CB1 cannabinoid receptors mediate the psychoactivity of cannabis, the most commonly abused illicit drug, and are also attractive therapeutic targets. Sustained CB1 receptor activation, as happens with heavy cannabis use, or if CB1 agonists are used therapeutically on a regular basis, leads to the development of behavioral tolerance - that is, more drug is required to produce the same effect. Earlier studies by us and others in simple preparations (e.g., cell culture models) indentified two serines in the C-terminus of the CB1 receptor whose phosphorylation desensitized CB1 signaling. To test the hypothesis that phosphorylation of these residues was necessary for the development of behavioral tolerance in an animal, we made a knockin mouse mutating these two residues to alanine (S426A/S430A). In preliminary experiments we have found that these mice, compared to wildtype mice are more sensitive to ?9tetrahydrocannabinol (THC), develop tolerance to repeated administration of THC more slowly and also recover from tolerance more rapidly. In the proposed work we will use these mice as a novel model system to address significant questions relating to the development and expression of tolerance to CB1 receptor agonists by completing three specific aims: 1. What is the role of CB1 receptor serines 426 and 430 during the development of tolerance to cannabinoids? By using our S426A/S430A """"""""knockin"""""""" mouse we will determine the role of phosphorylation of these two serines in the acute response to cannabinoids, in the development of cannabinoid tolerance and dependence, and in the severity of precipitated withdrawal. 2. How does preventing phosphorylation of serines 426 and 430 affect CB1 signaling during tolerance? In these experiments we will use biochemical and electrophysiological approaches (GTP?S autoradiography, MAP kinase activation, and whole cell patch clamp of cultured neurons and in slices) to assess the """"""""biochemical"""""""" signature of CB1 tolerance and its modification in the S426A/S430A mouse. 3. What is the mechanism of """"""""delayed"""""""" tolerance? In our preliminary studies we found that while tolerance was slowed in the S426A/S430A knockin mice, it still developed. This delayed tolerance is likely to be important in the adaptive responses to chronic CB1 receptor stimulation. In this aim we will take a """"""""discovery"""""""" approach to identify potential mechanisms underlying delayed tolerance. We will use microarrays to determine the transcriptional changes that take place as tolerance to THC develops in wildtype and S426A/S430A knockin mice. We will also use mass spectrometry to test the hypothesis that delayed tolerance is due to additional CB1 receptor phosphorylation. Tolerance in the clinical setting is significant and often difficult to manage problem. The completion of these studies will be helpful both for managing tolerance to CB1 agonists as it occurs in the therapeutic setting as well as for understanding the consequences of sustained and high intensity recreational cannabis use.

Public Health Relevance

Repeated use of cannabis, socially or therapeutically, leads to chronic adaptations in neurons where more drug is required to produce the same effect. This proposal tests the hypothesis that phosphorylation of two serines of the CB1 cannabinoid receptor is responsible for both tolerance and dependence that develop during chronic cannabis use. If these phosphorylation events underlie the adaptive changes that occur in neurons during cannabis use this will help us to better understand the implications of heavy cannabis use as well as sustained use of medical marijuana.

National Institute of Health (NIH)
National Institute on Drug Abuse (NIDA)
Research Project (R01)
Project #
Application #
Study Section
Molecular Neuropharmacology and Signaling Study Section (MNPS)
Program Officer
Pilotte, Nancy S
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Indiana University Bloomington
Schools of Arts and Sciences
United States
Zip Code
Leishman, Emma; Cornett, Ben; Spork, Karl et al. (2016) Broad impact of deleting endogenous cannabinoid hydrolyzing enzymes and the CB1 cannabinoid receptor on the endogenous cannabinoid-related lipidome in eight regions of the mouse brain. Pharmacol Res 110:159-72
Delgado-Peraza, Francheska; Ahn, Kwang H; Nogueras-Ortiz, Carlos et al. (2016) Mechanisms of Biased β-Arrestin-Mediated Signaling Downstream from the Cannabinoid 1 Receptor. Mol Pharmacol 89:618-29
Tung, Li-Wei; Lu, Guan-Ling; Lee, Yen-Hsien et al. (2016) Orexins contribute to restraint stress-induced cocaine relapse by endocannabinoid-mediated disinhibition of dopaminergic neurons. Nat Commun 7:12199
Xu, Changqing; Hermes, Douglas J; Mackie, Ken et al. (2016) Cannabinoids Occlude the HIV-1 Tat-Induced Decrease in GABAergic Neurotransmission in Prefrontal Cortex Slices. J Neuroimmune Pharmacol 11:316-31
Murataeva, Natalia; Dhopeshwarkar, Amey; Yin, Danielle et al. (2016) Where's my entourage? The curious case of 2-oleoylglycerol, 2-linolenoylglycerol, and 2-palmitoylglycerol. Pharmacol Res 110:173-80
Dhopeshwarkar, Amey; Mackie, Ken (2016) Functional Selectivity of CB2 Cannabinoid Receptor Ligands at a Canonical and Noncanonical Pathway. J Pharmacol Exp Ther 358:342-51
Carey, Lawrence M; Slivicki, Richard A; Leishman, Emma et al. (2016) A pro-nociceptive phenotype unmasked in mice lacking fatty-acid amide hydrolase. Mol Pain 12:
Lu, Hui-Chen; Mackie, Ken (2016) An Introduction to the Endogenous Cannabinoid System. Biol Psychiatry 79:516-25
Leishman, Emma; Mackie, Ken; Luquet, Serge et al. (2016) Lipidomics profile of a NAPE-PLD KO mouse provides evidence of a broader role of this enzyme in lipid metabolism in the brain. Biochim Biophys Acta 1861:491-500
Deng, Liting; Cornett, Benjamin L; Mackie, Ken et al. (2015) CB1 Knockout Mice Unveil Sustained CB2-Mediated Antiallodynic Effects of the Mixed CB1/CB2 Agonist CP55,940 in a Mouse Model of Paclitaxel-Induced Neuropathic Pain. Mol Pharmacol 88:64-74

Showing the most recent 10 out of 202 publications