Olfactory marker protein modulates the cAMP kinetics of the odour-induced response in cilia of mouse olfactory receptor neurons

Abstract

Olfactory marker protein (OMP), a phylogenetically conserved protein, is highly, and almost exclusively, expressed in vertebrate olfactory receptor neurons (ORNs). Although OMP is widely used as a marker for ORNs, its function has remained largely elusive. Here we used suction-pipette recordings from isolated ORNs of OMP(-/-) mice to investigate its role in olfactory transduction. Vertebrate olfactory transduction is initiated when odourants bind to receptor proteins to activate an adenylyl cyclase via a G protein-coupled signalling pathway. This leads to an increase in cAMP and the opening of a cyclic nucleotide-gated (CNG), non-selective cation channel which depolarizes the cells. Ca(2+) influx through the CNG channel in turn activates a Ca(2+)-activated Cl(-) channel, causing a Cl(-) efflux and further depolarization. In the absence of OMP, the time-to-transient-peak of the response, the latency to first spike, and the response termination were slowed 2- to 8-fold, indicating its role in regulating olfactory response kinetics and termination. This phenotype persisted in OMP(-/-) ORNs even in low external Ca(2+) solution chosen to prevent Cl(-) channel activation, suggesting OMP acts upstream of Cl(-) channel activation. Furthermore, the response kinetics in cilia are virtually indistinguishable between OMP(-/-) and wild-type ORNs when intracellular cAMP level was elevated by the phospho-diesterase inhibitor, IBMX, suggesting OMP acts upstream of cAMP production. Together, our results suggest a role for OMP in regulating the kinetics and termination of olfactory responses, implicating a novel mechanism for fast and robust response termination to ensure the temporal resolution of the odour stimulus. These observations also help explain the deficits in odour detection threshold and odour quality discrimination seen in the OMP(-/-) mice.

Figure 2. Averaged data for OMP^+/+ and OMP^−/− ORNs

A, maximal current (I_max) and current at the end of the 2 s cineole stimulation (I_2s). B, IBMX-induced peak current and current at the end of the 1 s IBMX (I_1s) exposure. Kinetic parameters of the cineole-induced (C) and IBMX-triggered response (D). The response delay was taken as the time of the first spike to be generated and t_20% represents the time for the response to fall to 20% of its value at the end of stimulation (2 s and 1 s for cineole and IBMX, respectively). The maximal firing frequency and number of spikes elicited by cineole or IBMX are shown in E and F, respectively. Symbols indicate values that are significantly different at the 0.05 (*) and 0.005 (**) level (Student's t test). Numbers in parentheses indicate the numbers of ORNs used in the analysis. Error bars represent s.e.m. Recordings included in this analysis were typically the first or second recording obtained from an ORN after it was sucked into the tip of the recording pipette to avoid possible stimulation-induced rundown. The cineole and IBMX concentrations were always 100 μm and 1 mm, respectively.

Figure 1. Suction pipette recordings of mouse olfactory receptor neurons

Fast wild-type (A) and slow OMP^−/− ORN (B) responses were recorded in response to a 2 s 100 μm cineole stimulation. C and D, suction pipette currents elicited by the phosphodiesterase inhibitor IBMX (1 mm for 1 s) in an OMP-positive and -negative ORN. All traces are filtered DC to 50 Hz to display the receptor current alone. E–F, the same recordings as in A–D at an expanded time scale, but filtered at DC to 5000 Hz to investigate action potential firing. The solution monitor at the top indicates the timing of the command pulse which initiated the solution change; the actual solution change occurred with a slight delay (see Methods).

Figure 4. The oscillatory response during prolonged stimulation

A, a wild-type ORN was exposed to 300 μm cineole for 60 s and exhibited a fast oscillatory response pattern with a mean oscillatory period of 0.64 s. The inset shows a recording from a different OMP-positive ORN, which was filtered DC to 5000 Hz. Action potentials are now visible at the rising phase of each repetitive oscillation. B, an OMP^−/− ORN shows drastically slowed oscillations when exposed to cineole (100 μm) with the mean oscillation period being 10.6 s.

Figure 3. Double pulse experiment to investigate recovery from adaptation

A wild-type (A) and an OMP^−/− ORN (B) were exposed twice to 100 μm cineole for 1 s with increasing recovery time between the odour exposures as indicated by the solution monitor at the top. C, the recovery time course as a function of the recovery time, the ordinate displays the peak current of the second response normalized to the first. Individual data points represent the average of 2–8 measurements. Cineole concentration was 100 μm.

Figure 5. Analysis of the falling phase of the suction pipette current using short cineole or IBMX stimulations

Wild-type (A) or OMP^−/− (C) ORNs were exposed to cineole and the falling phases of the suction pipette current fitted with single exponentials (grey line) with time constants of 26 and 260 ms, respectively. To minimize the contribution of the Ca²⁺-activated Cl⁻ conductance external Ca²⁺ was reduced to 20 μm (low Ca²⁺) for an OMP^+/+ (B) and an OMP^−/− (D) ORN. The fitted exponentials had time constants of 46 and 570 ms. Stimulation durations were chosen to generate peak currents of comparable magnitudes and were 40, 25, 50 and 25 ms in A–D, respectively, and chosen to elicit small currents, typically between 5 and 20 pA. Recordings in A and B are from the same OMP^+/+ ORN, and in C and D are from the same OMP^−/− ORN. Exposure to 1 mm IBMX for 50 ms in normal Ringer solution generated suction pipette currents with comparable time courses in wild-type (E) or OMP^−/− (G) neurons (time constants of 25 and 39 ms). In low Ca²⁺ Ringer solution the kinetics of the falling phase also did not change much and could be fitted with time constants of 40 ms in the wild-type (F) and 53 ms in the OMP^−/− ORN (G). Traces are in most cases averages of 5 recordings. Recordings in E–H are from different ORNs. Odourant or IBMX stimulations commenced at 40 ms.

Figure 6. Averaged time constants of the falling phases of cineole or IBMX-triggered responses

A, comparison of averaged time constants in OMP^+/+ and OMP^−/− ORNs when stimulated with cineole in either normal or low-Ca²⁺ Ringer solution. Values are averages of 8–10 cells. B, time constants of IBMX-triggered suction pipette currents in normal or low-Ca²⁺ Ringer solution. Symbols indicate values that are significantly different at the 0.05 (*) and 0.005 (**) level (Student's t test).