Peripheral injection of dexamethasone modulates anxiety related behaviors in mice: an interaction with opioidergic neurons.

Abstract

Stress and anxiety initiates a cascade of biochemical and endocrine event which results in behavioral and electrophysiological effects in both animals and humans. In this study, we investigated the effects of dexamethasone (DEX), as a synthetic glucocorticoid, and its interaction with opioidergic system on anxiety related behavior in mice. Young adult male mice were used in this study. A standard elevated plus-maze was used to determine anxiety levels in animal. Different doses of DEX (0.1, 0.5, 1, 2 and 10 mg/kg, SC) or vehicle was injected 30 min before of evaluation. Naloxone (1 and 2 mg/kg, IP) was injected 5 min before the DEX (0.5 and 1 mg/kg) administration. Results indicated that DEX at doses of 0.5 and 1 reduced and in dose of 10 mg/kg increased anxiety related behaviors significantly (P<0.05 in all cases). Also pretreatment of naloxone at doses of 1 and 2 mg/kg attenuated the effects of lower doses of DEX on anxiety related behaviors. Finding above indicated that peripheral administration of glucocorticoids induces biphasic effects on anxiety related behaviors: anxiolytic effects in lower doses and anxiogenic effects in a high dose. Data also revealed an involvement of opioidergic system in anxiolytic effects of glucocorticoids.

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Pak. J. Pharm. Sci., Vol.21, No.3, July 2008, pp.285-289
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PERIPHERAL INJECTION OF DEXAMETHASONE MODULATES ANXIETY
RELATED BEHAVIORS IN MICE: AN INTERACTION
WITH OPIOIDERGIC NEURONS

ABBAS ALI VAFAEI, ALI RASHIDY-POUR AND ABBAS ALI TAHERIAN
Physiology Research Center, Semnan University of Medical Sciences, Semnan, Iran

ABSTRACT
Stress and anxiety initiates a cascade of biochemical and endocrine event which results in behavioral and
electrophysiological effects in both animals and humans. In this study, we investigated the effects of
dexamethasone (DEX), as a synthetic glucocorticoid, and its interaction with opioidergic system on anxiety
related behavior in mice. Young adult male mice were used in this study. A standard elevated plus-maze was
used to determine anxiety levels in animal. Different doses of DEX (0.1, 0.5, 1, 2 and 10 mg/kg, SC) or vehicle
was injected 30 min before of evaluation. Naloxone (1 and 2 mg/kg, IP) was injected 5 min before the DEX (0.5
and 1 mg/kg) administration. Results indicated that DEX at doses of 0.5 and 1 reduced and in dose of 10 mg/kg
increased anxiety related behaviors significantly (P<0.05 in all cases). Also pretreatment of naloxone at doses of
1 and 2 mg/kg attenuated the effects of lower doses of DEX on anxiety related behaviors. Finding above
indicated that peripheral administration of glucocorticoids induces biphasic effects on anxiety related behaviors:
anxiolytic effects in lower doses and anxiogenic effects in a high dose. Data also revealed an involvement of
opioidergic system in anxiolytic effects of glucocorticoids.

Keywords: Glucocorticoids, anxiety, dexamethasone, opioid system, elevated plus maze, mice.


INTRODUCTION

Anxiety disorders are the most common type of
psychiatric disorders, with an incidence of 18.1% and a
lifetime prevalence of 28.8% (Kessler et al., 2005). The
term of anxiety is characterized by somatic, cognitive,
behavioral and perceptual symptoms. Also many
endocrine, autoimmune, metabolic and toxic disorders as
well as medi-cation adverse effects are known to generate
anxiety (Kaplan et al., 2004). In addition, anxiety is
experienced in situations where an individual has a need
that they see no means of satisfying, or is faced with the
threat of punishment that they see no means of avoiding
(Korneyev, 1997). Previous evidences indicate that
pretreatment with anti-anxiety agents attenuates various
behavioral consequences of stress and anxiety (Eisenberg,
1993). Many evidences indicate that stress and anxiety
initiates a cascade of biochemical and endocrine event
which results in behavioral and electrophysiological
effects in both animals and humans (Korneyev, 1997).
According to gray anti-anxiety affects are achieved
through the inhibition of noradrenergic, adrenergic and
serotonnergic neurons located in the locus ceruleus and
raphe nuclei. These neurons initiate a stress and anxiety
reaction through the stimulation of CRH release from the
paraventricular nucleus (Kaplan et al., 2004).

It seems that the hypothalamic-pituitary-adrenal (HPA)
axis plays a major role in the stress and anxiety reaction.
Cortisol is a steroid hormone which affects all cells and
maintains physiologic integrity within the body. Studies
over decades have consistently shown a positive
correlation between the severity of a stressor and the level
of cortisol in the bloodstream a (Fernandes et al., 1997).
There is now considerable evidence that dysregulation of
the HPA axis is implicated in the pathophysiological of
affective and anxiety disorders (Boyle et al., 2006;
Velisek, 2006).

Glucocorticoid hormones readily enter the brain and
activate adrenal steroid receptors. They modulate a
variety of behavioral responses through an interaction
with several neurotransmitter systems (Soravia et al.,
2006). The aim of this study was to determine the effects
of dexamethasone, as a synthetic glucocorticoid, and its
interaction with opioidergic system on anxiety related
behaviors in mice.

MATERIAL AND METHODS

Subjects
Young adult male albino mice (n=120), weighting
between 25-30 grams, were used in this experimental
study. They were housed in groups of ten in plastic cages
in a room with constant temperature (22°-24°) and natural
lighting (12h light-12h dark) conditions. Water and food
were freely available. The treatment of the animals was
approved by the Institute’s Ethical Committee, and
conformed to NIH guidelines and to the Animal
Protection Law of the Iran.

Corresponding author: e-mail: aavaf43@yahoo.com

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Pak. J. Pharm. Sci., Vol.21, No.3, July 2008, pp.285-289
286
Drug and Injection Procedure
Dexamethasone (Synopharm, Italy) as a specific GR
agonist (0.1, 0.5, 1, 2 and 10 mg/kg) or vehicle (VEL)
were systemic injected subcutaneously. DEX was
dissolved initially in 100% ethanol and diluted to a final
concentration of 2% ethanol in 0.9% saline. A 2%
ethanol solution in saline was used for vehicle control
injection. Also naloxone (Sigma Co.) as an opioidergic
antagonist (1 and 2 mg/kg IP) or saline (SAL) were
systemic injected IP. NAL was dissolved initially in SAL.
All drugs and solutions were freshly prepared before each
experiment. Doses of drug determined with our pilot
study and also a survey of other studies using these drugs
(Boyle et al., 2006; Korneyev, 1997, Soravia et al., 2006).

Behavioral test and apparatus
Elevation plus maze (EPM) task
Apparatus
The elevated plus maze test was made of wood with two
open arms (50 x 5 cm) and opposite closed arms of the
same size but with 40-cm high walls. The arms were
connected by a central square and thus formed a plus sign.
The apparatus was elevated 50 cm above the floor. Each
of mice was placed in the central square of the plus maze
facing an enclosed arm. The time spent in enclosed and
open arms was scored for 5 min. An arm entry was
defined as an animal entering the arm with all four feet
and the number of entries into open and enclosed arms
was scored (Zarrindast et al., 2001). This instrument is
commonly used to assess anxiety-like behavior in
laboratory animals. The open arms are perpendicular to
the closed arms, with the four arms intersecting to form
the shape of a plus sign. Security is provided by the
closed arms while the open arms over exploratory value.
Therefore, one might expect anxious rats to spend less
time in the open arms than those that are less fearful.
When placed in an elevated plus-maze for the first time, a
mice’s behavior is largely based on its anxiety level.
Normal mice's that have not received any anti-anxiety
drugs will become moderately anxious in this new
environment. Thus, they tend to prefer the closed arms
over the less secure open arms (Dawson and Tricklebank,
1995).

Meanwhile, mice’s treated with anti-anxiety drugs
(diazepam) tend to be less anxious, so they spend more
time in the open arms compared to normal mice’s, and
they are generally less active (Fernandes et al., 1997).
Two behavioral measures were used: the percentage of
time spent in the open arms and the ratio of open arm
entries to total entries during 5 min. More entries into the
open arms and more time spent in the open arms were
interpreted as indicating lower levels of anxiety.

Statistics
Data were analyzed by one-way and two-way analysis of
variance (ANOVA), followed by Tukey’s test for multiple
comparisons. Values of P<0.05 were considered
significant.

EXPERIMENTAL PROTOCOL

Experiment 1
The aim of Expt.1 was to determine the effect of systemic
injection of DEX on anxiety related behavior in EPM
task. Sixty mice were divided into six groups, SAL+VEL
(n =10), SAL+DEX (n=50 in five groups), and EPM
evaluation occurred in a single 5 min session according to
procedure described in Section 2. 30 min before
evaluation, control and treatments groups received
systemic injections of SAL (1 ml/kg IP) and VEL (1ml/kg
SC) or SAL (1 ml/kg IP) and DEX (0.1, 0.5, 1, 2 and
10mg/kg SC), respectively. EPM evaluation was assessed
during a 5 min.

Experiment 2
The aim of Expt.2 was to determine the role of the opioid
system on angiolytic effects of DEX. Sixty mice were
divided into six groups: NAL+VEL (n =20 in two
groups), and NAL+DEX (n=40 in four groups). EPM
evaluation occurred in a single 5 min session according to
procedure described in Section 2. Control and treatments
groups received systemic injections of NAL (1 and 2
mg/kg, IP) plus VEH (1 ml/kg SC). Treatments groups
received NAL (1 or 2 mg/kg IP) plus DEX (0.5 or 1
mg/kg SC). EPM evaluation was assessed during a 5 min.

RESULTS

Experiment 1
Analysis of data indicated that DEX–treated mice showed
anxiolytic responses in doses of 0.5 and 1 mg/kg. These
mice spent more percent time in open arms (P<0.02) and
higher number of entrances to open arm (P<0.01) than to
control groups. DEX at a higher dose (10 mg/kg) induced
an anxiogenic response in mice (fig.1 A and B). Thus,
DEX induces a biphasic effects on anxiety related
behaviors.

Experiment 2
Analysis of data (fig. 2A and B) indicated that
pretreatment of NAL at both doses attenuated the effects
of DEX on anxiety related behaviors. NAL alone did not
change the behavioral measures (time spent in open arm
and number of entrances to open arm) of anxiety related
behaviors in comparison with control groups, but
significantly reduced both measures in DEX-treated mice
(P<0.05). Thus, NAL attenuates anxiolytic effects of
DEX.

DISCUSSION

The major finding of the present experiment is that
systemic injection of the dexamethasone has a biphasic

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Vafaei et al.
Pak. J. Pharm. Sci., Vol.21, No.3, July 2008, pp.285-289
287
effects on anxiety related behaviors in mice: in lower
doses induces an anxiolytic while in a higher dose induces
an anxiogenic effects. Data also revealed an involvement
of opioidergic system in
glucocorticoids.

Previous study indicated that stress and anxiety activates
the hypothalamus-pituitary-adrenal axis and causes
release of glucocorticoids (Fernandes et al., 1997). It
seems that this sequence of events contributes to anxiety
induced behavioral responses (Korneyev, 1997). The
finding of the present experiments supports the hypothesis
that glucocorticoid receptors (GR) affect anxiety related
behaviors. We have found that systemic administration of
DEX modulates anxiety related behaviors, suggesting an
involvement of GR in anxiety behaviors. However, with a
systemic administration it is not possible to determine that
anatomical locations of GR that involved in anxiety. Our
finding consistent with previous studies showing that the
HPA axis plays a major role in the stress and anxiety
reaction. There is now considerable evidence that
dysregulation of HPA axis is implicated in the
pathophysiology of affective and anxiety disorders (Heim
et al., 2000, Holsboer, 2000).
anxiolytic effects of
Previous study indicated that many of neurotransmitter
systems have been implicated in the neurobiology of
anxiety disorders. There is growing evidence that
alterations in several neurotransmitter systems (such as
GABA, serotonin, adrenergic, glutamate, opioids and etc)
may be involved in general anxiety disorders (Nutt,
2001). Also many evidence indicates that these systems
and HPA may interact in influencing have behaviors
(Soravia et al., 2006). For example, a previous study
indicated that high levels of corticosteroids can also
down-regulate serotonin 5-HT1A receptor messenger
ribonucleic acid (mRNA) and 5-hydroxytryptamine (5-
HT) binding in the hippocampus (Lopez et al 1999),
suggesting an important and complex triangular
relationship among the HPA axis, glutamate, and
serotonin in the neural circuits implicated in stress and
anxiety responses. Also extensive preclinical evidence
suggests that glutamate serves an important role in
modulating actions of CRF and glucocorticoids,
particularly in brain regions important in anxiety and
mood regulation such as the amygdala, hypothalamus,
mono-aminergic brainstem nuclei, and hippocampus
(Mathew et al., 2001).

0
10
20
30
Persent of time spent
in open arm (%)
SAL+VEL
SAL+DEX 0.1mg
SAL+DEX 0.5mg
SAL+DEX 1mg
SAL+DEX 2mg
SAL+DEX 10mg
*
*
*
A
0
1
2
3
4
5
Mean of Entrance
Numbers
SAL+VEL
SAL+DEX 0.1mg
SAL+DEX 0.5mg
SAL+DEX 1mg
SAL+DEX 2mg
SAL+DEX 10mg
**
*
B
0
10
20
30
Persent of time spent in
open arm (%)
SAL+VEL
NAL 1+VEL
NAL 2+VEL
SAL+DEX 0.5
SAL+DEX 1
NAL 1+DEX 0.5
NAL 1+DEX 1
NAL 2+DEX 0.5
NAL 2+DEX 1
*
*
**
A
0
1
2
3
4
5
Mean of Entrance Numbers
SAL+VEL
NAL 1+VEL
NAL 2+VEL
SAL+DEX 0.5
SAL+DEX 1
NAL 1+DEX 0.5
NAL 1+DEX 1
NAL 2+DEX 0.5
NAL 2+DEX 1
*
*
**
B
Fig. 1: The effect of dexamethasone (0.1, 0.5, 1, 2 and 10
mg/kg, SC) on anxiety related behaviors in mice in Expt.1.
Mean ± SEM of percent of time spent in open arm (A) and
number of entrances (B) during a 5 min test. *P < 0.02 as
compared with control group.
Fig. 2: The effect of naloxone on anxiolytic effects of
dexamethasone in Expt. 2. Mean ± SEM of percent of
time spent in open arm (A) and number of entrances (B)
during a 5 min test.* P<0.01 as compared with saline +
VEH group. ** P<0.01 as compared with saline + DEX
groups. SAL: saline;
dexamethasone. N= 10 for each group.
NAL: Naloxone; DEX:

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Pak. J. Pharm. Sci., Vol.21, No.3, July 2008, pp.285-289
288
Our finding also indicated that naloxone did not affect
anxiety related behavior alone, but rather blocked the
angiolytic effects of dexamethasone. This finding
indicates involvement of naloxone sensitive pathway in
mediating the influences of glucocorticoids on anxiety.
There are two possible explanations for an interaction
between dexamethasone and naloxone on anxiety. First, it
is likely that dexamethasone activates the endogenous
opiate system and then that mediates their influences on
anxiety. Although, there are some evidences indicating
that the effects of DEX on some behaviors such as
analgesia and emotional memory mediated, at least in
part, by the endogenous opiate system (Rashidy-Pour et al
2004, Stevens and Pezulla 1989), but to our knowledge
there are no reports in literature concerning with such
mediation on anxiety. Another explanation for these
results might be that naloxone interacts with stress and
anxiety induced glucocorticoids or dexamethasone in
plasma membrane of target neurons in brain. As noted
above, anxiety modulate was found as soon as 30 min
after dexamethasone injection. This time is too fast to
explain genomic action of glucocorticoids via intracellular
receptors and it may reflect an involvement of membrane
receptors. In light of this idea, recently a membrane
receptor for glucocorticoids in brain of the roughskin
newt (Taricha granulosa) as an amphibian model has
been identified (Evans et al., 2000a), and this receptor
appears to be a G-protein coupled receptor super-family
(Orchinik et al., 1992). This receptor mediates the rapid
effects of stress or corticosterone on reproductive
behaviors and neural activity of the animal (Hua and
Chen 1989, Joles and de Kloet, 1989). Ligand binding
assays with radiolabel corticosterone revealed that this
receptor not only is the highly selective for corticosterone
and Cortisol, but also recognizes specific opiate ligands
such as dynorphine and naloxone. These opiate ligands
interact directly by competing for the same binding site
(Evans et al., 2000b). Therefore, based on these data,
opiate antagonists that bind to membrane receptor for
glucocorticoids should block the behavioral effects of
glucocorticoids. This idea supported by the present
findings, which indicate
deteriorating effects of dexamethasone on anxiety related
behavior.

CONCLUSION

In conclusion, the presents results provide evidence for
this hypothesis that peripheral administration of
glucocorticoids produce a biphasic effects on anxiety
related behavior in mice: in lower doses induce an
anxiolytic while in a higher dose induces an anxiogenic
effects. Further, the opiate system may mediate the
anxiolytic effects of glucocorticoids. Further studies are
required to determine the underlying mechanisms.

naloxone blocks, the
ACKNOWLEDGEMENTS

The authors would like to thank Mr. Hossain Miladi-
Gorgi and Mr. Hassan Sadeghi for their technical
assistance during this study. This work was supported by
a grant from Semnan University of Medical Sciences and
Health Services.

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