cAMP-independent responses of olfactory neurons in Xenopus laevis tadpoles and their projection onto olfactory bulb neurons

Abstract

We report on responses of olfactory receptor neurons (ORNs) upon application of amino acids and forskolin using a novel slice preparation of the olfactory epithelium of Xenopus laevis tadpoles. Responses were measured using the patch-clamp technique. Both amino acids and forskolin proved to be potent stimuli. Interestingly, a number of ORNs that responded to amino acids did not respond to forskolin. This suggests that some amino acids activate transduction pathways other than the well-known cAMP-mediated one. The differential processing of cAMP-mediated stimuli on the one hand and amino acid stimuli on the other was further elucidated by calcium-imaging of olfactory bulb neurons using a novel nose-olfactory bulb preparation of Xenopus laevis tadpoles. The projection pattern of amino acid-sensitive ORNs to olfactory bulb neurons differed markedly from the projection pattern of forskolin-sensitive ORNs. Olfactory bulb neurons activated by amino acids were located laterally compared to those activated by forskolin, and only a small proportion responded to both stimuli. The ensemble of neurons activated by forskolin was also activated by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) and the membrane-permeant cAMP analogue 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (pCPT-cAMP). We therefore conclude that sensory transduction of a number of amino acids is cAMP independent, and amino acid- and cAMP-mediated responses are processed differentially at the level of the olfactory bulb.

Figure 1. Slice of the olfactory mucosa and the olfactory bulb

A, image of the slice of the olfactory mucosa and the olfactory bulb. B, slice of the anterior part of the brain including the olfactory nerve (ON), the main olfactory bulb (MOB) and the accessory olfactory bulb (AOB) stained with propidium iodide. C, horizontal overview of the olfactory epithelium (PC, principal cavity and OE, olfactory epithelium). The neurons were backfilled through the nerve using biocytin-avidin staining (green fluorescence), and the slice was counterstained with propidium iodide (red fluorescence). D, higher magnification of C, showing ORNs with dendrites, dendritic knobs and cilia.

Figure 2. Odorant responses of different ORNs (stages 53 and 54) in the mucosa slice recorded in the on-cell configuration

The current traces from top to bottom show excitatory responses to the mixtures of long chain neutral (LCN), short chain neutral (SCN), alkaline (BAS), acidic (ACID) and aromatic (AROM) amino acids. In all of the submixtures the concentration of the individual amino acids was 200 μm.

Figure 3. Frequency histogram of spontaneous spiking activities of ORNs in the mucosa slice

Fifty-one ORNs (stages 49-54), recorded in the on-cell mode and tested for both forskolin and amino acids, were evaluated for this histogram.

Figure 4. Odorant responses of a single ORN in the mucosa slice from a tadpole (stage 53) recorded in on-cell configuration

The current traces from top to bottom show responses to: the mixture of amino acids (AA), the mixture of long chain neutral amino acids (LCN), l-methionine, the mixture of short chain neutral amino acids (SCN), l-glutamine, the mixture of alkaline amino acids (BAS) and to l-histidine. There was no response to the mixtures of aromatic (AROM) and acidic (ACID) amino acids. All amino acids were applied at a concentration of 200 μm. The traces are arranged according to the intensities of the responses to the different stimuli.

Figure 5. Correlation of odorant and forskolin responses

A, ORN (stage 53) excited by both l-glutamine (200 μm) and forskolin (50 μm). B, ORN (stage 51) excited by forskolin (50 μm) but not by the mixture of amino acids (AA, 200 μm). C, ORN (stage 54) excited by an amino acid (l-methionine, 200 μm) but not by forskolin (50 μm). D, occurrences of correlated and uncorrelated responses to forskolin and amino acids. Sixty-nine out of the 90 ORNs tested responded neither to amino acids nor to forskolin.

Figure 6. Comparison of changes of [Ca²⁺]_i in olfactory bulb neurons in response to stimulation of the olfactory mucosa with amino acids and forskolin

A, fluorescence image (excitation at 380 nm) of the olfactory bulb of a tadpole (stage 51) with outlines indicating the borders of the olfactory bulb, olfactory nerve, ventricle and the approximate borderlines between the mitral and granule cell layers and of the accessory olfactory bulb. Cell bodies of mitral and granule cells exhibit bright fluorescence. The box indicates the region of interest used for calcium imaging. B, ratio image at the time t + 0 s. C and D, spatial response patterns to stimulation with amino acids (100 μm, C, in red) and forskolin (100 μm, D, in green) expressed as stimulus-induced ratio changes. For identification of the location of olfactory bulb neurons, the ratio changes were superimposed on the fluorescence image shown in A. E, superimposed images of the responses to amino acids and forskolin. F, areas of overlap between the responses to stimulation with amino acids and forskolin. Note that only a small subset of neurons responded to both stimuli. G and H, time courses of calcium responses (expressed as ratio changes) in olfactory bulb neurons in the mitral cell layer to stimulation with amino acids (G) and forskolin (H). Three representative examples are shown in each case. The arrows indicate the time chosen for the spatial response patterns shown in the images in panels C -F.

Figure 7. Comparison of [Ca²⁺]_i responses in olfactory bulb neurons upon stimulation of the olfactory mucosa with amino acids, forskolin, IBMX and pCPT-cAMP

Background in A-D is as in the preceding figure. A, [Ca²⁺]_i activation pattern in response to the mixture of amino acids (100 μm, shown in red) superimposed on the background image. B-D, [Ca²⁺]_i activation patterns in response to forskolin (50 μm, B, green), IBMX (500 μm, C, blue) and pCPT-cAMP (2.5 mm, D, yellow) were superimposed on the image shown in A. The areas of overlap are shown in black.