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Journal of Neuroscience, Vol 14, 6744-6753, Copyright © 1994 by Society for Neuroscience
Distinct regulatory pathways control neurofilament expression and neurotransmitter synthesis in immortalized serotonergic neurons
LA White, MJ Eaton, MC Castro, KJ Klose, MY Globus, G Shaw and SR Whittemore
Miami Project, University of Miami School of Medicine, Florida 33136.
Following infection of dissociated embryonic day 13 rat medullary raphe
cells with a retrovirus encoding the temperature-sensitive mutant of SV40
large T-antigen (T-ag), a neuronal cell line, RN46A, was cloned by serial
dilution. At 33 degrees C, RN46A cells express nuclear T-ag
immunoreactivity and divide with a doubling time of 9 hr. Undifferentiated
RN46A cells express low levels of neuron-specific enolase (NSE) and low
(NF-L)-and medium (NF-M)- but not high (NF-H)- molecular-weight
neurofilament proteins. Under differentiation conditions, RN46A cells cease
dividing, take on a neuronal morphology, and express enhanced levels of NSE
and all three NF proteins. Elevation of intracellular cAMP levels increases
neurofilament protein expression, whereas activators of various other
intracellular second messenger systems have no effect. Differentiated RN46A
cells express low-affinity nerve growth factor (NGF) receptor (p75NGFR) and
are immunoreactive using an antibody that recognizes the carboxy-terminal
13 amino acids of all three trk proteins (pan-trk). Both immunoreactivities
could be potentiated by treatment with brain-derived neurotrophic factor
(BDNF), NGF, and adrenocorticotropic hormone, fragment 4-10 (ACTH4-10).
Differentiated RN46A cells express low levels of tryptophan hydroxylase
(TPH) immunoreactivity, which could be enhanced by treatment with ACTH4-10,
BDNF, or NGF. Low levels of serotonin immunoreactivity are detected in
differentiated RN46A cells, and this was potentiated by differentiating
RN46A cells with BDNF for 8 d and 40 mM KCl for days 4-8. HPLC analysis
confirmed these immunohistochemical data. RN46A cells should prove useful
to elucidate intracellular mechanisms that control neurofilament assembly
and 5-HT expression in differentiating raphe neurons.
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