The A2c gene is specific to and highly conserved in fishes and amphibians

Nucleotides released from food sources into environmental water are supposed to act as feeding cues for many fish species. However, it remains unknown how fish can sensitively detect those nucleotides. Here we discover a novel olfactory mechanism for ATP sensing in zebrafish. Upon entering into the nostril, ATP is efficiently converted into adenosine through enzymatic reactions of two ecto-nucleotidases expressed in the olfactory epithelium. Adenosine subsequently activates a small population of olfactory sensory neurons expressing a novel adenosine receptor A2c that is unique to fishes and amphibians. The information is then transmitted to a single glomerulus in the olfactory bulb and further to four regions in higher olfactory centers. These results provide conclusive evidence for a sophisticated enzyme-linked receptor mechanism underlying detection of ATP as a food-derived attractive odorant linking to foraging behavior that is crucial and common to aquatic lower vertebrates.

Nucleotides are essential for all living organisms as genetic materials, energy source, and intracellular and intercellular signaling molecules. In addition, it has been reported that various fishes and amphibians can sense nucleotides in environmental water as potential feeding stimulants or odorants []. For example, electrophysiological and anatomical studies revealed that ATP and other nucleotides induce excitatory responses of olfactory sensory neurons (OSNs) in the olfactory epithelium (OE) and neurons in the posterior olfactory bulb (OB) in channel catfish []. A neural activity imaging experiment with voltage-sensitive dye showed that nucleotides activate a latero-posterior portion of the OB in zebrafish []. A Caimaging analysis revealed that ATP activates both OSNs and supporting cells in the OE of Xenopus lavies []. However, it is largely unknown which olfactory receptor is activated by nucleotides, how the odor information of nucleotides is transferred to the OB and further to higher olfactory centers, and what behavioral responses fish actually show upon nucleotide stimulation. In the present study, we aim to elucidate the functional olfactory receptor and neural correlates that receive and transmit the information of nucleotides from the periphery to the central brain, evoking behavioral output responses in zebrafish.

Electrophysiological evidence for a chemotopy of biologically relevant odors in the olfactory bulb of the channel catfish.

Many olfactory cues pervade aquatic environment of fish and elicit various behavioral and endocrine responses that are essential for survival and reproduction. Fishes efficiently use the sense of smell for locating food sources, detecting and escaping from dangerous environment, communicating social information, and memorizing beneficial and detrimental contexts. The fish olfactory system is highly elaborated to receive, discriminate, and perceive various kinds of water-soluble chemicals such as amino acids, bile acids, amines, steroids, prostaglandins, and nucleotides [].

Taken together, these findings demonstrate that ATP is efficiently converted into adenosine by two ecto-nucleotidases to activate the A2c-expressing OSNs in the OE: (1) TNAP in non-sensory cells located close to the inlet naris would serve for rapid dephosphorylation of incoming ATP, and (2) CD73 and A2c co-expressed in the pear-shaped OSNs would serve for efficient detection of adenosine immediately after the conversion from AMP in a local environment.

Finally, in search of an antagonist of the zebrafish A2c receptor, we screened 12 compounds (caffeine, theophylline, XAC [xanthine amine congener], ACC, CGH2466, CGS15943, DPCPX, MRS1706, MRS1754, PSB36, PSB0788, and ZM241385) that have been reported to antagonize mammalian adenosine receptors, in cAMP production assay in the A2c-expressing CHO-K1 cells. Among the compounds tested, XAC effectively antagonized the adenosine-induced A2c activation ( Figure 7 B). XAC was also effective in antagonizing zebrafish A2aa and A2ab receptors, but not A2b receptor or OR114-1, an olfactory receptor for a female sex pheromone prostaglandin F] ( Figures 7 B and S6 B). When XAC was applied to the zebrafish nose, the ATP-, AMP-, and adenosine-induced Caincrease in lG2 was blocked in a concentration-dependent manner, while activations of lGx by alanine and dlG5 by cadaverine (polyamine) [] were unaffected ( Figures 7 C and S6 C). This result validates a crucial role of A2c in the lG2 activation by ATP, AMP, and adenosine.

We hypothesized that, upon entering into the fish nostril, ATP and related nucleotides are actively and rapidly converted into adenosine through enzymatic reactions. Hence we searched for extracellular ATP-dephosphorylating enzymes that are expressed in the zebrafish OE by in situ hybridization. Among 29 ecto-nucleotidase genes in zebrafish, two enzymes, ecto-5′-nucleotidase (CD73; nt5e) and tissue-non-specific alkaline phosphatase (TNAP; alpl), were expressed in distinct, small populations of cells in the OE ( Figure 6 B). Interestingly, CD73 was co-expressed with A2c in the pear-shaped OSNs ( Figure 6 B), whereas TNAP was present in non-neuronal cells predominantly located in the anterior OE close to the inlet naris ( Figure S5 ). Both TNAP and CD73 are glycosylphosphatidylinositol (GPI)-anchoring membrane proteins with a catalytic domain in the extracellular region []. TNAP catalyzes successive dephosphorylation steps from ATP to ADP, from ADP to AMP, and finally from AMP to adenosine, whereas CD73 specifically converts AMP to adenosine []. Expression of TNAP and CD73 in CHO-K1 cells together with A2c resulted in remarkable increase of cAMP production upon application of ATP, ADP, and AMP as well as adenosine ( Figure 6 C). Furthermore, Caimaging analysis revealed that the lG2 activation by ATP and AMP was drastically suppressed by the addition of TNAP inhibitor, 2,5-dimethoxy-N-(quinolin-3-yl)benzenesulfonamide (MLS0038949), and/or CD73 inhibitor, adenosine 5′-(α,β-methylene)diphosphate (AMPCP), whereas the lG2 activation by adenosine was not affected by those inhibitors ( Figure 7 A). These inhibitors did not alter the glomerular activation patterns and magnitudes by other odorants such as amino acids and bile acids ( Figure S6 A), indicating that they specifically inhibit the reaction of ecto-nucleotidases on adenine nucleotides.

(C) Effect of A2c antagonist (XAC) on lG2 activation by adenosine, AMP, and ATP, as assessed by Caimaging in OMP:Gal4FF;UAS:G-CaMP7 or OMP:Gal4FF;SAGFF27A;UAS:G-CaMP7 transgenic zebrafish. Left: representative Caresponses of lG2 upon stimulation with 1 μM adenosine (top row), AMP (middle row), and ATP (bottom low) in the presence or absence of 30 μM XAC. Right: concentration dependence of the suppressive effect of XAC on lG2 activation by 1 μM adenosine (blue), AMP (light blue), and ATP (red). Caincreases of dlG5 by cadaverine (green dot) and lGx by alanine (yellow dot) were not affected by 10 μM XAC. Values represent mean ± SEM (n = 3). Values of the ICfor ATP, AMP, and adenosine are 1.89 μM, 4.04 μM, and 3.29 μM, respectively. See also Figure S6 C.

(B) XAC is an antagonist of zebrafish A2c receptor. Adenosine (100 μM)-induced cAMP increase in A2c-expressing CHO-K1 cells is antagonized by XAC in a concentration-dependent manner (blue), whereas prostaglandin F(10 μM)-induced cAMP increase in OR114-1-expressing CHO-K1 cells is not affected (purple dot). The ICvalue of XAC for adenosine is 13.8 μM. See also Figure S6 B.

(A) Effects of TNAP and CD73 inhibitors (MLS0038949 and AMPCP) on lG2 activation by adenosine, AMP, and ATP, as assessed by Caimaging in OMP:Gal4FF;UAS:G-CaMP7 transgenic zebrafish. Left: representative Caresponses of lG2 upon stimulation with 1 μM adenosine (top row), AMP (middle row), and ATP (bottom low) in the presence or absence of 0.5 mM MLS0038949 and/or 0.25 mM AMPCP. Right: quantification of Caincrease in lG2. Values represent mean ± SEM (n = 3). Unpaired t test (AMP, MLS/AMPCP +/–, p = 0.00057; –/+, p = 0.0028; +/+, p = 0.0000012; ATP, +/–, p = 0.0087; +/+, p = 0.00000081).p < 0.01,p < 0.001. See also Figure S6 A.

Next we heterologously expressed zebrafish A2c receptor in Chinese hamster ovary (CHO)-K1 cells and investigated its ligand specificity by applying various compounds and measuring intracellular cAMP production. Contrary to our expectation, the A2c receptor was activated only by adenosine, but not at all by ATP and related nucleotides ( Figure 6 A). The median effective concentration (EC) of adenosine was 31.5 μM ( Figure 6 A). Thus, the A2c receptor displays an extremely narrow tuning specificity only to adenosine, suggesting that an additional piece of molecular machinery is required in fish OE, with which ATP can activate the A2c-expressing OSNs.

(C) Expression of TNAP and CD73 together with A2c in CHO-K1 cells results in A2c activation by AMP, ADP, and ATP as well as adenosine (1 mM) (n = 6). Unpaired t test (AMP, TNAP/CD73 +/–, p = 0.014; –/+, p = 0.0000044; +/+, p = 0.0000067; ADP, +/+, p = 0.0088; ATP, +/+, p = 0.0032).

(B) Double fluorescence in situ hybridization for A2c (magenta) and CD73 or TNAP (green) on OE sections (n = 3–4). Insets: magnified views of the boxed regions. Note the co-expression of A2c and CD73. Scale bar, 20 μm.

(A) Ligand specificity (left, n = 3) and sensitivity (right, n = 4) of A2c receptor examined by cAMP production assay in CHO-K1 cells. A2c is activated only by adenosine with high specificity and sensitivity. Unpaired t test (p = 0.00000016). ∗∗∗ p < 0.001.

Conversion of ATP to Adenosine by Two Ecto-nucleotidases Expressed in the OE

Figure 6 Conversion of ATP to Adenosine by Two Ecto-nucleotidases Expressed in the OE

We performed an extensive in silico database search for A2c orthologs in other animal species, based on amino acid sequence homology and syntenic conservation. Interestingly, A2c orthologous genes were found in all the fish species, both freshwater and seawater fishes, and amphibians whose genome sequences are available ( Figure 5 A; Table S2 ). On the chromosomes of these aquatic vertebrates, the A2c gene was located in the fourth intron of poly(ADP-ribose) polymerase-1 (parp1) gene on the complementary strand ( Figure S4 ). However, A2c gene was completely lost in terrestrial vertebrates (reptiles, birds, and mammals), as well as marine mammals such as whales and dolphins. In contrast, other adenosine receptor genes (A1, A2a, A2b, and A3) were commonly present in all vertebrates from fishes to mammals ( Figure 5 B; Table S3 ). These findings suggest a highly conserved and specific function of this unique receptor A2c in aquatic lower vertebrates.

(B) Neighbor-joining phylogenetic tree for adenosine receptors (A1, A2a, A2b, A2c, and A3) in fish (blue: zebrafish, spotted gar, medaka, fugu), amphibian (green: western clawed frog), reptile (brown: green anole), bird (orange: chicken), and mammals (red: mouse, human). Note that A2c genes are present only in fish and amphibian species. Bootstrap values were obtained from 500 replications. See Table S3 for the GenBank accession numbers and Ensembl gene numbers of individual genes. See also Figure S4

(A) A2c genes are present in all the fish and amphibian species whose genome sequences are available. See also Table S2 for the gene data.

The zebrafish genome harbors ∼140 OR-type, 6 V1R-type, ∼50 V2R-type, and ∼100 TAAR-type olfactory receptor genes []. To determine which olfactory receptor is expressed in the ATP-responding pear-shaped OSNs, we prepared 230 cRNA probes covering all the OR-, V1R-, V2R-, and TAAR-type olfactory receptors (including cross-hybridization) and performed double fluorescence in situ hybridization with a single or mixture of receptor probe(s) (one to seven receptors) and c-Fos probe (a neuronal activation marker) on OE sections of ATP-stimulated zebrafish. However, no overlapping signal was detected with c-Fos and these conventional olfactory receptors ( Figure 4 A; Table S1 ). We also examined all the 32 purinergic receptor genes including 9 P2Xs, 15 P2Ys, and 8 adenosine receptors that were annotated in the Ensembl zebrafish genome assembly Zv9, but revealed that none of them were expressed in OSNs (data not shown; Table S1 ). In the course of the database search for adenosine receptor orthologs in zebrafish genome, however, we noticed the presence of an unannotated, putative adenosine receptor with the highest homology to A2aa, A2ab, and A2b. We designated this novel gene as “A2c” and investigated its expression in the OE of ATP-stimulated zebrafish. Intriguingly, A2c mRNA was expressed in a small population of OSNs in the most superficial layer around the median raphe of the OE, and most of the A2c signals markedly overlapped with the c-Fos signals (93.7% ± 4.1%, n = 4) ( Figure 4 B). In addition, the A2c-expressing OSNs were positive for olfactory-specific signaling molecules, Golf and adenylyl cyclase III (ACIII) ( Figure 4 C). Furthermore, at 170 bp upstream of the zebrafish A2c gene, we found a putative Olf1/EBF-binding motif, which is commonly present in OSN-specific genes (data not shown) []. These results suggest that A2c is a strong candidate for the olfactory receptor recognizing adenine nucleotides and adenosine in the pear-shaped OSNs.

(C) Double fluorescence in situ hybridization for A2c (magenta) and Golf or ACIII (green) on OE sections (n = 3). A2c-positive OSNs express Golf and ACIII mRNA. Insets: magnified views of the boxed regions.

(B) Double fluorescence in situ hybridization for c-Fos (green) and A2c (magenta) on OE sections of 1 mM ATP-exposed fish (n = 4). Note that all c-Fos-positive OSNs express A2c mRNA. Inset: a magnified view of the boxed region.

(A) Double fluorescence in situ hybridization for c-Fos (green) and ORs, V1Rs, V2RL1, or TAARs (magenta) on OE sections of 1 mM ATP-exposed fish (n = 3). No double-positive OSNs are observed. See also Table S1

Next, the odor information represented on the OB glomerular array is decoded by telencephalic and diencephalic neurons in higher olfactory centers to elicit behavioral and physiological responses. We investigated ATP- and alanine-induced neural activation in higher brain centers by c-Fos in situ hybridization. ATP and alanine applications resulted in significant increase in the number of c-Fos-positive neurons in multiple brain areas. Among them, one telencephalic region, the supracommissural nucleus of ventral telencephalon (Vs), and two diencephalic regions, the posterior tuberal nucleus (PTN) and the dorsal zone of periventricular hypothalamus (Hd), were commonly activated by ATP and alanine ( Figure 3 ). In contrast, the posterior zone of dorsal telencephalon (Dp) and a small number of large-diameter neurons in the ventral-most part of the ventral zone of periventricular hypothalamus (Hv) tended to be activated by ATP, but not alanine ( Figure 3 ). These results indicate that ATP and alanine share several brain regions for activation, which may link those odorants to foraging behavior.

(B) Quantification of c-Fos-positive neurons in nine brain regions. Abbreviations for brain regions are as follows: Vs, supracommissural nucleus of ventral telencephalic area; Dp, posterior zone of dorsal telencephalic area; Hv, ventral zone of periventricular hypothalamus; Hd, dorsal zone of periventricular hypothalamus; Hc, central zone of periventricular hypothalamus; LH, lateral hypothalamic nucleus; ATN, anterior tuberal nucleus; PTN, posterior tuberal nucleus. Values represent median (n = 5). Wilcoxon rank sum test with Bonferroni’s correction (Vs, p = 0.024; Dp, p = 0.024; Hd, p = 0.024; PTN, p = 0.024 for vehicle versus ATP; Vs, p = 0.024; Hd, p = 0.048; PTN, p = 0.024 for vehicle versus alanine). ∗ p < 0.05.

(A) In situ hybridization with c-Fos cRNA probe on brain sections of zebrafish exposed to vehicle, ATP, and alanine. Vertical lines in the schematic zebrafish brain indicate the anterior-posterior positions of five coronal sections. Abbreviations for brain regions are as follows: Tel, telencephalon; TeO, optic tectum; Hy, hypothalamus; CCe, cerebellum. Red boxes indicate the locations of magnified views. Scale bar, 100 μm.

The odor information received by OSNs is then transmitted to the OB and represented as a topographic odor map on the two-dimensional array of olfactory glomeruli, spherical neuropil structures where the olfactory axons make synaptic connections with the second-order neurons. The zebrafish OB contains approximately 140 glomeruli of which 28 are identifiable and invariant across individuals []. To monitor the glomerular activation, we employed two different methods: pERK immunohistochemistry on whole-mount OB preparations ( Figures 2 B and S3 A) and G-CaMP calcium imaging in OMP:Gal4FF;UAS:G-CaMP7 or OMP:Gal4FF;SAGFF27A;UAS:G-CaMP7 transgenic zebrafish ( Figures 2 C and S3 B; Movie S2 ) []. A careful observation revealed that a single, large, identifiable glomerulus termed lG2 [] was specifically activated by adenine nucleotides (ATP, ADP, AMP) and adenosine, but not by other nucleotides, nucleosides, amino acids, or bile acids ( Figures 2 B and 2C and S3 A). Adenine and ribose were also ineffective, indicating that the minimal structural requirement for activating lG2 is adenosine. In contrast, alanine activated more anteroventrally located plural glomeruli (lGx) in the lateral OB that are distinct from lG2 ( Figure 2 B). These results are consistent with the earlier discovery in imaging studies using voltage- and Ca-sensitive dyes by Friedrich and Korsching []. When dose-response profiles of glomerular activation were compared among ATP, adenosine, and alanine by Caimaging, we found that the lG2 activation by ATP and adenosine was much more sensitive than the lGx activation by alanine by two orders of magnitude ( Figures 2 C and S3 B), corroborating the above-mentioned difference in effective concentrations for evoking attractive behavior ( Figures 1 D and S1 C).

Which type of OSNs is activated by ATP? In zebrafish nose, there are two major types (ciliated and microvillous) and two minor types (crypt and Kappe) of OSNs []. Zebrafish were exposed to ATP (nucleotide), alanine (amino acid), or taurocholic acid (bile acid), and the activated OSNs were detected with anti-phosphorylated ERK (pERK) antibody []. As previously reported [], alanine and taurocholic acid activated microvillous and ciliated OSNs that bear short and long dendrites, respectively ( Figure 2 A). In contrast, ATP stimulation induced ERK phosphorylation in a small number of extremely short dendrite-bearing pear-shaped OSNs that are located in the most superficial layer of the OE predominantly around the median raphe ( Figure 2 A). In spite of the superficial localization, these pear-shaped OSNs were positive for ciliated OSN markers such as OMP promoter-driven GFP, but negative for microvillous OSN markers such as TRPC2 promoter-driven gap-Venus ( Figure S2 ) []. Although crypt and Kappe OSNs are also located in the most superficial layer, both of them are negative for OMP promoter-driven GFP []. Thus, the morphological character and molecular expression of ATP-activated OSNs are unique and different from those of the four OSN types previously described.

(C) Ca 2+ imaging of OB glomeruli in OMP:Gal4FF;UAS:G-CaMP7 transgenic zebrafish. Top: representative Ca 2+ responses of lG2 upon stimulation with 10 μM ATP and related molecules. Scale bar, 50 μm. Bottom left graph: quantification of Ca 2+ increase in lG2. Values represent mean ± SEM (n = 3). Unpaired t test (adenosine, p = 0.015; AMP, p = 0.0088; ADP, p = 0.0082; ATP, p = 0.0025; ∗ p < 0.05, ∗∗ p < 0.01). Bottom right graph: dose-response relationship of glomerular Ca 2+ increase by ATP (red), adenosine (blue), and alanine (yellow, green). A response curve for the most sensitive lGx glomerulus to alanine is shown in yellow, while the averaged response of multiple lGx glomeruli to alanine is shown in green.

(B) pERK immunostaining of whole-mount OB. Top: whole-mount OB of 10 μM ATP-stimulated fish stained with anti-pERK (magenta) and anti-SV2 (green) antibodies. Bottom: lateral views of anti-pERK-labeled whole-mount OB of zebrafish exposed to various compounds. Adenine nucleotides and adenosine (10 μM) specifically activate lG2, whereas alanine (10 mM) activates multiple lateral glomeruli (asterisk), but not lG2. Closed arrowheads, pERK-positive lG2; open arrowheads, pERK-negative lG2. Abbreviations for glomerular clusters are as follows: dG, dorsal; dlG, dorsolateral; lG, lateral; mdG, mediodorsal; vmG, ventromedial; vpG, ventroposterior. Scale bar, 100 μm. (n = 3–6).

(A) ATP activates unique “pear-shaped” OSNs. pERK immunohistochemistry of zebrafish OE sections exposed to 10 μM ATP, 10 mM alanine, and 10 mM taurocholic acid (n = 3–5). The leftmost panel shows a low-magnification view of OE stimulated with ATP. Right panels are magnified views of OE stimulated with ATP (left), alanine (middle), and taurocholic acid (right). Red, pERK; cyan, DAPI. Scale bars, 100 μm (left), 50 μm (right).

We next examined behavioral response of zebrafish to ATP and adenosine in comparison with a well-known attractive odorant, alanine []. A single fish was acclimated in a test tank and then each compound at various concentrations was applied to one side of the tank. To quantify the degree of attraction, we calculated the preference index and the mean displacement scores for individual fish []. Zebrafish showed robust attractive responses to ATP and adenosine as well as to alanine ( Figures 1 B–1D and S1 Movie S1 ). Among those odorants tested, fish displayed more sensitive attraction to ATP and adenosine than alanine by two orders of magnitude ( Figures 1 D and S1 C). The attraction to ATP was completely abolished in anosmic fish in which the OE was surgically removed ( Figure 1 E), demonstrating that this behavior crucially depends on the olfaction. In contrast, the highest concentration of ATP was ineffective in attracting zebrafish ( Figure 1 D), which may be due to the activation of other sensory systems with peripheral ATP receptor (such as the trigeminal system) [] that evoke aversive response, resulting in a tug-of-war in behavioral outputs.

We first analyzed the composition and quantity of nucleotides and related molecules in the water containing brine shrimp (Artemia), an ordinary food for zebrafish in laboratories, by reverse-phase high-performance liquid chromatography (HPLC) ( Figure 1 A). Among the adenosine-related compounds (adenine, adenosine, AMP, ADP, and ATP), ATP was most abundantly present in the supernatant of brine shrimp-containing water. Therefore, we focused on ATP as a food-derived odor in the following behavioral, physiological, and molecular biological experiments.

(E) Anosmic fish show no attraction to ATP. Preference index (left) and mean displacement (right) are shown for sham-operated (n = 12) and OE-removed fish (n = 11). Wilcoxon signed-rank test for significant deviation from 0 (preference index, sham, p = 0.0068; mean displacement, sham, p = 0.0049; ∗∗ p < 0.01). Wilcoxon rank sum test for comparison between sham-operated and OE-removed fish (preference index, p = 0.027; mean displacement, p = 0.013; # p < 0.05).

(D) Concentration dependence of attractive response to ATP (top), adenosine (middle), and alanine (bottom) quantified as preference index (left) and mean displacement (right) (n = 9–11). Wilcoxon signed-rank test (preference index: ATP 1 μM, n = 11, p = 0.0098; ATP 10 μM, n = 11, p = 0.0098; adenosine 1 μM, n = 11, p = 0.001; adenosine 10 μM, n = 11, p = 0.001; adenosine 100 μM, n = 11, p = 0.001; alanine 100 μM, n = 9, p = 0.0039; mean displacement: ATP 1 μM, n = 11, p = 0.0049; ATP 10 μM, n = 11, p = 0.0098; adenosine 1 μM, n = 11, p = 0.001; adenosine 10 μM, n = 11, p = 0.001; adenosine 100 μM, n = 11, p = 0.001; alanine 100 μM, n = 9, p = 0.0078). ∗∗ p < 0.01.

(C) Top: positions of all individual fish (n = 11) plotted along the longitudinal axis of the tank every 1 s. ATP was applied at time 0 and remained in the tank thereafter. Middle: mean position (solid line) and SEM (shading) of the fish. Bottom: preference index in each time bin (30 s). Wilcoxon signed-rank test (post 0.5 to 1 min, p = 0.0098; post 1 to 1.5 min, p = 0.00098). ∗∗ p < 0.01, ∗∗∗ p < 0.001.

(B) A representative swimming path of a zebrafish upon ATP application. Individual panels represent swimming trajectory of a fish for every 30 s from 2 min before (left) and 2 min after (right) ATP application at time 0.

Discussion

In the present study, we found that a novel member of the adenosine receptor family, A2c, is an olfactory receptor expressed in the zebrafish OE. Furthermore, we revealed a pre-receptor event within the fish nostril, in which adenine nucleotides are actively converted into adenosine through enzymatic reactions. This sophisticated enzyme-receptor machinery should be beneficial for detecting ATP and its metabolites released from living organisms through a single olfactory receptor, thereby integrating the information into a specific glomerulus lG2.

To our knowledge, this is the first report demonstrating that the adenosine receptor plays a crucial role as an olfactory receptor. Based on the amino acid sequence homology and syntenic conservation ( Figure S4 ), we identified A2c gene orthologs in all the fish species (both freshwater and seawater fishes) and amphibian species whose genome sequences are available ( Figure 5 A; Table S2 ). This finding suggests that the A2c receptor is commonly used in aquatic lower vertebrates for detection of food source. In addition, the presence of A2c genes in sea lamprey and spotted gar indicates that the appearance of the first A2c gene occurred extremely early during the evolution of the vertebrate olfactory system. In contrast, the A2c gene is not present in reptiles, birds, or mammals, suggesting the loss of the A2c gene with the aquatic-to-terrestrial transition of vertebrate organisms during evolution.

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Korsching S.I. Crypt neurons express a single V1R-related ora gene. A2c and CD73 are expressed in a small population of OSNs with pear-shaped morphology. In spite of the expression of molecular markers of ciliated OSNs such as Golf, ACIII, and OMP [], these OSNs have an extremely short dendrite and are located in the superficial layer of OE. In addition to the ciliated and microvillous OSNs, zebrafish possess two minor types of OSNs, crypt cells and Kappe cells, both of which are located in the superficial layer []. However, the crypt and Kappe cells innervate two distinct mediodorsal glomeruli in the OB, mdG2 and mdG5, respectively [], which are different from lG2 innervated by the A2c-expressing OSNs. In addition, V1R4 is expressed in the crypt cells [], but not in the A2c-positive pear-shaped OSNs ( Figure 4 A). Based on these results, we propose designation of this fifth type of unique OSNs as the “pear OSNs.”

25 Reiten I.

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et al. Motile-cilia-mediated flow improves sensitivity and temporal resolution of olfactory computations. The A2c receptor recognizes only adenosine, but not ATP, related nucleotides or other nucleosides, whereas the lG2 glomerulus is activated by adenosine, AMP, ADP, and ATP. Our results indicate that ATP is enzymatically dephosphorylated and converted to adenosine by two GPI-anchoring ecto-nucletotidases, TNAP and CD73. TNAP in non-neuronal cells located close to the inlet of nose pit catalyzes the serial conversion of ATP to ADP, AMP, and adenosine, while CD73 co-expressed in A2c-positive pear OSNs dephosphorylates AMP to adenosine. How does ATP in the water environment efficiently reach the nose pit, enter into the nostril, and undergo subsequent enzymatic conversion to adenosine for activation of the A2c receptor? Recently, an interesting paper was published on the motile cilia-mediated directional flow in the zebrafish nose, which allows quick exchange of the content of the nasal mucus to facilitate the detection of odorants in stagnant environment []. We speculate that such a ciliary beating-based mechanism may be important for generating directional flow of ATP and its metabolites in the nose as well as effectively displacing mucus for highly sensitive responsiveness of the A2c receptor in vivo.

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Ngai J. The repertoire of olfactory C family G protein-coupled receptors in zebrafish: candidate chemosensory receptors for amino acids. The zebrafish OB contains nine glomerular clusters that are spatially segregated and invariant across individuals. The lateral cluster consists of dorsally located five identifiable glomeruli (lG1–lG5), ventrally located one identifiable glomerulus (lG6), and smaller, indistinguishable, multiple glomeruli (lGx) []. The present study revealed that adenosine and related nucleotides activate a single large glomerulus lG2 that is innervated by the pear OSNs. In contrast, amino acids activate multiple lGx in a combinatorial manner, which are innervated by microvillous OSNs []. Despite the fact that adenosine and amino acids bind different types of olfactory receptors (A2c and V2Rs) [] expressed in distinct types of OSNs (pear and microvillous), those axons innervate the nearby glomeruli in the lateral OB. Thus, it is likely that these lateral glomeruli, lG2 and lGx, might be responsible for relaying the information of food-associated olfactory cues synergistically. We hypothesize that topographically organized glomerular domains in the OB may play roles as functional units collecting ethologically relevant odorant or pheromone information and sending it to higher olfactory centers to evoke specific output responses.

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Cone R.D. Conserved neurochemical pathways involved in hypothalamic control of energy homeostasis. Beyond the OB, three brain regions (Vs, PTN, and Hd) are commonly activated by ATP and alanine. In contrast, Dp and a small population of neurons in the ventral-most Hv tend to be activated by ATP, but not alanine, suggesting a possibility of ATP-specific behavioral or physiological responses that we could not observe in the present study. The Dp and Vs are equivalent to the mammalian piriform cortex [] and central amygdala [], which receive direct inputs from the OB in fish [] and whose functions may be related to olfactory perception and emotion, respectively []. In addition, it has been recently reported that the central amygdala in mice mediates predatory hunting []. Thus, it is likely that the fish Vs may play a similar role in food searching upon activation by the olfactory stimuli. The PTN is a diencephalic nucleus that also receives massive inputs directly from the OB in zebrafish []. It is controversial which brain region in mammals corresponds to the fish PTN []. In sea lamprey, however, the PTN functions as a relay station transforming olfactory inputs into motor outputs []. Although mammalian homologs of the fish Hd and Hv cannot be precisely assigned at present, it is evident that the hypothalamic nuclei play a central role in feeding behavior []. Because the ventral Hv in zebrafish contains several types of neurons expressing feeding-related neuropeptides such as agouti-related peptide (AgRP) and proopiomelanocortin (POMC) [], we speculate that the ventral Hv activated by ATP might correspond to the arcuate nucleus, a feeding center, in mammals. Taken together, the activation of these brain regions by food-derived odorants leads to olfactory perception, emotion, motivation, and locomotion, all of which collectively and synergistically orchestrate the feeding behavior.