Advances in steroid research from the pioneering neurosteroid concept to metabolomics: New insights into pregnenolone function.

Advances in neuroendocrinology have led to major discoveries since the 19th century, identifying adaptive loops for maintaining homeostasis. One of the most remarkable discoveries was the concept of neurosteroids, according to which the brain is not only a target but also a source of steroid production. The identification of new membrane steroid targets now underpins the neuromodulatory effects of neurosteroids such as pregnenolone, which is involved in functions mediated by the GPCR CB1 receptor. Structural analysis of steroids is a key feature of their interactions with the phospholipid membrane, receptors and resulting activity. Therefore, mass spectrometry-based methods have been developed to elucidate the metabolic pathways of steroids, the ultimate approach being metabolomics, which allows the identification of a large number of metabolites in a single sample. This approach should enable us to make progress in understanding the role of neurosteroids in the functioning of physiological and pathological processes.

Endocannabinoid-dependent formation of columnar axonal projection in the mouse cerebral cortex.

Columnar structure is one of the most fundamental morphological features of the cerebral cortex and is thought to be the basis of information processing in higher animals. Yet, how such a topographically precise structure is formed is largely unknown. Formation of columnar projection of layer 4 (L4) axons is preceded by thalamocortical formation, in which type 1 cannabinoid receptors (CB1R) play an important role in shaping barrel-specific targeted projection by operating spike timing-dependent plasticity during development (Itami et al., J. Neurosci. 36, 7039-7054 [2016]; Kimura & Itami, J. Neurosci. 39, 3784-3791 [2019]). Right after the formation of thalamocortical projections, CB1Rs start to function at L4 axon terminals (Itami & Kimura, J. Neurosci. 32, 15000-15011 [2012]), which coincides with the timing of columnar shaping of L4 axons. Here, we show that the endocannabinoid 2-arachidonoylglycerol (2-AG) plays a crucial role in columnar shaping. We found that L4 axon projections were less organized until P12 and then became columnar after CB1Rs became functional. By contrast, the columnar organization of L4 axons was collapsed in mice genetically lacking diacylglycerol lipase α, the major enzyme for 2-AG synthesis. Intraperitoneally administered CB1R agonists shortened axon length, whereas knockout of CB1R in L4 neurons impaired columnar projection of their axons. Our results suggest that endocannabinoid signaling is crucial for shaping columnar axonal projection in the cerebral cortex.

Pharmaceutical targeting of the cannabinoid type 1 receptor impacts the crosstalk between immune cells and islets to reduce insulitis in humans.

AIMS/HYPOTHESIS: Insulitis, a hallmark of inflammation preceding autoimmune type 1 diabetes, leads to the eventual loss of functional beta cells. However, functional beta cells can persist even in the face of continuous insulitis. Despite advances in immunosuppressive treatments, maintaining functional beta cells to prevent insulitis progression and hyperglycaemia remains a challenge. The cannabinoid type 1 receptor (CB1R), present in immune cells and beta cells, regulates inflammation and beta cell function. Here, we pioneer an ex vivo model mirroring human insulitis to investigate the role of CB1R in this process. METHODS: CD4+ T lymphocytes were isolated from peripheral blood mononuclear cells (PBMCs) from male and female individuals at the onset of type 1 diabetes and from non-diabetic individuals, RNA was extracted and mRNA expression was analysed by real-time PCR. Single beta cell expression from donors with type 1 diabetes was obtained from data mining. Patient-derived human islets from male and female cadaveric donors were 3D-cultured in solubilised extracellular matrix gel in co-culture with the same donor PBMCs, and incubated with cytokines (IL-1ß, TNF-α, IFN-γ) for 24-48 h in the presence of vehicle or increasing concentrations of the CB1R blocker JD-5037. Expression of CNR1 (encoding for CB1R) was ablated using CRISPR/Cas9 technology. Viability, intracellular stress and signalling were assayed by live-cell probing and real-time PCR. The islet function measured as glucose-stimulated insulin secretion was determined in a perifusion system. Infiltration of immune cells into the islets was monitored by microscopy. Non-obese diabetic mice aged 7 weeks were treated for 1 week with JD-5037, then euthanised. Profiling of immune cells infiltrated in the islets was performed by flow cytometry. RESULTS: CNR1 expression was upregulated in circulating CD4+ T cells from individuals at type 1 diabetes onset (6.9-fold higher vs healthy individuals) and in sorted islet beta cells from donors with type 1 diabetes (3.6-fold higher vs healthy counterparts). The peripherally restricted CB1R inverse agonist JD-5037 arrested the initiation of insulitis in humans and mice. Mechanistically, CB1R blockade prevented islet NO production and ameliorated the ATF6 arm of the unfolded protein response. Consequently, cyto/chemokine expression decreased in human islets, leading to sustained islet cell viability and function. CONCLUSIONS/INTERPRETATION: These results suggest that CB1R could be an interesting target for type 1 diabetes while highlighting the regulatory mechanisms of insulitis. Moreover, these findings may apply to type 2 diabetes where islet inflammation is also a pathophysiological factor. DATA AVAILABILITY: Transcriptomic analysis of sorted human beta cells are from Gene Expression Omnibus database, accession no. GSE121863, available at https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSM3448161 .

Gut Microbiota-Mediated Alterations of Hippocampal CB1R Regulating the Diurnal Variation of Cognitive Impairment Induced by Hepatic Ischemia-Reperfusion Injury in Mice.

Patients suffering from hepatic ischemia-reperfusion injury (HIRI) frequently exhibit postoperative cognitive deficits. Our previous observations have emphasized the diurnal variation in hepatic ischemia-reperfusion injury-induced cognitive impairment, in which gut microbiota-associated hippocampal lipid metabolism plays an important role. Herein, we further investigated the molecular mechanisms involved in the process. Hepatic ischemia-reperfusion surgery was performed under morning (ZT0, 08:00) and evening (ZT12, 20:00). Fecal microbiota transplantation was used to associate HIRI model with pseudo-germ-free mice. The novel object recognition test and Y-maze test were used to assess cognitive function. 16S rRNA gene sequencing and analysis were used for microbial analysis. Western blotting was used for hippocampal protein analysis. Compared with the ZT0-HIRI group, ZT12-HIRI mice showed learning and short term memory impairment, accompanied by down-regulated expression of hippocampal CB1R, but not CB2R. Both gut microbiota composition and microbiota metabolites were significantly different in ZT12-HIRI mice compared with ZT0-HIRI. Fecal microbiota transplantation from the ZT12-HIRI was demonstrated to induce cognitive impairment behavior and down-regulated hippocampal CB1R and ß-arrestin1. Intraperitoneal administration of CB1R inhibitor AM251 (1 mg/kg) down-regulated hippocampal CB1R and caused cognitive impairment in ZT0-HIRI mice. And intraperitoneal administration of CB1R agonist WIN 55,212-2 (1 mg/kg) up-regulated hippocampal CB1R and improved cognitive impairment in ZT12-HIRI mice. In summary, the results suggest that gut microbiota may regulate the diurnal variation of HIRI-induced cognitive function by interfering with hippocampal CB1R.

Effects of CB1R inverse agonist, INV-202, in patients with features of metabolic syndrome. A randomized, placebo-controlled, double-blind phase 1b study.

AIMS: To evaluate the clinical safety, tolerability, and pharmacokinetic and pharmacodynamic profile of the novel cannabinoid receptor-1 (CB1R) inverse agonist, INV-202, in adults with features of metabolic syndrome. MATERIALS AND METHODS: This was a multicentre, randomized, double-blind, placebo-controlled, 28-day repeat-dose (INV-202 [25 mg] or placebo, once-daily oral tablet), parallel-group study in 37 participants aged 18 to 65 years (46% female, mean age 55 years, glycated haemoglobin 5.7% [39 mmol/mol], body mass index [BMI] 38.1 kg/m2 ) with features of metabolic syndrome and glucose intolerance. An oral glucose tolerance test (OGTT) was performed at baseline and at the end of the study. Lipid profiles, weight, waist circumference and biomarkers were assessed weekly. Statistical comparisons were performed post hoc. RESULTS: INV-202 was well tolerated with no serious or severe treatment-emergent adverse events; the most common events related to known effects of CB1R blockade in the gastrointestinal tract. INV-202 produced a significant mean weight loss of 3.5 kg (3.3% compared with placebo participants who gained a mean 0.6 kg [0.5%]). INV-202 also exhibited significant reductions in waist circumference and BMI (P ≤ 0.03). There was no significant difference in OGTT 0- to 3-hour area under the curve for INV-202 versus placebo: least squares mean 29.38 versus 30.25 h*mmol/L, with an INV-202: placebo ratio of 97.1% (95% confidence interval 90.2, 105.6; P = 0.43). CONCLUSIONS: INV-202 was well tolerated, producing a signal for rapid weight loss with improvements in other metabolic syndrome markers in this population. These findings support further exploration and long-term assessment of cardiometabolic effects.

Differential cannabinoid-like effects and pharmacokinetics of ADB-BICA, ADB-BINACA, ADB-4en-PINACA and MDMB-4en-PINACA in mice: A comparative study.

Despite synthetic cannabinoids' (SCs) prevalent use among humans, these substances often lack comprehensive pharmacological data, primarily due to their rapid emergence in the market. This study aimed to discern differences and causal factors among four SCs (ADB-BICA, ADB-BINACA, ADB-4en-PINACA and MDMB-4en-PINACA), with respect to locomotor activity, body temperature and nociception threshold. Adult male C57BL/6 mice received intraperitoneal injections of varying doses (0.5, 0.1 and 0.02 mg/kg) of these compounds. Three substances (including ADB-BINACA, ADB-4en-PINACA and MDMB-4en-PINACA) demonstrated dose- and time-dependent hypolocomotive and hypothermic effects. Notably, 0.1 mg/kg MDMB-4en-PINACA exhibited analgesic properties. However, ADB-BICA did not cause any effects. MDMB-4en-PINACA manifested the most potent and sustained effects, followed by ADB-4en-PINACA, ADB-BINACA and ADB-BICA. Additionally, the cannabinoid receptor 1 (CB1R) antagonist AM251 suppressed the effects induced by acute administration of the substances. Analysis of molecular binding configurations revealed that the four SCs adopted a congruent C-shaped geometry, with shared linker binding pockets conducive to robust steric interaction with CB1R. Essential residues PHE268 , PHE200 and SER173 within CB1R were identified as pivotal contributors to enhancing receptor-ligand associations. During LC-MS/MS analysis, 0.5 mg/kg MDMB-4en-PINACA exhibited the highest plasma concentration and most prolonged detection window post-administration. The study of SCs' pharmacological and pharmacokinetic profiles is crucial for better understanding the main mechanisms of cannabinoid-like effects induced by SCs, interpreting clinical findings related to SC uses and enhancing SCs risk awareness.

Regulation of the CB1R/AMPK/PGC-1α signaling pathway in mitochondria of mouse hearts and cardiomyocytes with chronic intermittent hypoxia.

PURPOSE: This study evaluated the effects of chronic intermittent hypoxia (CIH) at different times on the mitochondria of mouse hearts and H9C2 cardiomyocytes to determine the role of the cannabinoid receptor 1 (CB1R)/adenosine 5'-monophosphate-activated protein kinase (AMPK)/peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) signaling pathway. METHODS: Animal and cellular CIH models were prepared in an intermittent hypoxia chamber at different times. The cardiac function of mice was determined, and heart tissue and ultrastructural changes were observed. Apoptosis, reactive oxygen species (ROS), and mitochondrial membrane potential were detected, and MitoTracker™ staining was performed to observe cardiomyocyte mitochondria. Western blot, immunohistochemistry, and cellular immunofluorescence were also performed. RESULTS: In the short-term CIH group, increases in mouse ejection fraction (EF) and heart rate (HR); mitochondrial division; ROS and mitochondrial membrane potential; and the expression levels of CB1R, AMPK, and PGC-1α were observed in vivo and in vitro. In the long-term CIH group, the EF and HR increased, the myocardial injury and mitochondrial damage were more severe, mitochondrial synthesis decreased, the apoptosis percentage and ROS increased, mitochondrial fragmentation increased, membrane potential decreased, CB1R expression increased, and AMPK and PGC-1α expression levels decreased. Targeted blocking of CB1R can increase AMPK and PGC-1α, reduce damage attributed to long-term CIH in mouse hearts and H9C2 cells, and promote mitochondrial synthesis. CONCLUSION: Short-term CIH can directly activate the AMPK/PGC-1α pathway, promote mitochondrial synthesis in cardiomyocytes, and protect cardiac structure and function. Long-term CIH can increase CB1R expression and inhibit the AMPK/PGC-1α pathway, resulting in structural damage, the disturbance of myocardial mitochondria synthesis, and further alterations in the cardiac structure. After targeted blocking of CB1R, levels of AMPK and PGC-1α increased, alleviating damage to the heart and cardiomyocytes caused by long-term CIH.

Multiple cannabinoid signaling cascades powerfully suppress recurrent excitation in the hippocampus.

Recurrent excitatory neural networks are unstable. In the hippocampus, excitatory mossy cells (MCs) receive strong excitatory inputs from dentate granule cells (GCs) and project back onto the proximal dendrites of GCs. By targeting the ipsi- and contralateral dentate gyrus (DG) along the dorsoventral axis of the hippocampus, MCs form an extensive recurrent excitatory circuit (GC-MC-GC) whose dysregulation can promote epilepsy. We recently reported that a physiologically relevant pattern of MC activity induces a robust form of presynaptic long-term potentiation (LTP) of MC-GC transmission which enhances GC output. Left unchecked, this LTP may interfere with DG-dependent learning, like pattern separation-which relies on sparse GC firing-and may even facilitate epileptic activity. Intriguingly, MC axons display uniquely high expression levels of type-1 cannabinoid receptors (CB1Rs), but their role at MC-GC synapses is poorly understood. Using rodent hippocampal slices, we report that constitutively active CB1Rs, presumably via ßγ subunits, selectively inhibited MC inputs onto GCs but not MC inputs onto inhibitory interneurons or CB1R-sensitive inhibitory inputs onto GCs. Tonic CB1R activity also inhibited LTP and GC output. Furthermore, brief endocannabinoid release from GCs dampened MC-GC LTP in two mechanistically distinct ways: during induction via ßγ signaling and before induction via αi/o signaling in a form of presynaptic metaplasticity. Lastly, a single in vivo exposure to exogenous cannabinoids was sufficient to induce this presynaptic metaplasticity. By dampening excitatory transmission and plasticity, tonic and phasic CB1R activity at MC axon terminals may preserve the sparse nature of the DG and protect against runaway excitation.

Potential of CBD Acting on Cannabinoid Receptors CB1 and CB2 in Ischemic Stroke.

Stroke is one of the leading causes of death. It not only affects adult people but also many children. It is estimated that, every year, 15 million people suffer a stroke worldwide. Among them, 5 million people die, while 5 million people are left permanently disabled. In this sense, the research to find new treatments should be accompanied with new therapies to combat neuronal death and to avoid developing cognitive impairment and dementia. Phytocannabinoids are among the compounds that have been used by mankind for the longest period of history. Their beneficial effects such as pain regulation or neuroprotection are widely known and make them possible therapeutic agents with high potential. These compounds bind cannabinoid receptors CB1 and CB2. Unfortunately, the psychoactive side effect has displaced them in the vast majority of areas. Thus, progress in the research and development of new compounds that show efficiency as neuroprotectors without this psychoactive effect is essential. On the one hand, these compounds could selectively bind the CB2 receptor that does not show psychoactive effects and, in glia, has opened new avenues in this field of research, shedding new light on the use of cannabinoid receptors as therapeutic targets to combat neurodegenerative diseases such as Alzheimer's, Parkinson's disease, or stroke. On the other hand, a new possibility lies in the formation of heteromers containing cannabinoid receptors. Heteromers are new functional units that show new properties compared to the individual protomers. Thus, they represent a new possibility that may offer the beneficial effects of cannabinoids devoid of the unwanted psychoactive effect. Nowadays, the approval of a mixture of CBD (cannabidiol) and Δ9-THC (tetrahydrocannabinol) to treat the neuropathic pain and spasticity in multiple sclerosis or purified cannabidiol to combat pediatric epilepsy have opened new therapeutic possibilities in the field of cannabinoids and returned these compounds to the front line of research to treat pathologies as relevant as stroke.

Cannabidiol Protects against the Reinstatement of Oxycodone-Induced Conditioned Place Preference in Adolescent Male but Not Female Rats: The Role of MOR and CB1R.

Cannabidiol (CBD), a phytocannabinoid, appeared to satisfy several criteria for a safe approach to preventing drug-taking behavior, including opioids. However, most successful preclinical and clinical results come from studies in adult males. We examined whether systemic injections of CBD (10 mg/kg, i.p.) during extinction of oxycodone (OXY, 3 mg/kg, i.p.) induced conditioned place preference (CPP) could attenuate the reinstatement of CPP brought about by OXY (1.5 mg/kg, i.p.) priming in adolescent rats of both sexes, and whether this effect is sex dependent. Accordingly, a priming dose of OXY produced reinstatement of the previously extinguished CPP in males and females. In both sexes, this effect was linked to locomotor sensitization that was blunted by CBD pretreatments. However, CBD was able to prevent the reinstatement of OXY-induced CPP only in adolescent males and this outcome was associated with an increased cannabinoid 1 receptor (CB1R) and a decreased mu opioid receptor (MOR) expression in the prefrontal cortex (PFC). The reinstatement of CCP in females was associated with a decreased MOR expression, but no changes were detected in CB1R in the hippocampus (HIP). Moreover, CBD administration during extinction significantly potentialized the reduced MOR expression in the PFC of males and showed a tendency to potentiate the reduced MOR in the HIP of females. Additionally, CBD reversed OXY-induced deficits of recognition memory only in males. These results suggest that CBD could reduce reinstatement to OXY seeking after a period of abstinence in adolescent male but not female rats. However, more investigation is required.

Pharmacological Evaluation of Cannabinoid Receptor Modulators Using GRABeCB2.0 Sensor.

Cannabinoid receptors CB1R and CB2R are G-protein coupled receptors acted upon by endocannabinoids (eCBs), namely 2-arachidonoylglycerol (2-AG) and N-arachidonoyl ethanolamine (AEA), with unique pharmacology and modulate disparate physiological processes. A genetically encoded GPCR activation-based sensor that was developed recently-GRABeCB2.0-has been shown to be capable of monitoring real-time changes in eCB levels in cultured cells and preclinical models. However, its responsiveness to exogenous synthetic cannabinoid agents, particularly antagonists and allosteric modulators, has not been extensively characterized. This current study expands upon the pharmacological characteristics of GRABeCB2.0 to enhance the understanding of fluorescent signal alterations in response to various functionally indiscriminate cannabinoid ligands. The results from this study could enhance the utility of the GRABeCB2.0 sensor for in vitro as well as in vivo studies of cannabinoid action and may aid in the development of novel ligands.

A Comprehensive Exploration of the Multifaceted Neuroprotective Role of Cannabinoids in Alzheimer's Disease across a Decade of Research.

Alzheimer's disease (AD), a progressive neurodegenerative disorder, manifests through dysregulation of brain function and subsequent loss of bodily control, attributed to ß-amyloid plaque deposition and TAU protein hyperphosphorylation and aggregation, leading to neuronal death. Concurrently, similar cannabinoids to the ones derived from Cannabis sativa are present in the endocannabinoid system, acting through receptors CB1R and CB2R and other related receptors such as Trpv-1 and GPR-55, and are being extensively investigated for AD therapy. Given the limited efficacy and adverse effects of current available treatments, alternative approaches are crucial. Therefore, this review aims to identify effective natural and synthetic cannabinoids and elucidate their beneficial actions for AD treatment. PubMed and Scopus databases were queried (2014-2024) using keywords such as "Alzheimer's disease" and "cannabinoids". The majority of natural (Δ9-THC, CBD, AEA, etc.) and synthetic (JWH-133, WIN55,212-2, CP55-940, etc.) cannabinoids included showed promise in improving memory, cognition, and behavioral symptoms, potentially via pathways involving antioxidant effects of selective CB1R agonists (such as the BDNF/TrkB/Akt pathway) and immunomodulatory effects of selective CB2R agonists (TLR4/NF-κB p65 pathway). Combining anticholinesterase properties with a cannabinoid moiety may enhance therapeutic responses, addressing cholinergic deficits of AD brains. Thus, the positive outcomes of the vast majority of studies discussed support further advancing cannabinoids in clinical trials for AD treatment.

Potential Therapeutic Targets to Modulate the Endocannabinoid System in Alzheimer's Disease.

Alzheimer's disease (AD), the most common neurodegenerative disease (NDD), is characterized by chronic neuronal cell death through progressive loss of cognitive function. Amyloid beta (Aß) deposition, neuroinflammation, oxidative stress, and hyperphosphorylated tau proteins are considered the hallmarks of AD pathology. Different therapeutic approaches approved by the Food and Drug Administration can only target a single altered pathway instead of various mechanisms that are involved in AD pathology, resulting in limited symptomatic relief and almost no effect in slowing down the disease progression. Growing evidence on modulating the components of the endocannabinoid system (ECS) proclaimed their neuroprotective effects by reducing neurochemical alterations and preventing cellular dysfunction. Recent studies on AD mouse models have reported that the inhibitors of the fatty acid amide hydrolase (FAAH) and monoacylglycerol (MAGL), hydrolytic enzymes for N-arachidonoyl ethanolamine (AEA) and 2-arachidonoylglycerol (2-AG), respectively, might be promising candidates as therapeutical intervention. The FAAH and MAGL inhibitors alone or in combination seem to produce neuroprotection by reversing cognitive deficits along with Aß-induced neuroinflammation, oxidative responses, and neuronal death, delaying AD progression. Their exact signaling mechanisms need to be elucidated for understanding the brain intrinsic repair mechanism. The aim of this review was to shed light on physiology and pathophysiology of AD and to summarize the experimental data on neuroprotective roles of FAAH and MAGL inhibitors. In this review, we have also included CB1R and CB2R modulators with their diverse roles to modulate ECS mediated responses such as anti-nociceptive, anxiolytic, and anti-inflammatory actions in AD. Future research would provide the directions in understanding the molecular mechanisms and development of new therapeutic interventions for the treatment of AD.

Endocannabinoid signaling in microglia.

Microglia, the innate immune cells of the central nervous system (CNS), execute their sentinel, housekeeping and defense functions through a panoply of genes, receptors and released cytokines, chemokines and neurotrophic factors. Moreover, microglia functions are closely linked to the constant communication with other cell types, among them neurons. Depending on the signaling pathway and type of stimuli involved, the outcome of microglia operation can be neuroprotective or neurodegenerative. Accordingly, microglia are increasingly becoming considered cellular targets for therapeutic intervention. Among signals controlling microglia activity, the endocannabinoid (EC) system has been shown to exert a neuroprotective role in many neurological diseases. Like neurons, microglia express functional EC receptors and can produce and degrade ECs. Interestingly, boosting EC signaling leads to an anti-inflammatory and neuroprotective microglia phenotype. Nonetheless, little evidence is available on the microglia-mediated therapeutic effects of EC compounds. This review focuses on the EC signals acting on the CNS microglia in physiological and pathological conditions, namely on the CB1R, CB2R and TRPV1-mediated regulation of microglia properties. It also provides new evidence, which strengthens the understanding of mechanisms underlying the control of microglia functions by ECs. Given the broad expression of the EC system in glial and neuronal cells, the resulting picture is the need for in vivo studies in transgenic mouse models to dissect the contribution of EC microglia signaling in the neuroprotective effects of EC-derived compounds.

Pharmacologic antagonism of CB1 receptors improves electrophysiological alterations in Purkinje cells exposed to 3-AP.

INTRODUCTION: Although ataxia is associated with cerebellar dysfunction, little is known about the effects of 3-AP exposure on Purkinje cell electrophysiological properties. Here, we evaluated these parameters in cerebellar vermis brain slices. METHODS: Purkinje cells were exposed to artificial cerebrospinal fluid (aCSF) (control) or to 1 mM 3-acetylpyridine (3-AP) in the recording chamber. The effects of a cannabinoid agonist (WIN; 7.5 nmol) and a cannabinoid antagonist (AM; 20 nmol) were evaluated under both conditions. RESULTS: Exposure to 3-AP induced dramatic changes in cellular excitability that likely would affect Purkinje cell output. In whole-cell current clamp recordings, 3-AP-exposed Purkinje cells demonstrated a significantly higher frequency of action potentials, a larger afterhyperpolarization (AHP), and a larger rebound of action potentials. In addition, 3-AP caused a significant decrease in the interspike interval (ISI), half-width, and first spike latency. Remarkably, the action potential frequency, AHP amplitude, rebound, ISI, action potential halfwidth, and first spike latency were no longer different from controls in 3-AP cells treated with AM. Sag percentage, on the other hand, showed no significant difference under any treatment condition, indicating that cannabinoids' actions on 3-AP-mediated Purkinje cell changes may not include effects on neuronal excitability through changes of Ih. CONCLUSIONS: These data show that cannabinoid antagonists reduce the excitability of Purkinje cells following exposure to 3-AP and suggest their potential as therapeutics in cerebellar dysfunctions.

Focusing on the 5F-MDMB-PICA, 4F-MDMB-BICA synthetic cannabinoids and their primary metabolites in analytical and pharmacological aspects.

Nowadays, more and more new synthetic cannabinoids (SCs) appearing on the illicit market present challenges to analytical, forensic, and toxicology experts. For a better understanding of the physiological effect of SCs, the key issue is studying their metabolomic and psychoactive properties. In this study, our validated targeted reversed phase UHPLC-MS/MS method was used for determination of urinary concentration of 5F-MDMB-PICA, 4F-MDMB-BICA, and their primary metabolites. The liquid-liquid extraction procedure was applied for the enrichment of SCs. The pharmacological characterization of investigated SCs were studied by radioligand competition binding and ligand stimulated [35S]GTPγS binding assays. For 5F-MDMB-PICA and 4F-MDMB-BICA, the median urinary concentrations were 0.076 and 0.312 ng/mL. For primary metabolites, the concentration range was 0.029-881.02* ng/mL for 5F-MDMB-PICA-COOH, and 0.396-4579* ng/mL for 4F-MDMB-BICA-COOH. In the polydrug aspect, the 22 urine samples were verified to be abused with 6 illicit drugs. The affinity of the metabolites to CB1R significantly decreased compared to the parent ligands. In the GTPγS functional assay, both 5F-MDMB-PICA and 4F-MDMB-BICA were acting as full agonists, while the metabolites were found as weak inverse agonists. Additionally, the G-protein stimulatory effects of the full agonist 5F-MDMB-PICA and 4F-MDMB-BICA were reduced by metabolites. These results strongly indicate the dose-dependent CB1R-mediated weak inverse agonist effects of the two butanoic acid metabolites. The obtained high concentration of main urinary metabolites of 5F-MDMB-PICA and 4F-MDMB-BICA confirmed the relevance of their routine analysis in forensic and toxicological practices. Based on in vitro binding assays, the metabolites presumably might cause a lower psychoactive effect than parent compounds.

Blockade of endocannabinoid system by oxytocin attenuates memory deficits in oxycodone-treated rats.

Oxycodone is a highly prescribed opioid and its abuse has been rampant. Accumulating evidence shows that the cannabinoid CB1 receptor (CB1R) plays a key role in mediating rewarding effects to opioids. However, the downstream signalling of CB1R induced by oxycodone remains unclear. The neuropeptide oxytocin is well known as a potential remedy for drug addiction. Thus, our study aims to explore the mechanism of oxycodone-induced learning and memory deficits underlying the endocannabinoid system (ECS) and the effect of oxytocin. Rats were intraperitoneally injected with oxycodone once a day for eight consecutive day. Novel object recognition, resident-intruder and Morris Water Maze tests were employed to assess the cognitive, social and spatial memory of the rats after oxycodone withdrawal. The (co-)expression of CB1R, cyclin-dependent kinase 5 (Cdk5), regulatory protein p25, tau and phosphorylated tau was measured 1 day after the last behavioural test. The histopathological staining and synaptic density in the hippocampus were observed as well. We found that oxycodone upregulated the expression of p-GSK3ß, co-expression of p-Cdk5 and p25 through CB1R. This finding was accompanied by elevation of pSer396, pSer404 in the tau, and reduction of the number of neurons, dendritic spines and synaptic density in the hippocampus. Furthermore, i.c.v. treatment with oxytocin ameliorates memory deficits in oxycodone-treated rats through inhibition of the ECS. We propose further studies on the clinical use of this neuropeptide, which may potentially cure drug addiction.

Imaging and Genetic Tools for the Investigation of the Endocannabinoid System in the CNS.

As central nervous system (CNS)-related disorders present an increasing cause of global morbidity, mortality, and high pressure on our healthcare system, there is an urgent need for new insights and treatment options. The endocannabinoid system (ECS) is a critical network of endogenous compounds, receptors, and enzymes that contribute to CNS development and regulation. Given its multifaceted involvement in neurobiology and its significance in various CNS disorders, the ECS as a whole is considered a promising therapeutic target. Despite significant advances in our understanding of the ECS's role in the CNS, its complex architecture and extensive crosstalk with other biological systems present challenges for research and clinical advancements. To bridge these knowledge gaps and unlock the full therapeutic potential of ECS interventions in CNS-related disorders, a plethora of molecular-genetic tools have been developed in recent years. Here, we review some of the most impactful tools for investigating the neurological aspects of the ECS. We first provide a brief introduction to the ECS components, including cannabinoid receptors, endocannabinoids, and metabolic enzymes, emphasizing their complexity. This is followed by an exploration of cutting-edge imaging tools and genetic models aimed at elucidating the roles of these principal ECS components. Special emphasis is placed on their relevance in the context of CNS and its associated disorders.

Cannabinoids and Their Receptors in Skin Diseases.

The therapeutic application of cannabinoids has gained traction in recent years. Cannabinoids interact with the human endocannabinoid system in the skin. A large body of research indicates that cannabinoids could hold promise for the treatment of eczema, psoriasis, acne, pruritus, hair disorders, and skin cancer. However, most of the available data are at the preclinical stage. Comprehensive, large-scale, randomized, controlled clinical trials have not yet been fully conducted. In this article, we describe new findings in cannabinoid research and point out promising future research areas.

A Robust and Efficient FRET-Based Assay for Cannabinoid Receptor Ligands Discovery.

The identification of new modulators for Cannabinoid Receptors (CBRs) has garnered significant attention in drug discovery over recent years, owing to their manifold pathophysiological implications. In the context of hit identification, the availability of robust and sensitive high-throughput screening assays is essential to enhance the likelihood of success. In this study, we present the development and validation of a Tag-lite® binding assay designed for screening hCB1/hCB2 binding, employing a dual fluorescent ligand, CELT-335. Representative ligands for CBRs, exhibiting diverse affinity and functional profiles, were utilized as reference compounds to validate the robustness and efficiency of the newly developed Tag-lite® binding assay protocol. The homogeneous format, coupled with the sensitivity and optimal performance of the fluorescent ligand CELT-335, establishes this assay as a viable and reliable method for screening in hit and lead identification campaigns.