In vertebrates, muscle activity is dependent on acetylcholine (ACh) released from neuromuscular junctions (NMJs), and changes in cholinergic neurotransmission are linked to a variety of neuromuscular diseases, including congenital myasthenic syndromes (CMS). The storage and release of ACh depends on the activity of the Vesicular Acetylcholine Transporter (VAChT), a rate-limiting step for cholinergic neurotransmission whose loss of function mutations was shown to cause human congenital myasthenia. However, we know much less about increased VAChT activity, due to copy number variations, for example. Therefore, here we investigated the impact of increased VAChT expression and consequently ACh levels at the synaptic cleft of the diaphragm NMJs. We analyzed structure and function of nerve and muscles from a mouse model of cholinergic hyperfunction (ChAT-ChR2-EYFP) with increased expression of VAChT. Our results showed a significant increase of ACh released under evoked stimuli. However, we observed deleterious changes in synaptic vesicles cycle (impaired endocytosis and decrease in vesicles number), together with structural alterations of NMJs. Interestingly, ultrastructure analyses showed that synaptic vesicles from ChAT-ChR2-EYFP mice NMJs were larger, which might be related to increased ACh load. We also observed that these larger synaptic vesicles were less rounded in comparison with control. Finally, we showed that ChAT-ChR2-EYFP mice NMJs have compromised safety factor, possible due to the structural alterations we described. These findings reveal that physiological cholinergic activity is important to maintain the structure and function of the neuromuscular system and help to understand some of the neuromuscular adverse effects experienced by chronically increased NMJ neurotransmission, such as individuals treated with cholinesterase inhibitors.

在脊椎动物中，肌肉活动取决于神经肌肉接头（NMJ）释放的乙酰胆碱（ACh），胆碱能神经传递的变化与多种神经肌肉疾病有关，包括先天性肌无力综合症（CMS）。ACh的储存和释放取决于水泡乙酰胆碱转运蛋白（VAChT）的活性，这是胆碱能神经传递的限速步骤，其功能突变被证明可导致人类先天性肌无力。但是，例如由于拷贝数变化，我们对VAChT活性增加的了解却少得多。因此，在这里我们调查了增加的VAChT表达的影响，并因此调查了隔膜NMJs突触间隙中ACh的水平。我们分析了胆碱能亢进（ChAT-ChR2-EYFP）与VAChT表达增加的小鼠模型的神经和肌肉的结构和功能。我们的结果表明，诱发的刺激下乙酰胆碱释放明显增加。但是，我们观察到突触小泡周期的有害变化（内吞作用减弱和小泡数目减少），以及NMJ的结构改变。有趣的是，超微结构分析显示，来自ChAT-ChR2-EYFP小鼠NMJ的突触小泡较大，这可能与ACh负荷增加有关。我们还观察到，与对照相比，这些较大的突触囊泡较不圆。最后，我们表明ChAT-ChR2-EYFP小鼠NMJ损害了安全系数，这可能是由于我们描述的结构改变所致。