Jia Min, Li Min-Xu, Fields R Douglas, Nelson Phillip G
National Institute of Child Health and Human Development, The National Institutes of Health, Bethesda, Maryland 20892, USA.
Dev Neurobiol. 2007 Jun;67(7):924-32. doi: 10.1002/dneu.20402.
Electrical activity during early development affects the development and maintenance of synapses (Spitzer [2006]: Nature 4447:707-712), but the intercellular signals regulating maintenance of synapses are not well identified. At the neuromuscular junction, adenosine 5-triphosphate (ATP) is coreleased with acetylcholine at activated nerve terminals to modulate synaptic function. Here we use cocultured mouse motor neurons and muscle cells in a three-compartment cell culture chamber to test whether endogenously released ATP plays a role in activity-dependent maintenance of neuromuscular synapses. The results suggest that ATP release at the synapse counters the negative effect of electrical activity, thus stabilizing activated synapses. Confirming our previous work (Li et al. [2001]: Nat Neurosci 4:871-872), we found that in doubly innervated muscles, electrical stimulation induced heterosynaptic downregulation of the nonstimulated convergent input to the muscle fiber with no or little change of the stimulated inputs. However, in preparations that were stimulated in the presence of apyrase, an enzyme that degrades extracellular ATP, synapse downregulation of stimulated inputs was substantial and significant, and end plate potentials were reduced. Apyrase treatment for 20 h in the absence of stimulation did result in moderate diminution, but this was prevented by blocking spontaneous neural activity with tetrodotoxin. The P2 receptor blocker, suramin, also induced activity-dependent synapse diminution. The decrease in synaptic efficacy produced by prolonged stimulation in the presence of apyrase persisted for greater than 20 h, consistent with a developmental time-course and distinct from the rapid neuromodulatory actions of ATP that have been demonstrated by others. We conclude that extracellular ATP promotes stabilization of the neuromuscular junction and may play a role in activity-dependent synaptic modification during development.
早期发育过程中的电活动会影响突触的发育与维持（斯皮策[2006]：《自然》4447:707 - 712），但调节突触维持的细胞间信号尚未完全明确。在神经肌肉接头处，三磷酸腺苷（ATP）在激活的神经末梢与乙酰胆碱共同释放，以调节突触功能。在此，我们利用三室细胞培养室中共培养的小鼠运动神经元和肌肉细胞，来测试内源性释放的ATP是否在神经肌肉突触的活动依赖性维持中发挥作用。结果表明，突触处ATP的释放抵消了电活动的负面影响，从而稳定了激活的突触。与我们之前的研究结果一致（李等人[2001]：《自然神经科学》4:871 - 872），我们发现，在双重支配的肌肉中，电刺激会导致对肌肉纤维的非刺激汇聚输入的异突触下调，而刺激输入则无变化或仅有微小变化。然而，在存在可降解细胞外ATP的酶——腺苷三磷酸双磷酸酶的情况下进行刺激的实验中，刺激输入的突触下调显著且明显，终板电位降低。在无刺激的情况下，用腺苷三磷酸双磷酸酶处理20小时确实会导致适度减少，但用河豚毒素阻断自发神经活动可防止这种情况发生。P2受体阻滞剂苏拉明也会诱导活动依赖性突触减少。在存在腺苷三磷酸双磷酸酶的情况下，长时间刺激所产生的突触效能降低持续超过20小时，这与发育时间进程一致，且不同于其他人所证明的ATP的快速神经调节作用。我们得出结论，细胞外ATP促进神经肌肉接头的稳定，并可能在发育过程中的活动依赖性突触修饰中发挥作用。
