The objective of this study was to determine if mechanical force generated by airflow can stimulate the MOE using EOG recordings and to directly assess the role of cAMP signaling using AC3−/−
mice. Since the CNG responds to both cAMP and cGMP, the fact that CNG−/−
mice lack fluid-generated airflow responses cannot be taken as direct evidence for a role of cAMP signaling. The drug MDL 12330A cannot be used to implicate adenylyl cyclase activity in the airflow response because it is not specific to adenylyl cyclases (Rampe et al., 1987
; Gadea et al., 1999
) and it did not inhibit EOG responses caused by airflow or odorants in our study.
We discovered that airflow stimulates the MOE with progressively higher EOG responses as airflow increased. This sensitivity to airflow was desensitized by prior increases in cAMP caused by odorants or by a combination of forskolin and IBMX suggesting that odorant and airflow sensitivity may both depend on cAMP signaling. Indeed, the MOE from AC3−/− mice does not respond to airflow, thereby directly implicating cAMP signaling in airflow sensitivity.
One might argue that the EOG response to airflow is due to evaporation, cooling of the preparation, and activation of cold responsive sensory neurons. We think that this is unlikely since the nitrogen used in these experiments was humidified and pre-warmed to the same temperature as the MOE (22 °C). Furthermore, it has been established that cold-sensing neurons signal through TrpM8 channel (Latorre et al., 2011
) and do not depend on AC3 for EOG responses. Moreover, the EOG response to airflow of the MOE was not inhibited by the TrpM8 antagonist, SKF96365 (data not shown).
Since AC3 is expressed only in the olfactory cilia of OSN (Bakalyar and Reed, 1990
), we conclude that the cilia are most likely the primary organelle for airflow sensitivity. Sensing of mechanical force by cilia is not unique to olfactory cilia. For example, primary cilia in the apical surface of epithelia layer of the nephron can sense mechanical stress caused by fluid flow (Nauli et al., 2003
). Interestingly, these primary cilia also express AC3 (Pluznick et al., 2009
). Cilia in some sensory neurons of C. elegans also possess mechanosensitivity (Inglis et al., 2007
). Cilia on OSNs seem to have dual functions: the detection of odorants and airflow.
Although this study indicates that cAMP signaling is required for the airflow sensitivity of the MOE, the molecular sensor in the cilia is not known. Most likely membrane stretch generated by airflow is detected by a transmembrane protein. In principle, an odorant receptor, AC3, or a combination of these molecules could be the airflow-sensitive element. AC3 is a likely candidate because it is a transmembrane protein with two 6 transmembrane domains reminiscent of ion channels (Krupinski et al., 1989
). Moreover, adenylyl cyclase activity in vascular smooth muscle cells is sensitive to mechanical stretch (Mills et al., 1990
) and vascular smooth muscle also expresses AC3 (Wong et al., 2001
). Nevertheless, our data does not rule out a role of receptors in airflow sensitivity.
The threshold for airflow EOG responses in the mouse MOE preparation used in this study was between 0.03 to 0.06 l/min. The tidal volume of mice has been reported as 0.15–0.4 ml/breath (Fox et al 2007
). Mice usually breathe at 106–230 times per minute (Fox et al 2007
). From this one can estimate that the respiratory flow rate of mice would range from 0.03 l/min at the low end up to 0.18 l/min. We report measurable EOG changes at: 0.06 l/min; 0.17 l/mi; 0.35 l/min, 0.5 l/min or higher. Thus, the threshold flow rate for EOG responses that we obsevered is within the physiological sniffing range of mice. In addition, the maximal flow rate for sniffing with rats is reported as high as 0.5 l/min (Zhao et al., 2006
). Therefore, we also examined the rat MOE for EOG responses to airflow () and discovered that flow rates of 0.2 to 0.5 l/min generate a measurable EOG response (). Therefore, the threshold flow rate for EOG responses that we obsevered is within the physiological sniffing range of rats. Nevertheless, in our experiments the airflow was directed over an isolated section of the MOE and the cannula was 1 to 2 cm above the sample leading to uncertainties concerning the actual airflow at the surface of the sample. Also the velocity at the boundary layer of the intact MOE of the intact nose will be lower. Consequently, we cannot say unequivocally that the EOG responses to flow rates as low as 0.06 l/min in the isolated mouse MOE are physiologically relevant. However the observation that the EOG sensitivity to airflow was lost in AC3−/−
mice is important particularly since this enzyme activity is also required for odorant detection. The data showing that treatment of MOE preparations with odorants or agents which increase cAMP decreased EOG responses to airflow also supports the general hypothesis that the responses to airflow may be physiologically relevant.
Figure 8 Rat MOE is sensitive to airflow stimulation. A, top, EOG traces at different airflow rates up to 2 l/min are shown; bottom, bar graph (EOG amplitude) of airflow-sensitive responses at various flow rate. Recording site: middle of turbinate II; puff duration: (more ...)
Although the absolute value of the airflow-sensitive response is not very high, it may still affect the membrane potential and may facilitate depolarization of OSN, thereby promoting initiation of an action potential. On the other hand, OSN should not be too sensitive to airflow because it could increase noise during olfactory perception and interfere with the coding of odor information. Our data is consistent with the idea that airflow from respiration or sniff may cause a rhythmic oscillation in OSN. This would induce an up-phase and down-phase of membrane potential of OSN, which subsequently regulate coding of odor information, or provide an oscillatory drive to the olfactory bulb or olfactory cortex (Wachowiak, 2011
Neuron). This idea is in line with a number of observations suggesting that oscillations in the MOB and olfactory cortex are coupled with respiration (Grosmaitre et al., 2007
; Fontanini et al 2003
; Schaefer et al., 2006
; Carey and Wachowiak, 2011
In conclusion, the mechanical force exerted by respiration or sniffing may function synergistically or additively with odorants to promote the depolarization of OSN, an idea supported by other published studies (Scott, 2006
; Verhagen et al., 2007
; Oka et al., 2009
). Furthermore, airflow sensitivity of the MOE is detectable by EOG recordings and depends on cAMP signals generated by AC3.