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 border="0" cellpadding="0" cellspacing="0" width="2121">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:603px;">Name of Reagent/ Equipment</td>
			<td style="width:357px;">Company</td>
			<td style="width:192px;">Catalog Number</td>
			<td style="width:969px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:603px;">Heavy-duty utility cart</td>
			<td style="width:357px;">Rubbermaid</td>
			<td style="width:192px;">#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 height="21">
			<td height="21" style="height:21px;width:603px;">Rats, Long-Evans</td>
			<td style="width:357px;">Harlan</td>
			<td style="width:192px;">-</td>
			<td>Rats weighed 220-240g on arrival</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:603px;">Ohaus scale</td>
			<td style="width:357px;">Fisher</td>
			<td style="width:192px;">#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 height="21">
			<td height="21" style="height:21px;width:603px;">Beta-chips, aspen</td>
			<td style="width:357px;">Harlan</td>
			<td style="width:192px;">#7090A</td>
			<td>Bedding for home cage</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:603px;">Nylabone</td>
			<td style="width:357px;">Bio-Serv</td>
			<td style="width:192px;">#K3580</td>
			<td>Enrichment provided in the home cage</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:603px;">Autoclavable rodent chow</td>
			<td style="width:357px;">Agribrands, Charles River</td>
			<td style="width:192px;">#5075</td>
			<td>For feeding rats</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:603px;">Wire bar cage lid</td>
			<td style="width:357px;">Ancare</td>
			<td style="width:192px;">#R20SS2B</td>
			<td>Custom made with two slots for&#160; bottles</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:603px;">Cage bottom</td>
			<td style="width:357px;">Ancare</td>
			<td style="width:192px;">#R20PC</td>
			<td>To house rats</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:603px;">Stoppers</td>
			<td style="width:357px;">Ancare</td>
			<td style="width:192px;">#8.5</td>
			<td>Rubber stoppers to occlude opening in water bottles &#38; graduated cylinders; goes with ball point sipper tubes&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:603px;">Ball point tubes, 1.5&#34; length</td>
			<td style="width:357px;">Ancare</td>
			<td style="width:192px;">#TD-99</td>
			<td>Inserted in stoppers, enables rats to drink from water bottles and graduated cylinders</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:603px;">Water bottles</td>
			<td style="width:357px;">Ancare</td>
			<td style="width:192px;">#FSPC8HT</td>
			<td>473 ml (16 oz) capacity, needs to be occluded with stopper</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:603px;">Graduated cylinders, 100ml</td>
			<td style="width:357px;">Fisher</td>
			<td style="width:192px;">#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 height="21">
			<td height="21" style="height:21px;width:603px;">Mettler scale</td>
			<td style="width:357px;">Mettler Toledo</td>
			<td style="width:192px;">#MS6001S</td>
			<td>For weighing water bottles and ethanol cylinders</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:603px;">Polyethylene tubing, 1/32&#34; inner diameter,&#160; 1/32&#34; wall thickness, 3/32&#34; outside diameter</td>
			<td style="width:357px;">Fisher</td>
			<td style="width:192px;">#14-169-1A</td>
			<td>To connect 20 ml ethanol syringe to fluid port</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:603px;">Needle, 18g, 1.5&#34; length</td>
			<td style="width:357px;">Fisher</td>
			<td style="width:192px;">#B305199</td>
			<td>To connect polyethylene tubing to ethanol syringes; needs to be filed</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:603px;">Syringes, 20ml, Luer-Lok non-sterile</td>
			<td style="width:357px;">Fisher</td>
			<td style="width:192px;">#1482316J</td>
			<td>To contain ethanol on the fluid pumps; needs to be connected to the polyethylene tubing by a 18g 1 1/2 needle</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:603px;">Syringes, 60ml, Luer-Lok non-sterile</td>
			<td style="width:357px;">Fisher</td>
			<td style="width:192px;">#14-820-11</td>
			<td>To flush polyethylene tubing after PDT sessions</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:603px;">Single speed syringe pump, 3.33 rpm</td>
			<td style="width:357px;">Med Associates Inc.</td>
			<td style="width:192px;">#ENV-018MD</td>
			<td>To hold 20 ml ethanol syringes and deliver ethanol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:603px;">Fluid port</td>
			<td style="width:357px;">Med Associates Inc.</td>
			<td style="width:192px;">#ENV-254-CB</td>
			<td>To enable ethanol delivery during PDT sessions; connected to ethanol syringes with polyethylene tubing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:603px;">Port entry infrared detector</td>
			<td style="width:357px;">Med Associates Inc.</td>
			<td style="width:192px;">#ENV-215M</td>
			<td>Fixed on both sides of&#160; fluid port; need 2 per fluid port</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:603px;">Houselight, 28V, 100mA</td>
			<td style="width:357px;">Med Associates Inc.</td>
			<td style="width:192px;">#ENV-135M</td>
			<td>Provides general lighting in conditioning chamber</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:603px;">Clicker module</td>
			<td style="width:357px;">Med Associates Inc.</td>
			<td style="width:192px;">#ENV-225SM</td>
			<td>Auditory stimulus for conditioning chamber</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:603px;">White noise generator with speaker</td>
			<td style="width:357px;">Med Associates Inc.</td>
			<td style="width:192px;">#PHM-100</td>
			<td>Auditory stimulus for conditioning chamber</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:603px;">Bar floor</td>
			<td style="width:357px;">Med Associates Inc.</td>
			<td style="width:192px;">#ENV-009A-GF</td>
			<td style="width:969px;">To go with modular test chamber</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:603px;">Bench coat, absorbant</td>
			<td style="width:357px;">VWR</td>
			<td style="width:192px;">#89126-790</td>
			<td style="width:969px;">Absorbant paper to put in metal tray, needs to be cut to fit the metal tray</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:603px;">Polycarbonate sheet, 1/8&#34; thick, 12-5/8&#34;x10&#34;</td>
			<td style="width:357px;">Johnston Plastics</td>
			<td style="width:192px;">#30102515</td>
			<td style="width:969px;">To make smooth transparent floor insert used for context 1. Needs to be cut in 11.5&#34;x12.25&#34; pieces</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:603px;">Aluminium sheet perforated, 0.063&#34;thick, 48&#34;x96&#34;, 0.250&#34; holes x 0.375&#34; centers</td>
			<td style="width:357px;">Anica Steel</td>
			<td style="width:192px;">-</td>
			<td style="width:969px;">To make perforated floor insert used for context 2. Needs to be cut in 11.5&#34;x12.25&#34; pieces</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:603px;">Black cardboard, 0.053&#34; thick, 28&#34;x44&#34;</td>
			<td style="width:357px;">Omer DeSerres</td>
			<td style="width:192px;">#P1909</td>
			<td style="width:969px;">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&#34;x13.25&#34;. The panels can be joined by the narrower side using duct tape.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:603px;">All-purpose strength duct tape, 1.88&#34;x45yd.</td>
			<td style="width:357px;">Duck Tape</td>
			<td style="width:192px;">-</td>
			<td style="width:969px;">Adhesive material to connect the black cardboard panels used to cover the back wall, ceiling and door in context 1.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:603px;">Modular test chamber</td>
			<td style="width:357px;">Med Associates Inc.</td>
			<td style="width:192px;">#ENV-009A</td>
			<td>Conditioning chamber</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:603px;">Sound attenuating cubicles</td>
			<td style="width:357px;">Med Associates Inc.</td>
			<td style="width:192px;">#ENV-200R3AM</td>
			<td>Comes with installed 28V DC fan (#ENV-025F)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:603px;">Petri dish</td>
			<td style="width:357px;">Fisher</td>
			<td style="width:192px;">#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 height="21">
			<td height="21" style="height:21px;width:603px;">PCI interface package</td>
			<td style="width:357px;">Med Associates Inc.</td>
			<td style="width:192px;">#DIG-700P2-R2</td>
			<td>Connects tabletop interface cabinet to computer; includes 1 interface card, 1 Decode card, 1 ribbon cable and 1 28V DC power cable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:603px;">Large tabletop cabinet and power supply</td>
			<td style="width:357px;">Med Associates Inc.</td>
			<td style="width:192px;">#SG-6510D</td>
			<td>Used to contain the 12 cards included in the SmartCrtrl 8 Input, 16 Output package</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:603px;">SmartCtrl 8 Input, 16 Output package</td>
			<td style="width:357px;">Med Associates Inc.</td>
			<td style="width:192px;">#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 height="21">
			<td height="21" style="height:21px;width:603px;">Med PC IV software</td>
			<td style="width:357px;">Med Associates Inc.</td>
			<td style="width:192px;">#SOF-735</td>
			<td>Software to run programs. Needs to be installed on the computer.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:603px;">Monitor LG, 19&#34; LCD</td>
			<td style="width:357px;">Concordia computer store</td>
			<td style="width:192px;">#W1942TQ-BF</td>
			<td>Computer monitor; to go with computer running Med PC IV</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:603px;">HP Z200 tower workstation</td>
			<td style="width:357px;">Concordia computer store</td>
			<td style="width:192px;">#BZ788US#ABA</td>
			<td>Computer on which Med PC IV is run</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:603px;">Benzaldehyde, &#8805;99%</td>
			<td style="width:357px;">Les produits chimiques OMEGA Chemical Company Inc.</td>
			<td style="width:192px;">#B37-50</td>
			<td>Dilute in tap water for almond context odor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:603px;">Lemon oil, cold pressed, California</td>
			<td style="width:357px;">Sigma Aldrich</td>
			<td style="width:192px;">#W262528-1KG-K</td>
			<td>Dilute in tap water for lemon context odor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:603px;">Ethyl alcohol, 95%</td>
			<td style="width:357px;">Commercial Alcohols</td>
			<td style="width:192px;">-</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/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 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 ...

A Novel Procedure for Evaluating the Reinforcing Properties of Tastants in Laboratory Rats: Operant Intraoral Self-administration

This paper describes a novel method for studying the bio-behavioral basis of addiction to food. This method combines the surgical component of taste reactivity with the behavioral aspects of operant self-administration of drugs. Under very brief general anaesthesia, rats are implanted with an intraoral (IO) cannula that allows delivery of test solutions directly in the oral cavity. Animals are then tested in operant self-administration chambers whereby they can press a lever to receive IO infusions of test solutions. IO self-administration has several advantages over experimental procedures that involve drinking a solution from a spout&#160;or operant responding for solid pellets or solutions delivered in a receptacle. Here, we show that IO self-administration can be employed to study self-administration of high fructose corn syrup (HFCS). Rats were first tested for self-administration on a progressive ratio (PR) schedule, which assesses the maximum amount of operant behavior that will be emitted for different concentrations of HFCS (i.e.&#160;8%, 25%, and 50%). Following this test, rats self-administered these concentrations on a continuous schedule of reinforcement (i.e.&#160;one infusion for each lever press) for 10 consecutive days (1 session/day; each lasting 3 hr), and then they were retested on the PR schedule. On the continuous reinforcement schedule, rats took fewer infusions of higher concentrations, although the lowest concentration of HFCS (8%) maintained more variable self-administration. Furthermore, the PR tests revealed that 8% had lower reinforcing value than 25% and 50%. These results indicate that IO self-administration can be employed to study acquisition and maintenance of responding for sweet solutions. The sensitivity of the operant response to differences in concentration and schedule of reinforcement makes IO self-administration an ideal procedure to investigate the neurobiology of voluntary intake of sweets.

The current study evaluates a novel procedure for assessing the reinforcing effects of palatable solutions in laboratory rats: intraoral self-administration. To this end, operant responding (i.e.&#160;lever pressing) for intraoral infusions of sweet solutions at different concentrations was measured on continuous and progressive ratios schedules of reinforcement.

This paper describes a novel method for studying the bio-behavioral basis of addiction to food. This method combines the surgical component of taste ...

Fat Preference: A Novel Model of Eating Behavior in Rats

Obesity is a growing problem in the United States of America, with more than a third of the population classified as obese. One factor contributing to this multifactorial disorder is the consumption of a high fat diet, a behavior that has been shown to increase both caloric intake and body fat content. However, the elements regulating preference for high fat food over other foods remain understudied.
To overcome this deficit, a model to quickly and easily test changes in the preference for dietary fat was developed. The Fat Preference model presents rats with a series of choices between foods with differing fat content. Like humans, rats have a natural bias toward consuming high fat food, making the rat model ideal for translational studies. Changes in preference can be ascribed to the effect of either genetic differences or pharmacological interventions. This model allows for the exploration of determinates of fat preference and screening pharmacotherapeutic agents that influence acquisition of obesity.

Dietary fat content influences both energy intake and body fat composition in mammals. By examining rats&#8217; preference for high fat food in a series of choice experiments, it is possible to test genetic differences and pharmacological interventions on their preference for high fat food.

Obesity is a growing problem in the United States of America, with more than a third of the population classified as obese. One factor contributing to ...

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 ...

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 ...

The Rodent Psychomotor Vigilance Test (rPVT): A Method for Assessing Neurobehavioral Performance in Rats and Mice

The human Psychomotor Vigilance Test (PVT) is a widely used procedure for measuring changes in fatigue and sustained attention. The present article describes a rodent version of the PVT&#8212;termed the &#34;rPVT&#34;&#8212;that measures similar aspects of attention (i.e., performance accuracy, motor speed, premature responding, and lapses in attention). Data are presented that demonstrate both the short- and long-term usefulness of the rPVT when employed with laboratory rats. Rats easily learn the rPVT, and learning to perform the basic procedure takes less than two weeks of training. Once acquired, rat performances in the rPVT show a high degree of similarity to these same performance measures in the human PVT, including similarities in, lapses in attention, reaction times, vigilance decrements across session time (i.e., the human &#34;time-on-task&#34; effects), and the response-stimulus interval (RSI) effect described for humans. Thus the rPVT can be an extremely valuable tool for assessing the effects of a wide range of variables on sustained attention quite similar to human PVT performances, and thus can be useful for developing novel treatments for neurobehavioral dysfunctions.

The Rodent Psychomotor Vigilance Test rPVT: A Method for Assessing Neurobehavioral Performance in Rats and Mice

A rat version of the human Psychomotor Vigilance Test (PVT) is described that measures aspects of attention similar to those measured with the human PVT, including aspects of human vigilance such as performance accuracy, motor speed, premature responding, and lapses in attention.

The human Psychomotor Vigilance Test (PVT) is a widely used procedure for measuring changes in fatigue and sustained attention. The present article ...

Detecting Behavioral Deficits in Rats After Traumatic Brain Injury

With the increasing incidence of traumatic brain injury (TBI) in both civilian and military populations, TBI is now considered a chronic disease; however, few studies have investigated the long-term effects of injury in rodent models of TBI. Shown here are behavioral measures that are well-established in TBI research for times early after injury, such as two weeks, until two months. Some of these methods have previously been used at later times after injury, up to one year, but by very few laboratories. The methods demonstrated here are a short neurological assessment to test reflexes, a Beam-Balance to test balance, a Beam-Walk to test balance and motor coordination, and a working memory version of the Morris water maze that can be sensitive to deficits in reference memory. Male rats were handled and pre-trained to neurological, balance, and motor coordination tests prior to receiving parasagittal fluid percussion injury (FPI) or sham injury. Rats can be tested on the short neurological assessment (neuroscore), the beam-balance, and the Beam-Walk multiple times, while testing on the water maze can only be done once. This difference is because rats can remember the task, thus confounding the results if repeated testing is attempted in the same animal. When testing from one to three days after injury, significant differences are detected in all three non-cognitive tasks. However, differences in the Beam-Walk task were not detectable at later time points (after 3 months). Deficits were detected at 3 months in the Beam-Balance and at 6 months in the neuroscore. Deficits in working memory were detected out to 12 months after injury, and a deficit in a reference memory first appeared at 12 months. Thus, standard behavioral tests can be useful measures of persistent behavioral deficits after FPI.

The goal of the behavioral tests presented here is to detect functional deficits in rats after traumatic brain injury. Four specific tests are presented that detect deficits in behaviors to reflect the damage to specific brain areas at times extending to one year after injury.

With the increasing incidence of traumatic brain injury (TBI) in both civilian and military populations, TBI is now considered a chronic disease; however, ...

A New Method for Inducing a Depression-Like Behavior in Rats

Contagious depression is a phenomenon that is yet to be fully recognized and this stems from insufficient material on the subject. At the moment, there is no existing format for studying the mechanism of action, prevention, containment, and treatment of contagious depression. The purpose of this study, therefore, was to establish the first animal model of contagious depression.
Healthy rats can contract depressive behaviors if exposed to depressed rats. Depression is induced in rats by subjecting them to several manipulations of chronic unpredictable stress (CUS) over 5 weeks, as described in the protocol. A successful sucrose preference test confirmed the development of depression in the rats. The CUS-exposed rats were then caged with na&#239;ve rats from the contagion group (1 na&#239;ve rat/2 depressed rats in a cage) for an additional 5 weeks. 30 social groups were created from the combination of CUS-exposed rats and na&#239;ve rats.
This proposed depression-contagion protocol in animals consists mainly of cohabiting CUS-exposed and healthy rats for 5 weeks. To ensure that this method works, a series of tests are carried out - first, the sucrose preference test upon inducing depression to rats, then, the sucrose preference test, alongside the open field and forced-swim tests at the end of the cohabitation period. Throughout the experiment, rats are given tags and are always returned to their cages after each test.
A few limitations to this method are the weak differences recorded between the experimental and control groups in the sucrose preference test and the irreversible traumatic outcome of the forced swim test. These may be worth considering for suitability before any future application of the protocol. Nonetheless, following the experiment, na&#239;ve rats developed contagion depression after 5 weeks of sharing the same cage with the CUS-exposed rats.

This protocol describes a new model by which healthy rats could contract depression over a given time periodthrough contagion by exposure to chronic unpredictable stressed (CUS) rats.

Contagious depression is a phenomenon that is yet to be fully recognized and this stems from insufficient material on the subject. At the moment, there is ...

Testing for Metacognitive Responding Using an Odor-based Delayed Match-to-Sample Test in Rats

Metamemory involves the cognitive ability to assess the strength of one&#39;s memories. To explore the possibility of metamemory in non-human animals, numerous behavioral tasks have been created, many of which utilize an option to decline memory tests. To assess metamemory in rats, we utilized this decline-test option paradigm by adapting previous visual delayed-match-to-sample tests (DMTS)1,2 developed for primate species to an odor-based test suitable for rodents. First,&#160;rats are given a sample to remember by digging in a cup of scented sand. After a delay, the rat is presented with four distinctly scented cups, one of which contains the identical scent experienced during the sample; if this matching cup is selected, then the rat obtains a preferred, larger reward. Selection of any of the other three non-matching sand-filled scented cups results in no reward. Retention intervals are individually titrated such that subjects perform between 40 and 70% correct, therefore ensuring rats sometimes remember and sometimes forget the sample. Here, the operational definition of metamemory is the ability to distinguish between the presence and absence of memory through behavioral responding. Towards this end, on two-thirds of trials, a decline option is presented in addition to the four choice cups (choice trials). If the decline-test option- an unscented colored sand cup, is selected, the subject receives a smaller less-preferred reward and avoids the memory test. On the remaining third of trials, the decline-test option is not available (forced trials), causing subjects to guess the correct cup when the sample is forgotten. On choice tests, subjects that know when they remember should select the decline option when memory is weak rather than take the test and choose incorrectly. Therefore, significantly higher performance on chosen tests as compared to forced memory tests is indicative of the adaptive use of the decline-test response and metacognitive responding.

This protocol describes a method for investigating the possibility of metamemory, or memory awareness, in rodents. The odor-based delayed-matching-to-sample paradigm is a novel, ecologically-relevant behavioral test useful for determining the extent to which rodents can adaptively respond based on cognitively monitoring the strength of their memory states.

Metamemory involves the cognitive ability to assess the strength of one&#39;s memories. To explore the possibility of metamemory in non-human animals, ...

A Conflict Model of Reward-seeking Behavior in Male Rats

The present protocol describes a novel conflict task as a model of inhibitory control in rats. In this model, a natural rewarding stimulus (sexual stimulus) that represents a high-value reward, and the aversive stimuli (pins), are concurrently presented. The male rats have to climb or jump over the obstacle full of pins to approach and investigate the sexual partner. If the animal persists in their approaching behavior regardless of the aversive stimuli, it is considered as a maladaptive or risky reward-seeking behavior. The conflict task permits the evaluation of deficit in inhibitory control resulting from exposure to abused drug, such as morphine, or a stressful event. 
The main advantage of this model is that it provides a simple and quick way to discover the deficit in inhibitory control after exposure to opiate drugs or other stressful events. In addition to opiates, this behavioral model would also be useful for quickly discovering the inhibitory control deficits induced by other addictive drugs. However, the limitation is that the male rats' performance may be subject to exercising effects with repeated testing under this conflict task. In the future, one can hope that the individuals with the compulsive phenotype of reward-seeking behavior after exposure to opiates will be identified based on modifying this conflict model.

This conflict model is used to measure the impairment of inhibitory control after exposure to addictive drugs, or other factors that may influence inhibitory control. A sexual stimulus and an aversive obstacle are concurrently presented, thus male rats have to conquer the obstacle to approach the sexual reward.