All protocols were approved by the University of Cincinnati Institutional Animal Care and Use Committee and were consistent with NIH guidelines. Single-housed, male Long-Evans rats (250 g) from Harlan Labs (Indianapolis, IN) were housed in a temperature- and humidity-controlled vivarium with a 12-hour/12-hour light cycle (lights on at 06:00 h). All rats received normal rat chow (LM-485 Mouse/Rat sterilizable diet; Harlan-Teklad, Madison, WI) and water ad libitum
for the duration of the experiment. After a one-week period of acclimation, rats were randomly assigned to drink treatment groups of either 30% sucrose (Sigma Aldrich Co., St. Louis, MO) solution or water. Rats received a 14-d regimen of twice-daily (9:30 and 15:30 h), brief (maximum of 30 minutes), limited (up to 4 mL) access to their assigned drink solution in an additional sipper bottle on the homecage. Rats readily drank the sucrose in amounts near or at the maximum for the duration of the study, whereas the control rats drank little or none of their additional water (data not shown, see [10
] for typical intake). Drink treatment terminated on Day 14, after which rats no longer received access to their respective experimental drink solution. To test persistence of the sucrose effects, cohorts of animals were killed 1, 6, and 21 days after the snacking paradigm ended (corresponding to days 15, 20 and 35 after commencement of the sucrose delivery (see ). Groups of animals killed at each time point included: 1) Water - No restraint stress (n=12), 2) Sucrose - No restraint stress (n=12), 3) Water - With restraint stress (n=12), and 4) Sucrose - With restraint stress (n=13) (a total of 147 rats in the study). The `no restraint stress' groups did not receive a stress challenge, and were injected with pentobarbital and perfused with 0.9% saline followed by 4% paraformaldehyde for collection of brains. The `with restraint stress' groups received a 20-min restraint stress challenge and blood samples were taken by tail clip at 0, 20, 40, and 60 min after the onset of stress. Briefly, rats were placed into well-ventilated restraint tubes and 0-min tail clip blood samples (200 μl) were quickly collected into chilled tubes containing EDTA. The 0-min sample was completed in less than 3 min from first handling each rat's cage, thereby ensuring plasma ACTH and corticosterone levels that were reflective of the basal, unstressed state [17
]. Rats remained in the restrainers for 20 min, with a second tail blood collection occurring immediately prior to their removal from the restraint tubes (i.e., 20-min after the onset of restraint). At 40 and 60 min after the initiation of restraint, the rats were briefly returned to the restraint tubes (< 3 min) for collection of 40- and 60-min blood samples. It took less than 3 min to collect each post-stress blood sample. Blood samples were centrifuged (3000 g, 15 min, 4° C) and plasma was stored at −20° C until measurement of plasma corticosterone levels via radioimmunoassay (Corticosterone Double Antibody 125
I RIA Kit, MPBiomedicals, Solon, OH). Sipper “snack” intake, food intake, and body weight were monitored throughout the experiment. In this paradigm, sucrose rats typically decrease their chow intake isocalorically, resulting in no effect on body weight or fat depot weights [10
Brains were prepared for immunohistochemistry as described previously [18
]. Total FosB protein (FosB/deltaFosB) was detected by free floating immunocytochemistry using standard protocols with a rabbit polyclonal primary antibody against a protein corresponding to amino acids 75–100 of the human FosB/deltaFosB (FosB (H75) 1:300 from Santa Cruz Biotechnology (Santa Cruz, CA)). Western blot analysis with this FosB/deltaFosB antibody immunoreacts with two bands, one at 35–37 kD and one at 45 kD corresponding with truncated deltaFosB (a truncated splice variant of the full length FosB protein) and the full length FosB protein respectively [19
]. Primary antibody was detected using biotinylated goat anti-rabbit IgG (1:500 Vector Labs Burlingame, CA), incubated with avidin-horseradish peroxidase complex (1:500; ABC Elite Kit, Vector Laboratories), and reacted with 0.02% diaminobenzidine (DAB, Sigma-Aldrich, St. Louis, MO) for 15 minutes, resulting in a brown reaction product. Sections were mounted onto slides, dehydrated in a graded ethanol series, cleared with xylene, and coverslipped with DPX. An observer blind to the treatment group assignments counted positively-stained neurons in the BLA and NuAc using Image J software (NIH).
The number of rats used in these studies was based on a priori
power analyses using expected differences and variation based on our preliminary hormone and immunocytochemistry studies. Differences in the food intake, body weight and hormone data were determined using repeated measures two-way ANOVA. If group differences were present, then Fisher's least significant comparisons procedure was used to determine specific planned pairwise comparisons; no further adjustments were made to control for the experimentwise error rate. Differences in the numbers of FosB/deltaFosB-labeled cells between water and sucrose groups at each time point were determined using separate one-tailed t-tests based on the a priori
hypothesis that sucrose snacking will increase synaptic plasticity and synaptic activity. Outliers were removed only if they differed from the mean by more than 1.96 times the standard deviation and they were outside the lower or upper quartiles by more than 1.5 times the interquartile range [20
]. Data are shown as means ± SEM. Statistical significance was taken as p < 0.05.