Cannabidiol activation of vagal afferent neurons requires TRPA1.

Vagal afferent neurons abundantly express excitatory transient receptor potential (TRP) channels, which strongly influence afferent signaling. Cannabinoids have been identified as direct agonists of TRP channels, including TRPA1 and TRPV1, suggesting that exogenous cannabinoids may influence vagal signaling via TRP channel activation. The diverse therapeutic effects of electrical vagus nerve stimulation also result from administration of the nonpsychotropic cannabinoid, cannabidiol (CBD); however, the direct effects of CBD on vagal afferent signaling remain unknown. We investigated actions of CBD on vagal afferent neurons, using calcium imaging and electrophysiology. CBD produced strong excitatory effects in neurons expressing TRPA1. CBD responses were prevented by removal of bath calcium, ruthenium red, and the TRPA1 antagonist A967079, but not the TRPV1 antagonist SB366791, suggesting an essential role for TRPA1. These pharmacological experiments were confirmed using genetic knockouts where TRPA1 KO mice lacked CBD responses, whereas TRPV1 knockout (KO) mice exhibited CBD-induced activation. We also characterized CBD-provoked inward currents at resting potentials in vagal afferents expressing TRPA1 that were absent in TRPA1 KO mice, but persisted in TRPV1 KO mice. CBD also inhibited voltage-activated sodium conductances in A-fiber, but not in C-fiber afferents. To simulate adaptation, resulting from chronic cannabis use, we administered cannabis extract vapor daily for 3 wk. Cannabis exposure reduced the magnitude of CBD responses, likely due to a loss of TRPA1 signaling. Together, these findings detail a novel excitatory action of CBD at vagal afferent neurons, which requires TRPA1 and may contribute to the vagal mimetic effects of CBD and adaptation following chronic cannabis use.NEW & NOTEWORTHY CBD usage has increased with its legalization. The clinical efficacy of CBD has been demonstrated for conditions including some forms of epilepsy, depression, and anxiety that are also treatable by vagus nerve stimulation. We found CBD exhibited direct excitatory effects on vagal afferent neurons that required TRPA1, were augmented by TRPV1, and attenuated following chronic cannabis vapor exposure. These effects may contribute to vagal mimetic effects of CBD and adaptation after chronic cannabis use.

Long-term effects of maternal cannabis vapor exposure on emotional reactivity, social behavior, and behavioral flexibility in offspring.

The use of cannabis during pregnancy is a growing public health concern. As more countries implement legislation permitting recreational cannabis use, there is an urgent need to better understand its impact on fetal neurodevelopment and its long-term effects in exposed offspring. Studies examining effects of prenatal cannabis exposure typically employ injections of synthetic cannabinoids or isolated cannabis constituents that may not accurately model cannabis use in human populations. To address this limitation, we developed a novel e-cigarette technology-based system to deliver vaporized cannabis extracts to pregnant Long Evans rats. We used this model to determine effects of prenatal cannabis exposure on emotional, social, and cognitive endpoints of male and female offspring during early development and into adulthood. Dams were exposed to cannabis vapor (CANTHC: 400 mg/ml), vehicle vapor (VEH), or no vapor (AIR) twice daily during mating and gestation. Offspring exposed to CANTHC and VEH showed reduced weight gain relative to AIR offspring prior to weaning. CANTHC offspring made more isolation-induced ultrasonic vocalizations (USVs) on postnatal day 6 (P6) relative to VEH-exposed offspring, which is indicative of increased emotional reactivity. Male CANTHC offspring engaged in fewer social investigation behaviors than VEH-exposed male offspring during a social play test on P26. In adulthood, CANTHC-exposed offspring spent less time exploring the open arms of the elevated plus maze and exhibited dose-dependent deficits in behavioral flexibility in an attentional set-shifting task relative to AIR controls. These data collectively indicate that prenatal cannabis exposure may cause enduring effects on the behavioral profile of offspring.

Vaporized Cannabis Extracts Have Reinforcing Properties and Support Conditioned Drug-Seeking Behavior in Rats.

Recent trends in cannabis legalization have increased the necessity to better understand the effects of cannabis use. Animal models involving traditional cannabinoid self-administration approaches have been notoriously difficult to establish and differences in the drug used and its route of administration have limited the translational value of preclinical studies. To address this challenge in the field, we have developed a novel method of cannabis self-administration using response-contingent delivery of vaporized Δ9-tetrahydrocannabinol-rich (CANTHC) or cannabidiol-rich (CANCBD) whole-plant cannabis extracts. Male Sprague-Dawley rats were trained to nose-poke for discrete puffs of CANTHC, CANCBD, or vehicle (VEH) in daily 1 h sessions. Cannabis vapor reinforcement resulted in strong discrimination between active and inactive operanda. CANTHC maintained higher response rates under fixed ratio schedules and higher break points under progressive ratio schedules compared with CANCBD or VEH, and the number of vapor deliveries positively correlated with plasma THC concentrations. Moreover, metabolic phenotyping studies revealed alterations in locomotor activity, energy expenditure, and daily food intake that are consistent with effects in human cannabis users. Furthermore, both cannabis regimens produced ecologically relevant brain concentrations of THC and CBD and CANTHC administration decreased hippocampal CB1 receptor binding. Removal of CANTHC reinforcement (but not CANCBD) resulted in a robust extinction burst and an increase in cue-induced cannabis-seeking behavior relative to VEH. These data indicate that volitional exposure to THC-rich cannabis vapor has bona fide reinforcing properties and collectively support the utility of the vapor self-administration model for the preclinical assessment of volitional cannabis intake and cannabis-seeking behaviors.SIGNIFICANCE STATEMENT The evolving legal landscape concerning recreational cannabis use has increased urgency to better understand its effects on the brain and behavior. Animal models are advantageous in this respect; however, current approaches typically used forced injections of synthetic cannabinoids or isolated cannabis constituents that may not capture the complex effects of volitional cannabis consumption. We have developed a novel model of cannabis self-administration using response-contingent delivery of vaporized cannabis extracts containing high concentrations of Δ9 tetrahydrocannabinol (THC) or cannabidiol. Our data indicate that THC-rich cannabis vapor has reinforcing properties that support stable rates of responding and conditioned drug-seeking behavior. This approach will be valuable for interrogating effects of cannabis and delineating neural mechanisms that give rise to aberrant cannabis-seeking behavior.

The Lateral Habenula Directs Coping Styles Under Conditions of Stress via Recruitment of the Endocannabinoid System.

BACKGROUND: The ability to effectively cope with stress is a critical determinant of disease susceptibility. The lateral habenula (LHb) and the endocannabinoid (ECB) system have independently been shown to be involved in the selection of stress coping strategies, yet the role of ECB signaling in the LHb remains unknown. METHODS: Using a battery of complementary techniques in rats, we examined the localization of type-1 cannabinoid receptors (CB1Rs) and assessed the behavioral and neuroendocrine effects of intra-LHb CB1R manipulations. We further tested the extent to which the ECB system in the LHb is impacted following chronic unpredictable stress or social defeat stress, and whether manipulation of LHb CB1Rs can bias coping strategies in rats with a history of chronic stress. RESULTS: Electron microscopy studies revealed CB1R expression on presynaptic axon terminals, postsynaptic membranes, mitochondria, and glial processes in the rat LHb. In vivo microdialysis experiments indicated that acute stress increased the amount of 2-arachidonoylglycerol in the LHb, while intra-LHb CB1R blockade increased basal corticosterone, augmented proactive coping strategies, and reduced anxiety-like behavior. Basal LHb 2-arachidonoylglycerol content was similarly elevated in rats that were subjected to chronic unpredictable stress or social defeat stress and positively correlated with adrenal weight. Finally, intra-LHb CB1R blockade increased proactive behaviors in response to a novel conspecific, increasing approach behaviors irrespective of stress history and decreasing the latency to be attacked during an agonistic encounter. CONCLUSIONS: Alterations in LHb ECB signaling may be relevant for development of stress-related pathologies in which LHb dysfunction and stress-coping impairments are hallmark symptoms.

Sex- and hormone-dependent alterations in alcohol withdrawal-induced anxiety and corticolimbic endocannabinoid signaling.

Alcohol dependence is associated with anxiety during withdrawal. The endocannabinoid (ECB) system participates in the neuroendocrine and behavioral response to stress and changes in corticolimbic ECB signaling may contribute to alcohol withdrawal-induced anxiety. Moreover, symptoms of alcohol withdrawal differ between sexes and sexual dimorphism in withdrawal-induced ECB recruitment may be a contributing factor. Herein, we exposed intact male and female rats and ovariectomized (OVX) female rats with or without estradiol (E2) replacement to 6 weeks of chronic intermittent alcohol vapor and measured anxiety-like behavior, ECB content, and ECB-related mRNA in the basolateral amygdala (BLA) and ventromedial prefrontal cortex (vmPFC). Acute alcohol withdrawal increased anxiety-like behavior, produced widespread disturbances in ECB-related mRNA, and reduced anandamide (AEA) content in the BLA and 2-arachidonoylglycerol (2-AG) content in the vmPFC of male, but not female rats. Similar to males, alcohol-exposed OVX females showed reductions in Napepld mRNA in the BLA, decreased AEA content in the BLA and vmPFC, and reductions in all ECB-related genes measured in the vmPFC. Importantly, E2 replacement prevented withdrawal-induced alterations in ECB content (but not mRNA) in OVX females, and although alcohol-exposed OVX females failed to exhibit more anxiety compared to their respective control, chronic alcohol exposure abolished the anxiolytic properties of E2 in OVX rats. These data indicate that ovarian sex hormones (but not E2 alone) protect against withdrawal-induced alterations in corticolimbic ECB signaling but do not impart resilience to withdrawal-induced anxiety. Thus, the mechanisms implicated in the manifestation of alcohol withdrawal-induced anxiety are most likely sex-specific. This article is part of the Special Issue entitled "A New Dawn in Cannabinoid Neurobiology".

Impact of Roux-en-Y gastric bypass surgery on appetite, alcohol intake behaviors, and midbrain ghrelin signaling in the rat.

OBJECTIVE: Roux-en-Y gastric bypass (RYGB) surgery reduces appetite and stimulates new onset alcohol misuse; however, the genesis of these behavioral changes is unclear. This study is hypothesized that new onset alcohol intake is a behavioral adaptation that occurs secondary to reduced appetite and correlates with altered central ghrelin signaling. METHODS: Hedonic high-fat diet (HFD) intake was evaluated prior to the assessment of alcohol intake behaviors in RYGB and control rats. Measurements were also taken of circulating ghrelin and ghrelin receptor (GHSR) regulation of neuronal firing in ventral tegmental area (VTA) dopamine (DA) neurons. RESULTS: RYGB rats displayed reduced HFD intake relative to controls. Sham and RYGB rats consumed more alcohol and preferred lower concentrations of alcohol, whereas only RYGB rats escalated alcohol intake during acute withdrawal. Remarkably, GHSR activity, independent of peripheral ghrelin release, set the tonic firing of VTA DA neurons, a response selectively diminished in RYGB rats. CONCLUSIONS: This study indicates that gut manipulations lead to increased alcohol intake, whereas RYGB promotes behaviors that may maintain alcohol misuse. Reductions in hedonic feeding and diminished GHSR control of VTA firing further distinguish gut manipulation from complete bypass and present a potential mechanism linking reduced appetite with alcohol misuse after RYGB surgery.

Alcohol vapor exposure differentially impacts mesocorticolimbic cytokine expression in a sex-, region-, and duration-specific manner.

Alcohol exposure elicits the production of cytokines that regulate the host response to infection, immunity, inflammation, and trauma. Although increased production of pro-inflammatory cytokines has been linked to symptoms of alcoholism, few studies have evaluated whether cytokine expression changes across the development of alcohol dependence, or whether these changes are region and/or sex specific. In the present study, we subjected adult male and female rats to different regimens of alcohol vapor exposure (acute, subchronic, or chronic) and measured relative mRNA expression for tumor necrosis factor alpha (TNFα), interleukin-6 (IL-6), and chemokine (C-C motif) ligand 2 (CCL2) in reward-related brain regions. Results indicated that acute alcohol exposure increased TNFα mRNA expression in the basolateral amygdala (BLA), nucleus accumbens (NAc), and ventral tegmental area (VTA), whereas IL-6 expression was increased in the VTA, NAc, and ventromedial prefrontal cortex (vmPFC) only in males. After subchronic exposure (1week daily intermittent exposure, 14h on:10h off), TNFα expression remained elevated in the BLA, NAc, and VTA, while IL-6 expression was reduced in the male vmPFC. Chronic alcohol exposure (6week daily intermittent exposure, 14 h on: 10 h off) increased TNFα mRNA expression in the NAc and increased IL-6 mRNA in the vmPFC and NAc. Interestingly, chronic alcohol exposure also robustly increased CCL2 mRNA expression in the BLA and VTA in males but not females. Thus, alcohol vapor exposure elicits sex-, region-, and duration-specific cytokine alterations that may contribute to differences in the manifestation and progression of symptoms of alcohol dependence in male and female populations.

Sex differences in alcohol consumption and alterations in nucleus accumbens endocannabinoid mRNA in alcohol-dependent rats.

Chronic intermittent alcohol (CIA) exposure produces altered motivational states characterized by anxiety and escalated alcohol consumption during withdrawal. The endocannabinoid (ECB) system contributes to these symptoms, and sex differences in alcohol dependence, as well as bidirectional interactions between ECBs and gonadal hormones have been documented. Thus, we evaluated sex differences in alcohol consumption, anxiety-like behavior, and ECB mRNA expression in the nucleus accumbens (NAc) of alcohol-dependent rats during acute withdrawal. Male rats exposed to six weeks of CIA showed escalated alcohol consumption during acute withdrawal and reductions in NAc N-acyl phosphatidylethanolamine phospholipase D (NAPEPLD), DAG lipase alpha (DAGLα), and monoacylglycerol lipase (MAGL) mRNA. Intact alcohol-dependent female rats also escalated their consumption, but notably, this effect was also present in non-dependent females. No differences in NAc ECB mRNA were observed between CIA- and air-exposed females during acute withdrawal. However, when these data were analyzed according to estrous stage, significant differences in NAPEPLD and MAGL mRNA expression emerged in the NAc of air-exposed control rats, which were absent in alcohol-dependent females. We subsequently measured alcohol consumption and NAc ECB mRNA in ovariectomized (OVX) females with or without estradiol (E2) replacement during withdrawal. Neither E2 nor CIA altered alcohol consumption in OVX females. However, E2 reduced both DAGLα and MAGL mRNA, suggesting that E2 may influence the biosynthesis and degradation of 2-arachidonoylglycerol (2-AG) in the NAc. Collectively, these studies indicate sexual dimorphism in alcohol consumption in non-dependent rats and suggest that E2-mediated alterations in NAc ECB mRNA expression during withdrawal may be a mechanism by which sex differences in alcohol dependence emerge.