Gene Expression Profiling with Cre-Conditional Pseudorabies Virus Reveals a Subset of Midbrain Neurons That Participate in Reward Circuitry
Gene Expression Profiling with Cre-Conditional Pseudorabies Virus Reveals a Subset of Midbrain Neurons That Participate in Reward Circuitry
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DOI：
10.1523/jneurosci.3193-16.2017
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发表时间：
2017-04-12
影响因子：
5.3
通讯作者：
Friedman, Jeffrey M.
中科院分区：
文献类型：
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作者：
Pomeranz, Lisa E.;Ekstrand, Mats I.;Friedman, Jeffrey M.
中脑边缘多巴胺通路接受来自大脑许多区域的输入，作为神经系统的一部分，该神经系统检测奖励刺激并协调行为反应。因此，同时映射和分子定义这一复杂的多突触回路的组件的能力将促进我们对动机行为决定因素的理解。为了实现这一点，我们构建了伪狂犬病病毒(PRV)株，其中病毒的繁殖和荧光团的表达只有在接触Cre重组酶后才被激活。一旦在表达Cre的神经元中被激活，病毒就会连续标记突触前神经元链。GFP和mCherry跟踪病毒的双重注射同时照亮黑质纹状体和中脑边缘回路，且没有重叠，表明PRV的传播仅限于突触连接的神经元。为了从分子水平研究中脑边缘多巴胺神经元及其突触前传入，我们将Cre条件GFP病毒注射到(抗GFP)纳米体-L10转基因小鼠的NAC中，并免疫沉淀了逆行追踪后感染的神经元的翻译核糖体。对纯化的RNA的分析显示，在中缝背核和下丘脑外侧核的神经元中表达的转录产物丰富，投射到中脑边缘多巴胺回路。这些研究确定了中脑边缘多巴胺途径的重要输入，并进一步表明，PRV电路导向的翻译核糖体亲和纯化可以广泛应用于识别由复杂的多突触电路组成的分子定义的神经元。
The mesolimbic dopamine pathway receives inputs from numerous regions of the brain as part of a neural system that detects rewarding stimuli and coordinates a behavioral response. The capacity to simultaneously map and molecularly define the components of this complex multisynaptic circuit would thus advance our understanding of the determinants of motivated behavior. To accomplish this, we have constructed pseudorabies virus (PRV) strains in which viral propagation and fluorophore expression are activated only after exposure to Cre recombinase. Once activated in Cre-expressing neurons, the virus serially labels chains of presynaptic neurons. Dual injection of GFP and mCherry tracing viruses simultaneously illuminates nigrostriatal and mesolimbic circuitry and shows no overlap, demonstrating that PRV transmission is confined to synaptically connected neurons. To molecularly profile mesolimbic dopamine neurons and their presynaptic inputs, we injected Cre-conditional GFP virus into the NAc of (anti-GFP) nanobody-L10 transgenic mice and immunoprecipitated translating ribosomes from neurons infected after retrograde tracing. Analysis of purified RNA revealed an enrichment of transcripts expressed in neurons of the dorsal raphe nuclei and lateral hypothalamus that project to the mesolimbic dopamine circuit. These studies identify important inputs to the mesolimbic dopamine pathway and further show that PRV circuitdirected translating ribosome affinity purification can be broadly applied to identify molecularly defined neurons comprising complex, multisynaptic circuits.