Behavioural Brain Research 279 (2015) 7681

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Behavioural Brain Research

journal homepage: www.elsevier.com/locate/bbr

Research report

Fox urine exposure induces avoidance behavior in rats and activates

the amygdalar olfactory cortex
K.E.A. Wernecke a,d, , D. Vincenz b,d , S. Storsberg c,d , W. DHanis c,d , J. Goldschmidt b,d ,
M. Fendt a,d
a

Institute for Pharmacology and Toxicology, Otto-von-Guericke University Magdeburg, Magdeburg, Germany
Leibniz Institute for Neurobiology, Magdeburg, Germany
c
Institute for Anatomy, Otto-von-Guericke University Magdeburg, Magdeburg, Germany
d
Center for Behavioral Brain Sciences, Magdeburg, Germany
b

h i g h l i g h t s

Exposure to predator odors induces defensive behaviors in rats.

The AOC is interconnected with both olfactory systems and the defense circuit.
However, its role in predator-induced fear behavior is poorly understood.
Temporary inactivation of the AOC disrupts fox urine-evoked avoidance behavior.
Thus, we conclude that the AOC is critical for predator odor-induced fear behavior.

a r t i c l e

i n f o

Article history:
Received 30 October 2014
Received in revised form 6 November 2014
Accepted 8 November 2014
Available online 15 November 2014
Keywords:
Innate fear
Predator odor
Amygdala
Fox urine
Rat

a b s t r a c t
Predator odors represent a group of biologically-relevant chemosignals called kairomones. Kairomones
enable prey animals to recognize potential predatory threats in their environment and to initiate
appropriate defensive responses. Although the behavioral repertoire of anti-predatory responses (e.g.
avoidance, freezing, risk assessment) has been investigated extensively, our knowledge about the neural
network mediating these innate fear responses is rather limited. In the present study, the GABAA agonist
muscimol was bilaterally injected (2.6 nmol/0.3 l) into the amygdalar olfactory cortex (AOC), a brain
area that receives massive olfactory input from both olfactory systems and is strongly interconnected
with the medial hypothalamic defense circuit. Temporary inactivation of the AOC substantially disrupted
avoidance behavior of rats to fox urine that is strongly avoided under control conditions (saline injections). Taken together, these results demonstrate that the AOC is critically involved in fox urine-induced
fear behavior. This suggests that the AOC is part of a brain fear circuit that mediates innate fear responses
toward predatory odors.
2014 Elsevier B.V. All rights reserved.

1. Introduction
Many animal species rely on their sense to smell (olfaction),
i.e. the detection and discrimination of complex olfactory signals in
the surrounding, to guide their behaviors [1]. If these olfactory signals are transmitted between individuals of the same species, they

Corresponding author at: Institute for Pharmacology and Toxicology, Medical

Faculty of the Otto-von-Guericke University Magdeburg, Leipziger Strae 44, D39120 Magdeburg, Saxony-Anhalt, Germany. Tel.: +49 391 67 15880;
fax: +49 391 67 15869..
E-mail address: kerstin.wernecke@med.ovgu.de (K.E.A. Wernecke).
http://dx.doi.org/10.1016/j.bbr.2014.11.020
0166-4328/ 2014 Elsevier B.V. All rights reserved.

serve as important communication tools (pheromones) essential

for reproductive, territorial and maternal behaviors [10,16,17,28].
Predator odors are a distinct class of biologically-relevant olfactory signals. If recognized by prey animals, they signal the potential
presence of a predatorthereby alerting the prey animal and facilitating its survival. Such odors connected to a selective disadvantage
for the releaser while being benecial for the perceiving animal (of
another species) are termed kairomones [25,52].
Predator odors can induce an array of different anti-predatory
responses (reviewed in [3,15,60]). For example, when exposed
to odors from natural predators (e.g. from cat or ferret) or to
2,3,5-trimethyl-3-thiazoline (TMT), a compound isolated from fox
feces, rodents display avoidance and hiding behavior, freezing and

K.E.A. Wernecke et al. / Behavioural Brain Research 279 (2015) 7681

startle-potentiation. Non-defensive behaviors such as grooming

and overall locomotor activity are suppressed [8,18,21,39,40,64].
Rats will also engage in a variety of risk assessment behaviors
directed toward the odor source [6]. Since laboratory rodents
have been reared and bred without being exposed to predators
or predator odors for several hundred generations, it has been
suggested that the recognition of predator odors is mainly innate
[15,23].
Over the last years, our understanding of the neural systems
underlying innate fear to predator odors has been substantially
improved. The amygdala is important for both the detection of
threatening stimuli and the generation of defensive behaviors
[31,32,58]. However, the contribution of single amygdaloid subnuclei in processing predator odor-induced defensiveness is poorly
understood. Since the medial nucleus of the amygdala (MeA)
receives strong olfactory input [13,38], most of the research was
focused on this subnucleus. It was demonstrated that the MeA and
the basolateral nucleus of the amygdala (BLA) are crucial for both
cat odor- and TMT-induced fear behavior [5,33,44,59,63]. Lesions
or pharmacological inactivation of other subnuclei like the lateral
and central nucleus did not reliably affect predator odor-induced
behavioral changes [22,33,65].
In addition to the MeA, the amygdalar olfactory cortical group
(AOC) receives massive projections from both the main and accessory olfactory system [27,56]. The AOC, dened by Swanson and
Petrovich [56], includes the cortical nucleus of the amygdala (CoA),
the amygdalopiriform transition zone (APir) and the nucleus of
the lateral olfactory tract. The AOC heavily projects to the medial
hypothalamus [27,43,56], a region critical for the expression of
innate defensive behaviors [12]. Different studies emphasized the
importance of the AOC in sociosexual behaviors [35,41], although
a potential role in the processing of innate fear behavior has not
been studied yet.
In the present study, we investigated the role of the AOC in
predator odor-induced fear behavior in rats. Therefore, we temporary inactivated the AOC by local microinjections of the GABAA
receptor agonist muscimol. We then tested the effects of AOC
inactivation on fear behavior (avoidance behavior, general motor
activity) induced by exposing the animals to samples of fox urine
(cf. [21]).
2. Materials and methods
2.1. Subjects
Subjects were experimentally naive male Sprague-Dawley rats
(23 months old) weighing 220350 g at the time of the surgery.
Rats were bred and reared at the local animal facility (original
breeding stock: Taconic, Denmark). Animals were housed in groups
of 56 animals in standard Macrolon Type IV cages in temperaturecontrolled rooms (22 2 C) and maintained on a 12 h light/dark
cycle with lights on at 6:00 am. Water and food were available
ad libitum. All experiments were conducted during the light phase
between 8:00 am and 2:00 pm. The experiments were carried out in
accordance with the ethical guidelines of the National Institutes of
Health for the care and use of laboratory animals for experiments,
and were approved by the local authorities (Landesverwaltungsamt
Sachsen-Anhalt, Az. 42505-2-1172 UniMD).
2.2. Surgery
Rats were anesthetized with 2.02.5% isourane (5% for induction) in oxygen (2.4 l/min) and xed in a stereotactic frame with
blunt ear bars and with the incisor bar set at 3.3 mm relative to
the interaural line. Stainless steel guide cannulas (8.0 mm length;

77

0.7 mm outer diameter) were implanted bilaterally aiming at the

AOC (4.4 mm rostral, 4.8/5.5 mm lateral, 9.3 mm ventral to
bregma) [50]. The cannulas were xed to the skull with three
anchoring stainless steel screws and dental acrylic cement. After
surgery and between testing, stainless steel stylets (0.3 mm outer
diameter) were inserted into the guide cannulas to prevent occlusion. Rats were allowed one week to recover prior to the start of
behavioral testing.
2.3. Test apparatus
Testing took place in one of four identical testing boxes
(45 cm 45 cm 30 cm) constructed from opaque polyvinyl chloride. The boxes were placed inside an animal detection frame
(TSE Systems, Bad Homburg, Germany) with infrared light sensors
(14 mm distance between two sensors) which enables automatic
measurement of the locations of the rats and thereby their movements too. Each box was located in a sound-attenuating chamber
provided with four light sources (illumination: <10 lx) and an
observation camera. Four small glass bowls (44 mm diameter) were
placed and xed in each corner of the testing box.
2.4. Procedure for odor exposure
Rats were given three 10-min habituation sessions without odor
to familiarize themselves to the testing boxes (once per day on
different days). On the following days, each animal received (in
a pseudo-randomized order) bilateral microinjections of either
2.6 nmol muscimol (dissolved in 0.3 l saline) or 0.3 l of only
saline into the AOC. Solutions were injected at a rate of 0.2 l/min
using 10-l microsyringes (Hamilton, Bonaduz, Switzerland) and
a microinfusion pump (CMA Microdialysis, AB, Kista, Sweden)
connected to injection cannulas (0.3 mm outer diameter) via a
polyethylene tube. Following injections, the injection cannulas
were left in place for an additional minute to optimize diffusion into
the tissue. 1015 min later, the animals were placed into the exposure boxes and either exposed to 1 ml water or 1 ml fox urine (Main
Odor Solutions, Maine, USA). For this, the respective odor sample
was pipetted into a glass bowl in one corner. Each rat was tested
once per day and four times in total in a pseudo-randomized order
(saline injection/exposure to water, saline injection/exposure to
fox urine, muscimol injection/exposure to water, muscimol injection/exposure to fox urine) with the odor corner also changed in a
pseudo-randomized fashion. The rats behavior was monitored for
10 min by the infrared detectors (sample rate: 100 Hz). The TSE software automatically calculated the rats location for predened time
intervals and the general motor activity (distance traveled [in m],
number of rearings). Avoidance behavior was measured for each
minute by calculating the mean percentage of time the rat spent in
the quadrant (8 8 sensors) containing the test stimulus. The testing boxes were thoroughly cleaned with soapy water after each test
and ventilated with clean air.
2.5. Histology
After completion of behavioral testing, rats were sacriced
by carbon dioxide. Brains were rapidly removed and stored in
10% sucrose-formalin solution followed by 20% and 30% sucroseformalin solution for cryoprotection. Afterwards, serial 40 m
coronal sections were sliced with a cryostat (Leica, Nussloch,
Germany) and, after drying overnight, sections were Nissl-stained
with cresyl violet. The injection sites were veried under a
light microscope and mapped on schematic coronal sections
from employing the rat brain atlas of Paxinos and Watson
[50].

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2.6. Statistical analysis

Behavioral data were expressed as means standard errors
of the mean (SEM). Since data was not normally distributed
(DAgostino and Pearson omnibus test), avoidance behavior (mean
% time in odor quadrant) was analyzed using a non-parametric
analysis of variance (Friedman test) followed by Dunns multiple
comparisons test. For the analysis of the normally distributed motor
activity data, we accomplished a two-way repeated measures analysis of variance (ANOVA) using drug treatment (saline/muscimol)
and odor (water/fox urine) as within-subject factors. Post-hoc analysis was performed using Sidaks multiple comparisons test. For all
statistical evaluations, a p < 0.05 was considered statistically significant.
3. Results

Fig. 2. Photomicrograph showing a Nissl-stained coronal section with a representative injection site in the AOC.

3.1. Histology

3.2. Avoidance behavior

Fig. 1 shows a schematic reconstruction of the injection sites of

the rats receiving infusions of saline and muscimol into the area of
the AOC as veried by histological examination. A photomicrograph
of a Nissl-stained section from a representative injection site into
the AOC is shown in Fig. 2. Only animals with bilateral injections
localized within or very close to the AOC, i.e. inside the posterior division of the lateral/medial cortical or basolateral nucleus
of the amygdala (PLCo, PMCo, BLP), the posterolateral division of
the amygdalo-hippocampal area (AHiPL) or the amygdalopiriform
transition zone (APir), were included in the behavioral analysis
(n = 15).

Two rats were excluded from this analysis since they displayed
inappropriate behavioral responses toward fox urine after saline
injections (approach instead of avoidance behavior). Fig. 3 illustrates the mean percentage of time rats spent in the odor quadrant
during the 10-min exposure to water or to fox urine, respectively,
for the different treatment conditions. A non-parametric repeatedmeasure ANOVA showed that the different test conditions (odor
and treatment) signicantly affected the time spent in the odor
quadrant (Friedman test: Q = 18.28, p = 0.0004). Post-hoc pairwise
comparisons revealed that after saline injections into the AOC, rats
spent signicantly less time in the quadrant with the fox urine sample than in the quadrant with water (Dunns test: p < 0.001), i.e. the
quadrant with fox urine was avoided. After muscimol injections,
this avoidance response toward fox urine was signicantly reduced
(Dunns test, comparison saline-fox urine vs. muscimol-fox urine:
p < 0.001). Notably, the behavior during exposure to the control
odor water was not affected (Dunns test, comparison saline-water
vs. muscimol-water: p > 0.05).
3.3. General motor activity
The distance traveled (Fig. 4) of the rats was not affected by
exposure to fox urine (ANOVA, factor odor: F(1,14) = 1.31, p = 0.35)

Fig. 1. Reconstruction of the injection sites (lled triangles) on schematic diagrams of coronal brain sections [50]. Numbers indicate the caudal distance (in
millimeters) from bregma. Abbreviations: AHiAL, anterolateral part of the amygdalohippocampal area; AHiPL, posterolateral part of the amygdalo-hippocampal area;
AHiPM, posteromedial part of the amygdalo-hippocampal area; APir, amygdalopiriform transition area; BLP, posterior part of the basolateral amygdaloid nucleus;
BMP, posterior part of the basomedial amygdaloid nucleus; PLCo, posterolateral
cortical amygdaloid nucleus; PMCo, posteromedial cortical amygdaloid nucleus.

Fig. 3. Mean percentage of time (SEM) rats occupied the odor quadrant (chance
level = 25%), containing either water (black bars) or fox urine (white dotted bars)
samples. Rats received bilateral injections of either saline or muscimol into the
AOC. ** p < 0.01 comparison with the appropriate water control; ++ p < 0.01 comparison with the appropriate saline condition (Dunns multiple comparison tests
after signicant main effects in a Friedman test).

K.E.A. Wernecke et al. / Behavioural Brain Research 279 (2015) 7681

79

Fig. 4. General locomotor activity of rats, expressed as (A) the distance traveled in cm (+SEM) and (B) the number of rearings, during the 10-min exposure test under the
indicated treatments (saline/muscimol injections) and exposure conditions (water/fox urine). ** p < 0.01 compared with the appropriate saline condition (Sidaks multiple
comparison test after signicant main effects in an ANOVA).

but by muscimol injections (factor treatment: F(1,14) = 15.88,

p = 0.003). However, the interaction between odor and treatment clearly failed to reach statistical signicance (F(1,14) = 0.72,
p = 0.30) indicating that the weak increase of locomotor activity in muscimol-injected animals cannot explain the effects on
avoidance behavior described above. Regarding vertical activity
(number of rearings), the statistical analysis revealed that neither the treatment factor (F(1,14) = 0.71, p = 0.41) nor the odor factor
(F(1,14) = 0.0001, p = 0.99) exhibited signicant differences.
4. Discussion
The aim of the present study was to test whether the AOC is
involved in fox urine-induced fear behavior. Specically, we temporarily inhibited the AOC by local microinjections of the GABAA
receptor agonist muscimol and measured the effects of fox urine
exposure on avoidance behavior and locomotor activity. Our results
demonstrated that temporary inactivation of the AOC substantially
disrupted avoidance behavior of rats in response to fox urine that
is strongly avoided under the control condition (saline injection).
Also, muscimol treatment slightly increased the distance traveled
of rats exposed to both fox urine and water, while leaving horizontal
activity unaffected.
4.1. Fox urine-induced avoidance behavior
In the present study, saline-treated rats displayed robust avoidance behavior, i.e. approximately 6% of their time was spent in the
quadrant with fox urine (chance level would be 25%). Similar fearevoking properties of fox urine have been reported several times in
fox urine-exposed rats and mice showing higher levels of freezing,
suppressed locomotor activity, and elevated c-Fos immunoreactivity in brain regions involved in fear [20,21,26,30,34]. This data,
together with our results, indicate that fox urine functions as a
predatory signal that reliably induces fear behavior in rodents. In
contrast to fox urine, urine samples from other carnivore species
produce rather inconsistent effects. In nature, the urine of larger
carnivores (e.g. dingo, coyote, bobcat, wolf) has been proven to be
very effective repellents protecting forestry and agricultural areas
from feeding-related damage (reviewed in [3,9,46,49,53,55,57]).
Also, such urine samples robustly induce defensive behaviors under
laboratory conditions [21,24,47,67]. In contrast, urine samples of
smaller carnivores (e.g. cat, ferret) often failed to induce fear-like
behavioral effects [7,39]. Interestingly, Ferrero et al. [24] identied
a biogenic amine, 2-phenylethylamine (PEA) to be the key constituent of a carnivore urine blend that triggers innate avoidance

behavior in rodents. In comparison to the PEA-enriched urine of

larger carnivores, both the urine from cats and ferrets contain relative low levels of PEA which could be a likely explanation for the
absence of fear behavior to these odors. In line with this, Fendt [21]
observed that the urine of larger carnivores (e.g. bobcat, cougar)
induced even stronger avoidance responses than fox urine.
In the present experiment, rats exposed to water explored the
odor quadrant 24% of the time, which corresponds with the
chance level. However, the behavioral responses to the control odor
varied markedly among the animals tested. To minimize this interindividual variability, we habituated them to the testing boxes on
three days prior to testing.
As mentioned in the Introduction, two of the most widely used
predator odors in recent research are cat odor from fur/skin and
TMT. These odors not only elicit avoidance behavior as observed
in the present study with fox urine but also induce freezing and
risk assessment behaviors such as at back approaches or head
outs [8,15,19,40,45,64]. In the present study, fox urine-exposed rats
displayed only infrequent risk assessment (two animals) and no
freezing behavior. The lack of these behaviors in our study may be
caused by differences in the setup. For instance, Dielenberg and
McGregor [15] gave rats the opportunity to retreat into a hide box
and the Blanchards [8] established a semi-natural visible burrow
system. In contrast, we tested rats in rather simple testing boxes
that do not offer any possibility to hide. This suggests that a more
complex testing environment may support risk assessment. Freezing behavior, on the other hand, is expressed when the threatening
stimulus is very intense (e.g. foot shock, high concentration of TMT)
and inescapable (e.g. when tested in a small, conned test chamber)
[54,64]. Samples of fox urine, as used in the present study, may be
considered as relatively weak threatening stimuli. Taken together,
the experimental setup and the intensity of odor samples used in
the present study seem to specically induce avoidance behavior
and do not support risk assessment or freezing behavior.
4.2. Local muscimol injections
We used muscimol, a GABAA receptor agonist, to reversibly
inactivate neurons within the AOC. Local muscimol injections
lead to an unselective and rapid suppression of local neuron
activity which lasts between 1 and 2 h [4,37,62]. Such temporary
inactivations represent an important alternative to chronic lesions
because they allow within-subject designs. However, one issue of
this method is that it is difcult to determine the spatial spread of
the drug around the injection site. Injections of 0.3 l muscimol,
labeled with a dye, have been shown to diffuse 0.51 mm away
from the injection site [2]. Therefore, we assumed that muscimol

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K.E.A. Wernecke et al. / Behavioural Brain Research 279 (2015) 7681

injected very close to the AOC, i.e. into the AHiPL and BLP, will most
probably diffuse to the AOC, too. Due to this, we also included rats
with injection sites within the AHiPL and BLP into the behavioral
analysis of the present study. Otherwise, diffusion of muscimol
to the ventral hippocampus is unlikely since myelinated ber
tracts (alveus of the hippocampus, deep cerebral white matter)
that separate hippocampus and amygdala usually act as effective
diffusion barriers. Furthermore, unpublished data from our lab
showed that intraventricular muscimol injections induce strong
sedative effects, i.e. locomotor activity is decreased. Since we
observed a mild increase in locomotor activity in the present
study, a diffusion of muscimol from our injection sites into the
ventricular system is not very likely.

5. Role of the AOC

The main nding of the present study is that, in rats, temporary
inactivation of the AOC by local muscimol injections prevents
avoidance behavior toward fox urine. To the best of our knowledge, this is the rst study showing that the AOC is crucial for
predator odor-induced fear behaviors. Nevertheless, our ndings
are not surprising since the AOC is strongly interconnected with
both olfactory systems, as well as brain areas mediating fear
behaviors. Now the question arises how the AOC is implemented
into the brain circuit of innate fear? Olfactory information from
the main olfactory bulb is transmitted to the anterior (ACoA) and
posterior-lateral CoA (PLCo), as well as to the APir. Projections
from the accessory olfactory bulb, known to also convey predatorrelated pheromonal information [61], primarily terminate in the
posterior-medial CoA (PMCo) (and the MeA) [27,48,56]. Therefore,
the AOC could be regarded as the rst limbic/emotional brain
structure after the primary sensory brain regions, where olfactory
information is prossessed and integrated. With regard to our injection sites within both main olfactory- and vomeronasal-recipient
areas of the amygdala, we cannot say which olfactory system
plays a critical role in the processing of fox urine. Until recently,
little is known about how predator odor-derived kairomones are
detected and processed. Funk and Amir [26] observed that fox
urine induced high Fos expression in main olfactory pathways
that is strongly modulated by the circadian cycle. Also, both TMT
and lion urine-derived PEA seem to exert their aversion responses
by stimulating subsets of olfactory sensory neurons located in
the dorsal olfactory epithelium [24,29]. On the other side, Osada
et al. [47] demonstrated that volatile pyrazine analoges that were
identied in wolf urine and initiate defensive behaviors in mice
activate the accessory olfactory bulb, whereby the contribution
of the main olfactory system could not be excluded. Indeed, the
involvement of both olfactory systems is conceivable because there
is a complex network of interconnections between subregions
within the AOC. Especially, the PMCo and PLCo are good candidate
structures for olfactory-vomeronasal convergence, since both
subnuclei are strongly bidirectional connected [27,51].
In addition, the AOC provides via the BLP and BMP strong input
to the medial hypothalamic defense circuit, a network of distinct
medial hypothalamic subnuclei (anterior hypothalamic nucleus,
dorsomedial part of the ventromedial nucleus, dorsal premammillary nucleus) that plays a key role in initiating and regulating fear
behaviors [12,56]. Moreover, different studies using Fos immunohistochemistry and lesion methods have provided evidence that
these hypothalamic areas are involved in the control of antipredator defensiveness following exposure to both live predators or
their smell [5,14,36]. As a consequence, the AOC can modulate
fear behavior by directly affecting hypothalamic function which
in turn controls other brain areas involved in the expression of fear
behavior (e.g. periaqueductal gray).

Muscimol injections into the AOC further enhanced horizontal locomotor activity (distance traveled) during exposure to
both water and fox urine. However, vertical locomotor activity
(rearings) was not affected. We think that this increase in horizontal locomotor activity reects a general effect of AOC inactivation
on novelty-induced fearfulness, suggesting that the AOC is also part
of an amygdala network that is implicated in neophobic responses
toward novel but not frightening stimuli [42,66]. Consistent with
this view, bilateral excitotoxic lesions of of the BLA enhanced
exploratory behavior of rats to both fake and real cat hair [63] and
increased locomotion in a novel environment [11].
Taken together, the present study demonstrate that the fox
urine-induced avoidance response is diminished by temporary
inactivation of the AOC, an amygdala region that receives strong
olfactory input. This indicates that the AOC is part of an innate fear
circuit mediating fear responses toward predatory odors.
Acknowledgements
The authors thank Timothy French for language editing on the
manuscript and Kathrin Freke for her help in animal care. This study
was funded by the federal state of Saxony-Anhalt, Project: Center
for Behavioral Brain Sciences (CBBS).
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