Author Response
We are very thankful for the editors' and reviewers' thoughtful feedback and criticisms on our manuscript. We have carefully considered all of the comments and will provide a revised manuscript with detailed responses as soon as we can. In the meantime, we will make our best effort to conduct additional experiments to further support our conclusions.We greatly appreciate the time and consideration given to improving our work.
Reviewer #1 (Public Review):
Summary:
The question at hand is whether astrocytes contribute to the mechanism of long-term synaptic potentiation (LTP) at synaptic contacts between excitatory glutamatergic neurons and inhibitory neurons (E-I synapses). This is a legitimate query considering the immense body of work that has now established synaptic plasticity (LTP, LTD and spike-timing dependent plasticity) as an astrocyte-dependent process at excitatory synapses and, by contrast, the lack of knowledge on whether and how astrocytes control IN activity. Taking direct inspiration from that same body of work, authors recapitulate a number of experiments and approaches from prior seminal studies and provide evidence that E-I synapses in the stratum radiatum of the hippocampus display NMDAR-dependent plasticity, which can be suppressed by pharmacologically hindering astrocytes physiology, preventing astrocyte Ca2+ transients or blocking endocannabinoid CB1 receptors. Under any of these conditions, LTP can still be rescued by exogenously applying D-serine, a naturally occurring co-agonist of NMDARs primarily released by astrocytes. Coincidently, authors show that the conditions used to elicit LTP also cause a transient increase in NMDAR co-agonist site occupancy. Lastly, based on some evidence that gamma-CaMKII is predominantly expressed in INs rather than excitatory neurons, authors conducted AAV-mediated IN-specific gamma-CaMKII shRNA experiments and found that this is sufficient to suppress LTP at E-I synapses. They found that this approach also impairs contextual fear learning in behaving mice. Authors conclude that astrocytes gate LTP at E-I synapses via a mechanism wherein neuronal depolarization during LTP induction elicits endocannabinoid release which drives CB1-dependent astrocyte Ca2+ activity, causing the release of the NMDAR co-agonist D-serine (required for NMDAR activation).
Strengths:
This is an important question and the experimental work seems to have been conducted at high standards. The electrophysiology traces are impeccable, the experiments are well powered, including the behavioral testing, and multiple controls and validations are provided throughout. The figures are clear and easy to understand. Overall, the conclusions from the study are consistent, or partially consistent, by the findings.
We greatly appreciate you taking the time to evaluate our study thoroughly and provide such thoughtful feedback.
Main Weaknesses:
- A major point of concern is the lack of proper acknowledgment of the seminal studies that were mimicked in this manuscript, notably Henneberger et al, Nature 2010, Adamsky et al, Cell 2018; and Robin et al., Neuron 2017. The entire study design is a replica of these landmark studies: it isn't built upon or inspired from them, it exactly repeats the experiments and methods performed in them, coming dangerously close to being simply a hidden attempt to plagiarize published work. The resemblance goes as far as using an identical figure display (see Fig4.D vs Fig 2D of Ref#4). The issue is that authors frame the problem, scientist logic, reasoning, technical tricks, approaches, and interpretations as their own whereas, in reality, they were taken verbatim out of previous work and applied to a (shockingly) similar problem. The probity of the present study is thus in question. Authors need to clearly acknowledge, in all relevant instances, that the work presented here recapitulates the approach, reasoning and methodology used in past seminal studies that tackled the mechanisms of astrocyte regulation of LTP.
Thank you very much for your review and valuable comments on our manuscript. We greatly appreciate your concern regarding the proper acknowledgment of previous studies. We sincerely apologize for not adequately citing and acknowledging the seminal works in our manuscript. We highly value avoiding academic misconduct.
For the research design, although there are some similarities between our work and other studies, our key scientific questions and technical approaches are markedly different, as evidenced by our central hypothesis and experimental methods. We did not completely replicate their research design.
Regarding research methods, many basic techniques like electrophysiology, chemogenetic are common experimental methods, not patented by any one paper. Our choice of methods is based on the research needs, not to replicate a particular paper. But we recognize that there are similarities in our experimental methods, specifically the chemogenetic stimulation of astrocytes to induce de novo LTP, which has been inspired by previous studies (Van Den Herrewegen et al. Molecular Brain (2021), Adamsky et al. Cell (2018), Nam et al. Cell reports (2019)). We were also inspired by the previous work of Henneberger et al. in Nature (2010) to investigate whether stimulation, specifically we using TBS (theta burst stimulation), could transiently increase NMDA receptor-mediated synaptic responses.
For the similarity between our Fig. 4D and Fig. 2D of Ref. 4, it is primarily because both studies have the similar purpose(we monitored NMDA currents in interneurons, others monitored in pyramidal cells) using similar methods, but our figure layout follows a regular display pattern. Additionally, we would like to draw your attention to our previous studies, specifically Shen et al., Scientific Reports (2017), Supplementary figure 4, and Shen et al., Journal of Neurochemistry (2021), Supplementary figures 8 and 9. In these studies, we also employed a regular display pattern in our figure layouts. It is important to note that while there may be similarities in the figure arrangement, each study presents distinct findings and contributes to the broader understanding of the topic.Our use of a similar way to present data does not equal plagiarism. We apologize for any confusion caused by the lack of explicit citation and acknowledgment in our manuscript again. In the revised version, we will ensure to provide clear and detailed references to all relevant studies.
In terms of citations, we have cited Henneberger et al, Nature 2010, Adamsky et al, Cell 2018; and Robin et al., Neuron 2017.'s work in multiple places, indicating we have learned from their research ideas and findings. We will supplement any missing citations. But overall, our work has distinct differences and innovations.
We are not intended as a hidden attempt to plagiarize or simply replicate their methods. Rather, they are part of a deliberate effort to establish a comparable and reproducible experimental framework. Our study aims to validate and further explore the conclusions drawn by replicating the experiments of these seminal studies and deepening our understanding of the mechanisms of astrocyte regulation of LTPE-I.
We sincerely appreciate your review and guidance. We will carefully consider your criticism and incorporate more accurate and thorough citations in the revised version, ensuring proper respect and acknowledgment of the previous works.
- Relatedly, in past work, field recordings were used to monitor LTP in hippocampal slices (refs 4, 26 and others). This method captures indiscriminately all excitatory synapses where glutamate is released to cause AMPAR-dependent (and NMDAR) transmembrane flux of cations in the postsynaptic element, including E-I synapses and not just E-E synapse like the authors claim. Therefore, a strong argument can be made that there is no actual ground to differentiate the present results from past ones.
Thank you for your thoughtful comments regarding the differentiation of our results from previous studies. We appreciate the opportunity to address this issue and provide further clarification.
Indeed, in past studies, field recordings were commonly utilized to monitor long-term potentiation (LTP) in hippocampal slices. It is true that this method captures all flux of cations in excitatory synapses, inhibitory synapses and glia. This includes both excitatory-excitatory (E-E) and excitatory-inhibitory (E-I) synapses.
When using the LTP recording protocol, one limitation is that the experimenter cannot determine the exact contribution of E-E and E-I currents to the recorded current. Additionally, it is not possible to know, with the same induction protocol, the specific effects on E-E synapses versus E-I synapses. It is plausible that E-E synapses could undergo LTP, while E-I synapses could undergo LTD, or vice versa.
Thus, it becomes crucial to carefully dissect the functioning of E-I synapses and investigate how astrocytes modulate these synapses. Past field recordings have provided important insights, our selective interrogation of the astrocyte-E-I synapse interface represents a conceptual advance to delineate the nuanced modulation of distinct synaptic connections by astrocytes. We specifically focus on studying the modulation of E-I synapses by astrocytes and aim to elucidate the intricate dynamics and underlying mechanisms. By untangling the complex contributions of astrocytes to E-I synapse function and plasticity, we can unveil novel aspects of neuroglial interactions and advance our understanding of the fundamental principles governing neural network activity.
- There is a general lack of excitement about this study. One reason is that it replicates almost identically past work, as mentioned above. Another is that the scientific question and importance of the findings are not framed appropriately. The work is presented as an astrocyte-focused investigation, but it has very limited value to the astrocyte field. The findings are, in all accounts, identical to those unveiled by previous work especially because E-I synapses are, in fact, excitatory synapses. Where this study does bring value, however, is to the field of interneurons, but it would need to be reframed to shift the emphasis from astrocytes to E-I connections. Authors would need to elevate the text by framing their work around relevant considerations, such as IN diversity, mechanisms of LTP in IN subtypes, role of E-I connections in hippocampal circuit function, information processing, behavior, spatial learning, navigation, or grid cells activity etc...
We appreciate your insightful comments and concerns regarding the lack of excitement surrounding our study. We would like to clarify that while our study use similar certain methodologies, for example electrophysiology, chemogenetics and pharmacology, our research aims to provide a deeper understanding of the underlying mechanisms of how astrocytes regulate E-I synapses. We apologize if this replication aspect was not adequately highlighted in our manuscript, and we will make sure to emphasize the novel contributions of our study in the revised version.
Regarding the framing of our study, we recognize the importance of interneurons and the role of E-I connections in hippocampal circuit function, information processing, behavior, spatial learning, navigation, and other relevant aspects. However, the scientific question and scope of the study are to explore whether and how astrocytes modulate E-I synapses. We believe that this study brings value to the field of astrocyte-neuron interaction. Of course, this study also brings value to the field of interneurons. Perhaps the lack of excitement among audiences stems from the mechanisms for astrocytes modulating E-I and E-E synapses are the same.
- A clear weakness of the study is that it fails to consider the molecular and functional diversity of interneurons in the stratum radiatum and provides no insights or considerations related to it. Authors provide no information on what type of IN were patched, or the location of their cell body in the s.r., effectively treating all patched IN as a homogeneous ensemble of cells - which they are not. Relatedly, the study is extremely evasive on the importance of the results in the context of inhibitory interneurons. This renders the significance of the insights highly uncertain and dampens both the impact of the study and the excitement it generates. Hippocampal interneurons are very diverse in molecular identity, sub-anatomical location, morphology, projections, connectivity and functional importance. Some experts go as far as recognizing 29 subtypes in the CA1, including 9 in the stratum radiatum alone (based on the location of their soma). However, this is neither addressed nor acknowledged by the authors, with the exception of a statement (line 659) where they claim to have "focused on a subpopulation of interneurons in the stratum radiatum" without providing any precision or evidence to corroborate this assertion. This diversity, alone, could explain why not all cells showed LTP, or why the mechanisms authors describe in the radiatum do not seem to be at play in the oriens. Hence, carefully considering the diversity of INs in the present work is necessary. It would refine and augment the conclusions of the paper. Instead of a sub-region specificity, the study might fuel the notion of an IN subtype specificity of LTP mechanisms, which is more useful to the field.
Thank you very much for your review and valuable comments on our study. We agree with the point you raised regarding a clear weakness in our study, specifically the lack of consideration the diversity of interneurons in the stratum radiatum.
As the reviewer notes, there are many subtypes of interneurons in hippocampal region CA1 that likely contribute in distinct ways to circuit function. Unfortunately we did not gather information on the specific molecular or morphological identity of the interneurons we recorded from.This is a limitation of our study. We will add discussion of this issue as a caveat, and highlighted it as an opportunity for future work to dissect how long-term potentiation in interneurons regulated by astrocytes may differ across interneuron subpopulations. Thank you once again for your insightful comments.
- Authors take several shortcuts. Some of the conclusions are a leap from the experiments and are only acceptable due to the close analogy with very similar investigations conducted in the past that provided identical results. For instance, the present study provides no evidence of any sort that D-serine is involved - rather, it provides evidence that the pathway at hand contributes to increasing the occupancy of the co-agonist binding site of NMDARs. Considering the absence of work demonstrating that D-serine is the endogenous co-agonist of NMDARs at E-I synapses, most of the authors claims on D-serine are unfounded. This would necessitate using tools such as the canonical D-serine scavengers DAAS or DsDA, serine racemase KO mice etc. Similarly, authors provide no compelling evidence that endocannabinoid CB1 receptors involved in this pathway are located on astrocytes
Thank you for your insightful comments on our study. We appreciate your attention to detail and your concerns regarding our conclusions. We agree that further evidence is needed to establish the involvement of D-serine as the endogenous co-agonist of NMDARs at E-I synapses. We will take into consideration your suggestion of using tools such as D-serine scavengers to provide clearer evidence.
Regarding the involvement of endocannabinoid CB1 receptors on astrocytes in this pathway, we provide evidence that astrocytic calcium signaling could blocked by CB1 receptor antagonist AM251, as shown in figure 3.However, we agree that further research is necessary to accurately identify the localization of CB1 receptors. As part of our future investigations, we will take note of this limitation in our discussion and emphasize the need for additional studies to explore the precise location of CB1 receptors. In addition, we will endeavor to perform immunohistochemistry to identify the exact location of CB1 receptors in astrocytes.
Thank you once again for your valuable feedback. We will carefully address these concerns and make appropriate revisions to ensure the clarity and accuracy of our findings.
- An important caveat in this study is the protocol employed to induce LTP, which includes steps of sustained depolarization of the patched IN to -10mV. Neuronal depolarization is known to induce endocannabinoids production. In several instances, this was shown to 'activate' astrocytes and elicit the release of astrocyte-derived transmitters at nearby synapses. This implies that the endocannabinoid-dependent pathway described in the study is, most likely, artificially engaged by the protocol itself. Hence, the present work only provides evidence that an astrocyte-dependent, CB1-D-serine-pathway can be artificially called upon with this specific LTP protocol, but does not convincingly demonstrate that it is naturally occurring or necessary for plasticity at E-I synapses. Authors would need to thoroughly address this caveat by replicating some of their key findings (AM251, calcium-clamp, D-serine and CaMKII shRNA) using a protocol that does not entail the artificial depolarization of the patched interneuron.
Thank you for raising this important point. We agree that the sustained depolarization protocol we used to induce LTP could potentially engage endocannabinoid signaling and astrocyte activation. However, we observed that preventing astrocyte Ca2+ transients or blocking endocannabinoid CB1 receptors prevented the induction of LTP by this depolarization protocol suggests that this astrocyte-endocannabinoid-dependent pathway is necessary,
Importantly, synaptic depolarization of neurons can occur naturally during learning and memory. Though ‘artificial’ here, our protocol may mimic aspects of natural activity patterns that engage ‘endocannabinoid release’ and astrocyte involvement in plasticity.
Another limitation of our study is that we currently cannot conclusively determine the source of the CB1. We cannot distinguish whether the CB1 originates from neurons or astrocytes based on our current experiments. We will explicitly acknowledge this caveat in the discussion, noting that further experiments are needed to clarify the cellular origin of the CB1. Thank you for drawing our attention to this critical issue - we will refine the manuscript accordingly to more comprehensively and accurately present the study conclusions and limitations. Your feedback helps improve the rigor of our research.
- Reading and understanding are hindered by a rather vast array of issues with the text itself. It needs thorough editing for typos, misnomers, meaning-altering errors in syntax, and a number of issues with English.
Thank you very much for your review and feedback on our text. We highly appreciate your comments and take them seriously. We will carefully address the issues you mentioned and thoroughly edit the text to eliminate any typos, misnomers, syntax errors that may alter the meaning, and other English-related issues. We truly value your input and appreciate your patience as we work on these improvements.
Reviewer #2 (Public Review):
Summary:
This work explores the implication of astrocytes in the regulation of long-term potentiation of excitatory synapses onto inhibitory neurons in CA1 hippocampus. They found that astrocytes of a sub-region of CA1 regulate this plasticity through their activation of endocannabinoids that lead to the release of the NMDA receptor co-agonist, D-serine.
Strengths:
The experiments are well considered and conceptualized, and use appropriate tools to explore the role of astrocytes in the tripartite synapse. The results highlight a novel role of astrocytes in an important aspect of the synaptic regulation of the hippocampal circuit. There are extensive levels of analysis for each experimental group of evidence.
Thank you for your positive feedback on our study. We appreciate your recognition of the careful consideration and conceptualization of our experiments, as well as the use of appropriate tools to investigate the role of astrocytes in the tripartite synapse. We are pleased to hear that the results have highlighted a novel role of astrocytes in an important aspect of synaptic regulation in the hippocampal circuit.
Thank you for taking the time to review our work and for providing such positive feedback. We will continue to improve and refine our study based on your valuable comments.
Weaknesses:
The authors underscore and used an oversimplified view of the heterogeneity of interneuron populations and their selective roles in the hippocampal network. Also, there is an uneven level of astrocyte-selective tools used in the different experiments which creates an uneven strength of arguments and conclusions regarding the role of glial cells. Finally, the wording used by the authors often lead to some confusion or sense of overinterpretation
We appreciate the reviewer raising these important points about the characterization of interneuron and astrocyte populations in our study. We agree that oversimplifying or overlooking cellular heterogeneity could undermine the conclusions. In the revised manuscript, we will:
Add more detailed discussion of interneuron diversity. We will note this as an area for further study.
Review the wording used when describing results and conclusions, ensuring we avoid overstating interpretations of the data.
Thank you again for the thoughtful feedback.
