Sex and estrous phase influence functional connectivity during fear extinction
Description
We hypothesized that sex and estrous phase during fear extinction would influence neural activation, activity of catecholaminergic regions and overall functional connectivity during fear extinction. During fear extinction, proestrus and estrus females froze less than males and metestrus and diestrus females, exhibited reduced cFos in the prelimbic cortex and cingulate gyrus relative to males, had greater activity of the dorsal and ventral medial amygdala compared to males, displayed elevated cFos in ventral tegmental dopamine neurons compared to both groups, and demonstrated high overall functional connectivity distinguished by unique engagement of the dorsolateral striatum as a hub. Together, these findings reveal insight into how sex and estrous phase alter the neural processing of fear extinction and substantiate the dorsolateral striatum as a key region mediating fear extinction in proestrus and estrus females. This dataset contains behavioral freezing and locomotor data from cued fear conditioning and extinction tests, cFos data from the prefrontal cortex, nucleus accumbens, hippocampus, amygdala and dorsal striatum and cFos/tyrosine-hydroxylase co-expression in the ventral tegmental area and locus coeruleus following fear extinction in male and female adult Long Evans rats, as well as all R codes used to analyze the data.
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Vaginal lavages were collected for 7 days prior to the start of behavioral testing, as well as ~2 hours before and immediately after each behavioral test for verification. A blunt-tipped eye dropper containing ~0.5 mL sterile-filtered 0.2% PBS-Brij solution (Brij 35 Solution 30%) was used to gently flush the vagina. Samples were visualized under a 20X objective lens (Olympus BX53). Behavior was recorded and scored by Noldus EthoVision software and averaged with the scores of a human experimenter. Locomotor activity prior to the first CS presentation during extinction was also quantified using Noldus software. For fear conditioning, rats were placed into custom rectangular conditioning chambers (context A; 20” W x 10” D x 12” H) with shock grid floors (Coulbourn Instruments, Allentown, PA) housed within individual sound-attenuating cabinets illuminated by red lights. Rats were allowed 3min to explore the context before receiving 4 auditory CS presentations (10 second, 80 dB, 2 KHz), each co-terminating with a 1 second, 0.8-mA foot shock US, delivered on a 1min ITI. Rats remained in the conditioning chambers for 1min after the last shock before being transported back to their home cage. Conditioning chambers were cleaned with water between subjects. 24hr later, fear extinction took place in context B, where rats were placed into Plexiglass chambers that were either rectangular (15” W x 15” D x 20” H) with a textured floor or triangular (15” sides x 20” H) with a smooth floor. Extinction sessions were conducted in the same sound-attenuating cabinets, scented with vanilla, illuminated with bright white lights, and equipped with a fan to provide ventilation and background noise. There was a 3min exploration period before the CS was presented 20 times (1min ITI) without the foot shock. Rats remained in the chambers for 1min after the final CS presentation before returning to their home cages. Extinction chambers were cleaned with 10% ethanol between sessions. 90min after extinction, rats were anesthetized with pentobarbital and transcardially perfused with saline followed by 4% paraformaldehyde in 0.1 M phosphate buffer. Brains were post-fixed in 4% paraformaldehyde for 12hr, then frozen and stored at -80°C. Brain sections contained the prefrontal cortex, striatum, hippocampus/amygdala, substantia nigra/ventral tegmental area and the locus coeruleus. Rabbit anti-cFos was used at a dilution of 1:5000. Images were captured at 20X on an Olympus BX51 microscope in 4-6 hemispheres/rat using ImageJ. A 106μm x 80μm rectangle was drawn around the regions of interest, and cFos, tyrosine-hydroxylase and double-labeled neurons were quantified.
Institutions
- University of Colorado Denver
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Funders
- National Institutes of HealthUnited States