Manzini I and Schild D (2004), Classes and narrowing selectivity of olfactory ...

XB-ART-4052

J Gen Physiol 2004 Feb 01;1232:99-107. doi: 10.1085/jgp.200308970.

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Classes and narrowing selectivity of olfactory receptor neurons of Xenopus laevis tadpoles.

Manzini I , Schild D .

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In olfactory receptor neurons (ORNs) of aquatic animals amino acids have been shown to be potent stimuli. Here we report on calcium imaging experiments in slices of the olfactory mucosa of Xenopus laevis tadpoles. We were able to determine the response profiles of 283 ORNs to 19 amino acids, where one profile comprises the responses of one ORN to 19 amino acids. 204 out of the 283 response profiles differed from each other. 36 response spectra occurred more than once, i.e., there were 36 classes of ORNs identically responding to the 19 amino acids. The number of ORNs that formed a class ranged from 2 to 13. Shape and duration of amino acid-elicited [Ca2+]i transients showed a high degree of similarity upon repeated stimulation with the same amino acid. Different amino acids, however, in some cases led to clearly distinguishable calcium responses in individual ORNs. Furthermore, ORNs clearly appeared to gain selectivity over time, i.e., ORNs of later developmental stages responded to less amino acids than ORNs of earlier stages. We discuss the narrowing of ORN selectivity over stages in the context of expression of olfactory receptors.

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Species referenced: Xenopus laevis
Genes referenced: avd pc.1 sri tbxt.2

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	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 54, PC, principal cavity and OE, olfactory epithelium). The neurons were backfilled through the nerve using biocytin/avidin staining (green fluorescence), and then the slice was counterstained with propidium iodide (red fluorescence). Cell bodies of ORNs thus appear yellow. (B) Higher magnification of a part of the olfactory epithelium shown in A. Olfactory receptor neurons with dendrites, dendritic knobs, and cilia are easily recognizable. (C) Fluorescence image of a mucosa slice (stage 55, image acquired at rest) stained with Fluo-4. Cell bodies of amino acid–sensitive ORNs are encircled. The responses to amino acids of the ORNs indicated by arrows are shown in Fig. 2. (D–F) Sequence of three pseudocolored 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 C. (D) Image taken before the application of the amino acid mixture (t = 0 s). (E) Image taken at the peak of the response (t = 15 s). (F) Image taken after return to the base line fluorescence (t = 52 s). Bars: (A) 50 μm; (B–F) 20 μm.
	Figure 2. . Amino acid–induced changes in calcium-dependent fluorescence of four individual ORNs in a mucosa slice. (A) Time course of [Ca2+]i transients of an ORN (ORN #1 in Fig. 1 C) evoked by the application of amino acids. The traces show the responses to the mixture of 19 amino acids (AA), to the mixture of basic amino acids (BAS) and to L-histidine. No response to the mixtures of the long chain neutral (LCN), the short chain neutral (SCN), the aromatic (AROM), and the acidic (ACID) amino acids. No response to the remaining single amino acids of the BAS mixture. (B) ORN #2 (Fig. 1 C) responding to the mixture of AA, LCN, L-methionine, L-leucine (though slightly weaker), SCN, L-cysteine, L-glutamine, L-asparagine, 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 (Fig. 1 C) responding to the mixtures of AA, SCN, and to L-asparagine. No response to the other mixtures, nor to the remaining single amino acids of the SCN mixture. (D) ORN #4 (Fig. 1 C) responding to the mixture of AA and LCN, to L-isoleucine and to L-methionine, the mixture of SCN, and to L-glutamine. No response to the mixtures BAS, AROM, or ACID, nor to the remaining single amino acids of the responsive groups. All amino acids were applied at a concentration of 200 μM.
	Figure 3. . Different amino acids elicit distinguishable and highly reproducible [Ca2+]i transients in individual ORNs in a mucosa slice. Time courses of amino acid–induced [Ca2+]i transients of three individual ORNs (A–C). The traces show responses to successive applications of the amino acids L-cysteine and L-methionine (A); L-methionine, L-cysteine, and L-leucine (B); and L-glutamine and L-isoleucine (C). Shape and duration of the [Ca2+]i transients stayed approximately constant if the same amino acid was applied several times.
	Figure 4. . Response profiles of 283 olfactory receptor neurones to amino acids. 283 × 19 matrix representing the responses of the 283 ORNs each tested for 19 amino acids indicated by the common one-letter code for amino acids (first line). A 1 or a 0 in the matrix indicates whether or not a particular ORN responded to a particular amino acid (1 = response; 0 = no response). A response was assumed if the following two criteria were met: (a) the first two intensity values after stimulus arrival at the mucosa, I (t1) and I (t2), had to be larger than the maximum of the prestimulus intensities; (b) I (t2) > I (t1) with t2 > t1.
	Figure 5. . Histogram of response frequencies to amino acids. The histogram shows for each of the 19 amino acids used in how many ORNs (out of 283) a response was observed. Results are plotted as relative numbers, i.e., normalized to 283. As most of the ORNs (n = 283) responded to more than one amino acid, the sum of the response frequencies is much higher than 100%.
	Figure 6. . Histogram of the number of effective amino acids per ORN. Frequencies of ORNs (n = 283, 49 slices) that responded to a certain number n of amino acids (n out of 19 amino acids, Table I). Every possible number of effective stimuli, except 17, occurred. The distribution was, however, rather skewed, with small numbers of effective stimuli occurring much more often than larger numbers.
	Figure 7. . Histograms of the number of effective amino acids per ORN in two groups of ORNs of different stages. (A) Frequency distribution of ORNs (n = 143, 27 slices) responding to n stimuli, evaluated for earlier stages (stages 51, 52, and 53). (B) Frequency distribution of ORNs of later stages (n = 140, 22 slices, stages 54, 55, and 56). In the second subset (later stages) the individual ORNs clearly respond to less stimuli.

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