Investigating spatiotemporal dynamics of neuropeptide diffusion in Caenorhabditis elegans with an orthogonal peptide-sensor system
ORAL
Abstract
Neurons use neuropeptides (NPs) to signal extrasynaptically in nervous systems. However, the diffusion dynamics of NPs through extracellular space that govern these ‘wireless’ connections have yet to be determined, leaving open questions regarding the speed and extent of NP-mediated signaling.
To investigate NP dynamics, we employ 2-photon targeted optogenetic excitation coupled with state-of-the-art molecular GRAB (G protein-coupled Receptor-Activation-Based) sensors to acquire high-resolution, time-resolved mapping of peptide diffusion dynamics in the worm. GRAB sensors are well-characterized in mammals; our results present the first demonstration of their function in the worm in vivo. We designed C. elegans strains expressing GRABOT1.9 sensors and peptides for mammalian oxytocin (OT) and an optogenetic actuator (GUR-3+PRDX-2) to trigger NP release. Using an exogenous orthogonal peptide allows for precise measurements of NP dynamics without interactions from endogenous signaling. We then use 2-photon laser stimulation to excite NP-expressing neurons to release NPs while conducting high-speed imaging to track NP diffusion through the worm as they bind to GRAB sensors expressed throughout the nervous system.
Results demonstrating the feasibility of this novel system for measuring NP dynamics will be presented, showing that GRAB sensors can detect spatiotemporal dynamics of orthogonal NPs upon optogenetically-triggered release in C. elegans.
To investigate NP dynamics, we employ 2-photon targeted optogenetic excitation coupled with state-of-the-art molecular GRAB (G protein-coupled Receptor-Activation-Based) sensors to acquire high-resolution, time-resolved mapping of peptide diffusion dynamics in the worm. GRAB sensors are well-characterized in mammals; our results present the first demonstration of their function in the worm in vivo. We designed C. elegans strains expressing GRABOT1.9 sensors and peptides for mammalian oxytocin (OT) and an optogenetic actuator (GUR-3+PRDX-2) to trigger NP release. Using an exogenous orthogonal peptide allows for precise measurements of NP dynamics without interactions from endogenous signaling. We then use 2-photon laser stimulation to excite NP-expressing neurons to release NPs while conducting high-speed imaging to track NP diffusion through the worm as they bind to GRAB sensors expressed throughout the nervous system.
Results demonstrating the feasibility of this novel system for measuring NP dynamics will be presented, showing that GRAB sensors can detect spatiotemporal dynamics of orthogonal NPs upon optogenetically-triggered release in C. elegans.
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Presenters
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Pearl SL Thijssen
- Princeton University