A Procedure to Observe Context-induced Renewal of Pavlovian-conditioned Alcohol-seeking Behavior in Rats

This procedure examines the capacity of an alcohol associated context to trigger the renewal of Pavlovian alcohol seeking behavior. This is accomplished by first habituating ethanol experienced rats to conditioning chambers that have been configured into two unique contexts. The second step of the procedure is to conduct Pavlovian discrimination training sessions in one context where two different auditory cues signal either alcohol availability or the lack thereof.

The third step is to conduct extinction training. In the second context where both auditory cues are presented without alcohol, the final step is to return the rats to the original training context and present both auditory cues without alcohol. This manipulation causes a renewal of alcohol seeking behavior triggered by the auditory cue that was previously associated with alcohol.

The main advantage of this technique over existing procedures like instrumental conditioning, is that it provides an animal model of alcohol seeking behavior elicited by Pavlovian cues that predict alcohol. This method can help answer key questions in the addiction field, such as do context associated with alcohol provoke relapse Before training, habituate the rats to the testing room and the two environmental contexts that they will experience. Always transport the rats from the animal care facility to the testing room in a consistent manner.

In the first session, leave the rats in the testing room for 20 minutes with the room lights illuminated and the fans in the sound attenuating cubicles turned on after 24 hours. Conduct the second habituation session like the first, but now weigh and handle each rat. Conduct the third session 24 hours later.

For this, set up each conditioning chamber with one of two distinctive combinations of visual olfactory and tactile stimuli. To provide a consistent context, the olfactory stimulus should be the last contextual stimulus added to each chamber. To minimize co-mingling of different olfactory stimuli, close the doors of the chambers and the sound attenuating cubicle in which each chamber is housed.

Immediately after applying the olfactory stimulus for the third session, weigh each rat and place it into its conditioning chamber for 20 minutes. Half the rats should experience context one, and the other half should experience context. Two, to help maintain the vital need for consistency between the rats and their chambers, it is useful to mark the rats with a number that identifies a specific chamber.

One minute after all the rats have been placed in their chambers, illuminate the house light in the chambers During the 20 minutes, record all the fluid port entries made by the rats. Then turn off the lights, unload the rats, and return them to their care facility. Later on the same day, conduct a fourth habituation session nearly identical to the third one in which the context of the conditioning chambers is reversed.

Lastly, after each and every session, always wash the waste trays and floor panels of the conditioning chambers and the Petri dishes into which the olfactory contextual stimulus was applied. Use only unscented cleaning solutions on a weekly basis. Clean the chambers in their entirety for this training.

Divide the rats into two groups with the same average ethanol intake across their last two ethanol access sessions. One group is trained in context one and the other trained in context.Two. The context used for Pavlovian discrimination training is referred to as context A.During these sessions, the rats learn to distinguish between two different auditory cues, one paired with ethanol and one that is not.

So the first step is to prepare 20 milliliter syringes with ethanol. For the fluid ports, connect the tubing from the back of the fluid port to the loaded syringe. Using a blunt needle load each syringe into its own pump outside the sound attenuating cubicle.

Then prime the tubing by manually advancing the pump and wipe away excess ethanol from the fluid port. Now bring the rats to the laboratory testing room. As during habituation, weigh each rat before placing it into its assigned conditioning chamber.

Make sure to double check the context configuration within each chamber and be sure to load the correct rat in the correct chamber. A rat that's been exposed to the wrong context or the wrong CS plus may have to be dropped from the study. Once all the rats are weighed and loaded, start the computer program that controls the events of the training session.

Five minutes into the program, the house light in each chamber is illuminated in each session, 16, ten second bursts of white noise and 16, ten second bursts of clicking noise are presented. For each rat, designate one of these auditory stimuli for the ethanol and counterbalance these designations across the two conditioning contexts during the last six seconds of the Cs, plus, the pump delivers 0.2 milliliters of ethanol to the fluid port. Set the stimuli on a variable time 67 second schedule to make a one hour session.

Record all the fluid port entries starting 10 seconds pre Cs, during Cs, and 10 seconds post cs. Also, record the total number of fluid port entries made for the whole Pavlovian discrimination training session. When the session ends, turn off the house lights and return the rats to the animal care facility in the conditioning chambers.

Check the fluid ports to ensure that the rats consumed the 3.2 milliliters of ethanol delivered during the session. Remember to check the fluid ports after the rats have removed from the chambers to ensure they're consuming the ethanol rats that do not consume the ethanol and also show low levels of port entries during the CS plus may have to be dropped from the study. Clean the conditioning chambers as usual.

Clean the tubing by first flushing it with water, and then using an empty 60 milliliter syringe to pass air through it. Repeat the sessions daily until the rats discriminate between the two auditory stimuli as indicated by port entry responses. A selective increase in port entries to the CS plus that stabilizes within 16 to 20 sessions is anticipated.

For the extinction sessions. The conditioning chamber of every rat should be set up with the different context from the one used during Pavlovian discrimination training. The context in which extinction is conducted is referred to as Context B.Conduct the extinction sessions exactly as the discrimination sessions using the same computer program, but do not deliver any ethanol during the CS Plus.

For consistency, the pumps are loaded with empty 20 milliliter syringes connected to the chamber by ethanol free tubing. Perform the extinction sessions daily until the rats stop discriminating between CS plus and minus. This can take about eight sessions as always, clean the equipment as needed Before proceeding to the renewal test.

It is vital that there is a significant reduction in port entries during the CS plus at the end of extinction training compared to the beginning. The context induced renewal test uses the same chamber context configuration as used during the Pavlovian discrimination training, which is a different context from that of the extinction training. This single session test is performed identically to a Pavlovian discrimination training session, except that no ethanol is delivered and the syringes are empty.

Use the same computer program to present the auditory stimuli. The expected result is a renewal of alcohol seeking behavior that is selective to the CS plus clean the equipment at the end of the test. As before, when rats were given periodic intermittent access to ethanol in their home cages, ethanol consumption and ethanol preference increased to stable levels After eight to 12 training sessions.

During Pavlovian discrimination training, port entry responses to the CS plus increased across training sessions, there was a slight increase in responding to the CS minus, but the rats did learn to discriminate between the two auditory cues during extinction training, port entry responses to the CS plus gradually declined while responses to the CS minus remained low. When the rats were returned to the context associated with Pavlovian discrimination training, a selective increase in port entries occurred during the CS plus relative to the results after extinction training. By contrast, there was no change in response to the CS minus.

These results demonstrate that alcohol seeking behavior elicited by a discreet cue can be strongly modulated by environmental context. While attempting this procedure, it is important that the researcher does not use perfumes, colognes, or other scented products. Rats are highly olfactory creatures and olfactory cues associated with the researcher can be disruptive To the behavior in this task Following this method, other procedures like intracranial micro infusions can be performed in order to answer additional questions such as what brain regions are involved in.

Context induced renewal of Pavlovian conditioned alcohol seeking.