pH-sensitive fluorescent proteins have been widely used in various model organisms to study vesicle release and recycling. When targeted to the lumen of synaptic vesicles (SVs), these proteins are quenched due to acidification, which is essential for filling SVs with neurotransmitter. Activation of synaptic transmission leads to vesicle fusion with the plasma membrane and exposition of these fluorophores to the extracellular medium resulting in an increase in the emitted fluorescence. Subsequent acidification of SVs can thus be tracked by tagging of an integral SV protein to pH-sensitive proteins. Previous work by the Bargmann lab demonstrated that synaptic transmission and SV recycling in sensory neurons of live worms could be estimated by usage of the green-fluorescent pHluorin (1). However, while sensory neurons can be stimulated by application of the respective stimulus, this is not possible in motor neurons. To investigate the refilling of SV pools in this neuron type, we combined optogenetic methods to stimulate neuronal transmission with the novel red-fluorescent pH-sensitive protein pHuji (2). pHuji was inserted into the luminal loop of the integral SV protein synaptogyrin (SNG-1). This construct was co-expressed in cholinergic neurons with the blue light-gated ion channel channelrhodopsin-2 (ChR2). When supplemented with its fluorophore all-trans retinal (ATR), ChR2 can be used to depolarize neurons. We observed an increase in pHuji fluorescence after blue light illumination, only when animals were treated with ATR. Furthermore, the decay of pHuji fluorescence after stimulation is delayed in mutants of known SV recycling factors such as UNC-26 (synaptojanin) and UNC-57 (endophilin). Also, the increase in pHuji fluorescence is abolished in
snb-1 (synaptobrevin) SV fusion mutants. These results suggest that the rise and decay of fluorescence is an indicator for SV fusion and recycling. We now plan to investigate putative recycling factors through this assay we termed 'pOpsicle' for "pH-sensitive optogenetic reporter of synaptic vesicle recycling") by using semi-automatization based on microfluidic devices. We further want to explore differences in the recycling dynamics of cholinergic and GABAergic motor neurons and possibly other cell types. In sum, pOpsicle allows for a non-invasive and easily applicable investigation of the SV cycle. (1) Ventimiglia, D. and C. I. Bargmann (2017) Elife 6,
e31234. (2) Shen, Y et al. (2014) J Cell Biol 207(3): 419-432.