本文選題：海洛因　切入點：成癮　出處：《寧波大學》2017年碩士論文　論文類型：學位論文

【摘要】：目的前額皮層(PFc)、杏仁核到伏隔核(NAc)的谷氨酸能投射對NAc多巴胺(DA)的釋放具有調節(jié)作用,但這些環(huán)路是否參與海洛因成癮時獎賞和動機的改變還不清楚。本課題觀察激活腹內側前額皮層(vm PFc)或基底外側杏仁核(BLA)到伏隔核殼部(NAc shell)通路的代謝型谷氨酸受體2/3(m Glu R2/3)及谷氨酸釋放對海洛因成癮大鼠獎賞和動機的影響,從而為海洛因成癮的早期干預提供理論依據(jù)。方法實驗一:首先,大鼠在不同海洛因給藥劑量(0.0125,0.025,0.05,0.1mg/injection/kg)下進行連續(xù)14 d的自身給藥訓練;第15天,大鼠腹腔注射m Glu R2/3的激動劑LY379268(0,0.1,0.3,1 mg/kg),隨后固定比率1(FR1)程序下進行海洛因自身給藥測試,觀察LY379268濃度和海洛因劑量對獎賞的交互作用。其次,另有4組大鼠在固定的海洛因給藥劑量下進行連續(xù)14 d的訓練(0.05mg/injection/kg,下同);第15天,大鼠腹腔注射LY379268(0,0.1,0.3,1mg/kg),隨后累計比率(PR)程序下觀察LY379268對海洛因給藥動機的影響。最后,再有4組大鼠進行連續(xù)14 d的訓練;第15 d,大鼠腹腔注射生理鹽水、LY379268(1 mg/kg)、LY341495(1 mg/kg,m Glu R2/3的拮抗劑)或LY379268+LY341495,FR1程序下觀察LY379268和LY341495對海洛因獎賞的相互影響。實驗二:首先,大鼠進行連續(xù)14 d的自身給藥訓練;第15天,大鼠分別在大腦核團腹側被蓋區(qū)(VTA)、伏隔核核部(NAc core)或NAc shell雙側微注射不同濃度的LY379268(0,0.3,1 mg/ml),每側0.5μl,隨后FR1程序下進行自身給藥測試,觀察LY379268干預海洛因獎賞的中樞位點。其次,另3組大鼠進行連續(xù)14 d訓練;第15天,對照組在單側vm PFc和對側NAc shell給予生理鹽水,其它兩組在單側vm PFc或背內側前額皮層(dm PFc)注射GABA_A+GABA_B激動劑的混合液Muscimol+Baclofen(0.06+0.6 nmol/μl,),在對側NAc shell均注射LY379268(1 mg/ml),每側0.5μl,FR1程序下進行自身給藥測試。最后,兩組大鼠進行連續(xù)14 d的訓練;第15天,對照組在單側BLA和對側NAc shell給予生理鹽水,BLA-NAc shell組在單側BLA注射Muscimol+Baclofen和對側NAc shell注射LY379268,每側0.5μl,FR1程序下進行自身給藥測試。實驗三:首先,大鼠在單側vm PFc注射谷氨酸能光遺傳病毒或陰性對照(NC)病毒,術后進行連續(xù)14 d的自身給藥訓練。第15天,大鼠在FR1程序下測試vm PFc谷氨酸能神經(jīng)元激活對獎賞的影響。NC組和vm PFc激活組測試時均用波長470 nm、直流電頻率和功率5 m W的藍光刺激,每次持續(xù)1 h,間隔1 h。其次,大鼠在單側BLA注射上述病毒并在同側NAc shell埋置16通道電極。第15天,大鼠在FR1程序下測試BLA谷氨酸能神經(jīng)元激活對獎賞的影響或在PR程序下測試對動機的影響。NC組和BLA激活組測試時均用波長470 nm、頻率25 HZ和功率5 m W的藍光刺激,每次持續(xù)1 h,間隔1 h,同時在NAc shell神經(jīng)元末梢記錄刺激前后放電的變化。實驗四:大鼠單側NAc shell注射狂犬病毒(RV)。7 d后,大鼠灌流取腦,冰凍切片后顯微鏡下觀察神經(jīng)元逆向示蹤的表達。結果實驗一:首先,雙因素方差分析發(fā)現(xiàn)LY379268濃度(F_((3,106))=3.485,p0.05)和海洛因劑量(F_((3,106))=3.045,p0.05)存在統(tǒng)計學差異,但兩者沒有交互作用(p0.05)。海洛因劑量為0.5 mg/injection/kg時LY379268對獎賞的抑制效應最明顯,0.1和0.3 mg/kg LY379268組較對照組的注射針數(shù)均明顯減少(p0.05),1 mg/kg LY379268組較對照組極明顯減少(p0.01)。其次,單因素方差分析發(fā)現(xiàn),PR程序下各組完成最后一針海洛因注射所需有效鼻觸數(shù)(F_((3,25))=3.492,p0.05)和海洛因注射針數(shù)(F_((3,25))=5.373,p0.01)均有統(tǒng)計學差異。與對照組相比,1 mg/kg LY379268組完成最后一針的有效鼻觸數(shù)和注射針數(shù)均明顯減少(p0.05和p0.01)。最后,單因素方差分析發(fā)現(xiàn),各組有效鼻觸數(shù)(F_((3,27))=4.084,p0.05)和海洛因注射針數(shù)(F_((3,27))=4.203,p0.05)均有統(tǒng)計學差異。與對照組相比,LY379268組的有效鼻觸數(shù)和注射針數(shù)均明顯減少(p0.05);與LY379268組相比,LY341495+LY379268組的有效鼻觸數(shù)和注射針數(shù)均明顯增加(p0.05)。實驗二:首先,單因素方差分析發(fā)現(xiàn)VTA各組有效鼻觸數(shù)和注射針數(shù)均沒有統(tǒng)計學差異(p0.05);NAc core各組有效鼻觸數(shù)(F_((2,15))=8.26,p0.05)和注射針數(shù)(F_((2,15))=6.16,p0.05)均具有統(tǒng)計學差異,1 mg/kg LY379268組的有效鼻觸數(shù)和注射針數(shù)較對照組均明顯減少(p0.05);NAc shell各組有效鼻觸數(shù)(F_((2,16))=12.14,p0.01)和注射針數(shù)(F_((2,16))=11.70,p0.01)均具有統(tǒng)計學差異,與對照組相比,0.3 mg/kg LY379268組的有效鼻觸數(shù)和注射針數(shù)均明顯減少(p0.05),1 mg/kg LY379268組的有效鼻觸數(shù)和注射針數(shù)均極明顯的減少(p0.01)。其次,單因素方差分析發(fā)現(xiàn)各組有效鼻觸數(shù)(F_((2,15))=6.25,p0.05)和注射針數(shù)(F_((2,15))=5.19,p0.05)均有統(tǒng)計學差異。vm PFc-NAc shell組的有效鼻觸數(shù)和注射針數(shù)較對照組均明顯減少(p0.05)。最后,獨立樣本T檢驗發(fā)現(xiàn),與對照組相比,BLA-NAc shell組的有效鼻觸數(shù)和注射針數(shù)均沒有明顯差異(p0.05)。實驗三:首先,單因素方差分析表明FR1程序下vm PFc各組的有效鼻觸數(shù)(F_((2,15))=5.14,p0.05)、無效鼻觸數(shù)(F_((2,15))=5.32,p0.05)和注射針數(shù)(F_((2,15))=5.18,p0.05)均有統(tǒng)計學差異。與對照組相比,vm PFc激活組的有效鼻觸數(shù)和注射針數(shù)明顯減少(p0.05),無效鼻觸數(shù)明顯增加(p0.05);與NC組相比,vm PFc激活組的有效鼻觸數(shù)呈減少趨勢(p=0.063)。其次,單因素方差分析表明,FR1程序下BLA各組有效鼻觸數(shù)接近統(tǒng)計學差異(p=0.058),而注射針數(shù)沒有統(tǒng)計學差異(p0.05)。與NC組相比,BLA激活組的有效鼻觸數(shù)有減少趨勢(p=0.053)。最后,單因素方差分析表明,PR程序下BLA各組完成最后一針的有效鼻觸數(shù)和注射針數(shù)均沒有統(tǒng)計學差異(p0.05)。BLA激活組光刺激時NAc shell末梢記錄到明顯的神經(jīng)元放電。實驗四:顯微鏡下觀察到vm PFc和BLA均有神經(jīng)元胞體或軸突投射到NAc shell,且表達位置與核團注射LY379268或光遺傳病毒的位點一致。結論LY379268能明顯降低海洛因成癮大鼠的獎賞效應和給藥動機,且主要的中樞位點位于NAc shell;進一步實驗發(fā)現(xiàn)從vm PFc到NAc shell的m Glu R2/3激活參與了對海洛因獎賞和動機的抑制作用。光遺傳學研究發(fā)現(xiàn)刺激vm PFc谷氨酸能神經(jīng)元減少大鼠有效鼻觸的同時增加了無效鼻觸,可能是vm PFc到NAc shell谷氨酸能投射的激活干擾了大鼠獎賞記憶的提取;激活BLA到NAc shell的m Glu R2/3和谷氨酸能投射對海洛因成癮大鼠的獎賞和動機均沒有影響。
[Abstract]:The purpose of the prefrontal cortex, amygdala (PFc) to the nucleus accumbens (NAc) glutamatergic projection on NAc dopamine (DA) release has a regulatory role, but the loop is involved in heroin addiction when reward and motivation to change is not clear. This study observed the activation of ventromedial prefrontal cortex (VM PFc) or basolateral the amygdala (BLA) to the nucleus accumbens shell (NAc shell) metabotropic glutamate receptor 2/3 pathway (m Glu R2/3) effect on the reward and motivation of heroin addicted rats and the release of glutamate, so as to provide a theoretical basis for early intervention of heroin addiction. Methods: firstly, rats in different dosage of heroin the amount (0.0125,0.025,0.05,0.1mg/injection/kg) of 14 consecutive D self-administration training; fifteenth days, the rats received intraperitoneal injection of M Glu R2/3 LY379268 (0,0.1,0.3,1 mg/kg) agonist, then fixed ratio of 1 (FR1) of heroin from the program The body drug test, observation of interaction between LY379268 concentration and dose of heroin reward. Secondly, another 4 rats for 14 consecutive d training in fixed dose of heroin (0.05mg/injection/kg, the same below); fifteenth days, the rats received intraperitoneal injection of LY379268 (0,0.1,0.3,1mg/kg), then the cumulative effect ratio (PR) LY379268 of heroin administration motivation program. Finally, another 4 rats were trained for 14 d; fifteenth D rats received intraperitoneal injection of saline, LY379268 (1 mg/kg), LY341495 (1 mg/kg, m Glu R2/ 3 antagonist) or LY379268+LY341495, LY379268 and LY341495 observation of the mutual influence of heroin the reward FR1 program. Experiment two: first, the rats for 14 d self-administration training; fifteenth days, the rats were in brain nuclei of the ventral tegmental area (VTA), nucleus accumbens nucleus (NAc core) or NAc of bilateral shell micro injection of different concentrations of L Y379268 (0,0.3,1 mg/ml), each side 0.5 L, then the FR1 procedure is carried out under self administration test, observe the effect of LY379268 centralsites heroin reward. Secondly, the other 3 groups of rats for 14 consecutive d training; fifteenth days, the control group in the unilateral VM PFc and given the physiological saline water on the side of the NAc shell, other two groups of PFc or VM in the unilateral dorsal medial prefrontal cortex (DM PFc) injection of GABA_A+GABA_B excited Muscimol+Baclofen mixture agent (0.06+0.6 nmol/ L), on the side of the NAc shell were injected LY379268 (1 mg/ml), each side is 0.5 L, the FR1 procedure is carried out under self administration test. Finally, the two group the rats were trained for 14 d; fifteenth days, the control group received saline in unilateral BLA and contralateral NAc shell, BLA-NAc Shell Group in the unilateral BLA injection of Muscimol+Baclofen and NAc on the side of shell LY379268 injection, each side is 0.5 L, the FR1 procedure is carried out under self administration test. Experiment three: first, rat in VM PFc unilateral injection of glutamate can light genetic virus or negative control virus (NC), after 14 consecutive D self-administration training. Fifteenth days, rats can reward on neuronal activation effect of.NC group and VM PFc group were used to test the activation wavelength of 470 nm test VM PFc glutamic acid in the FR1 program. The frequency and power of DC 5 m W blue light stimulation, each lasting 1 h, interval 1 h. second, rats in the unilateral BLA injection of the virus and in the same side of the NAc shell embedded 16 channel electrode. For fifteenth days, rats can influence neuronal activation of reward or test in the PR process of motivation effect of.NC group and BLA group were used to test the activation wavelength of 470 nm test BLA glutamic acid in the FR1 program, the frequency of 25 HZ and 5 m power W light stimulation, each lasting 1 h, 1 h interval at the same time, the changes in peripheral stimulation before and after discharge of shell neurons recorded NAc. Experiment four: Rats with unilateral NAc shell Injection of rabies virus (RV).7 d after rat brain neurons, reverse tracing observation after frozen section under the microscope. Results: first, a double factor variance analysis showed that the concentration of LY379268 (F_ ((3106)) =3.485, P0.05 (F_) and heroin dose ((3106)) =3.045 P0.05), there was significant difference, but there is no interaction (P0.05) of heroin. When the dose was 0.5 mg/injection/kg the inhibitory effect of LY379268 on reward the most obvious, the needle number 0.1 and 0.3 mg/kg LY379268 group than in the control group were significantly reduced (P0.05), 1 mg/kg LY379268 group than in the control group decreased significantly (P0.01) Secondly, the single factor variance analysis showed that, the PR program was finally completed a heroin injection required effective nasal contact number (F_ ((3,25)) =3.492, P0.05) and heroin injection needles (F_ ((3,25)) =5.373, P0.01) were statistically significant. Compared with the control group, 1 mg/kg LY37926 8緇勫畬鎴愭渶鍚庝竴閽堢殑鏈夋晥榧昏Е鏁板拰娉ㄥ皠閽堟暟鍧囨槑鏄懼噺灝，

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