Ethovision
XT
(Noldus
Information
Technology,
Wageningen,
The
Netherlands)
Open-field
arena
(see
full
description
below)
Light-dark
box
(see
full
description
below)
Visual-cliff
arena
(see
full
description
below)
Hot
plate
analgesia
meter
(IITC
Life
Science
Inc,
catalog
#39,
Woodland
Hills
CA)
Tail
suspension
apparatus
(see
full
description
below)
Paper
cones
Masking
tape
Reagents
and
solutions
Ethanol
for
disinfecting
Procedure:
General
Mice
are
tested
in
order
of
perceived
stressfulness
of
the
test
(see
Workflow).
Mice
are
randomly
assigned
to
testing
groups
such
that
an
equal
number
of
male
and
female
mice
are
tested
each
day
(n=24
per
sex).
Mice
are
allowed
to
acclimate
at
least
6
wks
after
shipping
from
vendor.
For
the
open-field,
light-dark
box,
and
visual-cliff
tests,
mice
are
habituated
to
test
room
for
1
h
prior
to
testing;
for
the
hot
plate
and
tail-suspension
tests,
mice
are
habituated
30
min.
For
each
assay,
mice
are
removed
by
the
tail
then
returned
to
the
clean
side
of
a
duplex
home
cage
until
each
cage
mate
completes
testing.
A
single
experimenter
handles
mice
for
each
test.
Behavioral
measures
are
recorded
and
analyzed
by
real-time
video
tracking
using
Ethovision
XT.
Procedure:
Open-field
test
Apparatus:
opaque
Plexiglas
box
(39x39x39
cm)
with
a
dark
gray
floor,
illuminated
at
43
+/-
4
lux
in
a
10x15
ft
room;
zones
of
arena
are
delineated
as
follows
--
center:
10x10
cm;
corners:
4x4
cm;
periphery:
31x4
cm.
Each
mouse
is
placed
into
the
center
of
the
arena.
Behaviors
are
recorded
for
20
min.
Procedure:
Light-dark
box
test
Apparatus:
open-field
apparatus
with
an
insert
evenly
dividing
the
apparatus
into
light
and
dark
compartments
(light
side
illuminated
at
17
+/-
2
lux;
compartments
separated
by
a
sliding
door).
Mice
are
placed
into
the
dark
compartment
when
sliding
door
is
closed.
Sliding
door
is
open
and
the
lid
is
closed.
Behaviors
are
recorded
for
20
min.
Procedure:
Visual-cliff
avoidance
test
Apparatus:
open-field
box
with
clear
Plexiglas
bottom
that
is
secured
so
that
half
of
the
floor
overhangs
the
table-top
to
create
an
appearance
of
a
ledge
drop-off;
checkerboard
table
cloth
is
draped
from
the
table-top
to
the
floor
(serves
to
enhance
the
visual
appearance
of
the
cliff);
vertical
distance
between
box
floor
and
testing
room
floor
is
93
cm;
an
opaque
neutral
zone
(10x10
cm)
is
located
in
the
center
of
the
box
floor;
entire
field-of-view
is
covered
with
black
and
white
checkerboard
to
emphasize
changes
in
depth. 'Top' and 'bottom' refer to areas of the visual cliff, where 'top' is over the supported half of the apparatus and 'bottom' is over the suspended half.
Mice
are
placed
onto
the
neutral
center
region.
Behaviors
are
recorded
for
20
min.
Procedure:
Hot-plate
test
Apparatus:
hot
plate
(11x10.5
cm)
surrounded
by
a
clear
acrylic
cage
(15
cm
tall,
10
cm
diameter,
open
top
with
clear
acrylic
lid)
to
constrain
locomotion.
The
surface
of
the
hot
plate
is
set
to
55°C
as
measured
by
a
built-in
digital
thermometer.
Each
mouse
is
placed
on
the
hot
plate
and
timed
until
it
performs
either
of
two
behaviors
regarded
as
indicative
of
nociception:
hindpaw
lick
or
hindpaw
shake/flutter.
Latency
to
respond
is
measured
to
the
nearest
0.01
s.
If
there
is
no
response
within
30
s,
mice
are
removed
from
hot
plate
and
time
recorded
as
30
s.
Procedure:
Tail-suspension
test
Apparatus:
horizontal
ring-stand
bar
elevated
~30
cm
above
the
floor
of
the
apparatus.
A
paper
cone
is
placed
on
the
tail
of
each
mouse
to
limit
climbing
during
the
test
session.
Using
masking
tape,
mice
are
suspended
by
a
point
near
the
tip
of
the
tail
on
the
ring-stand
bar.
Behaviors
are
recorded
in
2
test
sessions
(consecutive
days,
5-min
trials).
Data
collected
by
investigator
Open
field
test
distance
traveled
in
first
4
min
(response
to
novelty)
distance
traveled
in
first
10
min
(response
to
novelty)*
total
distance
traveled
(general
locomotor
activity)
distance
traveled
(slope)*
total
time
in
corners*
percent
time
in
corners
percent
time
in
corners
(slope)*
total
time
in
periphery*
percent
time
in
periphery
percent
time
in
periphery
(slope)*
total
time
mobile*
total
time
immobile*
percent
time
mobile*
percent
time
immobile
percent
time
mobile
(log)*
total
time
in
center*
total
time
in
center
(log)*
percent
time
in
center
percent
time
in
center
(log)*
total
time
in
center
(slope)*
number
of
fecal
boli*
Light-dark
box
test
distance
traveled
in
light
compartment
(habituation)
number
of
light-dark
transitions
time
in
light
compartment
first
4
min
(response
to
novelty)
time
in
light
compartment*
percent
time
in
light
compartment
first
4
min
(response
to
novelty)*
percent
time
in
light
compartment
percent
time
in
light
compartment
(slope)*
time
in
light
compartment
(slope)*
distance
traveled
in
light
compartment
(slope)*
Visual-cliff
avoidance
test
distance
traveled
in
top
during
first
4
min
(response
to
novelty)
time
in
top
during
first
4min
(response
to
novelty)
total
distance
traveled
in
top
total
time
in
top
percent
time
in
top*
number
of
top
entries*
time
immobile
in
top*
mean
velocity
in
top
distance
traveled
in
bottom
during
first
4
min
(response
to
novelty)
time
in
bottom
during
first
4
min
(response
to
novelty)
total
distance
traveled
in
bottom
total
time
in
bottom
percent
time
in
bottom
number
of
bottom
entries*
time
immobile
in
bottom*
mean
velocity
in
bottom
number
of
top
to
bottom
transitions
total
distance
traveled*
total
time
immobile*
total
mean
velocity*
top
to
bottom
ratio
for
time
spent
during
first
4
min
(response
to
novelty)*