Lack of TRPM5-Expressing Microvillous Cells in Mouse Main Olfactory Epithelium Leads to Impaired Odor-Evoked Responses and Olfactory-Guided Behavior in a Challenging Chemical Environment

Odorants and pheromones evoke comparable EOG responses in control and Skn-1a^-/- mice housed under our standard conditions. A, Schematic drawing of a mouse heminose, showing the region where the EOG responses were recorded in the olfactory turbinate II. MOE, main olfactory epithelium; OB, olfactory bulb; VNO, vomeronasal organ; SO, septal organ. B, C, Representative images of MOE from control and Skn-1a^-/- mice, respectively, approximately corresponding to the region of EOG recording. The sections were stained with DAPI and imaged with additional weak transmitted light to review the morphology of the MOE in the area of olfactory turbinate II (OT II). Scale bar: 100 µm. D, Representative EOG traces. E, Average peak EOG responses to various stimuli (n = 6, mean ± SEM). Chemical stimuli were applied in exactly the same sequence for EOG recordings for both control and Skn-1a^-/- mice. The EOG responses to various odorants and pheromones at 100 µm were recorded first, followed by responses to the adenylyl cyclase activator forskolin (1 µm). Finally, the EOG responses to 2,5-dimethylpyrazine (2,5-DMP) and 2-heptanone (10 and 200 µm) were recorded. There was no significant difference in EOG amplitude in response to the same odorant between Skn-1a^-/- and control mice, except 2-heptanone at 200 µm (*, p < 0.05, t test, n = 6, mean ± SEM).

EOG responses in chemical-exposed Skn-1a^-/- mice are significantly reduced. A, Average peak EOG responses to odorants and pheromones at 100 µm in control mice. There was no significant difference in the response amplitude to the same stimuli between water- and chemical-exposed groups (p > 0.05, t test, n = 15 and 16, respectively). B, Average peak EOG responses to odorants at 100 µm in Skn-1a^-/- mice. The EOG responses obtained from the chemical-exposed group were significantly smaller than those from the water-exposed group (*, p < 0.05 t test, n = 15 and 18, respectively). C–F, Normalized EOG responses to 2-heptanone or 2,5-DMP at various concentrations (10, 100, and 200 µm), which are presented relative to the values of EOG responses at 200 µm of the same animals. There was no significant difference in the dose-dependent responses between the water- and chemical-exposed groups in control mice (p > 0.05, n = 4–6). In Skn-1a^-/- mice, normalized responses at 10 µm in chemical-exposed group are significantly smaller than responses of the vehicle group (*, p < 0.05, t test, n = 5–6).

Immunohistochemical examination of the posterior MOE in control and Skn-1a^-/- mice after 2-wk exposure. Posterior MOE sections at approximately the same region where EOG was recorded were obtained from control and Skn-1a^-/- mice exposed to either water or chemicals for 2 wks. The sections were immunoreacted with antibodies against GAP43 and OMP and stained with DAPI. Confocal images were taken from olfactory turbinate II. A–E, Control, water-exposed. F–J, Control, chemical-exposed. K–O, Skn-1a^-/- mouse, water-exposed. P–T, Skn-1a^-/- mouse, chemical-exposed. DAPI staining (blue; A, F, K, P); GAP43 immunoreactivity (red; B, G, L, Q); OMP immunoreactivity (cyan; C, H, M, R); GFP-positive TRPM5-MCs (green; D and I only, control mice); overlay of GAP 43, OMP, and GFP (E, J, O, T). Similar morphology and marker expression were found in four groups of mice, indicating there was no obvious tissue damage in both control and Skn-1a^-/- mice after chemical exposure in the posterior MOE regions where EOG recordings were performed. Scale bar: 50 µm.

Impaired olfactory ability to locate buried food in Skn-1a^-/- mice after chemical exposure. A, Photographs of the experimental setting for the buried food test. Left panel: side view, showing 5- to 6-cm wood chip bedding over a buried cookie piece (indicated by an arrow). Right panel: top view before a mouse began to search for the piece of cookie. B, C, Plot of the average time (latency) required for mice to locate the buried cookie. The latency was not significantly different between control and Skn-1a^-/- mice housed under our standard conditions (B; n = 14 and 13, WT and Skn-1a^-/-, respectively). After chemical exposure, Skn-1a^-/- mice took a significantly longer time to locate the buried cookie compared with the water-exposed group (*p < 0.05, t test, n = 14 and 9, respectively). There was no difference between the water- and chemical-exposed groups of control mice (C; n = 9 and 12, respectively).

Olfactory preference toward urine of the opposite sex is compromised in chemical-exposed Skn-1a^-/- mice. A, Schematic drawing of the T-maze apparatus used to test olfactory preference for urine. A single mouse was placed in the starting arm of the T-maze for a 5-min acclimation period before the gate was opened, which allowed the mouse to enter either arm. B–E, Plots of total sniff duration. Both control and Skn-1a^-/- male mice displayed a strong preference for the female urine samples before exposure (*, p < 0.05, **, p < 0.01, paired t test, n = 5–8). Control mice retained their strong preference for urine samples after exposure to either water or chemicals (*, p < 0.05, **, p < 0.01, paired t test, n = 5–8). In contrast, only the water-exposed Skn-1a^-/- mice maintained the strong preference (**, p < 0.01, paired t test, n = 6). Chemical-exposed Skn-1a^-/- mice no longer significantly preferred urine over water (p = 0.316, paired t test, n = 7). F, G, Plots of sniff preference (ratio of urine sniff duration over total sniff duration). No significant difference was found in the control mice pre- and post-exposure (p = 0.113 and 0.120, respectively, t test), or the pre-exposure Skn-1a^-/- mice group (p = 0.207, t test). However, a significant reduction was found in Skn-1a^-/- mice of chemical-exposed group compared with the water-exposed group (*, p < 0.05, t test).