Retinoic acid-gated BDNF synthesis in neuronal dendrites drives presynaptic homeostatic plasticity

Decision letter after peer review:

Thank you for submitting your article "Retinoic acid-gated BDNF synthesis in neuronal dendrites drives presynaptic homeostatic plasticity" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, including Dion K Dickman as Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Gary Westbrook as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Michael A Sutton (Reviewer #2).

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted the Essential Revisions section to help you prepare a revised submission.

Essential revisions:

This manuscript probes the mechanism of postsynaptic retinoic acid (RA) signaling on presynaptic function. BDNF has important roles in synaptic plasticity, but how retrograde BDNF signaling is controlled following synaptic inactivity is unclear. The authors use genetic tools to localize the action of different components of the pathway to pre- or post-synaptic compartments and use biochemical approaches to define a molecular link between retinoic acid and local translation of distinct BDNF transcripts. The findings presented here fill a gap in our knowledge regarding how presynaptic function is adaptively modulated by BDNF by highlighting the role of RA in this process. The experiments have been well-executed and the data provide compelling support for the model proposed by the authors. There are, however, some important issues to address:

1) Evaluate the relative infection efficiency in CA3 neurons: This is critical in interpreting the negative result with RARa deltion. The pre-synaptic Cre expression would require a high percentage of CA3 neurons infected. From Figure 3, it appears this is achieved, but some information on this point would be helpful. Also in the results text for Figure 1, it should mention the use of AAV to express Cre (this is mentioned for Figure 3 but not Figure 1).

2) Define and discuss what the change in mEPSC frequency means: Changes in mEPSC frequency may reflect multiple synaptic adaptations – changes in presynaptic release probability or alterations in synapse numbers. The experiments done clearly delineate pre- and/or postsynaptic roles for RARα, BDNF, and TrkB, but alone, mEPSC frequency changes could reflect either enhanced release probability, growth of new synaptic contacts, or AMPAR recruitment to silent synapses. The authors should explicitly address these potential alternative interpretations early on in the paper. There is substantial evidence from other studies that the mEPSC frequency changes under these conditions reflect altered presynaptic release as the authors suggest, and making contact with this literature would make the conclusions of this paper that much more compelling.

3) Discuss discrepancies with previous studies: The issue of age (of culture and animals) on whether this form of pre-synaptic plasticity occurs is important (although was also established in the earlier Wang paper). Some papers have reported frequency changes during HSP but many have reported no changes (including several from the Chen lab). Age/DIV seems to explain at least some of the difference, but I haven't gone through all the papers to check. It does seem that BDNF levels are increased even by 14DIV for slice cultures (Figure 2A). What age of slice cultures were used in early papers (like Aoto, 2008; this information was missing from that paper)? This has been a contentious issue over the years, and since this offers a potential resolution to the discrepancies, more discussion of it is warranted.

4) Assess mIPSC changes: It is noted in the discussion that BDNF can increase mIPSC frequency. Does this happen here? During HSP, at least post-synaptically, excitatory and inhibitory synapses are inversely regulated. But it is possible miniature frequency is increased for both types. Or maybe not – either way, it would be interesting to define. Perhaps mIPSC amplitude goes down, but maybe that also hasn't been shown at these older ages/DIV. Looking at mIPSCs would extend impact and improve the paper.

5) Improved discussion and integration with previous work in the field: The results demonstrate a role for RA in controlling local dendritic BDNF synthesis and suggest RARα binding as a mechanism. As the authors note, previous work has similarly implicated the mTORC1 signaling pathway in local BDNF translation in response to synaptic inactivity. Parallel findings have been observed at the Drosophila neuromuscular junction. The authors may want to speculate in the discussion on whether these two pathways work together or in parallel during homeostatic plasticity.

6) Respond to several points of clarification:

– Figures 1 and 3 include mEPSC traces where the background has been sharded to color coordinate with the respective histograms. This was likely done to make the figures easier to read, but the shading is somewhat distracting and really does the opposite. This is not a major issue as it does not affect the data or its interpretation, but the figures would be improved if either the traces themselves are colored or the shading is simply removed.

– The authors should consider superimposing the individual data points over the histograms for the electrophysiology data shown in Figure 1B-C and Figure 3B-F

– The model shown in Figure 3 —figure supplement 1 very nicely summarizes the RA pathway, but could also be used to include other work in the field that implicates other pathways (e.g., the mTORC1 pathway).

– In the introduction, the phrase 'runaway tendency' is vague and not obvious as to its meaning. Perhaps another phrase would be better.

– The figure labels could be improved. Pre-KO and post-KO can be misinterpreted –

"Pre-KO" could be misinterpreted as being before the Cre expression manifested. Maybe "pre-syn KO" and "post-syn KO" would be easier to understand when initially looking at the figures?

– The e-phys figures probably should show the data points (in the modern style) instead of just being bar graphs. And shouldn't the ephys statistics be 2-way ANOVAs instead of just t-tests? This likely won't impact the results but would be better.

Reviewer #1 (Recommendations for the authors):

Overall these findings fill a gap in our knowledge regarding how presynaptic function is adaptively modulated following postsynaptic inhibition and the connection between RA and BDNF in this process. The experiments in Figure 2 are well controlled and provide compelling evidence that RARalpha binds to BDNF mRNA transcripts, while Figures 1 and 3 localize activity in pre- vs post-synaptic compartments. A plausible model is presented in Figure S3 to describe how chronic inactivity at synapses initiates retrograde homeostatic signaling through RA and BDNF. This is a relatively brief manuscript and a question is raised about whether the specific results regarding RARalpha binding to BDNF mRNA to provide a sufficient enough advance beyond what was already known. However, the real advance here is in connecting RA to the previous work on retrograde BDNF signaling at presynaptic terminals, and separating this from what is known about postsynaptic control of AMPAR synthesis and receptor scaling. This provides an important foundation to understand the signaling systems that orchestrate pre- and post-synaptic responses to synaptic inactivity. Below are some important questions and experiments for the authors to consider.

1. Overall, the authors provide very good controls, both positive and negative, for BDNF mRNA binding to RARalpha. That being said, it seems a good control could be performed using their synaptoneurosome preparation: Add RA to synaptoneurosome preparations from the pre vs. postsynaptic RARalpha cKOs in Figure 1 to determine whether BDNF is still increased. This would be a great confirmation that postsynaptic RARalpha is necessary.

2. Can the authors further refine and/or separate the postsynaptic actions of glutamate receptor blockade/chronic inactivity between RA/RARalpha-mediated responses protein synthesis more generally? In terms of the presynaptic plasticity studied in this manuscript, both RA and BDNF seem to function in the same pathway, requiring modulation of BDNF protein synthesis by RARalpha. This retrograde signaling by BDNF is clearly distinct from the pathways that control postsynaptic homeostatic responses to inactivity, such as AMPAR synthesis and receptor scaling. Can the authors determine or define what responses are protein synthesis-dependent, RA/RARalpha-independent in the postsynaptic compartment?

3. Do the authors have any insights into what the increase in mEPSC frequency actually means for how presynaptic function is changing due to retrograde BDNF signaling gated by RA?

Reviewer #2 (Recommendations for the authors):

This paper examines the role of retinoic acid (RA) in regulating feedback control of presynaptic neurotransmitter release via postsynaptic synthesis and release of brain-derived neurotrophic factor (BDNF). The authors show that deletion of RA receptor α (RARα) in CA1 neurons of organotypic slices prevents the increase in mEPSC frequency that accompanies RA administration or chronic block of AMPA receptors in these cells. Importantly, deletion in presynaptic CA3 neurons has no effect, indicating a postsynaptic role for RARα. The authors then demonstrate that RARα (but not RARβ) binds specific BDNF transcripts (containing exons 2 and 6) that have been shown by previous studies to localize to distal dendrites, and that RA administration drives BDNF synthesis in isolated synaptic fractions (synaptoneurosomes). Finally, through conditional deletion experiments targeting either the pre- or postsynaptic compartment, the authors demonstrate a postsynaptic role for BDNF and a presynaptic role for its receptor TrkB in presynaptic compensation driven by RA or AMPAR blockade, revealing a transsynaptic feedback mechanism requiring postsynaptic release of BDNF and presynaptic BDNF-TrkB signaling.

The experiments have all been well executed with appropriate controls and the data provide compelling support for the conclusions of the paper. These results are important, as they reveal a homeostatic control mechanism regulating presynaptic function mediated by RA signaling that appears to operate in parallel with the well-established postsynaptic compensation mechanism first described by this group and validated by others. The findings have broad implications for our understanding of homeostatic regulation at synapses and are of significant general interest. I have just a few suggestions for the authors to consider in revising their paper.

1) Strictly speaking, changes in mEPSC frequency may reflect multiple synaptic adaptations – changes in presynaptic release probability or alterations in synapse numbers. The experiments done clearly delineate pre- and/or postsynaptic roles for RARα, BDNF, and TrkB, but alone, mEPSC frequency changes could reflect either enhanced release probability, growth of new synaptic contacts, or AMPAR recruitment to silent synapses. The authors should explicitly address these potential alternative interpretations early on in the paper. There is substantial evidence from other studies that the mEPSC frequency changes under these conditions reflect altered presynaptic release as the authors suggest, and making contact with this literature would make the conclusions of this paper that much more compelling.

2) The results demonstrate a role for RA in controlling local dendritic BDNF synthesis and suggest RARα binding as a mechanism. As the authors note, previous work has similarly implicated the mTORC1 signaling pathway in local BDNF translation in response to synaptic inactivity. The authors may want to speculate in the discussion on whether these two pathways work together or in parallel during homeostatic plasticity.

3) Figures 1 and 3 include mEPSC traces where the background has been sharded to color coordinate with the respective histograms. While I understand this was done to make the figures easier to read, the shading is somewhat distracting and really does the opposite. This is not a major issue as it does not affect the data or its interpretation, but I think the figures would be improved if either the traces themselves are colored or the shading is simply removed.

4) If possible, the authors should consider superimposing the individual data points over the histograms for the electrophysiology data shown in Figure 1B-C and Figure 3B-F.

5) The model shown in Figure 3 —figure supplement 1 very nicely summarizes the RA pathway, but could also be used to include other work in the field that implicates other pathways (e.g., the mTORC1 pathway).

Reviewer #3 (Recommendations for the authors):

The main issue is one of novelty. The basic findings have been made before, with this lab already showing the activity blockade increase in mEPSC frequency is RA dependent (and age dependent), and other papers (cited in the text but maybe underemphasized; Jakawich and Lindskog) have shown that this increase in frequency was BDNF dependent, released post-synaptically and signaling pre-synaptically. And the RAR is a known transcriptional regulator. This paper nicely links these elements together but it is confirming what is expected. The specific BDNF transcript involved (exon II and IV) is the novel finding.

However, it is noted that in the discussion that BDNF can increase mIPSC frequency. Does this happen here? During HSP, at least post-synaptically, excitatory and inhibitory synapses are inversely regulated. But it is possible miniature frequency is increased for both types. Or maybe not – either way, it would be interesting. I would assume that mIPSC amplitude would go down, but maybe that also hasn't been shown at these older ages/DIV. Looking at mIPSCs would add novelty and improve the paper.

The issue of age (of culture and animals) on whether this form of pre-synaptic plasticity occurs is important (although was also established in the earlier Wang paper). Some papers have reported frequency changes during HSP but many have reported no changes (including several from the Chen lab). Age/DIV seems to explain at least some of the difference, but I haven't gone through all the papers to check. It does seem that BDNF levels are increased even by 14DIV for slice cultures (Figure 2A). What age of slice cultures were used in early papers (like Aoto, 2008; this information was missing from that paper)? This has been a contentious issue over the years, and since this offers a potential resolution to the discrepancies, more discussion of it is warranted.

RA dependent plasticity at the early age (14 DIV) occurs rapidly, within a few hours. Here the CNQX treatment was for 36 hours. Is this longer time necessary? Is RA induction slower at 21DIV? Or is only the frequency change slow and the amplitude change faster? Some explanation of this would be appropriate.