Astrocytes mediate two forms of spike timing-dependent depression at entorhinal cortex-hippocampal synapses

Reviewer #1 (Public Review):

Summary:

The study characterized the cellular and molecular mechanisms of spike timing-dependent long-term depression (t-LTD) at the synapses between excitatory afferents from lateral (LPP) and medial (MPP) perforant pathways to granule cells (GC) of the dentate gyrus (DG) in mice.

Strengths:

The electrophysiological experiments are thorough. The experiments are systematically reported and support the conclusions drawn.
This study extends current knowledge by elucidating additional plasticity mechanisms at PP-GC synapses, complementing existing literature.

Weaknesses:

To more conclusively define the pivotal role of astrocytes in modulating t-LTD at MPP and LPP GC synapses through SNARE protein-dependent glutamate release, as posited in this study, the authors could adopt additional methods, such as alternative mouse models designed to regulate SNARE-dependent exocytosis, as well as optogenetic or chemogenetic strategies for precise astrocyte manipulation during t-LTD induction. This would provide more direct evidence of the influence of astrocytic activity on synaptic plasticity.

Reviewer #2 (Public Review):

Summary:

This work reports the existence of spike timing-dependent long-term depression (t-LTD) of excitatory synaptic strength at two synapses of the dentate gyrus granule cell, which are differently connected to the entorhinal cortex via either the lateral or medial perforant pathways (LPP or MPP, respectively). Using patch-clamp electrophysiological recording of tLTD in combination with either pharmacology or a genetically modified mouse model, they provide information on the differences in the molecular mechanism underlying this t-LTD at the two synapses.

Strengths:

The two synapses analyzed in this study have been understudied. This new data thus provides interesting new information on a plasticity process at these synapses, and the authors demonstrate subtle differences in the underlying molecular mechanisms at play. Experiments are in general well controlled and provide robust data that are properly interpreted.

Weaknesses:

- Caution should be taken in the interpretation of the results to extrapolate to adult brain as the data were obtained in P13-21 days old mice, a period during which synapses are still maturing and highly plastic.
- In experiments where the drug FK506 or thapsigargin are loaded intracellularly, the concentrations used are as high as for extracellular application. Could there be an error of interpretation when stating that the targeted actors are necessarily in the post-synaptic neuron? Is it not possible for the drug to diffuse out of the cell as it is evident that it can enter the cell when applied extracellularly?
- The experiments implicating glutamate release from astrocytes in t-LTD would require additional controls to better support the conclusions made by the authors. As the data stand, it is not clear how the authors identified astrocytes to load BAPTA and if dnSNARE expression in astrocytes does not indirectly perturb glutamate release in neurons.

Significance:

While this is the first report of t-LTD at these synapses, this plasticity process has been mechanistically well investigated at other synapses in the hippocampus and in the cortex. Nevertheless, this new data suggests that mechanistic differences in the induction of t-LTD at these two DG synapses could contribute to the differences in the physiological influence of the LPP and MPP pathways.

Reviewer #3 (Public Review):

Coatl et al. investigated the mechanisms of synaptic plasticity of two important hippocampal synapses, the excitatory afferents from lateral and medial perforant pathways (LPP and MPP, respectively) of the entorhinal cortex (EC) connecting to granule cells of the hippocampal dentate gyrus (DG). They find that these two different EC-DG synaptic connections in mice show a presynaptically expressed form of long-term depression (LTD) requiring postsynaptic calcium, eCB synthesis, CB1R activation, astrocyte activity, and metabotropic glutamate receptor activation. Interestingly, LTD at MPP-GC synapses requires ionotropic NMDAR activation whereas LTD at LPP-GC synapse is NMDAR independent. Thus, they discovered two novel forms of t-LTD that require astrocytes at EC-GC synapses. Although plasticity of EC-DG granule cell (GC) synapses has been studied using classical protocols, These are the first analysis of the synaptic plasticity induced by spike timing dependent protocols at these synapses. Interestingly, the data also indicate that t-LTD at each type of synapse require different group I mGluRs, with LPP-GC synapses dependent on mGluR5 and MPP-GC t-LTD requiring mGluR1.

The authors performed a detailed analysis of the coefficient of variation of the EPSP slopes, miniature responses and different approaches (failure rate, PPRs, CV, and mEPSP frequency and amplitude analysis) they demonstrate a decrease in the probability of neurotransmitter release and a presynaptic locus for these two forms of LTD at both types of synapses. By using elegant electrophysiological experiments and taking advantage of the conditional dominant-negative (dn) SNARE mice in which doxycycline administration blocks exocytosis and impairs vesicle release by astrocytes, they demonstrate that both LTD forms require the release of gliotransmitters from astrocytes. These data add in an interesting way to the ongoing discussion on whether LTD induced by STDP participates in refining synapses potentially weakening excitatory synapses under the control of different astrocytic networks. The conclusions of this paper are mostly well supported by data, but some aspects the results must be clarified and extended.

(1) It should be clarified whether present results are obtained with or without the functional inhibitory synapse activation. It is not clear if GABAergic synapses are blocked or not. If GABAergic synapses are not blocked authors must discuss whether the LTD of the EPSPs is due to a decrease in glutamatergic receptor activation or an increase in GABAergic receptor activation. Moreover, it should be recommended to analyze not only the EPSPs but also the EPSCs to address whether the decrease in synaptic transmission is caused by a decrease in the input resistance or by a decrease in the space constant (lambda).
(2) Authors show that Thapsigargin loaded in the postsynaptic neuron prevents the induction of LTD at both synapses. Analyzing the effects of blocking postsynaptic IP3Rs (Heparin in the patch pipette) and Ryanodine receptors (Ruthenium red in the patch pipette) is recommended for a deeper analysis of the mechanism implicated in the induction of this novel forms of LTD in the hippocampus.
(3) Authors nicely demonstrate that CB1R activation is required in these forms of LTD by blocking CB1Rs with AM251, however an interesting unanswered question is whether CB1R activation is sufficient to induce this synaptic plasticity. This reviewer suggests studying whether applying puffs of the CB1R agonist, WIN 55,212-2, could induce these forms of LTD.
(4) Finally, adding a last figure with a cartoon summarizing the proposed model of action in these novel forms of LTD would add a positive value and would help the reading of the manuscript, especially in those aspects related with the discussion of the results.

The extension of these results would improve the manuscript which provides interesting results showing two novel forms of presynaptic t-LTD in the brain synapses with different action mechanisms probably implicated in the different aspects of information processing.