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Dual processing of sulfated steroids in the olfactory system of an anuran amphibian.

Sansone A, Hassenklöver T, Offner T, Fu X, Holy TE, Manzini I - Front Cell Neurosci (2015)

Bottom Line: Chemical communication is widespread in amphibians, but if compared to later diverging tetrapods the available functional data is limited.Furthermore, we found that larval and adult animals excrete multiple sulfated compounds with physical properties consistent with sulfated steroids.Breeding tadpole and frog water including these compounds activated a large subset of sensory neurons that also responded to synthetic steroids, showing that sulfated steroids are likely to convey intraspecific information.

View Article: PubMed Central - PubMed

Affiliation: Institute of Neurophysiology and Cellular Biophysics, University of Göttingen Göttingen, Germany ; Center for Nanoscale Microscopy and Molecular Physiology of the Brain Göttingen, Germany.

ABSTRACT
Chemical communication is widespread in amphibians, but if compared to later diverging tetrapods the available functional data is limited. The existing information on the vomeronasal system of anurans is particularly sparse. Amphibians represent a transitional stage in the evolution of the olfactory system. Most species have anatomically separated main and vomeronasal systems, but recent studies have shown that in anurans their molecular separation is still underway. Sulfated steroids function as migratory pheromones in lamprey and have recently been identified as natural vomeronasal stimuli in rodents. Here we identified sulfated steroids as the first known class of vomeronasal stimuli in the amphibian Xenopus laevis. We show that sulfated steroids are detected and concurrently processed by the two distinct olfactory subsystems of larval Xenopus laevis, the main olfactory system and the vomeronasal system. Our data revealed a similar but partially different processing of steroid-induced responses in the two systems. Differences of detection thresholds suggest that the two information channels are not just redundant, but rather signal different information. Furthermore, we found that larval and adult animals excrete multiple sulfated compounds with physical properties consistent with sulfated steroids. Breeding tadpole and frog water including these compounds activated a large subset of sensory neurons that also responded to synthetic steroids, showing that sulfated steroids are likely to convey intraspecific information. Our findings indicate that sulfated steroids are conserved vomeronasal stimuli functioning in phylogenetically distant classes of tetrapods living in aquatic and terrestrial habitats.

No MeSH data available.


Related in: MedlinePlus

Sulfated steroid-induced calcium responses in the olfactory organ. (A) Schematic of a larval Xenopus laevis (stage: 52–53). The black rectangle outlines the olfactory system. An enlargement of the olfactory system is shown on the right hand side. The main olfactory epithelium (MOE) and the vomeronasal organ (VNO) are connected via the olfactory nerve (ON) to the main olfactory bulb (MOB) and the accessory olfactory bulb (AOB), respectively. (B) Olfactory organ of larval Xenopus laevis visualized by biocytin-streptavidin retrograde labeling of sensory neurons (MOE, green; VNO, magenta). (C) Time courses of sulfated steroid-induced [Ca2+]i transients of individual sensory neurons of the MOE (green traces, 2 different cells) and the VNO (magenta traces, 2 different cells). We recorded a total of 90 P mix-responsive sensory neurons in the MOE (18 slices) and 75 in the VNO (33 slices). We recorded a total of 30 E mix-responsive sensory neurons in the MOE (18 slices) and 5 in the VNO (32 slices). Sulfated steroid mixtures were applied at a concentration of 100–200 μM. All cells responded to high K+ solution, but did not respond upon application of standard bath solution.
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Figure 1: Sulfated steroid-induced calcium responses in the olfactory organ. (A) Schematic of a larval Xenopus laevis (stage: 52–53). The black rectangle outlines the olfactory system. An enlargement of the olfactory system is shown on the right hand side. The main olfactory epithelium (MOE) and the vomeronasal organ (VNO) are connected via the olfactory nerve (ON) to the main olfactory bulb (MOB) and the accessory olfactory bulb (AOB), respectively. (B) Olfactory organ of larval Xenopus laevis visualized by biocytin-streptavidin retrograde labeling of sensory neurons (MOE, green; VNO, magenta). (C) Time courses of sulfated steroid-induced [Ca2+]i transients of individual sensory neurons of the MOE (green traces, 2 different cells) and the VNO (magenta traces, 2 different cells). We recorded a total of 90 P mix-responsive sensory neurons in the MOE (18 slices) and 75 in the VNO (33 slices). We recorded a total of 30 E mix-responsive sensory neurons in the MOE (18 slices) and 5 in the VNO (32 slices). Sulfated steroid mixtures were applied at a concentration of 100–200 μM. All cells responded to high K+ solution, but did not respond upon application of standard bath solution.

Mentions: Sulfated steroids from the androgen, estrogen, pregnanolone and glucocorticoid families, potent murine vomeronasal ligands (see Supplementary Table 1 and Nodari et al., 2008; Isogai et al., 2011; Celsi et al., 2012), were tested for their chemosensory activity in larval Xenopus laevis. Responses were measured as stimulus-induced calcium increases of sensory neurons in acute slice preparations of the olfactory organ (Figures 1A,B). We found steroid-responsive sensory neurons in the VNO and the MOE (Figure 1C). Neurons responded to pregnanolone-derived (P mix) and estrogen-derived (E mix) steroids (for more information see Materials and Methods). Thereby, in both epithelia P mix responses were more frequent than E mix responses. None of the tested androgen-derived and glucocorticoid-derived steroids were effective (not shown). All cells that responded to sulfated steroids (VNO and MOE) also showed calcium transients upon stimulation with high K+ bath solution, indicating that they were sensory neurons (Dittrich et al., 2014). In both epithelia, sulfated steroid-induced calcium responses were dependent on the presence of extracellular Ca2+, indicating that the stimulus-induced intracellular Ca2+ increase is mainly due to Ca2+ entry through plasma membrane ion channels (Figure 2). Non-sulfated analogs of the employed steroids in most cases (>80%) failed to activate sulfated-steroid-responsive neurons (Figure 3).


Dual processing of sulfated steroids in the olfactory system of an anuran amphibian.

Sansone A, Hassenklöver T, Offner T, Fu X, Holy TE, Manzini I - Front Cell Neurosci (2015)

Sulfated steroid-induced calcium responses in the olfactory organ. (A) Schematic of a larval Xenopus laevis (stage: 52–53). The black rectangle outlines the olfactory system. An enlargement of the olfactory system is shown on the right hand side. The main olfactory epithelium (MOE) and the vomeronasal organ (VNO) are connected via the olfactory nerve (ON) to the main olfactory bulb (MOB) and the accessory olfactory bulb (AOB), respectively. (B) Olfactory organ of larval Xenopus laevis visualized by biocytin-streptavidin retrograde labeling of sensory neurons (MOE, green; VNO, magenta). (C) Time courses of sulfated steroid-induced [Ca2+]i transients of individual sensory neurons of the MOE (green traces, 2 different cells) and the VNO (magenta traces, 2 different cells). We recorded a total of 90 P mix-responsive sensory neurons in the MOE (18 slices) and 75 in the VNO (33 slices). We recorded a total of 30 E mix-responsive sensory neurons in the MOE (18 slices) and 5 in the VNO (32 slices). Sulfated steroid mixtures were applied at a concentration of 100–200 μM. All cells responded to high K+ solution, but did not respond upon application of standard bath solution.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4585043&req=5

Figure 1: Sulfated steroid-induced calcium responses in the olfactory organ. (A) Schematic of a larval Xenopus laevis (stage: 52–53). The black rectangle outlines the olfactory system. An enlargement of the olfactory system is shown on the right hand side. The main olfactory epithelium (MOE) and the vomeronasal organ (VNO) are connected via the olfactory nerve (ON) to the main olfactory bulb (MOB) and the accessory olfactory bulb (AOB), respectively. (B) Olfactory organ of larval Xenopus laevis visualized by biocytin-streptavidin retrograde labeling of sensory neurons (MOE, green; VNO, magenta). (C) Time courses of sulfated steroid-induced [Ca2+]i transients of individual sensory neurons of the MOE (green traces, 2 different cells) and the VNO (magenta traces, 2 different cells). We recorded a total of 90 P mix-responsive sensory neurons in the MOE (18 slices) and 75 in the VNO (33 slices). We recorded a total of 30 E mix-responsive sensory neurons in the MOE (18 slices) and 5 in the VNO (32 slices). Sulfated steroid mixtures were applied at a concentration of 100–200 μM. All cells responded to high K+ solution, but did not respond upon application of standard bath solution.
Mentions: Sulfated steroids from the androgen, estrogen, pregnanolone and glucocorticoid families, potent murine vomeronasal ligands (see Supplementary Table 1 and Nodari et al., 2008; Isogai et al., 2011; Celsi et al., 2012), were tested for their chemosensory activity in larval Xenopus laevis. Responses were measured as stimulus-induced calcium increases of sensory neurons in acute slice preparations of the olfactory organ (Figures 1A,B). We found steroid-responsive sensory neurons in the VNO and the MOE (Figure 1C). Neurons responded to pregnanolone-derived (P mix) and estrogen-derived (E mix) steroids (for more information see Materials and Methods). Thereby, in both epithelia P mix responses were more frequent than E mix responses. None of the tested androgen-derived and glucocorticoid-derived steroids were effective (not shown). All cells that responded to sulfated steroids (VNO and MOE) also showed calcium transients upon stimulation with high K+ bath solution, indicating that they were sensory neurons (Dittrich et al., 2014). In both epithelia, sulfated steroid-induced calcium responses were dependent on the presence of extracellular Ca2+, indicating that the stimulus-induced intracellular Ca2+ increase is mainly due to Ca2+ entry through plasma membrane ion channels (Figure 2). Non-sulfated analogs of the employed steroids in most cases (>80%) failed to activate sulfated-steroid-responsive neurons (Figure 3).

Bottom Line: Chemical communication is widespread in amphibians, but if compared to later diverging tetrapods the available functional data is limited.Furthermore, we found that larval and adult animals excrete multiple sulfated compounds with physical properties consistent with sulfated steroids.Breeding tadpole and frog water including these compounds activated a large subset of sensory neurons that also responded to synthetic steroids, showing that sulfated steroids are likely to convey intraspecific information.

View Article: PubMed Central - PubMed

Affiliation: Institute of Neurophysiology and Cellular Biophysics, University of Göttingen Göttingen, Germany ; Center for Nanoscale Microscopy and Molecular Physiology of the Brain Göttingen, Germany.

ABSTRACT
Chemical communication is widespread in amphibians, but if compared to later diverging tetrapods the available functional data is limited. The existing information on the vomeronasal system of anurans is particularly sparse. Amphibians represent a transitional stage in the evolution of the olfactory system. Most species have anatomically separated main and vomeronasal systems, but recent studies have shown that in anurans their molecular separation is still underway. Sulfated steroids function as migratory pheromones in lamprey and have recently been identified as natural vomeronasal stimuli in rodents. Here we identified sulfated steroids as the first known class of vomeronasal stimuli in the amphibian Xenopus laevis. We show that sulfated steroids are detected and concurrently processed by the two distinct olfactory subsystems of larval Xenopus laevis, the main olfactory system and the vomeronasal system. Our data revealed a similar but partially different processing of steroid-induced responses in the two systems. Differences of detection thresholds suggest that the two information channels are not just redundant, but rather signal different information. Furthermore, we found that larval and adult animals excrete multiple sulfated compounds with physical properties consistent with sulfated steroids. Breeding tadpole and frog water including these compounds activated a large subset of sensory neurons that also responded to synthetic steroids, showing that sulfated steroids are likely to convey intraspecific information. Our findings indicate that sulfated steroids are conserved vomeronasal stimuli functioning in phylogenetically distant classes of tetrapods living in aquatic and terrestrial habitats.

No MeSH data available.


Related in: MedlinePlus