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Nucleotides and amino acids are acknowledged categories of water-borne olfactory stimuli. In previous studies it has been shown that larvae of Xenopus laevis are able to sense amino acids. Here we report on the effect of ATP in the olfactory epithelium (OE) of Xenopus laevis tadpoles. First, ATP activates a subpopulation of cells in the OE. The ATP-sensitive subset of cells is almost perfectly disjoint from the subset of amino acid-activated cells. Both responses are not mediated by the well-described cAMP transduction pathway as the two subpopulations of cells do not overlap with a third, forskolin-activated subpopulation. We further show that, in contrast to amino acids, which act exclusively as olfactory stimuli, ATP appears to feature a second role. Surprisingly it activated a large number of sustentacular supporting cells (SCs) and, to a much lower extent, olfactory receptor neurons. The cells of the amino acid- and ATP-responding subsets featured differences in shape, size and position in the OE. The latencies to activation upon stimulus application differed markedly in these subsets. To obtain these results two technical points were important. We used a novel dextran-tetramethylrhodamine-backfilled slice preparation of the OE and we found out that an antibody to calnexin, a known molecular chaperone, also labels SCs. Our findings thus show a strong effect of ATP in the OE and we discuss some of the possible physiological functions of nucleotides in the OE.
Fig. 1. Stimulus-induced [Ca2+]i increases in individual olfactory receptor neurons (ORNs) in a slice of the olfactory epithelium (OE) of a Xenopus laevis tadpole.
(A) Overview of a horizontal slice of the OE of a Xenopus laevis tadpole (OE, olfactory epithelium; PC, principal cavity). The slice was stained with the calciumindicator
dye fluo-4 (green fluorescence, image acquired at rest). (B–D) The sequence of the three images shows the cells (black on grey background) of the same
slice responsive to application of ATP, amino acids and forskolin, respectively. Only cells showing a stimulus-correlated transient increase in calcium-dependent
fluorescence were counted. The time course of [Ca2+]i transients of the cells indicated by arrows (B–D) are shown in Fig. 2. (E) Schematic superposition of the
ATP-, amino acid- and forskolin-sensitive cells (shown in B–D). Cells sensitive to ATP (blue), amino acids (red), forskolin (green), ATP and amino acids (magenta),
and ATP and forskolin (cyan). (F) Occurrences of correlated and uncorrelated responses to ATP, amino acids and forskolin of all cells recorded plotted as a pie chart
(n ¼ 384 cells from 17 slices). The borders of the OE are traced for clarity in (A–E). ATP and amino acids were applied at a concentration of 200 lm, and forskolin
at a concentration of 100 lm. Scale bar, 20 lm.
Fig. 2. Stimulus-induced changes in calcium-dependent fluorescence of three individual cells in a slice of the olfactory epithelium. (A) Time courses of [Ca2+]i
transients of cell #1 (see Fig. 1B), evoked by the application of ATP, amino acids and forskolin. The traces show that this cell clearly responded to ATP, while it
showed no response to amino acids and forskolin. (B) Cell #2 (see Fig. 1C) responded to amino acids. No response to ATP and forskolin. (C) Cell #3 (see Fig. 1D)
responded to forskolin. No response to ATP and amino acids.
Fig. 3. Dextran-tetramethylrhodamine (d-TMR)-backfilled slices of the olfactory epithelium (OE) and correlation between cell type and responsiveness to ATP and
amino acids. (A) Slice of the OE of a Xenopus laevis tadpole where all cells responsive to application of ATP (black on grey background) are encircled.
(B) A second slice of the OE of a Xenopus laevis tadpole with its cells responsive to application of amino acids. In both slices only cells showing a stimuluscorrelated
transient increase in calcium-dependent fluorescence were counted. (C and D) d-TMR-backfilled olfactory receptor neurons (ORNs) of the slices shown in
(A and B), respectively. The contours of the cells responsive to ATP and amino acid (see A and B) were then superimposed onto these pictures. While in the slice
shown in (C) there is no overlap of d-TMR-backfilled ORNs and ATP-responsive cells, in the slice shown in (D) various cells responsive to amino acids overlap
d-TMR-backfilled cells. (E) The histograms summarize the results obtained in all of the d-TMR-backfilled slices tested for ATP and amino acids. While in eight
slices tested for their responsiveness to ATP just nine out of the 168 responsive cells were d-TMR-positive (blue bars; left-hand side), 32 out of the 52 cells sensitive
to amino acids (n ¼ 6 slices) were d-TMR-positive (red bars; right-hand side). (F) Example of one of the nine d-TMR-positive ORNs responsive to ATP (upper
part) and the respective time course of the [Ca2+]i transient of this cell evoked by the application of ATP (lower part). The borders of the OE are traced for clarity in
(A)–(D). ATP and amino acids were applied at a concentration of 200 lm in all of these experiments. Scale bars, 20 lm (C and D); 10 lm (F).
Fig. 4. Calnexin labels sustentacular supporting cells (SCs). (A–C) Image showing calnexin immunoreactivity (red fluorescence, A and C) of a biocytinbackfilled
⁄ avidin-stained (green fluorescence, B and C) slice of the olfactory epithelium (OE) of a Xenopus laevis tadpole. Note the localization of calnexin
immunoreactivity in the sustentacular cell layer and in cells in the basal cell layer (arrows). In the olfactory receptor cell layer only a few cells show positive calnexin
immunoreactivity (asterisks). The dashed lines in (A and B) indicate the border of the OE, the continuous line the approximate termination of the sustentacular cell
layer. (D) ATP-sensitive cells in an acute OE slice determined with fluo-4-calcium imaging. The dashed rectangle indicates one of the ATP-responding cells shown
at a higher magnification in (E). (F) Higher magnification of the dashed rectangle drawn in (C). The arrow marks an olfactory receptor neuron (ORN) dendrite.
The asterisk tags a typical calnexin-positive cell. (G) Typical sustentacular cell in the OE of Xenopus laevis tadpoles filled with biocytin through a patch pipette and
then stained with avidin. PC, principal cavity; SCL, sustentacular cell layer. Scale bars, 50 lm (A and D); 5 lm in (E–G).
Fig. 5. Latencies of activation upon stimulus application of cells responsive to ATP and amino acids in a slice of the olfactory epithelium (OE) of Xenopus laevis
tadpoles. (A) ATP-sensitive cells in an OE slice measured with fluo-4-calcium imaging. The dashed lines indicate the border of the mucosa. The responding cells
are numbered. (B) Amino acid-sensitive cells in the same OE slice as in (A). The responding cells are numbered. (C) Time courses of [Ca2+]i transients of the cells
marked in (A), ordered by increasing activation latencies. The straight vertical line going through all traces shows the arrival time of the stimulus to the OE. The
dotted line indicates the response latencies to ATP of the different cells. (D) Time courses of [Ca2+]i transients of the cells marked in (B). The straight vertical line
going through all traces shows the arrival time of the stimulus to the OE. PC, principal cavity. Scale bar, 50 lm.
