Abstract:
BACKGROUND: Hyperlocomotion induced by psychotomimetic agents is considered a proxy for positive psychotic-like symptoms and is used in antipsychotic drug screenings with rodents. Cannabinoids are psychotomimetic drugs in humans, and low doses of cannabinoid CB(1) receptor (CB1R) agonists induce hyperlocomotion in rodents. However, the cellular and molecular mechanisms underlying this effect remain unexplored so far. METHODS: We used a combination of pharmacological and genetic manipulations in cultured cells and adult male mouse models, together with an intracellular signaling detection method. Our multidisciplinary approach included locomotor activity (n = 10-12 mice/group), alphaLISA technology (n = 4-12 mice/group), transfection of the beta-arrestin 1 protein in STHdh (Q7/Q7) cells (n = 4-6/group) and a viral strategy in transgenic lines to selectively block GSK-3beta (glycogen synthase kinase-3beta) phosphorylation (n = 8-10 mice/group). We also used fluorescence in situ hybridization, real-time quantitative and droplet digital polymerase chain reaction, and tandem mass spectrometry. RESULTS: We showed that the hyperlocomotion induced by a low dose (0.3 mg/kg) of cannabinoid Delta(9)-tetrahydrocannabinol depends on the activation of plasma membrane CB1R located on dopamine D(2) receptor-positive striatopallidal projecting neurons. This activation regulates the beta-arrestin 1/Akt/GSK-3beta signaling pathway, which is required for the hyperlocomotor effect. The signaling-specific CB1R inhibitors, pregnenolone and its clinically developed analog, AEF0117, prevent GSK-3beta-dependent psychomotor stimulation induced by THC. CONCLUSIONS: This work highlights an intracellular mechanism of CB1R expressed by a selective key neuronal pathway mediating the psychotomimetic effect of THC and, by extension, low-dose cannabis consumption. This discovery will likely aid the development of unprecedented therapeutic modalities for the treatment of psychotic conditions.

Abstract:
Cannabis use disorder (CUD) is widespread, and there is no pharmacotherapy to facilitate its treatment. AEF0117, the first of a new pharmacological class, is a signaling-specific inhibitor of the cannabinoid receptor 1 (CB(1)-SSi). AEF0117 selectively inhibits a subset of intracellular effects resulting from Delta(9)-tetrahydrocannabinol (THC) binding without modifying behavior per se. In mice and non-human primates, AEF0117 decreased cannabinoid self-administration and THC-related behavioral impairment without producing significant adverse effects. In single-ascending-dose (0.2 mg, 0.6 mg, 2 mg and 6 mg; n = 40) and multiple-ascending-dose (0.6 mg, 2 mg and 6 mg; n = 24) phase 1 trials, healthy volunteers were randomized to ascending-dose cohorts (n = 8 per cohort; 6:2 AEF0117 to placebo randomization). In both studies, AEF0117 was safe and well tolerated (primary outcome measurements). In a double-blind, placebo-controlled, crossover phase 2a trial, volunteers with CUD were randomized to two ascending-dose cohorts (0.06 mg, n = 14; 1 mg, n = 15). AEF0117 significantly reduced cannabis' positive subjective effects (primary outcome measurement, assessed by visual analog scales) by 19% (0.06 mg) and 38% (1 mg) compared to placebo (P < 0.04). AEF0117 (1 mg) also reduced cannabis self-administration (P < 0.05). In volunteers with CUD, AEF0117 was well tolerated and did not precipitate cannabis withdrawal. These data suggest that AEF0117 is a safe and potentially efficacious treatment for CUD.ClinicalTrials.gov identifiers: NCT03325595 , NCT03443895 and NCT03717272 .

Abstract:
Bidirectional communication between neurons and astrocytes shapes synaptic plasticity and behavior. D-serine is a necessary co-agonist of synaptic N-methyl-D-aspartate receptors (NMDARs), but the physiological factors regulating its impact on memory processes are scantly known. We show that astroglial CB1 receptors are key determinants of object recognition memory by determining the availability of D-serine at hippocampal synapses. Mutant mice lacking CB1 receptors from astroglial cells (GFAP-CB1-KO) displayed impaired object recognition memory and decreased in vivo and in vitro long-term potentiation (LTP) at CA3-CA1 hippocampal synapses. Activation of CB1 receptors increased intracellular astroglial Ca(2+) levels and extracellular levels of D-serine in hippocampal slices. Accordingly, GFAP-CB1-KO displayed lower occupancy of the co-agonist binding site of synaptic hippocampal NMDARs. Finally, elevation of D-serine levels fully rescued LTP and memory impairments of GFAP-CB1-KO mice. These data reveal a novel mechanism of in vivo astroglial control of memory and synaptic plasticity via the D-serine-dependent control of NMDARs.

Abstract:
Cannabinoid-induced tetrad is a preclinical model commonly used to evaluate if a pharmacological compound is an agonist of the central type-1 cannabinoid (CB1) receptor in rodents. The tetrad is characterized by hypolocomotion, hypothermia, catalepsy, and analgesia, four phenotypes that are induced by acute administration of CB1 agonists exemplified by the prototypic cannabinoid delta-9-tetrahydrocannabinol (THC). This unit describes a standard protocol in mice to induce tetrad phenotypes with THC as reference cannabinoid. We provide typical results obtained with this procedure showing a dose effect of THC in different mouse strains. The effect of the CB1 antagonist rimonabant is also shown. This tetrad protocol is well adapted to reveal new compounds acting on CB1 receptors in vivo. (c) 2017 by John Wiley & Sons, Inc.

Abstract:
The endocannabinoid system is the target of the main psychoactive component of the plant Cannabis sativa, the Delta9 -tetrahydrocannabinol (THC). This system is composed by the cannabinoid receptors, the endogenous ligands, and the enzymes involved in their metabolic processes, which works both centrally and peripherally to regulate a plethora of physiological functions. This review aims at explaining how the site-specific actions of the endocannabinoid system impact on memory and feeding behavior through the cannabinoid receptors 1 (CB1 R). Centrally, CB1 R is widely distributed in many brain regions, different cell types (e.g. neuronal or glial cells) and intracellular compartments (e.g. mitochondria). Interestingly, cellular and molecular effects are differentially mediated by CB1 R according to their cell-type localization (e.g. glutamatergic or GABAergic neurons). Thus, understanding the cellular and subcellular function of CB1 R will provide new insights and aid the design of new compounds in cannabinoid-based medicine. Also watch the Video Abstract.

Abstract:
The endocannabinoid system is an important regulator of physiological functions. In the brain, this control is mainly exerted through the type-1-cannabinoid (CB1) receptors. CB1 receptors are abundant at neuron terminals where their stimulation inhibits neurotransmitter release. However, CB1 receptors are also expressed in astrocytes and recent studies showed that astroglial cannabinoid signalling is a key element of the tripartite synapse. In this review we discuss the different mechanisms by which astroglial CB1 receptors control synaptic transmission and plasticity. The recent involvement of astroglial CB1 receptors in the effects of cannabinoids on memory highlights their key roles in cognitive processes and further indicates that astrocytes are central active elements of high order brain functions.

Abstract:
The type-1-cannabinoid (CB1 ) receptor is amongst the most widely expressed G protein-coupled receptors in the brain. In few decades, CB1 receptors have been shown to regulate a large array of functions from brain cell development and survival to complex cognitive processes. Understanding the cellular mechanisms underlying these functions of CB1 is complex due to the heterogeneity of the brain cell types on which the receptor is expressed. Although the large majority of CB1 receptors act on neurons, early studies pointed to a direct control of CB1 receptors over astroglial functions including brain energy supply and neuroprotection. In line with the growing concept of the tripartite synapse highlighting astrocytes as direct players in synaptic plasticity, astroglial CB1 receptor signaling recently emerged as the mediator of several forms of synaptic plasticity associated to important cognitive functions. Here, we shortly review the current knowledge on CB1 receptor-mediated astroglial functions. This functional spectrum is large and most of the mechanisms by which CB1 receptors control astrocytes, as well as their consequences in vivo, are still unknown, requiring innovative approaches to improve this new cannabinoid research field.

Abstract:
Complex interactions between periphery and the brain regulate food intake in mammals. Cannabinoid type-1 (CB1) receptor antagonists are potent hypophagic agents, but the sites where this acute action is exerted and the underlying mechanisms are not fully elucidated. To dissect the mechanisms underlying the hypophagic effect of CB1 receptor blockade, we combined the acute injection of the CB1 receptor antagonist rimonabant with the use of conditional CB1-knockout mice, as well as with pharmacological modulation of different central and peripheral circuits. Fasting/refeeding experiments revealed that CB1 receptor signaling in many specific brain neurons is dispensable for the acute hypophagic effects of rimonabant. CB1 receptor antagonist-induced hypophagia was fully abolished by peripheral blockade of beta-adrenergic transmission, suggesting that this effect is mediated by increased activity of the sympathetic nervous system. Consistently, we found that rimonabant increases gastrointestinal metabolism via increased peripheral beta-adrenergic receptor signaling in peripheral organs, including the gastrointestinal tract. Blockade of both visceral afferents and glutamatergic transmission in the nucleus tractus solitarii abolished rimonabant-induced hypophagia. Importantly, these mechanisms were specifically triggered by lipid-deprivation, revealing a nutrient-specific component acutely regulated by CB1 receptor blockade. Finally, peripheral blockade of sympathetic neurotransmission also blunted central effects of CB1 receptor blockade, such as fear responses and anxiety-like behaviors. These data demonstrate that, independently of their site of origin, important effects of CB1 receptor blockade are expressed via activation of peripheral sympathetic activity. Thus, CB1 receptors modulate bidirectional circuits between the periphery and the brain to regulate feeding and other behaviors.

Abstract:
Type-1 cannabinoid (CB1) and leptin (ObR) receptors regulate metabolic and astroglial functions, but the potential links between the two systems in astrocytes were not investigated so far. Genetic and pharmacological manipulations of CB1 receptor expression and activity in cultured cortical and hypothalamic astrocytes demonstrated that cannabinoid signaling controls the levels of ObR expression. Lack of CB1 receptors also markedly impaired leptin-mediated activation of signal transducers and activators of transcription 3 and 5 (STAT3 and STAT5) in astrocytes. In particular, CB1 deletion determined a basal overactivation of STAT5, thereby leading to the downregulation of ObR expression, and leptin failed to regulate STAT5-dependent glycogen storage in the absence of CB1 receptors. These results show that CB1 receptors directly interfere with leptin signaling and its ability to regulate glycogen storage, thereby representing a novel mechanism linking endocannabinoid and leptin signaling in the regulation of brain energy storage and neuronal functions.

Abstract:
To maximize their chances of survival, animals need to rapidly and efficiently respond to aversive situations. These responses can be classified as active or passive and depend on the specific nature of threats, but also on individual fear coping styles. In this study, we show that the control of excitatory and inhibitory brain neurons by type-1 cannabinoid (CB(1)) receptors is a key determinant of fear coping strategies in mice. In classical fear conditioning, a switch between initially predominant passive fear responses (freezing) and active behaviors (escape attempts and risk assessment) develops over time. Constitutive genetic deletion of CB(1) receptors in CB(1)(-)/(-) mice disrupted this pattern by favoring passive responses. This phenotype can be ascribed to endocannabinoid control of excitatory neurons, because it was reproduced in conditional mutant mice lacking CB(1) receptors from cortical glutamatergic neurons. CB(1) receptor deletion from GABAergic brain neurons led to the opposite phenotype, characterized by the predominance of active coping. The CB(1) receptor agonist Delta(9)-tetrahydrocannabinol exerted a biphasic control of fear coping strategies, with lower and higher doses favoring active and passive responses, respectively. Finally, viral re-expression of CB(1) receptors in the amygdala of CB(1)(-)/(-) mice restored the normal switch between the two coping strategies. These data strongly suggest that CB(1) receptor signaling bimodally controls the spontaneous adoption of active or passive coping strategies in individuals. This primary function of the endocannabinoid system in shaping individual behavioral traits should be considered when studying the mechanisms of physiological and pathological fear.