Prefrontal cortex lesions and sex differences in fear extinction and perseveration

Abstract

Electrolytic lesions of the medial prefrontal cortex (PFCX) were examined using fear conditioning to assess the recall of fear extinction and performance in the Y-maze, open field, and object location/recognition in male and female Sprague-Dawley rats. Rats were conditioned to seven tone/footshocks, followed by extinction after 1-h and 24-h delays, revealing PFCX effects and sex differences during all phases of fear conditioning. In male rats, PFCX impaired 24-h recall of fear extinction to tone, which required the 1-h delay extinction and was not attributed to nonassociative factors. In contrast, sham and PFCX females increased freezing to tone following a 24-h delay, whether or not 1-h delay tone extinction was presented. Moreover, PFCX females failed to extinguish to tone, contrasting to the robust extinction to tone that was observed for sham females, PFCX, and sham males. Also, sex differences were found during acquisition, with sham females acquiring fear conditioning slower than PFCX females. By the last tone-shock presentation, sham and PFCX females showed a slight but significant reduction in freezing to tone relative to those of sham and PFCX males. Of the other behavioral measures, PFCX females maintained exploration of a novel object during object recognition when sham females habituated. PFCX did not influence other behaviors in the remaining tasks. These findings show important sex differences in PFC function, with the PFC influencing the recall of fear extinction in males and contributing to the acquisition and maintenance of fear extinction memory in females, perhaps through altering perseveration.

Figure 1.

Time line of PFCX, behavioral tasks, and brain collection.

Figure 2.

Coronal diagrams of the rat brain showing the extent of the PFCX in female rats that were included in the final analysis. The largest lesion is outlined in black and the smallest lesion is filled in solid black at each level. The infralimbic region was targeted. M2 = secondary motor cortex, Cg1 = anterior cingulate cortex, PrL = prelimbic cortex, MO = medial orbital cortex, VO = ventral orbital cortex, IL = infralimbic cortex, and DP = dorsal peduncular nucleus. The numbers in each diagram represent the distance anterior to bregma in millimeters.

Figure 3.

Coronal diagrams of the rat brain showing the extent of the PFCX in male rats. The largest lesion is outlined in black and the smallest lesion is filled in solid black at each level. The infralimbic region was targeted. M2 = secondary motor cortex, Cg1 = anterior cingulate cortex, PrL = prelimbic cortex, MO = medial orbital cortex, VO = ventral orbital cortex, IL = infralimbic cortex, and DP = dorsal peduncular nucleus. The numbers on the perimeter of each diagram represent the distance anterior to bregma in millimeters.

Figure 4.

Percentage of freezing to tone during fear conditioning and extinction in female and males rats. (A) Comparison of sham and PFCX females in the tone paired with footshock condition. During conditioning, PFCX females acquired freezing to tone faster than did sham rats. Sham and PFCX females froze more to the tone during the first block of extinction on day 2 than they did during the last block of extinction on day 1. During both extinction sessions, sham females decreased freezing to tone while PFCX females failed to extinguish. (B) Comparison of sham females with and without extinction experience on day 1. Sham females with and without an extinction experience on day 1 froze similarly to tone during extinction day 2. (C) Comparison of sham females in the paired condition to the sham females in the unpaired condition. Sham females in the unpaired condition froze less to tone than did the females in the paired condition during extinction on days 1 and 2. (D) Comparison of sham and PFCX males in the tone paired with footshock condition. PFCX male rats froze significantly more to the tone during the first block of extinction on day 2 than did sham males, despite similar freezing to tone during fear conditioning and extinction on day 1. (E) Comparison of the sham males with and without extinction experience on day 1. Day 1 extinction naïve males froze more to the tone during extinction on day 2 than did the sham males given the paired tones. (F) Comparison of sham males in the paired condition to the sham males in the unpaired condition. Unpaired males froze more to tone than did paired males during acquisition of fear conditioning and also showed a nonsignificant tendency to freeze more to tone than did paired males during extinction on day 2. Data for extinction trials was averaged into five blocks of three trials each for analyses and figures. Habit. = habituation trials; Cond. = conditioning trials; Extinct. = extinction trials. Means with different letters represent statistical significance. *P < 0.05. Data represent means±SEM.

Figure 5.

Percentage of freezing to tone following two unsignaled footshocks. (A) Sham females significantly increased freezing to tone following the two unsignaled footshocks. Changes in freezing in PFCX females were minimal, in part, because PFCX females maintained high freezing during extinction. (B) Male rats (sham and PFCX) significantly increased freezing to tone following the two unsignaled footshocks. Data represented as mean±SEM. *P < 0.05 compared with the last extinction trial of the same condition.

Figure 6.

Percentage of freezing to context at designated 30-sec periods during extinction to tone. (A) Diagram outlining the different time points sampled for freezing to context. Triangles represent arbitrary data points of tone presentation. The boxed letter “A” represents the 30 sec before the first tone (T1) presented during extinction on day 1. The boxed letter “B” represents the 30 sec immediately following T1. The boxed letter “C” represents the 30 sec before the last tone presentation (T15) during extinction on day 1. The boxed letter “D” represents the 30 sec before the first tone presentation (T16) on extinction day 2. (B) PFCX females froze significantly more to context than did the sham females after the first tone presentation during extinction (compare context B with context A). Sham and PFCX females froze similarly to context before the first tone presentation during extinction (see context A). (C) As extinction trials progressed on day 1, sham and PFCX females significantly increased freezing to context (see context C), but freezing to context on the second day was low for both groups (see context D). (D) Sham and PFCX males significantly increased freezing to context after the first extinction tone (T1) was presented (compare context B with context A). (E) As extinction trials progressed on day 1, sham males significantly increased freezing to context, while freezing to context was stable for PFCX males (see context C). However, freezing to context on the second day significantly decreased in sham males and remained stable and low for the PFCX males (see context D). Data represent means±SEM. *P < 0.05 compared with context A of the same treatment group. ⁺P < 0.05 compared with context A (collapsed across both sham and PFCX). ^#P < 0.05 compared with context C of the same treatment group.

Figure 7.

Y-maze performance following PFCX. (A) Difference scores during minutes 1 and 2 of Y-maze testing in PFCX and sham female rats were statistically similar. Extended preference for the novel arm was not seen. (B) Difference scores on the Y-maze were statistically similar for sham and PFCX male rats. Extended preference for the novel arm was not seen. Data represent means±SEM.

Figure 8.

Open field, object location, and object recognition. (A) Sham and PFCX females spent more time in the outer zone than in the inner zone of the open field. Since sham and PFCX were statistically similar, then PFCX did not alter anxiety levels. (B) PFCX and sham females spent similar amounts of time with the moved object across the 3 min of testing. Although there was no statistical difference between groups, the discrimination index, preference for the novel object, was positive for both groups in minutes 1 and 2 and reached chance by minute 3. (C) PFCX females explored the novel object more than the familiar object in minutes 1 and 2, while sham females explored the novel object more than the familiar object in minute 1 only. (D) Sham and PFCX males spent more time in the outer zone than in the inner zone of the open field. Since sham and PFCX were statistically similar, then PFCX did not alter anxiety levels. (E) Sham and PFCX males spent similar amounts of time with the moved object across the minutes of testing. (F) Sham and PFCX male rats spent similar amounts of time with the novel object across the minutes of testing. Although there was no statistical difference between groups, the discrimination index, preference for the novel object, was positive for both groups across all 3 min. Data represent means±SEM. *P < 0.05.