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

In this protocol, cue reactivity measures an animal's motivation to seek cocaine following absence from cocaine self-administration. Cue reactivity occurs when the rat is re-exposed to the context and cues previously associated with intravenous cocaine delivery. Cue reactivity is a measure for relapse risk.

The cue reactivity protocol described here is a pre-clinical means to test relapse modifying pharmacotherapies as well as investigate genetic and environmental factors that contribute to relapse risk. This is an operant task for drug-seeking behavior that aligns with many properties of human drug taking and relapse. Optimization of key parameters such as cocaine dose, delivery volume, and infusion duration have been made to establish phase validity and translational productive power.

For this protocol, use male Sprague Dawley rats that are roughly eight to nine weeks old which have been acclimated to the colony room for at least seven days. During the acclimation period, house the rats in pairs and handle them daily throughout the study. Provide unlimited access to food in their home cages throughout all phases of the study.

Implant jugular vein catheters using the method outlined by Kmiotek and collaborators in their JoVE video, then randomly assign the rats to saline or cocaine treatment groups. For training, program schedules for cocaine self-administration as described in the text protocol. Equip the training chambers with two retractable response levers, a stimulus light above each response lever, and a house light opposite the levers.

Deliver cocaine or saline from syringe infusion pumps outside of the cubicle. Use 23 gauge needles attached to the syringes and polyethylene tubing encased in a metal spring leash attached to the rat's catheters. Cocaine or saline is delivered after an active lever press, but not after inactive lever press.

Change the solutions daily. Use freshly made cocaine dilutions in sterile 0.9%sodium chloride tailored to each rat's weight. Load at least eight milliliters of solution into each rat's syringe.

Begin with training the rats on an FR1 schedule of reinforcement and progress to an FR5 schedule after three straight days of seven infusions per hour with less than 10%variability in the total number of infusions per session. It is imperative to analyze self-administration behavior data on a daily basis to determine when to advance an animal to FR5 and to check for loss of catheter patency. One of the most critical steps of the protocol is to monitor self-administration during the first 14 days of training to ensure the animal self-administer normally.

If self-administration dramatically decreases, the rat's catheter has likely lost patency. At the start of a session, check that there are no bubbles in the syringe. Attach the syringe to the polyethylene tubing and insert it into the infusion pump.

Carefully adjust the syringe so that the solution is evenly and completely distributed in the polyethylene tubing. Next, connect the rats to the delivery system and place them into their respective standard operant conditioning chambers housed in ventilated sound-attenuating cubicles with fans. Consistency throughout the experiment is vital.

Always place a rat into the same chamber and keep the active lever on the same side within that chamber. Over 14 daily 180-minute sessions, the rats have free access to the levers and will readily learn which lever provides stimulation provided the system is working. Each trigger of the active lever on a fixed ratio schedule of one releases 0.1 milliliters of solution over six seconds and simultaneously illuminates the cage light and the stimulus light above the active lever.

There is a one-second delay before solution is released. Following each cocaine or saline delivery, the stimulus light and infusion pump are deactivated for 20 seconds during which the house light remains on and pressing the lever does nothing but is recorded. When the light goes off, the lever is reactivated.

Any triggering of the inactive lever produces no scheduled consequences. At the end of that 180-minute session, use slow careful movements to remove the potentially aggressive rat from the chamber. Then flush its catheter.

Between sessions, carefully clean the chambers. Wipe down every surface with a 70%ethanol solution. At the end of the training sessions, put the rats into forced abstinence for 30 days with no access to the operant chamber.

Continue to handle the rats on a daily basis and monitor the general health. After the long abstinence period, allow the rats 60 minutes in the operant chamber immediately followed by brain harvesting. For this session, run the cue reactivity scheduled program.

Stagger the session start times to allow time to harvest brains between sessions. Importantly, operate the pump without the syringe attached so the auditory cue of the pump is still a part of the stimulus without the substance delivery. Begin this session as usual with the rat in the self-administration chamber in which it was trained.

In this test, when the active lever that previously delivered cocaine or saline is pressed, only the cues are delivered on an FR1 schedule. The inactive lever still does nothing. Record all the cue presentations, previously active lever presses and inactive lever presses as well as the latency to first lever press.

Using the described protocol, a study was conducted and rats were individually transitioned from FR1 to FR5 as they met criteria. In the cocaine-administering group, rats gradually increased number of infusions until they reached criterion, then they were transitioned from FR1 to FR5. Multiple output measures were gathered from the cocaine cue reactivity test.

For example, rats which previously self-administered cocaine pressed the previously active lever much more than rats which self-administered saline. Another important output measure is latency to first level press. When a change in this is observed, the animal may be physically stressed or have decreased motivation for cocaine self-administration.

Locomotor behavior tasks can be used to assess changes in physical health and differentiate motor impairment from motivational changes. After watching this video, you should have a good understanding of how to train rats to self-administer a drug, maintain catheter patency during self-administration, and assess cue reactivity to drug-associated cues following abstinence as a measure of relapse risk. While attempting this procedure, it's important to remember to carefully program the software, properly handle the animals, and regularly flush intravenous catheters.

Following this procedure, one can assess the cellular and molecular mechanisms that drive cocaine cue reactivity as well as test potential pharmacotherapuetics that modify cocaine cue reactivity.