SYNAPTIC FORMATION IN CUTURED SENSORY

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The project developed a basic technique to maintain lingual epithelium in culture, which allows us to study how synapses are made between gustatory nerve fibers and taste receptor cells. The glossophryngeal nerve ganglion and the lingual epithelium of the Mexican salamander, axolotl (ambystoma mexicanum) , were used. Neurons were isolated from the ganglion with enzymatic treatment and placed in L-15 culture medium. In 24-48 hours the neurons in culture started to extend processes, which grew into 1 mil meter in length by 2 weeks. Small pieces of the lingual epithelium containing taste buds were dissected out and kept in the medium. In one week the epithelial cells attached to the bottom of a culture dish and grew well. However, the taste bud cells lost a rosette-like arrangement distinct from general epithelial cells. Outside of the taste buds the glossopharyngeal nerve makes synapses with a few solitary cells. Electron-microscopic study revealed that those cells are of mechanosensory function. To maintain the taste bud cells of three-dimensional conformation pieces of the lingual epithelium were embedded in collagen gel, immersed in L-15 medium and kept at room temperature. The taste buds did not survive more than 24 hours, possibly due to paucity of oxygen. When the general epithelial cells were stripped off by collagenase, the taste buds were maintained for about 6 days. CO_2-incubator is required to achieve optimal temperature and sufficient oxygen for culturing the taste buds.