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

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

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International Journal of Developmental Neuroscience journal homepage: www.elsevier.com/locate/ijdevneu

Repeated restraint stress and corticosterone injections during late pregnancy alter GAP-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, Thailand Faculty of Allied Health Sciences, Christian University, Nakornpathom 73000, Thailand Department of Anatomy, Faculty of Medicine, Srinakhrinwirot University, Bangkok, Thailand d Center for Neuroscience, Faculty of Science, Mahidol University, Bangkok, Thailand e Department of Pharmacology, Faculty of Science, Mahidol University, Bangkok, Thailand b c

A R T I C L E I N F O

A B S T R A C T

Article history: Received 3 April 2009 Received in revised form 1 September 2009 Accepted 15 September 2009

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

Keywords: Prenatal stress Corticosterone GAP-43 Hippocampus Prefrontal coretx

1. Introduction Glucocorticoids have important roles in normal maturation of the 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 glucocorticoids in utero have widespread acute effects upon neuronal structure and may permanently alter developmental trajectories of hippocampus and other brain structures of the offspring (Matthews, 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-

* Corresponding author. Tel.: +66 2441 9321. E-mail address: [email protected] (N. Jutapakdeegul). 0736-5748/$36.00 ß 2009 ISDN. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijdevneu.2009.09.003

dent neuronal degeneration of hippocampal neuron and reduced hippocampal volume which persisted at 20 months of age (Uno et al., 1990). Childhood abuse has been associated with reduction in hippocampal volume in adults (Bremner et al., 1997; Stein et al., 1997; Driessen et al., 2000; Vythilingam et al., 2002), but not in children (Carrion et al., 2001; De Bellis et al., 2001). More importantly, animal studies have clearly indicated that exposure to variable types of stressors during development produces persistent behavioral defects that are associated with hormonal, neurotransmitter and functional changes resemble an array of psychopathological conditions (Huttunen, 1997; Heim et al., 2004; Howes et al., 2004). During postnatal period, there is a marked overproduction of axons, dendrites, synapses, and receptors (Rakic, 1991) followed by a period of rapid elimination, or pruning, between puberty and adulthood. Up to 50% of synapses and receptors are lost in both

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cortical (Huttenlocher, 1979; Andersen et al., 2000) and subcortical regions (Teicher et al., 1995). The time course and degree of pruning varies between regions. It is hypothesized that adaptation to stress in young animal, in which the neuronal development has not yet been completed, can be invariably different from that occur in adult animals. In fact, the timing of early life stress can interfere with specific developmental stages in specific brain area that might be selectively involved in the manifestation of the disease in later life. Virtually nothing is known about the effects of prenatal stress on the developmental trajectories of axons in the brain of the offspring. The hippocampus is a brain region that has protracted development (Giedd et al., 1996), and has high density of glucocorticoid receptors. It appears to be a brain region that especially vulnerable to the effects of stress. Early exposure to stress or corticosteroids can cause hippocampal atrophy, decreases in dendritic branching and vulnerable to the subsequent insult as adult (Sapolsky, 2000). On the opposite way, stroking pups with a soft brush or handling over the first week of life results in increased glucocorticoid receptor (GR) expression in the hippocampus and prefrontal cortex which can further modify HPA reactivity (Meaney and Aitken, 1985; Weaver et al., 2000; Jutapakdeegul et al., 2003). Much less is known about the effects of early life stress on the development of prefrontal cortex. The prefrontal cortex has a very protracted ontogeny (Alexander and Goldman, 1978) and is specifically activated by stressors (Bannon et al., 1983; Deutch et al., 1991), and in primates may have a higher density of glucocorticoid receptors than the hippocampus (Sanchez et al., 2000). In addition to their apparent sensitivity, the hippocampus and the prefrontal cortex play important roles in memory and executive function. We have hypothesized that prenatal stress could affect the development of hippocampus and prefrontal cortex, possibly resulting in accelerated but attenuated development. Growth-associated protein of 43 kDa (GAP-43) is an intracellular protein that contributes to the guiding mechanism of axonal outgrowth in embryo, establishment and reorganization of synaptic connections during development (Hrdina et al., 1998) and to the sprouting and axonal regeneration in adult (Benowitz and Routtenberg, 1997). GAP-43 is abundant in axonal growth cones of developing 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 et al., 2003). In PC 12 cells, nerve growth factor (NGF) elicits neurite outgrowth which is accompanied by increased expression of protein GAP-43 (Karns et al., 1987). Once growing axons have reached their targets and synaptogenesis is completed, protein GAP-43 levels decline sharply in most neurons (McGuire et al., 1988; Dani et al., 1991). Experimental and neuropathological lesions have been reported to associate with alteration of GAP-43 expression (Eastwood and Harrison, 1998). The aim of the present study was to test the hypothesis that early life exposure to stress hormone might alter normal developmental trajectories of brain and produce the delay effects on brain structure and function. Since the hippocampus and prefrontal cortex have been reported to be the potential targets of corticosteroid hormone, the present study quantitatively analyzed the effect of early life exposure to stress hormone on GAP-43 protein 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 transcardically 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 X100. 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 X100) 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.

N. Jutapakdeegul et al. / Int. J. Devl Neuroscience 28 (2010) 83–90 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 8C with 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 temperature. 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) was generally observed in the nerve fibers and neuropil. It was found throughout the neuronal processes of all neuronal cell types both

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pyramidal cells and non-pyramidal cells. The characteristic of GAP43 IR demonstrated a granular staining in the neuropil, and not observed in the cell body of all neuronal cell types (Fig. 1A). Specificity of GAP-43 antibody was tested by pre-absorption of the antibody with GAP-43 peptide and result in completely abolishes of 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 the prefrontal cortex of normal rat pups at different postnatal period from PND 0 to 21 (Fig. 2, control panel). In control rat pups, GAP-43 IR was observed throughout the PFC at very low levels at birth and remarkable increased during the second week of life. The density of GAP-43 reached the highest level during PND 7–14, then, progressively declined until PND 21 (data not shown). From PND 21, GAP-43 IR was slightly constant and reaches the adult level. We then examined whether prenatal stress (PS) alters the spatial and temporal pattern of GAP-43 expression in the prefrontal cortex of neonatal rat. We found that, maternal restrained stress increased of GAP-43 IR in the prefrontal cortex of rat pups at birth (Fig. 2, PS panel). The effect was markedly observed at PND 7 and 14, in which GAP-43 IR was increased throughout the cortical layer. We then observed GAP-43 IR in layer V in PFC under higher magnification (Fig. 3A–D), and measured the density of GAP-43 IR in this cortical layer. We found that, at PND 14, the percent density of GAP-43 IR in layer V of PFC was significantly increased in PS pups as compared with control pups (p < 0.0001). However, at PND 21, GAP-43 IR in layer V did not showed 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 obvious effect of PS on GAP-43 IR in the pup’s brain, we further examined the effect of PS on GAP-43 expression in the hippocampus at these time points. Visual inspection of GAP-43 IR at PND 7 and 14 showed the same results that maternal restrained stress increased GAP-43 expression in the dentate gyrus (DG), CA1 and CA3 region in the pup’s brain. In dentate gyrus, maternal restrained stress increased 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) and Stratum radiatum (Str. rad) of the PS pups as compared with control 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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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.

stress increased GAP-43 IR in the Stratum lucidum (Str. luc) that contain 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 expression in the pup’s brain was mediated by maternal exposure to stress hormone during pregnancy by treated pregnant rat with exogenous CORT during GD 14–21. From the previous results, we found that PND 7–14 is the peak period that exhibited highest level of GAP-43 IR both in the PFC and hippocampus, thus, we selected PND 14 to examine the effect of maternal CORT injection (40 mg/kg) on the expression of GAP-43 level in these brain areas by western blotting technique. Western blot analysis revealed that, at PND 14, GAP-43 expression in the pups born from CORT treated dam was significant higher (p  0.0001) than in the pups born from vehicle treated 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 the expression of GAP-43 in the brain of rat pups, immunocytochemical staining of GAP-43 in the prefrontal cortex of rat pups was examined at PND 60. The result showed that, as adult, pups born from PS dam exhibit lower GAP-43 IR as compared with control group (Fig. 6A–D). Then we measured the density of GAP-43 IR in cortical layer V between control and PS group, the result showed that the percent density of GAP-43 IR in PS group was significantly decreased (p < 0.001) as compared with control group at PND 60 (Fig. 6E). 4. Discussion During postnatal period, the trajectory of overproduction and pruning of axons, dendrites and synapses shapes the brain between puberty and adulthood. This studied was to ascertain whether this normal trajectory was affected by maternal stress or

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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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 bactin 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 bactin 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.

maternal exposure to corticosteroid hormone during pregnancy. We examined the effect of maternal restraint stress on GAP-43, the plasticity responsive protein, in developing rat brain at different postnatal periods and found that GAP-43 IR in the brain of prenatal stress pups were up-regulated during PND 7–14, however, when observed at PND 60, prenatal stress group exhibited lower GAP-43 IR as compare to control group. GAP-43 is a neurotrophin dependent membrane bound phosphoprotein found in the axon terminal and the growth cone of neurons (Perrone-Bizzozero et al., 1988; Tejero-Diez et al., 2000). GAP-43 is highly expressed in the nervous system during development (Jacobson et al., 1986) and phosphorylation of GAP-43 on Ser41 by PKC is important for various intracellular functions such as axonal path finding, synaptogenesis, as well as regulation of cytoskeletal organization in nerve ending (Benowitz and Routtenberg, 1997). Moreover, the time course of GAP-43 phosphorylation is correlated with the enhancement of neurotransmitter release during LTP induction (Ramakers et al., 1995). GAP-43 also contributes to axonal sprouting in several brain areas including the mossy fiber terminals of hippocampus (Aigner and Caroni, 1995). In the course of neural development, GAP-43 accumulates in axonal growth cones allowing them to navigate exactly to their appropriate targets. GAP-43 gene knock-out results in severe abnormalities in axonal path finding at certain ‘‘decision points’’ that lead to high rate mice lethality (90–95%) within 2 weeks after birth (Strittmatter et al., 1995; Zhang et al., 2000; Shen et al., 2002). Generally, neuronal GAP-43 expression declines dramatically as soon as

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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.

axons have reached their targets, however, it remains elevated in selected brain regions that retain a high level of synaptic plasticity (Neve et al., 1988). In mature neurons, GAP-43 resides in presynaptic area, where it regulates neurotransmitter release (Dekker et al., 1989; Hens et al., 1993). Taken together, GAP-43 is well-established markers of presynaptic axonal growth and can be used as an indicator for the plastic changes in the developing brain. During development, modest increase of GAP-43 mRNA in granule cell coincident with mossy fiber outgrowth in hippocampus (Meberg and Routtenberg, 1991). Cell-selective expression of GAP-43 was not restricted to the hippocampus, neurons containing biogenic amines, i.e., the substantia nigra pars compacta (dopamine), locus coeruleus (norepinephrine), and dorsal raphe (serotonin), also exhibited intense GAP-43 hybridization. Highly GAP-43 expression is apparent in many neurons having either neuromodulatory or memory storage functions suggested that GAP-43 is important for accelerating process outgrowth and synaptic remodeling, rather than directing growth itself (Meberg and Routtenberg, 1991; Aigner and Caroni, 1995). In the cerebral cortex, the development of axonal arbors is a critical step in the establishment of precise neural circuits. The period over the first 3 postnatal weeks spans the elaboration of thalamocortical and Cajal-Retzius axons and cortical synaptogenesis. The balance between growth and retraction favored overall growth. Excessive axonal branches and synaptic contacts are often formed during early development, and then, they are pruned or eliminated at later stages to create specific neuronal connections. In thalamocortical axons, both the addition of new branches and the degree of growth and retraction at individual tips diminished after the second postnatal week, suggesting that arbors reach a mature state around that time. The mechanisms by which prenatal stress can program neuronal development with long-term consequences are not well understood. Early stressors including parental separation are vulnerability factors for mood disorder and hippocampal involvement is prominent. In nonhuman primate, daily parental deprivation during infancy produces a pro-depressive state of increased basal activity and reactivity in stress systems and mild anhedonia that persists at least to adolescence. Early deprivation led to decreases in hippocampal GAP-43 mRNA, 5-HT1A receptor mRNA and binding and to increased vesicular GABA transporter mRNA; but did not affect hippocampal volume (Law et al., 2009). They conclude that early deprivation in the absence of subsequent

stressors has a long-term effect on the hippocampal expression of genes implicated in synaptic function and plasticity. The reduction of GAP-43 IR at PND 60 found in the present study correlated with the previous finding who showed that depression and stress are associated with neuronal atrophy and dendritic reorganization in hippocampus and prefrontal cortex (Cook and Wellman, 2004) and chronic antidepressant increases expression of plasticity related proteins, including GAP-43, in these brain areas (Sairanen et al., 2007). Using in situ hybridization studied, mRNA for synaptophysin and GAP-43 were shown to be slightly decreased in the hippocampus after chronic restraint stress in adult male rats for 5 days (1 h/day) and 21 days (6 h/day), respectively (Kuroda and McEwen, 1998; Thome et al., 2001). In contrast, some paper found no significant alteration in GAP-43 and synaptophysin after chronic restraint stress in adult male rats for 14 days (6 h/day) (Rosenbrock et al., 2005). These studies pay attention to the effects of restraint stress in adult rats where as our studied focus on the effect of maternal stress on the developing brain which may not totally be comparable. Our results suggest that stress during prenatal and early postnatal life produce differential effects from stress that occur in adult animals. To our knowledge, this is the first report that maternal stress causes a biphasic response to the axonal growth in the pup’s brain, early accelerated but attenuated in the later period. Cortical layer V of prefrontal cortex received the dopaminergic input from VTA (mesocortical DA pathway) and were a primary source of subcortical output and have collateral feedback with pyramidal neuron in layer III (DeFelipe and Farinas, 1992). Moreover, abnormal reduced in dendritic outgrowth in layer V pyramidal neurons has been reported in the PFC of patients with schizophrenia (Black et al., 2004). The reductions in GAP-43 and serotonin 1A receptor expressions in mood disorder and schizophrenia supporting the possibility that early developmental factors may contribute to disease vulnerability. Recent work has been reported that prenatal restraint stress induces significant increase in the expression of p38 MAPK in hippocampus of the offspring (Cai et al., 2008). This protein are a family of Ser/Thr kinases that regulate important cellular processes such as stress responses, differentiation, and cell-cycle control, moreover, it is activated in neuron in response to a variety of stimuli including oxidative stress, excitotoxicity, and inflammatory cytokines which could impose lasting effects on cellular signalling of offspring hippocampus.

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GAP-43 gene expression is known to be regulated at both the transcriptional and the postranscriptional levels. Ubiquitin (Ub) is well known for its role in targeting cytoplasmic proteins for degradation by the 26S proteasome. In neuronal cultures, the UPS has been reported to be one of the degradation mechanisms of GAP-43 (De Moliner et al., 2005). Proteasome inhibitors such as lactacystin and MG132 increased the cellular GAP-43 level and leading to the accumulation of polyubiquitinated forms of GAP-43. The ubiquitin proteasome pathway is also involved in the turnover of this protein in neurons (De Moliner et al., 2005). GAP-43 overexpression in the nervous system of adult transgenic mice is accompanied by enhanced learning and regenerative capabilities (Aigner and Caroni, 1995; Routtenberg et al., 2000). However, some data show that increased level of neuronal GAP-43 expression can lead to apoptosis. In particular, GAP-43 overexpression in transgenic mice induces a substantial loss of neurons in certain brain areas because of the apoptotic cell death (Aigner and Caroni, 1995). In contrast, GAP-43 gene knock-out leads to an increase in total number of neurons in the developing mouse brain (Gagliardini et al., 2000). The GAP-43-mediated neuronal death may be involved in the elimination of neurons that have not established the proper contacts with their targets during embryonic development (Wehrle et al., 2001). In adult animals, increase of GAP-43 has been reported to correlate with monoaminergic 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 can produce enduring morphological changes in the hippocampus and prefrontal cortex, which may not become evident until adulthood. Early life experience alters the development of neural connection and neurotransmitter systems in the developing brain which may contribute to neurodevelopmental disorders and psychiatric diseases in later life. 5. Conclusion In conclusion, the present study demonstrated that repeated maternal restraint stress increased GAP-43 in the postnatal rat brain during the second week of life but decreased after that. Abnormal axon sprouting and reorganization may lead to synaptic miswiring which may be functionally abnormal and lead to a defect in synaptic pruning at later stage of life. Further studies are needed in order to explain the neurobiological substrates that are affected by adverse event during early period of life that may be associated with the development of neuropsychiatric disorder as adult. Conflicts of interest The authors declare no conflicts of interest. Acknowledgements This study was supported by Mahidol University Research Thesis Scholarship from Faculty of Graduate Studies to SA, Mahidol University Research grant to NJ and TRF Senior Research Scholar Fellowship to PG. References Aigner, L., Caroni, P., 1995. Absence of persistent spreading, branching, and adhesion in GAP-43-depleted growth cones. J. Cell Biol. 128, 647–660. Alexander, G.E., Goldman, P.S., 1978. Functional development of the dorsolateral prefrontal cortex: an analysis utilizing reversible cryogenic depression. Brain Res. 143, 233–249. Andersen, S.L., Thompson, A.T., Rutstein, M., Hostetter, J.C., Teicher, M.H., 2000. Dopamine receptor pruning in prefrontal cortex during the periadolescent period in rats. Synapse 37, 167–169.

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