Abstract
Synaptic vesicles (SVs) undergo a cycle of biogenesis and membrane fusion to release transmitter, followed by recycling. How exocytosis and endocytosis are coupled is intensively investigated. We describe an all-optical method for identification of neurotransmission genes that can directly distinguish SV recycling factors in C. elegans, by motoneuron photostimulation and muscular RCaMP Ca2+ imaging. We verified our approach on mutants affecting synaptic transmission. Mutation of genes affecting SV recycling (unc-26 synaptojanin, unc-41 stonin, unc-57 endophilin, itsn-1 intersectin, snt-1 synaptotagmin) showed a distinct ‘signature’ of muscle Ca2+ dynamics, induced by cholinergic motoneuron photostimulation, i.e. faster rise, and earlier decrease of the signal, reflecting increased synaptic fatigue during ongoing photostimulation. To facilitate high throughput, we measured (3–5 times) ~1000 nematodes for each gene. We explored if this method enables RNAi screening for SV recycling genes. Previous screens for synaptic function genes, based on behavioral or pharmacological assays, allowed no distinction of the stage of the SV cycle in which a protein might act. We generated a strain enabling RNAi specifically only in cholinergic neurons, thus resulting in healthier animals and avoiding lethal phenotypes resulting from knockdown elsewhere. RNAi of control genes resulted in Ca2+ measurements that were consistent with results obtained in the respective genomic mutants, albeit to a weaker extent in most cases, and could further be confirmed by opto-electrophysiological measurements for mutants of some of the genes, including synaptojanin. We screened 95 genes that were previously implicated in cholinergic transmission, and several controls. We identified genes that clustered together with known SV recycling genes, exhibiting a similar signature of their Ca2+ dynamics. Five of these genes (C27B7.7, erp-1, inx-8, inx-10, spp-10) were further assessed in respective genomic mutants; however, while all showed electrophysiological phenotypes indicative of reduced cholinergic transmission, no obvious SV recycling phenotypes could be uncovered for these genes.
Highlights
Chemical synaptic transmission occurs at synaptic contacts, where a presynaptic neuron releases small neurotransmitters by regulated fusion of synaptic vesicles (SVs)[1,2,3]
2) SVs can be directly used for fusion, or they enter a pool of vesicles that are stored for future release [8, 9]. 3) Release involves approximation of the SV to the synaptic active zone membrane, and formation of protein complexes (e.g. SNARE complexes) that regulate and execute the fusion event, processes termed docking and priming [10]
Mutations generally affecting SV release exhibit stronger contraction than wildtype, which is due to compensatory higher excitability in muscle [28], and SV recycling mutants show a progressively decreasing contraction, as SVs become depleted (Fig 1C)
Summary
Chemical synaptic transmission occurs at synaptic contacts, where a presynaptic neuron releases small neurotransmitters by regulated fusion of synaptic vesicles (SVs)[1,2,3]. Exo- and endocytosis were speculated to be coordinated based on the elevated Ca2+ level during neuronal activation, but such Ca2+ sensors have not been identified as yet [21, 22] It is unclear how the SV proteins are sorted such that the new SV is equipped with the right complement of proteins, at endosomes or even the plasma membrane [20]. Specific proteins are involved in regulating these processes, but such proteins have not been described yet Absence of these proteins should result in a shortage of functional SVs, which should become limiting during sustained SV release at high rates, and this phenotype, indicative of SV recycling factors, could be used in screening approaches for such proteins
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