Jim Wager-Miller

Requirement of cannabinoid CB1 receptors in cortical pyramidal neurons for appropriate development of corticothalamic and thalamocortical projections

A role for endocannabinoid signaling in neuronal morphogenesis as the brain develops has recently been suggested. Here we used the developing somatosensory circuit as a model system to examine the role of endocannabinoid signaling in neural circuit formation. We first show that a deficiency in cannabinoid receptor type 1 (CB1R), but not G‐protein‐coupled receptor 55 (GPR55), leads to aberrant fasciculation and pathfinding in both corticothalamic and thalamocortical axons despite normal target recognition. Next, we localized CB1R expression to developing corticothalamic projections and found little if any expression in thalamocortical axons, using a newly established reporter mouse expressing GFP in thalamocortical projections. A similar thalamocortical projection phenotype was observed following removal of CB1R from cortical principal neurons, clearly demonstrating that CB1R in corticothalamic axons was required to instruct their complimentary connections, thalamocortical axons. When reciprocal thalamic and cortical connections meet, CB1R‐containing corticothalamic axons are intimately associated with elongating thalamocortical projections containing DGLβ, a 2‐arachidonoyl glycerol (2‐AG) synthesizing enzyme. Thus, 2‐AG produced in thalamocortical axons and acting at CB1Rs on corticothalamic axons is likely to modulate axonal patterning. The presence of monoglyceride lipase, a 2‐AG degrading enzyme, in both thalamocortical and corticothalamic tracts probably serves to restrict 2‐AG availability. In summary, our study provides strong evidence that endocannabinoids are a modulator for the proposed ‘handshake’ interactions between corticothalamic and thalamocortical axons, especially for fasciculation. These findings are important in understanding the long‐term consequences of alterations in CB1R activity during development, a potential etiology for the mental health disorders linked to prenatal cannabis use.

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.

Automated Solvent-Free Matrix Deposition for Tissue Imaging by Mass Spectrometry

The ability to analyze complex (macro) molecules is of fundamental importance for understanding chemical, physical, and biological processes. Complexity may arise from small differences in structure, large dynamic range, as well as a vast range in solubility or ionization, imposing daunting tasks in areas as different as lipidomics and proteomics. Here, we describe a rapid matrix application that permits the deposition of matrix-assisted laser desorption/ionization (MALDI) matrix solvent-free. This solvent-free one-step automatic matrix deposition is achieved through vigorous movements of beads pressing the matrix material through a metal mesh. The mesh (20 mum) produces homogeneous coverage of <12 microm crystals (DHB, CHCA matrixes) in 1 min, as determined by optical microscopy, permitting fast uniform coverage of analyte and possible high-spatial resolution surface analysis. Homogenous tissue coverage of <5 microm sized crystals is achieved using a 3 microm mesh. Solvent-free MALDI analysis on a time-of-flight (TOF) mass analyzer of mouse brain tissue homogenously covered with CHCA matrix subsequently provides a homogeneous response in ion signal intensity. Total solvent-free analysis (TSA) by mass spectrometry (MS) of tissue sections is carried out by applying the MALDI matrix solvent-free for subsequent ionization and gas phase separation for decongestion of complexity in the absence of any solvent using ion mobility spectrometry (IMS) followed by MS detection. Isobaric compositions were well-delineated using TSA by MS.

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.

COX-2 and fatty acid amide hydrolase can regulate the time course of depolarization-induced suppression of excitation

BACKGROUND AND PURPOSE Depolarization‐induced suppression of inhibition (DSI) and excitation (DSE) are two forms of cannabinoid CB1 receptor‐mediated inhibition of synaptic transmission, whose durations are regulated by endocannabinoid (eCB) degradation. We have recently shown that in cultured hippocampal neurons monoacylglycerol lipase (MGL) controls the duration of DSE, while DSI duration is determined by both MGL and COX‐2. This latter result suggests that DSE might be attenuated, and excitatory transmission enhanced, during inflammation and in other settings where COX‐2 expression is up‐regulated.

Differential signalling in human cannabinoid CB1 receptors and their splice variants in autaptic hippocampal neurones

BACKGROUND AND PURPOSE Cannabinoids such as Δ9‐ tetrahydrocannabinol, the major psychoactive component of marijuana and hashish, primarily act via cannabinoid CB1 and CB2 receptors to produce characteristic behavioural effects in humans. Due to the tractability of rodent models for electrophysiological and behavioural studies, most of the studies of cannabinoid receptor action have used rodent cannabinoid receptors. While CB1 receptors are relatively well‐conserved among mammals, human CB1 (hCB1) differs from rCB1 and mCB1 receptors at 13 residues, which may result in differential signalling. In addition, two hCB1 splice variants (hCB1a and hCB1b) have been reported, diverging in their amino‐termini relative to hCB1 receptors. In this study, we have examined hCB1 signalling in neurones.

Mutation of Putative GRK Phosphorylation Sites in the Cannabinoid Receptor 1 (CB1R) Confers Resistance to Cannabinoid Tolerance and Hypersensitivity to Cannabinoids in Mice

For many G-protein-coupled receptors (GPCRs), including cannabinoid receptor 1 (CB1R), desensitization has been proposed as a principal mechanism driving initial tolerance to agonists. GPCR desensitization typically requires phosphorylation by a G-protein-coupled receptor kinase (GRK) and interaction of the phosphorylated receptor with an arrestin. In simple model systems, CB1R is desensitized by GRK phosphorylation at two serine residues (S426 and S430). However, the role of these serine residues in tolerance and dependence for cannabinoids in vivo was unclear. Therefore, we generated mice where S426 and S430 were mutated to nonphosphorylatable alanines (S426A/S430A). S426A/S430A mutant mice were more sensitive to acutely administered delta-9-tetrahydrocannabinol (Δ9-THC), have delayed tolerance to Δ9-THC, and showed increased dependence for Δ9-THC. S426A/S430A mutants also showed increased responses to elevated levels of endogenous cannabinoids. CB1R desensitization in the periaqueductal gray and spinal cord following 7 d of treatment with Δ9-THC was absent in S426A/S430A mutants. Δ9-THC-induced downregulation of CB1R in the spinal cord was also absent in S426A/S430A mutants. Cultured autaptic hippocampal neurons from S426A/S430A mice showed enhanced endocannabinoid-mediated depolarization-induced suppression of excitation (DSE) and reduced agonist-mediated desensitization of DSE. These results indicate that S426 and S430 play major roles in the acute response to, tolerance to, and dependence on cannabinoids. Additionally, S426A/S430A mice are a novel model for studying pathophysiological processes thought to involve excessive endocannabinoid signaling such as drug addiction and metabolic disease. These mice also validate the approach of mutating GRK phosphorylation sites involved in desensitization as a general means to confer exaggerated signaling to GPCRs in vivo.

Mastering tricyclic ring systems for desirable functional cannabinoid activity.

There is growing interest in using cannabinoid receptor 2 (CB2) agonists for the treatment of neuropathic pain and other indications. In continuation of our ongoing program aiming for the development of new small molecule cannabinoid ligands, we have synthesized a novel series of carbazole and γ-carboline derivatives. The affinities of the newly synthesized compounds were determined by a competitive radioligand displacement assay for human CB2 cannabinoid receptor and rat CB1 cannabinoid receptor. Functional activity and selectivity at human CB1 and CB2 receptors were characterized using receptor internalization and [(35)S]GTP-γ-S assays. The structure-activity relationship and optimization studies of the carbazole series have led to the discovery of a non-selective CB1 and CB2 agonist, compound 4. Our subsequent research efforts to increase CB2 selectivity of this lead compound have led to the discovery of CB2 selective compound 64, which robustly internalized CB2 receptors. Compound 64 had potent inhibitory effects on pain hypersensitivity in a rat model of neuropathic pain. Other potent and CB2 receptor-selective compounds, including compounds 63 and 68, and a selective CB1 agonist, compound 74 were also discovered. In addition, we identified the CB2 ligand 35 which failed to promote CB2 receptor internalization and inhibited compound CP55,940-induced CB2 internalization despite a high CB2 receptor affinity. The present study provides novel tricyclic series as a starting point for further investigations of CB2 pharmacology and pain treatment.

The CB1 cannabinoid receptor C-terminus regulates receptor desensitization in autaptic hippocampal neurones

BACKGROUND AND PURPOSE The cannabinoid CB1 receptor is the chief mediator of the CNS effects of cannabinoids. In cell culture model systems, CB1 receptors both desensitize and internalize on activation. Previous work suggests that the extreme carboxy‐terminus of this receptor regulates internalization via phosphorylation of residues clustered within this region. Mutational analysis of the carboxy‐terminus of CB1 receptors has demonstrated that the last six serine/threonine residues are necessary for agonist‐induced internalization. However, the structural determinants of CB1 receptor internalization are also dependent on the local cellular environment. The importance of cell context on CB1 receptor function calls for an investigation of the functional roles of these residues in neurones.

AM841, a covalent cannabinoid ligand, powerfully slows gastrointestinal motility in normal and stressed mice in a peripherally restricted manner

Cannabinoid (CB) ligands have been demonstrated to have utility as novel therapeutic agents for the treatment of pain, metabolic conditions and gastrointestinal (GI) disorders. However, many of these ligands are centrally active, which limits their usefulness. Here, we examine a unique novel covalent CB receptor ligand, AM841, to assess its potential for use in physiological and pathophysiological in vivo studies.