Afferent activity to necklace glomeruli is dependent on external stimuli

Abstract

Background: The main olfactory epithelium (MOE) is a complex organ containing several functionally distinct subpopulations of sensory neurons. One such subpopulation is distinguished by its expression of the guanylyl cyclase GC-D. The axons of GC-D-expressing (GC-D+) neurons innervate 9-15 "necklace" glomeruli encircling the caudal main olfactory bulb (MOB). Chemosensory stimuli for GC-D+ neurons include two natriuretic peptides, uroguanylin and guanylin, and CO2. However, the biologically-relevant source of these chemostimuli is unclear: uroguanylin is both excreted in urine, a rich source of olfactory stimuli for rodents, and expressed in human nasal epithelium; CO2 is present in both inspired and expired air.

Findings: To determine whether the principal source of chemostimuli for GC-D+ neurons is external or internal to the nose, we assessed the consequences of removing external chemostimuli for afferent activity to the necklace glomeruli. To do so, we performed unilateral naris occlusions in Gucy2d-Mapt-lacZ +/- mice [which express a beta-galactosidase (beta-gal) reporter specifically in GC-D+ neurons] followed by immunohistochemistry for beta-gal and a glomerular marker of afferent activity, tyrosine hydroxylase (TH). We observed a dramatic decrease in TH immunostaining, consistent with reduced or absent afferent activity, in both necklace and non-necklace glomeruli ipsilateral to the occluded naris.

Conclusion: Like other MOB glomeruli, necklace glomeruli exhibit a large decrease in afferent activity upon removal of external stimuli. Thus, we conclude that activity in GC-D+ neurons, which specifically innervate necklace glomeruli, is not dependent on internal stimuli. Instead, GC-D+ neurons, like other OSNs in the MOE, primarily sense the external world.

Figure 1

Afferent activity to necklace glomeruli is dependent on exogenous stimulation of GC-D+ neurons. (A) RT-PCR of GC-D, PDE2 and uroguanylin from nasal epithelium cDNA of C57BL/6J mice with or without reverse transcriptase (RT). The identity of each product was confirmed by sequencing. (B) Immunohistochemical staining of horizontal sections of left (non-occluded side) and right (occluded side) MOBs of a Gucy2d-Mapt-lacZ ^+/- mouse. TH, green; DAPI, blue. Scale bar: 100 μM. (C) Immunohistochemical staining of left (non-occluded side) MOB of a naris occluded Gucy2d-Mapt-lacZ ^+/- mouse. Inset, higher magnification view of area represented in box. β-gal, red; TH, green; DAPI, blue. Scale bars 50 μM, 20 μM (inset). (D) Right (occluded side) MOB of same mouse as in (C). Inset, higher magnification view of area represented in box. β-gal, red; TH, green; DAPI, blue. Scale bars 50 μM, 20 μM (inset).