Reconstruction and quantitative evaluation of dopaminergic innervation of striatal neurons in dissociated primary cultures.

Repairing the nigrostriatal pathway is expected to become a future treatment strategy for Parkinson disease. Our aim is to establish an in vitro model for the quantitative analysis of the nigrostriatal projections of dopaminergic neurons using primary dissociated neruons. To form the mesencephalic cell region, mesencephalic cells derived from rat embryos were plated within an isolation wall, which was removed after cell adhesion to the coverslip. After incubation for 11 days, the dopaminergic neurites extending to the outside of the mesencephalic cell region were mainly axons. Treatment with glial cell line-derived neurotrophic factor for 11 days significantly promoted the outgrowth of dopaminergic axons from the mesencephalic cell region in a concentration-dependent manner. When striatal cells were plated outside the mesencephalic cell region, dopaminergic neurites were remarkably extended to the striatal cell region. Moreover, immunocytochemistry for tyrosine hydroxylase and synaptophysin revealed that dopaminergic axons formed synapses with striatal neurons. By contrast, spinal cells did not increase dopaminergic neurite outgrowth. These results indicate that the present method is valuable for evaluating nigrostriatal projections in vitro.