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Reversal learning and experimenter-administered chronic intermittent ethanol exposure in male rats
Kimberly A. Badanich1, Ph.D., Mackinzie E. Fakih1, Tatyana S. Gurina2, Emalie K. Roy1, Jessica L. Hoffman, M.A. 2, Adriana R. Uruena-Agnes, Ph.D. 2, Cheryl L. Kirstein, Ph.D2,3.
1. Department of Psychology, University of South Florida Sarasota-Manatee, Sarasota FL, 34243
2. Department of Psychology, University of South Florida, Tampa FL, 33620
3. Department of Physiology and Molecular Pharmacology, University of South Florida College of Medicine, Tampa FL, 33620
Address correspondence to:
Kimberly A. Badanich, Ph.D.
University of South Florida Sarasota-Manatee
8350 N. Tamiami Trail, B322, Sarasota FL, 34243
email: ; Phone: 941-359-4451; Fax: 941-359-4489
Caption: Information in this Supplementary Materials section includes a detailed description of the behavioral testing apparatus and a complete explanation of behavioral flexibility procedures.
Supplementary Materials: Online Resource 1
Testing Apparatus and Materials:The testing arena for assessing performance in discrimination tasks was a large rectangular clear plastic storage bin (60 x 43 x 33 cm) with a plastic lid (Sterilite Corporation, Townsend, MA). The arena was divided into three compartments: a start area, and two test chambers. A removable divider was used to split the arena in half, and the front half was designated as the start area. The remaining half of the arena contained an opaque divider placed perpendicular to the shortest wall of the arena to create the two test chambers (30 x 20 cm). During discrimination trials, a rodent food bowl (7.5 x 7.5 x 3.75 cm; PetSmart, Phoenix, AZ) was placed in the test chamber on each side of the divider. Both bowls were filled with rodent bedding and thoroughly mixed with different odors during discrimination trials. Digging media was obtained from the animal facility at The University of South Florida (Tampa, FL), while all odors were obtained from local grocery stores.
Behavioral Flexibility: Following the last treatment exposure, rats were gradually food restricted to 85% of their baseline body weightto motivate rats to search for a food reward in the behavioral flexibility task. Baseline body weights averaged 318+ 6 g for control rats and 332 + 8 g for CIE rats with no statistical group differences in body weight. Two days prior to behavioral flexibility testing, rats were habituated to the testing arena. Two rodent food bowls each filled with the same novel bedding (Tek Fresh) were placed inside the testing cage. Each bowl was baited with several pieces of Honey Nut Cheerios. To promote rats digging through the entire bowl’s contents, food rewards were placed near the bottom of the bowl, mixed into the bedding, and placed on top of the bedding.Rats were allowed to explore the testing arena and baited food bowls for 30 minutes.
On behavioral flexibility testing day, a simple discrimination training task was used to facilitate rats digging to the bottom of the correct bowl by successively increasing the depth of the buried food reward for 3 consecutive trials. During this training period, two rodent bowls were each filled with bedding (alpha-dri) and scented with different odors (garlic powder or ground cinnamon). Bedding in both bowls were additionally mixed with finely ground Honey Nut Cheerio powder to mask the scent of the food reward and to prevent rats from using the odor of the Cheerio to locate the reward. For the first trial, the food reward (1/2 Honey Nut Cheerio Loop) was placed on top of the bedding in one of the scented bowls (counterbalanced for garlic powder or ground cinnamon). The rat was placed inside the testing arena and allowed to inspect both bowls and locate the food reward. The second and third trials were conducted in the same manner except for the placement of the food reward. For the second trial, the food reward was placed further into the bedding and for the third trial the food reward was buried deep in the bedding. After the food reward was located and consumed on all 3 trials, the rat immediately proceeded with the simple discrimination learning task. Throughout the rest of the experiment (simple discrimination and reversal learning), the food reward remained buried at the same level as the third simple discrimination training trial.
The behavioral assay used in these experiments is a modified version of an attentional set-shifting task in rodents (Birrell and Brown, 2000). To examine the effects of CIE exposure on behavioral flexibility, rats were trained to complete two odor discrimination tasks: simple discrimination and reversal learning. Simple discrimination was performed as a continuation of the training task. Rats learned to discriminate between two odor-cued digging bowls, one of which was baited with a buried food reward (1/2 of a Honey Nut Cheerio Loop). Garlic powder and ground cinnamon were used as odor cues and alpha-dri was used as the bedding (Bissonette et al, 2008; Badanich et al, 2011). There were no significant preferences or aversions towards either odor. Each trial began by placing a rat in the start area of the testing arena and allowing the rat to make a choice between two odor cued bowls, one of which contained the buried food reward (e.g., baited garlic bowl vs. unbaited cinnamon bowl). The first four trials were exploratory and allowed the rat to dig in either of the bowls until the food reward was retrieved. Following these exploratory trials, rats were only allowed to dig in one bowl. If the rat dug in the unbaited bowl first, the rat was allowed to dig and determine no food reward was available. The rat was then removed from the testing chamber before it could dig in the other bowl, the trial was terminated, and recorded as an error.If the rat dug in the baited bowl first and obtained the food reward, the rat was allowed to eat the reward, the trial was terminated, and recorded as a correct choice. At the end of each trial, rats were returned to the homecage and allowed to rest for 30 sec and the next trial immediately started. Once rats reached a criterion of six consecutive correct trials, rats immediately proceeded to the reversal learning task where testing occurred in the same manner except that the previously unbaited bowl now contained the buried food reward (e.g., baited cinnamon bowl vs. unbaited garlic bowl). Once a criterion of six consecutive trials was met for the reversal learning task, rats were returned to their home cage and given free access to food. During simple discrimination and reversal learning, the odor concentration was kept constant between trials by mixing extra odor into the bedding after every few trials. Placement of the baited bowl in the testing apparatus was pseudo-randomized to prevent a side bias. Treatment groups were counterbalanced for direction of reversal by having some rats switch from garlic powder to ground cinnamon discriminations while others switched from cinnamon to garlic powder discriminations.