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

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.

Figure 1

(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.

Figure 2

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.

Figure 3

(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.

Figure 4

(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.