Behavioral discrimination and olfactory bulb encoding of odor plume intermittency
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eLife assessment
This important work addresses an interesting question for the vertebrate olfactory community of whether mice can discriminate odorant intermittency to help them navigate the environment. The data were collected and analyzed using solid methodology, however, the paper seems to fall short in demonstrating that animal is actually sensitive to intermittency but not other flow parameters. The work will be of interest to researchers working on sensory neurobiology and animal behavior.
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Abstract
In order to survive, animals often need to navigate a complex odor landscape where odors can exist in airborne plumes. Several odor plume properties change with distance from the odor source, providing potential navigational cues to searching animals. Here, we focus on odor intermittency, a temporal odor plume property that measures the fraction of time odor is above a threshold at a given point within the plume and decreases with increasing distance from the odor source. We sought to determine if mice can use changes in intermittency to locate an odor source. To do so, we trained mice on an intermittency discrimination task. We establish that mice can discriminate odor plume samples of low and high intermittency and that the neural responses in the olfactory bulb can account for task performance and support intermittency encoding. Modulation of sniffing, a behavioral parameter that is highly dynamic during odor-guided navigation, affects both behavioral outcome on the intermittency discrimination task and neural representation of intermittency. Together, this work demonstrates that intermittency is an odor plume property that can inform olfactory search and more broadly supports the notion that mammalian odor-based navigation can be guided by temporal odor plume properties.
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Author Response
Reviewer #2 (Public Review):
The study from Gumaste et al investigates whether mice can use changes of intermittency, a temporal odor feature, to locate an odor source. First, the study tries to demonstrate that mice can discriminate between low and high intermittency and that their performance is not affected by the odor used or the frequency of odor whiffs. Then, they show that there is a correlation between glomerular responses (OSNs and mitral cells) and intermittency. Finally, they conclude that sniffing frequency impacts the behavioral discrimination of intermittency as well as its neural representation. Overall, the authors seek to demonstrate that intermittency is an odor-plume property that can inform olfactory navigation.
The paper explored an interesting question, the use of intermittency of an odor plume …
Author Response
Reviewer #2 (Public Review):
The study from Gumaste et al investigates whether mice can use changes of intermittency, a temporal odor feature, to locate an odor source. First, the study tries to demonstrate that mice can discriminate between low and high intermittency and that their performance is not affected by the odor used or the frequency of odor whiffs. Then, they show that there is a correlation between glomerular responses (OSNs and mitral cells) and intermittency. Finally, they conclude that sniffing frequency impacts the behavioral discrimination of intermittency as well as its neural representation. Overall, the authors seek to demonstrate that intermittency is an odor-plume property that can inform olfactory navigation.
The paper explored an interesting question, the use of intermittency of an odor plume as a behavioral cue, which is a new and intriguing hypothesis. However, it falls short in demonstrating that the animal is actually sensitive to intermittency but not other flow parameters, and is missing some important details.
Major concerns
- One of the cornerstones of this paper consists in showing that mice are behaviorally able to distinguish among different intermittency values (high or low), across a variety of different stimuli and without confounds such as the number of whiffs or concentration. However, I could not find in the paper a convincing explanation of how these confounds were tested. It is clear that the authors repeat their measurements in different conditions (low or high concentration, and different whiff numbers) but it is not specified how: do the authors mix all stimuli in the same session, and so the animals simply generalize across all the stimuli and only consider intermittency for the behavioral choices? Or do authors repeat different sessions for different parameters? For example: do they perform two separate sessions with low concentration and high concentration? If this last one is the case, I would argue that this is not enough proof that animals generalize across concentrations, as the animals might simply use concentration as a cue and change the decision criteria at each session. Please clarify.
We appreciate the reviewer pointing out our oversight in including this information in the manuscript. Trials of the two gain values (which modulate the maximum concentration) are presented interleaved within a session. These trials are solely separated for post-session analysis to test the effect of gain on animal performance. To make this point clearer we have included the following text on line 952 of the manuscript:
“Additionally, trials of a gain of 0.5 and a gain of 1 are interwoven randomly during the session with each unique stimulus being presented at both a gain of 0.5 and 0.1. Thus, after the initial engagement trials, animals are presented with a total of 28 trials at a gain of 0.5 and 28 trials at a gain of 0.1.”
Additionally, to address one of the reviewer’s overarching points, that the manuscript “falls short in demonstrating that the animal is actually sensitive to intermittency but not other flow parameters,” we would like to highlight that through our olfactometer design (described in the Olfactometer Design subsection of the Methods section and illustrated in Figure 1C) the flow rate is held constant throughout the experiment. To further ensure that the animal is not using flowrate or other experimental conditions to perform the task, we tested all animals on a “no odor” condition in which the vial of odor is replaced with a vial of mineral oil. In this condition, their hit rate significantly lowered, as shown in Figure 2C and described in Lines 240- 245:
“Animals’ hit rate also significantly decreased when tested on the Go/No-Go task with the odor vial replaced with mineral oil (n=12 mice, two-sample t-test Naturalistic: odor hit rate = 0.87 ±0.01, no odor hit rate= 0.23 ±0.05, p<0.0001; two-sample t-test Binary Naturalistic: odor hit rate= 0.89±0.01, no odor hit rate= 0.18±0.07, p<0.0001; two-sample t-test Synthetic: odor hit rate= 0.86±0.007, no odor hit rate= 0.23±0.07, p<0.0001), confirming that mice are using odor to perform the task.”
- It looks to me that the measure of intermittency strongly depends on the set. What is the logic of setting a specific threshold? Do the results hold when this threshold changes within a reasonable range? The same questions (maybe even more important) go for the measure of glomerular intermittence. Unfortunately, a sensitivity analysis for both measures is missing, which makes it hard to interpret the results.
We assume the reviewer suggests that we could have tested discrimination at various Intermittency thresholds. This is indeed wat we did, though not by varying the threshold parametrically (due to abovementioned time constraints), but rather qualitatively/categorically. We tested our mice on 3 stimulus "types" (Figure 1F): actual continuous plume concentration traces (naturalistic), thresholded traces (binarized by threshold 0.1) and square wave (odor agnostic periodic binary). Further, each was tested at 2 gain levels. Figure 2B demonstrates mice discriminate similarly across these 3 widely differing stimuli, while traces were spanning most of the range of possible intermittencies. Reducing the threshold by 1 or 2 orders would skew the range of trials toward many more CS+ trials. We hence conclude that the mice are robustly discriminating and that the paradigm chosen and its associated constraints provide a reasonable test of "intermittency space".
We agree nonetheless that future work should address your suggestion directly by implementing an alternate paradigm. For example, in such a paradigm, mice may be trained to discriminate high vs low intermittencies at varying absolute levels (e.g. 1 vs 0.9 and 0.1 vs 0), etc., however that was well outside the scope of what we aimed to test.
See Figure 1- Supplement 1A. We varied the threshold half a log unit around the 0.1 threshold used in the neuro-behavioral research. As expected, the higher the odor threshold, the more left-shifted the curve. You can see that the monotonic relationship is qualitatively the same across thresholds.
- The logic of choosing the decision boundary for the discrimination task is not clear: low intermittency is considered to be below 0.15 and high intermittency is considered to be between 0.2 and 0.8. Do these values correspond to natural intermittency distribution? How were these values chosen?
Intermittency drops as function of distance from the source (downwind). It also has a close to normal (with kurtosis) distribution across wind, peaking at the center (see e.g. Crimaldi 2002, Connor 2018). So, animals may encounter any and all intermittencies (0-1). Given our Go/No-Go paradigm we had to set a CS-/CS+ boundary. Typically, to generate an adequate psychometric curve using this paradign, either the CS- or CS+ stimuli need to represent a wide range of values of which the animals are required to compare against a narrow range (or single value). Again, bounded by effective behavioral paradigm design, the number of CS+ and CS- trials need to be even in order to appropriately motivate animals to engage in the task. Thus, considering the entire range of intermittency values animals can encounter while navigating through a plume in conjunction with effective behavioral design, we arrived at our chosen values for low and high intermittency.
As you can see in Figure 1- Supplement 1A (and also reviewer #1, comment 2), I=0.15 is roughly at the knee where the monotonic decrease begins to asymptote. This is roughly true for all 3 concentration thresholds. Consequently, I=0.2-0.8 effectively samples the region where intermittency clearly relates to distance to the source, which is where we hypothesize animals.
- Only 2 odors were used in the whole study and some results were in disagreement between the two odors. By looking at only two odors it is very difficult to make a general conclusion about intermittency encoding in the OB.
We agree 2 odors are limited, but we were constrained in terms of number of tests that we could run on our cohort of animals. Nonetheless intermittency of both odors is clearly discriminable. As explained to comment 3 by Reviewer 1:
“We indeed considered several odorants and associated properties. Given time constrains we were limited to 2 stimuli of which we had to vary many parameters (type, I, gain, sniffing) in assessing both discrimination and neural processing.”
“Additionally, these two odorants recruit glomeruli in different regions of the dorsal olfactory bulb, have different functional groups and elicit different spatiotemporal response properties in the olfactory bulb (Figure 6- figure supplement 1A, stated on line 507). Both odorants are fruit-associated odors with neutral preference indices (Saraiva et al., 2016, Fletcher, 2012). Thus, while we do not explore a panel of odorants, we do explore the generalizability of intermittency processing with two distinct odorants.”
We decided to test 2 monomolecular odorants (2-heptanone and methyl valerate) as these have been widely used in rodent olfactory bulb imaging, providing distinct and clear glomerular response patterns. They are both fruity smelling odors, implying a relationship to edible food (at least, for humans). Methyl valerate is a methyl ester of pentanoic acid with a fruity (apple) smell and 2-Heptanone is a ketone with a fruity (green banana) smell.
- Assuming that all the above issues are resolved, one can conclude that intermittency can be perceived by an animal. The study puts a strong accent on the fact that this feature could be used for navigation. I understand that it is extremely hard to demonstrate that this feature is actually used for navigation, however, the analysis of relevance of this measure is missing. Even if it is used in navigation, most probably this would be in combination with other features, thus its relative importance needs to be discussed, or even better, established.
We fully appreciate the reviewers reasoning. Our approach indeed intended to establish a conditio sine qua non: if mice could not discriminate these stimuli they would likely not be able to use intermittency in general for navigation (at least for the odorants tested, for the intermittency ranges tested). We show however that they can, and hence they could use it. To demonstrate their use of intermittency alone or combined with other modalities or properties is well beyond the scope of this manuscript and we agree is a very interesting endeavor.
We discussed other temporal properties on line 58-71 and 657-664 and other general properties on lines 46-56. The relative roles were briefly addressed on lines 664-676 and we hesitate to speculate beyond this.
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eLife assessment
This important work addresses an interesting question for the vertebrate olfactory community of whether mice can discriminate odorant intermittency to help them navigate the environment. The data were collected and analyzed using solid methodology, however, the paper seems to fall short in demonstrating that animal is actually sensitive to intermittency but not other flow parameters. The work will be of interest to researchers working on sensory neurobiology and animal behavior.
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Reviewer #1 (Public Review):
Gumaste et al studied if a parameter of odor plumes, the intermittency can be detected by an animal species, such as mice that heavily rely on olfaction to navigate and search for food and mates, among other behaviors. They also ask if the animals can extract information from this to gain knowledge about the odor source. Intermittency is defined as the fraction of time an odorant is present at a sampled point within the odor plume space. Their findings could be summarized as follows: they found that animals are capable of detecting differences in intermittency levels and suggest that this parameter of odor plumes is important for odor-based navigation in mammals, as it has been seen in other animals such as flying insects. The authors used a combination of behavioral training while concomitantly performing …
Reviewer #1 (Public Review):
Gumaste et al studied if a parameter of odor plumes, the intermittency can be detected by an animal species, such as mice that heavily rely on olfaction to navigate and search for food and mates, among other behaviors. They also ask if the animals can extract information from this to gain knowledge about the odor source. Intermittency is defined as the fraction of time an odorant is present at a sampled point within the odor plume space. Their findings could be summarized as follows: they found that animals are capable of detecting differences in intermittency levels and suggest that this parameter of odor plumes is important for odor-based navigation in mammals, as it has been seen in other animals such as flying insects. The authors used a combination of behavioral training while concomitantly performing calcium imaging of olfactory receptor neurons (input to the olfactory bulb) and also mitral cells (output of the olfactory bulb). They found that mice are able to behaviorally discriminate between odor plumes of high and low intermittency. Interestingly, they found that the response of both input and output neurons of the olfactory bulb is capable to encode the intermittency experienced by the animals. The methods utilized in this work are very well suited for the kind of questions that the authors are asking. The combination of behavior and imaging, as opposed to only anesthetized imaging gives the authors a lot of power to interpret their data. A very relevant point is the generation of the olfactory stimuli that will be used to test the animals. The authors go to great lengths to generate more naturalistic odorant stimulations, as opposed to the typically used square pulses. Although there are some issues that can be addressed, the authors succeeded in answering the questions they set at the beginning of this work, and their conclusions are supported by their experiments. This work would generate interest among a relatively broad audience because the issue presented here (how the temporal structure of the odor plume affects the detection and encoding of an odorant) is novel in mice olfactory research.
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Reviewer #2 (Public Review):
The study from Gumaste et al investigates whether mice can use changes of intermittency, a temporal odor feature, to locate an odor source. First, the study tries to demonstrate that mice can discriminate between low and high intermittency and that their performance is not affected by the odor used or the frequency of odor whiffs. Then, they show that there is a correlation between glomerular responses (OSNs and mitral cells) and intermittency. Finally, they conclude that sniffing frequency impacts the behavioral discrimination of intermittency as well as its neural representation. Overall, the authors seek to demonstrate that intermittency is an odor-plume property that can inform olfactory navigation.
The paper explored an interesting question, the use of intermittency of an odor plume as a behavioral cue, …
Reviewer #2 (Public Review):
The study from Gumaste et al investigates whether mice can use changes of intermittency, a temporal odor feature, to locate an odor source. First, the study tries to demonstrate that mice can discriminate between low and high intermittency and that their performance is not affected by the odor used or the frequency of odor whiffs. Then, they show that there is a correlation between glomerular responses (OSNs and mitral cells) and intermittency. Finally, they conclude that sniffing frequency impacts the behavioral discrimination of intermittency as well as its neural representation. Overall, the authors seek to demonstrate that intermittency is an odor-plume property that can inform olfactory navigation.
The paper explored an interesting question, the use of intermittency of an odor plume as a behavioral cue, which is a new and intriguing hypothesis. However, it falls short in demonstrating that the animal is actually sensitive to intermittency but not other flow parameters, and is missing some important details.
Major concerns
One of the cornerstones of this paper consists in showing that mice are behaviorally able to distinguish among different intermittency values (high or low), across a variety of different stimuli and without confounds such as the number of whiffs or concentration. However, I could not find in the paper a convincing explanation of how these confounds were tested. It is clear that the authors repeat their measurements in different conditions (low or high concentration, and different whiff numbers) but it is not specified how: do the authors mix all stimuli in the same session, and so the animals simply generalize across all the stimuli and only consider intermittency for the behavioral choices? Or do authors repeat different sessions for different parameters? For example: do they perform two separate sessions with low concentration and high concentration? If this last one is the case, I would argue that this is not enough proof that animals generalize across concentrations, as the animals might simply use concentration as a cue and change the decision criteria at each session. Please clarify.
It looks to me that the measure of intermittency strongly depends on the set. What is the logic of setting a specific threshold? Do the results hold when this threshold changes within a reasonable range? The same questions (maybe even more important) go for the measure of glomerular intermittence. Unfortunately, a sensitivity analysis for both measures is missing, which makes it hard to interpret the results.
The logic of choosing the decision boundary for the discrimination task is not clear: low intermittency is considered to be below 0.15 and high intermittency is considered to be between 0.2 and 0.8. Do these values correspond to natural intermittency distribution? How were these values chosen?
Only 2 odors were used in the whole study and some results were in disagreement between the two odors. By looking at only two odors it is very difficult to make a general conclusion about intermittency encoding in the OB.
Assuming that all the above issues are resolved, one can conclude that intermittency can be perceived by an animal. The study puts a strong accent on the fact that this feature could be used for navigation. I understand that it is extremely hard to demonstrate that this feature is actually used for navigation, however, the analysis of relevance of this measure is missing. Even if it is used in navigation, most probably this would be in combination with other features, thus its relative importance needs to be discussed, or even better, established.
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Reviewer #3 (Public Review):
In this study, Gumaste et al. aim to determine whether mice can discriminate odor intermittency and whether the olfactory bulb encodes intermittency. Using a Go/No-Go task, the study first showed that mice can be trained to discriminate odor stimuli with a low versus high intermittency value. Next, the authors demonstrated that early olfactory processing in the OSNs and mitral/tufted cells encodes intermittency. Through calcium imaging of olfactory bulb glomeruli, they obtained the glomerular response properties across intermittency and demonstrated the effects of sniff frequency on the glomerular representation of intermittency. Although the results are expected based on previous literature, they do lend support to the notion that intermittency can be used for odor-guided navigation.
Strengths:
The …
Reviewer #3 (Public Review):
In this study, Gumaste et al. aim to determine whether mice can discriminate odor intermittency and whether the olfactory bulb encodes intermittency. Using a Go/No-Go task, the study first showed that mice can be trained to discriminate odor stimuli with a low versus high intermittency value. Next, the authors demonstrated that early olfactory processing in the OSNs and mitral/tufted cells encodes intermittency. Through calcium imaging of olfactory bulb glomeruli, they obtained the glomerular response properties across intermittency and demonstrated the effects of sniff frequency on the glomerular representation of intermittency. Although the results are expected based on previous literature, they do lend support to the notion that intermittency can be used for odor-guided navigation.
Strengths:
The counterbalanced olfactometer used in this study keeps the air flow constant while odor concentration changes. This design is very useful for experiments in which odor delivery needs to be precisely controlled.
In a Go/No-Go task, mice were successfully trained to discriminate CS+ versus CS- odor stimuli with high versus low intermittency values in three different stimulus types (termed naturalistic, binary naturalistic, and square wave).
The olfactory bulb glomerular activity (from either olfactory sensory neurons or mitral/tufted cells) was monitored while mice performing the behavioral tasks, supporting that intermittency coding could arise from early olfactory processing.
Weaknesses:
Alternative interpretations of the behavioral outcome could be better discussed. For instance, the odors delivered with high intermittency values may lead to higher odor concentrations that olfactory sensory neurons encounter in the mucus. Mice might discriminate the total amount of odors present in the mucus rather than intermittency.
The conclusion that intermittency encoding is odor specific and depends on the spatial patterning/intrinsic glomerular properties is only based on two odorants used in this study.
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