Ir56d-dependent fatty acid responses in Drosophila uncover taste discrimination between different classes of fatty acids
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Abstract
Chemosensory systems are critical for evaluating the caloric value and potential toxicity of food. While animals can discriminate between thousands of odors, much less is known about the discriminative capabilities of taste systems. Fats and sugars represent calorically potent and attractive food sources that contribute to hedonic feeding. Despite the differences in nutritional value between fats and sugars, the ability of the taste system to discriminate between different rewarding tastants is thought to be limited. In Drosophila , taste neurons expressing the ionotropic receptor 56d ( IR56d ) are required for reflexive behavioral responses to the medium-chain fatty acid, hexanoic acid. Here, we tested whether flies can discriminate between different classes of fatty acids using an aversive memory assay. Our results indicate that flies are able to discriminate medium-chain fatty acids from both short- and long-chain fatty acids, but not from other medium-chain fatty acids. While IR56d neurons are broadly responsive to short-, medium-, and long-chain fatty acids, genetic deletion of IR56d selectively disrupts response to medium-chain fatty acids. Further, IR56d+ GR64f + neurons are necessary for proboscis extension response (PER) to medium-chain fatty acids, but both IR56d and GR64f neurons are dispensable for PER to short- and long-chain fatty acids, indicating the involvement of one or more other classes of neurons. Together, these findings reveal that IR56d is selectively required for medium-chain fatty acid taste, and discrimination of fatty acids occurs through differential receptor activation in shared populations of neurons. Our study uncovers a capacity for the taste system to encode tastant identity within a taste category.
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###Reviewer #3:
In this manuscript Brown et al characterized fatty acid taste discrimination in Drosophila melanogaster. Fat taste is relatively poorly understood, but has critical implications for feeding and obesity research; thus, studies that advance our understanding of the molecular and physiological underpinning of this modality are important. The finding that Ir56d neurons enable organisms to discriminate between short, medium and long chain fatty acids but not to differentiate between types of medium chain fatty acids is certainly novel and interesting. It is also surprising but fascinating that this receptor is only required for the detection of medium fatty acids. The manuscript is well written and the figures presented in a clear and thoughtful manner. These findings lay out ground for future exciting work to investigate how …
###Reviewer #3:
In this manuscript Brown et al characterized fatty acid taste discrimination in Drosophila melanogaster. Fat taste is relatively poorly understood, but has critical implications for feeding and obesity research; thus, studies that advance our understanding of the molecular and physiological underpinning of this modality are important. The finding that Ir56d neurons enable organisms to discriminate between short, medium and long chain fatty acids but not to differentiate between types of medium chain fatty acids is certainly novel and interesting. It is also surprising but fascinating that this receptor is only required for the detection of medium fatty acids. The manuscript is well written and the figures presented in a clear and thoughtful manner. These findings lay out ground for future exciting work to investigate how sweet taste and fatty acid taste perception are selectively modulated by the brain since these gustatory neurons overlap and whether such discrimination is altered depending on the state of hunger.
Strengths:
Despite the overlapping nature of taste neurons in this case, i.e., Ir56d neurons being co-expressed with Gr64f - those that broadly label the sweet GRNs and the fact that Ir56d neurons are responsive to both sucrose and fatty acids; mutation in Ir56d results in loss of taste for hexanoic acid, but not sucrose. Authors use this taste discrimination to their advantage in combination with a robust aversive taste memory assay to address the question of differential fatty acid taste perception.
Authors rule out the potential involvement of olfaction in modulating taste perception.
Use of CRISPR-Cas9 to generate Ir56dGAL4 flies, implying accurate and targeted genome editing, provide validation to the results obtained when Ir56d expressing neurons are silenced. Additionally, use of the fly gustatory system for in-vivo Ca2+ imaging strengthens and corroborates the results at the physiological level, especially the rescue experiments.
Overall (minor) comments and questions:
Are the differences in taste discrimination between male and female flies?
Individual data points should be shown whenever possible for all figures (except PER because that would make it impossible to interpret).
Can the authors discuss how discriminating between different fatty acids types may be adaptive? Are they found in different food sources, some of which are "good" and some "bad"? Is there evidence from other organisms about this type of molecular discrimination in fatty acid taste?
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###Reviewer #2:
In the present paper Brown et al., study the ability of Drosophila melanogaster to discriminate between Fatty Acids (FAs) of different lengths. Using a combination of behavioral experiments, molecular biology and in vivo calcium imaging, the authors show that a subset of Ir56d expressing neurons are able to differentiate FAs. However, the Ir56d receptor is only necessary for the detection of medium-length FAs but not short- or long-. The paper explores in detail the role of the Ir56d receptor as FA detector, a role previously described by the authors in a previous paper Tauber et al 2017.
Major concerns:
I consider that the experiments are properly done, and so the statistical analysis, however gain in knowledge is very limited. So far, the authors can prove that flies can discriminate FAs of different lengths, being …
###Reviewer #2:
In the present paper Brown et al., study the ability of Drosophila melanogaster to discriminate between Fatty Acids (FAs) of different lengths. Using a combination of behavioral experiments, molecular biology and in vivo calcium imaging, the authors show that a subset of Ir56d expressing neurons are able to differentiate FAs. However, the Ir56d receptor is only necessary for the detection of medium-length FAs but not short- or long-. The paper explores in detail the role of the Ir56d receptor as FA detector, a role previously described by the authors in a previous paper Tauber et al 2017.
Major concerns:
I consider that the experiments are properly done, and so the statistical analysis, however gain in knowledge is very limited. So far, the authors can prove that flies can discriminate FAs of different lengths, being Ir56d the receptor detecting medium-length FAs, a result that expands the knowledge gained in Tauber et al 2017. In figure 3, the authors show that silencing Ir56d neurons using tetanus toxin expression, reduces dramatically PER to medium-length fatty acids, but not to short or long, pointing to a different set of neurons involved in their detection. However, the in vivo calcium imaging experiments show that Ir56d neurons also respond to short- and long- FAs. In this regard, I disagree with the statement at the abstract: Characterization of hexanoic acid-sensitive Ionotropic receptor 56d (Ir56d) neurons reveals broad responsive to short-, medium-, and long- chain fatty acids, suggesting selectivity is unlikely to occur through activation of distinct sensory neuron populations. In fact, I consider that selectivity would come from the activation of different subsets of gustatory neurons. It seems that Ir56d neurons could be a subset of the neurons that generally respond to FAs, providing the specificity for medium-length FAs. Other neurons, in addition to the Ir56d ones, might be responding to short- and long- FAs in an Ir56d independent manner.
I consider the authors should explore in deep how short- and long- FAs are actually detected, whether it depends on other Ionotropic Receptors (probably Ir25a and Ir76b might be involved (Ahn et al. 2017)) and which subset of gustatory neurons are actually responding to these compounds, considering they do not require Ir56d nor Ir56d neurons.
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###Reviewer #1:
This paper investigates fatty acid taste in flies and asks the broad question of whether flies can discriminate different compounds within a single taste modality. The authors' main finding is that flies can discriminate between long, medium, and short chain fatty acids using a previously established aversive memory taste paradigm. When they delve into the cellular and molecular basis of fatty acid detection they find that IR56d neurons respond to all three classes of fatty acids, but are required only for the behavioural responses to medium chain molecules. Similarly, CRISPR/Cas9 deletion of the IR56d receptor reveals that it too is required only for medium-chain fatty acid responses. Thus, different fatty acid classes presumably activate distinct, but partially overlapping subsets of appetitive taste neurons. In …
###Reviewer #1:
This paper investigates fatty acid taste in flies and asks the broad question of whether flies can discriminate different compounds within a single taste modality. The authors' main finding is that flies can discriminate between long, medium, and short chain fatty acids using a previously established aversive memory taste paradigm. When they delve into the cellular and molecular basis of fatty acid detection they find that IR56d neurons respond to all three classes of fatty acids, but are required only for the behavioural responses to medium chain molecules. Similarly, CRISPR/Cas9 deletion of the IR56d receptor reveals that it too is required only for medium-chain fatty acid responses. Thus, different fatty acid classes presumably activate distinct, but partially overlapping subsets of appetitive taste neurons. In general I think the paper is potentially interesting (see comment 1 below) and the data mostly supports the conclusions. However, there is some lack of attention to details that make some of the data hard to interpret (see minor comments).
Concerns:
The ability of flies to discriminate between different fatty acid classes is presented as the interesting finding, since, as the authors point out, discrimination between compounds within a taste modality is generally not thought to occur. On the surface I agree that this is interesting. However, in the authors' set up of the main question (line 101), they raise an important issue: "Is it possible that flies are capable of differentiating between tastants of the same modality, or is discrimination within a modality exclusively dependent on concentration?" This should be rephrased to replace "concentration" with "intensity" since not all tastants at the same concentration have the same intensity, and from a behavioural perspective it is intensity that matters. Given that, the authors don't do anything to demonstrate that their discrimination task does not depend on intensity, aside from the fact that 1% solutions of all the FA seem to give similar PER. They need to show more explicitly that this task is truly showing identity-based discrimination.
The second broad concern I have is over the nature of short and long chain fatty acid detection. Interpreting the discrimination results would be greatly aided if we knew what other neurons mediate the PER to these molecules. Is it the non-IR56d population of Gr64f neurons?
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##Preprint Review
This preprint was reviewed using eLife’s Preprint Review service, which provides public peer reviews of manuscripts posted on bioRxiv for the benefit of the authors, readers, potential readers, and others interested in our assessment of the work. This review applies only to version 1 of the manuscript.
###Summary:
The reviewers find fatty acid taste discrimination potentially interesting and agree that the experiments are performed to a high standard. One major concern is whether discrimination is based on intensity rather than quality. A second limitation is that the mechanism of FA detection is not greatly advanced beyond the authors' previous work: the cellular mechanisms for long and short chain FA detection remain unclear. The reviewers agreed that if the major concerns of Reviewer 1 were addressed, this …
##Preprint Review
This preprint was reviewed using eLife’s Preprint Review service, which provides public peer reviews of manuscripts posted on bioRxiv for the benefit of the authors, readers, potential readers, and others interested in our assessment of the work. This review applies only to version 1 of the manuscript.
###Summary:
The reviewers find fatty acid taste discrimination potentially interesting and agree that the experiments are performed to a high standard. One major concern is whether discrimination is based on intensity rather than quality. A second limitation is that the mechanism of FA detection is not greatly advanced beyond the authors' previous work: the cellular mechanisms for long and short chain FA detection remain unclear. The reviewers agreed that if the major concerns of Reviewer 1 were addressed, this manuscript would provide a broader understanding of fatty acid discrimination.
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