Functional analysis of the guanylyl cyclase type D signaling system in the olfactory epithelium

Abstract

The mammalian olfactory system recognizes a wide range of chemical stimuli. The majority of cells in the main olfactory epithelium (MOE) use a cAMP-mediated signaling system to transduce odor signals. However, a subset of MOE neurons instead expresses components of a cGMP signaling cascade, including the receptor guanylyl cyclase GC-D and the cyclic nucleotide-gated channel subunit CNGA3. We used a combination of molecular biological, physiological, and imaging approaches to characterize this neuronal population. Neurons expressing GC-D show excitatory responses to the natriuretic peptide hormones uroguanylin and guanylin, as well as to stimuli present in urine, that are dependent on both GC-D and CNGA3. Though all GC-D-expressing neurons are highly sensitive to these stimuli, individual cells are differentially tuned to either one or both of the peptides. Together, these findings suggest that neurons expressing GC-D are part of a specialized olfactory subsystem that is responsive to semiochemicals.

Figure

(A) X-gal histochemical labeling of the olfactory epithelium (left) and olfactory bulb (right; lateral view) of a Gucy2d −/− mouse reveals axons of GC-D+ neurons (blue) entering necklace glomeruli (arrows indicate two of the necklace glomerui). The location of the cribiform plate is indicated by the dashed line. (B) Dose dependency of peak responses to uroguanylin (UG) and guanylin (G) obtained from Gucy2d +/+, +/−, or−/− mice. Data points are normalized to the mean response of a given stimulus at 1 μM in Gucy2d +/+ mice. Insets: examples of the time course of peptide-induced responses in Gucy2d +/− or −/− mice (ligands 1 μM each). Scale bars: 40 μV, 1 s. (C) Patch clamp recording from individual dendritic knobs of GC-D cells. Stimulus-evoked discharges recorded from β-gal-positive knobs of Gucy2d +/− (left) or −/− (right) mice. KCl (60 mM); uroguanylin (UG; 1 μM); guanylin (G, 1 μM); dilute urine (1:100). Arrows: stimulus application. (D) Example of Ca²⁺ knob responses, assayed by confocal imaging of Ca²⁺ fluorescence, to UG (1 μM), G (1 μM), UG/G mixture (1 μM each), and urine (1:100). No responses were observed in GC-D cells from Gucy2d −/− mice. (E) Venn diagram indicating the proportions of GC-D+ neurons (n=27) responding to UG only, to G only, or to both peptides. Panels B-D reprinted, and panels A and E modified, with permission from ; copyright 2007, National Academy of Sciences.