Nerve-muscle interaction during synapse formation and the role of genes

突触传递是所有人类行为的基础，从“简单的”运动反射到“高级认知功能”。虽然在解决突触发育和传递的大致轮廓方面已经取得了相当大的进展，但分子事件仍然是模糊的。为了解决这个问题，我们必须有一个模型系统，我们可以在其中使用尽可能多的现代分析工具。我们选择的模型系统是果蝇，Drosophila melanogaster。本研究的最终目的是阐明突触形成的基因调控机制。在三年的资助期内，我们完成了以下工作：1）谷氨酸受体通道的离子渗透机制。我们与加州大学洛杉矶分校的Sergio Ciani教授合作，研究了果蝇谷氨酸受体通道的离子渗透机制，并根据Eyring的速率反应理论建立了一个模型。该模型能较好地拟合各种离子环境下的I-V曲线。F 关于我们 因此，该模型成功地扩展到哺乳动物NMDA受体通道。2)果蝇谷氨酸受体通道的动力学。昆虫谷氨酸受体通道具有较大的单位电导，适合动力学研究。我们已经详细研究了动力学。需要两个激动剂分子将通道打开到主要开放状态，而一个分子将其短暂打开。这些特性类似于ACh受体通道。解离常数在mM范围内。3)突触形成。用膜片钳技术研究了胚胎的突触传递。受精后16小时自发突触电流，微型突触电流，开始出现。受精后21小时，即孵化时，振幅逐渐增大。在此期间，受体聚集在突触下膜。为了观察受体分布，我们提出了针对受体亚基的抗体。甚至在神经-肌肉接触之前就发现了小的受体簇。这些受体簇在突触处受体聚集之前消散。4)一种在突触传递中有缺陷的新突变体。我们用P元件诱变产生了1000多个突变体，并进行了筛选。发现了一个突变体，其在突触前和突触后元件中存在缺陷。该基因定位于第二染色体上的5 9D-E。我们与东京大学的Yoshiki Hotta教授的小组合作，克隆了该基因，目前正在试图培养针对该基因产物的抗体。虽然我们离最终目标还很远，但我们已经为进一步发展奠定了坚实的基础。少

Synaptic transmission is the basis of all human behaviors ranging from "simple" motor reflexes to "higher cognitive functions. While considerable progress has been make to resolve the broad outlines of synaptic development and transmission, the molecular events are still vague. To address this problem we must have a model system in which we can use as many modern analytical tools as possible. The model system that we have chosen is the fruit fly, Drosophila melanogaster. The ultimate goal of this proposal to elucidate the gene control mechanism of synapse formation. During the tenure of the three year grant, we have accomplished the followings ; 1) Ion permeation mechanism of glutamate receptor channels. In collaboration with Professor Sergio Ciani at UCLA we have examined the ion permeation mechanism of Drosophila glutamate receptor channels and developed a model based on Eyring's rate reaction theory. I-V curves under a variety of ionic environment were well fitted with this model. F … More urthermore, this model was successfully extended to the mammalian NMDA receptor channel. 2) Kinetics of the Drosophila glutamate receptor channel. Insect glutamate receptor channel has a large unitary conductance and is suitable kinetic studies. We have examined the kinetics in detail. Two agonist molecules are required to open the channel to the main open state while one molecule opens it briefly. These properties are similar to that of the ACh receptor channel. The dissociation constants are in the mM range. 3) Synapse formation. Synaptic transmission was studied in embryos using patch-clamp techniques. 16 hours after fertilization spontaneous synaptic currents, miniature synaptic currents, start to appear. The amplitude increased gradually toward 21 hours after fertilization, which is the time of hatching. During this period receptors accumulate at the subsynaptic membrane. To visualize the receptor distribution we raised antibodies against a receptor subunit. Small receptor clusters were found even before nerve-muscle contact. These receptor clusters dissipate before accumulation of receptors at the synapse. 4) A novel mutant which has defects in synaptic transmission. We generated over 1000 mutants using P element mutagenesis and screened them. One mutant was found which has defects in pre- and postsynaptic elements. The gene was located at 5 9D-E on the second chromosome. In collaboration with Professor Yoshiki Hotta's group at University of Tokyo we cloned the gene and currently are trying to raise antibodies against the gene product. Although we are far from the final goal we have laid a solid ground for further development. Less