Glutamate receptor composition at Drosophila neuromuscular junctions depends on developmental stage and muscle identity

  1. Anne Sustar
  2. Chengjie Qiu
  3. Yu Xiong
  4. Dion Dickman
  5. John C. Tuthill

  • Curated by eLife

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    eLife Assessment

    This study provides important findings on the expression of glutamate receptor (GluR) subunits across developmental stages and muscle types in Drosophila. It shows that adult muscle differs in GluR composition from larval body wall muscles, which have been the focus of most past studies. The study, while convincing, could be strengthened by acknowledging that it relies on heterogeneous methods and the absence of positive signals to infer receptor loss, which limits confidence in some of its claims. The findings illuminate how Drosophila excites muscles in diverse tissue types at different life stages, and are of interest to researchers across neuroscience.

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Abstract

The neuromuscular junction (NMJ) of larval Drosophila is widely used for studying synaptic transmission. Larval body wall muscles express five ionotropic glutamate receptor (iGluR) subunits that assemble into two tetrameric complexes, with subunit composition determining the strength and plasticity of synaptic transmission. Because NMJ function has been extensively characterized in larvae, it is often assumed that adult fly NMJs have similar molecular composition, despite substantial differences between life stages. Here, we systematically compare glutamate receptor expression across larval and adult Drosophila muscles. We find that adult leg and flight muscles exhibit different iGluR expression than larvae, lacking several receptors previously considered essential for viability and NMJ function. Adjacent muscles within the adult femur express distinct iGluRs, suggesting specialization of flexor and extensor muscles. Finally, the glutamate-gated chloride channel (GluClα) is expressed extrasynaptically in adult but not larval muscle fibers. Our results reveal unexpected heterogeneity in glutamate receptor expression across muscles and developmental stages, challenging assumptions about the uniformity of neuromuscular function and demonstrating the need for muscle-specific analyses in flies and other animals.

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  1. eLife

    eLife Assessment

    This study provides important findings on the expression of glutamate receptor (GluR) subunits across developmental stages and muscle types in Drosophila. It shows that adult muscle differs in GluR composition from larval body wall muscles, which have been the focus of most past studies. The study, while convincing, could be strengthened by acknowledging that it relies on heterogeneous methods and the absence of positive signals to infer receptor loss, which limits confidence in some of its claims. The findings illuminate how Drosophila excites muscles in diverse tissue types at different life stages, and are of interest to researchers across neuroscience.

  2. eLife

    Reviewer #1 (Public review):

    Summary:

    The manuscript by Sustar et al. takes a methodical approach to document the types of glutamate receptor subunits that reside in Drosophila muscles, examining developmental stages spanning from larvae to adults. Prior work thoroughly documented the subunits operating in Drosophila larval body wall muscles. Most subsequent research focused on the glutamate receptor heterotetramers found in the body wall, composed of GluRIIA/C/D/E or GluRIIB/C/D/E subunits, along with auxiliary subunits like isoforms of Neto.

    For the current work, the authors report that the larval muscle glutamate receptor composition is not universal for all Drosophila muscles. They examine the following muscle systems: larval body wall, adult abdomen, adult leg coxa, and adult indirect flight. They also briefly examine adult muscle structures associated with the proboscis, neck, and haltere. The authors find that the receptor subunits in the adult abdomen (mostly) match those in the larval body wall. This makes sense given that the adult abdominal muscles are derived from the larval body wall. Yet not much else matches the larval body wall. For example, all (or most) of the GluRII-type subunits are missing from the adult indirect flight muscles. Leg muscles have GluRII-type subunits, but they do not have all of them expressed prominently, and they are missing GluRIIB. Additionally, leg muscles express a glutamate-gated chloride channel, which could be a source of inhibitory glutamatergic transmission. Interestingly, when it comes to non-abdominal adult muscles, one general theme seems to be an active promoter (GAL4 driver) for the kainate-type glutamate receptor called Clumsy. The authors propose that Clumsy could be key to understanding how functional GluR complexes are assembled in adult insects.

    Strengths:

    (1) Documenting the types of glutamate receptors that operate in diverse insect muscle systems is important because it uncovers fundamental information.

    (2) Much of the prior research focus has been on how the body wall muscle tetramers assemble and operate. It is a strength to demonstrate the other receptor solutions used by adult NMJs.

    (3) The work uses GAL4 drivers and immunohistochemistry (when possible) in combination to draw conclusions.

    (4) The muscle anatomical analyses are of high quality. This allows the research group to reach refined conclusions.

    (5) The confocal-level images of synaptic active zones and their apposed glutamate receptor clusters are of high quality.

    Weaknesses:

    (1) There is a strawman argument that is used repeatedly to highlight the significance of the work. The argument implies that the field broadly assumes (or "tacitly" assumes) that the larval body wall glutamate receptor composition extrapolates to all muscles of the fly, including the adult. This reviewer cannot find evidence that this assumption or argument has been explicitly promulgated by others. More likely, others have not examined these muscles directly, and thus, they have not speculated one way or the other.

    (2) Related - to the extent that there has been any tacit assumption about GluRIIC/D/E-anchored receptors being ubiquitous among adult muscles, tacit doubt was raised by Rivilin et al., 2004 (cited by the authors but not as a source of doubt) and by RNAseq datasets like FlyAtlas from 2022 (replicated in Figures s11 and s12). To be clear, the current analysis is better than a bulk transcript analysis from adult tissues. But rather than "overturning" a field or being paradigm-shifting, the current data seem confirmatory of FlyAtlas - and confirmatory of Rivlin et al., 2004, which explicitly concluded that larval and adult NMJs were different .

    (3) One can draw expression-level conclusions from these data. But genetic tests (e.g., would clumsy losses of function impair leg muscles?) could help the authors and the field draw stronger conclusions about the roles of some of these glutamate receptor gene products. The current dataset falls short of definitively establishing the function of alternate glutamate receptor modules.

    (4) The confocal synaptic images are of high quality. They are good enough that one could analyze how well Brp directly apposes a specific glutamate receptor subunit for all the associated imaging data underlying Figures S1-S8. No such analysis is done, but understanding what components seem to directly oppose the site of release could lead to better conclusions.

    Overall Assessment and Discussion:

    The data in this study are of high quality, and the results support the main conclusion: adult muscle glutamate receptor clusters do not recapitulate the "canonical" larval body wall clusters. This is important, and the data stand on their own. That is the most important part. This reviewer does have suggestions on how to put the current work in proper context; the current draft appears to overstate the novelty of the findings. Additionally, some sentences need editing for accuracy. None of those concerns impeach the excellent foundational data.

  3. eLife

    Reviewer #2 (Public review):

    Summary:

    This manuscript presents a broad survey of glutamate receptor composition at the neuromuscular junction in Drosophila across developmental stages and muscle types. The topic is clearly important, and the central observation-that adult muscles differ substantially from the canonical larval NMJ-is interesting and potentially impactful. The dataset is extensive and will likely be of value to the community. However, in my view, there are significant limitations in how the data are generated and interpreted, which at present reduce the strength of the conclusions.

    Strengths:

    The study addresses a relevant and timely question and provides a large and systematic dataset. The finding that adult muscles diverge from larval NMJ organization is compelling and challenges a widely held assumption in the field. The breadth of approaches, including genetic reporters, immunohistochemistry, endogenous tagging, and transcriptomic data, is, in principle, a strong aspect of the work and allows for a broad overview of receptor expression across tissues and developmental stages. Even in its current form, the manuscript provides useful descriptive information that will be of interest to the community.

    Weaknesses:

    A major concern is the reliance on a heterogeneous combination of detection methods (GAL4 reporters, antibody staining, endogenous tagging, and RNA), which are treated largely as equivalent lines of evidence. These approaches differ substantially in what they measure and in their sensitivity and specificity. While convergence across methods can in principle be convincing, here this convergence is often inferred from the shared absence of signal. This is problematic because all methods used are susceptible to false negatives for different reasons. As a result, the repeated conclusion that specific GluR subunits are "absent" from adult muscles, including those previously considered essential, is not fully justified by the data presented.

    This issue is not only theoretical. The manuscript itself seemingly contains examples where methods disagree, demonstrating that detection is incomplete and method-dependent. These discrepancies could be better integrated into the interpretation. Instead, negative results across methods are often taken as strong evidence for absence, which overstates the certainty of the findings.

    In addition, antibody validation appears to rely largely on prior work in larval tissue. Given the structural and biochemical differences in adult muscles, it is not clear that staining performance is equivalent, particularly in cases where the signal is weak or undetected. This further complicates the interpretation of negative results.

    More generally, the manuscript moves in several places from descriptive observations to functional or mechanistic implications that are not directly supported. The suggestion that adult muscles operate with fundamentally different receptor assemblies is intriguing, but remains speculative without functional validation. At a minimum, the distinction between observation and interpretation should be made more explicit.

    I thus think that the current conclusions need to be more carefully constrained. Ideally, the study would be strengthened by at least one functional experiment, such as electrophysiological recordings from adult NMJs or perturbation of candidate receptors like GluClα or Clumsy. This would help to anchor the expression data in synaptic function.

    In summary, this is an interesting and potentially important study, but the current manuscript somewhat overinterprets heterogeneous and partly indirect evidence. It will already be useful in its present form, but could be more convincing if the authors more rigorously account for methodological limitations and moderate their claims accordingly.

  4. eLife

    Reviewer #3 (Public review):

    The Sustar et al. manuscript catalogs glutamate receptor composition across distinct Drosophila NMJs: larval and adult abdominal NMJs, as well as NMJs on adult leg and flight muscles. This work is important and probably overdue. The larval NMJ is the exemplar NMJ in this system, and the identity of "essential" and "alternative" subunits at this stage is assumed by many to hold across developmental stages and NMJ types. Here, the authors show that there is surprising diversification among NMJ types and that the notion of essential/alternative subunits only holds true at larval NMJs.

    The study will generate interest in the Clumsy GluR subunit, which has not been well-characterized at all, but is widely expressed at adult NMJs. They also find striking extrasynaptic expression of glutamate-gated chloride channel GluRClalpha in adult leg and flight muscles, raising questions about its role. The study is interesting, logical, and well-written. The figures are clear, and the discussion was particularly thoughtful. I have a couple of comments that the authors could consider.

    (1) They cite Rivlin et al., (2004) in the Introduction as the sole previous study to investigate the molecular composition of adult NMJs, but do not mention this work again. In the Discussion, it would be helpful to compare/contrast their finding with those of the earlier work.

    (2) Were these analyses done in adults of consistent ages? It seems possible that the GluR subunit composition could be different in very young adults or in aged flies. The age of the animals should be mentioned in the Methods.

    (3) The broad expression of GluCl:V5 in adult leg and flight muscles is surprisingly robust and appears to light up the edges of all muscle fibers. Would the authors comment on the controls that were done to ensure that this staining is real and specific to animals carrying that V5 endogenous tag?

    (4) The snRNAseq data in Figure S12 differ a bit from the IHC/GAL4 data summarized in the table in Figure 2. In particular, the data suggests that Ukar and Grik are widely expressed in adult muscles. Is there a reason not to include an "snRNA seq" column in Figure 2 alongside the data from GAL4 lines and IHC? To my mind, it is about as reliable as GAL4 lines that often capture only a subset of the full expression pattern. In this case, the snRNAseq data suggest that Ukar/Grik are likely at adult flight muscle NMJs, which might be important since NMJ was negative for everything except Neto-beta by IHC.

  5. Version published to 10.64898/2026.03.17.712218 on bioRxiv