Grueneberg ganglion olfactory subsystem employs a cGMP signaling pathway

Abstract

The mammalian olfactory sense employs several olfactory subsystems situated at characteristic locations in the nasal cavity to detect and report on different classes of odors. These olfactory subsystems use different neuronal signal transduction pathways, receptor expression repertoires, and axonal projection targets. The Grueneberg ganglion (GG) is a newly appreciated olfactory subsystem with receptor neurons located just inside of the nostrils that project axons to a unique domain of interconnected glomeruli in the caudal olfactory bulb. It is not well understood how the GG relates to other olfactory subsystems in contributing to the olfactory sense. Furthermore, the range of chemoreceptors and the signal transduction cascade utilized by the GG have remained mysterious. To resolve these unknowns, we explored the molecular relationship between the GG and the GC-D neurons, another olfactory subsystem that innervates similarly interconnected glomeruli in the same bulbar region. We found that mouse GG neurons express the cGMP-associated signaling proteins phosphodiesterase 2a, cGMP-dependent kinase II, and cyclic nucleotide gated channel subunit A3 coupled to a chemoreceptor repertoire of cilia-localized particulate guanylyl cyclases (pGC-G and pGC-A). The primary cGMP signaling pathway of the GG is shared with the GC-D neurons, unifying their target glomeruli as a unique center of olfactory cGMP signal transduction. However, the distinct chemoreceptor repertoire in the GG suggests that the GG is an independent olfactory subsystem. This subsystem is well suited to detect a unique set of odors and to mediate behaviors that remained intact in previous olfactory perturbations.

Figure 1. The Grueneberg Ganglion (GG) is an unusual olfactory nerve at the rostral tip of the mouse nose

A) Schematic of the nasal cavity and septum of a mouse showing the olfactory subsystems. Both GG and GC-D subsystems (neurons as orange spots) project axons (orange lines) to a unique spatial domain containing sets of interconnected glomeruli in the caudal region of the olfactory bulb. The MOE is part of the MOS. The VNO is part of the AOS. Green lines demarcate anatomical locations in the nasal cavity; blue lines demarcate regions of the brain. B) In this medial view of a whole-mount preparation from an OMP-GFP mouse, GG neurons express olfactory marker protein (OMP, in white) and are arranged as clusters of cells comprising an arrowhead-shaped organ. C) OMP-expressing GG neurons (green) seen in thin section do not project cellular processes into the nasal cavity (n.c.). They are separated by a keratinized epithelium (KE, magenta) that is labeled with an antibody to particulate guanylyl cyclase E. D) En face view of proteolipid protein (PLP-GFP)-expressing glial cells in the nasal vestibule that ensheathe GG neurons. In thin sections (E–G): E) GG neurons visualized in nasal vestibules of OMP-GFP mice (GFP in green) positively stain for OMP protein (magenta). F) GFP-positive OSNs (green) in the OMP-GFP mouse MOE stain positively for OMP (magenta). G) OMP-GFP mice faithfully report expression of OMP in the glomerular layer (GL) of the olfactory bulb (GFP in green, OMP immunostaining in magenta). SO = septal organ; D = dorsal; V = ventral; R = rostral; C = caudal; ONL = olfactory nerve layer; EPL = external plexiform layer. Scale bars: B) 250 μm; C) 15 μm; D) 60 μm; E–F) 30 μm; G) 60 μm.

Figure 2. The GG expresses a cGMP-stimulated phosphodiesterase

A–B) Pde4a staining in thick sections from neonatal OMP-GFP mice in the MOE (A) and the rostral nasal vestibule (B). GG neurons correspond to OMP-positive cells in the nasal vestibule. C–D) Pde2a staining in the neonatal MOE (C) and nasal vestibule (D). The Pde2a antibody labeled GG neurons and vascular endothelial cells in the nasal vestibule. E–F) Immunoadsorption study with the Pde2a antibody in whole-mount preparations. Unblocked Pde2a antibody (magenta) labeled adult GG neurons (green) in the nasal vestibules of OMP-GFP mice (E), but there was no labeling when the antibody was blocked with the immunizing peptide (F). Scale bars: 40 μm.

Figure 3. Downstream cGMP signal transduction components are expressed in the GG

A) In OMP-GFP mice, GFP-fluorescing GG neurons (green) in the nasal vestibule express cGKII (magenta). On some neurons the cGKII antibody labels the GG neuron membrane (arrow). B) The cGKII antibody (red) labels the villus brush borders in the intestine (nuclei are in blue). C) CNGA3 antibody (red) labels photoreceptor segments (arrow) in thin sections from the mouse retina (nuclei are in blue). D) GG neurons (green) express CNGA3 (magenta) on unusual “whip-like” subcellular domains. E) Neurons of the GC-D olfactory subsystem, highlighted by GFP expression (green) in the GCD-iTG transgenic mouse, express CNGA3 (magenta) on their dendritic knobs (arrows). Scale bars: A) 20 μm B) 60 μm; C) 30 μm; D) 20 μm; E) 60 μm.

Figure 4. The GG does not express pGC-D

A) Pde2a immunolabeling in thick sections from the olfactory neuroepithelium of a transgenic GCD-iTG mouse. GFP-positive GC-D neurons (green) express Pde2a (magenta), consistent with their use of a cGMP signaling pathway. B) Pde2a-positive GG cells (magenta) in thick sections from the nasal vestibule are devoid of GFP (green) and, hence, pGC-D. Scale bars: 60 μm.

Figure 5. Particulate guanylyl cyclase expression in the GG

A–B) In whole-mount preparations, all GG neurons (green) express pGC-G (A, magenta) on unusual subcellular domains. In contrast, only a small, broadly-distributed subset of GG neurons (white asterisks) express pGC-A (B, magenta) on the same subcellular domains. C–D) Immunoadsorption study with the pGC-G antibody (magenta) and its antigenic blocking peptide on GG neurons (green). Labeling is observed when the antibody is unblocked (C) but is abolished when it is blocked (D). E–F) Blocking experiment with the pGC-A antibody (magenta) on GG neurons (green). Unblocked antibody labels the small GG neuron subset (E), blocked antibody does not (F). G–H) pGC-G is expressed in GG neurons (red asterisks) at all ages, from PD2/neonatal (G) to adult (H) ages. Gray-black areas indicate staining. I–J) A comparison on pGC-G expression the GG and GC-D olfactory subsystems of GCD-iTG mice reveals that pGC-G (magenta) is expressed in GG neurons of the nasal vestibule (I) but not in the GC-D neurons (green) of the caudal MOE (J). K) High magnification view of pGC-G-expressing “whip”-like subcellular domains (black staining, red arrows) in a whole-mount preparation. L) High magnification view of pGC-G localization (magenta) on a GG neuron (green) at different optical z planes (in μm) in thin sections. Labeled subcellular domains follow the surface contours of GG neurons. M) Immunoblot with pGC-G antibody on nasal vestibule lysate. A single 120 kDa band is labeled. Lanes correspond to the relative amount of protein loaded. Scale bars: A–B) 60 μm; C–F) 10 μm; G–H) 40 μm; I–J) 30 μm; K) 15 μm; L) 5 μm.

Figure 6. pGC-G expression in relation to the GG ensheathing cells

A) In whole-mount preparations, GFP-positive fibers (green) in the nasal vestibules of PLP-GFP mice co-localize with GFAP staining (magenta). B) In thin cross-sections of the nasal vestibule, GFP-positive cells (green) in PLP-GFP mice can be seen to envelop GG neurons stained with the OMP antibody (magenta). C) In thin sections, pGC-G (magenta) does not co-localize with the glial-like ensheathing cell population (green) visualized in PLP-GFP mice. Scale bars: A) 25 μm; B–C) 20 μm.

Figure 7. Ultrastructural view of GG neurons

A) Low magnification view of a single GG neuron. A cluster of ciliary basal bodies is visible in the yellow highlighted region, next to the nucleus [1]. The GG neuron is ensheathed by a glial cell [2]. B) The inset region of (A), magnified. Ciliary basal bodies [3] are in close proximity to a mitochondrion [4]. C–D) A closer view of the clusters of ciliary basal bodies [3]. These clusters are supported by a ring of actin filaments, visible in (D). E–F) Out of these basal bodies, long ciliary fibers [5] erupt and follow the membrane contour of the GG neuron. These fibers are held tight to the membrane by the glial-like ensheathing cells. Lower-right orange inset magnified areas outlined in red (F). Scale bars: A) 3 μm; B) 0.5 μm; C–D) 0.25 μm; E) 0.5 μm F) 1 μm (inset: 0.3 μm).

Figure 8. The cGMP signal transduction pathway in the GG

Binding of atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP), or other as-of-yet unidentified ligands to pGC-A or unknown ligands to pGC-G induces the conversion of GTP to cGMP by the particulate guanylyl cyclases. The produced cGMP can then open CNGA3 channels to depolarize the GG neuron, induce additional signaling through cGKII, or be degraded to GMP by Pde2a.