Cannabinoid Receptor Modulation of Synapses Received by Cerebellar Purkinje Cells

The high density of cannabinoid receptors in the cerebellum and the degradation of motor coordination produced by cannabinoid intoxication suggest that synaptic transmission in the cerebellum may be strongly regulated by cannabinoid receptors. Therefore the effects of exogenous cannabinoids on synapses received by Purkinje cells were investigated in rat cerebellar slices. Parallel fiber–evoked (PF) excitatory postsynaptic currents (EPSCs) were strongly inhibited by bath application of the cannabinoid receptor agonist WIN 55212-2 (5 μM, 12% of baseline EPSC amplitude). This effect was completely blocked by the cannabinoid CB1 receptor antagonist SR 141716. It is unlikely that this was the result of alterations in axonal excitability because fiber volley velocity and kinetics were unchanged and a cannabinoid-induced decrease in fiber volley amplitude was very minor (93% of baseline). WIN 55212-2 had no effect on the amplitude or frequency of spontaneously occurring miniature EPSCs (mEPSCs), suggesting that the effect of CB1 receptor activation on PF EPSCs was presynaptically expressed, but giving no evidence for modulation of release processes after Ca2+influx. EPSCs evoked by climbing fiber (CF) stimulation were less powerfully attenuated by WIN 55212-2 (5 μM, 74% of baseline). Large, action potential–dependent, spontaneously occurring inhibitory postsynaptic currents (sIPSCs) were either severely reduced in amplitude (<25% of baseline) or eliminated. Miniature IPSCs (mIPSCs) were reduced in frequency (52% of baseline) but not in amplitude, demonstrating suppression of presynaptic vesicle release processes after Ca2+ influx and suggesting an absence of postsynaptic modulation. The decrease in mIPSC frequency was not large enough to account for the decrease in sIPSC amplitude, suggesting that presynaptic voltage-gated channel modulation was also involved. Thus, while CB1 receptor activation reduced neurotransmitter release at all major classes of Purkinje cell synapses, this was not accomplished by a single molecular mechanism. At excitatory synapses, cannabinoid suppression of neurotransmitter release was mediated by modulation of voltage-gated channels in the presynaptic axon terminal. At inhibitory synapses, in addition to modulation of presynaptic voltage-gated channels, suppression of the downstream vesicle release machinery also played a large role.

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Mechanisms of Cannabinoid-Receptor–Mediated Inhibition of Synaptic Transmission in Cultured Hippocampal Pyramidal Neurons

Journal of Neurophysiology ◽

10.1152/jn.1999.82.3.1286 ◽

1999 ◽

Vol 82 (3) ◽

pp. 1286-1294 ◽

Cited By ~ 109

Author(s):

Jane M. Sullivan

Keyword(s):

Calcium Channels ◽

Synaptic Transmission ◽

Learning And Memory ◽

Neurotransmitter Release ◽

Cannabinoid Receptors ◽

Cannabinoid Receptor ◽

Receptor Activation ◽

Extracellular Calcium ◽

Paired Pulse ◽

Mediated Effects

Cannabinoids, such as marijuana, are known to impair learning and memory perhaps through their actions in the hippocampus where cannabinoid receptors are expressed at high density. Although cannabinoid receptor activation decreases glutamatergic synaptic transmission in cultured hippocampal neurons, the mechanisms of this action are not known. Cannabinoid receptor activation also inhibits calcium channels that support neurotransmitter release in these cells, making modulation of these channels a candidate for cannabinoid-receptor–mediated effects on synaptic transmission. Whole cell patch-clamp recordings of glutamatergic neurons cultured from the CA1 and CA3 regions of the hippocampus were used to identify the mechanisms of the effects of cannabinoids on synaptic transmission. Cannabinoid receptor activation reduced excitatory postsynaptic current (EPSC) size by ∼50% but had no effect on the amplitude of spontaneous miniature EPSCs (mEPSCs). This reduction in EPSC size was accompanied by an increase in paired-pulse facilitation measured in low (1 mM) extracellular calcium and by a decrease in paired-pulse depression measured in normal (2.5 mM) extracellular calcium. Together, these results strongly support the hypothesis that cannabinoid receptor activation decreases EPSC size by reducing release of neurotransmitter presynaptically while having no effect on postsynaptic sensitivity to glutamate. Further experiments were done to identify the molecular mechanisms underlying this cannabinoid-receptor–mediated decrease in neurotransmitter release. Cannabinoid receptor activation had no effect on the size of the presynaptic pool of readily releasable neurotransmitter-filled vesicles, eliminating reduction in pool size as a mechanism for cannabinoid-receptor–mediated effects. After blockade of Q- and N-type calcium channels with ω-agatoxin TK and ω-conotoxin GVIA; however, activation of cannabinoid receptors reduced EPSC size by only 14%. These results indicate that cannabinoid receptor activation reduces the probability that neurotransmitter will be released in response to an action potential via an inhibition of presynaptic Q- and N-type calcium channels. This molecular mechanism most likely contributes to the impairment of learning and memory produced by cannabinoids and may participate in the analgesic, antiemetic, and anticonvulsive effects of these drugs as well.

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Marijuana smoke induces severe pulmonary hyperresponsiveness, inflammation, and emphysema in a predictive mouse model not via CB1 receptor activation

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00354.2016 ◽

2017 ◽

Vol 313 (2) ◽

pp. L267-L277 ◽

Cited By ~ 10

Author(s):

Z. Helyes ◽

Á. Kemény ◽

K. Csekő ◽

É. Szőke ◽

K. Elekes ◽

...

Keyword(s):

Tobacco Smoke ◽

Airway Hyperresponsiveness ◽

Inflammatory Cell ◽

Cannabinoid Receptor ◽

Receptor Activation ◽

Cb1 Receptor ◽

Whole Body ◽

Myeloperoxidase Activity ◽

Tissue Destruction ◽

Cell Hyperplasia

Sporadic clinical reports suggested that marijuana smoking induces spontaneous pneumothorax, but no animal models were available to validate these observations and to study the underlying mechanisms. Therefore, we performed a systematic study in CD1 mice as a predictive animal model and assessed the pathophysiological alterations in response to 4-mo-long whole body marijuana smoke with integrative methodologies in comparison with tobacco smoke. Bronchial responsiveness was measured with unrestrained whole body plethysmography, cell profile in the bronchoalveolar lavage fluid with flow cytometry, myeloperoxidase activity with spectrophotometry, inflammatory cytokines with ELISA, and histopathological alterations with light microscopy. Daily marijuana inhalation evoked severe bronchial hyperreactivity after a week. Characteristic perivascular/peribronchial edema, atelectasis, apical emphysema, and neutrophil and macrophage infiltration developed after 1 mo of marijuana smoking; lymphocyte accumulation after 2 mo; macrophage-like giant cells, irregular or destroyed bronchial mucosa, goblet cell hyperplasia after 3 mo; and severe atelectasis, emphysema, obstructed or damaged bronchioles, and endothelial proliferation at 4 mo. Myeloperoxidase activity, inflammatory cell, and cytokine profile correlated with these changes. Airway hyperresponsiveness and inflammation were not altered in mice lacking the CB1 cannabinoid receptor. In comparison, tobacco smoke induced hyperresponsiveness after 2 mo and significantly later caused inflammatory cell infiltration/activation with only mild emphysema. We provide the first systematic and comparative experimental evidence that marijuana causes severe airway hyperresponsiveness, inflammation, tissue destruction, and emphysema, which are not mediated by the CB1 receptor.

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Activity-Based Detection of Cannabinoids in Serum and Plasma Samples

Clinical Chemistry ◽

10.1373/clinchem.2017.285361 ◽

2018 ◽

Vol 64 (6) ◽

pp. 918-926 ◽

Cited By ~ 18

Author(s):

Annelies Cannaert ◽

Jolien Storme ◽

Cornelius Hess ◽

Volker Auwärter ◽

Sarah M R Wille ◽

...

Keyword(s):

Cannabinoid Receptors ◽

Cannabinoid Receptor ◽

Synthetic Cannabinoids ◽

Receptor Activation ◽

Synthetic Cannabinoid ◽

New Psychoactive Substances ◽

Simple Liquid ◽

Analytical Sensitivity ◽

Monitoring Centre ◽

Screening Strategies

Abstract BACKGROUND Synthetic cannabinoids are the largest group of new psychoactive substances monitored by the European Monitoring Centre of Drugs and Drug Addiction. The rapid proliferation of novel analogs makes the detection of these new derivatives challenging and has initiated considerable interest in the development of so-called “untargeted” screening strategies to detect these compounds. METHODS We developed new, stable bioassays in which cannabinoid receptor activation by cannabinoids led to recruitment of truncated β-arrestin 2 (βarr2) to the cannabinoid receptors, resulting in functional complementation of a split luciferase, allowing readout via bioluminescence. Aliquots (500 μL) of authentic serum (n = 45) and plasma (n = 73) samples were used for simple liquid–liquid extraction with hexane:ethyl acetate (99:1 v/v). Following evaporation and reconstitution in 100 μL of Opti-MEM® I/methanol (50/50 v/v), 10 μL of these extracts was analyzed in the bioassays. RESULTS Truncation of βarr2 significantly (for both cannabinoid receptors; P = 0.0034 and 0.0427) improved the analytical sensitivity over the previously published bioassays applied on urine samples. The new bioassays detected cannabinoid receptor activation by authentic serum or plasma extracts, in which synthetic cannabinoids were present at low- or sub-nanogram per milliliter concentration or in which Δ9-tetrahydrocannabinol was present at concentrations >12 ng/mL. For synthetic cannabinoid detection, analytical sensitivity was 82%, with an analytical specificity of 100%. CONCLUSIONS The bioassays have the potential to serve as a first-line screening tool for (synthetic) cannabinoid activity in serum or plasma and may complement conventional analytical assays and/or precede analytical (mass spectrometry based) confirmation.

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CB1 Cannabinoid Receptor Inhibits Synaptic Release of Glutamate in Rat Dorsolateral Striatum

Journal of Neurophysiology ◽

10.1152/jn.2001.85.1.468 ◽

2001 ◽

Vol 85 (1) ◽

pp. 468-471 ◽

Cited By ~ 277

Author(s):

Gregory Gerdeman ◽

David M. Lovinger

Keyword(s):

Basal Ganglia ◽

Synaptic Transmission ◽

Neurotransmitter Release ◽

Cannabinoid Receptors ◽

Glutamate Release ◽

Brain Slices ◽

Cb1 Receptor ◽

Motor Deficits ◽

Dependent Manner ◽

Dorsolateral Striatum

CB1 cannabinoid receptors in the neostriatum mediate profound motor deficits induced when cannabinoid drugs are administered to rodents. Because the CB1 receptor has been shown to inhibit neurotransmitter release in various brain areas, we investigated the effects of CB1 activation on glutamatergic synaptic transmission in the dorsolateral striatum of the rat where the CB1 receptor is highly expressed. We performed whole cell voltage-clamp experiments in striatal brain slices and applied the CB1 agonists HU-210 or WIN 55,212–2 during measurement of synaptic transmission. Excitatory postsynaptic currents (EPSCs), evoked by electrical stimulation of afferent fibers, were significantly reduced in a dose-dependent manner by CB1 agonist application. EPSC inhibition was accompanied by an increase in two separate indices of presynaptic release, the paired-pulse response ratio and the coefficient of variation, suggesting a decrease in neurotransmitter release. These effects were prevented by application of the CB1 antagonist SR141716A. When Sr2+ was substituted for Ca2+ in the extracellular solution, application of HU-210 (1 μM) significantly reduced the frequency, but not amplitude, of evoked, asynchronous quantal release events. Spontaneous release events were similarly decreased in frequency with no change in amplitude. These findings further support the interpretation that CB1 activation leads to a decrease of glutamate release from afferent terminals in the striatum. These results reveal a novel potential role for cannabinoids in regulating striatal function and thus basal ganglia output and may suggest CB1-targeted drugs as potential therapeutic agents in the treatment of Parkinson's disease and other basal ganglia disorders.

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Inhibition of Inflammatory Hyperalgesia by Activation of Peripheral CB2Cannabinoid Receptors

Anesthesiology ◽

10.1097/00000542-200310000-00031 ◽

2003 ◽

Vol 99 (4) ◽

pp. 955-960 ◽

Cited By ~ 171

Author(s):

Aline Quartilho ◽

Heriberto P. Mata ◽

Mohab M. Ibrahim ◽

Todd W. Vanderah ◽

Frank Porreca ◽

...

Keyword(s):

Cannabinoid Receptor ◽

Thermal Hyperalgesia ◽

Receptor Activation ◽

Cb1 Receptor ◽

Cb2 Receptor ◽

Inflammatory Hyperalgesia ◽

Selective Antagonist ◽

Receptor Agonists ◽

Cb2 Receptors ◽

Cns Effects

Background Cannabinoid receptor agonists inhibit inflammatory hyperalgesia in animal models. Nonselective cannabinoid receptor agonists also produce central nervous system (CNS) side effects. Agonists selective for CB2 cannabinoid receptors, which are not found in the CNS, do not produce the CNS effects typical of nonselective cannabinoid receptor agonists but do inhibit acute nociception. The authors used the CB2 receptor-selective agonist AM1241 to test the hypothesis that selective activation of peripheral CB2 receptors inhibits inflammatory hyperalgesia. Methods Rats were injected in the hind paw with carrageenan or capsaicin. Paw withdrawal latencies were measured using a focused thermal stimulus. The effects of peripheral CB2 receptor activation were determined by using local injection of AM1241. CB2 receptor mediation of the actions of AM1241 was shown by using the CB2 receptor-selective antagonist AM630 and the CB1 receptor-selective antagonist AM251. Results AM1241 fully reversed carrageenan-induced inflammatory thermal hyperalgesia when injected into the inflamed paw. In contrast, AM1241 injected into the contralateral paw had no effect, showing that its effects were local. AM1241 also reversed the local edema produced by hind paw carrageenan injection. The effects of AM1241 were reversed by the CB2 receptor-selective antagonist AM630, but not by the CB1 receptor-selective antagonist AM251. AM1241 also inhibited flinching and thermal hyperalgesia produced by hind paw capsaicin injection. Conclusions Local, peripheral CB2 receptor activation inhibits inflammation and inflammatory hyperalgesia. These results suggest that peripheral CB2 receptors may be an appropriate target for eliciting relief of inflammatory pain without the CNS effects of nonselective cannabinoid receptor agonists.

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Activation of cannabinoid receptor 2 reduces inflammation in acute experimental pancreatitis via intra-acinar activation of p38 and MK2-dependent mechanisms

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00133.2012 ◽

2013 ◽

Vol 304 (2) ◽

pp. G181-G192 ◽

Cited By ~ 26

Author(s):

Thomas Michler ◽

Martin Storr ◽

Johannes Kramer ◽

Stefanie Ochs ◽

Antje Malo ◽

...

Keyword(s):

Acute Pancreatitis ◽

Western Blotting ◽

Map Kinases ◽

Cannabinoid Receptors ◽

Cannabinoid Receptor ◽

Endocannabinoid System ◽

Receptor Activation ◽

Myeloperoxidase Activity ◽

Wild Type ◽

Pancreatic Acini

The endocannabinoid system has been shown to mediate beneficial effects on gastrointestinal inflammation via cannabinoid receptors 1 (CB1) and 2 (CB2). These receptors have also been reported to activate the MAP kinases p38 and c-Jun NH2-terminal kinase (JNK), which are involved in early acinar events leading to acute pancreatitis and induction of proinflammatory cytokines. Our aim was to examine the role of cannabinoid receptor activation in an experimental model of acute pancreatitis and the potential involvement of MAP kinases. Cerulein pancreatitis was induced in wild-type, CB1−/−, and MK2−/− mice pretreated with selective cannabinoid receptor agonists or antagonists. Severity of pancreatitis was determined by serum amylase and IL-6 levels, intracellular activation of pancreatic trypsinogen, lung myeloperoxidase activity, pancreatic edema, and histological examinations. Pancreatic lysates were investigated by Western blotting using phospho-specific antibodies against p38 and JNK. Quantitative PCR data, Western blotting experiments, and immunohistochemistry clearly show that CB1 and CB2 are expressed in mouse pancreatic acini. During acute pancreatitis, an upregulation especially of CB2 on apoptotic cells occurred. The unselective CB1/CB2 agonist HU210 ameliorated pancreatitis in wild-type and CB1−/− mice, indicating that this effect is mediated by CB2. Furthermore, blockade of CB2, not CB1, with selective antagonists engraved pathology. Stimulation with a selective CB2 agonist attenuated acute pancreatitis and an increased activation of p38 was observed in the acini. With use of MK2−/− mice, it could be demonstrated that this attenuation is dependent on MK2. Hence, using the MK2−/− mouse model we reveal a novel CB2-activated and MAP kinase-dependent pathway that modulates cytokine expression and reduces pancreatic injury and affiliated complications.

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CB1 Cannabinoid Receptor Signaling and Biased Signaling

Molecules ◽

10.3390/molecules26175413 ◽

2021 ◽

Vol 26 (17) ◽

pp. 5413

Author(s):

Luciana M. Leo ◽

Mary E. Abood

Keyword(s):

Adverse Effects ◽

G Protein ◽

Conformational Changes ◽

Cannabinoid Receptor ◽

Therapeutic Potential ◽

Receptor Activation ◽

Cb1 Receptor ◽

Cb1 Cannabinoid Receptor ◽

Biased Signaling ◽

G Protein Coupled

The CB1 cannabinoid receptor is a G-protein coupled receptor highly expressed throughout the central nervous system that is a promising target for the treatment of various disorders, including anxiety, pain, and neurodegeneration. Despite the wide therapeutic potential of CB1, the development of drug candidates is hindered by adverse effects, rapid tolerance development, and abuse potential. Ligands that produce biased signaling—the preferential activation of a signaling transducer in detriment of another—have been proposed as a strategy to dissociate therapeutic and adverse effects for a variety of G-protein coupled receptors. However, biased signaling at the CB1 receptor is poorly understood due to a lack of strongly biased agonists. Here, we review studies that have investigated the biased signaling profile of classical cannabinoid agonists and allosteric ligands, searching for a potential therapeutic advantage of CB1 biased signaling in different pathological states. Agonist and antagonist bound structures of CB1 and proposed mechanisms of action of biased allosteric modulators are used to discuss a putative molecular mechanism for CB1 receptor activation and biased signaling. Current studies suggest that allosteric binding sites on CB1 can be explored to yield biased ligands that favor or hinder conformational changes important for biased signaling.

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Genetic Variation and Sex Mediate Differential Responses to ∆-9-tetrahydrocannabinol among Inbred Mice

10.21007/etd.cghs.2020.0516 ◽

2020 ◽

Author(s):

◽

Cory Parks ◽

Keyword(s):

Cannabinoid Receptors ◽

Cannabinoid Receptor ◽

Initial Response ◽

The United States ◽

Receptor Activation ◽

Major Constituent ◽

Mouse Strains ◽

Social Consequence ◽

Increased Risk

The plant Cannabis sativa has been used by people for both recreational and medicinal use for thousands of years, but scientific investigation of the plant and its components didn’t begin until the early nineteen hundreds when Cannabis components known as phytocannabinoids were characterized and later isolated. In the 1970’s, ∆9-tetrahydrocannabinol (THC) was isolated and recognized as the major constituent responsible for the psychoactive and intoxicating effects associated with consumption of cannabis. This opened the door for intensive research in the field that lead to the discovery of the endogenous cannabinoid system and its associated receptors, effectors of signaling, and biosynthetic enzymes. The primary cannabinoid receptor, cannabinoid receptor 1, is a G-protein coupled receptor (GPCR) that primarily associates with Gi/o proteins, giving it the properties of having mainly inhibitory actions by decreasing release of neurotransmitters and hormones. Upon receptor activation, the Gi/o protein disassociates with and inhibits adenylyl cyclase, decreasing cAMP production, a major second messenger of the cell. After stimulation by cannabinoids, cannabinoid receptors undergo a desensitization process where they are internalized by β-arrestins. This internalization subjects the receptors to intracellular trafficking during which the majority are degraded. This causes a decrease in surface levels of cannabinoid receptors and makes the cells less sensitive to agonists. Cannabis is among the most widely used psychoactive drugs in the world. In the United States, use and legalization of cannabis continues to grow. The spreading use and legalization of cannabis has the social consequence of a diminished sense of risk to the individual. This can be harmful in and of itself, but cannabis THC concentrations in the U.S. have tripled over the last 20 years, giving rise to a more potent drug, potentially increasing risk of adverse effects associated with use. Effects of acute, short term use include faulty judgment and perception, memory impairment, motor skill dysfunction, alteration of mood, and low levels of attention and alertness. Effects associated with more chronic, long term use include risk of dependence, an increased risk of developing psychotic disorders such as schizophrenia, and long-term cognitive impairment. For cannabis, and other drugs of abuse, initial response and/or tolerance to drug effects can predict later dependence and problematic use. In the work presented here, we identify sex and genetic (strain) differences in initial response and rapid tolerance to THC, the main psychoactive ingredient in cannabis, between highly genetically divergent inbred mouse strains—C57BL/6J (B6) and DBA/2J (D2). To identify variation in THC response we use the cannabinoid-induced tetrad test which quantifies the strength of agonist mediated cannabinoid receptor signaling by measuring the level of motor activity, nociception, and hypothermia elicited by receptor activation. We then extend our study of THC response variation to the BXD genetic reference population derived from B6 and D2 strains. Increasing the number of strains tested by tenfold (N=20) we detect significant strain and sex variation in THC response and use online tools to perform QTL mapping and correlation searches to begin to uncover potential genetic drivers of variation in response to THC.

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