The Role Of AMPA Receptor GluAl Subunit In Pain Hypersensitivity In Nitroglycerin-induced Migraine Mice Model

Migraine is the third most common and complex disease worldwide.It affects around 15%-18%of the population.This disorder typically manifests as attacks of one-sided or two-sided,often throbbing head pain that is worsened by movement and associated with nausea,in addition to sensitivity to light(photophobia)and sound(phonophobia)and so on.Also many migraineur may suffer migraine induced pain hypersensitivity,including hyperalgesia and allodynia,which may contribute to the severity and the chronicity of migraine headache.Despite its prevalence,the basic physiology of the molecules and mechanisms underlying migraine headache are still poorly understood,making the discovery of more effective treatments extremely difficult.Many studies focused on s of calcitonin gene-related peptide(CGRP)receptor and 5-HT receptor in migraine.Therefore,if AMPA receptor involves in migraine-like pain,what drives the activation of neuronal pain pathways in susceptible patients and the mechanisms underlying migraine induced pain hypersensitivity are still unknown.a-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor(AMPAR)are glutamate gated channels that mediate most fast excitatory transmission in the central nervous system(CNS).Because of their crucial role in regulating brain function,AMPAR are under tight regulatory processes that control their biosynthesis,membrane trafficking,degradation and various post-translational modifications(e.g.phosphorylation).Previous studies have demonstrated that AMPA receptor phosphorylation plays an important role in pain transmission in neuropathic pain.Thus,it is highly possible that AMPA receptor may play a key role in migraine induced pain hypersensitivity.Vasoconstriction and vasodilation occurring in conjunction with the neurological symptoms have been considered as cornerstones of the pathophysiology of migraine headache.More and more evidence shown that trigeminovascular system play an important role in migraine headache attack.One key component of the pathophysiology is the pain-producing innervation of the trigeminovascular system:the efferent projections from the spinal cord to the dura mater and large intracranial vessels by the first(ophthalmic)division of the trigeminal nerve,and the nerve’s afferent projection from these structures to the central trigeminal nucleus caudalis(Sp5C).This system is the pivotal relay center that passes nociceptive information from the cranial vasculature to the brainstem and higher processing centres.Furthermore,activation of this system can drive neurovascular changes involving a number of vasoactive neuropeptides that are thought to be involved in migraine and thus contribute to the symptoms.Experimental animal studies found neuronal activation in brainstem areas and the hypothalamus and thalamus during trigeminovascular nociception,and similar neuronal changes were observed in patients during an episode of migraine.PET of patients suffering spontaneous migraine attacks has highlighted neuronal activation in the medulla,midbrain and hypothalamus.These data were replicated in studies that used a human experimental migraine trigger,the nitric oxide donor nitroglycerin,in which activation was shown in the brainstem,thalamus and cortical structures.This study aims to investigate the role of AMPA receptor GluAl in NTG-induced migraine-like pain and the mechanisms underlying migraine induced hypersensitivity.Materials and methods1.Three different doses of NTG(1,5,10 mg/kg,i.p.)were used to induce migraine mice model.Different behavior tests were performed at different time points after NTG injection.Light aversive behavior and mouse grimace scale(MGS)were used to evaluate the migraine spontaneous pain,head or paw withdrawal threshold and UgoBasile orofacial pain testing apparatus were used to test hypersensitivity in NTG induced migraine model.Determine if there is any difference between male and female mice.2.Immunofluorescent technique was used to detect the expression of c-fos in Sp5C neurons after NTG injection,which can determine if the trigeminal system was activated by NTG.Immunofluorescent techniques was also used to detect the expression of AMPA subunit GluAl in Sp5C neurons.3.Western blot was used to detect the protein expression of AMPA receptor subunit GluAl and the level of GluAl Ser831/845 phosphorylation in Sp5C and trigeminal ganglia(TG).GluAl S831A mutant mice were employed to confirm the role of GluAl Ser831 phosphorylation in migraine induced hypersensitivity.4.Western blot was used to detect the expression change of AMPA receptor subunit GluAl in membrane section.Calcium imaging technique were used to detect the NTG induced calcium influx in cultured Sp5C neurons and 1-naphthyl acetyl spermine(NASPM),a selective Ca2+-permeable AMPA receptor channel blocker,was used to block this effect.In vivo,the role of Ca2+-permeable AMPA receptor in NTG induced migraine hypersensitivity was determined by intro-Sp5C injection of NASPM.5.Immunofluorescent double labeling was performed to detect the co-expression of TNFa receptor and AMPA subunit GluAl in Sp5C.6.Western blot were performed to identify the expression of TNFa.7.TNFα knockout mice were employed to identify the role of TNFa in NTG induced migraine hypersensitivity.8.To determine the effect of TNFa on regulation of AMPA receptor in NTG induced migraine hypersensitivity,Western blot was used to detect TNFa(icv)induced AMPA subunit GluA1 phosphorylation in Sp5C.Results1.NTG(10 mg/kg)induced migraine-like pain in C57BL/6 wild type(WT)mice.10 mg/kg NTG induced migraine spontaneous pain followed by migraine induced hypersensitivity as reported previously.Migraine spontaneous pain included that the time mice spent in dark room increased,while the times mice went through the door entering the light room decreased,and the MGS score increased in NTG induced migraine model(P<0.05 vs control).Migraine induced hypersensitivity included that the mechanical withdrawal threshold in head and hindpaws decreased significantly(P<0.05 vs control).5 mg/kg NTG only induced migraine hypersensitivity and mild migraine spontaneous pain.1 mg/kg NTG showed no effect on nether migraine spontaneous pain or migraine hypersensitivity.There is no significant difference between male and female WT mice in NTG induced migraine model.2.c-fos positive neurons were detected in Sp5C after NTG injection,which suggests that NTG can activate Sp5C neurons;AMPA receptor subunit GluAl express widely in Sp5C neurons.3.AMPA receptor phosphorylation play a key role in NTG induced migraine hypersensitivity.Western blot data showed that AMPA receptor GluAl Ser831 phosphorylation was enhanced in Sp5C after NTG injection(P<0.05 vs control).There is no change in GluAl Ser845 site.The results in spinal cord dorsal horn were similar with Sp5C.These results were consistent with the similar mechanical hypersensitivity in head and hindpaws.Targeted mutation of GluAl Ser831 site to prevent phosphorylation significantly inhibited NTG-induced migraine-like pain(P<0.05 vs WT mice),conform the effect of AMPA receptor phosphorylation on NTG induced migraine hypersensitivity.4.AMPA receptor GluAl Ser831 phosphorylation enhanced the calcium influx by regulating AMPA receptor trafficking.Western blot data showed that NTG promoted the AMPA receptor subunit GluAl inserting into membrane in Sp5C(P<0.05 vs control)while down-regulated GluA2 expression in membrane,which may then enhance calcium influx.Calcium imaging showed that NTG incubation caused a robust Ca2+ influx in cultured Sp5C-contained brainstem neurons(P<0.01 vs control),which was dramatically inhibited by GluAl S831A(serine at the 831 site of GluA1 is mutated to alanine)phospho-deficient mutation(P<0.01 vs WT mice),and treatment with NASPM,a selective Ca2+-permeable AMPA receptor channel blocker,dose-dependentuy blocked the NTG-evoked increase of Ca2+ influx in the cultured neurons(P<0.01 vs control).We further found that intra-Sp5C injection of NASPM significantly inhibited NTG-produced mechanical hypersensitivity(P<0.01 vs control).5.Immunofluorescent double labeling detected a large number of GluAl and TNFR positive signals in Sp5C,suggesting that TNFa may involve in the regulation of Ca2+-permeable AMPA receptor in NTG-produced mechanical hypersensitivity.6.NTG enhanced the expression of TNFa,in Sp5C.Western blot results showed that the expression of TNFa increased in Sp5C after NTG injection(P<0.05 vs control).7.TNFa involves in NTG-produced mechanical hypersensitivity.Compared with WT mice,TNFa KO mice showed less mechanical hypersensitivity after NTG administration(P<0.05).8.To determine the effect of TNFa on regulation of AMPA receptor in NTG induced migraine hypersensitivity,Western blot was used to detect the TNFa(icv)induced AMPA subunit GluAl phosphorylation in Sp5C.ConclusionIn conclusion,our study demonstrates that 1)NTG(i.p.)can induce migraine-like pain and pain hypersensitivity.2)Regulation of AMPA receptor GluAl phosphorylation in the Sp5C is an important molecular mechanism for NTG-induced migraine-like pain;phosphorylation-triggered AMPA receptor switch from Ca2+-impermeable to Ca2+-permeable may further upregulate AMPA receptor phosphorylation.3)NTG increasing TNFa expression may also involve in migraine induced pain hypersensitivity by enhancing GluA1 Ser831 phosphorylation in Sp5C.