The role of noradrenaline in the differentiation of amphibian embryonic neurons
Authors: Rowe Sally J. , Messenger Nigel J. , Warner Anne E. 
Published: April 2021

ABSTRACT
The possibility that monoamines might act as signalling molecules during the early development of the nervous system has been examined in embryos of the amphibian Xenopus laevis. The distributions of 5-hydroxytryptamine, dopamine, noradrenaline and their precursor, dopa, were determined from the fertilized egg up to the late neurula stages using High Performance Liquid Chromatography, formaldehyde-induced fluorescence and antibody staining. 5-hydroxytryptamine was not detected until the tail bud stage. The fertilized egg contained significant concentrations of dopa (10−6 M) and dopamine (10−7 M). Both monoamines persisted with little change in concentration up to the late neurula stage. Early neurula stage embryos contained very low levels of noradrenaline. Aldehyde-induced fluorescence showed that monoamines are localized in dorsal regions of the embryo, in ectoderm and mesoderm cells. Monoamines were not present in endoderm cells. Immunocytochemical staining showed dopamine predominantly in the ectoderm, except in future neural regions where it was found also in the mesoderm. Dopamine staining was always most intense in dorsal regions of the embryo. The consequences for subsequent neuronal differentiation of interfering with the biosynthesis and receptor binding of monoamines during neurulation was assayed. Neuronal differentiation was monitored quantitatively in cultures set up as the neural tube closed and qualitatively in intact tadpoles that were left to develop for two days after washout of test reagent. The number of neurons, the number of muscle cells and the total number of differentiated cells were counted after 18–24 hours of culture. Comparison of the number of neurons that differentiated from control and treated embryos showed that inhibition of dopamine –hydroxy lase, the enzyme catalysing the conversion of dopamine to noradrenaline, during the neural plate stages reduced substantially subsequent neuronal differentiation. The differentiation of myocytes and the total number of differentiated cells were not affected. Exogenous noradrenaline (10−6M) or dopamine (10−6M) could increase the number of neurons that differentiated subsequently in culture. Interfering with noradrenaline binding to receptors with receptor antagonists during neurulation showed that -adrenergic receptor antagonists reduced substantially the subsequent differentiation of neurons. The differentiation of myocytes and the total number of differentiated cells were not affected. The effect of adrenergic receptor antagonists was overcome by the simultaneous inclusion of noradrenaline or -receptor agonists, but not agonists at -adrenergic receptors. The quantitative reduction in the differentiation of neurons was paralleled by defects in the Central Nervous System of intact tadpoles. The grey matter (containing the cell bodies) was disorganized, although there were no signs of increased cell death, and there was a substantial reduction in the white matter, which contains outgrowing axons. Antagonists at -adrenergic receptors, dopamine receptors and acetylcholine receptors had no influence on the differentiation of neurons, whether assayed in tissue culture or in intact tadpoles. We suggest that during normal development endogenous noradrenaline binds to -adrenergic receptors on neural plate cells as part of the mechanism controlling the differentiation of CNS neurons.