de Castro Braulio M, De Jaeger Xavier, Martins-Silva Cristina, Lima Ricardo D F, Amaral Ernani, Menezes Cristiane, Lima Patricia, Neves Cintia M L, Pires Rita G, Gould Thomas W, Welch Ian, Kushmerick Christopher, Guatimosim Cristina, Izquierdo Ivan, Cammarota Martin, Rylett R Jane, Gomez Marcus V, Caron Marc G, Oppenheim Ronald W, Prado Marco A M, Prado Vania F
Molecular Brain Research Group, Robarts Research Institute, University of Western Ontario, P.O. Box 5015, 100 Perth Drive, London, Ontario N6A 5K8, Canada.
Mol Cell Biol. 2009 Oct;29(19):5238-50. doi: 10.1128/MCB.00245-09. Epub 2009 Jul 27.
The vesicular acetylcholine (ACh) transporter (VAChT) mediates ACh storage by synaptic vesicles. However, the VAChT-independent release of ACh is believed to be important during development. Here we generated VAChT knockout mice and tested the physiological relevance of the VAChT-independent release of ACh. Homozygous VAChT knockout mice died shortly after birth, indicating that VAChT-mediated storage of ACh is essential for life. Indeed, synaptosomes obtained from brains of homozygous knockouts were incapable of releasing ACh in response to depolarization. Surprisingly, electrophysiological recordings at the skeletal-neuromuscular junction show that VAChT knockout mice present spontaneous miniature end-plate potentials with reduced amplitude and frequency, which are likely the result of a passive transport of ACh into synaptic vesicles. Interestingly, VAChT knockouts exhibit substantial increases in amounts of choline acetyltransferase, high-affinity choline transporter, and ACh. However, the development of the neuromuscular junction in these mice is severely affected. Mutant VAChT mice show increases in motoneuron and nerve terminal numbers. End plates are large, nerves exhibit abnormal sprouting, and muscle is necrotic. The abnormalities are similar to those of mice that cannot synthesize ACh due to a lack of choline acetyltransferase. Our results indicate that VAChT is essential to the normal development of motor neurons and the release of ACh.
囊泡乙酰胆碱（ACh）转运体（VAChT）通过突触囊泡介导ACh的储存。然而，在发育过程中，不依赖VAChT的ACh释放被认为很重要。在此，我们构建了VAChT基因敲除小鼠，并测试了不依赖VAChT的ACh释放的生理相关性。纯合子VAChT基因敲除小鼠在出生后不久死亡，这表明VAChT介导的ACh储存对生命至关重要。实际上，从纯合子基因敲除小鼠大脑中获得的突触体不能响应去极化而释放ACh。令人惊讶的是，在骨骼肌神经肌肉接头处的电生理记录显示，VAChT基因敲除小鼠呈现出自发性微小终板电位，其幅度和频率降低，这可能是ACh被动转运到突触囊泡的结果。有趣的是，VAChT基因敲除小鼠的胆碱乙酰转移酶、高亲和力胆碱转运体和ACh的量大幅增加。然而，这些小鼠的神经肌肉接头发育受到严重影响。突变型VAChT小鼠的运动神经元和神经末梢数量增加。终板较大，神经出现异常发芽，肌肉坏死。这些异常与因缺乏胆碱乙酰转移酶而无法合成ACh的小鼠相似。我们的结果表明，VAChT对运动神经元的正常发育和ACh的释放至关重要。
