Female fear: influence of estrus cycle on behavioral response and neuronal activation

Abstract

Our observation that male rat's innate fear response differed with hormonal status, as well as the higher prevalence of fear and anxiety disorders in human females led to the current investigation of the impact of phases of the estrus cycle on innate fear responding. Female rats in different phases of the cycle were exposed to an innate fear-inducing stimulus (2,5-dihydro-2,4,5-trimethylthiazoline, TMT odor) and monitored for changes in behavior and brain activation. Behavioral data showed freezing responses to TMT were significantly enhanced during estrus as compared to other phases of the cycle. This data was supported by significant increases in pixel intensity in cortical and sub-cortical regions in estrus compared to proestrus and diestrus. Imaging results demonstrated significant increases in brain activation in the somatosensory and insular cortices when comparing estrus to diestrus. There were significant increases in neural activity in the bed nucleus of the stria terminals (BNST) and septum in estrus as compared to proestrus. Additionally, the hippocampus, hypothalamus, olfactory system, and cingulate cortex show significant increases in the estrus phase when compared to both diestrus and proestrus. Taken together, these results suggest that the female's hormonal status may be correlated with alterations in both neuronal and behavioral indices of fear.

Figure 1. Behavioral response to TMT and lemon scent across the estrus cycle

Error bars represent SEM. Time spent freezing, defined as an animal remaining motionless for at least 3 seconds was recorded. The estrus rat froze significantly longer than both the proestrus and diestrus phases (p=0.03). There were no significant differences between estrus phases in response to lemon scent (p>0.05). Additionally there were no significant differences between phases in freezing in response when no scent stimulus was presented (p>0.05).

Figure 2. TMT-elicited brain activation maps (2D) of positive BOLD signal response across the estrus cycle

Each subject was registered or aligned to a fully segmented rat brain atlas using MIVA (Medical Image Visualization and Analysis). Activated voxels (based on a t-test, thresholded at p <0.05) are overlaid onto the corresponding segmented atlas (2D).

Figure 3. Average number of significantly activated voxels across the estrus cycle in cortical areas

Error bars represent SEM (PFC, prefrontal cortex, RSC, retrosplenial cortex). The somatosensory cortex, insular cortex exhibits significant difference between diestrus and estrus (shown by *). The olfactory system and cingulate cortex exhibit significant differences between estrus and both pro and diestrus (represented by **).

Figure 4. Average number of significantly activated voxels across the estrus cycle in sub-cortical areas

Error bars represent SEM. (BNST, Bed nucleus stria terminalis; CPU- Caudate putamen, SN, Substantia nigra) The hypothalamus, hippocampus, BNST, and septum exhibit significant differences between the proestrus and estrus phases (*).

Figure 5. Time course showing signal change across estrus phases for the olfactory cortex

Error bars represent SEM. This figure demonstrates that there were no significant differences (p>0.05) in BOLD response during baseline imaging where no scent was presented.