Data_Sheet_3_pOpsicle: An all-optical reporter system for synaptic vesicle recycling combining pH-sensitive fluorescent proteins with optogenetic manipulation of neuronal activity.XLSX

pH-sensitive fluorescent proteins are widely used to study synaptic vesicle (SV) fusion and recycling. When targeted to the lumen of SVs, fluorescence of these proteins is quenched by the acidic pH. Following SV fusion, they are exposed to extracellular neutral pH, resulting in a fluorescence increase. SV fusion, recycling and acidification can thus be tracked by tagging integral SV proteins with pH-sensitive proteins. Neurotransmission is generally activated by electrical stimulation, which is not feasible in small, intact animals. Previous in vivo approaches depended on distinct (sensory) stimuli, thus limiting the addressable neuron types. To overcome these limitations, we established an all-optical approach to stimulate and visualize SV fusion and recycling. We combined distinct pH-sensitive fluorescent proteins (inserted into the SV protein synaptogyrin) and light-gated channelrhodopsins (ChRs) for optical stimulation, overcoming optical crosstalk and thus enabling an all-optical approach. We generated two different variants of the pH-sensitive optogenetic reporter of vesicle recycling (pOpsicle) and tested them in cholinergic neurons of intact Caenorhabditis elegans nematodes. First, we combined the red fluorescent protein pHuji with the blue-light gated ChR2(H134R), and second, the green fluorescent pHluorin combined with the novel red-shifted ChR ChrimsonSA. In both cases, fluorescence increases were observed after optical stimulation. Increase and subsequent decline of fluorescence was affected by mutations of proteins involved in SV fusion and endocytosis. These results establish pOpsicle as a non-invasive, all-optical approach to investigate different steps of the SV cycle.

pH敏感荧光蛋白（pH-sensitive fluorescent proteins）被广泛应用于突触囊泡（synaptic vesicle, SV）融合与回收过程的研究。当这类蛋白被靶向定位至突触囊泡的管腔时，其荧光会被酸性pH环境淬灭。突触囊泡发生融合后，这些蛋白会暴露于胞外的中性pH环境中，进而引发荧光信号增强。因此，通过将pH敏感蛋白与整合型突触囊泡蛋白进行标记，即可实现对突触囊泡融合、回收及酸化过程的追踪。通常而言，神经传递可通过电刺激激活，但该方法在小型完整活体动物中并不适用。此前的体内成像研究依赖于特定（感觉）刺激，这极大限制了可被研究的神经元类型。为克服上述局限，我们开发了一套全光学方法以实现突触囊泡融合与回收的刺激与可视化。我们将插入至突触囊泡蛋白突触泡蛋白（synaptogyrin）中的不同pH敏感荧光蛋白，与光门控通道视紫红质（channelrhodopsins, ChRs）相结合用于光学刺激，解决了光学串扰问题，从而实现了全光学研究方案。我们构建了两种不同版本的囊泡回收pH敏感光遗传报告蛋白（pOpsicle），并在完整秀丽隐杆线虫（Caenorhabditis elegans）的胆碱能神经元中对其进行了测试。第一种方案将红色荧光蛋白pHuji与蓝光门控的ChR2(H134R)相结合，第二种方案则将绿色荧光pHluorin与新型红移型通道视紫红质ChrimsonSA相结合。在两种方案中，光学刺激后均观测到了荧光信号增强。荧光信号的上升及随后的衰减过程，会受到参与突触囊泡融合与内吞作用的蛋白突变的影响。上述研究结果证实，pOpsicle是一种非侵入性的全光学研究方法，可用于探究突触囊泡循环的不同阶段。