Odor Detection in Manduca sexta Is Optimized when Odor Stimuli Are Pulsed at a Frequency Matching the Wing Beat during Flight

Sensory systems sample the external world actively, within the context of self-motion induced disturbances. Mammals sample olfactory cues within the context of respiratory cycles and have adapted to process olfactory information within the time frame of a single sniff cycle. In plume tracking insects, it remains unknown whether olfactory processing is adapted to wing beating, which causes similar physical effects as sniffing. To explore this we first characterized the physical properties of our odor delivery system using hotwire anemometry and photo ionization detection, which confirmed that odor stimuli were temporally structured. Electroantennograms confirmed that pulse trains were tracked physiologically. Next, we quantified odor detection in moths in a series of psychophysical experiments to determine whether pulsing odor affected acuity. Moths were first conditioned to respond to a target odorant using Pavlovian olfactory conditioning. At 24 and 48 h after conditioning, moths were tested with a dilution series of the conditioned odor. On separate days odor was presented either continuously or as 20 Hz pulse trains to simulate wing beating effects. We varied pulse train duty cycle, olfactometer outflow velocity, pulsing method, and odor. Results of these studies, established that detection was enhanced when odors were pulsed. Higher velocity and briefer pulses also enhanced detection. Post hoc analysis indicated enhanced detection was the result of a significantly lower behavioral response to blank stimuli when presented as pulse trains. Since blank responses are a measure of false positive responses, this suggests that the olfactory system makes fewer errors (i.e. is more reliable) when odors are experienced as pulse trains. We therefore postulate that the olfactory system of Manduca sexta may have evolved mechanisms to enhance odor detection during flight, where the effects of wing beating represent the norm. This system may even exploit temporal structure in a manner similar to sniffing

Odors Pulsed at Wing Beat Frequencies are Tracked by Primary Olfactory Networks and Enhance Odor Detection

Each down stroke of an insect's wings accelerates axial airflow over the antennae. Modeling studies suggest that this can greatly enhance penetration of air and air-born odorants through the antennal sensilla thereby periodically increasing odorant-receptor interactions. Do these periodic changes result in entrainment of neural responses in the antenna and antennal lobe (AL)? Does this entrainment affect olfactory acuity? To address these questions, we monitored antennal and AL responses in the moth Manduca sexta while odorants were pulsed at frequencies from 10–72 Hz, encompassing the natural wingbeat frequency. Power spectral density (PSD) analysis was used to identify entrainment of neural activity. Statistical analysis of PSDs indicates that the antennal nerve tracked pulsed odor up to 30 Hz. Furthermore, at least 50% of AL local field potentials (LFPs) and between 7–25% of unitary spiking responses also tracked pulsed odor up to 30 Hz in a frequency-locked manner. Application of bicuculline (200 μM) abolished pulse tracking in both LFP and unitary responses suggesting that GABAA receptor activation is necessary for pulse tracking within the AL. Finally, psychophysical measures of odor detection establish that detection thresholds are lowered when odor is pulsed at 20 Hz. These results suggest that AL networks can respond to the oscillatory dynamics of stimuli such as those imposed by the wing beat in a manner analogous to mammalian sniffing

The features of olfactory stimuli and the context of delivery affect their detection.

<p>Main effects of: stimulus protocol (pulsed vs continuous; <b>A</b>); duty cycle (<b>B</b>); the odorant used (<b>C</b>); and stimulus concentration (<b>D</b>). In all panels the Y-axis represents conditioned response probability. Error bars represent +/-1 SE. Inset letters indicated significant differences between means based on a Tukeys HSD (p<0.05). </p

Three measures of olfactometer output demonstrate that the 20 Hz odor pulse trains are preserved through primary olfactory input but the valve artifacts are not.

<p><b>A</b>,<b>C</b>,<b>E</b>. Mean response over two pulse cycles based on averaging 40 consecutive 100 ms samples from a 4 s 20 Hz pulse train for the two duty cycles: 10:40 ms (black), and 40:10 ms (red). Results from hotwire anemometry (same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081863#pone-0081863-g002" target="_blank">Figure 2D</a>; <b>A.</b>); photo ionization detection (C.); and electroantennogram (E.). Inset horizontal lines indicate pulse duration and are color coded to correspond to individual traces. Error bars are ±1 SD. <b>B,D,F</b>. Corresponding power spectral density (PSD) analyses showing the amount of oscillatory power as a function of frequency for the hotwire anemometry (<b>B</b>); photo ionization detector (<b>D</b>); and electroantennogram (<b>F</b>). Individual traces are color coded (as above) to correspond to the two duty cycles. In cases where the peak in the power spectra for a given duty cycle was occluded we highlight it with like colored arrowheads. Note that there are several spectral peaks in the anemometry results that are greatly reduced or absent in the PID and EAG results. </p

Pulsing method does not affect CR probability, olfactometer flow velocity does.

<p>Mean CR probability as a function of: concentration (<b>A</b>); stimulation protocol (<b>B</b>); olfactometer outflow velocity (<b>C</b>); and pulsing method (interleaved vs puffed; <b>D</b>). In all panels the Y-axis represents conditioned response (CR) probability. Error bars represent +/-1 SE. Inset letters indicated significant differences between means (Tukeys HSD; p<0.05). </p

Differences in stimulus duration do not account for differences in pulsed versus continuous stimuli.

<p>Main effect of stimulus protocol (pulsed vs continuous) for odor stimuli that were presented for the same duration either and the same concentration (<b>A</b>, <b>B</b>); or concentration corrected to 1/5^th to match the total odor delivered (<b>C</b>, <b>D</b>). In panels <b>B</b> and <b>D</b>, the main effect of stimulation protocol for each approach, is shown as a function of concentration to highlight that rates of increase in CR probability as a function of concentration are not significantly different. The Y-axis represents conditioned response (CR) probability. Error bars represent +/-1 SE. Inset letters indicate significant differences between means (Tukeys HSD; p<0.05). </p

Hotwire anemometry establishes that odor valve actuation causes stereotypic flow artifacts.

<p><b>A</b>. Raw hotwire anemometry traces in response to four consecutive pulses of odor using both the interleaving (black traces) or puffing (red traces) and an olfactometer flow of 30 cm/s (left panel) or 80 cm/s (right panel). Left and right panel also display 40:10 ms and 10:40 ms duty cycles respectively. Note that in all cases the cycle rate is on a 50 ms time scale (i.e. a pulsing frequency of 20 Hz). <b>B</b>. Three continuous stimuli of differing durations: 800 ms (black), 3200 ms (red), and 4000 ms (blue). Traces shown are averaged across 3 repeats each to eliminate noise associated with measurement error of the Mini CTA probe (see <b>A</b> for raw traces). Inset colored bars indicate stimulus duration for corresponding colored traces. <b>C</b>. Close up and aligned view of on and off response artifacts shown in B. D. Mean anemometry response over two pulse cycles (pulse durations are inset black bars along x-axis). Results based on averaging 40 consecutive 100 ms samples from a 4 s 20 Hz pulse train. Inset green trace represents the predicted integration of the on and off artifacts shown in B. Error bars are ±1 SD. Note that both duty cycles produce a unique anemometry artifact profile. Nevertheless, these profiles are accurately described as a simple linear summation of the on and off artifacts shown in <b>B</b>.</p

Schematic of the olfactometer configuration and stimulation protocols for conditioning (A) and testing (B-D).

<p><b>A</b>. For Pavlovian conditioning of all moths, the odor cartridge was connected to the normally closed output line (in gray) and the normally open output line (in red) was not used. The nozzle was placed 10 cm from the moth to provide enough distance to create an odor dispersion field (inset gray triangle) wide enough to cover most of the antennae. During each conditioning trial, odor (conditioning stimulus) was presented for 4 s; 3 s into odor presentation, the moth was presented with 0.75 m sucrose solution (unconditioned stimulus). <b>B</b>. In experiments 1-4 and 7 conditioned moths were tested with pulsed odor that was interleaved with clean air. This was achieved by connecting the normally open output line (red) with to the output of the normally closed line (gray) after the odor cartridge. In this configuration air is constantly flowing out the nozzle (inset gray and red triangle), independent of odor. <b>C</b>. In order to create oscillations in air flow velocity the normally open output line was not used (as in A). In both B and C the odor delivery nozzle was positioned approximately 2-3 mm in front of a single antenna, which was held in position at its base. This insured that the temporal structure of the pulse train was preserved and that further dilution of the odor with the surrounding air was minimized. <b>D</b>. Stimulation protocols used during testing. Shown are the first 200 ms (4 pulses) of the 4 s 20 Hz pulse trains for both duty cycles and stimulation protocols (interleaved and puffed).</p

Enhanced sensitivity to pulsed stimuli is the result of lower false positive rates.

<p>t-test comparison of blank responses elicited by the pulsed vs continuous stimuli. The Y-axis represents response probability and inset letters indicate significant differences between means based on a Tukeys HSD (p<0.05). Inset lines show mean differences between pulsed and continuous blanks by experimental subgroup (N = 10 sub-groupings). </p