научная статья по теме HYPOTHALAMIC NORADRENALINE SYNTHESIS, UPTAKE AND STORAGE IN RATS DURING ADAPTATION TO LONG-TERM INDIVIDUAL HOUSING Медицина и здравоохранение

Текст научной статьи на тему «HYPOTHALAMIC NORADRENALINE SYNTHESIS, UPTAKE AND STORAGE IN RATS DURING ADAPTATION TO LONG-TERM INDIVIDUAL HOUSING»

НЕЙРОХИМИЯ, 2013, том 30, № 2, с. 147-151

ЭКСПЕРИМЕНТАЛЬНЫЕ РАБОТЫ

YM 577

HYPOTHALAMIC NORADRENALINE SYNTHESIS, UPTAKE AND STORAGE IN RATS DURING ADAPTATION TO LONG-TERM INDIVIDUAL HOUSING1

© 2013 N. Spasojevic, P. Jovanovic, D. Stanisavljevic, B. Stefanovic, and S. Dronjak*

Institute of Nuclear Sciences "Vinca", Laboratory of Molecular Biology and Endocrinology, Belgrade, University of Belgrade, Belgrade, Serbia

Abstract—The brain monoamine systems play a crucial role in mediating social behaviors. In order to assess the effect of socially isolated rats on neurochemical substrates in hypothalamus we measured levels of tyrosine hydroxylase (TH), noradrenaline transporter (NET) and vesicular monoamine transporter 2 (VMAT 2). Our results show that 12 weeks of social isolation produced no significant changes in noradrenaline content in hypothalamus. The levels of TH and NET were elevated in these animals, whereas level of VMAT 2 was unchanged. The data suggests that both enhanced synthesis and reuptake may aid in the recovery of noradren-aline content in the hypothalamus. The adaptation of the hypothalamic noradrenaline to long period of stress may have great physiological importance and may represent mechanism designed to help the organism adapt to the deleterious effects of chronic indiviual housing.

Keywords: hypothalamus, noradrenaline, tyrosine hydroxylase, noradrenaline transporters, isolation stress, rats

DOI: 10.7868/S1027813313020052

INTRODUCTION

Activation of the sympathetic nervous system represent one of the early responses to stress. When animals are exposed to the long-term stress, it usually results in a reduction of sympathoneural responses. This process has been termed either adaptation or habitua-tion and neurochemical changes are likely to be involved in the phenomenon [1, 2]. The adaptation evoked by stressful stimuli serve to maintain homeostasis. However, prolonged activation of the stress response itself can become maladaptive [3]. The brain monoamine systems play a crucial role in mediating social behaviors. Social relationships are an important modulator of mental health. Social isolation might produce several negative long-term consequences for the animal. It has been demonstrated that isolated rats exhibit impairments in learning and memory tasks [4] as well as changes in the brain neurochemistry [5]. The brain noradrenaline is a major alarm system that leads to decrease in neurovegetative functions, such as eating and sleeping. The hypothalamus as a major inte-grative center of the neuroendocrine response recives innervation of noradrenaline-containing neurons [6]. Social isolation produced by individual housing affects monoamine in hypothalamus [7]. In contrast neuro-chemical, molecular events in noradrenergic neurons of hypothalamus have received little attention. Norad-

1 The article was submitted by the authors in English.

* Corresponding autor; address: P.O.B. 522-090, 11000 Belgrade, Serbia; phone/phax: 00381112455561; e-mail: sladj@vinca.ru.

renergic activity is dependent on the synthesis of noradrenaline, as determined by the rate-limiting enzyme tyrosine hydroxylase (TH), its reuptake through the noradrenaline transporter (NET), synaptic release, degradation and vesicular transport mediated by the vesicular monoamine transporter 2 (VMAT 2). The objective of the present study was to analyze influence of prolonged individual housing on noradrenaline synthesis, uptake and store in the hypothalamus. Therefore, we quantified the changes in noradrenaline content, TH, NET and VMAT 2 levels in the hypothalamus of individual housed rats during 12 weeks.

MATERIAL AND METHODS

Animals

Adult, 11-week-old Wistar rat males, maintained under standard laboratory conditions with water and food ad libitum in the 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 Ethical Committee of the "Vinca" Institute, Belgrade based on the Guide for Care and Use of Laboratory Animals of the National Institute of Health (Bethesda, MD, U.S.A). In the experiment we used 14 animals, which were divided into two groups. One group of animals was subjected to social isolation with a single animal per cage, while second group was naive controls which were group housed. After 12 weeks rats were decapitated, the right

and left hypothalamus rapidly dissected, frozen in liquid nitrogen and stored at —70°C until analyzed.

Radioenzymatic Assay

Hypothalamus were immersed into cold (4°C) perchloric acid (0.3 ^g of tissue per 30 ^L of 0.1 N HClO4), homogenized in a motor-driven homogeniz-er, the homogenates centrifuged (20000 r.p.m., 20 min, 4°C) and the supernatants (30 ^L) used for determination of catecholamines. Content of cate-cholamines in the tissues was determined using modified chromatographic method of Peuler and Johnson [8] 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.

Western Blot Analysis

Hypothalamic tissue were homogenized in 0.05 M sodium phosphate buffer pH 6.65. 15 ^g of hypothalamic protein extract separated by 10% SDS-poly-acrylamide gel electrophoresis were transferred to a supported nitrocellulose membrane (HybondTM C Extra, Amersham Bioscience, GE Healthcare, Buckinghamshire, UK). The membrane was blocked in 5% non-fat dry milk in Tris-buffered saline-Tween (TBST). All following washes and antibody incubations were also performed in TBST at ambient temperature on a shaker. For measuring TH, NET and VMAT 2 protein levels, a polyclonal anti-TH (N-Ter-minal) primary antibody, rabbit (dilution 1 : 1000, Ab-cam, Cambridge, UK), a polyclonal anti-NET primary antibody rabbit (dilution 1 : 1000, Abcam, Cambridge, UK) and polyclonal anti-VMAT 2 primary antibody, rabbit (dilution 1 : 5000, Abcam, Cambridge, UK) respectively, were used. Washed membrane was further incubated in the horseradish perox-idase conjugated secondary anti-rabbit antibody for luminol based detection (dilution 1 : 5000, GE Healthcare, Buckinghamshire, UK). Secondary antibody was then visualized by Amersham ECL Western Blotting Detection System (GE Healthcare, Buckinghamshire, UK). Western blot analysis was performed as previously described [9].

Statistical Analyses

The results are reported as means ± S.E.M. Significance of the differences in noradrenaline content and protein levels of the examined catecholamine biosyn-thetic enzyme and transporters of rats subjected to social isolation was estimated by One-way ANOVA test.

The Tukeypost-hoc test was used to evaluate the differences between the groups. Statistical significance was accepted atp < 0.05.

RESULTS

In the present study, we examined hypothalamic tissue samples from rats exposed to social isolation for 12 weeks. Under the examined stress conditions no significant changes of noradrenaline (NA) content were observed. (Fig. 1).

In contrast, One-way ANOVA analysis revealed significant stress-induced variations of TH protein levels in the hypotalamus. Stressed animals had 37% (p < 0.01) higher levels of TH protein in the hypotalamus, as compared to those found in the control animals (Fig. 2).

Analysis of the obtained data indicated that the applied stressful procedure considerably affected NET contents. Protein levels of NET were increased by 21% (p < 0.05) in hypothalamus of rats that suffered chronic stress. On the other hand, protein levels of VMAT 2 remained unchanged. (Fig. 3).

DISCUSSION

Social stress was reported to produce many changes in the brain, affecting neuronal structure and also inducing the stimulation of dopaminergic and noradr-energic neurons [10]. Previously we reported that 21-day individual housing reduced hypothalamic noradrenaline [11]. In our present study we observed unchanged hypothalamic noradrenaline during 12 weeks of social isolation. Recent report have suggested that different duration of stress may sometimes produce qualitatively different patterns of effects on physiology. Studies on the effect of long-term repeated stress on plasma catecholamine levels revealed significantly lower response of plasma adrenaline and noradrena-line to immobilization in 42 times immobilized rats in comparison to acutely stressed animal [12]. Similar findings concerning catecholamine stores were observed by Pol et al. [13] who found that chronic exposure to immobilization stress did not alter noradrena-line in the prefrontal cortex, hippocampus or hypothalamus but that 2-h immobilization significantly decreased noradrenaline in these brain area. Taken together all these data might suggesting adaptations of monoamine systems in hypothalamus to long-term individually housed rats. Such differences in hypothalamic noradrenaline content between 21-day and 12 weeks isolation stress could result from variations in neuronal synthesis and in variations in release at syn-aptic endings or uptake into nerve terminals. Therefore, we decided to examine protein levels of TH, NET and VMAT 2. Since TH is a rate-limiting step in the catecholaminergic synthesis [14], the modulation of TH have been expected to affect noradrenaline production in this area. Our results show that chronic iso-

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Fig. 1. Effects of chronic isolation stress on concentration of noradrenaline in hypothalamus of group- and individually housed rats. The values expressed in ^g/g fresh tissue are the means ± S.E.M. of 5—7 rats.

Fig. 2. Effects of chronic isolation stress on protein levels of tyrosine hydroxylase (TH) in hyp

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