PD123319

Role of angiotensin receptors in the medial amygdaloid nucleus in autonomic, baroreflex and cardiovascular changes evoked by chronic stress in rats

Willian Costa-Ferreira1,2 | Lucas Gomes-de-Souza1,2 | Carlos C. Crestani1,2
1 School of Pharmaceutical Sciences, São Paulo State University (UNESP), Araraquara, Brazil
2 Joint UFSCar-UNESP Graduate Program in Physiological Sciences, São Carlos, Brazil

Correspondence
Carlos C. Crestani, Laboratory of Pharmacology, School of Pharmaceutical Sciences, São Paulo State University – UNESP, Rodovia Araraquara-Jaú Km 01 (Campus Universitário), Campus Ville, 14800-903, Araraquara, SP, Brazil.
Email: [email protected]

Funding information
Conselho Nacional de Desenvolvimento Científico e Tecnológico, Grant/Award Number: 456405/2014-3 and 431339/2018-
0; Fundação de Amparo à Pesquisa do Estado de São Paulo, Grant/Award Number: 2015/05922-9 and 2017/19249-0; Scientific Support and Development Program of School of Pharmaceutical Sciences (UNESP)

Abstract

This study investigated the role of AT1, AT2 and Mas angiotensinergic receptors within the MeA in autonomic, cardiovascular and baroreflex changes evoked by a 10-day (1 hr daily) repeated restraint stress (RRS) protocol. Analysis of cardio- vascular function after the end of the RRS protocol indicated increased values of arterial pressure, without heart rate changes. Arterial pressure increase was not af- fected by acute MeA treatment after the RRS with either the selective AT1 receptor antagonist losartan, the selective AT2 receptor antagonist PD123319 or the selective Mas receptor antagonist A-779. Analysis of heart rate variability indicated that RRS increased the sympathetic tone to the heart, which was inhibited by MeA treatment with either losartan, PD123319 or A-779. Baroreflex function assessed using the pharmacological approach via intravenous infusion of vasoactive agents revealed a facilitation of tachycardia evoked by blood pressure decrease in chronically stressed animals, which was inhibited by MeA treatment with losartan. Conversely, barore- flex responses during spontaneous fluctuations of blood pressure were impaired by RRS, and this effect was not affected by injection of the angiotensinergic receptor antagonists into the MeA. Altogether, the data reported in the present study suggest an involvement of both angiotensinergic receptors present in the MeA in autonomic imbalance evoked by RRS, as well as an involvement of MeA AT1 receptor in the enhanced baroreflex responses during full range of blood pressure changes. Results also indicate that RRS-evoked increase in arterial pressure and impairment of barore- flex responses during spontaneous variations of arterial pressure are independent of MeA angiotensinergic receptors.
KEYWORDS
amygdala, angiotensin II, baroreflex, blood pressure, heart rate, Mas receptor
Abbreviations: BEI, baroreflex effectiveness index; HPA axis, hypothalamus–pituitary–adrenal axis; HR, heart rate; MAP, mean arterial pressure; MeA, medial amygdaloid nucleus; PI, pulse interval; RAS, renin–angiotensin system; RRS, repeated restraint stress; SAP, systolic arterial pressure.

1 | INTRODUCTION
Physiological changes during stressful events constitute prom- inent adaptive mechanisms in short term (Dhabhar, 2019; Sterling, 2012). Nevertheless, the frequent and/or prolonged exposure to these changes might lead to dysfunctions and diseases (Dhabhar, 2019; McEwen, 1998, 2016). In this sense, clinical and preclinical studies have linked repeated/ prolonged exposure to stressful events to cardiovascular dys- functions (Carnevali et al., 2017; Crestani, 2016; Esler, 2017; Golbidi et al., 2015; Kivimäki & Steptoe, 2018). Indeed, studies in rodents have shown cardiovascular and autonomic changes following exposure to chronic stressors, including: increased blood pressure and heart rate, cardiac autonomic imbalance, baroreflex impairment and cardiac and vascular dysfunctions (Carnevali et al., 2017; Crestani, 2016; Golbidi et al., 2015). However, the neurobiological mechanisms in- volved in these changes are still poorly understood.
Brain regions and circuits in the limbic forebrain, hypothal- amus and brainstem are responsible for mediating stress-evoked cardiovascular responses (Dampney, 2015; Myers, 2017). In this sense, the amygdaloid complex has been implicated in car- diovascular responses during aversive threats (Dampney, 2015; Myers, 2017), and the medial amygdaloid nucleus (MeA) was documented as a major amydgaloid sub-nucleus activated by psychological stressors (Cullinan et al., 1995; Dielenberg et al., 2001; Furlong et al., 2014; Krukoff & Khalili, 1997). Accordingly, the MeA has been described to be part of the cen- tral network controlling cardiovascular responses during acute sessions of stress (Fortaleza et al., 2009; Kubo et al., 2004). Nevertheless, despite evidence of morphological, neurochem- ical and functional changes within the MeA of chronically stressed animals (Bennur et al., 2007; Christiansen et al., 2011; Gray et al., 2010; Lau et al., 2017; Mozhui et al., 2010); an involvement of this amygdaloid nucleus in cardiovascular and autonomic dysfunctions evoked by chronic stressors has never been reported.
Angiotensinergic neurotransmission in the brain has been implicated in physiological and behavioural responses to aversive threats (Fontes et al., 2016; Jackson et al., 2018; Saavedra et al., 2004, 2011). In this sense, studies have con- sistently demonstrated that intracerebral administration of AT1 receptor antagonists decreases behavioural and physiological responses to emotional stress, including the cardiovascular changes (Mayorov, 2011). Thus, several authors have proposed that brain angiotensin II acting via AT1 receptor is a stress hor- mone, which present pro-stress actions (Fontes et al., 2016; Saavedra et al., 2004, 2011). Conversely, accumulating evi- dence indicates that angiotensin-(1–7)/Mas receptor axis of the renin–angiotensin system (RAS) attenuates physiological and behavioural stress responses, indicating an anti-stress effect (Fontes et al., 2016). An involvement of AT2 receptors in stress responses has also been reported, but the evidence are limited and the role of this receptor in responses to stress still needs to be determined (Saavedra & Armando, 2018).
The vast majority of studies that investigated the role of brain angiotensinergic receptors evaluated the responses to acute stressors. Therefore, although evidence that chronic stress alters distribution and expression of angiotensin II receptors in the brain (Aguilera et al., 1995; Castren & Saavedra, 1988; Du et al., 2013; Leong et al., 2002; McDougall et al., 2000; Milik et al., 2016), an involvement of brain angiotensinergic recep- tors in dysfunctions evoked by chronic stressors is still poorly understood. In this sense, previous studies provided evidence of a role of brain AT1 receptors in cardiovascular changes evoked by chronic stressors (Costa-Ferreira et al., 2016; Porter et al., 2004). However, the specific brain sites whereby angio- tensin II acts to evoke these changes have never been reported. Besides, to the best of our knowledge, the role of AT2 and Mas receptors in cardiovascular/autonomic dysfunctions evoked by chronic stressors has never been evaluated.
Angiotensinergic receptors (e.g. AT1, AT2 and Mas recep- tors), angiontensinogen and angiotensinergic terminals were identified within the MeA (Becker et al., 2007; von Bohlen und Halbach, 2005; de Kloet et al., 2016; Lenkei et al., 1997; Lynch et al., 1987), and the MeA seems to have the highest level of angiotensinogen within the amygdaloid complex (Lynch et al., 1987). However, it remains to be addressed the involvement of angiotensinergic neurotransmissions present in the MeA in modulation of autonomic and cardiovascular alterations evoked by chronic stress. Therefore, we tested the hypothesis that Mas, AT1 and AT2 receptors present in the MeA are involved in autonomic, cardiovascular and baroreflex changes evoked by repeated restraint stress (RRS) in male rats.

2 | MATERIALS AND METHODS

2.1 | Animals
Fifty-four male Wistar rats (8-week-old, weighing approxi- mately 250g) obtained from the animal breeding facility of the UNESP (São Paulo State University, Botucatu, São Paulo, Brazil) were used in this study. The rats were housed in collective plastic cages (four animals/cage) in tempera- ture-controlled rooms at 24°C with 12 hr light-dark cycle and free access to water and food. All procedures were approved by Local Ethical Committee for Use of Animals (approval #62/2015), which comply with Brazilian and international guidelines for animal use and welfare.

2.2 | Stereotaxic surgery
Animals were subjected to tribromoethanol anaesthe- sia (250 mg/kg, i.p.) and underwent stereotaxic surgery to implant bilateral cannulas directed to the MeA. The proce- dure was as previously described by our laboratory (Costa- Ferreira et al., 2019). As profylatic measurement, after surgery the rats were treated with a poly-antibiotic formu- lation (Pentabiotico®; Fort Dodge, Campinas, São Paulo, Brazil; 560 mg ml−1 kg−1, i.m.) and the non-steroidal anti-in- flammatory drug flunixin meglumine (Banamine®; Schering- Plough, Cotia, São Paulo, Brazil; 0.5 mg ml−1 kg−1, s.c.).

2.3 | Repeated restraint stress
The protocol of RRS was performed as previously described by our laboratory (Costa-Ferreira et al., 2016, 2019; Vieira, Duarte, Costa-Ferreira, Morais-Silva, et al., 2018). Briefly, the animals were restrained 1 hr daily (always starting at 9:00 a.m.) for 10 consecutive days.

2.4 | Femoral artery and vein catheter implant
Animals were subjected to tribromoethanol anaesthesia (250 mg/kg, i.p.) and underwent surgery procedure to implant polyethylene cannulas into the abdominal aorta via the fem- oral artery for cardiovascular recording. A second catheter was implanted into the femoral vein for drug administrations. The procedure was as previously described by our labora- tory (Costa-Ferreira, Gomes-de-Souza, et al., 2019; Costa- Ferreira et al., 2016; Crestani et al., 2006; Vieira, Duarte, Costa-Ferreira, Morais-Silva, et al., 2018). After surgery, the rats were subjected to profylatic treatments with antibiotic and anti-inflammatory drugs as described above (see stereo- taxic surgery description). The rats were housed in individual cages after the procedure.

2.5 | Cardiovascular recording
Mean arterial pressure (MAP) and heart rate (HR) values were obtained from pulsatile arterial pressure, which was recorded as previously described by our laboratory (Costa-Ferreira, Gomes-de-Souza, et al., 2019; Costa-Ferreira et al., 2016; Duarte et al., 2015a; Duarte Planeta & Crestani, 2015; Vieira, Duarte, Costa-Ferreira, Morais-Silva, et al., 2018).

2.6 | Drug microinjection into the brain
Microinjections into the brain were performed as previously described by our laboratory, and 100nl of the drugs was administrated per side (Costa-Ferreira, Gomes-de-Souza, et al., 2019; Fortaleza et al., 2015).

2.7 | Assessment of the autonomic activity
Power spectral analysis of systolic arterial pressure (SAP) and the pulse interval (PI) were used to analyse autonomic activity controlling the blood vessels and heart respectively. For this, beat-to-beat time series of SAP and PI were ex- tracted from the pulsatile arterial pressure signal. Using the software Cardioseries v2.4 (available on https://www.sites. google.com/site/cardioseries/), the overall variability of these series was calculated in the frequency domain. The power of the obtained oscillatory components from PI was quantified in two frequency bands: low frequency (LF, 0.20–0.75 Hz) and high frequency (HF, 0.75–3.0 Hz), whereas the power of the oscillatory components from SAP was quantified in LF (0.20–0.75 Hz). In order to minimize the effects of total power changes on the variability of each frequency band, the results of LF and HF from IP were expressed in nor- malized units (nu) (Electrophysiology, 1996). The normal- ized values were obtained by calculating the relative power of the LF and HF bands in proportion to the total spectrum power minus the very low frequency band power (<0.20 Hz; Electrophysiology, 1996). Oscillations of arterial pressure and PI at LF range are representative of the modulatory effects of sympathetic ac- tivity controlling vascular tonus and heart activity respec- tively; while oscillations of PI at HF range are associated with a parasympathetic modulation of the heart (Malliani et al., 1991; Ramaekers et al., 2002). The ratio between the powers of the LF and HF bands (LF/HF) of the PI was calcu- lated for determination of the cardiac sympathovagal balance (Almeida et al., 2015; Costa-Ferreira et al., 2016). 2.8 | Assessment of baroreflex activity Baroreflex function was assessed using (a) the sequence analysis technique and (b) the classical pharmacological approach. 2.8.1 | Classical pharmacological approach Arterial pressure changes were evoked by intravenous infu- sions of the selective α1-drenoceptor agonist phenylephrine (70 μg/ml at 0.4 ml min−1 kg) and the nitric oxide donor so- dium nitroprusside (100 μg/ml at 0.8 ml min−1 kg−1) using an infusion pump (K.D. Scientific, Holliston, MA; Almeida et al., 2019; Costa-Ferreira, Gomes-de-Souza, et al., 2019). Phenylephrine and sodium nitroprusside caused increase and decrease in blood pressure respectively. Infusions were ran- domized, and the second administration was not performed before the cardiovascular parameters returned to basal values (the interval between infusions was approximately 5 min). For assessment of baroreflex function, reflex HR re- sponses (ΔHR) evoked by intravenous infusion of phenyl- ephrine (vasoconstrictor agent) and sodium nitroprusside (vasodilator agent) were obtained each 10 mmHg of MAP change (ΔMAP), and data were analysed using linear regres- sion analysis (Almeida et al., 2019; Costa-Ferreira, Gomes- de-Souza, et al., 2019). The slope of the curves (i.e. gain) was used to evaluate the baroreflex activity. 2.8.2 | The sequence analysis technique The sequence method evaluated the baroreflex responses during physiological range of arterial pressure changes (i.e. without administration of vasoactive agents). The spontane- ous baroreflex sensitivity was obtained based on the slope of the linear regression between the SAP change and the reflex PI response. For this, the beat-to-beat values of SAP and PI were analysed using the software Cardioseries v2.4 (Costa-Ferreira, Gomes-de-Souza, et al., 2019; Reis-Silva et al., 2020) for identification of sequences in which the SAP increase was associated with PI lengthening (up sequence) or the SAP decrease was associated with PI shortening (down sequence). A baroreflex sequence was only used when the correlation coefficient (r) between the SAP and PI change was >0.8. The spontaneous baroreflex sensitivity was as- sessed based on the slope (ms/mmHg) of the linear regression between the SAP and PI. The baroreflex effectiveness index (BEI), which indicates the percentage of SAP changes that ef- fectively evokes reflex PI responses (Di Rienzo et al., 2001), was also evaluated in the sequence method (Costa-Ferreira, Gomes-de-Souza, et al., 2019; Reis-Silva et al., 2020).

2.9 | Drugs and solutions
A-779 (Tocris, Westwoods Business Park, Ellisville, MO), losartan potassium (Tocris), PD123319 ditrifluoroacetate (Tocris), sodium nitroprusside dihydrate (Sigma–Aldrich, St. Louis, MO, USA), phenylephrine hydrochloride (Sigma– Aldrich), urethane (Sigma-Aldrich) and 2,2,2-tribromoe- thanol (Sigma-Aldrich) were dissolved in saline (0.9% NaCl). The poly-antibiotic formulation (Pentabiotico®; Fort Dodge, Campinas, São Paulo, Brazil) and the non-steroidal anti-inflammatory drug flunixin meglumine (Banamine®; Schering-Plough, Cotia, São Paulo, Brazil) were used as pro- vided by the manufacture.

2.10 | Experimental design
Three days before the RRS onset, animals underwent stere- otaxic surgery for implantation of bilateral cannulas directed to the MeA. Afterwards, rats of RRS groups were subjected to daily 60 min sessions of restraint stress (always starting at 9:00 a.m.) for 10 consecutive days (Costa-Ferreira, Morais- Silva, et al., 2019; Costa-Ferreira et al., 2016; Vieira, Duarte, Costa-Ferreira, Morais-Silva, et al., 2018). Control animals were kept undisturbed, except for the cleaning of cages, at the animal facility for the same period that rats subjected to RRS, and were tested on the same day as the chronically stressed animals. After the last session of the RRS protocol, animals in all experimental groups underwent surgery for cannula- tion of femoral vein and artery, and spent the night in the recording room in their home cages. The recording room was temperature controlled (24°C) and acoustically isolated. The cardiovascular tests were performed 24 hr after the last stress-restraint session in unanaesthetized animals.
On the trial day, animals were connected to the cardio- vascular recording system and initially subjected to a 30-min period of arterial pressure and HR recording. Then, inde- pendent sets of control and RRS animals received bilateral microinjections into the MeA of the selective Mas receptor antagonist A-779 (0.1 nmol/100 nl), the selective AT1 re- ceptor antagonist losartan (1 nmol/100 nl), the selective AT2 receptor antagonist PD123319 (5 nmol/100 nl) or vehicle (sa- line, 100 nl; Costa-Ferreira, Gomes-de-Souza, et al., 2019; Oscar et al., 2015). Fifteen minutes later, phenylephrine and sodium nitroprusside were randomly infused for the classical pharmacological analysis of the baroreflex function. The as- sessment of the autonomic activity via power spectral anal- ysis and the sequence analysis technique of the baroreflex function were performed in the cardiovascular recording ob- tained from the period of 5–15 min after the MeA treatment. Figure 1 shows a schematic representation of the entire ex- perimental protocol.

2.11 | Histological determination of the microinjection sites
The rats were subjected to urethane anaesthesia (250 mg/ ml/200 g body weight, i.p.) at the end of experiments, and a marker of the microinjection sites (10% Evan’s blue dye, 100nl) was administrated into the brain. Then, the brain was obtained for determination of the microinjection sites, as pre- viously described by our laboratory (Costa-Ferreira, Gomes- de-Souza, et al., 2019; Crestani et al., 2006).

2.12 | Data analysis
The software GraphPad Prism (v.7.0, GraphPad Software Inc., La Jolla, CA) was used for analysis of all data, which were pre- sented as mean ± SEM. The parameters for analysis of auto- nomic activity and baroreflex function, as well as the baseline values of MAP and HR were compared using the two-way ANOVA, with stress (control vs. RRS) and treatment (vehicle vs. drugs) as independent factors, followed by the Bonferroni post-hoc test. The significance was set at p < .05. 3 | RESULTS Photomicrograph presenting microinjection sites of a repre- sentative animal into the MeA is showed in Figure 2. Figure 2 also presents diagrammatic representations indicating the mi- croinjection sites of vehicle, A-779, losartan and PD123319 into the MeA in control and RRS animals. 3.1 | Role of angiotensinergic receptors within the MeA in regulating arterial pressure and heart rate in control and RRS rats Values of MAP and HR obtained 24 hr after the last session of restraint stress are presented in Table 1. Analysis of the val- ues of MAP indicated a main effect of RRS (F(1,46) = 14.44, p < .05), which increased MAP, but without influence of MeA pharmacological treatments (F(3,46) = 1.61, p = .19) or RRS × treatment interaction (F(3,46) = 1.20, p = .32). Analysis of HR did not indicate effect of either RRS (F(1,46) = 0.50; p = .48) or pharmacological treatments (F(3,46) = 0.91, p = .44), or RRS × treatment interaction (F(3,46) = 0.61; p = .61). 3.2 | Role of angiotensinergic receptors within the MeA in regulating autonomic activity in control and RRS rats Power spectral analysis of SAP and PI is presented in Figure 3. 3.2.1 | Pulse interval Analysis of the PI oscillations at LF range indicated a main ef- fect of RRS (F(1,46) = 5.24, p = .0266) and a RRS × treatment interaction (F(1,46) = 7.35, p = .0004), but without effect of MeA pharmacological treatments (F(3,46) = 1.70, p = .1797). Post-hoc analysis revealed that RRS increased PI oscillations at LF range (p = .012) in animals treated with vehicle into the MeA. The effect of RRS in PI oscillation at LF range was not identified in animals treated with either losartan (p = .3412), PD123319 (p = .9465) or A-779 (p = .9720) in the MeA. Besides, values in RRS animals treated with either PD123219 were lower in relation to the respective vehicle group (p = .0101). Analysis of the PI oscillations at HF range did not indicate effect of either RRS (F(1,46) = 1.42, p = .2392) or pharmaco- logical treatments (F(3,46) = 1.75, p = .1685), or RRS × treat- ment interaction (F(3,46) = 2.39, p = .0802). However, analysis of LF/HF ratio indicated a RRS × pharmacological treatment interaction (F(3,46) = 6.48, p = .10009), but without main ef- fect of RRS (F(1,46) = 3.01, p = .0892) and pharmacological treatment (F(3,46) = 1.97, p = .1331). Such as identified for the oscillations at LH range, post hoc analysis revealed that RRS increased the LF/HF ratio in animals treated with vehicle into the MeA (p = .0014), and this effect was not detected in animals treated with losartan (p = .9810), PD123319 (p = .9988) or A-779 (p = .9002) in the MeA. Besides, values in RRS animals treated with either PD123219 (p = .0089) or A-779 (p = .0180) were lower in relation to the respective vehicle group. 3.2.2 | Systolic arterial pressure Analysis of the SAP oscillations at LF range indicated a main effect of RRS (F(1,46) = 4.11, p = .0484), but without influ- ence of pharmacological treatment (F(3,46) = 0.38, p = .7676) and RRS × treatment interaction (F(3,46) = 0.39, p = .7579). FIGURE 2 (Top) Diagrammatic representations (Paxinos & Watson, 1997) indicating administration sites into the MeA of vehicle (control: white circles, RRS: white triangle), A-779 (control: black circles, RRS: black triangles), losartan (control: light grey circles, RRS: light grey triangle) and PD123319 (control: dark grey circles, RRS: dark grey triangle) in control and RRS animals. (Bottom) A photomicrograph showing administration sites in the MeA of a representative animal. The administration sites are indicated by the arrows. IA, interaural coordinate; opt, optical tract TABLE 1 Basal values of mean arterial pressure (MAP) and heart rate (HR) and sample size (n) in control and RRS rats that received bilateral microinjection into the MeA of the selective AT1 receptor antagonist losartan (1 nmol/100 nl), the selective AT2 receptor antagonist PD123319 (5 nmol/100 nl) and the selective Mas receptor antagonist A-779 (0.1 nmol/100 nl) or vehicle (100 nl) Control RRS Treatment Vehicle Losartan PD123319 A-779 Vehicle Losartan PD123319 A-779 HR (bpm) 408 ± 18 381 ± 24 392 ± 19 398 ± 16 386 ± 17 387 ± 18 361 ± 12 409 ± 17 MAP (mmHg) 111 ± 3 106 ± 3 106 ± 2 110 ± 2 116 ± 3# 110 ± 2# 121 ± 5# 118 ± 3# n 8 7 7 7 6 6 6 7 Note: Values are mean ± SEM. #p < .05 indicating the main effect of stress, two-way ANOVA. FIGURE 3 Power spectral analysis of the SAP and PI in control and RRS animals treated with vehicle (saline, 100 nl, white bars), the selective Mas receptor antagonist A-779 (0.1 nmol/100 nl, black bars), the selective AT1 receptor antagonist losartan (1 nmol/100 nl, red bars) or the selective AT2 receptor antagonist PD123319 (5 nmol/100 nl, blue bars) into the MeA. (A) Power spectral analysis of the SAP oscillations at LF range. (B) Power spectral analysis of the PI oscillations at LF (left graph) and HF (middle graph) ranges and LF/HF ratio (right graph). Bars represent the mean ± SEM. #p < .05 in relation to the respective control group; *p < .05 in relation to the respective vehicle group. Two-way ANOVA followed by Bonferroni post-hoc test (n = 6–8/group, see Table 1) 3.3 | Role of angiotensinergic receptors within the MeA in regulating baroreflex function in control and RRS rats 3.3.1 | Pharmacological approach Results of the baroreflex function assessed using the pharma- cological approach are presented in Figure 4. Analysis of the gain for the tachycardiac response evoked by MAP decreases as consequence of intravenous infusion of sodium nitroprusside indicated main effects of RRS (F(1,46) = 50.77, p < .0001) and MeA pharmacological treatments (F(3,46) = 15.16, p < .0001), but without RRS × treatment interaction (F(3,46) = 1.23, p = .3092). Post-hoc analysis revealed that microinjection of the selective AT2 receptor antagonist PD123319 into the MeA of the control animals increased the reflex HR response dur- ing blood pressure decreases (p = .0127). Moreover, RRS in- creased reflex tachycardia in animals treated with vehicle into the MeA (p < .0001). Consistent with vehicle-treated rats, in- creased baroreflex tachycardic response was also identified in RRS animals that received either PD123319 (p < .05) or the se- lective Mas receptor antagonist A-779 (p < .001) into the MeA, when compared to the respective control groups. RRS-evoked enhance in reflex tachycardia was not identified in animals that received the selective AT1 receptor antagonist losartan into the MeA (p = .4486). Analysis of the gain for the reflex bradycardiac response evoked by MAP rise as consequence of intravenous infusion of phenylephrine did not indicate influence of either RRS (F(1,46) = 3.68, p = .0616) or MeA pharmacological treat- ments (F(3,46) = 2.20, p = .1012), or RRS × treatment interac- tion (F(3,46) = 1.02, p = .3947). FIGURE 4 Baroreflex function evaluated during arterial pressure changes caused by infusion of vasoactive agents (i.e. pharmacological approach) in control and RRS animals that received vehicle (saline, 100 nl, white circles and bars), the selective Mas receptor antagonist A-779 (0.1 nmol/100 nl, black circles and bars), the selective AT1 receptor antagonist losartan (1 nmol/100 nl, red circles and bars) and the selective AT2 receptor antagonist PD123319 (5 nmol/100 nl, blue circles and bars) into the MeA. (a) Linear regression curves of the baroreflex function correlating the mean arterial pressure change (ΔMAP) caused by intravenous infusion of the vasoactive agents (i.e. phenylephrine and sodium nitroprusside) and the reflex heart rate changes (ΔHR) in control (left graph) and RRS (right graph) animals. The circles in the curves represent the mean ± SEM. (b) Slope (i.e. gain) of the regression lines for the bradycardiac response evoked by MAP increase (slope bradycardia, right graph) and the tachycardia caused by MAP decrease (slope tachycardia, left graph). The bars represent the mean ± SEM. *p < .05 in relation to the respective vehicle group, #p < .05 in relation to the respective control group. Two-way ANOVA followed by Bonferroni post-hoc test (n = 6–8/ group, see Table 1) 3.3.2 | Sequence method The sequence method evaluated the baroreflex responses dur- ing physiological range of arterial pressure changes (i.e. without administration of vasoactive agents). Results of the sequence analysis technique are presented in Figure 5. Analysis of the up sequences (i.e. SAP increase associated with PI lengthening) indicated main effects of RRS (F(1,46) = 8.18, p = .0063), con- sistent with a decrease in baroreflex function, and MeA phar- macological treatments (F(3,46) = 2.94, p = .0428), but without RRS × treatment interaction (F(3,46) = 0.46, p = .7076). Post- hoc analysis did not reveal any specific difference between the experimental groups (p > .05). Analysis of the down sequences (i.e. SAP decrease as- sociated with PI shortening) and mean of all sequences (i.e. mean of up and downs sequences) indicated a main effect of RRS (down: F(1,46) = 11.93, p = .0012; mean: F(1,46) = 8.43, p = .0057), consistent with a decrease in baroreflex activity, but without influence of pharmacological treatments (down: F(3,46) = 1.24, p = .3048; mean: F(3,46) = 1.46, p = .2365) and RRS × treatment interaction (down: F(3,46) = 0.15, p = .9275; mean: F(3,46) = 0.26, p = .8512). Post-hoc analysis did not re- veal any specific difference between the experimental groups for the down sequences (p > .05) and the mean of all se- quences (p > .05). Analysis of the baroreflex effective index (BEI; i.e. the percentage of SAP changes that effectively evokes reflex PI responses) indicated a main effect of the MeA pharmacolog- ical treatments (F(3,46) = 15.1, p < .0001), but without influ- ence of RRS (F(1,46) = 2.73, p = .1049) and stress × treatment interaction (F(3,46) = 1.37, p = .2627). Post-hoc analysis re- vealed that administration into the MeA of the selective AT2 receptor antagonist PD123319 enhanced BEI in the control animals (p = .0045). 4 | DISCUSSION Results reported in the present study suggest for the first time a role of angiotensinergic receptors within the MeA in autonomic and cardiovascular changes evoked by aversive threats. Indeed, we identified that RRS increased PI oscil- lations at LF range and LF/HF ratio, which indicate an in- crease in sympathetic tone to the heart (Malliani et al., 1991; Ramaekers et al., 2002). The effects of RRS in PI oscilla- tions were not identified in animals treated with losartan, PD123319 or A-779 in the MeA, thus suggesting an in- volvement of both AT1, AT2 and Mas receptors in the MeA. Baroreflex function assessed via the pharmacological ap- proach provided evidence that RRS enhanced the reflex tach- ycardic response to blood pressure decrease, an effect that was not identified in animals that received the selective AT1 receptor antagonist losartan into the MeA. In addition, mi- croinjection of PD123319 into the MeA increased the reflex tachycardic response in both control and RRS animals, thus indicating a stress-independent inhibitory control of barore- flex function by MeA AT2 receptors. The sequence analysis technique suggested that RRS impaired the baroreflex func- tion during arterial pressure alterations within the narrow range of physiological variations, which was not affected by MeA pharmacological treatments. However, microinjection of PD123319 into MeA increased the BEI in control animals, thus reinforcing the data of the baroreflex pharmacological assessment indicating an inhibitory role of AT2 receptors in the MeA on baroreflex control. Data also indicated that RRS increased basal arterial pressure, but this effect was not af- fected by MeA pharmacological treatments. FIGURE 5 Baroreflex function evaluated by the sequence analysis technique in control and RRS animals treated with vehicle (saline, 100 nl, white bars), the selective Mas receptor antagonist A-779 (0.1 nmol/100 nl, black bars), the selective AT1 receptor antagonist losartan (1 nmol/100 nl, red bars) or the selective AT2 receptor antagonist PD123319 (5 nmol/100 nl, blue bars) into the MeA. (a) BEI (i.e. baroreflex effectiveness index). *p < .05 in relation to the respective vehicle group, two-way ANOVA followed by Bonferroni post-hoc test. (b) Spontaneous baroreflex sensitivity during decreases (down sequence, middle graph) and increases (up sequence, left graph) of systolic arterial pressure (SAP) and the mean of all sequences (i.e. average of up and down sequences, right graph). Bars represent the mean ± SEM. #p < .05, indicating the main effect of RRS; *p < .05, indicating the main effect of MeA pharmacological treatments. Two-way ANOVA (n = 6–8/ group, see Table 1) Neuroendocrine responses (e.g. sympathetic-adrenal med- ullary and hypothalamus–pituitary–adrenal [HPA] axis) have been well reported to habituate with repeated exposure to re- straint stress (Grissom & Bhatnagar, 2009; McCarty, 2016). This habituation process is proposed as a adaptative mecha- nism that limit the consequences of chronic stress (Bennett et al., 2018; Grissom & Bhatnagar, 2009; Herman, 2013; McCarty, 2016). However, recent evidence indicated that ha- bituation of the cardiovascular changes to restraint stress is less pronounced in relation to, for instance, the HPA axis re- sponse (Benini et al., 2020; Santos et al., 2020). Accordingly, several cardiovascular changes have been reported follow- ing repeated exposure to restraint stress, including increase in arterial pressure (Bruder-Nascimento et al., 2012, 2015; Duarte et al., 2015b; Duarte, Planeta, et al., 2015; Habib et al., 2015; Yang et al., 2014), without changes in HR (Daubert et al., 2012; Duarte et al., 2015b; Duarte, Planeta, et al., 2015). Although there is some inconsistency (for review, see Crestani, 2016; Nalivaiko, 2011), these above-mentioned studies support the increased arterial pressure identified in the present study in animals subjected to RRS. Results reported here are also in line with previous evidence that exposure to RRS protocols increased sympathetic tone to the heart and affected baroreflex activity (Conti et al., 2001; Costa-Ferreira et al., 2016; Duarte, Cruz, et al., 2015b; Duarte, Planeta, et al., 2015; Firmino et al., 2019; Porter et al., 2004; Vodička et al., 2020). Regarding the effect in baroreflex function, the increase in reflex tachycardia to arterial pressure decrease identified in the present study in RRS rats corroborates pre- vious evidence that RRS enhanced baroreflex-mediated HR responses (Conti et al., 2001; Duarte, Cruz, et al., 2015b; Firmino et al., 2019; Porter et al., 2004; Vieira et al., 2018). Facilitation of the reflex tachycardic response may be re- lated to the increased cardiac sympathetic activity, which are changes considered as poor prognosis for cardiovascular disease (Grassi et al., 2004). Therefore, as long-term small elevations of arterial pressure have been associated with in- creased cardiovascular risk (Collins et al., 1990), the arterial pressure, cardiac sympathetic activity and baroreflex changes evoked by RRS might be considered prominent risk factors for cardiovascular morbidity and mortality. Contrary to the facilitation of baroreflex responses identified by pharmacological approach, the sequence analysis technique indicated an impairment in this reflex during arterial pressure alterations within the narrow range of physiological variations in animals subjected to RRS. Our findings corroborate previous evidence that chronic stress differently affects baroreflex responses evoked during spontaneous variations and pronounced arterial pressure alterations (Almeida et al., 2015; Costa-Ferreira et al., 2016; Firmino et al., 2019). Besides, although some evidence that RRS does not affect baroreflex activity as- sessed by the sequence method (Costa-Ferreira et al., 2016; Daubert et al., 2012; Duarte, Planeta, et al., 2015), results documented here are in line with recent report that RRS de- creased spontaneous baroreflex gain (Firmino et al., 2019). Differences in the effect of RRS on baroreflex function assessed by pharmacological approach (i.e. facilitation) versus sequence method (i.e. impairment) are also in line with previous reports (Firmino et al., 2019), and might be explained by the possibility that different brain networks regulate reflex responses within the narrow range of physi- ological variations and during full range of arterial pressure changes (Crestani, 2016). The results of the present study further support this idea (see discussion below). Previous studies demonstrated that either intracerebroven- tricular or systemic (intraperitoneal) treatment with the AT1 receptor antagonist losartan completely prevented the barore- flex and autonomic changes evoked by RRS (Costa-Ferreira et al., 2016; Porter et al., 2004). In this sense, the findings reported in the present study provide evidence of the MeA as a brain site whereby angiotensin II activates AT1 receptors to affect the autonomic activity and baroreflex function in animals subjected to RRS. Regarding the effects in barore- flex function, previous studies indicated an involvement of the MeA in brain network controlling baroreflex function (Fortaleza et al., 2015), and we reported recently that con- trol of baroreflex function by MeA is mediated by activation of local AT2 and Mas receptors (Costa-Ferreira, Gomes-de- Souza, et al., 2019). Nevertheless, microinjection of losartan into the MeA of non-stressed rats did not affect baroreflex activity (either pharmacological assessment or the sequence analysis technique) in non-stressed animals (Costa-Ferreira, Gomes-de-Souza, et al., 2019). Taken together with data of the present study, it seems that RRS exposure induces the expression of AT1 receptors in MeA neurons involved in the central network controlling the baroreflex function, which in turn facilitate baroreflex responses. This idea is supported by previous evidence that exposure to RRS increased AT1 receptor in brainstem structures controlling cardiovascu- lar function, as well as in the hypothalamic paraventricular nucleus, median eminence and subfornical organ (Aguilera et al., 1995; Leong et al., 2002; McDougall et al., 2000). This increase in AT1 receptor expression might be medi- ated by activation of glucocorticoid receptors (Castren & Saavedra, 1989; Guo et al., 1995). However, to the best of our knowledge, the effect of chronic stress in expression of AT1 receptor within the MeA has never been reported. Therefore, further studies are necessary to address the mechanism re- lated to the involvement of MeA AT1 receptors in baroreflex change evoked by RRS. As stated above, the facilitation of reflex tachycardia by RRS is possibly related to the increase in cardiac sympathetic activity. This idea is further supported by evidence that AT1 receptors within the MeA is involved in both increased sym- pathetic tone to the heart and facilitation of reflex tachycardia evoked by RRS. However, autonomic changes do not neces- sarily result in changes in baroreflex function, as evidenced in the present study by findings that MeA treatment with the AT2 or Mas receptor antagonists inhibited the cardiac sympathetic response to RRS without affecting the barore- flex change. Despite the absence of influence in baroreflex function, AT2 and Mas receptors present within the MeA seem to play a prominent role in dysfunctions evoked by chronic stressors once increased cardiac sympathetic activ- ity is considered as poor prognosis for cardiovascular disease (Grassi, 2004). Interestingly, although our results indicate an involve- ment of MeA AT1 receptors in RRS-evoked changes in reflex HR responses during the full range of arterial pres- sure change (i.e. results of the pharmacological approach), the impairment of reflex responses during narrow range of physiological arterial pressure variations (i.e. sequence method data) seems to be independent of MeA angioten- sinergic receptors. Taken together with previous evidence that MeA treatment with losartan did not affect sponta- neous baroreflex gain in non-stressed rats (Costa-Ferreira, Gomes-de-Souza, et al., 2019), present finding provide fur- ther evidence that absence of control of reflex responses during spontaneous arterial pressure alterations by MeA AT1 receptor is stress independent. As stated above, the different impact of RRS in baroreflex function assessed via pharmacological approach and sequence method indi- cates different central networks mediating reflex responses during spontaneous alterations and full range of arterial pressure changes. In this sense, the evidence that MeA AT1 receptors is involved selectively in RRS-evoked change in reflex responses during full range of arterial pressure pro- vides further evidence of different central pathways me- diating reflex responses assessed via the pharmacological approach and the sequence method (Crestani, 2016). The MeA has no direct connections with medullary structures involved in tonic control of cardiovascular func- tion. Thus, the control of autonomic activity and cardio- vascular function by this amygdaloid nucleus is proposed to be mediated by connections with medial preoptic area (mPOA), posterior hypothalamus (PH) and bed nucleus of the stria terminalis (BNST); which, in turn, project to au- tonomic effectors neurons in the brainstem (Myers, 2017). Thus, the control of autonomic activity and baroreflex function by angiotensinergic neurotransmission within the MeA reported in the present study is possibly related to indirect connections with these brainstem-projecting structures (Myers, 2017; Ulrich-Lai & Herman, 2009). Accordingly, structures receiving inputs from the MeA (e.g. mPOA, BNST, and PH) have been described as part of central network of the baroreflex (Bauer et al., 1988; Crestani et al., 2006; Inui et al., 1995; Oliveira et al., 2017). This idea is further supported by recent report that BNST is involved in baroreflex changes evoked by chronic stress (Oliveira et al., 2020). Microinjection of the angiotensinergic receptor an- tagonists into the MeA did not change neither arterial pressure nor HR. Previous evidence indicated that MeA non-selective inhibition evoked by local treatment with either CoCl2 or muscimol did not influence basal cardio- vascular parameters (Fortaleza et al., 2009, 2015; Kubo et al., 2004). Furthermore, administration of cholinergic or noradrenergic receptor antagonists into the MeA also did not evoke changes on basal cardiovascular values (Fortaleza et al., 2009, 2012; Fortaleza et al., 2012). These results support our findings and indicate that MeA is not involved in tonic maintenance of HR and arterial pressure. In this sense, present study provides further evidence that even changes in basal cardiovascular parameters evoked by chronic stressors are not mediated by MeA. Besides, the results of the present study indicating a role of MeA AT1 receptor in facilitation of reflex tachycardic responses but not in basal arterial pressure increase suggest that these changes to RRS are not linked. Nevertheless, the arterial pressure increase might be related to the impairment of baroreflex function identified during physiological changes in arterial pressure. Indeed, decrease on baroreflex function has been proposed to be a prominent physiopathological mechanism of hypertension (Grassi et al., 2006; Honzíková & Fišer, 2009). Nevertheless, further studies are necessary to address the mechanisms related to the changes in arte- rial pressure and baroreflex responses during spontaneous blood pressure variations. In summary, present study provides initial evidence of an involvement of angiotensinergic receptors within the MeA in autonomic imbalance caused by RRS, as well as a role of MeA AT1 receptors in RRS-evoked enhance in reflex tachy- cardic response to blood pressure decrease. However, our re- sults indicate that the increase in arterial pressure as well as the impairment of baroreflex responses during spontaneous variations in arterial pressure evoked by chronic restraint stress are independent of MeA angiotensinergic receptors. Finally, present study provides new evidence that the inhibi- tory influence of MeA AT2 receptor in baroreflex function is independent of chronic stress exposure. 5 | COMPETING INTERESTS The authors declare no competing or financial interests. ACKNOWLEDGMENTS The present research was supported by grants from São Paulo Research Foundation (FAPESP) (grant #2015/05922-9 and 2017/19249-0), National Council for Scientific and Technological Development (CNPq) (grants #456405/2014-3 and 431339/2018-0) and Scientific Support and Development Program of School of Pharmaceutical Sciences (UNESP). CCC is a CNPq research fellow (process #305583/2015-8 and 304108/2018-9). The head schema in the Figure 1 was obtained from Luigi Petrucco (https://doi.org/10.5281/ze- nodo.3925903, https://scidraw.io). AUTHORS CONTRIBUTIONS Costa-Ferreira: Conceptualization, Methodology, Formal analysis, Investigation, Writing – original draft preparation, Visualization. Gomes-de-Souza: Methodology, Formal anal- ysis, Investigation, Writing – review and editing. Crestani: Conceptualization, Methodology, Resources, Data Curation, Writing – review and editing, Visualization, Supervision, Project administration, Funding acquisition. 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