Name: pavlovian conditioned approach training in rats
Uploaded: 2016-02-04T15:00:00
Description: Watch this Scientific Journal Video about Pavlovian Conditioned Approach Training in Rats at JoVE.com

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 wi...

<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. 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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>

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&#8242; (7.6 m)</td>
			<td>Med Associates, Inc.</td>
			<td>SG-210CB</td>
			<td>Based on individual needs, SG-210CB-50 (Universal Cable, 50&#8242; [15.2 m]) is also available</td>
		</tr>
		<tr>
			<td>18&#8243; (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&#8242; [61.0 cm] 3-Pin Mini-Molex) is also available</td>
		</tr>
		<tr>
			<td>Power Cable, 25&#8242; (7.6 m)&#160;</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&#160;</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, 45mg</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&#160;</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&#160;</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 ...

The overall goal of this procedure is to elicit Pavlovian conditioned approach behavior in rats. This procedure can help answer key questions in the field of behavioral neuroscience, such as why some individuals can resist the temptation to pursue reward cues while others cannot. The main advantage of this procedure is that it allows us to measure the incentive motivational value of cues separately from their predictive value.Demonstrating this procedure will be Christopher Fitzpatrick, a neuroscience graduate student from my laboratory. This protocol utilizes commercial operant conditioning chambers in sound-attenuating cabinets with ventilation fans to provide ambient noise. Within each chamber, equip a pellet magazine with an infrared sensor for head entries.Position it in the front wall of each chamber, three centimeters above the floor, and attach a dispenser to deliver 45-milligram banana-flavored food pellets. Next, in a counterbalanced manner, position a retractable lever about 2.5 centimeters to either side of the receptacle and about 3 centimeters above the floor. The lever should have an LED cue light to make it more attractive.To calibrate the lever, place a 10-gram weight on it and adjust the calibration screw until the weight pushes the lever down. Then, attach a red light to the roof of the chamber above the receptacle. This will increase the conspicuousness of the illuminated lever.This completes the chamber preparations. Order adult male Sprague Dawley rats that are between 250 and 300 grams, and pair-house them on arrival. Leave the rats undisturbed for at least two days in the housing colony to acclimate.The colony should be maintained on a 12-hour light-dark cycle, and the rats should have ad libitum access to food and water. After an initial acclimation period, handle each rat for at least one minute per day, for a couple of days. On the two days before pre-training, give each rat 15 banana-flavored pellets daily.Divide these pellets evenly on separate sides of the home cage. For this protocol, create a pre-training program. The programming is done in MEDState Notation.The manual is available online. Write a program to deliver 50 food pellets on a variable-time 30-second schedule five minutes after the start of the program. The interval is on average 30 seconds but varies randomly between zero and 60 seconds.During this time, the red house light should be on, but the retractable lever should remain retracted for the duration of the program. Do the pre-training during the light cycle the day before PCA training. Place each rat into an operant chamber.Close and latch the chamber door. Close the sound-attenuating cabinet door, and immediately run the program. As soon as the program is completed, remove the rats from the chambers.Record the number of pellets consumed. Any rats that do not consume all of the pellets should be excluded from further testing. In the software, write a training program to run 25 trials over about 40 minutes, beginning one minute after the start of the program.During each trial, have the lever illuminate and extend into the chamber for eight seconds. The interior red light is always on. When the lever retracts, have one food pellet dispense independently of the rat's response.Separate each trial on a variable-time 90-second schedule. The interval is on average 90 seconds but varies randomly between 30 and 150 seconds. Run the PCA program just as the pre-training was run.After removing the rats, record the number of pellets consumed, and exclude rats that do not consume all of the pellets. Now, using data collected by the program, calculate the PCA index scores. Male rats underwent five PCA training sessions, during which the number, latency, and probability of magazine entries and lever presses were recorded.For each daily PCA training session, PCA index scores were calculated from these variables. The PCA index scores from the last two training sessions were used to categorize the rats as sign trackers, goal trackers, or as having an intermediate phenotype. Sign trackers primarily approached and directed their behavior toward the reward-related lever cue.On the PCA index, their scores ranged from 5 to 1. In contrast, goal trackers primarily approached and directed their behavior toward the source of reward delivery, the pellet magazine. On the PCA index, their scores ranged from 5 to 1.The intermediate phenotype does not bias either behavior. After watching this video, you should have a good understanding of how to elicit Pavlovian conditioned approach in rats.

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Behavior

Stereotaxic Microinjection of Viral Vectors Expressing Cre Recombinase to Study the Role of Target Genes in Cocaine Conditioned Place Preference

Microinjecting recombinant adenoassociated viral (rAAV) vectors expressing Cre recombinase into distinct mouse brain regions to selectively knockout genes of interest allows for enhanced temporally- and regionally-specific control of gene deletion, compared to existing methods. While conditional deletion can also be achieved by mating mice that express Cre recombinase under the control of specific gene promoters with mice carrying a floxed gene, stereotaxic microinjection allows for targeting of discrete brain areas at experimenter-determined time points of interest. In the context of cocaine conditioned place preference, and other cocaine behavioral paradigms such as self-administration or psychomotor sensitization that can involve withdrawal, extinction and/or reinstatement phases, this technique is particularly useful in exploring the unique contribution of target genes to these distinct phases of behavioral models of cocaine-induced plasticity. Specifically, this technique allows for selective ablation of target genes during discrete phases of a behavior to test their contribution to the behavior across time. Ultimately, this understanding allows for more targeted therapeutics that are best able to address the most potent risk factors that present themselves during each phase of addictive behavior.

This article describes how to microinject viral vectors into mouse brain and then test in a conditioned place preference paradigm that includes an acquisition, extinction and reinstatement phase.

Microinjecting recombinant adenoassociated viral (rAAV) vectors expressing Cre recombinase into distinct mouse brain regions to selectively knockout genes ...

A Procedure to Study the Effect of Prolonged Food Restriction on Heroin Seeking in Abstinent Rats

In human drug addicts, exposure to drug-associated cues or environments that were previously associated with drug taking can trigger relapse during abstinence. Moreover, various environmental challenges can exacerbate this effect, as well as increase ongoing drug intake.
The procedure we describe here highlights the impact of a common environmental challenge, food restriction, on drug craving that is expressed as an augmentation of drug seeking in abstinent rats.
Rats are implanted with chronic intravenous i.v. catheters, and then trained to press a lever for i.v. heroin over a period of 10-12 days. Following the heroin self-administration phase the rats are removed from the operant conditioning chambers and housed in the animal care facility for a period of at least 14 days. While one group is maintained under unrestricted access to food (sated group), a second group (FDR group) is exposed to a mild food restriction regimen that results in their body weights maintained at 90% of their nonrestricted body weight. On day 14 of food restriction the rats are transferred back to the drug-training environment, and a drug-seeking test is run under extinction conditions (i.e.&#160;lever presses do not result in heroin delivery).
The procedure presented here results in a highly robust augmentation of heroin seeking on test day in the food restricted rats. In addition, compared to the acute food deprivation manipulations we have used before, the current procedure is a more clinically relevant model for the impact of caloric restriction on drug seeking. Moreover, it might be closer to the human condition as the rats are not required to go through an extinction-training phase before the drug-seeking test, which is an integral component of the popular reinstatement procedure.

A procedure that allows a demonstration of robust augmentation of drug seeking in food-restricted rats is described. Following heroin self-administration training, rats go through an abstinence period, in a drug-free environment, during which they are mildly food restricted. Drug seeking is then tested in the drug-associated environment.

In human drug addicts, exposure to drug-associated cues or environments that were previously associated with drug taking can trigger relapse during ...

Training Synesthetic Letter-color Associations by Reading in Color

Synesthesia is a rare condition in which a stimulus from one modality automatically and consistently triggers unusual sensations in the same and/or other modalities. A relatively common and well-studied type is grapheme-color synesthesia, defined as the consistent experience of color when viewing, hearing and thinking about letters, words and numbers. We describe our method for investigating to what extent synesthetic associations between letters and colors can be learned by reading in color in nonsynesthetes. Reading in color is a special method for training associations in the sense that the associations are learned implicitly while the reader reads text as he or she normally would and it does not require explicit computer-directed training methods. In this protocol, participants are given specially prepared books to read in which four high-frequency letters are paired with four high-frequency colors. Participants receive unique sets of letter-color pairs based on their pre-existing preferences for colored letters. A modified Stroop task is administered before and after reading in order to test for learned letter-color associations and changes in brain activation. In addition to objective testing, a reading experience questionnaire is administered that is designed to probe for differences in subjective experience. A subset of questions may predict how well an individual learned the associations from reading in color. Importantly, we are not claiming that this method will cause each individual to develop grapheme-color synesthesia, only that it is possible for certain individuals to form letter-color associations by reading in color and these associations are similar in some aspects to those seen in developmental grapheme-color synesthetes. The method is quite flexible and can be used to investigate different aspects and outcomes of training synesthetic associations, including learning-induced changes in brain function and structure.

Reading in color is a new method for training letter-color associations that are typically found only in grapheme-color synesthetes. It involves an implicit form of training that has potential for long-term associative training methods&#160;because the training is a byproduct of reading and any text can be colored.

Synesthesia is a rare condition in which a stimulus from one modality automatically and consistently triggers unusual sensations in the same and/or other ...

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 ...

Training Rats to Voluntarily Dive Underwater: Investigations of the Mammalian Diving Response

Underwater submergence produces autonomic changes that are observed in virtually all diving animals. This reflexly-induced response consists of apnea, a parasympathetically-induced bradycardia and a sympathetically-induced alteration of vascular resistance that maintains blood flow to the heart, brain and exercising muscles. While many of the metabolic and cardiorespiratory aspects of the diving response have been studied in marine animals, investigations of the central integrative aspects of this brainstem reflex have been relatively lacking. Because the physiology and neuroanatomy of the rat are well characterized, the rat can be used to help ascertain the central pathways of the mammalian diving response. Detailed instructions are provided on how to train rats to swim and voluntarily dive underwater through a 5 m long Plexiglas maze. Considerations regarding tank design and procedure room requirements are also given. The behavioral training is conducted in such a way as to reduce the stressfulness that could otherwise be associated with forced underwater submergence, thus minimizing activation of central stress pathways. The training procedures are not technically difficult, but they can be time-consuming. Since behavioral training of animals can only provide a model to be used with other experimental techniques, examples of how voluntarily diving rats have been used in conjunction with other physiological and neuroanatomical research techniques, and how the basic training procedures may need to be modified to accommodate these techniques, are also provided. These experiments show that voluntarily diving rats exhibit the same cardiorespiratory changes typically seen in other diving animals. The ease with which rats can be trained to voluntarily dive underwater, and the already available data from rats collected in other neurophysiological studies, makes voluntarily diving rats a good behavioral model to be used in studies investigating the central aspects of the mammalian diving response.

Detailed instructions are provided on how to train rats to voluntarily dive underwater through a 5 m long Plexiglas maze. Because the brains of rats have been very well characterized, voluntarily diving rats may help elucidate the central pathways of the mammalian diving response.

Underwater submergence produces autonomic changes that are observed in virtually all diving animals. This reflexly-induced response consists of apnea, a ...

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 ...

The Modified Hole Board - Measuring Behavior, Cognition and Social Interaction in Mice and Rats

This protocol describes the modified hole board (mHB), which combines features from a traditional hole board and open field and is designed to measure multiple dimensions of unconditioned behavior in small laboratory mammals (e.g., mice, rats, tree shrews and small primates). This paradigm is a valuable alternative for the use of a behavioral test battery, since a broad behavioral spectrum of an animal&#8217;s behavioral profile can be investigated in one single test.
The apparatus consists of a box, representing the &#8216;protected&#8217; area, separated from a group compartment. A board, on which small cylinders are staggered in three lines, is placed in the center of the box, representing the &#8216;unprotected&#8217; area of the set-up. The cognitive abilities of the animals can be measured by baiting some cylinders on the board and measuring the working and reference memory. Other unconditioned behavior, such as activity-related-, anxiety-related- and social behavior, can be observed using this paradigm. Behavioral flexibility and the ability to habituate to a novel environment can additionally be observed by subjecting the animals to multiple trials in the mHB, revealing insight into the animals&#8217; adaptive capacities.
Due to testing order effects in a behavioral test battery, na&#239;ve animals should be used for each individual experiment. By testing multiple behavioral dimensions in a single paradigm and thereby circumventing this issue, the number of experimental animals used is reduced. Furthermore, by avoiding social isolation during testing and without the need to food deprive the animals, the mHB represents a behavioral test system, inducing if any, very low amount of stress.

This protocol describes the modified hole board, which is a behavioral test set-up that comprises the characteristics of an open field and a traditional hole board. This set-up enables the differential analysis of unconditioned behavior of small laboratory mammals as well as the analysis of cognitive abilities.

This protocol describes the modified hole board (mHB), which combines features from a traditional hole board and open field and is designed to measure ...

Assessment of Cocaine-induced Behavioral Sensitization and Conditioned Place Preference in Mice

It is thought that rewarding experiences with drugs create strong contextual associations and encourage repeated intake. In turn, repeated exposures to drugs of abuse make lasting alterations in the brain function of vulnerable individuals, and these persistent alterations likely serve to maintain the maladaptive drug seeking and taking behaviors characteristic of addiction/dependence2. In rodents, reward experience and contextual associations are frequently measured using the conditioned place preference assay, or CPP, wherein preference for a previously drug-paired context is measured. Behavioral sensitization, on the other hand, is an increase in a drug-induced behavior that develops progressively over repeated exposures. Since sensitized behaviors can often be measured after several months of drug abstinence, depending on the dose and length of initial exposure, they are considered observable correlates of lasting drug-induced plasticity. Researchers have found these assays useful in determining the neurobiological substrates mediating aspects of addiction as well as assessing the potential of different interventions in disrupting these behaviors. This manuscript describes basic, effective protocols for mouse CPP and locomotor behavioral sensitization to cocaine.

This protocol is intended to enable researchers to conduct experiments designed to test these aspects of addiction using the conditioned place preference and locomotor behavioral sensitization assays.

It is thought that rewarding experiences with drugs create strong contextual associations and encourage repeated intake. In turn, repeated exposures to ...

A Method for Evaluating the Reinforcing Properties of Ethanol in Rats without Water Deprivation, Saccharin Fading or Extended Access Training

Operant oral self-administration methods are commonly used to study the reinforcing properties of ethanol in animals. However, the standard methods require saccharin/sucrose fading, water deprivation and/or extended training to initiate operant responding in rats. This paper describes a novel and efficient method to quickly initiate operant responding for ethanol that is convenient for experimenters and does not require water deprivation or saccharin/sucrose fading, thus eliminating the potential confound of using sweeteners in ethanol operant self-administration studies. With this method, Wistar rats typically acquire and maintain self-administration of a 20% ethanol solution in less than two weeks of training. Furthermore, blood ethanol concentrations and rewards are positively correlated for a 30 min self-administration session. Moreover, naltrexone, an FDA-approved medication for alcohol dependence that has been shown to suppress ethanol self-administration in rodents, dose-dependently decreases alcohol intake and motivation to consume alcohol for rats self-administering 20% ethanol, thus validating the use of this new method to study the reinforcing properties of alcohol in rats.

This protocol describes a novel and efficient method to quickly initiate operant responding for ethanol in rats that, contrary to standard methods, does not require water deprivation or saccharin/sucrose fading to initiate responding.

Operant oral self-administration methods are commonly used to study the reinforcing properties of ethanol in animals. However, the standard methods ...

Reinstatement of Drug-seeking in Mice Using the Conditioned Place Preference Paradigm

The present protocol describes the Conditioned Place Preference (CPP) as a model of relapse in drug addiction. In this model, animals are first trained to acquire a conditioned place preference in a drug-paired compartment, and after the post-conditioning test, they perform several sessions to extinguish the established preference. The CPP permits the evaluation of the conditioned rewarding effects of drugs related to environmental cues. Then, the extinguished CPP can be robustly reinstated by the non-contingent administration of a priming dose of the drug, and by exposure to stressful stimuli. Both methods will be explained here. When the animal reinitiates the behavioral response, a reinstatement of the conditioned reward is considered to have taken place.
The main advantages of this protocol are that it is non-invasive, inexpensive, and simple with good validity criteria. In addition, it allows the study of different environmental manipulations, such as stress or diet, which can modulate relapse into drug seeking behaviors. However, one limitation is that if the researcher aims to explore the motivation and primary reinforcing effects of the drug, it should be complemented with self-administration procedures, as they involve operant responses of animals.

This protocol describes the Conditioned Place Preference (CPP) as a model of relapse. This procedure permits the measurement of relapse in laboratory animals, considering the impact of drug-associated environmental cues as craving and relapse in abstaining addicts is currently the focus of drug-abuse treatment programs.

The present protocol describes the Conditioned Place Preference (CPP) as a model of relapse in drug addiction. In this model, animals are first trained to ...