NEUROBIOLOGIC STUDIES OF NEURONS AND GLIA IN CELL CULTURE

细胞培养中神经元和神经胶质细胞的神经生物学研究

• 批准号: 3756616
• 负责人: P G NELSON
• 金额: --
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3756616
• 关键词: acetylcholine; calcium channel; calcium flux; cell adhesion molecules; electrophysiology; gene expression; growth cones; hirudins; messenger RNA; muscle cells; neuromuscular function; polymerase chain reaction; protein biosynthesis; prothrombin; spinal ganglion; synapses; thrombin; tissue /cell culture; voltage gated channel

We have demonstrated activity-dependent synapse reduction in a 3- compartment tissue culture system in which two populations of cholinergic neurons converge to innervate a common population of muscle fibers. This synapse reduction is blocked by a specific thrombin inhibitor, hirudin; this implicates the action of thrombin in the process of synapse reduction. We now show that the source of the thrombin is the muscle or nerve cells (and not the serum usually present in the culture medium) by demonstrating activity-dependent synapse reduction in serum free medium. Exogenous thrombin produces synapse reduction even in the absence of neural activity and the thrombin induced reduction is blocked by hirudin. Thrombin takes some three days to produce synapse loss, while stimulation produces similar loss in only one day, so some synergy between thrombin action and nerve activity may occur. We have measured thrombin activity (hirudin sensitive proteolytic activity) in the supernatant media from inactive (tetrodotoxin silenced) and active (acetyl choline stimulated) muscle fibers. Activated muscle cells produce about twice as much thrombin as do inactive muscle. We have used semi-quantitative polymerase chain reaction methods to examine prothrombin mRNA levels in activated and inactive muscle cell cultures. After 48 hours of stimulation, prothrombin mRNA levels are about two times higher in activated than in inactive muscle cells. Thus in active muscle cells, and in correlation with the synapse loss that occurs with activation, thrombin secretion and the capacity for thrombin synthesis increases by some two fold. We have examined quantitatively the voltage-dependent Ca++ currents in dorsal root ganglion neurons, as a function of the activation of these cells. We find that the Ca++ currents are reduced substantially and significantly by 24 hours or more of electrical stimulation. This may be the basis for the adaptation of the growth cones of these cells to prolonged electrical stimulation that we had described earlier. mRNA for a cell adhesion molecule, L1, is strikingly down regulated by electrical stimulation of dorsal root ganglion neurons at only 0.1 Hz.

我们已经证明了活动依赖性突触减少在一个3- 隔室组织培养系统，其中两个群体的胆碱能 神经元会聚以神经支配共同的肌纤维群。 这 突触减少被特异性凝血酶抑制剂水蛭素阻断; 这暗示了凝血酶在突触形成过程中的作用 还原 我们现在表明凝血酶的来源是肌肉或 神经细胞（而不是通常存在于培养基中的血清）， 证明在无血清培养基中活性依赖性突触减少。 外源性凝血酶产生突触减少，即使在缺乏 神经活性和凝血酶诱导的减少被水蛭素阻断。 凝血酶需要大约三天的时间才能产生突触损失，而刺激 在一天内产生类似的损失，所以凝血酶和凝血酶之间的协同作用， 可能发生动作和神经活动。 我们测量了凝血酶活性 （水蛭素敏感的蛋白水解活性）在来自 非活性（河豚毒素沉默）和活性（乙酰胆碱刺激） 肌肉纤维 被激活的肌肉细胞产生大约两倍的 凝血酶和不活动的肌肉一样。 我们使用半定量 聚合酶链反应方法检测凝血酶原mRNA水平， 激活和非激活的肌肉细胞培养物。 48小时后 刺激，凝血酶原mRNA水平高出约两倍， 比不活跃的肌肉细胞更活跃。 因此在活跃的肌肉细胞中， 并且与伴随激活发生的突触损失相关， 凝血酶分泌和凝血酶合成的能力增加， 有些是双重的。 我们已经定量地研究了电压依赖性 背根神经节神经元Ca ~（++）电流随 激活这些细胞。 我们发现，Ca++电流减少 24小时或更长时间的电气 刺激. 这可能是适应生长的基础 长时间的电刺激， 前面描述的。一种细胞粘附分子L1的mRNA， 电刺激背根神经节神经元下调 只有0.1 Hz。