Pomeranz Lisa E, Ekstrand Mats I, Latcha Kaamashri N, Smith Gregory A, Enquist Lynn W, Friedman Jeffrey M
Laboratory of Molecular Genetics, Howard Hughes Medical Institute, Rockefeller University, New York, New York 10065.
Department of Biology, Graduate School of Arts and Science, New York University, New York, New York 10003.
J Neurosci. 2017 Apr 12;37(15):4128-4144. doi: 10.1523/JNEUROSCI.3193-16.2017. Epub 2017 Mar 10.
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 circuit-directed translating ribosome affinity purification can be broadly applied to identify molecularly defined neurons comprising complex, multisynaptic circuits. The mesolimbic dopamine circuit integrates signals from key brain regions to detect and respond to rewarding stimuli. To further define this complex multisynaptic circuit, we constructed a panel of Cre recombinase-activated pseudorabies viruses (PRVs) that enabled retrograde tracing of neural inputs that terminate on Cre-expressing neurons. Using these viruses and Retro-TRAP (translating ribosome affinity purification), a previously reported molecular profiling method, we developed a novel technique that provides anatomic as well as molecular information about the neural components of polysynaptic circuits. We refer to this new method as PRV-Circuit-TRAP (PRV circuit-directed TRAP). Using it, we have identified major projections to the mesolimbic dopamine circuit from the lateral hypothalamus and dorsal raphe nucleus and defined a discrete subset of transcripts expressed in these projecting neurons, which will allow further characterization of this important pathway. Moreover, the method we report is general and can be applied to the study of other neural circuits.
中脑边缘多巴胺通路接收来自大脑众多区域的输入，作为检测奖励性刺激并协调行为反应的神经系统的一部分。因此，能够同时绘制并从分子层面定义这个复杂多突触回路的组成部分，将推进我们对动机行为决定因素的理解。为实现这一目标，我们构建了伪狂犬病病毒（PRV）毒株，其中病毒传播和荧光团表达仅在暴露于Cre重组酶后才被激活。一旦在表达Cre的神经元中被激活，病毒会依次标记突触前神经元链。同时注射绿色荧光蛋白（GFP）和mCherry示踪病毒可同时照亮黑质纹状体和中脑边缘回路，且未显示出重叠，这表明PRV传播局限于突触连接的神经元。为从分子层面描绘中脑边缘多巴胺神经元及其突触前输入，我们将Cre条件性GFP病毒注射到（抗GFP）纳米抗体-L10转基因小鼠的伏隔核中，并从逆行示踪后被感染的神经元中免疫沉淀正在翻译的核糖体。对纯化RNA的分析揭示了在投射到中脑边缘多巴胺回路的背侧中缝核和外侧下丘脑神经元中表达的转录本的富集。这些研究确定了中脑边缘多巴胺通路的重要输入，并进一步表明PRV回路导向的翻译核糖体亲和纯化可广泛应用于识别构成复杂多突触回路的分子层面定义的神经元。中脑边缘多巴胺回路整合来自关键脑区的信号，以检测奖励性刺激并做出反应。为进一步定义这个复杂的多突触回路，我们构建了一组Cre重组酶激活的伪狂犬病病毒（PRV），能够对终止于表达Cre的神经元上的神经输入进行逆行示踪。使用这些病毒和Retro-TRAP（翻译核糖体亲和纯化），一种先前报道的分子描绘方法，我们开发了一种新技术，可提供有关多突触回路神经成分的解剖学和分子信息。我们将这种新方法称为PRV-回路-TRAP（PRV回路导向的TRAP）。使用该方法，我们确定了从外侧下丘脑和背侧中缝核到中脑边缘多巴胺回路的主要投射，并定义了在这些投射神经元中表达的离散转录本子集，这将有助于进一步表征这一重要通路。此外，我们报道的方法具有通用性，可应用于其他神经回路的研究。
