Accepted Manuscript

Hippocampal orexin receptor blocking prevented the stress induced social learn-
ing and memory deficits

Saeedeh Bahramzadeh Zoeram, Mahmoud Elahdadi Salmani, Taghi

Lashkarbolouki, Iran Goudarzi

PII: S1074-7427(18)30264-8
DOI: https://doi.org/10.1016/j.nlm.2018.11.009
Reference: YNLME 6964

To appear in: Neurobiology of Learning and Memory

Received Date: 8 April 2018

Revised Date: 14 October 2018
Accepted Date: 16 November 2018

Please cite this article as: Bahramzadeh Zoeram, S., Elahdadi Salmani, M., Lashkarbolouki, T., Goudarzi, I.,
Hippocampal orexin receptor blocking prevented the stress induced social learning and memory deficits,
Neurobiology of Learning and Memory (2018), doi: https://doi.org/10.1016/j.nlm.2018.11.009

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 Hippocampal orexin receptor blocking prevented the stress induced

social learning and memory deficits

Saeedeh Bahramzadeh Zoeram1, Mahmoud Elahdadi Salmani*1, Taghi Lashkarbolouki1, Iran

Goudarzi1

1. School of Biology, Damghan University, Damghan, Iran

*Corresponding author: Mahmoud Elahdadi Salmani, Ph.D., Damghan University, School of

Biology

Address: Damghan University, Cheshme Ali Road, Damghan, Iran.

Email: elahdadi@du.ac.ir ; elahdadi@gmail.com

Running Title:

Orexin receptor blocking prevents stress-induced disorders

Abbreviations:

CTRL; Control, OXR; Orexin Receptor, OX; Orexin; CSF; Cerebro-spinal Fluid, CRT;

Corticosterone, EPM; Elevated Plus Maze, OF; Open Field, HPA; Hypothalamic Pituitary

Axis, Central Nervous System; CNS, CRH; Corticotropin-Releasing Hormone, PVN;

Paraventricular Nucleus, LHA; Lateral Hypothalamic Area, ICV; IntraCerebroVentricular,

LTP; Long-term Potentiation, BNST; Bed Nucleus of Stria Terminalis, GR; Glucocorticoid

Receptor

1
Abstract

Stress as a homeostatic challenge leads to the malfunction of learning and memory processes,

namely social learning and memory. The orexin system is involved in stress responses

through connections to the hypothalamic-pituitary axis (HPA). In addition, the hippocampus,

a structure vulnerable to stress-induced changes, expresses orexin receptors 1 and 2 (OXr1

and OXr2) in various sub-regions. The present study is aimed at assessing the effects of

hippocampal orexin receptor blockade on social learning and memory impairments and

anxiety development following stress.

Male Wistar rats (220-250g) underwent cannula implantation in the hippocampus. Acute (two

mild electric shocks, 5.5 mA) and chronic stresses (ten days of restraint, 6 hours daily) were

applied with or without injection of orexin receptor antagonists (SB-334867 or TCS OX 29).

Sociability and social novelty in animals were assessed in a three-chamber social maze at the

end of stress application. Anxiety and exploratory behavior of animals were then examined,

with 20 min intervals, using the open field (OF) and elevated plus maze (EPM) tests,

respectively. Cisterna Magna cerebro-spinal fluid (CSF) was drained, before sacrifice, for

orexin (OX) assay and trunk blood was collected to measure the plasma corticosterone

(CRT).

Neither the acute nor the chronic stress could affect the sociability. The acute but not chronic

stress prevented the animal from sniffing the familiar caged rat in the novelty session, a

response which was reversed following the blockade of both OXRs. Furthermore, acute but

not chronic stress, led to increased anxiety and immobility behavior which were both

impeded by blocking the orexin receptor (OXR). Conversely, OX content in CSF increased

due to chronic restraint stress, an effect that was reversed by orexin blockade. Finally,

elevated plasma CRT was recorded in response to both acute and chronic stresses. The

2
observed increase in plasma CRT in chronically-stressed rats was abolished following

inhibition of OXRs, however a similar effect was not seen in the acute-stress group.

Our results identify hippocampal OXRs as potential candidates capable of preventing acute

stress-induced impairments of social novelty and anxiety behavior, and chronic stress-

induced plasma CRT and CSF orexin, changes. OXR manipulation may improve adaptation

to stress pathophysiology.

Keywords: Hippocampus, Orexin receptors, Stress, Anxiety, Social novelty

3
1. Introduction

Stress, as an inevitable part of our daily lives, leads to deficits in a variety of learning and

memory paradigms (Conrad, Galea, Kuroda, and McEwen, 1996; Luine, Villegas, Martinez,

and McEwen, 1994; Lupien and McEwen, 1997; McEwen and Sapolsky, 1995; Oei,

Everaerd, Elzinga, van Well, and Bermond, 2006), especially social learning and memory

(Van Der Kooij, Fantin, Rejmak, Grosse, Zanoletti, Fournier, Ganguly, Kalita, Kaczmarek,

and Sandi, 2014). Stress induced activation of hypothalamic-pituitary-adrenal (HPA) axis

results in the activation of downstream structures including paraventricular nucleus (PVN),

pituitary gland and adrenal tissue which leads to the production of stress hormones;

corticotropin-releasing hormone (CRH), adrenocorticotropic hormone (ACTH) and

corticosterone (CRT) (Smith and Vale, 2006; Tsigos and Chrousos, 2002), all of which

interefere with learning, memory and affect the related structures (Kim, Pellman, and Kim,

2015; Wingenfeld and Wolf, 2014).

CRH, secreted by the PVN of the hypothalamus (Carolyn, Christine, and Tallie, 1998) affects

orexinergic neurons of the lateral hypothalamus (LHA) through CRF receptors (CRFr1)

(Winsky-Sommerer, Boutrel, and Lecea, 2005; Winsky-Sommerer, Yamanaka, Diano,

Borok, Roberts, Sakurai, Kilduff, Horvath, and de Lecea, 2004). Orexin A (OXA), secreted

from LHA orexinergic neurons, plays a key role in the regulation of arousal/ wakefulness and

motivation (Sakurai, Nagata, Yamanaka, Kawamura, Tsujino, Muraki, Kageyama, Kunita,

Takahashi, and Goto, 2005; Tsujino and Sakurai, 2013; Yamanaka, Beuckmann, Willie,

Hara, Tsujino, Mieda, Tominaga, Yagami, Sugiyama, and Goto, 2003), and these neurons

project to multiple brain areas (Date, Ueta, Yamashita, Yamaguchi, Matsukura, Kangawa,

Sakurai, Yanagisawa, and Nakazato, 1999; Nambu, Sakurai, Mizukami, Hosoya,

Yanagisawa, and Goto, 1999; Peyron, Tighe, Van Den Pol, De Lecea, Heller, Sutcliffe, and

Kilduff, 1998) including brain stem and hippocampus (Nambu et al., 1999). Stimuli that

4
increase arousal/ wakefulness also often lead to an increase in stress and anxiety. Hence,

orexin function in the regulation of wakefulness suggests that this peptide may have a role in

the manifestation of behavioral and physiological signs of stress (Carter, Borg, and de Lecea,

2009; Winsky-Sommerer et al., 2005). In this regard, intracerebroventricular (ICV) injection

of OXA has evoked many stress-related behaviors such as stress-induced reinstatement of

cocaine-seeking behavior, increased time spent in the dark compartment in the light-dark test

and time spent on the closed arms in the elevated plus-maze test (Boutrel, Kenny, Specio,

Martin-Fardon, Markou, Koob, and de Lecea, 2005; Giardino and de Lecea, 2014; Suzuki,

Beuckmann, Shikata, Ogura, and Sawai, 2005) and alteration of autonomic processes such as

elevation of mean arterial blood pressure, heart rate, oxygen consumption, and body

temperature (Chen, Hwang, Chang, and Dun, 2000; Shirasaka, Nakazato, Matsukura,

Takasaki, and Kannan, 1999). In addition, prepro-orexin gene knockout mice have shown

reduced responses to acute and chronic stresses (Kayaba, Nakamura, Kasuya, Ohuchi,

Yanagisawa, Komuro, Fukuda, and Kuwaki, 2003). Conversely, acute stress application,

through CRH secretion affects orexinergic cells and increases orexin secretion (Winsky-

Sommerer et al., 2004).

Hippocampal areas involved in memory processes are also highly susceptible to stress effects

(Kim and Diamond, 2002). Stress and CRT have been shown to impair the induction of

hippocampal long-term potentiation (LTP) (Foy, Stanton, Levine, and Thompson, 1987;

Shors, Gallegos, and Breindl, 1997), alter hippocampal dendritic morphology (McEwen,

2000) and inhibit adult neurogenesis (Gould, McEwen, Tanapat, Galea, and Fuchs, 1997),

which can potentially have an impact on memory-related functioning (de Kloet, Oitzl, and

Joels, 1999; Kirschbaum, Wolf, May, Wippich, and Hellhammer, 1996) . Social memory, a

type of memory for recognition of persons and situations, is mediated by the hippocampus

5
and is also impaired by stress and glucocorticoid exposure (Takahashi, Ikeda, Ishikawa,

Tsukasaki, Nakama, Tanida, and Kameda, 2004; Tatomir, Micu, and Crivii, 2014).

Orexin peptide plays an important role in a variety of learning and memory processes

(Akbari, Motamedi, Naghdi, and Noorbakhshnia, 2008; Jaeger, Farr, Banks, and Morley,

2002; Sears, Fink, Wigestrand, Farb, De Lecea, and LeDoux, 2013). Orexin containing

terminals and orexinergic receptors are distributed in different brain regions related to

memory including hippocampal formation (Aitta-Aho, Pappa, Burdakov, and Apergis-

Schoute, 2016; Fadel and Burk, 2010; Peyron et al., 1998). Jaeger et al. showed that ICV

injection of orexin has improved T-maze avoidance and step-down passive avoidance

memory (Jaeger et al., 2002) and Akbari and coworkers in another study demonstrated that

blockade of hippocampal orexinergic receptors has impaired the spatial memory (Akbari,

Naghdi, and Motamedi, 2007). In opposition to these studies, Aou and colleagues showed

that OXA impaired spatial memory performance and CA1-Schaffer collateral LTP in rats

(Aou, Li, Li, Oomura, Shiraishi, Sasaki, Imamura, and Wayner, 2003).

Given the identified impacts of both stress and the orexinergic system on hippocampal

learning and memory, investigationg the role of hippocampal orexin signaling may provide

greater understanding of the relationship between learning and memory following stress.

Therefore, this study investigates the effect of hippocampal OXRs blockade on social

learning and memory impairment following stress.

6
2. Material and methods

2.1. Animals

In this study, male Wistar rats with an average weight of 200–250 g (purchased from Razi

Institute, Karaj, Iran) were used. All experiments were performed in accordance with the

National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH

Publication No. 23-80, revised 1996) and conformed to the research ethical standards for the

care and use of animals in Damghan University. Rats were housed five per cage and

maintained on a standard 12 h light/12 h dark cycle, with lights on at 07:00 am, and food and

water ad libitum.

2.2. Drugs

The orexin receptor 1 (OXr1) antagonist (SB334867; Tocris) and orexin receptor 2 (OXr2)

antagonist were injected (intra-hippocampal) respectively, in a dose of 50 and 40 nmol in a

volume of 0.5 µl. The SB334867 (SB) was dissolved in DMSO (Dimethyl sulfoxide) and

diluted with a 0.9% physiological saline solution to reach the appropriate concentration in

DMSO (2%). TCS OX 29 (TCS) used in this research was dissolved in saline. Ketamine (90

mg/kg, i.p.) and Xylazine (6.0 mg/kg, i.p.) were purchased from local markets in IRAN and

were used as anesthetics.

2.3. Surgery

Cannulae were surgically implanted to infuse OXr1 and OXr2 antagonists (in drug injection

groups) or saline/DMSO (in control animals and respective stress groups), bilaterally into

both hippocampi. Rats were anesthetized with a mixture of Ketamine and Xylazine and then

positioned in a stereotaxic apparatus (Stoelting Instruments, USA). Body temperature was

maintained stable using a towel pad. Stainless steel guide cannulae (23 gauges) fitted with

infusion cannulae (30 gauges) with 1 mm longer tip were positioned bilaterally above the

7
hippocampi based on coordinates from the atlas of Paxinos and Watson (Paxinos and Watson,

2007) for the rat brain (AP = −3.6 mm caudal to bregma, lat. = ±2.4 mm and DV = −3.5 mm

ventral from skull surface) (Goudarzi, Salmani, Lashkarbolouki, and Goudarzi, 2015). That

site was chosen in between of the hippocampal regions for the facilitation of drug diffusion

into the regions. The guide cannulae were fixed and armed on the skull using acrylic dental

cement using surgical screws. The infusion cannulae were replaced with dummy cannulae

and cut to extend 0.5 mm beyond the guide cannulae to prevent clogging. Tetracycline

antibiotic ointment was applied to the skin incision to prevent infections.

The infusion of SB, TCS or saline/DMSO into both hippocampi in awake, freely moving rats,

started one week following recovery from skull surgery. Every drug infusion was performed

using a 30 gauge cannula connecting to a 1μl Hamilton syringe through a polyethylene tubing

(PE-20). For each animal implanted with the cannula, the infusion took three minutes and the

cannula remained in the site for approximately 5 min to allow proper drug diffusion. Intra-

hippocampal infusions were single (for acute stress) or repeated (once every day during 10

days of chronic stress), 20 min before stress session. Although the insertion of the cannulae

were ensured during hippocampal extraction, the exact placement of the cannulae were

confirmed on three randomly selected brains per group, cut in 40 μm brain slices and Nissl

stained (Figure 2).

2.4. Stress paradigm

Acute stress included two foot-shocks with 0.7 mA intensities lasting two seconds, spaced by

5.5 min interval that was delivered using the shocking device (Noldus, Netherlands) equipped

with a grid floor. Chronic stress was applied using a Plexiglas restrainer, 6 hours a day for 10

days, starting at 8 am every day.

8
2.5. Experimental groups:

Animals used in this study divided into 8 experimental groups that 5 hippocampi out of each

group were randomly selected for molecular measurements [cerebro-spinal fluid (CSF) and

plasma CRT]. The group descriptions are as following:

1. Control (CTRL): the animals of the control (without surgery & stress exposure) and sham

groups (with surgery & saline/DMSO infusion & without stress exposure) were assessed

using behavioral test. Since their behaviors had no significant difference, results of the

behavioral tests of both groups were pooled together (n = 10-13).

2. Acute stress (A.STS): these animals (n = 8-12) were treated to acute stress (see above) on

day 7 following surgery. This stress was followed by the series of behavioral tests, outlined

previously. Social maze was tested 30 min following acute stress. Twenty minutes later, the

open field test and another 20 min later the elevated plus maze were performed (Figure 1).

3. Acute stress-TCS OX29 (A.STS.TCS); this group (n = 8-10) received the electric shock

after OXr2 antagonist infusion which was followed by the behavioral tests.

4. Acute stress-SB-334867 (A.STS.SB): animals in this group (n = 6-7) were injected with

OXr1antagonist and then received the electric shock. Thereafter, the behavior was assessed.

5. Chronic stress (CH.STS): the rats (n = 8-13) recovered from the surgery were immobilized

in a restrainer tube; 6 hours daily for 10 days. Social maze test was assessed 24h after the

termination of last session of immobilization stress. Twenty minutes later, the open field test

and another 20 min later the elevated plus maze was performed (Figure 1).

6. Chronic stress-TCS OX 29 (CH.STS.TCS): in this group (n = 7-9), animals received a ten-

day intra-hippocampal injection of TCS OX 29, and then immobilized for 6 hours daily up to

10 days of chronic restraining stress. Then, the behavioral assessment was accomplished.

9
7. Chronic stress- SB-334867 (CH.STS.SB): these animals (n = 6) received a chronic ten-day

infusion of SB-334867 before daily immobilization stress. The behavioral assessment was

performed at the end.

2.6. Social maze test

This maze was a Plexiglas box (40 ×60 × 90), divided into three equal size chambers, with a

cage-like wire cylinder space at the left and right compartments for stranger 1 and 2,

respectively. Behavioral test in this apparatus consists of three phases: habituation,

sociability, and social novelty. At first, the animal was placed in the empty central

compartment of the testing box and allowed to move freely and habituate for 10 min in the

three spaces. Next, in the sociability phase, an unknown rat (stranger1) was placed into the

left compartment of the testing box inside the wire cage, while the right compartment wire

cylinder remained free of any stranger. The time spent in each compartment from a total of

10 min was measured. Immediately after this step, another unknown rat (stranger2) was

placed in the empty wire cage on the opposite side and similarly, the time spent in each

chamber was measured during the 10 minutes. Apparatus and wire cage were washed with

water and dried with a towel and disinfected with 70% ethanol for each new animal. Also, at

the end of each day of testing, the apparatus was wiped down with 70% ethanol, and allowed

to air-dry. This test was performed to assess the role of the hippocampal orexinergic system

and stress interaction on social behavior.

2.7. Elevated plus maze (EPM) test

The elevated plus maze used for assessing anxiety-related behaviors (Walf and Frye, 2007)

was a plywood painted flat black apparatus. The plus-shaped maze is 53 cm high and consists

of two opposite facing open arms and two opposite facing closed arms. Each arm is 10 cm

10
wide and extends 50 cm in length from a 10 × 10 cm square in the middle. The open arms

had a 1 cm wide and ¼ cm high edging along their border preventing the rats from falling off

the platform. The closed arms were surrounded by 40 cm walls of thin wood. To increase

exploratory behavior, the rats put into a wooden box (60*60*40) for one minute and then

they were placed in the middle of the plus maze facing towards an open arm and their

behavior was visually monitored for 5 minutes. The result was expressed as the percent of

time spent in the open and closed arms. More time spent in the open arms is indicative of

reduced anxiety while more time spent in the closed arms signify anxious behaviors (Walf

and Frye, 2007).

2.8. Open field (OF) test

The open field test was conducted in an open square Plexiglas box (70*70*35); that the floor

of the open field was subdivided into 16 equal sized squares (4 centers, 12 peripheral). After

introducing the animal into the apparatus arena, behavior of the animal was assessed. The

time of staying immobile was evaluated as immobility and was quantified as inverse criteria

for the exploratory behavior of the animal.

2.9. Orexin measurement

In order to evaluate the level of orexin-A in the CSF, it was extracted from Cisterna Magna,

before sacrifice, and quickly stored in a -70 freezer for later assessing with an ELISA kit

(MBS762821, MyBiosource, USA). The method of extraction (Nirogi, Kandikere,

Mudigonda, Bhyrapuneni, Muddana, Saralaya, and Benade, 2009) was briefly as the

followings. After anaesthesia of the rat with the mentioned dose, the rat positioned in a

stereotaxic frame. The head was shaved and then fixed in the frame using ear bars with nose

flexed by 45º. After disinfection of the backhead with 70% alcohol, a 23 guage needle

connected to a polyethylene tube and syringe was inserted on the depressive surface between

11
occipital protuberances and the spine of the atlas. By feeling a change of resistance for needle

movement, a gentle aspiration allowed the CSF flowing to the syringe. The procedure was

done by a skillful person and the rare blood contaminated sample was avoided. On the testing

day, following the standard protocol provided with the kit, optical densities of the 96-well

microplate read using a spectrophotometer at 450 nm. The CSF orexin level was calculated as

the value of pg/ml. Intra-assay was <8% and inter-assay was <10%.

2.10. Plasma CRT measurement

Following the experiments, anesthetized animals, five animals per group, were decapitated

and trunk blood (2 ml), was collected into a syringe containing 0.25 ml sodium citrate

anticoagulant. Then, the specimen was centrifuged at 2320×g for 20 min and the supernatant

plasma was stored in -70˚C until assay. On the testing day, the concentration of CRT in

plasma was measured using an enzyme immune assay (EIA) competitive assay kit (Cayman

Chemical; cat, 500655). Briefly, supernatant (50 µl from 100 µl) were incubated for 2 hours

with CRT AchE (Acetylcholine esterase) tracer (50 µl) and CRT EIA antiserum (50 µl) in

22˚C in each well. Following the wash with 200 µl of washing buffer, 200 µl of Ellman`s

buffer (The substrate for AChE) was added and incubated for 60-90 min in the dark on a

horizontal shaker. Absorbance determined at 412 nm using a spectrophotometer and amounts

of CRT was estimated based on the standard curve. The sensitivity of the kit was 150 pg

using 1 ml of sample with a signal-to-noise ratio of 2:1.

2.11. Data analysis

ANOVA was used to compare averages between multiple independent groups, which was

followed by post-hoc comparisons using the Tukey test when the ANOVA was statistically

12
significant. All the statistical tests were performed by SPSS v.23 software. The minimum

level of significance was P < 0.05 and the data are presented as mean ± SEM.

13
3. Results

3.1. Acute intrahippocampal infusion of OXR antagonists prevents stress-induced decline

in social behavior

Animals in the different groups were subjected to three tests: Habituation, Sociability, and

social novelty. Assessing the results of all animal groups in habituation step using ANOVA

showed no significant difference in time spent in the three chambers in both acute and

chronic conditions. During habituation, most animals spent an equal amount of time

exploring the two chambers which shows an equal interest of chamber occupancy.

Comparing the behavioral results for sociability test in acute stressed (A.STS) and orexin

receptor antagonists (TCS or SB) infused rats with acute stress exposure (A.STS.TCS or

A.STS.SB) and control group using ANOVA showed no significant difference in time spent

at left [F (3, 39) = 2.208, P = 0.104] and right [F (3, 31) = 2.384, P = 0.09] chambers of the

social maze (Figure 3-A). Thereafter, the behavior was compared for social novelty in acutely

stressed animals (A.STS) and acutely stressed animals with orexin receptor antagonist

infusion (A.STS.TCS or A.STS.SB) with control groups using ANOVA, which showed a

significant difference in time spent in the both left [F (3, 33) = 27.350, P = 0.0005] and right

chambers [F (3, 34) = 12.201, P = 0.0005]. Tukey post-hoc comparison for social novelty

demonstrated a significant increase in time spent in the left chamber (with stranger 1; P <

0.001, n = 11) and a significant decrease in time spent in the right chamber (containing

stranger 2) for acutely stressed rats (P < 0.001, n = 11). Additionally, blocking OXr2 with

TCS OX 29 prevented the acute stress effect in both the left (stranger 1; P < 0.001, n=9) and

right (stranger 2; P < 0.001, n = 9) chambers. Similarly, blocking OXr1 with SB-334867

impeded the acute stress effect in the left chamber (stranger 1; P < 0.001, n = 6) and right

chamber (stranger 2; P < 0.001, n = 6, Figure 4-A).

14
Comparing the effect of chronic stress and OXR antagonists (both TCS and SB injected

groups) using ANOVA showed no significant difference in time spent in the left [F (3, 37) =

1.970, P = 0.137] or right [F (3, 35) = 2.524, P = 0.075] chamber in neither Sociability nor

social novelty (Figure 3-B and 4-B).

3.2. Acute intrahippocampal infusion of OXR antagonists reduced anxiety following stress

in the EPM test

Comparing the results of acute stress with and without the infusion of TCS or SB using

ANOVA showed significant differences in time spent on the open arms [F (3, 39) = 9.919, P

= 0.0005], and closed arms [F (3,40)= 15.180, P= 0.0005]. Tukey post-hoc demonstrated a

significant decrease in time spent on the open arms (P < 0.001), and an increase in the time

spent in the closed arms (P < 0.001) for acutely stressed rats (A.STS) with respect to control

animals. While TCS preceding the acute stress (A.STS.TCS), significantly increased the time

spent on the open arms (P < 0.01) and decreased the time spent in the closed arms (P < 0.01)

compared with acute stressed animals; infusion of SB before acute stress exposure

(A.STS.SB), significantly decreased the time spent only in the closed arms (P < 0.01) (Figure

5-A).

Chronic stress with or without TCS or SB infusion had no significant differences in time

spent on the open [F (3, 37) = 335, P = 0.800] and closed arms [F (3, 37) = 1.458, P = 0.243]

(Figure 5-B). While there was no significant difference in the time spent at the center due to

the acute stress conditions, Chronically stressed animals infused with TCS spent less time

(P<0.05) at the center (Figure 4-B).

3.3. Acute intrahippocampal infusion of OXR antagonists prevented stress-induced

immobility in the OF test

15
Assessing the effect of acute stress with or without hippocampal infusions of OXR

antagonists (TCS or SB) using ANOVA demonstrated significant differences in the duration

of immobility in the OF test [F (3, 33) = 10.169, P = 0.0005]. Tukey post-hoc comparison

revealed that acutely stressed (A.STS) rats had a significant increase in the immobility time

(P < 0.001) compared to control animals. Treatment of acutely stressed rats with TCS or SB

(A.STS.TCS or A.STS.SB) significantly decreased (P < 0.01) immobility time compared

with acutely stressed (A.STS) animals (Figure 6-A). Evaluating the immobility time of

chronic stress (CH.STS) with or without injection of TCS or SB (CH.STS.TCS or

CH.STS.SB) using ANOVA showed no significant difference [F (3,30) = 0.881, P = 0.463]

between these groups (Figure 6-B).

3.4. OXR antagonists restored the chronic stress-induced increase of the CSF orexin level

Evaluating the level of CSF orexin using ANOVA in chronically stressed (CH.STS) animals

with or without the infusion of orexin receptor antagonists (CH.STS.TCS or CH.STS.SB)

showed significant differences [F (3, 18) = 9.431, P = 0.001]. While, Tukey post-hoc

comparison revealed a significant increase of CSF orexin due to chronic stress treatment

(CH.STS, P < 0.05 compared with the control group), the infusion of TCS OX29

(CH.STS.TCS; P < 0.01) or SB-334867 (CH.STS.SB; P < 0.01) prevented the increase in

orexin values of chronically stressed animals (Figure 8-B).

3.5. OXR antagonists prevented chronic stress-induced increase in plasma CRT level

Comparing the results of plasma CRT level using ANOVA demonstrated a significant

difference between control animals, acutely stressed (A.STS) and acutely stressed with OXR

antagonists (A.STS.TCS or A.STS.SB) [F(3,19)=5.657, P=0.008]. Tukey test revealed that

acute stress (A.STS) significantly increased the plasma CRT level in respect to control

16
animals (P < 0.05); while intrahippocampal injection of OXR antagonists had no effect on the

CRT content (Figure 7-A). Comparing the results of the plasma CRT assay using ANOVA

revealed a significant difference in control and chronically stressed (CH.STS) rats with or

without an infusion of OXR antagonists [F (3,19)= 22.835, P=0.0005]. Tukey post-hoc

comparison showed that chronically stressed (CH.STS) animals had a significant increase in

the CRT level compared with control rats (P < 0.001). Chronically stressed animals with an

intrahippocampal injection of TCS (CH.STS.TCS) showed a significant decrease in the

plasma CRT level with respect to the chronically stressed (CH.STS) rats )P < 0.01).

Similarly, intrahippocampal injection of SB (CH.STS.SB) significantly decreased plasma

CRT level in the chronic stress group (CH.STS; P < 0.01, Figure 7-B).

17
4. Discussion

Our study investigated the effect of hippocampal OXR blockade on social learning and

memory impairment following stress. The results of the study demonstrated that acute stress-

induced anxiety and immobility of the animals in the EPM and OF tests, respectively, were

prevented by the blockade of hippocampal OXr1 and OXr2. Furthermore, social avoidance of

the unfamiliar animal in social novelty was counteracted by the blocking of OXR´s.

However, no effect due to chronic stress on social novelty, anxiety and immobility behaviors

was demonstrated. In addition, CSF orexin content increased following chronic restraint

stress, with the increase impeded by hippocampal TCS infusion. Finally, a rise in plasma

CRT due to both acute and chronic stresses was observed. Blockade of the receptors was only

seen to prevent this rise following chronic stress.

Stress exposure evokes a collection of behavioral symptoms such as anxiety and certain types

of emotional disorders (Christianson and Greenwood, 2014). Results of acute stress exposure

in our experiments also elicited anxiety behavior in EPM and reduction of exploratory

behavior in OF test which was in agreement with previous studies on the acute stress

increased anxiety behavior in animals (Atchley, 2010; Solomonow, 2015). Surprizingly and

in contradiction to some studies (Chiba, Numakawa, Ninomiya, Richards, Wakabayashi, and

Kunugi, 2012; Dhingra, Dhingra, Kumria, Chadha, Singh, Kumar, and Karan, 2014;

Greenwood, Thompson, Opp, and Fleshner, 2014; Negron-Oyarzo, Perez, Terreros, Munoz,

and Dagnino-Subiabre, 2014; Qin, Zhou, Pandey, Vecchiarelli, Stewart, Zhang, Lagace,

Brunel, Beique, Stewart, Hill, and Chen, 2015), chronic restraint stress did not change the

animal anxiety development and immobility behavior in EPM or OF tests. Accordingly, a

research showed that chronic restraint stress did not produce an increase in depressive

immobility and novelty feeding in a model of 4 weeks restraint (Zhu, Shi, Wang, Wang, and

Li, 2014). Therefore, it appears that chronic repeated stress could lead to a form of adaptation

18
to the stress effect which may be due to repeated sessions of the same stress paradigm (Sadler

and Bailey, 2016), that is potentially capable of reducing the responsiveness of the HPA

(Grissom, Iyer, Vining, and Bhatnagar, 2007). In this regard, we applied a type of unexpected

sequential stress to prevent this type of adaptation to repeated sessions of the same stress, but

surprisingly the same results were still produced (data not shown). This may indicate that

coping with stress is a rather more complex phenomenon than simply predicting a repeated

event. In the next experiment, acute stress reduced the social novelty and prevented the

animals from investigating the new stranger in the social maze, which is in line with the data

od reduction in exploratory behavior. Chronic stress exhibited a similar result on social

preference to EPM and OF with respect to controls. Accordingly, Eagle et al. have shown that

a single prolonged stress impaired both the social and object novelty in the rat, an impairment

that the authors believe that is connected to the lack of behavioral flexibility (Eagle,

Fitzpatrick, and Perrine, 2013). In contrast to acute stress induced increase of the encounter

with the present familiar (the previous stranger) rat, the chronic stress exhibited a similar

encountering time (time spent) with control and unlike other studies of chronic stress induced

behavioral impairments (Greenwood et al., 2014; Negron-Oyarzo et al., 2014), it was unable

to change the animal behavior.

Orexin is a major player in the regulation of wakefulness which suggests that this peptide

may have a role in the manifestation of behavioral and physiological signs of stress (Carter et

al., 2009). The hippocampus is one of the key brain regions involved in learning and

memory. It represents an important potential target in gaining a greater understanding of the

impact of stress on learning and anxiety given its susceptibility to stress and the widespread

expression of both types of OXRs within hippocampal subregions. Our data demonstrated

that blocking the hippocampal OXr1 or OXr2 could preclude the acute stress-induced

development of anxiety, immobility, and avoidance of new situations. Orexin neurons

19
mediate anxiety behavior and panic responses partially through actions on the bed nucleus of

stria terminalis (BNST) and central amygdala neurons (Lungwitz, Molosh, Johnson, Harvey,

Dirks, Dietrich, Minick, Shekhar, and Truitt, 2012). In turn, some areas of the hippocampus

such as ventral subiculum, the output of the hippocampus, and CA1 send their projections to

the BNST (Lebow and Chen, 2016). Thus, it is hypothesized that blocking OXRs may affect

the BNST involvement in anxiety as well as boosting hippocampal ability for learning, which

may happen at the level of hippocampal OXRs and their interaction with GRs.

Correspondingly, our data revealed orexin as playing a role in stress responses, while OXR

blockade prevented the stress effect on anxiety and learning. This is in addition to previous

studies where we demonstrated that acute stress has increased the expression of orexin in the

lateral hypothalamus (Mokhtarpour, Elahdadi Salmani, Lashkarbolouki, Abrari, and

Goudarzi, 2016).

Acute stress exposure, through CRH secretion, affects orexinergic neurons and increase

orexin production (Winsky-Sommerer et al., 2004). Acute stress did not alter the CSF orexin

in this study which indicates orexin may not be involved in acute stress effects on

hippocampal learning and anxiety. On the other hand, we demonstrated an increase in prepro-

orexin expression following the same acute stress in our a previous study (Mokhtarpour et al.,

2016). This shows that a longer time has been needed for orexin to flow in the CSF in

comparison to the synaptic induced neurotransmitter release which should be considered in

future studies. However, it was shown that blocking CRFr1 prevented the fear evoked by

acute shock stress but not the pre-pro orexin expression in the hypothalamus (Chen, Li, and

Kirouac, 2014) indicating differential effects of stress on some types of behavior and

molecular changes. Although, the acute stress increased the amount of plasma CRT, which

may be a result of HPA activation due to stress (Garabadu, Shah, Ahmad, Joshi, Saxena,

Palit, and Krishnamurthy, 2011), chronic treatment could increase CSF orexin as well as

20
plasma CRT following termination of repeated sessions of stress. Consecutive stress

applications not only increased the orexin content, but also augmented the amount of plasma

CRT, which is likely an HPA response to stressors of longer duration (Lowrance, Ionadi,

McKay, Douglas, and Johnson, 2016) that not only increase CRT from the axis but also

stimulate the PVN to LHA pathway to produce orexin. From another perspective, the healthy

negative feedback on the hypothalamus by adrenal CRT and hippocampus during acute stress

will decrease the HPA activity that, in turn, will decrease CRT plasma level after the primary

rise (ter Heegde, De Rijk, and Vinkers, 2015). Correspondingly, the chronic stress impact on

hippocampal structure and function may weaken the feedback and can result in a greater

production of CRT which persist for a longer duration, leading to impairment of different

aspects of declarative memory mainly through hippocampal damage (Tatomir et al., 2014).

The damage may partly be dependent on the impairment of glucocorticoid receptor (GR)

function, a key molecule in the negative regulation of the HPA axis (Zhu, Liu, Li, Liu, Chen,

Han, Wu, Jing, Zhou, Suh, Zhu, and Zhou, 2014).

Orexins, besides the central control of feeding and sleep, may also modulate the stress system

through the OXRs in PVN of the hypothalamus (Spinazzi, Andreis, Rossi, and Nussdorfer,

2006). It has even been shown that orexins increase in vitro secretion of human

pheochromocytoma cells by stimulation of OXr2. Our data revealed diminished plasma CRT

and CSF orexin following the blockade of either OXr1 or OXr2 in the hippocampus. As far

as orexins may modulate the HPA through peripheral or central parts (Spinazzi et al., 2006),

the blockade of hippocampal OXRs, in this study, may act through a hippocampus-LHA

pathway and not via the damaged hippocampal HPA feedback pathway in the chronic

conditions and, in turn, another pathway me be involved in this effect. The central

connections from the hippocampus to hypothalamus via the BNST has been shown to be

involved in mood and anxiety disorders (Lebow and Chen, 2016) is likely to mediate the

21
effect of OXR`s blockade on the orexin and CRT production, a pathway which needs more

attention in the future studies, or the interaction between GRs and OXRs at the level of the

hippocampus.

In conclusion, acute stress resulted in increased anxiety and immobility, while also causing

decreased social recognition of new strangers. Furthermore, chronic stress increased plasma

CRT and CSF orexin expression. The lack of effect of acute stress on molecular components,

and absence of chronic stress effects on behaviour, may be as a result of the impact of

adaptation on the anxiogenic effects of immobility stress during behavioral tests for chronic

stress (Sadler and Bailey, 2016) This may result in the employment of alternative pathways

for acute stress to change orexin expression, both of these findings represent interesting

avenues for further study.

The principle conclusion from this study is that hippocampal OXR blockade prevents acute

stress-induced behavioral, and chronic stress-induced plasma CRT and CSF orexin, changes.

OXR manipulation may improve adaptation to stress pathophysiology; further studies are

required to confirm if this effect is related to the interaction between OXR’s and GR’s in the

hippocampus.

5. Acknowledgment

The authors declare no conflict of interest. This work was financially supported by the

Cognitive Sciences and Technologies Council in IRAN [grant number; 2671]. The support of

Damghan University, as part of a thesis project with IRANDOC code 1253546 is also

appreciated. The authors would like to thank Dr. Mark Dunleavy (UX Consultant at Isobar

Madrid, Madrid, Spain and a former member of Laboratory of Molecular Neuropathology,

Centre for Integrative Biology, University of Trento Trento, Italy) and Dr Hossein Davari

(Damghan University, Iran) for English editing of the manuscript.

22
Legends

Figure 1. Timeline of the experiments. After 7 day recovery from surgery and cannulation,

intrahippocampal saline, OXr1 (SB334867) or OXr2 (TCS OX 29) receptor antagonists was

administered and the animals experienced acute (20 min later) or chronic (daily restraint upto

10 days; each antagonist injection was followed, 20 min later, by 6 hours stress) stresses.

Following 30 min in acute and 24 hours in chronic conditions, social maze test was

performed. Behavioral OF and EPM tests were done in 20 min intervals. Immediately after

behavioral observations, CSF and blood plasma were sampled and stored in -80 freezers until

orexin and CRT measurement, respectively.

Figure 2. Confirmation of cannula placement in the hippocampus. The injection site in a

coronal section modified from the atlas of Paxinos and Watson was shown at 3.6 mm

posterior to bregma. The black circle is the site of injection which is 0.7 mm lower than the

tip of cannula.

Figure 3. The effect of orexin receptor blocking and acute and chronic stresses on sociability.

Neither the acute nor the chronic stress and orexin receptor antagonists could change the

sociability in the social maze test.

CTRL (n = 11), control; A.STS (n = 12), acutely stressed; A.STS.TCS (n = 10), acutely

stressed with orexin receptor 2 antagonist infusion; A.STS.SB (n = 6-7), acutely stressed with

orexin receptor 1 antagonist infusion; CH.STS (n = 13), chronically stressed; CH.STS.TCS (n

= 8), chronically stressed that received orexin receptor 2 antagonist infusion; CH.STS.SB (n

= 6), chronically stressed with orexin receptor 1 antagonist infusion. Data are presented as

mean ± S.E.M.

23
Figure 4. The effect of OXR blocking on acute and chronic stress-induced change in social

novelty. The acute stress but not the chronic stress changed the time spent in the left (stranger

1) and right (stranger 2) chambers. Blocking the orexin receptor 1 and 2 prevented the acute

stress-induced effect on the time spent in the left and right chambers. ***P < 0.001 compared

with control, ††† P <0.001 compared with A.STS.

CTRL (n = 12), control; A.STS (n = 8-10), acutely stressed; A.STS.TCS (n = 8-10), acutely

stressed with orexin receptor 2 antagonist infusion; A.STS.SB (n = 6-7), acutely stressed with

orexin receptor 1 antagonist infusion; CH.STS (n = 13), chronically stressed; CH.STS.TCS (n

= 8), chronically stressed that received orexin receptor 2 antagonist infusion; CH.STS.SB (n

= 6), chronically stressed with orexin receptor 1 antagonist infusion. Data are presented as

mean ± S.E.M.

Figure 5. The effect of OXR blocking on acute and chronic stress-induced change in anxiety

behavior. The acute stress-induced decrease of time spent in the open arm and the increase in

the closed arm were restored to the control level following infusion of OXR antagonists. The

chronic stress and the receptor blocking had no effect. ***P < 0.001 compared with control,

††P < 0.01 compared with A.STS.

OA, open arms, CA, closed arms, CTRL (n = 12-13), control; A.STS (n = 11), acutely

stressed; A.STS.TCS (n = 10), acutely stressed with orexin receptor 2 antagonist infusion;

A.STS.SB (n = 7), acutely stressed with orexin receptor 1 antagonist infusion; CH.STS (n =

11-12), chronically stressed; CH.STS.TCS (n = 8-9), chronically stressed that received orexin

receptor 2 antagonist infusion; CH.STS.SB (n = 6), chronically stressed with orexin receptor

1 antagonist infusion. Data are presented as mean ± S.E.M.

24
Figure 6. The effect of OXR blocking on acute and chronic stress-induced change of

immobility. Acute stress could increase the immobility, while OXR antagonists restored the

effect to the control level. The chronic stress had no effect. ***P < 0.001, compared with

control, ††P < 0.01, compared with A.STS.

CTRL (n = 10), control; A.STS (n = 11), acutely stressed; A.STS.TCS (n = 8), acutely

stressed with orexin receptor 2 antagonist infusion; A.STS.SB (n = 6), acutely stressed with

orexin receptor 1 antagonist infusion; CH.STS (n = 8), chronically stressed; CH.STS.TCS (n

= 7), chronically stressed that received orexin receptor 2 antagonist infusion; CH.STS.SB (n

= 6), chronically stressed with orexin receptor 1 antagonist infusion. Data are presented as

mean ± S.E.M.

Figure 7. The effect of OXR blocking on acute and chronic stress induced changes in CSF

orexin. Non of the antagonist infusion could recover the orexin increased level in the acute

conditions, while the infusion of OXR antagonists on the chronic stress-induced increase of

orexin could prevent the effect. *P < 0.05 compared with control, ††P < 0.01 compared with

CH.STS.

CTRL (n = 5), control; A.STS (n = 5), acutely stressed; A.STS.TCS (n = 5), acutely stressed

with orexin receptor 2 antagonist infusion; A.STS.SB (n = 5), acutely stressed with orexin

receptor 1 antagonist infusion; CH.STS (n = 5), chronically stressed; CH.STS.TCS (n = 5),

chronically stressed that received orexin receptor 2 antagonist infusion; CH.STS.SB (n = 5),

chronically stressed with orexin receptor 1 antagonist infusion. Data are presented as mean ±

S.E.M.

Figure 8. The effect of OXR blocking on acute and chronic stress-induced change of plasma

CRT. Acute and chronic stresses increased the plasma level of CRT, while only the effect of

25
chronic stress was prevented due to OXR blocking. *P < 0.05, ***P < 0.001 compared with

control, ††P < 0.01, compared with CH.STS.

CTRL (n = 5), control; A.STS (n = 5), acutely stressed; A.STS.TCS (n = 5), acutely stressed

with orexin receptor 2 antagonist infusion; A.STS.SB (n = 5), acutely stressed with orexin

receptor 1 antagonist infusion; CH.STS (n = 5), chronically stressed; CH.STS.TCS (n = 5),

chronically stressed that received orexin receptor 2 antagonist infusion; CH.STS.SB (n = 5),

chronically stressed with orexin receptor 1 antagonist infusion. Data are presented as mean ±

S.E.M.

26
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Highlights

 Stress, either acute or chronic did not affect the sociability of animals.

 Acute stress reduction of novelty was impeded by orexin receptor

(OXR) blocking.

 The acute stress increase in anxiety and immobility was prevented by

OXR blocking.

 OXR blocking could hinder the chronic stress induced build up in

orexin production.

 OXR blocking could prevent chronic stress induced increase of

corticosterone.

 Hippocampal OXRs are probably good candidates to nullify stress

impairments.

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