SYNAPTIC FORMATION IN CUTURED SENSORY

该项目开发了一种在培养中维持舌上皮的基本技术，这使我们能够研究味觉神经纤维和味觉感受器细胞之间的突触是如何形成的。本研究使用了墨西哥火蜥蜴的舌咽神经节和舌上皮。用酶法分离神经节细胞，接种于L-15培养液中。培养24-48小时后，神经元突起开始延长，2周时突起长达1mil米。切取含有味蕾的舌上皮小片，放入培养液中保存。在一周内，上皮细胞附着在培养皿底部，生长良好。然而，味蕾细胞失去了与普通上皮细胞不同的玫瑰花样排列。在味蕾之外，舌咽神经与少数孤立细胞形成突触。电子显微镜研究表明，这些细胞具有机械感觉功能。为保持舌上皮三维构象的味蕾细胞，将舌上皮块包埋于胶原凝胶中，浸泡在L-15培养液中，室温保存。味蕾没有存活超过24小时，可能是因为氧气不足。当用胶原酶剥离一般上皮细胞时，味蕾可保持约6天。培养味蕾需要CO2培养箱来达到最适温度和充足的氧气。

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.