This work was supported by the National Institute on Alcohol Abuse and Alcoholism (RO1 AA014925 awarded to Dr. Patricia H. Janak at University of California, San Francisco, CA, USA), Fonds de recherche du Quebec &#8211; Sant&#233; (NC) and the Natural Sciences and Engineering Research Council (NC, 387224-2010). The open-access publication of this article was made possible by sponsorship from Med Associates Inc.

All procedures are approved by the Animal Research Ethics Committee at Concordia University and concur with recommendations from the Canadian Council on Animal Care.
1. Animals
<ol>
	<li>Obtain male Long-Evans rats (220 &#8211; 240 g on arrival).</li>
	<li>Maintain rats in a temperature (21 &#176;C) and humidity (44%) controlled animal care facility, on a 12 hr light-dark cycle with lights on at 7:00 am.</li>
	<li>Pair-house rats upon arrival. After 3 days, house rats individually in plastic shoebox cages with beta-chip bedding, a nylon bone, and free access to standard rat chow and water. Use cage lids that have double grommets to allow for the subsequent placement of a second bottle, in addition to the water bottle. NOTE: Rats may be individually housed as soon as they arrive in the animal care facility.</li>
	<li>Begin to handle rats daily. NOTE: As a guide, one week of handling is usually sufficient to acclimate them to being picked up and handled by the experimenter.</li>
</ol>
2. Intermittent Ethanol Access in the Home Cage
NOTE: Conduct intermittent ethanol access in the home cage to ensure that rats will drink physiologically relevant quantities of ethanol before the behavioral training phase of the experiment begins 17-19. Initiate this procedure during the light phase, although it could be initiated at any time during the light or dark cycle.
<ol>
	<li>Prepare two bottles for each rat.
	<ol>
		<li>Use a 100 ml plastic graduated cylinder as the ethanol bottle and a 473 ml standard plastic water bottle. Use a relatively small capacity ethanol bottle, filled almost to maximum volume capacity, to minimize loss of ethanol due to leakage and evaporation.</li>
		<li>Fill one bottle with 15% ethanol, made by diluting 95% ethanol in tap water. NOTE: Other ethanol concentrations, such as 10% or 20%, may be used in place of 15% ethanol.</li>
		<li>Fill the second bottle with tap water.</li>
		<li>Insert a rubber stopper, with a sipper tube that contains a ball bearing to minimize leakage, into each bottle.</li>
	</ol>
	</li>
	<li>Set up two control cages, treated identically to the home cages with the exception that the control cages do not contain rats or bedding.
	<ol>
		<li>Prepare ethanol and water bottles for the control cages as specified in steps 2.1.1 &#8211; 2.1.4.</li>
		<li>Use the measures obtained from the control cages to control for spillage and/or evaporation that may occur over the course of the ethanol access session (as described in steps 2.8.1 &#8211; 2.8.2).</li>
	</ol>
	</li>
	<li>Weigh all the ethanol and water bottles. Record all weights.</li>
	<li>Weigh all the rats. Record all weights.</li>
	<li>Place the ethanol bottle and the water bottle on each home cage and control cage at the same time. Leave both bottles on the cages for 24 hr.</li>
	<li>At the end of the 24 hr period, remove both bottles from the cages at the same time.</li>
	<li>Weigh all the ethanol and water bottles. Record all weights.</li>
	<li>To determine how much ethanol and water was consumed during the 24 hr period, subtract the weight of each bottle when it was removed from the home cage from the weight of the bottle when it was placed on the home cage 24 hr earlier for each rat.
	<ol>
		<li>Do the same for the two control cages, and then calculate the average amount of spillage for each fluid.</li>
		<li>Subtract the average amount of ethanol and water spillage, respectively, from the corresponding ethanol and water consumption measures for each rat.</li>
		<li>Use these spillage controlled difference measures to calculate ethanol consumption in g/kg, consumption of water, and ethanol preference.</li>
	</ol>
	</li>
	<li>To calculate ethanol consumption (g/kg), first convert the weight (g) of ethanol solution consumed into volume (ml) based on the density of the total solution (ethanol + water components), according to the formula Volume (ml) = Weight (g)/Density (g/ml). NOTE: The density of the solution will vary as a function of ethanol concentration.
	<ol>
		<li>Next, multiply the volume (ml) of ethanol solution consumed by the density (g/ml) of ethanol scaled for the specific percentage solution used, to yield the weight (g) of ethanol consumed.</li>
		<li>Divide the weight (g) of ethanol consumed by the rat weight (kg) to obtain the g/kg measure of ethanol consumption.</li>
	</ol>
	</li>
	<li>To calculate ethanol preference, express ethanol intake (ml) as a percentage of total fluid (ethanol + water, ml) intake.</li>
	<li>Return the water bottle only back to the rat&#8217;s home cage. For the next 24 hr, give the rats access to water but not ethanol. Instead of an ethanol bottle, place a pre-weighed second water bottle on the home cage.</li>
	<li>At the end of the 24 hr period, remove both water bottles from the cage at the same time.</li>
	<li>Weigh both sets of water bottles. Record all weights.</li>
	<li>Subtract the weight of each bottle when it was removed from the home cage from the weight of the bottle when it was placed on the home cage 24 hr earlier, subtracting out the average spillage measure obtained from control cages as described in steps 2.8.1 &#8211; 2.8.2.</li>
	<li>Use these spillage-controlled difference measures to calculate consumption of water.</li>
	<li>Begin the cycle again by providing both ethanol and water bottles to rats, as described in steps 2.1 through 2.14.
	<ol>
		<li>Repeat this cycle for multiple (about 12) sessions to induce high and stable levels of ethanol consumption. See Figure 1 for representative results of ethanol consumption (g/kg) in the home cage.</li>
		<li>Alternate the spatial position of the ethanol and water bottle on the cage lid across sessions to prevent the development of a side preference.</li>
		<li>Make a fresh ethanol solution once per week to ensure accurate and stable ethanol concentration across sessions. Minimize the impact of evaporation of ethanol over prolonged periods of time using this approach.</li>
	</ol>
	</li>
</ol>
3. Apparatus
<ol>
	<li>House each conditioning chamber inside a ventilated, sound-attenuating cubicle.</li>
	<li>Construct each&#160;conditioning chamber of a stainless steel metal rod floor, a clear polycarbonate ceiling, clear polycarbonate back wall and front door, and aluminum metal panel sidewalls.
	<ol>
		<li>Install a fluid port centrally near the base of the right metal sidewall. Equip the entrance to the fluid port with an infrared photocell to detect entries into the port.</li>
		<li>Connect polyethylene tubing from the back of the fluid port to a 20 ml syringe containing ethanol using a blunt-tipped needle. Place the syringe into a syringe pump located outside the sound-attenuating cubicle. For Pavlovian discrimination training sessions, load ethanol into the syringe before placing it into the pump. Prime the tubing by manually advancing the pump, and then wipe away excess ethanol from the fluid port.&#160;</li>
		<li>On the left sidewall, install a white houselight centrally near the ceiling of the chamber.</li>
		<li>Install a clicker (2 Hz cycle) and a white noise generator with speaker in the upper left corner of the left sidewall.</li>
	</ol>
	</li>
	<li>Outfit each conditioning chamber with a specific configuration of visual, olfactory, and tactile stimuli to create a distinctive context.
	<ol>
		<li>Use two different contexts, designated Context 1 and Context 2. Configure half of the conditioning chambers as Context 1 and the other half as Context 2.</li>
		<li>To create Context 1, place black cardboard panels over the ceiling and front and back walls of the chamber, spray lemon odor (10% v/v, made by diluting lemon oil in tap water) into a Petri dish and place it in the center of a metal waste tray under the floor of the chamber, and insert a clear polycarbonate floor panel over the metal rod floor. Line the waste tray with absorbent paper towel or bench liner.</li>
		<li>To create Context 2, leave the ceiling and front and back walls of the chamber uncovered, so that they are clear, spray almond odor (10% v/v, made by diluting benzaldehyde in tap water) into a Petri dish and place it centrally in a metal waste tray under the floor of the chamber, and insert a perforated metal floor panel over the metal rod floor. Line the waste tray with absorbent paper towel or bench liner.</li>
	</ol>
	</li>
</ol>
4. Habituation
Habituate rats to the behavioral training room and each of the two contexts within conditioning chambers. For this phase of the experiment, do not load ethanol into the 20 ml syringes.
<ol>
	<li>Habituate rats to the process of being transported from the animal care facility to the behavioral training room in the laboratory in two short habituation sessions.
	<ol>
		<li>In the first session, load the rats in their home cages onto a wheeled cart in the animal care facility, and transport them to the behavioral training room in the laboratory. Illuminate the room lights, turn on the fans in each sound attenuating cubicle, exit the room, close the door, and let the rats adjust to this new environment for 20 min, while remaining in their home cages on the cart. At the end of this 20 min period, return the rats to the animal care facility.</li>
		<li>In the second session, bring the rats to the behavioral training room in the laboratory. As before, leave the lights and cubicle fans on in the room. Remain in the room, with the door closed to minimize distractions, and handle and weigh each rat in the behavioral training room. Record the weights, and then return the rats to the animal care facility.</li>
	</ol>
	</li>
	<li>Habituate rats to the conditioning chambers in two short habituation sessions.
	<ol>
		<li>Transport the rats to the behavioral training room, record each rat&#8217;s weight, and load each rat into its designated conditioning chamber.</li>
		<li>In the first habituation session set up half the chambers as Context 1 and the remainder as Context 2.</li>
		<li>In the second habituation session set up each chamber with the context that was not used in the previous session.</li>
		<li>Conduct habituation sessions in a counterbalanced fashion, such that half of the rats are first exposed to Context 1, followed by Context 2, and the other half are first exposed to Context 2, followed by Context 1. Ideally, conduct both habituation sessions in the same day, although they could be spread across two days if necessary.</li>
		<li>For habituation sessions, use a computer program to illuminate the houselight 1 min&#160;after the program has been issued. Record the total number of fluid port entries made during a 20 min session. At the end of the session, program the houselight to turn off. Do not present any ethanol or cues during habituation sessions.</li>
	</ol>
	</li>
	<li>Remove rats from the chambers, return them to their home cages, and transport them back to the animal care facility.</li>
	<li>Clean the equipment (e.g., floor panels, waste trays, Petri dishes). See step 5.7 for further details.</li>
</ol>
5. Pavlovian Discrimination Training (PDT)
Train rats to behaviorally discriminate between one auditory cue that is paired with ethanol and a second auditory cue that is not. For this phase of the experiment, fill the 20 ml syringes with 15% ethanol.
<ol>
	<li>Outfit each conditioning chamber with a specific configuration of visual, olfactory, and tactile stimuli to create a distinctive context, as described in steps 3.3.2 and 3.3.3
	<ol>
		<li>Assign half of the rats to Context 1 and the other half to Context 2 in a counterbalanced fashion, based on ethanol consumption and preference averaged across the last 2 sessions of the home cage ethanol access procedure. Refer to the context used during PDT as Context A. NOTE: Each rat should only experience one context in this phase of training.</li>
		<li>Make sure that the olfactory stimulus is the last contextual stimulus to be added to each conditioning chamber. Close the doors of the chamber and the sound-attenuating cubicle to contain the olfactory stimulus and minimize the comingling of different odors.</li>
	</ol>
	</li>
	<li>Bring the rats into the behavioral training room, record each rat&#8217;s weight, and load each rat into its designated conditioning chamber.</li>
	<li>Initiate the computer program that controls the following sequence of events during each PDT session.
	<ol>
		<li>At 5 min after the program has been issued, illuminate the houselight.</li>
		<li>Present the white noise stimulus and the clicker stimulus 16 times each. Designate one of these auditory stimuli to be the CS+ and the other to be the CS-, in a counterbalanced fashion across both contexts.</li>
		<li>Make each CS+ and CS- trial last 10 sec.</li>
		<li>Deliver 0.2 ml ethanol into the fluid port during the last 6 sec of the CS+. Do not deliver any ethanol during the CS-.</li>
		<li>Make each PDT session last 58 min on average. Present the CS+ and the CS- each on a variable time 67 sec schedule.</li>
		<li>Record fluid port entries during the 10 sec CS interval (the 10 sec during CS presentation), the 10 sec pre-CS interval (the 10 sec immediately prior to CS onset), and the 10 sec post-CS interval (the 10 sec immediately following CS offset) for both CS+ and CS- presentations, as well as total fluid port entries for the entire session. Also record the duration of each port entry made during these intervals.</li>
		<li>At the end of the session, turn the houselight off.</li>
	</ol>
	</li>
	<li>Remove rats from the chambers, return them to their home cages, and transport them back to the animal care facility.</li>
	<li>Check the fluid ports to ensure that rats consumed the 3.2 ml of ethanol delivered in each PDT session.</li>
	<li>Flush the ethanol out of the polyethylene tubing with a 60 ml syringe containing a small amount of water. Then, pass 60 ml of air through the tubing using a second, empty syringe, to make sure it is dry.</li>
	<li>Clean the equipment. For daily cleaning, wash the floor panels, waste trays, and Petri dishes. Once a week, clean the entire conditioning chamber. Do not use scented cleaning products to avoid introducing unwanted odors into the experimental apparatus.</li>
	<li>Continue PDT sessions daily until rats show stable discrimination in their port entry responses to the CS+ vs. CS-. NOTE: Port entries made in response to the CS+ should increase over sessions, whereas port entries made in response to the CS- should remain low. The exact number of PDT sessions necessary to achieve discriminated conditioned responding can vary, but, as a guide, plan on approximately 20 sessions.</li>
</ol>
6. Extinction
<ol>
	<li>Outfit each conditioning chamber with visual, olfactory, and tactile stimuli to create a context that is distinct from the one the rats experienced during PDT sessions. For example, if Context 1 was used in PDT, configure the chamber as Context 2 for extinction sessions. Refer to the context used during extinction as Context B.</li>
	<li>Conduct extinction sessions identically to PDT sessions, but do not deliver any ethanol during the CS+.
	<ol>
		<li>Use the same computer program that was used during PDT to present the CS+ and CS-.</li>
		<li>To maintain consistency across phases of the experiment, load an empty syringe into the syringe pump, and leave the pump turned on. Make sure the polyethylene tubing does not contain ethanol before extinction sessions.</li>
	</ol>
	</li>
	<li>Continue extinction sessions daily until rats no longer show discriminated responding between the CS+ and CS-. NOTE: Port entries made in response to the CS+ should gradually decline across extinction sessions, until there is no difference between port entries made in response to the CS+ and port entries made in response to the CS-. The exact number of extinction sessions necessary to extinguish conditioned responding can vary, but as a guide, plan on approximately 8 sessions.</li>
	<li>Clean the equipment at the end of each session, as described in step 5.7.</li>
</ol>
7. Context-induced Renewal Test
<ol>
	<li>Equip each conditioning chamber with the visual, olfactory, and tactile stimuli to create the context that was experienced during PDT sessions. For example, if a given rat experienced Context 1 in PDT sessions, configure his chamber as Context 1 in the test session. Refer to the context used during the renewal test as Context A.</li>
	<li>Conduct the context-induced renewal test identically to PDT sessions, but do not deliver any ethanol during the renewal test. Present the 
	CS+ and CS- as before during PDT sessions, but withhold the ethanol.
	<ol>
		<li>Use the same computer program that was used during PDT to present the CS+ and CS-.</li>
		<li>To maintain consistency across phases, load an empty syringe into the syringe pump, and leave the pump turned on. Make sure the polyethylene tubing does not contain ethanol before the test session.</li>
	</ol>
	</li>
	<li>Observe context-induced renewal of Pavlovian-conditioned alcohol-seeking behavior. NOTE: This renewal effect will be evident by a selective increase in the number of port entries made in response to the CS+, with no change in port entry behavior made in response to the CS-, relative to CS+ and CS- responding observed at the end of extinction training.</li>
	<li>Clean the equipment at the end of the session, as described in step 5.7.</li>
</ol>

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V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Some+effects+of+two+temporal+variables+on+conditioned+suppression.">Some effects of two temporal variables on conditioned suppression.</a> J Exp Anal Behav. 1, 153-162 (1958).</li><li>Coleman, D. A., Hemmes, N. S., Brown, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relative+durations+of+conditioned+stimulus+and+intertrial+interval+in+conditioned+suppression.">Relative durations of conditioned stimulus and intertrial interval in conditioned suppression.</a> J Exp Anal Behav. 46, 51-66 (1986).</li><li>Schwarz-Stevens, K. S., Cunningham, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pavlovian+conditioning+of+heart+rate+and+body+temperature+with+morphine:+effects+of+CS+duration.">Pavlovian conditioning of heart rate and body temperature with morphine: effects of CS duration.</a> Behav Neurosci. 107, 1039-1048 (1993).</li><li>Palm, S., Roman, E., Nylander, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differences+in+voluntary+ethanol+consumption+in+Wistar+rats+from+five+different+suppliers.">Differences in voluntary ethanol consumption in Wistar rats from five different suppliers.</a> Alcohol. 45, 607-614 (2011).</li><li>Gauvin, D. V., Moore, K. R., Holloway, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Do+rat+strain+differences+in+ethanol+consumption+reflect+differences+in+ethanol+sensitivity+or+the+preparedness+to+learn.">Do rat strain differences in ethanol consumption reflect differences in ethanol sensitivity or the preparedness to learn.</a> Alcohol. 10, 37-43 (1993).</li><li>Goodwin, F. L., Amit, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relative+taste+thresholds+for+ethanol,+saccharin,+and+quinine+solutions+in+three+strains+of+rats+nonselected+for+ethanol:+a+comparative+study.">Relative taste thresholds for ethanol, saccharin, and quinine solutions in three strains of rats nonselected for ethanol: a comparative study.</a> Exp Clin Psychopharmacol. 8, 216-224 (2000).</li><li>Goodwin, F. L., Bergeron, N., Amit, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differences+in+the+consumption+of+ethanol+and+flavored+solutions+in+three+strains+of+rats.">Differences in the consumption of ethanol and flavored solutions in three strains of rats.</a> Pharmacol Biochem Behav. 65, 357-362 (2000).</li><li>Wilson, A. W., Neill, J. C., Costall, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strain+differences+in+ethanol+preference+and+reinforced+behaviour:+a+comparison+of+two-bottle+choice+and+operant+self-administration+paradigms.">Strain differences in ethanol preference and reinforced behaviour: a comparison of two-bottle choice and operant self-administration paradigms.</a> Behav Pharmacol. 8, 37-46 (1997).</li><li>Yap, C. S., Richardson, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extinction+in+the+developing+rat:+an+examination+of+renewal+effects.">Extinction in the developing rat: an examination of renewal effects.</a> Dev Psychobiol. 49, 565-575 (2007).</li><li>Carew, M. B., Rudy, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+functions+of+context+during+conditioning:+a+developmental+analysis.">Multiple functions of context during conditioning: a developmental analysis.</a> Dev Psychobiol. 24, 191-209 (1991).</li><li>Chaudhri, N., Sahuque, L. L., Janak, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ethanol+seeking+triggered+by+environmental+context+is+attenuated+by+blocking+dopamine+D1+receptors+in+the+nucleus+accumbens+core+and+shell+in+rats.">Ethanol seeking triggered by environmental context is attenuated by blocking dopamine D1 receptors in the nucleus accumbens core and shell in rats.</a> Psychopharmacology (Berl). 207, 303-314 (2009).</li></ol>

All experimental procedures were approved by the Concordia University Animal Research Ethics Committee and are in accordance with guidelines from the Canadian Council on Animal Care. The open-access publication of this article was made possible by sponsorship from Med Associates Inc.

Results from this procedure reveal that discrete environmental stimuli that routinely accompany alcohol delivery can acquire the capacity to drive alcohol-seeking behavior. They also demonstrate that contextual stimuli associated with the prior availability or absence of alcohol can guide conditioned behavioral responses to discrete alcohol-predictive cues.
Critical steps within the protocol
Contexts in the present task incorporate stimuli that are external to the rat. However, the interoceptive state of the rat at test can also constitute a &#8216;context&#8217; that could affect renewal20. It is therefore important to habituate rats to any procedures that may occur before the renewal test and change their interoceptive state, like intracranial microinfusions or systemic injections. Moreover, habituation to microinfusions or systemic injections should be conducted prior to both extinction and PDT sessions, to prevent such procedures from becoming associated with one specific phase of the experiment.
Temporal parameters, such as CS duration, CS-US interval, and intertrial interval (ITI), have long been known to influence conditioned responding in a variety of conditioning paradigms21-26. Similarly, temporal parameters may influence the observation of the renewal effect in our procedure. Any set of parameters other than those specified in the protocol should be experimentally validated in pilot studies before proceeding to employ them in larger investigations.
Modifications and troubleshooting
Previous research has found that the amount of ethanol that rats consume in the home cage can vary as a function of the supplier from which rats are purchased17,27. Supplier is therefore an important consideration for this phase of the study. Similarly, the choice of rat strain should be considered carefully, as there are numerous reports of strain differences in ethanol consumption28-31.
Prior work in an aversive conditioning paradigm has shown that the developmental age of rats influences the observation of renewal of a Pavlovian conditioned fear response32. The lack of a renewal effect in very young rats (younger than postnatal day 20) has been suggested to be due to impaired encoding of contextual information33. The developmental age of rats to be used in the present procedure should therefore be taken into consideration.
Renewal of Pavlovian conditioned alcohol-seeking behavior has been observed when 10%9,16, 15%14, or 20%15 ethanol solutions were used during the home cage ethanol access and PDT phases of the experiment. Researchers could use any of these concentrations of ethanol for the purposes of establishing this procedure in their laboratories.
During the acquisition of Pavlovian discrimination training it is important to check fluid ports after each session to ensure that all the ethanol that was delivered has been consumed. Leaving rats in the conditioning chambers for an additional 10 - 15 min after each session may be done early in PDT to encourage consumption of any ethanol that was not consumed during the session. The number of rats leaving unconsumed ethanol in the ports should decrease as PDT progresses. If by the end of PDT rats are either (a) not consuming all the ethanol and/or (b) not responding to the CS+ then these subjects should be dropped from the study. Most rats reliably respond more to the CS+ than the CS-. However, some rats may respond at high levels to both cues, thereby not showing evidence of discrimination. Rats that show high responding to both the CS+ and CS- during PDT are not dropped from the study. In our experience, these subjects show remarkable discrimination during the first extinction session in the absence of ethanol, suggesting that their high levels of responding to the CS- during PDT are driven by the presence of ethanol in the fluid port. These subjects also tend to show robust renewal effects.
Limitations of the procedure
Because the renewal test is conducted in the absence of ethanol, conditioned port entries elicited by the CS+ gradually diminish across the test session. Therefore, the overall amount of behavior generated at test, against which experimentally induced changes can be assessed, is fairly low. Because in this task responding tends to be higher at the start versus the end of the test session, it is important to examine the effects of experimental manipulations on conditioned responding on a trial-by-trial basis.
Entries into the fluid receptacle provide the main dependent measure in this task. However, an erroneous port entry might be registered if an animal is close enough to the fluid receptacle to break the infrared beam across the opening of the fluid port with its whiskers. While infrequent, erroneous responses are obvious as a dramatic increase in total port entries relative to the subject&#8217;s behavior on prior sessions. Videotaping the animals would facilitate the detection of possible erroneous responses, and allow for a more refined analysis of the behaviors exhibited by the rats throughout training and testing.
Significance of technique with respect to existing/alternative methods
Context-induced renewal of drug-seeking behavior is largely studied using an instrumental conditioning task in which the primary dependent variable is an operant response, such as a lever press8,34 or nose poke12. In the present procedure, the drug-seeking response is &#8216;conditioned approach&#8217; to the fluid port that is elicited by presentations of the CS+. This response is likely acquired through Pavlovian learning, which is the learning process that mediates the formation of associations between environmental stimuli and the pharmacological effects of drugs of abuse in humans.
Future applications
This task can be used in combination with neuropharmacology, optogenetics and neurochemistry to study the neural mechanisms that are involved in context-induced renewal of Pavlovian-conditioned alcohol-seeking behavior16. In addition, behavioral and neural mechanisms that regulate the acquisition and extinction of Pavlovian-conditioned alcohol-seeking behavior can be investigated. Lastly, this task can be used to explore manipulations of extinction that might prevent renewal, an important direction for translational research aimed at reducing the impact of drug associated environmental contexts on reactivity to drug predictive cues after cue exposure therapy in human addicts.

Remaining sober is a considerable challenge faced by individuals suffering from alcohol abuse disorders. Abstinence is a time of vulnerability to the behavioral, psychological and physiological impact of environmental stimuli that routinely accompany alcohol use, which can, through Pavlovian conditioning, become associated with inebriation1,2. Exposure to alcohol predictive cues can elicit conditioned responses such as alcohol craving, which may promote alcohol-seeking behaviors that facilitate relapse3,4.
The stereotypical sequences of behavior that lead to alcohol consumption can cause certain types of stimuli to be routinely experienced immediately before the pharmacological effects of alcohol intake. For example, the sight, smell and taste of alcohol are sensory properties of alcohol that reliably precede intoxication. In addition to such cues, which are referred to as &#8216;discrete&#8217; or &#8216;proximal&#8217; cues, environmental contexts in which drugs are regularly used can also provoke craving5,6. Exposure to physical locations in which drugs have previously been used may therefore be a critical trigger for relapse 7.
Animal models have been developed to study the neural mechanisms that mediate the impact of drug associated contexts on drug-seeking behavior8-13. The procedure described herein allows for the investigation of how contexts associated with alcohol consumption can modulate alcohol-seeking behavior that is elicited by a discrete, alcohol predictive Pavlovian cue.
Pavlovian discrimination training is conducted in a specific environmental context, where rats are trained to behaviorally distinguish between two auditory conditioned stimuli, a CS+ that is paired with alcohol, and a CS- that is not. Extinction sessions are then conducted in a different context, where responding to the CS+ diminishes as a result of alcohol being withheld. Subsequently, reexposure to the alcohol associated, Pavlovian training context triggers a selective increase in alcohol-seeking behavior elicited by the CS+, with no change in responding to the CS-. This result, which we have consistently replicated9,14-16, extends findings from instrumental conditioning procedures in which drug contexts have been found to stimulate the renewal of operant responses associated with drug delivery10,13.

<table><tbody><tr><td>Heavy-duty utility cart</td><td>Rubbermaid</td><td>#4520-88</td><td>Used to transport rats in their home cage from the colony room to the behavior room or other facilities</td></tr><tr><td>Rats, Long-Evans</td><td>Harlan</td><td></td><td>Rats weighed 220-240 g on arrival</td></tr><tr><td>Ohaus scale</td><td>Fisher</td><td>#S402421</td><td>For weighing rats; tare the bottom of a plastic container first, then put the rat in it to weigh it</td></tr><tr><td>Beta-chips, aspen</td><td>Harlan</td><td>#7090A</td><td>Bedding for home cage</td></tr><tr><td>Nylabone</td><td>Bio-Serv</td><td>#K3580</td><td>Enrichment provided in the home cage</td></tr><tr><td>Autoclavable rodent chow</td><td>Agribrands, Charles River</td><td>#5075</td><td>For feeding rats</td></tr><tr><td>Wire bar cage lid</td><td>Ancare</td><td>#R20SS2B</td><td>Custom made with two slots for&amp;nbsp; bottles</td></tr><tr><td>Cage bottom</td><td>Ancare</td><td>#R20PC</td><td>To house rats</td></tr><tr><td>Stoppers</td><td>Ancare</td><td>#8.5</td><td>Rubber stoppers to occlude opening in water bottles &amp;amp; graduated cylinders; goes with ball point sipper tubes&amp;nbsp;</td></tr><tr><td>Ball point tubes, 1.5&quot; length</td><td>Ancare</td><td>#TD-99</td><td>Inserted in stoppers, enables rats to drink from water bottles and graduated cylinders</td></tr><tr><td>Water bottles</td><td>Ancare</td><td>#FSPC8HT</td><td>473 ml (16 oz) capacity, needs to be occluded with stopper</td></tr><tr><td>Graduated cylinders, 100 ml</td><td>Fisher</td><td>#0300741</td><td>To contain ethanol during alcohol exposure in the home cage; spouted end needs to be cut off to enable occlusion with stoppers</td></tr><tr><td>Mettler scale</td><td>Mettler Toledo</td><td>#MS6001S</td><td>For weighing water bottles and ethanol cylinders</td></tr><tr><td>Polyethylene tubing, 1/32&quot; inner diameter,&amp;nbsp; 1/32&quot; wall thickness, 3/32&quot; outside diameter</td><td>Fisher</td><td>#14-169-1A</td><td>To connect 20 ml ethanol syringe to fluid port</td></tr><tr><td>Needle, 18 g, 1.5&quot; length</td><td>Fisher</td><td>#B305199</td><td>To connect polyethylene tubing to ethanol syringes; needs to be filed</td></tr><tr><td>Syringes, 20 ml, Luer-Lok non-sterile</td><td>Fisher</td><td>#1482316J</td><td>To contain ethanol on the fluid pumps; needs to be connected to the polyethylene tubing by a 18 G 1 1/2 needle</td></tr><tr><td>Syringes, 60 ml, Luer-Lok non-sterile</td><td>Fisher</td><td>#14-820-11</td><td>To flush polyethylene tubing after PDT sessions</td></tr><tr><td>Single speed syringe pump, 3.33 rpm</td><td>Med Associates Inc.</td><td>#ENV-018MD</td><td>To hold 20 ml ethanol syringes and deliver ethanol</td></tr><tr><td>Fluid port</td><td>Med Associates Inc.</td><td>#ENV-254-CB</td><td>To enable ethanol delivery during PDT sessions; connected to ethanol syringes with polyethylene tubing</td></tr><tr><td>Port entry infrared detector</td><td>Med Associates Inc.</td><td>#ENV-215M</td><td>Fixed on both sides of&amp;nbsp; fluid port; need 2 per fluid port</td></tr><tr><td>Houselight, 28 V, 100 mA</td><td>Med Associates Inc.</td><td>#ENV-135M</td><td>Provides general lighting in conditioning chamber</td></tr><tr><td>Clicker module</td><td>Med Associates Inc.</td><td>#ENV-225SM</td><td>Auditory stimulus for conditioning chamber</td></tr><tr><td>White noise generator with speaker</td><td>Med Associates Inc.</td><td>#PHM-100</td><td>Auditory stimulus for conditioning chamber</td></tr><tr><td>Bar floor</td><td>Med Associates Inc.</td><td>#ENV-009A-GF</td><td>To go with modular test chamber</td></tr><tr><td>Bench coat, absorbent</td><td>VWR</td><td>#89126-790</td><td>Absorbent paper to put in metal tray, needs to be cut to fit the metal tray</td></tr><tr><td>Polycarbonate sheet, 1/8&quot; thick, 12-5/8&quot; x 10&quot;</td><td>Johnston Plastics</td><td>#30102515</td><td>To make smooth transparent floor insert used for context 1. Needs to be cut in 11.5&quot; x 12.25&quot; pieces</td></tr><tr><td>Aluminium sheet perforated, 0.063&quot; thick, 48&quot; x 96&quot;, 0.250&quot; holes x 0.375&quot; centers</td><td>Anica Steel</td><td></td><td>To make perforated floor insert used for context 2. Needs to be cut in 11.5&quot; x 12.25&quot; pieces</td></tr><tr><td>Black cardboard, 0.053&quot; thick, 28&quot; x 44&quot;</td><td>Omer DeSerres</td><td>#P1909</td><td>To construct panels covering the ceiling, door and back wall of the conditioning chamber in context 1. Needs to be cut to size. Each chamber needs three panels measuring approximately 11.5&quot; x 13.25&quot;. The panels can be joined by the narrower side using duct tape.</td></tr><tr><td>All-purpose strength duct tape, 1.88&quot; x 45 yd.</td><td>Duck Tape</td><td></td><td>Adhesive material to connect the black cardboard panels used to cover the back wall, ceiling and door in context 1.</td></tr><tr><td>Modular test chamber</td><td>Med Associates Inc.</td><td>#ENV-009A</td><td>Conditioning chamber</td></tr><tr><td>Sound attenuating cubicles</td><td>Med Associates Inc.</td><td>#ENV-200R3AM</td><td>Comes with installed 28 V DC fan (#ENV-025F)</td></tr><tr><td>Petri dish</td><td>Fisher</td><td>#08748B</td><td>Bottom only, to contain the sprayed solution that provides the odor stimulus; needs to be centered in the metal tray in conditioning chambers</td></tr><tr><td>PCI interface package</td><td>Med Associates Inc.</td><td>#DIG-700P2-R2</td><td>Connects tabletop interface cabinet to computer; includes 1 interface card, 1 Decode card, 1 ribbon cable and 1 28 V DC power cable</td></tr><tr><td>Large tabletop cabinet and power supply</td><td>Med Associates Inc.</td><td>#SG-6510D</td><td>Used to contain the 12 cards included in the SmartCrtrl 8 Input, 16 Output package</td></tr><tr><td>SmartCtrl 8 Input, 16 Output package</td><td>Med Associates Inc.</td><td>#DIG-716P2</td><td>Used to connect and control the devices in the conditioning chamber. The package includes all the cables, the interface module and the connection panel.</td></tr><tr><td>Med PC IV software</td><td>Med Associates Inc.</td><td>#SOF-735</td><td>Software to run programs. Needs to be installed on the computer.</td></tr><tr><td>Monitor LG, 19&quot; LCD</td><td>Concordia computer store</td><td>#W1942TQ-BF</td><td>Computer monitor; to go with computer running Med PC IV</td></tr><tr><td>HP Z200 tower workstation</td><td>Concordia computer store</td><td>#BZ788US#ABA</td><td>Computer on which Med PC IV is run</td></tr><tr><td>Benzaldehyde, &amp;ge;99%</td><td>Les produits chimiques OMEGA Chemical Company Inc.</td><td>#B37-50</td><td>Dilute in tap water for almond context odor</td></tr><tr><td>Lemon oil, cold pressed, California</td><td>Sigma Aldrich</td><td>#W262528-1KG-K</td><td>Dilute in tap water for lemon context odor</td></tr><tr><td>Ethyl alcohol, 95%</td><td>Commercial Alcohols</td><td></td><td>Dilute in tap water to desired concentration</td></tr></tbody></table>

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.

Intermittent ethanol access in the home cage: Ethanol consumption (g/kg) and ethanol preference increase across sessions of intermittent ethanol access in the home cage, typically reaching stable levels within 8 &#8211; 12 sessions (Figure 1). In the data set shown in Figure 1, stable ethanol consumption averaged across the last two sessions of home cage ethanol access ranged from 1.33 &#8211; 6.44 g/kg for individual rats. The home cage ethanol consumption phase of the experiment can be stopped once the g/kg consumption has plateaued, and rats are maintained at this stable level of drinking for a number of sessions.
To analyze data from this phase, conduct separate repeated-measures analysis of variance (ANOVA) across the within-subject factor of session for ethanol consumption (g/kg) and ethanol preference.
Schematic of context-induced renewal procedure: Each phase of the experimental procedure (PDT, extinction, renewal test) is illustrated in Figure 2. See the figure caption for further details.
Response measures and data analysis: In all behavioral phases described below, port entry responses made during the CS+ and CS- are normalized with reference to pre-CS baseline port entry responses. To create the normalized CS measures, subtract the number of port entries made during the pre-CS baseline interval (the 10 sec immediately prior to CS onset) from the number of port entries made during the presentations of the CS+ and CS-. The normalized CS+ and normalized CS- measures serve as indicators of behavior elicited specifically by CS presentations, elevated above baseline levels.
Conduct separate repeated-measures ANOVA with the repeated within-subject factors of session and CS type (CS+ vs. CS-) for the PDT and extinction phases. For comparisons across phase (PDT, extinction, renewal test), use data averaged across the last two sessions of PDT and extinction and the one session of renewal test data in repeated-measures ANOVA. To follow up on significant main effects and interactions, use post hoc t-tests for independent or paired samples that are corrected for multiple comparisons. Criterion for significance is set at &#945; = 0.05.
PDT: Port entry responses to the CS+ increase across PDT sessions, whereas port entry responses to the CS- remain low and stable, overall. Note that port entry responses to the CS- may increase slightly in the early sessions of PDT training; however, port entries made in response to the CS- stabilize at a much lower level than those made in response to the CS+. Successful discrimination is evidenced by a statistically significant elevation in port entries during the CS+ compared to the CS- (Figure 3). This result indicates that Pavlovian-conditioned alcohol-seeking behavior has been established to the CS+, but not the CS-.
Extinction: Port entry responses to the CS+ gradually decline across extinction sessions. Port entry responses to the CS- remain low and stable across extinction sessions. Ideally, discriminated responding is abolished by the end of extinction training, as evidenced by the lack of a statistically significant difference between port entries to the CS+ and CS-. However, it is not uncommon for some degree of discriminated responding to remain, even at the end of extinction training. In this case, it is important to verify that port entry responses to the CS+ at the end of extinction training are statistically lower than port entry responses to the CS+ at the start of extinction, as well as at the end of PDT (Figure 3). This pattern of results indicates that Pavlovian-conditioned alcohol-seeking behavior has been significantly extinguished.
Context-induced renewal test: Port entry responses to the CS+ increase in the context-induced renewal test, relative to CS+ port entries observed at the end of extinction training. This increase is selective to the CS+, as port entries to the CS- remain unaltered. In addition, port entries to the CS+ are statistically elevated compared to port entries to the CS- in the context-induced renewal test (Figure 4). Statistical confirmation of comparisons of CS+ port entries between extinction and test, as well as comparisons of CS+ and CS- port entries within test, indicates a selective renewal of Pavlovian-conditioned alcohol-seeking behavior induced by the alcohol associated context at test.
<img alt="Figure 1" fo:content-width="4in" src="/files/ftp_upload/51898/51898fig1highres.jpg" width="400" /> 
Figure 1. Escalation of ethanol consumption and preference induced by intermittent ethanol access in the home cage. Mean &#177; SEM (A)&#160;ethanol consumption (g/kg) and (B)&#160;ethanol preference across 12 sessions of intermittent ethanol access in the home cage. Both measures typically increase across sessions and then stabilize. Stable ethanol consumption averaged across the last two sessions of home cage ethanol access ranged from 1.33 &#8211; 6.44 g/kg for individual rats. These data were obtained using male Long-Evans rats drinking 15% (v/v) ethanol.
<img alt="Figure 2" fo:content-width="4in" src="/files/ftp_upload/51898/51898fig2highres.jpg" width="400" /> 
Figure 2. A schematic of the experimental procedure. The procedure consists of three phases: Pavlovian discrimination training (PDT), extinction, and context-induced renewal test. During each PDT session, rats receive 16 trials of one auditory conditioned stimulus (CS+) that signals the delivery of ethanol and 16 trials of a second auditory stimulus (CS-) that is not associated with ethanol delivery. PDT sessions take place in Context A, composed of a specific configuration of visual, olfactory, and tactile stimuli. During extinction sessions, which are conducted in Context B, composed of a different set of visual, olfactory, and tactile stimuli, the CS+ and CS- are presented but ethanol is withheld. For the context-induced renewal test, rats are returned to Context A, and the CS+ and CS- are presented without ethanol. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/51898/51898fig2highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" fo:content-width="4in" src="/files/ftp_upload/51898/51898fig3highres.jpg" width="400" /> 
Figure 3. Acquisition and extinction of discriminated Pavlovian-conditioned responding for ethanol. Mean &#177; SEM normalized port entries made during the CS+ and CS- across PDT (sessions 1 &#8211; 18) in Context A and extinction (sessions 19 &#8211; 26) in Context B. These data were obtained using male Long-Evans rats, trained with 10% (v/v) ethanol as the unconditioned stimulus (US) that was presented in association with the CS+ during PDT. *p &#60;0.05, CS+ versus CS-. Figure originally published in Chaudhri, Sahuque, &#38; Janak (2008). Used with permission from Elsevier.
<img alt="Figure 4" fo:content-width="5in" src="/files/ftp_upload/51898/51898fig4highres.jpg" width="500" /> 
Figure 4. Context-induced renewal of Pavlovian-conditioned responding for ethanol. Mean &#177; SEM normalized port entries made during the CS+ and CS- in PDT sessions in Context A, extinction sessions in Context B, and renewal test in Context A. Data are averaged over the last two sessions for PDT and extinction; the renewal test is a single session. These data were obtained using male Long-Evans rats, trained with 10% (v/v) ethanol as the unconditioned stimulus (US) that was presented in association with the CS+ during PDT. *p &#60;0.05; **p &#60;0.001, CS+ versus CS-. #p &#60;0.05, CS+ PDT versus CS+ extinction. ^p &#60;0.05, CS+ extinction versus CS+ test. Figure originally published in Chaudhri, Sahuque, &#38; Janak (2008). Used with permission from Elsevier.

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

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 ...

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12.7K Views.  Concordia University. A procedure to study the capacity of an alcohol associated environmental context to trigger the renewal of alcohol-seeking behavior in rats is described.

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

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 ...

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.

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 ...

A General Method for Evaluating Incubation of Sucrose Craving in Rats

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and relapse to food taking 1. Food craving conceptualized in this way is akin to drug craving in response to drug-paired cues. A rich literature has been developed around understanding the behavioral and neurobiological determinants of drug craving; we and others have been focusing recently on translating techniques from basic addiction research to better understand addiction-like behaviors related to food 2-4.
As done in previous studies of drug craving, we examine sucrose craving behavior by utilizing a rat model of relapse. In this model, rats self-administer either drug or food in sessions over several days. In a session, lever responding delivers the reward along with a tone+light stimulus. Craving behavior is then operationally defined as responding in a subsequent session where the reward is not available. Rats will reliably respond for the tone+light stimulus, likely due to its acquired conditioned reinforcing properties 5. This behavior is sometimes referred to as sucrose seeking or cue reactivity. In the present discussion we will use the term "sucrose craving" to subsume both of these constructs.
In the past decade, we have focused on how the length of time following reward self-administration influences reward craving. Interestingly, rats increase responding for the reward-paired cue over the course of several weeks of a period of forced-abstinence. This "incubation of craving" is observed in rats that have self-administered either food or drugs of abuse 4,6. This time-dependent increase in craving we have identified in the animal model may have great potential relevance to human drug and food addiction behaviors.
Here we present a protocol for assessing incubation of sucrose craving in rats. Variants of the procedure will be indicated where craving is assessed as responding for a discrete sucrose-paired cue following extinction of lever pressing within the sucrose self-administration context (Extinction without cues) or as responding for sucrose-paired cues in a general extinction context (Extinction with cues).

Responding for food or drug-paired cues increases over the course of a period of abstinence and this may relate to an increased susceptibility to relapse behaviors. Here we detail a procedure for evaluating this "incubation of craving" in rats that have self-administered sucrose.

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and ...

Neuroscience

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 ...

A Conditioned Place Preference Protocol for Measuring Incubation of Craving in Rats

A major cause of repeated relapses is a craving for the drug. Drug craving increases progressively during the abstinence period, a phenomenon termed incubation of drug craving. Here, we describe a morphine conditioned place preference (CPP) protocol for measuring the incubation of craving in rats. In this protocol, a CPP paradigm mainly employing somatosensory cues is used to establish a long-term reward memory of morphine. A three-chamber CPP box that differs in the texture of the chamber floor is constructed. First, the animals are tested for their baseline preference to the two side chambers for three consecutive days. Then, they are injected intraperitoneally with morphine/saline and put into their non-preferred/preferred chamber for 45 min. After 6 days of conditioning, their preference to the side chambers is tested for 15 min at different time points after the last conditioning session. With this paradigm, the reward memory of morphine could last for at least 18 days. To test whether the above-mentioned protocol can model increased craving, the number of entrances into the two side chambers are counted during the abstinence period. The results show that the entrances increased, suggesting that the CPP paradigm could mimic the incubation of craving. Future studies can employ this model to study neural mechanisms underlying long-term memory and incubation of craving.

Here, a morphine conditioned place preference (CPP) protocol is described to measure incubation of craving in rats.

A major cause of repeated relapses is a craving for the drug. Drug craving increases progressively during the abstinence period, a phenomenon termed ...

A Procedure to Study Stress-Induced Relapse of Heroin Seeking after Punishment-Imposed Abstinence

The punishment-imposed abstinence procedure models the self-imposed abstinence that humans initiate due to the adverse consequences associated with drug-taking. This model has been implemented in experiments using different types of substances of abuse such as methamphetamine, cocaine, and alcohol. However, punishment-induced abstinence in heroin-trained animals has not been demonstrated. Furthermore, acute stress is a key trigger for relapse in humans and animal models. It was previously demonstrated that acute food deprivation robustly induced reinstatement of extinguished cocaine and heroin seeking. The procedure described here can be used to assess the effects of acute stress exposure on heroin seeking after punishment-imposed abstinence. A total of 8 rats were implanted with chronic intravenous (i.v.) catheters and trained to self-administer heroin (0.1 mg/kg/infusion) for 18 days under a seek-take chained schedule. Completing the seek link gave access to the take lever, which was paired with a heroin infusion. The seek lever was programmed with a variable interval 60 schedule of reinforcement (VI60), and the take lever was programmed with a fixed-ratio 1 reinforcement schedule (FR1). Following self-administration training, a mild foot shock was delivered on 30% of the completed seek links instead of the extension of the take lever. Footshock intensity was increased by 0.1 mA per daily session from 0.2 mA to 1.0 mA. Heroin-seeking tests were performed after 24 h of food deprivation (FD) or sated conditions. Rats under acute food deprivation condition robustly increased heroin seeking after punishment-imposed abstinence.

A procedure that demonstrates a robust acute food deprivation-induced relapse to heroin seeking after punishment-imposed abstinence is described. A punishment-imposed abstinence model was successfully implemented using the seek and take chain schedule for heroin self-administration. Heroin-seeking tests are then performed following 24 h of food-deprivation stress.

The punishment-imposed abstinence procedure models the self-imposed abstinence that humans initiate due to the adverse consequences associated with ...

The Use of Trace Eyeblink Classical Conditioning to Assess Hippocampal Dysfunction in a Rat Model of Fetal Alcohol Spectrum Disorders

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain undergoes rapid organizational change and is similar to accelerated brain changes that occur during the third trimester in humans. This model of fetal alcohol spectrum disorders (FASDs) produces severe brain damage, mimicking the amount and pattern of binge-drinking that occurs in some pregnant alcoholic mothers. We describe the use of trace eyeblink classical conditioning (ECC), a higher-order variant of associative learning, to assess long-term hippocampal dysfunction that is typically seen in alcohol-exposed adult offspring. At 90 days of age, rodents were surgically prepared with recording and stimulating electrodes, which measured electromyographic (EMG) blink activity from the left eyelid muscle and delivered mild shock posterior to the left eye, respectively. After a 5 day recovery period, they underwent 6 sessions of trace ECC to determine associative learning differences between alcohol-exposed and control rats. Trace ECC is one of many possible ECC procedures that can be easily modified using the same equipment and software, so that different neural systems can be assessed. ECC procedures in general, can be used as diagnostic tools for detecting neural pathology in different brain systems and different conditions that insult the brain.

Trace eyeblink classical conditioning (ECC) was used to assess hippocampal-dependent associative learning in adult rats that were administered a high concentration (11.9% v/v) of alcohol during early neonatal brain development. In general, ECC procedures are sound diagnostic tools for detecting brain dysfunction across many psychological and biomedical settings.

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain ...

A Protocol for Measuring Cue Reactivity in a Rat Model of Cocaine Use Disorder

Cocaine use disorder (CUD) follows a trajectory of repetitive self-administration during which previously neutral stimuli gain incentive value. Cue reactivity, the sensitivity to cues previously linked with the drug-taking experience, plays a prominent role in human craving during abstinence. Cue reactivity can be assessed as the attentional orientation toward drug-associated cues that is measurable as appetitive approach behavior in both preclinical and human studies. Herein describes an assessment of cue reactivity in rats trained to self-administer cocaine. Cocaine self-administration is paired with the presentation of discrete cues that act as conditioned reinforcers (i.e., house light, stimulus light, infusion pump sounds). Following a period of abstinence, lever presses in the cocaine self-administration context accompanied by the discrete cues previously paired with cocaine infusion are measured as cue reactivity. This model is useful to explore neurobiological mechanisms underlying cue reactivity processes as well as to assess pharmacotherapies to suppress cue reactivity and therefore, modify relapse vulnerability. Advantages of the model include its translational relevance, and its face and predictive validities.&#160;The primary limitation of the model is that the cue reactivity task can only be performed infrequently and must only be used in short duration (e.g., 1 hour), otherwise rats will begin to extinguish the pairing of the discrete cues with the cocaine stimulus. The model is extendable to any positively reinforcing stimulus paired with discrete cues; though particularly applicable to drugs of abuse, this model may hold future applications in fields such as obesity, where palatable food rewards can act as positively reinforcing stimuli.

Cue reactivity is conceptualized as sensitivity to cues linked with drug-taking experiences that contribute to craving and relapse in abstinent humans. Cue reactivity is modeled in rats by measuring attentional orientation toward drug-associated cues that results in appetitive approach behavior in a cue reactivity test following self-administration and forced abstinence.

Cocaine use disorder (CUD) follows a trajectory of repetitive self-administration during which previously neutral stimuli gain incentive value. Cue ...

A Prediction Error-driven Retrieval Procedure for Destabilizing and Rewriting Maladaptive Reward Memories in Hazardous Drinkers

Maladaptive reward memories (MRMs) can become unstable following retrieval under certain conditions, allowing their modification by subsequent new learning. However, robust (well-rehearsed) and chronologically old MRMs, such as those underlying substance use disorders, do not destabilize easily when retrieved. A key determinate of memory destabilization during retrieval is prediction error (PE). We describe a retrieval procedure for alcohol MRMs in hazardous drinkers that specifically aims to maximize the generation of PE and therefore the likelihood of MRM destabilization. The procedure requires explicitly generating the expectancy of alcohol consumption and then violating this expectancy (withholding alcohol) following the presentation of a brief set of prototypical alcohol cue images (retrieval + PE). Control procedures involve presenting the same cue images, but allow alcohol to be consumed, generating minimal PE (retrieval-no PE) or generate PE without retrieval of alcohol MRMs, by presenting orange juice cues (no retrieval + PE). Subsequently, we describe a multisensory disgust-based counterconditioning procedure to probe MRM destabilization by re-writing alcohol cue-reward associations prior to reconsolidation. This procedure pairs alcohol cues with images invoking pathogen disgust and an extremely bitter-tasting solution (denatonium benzoate), generating gustatory disgust. Following retrieval + PE, but not no retrieval + PE or retrieval-no PE, counterconditioning produces evidence of MRM rewriting as indexed by lasting reductions in alcohol cue valuation, attentional capture, and alcohol craving.

This manuscript describes a memory retrieval procedure for destabilizing robust reward memories and rewriting them with counterconditioning prior to their reconsolidation.

Maladaptive reward memories (MRMs) can become unstable following retrieval under certain conditions, allowing their modification by subsequent new ...

Intermittent-Access Two-Bottle Choice Drinking with Lick Detection in Rats

Modeling Alcohol Consumption in Rodents Using Two-Bottle Choice Home Cage Drinking and Microstructural Analysis

Two-bottle choice home cage drinking is one of the most widely used paradigms to study ethanol consumption in rodents. In its simplest form, animals are provided with access to two drinking bottles, one of which contains regular tap water and the other ethanol, with daily intake measured by change in bottle weight over the 24 h period. Consequently, this approach requires no specialized laboratory equipment. While such ease of implementation is likely the greatest contributor to its widespread adoption by preclinical alcohol researchers, the resolution of drinking data acquired using this approach is limited by the number of times the researcher measures bottle weight (e.g., once daily). However, the desire to examine drinking patterns in the context of overall intake, pharmacological interventions, and neuronal manipulations has prompted the development of home cage lick detection systems that can acquire data at the level of individual licks. Although a number of these systems have been developed recently, the open-source system, LIQ HD (Lick Instance Quantifier Home cage Device), has garnered significant attention in the field for its affordability and user friendliness. Although exciting, this system was designed for use in mice. Here, we review appropriate procedures for standard and lickometer-equipped two-bottle choice home cage drinking. We also introduce methods for adapting the LIQ HD system to rats including hardware modifications to accommodate larger cage size and a redesigned 3D printed bottle holder compatible with standard off-the-shelf drinking bottles. Using this approach, researchers can examine daily drinking patterns in addition to levels of intake in many rats in parallel thereby increasing the resolution of acquired data with minimal investment in additional resources. These methods provide researchers with the flexibility to use either standard bottles or a lickometer-equipped apparatus to interrogate the neurobiological mechanisms underlying alcohol drinking depending on their precise experimental needs.

This protocol describes a standard intermittent-access two-bottle choice home cage drinking paradigm to model alcohol consumption in rats. In addition, it provides step-by-step instructions to augment the standard protocol with a DIY lick detection system that enables microstructural analysis of drinking behavior.

Two-bottle choice home cage drinking is one of the most widely used paradigms to study ethanol consumption in rodents. In its simplest form, animals are ...