Local projections of layer Vb-to-Va are more effective in lateral than in medial entorhinal cortex
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Summary: The study addresses a fundamentally important question regarding the connectivity of LVb and LVa in the medial and lateral entorhinal cortex. The authors suggest that LVb to LVa connection exists in the LEC but not in the MEC. This finding would have important implications on studies investigating circuits functions between the hippocampus and the EC. All three reviewers found the central question important and the data novel. However, there are several technical issues that limit the robustness of the authors claim.
While the transgenic animal used in these experiments is elegant and novel, it only labels a subpopulation of the neurons. There is a possibility of selective labelling of neurons with distinct connectivity patterns. The authors would need to show that their approach is not leading to false negative results due to the selective visualization of those neurons that project more modestly to the LVa.
The specificity of the injection to the LEC/MEC should be better documented and potential spread to the perirhinal or postrhinal cortex carefully excluded.
The findings are presented as LVb to LVa connection did not exist at all in the MEC, however the data shows that the connection is there but it is significantly less dense than in the LEC. Given the graded finding, if the authors aim to show their central claim regarding the lack of mediation of hippocampo-cortical outputs by this connection in the MEC, this would require the addition of functional studies.
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Abstract
The entorhinal cortex, in particular neurons in layer V, allegedly mediate transfer of information from the hippocampus to the neocortex, underlying long-term memory. Recently, this circuit has been shown to comprise a hippocampal output recipient layer Vb and a cortical projecting layer Va. With the use of in vitro electrophysiology in transgenic mice specific for layer Vb, we assessed the presence of the thus necessary connection from layer Vb-to-Va in the functionally distinct medial (MEC) and lateral (LEC) subdivisions; MEC, particularly its dorsal part, processes allocentric spatial information, whereas the corresponding part of LEC processes information representing elements of episodes. Using identical experimental approaches, we show that connections from layer Vb-to-Va neurons are more effective in LEC compared with dorsal MEC. This indicates that the hippocampal-cortex output circuit is more effective in LEC, suggesting that episodic systems-consolidation may use LEC-derived information more than allocentric spatial information from MEC.
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Reviewer #3:
The present manuscript focuses on a subpopulation of layer 5 neurons in medial and lateral entorhinal cortex and its functional connections to target neurons in layers 2, 3 and 5. The authors show a difference in LVb-to-LVa connectivity between MEC and LEC. The results suggest that the entorhinal output circuit via LVb-to-LVa is present primarily in LEC.
The work relies on and is made possible by a newly described transgenic mouse (TG) where LVb neurons can be labeled and stimulated with light. The authors showed that these neurons are largely co-labeled with PCP4, a marker for LVb. They compared the apical dendritic extent from TG labeled cells (LVb) and Nac retrogradely labeled cells (LVa) in medial and lateral EC. The intrinsic electrophysiological properties of LVa and LVb neurons were measured and used for PCA showing …
Reviewer #3:
The present manuscript focuses on a subpopulation of layer 5 neurons in medial and lateral entorhinal cortex and its functional connections to target neurons in layers 2, 3 and 5. The authors show a difference in LVb-to-LVa connectivity between MEC and LEC. The results suggest that the entorhinal output circuit via LVb-to-LVa is present primarily in LEC.
The work relies on and is made possible by a newly described transgenic mouse (TG) where LVb neurons can be labeled and stimulated with light. The authors showed that these neurons are largely co-labeled with PCP4, a marker for LVb. They compared the apical dendritic extent from TG labeled cells (LVb) and Nac retrogradely labeled cells (LVa) in medial and lateral EC. The intrinsic electrophysiological properties of LVa and LVb neurons were measured and used for PCA showing segregation according to sublayer and region. The axonal distribution and translaminar local connections of LVb neurons form the TG mice were then examined. Cells were recorded in vitro and filled with biocytin, both from MEC and LEC, with multiple cells in the same slice, documented with high quality images. The study of the LVb translaminar connectivity via a direct comparison of postsynaptic responses in neurons in different layers in the same slice is the gold standard for this type of functional connectivity analysis. There is also an investigation of mixed excitatory-inhibitory postsynaptic response sequences, and evidence for a dorso-ventral gradient in LVb-to-LVa connectivity in MEC is given.
The study combines TG mice, immunolabeling, retrograde labeling, morphological analysis and in vitro electrophysiology with optogenetic photo-stimulation. While it builds on already published work by the same group and others, by comparing the local target neurons of LVb in MEC and in LEC, the manuscript provides a unique contribution to the literature on the laminar circuit organization in the Entorhinal Cortex. In view of the central position of this area in the hippocampal memory systems of the rodent brain, these results are of interest to a broader neuroscience audience. It is also a nice example of a bottom-up approach, where data on the entorhinal translaminar connectivity may influence and constrain theories of hippocampal-cortical processing.
Major Comments:
Almost all TG labelled neurons are positive for PCP4 but not so vice versa, only 45.9 and 30.P% of PCP4 + neurons in LEC and MEC are labeled in the TG mouse (page 5) leaving open the possibility that the TG mouse labels a (specific?) subset of LVb neurons. Did you test whether TG labeled LVb cells co-localize with Ctip2 ?
The direct comparison of translaminar connectivity of LVb neurons is very convincing. But if your main conclusion (title) concerns the difference of LVb-to-LVa connectivity between MEC and LEC, it would have been more appropriate to test that in the same slice. While the data strongly support conclusions on the laminar differences of LVb connectivity, the evidence for differences in LVb-to-LVa connectivity between MEC and LEC is a bit weaker and more indirect.
Postsynaptic responses (in mV) in LEC are about twice as high in amplitude as in MEC (Fig. 4E vs Fig 5E), across all layers. Please discuss possible reasons, and possible impact on the circuit function. Is the probability to initiate action potentials higher in LEC ?
Give the onset latencies of postsynaptic excitatory potentials induced by LVb photostimulation. Are latencies monosynaptic? Or also polysynaptic? Ideally this could be tested by applying a cocktail of TTX-4-AP.
Figure 4 S3, Fig 5 S2. Analysis of inhibition. What is the cut-off criteria to say inhibition is present or not? It might be more appropriate to give the I/E ratio.
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Reviewer #2:
The study investigates key components of the entorhinal circuits through which signals from the hippocampus are relayed to the neocortex. The question addressed is important but the stated claim that layer 5b (L5b) to layer 5a (L5a) connections mediate hippocampal-cortical outputs in LEC but not MEC appears to be an over-interpretation of the data. First, the experiments do not test hippocampal to L5a connections, but instead look at L5b to L5a connections. Second, the data provide evidence that there are L5b to L5a projections in LEC and MEC, which contradicts the claim made in the title. These projections do appear denser in LEC under the experimental conditions used, but possible technical explanations for the difference are not carefully addressed. If these technical concerns were addressed, and the conclusions modified …
Reviewer #2:
The study investigates key components of the entorhinal circuits through which signals from the hippocampus are relayed to the neocortex. The question addressed is important but the stated claim that layer 5b (L5b) to layer 5a (L5a) connections mediate hippocampal-cortical outputs in LEC but not MEC appears to be an over-interpretation of the data. First, the experiments do not test hippocampal to L5a connections, but instead look at L5b to L5a connections. Second, the data provide evidence that there are L5b to L5a projections in LEC and MEC, which contradicts the claim made in the title. These projections do appear denser in LEC under the experimental conditions used, but possible technical explanations for the difference are not carefully addressed. If these technical concerns were addressed, and the conclusions modified appropriately, then I think this study could be very important for the field and would complement well recent work from several labs that collectively suggests that information processing in deep layers of MEC is more complex than has been appreciated (e.g. Sürmeli et al. 2015, Ohara et al. 2018, Wozny et al. 2018, Rozov et al. 2020). Major Concerns:
An impressive component of the study is the introduction of a new mouse line that labels neurons in layer 5b of MEC and LEC. However, in each area the line appears to label only a subset (30-50%) of the principal cell population. It's unclear whether the unlabelled neurons have similar connectivity to the labelled neurons. If the unlabelled neurons are a distinct subpopulation then it's difficult to see how the experiments presented could support the conclusion that L5b does not project to L5a; perhaps there is a projection mediated by the unlabelled neurons? I don't think the authors need to include experiments to investigate the unlabelled population, but given that the labelling is incomplete they should be more cautious about generalising from data obtained with the line.
For experiments using the AAV conditionally expressing oChIEF-citrine, the extent to which the injections are specific to LEC/MEC is unclear. This is a particular concern for injections into LEC where the possibility that perirhinal or postrhinal cortex are also labelled needs to be carefully considered. For example, in Figure 3D it appears the virus has spread to the perirhinal cortex. If this is the case then axonal projections/responses could originate there rather than from L5b of LEC. I suggest excluding any experiments where there is any suggestion of expression outside LEC/MEC or where this can not be ruled out through verification of the labelling. Alternatively, one might include control experiments in which the AAV is targeted to the perirhinal and postrhinal cortex. Similar concerns should be addressed for injections that target the MEC to rule out spread to the pre/parasubiculum.
It appears likely from the biocytin fills shown that the apical dendrites of some of the recorded L5a neurons have been cut (e.g. Figure 4A, Figure 4-Supplement 1D, neuron v). Where the apical dendrite is clearly intact and undamaged synaptic responses to activation of L5b neurons are quite clear (e.g. Figure 4-Supplement 1D, neuron x). Given that axons of L5b cells branch extensively in L3, it is possible that any synapses they make with L5a neurons would be on their apical dendrites within L3. It therefore seems important to restrict the analysis only to L5a neurons with intact apical dendrites; a reasonable criteria would be that the dendrite extends through L3 at a reasonable distance (> 30 μm?) below the surface of the slice.
Throughout the manuscript the data is over-interpreted. Here are some examples:
The title over-extrapolates from the results and should be changed. A more accurate title would be along the lines of "Evidence that L5b to L5a connections are more effective in lateral compared to medial entorhinal cortex".
"the conclusion that the dorsal parts of MEC lack the canonical hippocampal-cortical output system" seems over-stated given the evidence (see comments above).
Discussion, para 1, "Our key finding is that LEC and MEC are strikingly different with respect to the hippocampal-cortical pathway mediated by LV neurons, in that we obtained electrophysiological evidence for the presence of this postulated crucial circuit in LEC, but not in MEC". This is misleading as there is also evidence for L5b to L5a connections in MEC, although this projection may be relatively weak. Recent work by Rozov et al. demonstrating a projection from intermediate hippocampus to L5a provides good evidence for an alternative model in which MEC does relay hippocampal outputs. This needs to be considered.
- What proportion of responses are mono-synaptic? How was this tested?
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Reviewer #1:
The current study by Ohara et al. describes differences in the connectivity patterns between LVb to LVa. The study builds on the authors previous study (Ohara et al., 2018) where they showed the intrinsic connectivity of LVb neurons in the MEC and LEC. The focus of the current study is the difference the authors observed in the strengths of connectivity between LVb and LVa in the MEC and LEC. The authors suggest that the in MEC Vb neurons do not provide substantial direct input to LVa neurons. The manuscript emphasizes the functional importance of difference as the authors suggest that "...hippocampal -cortex output circuit is present only in LEC, suggesting that episodic systems consolidation predominantly uses LEC-derived information and not allocentric spatial information from MEC." The study uses a newly developed mouse …
Reviewer #1:
The current study by Ohara et al. describes differences in the connectivity patterns between LVb to LVa. The study builds on the authors previous study (Ohara et al., 2018) where they showed the intrinsic connectivity of LVb neurons in the MEC and LEC. The focus of the current study is the difference the authors observed in the strengths of connectivity between LVb and LVa in the MEC and LEC. The authors suggest that the in MEC Vb neurons do not provide substantial direct input to LVa neurons. The manuscript emphasizes the functional importance of difference as the authors suggest that "...hippocampal -cortex output circuit is present only in LEC, suggesting that episodic systems consolidation predominantly uses LEC-derived information and not allocentric spatial information from MEC." The study uses a newly developed mouse line to investigate connectivity differences, this is a nice technical approach and the experimental data is of high quality. While the data is solid, the authors tend to over-interpret their findings from the functional point of view. While the observed difference is quite interesting, it is unclear what the impact is on information flow in the MEC and LEC and to which degree they differ functionally. The authors assume major differences and their work is framed based on these expected differences, but the manuscript does not provide data that would demonstrate functionally distinct features.
Major Comments:
Throughout the text the authors treat their findings as if it was 'all-or-none' i.e the LEC has a direct connection between LVb and LVa while the MEC does not. This does not seem to be the case based on their data, the data shows that connectivity in the MEC is less robust but it is definitely there. The difference seems to be quantitative and not qualitative.
Due to this problem, the authors seem to be over-interpreting their data by suggesting that the information flow must be significantly different conceptually in the LEC and the MEC and this would have important implications for memory consolidation. It is impossible to draw these conclusions based on the data presented, as there are no experiments investigating the functional, network level consequences of these connectivity differences.
The electrophysiology experiments provide information about the basic parameters of the investigated cells, but these lack a physiological context that would allow the authors to evaluate the consequences of these differences on information flow and/or processing in the MEC and the LEC.
The study is using a novel transgenic mouse line to differentiate between LVb and LVa neurons, while this is definitely a strength of the study, this strategy allows the authors to visualize ~50% of LEC and ~30% MEC neurons. Since the authors aim to prove a negative (MEC does not have direct connection) the fact that ~70% of the neurons are not labelled could be problematic.
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Summary: The study addresses a fundamentally important question regarding the connectivity of LVb and LVa in the medial and lateral entorhinal cortex. The authors suggest that LVb to LVa connection exists in the LEC but not in the MEC. This finding would have important implications on studies investigating circuits functions between the hippocampus and the EC. All three reviewers found the central question important and the data novel. However, there are several technical issues that limit the robustness of the authors claim.
While the transgenic animal used in these experiments is elegant and novel, it only labels a subpopulation of the neurons. There is a possibility of selective labelling of neurons with distinct connectivity patterns. The authors would need to show that their approach is not leading to false negative results due to …
Summary: The study addresses a fundamentally important question regarding the connectivity of LVb and LVa in the medial and lateral entorhinal cortex. The authors suggest that LVb to LVa connection exists in the LEC but not in the MEC. This finding would have important implications on studies investigating circuits functions between the hippocampus and the EC. All three reviewers found the central question important and the data novel. However, there are several technical issues that limit the robustness of the authors claim.
While the transgenic animal used in these experiments is elegant and novel, it only labels a subpopulation of the neurons. There is a possibility of selective labelling of neurons with distinct connectivity patterns. The authors would need to show that their approach is not leading to false negative results due to the selective visualization of those neurons that project more modestly to the LVa.
The specificity of the injection to the LEC/MEC should be better documented and potential spread to the perirhinal or postrhinal cortex carefully excluded.
The findings are presented as LVb to LVa connection did not exist at all in the MEC, however the data shows that the connection is there but it is significantly less dense than in the LEC. Given the graded finding, if the authors aim to show their central claim regarding the lack of mediation of hippocampo-cortical outputs by this connection in the MEC, this would require the addition of functional studies.
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