CB1 Receptor Negative Allosteric Modulators as a Potential Tool to Reverse Cannabinoid Toxicity

Peer reviewe

A biophysical model of endocannabinoid-mediated short term depression in hippocampal inhibition

Memories are believed to be represented in the synaptic pathways of vastly interconnected networks of neurons. The plasticity of synapses, that is, their strengthening and weakening depending on neuronal activity, is believed to be the basis of learning and establishing memories. An increasing number of studies indicate that endocannabinoids have a widespread action on brain function through modulation of synap–tic transmission and plasticity. Recent experimental studies have characterised the role of endocannabinoids in mediating both short- and long-term synaptic plasticity in various brain regions including the hippocampus, a brain region strongly associated with cognitive functions, such as learning and memory. Here, we present a biophysically plausible model of cannabinoid retrograde signalling at the synaptic level and investigate how this signalling mediates depolarisation induced suppression of inhibition (DSI), a prominent form of shortterm synaptic depression in inhibitory transmission in hippocampus. The model successfully captures many of the key characteristics of DSI in the hippocampus, as observed experimentally, with a minimal yet sufficient mathematical description of the major signalling molecules and cascades involved. More specifically, this model serves as a framework to test hypotheses on the factors determining the variability of DSI and investigate under which conditions it can be evoked. The model reveals the frequency and duration bands in which the post-synaptic cell can be sufficiently stimulated to elicit DSI. Moreover, the model provides key insights on how the state of the inhibitory cell modulates DSI according to its firing rate and relative timing to the post-synaptic activation. Thus, it provides concrete suggestions to further investigate experimentally how DSI modulates and is modulated by neuronal activity in the brain. Importantly, this model serves as a stepping stone for future deciphering of the role of endocannabinoids in synaptic transmission as a feedback mechanism both at synaptic and network level

Cannabinoid-mediated short-term plasticity in hippocampus

Endocannabinoids modulate both excitatory and inhibitory neurotransmission in hippocampus via activation of pre-synaptic cannabinoid receptors. Here, we present a model for cannabinoid mediated short-term depression of excitation (DSE) based on our recently developed model for the equivalent phenomenon of suppressing inhibition (DSI). Furthermore, we derive a simplified formulation of the calcium-mediated endocannabinoid synthesis that underlies short-term modulation of neurotransmission in hippocampus. The simplified model describes cannabinoid-mediated short-term modulation of both hippocampal inhibition and excitation and is ideally suited for large network studies. Moreover, the implementation of the simplified DSI/DSE model provides predictions on how both phenomena are modulated by the magnitude of the pre-synaptic cell's activity. In addition we demonstrate the role of DSE in shaping the post-synaptic cell's firing behaviour qualitatively and quantitatively in dependence on eCB availability and the pre-synaptic cell's activity. Finally, we explore under which conditions the combination of DSI and DSE can temporarily shift the fine balance between excitation and inhibition. This highlights a mechanism by which eCBs might act in a neuro-protective manner during high neural activity

Discovery of Prostamide F2α and Its Role in Inflammatory Pain and Dorsal Horn Nociceptive Neuron Hyperexcitability

It was suggested that endocannabinoids are metabolized by cyclooxygenase (COX)-2 in the spinal cord of rats with kaolin/λ-carrageenan-induced knee inflammation, and that this mechanism contributes to the analgesic effects of COX-2 inhibitors in this experimental model. We report the development of a specific method for the identification of endocannabinoid COX-2 metabolites, its application to measure the levels of these compounds in tissues, and the finding of prostamide F2α (PMF2α) in mice with knee inflammation. Whereas the levels of spinal endocannabinoids were not significantly altered by kaolin/λ-carrageenan-induced knee inflammation, those of the COX-2 metabolite of AEA, PMF2α, were strongly elevated. The formation of PMF2α was reduced by indomethacin (a non-selective COX inhibitor), NS-398 (a selective COX-2 inhibitor) and SC-560 (a selective COX-1 inhibitor). In healthy mice, spinal application of PMF2α increased the firing of nociceptive (NS) neurons, and correspondingly reduced the threshold of paw withdrawal latency (PWL). These effects were attenuated by the PMF2α receptor antagonist AGN211336, but not by the FP receptor antagonist AL8810. Also prostaglandin F2α increased NS neuron firing and reduced the threshold of PWL in healthy mice, and these effects were antagonized by AL8810, and not by AGN211336. In mice with kaolin/λ-carrageenan-induced knee inflammation, AGN211336, but not AL8810, reduced the inflammation-induced NS neuron firing and reduction of PWL. These findings suggest that inflammation-induced, and prostanoid-mediated, enhancement of dorsal horn NS neuron firing stimulates the production of spinal PMF2α, which in turn contributes to further NS neuron firing and pain transmission by activating specific receptors

Expression of G protein-coupled receptors and related proteins in HEK293, AtT20, BV2, and N18 cell lines as revealed by microarray analysis

<p>Abstract</p> <p>Background</p> <p>G protein coupled receptors (GPCRs) are one of the most widely studied gene superfamilies. Thousands of GPCR research studies have utilized heterologous expression systems such as human embryonic kidney cells (HEK293). Though often treated as 'blank slates', these cell lines nevertheless endogenously express GPCRs and related signaling proteins. The outcome of a given GPCR study can be profoundly influenced by this largely unknown complement of receptors and/or signaling proteins. Little easily accessible information exists that describes the expression profiles of the GPCRs in cell lines. What is accessible is often limited in scope - of the hundreds of GPCRs and related proteins, one is unlikely to find information on expression of more than a dozen proteins in a given cell line. Microarray technology has allowed rapid analysis of mRNA levels of thousands of candidate genes, but though often publicly available, the results can be difficult to efficiently access or even to interpret.</p> <p>Results</p> <p>To bridge this gap, we have used microarrays to measure the mRNA levels of a comprehensive profile of non-chemosensory GPCRs and over a hundred GPCR signaling related gene products in four cell lines frequently used for GPCR research: HEK293, AtT20, BV2, and N18.</p> <p>Conclusions</p> <p>This study provides researchers an easily accessible mRNA profile of the endogenous signaling repertoire that these four cell lines possess. This will assist in choosing the most appropriate cell line for studying GPCRs and related signaling proteins. It also provides a better understanding of the potential interactions between GPCRs and those signaling proteins.</p

Molecular Components and Functions of the Endocannabinoid System in Mouse Prefrontal Cortex

Background. Cannabinoids have deleterious effects on prefrontal cortex (PFC)-mediated functions and multiple evidences link the endogenous cannabinoid (endocannabinoid) system, cannabis use and schizophrenia, a disease in which PFC functions are altered. Nonetheless, the molecular composition and the physiological functions of the endocannabinoid system in the PFC are unknown. Methodology/Principal Findings. Here, using electron microscopy we found that key proteins involved in endocannabinoid signaling are expressed in layers V/VI of the mouse prelimbic area of the PFC: presynaptic cannabinoid CB1 receptors (CB1R) faced postsynaptic mGluR5 while diacylglycerol lipase alpha (DGL-alpha), the enzyme generating the endocannabinoid 2-arachidonoyl-glycerol (2-AG) was expressed in the same dendritic processes as mGluR5. Activation of presynaptic CB1R strongly inhibited evoked excitatory post-synaptic currents. Prolonged synaptic stimulation at 10Hz induced a profound long-term depression (LTD) of layers V/VI excitatory inputs. The endocannabinoid -LTD was presynaptically expressed and depended on the activation of postsynaptic mGluR5, phospholipase C and a rise in postsynaptic Ca2+ as predicted from the localization of the different components of the endocannabinoid system. Blocking the degradation of 2-AG (with URB 602) but not of anandamide (with URB 597) converted subthreshold tetanus to LTD-inducing ones. Moreover, inhibiting the synthesis of 2-AG with Tetrahydrolipstatin, blocked endocannabinoid-mediated LTD. All together, our data show that 2-AG mediates LTD at these synapses. Conclusions/Significance. Our data show that the endocannabinoid -retrograde signaling plays a prominent role in long-term synaptic plasticity at the excitatory synapses of the PFC. Alterations of endocannabinoid -mediated synaptic plasticity may participate to the etiology of PFC-related pathologies

Cannabidiol Inhibits Endocannabinoid Signaling in Autaptic Hippocampal Neurons

$\Delta^{9}$-Tetrahydrocannabinol (THC) and cannabidiol (CBD) are two main cannabinoid constituents of marijuana and hashish. The pharmacology of $\Delta^{9}$-THC has been extensively studied, whereas our understanding of the pharmacology of CBD has remained limited, despite excitement in CBD’s potential role in treating certain pediatric epilepsies and its reputation for attenuating some $\Delta^{9}$-THC–induced effects. It was established early on that CBD binds poorly to the orthosteric site of CB$_1$ or CB$_2$ cannabinoid receptors, and its actions were commonly attributed to other noncannabinoid receptor mechanisms. However, recent evidence suggests that CBD does indeed act at cannabinoid CB$_1$ receptors as a negative allosteric modulator (NAM) of CB$_1$ signaling. By altering the orthosteric signaling of a G protein–coupled receptor, allosteric modulators greatly increase the richness of G protein–coupled receptor pharmacology. We have recently surveyed candidate CB$_1$ NAMs in autaptic hippocampal neurons, a well-characterized neuronal model of endogenous cannabinoid signaling, and have now tested CBD in this model. We find that although CBD has no direct effect on excitatory transmission, it does inhibit two forms of endogenous cannabinoid-mediated retrograde synaptic plasticity: depolarization-induced suppression of excitation and metabotropic suppression of excitation, while not affecting signaling via GABA-B receptors. These results are consistent with the recently described NAM activity of CBD and suggest interesting possible mechanisms for CBD’s therapeutic actions

Cannabinoid CB1 receptors and ligands in vertebrate retina: localization and function of an endogenous signaling system.

CB1, a cannabinoid receptor enriched in neuronal tissue, was found in high concentration in retinas of rhesus monkey, mouse, rat, chick, goldfish, and tiger salamander by using a subtype-specific polyclonal antibody. Immunolabeling was detected in the two synaptic layers of the retina, the inner and outer plexiform layers, of all six species examined. In the outer plexiform layer, CB1 was located in and/or on cone pedicles and rod spherules. Labeling was detected in some amacrine cells of all species and in the ganglion cells and ganglion cell axons of all species except fish. In addition, sparse labeling was found in the inner and/or outer segments of the photoreceptors of monkey, mouse, rat, and chick. Using GC/MS to detect possible endogenous cannabinoids, we found 3 nmol of 2-arachidonylglycerol per g of tissue, but no anandamide was detectable. Cannabinoid receptor agonists induced a dramatic reduction in the amplitude of voltage-gated L-type calcium channel currents in identified retinal bipolar cells. The presence and distribution of the CB1 receptor, the large amounts of 2-arachidonylglycerol found, and the effects of cannabinoids on calcium channel activity in bipolar cells suggest a substantive role for an endogenous cannabinoid signaling system in retinal physiology, and perhaps vision in general

Controlled-Deactivation CB1 Receptor Ligands as a Novel Strategy to Lower Intraocular Pressure

Nearly half a century has passed since the demonstration that cannabis and its chief psychoactive component $\Delta^9$-THC lowers intraocular pressure (IOP). Elevated IOP remains the chief hallmark and therapeutic target for glaucoma, a condition that places millions at risk of blindness. It is likely that $\Delta^9$-THC exerts much of its IOP-lowering effects via the activation of CB1 cannabinoid receptors. However, the initial promise of CB1 as a target for treating glaucoma has not thus far translated into a credible therapeutic strategy. We have recently shown that blocking monoacylglycerol lipase (MAGL), an enzyme that breaks the endocannabinoid 2-arachidonoyl glycerol (2-AG), substantially lowers IOP. Another strategy is to develop cannabinoid CB1 receptor agonists that are optimized for topical application to the eye. Recently we have reported on a controlled-deactivation approach where the “soft” drug concept of enzymatic deactivation was combined with a “depot effect” that is commonly observed with $\Delta^9$-THC and other lipophilic cannabinoids. This approach allowed us to develop novel cannabinoids with a predictable duration of action and is particularly attractive for the design of CB1 activators for ophthalmic use with limited or no psychoactive effects. We have tested a novel class of compounds using a combination of electrophysiology in autaptic hippocampal neurons, a well-characterized model of endogenous cannabinoid signaling, and measurements of IOP in a mouse model. We now report that AM7410 is a reasonably potent and efficacious agonist at CB1 in neurons and that it substantially (30%) lowers IOP for as long as 5 h after a single topical treatment. This effect is absent in CB1 knockout mice. Our results indicate that the direct targeting of CB1 receptors with controlled-deactivation ligands is a viable approach to lower IOP in a murine model and merits further study in other model systems