Characterization by Gene Expression Analysis of Two Groups of Dopaminergic Cells Isolated from the Mouse Olfactory Bulb

SIMPLE SUMMARY: In the olfactory bulb, dopaminergic cells keep being replaced throughout life in all mammalians, including humans. These neurons can be easily identified in transgenic mice expressing eGFP under the tyrosine hydroxylase promoter, as they become fluorescent, and can also be isolated from other neurons using enzymatic dissociation and fluorescence-activated cell sorting (FACS), allowing investigation of gene expression in neurons with different levels of fluorescence. We demonstrated for the first time that fluorescence intensity correlates very well with the expression of genes typical of different stages of maturation in dopaminergic neurons. We propose that the FACS method used to isolate these neurons may be used to engineer new neurons for therapeutic purposes in widespread pathologies, such as Parkinson’s disease. ABSTRACT: The olfactory bulb (OB) is one of two regions of the mammalian brain which undergo continuous neuronal replacement during adulthood. A significant fraction of the cells added in adulthood to the bulbar circuitry is constituted by dopaminergic (DA) neurons. We took advantage of a peculiar property of dopaminergic neurons in transgenic mice expressing eGFP under the tyrosine hydroxylase (TH) promoter: while DA neurons located in the glomerular layer (GL) display full electrophysiological maturation, eGFP+ cells in the mitral layer (ML) show characteristics of immature cells. In addition, they also display a lower fluorescence intensity, possibly reflecting different degrees of maturation. To investigate whether this difference in maturation might be confirmed at the gene expression level, we used a fluorescence-activated cell sorting technique on enzymatically dissociated cells of the OB. The cells were divided into two groups based on their level of fluorescence, possibly corresponding to immature ML cells and fully mature DA neurons from the GL. Semiquantitative real-time PCR was performed to detect the level of expression of genes linked to the degree of maturation of DA neurons. We showed that indeed the cells expressing low eGFP fluorescence are immature neurons. Our method can be further used to explore the differences between these two groups of DA neurons.