title: A voltage-sensitive dye-based assay for the identification of differentiated neurons derived from embryonic neural stem cell cultures.
abstract: BACKGROUND
  Pluripotent and multipotent stem cells hold great therapeutical promise for the replacement of degenerated tissue in neurological diseases. To fulfill that promise we have to understand the mechanisms underlying the differentiation of multipotent cells into specific types of neurons. Embryonic stem cell (ESC) and embryonic neural stem cell (NSC) cultures provide a valuable tool to study the processes of neural differentiation, which can be assessed using immunohistochemistry, gene expression, Ca(2+)-imaging or electrophysiology. However, indirect methods such as protein and gene analysis cannot provide direct evidence of neuronal functionality. In contrast, direct methods such as electrophysiological techniques are well suited to produce direct evidence of neural functionality but are limited to the study of a few cells on a culture plate.
  
  
  RESULTS
  In this study we describe a novel method for the detection of action potential-capable neurons differentiated from embryonic NSC cultures using fast voltage-sensitive dyes (VSD). We found that the use of extracellularly applied VSD resulted in a more detailed labeling of cellular processes compared to calcium indicators. In addition, VSD changes in fluorescence translated precisely to action potential kinetics as assessed by the injection of simulated slow and fast sodium currents using the dynamic clamp technique. We further demonstrate the use of a finite element model of the NSC culture cover slip for optimizing electrical stimulation parameters.
  
  
  CONCLUSIONS
  Our method allows for a repeatable fast and accurate stimulation of neurons derived from stem cell cultures to assess their differentiation state, which is capable of monitoring large amounts of cells without harming the overall culture.
authors.affiliation: Neuronal Oscillations Laboratory, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Richardson N
authors.initials: RN
authors.lastname: Leão
authors.forename: Amilcar
authors.initials: A
authors.lastname: Reis
authors.forename: Amanda
authors.initials: A
authors.lastname: Emirandetti
authors.forename: Michalina
authors.initials: M
authors.lastname: Lewicka
authors.forename: Ola
authors.initials: O
authors.lastname: Hermanson
authors.forename: André
authors.initials: A
authors.lastname: Fisahn
mesh_terms: D000200:Action Potentials / Q000502:physiology; D000814:Aniline Compounds / Q000737:chemistry; D000818:Animals; D002454:Cell Differentiation; D019070:Cell Lineage; D002470:Cell Survival; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology / Q000196:embryology; D004396:Coloring Agents / Q000737:chemistry; D004594:Electrophysiology / Q000295:instrumentation / Q000379:methods; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000502:physiology; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry; D016257:Fura-2 / Q000737:chemistry; D006624:Hippocampus / Q000166:cytology; D051379:Mice; D008810:Mice, Inbred C57BL; D008959:Models, Neurological; D039902:Multipotent Stem Cells / Q000166:cytology* / Q000502:physiology; D058953:Neural Stem Cells / Q000166:cytology* / Q000502:physiology; D009474:Neurons / Q000166:cytology* / Q000502:physiology; D051381:Rats; D015203:Reproducibility of Results; D014966:Xanthenes / Q000737:chemistry
chemical_list: D000814:Aniline Compounds; D004396:Coloring Agents; C409648:Fluo 4; D005456:Fluorescent Dyes; D014966:Xanthenes; D016257:Fura-2