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Drug-induced conditioned place preference and

aversion in mice
Christopher L Cunningham, Christina M Gremel & Peter A Groblewski
Department of Behavioral Neuroscience and Portland Alcohol Research Center, Oregon Health & Science University, Portland, Oregon 97239-3098, USA.
Correspondence should be addressed to C.L.C. (cunningh@ohsu.edu)

Published online 16 November 2006; doi:10.1038/nprot.2006.279

This protocol describes the equipment and methods used to establish conditioned place preference (CPP) or aversion (CPA). Place
© 2006 Nature Publishing Group http://www.nature.com/natureprotocols

conditioning is a form of Pavlovian conditioning routinely used to measure the rewarding or aversive motivational effects of objects
or experiences (e.g., abused drugs). Here, we present a place conditioning procedure that has been used extensively to study the
motivational effects of ethanol and other abused drugs in mice. This protocol involves three phases: (i) habituation (or a pretest),
(ii) conditioning of an association between the drug and a tactile or visual stimulus and (iii) a test that offers a choice between the
drug-associated cue and a neutral cue. If the drug has motivational significance, mice will spend significantly more time (CPP) or less
time (CPA) in proximity to the drug-associated cue. Potential problems in the design and interpretation of place conditioning studies
are discussed. A typical experiment lasts 2 weeks.

INTRODUCTION
Place conditioning is most often used with rodents (rats, mice) to
study the positive (rewarding) or negative (aversive) motivational
effects of objects (e.g., food pellets, novel toys) or experiences (e.g.,
brain stimulation, drug intoxication, drug withdrawal, footshock,
illness, wheel running and copulation). The standard procedure is a
form of Pavlovian (classical) conditioning in which a distinctive
environmental cue (the positive conditioned stimulus or CS+)
becomes associated with a motivationally significant event (the
unconditioned stimulus or US). As a result of this association, the
CS is assumed to acquire the ability to evoke a conditioned
motivational response similar to that elicited by the US itself.
The procedure measures the valence of this conditioned motiva-
tional effect by offering the animal a choice between a spatial
location containing the CS+ and an adjacent spatial location
containing a different stimulus that has not been associated with
the US (the negative conditioned stimulus or CS
). When the US
is rewarding, rodents are more likely to approach and stay in
contact with the CS+ (CPP). In contrast, when the US is aversive,
rodents are more likely to escape and avoid contact with the CS+
(CPA).
Although many different types of events have been shown to be
effective USs for place conditioning, the technique has been most
widely used to study the motivational effects of various drugs,
especially abused drugs1. The principal advantages of place con-
ditioning over other procedures used to study the rewarding/
aversive effects of drugs are its (i) methodological simplicity (e.g.,
no need for complicated surgical procedures; simple, relatively
inexpensive equipment), (ii) potential for high throughput because
experiments are typically short (e.g., 2 weeks), (iii) ability to
measure rewarding and aversive effects in the same procedure,
(iv) ability to test drug-induced conditioned rewarding/aversive
effects in the absence of the target drug, thereby avoiding
confounds due to sensory/motor impairment, (v) sensitivity to
low-dose effects, (vi) ability to measure drug reward/aversion in
drug-naı̈ve animals (i.e., in the absence of tolerance or sensitiza-
tion), sometimes after only one conditioning trial, (vii) ability to
concurrently measure drug-induced changes in locomotor activity
(and tolerance/sensitization with repeated drug exposure), (viii)
adaptability for use with many different species (e.g., rodents, birds,
snakes) and genotypes within species, including genetically
modified animals, (ix) monophasic dose–effect curves and (x)
utility for studying the neurobiology of drug reward/aversion2,3.
Another important advantage is that the place conditioning pro-
cedure readily allows one to separately examine manipulations that
affect the initial learning (acquisition) of the cue–drug association
and manipulations that affect the performance (expression) of
approach/avoidance responses based on that learning4.
Despite its many advantages, there a few potential disadvantages
linked to the use of the place conditioning procedure. For example,
although dose–effect curves are generally monotonic, they are
sometimes step-like, showing little effect across a range of low
doses before suddenly showing a maximal effect across a range of
higher doses5. Nevertheless, meta-analyses have revealed graded
dose–effect curves for many different abused drugs, with the
notable exception of cocaine6. Another potential disadvantage is
the need to invest time in constructing an apparatus and stimuli
that do not produce an initial (unlearned) bias for one of the
stimulus alternatives. Even when an unbiased apparatus is used,
care must be taken in the experimental design and procedure to
provide adequate controls for evaluating or eliminating potential
confounding influences on expression of preference for the drug-
paired stimulus (e.g., stimulus novelty, alterations in locomotor
activity). Strategies for addressing these issues are discussed below.
Finally, because the experimenter (and not the subject) controls
drug exposure in place conditioning studies, the procedure has
sometimes been criticized as being less relevant to the study of drug
reinforcement than procedures in which animals self-administer
drug7. Although arguable, this viewpoint is not consistent with
contemporary approaches that recognize the important contribu-
tion of Pavlovian learning processes to drug addiction8,9.
The most commonly used alternative procedure for studying
the motivational effects of abused drugs is self-administration.
Although the behavior underlying self-administration models may
be classified as involving both drug-seeking and drug-taking

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behaviors, dissociation of these two behaviors is sometimes diffi-
cult3. It seems quite likely that drug-seeking behavior observed in
self-administration studies is under control of conditioned envir-
onmental cues that signal the availability of drug. Place condition-
ing is a useful model for studying the effects of environmental cues
associated with a drug experience2. Additional discussions of the
place conditioning technique and comparisons between place
conditioning and other measures of reward can be found in several
critical review papers1,6,10,11. Discussions of the behavioral and
neurobiological mechanisms underlying place conditioning are
also available elsewhere12–15.
© 2006 Nature Publishing Group http://www.nature.com/natureprotocols
Experimental design
Biased versus unbiased One of the most critical issues in the
design of place conditioning studies is whether the apparatus and
subject assignment procedure are ‘‘biased’’ or ‘‘unbiased.’’ As
discussed in more detail elsewhere1,2,11,16,17, use of a biased appa-
ratus or biased subject assignment procedure can severely compli-
cate interpretation of place conditioning studies. An apparatus is
considered ‘‘biased’’ when the average time that untrained animals
spend in each test compartment deviates from expectations based
on chance. Conversely, an apparatus is considered ‘‘unbiased’’ when
the average time spent in each compartment is consistent with
expectations based on chance. Thus, for example, a two-compart-
ment apparatus in which untrained animals spend an average of
50% time in each compartment would be considered unbiased.
One of the primary reasons for using an unbiased apparatus is to
avoid complications in measurement due to ‘‘floor’’ and ‘‘ceiling’’
effects16,17. Because learned preferences and aversions generally
appear to be superimposed on unlearned preferences (biases)16,
use of an unbiased apparatus should increase the ability to detect
both conditioned preferences and conditioned aversions.
Although it is linguistically convenient to describe bias as a
property of the apparatus, it is important to note that bias is
more appropriately viewed as a characteristic of the subject
population. That is, based on their average behavior, it is the
subject population that displays bias or lack of bias in a particular
apparatus. Appreciation of this point is critical when conducting
studies that compare subject populations that may differ in
their bias for a set of environmental cues (e.g., strain A versus
strain B; wild type versus knockout; sham versus lesion). Thus,
apparatus bias should be assessed for every subject population
under study.
It is also important to note that when an apparatus is labeled
unbiased, that does not mean that every individual within the
subject population distributes its behavior equally among the
stimulus alternatives. Indeed, as with most types of animal beha-
vior, time spent in each compartment approximates a normal
distribution16,18. Thus, even though the population mean might
indicate that an apparatus is unbiased, there will be individuals at
the tails of the distribution who will show preferences for one
compartment over another. To address this issue, we and others
recommend the use of an ‘‘unbiased’’ subject assignment procedure
in addition to using an unbiased apparatus3,16,17. The subject
assignment procedure is said to be unbiased when the specific
stimulus selected as the CS+ is assigned randomly, regardless of the
subject’s unlearned preference/aversion for that stimulus. Our
studies typically use a counterbalanced experimental design in
which one stimulus is randomly assigned as CS+ for half of the
PROTOCOL
subjects; the other stimulus serves as CS+ for the remaining
subjects. In contrast, the assignment procedure is considered biased
when the selection of the CS+ is based on the subject’s unlearned
preference or aversion for that stimulus. For example, some
experimenters will pair the US with whichever stimulus is initially
non-preferred. The interpretive danger in using a biased subject
assignment procedure is that US effects may then be attributed in
whole or in part to the ability of the US to reduce (or enhance) the
unlearned motivational response to the CS, rather than to the
presumed ability of the US to directly elicit rewarding or aversive
effects2,16. This interpretive complication is substantially reduced
when the stimulus serving as CS+ is assigned randomly in a
counterbalanced, unbiased subject assignment procedure.
Temporal parameters There are several important temporal
relationships in place conditioning procedures. These include (i)
the time interval between exposure to the drug and CS (‘‘inter-
stimulus interval’’), (ii) the duration of exposure to the CS (‘‘trial
duration’’) and (iii) the time interval between consecutive con-
ditioning trials (‘‘inter-trial interval’’). In most place conditioning
studies with drugs, the drug US is administered immediately before
the animal is placed into the conditioning compartment containing
the CS+. Thus, blood and brain drug concentrations are typically
rising during the initial part of the trial. However, depending on the
nature of the drug and route of administration, it may be necessary
to insert a short time delay between drug administration and
exposure to the CS to allow time for drug absorption19. The
order of exposure to the drug and CS can also have a profound
effect on the direction of the conditioned response. For example,
with ethanol, amphetamine and nicotine, pre-CS injection of drug
has been found to produce CPP, whereas post-CS injection of drug
has been found to produce CPA20–23.
As mentioned earlier, duration of exposure to the CS is an
important variable that can significantly affect the strength of CPP
and CPA. Previously, we found that a relatively short trial duration
(5 min) is most effective when using intraperitoneal (i.p.) ethanol
to induce CPP in DBA/2J mice24 and other mouse strains. How-
ever, we found that a much longer trial duration (60 min) is better
when cocaine is used to induce CPP in the same strain25. In
contrast, cocaine-induced CPP is unaffected by trial duration
across a wide range (15–60 min) in a different inbred strain
(C57BL/6J). Such findings underscore the importance of trial
duration.
In our experiments, we usually insert a delay of 24 h (or more)
between successive conditioning trials. Some investigators, how-
ever, expose subjects to two conditioning trials per day (e.g., drug
trial in the morning and vehicle trial in the afternoon). Bevins and
Cunningham17 discuss potential problems with this approach.
Test session duration is another temporal parameter that should
be considered. After using 60-min test sessions in many of our
earlier studies, we now use 30-min tests in most experiments.
Although the pattern of findings from the first 30 min is generally
similar to that seen over a 60-min session, we sometimes observed
a weakening of preference toward the end of the longer session.
In some cases, the longer test session was critical for observing
effects that might not have been detected in a shorter session. For
example, we found that naloxone’s ability to alter expression of
ethanol-induced CPP consistently emerged only during the second
half of a 60-min test session4,26. Had we ended our test after 30 min,
NATURE PROTOCOLS | VOL.1 NO.4 | 2006 | 1663
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we might not have discovered this effect. Thus, in this case, a longer
test session was necessary to detect an important effect.
General protocol
Our protocol for inducing CPP with ethanol typically consists of
three phases scheduled over a 2- to 3-week period: habituation (one
5-min session), conditioning (eight or more 5-min sessions) and
preference testing (one or more 30–60 min sessions). In some
experiments, we substitute a pretest session for the habituation
session (see below). All treatment groups are fully counterbalanced
for stimulus–drug assignment. That is, half of the treatment group is
assigned to the GRID+ conditioning subgroup (grid + drug, hole +
© 2006 Nature Publishing Group http://www.nature.com/natureprotocols
saline) and half is assigned to the GRID
conditioning subgroup
(hole + drug, grid + saline). Each group is also counterbalanced for
the order in which each type of trial is received (drug trial first versus
vehicle trial first) and left–right position of the stimulus alternatives
during testing (e.g., grid on the left side versus grid on the right side).
Our procedure is usually conducted 5 days per week with a 2-day
(weekend) break. One important issue that must be resolved is the
scheduling of cage changes, as most facilities will require that cages
be changed one or more times during a typical 2-week experiment.
As cage changing is a stressful event, we generally avoid changing
cages during the 72 h before a preference test. Moreover, we always
change cages after the mouse has received whatever treatment is
scheduled on cage-changing day. Most often, cage changes take
place while the mice are in the conditioning box and they are
returned to a clean cage.
Pretest (optional) Usually, we include a pretest session only when
working with new stimuli or a new mouse strain/line for which we
do not have good information on unconditioned preference for the
stimulus alternatives16,18. An alternative approach for collecting
this information is to include a separate control group that is
MATERIALS
REAGENTS
. Mice
. Drug
EQUIPMENT
. Needles and syringes
. Conditioning box
. Interchangeable floor cues (tactile or visual)
. Sound and light attenuating enclosure
. Photo-electronics and computer (or videocamera and recorder) for
recording side position and activity
REAGENT SETUP
Mice We typically use adult (8–10 weeks old) male mice of the DBA/2J strain
obtained from the Jackson Laboratory. This inbred strain has consistently
demonstrated CPP with many rewarding drugs, including ethanol12,16,18,28,
cocaine25 and morphine29. The same strain develops robust CPA in procedures
with aversive drugs like lithium chloride30 and naloxone4. Mice are 6 weeks old
when they are brought to the laboratory and are allowed to acclimatize to the
animal colony for 2 weeks before training. They are housed in groups of four in
polycarbonate cages (27.9  9.5  12.7 cm) with cob bedding at an ambient
temperature of 21±1 1C. Water and lab chow are available at all times in the
home cage. Experimental procedures are conducted during the light phase of a
12-h/12-h light/dark cycle (lights on at 0700 hours). Based on past experience,
we usually assign 12–16 mice to each conditioning subgroup to provide
sufficient statistical power to detect treatment effects in our studies.
The methods described here have also been used successfully to study drug-
induced place conditioning in outbred mouse strains (e.g., Swiss-Webster31),
other inbred stains (e.g., C57BL/6J29, BXD recombinant inbred strains32),
1664 | VOL.1 NO.4 | 2006 | NATURE PROTOCOLS
exposed to the vehicle only on all conditioning and test trials27.
Bevins and Cunningham17 address the advantages and disadvan-
tages of conducting a pretest. The procedure for this session is
identical to that described below for the preference test.
Habituation (day 1) This phase of the experiment precedes the
conditioning phase in order to reduce any effect of experimental
novelty by adapting mice to the procedure (including handling and
injections) and apparatus.
Conditioning (days 2–5, 8–11) This procedure typically consists of
four CS+ trials (trials in which drug is paired with one floor cue) and
four CS
trials (trials in which vehicle is paired with the other floor
cue). These trials occur on alternating days over 10 days (including a
2-day weekend break between conditioning trials 4 and 5). Although
most of our experiments have involved four conditioning trials of
each type, it is possible to induce CPP with ethanol after only two
trials of each type when other conditioning parameters are opti-
mized28. Number of compartments, trial duration and lighting
conditions depend on considerations discussed earlier. The proce-
dure described below assumes use of tactile cues in a non-illuminated
one-compartment training procedure with ethanol as the drug.
Preference test (day 12) Our place conditioning studies typically
conclude with a 30–60 min test that offers the animal a choice
between the CS+ and CS
. The apparatus is configured with one
cue on each side of the apparatus and the left/right position of the
cues is counterbalanced within each group. Test sessions are usually
preceded by an injection of vehicle (or no injection), unless we
are studying the effects of a drug pretreatment on the retrieval or
expression of the conditioned motivational response4. Primary
interest is in the duration or percentage of the session that the
animal spends in contact with each cue.
selectively bred mouse lines (e.g., HOT/COLD33, FAST/SLOW34, HTA/LTA
and STDRHI/STDRLO35), and various knockout and transgenic animal
models27,36–40. However, it is important to note that dose and temporal
parameters found to be optimal may be strain/line specific and may not
be optimal for other strains/lines25. Moreover, even within the same
strain, the optimal temporal parameters may vary widely across drugs.
This issue is discussed in more detail in the section on Experimental design.
! CAUTION Experimenters must comply with national regulations
concerning use of animals.
Route of drug administration Various routes of administration have been used
to produce CPP and CPA, with i.p. injections most commonly used. However,
intragastric infusions, intracranial ventricular infusions, intracranial microinjec-
tions, intravenous infusions, subcutaneous injections and oral self-administration
have also been used1. The choice of administration route will vary depending on
species, strain, drug, pharmacokinetics, technical skill and time constraints. All
factors should be considered when choosing the dosage and route of adminis-
tration. Because the route of administration can affect the time course of drug
absorption, it might be necessary to alter the temporal relationship between drug
and CS exposure in order to produce CPP. For example, in studies involving
intragastric infusion of ethanol, the time interval between ethanol infusion and CS
exposure critically determined whether CPP or CPA was obtained19.
EQUIPMENT SETUP
Conditioning box and enclosure Each conditioning box is 30 cm long  15
cm wide  15 cm high (interior dimensions). The long walls and lid are clear
acrylic (3.2-mm thick) and the end panels are 5052 sheet aluminum (2-mm
thick). These materials were selected because they are relatively inexpensive,
durable and can be easily sanitized to meet institutional standards for use with

laboratory animals. Depending on whether the apparatus will be illuminated
(e.g., in studies involving visual cues) and how the animal’s position will be
determined (discussed below), other approaches to box construction might be
considered (e.g., all acrylic, all aluminum, opaque acrylic).
We enclose each conditioning box in a separate ventilated, light- and
sound-attenuating enclosure (Coulbourn Instruments Model E10-20—inter-
ior dimensions: 56.1 cm wide  46.0 cm high  39.4 cm deep). The box is
carefully positioned within the enclosure to minimize air drafts from the ven-
tilation fan that may influence behavior. Although enclosures increase the
initial cost and the space required to conduct place conditioning studies, we
strongly recommend this approach in order to reduce the likelihood that
uncontrolled extraneous variables will influence the animal’s behavior during
testing (e.g., unexpected laboratory/building noises, experimenter movement,
sound/odor from other animals). We have found that expression of place pre-
ference can sometimes be profoundly disrupted by novel stimuli that seem
© 2006 Nature Publishing Group http://www.nature.com/natureprotocols
relatively innocuous to the experimenter (see ? TROUBLESHOOTING).
General activity and the animal’s position (left versus right side) within the
apparatus are detected via infrared light sources and detectors (total of six sets)
mounted opposite each other at 5-cm intervals along the long walls of the box,
positioned 2.2 cm above the floor. Transitions between sides are recorded when
all photobeams on one side are released and at least one beam on the opposite
side is broken. In our preparation, activity counts and time spent on each side
of the chamber are recorded every minute by computer (10-ms resolution).
The electronic circuits and computer software for continuous automated
recording of the animal’s position and activity were custom-made in our
laboratory at a time when there were few commercially available alternatives.
However, now there are several vendors that sell components (e.g., photode-
tector arrays and electronics) or complete turnkey systems for conducting
place conditioning studies (e.g., MED Associates Inc.; San Diego Instru-
ments). However, these systems vary considerably in the ease with which
stimulus and compartment configurations can be manipulated.
Conditioned stimuli: tactile stimuli Most of our published studies have
involved tactile cues presented on the floor of a non-illuminated box (Fig. 1).
The rationale for and advantages of using tactile cues are described else-
where18,41. The floor of each box consists of interchangeable halves made of one
of two textures. The ‘‘grid’’ floor consists of 2.3-mm stainless steel rods mounted
6.4-mm apart in acrylic rails. When positioned under the box, these rods are
parallel to the end panels. The ‘‘hole’’ floor consists of perforated 16-gauge
stainless steel with 6.4-mm round holes on 9.5-mm staggered centers mounted
on acrylic rails. Each half floor is constructed to be slightly larger than necessary
so that the conditioning box can be placed directly on top of the floor, which
facilitates manipulation of the location and type of tactile cue. The grid and hole
floors were initially selected based on studies showing that drug-naive mice of
several different strains spend an average of about 50% time on each floor type
during preference tests20,29. Thus, the apparatus can be considered ‘‘unbiased’’
for those strains16. For a further discussion of bias in the place conditioning
procedure, see the section on Experimental design.
In most of our published studies with tactile cues, we used a one-compart-
ment training procedure (see refs. 17,18 for discussions of one- versus
Figure 2 | Place conditioning apparatus with visual floor cues (stripes versus
dots).
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Figure 1 | Place conditioning apparatus with tactile floor cues (grid versus
hole).
multi-compartment procedures). That is, during each conditioning trial, both
sides of the box have the same floor texture and the mouse has free access
to the entire box. It is only during the final preference test that the box is
configured with a different texture on each side. Recently, however, we
successfully used a two-compartment training procedure with tactile cues18.
In the two-compartment procedure, the mouse is confined to one-half of
the box (using an acrylic barrier) during the conditioning trials and is
allowed access to the entire box only during the final choice test. The other
compartment is empty during conditioning trials using this procedure
(i.e., there is only one mouse at a time in the apparatus). Based on studies
to date, the magnitude of CPP produced by one- and two-compartment
procedures appears similar when tactile cues are used18. However, this
conclusion must be tempered by the fact that only one inbred strain
(DBA/2J) and one drug (ethanol) have been examined.
Conditioned stimuli: visual stimuli Recently, we completed our first series of
studies with visual CSs18. Our visual cues are black objects printed on white
paper (92 brightness) sealed in clear plastic laminating pouches (3-mm thick).
One cue consists of an irregular pattern of black circles (6.4-mm diameter)
printed in a staggered fashion with centers 9.5–12.7 mm apart (dots). The other
cue consists of 3.2-mm wide black lines printed at 6.4-mm intervals (edge-to-
edge) running parallel to the end panels of the box (stripes). These cues are
placed directly under the conditioning box and serve as the floor during training
and testing (Fig. 2). The floor texture is the same on both sides of the box (i.e.,
the smooth plastic covering). Pretests show that drug-naı̈ve DBA/2J mice spend
an average of about 50% time on each visual cue during choice tests, confirming
that our visual conditioning procedure is also unbiased18. The apparatus is
illuminated by a 4-inch ‘‘Mini Moon Lite’’ (AmerTac Model No. 73060, 3 VDC)
placed on the floor of the enclosure behind the conditioning box. White paper
(20 lb, 92 brightness) is taped to the outside of the clear acrylic walls of the
apparatus above the photodetector arrays to diffuse the light.
In contrast to procedures involving tactile cues, visual cues are effective CSs
for place conditioning only when used in a two-compartment procedure18.
That is, each visual cue must be assigned to a consistent spatial location (always
left side or always right side) during training/testing and the mouse must be
confined to that location (using an acrylic barrier) during the conditioning
phase. In a one-compartment procedure similar to that used in our tactile
cue experiments, we were unable to establish CPP with our visual cues18.
Conditioned stimuli: stimulus compounds In other laboratories, the stimuli
used for place conditioning are often compounds composed of elements from
several different modalities (e.g., visual, tactile and olfactory). In fact, one early
review suggested that using cues that differ on more than one stimulus
dimension might provide a more sensitive measure of place conditioning
(ref. 2, p. 268). However, the published data in support of this suggestion
are quite sparse. Although our own data on this issue are limited, we have not
found any evidence that a combination of visual and tactile cues is better than
either cue alone in our two-compartment procedure. Given the possibility
that different brain mechanisms may underlie conditioning with visual
and tactile cues and that individual mice within strains may differ in their
sensitivity to cues from each modality, experimenters are advised to give
careful consideration to their selection of stimuli in this procedure (for further
discussion of this issue, see ref. 18).
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Unconditioned stimuli As noted previously, the methods described here morphine16,25,29,42. Readers are directed to Tzschentke’s1 in-depth review
have been used successfully to study place conditioning induced by for a more complete listing of other drugs previously used in place
many different drugs including ethanol, cocaine, methamphetamine and conditioning studies.

PROCEDURE
Habituation (day 1)
1| Prepare all conditioning boxes with smooth, white paper floors (e.g., legal-size copy paper).
2| Weigh each mouse and immediately inject (i.p.) with vehicle. Place the mouse into the center of the box and close the
sound-attenuating chamber. Record activity.

3| After the 5-min habituation session is complete, remove the mouse from the apparatus and return it to its home cage.
© 2006 Nature Publishing Group http://www.nature.com/natureprotocols

Conditioning (days 2–5, 8–11)

4| Twenty-four hours after the habituation session, initiate the first conditioning session.
5| Prepare conditioning boxes with the appropriate floors (entirely grid or entirely hole floors for each box depending on
group, floor and order assignment).

6| Weigh each mouse and immediately inject i.p. with drug (if CS+ trial) or vehicle (if CS
trial). Place the mouse into the
center of the box and close the sound-attenuating chamber. Record activity during trial.
m CRITICAL STEP We strongly recommend that the time delay between opening or moving the home cage and placement of the
mouse in the conditioning box be kept as short as possible (e.g., 30–60 s). For reasons that are not entirely clear, we have found that
longer delays yield weaker and more variable place conditioning. This interference may be related to timing of stress axis activation
in relation to drug exposure or it may reflect some type of associative interference (e.g., overshadowing by pretrial cues). Ideally, the
home cage rack should be located within a few steps of the conditioning apparatus in order to minimize the time delay. In situations
where the animal colony is located at a distance from the equipment, we recommend moving the animals adjacent to the equipment
(in their home cage) and letting them rest at least 1 h before conditioning or testing.

7| After the 5-min conditioning trial is complete, remove the mouse from the apparatus and return it to its home cage. These
trials will occur at 48-h intervals (i.e., on days 2, 4, 8 and 10).

8| Twenty-four hours later (or 72 h later over weekend), prepare the boxes with the floor not used in the previous session.
9| Weigh each mouse and immediately inject i.p. with drug (if CS+ trial) or vehicle (if CS
trial). Place the mouse into the
center of the activity box and close the sound-attenuating chamber. Record activity during trial.

10| After the 5-min conditioning trial is complete, remove the mouse from the apparatus and return it to its home cage. These
trials will occur at 48-h intervals (i.e., days 3, 5, 9 and 11).

Preference test (day 12)

11| Twenty-four hours after the final conditioning session, prepare each conditioning box with half grid and half hole floors
(counterbalanced position).
12| Weigh each mouse and immediately inject i.p. with vehicle and place the mouse into the center of the box. Close the
sound-attenuating chamber and record test activity.
13| After the 30-min preference test session is complete, remove the mouse from the apparatus and return it to its home cage.
m CRITICAL STEP To control for odor, the floors and the inside of the box are wiped with a damp sponge (no soap) to disperse any
lingering localized odor and the litter paper beneath the floors is changed between subjects.

 TIMING
Below is an example of a timeline for the GRID+ conditioning subgroup (drug paired with the grid floor). This timeline
illustrates training for a group that receives the drug trial before the vehicle trial. Half of the GRID+ subgroup would receive the
vehicle trial before the drug trial (not shown).
Day 1: 5-min habituation session (or 30-min pretest session)
Day 2: 5-min CS+ trial (drug + grid floor)
Day 3: 5-min CS
trial (vehicle + hole floor)
Day 4: 5-min CS+ trial (drug + grid floor)
Day 5: 5-min CS
trial (vehicle + hole floor)

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 PROTOCOL

Days 6, 7: 2-day break from conditioning (weekend)

Day 8: 5-min CS+ trial (drug + grid floor)
Day 9: 5-min CS
trial (vehicle + hole floor)
Day 10: 5-min CS+ trial (drug + grid floor)
Day 11: 5-min CS
trial (vehicle + hole floor)
Day 12: 30-min drug-free test (both grid and hole floors)
Below is an example of a timeline for the GRID
conditioning subgroup (drug paired with the hole floor). This timeline
illustrates training for a group that receives the drug trial before the vehicle trial. Half of the GRID
subgroup would receive
the vehicle trial before the drug trial (not shown).
Day 1: 5-min habituation session (or 30-min pretest session)
Day 2: 5-min CS+ trial (drug + hole floor)
Day 3: 5-min CS
trial (vehicle + grid floor)
© 2006 Nature Publishing Group http://www.nature.com/natureprotocols

Day 4: 5-min CS+ trial (drug + hole floor)

Day 5: 5-min CS
trial (vehicle + grid floor)
Days 6, 7: 2 days break from conditioning (weekend)
Day 8: 5-min CS+ trial (drug + hole floor)
Day 9: 5-min CS
trial (vehicle + grid floor)
Day 10: 5-min CS+ trial (drug + hole floor)
Day 11: 5-min CS
trial (vehicle + grid floor)
Day 12: 30-min drug-free test (both grid and hole floors)

ANTICIPATED RESULTS
Data analyses
Historically, various dependent variables and comparisons have been used to define place conditioning. We generally use
analysis of variance (ANOVA) to examine (i) activity rates during conditioning trials, (ii) activity rates during the preference
test and (iii) time spent on the grid floor (seconds per minute) during the preference test. This last measure is the primary
index for deciding whether place conditioning has occurred. Using a fully counterbalanced unbiased experimental design,
a significant difference between the GRID+ and GRID
conditioning subgroups provides evidence of place conditioning16,43.
A significant interaction between conditioning subgroup and other treatment variables (e.g., strain, antagonist pretreatment)
provides evidence that the treatment variable altered CPP. Cunningham et al.16 offer a more complete discussion and
comparison of dependent variables commonly used in place conditioning studies.

Typical results
Following the above procedure, we define drug-induced CPP as a significant difference between the GRID+ and GRID

conditioning subgroups in an analysis of time spent on the grid floor. In other words, in a successful procedure, mice that
received drug pairings with the grid floor (GRID+ conditioning subgroup) will spend more time on the grid floor than mice
that received drug pairings with the hole floor (GRID
conditioning subgroup). This outcome is represented statistically as
a main effect of conditioning subgroup in the ANOVA. If a treatment variable has affected the expression of place preference
(i.e., increased or decreased the difference between GRID+ and GRID
conditioning subgroups), then a significant Treatment
Condition  Conditioning Subgroup
interaction should be observed in the
ANOVA. Appropriate post hoc statistical Figure 3 | Mean seconds per minute (+s.e.m.) 60 GRID+
spent on the grid floor during the 30-min post-
Mean time on grid floor (s min–1)

tests can then be used to identify 50

differences between conditioning
subgroups within a treatment group.
Figure 3 illustrates how the prefer-
ence test data are depicted graphically.
These data come from an unpublished
study with DBA/2J mice, in which
we examined the effects of extinction
after ethanol place conditioning.
After the habituation session, all mice
were initially exposed to our standard
counterbalanced conditioning protocol
(see PROCEDURE) using tactile cues in a Treatment  Conditioning Subgroup interaction (P o 0.001), indicating that the extinction manipulation
one-compartment procedure with 5-min
GRID–
extinction test session. During conditioning trials
(one-compartment procedure), mice in the GRID+ 40
conditioning subgroups received i.p. ethanol
(2 g kg
1) injections immediately before 5-min 30
exposure to the grid floor on CS+ trials; saline
was injected before exposure to the hole floor on 20
CS
trials. These floor–drug contingencies were
10
reversed for mice in the GRID
conditioning
subgroups. After conditioning, mice in the 0
Extinction groups received six 30-min exposures No-Extinction Extinction
Post-conditioning treatment group
to each CS after saline injection (one exposure to
each stimulus per day). Mice in the No-Extinction
groups were not exposed to the conditioning box or cues during this phase. Two-way ANOVA yielded
a significant main effect of conditioning subgroup (GRID+ versus GRID
) and a significant Extinction
reduced the expression of CPP. Each conditioning subgroup contained 12 mice.

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 PROTOCOL

TABLE 1 | Raw data from the experiment depicted in Figure 3.

No-Extinction groups Extinction groups

Subject no. GRID+ GRID
GRID+ GRID

1 1,263.6 407.7 672.9 1,577.7
2 904.8 531.9 1,002.6 1,368.9
3 1,621.8 691.5 564.6 687.3
4 1,307.7 661.8 1,574.4 575.7
5 1,537.5 595.5 274.5 393.6
6 1,332.6 357.6 453.3 425.7
7 1,312.2 516.0 1,421.1 1,544.7
8 1,467.0 624.9 1,145.4 618.9
© 2006 Nature Publishing Group http://www.nature.com/natureprotocols

9 1,370.7 322.5 381.3 998.7

10 1,345.8 522.6 1,415.4 738.3
11 816.0 518.7 534.9 502.5
12 958.2 533.4 1,010.1 1,140.6
Mean 1,269.8 523.7 870.9 881.1
Values represent the total number of seconds that individual mice spent on the grid floor during the 30-min choice test in each Treatment  Conditioning subgroup. Each value was obtained from a different mouse.
These values were converted to seconds per minute (value/30) for analysis and data plotting.

conditioning trials. Ethanol dose was 2 g kg
1 (i.p., 20% v/v in saline, 12.5 ml kg
1). After all mice had received four
conditioning trials of each type, the Extinction group was exposed to a series of trials intended to weaken expression of the
conditioned approach response. More specifically, on each of 6 consecutive days, these mice received two 30-min exposures
to the conditioning box. The CS+ floor was present on one of these trials whereas the CS
floor was present on the other trial
(counterbalanced order). Both trials were preceded by injection of saline (i.e., mice were never injected with ethanol during
the extinction phase). Mice assigned to the No-Extinction group were weighed daily but were not exposed to the conditioning
box or floor cues during this phase. Mice in this group were expected to display a robust preference for the ethanol-paired
floor despite the 8-day delay between the last conditioning trial and test session. Forty-eight hours after the last extinction
trial, both groups returned to the apparatus for a 30-min preference test.
As expected, mice in the No-Extinction group developed a strong CPP as indexed by the greater time spent on the grid floor
by the GRID+ subgroup than by the GRID
subgroup (Fig. 3). In contrast, mice in the Extinction group showed no evidence
of place conditioning (i.e., no difference between GRID+ and GRID
conditioning subgroups), indicating that the extinction
manipulation completely eliminated the expression of preference for the ethanol-paired floor. These observations were supported
by a two-way (Extinction Treatment  Conditioning Subgroup) ANOVA that yielded a significant main effect of conditioning
subgroup (F(1,44) ¼ 14.0, P o 0.001) and a significant interaction (F(1,44) ¼ 14.8, P o 0.001)). The significant interaction
provides statistical support for the conclusion that expression of place conditioning was reduced by the extinction
manipulation. The raw data for the test session are shown in Table 1.
Mean activity rates during the test session were similar in both groups (No-Extinction: 34.5±1.1 counts min
1; Extinction:
31.6±1.5 counts min
1; F(1,46) ¼ 2.4, P o 0.13). Thus, interpretation of the preference data is not complicated by group
differences in test activity. The importance of measuring test session activity when conducting place conditioning studies
is discussed in ref. 17.
Although the above study did not include any additional tests after the first post-extinction test, it is of interest to note that
several recent reports have shown that CPP can be reinstated by post-extinction exposure to the US drug, which may be useful
as a model of relapse to drug-seeking behavior44,45.

? TROUBLESHOOTING
Several factors might be involved when place conditioning is unexpectedly weak or absent. These factors generally fall into two
categories: procedural and experimental design issues.
If the experimenter is a novice with little or no prior experience in conducting place conditioning experiments, improper timing
and/or handling may be a cause for weak conditioned preference or aversion. As place conditioning is sensitive to temporal
parameters and outside disruptions (e.g., cage changes), consistency in handling, injecting and timing is imperative (see earlier
m CRITICAL STEP). Moreover, we have found that certain types of stressful handling can disrupt expression of CPA while not
affecting CPP36. Therefore, great care should be taken in implementing procedures in a timely, consistent manner while avoiding
excessive handling of the subjects. In our laboratory, only one well-trained experimenter typically handles all the animals for
a particular study.
In addition to experimenter-dependent procedural variations, several experimental design variables can influence the strength
of place conditioning, including strain, dose, drug type, stimulus selection (and bias), apparatus configuration (one versus two

1668 | VOL.1 NO.4 | 2006 | NATURE PROTOCOLS

 PROTOCOL

compartments) and temporal parameters (conditioning trial duration, inter-stimulus interval, inter-trial interval and preference
test duration). As noted previously, special care should be taken when selecting all of these experimental parameters.
Number of conditioning trials is an additional consideration when place conditioning is weak. Although we have typically found
four trials of each type (i.e., four CS+ trials and four CS
trials) to yield reliable place conditioning in most of our studies, it is
possible that four trials may not be sufficient to establish a robust conditioned response when using certain strains or drugs.
As strength of the stimulus–drug association increases as a function of the number of conditioning trials28, experimenters might
give additional conditioning trials if an initial test fails to show evidence of place conditioning. In fact, a previous study using
this protocol with Swiss-Webster mice showed significant place conditioning after six trials with low ethanol doses that did
not produce significant effects after four trials31.
Finally, as noted earlier, consideration should always be given to possible effects of locomotor activity variations during test
sessions. We have consistently found a negative genetic relationship between activity level during the test session and strength
of CPP32. That is, strains that show high levels of test session activity also tend to show weaker CPP. This inverse relationship
© 2006 Nature Publishing Group http://www.nature.com/natureprotocols

has also been observed among individuals within an inbred strain25. Thus, when weak place conditioning is observed in
combination with high levels of test session activity, one must consider the possibility that the low expression of preference
or aversion is the result of interference produced by high locomotor activity46.

ACKNOWLEDGMENTS We thank Laura Summers Bax for assistance in data
collection. We also thank the many research assistants, graduate students and
postdoctoral fellows who contributed to the evolution of our place conditioning
model over the last 18 years. Development of this protocol was supported by
NIH-NIAAA grants AA007702, AA010760, AA007468 and AA016041.
COMPETING INTERESTS STATEMENT The authors declare that they have no
competing financial interests.
Published online at http://www.natureprotocols.com
Reprints and permissions information is available online at http://npg.nature.com/
reprintsandpermissions
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