1. Ortho- and retronasal glomerular response patterns largely overlap
We first investigated how similar the retro- and orthonasal glomerular response patterns are. shows an example of averaged (n = 3) odor maps in the OB at their peak response for orthonasal 4% 2-hexanone (2hex), orthonasal 4% methyl valerate (MV) and retronasal 4% MV. Responses of glomerulus 1 and 12 vary strongly across the three conditions. Glomerulus 1 responded mainly to orthonasal MV, much less to the retronasal stimulation thereof, and not at all to orthonasal 2hex, whereas glomerulus 12 responded strongest to orthonasal 2hex. The average responses (mean ± sem) for these three stimuli at sixteen glomeruli appear in . Paired t-test between ortho- 2hex and ortho- MV responses revealed significant differences at nine glomeruli. Between ortho- and retronasal MV, however, responses were different significantly only at two glomeruli. Correlation analysis of the responses between ortho- MV and ortho- 2hex across all sixteen glomeruli showed a negative correlation (r = −0.61), while that between ortho- vs. retronasal MV exhibited a strong positive correlation (r = 0.74).
Figure 2 Ortho- and retronasal glomerular response patterns elicited by the same odor overlap but differ in terms of response size. A–E, an example from a rat: A, RLI image of the left bulb. B–D, averaged (n = 3) odor maps for ortho 4% 2-hexanone (more ...)
Across all 9 rats for 6 odors and air the glomerular pattern correlation between routes was 0.38 ± 0.09 (mean ± sem; n = 7), which increased to 0.45 ± 01 (n = 6) after removing putative outlier tolualdehyde (r = −0.03). The pattern similarity between different odors presented via the same route was 0.27 ± 0.03 (n = 42 odor-odor combinations), which decreased to 0.22 ± 0.04 (n = 30) after removing tolualdehyde. Response patterns were more similar for the same odorants between routes than for the same route between odors (p < 0.05) but only after removing tolualdehyde, the retronasal responses of which did not relate to the orthonasal ones. This was substantiated by using multidimensional scaling () to explore the response pattern similarities across all stimuli at once. Stimulus pairs are linked by lines. shows that odors are clearly differentiated along the hypothetical "odorant" axis, and are also differentiated (but to a lesser degree) along the "route" axis. The dotted line separates 5 of the six odorants by route.
Figure 3 Comparison of ortho- vs. retronasal response magnitudes and patterns for different odorants. A. The averaged ortho- and retronasal responses are shown, as well as their ratio to their left (mean ± sem, n = 6~9 rats). The magnitude of retronasal (more ...)
When we analyzed the similarity in response magnitudes between all ortho- and retronasal pairs in a single correlation this yielded an even higher correlation of r = 0.51 (893 pairs, ). We therefore conclude that ortho- vs. retronasal OB response patterns elicited by the same odorant differ less than response patterns elicited by two different odorants via the same route. In other words, odor route affects response patterns less than odorant identity.
To estimate the degree of overlap between ortho- and retronasal odor maps we arbitrarily set < 0.05% ΔF/F as noise and calculated for each stimulus the percentage of the total number of responsive glomeruli in an animal that was responsive to each route. On average (6 odorants, 9 rats, 779 glomerular responses) we found 69 ± 9% (mean ± sd) overlap between the two routes (). Remaining 20 ± 4% and 11 ± 6% glomeruli were activated, respectively, by ortho- and retronasal stimulation only. In terms of the number of glomeruli imaged per rat, 11.14 ± 2.37 (mean ± sd) responded to both routes while 2.48 ± 0.48 and 1.51 ± 0.73 glomeruli responded only to ortho- and retronasal stimulation, respectively.
2. Retronasal response amplitudes are smaller than orthonasal ones
We next asked how response magnitudes were affected by the routes of odorant delivery. compares retronasal response magnitudes to the corresponding orthonasal responses across six odorants and clean air, ordered by decreasing polarity (octanol : water partition coefficient, kow). shows both the mean glomerular response (first %ΔF/F averaged across glomeruli in each rat and then averaged across rats, mean ± sem; n = 5~9 rats) for each route and, the r/o ratio of %ΔF/F (averaged across rats; mean ± sem; n = 5~9) providing an estimate of the pair-wise retronasal response efficacy relative to the orthonasal response. Responses to the orthonasal 2-hexanone (2hex) (0.47 ± 0.05 %ΔF/F) were significantly larger than the retronasal responses (0.27 ± 0.10 %ΔF/F) (p < 0.05, r/o = 0.52 ± 0.19, n = 6). Ethyl butyrate (EB), showed relatively larger responses both ortho- (0.65 ± 0.07 %ΔF/F) and retronasally (0.37 ± 0.09 %ΔF/F), and the difference was significant (p < 0.05, r/o = 0.59 ± 0.14, n = 9). Methyl valerate (MV) responses were also robust orthonasally (0.60 ± 0.08 %ΔF/F) but less so retronasally (0.30 ± 0.08 %ΔF/F), showing a significant difference (p < 0.05, r/o = 0.61 ± 0.18, n = 8). Amyl acetate (AA) showed a significantly smaller (p < 0.05) retronasal response (0.14 ± 0.06 %ΔF/F) compared to an almost double orthonasal response (0.31 ± 0.04 %ΔF/F, r/o = 0.49 ± 0.18). Tolualdehyde (tolu) retronasal responses (0.10 ± 0.05 %ΔF/F) were also the smallest among the six odorants tested, and the orthonasal responses (0.26 ± 0.07 %ΔF/F) were significantly larger (p < 0.05, r/o = 0.34 ± 0.24, n = 5). Vinyl cyclohexane (VC), however, was only close to a significant difference in the response between the routes (ortho: 0.19 ± 0.05 %ΔF/F, retro: 0.11 ± 0.04 %ΔF/F, p = 0.07, r/o = 0.62 ± 0.20, n = 7). By contrast, clean air evoked responses of similar magnitude (ortho: 0.16 ± 0.03, retro: 0.13 ± 0.05 %ΔF/F, p = 0.22, r/o = 0.71 ± 0.36, n = 9). Across all ortho- vs. retronasal responses the orthonasal responses (0.39 ± 0.04 %ΔF/F) were significantly larger (p < 0.01, r/o = 0.63 ± 0.12, n = 9) than the retronasal responses (0.23 ± 0.04 %ΔF/F). The correlation analysis across all 893 ortho-retronasal stimulus pairs, shown in , similarly suggests that retronasal response amplitudes were 52% of the orthonasal magnitudes, which is close to the 63% suggested by the ratio above. These results indicate that, in general, retronasal responses to odorant stimuli are significantly smaller than orthonasal responses, despite identical odor concentrations and flow rate.
3. Volatility enhances retronasal response efficacy
To determine if there was a relationship between specific physicochemical properties of the odorants and their retronasal response efficacy relative to orthonasal responses, we plotted the odors and these properties relative to the mean retro/ortho ratio. We found that the relative retronasal efficacy (r/o ratio) did not correlate well with the lipophilicity/partition coefficient (P (log kow), r = 0.21, p = 0.34, ) or polar surface area (polar surface (Å2), r = − 0.10, p = 0.42, ), but did correlate strongly with vapor pressure (VP (mmHg), r = 0.92, p < 0.005, ), density ((g/ml), r = − 0.80, p < 0.05, ) and boiling point (BP (°C), r = − 0.93, p < 0.005, ). The orthonasal and retronasal response magnitudes at best only trended toward significant correlations with these properties (no shown).The ortho-retro pattern similarity was found to increase only with decreasing boiling point (r2 = 0.88, n = 6, p < 0.005) and with decreasing density (r2 = 0.79, n = 6, p < 0.01), and additionally with retro/ortho response ratio (r2 = 0.68, n = 6, p < 0.05).
Figure 4 Retronasal relative response efficacy correlates with the volatility of the odorants. The mean retro/ortho response ratio of for each odorant is plotted against vapor pressure (VP, mmHg; A), partition coefficient (P, logKow, B), density (g/ml, (more ...)
We'd like to point out that these findings were robust and not dependent on a hinge-like effect of the single high density and high boiling point odorant (), in that in a complimentary analysis based on correlation-derived slopes rather than ratios () the same results were obtained. There we also included two additional odorants, being the highly polar cyclohexane and highly non-polar mesitylene with continuous values for density and boiling point (0.95, 0.87 mg/ml, and 155°C and 164°C, respectively). In that analysis the relation between density and r/o ratio yielded r2 = 0.76 (p < 0.01) and the relation between boiling point and r/o ratio yielded r2 = 0.83 (p < 0.001). These two odors were otherwise excluded from analyses due to the low number of rats tested with them (4 rats).
As higher vapor pressure, lower density and lower boiling point tend to make an odorant more volatile, these results indicate that higher volatility increases especially retronasal efficacy and thereby increases the pattern similarity between the ortho- and retronasal response in the presynaptic dorsal OB.
4. Retronasal responses show slower but more consistent temporal dynamics than orthonasal responses
The temporal dynamics of calcium responses may contain information about odor quality at the level of the OB (Spors et al., 2006
; Junek et al., 2010
). Significant differences may exist between the response latencies of different glomeruli to the same odorant; and, for a given glomerulus, different odorants can evoke responses with different latencies (Spors et al., 2006
). To compare the temporal dynamics of ortho- vs. retronasal glomerular responses we examined the time course of the responses of each glomerulus (see Materials and Methods, and ). The averaged values of each parameter for each odorant (ordered by polarity) and each route are shown separately in . Temporal response parameters were relatively variable across orthonasally presented odorants, notably for t10
= 12.1, p < 10−7
, one-way ANOVA), t50
= 3.4, p < 0.01) and rise-time (F6,50
= 6.4, p < 10−4
). Retronasal dynamics barely varied between odorants, only for t50
= 3.3, p < 0.01). This is more clearly shown in , which shows the reconstituted sigmoidal plots for orthonasal (red) and retronasal (blue) odorants.
Figure 5 Temporal dynamics of ortho- vs. retronasal odor responses. A, response traces were fitted with a double sigmoidal function and the time of response onset (start), time to 10%, 50% and 90% of the response peak (t10, t50 and t90), the time of the response (more ...)
In order to determine if any of the temporal parameters differ between the ortho- and retronasal route we subtracted retro values from the ortho values for each odor and parameter in pair-wise fashion (). We found that the average duration of the lag between the stimulus onset and the response onset (start) was −439 ± 30 ms for ortho and −550 ± 22 ms for retronasal responses with a resulting retronasal delay (ortho minus retro, "retro delay") of 100 ± 39 ms relative to the orthonasal response (p < 0.0001, n = 57 (7 odors × 9 rats, 6 missing data points), paired 2-tailed t-test). The retronasal route was associated with slightly faster response from start to 10% of peak amplitude (retro delay t10, 42 ± 11 ms, (p < 0.0001), ), but similar from start to 50% (t50, 11 ± 7 ms, (p = 0.20)). The retronasal responses were consistently slower to reach 90% of peak (t90, −45 ± 22 ms, (p = 0.002)) and peak itself (tpeak, −147 ± 47 ms, (p = 0.0001)). On average the rise-time for retronasal response was longer than that for orthonasal response by 169 ± 49 milliseconds (p < 0.0001). Unpaired two-sided t-tests yielded the same conclusions.
We also performed two-way ANOVA to understand possible effects of route, odor and their interactions on each of the temporal parameters. Route showed a significant (F1,100>7.6, p < 0.01) effect on each of the temporal parameters except for t50 (p = 0.35), consistent with the t-tests above. Likewise, across both routes odor had an effect (F6,100 > 2.4, p < 0.05) on t-onset, t10, t50, and rise-time but not on t90 (p = 0.41) and t-peak (p = 0.20). The interaction between route and odor was significant only for the rise-time (F6,100 = 2.5, p < 0.05). A one-way ANOVA of odor effect on retro delay only showed an effect on rise time (F5,60 = 2.9, p < 0.05), as expected based on the 2-way ANOVA, and also t90 (F5,60 = 2.3, p < 0.05).
It should be noted that the response latencies we report are on the long side, but are not outside the bounds of prior measures. Response onset delay was ~0.5 sec, of which ~120ms was due to dead space in the olfactometer (see Methods). The remaining ~380ms are not unusual, as similar delays have been reported before using EOG. For example, see in (Scott, 2006
), where the ventrolateral response to isoamyl acetate at 200ml/min flow rate takes 300–400ms to develop.
We also would like to point out that despite having minimized the difference the dead-space of our olfactometer between both routes, it is possible that within the rat itself differences in dead space remain (i.e. between the odor mask or retronasal canula and OE). These could have accounted for the difference in onset delay between routes (), but do not explain the effect of route, or polarity on the other parameters from which this delay was subtracted (t10,t50, t90, tpeak and rise-time, , ). Indeed, we subtract onset delay from the other parameters and variability remains () as tested by ANOVA.
Figure 6 Retronasal delay (inverted values from for clarity) for the onset of the response and time to peak correlates with the polarity of odorants. A, mean retro delay for the onset of the response for each odorant is plotted against vapor pressure (VP), (more ...)
Overall, these observations suggest that the time course of retronasal responses is slower than that of orthonasal responses, including a longer onset latency, longer time to peak and longer rise-time. Moreover, the response dynamics varied less across odors presented retronasally than orthonasally.
5. Retronasal delay for the onset, time to peak and rise-time varies with the polarity of the odorants
We then asked if and how retronasal delays of correlated with specific odorant properties (). As shown in mean retro delay (which values are the inverted values of for clarity) for the onset of the response for each odorant was plotted against its partition coefficient (P), polar surface area (polar surface), vapor pressure (VP), density (d) and boiling point (BP). The response onset delay of the retronasal route versus the orthonasal route did not correlate with vapor pressure (r = 0.18, p = 0.37), density (r = 0.24, p = 0.32) or boiling point (r = −0.07, p = 0.45), but did correlate well with polar surface (r = 0.81, p < 0.05) and partition coefficient (r = −0.95, p < 0.005).
Relationship between retro-delay and odorant properties.
Similarly, the mean retro delay for time to peak also correlated with partition coefficient (r = −0.93, p < 0.005; ). Again, polar surface showed a positive correlation but no clear significance (r = 0.59, p = 0.11). Vapor pressure (r = 0.16, p = 0.38), boiling point (r = −0.16, p = 0.38) and density (r = 0.06, p = 0.46) did not appear to explain the variation in time to peak.
These data indicate that odorant polarity plays an important role in the length of the delay of retronasal relative to orthonasal responses. Increasing polar surface by 1 unit increases the response onset time by 3.7 ms and time to reach peak of retronasal responses by 5.2 ms, relative to orthonasal responses. Increasing the odorant's polarity by 1 log unit (i.e. decreasing P) increases the relative retronasal onset by ~49 ms and the time to peak by ~192ms. Polarity also correlated positively with relative (retro delay) rise-time and t10 nearly significantly (p = 0.06 and p =0.08, respectively). Given the larger variability in the dynamics of responses to orthonsasal odors (), we suggest that these effects of polarity are largely on orthonasal responses.
6. Ruling out effects of phase of breathing cycle and stimulus pressure on ortho- vs. retronasal responses
We recorded breathing and intranasal pressure signals from each animal for each route throughout the experiments. We tested whether glomerular responses to ortho- and retronasal stimulation had any unexpected relationships with these parameters. We did not expect effects of breathing, as stimulus onset was timed independently thereof. The double tracheotomized rats were breathing through a tracheal tube, uncoupled from the nose. The breathing frequency in animals remained uniform throughout the experiment (ortho: 1.60 ± 0.11 Hz, retro: 1.65 ± 0.14 Hz) (). We compared stimulus onset time from the preceding inspiration (ortho: 0.31 ± 0.02 s, retro: 0.31 ± 0.01 s), position of stimulus onset within a breath cycle (ortho: 0.47 ± 0.02, retro: 0.47 ± 0.01), range: 0 to 1), peak response time from the preceding inspiration (ortho: 1.61 ± 0.12 s, retro: 1.62 ± 0.12 s) and number of breath cycles until peak response (ortho: 2.56 ± 0.25, retro: 2.66 ± 0.31). As expected, there was no significant difference between ortho- and retronasal trials on these parameters, suggesting that the observed effects of route on glomerular responses were not influenced by the phase of breathing cycle on average.
Figure 7 No coupling of ortho and retronasal olfactory glomerular responses with breathing. A, an example of the time course of a glomerular response, breathing and artificial sniffing. Points of interest are marked with a, b, c and d. B, comparison of ortho and (more ...)
We also did not expect differences in intranasal pressure responses to ortho- and retronasal trials. Indeed, the difference in the intranasal pressure parameters (maximum pressure (Pmax), area under the pressure curve during odor stimulation (Pauc) and the time to Pmax could not explain the effect of route on the relative magnitudes of responses (). The ratios of paired averaged ortho- and retronasal responses for each stimulus for all animals ("r/o dF/F"; n = 106) did not correlate with averaged pressure ratios based on the same trials: Pmax explained 1%, Pauc 3% and time to Pmax 1% (). On average Pmax and Pauc were somewhat below unity (0.90 ± 0.01), indicating that the retronasal flow resistance was 10% higher than the orthonasal flow. No temporal differences in flow were found.
Figure 8 No correlation between intranasal pressure (flow-resistance) during artificial sniffing and effect of route on glomerular responses. A, scatter plot of retro/ortho response ratio vs. ortho/retro ratio of maximum pressure during stimulus delivery. B, scatter (more ...)