A dual infection pseudorabies virus conditional reporter approach to identify projections to collateralized neurons in complex neural circuits.
A dual infection pseudorabies virus conditional reporter approach to identify projections to collateralized neurons in complex neural circuits.
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DOI：
10.1371/journal.pone.0021141
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发表时间：
2011
期刊：
影响因子：
3.7
通讯作者：
Enquist LW
中科院分区：
文献类型：
--
作者：
Card JP;Kobiler O;Ludmir EB;Desai V;Sved AF;Enquist LW
伪狂犬病病毒（PRV）的复制和跨神经元运输被广泛用于确定啮齿动物大脑中神经回路的组织。在此我们报道一种双重感染方法，它突出了与在复杂网络中有侧支的神经元的连接。该方法结合了来自一种PRV重组体（PRV - 267）的Cre重组酶（Cre）表达以及来自第二种感染性PRV毒株（PRV - 263）的Cre依赖性报告基因表达。PRV - 267既表达Cre，又表达一种与病毒衣壳蛋白VP26融合的单体红色荧光蛋白（mRFP）（VP26 - mRFP），该蛋白在感染的细胞核中积累。PRV - 263携带一个Brainbow盒，并表达一种填充细胞质的红色（dTomato）报告基因。然而，在有Cre存在的情况下，dTomato基因从盒中被重组，消除了红色报告基因的表达，并释放出黄色（EYFP）或青色（mCerulean）细胞质报告基因的表达。我们使用一个特征明确的模型进行了原理验证实验，在该模型中，将重组病毒分别注射到左右肾脏会导致肾自主神经前网络中的神经元感染。专用于一个肾脏的神经元表达PRV - 263（细胞质dTomato）或PRV - 267（核VP26 - mRFP）所特有的报告基因。双重感染的神经元表达VP26 - mRFP以及通过Cre介导的Brainbow盒重组而激活的青色或黄色细胞质报告基因。在缺乏VP26 - mRFP的神经元中青色或黄色报告基因的差异表达为与双重感染神经元有突触连接的神经元提供了一种独特的标记，这种突触关系是使用其他双重感染追踪方法无法区分的。这些数据表明在多突触回路中Cre使能的条件性报告基因表达能够识别有侧支的神经元及其突触前伙伴。
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.
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影响因子：
5.4
作者：
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通讯作者：
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