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. 2023 Jan 26;17:974197. doi: 10.3389/fnins.2023.974197

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Copyright © 2023 Tyshynsky, Sensarma, Riedl, Bukowy, Schramm, Vulchanova and Osborn.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Illustration of glomerular scoring. (A) An image of native tdTomato fluorescence (magenta, tdTomato+ axons) and immunolabeling for nephrin (green, glomeruli) and calcitonin gene related peptide (CGRP) (cyan, peptidergic axons) in a renal slice from a TRPV1:Ai14 mouse. The image represents a maximum intensity projection of 6 optical sections collected 13.7 microns apart. While this image includes CGRP+ and tdTomato+ labeling for illustrative purposes, only images immunolabeled for one neuronal marker at a time were used during glomerular scoring. (B–E) A magnified inset from panel (A) illustrating the labeling of glomeruli and axons in the renal cortex (scale bars: 250 μm). (B) Nephrin+ labeling of glomerular podocytes. (C) Native tdTomato fluorescence. Examples of labeled axons are highlighted with arrowheads. The identity of globular cell-like profiles is unknown. They appeared to be associated with tubules and were not co-labeled with a pan-immune cell marker (anti-CD45, data not shown). They were clearly distinguished from axonal profiles and, therefore, did not affect the glomerular scoring. (D) CGRP+ labeling of afferent axons (some highlighted with arrowheads). (E) Composite image illustrating CGRP labeling and tdTomato fluorescence in afferent axons (some highlighted with arrowheads) in the renal cortex. (F–I) Examples of anatomical relationships between axons and glomeruli as observed while scoring individual glomeruli (scale bars: 50 μm). (F) Both CGRP+ and tdTomato+ axons (arrowheads) approach the glomerulus. (G) A tdTomato+ axon bundle (arrowhead) approaches the glomerulus, and although the cyan channel exhibits some labeling, its morphology is not consistent with the appearance of axons and is therefore not scored as an anatomical interaction between CGRP+ axons and the glomerulus. (H) A clear anatomical relationship between a CGRP+ axon bundle (arrowhead) and the glomerulus. Although there is faint labeling in the magenta channel, the signal to noise ratio is not high enough to count as a close apposition. (I) CGRP+ axon (arrowhead) surrounds the glomerulus extend beyond it. Scale bars: (A) 1000 μm, (B–E) 250 μm, (F–I) 50 μm.