Hefti F, Hartikka J, Eckenstein F, Gnahn H, Heumann R, Schwab M
Neuroscience. 1985 Jan;14(1):55-68. doi: 10.1016/0306-4522(85)90163-0.
Neurons dissociated from the septal area of fetal rat brains were grown in culture. Cholinergic neurons were identified by immunocytochemical visualization of choline acetyltransferase and cytochemical demonstration of acetyl cholinesterase. Choline acetyltransferase immunocytochemistry stained cell bodies and proximal processes while acetylcholinesterase cytochemistry visualized the entire neuron. Choline acetyltransferase-positive neurons could only be identified in cultures grown under conditions that produced the maximal choline acetyltransferase activity, measured biochemically. All of the choline acetyltransferase-positive neurons were double stained for acetylcholinesterase while only 6% of the acetylcholinesterase-positive cells were choline acetyltransferase negative in these cultures. These results indicate that acetylcholinesterase is a reliable marker for cholinergic cells in cultures of dissociated septal neurons. Being the more sensitive method, acetylcholinesterase staining was therefore used to identify cholinergic cells in cultures with choline acetyltransferase levels insufficient for immunocytochemical visualization of this enzyme. Addition of nerve growth factor or antibodies to nerve growth factor to the medium did not affect the number of cholinergic neurons surviving in culture. Furthermore, nerve growth factor and anti-nerve growth factor failed to influence the general morphological appearance and the number of processes of these neurons. However, nerve growth factor elevated the biochemically measured activity of choline acetyltransferase up to two-fold. The nerve growth factor-mediated increase in choline acetyltransferase activity was dose dependent with an ED50 of 10 ng/ml (4 X 10(-10) M). The increase was highly specific for nerve growth factor. It was blocked by anti-nerve growth factor, and epidermal growth factor, insulin and other control proteins failed to exert a similar effect. Nerve growth factor had to be present for at least 3 days in the culture medium to increase choline acetyltransferase activity, suggesting that the increase was due to an elevated choline acetyltransferase synthesis rather than to an activation of the enzyme.
从胎鼠脑隔区分离出的神经元在培养皿中生长。通过胆碱乙酰转移酶的免疫细胞化学可视化和乙酰胆碱酯酶的细胞化学鉴定来识别胆碱能神经元。胆碱乙酰转移酶免疫细胞化学染色细胞体和近端突起，而乙酰胆碱酯酶细胞化学则使整个神经元可视化。只有在生化测量显示产生最大胆碱乙酰转移酶活性的条件下培养的细胞中才能鉴定出胆碱乙酰转移酶阳性神经元。在这些培养物中，所有胆碱乙酰转移酶阳性神经元都被乙酰胆碱酯酶双重染色，而只有6%的乙酰胆碱酯酶阳性细胞是胆碱乙酰转移酶阴性。这些结果表明，乙酰胆碱酯酶是分离的隔区神经元培养物中胆碱能细胞的可靠标志物。由于乙酰胆碱酯酶染色是更敏感的方法，因此在胆碱乙酰转移酶水平不足以通过免疫细胞化学可视化该酶的培养物中，用乙酰胆碱酯酶染色来识别胆碱能细胞。向培养基中添加神经生长因子或抗神经生长因子抗体不会影响培养中存活的胆碱能神经元数量。此外，神经生长因子和抗神经生长因子未能影响这些神经元的一般形态外观和突起数量。然而，神经生长因子使生化测量的胆碱乙酰转移酶活性提高了两倍。神经生长因子介导的胆碱乙酰转移酶活性增加呈剂量依赖性，ED50为10 ng/ml（4×10⁻¹⁰ M）。这种增加对神经生长因子具有高度特异性。它被抗神经生长因子阻断，表皮生长因子、胰岛素和其他对照蛋白未能发挥类似作用。神经生长因子必须在培养基中至少存在3天才能增加胆碱乙酰转移酶活性，这表明这种增加是由于胆碱乙酰转移酶合成增加而不是酶的激活。
