Functional properties of neurons derived from fetal mouse neurospheres are compatible with those of neuronal precursors in vivo.
Neural stem cells can be propagated in culture as neurospheres, yielding neurons and glial cells upon differentiation. Although the neurosphere model is widely used, the functional properties of the neurosphere-derived neurons have been only partially characterized, and it is unclear whether repeated passaging alters their functional properties. In this study, we analyzed voltage- and transmitter-gated responses in neuron-like cells obtained by differentiating fetal mouse neurospheres at increasing passages in culture. We report that neurons fire overshooting action potentials in response to depolarizing currents up to passage 10 but loose this capability at later passages, as the density of voltage-gated Na(+) and K(+) currents decreases. In contrast, the immunoreactivity for the neuronal marker beta-tubulin remains unaltered up to passage 21, indicating that this marker is not representative of cell function. In almost all neurons, gamma-aminobutyric acid (GABA) evoked bicuculline-sensitive whole-cell currents, resulting from the activation of GABA(A) receptors, which appeared to be excitatory, insofar as the reversal potential of GABA-gated current was about -50 mV. Much smaller currents were elicited by the glutamatergic agonist AMPA, and only occasional responses to glycine were detected. In these functional aspects, neurosphere-derived neurons are similar to immature neurons differentiating in vivo. Therefore, at least for a limited number of passages in vitro, neurospheres provide an adequate model of in vivo neurogenesis.
Istituto Pasteur-Fondazione Cenci Bolognetti, Dipartimento di Fisiologia Umana e Farmacologia and Centro di Eccellenza BEMM, Universitá di Roma La Sapienza, Roma, Italy.
Francesca
F
Pagani
Clotilde
C
Lauro
Sergio
S
Fucile
Myriam
M
Catalano
Cristina
C
Limatola
Fabrizio
F
Eusebi
Francesca
F
Grassi
D000200:Action Potentials / Q000187:drug effects / Q000502:physiology; D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation / Q000502:physiology*; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002478:Cells, Cultured; D004558:Electric Stimulation; D018756:GABA Antagonists / Q000494:pharmacology; D058785:GABA-A Receptor Agonists; D058787:GABA-A Receptor Antagonists; D007473:Ion Channels / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D009474:Neurons / Q000166:cytology / Q000502:physiology*; D018408:Patch-Clamp Techniques; D015221:Potassium Channels / Q000187:drug effects / Q000378:metabolism; D011963:Receptors, GABA-A / Q000378:metabolism*; D015222:Sodium Channels / Q000187:drug effects / Q000378:metabolism; D018874:Spheroids, Cellular / Q000166:cytology / Q000502:physiology*; D013234:Stem Cells / Q000166:cytology / Q000502:physiology*; D005680:gamma-Aminobutyric Acid / Q000378:metabolism / Q000494:pharmacology
D018756:GABA Antagonists; D058785:GABA-A Receptor Agonists; D058787:GABA-A Receptor Antagonists; D007473:Ion Channels; D015221:Potassium Channels; D011963:Receptors, GABA-A; D015222:Sodium Channels; D005680:gamma-Aminobutyric Acid