Figure 1. Repeated restraint is an effective chronic stressor.

A) Rats were placed in a restraint chamber for 20 minutes, on 7 days over a 9 day period (gray). Control rats (black) experienced a similar degree of daily handling, but were not restrained. Following the last day of restraint, all rats were tested in the elevated plus maze. Some rats were then prepared for electrophysiology experiments. Remaining rats went through a fear conditioning and testing procedure. B) Repeated restraint stress decreased open arm entries (percent of open arm entries control 25.4 ± 2.7%, n=36, stress 15.6 ± 2.1%, n=34, p=0.0006, two-tailed t-test, t=3.58) and the time spent in open arms (percent of time in open arm, control 21.3 ± 2.9%, n=36, stress 12.8 ± 1.8%, n=34, p=0.016, two-tailed unpaired t-test, t=2.49), indicative of increased anxiety-like state. There was no significant change in the total number of arm entries (control 14.3 ± 0.9 arm entries, stress 13.5 ± 0.7 arm entries, p=0.485, two-tailed t-test, t=0.702). Single restraint did not significantly impact EPM exploration. C) Repeated restraint stress increased the weight of adrenal glands, a prototypical measure of the effectiveness of a stressor measured as raw weight (control 22.7 ± 0.7, n=12, stress 28.1 ± 1.2 mg, n=12, p=0.013, two-tailed unpaired t-test, t=2.92), or normalized to body weight (control 0.10 ± 0.005 mg/ kg, stress 0.13 ± 0.006 mg/ kg, p=0.002, two-tailed unpaired t-test, t=3.94). Single restraint did not significantly increase adrenal gland weight (normalized to body weight, control 0.10 ± 0.009, n=6, stress 0.11 ± 0.01, n=7, p=0.57, two-tailed unpaired t-test, t=0.61). * indicates significance at p<0.05.