Moy1_Protocol
Project
protocol
—
Contents
Workflow
and
sampling
Equipment
Reagents,
supplies,
and
solutions
Procedure
for
measuring
social
approach
using
an
automated
3-chambered
test
box
Data
Definitions
Reference
Workflow
and
sampling
Workflow
Step |
Procedure
performed |
Age
(wks) |
Apparatus
|
Time
(min) |
Data
collected |
1 |
Mice
are
brought
from
the
vivarium
to
the
test
room
to
acclimate
before
testing
begins |
6-7 |
- |
30 |
- |
2 |
A
mouse
is
placed
in
the
center
chamber
of
the
social
test
box
and
allowed
to
habituate
to
the
testing
conditions |
6-7 |
social
test
box/
habituation |
10 |
|
3 |
Following
a
period
of
habituation,
the
mouse
is
given
a
choice
between
proximity
to
an
unfamiliar
mouse
or
to
a
novel
object;
the
time
spent
with
the
conspecific
mouse
is
a
measure
of
sociability |
6-7 |
social
test
box/
sociability
test |
10 |
duration
of
chamber
occupancy,
number
of
entries
into
each
chamber,
and
duration
of
sniffing
each
cage
(occupied
or
empty)
|
4 |
After
sociability
test,
the
mouse
is
tested
again
for
preference
for
social
novelty
with
the
addition
of
a
second
conspecific
mouse |
6-7 |
social
test
box/
preference
for
social
novelty
test |
10 |
duration
of
chamber
occupancy,
number
of
entries
into
each
chamber,
and
duration
of
sniffing
each
occupied
cage
|
5 |
After
the
administration
of
sociability
and
social
novelty
preference
tests,
the
testing
chambers
are
cleaned
for
subsequent
testing |
6-7 |
social
behavior
test
box |
- |
- |
Equipment
•
Automated
three-chambered
apparatus
(National
Institutes
of
Mental
Health,
Bethesda,
MD,
USA)
made
of
clear
polycarbonate
for
social
behavior
test
Figure
1:
Social
behavior
test
box,
where
a
mouse
is
given
a
choice
between
staying
in
the
center
chamber,
spending
time
in
the
side
chamber
with
an
unfamiliar
mouse
(stranger
1),
or
spending
time
in
the
side
chamber
with
either
a
novel
object
or
a
newly
introduced
mouse
(stranger
2)
during
social
preference
tests.
Stranger
mice
are
enclosed
in
wire
cages.
- Activity
assessment
system
using
photocells
(National
Institutes
of
Mental
Health,
Bethesda,
MD,
USA)
- Customized
computer
software
developed
by
Dr.
Josephine
M.
Johns,
University
of
North
Carolina,
Chapel
Hill,
NC,
and
Dr.
Larry
W.
Means,
East
Carolina
University,
Greenville,
NC;
used
for
scoring
time
spent
sniffing
each
wire
cage
Reagents,
supplies,
solutions
- Disinfectant:
70%
isopropanol
used
to
clean
the
test
chambers
- Paper
towels
- Water
bottle
for
cleaning
test
chambers
Acclimation
to
test
conditions
Testing
is
conducted
during
the
light
phase
under
fluorescent
laboratory
lighting
(320-340
lx).
Mice
are
transported
from
the
vivarium
into
the
test
room
and
are
allowed
to
acclimate
for
at
least
30
min
and
habituate
to
testing
conditions
for
10
min.
Procedure
for
measuring
social
approach
using
an
automated
3-chambered
test
box
I.
Pre-testing
observations
a.
Following
arrival
to
the
animal
facility,
the
mice
are
subjected
to
a
series
of
observations
and
a
battery
of
behavioral
tests
for
at
least
a
week
(see
Moy
et
al.
2007-8).
b.
Included
in
the
series
of
observations
are
general
health
and
neurological
evaluations
by
a
single
observer
(see
Moy
et
al.
2007-8).
II.
Testing
for
sociability
and
preference
for
social
novelty
using
social
behavior
apparatus
Habituation
a.
An
empty
wire
cage
is
placed
within
each
of
the
side
chamber,
and
a
weighted
cup
is
placed
on
top
of
it
to
prevent
a
mouse
from
climbing
over
the
wire
cage
(see
Figure
1).
b.
With
the
doorways
into
the
two
side
chambers
opened,
the
test
mouse
is
placed
in
the
middle
chamber
and
allowed
to
explore
the
apparatus
for
10
min.
c.
Embedded
photocells
within
each
doorway
entrance
are
activated
to
automatically
track
the
number
of
entries
and
duration
spent
in
each
chamber.
d.
The
sociability
test
is
immediately
conducted
following
the
habituation
phase.
Investigator's
Notes:
"To
confirm
the
absence
of
a
side
preference
bias
for
either
of
the
two
side
chambers
of
the
social
test
box,
measures
were
taken
of
time
spent
in
each
side
during
the
10-min
habituation
period.
None
of
the
strains
showed
a
significant
preference
for
either
the
right
or
left
side
[no
main
effect
of
side;
p
>
0.05,
within
group
repeated
measures
analysis
for
each
strain].
In
addition,
separate
groups
of
C57BL/6J
mice
were
periodically
evaluated
in
the
social
behavior
task,
to
confirm
that
the
environmental
parameters
for
the
assay
had
not
changed
and
normal
tendencies
for
social
approach
could
still
be
observed.
Stranger
mice
were
adult
male
C57BL/6J
(JAX®),
and
were
housed
in
cages
separate
from
and
distant
to
the
cages
housing
the
subject
mice,
to
avoid
visual,
auditory,
and
olfactory
contact.
Strangers
have
no
previous
physical
contact
with
the
subjects,
and
were
kept
in
a
separate
location
from
the
subjects
on
the
day
of
testing.
Several
days
before
the
start
of
social
testing,
the
mice
serving
as
strangers
were
habituated
to
the
wire
cages
in
the
social
apparatus
for
5-10
min
per
day,
for
at
least
5
days.
Each
stranger
was
used
only
once
per
day,
and
the
strangers
for
the
sociability
test
and
the
social
novelty
tests
were
taken
from
separate
cages.
Containing
the
stranger
mouse
in
a
wire
cage
served
the
purpose
of
preventing
aggressive
and
sexual
interactions,
as
well
as
ensuring
that
all
social
approach
was
initiated
only
by
the
subject
mouse.
Previous
experiments
indicated
that
the
strain
of
the
stranger
did
not
change
the
social
approach
of
the
subject.
The
empty
wire
cage
served
as
a
control
for
the
properties
of
the
container,
in
addition
to
serving
as
the
novel
inanimate
object
with
no
social
valence
(see
Moy
et
al.
2007)."
III.
Sociability
test
a.
Shortly
after
the
habituation
period,
the
test
mouse
is
enclosed
in
the
center
compartment
of
the
social
test
box,
and
an
unfamiliar
mouse
(stranger1;
a
C57BL/6J
male)
is
enclosed
in
one
of
the
wire
cages
and
placed
in
one
of
the
side
chambers.
b.
The
location
for
stranger1
alternated
between
the
left
and
right
sides
of
the
social
test
box
across
subjects.
c.
Following
placement
of
stranger
1,
the
doors
are
re-opened,
and
the
mouse
subject
is
again
allowed
to
explore
the
entire
social
test
box
for
another
10-min
session.
d.
Measurements
are
automatically
recorded
of
the
amount
of
time
spent
and
the
number
of
entries
into
each
chamber
by
the
automated
testing
system.
e.
A
human
observer
scored
time
spent
sniffing
each
wire
cage,
using
a
computer
keypad
and
customized
software.
VI.
Preference
for
social
novelty
test
a.
Following
the
sociability
test,
each
mouse
is
again
tested
in
a
third
10-min
session
to
measure
preference
in
spending
time
with
a
new
stranger.
b.
The
test
mouse
is
again
briefly
enclosed
in
the
center
compartment.
c.
A
new
unfamiliar
mouse
(stranger2)
is
placed
in
the
wire
cage
that
had
been
empty
during
the
previous
10-min
session
(sociability
test).
d.
The
test
mouse
is
given
a
choice
between
the
first,
already-investigated,
now-familiar
mouse
(stranger1)
and
the
novel
unfamiliar
mouse
(stranger2).
e.
As
before,
measurements
are
automatically
recorded
of
the
amount
of
time
spent
and
the
number
of
entries
into
each
chamber
by
the
automated
testing
system.
f.
As
above,
a
human
observer
scored
time
spent
sniffing
each
wire
cage
in
both
side
chambers,
using
a
computer
keypad
and
customized
software.
g.
At
the
end
of
each
three-10
min
sessions,
the
chambers
are
cleaned
with
water
and
paper
towels
before
testing
another
mouse
subject.
h.
Each
wire
cage
is
used
only
once
per
day,
and
the
three-chambered
apparatus
is
cleaned
and
disinfected
at
the
end
of
the
day.
Data
collected
by
investigator
Using
an
automated
social
behavior
test
box,
the
following
measurements
were
obtained:
Measurement
description |
Sociability
test |
Preference
for
social
novelty
test
|
Units |
| Duration
of
occupancy
in
center-chamber |
yesa |
yesa |
s |
| Time
spent
in
chamber
with
empty
cage
(a
novel
object) |
yes |
not
applicable |
s |
| Time
spent
in
chamber
with
stranger
1
(a
novel
mouse) |
yesb |
yesb |
s |
| Time
spent
sniffing
empty
cage |
yes |
not
applicable |
s |
| Time
spent
sniffing
cage
with
stranger
1 |
yesc |
yesc |
s |
| Number
of
entries
to
chamber
with
empty
cage |
yes |
not
applicable |
n |
| Number
of
entries
to
chamber
with
stranger
1
in
cage |
yesd |
yesd |
n |
| Time
spent
in
chamber
with
stranger
2
(a
second
novel
mouse) |
not
applicable |
yes |
s |
| Time
spent
sniffing
cage
with
novel
stranger
2 |
not
applicable |
yes |
s |
|
Number
of
entries
to
chamber
with
novel
stranger
2
in
cage |
not
applicable |
yes |
n |
a,
b,
c
or
d:
These
measurements
are
similar
measurements,
but
recorded
under
two
distinct
tests.
|
|
only
Test age:
6-7wks
