Behavioural Brain Research 272 (2014) 196–204

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Behavioural Brain Research

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

Research report

Increased number of orexin/hypocretin neurons with high and

prolonged external stress-induced depression
Jaishree Jalewa a,1 , KongFatt Wong-Lin b , T. Martin McGinnity b , Girijesh Prasad b ,
Christian Hölscher a,∗,1
a
School of Biomedical Sciences, University of Ulster, Coleraine BT52 1SA, Northern Ireland, United Kingdom
b
Intelligent Systems Research Centre, University of Ulster, Magee Campus, Derry BT48 7JL, Northern Ireland, United Kingdom

h i g h l i g h t s

• 50 mg/kg per day (for 5 weeks) corticosterone induced depression in mice.

• The behavioural changes correlated with doses of corticosterone.
• A reduction of motor activity during the 12-h dark (wake) phase was seen.
• A ∼20% increase in orexin neurons in the lateral hypothalamus was found.
• An increase of Orexin neurons may underlie stress-induced depression.

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

Article history: It has been found that dysregulation in the orexin/hypocretin (Ox/HCRT) neuropeptide system in the
Received 27 December 2013 lateral hypothalamus (LHA) is known to affect sleep disorder, depression and motor activities. However,
Received in revised form 13 May 2014 to date there is no common agreement regarding the resulting specific changes induced in the Ox system.
Accepted 16 May 2014
In this study, we inject corticosterone to produce stress-induced depressed mice and investigate the Ox
Available online 24 May 2014
neuronal and corresponding behavioural changes. Different doses (10, 20, 50 mg/kgbw) of corticosterone
were injected in adult mice, and then were tested in the open field test, forced swim test, tail suspen-
Keywords:
sion test, elevated plus maze test and motor activity measurements to validate the depressed animal
Corticosterone
Depression
model. Significant dose-dependent behavioural changes were observed in correlation with the doses of
Orexin corticosterone. The effect is most significant and robust in the high 50 mg/kgbw dose group five weeks
Motor activity after injection. Interestingly, we found on average a reduction in motor activity during the 12-hour dark
phase (awake) of the depressed mice and no significant change during the light phase (asleep). Finally,
using confocal microscopy, immunofluorescence (IF) analysis shows a significant increase (∼20%) in the
number of Ox neurons in the LHA of the depressed mice as compared to the age-matched controls. This
study suggests that an increase in Ox neuronal signaling may be functionally linked to high and prolonged
external stress-induced depression.
© 2014 Elsevier B.V. All rights reserved.

1. Introduction depressed mood, anhedonia, cognitive dysfunctions, alteration in

Major depressive disorder (MDD) is a psychiatric disorder with
complex etiology and heterogenous symptoms characterized by
∗ Corresponding author at: Biomedical and Life Sciences, Faculty of Health and
Medicine, Lancaster University, Lancaster LA1 4YG, UK. Tel.: +44 0 1524 534870.
E-mail addresses: j.jalewa@lancaster.ac.uk (J. Jalewa), k.wong-lin@ulster.ac.uk
(K. Wong-Lin), tm.mcginnity@ulster.ac.uk (T.M. McGinnity), g.prasad@ulster.ac.uk
(G. Prasad), c.holscher@lancaster.ac.uk (C. Hölscher).
1
Present address: Division of Biomedical and Life Sciences, Faculty of Health and
Medicine, Lancaster University, Lancaster, LA1 4YG, United Kingdom.
weight and appetite, insomnia and hypersomnia [1,2]. A vicious
cycle between MDD and sleep disorders has been described
(dysregulation of orexin/hypocretin, Ox/HCRT) [3–8]. Ox neurons
comprising of neuropeptides orexin A (Ox-A) and orexin B (Ox-
B) are found predominantly in the lateral hypothalamus (LHA)
[9,10] and are known to function through OX1 R (HCRTR1) and
OX2 R (HCRTR2) G-protein coupled receptors, respectively [11–15].
Previous Ox studies were focused on the regulation of the sleep-
wake function and narcolepsy [16–18], but recent studies suggest
a further role in depression, emotion processing, energy homeosta-
sis, reward-seeking behaviour, and in the regulation of endocrine

http://dx.doi.org/10.1016/j.bbr.2014.05.030
0166-4328/© 2014 Elsevier B.V. All rights reserved.
 J. Jalewa et al. / Behavioural Brain Research 272 (2014) 196–204
functions [19–24]. Rainero et al. showed an association between
major depressive disorder and the HCRTR1 gene and suggested
an involvement of Ox in modulating affective disorders [25]. Fur-
ther, differential regulation of depression-like behaviour has been
shown, and the anti-depressant or pro-depressant like effects
observed appears to be due to the balance of OX1 R and OX2 R acti-
vation [26].
Moreover, some studies have shown Ox-induced locomotor
effects mediated via Ox1 R [27,28] and increased spontaneous phys-
ical activity by central administration of Ox-A [29,30]. In addition,
the role of Ox in regulating motor behaviour, postural muscle tone
and locomotor movements has been indicated by the presence of
Ox projections from the hypothalamus to the midbrain [31]. In spite
of the well-studied Ox role in different areas, its influence on motor
control still remains mostly unknown [32].
Modeling depression in animals is mostly induced by exter-
nal stimuli such as stress or emotional loss/removal of pups
from the mother, with few genetic mouse models of depression
available due to the fact that genetic links are poorly under-
stood [33]. Standard animal models of depression are induced by
external influences such as social defeats, neonatal administration
of antidepressants, electrical shocks, stress hormone (corticoste-
rone) injection, genetically alteration e.g. of 5-HT transporters,
and others [34]. In particular, both chronic mild stress and corti-
costerone (stress hormone) injection can induce depressive-like
state in rodents [35]. In stress-induced models, elevated lev-
els of cortisol have been reported due to stress exposure, and
research has shown that exogenous corticosteroid administration
leads to depression-like behaviour [36] and reduces mitochondrial
function [35].
Based on a recent review by Nollet and Leman (2013), Ox neu-
ronal and level changes seem to vary widely among experimental
studies in animals and humans [37]. Particularly, one study in
chronically stressed mice [38] showed a decrease in Ox neurons in
the LHA, while in another study (also on chronically stressed mice)
demonstrated no significant change of Ox neurons in the LHA but
an increase in number in the dorsomedial and perifornical hypo-
thalamic area [39]. A third study in Flinders Sensitive Line rats (a
genetic animal model of depression) [40] showed an increase of
∼16% more Ox neurons in the hypothalamus.
In this work, we employed the corticosterone-induced depres-
sion mouse model induced by injecting high levels of corticoste-
rone, once daily for five weeks [41]. We then investigated the effect
of depression on motor activity as well as the Ox system. Our anal-
ysis of the motor behaviour and rearing/jumping in the depressed
mice reveals a significant general decrease in the overall activ-
ity during the dark phase, thus indicating susceptibility of motor
activity to depression in the awake state. In addition, immunofluo-
rescence (IF) analysis of the Ox neurons using confocal microscopy
Fig. 1. Experimental design. Repeated corticosterone injections were administered in C57Bl/6 male mice for 5 weeks and behaviour tests were performed after 3 weeks and
5 weeks. Body weight was taken before and at the end of the study.
197
shows an increase of almost 20% in the hypothalamus of depressed
mice as compared to the age-matched control group.
2. Materials and methods
2.1. Animals
Twelve-week-old C57Bl/6J male mice from the Harlan labo-
ratory were used for the experiments described. Mice weighed
30 g ± 2 at the start of the experiment. Animals were maintained
on a 12/12 h light/dark cycle (lights on at 8:00A.M., off at 8:00P.M.),
in a temperature-controlled room (21.5 + 1 ◦ C). Animals were indi-
vidually caged, and received food and water ad libitum. Treatment
began after two weeks of acclimatization. All animal experiments
were licensed by the UK Home Office in accordance with the Ani-
mals (Scientific Procedures) Act of 1986 and in agreement with UK
and EU laws.
2.2. Experimental design and drug administration
Mice were randomly assigned to four experimental groups
(n = 8–10/group). Three groups were administered subcutaneously
with corticosterone (Sigma–Aldrich, C2505) (10 mg/kg body
weight (bw) (n = 8); 20 mg/kgbw (n = 10); 50 mg/kgbw (n = 10)) sus-
pended in corn oil (filtered through 0.22 ␮m membrane filter into
a previously sterilized conical tube) once a day while the con-
trol group (n = 8) was administrated with corn oil only. Analysis
of depression-like behaviour was performed using the open field
test (OFT), elevated plus maze, forced swim test (FST), tail sus-
pension test (TST) and motor activity measurements (CLAMS) at
3 weeks and 5 weeks (with continued injections for two more
weeks). Mice were weighed, terminally anaesthetized, perfused
and brains processed for quantification of Ox neurons (histology
studies) (Fig. 1).
2.3. Behavioural tasks
2.3.1. Open Field Test (OFT)
The open field test was conducted in a gray-colored aluminum
open-field arena (58 cm diameter; 31 cm high wall). The arena
was illuminated, from 2 m above, using a 60W lamp. The arena
was cleaned with 70% ethanol after each mouse trial to prevent
the build-up of olfactory cues. Mice received a session of 5 min
in the empty open-field. The mouse was placed in the middle of
the open-field and was free to explore it. During that time, motor
activity was recorded by total path length (m) and speed (cm/sec).
The number of rearing events (forepaws elevated from the floor)
was analysed as an index of exploratory behaviour. The anxiety
level was assessed by the percentage of time spent in the centre
198 J. Jalewa et al. / Behavioural Brain Research 272 (2014) 196–204
versus periphery of the arena (central ratio %) and the number of
grooming sessions. Grooming was defined as behaviour when the
mouse stopped running and start licking, chewing and scratching
at its fur. A video camera was fixed 2 m above the centre point of
the arena and was attached to a video recorder, monitor and a com-
puter. The movement of the animals in the open-field was tracked
using a computerised tracking system (Biosignals, New York) to
analyse the images. Grooming sessions were counted manually in
a double-blinded fashion.
2.3.2. Forced swim test (FST)
Mice were tested by placing in an opaque cylindrical tank
(height 0.3 m; diameter 0.24 m) containing tap water (24–25 ◦ C)
to a depth of 0.2 m. Mice were exposed to the tank for 10 min trial
on day 1 and after thorough drying on the paper towel returned
back to home cages. The following day, each mouse was allowed to
swim for a period of 5 min, and the duration of immobility (absence
of active, escape-oriented behaviours such as swimming, jumping,
rearing, sniffing, diving) was recorded during the last 4 min of the
test [42].
2.3.3. Tail suspension test (TST)
Mice were suspended on the edge of a shelf 0.4 m above a table-
top by adhesive tape, placed approximately 1 cm from the tip of
the tail [43]. They were allowed to hang for 6 min and the dura-
tion of immobility was recorded during the last 4 min of the test.
Mice were considered immobile only when they hung passively
and completely motionless.
2.3.4. Elevated plus maze [44]
The elevated plus maze consists of 4 arms (0.81 m long; 0.1 m
wide), two of which are open and two closed (by 0.38m high walls)
on a metal platform about 1.01m high from the floor [44]. Each
mouse was placed in the center of the 4 arms (facing towards
enclosed arm) and allowed to explore freely. The number of entries
in open/closed arms and time spent in the closed arm was recorded
for a 5 min period. An entry was considered if all the four paws of
a mouse were within the boundary of the arm.
2.3.5. Activity measurements
Activity of mice was recorded on the Columbus Instruments
Comprehensive Lab Animal Monitoring System (CLAMS). This sys-
tem records two different types of motor activity: locomotion (X
and Y axes) and exercise (Z axis). Oxymax/CLAMS detects animal
motion using infrared (IR) photocell technology. Coverage in a sin-
gle plane may be implemented with IR photocells located in the X
or XY direction. The height of the beams is such that they intersect
the animal midway vertically. Interruption of an IR beam accrue
one ‘count’ and when using infrared beams, data is presented in
terms of the number of ‘beam breaks’. Results were quantified as
XTotal (total counts: the total number of times IR beams were bro-
ken during an interval); Xamb (ambulatory counts: number of times
different beams were broken during an interval); ZTotal (placement
of IR photocells at a height above the animal for detecting rearing
or jumping). Mice were kept in the CLAMS cages and 12 h activity
from 20:00 h to 08:00 h was recorded after 3 and 5 weeks of daily
corticosterone injections.
2.3.6. Perfusion, fixation and sectioning
Mice were anaesthetised with pentobarbitone (0.3 ml; Euthanal,
Bayer AG, Leverkusen, Germany) and perfused transcardially with
0.1 M PBS (pH 7.4) buffer followed by ice-cold 4% paraformaldehyde
in PBS. The brains were removed and fixed in 4% paraformaldehyde
for at least 24 h and cryoprotected in a 30% sucrose solution in PBS
overnight at 4 ◦ C. Brains were then snap frozen using Envirofreez,
and coronal sections of 45 ␮m thickness were cut at a depth of
−0.34 mm to −2.80 mm bregma (according to the mouse brain atlas
by Paxinos and Franklin, 2004) using a Leica cryostat. Sections were
chosen according to the stereological rules, with the first section
taken at random and every fourth section afterward.
2.3.7. Immunohistochemistry
Single immunofluorescence (IF) staining of Ox neurons in the
hypothalamus was performed on 45 ␮m free-floating sections from
the left hemisphere of each brain. After blocking in 1% BSA and 5%
donkey normal serum in TBS buffer (pH 8, Sigma–Aldrich) to avoid
nonspecific antibody binding, sections were incubated in the pri-
mary antibody for Ox-A (C-19) (1:400, goat polyclonal, sc-8070,
Santa Cruz) overnight at 4 ◦ C. The sections were then incubated in
secondary antibody (1:800, donkey anti-goat Alexa 488 IgG, Molec-
ular probes) for one hour at room temperature. The sections were
mounted using Vectashield mounting medium (Vector Laborato-
ries) on the slides coated with 3-aminopropyl tri-ethoxy silane
(Sigma–Aldrich), followed by imaging. Negative control was per-
formed by omitting the primary antibody.
2.3.8. Confocal microscopy and orexin neuron quantification
Imaging was done using a confocal microscope (Leica Microsys-
tems; SP5 LAS IF Software). Fourteen sections were analyzed per
brain (control group and 50 mg/kgbw corticosterone/kgbw group;
n = 4/group). The number of all immunopositive Ox neurons (Ox-IR)
on the z-stack max image (taken for 30 ␮m thickness) was deter-
mined by blinded, manual counting, marked by green fluorescence
in the hypothalamic area (indicated by the location of third ventri-
cle (3 V)), using 10X objective [45].
2.3.9. Stereology
A modified version of the optical fractionator method was used
to quantify the number of Ox neurons in the hypothalamus. Esti-
mation of the total orexin neuron number was done using the
following formula [46–48]:
N = ˙Q × 1/f × number of series of sections
where N is the total number of neurons per brain, Q the number of
neurons counted per mouse, and f the fraction of the hypothalamic
area analyzed.
2.3.10. Fluorescence microscopy
All double IF stainings were visualized by Axio Scope 1 (Zeiss).
2.3.11. Statistics
Statistical analyses were performed using Prism 5 (GraphPad
Software Inc. USA) with the level of probability set at 95% and
the results are expressed as means ± SEM. Data were analyzed by
one-way or two-way ANOVA, followed by Bonferroni’s Multiple
Comparison post-hoc to measure the difference between groups.
Data for Ox neuron quantification was analysed by two-tailed
unpaired t-test.
3. Results
3.1. Significant gradual body weight changes were observed for
the high corticosterone dosages
Mice were weighed before start and at the end (before perfu-
sion) of the study (Fig. 1). After 5 weeks of corticosterone injections,
an increase (p < 0.001) in the weight of the 50 mg/kgbw group
(Fig. 2a) was observed. There was no significant weight change in
the 0, 10 and 20 mg/kgbw groups, indicating no effect of corn oil
on weight with an exception of the 50 mg/kgbw group where a sig-
nificant weight change was observed that may be due to either the
corticosterone induced depressive effect or lack of activity. There
was a 2.19 fold increase (Fig. 2b; p < 0.01) in the weight change
from start to finish (5 weeks) of the study when the 50 mg/kgbw
 J. Jalewa et al. / Behavioural Brain Research 272 (2014) 196–204
Fig. 2. Body weight change after 5 weeks of Corticosterone injections. a. The weight of each mouse was measured before and after study, ***p < 0.001. Two-way repeated
measure ANOVA followed by Bonferroni post hoc test. b. Change in the body weight in five weeks, **p < 0.01. One-way ANOVA followed by Bonferroni post hoc test.
group was compared to the 20 mg/kgbw group. More importantly,
the absolute weight gain was approximately 2 g in the 50 mg/kg
group whereas there was no weight gain in the control group.
3.2. Corticosterone- induced depression symptoms in a
dose-dependent way
After three weeks of daily corticosterone injections
(10 mg/kgbw) (n = 8); 20 mg/kgbw (n = 10); 50 mg/kgbw (n = 10),
open-field behaviour of mice was monitored including total path
length (m), speed (cm/sec), central ratio %, number of rearing events
and grooming sessions. There was no significant difference in the
open-field behaviour after 3 weeks of corticosterone treatment
therefore, repeated daily injections were continued for two more
weeks and all the parameters were observed again after five weeks.
There was a 1.65 and 2.12 fold increase in the number of grooming
sessions in the 20 and 50 mg/kgbw injected groups, respectively
(Fig. 3a) compared to the control group and a 1.3 fold increase
when compared between the 20 and 50 mg/kgbw injected groups
further indicates a dose dependent increase in the anxiety levels.
In the elevated plus maze test, we observed a 1.3 fold increase
in the total time spent in the closed arm when comparing the 20
and 50 mg/kgbw injected groups (Fig. 3b). Further, a decrease of a
factor of 0.52 (p < 0.001) and 0.61 (p < 0.001) in the number of open
arm entries and a consecutive 1.66 (p < 0.001) and 2.12 (p < 0.001)
fold increase in the total time spent in the closed arm by the 20
and 50 mg/kgbw injected groups, respectively (Fig. 3c) was found
when compared with the control group. Interestingly, not only the
number of open arm entries decreased with the increasing concen-
tration of corticosterone but there was also a significant decrease
by 60% (p < 0.05) in the closed arm entries of the plus maze in 20
and 50 mg/kgbw groups compared to the control group (Fig. 3c).
Hence, there was a general overall reduction in motor movements
in this test, and the time spent in closed arm had increased despite
the total number of closed arm entries decreasing. A decrease in
open arm entries indicates an increase in anxiety, and a decrease
in closed arm entries points towards likelihood of reduced motor
activity with depression.
3.3. A gradual development of depressed behaviour over time in
FST/TST with high corticosterone dosage
After repeated corticosterone injections, depression-like
behaviour of mice was assessed by the forced swim test (FST) and
199
tail suspension test (TST) at weeks 3 and 5. In general, repeated
corticosterone injections increased depression-like behaviour in
both the tests.
In the FST, there was a 1.8 and 2.9 fold increase in the float-
ing/immobility time (sec) in the groups injected with the 20 and
50 mg/kgbw corticosterone, respectively, compared to the control
groups (p < 0.001; Fig. 4a). Interestingly, a 1.4 fold change (p < 0.01)
was observed in the 50 mg/kgbw group when compared to the
20 mg/kgbw group (Fig. 4a), suggesting that the 50 mg/kgbw dose
to be the most influential.
Comparing immobility time (s) in the 10, 20 and 50 mg/kgbw
groups in the TST, a 0.65 fold increase (p < 0.0001) was found in
50 mg/kgbw group, compared to control group (Fig. 4b). Also, there
was 1.34 fold (p < 0.001) difference between the 20 and 50 mg/kgbw
groups, which further suggests that the latter dose was more potent
in inducing the depressed state.
Upon analyzing the 3-week with 5-week behavioural data we
found an increased effect of the continued repeated injections
(Fig. 5a, b). Overall, 50 mg/kgbw corticosterone injections increased
the floating time (p < 0.001) and immobility time (p < 0.05) in both
FST and TST behaviour tests, respectively, indicating the depressed
state of the mice. In FST, we observed a 1.61 fold increase in the
50 mg/kgbw compared to 20 mg/kgbw group when the change in
floating time from 3 to 5 weeks was analysed. In TST, we observed
a 3.34 fold increase in the 50 mg/kgbw compared to 20 mg/kgbw
group when change in immobility time from 3 to 5 weeks was ana-
lysed, indicating that injections of corticosterone for 5 weeks were
more promising as compared to 3 weeks.
3.4. Corticosterone induced a dose-dependent decrease in
locomotion and rearing/jumping
To study the effect of depressed state on the motor activ-
ity, we monitored locomotion (XTotal and Xamb counts) and
rearing/jumping (ZTotal counts) behaviour of the 0, 10, 20 and
50 mg/kgbw corticosterone injected groups of mice. Although
there was no significant difference in the locomotion and rear-
ing/jumping in the 10 and 20 mg/kgbw corticosterone injected
mice, we observed a 16, 33 and 45.6% dose dependent decrease
in the ZTotal counts for the 10, 20 and 50 mg/kgbw corticosterone
groups, respectively. A decrease (p < 0.05) in the locomotor activ-
ity (XTotal and Xamb counts) and rearing/jumping (ZTotal counts)
was observed during the dark phase of 12 h after 5 weeks of the
200 J. Jalewa et al. / Behavioural Brain Research 272 (2014) 196–204

Fig. 3. Anxiety-based depression tests after 5 weeks. Effects of repeated corticosterone injection on group 0, 10, 20 and 50 mice with daily injections of corn oil (n = 8),
10 mg corticosterone/kgbw (n = 8), 20 mg corticosterone/kgbw (n = 10), 50 mg corticosterone/kgbw (n = 10), respectively. a. Open-field test. The mean (+ S.E.M.) of number
of grooming sessions is shown. b and c. Elevated plus maze. Time spent in the closed arm of plus maze is shown in panel (b). Number of total entries (closed and open arm
entries) is shown in the panel (c) comparing with control. One-way ANOVA followed by Bonferroni post hoc test, *p < 0.05; **p < 0.01; ***p < 0.001.

50 mg/kgbw corticosterone injections (Fig. 6a–c). There was no sig-
nificant change in locomotor activity during the light phase.
3.5. An increase in expression of Ox neurons with high
corticosterone dosage
Based on our above behavioural results, the 50 mg/kgbw group
of mice appears to be suitable for an efficient assessment of depres-
sion in the context of this study. We then carried out quantification
of Ox-A neurons in the control and depression group. All Ox-A
immunopositive neurons were counted in the hypothalamus (left
hemisphere of brain) of the control and the 50 mg/kgbw group
of mice, under a 10X objective confocal microscope (z-stack max
images considered for counting). Estimation of the total Ox neuron
number was done by the modified version of the optical fractiona-
tor method (Table 1). Representative photomicrographs of the Ox
neurons at different magnifications (10X, 40X and 100X) are shown
in Fig. 7a–c. Calculated results showed a large increase in the num-
ber of Ox neurons in the 50 mg/kgbw group of mice when compared
to the control group (Fig. 7d), analysed by the two-tailed unpaired
t-test (P value = 0.0091). Specifically, an overall increase of 19.23%
was observed in the number of Ox neurons in this depression group.
 J. Jalewa et al. / Behavioural Brain Research 272 (2014) 196–204 201

Fig. 4. Despair-based depression tests. Effects of repeated corticosterone injection on group 0, 10, 20 and 50 mice with daily injections of corn oil (n = 8), 10 mg cortico-
sterone/kgbw (n = 8), 20 mg corticosterone/kgbw (n = 10), 50 mg corticosterone/kgbw (n = 10), respectively. a. Forced swim test (FST). The mean (+ S.E.M.) of floating time
in seconds is shown. b. Tail suspension test (TST). Immobility time in seconds is shown. One-way ANOVA followed by Bonferroni post hoc test, **p < 0.01; ***p < 0.001;
****p < 0.0001.

Fig. 5. Comparison of FST and TST (3 weeks v/s 5 weeks). Effects of repeated corticosterone injections for 3 and 5 weeks. a. Forced swimming test (FST). The mean (+ S.E.M.)
of floating time in seconds is shown. b. Tail suspension test (TST). Immobility time in seconds is shown. Two-way repeated measure ANOVA followed by Bonferroni post hoc
test, ***p < 0.001, *p < 0.05.

4. Discussion mouse model by injecting corticosterone and validated this by sev-

eral anxiety- and despair-based behaviour tests. Second, we studied
In the present study, we have investigated and quantified the the effect of depression on motor activity and Ox neurons in LHA.
effect of corticosterone-induced depression on motor activity and Our experiments showed that 20 mg/kgbw and 50 mg/kgbw
expression of Ox neurons in LHA. First, we employed a depression corticosterone doses resulted in increased anxiety as shown by
a significant increase in the number of grooming sessions (an
increase of grooming is typically interpreted as a reduction of anxi-
Table 1
ety) and a decrease in the number of open arm entries and increased
Effect of 50 mg corticosterone/kgbw on the number of Ox neurons.
total time spent in the closed arm in elevated plus maze test. Also,
Control Q N = Q × 1/1/2 × 3 corticosterone injections significantly increased the floating time
1 1105 6630 and immobility time in both FST and TST despair-behaviour tests,
2 1077 6462 respectively, indicating the depressed state of mice. Our analysis of
3 1205 7230 the locomotion (XTotal and Xamb counts) and rearing/jumping (ZTotal
4 1137 6822
counts) revealed a decrease in the overall activity of 50 mg/kgbw
50 mg Cort/kgbw
1 1252 7512 corticosterone group during the dark phase of 12 h and that the
2 1487 8922 50 mg/kgbw dose was the most effective in affecting motor activity
3 1350 8100 decrease compared to the 10 and 20 mg/kgbw doses of cortico-
4 1305 7830
sterone. In addition, other behavioural tests showed a significant
202 J. Jalewa et al. / Behavioural Brain Research 272 (2014) 196–204

Fig. 6. Effect of depression on the dark phase locomotor activity. Effects of repeated corticosterone injection on group 0, 10, 20 and 50 mice with daily injections of corn oil
(n = 8), 10 mg corticosterone/kgbw (n = 8), 20 mg corticosterone/kgbw (n = 10), 50 mg corticosterone/kgbw (n = 10), respectively. a. XTotal counts. The mean (+ S.E.M.) of XTotal
is shown. b. Xamb counts. The mean (+ S.E.M.) of Xamb is shown. c. ZTotal activity counts. The mean (+ S.E.M.) of ZTotal (rearing/jumping) is shown. One-way ANOVA followed by
Bonferroni post hoc test, *p < 0.05.

difference between the 20 mg/kgbw and 50 mg/kgbw group. Thus,
we consider 50 mg/kgbw corticosterone as the best dose for mod-
eling depression in mice.
A negative feedback loop between motor activity, e.g. exer-
cises, and 5-HT level (which plays a role in depression [49,50])
is known to exist, where exercise increases synthesis and release
of 5-HT by the serotonergic neurons that becomes active during
exercise, while increased 5-HT contributes to the buildup of cen-
tral fatigue [51]. Moreover, neuroanatomically, there is evidence
of direct interactions between the 5-HT neurons in the dorsal
raphe nucleus and Ox neurons in the LHA [22,52]. Physical exer-
cise has been identified as a helpful and preventive measure to

Fig. 7. Total number of orexin-A positive neurons in the hypothalamus in the control and experimental group. For both groups: control (wild type C57Bl/6) and experimental
(50 mg corticosterone/kgbw depression model) group, n = 4; number of sections per mouse = 14. a–c. Representative photomicrographs (confocal microscopic images) showing
orexin neurons in the hypothalamus (orexin-A (C-19)), a. 10X, b. 40X, c. 100X. Scale bars in the images a, b and c represents 500 ␮m, 100 ␮m and 50 ␮m respectively. d.
Orexin neuron quantification: Total number of neurons in the control group (6786 + SEM) and depression group (8091 + SEM). There is an increase of 19.23% in the number
of orexin neurons in depression mice group compared to the control group. Two-tailed unpaired t-test, P value = 0.0091 (**p < 0.01).
 J. Jalewa et al. / Behavioural Brain Research 272 (2014) 196–204
alleviate corticosterone- induced mitochondrial dysfunction asso-
ciated with depression-like states [35]. Recent studies indicate a
substantial role of exercise in improving depressive symptoms in
people diagnosed with depression [53,54]. Patients with chronic
illness show signs of physical inactivity and co-morbid depressive
symptoms, and exercise training in the patients of mild-to-
moderate depression has shown not only functional improvement,
but also reduced the depression [55]. It would be of interest to study
the neurophysiological changes due to exercise in mildly depressed
animals to evaluate the underlying neuronal mechanisms in further
detail.
Stress is known to increase cortisol levels and centrally released
corticotropin-releasing hormone as well as increased levels of cor-
tisol contribute to depression [56]. Glucocorticoids are known to
increase body weight and induce obesity by central stimulation of
appetite [57,58], and it has been shown in previous studies that
stress is a risk factor for weight gain [59–61]. Similarly, depression
can have strong effects on weight, either increasing the risk for obe-
sity/weight gain [62,63], decreasing weight/abdominal obesity [64]
or both weight gain and loss [65,66]. Taking the role of depression in
increase/decrease of body weight into account, we analyzed weight
changes of mice before the start and at the end of this study and
found a significant increase in weight in the depressed mice after 5
weeks provided sufficiently high corticosterone level (50 mg/kgbw
for our study) is injected. Several reports have demonstrated an
increase in body weight in depressed people, consistent with our
results. Recent findings showed an increased risk of weight gain in
women with prevalent and incidental depression when compared
to those without depression [67–69].
In addition, stress also leads to Ox neuronal activation [70] and
therefore we analyzed and quantified Ox neurons in the LHA of
depressed mice. There was an increase of ∼20% in the number of
Ox neurons in the hypothalamus of depressed mice as compared
to the age-matched control group. Although our results of Ox neu-
ronal number increase in the LHA was not consistent with some
previous studies of chronically stressed mice (Allard et al., 2004,
Ito et al., 2009, Allard et al., 2011), it was consistent (and quanti-
tatively similar) with the study results of (Mikrouli et al., 2011),
albeit the latter was performed with the Flinders Sensitive Line rat.
These contradictory results may be linked to the observation that
both hypoactivity and hyperactivity of Ox signaling pathways have
been found to be associated with depression [37]. As described pre-
viously, depression can have contradictory effects on weight gain
as well, either increasing weight to obesity or causing weight loss.
These discrepancies in the literature prompted the necessity for
additional investigations. Recently, one study has shown inhibited
locomotor activity in rats under the influence of Ox-A on the para-
ventricular nucleus of the midline thalamus [71]. However, Kiwaki
et al. in 2004 reported increased locomotor activity by Ox [28]. Also,
there is evidence of increased grooming mediated by OX1 R and
downstream 5-HT2C receptors [72]. In another study, intracerebro-
ventricular administration of Ox-A has been shown to cause dose
related increase in several locomotor behavioural parameters [73].
Overall, our Ox quantification data points to the possibility that
in cases of depression, where the 5-HT transmission is known to
decrease [49,50], it may lead to withdrawal of the control/inhibition
of Ox neurons [23,52]. An increase in the number of Ox neurons
not only indicates the influence of reduced serotonin (5-HT) levels
(depression) on the Ox system, but also suggests a link to the con-
trol of motor activity. Moreover, there is a complex bi-directional
connectivity between these brain regions, and future work, ide-
ally combining both experimental and computational approaches,
needs to address their emergent behaviour [52].
In conclusion, this study demonstrates an overall decrease in
the motor activity and an increase in the number of hypothalamic
Ox neurons in depressed mice. These results suggest that the state
203
of depression may lead/contribute to pathophysiology of psycho-
logical (sleep/wake) and physiological (motor) disorders, and the
complex bi-directional relationship between 5-HT and Ox suggests
that motor abnormality in the future may be used as a biomarker
for depression. Clarification of the physiological state of depres-
sion and its link with the Ox system along with impaired motor
activity will enable an understanding of whether depression leads
to motor abnormality or vice-versa, which may further open new
avenues for pharmaceutical intervention or treatments based on
sports and exercise.
Authors’ contributions
JJ designed and performed all experiments and drafted the
manuscript. KW-L and CH participated in the design and coordi-
nation of the study and helped to draft the manuscript. MM and GP
coordinated the study and critically reviewed the manuscript. All
authors read and approved the final manuscript.
Conflict of interests
The authors declare no conflict of interests.
Acknowledgement
The work has been supported by Centre of Excellence in Intel-
ligent Systems Project, ISRC, University of Ulster, Magee, Northern
Ireland, UK. We thank Alok Joshi for helpful discussions during the
course of the project.
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