Cannabinoid CB2 receptor (CB2) agonists are potential analgesics void of psychotropic effects. Peripheral immune cells, neurons and glia express CB2, however the involvement of CB2 from these cells in neuropathic pain remains unresolved. We explored spontaneous neuropathic pain through on-demand self-administration of the selective CB2 agonist JWH133 in wild-type and knockout mice lacking CB2 in neurons, monocytes or constitutively. Operant self-administration reflected drug-taking to alleviate spontaneous pain, nociceptive and affective manifestations. While constitutive deletion of CB2 disrupted JWH133-taking behavior, this behavior was not modified in monocyte-specific CB2 knockouts and was increased in mice defective in neuronal CB2 knockouts suggestive of increased spontaneous pain. Interestingly, CB2-positive lymphocytes infiltrated the injured nerve and possible CB2transfer from immune cells to neurons was found. Lymphocyte CB2depletion also exacerbated JWH133 self-administration and inhibited antinociception. This work identifies a simultaneous activity of neuronal and lymphoid CB2that protects against spontaneous and evoked neuropathic pain.

Background Neuropathic pain is a complex condition characterized by sensory, cognitive and affective symptoms that magnify the perception of pain. The underlying pathogenic mechanisms are largely unknown and there is an urgent need for the development of novel medications. The endocannabinoid system modulates pain perception and drugs targeting the cannabinoid receptor type 2 (CB2) devoid of psychoactive side effects could emerge as novel analgesics. An interesting model to evaluate the mechanisms underlying resistance to pain is the fragile X mental retardation protein knockout mouse (Fmr1KO), a model of fragile X syndrome that exhibits nociceptive deficits and fails to develop neuropathic pain. Methods A partial sciatic nerve ligation was performed to wild‐type (WT) and Fmr1KO mice having (HzCB2 and Fmr1KO‐HzCB2, respectively) or not (WT and Fmr1KO mice) a partial deletion of CB2 to investigate the participation of the endocannabinoid system on the pain‐resistant phenotype of Fmr1KO mice. Results Nerve injury induced canonical hypersensitivity in WT and HzCB2 mice, whereas this increased pain sensitivity was absent in Fmr1KO mice. Interestingly, Fmr1KO mice partially lacking CB2 lost this protection against neuropathic pain. Similarly, pain‐induced depressive‐like behaviour was observed in WT, HzCB2 and Fmr1KO‐HzCB2 mice, but not in Fmr1KO littermates. Nerve injury evoked different alterations in WT and Fmr1KO mice at spinal and supra‐spinal levels that correlated with these nociceptive and emotional alterations. Conclusions This work shows that CB2 is necessary for the protection against neuropathic pain observed in Fmr1KO mice, raising the interest in targeting this receptor for the treatment of neuropathic pain. Significance Neuropathic pain is a complex chronic pain condition and current treatments are limited by the lack of efficacy and the incidence of important side effects. Our findings show that the pain‐resistant phenotype of Fmr1KO mice against nociceptive and emotional manifestations triggered by persistent nerve damage requires the participation of the cannabinoid receptor CB2, raising the interest in targeting this receptor for neuropathic pain treatment. Additional multidisciplinary studies more closely related to human pain experience should be conducted to explore the potential use of cannabinoids as adequate analgesic tools.

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1Cannabinoid CB2 receptor (CB2r) agonists are potential painkillers void of 2 psychotropic effects. Peripheral immune cells, neurons and glia express CB2r, 3 however the involvement of CB2r from these cells in neuropathic pain remains 4 unresolved. We explored spontaneous neuropathic pain through on-demand self-5 administration of the selective CB2r agonist JWH133 in wild-type and knockout mice 6 lacking CB2r in neurons, monocytes or constitutively. Operant self-administration 7 reflected drug-taking to alleviate spontaneous pain, nociceptive and affective 8 manifestations. While constitutive deletion of CB2r disrupted JWH133-taking 9 behavior, this behavior was not modified in monocyte-specific CB2r knockouts and 10 was increased in mice defective in neuronal CB2r knockouts suggestive of increased 11 spontaneous pain. Interestingly, CB2r-positive lymphocytes infiltrated the injured 12 nerve and possible CB2r transfer from immune cells to neurons was found. 13 Lymphocyte CB2r depletion also exacerbated JWH133 self-administration and 14 inhibited antinociception. This work identifies a simultaneous activity of neuronal and 15 lymphoid CB2r that protects against spontaneous and evoked neuropathic pain. 16 3 Introduction 17Cannabinoid CB2 receptor (CB2r) agonists show efficacy in animal models of 18 chronic inflammatory and neuropathic pain, suggesting that they may be effective 19 inhibitors of persistent pain in humans (Bie et al., 2018; Maldonado et al., 2016; 20 Shang and Tang, 2017). However, many preclinical studies assess reflexive-21 defensive reactions to evoked nociceptive stimuli and fail to take into account 22 spontaneous pain, one of the most prevalent symptoms of chronic pain conditions in 23 humans (Backonja and Stacey, 2004; Mogil et al., 2010; Rice et al., 2018) that 24 triggers coping responses such as painkiller consumption. As a consequence, 25 conclusions drawn from animal models relying on evoked nociception may not 26 translate into efficient pharmacotherapy in humans (Huang et al., 2018; Mogil, 2009; 27 Percie du Sert and Rice, 2014), which underlines the need to apply more 28 sophisticated animal models with clear translational value. Operant paradigms in 29 which animals voluntarily self-administer analgesic compounds can provide high 30 translatability and also identify in the same experimental approach potential 31 addictive properties of the drugs (Mogil, 2009; Mogil et al., 2010; O'Connor et al., 32 2011). In this line, a previous work using a CB2r agonist, AM1241, showed drug-33 taking behavior in nerve-injured rats and not in sham-operated animals, suggesting 34 spontaneous pain relief and lack of abuse potential of CB2r agonists (Gutierrez et 35 al., 2011), although the possible cell populations and mechanisms involved remain36unknown. In addition, a recent multicenter study demonstrated off-target effects of 37 this compound on anandamide reuptake, calcium channels and serotonin, histamine 38 and kappa opioid receptors (Soethoudt et al., 2017).CB2r, the main ca...