Patterns of synaptic activity in neural networks recorded by light emission from synaptolucins

The emission of light, coupled to exocytosis, can in principle be utilized to monitor the activity of a large number of individual synapses simultaneously. To illustrate this concept, fusion proteins of Cypridina luciferase and synaptotagmin-I or VAMP-2/synaptobrevin (which we term “synaptolucins”) were expressed in cultured hippocampal neurons with the help of viral vectors. Synaptolucins were targeted to synaptic vesicles and, upon exocytosis, formed light-emitting complexes with their cognate luciferin, which was added to the extracellular medium. Photon emissions required a depolarizing stimulus, occurred from regions with high synaptic density as ascertained by vital staining of recycling synaptic vesicles, and were sensitive to Ca(2+) depletion and clostridial neurotoxins. The method can currently detect exocytosis of the readily releasable pool of synaptic vesicles at a hippocampal synapse, corresponding to about two dozen quanta, but has the potential for greater sensitivity.

从原理上而言，与胞吐作用偶联的光发射可用于同时监测大量单个突触的活动。为阐释这一研究理念，我们将海萤荧光素酶（Cypridina luciferase）与突触结合蛋白I（synaptotagmin-I）或VAMP-2/小突触泡蛋白（synaptobrevin）构建为融合蛋白，并将这类融合蛋白命名为synaptolucins（突触荧光素蛋白），随后通过病毒载体将其在培养的海马神经元中表达。突触荧光素蛋白可靶向定位于突触囊泡；当发生胞吐作用时，其可与添加至细胞外培养基中的对应荧光素结合形成发光复合物。该光子发射信号依赖于去极化刺激，仅在突触密度较高的区域产生，这一结论可通过循环突触囊泡的活体染色得以验证，且其信号对钙离子耗竭与梭菌神经毒素敏感。当前，该方法可在海马突触中检测到突触囊泡易释放池的胞吐作用，对应约二十余个量子单位，且具备进一步提升灵敏度的潜力。