Circuit Specific Effects of Morphine on VTA Inhibitory Neuroplasticity

项目总结 在成瘾的发展过程中，滥用药物诱导了整个奖赏通路的突触可塑性。 大脑的一部分。啮齿动物模型已经被用来证明这些变化在单一药物后开始发生 给药后，可增强腹侧被盖区(VTA)多巴胺能神经元的活性。 DA神经元的活动在很大程度上受兴奋性谷氨酸和抑制性GABA能输入的调节。 最近使用光遗传技术的研究提供了证据表明，DAR能细胞投射 不同的靶点表现出不同的药物诱导的谷氨酸能可塑性，并不同地参与了 对奖励行为的规范。尽管先前的研究表明，这种异质性存在于 VTA，这种光遗传电路水平分析独特地为研究人员提供了隔离输入/输出的能力 并直接确定其特定的突触属性。这里将使用这种方法来调查 吗啡对局部GABA能传入投射到三叉神经的DA能神经元突触可塑性的影响 区域：伏隔外侧核壳核(NAcSh)、内侧NAcSh和内侧前额叶皮质(MPFC)。 目的1-确定吗啡对VTA-GABA突触向NAcSh外侧投射的影响 假设：吗啡会降低GABA能传入外侧NAcSh的突触强度并阻断 LTPGABA在这些突触。AIM将通过将AAV2-DIO-YFP-ChR2注射到VTA来解决 用红色荧光逆行微球注射到VGAT CRE小鼠的外侧NAcSh内。这些动物将接受治疗 使用吗啡或生理盐水，局部GABA能连接到逆转录珠蛋白表达细胞的可塑性将 通过测量异步IPSC(AsIPSC)、微型IPSC(MIPSC)频率和幅度来确定， 配对脉冲比(PPR)和LTPGABA。 目的2-确定吗啡对VTA GABA突触内侧NAcSh投射的影响 假设：吗啡会降低或不改变内侧GABA能传入的突触强度 NAcSh和这些突触不表达LTPGABA。我将进行AAV2-DIO-YFP-ChR2的双重注射 VTA和红色荧光逆转录微球进入VGAT CRE小鼠NAcSh内侧。这些动物将接受治疗 使用吗啡或生理盐水，局部GABA能连接到逆转录珠蛋白表达细胞的可塑性将 通过测量asIPSCs、mIPSC频率和幅度、PPR和LTPGABA来确定。 目的3-确定吗啡对VTA GABA突触mPFC投射的影响 假设：吗啡会增加内侧NAcSh上GABA能传入的突触强度，但没有 对LTPGABA的影响。我会将AAV2-DIO-YFP-ChR2双重注射到VTA和红色荧光体内 逆转录-微球进入VGAT Cre小鼠的mPFC。这些动物将被注射吗啡或生理盐水，然后 通过测量将确定逆转录珠蛋白表达细胞上局部GABA能连接的可塑性 ASIPSCs、mIPSC频率和幅度、PPR和LTPGABA。

PROJECT SUMMARY During the development of addiction, drugs of abuse induce synaptic plasticity throughout the reward pathways of the brain. Rodent models have been used to show that these changes begin to occur after a single drug administration, enhancing the activity of dopaminergic (DAergic) neurons in the ventral tegmental area (VTA). DA neuron activity is largely regulated by excitatory glutamatergic inputs and inhibitory GABAergic inputs. Recent studies using optogenetic techniques have provided evidence indicating that DAergic cells that project to different targets display different drug-induced glutamatergic plasticity and are differentially involved in the regulation of reward behavior. Though previous studies had suggested that such heterogeneity existed in the VTA, this optogenetic circuit level analysis uniquely provides researchers with the ability to isolate input/output pairs and directly determine their specific synaptic properties. This approach will be used here to investigate morphine effects on synaptic plasticity of local GABAergic inputs onto DAergic neurons projecting to three regions: the lateral nucleus accumbens shell (NAcSh), medial NAcSh, and medial prefrontal cortex (mPFC). Aim 1 - Determine the effect of morphine on VTA GABA synapses onto lateral NAcSh projections Hypothesis: Morphine will decrease the synaptic strength of GABAergic inputs to the lateral NAcSh and block LTPGABA at these synapses. Aim will be addressed by combining AAV2-DIO-YFP-ChR2 injection into the VTA with red fluorescent retrobead injection into the lateral NAcSh of VGAT Cre mice. These animals will be treated with morphine or saline and the plasticity of local GABAergic connections onto retrobead expressing cells will be determined by measuring asynchronous IPSCs (asIPSCs), miniature IPSC (mIPSC) frequency and amplitude, paired pulse ratio (PPR), and LTPGABA. Aim 2 - Determine the effect of morphine of VTA GABA synapses onto medial NAcSh projections Hypothesis: Morphine will either decrease or not change the synaptic strength of GABAergic inputs on medial NAcSh and these synapses will not express LTPGABA. I will perform dual injections of AAV2-DIO-YFP-ChR2 into the VTA and red fluorescent retrobeads into the medial NAcSh of VGAT Cre mice. These animals will be treated with morphine or saline and the plasticity of local GABAergic connections onto retrobead expressing cells will be determined by measuring asIPSCs, mIPSC frequency and amplitude, PPR, and LTPGABA. Aim 3 - Determine the effect of morphine on VTA GABA synapses onto mPFC projections Hypothesis: Morphine will increase the synaptic strength of GABAergic inputs on medial NAcSh but have no effect on LTPGABA. I will perform dual injections of AAV2-DIO-YFP-ChR2 into the VTA and red fluorescent retrobeads into the mPFC of VGAT Cre mice. These animals will be treated with morphine or saline and the plasticity of local GABAergic connections onto retrobead expressing cells will be determined by measuring asIPSCs, mIPSC frequency and amplitude, PPR, and LTPGABA.