Electrophysiological evaluation of LUHMES cells as model of human dopaminergic neurons

Abstract

The loss of dopaminergic neurons in the substantia nigra plays an important role in the development of Parkinson's disease. The symptoms typically occur once ~80% of the neurons degenerated. This cell decay can be caused or promoted by genetic defects or environmental factors including chemical compounds like pesticides. For the proper testing of neurotoxic effects and for the development of neuroprotective drugs, assays based on animal primary cells lack predictivity as the correlation between animal and human data often is weak. Therefore, models based on human neuronal cells have the potential to overcome the limitations of animal models. One interesting neuronal cell line is the LUHMES (Lund human mesencephalic) line, which consists of immortalized fetal human mesencephalic precursor cells that can be differentiated into fully postmitotic dopaminergic neurons within one week. Electrophysiological properties of LUHMES derived neurons were investigated using manual and automated patch clamp as well as high-throughput calcium imaging for a functional characterization on both single cell and network level. LUHMES neurons were capable to generate spontaneous and stimulated action potentials. The underlying Na + channels were TTX-sensitive. Biophysical and pharmacological tests indicate the presence of the Nav 1.2 subtype. Furthermore, we checked for the presence of neurotransmitter receptors and compared them to data obtained by mRNA analysis from these cells. We found that several key receptor subtypes were expressed functionally in the cells, including dopamine, serotonin and acetylcholine receptors. Next, we investigated whether the neurons were capable of forming functional neuronal networks using a high-throughput calcium imaging system. While at rest cells were quiescent, oscillatory network activity was visible in the presence of neurotransmitter receptor agonists like serotonin and norepinephrine after modulation of extracellular K+ or Ca2+ concentrations. These oscillations were sensitive to dopamine (receptor) antagonists like Haloperidol or the anticonvulsive drug Phenytoin dose-dependently. The results show that differentiated cells derived from LUHMES cells electrophysiologically display neuronal characteristics and form functional networks. The capability of using increased throughput techniques including automated patch clamp and Ca imaging makes these cells attractive for the assessment of neurotox in a human dopaminergic cell system.