A General Method for Evaluating Incubation of Sucrose Craving in Rats

The overall aim of this experiment is to assess craving for sucrose in rats after either a short or protracted period of forced abstinence from sucrose self-administration. This is accomplished by first allowing rats to self-administer sucrose in 10 daily self-administration sessions. Then the rats are placed into a period of forced abstinence from sucrose self-administration simply by keeping them in their home cages for either one or 30 days.

The final step of the procedure is to return rats to the self-administration environment and allow them to respond to cues that were previously explicitly associated with sucrose, self-administration, but not for sucrose itself. Ultimately, the results show how the Vigo of responding for sucrose paired cues varies according to the length of forced abstinence. As we have previously reported, sucrose craving increases or incubates over a month of forced abstinence from sucrose.

Self-administration experiments may be conducted using this procedure to investigate behavioral and neurobiological substrates of the incubation of craving. The main advantage of this technique over existing methods is that the length of time following the final self-administration session can vary. This method can help answer key questions in the field of addiction research, in particular, how the length of a period of forced abstinence affects craving.

The Implications of using this technique extend toward therapy for relapse behaviors. In particular, as a vast majority of individuals have relapsed after an extended period of abstinence. Though this method can provide insight into sugar craving.

It can also be applied to other rewards such as drugs of abuse. This study uses male long Evans rats that are three months old at the start of the study. The rats are housed individually on a 12 hour reverse day night cycle with pelleted chow and water available ad libitum before beginning the procedure weigh all of the experimental and control animals thereafter.

Animals should be weighed each Monday, Wednesday, and Friday for the duration of the study. Lastly, begin water deprivation approximately 20 hours before beginning the sucrose craving study. This will encourage sucrose self-administration on the first day of training.

All training and testing takes place between oh 900 and 1300 hours, and the cohorts are always trained and tested at the same time daily in the testing room. The operant conditioning chambers are enclosed in sound attenuating cabinets with ventilation fans. The chambers used in this study have dimensions of 30 by 20 by 24 centimeters and contain two levers, a stationary lever, and a retractable lever.

Both levers are positioned 10 centimeters from the grid floor. In addition, the chambers contain a tone generator, a white stimulus light above the retractable lever, and a red house light on the opposite wall. To assess locomotor activity, the chambers are equipped with infrared photo beams in these chambers, the front beams are 10.5 centimeters from the side walls and the side beams are each six centimeters from the side walls.

Each beam is 4.5 centimeters above the stainless steel bar floor. Add fresh 10%sucrose solution to the infusion pump located on the top of each chamber. Program the associated software to activate the pump to deliver 0.2 milliliters of 10%sucrose solution into the receptacle to the right of the lever.

Also, using the associated software program the chamber, so that pressing the retractable or active lever also activates a compound stimulus consisting of a tone and the white light. The compound stimulus should last for five seconds and be followed by a 42nd timeout during which time presses on the active lever will be recorded but have no programmed consequence. The stationary or inactive lever is programmed to have no consequence when pressed, but to only record the number of presses.

Essentially, the software should record all presses on both of the levers and the number of infrared beam breaks made during each training session. On the first day of training, place the rat into the operant conditioning chamber for a total of two hours. During this time, the rat is allowed to move freely and may press the retractable lever for delivery of 10%Sucrose solution or the rat may press the inactive stationary lever.

All response and locomotive measures are recorded as time courses in two minute bins, but are usually reported as totals per session. Once two hours have elapsed, remove the rat and return it to the home cage. Ensure that the operant conditioning chamber is thoroughly cleaned to remove any residues, oral factory cues that may disrupt subsequent training sessions.

During the forced abstinence period, the rats remain in the home cage with pelleted chow and water available at libitum. The forced abstinence period can range for as long as necessary. A typical timeframe for incubation of craving studies is one to 30 days.

In addition, other parameters to be tested such as sucrose, satiation, or the effect of environmental enrichment may be administered under control conditions. During this period, two testing paradigms are presented here. The first of which illustrates extinction without cues.

First set up the operant conditioning chamber to replicate the training conditions with the exception that the tone and white light are not activated in response to the active lever being depressed. Program the chamber to run six of these one hour extinction sessions each separated by a five minute period wherein the house light is turned off and the active lever is retracted that run uninterrupted. Then after the forced abstinence period is complete, place the rat into the opera conditioning chamber, allow the rat to respond on the active lever in six one hour extinction sessions.

Behavioral extinction is indicated if active lever responding has been reduced to less than 20 presses per hour in the sixth extinction session. If extinction has not occurred, allow the rat to perform a seventh extinction session. The next session after another five minute delay is the one hour testing session where rats can then press for the tone plus light cue that was initially paired with each delivery of sucrose during training for the second testing procedure extinction with cues, the operant conditioning chamber is set up to replicate the training conditions.

With the exception that sucrose solution is not delivered upon depression of the active lever, the rat to be tested is placed in the operant conditioning chamber for the duration of two hours. Inactive lever pressing in conjunction with beam break data may also be used as a measure of non-specific motor activation. Since motoric versus motivational changes can potentially complicate the interpretation of Q reactivity behavior.

This figure illustrates typical data obtained from a group of 77 normal rats being trained to self-administer sucrose. According to the training paradigm outlined in this protocol, it can be seen that rats respond selectively on the active lever during training. Here, the data obtained from a testing session is shown.

The rats are seen to respond in extinction with the paired queue available extinction with cues. Again, the rats respond selectively on the active lever during testing and inactive responding and locomotor activity show some incubation, although the effect is most robust on active lever responding here, group sizes ranged from 11 to 12 per group, and a significant difference was seen between the day one and day 30 groups with a P value of less than 0.05. This figure shows the effect of sucrose satiation on sucrose Q reactivity after one or 30 days of forced abstinence, some rats received bottles of 10%sucrose in their home cages for the 17 hours immediately prior to testing using the extinction without cues.

Procedure, satiation reduced extinction without cues responding after either period of forced abstinence, but only reduced responding for the sucrose Q alone in rats that only had one day of forced abstinence group sizes are eight to nine per group. The asterisk indicates a significant difference from the day one group and the cross symbol indicates a significant difference from the control group where P is less than 0.05. These results illustrate that one month of environmental enrichment reduced sucrose Q reactivity in rats.

During the forced abstinence period, control rats remained in individual housing while the enriched cohort was moved into large cages with several toys in their environment, including PVC tubing. These rats were also given novel toys every Monday, Wednesday, and Friday. Rats in this study were tested using the extinction without Q'S procedure.

Environmental enrichment reduced incubated extinction without Q responding and responding for the sucrose Q alone behaviors to levels found after only one day of forced abstinence, each group consisted of eight rats. The asterisk indicates significant difference from the day one group and the cross symbol indicates a significant difference from the control group where P equals less than 0.05. Here rats were pretreated with a dose of the dopamine D one antagonist s CH 2 3 3 9 0 on the testing day and tested using the extinction with QS procedure.

It can be seen that systemic s CH 2 3 3 9 0 decrease sucrose seeking more effectively after one day of forced abstinence, each group consisted of eight to 12 rats per group. The asterisk indicates a significant difference from the day one group. The cross symbol indicates significant difference from the control group where P equals less than 0.05.

While attempting this procedure, it's important to remember to balance groups assigned to the forced abstinence conditions based upon their training behaviors. Following this procedure, other methods like drug self-administration can be performed to answer additional questions like the generality of incubation of craving effect After its development. This technique provided a way for researchers in the field of addiction to explore the neural substrates of abstinent dependent changes in craving in rats.

After watching this video, you should have a good understanding of how to conduct a study measuring abstinence dependent sucrose, craving in rats, assessed his rats responding for a Q previously associated with sucrose, self-administration.