Activation Of CB1R In The Ventrolateral Orbital Cortex Alleviates CPN Ligation-induced Neuropathic Pain And Negative Emotions

Chronic pain and mood disorder are prevalent psychiatric and neurological disorders.Chronic pain causes negative emotions like anxiety and depression.The pathogenesis of chronic pain and mood disorder is complex,which brings certain difficulties to the treatment of both disorders.Clinical studies have shown that patients with chronic pain have an increased likelihood of anxiety and depression as high as 80%,which is 4.9 times higher than the general population.Therefore,studying the mechanism and treatment of chronic pain and its related negative emotions is an urgent need.Humans have been using marijuana for centuries to relieve pain,and there is increasing evidence in the last decade that the endocannabinoid system(eCBs)plays a key role in nociceptive and emotional regulation.eCBs consist of cannabinoid receptors,endogenous ligands,and synthetic and degrading enzymes.The cannabinoid receptors mainly include cannabinoid 1 type receptors(CB1R)and cannabinoid 2 type receptors(CB2R).CB1R is mainly expressed in the presynaptic membrane and axons of the central nervous system and its activation can inhibit calcium channels and activate potassium channels,thereby inhibiting the release of neurotransmitters;CB2R is mainly expressed in peripheral nervous system and immune cells.The ventrolateral orbital cortex(VLO)is one of the subregions of the prefrontal cortex(PFC)and plays an important role in nociceptive,emotional and cognitive functions.VLO is a key brain region of the endogenous analgesic loop(spinal-Sm VLO-PAG-spinal cord),and a variety of receptors directly or indirectly regulate nociceptive effects by activating the brainstem descending inhibition system.A large number of CB1R is expressed in VLO and it is mainly expressed in γ-aminobutyric acid(GABA)neurons.However,whether CB1R in the VLO is involved in the regulation of nociception remains unknown.Studies have reported that the VLO contributes to the pain-induced negative emotions,and eCBs also plays an important role in the interaction between pain and the emotions.Clinical studies have also shown that cannabis can significantly relieve the anxiety and depression symptoms associated with pain in patients with chronic pain.However,it has not been reported whether VLO is involved in this cannabinoids effect of relieving neuropathic pain related mood disorders.In this study,a neuropathic pain model was established by ligation of common peroneal nerve(CPN)and allodynia was measured with the Von Frey filaments;the change of the spontaneous firing of pyramidal neurons in the VLO was observed by extracellular recording in vivo;the expression of CB1R in the VLO was measured with western blot method;CB1R knockout in the VLO was used with Cre virus;CB1R knockout efficiency in the VLO was measured with in situ hybridization method;anxiety-and depression-like behaviors were assessed by the elevated plus maze and the forced swimming test;The results are as follows:1.The spontaneous firing rate of the VLO pyramidal neurons reduced significantly after CPN ligation on day 13,which was increased by intraperitoneal injection of 20 μg/kg HU210.These results suggest that pyramidal neurons in VLO may be involved in mechanical allodynia induced by CPN ligation,and that intraperitoneal injection of HU210 can increase the activity of pyramidal neurons in the VLO in CPN-ligated mice.2.On the 13th day after CPN ligation,the expression of CB1R in VLO in the CPN ligation group was significantly higher than the SHAM group and the Control group.This result suggested that CB1R in the VLO is involved in mechanical allodynia induced by CPN ligation.3.On the 13th day after CPN ligation,microinjection of HU210(200 ng),a cannabinoid receptor agonist,into the VLO increased 50%PWT in mice;this effect was blocked by pre-microinjection of the CB1R antagonist AM281(50 ng).These results suggested that activation of the CB1R in VLO can attenuate mechanical allodynia induced by CPN ligation.4.On the 13th day after CPN ligation of VLO-GABA-CB1R-KO mice,50%PWT of VLO microinjection of HU210(200 ng)group mice was significantly lower than the WT group.These results suggested that analgesic effect of micro injection of HU210 into the VLO depends on CB1R on GABAergic neurons.5.On the 13th day after CPN ligation,mice in the CPN group spent significantly less time and entered less time into the open arms of the elevated plus maze,and showed more immobility time in the forced swimming test.Injected intraperitoneally with 20 μg/kg HU210,mice spent significantly more time and entered more times into the open arms in the elevated plus maze and showed less immobility time in the forced swimming test.Microinjected with 200 ng HU210 in the VLO,mice spent significantly more time and entered more times into the open arms in the elevated plus maze and showed less immobility time in the forced swimming test.These results suggested that intraperitoneal injection of 20 μg/kg HU210 or microinjection of 200 ng HU210 into the VLO can relieve anxiety-and depression-like behaviors caused by CPN ligation.Conclusion:Microinjection of HU210 into the VLO can alleviate neuropathic pain caused by CPN ligation,and this effect depends on CB1R on GABAergic neurons in the VLO;intraperitoneal injection and microinjection of HU210 into the VLO attenuates anxiety-and depression-like behaviors induced by CPN ligation in mice.