2.3 Playback trials

The evening before playback trials, females were individually housed in smaller test cages with access to multiple perches, food, grit, and water. For playback trials, females were rapidly transported one at a time to an acoustically isolated testing room and exposed in individual trials to either a unique 30 min playlist of several different male house sparrows singing with a few calls (n = 8 control females, n = 8 estradiol-treated females) or a unique mix of calls from local predators: Barn owls (Tyto alba), Eastern screech owls (Megascops asio), Great horned owls (Bubo virginianus), Broad-winged hawks (Buteo platypterus), Cooper’s hawks (Accipiter cooperii), Red-shouldered hawks (Buteo lineatus), Red-tailed hawks (Buteo jamaicensis), American kestrels (Falco sparverius), Loggerhead shrikes (Lanius ludovicianus), and Mississippi kites (Ictinia mississippiensis) (n = 8 control females, n = 9 estradiol-treated females). Therefore, treatment groups were as follows: n = 8 for Empty + Predator, n = 8 for Empty + Sparrow, n = 9 for Estradiol + Predator, and n = 8 for Estradiol + Sparrow. A female sparrow heard, on average, 31.7 ± 1.5 (range: 30–35) different male house sparrow sound files during conspecific playbacks or 58.5 ± 2.9 (range: 53–63) different predator sound files during predator playbacks. All 11 predator species were represented at least once in each predator playback playlist, and each had on average five different songs or calls per sound file. Sound files were obtained from the Macaulay Library (Cornell Lab of Ornithology, Ithaca, NY, United States) and the Borror Laboratory of Bioacoustics (The Ohio State University, Columbus, OH, United States). Each bird heard a unique playlist of different sound files played in a randomized order. No other sparrows were present in the testing room during trials. Loudness was standardized to 60 dBA from bird to speaker using a sound level pressure meter and we video recorded female behavior during the playback trials using a Logitech C615 portable webcam. A researcher began recording videos after transporting the individual cage, and then started the playback and immediately exited the room.

Previous work in songbirds has shown that IEG proteins peak ∼90 min after stimulus exposure (Goodson et al., 2005). Therefore, after the 30 min playback behavior trials, females were transported to a dark quiet room for 60 min before being deeply anesthetized with ketamine (80 mg/kg) and xylazine (20 mg/kg), doses shown to be appropriate for house sparrows (Muresan et al., 2008). Once animals were in a surgical plane of anesthesia, they were transcardially perfused with ice-cold heparinized saline and 0.1 M phosphate buffer containing 4% paraformaldehyde sequentially. Euthanasia was confirmed using rapid decapitation and brains extracted.