Optogenetic tools for clustering of synaptic vesicles to rapidly and reversibly silence neurons and for analysis of vesicle mobility
Authors:   , Rentsch Dennis 
Published: October 2024

Understanding the functional roles of cells in neuronal circuits and behavior requires the ability to control neuronal activity in an acute and precise way. The field of optogenetics offers a variety of molecular tools to excite or inhibit neurons with light. In the last decade, several strategies have been proposed for reversible silencing of neurotransmission. These tools vary widely in their mechanisms: ranging from opsin-based light-driven ion pumps or anion channels, which are known to hyperpolarize the cell, to alter ionic gradients or cellular biochemistry; over metabotropic receptors, to tools damaging the neurotransmitter release machinery, that allow only long-term silencing as their recovery requires de novo synthesis of targeted proteins. Therefore, the optogenetic toolbox still lacks tools that combine fast activation and fast reversibility with the ability of long-term silencing. In this study, the optogenetic tool optoSynC (optogenetic synaptic vesicle clustering) was characterized in depth. optoSynC utilizes the light-induced homo-oligomerization of Arabidopsis thaliana cryptochrome 2 (CRY2) for silencing synaptic transmission via clustering of synaptic vesicles (SVs). CRY2 was targeted to the SV membrane by fusion to the SV transmembrane protein synaptogyrin-1 (SNG-1). The silencing kinetics of optoSynC were determined with analyses of swimming and crawling behavior in Caenorhabditis elegans (C. elegans). Pan-neuronally expressed optoSynC reduced swimming locomotion by 80% within 30 s following photo-stimulation (τon ~7.2 s). Locomotion recovered within 15 min in darkness (τoff ~6.5 min). Analysis of crawling behavior indicated even faster activation within 2 s and an almost complete inhibition of the LITE-1-mediated escape response. This escape response occurs at high blue light intensities and results in an increased velocity, which was not detected after optoSynC activation. However, optoSynC can exert its full effect at significantly lower intensities (25 µW/mm²) and is so light-sensitive that it can even be activated at 1.4 µW/mm². optoSynC could be fully recovered and reactivated at least twice without decreased efficiency. With a combination of pharmacological analysis and optogenetics, it has been demonstrated that optoSynC can inhibit neurotransmitter release for several hours. To realize its full potential, optoSynC should be expressed in an sng-1 mutant background. Expression along with endogenous SNG-1 decreases the effectiveness of optoSynC by 50%. Specific expression of optoSynC in cholinergic or GABAergic neurons could robustly inhibit swimming behavior by 55% and 30%, respectively. Moreover, optoSynC can selectively inhibit individual neurons like the nociceptive neuron pair PVD. When PVD is photoactivated by Chrimson, a red-light activatable channelrhodopsin, forward locomotion increases. Activation of optoSynC reduced this behavior by 50%. Besides my experiments in C. elegans, my cooperation partners Dr. Holger Dill and Yilmaz Arda Ateş demonstrated silencing of neurotransmission with optoSynC in zebrafish, and Dr. Shigeki Watanabe and Brady D. Goulden in murine hippocampal neurons. The clustering of SVs as the mode of action of optoSynC could be confirmed using transmission electron microscopy. Analysis of micrographs of cholinergic synapses stimulated with and without light revealed that distances of neighboring synaptic vesicles in the cytosol were reduced by around 14% after photoactivation. Distances of docked SVs at the plasma membrane remained unaffected. However, near the dense projection, docked SVs accumulated, while other docked SVs were depleted after optoSynC activation. Activation of optoSynC increased the appearances of SVs and dense core vesicles (DCVs) in micrographs. It is unclear whether sizes became physically larger due to lateral pressure by CRY2 aggregates in the SV membrane or if oligomer formation only altered their appearance in micrographs. optoSynC did not accumulate in the plasma membrane to the extent that abnormal structures at the plasma membrane or dense projection were observed. Recycling of SVs remained unaffected by optoSynC as no unusual number of large vesicles was determined. Therefore, optoSynC inhibits neuronal activity mainly by clustering of cytosolic SVs. To study the precise sequence and timing of events of vesicle mobilization, it is necessary to introduce known stops in the synaptic vesicle cycle which can be achieved by optoSynC. The C. elegans mutation dyn-1(ts-) is a temperature-sensitive dynamin mutation that blocks the recycling of SVs from the plasma membrane and early endosomes at temperatures exceeding 25 °C. By expressing optoSynC in dyn-1(ts-) animals, a novel assay was established, enabling the transfer of SVs between different stages in the SV cycle. Cluster formation of reserve pool SVs blocks the SV cycle before the process of docking and priming begins, while the SV cycle is blocked after the fusion of SVs at high temperatures. It could be demonstrated that behavior returned 15 min after optoSynC activation while animals without optoSynC remained immobile. Unfortunately, the time scale of the recovery from inhibition by optoSynC is too long to effectively study vesicle mobilization.