Functional dopamine synthesis and homeostasis in differentiated neuronal cultures.

<p>A) Western blot analysis of proteins involved in dopamine synthesis and homeostasis in cells at different stages of maturation (EB: day 0, Rosette: day 16, Neuron: day 35). hiPSCs do not express TH, DAT or AADC but these were expressed as the cells differentiated in culture. DAT is expressed only in differentiated neurons. B) Immunostaining for dopamine in differentiated neurons. This labelling co-localised with TH expression. Scale bar: 70 µm. C) Dopamine content of differentiated neurons was measured by HPLC. Differentiated neurons (35 days total in culture) produced dopamine (48.98 pmol/mg ±0.389) and were responsive to L-DOPA (933.14 pmol/mg ±220.71), indicating high AADC activity. Data shown are from 4–6 wells each of 2 independent experiments and is expressed as mean ± SEM. D) Functional dopamine transporter activity was assessed by ³HDA uptake in differentiated neurons. DAT-specific uptake was demonstrated by inhibition by mazindol. Data are expressed as mean ± SEM.