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Some cells from cultured embryonic mouse hypothalamus were found to express aromatic-L-amino acid decarboxylase (EC 4.1.1.28) activity and serotonin uptake and storage. These neuron-like cells differed from serotoninergic neurons in cultured embryonic mouse brain stem since they did not contain tryptophan hydroxylase. We studied the effect of the serotonin agonist 8-hydroxy-2-[di-(n-propyl)amino]tetralin on neuronal differentiation of hypothalamic cells from 12-to 15-day embryos. Repeated treatment of cultures with the serotonin agonist for 10 days resulted in an increased number of serotonin cells containing high levels of decarboxylase activity. Both the increase in cell numbers and the elevated decarboxylase activity could be suppressed by the addition of the serotonin antagonist metergoline to the culture medium. These data show that serotonin (or an agonist), acting on specific receptors, can initiate and amplify its own synthesis in embryonic hypothalamic neurons, as observed in the primitive hypothalamic nerve cell line F7

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A mouse carbonic anhydrase (CA II) complementary(c) DNA probe was used for in situ hybridization on mouse brain cultured cells in order to follow CA II gene expression during brain development. An improved method was established using biotinated probes that resulted in a high sensitivity and an absence of background; this method could be combined with immunohistochemistry. Hypothalamic cells of embryonic day (ED) 12-14 mice were cultured for various periods. Chronologic appearance of CA II messenger(m)RNA and protein was studied. The CA II gene transcripts are detectable as early as ED 12-13, although the protein they encode is not detectable until ED 17-18. Gene expression is restricted to 0.1% of the total population. Northern blot analysis confirmed the presence of CA II transcripts in embryonic hypothalamus. At postnatal stage, the majority of glial cells express both the CA II mRNA and the protein. Our results favour the early appearance of a glial lineage in a precise area of the developing CNS. The precocity of CA II gene transcription makes in situ hybridization an invaluable approach in defining the onset of nerve cell lineages during embryonic development.