Angiotensinergic receptors in the medial amygdaloid nucleus differently modulate behavioral responses in the elevated plus-maze and forced swimming test in rats

Journal Pre-proof Angiotensinergic receptors in the medial amygdaloid nucleus differently modulate behavioral responses in the elevated plus-maze and forced swimming test in rats Beatriz Moreno-Santos (Conceptualization) (Formal analysis) (Investigation) (Methodology) (Visualization) (Writing - original draft), Camila Marchi-Coelho (Conceptualization) (Formal analysis) (Investigation) (Methodology) (Visualization) (Writing - original draft), Willian Costa-FerreiraConceptualization Formal analysis) (Investigation) (Methodology) (Visualization) (Writing - original draft), Carlos C. Crestani (Conceptualization) (Data curation) (Funding acquisition) (Methodology) (Project administration) (Resources)

PII:

S0166-4328(20)30646-X

DOI:

https://doi.org/10.1016/j.bbr.2020.112947

Reference:

BBR 112947

To appear in:

Behavioural Brain Research

Received Date:

22 May 2020

Revised Date:

1 September 2020

Accepted Date:

26 September 2020

Please cite this article as: Moreno-Santos B, Marchi-Coelho C, Costa-Ferreira W, Crestani CC, Angiotensinergic receptors in the medial amygdaloid nucleus differently modulate behavioral responses in the elevated plus-maze and forced swimming test in rats, Behavioural Brain Research (2020), doi: https://doi.org/10.1016/j.bbr.2020.112947

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Angiotensinergic receptors in the medial amygdaloid nucleus differently modulate behavioral responses in the elevated plus-maze and forced swimming test in rats Beatriz Moreno-Santos1#; Camila Marchi-Coelho1#; Willian Costa-Ferreira1,2#; Carlos C. Crestani1,2*[email protected] School of Pharmaceutical Sciences, São Paulo State University (UNESP), Araraquara, SP, Brazil

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Joint UFSCar-UNESP Graduate Program in Physiological Sciences, São Carlos, SP, Brazil

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* Correspondence to: Dr. Carlos C. Crestani, Laboratory of Pharmacology, School of Pharmaceutical Sciences, São Paulo State University - UNESP, RodoviaAraraquara-Jaú Km 01 (Campus Universitário), Campus Ville, 14800-903, Araraquara, SP, Brazil.

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Phone: +55 16 3301-6982 Fax: + 55 16 3301-6980

These authors contributed equally to this article.

ABSTRACT

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E-mail address:

The brain renin-angiotensin system (RAS) has been implicated in anxiety and depression

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disorders, but the specific brain sites involved are poorly understood. The medial amygdaloid

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nucleus (MeA) is involved in expression of behavioral responses. However, despite evidence of the presence of all angiotensinergic receptors in this amygdaloid nucleus, regulation of anxietyand depressive-like behaviors by angiotensinergic neurotransmissions within the MeA has never been reported. Thus, the present study aimed to investigate the role angiotensin II (AT1 and AT2 receptors) and angiotensin-(1-7) (Mas receptor) receptors present within the MeA in behavioral responses in the elevated plus-maze (EPM) and forced swimming test (FST). For this, male Wistar rats had cannula-guide bilaterally implanted into the MeA, and independent sets of

animals received bilateral microinjections of either the selective AT1 receptor antagonist losartan, the selective AT2 receptor antagonist PD123319, the selective Mas receptor antagonist A-779 or vehicle into the MeA before the EPM and FST. Treatment of the MeA with either PD123319 or A-779 decreased the EPM open arms exploration, while losartan did not affect behavioral responses in this apparatus. However, intra-MeA microinjection of losartan decreased immobility in the FST. Administration of either PD123319 or A-779 into the MeA did not affect the immobility during the FST, but changed the pattern of the active behaviors swimming and

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climbing. Altogether, these results indicate the presence of different angiotensinergic mechanisms within the MeA controlling behavioral responses in the FST and EPM.

Keywords: amygdala; angiotensin; anxiety; depression; rodents; stress.

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Abbreviations

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ACE, angiotensin converting enzyme; anxiety; depression; EPM, elevated plus-maze; FST, forced swimming test; MeA, medial amygdaloid nucleus; RAS, renin-angiotensin system.

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1. INTRODUCTION

The brain renin-angiotensin system (RAS) has been reported to play a role in behavioral

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responses during aversive threats [1–3]. Indeed, accumulating studies in rodents have demonstrated anxiolytic- and antidepressant-like effects following treatment with AT1 receptor antagonists [4–12]. Accordingly, case reports and observational studies in humans indicated

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reduced anxiety and depression in individuals treated with angiotensin converting enzyme

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(ACE)/angiotensin II/AT1 receptors pathway inhibitors [1,2,13]. Preclinical studies have also consistently demonstrated anxiolytic and antidepressant roles of the angiotensin ACE2/angiotensin-(1-7)/Mas receptor pathway [14–20]. Control of anxiety-like behavior by AT2 receptor is less clear, since although antagonism of this receptor inhibited the anxiogenic effect of angiotensin II [21], chronic i.c.v. administration of an AT2 receptor agonist evoked anxiolyticlike effects [22] and AT2 receptor-deficient mice presented increased anxiety-like behaviors [23]. A role of AT2 receptor in control of depressive-like behavior has also been reported recently [12].

Based on these pieces of evidence, the therapeutic use of RAS acting drugs in the treatment of stress-related diseases has been discussed [2,3,24]. However, the specific sites in the brain related to the behavioral control by angiotensinergic receptors are poorly understood. The amygdala is a limbic subcortical structure that plays a prominent role in control of physiological and behavioral responses during aversive situations [25–27]. It is subdivided into several nuclei, and the medial amygdaloid nucleus (MeA) has been reported as a major amygdaloid sub-nucleus activated by aversive stimuli [25], including the elevated plus-maze

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(EPM) and forced swimming test (FST) [28–32]. Decrease in immobility in the FST evoked by either systemic administration of antidepressant drugs or estradiol microinjected intra-MeA of ovariectomized rats was followed by decreased MeA neuronal activation [32,33]. A role of MeA in depressive-like behaviors was further supported by evidence that local MeA treatment with

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either tricyclic antidepressants, V1b receptor antagonist or minaprine, as well as estradiol in

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ovariectomized rats decreased immobility in the FST [33–35]. Besides, MeA catecholaminergic lesion inhibited the antidepressant-like effect evoked by systemic injection of tricyclic

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antidepressants and electroconvulsive shock [36,37]. A role of MeA in behavioral responses in the EPM was evidenced by demonstration that intra-MeA administration of either nitric oxide

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synthase inhibitors or neuropeptide S, as well as MeA activation and sustained neuropeptide S overexpression increased EPM open arm exploration [38–41]. Conversely, microinjection into

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the MeA of substance P, agonists of either NK1 or melanocortin-4 receptors or BDNF antisense oligodeoxynucleotide evoked anxiogenic-like effect in the EPM [42–46].

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Angiotensin-immunoreactive terminals, angiotensin II binding sites and expression of all

angiotensin receptors were identified within the MeA [47–50]. Besides, the MeA seems to have the highest level of angiotensinogen within the amygdaloid complex [51]. However, it remains to be addressed the involvement of angiotensinergic receptors present within the MeA in control of anxiety- and depressive-like behaviors. Thus, in the present study we investigated the role of AT1, AT2 and Mas receptors within the MeA in behavioral responses in the EPM and FST.

2. MATERIAL AND METHODS 2.1. Animals Eighty male Wistar rats (60-day-old, weighing 240-260g) were used in this study. The animals were obtained from the animal breeding facility of the São Paulo State UniversityUNESP (Botucatu, SP, Brazil) and were housed in collective plastic cages (4 rats/cage). The animals remained in a temperature-controlled room at 24°C with light-dark cycle 12:12 h (lights

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on between 7:00 a.m. and 7:00 p.m.) with free access to water and standard laboratory food in the Animal Facility of the Laboratory of Pharmacology-UNESP (Araraquara, SP, Brazil). The procedures and protocols of this study were approved by Local Ethical Committee for Use of Animals (approval # 12/2018), which complies with Brazilian and international guidelines for

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animal use and welfare.

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2.2. Surgery procedure

Five days before the experiment, the rats were subjected to inhalation anesthesia with

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isoflurane (2.0%) using a low-flow anesthesia system (Bonther, Ribeirão Preto, SP, Brazil). Then, after scalp anesthesia with 2% lidocaine, the skull was surgically exposed and stainless-

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steel guide cannulas (26 G, 15 mm long) directed to the MeA were bilaterally implanted using a stereotaxic apparatus (Stoelting, Wood Dale, IL, USA). Stereotaxic coordinates for cannula

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implantation in the MeA were chosen based on the rat brain atlas of Paxinos and Watson (1997): antero-posterior: +5.6 mm from interaural, lateral: +3.4 mm from the medial suture, ventral: −8.2

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mm from the skull. The incisor bar position was set at −3.2 mm. Dental cement was used to fix cannulas to the skull. After the surgery, the rats were treated with a poly-antibiotic formulation containing streptomycins and penicillins for prevent infection (560 mg/mL/kg, i.m.) and the nonsteroidal anti-inflammatory drug flunixin meglumine for post-operation analgesia (0.5 mg/mL/kg, s.c.). 2.3. Drug microinjection into the brain

Microinjections were performed into the brain using a 2-μL syringe (7002KH, Hamilton, USA), which was connected to the microinjection needle (33 G, Small Parts, USA) via a PE-10 tubing. The needle used for microinjection was 1 mm longer than the guide cannulas. The needles were carefully inserted into the guide cannula without restraining the animals, and drugs were injected in a final volume of 100 nL/side [53]. To avoid reflux, the needle was left in the guide cannula for 40 s after the microinjection before being removed. 2.4. Behavioral tests

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All behavioral tests were performed during the morning period in order to avoid circadian influence. 2.4.1. Elevated plus-maze (EPM)

The EPM test was used to assess anxiety-like behaviors [27,54,55]. The apparatus used

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for the test consisted of two open arms and two closed arms [(50 cm (length) x 10 cm (width) x

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0.25 cm (height)] joined perpendicularly and raised 50 cm from the ground. The closed arms were enclosed by 40cm high walls with no roof, and the open arms had a side border of 1 cm

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acrylic to prevent the animals from falling out the apparatus. The arms were connected by a common central platform [10 cm (length) x 10 cm (width)]. Rats were individually placed in the

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center of the apparatus and were allowed to explore freely the EPM for 5 minutes. The apparatus was cleaned with an alcohol solution (20%) before each session to prevent subsequent rat from

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being influenced by odors deposited by the previous one. The behavior was recorded by a camera connected to a microcomputer (Microsoft lifecam cinema HD), and the behaviors were analyzed

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using the behavioral tracking software ANY-maze (Stoelting, Wood Dale, IL, USA). Rodents avoid open arms and anxiolytic drugs usually increase the exploration of the

open arms, without interfering with the number of entries in the closed arms [54,55]. Therefore, behavioral measures were frequency of closed-arm entries (CE), percentage of open-arm entries (%OE), percentage of open-arm time (%OT) and total distance traveled [56–58]. 2.4.2. Forced swimming test (FST)

The FST was used to assess behavioral despair (depressive-like behavior) [27,59]. The test consisted of two sessions: pretest and test. In the pretest session animals were placed individually into a plastic cylinder (30 cm diameter by 40 cm height) filled with 30 cm of water at 24±1°C for 15 min. After the session, animals were gently wiped with a towel and returned to their home boxes. Twenty-four hours after the pretest session, the animals were re-exposed to the water-filled cylinder for 5 min (test session). The water was changed between animals in both sessions to prevent influence of the previous rat. The test session was recorded by a camera

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connected to a microcomputer (Microsoft lifecam cinema HD). All classical antidepressant drugs decrease immobility time in the test session [59]. Therefore, the total immobility (lack of movement with exception of that necessary to keep the head above the water surface) time and the latency to the first bout of immobility were analyzed

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during the test session. Additionally, total time of the active behaviors climbing (vertical

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movement of the forepaws directed towards the sides of the chamber), swimming (horizontal movement throughout the chamber) and diving (when the entire body was submerged) were also

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evaluated [60,61]. All behaviors were analyzed in a blinded manner using the behavioral analysis software X-Plo-Rat (the software can be freely downloaded at http://scotty.ffclrp.usp.br/X-Plo-

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Rat.html) [57,62,63]. 2.5. Experimental design

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The rats were brought to the experimental room in their own cage. The experimental room was temperature controlled (24°C) and acoustically isolated from the other rooms. Animals

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were given at least 60 min to adapt to the experimental room conditions, such as sound and illumination, before starting the experiment. 2.5.1. Effect of MeA pharmacological treatments with the angiotensinergic receptor antagonists on behavioral responses in the elevated plus-maze (EPM) Independent sets of animals received bilateral microinjection into the MeA of the selective AT1 receptor antagonist losartan (1nmol/100nL, n=10), the selective Mas receptor

antagonist A-779 (0.1nmol/100nL, n=10), the selective AT2 receptor antagonist PD123319 (5nmol/100nL, n=10) or vehicle (saline, 100nL, n=10). Ten minutes after the MeA treatment, animals were exposed to the EPM for 5 min. The dose of the antagonists was based on previous studies from our lab [53]. 2.5.2. Effect of MeA pharmacological treatments with the angiotensinergic receptor antagonists on behavioral responses in the forced swimming test (FST) Independent sets of animals received bilateral microinjection into the MeA of the

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selective AT1 receptor antagonist losartan (1nmol/100nL, n=10), the selective Mas receptor antagonist A-779 (0.1nmol/100nL, n=10), the selective AT2 receptor antagonist PD123319 (5nmol/100nL, n=10) or vehicle (saline, 100nL, n=10) [53]. Ten min after the MeA treatment, animals were subjected to the first session of the FST (i.e, pretest session), which lasted 15 min.

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Twenty-four h later, animals were exposed to the test session of the FST, which lasted 5 min.

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The treatment protocol was based on previous evidence that intra-brain pharmacological

the test session [62,64,65].

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treatment before the FST pretest session was effective in affecting behavioral responses during

2.6. Histological determination of the microinjection sites

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At the end of the experiments, animals were anesthetized with urethane (250 mg/mL/200 g body weight, i.p.) and 100 nL of 10% Evan’s blue dye was microinjected into the

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brain as a marker of the microinjection sites. They were then perfuse-fixed with intracardiac 0.9% NaCl followed by 10% formalin. Afterwards, the brains were removed and post-fixed in 10%

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formalin for at least 48h at 4°C. Then, serial 40 μm thick sections of the MeA region were cut with a cryostat (CM1900, Leica, Wetzlar, Germany). The actual placement of the microinjection needles was determined upon analysis of serial sections in a light microscopy according to the rat brain atlas of Paxinos and Watson [52]. 2.7. Drugs and solutions

Losartan potassium (selective AT1 receptor antagonist) (Tocris, Westwoods Business Park, Ellisville, MO, USA), A-779 (selective MAS receptor antagonist) (Tocris), PD123319 ditrifluoroacetate (selective AT2 receptor antagonist) (Tocris) and urethane (Sigma-Aldrich) were dissolved in saline (0.9% NaCl). Flunexin meglumine (Banamine®; Schering-Plough, Cotia, SP, Brazil), the poly-antibiotic preparation (Pentabiotico®; Fort Dodge, Campinas, SP, Brazil) and isoflurane (Isoforine®; Cristália, Itapira, SP, Brazil) were used as provided. 2.8. Data analysis

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Data were expressed as mean ± SEM. All data were analyzed using the software GraphPad Prism version 7.0 (GraphPad Software Inc., La Jolla, CA, USA). All behavioral parameters evaluated were analyzed using the one-way ANOVA. When statistical differences were identified by ANOVA, the Bonferroni post-hoc test was used to assess specific differences

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between the experimental groups. P<0.05 was assumed as significant.

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3. RESULTS

Photomicrograph of a coronal brain section depicting bilateral microinjection sites into

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the MeA of a representative animal is presented in the Fig. 1. Fig. 1 also shows diagrammatic representations showing the bilateral microinjection sites of losartan, A-779, PD123319 and

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vehicle into the MeA.

3.1. Effects of MeA pharmacological treatment with angiotensinergic receptor antagonists

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on behavioral responses in the elevated plus maze (EPM) Assessment of anxiety-like behavior in the EPM indicated effect of MeA

pharmacological treatments in percentage of time (F(3,36)=7.67, P=0.0004) and entries (F(3,36)=12.11, P<0.0001) in the open arms (Fig. 2). Post-hoc analysis revealed that bilateral microinjection into the MeA of either the selective AT2 receptor antagonist PD123319 (n=10) (time: P=0.0167, entries: P<0.0001) or the selective Mas receptor antagonist A-779 (n=10) (time: P=0.0438, entries: P=0.0264) decreased the time spent and entries in the open arms, when

compared with the vehicle group (n=10). The selective AT1 receptor antagonist losartan (n=10) did not affect open arms exploration (time: P>0.9999, entries: P>0.9999) (Fig. 2). Analysis did not indicate effect of the pharmacological treatments on number of entries in the closed arms (F(3,36)= 0.36; P=0.7804) and total distance traveled during the test (F(3,36)= 1.32; P=0.2892) (Fig. 2). 3.2. Effects of MeA pharmacological treatment with angiotensinergic receptor antagonists on behavioral responses in the forced swimming test (FST)

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Analysis indicated effect of MeA pharmacological treatments in the immobility time (F(3,36)=6.07, P=0.0019), latency to the first bout of immobility (F(3,36)=5.28, P=0.0040), swimming (F(3,36)=6.23, P=0.0016) and climbing (F(3,36)=6.12, P=0.0019) behaviors in the FST; but without affecting the diving (F(3,36)=2.33, P=0.090) (Fig. 3). Post-hoc analysis revealed that

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bilateral microinjection of the AT1 receptor antagonist losartan (n=10) into the MeA decreased

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the immobility time (P=0.0014) and increased the latency to the first immobility (P=0.0245), while MeA treatment with either the AT2 receptor antagonist PD123319 (n=10) (immobility

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time: P=0.1318; latency: P=0.6618) or the Mas receptor antagonist A-779 (n=10) (immobility time: P>0.9999; latency: P>0.9999) did not change these behaviors in the FST, when compared

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with the vehicle group (n=10) (Fig. 3). A tendency of increase in the climbing behavior (P=0.0585) without changing swimming (P>0.9999) was also observed in animals treated with

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losartan in the MeA (Fig. 3). Bilateral microinjection of either PD123319 (P=0.0021) or A-779 (P=0.01189) into the MeA increased the climbing behavior (Fig. 3). Besides, A-779 (P=0.0025),

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but not PD123319 (P=0.1837), decreased the swimming in the FST (Fig. 3).

4. DISCUSSION

The findings reported here provide further evidence of a role of MeA in central network controlling anxiety- and depressive-like behaviors. In this sense, our results indicate for the first time that control of these behavioral responses by MeA is mediated by local angiotensinergic

neurotransmissions. In this sense, a previous study reported greater MeA neuronal activation to an aversive stimulus in AT1 receptor-deficient mice in relation to wild animals [66], thus suggesting that AT1 receptor activation during aversive situations plays an inhibitory role in MeA neurons. Taken together with the present results, these previous findings indicated that the antidepressant-like effect observed in the present study in losartan-treated animals is possibly related to a greater MeA activation. This idea is in opposition to evidence that antidepressantlike effect in the FST evoked by systemic treatment with antidepressant drugs was followed by

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decreased MeA neuronal activation [32]. In this sense, a recent study provided evidence that antidepressant-like effect of losartan might be mediated by mechanisms beyond AT1 receptor blockade in the brain [12]. However, such as observed in the present report, previous studies have documented antidepressant-like effects following treatment with other AT1 receptor

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antagonists (e.g., valsartan, telmisartan and irbesartan) [67–69], and AT1 receptor polimorphisms

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have been linked to depression [70,71]. Therefore, we expect that effects reported in the present study were mediated by blockade of AT1 receptors within the MeA.

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Regarding the role of AT2 and Mas receptor in anxiety-like behavior, present study indicates that activation of these receptors within the MeA during EPM facilitates the open arm

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exploration. A recent study demonstrated anxiolytic-like effect in the EPM following activation of specific neuronal population within the MeA [39]. Therefore, the decreased EPM open arm

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exploration following treatment of the MeA with either PD123319 or A-779 might be mediated by a reduced MeA neuronal activation. As stated above, previous studies demonstrated greater

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stress-evoked MeA neuronal activation in AT1 receptor deficient mice [66]. Therefore, the idea that the effects of AT2 and Mas receptor antagonists are mediated by a decreased local neuronal activation is in line with the idea that these receptors are part of RAS contra-regulatory mechanisms to AT1 receptor-mediated effects [72,73]. Our findings are in line with previous evidence of anxiolytic role of brain ACE/angiotenin II/AT2 receptor and ACE2/angiotensin-(1-7)/Mas receptor pathways. The role

of the latter was evidenced by demonstration of increased anxiety-like behavior in Mas receptordeficient mice [17] as well as by evidence that i.c.v. administration of angiotensin-(1-7) reversed the anxiety-like state in the EPM of transgenic rats with low brain angiotensinogen and in transgenic (mRen2)27 hypertensive rats [14,15]. Besides, overexpression of either angiotensin(1-7) or ACE2 reduced anxiety-like behavior [19,20,74]. Regarding the AT2 receptor, chronic i.c.v. administration of a agonist of this receptor increased EPM open arms exploration [22], whereas AT2 receptor-deficient mice presented increased anxiety-like behaviors [23]. In this

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sense, our results provide the first evidence of the MeA as a brain site whereby AT2 and Mas receptor are activated to evoke anxiolytic-like effects. Regarding the AT1 receptor, present findings indicate that the well-stablished anxiolytic-like effects observed following systemic and i.c.v. administration of AT1 receptor antagonists seem to be mediated by brain sites other than

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MeA. In this sense, previous studies demonstrated anxiolytic-like effects after microinjection of

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AT1 receptor antagonists into the hippocampus, central amygdaloid nucleus and ventrolateral periaqueductal gray [75–77].

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The results of the present study indicating antidepressant-like effect in animals treated with losartan into the MeA are supported by previous evidence that systemic administration of

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AT1 receptor antagonists decreased immobility in the FST and inhibited chronic-stress evoked depression [9,11,12,68]. In this sense, the present study provides the first evidence of the MeA

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as a brain site whereby AT1 receptor antagonists act to evoke antidepressant-like effects. The absence of effect of MeA treatment with PD123319 is in line with previous evidence that AT2-

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receptor deficient mice did not present behavioral changes in the FST [23], thus indicating that AT2 receptor does not tonically control the behavior in this test. Regarding the Mas receptor, although report that intra-brain angiotensin-(1-7) administration reversed the depressive-like behavior in the FST of transgenic rats with low brain angiotensinogen and in transgenic (mRen2)27 hypertensive rats [14,15], changes in depressive-like behaviors following

antagonism of Mas receptor in depressive-like behavior in wild naïve animals has never been reported. The different roles of AT1 vs AT2 receptors in behavioral responses in EPM and FST is in line with evidence that these angiotensinergic receptors are not co-expressed in same neurons [49,72,78]. Indeed, a recent study identified little colocalization of the angiotensin II receptors in hypothalamic and brainstem structures [48]. Although colocalization of AT1 and AT2 receptors within the MeA has never been evaluated, the above-mentioned evidence together with results

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reported in the present study indicate that different neuronal populations within the MeA are involved in control of anxiety- and depressive-like behaviors. In this sense, MeA AT1 receptors seem to mediate the expression of depressive-like behaviors, whereas AT2 receptor activation presents anxiolytic-like effects.

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ACE/angiotensin II/AT2 receptor and ACE-2/angiotensin-(1-7)/Mas receptor pathways

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are described as protective RAS, which counteract the pro-stress effects of angiotensin II/AT1 receptor [72,73]. Indeed, accumulating evidence have demonstrated that AT2 and Mas receptor

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activation attenuates physiological and behavioral responses to stress [73,78]. In this sense, the present study provides the first evidence of the MeA as a site whereby AT2 and Mas receptors

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are activated to inhibit the expression of anxiety-like behaviors. Interaction between AT2 and Mas receptors has been reported. For instance, a recent study demonstrated that AT2 and Mas

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receptors may form heterodimers [79]. A direct interaction was further supported by evidence that effects of AT2 receptor activation were inhibited by the Mas receptor antagonist A-779 and,

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conversely, responses following Mas receptor activation were blocked by AT2 receptor antagonism [80,81]. Therefore, the effects of AT2 or Mas receptors in the present study might be mediated by an interaction between these receptors within the MeA. However, a number of studies have demonstrated effects of Mas and AT2 receptor activation independent of a direct interaction [81]. Anyway, the findings reported in the present study are in line with evidence of

cooperative role of AT2 and Mas receptors [72]. In this sense, present study provides the first evidence of this cooperation in control of anxiety-like behavior. Interestingly, although MeA treatment with the AT2 and Mas receptor antagonists did not affect the immobility in the FST, these treatments changed the pattern of active behaviors by increasing climbing and decreasing swimming. Despite not being related to depressive-like behavior (immobility was not affected), these findings are supported by previous evidence demonstrating that active behaviors were more sensitive to antidepressant drugs than immobility

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[60,61]. Previous pharmacological studies investigating effects of antidepressants drugs provided evidence that these active behaviors seem to be mediated by specific brain monoaminergic circuits [61,82,83]. In this sense, selective serotonin reuptake inhibitors (SSRIs) selectively increased swimming, whereas selective noradrenaline uptake inhibitors acted primarily

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increasing climbing [60,84–87]. Diving rarely occurs during the test and is not affected by

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antidepressant drugs [60], so that it is not usually explored. Taken together with these previous reports, our results indicate that AT2 and Mas receptors within the MeA seem to stimulate

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facilitatory inputs to serotonergic circuits and/or inhibit facilitatory drive to noradrenergic pathways involved in active behaviors in the FST. Additionally, the behavioral control in the

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FST by these angiotensinergic receptors might be mediated by facilitation of local serotoninergic neurotransmission and/or inhibition of noradrenergic mechanisms within the MeA. Accordingly,

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previous studies reported a role of MeA catecholaminergic neurotransmission in antidepressantlike effect of tricyclic antidepressants and electroconvulsive shock [36,37]. This idea is further

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supported by evidence that the control of climbing was demonstrated to be selectively mediated by ventral noradrenergic bundle (without influence of dorsal noradrenergic bundle) [88], which is the main source of noradrenergic inputs to the amygdala [89,90]. Interaction with serotoninergic mechanisms is supported by demonstration that antidepressant-like effect of the SSRI fluoxetine in the FST was followed by decreased MeA neuronal activation [32]. It is also worth noting that losartan treatment decreased immobility with correspondent increase in

climbing, thus indicating that this antidepressant-like effect is possibly mediated by modulation of brain noradrenergic mechanisms. In summary, the results reported here provide evidence of different angiotensinergic mechanisms within the MeA controlling behavioral responses in the FST and EPM. Indeed, the present findings indicate that local AT2 and Mas receptors within the MeA inhibit the expression of anxiety-like behaviors in the EPM. Conversely, our results indicate that AT1 receptor in the MeA is involved in behavioral despair in the FST. Finally, present findings indicate that MeA

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AT2 and Mas receptors control the pattern of active behaviors in the FST.

AUTHOR CONTRIBUTIONS

Beatriz Moreno-Santos: Conceptualization; Formal analysis; Investigation; Methodology;

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Visualization; Roles/Writing - original draft. Camila Marchi-Coelho: Conceptualization;

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Formal analysis; Investigation; Methodology; Visualization; Roles/Writing - original draft. Willian Costa-Ferreira: Conceptualization; Formal analysis; Investigation; Methodology;

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Visualization; Roles/Writing - original draft. Carlos C. Crestani: Conceptualization; Data curation; Funding acquisition; Methodology; Project administration; Resources; Visualization;

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Roles/Writing - original draft; Writing - review & editing. DECLARATIONS OF INTEREST

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None.

ACKNOWLEDGMENTS

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This work was supported by FAPESP (grants # 2017/19249-0), CNPq (grant #

456405/2014-3 and and 431339/2018-0) and Scientific Support and Development Program of School of Pharmaceutical Sciences (UNESP). WCF is a FAPESP PhD fellow (process # 2016/05218-2) and CMC is a FAPESP undergraduate fellow (process # 2018/23686-9). CCC is a CNPq research fellow (process # 305583/2015-8 and 304108/2018-9).

REFERENCES [1]

A. Bali, A.S. Jaggi, Angiotensin as stress mediator: role of its receptor and interrelationships among other stress mediators and receptors., Pharmacol. Res. 76 (2013) 49–57. https://doi.org/10.1016/j.phrs.2013.07.004.

[2]

J.M. Saavedra, Beneficial effects of Angiotensin II receptor blockers in brain disorders, Pharmacol. Res. 125 (2017) 91–103. https://doi.org/10.1016/j.phrs.2017.06.017. L.A. de Melo, A.F. Almeida-Santos, Neuropsychiatric Properties of the ACE2/Ang-(17)/Mas

Pathway:

A

Brief

Review.,

Protein

https://doi.org/10.2174/0929866527666191223143230. [4]

Pept.

Lett.

27

(2019).

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[3]

F.C. Kaiser, G.C. Palmer, A. V. Wallace, R.D. Carr, L. Fraser-Rae, C. Hallam, Antianxiety properties of the angiotensin II antagonist, DUP 753, in the rat using the elevated plus-

-p

maze, Neuroreport. 3 (1992) 922–924. https://doi.org/10.1097/00001756-199210000-

[5]

re

00026.

P.R. Gard, S.J. Haigh, P.T. Cambursano, C.A. Warrington, Strain differences in the

lP

anxiolytic effects of losartan in the mouse, Pharmacol. Biochem. Behav. 69 (2001) 35–40. https://doi.org/10.1016/S0091-3057(01)00491-9. J. Srinivasan, B. Suresh, M. Ramanathan, Differential anxiolytic effect of enalapril and

na

[6]

losartan in normotensive and renal hypertensive rats, Physiol. Behav. 78 (2003) 585–591.

[7]

ur

https://doi.org/10.1016/S0031-9384(03)00036-2. J. Pavel, J. Benicky, Y. Murakami, E. Sanchez-Lemus, J.M. Saavedra, Peripherally

Jo

administered angiotensin II AT1 receptor antagonists are anti-stress compounds in vivo, in: Ann. N. Y. Acad. Sci., 2008. https://doi.org/10.1196/annals.1410.006.

[8]

A. Ciobica, V. Bild, L. Hritcu, M. Padurariu, W. Bild, Effects of angiotensin II receptor antagonists on anxiety and some oxidative stress markers in rat, Open Med. 6 (2011) 331– 340. https://doi.org/10.2478/s11536-011-0010-8.

[9]

P. VIJAYAPANDI, A.N. NAGAPPA, Biphasic Effects of Losartan Potassium on Immobility

in

Mice,

YAKUGAKU

ZASSHI.

125

(2005)

653–657.

https://doi.org/10.1248/yakushi.125.653. [10]

J.M. Saavedra, I. Armando, C. Bregonzio, A. Juorio, M. Macova, J. Pavel, E. SanchezLemus, A Centrally Acting, Anxiolytic Angiotensin II AT1 Receptor Antagonist Prevents the Isolation Stress-Induced Decrease in Cortical CRF1 Receptor and Benzodiazepine Binding,

Neuropsychopharmacology.

54

(2006)

271–81.

https://doi.org/10.1038/sj.npp.1300921. [11]

P.R. Gard, A. Mandy, M.A. Sutcliffe, Evidence of a possible role of altered angiotensin

ro of

function in the treatment, but not etiology, of depression., Biol. Psychiatry. 45 (1999) 1030–4. [12]

C.R.A.F. Diniz, P.C. Casarotto, S.M. Fred, C. Biojone, E. Castrén, S.R.L. Joca, Antidepressant-like effect of losartan involves TRKB transactivation from angiotensin

-p

receptor type 2 (AGTR2) and recruitment of FYN, Neuropharmacology. 135 (2018) 163–

[13]

re

171. https://doi.org/10.1016/j.neuropharm.2018.03.011.

J. Vian, C. Pereira, V. Chavarria, C. Köhler, B. Stubbs, J. Quevedo, S.-W. Kim, A.F.

lP

Carvalho, M. Berk, B.S. Fernandes, The renin–angiotensin system: a possible new target for depression, BMC Med. 15 (2017) 144. https://doi.org/10.1186/s12916-017-0916-3. L.M. Kangussu, A.F. Almeida-Santos, M. Bader, N. Alenina, M.A.Ô.P. Fontes, R.A.S.

na

[14]

Santos, D.C. Aguiar, M.J. Campagnole-Santos, Angiotensin-(1-7) attenuates the anxiety

ur

and depression-like behaviors in transgenic rats with low brain angiotensinogen, Behav. Brain Res. (2013). https://doi.org/10.1016/j.bbr.2013.09.003. A.F. Almeida-Santos, L.M. Kangussu, F.A. Moreira, R.A.S. Santos, D.C. Aguiar, M.J.

Jo

[15]

Campagnole-Santos, Anxiolytic- and antidepressant-like effects of angiotensin-(1-7) in hypertensive transgenic (mRen2)27 rats, Clin. Sci. 130 (2016) 1247–1255. https://doi.org/10.1042/CS20160116.

[16]

W. Bild, A. Ciobica, Angiotensin-(1-7) central administration induces anxiolytic-like effects in elevated plus maze and decreased oxidative stress in the amygdala., J. Affect.

Disord. 145 (2013) 165–71. https://doi.org/10.1016/j.jad.2012.07.024. [17]

T. Walther, D. Balschun, J.-P. Voigt, H. Fink, W. Zuschratter, C. Birchmeier, D. Ganten, M. Bader, Sustained Long Term Potentiation and Anxiety in Mice Lacking the Mas Protooncogene,

J.

Biol.

Chem.

273

(1998)

11867–11873.

https://doi.org/10.1074/jbc.273.19.11867. [18]

X. Qi, L. Guzhva, Z. Yang, M. Febo, Z. Shan, K.K.W. Wang, A.W. Bruijnzeel, Overexpression of CRF in the BNST diminishes dysphoria but not anxiety-like behavior

ro of

in nicotine withdrawing rats, Eur. Neuropsychopharmacol. 26 (2016) 1378–1389. https://doi.org/10.1016/j.euroneuro.2016.07.007. [19]

L.M. Kangussu, A.F. Almeida-Santos, F.A. Moreira, M.A.P. Fontes, R.A.S. Santos, D.C. Aguiar, M.J. Campagnole-Santos, Reduced anxiety-like behavior in transgenic rats with

-p

chronically overproduction of angiotensin-(1–7): Role of the Mas receptor, Behav. Brain

[20]

re

Res. 331 (2017) 193–198. https://doi.org/10.1016/j.bbr.2017.05.026. D. Moura Santos, F. Ribeiro Marins, M. Limborço-Filho, M.L. de Oliveira, D. Hamamoto,

lP

C.H. Xavier, F.A. Moreira, R.A.S. Santos, M.J. Campagnole-Santos, M.A. Peliky Fontes, Chronic overexpression of angiotensin-(1-7) in rats reduces cardiac reactivity to acute and

dampens

anxious

behavior.,

Stress.

20

(2017)

189–196.

na

stress

https://doi.org/10.1080/10253890.2017.1296949. J.J. BRASZKO, M.M.W. , A. KU£AKOWSKA, Effects of angiotensin II and its receptor

ur

[21]

antagonists on motor activity and anxiety in rats., J. Physiol. Pharmacol. 54 (2003) 271–

Jo

281.

[22]

D. Pechlivanova, K. Petrov, P. Grozdanov, Z. Nenchovska, J. Tchekalarova, A. Stoynev, Intracerebroventricular infusion of angiotensin AT2 receptor agonist novokinin aggravates some diabetes-mellitus-induced alterations in Wistar rats, Can. J. Physiol. Pharmacol. 96 (2018) 471–478. https://doi.org/10.1139/cjpp-2017-0428.

[23]

S. Okuyama, T. Sakagawa, S. Chaki, Y. Imagawa, T. Ichiki, T. Inagami, Anxiety-like

behavior in mice lacking the angiotensin II type-2 receptor., Brain Res. 821 (1999) 150– 9. [24]

M.M. Gironacci, A. Vicario, G. Cerezo, M.G. Silva, The depressor axis of the renin– angiotensin system and brain disorders: a translational approach, Clin. Sci. 132 (2018) 1021–1038. https://doi.org/10.1042/CS20180189.

[25]

P.J. Davern, G.A. Head, Role of the medial amygdala in mediating responses to aversive stimuli leading to hypertension., Clin. Exp. Pharmacol. Physiol. 38 (2011) 136–43.

[26]

G.G. Calhoon, K.M. Tye, Resolving the neural circuits of anxiety, Nat. Neurosci. 18 (2015) 1394–1404. https://doi.org/10.1038/nn.4101.

[27]

ro of

https://doi.org/10.1111/j.1440-1681.2010.05413.x.

J. Muir, J. Lopez, R.C. Bagot, Wiring the depressed brain: optogenetic and chemogenetic

-p

circuit interrogation in animal models of depression, Neuropsychopharmacology. 44

[28]

re

(2019) 1013–1026. https://doi.org/10.1038/s41386-018-0291-6.

C. Troakes, C.D. Ingram, Anxiety behaviour of the male rat on the elevated plus maze:

lP

associated regional increase in c-fos mRNA expression and modulation by early maternal separation, Stress. 12 (2009) 362–369. https://doi.org/10.1080/10253890802506391. C.V. Dayas, K.M. Buller, J.W. Crane, Y. Xu, T.A. Day, Stressor categorization: acute

na

[29]

physical and psychological stressors elicit distinctive recruitment patterns in the amygdala

ur

and in medullary noradrenergic cell groups, Eur. J. Neurosci. 14 (2001) 1143–1152. https://doi.org/10.1046/j.0953-816x.2001.01733.x. G.E. Duncan, D.J. Knapp, G.R. Breese, Neuroanatomical characterization of Fos

Jo

[30]

induction in rat behavioral models of anxiety, Brain Res. 713 (1996) 79–91. https://doi.org/10.1016/0006-8993(95)01486-1.

[31]

A. Fodor, B. Klausz, O. Pintér, N. Daviu, C. Rabasa, D. Rotllant, D. Balazsfi, K.B. Kovacs, R. Nadal, D. Zelena, Maternal neglect with reduced depressive-like behavior and blunted c-fos activation in Brattleboro mothers, the role of central vasopressin, Horm.

Behav. 62 (2012) 539–551. https://doi.org/10.1016/j.yhbeh.2012.09.003. [32]

M. Silva, D.C. Aguiar, C.R.A. Diniz, F.S. Guimarães, S.R.L. Joca, Neuronal NOS Inhibitor and Conventional Antidepressant Drugs Attenuate Stress-induced Fos Expression in Overlapping Brain Regions, Cell. Mol. Neurobiol. 32 (2012) 443–453. https://doi.org/10.1007/s10571-011-9775-1.

[33]

C.M. Estrada, V. Ghisays, E.T. Nguyen, J.L. Caldwell, J. Streicher, M.B. Solomon, Estrogen signaling in the medial amygdala decreases emotional stress responses and in

ovariectomized

rats,

Horm.

Behav.

https://doi.org/10.1016/j.yhbeh.2017.12.002. [34]

98

(2018)

33–44.

ro of

obesity

N. Salomé, J. Stemmelin, C. Cohen, G. Griebel, Differential roles of amygdaloid nuclei in the anxiolytic- and antidepressant-like effects of the V1b receptor antagonist, SSR149415,

-p

in rats, Psychopharmacology (Berl). 187 (2006) 237–244. https://doi.org/10.1007/s00213-

[35]

re

006-0424-1.

K. Kazuaki, A. Hiroaki, A. Hironaka, O. Susumu, Effects of Minaprine and Sulpiride

lP

Injected into the Amygdaloid Nucleus on the Duration of Immobility in Rats Forced to Swim, Jpn. J. Pharmacol. (1990). https://doi.org/10.1254/jjp.53.411. H. Araki, K. Kawashima, Y. Uchiyama, H. Aihara, Involvement of amygdaloid

na

[36]

catecholaminergic mechanism in suppressive effects of desipramine and imipramine on

ur

duration of immobility in rats forced to swim, Eur. J. Pharmacol. 113 (1985) 313–318. https://doi.org/10.1016/0014-2999(85)90078-0. Kazuaki Kawashima, Hiroaki Araki, Yoshimi Uchiyama, Hironaka Aihara, Amygdaloid

Jo

[37]

catecholaminergic mechanisms involved in suppressive effects of electroconvulsive shock on duration of immobility in rat forced to swim, Eur. J. Pharmacol. 141 (1987) 1–6. https://doi.org/10.1016/0014-2999(87)90404-3.

[38]

D. Forestiero, C.M. Manfrim, F.S. Guimarães, R.M.W. de Oliveira, Anxiolytic-like effects induced by nitric oxide synthase inhibitors microinjected into the medial amygdala of rats,

Psychopharmacology (Berl). 184 (2006) 166–172. https://doi.org/10.1007/s00213-0050270-6. [39]

D.A. Adekunbi, X.F. Li, G. Lass, K. Shetty, O.A. Adegoke, S.H. Yeo, W.H. Colledge, S.L. Lightman, K.T. O’Byrne, Kisspeptin neurones in the posterodorsal medial amygdala modulate sexual partner preference and anxiety in male mice, J. Neuroendocrinol. 30 (2018) e12572. https://doi.org/10.1111/jne.12572.

[40]

T. Grund, I.D. Neumann, Neuropeptide S Induces Acute Anxiolysis by Phospholipase C-

ro of

Dependent Signaling within the Medial Amygdala, Neuropsychopharmacology. 43 (2018) 1156–1163. https://doi.org/10.1038/npp.2017.169. [41]

S. Tillmann, H.E. Skibdal, S.H. Christiansen, C.R. Gøtzsche, M. Hassan, A.A. Mathé, G. Wegener, D.P.D. Woldbye, Sustained overexpression of neuropeptide S in the amygdala

[42]

re

https://doi.org/10.1016/j.bbr.2019.03.039.

-p

reduces anxiety-like behavior in rats, Behav. Brain Res. 367 (2019) 28–34.

K. Ebner, N.M. Rupniak, A. Saria, N. Singewald, Substance P in the medial amygdala:

lP

Emotional stress-sensitive release and modulation of anxiety-related behavior in rats, Proc. Natl. Acad. Sci. 101 (2004) 4280–4285. https://doi.org/10.1073/pnas.0400794101. G.S. Bassi, M.C. de Carvalho, M.L. Brandão, Effects of substance P and Sar-Met-SP, a

na

[43]

NK1 agonist, in distinct amygdaloid nuclei on anxiety-like behavior in rats, Neurosci. Lett.

[44]

ur

569 (2014) 121–125. https://doi.org/10.1016/j.neulet.2014.03.065. J. Liu, J.C. Garza, W. Li, X.-Y. Lu, Melanocortin-4 receptor in the medial amygdala

Jo

regulates emotional stress-induced anxiety-like behaviour, anorexia and corticosterone secretion,

Int.

J.

Neuropsychopharmacol.

16

(2013)

105–120.

https://doi.org/10.1017/S146114571100174X.

[45]

S.C. Pandey, H. Zhang, A. Roy, K. Misra, Central and Medial Amygdaloid Brain-Derived Neurotrophic Factor Signaling Plays a Critical Role in Alcohol-Drinking and AnxietyLike

Behaviors,

J.

Neurosci.

26

(2006)

8320–8331.

https://doi.org/10.1523/JNEUROSCI.4988-05.2006. [46]

G.S. Bassi, M.C. Carvalho, R.C. Almada, M.L. Brandão, Inhibition of substance Pinduced defensive behavior via neurokinin-1 receptor antagonism in the central and medial but not basolateral nuclei of the amygdala in male Wistar rats, Prog. NeuroPsychopharmacology

Biol.

Psychiatry.

77

(2017)

146–154.

https://doi.org/10.1016/j.pnpbp.2017.03.026. [47]

L.K. Becker, G.M. Etelvino, T. Walther, R.A.S. Santos, M.J. Campagnole-Santos,

ro of

Immunofluorescence localization of the receptor Mas in cardiovascular-related areas of the rat brain, Am. J. Physiol. Circ. Physiol. 293 (2007) H1416–H1424. https://doi.org/10.1152/ajpheart.00141.2007. [48]

A.D. de Kloet, L. Wang, J.A. Ludin, J.A. Smith, D.J. Pioquinto, H. Hiller, U.M.

-p

Steckelings, D.A. Scheuer, C. Sumners, E.G. Krause, Reporter mouse strain provides a

re

novel look at angiotensin type-2 receptor distribution in the central nervous system, Brain Struct. Funct. 221 (2016) 891–912. https://doi.org/10.1007/s00429-014-0943-1. Z. Lenkei, M. Palkovits, P. Corvol, C. Llorens-Cortès, Expression of Angiotensin Type-1

lP

[49]

(AT1) and Type-2 (AT2) Receptor mRNAs in the Adult Rat Brain: A Functional Review,

Front.

Neuroendocrinol.

18

(1997)

383–439.

na

Neuroanatomical

https://doi.org/10.1006/frne.1997.0155. O. von Bohlen und Halbach, The renin-angiotensin system in the mammalian central

ur

[50]

nervous

system.,

Curr.

Protein

Pept.

Sci.

6

(2005)

355–71.

Jo

http://www.ncbi.nlm.nih.gov/pubmed/16101434.

[51]

K.R. Lynch, C.L. Hawelu-Johnson, P.G. Guyenet, Localization of brain angiotensinogen mRNA

by

hybridization

histochemistry.,

Brain

Res.

388

(1987)

149–58.

http://www.ncbi.nlm.nih.gov/pubmed/3476177. [52]

G. Paxinos, C. Watson, The Rat Brain in Stereotaxic Coordinates, Acad. Press. San Diego. 3rd (1997).

[53]

W. Costa-Ferreira, L. Gomes-de-Souza, C.C. Crestani, AT2 and MAS (but not AT1) angiotensinergic receptors in the medial amygdaloid nucleus modulate the baroreflex activity in rats, Pflügers Arch. - Eur. J. Physiol. 471 (2019) 1173–1182. https://doi.org/10.1007/s00424-019-02301-3.

[54]

A.P. Carobrez, L.J. Bertoglio, Ethological and temporal analyses of anxiety-like behavior: The elevated plus-maze model 20 years on, Neurosci. Biobehav. Rev. 29 (2005) 1193– 1205. https://doi.org/10.1016/j.neubiorev.2005.04.017. S. Pellow, P. Chopin, S.E. File, M. Briley, Validation of open:closed arm entries in an

ro of

[55]

elevated plus-maze as a measure of anxiety in the rat., J. Neurosci. Methods. 14 (1985) 149–67. [56]

J.O. Vieira, J.O. Duarte, W. Costa-Ferreira, G. Morais-Silva, M.T. Marin, C.C. Crestani,

-p

Sex differences in cardiovascular, neuroendocrine and behavioral changes evoked by

re

chronic stressors in rats, Prog. Neuro-Psychopharmacology Biol. Psychiatry. 81 (2018) 426–437. https://doi.org/10.1016/j.pnpbp.2017.08.014. J. Almeida, L.A. Oliveira, R. Benini, C.C. Crestani, Role of hippocampal nitrergic

lP

[57]

neurotransmission in behavioral and cardiovascular dysfunctions evoked by chronic social

[58]

na

stress, Nitric Oxide. 94 (2020) 114–124. https://doi.org/10.1016/j.niox.2019.11.004. M.K. Gouveia, T.T. Miguel, C. Busnardo, A.A. Scopinho, F.M.A. Corrêa, R.L. Nunes-

ur

De-Souza, C.C. Crestani, Dissociation in control of physiological and behavioral responses to emotional stress by cholinergic neurotransmission in the bed nucleus of the terminalis

Jo

stria

in

rats,

Neuropharmacology.

101

(2016)

379–388.

https://doi.org/10.1016/j.neuropharm.2015.10.018.

[59]

B. Petit-Demouliere, F. Chenu, M. Bourin, Forced swimming test in mice: a review of antidepressant

activity,

Psychopharmacology

(Berl).

177

(2005)

245–255.

https://doi.org/10.1007/s00213-004-2048-7. [60]

M.J. Detke, M. Rickels, I. Lucki, Active behaviors in the rat forced swimming test

differentially

produced

by

serotonergic

and

noradrenergic

antidepressants,

Psychopharmacology (Berl). 121 (1995) 66–72. https://doi.org/10.1007/BF02245592. [61]

J.F. Cryan, R.J. Valentino, I. Lucki, Assessing substrates underlying the behavioral effects of antidepressants using the modified rat forced swimming test, Neurosci. Biobehav. Rev. 29 (2005) 547–569. https://doi.org/10.1016/j.neubiorev.2005.03.008.

[62]

C.C. Crestani, F.H.F. Alves, F.M.A. Correa, F.S. Guimarães, S.R.L. Joca, Acute reversible inactivation of the bed nucleus of stria terminalis induces antidepressant-like effect in the

9081-6-30. [63]

ro of

rat forced swimming test, Behav. Brain Funct. 6 (2010) 30. https://doi.org/10.1186/1744-

J. Almeida, J.O. Duarte, L.A. Oliveira, C.C. Crestani, Effects of nitric oxide synthesis inhibitor or fluoxetine treatment on depression-like state and cardiovascular changes by

chronic

variable

stress

in

rats,

Stress.

-p

induced

18

(2015)

462–474.

[64]

re

https://doi.org/10.3109/10253890.2015.1038993.

A.A. Scopinho, M. Scopinho, S.F. Lisboa, F.M. de A. Correa, F.S. Guimarães, S.R.L.

lP

Joca, Acute reversible inactivation of the ventral medial prefrontal cortex induces antidepressant-like effects in rats, Behav. Brain Res. 214 (2010) 437–442.

[65]

na

https://doi.org/10.1016/j.bbr.2010.06.018.

S.R. Joca, F.S. Guimaraes, Inhibition of neuronal nitric oxide synthase in the rat

ur

hippocampus induces antidepressant-like effects, Psychopharmacol. 185 (2006) 298–305. https://doi.org/10.1007/s00213-006-0326-2. P.J. Davern, D. Chen, G. a. Head, C. a. Chavez, T. Walther, D.N. Mayorov, Role of

Jo

[66]

Angiotensin II Type 1A Receptors in Cardiovascular Reactivity and Neuronal Activation After

Aversive

Stress

in

Mice,

Hypertension.

54

(2009)

1262–1268.

https://doi.org/10.1161/HYPERTENSIONAHA.109.139741. [67]

G. Ping, W. Qian, G. Song, S. Zhaochun, Valsartan reverses depressive/anxiety-like behavior and induces hippocampal neurogenesis and expression of BDNF protein in

unpredictable chronic mild stress mice, Pharmacol. Biochem. Behav. 124 (2014) 5–12. https://doi.org/10.1016/j.pbb.2014.05.006. [68]

M. Ayyub, A. Najmi, M. Akhtar, Protective Effect of Irbesartan an Angiotensin (AT1) Receptor Antagonist in Unpredictable Chronic Mild Stress Induced Depression in Mice, Drug Res. (Stuttg). 67 (2016) 59–64. https://doi.org/10.1055/s-0042-118172.

[69]

U. Aswar, S. Chepurwar, S. Shintre, M. Aswar, Telmisartan attenuates diabetes induced depression

in

rats,

Pharmacol.

Reports.

69

(2017)

358–364.

[70]

ro of

https://doi.org/10.1016/j.pharep.2016.12.004. B. Bondy, T.C. Baghai, P. Zill, C. Schule, D. Eser, T. Deiml, P. Zwanzger, R. Ella, R. Rupprecht, Genetic variants in the angiotensin I-converting-enzyme (ACE) and angiotensin II receptor (AT1) gene and clinical outcome in depression, Prog. NeuroBiol.

Psychiatry.

(2005)

1094–1099.

Y.B. Saab, P.R. Gard, M.S. Yeoman, B. Mfarrej, H. El-Moalem, M.J. Ingram, Renin– angiotensin-system

gene

Psychopharmacology

polymorphisms

lP

[71]

re

https://doi.org/10.1016/j.pnpbp.2005.03.015.

29

-p

Psychopharmacology

Biol.

Psychiatry.

and

depression, 31

Prog.

(2007)

Neuro-

1113–1118.

[72]

na

https://doi.org/10.1016/j.pnpbp.2007.04.002. A.D. de Kloet, U.M. Steckelings, C. Sumners, Protective Angiotensin Type 2 Receptors the

Brain

and

Hypertension,

Curr.

Hypertens.

Rep.

19

(2017)

46.

ur

in

https://doi.org/10.1007/s11906-017-0746-x. M.A.P. Fontes, A. Martins Lima, R.A.S. Dos Santos, Brain angiotensin-(1–7)/Mas axis:

Jo

[73]

A new target to reduce the cardiovascular risk to emotional stress, Neuropeptides. 56 (2016) 9–17. https://doi.org/10.1016/j.npep.2015.10.003.

[74]

L. Wang, A.D. de Kloet, D. Pati, H. Hiller, J.A. Smith, D.J. Pioquinto, J.A. Ludin, S.P. Oh, M.J. Katovich, C.J. Frazier, M.K. Raizada, E.G. Krause, Increasing brain angiotensin converting enzyme 2 activity decreases anxiety-like behavior in male mice by activating

central

Mas

receptors,

Neuropharmacology.

105

(2016)

114–123.

https://doi.org/10.1016/j.neuropharm.2015.12.026. [75]

M.D.L.A. Marinzalda, P.A. Pérez, P.A. Gargiulo, B.S. Casarsa, C. Bregonzio, G. Baiardi, Fear-Potentiated Behaviour Is Modulated by Central Amygdala Angiotensin II ${\text{A}\text{T}}_{}$

1









Receptors

Stimulation, Biomed Res. Int. 2014 (2014) 1–7. https://doi.org/10.1155/2014/183248. K. Genaro, D. Fabris, H.A. Fachim, W.A. Prado, Angiotensin AT1 receptors modulate the

ro of

[76]

anxiogenic effects of angiotensin (5–8) injected into the rat ventrolateral periaqueductal gray, Peptides. 96 (2017) 8–14. https://doi.org/10.1016/j.peptides.2017.08.005. [77]

R. Tashev, M. Ivanova, Involvement of hippocampal angiotensin 1 receptors in anxiety-

[78]

re

https://doi.org/10.1016/j.pharep.2018.03.001.

-p

like behaviour of olfactory bulbectomized rats, Pharmacol. Reports. (2018).

J.M. Saavedra, I. Armando, Angiotensin II AT2 Receptors Contribute to Regulate the

lP

Sympathoadrenal and Hormonal Reaction to Stress Stimuli, Cell. Mol. Neurobiol. 38 (2018) 85–108. https://doi.org/10.1007/s10571-017-0533-x. J. Leonhardt, D.C. Villela, A. Teichmann, L.-M. Münter, M.C. Mayer, M. Mardahl, S.

na

[79]

Kirsch, P. Namsolleck, K. Lucht, V. Benz, N. Alenina, N. Daniell, M. Horiuchi, M. Iwai,

ur

G. Multhaup, R. Schülein, M. Bader, R.A. Santos, T. Unger, U.M. Steckelings, Evidence for Heterodimerization and Functional Interaction of the Angiotensin Type 2 Receptor and Receptor

Jo

the

MAS,

Hypertension.

69

(2017)

1128–1135.

https://doi.org/10.1161/HYPERTENSIONAHA.116.08814.

[80]

M. Garcia-Garrote, A. Perez-Villalba, P. Garrido-Gil, G. Belenguer, J.A. Parga, F. PerezSanchez, J.L. Labandeira-Garcia, I. Fariñas, J. Rodriguez-Pallares, Interaction between Angiotensin Type 1, Type 2, and Mas Receptors to Regulate Adult Neurogenesis in the Brain

Ventricular–Subventricular

Zone,

Cells.

8

(2019)

1551.

https://doi.org/10.3390/cells8121551. [81]

D. Villela, J. Leonhardt, N. Patel, J. Joseph, S. Kirsch, A. Hallberg, T. Unger, M. Bader, R.A. Santos, C. Sumners, U.M. Steckelings, Angiotensin type 2 receptor (AT2R) and receptor

Mas:

a

complex

liaison,

Clin.

Sci.

128

(2015)

227–234.

https://doi.org/10.1042/CS20130515. [82]

J.F. Cryan, A. Markou, I. Lucki, Assessing antidepressant activity in rodents: recent developments and future needs, Trends Pharmacol. Sci. 23 (2002) 238–245.

[83]

I. Lucki, The forced swimming test as a model for core and component behavioral effects of

antidepressant

drugs,

Behav.

Pharmacol.

https://doi.org/10.1097/00008877-199711000-00010.

8

(1997)

523–532.

D.A. Slattery, S. Desrayaud, J.F. Cryan, GABA B Receptor Antagonist-Mediated

-p

[84]

ro of

https://doi.org/10.1016/S0165-6147(02)02017-5.

re

Antidepressant-Like Behavior Is Serotonin-Dependent, J. Pharmacol. Exp. Ther. 312 (2005) 290–296. https://doi.org/10.1124/jpet.104.073536. S.D. Mague, A.M. Pliakas, M.S. Todtenkopf, H.C. Tomasiewicz, Y. Zhang, W.C. Stevens,

lP

[85]

R.M. Jones, P.S. Portoghese, W.A. Carlezon, Antidepressant-Like Effects of κ-Opioid

na

Receptor Antagonists in the Forced Swim Test in Rats, J. Pharmacol. Exp. Ther. 305 (2003) 323–330. https://doi.org/10.1124/jpet.102.046433. E. Estrada-Camarena, A. Fernández-Guasti, C. López-Rubalcava, Antidepressant-Like

ur

[86]

Effect of Different Estrogenic Compounds in the Forced Swimming Test,

Jo

Neuropsychopharmacology. 28 (2003) 830–838. https://doi.org/10.1038/sj.npp.1300097.

[87]

W.A. Carlezon, S.D. Mague, S.L. Andersen, Enduring behavioral effects of early exposure to

methylphenidate

in

rats,

Biol.

Psychiatry.

54

(2003)

1330–1337.

https://doi.org/10.1016/j.biopsych.2003.08.020. [88]

J.F. Cryan, M.E. Page, I. Lucki, Noradrenergic lesions differentially alter the antidepressant-like effects of reboxetine in a modified forced swim test, Eur. J. Pharmacol.

436 (2002) 197–205. https://doi.org/10.1016/S0014-2999(01)01628-4. [89]

J.A. Ricardo, E. Tongju Koh, Anatomical evidence of direct projections from the nucleus of the solitary tract to the hypothalamus, amygdala, and other forebrain structures in the rat, Brain Res. 153 (1978) 1–26. https://doi.org/10.1016/0006-8993(78)91125-3.

[90]

B. Le Foll, Neuropharmacology of Nicotine, in: Biol. Res. Addict., Elsevier Inc., 2013:

Jo

ur

na

lP

re

-p

ro of

pp. 561–571. https://doi.org/10.1016/B978-0-12-398335-0.00055-8.

FIGURE LEGENDS Figure 1 – (Top) Diagrammatic representations modified from rat brain atlas of Paxinos and Watson (1997) indicating bilateral microinjection sites into the medial amygdaloid nucleus (MeA) of vehicle (white circles), losartan (closed black circles), PD123319 (white triangle) and A-779 (black triangle). (Bottom) Photomicrograph of a coronal brain section depicting bilateral microinjection sites in the MeA of a representative animal. Arrows indicate the microinjection

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sites. IA, interaural coordinate; opt, optical tract.

ro of -p re lP na ur Jo Figure 2 - Anxiety-like behaviors evaluated in the elevated plus-maze (EPM). Percentage of time

spent (top left) and entries (top right) in open arms, number of entries in closed arms (bottom left) and total distance traveled (bottom right) in animals that received bilateral microinjection into the medial amygdaloid nucleus (MeA) of the selective AT1 receptor antagonist losartan (1nmol/100nL, n=10), the selective AT2 receptor antagonist PD123319 (5nmol/100nL, n=10), the selective Mas receptor antagonist A-779 (0.1nmol/100nL, n=10) or vehicle (saline, 100nL, n=10). The bars represent the mean±SEM. *P<0.05 vs vehicle group, one-way ANOVA

Figure 3 - Behaviors

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followed by Bonferroni post-hoc test.

evaluated in the forced swimming test (FST). Immobility time (top left), latency to the first bout

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of immobility (top right), climbing (bottom left), swimming (bottom middle) and diving (bottom right) in animals that received bilateral microinjection into the medial amygdaloid nucleus (MeA) of the selective AT1 receptor antagonist losartan (1nmol/100nL, n=10), the selective AT2 receptor antagonist PD123319 (5nmol/100nL, n=10), the selective Mas receptor antagonist A-779 (0.1nmol/100nL, n=10) or vehicle (saline, 100nL, n=10). The bars represent the mean±SEM. *P<0.05 vs vehicle group, one-way ANOVA followed by Bonferroni post-hoc test.

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