INCREASE IN THE NUMBER OF CB1 IMMUNOPOSITIVE NEURONS IN THE AMYGDALOID BODY AFTER ACUTE COLD STRESS EXPOSURE

Abstract

PURPOSE: According literature data the animal's response to stress depends not only upon the state and
conditions of the animal but also upon the nature of the stressor itself. It is known that stress have wide-
ranging effects on neuroendocrine, autonomic, immune, and hormonal function. Different research groups
have shown induction of acute physical stress by low temperature exposure which have been reported to
impair motor activity, modulate pain perception, anxiety and depression-like behaviors in the animals.
Considerable work has established the amygdaloid body as a key site involved in the generation of fear and
anxiety responses, the assignment of emotional salience to external stimuli, the coordination of affective,
autonomic, and behavioral responses. The aim of our study was to examine the effect of cold stress on density of CB1 receptors in the amygdaloid body.
METHODS: Immunocytochemistry and morphometric analysis were used to determined density of CB1
immunopositive neurons in the amygdaloid body of male Wistar rats exposed to acute cold stress.
RESULTS: Our morphometric studies reveal that cold stress significantly increased (around 40 %) density
of CB1 immunopositive neurons in the rat amygdaloid body compared with control group.
CONCLUSIONS: Our results confirm that temperature fluctuation induces stress and endocanabinoid
system is involved.

Full text

106 Trakia Journal of Sciences, Vol. 12, Suppl. 1, 2014
Trakia Journal of Sciences, Vol. 12, Suppl. 1, pp 106-109, 2014
Copyright © 2014 Trakia University
Available online at:
http://www.uni-sz.bg
ISSN 1313-7050 (print)
ISSN 1313-3551 (online)
INCREASE IN THE NUMBER OF CB1 IMMUNOPOSITIVE NEURONS IN
THE AMYGDALOID BODY AFTER ACUTE COLD STRESS EXPOSURE
E. Dzhambazova1, B. Landzhov2, L. Malinova2, Y. Kartelov2, S. Abarova2
1Department of Chemistry, Biochemistry, Physiology and Pathophysiology, Faculty of Medicine,
Sofia University St. Kl. Ohridski, Sofia, Bulgaria
2Department of Anatomy, Histology and Embryology, Faculty of of Medicine, Medical University,
Sofia, Bulgaria
ABSTRACT
PURPOSE: According literature data the animal's response to stress depends not only upon the state and
conditions of the animal but also upon the nature of the stressor itself. It is known that stress have wide-
ranging effects on neuroendocrine, autonomic, immune, and hormonal function. Different research groups
have shown induction of acute physical stress by low temperature exposure which have been reported to
impair motor activity, modulate pain perception, anxiety and depression-like behaviors in the animals.
Considerable work has established the amygdaloid body as a key site involved in the generation of fear and
anxiety responses, the assignment of emotional salience to external stimuli, the coordination of affective,
autonomic, and behavioral responses. The aim of our study was to examine the effect of cold stress on
density of CB1 receptors in the amygdaloid body.
METHODS: Immunocytochemistry and morphometric analysis were used to determined density of CB1
immunopositive neurons in the amygdaloid body of male Wistar rats exposed to acute cold stress.
RESULTS: Our morphometric studies reveal that cold stress significantly increased (around 40 %) density
of CB1 immunopositive neurons in the rat amygdaloid body compared with control group.
CONCLUSIONS: Our results confirm that temperature fluctuation induces stress and endocanabinoid
system is involved.
Key words: cold stress, endocannabinoids, amygdaloid body
INTRODUCTION
The term ‘stress’ generally indicates an internal
(i.e.,infection, psychological condition) or
external (i.e., physical danger or damage)
circumstance that threatens the homeostasis of
the organism. Thus, stress results in a
discrepancy, either real or perceived, between
the demands of a situation and the organism’s
resources (1). Exposure to a stressor is
immediately followed by somatic and neuro-
physiological reactions involving peripheral
organs such as adrenals, cardiovascular and
respiratory systems, metabolism, and also brain
____________________________
*Correspondence to: E. Dzhambazova, Department
of Chemistry, Biochemistry, Physiology and
Pathophysiology, Faculty of Medicine, Sofia
University St. Kl. Ohridski, Sofia, Bulgaria, е-mail:
elena.dzambazova@abv.bg
areas such as the prefrontal cortex, amygdala,
hippocampus, nucleus accumbens, and
hypothalamus (2). Considerable work has
established the amygdaloid body as a key site
involved in the generation of fear and anxiety
responses, the assignment of emotional salience
to external stimuli, the coordination of affective,
autonomic, and behavioral responses.
It is known that the endocannabinoid system
(ECS), formed by cannabinoid receptors (CB1
and CB2), their endogenous lipid ligands
(endocannabinoids, ECBs), and the machinery
for synthesis and degradation of ECBs plays a
key role in the psychoneuroendocrine responses
to stress. The ECS participates in multiple brain
circuits implicated in stress reactions, learning,
and extinction of fear, emotional regulation, and
reward processes. The endocannabinoid CB1

DZHAMBAZOVA E., et al.
Trakia Journal of Sciences, Vol. 12, Suppl. 1, 2014 107
receptors are found in many areas involved in
regulation of the stress response including the
paraventricular nucleus of the hypothalamus, the
pituitary, adrenal glands, and limbic-related
areas of the brain (2-4).
We have previously found that following acute
cold stress (CS) male Wistar rats showed a
pattern of decreased anxiety-like behavior
(reduced spontaneous and increased freezing
behavior in the open field test) (5). Because the
functional roles of amygdala and CB1 receptors
in mediation of stress-induced anxiety are
unclear, the aim of the current study was to to
examine the effect of cold stress on density of
CB1 receptors in the amygdaloid body.
MATERIALS AND METHODS
Animals and housing: Twelve experimentally
naive male Wistar rats (180-200g) served as
subjects. They were housed in a temperature-
controlled room (22 ± 2°C). Food and water
were available ad libitum.. The rats were
randomly divided in two experimental groups: a
cold stress (CS) group and a control group. All
experiments were performed during the light
period between 09:00 and 13:00 h. The
experimental procedures were carried out in
accordance with the institutional guidance and
general recommendations on the use of animals
for scientific purposes.
Acute model of cold stress: The animals were
placed in a refrigerating chamber at 4oC. The
control group was not submitted to 1 hour stress
procedure.
Immunohistochemistry and morphometric
analysis: The animals were anesthetized with
thiopental (40 mg/kg b.w.). Transcardial
perfusion was done with 4% paraformaldehyde
in 0.1 M phosphate buffer, pH 7.2. Postfixation
of the obtained brains was conducted in 4%
buffered solution (0.1 M phosphate buffer, pH
7.4) of paraformaldehyde overnight at 4ºC.
Small series of 40 µm thick coronal sections
were cut on а freezing microtome from bregma -
1.80 to bregma -4.16 according to stereotaxic
athlas of the rat brain (6). For
immunohistochemistry the free floating sections
were collected in Tris-HCl buffer 0.05M, pH 7.6
and preincubated for 1 h in 5% normal goat
serum in PBS. Afterwards, incubation of the
sections was performed in a solution of the
primary antibody for 48 hs at room temperature.
We used a polyclonal rabbit anti-CB1 antibody
(Santa Cruz, USA), in a dilution of 1:1000. Then
sections were incubated with biotinylated anti-
rabbit IgG (dilution, 1:500) for 2 hs and in a
solution of avidin-biotin-peroxidase complex
(Vectastain Elite ABC reagent; Vector Labs.,
Burlingame CA, USA; dilution 1:250) for 1 h.
This step was followed by washing in PBS and
then in 0.05 M Tris-HCl buffer, pH 7.6, which
preceded incubation of sections in a solution of
0.05% 3,3-diaminobenzidine (DAB, Sigma)
containing 0.01% H2O2 for 10 min at room
temperature for the visualization. Afterwards,
they were rinsed with 0.1 M Tris–HCl, pH 7.6
and thrice with 0.01 M PBS for 5 min and finally
the sections were mounted on gelatin-coated
glass, dried for 24 h and coverslipped with
Entellan. Morphometric analysis was performed
using a microanalysis system. Data of the entire
drawings were entered in computer programme
(Olympus CUE-2), recorded automatically,
calculated and compared by Student’s t-test.
RESULTS
А density of CB1 immunoreactive neurons was
examined on high magnification. We found CB1
immunoreaction in cell bodies, axons and
dendrites which was visualized as a brown color
in perikarya and fibers. The cells bodies were
comprised of several distinct shapes. Fine-
beaded fibers and many puncta were observed
around the neurons. CB1 receptor
immunoreactivity in the amygdala in both
groups of rats was diffuse and extensive. The
most distribution of immunoreactive product was
found around of the cell bodies. Great deal of
CB1 receptor immunoreactivity was found
outside of cell bodies labeling the processes of
the cells.
Our morphometric studies reveal differences
between expression of CB1 immunopositive
neurons in control and experimental group
(Figure 1A, B). Cold stress significantly
increased the density of CB1 immunopositive
neurons in the rat amygdala (p < 0.05) compared
with controls. The quantity of immunoreactive
cells in the rats, undergoing CS demonstrated
approximately 40% higher density of CB1
receptors than control group (Figure 1C).

DZHAMBAZOVA E., et al.
108 Trakia Journal of Sciences, Vol. 12, Suppl. 1, 2014
Figure 1. Photomicrograph of CB1 immunopositive neurons in the rat's amygdaloid body A. control group, B- cold
stress group (x100); C. Effect of cold stress (CS) on CB1 immunopositive neurons in male Wistar rat’s amygdaloid
body. Mean values ± S.E.M. are presented *P<0.05 vs. control
DISCUSSION
Activation of the hypothalamic–pituitary–
adrenal axis (HPA) by stress is thought to
promote physiological and behavioral changes so
that an organism can deal with a changing and
challenging environment. It has been established
that when animals are subjected to acute stress, a
wide range of physiological alterations take
place, and a short single intense exposure to
stress can produce profound changes in an
animal’s stress response and behavior (7). A
number of studies have revealed that various
stressors produce differential effects, which are
frequently referred to as stressor specific
response (1, 2, 8). Cold stress is one of the most
commonly employed animal models for studying
different aspects related to stress (9). Acute
change in temperature leads to stressful
conditions by activation of temperature
regulatory centre in the hypothalamus and
subsequently HPA axis. Different research
groups as well as our data have shown induction
of acute physical stress by low temperature
exposure which have been reported to impair
motor activity, modulate pain perception, anxiety
and depression-like behaviors in the animals (6,
10, 11). One of the main physiological
mechanisms that organize the response to a
stressful situation involves the amygdala - the
main gate through which processed sensory
information enters the emotional brain (3). The
amygdala is a limbic structure that receives
monoaminergic innervation (dopamine,
noradrenaline, serotonin, acetylcholine) as well
as nerve afferents releasing glutamate from
different areas of the brain (12, 13). Some
literature data showed that in limbic-related
areas of the brain CB1 receptors are found and
ECs participates in multiple brain circuits
implicated in stress reactions, learning, and
extinction of fear, emotional regulation, and
reward processes (3, 4). There is also evidence
that endocannabinoid content of the amygdala is
increased following exposure to the conditioned
stimulus (1, 2). Regardless of the precise nature
of the behavioral response to stress, the current
study found that cold stress demonstrated
approximately 40% higher density of CB1-
receptors in amygdaloid body compared with the
control group. Besides, our previous experiments
showed anxiogenic effect of cold stress in the
open field test (5). The exact mechanism of cold
stress due to endocannabinoid modulation of
emotional behavior in rats is unclear. We may
suggest the inhibition of wide range of
neurotransmitters including glutamate,
acetylcholine, norepinephrine, and GABA by
endogenous ligands (ECBs) for the CB1
receptors. Endocannabinoids are released by an
unknown mechanism and diffuse across the
synapse to activate receptors located on the axon
terminal, suppressing neuronal excitability and

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Trakia Journal of Sciences, Vol. 12, Suppl. 1, 2014 109
inhibiting neurotransmission. They are
inactivated rapidly by uptake (possibly carrier
mediated) followed by intracellular hydrolysis.
In general, the predominant effects of
endocannabinoid signaling are to constrain
activation of the HPA axis. (14, 15).
In conclusion, our findings revealed alterations
in CB1 receptor density that occurs in response
to cold exposure. May be this is one of the
mechanisms by which cold stress affects the
synaptic connectivity between amygdaloid
nuclei and leads to modulation of emotional
behavior, learning, and stress-response.
Acknowledgments: The research was supported
by Grant 14/2013 of the Medical University of
Sofia, Bulgaria.
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