Langenfeld-Oster B, Dorlöchter M, Wernig A
Department of Physiology, University of Bonn, Germany.
J Neurocytol. 1993 Jul;22(7):517-30. doi: 10.1007/BF01189040.
Repeated in vivo observations of vitally stained neuromuscular junctions allow direct monitoring of ongoing structural changes, although, normally occurring changes (remodelling) and those inflicted by the illumination itself (photodamage) need to be dissociated. In frog cutaneus pectoris muscles, stained in vivo with 4Di-2ASP twice within four to five weeks, growth only was observed in 14 out of 92 junctions (in 18 muscles), retraction only in 19, and both features simultaneously in 22 junctions, while 37 junctions showed no changes. The summed growth in a junction amounted to 5-42 microns, retraction to 5-52 microns, while overall changes in synaptic length were absent. This and the simultaneous occurrence of both, growth and retraction within a single junction, indicates junctional remodelling. Similar amounts of remodelling were observed in junctions illuminated for 180, 60 or 10-30 s, indicating that within this range and with the given optical system, blue light and 4Di-2ASP fluorescence were not harmful. Remodelling was not induced by the experimental procedure per se, since in in vitro preparations signs of sprouting (and retraction) were equally frequent in junctions of totally untreated muscles, in junctions vitally stained four to five weeks previously or vitally stained but not light-exposed in the same muscle. In endplates of mouse gluteus maximus muscles double stained with 4Di-2ASP and rhodamine-alpha-bungarotoxin, unusually large (> 10 microns) terminal sprouts and retraction with gain loss of ACh-receptors became prominent 9-29 days following illumination for more than 60 s. Frequently also, muscle fibre damage with local contracture and acute loss of stainability of axon terminals occurred; often followed by muscle fibre denervation. In contrast, no such changes occurred after lower illumination intensities (12% emission filter for green) or shorter exposure times (< 60 s); even at a fourth exposure performed within 53 days. Previously reported smaller changes indicating regular remodelling were not investigated here.
对经活体染色的神经肌肉接头进行反复的体内观察，可以直接监测正在发生的结构变化，不过，需要区分正常发生的变化（重塑）和光照本身造成的变化（光损伤）。在蛙胸皮肌中，在四到五周内用4Di - 2ASP进行两次活体染色，在92个接头（来自18块肌肉）中，仅观察到14个接头有生长，19个接头仅有回缩，22个接头同时出现这两种特征，而37个接头没有变化。一个接头的总生长量为5 - 42微米，回缩量为5 - 52微米，而突触长度没有总体变化。这以及单个接头内生长和回缩同时出现的情况，表明了接头重塑。在分别照射180秒、60秒或10 - 30秒的接头中观察到了相似程度的重塑，这表明在这个范围内以及使用给定的光学系统时，蓝光和4Di - 2ASP荧光并无危害。重塑并非由实验操作本身诱发，因为在体外制备中，完全未处理的肌肉的接头、四到五周前进行过活体染色的接头或在同一肌肉中进行过活体染色但未光照的接头，出现发芽（和回缩）迹象的频率相同。在用4Di - 2ASP和罗丹明 - α - 银环蛇毒素双重染色的小鼠臀大肌终板中，在照射超过60秒后的9 - 29天，异常大（> 10微米）的终末芽生以及伴随乙酰胆碱受体丧失的回缩变得明显。此外，还经常出现肌肉纤维损伤并伴有局部挛缩以及轴突终末染色性急性丧失的情况；随后常常是肌肉纤维去神经支配。相比之下，在较低光照强度（绿色发射滤光片为12%）或较短曝光时间（< 60秒）下，即使在53天内进行第四次曝光，也未出现此类变化。此前报道的表明正常重塑的较小变化在此未作研究。
