Biochemical and Biophysical Research Communications xxx (xxxx) xxx

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Biochemical and Biophysical Research Communications

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

Sleep deprivation and adrenalectomy lead to enhanced innate escape

response to visual looming stimuli
Yu-Ting Tseng a, 1, Binghao Zhao a, b, 1, Jingjing Liu a, 1, Hui Ding a, Feng Wang a,
Liping Wang a, *
a
Shenzhen Key Lab of Neuropsychiatric Modulation, Guangdong Provincial Key Laboratory of Brain Connectome and Behavior, CAS Key Laboratory of Brain
Connectome and Manipulation, CAS Center for Excellence in Brain Science and Intelligence Technology, The Brain Cognition and Brain Disease Institute,
Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen-Hong Kong Institute of Brain Science-Shenzhen Fundamental Research
Institutions, Shenzhen, 518055, China
b
University of the Chinese Academy of Sciences, Beijing, 100049, China

a r t i c l e i n f o a b s t r a c t

Article history: Optimal selections of innate behaviors that enable animals to adapt to particular conditions, whether
Received 29 March 2020 environmental or internal, remain poorly understood. We report that mice under acute (8 h) sleep
Accepted 13 April 2020 deprivation had an enhanced innate escape response and upregulation of c-fos expression in multiple
Available online xxx
brain areas that regulate wakefulness. By comparison, adrenalectomized mice under the same sleep
deprivation condition displayed an even more exaggerated escape response and these wake-regulating
Keywords:
brain areas were even more active. This suggests that acute sleep deprivation enhances innate escape
Sleep deprivation
response, possibly by altering wake state without causing significant anxiety. We also report that the
Innate escape response
HPA axis
hypothalamicepituitaryeadrenal axis feedback under sleep deprivation prevents an exaggerated escape
response by modulating wake-regulating brain areas. Taken together, our findings suggest that animals
prioritize escape response over sleep, as the need of both behaviors simultaneously increase. We also
provide an insight into the neural mechanisms underlying the interaction between sleep and innate
escape response.
© 2020 Published by Elsevier Inc.

1. Introduction predators is shown to induce innate escape responses in mice

Innate behaviors are crucial for the survival, including sleeping,
feeding, food foraging, reproductive behaviors, and escape re-
sponses, amongst others [1]. To maximize individual survival
probability, animals must optimize behavioral selections and pri-
oritize the most appropriate innate behavior to cope with envi-
ronmental threats and to fulfill internal demands. Animals in
nature often face a choice between staying vigilant to avoid envi-
ronmental threats or sleeping for internal recovery, where the
latter extends a greater vulnerability to predation. However, it is
not clear what behavioral selections would be made in challenging
situations with the simultaneous presence of increased internal
sleep demand and environmental predatory threat.
Looming stimuli mimicking threat from approaching aerial
* Corresponding author.
E-mail address: lp.wang@siat.ac.cn (L. Wang).
1
These authors contributed equally to this work.
[2e5]. Our previous work indicates that stress accompanying
anxiety states accelerate looming-induced escape responses [2],
suggesting the behavioral outputs of escape responses are influ-
enced by internal states. Human fMRI studies reveal that acute
sleep deprivation (SD) leads to altered neural activity in several
brain areas associated with arousal and anxiety regulation [6]. In
addition, sustained wake-state, altered during SD, is accompanied
by an increase in activity in wake-regulating brain areas [7]. In
animal models, whether sleep deprivation causes anxiety seems to
depend on the experimental paradigm used [8]. In this study, we
applied a relatively stress-free sweeping bar method [9], by which
the anxiety state triggered by stress is minimized. Therefore, we
were able to investigate how the innate escape response was
influenced by altered brain state under acute SD, without the in-
fluence of anxiety.
Dysregulation of the hypothalamicepituitaryeadrenal axis
(HPA axis) is associated with SD [10,11]. The HPA axis regulates its
own activity by a feedback mechanism that involves activation or

https://doi.org/10.1016/j.bbrc.2020.04.061
0006-291X/© 2020 Published by Elsevier Inc.

Please cite this article as: Y.-T. Tseng et al., Sleep deprivation and adrenalectomy lead to enhanced innate escape response to visual looming
stimuli, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.04.061
2 Y.-T. Tseng et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
inhibition of various brain areas [12], and its activation regulates
the fight-or-flight response [13]. As this response is necessary for
expeditious decisions to escape when animals are facing danger, it
suggests that HPA-axis activity may play a role in enhanced escape
response during acute SD. Here, we investigate HPA-axis contri-
bution to acute SD-enhanced innate escape responses, while
minimizing confounding effects of anxiety.
In this study, we demonstrate the impact of acute SD on
visually-evoked innate escape response by looming stimuli and the
involvement of multiple wake-regulating brain areas subserving
this effect. We also illustrate that the HPA axis under SD mediates
innate escape response, possibly by modulating the wake state.
Finally, our results also imply that animals prioritize escaping from
danger over sleeping whilst faced with a decision between the two.
2. Materials and methods
2.1. Animals
Male C57BL/6J mice (Hunan SJA laboratory animal Co., LTD.
Hunan, China) aged 8e12 weeks old) were maintained in standard
housing conditions on a 12/12 h day/night cycle with ad libitum
access to food and water. All behavioral tests were conducted
during the light cycle. All animal experiments were approved by
the Animal Care and Use Committees at the Shenzhen Institute of
Advanced Technology, Chinese Academy of Sciences.
2.2. Open-field test (OFT) and elevated plus maze (EPM)
Mice were tested in an open field [2] and allowed to freely
explore the chamber for 10 min. Behavioral parameters of anxiety-
like behavior during the last 5 min were recorded and analyzed by
Anymaze software (Stoelting, IL, USA). Following the OFT, each
mouse was placed in the central area of an EPM [14] facing one of
the two open arms and allowed to explore freely for 5 min.
Behavior were recorded and analyzed similarly to in the OFT.
2.3. Looming stimuli
The looming behavioral test was performed in the device
modified by Xing Yang and Qingqing Liu in our lab. This apparatus
consisted of an exploratory area (covering an area of about 2000
cm2) and an adjacently connected "refuge" (covering an area of
about 400 cm2). An LCD monitor was placed on the ceiling to
present looming stimuli. A looming stimulus was presented using
the parameters to mimic aerial predators in rodents/mice as pub-
lished in our previous work [5], which was manually triggered once
when mice entered the center of the exploratory area (covering an
area of about 500 cm2). Behavior was recorded and analyzed
manually.
2.4. Adrenalectomy
Adrenalectomies were performed similarly as described previ-
ously [15]. Briefly, mice were anesthetized, and two incisions were
made in the abdomen and adrenal glands removed. Sham-operated
mice underwent the same procedure except for the actual removal
of adrenal glands.
2.5. Sleep deprivation (SD)
We used a modified SD protocol based on previous work [16].
Briefly, SD was carried out by a sweeping bar chamber (LAIYUE,
Shenzhen), which is considered as a relatively stress-free method
[9]. We used a period of 8 h sleep deprivation where the chamber
Please cite this article as: Y.-T. Tseng et al., Sleep deprivation and adrenalectomy lead to enhanced innate escape response to visual looming
stimuli, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.04.061
was set to run from 9 a.m. to 5 p.m. Control mice were placed in the
chamber for the same time but without SD.
2.6. Immunohistochemistry
Immunohistochemistry was performed as described previously
by our group [5]. Briefly, brain sections (40 mm) were incubated in
blocking buffer and then incubated in primary antibody (rabbit
anti-c-fos, 1:500, Cell Signaling) overnight at 4
C. Subsequently,
sections were washed and then incubated with Alexa Fluor 594-
conjugated secondary antibodies at room temperature for 1 h.
Sections were mounted on ProLong Diamond antifade medium
with DAPI (ThermoFisher Scientific, P36971). Fluorescence slices
were photographed with an Olympus VS120 virtual microscopy
slide scanning system (Olympus, Japan).
2.7. Cell counting
To count c-fos-positive cells, ImagePro Plus 6.0 software was
used with particle analysis for constant regions of interest for each
brain area. This was done using manual thresholding and automatic
counting (ImageJ) in a section chosen randomly by an investigator
blind to experimental treatment.
2.8. Statistical analysis
All data were analyzed using GraphPad Prism 8.0 (GraphPad
Software, Inc.). Results are expressed as mean ± standard error
mean (S.E.M.). Statistical analyses of different experimental groups
in behavioral tests and c-fos expression were determined by un-
paired student’s t-test and one-way ANOVA, respectively.
****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05 were considered
as statistically significance in all tests.
3. Results
3.1. Acute SD enhances looming-induced escape responses
To investigate whether acute SD alters behavioral choice under
threatening signals such as predatory stimuli, we exposed sham-
adrenalectomy-operated mice under either acute SD for 8 h
(ShamSD group) [17] or no deprivation (Sham group) to looming
stimuli (Fig. 1A). The ShamSD group had shorter latency from the
onset of looming to the start of escape response (Fig. 1B) and
reduced time reaching the refuge (Fig. 1C) compared to the Sham
group. In addition, ShamSD mice had a higher amount of time spent
freezing in the refuge (Fig. 1E) compared to the Sham group. These
results indicate that innate escape responses evoked by predator-
mimicking threat signals are enhanced after acute SD.
Given that acute SD leads to dysregulation of the HPA axis, we
wondered whether the HPA axis activity was the driver of the
enhanced escape response. We performed adrenalectomies to
remove the secretion of glucocorticoids, thereby blocking HPA axis
feedback to the brain (Fig. 2A). We thus had another two groups:
adrenalectomized mice under normal sleep condition (AD group),
and adrenalectomized mice under acute SD condition (ADSD
group). Compared with the Sham group, the AD group showed an
enhanced trend in escape responses (Fig. 2BeE). However, the
ADSD group displayed significantly enhanced escape responses
when compared with the ShamSD group (Fig. 2FeI). Our results
suggest that altered HPA-axis feedback to the brain plays an
inhibitory role in the enhancement of escape response caused by
acute SD.
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Fig. 1. Acute SD enhances looming-induced escape response.

(A) Schematic illustrating surgery and experimental timeline. (BeE) Response latency (B), return time (C), time in the refuge (D) and freezing time in the refuge (E) in Sham and
ShamSD groups following looming stimuli (Sham group, n ¼ 12; ShamSD group, n ¼ 10). Data are presented as mean ± SEM. Unpaired Student’s t-test. *p < 0.05.

Fig. 2. ADSD mice show exaggerated escape responses evoked by looming.

(A) Schematic illustrating surgery and experimental timeline. (BeE) Response latency (B), return time (C), time in the refuge (D) and freezing time in the refuge (E) in Sham and AD
groups following looming stimuli (Sham group, n ¼ 11; AD group, n ¼ 31). (FeI) Response latency (F), return time (G), time in the refuge (H) and freezing time in the refuge (I) in
ShamSD and ADSD groups following looming stimuli (ShamSD group, n ¼ 13; ADSD group, n ¼ 16). Data are presented as mean ± SEM. Unpaired Student’s t-test. *p < 0.05.
**p < 0.01.

Please cite this article as: Y.-T. Tseng et al., Sleep deprivation and adrenalectomy lead to enhanced innate escape response to visual looming
stimuli, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.04.061
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3.2. Sleep-deprived mice do not show an increase in anxiety-like 3.3. Comparison of c-fos expression patterns under different
behaviors conditions

Acute stress accelerates looming-evoked escape responses [2],
and SD is considered a stressor [18]. We further tested whether the
SD protocol used in our looming test leads to anxiety by using an
open-field test (OFT) and an elevated plus maze (EPM) (Fig. 3A).
Compared with the Sham group, the ShamSD group had no sig-
nificant difference in the percentage of time spent in, or the total
moving distance inside, the central area of the OFT arena nor in the
open arms of EPM (Fig. 3BeC). This suggests that our acute SD
experimental paradigm does not significantly cause anxiety-like
behaviors in mice. To explore whether loss of the HPA-axis feed-
back results in anxiety-like behavioral changes, we examined mice
behavior after adrenalectomy in both OFT and EPM. There was no
difference between the Sham and AD groups (Fig. 3DeE) nor be-
tween the ShamSD group and ADSD groups (Fig. 3FeG), suggesting
that dysfunction of HPA-axis feedback may not alter anxiety states.
Together, our findings suggest that SD impacts the innate escape
response through a different mechanism than that caused by
anxiety.
To further investigate the brain areas underlying the regulation
of intensified escape response caused by acute SD, we examined c-
fos expression in mice brains following acute SD (Fig. 4AeB).
Expression of c-fos in multiple brain areas in the limbic system was
significantly higher in the ShamSD group compared to Sham group.
The limbic system is important for emotional processing and the
regions showing c-fos increase were the prelimbic cortex (PrL), the
cingulate cortex (Cg), the lateral septum (LS) and the basolateral
amygdala (BLA), whereas the bed nucleus of the stria terminalis
(BNST) was unaffected. The upregulation of c-fos expression was
also observed in the paraventricular thalamic nucleus (PVT) and in
the hypothalamus, such as the dorsomedial hypothalamic nucleus
(DMH), the lateral hypothalamic area (LH) and the paraventricular
nucleus (PVN). In the midbrain, we observed upregulation of c-fos
in the periaqueductal gray (PAG), the dorsal raphe nucleus (DR), the
laterodorsal tegmental nucleus (LDT), and the locus coeruleus (LC),
and downregulation of c-fos in the parabrachial nucleus (PB)
(Fig. 4C).

Fig. 3. SD mice do not display significant change in anxiety-like behaviors.

(A) Schematic illustrating surgery and experimental timeline. (B, D and F) Percentage of time in the center area (center/total area), percentage of total distance in the center area
(center/total areas), mean speed, and total distance in the OFT are presented as bar graphs. (C, E and G) Percentage of time in the open arm (open/total arms), percentage of total
distance in the open arms (open/total arms), mean speed, and total distance in the EPM are presented as bar graphs. Comparison between Sham and ShamSD groups (B and C: Sham
group, n ¼ 18; ShamSD group, n ¼ 8). Comparison between Sham and AD groups (D and E: Sham group, n ¼ 18; AD group, n ¼ 12). Comparison between ShamSD and ADSD groups
(F and G: ShamSD group, n ¼ 7; ADSD group, n ¼ 6). Data are presented as mean ± SEM. Unpaired Student’s t-test. **p < 0.01.

Please cite this article as: Y.-T. Tseng et al., Sleep deprivation and adrenalectomy lead to enhanced innate escape response to visual looming
stimuli, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.04.061
 Y.-T. Tseng et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx 5

Fig. 4. Comparison of c-fos expression under different conditions.

(A) Representative images showing c-fos expression in the PVT, LH and PVN in Sham, ShamSD, AD and ADSD groups. (B) Schematic illustrating distribution of c-fos expression.
Comparisons between ShamSD and Sham group (top), AD and Sham group (middle), and between ADSD and ShamSD group (bottom). (C) Quantitative c-fos expression in each brain
area (n ¼ 3 for each group). Cingulate cortex (Cg), prelimbic cortex (PrL), bed nucleus of the stria terminalis (BNST), lateral septum (LS), paraventricular thalamic nucleus (PVT),
dorsomedial hypothalamic nucleus (DMH), ventromedial hypothalamic nucleus (VMH), lateral hypothalamic area (LH), paraventricular nucleus (PVN), basolateral amygdala (BLA),
periaqueductal gray (PAG), dorsal raphe nucleus (DR), locus coeruleus (LC), laterodorsal tegmental nucleus (LDT), parabrachial nucleus (PB). Data are presented as mean ± SEM. One-
way ANOVA. *p < 0.05. **p < 0.01. ***p < 0.001. ****p < 0.0001.

To explore the brain areas regulated by HPA-axis feedback, we
examined c-fos expression in AD and ADSD groups. When
compared with the Sham group, the AD group displayed no sig-
nificant change of c-fos expression in any brain areas described
above (Fig. 4C), suggesting that loss of the HPA-axis feedback itself
does not alter the baseline activity of these brain areas. Interest-
ingly, comparison between the ADSD and ShamSD groups revealed
significantly increased c-fos expression in hypothalamic sub-
regions, such as the PVN, DMH, LH and ventromedial hypothalamic
nucleus (VMH). In addition, upregulation of c-fos expression was
also observed in the LS and PVT (Fig. 4C). These results imply that
these brain areas receive controls of the HPA-axis feedback under
acute SD.
4. Discussion
Behavioral selection between sleep and innate escape response
is critical for survival when animals are faced with the challenge of
simultaneously increased requirements for both. To our knowledge,
this present work is the first study illustrating that innate escape
response can be intensified by acute SD. We also provide evidence
that the HPA axis under sleep deprivation is involved in the inter-
action between sleep and innate escape response.
Evidence has shown that acute stress induced by multiple
stressors, including restraint, forced swim, and footshock, lead to
anxious brain states and an intensified visually-evoked escape
response [2]. Although sleep is also considered as a stressor, our
results show that 8 h of acute SD using a stress-free sweeping bar
method could minimize the generation of anxiety-like behavior in
mice [9]. Although somewhat surprising, these results are accor-
dant with previous work showing that 24 h of SD was not accom-
panied with an anxiety state in the open field test [19]. Therefore,
we were able to examine the behavioral change influenced by SD
without a confounding anxiety state. Interestingly, we observed
that SD led to elevated activity in the locus ceruleus (LC), an area in
which activity is induced upon acute stress and is involved in the
production of anxiety-like behaviors [2]. Given that acute SD pro-
longs the wake state [7], it is reasonable to suggest that LC activity
during acute SD are recruited for promoting wakefulness [20]. In
addition, we also show that acute SD did not change c-fos expres-
sion in BNST, which is strongly induced during anxiety [21].
Available evidence suggests that enhanced innate escape responses
due to acute SD may result from the changed wake state but not
from anxiety.
Previous work has reported that brain areas including PVN,
amygdala, PVT, LDT and cortex are upregulated under acute SD [22].
Our study not only shows a similar result but also provides a
comprehensive comparison demonstrating additional responsive

Please cite this article as: Y.-T. Tseng et al., Sleep deprivation and adrenalectomy lead to enhanced innate escape response to visual looming
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6 Y.-T. Tseng et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
brain areas under acute SD, including the LS, LH, DMH, PAG, DR, LC
and PB. As the LDT [23], BLA, PAG [24] and LC [2] mediate defensive
behavior, upregulated activity in these areas under acute SD may
hint at the mechanisms underlying the enhanced escape response.
In addition, changes in activity in those brain areas involving in
sleep-wake regulation, such as the PVN [25], PVT [26], LS [27], LH
[28], LDT, DMH, DR, PAG, LC and PB [29,30] may also contribute to
the enhanced escape response by altering the wake state.
Dysregulated secretion of HPA-axis-glucocorticoids into the
blood is a major factor contributing to the detrimental effect of
acute SD on our body [31,32]. After adrenalectomy to remove
glucocorticoid secretion to block HPA feedback, acute SD induced
an even more exaggerated escape response than it did in the group
without adrenalectomy. This indicates that glucocorticoid feedback
to the brain might function in preventing the exaggerated behav-
ioral reactivity caused by acute SD. Surprisingly, this study shows
that c-fos expression in the PVN was not different between the
Sham and AD groups, which is reflected by other work [33]. Like-
wise, we show that the AD and Sham groups did not have signifi-
cantly different levels of brain activity in other brain areas, as
indexed by c-fos. In contrast, we found that wake-regulating brain
areas, such as PVT, LH, DMH, LS, and PVN [25e28,34], were more
active in the ADSD group than in the ShamSD group. The VMH,
which is involved in processing escape responses, was also more
active [1]. Together, HPA-axis feedback under acute SD might
regulate innate escape response through the modulation of the
wake-regulating system, leading to the prevention of exaggerated
reactivity to negative stimuli.
This study provides evidence to suggest that escaping from
danger may be more critical for animals than other innate behav-
iors, such as sleeping, in challenging situations where these re-
sponses are in competition. The conservation of both the innate
escape responses to looming and in the underlying neural mech-
anisms across species, indicate their importance during the course
of evolution [35e39]. Prioritized natural selection of danger
avoidance over sleep may have occurred to ensure maximal sur-
vival for animals in the wild, even at the expense of damaging
health. However, the primordial purpose of this trade-off may not
necessarily be beneficial for modern humans, and instead may
result in a series of negative health consequences. For instance,
exaggerated reactivity to negative stimuli is often associated with
sleep disturbance [40]. Further investigations of the mechanisms
underlying the interaction between innate escape response and
sleep are needed, which will likely contribute to a better under-
standing of the neural mechanisms that subserve the pathology of
mental disorders associated with sleep disturbance.
Authors’ contributions
Y.T., contributed the study design; Y. T., B. Z., J.L., performed the
experiments; Y. T., B. Z., J.L., H. D. analyzed the data; Y. T., F. W., B. Z.
and J. L. drafted the manuscript; L.W. supported all aspects for this
project. The authors would like to thank Qingqing Liu and Xing
Yang for the modification of looming device.
Declaration of competing interest
All authors claim that there are no conflicts of interest.
Acknowledgments
This work was supported by the National Natural Science
Foundation of China (Grant Nos. 31930047 and 31630031 to L.W.),
the Guangdong Provincial Key Laboratory of Brain Connectome and
Behavior (Grant No. 2017B030301017 to L.W.), the International
Please cite this article as: Y.-T. Tseng et al., Sleep deprivation and adrenalectomy lead to enhanced innate escape response to visual looming
stimuli, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.04.061
Partnership Program of Chinese Academy of Sciences (Grant No.
172644KYS820170004 to L.W.), and also by the Chinese Academy of
Sciences Taiwan Young Talent Programme (Grant No.
2018TW2GB0002 to Y.T.), China Postdoctoral Science Foundation
(Grant No. 2019M653117 to Y.T.).
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Please cite this article as: Y.-T. Tseng et al., Sleep deprivation and adrenalectomy lead to enhanced innate escape response to visual looming
stimuli, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.04.061