"Juvenile stress" alters maturation-related changes in expression of the neural cell adhesion molecule L1 in the limbic system: relevance for stress-related psychopathologies.
ABSTRACT L1 is critically involved in neural development and maturation, activity-dependent synaptic plasticity, and learning processes. Among adult rats, chronic stress protocols that affect L1 functioning also induce impaired cognitive and neural functioning and heightened anxiety reminiscent of stress-induced mood and anxiety disorders. Epidemiological studies indicate that childhood trauma is related predominantly to higher rates of both mood and anxiety disorders in adulthood and is associated with altered limbic system functioning. Exposing rats to stress during the juvenile period ("juvenile stress") has comparable effects and was suggested as a model of induced predisposition for these disorders. This study examined the effects of juvenile stress on rats aversive learning and on L1 expression soon after exposure and in adulthood, both following additional exposure to acute stress and in its absence. Adult juvenile-stressed rats exhibited enhanced cued fear conditioning, reduced novel-setting exploration, and impaired avoidance learning. Furthermore, juvenile stress increased L1 expression in the BLA, CA1, DG, and EC both soon after the stressful experience and during adulthood. It appears that juvenile stress affects the normative maturational decrease in L1 expression. The results support previous indications that juvenile stress alters the maturation of the limbic system and further support a role for L1 regulation in the mechanisms that underlie the predisposition to exhibit mood and/or anxiety disorders in adulthood. Furthermore, the findings support the "network hypothesis," which postulates that information-processing problems within relevant neural networks might underlie stress-induced mood and anxiety disorders.
- SourceAvailable from: Ralf S Schmid[show abstract] [hide abstract]
ABSTRACT: The neural cell adhesion molecule L1 mediates the axon outgrowth, adhesion, and fasciculation necessary for proper development of synaptic connections. Mutations of human L1 cause an X-linked mental retardation syndrome termed CRASH (corpus callosum hypoplasia, retardation, aphasia, spastic paraplegia, and hydrocephalus), and L1 knock-out mice display defects in neuronal process extension resembling the CRASH phenotype. Little is known about the biochemical or cellular mechanism by which L1 performs neuronal functions. Here it is demonstrated that clustering of L1 with antibodies or L1 protein in rodent B35 neuroblastoma and cerebellar neuron cultures induced the phosphorylation/activation of the mitogen-activated protein kinases (MAPKs) and extracellular signal-regulated kinases 1 and 2. MAPK activation was essential for L1-dependent neurite outgrowth, because chemical inhibitors [2-(2'-amino-3'-methoxyphenyl)-oxanaphthalen-4-one and 1,4-diamino-2, 3-dicyano-1,4-bis(2-aminophenylthio)butadiene] of the MAPK kinase MEK strongly suppressed neurite outgrowth by cerebellar neurons on L1. The nonreceptor tyrosine kinase pp60(c-src) was required for L1-triggered MAPK phosphorylation, as shown in src-minus cerebellar neurons and by expression of the kinase-inactive mutant Src(K295M) in B35 neuroblastoma cells. Phosphatidylinositol 3-kinase (PI3-kinase) and the small GTPase p21(rac) were identified as signaling intermediates to MAPK by phosphoinositide and Rac-GTP assays and expression of inhibitory mutants. Antibody-induced endocytosis of L1, visualized by immunofluorescence staining and confocal microscopy of B35 cells, was blocked by expression of kinase-inactive Src(K295M) and dominant-negative dynamin(K44A) but not by inhibitors of MEK or PI3-kinase. Dynamin(K44A) also inhibited L1 antibody-triggered MAPK phosphorylation. This study supports a model in which pp60(c-src) regulates dynamin-mediated endocytosis of L1 as an essential step in MAPK-dependent neurite outgrowth on an L1 substrate.Journal of Neuroscience 07/2000; 20(11):4177-88. · 6.91 Impact Factor
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ABSTRACT: Neural recognition molecules were discovered and characterized initially for their functional roles in cell adhesion as regulators of affinity between cells and the extracellular matrix in vitro. They were then recognized as mediators or co-receptors which trigger signal transduction mechanisms affecting cell adhesion and de-adhesion. Their involvement in contact attraction and repulsion relies on cell-intrinsic properties that are modulated by the spatial contexts of their expression at particular stages of ontogenetic development, in synaptic plasticity and during regeneration after injury. The functional roles of recognition molecules in cell proliferation and migration, determination of developmental fate, growth cone guidance, and synapse formation, stabilization and modulation have been well documented not only by in vitro, but also by in vivo studies that have been greatly aided by generation of genetically altered mice. More recently, the functions of recognition molecules have been investigated under conditions of neural repair and manipulated using a broad range of genetic and pharmacological approaches to achieve a beneficial outcome. The principal aim of most therapeutically oriented approaches has been to neutralize inhibitory factors. However, less attention has been paid to enhancing repair by stimulating the stimulatory factors. When considering potential therapeutic strategies, it is worth considering that a single recognition molecule can possess domains that are conducive or repellent and that the spatial distribution of recognition molecules can determine the overall function: Recognition molecules may be repellent for neurite outgrowth when presented as barriers or steep-concentration gradients and conducive when presented as uniform substrates. The focus of this review will be on the more recent attempts to study the conducive mechanisms with the expectation that they may be able to tip the balance from a regeneration inhospitable to a hospitable environment. It is likely that a combination of the two principles, as multifactorial as each principle may be in itself, will be of therapeutic value in humans.Journal of Neurochemistry 06/2007; 101(4):865-82. · 3.97 Impact Factor
Article: Stress and the adolescent brain.[show abstract] [hide abstract]
ABSTRACT: During adolescence the brain shows remarkable changes in both structure and function. The plasticity exhibited by the brain during this pubertal period may make individuals more vulnerable to perturbations, such as stress. Although much is known about how exposure to stress and stress hormones during perinatal development and adulthood affect the structure and function of the brain, relatively little is known about how the pubertal brain responds to stress. Furthermore, it is not clear whether stressors experienced during adolescence lead to altered physiological and behavioral potentials in adulthood, as has been shown for perinatal development. The purpose of this review is to present what is currently known about the pubertal maturation of the hypothalamic-pituitary-adrenal (HPA) axis, the neuroendocrine axis that mediates the stress response, and discuss what is currently known about how stressors affect the adolescent brain. Our dearth of knowledge regarding the effects of stress on the pubertal brain will be discussed in the context of our accumulating knowledge regarding stress-induced neuronal remodeling in the adult. Finally, as the adolescent brain is capable of such profound plasticity during this developmental stage, we will also explore the possibility of adolescence as a period of interventions and opportunities to mitigate negative consequences from earlier developmental insults.Annals of the New York Academy of Sciences 01/2007; 1094:202-14. · 4.38 Impact Factor
‘‘Juvenile Stress’’ Alters Maturation-Related
Changes in Expression of the Neural Cell
Adhesion Molecule L1 in the Limbic
System: Relevance for Stress-Related
M.M. Tsoory,1,2*A. Guterman,1,2and G. Richter-Levin1,3
1Department of Psychology, University of Haifa, Haifa, Israel
2The Brain and Behavior Research Center, University of Haifa, Haifa, Israel
3The Institute for the Study of Affective Neuroscience (ISAN), University of Haifa, Haifa, Israel
L1 is critically involved in neural development and
maturation, activity-dependent synaptic plasticity, and
learning processes. Among adult rats, chronic stress
impaired cognitive and neural functioning and height-
ened anxiety reminiscent of stress-induced mood and
anxiety disorders. Epidemiological studies indicate that
childhood trauma is related predominantly to higher
rates of both mood and anxiety disorders in adulthood
and is associated with altered limbic system function-
ing. Exposing rats to stress during the juvenile period
(‘‘juvenile stress’’) has comparable effects and was sug-
gested as a model of induced predisposition for these
disorders. This study examined the effects of juvenile
stress on rats aversive learning and on L1 expression
soon after exposure and in adulthood, both following
additional exposure to acute stress and in its absence.
Adult juvenile-stressed rats exhibited enhanced cued
fear conditioning, reduced novel-setting exploration,
and impaired avoidance learning. Furthermore, juvenile
stress increased L1 expression in the BLA, CA1, DG,
and EC both soon after the stressful experience and
during adulthood. It appears that juvenile stress affects
the normative maturational decrease in L1 expression.
The results support previous indications that juvenile
stress alters the maturation of the limbic system and
further support a role for L1 regulation in the mecha-
nisms that underlie the predisposition to exhibit mood
and/or anxiety disorders in adulthood. Furthermore, the
postulates that information-processing problems within
relevant neural networks might underlie stress-induced
mood and anxiety disorders.
C 2009 Wiley-Liss, Inc.
Key words: animal
maturation; anxiety, depression
model;juvenile stress; L1;
The neural cell adhesion molecule L1, as with
some others immunoglobulins, is critically involved in
several fundamental processes of neurodevelopment,
including neurite outgrowth, adhesion, fasciculation,
migration, axon guidance, and myelination (Maness and
Schachner, 2007). In humans, mutations in the L1 gene
result in a spectrum of disorders (the CRASH syndrome)
including mental retardation (Lee, 2005). L1 deficient
mice show severe abnormalities in central and peripheral
nervous systems development, leading to an altered
behavioral phenotype (Law et al., 2003).
L1 expression regulation is critical for activity-
dependent synaptic plasticity processes and memory for-
mation in the adult brain. Interfering with L1 regulation
at different time points affects long-term potentiation
(LTP) induction and memory formation (Luthi et al.,
1994; Arami et al., 1996); increased hippocampal neural
activity results in rapid cleavage of L1 by neuropsin as a
precursor to further synaptic modification (Matsumoto-
Miyai et al., 2003). L1 expression levels remain increased
even 18 hr following learning, potentially supporting the
synaptic remodeling required for memory consolidation
(Tiunova et al., 1998; Weltzl and Stork, 2003).
L1 regulation in adulthood is affected by chronic
stress exposure or chronically elevated corticosterone
(CORT) levels and by acute stress while learning under
stressful conditions. Chronic stress-induced elevations in
L1 levels were suggested to represent activation of a
neuroprotective mechanism against the deleterious effects
of chronic stress exposure (Sandi et al., 2001; Venero
et al., 2002). Variations in L1 mRNA levels in several
Contract grant sponsor: EU’s PROMEMORIA; Contract grant number:
512012 (to G.R.-L.).
*Correspondence to: Michael Tsoory, PhD, Department of Psychology,
University of Haifa, Haifa 31905, Israel. E-mail: firstname.lastname@example.org
Received 20 January 2009; Revised 10 June 2009; Accepted 10 June
Published online 10 September 2009 in Wiley InterScience (www.
interscience.wiley.com). DOI: 10.1002/jnr.22203
Journal of Neuroscience Research 88:369–380 (2010)
' 2009 Wiley-Liss, Inc.
brain regions were negatively correlated with water
maze learning and positively correlated with plasma
CORT concentrations and anxiety indices (Venero
et al., 2004). An inverted U-shaped relationship between
L1 levels and cognitive functions was suggested to
account for the L1 transient elevation following learning
and for the chronic stress-induced L1 elevated levels
accompanied by emotional and cognitive impairments
Indeed, depressed patients exhibited increased L1
levels in the prefrontal cortex, although levels are
decreased in the parietooccipital cortex; treated patients
(antidepressants) exhibited higher parietooccipital cortex
L1 levels than untreated ones but lower levels than
nondepressed controls (Laifenfeld et al., 2005a). A
similar pattern appeared in chronically stressed rats
treated with antidepressants (Laifenfeld et al., 2005b).
Sandi and Bisaz (2007) suggested that altered L1 levels
may contribute to anomalies in neural network struc-
ture and function, which were suggested to underlie
stress-related psychopathologies (Castren, 2005).
Clinical studies and animal models suggest that early
life stress (ELS) dysregulates development and/or matura-
tion of neurocircuits pivotal to mediating stress and
emotion, thus augmenting the consequences of adversities
in adulthood and predisposing individuals to develop
stress-related psychopathologies (Sanchez et al., 2001;
Gutman and Nemeroff, 2003; Nemeroff, 2004). Several
studies indicated that exposing ‘‘juvenile’’ rats (?28 days)
to stressors impairs coping with stressors during adulthood
(Tsoory et al., 2008b) and alters maturation-related
expression levels of CAMs (neural cell adhesion mole-
cules) and GABAA receptor a subunit conformation
within the limbic system (Tsoory et al., 2008a; Jacobson-
Pick et al., 2008). Thus a novel ‘‘juvenile stress’’ ELS
model was proposed (Tsoory et al., 2008b).
Conditioned ablation of the L1 gene in the fore-
brain following PND (postnatal day) 22 produced adult
mice exhibiting decreased anxiety, increased synaptic
transmission, and altered spatial learning (Law et al.,
2003), suggesting a role for L1 also in maturation-related
processes. Therefore, this study examined the effects of
juvenile stress on maturational changes in L1 expression
and aversive learning in adulthood.
The first experiment assessed the effects of juvenile
stress on fear conditioning and L1 expression in the
basolateral amygdala (BLA) and hippocampus during
adulthood. The second experiment evaluated the effects
of juvenile stress on L1 expression in the BLA, CA1,
dentate gyrus (DG), and entorhinal cortex (EC) either
soon after the exposure or during adulthood, with or
without additional exposure to the stressful challenge of
the two-way shuttle avoidance task.
MATERIALS AND METHODS
Overall, 76 rats took part in the experiments (experi-
ment 1: n 5 16; experiment 2: n 5 60). After weaning at
PND 21, 22-day-old male Sprague-Dawley rats weighing 35–
49 g were deliverede by Harlan Laboratories (Jerusalem,
Israel). Three or four animals were maintained per cage in the
laboratory’s vivarium. After 5 days of acclimation, the
rats were randomly allocated to the different experimental
conditions of each of the experiments.
Housing conditions were as follows: lighting, 12 hr
light-dark cycle (on at 0700); temperature, 228C 6 28C; cage
dimensions, Floor 480 3 375 mm, height 210 mm (model
No. 1500U; TECNIPLAST S.p.a., Varese, Italy); cage bed-
ding, sterilized pine sawdust; diet, ad libitum water and solid
food pellets (Teklad Global Diet 2018S; Harlan Teklad Ltd.).
All stress procedures and subsequent behavioral assessments
were conducted in designated rooms away from the vivarium
and were approved by the institutional animal care commit-
tee, adhering to the NIH Guide for the care and use of laboratory
stress’’ on aversive learning and L1 expression during adult-
hood, rats were either exposed to juvenile stress (JS; n 5 8)
or not (C; n 5 8). During adulthood (at 9 weeks), both
groups underwent auditory fear conditioning. Twenty-four
hours following the conditioning, the rat’s freezing response
was assessed in the same context; 1 hr later, their freezing
responses to the CS (conditioned stimulus) were assessed in a
different context. Brain tissues were collected immediately
following the cue test for later assessment of L1 expression
Juvenile stress protocol.
exposing rats to a different stressor every day for 3 days. Stress
exposure took place at approximately midday (12:00–14:00)
in designated rooms (a different room each day). Protocols
(see below) were applied in parallel, so as not to isolate any
rat in its home cage. Upon completion of each of the first
two stress protocols, rats were returned to their home cage
and were not handled until the next day. After completion of
the day 3 stress protocol, rats were returned to their home
cage and were not handled again up until behavioral assess-
ments during adulthood, except for weekly cage maintenance.
The juvenile stress protocol consisted of: day 1 (PND
27), a 10-min forced swim; water temperature 228C 6 28C;
day 2 (PND 28), three 30-min trials of elevated platform stress
with an intertrial interval (ITI) of 60 min in the home cage;
day 3 (PND 29), 2-hr restraint (restraining box: 11 3 5 3 4
cm) at 258C under dim illumination.
Auditory fear conditioning.
provided two distinct contexts. Each was positioned in a
sound-attenuated, well-ventilated, and dimly lit (two 12-V
DC, 10-W bulbs) chamber (55 3 55 3 75 cm). Box A was
the conditioning and contextual test box, a clear Plexiglas
isosceles hexagonal enclosure with a metal grid floor through
which the unconditioned stimuli (US) of scrambled electric
foot shocks were delivered by an electrical shocker (precision
programmable animal shocker; model H13-16; Coulbourn).
Box B was the cue test box, a clear Plexiglas cylindrical
enclosure with a black Plexiglas floor. A computer timed the
To assess the effects of ‘‘juvenile
This protocol involves
Two boxes (A and B)
370Tsoory et al.
Journal of Neuroscience Research
presentation of the US and conditioned stimulus (CS) tones,
produced by small loudspeakers on both boxes’ ceilings. Mini-
ature digital cameras (Bosch, Portugal; B/W pinhole, model
VCM 3602) on the boxes’ ceilings transmitted information
(Freeze04), which compared the number of pixels’ difference
between adjacent frames obtained from successive digitized
images (1 Hz). If the difference was less than 0.05% of the
total number of pixels in a frame, the animal was considered
to be freezing in that 1-sec interval (Yee et al., 2004).
Conditioning (box A) consisted of a single session of
four CS-US pairings that followed a 2-min ‘‘silent’’ period.
CS was a 30-sec tone (4 kHz; 80 dB), US a 1-sec electric
foot shock (0.8 mA), which coterminated with the CS; inter-
trial interval (ITI) was randomly varying 120 sec 6 20%.
The contextual test (box A) consisted of a single 5-min
session 24 hr following the conditioning.
The cue test (box B; unfamiliar context) was 1 hr
following the contextual test. A single session of two CS
presentations followed a 2-min ‘‘silent’’ period. CS was a
30-sec tone (4 kHz; 80 dB); ITI was constant 120 sec. The
floors and walls of each chamber were cleaned with odorless
clean-wipes between sessions.
Limbic brain regions harvesting.
vesting immediately followed the cue test. Rats were
decapitated with a guillotine (Stoelting, Wood Dale, IL).
The left and right (pooled) basolateral amygdala (BLA) and
the hippocampus were collected, because numerous studies
have implicated the BLA and hippocampus in cue and con-
textual fear conditioning, respectively (Wiltgen et al., 2006;
LeDoux, 2007). These regions were dissected based on rat
brain coordinates (Paxinos and Watson, 1998). The sampled
stored (–808C) for later Western blotting analyses. For a full
description of the tissue harvesting and sample preparation
procedures, see Tsoory et al. (2008a).
Protein concentration was moni-
tored using the Bradford assay, and the equality of the protein
loaded was verified using a-extracellular signal-regulated kinase
type II (a-ERKII; Cell Signaling, Beverly, MA) antibody stain-
ing (primary, 1:1,000; secondary, a-rabbit 1:10,000; 12% acryl-
amide). Equal amounts of protein were loaded according to
ERKII normalization control. Specifically, ERKII was used as a
control protein, because, although many immunoglobulins,
including L1, can lead to transient ERKII activation (Silletti
et al., 2004), elevations in ERKII are not concomitantly
dependent on upstream overexpression of various MAK/Src
pathway activators but may rather result from increased degra-
dation of other factors within the aforementioned cascades.
Therefore, the feedback loop affecting ERKII may have non-
linear relationships, affecting both upstream and downstream
factors according to various cellular mechanisms reacting to
environmental stressors. Because both ERKII and actin, a major
component of the cytoskeletal matrix, are found downstream of
CAMs, ERKII was found to be a credible sensor of L1 expres-
sion levels in the tissues analyzed after the detailed behavioral
manipulation (Emrick et al., 2006; Lefloch et al., 2008).
Individual samples from each limbic region of each rat
(100–120 lg/ll of lysates) were separated by SDS-PAGE
Brain region har-
using an 8% resolving gel. After electrophoresis, gels were
transferred by wet transfer tanks to nitrocellulose membranes
and stained against L1 (primary, a-NCAM-L1 (H-200; Santa
Cruz Biotechnology, Santa, Cruz, CA; SC-15326, 1:1,000;
secondary, 1:10,000 a-rabbit; Biochem). Membranes were
stained by enhanced chemiluminescence (ECL) using Luminol
and b-coumaric acid. The mean band intensity was assessed
relatively in NIH Scion Image for total L1 based on the two
L1 isoforms: 150 and 250 kD for each lane.
The results are expressed as mean
6 SEM. Percentage freezing during the auditory fear condi-
tioning procedure, the contextual test, and the cue test were
analyzed by a two-way ANOVA for groups, the phases within
each of the procedures (repeated measure analysis), and the
interaction groups 3 phases. L1 expression was analyzed by
that juvenile stress affects aversive learning (fear conditioning)
and increases L1 expression 24 hr following the conditioning
(see Results), experiment 2 addressed the question of whether
the observed alterations in L1 expression relate to the juvenile
stress exposure or to the effects that juvenile stress may have
on subsequent exposure to stressors during adulthood. To that
end, rats (n 5 60) were randomly divided into six groups
(Table I); three groups were exposed to juvenile stress, and
three were not. Brain tissues were collected from juvenile rats
(PND 33) from two groups, one that was exposed to juvenile
stress (J-js; n 5 8) and one that was not (J-j0; n 5 8; Table I,
top). Rats from the remaining four groups [two that had been
exposed to juvenile stress (collectively termed A-JS; n 5 24)
and two that had not (collectively termed A-J0; n 5 20)]
were maintained into adulthood (9 weeks), at which point
their novel-setting exploration was assessed. Immediately fol-
lowing the exploration test, two of the adult groups (one that
had been exposed to juvenile stress and one that had not)
were exposed to ‘‘adulthood stress’’ (the challenge of learning
the two-way shuttle avoidance task; described below). Rats
from the remaining two groups were returned to their home
cages following the exploration test. Thus, the following four
groups of adult rats took part in experiment 2 (Table I, bot-
tom): adult rats exposed to stress during both juvenility and
adulthood (A-js 1 as; n 5 14), adult rats exposed only to
juvenile stress (A-js 1 a0; n 5 10), adult rats exposed only to
adulthood stress (A-j0 1 as; n 5 10), and adult rats not
exposed to any stress (A-j0 1 a0; n 5 10). As in experiment
1, brain tissues were collected from adult rats 24 hr following
the aversive task (avoidance learning/adulthood stress) for
groups A-js 1 as and A-j0 1 as; or 24 hr following the
exploration test for groups A-js 1 a0 and A-j0 1 a0.
Juvenile stress protocol.
experiment 1, with the exception that on day 3 either
restraint or foot shocks were utilized. Restraint was as in
experiment 1. Foot shocks consisted of six unconditioned
electric foot shocks of 1 sec, 0.8 mA; ITI 29 sec. No differen-
ces were observed between rats exposed to the foot shock or
After we established in experiment 1
The protocol was as in
‘‘Juvenile Stress’’ Alters L1 Expression371
Journal of Neuroscience Research
the restraint procedures in any of the measured indices, so the
two groups were pooled.
Novel-setting exploration and two-way shuttle
The assessment of novel setting explo-
ration and two-way shuttle avoidance learning (termed
‘‘avoidance learning’’ hereafter) allowed corroborating previ-
ous findings regarding the long-term consequences of juvenile
stress (Tsoory and Richter-Levin, 2006; Tsoory et al., 2008a),
and the avoidance task also served as a stressor in adulthood
Both novel-setting exploration and adulthood stress
exposure took place in the two-way shuttle avoidance appara-
tus (for a full description of the apparatus see Tsoory and
Novel-setting exploration was assessed by counting the
number of times the rats voluntarily shuttled between the
compartments of the two-way shuttle avoidance apparatus
(while it was in an inoperative mode) for 10 min (Tsoory and
Avoidance learning/adulthood stress cosisted of a single
100-trial session. CS was a 10 sec tone; US was an electric
foot shock (0.5 mA) delivered immediately following the CS
for a maximum of 10 sec; ITI was randomly varying 60 sec 6
20%. A PC kept track of the rat’s location and controlled CS
and US presentations. Rats could respond in one of three
ways: 1) avoidance, shuttling to the adjacent compartment
while the CS (tone) is on; this terminated the tone and initi-
ated an ITI, so that the rat avoided the electric shock; 2)
escape, shuttling to the adjacent compartment following shock
induction, terminating the shock and initiating an ITI, so that
the rat reduced the duration of its exposure to the foot shock;
3) escape failure, not shuttling to the adjacent compartment.
An ITI commenced following delivery of the full 10-sec foot
shock. Learning this task was evaluated based on the rats’
latency to shuttle to the adjunct compartment in each trial
(latencies were avoidance 5 10 sec; escape >10 sec, <20 sec;
escape failure 5 20), thus monitoring the gradual improvement
in shuttling (shorter latencies) throughout the session.
Limbic brain region harvesting.
sample preparation were as in experiment 1. The left and right
(polled) BLA, CA1, and DG subregions of the hippocampus
along with the EC were collected for several reasons: 1)
performance of the avoidance task depends on hippocampal
(Schwegler et al., 1981; Becker et al., 1997) and amygdalar
(Savonenko et al., 2003) function; 2) previous studies
indicated that the hippocampal formation–amygdala connec-
tions (BLA, CA1, DG, and EC) are related to the complex
interplay of stress exposure and learning and memory forma-
tion (Tsoory et al., 2008b); 3) juvenile stress affects CAMs
expression differentially in the CA1 and DG at PND 33
(Tsoory et al., 2008a).
Immunoblotting was performed as
in experiment 1.
The results are expressed as mean
6 SEM. Exploration was analyzed by Student’s t-tests. Avoid-
ance learning was analyzed by a two-way ANOVA for
groups, blocks of 10 trials (repeated measure analysis), and the
interaction groups 3 blocks, followed by Student’s t-tests per
block for between-subjects comparisons and within-subjects
contrasts comparing the first block with all the others. L1 lev-
els were analyzed within each brain region as follows: tissues
collected at PND 33 (Student’s t-tests); tissues collected during
adulthood [two-way ANOVA for juvenile stress, adulthood
stress, and the interaction juvenile stress 3 adulthood stress,
followed by Student’s t-tests (Bonferroni corrected)]; all the
collected tissues [two-way ANOVA for juvenile stress,
maturation, and the interaction juvenile-stress 3 maturation,
followed by Student’s t-tests (Bonferroni corrected)].
TABLE I. Design of Experiment 2: Timing of Procedures*
Adults (week 9)
A-js 1 as (14)
A-js 1 a0 (10)
A-j0 1 as (10)
A-j0 1 a0 (10)
*1, Procedure was applied; –, not applied; 0, not relevant for this group.
aGroups: J-js, juvenile rats (33 days old) exposed to ‘‘juvenile stress’’; J-j0, naı ¨ve juvenile rats (not exposed to
juvenile stress); A-js 1 as, adult rats (9 weeks old) exposed to both juvenile stress and ‘‘adulthood stress’’; A-js
1 a0, adult rats exposed to juvenile stress but not to adulthood stress; A-j0 1 as, adult rats not exposed to juve-
nile stress but exposed to adulthood stress; A-j0 1 a0, adult naı ¨ve rats not exposed to any stress. A-JS, adult rats
exposed to juvenile stress regardless of exposure to adulthood stress, i.e., groups A-js 1 a0 and A-js 1
as united; A-J0, adult rats not exposed to juvenile stress regardless of exposure to adulthood stress, i.e., groups
A-j0 1 a0 and A-j0 1 js united.
b‘‘Adulthood stress’’ was the challenge of learning the two-way shuttle avoidance task, performed immediately
following the exploration test.
cTissues harvesting 24 hr following the exploration test for groups A-js 1 a0 and A-j0 1 a0 or 24 hr following
adulthood stress for groups A-js 1 as and A-j0 1 as.
372 Tsoory et al.
Journal of Neuroscience Research
Juvenile stress affected auditory fear conditioning in
adulthood while increasing L1 expression levels in the
BLA and hippocampus.
Auditory fear conditioning in adulthood.
way ANOVA for groups, phases (of the procedure), and
the interaction groups 3 phases was conducted for the
percentage of time spent freezing during the condition-
ing, the context test, and the cue test.
Conditioning (Fig. 1a).
was observed for phases [F(8,112) 5 32.76; P < 0.01]
but not for groups [F(1,14) 5 0.79; n.s.]; the interaction
groups 3 phases was not significant [F(8,112) 5 1.70;
Context test (Fig. 1b).
was observed for phases [F(4,14) 5 7.30; P < 0.01] but
not for groups [F(1,14) 5 0.14; n.s.]; the interaction
groups 3 phases was not significant [F(8,112) 5 0.08;
Cue test (Fig. 1c). Significant main effects were
observed for groups [F(1,14) 5 10.47; P < 0.01] and for
phases [F(7,14) 5 51.04; P < 0.01.]; the interaction
groups 3 phases was not significant [F(7,14) 5 1.73;
n.s.]. Follow-up t-tests indicted that adult juvenile-
stressed rats exhibited significantly more freezing than
controls during both tone presentations [t(14) 5 3.25; P
< 0.01] and posttone ITIs [t(14) 5 2.68; P < 0.05 ],
though not prior to presentation of the first tone (in the
pretone phase) [t(14) 5 1.40; n.s.]. Additionally, adult
juvenile-stressed rats’ freezing during the first 2 min of
the context test and the first 2 min of the cue test
(‘‘silent’’ pretone phase) did not differ significantly [t(7)
5 1.05; n.s.], whereas control rats’ freezing was signifi-
cantly lower during the first 2 min of the cue test than
during the first 2 min of the context test [t(7) 5 2.48;
P < 0.05].
L1 expression during adulthood.
with controls, adult juvenile-stressed rats exhibited
increased total-L1 expression levels in both the BLA
[t(14) 5 7.84; P < 0.01] and the hippocampus [t(14) 5
7.18; P < 0.01] 24 hr following the fear conditioning
(Fig. 2). These differences were also evident when the
levels of each L1 isoform [expressed in arbitrary units
(au)] were examined separately, i.e., for L1-150 kD
[BLA: C, 19.84 6 4.02 au; JS, 96.14 6 11.03 au; t(14)
5 7.09; P < 0.01; hippocampus: C, 22.48 6 3.91 au;
JS, 95.41 6 9.52 au; t(14) 5 6.50; P < 0.01] and L1-
250 kD [BLA: C, 20.29 6 4.58 au; JS, 95.39 6 8.49
au; t(14) 5 8.29; P < 0.01; hippocampus: C, 19.85 6
4.00 au; JS, 89.50 6 7.38 au; t(14) 5 7.79; P < 0.01].
A significant main effect
A significant main effect
Novel-setting exploration (Fig. 3a).
stressed rats (A-JS: groups A-js 1 a0 and A-js 1 as
united) explored the shuttle box to a significantly lesser
extent [t(42) 5 5.29; P < 0.01] than did adult animals
not exposed to juvenile stress (A-J0: groups A-j0 1 a0
and A-j0 1 as united).
Avoidance learning (Fig. 3b).
hood stressed rats (A-js 1 as) exhibited poor avoidance
learning compared with rats exposed to stress only in
adulthood (A-j0 1 as). Two-way ANOVA for groups,
blocks of 10 trials (repeated measure), and the interaction
groups 3 blocks revealed significant main effects for
groups [F(1,31) 5 34.68; P < 0.01] and for blocks
[F(9,279) 5 23.12; P < 0.01]. The interaction groups 3
blocks was also significant [F(9,279) 5 4.91; P < 0.01].
Juvenile and adult-
Fig. 1. ‘‘Juvenile stress’’ affects auditory fear conditioning in adult-
hood. Percentage of time spent freezing during the fear conditioning
procedure (a; phases: pretone, CS (tone) presentations, and ITIs),
context test (b; phases: each minute of the test), and cue test (c;
phases: pretone, tone presentations, posttone ITIs). No significant dif-
ferences were detected between the groups during conditioning (a)
or in the contextual test (b); however, a significant (P < 0.01) groups
effect was found in the cue test. Subsequent t-tests (c) indicated that
adult juvenile-stressed rats exhibited significantly more freezing than
control animals (*P < 0.05, **P < 0.01) both during tone presenta-
tions (tones) and during the ITIs that followed (posttone ITIs).
‘‘Juvenile Stress’’ Alters L1 Expression 373
Journal of Neuroscience Research
Student’s t-tests per block showed that A-js 1 as rats’
shuttle latencies were significantly longer than those of
A-j0 1 as rats in all blocks.
Further analyses for blocks (repeated measure)
within these groups indicated significant main effects for
blocks in both groups [A-j0 1 as: F(9,126) 5 33.90; P
< 0.01; A-js 1 as: F(9,153) 5 4.39; P < 0.05]. How-
ever, within-subjects contrasts comparing the first block
(block 1) latency with that of the other blocks indicated
that, in the A-j0 1 as group, shuttle latencies were sig-
nificantly (P < 0.01) lower from block 1 in blocks 3–
10, whereas, in the A-js 1 as group, only blocks 5, 7, 9,
and 10 were significantly (P < 0.05) shorter than block
Total-L1 expression levels.
Assessing the effects of juvenile stress shortly after the
exposure (Fig. 4a).Soon after the exposure to juvenile
stress, at PND 33, J-js rats’ L1 levels were significantly
increased compared with those of J-j0 rats in all limbic
regions: BLA [t(14) 5 18.00; P < 0.01], CA1 [t(14) 5
23.69; P < 0.01], DG [t(14) 5 28.23; P < 0.01], and
EC [t(14) 5 5.74; P < 0.01].
Assessing the long-term effects of juvenile stress (Fig.
4b). Two-way ANOVA for juvenile stress, adulthood
stress, and their interaction compared total-L1 expression
levels within each brain region of the four adult groups
(A-j0 1 a0; A-j0 1 as; A-js 1 a0; A-js 1 as). These
analyses yielded significant main effects for juvenile stress
and adulthood stress in all examined regions (juvenile
stress: BLA [F(1,40) 5 4,851.37; P < 0.01], CA1
[F(1,40) 5 3,158.54; P < 0.01], DG [F(1,40) 5
2,848.28; P < 0.01], EC [F(1,40) 5 812.08; P < 0.01];
adulthood stress: BLA [F(1,40) 5 30.72; P < 0.01],
CA1 [F(1,40) 5 144.07; P < 0.01], DG [F(1,40) 5
13.07; P < 0.01], EC [F(1,40) 5 27.91; P < 0.01]).
The interaction juvenile stress 3 adulthood stress was
also significant in all examined regions (BLA [F(1,40) 5
58.55; P < 0.01], CA1 [F(1,40) 5 311.71; P < 0.01],
DG [F(1,40) 5 37.76; P < 0.01], EC [F(1,40) 5 24.46;
P < 0.01]).
Follow-up t-tests (Bonferroni corrected) indicated
that exposure to juvenile stress increased L1 levels in
adulthood among both rats not exposed to adulthood
stress (A-j0 1 a0 vs. A-js 1 a0: BLA [t(18) 5 46.28; P
< 0.01], CA1 [t(18) 5 45.99; P < 0.01], DG [t(18) 5
19.77; P < 0.01], EC [t(18) 5 10.83; P < 0.01]) and
those that were (A-j0 1 as vs. A-js 1 as: BLA [t(22) 5
52.40; P < 0.01]. CA1 [t(22) 5 33.58; P < 0.01]. DG
Fig. 2. ‘‘Juvenile stress’’ affects total L1 levels in the BLA and hippo-
campus in adulthood. Compared with control rats, adult juvenile-
stressed rats exhibited increased total L1 levels in both the BLA and
the hippocampus 24 hr following auditory fear conditioning (**P <
0.01). The insets depict representative bands of L1 isoforms (150 and
250 kD) and of ERKII (42 kD).
Fig. 3. ‘‘Juvenile stress’’ affects exploration and avoidance learning in
adulthood. a: Novel-setting exploration. Adult juvenile stressed rats
(A-JS) explored the shuttle box prior to avoidance learning to a sig-
nificantly lesser extent (**P < 0.01) than adult rats not exposed to it
(A-J0). b: Avoidance learning. Compared with A-j0 1 as rats, A-js
1 as rats exhibited significant (P < 0.01) impaired avoidance learning
throughout the task. A-js 1 as rats’ shuttle latencies were significantly
longer than those of A-j0 1 as rats in all blocks (**P < 0.01).
Within-subject contrasts indicated that A-j0 1 as rats’ shuttle laten-
cies significantly and substantially improved at an earlier phase of the
task compared with A-js 1 as rats (within-subject difference from
block 1:#P < 0.05,##P < 0.01).
374Tsoory et al.
Journal of Neuroscience Research
[t(22) 5 12.23; P < 0.01]. EC [t(22) 5 9.52; P <
0.01]). Adulthood stress increased L1 levels among rats
not exposed to juvenile stress in the BLA, CA1, and DG
but not EC (A-j0 1 a0 vs. A-j0 1 as: BLA [t(18) 5
11.09; P < 0.01], CA1 [t(18) 5 5.72; P < 0.01], DG
[t(18) 5 7.44; P < 0.01], EC [t(18) 5 0.28; n.s.]).
However, among adult juvenile stressed rats, adulthood
stress decreased L1 levels in the CA1 and EC, while not
affecting its expression in the BLA and DG (A-js 1 a0
vs. A-js 1 as: BLA [t(22) 5 1.37; n.s.], CA1 [t(22) 5
18.15; P < 0.01], DG [t(22) 5 1.72; n.s.], EC [t(22) 5
6.07; P < 0.01]).
Assessing the effects of juvenile stress and maturation
(from juvenility to adulthood) on L1 expression levels (Fig.
4c). Two-way ANOVA for maturation and juvenile
stress compared total-L1 expression levels within each
Fig. 4. ‘‘Juvenile stress’’ affects total L1 expression levels both soon
after the exposure and into adulthood. a: Juveniles (PND 33). Com-
pared with J-j0 rats, J-js rats exhibited significantly increased L1 lev-
els in the BLA, CA1, DG, and EC. **P < 0.01. b: Adults (9 weeks
old). Two-way ANOVA of L1 levels within each brain region indi-
cated significant (P < 0.01) main effects for juvenile stress, adulthood
stress, and the interaction juvenile stress 3 adulthood stress in all
examined regions. Follow-up t-tests (Bonferroni corrected) indicated
that juvenile stress increased L1 levels in adulthood among both rats
not exposed to adulthood stress (A-j0 1 a0 vs. A-js 1 a0; **P <
0.01) and those that were (A-j0 1 as vs. A-js 1 as;##P < 0.01).
Adulthood stress increased L1 levels among rats not exposed to juve-
nile stress in the BLA, CA1, and DG but not EC (A-j0 1 as vs. A-
j0 1 as;@@P < 0.01), whereas, among adult juvenile-stressed rats,
adulthood stress decreased L1 levels in the CA1 and EC while not
affecting the BLA and DG (A-js 1 a0 vs. A-js 1 as; yyP < 0.01). c:
Maturation. Two-way ANOVA of L1 levels within each brain
region indicated significant effects (P < 0.01) for maturation, for ju-
venile stress, and for the interaction maturation 3 juvenile stress.
Subsequent t-tests (Bonferroni corrected) indicated a significant and
substantial maturation-related decrease in L1 levels among both juve-
nile-stressed rats (J-js vs. A-JS) and those not exposed to it (J-j0 vs.
A-J0) in all examined regions (significantly different from matched-
group PND 33 rats,##P < 0.01). Rat that were exposed to juvenile
stress exhibited significantly increased L1 levels compared with those
that were not both during the juvenile stage (J-js vs. J-j0;
0.01) and in adulthood (A-JS vs. A-J0; **P < 0.01) in all examined
regions. d: The insets depict representative bands of L1 isoforms
(150 and 250 kD) and of ERKII (42 kD) across the groups and
‘‘Juvenile Stress’’ Alters L1 Expression375
Journal of Neuroscience Research
brain region between the following four groups: juvenile
rats (PND 33) exposed to juvenile stress or not (i.e.,
groups J-js and J-j0) and adult rats exposed to juvenile
stress or not, regardless of their exposure to adulthood
stress, i.e., A-JS (groups A-js 1 a0 and A-js 1 as united)
compared with A-J0 (groups A-j0 1 a0 and A-j0 1 as
united). This analysis indicated a significant maturation-
related decrease in L1 expression levels in all examined
regions, but exposure to juvenile stress significantly
increased L1 levels in all examined regions (maturation:
BLA [F(1,56) 5 1,190.55; P < 0.01], CA1 [F(1,56) 5
233.40; P < 0.01], DG[F(1,56) 5 1,349.92; P < 0.01],
EC [F(1,56) 5 39.74; P < 0.01]; juvenile stress: BLA
[F(1,56) 5 1,417.61; P < 0.01], CA1 [F(1,56) 5
432.06; P < 0.01], DG [F(1,56) 5 754.62; P < 0.01],
EC [F(1,56) 5 101.04; P < 0.01]). The interaction ju-
venile stress 3 maturation was significant in the BLA,
CA1, and DG but not in the EC (BLA [F(1,56) 5 5.71;
P < 0.05], CA1 [F(1,56) 5 11.76; P < 0.01], DG
[F(1,56) 5 102.74; P < 0.01], EC [F(1,56) 5 0.90;
Follow-up t-tests (Bonferroni corrected) comparing
L1 expression levels between juvenile and adult rats that
had or had not experienced juvenile stress indicated that
maturation was associated with a decrease in L1 expres-
sion levels in all examined regions (J-j0 vs. A-J0: BLA
[t(26) 5 24.23; P < 0.01], CA1 [t(26) 5 26.25; P <
0.01], DG [t(26) 5 19.07; P < 0.01] EC [t(26) 5 7.67;
P < 0.01]). Exposure to juvenile stress did not alter
these maturation-related decreases in any of the exam-
ined regions (J-js vs. A-JS: BLA [t(30) 5 24.21; P <
0.01], CA1 [t(30) 5 10.04; P < 0.01], DG [t(30) 5
41.49; P < 0.01], EC [t(30) 5 4.03; P < 0.01]),
although juvenile stressed rats of both ages exhibited sig-
nificantly higher L1 levels in all examined regions (J-j0
vs. J-js: BLA [t(14) 5 18.00; P < 0.01], CA1 [t(14) 5
23.69; P < 0.01], DG [t(14) 5 28.23; P < 0.01], EC
[t(14) 5 5.74; P < 0.01]; A-J0 vs. A-JS: BLA [t(42) 5
38.70; P < 0.01], CA1 [t(42) 5 16.53; P < 0.01], DG
[t(42) 5 15.13; P < 0.01], EC [t(42) 5 9.80; P <
Because exposure to adulthood stress was found to
affect differentially rats exposed to juvenile stress com-
pared with those that were not (see Results), further
analyses were conducted to describe better the matura-
tion related changes in L1 expression levels between rats
exposed to juvenile stress and those that were not. These
analyses took into account exposure to adulthood stress
and verified that its effects did not mask the effects of ju-
venile stress on maturation. These analyses, Bonferroni
corrected t-tests, compared L1 levels within each brain
region of juvenile rats (PND 33) that were or were not
exposed to juvenile stress with those of adult rats that
were or were not exposed to adulthood stress. These
comparisons indicated that, among rats not exposed to
juvenile stress, both adult rats that were not exposed to
adulthood stress (A-j0 1 a0) and those that were (A-j0
1 as) exhibited significantly lower levels of L1 compared
with juvenile rats (PND 33) that were not exposed to
juvenile stress (J-j0) in all examined regions (J-j0 vs. A-
j0 1 a0: BLA [t(16) 5 59.21; P < 0.01], CA1 [t(16) 5
35.06; P < 0.01], DG [t(16) 5 32.72; P < 0.01], EC
[t(16) 5 6.16; P < 0.01]; J-j0 vs. A-j0 1 as: BLA [t(16)
5 57.22; P < 0.01], CA1 [t(16) 5 31.42; P < 0.01],
DG [t(16) 5 16.39; P < 0.01], EC [t(16) 5 7.66; P <
0.01]. A similar pattern of effects was evident among rats
that were exposed to juvenile stress. Adult juvenile-
stressed rats that were not exposed to adulthood stress
(A-js 1 a0) exhibited lower expression levels of L1
compared with those of PND 33 juvenile-stressed rats
(J-js) in the BLA [t(16) 5 15.08; P < 0.01], CA1[t(16)
5 12.92; P < 0.01], DG [t(16) 5 31.31; P < 0.01] but
not EC [t(16) 5 1.67; n.s.]. Similarly, in comparison
with PND 33 juvenile-stressed rats (J-js), adult juvenile-
stressed rats that were also exposed to adulthood stress
(A-js 1 as) exhibited lower levels of L1 expression levels
in all examined regions (BLA [t(20) 5 16.43; P < 0.01],
CA1 [t(20) 5 22.08; P < 0.01], DG [t(20) 5 47.84;
P < 0.01], EC [t(20) 5 6.73; P < 0.01]).
Consistently with previous reports (Tsoory et al.,
2007, 2008a), adult juvenile-stressed rats exhibited
decreased novel-setting exploration and impaired avoid-
ance learning. Furthermore, adult juvenile-stressed rats
exhibited increased freezing in response to a conditioned
cue. Increased freezing responses were associated with
altered BLA activity (Rodriguez Manzanares et al., 2005;
Sekiguchi et al., 2008), so this increased freezing may
correspond to increased startle response among adult ju-
venile-stressed rats (Tsoory et al., 2007, 2008b). Collec-
tively, these altered behaviors indicate that the juvenile
stage (about PND 28 in rats) as a stress-sensitive age and
support Romeo and McEwen’s (2006) suggestion that,
as a result of ongoing plasticity during this period, the
maturing brain is especially vulnerable to perturbations
in key regulatory nuclei of stress responses, emotional
behavior, and cognitive functions.
The effects of juvenile stress on L1 levels also
suggest altered maturation of the limbic system. L1 is
processes (Maness and Schachner, 2007) and is poten-
tially relevant for maturation (Law et al., 2003). In this
study, comparing L1 levels between juvenile and adult
naı ¨ve rats indicated a maturation-related decrease in all
examined regions. A decrease was also evident among
juvenile-stressed rats; however, L1 levels were substan-
tially elevated soon after the exposure to juvenile stress
and remained significantly higher during adulthood
among juvenile-stressed rats compared with naı ¨ve and
Adult mice ablated of the L1 gene following PND
22 also displayed altered functionality of the limbic sys-
tem (Law et al., 2003). These mice exhibited increased
basal excitatory activity in the hippocampus CA1
subregion and altered spatial searching strategies related
to impairments in long-term memory of landmarks
376Tsoory et al.
Journal of Neuroscience Research
locations (Law et al., 2003). Although these mice exhib-
ited decreased anxiety indices in the open field and
elevated plus maze tests (EPM), the latter may reflect an
behavior and increased exploration of the EPM’s open
arms compared with both juvenile and adult mice (Macrı ´
et al., 2002; Laviola et al., 2003); this apparent reduced
anxiety was related to increased novelty-seeking behav-
ior among adolescent mice independent of correct
danger perception (Laviola et al., 2003). Thus, it may be
that altering maturation-related changes in L1 by either
conditional gene ablation (Law et al., 2003) or juvenile
stress dysregulates the limbic system maturation and
affects stress responses, emotional behavior, and cognitive
associated with impaired cognitive function and height-
ened anxiety (Sandi, 2004). L1 mRNA levels in the hip-
pocampus, thalamus, and striatum negatively correlated
with spatial navigation but positively correlated with
posttraining plasma CORT levels (Venero et al., 2004).
Because L1 stimulates neurite outgrowth and is involved
in repair processes in the lesioned CNS (Styren et al.,
1995; Jucker et al., 1996; Schachner, 1997), chronic
stress-induced elevated L1 levels in rats were proposed
to represent a neuroprotective mechanism triggered in
response to the deleterious effects of chronic stress, such
as different forms of neuronal atrophy (Sandi et al.,
2001; Venero et al., 2002; Sandi, 2004).
The current study may be the first to report
persistent increases in L1 expression following a non-
chronic stress procedure. In response to stressors, juve-
nile rats exhibit peak CORT levels similar to those of
adults, but the CORT response is prolonged because of
incomplete maturation of negative feedback systems
(Romeo and McEwen, 2006; McCormick and Math-
ews, 2007). It is possible that in the current study such
prolonged responses to juvenile stress triggered an L1
neuroprotective reaction. Alternatively, insofar as adult
juvenile-stressed rats exhibit higher basal CORT levels
than controls (Ilin and Richter-Levin, 2009), it may be
that in the current study a juvenile stress-induced state
provoked an L1 neuroprotective reaction against the
deleterious atrophic effects of chronic elevated CORT
levels. Several studies indicated direct involvement of
glucocorticoids in regulating NCAM and its polysialy-
lated form PSA-NCAM expression (Coughlan et al.,
1996; Coughlan and Breen, 1998; Georgopoulou and
Breen, 1999; Datson et al., 2001). Adult juvenile-stressed
rats were reported to exhibit altered expression ratio of
PSA-NCAM to NCAM (Tsoory et al., 2008a). It may
be that elevated CORT levels, either soon after the
exposure to juvenile stress or later during adulthood,
may directly or indirectly affect the regulation of
NCAM, PSA-NCAM, and L1. Collectively, these find-
ings suggest that juvenile stress dysregulates maturational
processes, including those involving CAMs’ regulation,
which may in turn affect stress responsiveness, thus
supporting a role for NCAM, PSA-NCAM, and L1
regulation in emotional regulation (Sandi and Bisaz,
The increased L1 levels among adult juvenile
stressed rats may relate to the impaired avoidance learn-
ing. L1 regulation is critical for memory formation
(Luthi et al., 1994; Weltzl and Stork, 2003). L1 levels
are increased in the hippocampus and in cortical regions
following the acquisition of various hippocampus-de-
pendent tasks (Merino et al., 2000; Venero et al., 2004),
suggesting that transient increases in L1 support synaptic
remodeling following learning (Knafo et al., 2005). This
study’s significant, though mild, increases in L1 levels in
the BLA and hippocampal subregions evident 24 hr
following avoidance learning (Fig. 4b; A-j0 1 a0 vs. A-
j0 1 as) may support this idea. The fact that adult
juvenile-stressed rats exhibited substantially higher L1
levels in all examined regions and performed poorly in
this task may relate to the suggested inverted U-shaped
relationship between L1 levels and cognitive functions
(Sandi, 2004); it is possible that mild increases in L1
levels are required for learning, whereas marked eleva-
tions in L1 levels preceding learning a task will disrupt
Overall, the data suggest that juvenile stress disrupts
maturation of the limbic system, possibly by affecting the
establishment of activity-dependent connectivity within
the neural circuitry. Sandi and Bisaz (2007) suggested
that alterations in CAM expression following chronic
stress relate to Castren’s (2005) ‘‘network hypothesis’’ of
mood and anxiety disorders. According to this hypothe-
sis, these disorders reflect problems in information
processing within particular neural networks within the
brain rather than a ‘‘chemical imbalance’’. Accordingly,
antidepressants and other treatments may function by
gradually improving information processing within these
affected neural networks (Castren, 2005).
It may be that altered ‘‘neural networks’’ result also
from exposure to stressors during critical periods of
development or maturation. For instance, reduced
hippocampal volume is particularly common in patients
with depression and posttraumatic stress disorder, who
suffered childhood trauma (Vythilingam et al., 2002;
Bremner et al., 2003). Similarly, in rats, chronic variable
stress during the peripubertal period induced hippocam-
pal volume deficits coupled with impaired cognitive
and hypothalamic-pituitary-adrenal axis functioning in
adulthood (Isgor et al., 2004).
Animal models of stress-induced mood and anxiety
disorders indicate neuronal atrophy, decreased neurogen-
esis, decreased hippocampal vascular endothelial growth
factor (VEGF), and decreased brain-derived neurotrophic
factor levels (BDNF; Castren et al., 2007). In contrast,
antidepressants restore neuronal atrophy, increase neuro-
genesis, increase VEGF and BDNF levels, and increase
insulin-like growth factor types 1 and 2, which activate
tyrosine kinase receptors coupled to similar signal trans-
duction pathways (Duman and Monteggia, 2006). The
‘‘Juvenile Stress’’ Alters L1 Expression377
Journal of Neuroscience Research
dysregulation of several members of the fibroblast
growth factor (FGF) family and their receptors was
found among depressed patients, and antidepressants
were reported to regulate FGF expression (Evans et al.,
2004; Riva et al., 2005). The activation of the FGF
receptor 1 tyrosine kinase by L1 was implicated in neu-
rite outgrowth (Tang et al., 2006). L1 may relate to
maturational processes (Law et al., 2003), so it is sug-
gested that stress-induced alterations in L1 expression,
early in life or later on, may relate to the dysregulation
of neurotrophic factors, potentially resulting in altered
neuronal connectivity. Indirect support is provided by
recent findings indicating that ‘‘enriched environment’’
rearing, which is known to restore the atrophic effects
of chronic stress exposure (Veena et al., 2009), also
restored the effects of juvenile stress on behavioral indi-
ces of anxiety and depression, basal CORT levels, and
L1 levels in the BLA (Ilin and Richter-Levin, 2009).
Furthermore, chronic antidepressant treatment known to
restore chronic stress-induced neuronal atrophy (Duman
and Monteggia, 2006) also increased PSA-NCAM
levels in the rat hippocampus, medial prefrontal cortex,
and piriform cortex (Sairanen et al., 2007), suggesting a
role for CAMs in antidepressant-induced elevations in
synaptic plasticity and connectivity within brain regions
associated with mood disorders.
It is noteworthy that this study did not assess the
underlying molecular mechanisms that may mediate the
observed elevations in L1 levels, nor did it evaluate the
effects that these elevated L1 levels might have on L1
intercellular functions or L1 homophilic and hetrophilic
interactions. Future research should address these issues;
they have been extensively investigated regarding L1
involvement in developmental processes and neuronal
repair (Loers and Schachner, 2007; Schmid and Maness,
2008; Zhang et al., 2008). Specifically, the role of the
Src family kinases in mediating the long-term effects of
juvenile stress should be investigated, because the latter
were implicated in regulating L1 adhesion in neurite
outgrowth, suggesting complementary and dynamic roles
for ankyrin and ezrin (Schmid et al., 2000; Gil et al.,
2000; Schaefer et al., 2002; Sakurai et al., 2008).
previous findings that related juvenile stress to induce a
predisposition for mood and anxiety disorder-like behav-
iors in adulthood (Tsoory et al., 2008b) and to altered
maturation of the limbic system (Jacobson-Pick et al.,
2008), including altered maturation-related changes in
CAM expression (Tsoory et al., 2008a). Collectively, the
data support a role for CAM dysregulation in ELS-
induced predisposition for mood and anxiety disorders,
which may result in altered neuronal connectivity and
Arami S, Jucker M, Schachner M, Welzl H. 1996. The effect of continu-
ous intraventricular infusion of L1 and NCAM antibodies on spatial
learning in rats. Behav Brain Res 81:81–87.
Becker A, Letzel K, Letzel U, Grecksch G. 1997. Kindling of the dorsal
and the ventral hippocampus: effects on learning performance in rats.
Physiol Behav 62:1265–1271.
Bremner JD, Vythilingam M, Vermetten E, Southwick SM, McGlashan
T, Nazeer A, Khan S, Vaccarino LV, Soufer R, Garg PK, Ng CK,
Staib LH, Duncan JS, Charney DS. 2003. MRI and PET study of defi-
cits in hippocampal structure and function in women with childhood
sexual abuse and posttraumatic stress disorder. Am J Psychiatry
Castren E. 2005. Is mood chemistry? Nat Rev Neurosci 6:241–246.
Castren E, Voikar V, Rantamaki T. 2007. Role of neurotrophic factors
in depression. Curr Opin Pharmacol 7:18–21.
Coughlan CM, Breen KC. 1998. Glucocorticoid induction of the
alpha2,6 sialyltransferase enzyme in a mouse neural cell line. J Neurosci
Coughlan CM, Seckl JR, Fox DJ, Unsworth R, Breen KC. 1996. Tis-
sue-specific regulation of sialyltransferase activities in the rat by cortico-
steroids in vivo. Glycobiology 6:15–22.
Datson NA, van der Perk J, de Kloet ER, Vreugdenhil E. 2001. Identifi-
cation of corticosteroid-responsive genes in rat hippocampus using serial
analysis of gene expression. Eur J Neurosci 14:675–689.
Duman RS, Monteggia LM. 2006. A neurotrophic model for stress-
related mood disorders. Biol Psychiatry 59:1116–1127.
Emrick MA, Lee T, Starkey PJ, Mumby MC, Resing KA, Ahn NG.
2006. The gatekeeper residue controls autoactivation of ERK2 via a
pathway of intramolecular connectivity. Proc Natl Acad Sci U S A
Evans SJ, Choudary PV, Neal CR, Li JZ, Vawter MP, Tomita H, Lopez
JF, Thompson RC, Meng F, Stead JD, Walsh DM, Myers RM, Bun-
ney WE, Watson SJ, Jones EG, Akil H. 2004. Dysregulation of the
fibroblast growth factor system in major depression. Proc Natl Acad Sci
U S A 101:15506–15511.
Georgopoulou N, Breen KC. 1999. Overexpression of the alpha2,6 (N)
sialyltransferase enzyme in human and rat neural cell lines is associated
with increased expression of the polysialic acid epitope. J Neurosci Res
Gil OD, Sakurai T, Bradley AE, Fink MY, Cassella MR, Kuo JA,
Felsenfeld DP. 2003. Ankyrin binding mediates L1CAM interactions
with static components of the cytoskeleton and inhibits retrograde
movement of L1CAM on the cell surface. J Cell Biol 162:719–730.
Gutman DA, Nemeroff CB. 2003. Persistent central nervous system
effects of an adverse early environment: clinical and preclinical studies.
Physiol Behav 79:471–478.
Ilin Y, Richter-Levin G. 2009. Enriched environment experience over-
comes learning deficits and depressive-like behavior induced by juvenile
stress. PLoS ONE 4:e4329.
Isgor C, Kabbaj M, Akil H, Watson SJ. 2004. Delayed effects of chronic
variable stress during peripubertal-juvenile period on hippocampal mor-
phology and on cognitive and stress axis functions in rats. Hippocampus
Jacobson-Pick S, Elkobi A, Vander S, Rosenblum K, Richter-Levin G.
2008. Juvenile stress-induced alteration of maturation of the GABAA
receptor alpha subunit in the rat. Int J Neuropsychopharmacol 11:891–
Jucker M, D’Amato F, Mondadori C, Mohajeri H, Magyar J, Bartsch U,
Schachner M. 1996. Expression of the neural adhesion molecule L1 in
the deafferented dentate gyrus. Neuroscience 75:703–715.
Knafo S, Barkai E, Libersat F, Sandi C, Venero C. 2005. Dynamics of
olfactory learning-induced up-regulation of L1 in the piriform cortex
and hippocampus. Eur J Neurosci 21:581–586.
Laifenfeld D, Karry R, Grauer E, Klein E, Ben-Shachar D. 2005a. Anti-
depressants and prolonged stress in rats modulate CAM-L1, laminin,
and pCREB, implicated in neuronal plasticity. Neurobiol Dis 20:432–
378 Tsoory et al.
Journal of Neuroscience Research
Laifenfeld D, Karry R, Klein E, Ben-Shachar D. 2005b. Alterations in
cell adhesion molecule L1 and functionally related genes in major
depression: a postmortem study. Biol Psychiatry 57:716–725.
Laviola G, Macri S, Morley-Fletcher S, Adriani W. 2003. Risk-taking
behavior in adolescent mice: psychobiological determinants and early
epigenetic influence. Neurosci Biobehav Rev 27:19–31.
Law JW, Lee AY, Sun M, Nikonenko AG, Chung SK, Dityatev A,
Schachner M, Morellini F. 2003. Decreased anxiety, altered place learn-
ing, and increased CA1 basal excitatory synaptic transmission in mice
with conditional ablation of the neural cell adhesion molecule L1.
J Neurosci 23:10419–10432.
LeDoux J. 2007. The amygdala. Curr Biol 17:R868–874.
Lee AY. 2005. The cell recognition molecule L1 and cognition. Chin
J Physiol 48:169–175.
Lefloch R, Pouyssegur J, Lenormand P. 2008. Single and combined
silencing of ERK1 and ERK2 reveals their positive contribution to
growth signaling depending on their expression levels. Mol Cell Biol
Loers G, Schachner M. 2007. Recognition molecules and neural repair.
J Neurochem 101:865–882.
Luthi A, Laurent JP, Figurov A, Muller D, Schachner M. 1994. Hippo-
campal long-term potentiation and neural cell adhesion molecules L1
and NCAM. Nature 372:777–779.
Macrı ´ S, Adriani W, Chiarotti F, Laviola G. 2002. Risk-taking during
exploration of a plus-maze is greater in adolescent than in juvenile or
adult mice. Anim Behav 64:541–546.
Maness PF, Schachner M. 2007. Neural recognition molecules of the im-
munoglobulin superfamily: signaling transducers of axon guidance and
neuronal migration. Nat Neurosci 10:19–26.
Matsumoto-Miyai K, Ninomiya A, Yamasaki H, Tamura H, Nakamura
Y, Shiosaka S. 2003. NMDA-dependent proteolysis of presynaptic ad-
hesion molecule L1 in the hippocampus by neuropsin. J Neurosci
McCormick CM, Mathews IZ. 2007. HPA function in adolescence: role
of sex hormones in its regulation and the enduring consequences of
exposure to stressors. Pharmacol Biochem Behav 86:220–233.
Merino JJ, Cordero MI, Sandi C. 2000. Regulation of hippocampal cell
adhesion molecules NCAM and L1 by contextual fear conditioning is
dependent upon time and stressor intensity. Eur J Neurosci 12:3283–
Nemeroff CB. 2004. Neurobiological consequences of childhood trauma.
J Clin Psychiatry 65(Suppl 1):18–28.
Paxinos G, Watson C. 1998. The rat brain in stereotaxic coordinates.
San Diego: Academic Press.
Riva MA, Molteni R, Bedogni F, Racagni G, Fumagalli F. 2005.
Emerging role of the FGF system in psychiatric disorders. Trends
Pharmacol Sci 26:228–231.
Rodriguez Manzanares PA, Isoardi NA, Carrer HF, Molina VA. 2005.
Previous stress facilitates fear memory, attenuates GABAergic inhibition,
and increases synaptic plasticity in the rat basolateral amygdala. J Neuro-
Romeo RD, McEwen BS. 2006. Stress and the adolescent brain. Ann N
Y Acad Sci 1094:202–214.
Sairanen M, O’Leary OF, Knuuttila JE, Castren E. 2007. Chronic antide-
pressant treatment selectively increases expression of plasticity-related
proteins in the hippocampus and medial prefrontal cortex of the rat.
Sakurai T, Gil OD, Whittard JD, Gazdoiu M, Joseph T, Wu J, Waksman
A, Benson DL, Salton SR, Felsenfeld DP. 2008. Interactions between
the L1 cell adhesion molecule and ezrin support traction-force genera-
tion and can be regulated by tyrosine phosphorylation. J Neurosci Res
Sanchez MM, Ladd CO, Plotsky PM. 2001. Early adverse experience as
a developmental risk factor for later psychopathology: evidence from
rodent and primate models. Dev Psychopathol 13:419–449.
Sandi C. 2004. Stress, cognitive impairment and cell adhesion molecules.
Nat Rev Neurosci 5:917–930.
Sandi C, Bisaz R. 2007. A model for the involvement of neural cell
adhesion molecules in stress-related mood disorders. Neuroendocrinol-
Sandi C, Merino JJ, Cordero MI, Touyarot K, Venero C. 2001. Effects
of chronic stress on contextual fear conditioning and the hippocampal
expression of the neural cell adhesion molecule, its polysialylation, and
L1. Neuroscience 102:329–339.
Savonenko A, Werka T, Nikolaev E, Zielinski K, Kaczmarek L. 2003.
Complex effects of NMDA receptor antagonist APV in the basolateral
amygdala on acquisition of two-way avoidance reaction and long-term
fear memory. Learn Mem 10:293–303.
Schachner M. 1997. Neural recognition molecules and synaptic plasticity.
Curr Opin Cell Biol 9:627–634.
Schaefer AW, Kamei Y, Kamiguchi H, Wong EV, Rapoport I,
Kirchhausen T, Beach CM, Landreth G, Lemmon SK, Lemmon V.
2002. L1 endocytosis is controlled by a phosphorylation-dephosphoryl-
ation cycle stimulated by outside-in signaling by L1. J Cell Biol 157:
Schmid RS, Maness PF. 2008. L1 and NCAM adhesion molecules as sig-
naling coreceptors in neuronal migration and process outgrowth. Curr
Opin Neurobiol 18:245–250.
Schmid RS, Pruitt WM, Maness PF. 2000. A MAP kinase-signaling
pathway mediates neurite outgrowth on L1 and requires Src-dependent
endocytosis. J Neurosci 20:4177–4188.
Schwegler H, Lipp HP, Van der Loos H, Buselmaier W. 1981.
Individual hippocampal mossy fiber distribution in mice correlates with
two-way avoidance performance. Science 214:817–819.
Sekiguchi M, Zushida K, Yoshida M, Maekawa M, Kamichi S, Yoshida
M, Sahara Y, Yuasa S, Takeda S, Wada K. 2008. A deficit of brain
dystrophin impairs specific amygdala GABAergic transmission and
enhances defensive behaviour in mice. Brain (in press).
Silletti S, Yebra M, Perez B, Cirulli V, McMahon M, Montgomery AM.
2004. Extracellular signal-regulated kinase (ERK)-dependent gene
expression contributes to L1 cell adhesion molecule-dependent motility
and invasion. J Biol Chem 279:28880–28888.
Styren SD, Miller PD, Lagenaur CF, DeKosky ST. 1995. Alternate
strategies in lesion-induced reactive synaptogenesis: differential expres-
sion of L1 in two populations of sprouting axons. Exp Neurol
Tang N, He M, O’Riordan MA, Farkas C, Buck K, Lemmon V, Bearer
CF. 2006. Ethanol inhibits L1 cell adhesion molecule activation of
mitogen-activated protein kinases. J Neurochem 96:1480–1490.
Tiunova A, Anokhin KV, Schachner M, Rose SP. 1998. Three time
windows for amnestic effect of antibodies to cell adhesion molecule L1
in chicks. Neuroreport 9:1645–1648.
Tsoory M, Richter-Levin G. 2006. Learning under stress in the adult rat
is differentially affected by juvenile or adolescent stress. Int J Neuropsy-
Tsoory M, Cohen H, Richter-Levin G. 2007. Juvenile stress induces a
predisposition to either anxiety or depressive-like symptoms following
stress in adulthood. Eur Neuropsychopharmacol 17:245–256.
Tsoory M, Guterman A, Richter-Levin G. 2008a. Exposure to stressors
during juvenility disrupts development-related alterations in the PSA-
NCAM to NCAM expression ratio: potential relevance for mood and
anxiety disorders. Neuropsychopharmacology 33:378–393.
Tsoory MM, Vouimba RM, Akirav I, Kavushansky A, Avital A,
Richter-Levin G. 2008b. Amygdala modulation of memory-related
processes in the hippocampus: potential relevance to PTSD. Prog Brain
‘‘Juvenile Stress’’ Alters L1 Expression 379
Journal of Neuroscience Research
Veena J, Srikumar BN, Mahati K, Bhagya V, Raju TR, Shankaranar-
ayana Rao BS. 2009. Enriched environment restores hippocampal cell
proliferation and ameliorates cognitive deficits in chronically stressed
rats. J Neurosci Res 87:831–843.
Venero C, Tilling T, Hermans-Borgmeyer I, Schmidt R, Schachner M,
Sandi C. 2002. Chronic stress induces opposite changes in the mRNA
expression ofthe cell adhesion
Venero C, Tilling T, Hermans-Borgmeyer I, Herrero AI, Schachner M,
Sandi C. 2004. Water maze learning and forebrain mRNA expression
of the neural cell adhesion molecule L1. J Neurosci Res 75:172–181.
Vythilingam M, Heim C, Newport J, Miller AH, Anderson E, Bronen
R, Brummer M, Staib L, Vermetten E, Charney DS, Nemeroff CB,
trauma associatedwith smaller
hippocampal volume in women with major depression. Am J Psychiatry
Weltzl H, Stork O. 2003. Cell adhesion molecules: key players in
memory consolidation? News Physiol Sci 18:147–150.
Wiltgen BJ, Sanders MJ, Anagnostaras SG, Sage JR, Fanselow MS. 2006.
Context fear learning in the absence of the hippocampus. J Neurosci
Yee BK, Hauser J, Dolgov VV, Keist R, Mohler H, Rudolph U, Feldon
J. 2004. GABA receptors containing the alpha5 subunit mediate the
trace effect in aversive and appetitive conditioning and extinction of
conditioned fear. Eur J Neurosci 20:1928–1936.
Zhang Y, Yeh J, Richardson PM, Bo X. 2008. Cell adhesion molecules
of the immunoglobulin superfamily in axonal regeneration and neural
repair. Restor Neurol Neurosci 26:81–96.
380 Tsoory et al.
Journal of Neuroscience Research