Incubation of conditioned fear in the conditioned suppression model in rats: role of food-restriction conditions, length of conditioned stimulus, and generality to conditioned freezing

Abstract

We recently adapted the conditioned suppression of operant responding method to study fear incubation. We found that food-restricted rats show low fear 2 days after extended (10 d; 100 30-s tone-shock pairings) fear training and high fear after 1-2 months. Here, we studied a potential mechanism of fear incubation: extended food-restriction stress. We also studied whether fear incubation is observed after fear training with a prolonged-duration (6-min) tone conditioned stimulus (CS), and whether conditioned freezing incubates after extended training in rats with or without a concurrent operant task. Conditioned fear was assessed 2 days and 1 month after training. In the conditioned suppression method, fear incubation was reliably observed in rats under moderate food-restriction conditions (18-20 g food/day) that allowed for weight gain, and after extended (10 d), but not limited (1 d), fear training with the 6-min CS. Incubation of conditioned freezing was observed after extended fear training in rats lever-pressing for food and, to a lesser degree, in rats not performing an operant task. Results indicate that prolonged hunger-related stress does not account for fear incubation in the conditioned suppression method, and that fear incubation occurs to a longer-duration (6-min) fear CS. Extended training also leads to robust fear incubation of conditioned freezing in rats performing an operant task and weaker fear incubation in rats not performing an operant task.

Figure 1

Fear incubation in rats maintained on mild food-restriction conditions of 20 g per day that lead to daily weight gain either with or without initial deprivation to 85% of free feeding weight. Data are mean±sem test suppression ratios and percent conditioned freezing of rats given 100 30-sec tone-shock pairings and tested 2 days and 1 month later. (A) Fear conditioning acquisition. * Different from Session 1, p<0.05. (B) Mean suppression ratios across the 4 tone trials. (C) Mean conditioned freezing across the 4 tone trials. (D) Trial-by-trial suppression ratios. (E) Trial-by-trial conditioned freezing. Left columns represent the rats whose body weight was decreased to 85% free feeding weight and then maintained on 20 g food per day (n=12). Right columns represent rats maintained on 20 g per day without initially decreasing their body weight to 85% (n=8). * Different from Day 2, p<0.05.

Figure 2

Fear incubation after limited (1 d) or extended (10 d) training with prolonged (6 min) tones: Mean (±sem) test suppression ratios of rats given 5 (1 session) or 50 (10 sessions) 6-min tones paired with shock and tested 2 days or 1 month later. (A) Fear conditioning acquisition. * and # Different from Session 1 or Session 10, respectively, p<0.05. (B) Mean suppression ratios across the first test trial. (C) Mean suppression ratios across the second test trial. (D) Minute-by-minute suppression ratios across the two test trials for the limited-training rats (n=24, 12 per test day). (E) Minute-by-minute suppression ratios across the two test trials for the extended-training rats (n=32, 16 per test day). Left columns represent between-subjects comparisons. Right columns represent within-subjects comparisons. * Different from Day 2, p<0.05; # different from the corresponding limited training group, p<0.05. For the minute by minute comparisons, asterisks represent significant differences found with Tukey’s HSD post hoc tests.

Figure 3

Fear incubation in a conditioned freezing procedure: Mean (±sem) conditioned freezing to 30-sec tones in rats that were not trained to lever press for food and were given 100 30-sec tone-shock pairings in the chambers. (A) Fear conditioning acquisition, 30-sec tone. * and ^# Different from Session 1 or Session 10, respectively, p<0.05. (B) Mean conditioned freezing across the 4 tone trials for the between-subjects comparisons. (C) Mean conditioned freezing across the 4 tone trials for the within-subjects comparisons. (D) Trial-by-trial conditioned freezing for the between-subjects comparison. (E) Trial-by-trial conditioned freezing for the within-subjects comparison. Left column represents rats maintained on unlimited food (n=24, 12 per test day). Right column represents the rats whose body weight was decreased to 85% free feeding weight and then maintained on 18–20 g food per day (n=24, 12 per test day). * Different from Day 2, p<0.05.