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

Kim Freeman, Wen Tao, Hongli Sun, Mark Soonpaa, Michael Rubart-von der Lohe

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16 Citations (Scopus)

Abstract

Background: Sympathetic 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 method: We 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. Results: Quantitative 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 methods: In 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. Conclusions: Our assay should be of general utility to unravel the mechanisms governing sympathetic axon spacing during development and disease.

Original languageEnglish (US)
Pages (from-to)48-61
Number of pages14
JournalJournal of Neuroscience Methods
Volume221
DOIs
StatePublished - Jan 15 2014

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Optical Imaging
Photons
Neurites
Axons
Adrenergic Fibers
Adrenergic Neurons
Fluorescence Microscopy
Transgenic Mice
Uncertainty
Anatomy
Myocardial Infarction
enhanced green fluorescent protein

Keywords

  • 3-D reconstruction
  • Enhanced green fluorescent protein
  • Heart
  • Sympathetic nervous system
  • Transgenic mice
  • Two-photon fluorescence microscopy

ASJC Scopus subject areas

  • Neuroscience(all)

Cite this

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title = "In situ three-dimensional reconstruction of mouse heart sympathetic innervation by two-photon excitation fluorescence imaging",
abstract = "Background: Sympathetic 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 method: We 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. Results: Quantitative 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 methods: In 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. Conclusions: Our assay should be of general utility to unravel the mechanisms governing sympathetic axon spacing during development and disease.",
keywords = "3-D reconstruction, Enhanced green fluorescent protein, Heart, Sympathetic nervous system, Transgenic mice, Two-photon fluorescence microscopy",
author = "Kim Freeman and Wen Tao and Hongli Sun and Mark Soonpaa and {Rubart-von der Lohe}, Michael",
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T1 - In situ three-dimensional reconstruction of mouse heart sympathetic innervation by two-photon excitation fluorescence imaging

AU - Freeman, Kim

AU - Tao, Wen

AU - Sun, Hongli

AU - Soonpaa, Mark

AU - Rubart-von der Lohe, Michael

PY - 2014/1/15

Y1 - 2014/1/15

N2 - Background: Sympathetic 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 method: We 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. Results: Quantitative 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 methods: In 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. Conclusions: Our assay should be of general utility to unravel the mechanisms governing sympathetic axon spacing during development and disease.

AB - Background: Sympathetic 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 method: We 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. Results: Quantitative 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 methods: In 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. Conclusions: Our assay should be of general utility to unravel the mechanisms governing sympathetic axon spacing during development and disease.

KW - 3-D reconstruction

KW - Enhanced green fluorescent protein

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KW - Sympathetic nervous system

KW - Transgenic mice

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