Figure 5.

Incorporation of catecholamine polymers into neuromelanin granules following exposure to precursor molecules in cultured neurons. Rat fetal Substantia nigra tissue was purchased commercially and induced to form dopaminergic neural networks according to published procedures [5]. Addition of control buffer solution (panels A and B, upper left frame) is shown compared with additions of 50 uM levodopa (l-DOPA; l-3,4-dihydroxyphenylalanine; Panels A and B; upper right frame) or 50 uM 6-hydroxy-levodopa (6-hydroxy l-DOPA; 2,4,5-trihydroxy-l-phenylalanine; Panels A and B; lower left frame) and a combination of both (panels A and B; lower right frame). Images were taken using an EVOS tabletop fluorescence microscope under visible light conditions (Panel A) or using the appropriate fluor filter (Panel B). Control cells are faint and difficult to see under these conditions due to the lack of accumulated melanin. Levodopa, which is presumably decarboxylated rapidly to dopamine upon entering the cell through the membrane as a neutral zwitterion, caused the cells to display an initial shocked period during catecholamine polymerization and accumulation into NM granules, recovering to a healthy state over a period of weeks. The 6-hydroxydopamine counterpart to levodopa (2,4,5-trihydroxy-l-phenylalanine) displayed similar characteristics but resulted in a noticeably more darkened pigmented granule. Dopamine appears less localized in cells that have accumulated melanin, with this effect being most pronounced in those cells treated with a combination of l-DOPA along with the 6-hydroxy counterpart.