Role of the basolateral amygdala in mediating the effects of the fatty acid amide hydrolase inhibitor URB597 on HPA axis response to stress
Introduction
Hypothalamic–pituitary–adrenal (HPA) axis is the main neuroendocrine system involved in the maintenance of homeostasis after stressful stimuli. The axis is composed of the paraventricular nucleus (PVN) of the hypothalamus, anterior pituitary gland and adrenal cortex (Herman and Cullinan, 1997). Stress stimulates parvocellular neurons in the PVN, to secrete the corticotropin-releasing hormone (CRH), which, in the anterior pituitary gland, drives the synthesis of adrenocorticotropic hormone (ACTH) from the precursor protein pro-opiomelanocortin (POMC). ACTH, released into the blood stream, reaches the adrenal cortex to stimulate corticosterone (CORT) secretion (Ulrich-Lai and Herman, 2009). CORT, in turn, both promotes mobilization of energy stores for rapid, adaptive responses to stress, and exerts a negative feedback to PVN and anterior pituitary to stop the further release of CRH and ACTH (Herman and Cullinan, 1997). Excitation of the HPA axis is driven by selected stress circuits and may vary depending on the type of stressor involved. Physical stress (hemorrhage, hypotension, respiratory distress) activates HPA axis through brainstem catecholaminergic pathways that project directly to CRH expressing neurons of the PVN, while psychological stressors (restraint, forced swimming) activate limbic forebrain structures like amygdala, hippocampus and the prefrontal cortex (Ulrich-Lai and Herman, 2009).
Several lines of evidences support the role of the endocannabinoid system as a modulator of the HPA (Haring et al., 2012, Patel et al., 2004) and of the behavioral responses to stress, including anxiety-related behaviors, mood tone and extinction of fear memories (Cota et al., 2007, Steiner et al., 2008).
The endocannabinoids (ECs) are an endogenous family of retrograde lipid messengers that activate CB1 and CB2 cannabinoid receptors (Piomelli, 2003). They are synthesized on demand through cleavage of membrane phospholipid precursors and are involved in various short-range signaling processes (Piomelli, 2003). The CB1 receptor is the most abundant G-protein-coupled receptor in the central nervous system, and it is expressed at presynaptic level to negatively control neurotransmitter release. Two principal ECs have been broadly characterized so far, namely N-arachidonoyl ethanolamide or anandamide (AEA) and 2- arachidonoylglycerol (2-AG) (Piomelli, 2003). Their actions are terminated by a putative uptake process, followed by degradation by fatty acid amide hydrolase (FAAH) and by monoacylglycerol lipase, respectively. FAAH is widely distributed in the rat brain, mostly in cell bodies juxtaposed to axon terminals that express CB1 receptors suggesting that FAAH activity has an important role in the postsynaptic inactivation of AEA (Piomelli, 2003).
The pharmacological manipulation of AEA tone in the brain has been shown to control several neurobehavioral aspects related to HPA axis activation. Pharmacological inhibition or genetic deletion of FAAH induces analgesia, enhance memory extinction and attenuate anxiety-like behavior via an increased activation of CB1 receptors (Bambico et al., 2010, Cassano et al., 2011, Moreira et al., 2008). Selective FAAH inhibitors that significantly increase the brain levels of AEA, but not 2-AG, are currently available. Among these is URB597, which inhibits FAAH activity both in vitro (IC50=4.6 nM in rat brain membranes and IC50=0.5 nM in intact neurons) and in vivo (ID50=0.15 mg/kg, intraperitoneally (i.p.), in the rat) (Kathuria et al., 2003). By increasing AEA tone and therefore indirectly activating CB1 receptors, URB597 has been shown to elicit marked anxiolytic-like and antidepressant-like responses in different behavioral paradigms, including the elevated zero maze test and the forced swimming test in adult rats, the isolation-induced ultrasonic vocalization test in rat pups, and the tail suspension test in adult mice (Kathuria et al., 2003). Such effects were not accompanied by the broad spectrum of cannabimimetic behavioral actions (Kathuria et al., 2003), and were associated to an increased firing of noradrenergic and serotonergic neurons, respectively, the locus coeruleus (LC) and the dorsal raphe of adult rats, and by an increase of the extracellular levels of serotonin in the hippocampus, an area receiving projections from dorsal raphe (Gobbi et al., 2005). These observations suggested that modulation of endogenous AEA tone, rather than administration of direct CB1 agonists, could represent a novel approach for the treatment of anxiety- and depression-related disorders. Moreover, inhibition of FAAH along with the blockade of transient receptor potential vanilloid type-1 (TRPV1) has been suggested to exert robust anxiolytic effect (John and Currie, 2012). The FAAH inhibition approach could be also useful to modulate the neurobehavioral responses to stress, thus providing a new approach to treat other stress-related neuropsychiatric conditions, such as the posttraumatic stress disorder (Varvel et al., 2007). This hypothesis is supported by the observation that AEA content in the amygdala of rodents subjected to acute restraint stress is significantly decreased, presumably as consequence of increased FAAH activity, and that amygdala AEA levels are indirectly correlated to blood CORT levels (Hill et al., 2009). Based on these considerations, in this study we tested the potential anti-stress effects of systemically administered URB597 to rats subjected to a forced swimming stress. Specifically, we investigated the impact of FAAH inhibition on markers of HPA-axis activation, such as plasma CORT levels, POMC mRNA in the pituitary gland and CRH mRNA in the hypothalamus, as well as on stress-induced neuronal activity in the PVN, the amygdala and the brainstem, as assessed by c-fos (a neuronal activity marker) mRNA induction.
This stress protocol is a modified version of the Porsolt forced swim test which was previously used in our laboratory to demonstrate the antidepressant-like properties of URB597 (Gobbi et al., 2005). Different from our previous study, here animals were not habituated to the test situation, but rather were faced directly to an inescapable 20-min stress. This protocol allowed us to investigate the modulatory effect of URB597 on coping responses to stress never experienced before. It has been clearly demonstrated that varying the environmental conditions and the stressful procedures can lead to different observations (Herman and Cullinan, 1997), thus demonstrating that often the pharmacological actions of a drug cannot be generalized from the specific experimental model used. Most of the available data on the effects of URB597 in acute and repeated stress exposure were generated from animals subjected to restraint stress (Hill et al., 2009, Patel et al., 2004). Therefore using a different experimental protocol of stress gave us also the opportunity to evaluate whether the anti-stress properties of URB597 are independent from the specific experimental context.
Finally, the current literature points at the BLA as a key player in the modulation of the stress response by ECs (Hill et al., 2009) as it is also suggested by the effects of BLA CB1 blockade by the CB1 antagonist AM251 on anxiogenic behavior (Dono and Currie, 2012). Therefore, we investigated also the role played by CB1 receptors in this area in mediating the effects of acute FAAH inhibition.
Section snippets
Animals
Male Wistar rats (300–350 g) from Harlan Laboratories, Italy were used in these experiments. Upon arrival, the animals were housed individually in a temperature and humidity controlled room with access to food and water ad libitum. The room was maintained at 12:12 h light/dark cycle, with light on at 06:30 h. All procedures met the guidelines from the Italian Ministry of Health, from the United States National Institutes of Health, detailed in the Guide for the Care of Laboratory Animals, and from
Forced swim induces CORT secretion
Plasma CORT levels were measured to confirm that 20 min of swimming stress activated the HPA axis and to evaluate the effect of URB597 on this parameter.
Stress exposure significantly increased plasma CORT levels in rats at both 20 (p<0.01) and 40 min (p<0.001). Moreover, one-way ANOVA revealed no significant effect of URB597 treatment on plasma CORT levels of stressed-animals at either time points (Figure 1B).
FAAH inhibition attenuates stress-induced CRH mRNA expression in PVN
The hybridization signal for CRH mRNA was evident in the PVN (Figure 1C) and
Discussion
In this study we showed that systemic administration of the FAAH inhibitor URB597 dampened the hypothalamic and pituitary responses to forced-swimming stress through a mechanism that, at least in part, involves the activation of CB1 receptors in the BLA. More specifically, URB597 treatment decreased 1) stress-induced CRH mRNA expression in PVN; 2) stress-induced POMC mRNA expression, dose dependently, in the pituitary gland; and 3) stress-induced c-fos mRNA in amygdala and PVN. Conversely,
Role of funding source
This study was supported by Sapienza intramural research (grant no. C26A13389Y) grant (Ricerche di Ateneo 2013) to SG and by Grant PRIN 2012 2012JTX3KL_002 from the Italian Ministry of University and Research to SG. Both grant providers had no further role in study design, in the collection, analysis, and interpretation of data, in the writing of the report, and in the decision to submit the paper for publication.
Contributors
Gaurav Bedse, Roberto Colangeli, Silvana Gaetani and Tommaso Cassano designed the study, and wrote protocol. Gaurav Bedse, Angelo M. Lavecchia and Adele Romano managed experimental part. Gaurav Bedse, Roberto Colangeli and Carlo Cifani managed the literature searches and analyses. Fabio Altieri managed all riboprobes required for this study.
Tommaso Cassano and Silvana Gaetani undertook the statistical analysis. Gaurav Bedse wrote the first draft of manuscript. All authors contributed to and
Conflict of interest
The authors have no actual or potential conflicts of interest including any financial, personal or other relationships with other people or organizations within three years of beginning the work submitted that could inappropriately influence (bias) our work.
No author׳s institution has contracts relating to this research through which it or any other organization may stand to gain financially now or in the future.
Acknowledgments
We thank to Dr. Jin Fu, University of California for providing all the riboprobes used for this study.
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2020, PsychoneuroendocrinologyCitation Excerpt :The patterns of c-fos mRNA measured in this region in the current study mirror those reported previously (Newsom et al., 2012), more clearly indicating that our initial findings were not mediated by the stress of the peripheral injections, supporting the interpretation that the eCB system affords constitutive inhibition of neural activity in this region. The basolateral amygdala has been the focus of much research on eCB involvement in HPA axis regulation (Bedse et al., 2014; Gray et al., 2015; Hill et al., 2009, 2010b; Ramikie and Patel, 2012). We previously reported systemic injection of 2.0 mg/kg AM251 to stimulate c-fos mRNA induction under basal conditions, but that this dose of the CB1R antagonist did not further potentiate acute response to loud noise stress (Newsom et al., 2012).
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These authors equally contributed to the study.