Analysis of molecular mechanism of neuronal circuit formation with newly generated interneurons in the mouse olfactory bulb

在本实验室生产的巢蛋白-绿色荧光蛋白转基因小鼠嗅球（OB）中，观察到两种表达gfp的细胞。在OB中，GFP表达弱的细胞（W型细胞）为未成熟的颗粒细胞，GFP表达强的细胞（S型细胞）为成熟的颗粒细胞，具有特征形态。通过实验调节小鼠的嗅觉感觉输入，我揭示了新颗粒细胞产生后存在一个关键时期，在这个时期，它们的生存受到嗅觉感觉体验的关键影响（PNAS, 2005）。在小鼠OB分散细胞体外建立神经元回路后，加入巢蛋白- gfp小鼠OB的神经元。然后发现表达gfp的W型颗粒细胞被纳入先前存在的神经元回路中。当药物抑制神经元活性时，未成熟颗粒细胞的死亡增加。因此，我成功地建立了一个体外系统，可以有效地分析新神经元与已有神经元回路的活动依赖性整合机制。一种新型的周围靶向颗粒细胞的鉴定-强GFP表达的S型细胞表达GABA和其他成熟颗粒细胞的标记蛋白。通过细胞内染料标记，我发现S型细胞的树突尖端与二尖瓣细胞的体细胞接触，OB的兴奋性突起神经元。电镜分析显示在接触部位存在互易突触。S型细胞被发现是一种新型的gaba能抑制颗粒细胞，通过相互突触相互作用特异性地向二尖瓣细胞体发送抑制（手稿正在准备中）。

In the olfactory bulb (OB) of nestin-GFP transgenic mice produced in our lab, two types of GFP-expressing cells were observed. The cells with weak GFP expression (type W cells) were immature granule cells in the OB, and the cells with strong GFP (type S cells) were mature granule cells with characteristic morphologies.1.Molecular analysis of integration of new neurons into preexisting neuronal circuitBy experimentally regulating the olfactory sensory input to mice, I revealed the existence of a critical period after the generation of new granule cells when their survival is crucially influenced by olfactory sensory experience (PNAS, 2005).Following the establishment of neuronal circuit in vitro from dispersed cells of mouse OB, neurons derived from the nestin-GFP mouse OB were added. Then the GFP-expressing type W granule cells were found to be incorporated into the preexisting neuronal circuit. When the neuronal activity was inhibited by drug application to the culture, cell death of the immature granule cells were enhanced. Thus I have succeeded in establishing an in vitro system where mechanism of activity-dependent integration of new neurons into preexisting neuronal circuit can be effectively analyzed.2.Identification of a novel type of perisomatic-targeting granule cellsType S cells with strong GFP expression expressed GABA and other marker proteins for mature granule cells. By intracellular dye labeling, I found that the dendritic tips of type S cells contacted with soma of mitral cells, the excitatory projecting neurons of the OB. Electron microscopic analysis revealed the existence of reciprocal synapses at the contact sites. Type S cells were found to be a novel type of GABAergic inhibitory granule cells that send inhibition specifically to mitral cell soma through the reciprocal synaptic interactions (manuscript in preparation).