Neonatal separation stress reduces glial fibrillary acidic protein- and S100β-immunoreactive astrocytes in the rat medial precentral cortex

Neonatal Separation Stress Reduces Glial FibrillaryAcidic Protein- and S100b-ImmunoreactiveAstrocytes in the Rat Medial Precentral Cortex

Kristina Musholt,1 Giovanni Cirillo,3 Carlo Cavaliere,3 Maria Rosaria Bianco,3

Joerg Bock,2 Carina Helmeke,1 Katharina Braun,1 Michele Papa3

1 Department of Zoology and Developmental Neurobiology, Institute of Biology,Otto von Guericke University Magdeburg, 39120 Magdeburg, Germany

2 Research Group \Structural Plasticity," Institute of Biology,Otto von Guericke University Magdeburg, 39120 Magdeburg, Germany

3 Laboratorio di Morfologia delle Reti Neuronali, Department of Medicina PubblicaClinica e Preventiva, Seconda Universita di Napoli, 80138 Napoli, Italy

Received 3 July 2008; revised 22 September 2008; accepted 22 October 2008

ABSTRACT: The interactions between the mother/

parents and their offspring provides socioemotional

input, which is essential for the establishment and main-

tenance of synaptic networks in prefrontal and limbic

brain regions. Since glial cells are known to play an im-

portant role in developmental and experience-driven

synaptic plasticity, the effect of an early adverse emo-

tional experience induced by maternal separation for 1

or 6 h on the expression of the glia specific proteins

S100b and glial fibrillary acidic protein (GFAP) was

quantitatively analyzed in anterior cingulate cortex, hip-

pocampus, and precentral medial cortex. Three animal

groups were analyzed at postnatal day 14: (i) separated

for 1 h; (ii) separated for 6 h; (iii) undisturbed (control).

Twenty-four hours after stress exposure, the stressed

brains showed significantly reduced numbers of S100b-immunoreactive (ir) cells in the anterior cingulate cor-

tex (6-h stress) and in the precentral medial cortex (1-

and 6-h stress). Significantly reduced numbers of

GFAP-ir cells were observed only in the medial precen-

tral cortex (1- and 6-h stress); no significant changes

were observed in the anterior cingulate cortex. No sig-

nificant changes of the two glial markers were observed

in the hippocampus. Double-labeling experiments with

GFAP and pCREB revealed pCREB labeling only in the

hippocampus, where the stressed brains (1 and 6 h) dis-

played significantly reduced numbers of GFAP/pCREB-

ir glial cells. The observed downregulation of glia-spe-

cific marker proteins is in line with our hypothesis that

emotional experience can alter glia cell activation in the

juvenile limbic system. ' 2009 Wiley Periodicals, Inc. Develop

Neurobiol 00: 000–000, 2009

Keywords: maternal separation; synaptic plasticity;

astrocyte; depression; limbic system

Correspondence to: Prof. M. Papa ([email protected]).Contract grant sponsor: Regione Campania; contract grant num-

bers: L.R. N.5 Bando 2003; Prog. Spec art 12 E.F. 2000 (to M.P.).Contract grant sponsor: Italian Minister of Research and Univer-

sity; contract grant number: PRIN2004 (to M.P.).Contract grant sponsor: CNR; contract grant number: Neurobio-

tecnologie 2003 (to M.P.).Contract grant sponsor: German Science Foundation; contract

grant number: SFB 779 (to K.B).

Contract grant sponsors: Associazione Levi-Montalcini (postdoc-toral fellowship to M.R.B.); Deutsche Studienstiftung (undergraduatefellowship to K.M.).' 2009 Wiley Periodicals, Inc.Published online in Wiley InterScience (www.interscience.wiley.com).DOI 10.1002/dneu.20694

1

INTRODUCTION

Environmental influences during sensitive periods of

early postnatal development have a strong impact

upon later development and behavior affecting the

growth and survival of dendrites, axons, synapses,

interneurons, neurons, and glia (Walsh, 1981; Rose-

nzweig and Bennett, 1996; Schrott, 1997; Joseph,

1999). These neural and synaptic changes within neu-

ronal networks accompany behavioral development

and, with respect to prefrontal and limbic regions,

determine cognitive as well as socioemotional capaci-

ties in adulthood. Despite the host of studies on expe-

rience-induced neuronal plasticity, the underlying

cellular and molecular mechanisms remain mostly

unclear, in particular with respect to the contribution

of glial cells. Based on the finding that the expression

of glia-derived growth factors such as brain-derived

neurotrophic factor (BDNF) are altered in response to

maternal separation (Roceri et al., 2004), we hypothe-

size that glial cells may play a crucial role in experi-

ence-induced neuronal and synaptic changes. It has

become more and more evident that glial cells play a

much more central role in nonsynaptic as well as

synaptic communication (Vernadakis, 1996; Volterra

and Meldolesi, 2005; Colangelo et al., 2008). Synap-

tic transmission can be modulated by dynamic

changes in the astrocytic coverage of synapses pro-

ducing such morphological changes that influence

synaptic transmission (Volterra and Meldolesi, 2005;

(Cavaliere et al., 2007; Giovannoni et al., 2007).

Astroglial changes have been described in some men-

tal disorders: Rajkowska (2000), Harrison (2002),

and Cotter et al. (2001) have reviewed the evidence

for reduction of glial cell number and packing density

in the prefrontal and anterior cingulate cortex in

major depressive disorder (MDD) and bipolar disor-

der. Miguel-Hidalgo et al. (2000), using glial fibril-

lary acidic protein (GFAP) as a selective immunohis-

tochemical marker of astroglia, found a reduction in

GFAP-stained cell count in the prefrontal cortex of

young (below 46 years) MDD patients. Muller et al.

(2001) reported a modest decrease in GFAP immu-

noreactivity in the hippocampal areas CA1 and CA2

in MDD with no parallel changes in neuronal density

or distribution in those areas. A study by Hamidi et

al. (2004) has shown oligodendrocyte deterioration in

the amygdala of patients with MDD. Experimental

animal models to study these events are of particular

interest, since astrocytes play vital roles in maintain-

ing neuroplasticity via multiple mechanisms includ-

ing support of synaptogenesis, synapse maintenance,

and secretion of neurotrophins (Newman, 2003;

Slezak and Pfrieger, 2003). Maternal separation or

deprivation in primates and rodents are classical de-

velopmental animal models, which are assumed to

mimic the etiology of anxiety disorders, including

depression (Heim et al., 2004; Sullivan et al., 2006).

Thus, in this study, the effect of early life time stress,

induced by maternal separation, on the number of

glial cells, and the expression of glia-specific proteins

was quantitatively analyzed in anterior cingulate cor-

tex, hippocampus, and prefrontal cortex.

METHODS

Animals

The study was carried out on the brains of 40 juvenile [post-

natal day (P) 14/15] male Wistar rats (Charles River, Italy),

weighing *50 g. Maternal separation stress was induced at

P 14, i.e., after the termination of the stress hyporesponsive

period of the HPA axis (Levine, 2002). Prior to exposure to

separation stress, the pups remained undisturbed with their

mothers and infants in standard cages with water and rat

diet pellets available to the mothers ad libitum, in an air-

conditioned room with an average room temperature of

228C. All experimental procedures were performed during

the light cycle and were approved by the Animal Ethics

Committee of The Second University of Naples. Animal

care was in compliance with Italian (D.L. 116/92) and EC

(O.J. of E.C. L358/1 18/12/86) regulations on the protection

of laboratory animals.

Maternal Separation

Maternal separation was carried out at 10 a.m. During the

isolation period, the male pups were kept in single cages

with acoustic and olfactory but no visual contact to their

siblings and dams. We choose 1 and 6 h of separation to

asses the very early changes in neuron–glia interaction and

the effects of neonatal isolation on brain plasticity. After

the isolation period, the pups were reunited with their

mothers and infants for 24 h, after which their brains were

prepared for the histological analysis.

Experimental Groups

Male pups were randomly allocated to one of the following

three experimental groups, each group representing animals

from eight different litters (an average of five pups per

mother and a mean ratio of 1:3 males versus females

infants):

Naive control animals (Ctr): These animals were kept

undisturbed with their mother (n ¼ 12). The animals

belonging to the control group (Ctr) were sacrificed at

P 14, i.e., immediately after the onset of the isolation

of their littermates to exclude any stressful experience

in this animal group.

2 Musholt et al.

Developmental Neurobiology

Maternal separation for 1 h (1-h group): As described

above, the pups were individually isolated from their

mothers and littermates for 1 h (n ¼ 14), after which

they were returned to their home cages and sacrificed

after 24 h.

Maternal separation for 6 h (6-h group): Pups were indi-

vidually isolated for 6 h from their mothers and litter-

mates (n ¼ 14), after which they were returned to their

home cages and sacrificed after 24 h.

Fixation and Sectioning

Pups were deeply anesthetized with an intraperitoneal

injection (300 mg/kg body weight) of chloral hydrate and

transcardially perfused with 30-mL phosphate-buffered

saline (PBS, pH 7.4) followed by 40-mL 4% paraformal-

dehyde in PBS. Brains were dissected out and postfixed

in the same fixative overnight at 48C. Series of 50-lmsections were cut on a Vibratome in a coronal plane and

collected in PBS containing 0.1% sodium azide. Sections

were alternatively stained for GFAP, S100b, Nissl, and

GFAP/pCREB.

Antibodies

Monoclonal mouse antibodies directed to GFAP (1:400;

Sigma-Aldrich) and monoclonal mouse antibodies directed

to the b-subunit of S100 (1:1000; Sigma-Aldrich) were

used for the single staining. Secondary antibodies were bio-

tinylated antimouse antibodies (1:200; Vector Labs). The

same monoclonal mouse antibodies directed to GFAP in

combination with polyclonal rabbit antibodies directed to

pCREB (1:200; Sigma-Aldrich) were used for the immuno-

fluorescent double-labeling. Secondary antibodies were

Alexa 546 (1:200, antimouse) and Alexa 488 (1:200, anti-

rabbit), respectively.

Immunocytochemistry

For the single-staining experiments, free-floating alternate

series of 50-lm-thick Vibratome sections were pretreated

in 0.75% glycine in PBS (pH 7.4), washed in PBS and

treated with 0.5% hydrogen peroxide (H2O2) in PBS,

washed three times in PBS, and preincubated in PBS

containing 10% normal serum (BSA) and 0.25% Triton

X-100 for 1 h at 48C. Sections were then incubated with

primary antibodies (GFAP 1:400, S-100b 1:1000) at 48Cby continuous shaking for 48 h. Sections were washed

six times in PBS and incubated with the appropriate bio-

tinylated secondary antibody (Vector Labs, Burlingame,

CA, 1:200 in PBS containing 10% normal serum) for 90

min at RT, washed three times in PBS, and processed by

using the Vectastain avidin–biotin peroxidase kit (Vector

Labs, Burlingame, CA) for 90 min at RT. The sections

were washed in 0.05 M Tris–HCl and reacted with 3,30-diaminobenzidine tetrahydrochloride (DAB; Sigma, 0.5

mg/mL Tris–HCl) and 0.01% hydrogen peroxide. Sec-

tions were then mounted on chrome-alume gelatin-coated

slides, dehydrated, and coverslipped. Adjacent sections

were Nissl-stained. A double-staining was performed as

previously reported (Papa et al., 2003). In brief, the tissue

sections were pretreated as stated above, preincubated in

PBS containing 5% BSA, 5% NSS, and 0.25% Triton X-

100 for 1 h at 48C, and then incubated with primary anti-

bodies (GFAP 1:400 + pCREB 1:200) at 48C by continu-

ous shaking for 24 h. Sections were washed six times in

PBS and incubated with the fluorescence-coupled second-

ary antibodies (antimouse, 546 nm + antirabbit, 488 nm,

1:200 in PBS containing 5% BSA and 5% NSS) for 120

min in the dark at RT. Afterwards, sections were washed

in PBS, mounted and coverslipped with Vectashield

Mounting Medium for Fluorescence.

Data Analysis

For each animal, between two and five sections for each

region were selected, representing the ventral and dorsal

anterior cingulate cortex (AC), the hippocampus (CA1-

region), and the precentral medial cortex (PrCm). Brain

sections were mounted on a Axioskope 2 light micro-

scope (Zeiss, Oberkochen, Germany), scanned into the

computer using a 103 objective by a Hamamatsu Digital

Camera C4247-95, controlled by a specific acquisition

program (HIPIC, Hamamatsu, Germany), and quantita-

tively analyzed by an acquisition board and a computer-

assisted image analysis system (MCID-M4; Imaging Res,

Canada). Animals were coded with a random letter and

number code so that the experimenter was unaware of

the experimental conditions during analysis. The identifi-

cation of brain regions was accomplished by inspection

of Nissl-stained adjacent sections (Van Eden and Uylings,

1985). The number of immunoreactive cells was deter-

mined using grain count analysis. Minimum and maxi-

mum gray levels were adjusted for each antibody and

region under visual control and applied for selective

labeling of the stained cells for measurement. For the

GFAP/pCREB double-staining image fusion of identical

images from two different channels (red and green) was

performed to detect colocalization of GFAP and pCREB.

As a consequence, only double-stained cells (as seen in

yellow) were counted. A signal could only be detected

for the hippocampus. The total number of immunoreac-

tive cells was counted for each measuring field of each

section and region. Cell density was calculated as cells/

mm2. Means were calculated for each animal and region

for statistical analysis, using the number of animals as n.Results were depicted in diagrams with the help of SIG-

MA.PLOT software (Jandel Scientific, Version 10.0 for

Windows). Data are expressed as means 6 SEM. Statisti-

cal analysis was carried out using SIGMA.STAT software

(Jandel Scientific, Version 3.0 for Windows). Data from

all the quantitative analyses were analyzed by one-way

ANOVA, using all pairwise Holm–Sidak method for mul-

tiple comparisons. Student’s t test was performed to com-

pare 1- or 6-h group with Ctr group.

Maternal Separation Affects Glia in Lymbic System 3

Developmental Neurobiology

RESULTS

In the present study, the number of GFAP-, S100-

and GFAP/pCREB-immunoreactive astrocytes in the

AC, the CA1 region of the hippocampus, and the

PrCm after 1 or 6 h of maternal separation was inves-

tigated and compared to the control group.

Glial Fibrillary Acidic Protein

In the AC, no change in reactive astrocytes, identified

by GFAP labeling, was found after 1 h (824.59 669.92 cells/mm2), or after 6 h of separation stress

(646.41 6 101.17 cells/mm2) compared to the Ctr

group (962.8 6 124.66 cells/mm2); p ¼ 0.534 and

0.101, respectively [Fig. 1(a,d)].

In the CA1 region, no changes of reactive glia

cells were observed after 1 h (1748.23 6 38.83 cells/

mm2) or after 6 h of separation stress (1519.88 672.27 cells/mm2) compared to the unstressed Ctr

group (1690.23 6 105.82 cells/mm2), p ¼ 0.945 and

0.181, respectively [Fig. 1(b,d)].

In the PrCm, a strong reduction of GFAP-labeled

glia was found after 1 h (1128.33 6 162.68 cells/

mm2), and even more after 6 h of separation (883.45

6 171.57 cells/mm2) compared to the Ctr group

(2463.1 6 214.73 cells/mm2); p < 0.001 in both

treated groups [Fig. 1(c,d)].

S100b

In the AC, again, the number of S100b-immunoreac-

tive glia decreased after maternal separation. While

the difference between the Ctr group (767.56 654.33 cells/mm2) and the 1-h separation group

(633.64 6 66.24 cells/mm2) was not statistically sig-

nificant (p ¼ 0.202), there was a significant reduction

of cells after 6 h of separation (544.51 6 53.37 cells/

mm2) (p ¼ 0.030) [Fig. 2(a,d)].

In the CA1 region, no effect of maternal separation

was found compared to the Ctr group. In CA1, the

numbers of S100b-stained cells was comparable after

1 h (1070.87 6 98.15 cells/mm2), as well as after 6 h

of separation stress (1101.44 6 71.22 cells/mm2),

and in the Ctr group (1203.67 6 107.54 cells/mm2);

p ¼ 0.295 and 0.281, respectively [Fig. 2(b,d)].

In the PrCm, a strong reduction of S100b-labeledglia was found after 1 h (1085.81 6 77.33 cells/

mm2), as well as after 6 h of separation stress (810.43

6 71.49 cells/mm2) compared to the unstressed Ctr

group (1486.52 6 143.01 cells/mm2); p < 0.001 in

both treated groups [Fig. 2(c,d)].

GFAP/pCREB

For the double fluorescence staining, the pCREB sig-

nal could only be detected in the CA1 region of hip-

pocampus. Here, the number of double-labeled glial

cells was significantly reduced after 1 h (80.41 618.21 cells/mm2) [Fig. 3(b,d)] as well as after 6 h of

separation stress (93.63 6 17.77 cells/mm2) [Fig.

3(c,d)], compared to the unstressed Ctr group (159.26

6 14.52 cells/mm2) [Fig. 3(a,d)]; p ¼ 0.018 and

0.048, respectively.

DISCUSSION

In line with our hypothesis that emotional experience

can alter glia cell activation in the juvenile limbic

system, the present study demonstrates that a single

exposure to separation stress, induced by maternal

separation, produces a rapid downregulation of the

expression of glia-specific proteins in the precentral

medial cortex, anterior cingulate cortex, and partly

also in the hippocampus. The brain areas, which dis-

play reduced density of GFAP-positive astrocytes in

response to separation stress, are implicated in both

mediation of stress-related behavior and in depres-

sion, with the latter evidence obtained in both imag-

ing (e.g., Vythilingam et al., 2002) and postmortem

studies (Harrison, 2002; Lucassen et al., 2006). Since

the level of GFAP expression is linked to the reactiv-

ity of astrocytes, and GFAP plays a role in maintain-

ing the morphology of astrocytes, the stress-induced

decrease in immunoreactivity toward GFAP seen in

the juvenile rats may be due to a rearrangement of

fibrillary astrocytes. This so-called \glial retraction"has been demonstrated in the hypothalamoneurophy-

sial system of adult animals (Hawrylak et al., 1998),

where it is correlated with the activation of neurons

caused by different stimuli, including dehydration,

lactation, parturition (Hatton and Yang, 1994), induc-

tion of maternal behaviors (Salm, 2000), and restraint

stress (Miyata et al., 1994). Mechanisms involved in

the rearrangement of astrocytes in the hypothalamo-

neurohyphysial system include the reorganization of

the astrocytic cytoskeleton, astrocytes undergoing

overall reorientation, astrocytes re-entering the cell

cycle, and possibly astrocytic death (Salm, 2000).

Whether similar mechanisms are involved in causing

the separation-induced reduction of GFAP immuno-

staining in the juvenile precentral medial, anterior

cingulate, and hippocampal regions requires further

analysis.

To shed more light on the question of whether the

differences in GFAP immunoreactivity reflect true

4 Musholt et al.

Developmental Neurobiology

Figure 1 GFAP immunostaining. This figure shows the GFAP immunostaining in the AC (a),

CA1 (b), and PrCm (c) regions in Ctr, and following 1 and 6 h of maternal separation. In AC (a), a

trend toward reduction of immunoreactivity was found after 6 h of separation. Both the number of

immunoreactive cells and the staining of astrocytic processes are reduced. In CA1 region (b), no

significant change of GFAP-immunoreactivity after maternal separation was found. In the PrCm

region (c), a significative reduction (p < 0.001) of immunoreactivity was found after 1 and 6 h of

separation. Scale bar ¼ 200 lm. The histogram on the bottom (d) shows the results obtained by

GFAP immunostaining. In the AC, after 6 h of separation, a tendency toward lower GFAP-immu-

noreactive glia numbers seems to emerge. In the CA1 region of the hippocampus, no difference

between Ctr and experimental groups was found. In the PrCm, GFAP-positive cells appear strongly

and significatively reduced after 1 and 6 h of treatment. Data is expressed as means 6 SEM. n ¼12 in the Ctr and n ¼ 14 in the experimental groups. [Color figure can be viewed in the online

issue, which is available at www.interscience.wiley.com.]

Maternal Separation Affects Glia in Lymbic System 5

Developmental Neurobiology

Figure 2 S100b immunostaining. This figure shows the S100b immunostaining in the AC (a),

CA1 (b), and PrCm (c) regions in Ctr, and following 1 and 6 h of maternal separation. In AC region

(a), a significant decrease (p ¼ 0.030) of immunoreactivity after 6 h of separation was found com-

pared to the Ctr group. In contrast to the GFAP staining, perikarya are better visualized, whereas

astrocytic processes appear shorter. In CA1 region (b), instead, no effect of maternal separation on

S100b staining was found. In the PrCm region (c), a significant decrease (p < 0.001) of immuno-

reactivity after 1 and 6 h of separation was found compared to the Ctr group. Scale bar ¼ 200 lm.

The histogram on the bottom (d) shows a significant reduction of immunoreactivity in the AC after

6 h of separation (p ¼ 0.030). In the hippocampus, no effect was found. In the PrCm, S100bpositive cells appear strongly and significatively reduced after 1 and 6 h of treatment. Data are

expressed as means 6 SEM. n ¼ 12 in the Ctr and n ¼ 14 in the experimental groups. [Color figure

can be viewed in the online issue, which is available at www.interscience.wiley.com.]

6 Musholt et al.

Developmental Neurobiology

Figure 3 Double immunolabeling GFAP/pCREB in the CA1 region of the hippocampus in Ctr

(a), 1 h of maternal separation (b), and 6 h of maternal separation (c) animals, respectively. In the

AC and the medial PrCm cortex, no pCREB signal could be detected. Only yellow signals, indicat-

ing a colocalization of GFAP and pCREB, were counted. A significant decrease of double-labeled

cells was found after 1 h as well as after 6 h of maternal separation. Scale bar ¼ 200 lm. The histo-

gram on the bottom (d) shows a significant decrease of double-labeled cells in the CA1 region of

the hippocampus after 1 h (p ¼ 0.018) as well as after 6 h (p ¼ 0.048) of separation. Data are

expressed as means 6 SEM. n ¼ 12 in the Ctr and n ¼ 14 in the experimental groups. [Color figure

can be viewed in the online issue, which is available at www.interscience.wiley.com.]

structural changes of astrocytic morphology, rather

than a reduction of GFAP-positive glial population,

we additionally used a different glia-specific marker.

S100b is a glia-specific protein of the EF-hand

calcium-binding family, which is expressed by both

protoplasmic and fibrillary astrocytes (Ogata and

Kosaka, 2002). As for GFAP-ir, our study revealed

that, in the juvenile precentral medial cortex, immu-

nostaining for S100b is significantly reduced after 6 h

of separation stress, indicating a decrease of astroglial

population in this area. These data are in line with

recent postmortem studies in major depressive disor-

der (Harrison, 2002), showing a reduction in the

packing density and number of glial cells in different

regions of the PrCm.

One of the putative mechanisms involved in these

stress-induced changes could be the activation of

plasticity-related proteins, such as the phosphoryla-

tion of cyclic AMP response element-binding protein

(pCREB). CREB is a constitutive transcription factor

and its regulation by phosphorylation is involved in

conditioning, learning, and memory in a variety of

species and conditions like a strong emotional event

(Yin et al., 1995; Alberini, 1999; Mayr and Mont-

miny, 2001). In our study, using colocalization of

GFAP and pCREB, we found a reduction of double-

labeled cells in the hippocampus after just 1 h of sep-

aration as well as after 6 h of separation, highlighting

a relative decrease of pCREB-positive glial cells.

This finding gains strength also in connection with

recent research, which suggests glial cells as a puta-

tive target for antidepressant treatments. For instance,

it has been shown that antidepressants, such as

norepinephrine selective reuptake inhibitor (desi-

pramine) or serotonin selective reuptake inhibitor

(fluoxetine), selectively increase the expression of

plasticity-related proteins, like pCREB, in the hippo-

campus and medial PFc of rats (Thome et al., 2000).

Furthermore, it has been found that the antidepressant

fluoxetine, a serotonin selective reuptake inhibitor

commonly used in the clinical practice, seems to act

increasing cell proliferation/neurogenesis in hippo-

campus and PFc by S100b modifications (Manev et

al., 2001, 2003), and that the activation of the recep-

tor for advanced glycation end products (RAGE) pro-

motes cell survival through increased expression of

the antiapoptotic protein Bcl-2 (Huttunen et al.,

2000).

Possible outcomes of the stress-evoked glial

changes in our experiments may include subsequent

decrease of glial glutamate transport and inhibition of

PKA-induced EAAT2 cell surface tracking following

the loss of GFAP (Hughes et al., 2004). These events

could determine the long-term potentiation, which is

modulated both by S100b (Nishiyama et al., 2002)

and CREB (Herdegen and Leah, 1998), probably

triggered by strong emotional events and responsible

of permanently altered brain circuitry/emotional

outcome.

Taken together, these results show that an emo-

tionally stressful event, such as the separation from

the mother and siblings, which has previously been

shown to affect neuronal and synaptic plasticity (Sul-

livan et al., 2006) in addition also involves glia plas-

ticity. Further investigations are required to determine

whether the very early changes found in our study

are transient or persistent, which mechanisms are

involved in these changes (with a focus on S100b-mediated events), and whether chronic separation or

separation during different developmental time win-

dows affect emotionality at adulthood leading to dif-

ferent behavioral and neuronal outcome (Si et al.,

2004). To understand the causal relationship of stress-

induced neuronal, synaptic, and glial changes, it will

be of crucial importance to focus on the interactions

between neurons, synapses, and glia in the juvenile

brain during critical developmental time windows.

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Developmental Neurobiology