High-speed odor transduction and pulse tracking by insect olfactory receptor neurons Paul Szyszka a,b,1 , Richard C. Gerkin a , C. Giovanni Galizia b , and Brian H. Smith a a School of Life Sciences, Arizona State University, Tempe, AZ 85287; and b Department of Biology, University of Konstanz, 78464 Konstanz, Germany Edited by Lynn M. Riddiford, Howard Hughes Medical Institute, Janelia Farm Research Campus, Ashburn, VA, and approved October 17, 2014 (received for review June 27, 2014) Sensory systems encode both the static quality of a stimulus (e.g., color or shape) and its kinetics (e.g., speed and direction). The limits with which stimulus kinetics can be resolved are well understood in vision, audition, and somatosensation. However, the maximum temporal resolution of olfactory systems has not been accurately determined. Here, we probe the limits of temporal resolution in insect olfaction by delivering high frequency odor pulses and measuring sensory responses in the antennae. We show that transduction times and pulse tracking capabilities of olfactory receptor neurons are faster than previously reported. Once an odorant arrives at the boundary layer of the antenna, odor transduction can occur within less than 2 ms and fluctuating odor stimuli can be resolved at frequencies more than 100 Hz. Thus, insect olfactory receptor neurons can track stimuli of very short duration, as occur when their antennae encounter narrow filaments in an odor plume. These results provide a new upper bound to the kinetics of odor tracking in insect olfactory receptor neurons and to the latency of initial transduction events in olfaction. olfaction | olfactory receptor neurons | odor transduction | temporal resolution | insect O dors carried in air plumes quickly break up into thin fila- ments that spread out across short distances from an odor source (1). The ability to track the temporal structure of filaments in an odor plume is essential for insects to segregate concurrent odors that arise from different sources (2–6). However, it is not clear whether signal transduction times and tracking rates of ol- factory receptor neurons (ORNs) are fast enough to allow animals to use the higher frequency components of information present in odor plumes. Insect odor-guided behavior is remarkably robust against the spatial and temporal variability inherent in olfactory stimuli. For example, moths and beetles use temporal stimulus cues to segregate concurrent odors from closely spaced sources (2–5), and honey bees can detect 6-ms asynchrony in the onset of concurrent odor stimuli and use this onset asynchrony to segregate concurrent odors (6, 7). These observations of fast temporal res- olution challenge the frequent notion that olfaction has slow in- tegration times relative to other senses. Olfactory transduction speed has never been measured directly, and estimates range from 10 to 30 ms (8–11). Previous studies suggest that the maximum pulse tracking frequency of ORNs is species specific and ranges from 5 to 50 Hz (12–19). However, these numbers do not match the high temporal resolution observed in behavioral studies (2–7). We tested the limits of olfactory transduction speed and pulse tracking in five different insect species by measuring ORN pop- ulation responses using electroantennogram (EAG) recordings. The amplitude and dynamics of EAG signals are proportional to the number of sensilla stimulated (20). They are also affected by receptor current amplitude, positions of the neurons relative to the recording electrode, and electrical properties of the antenna itself (21). With appropriate odorless controls for electrical and me- chanical artifacts, measuring EAG signals provides reliable esti- mates of transduction latencies and pulse tracking ability of ORNs. Results We constructed an odor delivery device capable of delivering high frequency pulses (Fig. 1A and Fig. S1). Using titanium tetrachloride (TiCl 4 ) in separate experiments to visualize stimuli, we estimated that this device delivers odor pulses to the antenna in 3.3 ± 0.3 ms (mean ± SD) after triggering the valve to open (Fig. 1C). We produced repetitive pulses at frequencies of up to 200 Hz. To control for responses to mechanical stimuli in experiments with olfactory stimuli, we alternated odor and blank (odorless) stimuli and subtracted a blank control from the pre- ceding odor-evoked EAG signal (Fig. S2). Odor-evoked mean EAG responses began between 1.6 and 26.4 ms after odors arrived at the antenna (Fig. 1B). EAG re- sponse latency depended on odor identity and decreased with increases in concentration (Fig. 1C). The shortest EAG response onset latencies ranged from 1.6 ms in locusts to 4.6 ms in moths, and there were no systematic differences between general odors and species-specific sex or alarm pheromones (Fig. 1C). To test antennal pulse tracking capability we applied a 1-s-long series of odor pulses at intervals ranging from 6 to 100 ms (167–10 Hz; Figs. 2 and 3). Pulse tracking capability decreased with increasing pulse frequency and differed between odor- ants and concentrations, and the maximum pulse tracking frequencies ranged from 50 Hz in the orange spotted cock- roach to 125 Hz in the honey bee and locust (Fig. 3 A and B; see Fig. S3A for variability across antennae). Filaments in natural odor plumes arrive at random intervals and persist for random durations (1). We mimicked this type of pattern by applying a 10-s-long broadband frequency stimulus train with random pulse durations and intervals (Fig. 4A). Insect EAG responses have approximately linear frequency response functions over a wide frequency range, and coherence analysis Significance How fast can animals smell? Whereas we know how fast our eyes are (in the cinema, images at 24 Hz fuse for humans, whereas our retina can resolve flickers at more than 100 Hz), olfactory perception is believed to be slow. After all, we take a sniff and later another one. Odor plumes in the air, however, can fluctuate at a millisecond time scale. Here, we show that insect olfactory receptor neurons can have response latencies shorter than 2 ms and resolve odorant fluctuations at more than 100 Hz. This high temporal resolution could facilitate odor- background segregation, and it has important implications for underlying cellular processes (transduction), ecology (odor rec- ognition), and technology (development of fast sensors). Author contributions: P.S., R.C.G., and B.H.S. designed research; P.S. performed research; P.S., R.C.G., C.G.G., and B.H.S. analyzed data; and P.S., R.C.G., C.G.G., and B.H.S. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. 1 To whom correspondence should be addressed. Email: [email protected]. This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1412051111/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1412051111 PNAS Early Edition | 1 of 6 NEUROSCIENCE
Transcript
High-speed odor transduction and pulse tracking byinsect olfactory receptor neuronsPaul Szyszkaa,b,1, Richard C. Gerkina, C. Giovanni Galiziab, and Brian H. Smitha
aSchool of Life Sciences, Arizona State University, Tempe, AZ 85287; and bDepartment of Biology, University of Konstanz, 78464 Konstanz, Germany
Edited by Lynn M. Riddiford, Howard Hughes Medical Institute, Janelia Farm Research Campus, Ashburn, VA, and approved October 17, 2014 (received forreview June 27, 2014)
Sensory systems encode both the static quality of a stimulus (e.g.,color or shape) and its kinetics (e.g., speed and direction). Thelimits with which stimulus kinetics can be resolved are wellunderstood in vision, audition, and somatosensation. However,the maximum temporal resolution of olfactory systems has notbeen accurately determined. Here, we probe the limits of temporalresolution in insect olfaction by delivering high frequency odorpulses and measuring sensory responses in the antennae. Weshow that transduction times and pulse tracking capabilities ofolfactory receptor neurons are faster than previously reported.Once an odorant arrives at the boundary layer of the antenna,odor transduction can occur within less than 2 ms and fluctuatingodor stimuli can be resolved at frequencies more than 100 Hz.Thus, insect olfactory receptor neurons can track stimuli of veryshort duration, as occur when their antennae encounter narrowfilaments in an odor plume. These results provide a new upperbound to the kinetics of odor tracking in insect olfactory receptorneurons and to the latency of initial transduction events inolfaction.
Odors carried in air plumes quickly break up into thin fila-ments that spread out across short distances from an odor
source (1). The ability to track the temporal structure of filamentsin an odor plume is essential for insects to segregate concurrentodors that arise from different sources (2–6). However, it is notclear whether signal transduction times and tracking rates of ol-factory receptor neurons (ORNs) are fast enough to allow animalsto use the higher frequency components of information present inodor plumes. Insect odor-guided behavior is remarkably robustagainst the spatial and temporal variability inherent in olfactorystimuli. For example, moths and beetles use temporal stimuluscues to segregate concurrent odors from closely spaced sources(2–5), and honey bees can detect 6-ms asynchrony in the onset ofconcurrent odor stimuli and use this onset asynchrony to segregateconcurrent odors (6, 7). These observations of fast temporal res-olution challenge the frequent notion that olfaction has slow in-tegration times relative to other senses. Olfactory transductionspeed has never been measured directly, and estimates range from10 to 30 ms (8–11). Previous studies suggest that the maximumpulse tracking frequency of ORNs is species specific and rangesfrom 5 to 50 Hz (12–19). However, these numbers do not matchthe high temporal resolution observed in behavioral studies (2–7).We tested the limits of olfactory transduction speed and pulse
tracking in five different insect species by measuring ORN pop-ulation responses using electroantennogram (EAG) recordings.The amplitude and dynamics of EAG signals are proportional tothe number of sensilla stimulated (20). They are also affected byreceptor current amplitude, positions of the neurons relative to therecording electrode, and electrical properties of the antenna itself(21). With appropriate odorless controls for electrical and me-chanical artifacts, measuring EAG signals provides reliable esti-mates of transduction latencies and pulse tracking ability of ORNs.
ResultsWe constructed an odor delivery device capable of deliveringhigh frequency pulses (Fig. 1A and Fig. S1). Using titaniumtetrachloride (TiCl4) in separate experiments to visualize stimuli,we estimated that this device delivers odor pulses to the antennain 3.3 ± 0.3 ms (mean ± SD) after triggering the valve to open(Fig. 1C). We produced repetitive pulses at frequencies of upto 200 Hz. To control for responses to mechanical stimuli inexperiments with olfactory stimuli, we alternated odor and blank(odorless) stimuli and subtracted a blank control from the pre-ceding odor-evoked EAG signal (Fig. S2).Odor-evoked mean EAG responses began between 1.6 and
26.4 ms after odors arrived at the antenna (Fig. 1B). EAG re-sponse latency depended on odor identity and decreased withincreases in concentration (Fig. 1C). The shortest EAG responseonset latencies ranged from 1.6 ms in locusts to 4.6 ms in moths,and there were no systematic differences between general odorsand species-specific sex or alarm pheromones (Fig. 1C).To test antennal pulse tracking capability we applied a 1-s-long
series of odor pulses at intervals ranging from 6 to 100 ms(167–10 Hz; Figs. 2 and 3). Pulse tracking capability decreasedwith increasing pulse frequency and differed between odor-ants and concentrations, and the maximum pulse trackingfrequencies ranged from 50 Hz in the orange spotted cock-roach to 125 Hz in the honey bee and locust (Fig. 3 A and B;see Fig. S3A for variability across antennae).Filaments in natural odor plumes arrive at random intervals
and persist for random durations (1). We mimicked this type ofpattern by applying a 10-s-long broadband frequency stimulustrain with random pulse durations and intervals (Fig. 4A). InsectEAG responses have approximately linear frequency responsefunctions over a wide frequency range, and coherence analysis
Significance
How fast can animals smell? Whereas we know how fast oureyes are (in the cinema, images at 24 Hz fuse for humans,whereas our retina can resolve flickers at more than 100 Hz),olfactory perception is believed to be slow. After all, we takea sniff and later another one. Odor plumes in the air, however,can fluctuate at a millisecond time scale. Here, we show thatinsect olfactory receptor neurons can have response latenciesshorter than 2 ms and resolve odorant fluctuations at more than100 Hz. This high temporal resolution could facilitate odor-background segregation, and it has important implications forunderlying cellular processes (transduction), ecology (odor rec-ognition), and technology (development of fast sensors).
Author contributions: P.S., R.C.G., and B.H.S. designed research; P.S. performed research;P.S., R.C.G., C.G.G., and B.H.S. analyzed data; and P.S., R.C.G., C.G.G., and B.H.S. wrotethe paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.1To whom correspondence should be addressed. Email: [email protected].
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1412051111/-/DCSupplemental.
www.pnas.org/cgi/doi/10.1073/pnas.1412051111 PNAS Early Edition | 1 of 6
provides a tool to measure the ORNs’ temporal resolution (15,16). We identified the antennae’s temporal resolution as themaximum stimulus frequencies at which the coherence betweenthe EAG response and the TiCl4 smoke signal was significant atthe 5 sigma level (Fig. 4B). The temporal resolution differedbetween odorants and increased with concentration (Fig. 4C; seeFig. S3B for variability across antennae). Across insects, themaximum temporal resolution ranged from 114 to 473 Hz (8.8-to 2.1-ms interpulse intervals).Odor sampling is ongoing during behavior, but ORNs adapt
over a variety of timescales (22, 23). We therefore asked whetherthe fast EAG kinetics we observed adapt across odor pulses andhow this adaptation would affect the maximum temporal reso-lution. For honey bee EAGs, the temporal resolution was higherduring the late odor response than during the initial odor re-sponse, whereas for the other insect species the temporal reso-lution appeared to be invariant to stimulus duration (Fig. 4D andFig. S4).To further demonstrate that insect antennae are capable of
resolving fast changes in odor concentration, we configured theodor delivery valve to play the notes of a children’s song aboutbees (Fig. S5 and Audio File S1), releasing a barrage of odorpulses (2-heptanone) at frequencies between 100 and 200 Hz(1/interval between pulse onsets) with each note (Audio File S2).We recorded the EAG during this song, and subtracted the cor-responding EAG when the same song was played by using blank
control pulses. In this blank-subtracted EAG, the original songwas clearly recognizable (Audio File S3). Thus, insect antennaecan follow olfactory stimuli in a frequency range high enoughto significantly overlap that of the human auditory system.
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Fig. 1. Olfactory transduction onset can occur inless than 2 ms. (A) Stimulus dynamics visualizedwith TiCl4 smoke during 20 Hz and 167 Hz pulseseries. A laser was positioned at the location of theantenna, and the resulting light reflectance of thesmoke was recorded with a photodiode (mean ±SEM). SE (SEM) is visualized as a shaded area. (B)Color-coded periodograms of visualized TiCl4 smokesignals show that the odor delivery device is capableof producing pulse frequencies between 10 and 167Hz. (C) TiCl4 smoke signals (red, n = 16) and EAGresponses to 2-heptanone (black) (mean ± SEM). ForEAG recordings, odor and blank stimuli were alter-nated, and the blank control was subtracted fromevery preceding odor-evoked EAG signal. Signalswere averaged across 10 recordings for each antenna.n, number of averaged antennae. The values at thevertical lines are the computed onset times in milli-seconds. (D) Summary of mean EAG response onsetsfor different insect species and odors. The TiCl4 smokesignal onset time (3.3 ms) was subtracted from theEAG onset times to get the real EAG onset. Numbersin the graph show the mean EAG onset. Experimentsare grouped at the bottom by species and thenodorant and dilution, with the number of antennaein parentheses. O, orange spotted cockroach.
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Fig. 2. EAG responses in different insect species. EAG responses (black, mean ±SEM) to 20, 50, 83, and 125 Hz pulse series and a continuous stimulus in threeinsect species are shown. TiCl4 smoke signals (red) show the stimulus dynamic.
2 of 6 | www.pnas.org/cgi/doi/10.1073/pnas.1412051111 Szyszka et al.
DiscussionOur data show that the initial olfactory transduction processrequires less than 2 ms. This short transduction time contradictsprevious studies that suggested olfactory transduction times be-tween 10 and 30 ms (8–11). What makes olfactory transductionfast? Olfactory transduction involves several steps, including dif-fusion of the odorant molecule through the antenna surface, itsbinding to an odorant-binding protein (OBP), diffusion throughthe aqueous sensillar lymph to an ORN dendrite, activation of anodor receptor, and opening of ion channels (11, 24, 25). Modelingstudies suggest that diffusion can delay the initial ORN responseby 10 ms (11, 26). Using available data about OBPs and thesensillum lymph, we estimate that >17% of the odorant moleculesshould reach the ORN dendrite by 3D diffusion alone within 1 msof encountering the lymph surface (SI Materials and Methods).Diffusion speed might be further increased by pore tubules thatconnect the sensillum pore with the ORN dendrite (8, 27, 28).Moreover, OBPs occur at high concentrations (10 mM) in thesensillum lymph of insects (29, 30), which improves ORN sensi-tivity (25) and might decrease transduction time.Insect olfactory transduction mechanisms include both iono-
tropic and metabotropic components (10, 24, 31, 32). Althoughionotropic transduction is believed to be faster, both trans-duction mechanisms are capable of high temporal resolution.For example, insect photoreceptor cells are metabotropic andreach temporal resolutions of 300 Hz (33). Unlike resolution,however, the transduction latencies of most metabotropic re-ceptors are at least one order of magnitude longer than the fewmillisecond short transduction latencies that we found. Metab-otropic olfactory receptors of vertebrates, for example, haveminimum transduction latencies between 50 and 150 ms (34, 35),and the fastest metabotropic receptors in photoreceptors of thefly have a minimum transduction latency of 20 ms (36). There-fore, the fast transduction latencies we found here—4.6 ms orless across all tested species—support the hypothesis that theprimary transduction mechanism for insect odor receptors isionotropic.Single ORNs typically have spike rates well below 350 Hz (8, 9,
22, 23). We found that EAG responses can follow odor fluctu-ations that exceed 350 Hz. It is therefore likely that higher pulsefrequencies are encoded by the volley principle (37): SingleORNs might respond to repetitive odor pulses intermittently sothat the combined ensemble activity is able to track frequenciesthat exceed the tracking capabilities of a single ORN.Previous studies reported maximum odor-tracking frequencies
between 5 and 50 Hz in EAG and single ORN recordings (12–14,16, 18, 38, 39) and between 10 and 30 Hz in olfactory interneuronsin the antennal lobe (17, 40, 41). These lower maximum pulsetracking frequencies might reflect the low-pass filter properties ofthe odor delivery devices. Odor delivery devices typically act asa low-pass filter because of the dead volume in the odor chamberof the device, and because odorants adhere to surfaces used toconstruct the device (23). In some studies, odor stimuli were givenas randomized fluctuating pulses and monitored at frequencies upto ∼100 Hz, still limiting the investigation of pulse tracking tovalues below its biological limits (15, 16).Fast olfactory transduction speeds could facilitate the recog-
nition of a target odor in the presence of background odors.Insects rely on olfaction to localize resources such as food,mates, or hosts (42). Finding an odor source poses a particularchallenge: Odor plumes break into thin filaments, and relevanttarget odors intermingle with background odors (43). Insects andslugs can exploit temporal differences in the arrival of odorantsto segregate a target odor from background odors that emanatefrom different sources (2, 3, 5–7, 44–46). In this task, fast ol-factory transduction might enable animals to resolve short dif-ferences in the arrival of a target odor and background odors.
We cannot directly extrapolate our results to mammalian ol-faction, because both olfactory transduction mechanisms andtemporal constraints differ across phyla. For example, mammalsimpose a sniffing rhythm onto odor samples that they takefrom the environment (47). Nevertheless, high temporal res-olution might be used by mammals to detect minor temporaldifferences in adsorption of odorants along the mucosal lining,which could allow odor identification based on odorant-spe-cific ORN response latencies (48).Finally, the fast temporal resolution of insect olfaction we
report here has implications for the development of artificialodor sensors for odor-source localization. The segregation ofa target odor from background odors via temporal stimulus cues(2–4, 6, 7, 49) requires fast sensor operation. Current artificialodor sensors, e.g., metal oxide sensors, have a temporal resolu-tion lower than that of the antennae investigated here (50). Thus,hybrids of engineered devices with biological sensors might im-prove the performance of new devices (51–53).
Materials and MethodsAnimals. Experiments were performed on adult orange spotted cockroachBlaptica dubia of both sexes, hissing cockroaches Gromphadorhina porten-tosa of both sexes, male locusts Schistocerca americana, female foragerhoney bees Apis mellifera, and male moth Manduca sexta 3–8 d afterhatching. Moth EAGs were recorded during the night.
Odorants. Odorants were used pure or diluted in mineral oil (Sigma-Aldrich).Odorant dilutions were prepared fresh every 4 wk. All dilutions are reportedas parts per one part of mineral oil. The odorants used were as follows: thehoney bee alarm pheromones and plant odors 2-heptanone (dilutions: 0.001,0.01, 0.1, undiluted) and isopentylacetat (0.032); a 1:1mixture of the syntheticM. sexta sex pheromone components E,Z-10,12-hexadecadienal and E,Z-11,13-pentadecadienal (Bom, 10 ng/μL); and the plant odors linalool (un-diluted) 1-hexanol (0.1, undiluted); 1-nonanol (undiluted); and lemon oil(0.1). Pure mineral oil served as blank control. One microliter of Bom and10 μL of all other odorant solutions were loaded onto a cellulose strip (Sugi,REF 31003; Kettenbach) located in a 3-mL syringe (Norm-Ject; Henke-Sass,Wolf), adjusted to 2.5-mL syringes were prepared daily.
Odorant Delivery Device. We built an odor delivery device with minutedead space and adsorbent surfaces to ensure that the odor dynamics atthe antenna reflected the dynamics of the valve as faithfully as possible(Fig. S1A). Odor stimuli were delivered with a three-way solenoid valve(LFAA1200118H; Lee) (Fig. S1A). Odor syringes were supplied with air viaa manifold that was designed to minimize cross-contamination. Themanifold was a Teflon septum equipped glass vial (20 mL of headspacevial; Schmidlin Labor and Service), which was connected to the odor sy-ringe and the valve via injection needles (1.20 × 40 mm, Sterican; Braun).The needles served as nonreturn valves. This odor delivery device wassupplied with pressurized, charcoal filtered, dry air. The air stream wasadjusted to 260 mL/min by a flow meter. The three-way solenoid valvecontrolled the odor pulses by diverting air from the manifold to theodorant syringe. The valve was switched with a spike-and-hold driver cir-cuit to minimize opening time. The valve was shielded with an iron cage. Astainless steel tube (inner diameter 1.2 mm) served as outlet. The air speedat the outlet of the steel tube was 383.15 cm/s.
The distance between antenna and the gate of the valve was 15.6 mm.The gate of the valve started moving 0.4 ms after the electrical triggersignal that switches the valve (Fig. S1B). Assuming a laminar airflow and nopressure changes, the odor would arrive 4.47 ms after the trigger signalthat operated the valve (0.4-ms valve delay plus 4.07-ms air travel time).For measuring the movement of the valve gate, a valve was cut open anda laser beam was directed in a 45° angle onto the gate and a photodiodewith a 0.2-mm pinhole was placed in the focus of the reflected laser beam.Movement of the valve caused changes in the light intensity and wasvisible as changes in the photodiode signal. The analog photodiode signalwas digitized with the same recording setup and settings that were usedfor the EAG recordings (see below).
The arrival time and dynamics of the odor stimulus were measured witha photodiode, a green laser, and TiCl4 smoke as tracer substance (Fig. S1C).The laser was positioned at the location of the antenna, perpendicular tothe airflow, and the reflectance of the smoke was recorded with a photo-diode. One microliter of TiCl4 solution was placed in an odor syringe. Before
starting the pulse sequence, the valve was opened for 1 s (from 2,000 to1,000 ms before pulse onset) to fill the valve with fresh smoke.
To estimate the effect of diffusion on the relative arrival times of odorantsand TiCl4 and, thus, on EAG onset, we calculated the time needed for a smallfraction (0.1%) of the molecules to reach the antenna by diffusion alone. TheSD or root-mean-square displacement of molecules along one dimension ina diffusion profile at time t is given by square root (2 × D × t). The diffusioncoefficient, D, in air for the odorant from our panel with the lowest-latencyEAG response, 2-heptanone, is ∼7.3 mm2/s. By definition, 0.1% of moleculeswill be found at least ∼2.33 SDs from the diffusion source, a distance we taketo be the 15.6 mm from the valve gate to the antenna. Solving gives a SD of6.7 mm, and a corresponding diffusion time of 6.7 mm2/(2 × 7.3 mm2/s) = 0.46 s.In contrast, the TiCl4 smoke reaches the antenna within 3.3 ms. Therefore,odorant delivery via diffusion is more than 100 times slower than odorantdelivery via airflow and, thus, for our odor delivery device, diffusion in airis not a relevant concern for the early EAG response.
Odorant Stimuli. Odorants were delivered as continuous 1-s long pulses, 1-slong trains of 3-ms-long pulses at frequencies of 10, 20, 33, 50, 67, 83, 100,125, and 167 Hz, or 10-s-long broadband frequency stimulus trains withtheoretically flat power spectrum. The power spectrum of the actualbroadband stimulus is colored because of the noninstantaneous impulseresponse of the valve (Fig. S1D). The broadband frequency stimulus wasgenerated by constructing a 12-bit M-sequence, scaling to achieve a mini-mum “on” time of 3 ms, and then selecting a random 10-s segment. Itconsisted of 12,330 pulses between 3 and 39 ms in length, with interpulseintervals (onset to onset) ranging from 56 to 6 ms.
The notes of the song “Biene” (Fig. S5 and Audio File S1) were encoded aspure tones, such that the first note “C5” was tuned to ∼“D3,” or 143 Hz.
These were played into the odor delivery device by opening and closing thevalve at the corresponding frequencies ranging between 100 and 200 Hz,with a fixed number of pulses representing the lengths of the notes (AudioFile S2). The resulting performance of the song by the valve thus matchesthe sheet music. EAG responses due to pressure from the air pulses wereaccounted for by recording in both the presence and the absence of anodorant (2-heptanone) loaded into the olfactometer and taking the differ-ence. The resulting subtracted EAG thus reflects a purely odor-driven response.This EAG response was averaged across four antennae (two honey bee and twomoth antennae), and band-pass filtered (40–400 Hz) to remove subauditory slowchanges in the EAG and to denoise the response. The resulting waveform wasupsampled andmultiplied by a constant to produce a sound file, but the spectralpeaks were unaltered (Audio File S3).
EAG Recordings. Antennae were cut and mounted with conductive gel (lu-bricating jelly; CVS pharmacy) between the two poles of a custom-made silverelectrode. For EAG recordings from honey bees, the scapus was cut at theflagellum joint and removed (Fig. S1A). For other insects, 5-mm-long antennapieces were used. Differences in the dynamic properties of the EAG responsesbetween insect species might reflect differences in the number of stimulatedORNs and difference in the electrical properties of the antenna preparations.Recordings started 15 min after mounting the antennae. The signal wasamplified, band-pass filtered to select the 0.1–9 kHz range, and digitizedat a sampling rate of 30 KHz with an extracellular recording system (Cheetah16;Neuralynx). Experiments were performed at 28 °C.
Data Analysis. Odor-evoked EAG signals were derived by subtracting EAGsignals in trials by using blank control stimulus from corresponding signals intrials with odor stimulus (Fig. S2A). Blanks recorded in trials before or after
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Frequency (Hz)
Stim
ulus
(Hz)
log(odor power/blank power)-0.6 (V²/Hz) 2.5
Stimulus83 Hz
100 Hz125 Hz
Stimulus67 Hz83 Hz
100 Hz
Stimulus67 Hz83 Hz
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Stimulus33 Hz50 Hz67 Hz
Min
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15 (
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12 (
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30 (
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10 (
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20 (
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20 (
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20 (
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0
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10
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Fig. 3. Antennal responses track pulses at 125 Hz ina 1-s-long odor stimulus. (A) Periodograms (mean ±SEM) of the EAG responses for the three highest re-solved pulse frequencies for different odors in dif-ferent insect species, and color-coded periodogramsfor all pulse series for the same species-odor combi-nation. A peak in the periodogram indicates that theEAG response followed that stimulus frequency. (B)Temporal resolution of EAG responses quantified asthe minimum interpulse interval in milliseconds(1/maximum pulse frequency) that an EAG responsecould follow. Minimum interpulse intervals weredetermined by locating the peak of the averageperiodogram in a ± 20-Hz window around the stim-ulus frequency. Pulse following was ascertained ifthe negative SE of the peak location rose above thepositive SE of the trough location within that win-dow. The corresponding pulse tracking frequenciesare given in parentheses. The number of antennaeis given in parentheses above the odorant.
4 of 6 | www.pnas.org/cgi/doi/10.1073/pnas.1412051111 Szyszka et al.
odor trials had similar properties, i.e., no long-term odor adaptation wasobserved. There was a small nonolfactory EAG signal that occurred beforethe odor reaches the antenna and that coincided with the closing of thevalve (Fig. S2B). The amplitudes of this nonolfactory signal ranged between12 and 36 μV compared with odor-evoked signals between 73 and 568 μV.We assume that these signals are inductive artifacts that arise from currentflow in the coil of the valve when the valve closes. The subtraction of theblank evoked from the odor-evoked EAG signal removed the stimulus arti-facts in the analyzed data (Fig. S2B).
Odor-evoked EAG signals were baseline-subtracted and valve-triggeredaverages were constructed by taking themean EAG response centered on theonset of the first pulse of each pulse train.
EAG response or TiCl4 signal onset times (Fig. 1 C and D) were determinedby using the continuous stimulus and the first pulse of each of the 10- to167-Hz stimuli. For each antenna or TiCl4 recording, the onset was defined asthe time point where the mean and SD of the signal to the first pulse in eachof 10 stimuli was greater than (and stayed above for at least 1 ms to avoidfalse onsets) 2.5 times the SD of the signal during the first 3 ms after valvetrigger (12 points).
Periodograms were estimated by using Welch’s method with 90% overlapand 1-s window size. Odor-evoked periodograms were calculated for singlerecordings by subtracting the periodogram derived from the EAG responseto the blank control from the periodogram derived from the EAG responseto the odor stimulus.
The periodogram in Fig. S1D was constructed from the final 9 s of 10-sstimuli to capture steady-state dynamics.
Coherence between the averaged, blank corrected responses to odorstimulus and to the averaged TiCl4 smoke signal (recorded on separate trialsin response to the same stimulus) was computed by using 265-ms segments(1,000 samples after 8× down-sampling) with 50% overlap. Blank sub-traction occurred in the time domain. Confidence intervals were computedby creating shuffles of the TiCl4 signal. The shuffle procedure consisted ofrandomizing phases in the frequency domain but maintaining an identicalpower spectrum. The SD of the coherence spectrum across shuffles was usedas an estimate of the SD of the unshuffled coherence. Coherograms werecomputed by using a sliding coherence estimate, with 1-s windows and 95%overlap. Within each window, the coherence was estimated by using 50-mssubwindows and 95% overlap.
ACKNOWLEDGMENTS. We thank Elizabeth Cash, Meghan E. Duell, Cécile P.Faucher, Hong Lei, Danielle Protas, and Zachary Shaffer for insects supply;Cécile P. Faucher and Hong Lei for Manduca sex pheromone; Cornelis Klokand Benjamin Paffhausen for technical support; and John G. Hildebrand,Ramón Huerta, Christopher Jernigan, Christoph J. Kleineidam, and IrinaSinakevitch for discussions. This work was financially supported by theBundesministerium für Bildung und Forschung Grant 01GQ0931 (to P.S.and C.G.G.) and by Deutsche Forschungsgemeinschaft Priority ProgramSPP1392 (to C.G.G.).
Fig. 4. Antennal responses track odor fluctuations inthe hundreds of Hertz range during a persistentbroadband stimulus. (A) TiCl4 smoke signals (red, n =12) and odor-evoked EAG responses (black, 20 Hzhigh-pass filtered, onset truncated, n = 30) (mean ±SEM) during 0–0.2 and 9.8–10 s of a 10-s-longbroadband frequency stimulus train with randompulse durations and intervals. (B) Coherence betweenmean EAG responses and TiCl4 smoke signals (black)and coherence between mean EAG response and ashuffled TiCl4 smoke signal (gray, mean ± 5 SD). Thefirst 1,000 and the last 100 ms of the 10-s-longbroadband stimulus were skipped to avoid onset/offset effects. Coherence was defined significantwhen it was larger than the coherence for the shuf-fled data plus 5 SD. Values at the vertical lines are themaximum frequencies at which the EAG responseshows significant coherence. (C) Temporal resolutionof EAG responses quantified as the minimum inter-pulse interval in milliseconds (1/maximum pulse fre-quency) at which the coherence was significant, i.e.,larger than the coherence for the shuffled data plus 5SD. The corresponding pulse tracking frequencies aregiven in parentheses. Experiments are grouped at thebottom by species and then odorant and dilution,with the number of antennae in parentheses. (D)Time-resolved, color-coded coherence between themean honey bee EAG response to undiluted 2-hep-tanone and the mean TiCl4 smoke signal (same dataas in Fig. 4A). The time-resolved coherence indicatesthe degree to which the EAG response is phase-locked to the fluctuations of the odor concentration,as opposed to merely matching the frequency. Sub-stantial coherence at high frequencies is visiblethroughout the odor presentation, indicating thattracking can persist for several seconds.
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Supporting InformationSzyszka et al. 10.1073/pnas.1412051111SI Materials and MethodsWe estimated the proportion of odorant molecules that couldreach the ORN dendrite within 1 ms via diffusion as follows: Weassumed a molecular mass of 15 kDa for the odorant/OBPcomplex (1) and estimated a radius r of 1.63 nm according toestablished relationships between volume and molecular massfor folded proteins (2). By reference to reported values for otheraqueous biological solutions, we assumed a dynamic viscosity ηof sensillum lymph of 0.002 Pa·s. The distance d between theORN dendrite and sensillum pore was taken to be 0.3 μm (3).Using the Stokes–Einstein equation, the diffusion constant D =k × T/(6 × π × η × r), where k is Boltzmann’s constant and T is
the temperature during experiments (25° C = 298 K). Plugging inthe values above gives D ∼5 × 10−11 m2·s−1. Concentration atdistance x in a profile along the dimension of interest evolves ac-cording to <x2>1/2 = (2 × D × t)1/2, where <x2>1/2 is the SD of thedisplacement. At t = 1 ms, this distance equals ∼0.316 μm. Takingthis value as the SD, and a target distance of 0.3 microns, the in-verse normal cumulative distribution function yields a quantile of0.171, implying that 17.1% of the molecules will have reachedthe target after 1 ms. Simulation using difference equations andapplying the actual boundary conditions (0 < x < 0.3 μm) givesan even larger proportion.
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3. Shanbhag SR, Muller B, Steinbrecht RA (1999) Atlas of olfactory organs of Drosophilamelanogaster - 1. Types, external organization, innervation and distribution of olfac-tory sensilla. Int J Insect Morphol 28(4):377–397.
Szyszka et al. www.pnas.org/cgi/content/short/1412051111 1 of 6
Fig. S1. The odor delivery device. (A) Scheme of the odor delivery device. 1, glass vial with Teflon septum, 20 mL; 2, injection needle, 1.2 × 40 mm; 3, tygontube, 1 mm inner diameter; 4, plastic syringe, 2.5 mL; 5, cellulose strip with odorant; 6, three-way solenoid valve; 7, steel tube, 1.2 mm inner diameter;8, antenna; 9, electrode. (B) Movement of the gate that opens the valve. The valve starts moving 0.4 ms after the electrical trigger signal measured by directinga laser beam onto the gate and measuring the reflected light with a photodiode. (C) Odor arrival at the antenna was visualized with TiCl4 smoke. A laser waspositioned perpendicular to the airflow at the location of the antenna, and the resulting light reflectance of the TiCl4 smoke was recorded with a photodiode.Numbers refer to A. (D) Periodogram of TiCl4 smoke signals during the final 9 s of the broadband stimulus (mean ± SD, n = 12).
Szyszka et al. www.pnas.org/cgi/content/short/1412051111 2 of 6
Fig. S2. Calculation of odor-evoked EAG responses. To exclude nonolfactory EAG signals, odor and blank control stimuli were alternated, and blank-evokedEAG signals (green) were subtracted from odor-evoked EAG signals (magenta). (A) EAG responses to 3-ms pulses of 2-heptanone delivered at 20-Hz pulses (grayvertical bars) and at different concentrations. The odor delivery device produced a short stimulus artifact (arrowhead) that coincided with the offset of thevalve trigger. The stimulus artifact was visible in the odor-evoked and blank evoked EAG signal (Ai), but not in the subtracted EAG responses (Aii). (B) EAGresponses to 3-ms and 1-s-long pulses of undiluted 2-heptanone. The stimulus artifact evoked by the stimulus offset, because it was visible 3 ms after the onsetof 3-ms pulses but not after the onset of 1-s pulses (Bi). The stimulus artifact was not visible in the subtracted EAG responses (Bii). (C) Honey bee EAG responsesto 3-ms pulses of undiluted 2-heptanone delivered at 100, 125, and 167 Hz (gray vertical bars) before (Ci) and after (Cii) subtraction of the blank-evoked EAGsignal. Odor-evoked EAG response could follow 125-Hz but not 167-Hz pulses (compare with power spectra in Fig. 3A). (Scale bars: 0.2 mV.)
Szyszka et al. www.pnas.org/cgi/content/short/1412051111 3 of 6
Fig. S3. Across-antenna variability of maximal temporal resolution of EAG responses. (A) Temporal resolution during a 1-s-long fixed frequency pulse train.Mean and SD of minimum resolvable interpulse intervals across antennae. Minimum resolvable interpulse interval (1/maximum pulse frequency) was ascer-tained if the value of the odor-evoked periodogram at the stimulus frequency was two times higher than the surrounding baseline ± 20 Hz. Odor-evokedperiodograms were calculated for single recordings by taking the periodogram of the EAG response to the odor stimulus and subtracting the correspondingperiodogram of the “blank” air EAG response. Numbers in the graph show the mean resolvable interpulse interval (1/frequency), and the corresponding pulsetracking frequencies are given in parentheses. The gray number above the graph shows the actual value of the truncated SD. At the x-axis label, the first valuein the parentheses indicates the total number of antennae, the second value indicates the number of antennae in which no response onset or minimuminterpulse interval could be detected. (B) Temporal resolution during a 10-s-long broadband frequency pulse train. Mean and SD of minimum resolvableinterpulse intervals across antennae. Minimum resolvable interpulse intervals were determined as in Fig. 4 B and C, except that the coherence between EAGresponse and TiCl4 smoke signal was calculated for each single antenna. Numbers in the graph show the mean resolvable interpulse interval, and the cor-responding pulse tracking frequencies are given in parentheses. The first value in the parentheses indicates the total number of antennae; the second valueindicates the number of antennae in which no minimum interpulse interval could be detected.
Szyszka et al. www.pnas.org/cgi/content/short/1412051111 4 of 6
Fig. S4. Temporal resolution of EAG responses during initial and late odor responses. (A) Coherence between EAG responses and smoke signals measuredduring the initial (0.2–2.7 s) and late (7.3–9.8 s) period of a 10-s-long broadband frequency stimulus train with random pulse durations. The values at thevertical lines show the maximum EAG frequency responses with significant coherence. (B) Temporal resolution of EAG responses quantified as the minimuminterpulse interval (1/maximum pulse frequency) at which the coherence is still significant.
Szyszka et al. www.pnas.org/cgi/content/short/1412051111 5 of 6
Fig. S5. Children’s song “Biene” by August Heinrich Hoffmann von Fallersleben, 1843. The note “F4” was replaced by “F5” to get a wider frequency range.Sheet music was generated in Musescore (musescore.org).
Audio File S1. Children`s song “Biene.” The audio file was generated in Musecore.
Audio File S1
Audio File S2. The song “Biene” transposed to notes in the 100- to 200-Hz range and played with the valve of the odor delivery device. Note that the songstopped prematurely in the second-to-last measure because of configuration of the stimulation software.
Audio File S2
Audio File S3. Odor-evoked EAG responses during the presentation of the song “Biene” with the odor delivery device (mean of 2 bee and 2 moth antennae,40–400 Hz band-pass filtered).
Audio File S3
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