The aim of this project is to culture neurons in the laboratory for use in studies of axoplasmic structure and transport. These cultured neurons are also to be used in connection with voltage clamp and patch clamp experiments of ionic channels and their conductances and gating mechanisms. The routine of culturing squid embryonic neurons has now been established. Eagle's media components dissolved in artificial sea water, 20% heat inactivated fetal bovine serum, and antibiotics when used with Primaria plastic dishes result in principally neurons (and some muscle cells) when incubated at 22 degrees C. Squid embryonic stages from 18 to 26 give most reliable results when head regions are dissected free of yolk. Indirect immunofluorescent assay with tetanus toxin specific for neurons showed most cells to be neuronal. Cells live in culture for over a month. Cells grow after subculturing but do not persist probably for lack of nerve growth factor. Somal diameters range from 5 to 40 microns; neurite processes extend to millimeters; cell morphologies vary from bipolar to pyramidal with six or seven neurites. Embryonic cells from two other molluscs, Hermissenda and Octopus, also grow under the same conditions.
