The enteric nervous system (ENS) supports diverse gut functions through circuits formed by molecularly distinct neuronal populations. While single-cell transcriptomic studies have clarified neuronal cell classes in the ENS, methods to define synaptic connectivity among those classes are limited. This study reports the development and implementation of mWmC, a fusion construct of wheat germ agglutinin (WGA) and mCherry, as a non-toxic, single-component viral tool for anterograde monosynaptic tracing of enteric circuits.
The authors aimed to validate mWmC for selective expression in enteric neurons, demonstrate transsynaptic transfer to postsynaptic neurons without non-neuronal labeling, map inter-organ connectivity from intestine to peripheral ganglia, and apply the tool to genetically defined interneuron populations. They also evaluated the temporal profile of labeling and produced a dual-reporter version to discriminate inputs and targets within the same tissue.
mWmC is a fusion of WGA, a known transsynaptic marker, and the fluorescent protein mCherry. The tracer was delivered to enteric neurons using adeno-associated virus (AAV)-mediated expression. Following AAV transduction, mWmC was reported to be expressed efficiently in targeted enteric neurons and transmitted selectively to postsynaptic neurons.
The study frames mWmC as a single-component, non-toxic tracer suitable for anterograde monosynaptic mapping within and beyond the gut. The authors highlight that the system links molecular classification from cell atlases with actual neuronal connectivity, enabling more mechanistic studies of ENS circuit function.
A critical validation step was assessment of cellular specificity. The authors report that mWmC transfer was observed selectively to postsynaptic neurons and that there was no detectable transfer to several non-neuronal cell types in the gut microenvironment. Specifically, mWmC labeling did not appear in enteric glia, interstitial cells of Cajal, blood vessels, or other mesenchymal cell types according to the observations reported in the preprint. This selective neuronal transfer supports use of the tracer for neuron-to-neuron circuit mapping in complex tissues.
To demonstrate utility beyond local ENS circuits, the authors traced intestinofugal enteric neurons and identified postsynaptic neurons in the celiac-superior mesenteric ganglia. This result shows that mWmC can reveal inter-organ circuits, mapping connections from the gut to peripheral autonomic ganglia. The report highlights this capacity as an important feature for defining how enteric circuits interface with systemic neural networks.
As a proof of principle for class-specific circuit mapping, the authors applied mWmC to two genetically defined myenteric interneuron populations. Using the tracer, they identified preferential postsynaptic targets for these interneuron populations and documented selective connectivity with distinct enteric neuron classes. These findings illustrate how mWmC can be combined with genetic targeting to resolve connectivity patterns of molecularly defined neuron types.
Time-course experiments reported in the preprint showed that transsynaptic labeling with mWmC became apparent between 4 and 10 days after expression and then reached a plateau. The authors interpret this temporal profile as consistent with monosynaptic transfer, supporting the tracer's mode of action. The preprint presents these kinetics as evidence that mWmC functions as a monosynaptic anterograde tracer in the ENS context.
To enhance interpretability, the group developed a dual-reporter version of the system. This variant allows simultaneous discrimination of input and target neurons within the same tissue preparation. The preprint reports that the dual-reporter design facilitates more detailed mapping of circuit architecture by enabling concurrent visualization of both presynaptic and postsynaptic populations.
The authors propose that mWmC provides a robust method to define circuit architecture in the ENS and to connect molecular cell atlases with functional wiring diagrams. By resolving selective connectivity patterns and inter-organ links, the tracer could help uncover circuit mechanisms underlying gut physiology.
The preprint does not report exhaustive quantitative metrics, behavioral or physiological consequences of mapped circuits, or extensive methodological parameters in this summary. Where such details are required for experimental implementation or interpretation, readers should consult the full preprint and supplementary materials referenced in the source.
The work reported funding from Hjärnfonden, the Swedish Research Council, the Knut and Alice Wallenberg Foundation, and the National Institute of Diabetes and Digestive and Kidney Diseases. The Competing Interest Statement notes that P.A. Muller and C.J. Millett have equity in Kallyope Inc.; the authors state that their employments did not influence study design, conduct, or interpretation.
Posted July 31, 2026. For full experimental details, datasets, and supplementary figures, refer to the original bioRxiv preprint (doi: https://doi.org/10.64898/2026.07.28.741165).