Department of Molecular and Cellular Biology and Center for Brain Science, Harvard University, Cambridge MA, USA.
Front Mol Neurosci. 2012 Feb 16;5:18. doi: 10.3389/fnmol.2012.00018. eCollection 2012.
In the "GFP reconstitution across synaptic partners" (GRASP) method, non-fluorescent fragments of GFP are expressed in two different neurons; the fragments self-assemble at synapses between the two to form a fluorophore. GRASP has proven useful for light microscopic identification of synapses in two invertebrate species, Caenorhabditis elegans and Drosophila melanogaster, but has not yet been applied to vertebrates. Here, we describe GRASP constructs that function in mammalian cells and implement a transgenic strategy in which a Cre-dependent gene switch leads to expression of the two fragments in mutually exclusive neuronal subsets in mice. Using a transgenic line that expresses Cre selectively in rod photoreceptors, we demonstrate labeling of synapses in the outer plexiform layer of the retina. Labeling is specific, in that synapses made by rods remain labeled for at least 6 months whereas nearby synapses made by intercalated cone photoreceptors on many of the same interneurons remain unlabeled. We also generated antisera that label reconstituted GFP but neither fragment in order to amplify the GRASP signal and thereby increase the sensitivity of the method.
在“跨突触伙伴 GFP 重建”（GRASP）方法中，非荧光 GFP 片段在两个不同的神经元中表达；这些片段在两个神经元之间的突触处自行组装，形成荧光团。GRASP 已被证明可用于鉴定两种无脊椎动物物种秀丽隐杆线虫和黑腹果蝇中的突触，但尚未应用于脊椎动物。在这里，我们描述了在哺乳动物细胞中起作用的 GRASP 构建体，并实施了一种转基因策略，其中 Cre 依赖性基因开关导致两种片段在小鼠中相互排斥的神经元亚群中表达。使用一种在杆状光感受器中特异性表达 Cre 的转基因系，我们证明了在视网膜外丛状层中突触的标记。标记是特异性的，因为杆状细胞形成的突触至少可以标记 6 个月，而在许多相同中间神经元上由插入型圆锥状光感受器形成的附近突触则保持未标记状态。我们还生成了针对重组 GFP 但不针对任何片段的抗血清，以放大 GRASP 信号，从而提高该方法的灵敏度。
