Department of Neurosciences, University of California, San Diego, La Jolla, California 92093, United States.
Institute of Pharmacology, Center for Physiology and Pharmacology, Medical University of Vienna, Vienna 1090, Austria.
ACS Chem Neurosci. 2021 Feb 17;12(4):626-639. doi: 10.1021/acschemneuro.0c00643. Epub 2021 Feb 1.
Communication between neurons relies on the release of diverse neurotransmitters, which represent a key-defining feature of a neuron's chemical and functional identity. Neurotransmitters are packaged into vesicles by specific vesicular transporters. However, tools for labeling and imaging synapses and synaptic vesicles based on their neurochemical identity remain limited. We developed a genetically encoded probe to identify glutamatergic synaptic vesicles at the levels of both light and electron microscopy (EM) by fusing the mini singlet oxygen generator (miniSOG) probe to an intralumenal loop of the vesicular glutamate transporter-2. We then used a 3D imaging method, serial block-face scanning EM, combined with a deep learning approach for automatic segmentation of labeled synaptic vesicles to assess the subcellular distribution of transporter-defined vesicles at nanometer scale. These tools represent a new resource for accessing the subcellular structure and molecular machinery of neurotransmission and for transmitter-defined tracing of neuronal connectivity.
神经元之间的通讯依赖于各种神经递质的释放，这些递质是神经元化学和功能特征的关键定义特征。神经递质由特定的囊泡转运体包装到囊泡中。然而，基于神经化学特征来标记和成像突触和突触小泡的工具仍然有限。我们通过将迷你单线态氧发生器（miniSOG）探针融合到囊泡谷氨酸转运体-2 的腔内环，开发了一种遗传编码探针，以在光学显微镜（LM）和电子显微镜（EM）水平上识别谷氨酸能突触小泡。然后，我们使用 3D 成像方法，即连续块面扫描 EM，结合深度学习方法对标记的突触小泡进行自动分割，以评估纳米尺度上转运体定义的囊泡的亚细胞分布。这些工具为研究神经传递的亚细胞结构和分子机制以及神经递质的传递性追踪提供了新的资源。
