The Imposition of, but not the Propensity for, Social Subordination Impairs Exploratory Behaviors and General Cognitive Abilities

Abstract

Imposed social subordination, such as that which accompanies physical defeat or alienation, has been associated with impaired cognitive function in both human and non-human animals. Here we examined whether domain-specific and/or domain-general learning abilities (c.f. general intelligence) are differentially influenced by the imposition of social subordination. Furthermore, we assessed whether the impact of subordination on cognitive abilities was the result of imposed subordination per se, or if it reflected deficits intrinsically expressed in subjects that are predisposed to subordination. Subordinate and dominant behaviors were assessed in two groups of CD-1 male mice. In one group (Imposed Stratification), social stratification was imposed (through persistent physical defeat in a colonized setting) prior to the determination of cognitive abilities, while in the second group (Innate Stratification), an assessment of social stratification was made after cognitive abilities had been quantified. Domain-specific learning abilities were measured as performance on individual learning tasks (odor discrimination, fear conditioning, spatial maze learning, passive avoidance, and egocentric navigation) while domain-general learning abilities were determined by subjects' aggregate performance across the battery of learning tasks. We observed that the imposition of subordination prior to cognitive testing decreased exploratory tendencies, moderately impaired performance on individual learning tasks, and severely impaired general cognitive performance. However, similar impairments were not observed in subjects with a predisposition toward a subordinate phenotype (but which had not experienced physical defeat at the time of cognitive testing). Mere colonization, regardless of outcome (i.e., stratification), was associated with an increase in stress-induced serum corticosterone (CORT) levels, and thus CORT elevations were not themselves adequate to explain the effects of imposed stratification on cognitive abilities. These findings indicate that absent the imposition of subordination, individuals with subordinate tendencies do not express learning impairments. This observation could have important ramifications for individuals in environments where social stratification is prevalent (e.g., schools or workplace settings).

Full text

(This is a sample cover image for this issue. The actual cover is not yet available at this time.)
This article appeared in a journal published by Elsevier. The attached
copy is furnished to the author for internal non-commercial research
and education use, including for instruction at the authors institution
and sharing with colleagues.
Other uses, including reproduction and distribution, or selling or
licensing copies, or posting to personal, institutional or third party
websites are prohibited.
In most cases authors are permitted to post their version of the
article (e.g. in Word or Tex form) to their personal website or
institutional repository. Authors requiring further information
regarding Elsevier’s archiving and manuscript policies are
encouraged to visit:
http://www.elsevier.com/copyright

Author's personal copy
Behavioural
Brain
Research
232 (2012) 294–
305
Contents
lists
available
at
SciVerse
ScienceDirect
Behavioural
Brain
Research
j
ourna
l
ho
me
pa
ge:
www.elsevier.com/locate/bbr
Research
report
The
imposition
of,
but
not
the
propensity
for,
social
subordination
impairs
exploratory
behaviors
and
general
cognitive
abilities
Danielle
Colas-Zelin, Kenneth
R.
Light,
Stefan
Kolata,
Christopher
Wass,
Alexander
Denman-Brice,
Christopher
Rios,
Kris
Szalk,
Louis
D.
Matzel∗
Program
in
Behavioral
Neuroscience,
Department
of
Psychology,
Rutgers
University,
Piscataway,
NJ
08854,
USA
a
r
t
i
c
l
e
i
n
f
o
Article
history:
Received
6
February
2012
Received
in
revised
form
5
April
2012
Accepted
9
April
2012
Available online xxx
Keywords:
Subordination
Aggression
Stress
Cognition
Social
status
General
intelligence
Stress
hormones
a
b
s
t
r
a
c
t
Imposed
social
subordination,
such
as
that
which
accompanies
physical
defeat
or
alienation,
has
been
associated
with
impaired
cognitive
function
in
both
human
and
non-human
animals.
Here
we
examined
whether
domain-specific
and/or
domain-general
learning
abilities
(c.f.
general
intelligence)
are
differen-
tially
influenced
by
the
imposition
of
social
subordination.
Furthermore,
we
assessed
whether
the
impact
of
subordination
on
cognitive
abilities
was
the
result
of
imposed
subordination
per
se,
or
if
it
reflected
deficits
intrinsically
expressed
in
subjects
that
are
predisposed
to
subordination.
Subordinate
and
dom-
inant
behaviors
were
assessed
in
two
groups
of
CD-1
male
mice.
In
one
group
(Imposed
Stratification),
social
stratification
was
imposed
(through
persistent
physical
defeat
in
a
colonized
setting)
prior
to
the
determination
of
cognitive
abilities,
while
in
the
second
group
(Innate
Stratification),
an
assessment
of
social
stratification
was
made
after
cognitive
abilities
had
been
quantified.
Domain-specific
learning
abilities
were
measured
as
performance
on
individual
learning
tasks
(odor
discrimination,
fear
condition-
ing,
spatial
maze
learning,
passive
avoidance,
and
egocentric
navigation)
while
domain-general
learning
abilities
were
determined
by
subjects’
aggregate
performance
across
the
battery
of
learning
tasks.
We
observed
that
the
imposition
of
subordination
prior
to
cognitive
testing
decreased
exploratory
tendencies,
moderately
impaired
performance
on
individual
learning
tasks,
and
severely
impaired
general
cognitive
performance.
However,
similar
impairments
were
not
observed
in
subjects
with
a
predisposition
toward
a
subordinate
phenotype
(but
which
had
not
experienced
physical
defeat
at
the
time
of
cognitive
testing).
Mere
colonization,
regardless
of
outcome
(i.e.,
stratification),
was
associated
with
an
increase
in
stress-
induced
serum
corticosterone
(CORT)
levels,
and
thus
CORT
elevations
were
not
themselves
adequate
to
explain
the
effects
of
imposed
stratification
on
cognitive
abilities.
These
findings
indicate
that
absent
the
imposition
of
subordination,
individuals
with
subordinate
tendencies
do
not
express
learning
impair-
ments.
This
observation
could
have
important
ramifications
for
individuals
in
environments
where
social
stratification
is
prevalent
(e.g.,
schools
or
workplace
settings).
© 2012 Elsevier B.V. All rights reserved.
1.
Introduction
Stress
has
been
shown
to
be
a
potent
modulator
of
the
ability
to
learn
and
to
express
memories.
However,
the
direction,
degree,
and
duration
of
stress
effects
on
cognitive
abilities
depends
greatly
on
the
characteristics
of
the
stressor,
the
type
of
learning
being
assessed,
and
the
social
structure
of
an
organism’s
environment
[for
reviews,
see:
1–7].
The
variability
in
reported
stress
effects
on
learning
highlights
the
need
to
focus
on
stressors
that
are
both
ethologically
relevant
and
conserved
across
both
human
and
non-
human
animal
species.
Numerous
mammalian
species,
including
humans,
live
in
complex
social
groups
and
are
subject
to
intense
∗Corresponding
author.
Tel.:
+1
848/445
5940;
fax:
+1
848/445
2263.
E-mail
address:
matzel@rci.rutgers.edu
(L.D.
Matzel).
and
often
unpredictable
stress
as
a
result
of
social
interactions.
As
such,
a
relatively
new
area
of
study
has
emerged
with
the
goal
of
investigating
the
learning
effects
induced
by
stressors
that
are
primarily
social
in
nature.
One
line
of
inquiry
has
focused
on
biobe-
havioral
and
learning
challenges
that
arise
consequent
to
social
subordination.
In
humans,
subordination
resulting
from
alienation
or
social
defeat
(e.g.,
bullying)
has
been
shown
to
exert
a
negative
influ-
ence
on
cognitive
performance
[8–15].
While
these
results
are
intriguing,
human
studies
of
social
stress
effects
are
limited.
In
the
laboratory,
researchers
employ
stressors
that
may
be
mild
in
comparison
to
the
stress
of
actual
life
events.
As
such,
the
results
from
these
studies
may
not
fully
demonstrate
the
impact
of
sub-
ordination
on
learning
performance.
Studies
that
examine
natural
instances
of
subordination
stress
in
humans
are
similarly
difficult
to
integrate
within
the
larger
phenomenon.
For
example,
these
0166-4328/$
–
see
front
matter ©
2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.bbr.2012.04.017

Author's personal copy
D.
Colas-Zelin
et
al.
/
Behavioural
Brain
Research
232 (2012) 294–
305 295
studies
often
rely
on
self-reports
or
personal
perceptions
of
subor-
dination,
which
may
not
accurately
reflect
actual
events.
Further,
prior
history
with
aggression,
environmental
factors,
and
impor-
tantly,
individual
predispositions,
may
interact
with
instances
of
social
subordination
and
may
complicate
the
interpretation
of
effects
on
cognitive
abilities
and
emotionality.
Lastly,
due
to
ethi-
cal
constraints,
it
is
difficult
to
probe
the
mechanisms
that
underlie
changes
in
cognition
in
human
participants.
Studies
of
laboratory
animals
provide
researchers
with
a
greater
degree
of
controllability
over
stressors
and
thus
allow
for
a
more
detailed
examination
of
the
physiological
substrates
that
may
underlie
alterations
in
cognition.
Animal
studies
examining
the
relationship
between
subor-
dination
and
cognitive
function
have
produced
mixed
results
[16–21,23–28,30].
Commonly,
two
types
of
social
subordination
have
been
examined,
i.e.,
imposed
subordination
or
the
innate
predisposition
toward
subordination
[16–21,23–28,30].
Imposed
subordination
refers
to
subordination
(e.g.,
antagonistic
encounters
with
conspecifics)
that
is
inflicted
upon
the
animals
prior
to
assess-
ment
of
cognitive
function.
In
contrast,
innate
subordination
(i.e.,
a
natural
predisposition
toward
subordination)
is
assessed
after
the
assessment
of
cognitive
abilities.
Imposed
subordination
has
typically
been
observed
to
nega-
tively
impact
learning
abilities
across
several
different
measures
[e.g.,
Spatial
learning
in
a
water
maze
or
radial
arm
maze:
16,
21–25;
reinforced
alternation:
17;
reference
and
working
memory:
18–20].
Despite
these
observations,
in
many
instances,
imposed
subordination
has
had
no
apparent
effect
on
learning
abilities
[21,23–29].
In
contrast
to
studies
of
imposed
subordination,
studies
of
innate
subordination
have
been
far
fewer
in
number.
Yet,
like
those
exam-
ining
imposed
subordination,
these
studies
have
also
provided
mixed
results
[25,30].
One
potential
reason
for
these
discrepant
results
is
the
variability
in
methodologies.
However,
it
is
also
possi-
ble
that
certain
forms
of
learning
and
memory
are
sensitive
to
social
subordination
while
others
are
spared.
Regardless
of
the
source
of
the
discrepancies,
the
mixed
findings
from
these
animal
studies
underscore
the
need
for
a
more
thorough
examination
of
whether
differences
in
learning
abilities
due
to
social
subordination
repre-
sent
an
innate
predisposition
toward
subordination
(of
animals
of
lower
cognitive
abilities)
or
whether
subordination-induced
learn-
ing
deficits
arise
in
response
to
imposed
subordination.
Studies
of
innate
and
imposed
subordination
to
date
have
focused
exclusively
on
domain-specific
learning
abilities
(e.g.,
spatial
learning).
Yet,
it
has
been
established
that
both
domain-
specific
(e.g.,
spatial
abilities)
as
well
as
domain-general
(general
intelligence)
factors
influence
cognition
[31].
In
humans,
general
intelligence
or
“g”
has
been
called
the
“single
most
dominant
cogni-
tive
trait
ever
discovered”
[32],
and
the
single
factor
that
underlies
g
is
purported
to
influence
all
domain-specific
learning
abilities.
Like
humans,
CD-1
outbred
mice
express
individual
differences
in
their
“general”
cognitive
abilities
such
that
performance
across
tasks
in
a
battery
of
diverse
learning
tests
is
positively
correlated.
Through
the
application
of
principal
components
analysis,
a
general
learn-
ing
factor
can
be
identified
that
accounts
for
25–48%
of
the
variance
in
the
performance
of
individual
mice.
This
general
learning
factor
in
mice
has
been
argued
to
be
structurally
and
psychometrically
analogous
to
general
intelligence
in
humans
[33–37].
To
date,
no
animal
studies
have
attempted
to
examine
the
relationship
between
social
subordination
and
general
learning
abilities.
Thus,
one
of
the
goals
of
the
current
experiment
was
to
determine
whether
an
individual
subjects’
domain-specific
and/or
domain-general
learning
abilities
are
altered
by
the
imposition
of
social
subordination
(in
a
colony
setting)
in
a
manner
similar
to
that
seen
in
previous
studies.
Additionally,
if
cognitive
differences
do
exist
in
animals
that
undergo
subordination,
we
would
deter-
mine
whether
they
reflect
the
imposition
of
subordination
or
if
they
represent
a
disposition
toward
poor
learning
in
animals
that
are
innately
disposed
to
subordination.
Domain-specific
learning
abilities
were
assessed
on
individual
learning
tasks
while
domain-general
learning
abilities
were
mea-
sured
as
the
aggregate
performance
across
a
battery
of
learning
tasks.
Stress-induced
levels
of
the
adrenal
hormone,
corticosterone
(CORT)
were
also
measured
since
prior
work
has
shown
a
dif-
ferential
activation
of
the
HPA
axis
in
subordinate
and
dominant
subjects
in
response
to
stress
[rats:
38,39;
mice:
40–44;
non-human
primates
44–49;
humans:
50,51].
Specifically,
it
is
has
been
sug-
gested
that
upregulation
of
HPA
activity,
such
as
that
seen
in
highly
stressed
animals,
may
lead
to
HPA
dysregulation
and
a
dysfunc-
tional
response
to
subsequent
stress
exposure.
Further,
CORT
has
been
implicated
as
a
possible
modulator
of
cognitive
function
[for
review
see:
1–7]
thereby
making
any
observation
of
differences
in
its
expression
of
particular
interest.
Behavioral
measures
of
stress/anxiety
also
vary
in
subordinate
animals
versus
dominant
subjects
[52–63].
Consequently,
we
assessed
performance
in
the
elevated
plus
maze
[EPM],
open
field
[OF]
and
light/dark
discrimi-
nation
tasks.
Lastly,
subjects
that
were
stratified
prior
to
cognitive
assessments
underwent
testing
in
a
battery
of
motor
tests
to
ensure
that
any
deficits
in
learning
performance
that
are
detected
are
not
the
result
of
motor
impairment.
2.
Materials
and
methods
2.1.
Animals
and
housing
Forty-eight
outbred,
male,
non-sibling
CD-1
mice
(Harlan
Sprague
Dawley
Inc.,
Indianapolis,
IN)
weighed
25–30
g
and
were
40–45
days
of
age
upon
arrival
in
our
laboratory.
Since
animals
were
obtained
pre-pubescence,
it
is
generally
accepted
that
they
would
not
had
yet
stratified
into
social
hierarchies.
Subjects
were
non-
littermates,
since
previous
work
has
revealed
that
aggressive
behaviors
are
more
readily
expressed
among
rodents
that
are
unrelated
[64].
Upon
arrival
and
prior
to
the
start
of
the
testing,
all
subjects
were
housed
individually
and
maintained
on
ad
libitum
food
and
water
(unless
noted
otherwise)
in
a
temperature-controlled
vivarium
on
a
12-h
light/dark
cycle.
They
were
allowed
to
acclimate
to
the
vivarium
and
were
handled
(removed
from
the
home
cage
and
held
by
the
experimenter
for
90
s/day)
for
three
weeks
prior
to
behavioral
testing
(which
began
at
approximately
68
days
of
age).
2.2.
Colonization
procedure
Subjects
were
randomly
assigned
to
one
of
two
colonization
conditions
(imposed
[IMP],
n
=
24,
or
innate
[INN],
n
=
24].
Subjects
in
the
imposed
(IMP)
group
were
housed
in
groups
of
three
(i.e.,
triads)
from
67–81
days
of
age.
This
imposed
group
colonization
took
place
prior
to
testing
in
the
learning
battery
so
that
the
effects
of
social
stratification
on
learning
performance
could
be
assessed.
Subjects
in
the
innate
(INN)
group
were
colonized
(at
163
days
of
age)
in
triads
after
comple-
tion
of
testing
in
the
learning
battery
(at
150
days
of
age)
so
that
the
relationship
between
innate
tendencies
toward
subordination/dominance
and
learning
perfor-
mance
could
be
examined
and
compared
with
any
relationships
between
these
factors
that
exists
as
a
result
of
the
imposition
of
subordinance
or
dominance
(i.e.,
imposed
group
performance)
prior
to
tests
of
learning.
In
both
conditions,
animals
in
each
triad
were
matched
for
body
weight
(to
within
±
1.2
g).
At
the
start
of
the
colonization
procedure,
subjects
were
transported
in
their
home
cages
to
an
isolated
testing
room
(300
lx).
To
examine
social
interactions,
three
subjects
were
placed
simultaneously
in
a
neutral
area,
i.e.,
a
novel
standard
shoebox
cage
lined
with
wood
shavings.
Behavior
was
observed
in
three
evenly
spaced
10-
min
sessions
during
the
light
cycle
(07:00–19:00)
and
three
10-min
sessions
during
the
dark
cycle
(19:00–7:00).
Between
observations
that
occurred
during
the
light
cycle
and
those
during
the
dark
cycle,
subjects
were
returned
to
their
home
cages.
Subjects
remained
housed
in
triads
until
the
termination
of
the
colonization
period
(14
days).
Rather
than
the
two
weeks
of
colonization
incurred
by
the
IMP
subjects
(where
it
was
the
intention
to
induce
subordination
prior
to
testing
in
the
cognitive
battery),
subjects
in
the
INN
group
(where
it
was
the
intention
to
assess
subordi-
nation
after
cognitive
abilities
had
been
determined)
were
colonized
for
only16
h
(after
the
completion
of
cognitive
testing).
This
was
done
as
it
was
determined
from
observations
of
group
IMP
that
stratification
of
the
colonized
animals
was
complete
after
only
16
h
of
interaction.
Thus
after
16
h,
social
stratification
could
be
accurately
estimated,
and
exposing
animals
in
Group
INN
to
additional
unnecessary
aggression
was
deemed
unwarranted.
Timelines
of
the
experimental
procedures
for
the
IMP
and
the
INN
groups
are
provided
in
Fig.
1.
All
behavioral
interactions
were
recorded
for
offline
measurement
as
detailed
below.

Author's personal copy
296 D.
Colas-Zelin
et
al.
/
Behavioural
Brain
Research
232 (2012) 294–
305
Fig.
1.
The
timeline
of
the
handling,
colonization,
exploratory,
sensorimotor,
learning
and
blood
collection
procedures
across
the
experimental
period
for
both
Groups
IMP
(colonized
before
the
learning
battery
tests)
and
INN
(colonized
after
the
learning
battery
tests).
Days
indicate
time
since
the
animal’s
day
of
birth.
2.3.
Assessment
of
social
dominance
Three
types
of
behavior
were
assessed
in
the
colonized
mice:
(1)
dominance-
related
behavior,
(2)
submission-related
behavior
and
(3)
affiliative
behavior.
Dominance-related
behaviors:
(1)
Number
of
bites
made:
A
bite
by
one
conspecific
directed
toward
any
area
of
the
body
(i.e.,
head/face
or
back/flanks)
of
another
conspecific
in
the
triad.
The
aver-
age
of
the
total
bites
made
across
all
six
observation
periods
was
the
measure
used
for
subsequent
statistical
analyses.
(2) Number
of
bites
received:
A
bite
received
by
an
individual
conspecific
in
the
triad
to
any
area
of
the
body
(head/face
or
back/flanks).
The
average
of
the
total
bites
received
across
all
six
observation
periods
was
the
measure
used
for
statistical
analyses.
(3)
Latency
to
first
attack:
The
time
(in
s)
that
elapsed
from
when
all
three
indi-
viduals
were
placed
together
on
Day
1
until
an
individual
subject
attacked/bit
another
subject
in
the
triad.
(4)
Total
number
of
tail
rattles:
A
tail
rattle
was
defined
as
a
“rapid
lateral
quivering
or
thrashing
of
the
tail”
[65].
Tail
rattles
have
been
reported
to
be
correlated
with
aggression
and
social
dominance
[66–68].
The
total
number
of
tail
rattles
summed
across
all
six
observation
periods
was
the
measure
used
for
statistical
analyses.
(5) Wounding:
After
the
last
observation
session
on
Day
1,
the
severity
of
bodily
wounds
was
ranked
for
each
individual
subject
using
a
scale
from
1
to
5
(1
being
the
least
severe
wounding
and
5
being
the
most
severe
wounding).
This
was
not
intended
to
be
an
absolute
scale,
but
rather,
was
indicative
of
the
relative
severity
of
wounds
in
the
animals
that
contributed
to
this
experiment.
Typically,
more
serious
wounding
is
seen
in
subordinate
subjects
while
dominants
are
often
spared
from
any
wounding.
Submission-related
behavior:
(1)
The
number
of
upright/sideways
defensive
rears:
The
defensive
rear
is
consid-
ered
a
sign
of
retreat
that
occurs
when
a
subject
rises
up
on
its
hindlimbs
with
its
“forearms
limp,
its
head
angled
upward,
and
its
ears
retracted”
[66,69–76].
We
summed
the
total
number
of
defensive
rears
across
all
six
observation
periods
for
statistical
analyses.
(2)
Wounding:
As
described
above,
depending
on
the
degree
of
wounding,
this
mea-
sure
could
be
indicative
of
a
dominant
or
submissive
status.
Typically,
more
serious
wounding
is
seen
in
subordinate
subjects
while
dominants
are
often
spared
extensive
physical
trauma.
Affiliative
behavior:
(1)
Total
time
spent
sniffing:
Sniffing
is
an
“introductory/investigatory
act”
that
was
defined
here
as
the
time
that
a
subjects’
nose
spent
in
contact
(or
within
2
mm)
with
any
area
of
the
body
of
another
conspecific
in
the
triad
[66]
while
engaged
in
sniffing.
The
sum
of
the
total
time
spent
sniffing
was
used
for
statistical
analyses.
(2)
Total
time
spent
huddling:
Huddling
was
defined
as
bodily
contact
between
two
or
more
mice
in
the
triad
during
a
period
of
rest
that
exceeded
60
s
in
duration.
The
sum
of
the
total
time
spent
huddling
was
the
measure
used
for
statistical
analyses.
Since
the
measures
of
behavioral
dominance
assessed
here
were
found
to
be
highly
related
(see
Results,
Section
3.1),
the
average
number
of
bites
made
was
used
to
categorize
animals
as
“dominant”
“mid
submissive”
or
“low
submissive”.
Only
the
behavioral
data
from
the
“dominant”
and
“low
submissive”
animals
from
each
triad
was
used
for
subsequent
statistical
analyses.
In
two
cases,
a
“low
submissive”
animal
died,
and
data
from
the
“mid
submissive”
from
the
same
triad
was
substituted
for
statistical
analysis.
2.4.
Learning
tasks
The
48
CD-1
mice
used
in
this
experiment
were
assessed
(in
two
independent
replications)
on
the
five
learning
tests
(i.e.,
Lashley
III
maze,
passive
avoidance,
spa-
tial
water
maze,
associative
fear
conditioning
and
odor
guided
discrimination)
which
make
up
core
tasks
previously
used
to
evaluate
general
learning
abilities.
These
tasks
were
chosen
so
that
they
placed
unique
sensory,
motor,
motivational
and
infor-
mation
processing
demands
on
the
animals.
The
order
of
testing
was
designed
so
as
to
provide
a
temporal
separation
between
any
two
tasks
that
are
motivated
by
food
deprivation
(to
prevent
excessive
physical
strain
and
to
minimize
any
poten-
tial
cross-task
influences
due
to
motivational
factors).
In
addition,
the
testing
order
was
designed
to
separate
tasks
based
on
similar
processes
or
motor
requirements
(e.g.,
mazes
of
a
similar
nature,
activity
vs.
passivity),
again
so
as
to
minimize
any
potential
transfer
between
tasks.
All
animals
were
assessed
on
tasks
in
the
follow-
ing
order:
Lashley
maze,
passive
avoidance,
odor
discrimination,
fear
conditioning
and
spatial
water
maze.
A
different
experimenter
tested
the
animals
on
each
of
the
learning
tasks,
and
these
experimenters
were
unaware
of
the
animals’
history
or
social
status.
In
all
learning
tasks,
the
animals’
performance
was
assessed
during
the
acqui-
sition
phase
of
learning
(i.e.,
prior
to
reaching
their
stable,
asymptotic
level
of
performance).
Thus
the
dependent
measure
for
each
task
was
representative
of
the
animals’
rate
of
learning
on
that
task,
and
these
measures
of
each
individual’s
per-
formance
could
be
ranked
(through
the
application
of
principal
component
analysis
and
the
resultant
factor
scores;
see
below)
relative
to
other
animals
in
the
sam-
ple.
To
quantify
an
animal’s
performance
in
tasks
in
which
there
were
multiple
training/test
trials,
performance
during
trials
that
fell
within
the
acquisition
phase
were
averaged.
In
tasks
in
which
there
was
only
one
test
trial
(i.e.,
passive
avoid-
ance),
training
parameters
were
used
that
were
previously
determined
to
result
in
sub-asymptotic
responding
by
most
animals.
To
characterize
the
general
cogni-
tive
performance
of
individual
animals,
the
performance
on
the
learning
tasks
was
subjected
to
a
principal
component
analysis,
which
groups
variables
into
“factors”
which
best
account
for
the
overall
pattern
of
correlations
between
them.
Based
only
on
data
in
the
learning
tasks,
a
single
factor
was
extracted
(which
accounted
for
29%
of
the
variance
across
all
tasks),
and
this
factor
yielded
a
factor
score
(analogous
to
an
average
of
z
scores
for
the
individual
animals’
performance
on
each
task)
for
each
animal
that
served
to
represent
its
general
cognitive
ability.
The
rationale
for
these
analysis
and
statistical
procedures
are
provided
in
detail
elsewhere
[31].
2.4.1.
Spatial
water
maze
This
task
requires
animals
to
locate
a
submerged
platform
in
a
round
pool
of
opaque
water.
Absent
distinct
intra-maze
cues,
animals’
performance
in
this
task
is
highly
dependent
on
the
interaction
of
extra-maze
spatial
cues.
The
animals
are
motivated
by
their
aversion
to
the
water.
The
latency
and
path
length
to
locate
the
platform
decreases
over
successive
trials,
despite
entering
the
pool
from
different
locations.
A
round
black
pool
(140
cm
diameter,
56
cm
deep)
was
filled
to
within
24
cm
of
the
top
with
water
made
opaque
by
the
addition
of
a
nontoxic,
water
soluble
black
paint.
A
hidden
11
cm
diameter
perforated
black
platform
was
in
a
fixed
location
1.5
cm
below
the
surface
of
the
water
midway
between
the
center
and
perimeter
of
the
pool.
The
pool
was
enclosed
in
a
ceiling-high
black
curtain
on
which
five
different
shapes
(landmark
cues)
were
variously
positioned
at
heights
(relative
to
water
surface)
ranging
from
24
to
150
cm.
Four
of
these
shapes
were
constructed
of

Author's personal copy
D.
Colas-Zelin
et
al.
/
Behavioural
Brain
Research
232 (2012) 294–
305 297
strings
of
white
LEDs
(spaced
at
2.5
cm
intervals)
and
include
an
“X”
(66
cm
arms
crossing
at
angles
40◦from
the
pool
surface),
a
vertical
“spiral”
(80
cm
long,
7
cm
diameter,
11
cm
revolutions),
a
vertical
line
(31
cm)
and
a
horizontal
line
(31
cm).
The
fifth
cue
was
constructed
of
two
adjacent
7-W
light
bulbs
(each
4
cm
diameter).
A
video
camera
was
mounted
180
cm
above
the
center
of
the
water
surface.
These
cues
provided
the
only
illumination
of
the
maze,
totaling
172
lx
at
the
water
surface.
On
the
day
prior
to
training,
each
animal
was
confined
to
the
escape
platform
for
5
min.
Training
was
conducted
on
the
two
subsequent
days.
On
Day
1
of
train-
ing,
animals
were
started
from
one
of
three
unique
locations
on
each
of
five
trials.
The
pool
was
conceptually
divided
into
four
quadrants,
and
one
starting
point
was
located
in
each
of
the
three
quadrants
that
did
not
contain
the
escape
platform.
The
starting
point
on
each
trial
alternated
between
the
three
available
quadrants.
An
animal
was
judged
to
have
escaped
from
the
water
(i.e.,
located
the
platform)
at
the
moment
at
which
all
four
paws
were
situated
on
the
platform,
provided
that
the
animal
remained
on
the
platform
for
at
least
5
s.
Each
animal
was
left
on
the
plat-
form
for
a
total
of
20
s,
after
which
the
trial
was
terminated.
Trials
were
spaced
at
10
min
intervals,
during
which
time
the
animals
were
held
in
their
home
cages.
On
each
trial,
a
90
s
limit
on
swimming
was
imposed,
at
which
time
any
animal
that
had
not
located
the
escape
platform
was
placed
onto
the
platform
by
the
experimenter,
where
it
remained
for
20
s.
The
time
it
took
for
the
animal
to
escape
(latency)
as
well
as
the
distance
traveled
(path
length)
to
reach
the
platform
were
recorded.
Animals
were
observed
from
a
remote
(outside
of
the
pool’s
enclosure)
video
monitor,
and
animals’
performance
was
recorded
on
videotape
for
subsequent
anal-
ysis.
Day
2
of
training
proceeded,
as
did
Day
1,
albeit
with
four
trials
only.
After
the
last
training
trial,
a
90
min
retention
period
began,
after
which
animals
were
tested
with
a
“probe”
trial.
On
the
probe
test,
the
escape
platform
was
removed
from
the
pool,
and
all
animals
were
started
from
the
first
position
for
that
day.
A
60
s
test
was
conducted
and
the
animals’
time
searching
in
the
target
quadrant
(that
in
which
the
escape
platform
was
previously
located)
and
non-target
quadrants
was
recorded.
2.4.2.
Lashley
III
maze
The
Lashley
III
maze
consisted
of
a
start
box,
four
interconnected
alleys
and
a
goal
box
containing
a
food
reward.
Previous
studies
have
shown
that
over
successive
trials,
the
latency
of
rodents
to
locate
the
goal
box
decreased,
as
does
their
number
of
errors
(i.e.,
wrong
turns
or
retracing).
A
Lashley
III
maze
scaled
for
mice
was
constructed
of
black
Plexiglas
and
a
goal
box
marked
by
white
electrical
tape
was
located
in
the
rear
portion
of
the
maze
where
a
45
mg
BioServe
(rodent
grain)
pellet
served
as
a
reinforcer.
Illumination
was
80
lx
at
the
floor
of
the
maze.
The
maze
was
isolated
behind
a
shield
of
white
Plexiglas
to
prevent
the
use
of
extra-maze
landmark
cues.
Food-deprived
animals
were
acclimated
and
trained
on
two
successive
days.
On
the
day
prior
to
acclimation,
all
animals
were
provided
with
three
food
pellets
in
their
home
cages
to
familiarize
them
with
the
novel
reinforcer.
On
the
acclimation
day,
each
mouse
was
placed
in
the
four
alleys
of
the
maze,
but
the
openings
between
the
alleys
were
blocked
so
that
the
animals
could
not
navigate
the
maze.
Each
animal
was
confined
to
the
start
and
subsequent
two
alleys
for
4
min,
and
for
6
min
in
the
last
(goal)
alley,
where
three
food
pellets
were
present
in
the
goal
box.
This
acclimation
period
promotes
stable
and
high
levels
of
activity
on
the
subsequent
training
day.
On
the
training
day,
each
animal
was
placed
in
the
start
box
and
allowed
to
traverse
the
maze
until
it
reached
the
goal
box
and
consumed
the
single
food
pellet
present
in
the
cup.
Upon
consuming
the
food,
the
animal
was
returned
to
its
home
cage
for
a
20
min
interval
(ITI)
during
which
the
apparatus
was
cleaned.
After
the
ITI,
the
mouse
was
returned
to
the
start
box
to
begin
the
next
trial,
and
the
sequence
was
repeated
for
five
trials.
Both
the
latency
and
errors
(i.e.,
a
turn
in
an
incorrect
direction,
including
those
which
result
in
path
retracing)
to
enter
the
goal
box
were
recorded
on
each
trial.
2.4.2.1.
Associative
fear
conditioning.
In
this
task
animals
received
a
tone
(CS)
paired
with
a
mild
foot
shock
(US).
Two
distinct
experimental
chambers
(i.e.,
contexts)
were
used,
each
of
which
was
contained
in
a
sound-
and
light
attenuating
enclosure.
These
boxes
were
designated
as
training
and
novel
contexts,
and
differed
as
follows:
The
training
chamber
(16.5
cm
×
26.5
cm
×
20
cm)
was
brightly
illuminated
(100
lx),
had
clear
Plexiglas
walls,
and
parallel
stainless-steel
rods
(5
mm,
10
mm
spacing)
form-
ing
the
floor.
The
novel
chamber
(23
cm
×
21.5
cm
×
19
cm)
was
dimly
illuminated
(6
lx)
and
all
of
the
walls
and
floor
were
constructed
of
clear
plexiglass.
In
both
boxes,
the
auditory
stimulus
(60
dB,
2.9
kHz)
was
delivered
by
a
piezoelectric
buzzer.
On
Day
1
subjects
were
acclimated
in
both
novel
and
training
contexts
for
a
20
min
period
in
each
box.
On
Day
2
subjects
received
an
18
min
training
session
in
the
training
chamber.
All
training
sessions
were
videotaped
for
subsequent
offline
scoring.
Subjects
received
three
tone/shock
presentations
at
4,
10
and
16
min
into
the
session.
The
CS
presentation
consisted
of
a
pulsed
(.7
s
on
.3
s
off)
20
s
tone.
Imme-
diately
following
the
tone
offset,
the
shock
US
(0.6-mA,
constant-current
footshock)
was
presented
for
500
ms.
Freezing
was
measured
during
the
20
s
before
(BASELINE
FREEZING),
during
(TONE
FREEZING)
and
after
(POST
SHOCK
FREEZING)
the
20
s
tone
presentation.
A
measure
for
freezing
during
the
training
period
(TRAINING
FREEZING)
was
calcu-
lated
by
subtracting
the
time
spent
freezing
in
baseline
from
the
time
spent
freezing
during
the
tone.
On
Day
3,
freezing
was
measured
during
a
5
min
session
in
the
novel
chamber
during
which
time,
tone,
but
no
shock
was
presented.
2.4.2.2.
Odor
discrimination
and
choice.
Rodents
rapidly
learn
to
use
odors
to
guide
appetitively
reinforced
behaviors.
In
a
procedure
based
on
one
designed
for
rats
[77],
mice
learned
to
navigate
a
square
field
in
which
unique
odor-marked
(e.g.,
almond,
lemon,
mint)
food
cups
were
located
in
three
corners.
Although
food
was
present
in
each
cup,
it
was
accessible
to
the
animals
in
only
one
cup,
the
one
marked
by
mint
odor.
An
animal
was
placed
in
the
empty
corner
of
the
field,
after
which
it
explored
the
field
and
eventually
retrieved
the
single
piece
of
available
food.
On
subsequent
trials,
the
location
of
the
food
cups
was
changed,
but
the
accessible
food
was
consistently
marked
by
the
same
odor
(mint).
On
successive
trials,
animals
required
less
time
to
retrieve
the
food
and
made
fewer
approaches
(i.e.,
“errors”)
to
those
food
cups
in
which
food
was
not
available.
Using
this
procedure,
errorless
performance
was
typically
observed
within
three
to
four
training
trials.
A
black
Plexiglas
60
cm
square
field
with
30
cm
high
walls
was
located
in
a
dimly
lit
(10
fc)
testing
room
with
a
high
ventilation
rate
(3
min
volume
exchange).
Three
4
cm
×4
cm
×2.0
cm
(l,
w,
h)
aluminum
food
cups
were
placed
in
three
corners
of
the
field.
A
food
reinforcer
(30
mg
portions
of
chocolate
flavored
puffed
rice)
was
placed
in
a
1.6
cm
deep,
1
cm
diameter
depression
in
the
center
of
each
cup.
The
food
in
two
of
the
cups
was
covered
(1.0
cm
below
the
surface
of
the
cup)
with
a
wire
mesh
so
that
it
was
not
accessible
to
the
animal,
while
in
the
third
cup
(the
“target”
cup),
the
food
could
be
retrieved
and
consumed.
A
cotton-tipped
laboratory
swab,
located
between
the
center
and
rear
corner
of
each
cup,
extended
vertically
3
cm
from
the
cups’
surface.
Immediately
prior
to
each
trial,
fresh
swabs
were
loaded
with
25
␮l
of
either
lemon,
almond,
or
mint
odorants
(McCormick
flavor
extracts).
The
mint
odor
was
always
associated
with
the
target
food
cup.
It
should
be
noted
that
in
pilot
studies,
the
odor
associated
with
food
was
counterbalanced
across
animals
and
no
dis-
cernible
differences
in
performance
could
be
detected
in
response
to
the
different
odors.
On
the
day
prior
to
test
animals
were
given
60
min
of
free
feeding
time
at
the
same
time
of
day
they
would
have
been
acclimated.
On
test
day,
animals
received
four
training
trials
in
the
field
with
three
food
cups
present.
On
each
trial,
an
animal
is
placed
in
the
empty
corner
of
the
field.
On
Trial
1,
the
reinforcing
food
was
available
to
the
animal
in
the
cup
marked
by
mint
odor.
An
additional
portion
of
food
was
placed
on
the
top
surface
of
the
same
cup
for
the
first
trial
only.
The
trial
continued
until
the
animal
retrieved
and
consumed
the
food
from
the
target
cup,
after
which
the
animal
was
left
in
the
chamber
for
an
additional
20
s
and
then
returned
to
its
home
cage
to
begin
a
6
min
ITI.
On
Trials
2–4,
the
location
of
the
food
cups
was
rearranged,
but
the
baited
cup
remained
consistently
marked
by
the
mint
odor.
Both
the
corner
location
of
the
mint
odor
and
its
position
relative
to
the
remaining
odors
was
changed
on
each
trial.
On
each
trial,
the
latency
to
retrieve
the
food
and
errors
was
recorded.
An
error
was
recorded
any
time
an
animal
made
contact
with
an
incorrect
cup,
or
its
nose
crossed
a
plane
parallel
to
the
perimeter
of
an
incorrect
cup.
Similarly,
an
error
was
recorded
when
an
animal
sampled
(as
above)
the
target
cup
but
did
not
retrieve
the
available
food.
2.4.3.
One-trial
passive
avoidance
A
chamber
illuminated
by
dim
(<20
lx)
red
light
was
used
for
training
and
testing.
Animals
were
confined
to
circular
(“safe”)
chamber
(10
cm
diameter,
8
cm
high).
The
walls
and
floor
of
this
chamber
were
white,
and
the
ceiling
was
translucent
orange.
The
floor
was
comprised
of
plastic
rods
(2
mm
diameter)
arranged
to
form
a
pattern
of
1
cm
square
grids.
A
clear
exit
door
(3
cm
square)
was
flush
with
the
floor
of
the
safe
compartment,
and
the
door
was
able
to
slide
horizontally
to
open
or
close
the
compartment.
The
bottom
of
the
exit
door
was
located
4
cm
above
the
floor
of
a
second
circular
chamber
(20
cm
diameter,
12
cm
high).
This
“unsafe”
chamber
had
a
clear
ceiling
and
a
floor
comprised
of
4
mm
wide
aluminum
planks
that
formed
a
pattern
of
1.5
cm
square
grids
oriented
at
a
45◦angle
relative
to
the
grids
in
the
safe
compartment.
When
an
animal
stepped
from
the
safe
compartment
through
the
exit
door
onto
the
floor
of
the
unsafe
compartment,
a
compound
aversive
stim-
ulus
comprised
of
a
bright
(550
lx)
white
light
and
“siren”
(60
dB
above
the
50
dB
background)
was
initiated.
Animals
learn
to
suppress
movement
to
avoid
contact
with
aversive
stimuli.
This
“passive
avoidance”
response
is
exemplified
in
step-down
avoidance
procedures,
where
commonly,
an
animal
is
placed
on
a
platform,
whereupon
stepping
off
of
the
platform
it
encounters
a
footshock.
Following
just
a
single
encounter
with
shock,
animals
are
subsequently
reluctant
to
step
off
of
the
safe
platform.
The
animals’
reluctance
to
leave
the
platform
is
believed
to
not
reflect
fear,
because
typical
fear
responses
are
not
expressed
in
animals
engaged
in
the
avoidance
response
[78,79].
Upon
stepping
off
the
platform,
animals
here
were
exposed
to
a
compound
of
bright
light
and
loud
oscillating
noise
rather
than
shock,
so
as
not
to
duplicate
stimuli
between
tasks
(see
fear
conditioning,
above).
Like
more
common
procedures,
our
variant
of
this
task
supports
learning
after
only
a
single
trial
(i.e.,
subsequent
step-
down
latencies
will
be
markedly
increased).
Animals
were
placed
on
the
platform
behind
the
exit
blocked
by
the
Plexiglas
door.
After
4
min
of
confinement,
the
door
was
retracted
and
the
latency
of
the
animal
to
leave
the
platform
and
make
contact
with
the
grid
floor
was
recorded.
Prior
to
training,
baseline
step-down
latencies
typically
range
from
8
to
20
s.
Upon
contact
with
the
floor,
the
door
to
the
platform
was
closed
and
the
aversive
stimulus
(light,
noise,
and
vibration)
was
presented
for
4
s,
at
which
time
the
platform
door
was
opened
to
allow
animals
to
return
to
the
platform,
where
they
were
again
confined
for
5
min.
At
the
end
of
this
interval,
the
door
was
opened
and
the
latency

Author's personal copy
298 D.
Colas-Zelin
et
al.
/
Behavioural
Brain
Research
232 (2012) 294–
305
of
the
animal
to
exit
the
platform
and
step
onto
the
grid
floor
(with
no
aversive
stimulation)
was
recorded.
The
ratio
of
post-training
to
pre-training
step-down
latencies
was
calculated
for
each
animal
and
this
served
to
index
learning.
It
has
been
determined
that
asymptotic
performance
is
apparent
in
group
averages
following
2–3
training
trials;
thus
performance
after
a
single
trial
reflects,
in
most
instances,
sub-asymptotic
learning.
2.5.
Exploratory
and
sensory/motor
testing
All
animals
underwent
seven
assessments
of
physical
characteristics
and
behav-
ioral
tendencies
[31,80,81].
These
included
activity
in
the
unwalled
areas
of
an
open
field
(a
common
measure
of
exploration),
exploration
in
a
light/dark
box
and
in
an
elevated
plus
maze
(potential
measures
of
anxiety),
latency
to
lick
a
paw
on
a
hot-
plate
test
(pain
sensitivity),
screen
hanging
(a
measure
of
paw
strength),
latency
to
fall
from
a
small
elevated
platform,
and
separately,
the
latency
to
fall
from
and
ability
to
maneuver
across
a
balance
beam
(measures
of
coordination).
2.5.1.
Exploratory
tasks
2.5.1.1.
Open
field.
A
square
field
(46
cm
×46
cm)
with
13
cm
high
walls
was
con-
structed
of
white
Plexiglas
and
was
located
in
a
brightly
lit
room
(400
lx)
with
a
background
noise
of
65
dBc.
The
field
was
conceptually
comprised
of
a
6
×
6
grid
(7.65
cm
quadrants),
where
20
of
the
quadrants
abutted
the
outer
walls
of
the
field
(i.e.,
“wall”
quadrants),
and
16
quadrants
were
displaced
from
the
walls
and
comprised
the
interior
(i.e.,
“open”
quadrants)
of
the
field.
Animals
were
placed
in
the
center
of
the
field.
After
20
s
had
elapsed
(during
which
the
animals
self-selected
a
“starting”
location),
the
animals’
behavior
was
monitored
for
4
min.
Throughout
this
time
the
animal’s
entries
into
walled
and
open
quadrants
were
recorded.
An
entry
was
recorded
whenever
both
front
paws
crossed
the
border
of
a
quadrant.
Both
total
activity
levels
(i.e.,
quadrant
entries
regardless
of
category)
as
was
the
percentage
of
entries
into
unwalled
(open)
quadrants
of
the
field
were
recorded.
It
should
be
noted
that
a
4-min
test
was
explicitly
chosen
because
changes
in
exploratory
behavior
(not
necessarily
simple
motor
activity)
were
not
detectable
over
time.
2.5.1.2.
Elevated
plus
maze.
The
maze
was
constructed
of
grey
Plexiglas
with
four
arms
in
the
form
of
a
“plus.”
Each
of
these
arms
was
6
cm
wide,
and
the
entire
maze
was
suspended
30
cm
above
a
black
surface.
Two
opposing
arms
of
the
maze
were
enclosed
in
8
cm
high,
grey
Plexiglas
walls,
while
the
two
remaining
arms
were
left
open.
The
maze
was
located
in
a
brightly
lit
room
(300
lx).
Animals
were
placed
in
the
center
of
the
maze
facing
a
closed
arm,
and
their
behavior
in
the
maze
was
recorded
in
1-min
blocks
for
4
min.
Of
particular
interest
was
their
total
number
of
arm
entries,
their
percent
of
total
arm
entries
that
were
into
open
arms,
closed
and
open
arm
entries
as
well
as
reentries
into
a
previously
occupied
arm.
2.5.1.3.
Light/dark
discrimination
test.
The
rectangular
box
used
in
this
task
(56
×15
×10,
l
×w
×
h)
was
constructed
from
black
Plexiglas
and
was
located
in
a
dimly
lit
room
(<50
lx).
The
box
was
divided
by
a
gray
Plexiglas
wall
to
create
two
equal
size
compartments
(28
cm
l).
The
animals
could
travel
between
compart-
ments
by
way
of
a
small
opening
in
the
dividing
wall
(3
cm
×
5
cm).
The
walls
of
one
compartment
were
painted
white
while
the
other
side
remained
black,
resulting
in
a
light
and
dark
side.
The
lid
on
the
apparatus
was
clear
above
the
light
side
of
the
box
and
was
opaque
on
the
dark
side.
The
lighting
was
arranged
so
that
a
60-
W
lamp
was
shined
directly
on
the
light
side
of
the
box,
resulting
in
a
differential
illumination
between
the
light
side
and
the
dark
side
(300
lx
versus
<50
lx).
The
animals
were
placed
in
the
dark
side
of
the
box
and
allowed
to
freely
explore
the
apparatus
for
5
min.
During
this
time
the
latency
to
enter
the
light
side
(front
and
hind
legs)
by
passing
through
the
door
between
compartments
was
recorded.
In
addition,
a
number
of
other
exploratory
measures
were
recorded
including
the
percent
of
total
time
spent
in
the
light
side
and
the
number
of
crossings
between
the
light
side
and
the
dark
side.
2.6.
Sensory/motor
tasks
2.6.1.
Balance
beam
Animals
were
placed
on
a
40
cm
×
0.7
cm
×
2
cm
(l
×
w
×
h)
beam
suspended
30
cm
above
the
ground.
The
beam
was
explicitly
designed
so
that
animals
do
not
typically
fall
from
it.
Instead,
movement
along
the
beam
was
the
variable
of
inter-
est,
as
movement
is
presumed
to
interact
with
balance.
In
a
4-min
test,
mice
exhibit
wide
variability
in
the
amount
of
movement
along
its
length.
2.6.2.
Hot
plate
test
of
pain
sensitivity
The
animals
were
placed
on
an
aluminum
plate
which
was
maintained
at
a
surface
temperature
of
52.6 ◦C.
The
animals’
latency
to
raise
a
hind
paw
and
to
either
lick
or
shake
the
paw
served
as
the
index
of
pain
sensitivity.
2.6.3.
Screen
hanging
test
of
grip
strength
The
animals
were
placed
on
the
underside
of
a
wire
mesh
screen
(7
mm
grids)
tilted
40◦from
vertical
and
suspended
24
cm
from
ground.
Both
the
latency
to
drop
from
the
screen
and
the
distance
moved
prior
to
dropping
from
the
screen
(cm/s;
180
maximum
test
duration)
were
recorded.
2.6.4.
Balance
pole
Animals
are
placed
on
a
platform
atop
a
4
mm
rod
coated
with
black
rubber
(shrink
tubing).
The
rod
was
suspended
30
cm
above
ground.
Latency
to
drop
from
the
rod
(an
index
of
balance)
was
recorded.
2.7.
Stress
procedure
and
assessment
of
plasma
CORT
After
colonization,
subjects
in
the
IMP
and
INN
groups
underwent
a
mild
stress
procedure.
For
the
IMP
group
this
procedure
occurred
114
days
after
colonization
and
for
the
INN
group
this
procedure
took
place
32
days
after
colonization.
Dur-
ing
the
interim
between
the
end
of
colonization
and
the
initiation
of
the
stress
procedure,
subjects
in
the
IMP
and
INN
groups
were
singly
housed.
To
inflict
the
stress,
animals
were
confined
on
a
10-cm
diameter
platform
ele-
vated
120
cm
above
the
floor
in
a
brightly
lit,
unfamiliar
room
for
a
6
min
period.
Ten
minutes
after
the
procedure
subjects
were
decapitated
to
collect
trunk
blood.
Plasma
CORT
levels
were
quantified
using
the
mouse
CORT
Enzyme
Immunoassay
(EIA)
kit
available
from
Cayman
Chemicals
(Ann
Arbor,
MI).
2.8.
Statistical
analyses
This
experiment
was
a
four-group
between
subjects
design
that
compared
behavioral
and
hormonal
measures
in
imposed
dominant
(IMP
DOM),
imposed
subordinate
(IMP
SUB),
innate
dominant
(INN
DOM)
and
innate
subordinate
(INN
SUB)
mice.
Statistical
comparisons
of
groups
were
conducted
using
either
anal-
ysis
of
variance
(ANOVA)
or
independent
samples
t-tests
to
examine
between
group
differences
in
general
learning
ability
and
performance
on
individual
learning
tasks.
Correlations
between
factor
scores
(an
estimate
of
an
animal’s
performance
on
a
factor
isolated
with
a
principal
component
analysis),
measures
of
behav-
ior
(exploratory,
social,
and
sensory/motor
function)
and
stress
reactivity
(stress
induced
CORT)
were
also
assessed.
3.
Results
3.1.
Social
behavior
data
To
examine
the
inter-relationships
between
the
measures
of
social
behavior
quantified
during
Day
1
observations,
a
Pear-
son’s
product-moment
correlation
matrix
was
created.
A
negative
correlation
was
observed
between
the
total
average
number
of
bites
made
and
the
number
of
wounds
received
[r
=
−.30,
n
=
46,
p
<
.05],
i.e.,
animals
that
made
more
bites
were
themselves
less
wounded.
There
was
also
a
positive
correlation
between
the
total
average
bites
made
and
total
tail
rattles
[r
=
.30,
n
=
46,
p
<
.05]
and
a
negative
correlation
between
total
tail
rattles
and
wound-
ing
[r
=
−.44,
n
=
46,
p
<
.05].
Wounding
and
time
spent
huddling
were
positively
correlated
[r
=
.46,
n
=
46,
p
<
.05]
while
time
spent
huddling
and
total
tail
rattles
were
inversely
correlated
[r
=
−.29,
n
=
46,
p
<
.05].
(It
is
notable
the
animals
characterized
as
“dom-
inant”
rarely
huddled
with
the
other
two
animals
of
the
triad,
i.e.,
only
the
submissive
and
mid-submissive
animals
engaged
in
huddling.)
Lastly,
there
was
a
positive
correlation
between
the
latency
to
attack
and
bites
received
[r
=
.31,
n
=
46,
p
<
.05].
These
results
indicate
that
both
dominant
and
subordinate
subjects
con-
sistently
express
behaviors
that
are
in
accord
with
previously
established
behavioral
phenotypes
for
submissive
and
dominant
rodents
[50,56,66–70,72,82–85].
Thus
the
dominant
and
submis-
sive
phenotypes
are
easily
recognized.
This
difference
can
be
best
summarized
qualitatively:
Animals
made
submissive
through
colonization
were
continuously
attacked
by
their
dominant
cage-
mates.
3.2.
Exploratory
data
Subjects
were
tested
prior
to
the
assessment
of
learning
abil-
ities
to
determine
whether
there
were
pre-existing
differences
in
exploratory
tendencies.
In
the
open
field
test,
there
was
a
trend
toward
significance
for
between-groups
differences
for
the
percent
of
internal
entries,
i.e.,
entries
into
unwalled
quadrants,

Author's personal copy
D.
Colas-Zelin
et
al.
/
Behavioural
Brain
Research
232 (2012) 294–
305 299
B
IM
P D
OMS
IM
P SUBS
IN
N DOMS
I
NN SUBS
Total Entries
0
20
40
60
80
100
120
140
160
180
200
Open Field: Total Entries
*
C
IM
P DOMS
IM
P SUBS
IN
N DOMS INN SUBS
Run Speed: cm/sec
0
5
10
15
20
25
30
35
40
Open Field: Running Spee
d
*D
IM
P DOMS
IM
P SUBS
IN
N DOM
S INN SUBS
% Entries into Open Arms
0
5
10
15
20
25
30
35
Elevated Plus Maze: % Open Entries
*
Open Field: % Unwalled Entries
IMP D
OMS
IMP SUBS
INN DOM INN SUBS
% Entries Into Internal Quadrants
0
5
10
15
20
25
A
*
*
Fig.
2.
(A)
Illustrated
is
the
percent
of
internal
(unwalled)
entries
in
the
Open
Field
for
the
IMP
DOMS,
IMP
SUBS,
INN
DOM,
and
INN
SUBS
groups.
IMP
DOMS
(which
were
colonized
prior
to
the
open
field
test)
had
a
higher
percentage
of
entries
in
the
internal
areas
of
the
open
field
than
IMP
SUBS
(p
=
.01).
Similarly,
INN
SUBS
(that
were
colonized
after
the
open
field
test)
had
a
higher
percentage
of
internal
entries
in
the
open
field
than
IMP
SUBS
(p
=
.05).
(B)
Open
Field
total
entries
(entries
in
the
internal
and
external
quadrants)
for
the
IMP
DOMS,
IMP
SUBS,
INN
DOM,
and
INN
SUBS
groups.
IMP
DOMS
were
more
active
(e.g.,
had
more
total
entries)
than
IMP
SUBS
(p
=
.04).
(C)
Open
Field
run
speed
(cm/s)
for
the
IMP
DOMS,
IMP
SUBS,
INN
DOM,
and
INN
SUBS
groups.
IMP
SUBS
had
a
slower
run
speed
(cm/s)
than
IMP
DOMS
(p
=
.03).
(D)
Elevated
plus
maze
percent
open
entries
for
the
IMP
DOMS,
IMP
SUBS,
INN
DOM,
and
INN
SUBS
groups.
Colonization
prior
to
the
elevated
plus
maze
test
promoted
fewer
entries
into
open
arms
in
animals
that
exhibited
submission,
and
a
tendency
toward
fewer
entries
in
animals
that
exhibited
dominance.
Asterisks
(*)
indicate
significant
comparisons.
[F(3,26)
=
2.67,
p
=
.07].
Post
hoc
comparisons
revealed
that
IMP
DOMS
(that
had
been
so
designated
based
on
previous
stratifi-
cation
in
a
colony)
had
a
higher
percentage
of
entries
in
the
internal/unwalled
areas
of
the
open
field
than
IMP
SUBS
[also
desig-
nated
based
on
previous
stratification
in
a
colony;
p
<
.01].
Similarly,
INN
SUBS
(designated
based
on
stratification
in
a
colony
assessed
after
the
completion
of
all
behavioral
testing)
had
a
higher
percent-
age
of
internal
entries
in
the
open
field
than
IMP
SUBS
[p
<
.05]
(Fig.
2A).
This
pattern
of
results
indicates
that
imposed
submission,
but
not
innate
submissive
tendencies,
tend
to
suppress
exploratory
behaviors.
A
separate
ANOVA
for
the
total
number
of
entries
(combined
walled
and
unwalled,
indicative
of
total
activity)
made
in
the
open
field
also
showed
a
trend
toward
significance
[F(3,26)
=
1.57,
p
=
.22].
Post
hoc
comparison
revealed
that
IMP
DOMS
were
more
active
(e.g.,
had
more
total
entries)
than
IMP
SUBS
[p
<
.05;
Fig.
2B].
Running
speed
in
the
open
field
also
showed
a
trend
toward
signifi-
cance
[(F(3,24)
=
1.74,
p
=
.19]
with
post
hoc
comparisons
indicating
that
IMP
SUBS
had
a
slower
running
speed
(cm/s)
than
IMP
DOMS
[p
<
.05;
Fig.
2C],
although
nominally,
this
effect
was
small.
Data
obtained
in
the
elevated
plus
maze
(after
completion
of
the
learning
battery)
followed
a
pattern
similar
to
that
in
the
open
field.
In
the
elevated
plus
maze
task,
there
was
a
trend
toward
significance
for
the
percent
of
open
entries
[F(3,23)
=
1.95,
p
=
.15].
A
post
hoc
test
showed
that
IMP
SUBS
had
a
significantly
lower
percentage
of
open
entries
than
did
INN
SUBS
[p
<
.05;
Fig.
2D].
There
were
no
other
significant
findings
for
measures
in
the
ele-
vated
plus
maze
(closed
entries:
[F(3,23)
=
.12,
n.s.],
open
entries:
[F(3,23)
=
1.41,
n.s.],
reentries:
[F(3,23)
=
1.32,
n.s.],
total
entries:
[F(3,23)
=
.65,
n.s.]).
Again,
this
pattern
suggests
that
the
impair-
ments
identified
here
are
due
to
imposition
of
subordination
and
are
not
innately
expressed
in
subjects
with
a
tendency
toward
sub-
ordination.
3.3.
Sensory/motor
data
There
were
no
significant
differences
between
groups
for
any
of
the
tests
of
sensory
or
motor
abilities
(balance
beam,
latency
to
fall
[F(3,26)
=
.33,
n.s.,
or
distance
traveled:
[F(3,26)
=
.94,
n.s.];
screen
hang,
latency
to
fall:
[F(3,26)
=
1.08,
n.s.]
or
number
of
grid
cross-
ings:
[F(3,26)
=
.52,
n.s.];
balance
pole,
latency
to
fall
[F(3,26)
=
.80,
n.s.];
hotplate,
latency
to
lick
a
hind
paw,
[F(3,26)
=
1.36,
n.s.]).
The
lack
of
significant
differences
between
groups
on
tests
of
sensory/motor
function
suggest
that
none
of
the
animals,
includ-
ing
those
that
were
stratified
(through
colonization)
prior
to
assessment,
suffered
from
gross
motor
impairments
as
a
result
of
wounding.
3.4.
Learning
battery
data
In
the
Lashley
III
maze,
mice
learned
the
task
as
evidenced
by
a
significant
reduction
of
errors
across
trials
[F(4,104)
=
11.97,

Author's personal copy
300 D.
Colas-Zelin
et
al.
/
Behavioural
Brain
Research
232 (2012) 294–
305
Trials
54321
Errors to Goal
5
0
10
15
20
25
30
35
40
IMP DOMS
IMP SUBS
INN DOMS
INN SUBS
**
Fig.
3.
Errors
across
trials
to
find
food
in
the
Lashley
III
maze
for
the
IMP
DOMS,
IMP
SUBS,
INN
DOM,
and
INN
SUBS
groups.
Mice
learned
the
task
as
evidenced
by
a
significant
reduction
of
errors
across
trials
(p
<
.001),
although
Group
IMP
SUBS
(which
were
colonized
and
exhibited
submission
prior
to
the
test)
exhibited
no
apparent
learning
and
took
longer
to
arrive
at
the
goal
box
than
IMP
DOMS
(p
=
.02).
Asterisks
(*)
indicate
significant
comparisons.
p
<
.0001].
However,
there
was
no
difference
between
groups
[F(3,26)
=
1.13,
n.s.]
nor
was
there
an
interaction
between
group
and
trial
[F(12,104)
=
.96,
n.s.].
Errors
to
reach
the
goal
box
in
the
Lashley
III
maze
also
decreased
across
trials
[F(4,104)
=
4.43,
p<.05],
and
group
differences
in
errors
were
seen
[F(3,26)
=
3.36,
p
<
.05]
with
IMP
SUBS
committing
more
errors
in
reaching
the
goal
box
than
IMP
DOMS
(p
<
.05.;
Fig.
3).
There
were
no
significant
group
by
trial
interaction
effects
[F(12,104)
=
.62,
n.s.].
In
the
associative
fear
conditioning
task,
training
levels
of
freez-
ing
(post-pre
freezing
in
s)
increased
across
trials
[F(2,52)
=
18.77,
p
<
.0001],
indicating
that
the
animals
learned
to
freeze
to
the
tone
as
result
of
its
pairing
with
foot
shock.
However
there
were
no
sig-
nificant
group
[F(3,26)
=
.31,
n.s.]
or
interaction
[F(6,52)
=
.33,
n.s.]
effects.
Similarly,
separate
repeated
measures
ANOVA’s
carried
out
for
freezing
assessed
in
the
absence
(baseline)
and
presence
(tone)
of
the
tone
were
significant
for
trial
only
(baseline:
[F(3,26)
=
.64,
p
<
.05];
tone:
[F(3,26)
=
.55,
p
<
.05]).
For
the
passive
avoidance
task,
ANOVA
revealed
a
significant
main
effect
for
group
for
baseline
step-down
latencies,
i.e.,
the
latency
to
step
into
a
novel
environment
prior
to
pairing
that
step
with
aversive
light
and
noise
[F(3,26)
=
17.52,
p
=
<.0001].
Planned
comparisons
demonstrated
that
IMP
SUBS
had
significantly
longer
baseline
step-down
latencies
compared
to
IMP
DOMS
[p
<
.0001]
and
INN
SUBS
[p
<
.0001]
(Fig.
4A).
A
prolonged
baseline
step-down
latency
has
previously
been
interpreted
as
indicative
of
a
reduction
in
exploratory
behavior
[80],
and
thus
the
long
baseline
latency
exhibited
by
Group
IMP
SUB
is
consistent
with
the
above
observa-
tions
of
reduced
exploration
in
this
group.
Given
this
difference
in
baseline
performance,
learning
in
the
passive
avoidance
task
was
assessed
by
computing
a
ratio
of
the
baseline
step-down
latency
and
the
post-training
step-down
latency,
where
higher
latencies
would
reflect
better
learning.
A
comparison
of
the
four
groups’
ratio
of
baseline
step-down
latencies
to
post-training
step-down
laten-
cies
was
significant
[F(3,26)
=
4.17,
p
<
.05].
Planned
comparisons
confirmed
that
IMP
SUBS
were
impaired
compared
to
IMP
DOMS
[p
<
.01]
and
INN
SUBS
[p
<
.05]
(Fig.
4B).
In
the
spatial
water
maze
task,
latencies
to
reach
the
platform
decreased
significantly
across
trials
[F(9,234)
=
6.50,
p
<
.0001].
Table
1
An
unrotated
principal
component
factor
analysis
of
the
primary
learning
battery
revealed
primary
and
secondary
factors
(general
learning
abilities)
explaining
29%
and
27%
of
the
total
variance,
respectively.
Factor
1
Factor
2
Passive
avoidance
0.75
−0.01
Lashley
maze 0.76
0.36
Morris
water
maze
0.09
0.83
Odor
discrimination
0.15
−0.66
Fear
conditioning
0.54
0.32
Eigenvalue 1.46
1.37
%
Variance
explained 29%
27%
However,
there
were
no
significant
group
[F(3,26)
=
1.16,
n.s.]
or
interaction
[F(27,234)
=
.47,
n.s.]
effects.
Path
length
to
the
plat-
form
also
decreased
across
the
ten
trials
[F(9,180)
=
5.80,
p
<
.0001]
and
there
were
significant
group
differences
for
path
length
to
the
platform
[F(3,20)
=
5.54,
p
<
.05],
although
planned
comparisons
revealed
a
significant
difference
between
IMP
SUBS
and
INN
SUBS
only
on
Trial
5.
There
was
no
effect
of
the
interaction
of
group
x
trial
[F(27,180)
=
.58,
n.s.].
In
the
odor
discrimination
task,
a
repeated-measures
ANOVA
for
errors
was
significant
for
trial
[F(3,78)
=
18.15,
p
<
.0001]
but
was
non-significant
for
group
[F(3,26)
=
.42,
n.s.]
or
interaction
[F(9,78)
=
.72,
n.s.].
Repeated-measures
ANOVA
for
latency
was
sig-
nificant
for
trial
[F(3,78)
=
25.21,
p
<
.0001],
however
there
were
no
significant
group
[F(3,26)
=
.67,
n.s.]
or
interaction
effects
[F(9,78)
=
.83,
n.s.].
3.5.
Aggregate
cognitive
performance
Significant
impairments
in
cognitive
performance
in
Group
IMP
SUB
relative
to
Group
INN
SUB
were
only
observed
in
three
of
the
five
cognitive
tasks,
and
in
the
water
maze,
that
difference
was
only
significant
for
a
single
trial.
However,
it
is
notable
that
a
tendency
toward
an
impairment
was
exhibited
by
Group
IMP
SUB
in
all
tasks.
Such
a
pattern
(i.e.,
consistent
directionality
of
effects)
is
best
described
by
factor-analytic
techniques.
An
unrotated
prin-
cipal
components
factor
analysis
of
the
performance
data
for
the
five
tasks
that
comprise
the
learning
battery
isolated
two
factors
that
accounted
for
a
total
of
56%
of
the
variance
in
performance.
Performance
from
all
of
the
learning
tasks
loaded
consistently
on
the
primary
factor,
which
accounted
for
29%
of
the
total
vari-
ance
(eigenvalue
=
1.46,
n
=
48;
Table
1).
Given
that
performance
from
all
learning
tasks
loaded
in
a
single
direction
on
this
factor,
this
factor
was
used
to
extract
factor
scores
to
represent
animals’
aggregate
performance
across
all
learning
tasks
(i.e.,
their
general
learning
ability).
(A
factor
score
is
analogous
to
an
average
of
an
animal’s
z
scores
on
each
task
that
comprised
the
factor,
where
the
z
scores
are
weighted
according
to
their
degree
of
loading
on
that
factor.)
ANOVA
of
factor
scores
revealed
a
main
of
effect
of
group,
[F(3,26)
=
5.21,
p
<
.01].
Post
hoc
comparisons
revealed
that
imposed
subordinates
(IMP
SUBS)
had
decrements
in
general
learn-
ing
performance
compared
to
imposed
dominants
(IMP
DOMS;
p
<
.01),
innate
dominants
(INN
DOMS;
p
<
.01),
and
innate
subordi-
nates
(INN
SUBS;
p
<
.01;
Fig.
5).
In
total,
this
analysis
suggests
that
the
imposition
of
submission,
but
not
the
innate
tendency
toward
submission,
is
associated
with
an
impairment
of
general
cognitive
performance.
The
second
factor
extracted
from
our
principal
component
anal-
ysis
accounted
for
27%
of
the
variance
(eigenvalue
=
1.37,
n
=
48;
Table
1).
Unlike
the
primary
factory,
this
secondary
factor
was
not
readily
interpretable
(i.e.,
no
obvious
pattern
of
factor
loadings
emerged)
and
will
not
be
further
considered
here.
We
conducted
a
second
principal
component
analysis
on
ani-
mals
in
the
IMP
groups
to
determine
the
relationship
between

Author's personal copy
D.
Colas-Zelin
et
al.
/
Behavioural
Brain
Research
232 (2012) 294–
305 301
Passive Av
oidance:
Baseline SDLs
IMP DOMS IMP SUBS INN DOMS INN SUBS
Latency (sec) to Step Prior to Training
0
5
10
15
20
25
30
35
40
A**
B
IMP D
OMS IMP SUBS INN DOMS
INN SUB
S
Post/Pre Step Down Latencies
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
Passive Avoidance: Learning
Ratios
*
*
Fig.
4.
(A)
Latency
to
step
from
platform
prior
to
pairing
the
step
with
aversive
stimulation
in
the
passive
avoidance
task
for
the
IMP
DOMS,
IMP
SUBS,
INN
DOM,
and
INN
SUBS
groups.
IMP
SUBS
had
significantly
longer
baseline
step-down
latencies
compared
to
IMP
DOMS
(p
<
.0001)
and
INN
SUBS
(p
<
.0001).
(B)
Ratio
of
the
latency
to
step
from
platform
before
and
after
pairing
the
step
with
aversive
stimulation
in
the
passive
avoidance
task
for
the
IMP
DOMS,
IMP
SUBS,
INN
DOM,
and
INN
SUBS
groups.
Subjects
in
the
IMP
SUB
group
exhibited
impaired
learning
relative
to
the
IMP
DOM
group
(p
=
.03).
Asterisks
(*)
indicate
significant
comparisons.
INN SUBSINN DOMS IMP SUBSIMP DOMS
Aggregate Learning Performance (factor scores)
-1.5
-1.0
-0.5
0.0
0.5
1.0
*
Fig.
5.
Factor
scores
representative
of
animals’
aggregate
le
for
the
IMP
DOMS,
IMP
SUBS,
INN
DOM,
and
INN
SUBS
groups.
IMP
SUBS
(who
had
been
colonized
and
exhibited
submission
prior
to
testing
in
the
learning
battery)
exhibited
significantly
worse
general
learning
performance
(indicated
by
higher
factor
scores)
than
IMP
DOMS
(p
=
.005),
INN
DOMS
(p
=
.002)
and
INN
SUBS
(p
=
.003).
Asterisks
(*)
indicate
significant
comparisons.
general
learning
and
measures
of
exploration
and
sensory/motor
function.
Previous
work
has
indicated
that
general
learning
abilities
are
directly
related
to
exploratory
tendencies
but
not
sen-
sory/motor
traits
[80].
This
unrotated
principal
component
factor
analysis
produced
a
factor
that
accounted
for
a
total
of
25%
of
the
variance
in
performance
(eigenvalue
=
4.42,
Table
2).
Similar
to
previous
reports
[80,86],
exploratory
measures
in
the
open
field
(%
internal
entries)
and
elevated
plus
maze
(closed,
open,
%
open
entries)
loaded
strongly
on
the
same
factor
with
performance
on
the
individual
learning
tasks.
The
low
magnitude
and
inconsistent
loading
of
other
measures
of
sensory/motor
performance
suggest
that
they
had
little
value
in
explaining
performance
on
this
fac-
tor.
This
later
result
indicates
that
sensory/motor
impairments
that
may
have
been
induced
by
the
colonization
procedure
(from
which
the
imposed
dominant
and
submissive
phenotypes
emerged)
had
little
value
in
explaining
the
learning
deficits
that
were
associated
with
this
treatment.
Table
2
An
unrotated
principal
component
factor
analysis
of
exploratory,
sensorimotor,
and
general
learning
abilities
revealed
only
a
primary
factor
explaining
25%
of
the
total
variance.
Factor
1
Open
field:
total
entries
0.68
Open
field:
%
internal
entries 0.46
Open
field:
run
speed
−0.62
Light/Dark
discrimination:
time
in
dark
−0.24
Light/Dark
discrimination:
latency
to
enter
light
−0.46
Balance
beam:
latency
to
fall −0.13
Balance
beam:
distance
travelled
0.16
Screen
Hang:
latency
to
fall
−0.06
Screen
Hang:
#
of
crossings −0.11
Balance
Pole:
latency
to
fall
0.26
Hotplate 0.10
EPM:
closed
entries
0.79
EPM:
open
entries
0.84
EPM:
reentries
0.10
EPM:
total
entries
0.89
EPM:
%
open
entries 0.82
G
Factor
1
−0.29
G
Factor
2
−0.27
Eigenvalue
4.42
%
Variance
Explained
25%
Lastly,
since
we
observed
group
differences
in
factor
scores
(indicative
of
general
learning
ability),
we
examined
how
individ-
ual
social
behaviors
were
related
to
general
learning
scores.
A
final
unrotated
principal
component
factor
analysis
of
general
learning
abilities
and
measures
from
the
dominance/social
behaviors
pro-
duced
a
factor
which
accounted
for
a
total
of
26%
of
the
variance
in
performance
(eigenvalue
=
2.11,
Table
3).
Behavioral
measures
linked
with
submission
(e.g.,
defensive
rears
and
huddling)
loaded
in
the
same
direction
as
general
learning
scores,
which
validates
the
conclusion
that
imposed
subordination
(but
not
the
predispo-
sition
toward
subordination)
is
associated
with
impaired
learning
abilities.
3.6.
Stress-induced
CORT
responses
A
between-groups
ANOVA
revealed
a
trend
toward
group
differences
in
the
stress-induced
CORT
elevation
of
the
four
groups
of
animals
[F(3,26)
=
1.45,
p
=
.25],
and
a
planned
comparison
post
hoc
test
confirmed
that
imposed
subordinates
(IMP
SUBS)
had

Author's personal copy
302 D.
Colas-Zelin
et
al.
/
Behavioural
Brain
Research
232 (2012) 294–
305
Table
3
An
unrotated
principal
component
factor
analysis
of
social
behaviors
and
general
learning
abilities
revealed
a
primary
and
secondary
factor
explaining
26%
and
18%
of
the
total
variance,
respectively.
Factor
1
Factor
2
G
Factor
1 0.40
0.15
G
Factor
2
0.52
0.27
Huddling
0.57
−0.10
Defensive
rears
0.12
0.85
Sniffing
−0.42
0.001
Bites
made −0.80
−0.23
Bites
received −0.67
0.37
Latency
to
attack −0.29
0.64
Eigenvalue
2.11
1.42
%
Variance
explained
26%
18%
significantly
lower
CORT
levels
than
innate
subordinates
[INN
SUBS;
p
<
.05]
(Fig.
6).
A
t-test
for
independent
samples
between
all
subjects
in
the
IMP
groups
and
subjects
in
the
INN
groups
was
sig-
nificant
[t(1,28)
=
2.08,
p
<
.05],
i.e.,
the
INN
groups
(those
who
were
made
subordinate
after
learning
was
assessed)
had
higher
levels
of
plasma
CORT
than
subjects
in
the
IMP
groups.
A
t-test
for
indepen-
dent
samples
between
all
subordinate
and
all
dominant
subjects
(regardless
of
status)
was
non-significant
[t(1,28)
=
.46,
n.s.].
Thus,
the
experience
of
triadic
colonization
leads
to
an
increase
in
stress
reactivity
(e.g.,
stress-induced
CORT
elevation
in
response
to
a
mild
stressor)
that
persists
for
at
least
one
month
after
removal
from
the
social
environment
(Group
INN),
but
appears
to
dissipate
within
three
months
(Group
IMP).
In
neither
case
was
the
stress-induced
CORT
elevation
sufficient
to
account
for
the
learning
impairments
associated
with
the
submissive
phenotype
(see
below).
In
this
regard
it
is
notable
that
although
colonization
(regardless
of
social
stature)
was
associated
with
increased
stress
reactivity
(and
resul-
tant
CORT
elevation),
only
animals
that
were
deemed
submissive
in
the
colonized
environment
exhibited
learning
impairments.
A
principal
components
analysis,
which
included
stress-induced
CORT
and
measures
of
dominance/submission
produced
a
factor
that
accounted
for
52%
of
the
variance
(eigenvalue
=
4.64,
Table
4).
Consistent
with
the
discussion
above,
analysis
of
the
structure
of
this
factor
suggests
that
dominance
measures
positive
for
agonis-
tic
involvement
(e.g.,
sniffing,
bites
made,
bites
received,
defensive
INN SUBSINN DOMSIMP SUBSIMP DOMS
CORT (ng/ml) in Response to Mild Stress
0
10
20
30
40
50
60
70
*
Fig.
6.
CORT
levels
(ng/ml)
for
the
IMP
DOMS,
IMP
SUBS,
INN
DOM,
and
INN
SUBS
groups.
Animals
were
confined
to
an
elevated
platform
in
a
bright
room
for
five
minutes
(which
prior
work
indicated
induced
a
moderate
stress
response)
either
124
days
(Groups
IMP
DOM
and
IMP
SUB)
or
32
days
after
colonization.
Regardless
of
phenotype,
colonization
within
approximately
one
month
of
the
CORT
assay
pro-
moted
an
increased
CORT
response
to
mild
stress,
which
had
dissipated
within
four
months.
Asterisks
(*)
indicate
significant
comparisons.
Table
4
An
unrotated
principal
component
factor
analysis
of
social
behaviors
and
CORT
lev-
els
revealed
a
primary
and
secondary
factor
explaining
52%
and
15%
of
the
total
variance,
respectively.
Dominance
Factor
1
Dominance
Factor
2
Huddling −0.31
0.06
Sniffing
0.42
−0.33
Bites
made
0.93
−0.02
Latency
−0.03
−0.79
Bites
received
0.10
−0.11
Defensive
rears 0.82
−0.33
Tail
rattling 0.87
0.29
Wounding 0.95
−0.05
CORT
0.51
0.62
Eigenvalue
4.64
1.33
%
Variance
explained
52%
15%
Table
5
An
unrotated
principal
component
factor
analysis
of
exploratory
and
sensorimotor
behaviors
and
CORT
levels
reveals
a
primary
and
secondary
factor
explaining
27%
and
15%
of
the
total
variance,
respectively.
Factor
1
Factor
2
Open
field:
total
entries
0.65
−0.16
Open
field:
%
internal
entries
0.42
−0.14
Open
field:
run
speed −0.58
−0.09
Light/Dark
discrimination:
time
in
dark
−0.18
0.36
Light/Dark
discrimination:
latency
to
enter
light −0.45
0.01
Balance
beam:
latency
to
fall
−0.21
0.64
Balance
beam:
distance
travelled
0.05
0.82
Screen
Hang:
latency
to
fall
−0.07
0.78
Screen
Hang:
#
of
crossings
−0.12
0.76
Balance
Pole:
latency
to
fall 0.33
0.03
Hotplate
0.12
−0.03
EPM:
closed
entries 0.80
0.06
EPM:
open
entries
0.86
0.20
EPM:
reentries
0.11
0.09
EPM:
total
entries 0.91
0.14
EPM:
%
open
entries
0.82
0.20
CORT 0.54
−0.18
Eigenvalue
4.53
2.60
%
Variance
Explained
27%
15%
rears,
tail
rattling,
wounding)
were
highly
related
to
stress-induced
CORT
levels.
One
could
conclude
that
the
experience
of
taking
part
in
an
agonistic
encounter,
regardless
of
whether
an
animal
was
sub-
ordinate
or
dominant,
is
related
to
stress-induced
CORT
reactivity.
A
final
principal
components
analysis
that
included
stress-
induced
CORT
and
sensorimotor
measures
produced
a
factor
that
accounted
for
27%
of
the
variance
(eigenvalue
=
4.53,
Table
5).
Open
field
(total
and
%
internal
entries)
and
elevated
plus
maze
(closed,
open,
%
open
entries)
measures
loaded
in
the
same
direction
of
stress-induced
CORT
levels,
a
pattern
of
results
that
we
have
pre-
viously
observed
[80].
4.
Discussion
Our
goal
here
was
to
assess
and
expand
upon
previous
reports
that
describe
the
effects
of
social
subordination
on
cognition,
and
to
determine
if
those
effects
reflected
the
impact
of
subordination
per
se,
or
whether
they
were
co-expressed
with
the
predisposi-
tion
to
adopt
a
subordinate
status.
To
that
end,
we
established
a
triadic
colony
model
(in
which
animals
adopted
dominant
or
sub-
missive
statures)
and
examined
the
impact
of
this
type
of
social
stress
on
both
domain-specific
and
domain-general
learning
pro-
cesses.
We
found
that
imposed,
but
not
innate
tendencies
toward
subordination
were
associated
with
significant
impairments
on
three
of
five
tests
of
learning,
although
other
tests
of
learning
were
statistically,
if
not
nominally,
spared.
However,
general
learning
abilities
(which
are
more
sensitive
to
overall
patterns
of
learning

Author's personal copy
D.
Colas-Zelin
et
al.
/
Behavioural
Brain
Research
232 (2012) 294–
305 303
performance)
were
severely
impaired
in
subjects
exposed
to
social
subordination
prior
to
the
learning
experience.
Importantly,
it
was
also
observed
that
the
tendency
to
adopt
a
subordinate
stature,
absent
the
actual
imposition
of
subordination,
was
not
in
itself
a
predisposing
factor
toward
learning
impairments.
Rather,
innately
subordinate
animals
upon
which
subordination
is
actually
imposed
appear
to
develop
impairments
in
general
learning
abilities.
This
later
conclusion
should
be
qualified
in
that
the
tendency
to
adopt
a
submissive
stature
may
have
been
dependent
on
the
composition
of
a
particular
triad
of
animals,
and
thus
may
not
be
an
absolute
(as
opposed
to
relative)
reflection
of
an
innate
phenotype.
That
is,
an
animal
that
adopted
a
submissive
stature
in
one
triad
may
have
been
dominant
in
another.
However,
the
nature
of
random
sam-
pling
makes
it
reasonable
to
assume
that
groups
of
animals
selected
from
those
that
adopted
a
submissive
stature
within
a
triad
are,
in
general,
representative
of
a
submissive
phenotype.
Nevertheless,
in
this
specific
case,
stature
of
any
given
animal
can
be
judged
relative
to
only
two
other
individuals.
In
addition
to
its
effect
on
cognitive
performance,
we
found
that
the
imposition
of
subordination
results
in
a
decrease
in
exploratory
tendencies
(in
the
open
field,
elevated
plus
maze,
and
the
step-down
avoidance
task).
It
is
often
difficult
to
dis-
tinguish
impairments
in
learning
from
impairments
that
reflect
differences
in
the
performance
of
learned
responses,
and
it
is
pos-
sible
that
that
the
imposition
of
subordination
induces
deficits
in
both
learning
and
performance.
Thus
we
must
fully
consider
whether
submission-induced
impairments
in
our
tests
of
learning
reflect
modifications
of
underlying
learning
processes
or
whether
they
are
the
consequence
of
variations
in
exploratory
tendencies
(which
might
indirectly
impact
learning
or
its
expression).
In
con-
sidering
performance
in
the
step-down
avoidance
task,
learning
was
assessed
with
a
ratio
of
pre-training
to
post-training
step-
down
latencies,
thus
normalizing
for
pre-existing
differences
in
the
tendency
to
step
off
the
safe
platform.
Despite
this
normalization
for
differences
in
baseline
step-down
latencies,
the
imposition
of
subordination
was
associated
with
impaired
performance
indica-
tive
of
learning.
The
inability
to
properly
form
associations
between
one’s
actions
and
negative
consequences
could
prove
detrimen-
tal
and
result
in
further
adverse
consequences
(including
further
reductions
in
exploration).
Thus
at
least
based
on
this
analysis,
it
does
not
appear
that
a
reduction
in
exploration
was
itself
sufficient
to
account
for
the
learning
deficit.
Consistent
with
this
conclusion,
we
have
previously
reported
that
directly
increasing
exploration,
either
through
repeated
exposure
to
novel
environments
[87]
or
through
the
pharmacological
manipulation
of
stress
reactivity
[86],
had
no
beneficial
impact
on
general
learning
abilities.
In
total,
these
results
suggest
that
the
impairment
of
general
learning
abilities
by
the
imposition
of
subordination
is
not
attributable
to
the
effects
of
this
treatment
on
exploratory
behaviors
[also
see
88],
although
it
is
entirely
possible
(if
not
likely)
that
these
phenotypic
tendencies
interact
in
ways
that
we
have
not
detected.
Similarly,
we
found
no
evidence
that
physical
injuries
that
accompanied
the
imposi-
tion
of
subordination
contributed
to
the
observed
learning
deficits,
i.e.,
sensory/motor
performance
accounted
for
little
of
the
variabil-
ity
in
learning.
This
is
not
surprising,
since
the
tests
of
learning
were
administered
more
than
30
days
after
the
termination
of
the
colonization
procedure,
allowing
sufficient
time
for
wound
heal-
ing
and
recovery
from
other
physical
injuries.
Nevertheless,
it
is
acknowledged
that
animals
that
adopted
a
submissive
stature
dur-
ing
colonization
were
exposed
to
rather
extreme
treatment
by
their
dominant
cohorts.
At
this
point
it
is
unknown
whether
a
less
severe
treatment
would
result
in
a
similar
impairment
of
general
cognitive
performance.
In
the
current
study,
we
also
found
that
that
stress-induced
CORT
levels
(i.e.,
stress
reactivity)
had
little
predictive
value
in
explaining
variations
in
general
learning
abilities,
suggesting
that
differences
in
individual
stress
reactivity
do
not
underlie
(under
the
present
conditions)
variations
in
general
learning
performance.
Similarly,
in
an
earlier
study
in
our
laboratory,
we
were
able
to
pharmacologically
disassociate
stress
reactivity
from
general
learn-
ing
abilities
[86].
Together,
this
data
suggests
that
the
relationship
between
general
learning
and
exploration
is
not
necessarily
medi-
ated
by
stress
reactivity.
To
more
fully
assess
the
relationship
between
CORT
and
learning
abilities
in
socially
stressed
animals,
we
would
need
to
quantify
circulating
CORT
levels
after
coloniza-
tion
but
prior
to
performance
in
learning
tasks
and
determine
if
these
measures
were
correlated,
and
if
so,
whether
the
modulation
of
glucocorticoids
contributes
to
the
decrements
in
general
learning
abilities
induced
by
the
imposition
of
subordination.
While
it
is
unlikely
that
stress
reactivity
(i.e.,
stress-induced
CORT
elevations)
can
broadly
explain
variations
in
general
learn-
ing
abilities,
it
is
still
possible
that
that
increases
in
glucocorticoid
expression
in
response
to
subordination
contribute
to
the
changes
in
general
learning
abilities
seen
in
this
study.
Indeed
structural
and
functional
changes
in
the
hippocampus
have
been
reported
subse-
quent
to
social
stress
[22,27,89,90].
However,
since
both
imposed
domination
and
imposed
submission
were
associated
with
ele-
vated
CORT
levels
(one,
but
not
four
months
after
colonization),
but
only
imposed
subordination
was
associated
with
impaired
general
learning
abilities,
it
appears
that
stress-induced
CORT
elevations
(at
the
low
levels
reported
here)
cannot
in
themselves
account
for
the
cognitive
deficits
that
arise
as
a
consequence
of
imposed
subordination.
It
is
possible
that
subordination
may
impact
learning
pro-
cesses
via
neurolophysiological
mechanisms
that
are
independent
of
corticosterone.
For
example,
it
has
been
observed
that
social
stress
restricts
dendritic
arborization
[91,92]
and
neurogenesis
[27,93,94].
Interestingly,
stress-related
effects
on
dendritric
atro-
phy
and
neurogenesis
can
be
prevented
by
pre-treatment
with
the
drug
tianeptine,
a
selective
serotonin
reuptake-enhancer
(SSRE)
[92].
If
the
stress
effects
on
hippocampal
morphology
and
spa-
tial
learning
are
prevented
by
treatment
with
SSREs,
it
is
plausible
that
other
stress-related
cognitive
deficits,
including
its
adverse
effects
on
general
learning
abilities
and
exploration
described
here,
could
be
alleviated
by
similar
pharmacological
treatments.
However,
in
the
social
environments
of
humans
(e.g.,
schools),
it
may
be
impractical
(if
not
ill-advised)
to
attempt
to
moderate
the
effect
of
imposed
submission
(e.g.,
bullying)
with
medication.
Thus,
alternative
treatments
must
be
explored.
Given
that
the
ten-
dency
toward
submission
is
not
itself
sufficient
to
promote
learning
deficits,
an
effective
strategy
might
be
to
implement
more
active
behavioral
intervention
to
eliminate
or
limit
exposure
to
subor-
dination
stress.
The
introduction
of
“anti-bullying”
programs
may
serve
to
reduce
the
occurrences
of
social
subordination
and
thereby
decrease
social
stress
and
ameliorate
its
negative
consequences
on
cognitive
abilities.
Cognitive
therapies
may
also
be
useful
in
counteracting
the
negative
effects
of
submission.
In
humans,
work-
ing
memory
training
has
been
shown
to
improve
cognition.
We
have
recently
developed
a
working
memory
training
regimen
in
mice
that
has
been
shown
to
successfully
increase
both
selective
attention
and
general
learning
performance
[35,95].
Thus
it
is
con-
ceivable
that
working
memory
training
administered
either
before
or
after
social
stress
might
prevent
or
alleviate
the
negative
effects
on
learning
which
result
from
subordination.
5.
Conclusions
The
present
results
underscore
the
detrimental
consequences
of
social
stress
on
cognition,
and
furthermore,
indicate
that
a
propensity
toward
submission
does
not
in
itself
(absent
the
impo-
sition
of
physical
defeat),
detrimentally
impact
cognitive
abilities.

Author's personal copy
304 D.
Colas-Zelin
et
al.
/
Behavioural
Brain
Research
232 (2012) 294–
305
The
social
stress
model
that
was
established
here
is
well
suited
to
probe
the
biological
basis
of
the
social
stress-related
learning
deficits
that
are
conserved
across
mammalian
species.
Further,
this
model
can
be
utilized
to
examine
how
potential
pharmacological
and
behavioral
interventions
may
be
instituted
in
order
to
improve
the
quality
of
life
for
individuals
who
suffer
due
to
the
stress
of
social
subordination.
Acknowledgements
This
work
was
supported
by
grants
from
the
National
Institute
of
Aging
(R01
AG029289)
and
the
Busch
Foundation
to
LDM.
References
[1] Shors
TJ.
Stressful
experience
and
learning
across
the
lifespan.
Annual
Review
of
Psychology
2006;57:55–85.
[2] Roozendaal
B.
Stress
and
memory:
opposing
effects
of
glucocorticoids
on
mem-
ory
consolidation
and
memory
retrieval.
Neurobiology
of
Learning
and
Memory
2002;78:578–95.
[3]
Roozendaal
B.
Systems
mediating
acute
glucocorticoid
effects
on
memory
con-
solidation
and
retrieval.
Progress
in
Neuro-Psychopharmacology
and
Biological
Psychiatry
2003;27(8):1213–23.
[4]
Joëls
M,
Pu
Z,
Wiegert
O,
Oitzl
MS,
Krugers
HJ.
Learning
under
stress:
how
does
it
work?
Trends
in
Cognitive
Sciences
2006;10(4):152–8.
[5] de
Kloet
ER,
Oitzl
MS,
Joëls
M.
Stress
and
cognition:
are
corticosteroids
good
or
bad
guys?
Trends
in
Neurosciences
1999;22(10):422–6.
[6]
Lupien
SJ,
McEwen
BS,
Gunnar
MR,
Heim
C.
Effects
of
stress
throughout
the
lifespan
on
the
brain,
behaviour
and
cognition.
Nature
Reviews
Neuroscience
2009;10(6):434–45.
[7]
McEwen
BS,
Sapolsky
RM.
Stress
and
cognitive
function.
Current
Opinion
in
Neurobiology
1995;5(2):5–16.
[8]
Baumeister
RF,
Twenge
JM,
Nuss
CK.
Effects
of
social
exclusion
on
cognitive
processes:
anticipated
aloneness
reduces
intelligent
thought.
Journal
of
Per-
sonality
and
Social
Psychology
2002;83(4):817–27.
[9]
Johnson
GM.
Student
alienation,
academic
achievement
and
WebCT
use.
Edu-
cational
Technology
&
Society
2005;8(2):179–89.
[10] Schmader
T,
Johns
M,
Forbes
C.
An
integrated
process
model
of
stereotype
threat
effects
on
performance.
Psychological
Review
2010;115(2):336–56.
[11]
Hoover
H,
Oliver
R,
Hazler
RJ.
Bullying:
perceptions
of
adolescent
victims
in
the
Midwestern
USA.
School
Psychology
International
1992;13(1):5–16.
[12] Olweus
D.
Aggressors
and
their
victims:
bullying
at
school.
In:
Disruptive
behaviours
in
schools.
New
York:
Wiley;
1984.
[13]
Knox
E,
Conti-Ramsden
G.
Bullying
risks
of
11-year-old
children
with
spe-
cific
language
impairment
(SLI):
does
school
placement
matter?
International
Journal
of
Language
and
Communication
Disorders
2003;38(1):1–12.
[14] Perry
DG,
Kusel
SJ,
Perry
LC.
Victims
of
peer
aggression.
Developmental
Psy-
chology
1988;24(6):807–14.
[15]
Horwood
J,
Salvi
G,
Thomas
K,
Duffy
L,
Gunnell
D,
Hollis
C,
et
al.
IQ
and
non-
clinical
psychotic
symptoms
in
12-year-olds:
results
from
the
ALSPAC
birth
Cohort.
The
British
Journal
of
Psychiatry
2008;193:185–91.
[16]
Alzoubi
KH,
Abdul-Razzak
KK,
Khabour
OF,
Al-Tuweiq
GM,
Alzubi
MA,
Alkadhi
KA.
Adverse
effect
of
combination
of
chronic
psychosocial
stress
and
high
fat
diet
on
hippocampus-dependent
memory
in
rats.
Behavioural
Brain
Research
2009;204(1):117–23.
[17]
Fitchett
AE,
Collins
SA,
Barnard
CJ,
Cassaday
HJ.
Subordinate
male
mice
show
long-lasting
differences
in
spatial
learning
that
persist
when
housed
alone.
Neurobiology
of
Learning
and
Memory
2005;84(3):247–51.
[18]
Krugers
HJ,
Douma
BR,
Andringa
G,
Bohus
B,
Korf
J,
Luiten
PG.
Exposure
to
chronic
psychosocial
stress
and
corticosterone
in
the
rat:
effects
on
spatial
discrimination
learning
and
hippocampal
protein
kinase
Cgamma
immunore-
activity.
Hippocampus
1997;7(4):427–36.
[19]
Ohl
F,
Fuchs
E.
Memory
performance
in
tree
shrews:
effects
of
stressful
expe-
riences.
Neuroscience
and
Biobehavioral
Reviews
1998;23(2):319–23.
[20]
Ohl
F,
Fuchs
E.
Differential
effects
of
chronic
stress
on
memory
processes
in
the
tree
shrew.
Cognitive
Brain
Research
1999;7:1379–87.
[21]
Kvist
B.
Learning
in
mice
selectively
bred
for
high
and
low
aggressiveness.
Psychological
Reports
1989;64(1):127–30.
[22]
Bodnoff
SR,
Humphreys
AG,
Lehman
JC,
Diamond
DM,
Rose
GM,
Meaney
MJ.
Enduring
effects
of
chronic
corticosterone
treatment
on
spatial
learning,
synap-
tic
plasticity,
and
hippocampal
neuropathology
in
young
and
mid-aged
rats.
Journal
of
Neuroscience
1995;15(1):61–9.
[23]
Touyarot
K,
Venero
C,
Sandi
C.
Spatial
learning
impairment
induced
by
chronic
stress
is
related
to
individual
differences
in
novelty
reactivity:
search
for
neu-
robiological
correlates.
Psychoneuroendocrinology
2004;29(2):290–305.
[24]
Francia
N,
Cirulli
F,
Chiarotti
F,
Antonelli
A,
Aloe
L,
Alleva
E.
Spatial
memory
deficits
in
middle-aged
mice
correlate
with
lower
exploratory
activity
and
a
subordinate
status:
role
of
hippocampal
neurotrophins?
European
Journal
of
Neuroscience
2006;23(3):711–28.
[25]
Barnard
CJ,
Luo
N.
Acquisition
of
dominance
status
affects
maze
learning
in
mice.
Behavioural
Processes
2002;60(1):53–9.
[26]
Fitchett
AE,
Barnard
CJ,
Cassaday
HJ.
Corticosterone
differences
rather
than
social
housing
predict
performance
of
T-maze
alternation
in
male
CD-1
mice.
Animal
Welfare
2009;18(1):21–31.
[27]
Buwalda
B,
Kole
MH,
Veenema
AH,
Huininga
M,
de
Boer
SF,
Korte
SM,
et
al.
Long-term
effects
of
social
stress
on
brain
and
behavior:
a
focus
on
hip-
pocampal
functioning.
Neuroscience
and
Biobehavioral
Reviews
2005;29(1):
83–97.
[28] Bartolomucci
A,
de
Biurrun
G,
Czéh
B,
van
Kampen
M,
Fuchs
E.
Selective
enhancement
of
spatial
learning
under
chronic
psychosocial
stress.
European
Journal
of
Neuroscience
2002;15(11):1863–6.
[29]
Moragrega
I,
Carrasco
MC,
Vicens
P,
Redolat
R.
Spatial
learning
in
male
mice
with
different
levels
of
aggressiveness:
effects
of
housing
conditions
and
nico-
tine
administration.
Behavioural
Brain
Research
2003;147(1–2):1–8.
[30] Spritzer
MD,
Meikle
DB,
Solomon
NG.
The
relationship
between
dominance
rank
and
spatial
ability
among
male
meadow
voles
(Microtus
pennsylvanicus).
Journal
of
Comparative
Psychology
2004;118(3):332–9.
[31]
Kolata
S,
Light
K,
Matzel
LD.
Domain
specific
and
domain
general
learning
factors
are
expressed
in
genetically
heterogeneous
CD-1
mice.
Intelligence
2008;36(6):619–29.
[32] Plomin
R.
Genetics
and
general
cognitive
ability.
Nature
1999;402:C25–9.
[33] Kolata
S,
Light
K,
Grossman
HC,
Hale
G,
Matzel
LD.
Selective
attention
is
a
pri-
mary
determinant
of
the
relationship
between
working
memory
and
general
learning
ability
in
outbred
mice.
Learning
and
Memory
2007;14(1):22–8.
[34]
Kolata
S,
Light
K,
Townsend
DA,
Hale
G,
Grossman
HC,
Matzel
LD.
Neurobiology
of
Learning
and
Memory
2005;84(3):241–6.
[35]
Light
KR,
Kolata
S,
Wass
C,
Denman-Brice
A,
Zagalsky
R,
Matzel
LD.
Working
memory
training
promotes
general
cognitive
abilities
in
genetically
heteroge-
neous
mice.
Current
Biology
2010;20(8):777–82.
[36]
Kolata
S,
Light
K,
Wass
CD,
Colas-Zelin
D,
Roy
D,
Matzel
LD.
A
dopaminergic
gene
cluster
in
the
prefrontal
cortex
predicts
performance
indicative
of
general
intelligence
in
genetically
heterogeneous
mice.
PLoS
ONE
2010;5(November
(11)):e14036.
[37]
Matzel
LD,
Kolata
S.
Selective
attention,
working
memory,
and
animal
intelli-
gence.
Neuroscience
and
Biobehavioral
Reviews
2010;34(January
(1)):23–30.
[38]
Hardy
MP,
Sottas
CM,
Ge
R,
McKittrick
CR,
Tamashiro
KL,
McEwen
BS,
et
al.
Trends
of
reproductive
hormones
in
male
rats
during
psychosocial
stress:
role
of
glucocorticoid
metabolism
in
behavioral
dominance.
Biology
of
Reproduc-
tion
2002;67(6):1750–5.
[39]
Monder
C,
Sakai
RR,
Miroff
Y,
Blanchard
DC,
Blanchard
RJ.
Reciprocal
changes
in
plasma
corticosterone
and
testosterone
in
stressed
male
rats
maintained
in
a
visible
burrow
system:
evidence
for
a
mediating
role
of
testicular
11
beta-
hydroxysteroid
dehydrogenase.
Endocrinology
1994;134(3):1193–8.
[40]
Dong
Q,
Salva
A,
Sottas
CM,
Niu
E,
Holmes
M,
Hardy
MP.
Rapid
glucocorticoid
mediation
of
suppressed
testosterone
biosynthesis
in
male
mice
subjected
to
immobilization
stress.
Journal
of
Andrology
2004;25(6):973–81.
[41]
Blanchard
DC,
Sakai
RR,
McEwen
B,
Weiss
SM,
Blanchard
RJ.
Subordination
stress:
behavioral,
brain,
and
neuroendocrine
correlates.
Behavioural
Brain
Research
1993;58(1–2):113–21.
[42] Blanchard
DC,
Spencer
RL,
Weiss
SM,
Blanchard
RJ,
McEwen
B,
Sakai
RR.
Visible
burrow
system
as
a
model
of
chronic
social
stress:
behavioral
and
neuroen-
docrine
correlates.
Psychoneuroendocrinology
1995;20(2):117–34.
[43] Veenema
AH,
Meijer
OC,
de
Kloet
ER,
Koolhaas
JM,
Bohus
BG.
Differences
in
basal
and
stress-induced
HPA
regulation
of
wild
house
mice
selected
for
high
and
low
aggression.
Hormones
and
Behavior
2003;43(1):197–204.
[44]
von
Holst
D.
Social
stress
in
tree
shrews:
problems,
results
and
goals.
Journal
of
Comparative
Physiology
1977;120:71–86.
[45]
Nock
BL,
Leshner
AI.
Hormonal
mediation
of
the
effects
of
defeat
on
agonistic
responding
in
mice.
Physiology
and
Behavior
1976;17(1):111–9.
[46]
Coe
C,
Mendoza
S,
Levine
S.
Social
stastus
constrains
the
stress
response
in
the
squirrel
monkey.
Physiology
and
Behavior
1979;23(4):633–8.
[47]
Sapolsky
RM.
Individual
differences
in
cortisol
secretory
patterns
in
the
wild
baboon:
role
of
negative
feedback
sensitivity.
Endocrinology
1983;113(6):2263–7.
[48] Bronson
FH.
Establishment
of
social
rank
among
grouped
male
mice:
relative
effects
on
circulating
FSH,
LH,
and
corticosterone.
Physiology
and
Behavior
1973;10(5):947–51.
[49]
Ely
DL,
Henry
JP.
Neuroendocrine
response
patterns
in
dominant
and
subordi-
nate
mice?
Hormones
and
Behavior
1978;10(2):156–69.
[50]
Wirth
MM,
Welsh
KM,
Schultheiss
OC.
Salivary
cortisol
changes
in
humans
after
winning
or
losing
a
dominance
contest
depend
on
implicit
power
motivation.
Hormones
and
Behavior
2006;49(3):346–52.
[51]
Blanchard
DC,
McKittrick
CR,
Hardy
MP,
Blanchard
RJ.
Effects
of
social
stress
on
hormones,
brain
and
behavior.
In:
Pfaff
DW,
Arnold
AP,
Etgen
AM,
Fahrbach
SE,
Rubin
RT,
editors.
Hormones,
brain
and
behavior,
vol.
1.
San
Diego:
Academic
Press;
2002.
p.
735–72.
[52]
Ruis
MAW,
te
Brake
JHA,
Buwalda
B,
de
Boer
SF,
Meerlo
P,
Korte
SM,
et
al.
Housing
familiar
male
wildtype
rats
together
reduces
the
long-term
adverse
behavioural
and
physiological
effects
of
social
defeat.
Psychoneuroendocrinol-
ogy
1999;24:285–300.
[53]
Menzaghi
F,
Heinrichs
SC,
Vargas-Cortes
M,
Goldstein
G,
Koob
GF.
IRI-514,
a
synthetic
peptide
analogue
of
thymopentin,
reduces
the
behavioral
response
to
social
stress
in
rats.
Physiology
and
Behavior
1996;60(2),
397–401(5).
[54]
Heinrichs
SC,
Menzaghi
F,
Pich
EM,
Baldwin
HA,
Rassnick
S,
Britton
KT,
et
al.
Anti-stress
action
of
a
corticotropin-releasing
factor
antagonist
on
behavioral
reactivity
to
stressors
of
varying
type
and
intensity.
Neuropsychopharmacology
1994;111(3):179–86.

Author's personal copy
D.
Colas-Zelin
et
al.
/
Behavioural
Brain
Research
232 (2012) 294–
305 305
[55]
Heinrichs
SC,
Pich
EM,
Miczek
KA,
Britton
KT,
Koob
GF.
Corticotropin-releasing
factor
antagonist
reduces
emotionality
in
socially
defeated
rats
via
direct
neu-
rotropic
action.
Brain
Research
1992;581(2):190–7.
[56]
Berton
O,
Aguerre
S,
Sarrieau
A,
Mormede
P,
Chaouloff
F.
Differential
effects
of
social
stress
on
central
serotonergic
activity
and
emotional
reactivity
in
Lewis
and
spontaneously
hypertensive
rats?
Neuroscience
1998;82(1):147–59.
[57]
Berton
O,
Durand
M,
Aguerre
S,
Mormède
P,
Chaouloff
F.
Behavioral,
neu-
roendocrine
and
serotonergic
consequences
of
single
social
defeat
and
repeated
fluoxetine
pretreatment
in
the
Lewis
rat
strain.
Neuroscience
1999;92(1):327–41.
[58]
Avgustinovich
DF,
Gorbach
OV,
Kudryavtseva
NN.
Comparative
analysis
of
anxiety-like
behavior
in
partition
and
plus-maze
tests
after
agonistic
inter-
actions
in
mice.
Physiology
and
Behavior
1997;61(1):37–43.
[59] Rodgers
RJ,
Cole
JC.
Anxiety
enhancement
in
the
murine
elevated
plus
maze
by
immediate
prior
exposure
to
social
stressors.
Physiology
and
Behavior
1993;53(2):383–8.
[60]
Haller
J,
Halász
J.
Mild
social
stress
abolishes
the
effects
of
isolation
on
anxiety
and
chlordiazepoxide
reactivity.
Psychopharmacology
1999;144(4):311–5.
[61]
Peres
RC,
Leite
JR.
The
influence
of
competitive
status
(winner/loser)
on
the
behavior
of
male
rats
in
three
models
of
anxiety.
Aggressive
Behavior
2002;28:164–71.
[62] Kudryavtseva
NN,
Bakshtanovskaya
IV,
Koryakina
LA.
Social
model
of
depres-
sion
in
mice
of
C57BL/6J
strain.
Pharmacology
Biochemistry
and
Behavior
1991;38(2):315–20.
[63]
Kudryavtseva
NN,
Madorskaya
IA,
Bakshtanovskaya
IV.
Social
success
and
voluntary
ethanol
consumption
in
mice
of
C57BL/6J
and
CBA/Lac
strains.
Phys-
iology
and
Behavior
1991;50(1):143–6.
[64] Zook
JM,
Adams
DB.
Competitive
fighting
in
the
rat.
Journal
of
Comparative
and
Physiological
Psychology
1975;88(1):418–23.
[65]
Schneider
R,
Hoffmann
HJ,
Schicknick
H,
Moutier
R.
Genetic
analysis
of
isolation-induced
aggression.
I.
Comparison
between
closely
related
inbred
mouse
strains.
Behavioral
and
Neural
Biology
1992;57:198–204.
[66]
Grant
EC,
Mackintosh
JH.
A
comparison
of
the
social
postures
of
some
common
laboratory
rodents.
Behaviour
1963;21(3–4):246–59.
[67]
Clark
LH,
Schein
MW.
Activities
associated
with
conflict
behavior
in
mice.
Ani-
mal
Behaviour
1966;14(1):44–9.
[68]
Burg
RD,
Slotnick
BM.
Response
of
colony
mice
to
intruders
with
different
fighting
experience.
Aggressive
Behavior
1983;9(1):49–58.
[69]
Miczek
KA,
Maxson
SC,
Fish
EW,
Faccidomo
S.
Aggressive
behavioral
pheno-
types
in
mice.
Behavioural
Brain
Research
2001;125(1–2):167–81.
[70] Ginsburg
B,
Allee
WC.
Some
effects
of
conditioning
on
social
dominance
and
subordination
in
inbred
strains
of
mice.
Physiological
Zoology
1942;15(4):
485–550.
[71] Miczek
KA,
O’Donnell
JM.
Intruder-evoked
aggression
in
isolated
and
nonisolated
mice:
effects
of
psychomotor
stimulants
and
L-dopa.
Psychophar-
macology
1978;57(1):47–55.
[72]
Miczek
KA,
Thompson
ML,
Shuster
L.
Opioid-like
analgesia
in
defeated
mice.
Science
1982;215(4539):1520–2.
[73] Miczek
KA.
Aggressive
and
social
stress
responses
in
genetically
modified
mice:
from
horizontal
to
vertical
strategy.
Psychopharmacology
1999;147(1):
17–9.
[74] Blanchard
RJ,
Takahashi
LK,
Fukunaga
KK,
Blanchard
DC.
Functions
of
the
vib-
rissae
in
the
defensive
and
aggressive
behavior
of
the
rat.
Aggressive
Behavior
1977;3:231–40.
[75]
Blanchard
DC,
Blanchard
RJ,
Lee
EM,
Nakamura
S.
Defensive
behaviors
in
rats
following
septal
and
septal–amygdala
lesions.
Journal
of
Comparative
and
Physiological
Psychology
1979;93(April
(2)):378–90.
[76]
Pellis
SM,
Pellis
VC,
Bekoff
Marc,
Byers
John
Alexander,
editors.
Animal
play:
evolutionary,
comparative,
and
ecological
perspectives.
Structure-function
interface
in
the
analysis
of
play
fighting.
New
York,
NY,
USA:
Cambridge
Uni-
versity
Press;
1998.
p.
115–40.
[77]
Sara
SJ,
Roullet
P,
Przybyslawski
J.
Consolidation
of
memory
for
odor-reward
association:
␤-adrenergic
receptor
involvement
in
late
phase.
Learning
and
Memory
1999;6:88–96.
[78]
Morris
RGM.
Pavlovian
conditioned
inhibition
of
fear
during
shuttlebox
avoid-
ance
behavior.
Learning
and
Motivation
1974;5:424–47.
[79]
Bolles
RC.
Avoidance
and
escape
learning:
simultaneous
acquisition
of
different
responses.
Journal
of
Comparative
and
Physiological
Psychology
1969;68(3):355–8.
[80]
Matzel
LD,
Townsend
DA,
Grossman
H,
Han
YR,
Hale
G,
Zappulla
M,
et
al.
Exploration
in
outbred
mice
covaries
with
general
learning
abilities
irrespec-
tive
of
stress
reactivity,
emotionality,
and
physical
attributes.
Neurobiology
of
Learning
and
Memory
2006;86(2):228–40.
[81]
Kolata
S,
Wu
J,
Light
K,
Schachner
M,
Matzel
LD.
Impaired
working
memory
duration
but
normal
learning
abilities
found
in
mice
that
are
con-
ditionally
deficient
in
the
close
homolog
of
L1.
Journal
of
Neuroscience
2008;28(50):13505–10.
[82]
Blanchard
RJ,
Wall
PM,
Blanchard
DC.
Problems
in
the
study
of
rodent
aggres-
sion.
Hormones
and
Behavior
2003;44(3):161–70.
[83]
Benton
D.
Is
the
concept
of
dominance
useful
in
understanding
rodent
behav-
ior?
Aggressive
Behavior
1982;8(2):104–7.
[84]
Uhrich
J.
The
social
hierarchy
in
albino
mice.
Journal
of
Comparative
Psychology
1938;25(2):373–413.
[85]
Brain
PF,
Hui
SE.
Variability
in
patterns
of
intra-specific
biting
attack
in
com-
monly
used
genetic
lines
of
laboratory
mice.
Scandinavian
Journal
of
Laboratory
Animal
Science
2003;3(30):113–27.
[86]
Grossman
HC,
Hale
G,
Light
K,
Kolata
S,
Townsend
DA,
Goldfarb
Y,
et
al.
Pharmacological
modulation
of
stress
reactivity
dissociates
general
learn-
ing
ability
from
the
propensity
for
exploration.
Behavioral
Neuroscience
2007;121(5):949–64.
[87]
Light
KR,
Kolata
S,
Hale
G,
Grossman
H,
Matzel
LD.
Up-regulation
of
exploratory
tendencies
does
not
enhance
general
learning
abilities
in
juvenile
or
young-
adult
outbred
mice.
Neurobiology
of
Learning
and
Memory
2008;90(2):317–29.
[88]
Montgomery
KC.
The
relation
between
fear
induced
by
novel
stimulation
and
exploratory
drive.
Journal
of
Comparative
and
Physiological
Psychology
1955;48(4):192–4.
[89] Whimbey.
AE,
Denenberg
VH.
Two
independent
behavioral
dimensions
in
open
field
performance.
Journal
of
Comparative
and
Physiological
Psychology
1967;63(3):500–4.
[90]
Von
Frijtag
JC,
Kamal
A,
Reijmers
LG,
Schrama
LH,
van
den
Bos
R,
Spruijt
BM.
Chronic
imipramine
treatment
partially
reverses
the
long-term
changes
of
hip-
pocampal
synaptic
plasticity
in
socially
stressed
rats?
Neuroscience
Letters
2001;309(3):153–6.
[91]
Magari˜
nos
AM,
McEwen
BS,
Flügge
G,
Fuchs
E.
Chronic
psychosocial
stress
causes
apical
dendritic
atrophy
of
hippocampal
CA3
pyramidal
neurons
in
subordinate
tree
shrews.
Journal
of
Neuroscience
1996;16(10):3534–40.
[92]
McKittrick
CR,
Magari˜
nos
AM,
Blanchard
DC,
Blanchard
RJ,
McEwen
BS,
Sakai
RR.
Chronic
social
stress
reduces
dendritic
arbors
in
CA3
of
hippocampus
and
decreases
binding
to
serotonin
transporter
sites.
Synapse
2000;36(2):85–94.
[93] Czéh
B,
Michaelis
T,
Watanabe
T,
Frahms
J,
De
Biurrun
G,
Van
Kampen
M,
et
al.
Stress-induced
changes
in
cerebral
metabolites,
hippocampal
volume,
and
cell
proliferation
are
prevented
by
antidepressant
treatment
with
tianeptine.
Pro-
ceedings
of
the
National
Academy
of
Sciences
of
the
United
States
of
America
2001;98(22):12796–801.
[94]
Gould
E,
Tanapat
P,
McEwen
BS,
Flügge
G,
Fuchs
E.
Proliferation
of
granule
cell
precursors
in
the
dentate
gyrus
of
adult
monkeys
is
diminished
by
stress.
Proceedings
of
the
National
Academy
of
Sciences
of
the
United
States
of
America
1998;95(6):3168–71.
[95]
Matzel
LD,
Light
KR,
Wass
C,
Colas-Zelin
D,
Denman-Brice
A,
Waddel
AC,
et
al.
Longitudinal
attentional
engagement
rescues
mice
from
age-related
cogni-
tive
declines
and
cognitive
inflexibility.
Learning
and
Memory
2011;18(April
(5)):345–56.