Novel strategy to selectively label excitatory and inhibitory neurons in the cerebral cortex of mice
Novel strategy to selectively label excitatory and inhibitory neurons in the cerebral cortex of mice
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DOI：
10.1016/j.jneumeth.2008.01.016
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发表时间：
2008-05-30
影响因子：
3
通讯作者：
Sik, Attila
中科院分区：
文献类型：
--
作者：
Basu, Kaustuv;Gravel, Claude;Sik, Attila
揭示神经系统之间的联系对于理解它们的功能至关重要。所有皮质区域的绝大多数神经元由兴奋性细胞组成，其活动由抑制性细胞控制。抑制性和兴奋性细胞的分布和投射模式是了解神经系统组织的关键信息。为了研究轴突投射，我们开发了一种方法，通过在含有钙/钙调蛋白依赖的蛋白激酶IIα的神经元中表达Cre重组酶的转基因小鼠，唯一地区分皮质中的兴奋性轴突和抑制性轴突。这些动物被注射了一种腺病毒载体，这种载体被设计成在非Cre表达的细胞中引导红色荧光蛋白的表达，在Cre阳性的神经元中引导绿色荧光蛋白的表达。我们在体外和体内证明，表达GFP的神经元是GABA免疫阴性(兴奋性)的，而表达RFP的细胞要么是GABA能神经元，要么是胶质细胞。病毒载体注射一周后，RFP和GFP信号在部分细胞中重叠，但在1个月后，两种信号显示完全分离。接种6个月后，GFP标记在轴突中清晰可见，而RFP仅在胞体和近端树突中保留。这项技术可以用来区分兴奋性轴突投射和抑制性轴突投射，是神经元回路分析的独特工具。(C)2008爱思唯尔B.V.版权所有..
Revealing the connections of neuronal systems is critical for understanding how they function. The vast majority of neurons in all cortical areas consist of excitatory cells whose activity is controlled by inhibitory cells. Distribution and projection patterns of inhibitory and excitatory cells are key information to understand the organization of the nervous system. To investigate axonal projections, we developed a method to uniquely distinguish excitatory axons from inhibitory ones in the cortex using transgenic mice expressing Cre recombinase in the Ca2+/calmodulin-dependent protein kinase II alpha-containing neurons. These animals were injected by an adenoviral vector engineered so that it directs red fluorescent protein expression in non-Cre-expressing cells, and green fluorescent protein in Cre-positive neurons. We demonstrated in vitro and in vivo that GFP-expressing neurons are GABA-immunonegative (excitatory), while the RFP-expressing cells are either GABAergic neurons or glial cells. One week after the viral vector injection RFP and GFP signals overlapped in a subset of cells but after 1 month, the two signals showed total segregation. Six months post-inoculation, GFP-labelling was clearly visible in axons but RFP remained only in somata and proximal dendrites. This technique can thus be used to differentiate excitatory axonal projections from inhibitory ones, and represent a unique tool in neuronal circuit analysis. (C) 2008 Elsevier B.V. All rights reserved..