| ObjectiveThis study will establish an animal anxiety model by using nicotinic receptor(n ACh R)antagonists in rodents or stopping chronic nicotine exposure to induce withdrawal.We also will use different models such as nicotine subcutaneous implantation pump administration model,animal intraperitoneal injection model and nicotine drinking water model,through corresponding behavioral methods,such as classic spontaneous activity and elevated cross experiment verify the most stable anxiety model and study the main brain areas related to anxiety which neurons are excitatory or which neurons are inhibitory? Because nicotine can induce addiction in humans and experimental animals,just like other members of the ligand-gated ion channel superfamily,nicotinic acetylcholine receptors(n ACh Rs)are composed of five transmembrane subunits.[3H]-nicotine has the high-affinity binding sites,the rate of dihydro-β-rubine hydrobromide(a4b2)is huge.Mecamylamine,a relatively non-specific non-competitive nicotinic acetylcholine receptor(n ACh R)antagonist,causes physical and emotional symptoms of nicotine withdrawal,while a competitive nicotinic acetylcholine receptor(n ACh R)antagonist Dihydro-β-Ruthine Hydrobromide(DHβE)only causes emotional signs of nicotine withdrawal.Studies have shown that withdrawal anxiety in animals is closely related to the amygdala or basolateral amygdala(BLA).The amygdala integrates and processes afferent information related to rewards and emotions.The destruction of regulatory neural circuits between the inner amygdala and basolateral amygdala(BLA)is caused by local gamma-aminobutyric acid(GABA)circuit and molecular changes of the main glutamatergic output neurons leading to abnormal neuroplasticity.These amygdala nucleus accumbens,prefrontal cortex,central nucleus and their connections with the midbrain are all related to the abuse of opioids and other acute drugs,positive enhancement or reward effects.Until recently,the classical theory of the function of the amygdala has been regarded the basolateral amygdala(BLA)and the central nucleus(Ce A)of the amygdala as the sensory input and output nuclei respectively.In the internal circuit of,the basolateral amygdala(BLA)is a key area of emotional learning and controls the central nucleus(Ce A)of the amygdala,which participates in the formulation of behavioral responses by projecting to the hypothalamus and brainstem.It is speculated that the emotional component of avoiding withdrawal may be more important in maintaining dependence on drugs of abuse including nicotine than the physical aspect of withdrawal,and the basolateral amygdala(BLA)may be the chief brain in regulating withdrawal anxiety.The change in the amount of food in the food reward experiment is used to verify whether the nicotine withdrawal anxiety is caused by the injection of the drug mecamylamine in C57BL/6J mice related to the gradual decrease in food intake under the stimulation of light and 85 decibels of sound.According to the clinical experience of Tongdu regulating mind and head Qi Street theory of Baihui on electroacupuncture treatment of smoking addiction and withdrawal anxiety symptoms has been done firstly by injecting Baihui acupoints through virus tracing method to explore the neural pathways from Baihui acupoint to the relevant brain regions.A nicotine drinking water model is established to compare the differences in anxiety behavior indicators of mice of the blank group,nicotine addiction group,withdrawal group and electroacupuncture group and the differences in c-Fos in the brain regions of the various groups.Then we explain whether the brain area is related to anxiety and electroacupuncture play a essential role in regulating this brain area.MethodsExperiment 1:the withdrawal model of C57BL/6J subcutaneously implanted nicotine sustained-release pump was constructed,of which the dose was 18mg/kg/d(base calculation,PH=7.2-7.4)for 14 consecutive days.On the 9th day according to the computer-generated numbers table random grouping,one group was given intraperitoneal injection of physiological saline and the other group was given mecamylamine solution,followed by drug cross use,for example,on the 12 th day,the mouse of the physiological saline group was given the mecamylamine solution and the mouse of the mecamylamine solution group was given the physiological saline for spontaneous activity and elevated cross test.On the 13 th day,the grouped administration was the same as that on the 12 th day.Take the brain 1.5 hours after administration and stain it for c-Fos to determine whether the brain area of the withdraw anxiety function was the amygdala(BLA).We also stain c-Fos and neurotransmitter glutamate,c-Fos and neurotransmitter γ-aminobutyric acid(GABA)to determine whether the subtypes of amygdala(BLA) neurons are mainly inhibitory neurons or excitatory neurons.Experiment 2:we first used C57BL/6J mice that had been injected with r AAVCa MKIIa-h M4D(Gi)-m Cherry-WPRE-h GH poly A into the bilateral basolateral amygdala(BLA).Four weeks later,an intraperitoneal injection model was established,1 mg/ml nicotine solution,twice a day for 14 consecutive days and train the mice to obtain stable amount of food as a baseline.According to the random number table generated by the computer,the mice were divided into two groups.They were given intraperitoneal injections of Clozapine N-oxide(CNO)or normal saline and they also were injected with mecamylamine solution for food reward experiments 10 minutes later.For 30 minutes,we could observe the changes in food intake under light and sound stimulation conditions.Experiment 3:The pseudorabies virus(PRV)was injected into the Baihui point of C57BL/6J mice and the brain was perfused at 120 h and the expression of PRV was observed by immunofluorescence method.Experiment 4:In the construction of nicotine drinking water experiment,according to the random number table generated by the computer,it was divided into blank group(2%saccharin sodium solution group),addiction group(nicotine solution group),withdrawal group(2% saccharin sodium solution group),electroacupuncture group,given 2/15 Hz,30minutes,once a day for three consecutive days.By electroacupuncture Baihui and Yintang points,we compared behavioral experiments of different groups of animals,spontaneous activity and elevated cross test in the central area of anxiety indicators,Central area residence time,total distance,number of open-arm entry,percentage of open-arm entry,open-arm stay time etc.Blood was collected from five groups of mice,brains were taken,plasma CORT was detected and the c-Fos protein was stained.Then we will compare different groups of CORT,c-Fos density value,verify the anti-anxiety effect of electroacupuncture in mice with withdrawal anxiety.ResultsExperiment 1 results:(1)In the subcutaneous nicotine implanted pump model,the number of times to enter the central area,the central area stay time,the total distance of the central area movement,there are no statistical differences between the saline group and the mecamylamine group(P>0.05).(2)Subcutaneous nicotine implanted pump model,c-Fos protein staining result: The abstinence chamber parachute nucleus(PVA)and the outer amygdala(BLA)brain region c-Fos density value is significantly higher than the saline group,respectively,by the pair t test comparison,n=6,t=3.185,df=5,*P=0.024;n=6,t=3.790,df=5,#P=0.013。(3)Subcutaneous nicotine implanted pump model,physiological saline group,GABA / c-Fos ratio and Glutamate/c-Fos ratio did not have significant difference in data(t=-1.467,df=5,GABA/ c-Fos vs Glutamate/c-Fos: P=0.202,paired t test).The mecamylamine solution group,GABA / c-Fos ratio and Glutamate/c-Fos ratio did not have significant difference in data(t=0.996,df=5,GABA/ c-Fos vs Glutamate/c-Fos P=0.365,paired t test).Experiment 2 Results:(1)In the nicotine solution abdominal injection model,under conditions(sound,light stimulation),the C57BL/6J mice of the saline group and the CNO group will obtain less on the amount of food after injection with mecamylamine solution,which will decreased gradually over time.Repeated measures analysis of variance,after Mauchly’s spherical hypothesis test,P=0.128,subject to the spherical hypothesis test,with withinsubjects effect test,time factor Greenhouse-Geisser F=59.009,****P=0.000,there is a significant difference.Time and Group effect,Greenhouse-Geisser F=0.476,P=0.752,there was no significant statistical difference in the interaction effect.(2)In the nicotine solution abdominal cavity injection model,the number of entries in the central area of the saline group and the mecamylamine group,the time of stay in the central area,and the total distance without statistical difference(P>0.05).Experiment 3 Results:Pseudo-rabies virus tracer neurons were mainly expressed in the amygdala(BLA)and the central core of the amygdala(Ce A)after 120 h.Experiment 4 Results:(1)In the spontaneous activity experiment of the nicotine solution drinking model,it was suggested that the number of entry in the central areas,the various groups was statistically different(P<0.05).Compared with the withdrawal group,the electroacupuncture group was **P=0.002.There was a statistical difference in the stay time in other central areas(P<0.05).Compared with the withdrawal group,the electroacupuncture group was ***P=0.001;The total distance indicated that different groups were not statistically different.In the elevated cross experiment,the number of open arm entrances in different groups were statistically different(P<0.05).Compared with the electroacupuncture group,the withdrawal group was ***P=0.001;The percentage of open arm entrances in different groups showed inconsistent normal distribution,using the Kruskal-Wallis test,the analysis results showed that ***P=0.001,which suggested that the percentage of open arm entry times in different groups was significantly different.Compared with the withdrawal group,the percentage of open arm entry times of the electroacupuncture group was **P=0.006;The result of open arm time showed that it was ANOVA,F=2.996,P=0.057;The result of electroacupuncture group was compared with the withdrawal group,**P=0.009.(2)In the drinking water model of nicotine solution,the serum CORT of the withdrawal group showed **P=0.006 compared with the blank group,the serum CORT of the withdrawal group showed *P=0.013 compared with the addiction group and the result between the needle group and the withdrawal group showed *P=0.022 was statistically significant.(3)In blank group,addiction group,withdrawal group and electroacupuncture group,c-Fos protein of mouse brain region staining result showed ANOVA,F=5.985,**P=0.004,indicating that there are significant statistical differences between different groups of c-Fos intensity(P<0.05).The withdrawal group was compared to the blank group,the result was ***P=0.001.The withdrawal group was compared with the addiction group,the result was **P =0.003 and the electroacupuncture group was compared with the withdrawal group,the result showed ##P =0.004.ConclusionCombined with the above results,it is proved that electroacupuncture at Baihui Yintang point may have a significant control effect on the withdrawal anxiety model mice constructed with the nicotine drinking experiment by regulating the brain’s basolateral amygdala(BLA)nerve signals. |
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