DOI: https://doi.org/10.22141/2224-0713.8.86.2016.90913

The Neurotrophic Factors Effects on Culturing N.Raphe Serotoninergic Neurons Population

I.G. Vasileva, N.P. Oleksenko, N.G. Chopic, O.I. Tsybko, O.S. Galanta, N.D. Snitsar, I.N. Shuba

Abstract


Background. Serotonergic neurons of n.raphe play a key role in the modulation of behavior, and their dysfunction is associated with severe neurological and psychiatric disorders. However, the molecular mechanisms underlying the differentiation of the nerve stem and progenitor cells and the specification of the serotonergic phenotype are not fully understood. The aim of our study was to determine the most effective neuroinductors to support the survivability and functional activity of serotonergic neurons and their determined precursors. Materials and methods. The research was carried out on the suspension culture of nerve cells, obtained from the n.raphe zone of fetal rat brain E16 gestation. Neuroinductors epidermal growth factor (EGF), fibroblast growth factor (FGFb), nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), retinoic acid and exogenic serotonin were tested in our study to determine their role in the serotonergic neurons differentiation. Such criteria were taking into account: the relevant genes expression (Nkx 2.2, Lmx 1b, Pet 1, Tph 1, Tph 2 and Sert), total serotonin content, and serotonergic neurons amount. Methods of reverse transcription polymerase chain reaction, enzyme-linked immunosorbent assay and hystochemical Falсk-Hillarp reaction were used in our work. Results. Our data demonstrated that cell population, received from the n.raphe zone of fetal rat brain E16 gestation, consists of all types of cells, taking part in the serotoninogenesis: from early mitotically-active progenitors to mature serotonergic neurons. So, it can be used as an experimental model of the neuroinductor activity testing in vitro. It is confirmed that standard culture condition supported already formed serotonergic neurons, but their generation from determined progenitors doesn’t take place. We have observed that in phase of cell expansion the presence of EGF and FGFb in n.raphe suspension culture stimulated the pool of early progenitors of serotoninogenesis. Another neuroinductors realized their effects in different ways. It has been established, that BDNF and NGF stimulated the serotonergic neurons amount increasing in the dissociated culture by 1.6 and 2 times compared with the control slices. Adding BDNF and retinoic acid leads to increased total serotonin content in the culture material. The Nkx2.2 genes expression, which is the marker of the mitosis-active serotonergic progenitors, is supported by BDNF and increased in the presence of EGF and FGFb. The gene Pet1 expression indicating the presence of serotonergic determined precursors is supported by all using factors, except exogenous serotonin. The gene Tph1 expression, marker of serotonin synthesis start, was observed in the presence of FGFb, EGF and NGF. Adding exogenic serotonin stimulated the earliest progenitors of serotogenesis, marked by the gene Nkx 2.2 expression. At the same time, it suppressed determined progenitors marked by Pet 1 and can be accumulated in the nerve cells of culture population by the reuptake way. Conclusions. So, our data show that neuroinductors, listed here, influence different phases of serotoninogenesis from the progenitor cell expansion to the serotonin synthesis in mature neurons and can be used for its regulation.


Keywords


serotonin; serotonergic neuron; nerve cell culture; nerve growth factor; brain derived neurotrophic factor; epidermal growth factor; fibroblast growth factor; retinoic acid

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