title: High-Throughput All-Optical Analysis of Synaptic Transmission and Synaptic Vesicle Recycling in Caenorhabditis elegans.
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.
authors: Buchmann Institute for Molecular Life Sciences (BMLS) and Institute of Biochemistry, Goethe-University, Max-von-Laue-Str. 9 and 15, 60438, Frankfurt, Germany.
authors: Sebastian
authors: S
authors: Wabnig
authors: Buchmann Institute for Molecular Life Sciences (BMLS) and Institute of Biochemistry, Goethe-University, Max-von-Laue-Str. 9 and 15, 60438, Frankfurt, Germany.
authors: Jana Fiona
authors: JF
authors: Liewald
authors: Buchmann Institute for Molecular Life Sciences (BMLS) and Institute of Biochemistry, Goethe-University, Max-von-Laue-Str. 9 and 15, 60438, Frankfurt, Germany.
authors: Szi-Chieh
authors: SC
authors: Yu
authors: Buchmann Institute for Molecular Life Sciences (BMLS) and Institute of Biochemistry, Goethe-University, Max-von-Laue-Str. 9 and 15, 60438, Frankfurt, Germany.
authors: Alexander
authors: A
authors: Gottschalk
mesh_terms: D000818:Animals; D017173:Caenorhabditis elegans / Q000235:genetics / Q000502:physiology*; D029742:Caenorhabditis elegans Proteins / Q000235:genetics*; D016000:Cluster Analysis; D004705:Endocytosis; D005089:Exocytosis; D008856:Microscopy, Fluorescence / Q000379:methods; D034622:RNA Interference; D059386:Reverse Genetics / Q000379:methods; D009435:Synaptic Transmission / Q000235:genetics / Q000502:physiology*; D013572:Synaptic Vesicles / Q000378:metabolism / Q000502:physiology*
chemical_list: D029742:Caenorhabditis elegans Proteins