GENE EXPRESSION CONTROLLING ADULT NEUROGENESIS OF DOPAMINERGIC NEURONES IN THE OLFACTORY BULB

The olfactory bulb (OB) is one of two regions of the mammalian central nervous system (CNS) which undergo continuous neuronal replacement during adulthood. New cells, originated in the subventricular zone and migrated in the OB following the rostral migratory stream, differentiate into interneurons in the granular (Gr), glomerular (GL), and external plexiform (EP) layers and synaptically integrate into the existing neural network. A significant fraction of the cells added in adulthood to the bulbar circuitry is constituted by dopaminergic (DA) neurons. We have exploited a peculiar property of DA neurones in a transgenic line of animals expressing eGFP (green fluorescent protein) under the tyrosine hydroxylase (TH) promoter: the intensity of the fluorescence reflects their degree of maturation. Mature DA neurons are known to be restricted to the glomerular layer (GL), but using this mice model, the presence of TH-GFP+ cells was detected in the mitral and external plexiform layers. Using the fluorescence-activated cell sorting after enzymatic dissociation of the OB, the fluorescent cells have been separated as a function of their level of fluorescence in three groups, corresponding to immature (mitral layer cells), intermediate (external plexiform layer cells) and fully mature DA neurones. During neurogenesis nerve cells are generated according to a precise spatio-temporal schedule. The specification of neural progenitors is subject to complex regulatory mechanisms at precise ontogenetic stages, needed to determine the neuronal types, the required quantities, and the specific location of their implant. Semiquantitative real time-PCR has been performed to detect the level of expression of several genes, showing how, and in which phase of development, changes of expression occur. We have first checked the expression of four genes, TH, AADC (L-Amino Acid Decarboxylase), DAT (Dopamine Trans-porter 1) and VMAT-2 (Vesicular Mono-amine Transporter 2). The first two are classical marker of DA neurons. DAT and VMAT-2 are also markers of the DA phenotype, but much more dependable than the first two, which can be expressed also by non-cathecolaminergic neurons. We show that all four genes are expressed in TH-GFP+ cells, and that their expression increases regularly with the progressive maturation of the cells. The differentiation of adult generated neuroblasts in dopa-minergic neurons is regulated by numerous different factors which have been divided in two groups: key regulators of the DA phenotype, and phenotype-independent regulators. Examples for each of this group are presented.