Immobilization and cold stress affect sympatho-adrenomedular system and pituitary-adrenocortical axis of rats exposed to long-term isolation and crowding

Abstract

Changes in plasma levels of noradrenaline (NA), adrenaline (A), adrenocorticotropic hormone (ACTH) and corticosterone (CORT), as well as in cytosol glucocorticoid receptor (GR) and heat shock protein 70 (Hsp 70) in hippocampus of adult rat males exposed to two long-term types of psychosocial stress, both under basal conditions and in response to immobilization and cold as heterotypic additional stressor were studied. Long-term isolation produced a significant elevation of basal plasma ACTH and CORT levels, but did not affect that of NA and A, while long-term crowding conditions did not elevate the basal plasma levels of these hormones. Long-term isolation of rats exposed to 2 h of immobilization or cold led to a significant elevation of plasma NA, A and CORT in comparison with the controls. Long-term crowding conditions and exposure of animals to immobilization or cold also resulted in an increased plasma NA, A and CORT levels, but to a lesser extent in comparison with the long-term isolation. At the same time, plasma ACTH was significantly more elevated in long-term crowded than in long-term isolated rats. Both kinds of long-term psychosocial stresses (isolation and crowding) had similar but less pronounced effects on cytosol GR and Hsp 70 concentrations in hippocampus comparing to acute immobilization and cold stress. It seems that long-term psychosocial stresses attenuate the effects of an additional stress on hippocampal GR and Hsp 70 concentrations. These data suggest that individual housing of rats appear to act as a stronger stressor than crowding conditions. When the animals suffering a long-term isolation were exposed to either acute immobilization or cold, a stronger activation of the sympatho-adrenomedullary system (SAS) was recorded in comparison with that found in the long-term crowded group subjected to short-term immobilization or cold. No significant differences in the activity of hypotalamo-pituitary-adrenal (HPA) axis were observed between long-term isolated and long-term crowded rats.

Full text

Immobilization and cold stress affect sympatho–adrenomedullary
system and pituitary–adrenocortical axis of rats exposed to
long-term isolation and crowding
Sladjana Dronjak*, Ljubica Gavrilovic
´, Dragana Filipovic
´, Marija B. Radojc
ˇic
´
Laboratory of Molecular Biology and Endocrinology, Institute of Nuclear Sciences ‘‘Vincˇa,’’ P.O. Box 522-090,
11001 Belgrade, Serbia and Montenegro
Received 11 June 2003; received in revised form 6 November 2003; accepted 28 January 2004
Abstract
Changes in plasma levels of noradrenaline (NA), adrenaline (A), adrenocorticotropic hormone (ACTH) and corticosterone (CORT), as
well as in cytosol glucocorticoid receptor (GR) and heat shock protein 70 (Hsp 70) in hippocampus of adult rat males exposed to two long-
term types of psychosocial stress, both under basal conditions and in response to immobilization and cold as heterotypic additional stressor
were studied. Long-term isolation produced a significant elevation of basal plasma ACTH and CORT levels, but did not affect that of NA and
A, while long-term crowding conditions did not elevate the basal plasma levels of these hormones. Long-term isolation of rats exposed to 2
h of immobilization or cold led to a significant elevation of plasma NA, A and CORT in comparison with the controls. Long-term crowding
conditions and exposure of animals to immobilization or cold also resulted in an increased plasma NA, A and CORT levels, but to a lesser
extent in comparison with the long-term isolation. At the same time, plasma ACTH was significantly more elevated in long-term crowded
than in long-term isolated rats. Both kinds of long-term psychosocial stresses (isolation and crowding) had similar but less pronounced effects
on cytosol GR and Hsp 70 concentrations in hippocampus comparing to acute immobilization and cold stress. It seems that long-term
psychosocial stresses attenuate the effects of an additional stress on hippocampal GR and Hsp 70 concentrations. These data suggest that
individual housing of rats appear to act as a stronger stressor than crowding conditions. When the animals suffering a long-term isolation
were exposed to either acute immobilization or cold, a stronger activation of the sympatho–adrenomedullary system (SAS) was recorded in
comparison with that found in the long-term crowded group subjected to short-term immobilization or cold. No significant differences in the
activity of hypotalamo–pituitary– adrenal (HPA) axis were observed between long-term isolated and long-term crowded rats.
D2004 Elsevier Inc. All rights reserved.
Keywords: Long-term psychosocial stress; Plasma catecholamines; Corticosterone; ACTH; Glucocorticoid receptor; Hsp 70
1. Introduction
The physiological responses to stress are initiated by the
activation of the sympatho –adrenomedullary system (SAS)
and the hypotalamo–pituitary–adrenal (HPA) axis, resulting
in the release of catecholamines and stress hormones, such as
glucocorticoids from the adrenal gland [1 – 3]. Corticoste-
roids act through specific mineralocorticoid and glucocorti-
coid receptors (GRs) localized in the brain structures
hippocampus and amygdala, involved in the regulation of
fear and anxiety [4]. The GR is a cytosol protein occurring as
a complex with several proteins of the heat shock family
(Hsp). Elevation of glucocorticoid level provokes both short-
and long-term effects in the brain. These changes indicate that
stress can affect hippocampal structure and function. On the
other hand, the hippocampus can also suppress stress reaction
through the feedback regulation of the HPA axis [5]. Protein
damage and misfolded protein structure are common denom-
inators of various stress types. These states lead to the
activation of Hsp chaperons, e.g., heat shock protein 70
(Hsp 70), that represents the major stress-induced chaperon
in mammals [6]. Although all stressors activate HPA and
SAS, the degree of their activation depends on stress dura-
tion, type and intensity. Social interaction is an important
source of stress. Mitsushima et al. [7,8] and Mizumo and
Kimura [9] reported a prominent nighttime increase in
0031-9384/$ – see front matter D2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.physbeh.2004.01.011
* Corresponding author. Tel.: +381-11455561; fax: +381-113440100.
E-mail address: sladj@vin.bg.ac.yu (S. Dronjak).
Physiology & Behavior 81 (2004) 409 – 415

acetylcholine release in the hippocampus of rats accommo-
dated in large cages, while this phenomenon was not ob-
served in rats living in small cages. They assumed that this
difference of nighttime acetylcholine release in the hippo-
campus of the latter could be connected to a significant
attenuation of locomotor activity. These authors [9] conclud-
ed that restriction of the environmental space itself, acts as a
kind of stress. Some others reported higher corticosteroid
levels as a result of crowded conditions [10,11]. On the other
hand, isolation, i.e., individual housing of experimental
animals, often termed as isolation stress, was shown to
decrease basal plasma corticosterone (CORT) level in rats
[12,13]. Studies on the effect of population density demon-
strated body weight gain inhibition, reduction of food intake,
atrophy of thymus and hypertrophy of the adrenals in
experimental animals exposed to either isolated or overpopu-
lated environment [14].
Taking into account the above studies, the aim of the
present work was to examine relationship between plasma
noradrenaline (NA), adrenaline (A), CORT, adrenocortico-
tropic hormone (ACTH) and hippocampal GR and Hsp70 in
adult rat males exposed to long-term social housing isola-
tion and crowding, both under basal conditions and in
response to short-term immobilization and cold as two
additional stressors. We were interested to learn whether
long-term (21 days) social housing isolation and crowding
would affect the activity of the SAS and HPA axis.
2. Materials and methods
Rat males of Wistar strain, weighing 330 –400 g were
used. They were offered water and food ad libitum. The
light schedule in the room was reversed, with lights on
between 7 p.m. and 7 a.m. Before being subjected to stress,
the animals were housed in groups of six individuals in wire
cages (width 32 cm, length 46 cm, height 22 cm). They
were divided into three groups. The first control group
consisted of six animals per cage. The rats that were
individually housed for the 21-day period represented the
second group. In the third group, 12 animals were housed
per cage, i.e., these were conditions of social crowding. The
rats of the second and third groups were housed in plastic
cages (width 21 cm, length 37 cm, height 13 cm). On the
day before blood sampling, cannule was inserted into the
tail artery under pentobarbital (40 mg/kg ip) anaesthesia.
This allowed the estimation of plasma catecholamines,
CORT and ACTH without stressing the animals during
manipulations. After the baseline blood collection, the rats
were immobilized or exposed to cold stress for 2 h. Blood
was collected 15, 30, 60 and 120 min after the onset of
immobilization. The animals exposed to cold for 2 h were
initially kept at ambient temperature and after the baseline
blood collection, the cages were carefully transferred into a
cold chamber at 4 jC and the blood was collected 30, 60
and 120 min later. The rats were killed by cervial disloca-
tion; hippocampi of six animals from each group were
excised and immediately cooled in an ice bath. They were
weighed and homogenized (1:2 w/v; 20 strokes; Potter –
Elvehjem teflon –glass homogenizer) in EPG buffer, pH 7.0,
supplemented with 2 mM DTT and protease-inhibitor cock-
tail. The homogenates were centrifuged (10 min, 10,000
rpm, 4 jC), the pellets taken as crude nuclei preparations,
and the supernatants recentrifuged (60 min, 45,000 rpm, 4
jC) to obtain purified cytosol fraction. Cytosol samples
mixed with denaturing buffer according to Laemmli [15]
were analysed either immediately or kept at 80 jC until
Fig. 1. The effects of acute immobilization on plasma level of NA and A
(pg/ml) in the controls, rats exposed to long-term isolation (LTI) and long-
term crowding (LTC). The values are means FS.E.M. of six to eight
animals. Statistical significance: * * P< .001 and * P< .05 as compared to
the control;
+
P< .001 LTI vs. LTC group.
Fig. 2. The effects of acute immobilization on plasma level of ACTH (pg/
ml) and CORT (ng/ml) in the controls, rats exposed to LTI or LTC. The
values are means FS.E.M. of six to eight rats. Statistical significance:
*P< .001 as compared to the control;
+
P< .001 LTI vs. LTC group.
S. Dronjak et al. / Physiology & Behavior 81 (2004) 409–415410

the analysis. Separation of proteins by SDS-PAGE and
quantification by immunoblotting were performed as previ-
ously described [16].
Immobilization stress was provoked as described by
Kvetnansky and Mikulaj [17]. Plasma catecholamines were
assayed by our modification of the radioenzymatic method
[18]. Plasma CORT was measured upon prior extraction
directly, using RIA commercial kits (ICN, Biochemicals,
Costa Mesa, CA, USA) and the values were expressed as ng
CORT/ml plasma. Plasma ACTH was determined by a
chemiluminescent method using IMMULITE automatic
analyzer (DPC, Los Angeles, CA, USA) and the values
were expressed as pg ACTH/ml plasma.
Statistical differences between experimental groups and
control were estimated by one-way ANOVA test.
3. Results
The effects of acute immobilization (2 h) on plasma
NA and A levels in the control and rats previously
exposed to long-term isolation and long-term crowding
are depicted in Fig. 1. As seen, none of these two
treatments affected the basal plasma level of either NA
or A. However, long-term immobilization led to a signif-
icant increase of NA and A contents in all three exper-
imental groups. In this group of animals, the increase of
plasma NA and A levels was more pronounced in all
experimental points compared to the other groups. In the
long-term crowded group, immobilization also led to
increased plasma catecholamine levels in comparison with
the corresponding controls, but this increase was lower
Fig. 3. The effects of immobilization on cytosol GR and Hsp 70 concentrations in hippocampi of rats exposed to LTI and LTC. The results are meansFS.E.M.
of three to six rats. Statistical significance in relation to the naive control are as follows: * P< .05; * * P< .01; * * * P< .001.
Fig. 4. The effects of acute cold stress on cytosolic GR and Hsp 70 concentrations in hippocampi of rats exposed to LTI or LTC. The results are
means FS.E.M. of three to six rats. Statistical significance in relation to the naive control are as follows: * P< .05; * * P< .01; * * * P< .001.
S. Dronjak et al. / Physiology & Behavior 81 (2004) 409–415 411

compared to that recorded in the group subjected to a
long-term isolation.
Basal plasma ACTH and CORT levels were significantly
elevated in the long-term isolation group (Fig. 2). Immobi-
lization elicited a conspicuous increase of plasma ACTH
and CORT in all investigated groups. The highest levels of
ACTH were found 15 and 30 min after the onset of
immobilization and reached about a 30-fold increase in
control and long-term crowded rats, while ACTH was
increased about 12-fold in the long-term isolation group.
Immobilization expressed a similar effect on plasma CORT
level in the control, long-term isolation and long-term
crowded groups and its concentrations were significantly
elevated in all investigated groups of rats.
From Fig. 3, it can be seen that long-term isolation
induced a more pronounced decrease in both cytosolic GR
and Hsp 70 concentrations in hippocampus comparing to
that resulting from long-term exposure to crowding. The
acute immobilization elicited the most pronounced decrease
of both hippocampal cytosol GR and Hsp 70 concentrations
in the control as compared to that found in the long-term
isolation or long-term crowded rats. The results depicted in
Fig. 4 clearly show that acute exposure of animals to cold
stress led to the most pronounced decrease of both hippo-
campal GR and Hsp 70 concentration in the controls. This
decrease was less pronounced in long-term isolation group
and the least expressed in long-term crowded rats.
From Fig. 5, it can be seen that acute exposure to cold
stress (2 h) resulted in an increase of NA level in all
experimental groups, the most pronounced effect being
recorded in long-term isolation group. Concentration of A
in the control after exposure to cold stress was approxi-
mately 1.5 times higher compared to the basal level. The
same cold treatment resulted in about three- and twofold
increase of plasma A level in long-term isolation group and
long-term crowded group, respectively, in comparison with
the basal levels.
Plasma levels of ACTH and CORT in control, long-term
isolation and long-term crowding rats after cold stress are
presented in Fig. 6. As shown, exposure to cold produced a
significant increase in ACTH level in the long-term crowded
group, somewhat less pronounced increase in the control
and surprisingly low elevation in the long-term isolation
group. In both long-term isolation and long-term crowded
groups, cold stress significantly elevated plasma CORT
comparing to the basal level. This treatment also led to
the elevation of plasma CORT in the controls, but it was less
pronounced than that observed in both long-term isolation
and long-term crowded rats.
4. Discussion
It has been found recently that housing conditions affect
the response of rats to chronic exposure to stress. Isolation
by itself appears to be a stressor for rat females and social
housing appears to reduce these adverse effects. In contrast,
rat males do not seem to benefit from social housing, as it
appears to augment negative effects of exposure [19]. In the
present study, we measured the level of plasma catechol-
amines, CORT, ACTH and cytosolic GR and Hsp 70
concentrations in hippocampi of rat males exposed to
isolation and crowding conditions as two different long-
term psychosocial stress types. These animals were also
exposed to immobilization and cold as heterotypic stressors.
Immobilization represents the strongest stress by combining
physical and emotional stress, whereas exposure to cold is
Fig. 5. The effect of acute cold stress on plasma levels of NA and A (pg/ml)
in the controls, rats exposed to LTI or LTC. The values are means FS.E.M.
of six to eight rats. Statistical significance: * * P< .001 and * P< .05 as
compared to the control;
+
P< .01 and
++
P< .001 LTI vs. LTC group.
Fig. 6. The effect of acute cold stress on plasma levels of ACTH (pg/ml)
and CORT (ng/ml) in the controls, rats exposed to LTI or LTC. The values
are means FS.E.M. of six to eight animals. Statistical significance:
*P< .01 and * * P< .001 as compared to the control;
+
P< .001 LTI vs.
LTC group.
S. Dronjak et al. / Physiology & Behavior 81 (2004) 409–415412

assumed to be a moderate stress. The studies on the effect of
acute exposure of rats to cold and immobilization indicated
the activation of the sympathoneural and adrenomedullary
systems, as measured by increased levels of plasma cat-
echolamines, adrenal TH activities and adrenal TH mRNA
[20–22]. Therefore, we have found it of interest to examine
the effects of these two stressors, differing in intensity, on
the activity of HPA axis and SAS in rats previously exposed
to long-term isolation and crowding conditions.
During long-term isolation, the basal plasma ACTH and
CORT levels were several-fold increased, whereas basal
plasma NA and A remained unchanged. We have also
observed no differences in the basal plasma levels of
catecholamines, ACTH and CORT between control and
long-term crowded rats. This indicates that long-term isola-
tion is a specific stimulus for the HPA axis, with no
significant effect on the activation of the SAS. These results
could be connected to the data of Armando et al. [23] who
found that 24-h isolation of rats in individual metabolic
cages resulted in increased levels of adrenal catecholamines,
tyrosine hydroxylase mRNA, aldosterone and CORT. More-
over, Nunez et al. [24] demonstrated that 4 days of isolation
stress provoked an increased CORT secretion. Miachon et
al. [12] showed that 13 weeks of isolation produced a
significant increase in catecholamine turnover in hippocam-
pus, cortex and cerebellum, accompanied by increased
ACTH and decreased CORT levels. The explanation for
the differences between these and our findings could be
sought for in different durations of the isolation stress in our
experiments and those performed by Miachon et al. [12].In
the present study, we decided to expose the animals to a
long-term 21-day stress because the data of several authors
suggested that this stress induced long-lasting behavioural
sequelae associated with reproducible neurochemical and
immunological modifications [25 –27].
It has been shown that immobilization stress resulted in
increased levels of plasma catecholamines and CORT ac-
companied by the activation of both sympatho – adrenome-
dullary and adrenocortical systems [28,29]. In the present
study, the highest elevation of plasma catecholamines was
observed in long-term isolation rats. It was elevated to a less
degree in long-term crowded rats and was the least in control
rats exposed to 2-h immobilization only. Previous studies
suggested that social defeat induced a much greater elevation
in NA and A concentrations, indicating a more extensive
involvement of both sympathoneural and adrenomedullary
systems [30]. In the long-term isolation and long-term
crowded groups, the plasma level of A, after reaching the
peak at about 15 min upon the onset of immobilization,
remained elevated during the whole period of immobiliza-
tion, while after immobilization of the control group, the A
level was reduced. The explanation for these findings might
be due to greater catecholamine stores, a more extensive or
longer period of catecholamine secretion, or reduced cate-
cholamine degradation and reuptake in long-term psychoso-
cially adapted rats. The exaggerated levels of both plasma
NA and A in rats exposed to psychosocial stress for 21 days
might also be a consequence of the readiness of such animals
to respond to altered quality or quantity of an additional
stressor. Immobilization (2 h) produced a significant increase
of plasma ACTH level in long-term crowded and control
rats, while this increase was lower in the animals exposed to
long-term isolation. Plasma CORT levels in the control,
long-term isolated and crowded groups were also increased
during immobilization, but no significant differences were
observed when these three groups were compared. Our
results clearly indicated immobilization-related activation
of HPA axis. We also showed that the exposure of rats to
long-term psychosocial stress (especially to long-term isola-
tion) to an additional stressor, such as immobilization,
produced a much more pronounced response of SAS and a
less pronounced response of HPA axis. Besides, the acute
immobilization elicited a significant decrease of both hippo-
campal cytosol GR and Hsp 70 concentrations in the controls
as compared to either long-term isolated or crowded groups.
In these cases, a decrease in the cytosolic GR concentration
most probably reflects an active negative feedback mecha-
nism, aimed at the restoration of homeostasis, by attenuating
the stress signal transduction via the GR [31].
Exposure of control, long-term isolated and long-term
crowded groups to cold stress for 2 h activated both SAS
and HPA axis and plasma NA levels were increased about 2-,
3.3- and 2.5-fold, respectively, in comparison with the
baseline values. At the same time, plasma A level was
elevated about 1.3-, 3- and 2-fold in control, long-term
isolated and long-term crowded groups, respectively. These
results demonstrate that exposure of rats to cold as an
additional stressor resulted in a more pronounced activation
of SAS in both long-term isolated and crowded groups in
comparison with the controls. These findings could be related
to the data of Zaretski et al. [32] who found that chronic stress
(footshock combined with randomized light flashes) led to an
increase and prolongation of the acute stress-induced NA
release. The results of the present study showed that the
exposure of long-term isolated and crowded rats to acute cold
stress was followed by significantly elevated levels of CORT.
In unstressed naive controls, cold also produced an increase
in CORT level, but it was less pronounced than in animals
exposed to long-term psychosocial stress. This is in agree-
ment with the findings of Hashiguchi et al. [33] who
suggested that CORT is especially sensitive to novel treat-
ment. Interestingly, cold as an additional stress, resulted in the
highest elevation of plasma ACTH in long-term crowded rats
(about 10-fold). This effect was somewhat less expressed in
the controls (about sixfold higher ACTH level) and the least
in long-term isolation group (about twofold). Cold stress
elicited the most pronounced decrease of both hippocampal
GR and Hsp 70 concentrations in the control as compared to
the groups exposed to long-term psychosocial stress. More-
over, exposure to cold for 2 h acted as a somewhat stronger
stressor than 2-h immobilization. However, long-term
stresses, such as 21-day isolation and 21-day crowding,
S. Dronjak et al. / Physiology & Behavior 81 (2004) 409–415 413

produced almost negligible decreases in cytosolic GR in
hippocampus of 10% and 5%, respectively. These results
could mean that the rats exposed for a long period of time to
the same stress, most probably experienced a partial disrup-
tion of the negative feedback regulation and acquired a partial
habituation to a high glucocorticoid level as suggested by
Mizoguchi et al. [34]. It should be noted that although long-
term stress-induced decrease in the cytosolic hippocampal
GR was negligible, long-term isolation seems to be a some-
what more potent stressor than long-term crowding. Further-
more, long-term isolated animals were more sensitive to
subsequent exposure to 2-h immobilization or cold (25%
and 30% decrease of cytosolic hippocampal GR content,
respectively). The animals exposed to long-term crowding
conditions and subjected to subsequent 2 h of immobilization
or cold, showed a 15% and 20% decrease in cytosolic GR
concentration, respectively. Thus, it seems that in long-term
isolated animals, negative feedback regulation of the GR
protein was less compromised than in long-term crowded
rats. The changes in cytosolic hippocampal Hsp 70 concen-
tration in these groups of animals closely followed the
changes in cytosol GR concentration. Taken together,
stress-induced increase in CORT concentration and subse-
quent down-regulation of both cytosolic GR and Hsp 70
confirm the involvement of the mechanism of a negative
feedback regulation at the level of GR-sensitive cells in the
hippocampus. The observed changes in the concentration of
these major stress-related cellular proteins in hippocampus
are most probably a part of a cellular mechanism serving to
restore disturbed equilibrium of an organism by switching off
or attenuating the stress signal.
The obtained results show that although long-term isola-
tion significantly elevated the basal plasma ACTH level,
exposure to novel stressors (immobilization or cold) pro-
duced the lowest increase in plasma ACTH in comparison
with that observed in either long-term crowded or control
rats.
Based on these results, it may be concluded that indi-
vidual housing of rats seems to act as a stronger stressor
than crowding conditions. Hurst et al. [35] found that
individual composition of groups, rather than size, had a
greater impact on the welfare of the rats. When long-term
isolated rats were exposed to additional stressors, a stronger
activation of SAS was observed in comparison with that
recorded in the long-term crowded group. Crowding as a
psychosocial stress seems to be the weakest stressor if
estimated by the activation of SAS but no significant
differences in the activity of HPA axis were observed when
the rats exposed to long-term isolation were compared to
those suffering in the long-term crowded conditions.
Acknowledgements
This work was supported by the Ministry for Science,
Development and Technology of Serbia, Grant #1953.
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