Skip to main content
NCBI home page
British Journal of Pharmacology logoLink to British Journal of Pharmacology
. 2016 Aug 22;173(19):2833–2844. doi: 10.1111/bph.13560

Involvement of endocannabinoid neurotransmission in the bed nucleus of stria terminalis in cardiovascular responses to acute restraint stress in rats

Lucas Gomes‐de‐Souza 1,2, Leandro A Oliveira 1,2, Ricardo Benini 1,2, Patrícia Rodella 1, Willian Costa‐Ferreira 1,2, Carlos C Crestani 1,2,✉
PMCID: PMC5055136  PMID: 27441413

Abstract

Background and Purpose

Endocannabinoid signalling has been reported as an important neurochemical mechanism involved in responses to stress. Previous studies provided evidence of endocannabinoid release in the bed nucleus of the stria terminalis (BNST) during aversive stimuli. Nevertheless, a possible involvement of this neurochemical mechanism in stress responses has never been evaluated. Therefore, in the present study we investigated the involvement of BNST endocannabinoid neurotransmission, acting via local CB1 receptors, in the cardiovascular responses to acute restraint stress in rats.

Experimental Approach

The selective CB1 receptor antagonist AM251 (1, 30 and 100 pmol 100 nL−1) and/or the fatty acid amide hydrolase (FAAH) enzyme inhibitor URB597 (30 pmol 100 nL−1) or the monoacylglycerol lipase (MAGL) enzyme inhibitor JZL184 (30 pmol 100 nL−1) was microinjected into the BNST before the acute restraint stress.

Key Results

Microinjection of AM251 into the BNST enhanced the tachycardia caused by restraint stress, without affecting the increase in arterial pressure and the sympathetic‐mediated cutaneous vasoconstrictor response. Conversely, the increased endogenous levels of AEA in the BNST evoked by local treatment with the FAAH enzyme inhibitor URB597 decreased restraint‐evoked tachycardia. Inhibition of the hydrolysis of 2‐arachidonoylglycerol (2‐AG) in the BNST by local microinjection of the MAGL enzyme inhibitor JZL184 also decreased the HR response. These effects of URB597 and JZL184 were abolished by BNST pretreatment with AM251.

Conclusions and Implications

These findings indicate an involvement of BNST endocannabinoid neurotransmission, acting via CB1 receptors, in cardiovascular adjustments during emotional stress, which may be mediated by the local release of either AEA or 2‐AG.

Abbreviations

2‐AG

2‐arachidonoylglycerol

AEA

anandamide or arachidonoylethanolamide

BNST

bed nucleus of the stria terminalis

CBD

cannabidiol

CRF

corticotropin‐releasing factor

DBP

diastolic blood pressure

FAAH

fatty acid amide hydrolase

HR

heart rate

MAGL

monoacylglycerol lipase

MBP

mean blood pressure

MPFC

medial prefrontal cortex

NTS

nucleus of the solitary tract

PAG

periaqueductal gray

PVN

hypothalamic paraventricular nucleus

RVLM

rostral ventrolateral medulla

TRPV1

transient receptor potential vanilloid 1

Δ9‐THC

Δ9‐tetrahydrocannabinol

Tables of Links

TARGETS
GPCRs a Enzymes b
CB1 receptor FAAH
MGL
LIGANDS
AM251 URB597
JZL184
Open in a new tab

These Tables list key protein targets and ligands in this article which are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Southan et al., 2016) and are permanently archived in the Concise Guide to PHARMACOLOGY 2015/16 (a,bAlexander et al., 2015a, 2015b).

Introduction

The bed nucleus of the stria terminalis (BNST) is a limbic structure localized in the rostral prosencephalon that is activated during aversive stimuli (Cullinan et al., 1995) and has a direct influence in physiological responses to stress (Crestani et al., 2013). Regarding the cardiovascular responses, the BNST has been reported to be involved in the control of BP and heart rate (HR) responses evoked by both conditioned and unconditioned aversive stimuli (Resstel et al., 2008a; Crestani et al., 2009). Additionally, it was reported that local microinjection of cannabidiol (CBD) (a phytocannabinoid of Cannabis sativa) into the BNST increased the tachycardia induced by acute restraint stress without affecting the pressor response (Gomes et al., 2013). However, CBD has a low affinity for cannabinoid receptors (Thomas et al., 1998). In fact, the changes in stress‐evoked cardiovascular and behavioral responses following microinjection of CBD into the BNST were mediated by local 5‐HT1A receptors (Gomes et al., 2012; Gomes et al., 2013). Therefore, despite the evidence that a phytocannabinoid has an effect in the BNST, a possible influence of local endocannabinoid neurotransmission in the control of physiological responses to stress has never been evaluated.

The endocannabinoid term refers to the set of endogenous ligands of the receptors activated by the Δ9‐tetrahydrocannabinol (Δ9‐THC). The two major endocannabinoids are the arachidonoylethanolamide/anandamide (AEA) and 2‐arachidonoylglycerol (2‐AG) (Pacher et al., 2006; Mechoulam and Parker, 2013). Both endocannabionids are synthesized “on demand” by the cleavage of phospholipids of postsynaptic membranes (Bisogno et al., 2005; Pacher et al., 2006). The endocannabinoids act as retrograde messengers by activating CB1 receptors on presynaptic terminals, which in turn suppresses the release of excitatory and inhibitory neurotransmitters (Katona and Freund, 2012; Mechoulam and Parker, 2013). There is evidence of the expression of the CB2 receptor in the brain, but its role has yet to be established (Mechoulam and Parker, 2013; Morena et al., 2016). The endocannabinoids can also target other sites (e.g., the orphan receptor GRP55, the transient receptor potential vanilloid 1 channel (TRPV1), PPARs and ion channels) (Pertwee et al., 2010). The endocannabinoids are rapidly taken up from synapses by a membrane transport process that is not still fully characterized (Mechoulam and Parker, 2013; Morena et al., 2016). In the cell, AEA is hydrolyzed by the enzymes fatty acid amide hydrolase (FAAH), whereas 2‐AG is hydrolyzed by monoacylglycerol lipase (MAGL) (Dinh et al., 2002; McKinney and Cravatt, 2005).

Cannabinoids induce complex cardiovascular changes by acting centrally and peripherally via mechanisms involving various receptors (Malinowska et al., 2012; Zubrzycki et al., 2014). Regarding the cardiovascular effects of central actions of cannabinoids, an increase in BP and sympathetic nerve activity mediated by local CB1 receptors was reported to follow the microinjection of cannabinoids into the periaqueductal gray (PAG) and rostral ventrolateral medulla (RVLM) (Padley et al., 2003; Dean, 2011; Malinowska et al., 2012). Additionally, a recent study demonstrated both depressor and pressor effects of cannabinoids in the hypothalamic paraventricular nucleus (PVN) (Grzeda et al., 2015). An involvement of endocannabinoid neurotransmission in stress‐evoked cardiovascular changes has also been reported. For instance, activation of CB1 receptors within the medial prefrontal cortex (MPFC) and PAG reduced the cardiovascular responses of contextual fear conditioning (Resstel et al., 2008b; Lisboa et al., 2010), whereas blockade of CB1 receptors in the PAG attenuated the sympathoexcitatory and pressor responses evoked by hypothalamic defense area stimulation (Dean, 2011). Despite these findings, the involvement of endocannabinoid neurotransmission in other limbic structures in the control of stress‐evoked cardiovascular responses has never been investigated.

CB1 receptors were identified in glutamatergic and GABA terminals within the BNST (Puente et al., 2010). Additionally, systemic treatment with the selective CB1 receptor antagonist AM251 increased BNST neural activation induced by an aversive stimulus (Newsom et al., 2012), indicating that endocannabinoid signalling in the BNST is activated during stressful events. Thus, this study aimed to evaluate the hypothesis that endocannabinoid neurotransmission within the BNST is involved in cardiovascular responses evoked by acute restraint stress in rats.

Methods

Animals

Animal studies are reported in compliance with the ARRIVE guidelines (Kilkenny et al., 2010; McGrath and Lilley, 2015).

Seventy‐four male Wistar rats weighing 240–260g (60‐days‐old) were used. Animals were obtained from the animal breeding facility of the State University Paulista–UNESP (Botucatu, SP, Brazil) and were housed in collective plastic cages (4 rats per cage) with sawdust on the bottom in a temperature‐controlled 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 07:00 h and 19:00 h) 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# 35/2014), which complies with Brazilian and international guidelines for animal use and welfare.

Surgical preparation

Five days before the trial, rats were anaesthetized with tribromoethanol (250 mg kg−1, i.p.). After scalp anaesthesia with 2% lidocaine, the skull was exposed and stainless‐steel guide cannulas (26G, 12mm‐long) were bilaterally implanted into the BNST at a position 1mm above the site of injection, using a stereotaxic apparatus (Stoelting, Wood Dale, IL, USA). Stereotaxic coordinates for cannula implantation into the BNST were: antero‐posterior=+8.6mm from interaural line; lateral=4.0 mm from the medial suture; dorso‐ventral=−5.8 mm from the skull with a lateral inclination of 23° (Paxinos and Watson, 1997). Cannulae were fixed to the skull with dental cement and one metal screw. After surgery, the animals received a poly‐antibiotic with streptomycins and penicillins (560 mg ml−1 kg−1, i.m.) to prevent infection and the non‐steroidal anti‐inflammatory drug flunixine meglumine (0.5 mg ml−1 kg−1, s.c.) for post‐operation analgesia.

One day before the trial, rats were anaesthetized with tribromoethanol (250 mg kg−1, i.p.) and a catheter (Clay Adams, Parsippany, NJ, USA) was inserted into the abdominal aorta through the femoral artery for cardiovascular recording. A catheter was tunnelled under the skin and exteriorized on the animal's dorsum. After surgery, flunixine meglumine (0.5 mg ml−1 kg−1, s.c.) was administered for post‐operation analgesia. The animals were kept in individual cages during the postoperative period and cardiovascular recording.

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 a digital acquisition board (PowerLab 4/30, ML866/P, ADInstruments, NSW, Australia). Mean (MBP), systolic (SBP) and diastolic (DBP) blood pressure and HR values were derived from the pulsatile blood pressure recording.

Tail skin temperature measurement

The cutaneous temperature of the tail was recorded using a thermal camera (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 (Vianna and Carrive, 2005; Cruz et al., 2012; Gouveia et al., 2016).

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 cannulae and were connected to a 2μL syringe (7002‐KH, Hamilton Co., Reno, NV, USA) using a PE‐10 tubing (Clay Adams, Parsippany, NJ, USA). Needles were carefully inserted into the guide cannulae without restraining the animals, and drugs were injected in a final volume of 100 nL (Crestani et al., 2009; Gomes et al., 2013; Gouveia et al., 2016).

Acute restraint stress

Restraint is one of the most commonly employed stressors to investigate stress‐evoked behavioural and physiological changes in laboratory animals (Campos et al., 2013). For acute restraint stress, each rat was placed 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 60 min (Crestani et al., 2009; Gomes et al., 2013), and immediately after the end of the stress exposure rats were returned to their home cages. Each rat was subjected to only one session of restraint in order to avoid habituation.

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 the experiments were started. The experimental room was temperature‐ controlled (24°C) and acoustically isolated from the other rooms. Cardiovascular recording began at least 30 min before the onset of the restraint, and was performed throughout the session of stress. The tail skin temperature was measured 10, 5 and 0 min before the restraint for baseline values, and at 10, 20, 40 and 60 min during restraint (Busnardo et al., 2013). Each animal received a single pharmacological treatment and was subjected to one session of restraint. In each protocol, animals were randomly distributed among the several experimental groups.

Effect of bilateral microinjection of AM251 into the BNST on cardiovascular responses to acute restraint stress

This protocol aimed to investigate the involvement of the CB1 receptor within the BNST in cardiovascular responses evoked by acute restraint stress. For this, independent groups of animals received bilateral microinjections into the BNST of different doses of AM251 (selective CB1 receptor antagonist) (1, 30 or 100 pmol 100 nL−1) or vehicle (20% DMSO diluted in saline) (100 nL) (Lisboa et al., 2008; Ferreira‐Junior et al., 2012). Ten minutes after the pharmacological treatment the animals underwent a 60 min session of restraint stress.

Effect of bilateral microinjection of AM251 and/or URB597 into the BNST on cardiovascular responses to acute restraint stress

This protocol aimed to investigate the effect of an increase in endogenous levels of AEA within the BNST on cardiovascular responses evoked by acute restraint stress, as well as the involvement of the CB1 receptor in AEA's effects. For this, independent groups of animals received bilateral microinjections into the BNST of AM251 (1 pmol 100 nL−1) or vehicle (20% DMSO diluted in saline) (100 nL) followed 5 min later by URB597 (FAAH enzyme inhibitor) (30 pmol 100 nL−1) or vehicle (20% DMSO diluted in saline) (100 nL). Five minutes after each pharmacological treatment the animals underwent a 60 min session of restraint stress.

Effect of bilateral microinjection of AM251 and/or JZL184 into the BNST on cardiovascular responses to acute restraint stress

This protocol aimed to investigate the effect of an increase in endogenous levels of 2‐AG within the BNST on cardiovascular responses evoked by acute restraint stress, as well as the involvement of the CB1 receptor in 2‐AG's effects. For this, independent groups of animals received bilateral microinjections into the BNST of AM251 (1 pmol 100 nL−1) or vehicle (20% DMSO diluted in saline) (100 nL) followed 5 min later by JZL184 (MAGL enzyme inhibitor) (30 pmol 100 nL−1) or vehicle (20% DMSO diluted in saline) (100 nL). Five minutes after each pharmacological treatment the animals underwent a 60 min session of restraint stress.

Histological determination of the microinjection sites

At the end of experiments, rats were anaesthetized with urethane (250 mg ml−1 200 g−1 body weight, 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 40μm‐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 (Paxinos and Watson, 1997).

Drugs and solutions

AM251 (N‐(piperidin‐1‐yl)‐5‐(4‐iodophenyl)‐1‐(2,4‐dichlorophenyl)‐4‐methyl‐1H‐pyrazole‐3‐carboxamide) (selective CB1 receptor antagonist) (Tocris, West‐woods Business Park, Ellisville, MO, USA), URB597 (cyclohexylcarbamic acid 3'‐(aminocarbonyl)‐[1,1'‐biphenyl]‐3‐yl ester) (selective FAAH enzyme inhibitor) (Tocris) and JZL184 (4‐[bis(1,3‐benzodioxol‐5‐yl)hydroxymethyl]‐1‐piperidinecarboxylic acid 4‐nitrophenyl ester) (MAGL enzyme inhibitor) (Tocris) were dissolved in 20% DMSO diluted in saline (NaCl 0.9%). Tribromoethanol (Sigma–Aldrich, St. Louis, MO, USA) and urethane (Sigma–Aldrich) were dissolved in saline. Flunixine meglumine (Banamine®; Schering‐Plough, Cotia, SP, Brazil) and the poly‐antibiotic preparation (Pentabiotico®; Fort Dodge, Campinas, SP, Brazil) were used as provided.

Data analysis

The data and statistical analysis comply with the recommendations on experimental design and analysis in pharmacology (Curtis et al., 2015).

Researchers were not blinded to treatment groups once all data were obtained via direct recording of physiological parameters, so that analysis did not include any subjective evaluation. Data are expressed as mean ± SEM. The basal values of MAP, HR, and cutaneous temperature were compared using one‐way ANOVA (AM251 results) or Student's t‐test (URB597 and JZL184 results). The time–course curves of cardiovascular and cutaneous temperature changes were analysed using 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.

Results

Diagrammatic representations showing the bilateral injection sites in the BNST of all animals used in the present study as well as a photomicrograph of a coronal brain section depicting microinjection sites in the BNST of a representative animal are presented in Figure 1.

Figure 1.

Figure 1

Open in a new tab

(Left) Photomicrograph of a coronal brain section from a representative rat showing bilateral sites of microinjection into the BNST. Arrows indicate the microinjection sites. (Right) Diagrammatic representation based on the rat brain atlas of Paxinos and Watson (1997) indicating the injection sites into the BNST of AM251 (black circles), URB597 (black squares), AM251+URB597 (grey squares), JZL184 (white squares), AM251+JZL184 (grey circles) and vehicle (white circles). 3V – third ventricle, IA – interaural coordinate; ac – anterior commissure; cc – corpus callosum; f – fornix; ic – internal capsule; LV – lateral ventricles; st – stria terminalis.

Effect of bilateral microinjection of AM251 into the BNST on cardiovascular responses to acute restraint stress

Bilateral microinjection into the BNST of the selective CB1 receptor antagonist AM251 (1, 30, and 100 pmol 100 nL−1) did not alter baseline values of either MBP, SBP, DBP, or HR (Table 1). Nevertheless, AM251 at the dose of 1 pmol (P < 0.05) reduced basal values of tail skin temperature (Table 1). Acute restraint stress increased MBP (F (34,630) = 6, P < 0.0001) and HR (F (34,630) = 15, P < 0.0001) and decreased the tail cutaneous temperature (F (6,126) = 20, P < 0.0001) (Figure 2). Treatment of the BNST with AM251 at the doses of 30 pmol (P < 0.0001) and 100 pmol (P < 0.0001) increased restraint‐evoked tachycardia (F (3,630) = 27, P < 0.0001). AM251 at the doses of 1 pmol (P < 0.0001) and 30 pmol (P < 0.006) enhanced the drop in tail skin temperature (F (3,126) = 11, P < 0.0001) (Figure 2), but this effect is possibly related to the reduction in basal values caused by pharmacological treatment (see Table 1), since analysis of restraint‐evoked skin temperature change did not indicate a significant effect of AM251 (P > 0.05) (Supporting Information Figure S1). Analysis of time‐course curve of MBP indicated an effect of CB1 receptor blockade within the BNST (F (3,630) = 5, P < 0.002), but post‐hoc analyses did not reveal an effect of AM251 at any dose (P > 0.05) (Figure 2).

Table 1.

Basal parameters of mean (MBP), systolic (SBP) and diastolic (DBP) blood pressure, heart rate (HR), and tail skin temperature after pharmacological treatment of the BNST with different doses of the selective CB1 receptor antagonist (AM251) and/or the FAAH enzyme inhibitor URB597 or the MAGL enzyme inhibitor JZL184.

Group n MBP (mmHg) SBP (mmHg) DBP (mmHg) HR (beats min‐1) Tail skin temperature (°C)
Vehicle 6 105 ± 3 124 ± 5 87 ± 4 380 ± 23 28 ± 0.3
AM251 ‐ 1 pmol 6 113 ± 2 122 ± 4 100 ± 2 418 ± 17 26 ± 0.5*
AM251 ‐ 30 pmol 5 104 ± 5 128 ± 4 86 ± 4 375 ± 35 27 ± 0.6
AM251 ‐ 100 pmol 5 108 ± 3 130 ± 5 90 ± 5 400 ± 33 28 ± 0.3
F (3,18)  = 1.6, P > 0.05 F (3,18)  = 0.6, P > 0.05 F (3,18)  = 3, P > 0.05 F (3,18)  = 0.6, P > 0.05 F (3,18)  = 5, P<0.01
Vehicle 7 107 ± 2 131 ± 3 91 ± 2 389 ± 9 27 ± 1
URB597 ‐ 30 pmol 7 101 ± 3 120 ± 7 84 ± 2 389 ± 1 26 ± 0.8
t=1.6, P > 0.05 t = 1.4, P > 0.05 t = 2.4, P > 0.05 t = 0.01, P > 0.05 t = 0.8, P > 0.05
Vehicle 6 111 ± 2 136 ± 6 87 ± 1 371 ± 18 28 ± 0.3
AM251 (1pmol) + URB597 (30 pmol) 6 113 ± 3 130 ± 3 91 ± 2 408 ± 25 27 ± 1.7
t = 0.5, P > 0.05 t = 0.8, P > 0.05 t = 1.8, P > 0.05 t = 1.6, P > 0.05 t = 0.9, P > 0.05
Vehicle 7 107 ± 4 131 ± 3 91 ± 2 399 ± 13 27 ± 0.8
JZL184 ‐ 30 pmol 7 107 ± 2 132 ± 4 89 ± 3 389 ± 12 29 ± 0.6
t = 0.06, P > 0.05 t = 0.2, P > 0.05 t = 0.5, P > 0.05 t = 0.5, P > 0.05 t = 3.4, P > 0.05
Vehicle 6 108 ± 4 138 ± 6 87 ± 1 402 ± 16 26 ± 0.4
AM251 (1 pmol) + JZL184 (30 pmol) 6 113 ± 4 127 ± 9 93 ± 8 417 ± 25 27 ± 1.7
t = 0.6, P > 0.05 t = 1.0, P > 0.05 t = 0.7, P > 0.05 t = 0.6, P > 0.05 t = 0.3, P > 0.05
Open in a new tab

Data are expressed as means ± S.E.M.

Figure 2.

Figure 2

Open in a new tab

Time course curves of mean blood pressure (MBP), heart rate (HR) and tail skin temperature (tail temperature) during pre‐stress period (basal) and restraint stress session (restraint, shaded area) in animals treated with different doses (1, 30, and 100 pmol · 100 nL−1; n = 5–6 per group) of the selective CB1 receptor antagonist AM251 or vehicle (20% DMSO diluted in saline, 100nL, n = 6) into the BNST. Circles represent the mean and bars the SEM. Effect of the different doses of AM251 is shown separately, but statistical analysis was carried out including all experimental groups. # P < 0.05 over the whole period compared to vehicle‐treated animals, ANOVA followed by post‐hoc t‐test with a Bonferroni correction.

Effect of bilateral microinjection of AM251 and/or URB597 into the BNST on cardiovascular responses to acute restraint stress

Bilateral microinjection into the BNST of the FAAH enzyme inhibitor URB597 (30 pmol 100 nL−1) alone or in combination with AM251 (1 pmol 100 nL−1) did not affect baseline values of either MBP, SBP, DBP, HR or tail skin temperature (Table 1). Acute restraint stress increased the MBP (F (35,432) = 7, P < 0.0001) and HR (F (35,432) = 8, P < 0.0001) and decreased the tail skin temperature (F (6,84) = 12, P < 0.0001) (Figure 3). Treatment of the BNST with URB597 reduced the increase in HR evoked by restraint (F (1,432) = 85, P < 0.0001), without affecting the drop in tail skin temperature (F (1,84) = 3, P > 0.05) (Figure 3). Analysis of the time‐course curve of MBP indicated an effect of URB597 (F (1,432) = 78, P < 0.0001) (Figure 3), but this effect is possibly related to a reduction in the values during the pre‐stress period since analysis of restraint‐evoked MBP changes did not indicate a significant effect of URB597 (P > 0.05) (Supporting Information Figure S2).

Figure 3.

Figure 3

Open in a new tab

(Left) Time course curves of mean blood pressure (MBP), heart rate (HR), and tail skin temperature (tail temperature) during pre‐stress period (basal) and restraint stress session (restraint, shaded area) in animals treated with the FAAH enzyme inhibitor URB597 (30 pmol 100 nL−1 , n = 7) or vehicle (20% DMSO diluted in saline, 100 nL, n = 7) into the BNST. (Right) Time course curves of MBP, HR and tail temperature during pre‐stress period (basal) and restraint session (restraint, shaded area) in animals treated with URB597 (30 pmol 100 nL−1) into the BNST after local pretreatment with the selective CB1 receptor antagonist AM251 (1 pmol 100 nL−1, n = 6) or vehicle (20% DMSO diluted in saline, 100 nL, n = 6). Circles represent the mean and bars the SEM. # P < 0.05 over the whole period compared to vehicle‐treated animals, ANOVA followed by post‐hoc t‐test with a Bonferroni correction.

Analysis of HR data identified an effect of treatment with URB597 in animals pretreated locally with AM251 (1 pmol 100 nL−1) (AM251+URB597) (F (1,353) = 35, P < 0.0001) (Figure 3), but this effect is possibly related to an increase in these values during the pre‐stress period since analysis of restraint‐evoked HR changes did not indicate a significant effect (P > 0.05) (Supporting Information Figure S2). Combined treatment with AM251+URB597 did not affect MBP (F (1,353) = 3, P > 0.05) or skin temperature values (F (1,80) = 1, P > 0.05) (Figure 3).

Effect of bilateral microinjection of AM251 and/or JZL184 into the BNST on cardiovascular responses to acute restraint stress

Bilateral microinjection into the BNST of the MAGL enzyme inhibitor JZL184 (30 pmol 100 nL−1) alone or in combination with AM251 did not affect baseline values of either MBP, SBP, DBP, HR or tail skin temperature (Table 1). Acute restraint stress induced an increase in the MBP (F (35,432) = 9, P < 0.0001) and HR (F (35,432) = 4, P < 0.0001) and decreased the tail skin temperature (F (7,96) = 5, P < 0.0001) (Figure 4). Treatment of the BNST with JZL184 reduced the restraint‐evoked increase in HR (F (1,432) = 146, P < 0.0001), without affecting the increase in MBP (F (1,432) = 1, P > 0.05) (Figure 4). Analysis indicated a significant effect of BNST treatment with JZL184 on the decrease in tail skin temperature (F (1,96) = 12, P < 0.002), but this effect is possibly related to changes in values during the pre‐stress period since analysis of restraint‐evoked tail temperature change did not indicate a significant effect of JZL184 (P > 0.05) (Supporting Information Figure S3).

Figure 4.

Figure 4

Open in a new tab

(Left) Time course curves of mean blood pressure (MBP), heart rate (HR) and tail skin temperature (tail temperature) during pre‐stress period (basal) and restraint stress session (restraint, shaded area) in animals treated with the MAGL enzyme inhibitor JZL184 (30 pmol 100 nL−1, n = 7) or vehicle (20% DMSO diluted in saline, 100 nL, n = 7) into the BNST. (Right) Time course curves of MBP, HR and tail temperature during pre‐stress period (basal) and restraint stress session (restraint, shaded area) in animals treated with JZL184 (30 nmol 100 nL−1) into the BNST after local pretreatment with the selective CB1 receptor antagonist AM251 (1 pmol 100 nL−1) (n = 6) or vehicle (20% DMSO diluted in saline, 100 nL, n = 6). Circles represent the mean and bars the SEM. # P < 0.05 over the whole period compared to vehicle‐treated animals, ANOVA followed by post‐hoc t‐test with a Bonferroni correction.

Analysis indicated that combined treatment of the BNST with AM251+JZL184 had an effect on the MBP (F (1,354) = 16, P < 0.0001), HR (F (1,354) = 6, P < 0.001) and tail skin temperature (F (1,80) = 27, P < 0.0001) following (Figure 4). However, all of these effects are possibly related to changes in values during the pre‐stress period since analysis of restraint‐evoked changes did not indicate a significant effect (P > 0.05) (Supporting Information Figure S3).

Discussion

The present study provides the first evidence of the involvement of endocannabinoid neurotransmission in the BNST in cardiovascular adjustments during emotional stress. The results showed that bilateral microinjection of the CB1 receptor antagonist AM251 into the BNST enhanced the HR increase caused by acute restraint stress, without affecting the BP increase and the sympathetic‐mediated cutaneous vasoconstriction response. Conversely, an increase in local endogenous levels of AEA or 2‐AG induced by BNST treatment with, respectively, the FAAH inhibitor URB597 or the MAGL inhibitor JZL184 attenuated the restraint‐evoked HR increase. These effects of URB597 and JZL184 were abolished by BNST pretreatment with AM251, thus confirming that control of cardiovascular responses during emotional stress by both AEA and 2‐AG within the BNST are mediated by activation of local CB1 receptors.

Facilitation of the tachycardiac response to restraint following BNST treatment with the CB1 receptor antagonist indicates an inhibitory role of BNST endocannabinoid signalling in cardiac responses during stress. Our findings corroborate the general idea that endocannabinoid neurotransmission acts to suppress physiological responses during stress (Hill and Tasker, 2012; Morena et al., 2016). Regarding the stress‐evoked cardiovascular responses, a previous study reported that blockade of CB1 receptors within the MPFC enhanced the pressor and tachycardiac response to contextual fear conditioning (Lisboa et al., 2010), thus providing evidence of an inhibitory role of endocannabinoid signalling in cardiovascular responses to stress. Local microinjection of either AEA or an AEA transporter inhibitor into the dorsolateral PAG also reduced the cardiovascular responses of contextual fear conditioning via local CB1 receptors (Resstel et al., 2008b). Conversely, Dean (2011) reported that blockade of CB1 receptors in the dorsal PAG attenuated the sympathoexcitatory and pressor responses to hypothalamic defense area stimulation in anaesthetized rats, suggesting that dorsal PAG endocannabinoid neurotransmission is involved in this defense‐like response. Opposite cardiovascular effects are evoked by cannabinoids in anaesthetized versus conscious animals (Malinowska et al., 2012), suggesting that the discrepancy in the findings in the PAG is possibly related to the anaesthesia. Therefore, the results of the present study are in line with previous evidence in conscious animals indicating that endocannabinoid neurotransmission acts to suppress the cardiovascular responses during defensive situations.

Results obtained with the endocannabinoid hydrolysis inhibitors indicated that control of cardiovascular responses during emotional stress by BNST endocannabinoid neurotransmission may be mediated by the action of either AEA or 2‐AG. Furthermore, the effects of both URB597 and JZL184 were completely abolished by pretreatment with the CB1 receptor antagonist AM251. Therefore, despite evidence that endocannabinoids can target other sites beyond the CB1 receptor (Pertwee et al., 2010), the present results provide evidence that the effects of both endocannabinoids within the BNST are mediated by activation of the CB1 receptor. Our findings are in line with previous findings showing that the release of both AEA and 2‐AG modulates synaptic transmission in the BNST via local CB1 receptors (Puente et al., 2011).

Previous studies reported a decrease in AEA during acute restraint stress in limbic structures such as the amygdala and hippocampus, whereas several studies have demonstrated that stress increases the endocannabinoid 2‐AG in brain (Morena et al., 2016). However, CB1 receptor antagonists did not affect per se the basal activity of BNST neurons (Massi et al., 2008; Puente et al., 2010), thus indicating an absence of tonic endocannabinoid activity within the BNST. Therefore, the control of the cardiovascular response by endocannabinoid signalling in the BNST during an aversive threat is likely to be mediated by an increase in the release of AEA and/or 2‐AG. Previous studies reported that changes in AEA release in the brain seem to occur rapidly during stress while a delayed release of 2‐AG has been observed (Morena et al., 2016). Endocannabinoids released within the BNST during aversive threats have never been investigated. Nevertheless, the present findings provide evidence that either URB597 or JZL184 affected both the initial and later phases of restraint‐evoked tachycardia, thus indicating that both AEA and 2‐AG are already released into the BNST at the beginning of a stress session. However, further studies assessing the endogenous levels of the endocannabinoids in the BNST during stress are necessary to clarify this hypothesis.

The majority of CB1 receptors are expressed on axon terminals, and their activation suppresses the release of other neurotransmitters into the synapse (Katona and Freund, 2012). Previous studies reported that local BNST treatment with either an α1‐adrenoceptor antagonist or a muscarinic ACh receptor antagonist enhanced the restraint‐evoked HR increase (Crestani et al., 2009; Gouveia et al., 2016). Additionally, a recent study documented that microinjection of corticotropin‐releasing factor (CRF) receptor antagonists into the BNST reduced cardiovascular responses to restraint (Oliveira et al., 2015). Considering these pieces of evidence, the control of the HR response during restraint by a BNST endocannabinoid is likely to be mediated by the inhibition of local CRF release rather than the control of noradrenaline or acetylcholine release. However, the possible control of local CRF release by BNST endocannabinoid signalling has never been investigated. CB1 receptors were identified in glutamatergic and GABAergic terminals within the BNST (Puente et al., 2010), but evidence for a role of these neurochemical mechanisms within the BNST in the control of cardiovascular responses to stress is missing. Therefore, further studies are necessary to elucidate the local neurochemical mechanism by which BNST endocannabinoid signalling interacts to control cardiovascular responses during an aversive threat.

Both branches of the autonomic nervous system participate in the control of cardiovascular function during stress (Carrive, 2006; Crestani et al., 2010; Dos Reis et al., 2014). Indeed, studies have reported the coactivation of cardiac sympathetic and parasympathetic activity during restraint (Crestani et al., 2009; Dos Reis et al., 2014). The BNST is directly connected with medullary structures involved with autonomic control, such as the nucleus of the solitary tract, nucleus ambiguous, and ventrolateral regions (Gray and Magnuson, 1987; Dong and Swanson, 2004). Taken together, these findings suggest that the inhibitory influence of BNST endocannabinoid signalling in restraint‐evoked tachycardia may be mediated by the facilitation of stimulatory inputs to medullary parasympathetic neurons and/or activation of inhibitory pathways to premotor sympathetic neurons.

The BNST has been proposed as a relay station connecting limbic structures such as the hippocampus, amygdala and medial prefrontal cortex (MPFC) with primary stress effector regions in the hypothalamus and brainstem (Ulrich‐Lai and Herman, 2009; Crestani et al., 2013; Adhikari, 2014). Accordingly, a MPFC lesion decreases activation of BNST neurons evoked by restraint stress (Figueiredo et al., 2003). CB1 receptors within the BNST were identified on excitatory presynaptic terminals from the infralimbic region of the MPFC (Massi et al., 2008), and local endocannabinoid signalling acts by inhibiting glutamatergic transmission from the MPFC in the BNST (Massi et al., 2008; Glangetas et al., 2013). Inhibition of the infralimbic region of the MPFC reduced restraint‐evoked tachycardia (Tavares et al., 2009), thus indicating a facilitatory influence of this cortical region in cardiac response during threat aversive. Therefore, it is possible that endocannabinoid release into the BNST during stress inhibits excitatory information from the infralimbic cortex, which in turn reduces HR response. However, glutamatergic and GABAergic inputs to the BNST also originate from amygdala and hippocampus (Myers et al., 2014), and BNST ablation inhibits physiological responses elicited by activation of these structures (Roder and Ciriello, 1993; Zhu et al., 2001). Thus, we cannot exclude the possibility that the control of cardiovascular responses to stress elicited by BNST endocannabinoid signalling is also mediated by a modulation of information coming from other limbic structures beyond the MPFC.

Convergent clinical and preclinical studies have provided evidence that emotional stress is a modifiable risk factor for several cardiovascular dysfunctions (Sgoifo et al., 2014; Cohen et al., 2015; Crestani, 2016). Substances that modulate endocannabinoid neurotransmission have been indicated as a promising strategy for the treatment of cardiovascular dysfunctions (Carnevali et al., 2016). In this context, the present findings provide additional evidence regarding the neurobiological mechanisms involved in the influence of endocannabinoid neurotransmission on stress‐evoked cardiovascular responses.

In summary, the present results indicate that endocannabinoid signalling within the BNST, acting via local CB1 receptors, plays an inhibitory role in the tachycardiac response to emotional stress. Our data also provide evidence that this effect may be mediated by the local release in the BNST of either AEA or 2‐AG.

Author contributions

L.G.S. and C.C.C. conceived and designed this research; L.G.S., L.A.O., R.B., P.R., and W.C.F. performed the experiments and analysed the data; L.G.S., L.A.O., R.B., P.R., W.C.F., C.C.C. interpreted the results of experiments; L.G.S. prepared the figures and drafted the manuscript; L.G.S. and C.C.C. edited and revised the manuscript; C.C.C. approved the final version of the manuscript.

Conflict of interest

The authors declare no conflicts of interest.

Declaration of transparency and scientific rigour

This Declaration acknowledges that this paper adheres to the principles for transparent reporting and scientific rigour of preclinical research recommended by funding agencies, publishers and other organisations engaged with supporting research.

Supporting information

Figure S1 Time course of changes in mean blood pressure (ΔMBP), heart rate (ΔHR), and tail skin temperature (Δtail temperature) induced by restraint stress in animals treated with different doses (1, 30, and 100 pmol/100 nL) of the selective CB1 receptor antagonist AM251 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.

Click here for additional data file. (753.2KB, pdf)

Figure S2 (Left) Time course of changes in mean arterial pressure (ΔMAP), heart rate (ΔHR), and tail skin temperature (Δtail temperature) induced by restraint stress in animals treated with the FAAH enzyme inhibitor URB597 (30 pmol /100nL) into the BNST. (Right) Tim1e course of ΔMAP, ΔHR, and Δ tail temperature induced by restraint in animals treated with URB597 (30 pmol /100nL) into the BNST after local pretreatment with the selective CB1 receptor antagonist AM251 (1pmol/100nL). 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.

Click here for additional data file. (787.2KB, pdf)

Figure S3 (Left) Time course of changes in mean arterial pressure (ΔMAP), heart rate (ΔHR), and tail skin temperature (Δtail temperature) induced by restraint stress in animals treated with the MAGL enzyme inhibitor JZL184 (30 pmol /100nL) into the BNST. (Right) Time course of ΔMAP, ΔHR, and Δtail temperature induced by restraint in animals treated with JZL184 (30nmol/100nL) into the BNST after local pretreatment with the selective CB1 receptor antagonist AM251 (1pmol/100nL). 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.

Click here for additional data file. (778.4KB, pdf)

Acknowledgements

The authors wish to thank Elisabete Lepera and Rosana Silva for technical assistance. This work was supported by FAPESP grant # 2015/05922‐9, CNPq grant # 456405/2014‐3, CAPES/MEC/Brazilian government, and PADC‐FCF/UNESP.

Gomes‐de‐Souza, L. , Oliveira, L. A. , Benini, R. , Rodella, P. , Costa‐Ferreira, W. , and Crestani, C. C. (2016) Involvement of endocannabinoid neurotransmission in the bed nucleus of stria terminalis in cardiovascular responses to acute restraint stress in rats. British Journal of Pharmacology, 173: 2833–2844. doi: 10.1111/bph.13560.

References

  1. Adhikari A (2014). Distributed circuits underlying anxiety. Front Behav Neurosci 8: 112. doi: 10.3389/fnbeh.2014.00112. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Alexander SPH, Davenport AP, Kelly E, Marrion N, Peters JA, Benson HE et al. (2015a). The Concise Guide to PHARMACOLOGY 2015/16: G protein‐coupled receptors. Br J Pharmacol 172: 5744–5869. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Alexander SPH, Fabbro D, Kelly E, Marrion N, Peters JA, Benson HE et al. (2015b). The Concise Guide to PHARMACOLOGY 2015/16: Enzymes. Br J Pharmacol 172: 6024–6109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bisogno T, Ligresti A, Di Marzo V (2005). The endocannabinoid signalling system: biochemical aspects. Pharmacol Biochem Behav 81: 224–238. [DOI] [PubMed] [Google Scholar]
  5. Blessing WW (2003). Lower brainstem pathways regulating sympathetically mediated changes in cutaneous blood flow. Cell Mol Neurobiol 23: 527–538. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Busnardo C, Alves FH, Crestani CC, Scopinho AA, Resstel LB, Correa FM (2013). Paraventricular nucleus of the hypothalamus glutamate neurotransmission modulates autonomic, neuroendocrine and behavioral responses to acute restraint stress in rats. Eur Neuropsychopharmacol 23: 1611–1622. [DOI] [PubMed] [Google Scholar]
  7. Campos AC, Fogaca MV, Aguiar DC, Guimaraes FS (2013). Animal models of anxiety disorders and stress. Rev Bras Psiquiatr 35 (Suppl 2): S101–S111. [DOI] [PubMed] [Google Scholar]
  8. Carnevali L, Rivara S, Nalivaiko E, Thayer JF, Vacondio F, Mor M et al. (2016). Pharmacological inhibition of FAAH activity in rodents: A promising pharmacological approach for psychological‐cardiac comorbidity? Neurosci Biobehav Rev. doi: 10.1016/j.neubiorev.2016.04.013. [DOI] [PubMed] [Google Scholar]
  9. Carrive P (2006). Dual activation of cardiac sympathetic and parasympathetic components during conditioned fear to context in the rat. Clin Exp Pharmacol Physiol 33: 1251–1254. [DOI] [PubMed] [Google Scholar]
  10. Cohen BE, Edmondson D, Kronish IM (2015). State of the Art Review: Depression, Stress, Anxiety, and Cardiovascular Disease. Am J Hypertens 28: 1295–1302. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Crestani CC (2016). Adolescent vulnerability to cardiovascular consequences of chronic emotional stress: Review and perspectives for future research. Neurosci Biobehav Rev. doi: 10.1016/j.neubiorev.2016.03.027. [DOI] [PubMed] [Google Scholar]
  12. Crestani CC, Alves FH, Gomes FV, Resstel LB, Correa FM, Herman JP (2013). Mechanisms in the bed nucleus of the stria terminalis involved in control of autonomic and neuroendocrine functions: a review. Curr Neuropharmacol 11: 141–159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Crestani CC, Alves FH, Tavares RF, Correa FM (2009). Role of the bed nucleus of the stria terminalis in the cardiovascular responses to acute restraint stress in rats. Stress 12: 268–278. [DOI] [PubMed] [Google Scholar]
  14. Crestani CC, Tavares RF, Alves FH, Resstel LB, Correa FM (2010). Effect of acute restraint stress on the tachycardiac and bradycardiac responses of the baroreflex in rats. Stress 13: 61–72. [DOI] [PubMed] [Google Scholar]
  15. Cruz FC, Engi SA, Leao RM, Planeta CS, Crestani CC (2012). Influence of the single or combined administration of cocaine and testosterone in autonomic and neuroendocrine responses to acute restraint stress. J Psychopharmacol 26: 1366–1374. [DOI] [PubMed] [Google Scholar]
  16. Cullinan WE, Herman JP, Battaglia DF, Akil H, Watson SJ (1995). Pattern and time course of immediate early gene expression in rat brain following acute stress. Neuroscience 64: 477–505. [DOI] [PubMed] [Google Scholar]
  17. Curtis MJ, Bond RA, Spina D, Ahluwalia A, Alexander SPA, Giembycz MA et al. (2015). Experimental design and analysis and their reporting: new guidance for publication in BJP. Br J Pharmacol 172: 3461–3471. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Dampney RA, Horiuchi J, McDowall LM (2008). Hypothalamic mechanisms coordinating cardiorespiratory function during exercise and defensive behaviour. Auton Neurosci 142: 3–10. [DOI] [PubMed] [Google Scholar]
  19. Dean C (2011). Endocannabinoid modulation of sympathetic and cardiovascular responses to acute stress in the periaqueductal gray of the rat. Am J Physiol Regul Integr Comp Physiol 300: R771–R779. [DOI] [PubMed] [Google Scholar]
  20. Dinh TP, Carpenter D, Leslie FM, Freund TF, Katona I, Sensi SL et al. (2002). Brain monoglyceride lipase participating in endocannabinoid inactivation. Proc Natl Acad Sci U S A 99: 10819–10824. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Dong HW, Swanson LW (2004). Organization of axonal projections from the anterolateral area of the bed nuclei of the stria terminalis. J Comp Neurol 468: 277–298. [DOI] [PubMed] [Google Scholar]
  22. Dos Reis DG, Fortaleza EA, Tavares RF, Correa FM (2014). Role of the autonomic nervous system and baroreflex in stress‐evoked cardiovascular responses in rats. Stress 17: 362–372. [DOI] [PubMed] [Google Scholar]
  23. Ferreira‐Junior NC, Fedoce AG, Alves FH, Correa FM, Resstel LB (2012). Medial prefrontal cortex endocannabinoid system modulates baroreflex activity through CB(1) receptors. Am J Physiol Regul Integr Comp Physiol 302: R876–R885. [DOI] [PubMed] [Google Scholar]
  24. Figueiredo HF, Bruestle A, Bodie B, Dolgas CM, Herman JP (2003). The medial prefrontal cortex differentially regulates stress‐induced c‐fos expression in the forebrain depending on type of stressor. Eur J Neurosci 18: 2357–2364. [DOI] [PubMed] [Google Scholar]
  25. Glangetas C, Girard D, Groc L, Marsicano G, Chaouloff F, Georges F (2013). Stress switches cannabinoid type‐1 (CB1) receptor‐dependent plasticity from LTD to LTP in the bed nucleus of the stria terminalis. J Neurosci 33: 19657–19663. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Gomes FV, Alves FH, Guimaraes FS, Correa FM, Resstel LB, Crestani CC (2013). 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: 1096–1104. [DOI] [PubMed] [Google Scholar]
  27. Gomes FV, Reis DG, Alves FH, Correa FM, Guimaraes FS, Resstel LB (2012). Cannabidiol injected into the bed nucleus of the stria terminalis reduces the expression of contextual fear conditioning via 5‐HT1A receptors. J Psychopharmacol 26: 104–113. [DOI] [PubMed] [Google Scholar]
  28. Gouveia MK, Miguel TT, Busnardo C, Scopinho AA, Correa FM, Nunes‐de‐Souza RL et al. (2016). Dissociation in control of physiological and behavioral responses to emotional stress by cholinergic neurotransmission in the bed nucleus of the stria terminalis in rats. Neuropharmacology 101: 379–388. [DOI] [PubMed] [Google Scholar]
  29. Gray TS, Magnuson DJ (1987). 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: 365–374. [DOI] [PubMed] [Google Scholar]
  30. Grzeda E, Schlicker E, Luczaj W, Harasim E, Baranowska‐Kuczko M, Malinowska B (2015). Bi‐directional CB1 receptor‐mediated cardiovascular effects of cannabinoids in anaesthetized rats: role of the paraventricular nucleus. J Physiol Pharmacol 66: 343–353. [PubMed] [Google Scholar]
  31. Hill MN, Tasker JG (2012). Endocannabinoid signaling, glucocorticoid‐mediated negative feedback, and regulation of the hypothalamic‐pituitary‐adrenal axis. Neuroscience 204: 5–16. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Katona I, Freund TF (2012). Multiple functions of endocannabinoid signaling in the brain. Annu Rev Neurosci 35: 529–558. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Kilkenny C, Browne W, Cuthill IC, Emerson M, Altman DG (2010). NC3Rs Reporting Guidelines Working Group. Br J Pharmacol 160: 1577–1579. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Lisboa SF, Reis DG, da Silva AL, Correa FM, Guimaraes FS, Resstel LB (2010). Cannabinoid CB1 receptors in the medial prefrontal cortex modulate the expression of contextual fear conditioning. Int J Neuropsychopharmacol 13: 1163–1173. [DOI] [PubMed] [Google Scholar]
  35. Lisboa SF, Resstel LB, Aguiar DC, Guimaraes FS (2008). Activation of cannabinoid CB1 receptors in the dorsolateral periaqueductal gray induces anxiolytic effects in rats submitted to the Vogel conflict test. Eur J Pharmacol 593: 73–78. [DOI] [PubMed] [Google Scholar]
  36. Malinowska B, Baranowska‐Kuczko M, Schlicker E (2012). Triphasic blood pressure responses to cannabinoids: do we understand the mechanism? Br J Pharmacol 165: 2073–2088. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Massi L, Elezgarai I, Puente N, Reguero L, Grandes P, Manzoni OJ et al. (2008). Cannabinoid receptors in the bed nucleus of the stria terminalis control cortical excitation of midbrain dopamine cells in vivo. J Neurosci 28: 10496–10508. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. McGrath JC, Lilley E (2015). Implementing guidelines on reporting research using animals (ARRIVE etc.): new requirements for publication in BJP. Br J Pharmacol 172: 3189–3193. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. McKinney MK, Cravatt BF (2005). Structure and function of fatty acid amide hydrolase. Annu Rev Biochem 74: 411–432. [DOI] [PubMed] [Google Scholar]
  40. Mechoulam R, Parker LA (2013). The endocannabinoid system and the brain. Annu Rev Psychol 64: 21–47. [DOI] [PubMed] [Google Scholar]
  41. Morena M, Patel S, Bains JS, Hill MN (2016). Neurobiological Interactions Between Stress and the Endocannabinoid System. Neuropsychopharmacology 41: 80–102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Myers B, Mark Dolgas C, Kasckow J, Cullinan WE, Herman JP (2014). Central stress‐integrative circuits: forebrain glutamatergic and GABAergic projections to the dorsomedial hypothalamus, medial preoptic area, and bed nucleus of the stria terminalis. Brain Struct Funct 219: 1287–1303. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Newsom RJ, Osterlund C, Masini CV, Day HE, Spencer RL, Campeau S (2012). Cannabinoid receptor type 1 antagonism significantly modulates basal and loud noise induced neural and hypothalamic‐pituitary‐adrenal axis responses in male Sprague‐Dawley rats. Neuroscience 204: 64–73. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Oliveira LA, Almeida J, Benini R, Crestani CC (2015). CRF1 and CRF2 receptors in the bed nucleus of the stria terminalis modulate the cardiovascular responses to acute restraint stress in rats. Pharmacol Res 95‐96: 53–62. [DOI] [PubMed] [Google Scholar]
  45. Pacher P, Batkai S, Kunos G (2006). The endocannabinoid system as an emerging target of pharmacotherapy. Pharmacol Rev 58: 389–462. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Padley JR, Li Q, Pilowsky PM, Goodchild AK (2003). Cannabinoid receptor activation in the rostral ventrolateral medulla oblongata evokes cardiorespiratory effects in anaesthetised rats. Br J Pharmacol 140: 384–394. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Paxinos G, Watson C (1997). The rat brain in stereotaxic coordinates, 3rd edn. Academic Press: Sidney, Australia. [Google Scholar]
  48. Pertwee RG, Howlett AC, Abood ME, Alexander SP, Di Marzo V, Elphick MR et al. (2010). International Union of Basic and Clinical Pharmacology. LXXIX. Cannabinoid receptors and their ligands: beyond CB(1) and CB(2). Pharmacol Rev 62: 588–631. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Puente N, Cui Y, Lassalle O, Lafourcade M, Georges F, Venance L et al. (2011). Polymodal activation of the endocannabinoid system in the extended amygdala. Nat Neurosci 14: 1542–1547. [DOI] [PubMed] [Google Scholar]
  50. Puente N, Elezgarai I, Lafourcade M, Reguero L, Marsicano G, Georges F et al. (2010). Localization and function of the cannabinoid CB1 receptor in the anterolateral bed nucleus of the stria terminalis. PLoS One 5: e8869. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Resstel LB, Alves FH, Reis DG, Crestani CC, Correa FM, Guimaraes FS (2008a). Anxiolytic‐like effects induced by acute reversible inactivation of the bed nucleus of stria terminalis. Neuroscience 154: 869–876. [DOI] [PubMed] [Google Scholar]
  52. Resstel LB, Lisboa SF, Aguiar DC, Correa FM, Guimaraes FS (2008b). Activation of CB1 cannabinoid receptors in the dorsolateral periaqueductal gray reduces the expression of contextual fear conditioning in rats. Psychopharmacology (Berl) 198: 405–411. [DOI] [PubMed] [Google Scholar]
  53. Roder S, Ciriello J (1993). Contribution of bed nucleus of the stria terminalis to the cardiovascular responses elicited by stimulation of the amygdala. J Auton Nerv Syst 45: 61–75. [DOI] [PubMed] [Google Scholar]
  54. Sgoifo A, Carnevali L, Grippo AJ (2014). The socially stressed heart. Insights from studies in rodents. Neurosci Biobehav Rev 39: 51–60. [DOI] [PubMed] [Google Scholar]
  55. Southan C, Sharman JL, Benson HE, Faccenda E, Pawson AJ, Alexander SP et al. (2016). The IUPHAR/BPS Guide to PHARMACOLOGY in 2016: towards curated quantitative interactions between 1300 protein targets and 6000 ligands. Nucl Acids Res 44: D1054–D1068. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Sterling P (2012). Allostasis: a model of predictive regulation. Physiol Behav 106: 5–15. [DOI] [PubMed] [Google Scholar]
  57. Tavares RF, Correa FM, Resstel LB (2009). Opposite role of infralimbic and prelimbic cortex in the tachycardiac response evoked by acute restraint stress in rats. J Neurosci Res 87: 2601–2607. [DOI] [PubMed] [Google Scholar]
  58. Thomas BF, Gilliam AF, Burch DF, Roche MJ, Seltzman HH (1998). Comparative receptor binding analyses of cannabinoid agonists and antagonists. J Pharmacol Exp Ther 285: 285–292. [PubMed] [Google Scholar]
  59. Ulrich‐Lai YM, Herman JP (2009). Neural regulation of endocrine and autonomic stress responses. Nat Rev Neurosci 10: 397–409. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Vianna DM, Carrive P (2005). Changes in cutaneous and body temperature during and after conditioned fear to context in the rat. Eur J Neurosci 21: 2505–2512. [DOI] [PubMed] [Google Scholar]
  61. Zhu W, Umegaki H, Suzuki Y, Miura H, Iguchi A (2001). Involvement of the bed nucleus of the stria terminalis in hippocampal cholinergic system‐mediated activation of the hypothalamo‐‐pituitary‐‐adrenocortical axis in rats. Brain Res 916: 101–106. [DOI] [PubMed] [Google Scholar]
  62. Zubrzycki M, Liebold A, Janecka A, Zubrzycka M (2014). A new face of endocannabinoids in pharmacotherapy. Part I: protective role of endocannabinoids in hypertension and myocardial infarction. J Physiol Pharmacol 65: 171–181. [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1 Time course of changes in mean blood pressure (ΔMBP), heart rate (ΔHR), and tail skin temperature (Δtail temperature) induced by restraint stress in animals treated with different doses (1, 30, and 100 pmol/100 nL) of the selective CB1 receptor antagonist AM251 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.

Click here for additional data file. (753.2KB, pdf)

Figure S2 (Left) Time course of changes in mean arterial pressure (ΔMAP), heart rate (ΔHR), and tail skin temperature (Δtail temperature) induced by restraint stress in animals treated with the FAAH enzyme inhibitor URB597 (30 pmol /100nL) into the BNST. (Right) Tim1e course of ΔMAP, ΔHR, and Δ tail temperature induced by restraint in animals treated with URB597 (30 pmol /100nL) into the BNST after local pretreatment with the selective CB1 receptor antagonist AM251 (1pmol/100nL). 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.

Click here for additional data file. (787.2KB, pdf)

Figure S3 (Left) Time course of changes in mean arterial pressure (ΔMAP), heart rate (ΔHR), and tail skin temperature (Δtail temperature) induced by restraint stress in animals treated with the MAGL enzyme inhibitor JZL184 (30 pmol /100nL) into the BNST. (Right) Time course of ΔMAP, ΔHR, and Δtail temperature induced by restraint in animals treated with JZL184 (30nmol/100nL) into the BNST after local pretreatment with the selective CB1 receptor antagonist AM251 (1pmol/100nL). 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.

Click here for additional data file. (778.4KB, pdf)

Articles from British Journal of Pharmacology are provided here courtesy of The British Pharmacological Society

RESOURCES