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Institute for Pharmacology and Toxicology, Otto-von-Guericke University Magdeburg, Magdeburg, Germany
Center of Behavioral Brain Sciences, Otto-von-Guericke University Magdeburg, Magdeburg, Germany
h i g h l i g h t s
g r a p h i c a l
a b s t r a c t
a r t i c l e
i n f o
Article history:
Received 19 October 2016
Received in revised form 6 December 2016
Accepted 7 December 2016
Available online 10 December 2016
Keywords:
Hypocretin
Light-dark box
Locomotor activity
Open eld
Predator odor avoidance
Unconditioned fear
a b s t r a c t
The loss of orexin neurons in humans leads to the disease narcolepsy, characterized by daytime sleepiness
and cataplexy. Recent data suggest that orexin is also involved in emotional processing. The goal of
the present study was to evaluate fear and safety learning as well as unconditioned fear (anxiety) in
orexin-decient animals. Orexin-decient mice are an established animal model used to investigate
the neuropathology and potential treatments for narcolepsy. Here, we present novel data showing that
orexin-decient mice express increased anxiety in the open eld, light-dark box test and carnivore odorinduced avoidance, but are normal in fear and safety learning. These ndings suggest an important role
of orexin in brain areas involved in anxiety.
2016 Published by Elsevier B.V.
1. Introduction
The neuropeptide orexin (also called hypocretin) plays an
important role in the regulation of sleep/wakefulness, feed-
daytime sleepiness, cataplexy (a loss of muscle tone), sleep paralysis, and hypnagogic hallucinations [3,9]. Similar symptoms are
observed in mice and dogs in which the orexin gene or the orexin2 receptor gene is decient or mutated [1012]. Thus, narcoleptic
mice and dogs are used as animal models of narcolepsy not only to
investigate the underlying neuropathology, but also to develop and
test pharmacological treatments for human narcolepsy [1317].
Recently, orexin has been linked to emotions and emotional
learning. Several studies demonstrated that orexin-1 receptors
are involved in the acquisition, consolidation and/or extinction of
conditioned fear [1820]. Furthermore, blockade or deciency of
orexin-1 receptors led to an anxiogenic- and depression-like phenotype in mice [2123]. Interestingly, orexin-2 receptors appear to
be involved only in contextual fear learning [18,20,23]. Notably, the
effects of orexin deciency on fear and anxiety were poorly investigated in laboratory rodents so far. Clinically, narcolepsy patients,
pathologically characterized by a loss of orexin, show a decreased
amygdala activation during fear conditioning [24] and express an
impaired startle response modulation by aversive slides [25] but
rate aversive slides as more unpleasant [26].
In the present study, we evaluated fear and anxiety behavior in
orexin-decient mice. To avoid interferences with the narcoleptic
phenotype of orexin-decient mice, we performed the experiments in the light period, when narcoleptic episodes are absent or
extremely rare, respectively [2730]. First, we measured locomotor
activity with the open eld test. Then, we characterized emotional
learning of orexin-decient mice in cued and contextual fear conditioning, as well as in safety conditioning, followed by measuring
the mices reactivity to aversive electric stimuli. Lastly, anxietylike behavior was assessed in the light-dark box paradigm and the
carnivore odor-avoidance test. The present data show that orexindecient mice exhibit increased anxiety-like behavior but show
normal behavior in fear and safety learning.
2. Material and methods
2.1. Animals
Orexin-decient mice (B6.129S6-Hcrttm1Ywa ) purchased from
the University of Texas, Dallas, USA [10] and backcrossed for more
than 10 generations with C57BL/6 mice were used for the present
study. In orexin-decient mice, the exon 1 of the prepro-orexin
gene is replaced by a nuclear lacZ/neomycin resistance cassette
which leads to an absence of orexin in these mice [10]. For genotyping, a Taqman assay was used. The mice were kept in groups
of up to four animals per cage in a humidity- and temperaturecontrolled room (5055%, 22 2 C) with a light cycle of 12 h on/off
periods (lights-on at 6:00 a.m.). Water and food were available
ad libitum. The experiments were performed during the lights-on
phase between 1:00 and 6:00 p.m. Animals were at an age of 23
months during the tests and if not stated differently, both genders
were used.
All animal experiments were performed in compliance with
international guidelines regarding the care and use of animals for
experimental procedures (2010/63/EU) and with conrmed ethical
approval (Landesverwaltungsamt Sachsen-Anhalt, 42502-2-1172
Uni MD).
2.2. Apparatus & behavioral protocol
2.2.1. Multiple tests and test order
All mice which were submitted to the open eld test were later
tested rst in the light-dark box and second in carnivore odorinduced fear. There was a break of 23 days between the different
test days. In the carnivore odor-induced fear paradigm, some addi-
211
tional naive mice were tested. For fear and safety learning, only
naive animals were used. Two days later, approximately half of
these mice were then tested for reactivity to electric stimuli.
2.2.2. Open eld
We used four identical black boxes (46 cm x 46 cm x 32 cm)
located in sound-attenuating chambers (background noise: 55 dB
SPL; illumination: 300 lx). The behavior of the mice was videotaped by an observation camera (CCTV camera, RS Components
GmbH, Mrfelden-Walldorf, Germany) mounted on the ceiling
of the chambers. A tracking system (EthoVision XT 11, Noldus,
Wageningen, Netherlands) recorded the spatiotemporal measures
of the mices movement and was used to collect and analyze behavioral data (distance travelled, inactivity, time spent in the center of
the open eld).
For this experiment, we placed each mouse (n = 30; 19 females,
31 males) into the center of a box and measured locomotor activity
for 10 min.
2.2.3. Fear learning
We used a computerized fear-conditioning system (TSE Systems, Bad Homburg, Germany). This setup consists of four identical
transparent Perspex boxes (46 cm 46 cm 32 cm), which were
surrounded by infrared animal detection sensor frames. Each box
was located in a sound-attenuating chamber provided with loudspeakers for the acoustic stimuli (background noise of 55 dB SPL
and the tone stimuli for fear conditioning), light sources (continuous illumination of ca. 10 lx), and a ventilation fan. The oor of
the boxes consisted of removable stainless steel grids (bars: 4 mm
diameter, distance: 9 mm) which were connected to a shock unit
and able to deliver foot shocks. Delivery of all stimuli was controlled
by the TSE Fear Conditioning software. To create a different test
context, we utilized four additional boxes as those described above
but made of black Perspex (including the oor). Movements of the
animals were detected by the infrared sensors (distance: 14 mm).
We dened freezing behavior as no infrared beam crosses for more
than 1 s. In addition, distance travelled was automatically recorded
during all phases of the experiments. The automatic measurement
of freezing in the TSE fear conditioning system was previously validated by demonstrating a high correlation with observer scoring
of freezing [31,32].
We placed the mice (n = 48; only males) individually into the
transparent boxes for fear conditioning. Sixty seconds later, the
rst of six pairings of a tone stimulus (10 kHz, 80 dB, 30 s; inter trial
interval: 90 s) and a scrambled foot shock (0.6 mA, during the last
two seconds of the tone stimulus) was presented. Thirty seconds
after the last pairing, we put the mouse back in the home cage.
On the next day, we placed the animals back into the conditioning boxes (transparent boxes) for about 10 min, and the freezing
response to the conditioning context was quantied (retention test
on contextual fear). The mice were then returned to the home cage.
Three hours later, we assessed the retention to cued fear. For this,
we put the mice into the black boxes, and after a habituation period
of 2 min, 10 tone stimuli (without foot shocks) were presented with
an interstimulus time (ISI) of 1 min (retention test on cued fear).
These retention tests on contextual and cued fear were repeated
on the two following days.
2.2.4. Safety learning
This experiment was also performed in the TSE fear conditioning
system.
First, the mice (n = 36; 24 females, 12 males) were habituated
to the transparent conditioning boxes for 2.5 min. On the two next
days, two trials of safety conditioning were performed. For each
trial, the mice were placed individually into the boxes and then
exposed to ve explicit unpairings of a tone stimulus (10 kHz, 85 dB,
212
Fig. 1. Open eld test: Depicted is (A) the mean distance travelled [ SEM] of wildtype (WT, n = 20), heterozygous (HT, n = 19) and homozygous (KO, n = 11) orexin-decient
mice as a function of time, as well as (B) number of inactivity epochs lasting longer than 1 s or 5 s, respectively, and (C) the time spent in the center of the open eld. Statistical
analyses revealed no genotype effects for distance travelled and number of inactivity epochs but a reduction of the time spent in the center in orexin-decient mice. * P < 0.05,
post-hoc Dunns multiple comparisons with wildtype mice after signicant main effects in Kruskal-Wallis test.
213
Fig. 2. Fear learning: Shown is the percent time spent with freezing during the fear conditioning procedure (A) and the retention tests on contextual fear (C + D) and cued
fear (E + F), as well as the locomotor reactivity to foot shocks during fear conditioning (B). Wildtype (WT, n = 16), heterozygous (HT, n = 14) and homozygous (KO, n = 18)
orexin-decient mice were tested. Statistical analyses revealed no signicant genotype effects but a trend for reduced locomotor reactivity to foot shocks in KO mice.
rst ve minutes of the open eld test (Fig. 1C); Kruskal-Wallis test:
H = 7.13, P = 0.03).
3.2. Fear learning
In our second experiment, we evaluated potential genotype
effects on fear learning (Fig. 2). We observed that freezing behavior of the mice was not affected by genotype, neither in the fear
conditioning phase (Fig. 2A; F2,45 = 0.81, P = 0.45) nor during the
retention tests either on contextual (Fig. 2C; F2,45 = 0.16, P = 0.86)
or cued fear (Fig. 2E; F2,45 = 1.60, P = 0.21). As expected, there was a
remarkable increase in freezing time during the fear conditioning
phase reecting fear learning (F5225 = 38.55, P < 0.0001). During the
retention tests, freezing time decreased reecting within-session
fear extinction (contextual fear: F9405 = 9.91, P < 0.0001; cued fear:
F9405 = 4.89, P < 0.0001). Notably, the genotype did not interact with
the factor time (Fs < 1.53, Ps > 0.13). Furthermore, baseline freezing levels were not affected by genotype (Fs < 1.41, Ps > 0.25). These
baseline freezing levels were measured in the periods before the
rst tone presentation of the fear conditioning session and of the
retention test on cued fear (indicated by p in Fig. 2A, E).
214
Importantly, the percent time (Fig. 5A) and the percent distance travelled by mice (Fig. 5B) in the bright compartment of the
light-dark box were reduced in orexin-decient mice (F2,49 = 3.55,
P = 0.04 and F2,49 = 3.22, P = 0.049, respectively). Both measures indicate increased anxiety. Post-hoc comparisons revealed signicant
differences between wildtype and homozygous orexin-decient
mice (ts > 2.38, Ps < 0.04) but not between wildtype and heterozygous orexin-decient mice (ts < 0.31, Ps < 0.93). Furthermore,
genotype did signicantly affect the number of transitions between
the bright and the dark compartment (Fig. 5C; F2,49 = 5.98,
P = 0.005). Notably, post-hoc comparisons with wildtype mice
revealed a signicant reduction in both heterozygous (t = 2.11,
P = 0.04) and homozygous (t = 3.37, P = 0.003) orexin-decient mice.
Lastly, there was a trend for an increase in the latency to enter
the bright compartment in orexin-decient mice (Fig. 5D, KruskalWallis test: H = 4.84, P = 0.09).
Fig. 3. Safety learning: Shown is the mean reactivity to the foot shocks during the
two conditioning phases (A), the freezing to the conditioning context and to safety
CS presentation during the retention test (B) and the percent inhibition by the safety
CS (C). Wildtype (WT, n = 10), heterozygous (HT, n = 14) and homozygous (KO, n = 12)
orexin-decient mice were tested. Statistical analyses revealed no genotype effects.
215
Fig. 4. Locomotor reactivity to foot shocks: Depicted is the mean distance moved of wildtype (WT, n = 19), heterozygous (HT, n = 13) and homozygous (KO, n = 15) orexindecient mice during the application of foot shocks with increasing intensities. Statistical analyses revealed no genotype effects.
Fig. 5. Light-dark box test: Wildtype (WT, n = 20), heterozygous (HT, n = 19) and homozygous (KO, n = 11) orexin-decient mice were tested. Depicted are the percent time
(A) and the percent distance travelled (B) in the bright compartment, the latency to enter the bright compartment (C), and the number of transitions between the two
compartments (D). Orexin-decient mice expressed behavioral changes indicating increased anxiety. This effect was more robust in homozygous than in heterozygous
orexin-decient mice. * P < 0.05, ** P < 0.01, post-hoc Dunnetts multiple comparisons with wildtype mice after signicant main effects in ANOVA.
Fig. 6. Carnivore odor induced fear: Contact duration with a lion urine sample and avoidance of the odor area close to the sample (A) was measured in wildtype (WT, n = 26),
heterozygous (HT, n = 24) and homozygous (KO, n = 14) orexin-decient mice. Water served as a control sample. Mice contacted lion urine much less than water indicating
avoidance (B). This avoidance behavior was less robust if time spent in the odor area was analyzed (C). Orexin-decient mice expressed signicantly more avoidance behavior
to lion urine. ** P < 0.01, post-hoc Dunnetts multiple comparisons with wildtype mice after signicant main effects in ANOVA.
216
ve minutes of the test (Fig. 1C). Importantly, the baseline inactivity/freezing levels in open eld and the fear and safety learning
experiments (Figs. 2 and 3) were not affected. In the open eld test,
all genotypes expressed short epochs of inactivity (Fig. 1B) but these
epochs never lasted longer than 10 s, implying that no narcoleptic
episodes were detected in this paradigm. This nding is of importance since narcoleptic episodes which are often observed during
the dark phase in orexin-decient mice last longer than 10 s [43].
Moreover, we did not observe narcoleptic episodes that may interfere with the behavioral measures in any of the other experiments.
This observation conrms published data showing that narcoleptic episodes in orexin-decient mice are absent or extremely rare,
respectively, during the light period [2730]. Furthermore, our data
also support the observation that orexin-decient mice do not
express more sleepiness during the light period [10,27].
The present study shows that fear and safety learning, as well
as within-session and between-session extinction of conditioned
fear, is not affected in orexin-decient mice (Figs. 2 and 3). Notably,
there are no published data on fear or safety learning in narcoleptic patients. However, both of these two learning processes have
been shown to be affected in human anxiety disorders [4448].
Recently, it was demonstrated that orexin receptors are involved in
fear extinction and fear learning. In particular, orexin-1 receptordecient mice showed decreased freezing times in all phases of
contextual and cued fear conditioning whereas orexin-2 receptordecient mice were only impaired in contextual fear conditioning
[18,20]. Treatment with specic orexin-1 or orexin-2 receptor
antagonists has very similar effects [20] suggesting that the behavioral changes in the transgenic mice are caused by the deciency
and not by compensatory mechanisms. In rats, blockade of both
orexin receptors by a dual antagonist decreases conditioned fear
[49,50]. However, based on the present data, lifelong deciency
of orexin does not affect fear conditioning. This observation is
supported by our safety learning experiment in which contextual
fear is inhibited by a learned safety signal. Clearly, contextual fear
was not impaired in this experiment but rather (non-signicantly)
increased in orexin-decient mice (Fig. 4B). Taken together, the
present data suggest that the mouse brain system responsible for
fear learning is not affected by orexin deciency or since literature demonstrates effects of orexin receptor deciency can
compensate for this deciency.
Similar to fear learning, safety learning was not affected in
orexin-decient mice. Here, we used a safety learning protocol with
explicit unpairings of the to-be-learned stimulus and the aversive
foot shock [cf.,46;51]. In the retention test, contextual fear was
induced by the conditioning context and the ability of the CS to
inhibit this contextual fear was measured. This paradigm was of
particular interest for us since very similar to fear extinction
a mechanism of fear inhibition is involved and fear inhibition is
impaired in several anxiety disorders [47,52,53]. However, our data
again suggest that orexin is either not involved in this type of fear
inhibition (by a safety cue) or the brain system responsible for fear
inhibition can compensate for orexin deciency.
Since there was a tendency for decreased reactivity to the electric foot shocks used for fear and safety learning (Fig. 2B), we
decided to perform a more specic test and tested the locomotor reactivity to foot shocks with different intensities. Again, we
observed a slightly decreased reactivity in orexin-decient mice
but this decrease was not signicant (Fig. 4). This suggests that
orexin is not involved in foot shock processing.
Last, we submitted orexin-decient mice to two different
paradigms of innate fear (anxiety), the light-dark box [54] and carnivore odor avoidance [55]. Both of these paradigms are regularly
used to evaluate the effects of genetic or pharmacological manipulations [5659]. It should be noted that protocol, parameters and
analyses in both behavioral paradigms can be easily modied in a
erally more fearful or anxious in fear and safety learning and the
carnivore odor avoidance. Furthermore, they were more responsive
to electric aversive stimuli and less active in the open eld. Only in
the light-dark box, no gender differences were observed. However,
it should be noted that the here observed gender effects were relatively modest and could be only detected as main effects, i.e. based
on large group sizes. To absolutely exclude interactions between
gender and genotype, presumably much larger group sizes have to
be used.
In summary, the present study shows for the rst time that
orexin-decient mice express increased anxiety in three different
paradigms of unconditioned fear but are normal in fear and safety
learning. Together with ndings from literature, this suggests an
important role of orexin in anxiety. Future studies should investigate fear and anxiety behavior in narcoleptic patients, as well
as evaluate whether orexin could be a target for the treatment of
anxiety disorders.
Acknowledgement
We are grateful to Timothy French and Judith Kreutzmann for
language editing and helpful comments on the manuscript.
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