Article Figures & Data
Figures
- Figure 1.
Timeline of methods and experimental groups. Three groups of mice were used in three different experimental paradigms. A, C57BL/6J mice received retrobead injections into the OB and were perfused at 2 d. B, C57BL/6J mice received AAVretro-GFP injections into the OB and were perfused at two weeks. C, Transgenic Ai9 reporter mice received AAVretro-Cre injections and were perfused at two weeks; *three of nine Ai9 mice received multiple injections of AAVretro-Cre throughout one OB (see Materials and Methods; Table 1).
- Figure 2.
Representative results from semi-automated cell-counting procedures. A, Representative results from semi-automated quantification of retrobead-labeled cells in aPCX. RB, retrobead. Bi, Representative results from semi-automated quantification of retrobead-labeled cells in the OT. Bii, Same section as shown in Bi, as a focused z-stack, indicating that many cells counted were likely non-neuronal fluorescent puncta. Z-stack included six steps, with 4 μm between each step. C, Representative results from semi-automated quantification of GFP-labeled cells in the aPCX following injection of AAVretro-GFP in the OB. D, Representative results from semi-automated quantification of tdTomato-labeled cells in the aPCX following injection of AAVretro-Cre in the OB. Arrows indicate counted cells. Boxed region indicates ROI used for quantification. All scale bars = 100 μm.
- Figure 3.
Retrobead injections to the OB indicate lack of OT to OB innervation. A, OB injection site. Dotted line indicates the glomerular layer. Scale bar = 500 μm. B, Retrobead labeling in the AON following OB injection. Box indicates region in D. Scale bar = 200 μm. C, aPCX and OT retrobead labeling. Boxes indicate regions in E, F. Scale bar = 500 μm. D, Enhanced view of boxed AON region in B. Scale bar = 100 μm. E, Enhanced view of boxed aPCX region in C. Scale bar = 100 μm. F, Enhanced view of boxed OT region in C. Scale bar = 100 μm. G, Estimated number of retrobead-labeled cells across all three brain regions. Each point represents one animal’s mean; n = 6 mice, four to six sections (4.78 ± 0.19; mean ± SEM) each. i.–iii., layers 1–3; D, dorsal; M, medial; L, lateral.
- Figure 4.
AAVretro-GFP tracing further supports lack of OT to OB innervation. A, OB injection site. Dotted line indicates the glomerular layer. Scale bar = 500 μm. B, AON GFP labeling following injection of AAVretro-GFP in the OB. Box indicates region in Figure 3D. Scale bar = 500 μm. C, aPCX and OT GFP labeling following injection of AAVretro-GFP in the OB. Boxes indicate regions in Figure 3E,F. Scale bar = 500 μm. D, Enhanced view of boxed AON region in B. Scale bar = 100 μm. E, Enhanced view of boxed aPCX region in C. Scale bar = 100 μm. F, Enhanced view of boxed OT region in C. Scale bar = 100 μm. G, Quantification of GFP-labeled cells across all three brain regions. Each point represents one animal’s mean; n = 6 mice, 4–12 sections (6.89 ± 0.54; mean ± SEM) each. H, GFP labeling in the OT following injection of AAVretro-GFP in the OT; n = 2 mice. VP, ventral pallidum; ICj, islands of Calleja; ICj, borders were approximated based on DAPI staining. Scale bar = 100 μm. i.–iii., layers 1–3; D, dorsal; M, medial; L, lateral.
- Figure 5.
AAVretro-Cre tracing in Ai9 reporter mouse verifies lack of OT to OB innervation. A, OB injection site. Dotted line indicates the glomerular layer. Scale bar = 500 μm. B, AON tdTomato labeling following injection of AAVretro-Cre in the OB of Ai9 mouse. Box indicates region in Figure 4D. Scale bar = 500 μm. C, aPCX and OT tdTomato labeling following injection of AAVretro-Cre in the OB of Ai9 mouse. Boxes indicate regions in Figure 4E,F. Scale bar = 500 μm. D, Enhanced view of boxed AON region in B. Scale bar = 100 μm. E, Enhanced view of boxed aPCX region in B. Scale bar = 100 μm. F, Enhanced view of boxed OT region in C. Scale bar = 100 μm. G, Quantification of tdTomato-labeled cells across all the three brain regions. Each point represents one animal’s mean; n = 4 mice, 5–13 sections/mouse (9.5 ± 0.68; mean ± SEM). H, OT neurons labeled following injection of AAVretro-Cre in the OT of Ai9 mouse; n = 2 mice. VP, ventral pallidum; ICj, islands of Calleja. ICj borders were approximated based on DAPI staining. Scale bar = 100 μm. i.–iii., layers 1–3; D, dorsal; M, medial; L, lateral.
- Figure 6.
Multiple injections of AAVretro-Cre in the OB of Ai9 reporter mouse reveals labeling in numerous OB-projecting structures, but not the OT. A, OB injection site. Dotted line indicates the glomerular layer. Scale bar = 500 μm. B, Intact brain following multiple injections of AAVretro-Cre in the OB of Ai9 reporter mouse shows strong tdTomato labeling in one OB and the PCX, but not the OT. Dotted line indicates the OT. Scale bar = 1 mm. C, aPCX and OT tdTomato labeling following injection of AAVretro-Cre in the OB of Ai9 mouse. Scale bar = 500 μm. D–K, tdTomato labeling in many regions following injection of AAVretro-Cre in the OB of Ai9 mouse. All scale bars = 100 μm. D, AON. E, Horizontal diagonal band of Broca (HDB) and magnocellular preoptic nucleus (MCPO). F, Posterior PCX (pPCX). G, Lateral hypothalamus (LH). H, Basolateral amygdala (BLA). I, Median raphe nucleus (MnR) and paramedian raphe nucleus (PMnR). J, Dorsal raphe nucleus (DR). K, Locus coeruleus (LC). f, fornix; ns, nigrostriatal bundle; IP, interpeduncular nucleus; VLPAG, ventrolateral periaqueductal gray; Su3, supraoculomotor cap; MPB, medial parabrachial nucleus; Bar, Barrington’s nucleus. i.–iii., layers 1–3; D, dorsal; M, medial; L, lateral; n = 3 mice.
- Figure 7.
Revised model for centrifugal inputs to the OB. The AON, aPCX, and OT receive input from the OB. The AON and aPCX send centrifugal input back to the OB, but based on our data, the OT does not.
Tables
Strain n Injected with Injection site Injection amount Figure C57BL/6J 4 (3 M, 1 F) Retrobeads OB (dispersed between 2400 and 800 μm ventral) 900 nl 2A–C, 3 C57BL/6J 2 M Retrobeads OB 200 nl 2A–C, 3 C57BL/6J 7 M AAVretro-GFP OB 200 nl 2D, 4A–G C57BL/6J 2 M AAVretro-GFP OT 500 nl 4H Ai9 6 M AAVretro-Cre OB 200 nl 2E, 5A–G Ai9 1 M, 1 F AAVretro-Cre OT 500 nl 5H Ai9 1 M, 2 sF AAVretro-Cre OB (1, 1.5, and 2 mm anterior the rhinal sinus, 1 mm lateral, 1.5 mm ventral) 200 nl × 3 sites = 600 nl total 6 Unless otherwise noted, OB injections were given at 1.5 mm anterior the rhinal sinus, 1 mm lateral, and 1.5 mm ventral, and OT injections were given at 1.5 mm anterior bregma, 1.2 mm lateral, and 4.8 mm ventral.
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