This study aimed to investigate the effects of chronic restraint stress (CRS) on the protein levels of dopamine-β-hydroxylase (DBH), noradrenaline transporter (NET), vesicular monoamine transporter 2 (VMAT2) and brain-derived neurotrophic factor (BDNF), as well as the concentration of noradrenaline (NA) in the rat hippocampus. The investigated parameters were quantified by Western blot analyses and ELISA kits. We found that CRS increased the protein levels of DBH by 30 %, VMAT2 by 11 %, BDNF by 11 % and the concentration of NA by 104 %, but decreased the protein levels of NET by 16 % in the hippocampus of chronically stressed rats. The molecular mechanisms by which CRS increased the hippocampal NA level are an important adaptive phenomenon of the noradrenergic system in the stress condition. © 2020 Charles University. All rights reserved

Gavrilović, Ljubica

Pajović, Snežana B.

Pavlović, Ivan

Pejić, Snežana

Popović, Nataša

Stojiljković, Vesna

Todorović, Ana

English

Popović, Nataša M.

Repository of the Vinča Nuclear Institute (VinaR)

Folia Biologica (Praha) 66, 81-84 (2020)Original ArticleChanges of Hippocampal Noradrenergic Capacity in Stress Condition(chronic restraint stress / noradrenaline / hippocampus / rats)L. GAVRILOVIĆ, N. POPOVIĆ, V. STOJILJKOVIĆ, S. PEJIĆ, A. TODOROVIĆ, I. PAVLOVIĆ, S. B. PAJOVIĆ“Vinča” Institute of Nuclear Sciences, National Institute of the Republic of Serbia, Laboratory of Molecular Biology and Endocrinology, University of Belgrade, Belgrade, SerbiaAbstract. This study aimed to investigate the effects of chronic restraint stress (CRS) on the protein levels of dopamine-β-hydroxylase (DBH), noradrenaline transporter (NET), vesicular monoamine transport-er 2 (VMAT2) and brain-derived neurotrophic fac-tor (BDNF), as well as the concentration of noradren-aline (NA) in the rat hippocampus. The investigated parameters were quantified by Western blot analy-ses and ELISA kits. We found that CRS increased the protein levels of DBH by 30 %, VMAT2 by 11 %, BDNF by 11 % and the concentration of NA by 104 %, but decreased the protein levels of NET by 16 % in the hippocampus of chronically stressed rats. The molecular mechanisms by which CRS in-creased the hippocampal NA level are an important adaptive phenomenon of the noradrenergic system in the stress condition.IntroductionThe hippocampus is a limbic structure of the brain, which is very sensitive to chronic stress (Eichenbaum, 2004; Tse et al., 2014). It is known that numerous dis-eases are related to the long-term adaptive response to stress. For example, stress has a major impact upon neu-rodegenerative diseases and mental disorders (Esch et Received March 6, 2019. Accepted March 5, 2020.This work was supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia, Con-tracts Nos. III 41027, OI 173041 and III41022.Corresponding author: Ljubica Gavrilović, Institute of Nuclear Sciences “Vinča”, Laboratory of Molecular Biology and Endo-crinology, P.O.B. 522-090, 11000 Belgrade, Serbia. Phone/Fax: 0038 1112455561; e-mail: gljubica@vin.bg.ac.rsAbbreviations: BDNF – brain-derived neurotrophic factor, CRS – chronic restraint stress, DBH – dopamine-β-hydroxylase, NA – noradrenaline, NET – noradrenaline transporter, VMAT2 – ve-sicular monoamine transporter 2.al., 2002). In this study, we applied chronic restraint stress (CRS) because CRS can exacerbate neurodegen-eration and provoke anxiety and depressive-like behav-iours in rats (Ferraz et al., 2011; Liu et al., 2013; Wang et al., 2014). Many studies have shown that monoamin-ergic signalling is the key mechanism for the modula-tion of brain functions (Arnsten and Goldman-Rakic, 1985). Therefore, monitoring of the changes of mono-amine level is important in the research of numerous diseases caused by chronic stress. However, data about the regulatory molecular mechanisms by which CRS changes noradrenergic capacity in the hippocampus are rather limited. Because of the significant role of no-radrenaline (NA) in the regulation of numerous hip-pocampal functions and hippocampal sensitivity to stress, in this work we investigated how CRS (2 h × 14 days) affected the protein levels of the key enzymes involved in NA synthesis (dopamine-β-hydroxylase-DBH), reuptake (noradrenaline transporter-NET), and storage (vesicular monoamine transporters-VMAT2), as well as the concentration of NA in the rat hippocampus. In addition, it is known that brain-derived neurotrophic factor (BDNF) modulates the activity of monoaminer-gic systems in the rat brain (Siuciak et al., 1996). There-fore, an additional aim of the study was to investigate how CRS affected the protein levels of BDNF. Detection of the regulatory molecular mechanisms by which CRS changes the NA level in the hippocampus may be very important in the research of numerous diseases caused by chronic stress.Material and MethodsAnimals and stress modelWistar 11-week-old male rats (300–340 g) were main-tained under standard laboratory conditions with water and food ad libitum and kept three to four per cage (Gavrilovic et al., 2010). In accordance with our previ-ous protocol (Popović et al., 2017), animals were divid-ed into two groups. The control group (N = 10) was not 82 Vol. 66exposed to stress. In the CRS group (N = 10), the ani-mals were exposed to chronic restraint stress. Restraint stress was achieved by placing each animal in a 25 × 7 cm plastic bottle, as described previously by Gamaro et al. (1999). The animals in these groups were exposed to 2 h of restraint stress every day at random times dur-ing the light period of the light/dark cycle to avoid ha-bituation during the experimental procedure of 14 days (Kim and Han, 2006). To reduce variance in the physi-ological parameters due to daily rhythms, the remaining animals were sacrificed at the same time point in the circadian cycle, between 9:00 and 11:00 am, i.e., one day after the last treatments. Animals were sacrificed under no-stress conditions by rapid decapitation. The hippocampi were rapidly dissected, frozen in liquid ni-trogen and stored at –70 °C until analysed.Western blot analysisThe protein concentration was determined using the BCA method (Thermo Scientific Pierce, Waltham, MA), described by Stich (1990). DBH, NET, VMAT2 and BDNF proteins were assayed by Western blot analysis as described previously by Gavrilović et al. (2013). Antibodies used for quantification of specific proteins were as follows: for DBH, ab63939 (Abcam, Cambridge, UK), for NET, ab41559 (Abcam), for VMAT2, ab70808 (Abcam), for BDNF, ab6201 (Abcam) and for β-actin, ab8227 (Abcam). After washing, the membranes were incubated in secondary anti-rabbit (Amersham ECLTM Western Blotting Analysis System, Little Chalpont, UK) antibodies conjugated to horseradish peroxidase. The secondary antibody was then visualized by the Western blotting enhanced chemiluminescent detection system (ECL, Amersham Biosciences). The membranes were exposed to ECL film (Amersham Biosciences). The re-sult was expressed in arbitrary units normalized in rela-tion to β-actin, which is in accordance with our previous protocol (Gavrilović et al., 2013). Noradrenaline measurementNoradrenaline concentration in the hippocampus frac-tions was determined using 3-CAT Research ELISA kits (Labor Diagnostica Nord, Nordhorn, Germany) accord-ing to the manufacturer’s protocol, as described previ-ously by Gavrilović et al. (2018). Absorbance was deter-mined at 450 nm using a microplate reader (Stat Fax 2100, Ramsey, MN). Values were expressed as ng of NA per g of tissues, which is in accordance with our previous protocol (Gavrilović et al., 2018).Data analysisThe data are presented as means ± S.E.M. Differences of gene expression of DBH, NET, VMAT2 and BDNF, as well as of NA concentration between control and CRS animals in the hippocampus, were analysed by t-test. Statistical significance was accepted at P < 0.05. Correlations of the protein levels (DBH, VMAT2 and BDNF) and NA levels were analysed by the Pearson test, using the Sigma Plot v10.0 (with SigmaStat inte-gration, San Jose, CA). ResultsChanges of DBH, NET, VMAT2 and BDNF protein levels in the hippocampusThe animals exposed to CRS showed increased levels of DBH protein by 30 % (P < 0.05, t-test, Fig. 1a), L. Gavrilović et al.Fig. 1. Effects of chronic restraint stress (CRS) on dopamine-ß-hydroxylase (DBH) [a], noradrenaline transporter (NET) [b], vesicular monoamine transporter 2 (VMAT2) [c], and brain-derived neurotrophic factor (BDNF) [d] protein levels in the hippocampus Data are shown as means ± S.E.M. of 10 rats. Statistical significance: *P < 0.05, CRS animals compared to control ani-mals (t-test)Vol. 66 83Hippocampal Noradrenergic CapacityVMAT2 protein by 11 % (P < 0.05, t-test, Fig. 1c) and BDNF protein by 11 % (P < 0.05, t-test, Fig. 1d) in the hippocampus, compared with the control animals. However, CRS significantly decreased the levels of NET protein by 16 % (P < 0.05, t-test, Fig. 1b) com-pared with the control animals. Changes of the NA concentration in the hippocampus We found that in the hippocampus, CRS significantly increased NA concentration by 104 % (P < 0.01, t-test, Fig. 2), compared with the control animals. In the con-trol animals, we did not record any significant correla-tion between the levels of VMAT2 protein and NA con-centration in the hippocampus. However, a significant positive correlation was found between the levels of VMAT2 protein and NA concentration (Pearson, r = 0.793; P < 0.05) in the hippocampus of the animals exposed to CRS. In addition, we found a significant positive corre-lation between the levels of DBH protein and NA con-centration (Pearson, r = 0.865; P < 0.01), as well as sig-nificant positive correlation between the levels of BDNF protein and NA concentration (Pearson r = 0.704; P < 0.05) in the hippocampus of animals exposed to CRS. DiscussionIn the present study, we found that CRS increased the hippocampal NA levels. In addition, we noted that the significant increase of hippocampal NA concentration coincided with the increased levels of DBH enzyme. This finding suggests the possible increase of conver-sion of neurotransmitter dopamine to NA in the hip-pocampus of the animals exposed to CRS. In addition, data about NA transmission are very important for un-derstanding changes of hippocampal noradrenergic ca-pacity in the stress condition. According to the literature data, the monoamine hypothesis of mood disorders states that depression is caused by insufficient signalling by monoamines (Carlson, 1988). It is known that the dy-namics of NA transmission is regulated by reuptake through NET (Blakely and Bauman, 2000). In this study, we found that CRS significantly decreased the protein levels of NET. It is possible that the reduced functional deficiency of NA is an adaptation to the prolonged exposure to stress. In addition, an important result in this work is that CRS increased the protein levels of VMAT2. We recorded a significant positive correlation between the levels of VMAT2 and NA in the hippocam-pus of chronically stressed rats. It is known that VMAT play a critical role not only in sorting, storage and re-lease of monoamines, but also in the fine-tuning of neu-ronal informational output (Eiden, 2000). Repeated stress increased expression of VMAT2 levels in many NA-synthesizing cells, indicating an adaptation to the prolonged exposure to a strong stressor, which could provide a mechanism for facilitating utilization of the enhanced noradrenergic capacity (Tillinger et al., 2010). In this study we also found that CRS significantly in-creased the protein levels of BDNF. The increased pro-tein levels of BDNF could restore neurohormone stocks highly solicited during chronic stress, thus playing a “protective” role during the stress (Nawa et al., 1993; Givalois et al., 2004a,b; Naert, 2006). We recorded that the increased concentrations of NA positively correlate with the protein levels of BDNF in the hippocampus of animals exposed to CRS. Our results confirm that the increased level of BDNF may play an important role in the adaptive physiological processes required by neu-rons to maintain the noradrenergic capacity in the condi-tions provoked by chronic stress. The molecular mecha-nisms by which CRS increases the hippocampal NA level represent an important adaptive phenomenon of the noradrenergic system in the stress condition. Our re-sults may be important in the research of numerous psy-chiatric diseases caused by chronic stress.Conflict of interestThe authors report no conflict of interest. ReferencesArnsten, A. F. T., Goldman-Rakic, P. S. (1985) Α-2 adrenergic mechanisms in prefrontal cortex associated with cognitive decline in aged nonhuman primates. Science 230, 1273-1276. Blakely, R. D., Bauman, A. (2000) Biogenic amine transport-ers: regulation in flux. Curr. Opin. Neurobiol. 10, 328-336.Carlson, N. R. (1988) Foundations of Physiological Psychol-ogy, Boston, Allyn and Bacon, Inc.Eichenbaum, H. (2004) Hippocampus: cognitive processes and neural representations that underlie declarative memo-ry. Neuron 44, 109.Eiden, L. E. 2000 The vesicular neurotransmitter transporters: current perspectives and future prospects. FASEB J. 14, 2396-2400. Fig. 2. Effects of chronic restraint stress (CRS) on the con-centrations of noradrenaline (NA) in the hippocampus Data are shown as means ± S.E.M. of 10 rats. Statistical significance: **P < 0.01, CRS animals compared to control animals (t-test)84 Vol. 66Esch, T., Stefano, G. B., Fricchione, G. L., Benson, H. 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Changes of Hippocampal Noradrenergic Capacity in Stress Condition

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Changes of Hippocampal Noradrenergic Capacity in Stress Condition

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