Overview of experimental design

Wild free-living adult chickadees of both sexes were live-captured during the non-breeding season and housed in flocks outdoors for 1 week prior to experimental trials. To experimentally test if predator-induced fear has enduring effects, individuals were housed solitarily in acoustic isolation chambers and exposed for 2 days to audio playbacks of the vocalizations of either predators (treatment group) or non-predators (control group)23,25,26,27,29,30,31, and then housed again in flocks outdoors for 7 days, during which time they were not exposed to any further experimental cues, but were instead exposed to natural sights and sounds and social interactions. Enduring effects on behaviour were then assessed in one set of individuals and effects on the brain were evaluated in a separate set, to ensure that effects on the brain were attributable to exposure to the cues heard 7 days previously and not the cues used in assessing behaviour.

To assess effects on behaviour, individuals were again housed solitarily in acoustic isolation chambers, and all were exposed for 15 minutes to playbacks of conspecific alarm calls (‘high zee’ calls33,34), a signal which, like hearing predator vocalizations, alerts the hearer to a predator danger33,34, but in the context of the experiment entailed individuals hearing cues (chickadee vocalizations) distinct from those they were exposed to 7 days previously. More fearful reactions in those individuals that previously heard predators, vs. non-predators, could thereby be interpreted as reflecting an enduring memory of fear, i.e., a heightened sensitivity to predator danger19, rather than a memory of the specific cues heard 7 days previously. To gauge the fearfulness of their reactions we measured the time each individual remained ‘vigilant and immobile’ (i.e., ‘freezing’) upon first hearing the alarm calls; ‘freezing’ being an anti-predator behaviour demonstrated in almost every type of animal15,16,33, which is commonly measured in animal model studies of PTSD19.

To determine if there were enduring effects on the brain we assayed ∆FosB expression to identify long-term neuronal activation30,31,32 in the avian homologues of the two brain regions most pertinent to PTSD in humans, the amygdala and hippocampus12,19,20,51,52. The amygdala is responsible for fear processing and the acquisition and expression of fear memories, as demonstrated by lesioning studies on laboratory rodents, and the fact that people with a damaged amygdala report not feeling fearful in response to a variety of fear-provoking stimuli, including life-threatening traumatic events51,52,53,54. The hippocampus is involved in forming declarative, episodic and spatial memories51,52. Whereas amygdala activation generally increases with the intensity of a trauma, the duration and magnitude of effects on the hippocampus can be complex and vary with what is measured51,52. ∆FosB is a protein produced by the FosB gene. It is a transcription factor, meaning it modifies the transcription of other genes. Whereas most transcription factors degrade within hours, ∆FosB is unusually stable and can continue to have effects for weeks, effects which include promoting resistance to the deleterious consequences of chronic stress32.

To be certain that enduring effects on the brain and behaviour, found in the main experiment, were directly attributable to the fear induced by hearing the various audio playbacks, we conducted a subsidiary experiment, testing the immediate effects of hearing predator vocalizations and conspecific alarm calls on short-term neuronal activation in the same brain areas examined in the main experiment. Additional wild free-living adult chickadees of both sexes were live-captured during the non-breeding season, housed in flocks outdoors for 1 week, and then housed solitarily in acoustic isolation chambers and exposed for 30 minutes to audio playbacks of either predator vocalizations, conspecific alarm calls signalling the highest level of predator danger (‘high zee’ calls33,34) or a lower level of predator danger (‘chick-a-dee’ calls33,34), or non-predator vocalizations. To quantify the immediate effects on short-term neuronal activation we assayed c-Fos expression in the two relevant brain areas; c-Fos being a short-lived transcription factor that degrades in a few hours, which is related to ∆FosB32.

Species, housing and playback procedures

The black-capped chickadee is a small (12 g) songbird resident year-round throughout southern Canada, which lives in small territorial flocks over winter. Male and female chickadees look and sound alike, and in the non-breeding season their behaviour is indistinguishable, such that the sexes can only be discriminated by a slight difference in wing length33. We included both sexes in our experiment for the purposes of obtaining an ecologically representative sample rather than to compare between the sexes, which we expected would not likely differ in their reactions to predators33. The immediate anti-predator responses of chickadees to playbacks of predator vocalizations and conspecific alarm calls have been well-studied33,34, as have the short-term neurobiological effects on the auditory processing areas of the brain resulting from hearing these signals of predator danger55.

Wild free-living chickadees were live-captured on the campus of Western University (London, Ontario, Canada) in the non-breeding season (September to March), and housed at the University’s Advanced Facility for Avian Research (http://birds.uwo.ca). Upon initial capture, and during the 7 days following the 2 days of cue exposure in the main experiment, individuals were housed in flocks in room-sized (2.1 × 2.4 × 3.7 m) outdoor aviaries on the Facility’s roof. During cue exposure individuals were housed solitarily, in small cages (25 × 30 × 37 cm), inside acoustic isolation chambers, to ensure that all they heard were the experimental cues.

In the main experiment, 27 individuals (15 males, 12 females) heard, during the 2 days of cue exposure, playlists matched for maximum amplitude and frequency and average decibel level, composed of the vocalizations of either, six predator species known to prey on chickadees (Cooper’s hawk, Accipiter cooperii; sharp-shinned hawk, Accipiter striatus; northern saw-whet owl, Aegolius acadicus; red-tailed hawk, Buteo jamaicensis; merlin, Falco columbarius; barred owl, Strix varia), or six non-threatening non-predator species (mallard, Anas platyrhynchos; wood frog, Lithobates sylvaticus; song sparrow, Melospiza melodia; downy woodpecker, Picoides pubescens; hairy woodpecker, Picoides villosus; red-breasted nuthatch, Sitta canadensis). All of these predator and non-predator species occur locally and their vocalizations would all be heard naturally by chickadees in the area. Vocalizations were broadcast for a total of 5 minutes per hour, during the 12 hours of daylight, at randomly selected intervals, with each species used one to four times every two hours, using different exemplars and call lengths each time. Individuals were randomly-assigned to treatment, balancing assignment between the treatments and sexes. To assess the enduring effects on behaviour, 7 days after predator cue exposure, 15 individuals were randomly-selected, balancing between the treatments and sexes, and exposed to playbacks of conspecific alarm calls signalling the highest level of predator danger (‘high zee’ calls33,34), for 15 seconds every minute, over a period of 15 minutes. All wild chickadees are familiar with conspecific alarm calls33,34. Individuals were filmed for 15 minutes before and during cue exposure, and the time they spent ‘freezing’15,16,19,33, operationally defined as immobile (stationary; not hopping, walking or flying) with their head upright and eyes open (vigilant; as opposed to, e.g. head down foraging)33, in the 1 minute before and after the start of the first playback33,56,57, was subsequently scored by an observer blind to which treatment each bird had received 7 days previously.

In the subsidiary experiment testing the immediate effects of hearing the playbacks on short-term neuronal activation, 22 individuals were randomly-assigned to treatment, balancing between treatments and sexes, and heard playbacks broadcast for 15 seconds every minute, over a period of 30 minutes, consisting of either predator (northern saw-whet owl) vocalizations, conspecific alarm calls (‘high zee’ or ‘chick-a-dee’ calls33,34) or non-predator (red-breasted nuthatch) vocalizations. Individuals hearing conspecific alarm calls necessarily heard the vocalizations of a single species (chickadees), which we matched by broadcasting a single predator and non-predator to the other individuals.

Predator and non-predator vocalizations were obtained from the Macaulay Library (www.macaulaylibrary.org) or xeno-canto (www.xeno-canto.org). Chickadee ‘high zee’ and ‘chick-a-dee’ alarm calls were obtained by recording captive wild-caught chickadees reacting to a taxidermic mount of a northern saw-whet owl. At least three exemplars of every vocalization were utilized. All sounds were edited using Audacity (www.audacityteam.org) to eliminate noise and standardize decibel levels. All playbacks were broadcast at a volume of 74 dB using all the same make and model of speaker and mp3 player.

This research was approved by Western University’s Animal Care Committee under protocol 2010-0245 and by Environment Canada under Scientific Permit CA-0244. All procedures were conducted in licensed and inspected facilities, and followed the guidelines set forth by the Canadian Council on Animal Care.

Neurobiological details and procedures

The nucleus taeniae of the amygdala (TnA) is the avian homologue of the mammalian medial amygdala35,36,58, and the hippocampus is homologous in birds and mammals35,36,59. To avoid any confusion concerning the relevance of our results to existing (i.e., mammalian) animal model studies of PTSD, we refer simply to the ‘amygdala’ when describing effects found in the nucleus taeniae of the amygdala. Immediate increases in activation in both the amygdala and the hippocampus have been reported previously in birds shown life-threatening visual cues35,36.

To assay ∆FosB and c-Fos expression in the amygdala and hippocampus in response to the audio playbacks used in our experiments, individuals were euthanized 7 days (∆FosB) or 90 minutes (c-Fos) after experimental cue exposure, using isoflurane, and perfused with 0.1 M phosphate buffered saline (pH 7.4) followed by 4% paraformaldehyde. Brains were removed, placed in 4% paraformaldehyde for a minimum of 24 h, then 30% sucrose for 24 h until saturated, and frozen at −80 °C. Brains were sectioned into 40 μm coronal slices using a cryostat at −20 °C. A series of sections were collected for Nissl staining to locate the brain regions of interest (see Supplementary Fig. S1), and two series were collected for immunohistochemistry. ∆FosB and c-Fos were labelled using commercial antibodies (Santa Cruz Biotechnology: FosB (102) rabbit IgG, sc-48; c-Fos (4) rabbit IgG, sc-52e) following established protocols30,31,60. Sections were processed free-floating in tissue culture wells. Sections were blocked in 0.05% H 2 O 2 followed by 10% normal serum and then incubated in the primary antibodies diluted (1:500) in phosphate buffered saline and 0.3% Triton (PBS/T). Following incubation in the primary antibody, sections were incubated in a biotinylated secondary antibody (diluted 1:500) followed by an avidin-biotin reaction (Vectastain Elite kit PK-6100, Vector Labs). Finally, immunoreactive cells were visualized using 3,3′-diaminobenzidine tetrachloride (SigmaFAST DAB). Sections were then mounted on microscope slides, dehydrated, cleared, and cover-slipped.

We captured z-stack images of each region of interest using a Leica Digital CCD (model 420D) camera mounted on a Leica DM5000B light microscope through X10 (amygdala) and X5 (hippocampus) objective lenses. We used Leica Application Suite to compile each picture as a z-stack from a series of images taken at a regular interval (0.63 mm) throughout the focal depth of the section to create images in which all cells were in focus60. The area of each region was measured in mm2 using ImageJ software calibrated to the relevant magnification. Each image was next converted from colour to 16-bit grayscale, the background subtracted and contrast enhanced, following which the ImageJ thresholding tool was used to convert ΔFosB or c-Fos positive nuclei to black against a white background, and the ImageJ count function was employed to quantify the number of positive cells/mm2 in each slice in each brain region of interest (see Supplementary Fig. S2). All image processing was conducted by an observer blind to which treatment each individual had received.

We confirmed that the enduring effects on ∆FosB expression in the amygdala and hippocampus demonstrated in response to predator-induced fear (Fig. 2) were specific to these regions, by additionally assaying ∆FosB expression in the medial caudal nidopallium (see Supplementary Fig. S1), an auditory processing area in the songbird brain55. We followed all the same procedures as just described regarding assaying ∆FosB expression in the amygdala and hippocampus 7 days after experimental cue exposure. In contrast to the significant effects seen in the amygdala and hippocampus (Fig. 2), there was no evidence of an enduring treatment effect on ∆FosB expression in the medial caudal nidopallium (F 1,8 = 1.3, P = 0.279, n = 6 predator and 6 non-predator individuals).

Statistical analyses

We conducted two-way ANOVAs with playback treatment and sex as fixed factors. In our test of enduring effects on behaviour our dependent variable was the change in time each individual spent ‘vigilant and immobile’, in the 1 minute before, vs. the 1 minute after, the start of the first conspecific alarm call; this being a repeated-measures value, which thereby controls for individual differences in fearfulness29,56,57. In our tests of effects on neuronal activation we calculated averages per individual of the number of ΔFosB or c-Fos immunoreactive cells/mm2 across all slices, in each brain area, and used these averages in our analyses. In testing effects on neuronal activation we conducted separate ANOVAs on each brain region (amygdala and hippocampus). Following our ANOVAs concerning the short-term effects on neuronal activation of hearing the various playback treatments in the subsidiary experiment, we conducted Dunnett’s post-hoc tests comparing each treatment with the control (non-predator vocalizations). Prior to analysis, all data were Box–Cox transformed and tested for normality and homogeneity of variances. All descriptive results reported (means ± SE) are untransformed or back transformed to the original units. Analogous to statistically controlling for individual variation by analyzing the change in each individual’s response in our test for an enduring treatment effect on behaviour, we included sex in our analyses to statistically account for this as a potential source of individual variation. There were no significant sex or treatment by sex effects (all p > 0.30), and we accordingly only report treatment effects in the Results.