All-optical analysis of electrical coupling in muscle ensembles reveals contributions of individual innexins to cell synchronization and locomotion

Abstract Gap junctions (GJs), formed by connexins in vertebrates and innexins in invertebrates, enable direct ion flow between adjacent cells. GJ coupling is typically analyzed by electrophysiological methods, which, however, are invasive and cannot be conducted in intact animals. Here, we used all-optical methods to non-invasively investigate electrical coupling in body wall muscle (BWM) cells of intact Caenorhabditis elegans . We analyzed the roles of specific innexins ( unc-9, inx-11, inx-16 ) in BWMs, using the genetically encoded fluorescent voltage indicator QuasAr2 to assess spontaneous muscle activity. unc-9 mutants showed strongly reduced coupling and asynchronous activity, and this was accompanied by severe locomotion defects. Overexpression of the murine connexin Cx36 increased synchronization but disrupted locomotion. inx-16 mutants exhibited increased excitability of individual muscle cells, likely due to reduced leak currents, while inx-11 mutants displayed only moderate defects. Patch-clamp recordings confirmed altered action potentials and increased input resistance in inx-16 mutants. Additionally, we established a cell-specific optogenetic voltage clamp (cOVC) method, directly revealing changes in junctional conductance in live, non-dissected animals. In sum, our findings demonstrate that a balanced level of GJ coupling is essential for coordinated muscle activity and proper motor behavior. Significance statement Coordination of cells in muscular organs requires electrical coupling via intercellular channels termed gap junctions (GJs). How this coupling contributes to coordinated activity like locomotion in an intact animal is difficult to assess, since commonly used electrophysiological methods require dissection, and are incompatible with behavior. Here, we developed all-optical electrophysiology methods to study GJ coupling in live C. elegans . Different innexin mutants, as well as overexpression of mammalian connexin Cx36, induced different locomotion defects and, compared to wild type, were accompanied by more or less electrical coupling and synchronicity of individual muscle cells. Loss of some innexins caused the ‘insulated’ muscle cells to be more excitable. Thus, GJ coupling is required for fine-tuning locomotion, and can be studied by voltage imaging.