GAD67Expression And Colocalization With BNOS In The Main Olfactory Bulb Of GAD67-GFP Knock In Mouse

GAD67-GFP knock-in mouse is being widely used as a model animals in biomedical experiments. When applied to detect the existence of GAD67, the advantage is apparent. Accordingly,GAD67-GFP knock-in mouse is usually being used in the study of neuron associated with GAD67such as GABAnergic nerons.GABA is an important inhibitory neurotransmitter. The initial researches have revealed that GABA appears in every cell layer of the olfactory bulb. To precisely describe the distribution of GABA in olfactory bulb, we used GAD67-GFP knock-in mouse as our experimental object. Nitric oxide(NO) is another important neurotransmitter.studies have shown that in the olfactory cortex, NO not only could coexist with GABA,but also could be an cooperative factor intriguing NMDA rcecptors. However, there is little about these in olfactory bulb.In our experiments, firstly we identified the genetype of the mouse by gene PCR technique. Secondly, we choosed the olfactory bulb of positive GAD67-GFP knock-in mouse to do the frozen sections. Nissl staining was performed to show the cellular framework of the olfactory bulb.Double staining with anti-GFP(GAD67-GFP) and anti-bNOS was performed to detect the percentage of GAD67in the cellular layers of olfactory bulb. Immunofluorescence double-labeling experiment was conducted to explore the distribution of bNOS and the colocalization of GAD67and bNOS. The results of Gene PCR identifying the genetype of the mouse indicate that GAD67-GFP knock-in mouse had the GFP gene assured by the appearance of750bp agarose gel electrophoresis band.Nissl staining results clearly showed that in the olfactory bulb of GAD67-GFP knock-in,there were three cellular layers from the outside to the inside as follows:Glomerular layer(GL), mitral cell layer(MCL), granule cell layer(GCL).Double staining of GAD67and DAPI showed that the ration of GAD67in three cellular layers were as follow:42.98±0.92%(GL),23.64±0.84%(MCL) and77.75±0.84%(GCL). Double-labeling immunofluorescence of GAD67and bNOS showed that bNOS mainly existing in GL and MCL colocolized in GL with a small ratio.