Ryan J. McLaughlin

Endogenous cannabinoid signaling is essential for stress adaptation

Secretion of glucocorticoid hormones during stress produces an array of physiological changes that are adaptive and beneficial in the short term. In the face of repeated stress exposure, however, habituation of the glucocorticoid response is essential as prolonged glucocorticoid secretion can produce deleterious effects on metabolic, immune, cardiovascular, and neurobiological function. Endocannabinoid signaling responds to and regulates the activity of the hypothalamic–pituitary–adrenal (HPA) axis that governs the secretion of glucocorticoids; however, the role this system plays in adaptation of the neuroendocrine response to repeated stress is not well characterized. Herein, we demonstrate a divergent regulation of the two endocannabinoid ligands, N-arachidonylethanolamine (anandamide; AEA) and 2-arachidonoylglycerol (2-AG), following repeated stress such that AEA content is persistently decreased throughout the corticolimbic stress circuit, whereas 2-AG is exclusively elevated within the amygdala in a stress-dependent manner. Pharmacological studies demonstrate that this divergent regulation of AEA and 2-AG contribute to distinct forms of HPA axis habituation. Inhibition of AEA hydrolysis prevented the development of basal hypersecretion of corticosterone following repeated stress. In contrast, systemic or intra-amygdalar administration of a CB1 receptor antagonist before the final stress exposure prevented the repeated stress-induced decline in corticosterone responses. The present findings demonstrate an important role for endocannabinoid signaling in the process of stress HPA habituation, and suggest that AEA and 2-AG modulate different components of the adrenocortical response to repeated stressor exposure.

Recruitment of Prefrontal Cortical Endocannabinoid Signaling by Glucocorticoids Contributes to Termination of the Stress Response

The mechanisms subserving the ability of glucocorticoid signaling within the medial prefrontal cortex (mPFC) to terminate stress-induced activation of the hypothalamic–pituitary–adrenal (HPA) axis are not well understood. We report that antagonism of the cannabinoid CB1 receptor locally within the mPFC prolonged corticosterone secretion following cessation of stress in rats. Mice lacking the CB1 receptor exhibited a similar prolonged response to stress. Exposure of rats to stress produced an elevation in the endocannabinoid 2-arachidonoylglycerol within the mPFC that was reversed by pretreatment with the glucocorticoid receptor antagonist RU-486 (20 mg/kg). Electron microscopic and electrophysiological data demonstrated the presence of CB1 receptors in inhibitory-type terminals impinging upon principal neurons within layer V of the prelimbic region of the mPFC. Bath application of corticosterone (100 nm) to prefrontal cortical slices suppressed GABA release onto principal neurons in layer V of the prelimbic region, when examined 1 h later, which was prevented by application of a CB1 receptor antagonist. Collectively, these data demonstrate that the ability of stress-induced glucocorticoid signaling within mPFC to terminate HPA axis activity is mediated by a local recruitment of endocannabinoid signaling. Endocannabinoid activation of CB1 receptors decreases GABA release within the mPFC, likely increasing the outflow of the principal neurons of the prelimbic region to contribute to termination of the stress response. These data support a model in which endocannabinoid signaling links glucocorticoid receptor engagement to activation of corticolimbic relays that inhibit corticosterone secretion.

Suppression of Amygdalar Endocannabinoid Signaling by Stress Contributes to Activation of the Hypothalamic-Pituitary-Adrenal Axis

Endocannabinoids inhibit hypothalamic–pituitary–adrenal (HPA) axis activity; however, the neural substrates and pathways subserving this effect are not well characterized. The amygdala is a forebrain structure that provides excitatory drive to the HPA axis under conditions of stress. The aim of this study was to determine the contribution of endocannabinoid signaling within distinct amygdalar nuclei to activation of the HPA axis in response to psychological stress. Exposure of rats to 30-min restraint stress increased the hydrolytic activity of fatty acid amide hydrolase (FAAH) and concurrently decreased content of the endocannabinoid/CB1 receptor ligand N-arachidonylethanolamine (anandamide; AEA) throughout the amygdala. In stressed rats, AEA content in the amygdala was inversely correlated with serum corticosterone concentrations. Pharmacological inhibition of FAAH activity within the basolateral amygdala complex (BLA) attenuated stress-induced corticosterone secretion; this effect was blocked by co-administration of the CB1 receptor antagonist AM251, suggesting that stress-induced decreases in CB1 receptor activation by AEA contribute to activation of the neuroendocrine stress response. Local administration into the BLA of a CB1 receptor agonist significantly reduced stress-induced corticosterone secretion, whereas administration of a CB1 receptor antagonist increased corticosterone secretion. Taken together, these findings suggest that the degree to which stressful stimuli reduce amygdalar AEA/CB1 receptor signaling contributes to the magnitude of the HPA response.

Regional alterations in the endocannabinoid system in an animal model of depression: effects of concurrent antidepressant treatment

It has been suggested that disturbances in endocannabinoid signaling contribute to the development of depressive illness; however, at present there is insufficient evidence to allow for a full understanding of this role. To further this understanding, we performed an analysis of the endocannabinoid system in an animal model of depression. Male rats exposed to chronic, unpredictable stress (CUS) for 21 days exhibited a reduction in sexual motivation, consistent with the hypothesis that CUS in rats induces depression‐like symptoms. We determined the effects of CUS, with or without concurrent treatment with the antidepressant imipramine (10 mg/kg), on CP55940 binding to the cannabinoid CB1 receptor; whole tissue endocannabinoid content; and fatty acid amide hydrolase (FAAH) activity in the prefrontal cortex, hippocampus, hypothalamus, amygdala, midbrain and ventral striatum. Exposure to CUS resulted in a significant increase in CB1 receptor binding site density in the prefrontal cortex and a decrease in CB1 receptor binding site density in the hippocampus, hypothalamus and ventral striatum. Except in the hippocampus, these CUS‐induced alterations in CB1 receptor binding site density were attenuated by concurrent antidepressant treatment. CUS alone produced a significant reduction in N‐arachidonylethanolamine (anandamide) content in every brain region examined, which was not reversed by antidepressant treatment. These data suggest that the endocannabinoid system in cortical and subcortical structures is differentially altered in an animal model of depression and that the effects of CUS on CB1 receptor binding site density are attenuated by antidepressant treatment while those on endocannabinoid content are not.

Opposing roles for the nucleus accumbens core and shell in cue-induced reinstatement of food-seeking behavior.

Reinstatement of previously extinguished instrumental responding for drug-related cues has been used as an animal model for relapse of drug abuse, and is differentially affected by inactivation of the core and shell subregions of the nucleus accumbens (NAc). To compare the roles of these subregions in reinstatement induced by cues associated with natural and drug rewards, the present study assessed the effects of inactivation of the NAc core and shell on cue-induced reinstatement of food-seeking behavior. Rats acquired a lever pressing response for food reward paired with a light/tone conditioned stimulus (CS). They were then subjected to extinction, where both food and the CS were withheld. Reinstatement of responding was measured during response-contingent presentations of the CS. Following saline infusions into the NAc core or shell, rats displayed a significant increase in lever pressing during reinstatement sessions. Inactivation of the core, induced by infusion of GABA agonists muscimol and baclofen, attenuated responding for the CS, but did not affect pavlovian approach toward the food receptacle. In contrast, inactivation of the shell had the opposite effect, potentiating responding relative to vehicle treatments. These data suggest that the NAc core and shell play opposing, yet complementary roles in mediating the influence that food-associated conditioned stimuli exert over behavior. The core enables reward-related stimuli to bias the direction and vigor of instrumental responding. In contrast, the shell facilitates alterations in behavior in response to changes in the incentive value of conditioned stimuli. The fact that the NAc core appears to play a similar role in cue-induced reinstatement induced by both natural and drug rewards suggests that this region of the ventral striatum may be a final common pathway through which both drug- and food-associated stimuli may influence the direction and magnitude of ongoing behavior.

Corticotropin-Releasing Hormone Drives Anandamide Hydrolysis in the Amygdala to Promote Anxiety

Corticotropin-releasing hormone (CRH) is a central integrator in the brain of endocrine and behavioral stress responses, whereas activation of the endocannabinoid CB1 receptor suppresses these responses. Although these systems regulate overlapping functions, few studies have investigated whether these systems interact. Here we demonstrate a novel mechanism of CRH-induced anxiety that relies on modulation of endocannabinoids. Specifically, we found that CRH, through activation of the CRH receptor type 1 (CRHR1), evokes a rapid induction of the enzyme fatty acid amide hydrolase (FAAH), which causes a reduction in the endocannabinoid anandamide (AEA), within the amygdala. Similarly, the ability of acute stress to modulate amygdala FAAH and AEA in both rats and mice is also mediated through CRHR1 activation. This interaction occurs specifically in amygdala pyramidal neurons and represents a novel mechanism of endocannabinoid–CRH interactions in regulating amygdala output. Functionally, we found that CRH signaling in the amygdala promotes an anxious phenotype that is prevented by FAAH inhibition. Together, this work suggests that rapid reductions in amygdala AEA signaling following stress may prime the amygdala and facilitate the generation of downstream stress-linked behaviors. Given that endocannabinoid signaling is thought to exert “tonic” regulation on stress and anxiety responses, these data suggest that CRH signaling coordinates a disruption of tonic AEA activity to promote a state of anxiety, which in turn may represent an endogenous mechanism by which stress enhances anxiety. These data suggest that FAAH inhibitors may represent a novel class of anxiolytics that specifically target stress-induced anxiety.

A critical role for prefrontocortical endocannabinoid signaling in the regulation of stress and emotional behavior

The prefrontal cortex (PFC) provides executive control of the brain in humans and rodents, coordinating cognitive, emotional, and behavioral responses to threatening stimuli and subsequent feedback inhibition of the hypothalamic-pituitary-adrenal (HPA) axis. The endocannabinoid system has emerged as a fundamental regulator of HPA axis feedback inhibition and an important modulator of emotional behavior. However, the precise role of endocannabinoid signaling within the PFC with respect to stress coping and emotionality has only recently been investigated. This review discusses the current state of knowledge regarding the localization and function of the endocannabinoid system in the PFC, its sensitivity to stress and its role in modulating the neuroendocrine and behavioral responses to aversive stimuli. We propose a model whereby steady-state endocannabinoid signaling in the medial PFC indirectly regulates the outflow of pyramidal neurons by fine-tuning GABAergic inhibition. Local activation of this population of CB1 receptors increases the downstream targets of medial PFC activation, which include inhibitory interneurons in the basolateral amygdala, inhibitory relay neurons in the bed nucleus of the stria terminalis and monoamine cell bodies such as the dorsal raphe nucleus. This ultimately produces beneficial effects on emotionality (active coping responses to stress and reduced anxiety) and assists in constraining activation of the HPA axis. Under conditions of chronic stress, or in individuals suffering from mood disorders, this system may be uniquely recruited to help maintain appropriate function in the face of adversity, while breakdown of the endocannabinoid system in the medial PFC may be, in and of itself, sufficient to produce neuropsychiatric illness. Thus, we suggest that endocannabinoid signaling in the medial PFC may represent an attractive target for the treatment of stress-related disorders.

Cannabinoids and emotionality: A neuroanatomical perspective

The endocannabinoid system has recently emerged as a promising therapeutic target for the treatment of stress-related emotional disorders. A growing literature base has collectively demonstrated that facilitation of endocannabinoid signaling promotes antidepressant- and anxiolytic-like responses in preclinical animal models, while disruption of this system profoundly affects emotion, cognition, and neuroendocrine functioning. Although these findings are encouraging, the role of endocannabinoid signaling within discrete corticolimbic brain structures is considerably complex. Consequently, researchers have recently shifted focus to examining the effects of local cannabinoid manipulations on emotion from a neuroanatomical standpoint. This review provides an overview of the site-specific effects of cannabinergic compounds in preclinical tests of emotionality, as well as the alterations in endocannabinoid signaling observed in animal models of depression. Broadly speaking, these studies indicate that CB(1) receptors in the medial prefrontal cortex and ventral hippocampus appear to be responsible for the antidepressant- and anxiolytic-like phenotype elicited by systemic CB(1) receptor agonists, which parallels biochemical studies showing that endocannabinoids are downregulated in these two regions following exposure to chronic stress. Conversely, CB(1) receptor activation within distinct amygdalar nuclei yields opposing effects on emotional behavior, such that local stimulation of CB(1) receptors in the basolateral amygdala and central amygdala promoting anxiogenesis and anxiolysis, respectively. Moreover, a series of elegant studies has revealed that cannabinoid transmission in the basolateral amygdala strongly modulates the acquisition and processing of associative fear memory via interactions with the medial prefrontal cortex. Given the crucial role of this corticolimbic network in regulating emotional behavior, it is palpable that alterations in endocannabinoid signaling within any of these structures could have profound implications for the pathophysiological development of affective illnesses. Accordingly, local pharmacological augmentation of endocannabinoid signaling within discrete corticolimbic subregions may serve as a promising therapeutic strategy for the treatment of these debilitating disorders.

Prefrontal cortical anandamide signaling coordinates coping responses to stress through a serotonergic pathway

The endocannabinoid system has recently emerged as a vital component of the stress response and is an appealing target for the treatment of mood and anxiety disorders. Additionally, corticolimbic endocannabinoid signaling is important for stress-induced regulation of emotional behavior. However, the mechanism by which this occurs remains elusive. Combining biochemical and behavioral analyses within the forced swim test, we examined whether stress-induced regulation of endocannabinoid signaling in the medial prefrontal cortex contributes to behavioral responses to stress, and whether these responses are dependent on serotonergic neurotransmission. Forced swim stress produced a rapid and pronounced reduction in medial prefrontal anandamide content, but had no effect on 2-arachidonoylglycerol content within this region. Local administration of the anandamide hydrolysis inhibitor URB597 (0.01μg) into the ventromedial region of the prefrontal cortex decreased passive coping responses and increased active behavioral strategies, a phenomenon which was blocked by local antagonism of the CB(1) receptor. Furthermore, local inhibition of anandamide hydrolysis within the medial PFC increased the firing rate of serotonergic neurons within the dorsal raphe, suggesting that prefrontal cortical endocannabinoid signaling may modulate stress coping behaviors through a regulation of serotonergic neurotransmission. Accordingly, serotonin depletion prevented the ability of inhibition of anandamide hydrolysis within the medial PFC to promote active stress coping responses. Collectively, these data argue that stress-induced changes in endocannabinoid signaling within the medial PFC modulate stress-coping behaviors through a regulation of serotonergic neurotransmission and provide a neuroanatomical framework by which we may understand the mechanisms subserving the antidepressant potential of the endocannabinoid system.

Local enhancement of cannabinoid CB1 receptor signalling in the dorsal hippocampus elicits an antidepressant-like effect.

Systemic administration of direct cannabinoid CB1 receptor agonists and inhibitors of the hydrolytic enzyme fatty acid amide hydrolase have been shown to elicit antidepressant effects. Moreover, the endocannabinoid system in the hippocampus is sensitive to both chronic stress and antidepressant administration, suggesting a potential role of this system in emotional changes associated with these regimens. The aim of this study was to determine if cannabinoid CB1 receptors in the hippocampus modulate emotionality in rats as assessed via the forced swim test. Male Sprague–Dawley rats were bilaterally implanted with cannulae directed at the dentate gyrus of the dorsal hippocampus and subsequently received three infusions of either the cannabinoid CB1 receptor agonist HU-210 (1 and 2.5 μg), the fatty acid amide hydrolase inhibitor URB597 (0.5 and 1 μg), the cannabinoid CB1 receptor antagonist AM251 (1 and 2.5 μg), or vehicle (dimethyl sulfoxide) and were assessed in the forced swim test. Infusion of both doses of HU-210 resulted in a dramatic reduction in immobility and increase in swimming behaviour, indicative of an antidepressant response, which was partially reversed by coadministration of AM251. No effect of URB597 administration or any effect following the administration of AM251 alone was, however, observed. These data indicate that activation of CB1 receptors in the dentate gyrus of the hippocampus results in an antidepressant-like response. Collectively, these data highlight the potential importance of changes in the hippocampal endocannabinoid system following stress or antidepressant treatment with respect to the manifestation and/or treatment of depression.