Figure 2. Primary Olfactory Pathway Activated by ATP and Related Molecules

(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).

(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).

(C) Ca2+ imaging of OB glomeruli in OMP:Gal4FF;UAS:G-CaMP7 transgenic zebrafish. Top: representative Ca2+ responses of lG2 upon stimulation with 10 μM ATP and related molecules. Scale bar, 50 μm. Bottom left graph: quantification of Ca2+ 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 Ca2+ 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.

Higher Brain Centers Activated by ATP and Alanine

(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.

(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.

ATP Activates OSNs Expressing a Novel Adenosine Receptor A2c

(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.

(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.

(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.

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

(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.

(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.

(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).

ATP activates a unique type of OSNs with pear-shaped morphology. Related to Figure 2. pERK immunohistochemistry of OE sections from OMP:GFP (top) and TRPC2:gap-Venus (bottom) transgenic zebrafish exposed to 10 μM ATP (n=2). ATP induces ERK phosphorylation specifically in pear-shaped OSNs that are OMP:GFP-positive, but TRPC2:gap-Venus-negative. Although these pear-shaped OSNs exhibits several hallmarks of ciliated OSNs (OMP:GFP, Golf, ACIII), they do not have a long dendrite and are located at the very apical layer of the OE. Scale bar, 5 μm.

Activation of distinct glomeruli by nucleotides, amino acids and, bile acids. Related to Figure2. (A) pERK immunostaining of whole-mount OB. Zebrafish were exposed to nucleotide mixture (ATP, IMP, TTP, 10 mM each), amino acid mixture (alanine, cysteine, histidine, lysine, methionine, phenylalanine, tryptophan, valine, 10 mM each), and bile acid mixture (glycocholic acid, taurocholic acid, taurodeoxycholic acid, 10 mM each) and their brains were subjected to immunohistochemistry with anti-pERK antibody. Amino acid mixture activates multiple glomeruli in the lateral cluster (lGx), while bile acid mixture activates dorsal and ventromedial glomerular clusters (dG and vmG). lG2 (arrowheads) is activated by nucleotide mixture, but not by amino acid and bile acid mixtures. Scale bars, 100 μm. (B) Ca2+ imaging of OB glomeruli in OMP:Gal4FF;SAGFF27A;UAS:G-CaMP7 transgenic zebrafish. Representative images of Ca2+ responses in lG2 and lGx upon stimulation with ATP, adenosine and alanine, respectively. Scale bar, 50 μm

TNAP is expressed in non-neuronal cells predominantly located in the anterior OE close to the inlet naris. Related to Figure 6.
(A) Double fluorescence in situ hybridization with cRNA probes for TNAP (green) and OMPs, TRPC2s, or V1Rs (magenta) on OE sections. No overlap is observed for TNAP signals with any OSN markers, indicating that TNAP is expressed in non-neuronal cells in the OE (n=3). Scale bars, 20 μm.
(B) Distribution of TNAP- and A2c-expressing cells in the zebrafish OE. Top left, a schematic diagram showing water flow through the zebrafish OE. Top right, a representative in situ hybridization image of TNAP expression in an OE section. Each OE section was divided into 10 compartments along the longitudinal axis and the numbers of TNAP and A2c-positive cells were counted. Bottom, a graph showing distribution of TNAP (red)- and A2c (blue)-expressing cells in the OE (n=3). Note that TNAP-expressing cells are predominantly located in the vicinity of the OE inlet, whereas A2c-expressing OSNs are observed in the middle portion between the inlet and outlet.

CD73/TNAP inhibitors and A2c antagonist attenuate the activation of lG2, but not other glomeruli. Related to Figure 7.
(A) Effects of TNAP and CD73 inhibitors (MLS0038949 and AMPCP) on glomerular activation by amino acid mixture, bile acid mixture and ATP in OMP:Gal4FF;SAGFF27A;UAS:G-CaMP7 transgenic zebrafish. Left panels, representative Ca2+ responses of the glomeruli (images and traces) upon stimulation with amino acid mixture (alanine, cysteine, histidine, lysine, methionine, phenylalanine, tryptophan, valine; 10 μM each) (top row), bile acid mixture (glycocholic acid, taurocholic acid, taurodeoxycholic acid; 10 & mu;M each) (middle row) and 1 μM ATP (bottom low) in the presence or absence of 0.5 mM MLS0038949 and 0.25 mM AMPCP. Amino acid mixture, bile acid mixture and ATP activate lGx, dG and lG2, respectively. Scale bar, 50 μm. Right graph, quantification of Ca2+ increase in the glomeruli. Values represent mean ± s.e.m. (n=3). Values for ATP are the same data from Figure 7A. Unpaired t-test (ATP, p=0.00000081). ***p < 0.001.
(B) Effects of XAC on 10 μM adenosine in A2c-, A2aa-, A2ab-, and A2b-expressing CHO-K1 cells. Antagonistic actions of XAC are observed for zebrafish A2c, A2aa and A2ab, but not A2b. IC50 values of XAC for A2c, A2aa, A2ab are 25.9 μM, 48.5 μM and 20.2 μM, respectively.
(C) Effect of A2c antagonist XAC on glomeruli activation by alanine, cadaverine and ATP in OMP:Gal4FF;SAGFF27A;UAS:G-CaMP7 transgenic zebrafish. Left panels, representative Ca2+ responses of the glomeruli (images and traces) upon stimulation with 10 μM alanine (top row), 10 μM cadaverine (middle row) and 1 μM ATP (bottom low) in the presence or absence of 10 μM XAC. Alanine, cadaverine and ATP activate lGx, dlG5 and lG2, respectively. Scale bar, 50 μm. Right graph, quantification of Ca2+ increase in the glomeruli. Values represent mean ± s.e.m. (n=3). Values for ATP, alanine and cadaverine are the same data from Figure 7C. Unpaired t-test (ATP, p=0.0016). ***p < 0.001.