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Novel stressors affected catecholamine stores in socially isolated normotensive and spontaneously hypertensive rats
Autonomic Neuroscience: Basic and Clinical 122 (2005) 38 – 44 www.elsevier.com/locate/autneu
Novel stressors affected catecholamine stores in socially isolated normotensive and spontaneously hypertensive rats Ljubica Gavrilovic, Natasa Spasojevic, Sladjana Dronjak * Institute of Nuclear Sciences ‘‘Vinca’’, Laboratory of Molekular Bilology and Endocrinology, P.O.Box 522-090, 1001 Belgrade, Serbia and Montenegro Received 16 May 2005; received in revised form 16 May 2005; accepted 30 July 2005
Abstract Catecholamines in some central (hypothalamus and hippocampus) and peripheral tissues (adrenal glands and heart auricles) of long-term socially isolated normotensive and spontaneously hypertensive rats exposed to novel immobilization stress were determined by a simultaneous single isotope radioenzymatic assay. Long-term isolation (21 days) produced depletion of hypothalamic norepinephrine (NE) stores and hippocampal dopamine (DA) stores in both normotensive and spontaneously hypertensive rats. Acute immobilization stress (2 h) significantly decreased NE and DA stores in hypothalamus and hippocampus of naive normotensive and spontaneously hypertensive rats controls. However, novel immobilization stress applied to normotensive rats previously subjected to long-term isolation produced no changes in catecholamine levels in hypothalamus, while resulting in somewhat higher depletion of NE stores in hypothalamus of spontaneously hypertensive rats treated in the same way. Novel immobilization stress decreased NE and DA stores in hippocampus of normotensive but was without effect on NE and DA stores of spontaneously hypertensive rats. Social isolation did not affect catecholamine stores in peripheral tissues but novel immobilization stress produced a significant decrease in catecholamine content. The results suggest that some central and peripherals tissues of spontaneously hypertensive rats and normotensive rats differ with regard to catecholamine content and that there are certain differences in their responsiveness to stress. D 2005 Elsevier B.V. All rights reserved. Keywords: Social isolation; Hippocampus; Hypothalamus; Adrenal gland; Catecholamines
1. Introduction Stress is considered to be one of the most important factors in the development of cardiovascular and mental diseases. Various stressors are known to activate the peripheral sympatho-adrenomedullary and limbic – hypothalamo – pituitary – adrenal system. Limbic circuits connecting, e.g. the hippocampus, amygdala and prefrontal cortex are sensitive to stressors such as restraint, fear or exposure to a novel environment. All these stressors act stimulating intralimbic processing of informations received from different sensory organs, and this processing depends on a previous experience. In contrast, physiological threats such as exposure to ether results in the activation of efferent viscelar pathways that are directly relayed to the para* Corresponding author. Tel./fax: +38 1118066514. E-mail address: [email protected] (S. Dronjak). 1566-0702/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.autneu.2005.07.010
ventricular nucleus of the hypothalamus (Fuchs and Flugge, 2003). Cortical and hippocampal noradrenergic afferents derive from the locus coeruleus, whereas hypothalamic afferents derive from the lateral tegmental noradrenergic system. Activation of limbic and hypothalamic brain structures is a major component of the stress reaction that integrates neuroendocrine and emotional components and thus, determines the magnitude and duration of the hormonal and neural stress response. Several authors found that acute exposure to stressful stimuli provokes the immediate release of norepinephrine (NE) in diverse brain regions (Shibasaki et al., 1995; Hellriegel and D’mello, 1997; Prieto et al., 2003). Also, it was shown that exposure to chronic stress can modify the function of the noradrenergic system (Nisenbaum and Abercrombie, 1993), while prior stress exposure was reported to alter the response to subsequent stressor, leading to the development of tolerance or sensitization (Natelson et al., 1998; Mcewen, 2000; Stam
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et al., 2000; Hajos-Korcsok et al., 2003). The distinct adaptive responses may be influenced by type, intensity and duration of the stressor. Social isolation of adult animals has been shown to enhance locomotion and exploratory behavior and anxiety in novel environments (Fone et al., 1996; Hall et al., 1998; Paulus et al., 1998; Rilke et al., 1998). We have found recently that rats previously exposed to social isolation for 21 days exhibited a significant increase of plasma NE and even greater increase of plasma epinephrine (E) in response to additional immobilization (Dronjak et al., 2004). Stress represents a major factor in pathogenesis of hypertension and stress-induced elevations in plasma concentration of E leading to cardiovascular stimulation and development of arterial hypertension (Esler et al., 2001). Spontaneously hypertensive rats have been used as a model for the studies of the mechanism(s) underlying the development of hypertension. Spontaneously hypertensive rats are hyperactive in a novel environment and hyperresponsive to environmental stimuli exhibiting an increased behavioral, sympathetic and cardiovascular responsiveness comparing to normotensive animals. Besides, sympathetic nervous system of SHR is overactive as compared to that of normotensive rats (Sohn et al., 2002). The adrenal gland represents one of the main components of the sympathetic nervous system. Adrenal gland medulla is innervated by sympathetic nerves, so that the adrenal nerve activity represents the sympathetic outflow to the adrenal medulla (Aoki et al., 1996). To estimate the influence of chronic social stress on the activity of the central and peripheral catecholaminergic system in normotensive and hypertensive animals, we compared the effect of immobilization as a novel stress applied to long-term socially isolated adult rats on catecholamine stores in some central (hippocampus and hypothalamus) and peripheral tissues (adrenal glands and heart auricles) of spontaneously hypertensive rats and normotensive Wistar rats.
2. Materials and methods Normotensive and spontaneously hypertensive 3-monthold Wistar rat males weighing 300– 340 g maintained under standard laboratory conditions with water and food ad libitum in groups of four individuals per cage were used. The care was taken to minimize the pain and discomfort of the animals according to the recommendations of the ‘‘Vinca’’ Institute which are in accordance with the Guide for Care and Use of Laboratory Animals of the National Institute of Health, Bethesda, MD, U.S.A. One group of animals was subjected to social isolation with a single animal per cage for the 21-day-period. After that, naive controls and the rats that suffered long-term isolation were exposed to acute immobilization stress by fixing all four limbs to a board with adhesive tape. The head was also fixed by a metal loop over the neck area, thus limiting the
39
motion of the head (Kvetnansky and Mikulaj, 1970). The rats were killed by decapitation after 2 h, hippocampi, hypothalami, heart auricles and adrenal glands were quickly dissected out and immersed into cold (4 -C) perchloric acid (0.3 Ag of tissue per 30 Al of 0.1 N HClO4). The tissues were homogenized in a motor-driven homogenizer, the homogenates centrifuged (a Janetzky K24 centrifuge, 20 000 r.p.m., 20 min, 4 -C) and the supernatants (30 Al) used for determination of catecholamines. Content of catecholamines in the tissues was determined using somewhat modified chromatographic method of Peuler and Johnson (1977) based on the conversion of catecholamines into the corresponding O-methylated derivatives by purified catechol-O-methyl-transferase (COMT) in the presence of S-adenosyl-l-(3H-methyl)-methionine. The O-methylated derivatives were extracted and oxidized into 3H-vaniline. Radioacitivites were measured using toluene-based scintillation cocktail in a LKB-Wallac model 1219 scintillation counter at an efficiency of 40% for tritium. Statistical analyses were performed by two-way factorial analysis of variance (ANOVA) for the normotensive and spontaneously hypertensive rats. To consider the influence of novelty stress, one-way ANOVA was applied individually to animals in the naive control and isolated group rats. The p < 0.05 level was chosen as the point of minimal acceptable statistical significance.
3. Results 3.1. Effect of novel immobilization stress on catecholamine levels in hypothalamus and hippocampus of spontaneously hypertensive and normotensive rats exposed to long-term isolation The results presented in Fig. 1. show that long-term isolation (21 days) produced a statistically significant ( p < 0.05) depletion of NE but not of dopamine (DA) in hypothalami of both spontaneously hypertensive rats and normotensive. Two hours of acute immobilization significantly decreased NE ( p < 0.001) and DA ( p < 0.01) stores in the hypothalamus of control spontaneously hypertensive rats and normotensive. However, novel immobilization stress produced no significant changes in hypothalamic catecholamine levels of normotensive previously exposed to 21-day-isolation, while resulting in a somewhat higher depletion ( p < 0.05) of NE stores in spontaneously hypertensive rats. Long-term isolation produced no significant changes in hippocampal NE level of either spontaneously hypertensive or normotensive rats, while a significant depletion ( p < 0.05) of DA stores in hippocampus of both groups was observed. Acute immobilization of spontaneously hypertensive and normotensive rats resulted in a significant decrease ( p < 0.001) of NE and DA stores in the hippo-
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L. Gavrilovic et al. / Autonomic Neuroscience: Basic and Clinical 122 (2005) 38 – 44 2.6 2.4 2.2
concentrations (ug/g)
2
+
1.8
a-NA contr. norm.
b-NA 21 days norm.
c-NA contr. hyper.
d-NA 21 days hyper.
e-DA contr. norm.
f-DA 21 days norm.
g-DA contr. hyper.
h-DA 21 days hyper.
+
*
1.6 ***
1.4
***
1.2 1 0.8 **
0.6
**
0.4 0.2 0
a
b
c
d
e
f
g
h
a
b
c
d
0
e
f
g
h
120 time (min)
Fig. 1. Concentrations of norepinephrine (NE) and dopamine (DA) in the hypothalami of normotensive and spontaneously hypertensive rats control and longterm isolated animals exposed to 2-h-immobilization. The values expressed in Ag/g fresh tissue are the means T S.E.M. of six rats. Statistical significance: *p < 0.05; **p < 0.01; ***p < 0.001, as compared to zero point; + p < 0.05 21 days isolation vs. naive control.
campus of naive controls. Acute immobilization stress applied to animals previously exposed to long-term isolation resulted in statistically significant changes in hippocampal NE stores of both normotensive ( p < 0.01) and spontaneously hypertensive rats ( p < 0.05), the changes being more prominent in the former comparing to the latter group (Fig. 2).
It is evident from the results depicted in Fig. 3, that longterm isolation produced no significant changes in E and NE
1.4
a-NA contr. norm.
b-NA 21 days norm.
1.3
c-NA contr. hyper.
d-NA 21 days hyper.
1.2
e-DA contr. norm.
f-DA 21 days norm.
1.1
g-DA contr. hyper. +
1 concentrations (ug/g)
3.2. Effect of novel immobilization stress on catecholamine levels in adrenal glands and heart auricles of spontaneously hypertensive and normotensive rats previously exposed to long-term isolation
h-DA 21 days hyper. *
+
0.9
**
0.8
***
f
g
***
***
0.7
*
** ***
0.6 0.5 0.4 0.3 0.2 0.1 0
a
b
c
d
e
f
g
h
a
0
b
c
d
e
h
120 time (min)
Fig. 2. Norepinephrine (NE) and dopamine (DA) levels in the hippocampi of normotensive and spontaneously hypertensive rats control and long-term isolated animals exposed to 2-h-immobilization as a novel stress. The values in Ag/g fresh tissue are the means T S.E.M. of six animals. Statistical significance: *p < 0.05; **p < 0.01; ***p < 0.001, as compared to zero point; + p < 0.05 21 days isolation vs. naive control.
L. Gavrilovic et al. / Autonomic Neuroscience: Basic and Clinical 122 (2005) 38 – 44
41
25
a-NA contr. norm. b-NA 21 days norm.
22.5
c-NA contr. hyper. d-NA 21 days hyper. e-A contr. norm. f-A 21 days norm.
concentrations (ug/g)
20 17.5 15
g-A contr. hyper. h-A 21 days hyper.
12.5 10
***
7.5
*** ***
5
***
2.5 0
a
b
c
d
e
f
g
h
***
***
a
b
*** c
** e
d
0
f
g
h
120 time (min)
Fig. 3. Epinephrine (E) and norepinephrine (NE) content in the adrenal glands of normotensive and spontaneously hypertensive rats control and long-term isolated animals additionally exposed to 2-h-immobilization. The values in Ag/g fresh tissue are the means T S.E.M. of six rats. Statistical significance: *p < 0.05; **p < 0.01; ***p < 0.001 as compared to zero point.
content in adrenal glands of both spontaneously hypertensive and normotensive rats. Immobilization stress led to a highly significant decrease ( p < 0.001) in the amount of E and NE in the adrenal glands of both naive control and long-term isolation groups of spontaneously hypertensive and normotensive rats. Relative decrease of NE and E level in adrenal glands of normotensive exceeded greatly that of sponta-
neously hypertensive rats exposed to the same period of immobilization following long-term isolation. The results presented in Fig. 4 show that long-term isolation did not affect NE level in heart auricles of either spontaneously hypertensive or normotensive rats. However, acute immobilization alone led to a significant decrease ( p < 0.001) in NE content of this tissue in control sponta-
3
a-NA contr. norm. b-NA 21 days norm. c-NA contr. hyper. d-NA 21 days hyper.
concentration (ug/g)
2.5
2
***
***
*** *** 1.5
1
0.5
0
a
d
c
d
a
0
b
c
d
120 time (min)
Fig. 4. Levels of norepinephrine (NE) in the heart auricles of normotensive and spontaneously hypertensive rats control and long-term isolated animals after 2h-exposure to immobilization. The values in Ag/g fresh tissue are the means T S.E.M. of six rats. Statistical significance: *p < 0.05; **p < 0.01; ***p < 0.001 as compared to zero point.
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L. Gavrilovic et al. / Autonomic Neuroscience: Basic and Clinical 122 (2005) 38 – 44
neously hypertensive and normotensive rats. Also, a significant depletion of NE stores in the heart auricles was observed after immobilization as a novel stress to long-term isolation of both spontaneously hypertensive and normotensive rats.
4. Discussion Our earlier studies showed that long-term social isolation of rats produced a significant elevation of basal plasma adrenocorticotropic hormone and corticosterone, but did not affect basal plasma NE and E levels, while the same treatment followed by acute immobilization as a novel stress led to a significant elevation of plasma NE and E (Dronjak et al., 2004). On the other hand, the results of microdialysis studies of Abercrombie et al. (1992) confirmed the sensitivity of central catecholamines to stressors. The same authors, reported significantly higher hippocampal NE and l-DOPA levels in chronically stressed rats ¨ in response to a novel stressor, in comparison with naNve acutely stressed animals (Abercrombie et al., 1992; Nisenbaum and Abercrombie, 1992). Also, Miura et al. (2002) demonstrated that chronic isolation of animals attenuated novelty stressinduced increase in DA turnover. This prompted us to investigate the effects of acute immobilization as a novel stress on catecholamine stores in some central and peripheral tissues of normotensive and hypertensive rats previously exposed to long-term social isolation. The results of the present study showed that acute 2-himmobilization produced a significant depletion of central and peripheral catecholamine stores in both normotensive and spontaneously hypertensive rats control groups of animals. Our findings are in accordance with the data of several others (Pol et al., 1992; Moore et al., 2001; Sanchez et al., 2003) who found that 2-h-immobilization significantly decreased NE and DA levels in hypothalamus and prefrontal cortex, as well as NE and E levels in the adrenal glands. Social stress was reported to produce many changes in the brain, affecting neuronal structure and also inducing the stimulation of noradrenergic neurons (Blanchard et al., 2001). Our results clearly demonstrated that long-term social isolation led to a significant depletion of NE in the hypothalamus but not in the hippocampus of both normotensive and spontaneously hypertensive rats. The hypothalamus is the brain structure that influences all of the limbic structures and hypothalamic neurons are responsive to information arising in the internal and external environment. This brain structure controls the visceromotor responses by arranging the equilibrium of sympathetic and parasympathetic outputs to the autonomic nervous system (Feenstra, 2000). The observed reduction of NE stores in the hypothalamus could indicate that neurons derived from lateral tegmental system respond more powerfully to stress than those in the hippocampus deriving from locus coeruleus. However, no significant reduction in NE stores
in the hippocampus were observed throughout the present study. This could be connected to the results of HajosKorcsok et al. (2003) and explained by habituation of rats to the chronic stressor. Namely, these authors have recently shown that an initial electric footshock induced a significant elevation of extracellular NA levels in the rat hippocampus (200% above the baseline) as measured by in vivo microdialysis method. A rapid habituation to this response was evident in the inability of a second footshock to evoke similar increase. Cuadra et al. (1999) observed a more prominent increase in frontal cortical DA release of chronically stressed rats after exposure to a novel restraint stressor as compared to that recorded in the controls that suffered only restraint stress. Our results are compatible with the findings of Fulford and Marsden (1997) who found no differences in the basal efflux of hippocampal NE between the rats exposed to isolation and group-reared ones. Brain DA levels change after acute stress and chronic exposure to stress may express different effects on this neurotransmitter. For example, acute stress was shown to lead to an increased extracellular level of DA in the prefrontal cortex and amygdala (Sorg and Kalivas, 1993; Jackson and Moghaddam, 2004), while chronic stress over several days reduced DA output and the number of active DA cells (Mangiavacchi et al., 2001; Moore et al., 2001). The results presented here also showed that acute immobilization of the control rats significantly decreased DA stores in the hypothalamus and hippocampus of both normotensive and spontaneously hypertensive rats. However, long-term isolation resulted in a reduction of hippocampal DA stores but did not affect hypothalamic ones. Several authors have reported similar results demonstrating that emotional changes, such as exposure to stress situation increased DA metabolite levels in the hippocampus and reduced DA neurotransmission and activity in the hypothalamus (Torres et al., 2002; Gamaro et al., 2003). Also, it is worth mentioning the results of Zaretskii et al. (1999) who showed that chronic stress led to an elevated and prolonged acute stress-induced release of NE but not of DA in the hypothalamus. Interestingly enough, we have observed that novel immobilization stress applied to spontaneously hypertensive rats previously exposed to 21-day-isolation resulted in a somewhat higher NE depletion in the hypothalamus but did not affect that in the hippocampus. These results are consistent with earlier studies demonstrating that enhanced hypothalamic noradrenergic activity can increase sympathetic outflow to the periphery, which may play the role in the pathogenesis of hypertension (Tsuda et al., 1991; Akine et al., 2003), while noradrenergic projections from the locus coeruleus do not appear to play a major role in cardiovascular stress response of SHR (Van Den Buuse et al., 2001). Besides, Krukoff et al. (1999) found a significantly higher number of double labeled neurons in the ventral medial parvocellular paraventricular nucleus of stressed spontaneously hypertensive rats as compared to WKY and SD rats. These results demonstrated that chronic elevation in
L. Gavrilovic et al. / Autonomic Neuroscience: Basic and Clinical 122 (2005) 38 – 44
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Table 1 Changes in catecholamine stores in normotensive and spontaneously hypertensive rats Central and peripheral tissues
Control 0’ NORM
Hypothalamus NA DA Hippocampus NA DA Adrenal glands NA A Heart auricles NA
Isolation IMMO HYPER
0’
NORM
HYPER
NORM
IMMO HYPER
NORM
HYPER
1.94 T 0.22 0.78 T 0.09
2.10 T 0.25 0.84 T 0.09
1.23 T 0.14 0.50 T 0.06
1.20 T 0.15 0.52 T 0.07
1.60 T 0.19 0.75 T 0.09
1.68 T 0.19 0.80 T 0.10
1.58 T 0.17 0.72 T 0.09
1.47 T 0.18+ 0.78 T 0.10
1.01 T 0.12 1.11 T 0.13
1.04 T 0.11 1.07 T 0.13
0.63 T 0.08 0.66 T 0.08
0.60 T 0.08 0.70 T 0.08
0.98 T 0.11 0.89 T 0.10
1.01 T 0.12 0.87 T 0.10
0.77 T 0.09+ 0.72 T 0.09
0.88 T 0.11 0.75 T 0.09
2.35 T 0.25 20.74 T 2.22
2.24 T 0.26 19.84 T 2.10
1.09 T 0.13 5.63 T 0.7+
1.13 T 0.13 3.84 T 0.5
2.39 T 0.26 21.01 T 2.3
2.49 T 0.27 19.91 T 2.20
1.26 T 0.15+ 5.95 T 0.7
1.52 T 0.16 4.79 T 0.6+
2.01 T 0.2
2.10 T 0.25
1.61 T 0.17
1.70 T 0.16
1.95 T 0.23
2.11 T 0.28
1.35 T 0.17
1.40 T 0.19
Catecholamine stores were measured as described in the text. Mean and S.E.M. for each of the two central and two peripheral tissues were calculated for each group. The number of animals used for each group was six. Statistical analysis were performed by two-way factorial ANOVA for normotensive and spontaneously hypertensive rats. NE—norepinephrine; E—epinephrine; DA—dopamine; IMMO—immobilization. For interaction of normotensive and spontaneously hypertensive rats :+ p < 0.05.
sympathetic activity of spontaneously hypertensive rats is associated with hyperreactiveness of the paraventricular nucleus. Based on these findings it can be concluded that enhanced hypothalamic noradrenergic activity can increase sympathetic outflow to the periphery, which may play a role in the pathogenesis of hypertension. The obtained results show that the influence of the previous exposure to a stressor on noradrenergic activity in the brain depends on the particular noradrenergic area of the brainstem studied and therefore, the sensitivity of the noradrenergic system to habituation appears to be regionspecific. Long-term social isolation of either normotensive or spontaneously hypertensive rats produced no significant changes in catecholamine levels of the examined peripheral tissues (Table 1). These results point to the adaptation of the sympathetic adrenomedullary system to long-term isolation. Sanchez and Pereira (2002) showed that exposure of animals to chronic intermittent stress results in a significant increase of catecholamine biosynthetic and storage capacities in peripheral tissues comparing to those of unstressed controls. Also, Kvetnansky et al. (2003) observed permanently increased adrenal tyrosine hydroxylase mRNA levels, tyrosine hydoxylase activity and protein levels in rats exposed to repeated immobilization stress (41 days). Novel immobilization stress applied throughout the present work to previously long-term isolated animals produced a highly significant decrease of NE content in the adrenal gland and heart auricles of both normotensive and spontaneously hypertensive rats and statistical significant ( p < 0.001) decrease of E in normotensive but less ( p < 0.05) in spontaneously hypertensive rats. These results suggest that mobilization of high stores of tissue catecholamines was accelerated after exposure of rats to a novel stressor. This readiness of long-term stressed animals to respond to novel
stressor together with increased levels of plasma NE and E is an important adaptive phenomenon of the sympatho-adrenomedullary system. These results clearly demonstrate that long-term social isolation led to a depletion of NE stores in the hypothalamus and of DA stores in the hippocampus of both normotensive and spontaneously hypertensive rats, while novel immobilization stress produced no further depletion in hypothalamic NE of normotensive and hippocampal DA of spontaneously hypertensive rats, but decreased NE stores in hypothalamus of spontaneously hypertensive rats and hippocampus of normotensive rats. At the same time, 21-day-social isolation did not affect catecholamine stores in peripheral tissues, but novel immobilization stress produced a significant decrease in the amount of catecholamine content in peripheral tissues of both normotensive and hypertensive rats. These data suggests that long-term isolation in normotensive and spontaneously hypertensive rats produces change in central catecholamine stores. Novel stress produced further depletion in peripheral and central tissues, especially in hypothalamus of hypertensive rats and hippocampi of normotensive rats.
Acknowledgement This work was supported by the Ministry for Science, Technology and Environmental Protection of Serbia, contract #1953.
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Miura, H., Qiao, H., Ohta, T., 2002. Attenuating effects of the isolated rearing condition on increased brain serotonin and dopamine turnover elicited by novelty stress. Brain Res. 926, 10 – 17. Moore, H., Rose, H.J., Grace, A.A., 2001. Chronic cold stress reduces the spontaneous activity of ventral tegmental dopamine neurons. Neuropsychopharmac. 24, 410 – 419. Natelson, B.H., Ottenweller, J.E., Cook, J.A., Pitman, D., Mccarty, R., Tapp, W.N., 1998. Effect of stressor intensity on habituation of the adrenocortical stress response. Physiol. Behav. 43, 41 – 46. Nisenbaum, L.K., Abercrombie, E.D., 1992. Enhanced tyrosine hydroxylation in hippocampus of chronically stressed rats upon exposure to a novel stressor. J. Neurochem. 58, 276 – 281. Nisenbaum, L.K., Abercrombie, E.D., 1993. Presynaptic alterations associated with enhancement of evoked release and synthesis of norepinephrine in hippocampus of chronically cold-stressed rats. Brain Res. 608, 280 – 287. Paulus, M.P., Bakshi, V.P., Geyer, M.A., 1998. Isolation rearing affects sequential organization of motor behavior in post-pubertal but not prepubertal Wister and Sprague – Dawley rats. Behav. Brain Res. 94, 271 – 280. Peuler, J.D., Johnson, A., 1977. Simultaneous single isotope radioenzymatic assay of plasma norepinephrine, epinephrine and dopamine. Life Sci. 21, 625 – 636. Pol, O., Campmany, L., Armario, A., 1992. Behavioral and neurochemical changes in response to acute stressors: influence of previous chronic exposure to immobilization. Pharmacol. Biochem. Behav. 42, 407 – 412. Prieto, M., Gomez, F.M., Giralt, M.T., 2003. Effects of acute, repeated and chronic variable stress on in vivo tyrosine hydroxylase activity and on a2 -adrenoceptor sensitivity in the rat brain. Stress 6, 281 – 287. Rilke, O., Jahkel, M., Oehler, J., 1998. Dopaminergic parameters during social isolation in low- and high-active mice. Pharmacol. Biochem. Behav. 60, 499 – 505. Sanchez, A., Pereira, O.C.M., 2002. Acute swimming stress induces changes in noradrenergic mechanisms in order to maintain the response pattern of the rat vas deferens to norepinephrine. Pharmacol. Res. 46, 55 – 60. Sanchez, A., Toledo-Pinto, E.A., Menezes, M.L., Pereira, O.C.M., 2003. Changes in norepinephrine and epinephrine concentrations in adrenal gland of the rats submitted of acute immobilization stress. Pharmacol. Res. 48, 607 – 613. Shibasaki, T., Tsumori, C., Hotta, M., Imaki, T., Yamada, K., Demura, H., 1995. The response pattern of noradrenaline release to repeated stress in the hypothalamic paraventricular nucleus differs according to the form of stress in rats. Brain Res. 670, 169 – 172. Sohn, H.S., Park, Y.N., Lee, S.R., 2002. Effect of immobilization stress on brain polyamine levels in spontaneously-hypertensive and Wistar – Kyoto rats. Brai Res. Bull. 57, 575 – 579. Sorg, B.A., Kalivas, P.W., 1993. Behavioral sensitization to stress and psychostimulants: role of dopamine and excitatory amino acids in the mesocorticolimbic system. Semin. Neurosci. 5, 343 – 350. Stam, R., Bruijnzeel, A.W., Wiegant, V.M., 2000. Long-lasting stress sensitization. Eur. J. Pharmacol. 405, 217 – 224. Torres, I.L.S., Gamaro, G.D., Vasconcellos, A.P., Silveira, R., Dalmaz, C., 2002. Effects of chronic restraint stress on feeding behavior and on monoamine levels in different brain structures in rats. Neurochem. Res. 27, 519 – 525. Tsuda, K., Tsuda, S., Masuyama, Y., Goldstein, M., 1991. Alterations in catecholamine release in the central nervous system of spontaneously hypertensive rats. Jpn. Heart J. 32, 701 – 709. Van Den Buuse, M., Lambert, G., Fluttert, M., Eikelis, N., 2001. Cardiovascular and behavioural responses to psychological stress in spontaneously hypertensive rats: effect of treatment with DSP-4. Behav. Brain Res. 119, 131 – 142. Zaretskii, D.V., Kalenikova, E.I., Livanova, L.M., Kuzmin, A.I., Zaretskaia, M.V., 1999. The activity of the monoaminergic systems of the rat hypothalamus in acute stress after chronic stressing. Zh. Vyssh. Nerv. Deiat. Im. I. P. Pavlova 49, 313 – 320.
Novel stressors affected catecholamine stores in socially isolated normotensive and spontaneously hypertensive rats Ljubica Gavrilovic, Natasa Spasojevic, Sladjana Dronjak * Institute of Nuclear Sciences ‘‘Vinca’’, Laboratory of Molekular Bilology and Endocrinology, P.O.Box 522-090, 1001 Belgrade, Serbia and Montenegro Received 16 May 2005; received in revised form 16 May 2005; accepted 30 July 2005
Abstract Catecholamines in some central (hypothalamus and hippocampus) and peripheral tissues (adrenal glands and heart auricles) of long-term socially isolated normotensive and spontaneously hypertensive rats exposed to novel immobilization stress were determined by a simultaneous single isotope radioenzymatic assay. Long-term isolation (21 days) produced depletion of hypothalamic norepinephrine (NE) stores and hippocampal dopamine (DA) stores in both normotensive and spontaneously hypertensive rats. Acute immobilization stress (2 h) significantly decreased NE and DA stores in hypothalamus and hippocampus of naive normotensive and spontaneously hypertensive rats controls. However, novel immobilization stress applied to normotensive rats previously subjected to long-term isolation produced no changes in catecholamine levels in hypothalamus, while resulting in somewhat higher depletion of NE stores in hypothalamus of spontaneously hypertensive rats treated in the same way. Novel immobilization stress decreased NE and DA stores in hippocampus of normotensive but was without effect on NE and DA stores of spontaneously hypertensive rats. Social isolation did not affect catecholamine stores in peripheral tissues but novel immobilization stress produced a significant decrease in catecholamine content. The results suggest that some central and peripherals tissues of spontaneously hypertensive rats and normotensive rats differ with regard to catecholamine content and that there are certain differences in their responsiveness to stress. D 2005 Elsevier B.V. All rights reserved. Keywords: Social isolation; Hippocampus; Hypothalamus; Adrenal gland; Catecholamines
1. Introduction Stress is considered to be one of the most important factors in the development of cardiovascular and mental diseases. Various stressors are known to activate the peripheral sympatho-adrenomedullary and limbic – hypothalamo – pituitary – adrenal system. Limbic circuits connecting, e.g. the hippocampus, amygdala and prefrontal cortex are sensitive to stressors such as restraint, fear or exposure to a novel environment. All these stressors act stimulating intralimbic processing of informations received from different sensory organs, and this processing depends on a previous experience. In contrast, physiological threats such as exposure to ether results in the activation of efferent viscelar pathways that are directly relayed to the para* Corresponding author. Tel./fax: +38 1118066514. E-mail address: [email protected] (S. Dronjak). 1566-0702/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.autneu.2005.07.010
ventricular nucleus of the hypothalamus (Fuchs and Flugge, 2003). Cortical and hippocampal noradrenergic afferents derive from the locus coeruleus, whereas hypothalamic afferents derive from the lateral tegmental noradrenergic system. Activation of limbic and hypothalamic brain structures is a major component of the stress reaction that integrates neuroendocrine and emotional components and thus, determines the magnitude and duration of the hormonal and neural stress response. Several authors found that acute exposure to stressful stimuli provokes the immediate release of norepinephrine (NE) in diverse brain regions (Shibasaki et al., 1995; Hellriegel and D’mello, 1997; Prieto et al., 2003). Also, it was shown that exposure to chronic stress can modify the function of the noradrenergic system (Nisenbaum and Abercrombie, 1993), while prior stress exposure was reported to alter the response to subsequent stressor, leading to the development of tolerance or sensitization (Natelson et al., 1998; Mcewen, 2000; Stam
L. Gavrilovic et al. / Autonomic Neuroscience: Basic and Clinical 122 (2005) 38 – 44
et al., 2000; Hajos-Korcsok et al., 2003). The distinct adaptive responses may be influenced by type, intensity and duration of the stressor. Social isolation of adult animals has been shown to enhance locomotion and exploratory behavior and anxiety in novel environments (Fone et al., 1996; Hall et al., 1998; Paulus et al., 1998; Rilke et al., 1998). We have found recently that rats previously exposed to social isolation for 21 days exhibited a significant increase of plasma NE and even greater increase of plasma epinephrine (E) in response to additional immobilization (Dronjak et al., 2004). Stress represents a major factor in pathogenesis of hypertension and stress-induced elevations in plasma concentration of E leading to cardiovascular stimulation and development of arterial hypertension (Esler et al., 2001). Spontaneously hypertensive rats have been used as a model for the studies of the mechanism(s) underlying the development of hypertension. Spontaneously hypertensive rats are hyperactive in a novel environment and hyperresponsive to environmental stimuli exhibiting an increased behavioral, sympathetic and cardiovascular responsiveness comparing to normotensive animals. Besides, sympathetic nervous system of SHR is overactive as compared to that of normotensive rats (Sohn et al., 2002). The adrenal gland represents one of the main components of the sympathetic nervous system. Adrenal gland medulla is innervated by sympathetic nerves, so that the adrenal nerve activity represents the sympathetic outflow to the adrenal medulla (Aoki et al., 1996). To estimate the influence of chronic social stress on the activity of the central and peripheral catecholaminergic system in normotensive and hypertensive animals, we compared the effect of immobilization as a novel stress applied to long-term socially isolated adult rats on catecholamine stores in some central (hippocampus and hypothalamus) and peripheral tissues (adrenal glands and heart auricles) of spontaneously hypertensive rats and normotensive Wistar rats.
2. Materials and methods Normotensive and spontaneously hypertensive 3-monthold Wistar rat males weighing 300– 340 g maintained under standard laboratory conditions with water and food ad libitum in groups of four individuals per cage were used. The care was taken to minimize the pain and discomfort of the animals according to the recommendations of the ‘‘Vinca’’ Institute which are in accordance with the Guide for Care and Use of Laboratory Animals of the National Institute of Health, Bethesda, MD, U.S.A. One group of animals was subjected to social isolation with a single animal per cage for the 21-day-period. After that, naive controls and the rats that suffered long-term isolation were exposed to acute immobilization stress by fixing all four limbs to a board with adhesive tape. The head was also fixed by a metal loop over the neck area, thus limiting the
39
motion of the head (Kvetnansky and Mikulaj, 1970). The rats were killed by decapitation after 2 h, hippocampi, hypothalami, heart auricles and adrenal glands were quickly dissected out and immersed into cold (4 -C) perchloric acid (0.3 Ag of tissue per 30 Al of 0.1 N HClO4). The tissues were homogenized in a motor-driven homogenizer, the homogenates centrifuged (a Janetzky K24 centrifuge, 20 000 r.p.m., 20 min, 4 -C) and the supernatants (30 Al) used for determination of catecholamines. Content of catecholamines in the tissues was determined using somewhat modified chromatographic method of Peuler and Johnson (1977) based on the conversion of catecholamines into the corresponding O-methylated derivatives by purified catechol-O-methyl-transferase (COMT) in the presence of S-adenosyl-l-(3H-methyl)-methionine. The O-methylated derivatives were extracted and oxidized into 3H-vaniline. Radioacitivites were measured using toluene-based scintillation cocktail in a LKB-Wallac model 1219 scintillation counter at an efficiency of 40% for tritium. Statistical analyses were performed by two-way factorial analysis of variance (ANOVA) for the normotensive and spontaneously hypertensive rats. To consider the influence of novelty stress, one-way ANOVA was applied individually to animals in the naive control and isolated group rats. The p < 0.05 level was chosen as the point of minimal acceptable statistical significance.
3. Results 3.1. Effect of novel immobilization stress on catecholamine levels in hypothalamus and hippocampus of spontaneously hypertensive and normotensive rats exposed to long-term isolation The results presented in Fig. 1. show that long-term isolation (21 days) produced a statistically significant ( p < 0.05) depletion of NE but not of dopamine (DA) in hypothalami of both spontaneously hypertensive rats and normotensive. Two hours of acute immobilization significantly decreased NE ( p < 0.001) and DA ( p < 0.01) stores in the hypothalamus of control spontaneously hypertensive rats and normotensive. However, novel immobilization stress produced no significant changes in hypothalamic catecholamine levels of normotensive previously exposed to 21-day-isolation, while resulting in a somewhat higher depletion ( p < 0.05) of NE stores in spontaneously hypertensive rats. Long-term isolation produced no significant changes in hippocampal NE level of either spontaneously hypertensive or normotensive rats, while a significant depletion ( p < 0.05) of DA stores in hippocampus of both groups was observed. Acute immobilization of spontaneously hypertensive and normotensive rats resulted in a significant decrease ( p < 0.001) of NE and DA stores in the hippo-
40
L. Gavrilovic et al. / Autonomic Neuroscience: Basic and Clinical 122 (2005) 38 – 44 2.6 2.4 2.2
concentrations (ug/g)
2
+
1.8
a-NA contr. norm.
b-NA 21 days norm.
c-NA contr. hyper.
d-NA 21 days hyper.
e-DA contr. norm.
f-DA 21 days norm.
g-DA contr. hyper.
h-DA 21 days hyper.
+
*
1.6 ***
1.4
***
1.2 1 0.8 **
0.6
**
0.4 0.2 0
a
b
c
d
e
f
g
h
a
b
c
d
0
e
f
g
h
120 time (min)
Fig. 1. Concentrations of norepinephrine (NE) and dopamine (DA) in the hypothalami of normotensive and spontaneously hypertensive rats control and longterm isolated animals exposed to 2-h-immobilization. The values expressed in Ag/g fresh tissue are the means T S.E.M. of six rats. Statistical significance: *p < 0.05; **p < 0.01; ***p < 0.001, as compared to zero point; + p < 0.05 21 days isolation vs. naive control.
campus of naive controls. Acute immobilization stress applied to animals previously exposed to long-term isolation resulted in statistically significant changes in hippocampal NE stores of both normotensive ( p < 0.01) and spontaneously hypertensive rats ( p < 0.05), the changes being more prominent in the former comparing to the latter group (Fig. 2).
It is evident from the results depicted in Fig. 3, that longterm isolation produced no significant changes in E and NE
1.4
a-NA contr. norm.
b-NA 21 days norm.
1.3
c-NA contr. hyper.
d-NA 21 days hyper.
1.2
e-DA contr. norm.
f-DA 21 days norm.
1.1
g-DA contr. hyper. +
1 concentrations (ug/g)
3.2. Effect of novel immobilization stress on catecholamine levels in adrenal glands and heart auricles of spontaneously hypertensive and normotensive rats previously exposed to long-term isolation
h-DA 21 days hyper. *
+
0.9
**
0.8
***
f
g
***
***
0.7
*
** ***
0.6 0.5 0.4 0.3 0.2 0.1 0
a
b
c
d
e
f
g
h
a
0
b
c
d
e
h
120 time (min)
Fig. 2. Norepinephrine (NE) and dopamine (DA) levels in the hippocampi of normotensive and spontaneously hypertensive rats control and long-term isolated animals exposed to 2-h-immobilization as a novel stress. The values in Ag/g fresh tissue are the means T S.E.M. of six animals. Statistical significance: *p < 0.05; **p < 0.01; ***p < 0.001, as compared to zero point; + p < 0.05 21 days isolation vs. naive control.
L. Gavrilovic et al. / Autonomic Neuroscience: Basic and Clinical 122 (2005) 38 – 44
41
25
a-NA contr. norm. b-NA 21 days norm.
22.5
c-NA contr. hyper. d-NA 21 days hyper. e-A contr. norm. f-A 21 days norm.
concentrations (ug/g)
20 17.5 15
g-A contr. hyper. h-A 21 days hyper.
12.5 10
***
7.5
*** ***
5
***
2.5 0
a
b
c
d
e
f
g
h
***
***
a
b
*** c
** e
d
0
f
g
h
120 time (min)
Fig. 3. Epinephrine (E) and norepinephrine (NE) content in the adrenal glands of normotensive and spontaneously hypertensive rats control and long-term isolated animals additionally exposed to 2-h-immobilization. The values in Ag/g fresh tissue are the means T S.E.M. of six rats. Statistical significance: *p < 0.05; **p < 0.01; ***p < 0.001 as compared to zero point.
content in adrenal glands of both spontaneously hypertensive and normotensive rats. Immobilization stress led to a highly significant decrease ( p < 0.001) in the amount of E and NE in the adrenal glands of both naive control and long-term isolation groups of spontaneously hypertensive and normotensive rats. Relative decrease of NE and E level in adrenal glands of normotensive exceeded greatly that of sponta-
neously hypertensive rats exposed to the same period of immobilization following long-term isolation. The results presented in Fig. 4 show that long-term isolation did not affect NE level in heart auricles of either spontaneously hypertensive or normotensive rats. However, acute immobilization alone led to a significant decrease ( p < 0.001) in NE content of this tissue in control sponta-
3
a-NA contr. norm. b-NA 21 days norm. c-NA contr. hyper. d-NA 21 days hyper.
concentration (ug/g)
2.5
2
***
***
*** *** 1.5
1
0.5
0
a
d
c
d
a
0
b
c
d
120 time (min)
Fig. 4. Levels of norepinephrine (NE) in the heart auricles of normotensive and spontaneously hypertensive rats control and long-term isolated animals after 2h-exposure to immobilization. The values in Ag/g fresh tissue are the means T S.E.M. of six rats. Statistical significance: *p < 0.05; **p < 0.01; ***p < 0.001 as compared to zero point.
42
L. Gavrilovic et al. / Autonomic Neuroscience: Basic and Clinical 122 (2005) 38 – 44
neously hypertensive and normotensive rats. Also, a significant depletion of NE stores in the heart auricles was observed after immobilization as a novel stress to long-term isolation of both spontaneously hypertensive and normotensive rats.
4. Discussion Our earlier studies showed that long-term social isolation of rats produced a significant elevation of basal plasma adrenocorticotropic hormone and corticosterone, but did not affect basal plasma NE and E levels, while the same treatment followed by acute immobilization as a novel stress led to a significant elevation of plasma NE and E (Dronjak et al., 2004). On the other hand, the results of microdialysis studies of Abercrombie et al. (1992) confirmed the sensitivity of central catecholamines to stressors. The same authors, reported significantly higher hippocampal NE and l-DOPA levels in chronically stressed rats ¨ in response to a novel stressor, in comparison with naNve acutely stressed animals (Abercrombie et al., 1992; Nisenbaum and Abercrombie, 1992). Also, Miura et al. (2002) demonstrated that chronic isolation of animals attenuated novelty stressinduced increase in DA turnover. This prompted us to investigate the effects of acute immobilization as a novel stress on catecholamine stores in some central and peripheral tissues of normotensive and hypertensive rats previously exposed to long-term social isolation. The results of the present study showed that acute 2-himmobilization produced a significant depletion of central and peripheral catecholamine stores in both normotensive and spontaneously hypertensive rats control groups of animals. Our findings are in accordance with the data of several others (Pol et al., 1992; Moore et al., 2001; Sanchez et al., 2003) who found that 2-h-immobilization significantly decreased NE and DA levels in hypothalamus and prefrontal cortex, as well as NE and E levels in the adrenal glands. Social stress was reported to produce many changes in the brain, affecting neuronal structure and also inducing the stimulation of noradrenergic neurons (Blanchard et al., 2001). Our results clearly demonstrated that long-term social isolation led to a significant depletion of NE in the hypothalamus but not in the hippocampus of both normotensive and spontaneously hypertensive rats. The hypothalamus is the brain structure that influences all of the limbic structures and hypothalamic neurons are responsive to information arising in the internal and external environment. This brain structure controls the visceromotor responses by arranging the equilibrium of sympathetic and parasympathetic outputs to the autonomic nervous system (Feenstra, 2000). The observed reduction of NE stores in the hypothalamus could indicate that neurons derived from lateral tegmental system respond more powerfully to stress than those in the hippocampus deriving from locus coeruleus. However, no significant reduction in NE stores
in the hippocampus were observed throughout the present study. This could be connected to the results of HajosKorcsok et al. (2003) and explained by habituation of rats to the chronic stressor. Namely, these authors have recently shown that an initial electric footshock induced a significant elevation of extracellular NA levels in the rat hippocampus (200% above the baseline) as measured by in vivo microdialysis method. A rapid habituation to this response was evident in the inability of a second footshock to evoke similar increase. Cuadra et al. (1999) observed a more prominent increase in frontal cortical DA release of chronically stressed rats after exposure to a novel restraint stressor as compared to that recorded in the controls that suffered only restraint stress. Our results are compatible with the findings of Fulford and Marsden (1997) who found no differences in the basal efflux of hippocampal NE between the rats exposed to isolation and group-reared ones. Brain DA levels change after acute stress and chronic exposure to stress may express different effects on this neurotransmitter. For example, acute stress was shown to lead to an increased extracellular level of DA in the prefrontal cortex and amygdala (Sorg and Kalivas, 1993; Jackson and Moghaddam, 2004), while chronic stress over several days reduced DA output and the number of active DA cells (Mangiavacchi et al., 2001; Moore et al., 2001). The results presented here also showed that acute immobilization of the control rats significantly decreased DA stores in the hypothalamus and hippocampus of both normotensive and spontaneously hypertensive rats. However, long-term isolation resulted in a reduction of hippocampal DA stores but did not affect hypothalamic ones. Several authors have reported similar results demonstrating that emotional changes, such as exposure to stress situation increased DA metabolite levels in the hippocampus and reduced DA neurotransmission and activity in the hypothalamus (Torres et al., 2002; Gamaro et al., 2003). Also, it is worth mentioning the results of Zaretskii et al. (1999) who showed that chronic stress led to an elevated and prolonged acute stress-induced release of NE but not of DA in the hypothalamus. Interestingly enough, we have observed that novel immobilization stress applied to spontaneously hypertensive rats previously exposed to 21-day-isolation resulted in a somewhat higher NE depletion in the hypothalamus but did not affect that in the hippocampus. These results are consistent with earlier studies demonstrating that enhanced hypothalamic noradrenergic activity can increase sympathetic outflow to the periphery, which may play the role in the pathogenesis of hypertension (Tsuda et al., 1991; Akine et al., 2003), while noradrenergic projections from the locus coeruleus do not appear to play a major role in cardiovascular stress response of SHR (Van Den Buuse et al., 2001). Besides, Krukoff et al. (1999) found a significantly higher number of double labeled neurons in the ventral medial parvocellular paraventricular nucleus of stressed spontaneously hypertensive rats as compared to WKY and SD rats. These results demonstrated that chronic elevation in
L. Gavrilovic et al. / Autonomic Neuroscience: Basic and Clinical 122 (2005) 38 – 44
43
Table 1 Changes in catecholamine stores in normotensive and spontaneously hypertensive rats Central and peripheral tissues
Control 0’ NORM
Hypothalamus NA DA Hippocampus NA DA Adrenal glands NA A Heart auricles NA
Isolation IMMO HYPER
0’
NORM
HYPER
NORM
IMMO HYPER
NORM
HYPER
1.94 T 0.22 0.78 T 0.09
2.10 T 0.25 0.84 T 0.09
1.23 T 0.14 0.50 T 0.06
1.20 T 0.15 0.52 T 0.07
1.60 T 0.19 0.75 T 0.09
1.68 T 0.19 0.80 T 0.10
1.58 T 0.17 0.72 T 0.09
1.47 T 0.18+ 0.78 T 0.10
1.01 T 0.12 1.11 T 0.13
1.04 T 0.11 1.07 T 0.13
0.63 T 0.08 0.66 T 0.08
0.60 T 0.08 0.70 T 0.08
0.98 T 0.11 0.89 T 0.10
1.01 T 0.12 0.87 T 0.10
0.77 T 0.09+ 0.72 T 0.09
0.88 T 0.11 0.75 T 0.09
2.35 T 0.25 20.74 T 2.22
2.24 T 0.26 19.84 T 2.10
1.09 T 0.13 5.63 T 0.7+
1.13 T 0.13 3.84 T 0.5
2.39 T 0.26 21.01 T 2.3
2.49 T 0.27 19.91 T 2.20
1.26 T 0.15+ 5.95 T 0.7
1.52 T 0.16 4.79 T 0.6+
2.01 T 0.2
2.10 T 0.25
1.61 T 0.17
1.70 T 0.16
1.95 T 0.23
2.11 T 0.28
1.35 T 0.17
1.40 T 0.19
Catecholamine stores were measured as described in the text. Mean and S.E.M. for each of the two central and two peripheral tissues were calculated for each group. The number of animals used for each group was six. Statistical analysis were performed by two-way factorial ANOVA for normotensive and spontaneously hypertensive rats. NE—norepinephrine; E—epinephrine; DA—dopamine; IMMO—immobilization. For interaction of normotensive and spontaneously hypertensive rats :+ p < 0.05.
sympathetic activity of spontaneously hypertensive rats is associated with hyperreactiveness of the paraventricular nucleus. Based on these findings it can be concluded that enhanced hypothalamic noradrenergic activity can increase sympathetic outflow to the periphery, which may play a role in the pathogenesis of hypertension. The obtained results show that the influence of the previous exposure to a stressor on noradrenergic activity in the brain depends on the particular noradrenergic area of the brainstem studied and therefore, the sensitivity of the noradrenergic system to habituation appears to be regionspecific. Long-term social isolation of either normotensive or spontaneously hypertensive rats produced no significant changes in catecholamine levels of the examined peripheral tissues (Table 1). These results point to the adaptation of the sympathetic adrenomedullary system to long-term isolation. Sanchez and Pereira (2002) showed that exposure of animals to chronic intermittent stress results in a significant increase of catecholamine biosynthetic and storage capacities in peripheral tissues comparing to those of unstressed controls. Also, Kvetnansky et al. (2003) observed permanently increased adrenal tyrosine hydroxylase mRNA levels, tyrosine hydoxylase activity and protein levels in rats exposed to repeated immobilization stress (41 days). Novel immobilization stress applied throughout the present work to previously long-term isolated animals produced a highly significant decrease of NE content in the adrenal gland and heart auricles of both normotensive and spontaneously hypertensive rats and statistical significant ( p < 0.001) decrease of E in normotensive but less ( p < 0.05) in spontaneously hypertensive rats. These results suggest that mobilization of high stores of tissue catecholamines was accelerated after exposure of rats to a novel stressor. This readiness of long-term stressed animals to respond to novel
stressor together with increased levels of plasma NE and E is an important adaptive phenomenon of the sympatho-adrenomedullary system. These results clearly demonstrate that long-term social isolation led to a depletion of NE stores in the hypothalamus and of DA stores in the hippocampus of both normotensive and spontaneously hypertensive rats, while novel immobilization stress produced no further depletion in hypothalamic NE of normotensive and hippocampal DA of spontaneously hypertensive rats, but decreased NE stores in hypothalamus of spontaneously hypertensive rats and hippocampus of normotensive rats. At the same time, 21-day-social isolation did not affect catecholamine stores in peripheral tissues, but novel immobilization stress produced a significant decrease in the amount of catecholamine content in peripheral tissues of both normotensive and hypertensive rats. These data suggests that long-term isolation in normotensive and spontaneously hypertensive rats produces change in central catecholamine stores. Novel stress produced further depletion in peripheral and central tissues, especially in hypothalamus of hypertensive rats and hippocampi of normotensive rats.
Acknowledgement This work was supported by the Ministry for Science, Technology and Environmental Protection of Serbia, contract #1953.
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