Multicolor sparse viral labeling and 3D digital tracing of enteric plexus in mouse proximal colon using a novel adeno‐associated virus capsid

• Published in: Neurogastroenterology and motility Vol. 33; no. 8; pp. e14014 - n/a
• Main Authors: Wang, Lixin, Challis, Collin, Li, Songlin, Fowlkes, Charless C., Ravindra Kumar, Sripriya, Yuan, Pu‐Qing, Taché, Yvette F.
• Format: Journal Article
• Language: English
• Published: England Wiley Subscription Services, Inc 01.08.2021
• Subjects: 3D imaging; adeno‐associated virus; Animals; Apposition; Axons; Colon; Colon - innervation; Colon - metabolism; colonic enteric neuron; Dependovirus; digital tracing; Enteric nervous system; Enteric Nervous System - metabolism; Enteric Nervous System - virology; Female; Fibers; Fluorophores; Gene Transfer Techniques; Green Fluorescent Proteins; Intravenous administration; Labeling; Male; Mice; mouse; Myenteric plexus; Nerve endings; Nervous system; Neurons; Regulatory sequences; Submucosal plexus; Submucous Plexus - metabolism; Submucous Plexus - virology; digital tracing; mouse; adeno-associated virus; 3D imaging; colonic enteric neuron
• ISSN: 1350-1925; 1365-2982
• DOI: 10.1111/nmo.14014

Background Intravenous administration of adeno‐associated virus (AAV) can be used as a noninvasive approach to trace neuronal morphology and links. AAV‐PHP.S is a variant of AAV9 that effectively transduces the peripheral nervous system. The objective was to label randomly and sparsely enteric plexus in the mouse colon using AAV‐PHP.S with a tunable two‐component multicolor vector system and digitally trace individual neurons and nerve fibers within microcircuits in three dimensions (3D). Methods A vector system including a tetracycline inducer with a tet‐responsive element driving three separate fluorophores was packaged in the AAV‐PHP.S capsid. The vectors were injected retro‐orbitally in mice, and the colon was harvested 3 weeks after. Confocal microscopic images of enteric plexus were digitally segmented and traced in 3D using Neurolucida 360, neuTube, or Imaris software. Key Results The transduction of multicolor AAV vectors induced random sparse spectral labeling of soma and neurites primarily in the myenteric plexus of the proximal colon, while neurons in the submucosal plexus were occasionally transduced. Digital tracing in 3D showed various types of wiring, including multiple conjunctions of one neuron with other neurons, neurites en route, and endings; clusters of neurons in close apposition between each other; axon–axon parallel conjunctions; and intraganglionic nerve endings consisting of multiple nerve endings and passing fibers. Most of digitally traced neuronal somas were of small or medium in size. Conclusions & Inferences The multicolor AAV‐PHP.S‐packaged vectors enabled random sparse spectral labeling and revealed complexities of enteric microcircuit in the mouse proximal colon. The techniques can facilitate digital modeling of enteric micro‐circuitry. Nerve cells and and fibers in the colon are labeled by spectral fluorescent molecules delivered by intravenous injection of multicolor viral vectors in mice. Individual nerve cells and fibers are digitally traced three dimensionally in microscopic images.