A short pre-conception bout of predation risk affects both children and grandchildren

Traumatic events that affect physiology and behavior in the current generation may also impact future generations. We demonstrate that an ecologically realistic degree of predation risk prior to conception causes lasting changes in the first filial (F1) and second filial (F2) generations. We exposed male and female mice to a live rat (predator stress) or control (non-predator) condition for 5 min. Ten days later, stressed males and females were bred together as were control males and females. Adult F1 offspring from preconception-stressed parents responded to a mild stressor with more anxiety-like behavior and hyperarousal than offspring from control parents. Exposing these F1 offspring to the mild stressor increased neuronal activity (cFOS) in the hippocampus and altered glucocorticoid system function peripherally (plasma corticosterone levels). Even without the mild stressor, F1 offspring from preconception-stressed parents still exhibited more anxiety-like behaviors than controls. Cross-fostering studies confirmed that preconception stress, not maternal social environment, determined offspring behavioral phenotype. The effects of preconception parental stress were also unexpectedly persistent and produced similar behavioral phenotypes in the F2 offspring. Our data illustrate that a surprisingly small amount of preconception predator stress alters the brain, physiology, and behavior of future generations. A better understanding of the ‘long shadow’ cast by fearful events is critical for understanding the adaptive costs and benefits of transgenerational plasticity. It also suggests the intriguing possibility that similar risk-induced changes are the rule rather than the exception in free-living organisms, and that such multigenerational impacts are as ubiquitous as they are cryptic.

free to travel around the arena. The subject was then briefly removed and a novel male C57BL6 mouse placed inside the metal cage. The subject was then returned to the arena for an additional 150 seconds. Video recordings were taken from above and scored by a researcher blind to treatment. Ratios were calculated to determine the frequency and amount of time spent interacting with the empty cage compared to when the cage contained a novel conspecific.

SUP 1.2 Hormone assay
Corticosterone concentrations were measured using a commercial ELISA kit (Arbor Assays Company, Ann Arbor, MI, US) with a minimum detection level of 16.9 pg/ml corticosterone. The kit measured total corticosterone in serum including the corticosterone combined with corticosteroid-binding globulin (CBG). The cross reactivity of the kit for cortisol was 0.38% (tested at 50% binding). Briefly, 50 μl of standards or samples were added in duplicate to wells of the microtiter plate. Assay buffer (75 μl) was added to the non-specific binding (NSB) wells and 50 μl of assay buffer was added to wells to act as maximum binding wells. Then, 25 μl of the DetectX Corticosterone Conjugate and 25 μl of the DetectX Corticosterone antibody (except the NSB wells) were added to each well and the titer plate was shaken for 1 h at room temperature. After the plate was washed using the wash solution and blot dried, 100 μl of tetramethylbenzidine (TMB) substrate was added to each well and incubated for 30 min at room temperature. The optical density (OD) of corticosterone was read at 450 nm wavelength using a plate reader within 15 minutes of the reaction being terminated by adding 50 μl of the stop solution. Corticosterone concentration was calculated using standard curves.

SUP 1.3 Immunostaining for F0 and F1 generation mice
Brain collection for immunostaining: Brain collection occurred 90 minutes (for c-FOS; 1,2 ), or two days (GR, FKBP5) after exposure 3,4 . Animals were anaesthetized with 15% urethane prepared in distilled water and, after a 1-minute saline (0.9%) pre-flush to remove all blood, transcardial perfused with ice-cold 4% paraformaldehyde (PFA in 0.1M phosphate buffer, pH 7.4). Brains were then removed and individually post-fixed in a 4% PFA solution. One day before sectioning, brains were immersed in 20% sucrose. Brains were stored at 4°C and all solutions were ice-cold to prevent tissue melting.
Slide preparation for analysis: A Leica CM3050 S cryostat machine (Leica Biosystems, Wetzlar, DE) was used to section all brain samples with a D-profile tungsten knife. The cryostat specimen temperature was set to -19 ± 3°C and the chamber temperature set to -17 ± 3°C. All slicing was conducted manually, with section and trimming thickness set to 30μm for all sections. Coronal sections were collected, prioritizing the cortex, ventricles, and hippocampus. All slides were transferred to slide storage boxes and kept in a -80°C environment until staining.
Immunohistochemistry for cFOS: Immunostaining followed previously published procedures 5 . During staining, slides were removed from the -80 o C storage and thawed at 4 o C for 10 minutes, followed by eight minutes at room temperature to further dry. Slide borders were marked with a PAP hydrophobic pen to create an antibody barrier. The primary antibody, rabbit cFOS (1/1000, Cell Signaling, Danvers, MA, US), was diluted in phosphate buffered saline (PBS) with PBS + 0.2% TritonX-100 and 2% normal goat serum. One mL of primary antibody was applied to each slide and incubated for 48 hours at 4 o C. Slides were then washed for 20 minutes with PBS before being incubated in the secondary antibody (biotinylated goat anti-rabbit diluted in PBS, 0.2%triton x-100 and 2% normal goat serum) for two hours. Washing procedures were repeated for 20 minutes before incubation in Vectastain avidin/biotinylated enzyme (A+B) solution for one hour (Vector Labs, Burlingame, CA, US). Washing was repeated and the reaction product visualized by adding 0.05% 3,3'-Diaminobenzidine (DAB) with 0.01% hydrogen peroxide in distilled water for five minutes. Sections were then washed with distilled water and air dried for 24 hours before being dehydrated with alcohol/xylene and cover slipped with Permount (Thermo Fisher Scientific, Waltham, MA, US).
c-FOS staining analysis: Images of sections were captured with a digital camera (Teledyne QImaging, Surry, BC, CA) connected to a bright field microscope (Olympus Life Science, Tokyo, JP) at 10x, 20x and 40x magnifications. Olympus cell Sens imaging software was used to process and store images. Representative fields consisted of eight subregions within the hippocampus (dentate gyrus and Cornu Ammonis 1-CA1) and two sub-regions (central and basolateral) within the amygdala. Within the dentate gyrus, we measured cFOS in the dorsal right hemisphere (DENTRH), dorsal left hemisphere (DENTLH), ventral right hemisphere (VDRH), and ventral left hemisphere (VDLH). Within the CA1 area, we measured cFOS in the dorsal right hemisphere (CA1RH), dorsal left hemisphere (CA1LH), ventral right hemisphere (VCA1RH), and ventral left hemisphere (VCA1LH). All sections were analyzed at a constant microscope light intensity. The intensity of cFOS staining was normalized with background OD and analyzed using ImageJ (National Institute of Health (NIH), Bethesda, MD, US).

SUP 1.4 SUP Experiment 1: Effect of two-minute rat exposure on mouse behavior
Sexually inexperienced male C57BL/6 mice, aged 7-8 weeks, were divided into two groups: PS (n=8) and C (n=8). All mice were habituated to the exposure chamber for five minutes per day for five consecutive days. On day six, mice in the PS group were exposed to a live rat in the exposure chamber for two minutes, while mice in the C group were exposed to an empty chamber for two min. Starting on day eight, all mice underwent a six-test behavioral battery (one test per day for six days). The behavioral battery started with the elevated plus maze, followed by the open field, light/dark box, acoustic startle test, forced swim test, and the social interaction test. Detailed descriptions of each test are provided in supplementary methods 1.1.

SUP 2.1 Two minute exposure to a rat does not increase subsequent anxiety-or depressive-like behavior.
As expected, mice exposed to a rat froze more often than mice exposed to an empty cage (F1,14=6.669, p=0.022]. Across all behavioral tests, there were no significant differences in the elevated plus maze, light/dark box, startle, forced swim test or social interaction test. There was a significant treatment effect on time in the center of the open field [F1,14=6.754, p=0.02]. Surprisingly, predator stressed mice spent more time in the center than control mice.

Literature Cited 1
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