A procedure that demonstrates a robust acute food deprivation-induced relapse to heroin seeking after punishment-imposed abstinence is described. A punishment-imposed abstinence model was successfully implemented using the seek and take chain schedule for heroin self-administration. Heroin-seeking tests are then performed following 24 h of food-deprivation stress.
The punishment-imposed abstinence procedure models the self-imposed abstinence that humans initiate due to the adverse consequences associated with drug-taking. This model has been implemented in experiments using different types of substances of abuse such as methamphetamine, cocaine, and alcohol. However, punishment-induced abstinence in heroin-trained animals has not been demonstrated. Furthermore, acute stress is a key trigger for relapse in humans and animal models. It was previously demonstrated that acute food deprivation robustly induced reinstatement of extinguished cocaine and heroin seeking. The procedure described here can be used to assess the effects of acute stress exposure on heroin seeking after punishment-imposed abstinence. A total of 8 rats were implanted with chronic intravenous (i.v.) catheters and trained to self-administer heroin (0.1 mg/kg/infusion) for 18 days under a seek-take chained schedule. Completing the seek link gave access to the take lever, which was paired with a heroin infusion. The seek lever was programmed with a variable interval 60 schedule of reinforcement (VI60), and the take lever was programmed with a fixed-ratio 1 reinforcement schedule (FR1). Following self-administration training, a mild foot shock was delivered on 30% of the completed seek links instead of the extension of the take lever. Footshock intensity was increased by 0.1 mA per daily session from 0.2 mA to 1.0 mA. Heroin-seeking tests were performed after 24 h of food deprivation (FD) or sated conditions. Rats under acute food deprivation condition robustly increased heroin seeking after punishment-imposed abstinence.
Relapse is the most challenging problem in the treatment of drug use1,2. However, only a handful of pharmacological treatments are approved to help avoid relapse in humans3. The opioid epidemic that North America is currently facing is a striking example of it, and it demands considering different approaches on animal models of relapse to opioids.
Acute stress has been shown to be a key trigger to relapse in humans4. One environmental stressor that is often associated with drug addiction is food deprivation. Drug users often choose to allocate resources towards obtaining drugs instead of food. Caloric deficit has been shown to be correlated with higher relapse to cigarettes5 and alcohol6 use. Due to ethical and practical issues, animal models have been developed over the last decades to facilitate research in the field. In animal models, acute food deprivation has been demonstrated to robustly reinstate extinguished heroin seeking7. Currently, most animal models of relapse are based on abstinence procedures that are either not representative of human abstinence (e.g., extinction-based models) or encompass only the small percentage of drug users that are forced to abstain due to incarceration or inpatient treatment (e.g., forced abstinence models). The main reason drug users choose to abstain is the negative consequences associated with drug-seeking and taking8. Punishment-imposed abstinence is an animal model that mimics the negative consequences associated with drug-seeking on self-imposed abstinence in humans. This model introduces an aversive stimulus, e.g., a mild footshock, with drug-seeking or taking, which leads the animal to stop taking the drug voluntarily. Another procedure that incorporates negative consequences for drug-seeking is the electrical barrier conflict model for drug abstinence and relapse9. The rat must cross an electrical barrier to perform the operant behavior linked with drug self-administration. The model was used successfully to demonstrate voluntary abstinence and relapse to psychostimulant and opioid drugs10,11. However, under the electrical barrier procedure, drug-seeking efforts are always associated with an aversive event, unlike the human condition. Moreover, drug-taking itself might overlap with the electrical footshock as the animal returns to the safe area following the infusion by crossing the barrier again.
Punishment-imposed abstinence has been used with other drugs of abuse such as cocaine12, alcohol13, methamphetamine14, remifentanil15, but it was never applied to heroin-trained animals. The model has been used to study relapse induced by priming14 and drug-associated cues16, but it was not integrated into a stress-induced relapse procedure. The procedure described here is used to demonstrate acute food deprivation-induced relapse to heroin seeking after punishment-imposed abstinence in male rats.
All rats are treated according to the guidelines of the Canadian Council on Animal Care. Approval for all the experimental procedures was granted by the Concordia University Animal Research Ethics Committee.
- Acquire Long Evans rats weighing 275-300 g (males) or 225-250 g (females). Double house rats in standard plastic cages with corncob bedding and shredded paper until surgery.
- Keep the rats on a reversed light-dark cycle (lights off at 9:30 AM, light on at 9:30 PM).
- Allow unlimited access to food and water, except during the food-deprivation test. Use regular rat chow (20.9% protein, 67.2% carbohydrates, and 11.8% fat).
2. Intravenous surgeries
- Build catheters and perform intravenous surgery as described in Sedki et al. 201317 with the following differences: 1) Use Isofluorane 2-2.5% for anesthesia, 2) Change the dose of penicillin administered during surgery to 60 000 IU/rat, via subcutaneous injection, and 3) Increase the dose of gentamicin in the mixture of heparin and gentamicin used to flush the catheters to 7.5 IU + 800 µg; 0.2- 0.3 mL.
- Inject ketoprofen or carprofen, an anti-inflammatory analgesic, intraperitoneally or subcutaneously (5 mg/kg) immediately after surgery (before recovery from the anesthesia) and on the following 3 days. During the 2 days recovery period, give mashed food and heat (if necessary) to help with the recovery. Weigh and supervise the animals daily to check for any abrupt loss of weight or any other signs of health issues.
3. Behavioral procedure
- Operant conditioning chambers:
- Set the operant conditioning chambers as described in Sedki et al. 201317.
- Habituation day:
- House the rats in the operant training chambers after recovery from surgery and allow animals to habituate to the chamber for 24 h before initiating any protocol. Keep animals housed in the operant chambers throughout the whole experiment.
- Do not attach the rats to the metal spring and do not initiate any experimental protocol (the cue-light, the tone, and the levers are not available during habituation day).
- Heroin self-administration:
- Attach the 5-up connector to the Tygon tube and the metal spring.
- Set up daily training sessions of 6 h of heroin self-administration (0.1 mg/kg/infusion) according to the procedure described below.
- Start daily training sessions on the onset of the dark phase of the reversed light cycle (around 9:30 AM in the representative results) with the extension of the seek lever (or take lever on the first 2 days; see below), as well as turning off the houselight on.
- Self-administration with only the take lever available under fixed-ratio 1 (FR1; 2 days)
- Program each trial to begin with the insertion of the take lever.
- Once the rat presses the take lever one time (FR1), retract the take lever, turn on the cue light above the lever and tone for a 20 s timeout period, turn off the houselight, and allow the delivery of a heroin infusion (0.1 mg/kg in 0.13 mL)
- Program a 30 s inter-trial interval in which no cues are presented, the houselight is turned off, and the lever is retracted. For the beginning of the next trial, insert the take lever, and turn on the houselight.
- Self-administration with seek-take chain under FR1 (5 days)
- Program the operant training system to insert only the seek lever and turn on the houselight to signal the beginning of the session. The take lever is in a retracted state.
- Once the rat presses one time (FR1) on the seek lever, the program retracts the seek lever and inserts the take lever.
- One press on the take lever (FR1) retracts the take lever and initiates the same procedure as in step 126.96.36.199 and a 30 s inter-trial interval.
- Program the operant training system to insert the seek lever at the conclusion of the inter-trial interval and turn on the houselight to signal the beginning of the next trial.
- Program the operant training system to initiate the inter-trial interval if a rat fails to accomplish the seek-take chain during 10 min (not pressing the seek or the take lever) to represent the end of a trial and the loss of the opportunity to administer the drug.
- Self-administration with seek-take chain under Variable Intervals (VI5, VI30, and VI60)
- Program all steps in section 3.3.5 but introduce a variable interval of 5 (VI5) as the new schedule of reinforcement on the seek lever, instead of FR1. Allow self-administration training under the VI5 schedule for 3 days.
- Set up the software to allow the first press on the seek lever to activate the VI5 schedule. For the VI5 schedule, let the software to randomly select an interval time from a list of 0.1 s, 5 s, and 10 s, which results in an average of 5 s interval. The first seek lever press after the selected interval has elapsed, retracts the seek lever and extends the take lever.
- Follow steps 188.8.131.52, 184.108.40.206., 220.127.116.11. for take lever and end of trial instructions.
- Next, train rats for 5 days under VI30 schedule for the seek link, followed by 3 days under VI60 schedule.
- For the VI30 schedule, allow the software to randomly select a time from a list of 15 s, 30 s, and 45 s, for an average of 30 s, whereas for the VI60 schedule, the software randomly selects a time from a list of 45 s, 60 s, and 75 s.
- Vary the inter-trial intervals along the different schedules of reinforcement. Start the inter-trial interval at 30 s during FR1 and VI5 but gradually increase to 7 min during VI30 and VI60.
- Punishment-imposed abstinence
- Start punishment sessions with insertion of seek lever and turning the houselight on. Allow punishment sessions for 8 days, with regular supervision to guarantee that animals do not show signs of abnormal pain or any health issues.
- Program the operant training system so that after completing the seek link under VI60, the seek lever retracts, the houselight is turned off, and a mild footshock is administered on 30% of the completed seek links instead of insertion of the take lever. The take lever extends on the other 70% of completed seek links, as in a normal self-administration trial.
- Program an inter-trial interval of 7 min with all cues and houselight off before inserting the seek lever and turning the houselight on to signal the beginning of the next trial.
- Set the footshock intensity at 0.2 mA and increase by 0.1 mA per punishment day until 1.0 mA.
- After rats press the take lever (on trials when it is available), repeat step 18.104.22.168 for retraction of the lever, delivery of the heroin infusion, and presentation of cues, followed by a 7 min inter-trial interval.
- Start the next trial with the insertion of the seek lever and turning the houselight on.
- If the rat does not press any lever for 10 min, follow step 22.214.171.124.
- Acute food deprivation (24 h)
- On the day following the last day of punishment-imposed abstinence, divide rats into two matched groups (according to body weight and the average number of seek lever and infusions over the last 3 days of self-administration training): Food Deprived (FD) and Sated.
- Remove food hoppers from the chambers (around 9:30 AM) for rats that undergo food deprivation-induced relapse test and keep food hoppers for the sated group that has unlimited access to food. Provide unlimited access to water in both groups and supervise animals to guarantee that they do not show any health problems during the food deprivation period.
- Food deprivation-induced heroin seeking tests
- The morning after the 24 h of food deprivation, perform a 3 h heroin-seeking test, under a VI60 schedule with an inter-trial interval of 7 min with no shock or heroin infusions being delivered. Return food hoppers to the food-deprived rats after the 3 h session.
- On the morning after the first heroin seeking test, remove food hoppers from the chambers of the rats that were sated on the first relapse test. Allow the rats that were previously food-deprived unlimited access to food (sated condition) before and during the test.
- Perform another 3 h heroin seeking test on the next day (after 24 h food deprivation), as in step 3.6.1.
- The day after the test euthanize the rats using a carbon dioxide chamber (100% CO2 at fill rate of 30-70% of the chamber volume per minute).
Male rats demonstrated an increase in seek lever presses as the schedule of reinforcement increased over the training days, and a reliable, consistent number of heroin infusions over training days (Figure 1). During punishment, rats decreased the number of seek lever presses and infusions with the increase of footshock intensity over 8 punishment days (Figure 2). Food-deprivation condition significantly increased heroin seeking after punishment-imposed abstinence (Feeding condition: F(1, 7) = 7.12, p = .0.03, d = 1.3; Figure 3A). There was no statistically significant effect for time nor for drug-seeking over time across feeding conditions (Time: F(2, 14) = 1.029, p = .38; Time x Feeding condition: F(2, 14) = 1.347, p = .29; Figure 3B).
Figure 1: Heroin self-administration (N = 8). Mean ± SEM of seek lever presses (in red) and the number of infusions (blue), over a gradual increase of reinforcement schedules (FR1, VI5, VI30, VI60) for 18 training days. Please click here to view a larger version of this figure.
Figure 2: Punishment-imposed abstinence (N = 8). Mean ± SEM seek lever responses over punishment days. Footshock intensity increased 0.1 mA per day from 0.2 mA to 1.0 mA, excluding 0.9 mA. Inserted graph is the mean (±SEM) infusions over the punishment days. Please click here to view a larger version of this figure.
Figure 3: Food deprivation-induced heroin seeking tests (N = 8). (A) Mean ± SEM seek lever presses under food-deprived and sated conditions during a 3 h heroin seeking test, under extinction conditions and no footshock punishment. Counterbalanced within-subject design. * Represents statistically significant difference, p = 0.032. (B) Mean ± SEM seek lever presses under food-deprived (FD) and sated conditions during the heroin relapse test (1 h segments). Please click here to view a larger version of this figure.
There are two important demonstrations in this paper. First, the validation of the punishment-imposed abstinence using the seek and take chain with heroin. Second, it was demonstrated that stress-induced relapse could be observed in a punishment-imposed abstinence procedure. These are important demonstrations because (i) The punishment-induced abstinence procedure more closely mimics the human condition as it results in voluntary abstinence, i.e., not due to extinction of drug-seeking or forced removal from the drug-taking environment18, (ii) A reliable relapse was demonstrated using an ecologically relevant stressor: acute food deprivation stress.
When implementing the protocol, care must be given to the proper training of the rats under the seek-take procedure that is more complex compared to the common continuous self-administration using one "active" lever. In addition, the punishment phase should be carefully adjusted to the rat strain used to prevent unnecessary pain and "over-punishment" that could result in a freezing response and would interfere with relapse tests. Note that the analgesic effects of heroin could decrease the response to footshock punishment; however, under the conditions described in this protocol, robust punishment-imposed abstinence from heroin self-administration was found. The conditions of this protocol aimed to separate as much as possible the time between a heroin infusion and the footshock (on punishment trials). To achieve this, the protocol included long inter-trial intervals (7 min) between infusion trials and foot shock trials.
The bimodal-like distribution found for drug-seeking under food deprivation is not unique when studying relapse. It reflects individual differences that are observed within humans with substance use experience19. These two separate subpopulations could be an advantage for future studies that aim to identify individual differences that predict stress-induced relapse.
In contrast to the training under the more common active/inactive levers procedures, where both drug-seeking and taking are associated with the same active lever, the seek-take chained procedure allows the examination of specific factors related to drug-seeking and drug-taking as two separate sets of actions. Only drug-seeking is punished, thus avoiding possible devaluation of the drug rewarding properties20.
One limitation of this study is the reduced drug intake under the VI60 schedule compared to studies using FR1. In addition, long inter-trial intervals of 7 min also reduce the available time during the session to access the drug. One option to overcome these limitations in the future would be to reduce the inter-trial interval or increase the session length. Another limitation of the model is the pairing physical stress (footshock) to drug-seeking, while human drug users mostly experience psychological stress. Yet, under the probabilistic introduction of the punishment, as set in the procedure, drug-seeking is attenuated by fear of the possible consequences, which can be perceived as a psychological stressor. The conditions in the protocol described here set up a motivational conflict that is experienced at every trial during the punishment phase and are central to the decision process in addiction21.
Relapse to heroin can occur even after a prolonged abstinence period22. Future studies should investigate the effect of exposure to food deprivation stress after longer punishment-imposed abstinence periods. Moreover, this paper was focused on the demonstration of relapse and not the study of individual differences in response to punishment ("punishment-resistant" and "punishment sensitive"23). Footshock intensity was, therefore, increased until 1.0 mA to guarantee that all rats achieved voluntary abstinence. It would be interesting to explore the development of individual differences in heroin-trained rats by keeping the footshock at lower intensities (e.g., 0.4 mA).
Here a relapse procedure is presented in which voluntary abstinence is reached due to negative consequences, and relapse is induced by an ecologically relevant environmental stressor. The food deprivation-induced relapse is robust and easily replicable. The procedure will allow a better understanding of the mechanism underlying relapse to heroin after voluntary abstinence.
The authors declare that they have no competing financial interests.
This work was supported by the Natural Sciences & Engineering Council Discovery Program (US: RGPIN-2016-06694).
|Anafen Injection 100 mg/mL Vial/50 mL||MERIAL Canada, Inc.||1938126||anti-inflammatory drug|
|Balance arm||Coulbourn Instruments||H29-01|
|Cannulae (22 G, 5-up)||Plastics One||C313G-5up|
|Environment connection board & Linc cable||Coulbourn Instruments||H03-04|
|Fixed speed infusion pump (3.3 RPM)||Coulbourn Instruments||A73-01-3.3|
|GE Marine Silicon||GE||SE-1134|
|Graphic State Notation 3||Coulbourn Instruments||GS3||Software|
|Habitest universal Linc||Coulbourn Instruments||H02-08|
|Heroin HCl||National Institute for Drug Abuse, Research Triangle Park, NC, USA|
|House light-Rat||Coulbourn Instruments||H11-01R|
|Isofluorane USP 99.9% Vial/250 mL||Fresenius Kabi Canada Ltd||2237518|
|Liquid Swivels, Plastic, 22 G||Lomir Biomedical, Inc.||RSP1|
|Rat test cage||Coulbourn Instruments||H10-11R-TC||Operant conditioning chambers|
|Retractable lever-Rat||Coulbourn Instruments||H23-17RA|
|Silastic tubing (ID 0.02, OD 0.037)||Fisher Scientific (Canada)||1118915A|
|Single high-bright cue-Rat||Coulbourn Instruments||H11-03R|
|Sound attenuation boxes||Concordia University||Home made|
|Stainless steal grid floor||Coulbourn Instruments||H10-11R-TC-SF|
|System controller 2||Coulbourn Instruments||SYS CTRL 2|
|System power base||Coulbourn Instruments||H01-01|
|Tone module 2.9 KHz||Coulbourn Instruments||H12-02R-2.9|
|Tygon tubing (ID 0.02, OD 0.060)||VWR||63018-044|
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