A pharmacological profile of the aldehyde receptor repertoire in rat olfactory epithelium

Abstract

Several lines of evidence suggest that odorants are recognized through a combinatorial process in the olfactory system; a single odorant is recognized by multiple receptors and multiple odorants are recognized by the same receptor. However few details of how this might actually function for any particular odour set or receptor family are available. Approaching the problem from the ligands rather than the receptors, we used the response to a common odorant, octanal, as the basis for defining multiple receptor profiles. Octanal and other aldehydes induce large EOG responses in the rodent olfactory epithelium, suggesting that these compounds activate a large number of odour receptors (ORs). Here, we have determined and compared the pharmacological profile of different octanal receptors using Ca(2+) imaging in isolated olfactory sensory neurones (OSNs). It is believed that each OSN expresses only one receptor, thus the response profile of each cell corresponds to the pharmacological profile of one particular receptor. We stimulated the cells with a panel of nine odorants, which included octanal, octanoic acid, octanol and cinnamaldehyde among others (all at 30microM). Cluster analysis revealed several distinct pharmacological profiles for cells that were all sensitive to octanal. Some receptors had a broad molecular range, while others were activated only by octanal. Comparison of the profiles with that of the one identified octanal receptor, OR-I7, indicated several differences. While OR-I7 is activated by low concentrations of octanal and blocked by citral, other receptors were less sensitive to octanal and not blocked by citral. A lower estimate for the maximal number of octanal receptors is between 33 and 55. This large number of receptors for octanal suggests that, although the peripheral olfactory system is endowed with high sensitivity, discrimination among different compounds probably requires further central processing.

Figure 1. Panel of odorants used to characterize the pharmacological profiles of receptors for octanal

The abbreviation, given for each compound, is that used in the subsequent figures.

Figure 2. Pharmacology of OR-I7-expressing cells and cells expressing other receptors for aldehydes

A, comparison of the responses to octanal and other odorants in cells expressing OR-I7. For this and the following figures odorants were applied for 8s, beginning at the time indicated by the arrow. Cells were activated by C7–C11 aldehydes (a) and the unsaturated aldehydes, CTR, 2-TO and 2,4-OD, but not by CIT, TMO, OAC and OOL (b). Ac, activity profile for a GFP– cell. This cell responded to octanal and 2-TO, but it also responded to OAC and OOL, while it did not respond to CTR and 2,4-OD. All compounds were tested at 30μm, except OAC and OOL (at 1mm). The calibration bar is 6%ΔF/F (vertical) and 1min (horizontal) and is the same for b and c. B, dose–response-relation for OAL in cells expressing OR-I7 (•) and a GFP– cell (▪). Responses were normalized to the largest response (10 and 100μm, respectively) and fitted to the Hill equation. Responses in OR-I7 are shifted to the left, indicating greater sensitivity for octanal. Inset, dose-dependent increases in calcium signal induced by OAL in a cell expressing OR-I7. The calibration bar is 4%ΔF/F (vertical) and 1min (horizontal).

Figure 3. Aldehyde receptors can discriminate closely related compounds

Pharmacological profiles of 59 GFP– cells, tested with a panel of 9 aldehydes. Responses are indicated by a red circle and no response, by a black, smaller circle. In this group only cell 4, outlined by a rectangle, exhibited the pharmacological profile of OR-I7; it was activated by all aldehydes tested except CIT. Other cells exhibited varying degrees of sensitivity. The lower section of the panel shows a group of 25 cells that did not respond to OAL, but responded to other aldehydes in the panel. These cells were all rather narrowly tuned. All compounds were tested at 30μm.

Figure 4. The isomers geranial and neral reduce the responses to octanal at some aldehyde receptors

A, GER (top traces) and NER (bottom traces) reversibly reduced the responses to octanal in cells expressing OR-I7. Both isomers were tested at 100μm, and in the cells shown here they did not generate a response (see Methods). Calibration bar, 6%ΔF/F (vertical) and 1min (horizontal). B, not all receptors for aldehydes were blocked by the isomers. Both isomers almost completely reduced the response to octanal in GFP+ cells (filled bars). In contrast, in GFP– cells, GER produced a small response and failed to inhibit the response to octanal (hatched bars; see text).

Figure 5. Cells exhibit distinct response profiles to a test panel of odorants

Odorant responses of a selected group of cells stimulated with a panel of 9 odorants. Responses to the different odorants were compared to a test application of OAL. Cells exhibited various profiles of activity, from narrowly tuned (cell 47) to broadly tuned (cell 1). In a few cells some of the components in the panel produced larger responses than octanal (i.e. CIN in cells 1 and 8). All odorants were tested at 30μm. For all cells the calibration bar is 6%ΔF/F (vertical) and 1min (horizontal).

Figure 6. Cluster analysis reveals several aldehyde receptor types

Cluster analysis of 55 octanal responding cells. Cell responses were transformed into a binary profile, which was then used for the cluster analysis (see Methods). Responses are indicated in red and no response in black. There were 33 distinct response profiles, and 10 of these profiles appeared in more than one cell. A few cells responded to all the odorants (cluster A), and a large number only to OAL (cluster D). Cluster C was stimulated only by octanal and TMO, while cell 27 had the expected profile of OR-I7. All odorants were tested at 30μm.

Figure 7. Selectivity of aldehyde receptors is maintained at different concentrations

Cluster analysis for cells tested, at 30μm (red) or 300μm (yellow), with a selected group of compounds from the panel. The number of cells included in the analysis is indicated by the labelled grid at the left of each panel, 178 cells in A and 146 cells in B. A, cluster analysis of the response profiles to 8-carbon compounds with different degrees of unsaturation and branching. All of the clusters, except one, included cells from both groups (30 or 300μm). B, cluster analysis of the response profiles to 8-carbon compounds with different functional groups. Cells that responded only to OOL and OAC were not observed at either concentration.