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SynLight:一种用于神经系统中同时标记活性区和细胞的双顺反子策略。

SynLight: a dicistronic strategy for simultaneous active zone and cell labeling in the nervous system.

作者信息

Aimino Michael A, Humenik Jesse, Parisi Michael J, Duhart Juan Carlos, Mosca Timothy J

机构信息

Department of Neuroscience, Vickie and Jack Farber Institute of Neuroscience, Thomas Jefferson University, Bluemle Life Sciences Building, Philadelphia, PA 19107.

出版信息

bioRxiv. 2023 Jul 17:2023.07.17.549367. doi: 10.1101/2023.07.17.549367.

Abstract

At synapses, chemical neurotransmission mediates the exchange of information between neurons, leading to complex movement behaviors and stimulus processing. The immense number and variety of neurons within the nervous system makes discerning individual neuron populations difficult, necessitating the development of advanced neuronal labeling techniques. In , Bruchpilot-Short and mCD8-GFP, which label presynaptic active zones and neuronal membranes, respectively, have been widely used to study synapse development and organization. This labeling is often achieved via expression of two independent constructs by a single binary expression system, but expression can weaken when multiple transgenes are expressed by a single driver. Ensuring adequate expression of each transgene is essential to enable more complex experiments; as such, work has sought to circumvent these drawbacks by developing methods that encode multiple proteins from a single transcript. Self-cleaving peptides, specifically 2A peptides, have emerged as effective sequences for accomplishing this task. We leveraged 2A ribosomal skipping peptides to engineer a construct that produces both Bruchpilot-Short and mCD8-GFP from the same mRNA, which we named SynLight. Using SynLight, we visualized the putative synaptic active zones and membranes of multiple classes of olfactory, visual, and motor neurons and observed correct separation of signal, confirming that both proteins are being generated separately. Furthermore, we demonstrate proof-of-principle by quantifying synaptic puncta number and neurite volume in olfactory neurons and finding no difference between the synapse densities of neurons expressing SynLight or neurons expressing both transgenes separately. At the neuromuscular junction, we determined that synaptic puncta number labeled by SynLight was comparable to endogenous puncta labeled by antibody staining. Overall, SynLight is a versatile tool for examining synapse density in any nervous system region of interest and allows new questions to be answered about synaptic development and organization.

摘要

在突触处,化学神经传递介导神经元之间的信息交换,从而产生复杂的运动行为和刺激处理。神经系统中神经元数量众多且种类各异,难以辨别单个神经元群体,因此需要开发先进的神经元标记技术。在[具体年份],分别标记突触前活性区和神经元膜的Bruchpilot-Short和mCD8-GFP已被广泛用于研究突触发育和组织。这种标记通常通过单一二元表达系统表达两个独立构建体来实现,但当单个驱动子表达多个转基因时,表达可能会减弱。确保每个转基因的充分表达对于开展更复杂的实验至关重要;因此,人们一直在努力开发从单个转录本编码多种蛋白质的方法来规避这些缺点。自我切割肽,特别是2A肽,已成为完成这项任务的有效序列。我们利用2A核糖体跳跃肽设计了一种构建体,该构建体可从同一mRNA产生Bruchpilot-Short和mCD8-GFP,我们将其命名为SynLight。使用SynLight,我们可视化了多类嗅觉、视觉和运动神经元的假定突触活性区和膜,并观察到信号的正确分离,证实两种蛋白质是分别产生的。此外,我们通过量化嗅觉神经元中的突触小点数和神经突体积来证明原理,发现表达SynLight的神经元与分别表达两个转基因的神经元的突触密度之间没有差异。在神经肌肉接头处,我们确定SynLight标记的突触小点数与抗体染色标记的内源性小点数相当。总体而言,SynLight是一种通用工具,可用于检查任何感兴趣的神经系统区域的突触密度,并能回答有关突触发育和组织的新问题。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1eb6/10370149/57def477f6ad/nihpp-2023.07.17.549367v1-f0001.jpg

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