Tiffany T.-Y. Lee

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.

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.

Endogenous cannabinoid signaling is required for voluntary exercise-induced enhancement of progenitor cell proliferation in the hippocampus

Voluntary exercise and endogenous cannabinoid activity have independently been shown to regulate hippocampal plasticity. The aim of the current study was to determine whether the endocannabinoid system is regulated by voluntary exercise and if these changes contribute to exercise‐induced enhancement of cell proliferation. In Experiment 1, 8 days of free access to a running wheel increased the agonist binding site density of the cannabinoid CB1 receptor; CB1 receptor‐mediated GTPγS binding; and the tissue content of the endocannabinoid anandamide in the hippocampus but not in the prefrontal cortex. In Experiment 2, the CB1 receptor antagonist AM251 (1 mg kg−1) was administered daily to animals given free access to a running wheel for 8 days, after which cell proliferation in the hippocampus was examined through immunohistochemical analysis of the cell cycle protein Ki‐67. Voluntary exercise increased proliferation of progenitor cells, as evidenced by the increase in the number of Ki‐67 positive cells in the granule cell layer of the dentate gyrus (DG) in the hippocampus. However, this effect was abrogated by concurrent treatment with AM251, indicating that the increase in endocannabinoid signaling in the hippocampus is required for the exercise‐induced increase in cell proliferation. These data demonstrate that the endocannabinoid system in the hippocampus is sensitive to environmental change and suggest that it is a mediator of experience‐induced plasticity.

Estrogenic regulation of limbic cannabinoid receptor binding.

Sex differences have been identified in many of the behavioral and physiological effects of cannabinoids. While estrogen has been linked to some of these variations, the effects of estrogen on cannabinoid receptor binding have not been characterized within regions of the brain specifically implicated in stress responsivity and emotional behavior. To examine sex differences, and the role of estradiol, in regulation of the cannabinoid receptor, we compared the binding site density of the cannabinoid receptor within the amygdala, hippocampus and hypothalamus in males, cycling females, ovariectomized (OVX) females and estradiol-treated OVX females (OVX+E). Our data reveal that males and OVX females have higher amounts of hypothalamic and lower amounts of amygdalar cannabinoid receptor binding relative to both cycling females and OVX+E females. Within the hippocampus, ovariectomy resulted in an upregulation of cannabinoid receptor binding. These data provide a putative biochemical mechanism mediating the observed behavioral and physiological sex differences in the effects of cannabinoids, particularly with respect to stress and emotional behavior.

Timing is everything: evidence for a role of corticolimbic endocannabinoids in modulating hypothalamic-pituitary-adrenal axis activity across developmental periods.

Growing evidence suggests that the endocannabinoid system is vital to ensuring normative maturation of the brain into adulthood. Endocannabinoid signaling contributes to guiding pro-neurogenic processes in early life and the development of neurotransmitter systems. Moreover, there is extensive evidence that recruitment of the endocannabinoid system is crucial in the regulation of neuroendocrine responses to stress via the hypothalamic-pituitary-adrenal (HPA) axis, and contributes to subsequent psychopathological consequences associated with emotionality and anxiety. These stress-induced physiological and behavioural sequelae are regulated by neural structures within the corticolimbic circuit, including the amygdala, hypothalamus, hippocampus, and prefrontal cortex. Based on evidence demonstrating endocannabinoid system involvement in both development and stress-induced changes in HPA axis function, it is reasonable to suggest that endocannabinoid signaling is an important mediator of interactions between stress responsivity and maturational stage. In this review, we discuss the ontogeny of the endocannabinoid system in the central nervous system, clinical and rodent models demonstrating short- and long-term effects of stress exposure, regulation of HPA axis responsivity by endocannabinoid signaling, as well as pharmacological and stress models indicating involvement of the endocannabinoid system in early post-natal and adolescent development on stress reactivity of the HPA, the corticolimbic system, and behaviour.

Temporal Changes in N-acylethanolamine Content and Metabolism Throughout the Peri-Adolescent Period

Fatty acid amide hydrolase (FAAH) regulates tissue concentrations of N‐acylethanolamines (NAEs), including the endocannabinoid, N‐arachidonylethanolamide (anandamide, AEA). FAAH activity and NAEs are widely distributed throughout the brain and FAAH activity regulates an array of processes including emotion, cognition, inflammation, and feeding. However, there is relatively little research describing how this system develops throughout adolescence, particularly within limbic circuits regulating stress and reward processing. Thus, this study characterized temporal changes in NAE content (AEA, oleoylethanolamine [OEA], and palmitoylethanolamide [PEA]) and FAAH activity across the peri‐adolescent period, in four corticolimbic structures (amygdala, hippocampus, prefrontal cortex, and hypothalamus). Brain tissue of male Sprague–Dawley rats was collected on postnatal days (PND) 25, 35, 45, and 70, representing pre‐adolescence, early‐ to mid‐adolescence, late adolescence, and adulthood, respectively. Tissue was analyzed for AEA, OEA, and PEA content as well as FAAH activity at each time point. AEA, OEA, and PEA exhibited a similar temporal pattern in all four brain regions. NAE concentrations were lowest at PND 25 and highest at PND 35. NAE concentrations decreased between PNDs 35 and 45 and increased between PNDs 45 and 70. FAAH activity mirrored the pattern of NAE content in which it decreased between PNDs 25 and 35, increased between PNDs 35 and 45, and decreased between PNDs 45 and 70. These age‐dependent patterns of NAE content and FAAH activity demonstrate temporal specificity to the development of this system and could contribute to alterations in stress sensitivity, emotionality, and executive function which also fluctuate during this developmental period. Synapse, 2013.

Age of stress exposure modulates the immediate and sustained effects of repeated stress on corticolimbic cannabinoid CB1 receptor binding in male rats

Chronic stress is known to modulate cannabinoid CB1 receptor binding densities in corticolimbic structures, in a region-dependent manner; however, the ontogeny of these changes and the degree to which they recover following exposure to stress have yet to be determined. To this extent, we examined both the immediate and sustained effects (following a 40-day recovery period) of a repeated restraint stress paradigm (30-min restraint/day for 10 days) on CB1 receptor binding in the prefrontal cortex (PFC), hippocampus and amygdala in both adolescent (stress onset at post-natal day [PND] 35) and adult (stress onset at PND 75) male Sprague-Dawley rats. Consistent with previous reports, we found that repeated stress in adult rats resulted in an increase in CB1 receptor binding in the PFC, a reduction in CB1 receptor binding in the hippocampus and no effect in the amygdala. Interestingly, adolescent rats exposed to repeated restraint stress did not show any change in hippocampal CB1 receptor density, but exhibited an upregulation of CB1 receptor binding in both the PFC and amygdala. In adults, a 40-day recovery period resulted in a normalization of CB1 receptor binding in the PFC, and surprisingly a pronounced upregulation of CB1 receptor binding in the hippocampus, possibly indicative of a rebound effect. Adolescents similarly exhibited this rebound increase in hippocampal CB1 receptor binding, despite a lack in immediate downregulation following repeated restraint. Of particular interest, adolescents exposed to stress were found to have a sustained downregulation of prefrontocortical CB1 receptors in adulthood, which may relate to some of the reported sustained behavioral effects of stress in adolescence. Collectively, these data indicate that the effects of chronic stress on cannabinoid CB1 receptor binding are modulated by the age of stress exposure and period of recovery following the cessation of stress.

Sex, drugs, and adult neurogenesis: Sex‐dependent effects of escalating adolescent cannabinoid exposure on adult hippocampal neurogenesis, stress reactivity, and amphetamine sensitization

Cannabinoid exposure during adolescence has adverse effects on neuroplasticity, emotional behavior, cognition, and reward sensitivity in adult rats. We investigated whether escalating doses of the cannabinoid receptor 1 (CB1R) agonist, HU‐210, in adolescence would affect adult hippocampal neurogenesis and behavioral processes putatively modulated by hippocampal neurogenesis, in adult male and female Sprague‐Dawley rats. Escalating doses of HU‐210 (25, 50, and 100 µg/kg), or vehicle were administered from postnatal day (PND) 35 to 46. Animals were left undisturbed until PND 70, when they were treated with 5‐bromo‐2‐deoxyuridine (BrdU; 200 mg/kg) and perfused 21 days later to examine density of BrdU‐ir and BrdU/NeuN cells in the dentate gyrus. In another cohort, hypothalamic‐pituitary‐adrenal (HPA) axis reactivity to an acute restraint stress (30 min; PND 75) and behavioral sensitization to d‐amphetamine sulfate (1‐2 mg/kg; PND 105‐134) were assessed in adulthood. Adolescent HU‐210 administration suppressed the density of BrdU‐ir cells in the dentate gyrus in adult male, but not adult female rats. Adolescent HU‐210 administration also induced significantly higher peak corticosterone levels and reminiscent of the changes in neurogenesis, this effect was more pronounced in adult males than females. However, adolescent cannabinoid treatment resulted in significantly higher stereotypy scores in adult female, but not male, rats. Thus, adolescent CB1R activation suppressed hippocampal neurogenesis and increased stress responsivity in adult males, but not females, and enhanced amphetamine sensitization in adult female, but not male, rats. Taken together, increased CB1R activation during adolescence results in sex‐dependent, long‐term, changes to hippocampal structure and function, an effect that may shed light on differing vulnerabilities to developing disorders following adolescent cannabinoid exposure, based on sex.

Sustained glucocorticoid exposure recruits cortico-limbic CRH signaling to modulate endocannabinoid function

Sustained exposure to stress or corticosteroids is known to cause changes in brain endocannabinoid (eCB) signaling, such that tissue contents of the eCBs N-arachidonylethanolamine (AEA) are generally reduced while 2-arachidonoylglycerol (2-AG) levels increase. These changes in eCB signaling are important for many of the aspects of chronic stress, such as anxiety, reward sensitivity and stress adaptation, yet the mechanisms mediating these changes are not fully understood. We have recently found that the stress-related neuropeptide corticotropin-releasing hormone (CRH), acting through the CRH type 1 receptor (CRHR1), can reduce AEA content by increasing its hydrolysis by the enzyme fatty acid amide hydrolase (FAAH) as well as increase 2-AG contents. As extra-hypothalamic CRH is upregulated by chronic corticosteroid or stress exposure, we hypothesized that increased CRH signaling through CRHR1 contributes to the effects of chronic corticosteroid exposure on the eCB system within the amygdala and prefrontal cortex. Male rats were exposed to 7 days of systemic corticosterone capsules, with or without concurrent exposure to a CRHR1 antagonist, after which we examined eCB content. Consistent with previous studies in the amygdala, sustained corticosterone exposure increases CRH mRNA in the prefrontal cortex. As was shown previously, FAAH activity was increased and AEA contents were reduced within the amygdala and prefrontal cortex following chronic corticosterone exposure. Chronic corticosterone exposure also elevated 2-AG content in the prefrontal cortex but not the amygdala. These corticosteroid-driven changes were all blocked by systemic CRHR1 antagonism. Consistent with these data indicating sustained increases in CRH signaling can mediate the effects of chronic elevations in corticosteroids, CRH overexpressing mice also exhibited increased FAAH-mediated AEA hydrolysis in the amygdala and prefrontal cortex compared to wild type. CRH overexpression increased 2-AG content in the amygdala, but not the prefrontal cortex. These data indicate that chronic elevations in CRH signaling, as is seen following exposure to chronic elevations in corticosterone or stress, drive persistent changes in eCB function. As reductions in AEA signaling mediate the effects of CRH and chronic stress on anxiety, these data provide a mechanism linking these processes.