iWAT (inguinal white adipose tissue) sympathetic innervation circuit pseudorabies viral tracing in reporter mice

Study purpose: We utilize pseudorabies virus (PRV) retrograde tracing in combination with reporter mice, iDISCO, and confocal/light-sheet microscopy to identify the location of pre- and post-ganglionic neurons as well as nerves that selectively innervate the iWAT in the mouse.

Data collection: High-resolution confocal/light-sheet microscopy images were collected.

Primary conclusion: None stated.

Curator's Notes

Experimental Design: Mice (ranging from 6 to 15 weeks old) were anesthetized with isoflurane/oxygen. A lower back incision was extended laterally on the right hind leg to reveal an adequate amount of iWAT (inguinal white adipose tissue) sufficient for injection. Ten individual injections were distributed across the right iWAT depot with green fluorescent protein-expressing PRV. 96 hours of viral incubation was allowed. After euthanasia, the peritoneal cavity was eviscerated to reveal the sympathetic chain ganglia.

A stereomicroscope was used to visually confirm the extent of viral labeling within the chain ganglia. Only mice with visible SChG (sympathetic chain ganglia) infection were included in further tissue dissection. The iWAT was removed for individual processing and excessive muscle mass was dissected from the spinal cord (SC). A laminectomy was performed in order to image sympathetic preganglionic neurons. The spine and SC were cut in half (roughly at the level of ribs 7–9) to accommodate the imaging capacity of both the light sheet and confocal microscope. Then, the tissue was postfixed overnight in formalin, and stored in phosphate-buffered saline (PBS)–azide (0.02% Na–azide in PBS) 4°C until iDISCO processing.

Immunohistochemical staining was performed following the iDISCO method against GFP, Tyrosine Hydroxylase, Calcitonin gene-related peptide, or CD31 where indicated. High-resolution images of relevant structures were captured via light-sheet microscopy.

• Purple color = Tyrosine hydroxylase
• Green color = PRV-GFP
• Yellow color = Co-localization
• Blue color = Calcitonin gene-related peptide or CD31 where indicated

Completeness: This dataset is a part of a larger study: Genetically-based neuro-modulation of adipose tissue functions.

Subjects & Samples: Female (n=8) and male (n=8) adult mice of mixed genetic background were used in this study.

Primary vs derivative data: Primary and derivative folders are organized by subject identification, then by the sample stained. The primary folder contains raw (.ims) microscopic images and AVI video files with 3D visualization of the imaged section. The derivative folder contains image data (JPEG2000 and OME-TIFF) derived from primary images (.ims).

Important notes: This dataset is currently undergoing image registration and will be updated once this process is complete.