前额皮层—杏仁核—伏隔核环路在海洛因奖赏动机转换中的作用和调控机制

发布时间：2018-03-11 21:44

  本文选题：海洛因　切入点：成瘾　出处：《宁波大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的前额皮层(PFc)、杏仁核到伏隔核(NAc)的谷氨酸能投射对NAc多巴胺(DA)的释放具有调节作用,但这些环路是否参与海洛因成瘾时奖赏和动机的改变还不清楚。本课题观察激活腹内侧前额皮层(vm PFc)或基底外侧杏仁核(BLA)到伏隔核壳部(NAc shell)通路的代谢型谷氨酸受体2/3(m Glu R2/3)及谷氨酸释放对海洛因成瘾大鼠奖赏和动机的影响,从而为海洛因成瘾的早期干预提供理论依据。方法实验一:首先,大鼠在不同海洛因给药剂量(0.0125,0.025,0.05,0.1mg/injection/kg)下进行连续14 d的自身给药训练;第15天,大鼠腹腔注射m Glu R2/3的激动剂LY379268(0,0.1,0.3,1 mg/kg),随后固定比率1(FR1)程序下进行海洛因自身给药测试,观察LY379268浓度和海洛因剂量对奖赏的交互作用。其次,另有4组大鼠在固定的海洛因给药剂量下进行连续14 d的训练(0.05mg/injection/kg,下同);第15天,大鼠腹腔注射LY379268(0,0.1,0.3,1mg/kg),随后累计比率(PR)程序下观察LY379268对海洛因给药动机的影响。最后,再有4组大鼠进行连续14 d的训练;第15 d,大鼠腹腔注射生理盐水、LY379268(1 mg/kg)、LY341495(1 mg/kg,m Glu R2/3的拮抗剂)或LY379268+LY341495,FR1程序下观察LY379268和LY341495对海洛因奖赏的相互影响。实验二:首先,大鼠进行连续14 d的自身给药训练;第15天,大鼠分别在大脑核团腹侧被盖区(VTA)、伏隔核核部(NAc core)或NAc shell双侧微注射不同浓度的LY379268(0,0.3,1 mg/ml),每侧0.5μl,随后FR1程序下进行自身给药测试,观察LY379268干预海洛因奖赏的中枢位点。其次,另3组大鼠进行连续14 d训练;第15天,对照组在单侧vm PFc和对侧NAc shell给予生理盐水,其它两组在单侧vm PFc或背内侧前额皮层(dm PFc)注射GABA_A+GABA_B激动剂的混合液Muscimol+Baclofen(0.06+0.6 nmol/μl,),在对侧NAc shell均注射LY379268(1 mg/ml),每侧0.5μl,FR1程序下进行自身给药测试。最后,两组大鼠进行连续14 d的训练;第15天,对照组在单侧BLA和对侧NAc shell给予生理盐水,BLA-NAc shell组在单侧BLA注射Muscimol+Baclofen和对侧NAc shell注射LY379268,每侧0.5μl,FR1程序下进行自身给药测试。实验三:首先,大鼠在单侧vm PFc注射谷氨酸能光遗传病毒或阴性对照(NC)病毒,术后进行连续14 d的自身给药训练。第15天,大鼠在FR1程序下测试vm PFc谷氨酸能神经元激活对奖赏的影响。NC组和vm PFc激活组测试时均用波长470 nm、直流电频率和功率5 m W的蓝光刺激,每次持续1 h,间隔1 h。其次,大鼠在单侧BLA注射上述病毒并在同侧NAc shell埋置16通道电极。第15天,大鼠在FR1程序下测试BLA谷氨酸能神经元激活对奖赏的影响或在PR程序下测试对动机的影响。NC组和BLA激活组测试时均用波长470 nm、频率25 HZ和功率5 m W的蓝光刺激,每次持续1 h,间隔1 h,同时在NAc shell神经元末梢记录刺激前后放电的变化。实验四:大鼠单侧NAc shell注射狂犬病毒(RV)。7 d后,大鼠灌流取脑,冰冻切片后显微镜下观察神经元逆向示踪的表达。结果实验一:首先,双因素方差分析发现LY379268浓度(F_((3,106))=3.485,p0.05)和海洛因剂量(F_((3,106))=3.045,p0.05)存在统计学差异,但两者没有交互作用(p0.05)。海洛因剂量为0.5 mg/injection/kg时LY379268对奖赏的抑制效应最明显,0.1和0.3 mg/kg LY379268组较对照组的注射针数均明显减少(p0.05),1 mg/kg LY379268组较对照组极明显减少(p0.01)。其次,单因素方差分析发现,PR程序下各组完成最后一针海洛因注射所需有效鼻触数(F_((3,25))=3.492,p0.05)和海洛因注射针数(F_((3,25))=5.373,p0.01)均有统计学差异。与对照组相比,1 mg/kg LY379268组完成最后一针的有效鼻触数和注射针数均明显减少(p0.05和p0.01)。最后,单因素方差分析发现,各组有效鼻触数(F_((3,27))=4.084,p0.05)和海洛因注射针数(F_((3,27))=4.203,p0.05)均有统计学差异。与对照组相比,LY379268组的有效鼻触数和注射针数均明显减少(p0.05);与LY379268组相比,LY341495+LY379268组的有效鼻触数和注射针数均明显增加(p0.05)。实验二:首先,单因素方差分析发现VTA各组有效鼻触数和注射针数均没有统计学差异(p0.05);NAc core各组有效鼻触数(F_((2,15))=8.26,p0.05)和注射针数(F_((2,15))=6.16,p0.05)均具有统计学差异,1 mg/kg LY379268组的有效鼻触数和注射针数较对照组均明显减少(p0.05);NAc shell各组有效鼻触数(F_((2,16))=12.14,p0.01)和注射针数(F_((2,16))=11.70,p0.01)均具有统计学差异,与对照组相比,0.3 mg/kg LY379268组的有效鼻触数和注射针数均明显减少(p0.05),1 mg/kg LY379268组的有效鼻触数和注射针数均极明显的减少(p0.01)。其次,单因素方差分析发现各组有效鼻触数(F_((2,15))=6.25,p0.05)和注射针数(F_((2,15))=5.19,p0.05)均有统计学差异。vm PFc-NAc shell组的有效鼻触数和注射针数较对照组均明显减少(p0.05)。最后,独立样本T检验发现,与对照组相比,BLA-NAc shell组的有效鼻触数和注射针数均没有明显差异(p0.05)。实验三:首先,单因素方差分析表明FR1程序下vm PFc各组的有效鼻触数(F_((2,15))=5.14,p0.05)、无效鼻触数(F_((2,15))=5.32,p0.05)和注射针数(F_((2,15))=5.18,p0.05)均有统计学差异。与对照组相比,vm PFc激活组的有效鼻触数和注射针数明显减少(p0.05),无效鼻触数明显增加(p0.05);与NC组相比,vm PFc激活组的有效鼻触数呈减少趋势(p=0.063)。其次,单因素方差分析表明,FR1程序下BLA各组有效鼻触数接近统计学差异(p=0.058),而注射针数没有统计学差异(p0.05)。与NC组相比,BLA激活组的有效鼻触数有减少趋势(p=0.053)。最后,单因素方差分析表明,PR程序下BLA各组完成最后一针的有效鼻触数和注射针数均没有统计学差异(p0.05)。BLA激活组光刺激时NAc shell末梢记录到明显的神经元放电。实验四:显微镜下观察到vm PFc和BLA均有神经元胞体或轴突投射到NAc shell,且表达位置与核团注射LY379268或光遗传病毒的位点一致。结论LY379268能明显降低海洛因成瘾大鼠的奖赏效应和给药动机,且主要的中枢位点位于NAc shell;进一步实验发现从vm PFc到NAc shell的m Glu R2/3激活参与了对海洛因奖赏和动机的抑制作用。光遗传学研究发现刺激vm PFc谷氨酸能神经元减少大鼠有效鼻触的同时增加了无效鼻触,可能是vm PFc到NAc shell谷氨酸能投射的激活干扰了大鼠奖赏记忆的提取;激活BLA到NAc shell的m Glu R2/3和谷氨酸能投射对海洛因成瘾大鼠的奖赏和动机均没有影响。
[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缁勫畬鎴愭渶鍚庝竴閽堢殑鏈夋晥榧昏Е鏁板拰娉ㄥ皠閽堟暟鍧囨槑鏄惧噺灏，

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