MORPHOLOGICAL AND TRANSMITTER RELEASE PROPERTIES ARE CHANGED WHEN SYMPATHETIC NEURONS ARE CULTURED IN LOW CA2-MEDIUM( CULTURE)

The stimulated elevation of [Ca2+](i) can either promote neuronal surv ival or lead to Ca2+-mediated neurotoxicity. Similarly, growth cone mo bility and neurite outgrowth may be promoted or arrested by elevated [ Ca2+](i). We examined survival, development and transmitter release pr operties of chick sympathetic neurons maintained in culture medium con taining varying concentrations of Ca2+. Neurons maintained in medium w ith no added Ca2+ or as low as 0.1 mM external Ca2+ show a dramatic ch ange in growth and development compared to neurons kept in 1-2 mM Ca2-containing medium. Furthermore, neurons in Ca2+-free medium(+100 mu M 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+ me dium (0.6-2 mM) exhibited thick neurites, aggregated cell bodies, and neurites began to detach after six to eight days in culture. Neurons i n low Ca2+ medium (no added Ca2+ to 0.3 mM) grew as single cells with extensive, thin branching neurites, remained firmly attached to the su bstrate 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 h igh (and low) Ca2+ medium over the next six days, demonstrating the pl asticity of effects of external Ca2+. The above characteristic changes in growth of sympathetic neurons in low Ca2+ medium occurred when neu rons were supported by 35 mM KCl or 30 nM phorbol 12,13-dibutyrate ins tead of nerve growth factor. The uptake and retention of tritiated nor epinephrine in neurons grown in low or high Ca2+ medium were similar. However, basal release of [H-3]norepinephrine in neurons maintained in low Ca2+ medium was one-third of that in neurons kept in high Ca2+ me dium. Futhermore, electrically stimulated (10 pulses at 1 or 10 Hz) [H -3]norepinephrine release from neurons grown in high Ca2+ had a high f ractional release (>1%) which did not change during six days in cultur e. On the other hand, fractional release in neurons grown in low Ca2medium for six to IO days decreased about 50% and 75%, respectively, c ompared to release after two days in culture. The resulting low fracti onal release (<0.5%) is characteristic of sympathetic neurons in neuro effector organs. Besides the practical advantage of availability of ne urons grown in low Ca2+ medium to study transmitter release and morpho logical characteristics over extended periods of time, our study shows that some transmitter release properties of these neurons resemble th ose of neurons growing in mature sympathetic neuroeffector organs. It is reasonable to propose that extracellular Ca2+ may have important co nsequences in the development and expression of transmitter release fu nction.