Dual functions of mammalian olfactory sensory neurons as odor detectors and mechanical sensors

Abstract

Most sensory systems are primarily specialized to detect one sensory modality. Here we report that olfactory sensory neurons (OSNs) in the mammalian nose can detect two distinct modalities transmitted by chemical and mechanical stimuli. As revealed by patch-clamp recordings, many OSNs respond not only to odorants, but also to mechanical stimuli delivered by pressure ejections of odor-free Ringer solution. The mechanical responses correlate directly with the pressure intensity and show several properties similar to those induced by odorants, including onset latency, reversal potential and adaptation to repeated stimulation. Blocking adenylyl cyclase or knocking out the cyclic nucleotide-gated channel CNGA2 eliminates the odorant and the mechanical responses, suggesting that both are mediated by a shared cAMP cascade. We further show that this mechanosensitivity enhances the firing frequency of individual neurons when they are weakly stimulated by odorants and most likely drives the rhythmic activity (theta oscillation) in the olfactory bulb to synchronize with respiration.

Figure 1

The septal organ neurons respond to both chemical and mechanical stimuli. (a) Schematic drawing shows the recording configuration. The distance between the recorded knob and the puffing pipette opening is defined as the puffing distance (d). (b,c) In voltage-clamp mode, inward currents were elicited by mix 1 (100 μM) and Ringer solution puffs from a single neuron with the puffing distance d =20 μm (b) or 70 μm (c). The holding potential was −60 mV. (d) The peak currents induced by mix 1 or Ringer puffs are averaged from nine neurons. Star (*) marks significant difference in paired t-test (P < 0.01). (e) The responses induced by Ringer puffs show variation. The inward currents were induced by Ringer puffs via different barrels of puffing pipettes and the peak currents were normalized to the mean obtained from individual neurons. The frequency distribution is plotted with a bin width of 0.1 and fitted by a Gaussian distribution (smoothed line). The mean of the normalized peak currents is 1.0 and the s.d. is 30%. The puffing pressure was 20 p.s.i. for all recordings.

Figure 2

The septal organ neurons respond to diverse odorants and mechanical stimulation. (a) A single neuron responded to multiple odorants (except (+)-limonene) at 300 μM. Inward currents were elicited by odor and Ringer solution puffs in voltage-clamp mode. The same Ringer response (gray) was shown with each odorant response. (b–d) A non-responsive neuron showed normal functional properties. In voltage-clamp mode, a single neuron did not respond to mix 1 (100 μM) or Ringer puffs, but responded to a cocktail of IBMX and forskolin (b). In the same neuron, voltage-gated ionic currents were elicited by voltage steps from −40 to +60 mV (c) and action potentials were elicited by injecting a depolarizing current (6 pA) (d). The dashed line marks −60 mV. (e,f) The septal organ neurons responded to octanoic acid with a low threshold and a wide dynamic range. (e) Inward currents from a single neuron (filled squares in f) were elicited by puffing octanoic acid at different concentrations (10⁻¹²–10⁻⁴ M) in voltage-clamp mode. The gray trace indicates the response induced by puffing Ringer. (f) The dose-response curves (peak current I versus concentration C) are plotted and fitted with the modified Hill equation I = I_baseline + I_max/(1 + (K_1/2/C)ⁿ), where n = 0.5 ± 0.1 (n = 4, mean ± s.e.m.) and K_1/2 = 3.2 ± 2.3 μM. The holding potential was −60 mV for all recordings in voltage-clamp mode and the puffing pressure was 20 p.s.i. for all recordings.

Figure 3

OSNs from both the septal organ and the main olfactory epithelium show mechanical responses. (a,b) Inward currents from a single neuron within the septal organ (SO; a) or within the main olfactory epithelium (MOE; b) were induced by Ringer solution puffs at different pressures in p.s.i. in voltage-clamp mode. The holding potential was −60 mV for both neurons. (c) The normalized peak currents induced by Ringer puffs are plotted against the pressure and fitted with the Boltzmann equation. (d,e) The currents induced by Ringer puffs reversed at 0 mV in voltage-clamp mode. (d) The currents were induced by puffing Ringer at varying holding potentials indicated next to each trace. (e) The peak currents induced by mix 1 and Ringer puffs from four cells are plotted against the holding potentials. The puffing pressure was 20 p.s.i. for recordings in d and e. Error bars, s.e.m.

Figure 4

The cAMP cascade and CNG channels underlie the mechanical responses of the OSNs. (a) The inward currents induced by Ringer solution puffs in voltage-clamp mode were reversibly blocked by 50 μM of MDL12330A, an adenylyl cyclase inhibitor. (b) The inward currents induced by Ringer puffs were first obtained in Ca²⁺-free solution and then after perfusion of normal Ringer with 2 mM Ca²⁺ (2 or 2.5 min). (c–e) The mechanical responses showed adaptation during repetitive stimuli. (c) The inward current was induced by Ringer puffs at 0.5 Hz in voltage-clamp mode. (d) The depolarization was induced by Ringer puffs at 0.5 Hz in current-clamp mode. (e) A septal organ neuron responded to alternate Ringer puffs at 1 Hz in current-clamp mode. (f–h) Both odorant and mechanical responses were eliminated in Cnga2^−/y mice. (f) A single septal organ neuron did not respond to odorant, Ringer or IBMX+forskolin puffs in voltage-clamp mode. (g) In the same neuron as in f, voltage-gated ionic currents were elicited by voltage steps from −40 to +60 mV from a holding potential of −60 mV. (h) In current-clamp mode, action potentials were elicited by injecting a depolarizing current of 10 pA into the same neuron. The holding potential was −60 mV for all recordings in voltage-clamp mode. The dashed lines mark −60 mV. The puffing pressure was 20 p.s.i. for all recordings.

Figure 5

Increasing puffing pressure enhances the odorant responses in individual OSNs. (a) In voltage-clamp mode, inward currents were induced by 1 μM octanoic acid puffs at different pressures indicated next to each trace. (b) Inward currents were induced by 100 μM mix 1 puffs at different pressures. The holding potential was −60 mV for both neurons. (c) In current-clamp mode, action potentials were elicited by 1 μM octanoic acid puffs with increasing pressures. The rectangle indicates the time window in which the firing frequency is averaged. The dashed lines mark −60 mV. (d) The firing frequencies are averaged for the responses induced by odorant stimuli with increasing pressures. The neurons were either ‘weakly stimulated’ by 1 μM octanoic acid (n = 5) or ‘strongly stimulated’ by saturating odorants (100 μM mix 1 or 300 μM octanoic acid, n = 4). Error bars, s.e.m.

Figure 6

The rhythmic activity (theta band) in the olfactory bulb uncouples from respiration in Cnga2^−/y mice. (a,b) In wild-type (WT) olfactory bulbs, oscillatory field potentials (F.P.) (black) were recorded at 100 μm (a) or 500 μm (b). (c,d) Field potentials (black) were recorded at the depths of 100 μm (c) or 500 μm (d) in Cnga2^−/y mice. Gray traces (Resp.) indicate the respiratory rhythm. The averaged field potential within one respiratory cycle is shown in the right column of each panel. To calculate the averaged traces, the field potentials within individual respiratory cycles were truncated, normalized to the same length, and then averaged over 200 s. (e) The cross-correlation coefficients between the field potentials and respiratory rhythms are averaged for the recordings obtained at 100 μm or 500 μm. At both depths, the coefficients are significantly higher in wild-type (n = 6) than in Cnga2^−/y bulbs (n = 4) with P < 0.001 in t-test (marked by **). Error bars, s.e.m.