Brown2
Protocol
Project
protocol
—
Contents
Definitions
Overall
workflow
Equipment
common
to
multiple
procedures
Notes
for
all
procedures
in
this
project
Procedure:
Barnes
Circular
Maze
(BCM)
for
testing
visuo-spatial
learning
and
memory
Procedure:
Rotarod
(ROD)
for
measuring
neuromuscular
coordination,
cerebellar
function,
and
motor
learning
Procedure:
Olfactory
discrimination
task
(ODT)
to
measure
olfactory
discrimination
learning
and
memory
Procedure:
Morris
Water
Maze
(MWM)
Procedure:
Visual
Water
Box
(VWB)
to
assesses
visual
discrimination,
pattern
discrimination
and
visual
acuity
Data
References
Overall
workflow
The
majority,
but
not
all
mice
were
tested
for
all
procedures.
Mice
were
not
pretreated
with
drugs,
special
diet,
or
exposed
to
other
environmental
test
conditions.
*
Week-1
is
devoted
to
testing
anxiety
and
exploratory
behavior
(Brown1).
Equipment
common
to
multiple
procedures
- Camera-based
computer
tracking
system:
Limelight
and
Watermaze,
Actimetrics;
IBM
PC
computer)
fixed
to
the
ceiling
2.1
m
above
the
apparatus
for
measuring
latency
and
distance
traveled.
- Video
camcorder:
(Hitachi,
VM-7500LA)
located
150
cm
above
the
apparatus
for
recording
trials
for
subsequent
analysis
(with
Hindsight).
- Hindsight
event
recording
software:
(MS-dos,
version
1.5,
Scott
Weiss)
for
scoring
behavior.
Notes
for
all
procedures
in
this
project
- Mice
are
transported
to
the
test
room
in
their
home
cage.
- Mice
are
handled
by
the
base
of
the
tail
or
with
a
500
ml
plastic
container
when
moved
in
and
out
of
the
apparatus
- An
observer
records
the
behavior
of
each
mouse
from
a
distance
of
1
m.
- Each
trial
is
recorded
with
a
video
camera
positioned
above
the
apparatus
(for
subsequent
computer
analysis).
- The
apparatus
is
thoroughly
cleaned
with
70%
EtOH
and
allowed
to
dry
between
tests.
Definitions
----------------------------------
Barnes
Circular
Maze
(BCM)
Purpose:
To
measure
strain
differences
in
visuo-spatial
learning
and
memory
in
14
inbred
strains
of
mice.
Workflow
and
Sampling
Workflow
Day |
Phase |
Trials
|
Time/Trial |
Inter-trial
interval |
Escape |
Aversive
stimulus
|
1 |
Habituation |
4 |
5
min |
20
min |
Yes |
Bright
lights |
2-5 |
Acquisition
training |
4 |
5
min |
20
min |
Yes |
Bright
lights,
buzzer |
6-9 |
Reversal
training |
4 |
5
min |
20
min |
Yes |
Bright
lights,
buzzer |
10 |
Probe
trial |
1 |
5
min |
- |
No |
Bright
lights |
Equipment
Barnes
Circular
Maze:
The
Barnes
maze
was
originally
developed
by
Carol
Barnes
as
a
method
for
testing
visuo-spatial
learning
in
aging
rats
(Barnes
1979;
Barnes,
Nadel,
&
Honig,
1980).
The
Barnes
Maze
for
mice
used
here
is
a
modified
version
of
that
developed
by
Pompl
et
al.
(1999).
In
this
maze
mice
are
motivated
by
aversive
bright
light
and
loud
noise
to
locate
a
hole
which
leads
to
a
dark
escape
box
beneath
the
maze.
The
Barnes
Maze
was
a
69
cm
diameter
circular
disk
constructed
from
2
cm
thick
plywood
and
painted
white
with
non-toxic
oil-based
paint.
A
15
cm
high
wall
made
from
white
polyethylene
was
placed
around
the
edge
of
the
disk.
There
were
16,
4.45
cm
diameter
circular
holes
equally
spaced
around
the
edge
of
the
maze
and
these
holes
were
located
1.3
cm
from
the
wall.
The
circular
disk
was
placed
on
top
of
a
circular
platform
that
was
89
cm
in
diameter
and
1
cm
thick.
The
platform
had
four
legs
screwed
into
the
bottom
that
raised
the
maze
48.4
cm
above
the
floor.
The
holes
on
the
circular
disk
went
through
this
platform
and
these
holes
were
labeled
from
1
to
16
on
the
outside
of
the
wall.
An
escape
box
(13
x
29
x
14
cm)
made
of
white
plastic
could
be
placed
under
four
different
escape
holes
(holes
4,
8,
12
and
16).
A
staircase
made
of
plywood
was
placed
in
the
escape
box
to
ease
the
descent
of
mice
from
the
test
arena
into
the
escape
box.
This
staircase
had
five
stairs
(each
1.8
cm
high,
1.3
cm
long
and
10
cm
wide)
and
the
top
of
the
stairs
was
4
cm
below
the
escape
hole.
The
bottom
of
the
staircase
was
18
cm
long
and
10
cm
wide,
while
the
top
of
the
staircase
was
7.7
cm
long
and
10
cm
wide.
The
entire
set
of
stairs
was
9.0
cm
high.
The
stairs
rested
on
a
piece
of
1
cm
thick
plywood
in
the
bottom
of
the
plastic
escape
box.
The
top
of
the
staircase
was
painted
white,
while
the
rest
of
the
stairs
were
painted
black.
A
blue
polyvinyl
chloride
(PVC)
tube
that
was
8
cm
in
diameter
and
12.5
cm
high
was
placed
in
center
of
the
maze
floor
and
mice
were
placed
into
this
tube
before
each
trial.
A
light
stand
held
two
150-Watt
light
bulbs
(emitted
2580
lux)
76
cm
above
the
maze
platform
to
provide
aversive
bright
light.
Also
a
buzzer
powered
by
a
9-volt
battery
was
attached
to
the
light
stand
and
hung
20
cm
above
the
center
of
the
maze.
The
buzzer
emitted
a
372
kHz
sound
at
89
decibels
directly
under
the
buzzer
(86
decibels
at
the
wall).
The
buzzer
was
turned
on
and
off
by
the
experimenter
using
a
push
button.
Figure
1
is
a
schematic
illustration
of
the
Barnes
circular
maze
apparatus.
Visual
cues:
Three
intra-maze
visual
cues
(geometric
shapes)
were
taped
to
the
inside
of
the
wall
in
semi-random
locations,
so
that
no
cue
was
located
directly
above
an
escape
hole.
Also
objects
around
the
room
(room
walls,
shelves,
desk,
computer,
light
stand
etc.)
served
as
extra-maze
visual
cues.
Environmental
test
conditions
The
Barnes
maze
was
located
in
the
same
laboratory
room
(1.8
x
4.5
m)
where
testing
on
the
elevated
zero
maze,
elevated
plus
maze,
light-dark
box
and
open
field
test
previously
occurred.
This
room
had
a
video
camcorder
mounted
on
a
bracket
150
cm
above
the
center
of
the
maze
to
videotape
test
trials
for
later
analysis.
The
room
was
kept
dark
except
for
the
bright
lights
above
of
the
Barnes
maze.
Procedure:
Barnes
Circular
Maze
(BCM)
See
details
common
to
all
procedures
in
this
project,
and
see
Table
1
Barnes
maze
for
a
greater
detail
of
the
test.
Before
testing,
mice
are
randomly
assigned
to
one
of
four
different
escape
hole
locations,
which
determined
where
the
escape
hole
would
be
located
throughout
testing
on
the
Barnes
maze.
Mice
first
completed
habituation
training
(Day
1),
which
consisted
of
four
trials.
In
each
trial
mice
are
placed
next
to
their
escape
hole,
under
an
inverted
2
L
glass
beaker.
Following
habituation
training,
mice
completed
acquisition
training
for
4
days
(4
trials
per
day).
On
each
trial,
mice
are
released
from
the
start
tube
and
are
given
5
minutes
to
enter
the
escape
hole.
Following
acquisition
training,
mice
completed
4
days
of
reversal
training
where
the
escape
hole
is
moved
to
the
opposite
side
of
the
maze.
During
the
probe
trial
no
escape
box
is
present
and
the
trial
is
videotaped
and
later
analyzed
using
the
Hindsight
event
recorder
program.
The
maze
is
divided
into
four
quadrants,
and
the
time
spent
in
each
quadrant
is
measured.
Memory
for
the
location
of
the
escape
hole
is
assessed
with
the
%
time
in
the
quadrant
where
the
escape
hole
is
located
in
previous
training.
Data
collected
by
investigator
The
numbers
of
errors
made
(head-dips
into
incorrect
holes)
and
latency
to
find
the
escape
hole
were
recorded
on
each
trial
of
acquisition
and
reversal
training
to
assess
learning.
Also,
distance
traveled
during
each
trial
on
the
last
day
(day
4)
of
reversal
training
was
used
to
assess
learning.
Entries
and
time
spent
in
the
correct,
right,
left,
and
opposite
quadrants
were
recorded
during
the
probe
trial
to
assess
memory.
Also
the
number
of
nose
pokes
into
the
acquisition
and
reversal
escape
holes
during
the
probe
trial
were
recorded
to
assess
memory.
Distance
traveled
was
obtained
with
analysis
of
the
video
recorded
trials,
by
placing
transparent
plastic
sheets
over
a
television
screen,
and
tracing
the
movement
of
each
mouse
with
a
marker.
The
distance
traveled
and
the
diameter
of
the
maze
on
the
plastic
transparencies
(Scalex,
PlanWheel
XL)
is
then
used
to
determine
the
distance
traveled
in
the
actual
maze
using
the
formula
[(Real
Maze
Diameter
÷
Transparent
Sheet
Maze
Diameter)
X
Tracing
Distance].
Distance
traveled
can
also
be
obtained
using
a
video-camera
based
tracking
system
such
as
Limelight
(Actimetrics).
Investigator's
notes:
The
Barnes
maze
was
originally
designed
for
rats
and
the
original
apparatus
design
is
often
altered
when
the
maze
is
used
with
mice.
For
example,
the
maze
used
here
has
a
smaller
size
(69
cm
diameter)
and
has
fewer
holes
(16)
than
the
original
design
for
rats
(122
cm
diameter
and
18
holes).
Also,
a
wall
and
intra-maze
cues
have
been
added
around
the
edge
of
the
maze.
Other
designs
have
been
used
with
mice
that
have
more
holes
(40)
(Bach
et
al.,
1995;
Fox
et
al.,
1998),
or
use
different
stimuli
to
provide
aversive
motivation
(tactile
fan
or
auditory
buzzer)
(Pompl
et
al.,
1999).
Little
research
has
been
completed
to
determine
if
results
using
one
Barnes
maze
design
can
generalize
to
other
Barnes
maze
designs.
Therefore,
differences
in
apparatus
design
should
be
considered
when
comparing
results
from
previous
studies
to
the
present
database.
The
validity
of
the
Barnes
maze
design
used
in
the
present
database
has
been
questioned.
We
found
that
C57BL/6J
mice
on
this
design
may
not
use
visual
cues
to
locate
the
escape
hole
as
performance
of
mice
did
not
change
when
extra-maze
visual
cues
were
not
visible,
or
when
the
location
of
the
escape
hole
was
moved.
Also,
Garcia
et
al.
(2004)
found
that
the
performance
of
blind
mice
did
not
differ
from
sighted
mice
on
this
design.
Therefore,
results
from
this
Barnes
maze
design
should
be
considered
in
conjunction
with
results
from
another
visuo-spatial
learning
and
memory
test
(i.e.
Morris
water
maze).
---------------------------------------------
Rotarod
(ROD)
Purpose:
To
measure
strain
differences
in
neuromuscular
coordination
and
motor
learning
using
the
rotarod
apparatus.
Equipment
Rotarod:
The
rotarod
apparatus
(AccuRotor
Rotarod;
Accuscan
Instruments
Inc.
Columbus,
Ohio
(number:
AI9612AR04/M))
was
capable
of
accommodating
four
mice
at
the
same
time,
with
each
mouse
separated
by
a
15
cm
high
white
Plexiglas
circular
divider.
The
space
between
each
divider
was
11
cm.
The
rotating
rod
was
acrylic
and
measured
3
cm
in
thickness.
There
was
a
drop
of
39
cm
from
the
rod
to
the
individual
holding
chambers
that
were
directly
below
each
section
of
the
rotating
rod.
Four
automatic
timers
were
built
into
the
apparatus
and
these
timers
started
the
moment
the
rod
was
set
into
motion
at
the
start
of
each
trial,
and
turned
off
automatically
when
an
animal
fell
into
the
holding
chamber
below
the
rod.
Figure
2
is
an
example
of
a
Rotarod
apparatus.
Note
the
mice
positioned
in
different
trails.
Environmental
test
conditions
The
Rotarod
was
located
in
a
laboratory
room
measuring
112
cm
x
260
cm.
The
room
was
illuminated
with
a
single
60-Watt
red
light
bulb.
Procedure:
Rotarod
a.
See
details
common
to
all
procedures
in
this
project,
Table
2
Rotarod,
and
Brown
and
Wong
(2007)
for
greater
details
of
this
test.
b.
Mice
are
tested
in
squads
of
4
and
are
tested
during
the
dark
phase
of
the
Light/Dark
cycle.
c.
For
each
trial,
four
mice
are
placed
on
the
rod
facing
the
direction
opposite
to
the
rod's
motion.
The
speed
of
the
rod
increased
from
0
rotation
per
min
(rpm)
to
a
maximum
speed
of
48
rpm
over
each
6
min
trial.
d.
Between
trials
mice
are
given
1
min
to
rest
in
the
holding
chambers
until
the
next
trial.
e.
To
measure
motor
learning,
mice
completed
six
trials
per
day
for
seven
days.
Data
collected
by
investigator
Days
1-7
of
sensorimotor
learning:
the
latency
for
mice
to
fall
off
the
accelerating-rotating
rod,
and
body
weight
data
were
recorded.
Investigator
notes:
The
Rotarod
is
a
measure
of
neuromuscular
co-ordination,
fatigue,
and
learning
(LeMarec
&
Lalonde,
1997).
Rotarod
(or
Roto
rod)
performance
has
often
been
used
as
a
method
to
study
the
effects
of
drugs
on
neuromuscular
co-ordination
(Hymson
&
Hynes,
1982),
a
measure
of
cerebellar
function
(Caston
et
al.,
1998)
and
a
measure
of
motor
learning
(Hyde
et
al.,
2001).
The
Rotarod
can
rotate
at
a
constant
speed
or
accelerate
over
time.
The
accelerating
Rotarod
is
better
at
detecting
drug
effects
and
a
better
model
of
motor
learning
than
the
constant
speed
Rotarod
(Bogo
et
al.,
1981).
The
Procedure
as
described
here
is
a
sensorimotor
learning
task.
The
latency
for
the
mice
to
fall
off
the
rod
will
increase
over
trials
and
over
days
as
the
animal
becomes
more
proficient
with
maneuvering
on
the
apparatus
and
can
stay
on
longer.
Rotarod
performance
is
sensitive
to
body
weight
with
mice
of
smaller
strains
having
longer
latencies
to
fall
then
mice
of
larger
strains
and
females
having
a
longer
latency
then
males.
Thus
it
is
important
to
record
body
weights
and
to
use
an
analysis
of
co-variance
with
weight
as
the
co-variate
to
examine
strain
x
sex
differences
(McFadyen
et
al.,
2003;
Brown,
unpublished).
-------------------------------------
Olfactory
discrimination
task
(ODT)
Purpose:
To
measure
olfactory
discrimination
learning
and
memory
by
using
a
classical
conditioning
paradigm.
Workflow
and
Sampling
Workflow
Day |
Phase |
Trials
(#/day) |
Trial
duration |
Inter-trial
interval |
Data
recorded |
1-3 |
Food
restriction |
- |
- |
- |
bw |
4-7 |
Training |
4 |
10
min |
5
min |
bw |
8 |
Odor
discrimination |
1
(habituation) |
2
min |
- |
bw,
time
spent
(L
or
R) |
8 |
Odor
discrimination |
1
(discrimination) |
3
min |
- |
bw,
time
spent
digging |
Equipment
The
odor
discrimination
test
apparatus
consisted
of
a
69
x
20
x
20
cm
box
made
of
3
mm
acrylic
and
was
divided
into
three
compartments
of
equal
size
(23
x
20
x
20
cm)
by
two
walls.
These
walls
had
6
x
5.5
cm
openings
located
at
floor
level,
and
allowed
mice
to
move
between
compartments
of
the
apparatus.
Before
each
trial,
the
floor
of
the
test
apparatus
was
covered
with
1000
ml
of
Pro-chip
(P.W.
I
Industries)
and
acrylic
doors
were
placed
over
the
openings
in
the
walls
between
the
chambers.
These
doors
were
removed
at
the
start
of
each
test
trial.
Odor
training
apparatus:
Training
trials
were
completed
in
polycarbonate
cages
with
stainless
steel
wire
tops,
identical
to
the
cages
mice
were
housed
in.
Figure
3
illustrates
the
typical
layout
of
an
odor
discrimination
preference
apparatus.
Odor
pot:
A
diagram
of
the
odor
pot
used
to
present
the
odors
during
training
and
testing
(From
Schellink
2001
a)
is
shown
below.
Odor
pots
contained
rose
or
lemon
scent
(15%,
3
ml
of
phenyl
acetate
(rose)
or
linalool
(lemon)
in
17
ml
of
propylene
glycol),
6-8
small
pieces
of
sugar
(cut
from
sugar
cubes),
and
pine
chip
bedding.
Odors
were
stored
in
1.5
ml
Eppendorf
tubes
at
–80°C
to
avoid
degradation.
The
odor
pots
were
covered
with
perforated
Petri
dishes
to
prevent
physical
contact
with
odorants,
while
allowing
mice
to
smell
the
odors.
Figure
4
is
a
schematic
diagram
of
the
odor
pot
setup
from
Schellink
2001
a.
Reagents,
supplies,
solutions
- Pots:
trimmed
plastic
beverage
cups
(1.5
cm
in
height
and
6.25
cm
diameter).
- Odors:
Phenyl
acetate
(Rose)
and
Linalool
(Lemon)
(Aldrich
Chemicals).
- Diluent:
propylene
glycol
(Caledon
Chemical
Co.).
- Adsorbent
paper:
Whatman®
no.1
filter
paper
(55
mm
diameter).
- Reinforcement:
sugar
cubes
pre-cut
with
razor
blade.
- Digging
medium:
pine
chips
(Pro-chip,
P.W.
I.
Industries)
bedding.
Procedure:
Olfactory
discrimination
task
(ODT)
See
details
common
to
all
procedures
in
this
project,
Table
3
olfactory
discrimination,
and
see
Schellinck
et
al
(2001a
&
2001b)
for
more
in-depth
details
of
the
task.
a.
Three
days
before
training
mice
are
placed
under
food
restriction
until
each
mouse's
body
weight
is
reduced
to
85-90%
of
its
body
weight
when
food
is
available
ad
libitum.
During
food
restriction
mice
are
weighed
and
fed
more
or
less
than
3g
of
food
to
maintain
their
body
weight
at
85-90%
of
their
ad-libitum
weight.
Mice
are
fed
shortly
after
lights
off
(9:00
am).
Food
restriction
is
continued
throughout
training.
After
the
completion
of
the
discrimination
test
(day
8),
food
is
again
made
available
ad
libitum.
b.
Mice
are
weighed
each
day
before
training
trials,
and
fed
3
hours
after
each
day's
training
is
finished
(~4:00
pm).
c.
Training
occurred
in
three
separate
rooms,
one
for
the
rose
odor,
one
for
the
lemon
odor,
and
one
neutral.
Mice
are
kept
in
the
neutral
room
during
inter-trial
intervals.
d.
Half
of
the
mice
are
trained
with
the
rose
odor
paired
with
sugar
(CS+)
and
the
lemon
odor
not
paired
with
sugar
(CS-).
The
other
half
of
the
mice
are
trained
with
the
lemon
odor
paired
with
sugar
(CS+)
and
the
rose
odor
not
paired
with
sugar
(CS-).
In
each
training
trial
an
odor
pot
is
placed
in
the
center
of
the
back
third
of
the
training
cage.
Each
mouse
received
four
10-min
training
trials
per
day
on
each
of
4
days.
Two
trials
are
completed
with
the
rose
odor
and
two
trials
are
completed
with
the
lemon
odor
and
these
trials
are
presented
in
a
pseudo-random
order.
e.
The
odor
discrimination
test
is
done
in
a
novel
room
to
avoid
any
influence
of
contextual
cues
from
rooms
used
during
odor
training.
f.
During
the
habituation
trial
a
pot
(no
odors
or
sugar)
is
placed
in
each
of
the
end
compartments.
Each
mouse
is
placed
in
the
center
compartment
and
are
allowed
to
explore
the
apparatus
for
3
min.
g.
To
prevent
the
use
of
visual
cues,
the
apparatus
is
rotated
180
degrees
for
the
preference
test.
Odor
pots
(without
sugar)
containing
rose
and
lemon
odors
are
placed
in
each
of
the
end
compartments.
During
the
discrimination
trial
each
mouse
is
placed
in
the
center
chamber,
and
is
allowed
to
explore
the
apparatus
for
3
min.
h.
The
time
spent
digging
in
the
rose
and
lemon
odor
pots
is
measured
to
assess
memory
for
the
odor
that
is
paired
with
sugar.
This
test
phase
is
sensitive
to
the
level
of
food
restriction
in
mice
(Forestell
et
al.,
2001).
i.
Bedding
and
odor
pots
are
replaced
between
test
trials,
and
the
odor
discrimination
apparatus
is
cleaned
between
trials.
Data
collected
by
investigator
In
addition
to
behavioral
measures:
body
weight
on
each
day
of
training,
body
weight
before
training
began
with
food
ad
libitum,
body
weight
on
the
day
of
odor
discrimination
testing.
During
the
habituation
trial
time
spent
in
each
compartment
was
measured
to
determine
pre-existing
preferences
for
different
compartments
of
the
apparatus.
Memory
for
odor-sugar
pairing
was
measured
in
the
odor
discrimination
test
with
time
spent
digging
at
the
Cs+
odor
pot,
time
spent
digging
at
the
Cs-
odor
pot,
and
percentage
of
the
total
time
digging
that
each
mouse
dug
at
the
Cs+
odor
pot.
Investigator
notes:
Mice
were
placed
under
a
food
restricted
diet
so
that
they
are
motivated
to
obtain
sugar
during
training
and
during
the
odor
preference
test.
If
mice
are
not
food
deprived
during
the
odor
preference
test,
they
do
not
accurately
discriminate
between
the
two
odors
(Forestall
et
al.,
2001).
Therefore,
the
body
weight
of
mice
on
the
preference
test
should
be
considered
when
interpreting
the
percent
time
digging
in
the
Cs+
odor
pot.
------------------------------------------
Morris
Water
Maze
(MWM)
Purpose:
To
test
visuo-spatial
learning
and
memory
on
the
Morris
Water
Maze.
Workflow
and
Sampling
Workflow
Day |
Phase |
Trial
(#/day) |
Duration |
Inter-trial
interval |
Platform/location |
1-3
|
Acquisition |
4 |
1
min |
10
min |
Invisible,
north
west
|
4-6 |
Reversal |
4 |
1
min |
10
min |
Invisible,
south
east |
7 |
Probe |
1 |
1
min |
10
min |
none |
8 |
Visible
platform |
4 |
1
min |
10
min |
Visible,
south
west |
Equipment
- The
Morris
water
maze
(MWM)
was
originally
designed
to
assess
visuo-spatial
learning
and
memory
in
rats
(Morris,
1984).
A
modified
version
of
the
MWM
for
mice
(Paylor
et
al.,
1996)
was
used
and
consisted
of
a
circular
polypropylene
pool
(Canadian
Tire
"Pelican"
pool)
that
was
110
cm
in
diameter
and
20
cm
in
height.
The
pool
was
filled
with
water
to
a
dept
of
14
cm,
and
was
kept
at
a
20±1°C
temperature.
- The
water
was
made
opaque
with
500
ml
of
non-toxic
white
liquid
tempura
paint
(Schola,
Marieville)
to
camouflage
the
white
escape
platform.
- A
Plexiglas
cylinder
(13.75
cm
x
9
cm
diameter)
was
used
as
the
escape
platform
in
the
maze.
The
cylinder
was
filled
with
water
to
weigh
it
down
in
the
pool.
The
escape
platform
sat
0.5
cm
below
the
surface
of
the
water.
A
removable
red
and
yellow
striped
top
(3
cm
x
9
cm
in
diameter)
with
a
colorful
flag
erected
in
the
center
could
be
placed
on
the
top
of
the
escape
platform
to
make
the
escape
platform
visible
in
the
water.
- A
video
camera-based
computer
(IBM
PC)
tracking
system
(WaterMaze,
Actimetrics)
mounted
to
the
ceiling
2.1
m
above
the
pool,
was
used
to
record
the
mice
water
maze
performance.
- Visual
cues:
Extra-maze
visual
cues
around
the
maze
consisted
of
a
table,
computer,
and
posters
placed
along
the
walls
of
the
test
room.
- The
pool
is
divided
into
four
quadrants:
Northwest,
Northeast,
Southwest
and
Southeast.
Boundaries
of
these
quadrants
are
marked
on
the
edges
of
the
pool
with
masking
tape
and
labeled:
North,
South,
East
and
West.
Figure
5
is
a
photographic
image
of
a
Morris
water
maze.
Environmental
test
conditions
The
pool
is
located
in
a
room
measuring
5.2
x
2.4
m,
and
the
test
room
was
diffusely
lit
with
white
light
(30
lux).
Procedure:
Morris
Water
Maze
(MWM)
See
details
common
to
all
procedures
in
this
project,
and
see
Table
4A
Morris
water
maze
and
Brown
and
Wong
(2007)
for
specifics.
a.
Mice
are
tested
in
squads
of
4
and
each
mouse
is
placed
in
a
clean
empty
cage
during
testing.
Paper
towel
is
torn
and
placed
in
the
bottom
of
the
cage
to
allow
mice
to
dry
quickly
between
trials.
The
paper
towel
is
replaced
when
it
became
completely
wet.
b.
Mice
completed
3
days
of
acquisition
training
with
4
trials
per
day.
During
each
trial
the
mice
are
placed
in
the
pool
at
one
of
four
different
start
locations
(North,
South,
East
and
West),
and
are
given
60
s
to
find
the
hidden
escape
platform.
To
prevent
mice
from
developing
fixed
motor
patterns,
the
start
locations
varied
across
trials
based
on
a
latin
square
design
(see
Table
4B
Morris
water
maze).
c.
Following
acquisition
training,
mice
completed
3
days
of
reversal
training
(4
trials
per
day),
where
the
escape
platform
is
moved
to
the
opposite
side
of
the
maze.
d.
During
the
probe
trial
(day
8),
the
escape
platform
is
not
present.
The
maze
is
divided
into
4
quadrants
using
the
Watermaze
(Actimetrics)
software,
and
the
time
spent
in
each
quadrant
is
recorded.
e.
The
day
following
the
probe
trial,
mice
completed
visible
platform
training
where
a
striped
top
was
placed
on
top
of
the
escape
platform
so
that
the
escape
platform
was
visible.
Mice
completed
one
day
of
visible
platform
training
with
four
trials.
Data
collected
by
investigator
During
acquisition
and
reversal
training
learning
was
assessed
with
the
measures
of
swim
latency
(time
to
climb
onto
the
escape
platform)
and
distance
traveled
to
reach
the
escape
platform.
Average
velocity
and
the
duration
and
frequency
of
thigmotaxic
behavior
(9-cm-wide
corridor)
were
also
recorded.
During
the
probe
trial
time
spent
in
each
quadrant
(Northeast,
Southeast,
Northwest,
Southwest)
was
recorded
and
memory
was
assessed
with
time
spent
in
the
quadrant
that
contained
the
escape
platform
during
reversal
training
(southeast
or
correct
quadrant).
Memory
was
also
assessed
with
the
number
of
times
each
mouse
crossed
the
location
where
the
escape
platform
was
located
during
reversal
training
(annulus
reversal
crossing)
or
acquisition
training
(annulus
acquisition
crossing).
Overall
distance
traveled
is
determined
by
the
formula:
[Real
Maze
Diameter/
Transparent
Sheet
Maze
Diameter)
X
Tracing
Distance].
Investigator
notes:
The
swim
path
analysis
can
be
used
to
determine
whether
mice
are
developing
a
spatial
strategy
for
learning
the
maze
or
are
using
non-spatial
strategies,
such
as
circular
swimming
or
thigmotaxis.
Analysis
of
swim
speed
may
be
necessary
to
control
for
locomotor
differences
between
strains
(Wolfer
et
al.,
1998).
Wolfer
et
al.,
1998
found
3
factors
which
accounted
for
81%
of
the
observed
variability
in
the
scores
of
mice
in
the
Morris
water
maze:
thigmotaxis
(49%),
passivity
(19%)
and
memory
(13%).
Thigmotaxis
is
associated
with
swimming
along
the
wall
of
the
pool;
while
passivity
means
floating
in
the
pool,
with
a
slow
swimming
speed;
and
memory
represents
the
time
spent
in
the
target
quadrant
during
the
probe
trial.
----------------------------
Visual
Water
Box
(VWB)
Purpose:
To
test
visual
discrimination,
pattern
discrimination
and
visual
acuity
in
mice.
Workflow
and
Sampling
Mice
are
first
gradually
trained
to
locate
a
hidden
platform
below
a
screen
displaying
a
vertical
grating
of
low
spatial
frequency
(S+)
(see
Wong
&
Brown,
2006).
Workflow
Day |
Phase |
Trial
(#/day) |
Duration |
Inter-trial
interval |
Gratings
(S+) |
1
|
Pre-training |
12 |
1
min |
3
min |
Vertical,
0.17
(c/deg) |
2-9 |
Visual
detection |
8 |
1
min |
3
min |
Vertical,
0.17
(c/deg) |
10-17 |
Pattern
discrimination |
8 |
1
min |
3
min |
Vertical*,
horizontal,
0.17
(c/deg) |
18-25 |
Visual
acuity |
8 |
1
min |
3
min |
Vertical,
0.17
to
0.64
(c/deg) |
*Only
the
vertical
grating
is
paired
with
the
hidden
platform.
Equipment
The
visual
water
box
combined
the
basic
principles
of
a
discrimination
box
(Yerkes,
1907)
and
the
Morris
water
maze
(Morris,
1984).
The
apparatus
consisted
of
a
trapezoidal-shaped
pool
made
from
6
mm
clear
Plexiglas,
and
had
55
cm
high
walls
around
the
edge.
The
walls
on
the
smallest
and
longest
sides
of
the
pool
were
painted
black
to
reduce
reflections.
The
wall
at
the
wide
edge
of
the
pool
remained
clear,
so
that
two
adjacent
computer
monitors
placed
behind
this
wall
were
visible.
A
black
Plexiglas
divider
(41
x
40
cm)
was
placed
in
guides
between
the
computer
screens,
and
extended
from
the
clear
wall,
bisecting
the
end
of
the
pool
along
its
midline.
A
black
release
alley
(35
cm
long
x
7
cm
wide
x
20
cm
high)
was
placed
at
the
narrow
end
of
the
pool.
A
movable
clear
Plexiglas
escape
platform
(37cm
long
x
13cm
wide
x
14cm
high)
was
placed
below
the
computer
screen
displaying
the
positive
(S+)
visual
stimulus.
The
pool
and
computer
monitors
were
placed
on
a
solid
table
(146
cm
long
x
100
cm
wide
x
46
cm
high).
The
pool
was
filled
with
tepid
(22
°C)
water
to
a
depth
of
15
cm.
Reflections
from
the
computer
screens
on
the
surface
of
the
water
made
the
platform
invisible
from
water
level.
Grating
setup:
Gratings
were
displayed
on
two
identical
17-inch
monitors
(Accu
Sync
70
NEC
3)
that
face
into
the
wide
end
of
the
pool,
and
the
bottom
of
the
monitors
were
at
water
level.
A
customized
computer
program
(developed
by
Stephen
Turner
and
modeled
after
the
Vista
program
used
by
Prusky
et
al,
(2000))
controlled
the
visual
stimuli.
Figure
6
depicts
the
schematic
layout
of
the
visual
water
box.
Note
that
lines
are
drawn
for
illustrative
purposes.
Environmental
test
conditions
Testing
was
completed
during
the
active
(dark)
phase
of
the
Light/Dark
cycle
without
any
other
lights
present,
except
that
from
the
computer
screen.
Procedure:
Visual
Water
Box
(VWB)
See
details
common
to
all
procedures
in
this
project,
and
see
Table
5
visual
water
box,
and
Wong
&
Brown,
2007
for
specific
details
of
the
test.
a.
Mice
are
trained
in
groups
of
4
and
given
an
inter-trial
interval
of
about
3
min.
Mice
completed
4
phases
of
testing:
pre-training,
visual
discrimination,
pattern
discrimination
and
visual
acuity.
b.
During
pre-training
(day
1),
mice
are
shaped
to
locate
a
hidden
platform
situated
below
a
computer
monitor
displaying
a
low
spatial
frequency
vertical
grating.
The
positive
stimulus
(S+,
hidden
platform)
alternated
between
the
left
and
the
right
side
of
the
water
box
divider
during
testing.
The
negative
stimulus
(S-)
is
a
grey
screen
and
no
escape
platform
is
placed
beneath
it.
c.
Mice
that
did
not
find
the
escape
platform
within
1
min
are
guided
to
the
escape
platform.
Mice
are
allowed
to
stay
on
the
platform
for
10
s
before
being
returned
to
the
holding
cages.
d.
Visual
discrimination
testing
(days
2-9)
is
completed
following
pre-training,
and
consisted
of
8
days
of
training
(8
trials/
day).
During
visual
discrimination
the
escape
platform
is
located
under
the
monitor
displaying
vertical
gratings
(0.17
c/deg)
(S+),
whereas
no
escape
platform
is
located
under
the
monitor
displaying
a
grey
screen
(S-).
A
trial
is
correct
if
an
animal
swam
to
the
escape
platform
without
entering
the
negative
stimulus
arm.
If
an
animal
broke
the
plane
perpendicular
to
the
end
of
the
divider
on
the
side
of
the
tank
with
the
monitor
displaying
the
S-
stimulus,
the
trial
is
recorded
as
an
error.
A
trial
is
also
incorrect
if
mice
did
not
find
the
escape
platform
in
less
than
1
min.
e.
If
a
mouse
made
an
error,
the
mouse
is
required
to
run
another
trial
(error
trial)
before
being
returned
to
its
holding
cage.
Only
one
error
trial
is
completed
for
each
training
trial.
f.
Pattern
discrimination
training
(days
10-17)
is
completed
following
visual
discrimination
training
and
consisted
of
8
days
of
training
(8
trials/day).
During
pattern
discrimination
the
escape
platform
is
located
under
a
monitor
displaying
vertical
gratings
(S+)
(0.17
c/deg),
whereas
no
escape
platform
is
located
under
a
monitor
displaying
horizontal
gratings
(S-).
g.
Visual
acuity
testing
(days
18-25)
is
completed
following
pattern
discrimination
testing,
and
consisted
of
8
days
of
testing
(8
trials/day).
On
each
day
the
escape
platform
is
located
under
the
monitor
displaying
vertical
gratings
(S+),
whereas
no
escape
platform
was
located
under
the
monitor
displaying
a
grey
screen
(S-).
The
spatial
frequency
of
the
vertical
gratings
ranged
from
0.17
to
0.64
c/deg
across
trials
for
each
day
of
testing.
h.
Testing
is
accomplished
efficiently
and
timely
to
prevent
hypothermia
and
exhaustion
in
the
mice.
Data
collected
by
investigator
For
visual
discrimination
and
pattern
discrimination
the
percentage
of
correct
trials
for
each
day
(8
trials
per
day)
and
latency
to
reach
the
platform
were
recorded
as
measures
of
learning.
Data
from
error
trials
were
not
used.
If
a
mouse
performed
above
70%
(~6/8)
correct
response
on
two
consecutive
days,
it
met
the
criteria
for
that
phase.
For
visual
acuity,
the
percent
correct
at
each
spatial
frequency
was
recorded.
The
visual
acuity
was
defined
as
the
highest
spatial
frequency
at
which
mice
made
more
than
70%
correct
choices
(~6/8
trials).
Latency
to
find
the
escape
platform
was
also
recorded
for
each
spatial
frequency.
Investigator
notes:Prolonged
testing
does
not
guarantee
accurate
results,
because
animals
can
get
hypothermic
and
tired.
Therefore,
the
holding
cages
of
the
mice
were
placed
under
a
standard
60W
light
bulb
as
a
source
of
heat
and
the
holding
cages
were
lined
with
paper
towel
to
dry
mice
between
trials.
However,
mice
appeared
to
be
visibly
cold
or
tired,
they
were
allowed
to
rest
before
continuing
the
experiment.
----------------------------------
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suggested
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Pubmed:
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maze.
Behavioral
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Pubmed:
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S.J,
Justice,
A.,
McConlogue,
L.,
Games,
D.,
Freedman,
S.B.
&
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(2000).
A
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Nature,
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Behavior
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Pubmed:
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Pubmed:
11516773
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Psychological
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6045339
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D.,
Clark,
V.,
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R.
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Wehner,
J.
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Enhancement
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F1
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of
genetic
backgrounds
for
single
gene
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and
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X
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S.
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The
Atlantis
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a
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Data available through MPD
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PubMed 20801160
MGI
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Genes Brain Behav. 2006 Jul;5(5):389-403.
PubMed 16879633
MGI
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Web resources
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Test age:
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