Kirischuk S, Veselovsky N, Grantyn R
Entwicklungsphysiologie, Institut für Physiologie der Charité, Medizinische Fakultät (Charité) der Humboldt-Universität zu Berlin, Germany.
Pflugers Arch. 1999 Oct;438(5):716-24. doi: 10.1007/s004249900075.
Most central neurons contact their dendritic targets at several sites. However, it is not known whether all synapses formed by a single parent axon make the same contribution to the postsynaptic response. In order to answer this question it is necessary to isolate the synaptic currents generated by individual axon terminals. This paper describes a method that was designed to activate transmitter release from solitary synaptic boutons in culture. Neurons from the embryonic rat superior colliculus were grown at low density and double-loaded with a fluorescent marker of synaptic vesicles (FM1-43 or RH414) and a fluorescent Ca2+ indicator (Fura-2, Mag-fura-2, Oregon Green BAPTA-1 or Oregon Green BAPTA-5N). Action potential generation was blocked by tetrodotoxin. Appropriate synaptic boutons were selected under phase-contrast and fluorescence illumination at a magnification of 1000. They were activated by short electrical pulses via a fine-tipped glass pipette filled with bath solution. Presynaptic Ca2+ transients were measured in a region delineated by the FM1-43/RH414 fluorescence. By simultaneous presynaptic Ca2+ imaging and whole-cell recording of postsynaptic responses to single depolarizing pulses, the quantitative relationships between pre- and postsynaptic parameters of synaptic strength in a small synapse of central origin could, for the first time, be analysed. The experiments showed that the average postsynaptic currents depend strongly on the size of the presynaptic Ca2+ transients. However, at any level of presynaptic Ca2+ concentration postsynaptic responses fluctuated in amplitude.
大多数中枢神经元在多个位点与它们的树突靶点相接触。然而，单个母轴突形成的所有突触是否对突触后反应有相同的贡献尚不清楚。为了回答这个问题，有必要分离单个轴突终末产生的突触电流。本文描述了一种旨在激活培养物中孤立突触小体释放神经递质的方法。将胚胎大鼠上丘的神经元以低密度培养，并同时装载突触小泡的荧光标记物（FM1-43或RH414）和荧光Ca2+指示剂（Fura-2、Mag-fura-2、Oregon Green BAPTA-1或Oregon Green BAPTA-5N）。河豚毒素阻断动作电位的产生。在相差显微镜和荧光显微镜下，以1000倍放大倍数选择合适的突触小体。通过一根充满浴液的细尖玻璃微管施加短电脉冲来激活它们。在FM1-43/RH414荧光划定的区域内测量突触前Ca2+瞬变。通过同时进行突触前Ca2+成像和对单个去极化脉冲的突触后反应的全细胞记录，首次能够分析中枢起源的小突触中突触强度的突触前和突触后参数之间的定量关系。实验表明，平均突触后电流强烈依赖于突触前Ca2+瞬变的大小。然而，在任何突触前Ca2+浓度水平下，突触后反应的幅度都会波动。
