Exposure to pesticides, such as rotenone or paraquat, increases the risk of Parkinson's disease (PD). Various studies revealed the link between pesticide toxicity and cellular pathology in PD such as alpha-synuclein (alpha-Syn) aggregation and neuronal death. Recently, we demonstrated region-specific astrocyte-microglia interaction promoted rotenone-induced non-cell-autonomous dopaminergic neurodegeneration. In this study, we examined effects of regional difference in glial cells on neuronal alpha-Syn expression and neurotoxicity. We prepared mesencephalic neuronal culture and glial cell culture (astrocyte+microglia) from mesencephalon or striatum of SD rats embryos (E15). Treatment with conditioned media from mesencephalic, but not striatal, glial cell culture upregulated alpha-Syn expression in mesencephalic neurons, which were dopaminergic, serotonergic and GABAergic neurons. Conditioned media from rotenone-exposed mesencephalic, but not striatal, glial cells increased alpha-Syn expression in dopaminergic and serotonergic neurons; however, neuronal damage was observed only in the dopaminergic neurons. These results suggest that regional specificity of glial cells could contribute to neuronal alpha-Syn expression, and that some factors in addition to alpha-Syn accumulation are involved in dopaminergic neurodegeneration.