Alex Straiker

JWH018, a common constituent of ‘Spice’ herbal blends, is a potent and efficacious cannabinoid CB1 receptor agonist

Background and purpose:  ‘Spice’ is an herbal blend primarily marketed in Europe as a mild hallucinogen with prominent cannabis‐like effects and as a legal alternative to cannabis. However, a recent report identified a number of synthetic additives in samples of ‘Spice’. One of these, the indole derivative JWH018, is a ligand for the cannabinoid receptor 1 (CB1) cannabinoid receptor and inhibits cAMP production in CB1 receptor‐expressing CHO cells. Other effects of JWH018 on CB1 receptor‐mediated signalling are not known, particularly in neurons. Here we have evaluated the signalling pathways activated by JWH018 at CB1 receptors.

Depolarization-induced suppression of excitation in murine autaptic hippocampal neurones

Depolarization‐induced suppression of excitation and inhibition (DSE and DSI) appear to be important forms of short‐term retrograde neuronal plasticity involving endocannabinoids (eCB) and the activation of presynaptic cannabinoid CB1 receptors. We report here that CB1‐dependent DSE can be elicited from autaptic cultures of excitatory mouse hippocampal neurones. DSE in autaptic cultures is both more robust and elicited with a more physiologically relevant stimulus than has been thus far reported for conventional hippocampal cultures. An additional requirement for autaptic DSE is filled internal calcium stores. Pharmacological experiments favour a role for 2‐arachidonyl glycerol (2‐AG) rather than arachidonyl ethanolamide (AEA) or noladin ether as the relevant endocannabinoid to elicit DSE. In particular, the latter two compounds fail to reversibly inhibit EPSCs, a quality inconsistent with the role of bona fide eCB mediating DSE. Δ9‐Tetrahydrocannabinol (Δ9‐THC) fails to inhibit EPSCs, yet readily occludes both DSE and EPSC inhibition by a synthetic CB1 agonist, WIN 55212‐2. With long‐term exposure (∼18 h), Δ9‐THC also desensitizes CB1 receptors. Lastly, a functional endocannabinoid transporter is necessary for the expression of DSE.

Incensole acetate, an incense component, elicits psychoactivity by activating TRPV3 channels in the brain

Burning of Boswellia resin as incense has been part of religious and cultural ceremonies for millennia and is believed to contribute to the spiritual exaltation associated with such events. Transient receptor potential vanilloid (TRPV) 3 is an ion channel implicated in the perception of warmth in the skin. TRPV3 mRNA has also been found in neurons throughout the brain; however, the role of TRPV3 channels there remains unknown. Here we show that incensole acetate (IA), a Boswellia resin constituent, is a potent TRPV3 agonist that causes anxiolytic‐like and antide‐pressive‐like behavioral effects in wild‐type (WT) mice with concomitant changes in c‐Fos activation in the brain. These behavioral effects were not noted in TRPV3−/− mice, suggesting that they are mediated via TRPV3 channels. IA activated TRPV3 channels stably expressed in HEK293 cells and in keratinocytes from TRPV3+/+ mice. It had no effect on keratinocytes from TRPV3−/− mice and showed modest or no effect on TRPV1, TRPV2, and TRPV4, as well as on 24 other receptors, ion channels, and transport proteins. Our results imply that TRPV3 channels in the brain may play a role in emotional regulation. Furthermore, the biochemical and pharmacological effects of IA may provide a biological basis for deeply rooted cultural and religious traditions.—Moussaieff, A., Rimmerman, N., Bregman, T., Straiker, A., Felder, C. C., Shoham, S., Kashman, Y., Huang, S. M., Lee, H., Shohami, E., Mackie, K., Caterina, M. J., Walker, J. M., Fride, E., Mechoulam, R. Incensole acetate, an incense component, elicits psychoactivity by activating TRPV3 channels in the brain. FASEB J. 22, 3024–3034 (2008)

Functional Selectivity in CB2 Cannabinoid Receptor Signaling and Regulation: Implications for the Therapeutic Potential of CB2 Ligands

Receptor internalization increases the flexibility and scope of G protein-coupled receptor (GPCR) signaling. CB1 and CB2 cannabinoid receptors undergo internalization after sustained exposure to agonists. However, it is not known whether different agonists internalize CB2 to different extents. Because CB2 is a promising therapeutic target, understanding its trafficking in response to different agonists is necessary for a complete understanding of its biology. Here we profile a number of cannabinoid receptor ligands and provide evidence for marked functional selectivity of cannabinoid receptor internalization. Classic, aminoalkylindole, bicyclic, cannabilactone, iminothiazole cannabinoid, and endocannabinoid ligands varied greatly in their effects on CB1 and CB2 trafficking. Our most striking finding was that (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl) pyrrolo-[1,2,3-d,e]-1,4-benzoxazin-6-yl]-1-naphthalenyl-methanone (WIN55,212-2) (and other aminoalkylindoles) failed to promote CB2 receptor internalization, whereas 5-(1,1-dimethylheptyl)-2-(5-hydroxy-2-(3-hydroxypropyl)cyclohexyl)phenol (CP55,940) robustly internalized CB2 receptors. Furthermore, WIN55,212-2 competitively antagonized CP55,940-induced CB2 internalization. Despite these differences in internalization, both compounds activated CB2 receptors as measured by extracellular signal-regulated kinase 1/2 phosphorylation and recruitment of β-arrestin2 to the membrane. In contrast, whereas CP55,940 inhibited voltage-gated calcium channels via CB2 receptor activation, WIN55,212-2 was ineffective on its own and antagonized the effects of CP55,940. On the basis of the differences we found between these two ligands, we also tested the effects of other cannabinoids on these signaling pathways and found additional evidence for functional selectivity of CB2 ligands. These novel data highlight that WIN55,212-2 and other cannabinoids show strong functional selectivity at CB2 receptors and suggest that different classes of CB2 ligands may produce diverse physiological effects, emphasizing that each class needs to be separately evaluated for therapeutic efficacy.

Monoacylglycerol lipase limits the duration of endocannabinoid-mediated depolarization-induced suppression of excitation in autaptic hippocampal neurons

Depolarization-induced suppression of excitation (DSE) is a major form of cannabinoid-mediated short-term retrograde neuronal plasticity and is found in numerous brain regions. Autaptically cultured murine hippocampal neurons are an architecturally simple model for the study of cannabinoid signaling, including DSE. The transient nature of DSE—tens of seconds—is probably determined by the regulated hydrolysis of the endocannabinoid 2-arachidonoyl glycerol (2-AG). No less than five candidate enzymes have been considered to serve this role: fatty acid amide hydrolase (FAAH), cyclooxygenase-2 (COX-2), monoacylglycerol lipase (MGL), and α/β-hydrolase domain (ABHD) 6 and 12. We previously found that FAAH and COX-2 do not have a role in determining the duration of autaptic DSE. In the current study, we found that two structurally distinct inhibitors of MGL [N-arachidonoyl maleimide and 4-nitrophenyl 4-(dibenzo[d][1,3]dioxol-5-yl(hydroxy)methyl)piperidine-1-carboxylate (JZL184)] prolong DSE in autaptic hippocampal neurons, whereas inhibition of ABHD6 by N-methyl-N-[[3-(4-pyridinyl)phenyl]methyl]-4′-(aminocarbonyl)[1,1′-biphenyl]-4-yl ester, carbamic acid (WWL70) had no effect. In addition, we developed antibodies against MGL and ABHD6 and determined their expression in autaptic cultures. MGL is chiefly expressed at presynaptic terminals, optimally positioned to break down 2-AG that has engaged presynaptic CB1 receptors. ABHD6 is expressed in two distinct locations on autaptic islands, including a prominent localization in some dendrites. In summary, we provide strong pharmacological and anatomical evidence that MGL regulates DSE in autaptic hippocampal neurons and, taken together with other studies, emphasizes that endocannabinoid signaling is terminated in temporally diverse ways.

Metabotropic suppression of excitation in murine autaptic hippocampal neurons

Depolarization‐induced suppression of excitation (DSE) and inhibition (DSI) are forms of short‐term neuronal plasticity involving postsynaptic release of an endocannabinoid and the activation of presynaptic cannabinoid CB1 receptors. We have recently reported that CB1‐dependent DSE can be elicited in autaptic cultures of excitatory hippocampal neurons of the mouse. We now report that the same preparation exhibits a parallel Gq‐coupled receptor‐dependent production of endocannabinoids causing retrograde inhibition, also via CB1 receptors, which we will refer to as metabotropic suppression of excitation (MSE). We tested a spectrum of Gq‐coupled receptor agonists and found that both muscarinic and metabotropic glutamate receptors (group I) mediate retrograde inhibition via CB1 receptors in autaptic hippocampal neurons. Thus these neurons possess not only the pre‐ and postsynaptic machinery necessary for DSE but also that for MSE. This permitted a closer examination of MSE and its interaction with other aspects of the endocannabinoid retrograde signalling machinery: MSE mimics and occludes DSE and is itself occluded by the endocannabinoid 2‐arachidonoyl glycerol (2‐AG), consistent with 2‐AG as a likely mediator of MSE. In contrast to DSE, MSE undergoes heterologous desensitization over the time course of minutes. In keeping with data reported for metabotropic suppression of inhibition (MSI) and DSI in the hippocampus, subthreshold MSE and DSE act synergistically. We additionally found that Δ9‐tetrahydrocannabinol, which has been shown to attenuate DSE, antagonizes MSE. Finally, we have distinguished a neuronal subpopulation that exhibits DSE and a differential complement of MSE‐mediating Gq‐coupled receptors, making possible contrasting studies of MSE. Autaptic endocannabinoid signalling is rich, robust and complex in a deceptively simple package, including a previously unreported postsynaptic mechanism of adaptation in addition to known presynaptic CB1 desensitization. These adaptive sites offer novel targets for modulation of endogenous cannabinoid signalling.

Parsing the players: 2‐arachidonoylglycerol synthesis and degradation in the CNS

The endogenous cannabinoid signalling system, composed of endogenous cannabinoids, cannabinoid receptors and the enzymes that synthesize and degrade the endogenous cannabinoids, is much more complex than initially conceptualized. 2‐Arachidonoylglycerol (2‐AG) is the most abundant endocannabinoid and plays a major role in CNS development and synaptic plasticity. Over the past decade, many key players in 2‐AG synthesis and degradation have been identified and characterized. Most 2‐AG is synthesized from membrane phospholipids via sequential activation of a phospholipase Cβ and a diacylglycerol lipase, although other pathways may contribute in specialized settings. 2‐AG breakdown is more complicated with at least eight different enzymes participating. These enzymes can either degrade 2‐AG into its components, arachidonic acid and glycerol, or transform 2‐AG into highly bioactive signal molecules. The implications of the precise temporal and spatial control of the expression and function of these pleiotropic metabolizing enzymes have only recently come to be appreciated. In this review, we will focus on the primary organization of the synthetic and degradative pathways of 2‐AG and then discuss more recent findings and their implications, with an eye towards the biological and therapeutic implications of manipulating 2‐AG synthesis and metabolism.

GPR55, a G-Protein Coupled Receptor for Lysophosphatidylinositol, Plays a Role in Motor Coordination

The G-protein coupled receptor 55 (GPR55) is activated by lysophosphatidylinositols and some cannabinoids. Recent studies found prominent roles for GPR55 in neuropathic/inflammatory pain, cancer and bone physiology. However, little is known about the role of GPR55 in CNS development and function. To address this question, we performed a detailed characterization of GPR55 knockout mice using molecular, anatomical, electrophysiological, and behavioral assays. Quantitative PCR studies found that GPR55 mRNA was expressed (in order of decreasing abundance) in the striatum, hippocampus, forebrain, cortex, and cerebellum. GPR55 deficiency did not affect the concentrations of endocannabinoids and related lipids or mRNA levels for several components of the endocannabinoid system in the hippocampus. Normal synaptic transmission and short-term as well as long-term synaptic plasticity were found in GPR55 knockout CA1 pyramidal neurons. Deleting GPR55 function did not affect behavioral assays assessing muscle strength, gross motor skills, sensory-motor integration, motor learning, anxiety or depressive behaviors. In addition, GPR55 null mutant mice exhibited normal contextual and auditory-cue conditioned fear learning and memory in a Pavlovian conditioned fear test. In contrast, when presented with tasks requiring more challenging motor responses, GPR55 knockout mice showed impaired movement coordination. Taken together, these results suggest that GPR55 plays a role in motor coordination, but does not strongly regulate CNS development, gross motor movement or several types of learned behavior.

Architecture of cannabinoid signaling in mouse retina.

Cannabinoid receptors and their ligands constitute an endogenous signaling system that is found throughout the body, including the eye. This system can be activated by Δ9‐tetrahydrocannabinol, a major drug of abuse. Cannabinoids offer considerable therapeutic potential in modulating ocular immune and inflammatory responses and in regulating intraocular pressure. The location of cannabinoid receptor 1 (CB1) in the retina is known, but recently a constellation of proteins has been identified that produce and break down endocannabinoids (eCBs) and modulate CB1 function. Localization of these proteins is critical to defining specific cannabinoid signaling circuitry in the retina. Here we show the localization of diacylglycerol lipase‐α and ‐β (DGLα/β), implicated in the production of the eCB 2‐arachidonoyl glycerol (2‐AG); monoacylglycerol lipase (MGL) and α/β‐hydrolase domain 6 (ABHD6), both implicated in the breakdown of 2‐AG; cannabinoid receptor‐interacting protein 1a (CRIP1a), a protein that may modulate CB1 function; and fatty acid amide hydrolase (FAAH) and N‐acylethanolamine‐hydrolyzing acid amidase (NAAA), which have been shown to break down the eCB anandamide and related acyl amides. Our most prominent finding was that DGLα is present in postsynaptic type 1 OFF cone bipolar cells juxtaposed to CB1‐containing cone photoreceptor terminals. CRIP1a is reliably presynaptic to DGLα, consistent with a possible role in cannabinoid signaling, and NAAA is restricted to retinal pigment epithelium, whereas DGLβ is limited to retinal blood vessels. These results taken together with previous anatomical and functional studies define specific cannabinoid circuitry likely to modulate eCB signaling at the first synapse of the retina as well as in the inner plexiform layer. J. Comp. Neurol. 518:3848–3866, 2010.

Cannabinoid signaling in inhibitory autaptic hippocampal neurons.

Depolarization-induced suppression of excitation and inhibition (DSE/DSI) appears to be an important form of short-term retrograde neuronal plasticity involving endocannabinoids (eCBs), the activation of presynaptic cannabinoid CB1 receptors, and the suppression of neurotransmitter release. Using murine autaptic hippocampal cultures, we have distinguished five populations of autaptic inhibitory neurons that exhibit differential cannabinoid responses, including three temporally distinct forms of DSI. One remaining population responded to cannabinoids but did not have DSI while a fifth had neither DSI nor cannabinoid responses. Of the two chief candidate eCBs, 2-AG reversibly inhibited inhibitory post synaptic currents (IPSCs) while anandamide did so irreversibly, the latters action inconsistent with a role as a bona fide eCB mediator of DSI. The duration of depolarization necessary to elicit the two most prominent forms of DSI (effective dose (ED-50) approximately 210, approximately 280 ms) was far less than for autaptic DSE. However the nearly identical concentration response for 2-AG to inhibit excitatory postsynaptic currents (EPSCs) and IPSCs indicates that this difference is not due to differential cannabinoid receptor sensitivity. Interestingly, of the two populations exhibiting prominent DSI, one had a substantially faster recovery time course both after DSI and 2-AG, this despite being cultured under identical conditions. Several enzymes have been proposed to play a role in 2-AG breakdown, presumably determining the time course of DSI: fatty acid amide hydrolase (FAAH), cyclooxygenase-2 (COX-2), monoacyl glycerol lipase (MGL), and alpha/beta-hydrolase domains 6 and 12 (ABHD6 and ABHD12). We tested the impact on DSI duration by blockers of FAAH, COX-2, MGL and ABHD6. Notably, the population with slow DSI was regulated only by MGL, whereas the fast DSI population was regulated by both MGL and COX-2. This suggests that the faster DSI time course may occur as a result of the concerted action of multiple enzymes, which may represent a more general mechanism for regulation of the duration of different forms of DSI and DSE.