A subscription to JoVE is required to view this content. Sign in or start your free trial.

Method Article

Pavlovian Conditioned Approach Training in Rats

10.9K views

⸱

DOI:

10.3791/53580

⸱

February 4th, 2016

 , 
1Neuroscience Graduate Program, University of Michigan, 2Department of Psychiatry, University of Michigan

In This Article

Summary

Here, we present a protocol to elicit Pavlovian conditioned approach behavior in rats. This procedure can be used to measure individual differences in the tendency to approach and attribute incentive salience to reward-related cues and investigate addiction vulnerability.

Abstract

Cues that are contingently paired with unconditioned, rewarding stimuli can acquire rewarding properties themselves through a process known as the attribution of incentive salience, or the transformation of neutral stimuli into attractive, "wanted' stimuli capable of motivating behavior. Pavlovian conditioned approach (PCA) develops after the response-independent presentation of a conditioned stimulus (CS; e.g., a lever) that predicts the delivery of an unconditioned stimulus (US; e.g., a food pellet) and can be used to measure incentive salience. During training, three patterns of conditioned responses (CRs) can develop: sign-tracking behavior (CS-directed CR), goal-tracking behavior (US-directed CR), and an intermediate response (both CRs). Sign-trackers attribute incentive salience to reward-related cues and are more vulnerable to cue-induced reinstatement of drug-seeking as well as other addiction-related behaviors, making PCA a potentially valuable procedure for studying addiction vulnerability. Here, we describe materials and methods used to elicit PCA behavior from rats as well as analyze and interpret PCA behavior in individual experiments.

Introduction

The transition of drug use to addiction involves complex interactions between Pavlovian and instrumental learning1,2. During drug-taking and drug-seeking behaviors, actions and outcomes are learned through instrumental processes; however, relationships between stimuli (e.g., drug-related cues) and rewards are also learned through Pavlovian processes. Pavlovian cues acquire predictive value, but they can also acquire incentive motivational value3, whereby they become attractive, desirable, and capable of promoting and maintaining reward-seeking behavior4.

Many procedures investigating reward learning in animals, however, do not permit the disentanglement of predictive versus incentive motivational learning. In drug self-administration procedures, for example, both instrumental and Pavlovian contingencies are typically employed, such that rats learn to perform an action (e.g., nose-poking, lever-pressing, etc.) in order to receive an outcome (i.e., intravenous drug infusion). The rewarding outcome is also paired with Pavlovian stimuli (e.g., illumination of a nose-poke port, cue light presentation, sound of the infusion pump, interoceptive feeling of fluid delivery into the bloodstream, etc.). It is often unclear in these procedures whether cues are supporting goal-directed actions simply because of their predictive relationship with the reward, or whether cues have acquired incentive motivational properties of their own.

In order to experimentally isolate the incentive motivational value from the predictive value of Pavlovian stimuli, our laboratory uses a Pavlovian conditioned approach (PCA) procedure in order to identify rats that attribute incentive salience to reward-related cues. During a training session, a conditioned stimulus (CS; e.g., a lever) response-independently predicts the delivery of an unconditioned stimulus (US; e.g., a food pellet). Over the course of multiple training sessions, three phenotypes develop: sign-tracking (CS-directed conditioned response [CR]), goal-tracking (US-directed CR), and an intermediate response (both CRs). For goal-trackers (GTs), the CS is utilized as a predictor of reward delivery; however, for sign-trackers (STs), the CS is attributed with incentive salience, becoming attractive and desirable. In this review, we outline the equipment, setup, and data processing necessary to perform a PCA procedure. In addition, we provide representative results of PCA training, outline important experimental considerations, and discuss the putative utility of PCA procedures in investigating addiction and other neuropsychiatric disorders.

Access restricted. Please log in or start a trial to view this content.

Protocol

All procedures have been approved by the University Committee on the Use and Care of Animals (UCUCA; University of Michigan, Ann Arbor, MI).

1. Equipment and Software

  1. Obtain commercial operant chambers and devices. See Materials Table.
  2. Conduct training in rat operant conditioning chambers (24.1 cm width × 20.5 cm depth x 29.2 cm height), situated in sound-attenuating cabinets with ventilation fans to provide ambient noise.
  3. Equip each chamber with a pellet magazine (receptacle), located centrally in the 21.6 cm wide front wall of the operant chamber 3 cm above the stainless steel grid floor. Ensure that the pellet magazine contains an infrared sensor that is capable of detecting head entries into the device.
  4. Connect the pellet magazine to a pellet dispenser, which delivers banana-flavored food pellets (45 mg).
  5. In a counter-balanced manner, position one retractable lever (CS) approximately 2.5 cm on either side of the pellet receptacle (left or right) 3 cm above the stainless steel grid floor.
  6. Ensure that the lever contains an LED cue light within it to make the extended lever more visually conspicuous.
  7. If using a retractable lever, calibrate the lever by placing a 10 g calibration weight on top of the lever, and then adjusting the calibration screw (located on top of the device) with a hex nut wrench until the weight by itself is able to depress the lever.
  8. Equip a red house-light at the top of the chamber on the wall opposite the magazine receptacle.
    Note: A red house light is used to increase the conspicuousness of the illuminated lever.

2. PCA Training

  1. Pair-house adult male Sprague Dawley rats (250-300 g) upon arrival and leave them undisturbed for at least two days in the housing colony, which is maintained on a 12 hr light/dark cycle.
  2. Gently handle each rat for at least 1 min per day for two days in order to acclimate the rats to human contact. Provide rats ad libitum access to food and water in the housing colony.
  3. Familiarize rats with the banana-flavored food pellets by placing food pellets in their home cages (approximately 15 pellets per rat) for two days, starting three days prior to PCA training.
    Note: If rats are pair-housed, it is useful to divide the pellets evenly on two sides of the home cage.
  4. Pretraining Program
    Note: All of our programing is performed using MEDState Notation (for Med Associates, software), and the manual is available online (mednr.com/pdf/doc003_MedPCProgramr.pdf). Programs are written as unformatted text documents (e.g., ASCII or DOS text) using MedState Notation, saved with the proper extension (i.e., [filename].mpc), then translated using the MED-PC program, Trans IV. All of these steps are provided in more detail in the aforementioned programming manual.
    1. Create a pretraining program that delivers fifty food pellets on a variable time (VT) 30 sec schedule 5 min after the start of the program (i.e., one food pellet is delivered on average every 30 sec, but the actual delivery varies randomly between 0-60 sec).
    2. Write the pretraining program such that the red house light is on, but the lever remains retracted for the duration of the (approximately 25 min) program.
    3. Perform pretraining and all subsequent experimentation during the light cycle.
    4. One day prior to PCA training, place each rat into an operant chamber, close and latch the chamber door, and close the sound-attenuating cabinet door.
    5. Start the pretraining program as soon as possible after placing each rat in its chamber.
    6. Remove each rat from its chamber as soon as possible after the program ends.
    7. Record the number of pellets consumed and exclude rats that do not consume all of the pellets from further testing.
  5. Create a PCA training program that delivers 25 trials in approximately 40 min, beginning 1 min after the start of the program.
    1. Write the program so that during each trial the lever is extended into the chamber and illuminated for 8 sec.
    2. Program lever retraction to be followed immediately by the response-independent delivery of one food pellet into the pellet magazine.
    3. Separate each trial on a VT 90 sec schedule (i.e., lever extension and subsequent food pellet delivery occurs on average every 90 sec, but the actual delivery varies randomly between 30-150 sec).
    4. Place each rat into a conditioning chamber, close and latch the chamber door, and close the sound-attenuating cabinet door.
    5. Start the PCA training program as soon as possible after placing each rat in its chamber.
    6. Remove each rat from its chamber as soon as possible after the program ends.
    7. Exclude rats that do not consume all of the pellets from further testing.

3. Data Processing

  1. Transfer output information to a spreadsheet (e.g., a spreadsheet document).
  2. Create a profile that extracts identifying information (subject number, start date, start time, and box number) as well as output information (number and latency of lever presses and magazine entries).
    Note: In the experiments, non-CS magazine entries are also extracted by the profile. Define non-CS magazine entries as entries into the magazine that occur outside of the 8 sec CS period and during the VT 90 sec (i.e., 60-120 sec) period during which the lever is not extended.
  3. Further process the data by capping the lever press and magazine entry latencies at 8 sec (i.e., the duration of CS presentation). This can be achieved in spreadsheet through the IF function: IF(X >8,8,X).
  4. Calculate scores for response bias (i.e., total number of lever presses and magazine entries for a session; [lever presses - magazine entries] / [lever presses + magazine entries]), latency score (i.e., average latency to perform a lever press or magazine entry during a session; [magazine entry latency - lever press latency]/8), and probability difference (i.e., proportion of lever presses or magazine entries; [lever press probability - magazine entry probability])5.
    Note: With these formulas, all scores should range between 1.0 and -1.0.
  5. Calculate a PCA index score for each rat by averaging its response bias, latency score, and probability difference within each individual session.
    Note: The PCA index score should range between 1.0 and -1.0.

Access restricted. Please log in or start a trial to view this content.

Results

We have found that 5-7 daily sessions of PCA training is sufficient to phenotype rats as STs, GTs, and intermediate responders (IRs), although rats can be trained further based on the needs of a particular laboratory or experiment. Phenotyping is based on PCA index scores, which are calculated by averaging the response bias, latency score, and probability difference of individual sessions as previously described in the protocol. Phenotypes are determined by averaging the PCA index scores ...

Access restricted. Please log in or start a trial to view this content.

Discussion

PCA training can be used to determine individual variation in the tendency to attribute incentive salience to conditioned cues, which has been argued to be an important component of addiction vulnerability. For example, it has previously been demonstrated that STs attribute incentive salience to both food and drug cues6, and both food- and drug-related cues activate similar brain regions within an amygdalo-striatal-thalamic circuit in STs7. Moreover, STs are more impulsive8, vulnerable to...

Access restricted. Please log in or start a trial to view this content.

Disclosures

The authors have nothing to disclose.

Acknowledgements

This work was funded by the University of Michigan Department Of Psychiatry (U032826; JDM), the National Institute on Drug Abuse (NIDA; K08 DA037912-01; JDM) and the Department of Defense (DoD) National Defense Science and Engineering Graduate (NDSEG) Fellowship (CJF).

Access restricted. Please log in or start a trial to view this content.

Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Standard Modular Operant Test Chamber with Modified Top for RatMed Associates, Inc.ENV-008CTTop has access slot for drug delivery leashes or other harness systems; comes with ENV-008CTC (Cover Insert) for access slot
Expanded PVC Sound Attenuating CubicleMed Associates, Inc.ENV-022V
PCI Operating Package for up to Sixteen ChambersMed Associates, Inc.MED-SYST-16Based on the number of operant chambers, MED-SYST-8 (PCI Operating Package for up to Eight Chambers) is also available
SmartCtrlMed Associates, Inc.DIG-716P2
Universal Cable, 25′ (7.6 m)Med Associates, Inc.SG-210CBBased on individual needs, SG-210CB-50 (Universal Cable, 50′ [15.2 m]) is also available
18″ (45.7 cm) 3-Pin Mini-MolexMed Associates, Inc.SG-216ABased on individual needs, SG-216A-2 (2′ [61.0 cm] 3-Pin Mini-Molex) is also available
Power Cable, 25′ (7.6 m) Med Associates, Inc.SG-210CP-25
Stainless Steel Grid Floor for RatMed Associates, Inc.ENV-005Based on preference, ENV-005A (Stainless Steel Grid Floor for Mouse) is also available 
Reusable Waste PanMed Associates, Inc.ENV-007-P3
Filler Panel Package for Standard Modular Test ChamberMed Associates, Inc.ENV-008-FP
Modular Pellet Dispenser for Rat, 45 mgMed Associates, Inc.ENV-203M-45
Pellet Receptacle, Trough TypeMed Associates, Inc.ENV-200R2MBased on preference, ENV-200R1M (Pellet Receptable, Cup Type) is also available 
Retractable LeverMed Associates, Inc.ENV-112CMCalibrated to 25 g; needs to be adjusted to 10 g before Pavlovian conditioned approach training
Retractable Lever Cue LightMed Associates, Inc.ENV-112CML
House Light for RatMed Associates, Inc.ENV-215M
100 mA Replacement Bulbs, Pack of 10Med Associates, Inc.SG-800RRed-colored; for use with ENV-215M (House Light for Rat)
Pellets, 45 mg, Primate Purified Diet, Banana FlavorBio-ServF005950,000/box 

References

  1. Everitt, B. J., Robbins, T. W. Neural systems of reinforcement for drug addiction: from actions to habits to compulsion. Nature Neurosci. 8 (11), 1481-1489 (2005).
  2. Everitt, B. J. Neural and psychological mechanisms underlying compulsive drug seeking habits and drug memories--indications for novel treatments of addiction. Eur J Neurosci. 40 (1), 2163-2182 (2014).
  3. Robinson, T. E., Berridge, K. C. The neural basis of drug craving: an incentive-sensitization theory of addiction. Brain Res Brain Res Rev. 18 (3), 247-291 (1993).
  4. Milton, A. L., Everitt, B. J. The psychological and neurochemical mechanisms of drug memory reconsolidation: implications for the treatment of addiction. Eur J Neurosci. 31 (12), 2308-2319 (2010).
  5. Meyer, P. J., et al. Quantifying individual variation in the propensity to attribute incentive salience to reward cues. PloS One. 7 (6), e38987(2012).
  6. Yager, L. M., Robinson, T. E. A classically conditioned cocaine cue acquires greater control over motivated behavior in rats prone to attribute incentive salience to a food cue. Psychopharmacology (Berl). 226 (2), 217-228 (2013).
  7. Yager, L. M., Pitchers, K. K., Flagel, S. B., Robinson, T. E. Individual variation in the motivational and neurobiological effects of an opioid cue. Neuropsychopharmacology. 40 (5), (2015).
  8. Lovic, V., Saunders, B. T., Yager, L. M., Robinson, T. E. Rats prone to attribute incentive salience to reward cues are also prone to impulsive action. Behav Brain Res. 223 (2), 255-261 (2011).
  9. Saunders, B. T., Robinson, T. E. Individual variation in the motivational properties of cocaine. Neuropsychopharmacology. 36 (8), 1668-1676 (2011).
  10. Saunders, B. T., Yager, L. M., Robinson, T. E. Cue-evoked cocaine 'craving': role of dopamine in the accumbens core. J Neurosci. 33 (35), 13989-14000 (2013).
  11. Kearns, D. N., Gomez-Serrano, M. A., Weiss, S. J., Riley, A. L. A comparison of Lewis and Fischer rat strains on autoshaping (sign-tracking), discrimination reversal learning and negative auto-maintenance. Behav Brain Res. 169 (2), 193-200 (2006).
  12. Fitzpatrick, C. J., et al. Variation in the form of Pavlovian conditioned approach behavior among outbred male Sprague-Dawley rats from different vendors and colonies: sign-tracking vs. goal-tracking. PloS One. 8 (10), e75042(2013).
  13. Anderson, R. I., Spear, L. P. Autoshaping in adolescence enhances sign-tracking behavior in adulthood: impact on ethanol consumption. Pharmacol Biochem Behav. 98 (2), 250-260 (2011).
  14. Pitchers, K. K., et al. Individual variation in the propensity to attribute incentive salience to a food cue: Influence of sex. Behav Brain Res. 278, 462-469 (2015).
  15. Anderson, R. I., Bush, P. C., Spear, L. P. Environmental manipulations alter age differences in attribution of incentive salience to reward-paired cues. Behav Brain Res. 257, 83-89 (2013).
  16. Beckmann, J. S., Bardo, M. T. Environmental enrichment reduces attribution of incentive salience to a food-associated stimulus. Behav Brain Res. 226 (1), 331-334 (2012).
  17. Burns, M., Domjan, M. Topography of spatially directed conditioned responding: effects of context and trial duration. J Exp Psychol Anim Behav Process. 27 (3), 269-278 (2001).
  18. Christie, J. Spatial contiguity facilitates Pavlovian conditioning. Psychon Bull Rev. 3 (3), 357-359 (1996).
  19. Meyer, P. J., Cogan, E. S., Robinson, T. E. The form of a conditioned stimulus can influence the degree to which it acquires incentive motivational properties. PloS One. 9 (6), e98163(2014).
  20. Beckmann, J. S., Chow, J. J. Isolating the incentive salience of reward-associated stimuli: value, choice, and persistence. Learn Mem. 22 (2), 116-127 (2015).
  21. Flagel, S. B., et al. A selective role for dopamine in stimulus-reward learning. Nature. 469 (7328), 53-57 (2011).
  22. Saunders, B. T., Robinson, T. E. The role of dopamine in the accumbens core in the expression of Pavlovian-conditioned responses. Eur J Neurosci. 36 (4), 2521-2532 (2012).
  23. Clark, J. J., Collins, A. L., Sanford, C. A., Phillips, P. E. Dopamine encoding of Pavlovian incentive stimuli diminishes with extended training. J Neurosci. 33 (8), 3526-3532 (2013).
  24. Anselme, P., Robinson, M. J., Berridge, K. C. Reward uncertainty enhances incentive salience attribution as sign-tracking. Behav Brain Res. 238, 53-61 (2013).
  25. Robinson, M. J., Anselme, P., Fischer, A. M., Berridge, K. C. Initial uncertainty in Pavlovian reward prediction persistently elevates incentive salience and extends sign-tracking to normally unattractive cues. Behav Brain Res. 266, 119-130 (2014).
  26. Gallistel, C. R., Gibbon, J. Time, rate, and conditioning. Psychol Rev. 107 (2), 289-344 (2000).
  27. Morrow, J. D., Maren, S., Robinson, T. E. Individual variation in the propensity to attribute incentive salience to an appetitive cue predicts the propensity to attribute motivational salience to an aversive cue. Behav Brain Res. 220 (1), 238-243 (2011).
  28. Morrow, J. D., Saunders, B. T., Maren, S., Robinson, T. E. Sign-tracking to an appetitive cue predicts incubation of conditioned fear in rats. Behav Brain Res. 276, 59-66 (2015).
  29. Saunders, B. T., O'Donnell, E. G., Aurbach, E. L., Robinson, T. E. A cocaine context renews drug seeking preferentially in a subset of individuals. Neuropsychopharmacology. 39 (12), 2816-2823 (2014).

Access restricted. Please log in or start a trial to view this content.

Reprints and Permissions

Tags

Operant Conditioning ChambersSign Tracking BehaviorGoal Tracking BehaviorFood Pellet DeliveryLever Press RecordingMagazine Entry AnalysisPCA Index ScoresRat Behavioral PhenotypesIncentive Salience Measurement
Pavlovian Conditioned Approach Training in Rats
Contact Us
Recommend to Library

Privacy

Terms of Use

Policies

Copyright © 2026 MyJoVE Corporation. All rights reserved.