ARTICLE https://doi.org/10.1038/s41467-020-19780-z OPEN Endocannabinoid signaling regulates the reinforcing and psychostimulant effects of ketamine in mice Wei Xu1,3, Hongchun Li1,3, Liang Wang1, Jiamei Zhang1, Chunqi Liu1, Xuemei Wan1, Xiaochong Liu1, Yiming Hu1, ✉ Qiyao Fang1, Yuanyuan Xiao1, Qian Bu1, Hongbo Wang2, Jingwei Tian2, Yinglan Zhao1 & Xiaobo Cen 1 The abuse potential of ketamine limits its clinical application, but the precise mechanism remains largely unclear. Here we discovered that ketamine significantly remodels the 1234567890():,; endocannabinoid-related lipidome and activates 2-arachidonoylglycerol (2-AG) signaling in the dorsal striatum (caudate nucleus and putamen, CPu) of mice. Elevated 2-AG in the CPu is essential for the psychostimulant and reinforcing effects of ketamine, whereas blockade of the cannabinoid CB1 receptor, a predominant 2-AG receptor, attenuates ketamine-induced remodeling of neuronal dendrite structure and neurobehaviors. Ketamine represses the transcription of the monoacylglycerol lipase (MAGL) gene by promoting the expression of PRDM5, a negative transcription factor of the MAGL gene, leading to increased 2-AG pro- duction. Genetic overexpression of MAGL or silencing of PRDM5 expression in the CPu robustly reduces 2-AG production and ketamine effects. Collectively, endocannabinoid signaling plays a critical role in mediating the psychostimulant and reinforcing properties of ketamine. 1 National Chengdu Center for Safety Evaluation of Drugs, State Key Laboratory of Biotherapy/Collaborative Innovation Center for Biotherapy, West China Hospital, Sichuan University, 610041 Chengdu, People’s Republic of China. 2 Ministry of Education, Collaborative Innovation Center of Advanced Drug Delivery System and Biotech Drugs in Universities of Shandong, Yantai University, 264005 Yantai, People’s Republic of China. 3These authors contributed ✉ equally: Wei Xu, Hongchun Li. email: [email protected] NATURE COMMUNICATIONS | (2020) 11:5962 | https://doi.org/10.1038/s41467-020-19780-z | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-19780-z etamine, a noncompetitive antagonist of the N-methyl-D- Results Kaspartate (NMDA) receptor, has been widely used as a Ketamine significantly elevates the 2-AG level in the dorsal dissociative anesthetic agent, effective analgesic, procedural striatum. To delineate changes in the lipidome of the brain after sedative, and refractory antidepressant1. However, the reinforcing ketamine exposure, we initially performed untargeted lipidomic and rewarding properties of ketamine make it a substance analysis of the brains of mice that received an intraperitoneal (i. with abuse potential and limit its clinical use. The nonmedical p.) injection of 15 mg/kg ketamine or saline for 7 consecutive use of ketamine has steadily increased worldwide. “Special K”, days. The dose of 15 mg/kg ketamine in mice is almost equivalent mainly made of ketamine, is one of the top three most abused to recreational use in humans and is similar to the dose applied in synthetic drugs in China, causing severe individual and social other animal studies29–31. The PFC, nucleus accumbens (NAc), problems2,3. CPu (dorsal striatum), and hippocampus (Hipp), which are cri- The psychostimulant and reinforcing effects of ketamine are tically involved in various stages of the addiction cycle32, were thought to be attributed to the inhibition of NMDA receptors on collected for untargeted lipidomic analysis using liquid chroma- GABAergic neurons, as well as the disinhibition of excitatory tography coupled with tandem mass spectrometry (LC/MS–MS). neurons, causing excessive glutamate and dopamine release in the Considering the important role of emotion in drug use vulner- prefrontal cortex (PFC) and limbic striatal regions4–6. Mechan- ability and the close link between the amygdala and emotion33, istic studies have suggested that several molecules and signaling we also collected the central nucleus of the amygdala (ACe) for cascades are involved in the neurochemical mechanisms of lipidomic analysis. The major lipid classes were separated well ketamine dependence, such as extracellular signal-regulated with high resolution (Supplementary Fig. 1a). Well-fitted ortho- kinase (ERK), cAMP-responsive element binding protein gonal projections to latent structures discriminant analysis (CREB), brain-derived neurotrophic factor, and glycogen syn- models were constructed, and clear separations for each treat- thase kinase 3β, as well as changes in membrane receptors7,8. ment group were obtained in most brain regions (Supplementary Despite these advances, the downstream effects of ketamine have Fig. 1b, c). The apparent separation illustrated that the saline not been fully elucidated. group and ketamine group had profoundly different lipid profiles. Addiction is characterized by heightened reinforcement The CPu presented the most obvious lipid alterations, with from drug-paired stimuli9. The endocannabinoid system (ECS) changes in 174 lipids in 30 subclasses, and the Hipp showed the plays a crucial role in regulating the drug reinforcement and fewest lipid alterations (Fig. 1a), indicating that ketamine may the neurobehavioral effect of abuse substances, such as cocaine, preferentially affect the CPu. The detailed lipid alterations are morphine, heroin, and methamphetamine10–14.TheECS shown in Supplementary Data 1 and Data 2, and the top ten comprises G-protein-coupled receptors (CB1R and CB2R) and lipids with the most significant elevation in each brain region are small neuromodulatory lipid ligands (N-arachidonoyl listed (Supplementary Fig. 1d). Although several lipids, such as ethanolamide and 2-arachidonoylglycerol (2-AG)), as well as phosphatidylcholines (PCs) and PEs, decreased evidently in the the biosynthetic and metabolic enzymes that synthesize CPu in the ketamine group compared with the saline group and degrade these ligands, respectively13. Endocannabinoids (Supplementary Data 1), their alterations varied in different brain (eCBs) mediate retrograde synaptic signaling to suppress regions. Interestingly, a few lipid molecules of eCBs exhibited neurotransmitter release at excitatory and inhibitory synapses, brain region-specific changes. Ketamine-treated mice showed with both short- and long-term effects15.ActivationofeCB significantly higher levels of anandamide (AEA) in the CPu, ACe, signaling suppresses both excitatory and inhibitory signaling and PFC than saline-treated mice; moreover, 2-AG levels were within some neuronal circuits16, whereas blockade of eCB obviously higher in the CPu and NAc of ketamine-treated mice signaling attenuates addiction-related behaviors17–20. In addi- (Fig. 1b). In light of the critical roles of eCBs in drug reward, we tion, 2-AG and CB1R mediate neuronal morphology and focused on their potential function in mediating ketamine- growth, suggesting that eCB is involved in neuroplasticity10,21. seeking behaviors. Furthermore, chronic ketamine exposure modulates synaptic Considering the identification uncertainty in untargeted plasticity by increasing the dendritic spine density22–24.How- lipidomic analysis, we performed targeted ultra-performance ever, the contribution of eCBs to ketamine-seeking behaviors liquid chromatography tandem mass spectrometry (UPLC/MS/ and neuroplasticity has remained largely unknown. MS) to quantitatively validate the changes in eCBs in the CPu, PR domain proteins (PRDMs) are a subfamily of kruppel-like Hipp, PFC, and NAc in two ketamine addiction models: zinc-finger gene products that are known to be involved in cell locomotor activity and self-administration. We further used differentiation and growth25. PRDM5, an identified member of deuterated AEA (d4-AEA), deuterated 2-AG (d5-2-AG), and the PRDM family, is a sequence-specific transcriptional repressor deuterated palmitoylethanolamide (PEA) as internal standards encompassing an N-terminal PR domain without histone for comparison with these altered lipids and confirmed that the methyltransferase activity and C2H2 zinc-finger domains that alterations in 2-AG and AEA were specific (Supplementary mediate protein–DNA interactions26. A study showed that Fig. 2a–d). However, PEA, another ligand of eCB, was below the PRDM5 may function as a putative transcriptional repressor of detection limit. We therefore focused on AEA and 2-AG in the the Mgll gene encoding monoacylglycerol lipase (MAGL)27, and following studies. play a role in central nervous system pathophysiology after spinal The locomotor activity paradigm is used to measure the cord injury28. psychomotor and psychostimulant effects of a drug by assessing In the present study, we show that eCBs in the dorsal striatum moving distance after drug exposure. Compared to the saline (caudate nucleus and putamen, CPu) play a critical role in group, the ketamine (15 mg/kg, i.p.) group exhibited a significant ketamine addiction, and that blockade of CB1R signaling or increase in moving distance (Supplementary Fig. 3a–c), demon- overexpression of MAGL attenuates ketamine-induced behaviors strating the psychostimulant and psychomotor properties of and dendritic remodeling. PRDM5 functions as a transcriptional ketamine. At the end of the test, we quantitatively measured AEA repressor of MAGL, which governs the hydrolysis of 2-AG in and 2-AG levels in different brain regions by LC/MS–MS. response to ketamine. Genetic manipulation of Mgll or Prdm5 Consistent with the results from the untargeted lipidomic prevents ketamine-induced