PROTOCOL

Two-day radial-arm water maze learning and memory

task; robust resolution of amyloid-related memory
deficits in transgenic mice
Jennifer Alamed1, Donna M Wilcock1, David M Diamond1,2, Marcia N Gordon1 & Dave Morgan1
1Alzheimer’s Research Laboratory, Department of Molecular Pharmacology and Physiology, School of Basic Biomedical Sciences, College of Medicine and 2Department of

Psychology, University of South Florida, Tampa, Florida 33612, USA. Correspondence should be addressed to D.M. (dmorgan@health.usf.edu)

Published online 9 November 2006; doi:10.1038/nprot.2006.275

© 2006 Nature Publishing Group http://www.nature.com/natureprotocols

The radial arm water maze (RAWM) contains six swim paths (arms) extending out of an open central area, with an escape platform
located at the end of one arm (the goal arm). The goal arm location remains constant for a given mouse. On day 1, mice are trained
for 15 trials (spaced over 3 h), with trials alternating between visible and hidden platform. On day 2, mice are trained for 15 trials
with the hidden platform. Entry into an incorrect arm is scored as an error. The RAWM has the spatial complexity and performance
measurement simplicity of the dry radial arm maze combined with the rapid learning and strong motivation observed in the Morris
water maze without requiring foot shock or food deprivation as motivating factors. With two sessions each day, 16 mice can be tested
over 2 days.

INTRODUCTION
Behavioral testing of transgenic mouse models of Alzheimer disease
is essential for establishing the progression of mnemonic deficits as
mice age and pathology accumulates. In addition, behavioral out-
comes are useful for assessing clinically relevant outcomes of
potential therapeutics. There are an increasing number of memory
tasks that are sensitive to amyloid accumulation in amyloid pre-
cursor protein (APP) transgenic mice, including the Morris water
maze, the Y-maze, fear conditioning, conditioned food avoidance
and object recognition testing1–4. However, we have found the
RAWM to be the most reliable task for detecting memory deficits in
APP transgenic mice and to robustly discriminate between mice
that learn well and those that learn poorly5–8.
The RAWM is a hybrid of the Morris water maze and a radial
arm maze, which takes advantage of the simple motivation pro-
vided by immersion into water, together with the benefits of scoring
errors (rather than time or proximity to platform location)
associated with the radial arm maze9,10. The scoring is sufficiently
straightforward that video recording of the sessions is not required.
The mouse version of this maze uses a circular pool with six swim
alleys (arms) radiating out from an open central area with a hidden
escape platform submerged at the end of one goal arm (Fig. 1). In
the reference memory version of the task described here, the goal
arm is held constant for all trials, with a different start arm on
successive trials. A working memory version of the task described
by us previously5 varied the goal arm location on each day, but
required 10–15 days of training for nontransgenic mice to reach the
learning criterion. In both instances, the mouse is required to use
visual cues placed around the room to locate the goal arm9.
continuous testing required for mice to learn the procedural
requirements of the task was taxing on both the mice and the
investigators. To shorten the task, we developed a 2-day protocol,
which shows robust cognitive deficits in aged APP transgenic
(Tg2576) mice whereas nontransgenic mice are typically able to
learn the task to criterion. These changes were comparable in
magnitude to those obtained previously with the working memory
version of the task. If additional discrimination of learning capacity
is deemed necessary, mice can be tested for reversal on day 3 and
possibly day 4 by specifying a new goal arm for each mouse. After
RAWM testing is completed, mice are administered a visible plat-
form task in an open pool to ensure that all of the mice are able to
swim and see the platform. This is important as many inbred
mouse lines (CBA/J, SJL/J, FVB/NJ, BUB/BnJ, some Swiss Webster)
inherit the retinal degeneration mutation (rd1), which, when
homozygous, causes blindness at an early age11,12.
60° 60°
cm
30–
35
40 cm
60° 60°
m

Although the working memory version of the task succeeded in

5c
–1

identifying memory deficits in APP mice, the multiple days of 20–25 cm

13

60° 60°

Figure 1 | A schematic of the RAWM alley and inserts dimensions. The inserts
should measure 30–35 cm on each leg and be bent to an angle of 601. The
alleys should measure 20–25 cm across and 30–35 cm long. The central
swimming area should be 36–40 cm in diameter. The size of the escape
platform is also included for reference.

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The first day of the 2-day protocol is a training day where mice
are shaped to identify platform location by alternating between a
visible and a hidden platform in the goal arm. The final trials on
day 1 and all 15 trials on day 2 use a hidden escape platform to force
mice to use a spatial strategy to identify the goal arm location. To
MATERIALS
REAGENTS
Mice We have used APP mice derived from the Tg2576 line1 and mice with
both the APP transgene and a presenilin-1 mutant transgene5. We typically test
these mice on the balance beam, Y-maze and rotorod in the week before water
maze testing. This serves to acclimatize mice to the experimenter and permits us
© 2006 Nature Publishing Group http://www.nature.com/natureprotocols
to identify mice with severe motoric abnormalities that would preclude water
maze testing. ! CAUTION All animals must be used in a manner that is compliant
with national and local animal care and use guidelines
EQUIPMENT
. Child’s wading pool or other vertically walled container with an interior
diameter of 0.9–1.2 m and a depth of 23–25 cm. Larger sizes may be used,
but we have no experience other than with pools of this diameter
. Six identical metal or black plastic inserts (approximately 20–23 cm
high by 60–75 cm long before bending (depending on pool diameter and
depth) bent at a 601 angle (forming the shape of a V). ! CAUTION The
inserts should rise above the level of the water by at least 6 cm to restrict
mice from climbing over the inserts and gaining access to blocked areas
of the pool
. Black marine paint and primer
. An elevated platform for the pool to rest on (approximately 75 cm tall)
. Black painter’s tarp (2–6 ml thickness) if the pool is not a solid
color or painted a solid color and duct tape (available at local hardware
stores)
. Black and white electrician’s tape, for marking the visible platform
. Black spray paint
. Two circular/cylindrical objects with holes in them to allow submersion.
One should be slightly deeper than the other to allow one to be visible
and one hidden (we use terra cotta plant pots)
. On each of the four surrounding walls and the ceiling, different visual cues
should be used. Shower curtains are useful for creating these visual cues.
A large black square spray painted on a light-colored wall can also be used
on one side
. A score sheet for recording the number of arm entries, which also specifies
the goal arm location for each mouse and the start arm locations for each
trial. m CRITICAL It is important to remember that each mouse has the same
goal arm throughout testing, whereas its start arm varies on each trial. The
goal arm will be in a different location for successive mice to preclude the
use of intramaze odor cues
. Plastic (water-resistant) clipboard for score sheets
. Ceiling hooks, metal cable for hanging shower curtains
. Clear 100% silicone sealant
. Epoxy/resin glue
. Terra cotta gardening pots (3) with 10 cm height and 13 cm diameter and a
clay saucer with a diameter of 10–15 cm (make sure it fits onto the narrow
end of plant pot)
. A water-resistant/waterproof stop watch or timer able to count in seconds
. Towels and dark-colored washcloths
. Heat source such as a heat lamp, clamp on reflector light (25 cm diameter)
with incandescent 60 W bulb or brooder light/heat lamp designed for
poultry
. A visible platform (we use white LEGOs) for the third day of visual
platform testing
. A pool thermometer to ensure consistent water temperature
Figure 2 | Platforms used in RAWM testing. The first platform on the left is
for open pool testing. The water level reaches about 2 mm below to top of
the platform. The platform in the center is the hidden platform used in the
RAWM. The third platform on the right is the visible platform used in the
radial water maze.
1672 | VOL.1 NO.4 | 2006 | NATURE PROTOCOLS
avoid the learning limitations imposed by massed practice and to
avoid fatigue that may result from consecutive trials, we have
established a spaced practice training schedule by running the
mice in cohorts of 4–5 and alternating different cohorts through
the 15 training trials over a 3-h testing period each day.
EQUIPMENT SETUP
Pool The pool should be painted black with marine paint to remove any visual
cues present if a pool pattern exists. Follow the manufacturer’s recommenda-
tions for optimal longevity of the paint (may need a primer). If marine paint is
not available, black painter’s tarp can be cut to fit the pool, but must be changed
regularly for cleanliness. ! CAUTION Paint should be applied in a well-ventilated
area and a respirator should also be worn.
Inserts Aluminum or other metals can be used for inserts if stainless steel
or plastic is not available. However, other metals will pit and corrode over time
in the water and may not meet animal housing protocols. For a pool with a
diameter of 1 m, the metal or plastic for the inserts should be cut into six strips
20–25 cm wide (depending on the depth of the pool; inserts should be slightly
lower than the walls of the pool so that mice can locate external cues, but high
enough that they cannot climb over them while swimming) and 70 cm long and
then bent in the center to 601angle. Once bent, the sides of each insert should be
approximately 35 cm long. Larger or smaller pools will require longer or shorter
inserts. If using stainless steel, no other support is needed. If using softer, more
flexible metal or plastic, it is strongly suggested that a cross-support beam be added
to one side of the insert to keep the legs of the insert stable. This can also serve
as a site where weights can be placed to hold the inserts stable in the pool. Place
the inserts into the pool so that there are six alleys of approximately 18–20 cm
width and a clear center area of approximately 36–40 cm diameter (Fig. 1).
? TROUBLESHOOTING If the edges of the inserts are not flush with the vertical
side of the pool, duct tape can be applied and cut to fill in the gap (to prevent
mice from getting into the center of the inserts). Visual cues should be hung
or placed on the walls around the pool. m CRITICAL The experimenter serves
as a visual cue on one side of the pool and should stay in this place as much
as possible.
Platform The pool should rest on a raised plastic platform of about 75 cm to
allow easier access by the experimenter. ! CAUTION Materials used to construct
the platform should be durable, easily cleaned and able to support 20 gallons of
water (or about 160 pounds). Check with your animal housing specialists for
allowable materials.
Pool thermometer A pool thermometer is required to monitor pool
temperature (the pool temperature should typically be 20.5 1C or 69 1F).
Terra cotta pots Terra cotta pots (10 cm diameter) can be used for all platforms
(Fig. 2). Spray paint the first with black using regular spray paint and allow to
dry. Glue a 10–15 cm clay saucer (we use the base of a terra cotta pot) to the
bottom of the second pot using 100% clear silicone sealant to raise it above the
top of the water. Mark with alternating black and white stripes using an electrical
tape. The electrical tape may be glued to the pot using 100% clear silicone sealant
for added adhesiveness and silicone ‘‘feet’’ can be added to the pot rim to
add additional height to the pot if needed. Also, spray paint the third pot
black and then glue white LEGOs (copyright) to the top using epoxy/resin
for raised visual cues.
 PROTOCOL

PROCEDURE
Preparation
1| A day or two before testing is to start, the pool should be inspected for any damage or leaks.
2| If the pool is painted black, fill with water to approximately 10 cm from the top. If the pool is not of a solid color, then
cover the pool with black painter’s tarp and add water.
! CAUTION The water should be high enough to just cover the hidden platform by about 0.5 cm, but low enough that the
visible platform is not covered.
3| Add the pool inserts and measure each alley to ensure they are all of a similar width. Adjust as necessary: if using painter’s
tarp, the stainless inserts will hold the tarp in place; if using lighter material inserts, small weights (filled mouse cage water
bottles work) may need to be placed on the insert cross-support beam to hold the insert and tarp in place. Trim excess tarp off
and tape to the exterior of the pool using duct tape (Fig. 3).
© 2006 Nature Publishing Group http://www.nature.com/natureprotocols

4| Place pool thermometer in the pool in an area not visible to the mice (behind the inserts).
5| Add the visible and hidden platforms, so they may equilibrate to pool temperature.
6| Assign each alley a number or letter (1–6 or A–F). When first performing this task, it may be helpful to place a small piece
of white tape with the label for the alley on the pool edge (out of sight of the mouse).
7| Prepare the score sheets for each day and each group of mice. Typically, data for eight mice will fit in one sheet of paper (Fig. 4).
’ PAUSE POINT Once the pool is set up, the investigator may leave the pool in this condition indefinitely. Be certain to check
the pool temperature before resuming with the testing procedure, and make certain water is clean.

Day 1
8| Divide eight mice into two cohorts of four, which are mixed with respect to experimental treatment and/or genotype.
The tester should be unaware of the experimental treatments for specific mice. The simplest way to achieve this ‘‘blinding’’
procedure is for someone else to color or number/letter code the cages of each experimental group. It is important, however,
that experimental groups be intentionally balanced across the days and cohorts of testing to minimize time of day or other
cohort-specific effects.
9| Fill in the mouse identification numbers in the appropriate slots on the score sheet.
10| Place each mouse into a cage empty of bedding and lined with a dark dry terry cloth washcloth.
11| Place the cages under a heat source while testing. Each lamp should warm four cages. Make sure the mice have an area
they can retreat to if they get too warm.

Trial 1: Visible platform

12| Place visible platform in goal location for mouse being tested as specified on the score sheet.

13| Beginning with the visible escape platform in the

assigned arm, place mouse 1 of cohort 1 gently into the pool
4
near the perimeter of wall of the first start arm (specified on
the score sheet) and facing the center of the pool.
3 5
14| Begin timing. The trial should last up to 60 s.
15| Count the number of incorrect arm entries made. Incorrect
arm entries occur when the mouse selects an arm that is not
the goal arm. Entries into the goal arm are not counted as
errors, even if the platform is not located. An entry is consi-
2
dered to occur when all four legs of the mouse have entered 6

the alley completely and the mouse is parallel to the alley

walls. Failure to select an arm after 15 s is counted as an error.
16| After 1 min, if the platform has not been located, guide
the mouse gently through the water by placing a hand behind 1

the mouse to direct its swimming direction toward the platform.

Figure 3 | RAWM after completing setup. Labels can be placed on the outer
17| Place the mouse on the platform. edges of the pool out of sight of the mice in the pool.

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Anim. t1 Visible t2 Hidden t3 Visible t4 Hidden t5 Visible t6 Hidden

G # S T E N S T E N S T E N S T E N S T E N S T E N
6 CR-1 1 60 4 s 3 60 4 s 5 60 4 s 2 4 1
2 CR-2 3 60 6 5 55 7 6 60 4 4 1 3
4 CR-3 5 45 3 2 60 4 6 55 6 1 3 5
1 CR-4 2 45 2 4 34 2 6 22 1 3 5 2
5 4 1 3 6 2 4
3 1 6 5 2 4 1
6 CR-5 3 60 9 5 60 11 2 60 7 4 1 3
2 CR-6 5 60 4 fl 6 55 3 fl 4 60 4 fl 1 3 5
4 CR-7 5 50 4 2 45 5 6 52 3 1 3 5
1 CR-8 2 7 0 4 55 4 6 37 2 3 5 2
5 4 1 3 6 2 4
3 1 6 5 2 4 1
© 2006 Nature Publishing Group http://www.nature.com/natureprotocols

t7 Visible t8 Hidden t9 Visible t10 Hidden t11 Visible t12 Hidden

G # S T E N S T E N S T E N S T E N S T E N S T E N
6 CR-1 3 5 2 4 1 3
2 CR-2 5 6 4 1 3 5
4 CR-3 2 6 1 3 5 2
1 CR-4 4 6 3 5 2 4
5 4 1 3 6 2 4
3 1 6 5 2 4 1
6 CR-5 3 5 2 4 1 3
2 CR-6 5 6 4 1 3 5
4 CR-7 5 2 6 1 3 5
1 CR-8 2 4 6 3 5 2
5 4 1 3 6 2 4
3 1 6 5 2 4 1

t13 Hidden t14 Hidden t15 Hidden

G # S T E N S T E N S T E N
6 CR-1 5 2 4
2 CR-2 6 4 1
4 CR-3 6 1 3
1 CR-4 6 3 5
5 3 6 2
3 1 6 5
6 CR-5 3 5 2
2 CR-6 5 6 4
4 CR-7 5 2 6
1 CR-8 2 4 6
5 4 1 3
3 1 6 5
Note : T = time, E = error, N = notes, S = start arm cl = climber, h = hanger, f = floater, s = spins
G = goal location, N = animal number

Figure 4 | Image of a score sheet for the RAWM task. The goal location column is labeled G and stays the same for each mouse. The time recorded goes into
T, number of errors goes into E (which changes for each trial) and any notes pertaining to a mouse go into N (such as floater, spinner, jumper, climber, hanger,
lesions, tumors, cloudy eyes, etc.). Notes for abbreviations can be included at the bottom of the score sheet. A score sheet file is included as Supplementary material.

18| Whether the mouse was guided or the mouse located the platform within the 1 min, allow the mouse to stay on the plat-
form for 15 s. Record the number of errors and time to complete the trial on the score sheet.
19| Remove the mouse and gently towel dry before placing back into its cage under a heat source, such as a heat lamp.
20| Move visible platform to new goal location assigned to the next mouse as specified on the score sheet.
21| Take mouse 2 of cohort 1 (or the next mouse) and repeat Steps 12–20 ensuring that the escape platform has been moved
to its correct location for this mouse.
m CRITICAL STEP It is essential that the tester remains in the same position throughout testing, as the tester acts as an addi-
tional visual cue for the mice.
22| Repeat Steps 12–21 for mice 3 and 4 from cohort 1.

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Trial 2: Hidden platform

23| After all four mice in the first cohort have had a trial, switch platforms from visible to hidden.
24| Place hidden platform in goal arm location appropriate for the mouse being tested.

25| Beginning with mouse 1 cohort 1, repeat Steps 12–21.

26| Repeat Steps 12–21 for mice 2–4 from cohort 1.

Trial 3: Visible platform

27| After all four mice in the first group have had a trial, switch platforms from hidden to visible.

28| Beginning with mouse 1 of cohort 1, place the visible platform again in its assigned arm and perform the test as in
© 2006 Nature Publishing Group http://www.nature.com/natureprotocols

Steps 12–21.
29| Repeat for mice 2–4 from cohort 1.

Trial 4: Hidden platform

30| After all four mice in the first cohort complete trial 3, switch platforms from visible to hidden.

31| Beginning with mouse 1 of cohort 1, place the hidden platform again in its assigned arm and perform the test as in
Steps 12–21.
32| Repeat for mice 2–4 from cohort 1.

Trial 5: Visible platform

33| After all four mice in the first cohort have completed trial 4, switch platforms from hidden to visible.

34| Beginning with mouse 1 of cohort 1, place the visible platform again in its assigned arm and perform the test as in
Steps 12–21.

35| Repeat for mice 2–4 from cohort 1.

Trial 6: Hidden platform

36| After all four mice in the first cohort have completed trial 5, switch platforms from visible to hidden.

37| Beginning with mouse 1 of cohort 1, place the hidden platform again in its assigned arm and perform the test as in
Steps 12–21.

38| Repeat for mice 2–4 from cohort 1. At this point, mice in cohort 1 have received six trials.

39| Keeping the mice from cohort 1 warm, repeat Steps 12–38 with the mice from the second cohort for trials 1–6. This allows
the mice from cohort 1 to rest while mice from cohort 2 are tested.

40| Keeping the mice from cohort 2 warm, repeat Steps 12–38 with the mice from the first cohort for trials 7–12. This is now
cohort 2’s rest period.

41| Keeping the mice from cohort 1 warm, repeat Steps 13–38 with the mice from cohort 2 for trials 7–12. This allows the mice
from cohort 1 to rest while mice from cohort 2 are tested.

42| Repeat Steps 12–29 again for cohort 1 ensuring that this time all trials are performed using the hidden platform and mice
only receive three trials.

43| Repeat Steps 12–29 again for cohort 2 ensuring that all trials are performed using the hidden platform and mice only
receive three trials.

44| Allow mice to dry off and warm up before placing back in home cages.

45| Clean pool of any debris left by the mice.

’ PAUSE POINT This can be for up to 21 h. Often one set of 8–10 mice can be tested in the morning and a second set in the
afternoon. In different sets of mice, try to shuffle the sequence of the experimental group to avoid having mice from the same
experimental group going first or last in all cohorts.

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Day 2
46| Repeat day 1 Steps 12–45 using the same mice in exactly the same groups except this time using only the hidden platform
for all 15 trials again in blocks of six, six and three.
47| Calculate the average errors to find the platform for each mouse in blocks of three trials. Average trials 1–3, 4–6, 7–9,
10–12 and 13–15 for each day of testing separately.
48| At this stage, more days of testing can be added if many mice have not reached a criterion of one or fewer errors in the
final block(s) of three trials. In most of our studies, this extent of training is adequate to discriminate mice learning normally
from those with deficits. However, there are two options one may consider for additional training. If control mice have not
reached or come close to the learning criterion, additional training may be necessary (option A). Conversely, if all groups
learned and increased task difficulty is desired, this may be achieved by performing reversal training with a new platform loca-
tion for each mouse (option B).
© 2006 Nature Publishing Group http://www.nature.com/natureprotocols

Option A: Additional hidden platform training

(i) Repeat Steps 46–48 on day 3 (identical to day 2 of testing) with the animals receiving 15 additional trials using a hidden
escape platform and having the same platform locations as days 1 and 2 of testing.
Option B: Reversal training
(i) Extend testing by adding a third day of testing with the mice receiving 15 additional trials using only a hidden platform
but having new platform locations to learn (reversal training), repeating all Steps on day 2 (Steps 46–48). The new goal
arm should be at least two alleys away from the previous location for each mouse. For example, a mouse that had alley
one as the goal arm would change to alleys 3, 4 or 5 but not in alley 2 or 6 (neighbors).
(ii) If, after a third day of testing, the mice have not reached a learning criterion of less than one error on average for the
last block, a fourth day of testing may be added for option A or B. The fourth day would be exactly the same as day 3,
using all hidden platforms with the same locations as for day 3.
’ PAUSE POINT This can be for a period of up to 21 h, or until a second cohort is tested.
Open pool task with visible platform
49| This task is used to confirm that deficits found in the hidden platform task are not caused by sensory deficits or
performance deficits in the mice. It should be noted that some APP transgenic mice exhibit an initial neophobia to the visible
platform and appear to avoid it. Although this may initially cause longer escape latencies, these largely disappear over the 15
trials of visible platform training. First, remove the inserts from the pool used to perform the RAWM.
50| Using lines drawn on small pieces of tape on the edge of the pool, divide it into equal quadrants.
51| Remove as many extra-maze cues as possible.
52| Prepare a score sheet with the mice in the same cohorts as for the RAWM testing (Fig. 5).
53| Place flagged platform in the quadrant location for mouse being tested. The platform should be near the middle of the
quadrant. Fill pool to about 2 mm below the top of the pot.
m CRITICAL STEP It is important to remember that each mouse has the same start position throughout testing whereas its goal
position varies on each trial. The goal position will be in a different location for successive mice.
54| Place mouse 1 of cohort 1 gently into a quadrant (by the quadrant marker), as assigned on the score sheet, facing the wall
of the pool and start the stop watch. Record how long it takes the mouse to find the platform. Record the escape latency. Gently
guide the mouse to the platform if the mouse does not escape within 60 s.
55| Gently dry the mouse with a small towel and place in its cage under a heat source.
56| Move platform to quadrant location for the next mouse in the cohort.
57| Repeat Step 54–56 for mice 2–4 of cohort 1.
58| After all four mice in the cohort have had a trial, begin again with mice 1–4 of cohort 1 and repeat Step 54–56 for each mouse.
59| Repeat until the mice in cohort 1 have performed six trials.
60| Repeat Steps 54–59 for the mice from cohort 2.
61| Repeat Steps 54–60 until all mice from both cohorts have received 15 trials. The last block of testing will only contain
three trials as in the RAWM testing period.
62| Clean the pool before putting mice away.
63| Calculate the average time to find the platform for each mouse in blocks of three trials.

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Trial 1 Trial 2 Trial 3 Trial 4 Trial 5 Trial 6

Mouse g s t n g s t n g s t n g s t n g s t n g s t n
CR-1 3 1 20 2 1 26 4 1 19 1 1 3 1 4 1
CR-2 2 3 55 4 3 42 1 3 48 3 3 4 3 2 3
CR-3 4 2 13 1 2 16 3 2 8 4 2 2 2 3 2
CR-4 1 4 6 3 4 11 4 4 7 2 4 3 4 1 4
3 1 4 1 2 1 3 1 1 1 4 1
4 3 2 3 3 3 1 3 4 3 2 3
CR-5 2 2 40 3 2 45 1 2 37 4 2 2 2 4 2
CR-6 3 4 33 1 4 29 4 4 22 2 4 4 4 3 4
CR-7 3 1 27 2 1 17 4 1 12 1 1 3 1 4 1
CR-8 2 3 5 4 3 15 1 3 9 3 3 4 3 2 3
4 2 1 2 3 2 4 2 2 2 3 2
© 2006 Nature Publishing Group http://www.nature.com/natureprotocols

1 4 3 4 4 4 2 4 3 4 1 4

Trial 7 Trial 8 Trial 9 Trial 10 Trial 11 Trial 12

Mouse g s t n g s t n g s t n g s t n g s t n g s t n
CR-1 2 1 3 1 1 1 4 1 2 1 4 1
CR-2 3 3 1 3 4 3 2 3 4 3 3 3
CR-3 1 2 4 2 2 2 4 2 3 2 1 2
CR-4 4 4 2 4 4 4 3 4 1 4 2 4
2 1 4 1 3 1 1 1 2 1 1 1
4 3 3 3 1 3 2 3 1 3 2 3
CR-5 3 2 1 2 2 2 1 2 2 2 4 2
CR-6 1 4 2 4 1 4 2 4 4 4 1 4
CR-7 2 1 3 1 1 1 4 1 2 1 4 1
CR-8 3 3 1 3 4 3 2 3 4 3 3 3
1 2 4 2 2 2 4 2 3 2 1 2
4 4 2 4 4 4 3 4 1 4 2 4

Trial 13 Trial 14 Trial 15

Mouse g s t n g s t n g s t n
CR-1 3 1 1 1 2 1
CR-2 1 3 2 3 1 3
CR-3 2 2 1 2 2 2
CR-4 1 4 2 4 4 4
2 1 4 1 1 1
4 3 1 3 3 3
CR-5 1 2 3 2 4 2
CR-6 3 4 4 4 2 4
CR-7 3 1 1 1 2 1
CR-8 1 3 2 3 1 3
2 2 1 2 2 2
1 4 2 4 4 4
Note : T = time, E = error, N = notes, S = start arm cl = climber, h = hanger, f = floater, s = spins
G = goal location, N = animal number

Figure 5 | Image of a score sheet for the open pool task. The goal location is labeled as G and changed for each trial. The start location is labeled as S and
stays the same for each trial. The time recorded for each trial goes into the column labeled T. Notes for any of the mice can go into the column labeled N. A file
is included as supplemental material.

64| Average trials 1–3, 4–6, 7–9, 10–12 and 13–15.

! CAUTION Mice with long escape latencies (420 s) on the last block of trials may have a swimming or visual deficit, which
would have affected their performance in the RAWM. These mice are excluded from RAWM analyses.
? TROUBLESHOOTING

TIMING
Step 1: 10–20 min if the pool is intact
Steps 2–7: about 30 min day1
Steps 8–45: approximately 3.5 h day1 to test eight mice on day 1; a second group of eight mice would take an additional 3.5 h

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? TROUBLESHOOTING Figure 6 | Performance 6

Biological of mice lacking normal 5

If mice are not swimming or swimming in circles, try poking vision. Eight-month-old 4
mice derived from
them gently, dripping some water on their heads or dunking

Errors
breeding APP Tg2576 3
them briefly underwater. If a large percentage of the mice are mice with tauP301L 2
not swimming, lowering the water temperature may act as a mice13 were tested for
transgene status and 1
greater motivating factor. Mice that fail to swim on a majority
of the trials are excluded from data analysis. Mice that fail to the rd1 mutation by 0
1 2 3 4 5 6 7 8 9 10
genotyping. Data
swim occasionally end up with a score of four errors on the shown here are for mice
Day 1 Day 2
Blocks
trial owing to assignment of one error for each 15 s without that are nontransgenic
an arm entry. (solid squares; n ¼ 8)
If a mouse starts to swim underwater, manually bring the or homozygous for the rd1 mutation (open circles; n ¼ 8). *P o 0.05
© 2006 Nature Publishing Group http://www.nature.com/natureprotocols

compared to nontransgenic mice. Data are mean ± s.e.m.

mouse to the surface after 10 s. Mice that persistently swim
underwater should be removed from the data analysis (we had one mouse that would swim to the center of the pool, submerge,
spin around to find the platform and surface to swim to the platform).
If a mouse is not able to climb onto the platform, gently help the mouse climb up after ascertaining that the platform has
truly been found based on the orientation of the mouse. If after several trials the mouse still needs help climbing up, continue
to help but note down difficulties on the score sheet.
If a mouse continuously tries to climb the pool walls or hangs on the inserts, try to encourage swimming behavior by gently
pushing the mouse away from the pool walls and inserts. Be sure to make a note on the score sheet if the mouse continues to
try and climb out of the pool.
If a mouse fails to wait on the platform for 15 s, gently place the mouse back onto the platform. If the mouse is jumping off,
the experimenter may need to restrain the mouse to the platform without blocking visual cues as much as possible. This is gen-
erally done by holding the tail of the mouse. Another option is to place a hand in front of the mouse just before the mouse
jumps as a distraction.
Make sure to note down on the score sheet any mouse that exhibits atypical behavior such as swimming in circles, jumping,
refusing to stay on platform, floating or climbing.
Technical
If there is a leak in the pool, then silicone or epoxy resin can be used to seal the pool depending on the size of the leak.
If inserts are displaced when mice swim or platforms are moved, add small water-resistant weights to hold the inserts in place.

ANTICIPATED RESULTS
Data analysis
The number of errors should be entered into the preferred statistical program (we use Statview by SAS; Chicago, IL), and then
averages for each mouse should be calculated using the three-trial blocks as specified above for each day that testing was
performed. Mice can then be sorted into appropriate experimental treatment and/or genotype groups to allow collection of
descriptive statistics (mean, SEM, etc.) and graphing. ANOVAs
Figure 7 | Results of 8
and post hoc mean tests may then be performed on the 7
2-day-RAWM testing in
resulting group data. We strongly urge investigators to use a APP transgenic mice. 6
single measurement when calculating their results rather than Data are taken from 5
Errors

seeking a measure that yields a significant result by analyzing Wilcock et al.7 with 4

multiple measurement methods (errors, time, trial 1 minus permission. These data 3
show RAWM results 2
trial 5, etc.). Robust effects should be significant irrespective
from four groups of 26- 1
of the measurement used. Moreover, it is preferable to report month-old mice. The 0
1 2 3 4 5 6 7 8 9 10
the results for individual tests, rather than aggregating them triangles are data from Blocks
into a discriminant function analysis, where one unusual test nontransgenic mice Day 1 Day 2

result can have an untoward impact on the final outcomes
(such analysis is acceptable as an adjunct to reporting
individual values). We have found errors to be the most
sensitive measure and have consistently used that
measurement in all our publications.
Expected outcomes
Mice that have learned the RAWM will exhibit performance
errors of 1 or less, averaged over three trials near the end of
the second day (Figs. 6 and 7). Mice that fail to learn well
that show improving
performance over
blocks (P o 0.01, ANOVA), reaching the learning criterion (o 1 error) by
block 8–9 on day 2, whether treated with anti-Ab antibody (filled triangles)
or control antibody (open triangles). APP transgenic mice that were treated
with a control antibody for 5 months (open circles; weekly injections of
10 mg kg1, see ref. 7). These mice fail to reach criterion over the 2 days
of testing. The filled circles represent APP transgenic mice treated for
5 months with an antibody directed against the Ab peptide. These mice
also reach the learning criterion by the end of day 2. *P o 0.05; **P o 0.01
between APP anti-Ab antibody treated and APP control antibody treated.
Data presented are mean ± s.e.m.

1678 | VOL.1 NO.4 | 2006 | NATURE PROTOCOLS


will exhibit 3–5 errors throughout the training session, without apparent improvement over trials. Mice that are not
having swimming or vision difficulties usually find the platform in 45 s or less in the open pool. Mice that score over 45 s
throughout the visible platform testing may be physically or visually impaired, unable to learn intramaze cues or be phobic
of the ‘‘flagged’’ platform.
Note: Supplementary information is available via the HTML version of this article.
ACKNOWLEDGMENTS We have been supported by NIH awards AG15490, AG18478,
AG25509, AG25711 and NS48335. D.W. was a Benjamin Scholar in Alzheimer’s
Research.
COMPETING INTERESTS STATEMENT The authors declare that they have no
competing financial interests.
© 2006 Nature Publishing Group http://www.nature.com/natureprotocols
Published online at http://www.natureprotocols.com
Reprints and permissions information is available online at http://npg.nature.com/
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