This video demonstrates a procedure to systematically study the relationship between stress and relapse to cocaine seeking. In laboratory rats, rats are first trained to self-administer cocaine intravenously by performing a lever press response over multiple daily sessions during the dark or active phase of a 12 hour light dark cycle. Next, the cocaine reinforced lever press response is extinguished over multiple sessions by withholding response contingent reinforcement.
The extinguished lever pressing behavior is then reinstated by exposing animals to intermittent foot shock immediately before being given access to the previously cocaine reinforced lever. Although foot shock induced reinstatement of cocaine seeking is an effect that has been reliably achieved by several research groups, it's one that can be sensitive to certain parametal variables. The protocol we will describe reflect our successful efforts to optimize these parameters for achieving reliable and robust foot shock induced reinstatement of cocaine seeking in rats.
The methods we describe here today and variations of it can be used to examine key questions in the field of drug addiction, and in particular, it can be used to examine the fundamental behavioral and neurobiological mechanisms mediating the relationship between stress and relapse to drug seeking. The equipment used to study foot shock induced reinstatement of cocaine seeking includes a set of standard operant chambers each contained within a sound attenuating enclosure. All equipment is supplied by Med Associates Incorporated berated.
Each operant chamber is equipped with two retractable levers, both elevated 6.5 centimeters above a stainless steel rod floor. A white stimulus light is located just above one lever. The so-called active lever and responses on that lever result in the simultaneous illumination of the stimulus light, and activation of an infusion pump situated on a shelf just outside the upper end chamber.
Within the sound attenuating enclosure, the other lever, the so-called inactive lever, is left extended in the chamber. Throughout all experimental procedures, responding on this lever is without consequence. Finally, each chamber is equipped to deliver constant current, intermittent and escapable electric foot shock through a scrambler to the steel rod floor.
All experimental conditions as well as data recording and consolidation are accomplished via interfaces between the test chambers and a central computer. Operating mid associates software. Before each daily session, which occurs during the dark or active phase of the light dark cycle, weigh the rats in the housing room and then transfer them to the operant chambers.
24 hours before the first cocaine self-administration session allow the rats to habituate to the operant chambers during a single two hour session. During this session, the active lever is retracted and the inactive lever is extended at the start of daily training sessions, connect a spring and tubing assembly to the skull mounted cannula and place the rat in the operant chamber. The cannula is connected to a celesta catheter that has been surgically implanted in the right jugular vein to permit intravenous drug delivery.
Allow the rats to self-administer cocaine during daily three hour sessions for a total of eight to 10 days. Precede each training session by a five minute acclimatization period during which animals are placed in the operant chambers with the active lever retracted. To begin a training session, extend the active lever into the chamber and illuminate the stimulus light for 30 seconds.
In addition, activate the house light at this time and have it remain illuminated throughout the session. Together these events signal the availability of cocaine during the session. Responses on the active lever are reinforced by a three second infusion of cocaine on a fixed ratio, one schedule of reinforcement concurrent with activation of the infusion, pump and drug delivery is a 22nd activation of the stimulus light above the active lever.
This 22nd light presentation corresponds to a time out period during which additional active lever responses are recorded but not reinforced. During each three hour session, animals are allowed a maximum of 50 infusions to reduce the likelihood of overdose. At the end of each daily session, flush the rat's catheters with 0.2 to 0.3 milliliters of heparin dextro solution and then return them to their home cage.
After the final cocaine self-administration session, leave the rats undisturbed in their home cage for a minimum of five to seven days When this time has passed. Transfer the rats to the operant chambers for extinction training consistent with the conditions for training. Precede each day of extinction training by a five minute acclimatization period during which animals are placed in the operant chambers with the active lever retracted.
Then give the rats four 60 minute extinction sessions separated by 30 minute intervals. Each 60 minute session is initiated by the same events that occur at the start of training sessions throughout the sessions. Maintain all conditions that were present during training except no longer reinforce active lever responses with cocaine infusions during the 30 minute intervals that separate each 60 minute extinction session.
Retract the active lever and extinguish the house light at the end of the last 60 minute session. Return the rats to their home cage. These steps are repeated for three consecutive days.
Transfer the rats to the operant chambers as is done during the extinction phase consistent with the conditions for training and extinction. Provide a five minute acclimatization period during which rats are placed the operant chambers with the active lever retracted. Then give the rats two 60 minute extinction sessions separated by a 30 minute interval identical to those given during the extinction phase.
If after the second 60 minute session, the total number of responses on the previously active lever is less than 15, give the animals a test for reinstatement after an additional 30 minute interval during which foot shock or sham foot shock is administered. Beginning 10 minutes after the second 60 minute extinction session, expose animals to 20 minutes of intermittent electric foot shock, stress or sham foot shock in the operant chamber. Administer foot shock during a time when the active lever is retracted and house and stimulus lights are extinguished.
Immediately following termination of the foot shock session extended the active lever and allow the rat to lever press under extinction conditions. This period of time is defined as the reinstatement test session and is typically one to three hours in duration. At the end of the test session, remove the rat from the chamber and return it to its home cage.
Now we'll show some representative results. Shown here is a typical cocaine self-administration training curve that includes total number of infusions and total number of active and inactive liver presses. Rats generally acquire cocaine self-administration within one to three sessions, and once the behavior is established, maintain a stable rate of drug intake over subsequent sessions on day one of extinction.
When responses are non reinforced for the first time, the total number of responses on the active lever is initially very high, but gradually decreases over the four extinction sessions on days two and three of extinction. Some recovery of responding may be observed, however, responses are generally fewer than at the start of the previous day of extinction and extinction generally proceeds more rapidly over the four sessions for that day. Non reinforced responding on the active lever.
After 20 minutes of exposure to intermittent foot shock, stress induces an increase in responding on the previously active lever relative to the inactive lever. Foot shock also induces an increase in responding relative to that observed. Under conditions in which sham foot chalk is administered Once mastered, the procedures described can be completed in approximately six weeks.
In our experience, success is optimized when the procedure is carried out systematically and by well-trained hands. The procedures we've described here can be readily adapted to accommodate various pharmacological or neurochemical manipulations targeting specific neuronal substrates. With the addition of such manipulations, the foot shock inductory and statement procedure can serve as a powerful tool for characterizing the neurobiology of drug taking extinction and relapse to drug seeking by stress.
