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 (T 3 )

1 Development has been studied in re‐aggregate cultures derived from the 16 day foetal rat brain and the effects of triiodothyronine (T 3 ) 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 30n m T 3 . 2 The muscarinic cholinoceptor, measured by specific binding of [ 3 H]‐quinuclidinyl benzitate ([ 3 H]‐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 [ 3 H]‐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 n m ). 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. T 3 treatment of cultures grow in S − medium significantly enhanced the developmental rate of increase of ChAT activity. 4 The characteristics of [ 3 H]‐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 μ m HC3). Metabolism studies showed virtually no conversion to [ 3 H]‐acetylcholine ([ 3 H]‐ACH) in re‐aggregates grown in either the S + , S − or T 3 containing media. However, a small amount of [ 3 H]‐choline was incorporated into phosphorylcholine. T 3 treatment had no effect on this metabolic profile. 5 The kinetics of [ 3 H]‐choline uptake by the re‐aggregates was also studied in the re‐aggregate cultures (after 12 and 22 days in vitro ) using [ 3 H]‐choline at 0.05–100 μ m. 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 μ m . Unlike ChAT and [ 3 H]‐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 [ 3 H]‐choline uptake does not seem to be associated with cholinergic cells under these culture conditions, and is unaffected by thyroid hormone treatment.