Reinstatement of Drug-seeking in Mice Using the Conditioned Place Preference Paradigm

The overall goal of this procedure is to evaluate reinstatement of drug-seeking behavior in laboratory animals considering the impact of the drug associated environmental cues and stress. This method can help answer key question in the drug addiction field, such as the role of different environmental manipulation that can modulate relapsing to drug-seeking. The main advantage of this technique is that the CPP reinstatement is considered to reflect the reactivation of the incentive motivational value of the context stimuli paired with the drug consistent in the reappearance of the approach behavior to the context.

The implications of this technique extend toward the ethology of drug relapse, because it is a useful and sensitive way to assess different environmental manipulations, which are the main triggers of human relapse. New to this method will struggle because the reinstatement model is very sensitive to environmental change. Room and environment condition should stay constant during the procedure, especially with mates of the opposite sex, noises, or light conditions.

Responses to the drug and stress-induced reinstatement is performed by different procedures that can affect the results. Especially for this reason, it is important to show social defeat as a determinant factor of reinstatement. To begin, handle mice for one to two minutes each three days prior to testing.

Set up identical boxes, testing room, and cocaine solution, as directed in the written protocol. Every day, bring the mice to the testing room in their home cages. Allow the mice to sit undisturbed for 15 minutes, as the habituation period to all test-related noises and stimuli.

Ensure the guillotine doors are removed from the cage, and begin setting up the computer. Enter the mouse's ID, and press the trial and start command. Then, place the mouse in the middle chamber of the box, and maintain minimal noise in the testing room.

When the trial is complete, gather the mice back to their home cages, and save the data on the computer. Allow each mouse to access both compartments of the boxes for 15 minutes per day, for three consecutive days. On the third day, record the amount of time each mouse spends in each compartment.

Next, assign half of the mice to receive the drug in one compartment and the other half in the other compartment. First, weigh the mice to determine the appropriate cocaine dosage. Then, prepare syringes with the appropriate dosage of cocaine or saline.

Inject each mouse at a time intraperitoneally, and gently place it into its assigned compartment. After four hours, inject the cocaine dose, and confine the mouse to its drug-paired compartment for 30 minutes. Ensure that the guillotine doors separating the two compartments is closed.

On the post-conditioning test day, bring the animals to the testing room, and allow them to habituate for 15 minutes. Remove the guillotine doors, separating the two compartments and set up the computer program. Enter the animal IDs and press the start command.

Record the amount of time spent by the untreated mice in compartment of the box during the 15 minute observation period. When the observation period is over, gather the mice back into their home cages, and save the data on the computer. Each week, conduct an extinction session with all of the mice that have demonstrated a preference for the drug-paired compartment.

First, remove the guillotine door from the box, and place the animal in the box for 15 minutes. When a whole group demonstrates no preference for the drug-paired compartment, repeat the test 24 hours later to confirm the extinction. For drug-induced reinstatement, bring the animals to a different room 24 hours after extinction has been confirmed.

Then, inject them with half the previous dose of cocaine, and return them to their vivarium for 15 minutes. Next, take the mice into the testing room, and place them into the box for 15 minutes. Record the amount of time each mouse spends in each compartment, and save the data on the computer.

To evaluate the effects of stress-induced reinstatement, take the mice to a different room 24 hours after extinction has been confirmed. Next, place an experimental mouse into a plastic cage with an aggressive opponent mouse for 10 minutes. After the antagonistic encounter, remove the experimental mice, and bring them to the testing room.

Finally remove the guillotine door, and keep the mice in the box for 15 minutes. Regarding social stress, we should confirm that the animal has actually been defeated. A positive social encounter, that does not end in defeat shows protective effects, blocking the reinstatement of the place preference of cocaine.

Using the CPP protocol, male mice in this study were subjected to varying environmental manipulations to measure relapse from cocaine. Priming induced reinstatement was subjected to subsequent decreasing priming doses until the priming dose was confirmed to be ineffective. As shown in this figure, priming with 12.5, 6.25, 3.125, and 1.56 milligrams per kilogram of cocaine had a significant effect on reinstatement.

Mice were subjected to a social interaction, in which the experimental animals were defeated by an aggressive opponent to measure stress-induced reinstatement. Being exposed to an aggressive animal and defeated 15 minutes before the test led to reinstating previously extinguished preference. Mice that suffered repeated social defeat before the CPP procedure, demonstrated modified rewarding effects of subthreshold doses of cocaine.

After the extinction of preference, socially defeated mice reinstated their preference with the priming dose of only 0.5 milligrams per kilogram of cocaine. It is important to remember that the key point of the crack addiction research is that the development of treatments that diminish craving and consequently reduce vulnerability to relapse. Other methods, like intravenous self-administration, can be performed in order to answer additional questions like compulsivity or motivation to obtain the drug.

After this development, this technique paved the way for researchers in the field of the drug addiction to explore reinstatement into drug-seeking in mice. After watching this video, you should have a good understanding of how to induce relapse through different environment manipulations, such as context or drug cues, which re-establish the place preference.