Pharmacological Research 95–96 (2015) 53–62 Contents lists available at ScienceDirect Pharmacological Research journal homepage: www.elsevier.com/locate/yphrs CRF1 and CRF2 receptors in the bed nucleus of the stria terminalis modulate the cardiovascular responses to acute restraint stress in rats Leandro A. Oliveira a,b , Jeferson Almeida a,b , Ricardo Benini a , Carlos C. Crestani a,b,∗ a b Laboratory of Pharmacology, School of Pharmaceutical Sciences, Univ. Estadual Paulista – UNESP, Araraquara, SP, Brazil Joint UFSCar-UNESP Graduate Program in Physiological Sciences, São Carlos, SP, Brazil a r t i c l e i n f o Article history: Received 5 January 2015 Received in revised form 16 March 2015 Accepted 16 March 2015 Available online 28 March 2015 Keywords: Autonomic activity Bed nucleus stria terminalis (BSNT) Corticotropin releasing factor receptors Emotional stress Extended amygdala Neuropeptides Urocortin a b s t r a c t The corticotropin-releasing factor (CRF) is involved in behavioral and physiological responses to emotional stress through its action in several limbic structures, including the bed nucleus of the stria terminalis (BNST). Nevertheless, the role of CRF1 and CRF2 receptors in the BNST in cardiovascular adjustments during aversive threat is unknown. Therefore, in the present study we investigated the involvement of CRF receptors within the BNST in cardiovascular responses evoked by acute restraint stress in rats. For this, we evaluated the effects of bilateral treatment of the BNST with selective agonists and antagonists of either CRF1 or CRF2 receptors in the arterial pressure and heart rate increase and the decrease in tail skin temperature induced by restraint stress. Microinjection of the selective CRF1 receptor antagonist CP376395 into the BNST reduced the pressor and tachycardiac responses caused by restraint. Conversely, BNST treatment with the selective CRF1 receptor agonist CRF increased restraint-evoked arterial pressure and HR responses and reduced the fall in tail skin temperature response. All effects of CRF were inhibited by local BNST pretreatment with CP376395. The selective CRF2 receptor antagonist antisalvagine-30 reduced the arterial pressure increase and the fall in tail skin temperature. The selective CRF2 receptor agonist urocortin-3 increased restraint-evoked pressor and tachycardiac responses and reduced the drop in cutaneous temperature. All effects of urocortin-3 were abolished by local BNST pretreatment with antisalvagine-30. These findings indicate an involvement of both CRF1 and CRF2 receptors in the BNST in cardiovascular adjustments during emotional stress. © 2015 Elsevier Ltd. All rights reserved. 1. Introduction The maintenance of homeostasis and adaptation to aversive situations requires an appropriate and coordinated set of physiological adjustments, including neuroendocrine and autonomic responses [1–3]. The autonomic nervous system provides the initial response to stress, which is characterized by blood pressure and heart rate (HR) increase, redistribution of blood flow (reduction to skin and viscera and increase for skeletal muscle), and modulation Abbreviations: BNST, bed nucleus of the stria terminalis; CRF, corticotropinreleasing fator; HPA axis, hypothalamic–pituitary–adrenal axis; HR, heart rate; MAP, mean arterial pressure; Ucn1, urocortin 1; Ucn2, urocortin 2; Ucn3, urocortin 3. ∗ Corresponding author at: Laboratory of Pharmacology, Department of Natural Active Principles and Toxicology, School of Pharmaceutical Sciences, São Paulo State University – UNESP, Rodovia Araraquara-Jau Km 01 (Campus Universitário), 14801902, Caixa Postal 502, Araraquara, SP, Brazil. Tel.: +55 16 3301 6982; fax: +55 16 3301 6980. E-mail address: cccrestani@yahoo.com.br (C.C. Crestani). http://dx.doi.org/10.1016/j.phrs.2015.03.012 1043-6618/© 2015 Elsevier Ltd. All rights reserved. of baroreflex activity [1,4–6]. The cutaneous vasoconstriction leads to a rapid fall in the skin temperature during stress [7,8]. Physiological adjustments during emotional stress are mediated by activation of limbic structures through action of several neurochemical mechanisms [3,9]. The bed nucleus of the stria terminalis (BNST) is a limbic structure localized in the rostral prosencephalon, which is a component of the “extended amygdala” (continuum formed by the BNST and centromedial amygdala) [10]. The BNST is activated during aversive threat [11] and has a direct influence in cardiovascular, neuroendocrine and behavioral responses to stress [12,13]. Regarding the cardiovascular responses, previous findings from our group demonstrated that reversible inactivation of the BNST enhanced the HR increase evoked by acute restraint stress without affecting the blood pressure increase [14]. Conversely, BNST ablation attenuated the freezing behavior and the increase in blood pressure and HR induced by contextual fear conditioning [15], thus indicating that the role of the BNST in neurobiological mechanisms of emotional stress depends on the paradigm of stress. Nevertheless, although the above findings indicate a role of the BNST in the modulation of stress-evoked autonomic responses, the 54 L.A. Oliveira et al. / Pharmacological Research 95–96 (2015) 53–62 local neurochemical mechanisms involved in this control was not fully elucidated. The corticotropin-releasing factor (CRF) is a neuropeptide released in numerous limbic structures during aversive stimuli [9], which is involved in behavioral and physiological responses to emotional stress [16–18]. The CRF system in mammals is composed by the CRF and other three CRF-like peptides, denominated urocortin (Ucn) 1, Ucn2, and Ucn3 [16,19]. The effects of CRF and Ucns are mediated by two receptors, denominated CRF1 and CRF2 , and a CRF binding protein [16,19]. The ligands present differences in the binding profile to CRF receptors. For instance, CRF has 10fold higher affinity for CRF1 than for CRF2 receptors, while Ucn2 and Ucn3 bind with 100-fold higher affinities to the CRF2 receptor [19]. Ucn1 has similar affinities for both receptors [19]. High density of CRF-containing terminals, arising mainly from the central nucleus of the amygdala [20,21], and moderate to dense Ucn1- and Ucn3-immunoreactive fibers [22,23] were found within BNST. Also, populations of CRF- and Ucn3-containing neurons have been identified in the BNST [23–25]. Both CRF receptors are expressed within the BNST [26,27]. A role of BNST CRF neurotransmission in behavioral response to aversive threats is well described [13]. However, the participation of this signaling mechanism in control of cardiovascular function is poorly understood [12]. Nijsen et al. [28] reported that BNST treatment with a nonselective CRF receptor antagonist enhanced the tachycardic response induced by contextual fear conditioning. However, the subtype of CRF receptor within the BNST involved in control of stressevoked cardiovascular responses have never been investigated. Furthermore, although evidence that BNST differently modulate the responses to conditioned vs unconditioned aversive stimuli [14,15], a possible role of local CRF signaling in the BNST in control of cardiovascular responses to innate stress is unknown. Therefore, in the present study we tested the hypothesis that CRF receptors in the BNST are involved in cardiovascular responses to acute restraint stress in rats. To this end, we investigated the effects of BNST treatment with either selective CRF1 or CRF2 receptor antagonists and agonists on the pattern of cardiovascular responses (arterial pressure and HR increase, and decrease in tail skin temperature) evoked by acute restraint stress. 2. Material and methods stereotaxic apparatus (Stoelting, Wood Dale, IL, USA). Stereotaxic coordinates for cannula implantation into the BNST were antero-posterior = +8.6 mm from interaural; lateral = 4.0 mm from the medial suture, ventral = −5.8 mm from the skull with a lateral inclination of 23◦ [29]. Cannulas were fixed to the skull with dental cement and one metal screw. After surgery, the animals received a poly-antibiotic (Pentabiotico® , Fort Dodge, Campinas, SP, Brazil), with streptomycins and penicillins, to prevent infection and the non-steroidal anti-inflammatory flunixine meglumine (Banamine® , Schering Plough, Cotia, SP, Brazil) for post-operation analgesia. One day before the experiment, rats were anesthetized with tribromoethanol (250 mg/kg, i.p.) and a catheter (Clay Adams, Parsippany, NJ, USA) filled with a solution of heparin (50 UI/ml, Hepamax-S® , Blausiegel, Cotia, SP, Brazil) diluted in saline (0.9% NaCl) was inserted into the abdominal aorta through the femoral artery for cardiovascular recording. Catheter was tunneled under the skin and exteriorized on the animal’s dorsum. After surgery, the non-steroidal anti-inflammatory flunixine meglumine (Banamine® , Schering Plough, Cotia, SP, Brazil) was administered for post-operation analgesia. The animals were kept in individual cages during the postoperative period and cardiovascular recording. 2.3. Blood pressure and heart rate recording The catheter implanted into the femoral artery was connected to a pressure transducer (DPT100, Utah Medical Products Inc., Midvale, UT, USA). Pulsatile blood pressure was recorded using an amplifier (Bridge Amp, ML224, ADInstruments, Australia) and an acquisition board (PowerLab 4/30, ML866/P, ADInstruments, NSW, Australia) connected to a personal computer. Mean arterial pressure (MAP) and HR values were derived from the pulsatile arterial pressure. 2.4. Tail cutaneous temperature measurement The tail skin temperature was recorded using a thermal câmera; the Multi-Purpose Thermal Imager (IRI4010, Infra Red Integrated Systems Ltd., Northampton, UK). The temperature was measured on five points of the animal’s tail and the mean value was calculated for each recording [7,8]. 2.1. Animals 2.5. Drugs and solutions One hundred twenty-two male Wistar rats weighting 240–260 g (60-days-old) were used. Animals were obtained from the animal breeding facility of the Univ. Estadual Paulista – UNESP (Botucatu, SP, Brazil) and were housed in plastic cages in a temperaturecontrolled room at 24 ◦ C in the Animal Facility of the Laboratory of Pharmacology, School of Pharmaceutical Sciences – UNESP. They were kept under a 12:12 h light–dark cycle (lights on between 6:00 am and 6:00 pm) with free access to water and standard laboratory food. Housing conditions and experimental procedures were approved by the Ethical Committee for Use of Animals of the School of Pharmaceutical Science/UNESP (approval number: 05/2013), which complies with Brazilian and international guidelines for animal use and welfare. 2.2. Surgical preparation Five days before the trial, rats were anesthetized with tribromoethanol (250 mg/kg, i.p.). After scalp anesthesia with 2% lidocaine the skull was exposed and stainless-steel guide cannulas (26G, 12 mm-long) were bilaterally implanted into the BNST at a position 1 mm above the site of injection, using a CP376395 (selective CRF1 receptor antagonist) (Tocris, Westwoods Business Park, Ellisville, MO, USA), antisalvagine-30 (selective CRF2 receptor antagonist) (Tocris), corticotrophin-releasing factor (selective CRF1 receptor agonist) (Tocris), urocortin 3 (selective CRF2 receptor agonist) (Sigma–Aldrich, St. Louis, MO, USA), tribromoethanol (Sigma–Aldrich) and urethane (Sigma–Aldrich) were dissolved in saline (NaCl 0.9%). Flunexine meglumine (Banamines, Schering-Plough, Brazil) and the poly-antibiotic preparation of streptomycins and penicillins (Pentabioticos, Fontoura-Wyeth, Brazil) were used as provided. 2.6. Drug microinjection into the BNST The needles (33G, Small Parts, Miami Lakes, FL, USA) used for microinjection into the BNST were 1 mm longer than the guide cannulas and were connected to a 2 ␮L syringe (7002-KH, Hamilton Co., Reno, NV, USA) through a PE-10 tubing (Clay Adams, Parsippany, NJ, USA). Needles were carefully inserted into the guide cannulas without restraining the animals, and drugs were injected in a final volume of 100 nL [14,15]. L.A. Oliveira et al. / Pharmacological Research 95–96 (2015) 53–62 55 2.7. Acute restraint stress 2.10. Data analysis Animals were submitted to restraint by placing each rat in a plastic cylindrical restraint tube (diameter 6.5 cm, length 15 cm), ventilated by holes (1 cm diameter) that made up approximately 20% of the tube surface. Restraint lasted 30 min [4,7], and immediately after the end of the stress exposure, rats were returned to their home cages. Each rat was submitted to only one session of restraint in order to avoid habituation. Data were expressed as mean ± standard error of the mean (SEM). The basal values of MAP, HR, tail skin temperature were compared using the one-way ANOVA (agonists or antagonists alone) or Student’s t-test (agonists and antagonists in combination). The time–course of changes in the MAP, HR, and tail cutaneous temperature were analyzed using the two-way ANOVA, with treatment as main factor and time as repeated measurement. A post hoc t-test with a Bonferroni correction was used for identification of differences between the groups. Results of statistical tests with P < 0.05 were considered significant. 2.8. Experimental protocols Animals were brought to the experimental room in their own cages. Animals were allowed at least 60 min to adapt to the experimental room conditions, such as sound and illumination, before starting the experiments. The experimental room was temperature controlled (24 ◦ C) and acoustically isolated from the other rooms. Cardiovascular recordings began at least 30 min before the onset of the restraint and were performed throughout the period of exposure to the restraint stress. The tail skin temperature was measured 10, 5 and 0 min before the restraint for baseline values, and at every 5 min during restraint. Each animal received a single pharmacological treatment and was submitted to one session of restraint. 2.8.1. Effect of bilateral microinjection into the BNST of CP376395 and/or CRF in cardiovascular responses to acute restraint stress This protocol aimed to investigate the involvement of the CRF1 receptor within the BNST in cardiovascular responses evoked by acute restraint stress. For this, independent groups of animals received bilateral microinjection into the BNST of different doses of CP376395 (selective CRF1 receptor antagonist) (0.5, 2.75 or 5 nmol/100 nL), CRF (selective CRF1 receptor agonist) (0.01, 0.07 or 0.15 nmol/100 nL) or vehicle (saline) (100 nL) [28,30]. An additional group received bilateral microinjections of CP376395 (0.5 nmol/100 nL) and 5 min later CRF (0.07 nmol/100 nL). Ten minutes after pharmacological treatment of the BNST, the animals underwent a 30 min session of restraint stress. 2.8.2. Effect of bilateral microinjection into the BNST of antisalvagine-30 and/or urocortin 3 in cardiovascular responses to acute restraint stress This protocol aimed to investigate the involvement of the CRF2 receptors within the BNST in autonomic responses to restraint stress. Thus, independent groups of animals received bilateral microinjection into the BNST of increasing doses of antisalvagine-30 (selective CRF2 receptor antagonist) (0.5, 2.75 or 5 nmol/100 nL), Ucn3 (0.01, 0.07 or 0.15 nmol/100 nL) or vehicle (saline) (100 nL) [30,31]. An additional group received bilateral microinjection of antisalvagine-30 (0.5 nmol/100 nL) and 5 min later UCN3 (0.15 nmol/100 nL). Ten minutes after pharmacological treatment of the BNST, the animals were submitted to a 30 min session of restraint stress. 2.9. Histological determination of the microinjection sites At the end of experiments, animals were anesthetized with urethane (1.25 g/kg, i.p.) and 100 nL of 1% Evan’s blue dye was injected into the brain as a marker of the injection site. Brains were removed and post-fixed in 10% formalin for at least 48 h at 4 ◦ C. Then, serial 40 ␮m-thick sections of the BNST region were cut with a cryostat (CM1900, Leica, Wetzlar, Germany). The actual placement of the microinjection needles was determined according to the rat brain atlas of Paxinos and Watson [29] by analyzing the serial sections under a light microscopy. 3. Results Diagrammatic representations showing the bilateral injection sites in the BNST of all animals used in the present study are presented in Fig. 1. 3.1. Effect of bilateral microinjection into the BNST of CP376395 and/or CRF in cardiovascular responses to acute restraint stress CP376395 – Bilateral microinjection into the BNST of the selective CRF1 receptor antagonist CP376395 (0.5, 2.75 or 5 nmol/100 nL) did not alter baseline values of either MAP, HR or tail skin temperature (Table 1). Acute restraint stress induced an increase in the MAP (F(19,380) = 16, P < 0.0001) and HR (F(19,380) = 20, P < 0.0001) and decreased the tail skin temperature (F(8,171) = 9, P < 0.0001) (Fig. 2). Treatment of the BNST with CP376395, at the doses of 2.75 nmol (PAM: P < 0.0001 and HR: P < 0.0001) and 5 nmol (PAM: P < 0.0001 and HR: P < 0.0001), reduced the restraint-evoked increase in the MAP (F(3,380) = 24, P < 0.0001) and HR (F(3,380) = 5, P < 0.006) without affecting the decrease in tail skin temperature (F(3,171) = 0.9, P > 0.05) (Fig. 2). Moreover, there was a significant interaction between treatment and time for the HR response (F(57,380) = 2, P < 0.05), but not for MAP (F(57,380) = 1, P > 0.05) and tail temperature (F(24,171) = 0.1, P > 0.05). Restraint-evoked MAP (F(1,160) = 1, P > 0.05), HR (F(1,160) = 0.1, P > 0.05) and tail temperature (F(1,72) = 3, P > 0.05) responses were not affected when CP376395 was microinjected into structures surrounding the BNST, such as the anterior commissure, fornix and internal capsule (n = 4, data not shown). CRF – Bilateral treatment of the BNST with the selective CRF1 receptor agonist CRF (0.01, 0.07 or 0.15 nmol/100 nL) did not affect baseline values of either MAP, HR or tail skin temperature (Table 1). Restraint caused an increase in the MAP (F(19,340) = 48, P < 0.0001) and HR (F(19,340) = 26, P < 0.0001) and decreased the tail skin temperature (F(8,153) = 6, P < 0.0001) (Fig. 3). Bilateral microinjection of CRF into the BNST at the dose of 0.07 nmol (MAP: P < 0.0001; HR: P < 0.0001) enhanced the restraint-evoked increase in the MAP (F(3,340) = 17, P < 0.0001) and HR (F(3,340) = 18, P < 0.0001). Furthermore, CRF at all doses (0.01 nmol: P < 0.0001, 0.07 nmol: P < 0.0001, 0.15 nmol: P < 0.0001) reduced the tail skin temperature response (F(3,153) = 30, P < 0.0001) (Fig. 3). Analysis also identified interaction between treatment and time for the tail skin temperature response (F(24,153) = 3, P < 0.001), but not for MAP (F(57,320) = 1, P > 0.05) and HR (F(57,320) = 0.8, P > 0.05). The increase in the MAP (F(1,160) = 1, P > 0.05) and HR (F(1,160) = 3, P > 0.05), and the decrease in tail temperature (F(1,72) = 0.6, P > 0.05) induced by restraint were not affected when CRF was microinjected into structures surrounding the BNST, such as the anterior commissure, internal capsule and fornix (n = 4, data not shown). CP376395 + CRF – Combined treatment of the BNST with CP376395 at the dose of 0.5 nmol and 0.07 nmol of CRF did not affect baseline values of either MAP, HR or tail skin temperature 56 L.A. Oliveira et al. / Pharmacological Research 95–96 (2015) 53–62 Fig. 1. Diagrammatic representation based on the rat brain atlas of Paxinos and Watson [29] indicating the injection sites into the BNST of CP376395 (black circles), CRF (gray circles), CP376395 + CRF (white circles), antisalvagine-30 (ASV-30) (black squares), Ucn3 (gray squares), ASV-30 + Ucn3 (white squares) and vehicle (white triangles). 3V – third ventricle, IA – interaural coordinate; ac – anterior commissure; cc – corpus callosum; f – fornix; ic – internal capsule; LV – lateral ventricle; LSV – lateral septal ventral; sm – stria medullaris, st – stria terminalis. (Table 1). Nevertheless, the effects of CRF (0.07 nmol) in restraintevoked changes in the MAP (F(1,160) = 1.3, P > 0.05), HR (F(1,160) = 0.9, P > 0.05), and tail skin temperature (F(1,72) = 2, P > 0,05) were completely inhibited by BNST pretreatment with CP376395 (Fig. 3). 3.2. Effect of bilateral microinjection into the BNST of antisalvagine-30 and/or urocortin 3 in cardiovascular responses to acute restraint stress Antisalvagine-30 – Bilateral treatment of the BNST with the selective CRF2 receptor antagonist antisalvagine-30 (0.5, 2.75 or 5 nmol/100 nL) did not alter baseline values of either MAP, HR or tail skin temperature (Table 2). Restraint caused an increase in the MAP (F(19,340) = 21, P < 0.0001) and HR (F(19,340) = 11, P < 0.0001) and reduced the tail skin temperature (F(8,153) = 4, P < 0.0002) (Fig. 4). Bilateral microinjection into the BNST of antisalvagine-30 at the dose of 5 nmol (MAP: P < 0.0001, temperature: P < 0.01) reduced the MAP (F(3,340) = 12, P < 0.0001) and tail skin temperature (F(3,153) = 6, P < 0.0005) changes to restraint stress without affecting the tachycardiac response (F(3,340) = 2, P > 0.05) (Fig. 4). Statistical analysis did not identify interaction between treatment and time for measures of MAP (F(57,340) = 0.8, P > 0.05), HR (F(57,340) = 0.5, P > 0.05) and tail skin temperature (F(24,153) = 0.3, P > 0.05). Restraint-evoked changes in the MAP (F(1,160) = 3, P > 0.05), HR (F(1,160) = 0.2, P > 0.05), and tail temperature (F(1,72) = 0.6, P > 0.05) were not affected when antisalvagine-30 was microinjected into structures surrounding the BNST, such as such as the anterior commissure, internal capsule and fornix (n = 4, data not shown). Urocortin 3 – Bilateral microinjection into the BNST of the selective CRF2 receptor agonist Ucn3 (0.01, 0.07 or 0.15 nmol/100 nL) did not affect baseline values of either MAP (F(3,16) = 2.5, P > 0.05) or HR (F(3,16) = 2, P > 0.05) (Table 2). However, Ucn3 at the dose of 0.07 nmol (P < 0.05) evoked a reduction in basal tail skin temperature (Table 2). Restraint increased MAP (F(19,340) = 26, P < 0.0001) and HR (F(19,340) = 21, P < 0.0001) and reduced the tail skin temperature (F(8,153) = 6, P < 0.0001) (Fig. 5). Treatment of the BNST with Ucn3 at all doses (MAP: P < 0.0001, HR: P < 0.0001) enhanced the restraint-evoked increase in the MAP (F(3,340) = 17, P < 0.0001) and HR (F(3,340) = 11, P < 0.0001). Furthermore, 0.07 nmol (P < 0.0001) and 0.15 nmol (P < 0.0001) of Ucn3 reduced the decrease in the tail cutaneous temperature induced by restraint stress (F(3,153) = 23, P < 0.0001) (Fig. 5). Analysis identified interaction between treatment and time for tail temperature (F(24,153) = 2, P < 0.03), but not for MAP (F(57,340) = 0.6, P > 0.05) and HR (F(57,340) = 0.8, P > 0.05). The restraint-evoked changes in the MAP (F(1,140) = 0.04, P > 0.05), HR (F(1,140) = 1.7, P > 0.05), and tail temperature (F(1,63) = 1.4, P > 0.05) were not affected when Ucn3 was microinjected into structures surrounding the BNST, such as the anterior commissure, internal capsule and fornix (n = 3, data not shown). Antisalvagine-30 + urocortin 3 – Combined treatment of the BNST with antisalvagine-30 at the dose of 0.5 nmol and 0.15 nmol of Ucn3 did not affect baseline values of either MAP, HR or tail skin temperature (Table 2). Nevertheless, the effects of Ucn3 (0.15 nmol) in restraint-evoked changes in the MAP (F(1,160) = 0.3, P > 0.05), HR (F(1,160) = 3, P > 0.05), and tail skin temperature (F(1,72) = 3, P > 0.05) were completely inhibited by BNST pretreatment with antisalvagine-30 (Fig. 5). Table 1 Basal parameters of mean arterial pressure (MAP), heart rate (HR) and tail skin temperature after pharmacological treatment of the BNST with different doses of selective CRF1 receptor agonist (CRF) and/or antagonist (CP376395). Group n MAP (mmHg) HR (bpm) Tail skin temperature (◦ C) Vehicle CP376395 – 0.5 nmol CP376395 – 2.75 nmol CP376395 – 5.0 nmol 6 6 5 6 103 ± 4 109 ± 3 98 ± 2 107 ± 4 F(3,22) = 3, P > 0.05 387 ± 20 412 ± 22 384 ± 11 391 ± 9 F(3,22) = 0.5, P > 0.05 28.0 ± 1 28.0 ± 1 27.2 ± 1 27.2 ± 1 F(3,22) = 0.5, P > 0.05 Vehicle CRF – 0.01 nmol CRF – 0.07 nmol CRF – 0.15 nmol 6 5 5 5 105 ± 5 103 ± 3 102 ± 4 114 ± 4 F(3,20) = 2, P > 0.05 390 ± 29 360 ± 2 360 ± 14 402 ± 14 F(3,20) = 1, P > 0.05 26.0 ± 0.3 26.1 ± 0.6 24.8 ± 0.7 26.0 ± 0.6 F(3,20) = 1, P > 0.05 Vehicle CP376395(0.5 nmol) + CRF(0.07 nmol) 5 5 112 ± 4 108 ± 4 t = 0.7, P > 0.05 369 ± 8 363 ± 15 t = 0.3, P > 0.05 28.0 ± 0.5 28.6 ± 0.9 t = 0.6, P > 0.05 57 L.A. Oliveira et al. / Pharmacological Research 95–96 (2015) 53–62 Fig. 2. Time course of changes in mean arterial pressure (MAP), heart rate (HR), and tail skin temperature (tail temperature) induced by restraint stress in animals treated with different doses (0.5, 2.75, and 5.0 nmol/100 nL) of the selective CRF1 receptor antagonist CP376395 into the BNST. Shaded area indicates the period of restraint. Circles represent the mean and bars the SEM. *P < 0.05 over the whole restraint period compared to vehicle-treated animals, ANOVA followed by post hoc t-test with a Bonferroni correction. 4. Discussion The present results provide the first evidence that both CRF1 and CRF2 receptors in the BSNT are involved in cardiovascular adjustments during emotional stress. We have demonstrated that bilateral microinjection of the selective CRF1 receptor antagonist CP376395 into the BNST decreased the pressor and tachycardiac responses caused by acute restraint stress. Conversely, BNST Table 2 Basal parameters of mean arterial pressure (MAP), heart rate (HR) and tail skin temperature after pharmacological treatment of the BNST with different doses of selective CRF2 receptor agonist (Ucn3) and/or antagonist (antisalvagine-30). Group n MAP (mmHg) HR (bpm) Tail skin temperature (◦ C) Vehicle Antisalvagine-30 – 0.5 nmol Antisalvagine-30 – 2.75 nmol Antisalvagine-30 – 5.0 nmol 6 5 5 5 101 ± 2 101 ± 3 114 ± 4 98 ± 6 F(3,20) = 3, P > 0.05 370 ± 25 405 ± 28 360 ± 10 404 ± 28 F(3,20) = 1, P > 0.05 28.3 ± 0.9 28.9 ± 1.0 30.7 ± 0.8 26.8 ± 1.8 F(3,20) = 1, P > 0.05 Vehicle UCN3 – 0.01 nmol UCN3 – 0.07 nmol UCN3 3 – 0.15 nmol 6 5 5 5 115 ± 4 102 ± 6 118 ± 4 108 ± 6 F(3,20) = 2, P > 0.05 416 ± 16 383 ± 9 451 ± 27 440 ± 27 F(3,20) = 2, P > 0.05 27.8 ± 0.6 27.2 ± 0.9 24.9 ± 0.4* 26.4 ± 0.4 F(3,20) = 5, P < 0.01 Vehicle Antisalvagine-30(0.5 nmol) + UCN3(0.15 nmol) 6 5 110 ± 3 109 ± 2 t = 0.3, P > 0.05 377 ± 10 397 ± 9 t = 1.5, P > 0.05 28.0 ± 0.6 28.6 ± 0.4 t = 1.0, P > 0.05 58 L.A. Oliveira et al. / Pharmacological Research 95–96 (2015) 53–62 Fig. 3. (Left) Time course of changes in mean arterial pressure (MAP), heart rate (HR), and tail skin temperature (tail temperature) induced by restraint stress in animals treated with different doses (0.01, 0.07, and 0.15 nmol/100 nL) of the selective CRF1 receptor agonist CRF into the BNST. (Right) Time course of MAP, HR, and  tail temperature induced by restraint in animals treated with 0.07 nmol/100 nL of CRF into the BNST after local pretreatment with the selective CRF1 receptor antagonist CP376395 (0.5 nmol/100 nL). Shaded area indicates the period of restraint. Circles represent the mean and bars the SEM. *P < 0.05 over the whole restraint period compared to vehicle-treated animals, ANOVA followed by post hoc t-test with a Bonferroni correction. treatment with the selective CRF1 receptor agonist CRF increased restraint-evoked arterial pressure and HR responses and reduced the fall in tail skin temperature. All effects of CRF were inhibited by local BNST pretreatment with CP376395, thus confirming the CRF effects were mediated by activition of local CRF1 receptor. Bilateral microinjection of the selective CRF2 receptor antagonist antisalvagine-30 into the BNST reduced the arterial pressure increase and the drop in tail skin temperature. Bilateral treatment of the BNST with the selective CRF2 receptor agonist Ucn3 increased restraint-evoked pressor and tachycardiac responses and reduced the fall in cutaneous temperature. All effects of Ucn3 were abolished by local BNST pretreatment with antisalvagine-30. In contrast with present findings, some evidence has indicated an opposing role of CRF1 and CRF2 receptors in regulating physiological responses to stress. For instance, studies using mice deficient to either CRF1 or CRF2 receptors have indicated an involvement of CRF1 receptors in the activation of the hypothalamic–pituitary–adrenal (HPA) axis during stress, whereas CRF2 receptors inhibit it [16,32]. Although a facilitatory role of CRF1 receptor was further supported by pharmacological studies [16,32], a possible inhibitory influence of CRF2 receptors is controversy once central administration of a selective CRF2 receptor antagonist produced little effect in adrenocorticotropic hormone (ACTH) to restraint stress [33]. Previous studies demonstrated that central administration of nonselective CRF receptor antagonists reduced stress-evoked cardiovascular responses [34,35]. However, to the best of our knowledge, present study is the first to investigate the specific role of CRF1 and CRF2 receptors in cardiovascular adjustments to emotional stress. It has been reported a regionalization in the BSNT in control of neuroendocrine responses to stress, with rostral regions involved in activation of the HPA axis and posterior division inhibiting it [12]. Consistent with evidence that anterior division is the critical BNST region involved in autonomic control [12], most of our microinjection sites reached regions of the BSNT anterior division. Therefore, centralization of the microinjection sites in rostral regions of the BNST may have contributed to identification of a similar role of CRF1 and CRF2 receptors in the present study. However, the expression of both CRF receptors was reported in rostral regions of the BNST [26,27], thus supporting present findings. Furthermore, CRF-containing terminals (though to be the endogenous CRF1 ligand) as well as Ucn1- and Ucn3immunoreactive fibers (thought to be, together with Ucn2, the endogenous ligands of the CRF2 receptors) were identified in BNST anterior division [22,23]. A similar role of CRF receptors in the BNST is further supported by demonstration that aversive effects of the CRF in the BNST are mediated by activation of both CRF1 and CRF2 receptors [30]. Thus, CRF1 and CRF2 receptors seem to work sinergically within the BNST. A previous study conducted by our group demonstrated that bilateral BNST neurotransmission inhibition evoked by local treatment with CoCl2 enhanced the HR increase evoked by restraint stress without affecting the arterial pressure response [14]. Similar effect was observed after BNST treatment with a selective ␣1 -adrenoceptor antagonist (i.e., facilitation of restraint-evoked tachycardiac response without affecting pressor effect) [14], suggesting that the inhibitory role of the BNST in HR response to restraint stress is mediated, at least in part, by the local action of noradrenergic mechanisms. In contrast, present study indicates L.A. Oliveira et al. / Pharmacological Research 95–96 (2015) 53–62 59 Fig. 4. Time course of changes in mean arterial pressure (MAP), heart rate (HR), and tail skin temperature (tail temperature) induced by restraint stress in animals treated with different doses (0.5, 2.75, and 5.0 nmol/100 nL) of the selective CRF2 receptor antagonist antisalvagine-30 into the BNST. Shaded area indicates the period of restraint. Circles represent the mean and bars the SEM. *P < 0.05 over the whole restraint period compared to vehicletreated animals, ANOVA followed by post hoc t-test with a Bonferroni correction. that activation of CRF1 receptor in the BNST during restraint stress play a facilitatory role in tachycardiac response. Noradrenaline has predominantly an inhibitory influence in the activity of BNST neurons [36], which is mediated by a facilitation of local GABAergic neurotransmission and inhibition of glutamatergic inputs [37–41]. In contrast, CRF1 receptor activation enhances excitatory neurotransmission in the BNST [42,43]. These pieces of evidence support the findings of an opposite influence of BNST CRF1 receptor and ␣1 -adrenoceptors in the restraint-evoked tachycardic response. Present findings bring the first evidence of an influence of the BNST in the arterial pressure and skin temperature responses induced by an unconditioned aversive stimulus. Conversely to the reduction in restraint-evoked HR increase following the blockade of CRF receptors, Nijsen et al. [28] demonstrated that BNST treatment with a nonselective CRF receptor antagonist enhanced the tachycardia evoked by contextual fear conditioning. Taken together, these results indicate that CRF neurotransmission in the BNST display distinct roles in control of cardiovascular adjustments during conditioned vs unconditioned aversive stimuli. This finding is in line with previous data demonstrating that either nonselective inhibition of BNST neurotransmission caused by local treatment with CoCl2 [14,15] or BNST treatment with cannabidiol (a component of Cannabis sativa) [44,45] caused opposite effects in cardiovascular responses to contextual fear conditioning and restraint stress. Both the sympathetic and parasympathetic nervous systems are directly responsible for cardiovascular adjustments during stress. For instance, stress-evoked tachycardia is abolished by blockade of cardiac sympathetic activity while inhibition of cardiac parasympathetic activity increases this response [46,47], thus suggesting a coactivation of cardiac sympathetic and parasympathetic activity during aversive threats. The pressor response is mediated by a vasoconstriction in splanchnic, renal and cutaneous vascular territories [5,6] through activation of ␣1 -adrenoreceptors in vascular smooth muscle [47]. The vasoconstriction reduces the blood flow in cutaneous beds [6], which causes a fall in skin temperature [7,8]. It has been reported that intracerebroventricular administration of CRF evokes a sympathetic-mediated increase in blood pressure and HR [48,49]. Also, the reduction in stress-evoked tachycardia caused by intracerebroventricular administration of a nonselective CRF receptor antagonist was abolished in animals systemically treated with a blocker of cardiac parasympathetic activity [34], whereas 60 L.A. Oliveira et al. / Pharmacological Research 95–96 (2015) 53–62 Fig. 5. (Left) Time course of changes in mean arterial pressure (MAP), heart rate (HR), and tail skin temperature ( tail temperature) induced by restraint stress in animals treated with different doses (0.01, 0.07, and 0.15 nmol/100 nL) of the selective CRF2 receptor agonist Ucn3 into the BNST. (Right) Time course of MAP, HR, and  tail temperature induced by restraint stress in animals treated with 0.15 nmol/100 nL of Ucn3 into the BNST after local pretreatment with the selective CRF2 receptor antagonist antisalvagine-30 (0.5 nmol/100 nL). Shaded area indicates the period of restraint. Circles represent the mean and bars the SEM. *P < 0.05 over the whole restraint period compared to vehicle-treated animals, ANOVA followed by post hoc t-test with a Bonferroni correction. intracerebroventricular injection of CRF reduced the restraintevoked activation of dorsal motor nucleus of the vagus [50], indicating that CRF control of HR during aversive threat may also be mediated by an inhibition of cardiac parasympathetic activity. Direct projections from the BNST reach medullary structures involved with autonomic control, including the nucleus of the solitary tract, nucleus ambiguus, and ventrolateral regions [51,52]. Thus, activation of CRF receptors within the BNST can modulate the cardiovascular activity during restraint through a facilitation of inhibitory inputs to medullary parasympathetic neurons and/or activation of facilitatory pathways to premotor sympathetic neurons. Results obtained with the CRF receptor antagonists provided evidence that physiological release of CRF and CRF-related peptides within the BNST during restraint modulates the tachycardia through activation of local CRF1 receptors while CRF2 receptors mediates the tail skin temperature responses. Moreover, both receptors control the increase in arterial pressure. Nevertheless, BNST treatment with CRF and Ucn3 affected both blood pressure, HR and skin temperature responses. Therefore, although the specificity of CRF1 and CRF2 receptors in the control of restraint-evoked cardiovascular responses, both receptors in the BNST are able to modulate arterial pressure, HR, and cutaneous vasoconstriction responses during emotional stress. Furthermore, CRF at the dose of 0.07 nmol affected restraint-evoked cardiovascular responses, but microinjection of a higher dose (0.15 nmol) did not evoke any change in restraint responses, which indicates an inverted Ushaped dose–response for CRF. As stated above, CRF1 receptor has an excitatory influence in the activity of BNST neurons by enhancing local glutamatergic neurotransmission [42,43]. However, it has been reported that CRF1 receptor also increases GABAergic neutransmission in the BNST [53,54]. Nevertheless, electrophysiological studies have demonstrated that increasing doses of CRF evoked greater effects in glutamatergic transmission (maximum increase of 165–200% with 1 ␮M CRF) [42,43] than in GABAergic neurotransmission (maximum increase of ∼115% with 1 ␮M CRF) [53], indicating that control of GABAergic transmission by CRF1 receptors occurs with reduced potency relative to the modulation of excitatory neurotransmission. Therefore, it is possible that at the higher dose CRF facilitation of GABAergic neurotransmission buffers the increase in local excitatory neurotransmission, thus precluding the emergence of changes in restraint-evoked cardiovascular changes. Unexpectedly, CRF2 receptor agonist and antagonist evoked similar effects in the fall in tail skin temperature caused by restraint. However, Ucn3 reduced basal values of tail skin temperature, thus indicating that some degree of basal vasoconstriction caused by BNST treatment with Ucn3 may have contributed to the decrease in restraint-evoked change in tail skin temperature. Also, it has been demonstrated that activation of CRF2 receptors by Ucn3 in the central nervous system decreases sympathetic nerve activity [31]. Therefore, a second possibility is that although activation of BNST CRF2 receptor by physiological release of CRF-like peptides during restraint contribute to cutaneous vasoconstriction (evidenced by reduction in the fall in skin temperature following treatment with antisalvagine-30), exogenous administration of Ucn3 may recruit a neural circuitry that decrease sympathetic nerve activity to cutaneous beds, thus buffering stress-evoked vasoconstriction in cutaneous beds. L.A. Oliveira et al. / Pharmacological Research 95–96 (2015) 53–62 In summary, the present results provide evidence that both CRF1 and CRF2 receptors within the BNST modulate the cardiovascular responses during emotional stress. Our data suggest that BSNT CRF1 receptor is involved in the mediation of the pressor and tachycardiac responses induced by restraint stress, whereas local CRF2 receptors control the arterial pressure increase and sympatheticmediated cutaneous vasoconstriction during restraint. Although the results obtained with the antagonists indicate a specificity of CRF1 (control of arterial pressure and HR responses) and CRF2 (control of arterial pressure and cutaneous vasoconstriction responses) receptors in the control of restraint-evoked cardiovascular responses, treatment with CRF receptor agonists indicated that both receptors are able to modulate arterial pressure, HR, and cutaneous vasoconstriction responses during aversive threats. Acknowledgments The authors wish to thank Elisabete Lepera and Rosana Silva for technical assistance. This work was supported by FAPESP grants # 2012/14376-0 and 2012/50549-6; and PADC-FCF UNESP. References [1] R.A. Dampney, J. Horiuchi, L.M. McDowall, Hypothalamic mechanisms coordinating cardiorespiratory function during exercise and defensive behaviour, Auton. Neurosci. 142 (2008) 3–10. [2] P. Sterling, Allostasis, A model of predictive regulation, Physiol. Behav. 106 (2012) 5–15. [3] Y.M. Ulrich-Lai, J.P. Herman, Neural regulation of endocrine and autonomic stress responses, Nat. Rev. Neurosci. 10 (2009) 397–409. [4] C.C. Crestani, R.F. Tavares, F.H. Alves, L.B. Resstel, F.M. Correa, Effect of acute restraint stress on the tachycardiac and bradycardiac responses of the baroreflex in rats, Stress 13 (2010) 61–72. [5] J.C. Schadt, E.M. Hasser, Hemodynamic effects of acute stressors in the conscious rabbit, Am. J. Physiol. 274 (1998) R814–R821. [6] W.W. Blessing, Lower brainstem pathways regulating sympathetically mediated changes in cutaneous blood flow, Cell. Mol. Neurobiol. 23 (2003) 527–538. [7] F.C. Cruz, S.A. Engi, R.M. Leao, C.S. Planeta, C.C. Crestani, Influence of the single or combined administration of cocaine and testosterone in autonomic and neuroendocrine responses to acute restraint stress, J. Psychopharmacol. 26 (2012) 1366–1374. [8] C. Busnardo, F.H. Alves, C.C. Crestani, A.A. Scopinho, L.B. Resstel, F.M. Correa, Paraventricular nucleus of the hypothalamus glutamate neurotransmission modulates autonomic, neuroendocrine and behavioral responses to acute restraint stress in rats, Eur. Neuropsychopharmacol. 23 (2013) 1611–1622. [9] M. Joels, T.Z. Baram, The neuro-symphony of stress, Nat. Rev. Neurosci. 10 (2009) 459–466. [10] J. de Olmos, C.A. Beltamino, G.F. Alheid, Amygdala and extended amygdala of the rat: a cytoarchitectonical, fibroarchitectonial and chemoarchitectonical survey, in: G. Paxinos (Ed.), The Rat Nervous System, 3rd ed., Elsevier, Amsterdam, 2004. [11] W.E. Cullinan, J.P. Herman, D.F. Battaglia, H. Akil, S.J. Watson, Pattern and time course of immediate early gene expression in rat brain following acute stress, Neuroscience 64 (1995) 477–505. [12] C.C. Crestani, F.H. Alves, F.V. Gomes, L.B. Resstel, F.M. Correa, J.P. Herman, Mechanisms in the bed nucleus of the stria terminalis involved in control of autonomic and neuroendocrine functions: a review, Curr. Neuropharmacol. 11 (2013) 141–159. [13] D.L. Walker, L.A. Miles, M. Davis, Selective participation of the bed nucleus of the stria terminalis and crf in sustained anxiety-like versus phasic fearlike responses, Prog. Neuropsychopharmacol. Biol. Psychiatry 33 (2009) 1291–1308. [14] C.C. Crestani, F.H. Alves, R.F. Tavares, F.M. Correa, Role of the bed nucleus of the stria terminalis in the cardiovascular responses to acute restraint stress in rats, Stress 12 (2009) 268–278. [15] L.B. Resstel, F.H. Alves, D.G. Reis, C.C. Crestani, F.M. Correa, F.S. Guimaraes, Anxiolytic-like effects induced by acute reversible inactivation of the bed nucleus of stria terminalis, Neuroscience 154 (2008) 869–876. [16] T.L. Bale, W.W. Vale, Crf and crf receptors: role in stress responsivity and other behaviors, Annu. Rev. Pharmacol. Toxicol. 44 (2004) 525–557. [17] G.F. Koob, S.C. Heinrichs, A role for corticotropin releasing factor and urocortin in behavioral responses to stressors, Brain Res. 848 (1999) 141–152. [18] A. Stengel, Y. Tache, Corticotropin-releasing factor signaling and visceral response to stress, Exp. Biol. Med. (Maywood) 235 (2010) 1168–1178. [19] R.L. Hauger, D.E. Grigoriadis, M.F. Dallman, P.M. Plotsky, W.W. Vale, F.M. Dautzenberg, International union of pharmacology. Xxxvi. Current status of the nomenclature for receptors for corticotropin-releasing factor and their ligands, Pharmacol. Rev. 55 (2003) 21–26. 61 [20] M.A. Beckerman, T.A. Van Kempen, N.J. Justice, T.A. Milner, M.J. Glass, Corticotropin-releasing factor in the mouse central nucleus of the amygdala: ultrastructural distribution in nmda-nr1 receptor subunit expressing neurons as well as projection neurons to the bed nucleus of the stria terminalis, Exp. Neurol. 239 (2013) 120–132. [21] M. Sakanaka, T. Shibasaki, K. Lederis, Distribution and efferent projections of corticotropin-releasing factor-like immunoreactivity in the rat amygdaloid complex, Brain Res. 382 (1986) 213–238. [22] J.C. Bittencourt, J. Vaughan, C. Arias, R.A. Rissman, W.W. Vale, P.E. Sawchenko, Urocortin expression in rat brain: evidence against a pervasive relationship of urocortin-containing projections with targets bearing type 2 crf receptors, J. Comp. Neurol. 415 (1999) 285–312. [23] C. Li, J. Vaughan, P.E. Sawchenko, W.W. Vale, Urocortin iii-immunoreactive projections in rat brain: partial overlap with sites of type 2 corticotrophin-releasing factor receptor expression, J. Neurosci. 22 (2002) 991–1001. [24] C.F. Phelix, W.K. Paull, Demonstration of distinct corticotropin releasing factor – containing neuron populations in the bed nucleus of the stria terminalis. A light and electron microscopic immunocytochemical study in the rat, Histochemistry 94 (1990) 345–364. [25] L.W. Swanson, P.E. Sawchenko, J. Rivier, W.W. Vale, Organization of ovine corticotropin-releasing factor immunoreactive cells and fibers in the rat brain: an immunohistochemical study, Neuroendocrinology 36 (1983) 165–186. [26] K. Van Pett, V. Viau, J.C. Bittencourt, R.K. Chan, H.Y. Li, C. Arias, G.S. Prins, M. Perrin, W. Vale, P.E. Sawchenko, Distribution of mRNAs encoding crf receptors in brain and pituitary of rat and mouse, J. Comp. Neurol. 428 (2000) 191–212. [27] J. Dabrowska, R. Hazra, T.H. Ahern, J.D. Guo, A.J. McDonald, F. Mascagni, J.F. Muller, L.J. Young, D.G. Rainnie, Neuroanatomical evidence for reciprocal regulation of the corticotrophin-releasing factor and oxytocin systems in the hypothalamus and the bed nucleus of the stria terminalis of the rat: implications for balancing stress and affect, Psychoneuroendocrinology 36 (2011) 1312–1326. [28] M.J. Nijsen, G. Croiset, M. Diamant, D. De Wied, V.M. Wiegant, Crh signalling in the bed nucleus of the stria terminalis is involved in stress-induced cardiac vagal activation in conscious rats, Neuropsychopharmacology 24 (2001) 1–10. [29] G. Paxinos, C. Watson, The Rat Brain in Stereotaxic Coordinates, 3rd ed., Academic Press, Sydney, Australia, 1997. [30] L.L. Sahuque, E.F. Kullberg, A.J. McGeehan, J.R. Kinder, M.P. Hicks, M.G. Blanton, P.H. Janak, M.F. Olive, Anxiogenic and aversive effects of corticotropin-releasing factor (crf) in the bed nucleus of the stria terminalis in the rat: role of crf receptor subtypes, Psychopharmacology (Berl.) 186 (2006) 122–132. [31] T. Nakamura, K. Kawabe, H.N. Sapru, Cardiovascular responses to microinjections of urocortin 3 into the nucleus tractus solitarius of the rat, Am. J. Physiol. Heart Circ. Physiol. 296 (2009) H325–H332. [32] J.M. Reul, F. Holsboer, Corticotropin-releasing factor receptors 1 and 2 in anxiety and depression, Curr. Opin. Pharmacol. 2 (2002) 23–33. [33] M.A. Pelleymounter, M. Joppa, N. Ling, A.C. Foster, Pharmacological evidence supporting a role for central corticotropin-releasing factor (2) receptors in behavioral, but not endocrine, response to environmental stress, J. Pharmacol. Exp. Ther. 302 (2002) 145–152. [34] M.J. Nijsen, G. Croiset, M. Diamant, R. Stam, P.J. Kamphuis, A. Bruijnzeel, D. de Wied, V.M. Wiegant, Endogenous corticotropin-releasing hormone inhibits conditioned-fear-induced vagal activation in the rat, Eur. J. Pharmacol. 389 (2000) 89–98. [35] T. Nakamori, A. Morimoto, N. Murakami, Effect of a central crf antagonist on cardiovascular and thermoregulatory responses induced by stress or il-1 beta, Am. J. Physiol. 265 (1993) R834–R839. [36] J.H. Casada, N. Dafny, Responses of neurons in bed nucleus of the stria terminalis to microiontophoretically applied morphine, norepinephrine and acetylcholine, Neuropharmacology 32 (1993) 279–284. [37] E.C. Dumont, J.T. Williams, Noradrenaline triggers gabaa inhibition of bed nucleus of the stria terminalis neurons projecting to the ventral tegmental area, J. Neurosci. 24 (2004) 8198–8204. [38] R.E. Egli, T.L. Kash, K. Choo, V. Savchenko, R.T. Matthews, R.D. Blakely, D.G. Winder, Norepinephrine modulates glutamatergic transmission in the bed nucleus of the stria terminalis, Neuropsychopharmacology 30 (2005) 657–668. [39] M.I. Forray, G. Bustos, K. Gysling, Noradrenaline inhibits glutamate release in the rat bed nucleus of the stria terminalis: in vivo microdialysis studies, J. Neurosci. Res. 55 (1999) 311–320. [40] M. Krawczyk, F. Georges, R. Sharma, X. Mason, A. Berthet, E. Bezard, E.C. Dumont, Double-dissociation of the catecholaminergic modulation of synaptic transmission in the oval bed nucleus of the stria terminalis, J. Neurophysiol. 105 (2011) 145–153. [41] A.D. Shields, Q. Wang, D.G. Winder, Alpha2a-adrenergic receptors heterosynaptically regulate glutamatergic transmission in the bed nucleus of the stria terminalis, Neuroscience 163 (2009) 339–351. [42] T.L. Kash, W.P. Nobis, R.T. Matthews, D.G. Winder, Dopamine enhances fast excitatory synaptic transmission in the extended amygdala by a crf-r1dependent process, J. Neurosci. 28 (2008) 13856–13865. [43] Y. Silberman, R.T. Matthews, D.G. Winder, A corticotropin releasing factor pathway for ethanol regulation of the ventral tegmental area in the bed nucleus of the stria terminalis, J. Neurosci. 33 (2013) 950–960. [44] F.V. Gomes, F.H. Alves, F.S. Guimaraes, F.M. Correa, L.B. Resstel, C.C. Crestani, Cannabidiol administration into the bed nucleus of the stria terminalis alters cardiovascular responses induced by acute restraint stress through 5-ht(1)a receptor, Eur. Neuropsychopharmacol. 23 (2013) 1096–1104. 62 L.A. Oliveira et al. / Pharmacological Research 95–96 (2015) 53–62 [45] F.V. Gomes, D.G. Reis, F.H. Alves, F.M. Correa, F.S. Guimaraes, L.B. Resstel, Cannabidiol injected into the bed nucleus of the stria terminalis reduces the expression of contextual fear conditioning via 5-ht1a receptors, J. Psychopharmacol. 26 (2012) 104–113. [46] P. Carrive, Dual activation of cardiac sympathetic and parasympathetic components during conditioned fear to context in the rat, Clin. Exp. Pharmacol. Physiol. 33 (2006) 1251–1254. [47] D.G. Dos Reis, E.A. Fortaleza, R.F. Tavares, F.M. Correa, Role of the autonomic nervous system and baroreflex in stress-evoked cardiovascular responses in rats, Stress 17 (2014) 362–372. [48] C.L. Grosskreutz, M.J. Brody, Regional hemodynamic responses to central administration of corticotropin-releasing factor (crf), Brain Res. 442 (1988) 363–367. [49] M.J. Nijsen, G. Croiset, R. Stam, A. Bruijnzeel, M. Diamant, D. de Wied, V.M. Wiegant, The role of the crh type 1 receptor in autonomic responses to corticotropin-releasing hormone in the rat, Neuropsychopharmacology 22 (2000) 388–399. [50] L. Wang, S. Cardin, V. Martinez, Y. Tache, Intracerebroventricular crf inhibits cold restraint-induced c-fos expression in the dorsal motor nucleus of the vagus and gastric erosions in rats, Brain Res. 736 (1996) 44–53. [51] H.W. Dong, L.W. Swanson, Organization of axonal projections from the anterolateral area of the bed nuclei of the stria terminalis, J. Comp. Neurol. 468 (2004) 277–298. [52] T.S. Gray, D.J. Magnuson, Neuropeptide neuronal efferents from the bed nucleus of the stria terminalis and central amygdaloid nucleus to the dorsal vagal complex in the rat, J. Comp. Neurol. 262 (1987) 365–374. [53] T.L. Kash, D.G. Winder, Neuropeptide y and corticotropin-releasing factor bidirectionally modulate inhibitory synaptic transmission in the bed nucleus of the stria terminalis, Neuropharmacology 51 (2006) 1013–1022. [54] J.G. Oberlander, L.P. Henderson, Corticotropin-releasing factor modulation of forebrain gabaergic transmission has a pivotal role in the expression of anabolic steroid-induced anxiety in the female mouse, Neuropsychopharmacology 37 (2012) 1483–1499.