Morphological and transmitter release properties are changed when sympathetic neurons are cultured in low Ca2+ culture medium.
The stimulated elevation of [Ca2+]i can either promote neuronal survival or lead to Ca(2+)-mediated neurotoxicity. Similarly, growth cone mobility and neurite outgrowth may be promoted or arrested by elevated [Ca2+]i. We examined survival, development and transmitter release properties of chick sympathetic neurons maintained in culture medium containing varying concentrations of Ca2+. Neurons maintained in medium with no added Ca2+ or as low as 0.1 mM external Ca2+ show a dramatic change in growth and development compared to neurons kept in 1-2 mM Ca(2+)-containing medium. Furthermore, neurons in Ca(2+)-free medium (+ 100 microM EGTA) survived up to 24 h and, following change to 0.1 mM Ca2+, grew neurites and survived for several weeks. Neurons grown in high Ca2+ medium (0.6-2 mM) exhibited thick neurites, aggregated cell bodies, and neurites began to detach after six to eight days in culture. Neurons in low Ca2+ medium (no added Ca2+ to 0.3 mM) grew as single cells with extensive, thin branching neurites, remained firmly attached to the substrate and survived for several weeks. Neurons initially plated in 0.1 mM Ca2+ (or 2 mM Ca2+) medium and switched over to 2 mM (or 0.1 mM) Ca2+ medium after two days acquired the characteristic morphology of high (and low) Ca2+ medium over the next six days, demonstrating the plasticity of effects of external Ca2+. The above characteristic changes in growth of sympathetic neurons in low Ca2+ medium occurred when neurons were supported by 35 mM KCl or 30 nM phorbol 12,13-dibutyrate instead of nerve growth factor. The uptake and retention of tritiated norepinephrine in neurons grown in low or high Ca2+ medium were similar. However, basal release of [3H]norepinephrine in neurons maintained in low Ca2+ medium was one-third of that in neurons kept in high Ca2+ medium. Furthermore, electrically stimulated (10 pulses at 1 or 10 Hz) [3H]norepinephrine release from neurons grown in high Ca2+ had a high fractional release (> 1%) which did not change during six days in culture. On the other hand, fractional release in neurons grown in low Ca2+ medium for six to 10 days decreased about 50% and 75%, respectively, compared to release after two days in culture. The resulting low fractional release (< 0.5%) is characteristic of sympathetic neurons in neuroeffector organs.(ABSTRACT TRUNCATED AT 400 WORDS)
Wakade
T D
TD
Department of Pharmacology, School of Medicine, Wayne State University, Detroit, MI 48201, USA.
Przywara
D A
DA
Kulkarni
J S
JS
Wakade
A R
AR
D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002470:Cell Survival / Q000187:drug effects*; D002478:Cells, Cultured; D002642:Chick Embryo; D004305:Dose-Response Relationship, Drug; D009474:Neurons / Q000187:drug effects; D018377:Neurotransmitter Agents / Q000378:metabolism*; D009638:Norepinephrine / Q000378:metabolism; D013564:Sympathetic Nervous System / Q000254:growth & development*; D013997:Time Factors
D018377:Neurotransmitter Agents; D002118:Calcium; D009638:Norepinephrine