Sympathetic innervation of inguinal white adipose tissue in the mouse

Figures

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  FIGURE 1. Ventral views of sympathetic chain and prevertebral ganglia as seen under the fluorescence dissecting microscope (a–c) and in the light sheet microscope after iDISCO processing (d). (a,a1) TH‐EGFP reporter mouse (male, 8 weeks old, 0.5× objective) with ventral view of the SChG (T4–T8) and abdominal prevertebral sympathetic ganglia (celiac superior mesenteric complex) at low magnification. Note that the lower SChG caudal to T8 are obscured by overlying fat tissue. The celiac superior mesenteric complex depicts the fused celiac and superior mesenteric ganglia as well as the left suprarenal, the left aorticorenal ganglia (a1). (b,b1) TH‐tomato mouse (male, 12 weeks old, 0.5× objective) with ventral views of lower thoracic SChG (T10–T13) and upper lumbar SChG L1–L3. The lumbar SChG can be seen after moving the overlying prevertebral ganglia to the side. In (b1) the celiac superior‐mesenteric complex is seen undisturbed, showing several details, for example, splanchnic nerves and aorticorenal ganglion. Note several non‐specifically labeled structures such as ribs and muscle fibers. (c) Light sheet microscope extended focus view of cleared tissue block (iDISCO, 1.6× zoom factor) with…

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  FIGURE 2. Sympathetic postganglionic innervation of iWAT. (a) Mice were injected with the retrograde, transsynaptic tracer PRVGFP into the right iWAT pad. Perfusion and tissue harvest was performed 3–4 days postviral injection. Whole body iDISCO for TH peptide (b and c) and PRV ((b1) and (c1) imaged under a confocal microscope (10× objective) of a 12‐week‐old female mouse). In the merged image (b2, c2) retrogradely labeled postganglionic sympathetic neurons can be seen in the right chain ganglia, T13 and L1. (d) Summary of ganglionic labeling (positive cases/total number of mice) showing most consistent labeling in T12 to L1. iWAT, inguinal white adipose tissue; PRV, pseudorabies virus; TH, tyrosine hydroxylase

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  FIGURE 3. PRV labeling is occasionally found in dorsal root ganglia. Example of PRV labeling (green) in DRG at level T11 (a), T12 (b), T13 (c) and L1 (d). DRG neurons were easily identified by the existence of few, but consistent TH‐positive neurons (red). PRV‐positive DRG neurons were consistently not co‐localized with TH. Light sheet images, 0.63× zoom factor. DRG, dorsal root ganglia; PRV, pseudorabies virus; TH, tyrosine hydroxylase [Color figure can be viewed at wileyonlinelibrary.com]

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  FIGURE 4. PRV injections into iWAT exclusively labels the sympathetic chain ganglia and IML. (a–c) Negative control “drip‐on” tests were conducted in five mice. PRV administered onto the iWAT via this method provided no evidence of PRV labeling in the sympathetic chain ganglia ((a,a1) light sheet microscope, 0.63× zoom factor), celiac ganglion ((b,b1) dissecting microscope, 0.5× objective), or iWAT ((c,c1) light sheet microscope, 0.63× zoom factor). IML, intermediolateral nucleus of the spinal cord; iWAT, inguinal white adipose tissue; PRV, pseudorabies virus

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  FIGURE 5. Preganglionic sympathetic neurons related to inguinal white adipose tissue (iWAT) in the spinal cord in iDISCO preparation. (a) Most preganglionic neurons‐labeled transsynaptically with pseudorabies virus (PRV) from iWAT are located in the intermediolateral nucleus (IML, white arrows), while a significant number is detected in the intercalated (IC, filled arrow heads) and central autonomic nuclei (CA, open arrowheads); confocal microscope, 10× objective. (b) In a 3D stack with overlaying tyrosine hydroxylase (TH) immunofluorescence, the levels of sympathetic chain ganglia (red) serve as proxies for spinal cord segments. The location of dorsal root ganglia (DRG) and according sympathetic chain ganglia (SCHG) is indicated, but only faintly visible. Light sheet microscopy, 0.63× zoom factor. (b1) In a slice image, the individual dorsal root ganglia and according dorsal roots (white arrows) are visualized as they enter the spinal cord (SC), which defines the middle of the according spinal cord segment. Light sheet microscopy, 0.63× zoom factor. Note that lower sympathetic chain ganglia may serve as a proxy for spinal segments due to the increasing elongation of spinal roots before…

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  FIGURE 6. Preganglionic and postganglionic innervation of iWAT. (a) Diagrammatic representation of spinal cord segments (SCS) with PRV‐positive preganglionic neurons (n positive/n total number of mice) in IML, IC, and CA. Preganglionic iWAT innervation is observed from T5 to T12, but is most consistent across animals from T7 to T10. (b): Diagrammatic synopsis of preganglionic (green) and postganglionic (red) iWAT innervation. Note that the diagram only includes consistent pre‐ and postganglionic labeling sites. Also note the significant segmental caudal shift of postganglionic PRV labeling with respect to preganglionic innervation. CA, central autonomic; IC, intercalated; IML, intermediolateral nucleus of the spinal cord; iWAT, inguinal white adipose tissue; PRV, pseudorabies virus [Color figure can be viewed at wileyonlinelibrary.com]

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  FIGURE 7. Sympathetic chain ganglia T11–T13 provide sympathetic innervation to dorsolumbar WAT via lateral cutaneous nerves. (a) Distribution of lateral cutaneous rami of intercostal nerves T11–T13 to dorsolateral iWAT (dl) as seen under the stereomicroscope (0.5× objective). Yellow dots indicate exit of dorsal cutaneous rami T11–T13 through the external oblique abdominal muscle (exobl) and fascia as landmarks for spinal nerve identification. (b) Scheme of lower thoracic spinal nerve branching emphasizing lateral cutaneous branches to iWAT. iWAT, inguinal white adipose tissue; WAT, white adipose tissue [Color figure can be viewed at wileyonlinelibrary.com]

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  FIGURE 8. Peripheral nerves to right iWAT in a 3 week old female TH:tomato mouse in brightfield (a) and fluorescence (b–d) stereomicroscope, ventral view. (a) Brightfield overview (0.5× objective) of the right dorsolateral (dl) and inguinal (ing) portions of iWAT. The thoracoepigastric vein (thev) is visible at the cranial tip of the dl and the superficial ilium circumflex vein (sicv) is visible at the dl/ing iWAT junction. The femoral nerve (FN) appears upon its passage under the inguinal ligament (dotted) through the muscular lacuna. Medially, the femoral artery and vein (avf) are passing through the vascular lacuna. (b) The upper rectangle in (a) imaged at higher magnification under the fluorescent microscope (0.5× objective). Intercostal (subcostal) T13, iliohypogastric (IHG), the fine ilioinguinal (ILI, open arrowheads) and lateral cutaneous femoral (LCFN) nerves and the iliolumbar artery and vein (avil) travel to the dorsolateral portion. Dashed contour indicates lymph node (ly) at dl/ing junction; exobl denotes external oblique abdominal muscle and fascia. (c): Lower rectangle of (a) imaged at higher magnification under the fluorescent microscope (0.5× objective). The LCFN is seen…

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  FIGURE 9. iDISCO tissue clearing shows main innervation branches to iWAT. (a) Confocal microscope images (5× objective) of an iWAT depot, stained and cleared with iDISCO for TH‐peptide (red = sympathetic innervation) and CD31 (green = vasculature and lymph node (ly)). The dorsolateral innervation shows several incoming branches (asterisk) that cannot be conclusively assigned to individual nerves, but should represent lateral cutaneous rami from T11 to T13. In the inguinal portion one prominent incoming branch can be consistently identified that we conclude is the anterior cutaneous femoral nerve (ACFN), which is also the most prominent nerve visible under the stereomicroscope. (b): Schematic drawing of the lumbar plexus that provides innervation to the thigh, including adipose tissue, vasculature and skin. GFN, genitofemoral nerve; ICN, intercostal nerve; iWAT, inguinal white adipose tissue; other abbreviations see legend to Figure 8

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  FIGURE 10. Varicose innervation density depends on white versus brown adipocyte innervation. (a) Hematoxilin and eosin staining (stains cytoplasmatic content purple) of a paraffin, longitudinal section (5 μm) of iWAT shows the prominent lymph node (ly) at the junction of dorsolumbar (dl) and inguinal (ing) portions of iWAT (confocal microscopy, 5× objective). Areas with mainly white adipocytes appear light purple (rectangle to (b)), due to the reduced cytoplasmatic content of white adipocytes. Areas with darker purple stain depict areas with increased number of brown adipocytes (beige islands, purple arrows and rectangle to (c)), which contain more coplasm than white adipocytes. The upper square depicts an area with mostly white adipocytes. Staining of an adjacent section with TH‐peptide (b) and the brown adipocyte marker Uncoupling protein 1 (UCP1) (b1) shows that low TH‐positive varicosities are found among UCP1‐negative white adipocytes. The lower square depicts an area with mostly brown adipocytes. Staining of an adjacent section with TH‐peptide (c) and UCP1 (c1) shows that high TH‐positive varicosities are found among UCP1‐positive brown adipocytes. (d) Confocal image (5× objective) of…