Cannabinoid CB1 Receptor Research Articles

Investigating the Protective Effects of Luteolin and Gallic Acid from Luffa acutangula L. (Roxb.) amara (Roxb.) C. B. Clarke. Fruit Pericarp Against Alcohol-Induced Liver Toxicity: Extraction, Bioactivity-Guided Fractionation, Molecular Docking, and Dynamics Studies.

Luffa acutangula var. amara (Roxb.) C.B. Clarke, known as 'Katu Koshataki' in Ayurveda, is a traditional medicinal plant in India. The juice of these fruits is recommended for chronic alcohol-induced liver diseases. This study aimed to validate the hepatoprotective and antioxidant properties of Luffa acutangula var. amara fruit pericarp ethanolic extract (EOLA) against ethanol-induced chronic hepatotoxicity in rats, isolate bioactive phytochemicals and in silico evaluation. Ethanolic extract of L. acutangula var. amara fruit pericarp was evaluated in chronic alcohol-induced hepatitis (20% ethanol orally twice daily for 28 days) at doses of 100 and 200 mg/kg p.o. Additionally, in vivo anti-inflammatory and antioxidant potential were assessed. Luteolin and gallic acid were isolated using bioactivity guided fractionation. Furthermore, these phytochemicals were assessed for hepatoprotective potential using molecular docking and molecular dynamics studies against different targets. The EOLA demonstrated efficacy in stabilizing elevated biochemical markers and antioxidant levels associated with ethanol-induced hepatotoxicity in rats, and it preserved the normal architecture of hepatocytes in histological analyses compared to the ethanol control group. The EOLA was subjected to fractionation using hexane: chloroform (8:2→2:8), followed by chloroform: methanol (8:2→2:8) to yield a total eight fractions. The fractions 1, 2, and 3 showed potent antioxidant potential, yielding luteolin and gallic acid as LAC-1 and LAC-3 from fractions 1 and 8, respectively. Gallic acid and luteolin were investigated using molecular docking and molecular dynamic simulation techniques to assess their interactions with key proteins such as Cytochrome P450 2C9, Superoxide Dismutase, Glutathione Peroxidase, Glutathione S-Transferase, Catalase, Peroxisome Proliferator-Activated Receptor-ϒ, Vanin-1, and Cannabinoid Receptor CB2. The metabolites exhibit strong binding stability, with a root mean square deviation range of less than 2.8Å. It can be concluded that the fruit pericarp of L. acutangula var. amara could be used as a functional food for reducing the liver damage caused by alcohol and other factors.

Role of peroxisome proliferator-activated receptors α and γ in mediating the beneficial effects of β-caryophyllene in a rat model of fragile X syndrome.

β-Caryophyllene (BCP) is a naturally occurring sesquiterpene found in numerous plant species, including Cannabis sativa. BCP has shown a high safety profile and a wide range of biological functions, including beneficial effects in neurodegenerative and inflammatory diseases. Here, we used behavioral, pharmacological, and in-silico docking analyses to investigate the effects and mechanism of action of BCP in Fragile X Syndrome (FXS), the most common inherited cause of Autism Spectrum Disorder (ASD) and intellectual disability. To this aim, we used the recently validated Fmr1-Δexon 8 rat model of FXS, that is also a genetic rat model of ASD. Acute and repeated oral administration of BCP rescued the cognitive deficits displayed by Fmr1-Δexon 8 rats, without inducing tolerance after repeated administration. These beneficial effects were mediated by activation of hippocampal peroxisome proliferator-activated receptors (PPARs) α and γ, and were mimicked by the PPARα agonist Fenofibrate and the PPARγ agonist Pioglitazone. Conversely, CB2 cannabinoid receptors were not involved. Docking analyses further confirmed the ability of BCP to bind rat PPARs. Together, our findings demonstrate that hippocampal PPARs α and γ play a role in the cognitive deficits observed in a rat model of FXS, and provide first preclinical evidence about the efficacy and mechanism of action of BCP in neurodevelopmental disorders.

INTERACTION BETWEEN THE DOPAMINERGIC AND ENDOCANNABINOID SYSTEMS PROMOTES PERIPHERAL ANTINOCICEPTION.

Dopamine has been widely related to pain modulation, at central and peripheral levels. In this study we aimed to investigate the mechanisms involved in peripheral antinociception, evaluating the interaction between the dopaminergic and endocannabinoid systems in this event. Male Swiss mice (30-40 g) were pre-sensitized by administration of the hyperalgesic PGE2 (2 μg/paw). The nociceptive threshold was measured using the paw withdrawal test. Dopamine (80 ng/paw) promoted antinociception. This effect was reversed by the CB1 and CB2 cannabinoid receptor antagonists AM251 (20, 40, and 80 μg/paw) and AM630 (25, 50, and 100 μg/paw). JZL (4 μg/paw), an inhibitor of the degradation of the 2-arachidonylglycerol (2-AG), potentiated the antinociceptive action of the submaximal dose of dopamine (5 ng/paw). While anandamide degradation and reuptake inhibitors (MAFP 0.5 μg/paw and VDM11 2.5 μg/paw) did not promote changes in intermediate antinociception induced by dopamine. Anandamide at a submaximal dose (12.5 ng/paw) promoted intermediate antinociception that was not potentiated by the administration of the dopamine reuptake inhibitor GBR 12783 (16 μg/paw). In contrast, the administration of GBR potentiated the intermediate antinociception induced by a submaximal dose of 2-AG (10 μg/paw). Furthermore, the dopaminergic receptor antagonists D2 Remoxipride (4 μg/paw) and D3 U99194 (16 μg/paw) reversed the antinociception mediated by the maximum dose of this endocannabinoid (20 μg/paw). In contrast, the D4 receptor antagonist L-745,870 (16 μg/paw) did not change the nociceptive threshold. In this way, we demonstrate the interaction between the dopaminergic and endocannabinoid systems to promote analgesia peripherally.

Selective blockade of cannabinoid receptors influence motoneuron survival and glial reaction after neonatal axotomy

Sciatic nerve crush in neonatal rats leads to an extensive death of motor and sensory neurons, serving as a platform to develop new neuroprotective approaches. The endocannabinoid system plays important neuromodulatory roles and has been involved in neurodevelopment and neuroprotection. The present work investigated the role of the cannabinoid receptors CB1 and CB2 in the neuroprotective response after neonatal axotomy. CB1 and CB2 antagonists (AM251 and AM630, respectively) were used after sciatic nerve crush in 2-day-old Wistar rats. Five days after lesion and treatment, the rats were perfused, and the spinal cords and dorsal root ganglia (DRG) were obtained and processed to investigate neuronal survival and immunohistochemistry changes, or RT-qPCR analysis. Motoneuron survival analysis showed that blocking CB2 alone or in combination with CB1 was neuroprotective. This effect was associated with a decrease in astrogliosis and microglial reaction. Interestingly, Cnr1 (CB1) and Bdnf gene transcripts were downregulated in the spinal cords of the antagonist-treated groups. Despite no intergroup difference regarding neuronal survival in the DRG, the simultaneous blockade of CB1 and CB2 receptors led to an increased expression of both Cnr1 and Cnr2, combined with Gdnf upregulation. The results indicate that the selective antagonism of cannabinoid receptors facilitates neuroprotection and decreases glial reactivity, suggesting new potential treatment approaches.

Design of Small Non-Peptidic Ligands That Alter Heteromerization between Cannabinoid CB1 and Serotonin 5HT2A Receptors.

Activation of cannabinoid CB1 receptors (CB1R) by agonists induces analgesia but also induces cognitive impairment through the heteromer formed between CB1R and the serotonin 5HT2A receptor (5HT2AR). This side effect poses a serious drawback in the therapeutic use of cannabis for pain alleviation. Peptides designed from the transmembrane helices of CB1R, which are predicted to bind 5HT2AR and alter the stability of the CB1R-5HT2AR heteromer, have been shown to avert CB1R agonist-induced cognitive impairment while preserving analgesia. Using these peptides as templates, we have now designed nonpeptidic small molecules that prevent CB1R-5HT2AR heteromerization in bimolecular fluorescence complementation assays and the heteromerization-dependent allosteric modulations in cell signaling experiments. These results provide proof-of-principle for the design of optimized ligand-based disruptors of the CB1R-5HT2AR heteromer, opening new perspectives for in vivo studies.

Role of the Endocannabinoid System in Gonadal Development: Implications for Endocrine Disruption and Reproductive Toxicity.

The endocannabinoid system (ECS) plays a pivotal role in reproductive physiology, including gonadal development, though its influence on testis and ovary development has only recently gained attention. The ECS comprises lipid-derived ligands such as anandamide (AEA) and 2-arachidonoylglycerol (2-AG), along with cannabinoid receptors CB1 and CB2, which are expressed in various gonadal cells. Emerging research indicates that ECS signaling is critical for testosterone synthesis and gonadal cell proliferation and differentiation. This review explores the expression and function of ECS components in developing gonads, highlighting the differential roles of CB1 and CB2 receptors in species-specific contexts. Furthermore, the ECS has been suggested to be involved in the adverse effects of endocrine-disrupting chemicals (EDCs) on reproductive development. EDCs, such as phthalates, may interfere with ECS signaling, potentially leading to reproductive abnormalities that resemble the human Testicular Dysgenesis Syndrome (TDS). Understanding the molecular interactions between EDCs and the ECS could reveal novel mechanisms underlying reproductive toxicities. Future research should focus on the detailed localization and temporal expression of ECS components in fetal gonads, the mechanisms of cannabinoid-mediated testosterone inhibition, and the potential direct interaction of EDCs with the ECS. This knowledge could be crucial for developing strategies to mitigate reproductive health risks associated with EDC exposure.

Impact of Omega-3 on Endocannabinoid System Expression and Function, Enhancing Cognition and Behavior in Male Mice.

Background/Objectives: Omega-3 long-chain polyunsaturated fatty acids (PUFAs) support brain cell membrane integrity and help mitigate synaptic plasticity deficits. The endocannabinoid system (ECS) is integral to synaptic plasticity and regulates various brain functions. While PUFAs influence the ECS, the effects of omega-3 on the ECS, cognition, and behavior in a healthy brain remain unclear. Methods and Results: Here, we demonstrate that hippocampal synaptosomes from male mice fed an omega-3-rich diet exhibit increased levels of cannabinoid CB1 receptors (~30%), phospholipase C β1 (PLCβ1, ~30%), monoacylglycerol lipase (MAGL, ~30%), and cannabinoid receptor-interacting protein 1a (Crip1a, ~60%). Conversely, these synaptosomes show decreased levels of diacylglycerol lipase α (DAGLα, ~40%), synaptosomal-associated protein 25kDa (SNAP-25, ~30%), and postsynaptic density protein 95 (PSD-95, ~40%). Omega-3 intake also reduces Gαo and Gαi3 levels, though receptor-stimulated [35S]GTPγS binding remains unaffected. Stimulation of the medial perforant path (MPP) induced long-term potentiation (LTP) in omega-3-fed mice. This LTP was dependent on group I metabotropic glutamate receptors (mGluR), 2 arachidonoylglycerol (2-AG), CB1 receptors, N-type Ca2+ channels, and actin filaments. Behaviorally, omega-3-fed mice displayed reduced exploratory behavior and significantly improved object discrimination in the novel object recognition test (NORT). They also spent more time in open arms and exhibited reduced freezing time in the elevated plus maze (EPM), indicative of reduced anxiety-like behavior. Conclusions: Our findings suggest that omega-3 leverages the ECS to enhance brain function under normal conditions.

The role of the endocannabinoid system in the pathogenesis and treatment of epilepsy.

Epilepsy is a group of chronic neurological brain disorders characterized by recurrent spontaneous unprovoked seizures, which are accompanied by significant neurobiological, cognitive, and psychosocial impairments. With a global prevalence of approximately 0.5-1 % of thepopulation, epilepsy remains a serious public health concern. Despite the development and widespread use of over 20 anticonvulsant drugs, around 30 % of patients continue to experience drug-resistant seizures, leading to asubstantial reduction in quality of life and increased mortality risk. Given the limited efficacy of current treatments, exploring new therapeutic approaches is critically important. In recent years, Gi-protein-coupled receptors, particularly cannabinoid receptors CB1 and CB2, have garnered increasing attention as promising targets for the treatment seizures and prevention of epilepsy. Emerging evidence suggests a significant role of the cannabinoid system in modulating neuronal activity and protecting against hyperexcitability, underscoring the importance of further research inthis area. This review provides up-to-date insights into the pathogenesis and treatment of epilepsy, with a special focus on the role of the cannabinoid system, highlighting the need for continued investigation to develop more effective therapeutic strategies.

A Sex-Dependent Cannabinoid CB1 Receptor Role in Circadian Tearing of the Mouse.

We have shown that cannabinoid CB1 receptors regulate both salivation and tearing, but for tearing, this regulation is sex dependent with opposing effects by sex. We investigated a potential interplay of circadian and cannabinoid regulation of tearing. We measured cannabinoid and circadian regulation of tearing in CD1 strain mice as well as CB1 receptor protein expression using immunohistochemistry. We now report that CD1 strain mice have a circadian variation in basal tearing, differing by sex in terms of phase and amplitude. The amplitude of circadian variation in females is substantially dampened relative to males. Male CB1 receptor knockout mice do not differ from strain controls, but in female CB1 knockouts, the amplitude is enhanced and resembles that of WT males. This increased tearing is mimicked by the CB1 antagonist SR141716 (4 mg/kg, intraperitoneally [IP]), suggesting that tonic CB1 activation dampens female circadian tearing. Consistent with this, the cannabinoid receptor agonist CP55940 (0.5 mg/kg, IP) decreases tearing during the rest phase but increases tearing during the active phase in females. CB1 protein expression also differs by sex. While both males and females have CB1 receptors in parasympathetic inputs to the lacrimal gland, in female lacrimal glands, CB1 is also detected in myoepithethial cells. Mice have a sex-dependent circadian cycle of tearing. The endogenous cannabinoid signaling system appears to mediate some circadian effects, albeit in a sex-dependent manner and via distinct cellular targets.

Basal forebrain-lateral habenula inputs and control of impulsive behavior.

Deficits in impulse control are observed in several neurocognitive disorders, including attention deficit hyperactivity (ADHD), substance use disorders (SUDs), and those following traumatic brain injury (TBI). Understanding brain circuits and mechanisms contributing to impulsive behavior may aid in identifying therapeutic interventions. We previously reported that intact lateral habenula (LHb) function is necessary to limit impulsivity defined by impaired response inhibition in rats. Here, we examine the involvement of a synaptic input to the LHb on response inhibition using cellular, circuit, and behavioral approaches. Retrograde fluorogold tracing identified basal forebrain (BF) inputs to LHb, primarily arising from ventral pallidum and nucleus accumbens shell (VP/NAcs). Glutamic acid decarboxylase and cannabinoid CB1 receptor (CB1R) mRNAs colocalized with fluorogold, suggesting a cannabinoid modulated GABAergic pathway. Optogenetic activation of these axons strongly inhibited LHb neuron action potentials and GABA release was tonically suppressed by an endogenous cannabinoid in vitro. Behavioral experiments showed that response inhibition during signaled reward omission was impaired when VP/NAcs inputs to LHb were optogenetically stimulated, whereas inhibition of this pathway did not alter LHb control of impulsivity. Systemic injection with the psychotropic phytocannabinoid, Δ9-tetrahydrocannabinol (Δ9-THC), also increased impulsivity in male, and not female rats, and this was blocked by LHb CB1R antagonism. However, as optogenetic VP/NAcs pathway inhibition did not alter impulse control, we conclude that the pro-impulsive effects of Δ9-THC likely do not occur via inhibition of this afferent. These results identify an inhibitory LHb afferent that is controlled by CB1Rs that can regulate impulsive behavior.

PLAAT5 as an N-acyltransferase responsible for the generation of anti-inflammatory N-acylethanolamines in testis

N-Acylethanolamines (NAEs) are a class of lipid mediators that exhibit anti-inflammatory and appetite-suppressive activities. Among them, palmitoylethanolamide (PEA) and arachidonoylethanolamide (AEA) bind to peroxisomal proliferator-activated receptor (PPAR) α and cannabinoid receptor CB1, respectively. N-Acyl-phosphatidylethanolamine (NAPE) as a precursor of NAEs is biosynthesized from membrane phospholipids by N-acyltransferases, which consist of group IVE cytosolic phospholipase A2ε (cPLA2ε) and PLAAT (phospholipase A and acyltransferase) family enzymes. While cPLA2ε is responsible for the production of NAEs not only in specific tissues, including muscle, skin, and the stomach, but also under pathological conditions, such as psoriasis and brain ischemia, the involvement of the PLAAT family in vivo remains unclear. Considering the specific expression of PLAAT5 in testes, we investigated the potential role of PLAAT5 in the formation of NAEs in testes using PLAAT5-deficient (Plaat5−/−) mice. High-performance liquid chromatography coupled with tandem mass spectrometry showed that PLAAT5 deficiency decreased the total level of NAEs by 61 %, with PEA and AEA being reduced by 64 % and 87 %, respectively. Following a treatment with cadmium chloride, an environmental toxin that induces testicular inflammation, the expression of inflammatory genes (Il6, Tnf, and Nos2) in testes was markedly higher in Plaat5−/− mice than in Plaat5+/+ mice, and their expression was attenuated by the administration of PEA and AEA. Furthermore, these anti-inflammatory effects were canceled by a co-treatment with the antagonists of PPARα or CB1. These results suggest that PLAAT5 is responsible for the biosynthesis of anti-inflammatory NAEs in testes.

The synthetic cannabinoid agonist WIN 55,212-2 reduces experimental pruritus via CB2 receptor activation

Pruritus (i.e., the experience that evokes a desire to scratch) is an adaptive process that can become maladaptive, leading to a persistent scratch-itch cycle that potentiates pruritus and increases the risk of infection. Cannabinoid drugs have been reported to decrease pruritus, but often at doses that also decrease locomotor activity, which confounds assessments of utility. To determine the utility of cannabinoids in treating pruritus without undesirable adverse effects, the current preclinical study investigated a range of doses of the synthetic cannabinoid agonist, WIN 55,212-2, and two minor Cannabis phytoconstituents, Δ8-tetrahydrocannabinol and β-caryophyllene, in experimentally induced pruritus in male and female C57BL/6J adult mice. WIN 55,212-2 reduced compound 48/80-induced scratching, and this antipruritic effect was prevented by either chemically blocking (via SR144528 antagonism) or genetically deleting the CB2 cannabinoid receptor. The CB2 receptor selective agonist, JWH-133, also attenuated compound 48/80-induced scratching, while the CB1 positive allosteric modulator, ZCZ011, had no effect. Similarly, the minor phytocannabinoid Δ8-tetrahydrocannabinol reduced scratching at doses that did not affect locomotor activity. In contrast, the sesquiterpene cannabis constituent β-caryophyllene induced scratching, acting as a pruritogen. These preclinical data support the continuing investigation of cannabinoid receptor modulation as a potential therapeutic strategy for pruritus.

Possible Mechanism for Perception of Auditory-Verbal Hallucinations in Schizophrenia and Approaches to Their Weakening

We put forward a hypothesis that the processing and perception of auditory-verbal hallucinations in schizophrenia — internally generated speech, occurs in the same neural circuits as speech from external sources. These topographically organized cortico—basal ganglia—thalamocortical neural circuits include auditory, language, and frontal neocortical areas. It follows from our proposed mechanism for sound processing, that the increased action on dopamine D2 receptors on striatal neurons, which is considered the cause of hallucinations, should lead to a determined reorganization of activity in these neural circuits. As a result of this reorganization, the inhibition of neurons in the thalamic nuclei, including the internal geniculate body, should decrease synergistically through the direct and indirect pathways in the basal ganglia, and the excitation of connected with them cortical neurons, in the activity of which speech is represented, should increase. From this mechanism it follows that in order to weaken the perception of auditory-verbal hallucinations, it is necessary to increase the inhibition of thalamic neurons. Taking into account the known data on the distribution of receptors of different types on neurons in the striatum, thalamus and neocortex, as well as the previously formulated unified modification rules for the effectiveness of synaptic transmission in different structures, we proposed that agonists of adenosine A1 and muscarinic M4 receptors located on striatonigral spiny cells, giving rise to a direct disinhibitory pathway through the basal ganglia as well as antagonists of delta-opioid and cannabinoid CB1 receptors, located on striatopallidal spiny cells, giving rise to an indirect inhibitory pathway through the basal ganglia may be useful to weaken the perception of hallucinations. In addition, activation of A1 receptors can directly weaken the activity of neurons in the thalamus and neocortex due to the induction of long-term depression in the efficiency of their excitation. Inactivation of cannabinoid CB1 receptors on the projection GABAergic cells of the reticular thalamic nucleus may enhance their inhibitory effect on neurons of different thalamic nuclei. Since the proposed substances only indirectly affect the dopaminergic system, their use should not cause such pronounced side effects as D2 receptor antagonists, antipsychotics that are widely used to suppress auditory-verbal hallucinations.

Cannabinoid-like compounds found in non-cannabis plants exhibit antiseizure activity in genetic mouse models of drug-resistant epilepsy.

The cannabinoid cannabidiol has established antiseizure effects in drug-resistant epilepsies such as Dravet syndrome and Lennox-Gastaut syndrome. Amorfrutin 2, honokiol, and magnolol are structurally similar to cannabinoids (cannabis-like drugs) but derive from non-cannabis plants. We aimed to study the antiseizure potential of these compounds in various mouse seizure models. In addition, we aimed to characterize their molecular pharmacology at cannabinoid CB1 and CB2 receptors and at T-type calcium channels, which are known targets of the cannabinoids. Brain and plasma pharmacokinetic profiles were determined. Antiseizure activity was assessed against hyperthermia-induced seizures in a Scn1a+/- mouse model of Dravet syndrome. We then elaborated on the most promising compounds in the maximal electroshock (MES) test in mice and the Gabrb3+/D120N mouse model of Lennox-Gastaut syndrome. Fluorescence-based assays were used to examine modulatory activity at cannabinoid CB1 and CB2 receptors and T-type calcium channel subtypes CaV3.1, CaV3.2, and CaV3.3 overexpressed in mammalian cells. Automated patch-clamp electrophysiology was then used to confirm inhibitory activity on CaV3.1, CaV3.2, and CaV3.3 channels. Magnolol and honokiol had high brain-to-plasma ratios (3.55 and 7.56, respectively), unlike amorfrutin 2 (0.06). Amorfrutin 2 and magnolol but not honokiol significantly increased the body temperature threshold at which Scn1a+/- mice had a generalized tonic-clonic seizure. Both amorfrutin 2 and magnolol significantly decreased the proportion of mice exhibiting hindlimb extension in the MES test. Furthermore, magnolol reduced the number and duration of atypical absence seizures in Gabrb3+/D120N mice. The three compounds inhibited all T-type calcium channel subtypes but were without specific activity at cannabinoid receptors. We show for the first time that amorfrutin 2 and magnolol display novel antiseizure activity in mouse drug-resistant epilepsy models. Our results justify future drug discovery campaigns around these structural scaffolds that aim to develop novel antiseizure drugs for intractable epilepsies.

A sleepy cannabis constituent: cannabinol and its active metabolite influence sleep architecture in rats

Medicinal cannabis is being used worldwide and there is increasing use of novel cannabis products in the community. Cannabis contains the major cannabinoids, Δ9-tetrahydrocannabinol (Δ9-THC) and cannabidiol (CBD), but also an array of minor cannabinoids that have undergone much less pharmacological characterization. Cannabinol (CBN) is a minor cannabinoid used in the community in “isolate’ products and is claimed to have pro-sleep effects comparable to conventional sleep medications. However, no study has yet examined whether it impacts sleep architecture using objective sleep measures. The effects of CBN on sleep in rats using polysomnography were therefore examined. CBN increased total sleep time, although there was evidence of biphasic effects with initial sleep suppression before a dramatic increase in sleep. CBN increased both non-rapid eye movement (NREM) and rapid eye movement (REM) sleep. The magnitude of the effect of CBN on NREM was comparable to the sleep aid zolpidem, although, unlike CBN, zolpidem did not influence REM sleep. Following CBN dosing, 11-hydroxy-CBN, a primary metabolite of CBN surprisingly attained equivalently high brain concentrations to CBN. 11-hydroxy-CBN was active at cannabinoid CB1 receptors with comparable potency and efficacy to Δ9-THC, however, CBN had much lower activity. We then discovered that the metabolite 11-hydroxy-CBN also influenced sleep architecture, albeit with some subtle differences from CBN itself. This study shows CBN affects sleep using objective sleep measures and suggests an active metabolite may contribute to its hypnotic action.

A combination of peripherally restricted CB1 and CB2 cannabinoid receptor agonists reduces bladder afferent sensitisation in cystitis

Cannabinoid agonists can potentially ameliorate lower urinary tract symptoms (LUTS), including pain associated with interstitial cystitis/bladder pain syndrome (IC/BPS). This study aims to determine the contributions of the cannabinoid 1 receptors (CB1Rs) and CB2Rs in regulating the activity of different functional classes of afferents, comparing normal healthy bladder with bladders from guinea pigs with protamine/zymosan-induced cystitis. The mechanosensitivity of different functional afferent classes was determined by ex vivo single-unit extracellular recordings.Peripherally restricted CB1R preferential agonists, ACEA and PrNMI and peripherally restricted CB2R selective agonists, 4Q3C and olorinab all reduced the mechanosensitivity of mucosal bladder afferents. The potency and efficacy of these synthetic cannabinoid agonists were significantly increased in cystitis compared to controls. Combined application of CB1R agonists, ACEA or PrNMI with the CB2R agonist, 4Q3C produced additive inhibitory effects. ACEA and PrNMI also inhibited the stretch-induced firing of high-threshold muscular bladder afferents in animals with cystitis. In contrast, low- and high-threshold muscular-mucosal bladder afferents were unaffected by the CB1R and CB2R agonists in control and cystitis. Our data indicated that peripherally restricted CB1R and CB2R agonists effectively reduce the sensitisation of probable nociceptive afferents in the bladder in cystitis. The findings also suggest a potential benefit of simultaneously targeting both the CB1Rs and CB2Rs to ameliorate LUTS in cystitis.

CB1 receptors in NG2 cells mediate cannabinoid-evoked functional myelin regeneration

Defects in myelin homeostasis have been reported in many neuropathological conditions. Cannabinoid compounds have been shown to efficiently promote myelin regeneration in animal models of demyelination. However, it is still unknown whether this action relies mostly on a cell autonomous effect on oligodendroglial-lineage-NG2 cells. By using conditional genetic mouse models, here we found that cannabinoid CB1 receptors located on NG2 cells are required for oligodendroglial differentiation and myelin regeneration after demyelination. Selective CB1 receptor gene depletion in NG2 cells following toxin-induced demyelination disrupted oligodendrocyte regeneration and functional remyelination and exacerbated axonal damage. These deficits were rescued by pharmacological blockade of the RhoA/ROCK/Cofilin pathway. Conversely, tetrahydrocannabinol administration promoted oligodendrocyte regeneration and functional remyelination in wild-type but not Ng2-CB1-deficient mice. Overall, this study identifies CB1 receptors as essential modulators of remyelination and support the therapeutic potential of cannabinoids for promoting remyelination in neurological disorders.

Nerve injury inhibits Oprd1 and Cnr1 transcription through REST in primary sensory neurons

The transcription repressor REST in the dorsal root ganglion (DRG) is upregulated by peripheral nerve injury and promotes the development of chronic pain. However, the genes targeted by REST in neuropathic pain development remain unclear. The expression levels of four opioid receptor genes (Oprm1, Oprd1, Oprl1 and Oprk1) and the cannabinoid CB1 receptor (Cnr1) gene in the DRG regulate nociception. In this study, we determined the role of REST in controlling their expression in the DRG induced by spared nerve injury (SNI). SNI induced chronic pain hypersensitivity in wild-type mice and was accompanied by increased levels of Rest transcript and protein. Transcriptomic analyses of wild-type mouse DRGs suggested that SNI upregulates the expression of Rest transcripts and downregulates the transcripts of all four opioid receptor genes and the Cnr1 gene. Quantitative reverse transcription polymerase chain reaction analyses of these tissues validated these results. Analysis of publicly available bioinformatic data suggested that REST binds to the promoter regions of Oprm1 and Cnr1. Chromatin immunoprecipitation analyses indicated the presence of REST at these promoters. Full-length Rest conditional knockout in primary sensory neurons reduced SNI-induced pain hypersensitivity and rescued the SNI-induced reduction in the expression of Oprd1 and Cnr1 in mouse DRG. Our results suggest that nerve injury represses the transcription of at least the Oprd1 and Cnr1 genes via REST in primary sensory neurons and that REST is a potential therapeutic target for neuropathic pain. Thus, inhibiting REST activity could potentially reduce chronic neuropathic pain and augment opioid/cannabinoid analgesic actions by increasing the transcription of Oprd1 and Cnr1 genes in DRG neurons.

Cannabinoid regulation of sex-dependent murine odorant-stimulated salivation

Salivation is easily taken for granted, but without normal salivation, simple essential tasks such as chewing and swallowing become difficult, with consequences for quality of life, nutrition and oral health. Many important drug classes cause dry mouth as a side effect, contributing substantially to patient non-compliance. Available treatments are mostly palliative. Cannabis user complaints of dry mouth prompted a study that showed that basal salivation is likely regulated by cannabinoid CB1 receptors on neurons that innervate the submandibular gland. But what about stimulated salivation? The adjoining parotid gland releases saliva in response to olfactory or other cues and contributes a large portion of the net salivation in humans. We investigated cannabinoid regulation of stimulated salivation using functional and protein-expression studies in mice. In developing a model of stimulated salivary responses to food-related odorants in mice, we noted sex-dependent responses to food-related cues. Only male mice learned to salivate in response to the odor of peanut butter while only female mice responded to a chocolate hazelnut spread. Both males and females responded to sugar or marmite. Testing peanut butter, we found that the cannabinoid receptor agonist CP55940 (0.5 mg/kg, IP) lowered baseline salivation, as shown previously, but also prevented the odorant-induced increase in salivation. CB1 receptors are expressed in axons innervating the parotid gland, paralleling our findings in the submandibular gland. Notably, we also found that CB1 deletion impaired some responses (those to peanut butter and chocolate hazelnut spread) but not others (sugar or marmite). In mice, the CB1 antagonist SR141716 (4 mg/kg, IP) prevented a previously learned salivary response to peanut butter. We find that CB1 receptors are expressed in a subset of glomeruli in coronal sections of olfactory bulb that may serve as a site of action for scent-specific effects of CB1 receptors. We additionally observe CB1 expression in accessory olfactory bulb. In summary, we find a novel sex-dependence in responses to a subset of food-related odorant cues and that cannabinoid receptors regulate some of these responses. We propose that CB1 receptors act at the parotid gland to inhibit stimulated salivation but also in the olfactory system, where functional CB1 receptors are required for salivary responses to specific appetitive odors.

Tuning the properties of peptide imprinted nanoparticles for protein immunoprecipitation using magnetic streptavidin beads

Maximizing the binding properties of thermoresponsive molecularly imprinted nanoparticles (MIN) was aimed to explore their feasibility as antibody substitutes in protein immunoprecipitation (IPP) with magnetic streptavidin beads (MSB). Thermoresponsive MIN targeting the cannabinoid CB1 receptor were produced by epitope imprinting through solid-phase synthesis. It was intended to determine how different variables influenced physicochemical features, binding behaviour and immunoprecipitation of the target recombinant glutathione S-transferase tagged fusion protein (GST-CTer). Such variables included the cross-linking degree of MIN, and variables like pH, temperature or the use of Tween-20 for binding and IPP experiments. The cross-linker (CL) amount influenced the coil-to-globule transition of thermoresponsive MIN, making the lower critical solution temperature (LCST) decrease from 37.2 °C using 5% of CL, to 29.0 °C using 25%, also suggesting higher plasticity on the former. Temperature influence on size was corroborated by dynamic light scattering, observing size reductions from 250–450 nm (RT) to 70–100 nm (> LCST) for MIN produced with 5–15% of CL. However, binding behaviour did not clearly improve for more than 10% CL. Further experiments revealed that temperature and pH control were critical for efficient binding and release, selecting 40 °C and pH 5 as appropriate. Following binding experiments, the GST-CTer-MIN complex was successfully immunoprecipitated using MSB, achieving an IPP efficiency of 11.48% over the initial input protein concentration, which was calculated after SDS-PAGE separation and Western blot analysis. The methodology may be exploited for selective protein extraction and quantification from complex tissue homogenates.Graphical