Novel organization of microtubules in cultured central nervous system neurons: formation of hairpin loops at ends of maturing neurites.

Using the high voltage electron microscope, we have examined cultured embryonic neurons in order to understand better the organization of microtubules in developing neurites. We found that, in embryonic chick retina neurons, microtubules were abundant in the ends of neurites and showed an unusual pattern of organization. Most striking was the presence of microtubule loops; after entering the flattened region of a growth cone, microtubules frequently made tight 180 degrees turns. Occasionally these looping microtubules re-entered the neurite and returned in the direction of the cell body. Positive identification of the loop structures as microtubules was made by specific immunocytochemical labeling. Quantitative analysis showed that more than half of the retina neurons that were dissociated on embryonic day 8 and kept in culture for 4 to 6 days (E8C4 and E8C6) contained at least one microtubule that made a 180 degrees turn at flat regions along or at the tips of neurites. The area within the loops typically contained larger membranous organelles, whereas only small vesicles were seen outside the loops. Fine filaments were seen to interconnect the loops at various places, suggesting the possibility that they played a role in maintaining the shape of microtubule loops. Examination of other neurons showed that tight microtubule loops were prominent in chick spinal cord neurons, but they were rarely seen in neurons of the sympathetic ganglia or dorsal root ganglia or in NG108-15 cloned cells. Developmentally, no loops were observed in E8C1 retina neurons, but retina neurons dissociated from older embryos (12 days) did show loops after 1 day in culture; these data suggest that microtubule loops may be abundant around embryonic day 12 to 13 in the chick retina. The possible significance of this unusual microtubule organization to the control of neurite growth and bidirectional transport is discussed.