Enriched rat primary ventral mesencephalic neurons as an in-vitro culture model

Abstract

Primary ventral mesencephalic (VM) neuronal cultures are gaining importance in the study of molecular mechanisms leading to Parkinson's disease and development of treatment strategies. In practice, these neurons are cocultured with glial cells, making assessment of neuronal specific proteomic and genomic analyses difficult. Hence, development of VM neuron-enriched culture is indispensable for such analyses. In the current study, VM neurons with less than 5% of glial cells in culture were found to survive for 9 days in vitro (DIV), followed by a sudden death phase resulting in less than 5% of neuronal viability. Analysis of expression of precursor and mature neuronal markers, Nestin and MAP-2, respectively, has shown that these VM neurons attain maturity at the 7th DIV both in the presence and in the absence of glial cells. This VM neuron-enriched culture was shown to be rich in dopaminergic neurons from 7th DIV and survived up to 10th DIV. Reactive oxygen species and DNA damage estimated using CMH2CDFDA dye and comet assay, respectively, showed an increase in their respective levels at 9th DIV. Furthermore, expression of topoisomerase II β, a key player in neuronal development, was found to increase until 9th DIV, followed by a sudden decrease on 10th DIV. In conclusion, the above results provide a good working model of VM neurons in vitro along with 7th DIV as an ideal time period to study and evaluate the pro/antisurvival effects of various compounds on VM neurons.