Cannabinoids regulate an insula circuit controlling water intake.

The insular cortex, or insula, is a large brain region involved in the detection of thirst and the regulation of water intake. However, our understanding of the topographical, circuit, and molecular mechanisms for controlling water intake within the insula remains parcellated. We found that type-1 cannabinoid (CB1) receptors in the insular cortex cells participate in the regulation of water intake and deconstructed the circuit mechanisms of this control. Topographically, we revealed that the activity of excitatory neurons in both the anterior insula (aIC) and posterior insula (pIC) increases in response to water intake, yet only the specific removal of CB1 receptors in the pIC decreases water intake. Interestingly, we found that CB1 receptors are highly expressed in insula projections to the basolateral amygdala (BLA), while undetectable in the neighboring central part of the amygdala. Thus, we recorded the neurons of the aIC or pIC targeting the BLA (aIC-BLA and pIC-BLA) and found that they decreased their activity upon water drinking. Additionally, chemogenetic activation of pIC-BLA projection neurons decreased water intake. Finally, we uncovered CB1-dependent short-term synaptic plasticity (depolarization-induced suppression of excitation [DSE]) selectively in pIC-BLA, compared with aIC-BLA synapses. Altogether, our results support a model where CB1 receptor signaling promotes water intake by inhibiting the pIC-BLA pathway, thereby contributing to the fine top-down control of thirst responses.

Single cell tracing of Pomc neurons reveals recruitment of 'Ghost' subtypes with atypical identity in a mouse model of obesity.

The hypothalamus contains a remarkable diversity of neurons that orchestrate behavioural and metabolic outputs in a highly plastic manner. Neuronal diversity is key to enabling hypothalamic functions and, according to the neuroscience dogma, it is predetermined during embryonic life. Here, by combining lineage tracing of hypothalamic pro-opiomelanocortin (Pomc) neurons with single-cell profiling approaches in adult male mice, we uncovered subpopulations of 'Ghost' neurons endowed with atypical molecular and functional identity. Compared to 'classical' Pomc neurons, Ghost neurons exhibit negligible Pomc expression and are 'invisible' to available neuroanatomical approaches and promoter-based reporter mice for studying Pomc biology. Ghost neuron numbers augment in diet-induced obese mice, independent of neurogenesis or cell death, but weight loss can reverse this shift. Our work challenges the notion of fixed, developmentally programmed neuronal identities in the mature hypothalamus and highlight the ability of specialised neurons to reversibly adapt their functional identity to adult-onset obesogenic stimuli.

The CB1 receptor interacts with cereblon and drives cereblon deficiency-associated memory shortfalls.

Cereblon/CRBN is a substrate-recognition component of the Cullin4A-DDB1-Roc1 E3 ubiquitin ligase complex. Destabilizing mutations in the human CRBN gene cause a form of autosomal recessive non-syndromic intellectual disability (ARNSID) that is modelled by knocking-out the mouse Crbn gene. A reduction in excitatory neurotransmission has been proposed as an underlying mechanism of the disease. However, the precise factors eliciting this impairment remain mostly unknown. Here we report that CRBN molecules selectively located on glutamatergic neurons are necessary for proper memory function. Combining various in vivo approaches, we show that the cannabinoid CB1 receptor (CB1R), a key suppressor of synaptic transmission, is overactivated in CRBN deficiency-linked ARNSID mouse models, and that the memory deficits observed in these animals can be rescued by acute CB1R-selective pharmacological antagonism. Molecular studies demonstrated that CRBN interacts physically with CB1R and impairs the CB1R-Gi/o-cAMP-PKA pathway in a ubiquitin ligase-independent manner. Taken together, these findings unveil that CB1R overactivation is a driving mechanism of CRBN deficiency-linked ARNSID and anticipate that the antagonism of CB1R could constitute a new therapy for this orphan disease.

Striatopallidal cannabinoid type-1 receptors mediate amphetamine-induced sensitization.

Repeated exposure to psychostimulants, such as amphetamine, causes a long-lasting enhancement in the behavioral responses to the drug, called behavioral sensitization.1 This phenomenon involves several neuronal systems and brain areas, among which the dorsal striatum plays a key role.2 The endocannabinoid system (ECS) has been proposed to participate in this effect, but the neuronal basis of this interaction has not been investigated.3 In the CNS, the ECS exerts its functions mainly acting through the cannabinoid type-1 (CB1) receptor, which is highly expressed at terminals of striatal medium spiny neurons (MSNs) belonging to both the direct and indirect pathways.4 In this study, we show that, although striatal CB1 receptors are not involved in the acute response to amphetamine, the behavioral sensitization and related synaptic changes require the activation of CB1 receptors specifically located at striatopallidal MSNs (indirect pathway). These results highlight a new mechanism of psychostimulant sensitization, a phenomenon that plays a key role in the health-threatening effects of these drugs.

Mitochondria-targeted melatonin photorelease supports the presence of melatonin MT1 receptors in mitochondria inhibiting respiration.

The presence of signaling-competent G protein-coupled receptors in intracellular compartments is increasingly recognized. Recently, the presence of Gi/o protein-coupled melatonin MT1 receptors in mitochondria has been revealed, in addition to the plasma membrane. Melatonin is highly cell permeant, activating plasma membrane and mitochondrial receptors equally. Here, we present MCS-1145, a melatonin derivative bearing a triphenylphosphonium cation for specific mitochondrial targeting and a photocleavable o-nitrobenzyl group releasing melatonin upon illumination. MCS-1145 displayed low affinity for MT1 and MT2 but spontaneously accumulated in mitochondria, where it was resistant to washout. Uncaged MCS-1145 and exogenous melatonin recruited ß-arrestin 2 to MT1 in mitochondria and inhibited oxygen consumption in mitochondria isolated from HEK293 cells only when expressing MT1 and from mouse cerebellum of WT mice but not from MT1-knockout mice. Overall, we developed the first mitochondria-targeted photoactivatable melatonin ligand and demonstrate that melatonin inhibits mitochondrial respiration through mitochondrial MT1 receptors.

Early Administration of the Phytocannabinoid Cannabidivarin Prevents the Neurobehavioral Abnormalities Associated with the Fmr1-KO Mouse Model of Fragile X Syndrome.

Phytocannabinoids, including the non-addictive cannabis component cannabidivarin (CBDV), have been reported to hold therapeutic potential in several neurodevelopmental disorders (NDDs). Nonetheless, the therapeutic value of phytocannabinoids for treating Fragile X syndrome (FXS), a major NDD, remains unexplored. Here, we characterized the neurobehavioral effects of CBDV at doses of 20 or 100 mg/kg in the Fmr1-knockout (Fmr1-KO) mouse model of FXS using two temporally different intraperitoneal regimens: subchronic 10-day delivery during adulthood (Study 1: rescue treatment) or chronic 5-week delivery at adolescence (Study 2: preventive treatment). Behavioral tests assessing FXS-like abnormalities included anxiety, locomotor, cognitive, social and sensory alterations. Expression of inflammatory and plasticity markers was investigated in the hippocampus and prefrontal cortex. When administered during adulthood (Study 1), the effects of CBDV were marginal, rescuing at the lower dose only the acoustic hyper-responsiveness of Fmr1-KO mice and at both doses their altered hippocampal expression of neurotrophins. When administered during adolescence (Study 2), CBDV at both doses prevented the cognitive, social and acoustic alterations of adult Fmr1-KO mice and modified the expression of several inflammatory brain markers in both wild-type littermates and mutants. These findings warrant the therapeutic potential of CBDV for preventing neurobehavioral alterations associated with FXS, highlighting the relevance of its early administration.

Type-1 cannabinoid receptors and their ever-expanding roles in brain energy processes.

The brain requires large quantities of energy to sustain its functions. At the same time, the brain is isolated from the rest of the body, forcing this organ to develop strategies to control and fulfill its own energy needs. Likely based on these constraints, several brain-specific mechanisms emerged during evolution. For example, metabolically specialized cells are present in the brain, where intercellular metabolic cycles are organized to separate workload and optimize the use of energy. To orchestrate these strategies across time and space, several signaling pathways control the metabolism of brain cells. One of such controlling systems is the endocannabinoid system, whose main signaling hub in the brain is the type-1 cannabinoid (CB1 ) receptor. CB1 receptors govern a plethora of different processes in the brain, including cognitive function, emotional responses, or feeding behaviors. Classically, the mechanisms of action of CB1 receptors on brain function had been explained by its direct targeting of neuronal synaptic function. However, new discoveries have challenged this view. In this review, we will present and discuss recent data about how a small fraction of CB1 receptors associated to mitochondrial membranes (mtCB1 ), are able to exert a powerful control on brain functions and behavior. mtCB1 receptors impair mitochondrial functions both in neurons and astrocytes. In the latter cells, this effect is linked to an impairment of astrocyte glycolytic function, resulting in specific behavioral outputs. Finally, we will discuss the potential implications of (mt)CB1 expression on oligodendrocytes and microglia metabolic functions, with the aim to encourage interdisciplinary approaches to better understand the role of (mt)CB1 receptors in brain function and behavior.

Cannabidiol for Oral Health: A New Promising Therapeutical Tool in Dentistry.

The medical use of cannabis has a very long history. Although many substances called cannabinoids are present in cannabis, Δ9tetrahydrocannabinol (Δ9-THC), cannabidiol (CBD) and cannabinol (CBN) are the three main cannabinoids that are most present and described. CBD itself is not responsible for the psychotropic effects of cannabis, since it does not produce the typical behavioral effects associated with the consumption of this drug. CBD has recently gained growing attention in modern society and seems to be increasingly explored in dentistry. Several subjective findings suggest some therapeutic effects of CBD that are strongly supported by research evidence. However, there is a plethora of data regarding CBD's mechanism of action and therapeutic potential, which are in many cases contradictory. We will first provide an overview of the scientific evidence on the molecular mechanism of CBD's action. Furthermore, we will map the recent developments regarding the possible oral benefits of CBD. In summary, we will highlight CBD's promising biological features for its application in dentistry, despite exiting patents that suggest the current compositions for oral care as the main interest of the industry.

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.

Control of a hippocampal recurrent excitatory circuit by cannabinoid receptor-interacting protein Gap43.

The type-1 cannabinoid receptor (CB1R) is widely expressed in excitatory and inhibitory nerve terminals, and by suppressing neurotransmitter release, its activation modulates neural circuits and brain function. While the interaction of CB1R with various intracellular proteins is thought to alter receptor signaling, the identity and role of these proteins are poorly understood. Using a high-throughput proteomic analysis complemented with an array of in vitro and in vivo approaches in the mouse brain, we report that the C-terminal, intracellular domain of CB1R interacts specifically with growth-associated protein of 43 kDa (GAP43). The CB1R-GAP43 interaction occurs selectively at mossy cell axon boutons, which establish excitatory synapses with dentate granule cells in the hippocampus. This interaction impairs CB1R-mediated suppression of mossy cell to granule cell transmission, thereby inhibiting cannabinoid-mediated anti-convulsant activity in mice. Thus, GAP43 acts as a synapse type-specific regulatory partner of CB1R that hampers CB1R-mediated effects on hippocampal circuit function.

COVID-19 on Oral Health: A New Bilateral Connection for the Pandemic.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and transmission are generally known to be produced by respiratory droplets and aerosols from the oral cavity (O.C.) of infected subjects, as stated by the World Health Organization. Saliva also retains the viral particles and aids in the spread of COVID-19. Angiotensin-converting enzyme Type 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) are two of the numerous factors that promote SARS-CoV-2 infection, expressed by O.C. structures, various mucosa types, and the epithelia of salivary glands. A systemic SARS-CoV-2 infection might result from viral replication in O.C. cells. On the other hand, cellular damage of different subtypes in the O.C. might be associated with various clinical signs and symptoms. Factors interfering with SARS-CoV-2 infection potential might represent fertile ground for possible local pharmacotherapeutic interventions, which may confine SARS-CoV-2 virus entry and transmission in the O.C., finally representing a way to reduce COVID-19 incidence and severity.

Expression of Functional Cannabinoid Type-1 (CB1) Receptor in Mitochondria of White Adipocytes.

Via activation of the cannabinoid type-1 (CB1) receptor, endogenous and exogenous cannabinoids modulate important biochemical and cellular processes in adipocytes. Several pieces of evidence suggest that alterations of mitochondrial physiology might be a possible mechanism underlying cannabinoids' effects on adipocyte biology. Many reports suggest the presence of CB1 receptor mRNA in both white and brown adipose tissue, but the detailed subcellular localization of CB1 protein in adipose cells has so far been scarcely addressed. In this study, we show the presence of the functional CB1 receptor at different subcellular locations of adipocytes from epididymal white adipose tissue (eWAT) depots. We observed that CB1 is located at different subcellular levels, including the plasma membrane and in close association with mitochondria (mtCB1). Functional analysis in tissue homogenates and isolated mitochondria allowed us to reveal that cannabinoids negatively regulate complex-I-dependent oxygen consumption in eWAT. This effect requires mtCB1 activation and consequent regulation of the intramitochondrial cAMP-PKA pathway. Thus, CB1 receptors are functionally present at the mitochondrial level in eWAT adipocytes, adding another possible mechanism for peripheral regulation of energy metabolism.

Forgetting in obesity: The pregnenolone link.

Cognitive dysfunction is often diagnosed in people with obesity and associated metabolic disorders. In the latest issue of Cell Metabolism, Ramírez et al. highlight an impaired production of the neurosteroid pregnenolone in the hypothalamus as a mechanism for obesity-induced cognitive impairment in both rodent models and patients with obesity.

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.

Functional heterogeneity of POMC neurons relies on mTORC1 signaling.

Hypothalamic pro-opiomelanocortin (POMC) neurons are known to trigger satiety. However, these neuronal cells encompass heterogeneous subpopulations that release γ-aminobutyric acid (GABA), glutamate, or both neurotransmitters, whose functions are poorly defined. Using conditional mutagenesis and chemogenetics, we show that blockade of the energy sensor mechanistic target of rapamycin complex 1 (mTORC1) in POMC neurons causes hyperphagia by mimicking a cellular negative energy state. This is associated with decreased POMC-derived anorexigenic α-melanocyte-stimulating hormone and recruitment of POMC/GABAergic neurotransmission, which is restrained by cannabinoid type 1 receptor signaling. Electrophysiology and optogenetic studies further reveal that pharmacological blockade of mTORC1 simultaneously activates POMC/GABAergic neurons and inhibits POMC/glutamatergic ones, implying that the functional specificity of these subpopulations relies on mTORC1 activity. Finally, POMC neurons with different neurotransmitter profiles possess specific molecular signatures and spatial distribution. Altogether, these findings suggest that mTORC1 orchestrates the activity of distinct POMC neurons subpopulations to regulate feeding behavior.

Identification of BiP as a CB1 Receptor-Interacting Protein That Fine-Tunes Cannabinoid Signaling in the Mouse Brain.

Cannabinoids, the bioactive constituents of cannabis, exert a wide array of effects on the brain by engaging Type 1 cannabinoid receptor (CB1R). Accruing evidence supports that cannabinoid action relies on context-dependent factors, such as the biological characteristics of the target cell, suggesting that cell population-intrinsic molecular cues modulate CB1R-dependent signaling. Here, by using a yeast two-hybrid-based high-throughput screening, we identified BiP as a potential CB1R-interacting protein. We next found that CB1R and BiP interact specifically in vitro, and mapped the interaction site within the CB1R C-terminal (intracellular) domain and the BiP C-terminal (substrate-binding) domain-α. BiP selectively shaped agonist-evoked CB1R signaling by blocking an "alternative" Gq/11 protein-dependent signaling module while leaving the "classical" Gi/o protein-dependent inhibition of the cAMP pathway unaffected. In situ proximity ligation assays conducted on brain samples from various genetic mouse models of conditional loss or gain of CB1R expression allowed to map CB1R-BiP complexes selectively on terminals of GABAergic neurons. Behavioral studies using cannabinoid-treated male BiP+/- mice supported that CB1R-BiP complexes modulate cannabinoid-evoked anxiety, one of the most frequent undesired effects of cannabis. Together, by identifying BiP as a CB1R-interacting protein that controls receptor function in a signaling pathway- and neuron population-selective manner, our findings may help to understand the striking context-dependent actions of cannabis in the brain.SIGNIFICANCE STATEMENT Cannabis use is increasing worldwide, so innovative studies aimed to understand its complex mechanism of neurobiological action are warranted. Here, we found that cannabinoid CB1 receptor (CB1R), the primary molecular target of the bioactive constituents of cannabis, interacts specifically with an intracellular protein called BiP. The interaction between CB1R and BiP occurs selectively on terminals of GABAergic (inhibitory) neurons, and induces a remarkable shift in the CB1R-associated signaling profile. Behavioral studies conducted in mice support that CB1R-BiP complexes act as fine-tuners of anxiety, one of the most frequent undesired effects of cannabis use. Our findings open a new conceptual framework to understand the striking context-dependent pharmacological actions of cannabis in the brain.

Cannabinoids Drugs and Oral Health-From Recreational Side-Effects to Medicinal Purposes: A Systematic Review.

BACKGROUND: marijuana, the common name for cannabis sativa preparations, is one of the most consumed drug all over the world, both at therapeutical and recreational levels. With the legalization of medical uses of cannabis in many countries, and even its recreational use in most of these, the prevalence of marijuana use has markedly risen over the last decade. At the same time, there is also a higher prevalence in the health concerns related to cannabis use and abuse. Thus, it is mandatory for oral healthcare operators to know and deal with the consequences and effects of cannabis use on oral cavity health. This review will briefly summarize the components of cannabis and the endocannabinoid system, as well as the cellular and molecular mechanisms of biological cannabis action in human cells and biologic activities on tissues. We will also look into oropharyngeal tissue expression of cannabinoid receptors, together with a putative association of cannabis to several oral diseases. Therefore, this review will elaborate the basic biology and physiology of cannabinoids in human oral tissues with the aim of providing a better comprehension of the effects of its use and abuse on oral health, in order to include cannabinoid usage into dental patient health records as well as good medicinal practice. METHODS: the paper selection was performed by PubMed/Medline and EMBASE electronic databases, and reported according to the PRISMA guidelines. The scientific products were included for qualitative analysis. RESULTS: the paper search screened a total of 276 papers. After the initial screening and the eligibility assessment, a total of 32 articles were considered for the qualitative analysis. CONCLUSIONS: today, cannabis consumption has been correlated to a higher risk of gingival and periodontal disease, oral infection and cancer of the oral cavity, while the physico-chemical activity has not been completely clarified. Further investigations are necessary to evaluate a therapeutic efficacy of this class of drugs for the promising treatment of several different diseases of the salivary glands and oral diseases.

Hypothalamic bile acid-TGR5 signaling protects from obesity.

Bile acids (BAs) improve metabolism and exert anti-obesity effects through the activation of the Takeda G protein-coupled receptor 5 (TGR5) in peripheral tissues. TGR5 is also found in the brain hypothalamus, but whether hypothalamic BA signaling is implicated in body weight control and obesity pathophysiology remains unknown. Here we show that hypothalamic BA content is reduced in diet-induced obese mice. Central administration of BAs or a specific TGR5 agonist in these animals decreases body weight and fat mass by activating the sympathetic nervous system, thereby promoting negative energy balance. Conversely, genetic downregulation of hypothalamic TGR5 expression in the mediobasal hypothalamus favors the development of obesity and worsens established obesity by blunting sympathetic activity. Lastly, hypothalamic TGR5 signaling is required for the anti-obesity action of dietary BA supplementation. Together, these findings identify hypothalamic TGR5 signaling as a key mediator of a top-down neural mechanism that counteracts diet-induced obesity.

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.