Margiotta J F, Howard M J
Department of Physiology and Biophysics, Mount Sinai School of Medicine, New York, New York 10029.
Dev Biol. 1994 May;163(1):188-201. doi: 10.1006/dbio.1994.1135.
We have examined the development of acetylcholine (ACh) responses in chick sensory neurons and their regulation by growth factors. Between Embryonic Days E13 and E18 in vivo, the proportion of freshly dissociated dorsal root ganglion (DRG) neurons that were appreciably sensitive to 500 microM ACh increased from approximately 40 to > 90% and the size of the ACh response per unit membrane area increased nearly 10-fold. Nerve growth factor (NGF) supports the survival, growth, and differentiation of central, sympathetic, and sensory neurons. NGF also promotes ACh sensitivity in PC12 cells and nodose ganglion neurons grown in cell culture. When E13 chick DRG neurons were maintained in culture with NGF for 4-6 days, however, only approximately 50% had appreciable ACh sensitivity, and large responses comparable to those observed at E18 in vivo were rare. Chick-eye extract contains a 21.5-kDa trophic factor termed growth-promoting activity (GPA) that supports survival of sympathetic and DRG neurons for 24 hr in culture and an activity of approximately 50 kDa that promotes increases in choline acetyltransferase activity, ACh sensitivity, and ACh receptors (AChRs) in ciliary ganglion neurons (Nishi and Berg, 1981; Halvorsen et al., 1991). In the present study, whole-eye extract supported full survival of E13 DRG neurons for up to 6 days in culture and promoted ACh sensitivity in > 90% of the neurons tested. GPA-containing eye-extract fractions and NGF individually supported full DRG neuron survival for up to 6 days in culture, but neither promoted ACh sensitivity. Adding eye-extract fractions containing material of approximately 50 kDa for 4-6 days to either GPA- or NGF-supplemented media led to appreciable ACh sensitivity in approximately 90% of the DRG neurons tested. The levels of ACh sensitivity observed in DRG neurons maintained either in whole-eye extract or in fractions containing 50-kDa material were close to those observed at an equivalent developmental age in vivo. The results indicate that components in the 50-kDa eye-extract fraction promote the ACh sensitivity of DRG neurons in culture and suggest that similar factors may influence the appearance of AChRs on the neurons in vivo.
我们研究了鸡感觉神经元中乙酰胆碱（ACh）反应的发育及其受生长因子的调节。在胚胎期第13天（E13）至第18天（E18）的体内，对500微摩尔ACh有明显反应的新鲜解离背根神经节（DRG）神经元的比例从约40%增加到>90%，且单位膜面积的ACh反应大小增加了近10倍。神经生长因子（NGF）支持中枢、交感和感觉神经元的存活、生长及分化。NGF还可促进PC12细胞和细胞培养中生长的结状神经节神经元的ACh敏感性。然而，当将E13鸡DRG神经元与NGF一起在培养中维持4 - 6天时，只有约50%的神经元有明显的ACh敏感性，且很少有与E18体内观察到的大反应相当的情况。鸡眼球提取物含有一种名为生长促进活性（GPA）的21.5千道尔顿营养因子，它能在培养中支持交感和DRG神经元存活24小时，还有一种约50千道尔顿的活性物质，可促进睫状神经节神经元中胆碱乙酰转移酶活性、ACh敏感性及ACh受体（AChRs）的增加（西和伯格，1981；哈尔沃森等人，1991）。在本研究中，全眼球提取物在培养中支持E13 DRG神经元完全存活长达6天，并使>90%受试神经元的ACh敏感性增加。含GPA的眼球提取物组分和NGF单独在培养中支持DRG神经元完全存活长达6天，但均未促进ACh敏感性。向补充了GPA或NGF的培养基中添加含约50千道尔顿物质的眼球提取物组分4 - 6天，导致约90%受试DRG神经元有明显的ACh敏感性。在全眼球提取物或含50千道尔顿物质的组分中维持培养的DRG神经元中观察到的ACh敏感性水平接近在体内相同发育年龄时观察到的水平。结果表明，50千道尔顿眼球提取物组分中的成分可促进培养中DRG神经元的ACh敏感性，并提示类似因子可能在体内影响神经元上AChRs的出现。
