Anxiety induced by prenatal stress is associated with suppression of hippocampal genes involved in...

Anxiety induced by prenatal stress is associated with suppressionof hippocampal genes involved in synaptic function

Y. Bogoch,* Y. N. Biala,� M. Linial* and M. Weinstock�

*Department of Biological Chemistry, Life Science Institute, The Hebrew University, Jerusalem, Israel

�Department of Pharmacology, School of Pharmacy, The Hebrew University, Jerusalem, Israel

Abstract

Exposure of pregnant women or animals to stress during a

critical period of foetal brain development increases the like-

lihood of anxiety, depression and learning deficits that are

associated with structural alterations in the offspring hippo-

campus. In this study, we report the effect of gestational

stress in rats on anxiogenic behaviour and hippocampal gene

expression of their 23-day-old female offspring. As the rat

brain continues to develop after birth, we also used the pro-

cedure of handling (H) during the first 10 days of life to reverse

the anxiogenic behaviour of prenatally stressed (PS) rats. By

means of micro-array analysis on hippocampal extracts, we

found that the expression of about 6.1% of 9505 valid genes

was significantly altered by prenatal stress (p < 0.05). Of

these, 48% were over-expressed and 52% under-expressed.

The latter included �300 genes that participate in axonal

growth, regulation of ion channels and transporters, trafficking

of synaptic vesicles and neurotransmitter release. About 30%

of the genes that were down-regulated in PS rats were re-

stored to control levels by H. These include genes that play a

role in pre-synaptic organization and function. Our results

provide a possible relationship between hippocampal gene

expression and changes in behaviour resulting from prenatal

stress.

Keywords: anxiety, exocytosis, gene annotation, handling,

micro-array, synapse.

J. Neurochem. (2007) 101, 1018–1030.

Several reports have linked prenatal stress to a greaterincidence of anxiety, affective disorders, attention deficitsand schizophrenia (Weinstock 1997; van Os and Selten 1998;Geddes 1999; Linnet et al. 2003; Maccari et al. 2003). Inrodents and non-human primates, prenatal stress alsoincreases the incidence of attention deficits (Schneider1992) and induces anxiogenic and depressive-like behaviour,characterized by increased fear of novelty and impairedcoping under adversity (Fride et al. 1986; Fride and Wein-stock 1988) and learned helplessness and anhedonia,respectively (Fride et al. 1986; Alonso et al. 1991; Schneider1992; Keshet and Weinstock 1995; Milberger et al. 1997). Achronic anxiety state in association with excessive fear ofnovelty has been linked to alterations in the size of the lateralamygdaloid nucleus in humans (De Bellis et al. 2000) andprenatally stressed (PS) rats (Salm et al. 2004) and also in thesize of the synaptic area of the mossy fibres in the dentategyrus (Belzung 1992). PS non-human primates and rats alsoshow a number of structural changes in the hippocampus,including a decrease in hippocampal synapses (Hayashi et al.1998), the number of granule cells in the dentate gyrus

(Schmitz et al. 2002) and in neurogenesis (Lemaire et al.2000; Coe et al. 2003).

Because a considerable amount of brain developmentoccurs after birth in the rat, it is possible to influence theplasticity of the nervous system and behaviour by an earlyenvironmental manipulation known as handling (H). Thisprocedure, first described by Levine et al. (1967), involvesremoval of the pups from the maternal nest for periods of3–15 min during the first 10–14 days of life. Its effect onbehaviour was detected when compared with that of rats

Received May 25, 2006; revised manuscript received August 30, 2006;accepted November 21, 2006.Address correspondence and reprint requests to Marta Weinstock,

Department of Pharmacology, School of Pharmacy, Hebrew University,Ein Kerem, Jerusalem 91120, Israel. E-mail: [email protected] used: CH, Control handled; CNH, Control non-han-

dled; cRNA, complementary RNA; EPM, elevated plus maze; GO, geneontology; H, Handling; PS, prenatally stressed; PSH, prenatally stressedhandled; PSNH, prenatally stressed non-handled; RT-PCR, reversetranscriptase PCR; SNARE, soluble N-ethylmaleimide-sensitive factorreceptor.

Journal of Neurochemistry, 2007, 101, 1018–1030 doi:10.1111/j.1471-4159.2006.04402.x

1018 Journal Compilation � 2007 International Society for Neurochemistry, J. Neurochem. (2007) 101, 1018–1030� 2007 The Authors

from which all contact with humans had been withheldduring the pre-weaning period (Levine et al. 1967; Ader1970). In later studies, H was found to prevent thehyperactivity induced by isolation stress in adult rats(Gentsch et al. 1988) and the increased fear of novelty inPS rats (Wakshlak and Weinstock 1990).

The aim of the present study was to identify the molecularprocesses in the hippocampus underlying anxiety induced byprenatal stress. As the incidence of anxiety and depressionand alterations in hippocampal structure are more prevalentin females (Alonso et al. 1991; Kuehner 1999; Schmitz et al.2002; Gue et al. 2004; Zhu et al. 2004), we looked forchanges induced by prenatal stress in genes and proteins inrats of this gender. To enable us identify the gene changesthat could be linked to anxiogenic behaviour, we determinedwhether any of them were reversed by H.

Materials and methods

Animals and treatments

All experiments were carried out according to the guidelines of the

University Committee for Institutional Animal Care, based on those

of the National Institutes of Health, USA. Female pathogen-free

(SPF) Wistar rats weighing 280–300 g (Harlan Biotech, Jerusalem,

Israel) on day 1 of pregnancy (detected by the presence of a vaginal

plug) were randomly allocated to stress and control (10 rats each)

groups, as previously described (Poltyrev and Weinstock 2004).

Maternal treatmentFrom day 17 to day 22 of gestation, rats were stressed daily in a

random order by three different stressors, restraint, forced swim and

saline injections once on each of 2 of 6 days, to prevent the rats

from adapting to them. Stress was applied to the rats during this

period because it is when key areas of the limbic system develop

(Bayer et al. 1993). Control pregnant females were left undisturbed

in their home cages. The length of gestation was 22.5 days.

Postnatal treatmentAfter birth, all litters were culled to eight pups (no more than three of

one sex and five of the other). From day 1 to day 10 of age, the pups

from half the control and half the stressed litters were handled by

removing them from the maternal cage for only 3 min as described

in Wakshlak and Weinstock (1990). As shown in our earlier study,

this manoeuvre does not significantly affect the behaviour of

controls, if they are handled in the usual manner by experimenters.

The pups were weaned at the age of 21 days and housed in groups of

two to three, according to sex and prenatal treatment. All subsequent

experiments described in the following paragraphs were performed

on the female offspring shortly after weaning in order to avoid the

effect of additional environmental factors on gene expression.

Anxiogenic behaviour

Anxiogenic behaviour was tested in the elevated plus maze (EPM)

at the age of 12 weeks in 11–12 females of each group of control

non-handled (CNH), PS non-handled (PSNH), control handled (CH)

and PS handled (PSH) rats, as previously described (Poltyrev and

Weinstock 2004). The EPM has two open and two closed arms

presenting the rat with a conflict between the desire to explore a

novel situation and its fear of height and open spaces (Handley and

Mithani 1984). Anxiolytic agents like diazepam increase, and

anxiogenic agents decrease the time spent in the open arms of the

maze (Pellow and File 1986). It has been shown that PS rats make

fewer entries into, and spend less time in the open arms of the maze

than controls (Fride and Weinstock 1988; Vallee et al. 1997). Thisbehaviour is consistent with increased anxiety, can be detected more

readily in females at the age of 23–30 days (Fig. 1) and persists into

adulthood (Zagron and Weinstock, unpublished data).

Gene expression in hippocampus

At the age of 23 days, three rats were chosen randomly from each

group of CNH, PSNH, CH and PSH litters. They were decapitated

and the whole hippocampi rapidly dissected out, snap frozen in liquid

nitrogen and then stored at –70 C until RNA isolation. Total RNA of

hippocampus was prepared with Triazol and subsequently cleaned by

the Rneasy Mini Kit (Qiagen, Valencia, CA, USA). RNA quality and

quantity were measured by spectrophotometer with A260/A280 of

1.85–2.1. The RNA was prepared for hybridization using the

protocol recommended by Affymetrix (Santa Clara, CA, USA).

Briefly, total RNA (10 lg) was first reverse transcribed using a T7-

Oligo (O’Donovan et al. 1999). Following Rnase H-mediated

second-strand cDNA synthesis, double-stranded cDNAwas purified

to serve as a template in the subsequent in vitro transcription reaction.The reaction was carried out in the presence of T7 RNA polymerase

and a biotinylated nucleotide analogue and ribonucleotide mix for

complementary RNA (cRNA) amplification and biotin labelling

(BioArray Labeling Kit; Enzoi Diagnostics, Farmingdale, NY, USA).

The biotinylated cRNA targets were cleaned up, fragmented and

hybridized to GeneChip expression arrays (Affymetrix) RAE 230A.

After washing, the chips were stained with a streptavidin–phyco-

erythrin conjugate for fluorescence detection. Hybridization on a test

chip and analysis for the quality of the labelled cRNA were

performed for each of the cRNA samples prior to the hybridization

experiment. Ratios of 3¢/5¢ for glyceraldehyde-3-phosphate dehy-

drogenase and ß-actin, used as a measure of the quality of the RNA

and not as a standard, were confirmed to be within acceptable limits

as recommended by Affymetrix. BioB spike controls were found to

be present on all the arrays. When scaled to a target intensity of 150

Males Females 0

20

40

60

80

100

120

Tim

e in

ope

n ar

ms

(s)

*

Fig. 1 Time spent by young PS and control male and female rats in

the open arms of the EPM. Open bars: control; Grey bars: PS group.

*Significantly different from control, p < 0.01. Each experimental group

comprises eight rats.

Prenatal stress, anxiety and hippocampal genes 1019

� 2007 The AuthorsJournal Compilation � 2007 International Society for Neurochemistry, J. Neurochem. (2007) 101, 1018–1030

(using Affymetrix Microarray Suite 5.0 array analysis software),

scaling factors for all arrays were found to be within acceptable limits

(1.5–2.8), as were the background, Q values and mean intensities.

Reverse transcriptase PCR

Reverse transcriptase (RT)-PCR was performed to validate the

changes in a number of genes from the micro-array using RNA that

was identical to that used for the GeneChip experiment and RNA

prepared from animals taken from the same groups. All reactions were

performed on the same cDNA preparation. For each reaction, the

number of PCR cycles was optimized to match the dynamic range of

the reaction (22–32 cycles). All reaction products were designed to

range between 190 and 270 nucleotides. Pairs of oligonucleotides for

two genes with a difference in amplimer length larger than 50

nucleotides were included in each reaction to reduce experimental

variation. Each set of oligonucleotides was tested at least twice.

Samples were treated for 15 min with 10 U of Rnase-free Dnase

(Qiagen) at 25 �C prior to the RT reaction. The relative amounts of

mRNA were normalized to L19 (housekeeping gene of the large

subunit of the ribosome). Oligonucleotide primers used are for

forward (F) and reverse (R) for the following genes: Stathmin like-2 F:

cggaagctccacgaactcta, R: tagcctcacggttttcctta; SV2b F: aactcccaag-

caaatggatg, R: atcctggaaataccggatca, Rim1 F: cgtcgtcggacacctaagag,

R: tgccctggtaagacttgtg; Synaptotagmin 5 F: gtctgaagaaacggaagacct,

R: cccttggtaaagctgcttag; L19 F: ctgaaggtcaaagggaatgtg, R: ggacagag-

tcttgatgatctc; Rab3a F: cgccagcttgtctcagtttag, R: ggctgtggtgatggttcgg-

ta; Staufen F: tgctgcagaagctatgctgt, R: gcctagagttgtgccagagg.

Western blot

Following separation by sodium dodecyl sulfate (SDS) – polyacryl-

amide gel electrophoresis, extracts of the whole hippocampus from

23-day-old littermates of the rats used for the gene arraywere prepared

for western analyses. Cell lysates were homogenized in solubilization

buffer [Tris 50 mmol/L, NaCl 150 mmol/L, 1% (v/v) NP-40]

supplemented with protease inhibitors (1 mmol/L phenyl methyl

sulphonyl fluoride, 5 lmol/L leupeptin, 10 lg/mL aprotinin and

10 mmol/L EDTA) on ice. The extracts (1 mL/g tissue) were

centrifuged (14 000 g, 20 min, 4�C) and used for separation on

12.5% SDS gels. Protein concentrations were determined by BCA

(Pierce, Rockford, IL, USA) and blotted on nitrocellulosemembranes.

Major synaptic proteins were detected by means of a set of

antibodies to synaptophysin (Sigma, St Louis, MO, USA), syna-

ptojanin (Chemicon, Temecula, CA, USA), synapsin (Chemicon),

synaptotagmin 1 (Alomone Labs, Jerusalem, Israel) and SNAP-25

(kindly contributed by N. Takahashi, Japan). Secondary antibodies

used to detect monoclonal and polyclonal antibodies were Goat amouse and Goat a rabbit coupled to HRP (Jackson, West Grove, PA,

USA). b-tubulin (Sigma) was used for normalization, as it was

unchanged in all three experimental groups. Each experiment was

repeated at least twice. Western blots were quantified and normal-

ized according to controls. Detection method used was enhanced

chemiluminescence based and quantification was performed by

means of ImageGauge.

Data analysis

Differences in behavioural parameters in the EPM between the four

groups of rats were analysed by ANOVA for factorsMATERNAL (stress

or control) and NEONATAL treatment (handled or non-handled).

Duncan’s post hoc test was appliedwhen appropriate, and a differenceof p < 0.05 was considered to be statistically significant.

Changes in gene expression were analysed by means of

Microarray Suite 5.0. We included genes that showed a differential

expression in intensity of >500. We considered genes to be valid for

analysis, if the gene probe was defined as Present (P) according to

Microarray Suite 5.0 for at least two of three samples in each

experimental group. Most (86%) of the genes were assigned P for all

three samples. Genes that did not have a signal intensity of at least

20 in all of the samples were disregarded. Significance of the

difference between CNH and PSNH, PSNH and PSH, and CNH and

CH was tested by Student’s t-test. Only genes that showed a

difference in expression greater than 1.25-fold and statistical

difference of p < 0.05 between groups were considered.

Functional annotation was performed by means of the statistical

model implemented in DAVID (Dennis et al. 2003) and GOTM(Zhang et al. 2004). DAVID is a web-based application that

translates the list of probes to functional annotations that integrate

data from the Gene Ontology (GO) Consortium (Camon et al.2004). These tools apply a statistical model based on annotation

sources (i.e. GO) to rank the coregulated functional categories

among the differentially expressed genes. Ranking was performed

according to a modified Fisher’s exact test referred to as ‘enrichment

score’ (Dennis et al. 2003). The p-value for the ‘enrichment score’

is the probability of finding a certain number of genes that have a

specific annotation by chance (given the number of genes in the set

with this annotation, the total number of genes in the database and

the number of genes in the database with this annotation). Changes

in gene expression between any pair of the experimental groups

were tested for enrichment in functional annotations. No such

functional enrichment was found among genes in the CH group

compared with CNH. Although all gene changes resulting from

prenatal stress were analysed, the major focus was on those that

showed a significantly reduced expression which was restored by H.

Significance of the difference among the various groups in the

levels of the proteins from the western blots of hippocampal extracts

was analysed by ANOVA.

Results

Effect of prenatal stress and handling on behaviour

in the EPM

Adult PSNH displayed greater fear than CNH rats in theEPM. This was indicated by the shorter amount of time spentin the open arms and the smaller ratios of the number of open/total arms entries (Fig. 2). There was a significant interactionbetween maternal and neonatal treatment (F(1,43) = 4.61,p < 0.05), as H had a greater effect on PS than on control rats.A post hoc analysis showed that the ratio of open/total armsentries was significantly lower in PSNH rats than in eitherCNH or PSH (p < 0.05), and that there was no significantdifference between these ratios in CNH, PSH or CH rats.

General gene analysis

DNA GeneChip technology (Affymetrix) was used tocompare transcriptional profiles of PSNH and CNH and the

1020 Y. Bogoch et al.

Journal Compilation � 2007 International Society for Neurochemistry, J. Neurochem. (2007) 101, 1018–1030� 2007 The Authors

effect of H on them. The RAE 230A micro-array includes16 000 probes (representing �12 000 genes). Of 9505 validgenes (see Materials and methods section), 6.1% showed asignificant difference between the CNH and PSNH groups(p < 0.05). Of these genes, 48% were over-expressed and52% under-expressed in PSNH relative to CNH (total of 582genes). These gene sets were derived from a merger of thetop lists of independent analysis tools and included theoverlap between the lists (see Materials and methodssection). An unsupervised classification algorithm for twogroups completely separated CNH from PSNH rats. No suchseparation was achieved between CNH and CH, indicating

that H did not cause similar significant gene changes incontrol offspring.

Functional characterization of genes with reduced

expression in prenatally stressed rats

While the difference in behaviour between PS and controloffspring is more readily detected in intimidating situationslike the EPM (Fride and Weinstock 1988; Vallee et al.1997) and in the defensive withdrawal test (Ward et al.2000), it should be pointed out that the analysis of geneswas made in naıve rats. In addition, about 4 weeks hadelapsed between the exposure of the foetus to theconsequences of maternal stress and the measurement ofchanges in gene expression at 23 days of age. It cantherefore be expected that the absolute fold change in geneexpression under resting conditions would be relativelysmall. A common practice in such instances is to searchfor statistically sound coherence in cellular processes andpathways that accentuate such small changes. This wasaccomplished by means of functional categorization by GO(Camon et al. 2004), which allowed the unification ofgenes by their cellular biochemical process and cellularlocalization. Functional categorization by GO is availablefor about 40% of the genes and enabled us to reveal thecellular and biochemical functions of the genes thatshowed a significantly decreased expression as a result ofprenatal stress. Most of these genes are involved in the

Non-handled Handled 0.00

0.10

0.20

0.30

0.40

0.50 O

pen/

tota

l arm

s en

trie

s

*

#

Fig. 2 Behaviour of adult handled and non-handled control and PS

female rats in the EPM. Open bars: control; Grey bars: PS group.

*Significantly different from all other groups, p < 0.05. Each experi-

mental group comprises 11–12 rats.

Table 1 Enrichment of molecular function

categories based on GO annotation for the

genes that are significantly changed in

PSNH compared with CNH

Gene ontology terms Group Down (%) Down p-valuea Up (%) Up p-valuea

Transport A 15.0 0.00009 15.1 0.0017

Vesicle-mediated transport B 4.0 0.0017 4.3 0.0083

Cell physiological process A 20.4 0.0022 42.7 0.045

Cell growth and maintenance A 18.1 0.0035

Calcium ion transport C 2.7 0.0067

Cation transport C 5.3 0.0125 3.6 0.07

Cell–cell signalling A 4.4 0.0125

Synaptic transmission B 4.0 0.0130

Synaptic vesicle transport B 1.8 0.0131

Neuromuscular physiology A 4.0 0.0165

Transmission nerve impulse A 4.0 0.0165

Inorganic cation transport C 2.7 0.0216

Ion transport C 6.6 0.0234 5.7 0.0049

Metal ion transport C 4.4 0.0262 3.2 0.04

Organismal movement B 4.0 0.0297

Intracellular transport B 3.5 0.0363 5.4 0.007

Endocytosis B 1.8 0.0455 1.8 0.0455

Actin cytoskeleton A 3.23 0.001

Calmodulin binding B 2.51 0.0016

Actin binding A 2.51 0.024

aFisher’s test p-value implemented in enrichment score according to DAVID software with a

threshold of 0.05 (see Methods section). The functional groups are A, maintenance of growth; B,

trafficking of the secretory organelles and regulation of neurotransmitter release; C, membrane

potential and physiology of the neurons. Down and Up refer to down- and up-regulated genes in

PSNH relative to CNH, respectively.



following processes: (i) neuronal growth and structuralplasticity; (ii) regulation of membrane potential, mainly ionchannels and transporters; (iii) trafficking of synapticvesicle and the neurotransmitter release machinery. Table 1shows the degree of statistical enrichment according to GO

Table 2 Genes showing reduced expression following PS that was

reversed by H

IDa Gene name

1372847_at ACN9 homolog (Saccharomyces cerevisiae)

(predicted)

1368587_at Apolipoprotein C-I

1369122_at Bcl2-associated X protein

1373081_at Brain-specific angiogenesis inhibitor 1-associated

protein 2

1368437_at Carbonic anhydrase 4

1374252_at Centrosomal protein 1 (predicted)

1371395_at Chromobox homolog 3 (HP1 gamma homolog,

Drosophila)

1373256_at Chromodomain helicase DNA-binding protein 3

(predicted)

1372132_at CNDP dipeptidase 2 (metallopeptidase M20 family)

(predicted)

1368584_a_at Complexin 2

1370123_a_at Cortactin isoform B

1372514_s_at Dynein, axonemal, light chain 4 (predicted)

1374016_at Endothelial differentiation, lysophosphatidic acid

GPCR, 2

1370503_s_at Erythrocyte protein band 4.1-like 3

1376578_at Euchromatic histone methyltransferase 1 (predicted)

1367766_at Expressed in non-metastatic cells 2

1372662_at F-box protein 34 (predicted)

1374103_at Frequenin homolog (Drosophila)

1373170_at G protein pathway suppressor 2 (predicted)

1367954_at Glial cell line-derived neurotrophic factor family

receptor alpha 1

1371392_at Glucose phosphate isomerase

1370240_x_at Haemoglobin alpha, adult chain 1

1369868_at Implantation-associated protein

1388240_a_at Integrin alpha 7

1373500_at Leucine-rich PPR-motif containing (predicted)

1375726_at LIM domain only protein 7

1367923_at Lipidosin

1390827_at MAD homolog 3 (Drosophila)

1371607_at Microtubule-associated protein 4

1374468_at Myeloid differentiation primary response gene 88

1369948_at Nerve growth factor receptor-associated

(TNFRSF16) protein 1

1374075_at NSF attachment factor, SNAP, gamma (predicted)

1368261_at Neurexin 3

1371812_at P55

1388076_at PAI-1 mRNA-binding protein

1370214_at Parvalbumin

1371455_at Phosphomannomutase 1 (predicted)

1373243_at Phosphomevalonate kinase (predicted)

1388847_at Positive cofactor 2, multiprotein complex, Q-rich-

associated protein

1370187_at Propionyl coenzyme A carboxylase, beta polypeptide

1370746_at Protein kinase, cAMP dependent, catalytic, beta

(predicted)

1373741_at Pseudouridine synthase 1 (predicted)

1369564_at Rad- and gem-related GTP-binding protein 2

Table 2 Continued

IDa Gene name

1367596_at Ribosomal protein S26

1372240_at Sarcoglycan, alpha (dystrophin-associated

glycoprotein) (predicted)

1368058_at Scaffold attachment factor B

1370116_at Septin 3

1371425_at Serine/arginine repetitive matrix 1 (predicted)

1371487_at SH3 domain binding glutamic acid-rich protein-like 3

(predicted)

1373605_at Similar to 106 kDa O-GlcNAc transferase-interacting

(predicted)

1377656_at Similar to 2600016B03Rik protein (predicted)

1374362_at Similar to 4930566A11Rik protein (predicted)

1373958_at Similar to ALEX3 protein

1373945_at Similar to bruno-like 5, RNA-binding protein

1372157_at Similar to CGI-143 protein (predicted)

1371399_at Similar to over-expressed breast tumour protein

1371863_at Similar to phosphatidylglycerophosphate synthase

(predicted)

1370925_at Similar to potential phospholipid-transporting

ATPase IIB

1372054_at Similar to sex-determination protein homolog Fem1a

1369160_a_at Solute carrier family 4, sodium bicarbonate

cotransporter, 7

1368391_at Solute carrier family 7 (cationic amino acid

transporter), 1

1370042_at Stathmin-like 2

1369251_a_at Synapsin I

1369628_at Synaptic vesicle glycoprotein 2b

1368276_at Synaptophysin

1373865_at Synaptosomal-associated protein, 91 kDa homolog

(mouse)

1369058_at Synaptotagmin 3

1368417_at Synaptotagmin 5

1370691_a_at Thyroid hormone receptor alpha

1371695_at Translocated promoter region (predicted)

1373495_at Ubiquitin-conjugating enzyme E2, J1 (predicted)

1367481_at Vacuolar protein sorting 28 (yeast) (predicted)

1368641_at Wingless-related MMTV integration site 4

1372483_at Zinc finger protein 469 (predicted)

1390649_at Zinc transporter ZnT-3

aID, the Affymetrix GeneChip ID. The list is sorted alphabetically. The

following probes showed a similar trend in their relative expression, but

are associated with un-annotated ESTs supported sequences:

1371321_at, 1371465_at, 1371512_at, 1371954_at, 1372623_at,

1375108_at, 1379073_at, 1372313_at, 1372901_at, 1371472_at,

1372719_at, 1374968_at, 1375192_at, 1376400_at, 1384204_at,

1390132_at, 1391437_at, 1367508_at.



annotations (p < 0.05) for genes that were decreased orincreased, respectively, by prenatal stress compare withcontrol.

Functional characterization of genes that showed

recovery of their levels of expression following handling

The expression of about 30% of the genes (total of 93 genes),which were down-regulated by prenatal stress (Table 2) andwere significantly up-regulated by H satisfied the statisticalcriteria (p < 0.05). By contrast, these same genes remainedunchanged in the CNH group.

These genes were reanalysed by GOTM (Zhang et al.2004), a tool that presents a subset of the GO hierarchy that isassociated with statistically enriched functional categories.The results of the analysis are shown in Table 3.

The enrichment in GO annotations for the genes that wereanalysed is shown by a directed acyclic graph based on thehierarchy of these annotations. These genes play a role intrafficking in the synapse and in the neurotransmitter releasemachinery (Fig. 3; Table 3). By contrast, other genes thatwere down-regulated and linked to growth and electricalproperties of neurons were not restored by H. (Table 1).

Table 3 Enriched functional categories

according to GOTM of genes, the expres-

sion of which was altered in PSNH and

restored by H

Go term Obsa Expb Ratioc p-valued

Down-regulated (93 genes)

Biological process

Transmission of nerve impulse 7 2.37 2.95 0.008718

Synaptic transmission 7 2.25 3.11 0.006539

Regulation of neurotransmitter levels 4 0.66 6.06 0.004077

Neurotransmitter secretion 3 0.43 6.98 0.008711

Membrane organization and biogenesis 3 0.21 14.29 0.001184

Secretory pathway 6 1.34 4.48 0.002005

Exocytosis 4 0.57 7.02 0.002403

Calcium ion-dependent exocytosis 2 0.14 14.29 0.008633

Vesicle-mediated transport 7 2.41 2.9 0.009429

Synaptic vesicle transport 3 0.3 10 0.003289

Secretion 6 1.75 3.43 0.007488

Molecular function

Death receptor binding 2 0.04 50 0.000460

SNARE binding 2 0.15 13.33 0.009689

Syntaxin binding 2 0.12 16.67 0.006595

Cellular component

Cell projection 5 1.11 4.5 0.004762

Neuron projection 3 0.42 7.14 0.008148

Axon 2 0.15 13.33 0.009680

Cytoplasmic vesicle 5 1.33 3.76 0.009904

Cytoplasmic membrane-bound vesicle 5 1.33 3.76 0.009904

Coated vesicle 5 0.82 6.1 0.001243

Clathrin-coated vesicle 5 0.72 6.94 0.000697

Synaptic vesicle 5 0.44 11.36 0.000070

Synaptic vesicle membrane 2 0.12 16.67 0.006588

Synapse 5 0.99 5.05 0.002856

Up-regulated (125 genes)

Biological process

Transmembrane receptor protein tyrosine

phosphatase signalling pathway

2 0.11 18.18 0.005

Transport 27 16.16 1.62 0.003

Localization 29 18.5 1.57 0.004

Establishment of localization 29 18.41 1.58 0.003

Molecular function

Transmembrane receptor protein

tyrosine phosphatase activity

2 0.1 20 0.004

Cellular component

None

aObserved gene number in the GO category; bExpected gene number in the GO category; cThe

Exp/Obs enrichment. dSignificance of enrichment for the GO category that show a significant

p-value of <0.01.



Table 3 also shows the enrichment in GO annotations forthe 125 genes that were up-regulated by stress and reversedby H. As can be seen, they conform to only a small numberof GO terms that are not associated to any specific process,unlike those genes that were down-regulated by prenatalstress and reversed by H.

Relative expression of genes associated with the

pre-synaptic secretory apparatus

The prevalent functional category that was restored by H isassociated with the structure and function of the synapses.RT-PCR of various genes that have been linked to vesicletrafficking and organization of the pre-synaptic secretoryproteins, e.g. stathmin like-2, rab3A, SV2b and to a lesserextent synaptotagmin 5, validated the results obtained fromthe GeneChip, which were changed by prenatal stress and H,as well as those that were unchanged. The latter groupincluded genes that are involved in dendritic maturation(staufen) and in the regulation of the active zone (Rim1).Results of the RT-PCR reactions and the expression profilefor eight representative genes are shown (Fig. 4).

Change in expression of major pre-synaptic proteins

Prenatal stress significantly reduced the levels of synapto-physin, synaptotagmin 1 and synaptojanin as shown bywestern analysis (Fig. 5). H only restored the expression ofthe first two proteins (Figs 5a and b). By contrast, there wereno detectable changes in synapsin 1 and rab3A proteins,despite changes in the levels of gene expression. Synapsin 1is associated with a pre-synaptic cytoskeletal adaptor ofsynaptic vesicles and rab3A, with the regulation of theirpriming (Sudhof 2004). In agreement with the data from theGeneChip analysis, there was no difference in the expressionof soluble N-ethylmaleimide-sensitive factor receptors

(SNAREs), syntaxin 1A, SNAP-25 and VAMP-2 in PSNH,PSH or CNH and CH rats. These results indicate that somebut not all the changes in proteins match the profile of thoseat the mRNA level. They also show that the effects ofprenatal stress and H are very specific and cannot beattributed to a global change in synaptic proteins or in thenumber of synapses.

Discussion

The major new finding in this study is the identification ofspecific genes and proteins in the hippocampus of naıvejuvenile female rats that show altered expression as a resultof prenatal stress. The hippocampus was chosen because ofits important role in the aetiology of anxiety and depressionand because it shows specific structural changes both inhumans (Campbell and MacQueen 2004) and in experimen-tal animal models of these conditions (Lemaire et al. 2000;Coe et al. 2003). By performing the study in weanling rats,we attempted to limit any gene changes to those resultingfrom increased levels of stress hormones in the maternalmilieu and early postnatal rearing influences. We found thatdifferences in the behaviour of PS females from that ofcontrols in the EPM could be detected at this early age with asmaller variance than that in adults, probably because of theabsence of an influence of the oestrus cycle (Marcondeset al. 2001).

Having identified a number of genes that were altered byprenatal stress, we focussed on the genes that no longerdiffered from those of controls in rats that had been subjectedto H, as this manipulation prevented the development ofanxiogenic behaviour. Of 582 genes that were altered byprenatal stress, 52% were under-expressed and 48% over-expressed relative to controls. We did not include these genes

Biological process

Physiological process

Cellular process

Organismal physiological process

Localization

Cell communication Cellular physiological

process

Neurophysiological process

Cell–cell signaling

Cell organization & biogenesis

Transmission of nerve impulse

Secretion Transport Membrane organization

and biogenesis

Synaptic transmission Secretory pathway

Regulation of neurotransmitter

levels

Regulated secretory pathway Synaptic vesicle

transport

Exocytosis

Neurotransmitter secretion Calcium ion-dependant exocytosis

Establishment of localization

Vesicle mediated transport

Fig. 3 A diacyclic graph according to

GOTM. In bold and grey background are

the enriched gene ontology (GO) (p < 0.01)

belonging to ‘biological process’ category.

For details see Table 2. Only 42.5% of the

genes differentially expressed between the

CNH and PSNH rats are annotated by GO.



in the present analysis, as they were unaltered by H andtherefore less likely to contribute to the anxiogenic beha-viour.

Genes that were down-regulated by PS relative tocontrols were found by GO annotation to fall into threemain functional categories. These participate in; (i) devel-opment and axonal growth; (ii) function of ion channels,transporters and proteins that define the physiology of the

neurons and (iii) trafficking of secretory organelles andregulation of neurotransmitter release (Table 1). About30% of them were fully restored by H and are mainlyassociated with trafficking and regulation of neurotrans-mitter release.

Several complementary processes could be involved in theprevention of the anxiogenic effect of prenatal stress by H: (i)induction of new synapses; (ii) acceleration of axonaltransport to the nerve terminal; (iii) reshaping of post-synaptic sites; (iv) reshaping of pre-synaptic sites; (v)activation of neurotransmitter release. However, our resultsshow that H mainly affects processes (iv) and (v). It is alsoevident that the genes affected by H are not restricted tosynaptic vesicle proteins (i.e. SV2b, synaptophysin), butinclude synaptic vesicle reorganizer proteins (i.e. synapsin)and those that directly regulate neurotransmitter release (i.e.synaptotagmin, rab3A, neurexin, complexin). Table 4 showsthe role and the interacting partners for 22 representativegenes that are associated with the GO term synaptic vesiclesand synaptic transmission.

Stathmin2

L19

L19

Rab3A

RimsStaufen

Tagmin5

Sv2b

0

0.2

0.4

0.6

0.8

1

1.2

1.4

Rel

ativ

e ex

pres

sion

L19StathminRab3bTagmin5Sv2bRimsStaufenArc

PSHPSNHCNH(a)

(b)

PSHPSNHCNH

Fig. 4 Reverse transcriptase PCR (RT-PCR) for selected genes.

RT-PCR reaction products are shown following separation in 1.5%

agarose gels and ethidium bromide staining. (a) Representative re-

sults for a number of selected genes – stathmin-like 2, rab3A, syn-

aptotagmin 5 (tagmin 5), SV2b, staufen (RNA localization) and Rims

(Rab2 interaction, active zone protein) are shown. The level of the

housekeeping ribosomal L19 gene is used for calibration and as a

reference to test the relative expression. (b) The expression levels of

specific amplimers were determined by densitometry relative to L19

expression. The plot summarizes results for the genes shown in (a)

and Arc (activity-dependent cytoskeleton-associated gene). Each

gene from each group (CNH, PSNH and PSH) was measured twice

and the average value is plotted.

β -tubulin

Synaptophysin

SNAP-25

Synaptotagmin-1

Synaptojanin

(a)

0

20

40

60

80

100

120

Ch

ang

e (%

)

(b)

*

#

PSH PSNH CNH

PSH PSNH CNH

Fig. 5 Western blot analysis of hippocampal protein extracts from

CNH, prenatally stressed non-handled (PSNH) and PSH groups. (a)

b-Tubulin is used as a reference for unchanged protein. SNAP-25

showed no difference in expression in the different groups. Synapto-

physin, synaptotagmin 1 and synaptojanin showed a significant

reduction in PSNH, but were not reversed by H. (b) The average

differential expression (triplicate) for synaptophysin is shown. Signifi-

cantly different from the CNH, *p < 0.005; significantly different from

the PSNH #p < 0.01. There was no significant difference between the

CH, CNH and PSH groups.



Tab

le4

Are

pre

senta

tive

list

of

genes

that

show

ed

reduced

expre

ssio

nas

are

sult

of

pre

nata

lstr

ess

No.

inF

ig.

6a

Gene

nam

es

Gene

sym

bol

No.

infa

mily

bR

efe

rence

Cellu

lar

pro

cess

(pro

tein

inte

raction)

1S

Vgly

copro

tein

2B

SV

2B

3Lazzell

et

al.

(2004)

Synaptic

vesic

leexocyto

sis

(synapto

tagm

in1)

2S

ynapto

tagm

in5

SY

T5

16

Ste

vens

and

Sulli

van

(2003)

Ca

2+

sensor,

traffi

ckin

g,

exo–endocyto

sis

(Rab27,

Ca

2+

channel)

3S

ynapto

tagm

in3

SY

T3

16

Ste

vens

and

Sulli

van

(2003)

Ca

2+

sensor,

exo–endocyto

sis

(Syt9

,C

a2+

channeland

neure

xin

)

4S

ynapto

physin

/p38

SY

N4

Valtort

aet

al.

(2004)

Synaptic

vesic

leta

rgeting,

bio

genesis

,exo-e

ndocyto

sis

(Vam

p,

physophili

n)

5V

esic

le-a

ssocia

ted

mem

bra

ne

pro

tein

1V

AM

P1

8S

udhof

et

al.

(1989)

Vesic

le-m

edia

ted

transport

(Snap25,

synta

xin

1)

6V

esic

le-a

ssocia

ted

mem

bra

ne

pro

tein

7V

AM

P7

8M

art

inez-A

rca

et

al.

(2003)

Vesic

le-m

edia

ted

transport

(Snap25,

Vam

p2)

7B

locked

early

intr

ansport

1B

ET

11

Mossessova

et

al.

(2003)

ER

toG

olg

itr

ansport

(Sec22)

8H

untingtin-a

ssocia

ted

pro

tein

1H

AP

11

Kittler

et

al.

(2004)

Synaptic

transm

issio

n(h

untingtin,

cynactin

1)

9P

ota

ssiu

mvoltage-g

ate

d

channelsubfa

mily

H

KC

NH

18

Jeng

et

al.

(2005)

Ion

transport

(pota

ssiu

mchannelre

gula

tor

1)

10

Rab2

RA

B2

40

Short

et

al.

(2001)

ER

toG

olg

ivesic

le-m

edia

ted

transport

(GD

Pdis

socia

tion

inhib

itor

2)

11

c-S

NA

PN

AP

G3

Chen

et

al.

(2001)

Fusio

n,

vesic

letr

affi

ckin

g(G

AF

-1,

NS

Fand

RIP

11)

12

Com

ple

xin

2/s

ynaphin

CP

LX

24

McM

ahon

et

al.

(1995)

Synaptic

vesic

leexocyto

sis

(SN

AR

Es,

synta

xin

)

13

Fre

quenin

hom

olo

gF

RE

Q5

O’C

alla

ghan

et

al.

(2005)

Ca

2+

bin

din

g,

transm

issio

nre

gula

tion

(KC

ND

2)

14

Cort

actin

isofo

rmB

CT

TN

1R

acz

and

Wein

berg

(2004)

Pro

tein

inte

raction

(d-c

ate

nin

,dynam

in1)

15

Sta

thm

in-lik

e2/S

CG

10

ST

MN

24

Liu

et

al.

(2002)

Neurite

outg

row

th,

cyto

skele

ton

(RG

S6)

16

Kin

esin

1K

IF5b

25

Kanaiet

al.

(2004)

Mic

rotu

bule

-based

movem

ent

(merlin

)

17

Bra

in-s

pecifi

cangio

genesis

inhib

itor

1-a

ssocia

ted

pro

tein

2

BA

P2

2O

da

et

al.

(1999)

Neurite

outg

row

th,

cyto

skele

ton

(Atn

1,

Bal1

,

Enah,

Eps8,

Shank1–3

and

Wasf1

–2)

18

Synapsin

1S

YN

13

Paggiand

Petr

ucci(1

992)

Cyto

skele

ton

(Nos1,

Rab3

and

F-a

ctin)

19

Neure

xin

1N

RX

N1

4D

ean

and

Dre

sbach

(2006)

Axon

guid

ance

(neuro

ligin

1)

20

SN

AP

91/A

P180

SN

AP

91

4S

ousa

et

al.

(1992)

Endocyto

sis

(cla

thrin,

AP

2)

21

Am

phip

hysin

AM

PH

1S

hang

et

al.

(2004)

Synaptic

transm

issio

n(d

ynam

in1)

22

Synapto

janin

1S

YN

J1

Irie

et

al.

(2005)

Synaptic

vesic

leendocyto

sis

(am

phip

hysin

)

aG

enes

liste

dare

those

that

were

resto

red

by

H(b

old

,gre

ybackgro

und)

and

the

rest

were

unaff

ecte

dby

H.

bT

he

num

bers

of

fam

ilym

em

bers

that

are

dete

cte

din

mam

mals

,exclu

din

galtern

ative

splic

ed

variants

.



The assembly of a functional synapse is a fully coordi-nated sequential process that is sensitive to cues from theenvironment (Ziv and Garner 2004). Neurotransmitter releaseis needed for the fine tuning and maintenance of synapticconnections (Verhage et al. 2000) and may be modulated bya few genes such as synaptotagmin 1, synaptophysin andSV2b, all of which were restored by H. These major synapticvesicle proteins participate in multiple protein–protein inter-actions in the synapse (Table 4). Their relative amountsdetermine whether a synaptic vesicle will be engaged into aproductive cycle of docking and fusion. Synaptophysindetermines the accessibility of VAMP-2 to the SNAREcomplex (McMahon et al. 1996) and SV2b determines theaccessibility of synaptotagmin 1 to the SNAREs and the Ca2+

sensitivity of the fusion complexes (Lazzell et al. 2004). Amodel that shows the location and function of representativegenes that were altered by prenatal stress and H is shown(Fig. 6). Although the data do not reveal the identity of the

synapses and neurotransmitters, other studies have shownthat alterations occur in the release of noradrenaline,dopamine, serotonin and glutamate in PS rats (Weinstock2001; Berger et al. 2002).

Several groups of genes down-regulated by prenatal stresswere not restored by H. These include genes that areimportant for axonal transport [i.e. myosin V, kinesin-likeproteins (Okada et al. 1995)], shaping of the post-synapticsites, neurotransmitter receptors, scaffolding proteins con-taining PDZ domains (Kim and Sheng 2004) and neuronalSNAREs. SNARE proteins are transported in vesicles that donot contain other synaptic vesicle proteins (Okada et al.1995) and their accumulation in the growth cone precedessynaptic vesicle protein transport and the maturation of afunctional synapse (Igarashi et al. 1997). The highest changein expression in PSNH relative to CNH was obtained for ionchannels including pre-synaptic voltage-gated Ca2+ type P/Qand several K+ channels. These ion channels regulate theelectrical properties of the neuron, and their down-regulationsuggests a potential decrease in excitability and electricalproperties of the newly formed synapses. However, thesealso were unaffected by H, suggesting that they are lessimportant in determining anxiogenic behaviour in PS rats.Other genes that failed to show a significant level of recoveryfollowing H include synaptojanin, ampiphysin, caveolin thatplay a central role in synaptic vesicle endocytosis (Fig. 6). Ina mature synapse, endocytosis is coupled to the exocytoticmachinery (Linial and Parnas 1996). It is possible that H didnot restore the activity of the endocytotic apparatus, becauseit matures at a later age than that at which our experimentswere performed. Indeed, endocytotic proteins were shown tobe expressed long after the biogenesis of the synapticvesicles and following their accumulation in the synapse(Grabs et al. 2000). We will investigate this possibility bytesting the level of gene expression of this machinery at alater stage of development (12 weeks).

Similar to other studies (Weaver et al. 2006), wholehippocampus was used to monitor changes in gene expres-sion, enabling us to detect genes, proteins and processes thatare differentially expressed as a result of prenatal stress andH. We expect to gain additional information by performingthe analysis in discrete hippocampal regions (Lein et al.2004). To our knowledge, there is only one other report ofthe effect of prenatal stress on gene expression that wasperformed on RNA from the prefrontal cortex of adult males.Its aim was to determine whether there were any similaritiesin the molecular changes that were found in PS rats to thoseseen in schizophrenic and bipolar patients (Kinnunen et al.2003). In spite of the differences in experimental design,gender of the animals, their age and the brain area from thosein the current study, some notable similarities were seenamong the genes that showed a decreased expression as aresult of prenatal stress. These include complexin 2, BDNF,synapsin and ionotropic NMDA 2A receptor. In contrast to

Plasma membrane & endocytosis related proteins

Synaptic vesicle/secretory vesicle membrane proteins

SNARE regulators & ion channels

Cytoskeleton related proteins

Synaptic vesicle

15

17

16

18

Transport organelle

1 2

34567

9

810

11

12

13

14

19

2120

Fig. 6 Schematic representation of a pre-synaptic site indicating

genes that were detected in the current study. A scheme of a synaptic

vesicle and a generic transport organelle is shown. Various proteins

are marked according to their location and function. In bold and grey

are shown those genes that showed a reduced expression as a result

of prenatal stress that was restored by H. The rest were down-regu-

lated by prenatal stress but not restored by H. For additional infor-

mation of these genes see Table 4.



those reported (Kinnunen et al. 2003), we did not detect asignificant change in the expression of genes that make upthe post-synaptic density.

In conclusion, our study shows that prenatal stressoccurring during a critical period of foetal developmentalters the expression of a well-defined set of hippocampalgenes in association with permanent changes in behaviour.Using the procedure of neonatal H, we were able to focus ona defined set of genes, the expression of which was restoredto control levels in association with normalization ofbehaviour. The genes and proteins identified in this wayplay an important role in regulating the rate of pre-synapticmaturation. It remains to be determined whether the changesinduced by prenatal stress in gene expression and behaviourresult from exposure of the foetal brain to excess levels ofmaternal stress hormones and/or changes in maternal atten-tion during the early postnatal period.

Acknowledgements

We thank Yaniv Bledi for helpful suggestions. Analysis of the

Affymetrix data was performed at the Genomic Sequencing Centre

at the Hebrew University. Y.B awarded a fellowship from SCCB, the

Sudarsky Centre for Computational Biology, The Hebrew Univer-

sity of Jerusalem.

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