (PCP-treated group: neurons94.0%, glia 3.0%, nonneurons and nonglia 3.0%; Con-trol group: neurons 92.0%, glia 3.5%, nonneurons andnonglia 4.5%).

Survival of BrdU-positive cell in theGCL of offspring between PCP-treated

and the control groups

Within 5 weeks (PD56) after BrdU injections atPD18-20, new cells generated from cell divisions atPD21 developed into mature cells. Analysis of the sur-vival of newly generated cells at PD56 found a signifi-cant increase of BrdU-positive cells in the GCL in PCP-treated group, compared with control group (174.2%,

P 5 0.012; n 5 4) (Fig. 5). This finding applied to thenumber of surviving BrdU-positive cells.

DISCUSSION

Repeated treatment of PCP causes hyperlocomo-tion, stereotyped behaviors, and serotonin-related

Fig. 2. Counts of BrdU-positive cells in the GCL of offspring atPD21 between PCP-treated and the control groups. Dams receivedrepeated dorsal subcutaneous injections of PCP (5 mg/kg) once dailyfor 14 consecutive days during the last 2 weeks of gestation. After abirth, all pups received subcutaneous injections of BrdU (50 mg/kg)total three times, once on each PD18, PD19, and PD20. At PD21,

numbers of BrdU-positive cells in the GCL in male and female off-spring of the PCP-treated group were significantly increased by�77.5% in males and 77.7% in females compared with those in thecontrol group (n 5 8). This increase in BrdU-positive cells was sig-nificantly found in both males (P 5 0.00012) and females (P 50.00043).

Fig. 3. Locomotor activities of pups at PD21 between PCP-treated and the control groups. Pups were individually placed in atransparent 35 3 35 3 25 cm plastic cage. After 30 min of acclimati-zation, pups received a PCP injection, and then locomotor activitieswere assessed for 60 min using a Supermex behavior-analyzing sys-tem. In the Supermex method, scores are measured using a PCinterfaced with body-temperature-sensitive sensors. The sensormonitors motion in multiple zones of the cage through an array ofFresnel lenses placed above the cage. At PD21, locomotor activitiesof pups in the PCP-treated group were significantly decreased by28.9% compared with the Control group.

733PHENCYCLIDINE ON HIPPOCAMPAL NEUROGENESIS OF PUPS

Synapse

behaviors. In this study, we counted locomotor activ-ity to examine the development of hyperlocomotionduring gestation. Behavioral assessments of damsduring the last 2 weeks of pregnancy were done ondays 1, 5, and 10 of repeated PCP administration. Ina preliminary experiment, we confirmed that Super-mex could monitor all spontaneous movement by aPC interfaced with body-temperature-sensitive sen-sors, which monitors motion in multiple zones of thecage through an array of Fresnel lenses placed abovethe cage (Liu et al., 2006). Counts of locomotor activ-ities of dams in PCP-treated group (n 5 3) rangedfrom 11323.0 6 3414.5 to 15833.0 6 2812.2 countsamong the 3 days measured. They increased three tofourfold compared with those (3710.0 6 691.3 to3925.0 6 554.1 counts) previously observed in physio-logical saline-treated adult rats (Liu et al., 2006). Inthis study, we confirmed that dams received repeated

PCP produced hyperlocomotion during the last 2weeks.

In the previous study of analysis of early postnatalneurogenesis, Namba et al. (2005) demonstrated thatthe distribution pattern of Ki67-positive cells, whichis also a marker of proliferation, gradually changedand a majority of BrdU-positive cells were detected inthe inner half of the GCL at PD19. We confirmed thata relatively dense population of Ki67-positive prolifer-ating cells was visible in the hilus at PD7 and PD14and the dense population of Ki67-positive cells hadalmost completely disappeared from the hilus andwere localized mainly in the GCL at PD21 in a pre-liminary experiment. This suggests that the patternof distribution of BrdU-positive cells in the hippocam-pus but it’s not clear how this is nearly identical tothat in adult neurogenesis of rats. The pattern ofBrdU-positive cell incorporation in the GCL at PD21

Fig. 4. Phenotype of BrdU-positive cells 5 weeks after BrdUinjection at PD21. At 5 weeks after BrdU injections on PD18-20,newly formed cells differentiate into mature neurons or glia andintegrate into the existing hippocampal circuitry. Triple-labeling (D)was performed in the dentate gyrus of the control rats using confo-cal microscopy. Cells were labeled for BrdU (red) (A), neuronal

marker NeuN (blue) (B), and glial marker S-100b (green) (C). Con-cerning cell differentiation of BrdU-positive cells in the GCL atPD56, no difference in ratio of neurons to glia was seen betweenPCP-treated and Control groups. (PCP-treated group: neurons94.0%, glia 3.0%, nonneurons and nonglia 3.0%; Control group:neurons 92.0%, glia 3.5%, nonneurons and nonglia 4.5%).

734 A. TANIMURA ET AL.

Synapse

in this study did not differ from that observed withsimilar experiments in adult rats (Namba et al.,2005). For this reason, we performed quantitativeanalysis in the GCL at 3 weeks after birth.

The characteristic finding is a marked increase inBrdU-positive cells of the GCL at PD21 in pups fromthe PCP-treated group compared with those from theControl group. At PD56, 5 weeks after BrdU injec-tions, the number of surviving BrdU-positive cellswas also increased. In addition, there was no signifi-cant difference in phenotype of BrdU-positive cellsbetween two groups. Consequently, these findingsreflect that cell differentiation and cell survival inpups were not influenced by prenatal stress withrepeated PCP administrations. Many biochemical andphysiological conditions could influence cell prolifera-tion in the GCL. We have previously demonstrated atransient reduction in cell proliferations in the GCLfor PCP-treated rats showing behavioral sensitization(Liu et al., 2006). This would possibly result from ad-aptation to new pharmacological conditions, such asdisturbances of dopamine or serotonergic systems, ornew stressful physiological conditions. Interestingly,the findings of this study are opposite to the decreasein number of cell proliferations in PCP-treated adultrats. Although PCP easily crosses the placenta, incon-sistencies in cell proliferations in the GCL were seenbetween these two studies. Pharmacological studieshave shown no significant changes in dopaminergic orserotonergic receptor bindings and concentrations,except for PCP binding, in the fetal whole brain ongestational day 21 from dams receiving repeated PCPtreatment (Ali et al., 1989). In addition, no significant

effects of prenatal PCP exposure have been seen ondopamine, serotonin, TCP binding, or dopaminerelease in striatum at PD21 (Ali et al., 1993). How-ever, an increased acetylcholine release to PCP chal-lenge injection was revealed in the frontal cortex ofrat pups at PD21 (Howard and Takeda, 1990). PCP-induced supersensitivity of the cholinergic systemwould be relevant to data with increased binding ofmuscarinic cholinergic receptors in the septohippo-campal innervation of adult rats following prenatalexposure to PCP by Yanai et al. (1992). Nabeshimaet al. (1988) also reported recovery of PCP-inducedamnesia by physostigmine. Recently, Glenn et al.(2007) suggested that prenatal choline intake hasenduring effects on adult hippocampal neurogeneis.Kaneko et al. (2006) demonstrated that the choliner-gic system regulates survival of newborn neurons inthe adult dentate gyrus. Together with these find-ings, this raises the possibility that alterations tothe cholinergic system may influence the develop-ment of hippocampal neurogenesis. As another ex-planation for the present results, hyperlocomotion inpups during early development may increase cellproliferation in the hippocampus, since running hasbeen reported to enhance hippocampal neurogenesisin rodents (Van Praag et al., 1999). Our behavioralresults, however, showed a marked decrease in loco-motor activities at PD21 in pups. Thus, such a be-havioral change could not explain enhancement ofhippocampal neurogenesis.

Another possible explanation is that a transientincrease in cell proliferation may occur during theearly postnatal period following prenatal stress. Bick-Sander et al. (2006) demonstrated that a decrease incell proliferation during embryogenesis in offspringinduced by voluntary running in pregnant dams wasfollowed by a postnatal 40% increase in hippocampalneurogenesis in offspring mice. Such a biphasic phe-nomenon has also been reported in a previous paper(King et al., 2004) in which prenatally malnourishedrats showed decreased cells tagged in the fascia den-tate at PD7 and increase of cells at PD30. Jebelliet al. (2002) reported increased neurodegeneration inthe entorhinalcortex and subiculum of rats with pre-natal exposure to PCP during the second trimester.These findings suggest a transient increase in cellproliferation during early development as a reboundphenomenon.

Conversely, induction of a long-term or life-longreduction in hippocampal neurogenesis has beenreported following prenatal stress in rats (Lemaireet al., 2000). Differences between the underlyingmechanisms remain unelucidated, whereas growingevidence suggests that dysregulation of the hypo-thalamic-pituitary-adrenal (HPA) axis includingglucocorticoid levels resulting from prenatal stress(Weinstock, 2005) can inhibit cell proliferation in

Fig. 5. Survival of BrdU-positive cell in the GCL of offspringbetween PCP-treated and the control groups. Within 5 weeks afterBrdU injections, new cells generated from cell divisions at PD21developed into mature cells. Analysis of the survival of newly gener-ated cells at PD56 found a significant increase of BrdU-positive cellsin the GCL in PCP-treated group, compared with control group(174.2%, P 5 0.012; n 5 4) (Fig. 5).

735PHENCYCLIDINE ON HIPPOCAMPAL NEUROGENESIS OF PUPS

Synapse

neonatal rats (Lemaire et al., 2000; Tanapat et al.,1998). In addition, the potential limitation of thisstudy is that any major effects on the development ofCNS organ systems, including HPA axis would not beeliminated. In any case, these biological or physiologi-cal changes by PCP treatment would affect develop-ment of hippocampal neurogenesis and locomotor ac-tivity in the prepuberty of offspring. Further investi-gations are needed to clarify any impairment tolearning and memory processes in offspring from ratswith prenatal PCP administration.

REFERENCES

Adams B, Moghaddam R. 1998. Corticolimbic dopamine neurotrans-mitter is temporally dissociated from the cognitive and locomotoreffects of phencyclidine. J Neurosi 18:5545–5554.

Ali SF, Ahmad G, Slikker W Jr, Bondy SC. 1989. Effects of gesta-tional exposure to phencyclidine: Distribution and neurochemicalalterations in maternal and fetal brain. Neurotoxicology 10:383–392.

Ali SF, Holson RR, Newport GD, Slikker W Jr, Bowyer JF. 1993. De-velopment of dopamine and N-methyl-D-aspartate systems in ratbrain: The effect of prenatal phencyclidine exposure. Dev BrainRes 73:25–33.

Bick-Sander A, Steiner B, Wolf SA, Babu H, Kempermann G. 2006.Runnin in pregnacy transiently increases postnatal hippocampalneurogenesis in the offspring. Proc Natl Acad Sci USA 103:3852–3857.

Castner SA, Goldman-Rakic PS, Williams GV. 2004. Animal modelsof working memory: Insight for targeting cognitive dysfunction inschizophrenia. Psychopharmacology 174:111–125.

Chasnoff IJ, Burns KA, Burns WJ, Schnoll SH. 1986. Prenatal drugexposure: Effects on neonatal and infant growth and development.Neurobehav Toxicol Teratol 8:357–362.

Chasnoff IJ, Burns WJ, Hatcher RP, Burns KA. 1983. Phencycli-dine: Effects on the fetus and neonate. Dev Pharmacol Ther6:404–408.

Fico TA, Vanderwende C. 1988. Phencyclidine during pregnancy: Fe-tal brain levels and neurobehavioral effects. Neurotoxicol Teratol10:349–354.

Glenn MJ, Gibson EM, Kirby ED, Mellott TJ, Blusztajn JK,Williams CL. 2007. Prenatal choline availability modulateshippocampal neurogenesis and neurogenic responses to enrichingexperiences in adult female rats. Eur J Neurosci 25:2473–2482.

Golden NL, Kuhnert BR, Sokol RJ, Martier S, Williams T. 1987.Neonatal manifestations of maternal phencyclidine exposure. JPerinat Med 15:185–191.

Howard SG, Takeda H. 1990. Effect of prenatal exposure to phency-clidine on the postnatal development of the cholinergic system inthe rat. Dev Neurosci 12:204–209.

Jebelli AK, Doan N, Ellison G. 2002. Prenatal phencyclidine inducesheightened neurodegeneration in rats in some brain regions, espe-cially during 2nd trimester, but possible anti-apoptotic effects inothers. Pharmacol Toxicol 90:20–25.

Kaneko N, Okano H, Sawamoto K. 2006. Role of the cholinergic sys-tem in regulating survival of newborn neurons in the adult mousedentate gyrus and olfactory bulb. Genes Cells 11:1145–1159.

King RS, De Bassio WA, Kemper TL, Rosene DL, Tonkiss J, GallerJR, Blatt GJ. 2004. Effects of prenatal protein malnutrition andacute postnatal stress on granule cell genesis in the fascia dentateof neonatal and juvenile rats. Dev Brain Res 150:9–15.

Lemaire V, Koehl M, Le Moal M, Abrous DN. 2000. Prenatal stressproduces learning deficits associated with an inhibition of neuro-genesis in the hippocampus. Proc Natl Acad Sci USA 97:11032–11037.

Le Pen G, Grottick AJ, Higgins GA, Moreau J-L. 2003. Phencycli-dine exacerbates attentional deficits in a neurodevelopmental ratmodel for schizophrenia. Neuropsychopharmacology 28:1799–1809.

Liu J, Suzuki T, Seki T, Namba T, Tanimura A, Arai H. 2006.Effects of repeated phencyclidine administration on adult hippo-campal neurogenesis in the rat. Synapse 60:56–68.

Marble RD, Thomas RG, Sterling ML. 1980. Screening for angeldust in newborns. Pediatrics 66:334.

Martin JR, Berman MH, Krewsun I, Small SF. 1979. Phencyclidine-induced stereotyped behavior and serotonergic syndrome in rat.Life Sci 24:1699–1704.

Mvula MM, Miller JM, Ragan FA. 1999. Relationship of phencycli-dine and pregnancy outcome. J Reprod Med 44:1021–1024.

Nabeshima T, Hiramatsu M, Yamaguchi K, Kasugai M, Ishizaki K,Kawashima K, Itoh K, Ogawa S, Katoh A, Furukawa H,Kameyama T. 1988. Effects of prenatal administration of phency-clidine on the learning and memory processes of rat offspring. JPharmacobiodyn 11:816–823.

Nabeshima T, Yamaguchi K, Hiramatsu M, Ishizaki K, FurukawaH, Kameyama T. 1987. Effects of prenatal and perinatal adminis-tration of phencyclidine on the behavioral development of rat off-spring. Pharmacol Biochem Behav 28:411–418.

Namba T, Mochizuki H, Onodera M, Mizuno Y, Namiki H, Seki T.2005. The fate of neural progenitor cells expressing astrocytic andradial glial markers in the postnatal rat dentate gyrus. Eur JNeurosci 22:1928–1941.

Nicholas JM, Lipshitz J, Schreibner EC. 1982. Phencyclidine: Itstransfer across the placenta as well as into breast milk. Am JObstet Gynecol 18:143–146.

Rahbar FR, Fomufod A, White D, Westney LS. 1993. Impact ofintrauterine exposure to phencyclidine (PCP) and cocaine on neo-nates. J Natl Med Assoc 85:349–352.

Strauss AA, Modanlou HD, Bosu SK. 1981. Neonatal manifestationsof maternal phencyclidine (PCP) abuse. Pediatrics 68:550–552.

Tanapat P, Galea LA, Gould E. 1998. Stress inhibits the prolifera-tion of granule cell precursors in the developing dentate gyrus.Int J Dev Neurosci 16:235–239.

Van Dyke DC, Fox AA. 1990. Fetal drug exposure and its possibleimplications for learning in the preschool and school-age popula-tion. J Learn Disabil 23:160–163.

Van Praag H, Christie BR, Sejnowski TJ, Gage FH. 1999. Runninenhances neurogenesis, learning, and long-term potentiation inmice. Proc Natl Acad Sci USA 96:13427–13431.

Weinstock M. 2005. The potential influence of maternal stress hor-mones on development and mental health of the offspring. BrainBehav Immun 19:296–308.

Yanai J, Avraham Y, Lewy S, Maslaton J, Pick CG, Rogel-Fuchs Y,Zahalka EA. 1992. Alterations in septohippocampal cholinergicinnervations and related behaviors after early exposure to heroinand phencyclidine. Dev Brain Res 69:207–214.

736 A. TANIMURA ET AL.

Synapse