Olfactory marker protein is critical for functional maturation of olfactory sensory neurons and development of mother preference

Abstract

Survival of many altricial animals critically depends on the sense of smell. Curiously, the olfactory system is rather immature at birth and undergoes a maturation process, which is poorly understood. Using patch-clamp technique on mouse olfactory sensory neurons (OSNs) with a defined odorant receptor, we demonstrate that OSNs exhibit functional maturation during the first month of postnatal life by developing faster response kinetics, higher sensitivity, and most intriguingly, higher selectivity. OSNs expressing mouse odorant receptor 23 (MOR23) are relatively broadly tuned in neonates and become selective detectors for the cognate odorant within 2 weeks. Remarkably, these changes are prevented by genetic ablation of olfactory marker protein (OMP), which is exclusively expressed in mature OSNs. Biochemical and pharmacological evidence suggests that alteration in odorant-induced phosphorylation of signaling proteins underlie some of the OMP(-/-) phenotypes. Furthermore, in a novel behavioral assay in which the mouse pups are given a choice between the biological mother and another unfamiliar lactating female, wild-type pups prefer the biological mother, while OMP knock-out pups fail to show preference. These results reveal that OSNs undergo an OMP-dependent functional maturation process that coincides with early development of the smell function, which is essential for pups to form preference for their mother.

Figure 1.

Wild-type but not OMP^−/− MOR23 neurons develop faster response kinetics during the first month. A, Analysis of odorant-induced transduction currents under voltage-clamp mode. The latency (1) is the time between the onset of the stimulus and the starting point of the response. The rise time (2) is the time it takes for the current to reach 90% of the peak (3) from the starting point of the response. The decay time (4) is the time it takes for the current to return to 10% of the peak from the peak. Since some responses take minutes to return to the baseline, a “residual” current (5) is measured 10 s after the stimulation. B, Inward currents (normalized to the same peak) were elicited by lyral pulses from MOR23 neurons under different conditions: WT P0 (thin blue), WT P30 (thick blue), OMP^−/− P0 (thin red) and OMP^−/− P30 (thick red). Representative traces from four single neurons were aligned at the start of the responses. C, D, The rise time (C) and the ratio of the residual current to the peak (D) are summarized for the four conditions: WT P0, WT P30, OMP^−/− P0, and OMP^−/− P30. The holding potential was −65 mV for all neurons. The output of the statistical analysis using three-way ANOVA is detailed in Figure 2D.

Figure 2.

The response kinetics of MOR23 neurons depends on age, OMP status, and concentration. A–C, Summary of the rise time (A), the decay time (B), and the ratio of the residual current to the peak (C) under different conditions: WT P0, WT P30, OMP^−/− P0, and OMP^−/− P30. All neurons were recorded under voltage-clamp mode with a holding potential of −65 mV. Not shown are P7 data, which generally fall in between P0 and P30 data. D, Comparison of all five parameters is performed by three-way ANOVA tests based on age (coded as an ordinal variable with levels 1, 2, and 3 corresponding to P0, P7, and P30, respectively), OMP status (coded as a categorical variable), and concentration (coded as an ordinal variable with level 1, 2 and 3 corresponding to 1, 10 and 100 μm, respectively). Significant p values are in bold. A significant p value in OMP status indicates difference between WT and OMP^−/− mice. A significant p value in age (or concentration) indicates that an increment in age (or concentration) increases or decreases the parameter. Other significant differences not detailed in the main text include a longer latency of the responses in OMP^−/− neurons than in WT neurons and a decrease in peak currents with age.

Figure 3.

Deletion of OMP alters ACIII signaling. A, The signaling proteins from the olfactory epithelia obtained from WT and OMP^−/− P30 mice were stained by specific antibodies in Western blots. Three of the five tested proteins are shown here. Note that ACIII protein has a smear-like band according to the manufacture's datasheet. B, The expression levels of three signaling proteins in OMP^−/− mice are normalized to those in WT animals. The ACIII level in the olfactory epithelium was ∼75% higher in OMP^−/− mice (n = 5 samples) with adjusted p < 0.05 (*) in t test with Bonferroni correction. C, Schematic drawing illustrates that phosphorylation of ACIII by CaMKII inhibits its enzyme activity, while dephosphorylation by phosphatase resets its activity. Phosphorylation and dephosphorylation are represented by green and magenta, respectively. D, OMP deletion reduced odorant-induced ACIII phosphorylation. ACIII phosphorylation was measured by incubating purified olfactory cilia with (+) or without (−) an odorant mixture with [γ-³²P]ATP for 1 min. ACIII protein was immunoprecipitated and resolved with SDS-PAGE, and radiolabeled phospho-ACIII was visualized on x-ray film. Basal ACIII phosphorylation was ∼10% higher in OMP^−/− mice. Odorant-induced phosphorylation of ACIII increased by ∼2.3-fold in WT animals, and only ∼1.5 in OMP^−/− animals. Similar results were obtained from two samples. Considering that ACIII protein is upregulated in OMP^−/− mice (A, B), the reduction in phosphorylated ACIII in OMP^−/− mice is even more significant. E, Block of ACIII phosphorylation via inhibition of CaMKII slows odorant response decay in WT-MOR23 neurons from P30 mice. Inward currents (normalized to the same peak) were elicited by brief lyral (10 μm) pulses in the presence of 1 μm AIP (magenta) and after washout (green). F, Block of ACIII dephosphorylation via inhibiting phosphatase activity hastens odor response decay in OMP^−/− MOR23 neurons from P30 mice. Inward currents (normalized to the same peak) were elicited by brief lyral (10 μm) pulses in the presence of 1 μm OA (green) and after washout (magenta). The holding potential was −65 mV for all neurons. G, The ratio of the residual current to the peak is summarized for the four conditions in E and F. Unpaired Student's t test (assuming equal variances and two-tail) is used: *p < 0.05 and **p < 0.01.

Figure 4.

Wild-type but not OMP^−/− MOR23 neurons increase the sensitivity to lyral during the first month. A–D, Inward currents were induced by lyral at various concentrations (0.01–100 μm) under different conditions: WT P0 (A), WT P30 (B), OMP^−/− P0 (C), and OMP^−/− P30 (D). The holding potential was −65 mV for all neurons. The traces were from four individual neurons. E, The dose–response curves are plotted for different conditions. F, The K_1/2 value is summarized for different conditions. We performed Bonferroni multiple comparisons for logK_1/2 among the four groups. MOR23 has significantly lower logK_1/2 compared with the other three groups (***adjusted p < 0.001 for all 3 comparisons), whereas there is no difference among the other three. For easier comprehension, the mean K_1/2 ± 95% confidence interval (CI) is plotted here.

Figure 5.

Wild-type but not OMP^−/− MOR23 neurons increase the selectivity during the first month. A–F, Odorant-induced transduction currents were recorded in MOR23 neurons under different conditions: WT P0 (A), WT P9–P15 (B), WT P30 (C), OMP^−/− P0 (D), OMP^−/− P9–P15 (E) and OMP^−/− P30 (F). Each scatter plot in the bottom row shows the peak currents induced by different stimuli in individual cells under the corresponding condition. Each color represents a single cell. When a peak current exceeds 200 pA, it is plotted at 200 pA for the purpose of visualization. All odorants were delivered at 100 μm (except two cells in WT P0 tested by 10 μm) and the holding potential was −65 mV for all neurons.

Figure 6.

Wild-type but not OMP^−/− pups prefer the biological mother. A, B, WT pups preferred to suckle or huddle with the biological mother over another unfamiliar lactating female (A), while OMP^−/− mice failed to show preference (B). The nose of each pup is marked by a dot and its location relative to the midline (dashed line) between the two anesthetized mothers is used to assign the pup's preference. C, Summary of mother preference for WT and OMP^−/− pups. The numbers in parentheses indicate the total number of pups tested in each group. Statistical analysis was performed using a χ² test. The choice between a lactating and a nonlactating female (both are unfamiliar) was tested from two litters of each genotype.