Replating improves whole cell voltage clamp recording of human fetal dorsal root ganglion neurons.
The whole cell patch clamp technique allows recording of membrane currents in an entire cell under voltage clamp conditions. However, technical difficulties arise in large cells bearing extensive processes, such as human fetal dorsal root ganglion (DRG) neurons in culture. In order to improve space clamp conditions, human fetal DRG neurons cultured for 1-2 weeks were enzymatically detached and replaced in new dishes, yielding round or oval cells with absent or short processes at 24 h in culture. Current clamp recordings demonstrated no difference in action potential parameters of the replated cells compared to control non-replated cells. Analysis of passive properties showed a reduction of 40% in mean specific membrane capacitance and a 57% increase in mean specific membrane resistance in the replated cells, consistent with the decrease of cell membrane surface area. Whole cell voltage clamp studies demonstrated great improvement of the space clamp, indicating that more efficient voltage clamp conditions can be achieved in neurons in culture by eliminating neurites through replating.
Caviedes
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Laboratory of Neurosciences, National Institutes on Aging, National Institutes of Health, Bethesda, MD 20892.
Ault
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Rapoport
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D004314:Down Syndrome / Q000503:physiopathology; D004553:Electric Conductivity; D004594:Electrophysiology / Q000295:instrumentation / Q000379:methods; D005260:Female; D005333:Fetus; D005727:Ganglia, Spinal / Q000502:physiology* / Q000503:physiopathology; D006801:Humans; D008564:Membrane Potentials; D009474:Neurons / Q000502:physiology*; D011247:Pregnancy; D012016:Reference Values