PROJECT SUMMARY Pain management is a major public health problem in the United States. Opioids like morphine are widely used to treat severe pain, but they possess limitations such as abuse liability and potentially lethal respiratory depression that have fed into the current opioid public health crisis. The endocannabinoid system, which includes cannabinoid receptors (CBRs), endogenous cannabinoid (eCB) lipid neurotransmitters, and enzymes responsible for the synthesis and degradation of eCBs, has emerged as a promising source of targets for development of candidate non-opioid analgesics. In particular, the enzyme monoacylglycerol lipase (MAGL) breaks down the eCB agonist 2-arachidonoylglycerol (2-AG), and MAGL inhibition can increase 2-AG levels, increase CBR activation, and potentially inhibit pain-related activity in neural pathways that mediate different pain behaviors. This F31 application proposes research that will evaluate the effectiveness of the MAGL inhibitor MJN110 to alleviate a battery of pain-related behavioral and neurochemical endpoints in adult male and female mice following intraperitoneal administration of dilute lactic acid (IP acid) as an acute, visceral, and physiologically relevant noxious stimulus. Aim 1 will determine MJN110 effectiveness to alleviate IP acid- induced sensory and affective pain-related behaviors mediated by spinal and central pain-processing pathways. The effects of MJN110 will be compared to clinically effective positive-control analgesics (the mu opioid receptor agonist oxycodone and the cyclooxygenase inhibitor ketoprofen), non-analgesic negative controls that nonspecifically depress or stimulate general behavior (the GABA-A receptor positive allosteric modulator diazepam and dopamine/norepinephrine transporter inhibitor amphetamine), and the direct CBR agonist ∆9-tetrahydrocannabinol (THC). Aim 2 will determine MJN110 effectiveness to inhibit IP acid-induced activation of sensory and affective pain pathways. Neuronal activation will be assessed with immunohistochemical and fluorescent in situ hybridization of the proto-oncogene cFos, and the chemical phenotype of cFos-positive neurons will be determined using double-labeling with antibodies selective for markers in glutamaterigic vs. GABAergic neurons. Aim 3 will use high-performance liquid chromatography- mass spectrometry to determine MJN110 effectiveness to modulate pain-related changes lipidomic profiles of fatty acids, oxilipins, and N-acylethanolimines in sensory and affective pain pathways. Overall, the proposed research will provide a detailed evaluation of the antinocicpetive effectiveness of MJN110 for acute noxious stimuli, as well as improve our understanding of the eCB system’s role in acute inflammatory pain signaling and antinociception through comparative analyses of clinically-relevant pain-related behaviors and neurochemical and lipidomic correlates.