Nolan D. Hartley

Substrate-selective COX-2 inhibition decreases anxiety via endocannabinoid activation

Augmentation of endogenous cannabinoid (eCB) signaling represents an emerging approach to the treatment of affective disorders. Cyclooxygenase-2 (COX-2) oxygenates arachidonic acid to form prostaglandins, but also inactivates eCBs in vitro. However, the viability of COX-2 as a therapeutic target for in vivo eCB augmentation has not been explored. Using medicinal chemistry and in vivo analytical and behavioral pharmacological approaches, we found that COX-2 is important for the regulation of eCB levels in vivo. We used a pharmacological strategy involving substrate-selective inhibition of COX-2 to augment eCB signaling without affecting related non-eCB lipids or prostaglandin synthesis. Behaviorally, substrate-selective inhibition of COX-2 reduced anxiety-like behaviors in mice via increased eCB signaling. Our data suggest a key role for COX-2 in the regulation of eCB signaling and indicate that substrate-selective pharmacology represents a viable approach for eCB augmentation with broad therapeutic potential.

Central anandamide deficiency predicts stress-induced anxiety: behavioral reversal through endocannabinoid augmentation

Stress is a major risk factor for the development of mood and anxiety disorders; elucidation of novel approaches to mitigate the deleterious effects of stress could have broad clinical applications. Pharmacological augmentation of central endogenous cannabinoid (eCB) signaling may be an effective therapeutic strategy to mitigate the adverse behavioral and physiological consequences of stress. Here we show that acute foot-shock stress induces a transient anxiety state measured 24 h later using the light–dark box assay and novelty-induced hypophagia test. Acute pharmacological inhibition of the anandamide-degrading enzyme, fatty acid amide hydrolase (FAAH), reverses the stress-induced anxiety state in a cannabinoid receptor-dependent manner. FAAH inhibition does not significantly affect anxiety-like behaviors in non-stressed mice. Moreover, whole brain anandamide levels are reduced 24 h after acute foot-shock stress and are negatively correlated with anxiety-like behavioral measures in the light–dark box test. These data indicate that central anandamide levels predict acute stress-induced anxiety, and that reversal of stress-induced anandamide deficiency is a key mechanism subserving the therapeutic effects of FAAH inhibition. These studies provide further support that eCB-augmentation is a viable pharmacological strategy for the treatment of stress-related neuropsychiatric disorders.

Prefrontal inputs to the amygdala instruct fear extinction memory formation

Traumatic fear memories are extinguished by a discrete brain circuit comprising inputs from the prefrontal cortex to the amygdala. Persistent anxiety after a psychological trauma is a hallmark of many anxiety disorders. However, the neural circuits mediating the extinction of traumatic fear memories remain incompletely understood. We show that selective, in vivo stimulation of the ventromedial prefrontal cortex (vmPFC)–amygdala pathway facilitated extinction memory formation, but not retrieval. Conversely, silencing the vmPFC-amygdala pathway impaired extinction formation and reduced extinction-induced amygdala activity. Our data demonstrate a critical instructional role for the vmPFC-amygdala circuit in the formation of extinction memories. These findings advance our understanding of the neural basis of persistent fear, with implications for posttraumatic stress disorder and other anxiety disorders.

Multiple Mechanistically Distinct Modes of Endocannabinoid Mobilization at Central Amygdala Glutamatergic Synapses

The central amygdala (CeA) is a key structure at the limbic-motor interface regulating stress responses and emotional learning. Endocannabinoid (eCB) signaling is heavily implicated in the regulation of stress-response physiology and emotional learning processes; however, the role of eCBs in the modulation of synaptic efficacy in the CeA is not well understood. Here we describe the subcellular localization of CB1 cannabinoid receptors and eCB synthetic machinery at glutamatergic synapses in the CeA and find that CeA neurons exhibit multiple mechanistically and temporally distinct modes of postsynaptic eCB mobilization. These data identify a prominent role for eCBs in the modulation of excitatory drive to CeA neurons and provide insight into the mechanisms by which eCB signaling and exogenous cannabinoids could regulate stress responses and emotional learning.

Dissociable effects of CB1 receptor blockade on anxiety-like and consummatory behaviors in the novelty-induced hypophagia test in mice

RationaleCentral CB1 cannabinoid receptors regulate anxiety-like and appetitive consummatory behaviors. Pharmacological antagonism/inverse-agonism of CB1 receptors increases anxiety and decreases appetitive behaviors; however, neither well-defined dose nor context dependence of these effects has been simultaneously assessed in one behavioral assay.ObjectivesWe sought to determine the context and dose dependence of the effects of CB1 receptor blockade on anxiety-like and consummatory behaviors in a model that allowed for simultaneous detection of anxiety-like and consummatory-related behaviors.MethodsWe determined the effects of the CB1 receptor antagonist/inverse-agonist, rimonabant, in the novelty-induced hypophagia (NIH) assay in juvenile male ICR mice.ResultsRimonabant dose-dependently decreased consumption of a palatable reward solution completely independent of contextual novelty. Grooming and scratching behavior was also increased by rimonabant in a context-independent manner. In contrast, rimonabant increased feeding latency, a measure of anxiety-like behaviors, only in a novel, mildly anxiogenic context. The effects of rimonabant were specific since no effects of rimonabant on despair-like behavior were observed in the tail suspension assay. Blockade of CB2 receptors had no effect on novelty-induced increases in feeding latency or palatable food consumption.ConclusionsOur findings indicate that CB1 receptor blockade decreases the hedonic value of palatable food irrespective of environmental novelty, whereas the anxiogenic-like effects are highly context-dependent. Blockade of CB2 receptors does not regulate either anxiety-like or consummatory behaviors in the NIH assay. These findings suggest that rimonabant modulates distinct and dissociable neural processes regulating anxiety and consummatory behavior to sculpt complex and context-dependent behavioral repertories.

Endocannabinoid signalling modulates susceptibility to traumatic stress exposure

Stress is a ubiquitous risk factor for the exacerbation and development of affective disorders including major depression and posttraumatic stress disorder. Understanding the neurobiological mechanisms conferring resilience to the adverse consequences of stress could have broad implications for the treatment and prevention of mood and anxiety disorders. We utilize laboratory mice and their innate inter-individual differences in stress-susceptibility to demonstrate a critical role for the endogenous cannabinoid 2-arachidonoylglycerol (2-AG) in stress-resilience. Specifically, systemic 2-AG augmentation is associated with a stress-resilient phenotype and enhances resilience in previously susceptible mice, while systemic 2-AG depletion or CB1 receptor blockade increases susceptibility in previously resilient mice. Moreover, stress-resilience is associated with increased phasic 2-AG-mediated synaptic suppression at ventral hippocampal-amygdala glutamatergic synapses and amygdala-specific 2-AG depletion impairs successful adaptation to repeated stress. These data indicate amygdala 2-AG signalling mechanisms promote resilience to adverse effects of acute traumatic stress and facilitate adaptation to repeated stress exposure.

Functional Redundancy Between Canonical Endocannabinoid Signaling Systems in the Modulation of Anxiety

BACKGROUND Increasing the available repertoire of effective treatments for mood and anxiety disorders represents a critical unmet need. Pharmacological augmentation of endogenous cannabinoid (eCB) signaling has been suggested to represent a novel approach to the treatment of anxiety disorders; however, the functional interactions between two canonical eCB pathways mediated via anandamide (N-arachidonylethanolamine [AEA]) and 2-arachidonoylglycerol (2-AG) in the regulation of anxiety are not well understood. METHODS We utilized pharmacological augmentation and depletion combined with behavioral and electrophysiological approaches to probe the role of 2-AG signaling in the modulation of stress-induced anxiety and the functional redundancy between AEA and 2-AG signaling in the modulation of anxiety-like behaviors in mice. RESULTS Selective 2-AG augmentation reduced anxiety in the light/dark box assay and prevented stress-induced increases in anxiety associated with limbic AEA deficiency. In contrast, acute 2-AG depletion increased anxiety-like behaviors, which was normalized by selective pharmacological augmentation of AEA signaling and via direct cannabinoid receptor 1 stimulation with Δ9-tetrahydrocannabinol. Electrophysiological studies revealed 2-AG modulation of amygdala glutamatergic transmission as a key synaptic correlate of the anxiolytic effects of 2-AG augmentation. CONCLUSIONS Although AEA and 2-AG likely subserve distinct physiological roles, a pharmacological and functional redundancy between these canonical eCB signaling pathways exists in the modulation of anxiety-like behaviors. These data support development of eCB-based treatment approaches for mood and anxiety disorders and suggest a potentially wider therapeutic overlap between AEA and 2-AG augmentation approaches than was previously appreciated.

2-arachidonoylglycerol signaling impairs short-term fear extinction.

Impairments in fear extinction are thought to be central to the psychopathology of posttraumatic stress disorder, and endocannabinoid (eCB) signaling has been strongly implicated in extinction learning. Here we utilized the monoacylglycerol lipase inhibitor JZL184 to selectively augment brain 2-AG levels combined with an auditory cue fear-conditioning paradigm to test the hypothesis that 2-AG-mediated eCB signaling modulates short-term fear extinction learning in mice. We show that systemic JZL184 impairs short-term extinction learning in a CB1 receptor-dependent manner without affecting non-specific freezing behavior or the acquisition of conditioned fear. This effect was also observed in over-conditioned mice environmentally manipulated to re-acquire fear extinction. Cumulatively, the effects of JZL184 appear to be partly due to augmentation of 2-AG signaling in the basolateral nucleus of the amygdala (BLA), as direct microinfusion of JZL184 into the BLA produced similar results. Moreover, we elucidate a short ~3-day temporal window during which 2-AG augmentation impairs extinction behavior, suggesting a preferential role for 2-AG-mediated eCB signaling in the modulation of short-term behavioral sequelae to acute traumatic stress exposure.

Cyclooxygenase-2 inhibition reduces stress-induced affective pathology

Mood and anxiety disorders are the most prevalent psychiatric conditions and are exacerbated by stress. Recent studies have suggested cyclooxygenase-2 (COX-2) inhibition could represent a novel treatment approach or augmentation strategy for affective disorders including anxiety disorders and major depression. We show that traditional COX-2 inhibitors and a newly developed substrate-selective COX-2 inhibitor (SSCI) reduce a variety of stress-induced behavioral pathologies in mice. We found that these behavioral effects were associated with a dampening of neuronal excitability in the basolateral amygdala (BLA) ex vivo and in vivo, and were mediated by small-conductance calcium-activated potassium (SK) channel and CB1 cannabinoid receptor activation. Taken together, these data provide further support for the potential utility of SSCIs, as well as traditional COX-2 inhibitors, as novel treatment approaches for stress-related psychiatric disorders. DOI: http://dx.doi.org/10.7554/eLife.14137.001

Sigma‐1 receptor ligands inhibit catecholamine secretion from adrenal chromaffin cells due to block of nicotinic acetylcholine receptors

Adrenal chromaffin cells (ACCs) are the neuroendocrine arm of the sympathetic nervous system and key mediators of the physiological stress response. Acetylcholine (ACh) released from preganglionic splanchnic nerves activates nicotinic acetylcholine receptors (nAChRs) on chromaffin cells causing membrane depolarization, opening voltage‐gated Ca2+ channels (VGCC), and exocytosis of catecholamines and neuropeptides. The serotonin transporter is expressed in ACCs and interacts with 5‐HT1A receptors to control secretion. In addition to blocking the serotonin transporter, some selective serotonin reuptake inhibitors (SSRIs) are also agonists at sigma‐1 receptors which function as intracellular chaperone proteins and can translocate to the plasma membrane to modulate ion channels. Therefore, we investigated whether SSRIs and other sigma‐1 receptor ligands can modulate stimulus‐secretion coupling in ACCs. Escitalopram and fluvoxamine (100 nM to 1 μM) reversibly inhibited nAChR currents. The sigma‐1 receptor antagonists NE‐100 and BD‐1047 also blocked nAChR currents (≈ 50% block at 100 nM) as did PRE‐084, a sigma‐1 receptor agonist. Block of nAChR currents by fluvoxamine and NE‐100 was not additive suggesting a common site of action. VGCC currents were unaffected by the drugs. Neither the increase in cytosolic [Ca2+] nor the resulting catecholamine secretion evoked by direct membrane depolarization to bypass nAChRs was altered by fluvoxamine or NE‐100. However, both Ca2+ entry and catecholamine secretion evoked by the cholinergic agonist carbachol were significantly reduced by fluvoxamine or NE‐100. Together, our data suggest that sigma‐1 receptors do not acutely regulate catecholamine secretion. Rather, SSRIs and other sigma‐1 receptor ligands inhibit secretion evoked by cholinergic stimulation because of direct block of Ca2+entry via nAChRs.