Nakada T et al. (2014), Expression of G proteins in the olfactory recep...

XB-ART-48894

J Comp Neurol 2014 Oct 15;52215:3501-19. doi: 10.1002/cne.23619.

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Expression of G proteins in the olfactory receptor neurons of the newt Cynops pyrrhogaster: their unique projection into the olfactory bulbs.

Nakada T , Hagino-Yamagishi K , Nakanishi K , Yokosuka M , Saito TR , Toyoda F , Hasunuma I , Nakakura T , Kikuyama S .

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We analyzed the expression of G protein α subunits and the axonal projection into the brain in the olfactory system of the semiaquatic newt Cynops pyrrhogaster by immunostaining with antibodies against Gαolf and Gαo , by in situ hybridization using probes for Gαolf , Gαo , and Gαi2 , and by neuronal tracing with DiI and DiA. The main olfactory epithelium (OE) consists of two parts, the ventral OE and dorsal OE. In the ventral OE, the Gαolf - and Gαo -expressing neurons are located in the apical and basal zone of the OE, respectively. This zonal expression was similar to that of the OE in the middle cavity of the fully aquatic toad Xenopus laevis. However, the Gαolf - and Gαo -expressing neurons in the newt ventral OE project their axons toward the main olfactory bulb (MOB) and the accessory olfactory bulb (AOB), respectively, whereas in Xenopus, the axons of both neurons project solely toward the MOB. In the dorsal OE of the newt, as in the principal cavity of Xenopus, the majority of the neurons express Gαolf and extend their axons into the MOB. In the vomeronasal organ (VNO), the neurons mostly express Gαo . These neurons and quite a few Gαolf -expressing neurons project their axons toward the AOB. This feature is similar to that in the terrestrial toad Bufo japonicus and is different from that in Xenopus, in which VNO neurons express solely Gαo , although their axons invariably project toward the AOB. We discuss the findings in the light of diversification and evolution of the vertebrate olfactory system.

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Species referenced: Xenopus laevis
Genes referenced: eno2
GO keywords: olfactory receptor activity [+]
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	Figure 1. Specificity of anti-Gαolf and anti-Gαo antibodies. Tissue extract from the mixture of olfactory epithelium (OE) and vomeronasal epithelium (VNE) of the female newt Cynops pyrrhogaster was subjected to western blot analysis. Lane 1, stained with anti-Gαolf. Lane 2, negative control with anti-Gαolf omitted. Lane 3, stained with anti-Gαo. Lane 4, negative control with anti-Gαo omitted. Arrows indicate the specific bands.
	Figure 2. Partial nucleotide sequences of a cDNAs encoding the olfactory G proteins. The nucleotide sequences of partial cDNAs for C. pyrrhogaster (Cp) Gαolf, Gαo, and Gαi2 that correspond to X. laevis (Xl) Gαolf (GenBank accession number AJ296281), Gαo (GenBank accession number X14636), and Gαi2 (GenBank accession number NM001097056), respectively. Identical nucleotides in Cynops and Xenopus cDNAs are in black boxes.
	Figure 3. The nasal cavity of the C. pyrrhogaster. A: Coronal section of the middle portion of the nasal cavity stained with hematoxylin and eosin. Lateral view is given on the right and ventral view is at the bottom. The nasal cavity is a pair of flattened single chambers and consists of the main nasal chamber (MNC) and lateral nasal sinus (LNS). B–D: Higher magnifications of the olfactory epithelium (OE; B: ventral, C: dorsal) and vomeronasal organ (VNO; D) in boxes b, c, and d, respectively, in A. Note the ridges of nonsensory epithelium (NSE; arrowheads in B) separating the clusters of sensory epithelial cells. E: Dorsal view of the telencephalon exposed to label the olfactory nerve bundle (ONB) and nasal epithelium with DiI placed on the main olfactory bulb (MOB) and accessory olfactory bulb (AOB). Dark materials on the rostral end of the telencephalon are DiI crystals placed in excess. F: Coronal section of the middle portion of the nasal cavity. Note that the ventral, dorsal OE and VNO were divided by the distinct NSE. G: Sensory neurons of the ventral OE often interspaced with NSE. H: Sensory neurons of the dorsal OE. I: Sensory neurons of the VNO. VN, vomeronasal nerves. G–H are from a specimen treated as in E. Dashed lines indicate the outlines of the epithelia. Scale bar = 500 μm in A; 100 μm in B–D; 1 mm in E; 200 μm in F; 50 μm in G–H.
	Figure 4. Distribution of Gαolf- and Gαo-immunoreactive cells in the nasal cavity of C. pyrrhogaster. A: Distribution of Gαolf- (green) and Gαo-expressing neurons (magenta) in the middle portion of the cavity: This region consists of three types of sensory epithelium surrounding the main nasal cavity (MNC) or the lateral nasal sinus (LNS): the ventral olfactory epithelium (OE), dorsal OE, and vomeronasal epithelium. This section was counterstained with 4′-6-diamidino-2-phenylindole (DAPI; blue). Asterisk represents the nonsensory epithelium (NSE) interspacing the dorsal and ventral OE. B: Higher magnification image of the ventral OE: Clusters of sensory neurons constituting a double-layered structure. Gαolf-immunoreactive cell bodies are situated in the apical side and Gαo-immunoreactive cell bodies in the basal side of the cluster. C: The dorsal OE: Sensory epithelium consists of a large number of Gαolf-immunoreactive neurons (green) situated on the apical side and a small number of Gαo-immunoreactive neurons on the basal side (magenta; arrowheads). D: The vomeronasal organ (VNO): Sensory epithelium consists of abundant Gαo-immunoreactive neurons (magenta) and a few Gαolf-immunoreactive neurons (green; outlined arrowhead). Co-localization of the Gαolf and Gαo signals were not observed in any epithelium examined. E: Mean number of immunopositive neurons per section in each epithelium. Each column and vertical bar represents the mean of measurements of sections from eight different animals and SEM, respectively. Asterisk represents significant difference between the numbers of the Gαolf- and Gαo-immunoreactive cells within each epithelium. Values with different letters differ significantly at the 5% level in numbers of G protein–positive cells among the epithelia. Scale bar = 500 μm in A; 100 μm in B–D.
	Figure 5. Expression of Gαolf, Gαo, and Gαi2 mRNAs in the nasal epithelium as demonstrated by in situ hybridization. A–I: Sections of the olfactory epithelium (OE); ventral (A–C), dorsal (D–F), and the vomeronasal organ (VNO; G–I) were hybridized with an antisense Gαolf, Gαo, or Gαi2 probe. J–L: As negative controls, sections of the ventral olfactory epithelium were hybridized with a sense Gαolf, Gαo, or Gαi2 probe. Gαolf-expressing cells lined the apical zones of the neuroepithelium (a two-headed arrow in A and D) and the VNO (arrow in G), whereas Gαo-expressing cells were located in the basal zones of the neuroepithelium (double-headed arrow in B, E, and H). A small number of Gαi2-expressing cells were also observed in the ventral OE and a lower number in the dorsal OE (arrows in C and F), but no signals were found in the VNO (I). No positive signals were seen in the ventral OE when hybridized with sense probes (J–L). Dashed lines indicate the boundaries of the neuroepithelia. Scale bar = 50 μm in A (applies to a–L).
	Figure 6. Double-labeling immunohistochemistry for G proteins and acetylated tubulin (AcT). A: Merged image of Gαolf- (green) and AcT-immunoreactive signals (magenta) in the ventral olfactory epithelium (OE). B: Merged image of Gαo- (green) and AcT-immunoreactive signals (magenta) in the ventral OE. C: Merged image of Gαolf- (green) and AcT-immunoreactive signals (magenta) in the dorsal OE. D: Merged image of Gαo- (green) and AcT-immunoreactive signals (magenta) in the dorsal OE. E: Merged image of Gαolf- (green) and AcT-immunoreactive signals (magenta) in the vomeronasal organ (VNO). No significant AcT- and Gαolf-immunoreactive signals at the epithelial surface (dashed line) of the VNO were observed. F: Merged image of Gαo- (green) and AcT-immunoreactive signals (magenta) in the VNO. The white-colored image at the surface of the ventral and dorsal neuroepithelial regions (A, C) indicates the overlapping of immunoreactive signals on the cilia labeled with anti-AcT with those labeled with anti-Gαolf. Gαo-immunoreactive olfactory knob-like structures are invariably immunonegative for AcT (B, F). Scale bar = 10 μm in A (applies to A–F).
	Figure 7. Transmission immunoelectron microscopic analysis of the ventral (A–D) and dorsal olfactory epithelia (OE; E,F), and the vomeronasal epithelium (G–I). A,B: On the ciliated olfactory knobs (CK) located in the ventral OE, Gαolf-immunoreactive signals were observed on the cilia (arrows in A) projecting from the CK, whereas no Gαo-immunoreactive signals were observed on the cilia and the immunoreactive signals for Gαo were observed on the microvilli (arrowheads in B). C,D: In close vicinity to the microvillous olfactory knobs (MK) located in the ventral OE, Gαolf immunoreactivities were observed on the cilia (arrow in C) but not on the microvilli (arrowheads in C) projecting from the MK, whereas Gαo immunoreactivities were observed on the microvilli (arrowheads in D) but not on the cilia (arrows in D). E,F: Gαolf immunoreactivities were observed on the cilia (arrows in E) projecting from the CK, whereas Gαo-immunoreactive signals were not observed on the cilia (arrows in F). G: In the vomeronasal organ (VNO), Gαolf immunoreactivities were observed neither on the cilia (arrow) nor on the microvilli (arrowhead) located in the apical portion of the sensory epithelium. H: Cells with Gαolf-immunoreactive signals (outlined arrows) were rarely encountered in the VNO. N, nucleus. I: Abundant Gαo-immunoreactive signals were observed on the microvillar surface (arrowheads) in the VNO. No gold particles were observed on the cilia (arrow). Scale bar = 500 nm in A–I.
	Figure 8. The axonal projections of the sensory neurons. A: Coronal section of the nasal cavities. This region consists of three types of sensory organs: the ventral olfactory epithelium (OE), dorsal OE, and vomeronasal organ (VNO). B: Horizontal section of the telencephalon stained with hematoxylin and eosin. Lateral view (l) is on the right side and caudal view (c) is at the bottom. Glomeruli in both the main olfactory bulb (MOB) and accessory olfactory bulb (AOB) were eosinophilic. M, medial; r, rostral. C: The cross-sectioned upper jaw with the dorsal OE and VNO surgically exposed, embedded in agar. The neural tracers DiI and DiA were placed on the indicated positions (circled) of the VNO and dorsal OE, respectively. D: The VNO neurons labeled with DiI (magenta) projected their axons exclusively toward the AOB, whereas the dorsal OE neurons labeled with DiA (green) projected their axons to the MOB but not to the AOB. ONB, olfactory nerve bundle. E: The cross-sectioned upper jaw with the dorsal OE and ventral OE exposed. DiI and DiA were placed on the indicated positions (circled) of the ventral OE and dorsal OE, respectively. F: Projection of the sensory neurons in the OE to the MOB and AOB. The dorsal OE sensory neurons labeled with DiA (green) projected their axons to the MOB but not to the AOB, and the ventral OE sensory neurons labeled with DiI (magenta) projected their axons to the middle and caudal MOB and the AOB. Note the difference in the distribution between the axon terminals of the dorsal OE and those of the ventral OE. ONB contained axons of both the dorsal and ventral sensory neurons labeled with DiA and DiI, respectively. Axons projecting toward the AOB were derived from the ventral OE. G: The cross-sectioned upper jaw with the ventral OE and VNO exposed. DiI and DiA were placed on the indicated positions (circled) of the ventral OE and VNO, respectively. H: The ventral OE neurons labeled with DiI (magenta) projected their axons to the caudal MOB and the AOB, whereas the VNO neurons labeled with DiA (green) projected their axons exclusively toward the AOB. There were no apparent differences in the distribution pattern between the axon terminals of the ventral OE and those of the VNO. Sections in D, F, and H were counterstained with DAPI (blue). Dashed lines indicate the area of the glomeruli. Scale bar = 2 mm in A and C (applies to C, E, G); 300 μ in B and D (applies to D,F,H).
	Figure 9. Distribution of Gαolf- and Gαo-expressing neurons in the brain. A: Horizontal section of the telencephalon: Gαolf-immunoreactive fibers (green) converged on the glomeruli of the main olfactory bulb (MOB). Gαo-immunoreactive fibers (magenta) converged on the glomeruli of the accessory olfactory bulb (AOB). B: Nonmerged image of Gαolf-immunoreactive fibers. Note that a small number of immunoreactive fibers was observed in the AOB (arrow). C: Nonmerged image of Gαo-immunoreactive fibers. The fibers did not project into the MOB. Sections in A were counterstained with DAPI (blue). Dashed lines indicate the area of the glomeruli. Scale bar = 500 μm in A (applies to A–C).
	Figure 10. Retrograde tracing of Gαolf- and Gαo-expressing neurons from the accessory olfactory bulb (AOB). A: Sensory epithelial cells labeled by DiI placed on the AOB. The labeled cells were localized in the ventral olfactory epithelium (OE) and vomeronasal organ (VNO) but not in the dorsal OE. B–G: Higher magnification of the ventral OE. DiI labeled the olfactory sensory neuron clusters in the ventral OE (B, E). Gαolf-immunoreactive cells were situated in the apical area (C). Merged image of DiI labeled and Gαolf-immunoreactive cells: co-localization of DiI and Gαolf signals was not observed in the ventral OE (D). Gαo-immunoreactive cells were distributed in the basal area (F). Merged image of DiI-labeled and Gαo-immunoreactive cells in the ventral OE (G): co-localization of DiI and Gαo signals was evidenced. H–M: Higher magnification of the VNO. DiI-labeled cells were distributed in the whole area of the VNO (H, K). A small number of Gαolf-immunoreactive cells were found in the VNO (I). Merged image of DiI-labeled and Gαolf-immunoreactive cell in the VNO (J): among the dominantly existing DiI-labeled cells, quite a few cells were also Gαolf-immunoreactive (arrows). DiI-labeled cells (K) and Gαo-immunoreactive cells (L) were widely distributed in the VNO. Merged image of DiI-labeled and Gαo-immunoreactive cells (M): DiI-labeled cells coincided well with the cells exhibiting Gαo-immunoreactive signals. Dashed lines indicate the outlines of the sensory epithelia. Scale bar = 200 μ in A, B (applies to B–D), E (applies to E–G), H (applies to H–J), and K (applies to K–M).
	Figure 11. Schematic representation of diversified olfactory system in amphibians. Gαolf (green)- and Gαo (magenta)-expressing olfactory receptor neurons in the OE and/or VNO and their axonal projection toward MOB and/or AOB in X. laevis, C. pyrrhogaster, and B. japonicus. For comparison, the pattern of distribution and projection of Gαolf- and Gαo-expressing neurons in the goldfish, Carassius auratus, is also presented. OE, olfactory epithelium; MOB, main olfactory bulb; VNO, vomeronasal organ; AOB, accessory olfactory bulb. References: C. auratus: Hansen et al., 2004, 2005. X. laevis: Saito and Taniguchi, 2000; Hagino-Yamagishi et al., 2004; Date-Ito et al., 2007. C. pyrrhogaster: this study. B. japonicus: Hagino-Yamagishi and Nakazawa, 2011.