Selective expression of factors preventing cholinergic dedifferentiation.
Chicken retina neurons from 8-9-day-old embryos developed prominent cholinergic properties after several days in stationary dispersed cell (monolayer) culture. These cells accumulated [3H]choline by a high-affinity, hemicholinium-sensitive transport system, converted [3H]choline to [3H]-acetylcholine [( 3H]ACh), released [3H]ACh in response to depolarization stimuli, and developed choline acetyltransferase (ChAT) activity to levels comparable to those of the intact retina. The cholinergic state, however, was not permanent. After 7 days in culture, the capacity for [3H]ACh release decreased drastically and continued to diminish with longer culture periods. Loss of this capacity seemed not to be due to loss of cholinergic neurons, because high-affinity choline uptake was unchanged. However, a substantial decrease of ChAT activity was observed as a function of culture age, and probably accounted for the low level of ACh synthesis in long-lasting cultures. The loss of ChAT activity could be prevented in at least two different ways: (a) Maintaining the neurons in rotary (aggregate) rather than stationary culture completely blocked the loss of enzyme activity and gave a developmental profile identical to the known "in situ" pattern of differentiation; and (b) Conditioned medium from aggregate cultures significantly reduced the drop in ChAT activity of neurons maintained in stationary, dispersed cell cultures. Activity that stabilized cholinergic differentiation was nondialyzable, heat-sensitive, and not mimicked by functional nerve growth factor. Production of activity by aggregates was developmentally regulated; medium obtained from aggregates after 3 days in culture had no effect on cholinergic differentiation, whereas medium obtained from aggregates between 6 and 10 days in culture produced a fivefold increase of ChAT in monolayers.(ABSTRACT TRUNCATED AT 250 WORDS)
Department of Neurobiology and Physiology, Northwestern University Institute for Neuroscience, Evanston, IL 60208.
F G
FG
DeMello
M C
MC
DeMello
R
R
Hudson
W L
WL
Klein
D000109:Acetylcholine / Q000378:metabolism; D000818:Animals; D002449:Cell Aggregation; D002454:Cell Differentiation; D002642:Chick Embryo; D002794:Choline / Q000378:metabolism; D002795:Choline O-Acetyltransferase / Q000378:metabolism; D003470:Culture Media; D003584:Cytological Techniques; D009414:Nerve Growth Factors / Q000494:pharmacology; D009474:Neurons / Q000166:cytology* / Q000378:metabolism; D010275:Parasympathetic Nervous System / Q000166:cytology*; D012160:Retina / Q000166:cytology / Q000378:metabolism*; D013997:Time Factors
D003470:Culture Media; D009414:Nerve Growth Factors; D002795:Choline O-Acetyltransferase; D002794:Choline; D000109:Acetylcholine