Intracellular sodium and the differentiation of amphibian embryonic neurones

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Abstract
The mechanism of the inhibition of neural differentiation produced by inhibiting the Na pump with cardiac glycosides during the mid-neural fold stages of development of the amphibian embryo was studied. Neural differentiation was assessed quantitatively by counting the number of neurons that undergo primary differentiation in tissue culture, as a proportion of the total number of differentiated cells. Inhibition of the Na pump by lowering the extracellular K concentration ([Ko]) to 0 during the mid-neural fold stages inhibited neural differentiation. Raising the extracellular Ca concentration to 10 mM during treatment with strophanthidin protected differentiating neurons from the effects of the Na pump inhibitor. Lowering [Ca]o to 0.05 mM potentiated the effect of low doses of glycoside. In the presence of high extracellular Ca and 5 .times. 10-6 M-strophanthidin, the membrane potential of neural plate cells remained close to the levels recorded at the beginning of neurulation; the normal increase in resting potential was not restored. Addition of 10 mM-Sr2+ to the bathing medium also protected nerve cells against the inhibition produced by strophanthidin; Sr2+ was less effective than Ca2+. Addition of either 10 mM-Mg2+ or Mn2+ had no efect on the inhibition of differentiation produced by strophanthidin. Addition of Mn2+ along with high Ca2+ prevented Ca from exerting its protective effect. The eyes of embryos were treated with high Ca2+ together with strophanthidin during neurulation and then allowed to grow into tadpoles developed normally. When Mn2+ was added, the eyes were disrupted similarly to those of embryos treated with strophanthidin alone. Replacement of extracellular Na with equimolar amounts of choline or Li prevented the cardiac glycoside from inhibiting neural differentiation. The protection afforded by lowering [Na]o was abolished when [Ca]o was simultaneously lowered to 0.05 mM. Tadpoles from embryos treated with low extracellular Na and strophanthidin during neurulation had normal eyes compared to those treated with strophanthidin alone. Measurement of the intracellular Na concentration ([Na]i) with Na-sensitive micro-electrodes put [Na]i at .apprx. 30 mM before the neural folds lift. As the Na pump is activated (stages 14 1/2-15)[Na]i in the neural plate falls; by the end of the mid-neural fold stage it is < 10 mM. Addition of 5 .times. 10-6 M-strophanthidin to the bathing fluid before activation of the Na pump prevented the fall in [Na]i; in embryos where [Na]i had begun to drop strophanthidin produced a rise to .apprx. 30 mM. When 10mM-Ca was present along with strophanthidin [Na]i fell to .apprx. 17 mM during neurulation, despite inhibition of the Na pump. It is unlikely that either abolition of the normal increase in resting potential or a fall in gap junction permeability is responsible for the reduction in neural differentiation produced by blocking the Na pump during neurulation. Strophanthidin evidently achieves its efect by preventing the fall in [Na]i that occurs during normal neurulation because of activation of the Na pump.