Uncoupling stimulus specificity and glomerular position in the mouse olfactory system

Abstract

Sensory information is often mapped systematically in the brain with neighboring neurons responding to similar stimulus features. The olfactory system represents chemical information as spatial and temporal activity patterns across glomeruli in the olfactory bulb. However, the degree to which chemical features are mapped systematically in the glomerular array has remained controversial. Here, we test the hypothesis that the dual roles of odorant receptors, in axon guidance and odor detection, can serve as a mechanism to map olfactory inputs with respect to their function. We compared the relationship between response specificity and glomerular position in genetically-defined olfactory sensory neurons expressing variant odorant receptors. We find that sensory neurons with the same odor response profile can be mapped to different regions of the bulb, and that neurons with different response profiles can be mapped to the same glomeruli. Our data demonstrate that the two functions of odorant receptors can be uncoupled, indicating that the mechanisms that map olfactory sensory inputs to glomeruli do so without regard to stimulus specificity.

Fig. 1. Screening M71 and M72-OSNs with odorant mixtures

(A) Diagram of the experimental approach. OSNs that express a defined OR are labeled with GFP in gene-targeted mice. The coding sequence for tauGFP (green) is inserted after the intact OR coding sequence (grey box) and downstream of an internal ribosome entry site (IRES). Confocal image shows green fluorescent OSNs in the nasal cavity. Anterior (a) and dorsal (d) are indicated. Scale bar = 500 μm. Diagram of the recording configuration (right). Perforated patch recordings were made from the dendrites of semi-intact OSNs. (B) Responses elicited by 35 odorant mixtures tested at 50μM for each odorant in the mixture. + indicates repeatable response, ± small unreliable response, − negative response, ND not tested. (Right) Typical current traces from M71 and M72 OSNs elicited by the odorant mixtures. Identified ligands from the mixes are shown in bold.

Fig. 2. M71 and M72 have distinct response profiles to individual odorants

(A) Current recordings in M71 and M72 OSNs in response to acetophenone and its analogues at 10 μM. (B) Dot plot showing the average response of M71 and M72 OSNs to a set of individual odorants. Size of the dot is proportional to current amplitude. Data are averages from ≥3 OSNs. All odorants were 10 μM except 2-amino acetophenone (1 μM). Odorant structures are shown (right).

Fig. 3. Analyzing glomerular position and response specificity in M71/M72 hybrids

(A) Schematic of the ORs M71 and M72 and two Hybrids D and J showing the location of the amino acid substitution. M71-derived sequences are illustrated as red, M72 as blue. (B) Confocal images of the dorsal bulbs in mice expressing M71-HybJ and M71 (top panels), or M71-HybJ and M72 (bottom panels). Axons expressing M71-HybJ (green) form a glomerulus that is close to, but distinct from, M71 glomeruli (red). The M71-HybJ glomeruli are also separate from M72. (C) Confocal images of the dorsal bulbs in mice expressing M71-HybD and M71. Axons expressing M71-HybD (green) project with axons expressing M71 (red). (D) Average dose-response data for M72-OSNs and M71-HybJ-OSNs to the M72-selective ligand, methyl salicylate (top) or the M71/M72 common ligand, 4-methyl acetophenone (bottom). Data are mean ± SEM. EC₅₀ values and number of recorded OSNs are indicated. (E) Average dose response data for M71, M72, and M71-HybD to three different odorants: the M71/M72 common ligand 4-methyl acetophenone, and the M71-selective ligands ethyl maltol and coumarin (mean ± SEM). EC₅₀ values and number of recorded OSNs are indicated. (F) Dot plot showing the average response amplitudes elicited by a panel of odorants in OSNs expressing M71, IRES-M71, M72, M71-HybJ and M71-HybD alleles. Each row shows responses for a given odorant. The size of the dot is proportional to the response amplitude. Odorants were presented at 10 μM, except 2-aminoacetophenone (1μM).

Fig. 4. Axon guidance and odorant specificity in inter-class odorant receptor swaps

(A) Confocal images of the dorsal bulbs in mice expressing M72 (left) and M72→S50 (right) showing projections to glomeruli in different domains of the dorsal bulb. (B) Response amplitudes to a set of odorants in M72- (n=5) and M72→S50 (n=5) OSNs. Response profiles are not significantly different. (C–D) Average dose-response data for M72- and M72→S50 OSNs to two known M72 ligands, methyl salicylate and acetophenone (mean ± SEM). Measured EC₅₀ values and number of OSNs recorded are indicated and are not statistically different between the two populations (t-test, p>0.05).

Fig. 5. Axon guidance and odorant specificity with reduced odorant receptor expression

(A) Diagram of M71 and IRES-M71 alleles. IRES-M71 expressing cells are labeled with GFP and RFP. (B) Confocal image of the medial olfactory bulb in a mouse expressing M71 (green) and IRES-M71 (yellow) mutations showing dramatic anterior shift in glomerular position with the IRES mutation. Distance between glomeruli is 1.52mm. (C) Confocal images of M71 (green) and IRES-M71 (green and red) OSNs stained with an antibody against M71 (blue). M71 expression is high in the dendritic knob of M71 OSNs. Images were taken from the sections derived from the same tissue using identical imaging settings. Scale bar = 20 μm. (D) Quantification of mean pixel values from M71 fluorescence measured from knobs and cilia in epithelial sections. M71 = 1,436 ± 130 (SEM, n=34 cells); IRES-M72 = 454 ± 75 (n= 22 cells). M71 expression is reduced by 68% (p<0.01, t-test). (E) Average dose-response data for M71- and IRES-M71 OSNs showing no difference for 2-amino acetophenone and a shift for 4-methyl acetophenone (M71 data same as in Fig. 3E).