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 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.
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 ‘discrete’ or ‘proximal’ 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.
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
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.
3. Apparatus
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.
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.
6. Extinction
7. Context-induced Renewal Test
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 – 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 – 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 α = 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.
Figure 1. Escalation of ethanol consumption and preference induced by intermittent ethanol access in the home cage. Mean ± SEM (A) ethanol consumption (g/kg) and (B) 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 – 6.44 g/kg for individual rats. These data were obtained using male Long-Evans rats drinking 15% (v/v) ethanol.
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. Please click here to view a larger version of this figure.
Figure 3. Acquisition and extinction of discriminated Pavlovian-conditioned responding for ethanol. Mean ± SEM normalized port entries made during the CS+ and CS- across PDT (sessions 1 – 18) in Context A and extinction (sessions 19 – 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 <0.05, CS+ versus CS-. Figure originally published in Chaudhri, Sahuque, & Janak (2008). Used with permission from Elsevier.
Figure 4. Context-induced renewal of Pavlovian-conditioned responding for ethanol. Mean ± 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 <0.05; **p <0.001, CS+ versus CS-. #p <0.05, CS+ PDT versus CS+ extinction. ^p <0.05, CS+ extinction versus CS+ test. Figure originally published in Chaudhri, Sahuque, & Janak (2008). Used with permission from Elsevier.
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 ‘context’ 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’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 ‘conditioned approach’ 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.
The authors have nothing to disclose.
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 – Santé (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.
Name of Reagent/ Equipment | Company | Catalog Number | Comments/Description |
Heavy-duty utility cart | Rubbermaid | #4520-88 | Used to transport rats in their home cage from the colony room to the behavior room or other facilities |
Rats, Long-Evans | Harlan | – | Rats weighed 220-240g on arrival |
Ohaus scale | Fisher | #S402421 | For weighing rats; tare the bottom of a plastic container first, then put the rat in it to weigh it |
Beta-chips, aspen | Harlan | #7090A | Bedding for home cage |
Nylabone | Bio-Serv | #K3580 | Enrichment provided in the home cage |
Autoclavable rodent chow | Agribrands, Charles River | #5075 | For feeding rats |
Wire bar cage lid | Ancare | #R20SS2B | Custom made with two slots for bottles |
Cage bottom | Ancare | #R20PC | To house rats |
Stoppers | Ancare | #8.5 | Rubber stoppers to occlude opening in water bottles & graduated cylinders; goes with ball point sipper tubes |
Ball point tubes, 1.5" length | Ancare | #TD-99 | Inserted in stoppers, enables rats to drink from water bottles and graduated cylinders |
Water bottles | Ancare | #FSPC8HT | 473 ml (16 oz) capacity, needs to be occluded with stopper |
Graduated cylinders, 100ml | Fisher | #0300741 | To contain ethanol during alcohol exposure in the home cage; spouted end needs to be cut off to enable occlusion with stoppers |
Mettler scale | Mettler Toledo | #MS6001S | For weighing water bottles and ethanol cylinders |
Polyethylene tubing, 1/32" inner diameter, 1/32" wall thickness, 3/32" outside diameter | Fisher | #14-169-1A | To connect 20 ml ethanol syringe to fluid port |
Needle, 18g, 1.5" length | Fisher | #B305199 | To connect polyethylene tubing to ethanol syringes; needs to be filed |
Syringes, 20ml, Luer-Lok non-sterile | Fisher | #1482316J | To contain ethanol on the fluid pumps; needs to be connected to the polyethylene tubing by a 18g 1 1/2 needle |
Syringes, 60ml, Luer-Lok non-sterile | Fisher | #14-820-11 | To flush polyethylene tubing after PDT sessions |
Single speed syringe pump, 3.33 rpm | Med Associates Inc. | #ENV-018MD | To hold 20 ml ethanol syringes and deliver ethanol |
Fluid port | Med Associates Inc. | #ENV-254-CB | To enable ethanol delivery during PDT sessions; connected to ethanol syringes with polyethylene tubing |
Port entry infrared detector | Med Associates Inc. | #ENV-215M | Fixed on both sides of fluid port; need 2 per fluid port |
Houselight, 28V, 100mA | Med Associates Inc. | #ENV-135M | Provides general lighting in conditioning chamber |
Clicker module | Med Associates Inc. | #ENV-225SM | Auditory stimulus for conditioning chamber |
White noise generator with speaker | Med Associates Inc. | #PHM-100 | Auditory stimulus for conditioning chamber |
Bar floor | Med Associates Inc. | #ENV-009A-GF | To go with modular test chamber |
Bench coat, absorbant | VWR | #89126-790 | Absorbant paper to put in metal tray, needs to be cut to fit the metal tray |
Polycarbonate sheet, 1/8" thick, 12-5/8"x10" | Johnston Plastics | #30102515 | To make smooth transparent floor insert used for context 1. Needs to be cut in 11.5"x12.25" pieces |
Aluminium sheet perforated, 0.063"thick, 48"x96", 0.250" holes x 0.375" centers | Anica Steel | – | To make perforated floor insert used for context 2. Needs to be cut in 11.5"x12.25" pieces |
Black cardboard, 0.053" thick, 28"x44" | Omer DeSerres | #P1909 | 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"x13.25". The panels can be joined by the narrower side using duct tape. |
All-purpose strength duct tape, 1.88"x45yd. | Duck Tape | – | Adhesive material to connect the black cardboard panels used to cover the back wall, ceiling and door in context 1. |
Modular test chamber | Med Associates Inc. | #ENV-009A | Conditioning chamber |
Sound attenuating cubicles | Med Associates Inc. | #ENV-200R3AM | Comes with installed 28V DC fan (#ENV-025F) |
Petri dish | Fisher | #08748B | Bottom only, to contain the sprayed solution that provides the odor stimulus; needs to be centered in the metal tray in conditioning chambers |
PCI interface package | Med Associates Inc. | #DIG-700P2-R2 | Connects tabletop interface cabinet to computer; includes 1 interface card, 1 Decode card, 1 ribbon cable and 1 28V DC power cable |
Large tabletop cabinet and power supply | Med Associates Inc. | #SG-6510D | Used to contain the 12 cards included in the SmartCrtrl 8 Input, 16 Output package |
SmartCtrl 8 Input, 16 Output package | Med Associates Inc. | #DIG-716P2 | Used to connect and control the devices in the conditioning chamber. The package includes all the cables, the interface module and the connection panel. |
Med PC IV software | Med Associates Inc. | #SOF-735 | Software to run programs. Needs to be installed on the computer. |
Monitor LG, 19" LCD | Concordia computer store | #W1942TQ-BF | Computer monitor; to go with computer running Med PC IV |
HP Z200 tower workstation | Concordia computer store | #BZ788US#ABA | Computer on which Med PC IV is run |
Benzaldehyde, ≥99% | Les produits chimiques OMEGA Chemical Company Inc. | #B37-50 | Dilute in tap water for almond context odor |
Lemon oil, cold pressed, California | Sigma Aldrich | #W262528-1KG-K | Dilute in tap water for lemon context odor |
Ethyl alcohol, 95% | Commercial Alcohols | – | Dilute in tap water to desired concentration |