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Year:: 2022
Contributor:: McLean, Lily (Creator); Prakash, Mansi (Creator); Crespo, Emmanuel L. (Creator); Hochgeschwender, Ute (Creator)
Subject:: Bioluminescence; interluminescence; Lambert-Eaton Myasthenic Syndrome; neuromuscular junction
DOI: https://doi.org/10.26300/z1az-6m95

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Abstract:: Interluminescence is a method for selective modulation of synaptic communication between genetically defined partners. Bioluminescent light from a pre-synaptic released luciferase activates a post-synaptic localized optogenetic channel, thereby allowing synapse-specific modulation of selected partners under experimenter-controlled introduction of a luciferin. Here we applied the Interluminescence strategy to control neuron-muscle communication at the neuro-muscular junction (NMJ-Int) via this ‘optical synapse.’ The NMJ is a specialized peripheral synapse that translates the action potential of the presynaptic motor neuron to the contraction of the postsynaptic muscle fiber through the release of the neurotransmitter acetylcholine (ACh) and is thus essential for our physical mobility and daily life. Deficits in NMJ formation and maintenance cause several NMJ disorders (NMJDs), including Lambert–Eaton Myasthenic Syndrome (LEMS), Congenital Myasthenic Syndrome (CMS), Duchenne muscular dystrophy (DMD) and Myasthenia Gravis (MG). In a translational context, the NMJ-Int application carries the potential to rescue trans-synaptic neuro-muscular events when the traditional NMJ synaptic milieu is compromised by therapeutically correcting pathologically diminished or overactive muscle responses to neural input. To test bioluminescence control of neuron-muscle communication we set up an in vitro system by co-culturing embryonic spinal cord explants transduced with viral vectors expressing the luciferase and stable lines of C2C12, an immortalized mouse myoblast line, which expresses three different opsin constructs: ChR2(CS) (Step-function opsin; excitatory), CheRiff (excitatory) and hGtACR2 (inhibitory). We then tested the impact of Interluminescence on communication between spinal cord motor neurons and C2C12 skeletal muscle cells by recording the muscular contractions of opsin expressing myofibers, before and after application of the luciferin coelenterazine (CTZ) and vehicle. Images were acquired using an inverted microscope (Zeiss Axio Observer). The contractions of myotubes were recorded using a high-speed camera at 20X magnification under phase-contrast microscopy (Hamamatsu Orca-Flash4.0 V2 sCMOS camera; Andor iXon Ultra 888 EMCCD camera). Recordings looked at the excitatory opsin ChR2(CS) for experimental data and plain NMJ for control data. Myotube contractions were analyzed before and after LED stimulation or CTZ stimulation for experimental groups; vehicle addition was used as our control. The image analysis tool MUSCLEMOTION was used to analyze recordings in the open-source software Fiji. Using an automated open-source software tool increases the efficiency of image analysis compared to manual human analysis and enables quantitative analysis of spontaneous and stimulated myotube contractions.
Notes:: This research was supported by the the National Science Foundation (NSF) and its Division of Biological Infrastructure under Award No. 1707352 and the Chemical, Bioengineering, Environmental and Transport Systems (CBET) Division under award no. CBET-1464686

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Use and Reproduction: Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0)
Rights: In Copyright
Restrictions on Use: All Rights Reserved

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McLean, Lily, Prakash, Mansi, Crespo, Emmanuel L., et al., "Controlling neuron-muscle communication with biological light" (2022). NeuroNex Data and Research Products. Brown Digital Repository. Brown University Library. https://doi.org/10.26300/z1az-6m95

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NeuroNex Data and Research Products

This collection contains publicly available research products, including data, codes, and publications, among others. The NeuroNex Technology Hub is supported by the National Science Foundation (NSF) and its Division of Biological Infrastructure under Award No. 1707352.
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Controlling neuron-muscle communication with biological light