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  1. Author Response:

    Reviewer #1 (Public Review):

    Lenz et al have shown that IP injection of atRA does not affect sEPSC amplitude, sIPSC amplitude and frequency in the denta gyrus of both ventral and dorsal hippocampus. Interestingly, they observed a strong promoting effect of atRA on sEPSC frequency in the denta gyrus of dorsal, but not ventral, hippocampus. Lastly, they did not observe an difference in I/O in vivo, but did observe enhanced in vivo LTP in denta gyrus of mice injected with atRA which is abolished in the synaptopodin KO mice. The effect of atRA on LTP is very interesting as on sEPSC frequency in dorsal denta gyrus.

    1. I do not agree with the authors' claim that atRA does not have a major effect on excitatory synaptic transmission. It seems that the sEPSC frequency increase by ~100%. Even if the 4 outlier points are excluded, the rest of the data points still clearly indicate an increase of sEPSC frequency.

    We agree with the reviewer, that this point warrants further discussion. We have highlighted out findings in the revised version of the manuscript.

    The abstract (lines 30-32) of the revised manuscript now read: “No major changes in synaptic transmission were observed in the ventral hippocampus while a significant increase in both sEPSC frequencies and synapse numbers were evident in the dorsal hippocampus 6 hours after atRA administration."

    Lines 392-395: “Nevertheless, the results of the present study demonstrate increased sEPSC frequencies and synapse numbers in the dorsal hippocampus of atRA-treated animals, thereby confirming that atRA targets excitatory synapses in the dorsal hippocampus.”

    What is the possible explanation of increased sEPSC frequency by atRA in dorsal region? Increased excitability of presynaptic neurons? (use TTX to decipher this?) Increased spine density? It seems that the authors did dye fill already… Count spine density? AND/OR increased glutamate release probability? (PPR measurement?) Did the authors perform I/O measurement in slice?

    It is imperative that the authors tackle this issue head on.

    We thank the reviewer for these important comments. To further address this issue, additional experiments were performed and structural properties of asymmetric synapses were assessed in the molecular layer of the dorsal hippocampus using transmission electron microscopy (see new Figure 6). Indeed, these experiments revealed no significant difference in the morphological properties of individual asymmetric synapses, i.e., PSD length and presynaptic vesicle numbers. However, an increase in the number of PSDs per area was observed, which may reflect -at least in part- increased sEPSC frequencies in our experiments.

    Lines 302-316: “Next, transmission electron microscopy was used to assess the structural properties of excitatory synapses in the outer two thirds of the molecular layer in the dorsal hippocampus which is the layer of the major excitatory input from the entorhinal cortex (Figure 6). Cross sections of asymmetric synapses, i.e., the numbers and length of postsynaptic densities (PSD) and presynaptic vesicle counts, were quantified in control and atRA-treated mice (Figure 6A). It is well-established that PSD length in synaptic cross sections correlates to synaptic strength [43]. In agreement with our electrophysiological recordings, which showed no significant difference in the sEPSC amplitude between the groups (c.f., Figure 1D), PSD lengths did not significantly change in the atRA-treated group (Figure 6B). However, a robust increase in the number of PSDs per area was detected, and presynaptic vesicle counts were not significantly different between the two groups (Figure 6B, C). These results indicate that the structural properties of synapses are not affected by atRA, and that increased synapse numbers may explain the increased sEPSC frequencies in the dorsal hippocampus of atRA-treated mice.”

    Lines 373-379: “In the present study, however, we did not observe changes in excitatory synaptic strength in dentate granule cells in either the ventral or the dorsal hippocampus. Specifically, no changes in the sEPSC amplitudes were observed [12]. Consistent with these findings no major changes in the ultrastructural properties of excitatory synapses, i.e., PSD lengths and presynaptic vesicle counts of asymmetric synapses, were observed between the two groups. Interestingly, ultrastructural analysis revealed an increase in the number of asymmetric synapses in the dorsal hippocampus of atRA-treated animals.”

    1. The author need to specify which part of the denta gyrus for their in vivo study, as they discovered difference between ventral and dorsal in sEPSC frequency in slice preparation.

    Done! Thank you!

    Lines 183-186 now read: “Then a tungsten recording electrode (TM33B01KT, World Precision Instruments) was lowered in 0.1 mm increments while monitoring the waveform of the field excitatory postsynaptic potential (fEPSP) in response to 500 µA test pulses until the granule cell layer in the dorsal part of the hippocampus was reached (1.7-2.2 mm below the surface).”

    Lines 324-326 now read: “To test the effects of atRA on the ability of neurons to express synaptic plasticity, long-term potentiation (LTP) experiments on perforant path synapses to dentate granule cells were carried out in the dorsal hippocampus of anesthetized mice (Figure 7A).”

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  2. Evaluation Summary:

    All-trans retinoic acid (atRA) is a potent regulator of synaptic function known to be critical for certain forms of homeostatic plasticity. Previous work by the Vlachos group established that atRA also modulates synaptic function in human cortex and linked the synaptic effects of atRA to the spine apparatus protein synaptopodin. As a follow up study, the present work investigated the effect of atRA in the hippocampus. The authors found that atRA can play a key role in modulating enduring forms of synaptic plasticity in the dentate gyrus of the hippocampus even when it does not seem to drive overt changes in basal synaptic function, and this "metaplasticity"-related effects also require synaptopodin. Together, these studies establish a critical role of atRA in modulating synaptic transmission and plasticity at multiple regions of the human brain.

    (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 #2 agreed to share their name with the authors.)

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  3. Reviewer #1 (Public Review):

    Lenz et al have shown that IP injection of atRA does not affect sEPSC amplitude, sIPSC amplitude and frequency in the denta gyrus of both ventral and dorsal hippocampus. Interestingly, they observed a strong promoting effect of atRA on sEPSC frequency in the denta gyrus of dorsal, but not ventral, hippocampus. Lastly, they did not observe an difference in I/O in vivo, but did observe enhanced in vivo LTP in denta gyrus of mice injected with atRA which is abolished in the synaptopodin KO mice. The effect of atRA on LTP is very interesting as on sEPSC frequency in dorsal denta gyrus.

    1. I do not agree with the authors' claim that atRA does not have a major effect on excitatory synaptic transmission. It seems that the sEPSC frequency increase by ~100%. Even if the 4 outlier points are excluded, the rest of the data points still clearly indicate an increase of sEPSC frequency.

    What is the possible explanation of increased sEPSC frequency by atRA in dorsal region? Increased excitability of presynaptic neurons? (use TTX to decipher this?) Increased spine density? It seems that the authors did dye fill already... Count spine density? AND/OR increased glutamate release probability? (PPR measurement?) Did the authors perform I/O measurement in slice?

    It is imperative that the authors tackle this issue head on.

    1. The author need to specify which part of the denta gyrus for their in vivo study, as they discovered difference between ventral and dorsal in sEPSC frequency in slice preparation.
    Read the original source
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  4. Reviewer #2 (Public Review):

    The authors explore the effect of all-trans retinoic acid (atRA) on synaptic function and plasticity in the dentate gyrus of the hippocampus. Previous studies have established that atRA is a critical synaptic regulator that underlies an important form of homeostatic synaptic plasticity. This study builds on recent work by the same group showing that atRA enhances synaptic function in layer 2/3 pyramidal neurons of cortex in both mice and humans, and that atRA's synaptic regulating effects are critically dependent on synaptopodin, a protein that is specifically localized to the spine apparatus. In the present study, the authors used systemic treatment of atRA (via i.p. injection) and then monitored synaptic and intrinsic properties of dentate granule neurons in both dorsal and ventral hippocampus. Under these conditions, they found very little effect of systemic atRA on basal synaptic properties or intrinsic excitability of dentate granule neurons, but observed a striking enhancement of long-term potentiation (LTP) of inputs onto granule cells recorded in vivo in anesthetized mice. In the absence of atRA, LTP was induced but diminished over the next 60 min - systemic atRA rendered this LTP significantly more persistent over this time period. This plasticity modulating effect of atRA is absent in synaptopodin knockout mice, suggesting some mechanistic overlap in the plasticity modulating and synapse enhancing actions of atRA. This work represents a significant advance in demonstrating atRA can modulate enduring forms of synaptic plasticity even in the absence of overt regulation of basal synaptic function.

    The experiments presented in this paper have all been well executed and the data presented justify the conclusions made in the paper. The authors also provide a thoughtful discussion of potential limitations of the approaches and outline follow-up experiments that will be informative. In particular, the systemic administration of atRA used in the study has the advantage of examining atRA actions in vivo, but the effective concentration of atRA in different brain regions that follows is unclear. The authors demonstrate that systemic atRA alters gene expression in the hippocampus, an important finding that clearly shows systemic atRA reaches the brain in sufficient concentrations. Yet, whether basal synaptic properties or intrinsic excitability of granule neurons might be altered by different local atRA concentrations remains an open question that should be addressed in future studies. Still, the data clearly demonstrate that atRA can modulate enduring forms of synaptic plasticity in the absence of overt changes in basal synaptic function.

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