Postnatal hypofunction of N-methyl-D-aspartate receptors alters perforant path synaptic plasticity and filtering and impairs dentate gyrus-mediated spatial discrimination.

BACKGROUND AND PURPOSE: Transient hypofunction of the NMDA receptor represents a convergence point for the onset and further development of psychiatric disorders, including schizophrenia. Although the cumulative evidence indicates dysregulation of the hippocampal formation in schizophrenia, the integrity of the synaptic transmission and plasticity conveyed by the somatosensorial inputs to the dentate gyrus, the perforant pathway synapses, have barely been explored in this pathological condition. EXPERIMENTAL APPROACH: We identified a series of synaptic alterations of the lateral and medial perforant paths in animals postnatally treated with the NMDA antagonist MK-801. This dysregulation suggests decreased cognitive performance, for which the dentate gyrus is critical. KEY RESULTS: We identified alterations in the synaptic properties of the lateral and medial perforant paths to the dentate gyrus synapses in slices from MK-801-treated animals. Altered glutamate release and decreased synaptic strength precede an impairment in the induction and expression of long-term potentiation (LTP) and CB1 receptor-mediated long-term depression (LTD). Remarkably, by inhibiting the degradation of 2-arachidonoylglycerol (2-AG), an endogenous ligand of the CB1 receptor, we restored the LTD in animals treated with MK-801. Additionally, we showed for the first time, that spatial discrimination, a cognitive task that requires dentate gyrus integrity, is impaired in animals exposed to transient hypofunction of NMDA receptors. CONCLUSION AND IMPLICATIONS: Dysregulation of glutamatergic transmission and synaptic plasticity from the entorhinal cortex to the dentate gyrus has been demonstrated, which may explain the cellular dysregulations underlying the altered cognitive processing in the dentate gyrus associated with schizophrenia.

Anandamide and WIN 55212-2 Afford Protection in Rat Brain Mitochondria in a Toxic Model Induced by 3-Nitropropionic Acid: an In Vitro Study.

Mitochondrial dysfunction plays a key role in the development of neurodegenerative disorders. In contrast, the regulation of the endocannabinoid system has been shown to promote neuroprotection in different neurotoxic paradigms. The existence of an active form of the cannabinoid receptor 1 (CB1R) in mitochondrial membranes (mitCB1R), which might exert its effects through the same signaling mechanisms as the cell membrane CB1R, has been shown to regulate mitochondrial activity. Although there is evidence suggesting that some cannabinoids may induce protective effects on isolated mitochondria, substantial evidence on the role of cannabinoids in mitochondria remains to be explored. In this work, we developed a toxic model of mitochondrial dysfunction induced by exposure of brain mitochondria to the succinate dehydrogenase inhibitor 3-nitropropionic acid (3-NP). Mitochondria were also pre-incubated with the endogenous agonist anandamide (AEA) and the synthetic CB1R agonist WIN 55212-2 to evaluate their protective effects. Mitochondrial reduction capacity, reactive oxygen species (ROS) formation, and mitochondrial swelling were assessed as toxic markers. While 3-NP decreased the mitochondrial reduction capacity and augmented mitochondrial ROS formation and swelling, both AEA and WIN 55212-2 ameliorated these toxic effects. To explore the possible involvement of mitCB1R activation on the protective effects of AEA and WIN 55212-2, mitochondria were also pre-incubated in the presence of the selective CB1R antagonist AM281, which completely reverted the protective effects of the cannabinoids to levels similar to those evoked by 3-NP. These results show partial protective effects of cannabinoids, suggesting that mitCB1R activation may be involved in the recovery of compromised mitochondrial activity, related to reduction of ROS formation and further prevention of mitochondrial swelling.

Activation of the cannabinoid type 2 (CB2) receptor improves cardiac contractile performance in fish, Brycon amazonicus.

In addition to their well-known classical effects, cannabinoid CB1 and CB2 receptors have also been involvement in both deleterious and protective actions on the heart under various pathological conditions. While the potential therapeutic applications of the endocannabinoid system in the context of cardiovascular function are indeed a viable prospect, significant debate exists within the literature regarding whether CB1, CB2, or a combination of both receptors exert a favorable influence on cardiac function. Hence, the aim of this study was to investigate the effects of CB1 + CB2 or CB2 agonists on cardiac excitation-contraction (E-C) coupling, utilizing fish (Brycon amazonicus) as an experimental model. The CB2 agonist elicited marked positive inotropic and lusitropic responses in isolated ventricular myocardium, induced cyclic adenosine 3',5'-monophosphate (cAMP) production, and upregulated critical Ca2+ handling proteins, such as sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) and Na+/Ca2+ exchanger (NCX). Our current study demonstrated, for the first time, that CB2 receptor activation-induced effects improved the efficiency of Ca2+ cycling, excitation-contraction coupling (E-C coupling), and cardiac performance in under physiological conditions. Hence, CB2 receptors could be considered a potential therapeutic target for modulating cardiac contractile dysfunctions.

Anandamide reduces the migration of lymphocytes to the intestine by CB2 activation and reduces TNF-α in the target organs, protecting mice from graft-versus-host disease.

Graft-versus-host disease (GVHD) is a serious inflammatory illness that often occurs as a secondary complication of bone marrow transplantation. Current therapies have limited effectiveness and fail to achieve a balance between inflammation and the graft-versus-tumor effect. In this study, we investigate the effects of the endocannabinoid anandamide on the complex pathology of GVHD. We assess the effects of an irreversible inhibitor of fatty acid amine hydrolase or exogenous anandamide and find that they increase survival and reduce clinical signs in GVHD mice. In the intestine of GVHD mice, treatment with exogenous anandamide also leads to a reduction in the number of CD3+, CD3+CD4+, and CD3+CD8+ cells, which reduces the activation of CD3+CD4+ and CD3+CD8+ cells, as assessed by enhanced CD28 expression, a T cell co-stimulatory molecule. Exogenous AEA was also able to reduce TNF-α and increase IL-10 in the intestine of GVHD mice. In the liver, exogenous AEA reduces injury, TNF-α levels, and the number of CD3+CD8+ cells. Interestingly, anandamide reduces Mac-1α, which lowers the adhesion of transplanted cells in mesenteric veins. These effects are mimicked by JWH133-a CB2 selective agonist-and abolished by treatment with a CB2 antagonist. Furthermore, the effects caused by anandamide treatment on survival were related to the CB2 receptor, as the CB2 antagonist abolished it. This study shows the critical role of the CB2 receptor in the modulation of the inflammatory response of GVHD by treatment with anandamide, the most prominent endocannabinoid.

Motifs in Natural Products as Useful Scaffolds to Obtain Novel Benzo[d]imidazole-Based Cannabinoid Type 2 (CB2) Receptor Agonists.

The endocannabinoid system (ECS) constitutes a broad-spectrum modulator of homeostasis in mammals, providing therapeutic opportunities for several pathologies. Its two main receptors, cannabinoid type 1 (CB1) and type 2 (CB2) receptors, mediate anti-inflammatory responses; however, their differing patterns of expression make the development of CB2-selective ligands therapeutically more attractive. The benzo[d]imidazole ring is considered to be a privileged scaffold in drug discovery and has demonstrated its versatility in the development of molecules with varied pharmacologic properties. On the other hand, the main psychoactive component of Cannabis sativa, delta-9-tetrahydrocannabinol (THC), can be structurally described as an aliphatic terpenoid motif fused to an aromatic polyphenolic (resorcinol) structure. Inspired by the structure of this phytocannabinoid, we combined different natural product motifs with a benzo[d]imidazole scaffold to obtain a new library of compounds targeting the CB2 receptor. Here, we synthesized 26 new compounds, out of which 15 presented CB2 binding and 3 showed potent agonist activity. SAR analysis indicated that the presence of bulky aliphatic or aromatic natural product motifs at position 2 of the benzo[d]imidazoles ring linked by an electronegative atom is essential for receptor recognition, while substituents with moderate bulkiness at position 1 of the heterocyclic core also participate in receptor recognition. Compounds 5, 6, and 16 were further characterized through in vitro cAMP functional assay, showing potent EC50 values between 20 and 3 nM, and compound 6 presented a significant difference between the EC50 of pharmacologic activity (3.36 nM) and IC50 of toxicity (30-38 µM).

CB1R chronic intermittent pharmacological activation facilitates amphetamine seeking and self-administration and changes in CB1R/CRFR1 expression in the amygdala and nucleus accumbens in rats.

Patterns of drug ingestion may have a dissimilar impact on the brain, and therefore also the development of drug addiction. One pattern is binge intoxication that refers to the ingestion of a high amount of drug on a single occasion followed by an abstinence period of variable duration. In this study, our goal was to contrast the effect of continuous low amounts with intermittent higher amounts of Arachidonyl-chloro-ethylamide (ACEA), a CB1R agonist, on amphetamine seeking and ingestion, and describe the effects on the expression of CB1R and CRFR1 in the central nucleus of the amygdala (CeA) and in the nucleus accumbens shell (NAcS). Adult male Wistar rats were treated with a daily administration of vehicle or 20 µg of ACEA, or four days of vehicle followed by 100 µg of ACEA on the fifth day, for a total of 30 days. Upon completion of this treatment, the CB1R and CRFR1 expression in the CeA and NAcS was evaluated by immunofluorescence. Additional groups of rats were evaluated for their anxiety levels (elevated plus maze, EPM), amphetamine (AMPH) self-administration (ASA) and breakpoint (A-BP), as well as AMPH-induced conditioned place preference (A-CPP). Results indicated that ACEA induced changes in the CB1R and CRFR1 expression in both the NAcS and CeA. An increase in anxiety-like behavior, ASA, A-BP and A-CPP was also observed. Since the intermittent administration of 100 µg of ACEA induced the most evident changes in most of the parameters studied, we concluded that binge-like ingestion of drugs induces changes in the brain that may make the subject more vulnerable to developing drug addiction.

Activation of cannabinoid type 1 receptor (CB1) modulates oligodendroglial process branching complexity in rat hippocampal cultures stimulated by olfactory ensheathing glia-conditioned medium.

The endocannabinoid system (ECS) refers to a complex cell-signaling system highly conserved among species formed by numerous receptors, lipid mediators (endocannabinoids) and synthetic and degradative enzymes. It is widely distributed throughout the body including the CNS, where it participates in synaptic signaling, plasticity and neurodevelopment. Besides, the olfactory ensheathing glia (OEG) present in the olfactory system is also known to play an important role in the promotion of axonal growth and/or myelination. Therefore, both OEG and the ECS promote neurogenesis and oligodendrogenesis in the CNS. Here, we investigated if the ECS is expressed in cultured OEG, by assessing the main markers of the ECS through immunofluorescence, western blotting and qRT-PCR and quantifying the content of endocannabinoids in the conditioned medium of these cells. After that, we investigated whether the production and release of endocannabinoids regulate the differentiation of oligodendrocytes co-cultured with hippocampal neurons, through Sholl analysis in oligodendrocytes expressing O4 and MBP markers. Additionally, we evaluated through western blotting the modulation of downstream pathways such as PI3K/Akt/mTOR and ERK/MAPK, being known to be involved in the proliferation and differentiation of oligodendrocytes and activated by CB1, which is the major endocannabinoid responsive receptor in the brain. Our data show that OEG expresses key genes of the ECS, including the CB1 receptor, FAAH and MAGL. Besides, we were able to identify AEA, 2-AG and AEA related mediators palmitoylethanolamide (PEA) and oleoylethanolamide (OEA), in the conditioned medium of OEG cultures. These cultures were also treated with URB597 10-9 M, a FAAH selective inhibitor, or JZL184 10-9 M, a MAGL selective inhibitor, which led to the increase in the concentrations of OEA and 2-AG in the conditioned medium. Moreover, we found that the addition of OEG conditioned medium (OEGCM) enhanced the complexity of oligodendrocyte process branching in hippocampal mixed cell cultures and that this effect was inhibited by AM251 10-6 M, a CB1 receptor antagonist. However, treatment with the conditioned medium enriched with OEA or 2-AG did not alter the process branching complexity of premyelinating oligodendrocytes, while decreased the branching complexity in mature oligodendrocytes. We also observed no change in the phosphorylation of Akt and ERK 44/42 in any of the conditions used. In conclusion, our data show that the ECS modulates the number and maturation of oligodendrocytes in hippocampal mixed cell cultures.

The mystery behind the apprehensions of the selective cannabinoid receptor type-2 agonist BZO-HEXOXIZID (MDA-19) as a drug of abuse.

PURPOSE: MDA-19 or BZO-HEXOXIZID (N'-[(3Z)-1-(1-hexyl)-2-oxo-1,2-dihydro-3H-indol-3-ylidene]-benzohydrazide), in a more recent nomenclature, was first synthesized in 2008 as a selective type-2 cannabinoid receptor (CB2) agonist due to its potential to treat neuropathic pain. In Brazil, this substance was identified in a series of 53 apprehensions between September 2021 and February 2022. Nevertheless, what intrigues toxicologists is that BZO-HEXOXIZID does not exert significant type-1 cannabinoid receptor (CB1) agonism-which is responsible for the well-known psychoactivity of Δ-9-tetrahydrocannabinol. Thus, the objective of this work is to report the first apprehension and identification of BZO-HEXOXIZID in Brazil and to discuss pharmacologically the possible reasons why a CB2 agonist has been incorporated to the illicit market. METHODS: Suspected seized samples were sent to the Laboratory of the Scientific Police of the State of Sao Paulo. After the screening, samples were confirmed for the presence of BZO-HEXOXIZID using chromatography gas-mass spectrometry, Fourier-transform infrared spectroscopy and nuclear magnetic resonance techniques. RESULTS: Of the 53 samples analyzed, 25 contained only BZO-HEXOXIZID and 28 with mixtures, of which 11 with the CB1 agonist ADB-BUTINACA. Other substances were found in association such as cocaine and caffeine. CONCLUSIONS: BZO-HEXOXIZID was detected in a series of seized materials for the first time in Brazil. Nevertheless, there are still unanswered questions regarding the use of this selective CB2 agonist as a drug of abuse.

ß-caryophyllene, a cannabinoid receptor 2 agonist, decreases the motivational salience and conditioning place preference for palatable food in female mice.

ß-caryophyllene (BCP) is a cannabinoid receptor CB2 agonist plant-derived terpenoid found in different essential oil plants, including rosemary, black pepper, copaiba and cannabis. It has GRAS (generally recognized as safe) status and is approved by the FDA (Food and Drug Administration) for food use. BCP displays agonist activity on the CB2 receptor and is a potential therapeutic target in several neuropsychiatric disorders, including anxiety and drug addiction. Unlike CB1 receptors, activation of the CB2 receptors is devoid of psychotomimetic and addictive properties. In this regard, this study aimed to evaluate the effects of BCP on incentive salience ("wanting") performance and motivational properties elicited by sweetened palatable foods in female Swiss mice. After 9 days of training for incentive salience performance for a sweet reward (hazelnut cream with chocolate), food-restricted mice received a systemic injection of BCP (50 and 100 mg/kg) before testing over 3 days. Moreover, independent groups of female mice were tested on sweet reward-induced conditioned place preference (CPP) for 22 consecutive days. To evaluate BCP effects on the expression of seeking behaviour for sweetened food, mice received a single intraperitoneal injection of BCP (50 mg/kg) 30 min before testing on the CPP task. BCP significantly decreased the incentive performance for a sweet reward compared with the control group in a CB2 receptor-dependent manner. Also, BCP suppressed the expression of sweet reward-CPP. Altogether, these preclinical data demonstrate the potential role of BCP in treating disorders associated with food addiction-like behaviour.

The cannabinoids mechanism of action: an overview / Mecanismo de ação dos canabinoides: visão geral

ABSTRACT BACKGROUND AND OBJECTIVES: The discovery of the psychoactive agent of Cannabis sativa (tetrahydrocannabinol - THC) in the second half of the 20th century originated the research that later came to identify dozens of other substances from this plant, including cannabinoids, terpenes and flavonoids. Ensuing description of their interaction sites in animals and humans, together with endogenous ligands, transport proteins as well as synthesis and degradation enzymes, revealed what came to be known as the endocannabinoid system. Several receptors participate in this system. CONTENTS: The first receptors to be discovered were called CB1 and CB2, both are G protein-coupled (GPCR). It is noteworthy that CB1 receptors are among the most abundant and widely distributed GPCR in the mammalian brain, with marked expression in basal ganglia, cerebellum and hippocampus, for instance; on the other hand, they are scarce in areas of the brainstem related to breathing control. In light of the multiplicity of pharmacological effects of cannabinoids, concomitant with the lack of more clarifying studies on their mechanisms of action despite the great interest in research on their therapeutic application, it is necessary to deepen the knowledge in this area. CONCLUSION: Considering the literature research conducted for the composition of this article, it is possible to conclude that cannabinoids have a broad spectrum of action mechanisms in the human body, and that more robust clinical studies are needed to better understand their broad therapeutic potential.

RESUMO JUSTIFICATIVA E OBJETIVOS: A descoberta do princípio psicoativo da Cannabis sativa (tetrahidrocanabinol - THC) na segunda metade do século XX inaugurou pesquisas que posteriormente vieram a identificar dezenas de outras substâncias a partir dessa planta, incluindo canabinoides, terpenos e flavonoides. A subsequente descrição dos sítios de interação dessas substâncias em animais e humanos, assim como seus ligantes endógenos, proteínas de transporte e enzimas de síntese e degradação, revelou o que veio a ser conhecido como sistema endocanabinoide. Diversos receptores participam deste sistema. CONTEÚDO: Os primeiros receptores a serem descobertos foram denominados CB1 e CB2, ambos são acoplados à proteína G (GPCR). É importante ressaltar que os receptores CB1 estão entre os GPCRs mais abundantes e amplamente distribuídos do encéfalo de mamíferos, com marcada expressão, por exemplo, em gânglios da base, cerebelo e hipocampo; em contrapartida, são escassos em áreas do tronco cerebral relacionadas ao controle da respiração. Diante da multiplicidade de efeitos farmacológicos dos canabinoides, concomitante à falta de estudos mais esclarecedores sobre seus mecanismos de ação apesar do grande interesse na pesquisa de sua aplicação terapêutica, é preciso aprofundar o conhecimento nessa área. CONCLUSÃO: Considerando as pesquisas bibliográficas realizadas para a composição deste artigo, é possível concluir que os canabinoides possuem um amplo espectro de mecanismos de ação no organismo humano, e que mais estudos clínicos robustos são necessários para que seja possível entender melhor o seu amplo potencial terapêutico.

Synthetic cannabinoid receptor agonists profile in infused papers seized in Brazilian prisons.

PURPOSE: Synthetic cannabinoid receptor agonists (SCRAs) are a class of varied compounds that mimic the effects of natural cannabinoids found in cannabis. Because they have a wide range of diverse structures, they vary widely in their potency. The abuse of new psychoactive substances (NPS) in prisons was reported in many European countries and in the USA. In the present study, we have described the identification of SCRAs in 56 infused paper sheet samples, seized mainly in Brazilian prisons between 2016 and 2020. METHODS: The materials were seized by local or federal law enforcement and analyzed by São Paulo State Police or Brazilian Federal Police using gas chromatography-mass spectrometry, attenuated total reflection-Fourier transform infrared spectroscopy, liquid chromatography-high-resolution mass spectrometry or nuclear magnetic resonance spectrometry. RESULTS: Most of these samples (87.5%) were seized in 2019-2020; seven different SCRAs were identified in samples, and the most frequently identified substances were MDMB-4en-PINACA (23.6%) and 5F-MDMB-PICA (36.4%), the newest SCRAs emerging recently. CONCLUSIONS: As observed in Europe and the USA, Brazil also shows the prevalence of indazole-3-carboxamides and indole-3-carboxamides among SCRAs seizures in the prison system. This phenomenon is spreading all over the world at this moment. These data on the prevalence could help to alert judicial authorities to shutting down the introduction of NPS, including SCRAs, into prisons to ensure safety and security for avoiding health risks of prisoners and staff, leading to positive effects in this population. To our knowledge, this is the first demonstration of SCRAs smuggling into prisons in Latin America.

Immune challenges upregulate the expression of cannabinoid receptors in cultured human odontoblasts and gingival fibroblasts.

Odontoblasts and gingival fibroblasts play essential roles in the physiological and pathological processes of dental tissue. Cannabinoid receptors (CB1 and CB2) are involved in analgesia by modulating the función of calcium channels that inhibit the synthesis of some neurotransmitters. A better understanding of the physiology of these receptors would provide the possibility of using them as therapeutic targets in controlling dental pain. The aim of this study was to evaluate the presence and activity of cannabinoid receptors in human odontoblast-like cells (OLC) and human gingival fibroblasts (HGF). CB1 and CB2 transcription was analyzed by real-time PCR, proteins were detected by immunofluorescence, and functional cannabinoid receptors were evaluated by measuring intracellular calcium concentration after stimulation with cannabidiol (CBD) and pre-treatment with a CB1 antagonist, a CB2 inverse agonist and a TRPV1 antagonist. Transcripts for CB1 and CB2 were found in both odontoblasts and gingival fibroblasts. Cannabidiol induced an increase in [Ca2+]i in both cells types, but surprisingly, pre-treatment with selective cannabinoid antagonists attenuated this effect, suggesting a functional communication between specific cannabinoid receptors and other CBD target receptors. In conclusion, human odontoblasts and gingival fibroblasts express functional CB1 and CB2 cannabinoid receptors, which could be modulated to improve the treatment of pain or dental sensitivity.

Los odontoblastos y los fibroblastos gingivales desempeñan funciones esenciales en los procesos fisiológicos y patológicos de los tejidos dentales. Los receptores cannabinoides (CB1 y CB2) participan en la analgesia mediante la modulación de la función de canales de calcio que inhiben la síntesis de algunos neurotransmisores. Un mejor conocimiento de su fisiología abre la posibilidad de utilizar estos receptores como dianas terapéuticas en el control del dolor dental. Este trabajo tuvo como objetivo evaluar la presencia y la actividad de los receptores cannabinoides en células humanas similares a los odontoblastos (OLC) y en fibroblastos gingivales humanos (HGF). Se analizó la transcripción de CB1 y CB2 por PCR en tiempo real, la detección de las proteínas por inmunofluorescencia y se evaluaron los receptores cannabinoides funcionales midiendo las concentraciones de calcio intracelular, tras la estimulación con cannabidiol (CBD) y el pretratamiento con un antagonista de CB1, un agonista inverso de CB2 y un antagonista de TRPV1. Se encontraron mensajeros para CB1 y CB2 tanto en odontoblastos como en fibroblastos gingivales. El cannabidiol indujo un aumento de la [Ca2+]i en ambos tipos de células, pero sorprendentemente el pretratamiento con antagonistas cannabinoides selectivos atenuó este efecto, lo que sugiere una comunicación funcional entre receptores cannabinoides específicos y otros receptores diana del CBD. En conclusión, los odontoblastos humanos y los fibroblastos gingivales expresan receptores cannabinoides CB1 y CB2 funcionales, que podrían ser modulados para mejorar el tratamiento del dolor o la sensibilidad dental.

Reference gene validation for the relative quantification of cannabinoid receptor expression in human odontoblasts via quantitative polymerase chain reaction.

Objective: The aim of this study was to identify and validate the reference genes in cultured human odontoblasts to quantify their cannabinoid receptor transcripts. Methods: The most stably transcribed genes in cultured human odontoblast cells were identified using the RefGenes tool and were selected for real-time polymerase chain reaction (PCR) amplification. Human odontoblast cells were differentiated from mesenchymal stem cells using a transforming growth factor-ß-supplemented differentiation medium, and total RNA was purified. Reverse transcription-quantitative PCR and relative quantification analyses were performed using the Schefe's method. The relative expression dataset was analyzed to select the most stable genes. Results: The analysis showed that the transcripts of cholinergic receptor nicotinic beta 2 subunit, LIM homeobox transcription factor 1 beta, and family with sequence similarity 223 member B presented the lowest standard deviation (SD) in expression (SD: 0.2, 0.17, and 0.16, respectively). These genes showed similar expression levels as the target genes (cannabinoid receptors). Significant differences were found in the relative expression levels of cannabinoid receptors using the selected genes compared to those calculated using beta actin transcripts as references (p < 0.05). Conclusions: The strategy reported here for searching and verifying new reference genes will aid in the accurate and reliable expression of cannabinoid receptors in human odontoblast cells.

Immune challenges upregulate the expression of cannabinoid receptors in cultured human odontoblasts and gingival fibroblasts / Los desafíos inmunológicos regulan la expresión de los receptores cannabinoides en cultivos de odontoblastos y fibroblastos gingivales humanos

ABSTRACT Odontoblasts and gingival fibroblasts play essential roles in the physiological and pathological processes of dental tissue. Cannabinoid receptors (CB1 and CB2) are involved in analgesia by modulating the función of calcium channels that inhibit the synthesis of some neurotransmitters. A better understanding of the physiology of these receptors would provide the possibility of using them as therapeutic targets in controlling dental pain. The aim of this study was to evaluate the presence and activity of cannabinoid receptors in human odontoblast-like cells (OLC) and human gingival fibroblasts (HGF). CB1 and CB2 transcription was analyzed by real-time PCR, proteins were detected by immunofluorescence, and functional cannabinoid receptors were evaluated by measuring intracellular calcium concentration after stimulation with cannabidiol (CBD) and pre-treatment with a CB1 antagonist, a CB2 inverse agonist and a TRPV1 antagonist. Transcripts for CB1 and CB2 were found in both odontoblasts and gingival fibroblasts. Cannabidiol induced an increase in [Ca2+]i in both cells types, but surprisingly, pre-treatment with selective cannabinoid antagonists attenuated this effect, suggesting a functional communication between specific cannabinoid receptors and other CBD target receptors. In conclusion, human odontoblasts and gingival fibroblasts express functional CB1 and CB2 cannabinoid receptors, which could be modulated to improve the treatment of pain or dental sensitivity.

RESUMEN Los odontoblastos y los fibroblastos gingivales desempeñan funciones esenciales en los procesos fisiológicos y patológicos de los tejidos dentales. Los receptores cannabinoides (CB1 y CB2) participan en la analgesia mediante la modulación de la función de canales de calcio que inhiben la síntesis de algunos neurotransmisores. Un mejor conocimiento de su fisiología abre la posibilidad de utilizar estos receptores como dianas terapéuticas en el control del dolor dental. Este trabajo tuvo como objetivo evaluar la presencia y la actividad de los receptores cannabinoides en células humanas similares a los odontoblastos (OLC) y en fibroblastos gingivales humanos (HGF). Se analizó la transcripción de CB1 y CB2 por PCR en tiempo real, la detección de las proteínas por inmunofluorescencia y se evaluaron los receptores cannabinoides funcionales midiendo las concentraciones de calcio intracelular, tras la estimulación con cannabidiol (CBD) y el pretratamiento con un antagonista de CB1, un agonista inverso de CB2 y un antagonista de TRPV1. Se encontraron mensajeros para CB1 y CB2 tanto en odontoblastos como en fibroblastos gingivales. El cannabidiol indujo un aumento de la [Ca2+]i en ambos tipos de células, pero sorprendentemente el pretratamiento con antagonistas cannabinoides selectivos atenuó este efecto, lo que sugiere una comunicación funcional entre receptores cannabinoides específicos y otros receptores diana del CBD. En conclusión, los odontoblastos humanos y los fibroblastos gingivales expresan receptores cannabinoides CB1 y CB2 funcionales, que podrían ser modulados para mejorar el tratamiento del dolor o la sensibilidad dental.

Interactions between the Nicotinic and Endocannabinoid Receptors at the Plasma Membrane.

Compartmentalization, together with transbilayer and lateral asymmetries, provide the structural foundation for functional specializations at the cell surface, including the active role of the lipid microenvironment in the modulation of membrane-bound proteins. The chemical synapse, the site where neurotransmitter-coded signals are decoded by neurotransmitter receptors, adds another layer of complexity to the plasma membrane architectural intricacy, mainly due to the need to accommodate a sizeable number of molecules in a minute subcellular compartment with dimensions barely reaching the micrometer. In this review, we discuss how nature has developed suitable adjustments to accommodate different types of membrane-bound receptors and scaffolding proteins via membrane microdomains, and how this "effort-sharing" mechanism has evolved to optimize crosstalk, separation, or coupling, where/when appropriate. We focus on a fast ligand-gated neurotransmitter receptor, the nicotinic acetylcholine receptor, and a second-messenger G-protein coupled receptor, the cannabinoid receptor, as a paradigmatic example.

The controversial role of the vagus nerve in mediating ghrelin's actions: gut feelings and beyond.

Ghrelin is a stomach-derived peptide hormone that acts via the growth hormone secretagogue receptor (GHSR) and displays a plethora of neuroendocrine, metabolic, autonomic and behavioral actions. It has been proposed that some actions of ghrelin are exerted via the vagus nerve, which provides a bidirectional communication between the central nervous system and peripheral systems. The vagus nerve comprises sensory fibers, which originate from neurons of the nodose and jugular ganglia, and motor fibers, which originate from neurons of the medulla. Many anatomical studies have mapped GHSR expression in vagal sensory or motor neurons. Also, numerous functional studies investigated the role of the vagus nerve mediating specific actions of ghrelin. Here, we critically review the topic and discuss the available evidence supporting, or not, a role for the vagus nerve mediating some specific actions of ghrelin. We conclude that studies using rats have provided the most congruent evidence indicating that the vagus nerve mediates some actions of ghrelin on the digestive and cardiovascular systems, whereas studies in mice resulted in conflicting observations. Even considering exclusively studies performed in rats, the putative role of the vagus nerve in mediating the orexigenic and growth hormone (GH) secretagogue properties of ghrelin remains debated. In humans, studies are still insufficient to draw definitive conclusions regarding the role of the vagus nerve mediating most of the actions of ghrelin. Thus, the extent to which the vagus nerve mediates ghrelin actions, particularly in humans, is still uncertain and likely one of the most intriguing unsolved aspects of the field.

Characterization of hypotensive and vasorelaxant effects of PHAR-DBH-Me a new cannabinoid receptor agonist.

The effect of PHAR-DBH-Me, a cannabinoid receptor agonist, on different cardiovascular responses in adult male rats was analyzed. The blood pressure was measured directly and indirectly. The coronary flow was measured by Langendorff preparation, and vasomotor responses induced by PHAR-DBH-Me in aortic rings precontracted with phenylephrine (PHEN) were analyzed. The intravenous injection of the compound PHAR-DBH-Me (0.018-185 µg/kg) resulted in decreased blood pressure; maximum effect was observed at the dose of 1,850 µg/kg. A concentration-dependent increase in the coronary flow was observed in a Langendorff preparation. In the aortic rings, with and without endothelium, pre-contracted with PHEN (10-6 M), the addition of PHAR-DBH-Me to the superfusion solution (10-12-10-5 M), produced a vasodilator response, which depends on the concentration and presence of the endothelium. L-NAME inhibited these effects. Addition of CB1 receptor antagonist (AM 251) did not modify the response, while CB2 receptor antagonist (AM630) decreased the potency of relaxation elicited by PHAR-DBH-Me. Indomethacin shifted the curve concentration-response to the left and produced an increase in the magnitude of the maximum endothelium dependent response to this compound. The maximum effect of PHAR-DBH-Me was observed with the concentration of 10-5 M. These results show that PHAR-DBH-Me has a concentration-dependent and endothelium-dependent vasodilator effect through CB2 receptor. This vasodilation is probably mediated by the synthesis/release of NO. On the other hand, it is suggested that PHAR-DBH-Me also induces the release of a vasoconstrictor prostanoid.

Nanoscale Sub-Compartmentalization of the Dendritic Spine Compartment.

Compartmentalization of the membrane is essential for cells to perform highly specific tasks and spatially constrained biochemical functions in topographically defined areas. These membrane lateral heterogeneities range from nanoscopic dimensions, often involving only a few molecular constituents, to micron-sized mesoscopic domains resulting from the coalescence of nanodomains. Short-lived domains lasting for a few milliseconds coexist with more stable platforms lasting from minutes to days. This panoply of lateral domains subserves the great variety of demands of cell physiology, particularly high for those implicated in signaling. The dendritic spine, a subcellular structure of neurons at the receiving (postsynaptic) end of central nervous system excitatory synapses, exploits this compartmentalization principle. In its most frequent adult morphology, the mushroom-shaped spine harbors neurotransmitter receptors, enzymes, and scaffolding proteins tightly packed in a volume of a few femtoliters. In addition to constituting a mesoscopic lateral heterogeneity of the dendritic arborization, the dendritic spine postsynaptic membrane is further compartmentalized into spatially delimited nanodomains that execute separate functions in the synapse. This review discusses the functional relevance of compartmentalization and nanodomain organization in synaptic transmission and plasticity and exemplifies the importance of this parcelization in various neurotransmitter signaling systems operating at dendritic spines, using two fast ligand-gated ionotropic receptors, the nicotinic acetylcholine receptor and the glutamatergic receptor, and a second-messenger G-protein coupled receptor, the cannabinoid receptor, as paradigmatic examples.

ZnO Nanoparticles Induce Dyslipidemia and Atherosclerotic Lesions Leading to Changes in Vascular Contractility and Cannabinoid Receptors Expression as Well as Increased Blood Pressure.

ZnO nanoparticles (ZnONPs) have been shown to have therapeutic potential in some diseases such as diabetes and cancer. However, concentration-dependent adverse effects have also been reported. Studies which evaluate the effects of ZnONPs on the cardiovascular system are scarce. This study aimed to evaluate the cardiovascular effects of a low dose of ZnONPs administered chronically in healthy rats. Changes in dyslipidemia biomarkers, blood pressure, aortic wall structure, vascular contractility, and expression of cannabinoid receptors in the aorta wall were evaluated. Healthy rats were divided into two groups: control or treated (one, two, and three months). The treated rats received an oral dose of 10 mg/kg/day. The results showed that treatment with ZnONPs induced dyslipidemia from the first month, increasing atherosclerosis risk, which was confirmed by presence of atherosclerotic alterations revealed by aorta histological analysis. In in vitro assays, ZnONPs modified the aorta contractile activity in response to the activation of cannabinoid receptors (CB1 and CB2). The expression of CB1 and CB2 was modified as well. Moreover, ZnONPs elicited an increase in blood pressure. In conclusion, long-time oral administration of ZnONPs induce dyslipidemia and atherosclerosis eliciting alterations in aorta contractility, CB1 and CB2 receptors expression, and an increase in blood pressure in healthy rats.

Cnr2 Is Important for Ribbon Synapse Maturation and Function in Hair Cells and Photoreceptors.

The role of the cannabinoid receptor 2 (CNR2) is still poorly described in sensory epithelia. We found strong cnr2 expression in hair cells (HCs) of the inner ear and the lateral line (LL), a superficial sensory structure in fish. Next, we demonstrated that sensory synapses in HCs were severely perturbed in larvae lacking cnr2. Appearance and distribution of presynaptic ribbons and calcium channels (Cav1.3) were profoundly altered in mutant animals. Clustering of membrane-associated guanylate kinase (MAGUK) in post-synaptic densities (PSDs) was also heavily affected, suggesting a role for cnr2 for maintaining the sensory synapse. Furthermore, vesicular trafficking in HCs was strongly perturbed suggesting a retrograde action of the endocannabinoid system (ECs) via cnr2 that was modulating HC mechanotransduction. We found similar perturbations in retinal ribbon synapses. Finally, we showed that larval swimming behaviors after sound and light stimulations were significantly different in mutant animals. Thus, we propose that cnr2 is critical for the processing of sensory information in the developing larva.