In situ three-dimensional reconstruction of mouse heart sympathetic innervation by two-photon excitation fluorescence imaging.

backgroundSympathetic nerve wiring in the mammalian heart has remained largely unexplored. Resolving the wiring diagram of the cardiac sympathetic network would help establish the structural underpinnings of neurocardiac coupling. NEW

methodWe used two-photon excitation fluorescence microscopy, combined with a computer-assisted 3-D tracking algorithm, to map the local sympathetic circuits in living hearts from adult transgenic mice expressing enhanced green fluorescent protein (EGFP) in peripheral adrenergic neurons.

resultsQuantitative co-localization analyses confirmed that the intramyocardial EGFP distribution recapitulated the anatomy of the sympathetic arbor. In the left ventricular subepicardium of the uninjured heart, the sympathetic network was composed of multiple subarbors, exhibiting variable branching and looping topology. Axonal branches did not overlap with each other within their respective parental subarbor nor with neurites of annexed subarbors. The sympathetic network in the border zone of a 2-week-old myocardial infarction was characterized by substantive rewiring, which included spatially heterogeneous loss and gain of sympathetic fibers and formation of multiple, predominately nested, axon loops of widely variable circumference and geometry. COMPARISON WITH EXISTING

methodsIn contrast to mechanical tissue sectioning methods that may involve deformation of tissue and uncertainty in registration across sections, our approach preserves continuity of structure, which allows tracing of neurites over distances, and thus enables derivation of the three-dimensional and topological morphology of cardiac sympathetic nerves.

conclusionsOur assay should be of general utility to unravel the mechanisms governing sympathetic axon spacing during development and disease.