Modeling Appetitive UNIT 8.

25
Pavlovian-Instrumental Interactions
in Mice
Eoin C. O’Connor,1 David N. Stephens,1 and Hans S. Crombag1
1
Behavioral and Clinical Neuroscience Research Group, School of Psychology,
The University of Sussex, Brighton, East Sussex, United Kingdom

ABSTRACT
In appetitive Pavlovian associative learning, a stimulus (conditioned stimulus, CS) that
has been associated with the delivery of a reinforcing event (unconditioned stimulus,
US; e.g., food) can subsequently elicit or modulate goal-directed instrumental behav-
iors. For example, a Pavlovian CS can serve to reinforce (novel) instrumental behavior
(conditioned reinforcement or CRf), or it can energize and potentiate ongoing instru-
mental responses when presented non-contingently (Pavlovian-instrumental transfer or
PIT). Notably, these different effects of a Pavlovian CS on instrumental behavior are
mediated by dissociable psychological and neurobiological mechanisms. Given the crit-
ical role that Pavlovian-instrumental interactions play in regulating motivated behavior
and maladaptive manifestations of motivation such as eating disorders and addictions,
understanding the underlying psychological and neurobiological mechanisms will be
important. This unit describes behavioral protocols that produce robust and reliable PIT
and CRf in mice and that open the door for future studies using transgenic approaches
into the molecular mechanisms underlying associative learning and motivation. Curr.
Protoc. Neurosci. 53:8.25.1-8.25.27. C 2010 by John Wiley & Sons, Inc.

Keywords: associative learning r behavior r classical conditioning r operant

conditioning r incentive learning r reward r transgenic r knockout r mouse

INTRODUCTION
Although Pavlovian, or classical conditioning, is procedurally quite different from in-
strumental, or operant conditioning, it has long been understood that these two forms of
associative learning closely interact. In particular, in Pavlovian conditioning, a stimulus
that is paired with the delivery of a reinforcing unconditioned stimulus (US; e.g., food, wa-
ter, or sex) acquires qualities that enable the now conditioned stimulus (CS) to modulate
goal-directed instrumental behavior in a variety of ways (Mackintosh, 1974; Rescorla,
1988; Dickinson and Balleine, 1994). For example, the Pavlovian cue may come to serve
as a conditioned incentive to energize or potentiate an existing and ongoing instrumental
response habit, as modeled in the laboratory by the Pavlovian-instrumental transfer pro-
cedure (e.g., Estes, 1948; Morse and Skinner, 1958; Lovibond, 1981, 1983). Additionally,
a Pavlovian cue may come to substitute for its referent US by serving as a reinforcer to
support the learning of a novel instrumental response, as modeled by the conditioned
reinforcement procedure (e.g., Hyde, 1976). Thus, aside from becoming an elicitor of
approach and/or consummatory responses towards the US (cue-potentiated feeding: e.g.,
Zambie, 1973; Weingarten, 1983; Galarce et al., 2007) or the CS (sign-tracking: e.g.,
Brown and Jenkins, 1968; Tomie, 1996), Pavlovian cues associated with appetitive events
can also directly affect goal-directed, instrumental actions. Notably, these different ef-
fects are both neurobiologically and psychologically dissociable (Everitt et al., 2000;
Holland and Gallagher, 2003).
Behavioral
Neuroscience

Current Protocols in Neuroscience 8.25.1-8.25.27, October 2010 8.25.1

Published online October 2010 in Wiley Online Library (wileyonlinelibrary.com).
DOI: 10.1002/0471142301.ns0825s53 Supplement 53
Copyright C 2010 John Wiley & Sons, Inc.
 There has been a recent resurgence of interest in Pavlovian conditioning, particularly
within the field of drug addiction research, where the transition from initial drug use to
compulsive drug taking is thought to depend, in part, on interactions between Pavlovian
and instrumental learning processes (Robinson and Berridge, 1993; Everitt and Robbins,
2005). Although the neural circuitry mediating these interactions is relatively well char-
acterized (see review by Everitt et al., 2000), the molecular mechanisms involved, which
may offer a target for the treatment of addictive behaviors, are less well understood.
Importantly, the increased availability of genetically modified mouse models provides
an opportunity to further the understanding of the role of those mechanisms in appetitive
learning and motivated behavior.

This unit describes behavioral protocols that produce reliable and robust Pavlovian dis-
crimination learning in mice and allow subsequent assessment of the Pavlovian cue’s abil-
ity to potentiate a goal-directed (instrumental) response (Pavlovian-instrumental transfer
or PIT: see Basic Protocol), as well as the CS’s ability to serve as a conditioned re-
inforcer, i.e., to support the acquisition of a novel instrumental response (conditioned
reinforcement or CRf: see Alternate Protocol). Notably, a large number of experimental
parameters exist in these procedures and these can both quantitatively and qualitatively
alter the results of experiments. These parameters are highlighted so the experimenter can
both appreciate the intricacies of these procedures and determine the most appropriate
experimental design for their requirements.

STRATEGIC PLANNING
All behavioral training and testing procedures are performed in commercially available
conditioning chambers. The hinged front door, ceiling, and rear wall are constructed
from clear polycarbonate, and the side walls consist of removable aluminum panels
allowing the easy removal or insertion of response manipulanda (e.g., levers or nose-
poke devices), auditory and visual stimulus modules, and reinforcer delivery devices.
The floor of each conditioning chamber is constructed from 22 stainless steel rods (3.3
mm in diameter, spaced 5.7 mm center-to-center). A waste collection tray filled with
bedding material is located underneath the raised floor. Each chamber (21.6 × 17.8 ×
12.7–cm, although use of similar procedures in chambers of different dimensions have
been successful) is located inside a sound isolating and light-resistant enclosure fitted
with an exhaust fan to provide constant ventilation and low-level background noise and
an incandescent light bulb that provides low-level background lighting. Enclosures are
available from behavioral laboratory equipment manufacturers, although good (and often
better) sound-attenuation may be achieved by custom building enclosures.

For the protocols described in this unit, retractable response levers (see Basic Protocol) or
infrared nose-poke detector ports (see Alternate Protocol) are used and are located ∼5 cm
from either side of the reward delivery receptacle. The type of response-manipulandum
used may be chosen for convenience or rapidity and reliability in the acquisition of the
instrumental response that in turn is likely to depend on the mouse strain/model. Relative
to levers, nose-poke devices typically yield high (unconditioned) baseline response levels
that may facilitate more rapid acquisition of robust instrumental response levels. The
authors have successfully used retractable levers or nose-poke ports for either protocol
in this unit.

Located between the two instrumental response devices is a recessed receptacle for
the delivery of liquid or solid food reinforcers. An infrared photobeam is positioned at
the entrance of the receptacle to allow for the detection of head-entry responses. Food
Pavlovian-
Instrumental reinforcement can be achieved using different delivery devices such as precision pellet
Interactions in dispensers, motorized liquid dippers, or syringe drivers/pumps. Any of these may be used
Mice
but each offers specific advantages and disadvantages. Most commonly used are pellet
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dispensers, based on the original Ralph Gerbrands design, that deliver pellets, which are
available in different sizes, flavors, palatability and/or nutrient composition. The number
of different types of pellets available makes pellet-dispensing devices a versatile option.

Motorized dipper mechanisms deliver pre-determined amounts of a liquid reinforcer

(e.g., sucrose solution) via a small metal cup that is elevated through a hole located in
the bottom of the recessed receptacle. An important advantage of the liquid dipper is that
reinforcement can be terminated by lowering the dipper cup from the receptacle, allowing
for more precise control over reinforcer availability. The use of liquid food reinforcement
also provides flexibility in the composition of reinforcement along nutrient, sensory,
and/or hedonic dimensions.

In the protocols described in this unit, liquid reinforcement is delivered using syringe
driver/pumps and polyethylene (PE) or Tygon infusion lines. In addition to the flexibility
of liquid reinforcer composition, syringe pumps allow for precise delivery of varying
amounts of liquid reinforcement at varying delivery speeds. An important advantage
of this delivery method, especially with respect to Pavlovian conditioning, is that by
positioning the syringe pump outside the sound-isolating enclosure, reinforcer delivery
is achieved in the absence of any auditory signals, otherwise produced by a motorized
dipper or pellet dispenser, which could interact with Pavlovian conditioning.

The protocols described in this unit use simple auditory stimuli. Stand-alone tone genera-
tors or programmable soundcards are available for presenting broadband white noise and
pure tones with easily varied frequency (pitch in Hz), intensity (loudness in dB), and pre-
sentation parameters (e.g., continuous or intermittent). Additionally, physically separate
white noise, clicker (relay), or tone (sonalert) modules are available that are generally
less expensive but offer less flexibility. Visual stimuli may also be used, either instead of,
or alongside an auditory cue; such as a discrete cue light positioned on the chamber wall
or the ambient enclosure (house) light. The physical nature of the stimuli (e.g., auditory
or visual, continuous or intermittent, discrete or ambient) can markedly alter the degree
and content of conditioning (see Commentary; e.g., Holland, 1977). Thus, it is of critical
importance that the stimuli that serve as the to-be-conditioned CSs are counterbalanced
across experimental conditions. Consideration is also given to the location of the stimuli,
relative to the response devices and the reinforcer delivery location, which can both
quantitatively and qualitatively affect conditioning performance (Tomie, 1996). Where
possible, separate stimulus modules are positioned in close proximity to each other
(Fig. 8.25.1).

Control over experimental events and data recording are performed using a Windows-
based Med-PC IV application, running procedures written in State Notation Language
(SNL). The principal data measures recorded to assess behavioral performance are the
number of instrumental (lever press or nose-poke) responses and approach responses
(head-entry) made into the reinforcement delivery receptacle. These receptacle approach
responses consist of two separately quantified and recorded measures. First, the number
of approach responses is indicated by the number of discrete interruptions (on and off)
of the infrared photobeam located inside the receptacle. Second, the duration of each
photobeam interruption provides an index of the amount of time spent in the receptacle
for each approach response.

In addition to automated capture of performance data described above, conditioning

and testing sessions may be video-recorded to allow for quantitative and qualitative
assessment of additional behaviors that are relevant to assess overall performance dur-
ing sessions. Such behaviors include locomotor activity levels and unconditioned or
conditioned orienting and approach responses to both cues and the location of rein- Behavioral
forcer delivery (Holland, 1977). Such measures are especially important when assessing Neuroscience

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 A B

conditioning
chamber houselight

sound isolating
enclosure

video recording

C D
video board camera
syringe pump
syringe

auditory stimuli
modules
fan
grid floor

retractable
lever

door chamber door

waste tray

E F
cue light
cue light

left
retractable lever reinforcer receptacle left reinforcer receptacle
with IR photobeam nose-poke port containing IR
photobeam

right
retractable lever right
nose-poke port

Figure 8.25.1 Photographs of typical equipment used for studying appetitive Pavlovian and instrumental interactions.
(A) Set of four conditioning chambers (Med-Associates) located in sound-isolating enclosures. (B) Video monitoring and
digital recording system showing the inside of four chambers. (C) Components of conditioning chamber using auditory
stimuli and reinforcer delivery by a syringe pump. (D) Close-up of conditioning chamber showing positioning of video
camera. (E) Conditioning chamber configuration for instrumental training and testing for Pavlovian-instrumental transfer.
(F) Conditioning chamber configuration for testing for conditioned reinforcement (CRf).

Pavlovian-
Instrumental
Interactions in
Mice

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Supplement 53 Current Protocols in Neuroscience
performance differences as a function of mouse strain and/or genotype, or following
pharmacological challenges. In the authors’ laboratories, each sound attenuating enclo-
sure is therefore outfitted with a board or bullet CCTV camera that is routed, via quad or
multiplexing splitters, to low-cost, digital HD/DVD recorders (Fig. 8.25.1B). The digital
recording devices are interfaced with the experimental acquisition software allowing for
time and/or event-triggered recording.

Typically, eight to twelve subjects are required for each experimental and control group
(e.g., mutant versus wild type) or treatment condition (e.g., drug versus vehicle treat-
ment) to achieve appropriate statistical power. The number of mice that can be run per
session will depend on the number of conditioning chambers available. To control for
unintended physical differences between conditioning chamber conditions (e.g., ambient
noise, lighting), mice are trained and tested in the same, pre-assigned chamber for the
duration of an experiment and equal numbers of mice in each experimental condition
(e.g., strain, genotype, pharmacological treatment) are assigned to each conditioning
chamber. The approximate time of day that training/test sessions are conducted is held
consistent across the experiment and counterbalanced across groups and conditions.

PAVLOVIAN-INSTRUMENTAL TRANSFER BASIC

PROTOCOL
The following protocol produces robust and reliable Pavlovian and instrumental (discrim-
inative) conditioning and enables subsequent assessment of the Pavlovian-instrumental
transfer effect in mice. The parameters of the protocol have been established with the
C57BL/6J mouse strain, commonly used in behavioral neuroscience research, but the
authors have successfully used other strains and/or mutant models for the procedures
described. The protocol consists of four distinct phases: (1) a brief training phase de-
signed to familiarize the mice with the conditioning chamber and train them to locate and
approach the reinforcement delivery receptacle and consume liquid sucrose solution; (2)
a Pavlovian discrimination training phase in which mice are conditioned to associate one
discrete auditory stimulus with reinforcer delivery (CS+) and a second (control) stimulus
with the absence of reinforcement (CS–); (3) an instrumental training phase designed to
train mice to selectively and reliably respond on one of two available levers to receive
food reinforcement; and (4) the PIT test phase in which the effects of non response-
contingent Pavlovian cue presentations on instrumental responding are assessed. The
test is conducted in the absence of the primary reinforcer (i.e., under extinction condi-
tions) to exclude the possibility of CS interactions with the US (e.g., cue-potentiated
feeding). The different phases of the Basic Protocol are shown in Table 8.25.1.

Housing and feeding

Mice are best housed in groups of two to four per cage and, in the case of genetically
altered mice, with littermates. Note that dominance hierarchies and some strains and/or
genetic mutations can result in occasional aggressive behavior requiring individual hous-
ing, though this may affect behavioral performance in ways that are not fully understood.
In the case that an experimental group needs to be housed as singletons, any comparator
groups will need similar treatment. Other than during training and test sessions, mice
are housed in cages kept in a climate-controlled facility with a 12-hr light:dark cycle.
All behavioral procedures are conducted during the light phase of the day. Starting at
least 7 days prior to the first training phase and for the duration of the experiment, mice
are maintained at 85% to 90% of their free-feeding weight by restricting their access
to the normal diet. Food restriction is achieved by either limiting the amount of food
(number and size of laboratory ‘chow’ pellets) given each day and/or by time-limiting the
availability of food (e.g., 1 to 2 hr) in the home cages. In the case of group-housed mice,
restriction by limiting the amount of food available may result in aggressive behaviors Behavioral
and unequal opportunity for mice to feed due to hierarchy dominance, in which case Neuroscience

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Table 8.25.1 Training and Test Phases of the Basic Protocol

Days 1 and 2: Receptacle
approach training
Days 3 to 14: Pavlovian
discrimination conditioning
Days 15 to 27: Instrumental
conditioning
Day 28: Pavlovian discrimination At the end of the instrumental conditioning phase, a single Pavlovian conditioning
conditioning ‘reminder’ session session is conducted to verify and re-establish robust Pavlovian discriminated
responding
Day 29: Test for
Pavlovian-instrumental transfer
Mice undergo a brief reinforcer delivery–receptacle training phase designed to
familiarize (habituate) them with the conditioning chamber, train them to approach
the reinforcement delivery location, and consume the reinforcer. No auditory stimuli
or levers are presented during these sessions. The principal behavioral measures
recorded are the number of receptacle head entries (i.e., infrared beam breaks) and
the duration of each head entry.
Twenty-four hours after the approach training session, the Pavlovian training phase
starts, during which mice are trained to discriminate between one auditory stimulus
(CS+) paired with the delivery of sucrose delivery (US) and a second stimulus
(CS–) that is never paired with reinforcement. Principal response measures are
approach responses (head entry rate and duration), and these are recorded separately
for each of three possible stimulus events that occur during each conditioning
session: (1) sucrose-paired stimulus (CS+) trials; (2) non-reinforced stimulus (CS–)
trials; and (3) intervening time periods (inter-trial interval or ITI). Additionally, the
latency (in seconds) for the mouse to make the first approach response (head entry)
following the onset of each CS+ trial is measured.
Twenty-four hours after Pavlovian conditioning, mice undergo training to selectively
respond at high and stable levels on one lever to receive sucrose reinforcement. A
second (control) lever is available but responding on this lever does not result in
sucrose delivery. Principal response measures are the number of responses on the
reinforced and non-reinforced levers, the number of reinforcers earned, and the
number and duration of head entries into the food receptacle.
The test of transfer is performed 24 hr after the Pavlovian conditioning reminder
session. Lever responding on both reinforced and non-reinforced levers is recorded
for each of the three possible stimulus events that occur during the test: (1) CS+
trials, (2) CS– trials, and (3) ITI. Additionally, head-entry responses into the food
receptacle are recorded during each stimulus event.

food-restriction by time-limiting access is preferred. Feeding is conducted following

training sessions and the time between session-end and food availability is semi-randomly
varied to minimize the impact on performance of mice ‘anticipating’ food availability
(i.e., expectation of imminent daily chow access may decrease performance during ses-
sions). Mice are weighed immediately prior to each session to monitor and maintain the
85% to 90% target weight by daily comparison to pre-restriction free-feeding weight.
If the length of the food-restriction period exceeds 2 weeks, growth charts (available
from most commercial mouse suppliers) are referenced to adjust the 85% to 90% target
weights.

Limiting access to food can be poorly tolerated by some mouse strains and/or as a con-
sequence of select genetic mutations. Mouse health should be closely and continuously
monitored throughout the period of restriction and especially during the first few days
following commencement of food restriction. The degree of restriction is adjusted as
necessary.

When using unfamiliar/novel foods (e.g., sucrose or sweetened milk), neophobia may
adversely affect (retard) behavioral performance during the initial phases of training and
it is possible that the level of neophobia may vary as a function of mouse strain and/or
Pavlovian-
Instrumental mutations (e.g., Amico et al., 2005). Familiarizing mice with the reinforcer, by providing
Interactions in a small quantity of the food in the home cage on 2 consecutive days prior to training,
Mice
aids to reduce neophobia-dependent variability during performance.
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NOTE: All protocols using live animals must first be reviewed and approved by an
Institutional Animal Care and Use Committee and must follow approved federal, national,
and local procedures for the care and use of laboratory animals.

Materials
20% (w/v) sucrose solution made fresh daily (or 14 mg dust-free precision pellets
for pellet dispenser; e.g., Bio-serve)
70% ethanol
Eight to twelve mice per experimental group (e.g., male, at least 8 weeks old,
C57BL/6J mice; Jackson Laboratories)
A personal computer running MS Windows, Delphi compiler, and MED-PC IV,
and interfaced with the conditioning chambers
Investigator-programmed State Notation procedures, written using a plain text (txt)
editing (e.g., Windows Notepad or Mac OS Textedit) and Delphi compiler
software
Conditioning chambers (Med-Associates)
20-ml plastic syringes and dull 16-G hypodermic needles
Single syringe liquid infusion pumps (PHM-100; Razel Scientific
Instruments/Med-Associates) and hypodermic tubing (polyethylene (PE) or
Tygon)
Two retractable levers (ENV-310M; Med-Associates)
Liquid delivery receptacle (ENV-303LP; Med-Associates)
Auditory stimulus module(s) to present 3 kHz tone or white-noise (ANL-926;
Med-Associates)
Decibel (dB) meter (e.g., Radio Shack) for calibration of auditory stimuli (10 dB
above background)
Perform receptacle approach training (days 1 to 2)
1. Turn on all equipment.
Note that this step can be performed immediately prior to loading the syringes with the
sucrose solution (step 4).

2. Prepare fresh 10% (w/v) sucrose solution with water.

To determine an effective sucrose (or other liquid foods) concentration for different
mouse strains and/or mutant models, separate intake-concentration (pilot) studies may
be conducted. These consist of 20- to 30-min sessions during which individual mice have
free-access to different concentrations of liquid solution (e.g., 0%, 5%, 10%, 15%, and
30% sucrose, w/v) and intake is recorded. The sucrose concentration that supports the
greatest intake and no significant differences between strains and/or mutant models is
then used for behavioral training procedures.

3. Clean the inside of the conditioning chambers with a 70% ethanol solution.
If precision pellet dispensers are used, retrieve any pellets left from previous sessions
before starting a new group of mice. This is also critical for assessing previous groups’
intake of reinforcers.
4. Fill 20-ml syringes with sucrose solution, attach dull 16-G needles and infusion
lines and mount each syringe on the syringe pump. Activate the pump to remove
solution and/or air until the entire infusion line is filled with fresh sucrose solution
and sucrose enters the delivery cup (priming).
5. Using the Delphi compiler software, compile the investigator-programmed State
Notation procedure for stimulus-delivery and data acquisition to an executable
Med-PC IV program.
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 6. Start the Med-PC IV program and load the procedure for each conditioning chamber
used. Enter data output file, mouse i.d., and group label information for each chamber.
7. Transport mice in their housing chambers from the animal holding facility to the
testing room.
8. Weigh each mouse and ensure that recorded weight approximates target weight.
The quantity of food or the duration of access to food can be adjusted if mice are not
close to their target weight. Note that Med-PC allows for weights to be recorded in the
data output file by using the Named Variables function.

9. Place the mouse into the pre-assigned conditioning chamber and start program.
10. Turn on enclosure light and activate the syringe pump every 30 sec on average (using
a random interval or RI schedule with a minimum and maximum time interval of 15
and 45 sec, respectively) to deliver a small volume of sucrose solution (∼50 μl) into
the receptacle cup.
The infusion duration required to deliver a set amount of liquid depends on the syringe
pump speed (rpm) and the particular syringe used. For example, a 3.33-rpm infusion pump
and a 20-ml syringe delivers 50 μl of solution over ∼1.5 sec. Flow-rate information
(in ml/min) as a function of pump motor (rpm) and syringe type and size is available
from the Razel Scientific Instruments Web site, but should nevertheless be tested using a
micropipettor or weighing to measure output volume.

11. Record receptacle approach responses throughout the session.

12. Terminate program after 20 min and remove mice from the chambers and return
them to the holding facility.
13. Obtain the next set of animals and repeat steps 3 through 12.
14. Turn equipment off at the end of the day and flush infusion lines with distilled water
and/or 70% ethanol.
15. Repeat procedure for a total of 2 days.
Perform Pavlovian discrimination training (days 3 to 14)
16. Turn on all equipment and clean the chambers with 70% ethanol solution.
17. Calibrate the loudness of all auditory stimulus cues, setting the sound intensity to
∼10 to 15 dB above ambient background noise levels produced by the enclosure
fans and other environmental sources.
Setting auditory cue loudness too low may retard or prevent discriminated Pavlovian
conditioning, whereas setting loudness too high increases the likelihood of (unwanted)
motor and emotional responses including startle and/or freezing responses that may
similarly interfere with performance. Over time and with experience, overall loudness
levels and/or relative levels between cues may be adjusted if discriminated approach
performance is weak.
18. Fill 20-ml syringes with fresh 10% sucrose solution and prime infusion lines (see
steps 2 and 4).
19. Assign mice to a Pavlovian stimulus condition so that the physical nature of the CS+
and CS– is counterbalanced across all experimental conditions (Table 8.25.2). Thus,
an equal number of mice from each experimental group (e.g., strain, genotype, and/or
pharmacological treatment condition) must be assigned to the following conditions:
Pavlovian- (a) intermittent (1 sec on, 1 sec off) 3-kHz pure tone paired with sucrose (CS+)
Instrumental and a continuous, broad-band white noise (WN) unpaired with reinforcement (CS–);
Interactions in
Mice or (b) WN paired with sucrose and tone unpaired with reinforcement (see Strategic
Planning and Commentary sections for further discussion).
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Table 8.25.2 Fully Counterbalanced Design Across Pavlovian and Instrumental Conditioning Phasesa

Group Pavlovian trainingb Instrumental trainingb PIT test

A WN: sucrose (CS+) Left lever: sucrose (reinforced lever) Left lever: CS+, CS–, ITI
Tone: Ø (CS–) Right lever: Ø (control lever) Right lever: CS+, CS–, ITI
B WN: sucrose (CS+) Left lever: Ø (control lever) Left lever: CS+, CS–, ITI
Tone: Ø (CS–) Right lever: sucrose (reinforced lever) Right lever: CS+, CS–, ITI
C WN: Ø (CS–) Left lever: sucrose (reinforced lever) Left lever: CS+, CS–, ITI
Tone: sucrose (CS+) Right lever: Ø (control lever) Right lever: CS+, CS–, ITI
D WN: Ø (CS–) Left lever: Ø (control lever) Left lever: CS+, CS–, ITI
Tone: sucrose (CS+) Right lever: sucrose (reinforced lever) Right lever: CS+, CS–, ITI
a Controlling for potential variations due to the physical identity of the CSs (tone or white noise, WN) and the location of the reinforced lever
in the conditioning chamber (left or right of the reinforcement receptacle).
b Ø refers to the absence of US reinforcement.

20. Ensure that no levers are present at this stage.

21. Compile investigator-programmed State Notation procedure for reinforcer and
stimulus-delivery and for data acquisition (see step 5).
22. Start Med-PC IV program and load the procedure for each conditioning chamber
used. Enter data output file, mouse i.d., and group label information for each chamber.
23. Transport mice in their housing chambers from the animal holding facility to the
testing room.
24. Weigh each mouse, record, and compare weight to 90% target weight.
25. Place the mouse into the pre-assigned conditioning chamber and start program.
26. Turn on enclosure light and present five conditioning trials in which the auditory
cue is paired with delivery of sucrose (CS+), and five trials in which the alternate
auditory cue is presented but no sucrose is delivered (CS–).
CS+ and CS– trials are presented in a pseudo-randomly determined order, such that
the order of stimulus presentations is randomly determined within each session, but a
maximum of five CS+ and five CS– trials should occur. Both the intermitted tone and
white-noise cues are 2 min in duration. During each of the CS+ presentations, 50 μl
sucrose solution is infused into the receptacle cup according to a random-interval (RI)
schedule that delivers reinforcement, on average, every 30 sec (varying between 15 and
45 sec). Thus, on average, four sucrose deliveries occur during each CS+ trial. Stimulus
trials are separated by inter-trial-interval (ITI) periods lasting on average 120 sec and
ranging in duration between 60 and 180 sec. Thus, mice are presented with a total of ten
2-min trials (five CS+ and five CS–) separated by nine 2-min (on average) ITI periods,
yielding a ∼40-min session.

27. Record separately the number of receptacle approach responses for the CS+ trials,
CS– trials, and ITI periods. Additionally, latencies to make the first approach response
following onset of the CS+ are measured.
28. After the end of the session, remove mice from the chambers, return them to their
home cages in the housing facility, and clean each chamber with 70% ethanol
solution.
29. Obtain the next group of mice and repeat steps 18 to 28.
30. Turn equipment off at the end of the day.
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 31. Repeat the procedure for a total of 10 to 12 days while monitoring behavioral
performance daily.
Approach responses (receptacle head entry rates, duration, and latency to make first head
entry) are analyzed for discriminated performance by comparing responses during CS+
to CS– and ITI periods (see Commentary section).

Perform instrumental conditioning (days 15 to 27)

32. Turn on all equipment.
33. Verify that the retractable lever devices (one located on each side of the reinforcement
delivery receptacle) operate in good order and that levers easily depress and lift.
34. Assign mice to instrumental lever training groups. Mice may show baseline prefer-
ences for specific locations within the chamber (e.g., because of lighting differences).
It is, therefore, critical that the physical location of the lever resulting in response-
contingent sucrose delivery (left or right side of the reinforcer receptacle) is fully
counterbalanced across all conditions and groups (see Table 8.25.2). Thus, within
each experimental group (e.g., strain, genotype, or pharmacological treatment) and
Pavlovian condition (Table 8.25.2, groups A through C), for half the mice, presses
on the left lever produce sucrose delivery and right (control) lever presses have no
programmed consequences. This response-contingency is reversed for the remaining
animals.
35. Clean the conditioning chambers with 70% ethanol, fill syringes with fresh 10%
sucrose solution, and prime infusion lines (see step 4).
36. Compile investigator programmed State Notation procedure for lever operation,
reinforcement schedule, and data acquisition (see step 5).
37. Start the Med-PC IV program and load the procedure for each conditioning chamber
used. Enter data output file, mouse i.d., and group label information for each chamber.
38. Transport mice to the testing room, weigh each mouse, record and compare weight
to 85% to 90% target weight.
39. Place the mouse into the pre-specified conditioning chamber and start program.
40. Turn on the enclosure light and insert (activate) both levers into the conditioning
chamber. Each single press on the lever designated as the reinforced lever results in
immediate activation of the syringe pump and delivery of ∼50 μl of sucrose solution
into the delivery receptacle (i.e., a continuous or fixed ratio FR1 schedule of rein-
forcement). Lever presses occurring during a 10-sec period immediately following
sucrose delivery are recorded, but not reinforced with additional sucrose delivery
(time-out period). Presses on the control lever are recorded throughout the session
but have no programmed consequences.
41. Retract levers and terminate the program 45 min after the session start or once 30
response-contingent US reinforcers have been delivered (whichever occurs first).
Remove mice from the chamber and transport back to the housing facility.
42. Obtain the next set of mice for instrumental lever press training and repeat steps 33
to 41.
43. Turn off equipment at the end of the day.
44. Record and analyze lever-pressing rates on both levers to verify higher levels of
Pavlovian- responding on the reinforced lever than on the non-reinforced control lever. If a
Instrumental
Interactions in substantial percentage of mice (>10%) respond equally on both levers, repeat steps
Mice 32 to 43.
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Supplement 53 Current Protocols in Neuroscience
 In some instances (e.g., particular mouse strains and/or genetic mutations), mice may
show slow acquisition of lever responding during early stages of training. To aid lever
press acquisition, levers may be baited by placing a small amount of sucrose and/or
laboratory chow (pureed by adding water) on both levers to increase the probability of
mice sampling the levers. Additionally, mice that fail to lever press following two to three
training sessions may be trained on a single overnight session, consisting of a series of
1-hr sessions (separated by 1-hr time periods between consecutive sessions). While these
methods typically help instrumental training in most situations, they introduce potential
confounds and should be applied equally for all experimental conditions.
45. Once the majority of mice (>90%) lever-press at high levels on the reinforced
lever, alter the programmed schedule-of-reinforcement for the next session to train
mice to lever press on a progressively more lean, variable interval (VI) schedule
of reinforcement. The progression to a more sparse/lean schedule depends on high
(>10 reinforcers earned) and stable (across a minimum of two consecutive sessions)
response rates on the reinforced (but not control) lever. Specifically, following the
continuous (FR1) schedule, rapidly progress mice to a VI30 and then VI60 schedule
such that sucrose delivery is contingent on lever-presses made on average every 30
(range 15 to 45 sec) or 60 (range 30 to 90 sec) sec, respectively. Interval schedule
sessions are reduced to 30 min when the schedule is increased from FR1 and the
limit placed on the maximum number of reinforcers that can be earned in a single
session is removed.
Additional sessions may be conducted if required and intervening VI schedules (e.g.,
VI15, VI45 sec) may be inserted to aid the acquisition of instrumental responding in mice
that do not readily respond under leaner schedules of reinforcement.

46. Train mice until stable, high and asymptotic levels of VI60 schedule lever pressing
on the reinforcement lever and low control lever response levels are observed before
moving to the testing phase.
Perform Pavlovian discrimination conditioning (reminder) session (day 28)
47. Repeat steps 16 through 30.
Test for Pavlovian-instrumental transfer (day 29)
48. Turn on all equipment and verify, re-calibrate if necessary, the loudness of the
auditory cues and proper operation of the retractable levers (see steps 17 and 33,
respectively).
49. Clean the conditioning chambers with 70% ethanol solution.
50. Compile investigator-programmed State Notation procedure for lever operation,
stimulus presentations, and data acquisition (see step 5).
51. Start the Med-PC IV program and load the procedure for each conditioning chamber
used. Enter data output file, mouse i.d., and group label information for each chamber.
To ‘bias’ the test towards putative interactions of Pavlovian cue presentations on in-
strumental behavior rather than on reinforcement-related variables, the session is best
conducted in the absence of reinforcement (i.e., under extinction conditions).

52. Transport mice to the testing room, weigh each mouse, record, and compare weight
to 90% target weight.
53. Place the mouse into the pre-assigned conditioning chamber and start program.
54. Turn on the enclosure light and insert both levers into the conditioning chamber—
presses on either lever have no programmed consequences. At 2 min after insertion of
the levers, present five trials of the 2-min WN and five trials of the 2-min pure tone
Behavioral
cue in alternating order and with equal probability of the WN or tone occurring Neuroscience

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Current Protocols in Neuroscience Supplement 53
 first. Each cue presentation trial is separated by a 2-min fixed interval (FI)
period.
55. Following the last cue presentation (32 min after session start) remove mice and
return them to the housing facility.
56. Repeat steps 49 to 55 for the remaining groups.
57. Turn off equipment at the end of the day.
Analyze data
58. PIT is demonstrated by an increase in instrumental response rates in the presence
of the previously food-paired stimulus (CS+). A decrease, or no change, in the rate
of instrumental responding may be seen in the presence of the unpaired stimulus
(CS–; see Commentary). A number of approaches are used for analyzing PIT data.
The critical comparison to assess PIT is between lever-press response rates during
CS+ versus CS– trials (e.g., Dickinson et al., 2000) as this isolates CS+ effects on
behavior that are uniquely due to an associative history with the US rather than to non-
associative processes (Rescorla, 1967). Variability in baseline response rates may be
incorporated by calculating absolute elevation scores through subtracting responses
made during ITI periods preceding CS presentation from CS trial response rates.
Alternatively, elevation ratios may be calculated by dividing the number of CS-
trial responses by responses made during preceding ITI periods. Both approaches
facilitate between-group comparisons when significant differences in baseline lever-
response levels exist (e.g., due to strain and/or genotype). Finally, it is critical that
the temporal distribution (time-course) of responding across the PIT test session
is assessed and/or analyzed as overall CS-specific response levels is likely to vary
strongly over the course of the test sessions and within CS trial periods (Holland and
Gallagher, 2003; Crombag et al., 2008a).

ALTERNATE CONDITIONED REINFORCEMENT

PROTOCOL
In addition to Pavlovian-instrumental interactions through the ability of a Pavlovian CS to
modulate existing instrumental responding (see Basic Protocol), Pavlovian cues acquire
reinforcing properties and can support the acquisition of a novel instrumental response.
This protocol outlines a simple and reliable methodology and design for assessing these
conditioned reinforcing effects of Pavlovian cues in mice.

As in the Basic Protocol, following a brief initial training phase to approach the reinforcer
delivery receptacle and consume sucrose (receptacle approach training), mice are then
trained to discriminate between two differentially reinforced auditory cues (Pavlovian
discrimination training). In the test for conditioned reinforcement, conducted in the
absence of sucrose reinforcement (i.e., under extinction conditions), two operant response
devices (e.g., retractable levers or, as described in this protocol, nose-poke detectors) are
made available. Responding on one manipulandum produces a brief presentation of
the CS+ and responding on the alternate manipulandum produces the CS–. Thus, this
protocol differs procedurally from the Basic Protocol in that: (1) presentation of the
CS+ and CS– are dependent on the mouse performing an instrumental response (i.e.,
response-contingent) and, (2) instrumental behavior has not been previously established
under primary reinforcement conditions and is entirely novel.

Notably, the optimal CS-US pairing conditions for the Pavlovian conditioning phase are
also different from those used for the Basic Protocol in respect to the duration of the CS
Pavlovian- and its temporal relationship with reinforcement. The authors have previously found that
Instrumental
Interactions in using a short (10-sec) stimulus that was closely associated with sucrose-reinforcement
Mice produces more reliable and robust conditioned reinforcement compared to the longer
8.25.12
Supplement 53 Current Protocols in Neuroscience
(2-min) CS that was more ‘loosely’ associated with reinforcement as in the Basic Protocol
(see Commentary; Crombag et al., 2008a).

The testing for conditioned reinforcement (day 15) in this protocol takes place 24 hr after
the last Pavlovian discrimination training session; all the mice undergo a test session to
assess the conditioned reinforcing properties of the Pavlovian cue. The critical measure
in the test of conditioned reinforcement is the number of nose-poke responses reinforced
by presentations of the CS+ versus the number of nose-poke responses that result in
presentations of the CS–. Approach responses to the receptacle (entries and duration) are
also recorded.

Additional Materials (also see Basic Protocol)

Two operant nose-poke modules (Med-Associates)
Investigator-programmed State Notation procedures, written using Med-PC IV
software, for experimental variable control and data acquisition
Perform receptacle approach training (days 1 and 2)
1. Follow Basic Protocol, steps 1 through 15.
Perform Pavlovian discrimination conditioning (days 3 to 14)
2. Follow Basic Protocol, steps 16 to 18.
3. Assign mice to Pavlovian stimulus conditions so that the physical nature of the
CS+ and CS– is counterbalanced across all experimental conditions (Table 8.25.3).
Thus, for each experimental group (e.g., genotype or pharmacological treatment
condition), an equal number of mice must be assigned to the following conditions:
(1) intermittent (1 sec on, 1 sec off) 3-kHz pure tone paired with sucrose (CS+) and
WN with no reinforcement (CS–); or (2) WN paired with sucrose and tone with no
reinforcement (see Strategic Planning and Commentary).
4. Follow Basic Protocol, steps 20 to 25.
5. Turn on enclosure light and in a pseudo-random order, present 20 conditioning trials
in which one auditory cue is paired with 10% sucrose delivery (CS+) and 20 trials in
which the alternate auditory cue is presented and no sucrose is delivered (CS–). Both
auditory cues are 10 sec in duration. During CS+ trials, infuse 50 μl sucrose solution
into the food-cup receptacle 5 sec following the cue onset. Do not pair CS– trials
with sucrose delivery. Trials are separated by inter-trial-interval (ITI) time periods
averaging 60 sec (and ranging from 30 to 90 sec).

Table 8.25.3 Fully Counterbalanced Design Across Pavlovian Conditioning and

the Conditioned Reinforcement Test Phasesa

Group Pavlovian trainingb Test for conditioned reinforcement

A WN: sucrose (CS+) Left nose-poke: CS+

Tone: Ø (CS–) Right nose-poke: CS–
B WN: Ø (CS–) Left nose-poke: CS+
Tone: sucrose (CS+) Right nose-poke: CS–
C WN: sucrose (CS+) Left nose-poke: CS–
Tone: Ø (CS–) Right nose-poke: CS+
D WN: Ø (CS–) Left nose-poke: CS–
Tone: sucrose (CS+) Right nose-poke: CS+
a Controlling for physical nature of the CSs (tone or white noise, WN) and the location of the nose-
poke port (left or right of reinforcement receptacle) during the test for conditioned reinforcement. Behavioral
b Ø refers to the absence of US reinforcement. Neuroscience

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Current Protocols in Neuroscience Supplement 53
 6. Record separately the number of receptacle approach responses for the CS+ trials,
CS– trials, and ITI periods. Additionally, latencies to make the first approach response
following onset of the CS+ are measured.
7. After the session ends (∼40 min), remove and return mice to their home cages; clean
each chamber with 70% ethanol, and obtain the next group of mice and repeat steps
4 through 7.
8. Turn equipment off at the end of the day.
9. Repeat the procedure for a total of 10 to 12 days while monitoring behavioral
performance daily. Approach responses (receptacle head entry rates, duration, and
latency to make first head entry) are analyzed for discriminated performance by
comparing responses during CS+ to CS– and ITI periods (see Commentary).
Test for conditioned reinforcement (day 15)
10. Turn on all equipment and clean the conditioning chambers with 70% ethanol solu-
tion.
11. Assign mice to groups such that the physical location of instrumental nose-poke
manipulanda (left or right of the reinforcement delivery receptacle) is fully counter-
balanced (Table 8.25.3). Thus, within each experimental group (e.g., strain and/or
genotype or pharmacological treatment) and Pavlovian condition (groups A and
B; Table 8.25.3), for half of the mice nose-pokes into the left port result in CS+
presentations and nose-pokes into the right port result in CS– presentations. This
response-outcome contingency is reversed for the remaining mice.
12. Repeat Basic Protocol, steps 35 to 39.
13. Turn on enclosure light and following each nose-poke response into the CS+ port,
present the (previously sucrose paired) CS+ for 3 sec. Each nose-poke response
into the alternate port is followed by a 3-sec presentation of the CS–. Nose-poke
responses made during the 3-sec presentation periods are recorded but do not result
in additional cue presentations.
14. Record the number of responses into the CS+ and CS– producing nose-poke ports,
as well as approach behaviors to the receptacle during the test session.
15. Terminate the session after 60 min, remove mice and return to their home cages, and
clean each chamber with 70% ethanol.
16. Obtain the next group of mice and repeat steps 12 to 15.
Analyze data
17. The ability of the Pavlovian CS+ to function as a conditioned reinforcer is assessed
by comparing responses into the CS+ versus CS− nose-poke port during the test
session. Responding for conditioned reinforcement may vary over the course of
the test session and responding at different time points (e.g., every 15 min) of the
session is analyzed. Analysis of differences between groups (e.g., due to strain and/or
genotype) in the magnitude of conditioned reinforcement is aided by calculating
difference scores (CS+ minus CS− nose-poke port responses).

COMMENTARY
Background Information lus (CS) and the unconditioned stimulus (US).
Contemporary learning theory describes These associative relations developed during
Pavlovian- Pavlovian-associative learning in terms of the conditioning allow the CS to activate the US
Instrumental
Interactions in establishment of associations between inter- representation and substitute for the US in
Mice nal representations of the conditioned stimu- a wide variety of emotional, cognitive, and
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Supplement 53 Current Protocols in Neuroscience
behavioral functions. For instance, the CS may
acquire the ability to engage the response cir-
cuitry originally controlled by the US, and
elicit a conditioned response (CR) similar in
form to the unconditioned response (UR)—an
evocation traditionally emphasized by animal
learning scientists and Pavlov himself (Pavlov,
1927). Additionally, a CS can come to pro-
duce emotional and motivational effects that
allow it to substitute for its referent (the US)
by serving as a reinforcer for new learning
(e.g., Hyde, 1976) and/or modify the activ-
ity of other behavioral systems including in-
strumental response habits (e.g., Estes, 1948;
Lovibond, 1981, 1983). Which of these dif-
ferent effects predominate and are expressed
at a given time depends not only on condi-
tions at the time of testing (e.g., whether a
CS+ is contingent on a response output), but
also on the parameters of conditioning. Indeed,
seemingly small variations in Pavlovian train-
ing conditions (e.g., the physical nature of the
CS and/or US and CS-US pairing parameters)
may bias CS-activated representations towards
particular types of consequences for behav-
ior and away from others (Konorski, 1967;
Lovibond, 1981; Holland, 1990; Delamater
and Oakeshott, 2007; Crombag et al., 2008a;
Delamater and Holland, 2008). It is against
this empirical and theoretical background
that behavioral neuroscientists are renewing
their interest in understanding the psychol-
ogy and neurobiology of Pavlovian associative
learning.
Beyond the more basic perspective of un-
derstanding the psychology of associative
learning, Pavlovian associations are widely
believed to contribute significantly to the de-
velopment and maintenance of different psy-
chopathologies, including certain eating disor-
ders and addictions (Robinson and Berridge,
1993; Everitt and Robbins, 2005; Hyman et al.,
2006). For instance, drug addicts exposed to
stimuli previously associated with their drug
taking habit (e.g., drug paraphernalia or en-
vironmental context) often report physiolog-
ical changes and increases in cravings for
the drug (e.g., Wikler, 1948; O’Brien et al.,
1992; Childress et al., 1993). Moreover, when
asked, they report that encounters with these
cues contributed to their relapse episodes (e.g.,
Shiffman, 1982; Wallace, 1989). Thus, un-
derstanding how appetitive Pavlovian associa-
tions are established, what is learned during
conditioning, and the consequences CS-
activated representations have on behav-
ior should provide valuable insights to the
Current Protocols in Neuroscience
psychobiological mechanisms of numerous
clinical disorders.
At the level of brain systems mediating
Pavlovian-instrumental interactions, a number
of brain lesioning investigations in rats have
identified a fairly well circumscribed fore-
brain neural circuitry, involving regions within
the amygdala, nucleus accumbens and pre-
frontal cortex (Everitt et al., 2000). For in-
stance, the ability of a food-associated cue
to enhance ongoing instrumental responding
(PIT; see Basic Protocol) is disrupted by se-
lective lesions of either the central nucleus of
the amygdala or the shell region of the nu-
cleus accumbens (e.g., Blundell et al., 2001;
Hall et al., 2001; Holland and Gallagher, 2003;
Murschall and Hauber, 2006). Conversely, le-
sions of the accumbens core (e.g., Corbit et al.,
2001; Ito et al., 2004), the basolateral nu-
cleus of the amygdala (Cador et al., 1989;
Holland and Gallagher, 2003; Burke et al.,
2007), as well as orbitofrontal cortex (e.g.,
Ostlund and Balleine, 2007; Burke et al., 2008)
interfered with responding for conditioned re-
inforcement. Thus, a simple model proposes
that direct and/or indirect (presumably gluta-
matergic) connections between the amygdala,
nucleus accumbens, and prefrontal cortex,
and midbrain tegmentum dopaminergic inputs
(Murschall and Hauber, 2006; El-Amamy and
Holland, 2007) form a critical neuroanatom-
ical locus for the consequences of Pavlovian
cues on goal-directed instrumental behavior.
Moreover, depending on whether these cues
are exerting their effects by serving as condi-
tioned reinforcers, or by potentiating already
established instrumental responding when pre-
sented non-contingently, different and exper-
imentally dissociable neural pathways within
this larger forebrain circuitry are engaged.
The type of information about the US that
is coded and is accessible by CS-activated rep-
resentations can be quite detailed. Thus, apart
from more general motivational and/or emo-
tional features, separately learned associations
may be established during the conditioning
process that involve much more detailed as-
pects of the US (e.g., specific sensory fea-
tures, such as smell or taste). Moreover, the
extent to which these general motivational or
outcome-specific aspects are coded and acces-
sible to the CS depends, in part, on the cir-
cumstances or parameters during condition-
ing (Rescorla and Solomon, 1967; Colwill and
Rescorla, 1988; Holland, 1990). For example,
the Pavlovian-instrumental transfer effect may
be general (see Basic Protocol) such that the Behavioral
Neuroscience
8.25.15
Supplement 53
 Pavlovian cue is able to potentiate instrumental
responding beyond the specific referent food
US to other food USs, or may be outcome-
specific such that the CS increases responding
preferentially for the specific US. These two
forms of Pavlovian transfer rely on separate
representations, which is supported by the fact
that whereas lesions of central nucleus of the
amygdala abolish the general transfer effect,
the specific transfer effect is abolished by le-
sions of the basolateral amygdala (e.g., Hol-
land, 2004; Corbit and Balleine, 2005; Corbit
et al., 2007). Similar analysis of conditioned
reinforcement serving in general and outcome-
specific ways has been conducted (Burke et al.,
2007, 2008).
The rapidly expanding development of
transgenic and knockout mouse models is
providing behavioral neuroscientists with
new tools to begin understanding Pavlovian-
instrumental interactions from an increasingly
mechanistic perspective. In particular, the abil-
ity to genetically target cellular and molec-
ular processes involved in the establishment
and maintenance of synaptic plasticity is pro-
viding new insights into the relations be-
tween mechanisms regulating synaptic change
and specific effects of Pavlovian cues on
instrumental behaviors. For example, select
deletion of the AMPA receptor GluR2 sub-
unit (gria2) results in deficits in Pavlovian-
instrumental transfer while actually enhancing
conditioned reinforcement. Conversely, dele-
tion of the GluR1 subunit (gria1) results in
severe deficits in responding for a conditioned
reinforcer while leaving the Pavlovian trans-
fer effect unaffected (Mead and Stephens,
2003a,b). Moreover, knock-in mutations that
prevent phosphorylation at a select serine site
on the GluR1 subunit produce the same out-
come. Specifically, mice with mutations of
the GluR1 serine 831 site, normally phospho-
rylated by Ca2+ /calmodulin-dependent pro-
tein kinase II (CaMKII) or protein kinase C
(PKC), but not the protein kinase A phos-
phorylated GluR1 serine 845, fail to show
conditioned reinforcement but do show nor-
mal Pavlovian-instrumental transfer (Crom-
bag et al., 2008b,c). Thus, much like the previ-
ously mentioned lesioning studies in rats, ge-
netically modified mouse models have pointed
to a dissociation in the neural mechanisms that
underpin specific Pavlovian-instrumental in-
teractions.
Pavlovian- In summary, it is apparent that traditional
Instrumental accounts of Pavlovian conditioning, as a shift
Interactions in of control over response-output machinery
Mice
8.25.16
Supplement 53
from the US to the CS, fail to capture the vari-
ety of consequences that Pavlovian CSs have
for behaviors. Not surprisingly, the intricacy
of the underlying psychological processes is
reflected at the neurobiological level showing
that separate and dissociable anatomical sites
and synaptic mechanisms mediate different
forms of Pavlovian-instrumental interactions.
Critical Parameters and
Troubleshooting
Physical nature of US and CS
A number of different food reinforcers and
delivery devices are available such as pellets
delivered from dispensers or liquid solutions
using dippers or syringe pumps. Within the
context of the current protocols, the choice be-
tween these options is in large part a practical
one, although auditory feedback from the de-
livery device (e.g., a pellet falling or motorized
dipper activation) will interact with condition-
ing by providing competing cues. This issue
is easily avoided when using syringe pumps
that are placed outside the sound-isolation
chamber.
A number of options are available in the
choice of the stimuli used to serve as the CS+
and CS–, including different auditory cues
(e.g., tone versus white noise), visual cues,
or a combination of stimulus modalities. An
obvious but critical parameter for this is that
stimuli need to be sufficiently salient and dis-
criminable from background noise levels (am-
bient noise and light levels) as well as from
each another. In general, more intense stimuli
enter more readily into associations and ac-
quire CS properties, but are also more likely
to elicit unconditioned responses (e.g., star-
tle) that could adversely affect with condi-
tioning. However, stimulus saliency or asso-
ciability is determined by factors additional
to psychophysical ones, including the nature
of the US, modality of the CS, and past ex-
periences. Additionally, these factors may in-
fluence the type of conditioned response that
develops (Holland, 1977, 1980a,b). It is there-
fore essential to counterbalance the physical
stimuli that serve as the CS+ and CS– across
all conditions (see below). Additionally, as
it is possible that physically different would-
be CSs could produce different unconditioned
and conditioned responses in different mouse
strains and/or mutant models (see Genetically
Altered Mice, below), pilot experiments may
be necessary to determine the most appropriate
stimulus conditions.
Current Protocols in Neuroscience
Proper control conditions
The inclusion of a CS that is not paired with
reward delivery (CS–) during the Pavlovian
conditioning phase is essential since it pro-
vides the critical comparison to isolate CS+
effects on behavior that are uniquely due to
an associative history with the US, rather than
to non-associative processes such as sensitiza-
tion or pseudo-conditioning (Rescorla, 1967;
Winterbauer and Balleine, 2007). A variety of
different approaches have been developed to
establish the CS– control condition and po-
tential advantages and shortcomings of each
are discussed in detail elsewhere (Rescorla,
1967, 1988). For example, the explicitly un-
paired discrimination procedure described in
this unit may lead to the CS– acquiring con-
ditioned inhibitory qualities that could sup-
press instrumental responding when assessing
Pavlovian-instrumental transfer effects. Dif-
ferences between instrumental response rates
between CS+ to CS– trials could therefore re-
flect the net sum of potentiation by the CS+
and inhibition by CS–. Comparing response
rates during CS– trials with ITI periods will
indicate inhibitory contribution by the CS– to
the transfer effects.
Instrumental schedule-of-reinforcement
Random and intermittent reinforcement
schedules, such as variable interval (VI) or
random ratio (RR) schedules, are most suit-
able for establishing an instrumental response
baseline to assess PIT (see Basic Protocol)
for a number of reasons. First, these sparse
or lean intermittent schedules maintain rates
of responding that are more resistant to ex-
tinction during testing conducted in the ab-
sence of reinforcement and provide a (rel-
atively) stable baseline response rate from
which to assess modulatory influences by a
CS. However, high, sustained instrumental re-
sponse rates may come to obscure any rate-
enhancing effects by a CS (ceiling effect). Re-
ducing baseline response rates somewhat by
introducing a brief extinction period (e.g., 5
min) prior to the first stimulus presentation
trial, often aids the observation of transfer ef-
fects (Dickinson et al., 2000). From a more the-
oretical perspective, some evidence exists that
VI schedules promote instrumental response
habits that become increasingly stimulus-
dependent (i.e., stimulus-response learning)
and less dependent on response-outcome as-
sociations (Dickinson and Nicholas, 1983;
Dickinson et al., 1983, 1995). To the extent
that the transfer effect is dependent upon gen-
eral motivational properties of the Pavlovian
Current Protocols in Neuroscience
cue, its interaction with instrumental respond-
ing is thought to become more pronounced
when responding is stimulus-driven, rather
than outcome-dependent (Pearce and Hall,
1978; Holland, 2004).
Although the test session to assess PIT
or CRf is best conducted in the absence of
the primary reinforcer, intermittent food USs
could be given during testing sessions (e.g.,
Lovibond, 1981); the authors have used such
schedules with success in mice. Instrumen-
tal second-order reinforcement procedures, in
which responding is maintained by response-
dependent contingent cue presentations and
reinforcement is intermittently presented, also
combine responding that is reinforced by both
the primary reinforcer and the conditioned re-
inforcer (Ferster and Skinner, 1957; Kelleher,
1966). However, delivering US reinforcement
during tests for PIT or CRf introduces the pos-
sibility of CS interactions with the US (e.g.,
cue-potentiated feeding, Weingarten, 1983;
see also Wyvell and Berridge, 2000; Galarce
et al., 2007).
Counterbalancing
It is critically important to counterbal-
ance the physical stimuli that serve as the
CS+ and CS– for Pavlovian conditioning (see
Tables 8.25.2 and 8.25.3). Counterbalancing
ensures that any differential effects of the
CS+ and CS– during the tests for Pavlovian-
instrumental transfer (see Basic Protocol)
or conditioned reinforcement (see Alternate
Protocol) are not due to intrinsic differences
in the salience or associability of the stim-
uli. When using tone and white noise stimuli,
within each relevant condition these serve as
the CS+ and CS–, respectively, for half the
mice and for the remaining mice this relation is
reversed.
Similarly, the location (left or right of the
reinforcement receptacle) of the retractable
levers associated with reinforcement during
instrumental training (see Basic Protocol) or
nose-poke on the test for conditioned rein-
forcement (see Alternate Protocol) need to
be counterbalanced. This counterbalancing en-
sures that differential responding does not de-
velop as a function of pre-existing or de-
veloped preferences for locations within the
chamber (e.g., because of ambient light con-
ditions).
Performance differences in training and
testing as a function of the physical cue
and/or location of the response devices should
nonetheless be monitored to ensure these Behavioral
Neuroscience
are not introducing significant variability. If
8.25.17
Supplement 53
Table 8.25.4 List of Common Problems and Potential Solutions
Problem Possible cause
Mice fail to consume Neophobia
reinforcers
Inadequate level of food
restriction
Reduced reward-sensitivity in
particular strain and/or genotype consumption of different reinforcers or different dilutions
Too much food is delivered
during each session, inducing
satiety
Reinforcer delivery device does
not function
Mice fail to show Sensory deficit in mouse (e.g.,
Pavlovian discrimination deaf or blind)
conditioning
Stimuli lack saliency
Stimuli are aversive
Stimuli do not function
Reinforcer delivery device does
not function
Mice fail to consume reinforcer
Mice fail to respond on Motor impairment in mouse
instrumental levers for
food reinforcement
Failure to sample levers
Levers do not function
Reinforcer delivery device does
not function
Mice fail to consume reinforcer
Other
8.25.18
Supplement 53
Solution
Provide mice with a small quantity of the reinforcer in
their home cage prior to commencing training; confirm
that the reinforcer has been consumed in the home cage
Check body weights and adjust food restriction regime as
needed
Perform reinforcer intake pilot studies to compare
of the reinforcer. Limit the number of CS-US
presentations in each conditioning session. Limit the
number of reinforcers that can be earned in a single
instrumental training session.
Increase the delay time until feeding following the
experimental session (increasing the degree of food
deprivation may have deleterious effects on performance
and should be undertaken with caution)
Check operation and repair if necessary
Assess sensory abilities of mice (see Crawley, 2000);
Use alternative stimuli; if mice are auditorily impaired
(use visual cues) or visually impaired (use auditory cues)
Increase loudness (dB) for auditory stimuli. For visual
cues, decrease ambient light and/or increase stimulus
light. Alter presentation parameters of stimuli
(intermittent versus continuous).
For auditory cues, decrease loudness. For visual cues,
increase ambient light and/or decrease cue light output.
Alter presentation parameters of the stimuli (intermittent
versus continuous).
Check operation and repair if necessary
Check operation and repair if necessary
See problem 1
Assess motor capabilities of mice (see, Crawley, 2000).
Consider using alternative response input (e.g., nose
pokes). Bait levers with sucrose and/or chow paste.
Perform initial training session during dark phase when
exploratory activity is greater.
Check operation and repair if necessary
Check operation and repair if necessary
See problem 1
Conduct one overnight procedure consisting of multiple
1-hr sessions on continuous reinforcement schedule
continued
Current Protocols in Neuroscience
Table 8.25.4 List of Common Problems and Potential Solutions, continued

Problem Possible cause Solution

Mice do not show
Pavlovian-instrumental
transfer (PIT)
Mice do not show
responding for
conditioned
reinforcement (CRf)
Instrumental responding cannot
be sufficiently modulated due to
a ‘ceiling’ effect; i.e., mice show
persistent high rates of
responding
Instrumental responding cannot
be sufficiently modulated due to
a ‘floor’ effect; i.e., mice rapidly
extinguish responding
The transfer effect is transient
and is masked by null-effects at
certain time points
Mice failed to learn the
Pavlovian discrimination
Response levers or stimuli do not Check operation and repair, if necessary
function
Mice do not explore the novel
response inputs
The conditioned reinforcement
effect is transient and is masked
by null-effects at certain time
points
Mice have failed to learn the
Pavlovian discrimination
Response levers or stimuli do not Check operation and repair if necessary
function
Provide a longer extinction period prior to presentation
of the first stimulus. Consider training on an alternative
schedule of reinforcement that reduces baseline response
rates.
Provide a shorter extinction period prior to presentation
of the first stimulus. Consider training on an alternative
schedule of reinforcement that increases baseline
response rates. Provide fewer CS– conditioning trials,
such that the CS– does not further inhibit responding.
Analyze response patterns separately for each stimulus
trial (PIT). Examine responding over time-course (e.g.,
per 10 sec) during trial and inter-trial interval periods.
See problems 1 and 2
Increase the duration of the test, or commence recording
only after first response has been made. If using levers,
consider using nose-poke ports that are more readily
explored.
Analyze time-course (e.g., 15 min) of responding across
the test session
See problems 1 and 2

differences are small and independent of crit-
ical comparison conditions, the results can be
collapsed across stimulus and lever-location
conditions.
CS-US pairings
A critical procedural difference between
PIT and CRf is in the parameters of the CS-US
pairings during Pavlovian conditioning. The
two procedures differ not only with respect
to the duration of the CS (2 min for PIT and
10 sec for CRf) but also in the temporal re-
lationship between the CS and US reinforcer
presentation. Specifically, in the 2-min con-
dition (see Basic Protocol), CS-US pairings
were more variable, occurring on average ev-
ery 30 sec during the 2-min CS period (see
Basic Protocol). In contrast, in the 10-sec con-
dition (see Alternate Protocol), reinforcement
always occurred 5 sec after CS-onset and co-
terminated. This difference in CS-US pairing
Current Protocols in Neuroscience
parameters turns out to be critical in that the
2-min condition produces a robust PIT effect
but fails to support CRf, whereas the 10-sec CS
condition produces robust CRf but fails to pro-
duce PIT (Crombag et al., 2008a). A sizable
empirical literature exists exploring the role
of CS-US relations in Pavlovian conditioning
(e.g., Mowrer and Aiken, 1954; Morse and
Skinner, 1958; Azrin and Hake, 1969; Lovi-
bond, 1981; Delamater and Holland, 2008)
and potential accounts for these effects are
discussed elsewhere (Konorski, 1967; Wagner
and Brandon, 1989; Holland, 1990; Crombag
et al., 2008a; Delamater and Holland, 2008).
Genetically altered mice
Strain and/or genotype-related changes in
sensory/perceptual processing, metabolic pro-
cesses, gross and fine motoric function, ac- Behavioral
Neuroscience
tivity levels, and general motivational state
8.25.19
Supplement 53
 can affect performance at various stages in
the protocols described in this unit. For ex-
ample, differences in sensory and/or hedonic
processing may affect consumption patterns
during instrumental and/or Pavlovian training
phases. As noted, consumption studies may be
conducted to assess intake differences using
food reinforcers with varying levels of palata-
bility and/or nutritional (caloric) content. Fur-
thermore, strain and/or genotype-related varia-
tions in levels of neophobia (e.g., Amico et al.,
2005) may be ameliorated by pre-feeding mice
small amounts of the US in their home cages.
Deficits in gross or fine motor function may
interfere with instrumental response perfor-
mance, as well as US consumption patterns.
In the former case, the use of nose-poke de-
vices instead of lever manipulanda may re-
duce these differences. Conversely, some mu-
tations (e.g., in gria2) may cause a hyperac-
tive phenotype (Mead et al., 2005) in which
case nose-poke devices may exaggerate po-
tential response differences. Obvious issues
are raised by strain- and/or genotype-related
deficits in auditory and visual function that are
less easily resolved but should be considered
and assessed independently where differences
in Pavlovian discrimination performance are
observed.
Finally, a much more difficult rubric of po-
tential strain- and/or genotype-related deficits
to consider involve differences in general mo-
tivational function, as these are difficult to sep-
arate from potential effects on PIT or CRf per-
formance. While there are no straightforward
solutions to this, it is critical to monitor and

A B
30 30
CS⫹ CS⫺

WN
Approaches/2 min

20 Approaches/2 min 20 tone

collapsed

10 10

0 0

1 3 6 9 12 1 3 6 9 12

C D
30 100
ITI
CS⫹
75
Approaches/2 min

Approach percent

20

50
10

25

0
0
1 3 6 9 12 1 3 6 9 12
Pavlovian training session

Figure 8.25.2 Actual data (unpub. observ.) obtained from C57BL/6J mice showing receptacle
approach behavior during Pavlovian discrimination training (see Basic Protocol). Number of head
Pavlovian- entry counts during stimulus trials, combined and separated by physical stimulus, for (A) reinforced
Instrumental CS+ trials and (B) non-reinforced CS–, and (C) for ITI periods. (D) Percentage of head-entries
Interactions in made during CS+ trials (CS+/total head entries) for each Pavlovian training session. Data points
Mice present averages (±SEM).
8.25.20
Supplement 53 Current Protocols in Neuroscience
analyze mouse performance across all stages ber (rate) and duration of head entries into
of training and testing, and in response to the the receptacle. The briefness of this stage pre-
various instrumental (schedules of reinforce- cludes meaningful analysis of an acquisition
ment) and stimulus (CS+, CS–, and IT) period measure and casual observation suffices. Nev-
conditions. For example, when testing for PIT, ertheless, substantial differences in approach
a mutant mouse that shows reduced lever re- responses between different strains and/or
sponding during CS+ trials, as well as during genotypes may provide an early indication of
CS– and ITI trials, and/or shows reduced re- important differences in behavior that should
ceptacle approach responses, is likely a reflec- be closely monitored in subsequent stages.
tion of a general deficit in motivational state During Pavlovian conditioning training,
or motor function. Similarly, during tests for mice should show overall high levels of
CRf, concomitant changes in nose-poke re- receptacle approach responding (head-entry
sponding for the CS+, CS–, and overall lev- and duration), and with training, responding
els of approach responding may signal gen- should progressively become more restricted
eralized deficits rather than select effects on to the US reinforced CS+ trials versus the
conditioned reinforcement. Table 8.25.4 pro- non-reinforced CS– trials or inter-trial in-
vides a list of potential solutions for common terval (ITI) periods (Fig. 8.25.2). Head en-
problems that may arise at various stages dur- try rates (Fig. 8.25.2A) during CS+ trials
ing the protocols described in this unit. For typically increase or remain relatively sta-
additional considerations, when using mutant ble across Pavlovian training sessions (some
mice in behavioral research, see Picciotto and small fluctuations may be seen). Recepta-
Wickman (1998); Crawley (2000); Stephens cle approach responses during non-reinforced
et al. (2002); Brown (2007); and Crusio et al. CS– trials (Fig. 8.25.2B) or inter-trial inter-
(2009). val periods (Fig. 8.25.2C) should rapidly de-
crease over training to asymptotic levels ap-
proaching zero. Approach responses during
Anticipated Results stimulus (CS+ or CS–) presentations should
Basic Protocol not significantly vary depending on the phys-
The brief receptacle approach-training ical identity (tone or WN) of the stimuli
phase should readily produce high levels of used (Fig. 8.25.2A,B). Calculated percent-
approach responses, both in terms of the num- ages for approach responses (i.e., receptacle

A B
25 1.5 15
reinforced lever reinforcers
non-reinforced lever cup time
Number of reinforcers/min

20
Approach duration/min
Lever presses/min

1.0 10
15

10
0.5 5
5

0 0.0 0
1 3 5 7 9 12 1 3 5 7 9 12
Session no. Session no.

FR1 VI30 VI60 FR1 VI30 VI60

schedule of reinforcement schedule of reinforcement

Figure 8.25.3 Actual data (unpub. observ.) obtained from C57BL/6J mice showing lever pressing
rates and head entry durations during instrumental training for PIT (see Basic Protocol). (A) Rate
of presses on the US reinforced and control lever across training sessions with increasingly sparse
VI schedules of reinforcement. (B) Number of response-contingent US-reinforcer deliveries and Behavioral
duration of receptacle approaches. Data points present averages (±SEM). Neuroscience

8.25.21
Current Protocols in Neuroscience Supplement 53
 A B
80 50
reinforcement lever
CS⫹
CS⫺ control lever
40

Lever presses/2 min

Lever presses/2 min

60 ITI
30
40
20

20
10

0 0
1 2 3 4 5 CS⫹ CS⫺ ITI
Trial

C D
15 15

Approach duration (sec/2 min)

Approach entries/2 min

10
10

5
5

0
0
CS⫹ CS⫺ ITI CS⫹ CS⫺ ITI

Figure 8.25.4 Actual data (unpub. observ.) obtained from C57BL/6J mice showing results from a
test session for Pavlovian-instrumental transfer (see Basic Protocol). (A) Number of lever presses
per trial on the previously reinforced lever during CS+ trials, CS– trials, and ITI periods. (B)
Number presses on the previous reinforced and control levers, averaged across the test session,
during CS+ trials, CS– trials, and ITI periods. (C) Number of head entry approach responses and
(D) duration of head approaches during CS+, CS–, and ITI periods. Data points and bars present
averages (±SEM).

head entries) during CS+ (CS+/total ap-
proaches) and CS– (CS–/total approaches) tri-
als (Fig. 8.25.2D) should rapidly diverge with
the percent of approach responses during CS+
trials increasing during the earlier stages of
Pavlovian training and stabilizing at high lev-
els (>70%) during later sessions of Pavlo-
vian training. Latency measures for CS+ tri-
als should show a progressive decrease to low
asymptotic levels with training (not shown).
During instrumental lever training, re-
sponse rates (presses/min) on the US rein-
forced lever should rapidly increase over ses-
sions, and with increases in schedule require-
ments (FR1, VI30 to VI60) should reach stable
Pavlovian- (<15% variability across thee sessions) and
Instrumental
Interactions in high (>10 presses/min or 300 presses/session)
Mice levels or responding (Fig. 8.25.3A). Lever
presses on the non-reinforced control lever
should remain low and stable across training
sessions. During the instrumental training ses-
sions, approach responses to the US deliv-
ery receptacle and the number of response-
contingent US deliveries should remain high
and stable across sessions, indicating con-
sumption of the reinforcer (Fig. 8.25.3B).
The results during the test session of the
PIT experiment should show greater respond-
ing on the previously US-reinforced lever dur-
ing CS+ trials compared to CS– trials and ITI
periods (Fig. 8.25.4). The time course of lever
pressing responses should show that overall
lever response rates decrease in absence of
reinforcement (Fig. 8.25.4A) that may result
in variations in the magnitude of lever press-
ing differences between CS+ and CS– trials.

8.25.22
Supplement 53 Current Protocols in Neuroscience
 7
Lever presses/10 sec 4
0
on off on
4 min
Figure 8.25.5 Actual data obtained from C57BL/6J mice showing a ‘fine-grained’ time-course
analysis of lever responding during ten consecutive CS+ trials and ITI periods. Data points present
averages (±SEM). Data modified from Crombag et al. (2008a).
Inspection of responding on the previously re-
inforced lever averaged over the full session
should show that responses during CS+ trials
are markedly greater compared to CS– and ITI
trials (Fig. 8.25.4B). Lever responding during
CS– trials may show a slight decrease rela-
tive to ITI trials suggestive of an inhibitory
effect by the CS– (Fig. 8.25.4A,B). Respond-
ing on the control lever should be low and not
vary as a function of trial type (Fig. 8.25.4B).
More detailed time-course analysis of lever re-
sponse levels during CS+ trials over the ses-
sion should show strong CS+ stimulus depen-
dency of lever response levels; increasing and
decreasing during CS+ trials and ITI (or CS–)
periods, respectively (Fig. 8.25.5).
Approach responses into the receptacle
(rates and duration of head entries) during the
test for PIT should be markedly increased dur-
ing CS+ versus CS– and ITI trials indicat-
ing discriminated control. Failure to show dis-
criminated approach responses may indicate
generalized deficits. However, in some cases,
lever responding may compete with recepta-
cle approach responses such that few, if any,
approach responses are performed during the
test of transfer.
Alternate Protocol
Results during the receptacle approach and
Pavlovian discrimination conditioning phases
should roughly be the same as those de-
scribed for the Basic Protocol except that
approach rates may initially be lower be-
cause of the shorter duration of the stimuli
Current Protocols in Neuroscience
off on off on off on off on off on off on off
8 min 12 min 16 min 20 min 24 min 28 min 32 min
Session time
(Fig. 8.25.6A). Calculated percentages for ap-
proach responses (head-entry) during CS+
and CS– trials should again rapidly diverge,
increasing to stable and high percentage lev-
els (>70%) during CS+ trials and decreasing
with training during CS– trials (Fig. 8.25.6B).
Latency measures for CS+ trials should show
a progressive decrease to low asymptotic lev-
els with training (not shown).
The results from the test session for condi-
tioned reinforcement should show that nose-
pokes into the port that result in brief pre-
sentations of the CS+ are markedly greater
compared to nose-poke into the port producing
the CS– (Fig. 8.25.6C). More precise inspec-
tion of the time-course of nose-poke respond-
ing during the test session shows that higher
rates of responding for the CS+ versus CS–
can persist for the duration of a 1-hr test session
(Fig. 8.25.6D), but on occasions, conditioned
reinforcement effects may vary dramatically
during the session.
Time Considerations
The typical Pavlovian-instrumental trans-
fer experiment (see Basic Protocol) will take
∼23 days, but additional time should be
allowed where a strain and/or genotype fails to
acquire robust Pavlovian conditioning and/or
instrumental responding requiring additional
training sessions. The conditioned reinforce-
ment procedure (see Alternate Protocol) is
typically completed over 13 days but, as Behavioral
with PIT, the duration of the Pavlovian Neuroscience
8.25.23
Supplement 53
 A B
25 100
CS⫹
20

Approach entries/min
CS⫹ 75

% Approach CS⫹
15

50
10

5 25
CS⫺ CS⫺
0
0
1 3 5 7 9 11 1 3 5 7 9 11
Session no. Session no.

C D
8 2.0

CS⫹
6 1.5
Nose-pokes/min

Nose-pokes/min
4 1.0

2 0.5
CS⫺

0 0.0
CS⫹ CS⫺ 15 30 45 60
Stimulus Session time (min)

Figure 8.25.6 Actual data (unpub. observ.) obtained from C57BL/6J mice showing receptacle
approach behavior during Pavlovian discrimination training and test results for conditioned re-
inforcement (see Alternate Protocol). Number of head entry counts during (A) reinforced CS+
trials and non-reinforced CS– trials. (B) Percentage of head-entries made during CS+ and CS–
trials (CS+/total head entries) for each Pavlovian training session. (C) Number of nose-poke
response resulting in CS+ and CS– presentations. (D) Time course (15-min intervals) of nose-
poke responses resulting in CS+ and CS– presentations. Data points and bars present averages
(±SEM).

conditioning procedure may vary depending
on performance. Eight to twelve mice are typ-
ically required per experimental group to yield
sufficient statistical power. In the authors’ lab-
oratory, eight conditioning chambers are nor-
mally used for a single experiment. Approxi-
mately 15 min should be allowed for checking
and cleaning equipment, preparing syringes,
and loading one group of mice into eight cham-
bers. A single experimenter therefore can com-
fortably run four 1-hr sessions, using eight
experimental chambers in 5 hr. Due to the na-
ture of operant experiments, once a session has
Pavlovian- been started the experimenter has little to do
Instrumental except wait for the session to finish, and this
Interactions in
Mice
time can be used for other activities. However,
if cameras are available inside the chambers,
regular inspection of session progress, and ca-
sual observation of mice for short periods of
time are highly advised.
Acknowledgments
The authors thank G. Collins, F. Passell,
and G. Zaksaite for contributing data to
this manuscript. E.C. O’Connor holds a stu-
dentship from the BBSRC and Pfizer Inc. Re-
search in D.N. Stephens’ laboratory is sup-
ported by the UK Medical Research Council
and H.S. Crombag is supported by a European
council FP7 Marie Curie award.

8.25.24
Supplement 53 Current Protocols in Neuroscience
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