Estradiol shifts interactions between the infralimbic cortex and central amygdala to enhance fear extinction memory in female rats

Abstract

There is growing evidence that estradiol (E2) enhances fear extinction memory consolidation. However, it is unclear how E2 influences the nodes of the fear extinction network to enhance extinction memory. This study begins to delineate the neural circuits underlying the influence of E2 on fear extinction acquisition and consolidation in female rats. After fear conditioning (day 1), naturally cycling female rats underwent extinction learning (day 2) in a low-E2 state, receiving a systemic administration of either E2 or vehicle prior to extinction training. Extinction memory recall was then tested 24 hr later (day 3). We measured immediate early gene c-fos expression within the extinction network during fear extinction learning and extinction recall. During extinction learning, E2 treatment increased centrolateral amygdala c-fos activity and reduced lateral amygdala activity relative to vehicle. During extinction recall, E2-treated rats exhibited reduced c-fos expression in the centromedial amygdala. There were no group differences in c-fos expression within the medial prefrontal cortex or dorsal hippocampus. Examining c-fos ratios with the infralimbic cortex (IL) revealed that, despite the lack of group differences within the IL, E2 treatment induced greater IL activity relative to both prelimbic cortex and central amygdala (CeA) activity during extinction memory recall. Only the relationship between IL and CeA activity positively correlated with extinction retention. In conclusion, E2 appears to modify interactions between the IL and the CeA in females, shifting from stronger amygdalar modulation of fear during extinction learning to stronger IL control during extinction recall. © 2016 Wiley Periodicals, Inc.

Keywords: PTSD; anxiety; c-fos; central amygdala; centromedial amygdala; estradiol; estrous cycle; fear; fear extinction; female; infralimbic cortex; sex differences; ventromedial prefrontal cortex.

Figure 1

Timeline of experiment and effect of estradiol on freezing behavior. All animals underwent a 3-day behavioral protocol. Habituation and fear conditioning took place on day 1, fear extinction training on day 2, and extinction recall on day 3. Extinction training occurred during the metestrus phase of the estrous cycle, and subcutaneous vehicle or estradiol injections (INJ) were performed 30 minutes (30”) prior to the start of the session. The POST-EXT group was sacrificed 1h after the extinction session, while the POST-REC group was sacrificed 1h after the recall session. Estradiol does not influence freezing behavior during fear conditioning and extinction, but does enhance extinction recall. POST-EXT and POST-REC groups (Vehicle, n=28; Estradiol, n=24) were combined for both the conditioning and extinction phases. The extinction recall phase is represented by the POST-REC group (Vehicle, n=14; Estradiol, n=14). *p≤0.05.

Figure 2

Photomicrographs of c-fos stained sections and average c-fos positive cell counts. The first column consists of images containing the regions of interest (ROIs) bordered by dashed lines. The second column displays the c-fos positive cell counts for the POST-EXT group, and the third column for the POST-REC group. A. Quantification of c-fos positive expression within the PL and IL regions of the mPFC as outlined by the dashed lines in the image are represented in the graphs. There were no significant effects of drug in the mPFC (POST-EXT: Vehicle, n=8; Estradiol, n=8; POST-REC: Vehicle, n=8; Estradiol, n=9). B. C-fos expression was measured within the LA, BLA, CeL, and CeM. During extinction, estradiol increased CeL activity, while reducing LA and BLA activity (Vehicle, n=8; Estradiol, n=8). During recall, estradiol reduced neuronal activation in the CeM and LA (Vehicle, n=8; Estradiol, n=9). C. Hippocampal c-fos expression was counted for the dorsal subregions: DG (POST-EXT: Vehicle, n=8; Estradiol, n=8; POST-REC: Vehicle, n=5; Estradiol, n=9), CA1 (POST-EXT: Vehicle, n=8; Estradiol, n=8; POST-REC: Vehicle, n=6; Estradiol, n=9), and CA3 (POST-EXT: Vehicle, n=7; Estradiol, n=7; POST-REC: Vehicle, n=6; Estradiol, n=7). There were no differences in c-fos activity with estradiol administration. PL=prelimbic; IL=infralimbic; LA=lateral amygdala; BLA=basolateral amygdala; CeL=centrolateral amygdala; CeM=centromedial amygdala; DG=dentate gyrus; CA1=cornus ammonis; CA3=cornus ammonis. White bars represent vehicle-treated animals. Black bars represent estradiol-treated animals. *p≤0.05.

Figure 3

Photomicrographs of the centrolateral amygdala comparing c-fos expression with vehicle vs. estradiol treatment in POST-EXT. The ROI for the CeL is zoomed in to 20X magnification (right column) from 4X magnification (left column) to visualize the pronounced increase in c-fos immunoreactivity following estradiol administration.

Figure 4

IL/PL c-fos activity ratio of POST-EXT and POST-REC groups and correlation with extinction retention index. IL/PL c-fos activity ratio is significantly higher in the estradiol group during extinction recall, but not during extinction learning. The IL/PL c-fos ratio is not correlated with how much extinction was conserved (ERI). White bars represent vehicle-treated animals. Black bars represent estradiol-treated animals. White dots in the correlation graphs represent vehicle-treated animals, and black dots represent the estradiol-treated animals. POST-EXT: Vehicle, n=8; Estradiol, n=8. POST-REC: Vehicle, n=8; Estradiol, n=9. *p≤0.05.

Figure 5

C-fos activity ratios with the IL and subregions of the amygdala and their correlations with extinction retention index. A. Estradiol did not alter the IL/CeM c-fos ratio in the POST-EXT group; however, it increased it in the POST-REC group. The IL/CeM ratio was positively correlated with ERI. B. The IL/CeL c-fos ratio was significantly reduced in the POST-EXT group but significantly increased in the POST-REC group. The IL/CeL c-fos ratio was also positively correlated with ERI. C and D. There were no differences in IL/BLA and IL/LA c-fos ratios with estradiol treatment, and similarly, no correlations were found between both ratios with ERI. White bars represent vehicle-treated animals. Black bars represent estradiol-treated animals. White dots in the correlation graphs represent vehicle-treated animals, and black dots represent the estradiol-treated animals. POST-EXT: Vehicle, n=8; Estradiol, n=8. POST-REC: Vehicle, n=8; Estradiol, n=9. *p≤0.05.

Figure 6

No extinction control behavior and c-fos. A. A separate group of animals underwent the same 3-day protocol as the POST-REC group but did not receive extinction training following the vehicle (n=6) or estradiol (n=6) injection. There was no difference between drug treatments in percent freezing in fear conditioning or recall. INJ= vehicle or estradiol injection. B. Estradiol did not influence neuronal activation in any subregion of the mPFC, amygdala, or dorsal hippocampus. C. In contrast to the POST-REC group, the no extinction control group did not exhibit any differences in c-fos activity ratios with the IL (IL/PL, IL/CeM, IL/CeL). White bars represent vehicle-treated animals. Black bars represent estradiol-treated animals.

Figure 7

Estradiol modulation of fear extinction memory and circuitry. Based on the findings in this study, we propose that estradiol’s modulatory effects on fear extinction memory consolidation are driven by the IL-amygdala circuit of the network. In the case of the metestrus females that received vehicle injections and thus had less estradiol during extinction training, the glutamatergic connections (blue) between the IL and ITC and GABAergic projections (black) to the CeA are weak, as indicated by the dashed lines. This leads to greater fear expression during fear extinction recall (red). Estradiol administration strengthens these connections (indicated by the solid lines) to reduce fear expression during recall (green) and enhance extinction retention. More specifically, it appears that with increasing estradiol levels, there is a shift in neuronal activation from stronger CeA to IL activity (red) to stronger IL to CeA activity (green). This shift is correlated with more and less fear during extinction recall, respectively. PL=prelimbic cortex; IL=infralimbic cortex; BLA=basolateral amygdala; CeL=centrolateral amygdala; CeM=centromedial amygdala; ITCv=ventral intercalated cells of the amygdala; CeA=central amygdala; E2=estradiol.