HIPPOCAMPUS 22:577–589 (2012)
Differential Expression of Molecular Markers of Synaptic Plasticity in
the Hippocampus, Prefrontal Cortex, and Amygdala in Response to
Spatial Learning, Predator Exposure, and Stress-Induced Amnesia
Phillip R. Zoladz,1 Collin R. Park,2,3,4 Joshua D. Halonen,2,3,4 Samina Salim,5
Karem H. Alzoubi,6 Marisa Srivareerat,5 Monika Fleshner,7
Karim A. Alkadhi,5 and David M. Diamond2,3,4,8*
ABSTRACT:
We have studied the effects of spatial learning and predator
stress-induced amnesia on the expression of calcium/calmodulin-dependent
protein kinase II (CaMKII), brain-derived neurotrophic factor (BDNF) and
calcineurin in the hippocampus, basolateral amygdala (BLA), and medial
prefrontal cortex (mPFC). Adult male rats were given a single training session in the radial-arm water maze (RAWM) composed of 12 trials followed
by a 30-min delay period, during which rats were either returned to their
home cages or given inescapable exposure to a cat. Immediately following
the 30-min delay period, the rats were given a single test trial in the RAWM
to assess their memory for the hidden platform location. Under control (no
stress) conditions, rats exhibited intact spatial memory and an increase in
phosphorylated CaMKII (p-CaMKII), total CaMKII, and BDNF in dorsal
CA1. Under stress conditions, rats exhibited impaired spatial memory and a
suppression of all measured markers of molecular plasticity in dorsal CA1.
The molecular profiles observed in the BLA, mPFC, and ventral CA1 were
markedly different from those observed in dorsal CA1. Stress exposure
increased p-CaMKII in the BLA, decreased p-CaMKII in the mPFC, and had
no effect on any of the markers of molecular plasticity in ventral CA1. These
findings provide novel observations regarding rapidly induced changes in the
expression of molecular plasticity in response to spatial learning, predator exposure, and stress-induced amnesia in brain regions involved in different
aspects of memory processing. V 2011 Wiley Periodicals, Inc.
C
KEY WORDS:
corticosterone; calcium/calmodulin-dependent protein
kinase II (CaMKII); brain-derived neurotrophic factor (BDNF); stress;
memory; amnesia
INTRODUCTION
The effects of stress on learning and memory have been studied in
the context of functional alterations of brain regions that are important
1
Department of Psychology & Sociology, Ohio Northern University, Ada,
Ohio; 2 Research & Development Service, James A. Haley VA Hospital,
Tampa, Florida; 3 Department of Psychology, University of South Florida,
Tampa, Florida; 4 Center for Preclinical & Clinical Research on PTSD,
University of South Florida, Tampa, Florida; 5 Department of Pharmacological & Pharmaceutical Sciences, College of Pharmacy, University of
Houston, Houston , Texas; 6 Department of Clinical Pharmacy, Jordan
University of Science and Technology, Irbid, Jordan; 7 Department of
Integrative Physiology & Center for Neuroscience, University of Colorado, Boulder, Colorado; 8 Department of Molecular Pharmacology &
Physiology, University of South Florida, Tampa, Florida
Grant sponsors: Merit Review and Research Career Scientist Awards
(Department of Veterans Affairs), SGP grant (University of Houston).
*Correspondence to: David Diamond, Department of Psychology, University of South Florida, 4202 E. Fowler Ave. PCD 4118G, Tampa, FL
33620. E-mail: ddiamond@usf.edu
Accepted for publication 6 December 2010
DOI 10.1002/hipo.20922
Published online 2 May 2011 in Wiley Online Library
(wileyonlinelibrary.com).
C 2011
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WILEY PERIODICALS, INC.
for cognition, such as the hippocampus, prefrontal
cortex (PFC), and amygdala (Nathan et al., 2004; Diamond et al., 2007; Ramos and Arnsten, 2007; Sandi
and Pinelo-Nava, 2007; Joels and Baram, 2009; van
Stegeren, 2009; Wolf, 2009; Segal et al., 2010).
Numerous studies have shown that stress impairs performance on hippocampus- and PFC-dependent tasks
and induces morphological alterations within each
brain region (Kim and Diamond, 2002; Conrad, 2006;
Lupien et al., 2007; McEwen, 2007; Holmes and Wellman, 2009; Liston et al., 2009; McLaughlin et al.,
2009). Electrophysiological studies have provided findings consistent with this work with the well-described
finding that stress blocks the induction of long-term
potentiation (LTP), a form of synaptic plasticity
hypothesized to underlie memory formation, in the
hippocampus and PFC (Kim et al., 2006; Diamond
et al., 2007). In contrast, acute stress significantly
enhances LTP in the amygdala (Vouimba et al., 2004;
Maroun, 2006; Vouimba et al., 2006), a brain region
which is critically involved in the processing of emotional memories (LeDoux, 2000; McGaugh, 2004).
The effects of stress on these brain structures often
depend on the duration of the stress (e.g., acute versus
chronic) and the specific subregion of the structures
being examined. For instance, chronic stress work has
focused on describing alterations of hippocampal morphology primarily in CA3 (McEwen, 2007; McLaughlin et al., 2009) and a suppression of neurogenesis in
the dentate gyrus (Fuchs et al., 2006; Lucassen et al.,
2009). Acute stress manipulations, by contrast, have
revealed the susceptibility of the CA1 region to exhibit
an impairment of electrophysiological plasticity, such as
primed burst and long-term potentiation (LTP; Kim
et al., 2006; Diamond et al., 2007; Joels and Krugers,
2007). That stress exerts different effects on these hippocampal subregions, as well as other brain areas, may
explain why stress exerts such complex effects on cognition (Kim et al., 2006; Diamond et al., 2007; Wolf,
2009; Schwabe et al., 2010). Indeed, the differential
susceptibility of each region of the hippocampus to
stress may be related to the more general finding that
the subregions of the hippocampus are differentially
involved in processes underlying learning and memory
(Bannerman et al., 2004; Kesner et al., 2004; Barkus
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ZOLADZ ET AL.
et al., 2010; Fanselow and Dong, 2010; Maggio and Segal,
2010b; Segal et al., 2010).
With regards to the PFC, researchers have typically focused
on stress-induced alterations of the medial portion of this structure, which includes the cingulate, prelimbic, and infralimbic
cortices (Holmes and Wellman, 2009). Studies have shown that
stress significantly alters dendritic branching and length, as well
as levels of several neurotransmitters (e.g., glutamate, dopamine, norepinephrine), in the medial PFC (mPFC) (Moghaddam and Jackson, 2004; Ramos and Arnsten, 2007; Holmes
and Wellman, 2009). As with the CA1 region of the hippocampus, acute stress has been shown to impair the induction of
LTP in the PFC (Maroun and Richter-Levin, 2003; Jay et al.,
2004; Rocher et al., 2004; Richter-Levin and Maroun, 2010).
These findings highlight the importance of taking the duration
of the stressor and the specific subregions into account when
examining the effects of stress on memory and brain function.
Related research has demonstrated that activation of the
basolateral amygdala (BLA) contributes to the stress-induced
modulation of hippocampal and mPFC function. Damage to,
or inactivation of, the BLA blocks the inhibitory effects of
stress on hippocampus-dependent memory and synaptic plasticity (Kim et al., 2001, 2005). Complementary work has shown
that electrical stimulation of the BLA can mimic the stressinduced impairment of LTP in CA1 (Akirav and Richter-Levin,
1999; Vouimba and Richter-Levin, 2005; Tsoory et al., 2008),
and can block LTP in the mPFC, as well (Maroun and
Richter-Levin, 2003; Richter-Levin and Maroun, 2010). These
findings support the hypothesis that the stress-induced impairment of memory is generated by an amygdala-mediated
suppression of synaptic plasticity in CA1 and the mPFC.
Studies have also shown that stress exerts a profound impact
on molecular mechanisms underlying synaptic plasticity (Kim
et al., 2006; Howland and Wang, 2008; Pittenger and Duman,
2008; Bisaz et al., 2009). In one example, stress reduced the
expression of phosphorylated calcium/calmodulin-dependent
protein kinase II (p-CaMKII; Gerges et al., 2004), an enzyme
which is critical to LTP induction and memory formation
(Lisman et al., 2002), and increased expression of calcineurin
in the rat hippocampus, effects that were both associated with
impaired hippocampal LTP (Gerges et al., 2004). It is also
well-established that stress reduces hippocampal expression of
brain-derived neurotrophic factor (BDNF), which has been
associated with impaired hippocampus-dependent memory
(Radecki et al., 2005; Duman and Monteggia, 2006). In contrast, stress or a fear-provoking experience activates molecular
plasticity in the amygdala (Pare, 2003; Monfils et al., 2007;
Zoladz and Diamond, 2008; Ilin and Richter-Levin, 2009),
which exerts an inhibitory influence on hippocampal plasticity
(Akirav and Richter-Levin, 1999; Akirav and Richter-Levin,
2006). Therefore, understanding how stress differentially affects
molecular plasticity in the hippocampus, amygdala and PFC
could enhance our understanding of the complexity of how
stress affects learning and memory.
Although numerous studies have reported that memory and
stress alter plasticity-related protein expression (e.g., Izquierdo
Hippocampus
et al., 2006; Izquierdo et al., 2008; Bisaz and Sandi, 2010),
few studies have contrasted molecular plasticity in animals
having intact memory with those rendered amnestic by stress.
Our group has shown that stress impairs rat spatial memory
(Diamond et al., 1996, 1999; Sandi et al., 2005; Campbell
et al., 2008; Park et al., 2008; Zoladz et al., 2008) and suppresses synaptic plasticity in CA1 (Diamond et al., 1990, 1994;
Mesches et al., 1999; Vouimba et al., 2006). We have also
reported that the predator stress-induced impairment of spatial
memory was associated with a rapid reduction of levels of
hippocampal neural cell adhesion molecules (NCAMs), which
are proteins that are critically involved in forebrain development
and synaptic plasticity (Sandi et al., 2005). Here, we have
extended these findings by examining the influence of spatial
learning and the acute stress-induced impairment of spatial
memory on the expression of CaMKII, BDNF, and calcineurin
in the CA1 region of the hippocampus, BLA, and mPFC.
MATERIALS AND METHODS
Subjects
The subjects were adult male Sprague-Dawley rats (250–275 g;
Charles River Laboratories) housed on a 12 h/12 h light dark
schedule (lights on at 0700 h) in Plexiglas cages (2 per cage) with
food and water provided ad libitum. Colony room temperature
and humidity were maintained at 208C 6 18C and 60% 6 3%,
respectively. All rats were given 1 week to acclimate to the colony
room environment before any experimental manipulations took
place. The rats were brought to the laboratory’s water maze training room and handled for 2–3 min each during the last 3 days of
the 1-week acclimation period. Behavioral manipulations were
conducted between 0800 and 1300 h and were always preceded
by 30 min of acclimation to the testing environment. All experimental procedures were approved by the Institutional Animal
Care and Use Committee at the University of South Florida.
Stress Manipulation
To induce predator stress, rats were first placed in Plexiglas
boxes (28 3 9 3 14 cm), with multiple air holes in the top.
The rats within the boxes were then placed in a large cage
(57 3 57 3 76 cm), which contained an adult female cat, for
30 min. The Plexiglas box prevented any physical contact
between the cat and rats but enabled the rats to be exposed to
all other sensory stimuli (e.g., sight, smell, and sound) associated
with the cat. Moist cat food was smeared on top of the Plexiglas
box which kept the cat’s attention directed toward the rats.
Water Maze Apparatus and Training Procedures
The radial-arm water maze (RAWM) is a hippocampus-dependent task (Diamond et al., 1999) which has been described
at length in our previous publications (Diamond et al., 1999;
Sandi et al., 2005; Campbell et al., 2008; Park et al., 2008;
MOLECULAR MARKERS OF MEMORY AND STRESS-INDUCED AMNESIA
Zoladz et al., 2008). Briefly, the RAWM consists of a black,
galvanized round tank (168 cm diameter, 56 cm height, 43 cm
depth) filled with water (228C). Six V-shaped stainless steel
inserts (54 cm height, 56 cm length) were placed in the tank
to produce six swim arms radiating from an open central area.
A black, plastic platform (12 cm diameter) was placed 1 cm
below the surface of the water at the end of one arm (referred
to as the ‘‘goal arm’’). At the start of each trial, rats were
released into one arm (referred to as the ‘‘start arm’’) facing the
center of the maze. If a rat did not locate the hidden platform
within 1 min, it was gently guided to the platform by the
experimenter. Once a rat found or was guided to the platform,
it was left there undisturbed for 15 s.
Spatial learning and memory was measured by counting the
number of arm entry errors that rats made on each trial. An
arm entry was operationally defined as a rat passing at least
halfway down one of the arms that did not contain the hidden
platform or, very rarely, when a rat entered and exited the goal
arm without climbing onto the platform.
Rats receiving water maze training were given a single training
session composed of 12 trials in the RAWM, followed by a 30min delay period. During the delay period, the rats were either
returned to their home cages or were administered predator
stress (as described above) for 30 min. Immediately following
the delay period, rats were given a single test trial in the RAWM
to assess their memory for the hidden platform location.
Experimental Design
A total of five groups of rats were studied (8 rats/group).
Two groups were given water maze training, as described above.
Rats in one trained group (Train–No Stress) were placed in
their home cages during the 30-min delay period, and rats in
the other trained group (Train–Stress) were given predator
stress during the delay period. A third group of rats (Water
Maze–Yoked) was given an amount of swim time in the water
maze which was equivalent to the amount of swim time the
trained groups spent in the maze. That is, the rats in the Water
Maze–Yoked group received a total of 13 trials with the mean
water exposure time equal to that of the mean on each trial of
the trained groups, but the rats in this group were not given the
opportunity to learn the location of the hidden platform. For
the Water Maze–Yoked group, a hidden platform was located in
the water maze at the end of one arm; however, if a rat located
the platform on any trial, the platform was then moved to the
opposite side of the maze on the next trial. Whereas rats in the
normal trained groups could find the platform in the same location on all 13 trials, rats in the Water Maze–Yoked group found
the hidden platform on an average of only 1.38 6 0.26 trials.
Thus, the Water Maze–Yoked paradigm facilitated purposive
swimming behavior by rats, but since the platform was not in a
constant location, rats in this group were unable to form a spatial memory of the location of the platform.
A fourth group of rats (No Train–Stress) was given predator
stress only. Rats in this group were brought to the water maze
training room, where they remained for a period of time which
579
was yoked to the duration of water maze training in the two
maze-trained groups. Then, these rats were exposed to a cat for
30 min, as described above. The fifth group (No Train–Home
Cage) was not given water maze training or predator stress
exposure. Rats in this group were given routine handling and
were brought to the water maze training room, where they
remained for a period of time yoked to the duration of water
maze training and testing in the two trained groups.
Tissue Preparation and Serum
Corticosterone Assay
Immediately following the memory test trial (or an equivalent period of time in the No Train–Stress and No Train–
Home Cage groups), each rat was rapidly decapitated; a sample
of trunk blood was collected in a microcentrifuge tube and
allowed to clot at room temperature. The brain was quickly
removed from the skull and then dissected following coordinates
defined by Paxinos and Watson (1998). Brain regions were collected and stored in a microcentrifuge tube at 2808C immediately after dissection. All dissections were performed on a cold
plate which was maintained at 228C for the duration of the
procedure. First, the entire cerebellum was removed, and the
brain was separated into left and right hemispheres. Second,
the mPFC, including cingulate and prelimbic cortices, was
isolated from each hemisphere. Next, the hippocampus was
removed from each hemisphere and divided into its dorsal and
ventral components. A magnifying headset was then used to subdivide each section into CA3, CA1, and dentate gyrus. Finally,
the left and right hemispheres were sectioned coronally between
the rostral-caudal dimensions of the BLA, which was removed
bilaterally from the resulting section with a micropunch tool.
The locations of those dissected brain regions whose findings
are presented in the current manuscript (i.e., CA1, BLA, and
mPFC) are illustrated in Figure 1. Brain tissue samples were
shipped frozen to the University of Houston, Texas, and stored
at 2808C until they were assayed by co-authors (S.S., K.H.A.,
M.S.) who were blind to the behavioral manipulations. For
some rats, there was not a sufficient amount of tissue to allow
for an accurate quantification of the plasticity-related proteins;
these samples were not included in the analyses.
The clotted trunk blood was centrifuged (3,000 rpm for 5
min) and then the serum was extracted and stored at 2808C
until it was shipped frozen to the University of Colorado,
Boulder, where it was assayed for corticosterone with an
Enzyme ImmunoAssay kit from Assay Design (cat#901-097,
Ann Arbor, MI) by a co-author (M.F.) who was blind to the
behavioral manipulations. All samples were diluted 1:50 and
assayed per kit instructions.
Quantification of CaMKII, BDNF, and
Calcineurin by Western Blot Analysis
Brain tissue samples were suspended in 200–800 ll of icecold hypotonic lysis buffer (50 mM Tris-HCl pH 7.4, 4 mM
EDTA, 100 lg/ml phenylmethylsulfonyl fluoride, 1 lg/ml
Hippocampus
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ZOLADZ ET AL.
FIGURE 1.
Brain regions which were assayed in the current
study. The regions are outlined in illustrations taken from Paxinos
and Watson (1998) and were selected based on previous research
establishing their involvement in the effects of acute stress on learn-
ing, memory, and synaptic plasticity. The mPFC (labeled A) included
the cingulate and prelimbic cortices. The hippocampus was divided
into the dorsal and ventral CA1 (dorsal pictured and labeled B).
The BLA (labeled C) was dissected with a micropunch tool.
leupeptin, 1 lg/ml aprotinin, and 1 lg/ml pepstatin) plus protease inhibitor cocktail. The tissues were homogenized using
PRO200 post-mounted laboratory homogenizer with a 5X 75
mm (DXL) flat style probe for 20 s, with 10 s pulse at 12,000
rpm. The homogenized tissue suspension was subsequently
centrifuged at 1,000 rpm for 10 min to remove cellular debris.
The protein concentrations of the homogenates were determined by the Pierce bicinchoninic acid protein detection kit
(Pierce, Rockford, IL Cat# 232009) using BCA protein assay
reagent A (Cat# 23223) and reagent B (Cat# 23224) (Smith
et al., 1985). The homogenates were subjected to SDS-PAGE
using the high-throughput E-PAGETM 48 Protein electrophoresis System (Invitrogen Corp, #EP048-08). Equal amounts of
protein (10 lg) were incubated with 2.5 ll E-PAGETM loading
buffer and 1 ll NuPAGE1 sample reducing agent in a final
volume of 10 ll per tube and heated at 708C for 10 min, as
recommended by the manufacturer. The prepared samples were
then loaded onto wells of the pre-cast gel. The proteins were
transferred to PVDF membranes using the buffer-less, dry iBlot
gel transfer system (Invitrogen Corp, #IB4010-01). The levels
of different proteins were determined by immunoblotting using
the fast speed SNAP i.dTM protein detection system (Millipore
Corp, #WBAVDBH01). The blots were stripped using 0.2 N
sodium hydroxide solution for 5 min and subjected to a quick
distilled water wash before re-probing with the next antibody.
The immunoreactive bands were detected with a primary antibody and horseradish peroxidase-conjugated secondary antibody, and the blots were developed using a chemiluminescence
reagent prepared by adding para-coumaric acid and luminol in
100 mM Tris-HCl, pH 8.5, and hydrogen peroxide solution.
Chemiluniscence was detected using an Alpha Innotech imaging
system and the protein bands quantified by densitometry using
Fluorchem FC8800 software. Glyceraldehyde 3-phosphate
dehydrogenase (GAPDH) was used as a loading control for each
gel. Bands of each test protein detected via chemiluniscence on an
Alpha Innotech imaging system were densitized using Fluorchem
FC8800 software. Next, the gels were stripped and probed for
GAPDH, following which the bands of GAPDH were densitized
Hippocampus
MOLECULAR MARKERS OF MEMORY AND STRESS-INDUCED AMNESIA
581
as before. Finally, test protein bands were normalized against
GAPDH.
Levels of phosphorylated CaMKII (p-CaMKII) were detected
using a mouse monoclonal antibody (22B1, sc-32289; at 1:400
dilution). The blots were stripped and then probed for detection of total CaMKII using an anti-CaMKII rabbit polyclonal
antibody (M-176, sc-9035; using 1:400 dilution), as previously
reported (Gerges et al., 2004). Protein levels of brain-derived
neurotrophic factor (BDNF) and calcineurin in different brain
regions were detected using an anti-BDNF rabbit polyclonal
antibody (N-20, sc-546; 1:100 dilution) and anti-calcineurin/
PP2B (Upstate #07-069; 1:400 dilution) antibodies, respectively.
GAPDH used as a loading control was detected by probing
with the mouse anti-GAPDH monoclonal antibody (MAB374;
1:2,000 dilution) (Alzoubi et al., 2006, 2008; Srivareerat et al.,
2008). All data were normalized to GAPDH levels.
Statistics
A mixed-model, two-way analysis of variance (ANOVA;
Sigmastat, SPSS, GraphPad) was used to analyze RAWM
performance during the 12-trial acquisition phase, with group and
trial serving as the factors. Performance on the single memory test
trial was analyzed with an independent samples t test. The levels of
serum corticosterone, p-CaMKII, total CaMKII, BDNF, and calcineurin from all groups were analyzed with separate one-way
ANOVAs, followed by Holm-Sidak post hoc tests when appropriate. Outlier data greater than three standard deviations from the
exclusive group means were eliminated from analyses (<1% of the
data were outliers). Alpha was set at 0.05 for all analyses, and all
data in the text and figures are presented as Mean 6 SEM.
RESULTS
Spatial Learning, Memory, and Stress-Induced
Amnesia
The learning curves and memory performance of the two
groups given water maze training are illustrated in Figure 2. A
mixed-model, two-way ANOVA was used to analyze the water
maze performance of both groups across the 12-trial acquisition
phase. This analysis revealed a significant main effect of trial,
F(11, 154) 5 11.07, P < 0.001, indicating that the rats from
both groups successfully acquired the task, as indicated by a
reduction of errors as the trials progressed. Both groups’ performance during the acquisition phase was statistically equivalent, as there was no significant main effect of group, F(1, 14)
5 0.02, P 5 0.90, and the Group 3 Trial interaction was not
significant, F(11, 154) 5 0.60, P 5 0.83.
An independent samples t test was used to compare the
two groups’ performance on the memory test trial, which followed
the 30-min delay period during which the Train–Stress group was
exposed to a cat. This analysis revealed that the Train–Stress group
made significantly more arm entry errors on the retention trial
FIGURE 2.
Exposing rats to a cat during the 30-min delay
period between acquisition and memory testing (RT 5 retention
trial) impaired their retrieval of the hidden platform location.
Performance during the acquisition phase (Trials 1–12) is presented in two-trial blocks. Both groups learned the location of the
hidden platform at an equivalent rate. Rats given home cage exposure during the 30-min delay period exhibited intact memory on
the RT. In contrast, rats exposed to a cat during the 30-min delay
period displayed impaired memory, as indicated by a significant
increase in arm entry errors on the RT. The data are presented as
Mean 6 SEM, N 5 8 rats/group. The dashed line at 2.5 errors indicates chance level of performance (Diamond et al., 1999). *P < 0.05
relative to the Train–No Stress group.
than the Train–No Stress group, t(14) 5 3.39, P 5 0.004 (twotailed). This finding replicates our well-established work demonstrating that acute predator stress impairs spatial memory (Diamond et al., 1999; Sandi et al., 2005; Campbell et al., 2008; Park
et al., 2008; Zoladz et al., 2008).
Serum Corticosterone Levels
The analysis of serum corticosterone levels revealed a significant effect of group, F(4, 34) 5 14.74, P < 0.001 (Fig. 3).
Holm-Sidak post hoc tests indicated that the Train–Stress
group exhibited significantly higher corticosterone levels than
all other groups, and the Train–No Stress, No Train–Stress,
and Water Maze–Yoked groups all displayed significantly higher
corticosterone levels than the No Train–Home Cage group
(all P’s < 0.05). Thus, RAWM training in conjunction with
predator stress had an additive effect on serum corticosterone
levels, resulting in the greatest levels in the Train–Stress group.
CaMKII, BDNF, and Calcineurin
Expression in the Hippocampus
Immediate post-memory test levels of CaMKII, BDNF, and
calcineurin were compared across all five experimental groups
for the dorsal and ventral CA1 regions of the hippocampus.
Hippocampus
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ZOLADZ ET AL.
BDNF, F(4, 35) 5 0.17, P 5 0.95, or calcineurin, F(4, 28) 5
0.08, P 5 0.98, in the BLA.
CaMKII, BDNF, and Calcineurin Expression
in the Prefrontal Cortex
In the mPFC, there was a significant effect of group for the
expression of p-CaMKII, F(4, 34) 5 3.18, P 5 0.03. HolmSidak post hoc tests indicated that the No Train–Stress group
exhibited significantly lower p-CaMKII levels than every other
group (P’s < 0.05; Fig. 7). There was no significant effect of
group for levels of total CaMKII, F(4, 35) 5 0.92, P 5 0.47,
BDNF, F(4, 19) 5 0.54, P 5 0.71, or calcineurin, F(4, 20) 5
1.46, P 5 0.25, in the mPFC.
FIGURE 3.
Serum corticosterone levels from all five experimental groups. The Water Maze–Yoked, No Train–Stress, and Train–No
Stress groups each exhibited significantly greater corticosterone
levels than the No Train–Home Cage group. The Train–Stress group
displayed significantly greater corticosterone levels than every other
group, indicating that predator stress and water maze exposure had
an additive effect on HPA axis activation. The data are presented as
the Mean 6 SEM, N 5 7–8 rats/group. *P < 0.05 relative to the
Home Cage group. **P < 0.05 relative to every other group.
There was a significant effect of group for the levels of p-CaMKII, F(4, 34) 5 4.02, P 5 0.009, total CaMKII, F(4, 34) 5
5.09, P 5 0.003, and BDNF, F(4, 29) 5 4.26, P 5 0.008, in
dorsal CA1. In each case, Holm-Sidak post hoc tests indicated
that the Train–No Stress groups exhibited significantly greater
levels than every other group (P’s < 0.01; Fig. 3). Thus,
RAWM training led to a significant increase in p-CaMKII,
total CaMKII, and BDNF levels in the dorsal CA1 region of
the hippocampus, which was blocked by exposure to predator
stress. There was no significant effect of group for the analysis
of calcineurin levels in the dorsal CA1, F(4, 21) 5 0.05,
P 5 0.99.
There was no significant effect of group for the levels
of p-CaMKII, F(4, 30) 5 0.58, P 5 0.68, total CaMKII,
F(4, 31) 5 0.09, P 5 0.98, BDNF, F(4, 24) 5 0.39, P 5 0.82,
or calcineurin, F(4, 24) 5 1.76, P 5 0.17, in the ventral CA1
region of the hippocampus (Fig. 4). These findings indicate that
the training-induced changes in CaMKII and BDNF levels were
selective to the dorsal region of CA1 (Fig. 5).
CaMKII, BDNF, and Calcineurin Expression
in the Amygdala
In the BLA, there was a significant effect of group for the
expression of p-CaMKII levels, F(4, 30) 5 6.39, P < 0.001.
Holm-Sidak post hoc tests indicated that the Train–Stress and
No Train–Stress groups displayed significantly greater p-CaMKII levels than every other group (P’s < 0.01; Fig. 6). Thus,
predator exposure, independent of water maze training, was
associated with a significant increase in the phosphorylation of
CaMKII in the BLA. There was no significant effect of group
for the levels of total CaMKII, F(4, 29) 5 0.32, P 5 0.86,
Hippocampus
DISCUSSION
The primary purpose of this work was to identify rapid
changes in levels of p-CaMKII, total CaMKII, calcineurin, and
BDNF in the hippocampus, BLA, and mPFC in response to
learning, predator exposure, and predator stress-induced amnesia. We have found that within 40 min of the initiation of
water maze training, there was a significant increase in the
expression of p-CaMKII, total CaMKII, and BDNF in dorsal
CA1, but not in the BLA, mPFC, or ventral CA1. The dorsal
CA1-specific expression of plasticity occurred only when training and memory testing occurred under control (non-stress)
conditions. That is, when the spatial memory consolidation
process was permitted to develop unimpeded by a post-learning
stress experience, the dorsal CA1 expressed a rapid up-regulation of proteins which are known to be essential for synaptic
plasticity and stabilization of the memory trace. However,
when the rats were exposed to a cat immediately after training,
their 30 min memory of the platform location was impaired
and the expression of plasticity in their dorsal CA1 was
suppressed. The differential expression of molecular plasticity
in the dorsal CA1 under the stress versus control conditions
indicates that processes involved in the consolidation of spatial
memory were rapidly activated in dorsal CA1 by water maze
training, only to be suppressed by post-training stress.
Importantly, we have demonstrated that the increased levels
of molecular markers of synaptic plasticity were generated by
spatial learning, itself, and were not produced merely as a
consequence of water exposure; rats that were given an equivalent
amount of water maze exposure, but were not trained to learn the
location of a hidden platform, did not express an increase in
molecular markers of synaptic plasticity in the hippocampus (or
in any other structure). Thus, the finding of a water maze training-induced increase of CaMKII and BDNF expression in the rat
hippocampus is consistent with other work in rodents reporting
significant increases in hippocampal expression of these proteins
following training on hippocampus-dependent tasks (Cammarota
et al., 2002; Bekinschtein et al., 2008). That water maze training
led to increased CaMKII and BDNF expression only in the
MOLECULAR MARKERS OF MEMORY AND STRESS-INDUCED AMNESIA
583
FIGURE 4.
Expression of (A) p-CaMKII, (B) total CaMKII, (C)
BDNF, and (D) calcineurin in the dorsal CA1 region of the rat hippocampus. The expression of each protein is presented as the amount of
immunoreactivity relative to the loading control, glyceraldehyde 3-phosphate dehydrogenase (GAPDH). RAWM training (Train–No Stress) led
to a significant increase in the expression of p-CaMKII, total CaMKII,
and BDNF, which was blocked by predator stress (Train–Stress). Neither
swim stress (Water Maze–Yoked) nor predator stress (No Train–Stress),
alone, had any significant effect on the expression of these proteins.
There were no significant group differences for the expression of calcineurin. The data are presented as Mean 6 SEM, N 5 5–8 rats/group.
*P < 0.05 relative to every other group.
dorsal CA1 is important because it indicates that the effect is
both structure and subregion specific.
The second and entirely novel finding of the present study is
that exposing rats to a cat not only impaired their spatial memory, but also blocked each of the training-induced increases of
plasticity-related protein expression in dorsal CA1. Whereas
other studies have shown that stress alters the expression of
CaMKII and BDNF in the hippocampus (Gerges et al., 2004;
Suenaga et al., 2004; Duman and Monteggia, 2006), the present findings are the first to show that a purely psychological
stressor, inescapable exposure to a predator, impaired spatial
memory, and suppressed the learning-induced activation of
CaMKII and BDNF in the dorsal CA1.
Our finding of an increase in total levels of CaMKII in the
dorsal CA1 with spatial learning contrasts with that of Pollak
et al. (2005), who reported that spatial learning in the Morris
water maze led to significantly greater p-CaMKII in the hippocampus without affecting total hippocampal CaMKII protein
expression. However, in addition to employing a different water
maze training paradigm, species (rat vs. mouse) and apparatus
that were utilized in the present study, Pollak and colleagues
observed their learning-induced alteration of p-CaMKII expression 24 h after the final day of water maze training and did
not distinguish between the levels of CaMKII present in the
different subregions of the hippocampus. Thus, differences in
methodology employed by the two studies are likely to explain
why we observed a learning-induced increase not only in
p-CaMKII but also in total CaMKII and in dorsal CA1.
The finding that spatial learning increased CaMKII and
BDNF expression in the dorsal, but not ventral, CA1, is consistent with a large body of research indicating a dissociation
between the functional roles of the dorsal and ventral hippocampus (Bannerman et al., 2004; Barkus et al., 2010; Fanselow
and Dong, 2010; Maggio and Segal, 2010b; Segal et al.,
2010). Specifically, our finding supports the hypothesis that it
is the dorsal, but not ventral, region of the hippocampus which
is involved in the encoding of spatial information. Moreover,
the absence of learning or stress-induced changes in molecular
plasticity in the ventral CA1 that we have described here is
consistent with electrophysiological work indicating that the
Hippocampus
584
ZOLADZ ET AL.
FIGURE 5.
Expression of (A) p-CaMKII, (B) total CaMKII, (C)
BDNF, and (D) calcineurin in the ventral CA1 region of the rat hippocampus. The expression of each protein is presented as the amount
of immunoreactivity relative to the loading control, glyceraldehyde 3phosphate dehydrogenase (GAPDH). There were no significant
group differences for the expression of any plasticity-related protein
in ventral CA1. This finding is important because it indicates that the
RAWM training-induced increase of p-CaMKII, total CaMKII, and
BDNF expression was selective to the dorsal CA1 hippocampal
region. The data are presented as Mean 6 SEM, N 5 5–8 rats/group.
ventral CA1, with the exception of its extreme ventral pole
(Maggio and Segal, 2007a; Vlachos et al., 2008), is less amenable to express synaptic plasticity than is the dorsal CA1 (Papatheodoropoulos and Kostopoulos, 2000a,b; Maruki et al.,
2001; Maggio and Segal, 2007a; Ravassard et al., 2009). Thus,
our findings are consistent with the view that the ventral
hippocampus appears be more involved with short-term coping
responses involved in emotionality (Maggio and Segal, 2007a),
than with spatial or emotional memory storage.
It is important to emphasize, however, that our findings do
not indicate that the ventral hippocampus is not involved in
stress responsivity or in memory. Indeed, there is an extensive
literature linking the ventral hippocampus to the modulation
of the hypothalamic-pituitary-adrenal axis (Casady and Taylor,
1976; Nettles et al., 2000), as well as to elements of fear conditioning, such as in the expression of anxiety and unconditioned
fear (Esclassan et al., 2009; Barkus et al., 2010; McEown and
Treit, 2010). Moreover, the ventral hippocampus is sensitive to
behavioral stress and hormonal modulation of activity and
synaptic plasticity, but unlike the dorsal CA1, LTP in the ventral CA1 is enhanced by stress or corticosterone (Maggio and
Segal, 2007a,b, 2009a,b, 2010a). Furthermore, the ventral
CA1, unlike the dorsal CA1, expresses an NMDA-receptor
independent (L-type calcium channel) form of LTP which is
enhanced by stress (Maggio and Segal, 2010a). Therefore, our
findings of a lack of evidence of molecular plasticity in the ventral CA1 in response to stress and learning should be interpreted conservatively, to indicate only that the ventral CA1
does not appear to contribute to the storage of spatial information, and that elevated levels of corticosterone, as well as the
predator-evoked impairment of memory, do not activate the
molecular markers measured in this study. It is possible, however, that unconditioned fear-evoked responses to predator
exposure may be expressed only in the extreme temporal pole
of the ventral hippocampus (Maggio and Segal, 2007a). Thus,
a more extensive behavioral analysis and a localized analysis
of subregions within the ventral hippocampus may reveal a
contribution of this region to stress-induced amnesia.
The predator stress-induced impairment of spatial memory
and suppression of molecular plasticity in the dorsal CA1
could be related to the stress-induced activation of the hypothalamic-pituitary-adrenal (HPA) axis, as the Train–Stress
Hippocampus
MOLECULAR MARKERS OF MEMORY AND STRESS-INDUCED AMNESIA
585
FIGURE 6.
Expression of (A) p-CaMKII, (B) total CaMKII,
(C) BDNF, and (D) calcineurin in the BLA. The expression of
each protein is presented as the amount of immunoreactivity relative to the loading control, glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Cat exposure, independent of RAWM training,
led to a significant increase in the expression of p-CaMKII. Those
groups given water maze exposure in the absence of predator stress
(i.e., Water Maze–Yoked and Train–No Stress) did not display such
an increase, indicating that cat exposure is qualitatively different
from swim stress. There were no significant group differences for
the expression of total CaMKII, BDNF, or calcineurin. The data
are presented as Mean 6 SEM, N 5 5–8 rats/group. *P < 0.05 relative to the No Train–Home Cage, Water Maze–Yoked, and Train–
No Stress groups.
group exhibited greater levels of serum corticosterone than any
other group. There is a large body of work indicating that
glucocorticoids can impair hippocampus-dependent learning
and memory and hippocampal synaptic plasticity (Kim et al.,
2006; Diamond et al., 2007; Kim and Haller, 2007; Lupien
et al., 2007; Henckens et al., 2009; Joels and Baram, 2009).
Mechanistic studies indicate that prolonged stress or glucocorticoid administration produces a rapid modulation of the
hippocampal glutamatergic system (Lowy et al., 1993; Lowy
et al., 1995; Raudensky and Yamamoto, 2007; Joels et al.,
2008; Prager and Johnson, 2009; McEwen et al., 2010), which
can ultimately result in an impairment of hippocampal synaptic
plasticity (Diamond et al., 2007; Joels and Baram, 2009). Investigators have also shown that, in hippocampal cells, corticosterone augments intracellular calcium levels (Joels et al., 2009),
while simultaneously reducing the expression of BDNF and
CaMKII (Schaaf et al., 1998; Sun et al., 2004). Moreover, recent
work has suggested that corticosterone might enhance, as well as
impair, hippocampal synaptic plasticity through glucocorticoid
receptor-dependent alterations of AMPA receptor trafficking
(Groc et al., 2008; Martin et al., 2009). Thus, cat exposure may
impair spatial memory by interfering with the expression of plasticity-related proteins (e.g., CaMKII) in the hippocampus via
activation of the HPA axis, in conjunction with excessive activation of the glutamatergic system.
Our third primary finding was that predator stress, independent of water maze exposure, led to a significant increase of
p-CaMKII expression in the BLA. This effect, unlike the stressinduced suppression of plasticity in CA1, cannot be explained
solely by the stress-induced activation of the HPA axis. The group
given cat exposure, alone, exhibited corticosterone levels which
were equivalent to those found in the two groups given water
maze exposure (Train–No Stress and Water Maze–Yoked). Despite
the equivalence of the corticosterone levels in these three groups,
only the group given cat exposure, alone, exhibited an increase in
p-CaMKII expression in the BLA. Moreover, the two groups
exposed to the cat exhibited an equivalent increase in p-CaMKII
expression, despite having differences in their corticosterone levels.
Hippocampus
586
ZOLADZ ET AL.
FIGURE 7.
Expression of (A) p-CaMKII, (B) total CaMKII,
(C) BDNF, and (D) calcineurin in the mPFC. The expression of
each protein, is presented as the amount of immunoreactivity relative to the loading control, glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Cat exposure, alone, led to a significant decrease
in the expression of p-CaMKII. This decrease was counteracted by
the presence of RAWM training, as the Train–Stress group did not
show a reduction of p-CaMKII expression. There were no significant group differences for the expression of total CaMKII, BDNF,
or calcineurin. The data are presented as Mean 6 SEM, N 5 5
rats/group. *P < 0.05 relative to every other group.
Thus, there appears to be a unique feature of predator exposure,
independent of water maze training and corticosterone levels,
which activates mechanisms of plasticity in the amygdala.
It is important to emphasize that the predator stress-induced
increase in p-CaMKII expression in the BLA is noteworthy
because it indicates that cat exposure did not merely result
in increased amygdala activity; rather, cat exposure specifically
led to increased expression of plasticity-related molecules in the
amygdala. This finding potentially links the induction of
molecular mechanisms of synaptic plasticity in the BLA
described here to work demonstrating long-lasting predator
stress-induced modulation of multiple forms of plasticity in the
amygdala (Vouimba et al., 2004; Adamec et al., 2005; Blundell
and Adamec, 2007; Mitra et al., 2009), and further, to the persistent enhancement of amygdala activity in traumatized people
(Debiec and LeDoux, 2006; Etkin and Wager, 2007; Koenigs
and Grafman, 2009; Milad et al., 2009).
Consistent with previous work (Woodson et al., 2003), our
findings reveal that predator stress is qualitatively different from
other stressors, such as swim stress, in that predator exposure
was the only manipulation that activated molecular plasticity
within the amygdala. Moreover, the finding that predator stress
led to increased p-CaMKII in the BLA is consistent
with previous research revealing that acute stress enhances
synaptic plasticity in the amygdala (Vouimba et al., 2004;
Vouimba et al., 2006). Prior work has also shown that amygdala
activation can result in the impairment of hippocampal synaptic
plasticity (Akirav and Richter-Levin, 1999), and that the suppression of amygdala activity blocks stress effects on CA1 (Kim
et al., 2001, 2005). These findings are consistent with the hypothesis that the stress-induced enhancement of plasticity in the
BLA we have observed here contributed to the suppression of
plasticity in CA1. Direct manipulations of amygdala functioning
during water maze training and predator exposure, with measurements of their influence on the expression of plasticity in the
hippocampus, would provide a test of this hypothesis.
Our final observation revealed that spatial learning did not
affect molecular plasticity in the mPFC, but we did find that
Hippocampus
MOLECULAR MARKERS OF MEMORY AND STRESS-INDUCED AMNESIA
predator stress, alone, reduced p-CaMKII in this cortical
region. Interestingly, the group of rats given water maze training, in addition to cat exposure, did not exhibit this reduction,
suggesting that spatial learning, perhaps via a training-induced
translocation of CaMKII from the cytoplasm to the post-synaptic density (Mullasseril et al., 2007), prevented the predator
stress-induced decrease of p-CaMKII expression.
An alternative perspective on our PFC findings is based on
work by Czeh et al. (2008), who showed that stress exerted
different effects on different subregions of the mPFC, effects
that, in some cases, could potentially cancel each other out
(however, see Cerqueira et al., 2005, 2007a,b for evidence that
stress-induced morphological alterations in the mPFC are
fairly consistent across subregions). Since we assayed more
than one subregion of the mPFC in the present experiment
(i.e., cingulate and prelimbic cortices), it is possible that a
differential influence of stress on the expression of plasticity in
the different subdivisions of the mPFC may have contributed
to the overall absence of effects of stress in this region. Nevertheless, the finding of reduced CaMKII expression in the
mPFC following cat exposure supports the notion that stress
rapidly suppresses synaptic plasticity in the PFC, potentially
underlying the stress-related impairment of working memory,
which is dependent upon this brain region (Maroun and
Richter-Levin, 2003; Rocher et al., 2004; Arnsten, 2009;
Holmes and Wellman, 2009).
Taken together, the present findings reveal structure-specific
molecular plasticity in response to spatial learning, cat exposure
and stress-induced amnesia. Spatial learning induced a rapid
increase in total and phosphorylated CaMKII and BDNF in the
dorsal CA1 region of the rat hippocampus, which was blocked
in predator-exposed rats with impaired spatial memory. In addition, cat exposure, with or without RAWM training, increased
p-CaMKII expression in the BLA, which indicates that an
intense fear-provoking experience generates memory-related plasticity intrinsic to amygdala circuitry. Finally, we have found that
cat exposure, alone, suppressed the expression of p-CAMKII in
the mPFC. The rapid acute stress-induced alterations of the different forms of molecular plasticity described here may underlie
the long-lasting effects of stress on morphological, molecular,
and synaptic forms of plasticity in the hippocampus, amygdala,
and PFC (Adamec et al., 2005; McEwen, 2006; Diamond et al.,
2007; Kozlovsky et al., 2007; Segal et al., 2010).
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