Differentiation of hippocampal progenitor cells in vitro: temporal expression of intercellular coupling and voltage- and ligand-gated responses.
Mechanisms regulating the expression of intercellular coupling, development of membrane excitability, and cellular responsiveness to neurotransmitters during neuronal ontogeny are largely unknown. To define the temporal relationship among these properties during neurogenesis, murine embryonic hippocampal progenitor cells immortalized with a temperature-sensitive allele of the SV40 large T-antigen were examined during successive stages of neuronal differentiation in vitro using patch clamp, dye coupling, and Ca2+ imaging techniques. Electrotonic and dye coupling between untreated neuroblasts were frequent in cells maintained at the temperature (39 degrees C) nonpermissive for T-antigen expression. However, as neuroblasts differentiated into neurons under the influence of interleukin-7 added alone or concurrently with transforming growth factor-alpha after basic fibroblast growth factor, both junctional conductance and the extent of dye coupling progressively decreased. Voltage-dependent inward currents were present within 2 to 6 days after differentiating treatments began. During intermediate developmental stages (3 to 5 days in culture), cells became responsive to GABA (> or = 100 microM) but not to glutamate, glycine, or to acetylcholine (< or = 1 mM), as indicated by [Ca2+]i measurements and patch clamp recordings. In contrast, voltage- and ligand-gated responses but not electronic coupling were frequently observed in mature neuronal primary cultures. Together, these results indicate that certain cytokines may orchestrate the progressive expression of functional neuronal phenotypes in vitro, in which the gradual disappearance of intercellular coupling parallels the onset of voltage-dependent responses and both of which precede the expression of neurotransmitter chemosensitivity.
Department of Neuroscience, Albert Einstein College of Medicine, Bronx, New York 10461.
R
R
Rozental
M F
MF
Mehler
M
M
Morales
A F
AF
Andrade-Rozental
J A
JA
Kessler
D C
DC
Spray
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002450:Cell Communication; D002454:Cell Differentiation; D002478:Cells, Cultured; D005260:Female; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D017629:Gap Junctions / Q000502:physiology; D006624:Hippocampus / Q000196:embryology*; D007150:Immunohistochemistry; D015851:Interleukin-7 / Q000494:pharmacology; D051379:Mice; D016900:Neurofilament Proteins / Q000032:analysis; D009474:Neurons / Q000502:physiology*; D011247:Pregnancy; D013234:Stem Cells / Q000502:physiology*
D015851:Interleukin-7; D016900:Neurofilament Proteins; D016222:Fibroblast Growth Factor 2; D002118:Calcium