Development of cholinergic neurones in cultures of rat superior cervical ganglia. Role of calcium and macromolecules

Superior cervical ganglia from 3-day-old rats were grown in vitro for 14 days. Addition of an extract of rat heart to the culture medium led to no change in the activity of tyrosine hydroxylase but a doubling of the activity of choline acetyltransferase in the ganglia.To see whether this effect was due to a change in the concentration of unbound Ca²⁺, the calcium-binding capacity of extracts of adult rat heart was determined in a dialysis-binding study. Both cardiac extracts and serum lowered the levels of free calcium in the medium, but by relatively small amounts. When ganglia were grown in media containing 0, 2.5 and 6 mm Ca²⁺, there was no change in the ratio of choline acetyltransferase activity to tyrosine hydroxylase activity, although the activities of both enzymes increased at lower Ca²⁺ concentrations.When superior cervical ganglia were grown in Rose chambers under cellophane strips, the choline acetyltransferase/tyrosine hydroxylase ratio was higher than when superior cervical ganglia were loose in the medium. When a larger number of ganglia was grown under cellophane this further increased the ratio, while positioning of the ganglia so that substances could diffuse out from under the cellophane decreased the choline acetyltransferase/tyrosine hydroxylase ratio. When ganglia from 3-day-old rats were grown side by side with ganglia from 3-week-old rats, there was an even greater effect on the ratio of the two enzyme activities.We conclude that the ability of cardiac extracts to increase the ratio of choline acetyltransferase activity to tyrposine hydroxylase activity in ganglia is not due to changes in the Ca²⁺ concentration in the medium, but to the presence of a non-dialysable macromolecule. A similar factor also seems to be secreted by the ganglion itself.