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).

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
<ol>
	<li>Obtain commercial operant chambers and devices. See Materials Table.</li>
	<li>Conduct training in rat operant conditioning chambers (24.1 cm width &#215; 20.5 cm depth x 29.2 cm height), situated in sound-attenuating cabinets with ventilation fans to provide ambient noise.</li>
	<li>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.</li>
	<li>Connect the pellet magazine to a pellet dispenser, which delivers banana-flavored food pellets (45 mg).</li>
	<li>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.</li>
	<li>Ensure that the lever contains an LED cue light within it to make the extended lever more visually conspicuous.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
2. PCA Training
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.
	<ol>
		<li>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).</li>
		<li>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.</li>
		<li>Perform pretraining and all subsequent experimentation during the light cycle.</li>
		<li>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.</li>
		<li>Start the pretraining program as soon as possible after placing each rat in its chamber.</li>
		<li>Remove each rat from its chamber as soon as possible after the program ends.</li>
		<li>Record the number of pellets consumed and exclude rats that do not consume all of the pellets from further testing.</li>
	</ol>
	</li>
	<li>Create a PCA training program that delivers 25 trials in approximately 40 min, beginning 1 min after the start of the program.
	<ol>
		<li>Write the program so that during each trial the lever is extended into the chamber and illuminated for 8 sec.</li>
		<li>Program lever retraction to be followed immediately by the response-independent delivery of one food pellet into the pellet magazine.</li>
		<li>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).</li>
		<li>Place each rat into a conditioning chamber, close and latch the chamber door, and close the sound-attenuating cabinet door.</li>
		<li>Start the PCA training program as soon as possible after placing each rat in its chamber.</li>
		<li>Remove each rat from its chamber as soon as possible after the program ends.</li>
		<li>Exclude rats that do not consume all of the pellets from further testing.</li>
	</ol>
	</li>
</ol>
3. Data Processing
<ol>
	<li>Transfer output information to a spreadsheet (e.g., a spreadsheet document).</li>
	<li>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.</li>
	<li>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 &#62;8,8,X).</li>
	<li>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.</li>
	<li>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.</li>
</ol>

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

The authors have nothing to disclose.

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...

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.

<table><tbody><tr><td>Standard Modular Operant Test Chamber with Modified Top for Rat</td><td>Med Associates, Inc.</td><td>ENV-008CT</td><td>Top has access slot for drug delivery leashes or other harness systems; comes with ENV-008CTC (Cover Insert) for access slot</td></tr><tr><td>Expanded PVC Sound Attenuating Cubicle</td><td>Med Associates, Inc.</td><td>ENV-022V</td><td></td></tr><tr><td>PCI Operating Package for up to Sixteen Chambers</td><td>Med Associates, Inc.</td><td>MED-SYST-16</td><td>Based on the number of operant chambers, MED-SYST-8 (PCI Operating Package for up to Eight Chambers) is also available</td></tr><tr><td>SmartCtrl</td><td>Med Associates, Inc.</td><td>DIG-716P2</td><td></td></tr><tr><td>Universal Cable, 25&amp;prime; (7.6 m)</td><td>Med Associates, Inc.</td><td>SG-210CB</td><td>Based on individual needs, SG-210CB-50 (Universal Cable, 50&amp;prime; [15.2 m]) is also available</td></tr><tr><td>18&amp;Prime; (45.7 cm) 3-Pin Mini-Molex</td><td>Med Associates, Inc.</td><td>SG-216A</td><td>Based on individual needs, SG-216A-2 (2&amp;prime; [61.0 cm] 3-Pin Mini-Molex) is also available</td></tr><tr><td>Power Cable, 25&amp;prime; (7.6 m)&amp;nbsp;</td><td>Med Associates, Inc.</td><td>SG-210CP-25</td><td></td></tr><tr><td>Stainless Steel Grid Floor for Rat</td><td>Med Associates, Inc.</td><td>ENV-005</td><td>Based on preference, ENV-005A (Stainless Steel Grid Floor for Mouse) is also available&amp;nbsp;</td></tr><tr><td>Reusable Waste Pan</td><td>Med Associates, Inc.</td><td>ENV-007-P3</td><td></td></tr><tr><td>Filler Panel Package for Standard Modular Test Chamber</td><td>Med Associates, Inc.</td><td>ENV-008-FP</td><td></td></tr><tr><td>Modular Pellet Dispenser for Rat, 45 mg</td><td>Med Associates, Inc.</td><td>ENV-203M-45</td><td></td></tr><tr><td>Pellet Receptacle, Trough Type</td><td>Med Associates, Inc.</td><td>ENV-200R2M</td><td>Based on preference, ENV-200R1M (Pellet Receptable, Cup Type) is also available&amp;nbsp;</td></tr><tr><td>Retractable Lever</td><td>Med Associates, Inc.</td><td>ENV-112CM</td><td>Calibrated to 25 g; needs to be adjusted to 10 g before Pavlovian conditioned approach training</td></tr><tr><td>Retractable Lever Cue Light</td><td>Med Associates, Inc.</td><td>ENV-112CML</td><td></td></tr><tr><td>House Light for Rat</td><td>Med Associates, Inc.</td><td>ENV-215M</td><td></td></tr><tr><td>100 mA Replacement Bulbs, Pack of 10</td><td>Med Associates, Inc.</td><td>SG-800R</td><td>Red-colored; for use with ENV-215M (House Light for Rat)</td></tr><tr><td>Pellets, 45 mg, Primate Purified Diet, Banana Flavor</td><td>Bio-Serv</td><td>F0059</td><td>50,000/box&amp;nbsp;</td></tr></tbody></table>

pavlovian conditioned approach training in rats

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, &#34;wanted&#39; 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.

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 ...

Watch this Scientific Journal Video about Pavlovian Conditioned Approach Training in Rats at JoVE.com

Pavlovian Conditioned Approach Training in Rats

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.

Cues that are contingently paired with unconditioned, rewarding stimuli can acquire rewarding properties themselves through a process known as the ...

pavlovian-conditioned-approach-training-in-rats

Research

JoVE Journal

Behavior

10.9K Views.  University of Michigan. 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. 

Video: Pavlovian Conditioned Approach Training in Rats

A Fully Automated Rodent Conditioning Protocol for Sensorimotor Integration and Cognitive Control Experiments

Rodents have been traditionally used as a standard animal model in laboratory experiments involving a myriad of sensory, cognitive, and motor tasks. Higher cognitive functions that require precise control over sensorimotor responses such as decision-making and attentional modulation, however, are typically assessed in nonhuman primates. Despite the richness of primate behavior that allows multiple variants of these functions to be studied, the rodent model remains an attractive, cost-effective alternative to primate models. Furthermore, the ability to fully automate operant conditioning in rodents adds unique advantages over the labor intensive training of nonhuman primates while studying a broad range of these complex functions.
Here, we introduce a protocol for operantly conditioning rats on performing working memory tasks. During critical epochs of the task, the protocol ensures that the animal's overt movement is minimized by requiring the animal to 'fixate' until a Go cue is delivered, akin to nonhuman primate experimental design. A simple two alternative forced choice task is implemented to demonstrate the performance. We discuss the application of this paradigm to other tasks.

A fully automated protocol for rodent operant conditioning is proposed. The protocol relies on precise temporal control of behavioral events to investigate the extent to which this control influences neural activity underlying sensorimotor integration and cognitive control experiments.

Rodents have been traditionally used as a standard animal model in laboratory experiments involving a myriad of sensory, cognitive, and motor tasks. ...

A Procedure to Observe Context-induced Renewal of Pavlovian-conditioned Alcohol-seeking Behavior in Rats

Environmental contexts in which drugs of abuse are consumed can trigger craving, a subjective Pavlovian-conditioned response that can facilitate drug-seeking behavior and prompt relapse in abstinent drug users. We have developed a procedure to study the behavioral and neural processes that mediate the impact of context on alcohol-seeking behavior in rats. Following acclimation to the taste and pharmacological effects of 15% ethanol in the home cage, male Long-Evans rats receive Pavlovian discrimination training (PDT) in conditioning chambers. In each daily (Mon-Fri) PDT session, 16 trials each of two different 10 sec auditory conditioned stimuli occur. During one stimulus, the CS+, 0.2 ml of 15% ethanol is delivered into a fluid port for oral consumption. The second stimulus, the CS-, is not paired with ethanol. Across sessions, entries into the fluid port during the CS+ increase, whereas entries during the CS- stabilize at a lower level, indicating that a predictive association between the CS+ and ethanol is acquired. During PDT each chamber is equipped with a specific configuration of visual, olfactory and tactile contextual stimuli. Following PDT, extinction training is conducted in the same chamber that is now equipped with a different configuration of contextual stimuli. The CS+ and CS- are presented as before, but ethanol is withheld, which causes a gradual decline in port entries during the CS+. At test, rats are placed back into the PDT context and presented with the CS+ and CS- as before, but without ethanol. This manipulation triggers a robust and selective increase in the number of port entries made during the alcohol predictive CS+, with no change in responding during the CS-. This effect, referred to as context-induced renewal, illustrates the powerful capacity of contexts associated with alcohol consumption to stimulate alcohol-seeking behavior in response to Pavlovian alcohol cues.

A procedure to study the capacity of an alcohol associated environmental context to trigger the renewal of alcohol-seeking behavior in rats is described.

Environmental contexts in which drugs of abuse are consumed can trigger craving, a subjective Pavlovian-conditioned response that can facilitate ...

Protocol for Studying Extinction of Conditioned Fear in Naturally Cycling Female Rats

Extinction of conditioned fear has been extensively studied in male rodents. Recently, there have been an increasing number of studies indicating that neural mechanisms for certain behavioral tasks and response behaviors are different in females and males. Using females in research studies can represent a challenge because of the variation of gonadal hormones during their estrous cycle. This protocol describes well-established procedures that are useful in investigating the role of estrogen in fear extinction memory consolidation in female rats. Phase of the estrous cycle and exogenous estrogen administration prior to extinction training can influence extinction recall 24 hr later. The vaginal swabbing technique for estrous phase identification described here aids the examination and manipulation of naturally cycling gonadal hormones. The use of this basic rodent model may further delineate the mechanisms by which estrogen can modulate fear extinction memory in females.

Gonadal hormones such as estrogen modulate memory formation in a number of experimental paradigms including fear extinction memory. This protocol describes a set of methods for investigating the influence of gonadal hormones specifically during extinction in naturally cycling females, including estrous cycle monitoring and exogenous estrogen administration.

Extinction of conditioned fear has been extensively studied in male rodents. Recently, there have been an increasing number of studies indicating that ...

Signal Attenuation as a Rat Model of Obsessive Compulsive Disorder

In the signal attenuation rat model of obsessive-compulsive disorder (OCD), lever-pressing for food is followed by the presentation of a compound stimulus which serves as a feedback cue. This feedback is later attenuated by repeated presentations of the stimulus without food (without the rat emitting the lever-press response). In the next stage, lever-pressing is assessed under extinction conditions (i.e., no food is delivered). At this stage rats display two types of lever-presses, those that are followed by an attempt to collect a reward, and those that are not. The latter are the measure of compulsive-like behavior in the model. A control procedure in which rats do not experience the attenuation of the feedback cue serves to distinguish between the effects of signal attenuation and of extinction. The signal attenuation model is a highly validated model of OCD and differentiates between compulsive-like behaviors and behaviors that are repetitive but not compulsive. In addition the measures collected during the procedure eliminate alternative explanations for differences between the groups being tested, and are quantitative, unbiased and unaffected by inter-experimenter variability. The major disadvantages of this model are the costly equipment, the fact that it requires some technical know-how and the fact that it is time-consuming compared to other models of OCD (11 days). The model may be used for detecting the anti- or pro-compulsive effects of pharmacological and non-pharmacological manipulations and for studying the neural substrate of compulsive behavior.

The goal of the protocol described in this paper is to induce compulsive-like behavior in rats for the study of obsessive-compulsive disorder (OCD). This behavior is precipitated by attenuating a signal indicating that a lever-press response was effective in producing food.

In the signal attenuation rat model of obsessive-compulsive disorder (OCD), lever-pressing for food is followed by the presentation of a compound stimulus ...

A Fully Automated and Highly Versatile System for Testing Multi-cognitive Functions and Recording Neuronal Activities in Rodents

We have developed a fully automated system for operant behavior testing and neuronal activity recording by which multiple cognitive brain functions can be investigated in a single task sequence. The unique feature of this system is a custom-made, acoustically transparent chamber that eliminates many of the issues associated with auditory cue control in most commercially available chambers. The ease with which operant devices can be added or replaced makes this system quite versatile, allowing for the implementation of a variety of auditory, visual, and olfactory behavioral tasks. Automation of the system allows fine temporal (10 ms) control and precise time-stamping of each event in a predesigned behavioral sequence. When combined with a multi-channel electrophysiology recording system, multiple cognitive brain functions, such as motivation, attention, decision-making, patience, and rewards, can be examined sequentially or independently.

In this report, we present a fully automated and highly versatile system capable of simultaneously testing multi-cognitive behaviors and recording neuronal activities for rodents.

We have developed a fully automated system for operant behavior testing and neuronal activity recording by which multiple cognitive brain functions can be ...

Neuroscience

Using Clicker Training and Social Observation to Teach Rats to Voluntarily Change Cages

Cage cleaning is a routinely performed husbandry procedure and is known to induce stress in laboratory rats. As stress can have a negative impact on well-being and can affect the comparability and reproducibility of research results, the amount of stress experienced by laboratory animals should be minimized and avoided when possible. Further, the direct contact between the rat and animal caretaker during the cage change bears hygiene risks and therefore possibly negatively impacts the well-being of the rats and the quality of the research. 
Our protocol aims to improve the routinely performed cage changing procedure. For this reason, we present a feasible protocol that enables rats to learn via clicker training and observation to voluntarily change to a clean cage. This training helps to reduce stress caused by the physical disturbance and handling associated with the cage changes and concurrently enables a reduction in direct contact between animal and animal caretaker after the training phase is completed.
The implementation of clicker training to rats is fast and easy. Rats are generally interested in the training and efficiently learn the desired behavior, which entails changing cages through a pipe. Even without training, the rats learn to perform the desired behavior by observation, as 80% of the observational learning group successfully changed cages when tested. The training further helps to establish a relationship of trust between trainer and animal. As hygiene and well-being are both very important in animal experiments, this protocol might also help to improve high-quality research.

This protocol introduces a method of cage change for rats via clicker training. Rats learn the desired behavior not only by direct training but also by observational learning. The implementation of this fast and easy protocol might help to improve well-being and hygiene in rodent facilities.

Cage cleaning is a routinely performed husbandry procedure and is known to induce stress in laboratory rats. As stress can have a negative impact on ...

Fear Incubation Using an Extended Fear-Conditioning Protocol for Rats

Emotional memory has been primarily studied with fear-conditioning paradigms. Fear conditioning is a form of learning through which individuals learn the relationships between aversive events and otherwise neutral stimuli. The most-widely utilized procedures for studying emotional memories entail fear conditioning in rats. In these tasks, the unconditioned stimulus (US) is a footshock presented once or several times across single or several sessions, and the conditioned response (CR) is freezing. In a version of these procedures, called cued fear conditioning, a tone (conditioned stimulus, CS) is paired with footshocks (US) during the training phase. During the first test, animals are exposed to the same context in which training took place, and freezing responses are tested in the absence of footshocks and tones (i.e., a context test). During the second test, freezing is measured when the context is changed (e.g., by manipulating the smell and walls of the experimental chamber) and the tone is presented in the absence of footshocks (i.e., a cue test). Most cued fear conditioning procedures entail few tone-shock pairings (e.g., 1-3 trials in a single session). There is a growing interest in less common versions involving an extensive number of pairings (i.e., overtraining) related to the long-lasting effect called fear incubation (i.e., fear responses increase over time without further exposure to aversive events or conditioned stimuli). Extended fear-conditioning tasks have been key to the understanding of fear incubation&#8217;s behavioral and neurobiological aspects, including its relationship with other psychological phenomena (e.g., post-traumatic stress disorder). Here, we describe an extended fear-conditioning protocol that produces overtraining and fear incubation in rats. This protocol entails a single training session with 25 tone-shock pairings (i.e., overtraining) and a comparison of conditioned freezing responses during context and cue tests 48 h (short-term) and 6 weeks (long-term) after training.

We describe an extended fear-conditioning protocol that produces overtraining and fear incubation in rats. This protocol entails a single training session with 25 tone-shock pairings (i.e., overtraining) and a comparison of conditioned freezing responses during context and cue tests 48 h (short-term) and 6 weeks (long-term) after training.

Emotional memory has been primarily studied with fear-conditioning paradigms. Fear conditioning is a form of learning through which individuals learn the ...

A Novel Pavlovian Fear Conditioning Paradigm to Study Freezing and Flight Behavior

Fear- and anxiety-related behaviors significantly contribute to an organism&#8217;s survival. However, exaggerated defensive responses to perceived threat are characteristic of various anxiety disorders, which are the most prevalent form of mental illness in the United States. Discovering the neurobiological mechanisms responsible for defensive behaviors will aid in the development of novel therapeutic interventions. Pavlovian fear conditioning is a widely used laboratory paradigm to study fear-related learning and memory. A major limitation of traditional Pavlovian fear conditioning paradigms is that freezing is the only defensive behavior monitored. We recently developed a modified Pavlovian fear conditioning paradigm that allows us to study both conditioned freezing and flight (also known as escape) behavior within individual subjects. This model employs higher intensity footshocks and a greater number of pairings between the conditioned stimulus and unconditioned stimulus. Additionally, this conditioned flight paradigm utilizes serial presentation of pure tone and white noise auditory stimuli as the conditioned stimulus. Following conditioning in this paradigm, mice exhibit freezing behavior in response to the tone stimulus, and flight responses during the white noise. This conditioning model can be applied to the study of rapid and flexible transitions between behavioral responses necessary for survival.

Defensive behavioral responses are contingent upon threat intensity, proximity, and context of exposure. Based on these factors, we developed a classical conditioning paradigm that elicits clear transitions between conditioned freezing and flight behavior within individual subjects. This model is crucial for the understanding the pathologies involved in anxiety, panic, and post-traumatic stress disorders.

Fear- and anxiety-related behaviors significantly contribute to an organism&#8217;s survival. However, exaggerated defensive responses to perceived threat ...

An Inescapable Cat Odor Exposure Protocol for Studying Innate and Contextual Threat Conditioning in Rats

Animals respond to threatening situations by exhibiting a number of defensive behaviors, including avoidance, freezing, and risk assessment. An animal model with an ethological approach offers a deeper insight into the biological mechanisms underlying threat responses. This paper describes a methodology to measure defensive behaviors toward both innate and learned aversive stimuli in rats. Animals were individually exposed to predator odor in an inescapable chamber to elicit a measurable, sustained, defensive state. The experimental design involved placing a rat in a familiar chamber for 10 min followed by exposure to cat odor for another 10 min in the same context. The next day, the rats were re-exposed for 10 min to the same context chamber where cat odor exposure occurred. Sessions were video-recorded and defensive behaviors were assessed on both days. 
The behavioral test was coupled with reversible functional inactivation and c-Fos immunohistochemistry techniques to determine the role of the interoceptive cortex in threat responses. Rats exposed to cat odor on the first day and re-exposed to the context chamber on the second day displayed higher levels of defensive behaviors, and that cat odor elicited a robust increase in the neural activity of the interoceptive cortex. Moreover, muscimol inactivation of the interoceptive cortex reduced the expression of defensive behaviors in response to cat odor and impaired contextual threat memory. These results show that this behavioral assay is a useful tool for studying neural mechanisms of defensive behaviors and may offer insight into mechanisms that mediate fear in humans and its related disorders.

Cat skin/fur odor exposure provides a reliable method to investigate neural circuits and mechanisms of defensive responses in rodents and may offer insight into mechanisms that mediate fear in humans. Here, we describe a protocol for investigating the role of the interoceptive cortex in threat responses in rats.

Animals respond to threatening situations by exhibiting a number of defensive behaviors, including avoidance, freezing, and risk assessment. An animal ...

Classical Conditioning

Associative learning, a core principle in behavioral psychology, involves forming connections between events and facilitating learned responses. This concept is vividly illustrated by classical conditioning, a process extensively studied by the Russian physiologist Ivan Pavlov. Pavlov&#39;s pioneering research on dogs&#39; digestive systems led to the discovery that behaviors can be learned through association, laying the groundwork for classical conditioning.
Ivan Pavlov observed that dogs salivated not only when presented with food but also in response to stimuli associated with feeding, such as the sight of empty food bowls or the sound of laboratory assistant&#39;s footsteps. He systematically explored this phenomenon by pairing a neutral stimulus (a bell) with an unconditioned stimulus (meat powder) that naturally elicited salivation. Initially, the bell, as a neutral stimulus, did not provoke any salivation. However, after repeated pairings with the meat powder, the dogs began to salivate at the sound of the bell alone. This experiment demonstrated that the previously neutral stimulus had become a conditioned stimulus, eliciting a conditioned response of salivation.
Classical conditioning involves the association of a neutral stimulus with an unconditioned stimulus, resulting in a newly learned response. The process unfolds in several stages:
<ul>
	<li>&#8226; Before Conditioning: The unconditioned stimulus (meat powder) naturally triggers an unconditioned response (salivation). The neutral stimulus (bell) does not elicit salivation.</li>
	<li>&#8226; During Conditioning: The neutral stimulus (bell) is repeatedly paired with the unconditioned stimulus (meat powder), which continues to evoke the unconditioned response (salivation).</li>
	<li>&#8226; After Conditioning: The neutral stimulus (bell) transforms into a conditioned stimulus, which alone elicits a conditioned response (salivation).</li>
</ul>
This paradigm shift from a neutral to a conditioned stimulus and from an unconditioned to a conditioned response emphasizes the associative learning process fundamental to classical conditioning.
Pavlov&#39;s work on classical conditioning has far-reaching implications beyond the laboratory. It provides a framework for understanding how associative learning shapes behavior in various contexts. For example, in educational settings, teachers can use classical conditioning principles to create positive learning environments by associating learning activities with pleasant experiences. In therapeutic contexts, techniques such as systematic desensitization leverage classical conditioning to treat phobias by gradually associating the feared object with a sense of calm.
Overall, classical conditioning exemplifies the intricate process by which associations between stimuli lead to learned responses, contributing significantly to our understanding of behavior and learning mechanisms.

While studying dogs&#39; digestive systems, Ivan Pavlov, a Russian physiologist, discovered that behaviors can be learned through association.
He noticed ...