A General Method for Evaluating Incubation of Sucrose Craving in Rats

1. Animal Subjects

1. Male Long-Evans rats are 3 months old at the start of a study. Rats are Simonsen-derived (Gilroy, California, USA) bred in the Western Washington University vivarium and are housed individually on a 12-h reverse day/night cycle (lights off at 0700) with Purina Mills Inc. Mazuri Rodent Pellets (Gray Summit, MO, USA) and water available ad libitum.
2. All training and testing takes place between 0900-1300 with cohorts of rats always trained and tested at the same time daily.
3. Rats are weighed each Monday, Wednesday, and Friday for the duration of the study.
4. Immediately prior to the training phase, rats are deprived of water for approximately 20 h to encourage sucrose self-administration on the first day of training.

2. Behavioral Procedures

All operant behavioral procedures use standard operant conditioning chambers (Med Associates Inc., St. Albans, VT, USA) with an infusion pump (Razel Scientific Instruments, St. Albans, VT, USA) located on top of each operant conditioning chamber. Operant conditioning chambers are enclosed in sound-attenuating cabinets with ventilation fans (Western Washington University, USA). All experimental conditions and data collection are controlled by Med PC IV software (Med Associates Inc., St. Albans, VT, USA).

Operant conditioning chambers (30 x 20 x 24 cm) contain two levers (one stationary and one retractable), a tone generator, a white stimulus light above the retractable lever, and a red house light on the opposite wall. The infusion pump delivers sucrose into a reward receptacle to the right of the active lever. Levers are 10 cm from the grid floor. Four infrared photobeams crisscross each chamber. The front beams are each 10.5 cm from the side walls and the side beams each 6 cm from the side walls. Each beam is 4.5 cm above the stainless steel bar floor. The beams are set to count the number of complete breaks. The total number of beam breaks are recorded during Training and craving Testing.

With the exception of water restriction prior to the first Training session, rats are provided food and water ad libitum both in home cages and in the operant conditioning chambers. Chambers are in a dedicated test room that, as with the vivarium, is on a reverse light cycle. Rats are transported in home cages between the rooms on a cart that is covered with light blocking fabric. At the end of each session, rats are returned to home cages.

There are three phases to the typical experiment: Training, Forced Abstinence, and Testing. Testing is the session wherein craving is assessed. We assess craving in two general testing procedures, depending on the study. These two procedures are described in sections 2.3.1 and 2.3.2.

1. Training
   1. In the typical experiment, rats spend 2 h/day for 10 consecutive days in operant conditioning chambers where they are allowed to press the retractable (active) lever for a 0.2 ml delivery of 10 % sucrose solution into the receptacle to the right of the lever.
   2. This response also activates a compound stimulus consisting of a tone (2 kHz, 15 dB over ambient noise) and the white light. The compound stimulus lasts for 5 s and is followed by a 40-s time out, during which presses on the active lever are recorded but have no programmed consequence.
   3. A response on the inactive (stationary) lever has no programmed consequence, but presses are recorded. The numbers of beam breaks are recorded during Training and Testing. All response and locomotor measures are recorded as time courses (2 minute bins), but are usually reported as totals per session.
2. Forced Abstinence. Rats remain in home cages for the duration of forced abstinence. The typical forced abstinence periods we use are to have rats tested the day immediately following the final day of Training (Day 1 of forced abstinence) or 30 days following the final day of Training (Day 30 of forced abstinence).
3. Testing
   1. Extinction without cues. In this Testing procedure, rats are first allowed to press the active lever in conditions identical to Training with the exception that the response has no consequences and the session duration is 1 h. Rats experience at least six of these extinction sessions on the Test day. Each extinction session is separated by a 5 minute period where the active lever is retracted and the house light is turned off. “Extinction” is indicated if active lever responding has been reduced to less than 20 presses/h. Some rats require a seventh session. The next session (after another 5 minute delay) is the Testing session where rats can then press for the tone+light cue that was initially paired with each delivery of sucrose during Training.
   2. Extinction with cues. In this abbreviated Testing procedure, rats are allowed to press the active lever in conditions identical to Training except that sucrose is not delivered.
4. Inactive lever pressing is used as a measure of lever discrimination. It is also, along with photobeam breaks, used as a measure of non-specific motor activation. Motoric vs. motivational changes complicate the interpretation of craving behavior. The inactive lever responding and photobeam break data are useful in clarifying motor vs. motivation contributions to craving, as are comparison groups in pharmacological studies where rats responding at a high rate for sucrose or another food are challenged with a potential craving affecting compound (e.g. ^7,8).

3. Representative Manipulations to Affect Sucrose Craving after Variable Periods of Forced Abstinence

The general method described here provides a way to probe the behavioral and neurobiological determinants of craving after variable periods of forced abstinence. Described here are methods related to Representative Results provided in section 4.

1. Satiation. Some rats received bottles of 10 % sucrose in their home cages for the 17 h immediately prior to Testing 8. Rats in this study were tested using the Extinction without cues procedure.
2. Environmental Enrichment. During the forced-abstinence period, some rats remained in individual housing (Controls) while others were moved into a large (36 ” width x 20 ” depth x 40 ” height; Quality Cage Products, Portland, OR, USA) cage with three cohorts (Enriched). The Enriched animals had several toys in their environment including PVC tubing and were also given novel toys every M,W,F ¹⁰. Rats in this study were tested using the Extinction without cues procedure.
3. Pharmacological challenge. On the Testing day, rats were pretreated with a dose of the dopamine D1 antagonist SCH 23390. Rats in this study were tested using the Extinction with cues procedure.

4. Representative Results:

Figure 1 Representative Training and Testing data for an incubation of sucrose craving experiment.

Data points indicate means ± SEMs. Group size for Figure 1a is 77 rats and for Figure 1b group sizes ranged from 11-12 per group. * significant difference from Day 1 group, p < 0.05. This figure is adapted from data in ⁸ with permission of Springer.

Figure 2 Effect of sucrose satiation on sucrose craving after 1 or 30 days of forced abstinence. Group sizes are 8-9 per group. * significant difference from Day 1 group, † significant difference from Control group, p < 0.05. This figure is adapted from ⁹ with permission of Elsevier.

Figure 3 One month of environmental enrichment reduced sucrose craving. Group sizes are each 8 rats. * significant difference from Day 1, † significant difference from Control group, p < 0.05. This figure is adapted from ¹⁰ with permission of Wolters Kluwer Health.

Figure 4 Systemic SCH 23390 decreased sucrose craving more effectively after one day of forced abstinence. Group sizes are 8-12 per group. * significant difference from Day 1 group, † significant difference from 0 dose group, p < 0.05. This figure is adapted from ⁸ with permission of Springer.

1. Figure 1 indicates representative Training and Testing data for rats self-administering sucrose (Figure 1 TOP) and then responding in Extinction with the sucrose-paired cue available (Figure 1 BOTTOM). As shown, rats respond selectively on the active lever during Training and during Testing. Interestingly, inactive responding and locomotor activity show some incubation, although the effect is most robust on active lever responding.
2. Figure 2 shows the effect of satiation with sucrose on sucrose craving after 1 or 30 days of forced abstinence. Satiation reduced Extinction without cues responding after either period of forced abstinence, but only reduced responding for the sucrose cue alone in rats that had only one day of forced abstinence.
3. As shown in Figure 3, environmental enrichment reduced incubated Extinction without cue responding and responding for the sucrose cue alone behaviors to levels found after only one day of forced abstinence.
4. Figure 4 depicts an abstinence-dependent effect of SCH 23390 on Extinction with cues responding. SCH 23390 was most effective at reducing sucrose craving after 1, vs. 30, days of forced abstinence.