Botulinum neurotoxin accurately separates tonic vs. phasic transmission and reveals heterosynaptic plasticity rules in Drosophila
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Evaluation Summary:
The authors describe a new tool to completely block both evoked and spontaneous release in a selective manner using the GAL4 system in Drosophila. Surprisingly, they see no effects on either pre or postsynaptic development when both evoked and spontaneous release are blocked, in contrast to prior studies in the field. Overall, the results are provocative and will be of interest to the field. The study contradicts a number of published works, so it is important for the field to see the data and be able to evaluate it themselves.
(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 and Reviewer #2 agreed to share their name with the authors.)
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Abstract
In developing and mature nervous systems, diverse neuronal subtypes innervate common targets to establish, maintain, and modify neural circuit function. A major challenge towards understanding the structural and functional architecture of neural circuits is to separate these inputs and determine their intrinsic and heterosynaptic relationships. The Drosophila larval neuromuscular junction is a powerful model system to study these questions, where two glutamatergic motor neurons, the strong phasic-like Is and weak tonic-like Ib, co-innervate individual muscle targets to coordinate locomotor behavior. However, complete neurotransmission from each input has never been electrophysiologically separated. We have employed a botulinum neurotoxin, BoNT-C, that eliminates both spontaneous and evoked neurotransmission without perturbing synaptic growth or structure, enabling the first approach that accurately isolates input-specific neurotransmission. Selective expression of BoNT-C in Is or Ib motor neurons disambiguates the functional properties of each input. Importantly, the blended values of Is+Ib neurotransmission can be fully recapitulated by isolated physiology from each input. Finally, selective silencing by BoNT-C does not induce heterosynaptic structural or functional plasticity at the convergent input. Thus, BoNT-C establishes the first approach to accurately separate neurotransmission between tonic vs. phasic neurons and defines heterosynaptic plasticity rules in a powerful model glutamatergic circuit.
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Author Respose
Reviewer #1 (Public Review):
This manuscript reports a new genetically encoded neuronal silencer BoNT-C. They show that it fully blocks neurotransmission in two classes of Drosophila motor neurons (Is and 1b; tonic and phasic, respectively). They also update a GCaMP postsynaptic reporter SynapGCaMP to express GCaMP8f instead of 6f. They selectively silence 1b or 1s neurons to disambiguate the neurotransmission properties of each neuron. Finally, they show that silencing either 1b or 1s neurons does not induce heterosynaptic structural or functional plasticity (only neuron ablation triggers plasticity). The data are convincing. The new silencing tool will be widely used.
We thank this reviewer for his positive assessment of our study and for highlighting the utility of the new silencing tool presented in this study.
Re…
Author Respose
Reviewer #1 (Public Review):
This manuscript reports a new genetically encoded neuronal silencer BoNT-C. They show that it fully blocks neurotransmission in two classes of Drosophila motor neurons (Is and 1b; tonic and phasic, respectively). They also update a GCaMP postsynaptic reporter SynapGCaMP to express GCaMP8f instead of 6f. They selectively silence 1b or 1s neurons to disambiguate the neurotransmission properties of each neuron. Finally, they show that silencing either 1b or 1s neurons does not induce heterosynaptic structural or functional plasticity (only neuron ablation triggers plasticity). The data are convincing. The new silencing tool will be widely used.
We thank this reviewer for his positive assessment of our study and for highlighting the utility of the new silencing tool presented in this study.
Reviewer #2 (Public Review):
The conclusions of this paper are properly supported by the provided data.
Overall this work opens a new window to examine novel aspects of heterosynaptic structural and functional plasticity.
We also thank this reviewer for his positive assessment of our study and for putting the importance of our findings in context.
Reviewer #3 (Public Review):
The strength of the manuscript by Han et al. is the comprehensive characterization of BoNT-C, showing that it truly abolishes all evoked and mini responses without structural alteration of the NMJ. Based on this, the authors then show that ablation of all neurotransmission in either Ib or Is does not cause any compensatory changes (neither functional nor structural) in the 'other' (i.e. looking at Is when silencing Ib or looking at Ib when silencing Is).
The weakness of the manuscript lies in the modest gain over the previous work. Specifically, Aponte-Santiago had already shown that many parameters are not changed (in Ib when Is is perturbed, or in Is when Ib is perturbed), including that 'the Is terminal failed to show functional or structural changes following loss of the coinnervating Ib input' (quote from 2020 paper). Hence, the only major difference is that Han et al now show that Ib also does not really change when Is is silenced. Aponte-Santiago also clearly showed a ~50% EJP reduction when Is or Ib are perturbed alone, and adding these two equals wild type. The highly emphasized finding of Han et al. that (quote) ' composite values of Is and Ib neurotransmission can be fully recapitulated by isolated physiology from each input' quite obviously follows from the one key finding that one does not affect the other, as mentioned above in the strengths. The wording is a bit odd, but really adding Is (with Ib perturbed) and Ib (with Is perturbed) inputs is really not adding much over either the main finding nor the previous work.
We thank this reviewer for his/her/their assessment of our study and for highlighting the strengths in characterizing the impact of BoNT-C expression at the NMJ. We also understand and appreciate the criticisms raised. It is important to note from the outset that the motivation and central goal of this study was not primarily to mechanistically dissect heterosynaptic plasticity between tonic and phasic motor inputs at the Drosophila NMJ. Rather, it was to develop an approach that would, for the first time, enable accurate isolation of complete neurotransmission from entire MN-Is or MN-Ib NMJs (both miniature and evoked transmission). By the reviewer’s own admission, we were entirely successful at achieving this central goal in our comprehensive characterization of BoNT-C.
Next, the reviewer raises the valid question about whether this achievement is a significant advance over previous work, and discusses recent experimental findings regarding heterosynaptic plasticity at the fly NMJ. We want to emphasize here that having a tool that is capable, for the first time, of accurately discriminating complete transmission from Is vs Ib alone is a major advance, one that it is not clear the reviewer sufficiently appreciates. As summarized in Fig. 1, no previous attempts have been successful in accurately isolating synaptic transmission between Is vs Ib synapses. In particular, no previous approach was capable of isolating miniature activity from Is vs Ib, and as we show in our manuscript, miniature events exhibit major differences between the two inputs. Thus, without isolating miniature transmission, one cannot know baseline synaptic function in Is vs Ib nor whether heterosynaptic functional plasticity has been induced. Further, we detail major confounds with some of the previous approaches the reviewer alludes to in prior studies, including selective optogenetic stimulation.
Finally, the reviewer discusses at length recent findings regarding heterosynaptic plasticity and questions whether the new insights revealed by BoNT-C provides a sufficient advance. In particular, the reviewer refers to previous work published in 2020 and 2021, where important initial insights into Is vs Ib structure and transmission after differential manipulations to either input was reported. The reviewer appears to believe that it was settled in these studies that no heterosynaptic functional plasticity was induced.
However, a critical point that the reviewer appears not to appreciate is that while the two previous studies on heterosynaptic plasticity at the Drosophila NMJ were able to assess structural plasticity (AponteSantiago et al., 2020; Wang et al., 2021), no accurate or quantitative conclusions can be made about heterosynaptic functional plasticity from these studies. This is due to the authors not knowing what baseline synaptic function is at Is vs Ib (miniature frequency, miniature amplitude, and evoked transmission), so that in their manipulations they cannot accurately determine whether any functional changes are observed after their manipulations. Further complicating the interpretation of the previous studies is that at the muscle 1 NMJ (2020 study), like the muscle 4 NMJ (2021 study), ~30% of these NMJs fail to be innervated by a Is input in wild-type larvae. This major confound makes it difficult to know how or whether adaptive plasticity is induced in wild-type NMJs with or without Is innervation (since, interestingly, evoked transmission does not appear to change in wild-type m1 or m5 NMJs with or without a Is input), and then to determine whether any heterosynaptic plasticity is induced. Indeed, we have also struggled with how to accurately determine whether synaptic function changes compared to baseline throughout our studies at earlier stages, despite the fact that the muscle 6/7 NMJ we use in our study does not suffer from the variable Is innervation confounds observed at muscle 4 (Wang et al., 2021) and muscle 1 (Aponte-Santiago et al., 2020).
Respectfully, we contend that the only way one can accurately and quantitatively determine baseline synaptic transmission (miniature amplitude, frequency, evoked, quantal content), and whether any changes are observed following manipulations to Is or Ib, is to fully and accurately recapitulate wild type (blended Is+Ib) neurotransmission from isolated Is vs Ib transmission. This is why we believe the data shown in Fig. 7 (and also Fig. S7 in the revised manuscript) is so important. It is true that numerous previous studies established relative and qualitative differences between Is vs Ib (miniature events are larger at Is relative to Ib, Is drives larger depolarization in response to single synaptic stimulation over Ib, etc). However, in no case did previous studies accurately assess baseline Is vs Ib synaptic function from entire inputs, and therefore could not conclude with certainty whether heterosynaptic functional plasticity was induced.
On a different but somewhat similar topic, UAS-BoNT-C is not a new tool. I am a bit put off by the wording ' We have developed a botulinum neurotoxin, BoNT-C...'. More on this and the way the previous BoNT-C paper (Backhaus et al., 2016) is cited in the detail comments below in the recommendations for the authors.
We understand these points raised by the reviewer. Our BoNT-C transgenic line is indeed a new tool, the only one in which synaptic transmission has ever been electrophysiologically characterized and shown to completely silence synaptic transmission in Drosophila. That being said, in retrospect, we can appreciate that the term “developed” might imply a level of innovation that reasonable people can disagree about. We have therefore elected to change the apparently offensive wording to “We have employed a botulinum neurotoxin, BoNT-C…” in the abstract of the revised manuscript.
Additionally, the manuscript does not really dive into an analysis of phasic versus tonic functions (that's just a correlation with the Is and Ib dominant modes of function).
We absolutely agree that selective silencing by BoNT-C now enables a rigorous study of tonic vs phasic neurotransmission at MN-Is vs MN-Ib NMJs, but that in the current manuscript we have not focused on this interesting question. We have adopted the convention the field has used to classify MN-Is and MN-Ib subtypes based on their apparent firing modes as “phasic” vs “tonic”, but like previous studies, we have not analyzed these functional distinctions on a deeper level. Although the focus of the current manuscript was to establish the properties of BoNT-C and highlight its utility as a tool for the field, we are now in the process of preparing an entirely new manuscript focused on just this reviewer’s question about the differences in tonic vs phasic synaptic physiology. This eight-figure manuscript will be entitled “Electrophysiological properties and nanoscale distinctions that define tonic vs phasic glutamatergic synapses” and is focused on the central question raised by the reviewer - how and why synaptic transmission differs between tonic vs phasic inputs. While this interesting question is outside the scope of the current manuscript, we will submit this new manuscript within the next few months, which is based on new experimental insights now enabled by selective BoNT-C silencing established in the current manuscript.
Finally, since the authors show that loss of Is or Ib function does not cause any change in the other, we are left to wonder what actually DOES cause heterosynaptic plasticity. TNT or rpr DO cause some heterosynaptic plasticity and they also DO cause some structural changes - but whether the structural changes themselves are important here remains unclear. Substantial progress would have been to take the starting point that BoNT-C does not cause heterosynaptic plasticity, and then identify the signal that does (is it morphology? or signaling between Is and Ib? Or with the muscle?).
We certainly agree with the reviewer that understanding how heterosynaptic plasticity is induced is an important question and worthy of additional investigation. As stated above, the focus of our current study was to establish the tool, BoNT-C, that will now enable a variety of fascinating and important future studies, both at understanding how and why synaptic strength differs between tonic vs phasic synapses and also how heterosynaptic plasticity signaling occurs at the NMJ. It required substantial time and experimental effort to establish that BoNT-C works to cleanly silence transmission without inducing structural and functional plasticity in the current manuscript (Figures 1-7 and several supplemental figures). Respectfully, we believe it is unreasonable to expect all of this data to be relegated to a “starting point” to then go on and probe heterosynaptic plasticity in more detail, all compressed into a single paper.
It appears this reviewer is particularly interested in heterosynaptic plasticity, which we agree is a fascinating topic. First, we should clarify that in our experiments, TNT expression does NOT induce any heterosynaptic structural or functional plasticity (see Figures 6 and Table S2), at least in our studies at m6/7, m12/13, and m4 NMJs. Rather, TNT expression alters synaptic structure in the neuron in which it is expressed (“intrinsic structural plasticity”, Fig. 6), but does not induce any changes to the convergent input. Hence, the only evidence for actual heterosynaptic plasticity is the rather minor adaptations in synaptic structure and function observed following ablation of Is motor inputs (Fig. 6 and 8).
In addition to the important insights revealed by BoNT-C in accurately distinguishing tonic vs phasic transmission outlined above, it appears that the reviewer does not fully appreciate the mechanistic constraints that the new BoNT-C tool reveals about heterosynaptic signaling. We would therefore like to highlight the key insights our study has revealed specifically about heterosynaptic plasticity. First, we show that at the muscle 6/7 NMJ, loss of MN-Ib completely eliminates Is innervation – this was not the finding reported in the 2020 study (Ib ablation was not reported in the 2021 study). Rather, AponteSantiago et al. 2020 reported that elimination of Ib did not trigger compensatory changes in active zone or bouton numbers of the Is input, no were compensatory increases in the Is EPSP reported. This may be due to the confounding variable Is innervation at the muscle 1 and muscle 4 NMJs used in the previous studies. Second, to what extent miniature transmission changes after manipulating activity from Is vs Ib could not be accurately assessed in previous studies because spontaneous activity persists following TNT expression as does innervation following rpr.hid expression. Third, and perhaps most important, our study is the only one that can demonstrate no heterosynaptic functional plasticity is induced by the physical presence but functional silencing of neurotransmitter release between tonic vs phasic inputs at NMJs with consistent innervation by both Is and Ib inputs.
It is clear to us now that we did not do a sufficient job of emphasizing these advances our study has now revealed about the baseline and heterosynaptic relationships between Is vs Ib. We have added additional details throughout the revised manuscript to ensure these insights are highlighted in an effort for the reader to better appreciate the importance of this study.
Overall, while an initial reading of the manuscript sounded rather exciting, a deeper analysis of the work in context of the literature of the last few years diminishes my enthusiasm for the novelty and progress provided.
We have responded to the major criticisms raised by this reviewer above and hope that he/she/they can more fully appreciate the importance of the new tool we developed, the impact it will have on the field in opening new studies on tonic vs. phasic transmission, and establishing the rules of heterosynaptic plasticity between convergent tonic and phasic inputs on common targets.
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Evaluation Summary:
The authors describe a new tool to completely block both evoked and spontaneous release in a selective manner using the GAL4 system in Drosophila. Surprisingly, they see no effects on either pre or postsynaptic development when both evoked and spontaneous release are blocked, in contrast to prior studies in the field. Overall, the results are provocative and will be of interest to the field. The study contradicts a number of published works, so it is important for the field to see the data and be able to evaluate it themselves.
(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 and Reviewer #2 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
This manuscript reports a new genetically encoded neuronal silencer BoNT-C. They show that it fully blocks neurotransmission in two classes of Drosophila motor neurons (Is and 1b; tonic and phasic, respectively). They also update a GCaMP postsynaptic reporter SynapGCaMP to express GCaMP8f instead of 6f. They selectively silence 1b or 1s neurons to disambiguate the neurotransmission properties of each neuron. Finally, they show that silencing either 1b or 1s neurons does not induce heterosynaptic structural or functional plasticity (only neuron ablation triggers plasticity). The data are convincing. The new silencing tool will be widely used.
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Reviewer #2 (Public Review):
In this manuscript, Han et al. investigate the intrinsic and heterosynaptic plasticity in motor neurons with emphasis on both structure and function of neuromuscular junctions (NMJs) in fruit fly larvae. The larval NMJs have been extensively used by multiple labs as a model to study both the structure and function of synapses, shedding light on mechanisms of heterosynaptic plasticity. The body wall larval muscles in Drosophila are coinnervated by two types of glutamatergic excitatory motor neurons: Ib and Is. Ib MNs establish big synaptic buttons and exhibit a tonic firing pattern, while Is MNs make smaller synaptic buttons and have a phasic firing pattern.
First, the authors develop a new genetic tool (BoNT-C) to selectively suppress neurotransmission in either Ib or Is synapses without affecting the …
Reviewer #2 (Public Review):
In this manuscript, Han et al. investigate the intrinsic and heterosynaptic plasticity in motor neurons with emphasis on both structure and function of neuromuscular junctions (NMJs) in fruit fly larvae. The larval NMJs have been extensively used by multiple labs as a model to study both the structure and function of synapses, shedding light on mechanisms of heterosynaptic plasticity. The body wall larval muscles in Drosophila are coinnervated by two types of glutamatergic excitatory motor neurons: Ib and Is. Ib MNs establish big synaptic buttons and exhibit a tonic firing pattern, while Is MNs make smaller synaptic buttons and have a phasic firing pattern.
First, the authors develop a new genetic tool (BoNT-C) to selectively suppress neurotransmission in either Ib or Is synapses without affecting the intrinsic structure of the pre and post-synaptic components of the NMJs. Compared to previously used methods (e.g. using Kir2.1, TNT, or apoptotic ablation), this is a significant advance in the field. Then, the authors take advantage of this tool to study how BoNT-C mediated silencing of Is inputs affects the structure/function of Ib inputs and vice versa. In parallel with using BoNT-C, the authors perform similar experiments in animals where Is or Ib NMJs are being perturbed by TNT or apoptotic ablation, enabling them to accomplish a nice comparative analysis of data collected from different animal groups. The authors make two significant conclusions as below:
Synaptic assembly, composition, and growth of Drosophila NMJs (both pre and post-synapse components) are not dependent on neurotransmitter release or firing patterns of motor neurons (phasic vs tonic).
Heterosynaptic structural or functional plasticity is induced only when MN-Is or MN-Ib innervation is physically absent. Such Heterosynaptic plasticity is not observed when they use BoNT-C to functionally silence (but not structurally perturb) MN-Is or MN-Ib motor neurons.
The conclusions of this paper are properly supported by the provided data. Overall this work opens a new window to examine novel aspects of heterosynaptic structural and functional plasticity.
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Reviewer #3 (Public Review):
The strength of the manuscript by Han et al. is the comprehensive characterization of BoNT-C, showing that it truly abolishes all evoked and mini responses without structural alteration of the NMJ. Based on this, the authors then show that ablation of all neurotransmission in either Ib or Is does not cause any compensatory changes (neither functional nor structural) in the 'other' (i.e. looking at Is when silencing Ib or looking at Ib when silencing Is).
The weakness of the manuscript lies in the modest gain over the previous work. Specifically, Aponte-Santiago had already shown that many parameters are not changed (in Ib when Is is perturbed, or in Is when Ib is perturbed), including that 'the Is terminal failed to show functional or structural changes following loss of the coinnervating Ib input' (quote …
Reviewer #3 (Public Review):
The strength of the manuscript by Han et al. is the comprehensive characterization of BoNT-C, showing that it truly abolishes all evoked and mini responses without structural alteration of the NMJ. Based on this, the authors then show that ablation of all neurotransmission in either Ib or Is does not cause any compensatory changes (neither functional nor structural) in the 'other' (i.e. looking at Is when silencing Ib or looking at Ib when silencing Is).
The weakness of the manuscript lies in the modest gain over the previous work. Specifically, Aponte-Santiago had already shown that many parameters are not changed (in Ib when Is is perturbed, or in Is when Ib is perturbed), including that 'the Is terminal failed to show functional or structural changes following loss of the coinnervating Ib input' (quote from 2020 paper). Hence, the only major difference is that Han et al now show that Ib also does not really change when Is is silenced. Aponte-Santiago also clearly showed a ~50% EJP reduction when Is or Ib are perturbed alone, and adding these two equals wild type. The highly emphasized finding of Han et al. that (quote) ' composite values of Is and Ib neurotransmission can be fully recapitulated by isolated physiology from each input' quite obviously follows from the one key finding that one does not affect the other, as mentioned above in the strengths. The wording is a bit odd, but really adding Is (with Ib perturbed) and Ib (with Is perturbed) inputs is really not adding much over either the main finding nor the previous work. On a different but somewhat similar topic, UAS-BoNT-C is not a new tool. I am a bit put off by the wording ' We have developed a botulinum neurotoxin, BoNT-C...'. More on this and the way the previous BoNT-C paper (Backhaus et al., 2016) is cited in the detail comments below in the recommendations for the authors. Additionally, the manuscript does not really dive into an analysis of phasic versus tonic functions (that's just a correlation with the Is and Ib dominant modes of function). Finally, since the authors show that loss of Is or Ib function does not cause any change in the other, we are left to wonder what actually DOES cause heterosynaptic plasticity. TNT or rpr DO cause some heterosynaptic plasticity and they also DO cause some structural changes - but whether the structural changes themselves are important here remains unclear. Substantial progress would have been to take the starting point that BoNT-C does not cause heterosynaptic plasticity, and then identify the signal that does (is is morphology? or signaling between Is and Ib? Or with the muscle?).
Overall, while an initial reading of the manuscript sounded rather exciting, a deeper analysis of the work in context of the literature of the last few years diminishes my enthusiasm for the novelty and progress provided.
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