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

Reviewer #1 (Public review):

Summary:

This study aims to reveal the contribution of individual gap junction proteins to the signal transmission and connectivity of living C. elegans animals in a completely non-invasive way through all-optical electrophysiology. The authors achieve this by simultaneous expression of bipoles, an excitatory/inhibitory light-activated actuator and Quasar2, a genetically encoded voltage dye. With this study, the authors extend their previous efforts to leverage the strength of optogenetic neurophysiology and set a new standard in this domain. In addition, they adapted their established methods to perform cell-specific optogenetic voltage clamp and revealed changes in gap junction connectivity. They also find that increasing excitability in innexin mutants is indicative of a reduction in gap-junction connectivity and current leaks.

Strengths:

This is an extremely strong manuscript, a technical feat and tour de force to infer junctional coupling through all-optical electrophysiology. The establishment of the voltage clamp method is powerful and allows researchers to obtain not only tight control over voltage signals but also permits the investigation of gap junction function in response to positive and negative voltage steps in a completely non-invasive fashion. This will be a new paradigm for investigating muscle electrophysiology in future.

Weaknesses:

This is a strong pioneering study, and I found very few technical weaknesses. The correlation quantification is relatively weak to establish connective causality, as a shared upstream input may lead to a similar perceived correlation. This is especially concerning for an average lag time of ~0, and the authors may want to investigate if there is unchanged connectivity in an unc-31 or unc-13 mutant. Conceptually, the local connectivity is scaled to account for behaviour: future studies may wish to perform this method on moving animals, and in specific neuronal populations, where a non-invasive optogenetic voltage clamp method will truly shine.

Reviewer #2 (Public review):

Summary:

This technically sophisticated study combines behavioral analysis, voltage imaging, electrophysiology, and a newly developed cell-specific optogenetic voltage clamp (cOVC) approach to investigate gap-junction (GJ)-mediated coupling in C. elegans body-wall muscle cells. The work explores the coordination of muscle cells and systems physiology and introduces a method with potential utility beyond the nematode system studied.

Strengths:

The main strength of the work is the development and application of the cOVC method. This approach enables minimally invasive in vivo assessment of cell-to-cell electrical coupling in intact animals. This technique represents a meaningful advance over traditional electrophysiological techniques that require dissection or cell isolation.

With respect to the GJ biology and function, the authors support their conclusions by integrating additional independent experimental approaches. Findings from behavioural/locomotion assays, voltage imaging, patch-clamp recordings, and cOVC measurements are generally consistent, particularly for unc-9 mutants, which show reduced synchronization of muscle cells, impaired electrical coupling, and severe locomotor defects.

The gain-of-function experiment using murine Cx36 suggests that more or less electrical coupling can disrupt (normal) locomotion.

Weaknesses:

The main issue of this otherwise excellent manuscript relates to interpretation rather than experimental quality. Throughout the manuscript, increased correlation is often interpreted as evidence of increased electrical coupling. Are correlation, synchrony, and conductance equivalent measures? If not, how would this affect these correlations? Furthermore, could broader action potentials and altered excitability also increase correlation values? This concern could be addressed through a discussion of this limitation.

Similarly, the proposed mechanism that reduced GJ coupling increases excitability through reduced leak currents is plausible but not directly demonstrated. Are alternative explanations, e.g., compensatory changes in ion-channel expression or gap-junction composition, possible? These could also be considered to improve the balance of this work.

The conclusions regarding Cx36 overexpression would also benefit from more cautious wording, as developmental or localization effects have not been excluded.

However, the experimental dataset is very strong. In my opinion, no major additional studies are needed. Direct analysis of compensatory changes in innexin expression or localization could strengthen the interpretation of the proposed mechanism. Overall, the study is of high technical quality, contains a notable methodological advance, and provides important insights into muscle synchronization and GJ biology.