This study used 76 male Wistar rats (230–250 g; 60 days old) provided by the Central Animal Facility of the UNESP (Botucatu, SP, Brazil). All rats had free access to standard laboratory food and water and were kept in acclimatized rooms (temperature at 24 ± 2°C and humidity at 50 ± 5%) with lights on between 7:00 and 19:00 h. Experimental procedures were approved by the Institutional Ethical Committee for Use of Animals (approval # 38/2018).

Animals were brought to the trial room in their own home-cages and were allowed to adapt to the experimental room conditions (at least 60 min) before beginning the tests. The trial room was temperature-controlled (24°C) and isolated from the other rooms.

For restraint stress, animals were kept for 60 min into a cylindrical plastic tube, as previously described (Alves et al., 2010, 2014). Animals were subjected to only one restraint session to avoid the habituation process (Benini et al., 2019; Santos et al., 2020).

The procedures for the assessment of Fos-positive cells were as previously described (Gomes-de-Souza et al., 2021). Briefly, 90 min after the onset of the restraint stress (i.e., 30 min following the termination of the restraint stress session), the rats were anesthetized (urethane, 1.2 g/kg, i.p.) and subjected to perfusion. Later, the brain was obtained and fixed [paraformaldehyde and 30% sucrose solution in phosphate-buffered saline (PBS)]. Then, the brains were sectioned into 35-μm frontal cuts (CM1900, Leica, Wetzlar, Germany) and the slices containing the anterior, rostral posterior, and caudal posterior subregions of the IC were obtained. The IC subregions were determined according to Paxinos and Watson (1997). The slices were washed (PBS) and incubated for 1 h at room temperature in a blocking solution (0.25% Triton X-100 and 3% goat serum in PBS). Next, the slices were incubated for 24 h at 4°C with an anti-Fos primary antibody produced in rabbits (cat.# 5348; dilution 1:2,000; Cell Signaling, Danvers, MA, United States). Later, the slices were washed (PBS) and incubated for 2 h at room temperature with biotinylated antirabbit secondary antibody (cat.# BA-1000; dilution 1:600; Vector Laboratories, Burlingame, CA, United States). The slices were then washed (PBS) and incubated for 1 h in avidin–biotin–peroxidase solution [cat.# PK-6100; Vectastain Elite ABC-HRP Kit, Peroxidase (Standard); Vector Laboratories, Burlingame, CA, United States]. Then, the slices were washed and incubated for 7 min in 3, 3’-diaminobenzidine tetrahydrochloride hydrate (cat.# D5637, Sigma-Aldrich, St Louis, MO, United States). Finally, the slices were washed and mounted on gelatinized slides.

Images of the right and left hemispheres were captured in each slice, and the analysis was performed in at least two slices per animal. The number of Fos-immunoreactive cells was calculated from a fixed area of each IC subregion using the ImageJ software. The results are presented as the number of Fos-immunoreactive neurons/mm².

Five days before the experiments, rats were anesthetized with tribomoethanol at a dose of 250 mg/kg i.p. The animal’s head was shaved and then fixed to a stereotaxic apparatus (Stoelting, Wood Dale, IL, United States), and the surgical field was cleaned with 70% alcohol and iodine. A solution of lidocaine with vasoconstrictor (2% lidocaine + 3% phenylephrine, Harvey Química Farmacêutica, Catanduva, SP, Brazil) was administrated for scalp anesthesia and bleeding reduction. Then, the skull was exposed, and the region was cleaned with saline and hydrogen peroxide (10%). Bilateral cannulas were then directed to either the anterior, rostral posterior, or caudal posterior subregions of the IC. The coordinates used for implanting the guide cannulas were anteroposterior, +11.7 mm (anterior IC); +9.1 mm (rostral posterior IC) or +7.0 mm (caudal posterior IC) in relation to the interaural; lateral, +4 mm (anterior IC), +5 mm (rostral posterior IC), or +6.2 mm (caudal posterior IC) from the middle suture; vertical, −4.5 mm (anterior IC), −5.0 mm (rostral posterior IC), or −6.0 mm (caudal posterior IC) in relation to the skull (Paxinos and Watson, 1997). Cannulas were implanted without lateral angulation, and incisor bar was set at −3.2 mm. Two holes were made in the skull using a dental drill through which the bilateral cannulas (26 G, 12 mm) were introduced. The cannulas were fixed to the skull with a self-curing acrylic resin (Simples, DFL, Ind. Com., Rio de Janeiro, RJ, Brazil) and stainless-steel screws were previously implanted in the skull to fix the resin. A 0.2-mm-diameter mandrel was introduced into the cannula to prevent it from clogging. At the end of the surgery, the rats were treated with a veterinary antibiotic intramuscularly (560 mg/ml/kg) and the non-steroidal anti-inflammatory flunixin meglumine by the subcutaneous route (0.5 mg/ml/kg).

Twenty-four hours before the trial, the rats were again anesthetized (tribromoethanol, 250 mg/kg, i.p.) and a polyethylene catheter was implanted into the femoral artery for arterial pressure and HR recording. The cannula consisted of a PE-10 polyethylene segment (4–5 cm) welded to a PE-50 polyethylene segment (12–13 cm) (Clay Adams, Parsippany, NJ, United States). The cannulas were filled with a saline solution containing anticoagulant (50 IU/ml of heparin; Hepamax-S, Blausiegel, Cotia, SP, Brazil) and occluded with a metal pin before the implantation. The catheter was exteriorized in the animal’s dorsal region and fixed to the skin by surgical suture.

Animals were individually housed during the postoperative period and trials. At the end of the surgery, the animals were treated with the non-steroidal anti-inflammatory flunixin meglumine by the subcutaneous route (0.5 mg/ml/kg).

Cardiovascular parameters (i.e., arterial pressure and HR) were recorded as previously described (Almeida et al., 2015; Oliveira et al., 2015). Briefly, the arterial catheter (implanted into the femoral artery) was connected to a pressure transducer and pulsatile arterial pressure (PAP) was recorded using an amplifier connected to an acquisition system (ADInstruments, Australia). The mean arterial pressure (MAP) and HR were calculated from the PAP signal.

Tail skin temperature recording was performed as previously described (Oliveira et al., 2015; Gomes-de-Souza et al., 2021). Briefly, the recordings were performed using a thermal camera (IRI4010, InfraRed Integrated Systems Ltd., Northampton, United Kingdom) and the temperature was obtained at five sites along the tail, and then the mean was obtained for each record (Oliveira et al., 2015; Gomes-de-Souza et al., 2021).

CoCl₂ (non-selective synaptic inhibitor) (cat. # 232696; Sigma-Aldrich, St Louis, Missouri, United States), tribromoethanol (cat. # T48402, Sigma-Aldrich), and urethane (cat. # U2500, Sigma-Aldrich) were dissolved in saline solution (0.9% NaCl). The antibiotic (Pentabiotico, Fort-Dodge, Campinas, SP, Brazil) and the non-steroidal anti-inflammatory flunixin meglumine (Banamine, Schering Plough, Cotia, SP, Brazil) were used as supplied by the manufacturers.

The injection needles (33 G, Small Parts, Miami Lakes, FL, United States) used for intra-brain pharmacological treatment (1 mm longer than the guide-cannulas) were connected to a microsyringe (7002-KH, Hamilton Co., Reno, NV, United States) using a polyethylene tube (PE-10). Vehicle and CoCl₂ were microinjected in a final volume of 100 nl/side (Alves et al., 2010; Marins et al., 2016).

Following the termination of each experiment, the rats subjected to intra-brain pharmacological treatment were anesthetized (urethane, 1.2 g/kg, i.p.) and 1% Evan’s blue dye was bilaterally microinjected (100 nl/side) in the brain to determine the injection sites. Then, the brain was removed, fixed in 10% formalin, and subsequently sectioned into 50-μm frontal cuts (CM1900, Leica, Wetzlar, Germany). Microinjection sites were determined in a microscope (Zeiss Axioskop 2) based on Paxinos and Watson (1997).

Data were expressed as mean ± standard error of the mean (SEM). Baseline parameters of MAP, HR, and tail skin temperature, as well as the number of Fos-immunoreactive cells, were analyzed using Student’s t-test. Two-way analysis of variance (ANOVA), with treatment as an independent factor and time as a repeated measure, was used to analyze the time-course curves. The mean of cardiovascular and tail skin temperature responses during the restraint and recovery periods was also obtained and analyzed using the two-way ANOVA (treatment as an independent factor and time as a repeated measure). P < 0.05 was assumed to be significant.

Figures 2A,B also presents coronal brain sections of representative animals showing Fos-immunoreactive cells in the anterior, rostral posterior, and caudal posterior subregions of the IC of control animals and rats subjected to restraint stress, as well as an indication of the IC locations where the analysis was performed. Analysis indicated that exposure to a 60-min session of restraint stress (n = 9) decreased the number of Fos-positive cells in the anterior (t = 2.44, df = 15, p = 0.0276) (Figure 2C) and caudal posterior (t = 2.27, df = 15, p = 0.0388) (Figure 2E) subregions of the IC (Figure 2) when compared with control animals (n = 8). Conversely, increased number of Fos-immunoreactive cells was found in the rostral posterior IC following acute restraint stress (t = 2.95, df = 15, p = 0.0106) (Figure 2D).

Analysis of the basal parameters indicated that bilateral microinjection of the non-selective synaptic inhibitor CoCl₂ (1 mM/100 nl) into either the rostral posterior or caudal posterior subregions of the IC did not affect the baseline values of MAP, HR, and tail skin temperature (Table 1). However, analysis of basal parameters in animals that received CoCl₂ into the anterior IC indicated enhanced tail skin temperature values when compared with vehicle-treated animals, without changes of MAP and HR (Table 1).

Analysis of the time-course curves indicated that an acute 60-min session of restraint stress increased MAP [time factor: F_{(65, 975)} = 14.52; p < 0.0001] and HR [time factor: F_{(65, 975)} = 11.06; p < 0.0001], and decreased tail skin temperature [time factor: F_{(13, 195)} = 16.79; p < 0.001] (Figures 3A–C). Treatment of the anterior subregion of the IC with the non-selective synaptic inhibitor CoCl₂ (1 mM/100 nl, n = 9) did not affect the restraint-evoked increase in MAP [F_{(1, 15)} = 0.90, p = 0.3633] and HR [F_{(1, 15)} = 0.3, p = 0.5691], as well as the drop in tail skin temperature [F_{(1, 15)} = 2.23, p = 0.1608] when compared with animals treated locally with vehicle (100 nl, n = 8) (Figures 3A–C). We identified an interaction between time and treatment for MAP [F_{(65, 975)} = 1.82, p = 0.0004], but not for HR [F_{(65, 975)} = 0.68, p = 0.9786] and tail skin temperature [F_{(13, 195)} = 1.75, p = 0.0624] values.

Analysis of mean responses during the restraint and recovery periods indicated differences in the values during restraint and recovery periods for MAP [time factor: F_{(1, 15)} = 13.21, p = 0.0025), HR [time factor: F_{(1, 15)} = 25.40, p = 0.0002], and tail skin temperature [time factor: F_{(1, 15)} = 5.33, p = 0.0363] (Figures 3D–F). However, two-way ANOVA did not indicate neither effect of the bilateral microinjection of CoCl₂ into the anterior IC [MAP: F_{(1, 15)} = 0.80, p = 0.3879; HR: F_{(1, 15)} = 0.69, p = 0.4865; and skin temperature: F_{(1, 15)} = 2.38, p = 0.1213]nor treatment × time interaction [MAP: F_{(1, 15)} = 0.47, p = 0.5041; HR: F_{(1, 15)} = 0.01, p = 0.9206; and skin temperature: F_{(1, 15)} = 0.59, p = 0.6354] for all measurements (Figures 3D–F).

Analysis of the time-course curves indicated that acute restraint stress increased MAP [time factor: F_{(65, 1, 365)} = 9.5, p < 0.0001] and HR [time factor: F_{(65, 1, 365)} = 12.3, p < 0.0001] and decreased the tail skin temperature values [time factor: F_{(13, 273)} = 15.8, p < 0.0001] (Figures 4A–C). Treatment of the rostral posterior subregion of the IC with CoCl₂ (1 mM/100 nl, n = 12) decreased the restraint-evoked MAP increase [F_{(1, 21)} = 5.3, p = 0.0313], without affecting the tachycardia [F_{(1, 21)} = 4.1, p = 0.0561] and the reduction in tail skin temperature [F_{(1, 21)} = 0.05, p = 0.8411]; when compared with animals treated locally with vehicle (100 nl, n = 11) (Figures 4A–C). Analysis also indicated an interaction between time and treatment for MAP [F_{(65, 1, 365)} = 1.63, p = 0.0013], but not for HR [F_{(65, 1, 365)} = 0.6, p = 0.9827] and tail skin temperature [F_{(13, 273)} = 0.4, p = 0.9708].

Analysis of the mean Δ MAP during the restraint and recovery periods indicated effect of the CoCl₂ microinjection into the rostral posterior IC [F_{(1, 21)} = 8.47, p = 0.0081], but without significant differences in values during restraint and recovery periods [time factor: F_{(1, 21)} = 2.62, p = 0.1216] and interaction between treatment and time [F_{(1, 21)} = 2.35, p = 0.1414] (Figure 4D). Bonferroni post-hoc analysis revealed that MAP values during the restraint (p = 0.0045), but not during the recovery period (p = 0.0603), were reduced in animals treated with CoCl₂ in the rostral posterior IC (Figure 4D). Analysis of HR values indicated effect of time [F_{(1, 21)} = 18.12, p = 0.0004], but without influence of CoCl₂ treatment [F_{(1, 21)} = 4.20, p = 0.0531] and interaction between the factors [F_{(1, 21)} = 0.005, p = 0.9481] (Figure 4E). Analysis of tail skin temperature did not indicate effect of either time [F_{(1, 21)} = 1.29, p = 0.2707] or treatment [F_{(1, 21)} = 0.03, p = 0.8557], or interaction between the factors [F_{(1, 21)} = 0.002, p = 0.9653] (Figure 4F).

Analysis of the time-course curves indicated that acute restraint stress caused an increase on MAP [time factor: F_{(65, 1,105)} = 11.6, p < 0.0001] and HR [time factor: F_{(65, 1,105)} = 6.3, p < 0.0001] and decreased the values of tail skin temperature [time factor: F_{(13, 221)} = 13.5, p < 0.0001] (Figures 5A–C). Bilateral microinjection of CoCl₂ (1 mM/100 nl, n = 9) into the caudal posterior IC decreased the tachycardia to restraint stress [F_{(1, 17)} = 11.3, p = 0.0036], without affecting the MAP [F_{(1, 17)} = 0.11, p = 0.7462] and tail skin temperature [F_{(1, 17)} = 1.4, p = 0.2640] responses when compared to vehicle-treated animals (100 nl, n = 10) (Figures 5A–C). Analysis did not indicate a time × treatment interaction for either MAP [F_{(65, 1,105)} = 1.4, p = 0.0686], HR [F_{(65, 1,105)} = 1.1, p = 0.2151], or tail skin temperature [F_{(13, 221)} = 0.9, p = 0.5022].

Analysis of the mean Δ HR during the restraint and recovery periods indicated effect of the microinjection of CoCl₂ into the caudal posterior IC [F_{(1, 17)} = 11.85, p = 0.0029], without differences in values during restraint and recovery periods [time factor: F_{(1, 17)} = 3.85, p = 0.0602] and interaction between treatment and time [F_{(1, 17)} = 0.23, p = 0.4569] (Figure 5E). Bonferroni post-hoc analysis revealed that HR values during restraint (p = 0.0076) and recovery (p = 0.0294) periods were reduced in animals treated with CoCl₂ into the caudal posterior IC (Figure 5E). Analysis of MAP values indicated effect of time [F_{(1, 17)} = 8.63, p = 0.0092], without treatment influence [F_{(1, 17)} = 0.12, p = 0.7243] and interaction between the factors [F_{(1, 17)} = 0.75, p = 0.3964] (Figure 5D). Analysis of tail skin temperature did not indicate effect of either time [F_{(1, 17)} = 3.57, p = 0.0763] or treatment [F_{(1, 17)} = 1.32, p = 0.2683], or interaction between the factors [F_{(1, 17)} = 0.35, p = 0.5648] (Figure 5F).