Optimization of serum-free culture conditions for growth of embryonic rat cholinergic basal forebrain neurons.
The objective of the present study was to optimize conditions for culturing embryonic rat basal forebrain neurons in serum-free defined medium to be used in investigations of cholinergic neuron function and responsiveness to neurotrophic factors. It was determined that a combination of neurobasal medium (NB) and DMEM/F12 medium (DM:F12) maintained culture viability, basal choline acetyltransferase (ChAT) activity and responsiveness of these neurons to nerve growth factor (NGF) better than growth of neurons in either medium alone; all media tested contained N2 supplements. While NB which was developed initially for culturing embryonic rat hippocampal neurons supported the growth of basal forebrain neurons, they had reduced ChAT activity and did not respond to NGF with enhanced cholinergic neuronal enzyme activity. On the other hand, DM:F12 did not consistently support survival of the neurons until assay of ChAT activity on day 6 in vitro; surviving cultures were compromised in their cholinergic capacity either under basal or NGF-enhanced conditions. Cultures grown in the combined media responded to brain-derived neurotrophic factor (BDNF), but not ciliary neurotrophic factor (CNTF), at concentrations up to 100 ng/ml with increased ChAT activity as predicted from the literature. These findings suggest that the nutrient composition of the medium is important in promoting expression of the cholinergic neuronal phenotype and that growth factor supplementation alone is insufficient to compensate for inadequate nutrient composition.
Pongrac
J L
JL
Department of Physiology, University of Western Ontario, London, Canada.
Rylett
R J
RJ
D000818:Animals; D019208:Brain-Derived Neurotrophic Factor / Q000494:pharmacology; D018929:Cell Culture Techniques / Q000379:methods; D002455:Cell Division / Q000187:drug effects; D002795:Choline O-Acetyltransferase / Q000378:metabolism*; D020934:Ciliary Neurotrophic Factor; D016895:Culture Media, Serum-Free; D004622:Embryo, Mammalian; D006801:Humans; D007150:Immunohistochemistry; D009414:Nerve Growth Factors / Q000494:pharmacology* / Q000502:physiology; D009419:Nerve Tissue Proteins / Q000494:pharmacology; D016501:Neurites / Q000187:drug effects / Q000502:physiology; D009474:Neurons / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology; D016548:Prosencephalon / Q000166:cytology* / Q000196:embryology; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000494:pharmacology
D019208:Brain-Derived Neurotrophic Factor; D020934:Ciliary Neurotrophic Factor; D016895:Culture Media, Serum-Free; D009414:Nerve Growth Factors; D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins; D002795:Choline O-Acetyltransferase