Tse F W, Marin L, Atwood H L
Department of Physiology, University of Toronto, Ont., Canada.
J Neurosci Methods. 1987 Sep;21(1):17-29. doi: 10.1016/0165-0270(87)90099-9.
A technique to label active synaptic terminals, whose electrophysiology had been monitored by a macro-patch electrode, was developed for a crustacean neuromuscular preparation. The active synaptic terminals were labeled by release-dependent uptake of horseradish peroxidase (HRP) into synaptic vesicles. The focal labeling technique involved the following steps: (1) locating a site where evoked synaptic currents could be recorded at a subset of neuromuscular synapses by a macro-patch electrode; (2) reducing synaptic transmission by bathing the preparation in a solution containing low [Ca2+] and high [Mg2+]; (3) introducing HRP as an extracellular marker into the solution bathing the preparation; (4) restoring synaptic release focally by ejection of a solution containing Ca2+ from the macro-patch electrode. The muscle fibre with labeled synapses was fixed for electron microscopy and processed for HRP histochemistry. The distribution of HRP-labeled vesicles was documented by electron microscopy and semi-serial sectioning. A significant increase in labeled vesicles was found within a maximum radius of 10-15 micron from the lumen of the macro-path electrode. This maximum radius of labeling set an upper limit to the number of active synapses recorded by the macro-patch electrode.
针对一种甲壳类神经肌肉标本，开发了一种标记活跃突触终末的技术，该突触终末的电生理学已通过一个大膜片电极进行监测。活跃的突触终末通过依赖释放的辣根过氧化物酶（HRP）摄取到突触小泡中进行标记。局灶性标记技术包括以下步骤：（1）通过大膜片电极在神经肌肉突触的一个子集中找到一个可记录诱发突触电流的部位；（2）将标本置于含有低[Ca2+]和高[Mg2+]的溶液中浸泡以减少突触传递；（3）将HRP作为细胞外标记物引入浸泡标本的溶液中；（4）通过从大膜片电极喷射含有Ca2+的溶液来局部恢复突触释放。对具有标记突触的肌纤维进行固定用于电子显微镜检查，并进行HRP组织化学处理。通过电子显微镜和半连续切片记录HRP标记小泡的分布。在距大膜片电极管腔最大半径10 - 15微米范围内发现标记小泡显著增加。这种最大标记半径为大膜片电极记录的活跃突触数量设定了上限。
