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ARTICLE OPEN
Sounds of danger and post-traumatic stress responses in
wild rodents: ecological validity of a translational model
of post-traumatic stress disorder
Hagit Cohen
1,2
✉, Michael A. Matar
1
, Doron Todder
1
, Carmit Cohen
1
, Joseph Zohar
3
, Hadas Hawlena
4
and Zvika Abramsky
5
© The Author(s) 2023
In the wild, animals face a highly variable world full of predators. Most predator attacks are unsuccessful, and the prey survives.
According to the conventional perspective, the fear responses elicited by predators are acute and transient in nature. However, the
long-term, non-lethal effects of predator exposure on prey behavioral stress sequelae, such as anxiety and post-traumatic
symptoms, remain poorly understood. Most experiments on animal models of anxiety-related behavior or post-traumatic stress
disorder have been carried out using commercial strains of rats and mice. A fundamental question is whether laboratory rodents
appropriately express the behavioral responses of wild species in their natural environment; in other words, whether behavioral
responses to stress observed in the laboratory can be generalized to natural behavior. To further elucidate the relative contributions
of the natural selection pressures influences, this study investigated the bio-behavioral and morphological effects of auditory
predator cues (owl territorial calls) in males and females of three wild rodent species in a laboratory set-up: Acomys cahirinus;
Gerbillus henleyi; and Gerbillus gerbillus. Our results indicate that owl territorial calls elicited not only “fight or flight”behavioral
responses but caused PTSD-like behavioral responses in wild rodents that have never encountered owls in nature and could cause,
in some individuals, enduring physiological and morphological responses that parallel those seen in laboratory rodents or
traumatized people. In all rodent species, the PTSD phenotype was characterized by a blunting of fecal cortisol metabolite response
early after exposure and by a lower hypothalamic orexin-A level and lower total dendritic length and number in the dentate gyrus
granule cells eight days after predator exposure. Phenotypically, this refers to a significant functional impairment that could affect
reproduction and survival and thus fitness and population dynamics.
Molecular Psychiatry; https://doi.org/10.1038/s41380-023-02240-7
INTRODUCTION
Post-traumatic stress disorder (PTSD) is a potentially chronic
impairing disorder involving cognitive, emotional, and physiolo-
gical failure to adequately process and/or recover from exposure
to a traumatic experience [1]. The memories of a traumatic event
remain vivid, chronically active, and disruptive over long periods
of time, together with the emotions at the time of the event,
shaping symptoms such as intrusive thoughts, physiological
hyperarousal, active avoidance of traumatic reminders, and
negative alterations in cognitions and mood.
From an evolutionary-ecological perspective, memory is dis-
tinctly advantageous for survival. An organism’s ability to form
and retain a record, especially of threatening events, and to
accumulate and maintain this information to allow for ready
access and ongoing updating, that is, to form memories, confers
the ability to anticipate danger and prepare for or avoid it. In
natural habitats, animals are required to continually assess and
evaluate the potential risks they face while balancing the need for
food acquisition with the need for safety, considering the dynamic
changes in resource availability, competition intensity, and
predation risk. Therefore, animals have evolved a variety of
mechanisms for evaluating the potential risk of predation,
implementing antipredator decision-making strategies, and devel-
oping adaptive behavioral responses to these conditions [2–7]. For
example, when faced with predation risk, some prey individuals
may alter their selection of micro or macro-habitats, seeking
concealment, while others may exhibit morphological changes
such as alteration of coloration, to enhance camouflage and
increase the trade-off between food acquisition and safety [6].
According to the conventional perspective, the fear responses
elicited by predators are acute and transient in nature [8]. The prey
identifies the predator and initiates a behavioral response (fleeing,
fighting, or freezing). Following the encounter, the animal either
survives or dies. If survival occurs, it is commonly assumed that the
animal resumes its normal activities [9] and physiological
equilibrium is restored. However, studies on commercial strains
of rats and mice have shown that non-lethal, long-lasting effects
of predators, such as fear, anxiety, or post-traumatic responses,
Received: 1 February 2023 Revised: 17 August 2023 Accepted: 24 August 2023
1
Faculty of Health Sciences, Ben-Gurion University of the Negev, Israel & Ministry of Health, Anxiety and Stress Research Unit, Beer-Sheva Mental Health Center, Beer-Sheva, Israel.
2
Department of Psychology, Ben-Gurion University of the Negev, Beer-Sheva, Israel.
3
Post-Trauma Center, Sheba Medical Center, Tel Aviv University, Tel Aviv 52621, Israel.
4
Mitrani Department of Desert Ecology, The Jacob Blaustein Institutes for Desert Research, Ben-Gurion University of the Negev, Midreshet Ben-Gurion Israel, Sde Boker 8499000,
Israel.
5
Department of Life Sciences and Ramon Science Center, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel. ✉email: hagitc@bgu.ac.il
www.nature.com/mp
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can have a significant impact on individual morphology, behavior,
and reproductive success [10–14]. A fundamental question is thus
the extent to which experimental findings using a commercial
strain of rats or mice can be generalized to real-world situations. In
particular, it is not known if there are “post-trauma”symptoms for
animals in the wild or which behavioral stress responses observed
in the laboratory appear in wild animals in the field [9,15]. Should
we expect that in natural communities the non-lethal enduring
effects of predators will be significant as well, and whether these
effects will have a greater impact on wild prey populations (and
on predators) in terms of individual behavior, population
dynamics, and reproductive success compared to the non-lethal
short-term effects and the lethal effects.
To fill the above knowledge gaps, we investigated the effects of
predator stress on the behavioral, molecular, and morphological
responses of wild rodent species in a laboratory setup. We studied
males and females of three rodent species: Acomys Cahirinus
(A-Cahirinus); Gerbillus Henleyi (G-Henleyi); and Gerbillus Gerbillus (G-
Gerbillus) (Supplement #1).
Gerbils are particularly interesting for the study of PTSD as their
adrenal glands are comparable to the adrenals of the humans, due
to their high concentration of ascorbic acid and their secretion of
cortisol, unlike most rodents [16–18].
The goal of this study was to evaluate the bio-behavioral and
morphological effects of predator cues (playback of tape-recorded
owl calls) in males and females of three wild rodent species. To
this end, we used (a) the cut-off behavioral criteria (CBC)
classification model. In this model, populations of exposed rodents
are classified based on the degree of their individual behavioral
response, creating three distinct groups: “extreme,”“partial,”or
“minimal”behavioral response (EBR, PBR, and MBR, respectively)
(Supplement #2), (b) measured fecal cortisol metabolite (FCM)
levels 1–2 h after predator stress, (c) examined orexin-A levels in
the hypothalamus eight days after exposure, and (d) we evaluated
changes in the cytoarchitecture of the hippocampus using the
Golgi-Cox method.
MATERIALS AND METHODS
Animals
We used two gerbilline species (G-Henleyi and G-Gerbillus) and one murine
species (A-Cahirinus), which are burrow dwellers of the Negev Desert in
Israel. Twenty-three G-Henleyi (10 females and 13 males), fifteen G-Gerbillus
(8 females and 7 males), and thirty-five A-Cahirinus (19 females and 16
males) specimens were used in this study.
All rodents were descendants of wild rodents captured in the vicinity of
the Negev Desert in Southern Israel. After trapping, the animals were
transported to Midreshet Ben-Gurion, where they were housed in pairs
(male and female) and bred for 6–8 generations. Considering the small
number of generations compared to the evolution of the species themselves
[19,20], these rodents, which were kept and bred under semi-natural
conditions, can be considered wild rather than domesticated [20].
All rodents used in the experiments were adults and were acclimatized
to the conditions to which they were later exposed for at least one month
prior to the habituation experiments. Rodents were housed in pairs in
plastic cages (60 × 50 × 40 cm) under controlled temperature (25 ± 1 °C)
and humidity (30 ± 5%) conditions, with a photoperiod of 12D:12 L (lights
on at 0700 h) and with sawdust and dried grass as bedding material. They
were provided daily with millet seeds ad libitum and fresh alfalfa
(Medicago sp.) as a water source following Hawlena et al. [21]. All
experimental procedures were conducted between 13:00–16:00.
Experimental design
The base level of anxiety-related behavior (and the intensity of the
response to stressor) were determined using the open field test (OFT). This
test was chosen over the elevated plus maze (EPM) because repeated
testing on the same animals reduces its validity [22]. After being exposed
to predator cues for 10 min, fresh feces were collected to test for cortisol
levels. Behavioral tests were conducted using the OFT, EPM, and ASR tests
on day 7. Exploratory behavior in the EPM serves as the main platform for
the assessment of overall behavior, and the ASR paradigm provides a
precise quantification of hyperalertness in terms of the magnitude of
response and habituation to the stimulus. These data were used to classify
the animals into behavioral response groups. The rats were then sacrificed,
and brains were collected for morphology analysis. The baseline FCM
levels were evaluated in G-Gerbillus specimens.
Stress exposure
Individual rodents were exposed to tape-recorded territorial calls of owls.
Stress was induced by placing the test animals in a plastic cage
(40 × 40 × 40 cm) that was situated on a yard paving stone for 10 min in
a closed environment. Sound was transmitted into the arena using a small
loudspeaker (2.5 inch, 10 Ω, 0.2 W max) placed on the rear wall, 45 cm
above the floor, and connected to a cassette recorder. The sound levels of
the tape-recorded owl calls were standardized to approximately 60–70 dB.
After 10 min of playback of the owl calls, we turned off the recording
device and left the rodents in the arena for another 5 min of silence. This
period simulates the hunting pattern of the owl, which, following a period
of territorial calling, flies to a hunting perch where it waits in complete
silence to pounce on its prey [23].
Behavioral assessments
All behavioral tests were video-recorded using the ETHO-VISION program
(Noldus), by an investigator blinded to the experimental protocol.
Behaviors of specimens were assessed using OFT, EPM and ASR, as
described previously [24–26]. Detailed protocols are described in
Supplementary Information #3:1.
Fecal cortisol metabolites
This non-invasive technique for measuring steroid metabolites in fecal
samples has been established in an increasing number of species [27–33].
Fresh fecal samples were collected, and any feces contaminated with urine
was excluded from the analysis. The fecal samples were placed in 2 mL
microfuge tubes and immediately frozen at −20 °C for subsequent
analysis. Hormones were extracted using the method described by
Gutman et al. [34]. Fecal cortisol metabolites were measured using a
radioimmunoassay kit (ICN Biomedical, Inc.) in duplicate.
Brains
24-hours after the behavioral tests, between 12:00 and 14:00, the animals
were deeply anesthetized and perfused intracardially with saline. The
brains were immediately removed, and the hypothalamus was dissected.
Each hypothalamus was washed in saline, weighed, and frozen at −80 °C
until later use. Brains (without hypothalamus) were prepared using a rapid
Golgi kit (FD Neurotechnologies, USA) according to the manufacturer’s
instructions (Supplementary Information 3:2).
Orexin-A levels in the hypothalamus
The hypothalamus samples were homogenized (PRO Scientific Inc., CT,
USA) at 24,000 rpm for 2 min in ice-cold PBS and centrifuged at 15,000 rpm
for 30 min at 4 °C. The supernatant was separated and stored at −80 °C.
Orexin-A levels in brain tissue were measured using a commercially
available enzyme-linked immunoassay kit (MyBioSource, Inc., San Diego,
CA, USA) according to the manufacturer’s protocol, in duplicates.
Statistical analyses
All data are expressed as mean±standard error of the mean (SEM), and
statistical analyses were performed using a two-way analysis of variance
(ANOVA). For the OFT, statistical analyses were performed using repeated
measures (RM)-ANOVA. Where significant group effects were detected, the
Bonferroni test assessed significant post-hoc differences between groups.
The prevalence of PTSD-classified groups was tested in relation to rodent
species group or sex using cross-tabulation, chi-square tests, and logistic
regression analysis. To gain an additional understanding of the relationship
between behavioral and molecular/morphology measures, Pearson’s
correlation analysis was performed.
RESULTS
To preclude the possible effects of basal pre-trauma anxiety,
which can be a risk factor for the development and persistence of
H. Cohen et al.
2
Molecular Psychiatry
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PTSD, animals were first evaluated in the OFT under basal
conditions. We found that the level of anxiety-related behavior
was low: the path length or thigmotaxis all indicated a low level of
anxiety-related behavior without any differences among the
species or between sexes. In all species, exposure to the owl cues
reduced the percentage of time spent in the inner and middle
zones and increased the time spent in the outer zone, which are
common indices of anxiety-related behavior (Supplementary
Information #4:0).
In terms of anxiety index (Fig. 1A), two-way ANOVA revealed a
significant effect of species (F(2,67) =10.2, p< 0.00015, respec-
tively) and sex (F(2,67) =4.9, p< 0.035, respectively). Bonferroni
test confirmed that female A-Cahirinus exposed to predator stress
spent significantly less time in the open arms, entered the open
arms less frequently, and exhibited a higher anxiety index than
female G-Gerbillus (p< 0.005, p< 00002, and p< 0.0035, respec-
tively) (Supplementary Information #4:1 for all EPM parameters).
No species or sex differences in the ASR or startle habituation
(Fig. 1B, C) were observed.
Relative prevalence rates according to CBC-classification
The species didn’t differ in their overall responses to imposed
predator cue. There were no significant differences in the
prevalence of EBR (PTSD phenotype) (Fig. 1D), MBR (Fig. 1E), or
PBR (Fig. 1F) among species or between sexes. No predictor
variable reached significance.
The prevalence of EBR among A-Cahirinus females was 26.3%
(5/19), and 25.0% (4/16) among A-Cahirinus males. Moreover, in
A-Cahirinus, 2 females (10.5%) and 1 male (6.25%) fulfilled the
criteria for MBR, and 12 females (63.15%) and 11 males (68.75%)
were classified as PBR. In G-Henleyi, the prevalence of EBR in
females was 20.0% (2/10) and 23.0% (3/13) among males.
Moreover, 3 females (30.0%) and 1 male (7.69%) fulfilled the
criteria for MBR, and 5 females (50.0%) and 9 males (69.23%) were
classified as PBR. In G-Gerbillus, the prevalence of EBR females was
12.5% (1/8) and 42.85% (3/7) among males, without significant
differences, likely due to the small sample size. Moreover, 3
females (37.5%) and 1 male (14.3%) fulfilled the criteria for MBR,
and 4 females (50.0%) and 3 males (37.5%) were classified as PBR.
In G-Gerbillus, although a χ2 analysis indicated that sex did not
affect the prevalence of the extremes in the behavioral response
to stress (Fisher exact p=0.023, not significant), marked
behavioral differences were evident between female and male
rodents. Male G-Gerbillus showed a higher prevalence of EBR than
female rats.
Overall, the prevalence of EBR in A-Cahirinus species was 25.71%
(9/35), while in G-Henleyi or G-Henleyi species, it was 21.7% (5/23)
and 26.67% (4/15), respectively.
FCM
In A-Cahirinus and G-Henleyi, measurements were made only after
exposure to stress, that is, without basal level measurements.
Overall, two-way ANOVA revealed a significant effect of species on
FCM levels (F(1,67) =6.85, p< 0.015).
In A-Cahirinus, FCM concentrations were not homogeneous and
wide scattering was detected (Fig. 2A). No significant differences
were found in FCM concentrations between females and males,
but there was a trend of lower FCM concentrations in males
(F(1,29) =2.9, p=0.09). Looking at FCM concentrations between
PTSD-classified groups (Fig. 2B), two-way ANOVA revealed a
1)
FMFMFM
0.0
0.2
0.4
0.6
0.8
1.0
)U.A(xednIyteixnA
p<0.0035
A.Cahirinus G. Henleyi G.Gerbilus
FMFMFM
0
200
400
600
800
1000
)U.A(esnopseReltratS
A.Cahirinus G. Henleyi G.Gerbilus
FMFMFM
0
10
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30
40
50
60
70
80
)%(noitautibaHeltratS
A.Cahirinus G. Henleyi G.Gerbilus
A) B) C)
D) E) F)
FM FM FM
0
10
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30
40
50
60
70
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90
100
RBMfoecnelaverP
A.Cahirinus G. Henleyi G.Gerbilus
FM FM FM
0
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RBEfoecnelaverP
A.Cahirinus G. Henleyi G.Gerbilus
FM FM FM
0
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RB
Pf
o
ecne
lave
r
P
A.Cahirinus G. Henleyi G.Gerbilus
Fig. 1 The long-term effects of predator cue exposure on behavior. The top panel (1) depicts the experimental protocol. The red circle
signifies the behavioral test performed, for which the results are shown. AAnxiety Index which integrates the measured EPM behavioral
measures. BStartle amplitude in the acoustic startle response (ASR) paradigm. CPercentage of startle habituation in the ASR paradigm.
DPrevalence of extreme behavioral response (EBR) (in percentages), EPrevalence of minimal behavioral response (MBR) (in percentages),
FPrevalence of partial behavioral response (PBR) (in percentages). Owl territory calls had long-lasting influences on rodent behavior; all
species reacted significantly to predator cue stress in terms of anxiety-related behavior in the EPM and ASR paradigms 7 days after exposure.
In addition, predator cue exposure causes post-traumatic stress disorder (PTSD)-like behavioral responses in wild rodents that have never
encountered owls. Data are presented as data points and mean ± SEM and percentage.
H. Cohen et al.
3
Molecular Psychiatry
Content courtesy of Springer Nature, terms of use apply. Rights reserved
significant difference in FCM concentrations between groups
(F(2,29) =9.7, p< 0.0006). In females, Bonferroni post-hoc tests
confirmed significantly lower FCM concentrations in EBR than in
PBR (p< 0.0003) and MBR (p< 0.004) rodents. In males, Bonferroni
tests confirmed significantly lower FCM concentrations in EBR
than in PBR (p< 0.05) rodents.
In G-Henleyi, no significant differences were found in FCM levels
between females and males (Fig. 2C). Two-way ANOVA revealed a
significant effect of groups (F(2,17) =8.3, p< 0.0035) on FCM
concentrations (Fig. 2D). In females, Bonferroni tests confirmed
significantly lower FCM concentrations in EBR than in PBR
(p< 0.005) and MBR (p< 0.05) rodents. In males, Bonferroni post-
hoc tests confirmed significantly lower FCM concentrations in EBR
than in PBR (p< 0.03).
In G-Gerbillus,wefirst monitored baseline FCM concentrations
and subsequently measured the post-stress FCM response. RM-
ANOVA revealed a significant effect of sex (F(1,16) =8.3, p< 0.015)
and stress (F(1,16) =26.2, p< 0.00025) (Fig. 2E). In both females
and males, stress significantly increased FCM concentration
relative to the baseline conditions. Bonferroni tests indicated that
all exposed rodents exhibited significantly elevated mean FCM
concentrations compared with those in the control group
(females: p< 0.0001 and males: p< 0.015). Moreover, both under
baseline conditions and stress conditions the FCM concentrations
were significantly higher in females than in males (p< 0.003 and
p< 0.05, respectively). Two-way ANOVA revealed a significant
effect of groups (F(2,9) =7.76, p< 0.015) on FCM concentrations
(Fig. 2F). No effects were observed for sex, but there was a trend of
lower FCM concentrations in males (F(1,9) =3.6, p=0.09). In
females, Bonferroni tests confirmed significantly lower FCM
concentrations in EBR than in PBR (p< 0.05) and MBR (p< 0.01).
In males, Bonferroni tests confirmed significantly lower FCM
concentrations in EBR than in PBR (p< 0.02).
We conducted regression analyses to further understand the
relationship between FCM levels and behavioral measures,
regardless of CBC classification (Table 1). In A-Cahirinus,
G-Henleyi and G-Gerbillus Pearson’s correlation analysis revealed
that FCM concentrations were significantly and negatively
correlated with the anxiety index.
Brain orexin-A levels
Changes in total orexin-A levels in rodent brains following stress
exposure are shown in Fig. 3. Overall, no significant differences
were found in orexin-A levels among species or between sexes.
In A-Cahirinus, no significant differences were found in orexin-A
levels between females and males (Fig. 3A). Two-way ANOVA
revealed a significant effect of groups (F(2,29) =38.2, p< 0.00016)
(Fig. 3B). In both sexes, Bonferroni tests confirmed significantly
lower orexin-A levels in EBR than in PBR (p< 0.0001 for both
females and males) and MBR (females: p< 0.0025 and males:
p< 0.035).
In G-Henleyi, no significant differences were found in orexin-A
levels between females and males (Fig. 3C). Two-way ANOVA
revealed a significant effect of groups (F(2,17) =15.2, p< 0.0002)
Female Male
0
50
100
150
200
250
300
350
400
(slevellositroclaceF )Ld/g
A.Cahirinus: 1-2 h after predator exposure
Female Male
0
50
100
150
200
250
300
G. Henleyi: 1-2 h after predator exposure
(s
leve
ll
os
it
roc
lace
F)Ld/g
Female Male Female Male
0
20
40
60
80
100
120
140
160
180
200
220
Baseline 1-2 h after stress
G. Gerbilus
(slevellositroclaceF )Ld/g
P<0.05
P<0.003
P<0.015
P<0.0001
EBR PBR MBR EBR PBR MBR
0
50
100
150
200
250
300
350
p<0.05
p<0.0003
A.Cahirinus: 1-2 h after predator exposure
p<0.004
slevellositroClaceF(g/dL)
Females Males
EBR PBR MBR EBR PBR MBR
0
50
100
150
200
250
300
350
Females Males
p<0.02
p<0.005
G. Henleyi: 1-2 h after predator exposure
p<0.03
slevellositroClaceF(g/dL)
EBR PBR MBR EBR PBR MBR
0
50
100
150
200
250
300
350
Females Males
p<0.05 p<0.02
G. Gerbilus: 1-2 h after predator exposure
p<0.01
slevellositroClaceF(g/dL)
1)
A)
B)
C)
D)
E)
F)
Fig. 2 The effects of predator cue exposure on fecal cortisol metabolite (FCM) levels. The top panel (1) depicts the experimental protocol.
The red circle signifies the behavioral test performed, for which the results are shown. AThe FCM levels, collected 1–2 h after predator stress
exposure, in female and male Acomys cahirinus.BThe FCM levels in Acomys cahirinus according to the affected groups (classified using the cut-
off behavioral criteria (CBC) method). CThe FCM levels, collected 1–2 h after predator stress exposure, in female and male Gerbillus henleyi.
DThe FCM levels in Gerbillus henleyi according to the affected groups (classified using the CBC method). EThe FCM levels, collected 1–2 h after
predator stress exposure, in female and male Gerbillus gerbillus.FThe FCM levels in Gerbillus gerbillus according to the affected groups
(classified using the CBC method). Although we tested basal FCM concentrations in G. gerbillus before predator exposure and found that the
FCM concentrations were relatively low and significantly elevated in response to the stressor compared to their baseline levels, it is still
unclear whether the poor FCM response to predator stress and the dysregulation observed in the acute aftermath of trauma represent an
existing pre-trauma vulnerability trait or develops from the exposure to the trauma itself. Data are presented as data points and mean ± SEM.
H. Cohen et al.
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Molecular Psychiatry
Content courtesy of Springer Nature, terms of use apply. Rights reserved
(Fig. 3D). In females, Bonferroni tests confirmed significantly lower
orexin-A levels in EBR than in MBR (p< 0.05). In males, Bonferroni
post-hoc tests confirmed significantly lower orexin-A levels in EBR
than in PBR (p< 0.0015).
In G-Gerbillus, no significant differences were found in orexin-A
levels between females and males (Fig. 3E). Two-way ANOVA
revealed a significant effect of groups (F(2,9) =14.4, p< 0.002) on
orexin-A levels (Fig. 3F). In males, Bonferroni tests confirmed
significantly lower orexin-A levels in EBR than in PBR (p< 0.015).
In A-Cahirinus,G-Henleyi and Gerbillus-G Pearson’s correlation
analysis revealed that orexin-A levels were significantly and
negatively correlated with the anxiety index (Table 1).
Morphology of DG granular neurons
Overall, no significant differences were found in the total dendritic
length or number among species or between sexes (Figs. 4–5).
In A-Cahirinus, no significant differences were found in the total
dendritic length (Fig. 4A) or number (Fig. 4C) between females
and males. Eight days after predator stress, the total dendritic
length (Fig. 4B) and total dendritic number (Fig. 4D) were
significantly lower in both female and male EBR animals than in
the PBR group (p< 0.0001 and p< 0.00025, respectively).
In G-Henleyi, no significant differences were found in the total
dendritic length (Fig. 4E) or number (Fig. 4G) between females and
males. The total dendritic length (Fig. 4F) and the total dendritic
number (Fig. 4H) were significantly lower in both female and male
EBR animals than in the PBR (p< 0.0001 and p< 0.0002,
respectively) and MBR (p< 0.0001 and p< 0.02, respectively)
groups.
In G-Gerbillus, no significant differences were found in the total
dendritic length (Fig. 4I) or number (Fig. 4K) between females and
males. The total dendritic length (Fig. 4J) and the total dendritic
number (Fig. 4L) were significantly lower in both female and male
EBR animals than in the PBR (p< 0.0065 and p< 0.00015,
respectively) and MBR (p< 0.0001 and p< 0.00065, respectively)
groups.
Female Male
0
20
40
60
80
100
120
140
160
180
200
)gm/gp(A-nixerOcimalahtopyH
A.Cahirinus: 8 days after predator exposure
Female Male
0
20
40
60
80
100
120
140
160
180
200
G. Henleyi: 8 days after predator exposure
)gm/gp(A-nixerOcimalahtopyH
Female Male
0
20
40
60
80
100
120
140
160
180
200
)gm/gp(A-nixerOcimalahtopyH
G. Gerbilus: 8 days after predator exposure
EBR PBR MBR EBR PBR MBR
0
20
40
60
80
100
120
140
160
180
200
p<0.0025
p<0.0001
p<0.0001
A.Cahirinus: 8 days after predator exposure
p<0.035
A-
ni
x
er
Ocim
ala
ht
o
p
y
H(l
m
/
g
p)
Females Males
EBR PBR MBR EBR PBR MBR
0
20
40
60
80
100
120
140
160
180
200
Females Males
p<0.05
G. Henleyi: 8 days after predator exposure
p<0.0015
A-nixerOcimalahtopyH(lm/gp)
EBR PBR MBR EBR PBR MBR
0
20
40
60
80
100
120
140
160
180
200
Females Males
G. Gerbilus: 8 days after predator exposure
A-nixerOcimalahtopyH(lm/gp)
p<0.015
1)
A)
B)
C) E)
D) F)
Fig. 3 The long-term effects of predator cue exposure on hypothalamic levels of orexin A. (1) The top panel depicts the experimental
protocol. The red circle signifies the test performed whose results are shown. AHypothalamic levels of orexin-A (ORX-A) in females and males
Acomys cahirinus species. BHypothalamic levels of ORX-A in Acomys cahirinus species according to the affected groups (according to the cut-
off behavioral criteria (CBC) method). CHypothalamic levels of ORX-A in females and males Gerbillus henleyi species. DHypothalamic levels of
ORX-A in Gerbillus henleyi species according to the affected groups (according to the CBC method). EHypothalamic levels of ORX-A in females
and males Gerbillus gerbillus species. FHypothalamic levels of ORX-A in Gerbillus gerbillus species according to the affected groups (according
to the CBC method). Data are presented as data points and mean ± SEM.
Table 1. Correlation analysis between anxiety index and fecal cortisol levels.
Anxiety index
Acomys Cahirinus Gerbillus Henleyi Gerbillus Gerbillus
Fecal cortisol levels (µg/dL) r=−0.57, p< 0.0004, n=35 r=−0.42, p< 0.05, n=23 r=−0.8, p< 0.0004, n=15
Orexin-A levels (pg/ml) r=−0.53, p< 0.001, n=35 r=−0.61, p< 0.0025, n=23 r=−0.81, p< 0.0003, n=23
DG dendritic Number r=−0.68, p< 0.0025, n=24 r=−0.61, p< 0.003, n=23 r=−0.89, p< 0.0001, n=15
DG dendritic Length (µm) r=−0.57, p< 0.004, n=24 r=−0.55, p< 0.007, n=23 r=−0.75, p< 0.002, n=15
Marked correlations are significant at p< 0.05.
H. Cohen et al.
5
Molecular Psychiatry
Content courtesy of Springer Nature, terms of use apply. Rights reserved
In A-Cahirinus,G-Henleyi and Gerbillus-G Pearson’s correlation
analysis revealed that dendritic number and length were
significantly and negatively correlated with the anxiety index
(Table 1).
DISCUSSION
This study aimed to explore the application of our standard model
and procedures for translational studies of PTSD in laboratory
rodents to three groups of wild rodents of both sexes, specifically
A-Cahirinus,G-Henleyi, and G-Gerbillus, in an adapted format. This
study employed recordings of owl territorial calls as the trigger, to
which they had never previously been exposed in their lifetime.
The results indicate that this evolutionary trauma cue indeed
elicited not only the acute “fight or flight”responses to the
potential predator threat, but also significant long-term beha-
vioral, neurobiological, and morphological sequelae that are
completely in line with our previous findings in laboratory
rodents. The results similarly mirror findings from clinical studies
in traumatized patients, changes that could significantly compro-
mise functions related to survival and reproduction.
All species reacted to the playback of owl vocalizations
significantly in terms of anxiety-related behavior in the EPM,
OFT, and ASR paradigms and in the overall pattern of resultant
FCM concentrations, orexin-A levels, and DG dendritic arboriza-
tion, validating the potentially traumatizing effect of the stressor.
However, bio-behavioral stress responses showed extensive
individual phenotypic heterogeneity at the baseline and after
predator exposure within the species. Marked behavioral (i.e.,
phenotypic) differences were evident in all paradigms among
individuals within each species after stress exposure.
Female Male
0
5
10
15
20
25
30
35
40
rebmunciti
r
dn
e
d
l
atoT
A.Cahirinus
Female Male
0
5
10
15
20
25
30
35
40
G. Henleyi:
rebmuncitirdnedlatoT
Female Male
0
5
10
15
20
25
30
35
40
rebmuncitirdnedlatoT
G.Gerbilus
EBR PBR MBR EBR PBR MBR
0
5
10
15
20
25
30
35
40
p<0.00025
p<0.0001
A.Cahirinus
rebmuncitirdnedlatoT
Females Males
EBR PBR MBR EBR PBR MBR
0
5
10
15
20
25
30
35
40
p<0.02
p<0.0002
p<0.0001
G. Henleyi
p<0.0001
rebmuncitirdnedlatoT
Females Males
EBR PBR MBR EBR PBR MBR
0
5
10
15
20
25
30
35
40
p<0.0001
p<0.00065
p<0.0015
p<0.0065
G. Gerbilus
rebmuncitirdnedlatoT
Females Males
Female Male
0
300
600
900
1200
1500
1800
2100
2400
2700
3000
(htgnelcitirdnedlatoT m)
G.Gerbilus
Female Male
0
300
600
900
1200
1500
1800
2100
2400
2700
3000
G. Henleyi
(htgnelcitirdnedlatoT m)
Female Male
0
300
600
900
1200
1500
1800
2100
2400
2700
3000
(
ht
g
nelci
t
ird
n
edla
t
o
Tm)
A.Cahirinus
EBR PBR MBR EBR PBR MBR
0
300
600
900
1200
1500
1800
2100
2400
2700
3000
p<0.0003
p<0.0001
p<0.0001
A.Cahirinus
htgnelcitirdnedlatoT(m)
Females Males
EBR PBR MBR EBR PBR MBR
0
300
600
900
1200
1500
1800
2100
2400
2700
3000
p<0.0075
p<0.005
p<0.0007
G. Henleyi
p<0.0005
ht
g
nelci
t
i
r
dn
e
dlatoT (m)
Females Males
EBR PBR MBR EBR PBR MBR
0
300
600
900
1200
1500
1800
2100
2400
2700
3000
p<0.04
G. Gerbilus
htgnelcitirdnedlatoT(m)
Females Males
1)
A) B) C) D)
E) F) G) H)
I) J) K) L)
Fig. 4 The long-term effects of predator cue exposure on morphology of dentate gyrus (DG) granular neurons. (1) The top panel depicts
the experimental protocol. The red circle signifies the test performed, for which the results are shown. AQuantitative analysis of total
dendritic length (μm), and (C) total dendritic number of dentate gyrus granule cells from the suprapyramidal blade in female and male Acomys
cahirinus,Gerbillus henleyi (E,G, respectively), and Gerbillus gerbillus (I,K, respectively). BQuantitative analysis of total dendritic length (μm), and
(D) total dendritic number of dentate gyrus granule cells according to the affected groups in Acomys cahirinus,Gerbillus henleyi
(F,Hrespectively), and Gerbillus gerbillus (J,L, respectively). Data are presented as data points and mean ± SEM.
Fig. 5 Computer-generated plots of reconstructions of the dendritic
trees from granule cells of female and male Acomys cahirinus,
Gerbillus henleyi, and Gerbillus gerbillus.
H. Cohen et al.
6
Molecular Psychiatry
Content courtesy of Springer Nature, terms of use apply. Rights reserved
Separating out the more clearly affected animals using CBC
enabled a more precise assessment of the magnitude and
character of the predator effect [25]. Accordingly, the prevalence
of severely behaviorally affected animals (PTSD phenotype) in
A-Cahirinus was 26.3% in females and 25% in males, whereas in
G-Henleyi it was 20% in females and 23% in males. The prevalence
of the PTSD phenotype in G-Gerbillus was 12.5% in females and
41.85% in males. Nevertheless, there were no statistically
significant differences in the prevalence rates of PTSD phenotype
among species or between sexes, but it is possible that the
absence of species and sex differences is a result of the low
statistical power due to the limited sample size. Future studies
should use larger sample sizes. Taken together, predator cue
exposure induces PTSD-like symptoms in wild rodents, similar to
those in laboratory rats [35–37] or mice [38].
Our results corroborate previous results on possible PTSD-like
symptoms in adult female wild captive wolves (Canis lupus),
elephants, chimpanzees and birds (black-capped chickadees,
Poecile atricapillus)[39–42].
HPA-axis
Retrospective analysis of FCM concentrations collected 1–2 hours
after predator exposure showed that among all wild rodent
species, the PTSD phenotype individuals were typified by a
blunting of the FCM response to the stressor, which was
significantly different from those observed in less or non-
affected groups. As expected, rodents with less extreme patterns
of behavioral responses (partial or minimal behavioral disruption)
displayed a significant increase in FCM stress response. The
validity of these findings was supported by the results of the
Pearson correlation analysis performed irrespective of the CBC
classification, which revealed that FCM concentrations were
significantly and negatively correlated with the anxiety index,
indicating that lower FCM concentrations shortly after exposure
predicted higher anxiety levels overall.
The blunted cortisol response indicates an underlying dysfunc-
tion in the overall dynamic modulation action of the HPA axis in
rodents with the PTSD phenotype. This blunted FCM concentra-
tion could prolong the availability of norepinephrine to synapses
in both the periphery and brain [43–45], which, in turn, might
affect the consolidation of the memory of the traumatic event.
Adrenergic activation in the face of low cortisol levels facilitates
fear memory in animals [46]. Additionally, glucocorticoids play an
important role during foraging, as their release controls appetite
and food intake [47]. Allenby’s gerbils implanted with cortisol
foraged longer, but harvested food more slowly due to greater
vigilance and apprehension than placebo-treated gerbils [48]. The
authors suggested that glucocorticoids affect energy acquisition
and provide a physiological context to explain how foragers
manage risks and the trade-off between food and safety [48].
Gutman et al. [34] studied FCM levels and foraging of nocturnal
A-Cahirinus and diurnal Acomys russatus and found that both
species exhibited high FCM concentrations and reduced foraging
when the moon was full, suggesting that reduced foraging may
be mediated by increased glucocorticoid concentrations [34]. Tree
lizards treated with exogenous glucocorticoids responded more
quickly to predator stress and hid for a longer duration (decreased
feeding effort/reduced foraging) than did control lizards [49].
Moreover, the HPA axis of wild strain birds reacted more quickly to
capture and handling (corticosterone levels increase within 250 s)
and more strongly (higher corticosterone peak values) than the
domesticated strain [50]. Together with our previous results and
the ecology literature (which is not limited to laboratory rodents),
a clearer picture of the HPA axis as a contributing factor to the
development of PTSD is starting to emerge. Based on this
evidence, we hypothesize that during an acute threat, the quick
initiation and strong modulation action of the HPA axis should
also entail rapid termination of the physiological stress response
through negative feedback inhibition (short-term duration) once
the treatment has passed [51]. Rapid return to the basal state
renews foraging behavior via its effect on locomotor activity,
which leads to better foraging capacity and increased dispersal
[51–53]. Increased dispersal may be an optimal strategy to escape
stressful small-scale events that persist over long periods of time
[52]. All the factors mentioned above could lead to a return to
routine, that is, faster recovery and preparedness to deal with
subsequent stressors. Quick initiation accompanied by rapid
termination of the HPA axis can directly or indirectly modify
behavior and enhance individual survival. In contrast, in the
absence of a rapid and strong HPA-axis response, rodents with the
PTSD phenotype are at greater risk of predation. However,
because predators represent an emergency that may require
split-second responses for survival, a delayed, slower, or faulty
release of glucocorticoid modulation action may result in costly or
even fatal consequences.
In G-Gerbillus, we found significant intersexual differences in
FCM concentrations, with females demonstrating significantly
higher levels than males. This is interesting because, in this
species, we also observed fewer PTSD phenotypes in females than
in males. These findings imply that higher levels of cortisol could
be protective against the development of PTSD and might explain
the disparity in findings in rodent and human studies. Female
mice and rats generally demonstrate higher plasma and adrenal
corticosterone levels and more adaptive stress responses [54–60],
whereas men (humans) have higher basal cortisol levels
associated with a lower prevalence of stress-related psychopathol-
ogy [61,62]. Therefore, one might hypothesize that higher basal
cortisol levels are protective against the development of PTSD.
As the HPA axis may serve as a key mechanism of development,
modulating growth, and maintenance across a diverse array of
taxa, a prolonged glucocorticoid stress response bears the risk of
high stress-induced physiological and energetic costs (depletion
of energetic stores) [50–52,63], which could also impair
reproduction.
In the wild, individual basal body condition, which is an
important factor in determining survival, is also a major
component of physiological stress responses [52]. A factor that
is less considered in laboratory conditions (according to ethics
instructions, individuals who lose weight or show signs of body
neglect or illness are excluded from studies).
Orexin-A levels
Next, we analyzed harvested brains to assess the consequences of
predator stress on orexin-A levels in the hypothalamic nucleus.
Orexin neurons, which are considered “multi-tasking”neurons,
receive a variety of signals related to environmental, physiological,
and emotional stimuli and project broadly to the entire central
nervous system [64–67]. Orexin neurons orchestrate various
aspects of survival via regulation of feeding behavior (energy
homeostasis) and sleep-wakefulness [67]. We found that eight
days after stress exposure, orexin-A levels in the hypothalamus
were downregulated in animals whose behaviors were severely
affected by the stressor (PTSD phenotype) in all three species but
did not change in the PBR and MBR groups in all species.
Moreover, there was a striking negative correlation between the
severity of the anxiety index and changes in the hypothalamus
orexin-A levels in all three species. Pearson’s correlation analysis
revealed that the downregulation of orexin-A levels was
significantly correlated with an increased degree of anxiety-
related behaviors, assessed by EPM, across all samples in each
species.
The reduction in hypothalamic orexin-A levels eight days after
stress exposure in PTSD phenotype rodents may have profound
ecological implications. The orexinergic system initiates, coordi-
nates, and maintains survival behaviors and survival-related
processes (unified orexinergic survival theory) [68]. When the
H. Cohen et al.
7
Molecular Psychiatry
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orexinergic system is activated in response to stress or alerting
stimuli, orexin neurons can initiate (and maintain) behavioral
stress responses by activating arousal, sensory, somatomotor,
visceromotor, hormonal, and other systems, enabling animals to
better prepare for, respond to, and cope with the acute demands
of physical and emotional threats to re-establish homeostasis [69].
In the wild, animals exhibit reactionary prey behaviors when
orexinergic neurons in the lateral hypothalamus of prey animals
are activated by adequate exteroceptive inputs detailing the
presence of a hungry predator [68]. Accordingly, adequate survival
responses require that animals maximize the functioning of their
sensory systems to critically evaluate their environmental changes,
such as threats or other life-threatening circumstances (i.e., food
deprivation) [68]. Thus, the decreased levels of hypothalamic
orexin-A in the PTSD phenotype eight days after predator stress
across all three species may affect survival behaviors and survival-
related processes, such as reduced alertness to potential threats,
diminished ability to respond proportionally to threats, which
together may increase predation risk.
These findings are supported by our previous report that the
orexinergic system orchestrates various aspects of survival
behaviors in response to predator stress and is related to the
pathophysiology of PTSD [69]. Our findings are also supported by
previous studies that investigated orexin expression in patients
with PTSD [70]. It has been demonstrated that cerebrospinal fluid
(CSF) and plasma orexin-A levels are significantly lower in patients
with PTSD than in healthy controls, and CSF orexin-A levels are
strongly and negatively correlated with PTSD severity, as
measured by the Clinician-Administered PTSD Scale, in patients
with PTSD [70].
Morphological changes
The morphological characteristics of DG granule cells were
evaluated using the Golgi-Cox method in all animals. All wild
rodent species whose behavior was extremely disrupted (PTSD
phenotype) selectively displayed significantly lower total dendritic
length along the DG neurons. The implications of these results are
that rodents with the PTSD phenotype were characterized by
severe atrophy in the DG subregion. Since the dendritic arbor is
responsible for receiving and consolidating neuronal information
input [71–73], the reduced dendritic arbor in the DG in PTSD
phenotype rodents can have considerable consequences for the
functional properties of cells and neuronal circuitry, including
decreased synaptic plasticity and synaptic strength and impaired
stabilization of synaptic connectivity, which may in turn lead to
vulnerability to psychopathology.
In contrast, rodents whose behavior was minimally affected or
unaffected (MBR) displayed significantly longer total dendritic
lengths and longer dendritic branches along the DG neurons. In all
wild rodent species that displayed partial behavioral responses,
the morphological response was intermediate or identical to that
of the MBR group. These findings were consistent with our
previous findings in rats [73]. Thus, these results suggest that
predator cue exposure leads to dendritic atrophy of dentate
granule cells, which probably decreases the amount of informa-
tion that neurons can obtain from the environment.
One limitation of this study is the relatively small sample sizes
employed. To establish the long-term, non-lethal effects of
predators on wild rodents, further research incorporating larger
sample sizes and longitudinal follow-up tests is necessary.
In summary, in wild rodent species, the prevalence of PTSD
phenotype was found to be 21.7% to 26.7% of the total
population, similar to that seen in laboratory rodents or
individuals with a history of trauma. In all three rodent species,
individuals who developed the PTSD phenotype to the predator
stimuli were characterized by a blunting of the FCM response, a
lower hypothalamic orexin-A level, and lower total dendritic
length in the DG granule cells eight days after exposure.
Phenotypically, this results in a significant functional impairment,
potentially impacting reproduction and survival, mediating
indirect effects of predators on prey demographics.
Allen et al. [74] experimentally manipulated fear in free-living
wild songbird populations over three annual breeding seasons by
intermittently broadcasting playbacks of either predator or non-
predator vocalizations and comprehensively quantified the effects
on all components of population growth, together with evidence
of a transgenerational impact on offspring survival as adults. They
found that fear itself significantly reduced the population growth
rate by causing cumulative, compounding adverse effects on
fecundity and every component of offspring survival, resulting in
predator playback parents producing 53% fewer recruits to the
adult breeding population [74].
CONCLUSIONS
Although PTSD is defined in terms of human responses, several
behavioral, physiological, and neurobiological survival responses
are shared between humans and other mammals (i.e., learning
and remembering about danger and responding to or avoiding
situations that present life-threatening risks), suggesting that just
as the adapted stress/threat response has evolutionary roots, the
post-traumatic stress syndrome has evolutionary roots as well.
Gerbils, wolves, elephants, and marine iguanas may seem
evolutionarily far from humans, but there are compelling scientific
reports to hypothesize that the ecology of fear [2], which may
manifest differently across different taxa, may be largely the same.
Contrary to the hypothesis that the impact of a predator on an
individual prey is either fatal or transitory (i.e., “fight or flight”), it
seems that some individuals that survive predation stress do not
continue their lives as before, but display long-lasting behavioral
and morphological dysfunction, that resemble human PTSD
responses, which could affect reproduction and survival. The
finding that PTSD symptoms may affect 1 in every 5 wild rodents,
which could negatively impact their fitness, has significant
ramifications for study animal behavior and population dynamics.
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AUTHOR CONTRIBUTIONS
CH, ZJ, AZ contributed to conception and design of the study. CH, MM, CC organized
the database. CH, MM, CC conducted the experiments. CH performed the statistical
analysis. CH and MM wrote the first draft of the manuscript.CH, MM, TD, CC, ZJ, HH and
AZ contributed to manuscript revision and read and approved the submitted version.
COMPETING INTERESTS
The authors declare no competing interests.
ADDITIONAL INFORMATION
Supplementary information The online version contains supplementary material
available at https://doi.org/10.1038/s41380-023-02240-7.
Correspondence and requests for materials should be addressed to Hagit Cohen.
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