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

It is the first time that stress-induced relapse to heroin is shown after punishment-imposed abstinence. These methods could help us understand the neuronal mechanism underlying stress-induced relapse in humans after voluntary abstinence. By using food deprivation and punishment imposed abstinence, we are more closely mimicking the human condition and bringing ecological validity to animal models of relapse.

Begin by setting the operant conditioning chambers as described in the manuscript. After recovery from the surgery, habituate the rat in the operant training chamber for 24 hours before initiating the protocol without attaching the rat to the metal spring. Place the rat housed in the operant chambers throughout the experiment.

Next, attach the 5up connector to the Tygon tube and the metal spring. Start the training session on the onset of the dark phase of the reversed light cycle. For self-administration of heroin, the take lever should be available under fixed ratio one, or FR one.

Program each trial to begin with the insertion of the take lever and turning the house light on. Once the rat presses the take lever one time, retract the take lever, turn on the cue light above the lever, and tone for a 20-second timeout period. Turn off the house light, and allow the delivery of 0.13 milliliters of heroin infusion at a dose of 0.1 milligram per kilogram.

Then program a 30-seconds intertrial interval in which no cues are presented, the house light is turned off, and the lever is retracted. For the next trial, insert the take lever, and turn on the house light. For the self-administration with the seek take chain under FR one, program the operant training system to insert only the seek lever and turn on the house light to signal the beginning of the session with the take lever in a retracted state.

Once the rat presses one time on the seek lever, the program retracts the seek lever and inserts the take lever. One press on the take lever retracts the take lever and initiates the procedure of turning on the cue light, and a 30-seconds intertrial interval, as demonstrated earlier. After the intertrial interval, program the operant training system to insert the seek lever and turn on the house light to signal the beginning of the next trial.

Next, program all steps for self-administration with a seek take chain under fixed ratio one, as demonstrated earlier with introducing a variable interval of five for three days as the new schedule of reinforcement on the seek lever. Set up the software to allow the first press on the seek lever to activate variable interval five schedules. Allow the software to randomly select an interval time from a list of 0.1, five, and 10 seconds, resulting in an average of five seconds interval.

After the selected interval has elapsed, the first seek lever press retracts the seek lever and extends the take lever. After following the steps for the take lever, and as the trial ends, train the rat for five days under variable interval 30 schedule for the seek link. To do so, allow the software to randomly select a time from a list of 15, 30, and 45 seconds for an average of 30 seconds.

Then, train rats for three days under variable interval 60 schedules, allowing the software to randomly select a time from a list of 45, 60, and 75 seconds. For punishment-imposed abstinence, conduct punishment sessions for eight days with regular supervision to guarantee that rats do not show signs of abnormal pain or any health issues. Start punishment sessions with insertion of seek lever and turning the house light on.

Program the operant training system so that after completing the seek link under variable interval 60, the seek lever retracts, the house light is turned off, and a mild foot shock is administered to the rat 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 regular self-administration trial. After programming an intertrial interval of seven minutes, set the foot shock intensity at 0.2 milliamperes, and increase by 0.1 milliamperes per punishment day until 1.0 milliamperes.

Once the rat presses the take lever, repeat the fixed ratio one step for retraction of the lever, delivery of heroin infusion, and presentation of the cues followed by seven minutes intertrial interval. After the last day of the punishment-imposed abstinence, remove food hoppers from the chambers for rats that undergo food deprivation-induced relapse test, and keep food hoppers for the sated group. Provide unlimited water access in both the groups and supervise the animals’health.

After 24 hours of food deprivation, proceed for a three-hour food deprivation-induced heroin-seeking test under a variable interval 60 schedule with an intertrial interval of seven minutes with no shock or heroin infusions being delivered. Following the session, return food hoppers to the food deprived rats. The next day, remove food hoppers from the chambers of the rats that were sated on the first relapse test.

Allow the previously food-deprived rats unlimited access to food before and during the test. After 24-hour food deprivation, perform three-hour heroin-seeking test, as demonstrated previously. In the study, male rats demonstrated an increase in seek lever presses as the schedule of reinforcement increased.

A reliable, consistent number of heroin infusions was observed over the training days. During the punishment, rats decreased the number of seek lever presses and infusions with the increase of foot shock intensity over eight punishment days. In the heroin-seeking test, the food deprivation condition significantly increased heroin-seeking after punishment-imposed abstinence, and no statistically significant effect was observed for time nor for drug seeking over time across feeding conditions It is important to take the time to train the animals to learn in the basic seek and take chain under FR one.

Without this step, animals will not be able to move forward in the experiments. After implementing the model, we can now design experiments that look at the neurobiology of stress-induced relapse after voluntary abstinence.