Study On The Signaling Pathway Of DOM Activated 5-HT_2AR Induced Head Twitch Response In Mice

Background:2,5-dimethoxy-4-methylamphetamine（DOM）is a powerful psychoactive substance that can induce profound alterations of human consciousness,emotion,and cognition.DOM is also a type of new drug that seriously endangers public health and property safety.In recent years,numerous studies have shown that DOM has good medical value.Studies have found that the major target of DOM for hallucinogenic effects in the human is 5-hydroxytryptamine 2A Receptor(5-HT_2AR),but the precise molecular regulation mechanism followed the receptor activation is still unclear.In order to avoid the disadvantages of hallucinogens,we must first clarify the mechanism of their action.The head twitch response（HTR）in rodents is a rapid rhythmic paroxysmal side-to-side rotational head movement after administration of DOM,HTR in mice are highly correlated with the hallucinogenic effects of DOM in humans.Some 5-HT_2AR agonists,such as lisuride（LIS）and ergotamine（ERG）,have not been reported to cause hallucinations in humans,nor do they cause HTR in mice.The researchers speculate that this difference may be related to the biased 5-HT_2AR signaling.In addition to coupling the classical Gα_q/11-PLC_βsignaling pathway,5-HT_2AR can also couple with the Gα_i/o,Gα_12/13 andβ-arrestin2 signaling pathways.However,the post-receptor signaling pathway that mainly mediates the HTR and induces hallucinations is still unclear.Objective:To demonstrate the key molecular pathways of DOM-induced HTR in mice.Illustrate the post-receptor molecular mechanism of the special pharmacological effects induced by DOM,that will provide the basis for the design of new compounds with therapeutic effects and without hallucinogenic effects.Methods:（1）Demonstration of the target of DOM-induced HTR in miceTwo drugs including DOM and LIS were used.The effects of DOM and LIS on the motor ability of C57BL/6J mice were evaluated by a locomotor activity model.The HTR model was used to evaluate the effects of DOM and LIS on the HTR of C57BL/6J mice.M100907,the 5-HT_2AR highly selective antagonist,was pretreated to demostrate that 5-HT_2AR is a key target of DOM-induced HTR in mice.（2）The signaling pathway mechanism of 5-HT_2AR-mediated HTR in DOM（1）Gα_q/11 inhibitor（YM-254890）,calcium store inhibitor（TMB-8,heparin）and L-type calcium channel blocker（nimodipine）were administered respectively to explore the effect of Gα_q/11 signaling pathway on DOM-induced HTR in mice.HEK293 cells transiently or stably transfected with 5-HT_2AR were used to explore the effects of DOM or LIS on intracellular IP1（HTRF IP1 assay）,Ca²⁺（FLIPR Calcium6 experiment）,c AMP(Glo Sensor^TMc AMP assay).In HEK293 cells transiently or stably transfected with 5-HT_2AR,YM-254890 was administered to explore the effect of Gα_q/11 signaling pathway on the increase of intracellular IP1,Ca²⁺and c AMP induced by DOM.（2）The mice were given the Gα_i/o inhibitor PTX to explore the effect of Gα_i/osignaling pathway on the DOM-induced HTR in mice.（3）Mice were given a Gα_s inhibitor（melittin）or c AMP analog（8-Br-c AMP）to explore the effect of the Gα_s signaling pathway on the DOM-induced HTR in mice.Transient or stably transfected 5-HT_2AR HEK293 cells were administered with melittin to explore the effect of Gα_s signaling pathway on the increase of intracellular c AMP,IP1 and Ca²⁺induced by DOM.（4）Mice were given Gβγinhibitor（gallein）or overexpressed Gβγinhibitory peptide(βARK_CT)in the brain to explore the effect of Gβγsignaling pathway on DOM-induced HTR in mice.Transient or stably transfected 5-HT_2AR HEK293 cells were administered with gallein to explore the effect of Gβγsignaling pathway on the increase of intracellular c AMP,IP1 and[Ca²⁺]_i induced by DOM.（5）β-arrestin2 knockout mice were used to explore the effect ofβ-arrestin2signaling pathway on the DOM-induced HTR in mice.Results:（1）Demonstration of the target of DOM-induced HTR in mice（1）DOM had a tendency to increase the locomotor activity of mice at low doses,but LIS had no effect at low doses.DOM and LIS significantly attenuated the locomotor activity of mice at high doses compared with the vehicle group（P<0.05）.（2）Compared with the vehicle group,DOM induced a strong HTR in mice（P<0.001）,LIS did not.The highly selective 5-HT_2AR antagonist M100907dose-dependently reduced the HTR induced by DOM（2.50 mg/kg,i.p.）in mice;M100907（10.00μg/kg,i.p.）,which had no effect on the locomotor activity and HTR of mice,could significantly inhibit the HTR induced by DOM（P<0.05）.（2）The signaling pathway of 5-HT_2AR-mediated HTR（1）The relationship between Gα_q/11 signaling pathway and HTR in mice:The Gα_q/11 inhibitor YM-254890（0.10 nmol/5μL,i.c.v.）could significantly reduce the HTR induced by DOM（P<0.05）;Intracellular calcium store inhibitors TMB-8（20.00μg/5μL,i.c.v.）and heparin（5.00 nmol/5μL,i.c.v.）could significantly reduce the HTR induced by DOM（P<0.05）;calcium channel antagonist nimodipine（4.00mg/kg,i.p.）could significantly reduce the HTR induced by DOM（P<0.01）.In stably transfected 5-HT_2AR-HEK293T cell line,LIS(10^-10～10^-5 M)up-regulated the intracellular IP1 content to 42%.DOM significantly up-regulated the intracellular IP1content at the concentration of 10^-9.5-10^-5 M.At the concentration was 10^-7 M,the IP1was up-regulated by DOM and reached the maximum 114%,respectively.M100907（1μM）significantly inhibited the up-regulation of IP1 induced by DOM（P<0.001）.In stably transfected 5-HT_2AR-HEK293T cell line,LIS(10^-9～10^-5 M)had no effect on intracellular[Ca²⁺]_i.DOM concentration-dependently(10^-9～10^-5 M)up-regulated intracellular[Ca²⁺]_i.At the concentration 10^-5 M,the intracellular[Ca²⁺]_i reach the peak activation ratios 100%under the DOM addition.M100907（1μM）significantly inhibited the up-regulation of[Ca²⁺]_i levels induced by DOM（P<0.001）.LIS(10^-10～10^-5 M)had no effect on the intracellular c AMP content of HEK293 transiently transfected with 5-HT_2AR,whereas DOM could significantly increase the intracellular c AMP content.When the DOM concentration was 10^-5 M,the c AMP content reached its peak,which was 90%higher than that of the vehicle group.M100907（1μM）significantly inhibited the DOM-induced increases in c AMP levels（P<0.001）.YM-254890（1μM）significantly inhibited the increase of c AMP accumulation induced by DOM.At the cellular level,YM-254890（1μM）could significantly inhibit the up-regulation of c AMP induced by DOM,and completely antagonize the increase of IP1 and[Ca²⁺]_i induced by DOM.（2）The relationship between Gα_i/o signaling pathway and HTR in mice:The Gα_i/o inhibitor PTX（0.25μg/5μL,i.c.v.）had no effect on the total number of HTR induced by DOM in the low-dose group,but significantly increased the DOM-induced HTR at the middle and high dose.（3）The relationship between Gα_s signaling pathway and HTR in mice:The Gα_sinhibitor melittin（0.05 nmol/5μL,i.c.v.）significantly reduced the HTR induced by DOM（P<0.05）.The analog of c AMP,8-Br-c AMP（10.00μg/5μL,i.c.v.）slightly increased the number of HTR induced by DOM.Melittin（1μM）can completely inhibit the increase of c AMP accumulation induced by DOM.Melittin（10μM）significantly inhibit the increase of IP1 induced by DOM.Melittin（1μM）can partially inhibit the increase of[Ca²⁺]_i induced by DOM.（4）The relationship between Gβγsignaling pathway and HTR in mice:The Gβγinhibitor gallein dose-dependently inhibited the HTR induced by DOM（2.50 mg/kg,i.p.）.Gallein（2.50 mg/kg,i.p.）inhibited the HTR induced by different doses of DOM（P<0.05）.The Gβγinhibitory peptideβARK_CT also significantly reduced the HTR induced by DOM（P<0.05）.Gallein 10μM significantly inhibited the increase of c AMP accumulation,IP1 and[Ca²⁺]_i induced by DOM.（5）The relationship betweenβ-arrestin2 signaling pathway and HTR in mice:The number of HTR induced by DOM inβ-arrestin2 knockout（HOM）mice was significantly decreased compared with wild-type（WT）mice,（P<0.05）.Conclusion:（1）DOM can obviously induce HTR in mice and up-regulate the levels of intracellular c AMP,IP1and[Ca²⁺]_i whereas LIS can not.（2）DOM induced mice HTR by 5-HT_2AR activation through Gα_q/11,Gα_s,and Gβγthree signaling pathways,with the up-regulation of c AMP,IP1 and[Ca²⁺]_i levels.c AMP,IP1,Ca²⁺play important role in 5-HT_2AR mediated HTR in mice.β-arrestin2signaling pathway also invovled in HTR induced by DOM.（3）The Gα_i/osignaling pathway might play a negative regulatory role in the DOM-induced HTR.Innovation:（1）We found that the Gα_s and Gβγsignaling pathways mediate the DOM-induced HTR.We found that Gα_s and Gβγsignaling pathways mediate the up-regulation of c AMP,IP1,[Ca²⁺]_i levels.These results provide a theoretical basis for the elucidation of the HTR mechanism of DOM.（2）Gα_i/o signaling pathway inhibits the HTR induced by high doses of DOM,but the mechanism needs further study.