Development of markers for cholinergic neurones in re-aggregate cultures of foetal rat whole brain in serum-containing and serum-free media: effects of triiodothyronine (T3)

Abstract

1 Development has been studied in re-aggregate cultures derived from the 16 day foetal rat brain and the effects of triiodothyronine (T₃) investigated. Cultures were maintained in either a medium containing 10% serum (S+), or in serum-free culture medium (S⁻) or in serum-free medium containing 30nm T₃. 2 The muscarinic cholinoceptor, measured by specific binding of [³H]-quinuclidinyl benzitate ([³H]-QNB) at 9 and 14 days in vitro, was at a lower level in the serum-free cultured cells compared with those in serum-containing culture medium (S⁺). In cultures in the latter medium, receptor concentration at day 14 was of a similar magnitude to that in rat brain at an equivalent postnatal age. Binding increased with development from 9 to 14 days in vitro in the S⁺ medium but not in the S⁻ medium. T3 treatment caused an 85% increase in [³H]-QNB binding compared with the cultures in S⁻ medium at day 14 to a level equivalent to that found in the cells grown in S⁺ medium. This increase was reflected in the B_max but not in the K_D (approx. 0.1 nm). 3 Choline acetytransferase (ChAT) activity developed more slowly in the S⁻ medium than in the S⁺ medium where the specific activity approximated values obtained in vivo. T₃ treatment of cultures grow in S⁻ medium significantly enhanced the developmental rate of increase of ChAT activity. 4 The characteristics of [³H]-choline uptake and metabolism in the cultures was examined. Uptake was strictly Na⁺-independent but was energy-dependent, and inhibited by 2, 4′-dinitrophenol (2, 4′-DNP) and cooling (0–4°C). Neither iodoacetate nor ouabain had any effect on the amount of uptake. Hemicholinium (HC3) was a potent inhibitor of uptake (70% inhibition at 10 μm HC3). Metabolism studies showed virtually no conversion to [³H]-acetylcholine ([³H]-ACH) in re-aggregates grown in either the S⁺, S⁻ or T₃ containing media. However, a small amount of [³H]-choline was incorporated into phosphorylcholine. T₃ treatment had no effect on this metabolic profile. 5 The kinetics of [³H]-choline uptake by the re-aggregates was also studied in the re-aggregate cultures (after 12 and 22 days in vitro) using [³H]-choline at 0.05–100 μm. Both Eadie-Hofstee transformation and least-squares analysis of the data showed that the uptake comprised only a single low-affinity component with an apparent K_t = approx. 50 μm. Unlike ChAT and [³H]-QNB binding, there appeared to be no difference between the uptake in the different culture conditions. 6 It is concluded that the differentiation of cholinergic neurones and muscarinic receptors in serum-free cultured re-aggregates from foetal rat brain is enhanced by thyroid hormone treatment. The development of [³H]-choline uptake does not seem to be associated with cholinergic cells under these culture conditions, and is unaffected by thyroid hormone treatment.