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. 2021 Dec 9;14:790466. doi: 10.3389/fnmol.2021.790466

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Copyright © 2021 Ruff, Peters, Matsumoto, Ihle, Morales, Gaitanos, Yonehara, del Toro and Klein.

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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MTN-projecting Flrt3-RGCs are a homogeneous downward ON-DSGC subpopulation. (A) Stereotaxic injection of AAV2^{retro–cre–Tomato} in the MTN of Flrt3-CreERT2 mice to identify Flrt3-RGCs projecting to the MTN. The image on the right shows a representative MTN-injection site after 3 weeks of viral expression. N = 4 mice. (B) Whole mount retina showing the distribution of MTN-projecting Flrt3-RGCs. (C) Normalized density profile of MTN-projecting Flrt3-RGCs. N = 50 ROIs from 11 retinas and 8 animals. (D) Regularity index plot for Flrt3-RGC, Flrt3-MTN-projecting-RGC and Flrt3-amacrine cells. N = 4–11 ROI from different retinas of 3–4 mice (as shown in (B). One-way ANOVA Tukey’s Multiple Comparison Test, *P < 0.05. (E) Average nearest neighbor distance (μm), calculated from same ROIs as in (D). One-way ANOVA Tukey’s Multiple Comparison Test, ***P < 0.001. (F) Orthogonal section through the IPL to visualize the stratification pattern of MTN-projecting Flrt3-RGCs. The right panel shows the average normalized stratification pattern of Calbindin (orange), mono- (green, n = 16 cells from 3 animals, 26%) and bistratified MTN-projecting Flrt3-RGCs (magenta, n = 46 cells from 3 animals, 74%). (G) Immunostaining for CART, Calbindin and Parvalbumin in a retinal sections of MTN injected mice from (A). Tomato signal (magenta) shows MTN-projecting Flrt3-RGCs. (H) Fraction (%) of MTN-projecting Flrt3-RGCs positive for CART, Calbindin and Parvalbumin. N = 60–66 ROIs for each marker from 3 retinas. (I) Normalized stratification patterns of mono- (green) and bistratified (red) MTN-Flrt3-RGCs in the inner plexiform layer, from (F). N = 46 cells from 3 animals.