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

在本实验室培育的Nestin-GFP转基因小鼠的嗅球中，观察到两种类型的GFP表达细胞。1.新神经元整合到原有神经元回路的分子分析通过对小鼠嗅觉传入的实验调节，我发现在新颗粒细胞产生后存在一个关键期，此时其存活受到嗅觉经验的关键影响(PNAS，2005)。然后发现表达GFP的W型颗粒细胞被整合到先前存在的神经元回路中。当药物抑制神经元活性时，幼稚颗粒细胞的细胞死亡增加。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活性依赖性整合到原有神经元回路的机制。2.新型周体靶向颗粒细胞的鉴定绿色荧光蛋白表达强的S细胞表达GABA等成熟颗粒细胞的标志蛋白。通过细胞内染料标记，发现S细胞的树突尖端与OB的兴奋性投射神经元--二尖瓣细胞胞体接触。电子显微镜分析显示，接触部位存在相互突触。S细胞是一种新型的GABA能抑制颗粒细胞，它通过相互突触相互作用对二尖瓣细胞胞体产生抑制作用(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).