Glomerular input patterns in the mouse olfactory bulb evoked by retronasal odor stimuli

Abstract

Background: Odorant stimuli can access the olfactory epithelium either orthonasally, by inhalation through the external nares, or retronasally by reverse airflow from the oral cavity. There is evidence that odors perceived through these two routes can differ in quality and intensity. We were curious whether such differences might potentially have a neural basis in the peripheral mechanisms of odor coding. To explore this possibility, we compared olfactory receptor input to glomeruli in the dorsal olfactory bulb evoked by orthonasal and retronasal stimulation. Maps of glomerular response were acquired by optical imaging of transgenic mice expressing synaptopHluorin (spH), a fluorescent reporter of presynaptic activity, in olfactory nerve terminals.

Results: We found that retronasally delivered odorants were able to activate inputs to multiple glomeruli in the dorsal olfactory bulb. The retronasal responses were smaller than orthonasal responses to odorants delivered at comparable concentrations and flow rates, and they displayed higher thresholds and right-shifted dose-response curves. Glomerular maps of orthonasal and retronasal responses were usually well overlapped, with fewer total numbers of glomeruli in retronasal maps. However, maps at threshold could be quite distinct with little overlap. Retronasal responses were also more narrowly tuned to homologous series of aliphatic odorants of varying carbon chain length, with longer chain, more hydrophobic compounds evoking little or no response at comparable vapor levels.

Conclusions: Several features of retronasal olfaction are possibly referable to the observed properties of glomerular odorant responses. The finding that retronasal responses are weaker and sparser than orthonasal responses is consistent with psychophysical studies showing lower sensitivity for retronasal olfaction in threshold and suprathreshold tests. The similarity and overlap of orthonasal and retronasal odor maps at suprathreshold concentrations agrees with generally similar perceived qualities for the same odorant stimuli administered by the two routes. However, divergence of maps near threshold is a potential factor in perceptual differences between orthonasal and retronasal olfaction. Narrower tuning of retronasal responses suggests that they may be less influenced by chromatographic adsorption effects.

Figure 1

Schematic diagrams of the odor presentation system. A: Experimental set-up for orthonasal odor presentation. The olfactometer was connected to an odor nozzle and presented odor stimuli to the mouse’s nose. The upper cannula was connected to a flow meter and a vacuum pump, which controlled nasal airflow rates. The lower cannula accessed the lower trachea and remained open for normal breathing. B: Experimenatal set-up for retronasal odor presentation. The olfactometer was connected to a three-way valve and presented odor stimuli through the upper cannula. Our set-up permitted reciprocal switching of orthonasal and retronasal delivery modes in the same animal.

Figure 2

Odor-evoked spH responses to retronasal airflow in the dorsal olfactory bulb. A: Resting fluorescence image of the dorsal olfactory bulb showing spH-labeled glomeruli visible through thinned bone. No glomerular responses were observed in the absence of odor stimuli. Scale bar = 200 μm. B: Glomerular response maps to 50% v/v eugenol presented orthonasally or retronasally under different flow rates and stimulus durations. The pseudocolored images show % change in spH fluorescence from resting fluorescence intensity recorded before stimulus onset. Images were obtained from the same animal preparation. The three numbered white arrows indicate the glomeruli for which odor-evoked responses were analyzed in panels C and D. C: The amplitudes of orthonasal and retronasal responses at different flow rates and stimulus durations. Response amplitudes were quantified for glomeruli #1 – #3 corresponding to numbered arrows in panel B. Each value is the mean ± SE of 3 trials. D: Normalized response traces evoked by 10 s (left) and 30 s (right) stimulus pulses in glomerulus #1. The shaded regions indicate the duration of odor pulses. Red lines indicate orthonasal response traces at a flow rate of 150 ml/min. Blue and green lines indicate retronasal response traces at 150 and 300 ml/min, respectively.

Figure 3

Comparison of dose–response relationships for retronasal and orthonasal airflow. A: Odor maps from a single mouse, elicited with increasing concentrations of valeric acid (0.001% – 50% v/v). Retronasal stimuli were presented for 30 s at 300 ml/min (upper panels). Orthonasal stimuli were delivered for 10 s at 150 ml/min (lower panels). White arrow heads indicate glomeruli that exhibited responses at near-threshold concentrations for each airflow pathway. Scale bar = 200 μm. B: Spatial arrangement of activated glomeruli. Open circles show the positions of glomeruli whose responses were increased by 0.5% from resting fluorescence. Among open circles, numbered and colored circles indicate the numbered glomeruli that exhibited either or both retronasal and orthonasal responses at near-threshold concentrations in panel A (retronasal: green, #1 – #4; both: magenta, #5; orthonasal: red, #6 – #9). C: The dose–response relationship to retronasal (left) and orthonasal (right) odor. The response amplitudes were analyzed for glomeruli #1 – #9 that correspond to the number in panel B. Each value is the mean of 3 trials. Note: the apparent hotspot in upper right quadrant of 0.001% v/v retronasal map was a spurious signal from a blood vessel.

Figure 4

Overlap between orthonasal and retronasal response patterns. A: Glomerular activity patterns of 5 different odorants presented orthonasally or retronasally in the same animal. Eugenol (50% v/v for both stimulation modes), methyl isoeugenol (50% v/v for both), valeric acid (50% v/v for both), hexanal (0.1% v/v for orthonasal, 10% v/v for retronasal), methyl benzoate (50% v/v for both) were used for spH imaging. Orthonasal stimuli were delivered for 10 s at 150 ml/min. Retronasal stimuli were delivered for 30 s at 300 ml/min. Response maps for each stimulus mode are rendered in different colors (orthonasal: red, retronasal: green). Scale bar = 200 μm. B: Overlay of orthonasal and retronasal representations expressed in different color channels to indicate the glomeruli responding to one or both airflow pathways (orthonasal only: red; retronasal only: green; both: yellow).

Figure 5

Glomerular activity patterns of aliphatic aldehydes presented by the retronasal or orthonasal pathway. A: Response maps evoked by a homologous series of aliphatic aldehydes (C4 – C8) from a single mouse. The left-most panel shows the resting fluorescence image. Scale bar = 200 μm. Retronasal stimuli were delivered for 30 s at 300 ml /min (upper panels). Orthonasal stimuli were delivered for 10 s at 150 ml /min (lower panels). Percent dilutions in mineral oil are given in each panel. B: Overlay of retronasal and orthonasal response maps expressed in different color channels to indicate glomeruli responding to one or both airflow pathways (retronasal only: green, orthonasal only: red, both: yellow). C: Dot-plot representation of responses of the glomeruli numbered in panel B. Dots correspond to > 0.5% increase in response amplitudes. Black and gray filled circles show retronasal and orthonasal responses, respectively. D: Vapor pressure and log P of test aldehydes (log P = index of hydrophobicity).

Figure 6

Mapping of glomerular responses to aliphatic acids (C3 – C7). Odor maps were obtained from a single mouse. Retronasal stimulus was delivered for 30 s at 300 ml/min (upper panels), orthonasal stimulus for 10 s at 150 ml/min (lower panels). Percent dilutions in mineral oil are given in each panel. The resting fluorescence of olfactory bulb is shown (leftmost panel). Scale bar = 200 μm. No retronasal responses were found for hexanoic acid and heptanoic acid.