Detection and localization of 14-3-2 protein in primary cultures of embryonic rat brain

The development of embryonic rat brain in cell cultures was studied by an immunocytochemical method based on the detection of 14-3-2 protein (neuron-specific enolase or NSE), a neuron-specific protein. This protein was already present in undifferentiated neurons (less than 5 days in culture), being dispersed throughout the cytoplasm, though seemingly concentrated in the vicinity of polyribosomal structures. It was not found in nuclei, in mitochondria or in the Golgi apparatus. During neuron differentiation, the location of 14-3-2 protein was related to neurite development insofar as it was detected along the axon and even in what could be taken to be the presynaptic region of numerous interneuron contacts. In contact areas, a thickening of the junction membrane was observed but the presence of 14-3-2 protein was always unilateral demonstrating the absence of a true synapse and reflecting the halt in neurite development observed after 15 days in culture. The presence of 14-3-2 protein in the cell cultures was confirmed by a microcomplement fixation assay. The protein detected in cell cultures had the same immunological properties as that found in the 17-day-old embryo, but was slightly different from that found in adult rat brain. This observation can be confronted with the lack of neuron maturation in the immunocytochemical studies.