cAMP-independent olfactory transduction of amino acids in Xenopus laevis tadpoles

Abstract

Whether odorants are transduced by only one or more than one second messenger has been a long-standing question in olfactory research. In a previous study we started to address this question mainly by using calcium imaging in the olfactory bulb. Here, we present direct evidence for our earlier conclusions using the calcium imaging technique in the mucosa slice. The above question can now unambiguously be answered. We show that some olfactory receptor neurons (ORNs) respond to stimulation with amino acids with an increase of the intracellular calcium concentration [Ca2+]i. In order to see whether or not these responses were mediated by the cAMP transduction pathway we applied forskolin or the membrane-permeant cAMP analogue pCPT-cAMP to the olfactory epithelium. The ensemble of ORNs that was activated by amino acids markedly differed from the ensemble of neurons activated by forskolin or pCPT-cAMP. Less than 6 % of the responding ORNs showed a response to both amino acids and the pharmacological agents activating the cAMP transduction pathway. We conclude that ORNs of Xenopus laevis tadpoles have both cAMP-dependent and cAMP-independent olfactory transduction pathways and that most amino acids are transduced in a cAMP-independent way.

Figure 1. Slice of the olfactory epithelium of a Xenopus laevis tadpole and amino acid-induced [Ca²⁺]_i increases in individual ORNs in a mucosa slice

A, overview of a horizontal slice of the olfactory epithelium of a Xenopus laevis tadpole (stage 52, PC, principal cavity, OE, olfactory epithelium and ON, olfactory nerve). The neurons were backfilled through the nerve using biocytin-avidin staining (green fluorescence), and then the slice was counterstained with propidium iodide (red fluorescence). B, fluorescence image of a mucosa slice (stage 52, image acquired at rest) stained with fluo-4. Amino acid-sensitive ORNs are encircled. * ORNs showing high basal fluorescence levels at rest. The responses to amino acids of the ORNs indicated by arrows are shown in Fig. 2. C-E, sequence of three pseudocoloured images of the slice showing that stimulation with a mixture of amino acids (200 μM, each) transiently increases calcium-dependent fluorescence in the ORNs encircled in B. C, before the application of the amino acid mixture (time (t) 0 s). D, at the peak of the response (t, 16 s) and E, after return to the basal fluorescence level (t, 50 s). Scale bars: 50 μM in A and 20 μM in B-E.

Figure 2. Amino acid-induced changes in calcium-dependent fluorescence of three individual ORNs in a mucosa slice

A, time course of [Ca²⁺]_i transients of ORN 1 (see Fig. 1B) evoked by the application of amino acids. The traces show responses to the mixture of 19 amino acids (AA), to the mixture of short chain neutral amino acids (SCN) and to L-glycine. No response to the mixtures of the long chain neutral (LCN), the basic (BAS), the aromatic (AROM) and the acidic (ACID) amino acids. No response to the remaining single amino acids of the SCN mixture. B, ORN 2 (see Fig. 1B) responded to the mixture of AA, the mixture of LCN, to L-methionine and, though slightly weaker, to L-isoleucine, the mixture of SCN, to L-alanine, the mixture of BAS and to L-arginine. No response to the mixtures AROM or ACID, nor to the remaining single amino acids of the responsive groups. C, ORN 3 (see Fig. 1B) responded to the mixtures of AA and LCN; to L-leucine and L-methionine the mixture of SCN; to L-cysteine, L-alanine and L-threonine the mixture of BAS; to L-arginine and, though slightly weaker, to L-histidine the mixture of AROM and to L-tryptophan. No response to the ACID mixture, nor to the remaining single amino acids of the responsive mixtures. All amino acids were applied at a concentration of 200 μM.

Figure 3. Influence of TTX on odorant-induced [Ca²⁺]_i transients, spike-associated currents and sodium currents of Xenopus laevis tadpole ORNs

A, L-glutamine (200 μM) -induced [Ca²⁺]_i transient of an individual ORN (stage 53) of a mucosa slice. B, 5 min after the addition of 2 μM TTX to the bath solution the L-glutamine-induced [Ca²⁺]_i transient was clearly smaller but still present. With TTX the slope of the transient was smaller. C, after a wash-out time of 12 min the L-glutamine-induced [Ca²⁺]_i transient recovered completely. D, relative decrease of odorant-induced [Ca²⁺]_i transients after addition of TTX (2 μM) to the bath solution plotted as a histogram (n = 59 ORNs). E, current traces showing spike-associated currents of an ORN (stage 54) of a mucosa slice recorded in the on-cell configuration of the patch-clamp technique. Less than 15 s after the addition of TTX (2 μM) to the bath solution (see arrow in the upper trace) the spike-associated currents are completely blocked. As long as TTX was present in the bath solution the spike-associated currents did not recover (middle trace). Thirteen minutes after the beginning of wash-out the spike-associated currents are almost completely recovered (bottom trace). F, voltage-activated sodium currents of an ORN in the slice preparation recorded in the voltage-clamp configuration of the patch-clamp technique. To block potassium currents, an intracellular solution containing caesium instead of potassium was used (see solutions in Methods). The holding potential was -80 mV. The current responses were evoked by depolarizing voltage steps to -30 mV given every 3 s. After the first depolarizing step TTX (2 μM) was added to the bath solution. 15 s after the beginning of TTX application the current was completely blocked. G, recovery of the current after 9 min of wash-out.

Figure 4. Comparison of changes of calcium-dependent fluorescence in olfactory receptor neurons of a mucosa slice in response to stimulation with forskolin, pCPT-cAMP and amino acids

Sequences of pseudocoloured images of a mucosa slice (stage 53) showing that stimulation with forskolin (50 μM, A-C), pCPT-cAMP (2.5 mM, D -F) and the mixture of amino acids (200 μM, each, G-I) transiently elevates calcium-dependent fluorescence in two different ensembles of ORNs (encircled in B, E and H). The upper images show the fluorescence images before application (t 0 s), the images in the middle at the peak of the response (t 30 s in B and E, t 16 s in H) and the bottom images after return to the basal fluorescence levels (t 230 s in C and F, t 60 s in I). J, schematic diagram showing superimposition of the forskolin and pCPT-cAMP-sensitive ORNs and the amino acid-sensitive ORNs (encircled in B, E and H). ORNs sensitive to forskolin and pCPT-cAMP (green), to amino acids (red) and both (yellow). Only one ORN showed a response to both stimuli. The responses of the ORNs indicated with arrows are shown in Fig. 5. K, occurences of correlated and uncorrelated responses to forskolin and amino acids plotted as a histogram (n = 44 slices). Scale bar 20 μM.

Figure 5. Time courses of calcium-dependent fluorescence changes in ORNs upon stimulation with amino acids, forskolin and pCPT-cAMP

A, ORN 1 (see Fig. 4J) was responsive to L-asparagine (red trace) but insensitive to forskolin (green trace) and pCPT-cAMP (black trace). B, ORN 2 (see Fig. 4J) was responsive to forskolin and pCPT-cAMP (green and black trace, respectively) but insensitive to the mixture of amino acids (red trace). C, ORN 3 (see Fig. 4J) responded to all three stimuli applied (L-alanine, red trace; forskolin, green trace and pCPT-cAMP, black trace).Amino acids, forskolin and pCPT-cAMP were applied at a concentration of 200, 50 and 2.5 mM, respectively.