Freezing response in Context B in Experiment 2. Data are presented as mean + SEM percent time. The x-axes indicate the estrous cycle phase classification at the time of trauma or no trauma exposure in Context A on experimental Day 3 (A,B), or at the time of mild stress exposure on Day 4 (C,D). Individual data points represent the estrous phases: M-D = solid triangles, P-E = open circles. (A) During the 5 min single shock session in Context B, rats in the 15-shock group exhibited significantly greater freezing than those in the 0-shock control group. Regardless of shock group, freezing increased from the pre- to post-shock timepoint (each consisting of 32 sec windows averaged across four samplings). However, an interaction between Day 3 estrous cycle phase and timepoint revealed that P-E rats (open circles) deviate from this pattern, showing no significant change in freezing behavior from pre- to post-shock. (B) During the 12 min SEFL Test 1 (left panel) and Test 2 (right panel), both conducted in Context B, the 15-shock groups showed elevated freezing compared to 0-shock controls. These differences persisted despite an overall reduction in freezing behavior from Test 1 to Test 2. An interaction between cycle phase, shock group, and test day indicated that this reduction in the 15-shock group was driven by P-E females (open circles). Additionally, a cycle phase difference (M-D vs. P-E) was apparent only at Test 1 (24 h test day), with P-E rats freezing significantly more than M-D within the shock group. (C) Reclassifying cycle phase based on Day 4 (the day of single shock) revealed similar main effects as in A. However, a significant cycle phase, group, and timepoint interaction emerged: P-E rats in the 15-shock group showed a significant increase from pre- to post-shock, whereas M-D rats did not, regardless of shock history. (D) When using Day 4 mild stress classifications, no effect of cycle phase was observed during the SEFL tests. Rats in the 15-shock group froze more than controls across both test days, and freezing significantly decreased from Test 1 to Test 2. (*) P < 0.05.
