Figure 1.
Nonoverlapping expression of PDE2 and OMP in necklace glomeruli and MOE.
(A) The mouse main and accessory olfactory systems. The main system (bright green) includes the main olfactory epithelium (MOE) and main olfactory bulb (MOB). GC-D+ neurons (red stippling) are dispersed amongst canonical OSNs in restricted regions of the MOE. Necklace glomeruli (NGs; red circles) are in the caudal MOB. The accessory system (dark green) is comprised of the vomeronasal organ (VNO) and accessory olfactory bulb (AOB). Adapted from [3], with permission from Elsevier. (B) NGs in paired MOBs of an OMP-tauEGFP+/−::Gucy2d-Mapt-lacZ+/− mouse. EGFP, green; PDE2, red. Arrowhead: canonical MOB glomerulus; arrows: necklace glomeruli; box, shown in (C). (C) Orthogonal views (right, bottom; vertical, horizontal lines are planes of optical section) of a single NG confocal z-stack showing little overlap of EGFP-positive (green) and PDE2-positive (red) axons. (D) MOB section from Gucy2d-Mapt-lacZ+/− mouse, immunostained for OMP (green), β-gal (red). Arrows: OMP-positive axons; arrowheads: β-gal-positive axons. (E) MOE dorsal recess of OMP-tau-EGFP+/− mouse shows clusters of PDE2-positive/EGFP-negative (red) and PDE2-negative/GFP-positive (green) neurons. Yellow box: area magnified 2-fold, inset (F) Single PDE2-positive OSN (red) in the MOE of an OMP-tau-EGFP+/− mouse. EGFP, green; box, shown in (G). (G) Orthogonal views in MOE of single PDE-positive/EGFP-negative (red) neuron and multiple PDE2-negative/EGFP-positive (green) neurons. Confocal z-stack (B,C,E–G); single optical section (D). Scale bars: 50 µm (B–D); 10 µm (E–G). Blue, DAPI.
Figure 2.
Intrabulbar connections with necklace glomeruli.
(A) Brightfield (left) and fluorescent (right) whole mount images after X-gal histochemistry of a necklace glomerulus (NG; arrowhead) injected with DiI (arrow). Scale bar, 25 µm. (B) DiI labeled cells associated with a DiI-injected NG (asterisk). Arrow, periglomerular cell; arrowhead, mitral cell. Scale bar, 25 µm. (C) Distribution of labeled cell bodies (green) from a representative NG injection. Red lines indicate position of the injection point within the cell cluster and on the shadows (gray) of the generic MOB schematic (yellow). Each grid square = 500 µm2. (D) Labeled cell processes innervating glomeruli (NGs, blue; non-NGs, black) after DiI injections of single NGs (n = 7). (E) Mean percentage of labeled processes innervating NGs after DiI injection of NGs (blue) or non-NGs (gray). No significant differences between groups by Student's t test. (F) NGs and non-NGs innervated by labeled processes after DiI injections of single NGs (n = 7). (G) Necklace glomeruli as a percentage (mean) of total glomeruli innervated by labeled processes after DiI injection of NGs (blue) or non-NGs (gray). No significant differences between groups by Student's t test. (H) Distribution of innervated glomeruli (NGs, blue; non-NGs, black) from same NG injection as in (C). All error bars, s.e.m.
Figure 3.
Schematic of sensory neuron and interneuron innervation of necklace glomeruli.
The MOE contains numerous sensory neurons (top), including: canonical OSNs that all express OMP but which express different ORs (orange, brown); GC-D/PDE2-expressing OSNs (red); and OMP-positive/PDE4A-negative neurons (green). Canonical OSNs expressing the same OR converge on the individual canonical glomeruli (orange, brown circles) in the MOB, providing homogeneous sensory input to those structures. GC-D/PDE2-positive neurons innervate necklace glomeruli (blue circles), as do OMP-positive/PDE4A-negative neurons. Juxtaglomerular interneurons in the glomerular layer (yellow ovals) connect individual necklace glomeruli with other necklace glomeruli and with near and distant canonical glomeruli. Mitral cells (gray, bottom) innervating necklace glomeruli display a typical MOB mitral cell morphology.