Multicolor adeno-associate virus labeling and 3D digital tracing of enteric plexus in mouse proximal colon v1

Abstract

Using a multicolor adeno-associate virus system to label the colonic enteric nervous system for digital tracing of individual neurons and nerve fibers in microcircuits in three-dimensions (3D). The methods include viral vectors retro-orbital injection in mice, preparation of colon tissues, microscopy and 3D digital tracing.

Transduction of Systemically Administered Adeno-Associated Virus in the Colonic Enteric Nervous System and c-Kit Cells of Adult Mice

“…The AAV types, suppliers, and doses used are detailed in Table 1 . Mice were anesthetized with 2.5% isoflurane in oxygen and injected unilaterally into the retro-orbital venous sinus with the vectors diluted in phosphate-buffer saline (PBS) (60 μl/mouse) as in our previous study (Wang et al, 2020 ). The injection was performed using a BD Veo Insulin Syringe with BD Ultra-Fine 6 mm × 31 G needle (324911, Becton, Dickinson and Company, Franklin Lakes, NJ).…”

“…The colon was flat-pinned on a Sylgard™ 184 silicone elastomer (Electron Microscopy Science, Hatfield, PA), and fixed in 4% paraformaldehyde in 0.1M phosphate buffer. To compare the labeling of the proximal colon with other GI segments, we also collected the esophagus, gastric corpus, gastric antrum, duodenum, jejunum, and ileum; as well as the nodose ganglia of the vagus, dorsal root ganglia at L1 and L6, celiac-superior mesenteric ganglion, and pelvic ganglia from some mice as previously (Wang et al, 2020 ). For 3D imaging, the colon with the whole thickness was cleared using passive CLARITY with the hydrogel containing 4% acrylamide without paraformaldehyde (A4F0) (Yang et al, 2014 ), or pretreated with 2% Triton X 100–0.01M phosphate buffered saline (TPBS) to enhance penetration of the primary antibodies.…”

“…AAV8 and 9 showed higher transduction efficiency than AAV1, 5, and 6 in the mouse enteric nervous system (Gombash et al, 2014 ). In adult mice, we performed iv injection of a multicolor AAV vector system for sparse neural tracing, and found that the transduction was prominent in enteric neurons of the proximal colon, while few in the distal colon (Wang et al, 2020 ). Another study using tail vein injection of AAV2/9 at low doses to label sparsely the colonic enteric neurons in adult mice addressed the projection orientation and length of individual neurons and did not present evidence of different segmental distributions of AAV2/9-transduced neurons (Li et al, 2019 ).…”