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Quantitative imaging of synaptic vesicle localization and abundance using fluorescently labeled synaptic vesicle associated proteins like GFP::SNB-1 is a well-established method for measuring changes in synapse structure at neuromuscular junctions (NMJ) in C. elegans. To date, however, the ability to easily and reproducibly measure key parameters at the NMJ – maximum intensity, size of GFP::SNB-1 puncta, density of puncta – has relied on the use of expensive, customizable software that requires coding skills to modify, precluding widespread access and thus preventing standardization within the field. We carried out a comparative evaluation of a new, open-source Fiji puncta plugin versus traditional Igor-based analysis of GFP::SNB-1 imaging data taken of cholinergic motor neurons in the dorsal nerve cord of loss of function mutants in fshr-1, which encodes a G protein-coupled receptor known to impact GFP::SNB-1 accumulation. We analyzed images taken on a widefield fluorescence microscope, as well as on a spinning disk confocal microscope. Our data demonstrate strong concordance between the differences in GFP::SNB-1 localization in fshr-1 mutants compared to wild type worms across both analysis platforms (Fiji and Igor), as well as across microscope types (widefield and confocal). These data also agree with previously published observations related to synapse number and GFP::SNB-1 intensity in fshr-1 and wild type worms. Based on these findings, we conclude that the Fiji platform is viable as a method for analyzing synaptic vesicle localization and abundance at cholinergic dorsal nerve cord motor NMJs and expect the Fiji puncta plugin to be of broad utility in imaging across a variety of imaging platforms and synaptic markers.
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This content will become publicly available on November 19, 2025
Cell non-autonomous signaling through the conserved C. elegans glycoprotein hormone receptor FSHR-1 regulates cholinergic neurotransmission
Modulation of neurotransmission is key for organismal responses to varying physiological contexts such as during infection, injury, or other stresses, as well as in learning and memory and for sensory adaptation. Roles for cell autonomous neuromodulatory mechanisms in these processes have been well described. The importance of cell non-autonomous pathways for inter-tissue signaling, such as gut-to-brain or glia-to-neuron, has emerged more recently, but the cellular mechanisms mediating such regulation remain comparatively unexplored. Glycoproteins and their G protein-coupled receptors (GPCRs) are well-established orchestrators of multi-tissue signaling events that govern diverse physiological processes through both cell-autonomous and cell non-autonomous regulation. Here, we show that follicle stimulating hormone receptor, FSHR-1, the soleCaenorhabditis elegansortholog of mammalian glycoprotein hormone GPCRs, is important for cell non-autonomous modulation of synaptic transmission. Inhibition offshr-1expression reduces muscle contraction and leads to synaptic vesicle accumulation in cholinergic motor neurons. The neuromuscular and locomotor defects infshr-1loss-of-function mutants are associated with an underlying accumulation of synaptic vesicles, build-up of the synaptic vesicle priming factor UNC-10/RIM, and decreased synaptic vesicle release from cholinergic motor neurons. Restoration of FSHR-1 to the intestine is sufficient to restore neuromuscular activity and synaptic vesicle localization tofshr-1-deficient animals. Intestine-specific knockdown of FSHR-1 reduces neuromuscular function, indicating FSHR-1 is both necessary and sufficient in the intestine for its neuromuscular effects. Re-expression of FSHR-1 in other sites of endogenous expression, including glial cells and neurons, also restored some neuromuscular deficits, indicating potential cross-tissue regulation from these tissues as well. Genetic interaction studies provide evidence that downstream effectorsgsa-1/GαS,acy-1/adenylyl cyclase andsphk-1/sphingosine kinase and glycoprotein hormone subunit orthologs, GPLA-1/GPA2 and GPLB-1/GPB5, are important for intestinal FSHR-1 modulation of the NMJ. Together, our results demonstrate that FSHR-1 modulation directs inter-tissue signaling systems, which promote synaptic vesicle release at neuromuscular synapses.
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- Award ID(s):
- 2116348
- PAR ID:
- 10556059
- Author(s) / Creator(s):
- ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; more »
- Editor(s):
- Hart, Anne C
- Publisher / Repository:
- PLOS
- Date Published:
- Journal Name:
- PLOS Genetics
- Volume:
- 20
- Issue:
- 11
- ISSN:
- 1553-7404
- Page Range / eLocation ID:
- e1011461
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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