NEUROBIOLOGIC STUDIES OF NEURONS AND GLIA IN CELL CULTURE

我们一直在研究 小鼠神经肌肉接头处的依赖性突触消除 体外 凝血酶和凝血酶受体是必不可少的 消除，消除被特定的蛋白激酶阻断 C抑制剂calphostin C，以及更一般的激酶抑制剂， 星形孢菌素和H-7。 蛋白激酶A抑制剂H-89没有 对活动依赖性突触消除过程的阻断作用。 B. 我们研究了凝血酶原（PT）、凝血酶受体（THR）和凝血酶受体（THR）mRNA的表达， (ThR)和内源性蛋白酶抑制剂蛋白酶-连接蛋白1（PN-1） 在发育过程中和成年小鼠肌肉去神经后。 PT和 ThR在给药后的前三周显著下调， 出生时，PN-1在最大产程期间呈一过性下降， 自然发生的突触消除（出生后10-15天）。 PT和 ThR在去神经后显著增加，而PN-1 降至对照组的50%。 消除期间PN-1的减少 周期可能导致凝血酶（或其他蛋白水解）增强 活性，我们的体外结果表明这会导致突触丢失。 C. 鸡胚实验表明， （脑源性神经营养因子或BDNF）和丝氨酸蛋白酶 （可能是凝血酶）参与了同侧的损失， 视网膜顶盖投影在此准备。 全身水蛭素或PN-1 产生一个显着减缓损失的投射，而BDNF 注射到眼睛里就能完全防止失血 D. 进一步的实验 已经描述了视网膜轴突生长锥的行为， 视锥细胞遇到鸡顶盖或小鼠丘中的靶细胞， 分析细胞表面的细胞和地形分布 引导分子 我们开发了一个模型， 肝配蛋白-A2和肝配蛋白-A5的mRNA表达的分布（已知 排斥导向分子）与生长锥行为， 小鼠视网膜靶细胞发育模式的差异 还有小鸡

We have continued studies of the mechanisms involved in activity dependent synapse elimination at the mouse neuromuscular junction in vitro. Thrombin and the thrombin receptor are essential for this elimination and the elimination is blocked by a specific protein kinase C inhibitor calphostin C, as well as the more general kinase inhibitors, staurosporine and H-7. The protein kinase A inhibitor, H-89, has no blocking effect on the activity dependent synapse elimination process. b. We studied expression of mRNA for prothrombin (PT), thrombin receptor (ThR), and the endogenous protease inhibitor protease-nexin 1 (PN-1) during development and after denervation of adult mouse muscle. PT and ThR were markedly down regulated during the first three weeks after birth, but PN-1 showed a transient decrease during the period of maximal naturally occurring synapse elimination (postnatal days 10-15). PT and ThR were substantially increased following denervation while PN-1 decreased to 50% of control. The decrease in PN-1 during the elimination period may result in an enhanced thrombin (or other proteolytic) activity, which our in vitro results indicate would induce synapse loss. c. Experiments with chick embryos indicate that both a trophic factor (brain derived neurotrophic factor or BDNF) and serine proteases (probably thrombin) are involved in the loss of the ipsilateral retinotectal projection in this preparation. Systemic hirudin or PN-1 produce a significant slowing of the loss of projection while BDNF injected in the eye prevents loss completely. d. Further experiments have been done describing retinal axon growth cone behavior as the growth cones encounter target cells in the chick tectum or mouse colliculus and analyzing the cellular and topographic distribution of cell surface guidance molecules. We have developed a model incorporating the cellular distribution of mRNA expression for ephrin-A2 and ephrin-A5 (known repulsive guidance molecules) with growth cone behaviors which accounts for differences in the patterns of retinotarget development in the mouse and chick.