Virmani Tuhin, Ertunc Mert, Sara Yildirim, Mozhayeva Marina, Kavalali Ege T
Center for Basic Neuroscience, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9111, USA.
J Neurosci. 2005 Nov 23;25(47):10922-9. doi: 10.1523/JNEUROSCI.3766-05.2005.
Using electrophysiology and styryl dye imaging, we studied the effect of phorbol 12-myristate 13-acetate (PMA) on activity-dependent and spontaneous vesicle recycling. In electrophysiological experiments, we found that the PMA effect depended on the maturational state of the synapses. Spontaneous neurotransmitter release from nascent synapses without a functional readily releasable pool (RRP) was unresponsive to PMA application. In contrast, mature synapses responded robustly to PMA application, consistent with previous studies. Using styryl dye imaging, we found that there was a PMA-dependent increase in the size of the RRP when PMA was present before, during, or after activity-dependent dye uptake, suggesting that this effect involves an increase in the population of the RRP by vesicles recruited from the reserve pool. Additionally, we found that when PMA was present during spontaneous dye uptake, there was an increase in dye labeling, and these additional dye-loaded vesicles showed rapid destaining in response to strong stimulation and were also releasable by hypertonic sucrose. In contrast, these observations were not reproducible when PMA treatment was performed after spontaneous dye uptake and extracellular dye washout. Together, these findings suggest that the increased spontaneous neurotransmission in the presence of PMA was attributable to release of vesicles from the RRP rather than an effect of PMA on the spontaneously recycling pool. Thus, the phorbol esters selectively regulate the activity-dependent pool of vesicles, indicating that priming mechanisms that prepare vesicles for fusion, which are targeted by phorbol esters, are different for the spontaneous and evoked forms of fusion.
利用电生理学和苯乙烯基染料成像技术，我们研究了佛波醇12 - 肉豆蔻酸酯13 - 乙酸酯（PMA）对活动依赖性和自发性囊泡循环的影响。在电生理实验中，我们发现PMA的作用取决于突触的成熟状态。来自新生突触且没有功能性的易释放池（RRP）的自发性神经递质释放对PMA的应用无反应。相比之下，成熟突触对PMA的应用反应强烈，这与先前的研究一致。利用苯乙烯基染料成像，我们发现当在活动依赖性染料摄取之前、期间或之后存在PMA时，RRP的大小会出现PMA依赖性增加，这表明这种效应涉及从储备池中募集的囊泡增加了RRP的数量。此外，我们发现当在自发性染料摄取期间存在PMA时，染料标记增加，并且这些额外的染料加载囊泡在受到强刺激时显示出快速脱色，并且也可被高渗蔗糖释放。相比之下，当在自发性染料摄取和细胞外染料洗脱后进行PMA处理时，这些观察结果无法重现。总之，这些发现表明在PMA存在下自发性神经传递增加归因于从RRP释放囊泡，而不是PMA对自发循环池的影响。因此，佛波醇酯选择性地调节活动依赖性囊泡池，表明为融合准备囊泡的引发机制，即佛波醇酯的作用靶点，对于自发融合和诱发融合形式是不同的。
