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Repeated restraint stress and corticosterone injections during late pregnancy alterGAP-43 expression in the hippocampus and prefrontal cortex of rat pups

Nuanchan Jutapakdeegul a,*, Szeifoul Afadlal a, Nongnuch Polaboon b, Pansiri Phansuwan-Pujito c,Piyarat Govitrapong a,d,e

a Neuro-Behavioral Biology Center, Institute of Molecular Biosciences, Mahidol University, Nakornpathom 73170, Thailandb Faculty of Allied Health Sciences, Christian University, Nakornpathom 73000, Thailandc Department of Anatomy, Faculty of Medicine, Srinakhrinwirot University, Bangkok, Thailandd Center for Neuroscience, Faculty of Science, Mahidol University, Bangkok, Thailande Department of Pharmacology, Faculty of Science, Mahidol University, Bangkok, Thailand

1. Introduction

Glucocorticoids have important roles in normal maturation ofthe developing brain such as maturation of nerve terminal,remodeling axons and dendrites, and the cell survival (Meyer,1983; Korte, 2001). However, exposure to high level of gluco-corticoids in utero have widespread acute effects upon neuronalstructure and may permanently alter developmental trajectories ofhippocampus and other brain structures of the offspring (Mat-thews, 2000; McEwen, 2000; Weinstock, 2001; Welberg et al.,2001; Antonow-Schlorke et al., 2003). In rhesus monkeys,treatment with antenatal dexamethasone caused a dose depen-

dent neuronal degeneration of hippocampal neuron and reducedhippocampal volume which persisted at 20 months of age (Unoet al., 1990). Childhood abuse has been associated with reductionin hippocampal volume in adults (Bremner et al., 1997; Stein et al.,1997; Driessen et al., 2000; Vythilingam et al., 2002), but not inchildren (Carrion et al., 2001; De Bellis et al., 2001). Moreimportantly, animal studies have clearly indicated that exposure tovariable types of stressors during development produces persis-tent behavioral defects that are associated with hormonal,neurotransmitter and functional changes resemble an array ofpsychopathological conditions (Huttunen, 1997; Heim et al., 2004;Howes et al., 2004).

During postnatal period, there is a marked overproduction ofaxons, dendrites, synapses, and receptors (Rakic, 1991) followed bya period of rapid elimination, or pruning, between puberty andadulthood. Up to 50% of synapses and receptors are lost in both

Int. J. Devl Neuroscience 28 (2010) 83–90

A R T I C L E I N F O

Article history:

Received 3 April 2009

Received in revised form 1 September 2009

Accepted 15 September 2009

Keywords:

Prenatal stress

Corticosterone

GAP-43

Hippocampus

Prefrontal coretx

A B S T R A C T

In the offspring of prenatal stress animals, overactivity and impaired negative feedback regulation of the

hypothalamic–pituitary–adrenal axis are consistent finding. However, little was known about how

prenatal stress can permanently alter developmental trajectories of pup’s brain. Growth-associated

protein-43 (GAP-43) is a presynaptic membrane phosphoprotein whose expression increases during

developmental events such as axonal outgrowth or remodeling and synaptogenesis. Phosphorylation of

GAP-43 by protein kinase C was correlated with enhanced axonal growth and transmitter release. In

adult animals, increase of GAP-43 correlated with monoaminergic deficit in neuropsychiatric disorders.

The present study examines the effects of repeated maternal restraint stress on the level of GAP-43 in the

brain of rat pups. The results showed that prenatal stress significantly increased GAP-43 level in the PFC

of rat pup during PND 7–14 as compared to control but not significant difference when observed at PND

21. Increased GAP-43 expression was also observed in the pup’s hippocampus during the same postnatal

periods. However, when observed at PND 60, pups born from stressed mother showed a significant lower

(p < 0.001) GAP-43 expression as compare with control group. These changes indicate the direct effect of

corticosteroid hormone, since repeated maternal injection with corticosterone (CORT, 40 mg/kg) during

GD 14–21 also gave the same results. PND 7–14 is the peak period of synaptogenesis in these brain areas

and abnormal axon sprouting and reorganization may lead to a defect in synaptic pruning at later stage

of life. The results suggested that maternal stress is harmful to the developing brain and upregulation of

GAP-43 indicated a protective mechanism against the toxicity of maternal stress hormone. Prenatal

stress alter the normal developmental trajectories in the pup’s brain may underlies the mechanism link

between early life stress and neuropsychopathology in later life.

� 2009 ISDN. Published by Elsevier Ltd. All rights reserved.

* Corresponding author. Tel.: +66 2441 9321.

E-mail address: grnjg@mahidol.ac.th (N. Jutapakdeegul).

Contents lists available at ScienceDirect

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journa l homepage: www.e lsev ier .com/ locate / i jdevneu

0736-5748/$36.00 � 2009 ISDN. Published by Elsevier Ltd. All rights reserved.

doi:10.1016/j.ijdevneu.2009.09.003

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cortical (Huttenlocher, 1979; Andersen et al., 2000) and subcorticalregions (Teicher et al., 1995). The time course and degree of pruningvaries between regions. It is hypothesized that adaptation to stressin young animal, in which the neuronal development has not yetbeen completed, can be invariably different from that occur in adultanimals. In fact, the timing of early life stress can interfere withspecific developmental stages in specific brain area that might beselectively involved in the manifestation of the disease in later life.Virtually nothing is known about the effects of prenatal stress on thedevelopmental trajectories of axons in the brain of the offspring.

The hippocampus is a brain region that has protracteddevelopment (Giedd et al., 1996), and has high density ofglucocorticoid receptors. It appears to be a brain region thatespecially vulnerable to the effects of stress. Early exposure to stressor corticosteroids can cause hippocampal atrophy, decreases indendritic branching and vulnerable to the subsequent insult as adult(Sapolsky, 2000). On the opposite way, stroking pups with a softbrush or handling over the first week of life results in increasedglucocorticoid receptor (GR) expression in the hippocampus andprefrontal cortex which can further modify HPA reactivity (Meaneyand Aitken, 1985; Weaver et al., 2000; Jutapakdeegul et al., 2003).Much less is known about the effects of early life stress on thedevelopment of prefrontal cortex. The prefrontal cortex has a veryprotracted ontogeny (Alexander and Goldman, 1978) and isspecifically activated by stressors (Bannon et al., 1983; Deutchet al., 1991), and in primates may have a higher density ofglucocorticoid receptors than the hippocampus (Sanchez et al.,2000). In addition to their apparent sensitivity, the hippocampusand the prefrontal cortex play important roles in memory andexecutive function. We have hypothesized that prenatal stress couldaffect the development of hippocampus and prefrontal cortex,possibly resulting in accelerated but attenuated development.

Growth-associated protein of 43 kDa (GAP-43) is an intracellularprotein that contributes to the guiding mechanism of axonaloutgrowth in embryo, establishment and reorganization of synapticconnections during development (Hrdina et al., 1998) and to thesprouting and axonal regeneration in adult (Benowitz and Routten-berg, 1997). GAP-43 is abundant in axonal growth cones ofdeveloping CNS when axons are actively growing (McGuire et al.,1988; Dani et al., 1991) and in sprouting axons of the adult CNS(Benowitz et al., 1990) as well as in presynaptic nerve terminals(Eastwood and Harrison, 2001; Chen etal., 2003). InPC 12 cells,nervegrowth factor (NGF) elicits neurite outgrowth which is accompaniedby increased expression of protein GAP-43 (Karns et al., 1987). Oncegrowing axons have reached their targets and synaptogenesis iscompleted, protein GAP-43 levels decline sharply in most neurons(McGuire et al., 1988; Dani et al., 1991). Experimental andneuropathological lesions have been reported to associate withalteration of GAP-43 expression (Eastwood and Harrison, 1998).

The aim of the present study was to test the hypothesis thatearly life exposure to stress hormone might alter normaldevelopmental trajectories of brain and produce the delay effectson brain structure and function. Since the hippocampus andprefrontal cortex have been reported to be the potential targets ofcorticosteroid hormone, the present study quantitatively analyzedthe effect of early life exposure to stress hormone on GAP-43protein expression in these brain areas of the rat offspring.

2. Experimental procedures

2.1. Experimental animals

Adult female Sprague Dawley rats, weighing 200–250 g, and their offspring were

used in this experiment. In order to avoid sex difference in the effect of prenatal

stress, equal numbers of male and female pups in each group were used in this

experiment. Rats were obtained from the National Experimental Animals Center of

Mahidol University, Salaya, Thailand and housed in single housing condition in a

temperature- and humidity-controlled environment and maintained on a 12-h

light/dark cycle with free access to food and water. Each pregnant female was

weight on gestation day (GD) 7–21 before any other manipulation. On the morning

of GD 21, each pregnant female was received nesting material, and there after the

cage was checked twice daily for the appearance of a litter. The day a litter was

discovered was designated as postnatal day 0 (PND 0) and the length of gestation

was noted. All procedures were carried out in accordance with the NIH Guidelines

on the Care and Use of Animals and the animal study protocol were approved by the

Experimental Animal Ethics Committee of The Institute of Molecular Biosciences,

Mahidol University, Thailand. Every effort was taken to minimize the number of

animals used in the experiment.

2.2. Maternal restraint stress

Pregnant rats were randomly divided into two groups: (1) control group, and (2)

prenatal stress (PS) group. In this experiment, pregnant rats in PS group were

restrained by placing them individually into the restrainer, a Plexiglas cylindrical

cage, in which the diameter and length can be adjusted to accommodate the size of

each animal. This will result in restricted mobility and aggression. Restraint stress

for many hours has been widely accepted as an animal model to induce both

psychological and physical stress by increasing corticosteroid or stress hormone

(Zuena et al., 2008). The immobilization was performed during the dark phase of the

cycle. Each pregnant rat was restrained for 4 h per day during GD 14–21 as

previously reported (Beyer and Chernoff, 1986; Ramakers et al., 1995; Miyahara

et al., 2000; Yamamoto et al., 2003; Zaidi et al., 2003; Conrad et al., 2004;

Rosenbrock et al., 2005; Peruzzo et al., 2008). During restraint period, the animal’s

behavior were observed every half an hour, if pregnant rats show sign of restless or

suffering, it will be free and not include in the experiment. Control mothers were

left undisturbed for the duration of their pregnancies as previously described (Cai

et al., 2008; Zuena et al., 2008; Fumagalli et al., 2009; Lucassen et al., 2009).

Gestation days 14–21 were selected because this is the most sensitive period to the

teratogenic effects of prenatal stress, moreover, this is the period of neurogenesis of

pyramidal and non-pyramidal cells in the cortex (Fride and Weinstock, 1984).

2.3. Maternal corticosterone treatment

Pregnant rats were randomly divided into two groups: (1) control group, and (2)

corticosterone treatment (CORT) group. Corticosterone (C2505, Sigma–Aldrich Inc.,

USA) was freshly prepared prior to use by suspension in pure sesame oil. Pregnant

rat in CORT group were injected intrasubcutaneously with CORT (40 mg/kg) during

GD 14–21 while pregnant rats in control group were received intrasubcutaneously

injection with equivalent volume of vehicle. The injections were done at the

beginning of dark phase of the cycle. Rat pups at PND 7 and 14 (n = 3 for each group)

were used for western blotting studies.

2.4. Immunohistochemical studied of GAP-43

The prefrontal cortex and hippocampus of rat pups at PND 0, 7, 14, 21 and 60

(n = 8 for each group) were used for study of GAP-43 immunoreactivity (IR). Rats

were deeply anesthetized with sodium pentobarbital (30 mg/kg) and transcardi-

cally perfused with 0.1 M phosphate-buffered saline (PBS, pH 7.4), followed by 4%

paraformaldehyde in 0.1 M phosphate buffer. Brains were rapidly removed and

postfixed in the same fixative at 4 8C overnight, then cryoprotected with 30%

sucrose in 0.1 M PBS, at 4 8C. Coronal sections (30 mm) were cut with cryostat. Free

floating sections were kept in 0.1 M PBS at 4 8C for immunoperoxidase staining.

Immunostaining was performed using mouse monoclonal anti-growth-associated

protein-43 antibody (G9264, Sigma–Aldrich Inc., USA) which recognizes an epitope

present on kinase C domain in the N terminal of GAP-43 protein. First of all, the

sections were rinsed 2 � 5 min with 0.1 M PBS, then pre-treated with 1% H2O2 for

10 min and rinsed for 5 min with 0.1 M PBS containing 1% BSA and 0.3% Triton X-

100. Then, the sections were washed 3 � 10 min in 0.1 M PBS and blocked with 5%

normal horse serum diluted in PBS-A (PBS containing 0.25% BSA and 0.1% Triton X-

100) for 30 min at room temperature. Sections were then incubated with primary

antibodies (1:12,000) at 4 8C overnight and washed with PBS prior to incubation

with biotinylated goat anti-mouse IgG (SC-2039, Santa Cruz Biotechnology Inc.,

USA) (1:200), for 30 min at room temperature followed by PBS washed. Sections

were then incubated for 1 h at room temperature in avidin–biotin complex (1:50)

(Vectastain Elite ABC kit; Vectastain1, Vector Laboratories, USA), rinsed three times

in PBS and reacted for peroxidase activity with 0.025% DAB solution (Sigma–Aldrich

Inc., USA) containing 0.01% H2O2 in 0.05 M Tris–HCl buffer (pH 7.6) for 10 min.

Finally, sections were rinsed with distilled water for 2 � 5 min, dehydrated in an

ethanol gradient, cover slip and observed under the light microscope. Tissue

sections from both control and PS group were processed in the same condition and

were stained at the same time throughout the studied. The specificities of antisera

against GAP-43 were performed by absorption control on adjacent sections.

Sections were treated with immunoperoxidase as described above, except that the

primary antibody solution was substituted with the pre-absorbed solution

consisting of the mixture of the specific antiserum diluted 1:12,000 with

1000 mM synthetic peptides of GAP-43 and shaken overnight at 4 8C. GAP-43

immunogen peptide (SC-4507) corresponding to amino acids 1–100 of GAP-43 was

purchased from Santa Cruz Biotechnology Inc., USA.

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2.5. Western blot analysis of GAP-43

Brain regions were immediately dissected out, frozen on dry ice and stored at

�80 8C. Dissections were performed according to The Rat Brain in Stereotaxic

Coordinates (Paxinos and Watson, 2007). In details, the prefrontal cortex was

dissected from 2-mm thick slices (prefrontal cortex defined as Cg1, Cg3, and IL

subregions corresponding to the Plates 6–9 (approximately weight 8 mg), whereas

hippocampus (including both ventral and dorsal parts) was dissected from the

whole brain. The protein concentration was determined by Lowry method.

Appropriate amount of protein samples (10 mg) were denatured in sample buffer

(62.5 mm Tris–HCl pH 6.8, 2% SDS, 10% glycerol, 2% mercaptoethanol, and 0.01%

bromophenol blue) at 100 8C for 5 min. Proteins were loaded onto 10% SDS-PAGE

and then electrophophoretically transferred to a nitrocellulose membranes

(Amersham Bioscience, Piscataway, NJ, USA). The transfer efficiency was checked

by Ponceau-S red staining. The membrane was washed with Tris-buffered saline

(TBS) for 5 min, then incubated in blocking buffer (5% nonfat milk in TBS containing

0.1% Tween-20, TBST) for 1 h at room temperature and incubated overnight at 4 8Cwith rabbit polyclonal anti-GAP-43 antibody (AB-5312, Chemicon International

Inc., Temecula, CA, USA) (1:2000) or mouse polyclonal anti-b-actin antibody

(1:2000) from Chemicon International Inc., Temecula, CA, USA. Then, membranes

were washed three times with TBST and incubated in a 1:5000 dilution of

peroxidase conjugated horseradish secondary antibody for 1 h at room tempera-

ture. After that, they were washed three times with TBST and incubated with ECL

(Amersham Biosciences, Piscataway, NJ, USA) for 5 min and the band density was

captured on an X-ray film (Kodak, Rochester, NY, USA). The immunoblot band

densities were quantified using Scion image program (National Institutes of Health,

Bethesda, MD, USA).

2.6. Image analysis

The stained sections were observed under a light microscope (the Nikon Eclipse

E400, Nikon, Japan) and were photographed. In both control and PS group, the

pictures were analyzed in the same way. In all cases, the photographs were taken

from the middle of each layer and three continuous, non-overlapping frames, were

photographed in each layer from each section with total 10 sections for each rat.

Thus, each mean value represented the mean value of 30 photographs from each rat

(n = 8 rats for each group). Photographs from the selected area were captured by

CCD color camera and were transformed into digits. Total areas of each frame were

estimated using Adobe Photoshop 9.0 and the percent density of GAP-43 IR were

measured from each picture using UTHSCSA Image Tools software (Version 3.0)

which can be downloaded from: http://ddsdx.uthscsa.edu/dig/download.html.

2.7. Statistical analysis

The data were expressed as the mean � S.E.M. Changes produced by maternal

stress were analyzed in the different brain regions. Difference between control and PS

group in the same brain region was analyzed by Student’s t-test (unpaired, unless

otherwise stated). A probability level of p < 0.05 was considered statistically

significant difference between the two sets of data.

3. Results

3.1. Characteristic of GAP-43 immunostaining

In the cortex of neonatal rat, GAP-43 immunoreactivity (IR) wasgenerally observed in the nerve fibers and neuropil. It was foundthroughout the neuronal processes of all neuronal cell types both

pyramidal cells and non-pyramidal cells. The characteristic of GAP-43 IR demonstrated a granular staining in the neuropil, and notobserved in the cell body of all neuronal cell types (Fig. 1A).Specificity of GAP-43 antibody was tested by pre-absorption of theantibody with GAP-43 peptide and result in completely abolishesof the immunostaining as shown in Fig. 1B.

3.2. Maternal restrained stress increased GAP-43 expression in the

prefrontal cortex of rat pups during PND 7–14

We performed an immunohistological staining of GAP-43 in theprefrontal cortex of normal rat pups at different postnatal periodfrom PND 0 to 21 (Fig. 2, control panel). In control rat pups, GAP-43IR was observed throughout the PFC at very low levels at birth andremarkable increased during the second week of life. The density ofGAP-43 reached the highest level during PND 7–14, then,progressively declined until PND 21 (data not shown). FromPND 21, GAP-43 IR was slightly constant and reaches the adultlevel.

We then examined whether prenatal stress (PS) alters thespatial and temporal pattern of GAP-43 expression in theprefrontal cortex of neonatal rat. We found that, maternalrestrained stress increased of GAP-43 IR in the prefrontal cortexof rat pups at birth (Fig. 2, PS panel). The effect was markedlyobserved at PND 7 and 14, in which GAP-43 IR was increasedthroughout the cortical layer. We then observed GAP-43 IR in layerV in PFC under higher magnification (Fig. 3A–D), and measured thedensity of GAP-43 IR in this cortical layer. We found that, at PND14, the percent density of GAP-43 IR in layer V of PFC wassignificantly increased in PS pups as compared with control pups(p < 0.0001). However, at PND 21, GAP-43 IR in layer V did notshowed any significant difference between control and PS group.

3.3. Maternal restrained stress increased GAP-43 expression in the

hippocampus of rat pups

Since PND 7–14 was the period that we can observe an obviouseffect of PS on GAP-43 IR in the pup’s brain, we further examinedthe effect of PS on GAP-43 expression in the hippocampus at thesetime points. Visual inspection of GAP-43 IR at PND 7 and 14showed the same results that maternal restrained stress increasedGAP-43 expression in the dentate gyrus (DG), CA1 and CA3 regionin the pup’s brain. In dentate gyrus, maternal restrained stressincreased GAP-43 IR in the molecular layer (mol) and hilar region(hil) as compared with control pups (Fig. 4A and B). In CA1 region,GAP-43 IR was increased both in Stratum orients (Str. or) andStratum radiatum (Str. rad) of the PS pups as compared withcontrol pups (Fig. 4C and D). In CA3 region, maternal restrained

Fig. 1. Characteristic of GAP-43 immunoperoxidase staining (1:12,000) in prefrontal cortex of PND 7 rat pups. GAP-43 immunostaining demonstrated a granular staining

pattern in the neuropil, while most of neuronal cell bodies were not stained (A). The specificities of antisera against GAP-43 were performed with absorption control on

adjacent sections by pre-absorbed with 100 mM of synthetic peptides of GAP-43, the result shows no immunostaining at all (B).

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stress increased GAP-43 IR in the Stratum lucidum (Str. luc) thatcontain the perforant path axon (PP) and the mossy fiber (MF)terminals synapse onto CA3 pyramidal cell (Fig. 4E and F).

3.4. Prenatal CORT exposure increased GAP-43 expression in the PFC

and hippocampus of rat pups

We further investigate whether changed in GAP-43 expressionin the pup’s brain was mediated by maternal exposure to stresshormone during pregnancy by treated pregnant rat with exogen-ous CORT during GD 14–21. From the previous results, we foundthat PND 7–14 is the peak period that exhibited highest level ofGAP-43 IR both in the PFC and hippocampus, thus, we selected PND14 to examine the effect of maternal CORT injection (40 mg/kg) onthe expression of GAP-43 level in these brain areas by westernblotting technique. Western blot analysis revealed that, at PND 14,GAP-43 expression in the pups born from CORT treated dam wassignificant higher (p � 0.0001) than in the pups born from vehicletreated dam, both in the prefrontal cortex and the hippocampus(Fig. 5).

3.5. Long-term effect of prenatal stress on the expression of GAP-43 in

the prefrontal cortex of rat pups

To investigate whether PS can exert long-term effect on theexpression of GAP-43 in the brain of rat pups, immunocytochem-ical staining of GAP-43 in the prefrontal cortex of rat pups wasexamined at PND 60. The result showed that, as adult, pups bornfrom PS dam exhibit lower GAP-43 IR as compared with controlgroup (Fig. 6A–D). Then we measured the density of GAP-43 IR incortical layer V between control and PS group, the result showedthat the percent density of GAP-43 IR in PS group was significantlydecreased (p < 0.001) as compared with control group at PND 60(Fig. 6E).

4. Discussion

During postnatal period, the trajectory of overproduction andpruning of axons, dendrites and synapses shapes the brainbetween puberty and adulthood. This studied was to ascertainwhether this normal trajectory was affected by maternal stress or

Fig. 2. Photomicrographs showed GAP-43 immunostaining in prefrontal cortex sections compared between control and prenatal stress (PS) at different postnatal staged from

PND 0, 7 and 14. Dash lines represents border between each cortical layer. I–VI represent the cortical layers 1–6, respectively. Scale bar = 100 mm.

Fig. 3. (Left) Photomicrograph showed GAP-43 immunostaining in layer V of prefrontal cortex of rat pups compared between control and PS group at PND 14 (A and B) and

PND 21 (C and D), respectively. (Right) Bar graph showed the %density of GAP-43 IR in layer V of rat prefrontal cortex compared between control and PS group at PND 7 and 21.

The percent densities of GAP-43 IR were measured using Image Tools software as described in Section 2. Each value represents mean � S.E.M., with n = 8 for each group.

Asterisks indicate significant difference compared with control (***p < 0.0001).

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maternal exposure to corticosteroid hormone during pregnancy.We examined the effect of maternal restraint stress on GAP-43, theplasticity responsive protein, in developing rat brain at differentpostnatal periods and found that GAP-43 IR in the brain of prenatalstress pups were up-regulated during PND 7–14, however, whenobserved at PND 60, prenatal stress group exhibited lower GAP-43IR as compare to control group. GAP-43 is a neurotrophindependent membrane bound phosphoprotein found in the axonterminal and the growth cone of neurons (Perrone-Bizzozero et al.,1988; Tejero-Diez et al., 2000). GAP-43 is highly expressed in thenervous system during development (Jacobson et al., 1986) andphosphorylation of GAP-43 on Ser41 by PKC is important forvarious intracellular functions such as axonal path finding,synaptogenesis, as well as regulation of cytoskeletal organizationin nerve ending (Benowitz and Routtenberg, 1997). Moreover, thetime course of GAP-43 phosphorylation is correlated with theenhancement of neurotransmitter release during LTP induction(Ramakers et al., 1995). GAP-43 also contributes to axonalsprouting in several brain areas including the mossy fiberterminals of hippocampus (Aigner and Caroni, 1995). In the courseof neural development, GAP-43 accumulates in axonal growthcones allowing them to navigate exactly to their appropriatetargets. GAP-43 gene knock-out results in severe abnormalities inaxonal path finding at certain ‘‘decision points’’ that lead to highrate mice lethality (90–95%) within 2 weeks after birth (Stritt-matter et al., 1995; Zhang et al., 2000; Shen et al., 2002). Generally,neuronal GAP-43 expression declines dramatically as soon as

Fig. 4. Photomicrographs illustrated GAP-43 immunostaining in dentate gyrus (DG), CA1 and CA3 of the hippocampus compared between control and PS group at PND 7 rat

pups. Abbreviation: gcl, granule cell layer; mol, molecular layer; hil, hilus; Str. or, Stratum oriens; Str. py, Stratum pyramidale; Str. rad, Stratum radiatum, Str. luc, Stratum

lucidum. Scale bar = 50 mm (A–D) and 100 mm (E and F), respectively.

Fig. 5. (A) Representative Western blots obtained by using anti-GAP-43 antibody.

The level of GAP-43 protein was determined in the proteins lysates from the

prefrontal cortex (PFC) and hippocampal (HP) tissues from PND 14 pups. The b-

actin levels form both regions are shown below and were used to normalize the

data. (B) Mean gray level ratios (means � S.E.M.) of GAP-43 signal normalized with b-

actin signal, n = 3 for each group. White bar represents control group and stripped bar

represents CORT injection group. *p � 0.01 and ***p � 0.0001 compared with the

control group.

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axons have reached their targets, however, it remains elevated inselected brain regions that retain a high level of synaptic plasticity(Neve et al., 1988). In mature neurons, GAP-43 resides inpresynaptic area, where it regulates neurotransmitter release(Dekker et al., 1989; Hens et al., 1993). Taken together, GAP-43 iswell-established markers of presynaptic axonal growth and can beused as an indicator for the plastic changes in the developing brain.

During development, modest increase of GAP-43 mRNA ingranule cell coincident with mossy fiber outgrowth in hippocam-pus (Meberg and Routtenberg, 1991). Cell-selective expression ofGAP-43 was not restricted to the hippocampus, neurons containingbiogenic amines, i.e., the substantia nigra pars compacta (dopa-mine), locus coeruleus (norepinephrine), and dorsal raphe(serotonin), also exhibited intense GAP-43 hybridization. HighlyGAP-43 expression is apparent in many neurons having eitherneuromodulatory or memory storage functions suggested thatGAP-43 is important for accelerating process outgrowth andsynaptic remodeling, rather than directing growth itself (Mebergand Routtenberg, 1991; Aigner and Caroni, 1995).

In the cerebral cortex, the development of axonal arbors is acritical step in the establishment of precise neural circuits. Theperiod over the first 3 postnatal weeks spans the elaboration ofthalamocortical and Cajal-Retzius axons and cortical synaptogen-esis. The balance between growth and retraction favored overallgrowth. Excessive axonal branches and synaptic contacts are oftenformed during early development, and then, they are pruned oreliminated at later stages to create specific neuronal connections.In thalamocortical axons, both the addition of new branches andthe degree of growth and retraction at individual tips diminishedafter the second postnatal week, suggesting that arbors reach amature state around that time.

The mechanisms by which prenatal stress can programneuronal development with long-term consequences are not wellunderstood. Early stressors including parental separation arevulnerability factors for mood disorder and hippocampal involve-ment is prominent. In nonhuman primate, daily parental depriva-tion during infancy produces a pro-depressive state of increasedbasal activity and reactivity in stress systems and mild anhedoniathat persists at least to adolescence. Early deprivation led todecreases in hippocampal GAP-43 mRNA, 5-HT1A receptor mRNAand binding and to increased vesicular GABA transporter mRNA;but did not affect hippocampal volume (Law et al., 2009). Theyconclude that early deprivation in the absence of subsequent

stressors has a long-term effect on the hippocampal expression ofgenes implicated in synaptic function and plasticity.

The reduction of GAP-43 IR at PND 60 found in the present studycorrelated with the previous finding who showed that depressionand stress are associated with neuronal atrophy and dendriticreorganization in hippocampus and prefrontal cortex (Cook andWellman, 2004) and chronic antidepressant increases expressionof plasticity related proteins, including GAP-43, in these brainareas (Sairanen et al., 2007). Using in situ hybridization studied,mRNA for synaptophysin and GAP-43 were shown to be slightlydecreased in the hippocampus after chronic restraint stress inadult male rats for 5 days (1 h/day) and 21 days (6 h/day),respectively (Kuroda and McEwen, 1998; Thome et al., 2001). Incontrast, some paper found no significant alteration in GAP-43 andsynaptophysin after chronic restraint stress in adult male rats for14 days (6 h/day) (Rosenbrock et al., 2005). These studies payattention to the effects of restraint stress in adult rats where as ourstudied focus on the effect of maternal stress on the developingbrain which may not totally be comparable. Our results suggestthat stress during prenatal and early postnatal life producedifferential effects from stress that occur in adult animals. Toour knowledge, this is the first report that maternal stress causes abiphasic response to the axonal growth in the pup’s brain, earlyaccelerated but attenuated in the later period.

Cortical layer V of prefrontal cortex received the dopaminergicinput from VTA (mesocortical DA pathway) and were a primarysource of subcortical output and have collateral feedback withpyramidal neuron in layer III (DeFelipe and Farinas, 1992).Moreover, abnormal reduced in dendritic outgrowth in layer Vpyramidal neurons has been reported in the PFC of patients withschizophrenia (Black et al., 2004). The reductions in GAP-43 andserotonin 1A receptor expressions in mood disorder and schizo-phrenia supporting the possibility that early developmentalfactors may contribute to disease vulnerability. Recent work hasbeen reported that prenatal restraint stress induces significantincrease in the expression of p38 MAPK in hippocampus of theoffspring (Cai et al., 2008). This protein are a family of Ser/Thrkinases that regulate important cellular processes such as stressresponses, differentiation, and cell-cycle control, moreover, it isactivated in neuron in response to a variety of stimuli includingoxidative stress, excitotoxicity, and inflammatory cytokineswhich could impose lasting effects on cellular signalling ofoffspring hippocampus.

Fig. 6. Photomicrograph showed GAP-43 immunostaining in the prefrontal cortex of PND 60 rat pups compared between control (A and C) and PS group (B and D). Scale

bar = 100 mm (A and B) and 40 mm (C and D), respectively. (E) The %density of GAP-43 IR were measured using Image Tools software as described in Section 2 and compared

between control and PS group at PND 60. Each value represents mean � S.E.M. Asterisks indicate significant difference compared with control with **p < 0.001.

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GAP-43 gene expression is known to be regulated at both thetranscriptional and the postranscriptional levels. Ubiquitin (Ub) iswell known for its role in targeting cytoplasmic proteins fordegradation by the 26S proteasome. In neuronal cultures, the UPShas been reported to be one of the degradation mechanisms ofGAP-43 (De Moliner et al., 2005). Proteasome inhibitors such aslactacystin and MG132 increased the cellular GAP-43 level andleading to the accumulation of polyubiquitinated forms of GAP-43.The ubiquitin proteasome pathway is also involved in the turnoverof this protein in neurons (De Moliner et al., 2005). GAP-43 over-expression in the nervous system of adult transgenic mice isaccompanied by enhanced learning and regenerative capabilities(Aigner and Caroni, 1995; Routtenberg et al., 2000). However,some data show that increased level of neuronal GAP-43expression can lead to apoptosis. In particular, GAP-43 over-expression in transgenic mice induces a substantial loss of neuronsin certain brain areas because of the apoptotic cell death (Aignerand Caroni, 1995). In contrast, GAP-43 gene knock-out leads to anincrease in total number of neurons in the developing mouse brain(Gagliardini et al., 2000). The GAP-43-mediated neuronal deathmay be involved in the elimination of neurons that have notestablished the proper contacts with their targets duringembryonic development (Wehrle et al., 2001). In adult animals,increase of GAP-43 has been reported to correlate with mono-aminergic deficit in neuropsychopathology (Sower et al., 1995;Blennow et al., 1999; Rekart et al., 2004; Valdez et al., 2007).

Taken together, these finding indicated that maternal stress canproduce enduring morphological changes in the hippocampus andprefrontal cortex, which may not become evident until adulthood.Early life experience alters the development of neural connectionand neurotransmitter systems in the developing brain which maycontribute to neurodevelopmental disorders and psychiatricdiseases in later life.

5. Conclusion

In conclusion, the present study demonstrated that repeatedmaternal restraint stress increased GAP-43 in the postnatal rat brainduring the second week of life but decreased after that. Abnormalaxon sprouting and reorganization may lead to synaptic miswiringwhich may be functionally abnormal and lead to a defect in synapticpruning at later stage of life. Further studies are needed in order toexplain the neurobiological substrates that are affected by adverseevent during early period of life that may be associated with thedevelopment of neuropsychiatric disorder as adult.

Conflicts of interest

The authors declare no conflicts of interest.

Acknowledgements

This study was supported by Mahidol University ResearchThesis Scholarship from Faculty of Graduate Studies to SA, MahidolUniversity Research grant to NJ and TRF Senior Research ScholarFellowship to PG.

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