Department of Neuroscience, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America.
PLoS One. 2011;6(6):e21141. doi: 10.1371/journal.pone.0021141. Epub 2011 Jun 16.
Replication and transneuronal transport of pseudorabies virus (PRV) are widely used to define the organization of neural circuits in rodent brain. Here we report a dual infection approach that highlights connections to neurons that collateralize within complex networks. The method combines Cre recombinase (Cre) expression from a PRV recombinant (PRV-267) and Cre-dependent reporter gene expression from a second infecting strain of PRV (PRV-263). PRV-267 expresses both Cre and a monomeric red fluorescent protein (mRFP) fused to viral capsid protein VP26 (VP26-mRFP) that accumulates in infected cell nuclei. PRV-263 carries a Brainbow cassette and expresses a red (dTomato) reporter that fills the cytoplasm. However, in the presence of Cre, the dTomato gene is recombined from the cassette, eliminating expression of the red reporter and liberating expression of either yellow (EYFP) or cyan (mCerulean) cytoplasmic reporters. We conducted proof-of-principle experiments using a well-characterized model in which separate injection of recombinant viruses into the left and right kidneys produces infection of neurons in the renal preautonomic network. Neurons dedicated to one kidney expressed the unique reporters characteristic of PRV-263 (cytoplasmic dTomato) or PRV-267 (nuclear VP26-mRFP). Dual infected neurons expressed VP26-mRFP and the cyan or yellow cytoplasmic reporters activated by Cre-mediated recombination of the Brainbow cassette. Differential expression of cyan or yellow reporters in neurons lacking VP26-mRFP provided a unique marker of neurons synaptically connected to dual infected neurons, a synaptic relationship that cannot be distinguished using other dual infection tracing approaches. These data demonstrate Cre-enabled conditional reporter expression in polysynaptic circuits that permits the identification of collateralized neurons and their presynaptic partners.
伪狂犬病毒（PRV）的复制和跨神经元转运被广泛用于定义啮齿动物大脑中神经回路的组织。在这里，我们报告了一种双重感染方法，该方法突出了与在复杂网络中侧支化的神经元的连接。该方法结合了 PRV 重组体（PRV-267）中的 Cre 重组酶（Cre）表达和来自第二种感染 PRV 株（PRV-263）的 Cre 依赖性报告基因表达。PRV-267 表达 Cre 和与病毒衣壳蛋白 VP26（VP26-mRFP）融合的单体红色荧光蛋白（mRFP），该蛋白在感染的细胞核中积累。PRV-263 携带 Brainbow 盒并表达红色（dTomato）报告基因，该报告基因充满细胞质。但是，在 Cre 的存在下，dTomato 基因从盒中重组，消除了红色报告基因的表达，并释放了黄色（EYFP）或青色（mCerulean）细胞质报告基因的表达。我们使用一个经过充分验证的模型进行了原理验证实验，其中将重组病毒分别注射到左肾和右肾中会导致肾前自主网络中的神经元感染。专门用于一个肾脏的神经元表达 PRV-263（细胞质 dTomato）或 PRV-267（核 VP26-mRFP）的独特报告基因。双重感染的神经元表达 VP26-mRFP 和通过 Cre 介导的 Brainbow 盒重组激活的青色或黄色细胞质报告基因。在缺乏 VP26-mRFP 的神经元中表达青色或黄色报告基因，为通过突触连接到双重感染神经元的神经元提供了独特的标记，而这种突触关系无法通过其他双重感染示踪方法来区分。这些数据证明了在多突触回路中 Cre 启用条件性报告基因表达，这允许鉴定侧支神经元及其突触前伙伴。
