First-order visual interneurons distribute distinct contrast and luminance information across ON and OFF pathways to achieve stable behavior

  1. Madhura D Ketkar
  2. Burak Gür
  3. Sebastian Molina-Obando
  4. Maria Ioannidou
  5. Carlotta Martelli
  6. Marion Silies

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    Evaluation Summary:

    This paper will be of interest to neuroscientists studying visual processing and is also more broadly relevant to understanding how sensory systems process information. The paper reveals several new insights into how first-order interneurons in the fly visual system encode visual features that help guide behavior.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

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Abstract

The accurate processing of contrast is the basis for all visually guided behaviors. Visual scenes with rapidly changing illumination challenge contrast computation because photoreceptor adaptation is not fast enough to compensate for such changes. Yet, human perception of contrast is stable even when the visual environment is quickly changing, suggesting rapid post receptor luminance gain control. Similarly, in the fruit fly Drosophila , such gain control leads to luminance invariant behavior for moving OFF stimuli. Here, we show that behavioral responses to moving ON stimuli also utilize a luminance gain, and that ON-motion guided behavior depends on inputs from three first-order interneurons L1, L2, and L3. Each of these neurons encodes contrast and luminance differently and distributes information asymmetrically across both ON and OFF contrast-selective pathways. Behavioral responses to both ON and OFF stimuli rely on a luminance-based correction provided by L1 and L3, wherein L1 supports contrast computation linearly, and L3 non-linearly amplifies dim stimuli. Therefore, L1, L2, and L3 are not specific inputs to ON and OFF pathways but the lamina serves as a separate processing layer that distributes distinct luminance and contrast information across ON and OFF pathways to support behavior in varying conditions.

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  1. Version published to 10.7554/elife.74937 on eLife
  2. eLife

    Author Response

    Reviewer #2 (Public Review):

    The visual system must extract two basic features of visual stimuli: luminance, which we perceive as brightness, and contrast, the change in luminance over space or time (this paper focuses on changes over time). Contrast is separately processed by ON and OFF pathways, which encode luminance increments or decrements, respectively. Contrast must be robustly detected even if the overall luminance changes rapidly, as might occur if an animal is moving in and out of shadows. This paper addresses how such a luminance correction occurs in the fly.

    In the fly, three types of first-order interneurons - L1, L2, and L3 - transmit information from photoreceptors to the medulla, where ON and OFF encoding emerges. Previous work suggested that all three interneurons primarily encode contrast signals and that they project to distinct pathways: L1 to the ON pathway and L2 and L3 to the OFF pathway. Ketkar et al. show that, contrary to this model, these interneurons encode both contrast and luminance in specific ways and are not cleanly segregated into ON versus OFF inputs.

    This study reveals several new insights into early visual processing that are interesting and well-supported by the data:

    1. The authors show that behavioral responses to ON stimuli can compensate for rapid changes in luminance. However, the purported sole input to the ON pathway, L1, shows activity that is highly dependent on luminance. This suggests that a luminance correction must arise downstream of L1. These results are analogous to findings previously made by the same group regarding the OFF pathway (Ketkar et al., 2020). The previous paper showed that L2 provides contrast information to the OFF pathway, and L3 provides luminance information to allow for a luminance correction in downstream contrast encoding. But unlike the multiple inputs to the OFF pathway, the ON pathway was thought to only receive input from L1, provoking the question of whether L1 is able to provide both contrast and luminance information.
    1. Using well-designed calcium imaging studies, the authors surveyed the responses of the three interneurons and found that they encode different stimulus features: L1 encodes both contrast and luminance, L2 purely encodes contrast, and L3 purely encodes luminance (with a different dependence than L1). These are interesting and important findings revealing how both contrast and luminance encoding are distributed across the three interneurons.
    1. Using neuronal manipulations, the authors dissected the contributions of the three interneurons to ON and OFF behavior under changing luminance. These experiments showed that L1 and L3 are required for the luminance correction in the behavior. Moreover, the finding that all three interneurons contribute to both ON and OFF behavior contrasts with the existing model of segregated pathways. Thus, this paper could change the way we think about early visual processing in the fly: rather than relaying similar information to distinct downstream pathways, first-order interneurons relay distinct information to common pathways.

    Overall, the major claims of this paper are important and supported by the experiments. There are just a few concerns that I would note:

    Thank you for the overall positive evaluation of our work, as well as for the constructive criticism, which we are going to address below.

    1. The authors state that they have shown luminance invariance in ON behavior (e.g. line 376-377 of the Discussion), but this is not entirely accurate: the ON behavior decreases as luminance increases. This is still an interesting effect since it's the opposite of what L1 activity does, so it's clear that the circuit is implementing a luminance correction, but it is not "luminance invariance".

    As pointed out in response to essential comment #2, we carefully edited the manuscript to talk about ‘near’ luminance invariance, or data approaching luminance invariance. More prominently, we rephrased the text to highlight the need for a luminance gain to scale behavioral responses to contrast, even if the resulting behavior is not entirely luminance invariant.

    1. The visual stimuli presented for most imaging experiments (full-field) are not the same as those presented for behavior (moving edges). It is possible neuronal responses and their encoding of luminance and contrast may differ if tested with the moving edge stimuli (if so, this would be concerning). The authors did image L1 with both types of stimuli and could compare these responses. Also, testing behavior at 34º and imaging at 20º presents a possible discrepancy in comparing these data.

    We use moving ON edges in Figure 1, and these data suggest that the transient response of L1 scales with step changes in luminance, consistent with data in Figure 2B. Although we did not point this out in the paper, the L1 responses in Figure 1 also decay to different response levels, consistent with the luminance-sensitive component that static stimuli reveal in Figure 2. Furthermore, for other ongoing projects in the lab, we have for example measured physiological responses in L2 with the same stimuli used in behavior, and there is no discrepancy with the data reported here. Overall, there is no reason to believe, following a vast amount of literature in Drosophila and other flies, that LMCs would respond any different to moving vs. static stimuli.

    We can additionally point out that the behavioral data of L3 silencing (at 34ºC) nicely correlate with physiological contrast responses of L1 and L2 (at 20ºC, predicted from electrophysiological recordings for LMCs in Ketkar et al. 2020, measured for L1 here). Many previous studies, for example in motion detection, have linked data from physiological recordings at room temperature with behavioral experiments done at higher temperature (e.g., Ammer et al., 2015; Clark et al., 2011; Creamer et al., 2019; Fisher et al., 2015; Leonhardt et al., 2017; Salazar-Gatzimas et al., 2016; Serbe et al., 2016; Silies et al., 2013; Strother et al., 2017). We therefore do not think that these are major concerns.

    1. I find it puzzling that silencing L1 has little effect on ON behavior at 100% contrast and varying luminance (Figure 3A), but severely affects ON behavior to 100% contrast (and lower values) when different contrasts are interleaved (Figure S1). The authors note this but do not provide a clear explanation of why this might be the case. Aside from mechanism, it is not clear whether the difference is due to varying luminance in the first experiment or varying contrast in the second one (e.g. they could test 100% contrast without varying luminance).

    The two stimulus sets used here do not allow us to pinpoint why the L1 silencing phenotype differs between them, since they comprise more than one difference as discussed above (see point 4) in “Essential Revisions”). We now include two additional experiments that dissect the role of different stimulus parameters (Supp. Figure 2). To understand whether the difference is due to varying luminance, we tested responses to ON edges of fixed (100%) contrast and luminance at the same stimulus parameters (motion duration, speed) as used in Figure 3, and did not find reduced turning responses when silencing L1. Thus, varying luminance does not change the effect of L1 on ON behavior. However, when repeating this experiment with a bright inter-stimulus interval, L1 silencing lead to a strong response deficit. Therefore, differences in the interval luminance explain the differences in the L1 silencing phenotype observed not only in this study but also across studies. Although we hypothesize a role of contrast adaptation that may function differently with altered contrast statistics, a more detailed investigation would be necessary to understand the mechanism. Nevertheless, our experiments allow us to conclude that L1 is not the sole major input to the ON pathway, even though it is required under certain stimulus conditions.

    1. I do not entirely agree with the authors' interpretation of the L1 ort rescue experiment for OFF behavior. They state that rescue flies "responded similarly to positive controls". However, the graph shows that the rescue flies generally fall in between the mutant and heterozygote control flies; they resemble the controls at low luminance but resemble the mutants at high luminance. One may conclude that L1 is sufficient to enhance OFF behavior at low luminance, but it is a stretch to say it's a complete rescue.

    Sorry, we just meant to say that they “responded similarly to positive controls (...) at low luminance”, but the sentence was badly written. We corrected this to: “L1 ort rescue flies responded similarly to positive controls at low luminances, rescuing responses to OFF edges at dim backgrounds.”

    1. The authors typically use t-tests to analyze experiments with 2 variables (genotype and luminance) and 3 or more conditions per variable. This is not the most appropriate statistical test; typically one would use a two-way ANOVA. At the least, it should be clear whether they are performing corrections for multiple comparisons if performing many t-tests on the same dataset.

    Thank you for the suggestion, we now use a two-way ANOVA followed by corrected pairwise comparisons and state this clearly in the figure captions (also addressed above in essential comment #5).

    Reviewer #3 (Public Review):

    Ketkar et al combine calcium imaging and behavioral experiments to investigate the encoding of luminance and contrast in 3 first-order interneurons in the Drosophila lamina: L1, L2, and L3, as well as the role of these signals in moving ON edge behavior across luminance. The behavioral experiments are well performed. The rescue experiments are particularly interesting. Together with silencing they support and nicely extend previous work showing that L1/2/3 are not simply segregated between ON and OFF pathways. My main issue is the link that the authors make between the cellular responses and the behaviors performed and therefore the overall conclusions and claims of the paper about the roles of contrast vs luminance encoding of each neuron type (particularly L1) in the behaviors.

    Major concerns:

    1. The authors state that the main behavior they study, namely optomotor response to moving light edges at 100% contrast, is "luminance invariant". A strict definition of this would be that behavioral responses are constant with increasing luminance. However, there are very few plots in this paper where this is the case. In almost all examples, the response is decreasing with respect to increasing luminance. The authors do qualify a "nearly" invariant behavior, but this does not change the fact that interpretation of the data in the context of the framing of the paper is often problematic.

    We thank the reviewer for this critical comment. The main point (that we apparently failed to make clear enough) is that there is a clear requirement for a luminance gain. Physiological LMC responses measured using calcium imaging to ON stimuli in Figure 1, or predicted from previous electrophysiological recordings to OFF stimuli in (Ketkar et al., 2020) cannot account for any of the (control) behavioral data. We now edited the text to tone down statements about luminance invariance, and instead highlighted the need for a luminance gain.

    1. The manuscript would benefit from clear definitions of luminance and contrast, as well as an explanation of how contrast and luminance sensitivity can be inferred from experiments. In particular, the authors use transient vs. sustained response properties in L1, L2, and L3 as indicators of contrast and luminance sensitivity, but this is not stated clearly. It would be important to explain this to the reader early on.

    We now added definitions of general terms to the introduction and added data and analysis to the manuscript (Figure S1, and Figure 2B-D) to more clearly test which component of the neurons’ responses encode contrast or luminance.

    1. In the manuscript, it is often stated that "calcium imaging experiments reveal that each first order interneuron is unique in its contrast and luminance encoding properties" (line 110). This was shown clearly for L2 and L3 in their previous work in Ketkar et al. 2020, with a welldesigned two-step stimulus that was able to tease apart contrast vs. luminance invariance. Unfortunately it does not seem that this level of experimental detail and analysis is applied to L1 here. In particular, the authors state " L1 encodes both contrast and luminance in distinct response components." Line 112, in the summary of their findings. I would not agree that the authors have actually shown this properly in this manuscript.

    Addressed above, in point 6 of “Essential Revisions”

    1. The results as they are stated, are at times not well supported by the data. The manuscript would benefit from a careful assessment of the accuracy and precision of the language used to interpret the data. Sometime just moving some conclusions to the discussion and explaining the assumptions made to reach a particular conclusion would be enough. A few of examples:

    We carefully edited the entire manuscript, in addition to addressing the specific points below.

    o Figure 2: "Lamina neuron types L1-L3 are differently sensitive to contrast and luminance". It is overall true that from the raw traces, the response are different. However the quantification in C-E only pertains to luminance.

    As stated above, we now did further analysis on the contrast encoding properties of L1 and L2 and pointed out the major differences between these neurons (Figure 2B-D).

    o Figure 3: "L1 is not required but sufficient for ON behavior across luminance". The data convincingly shows this. I would however point out that the statement "this data [..] highlights its behavioral relevant role of its luminance component" line 231 is an overstatement.

    We deleted this statement at the end of the paragraph.

    o Figure 6: "L1 luminance signal is required and sufficient for OFF behavior" the data presented shows convincingly that when L1 is inactive the behavior becomes (more) intensity variant. However, it does not show that it is the "luminance signal" in L1 that is required for this effect. In general, because L1 has a sustained and a transient response, it is difficult to strictly implicate one or the other in supporting any behavior, short of manipulating L1 to make it fully transient or fully sustained.

    We agree. The figure title now reads “L1 function is required and sufficient for OFF behavior”.

    o It is often not clear which conclusions stem from this work and which from their previous work Ketkar et al. 2020, or even other previous work on contrast sensitivity in particular. Clarifying this might help with my concern about statements not well supported by the data in this paper, and also justify their overall novelty. In general the manuscript assumes familiarity with this previous work, which is not always helpful for the reader.

    As stated above, we now more clearly separate previous findings from novel findings in the abstract, and throughout the text. We also expanded the introduction to better explain the core concepts that are needed to understand this work, without having read Ketkar et al. 2020.

  3. eLife

    Evaluation Summary:

    This paper will be of interest to neuroscientists studying visual processing and is also more broadly relevant to understanding how sensory systems process information. The paper reveals several new insights into how first-order interneurons in the fly visual system encode visual features that help guide behavior.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

  4. eLife

    Reviewer #1 (Public Review):

    In this manuscript, Ketkar et al. describe the role of Lamina L1, L2 and L3 neurons in processing contrast and luminance information in the Drosophila visual system. To this end, they combine calcium imaging to quantify the sensory responses of the three neuron types with neurogenetic silencing to unravel their role in modulating the fly's behavioral responses to moving OFF and ON edges of varying luminance. The main conclusions of the study are that L1, L2, and L3 each encode different aspects of luminance and contrast information, and that they each make complex contributions to visual processing in both the ON and OFF pathway. Together, the three neuron types enable the fly to respond equally well to high-contrast stimuli over a large luminance range. Overall, this is an important study complementing and significantly expanding earlier work on luminance and contrast processing in the (fly) visual system. Given how well-suited and established the Drosophila visual system is as a model for visual processing in general, this paper should be highly relevant to a broad neuroscience readership.

    The strengths of the paper are that the authors combine calcium imaging of individual neuron types with behavioral experiments in which they silence these same neuron types individually and in combinations, using comparable visual stimuli in all experiments. The authors test for behavioral necessity and sufficiency of L1, L2, and L3 neurons by using straight-forward silencing of individual neuron types as well as broad silencing of multiple neuron types combined with rescuing individual types. Hence, the authors either silence single neurons, or all neurons other than the target for that particular experiment. This approach allows them to identify the behavioral contributions of L1, L2, and L3 neurons both individually and in combinations. The calcium imaging experiments allow the authors to define which aspects of the visual stimuli each neuron type is encoding. Thus, we get a clear picture of the contributions L1, L2, and L3 make to processing high contrast stimuli in different luminance regimes.

    The quality of the data and analysis is high, the data presentation is clear, and the conclusions are convincing. The main area of this manuscript that would benefit from improvements is the writing and presentation.

  5. eLife

    Reviewer #2 (Public Review):

    The visual system must extract two basic features of visual stimuli: luminance, which we perceive as brightness, and contrast, the change in luminance over space or time (this paper focuses on changes over time). Contrast is separately processed by ON and OFF pathways, which encode luminance increments or decrements, respectively. Contrast must be robustly detected even if the overall luminance changes rapidly, as might occur if an animal is moving in and out of shadows. This paper addresses how such a luminance correction occurs in the fly.

    In the fly, three types of first-order interneurons - L1, L2, and L3 - transmit information from photoreceptors to the medulla, where ON and OFF encoding emerges. Previous work suggested that all three interneurons primarily encode contrast signals and that they project to distinct pathways: L1 to the ON pathway and L2 and L3 to the OFF pathway. Ketkar et al. show that, contrary to this model, these interneurons encode both contrast and luminance in specific ways and are not cleanly segregated into ON versus OFF inputs.

    This study reveals several new insights into early visual processing that are interesting and well-supported by the data:

    1. The authors show that behavioral responses to ON stimuli can compensate for rapid changes in luminance. However, the purported sole input to the ON pathway, L1, shows activity that is highly dependent on luminance. This suggests that a luminance correction must arise downstream of L1. These results are analogous to findings previously made by the same group regarding the OFF pathway (Ketkar et al., 2020). The previous paper showed that L2 provides contrast information to the OFF pathway, and L3 provides luminance information to allow for a luminance correction in downstream contrast encoding. But unlike the multiple inputs to the OFF pathway, the ON pathway was thought to only receive input from L1, provoking the question of whether L1 is able to provide both contrast and luminance information.

    2. Using well-designed calcium imaging studies, the authors surveyed the responses of the three interneurons and found that they encode different stimulus features: L1 encodes both contrast and luminance, L2 purely encodes contrast, and L3 purely encodes luminance (with a different dependence than L1). These are interesting and important findings revealing how both contrast and luminance encoding are distributed across the three interneurons.

    3. Using neuronal manipulations, the authors dissected the contributions of the three interneurons to ON and OFF behavior under changing luminance. These experiments showed that L1 and L3 are required for the luminance correction in the behavior. Moreover, the finding that all three interneurons contribute to both ON and OFF behavior contrasts with the existing model of segregated pathways. Thus, this paper could change the way we think about early visual processing in the fly: rather than relaying similar information to distinct downstream pathways, first-order interneurons relay distinct information to common pathways.

    Overall, the major claims of this paper are important and supported by the experiments. There are just a few concerns that I would note:

    1. The authors state that they have shown luminance invariance in ON behavior (e.g. line 376-377 of the Discussion), but this is not entirely accurate: the ON behavior decreases as luminance increases. This is still an interesting effect since it's the opposite of what L1 activity does, so it's clear that the circuit is implementing a luminance correction, but it is not "luminance invariance".

    2. The visual stimuli presented for most imaging experiments (full-field) are not the same as those presented for behavior (moving edges). It is possible neuronal responses and their encoding of luminance and contrast may differ if tested with the moving edge stimuli (if so, this would be concerning). The authors did image L1 with both types of stimuli and could compare these responses. Also, testing behavior at 34º and imaging at 20º presents a possible discrepancy in comparing these data.

    3. I find it puzzling that silencing L1 has little effect on ON behavior at 100% contrast and varying luminance (Figure 3A), but severely affects ON behavior to 100% contrast (and lower values) when different contrasts are interleaved (Figure S1). The authors note this but do not provide a clear explanation of why this might be the case. Aside from mechanism, it is not clear whether the difference is due to varying luminance in the first experiment or varying contrast in the second one (e.g. they could test 100% contrast without varying luminance).

    4. I do not entirely agree with the authors' interpretation of the L1 ort rescue experiment for OFF behavior. They state that rescue flies "responded similarly to positive controls". However, the graph shows that the rescue flies generally fall in between the mutant and heterozygote control flies; they resemble the controls at low luminance but resemble the mutants at high luminance. One may conclude that L1 is sufficient to enhance OFF behavior at low luminance, but it is a stretch to say it's a complete rescue.

    5. The authors typically use t-tests to analyze experiments with 2 variables (genotype and luminance) and 3 or more conditions per variable. This is not the most appropriate statistical test; typically one would use a two-way ANOVA. At the least, it should be clear whether they are performing corrections for multiple comparisons if performing many t-tests on the same dataset.

  6. eLife

    Reviewer #3 (Public Review):

    Ketkar et al combine calcium imaging and behavioral experiments to investigate the encoding of luminance and contrast in 3 first-order interneurons in the Drosophila lamina: L1, L2, and L3, as well as the role of these signals in moving ON edge behavior across luminance. The behavioral experiments are well performed. The rescue experiments are particularly interesting. Together with silencing they support and nicely extend previous work showing that L1/2/3 are not simply segregated between ON and OFF pathways. My main issue is the link that the authors make between the cellular responses and the behaviors performed and therefore the overall conclusions and claims of the paper about the roles of contrast vs luminance encoding of each neuron type (particularly L1) in the behaviors.

    Major concerns:

    1. The authors state that the main behavior they study, namely optomotor response to moving light edges at 100% contrast, is "luminance invariant". A strict definition of this would be that behavioral responses are constant with increasing luminance. However, there are very few plots in this paper where this is the case. In almost all examples, the response is decreasing with respect to increasing luminance. The authors do qualify a "nearly" invariant behavior, but this does not change the fact that interpretation of the data in the context of the framing of the paper is often problematic.

    2. The manuscript would benefit from clear definitions of luminance and contrast, as well as an explanation of how contrast and luminance sensitivity can be inferred from experiments. In particular, the authors use transient vs. sustained response properties in L1, L2, and L3 as indicators of contrast and luminance sensitivity, but this is not stated clearly. It would be important to explain this to the reader early on.

    3. In the manuscript, it is often stated that "calcium imaging experiments reveal that each first order interneuron is unique in its contrast and luminance encoding properties" (line 110). This was shown clearly for L2 and L3 in their previous work in Ketkar et al. 2020, with a well-designed two-step stimulus that was able to tease apart contrast vs. luminance invariance. Unfortunately it does not seem that this level of experimental detail and analysis is applied to L1 here. In particular, the authors state " L1 encodes both contrast and luminance in distinct response components." Line 112, in the summary of their findings. I would not agree that the authors have actually shown this properly in this manuscript.

    4. The results as they are stated, are at times not well supported by the data. The manuscript would benefit from a careful assessment of the accuracy and precision of the language used to interpret the data. Sometime just moving some conclusions to the discussion and explaining the assumptions made to reach a particular conclusion would be enough. A few of examples:

    o Figure 2: "Lamina neuron types L1-L3 are differently sensitive to contrast and luminance". It is overall true that from the raw traces, the response are different. However the quantification in C-E only pertains to luminance.
    o Figure 3: "L1 is not required but sufficient for ON behavior across luminance". The data convincingly shows this. I would however point out that the statement "this data [..] highlights its behavioral relevant role of its luminance component" line 231 is an overstatement.
    o Figure 6: "L1 luminance signal is required and sufficient for OFF behavior" the data presented shows convincingly that when L1 is inactive the behavior becomes (more) intensity variant. However, it does not show that it is the "luminance signal" in L1 that is required for this effect. In general, because L1 has a sustained and a transient response, it is difficult to strictly implicate one or the other in supporting any behavior, short of manipulating L1 to make it fully transient or fully sustained.
    o It is often not clear which conclusions stem from this work and which from their previous work Ketkar et al. 2020, or even other previous work on contrast sensitivity in particular. Clarifying this might help with my concern about statements not well supported by the data in this paper, and also justify their overall novelty. In general the manuscript assumes familiarity with this previous work, which is not always helpful for the reader.

  7. Version published to 10.1101/2021.11.03.467156v1 on bioRxiv