Effect of chronic stress on cardiovascular and ventilatory responses activated by both chemoreflex and baroreflex in rats

Living organisms are constantly subjected to a wide range of stressful stimuli that affect many physiological processes (Marin et al., 2007). Stress can affect the function of the immune system by modulating processes in the central nervous system Nakata et al. (1993); moreover, stress can alter and activate endocrine processes associated with the hypothalamus, pituitary and adrenal, and adrenergic system (Lupien and McEwen, 1997; Yaribeygi et al., 2017). In addition, changes in cardiovascular variables, such as blood pressure (BP) and heart rate (f_H), also occur. Because of this, psychological stress has been recognized as a risk factor for several cardiovascular dysfunctions (Inoue, 2014). Although BP and f_H changes are common after exposure to stress, they can vary according to different stressors (Crestani et al., 2010; Crestani, 2016).

Studies of the physiological effects of stress have commonly used different psychological stress protocols, involving daily exposure to the same type of stressor (homotypic) or different aversive stimuli (heterotypic) (Magariños and McEwen, 1995; Crestani, 2016; Duarte et al., 2015a; Vieira et al., 2018). In repeated restraint stress (RRS) models, animals are subjected to the homotypic stressor, while in chronic variable stress (CVS) models, animals are subjected to heterotypic stressors to investigate physiological, behavioral and somatic responses to stress (Ulrich-Lai and Herman, 2009; Grippo et al., 2002; Magariños and McEwen, 1995; Willner, 1997). Additionally, some studies have shown that RRS and CVS in rats may have a different impact on their behavior, body weight and plasma corticosterone secretion (Marin et al., 2007; Ortiz et al., 1996; Martí et al., 1994; Crestani, 2016).

Psychological stress can affect the body through two main pathways: the hypothalamic–pituitary–adrenal (HPA) axis and autonomic nervous system (Kim et al., 2018; Marques et al., 2010). It is known that one of the main endocrine responses to stress is activation of the HPA axis, which results in a rapid increase in circulating glucocorticoids (Aguilera, 1998; Levine, 2005). Both physiological and psychological stress activate the HPA axis, resulting in the release of corticotrophin releasing hormone by the hypothalamus in the hypothalamic–pituitary circulation, thereby stimulating the release of adrenocorticotropic hormone (ACTH) from the pituitary into the general circulation. This in turn stimulates systemic release of glucocorticoids by the cortex of the adrenal gland (Myers et al., 2014; Kolber et al., 2008). In the same way, the autonomic nervous system quickly promotes physiological changes through the sympathetic nervous system and parasympathetic nervous system, via a decrease of the parasympathetic response and activation of the sympathetic response (Porges, 1995).These changes are followed by several alterations in the central nervous system, including the release of noradrenaline from the locus coeruleus (Curtis et al., 1997).

Chronic stress protocols are capable of modifying HPA axis activity, besides stimulating separate regions of the nervous system to elicit appropriate responses (Scharf and Schmidt, 2012). A study performed by Cruz et al. (2012) showed that animals subjected to RRS present a habituation in the release of corticosterone in response to a new acute exposure to restraint stress, whereas animals exposed to CVS presented a sustained elevation in corticosterone levels (Magariños and McEwen, 1995; Gadek-Michalska and Bugajski, 2003). However, chronic activation of the HPA axis is capable of promoting physiological changes, resulting in hypertension, immunosuppression and behavioral changes (McEwen, 2000; McEwen et al., 2012). In addition, cardiovascular alterations caused by chronic stress are followed by modifications in baroreflex activity (Duarte et al., 2015a), a neuronal mechanism that is responsible for maintaining the BP at homeostatic levels by influencing variables such as f_H. Studies have demonstrated that adult rodents exposed to CVS had impairment in baroreflex activity (Grippo et al., 2008). Nevertheless, modifications in baroreflex activity and autonomic imbalance have also been reported in adult rats exposed to RRS (Haibara et al., 1999). According to Engi et al. (2012), impairment of baroreflex activity may contribute to the pathogenesis of various cardiovascular diseases. In this context, efficient reversal of stress-induced autonomic changes may be important to reduce the chance of long-term cardiovascular pathologies (Crestani et al., 2010).

In addition to the baroreflex, the chemoreflex is a relevant neural mechanism responsible for evoking respiratory and cardiovascular responses as a result of specific hypoxic or hypercapnia conditions (Fitzgerald, 2000). A widely used animal model of acute and transient cytotoxic hypoxia is the systemic infusion of low doses of potassium cyanide (KCN) (Haibara et al., 1995; Olivan et al., 2001; Kuntze et al., 2016; Granjeiro et al., 2011). The physiological responses characteristic of activation of the chemoreflex, using such a model, is tachypnea accompanied by a BP rise and bradycardia (Franchini and Krieger, 1993; Haibara et al., 1999; Fernandes et al., 2005; Kuntze et al., 2016), followed by behavioral responses characterized by arousal and increased exploration of the environment (Franchini and Krieger, 1993).

Some studies have shown that stress interferes with respiratory activity and that early stress exposure alters the developmental trajectory of the respiratory control system (Bavis and Mitchell, 2008; Cayetanot et al., 2009). According to Genest et al. (2004), maternal separation in early life induces ventilatory alterations in rats, such as altered minute ventilation (V_E) and increased BP. Studies have also reported that animals undergoing neonatal stress have similar respiratory and cardiovascular changes to those in patients who have obstructive sleep syndrome, and become more sensitive to chemoreflex activation (Genest et al., 2004; Gulemetova et al., 2013). Most of the studies cited above demonstrate the effect of stress early in life or in adolescent animals. However, there are no data in the literature demonstrating the effect of the different types of stress in the autonomous baroreflex and chemoreflex mechanisms of adult animals.

According to the evidence of cardiovascular and autonomic changes induced by chronic stressors, the hypothesis of the present study was that different types of chronic stress (RRS and CVS) in adult animals alter the cardiovascular and ventilatory responses controlled by the autonomic system such as the baroreflex and chemoreflex, as well as interfering with heart rate variability. Based on this, our purpose was to investigate the cardiovascular and respiratory consequences of exposure to RRS and CVS in adult animals.

A total of 51 adult male Wistar rats (aged 60 days) were used in the present study. All animals were kept in the Animal Care Unit of the School of Medicine of Ribeirão Preto, University of São Paulo Ribeirão Preto (USP). The animals were housed in groups of five per cage in a temperature-controlled room (24±1°C) under a 12 h light/dark cycle (lights on at 06:00 h) and were given water and food ad libitum. All experiments were performed in accordance with Ethical Principles for Animal Experimentation followed by the Brazilian Committee for Animal Experimentation (COBEA) and approved by the Committee of Ethics in Animal Research of the School of Medicine of Ribeirão Preto, University of São Paulo (number 175/2014).

A schematic representation of the complete protocol is presented in Fig. 1. The chronic stress regimens were based on protocols previously used and modified accordingly (Marin et al., 2007; Cruz et al., 2012; Duarte et al., 2015b). Two animal models of stress were chosen. RRS was used as a homotypic stressor, whereas CVS was used as a heterotypic stressor. A total of 31 animals were submitted to stress sessions. Animals of the RRS group were restrained in plastic cylindrical tubes, 17 cm long and 7.5 cm high, for 1 h daily starting at 10:00 h for 14 consecutive days. Animals of the CVS group were exposed to different stressors on variable schedules for 14 consecutive days (Table 1). The protocols were initiated 7 days apart from each other, so that the experiments performed with the respective groups also complied with this interval. Animals of the control group were left undisturbed, except for cleaning of the cages. All tests described below, except for the behavioral tests, were carried out on the same animals.

Adult animals were randomly divided into three groups: (1) control, (2) RRS and (3) CVS; the mass of the animals was measured every day before the stress protocol. Animals of the CVS and RRS groups were subjected to daily sessions of stress for 14 consecutive days. On the 14th day, after the last session of stress, animals in all experimental groups were prepared for surgery; the tests were performed 24 h later. On test days, animals were transferred to the experimental room in their home box and allowed to adapt for 30 min to experimental room conditions before experiments began. In the sequence, a 10 min period of basal cardiovascular activity was recorded for power spectral analysis and spontaneous baroreflex sensitivity. Afterwards, we performed the baroreflex and chemoreflex stimulation following the methods mentioned above (Fig. 1).

This test was developed with an independent group of 20 animals. At the end of the chronic stress protocol, ingestion of sucrose (2% solution) was evaluated as a measure of anhedonia (decrease in or absence of the ability to experience pleasure) through a preference test with sucrose. Therefore, the protocol was carried out over 2 days. On each day, the animals were treated individually. On the first day, individual animals were habituated to samples for a period of 5 h. The following day (testing day), the two bottles containing either pure water or sucrose solution were first weighed according to protocol (initial mass) (Willner, 1997) then offered to rats for a period of 10 h; they were then weighed again (final mass). Consumption of both water and sucrose solution was calculated as the difference between initial and final mass. The preference for sucrose was calculated as a percentage by dividing sucrose consumption (in grams) by total consumption (i.e. water consumption+sucrose consumption in grams) and multiplying it by 100.

Animals were anesthetized with tribromoethanol (250 mg kg⁻¹, i.p.) and a catheter (a 4 cm segment of PE-10 that was heat-bound to a 13 cm segment of PE-50; Clay Adams, Parsippany, NJ, USA) was inserted into the abdominal aorta through the femoral artery for BP recording. A second catheter was implanted into the femoral vein for vasoactive drug infusion. Both catheters were tunneled under the skin and exteriorized on the animal's dorsum. After surgery, the animals were given a polyantibiotic preparation of streptomycin and penicillin (0.3 ml, i.m.; Pentabiotic, 80,000 IU; Fort Dodge, Campinas, SP, Brazil) to prevent infection and also received the non-steroidal anti-inflammatory flunixine meglumine (2.5 mg kg⁻¹, s.c., Banamine; Intervet Schring-Plough, Cruzeiro, SP, Brazil) for post-operative care.

The pulsatile arterial pressure of freely moving animals was recorded using an ML870 preamplifier (LabChart) and an acquisition board (PowerLab, ADInstruments, Bella Vista, NSW, Australia) connected to a computer. Mean arterial pressure (MAP) and heart rate (f_H) values were derived from pulsatile recordings and processed on-line.

Power spectral analysis of systolic arterial pressure (SAP) and pulse interval (PI) was employed to analyze autonomic activity controlling the blood vessels and heart, respectively. For this purpose, relatively stable 10 min duration segments of the pulsatile arterial pressure signals were used in order to extract PI and SAP. Using fast Fourier transform spectral analysis (Software Cardioseries v2.4, https://www.sites.google.com/site/cardioseries/home), the overall variability of these series was calculated in the time and frequency domain. The time domain analysis consisted of calculating the variance in the entire time series. For frequency domain analysis, the PI spectra were integrated into low (LF: 0.20–0.75 Hz) and high (HF: 0.75–3.00 Hz) frequency bands, whereas the SAP spectra were integrated only into the LF band (0.20–0.75 Hz).

The baroreflex sensitivity was calculated in the time domain as follows. In the time series, the SAP and PI were processed by Cardioseries v2.4 software (https://www.sites.google.com/site/cardioseries/home). For this, beat to beat values of SAP and PI were analyzed for identification of sequences in which SAP increases were associated with PI lengthening (up sequence) or SAP decreases were associated with PI shortening (down sequence). Differences were considered only when changes greater than or equal to 1 mmHg (SAP) and 1 ms (PI) were observed. The spontaneous baroreflex sensitivity was determined using the average slope of the linear regression between the SAP and PI values found for each sequence.

The baroreflex was activated by phenylephrine (α1-adrenoceptor agonist; 50 μg kg⁻¹, 0.34 ml min⁻¹) or sodium nitroprusside (SNP; NO donor; 50 μg kg⁻¹, 0.8 ml min⁻¹) infusion using an infusion pump (KD Scientific, Holliston, MA, USA). The phenylephrine or SNP infusion lasted 30–40 s and caused, respectively, an increase and a decrease in BP (Alves et al., 2009).

Baroreflex curves were constructed by matching variation in MAP (ΔMAP) with f_H responses (Δf_H). Paired values for ΔMAP and Δf_H were plotted to create sigmoid curves for each rat, which were used to determine baroreflex activity (Resstel et al., 2004). To analyze bradycardic and tachycardic responses separately, f_H values matching 10, 20, 30 and 40 mmHg of MAP changes were calculated (Alves et al., 2009). Values were plotted to create linear regression curves for each rat, and their slopes were compared to determine changes in baroreflex gain.

The chemoreflex was activated by systemic bolus injections of 0.1 ml KCN i.v. (40 μg per rat; Merck, Darmstadt, Germany) following procedures described by Franchini and Krieger (1993) and experimental conditions previously validated (Granjeiro et al., 2011).

The magnitude of changes in MAP and f_H in response to chemoreflex activation was quantified at the peak, and the duration of the responses was recorded (Granjeiro et al., 2011; Kuntze et al., 2016).

The respiratory frequency (f_R) response induced by chemoreflex activation was evaluated by the whole-body plethysmography method for small animals described by Bartlett and Tenney (1970). Rats were kept inside a 6 l Plexiglas^® chamber (with sealed exit ports for catheters) which allowed them to move freely. After the chamber was closed for f_R recording, pressure oscillations originated by breathing movements were detected by a differential pressure transducer (ML141 Spirometer, PowerLab, ADInstruments). f_R was expressed as the number of cycles per minute, which was calculated from the extrapolation of the plestimographic signal, regarding the volume calibration accomplished for each measurement (1 ml injection of air into the chamber). Respiratory cycles were acquired 20 s before and 20 s after chemoreflex activation with KCN (i.v.) and were divided into 2 s intervals by the acquisition software program (PowerLab, ADInstruments) with manual corrections, when necessary. Equations described by Drorbaugh and Fenn (1955) allowed calculation of the tidal volume (V_T), and V_E was obtained as the product of f_R and V_T. The V_T and V_E parameters were only calculated 10 points before chemoreflex activation (where the 10 points correspond to 20 s prior to chemoreflex activation with KCN, divided into 2 s intervals for analysis) to avoid incorrect values given that vigorous behavioral responses are evoked by KCN injection.

All the data presented a normal distribution and homogeneity of variance. Data are expressed as means±s.e.m. Results were analyzed using one-way analysis of variance (ANOVA) followed by Bonferroni's post hoc test for identification of differences between the groups from GraphPad Prism software version 7 (San Diego, CA, USA). Comparison of the effect of stress on body mass was realized using two-way ANOVA, with stress (control, RRS and CVS) as independent factors, and time as a repeated measurement. Results of statistical tests with P<0.05 were considered significant.

The animals subjected to CVS and RRS had a lower preference for sucrose solution when compared with the control group (control 97.31±0.66%, RRS 81.13±5.84%, CVS 78.39±4.05%; F_2,17=7.07, P<0.05) (Fig. 2A).

Analysis of body mass revealed that animals on the RRS and CVS protocols presented a reduction in body mass when compared with control groups (control 400±20.76 g, RRS 375±15.02 g, CVS 376.7±14.05 g; F_2,28=9.298, P<0.05). Similarly, there was a main effect of time (F_2,56=267.9, P<0.05), especially from the 8th day of stress (Fig. 2B).

There was no change in basal levels of either MAP (F_2,30=0.2671, P>0.05) or f_H (F_2,28=0.05448, P>0.05) between the different groups (Fig. 3).

We found no difference in PI (F_2,28=0.7181, P>0.05) and in total PI variability (F_2,28=0.8925, P>0.05) among the stress and control groups (Fig. 4A,B). However, RRS and CVS were able to attenuate both LF (F_2,28=7.883, P<0.05) and HF band power (F_2,28=7.299; P<0.05) (Fig. 4C,D). In addition, it was verified that chronic stress was able to decrease the power of the LF bands (F_2,28=5.280, P<0.05), increase the power of the HF bands (F_2,28=5.280, P<0.05) (Fig. 4E,F) and decrease the band power ratio when compared with the control group (F_2,28=4.307, P<0.05) (Fig. 4G).

We observed no difference in SAP (F_2,28=1.267, P>0.05) or in total SAP variability (F_2,28=0.3351, P>0.05) among the groups (Fig. 5A,B). The RRS group showed an increase in LF band power (F_2,28=3.488, P<0.05) when compared with the control group (Fig. 5C).

Baroreflex activity assessed by the sequence analysis technique indicated that both RRS and CVS caused a reduction in total gain (F_2,28=12.77, P<0.05) (Fig. 6A). In addition, chronic stress also decreased the number of up sequences (F_2,28=12.74, P<0.05) and the number of down sequences (F_2,28=6.024, P<0.05) (Fig. 6B,C).

RRS increased the slope of the linear regression of bradycardic (control −2.07±0.25, RRS −2.99±0.35; F_2,25=4.090, P<0.05) and tachycardic responses (control −1.59±0.18, RRS=−2.45±0.34; F_2,25=3.407, P<0.05) (Fig. 7A). However, there was no significant difference between the CVS group and the control group in bradycardic (control −2.07±0.25, CVS −2.04±0.17) and tachycardic responses (control −1.59±0.18, CVS −1.46±0.27). Apart from BP₅₀ (median blood pressure, which is the value of MAP when 50% of the f_H is altered), the sigmoid curve parameters (gain, lower and upper f_H plateau and f_H plateau range) were increased in the RRS group (Table 2).

The RRS and CVS groups showed a decrease in the magnitude of the pressor response to chemoreflex activation when compared with the control group (F_2,28=10.32, P<0.05) (Fig. 8A). However, there was no significant difference between groups regarding the bradycardic response (F_2,28=1.948, P>0.05) (Fig. 8B).

Both chronic stress groups showed a greater average maximum increase of f_R due to activation of the chemoreflex when compared with the control group (F_2,28=5.317, P<0.05) (Fig. 8C). Regarding the baseline ventilatory parameters, recorded before chemoreflex activation, both the RRS and CVS groups presented attenuation in the V_T (F_2,28=8.154, P<0.05; Fig. 8D) and V_E (F_2,28=6.411, P<0.05; Fig. 8E) response when compared with the control group. Additionally, basal f_R was increased only in the CVS group (F_2.28=4365, P<0.05) (Fig. 8F).

The hypothesis of the present study was that different types of chronic stress (RRS and CVS) alter the cardiovascular and ventilatory responses controlled by the autonomic system such as the baroreflex and chemoreflex, as well as interfering in heart rate variability. Our main findings were: (1) RRS but not CVS was able to potentiate tachycardic and bradycardic responses to baroreflex activation induced by vasoactive drugs; (2) chronic stress decreased the variability of PI and spontaneous baroreflex sensitivity and decreased the magnitude of the chemoreflex-induced BP rise; and (3) RRS and CVS decreased the basal respiratory V_T and V_E parameters.

Our results also corroborate with the literature data, which demonstrate that animals subjected to chronic stress protocols (RRS and CVS) present a lower body mass gain over time when compared with control animals that did not receive any type of stress. In addition, both protocols promoted a decrease in the sucrose preference test. The preference for sucrose has been used as a marker of chronic stress exposure in over 50 studies, indicating a depressed-like behavior in animals (Willner, 2005).

In addition to these changes in mass and behavior, several studies have shown that stress can modify the circulatory system activity, depending on the type of stressor involved (Esch et al., 2002; Crestani, 2016; Duarte et al., 2015b), as stress exposure increases MAP and f_H (Grippo et al., 2008; Tavares and Correa, 2006; Irvine et al., 1997; Carrive, 2006; Barr; et al., 2004; Duarte et al., 2015b). Some studies compared the impact on arterial pressure in RRS versus CVS and observed a small pressor effect after 10 days. However, our findings demonstrate that chronic stress was not able to change the baseline parameters of MAP and f_H, which complies with previous observations (Grippo et al., 2006; Grippo et al., 2003). According to Crestani (2016), cardiovascular dysfunction induced by chronic stressors may be influenced by age; thus, differences in rat strains and age could explain these discrepancies.

In addition, cardiovascular alterations caused by chronic stress may reflect baroreflex impairment (Duarte et al., 2015a). Changes in the baroreflex were reported after exposure of different chronic stressors (Almeida et al., 2015; Cruz et al., 2012; Duarte et al., 2015a; Crestani, 2016). Based on this, our results demonstrated that RRS but not CVS was able to increase the magnitude of both tachycardic and bradycardic components of the cardiac baroreflex. Therefore, there was a greater variation of the f_H in response BP variation induced by RRS. However, the effect of chronic stress on the baroreflex seems to be dependent on the duration and type of stress. Differently from our results, in another study, a CVS protocol for 14 days modified baroreflex activity, but this protocol was performed in adolescent animals (Almeida et al., 2015). Other studies have shown that the 4 week CVS protocol did not modify baroreflex activity in adult animals (Grippo et al., 2008; Simas et al., 2018), corroborating our findings. Few studies to date have investigated the influence of predictability by directly comparing cardiovascular and autonomic changes induced by predictable versus unpredictable stress protocols (Almeida et al., 2015; Cruz et al., 2012; Duarte et al., 2015a; Crestani, 2016). Most results are contradictory and this may be related to the age difference, rat strain, type of stress and intensity of aversive stimulus. In a study by De Boer et al. (1989) it was shown that regular, but not irregularly, applied noise increased plasma noradrenaline concentration, while noradrenaline response to noise decreased in animals subjected to irregular protocols (De Boer et al., 1989). In contrast, it has been shown that although repeated exposure to restraint stress indicates a decrease in pressure and the tachycardic response (Bechtold et al., 2009), other studies have reported similar cardiovascular responses during both acute and repeated chronic exposure (Conti et al., 2001; Daubert et al., 2012), suggesting that there may not be a cardiovascular habituation. Thus, it is possible that RRS promotes a higher cardiovascular demand, which leads to baroreflex impairment due to increased BP and f_H during daily stress exposure, which does not happen in the CVS protocol. Reports in the literature demonstrate that exacerbated tachycardic responses constitute an important risk factor for myocardial ischemia and sudden death (Palatini and Julius, 1997). Thus, the potentiation of tachycardic reflex responses after RRS may be a risk factor for cardiovascular disease. However, facilitation of bradycardic responses may serve as a mechanism for attenuation of cardiac changes induced by chronic stress (Duarte et al., 2015a), which may contribute to the lack of effect on the basal levels of MAP and f_H.

It is well established that the baroreceptor reflex is a homeostatic response modulated by the autonomic nervous system. In mammals, baroreceptors are present in nerve endings found in the carotid sinus and aortic arch (Papademetriou et al., 2011). However, structures of the limbic system and forebrain have been shown to connect to brainstem regions responsible for modulation in autonomic responses (Berntson and Cacioppo, 2007).

In this context, studies have shown that the stimulation or inhibition of these limbic structures, such as the medial prefrontal cortex (Resstel et al., 2004; Resstel et al., 2006; Lagatta et al., 2015), anterior hypothalamus (Simon et al., 1985) or posterior hypothalamus (Bauer et al., 1988), hippocampus (Ferreira-Junior et al., 2017) and periaqueductal gray matter (Pelosi et al., 2007), is able to influence the function of the baroreflex. Similarly, chronic stress causes structural remodeling in neurons in both the hippocampus and the prefrontal cortex (Cook and Wellman, 2004; Radley et al., 2004; McEwen et al., 2016). Thus, changes observed in the baroreflex response may reflect the alterations induced by chronic stress in these structures.

Stressors are often associated with an increase in sympathetic cardiac control, or a decrease in parasympathetic activity, or both (Berntson and Cacioppo, 2007). On account of this, there is an increasing interest in the study of heart rate variability among researchers from diverse fields. Several studies have investigated the association between stress and the decrease of heart rate variability (HRV) (Clays et al., 2011; Kang et al., 2004; Kim et al., 2018; Duarte et al., 2015b). It is noteworthy to mention that the analysis of f_H and BP fluctuations reflects the ability of the regulatory mechanisms to respond to a wide variety of scenarios. It is well known that the higher the HRV, the better is the capacity to adjust f_H level accordingly to internal and/or external stimuli. However, the lower the BP variability, the better are the regulatory mechanisms that monitor BP oscillations on a beat-to-beat basis (Kim et al., 2018). In our study, chronic stress promoted alterations in sensitivity of the spontaneous baroreflex and HRV. RRS animals showed an increase in the LF band of the SAP spectrum. LF oscillations of both SAP and PI are associated with cardiac sympathetic modulation (Malik and Camm, 1995). Despite the enhancement in the LF band of the SAP spectrum, it was not enough to modify BP. There were no changes in PI, but the chronic stress protocols decreased the potency of the LF and HF bands, as well as the power ratio (LF/HF) of these bands. The LF oscillations are mediated, essentially, by the sympathetic nervous system, and depend on the integrity of the baroreflex (Malik and Camm, 1995). In contrast, HF oscillations are related to the parasympathetic nervous activity (Malik and Camm, 1995). Despite sympathetic and parasympathetic potentiation of baroreflex activation with vasoactive drug infusion, the LF (sympathetic) and HF (parasympathetic) frequency bands of the PI spectrum were decreased.

Several studies in the literature report that chronic stress promotes increased sympathetic basal activity and alters baroreflex sensitivity (Grassi et al., 2004; Malliani et al., 1991; Duarte et al., 2015b). Recent work by Duarte et al. (2015b) demonstrated that chronic stress in adolescent animals increases the power of the LF band, the HF band and the LF/HF ratio of the PI, indicating an enhancement in both cardiac sympathetic and parasympathetic drives. In addition, regarding the sensitivity of the spontaneous baroreflex, the animals subjected to the RRS protocol presented an increase in the total baroreflex sequence gains, demonstrating an increase in the spontaneous baroreflex sensitivity. According to Grassi et al. (2004), alterations in baroreflex activity are related to hyperactivity of sympathetic tone.

Our results also show that in addition to changes in the potency of the LF and HF bands, chronic stress was also able to decrease baroreflex sensitivity analyzed by the sequence method. These results are different to the aforementioned data. Nevertheless, it is important to highlight that sympathetic hyperactivity (Mortara et al., 1994), baroreflex deficiency (Casadei et al., 1996) or low sinus node responsiveness (Malik and Camm, 1993) may reduce the LF component. This reduction in the LF band, the LF/HF ratio, in addition to the decrease in spontaneous baroreflex sensitivity, can probably be explained by a sympathetic hyperactivity, making LF oscillations less evident in the PI spectrum. For example, in the case of patients with the most severe stages of congestive heart failure (van de Borne et al., 1997), the neurohormonal excitation displays a reduction of the heart rate modulation such that only small fluctuations that are synchronous during respiratory activity are detectable (Mortara et al., 1994). In the same sense, Borchini et al. (2018) demonstrated that a high level of stress in the workplace for a period of 1 year is associated with consistent reductions of both HF and LF power and LF/HF ratio. Moreover, LF and HF values in rest periods are also reduced or absent (Borchini et al., 2018). As previously outlined, various limbic structures are involved in the autonomic system (Resstel et al., 2004, 2006; Lagatta et al., 2015; Bauer et al., 1988; Ferreira-Junior et al., 2017; Simon et al., 1985). Recently, a meta-analysis study conducted by Thayer et al. (2012) showed that several areas, including the amygdala and the medial prefrontal cortex, which are involved in the perception of threat and safety, are also associated with HRV (Thayer et al., 2012). Based on observations of stress-associated HRV and existing neurobiological evidence, HRV may be used as an objective assessment of stress and mental health. In addition, the impairment of these mechanisms by chronic stress may be the cause of the development of several cardiovascular pathologies.

Another important autonomic response that may be altered by chronic stress is the chemoreflex. This mechanism mediates cardiorespiratory responses (Fitzgerald, 2000; Paula-Ribeiro and Rocha, 2016), by maintaining arterial blood gases and acid–base balance within normal limits. However, dysfunction in the chemoreflex may impair ventilatory control and may play an important role in the pathophysiology of highly prevalent diseases, like chronic heart failure, hypertension and chronic obstructive pulmonary disease (COPD) (Paula-Ribeiro and Rocha, 2016). Our study demonstrated that RRS and CVS reduce the magnitude of the pressor response without altering the fall in f_H. This is in accordance with previous evidence supporting the concept that the sympathetic and parasympathetic components of the chemoreflex are mediated by different neural pathways and independent neurochemical mechanisms (Franchini and Krieger, 1993; Haibara et al., 1995).

Apart from their role in baroreflex control, supramedullary structures are also able to modulate cardiovascular responses activated by the chemoreflex, as inhibition of the pre-limbic cortex with cobalt chloride decreased the pressor response in awake animals without altering the bradycardic response when compared with the control group (Granjeiro et al., 2011). Additionally, the ventral hippocampus is involved in the modulation of the parasympathoexcitatory component of the peripheral chemoreflex (Kuntze et al., 2016). Chronic stress-activated regions for both RRS and CVS have been identified in the dentate gyrus, through an increase of FosB/DFosB neuronal activity marker. Additionally, CVS chronically activates the medial prefrontal cortex (Flak et al., 2012). Based on these data, chronic stress probably triggers changes in these brain areas, which in turn may impact alterations in cardiovascular responses to chemoreflex activation.

In addition to cardiovascular alterations during chemoreflex activation, chronic stress led to basal respiratory changes, such as decreased V_T and V_E and increased magnitude of f_R in response to chemoreflex activation. Additionally, both chronic stress groups displayed an increment in the mean of the maximal response of f_R. It is known that the hypoxic ventilatory response is not the result of a single mechanism, but rather is an interaction between several different mechanisms. These mechanisms depend on specific evoking stimuli, such as intensity and exposure to hypoxia, the time course of the response (seconds to years) and the effects on ventilation components (tidal volume versus frequency) (Powell et al., 1998).

Some studies have shown that stress interferes with respiratory activity and that early stress exposure alters the developmental trajectory of the respiratory control system (Bavis and Mitchell, 2008; Cayetanot et al., 2009). According to Genest et al. (2004), early-life maternal separation induces modifications in V_E, besides altering the central and peripheral chemoreflex components of respiratory control (Kinkead et al., 2001). However, although there are reports in the literature evidencing respiratory changes caused by chronic stress in adult life, animal studies on the subject are still scarce.

In summary, the mechanisms of the baroreflex and chemoreflex play a major role in the control of the cardiovascular system via the profound influence they exert on autonomic outflow. In this way, the findings of the present study demonstrate the impact of chronic stress not only on depressive-like behavior but also on alterations of the autonomic baroreflex response and cardiocirculatory variability (SAP and PI). Additionally, our study is one of the first to show that chronic stress decreases the magnitude of the pressor response and potentiates respiratory responses to chemoreflex activation, which can trigger cardiovascular and respiratory pathologies. These alterations seem to constitute important mechanisms in the etiology of chronic stress in cardiovascular modifications. Moreover, stress is a risk factor for cardiovascular disease; however, the mechanisms linking chronic stress to increased risk of cardiac events may be partially attributed to direct or indirect effects on autonomic function. Thus, the present results provide evidence that the autonomic alterations could reflect important mechanisms underlining the etiology of cardiovascular diseases which are associated with chronic stress exposure.

The authors wish to thank L. H. A. Camargo and O. Mesquita for technical help.