Signaling-specific inhibition of the CB1 receptor for cannabis use disorder: phase 1 and phase 2a randomized trials.

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 (CB1-SSi). AEF0117 selectively inhibits a subset of intracellular effects resulting from Δ9-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 .

Mitochondrial cannabinoid receptors gate corticosterone impact on novel object recognition.

Corticosteroid-mediated stress responses require the activation of complex brain circuits involving mitochondrial activity, but the underlying cellular and molecular mechanisms are scantly known. The endocannabinoid system is implicated in stress coping, and it can directly regulate brain mitochondrial functions via type 1 cannabinoid (CB1) receptors associated with mitochondrial membranes (mtCB1). In this study, we show that the impairing effect of corticosterone in the novel object recognition (NOR) task in mice requires mtCB1 receptors and the regulation of mitochondrial calcium levels in neurons. Different brain circuits are modulated by this mechanism to mediate the impact of corticosterone during specific phases of the task. Thus, whereas corticosterone recruits mtCB1 receptors in noradrenergic neurons to impair NOR consolidation, mtCB1 receptors in local hippocampal GABAergic interneurons are required to inhibit NOR retrieval. These data reveal unforeseen mechanisms mediating the effects of corticosteroids during different phases of NOR, involving mitochondrial calcium alterations in different brain circuits.

Astroglial cannabinoid signaling and behavior.

In neuroscience, the explosion of innovative and advanced technical accomplishments is fundamental to understanding brain functioning. For example, the possibility to distinguish glial and neuronal activities at the synaptic level and/or the appearance of new genetic tools to specifically monitor and manipulate astroglial functions revealed that astrocytes are involved in several facets of behavioral control. In this sense, the discovery of functional presence of type-1 cannabinoid receptors in astrocytes has led to identify important behavioral responses mediated by this specific pool of cannabinoid receptors. Thus, astroglial type-1 cannabinoid receptors are in the perfect place to play a role in a complex scenario in which astrocytes sense neuronal activity, release gliotransmitters and modulate the activity of other neurons, ultimately controlling behavioral responses. In this review, we will describe the known behavioral implications of astroglial cannabinoid signaling and highlight exciting unexplored research avenues on how astroglial cannabinoid signaling could affect behavior.

Gene Expression Analysis of the Endocannabinoid System in Presymptomatic APP/PS1 Mice.

Alzheimer's disease (AD) is the most common type of dementia and neurodegeneration. The actual cause of AD progression is still unknown and no curative treatment is available. Recently, findings in human samples and animal models pointed to the endocannabinoid system (ECS) as a promising therapeutic approach against AD. However, the specific mechanisms by which cannabinoid drugs induce potential beneficial effects are still undefined. For this reason, it is required a full characterization of the ECS at different time points of AD progression considering important factors such as sex or the analysis of different brain regions to improve future cannabinoid-dependent therapies in AD. Thus, the main aim of the present study is to expand our knowledge of the status of the ECS in a presymptomatic period (3 months of age) using the AD mouse model APP/PS1 mice. First, we evaluated different behavioral domains including anxiety, cognitive functions, and social interactions in male and female APP/PS1 mice at 4 months of age. Although a mild working memory impairment was observed in male APP/PS1 mice, in most of the behaviors assessed we found no differences between genotypes. At 3 months of age, we performed a characterization of the ECS in different brain regions of the APP/PS1 mice considering the sex variable. We assessed the expression of the ECS components by quantitative Real-Time Polymerase Chain Reaction in the hippocampus, prefrontal cortex, hypothalamus, olfactory bulb, and cerebellum. Interestingly, gene expression levels of the type-1 and type-2 cannabinoid receptors and the anabolic and catabolic enzymes, differed depending on the brain region and the sex analyzed. For example, CB1R expression levels decreased in both hippocampus and prefrontal cortex of male APP/PS1 mice but increased in female mice. In contrast, CB2R expression was decreased in females, whereas males tended to have higher levels. Overall, our data indicated that the ECS is already altered in APP/PS1 mice at the presymptomatic stage, suggesting that it could be an early event contributing to the pathophysiology of AD or being a potential predictive biomarker.

Metabolomics and integrated network analysis reveal roles of endocannabinoids and large neutral amino acid balance in the ayahuasca experience.

There has been a renewed interest in the potential use of psychedelics for the treatment of psychiatric conditions. Nevertheless, little is known about the mechanism of action and molecular pathways influenced by ayahuasca use in humans. Therefore, for the first time, our study aims to investigate the human metabolomics signature after consumption of a psychedelic, ayahuasca, and its connection with both the psychedelic-induced subjective effects and the plasma concentrations of ayahuasca alkaloids. Plasma samples of 23 individuals were collected both before and after ayahuasca consumption. Samples were analysed through targeted metabolomics and further integrated with subjective ratings of the ayahuasca experience (i.e., using the 5-Dimension Altered States of Consciousness Rating Scale [ASC]), and plasma ayahuasca-alkaloids using integrated network analysis. Metabolic pathways enrichment analysis using diffusion algorithms for specific KEGG modules was performed on the metabolic output. Compared to baseline, the consumption of ayahuasca increased N-acyl-ethanolamine endocannabinoids, decreased 2-acyl-glycerol endocannabinoids, and altered several large-neutral amino acids (LNAAs). Integrated network results indicated that most of the LNAAs were inversely associated with 9 out of the 11 subscales of the ASC, except for tryptophan which was positively associated. Several endocannabinoids and hexosylceramides were directly associated with the ayahuasca alkaloids. Enrichment analysis confirmed dysregulation in several pathways involved in neurotransmission such as serotonin and dopamine synthesis. In conclusion, a crosstalk between the circulating LNAAs and the subjective effects is suggested, which is independent of the alkaloid concentrations and provides insights into the specific metabolic fingerprint and mechanism of action underlying ayahuasca experiences.

Special issue editorial: Cannabinoid signalling in the brain: New vistas.

The endocannabinoid system is widely expressed both in the brain and in the periphery. This system regulates a plethora of physiological functions and is composed of cannabinoid receptors, their endogenous ligands, and the enzymes involved in their metabolic processes. In the last few years, the development of new imaging and molecular tools has demonstrated that these receptors are distributed in many cell types (e.g., neuronal or glial cells) and intracellular compartments (e.g., mitochondria). Interestingly, cellular or molecular effects are differentially mediated by cannabinoid receptors according to their specific localization in different cell-types or in different subcellular locations. Moreover, the endocannabinoid system is also expressed throughout the body where it can serve to modulate the connection between the brain and the periphery. Finally, better understanding of the cannabinoid receptors structure and pharmacology has led researchers to propose interesting and new allosteric modulators of synaptic communication. The latest advances and innovative research in the cannabinoid field will provide new insights and better approaches to improve its interesting potential therapeutic profile. This special issue intends to bring together a series of empirical papers, targeted reviews and opinions from leaders in the field that will highlight the new advances in cannabinoid research.

Astroglial ER-mitochondria calcium transfer mediates endocannabinoid-dependent synaptic integration.

Intracellular calcium signaling underlies the astroglial control of synaptic transmission and plasticity. Mitochondria-endoplasmic reticulum contacts (MERCs) are key determinants of calcium dynamics, but their functional impact on astroglial regulation of brain information processing is unexplored. We found that the activation of astrocyte mitochondrial-associated type-1 cannabinoid (mtCB1) receptors determines MERC-dependent intracellular calcium signaling and synaptic integration. The stimulation of mtCB1 receptors promotes calcium transfer from the endoplasmic reticulum to mitochondria through a specific molecular cascade, involving the mitochondrial calcium uniporter (MCU). Physiologically, mtCB1-dependent mitochondrial calcium uptake determines the dynamics of cytosolic calcium events in astrocytes upon endocannabinoid mobilization. Accordingly, electrophysiological recordings in hippocampal slices showed that conditional genetic exclusion of mtCB1 receptors or dominant-negative MCU expression in astrocytes blocks lateral synaptic potentiation, through which astrocytes integrate the activity of distant synapses. Altogether, these data reveal an endocannabinoid link between astroglial MERCs and the regulation of brain network functions.

Neural Substrates of Incidental Associations and Mediated Learning: The Role of Cannabinoid Receptors.

The ability to form associations between different stimuli in the environment to guide adaptive behavior is a central element of learning processes, from perceptual learning in humans to Pavlovian conditioning in animals. Like so, classical conditioning paradigms that test direct associations between low salience sensory stimuli and high salience motivational reinforcers are extremely informative. However, a large part of everyday learning cannot be solely explained by direct conditioning mechanisms - this includes to a great extent associations between individual sensory stimuli, carrying low or null immediate motivational value. This type of associative learning is often described as incidental learning and can be captured in animal models through sensory preconditioning procedures. Here we summarize the evolution of research on incidental and mediated learning, overview the brain systems involved and describe evidence for the role of cannabinoid receptors in such higher-order learning tasks. This evidence favors a number of contemporary hypotheses concerning the participation of the endocannabinoid system in psychosis and psychotic experiences and provides a conceptual framework for understanding how the use of cannabinoid drugs can lead to altered perceptive states.

Subcellular specificity of cannabinoid effects in striatonigral circuits.

Recent advances in neuroscience have positioned brain circuits as key units in controlling behavior, implying that their positive or negative modulation necessarily leads to specific behavioral outcomes. However, emerging evidence suggests that the activation or inhibition of specific brain circuits can actually produce multimodal behavioral outcomes. This study shows that activation of a receptor at different subcellular locations in the same neuronal circuit can determine distinct behaviors. Pharmacological activation of type 1 cannabinoid (CB1) receptors in the striatonigral circuit elicits both antinociception and catalepsy in mice. The decrease in nociception depends on the activation of plasma membrane-residing CB1 receptors (pmCB1), leading to the inhibition of cytosolic PKA activity and substance P release. By contrast, mitochondrial-associated CB1 receptors (mtCB1) located at the same terminals mediate cannabinoid-induced catalepsy through the decrease in intra-mitochondrial PKA-dependent cellular respiration and synaptic transmission. Thus, subcellular-specific CB1 receptor signaling within striatonigral circuits determines multimodal control of behavior.

Protein Kinase C-Gamma Knockout Mice Show Impaired Hippocampal Short-Term Memory While Preserved Long-Term Memory.

The brain encodes, stores, and retrieves relevant information in the form of memories that are classified as short-term (STM) and long-term memories (LTM) depending on the interval between acquisition and retrieval. It is classically accepted that STM undergo a consolidation process to form LTM, but the molecular determinants involved are not well understood. Among the molecular components relevant for memory formation, we focused our attention on the protein kinase C (PKC) family of enzymes since they control key aspects of the synaptic plasticity and memory. Within the different PKC isoforms, PKC-gamma has been specifically associated with learning and memory since mice lacking this isoform (PKC-gamma KO mice) showed mild cognitive impairment and deficits in hippocampal synaptic plasticity. We now reveal that PKC-gamma KO mice present a severe impairment in hippocampal-dependent STM using different memory tests including the novel object-recognition and novel place-recognition, context fear conditioning and trace fear conditioning. In contrast, no differences between genotypes were observed in an amygdala-dependent test, the delay fear conditioning. Strikingly, all LTM tasks that could be assessed 24 h after acquisition were not perturbed in the KO mice. The analysis of c-Fos expression in several brain areas after trace fear conditioning acquisition showed a blunted response in the dentate gyrus of PKC-gamma KO mice compared with WT mice, but such differences between genotypes were absent when the amygdala or the prefrontal cortex were examined. In the hippocampus, PKC-gamma was found to translocate to the membrane after auditory trace, but not after delay fear conditioning. Together, these results indicate that PKC-gamma dysfunction affects specifically hippocampal-dependent STM performance and disclose PKC-gamma as a molecular player differentially involved in STM and LTM processes.

Sex-dependent pharmacological profiles of the synthetic cannabinoid MMB-Fubinaca.

Synthetic cannabinoids have emerged as novel psychoactive substances with damaging consequences for public health. They exhibit high affinity at the cannabinoid type-1 (CB1 ) receptor and produce similar and often more potent effects as other CB1 receptor agonists. However, we are still far from a complete pharmacological understanding of these compounds. In this study, by using behavioral, molecular, pharmacological, and electrophysiological approaches, we aimed at characterizing several in vitro and in vivo pharmacological effects of the synthetic cannabinoid MMB-Fubinaca (also known as AMB-Fubinaca or FUB-AMB), a particular synthetic cannabinoid. MMB-Fubinaca stimulates CB1 receptor-mediated functional coupling to G-proteins in mouse and human brain preparations in a similar manner as the CB1 receptor agonist WIN55,512-2 but with a much greater potency. Both drugs similarly activate the CB1 receptor-dependent extracellular signal-regulated kinase (ERK) pathway. Notably, in vivo administration of MMB-Fubinaca in mice induced greater behavioral and electrophysiological effects in male than in female mice in a CB1 receptor-dependent manner. Overall, these data provide a solid pharmacological profiling of the effects of MMB-Fubinaca and important information about the mechanisms of action underlying its harmful impact in humans. At the same time, they reinforce the significant sexual dimorphism of cannabinoid actions, which will have to be taken into account in future animal and clinical studies.

A Novel Cortical Mechanism for Top-Down Control of Water Intake.

Water intake is crucial for maintaining body fluid homeostasis and animals' survival [1-4]. In the brain, complex processes trigger thirst and drinking behavior [1-5]. The anterior wall of the third ventricle formed by the subfornical organ (SFO), the median preoptic nucleus, and the organum vasculosum of the lamina terminalis (OVLT) constitute the primary structures sensing thirst signals and modulating water intake [6-10]. These subcortical regions are connected with the neocortex [11]. In particular, insular and anterior cingulate cortices (IC and ACC, respectively) have been shown to receive indirect innervations from the SFO and OVLT in rats [11] and to be involved in the control of water intake [12-15]. Type-1 cannabinoid receptors (CB1) modulate consummatory behaviors, such as feeding [16-26]. However, the role of CB1 receptors in the control of water intake is still a matter of debate [27-31]. Here, we show that endogenous activation of CB1 in cortical glutamatergic neurons of the ACC promotes water intake. Notably, presynaptic CB1 receptors of ACC glutamatergic neurons are abundantly located in the basolateral amygdala (BLA), a key area in the regulation of water intake. The selective expression of CB1 receptors in the ACC-to-BLA-projecting neurons is sufficient to stimulate drinking behavior. Moreover, chemogenetic stimulation of these projecting neurons suppresses drinking behavior, further supporting the role of this neuronal population in the control of water intake. Altogether, these data reveal a novel cortico-amygdalar mechanism involved in the regulation of drinking behavior.

Specific Hippocampal Interneurons Shape Consolidation of Recognition Memory.

A complex array of inhibitory interneurons tightly controls hippocampal activity, but how such diversity specifically affects memory processes is not well understood. We find that a small subclass of type 1 cannabinoid receptor (CB1R)-expressing hippocampal interneurons determines episodic-like memory consolidation by linking dopamine D1 receptor (D1R) signaling to GABAergic transmission. Mice lacking CB1Rs in D1-positive cells (D1-CB1-KO) display impairment in long-term, but not short-term, novel object recognition memory (NOR). Re-expression of CB1Rs in hippocampal D1R-positive cells rescues this NOR deficit. Learning induces an enhancement of in vivo hippocampal long-term potentiation (LTP), which is absent in mutant mice. CB1R-mediated NOR and the associated LTP facilitation involve local control of GABAergic inhibition in a D1-dependent manner. This study reveals that hippocampal CB1R-/D1R-expressing interneurons control NOR memory, identifying a mechanism linking the diversity of hippocampal interneurons to specific behavioral outcomes.

Glucose metabolism links astroglial mitochondria to cannabinoid effects.

Astrocytes take up glucose from the bloodstream to provide energy to the brain, thereby allowing neuronal activity and behavioural responses1-5. By contrast, astrocytes are under neuronal control through specific neurotransmitter receptors5-7. However, whether the activation of astroglial receptors can directly regulate cellular glucose metabolism to eventually modulate behavioural responses is unclear. Here we show that activation of mouse astroglial type-1 cannabinoid receptors associated with mitochondrial membranes (mtCB1) hampers the metabolism of glucose and the production of lactate in the brain, resulting in altered neuronal functions and, in turn, impaired behavioural responses in social interaction assays. Specifically, activation of astroglial mtCB1 receptors reduces the phosphorylation of the mitochondrial complex I subunit NDUFS4, which decreases the stability and activity of complex I. This leads to a reduction in the generation of reactive oxygen species by astrocytes and affects the glycolytic production of lactate through the hypoxia-inducible factor 1 pathway, eventually resulting in neuronal redox stress and impairment of behavioural responses in social interaction assays. Genetic and pharmacological correction of each of these effects abolishes the effect of cannabinoid treatment on the observed behaviour. These findings suggest that mtCB1 receptor signalling can directly regulate astroglial glucose metabolism to fine-tune neuronal activity and behaviour in mice.

An Alternative Maze to Assess Novel Object Recognition in Mice.

The novel object recognition (NOR) task is a behavioral test commonly used to evaluate episodic-like declarative memory and it relies on the innate tendency of rodents to explore novelty. Here we present a maze used to evaluate NOR memory in mice that reduces the time of the assay while improving reliability of the measurements by increasing the exploratory behavior. This memory test, being performed in a two-arms maze, is suitable for several strains of mice (including inbreed and outbreed) and does not require extended training sessions allowing an accurate temporal assessment of memory formation. This particular maze increases the mouse exploration time and reduces variability compared to other arenas used before to assess NOR. As both long- and short-term NOR memory can be easily and accurately quantified using this paradigm, this improved methodology can be easily applied to study pharmacological, genetic or age-related modulation of cognitive function.

CB1 Receptors in the Anterior Piriform Cortex Control Odor Preference Memory.

The retrieval of odor-related memories shapes animal behavior. The anterior piriform cortex (aPC) is the largest part of the olfactory cortex, and it plays important roles in olfactory processing and memory. However, it is still unclear whether specific cellular mechanisms in the aPC control olfactory memory, depending on the appetitive or aversive nature of the stimuli involved. Cannabinoid-type 1 (CB1) receptors are present in the aPC (aPC-CB1), but their potential impact on olfactory memory was never explored. Here, we used a combination of behavioral, genetic, anatomical, and electrophysiological approaches to characterize the functions of aPC-CB1 receptors in the regulation of appetitive and aversive olfactory memory. Pharmacological blockade or genetic deletion of aPC-CB1 receptors specifically impaired the retrieval of conditioned odor preference (COP). Interestingly, expression of conditioned odor aversion (COA) was unaffected by local CB1 receptor blockade, indicating that the role of aPC endocannabinoid signaling is selective for retrieval of appetitive memory. Anatomical investigations revealed that CB1 receptors are highly expressed on aPC GABAergic interneurons, and ex vivo electrophysiological recordings showed that their pharmacological activation reduces miniature inhibitory post-synaptic currents (mIPSCs) onto aPC semilunar (SL), but not pyramidal principal neurons. COP retrieval, but not COA, was associated with a specific CB1-receptor-dependent decrease of mIPSCs in SL cells. Altogether, these data indicate that aPC-CB1 receptor-dependent mechanisms physiologically control the retrieval of olfactory memory, depending on odor valence and engaging modulation of local inhibitory transmission.

The motivation for exercise over palatable food is dictated by cannabinoid type-1 receptors.

The lack of intrinsic motivation to engage in, and adhere to, physical exercise has major health consequences. However, the neurobiological bases of exercise motivation are still unknown. This study aimed at examining whether the endocannabinoid system (ECS) is involved in this process. To do so, we developed an operant conditioning paradigm wherein mice unlocked a running wheel with nose pokes. Using pharmacological tools and conditional mutants for cannabinoid type-1 (CB1) receptors, we provide evidence that CB1 receptors located on GABAergic neurons are both necessary and sufficient to positively control running motivation. Conversely, this receptor population proved dispensable for the modulation of running duration per rewarded sequence. Although the ECS mediated the motivation for another reward, namely palatable food, such a regulation was independent from CB1 receptors on GABAergic neurons. In addition, we report that the lack of CB1 receptors on GABAergic neurons decreases the preference for running over palatable food when mice were proposed an exclusive choice between the two rewards. Beyond providing a paradigm that enables motivation processes for exercise to be dissected either singly or in concurrence, this study is the first to our knowledge to identify a neurobiological mechanism that might contribute to sedentary behavior.