OPTICAL MONITORING OF TRANSMITTER RELEASE AND SYNAPTIC VESICLE RECYCLING AT THE FROG NEUROMUSCULAR-JUNCTION
OPTICAL MONITORING OF TRANSMITTER RELEASE AND SYNAPTIC VESICLE RECYCLING AT THE FROG NEUROMUSCULAR-JUNCTION
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DOI：
10.1113/jphysiol.1993.sp019472
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发表时间：
1993-01-01
影响因子：
5.5
通讯作者：
BEWICK, GS
中科院分区：
文献类型：
--
作者：
BETZ, WJ;BEWICK, GS
1.用荧光染料FM 1 -43负载蛙皮心运动神经末梢，沿着末梢长度方向产生一系列离散的斑点，每个斑点明显标记一簇突触小泡。终端成像2-10分钟，因为它们在重复神经刺激期间脱色。同时记录这些终末支配的肌纤维的终板电位（EPP），其振幅之和提供了累积递质释放的量度.任一末端的单个荧光点的初始亮度不同，但以相似的分数率褪色。在2-30 Hz范围内，随着刺激频率的增加，累积递质释放率和褪色率增加。然而，在40 Hz时，递质释放和脱色都比30 Hz时慢。在26个实验中，定量比较了染料损失率和递质释放率。当缩放总EPP的时间过程以拟合脱色前30-60 s期间染料损失的时间过程时，两条曲线通常在稍后的时间发散，染料损失曲线低于总EPP曲线。因此，假设染料损失和发射器释放在早期是成比例的，在稍后的时间，染料损失的速率相对于发射器释放的速率降低。在2至30 Hz的刺激频率下，结果可以通过一个简单的模型拟合，其中囊泡在胞吐过程中失去染料，并且在固定的再循环“停滞时间”之后，它们重新进入囊泡池，与其他囊泡随机混合。与较低频率的刺激不同，在40 Hz时，染料损失和EPP振幅曲线的总和并没有显着发散。刺激期持续约2分钟。根据囊泡再循环模型解释，这表明囊泡再循环在40 Hz下受到抑制。该模型导致预测的相对数量，N，囊泡（标记和未标记）在终端在任何时候在刺激。N的计算值在小于再循环“死时间”的时间降低。然后反映囊泡池中再循环囊泡的外观。从N和记录的EPP振幅的估计，每次电击释放的囊泡的分数，F，可以计算在整个刺激期间。在低刺激频率（2 - 5赫兹）。在初始的快速下降后，F在6 min内缓慢地单调下降约50%。在较高的刺激频率下，观察到不同的过程。高频刺激开始后约20 s，F的下降速度减慢，在某些情况下逆转并开始增加。在10-20 Hz下，F在刺激的第二分钟期间大约加倍。这种囊泡释放的可能性增加可能产生强直后增强现象。结果表明，染料释放在重复神经刺激期间的早期提供了递质释放的准确测量。在以后的时间，光学和电生理记录之间的分歧可以解释一个模型，其中的“死时间”后，胞吐，囊泡在随机位置回收到池中预先存在的囊泡。
1. Frog cutaneous pectoris motor nerve terminals were loaded with the fluorescent dye FM1-43, which produced a series of discrete spots along the length of terminals, each spot evidently marking a cluster of synaptic vesicles. Terminals were imaged for 2-10 min as they destained during repetitive nerve stimulation. Endplate potentials (EPPs) were recorded simultaneously from the muscle fibres innervated by these terminals; their summed amplitudes provided a measure of cumulative transmitter release.2. Individual fluorescent spots in any one terminal varied in initial brightness but destained at similar fractional rates.3. The rates of cumulative transmitter release and destaining increased with stimulus frequency in the range 2-30 Hz. At 40 Hz, however, both transmitter release and destaining were slower than at 30 Hz.4. In twenty-six experiments, rates of dye loss and transmitter release were compared quantitatively. When the time course of summed EPPs was scaled to fit the time course of dye loss during the first 30-60 s of destaining, the two curves usually diverged at later times, the dye loss curve failing below the summed EPP curve. Thus, assuming that dye loss and transmitter release are proportional at early times, at later times the rate of dye loss decreases relative to the rate of transmitter release.5. At stimulus frequencies from 2 to 30 Hz, the results could be fitted by a simple model in which vesicles lose their dye during exocytosis and, after a fixed recycle 'dead time', they re-enter the vesicle pool, mixing randomly with other vesicles.6. Unlike stimulation at lower frequencies, at 40 Hz dye loss and summed EPP amplitude curves did not significantly diverge. Stimulation periods lasted up to about 2 min. Interpreted according to the model of vesicle recycling, this suggests that vesicle recycling is inhibited at 40 Hz.7. The model led to predictions about the relative number, N, of vesicles (labelled and unlabelled) in the terminal at any time during stimulation. The calculated value of N decreased at times less than the recycle 'dead time'. and then reflecting the appearance of recycled vesicles in the vesicle pool.8. From estimates of N and recorded EPP amplitudes, the fraction of vesicles released per shock, F, could be calculated during the entire stimulation period. At low stimulus frequencies (2 5 Hz). after an initial rapid fall, F decreased slowly and monotonically by about 50% in 6 min. At higher stimulus frequencies, a different process was observed. About 20 s after high-frequency stimulation began, the decline in F slowed, and in some cases reversed and began to increase. At 10-20 Hz, F approximately doubled during the second minute of stimulation. This increased probability of vesicle release may produce the phenomenon of post-tetanic potentiation.9. The results suggest that dye release gives an accurate measure of transmitter release at early times during repetitive nerve stimulation. At later times, divergence between optical and electrophysiological recordings can be explained by a model in which a 'dead time' follows exocytosis, after which vesicles recycle at random positions into the pool of pre-existing vesicles.